Myocardial Scaffold-based Cardiac Tissue Engineering: Application of Coordinated Mechanical and Electrical Stimulations

PubMed Central

Wang, Bo; Wang, Guangjun; To, Filip; Butler, J. Ryan; Claude, Andrew; McLaughlin, Ronald M.; Williams, Lakiesha N.; de Jongh Curry, Amy L.; Liao, Jun

2013-01-01

Recently, we have developed an optimal decellularization protocol to generate 3D porcine myocardial scaffolds, which preserved natural extracellular matrix structure, mechanical anisotropy, and vasculature templates, and also showed good cell recellularization and differentiation potential. In this study, a multi-stimulation bioreactor was built to provide coordinated mechanical and electrical stimulations for facilitating stem cell differentiation and cardiac construct development. The acellular myocardial scaffolds were seeded with mesenchymal stem cells (106 cells/ml) by needle injection and subjected to 5-azacytidine treatment (3 μmol/L, 24 h) and various bioreactor conditioning protocols. We found that, after 2-day culture with mechanical (20% strain) and electrical stimulation (5 V, 1 Hz), high cell density and good cell viability were observed in the reseeded scaffold. Immunofluorescence staining demonstrated that the differentiated cells showed cardiomyocyte-like phenotype, by expressing sarcomeric α-actinin, myosin heavy chain, cardiac troponin T, connexin-43, and N-cadherin. Biaxial mechanical testing demonstrated that positive tissue remodeling took place after 2-day bioreactor conditioning (20% strain + 5 V, 1 Hz); passive mechanical properties of the 2-day and 4-day tissue constructs were comparable to the tissue constructs produced by stirring reseeding followed by 2-week static culture, implying the effectiveness and efficiency of the coordinated simulations in promoting tissue remodeling. In short, the synergistic stimulations might be beneficial not only for the quality of cardiac construct development, but also for patients by reducing the waiting time in future clinical scenarios. PMID:23923967

Fiber-Based Tissue Engineering: Progress, Challenges, and Opportunities

PubMed Central

Tamayol, Ali; Akbari, Mohsen; Annabi, Nasim; Paul, Arghya; Khademhosseini, Ali; Juncker, David

2013-01-01

Tissue engineering aims to improve the function of diseased or damaged organs by creating biological substitutes. To fabricate a functional tissue, the engineered construct should mimic the physiological environment including its structural, topographical, and mechanical properties. Moreover, the construct should facilitate nutrients and oxygen diffusion as well as removal of metabolic waste during tissue regeneration. In the last decade, fiber-based techniques such as weaving, knitting, braiding, as well as electrospinning, and direct writing have emerged as promising platforms for making 3D tissue constructs that can address the above mentioned challenges. Here, we critically review the techniques used to form cell-free and cell-laden fibers and to assemble them into scaffolds. We compare their mechanical properties, morphological features and biological activity. We discuss current challenges and future opportunities of fiber-based tissue engineering (FBTE) for use in research and clinical practice. PMID:23195284

Bioprinting a cardiac valve.

PubMed

Jana, Soumen; Lerman, Amir

2015-12-01

Heart valve tissue engineering could be a possible solution for the limitations of mechanical and biological prostheses, which are commonly used for heart valve replacement. In tissue engineering, cells are seeded into a 3-dimensional platform, termed the scaffold, to make the engineered tissue construct. However, mimicking the mechanical and spatial heterogeneity of a heart valve structure in a fabricated scaffold with uniform cell distribution is daunting when approached conventionally. Bioprinting is an emerging technique that can produce biological products containing matrix and cells, together or separately with morphological, structural and mechanical diversity. This advance increases the possibility of fabricating the structure of a heart valve in vitro and using it as a functional tissue construct for implantation. This review describes the use of bioprinting technology in heart valve tissue engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

Rapid construction of mechanically- confined multi- cellular structures using dendrimeric intercellular linker.

PubMed

Mo, Xuejun; Li, Qiushi; Yi Lui, Lena Wai; Zheng, Baixue; Kang, Chiang Huen; Nugraha, Bramasta; Yue, Zhilian; Jia, Rui Rui; Fu, Hong Xia; Choudhury, Deepak; Arooz, Talha; Yan, Jie; Lim, Chwee Teck; Shen, Shali; Hong Tan, Choon; Yu, Hanry

2010-10-01

Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications. 2010 Elsevier Ltd. All rights reserved.

The materials used in bone tissue engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tereshchenko, V. P., E-mail: tervp@ngs.ru; Kirilova, I. A.; Sadovoy, M. A.

Bone tissue engineering looking for an alternative solution to the problem of skeletal injuries. The method is based on the creation of tissue engineered bone tissue equivalent with stem cells, osteogenic factors, and scaffolds - the carriers of these cells. For production of tissue engineered bone equivalent is advisable to create scaffolds similar in composition to natural extracellular matrix of the bone. This will provide optimal conditions for the cells, and produce favorable physico-mechanical properties of the final construction. This review article gives an analysis of the most promising materials for the manufacture of cell scaffolds. Biodegradable synthetic polymers aremore » the basis for the scaffold, but it alone cannot provide adequate physical and mechanical properties of the construction, and favorable conditions for the cells. Addition of natural polymers improves the strength characteristics and bioactivity of constructions. Of the inorganic compounds, to create cell scaffolds the most widely used calcium phosphates, which give the structure adequate stiffness and significantly increase its osteoinductive capacity. Signaling molecules do not affect the physico-mechanical properties of the scaffold, but beneficial effect is on the processes of adhesion, proliferation and differentiation of cells. Biodegradation of the materials will help to fulfill the main task of bone tissue engineering - the ability to replace synthetic construct by natural tissues that will restore the original anatomical integrity of the bone.« less

Monitoring fibrous scaffold guidance of three-dimensional collagen organisation using minimally-invasive second harmonic generation.

PubMed

Delaine-Smith, Robin M; Green, Nicola H; Matcher, Stephen J; MacNeil, Sheila; Reilly, Gwendolen C

2014-01-01

The biological and mechanical function of connective tissues is largely determined by controlled cellular alignment and therefore it seems appropriate that tissue-engineered constructs should be architecturally similar to the in vivo tissue targeted for repair or replacement. Collagen organisation dictates the tensile properties of most tissues and so monitoring the deposition of cell-secreted collagen as the construct develops is essential for understanding tissue formation. In this study, electrospun fibres with a random or high degree of orientation, mimicking two types of tissue architecture found in the body, were used to culture human fibroblasts for controlling cell alignment. The minimally-invasive technique of second harmonic generation was used with the aim of monitoring and profiling the deposition and organisation of collagen at different construct depths over time while construct mechanical properties were also determined over the culture period. It was seen that scaffold fibre organisation affected cell migration and orientation up to 21 days which in turn had an effect on collagen organisation. Collagen in random fibrous constructs was deposited in alternating configurations at different depths however a high degree of organisation was observed throughout aligned fibrous constructs orientated in the scaffold fibre direction. Three-dimensional second harmonic generation images showed that deposited collagen was more uniformly distributed in random constructs but aligned constructs were more organised and had higher intensities. The tensile properties of all constructs increased with increasing collagen deposition and were ultimately dictated by collagen organisation. This study highlights the importance of scaffold architecture for controlling the development of well-organised tissue engineered constructs and the usefulness of second harmonic generation imaging for monitoring collagen maturation in a minimally invasive manner.

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

PubMed Central

Skardal, Aleksander; Devarasetty, Mahesh; Kang, Hyun-Wook; Seol, Young-Joon; Forsythe, Steven D.; Bishop, Colin; Shupe, Thomas; Soker, Shay; Atala, Anthony

2016-01-01

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening. Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types. PMID:27166839

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink.

PubMed

Skardal, Aleksander; Devarasetty, Mahesh; Kang, Hyun-Wook; Seol, Young-Joon; Forsythe, Steven D; Bishop, Colin; Shupe, Thomas; Soker, Shay; Atala, Anthony

2016-04-21

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening. Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

Modeling the impact of scaffold architecture and mechanical loading on collagen turnover in engineered cardiovascular tissues.

PubMed

Argento, G; de Jonge, N; Söntjens, S H M; Oomens, C W J; Bouten, C V C; Baaijens, F P T

2015-06-01

The anisotropic collagen architecture of an engineered cardiovascular tissue has a major impact on its in vivo mechanical performance. This evolving collagen architecture is determined by initial scaffold microstructure and mechanical loading. Here, we developed and validated a theoretical and computational microscale model to quantitatively understand the interplay between scaffold architecture and mechanical loading on collagen synthesis and degradation. Using input from experimental studies, we hypothesize that both the microstructure of the scaffold and the loading conditions influence collagen turnover. The evaluation of the mechanical and topological properties of in vitro engineered constructs reveals that the formation of extracellular matrix layers on top of the scaffold surface influences the mechanical anisotropy on the construct. Results show that the microscale model can successfully capture the collagen arrangement between the fibers of an electrospun scaffold under static and cyclic loading conditions. Contact guidance by the scaffold, and not applied load, dominates the collagen architecture. Therefore, when the collagen grows inside the pores of the scaffold, pronounced scaffold anisotropy guarantees the development of a construct that mimics the mechanical anisotropy of the native cardiovascular tissue.

A modular approach to creating large engineered cartilage surfaces.

PubMed

Ford, Audrey C; Chui, Wan Fung; Zeng, Anne Y; Nandy, Aditya; Liebenberg, Ellen; Carraro, Carlo; Kazakia, Galateia; Alliston, Tamara; O'Connell, Grace D

2018-01-23

Native articular cartilage has limited capacity to repair itself from focal defects or osteoarthritis. Tissue engineering has provided a promising biological treatment strategy that is currently being evaluated in clinical trials. However, current approaches in translating these techniques to developing large engineered tissues remains a significant challenge. In this study, we present a method for developing large-scale engineered cartilage surfaces through modular fabrication. Modular Engineered Tissue Surfaces (METS) uses the well-known, but largely under-utilized self-adhesion properties of de novo tissue to create large scaffolds with nutrient channels. Compressive mechanical properties were evaluated throughout METS specimens, and the tensile mechanical strength of the bonds between attached constructs was evaluated over time. Raman spectroscopy, biochemical assays, and histology were performed to investigate matrix distribution. Results showed that by Day 14, stable connections had formed between the constructs in the METS samples. By Day 21, bonds were robust enough to form a rigid sheet and continued to increase in size and strength over time. Compressive mechanical properties and glycosaminoglycan (GAG) content of METS and individual constructs increased significantly over time. The METS technique builds on established tissue engineering accomplishments of developing constructs with GAG composition and compressive properties approaching native cartilage. This study demonstrated that modular fabrication is a viable technique for creating large-scale engineered cartilage, which can be broadly applied to many tissue engineering applications and construct geometries. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Bioreactor to Identify the Driving Mechanical Stimuli of Tissue Growth and Remodeling.

PubMed

van Kelle, Mathieu A J; Oomen, Pim J A; Bulsink, Jurgen A; Janssen-van den Broek, Marloes W J T; Lopata, Richard G P; Rutten, Marcel C M; Loerakker, Sandra; Bouten, Carlijn V C

2017-06-01

Tissue growth and remodeling are essential processes that should ensure long-term functionality of tissue-engineered (TE) constructs. Even though it is widely recognized that these processes strongly depend on mechanical stimuli, the underlying mechanisms of mechanically induced growth and remodeling are only partially understood. It is generally accepted that cells sense mechanical changes and respond by altering their surroundings, by means of extracellular matrix growth and remodeling, in an attempt to return to a certain preferred mechanical homeostatic state. However, the exact mechanical cues that trigger cells to synthesize and remodel their environment remain unclear. To identify the driving mechanical stimuli of these processes, it is critical to be able to temporarily follow the mechanical state of developing tissues under physiological loading conditions. Therefore, a novel "versatile tissue growth and remodeling" (Vertigro) bioreactor was developed that is capable of tissue culture and mechanical stimulation for a prolonged time period, while simultaneously performing mechanical testing. The Vertigro's unique two-chamber design allows easy, sterile handling of circular 3D TE constructs in a dedicated culture chamber, while a separate pressure chamber facilitates a pressure-driven dynamic loading regime during culture. As a proof-of-concept, temporal changes in the mechanical state of cultured tissues were quantified using nondestructive mechanical testing by means of a classical bulge test, in which the tissue displacement was tracked using ultrasound imaging. To demonstrate the successful development of the bioreactor system, compositional, structural, and geometrical changes were qualitatively and quantitatively assessed using a series of standard analysis techniques. With this bioreactor and associated mechanical analysis technique, a powerful toolbox has been developed to quantitatively study and identify the driving mechanical stimuli of engineered tissue growth and remodeling.

Mechanical properties and structure-function relationships of human chondrocyte-seeded cartilage constructs after in vitro culture.

PubMed

Middendorf, Jill M; Griffin, Darvin J; Shortkroff, Sonya; Dugopolski, Caroline; Kennedy, Stephen; Siemiatkoski, Joseph; Cohen, Itai; Bonassar, Lawrence J

2017-10-01

Autologous Chondrocyte Implantation (ACI) is a widely recognized method for the repair of focal cartilage defects. Despite the accepted use, problems with this technique still exist, including graft hypertrophy, damage to surrounding tissue by sutures, uneven cell distribution, and delamination. Modified ACI techniques overcome these challenges by seeding autologous chondrocytes onto a 3D scaffold and securing the graft into the defect. Many studies on these tissue engineered grafts have identified the compressive properties, but few have examined frictional and shear properties as suggested by FDA guidance. This study is the first to perform three mechanical tests (compressive, frictional, and shear) on human tissue engineered cartilage. The objective was to understand the complex mechanical behavior, function, and changes that occur with time in these constructs grown in vitro using compression, friction, and shear tests. Safranin-O histology and a DMMB assay both revealed increased sulfated glycosaminoglycan (sGAG) content in the scaffolds with increased maturity. Similarly, immunohistochemistry revealed increased lubricin localization on the construct surface. Confined compression and friction tests both revealed improved properties with increased construct maturity. Compressive properties correlated with the sGAG content, while improved friction coefficients were attributed to increased lubricin localization on the construct surfaces. In contrast, shear properties did not improve with increased culture time. This study suggests the various mechanical and biological properties of tissue engineered cartilage improve at different rates, indicating thorough mechanical evaluation of tissue engineered cartilage is critical to understanding the performance of repaired cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2298-2306, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Combined effects of scaffold stiffening and mechanical preconditioning cycles on construct biomechanics, gene expression, and tendon repair biomechanics.

PubMed

Nirmalanandhan, Victor Sanjit; Juncosa-Melvin, Natalia; Shearn, Jason T; Boivin, Gregory P; Galloway, Marc T; Gooch, Cynthia; Bradica, Gino; Butler, David L

2009-08-01

Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases both construct stiffness and the biomechanical properties of the repair tissue after surgery. When optimized using response surface methodology, our results indicate that a mechanical stimulus with three components (2.4% strain, 3000 cycles/day, and one cycle repetition) produced the highest in vitro linear stiffness. Such positive correlations between construct and repair stiffness after surgery suggest that enhancing structural stiffness before surgery could not only accelerate repair stiffness but also prevent premature failures in culture due to poor mechanical integrity. In this study, we examined the combined effects of scaffold crosslinking and subsequent mechanical stimulation on construct mechanics and biology. Autologous tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 15 New Zealand white rabbits on type I collagen sponges that had undergone additional dehydrothermal crosslinking (termed ADHT in this manuscript). Both constructs from each rabbit were mechanically stimulated for 8h/day for 12 consecutive days with half receiving 100 cycles/day and the other half receiving 3000 cycles/day. These paired MSC-collagen autologous constructs were then implanted in bilateral full-thickness, full-length defects in the central third of rabbit patellar tendons. Increasing the number of in vitro cycles/day delivered to the ADHT constructs in culture produced no differences in stiffness or gene expression and no changes in biomechanical properties or histology 12 weeks after surgery. Compared to MSC-based repairs from a previous study that received no additional treatment in culture, ADHT crosslinking of the scaffolds actually lowered the 12-week repair stiffness. Thus, while ADHT crosslinking may initially stiffen a construct in culture, this specific treatment also appears to mask any benefits of stimulation among repairs postsurgery. Our findings emphasize the importance of properly preconditioning a scaffold to better control/modulate MSC differentiation in vitro and to further enhance repair outcome in vivo.

Temporal development of near-native functional properties and correlations with qMRI in self-assembling fibrocartilage treated with exogenous lysyl oxidase homolog 2.

PubMed

Hadidi, Pasha; Cissell, Derek D; Hu, Jerry C; Athanasiou, Kyriacos A

2017-12-01

Advances in cartilage tissue engineering have led to constructs with mechanical integrity and biochemical composition increasingly resembling that of native tissues. In particular, collagen cross-linking with lysyl oxidase has been used to significantly enhance the mechanical properties of engineered neotissues. In this study, development of collagen cross-links over time, and correlations with tensile properties, were examined in self-assembling neotissues. Additionally, quantitative MRI metrics were examined in relation to construct mechanical properties as well as pyridinoline cross-link content and other engineered tissue components. Scaffold-free meniscus fibrocartilage was cultured in the presence of exogenous lysyl oxidase, and assessed at multiple time points over 8weeks starting from the first week of culture. Engineered constructs demonstrated a 9.9-fold increase in pyridinoline content, reaching 77% of native tissue values, after 8weeks of culture. Additionally, engineered tissues reached 66% of the Young's modulus in the radial direction of native tissues. Further, collagen cross-links were found to correlate with tensile properties, contributing 67% of the tensile strength of engineered neocartilages. Finally, examination of quantitative MRI metrics revealed several correlations with mechanical and biochemical properties of engineered constructs. This study displays the importance of culture duration for collagen cross-link formation, and demonstrates the potential of quantitative MRI in investigating properties of engineered cartilages. This is the first study to demonstrate near-native cross-link content in an engineered tissue, and the first study to quantify pyridinoline cross-link development over time in a self-assembling tissue. Additionally, this work shows the relative contributions of collagen and pyridinoline to the tensile properties of collagenous tissue for the first time. Furthermore, this is the first investigation to identify a relationship between qMRI metrics and the pyridinoline cross-link content of an engineered collagenous tissue. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Enhanced nutrient transport improves the depth-dependent properties of tri-layered engineered cartilage constructs with zonal co-culture of chondrocytes and MSCs.

PubMed

Kim, Minwook; Farrell, Megan J; Steinberg, David R; Burdick, Jason A; Mauck, Robert L

2017-08-01

Biomimetic design in cartilage tissue engineering is a challenge given the complexity of the native tissue. While numerous studies have generated constructs with near-native bulk properties, recapitulating the depth-dependent features of native tissue remains a challenge. Furthermore, limitations in nutrient transport and matrix accumulation in engineered constructs hinders maturation within the central core of large constructs. To overcome these limitations, we fabricated tri-layered constructs that recapitulate the depth-dependent cellular organization and functional properties of native tissue using zonally derived chondrocytes co-cultured with MSCs. We also introduced porous hollow fibers (HFs) and HFs/cotton threads to enhance nutrient transport. Our results showed that tri-layered constructs with depth-dependent organization and properties could be fabricated. The addition of HFs or HFs/threads improved matrix accumulation in the central core region. With HF/threads, the local modulus in the deep region of tri-layered constructs nearly matched that of native tissue, though the properties in the central regions remained lower. These constructs reproduced the zonal organization and depth-dependent properties of native tissue, and demonstrate that a layer-by-layer fabrication scheme holds promise for the biomimetic repair of focal cartilage defects. Articular cartilage is a highly organized tissue driven by zonal heterogeneity of cells, extracellular matrix proteins and fibril orientations, resulting in depth-dependent mechanical properties. Therefore, the recapitulation of the functional properties of native cartilage in a tissue engineered construct requires such a biomimetic design of the morphological organization, and this has remained a challenge in cartilage tissue engineering. This study demonstrates that a layer-by-layer fabrication scheme, including co-cultures of zone-specific articular CHs and MSCs, can reproduce the depth-dependent characteristics and mechanical properties of native cartilage while minimizing the need for large numbers of chondrocytes. In addition, introduction of a porous hollow fiber (combined with a cotton thread) enhanced nutrient transport and depth-dependent properties of the tri-layered construct. Such a tri-layered construct may provide critical advantages for focal cartilage repair. These constructs hold promise for restoring native tissue structure and function, and may be beneficial in terms of zone-to-zone integration with adjacent host tissue and providing more appropriate strain transfer after implantation. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Mechanical Stimulation of Adipose-Derived Stem Cells for Functional Tissue Engineering of the Musculoskeletal System via Cyclic Hydrostatic Pressure, Simulated Microgravity, and Cyclic Tensile Strain.

PubMed

Nordberg, Rachel C; Bodle, Josie C; Loboa, Elizabeth G

2018-01-01

It is critical that human adipose stem cell (hASC) tissue-engineering therapies possess appropriate mechanical properties in order to restore function of the load bearing tissues of the musculoskeletal system. In an effort to elucidate the hASC response to mechanical stimulation and develop mechanically robust tissue engineered constructs, recent research has utilized a variety of mechanical loading paradigms including cyclic tensile strain, cyclic hydrostatic pressure, and mechanical unloading in simulated microgravity. This chapter describes methods for applying these mechanical stimuli to hASC to direct differentiation for functional tissue engineering of the musculoskeletal system.

Preparation of acellular myocardial scaffolds with well-preserved cardiomyocyte lacunae, and method for applying mechanical and electrical simulation to tissue construct.

PubMed

Wang, Bo; Williams, Lakiesha N; de Jongh Curry, Amy L; Liao, Jun

2014-01-01

Cardiac tissue engineering/regeneration using decellularized myocardium has attracted great research attention due to its potential benefit for myocardial infarction (MI) treatment. Here we describe an optimal decellularization protocol to generate 3D porcine myocardial scaffolds with well-preserved cardiomyocyte lacunae and a multi-stimulation bioreactor that is able to provide coordinated mechanical and electrical stimulation for facilitating cardiac construct development.

Engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization.

PubMed

de Jonge, Nicky; Baaijens, Frank P T; Bouten, Carlijn V C

2013-10-28

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner. Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

A novel bioreactor for the generation of highly aligned 3D skeletal muscle-like constructs through orientation of fibrin via application of static strain.

PubMed

Heher, Philipp; Maleiner, Babette; Prüller, Johanna; Teuschl, Andreas Herbert; Kollmitzer, Josef; Monforte, Xavier; Wolbank, Susanne; Redl, Heinz; Rünzler, Dominik; Fuchs, Christiane

2015-09-01

The generation of functional biomimetic skeletal muscle constructs is still one of the fundamental challenges in skeletal muscle tissue engineering. With the notion that structure strongly dictates functional capabilities, a myriad of cell types, scaffold materials and stimulation strategies have been combined. To further optimize muscle engineered constructs, we have developed a novel bioreactor system (MagneTissue) for rapid engineering of skeletal muscle-like constructs with the aim to resemble native muscle in terms of structure, gene expression profile and maturity. Myoblasts embedded in fibrin, a natural hydrogel that serves as extracellular matrix, are subjected to mechanical stimulation via magnetic force transmission. We identify static mechanical strain as a trigger for cellular alignment concomitant with the orientation of the scaffold into highly organized fibrin fibrils. This ultimately yields myotubes with a more mature phenotype in terms of sarcomeric patterning, diameter and length. On the molecular level, a faster progression of the myogenic gene expression program is evident as myogenic determination markers MyoD and Myogenin as well as the Ca(2+) dependent contractile structural marker TnnT1 are significantly upregulated when strain is applied. The major advantage of the MagneTissue bioreactor system is that the generated tension is not exclusively relying on the strain generated by the cells themselves in response to scaffold anchoring but its ability to subject the constructs to individually adjustable strain protocols. In future work, this will allow applying mechanical stimulation with different strain regimes in the maturation process of tissue engineered constructs and elucidating the role of mechanotransduction in myogenesis. Mechanical stimulation of tissue engineered skeletal muscle constructs is a promising approach to increase tissue functionality. We have developed a novel bioreactor-based 3D culture system, giving the user the possibility to apply different strain regimes like static, cyclic or ramp strain to myogenic precursor cells embedded in a fibrin scaffold. Application of static mechanical strain leads to alignment of fibrin fibrils along the axis of strain and concomitantly to highly aligned myotube formation. Additionally, the pattern of myogenic gene expression follows the temporal progression observed in vivo with a more thorough induction of the myogenic program when static strain is applied. Ultimately, the strain protocol used in this study results in a higher degree of muscle maturity demonstrated by enhanced sarcomeric patterning and increased myotube diameter and length. The introduced bioreactor system enables new possibilities in muscle tissue engineering as longer cultivation periods and different strain applications will yield tissue engineered muscle-like constructs with improved characteristics in regard to functionality and biomimicry. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Mechanical testing of hydrogels in cartilage tissue engineering: beyond the compressive modulus.

PubMed

Xiao, Yinghua; Friis, Elizabeth A; Gehrke, Stevin H; Detamore, Michael S

2013-10-01

Injuries to articular cartilage result in significant pain to patients and high medical costs. Unfortunately, cartilage repair strategies have been notoriously unreliable and/or complex. Biomaterial-based tissue-engineering strategies offer great promise, including the use of hydrogels to regenerate articular cartilage. Mechanical integrity is arguably the most important functional outcome of engineered cartilage, although mechanical testing of hydrogel-based constructs to date has focused primarily on deformation rather than failure properties. In addition to deformation testing, as the field of cartilage tissue engineering matures, this community will benefit from the addition of mechanical failure testing to outcome analyses, given the crucial clinical importance of the success of engineered constructs. However, there is a tremendous disparity in the methods used to evaluate mechanical failure of hydrogels and articular cartilage. In an effort to bridge the gap in mechanical testing methods of articular cartilage and hydrogels in cartilage regeneration, this review classifies the different toughness measurements for each. The urgency for identifying the common ground between these two disparate fields is high, as mechanical failure is ready to stand alongside stiffness as a functional design requirement. In comparing toughness measurement methods between hydrogels and cartilage, we recommend that the best option for evaluating mechanical failure of hydrogel-based constructs for cartilage tissue engineering may be tensile testing based on the single edge notch test, in part because specimen preparation is more straightforward and a related American Society for Testing and Materials (ASTM) standard can be adopted in a fracture mechanics context.

A 3D bioprinting system to produce human-scale tissue constructs with structural integrity.

PubMed

Kang, Hyun-Wook; Lee, Sang Jin; Ko, In Kap; Kengla, Carlos; Yoo, James J; Atala, Anthony

2016-03-01

A challenge for tissue engineering is producing three-dimensional (3D), vascularized cellular constructs of clinically relevant size, shape and structural integrity. We present an integrated tissue-organ printer (ITOP) that can fabricate stable, human-scale tissue constructs of any shape. Mechanical stability is achieved by printing cell-laden hydrogels together with biodegradable polymers in integrated patterns and anchored on sacrificial hydrogels. The correct shape of the tissue construct is achieved by representing clinical imaging data as a computer model of the anatomical defect and translating the model into a program that controls the motions of the printer nozzles, which dispense cells to discrete locations. The incorporation of microchannels into the tissue constructs facilitates diffusion of nutrients to printed cells, thereby overcoming the diffusion limit of 100-200 μm for cell survival in engineered tissues. We demonstrate capabilities of the ITOP by fabricating mandible and calvarial bone, cartilage and skeletal muscle. Future development of the ITOP is being directed to the production of tissues for human applications and to the building of more complex tissues and solid organs.

A novel bioprinting method and system for forming hybrid tissue engineering constructs.

PubMed

Shanjani, Y; Pan, C C; Elomaa, L; Yang, Y

2015-12-18

Three dimensional (3D) bioprinting is a promising approach to form tissue engineering constructs (TECs) via positioning biomaterials, growth factors, and cells with controlled spatial distribution due to its layer-by-layer manufacturing nature. Hybrid TECs composed of relatively rigid porous scaffolds for structural and mechanical integrity and soft hydrogels for cell- and growth factor-loading have a tremendous potential to tissue regeneration under mechanical loading. However, despite excessive progress in the field, the current 3D bioprinting techniques and systems fall short in integration of such soft and rigid multifunctional components. Here we present a novel 3D hybrid bioprinting technology (Hybprinter) and its capability enabling integration of soft and rigid components for TECs. Hybprinter employs digital light processing-based stereolithography (DLP-SLA) and molten material extrusion techniques for soft and rigid materials, respectively. In this study, poly-ethylene glycol diacrylate (PEGDA) and poly-(ε-caprolactone) (PCL) were used as a model material for soft hydrogel and rigid scaffold, respectively. It was shown that geometrical accuracy, swelling ratio and mechanical properties of the hydrogel component can be tailored by DLP-SLA module. We have demonstrated the printability of variety of complex hybrid construct designs using Hybprinter technology and characterized the mechanical properties and functionality of such constructs. The compressive mechanical stiffness of a hybrid construct (90% hydrogel) was significantly higher than hydrogel itself (∼6 MPa versus 100 kPa). In addition, viability of cells incorporated within the bioprinted hybrid constructs was determined approximately 90%. Furthermore, a functionality of a hybrid construct composed of porous scaffold with an embedded hydrogel conduit was characterized for vascularized tissue engineering applications. High material diffusion and high cell viability in about 2.5 mm distance surrounding the conduit indicated that culture media effectively diffused through the conduit and fed the cells. The results suggest that the developed technology is potent to form functional TECs composed of rigid and soft biomaterials.

No-cost manual method for preparation of tissue microarrays having high quality comparable to semiautomated methods.

PubMed

Foda, Abd Al-Rahman Mohammad

2013-05-01

Manual tissue microarray (TMA) construction had been introduced to avoid the high cost of automated and semiautomated techniques. The cheapest and simplest technique for constructing manual TMA was that of using mechanical pencil tips. This study was carried out to modify this method, aiming to raise its quality to reach that of expensive ones. Some modifications were introduced to Shebl's technique. Two conventional mechanical pencil tips of different diameters were used to construct the recipient blocks. A source of mild heat was used, and blocks were incubated at 38°C overnight. With our modifications, 3 high-density TMA blocks were constructed. We successfully performed immunostaining without substantial tissue loss. Our modifications increased the number of cores per block and improved the stability of the cores within the paraffin block. This new, modified technique is a good alternative for expensive machines in many laboratories.

Bone scaffolds with homogeneous and discrete gradient mechanical properties.

PubMed

Jelen, C; Mattei, G; Montemurro, F; De Maria, C; Mattioli-Belmonte, M; Vozzi, G

2013-01-01

Bone TE uses a scaffold either to induce bone formation from surrounding tissue or to act as a carrier or template for implanted bone cells or other agents. We prepared different bone tissue constructs based on collagen, gelatin and hydroxyapatite using genipin as cross-linking agent. The fabricated construct did not present a release neither of collagen neither of genipin over its toxic level in the surrounding aqueous environment. Each scaffold has been mechanically characterized with compression, swelling and creep tests, and their respective viscoelastic mechanical models were derived. Mechanical characterization showed a practically elastic behavior of all samples and that compressive elastic modulus basically increases as content of HA increases, and it is strongly dependent on porosity and water content. Moreover, by considering that gradients in cellular and extracellular architecture as well as in mechanical properties are readily apparent in native tissues, we developed discrete functionally graded scaffolds (discrete FGSs) in order to mimic the graded structure of bone tissue. These new structures were mechanically characterized showing a marked anisotropy as the native bone tissue. Results obtained have shown FGSs could represent valid bone substitutes. Copyright © 2012 Elsevier B.V. All rights reserved.

Predicting cell viability within tissue scaffolds under equiaxial strain: multi-scale finite element model of collagen-cardiomyocytes constructs.

PubMed

Elsaadany, Mostafa; Yan, Karen Chang; Yildirim-Ayan, Eda

2017-06-01

Successful tissue engineering and regenerative therapy necessitate having extensive knowledge about mechanical milieu in engineered tissues and the resident cells. In this study, we have merged two powerful analysis tools, namely finite element analysis and stochastic analysis, to understand the mechanical strain within the tissue scaffold and residing cells and to predict the cell viability upon applying mechanical strains. A continuum-based multi-length scale finite element model (FEM) was created to simulate the physiologically relevant equiaxial strain exposure on cell-embedded tissue scaffold and to calculate strain transferred to the tissue scaffold (macro-scale) and residing cells (micro-scale) upon various equiaxial strains. The data from FEM were used to predict cell viability under various equiaxial strain magnitudes using stochastic damage criterion analysis. The model validation was conducted through mechanically straining the cardiomyocyte-encapsulated collagen constructs using a custom-built mechanical loading platform (EQUicycler). FEM quantified the strain gradients over the radial and longitudinal direction of the scaffolds and the cells residing in different areas of interest. With the use of the experimental viability data, stochastic damage criterion, and the average cellular strains obtained from multi-length scale models, cellular viability was predicted and successfully validated. This methodology can provide a great tool to characterize the mechanical stimulation of bioreactors used in tissue engineering applications in providing quantification of mechanical strain and predicting cellular viability variations due to applied mechanical strain.

Challenges in engineering osteochondral tissue grafts with hierarchical structures.

PubMed

Gadjanski, Ivana; Vunjak-Novakovic, Gordana

2015-01-01

A major hurdle in treating osteochondral (OC) defects is the different healing abilities of two types of tissues involved - articular cartilage and subchondral bone. Biomimetic approaches to OC-construct engineering, based on recapitulation of biological principles of tissue development and regeneration, have potential for providing new treatments and advancing fundamental studies of OC tissue repair. This review on state of the art in hierarchical OC tissue graft engineering is focused on tissue engineering approaches designed to recapitulate the native milieu of cartilage and bone development. These biomimetic systems are discussed with relevance to bioreactor cultivation of clinically sized, anatomically shaped human cartilage/bone constructs with physiologic stratification and mechanical properties. The utility of engineered OC tissue constructs is evaluated for their use as grafts in regenerative medicine, and as high-fidelity models in biological research. A major challenge in engineering OC tissues is to generate a functionally integrated stratified cartilage-bone structure starting from one single population of mesenchymal cells, while incorporating perfusable vasculature into the bone, and in bone-cartilage interface. To this end, new generations of advanced scaffolds and bioreactors, implementation of mechanical loading regimens and harnessing of inflammatory responses of the host will likely drive the further progress.

Nanoengineered Ionic-Covalent Entanglement (NICE) Bioinks for 3D Bioprinting.

PubMed

Chimene, David; Peak, Charles W; Gentry, James L; Carrow, James K; Cross, Lauren M; Mondragon, Eli; Cardoso, Guinea B; Kaunas, Roland; Gaharwar, Akhilesh K

2018-03-28

We introduce an enhanced nanoengineered ionic-covalent entanglement (NICE) bioink for the fabrication of mechanically stiff and elastomeric 3D biostructures. NICE bioink formulations combine nanocomposite and ionic-covalent entanglement (ICE) strengthening mechanisms to print customizable cell-laden constructs for tissue engineering with high structural fidelity and mechanical stiffness. Nanocomposite and ICE strengthening mechanisms complement each other through synergistic interactions, improving mechanical strength, elasticity, toughness, and flow properties beyond the sum of the effects of either reinforcement technique alone. Herschel-Bulkley flow behavior shields encapsulated cells from excessive shear stresses during extrusion. The encapsulated cells readily proliferate and maintain high cell viability over 120 days within the 3D-printed structure, which is vital for long-term tissue regeneration. A unique aspect of the NICE bioink is its ability to print much taller structures, with higher aspect ratios, than can be achieved with conventional bioinks without requiring secondary supports. We envision that NICE bioinks can be used to bioprint complex, large-scale, cell-laden constructs for tissue engineering with high structural fidelity and mechanical stiffness for applications in custom bioprinted scaffolds and tissue engineered implants.

Mechanically robust cryogels with injectability and bioprinting supportability for adipose tissue engineering.

PubMed

Qi, Dianjun; Wu, Shaohua; Kuss, Mitchell A; Shi, Wen; Chung, Soonkyu; Deegan, Paul T; Kamenskiy, Alexey; He, Yini; Duan, Bin

2018-05-26

Bioengineered adipose tissues have gained increased interest as a promising alternative to autologous tissue flaps and synthetic adipose fillers for soft tissue augmentation and defect reconstruction in clinic. Although many scaffolding materials and biofabrication methods have been investigated for adipose tissue engineering in the last decades, there are still challenges to recapitulate the appropriate adipose tissue microenvironment, maintain volume stability, and induce vascularization to achieve long-term function and integration. In the present research, we fabricated cryogels consisting of methacrylated gelatin, methacrylated hyaluronic acid, and 4arm poly(ethylene glycol) acrylate (PEG-4A) by using cryopolymerization. The cryogels were repeatedly injectable and stretchable, and the addition of PEG-4A improved the robustness and mechanical properties. The cryogels supported human adipose progenitor cell (HWA) and adipose derived mesenchymal stromal cell adhesion, proliferation, and adipogenic differentiation and maturation, regardless of the addition of PEG-4A. The HWA laden cryogels facilitated the co-culture of human umbilical vein endothelial cells (HUVEC) and capillary-like network formation, which in return also promoted adipogenesis. We further combined cryogels with 3D bioprinting to generate handleable adipose constructs with clinically relevant size. 3D bioprinting enabled the deposition of multiple bioinks onto the cryogels. The bioprinted flap-like constructs had an integrated structure without delamination and supported vascularization. Adipose tissue engineering is promising for reconstruction of soft tissue defects, and also challenging for restoring and maintaining soft tissue volume and shape, and achieving vascularization and integration. In this study, we fabricated cryogels with mechanical robustness, injectability, and stretchability by using cryopolymerization. The cryogels promoted cell adhesion, proliferation, and adipogenic differentiation and maturation of human adipose progenitor cells and adipose derived mesenchymal stromal cells. Moreover, the cryogels also supported 3D bioprinting on top, forming vascularized adipose constructs. This study demonstrates the potential of the implementation of cryogels for generating volume-stable adipose tissue constructs and provides a strategy to fabricate vascularized flap-like constructs for complex soft tissue regeneration. Copyright © 2018. Published by Elsevier Ltd.

Combined Effects of Scaffold Stiffening and Mechanical Preconditioning Cycles on Construct Biomechanics, Gene Expression, and Tendon Repair Biomechanics

PubMed Central

Juncosa-Melvin, Natalia; Shearn, Jason T.; Boivin, Gregory P.; Galloway, Marc T.; Gooch, Cynthia; Bradica, Gino; Butler, David L.

2009-01-01

Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases both construct stiffness and the biomechanical properties of the repair tissue after surgery. When optimized using response surface methodology, our results indicate that a mechanical stimulus with three components (2.4% strain, 3000 cycles/day, and one cycle repetition) produced the highest in vitro linear stiffness. Such positive correlations between construct and repair stiffness after surgery suggest that enhancing structural stiffness before surgery could not only accelerate repair stiffness but also prevent premature failures in culture due to poor mechanical integrity. In this study, we examined the combined effects of scaffold crosslinking and subsequent mechanical stimulation on construct mechanics and biology. Autologous tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 15 New Zealand white rabbits on type I collagen sponges that had undergone additional dehydrothermal crosslinking (termed ADHT in this manuscript). Both constructs from each rabbit were mechanically stimulated for 8 h/day for 12 consecutive days with half receiving 100 cycles/day and the other half receiving 3000 cycles/day. These paired MSC–collagen autologous constructs were then implanted in bilateral full-thickness, full-length defects in the central third of rabbit patellar tendons. Increasing the number of in vitro cycles/day delivered to the ADHT constructs in culture produced no differences in stiffness or gene expression and no changes in biomechanical properties or histology 12 weeks after surgery. Compared to MSC-based repairs from a previous study that received no additional treatment in culture, ADHT crosslinking of the scaffolds actually lowered the 12-week repair stiffness. Thus, while ADHT crosslinking may initially stiffen a construct in culture, this specific treatment also appears to mask any benefits of stimulation among repairs postsurgery. Our findings emphasize the importance of properly preconditioning a scaffold to better control/modulate MSC differentiation in vitro and to further enhance repair outcome in vivo. PMID:19191501

I-Wire Heart-on-a-Chip II: Biomechanical analysis of contractile, three-dimensional cardiomyocyte tissue constructs.

PubMed

Schroer, Alison K; Shotwell, Matthew S; Sidorov, Veniamin Y; Wikswo, John P; Merryman, W David

2017-01-15

This companion study presents the biomechanical analysis of the "I-Wire" platform using a modified Hill model of muscle mechanics that allows for further characterization of construct function and response to perturbation. The I-Wire engineered cardiac tissue construct (ECTC) is a novel experimental platform to investigate cardiac cell mechanics during auxotonic contraction. Whereas passive biomaterials often exhibit nonlinear and dissipative behavior, active tissue equivalents, such as ECTCs, also expend metabolic energy to perform mechanical work that presents additional challenges in quantifying their properties. The I-Wire model uses the passive mechanical response to increasing applied tension to measure the inherent stress and resistance to stretch of the construct before, during, and after treatments. Both blebbistatin and isoproterenol reduced prestress and construct stiffness; however, blebbistatin treatment abolished subsequent force-generating potential while isoproterenol enhanced this property. We demonstrate that the described model can replicate the response of these constructs to intrinsic changes in force-generating potential in response to both increasing frequency of stimulation and decreasing starting length. This analysis provides a useful mathematical model of the I-Wire platform, increases the number of parameters that can be derived from the device, and serves as a demonstration of quantitative characterization of nonlinear, active biomaterials. We anticipate that this quantitative analysis of I-Wire constructs will prove useful for qualifying patient-specific cardiomyocytes and fibroblasts prior to their utilization for cardiac regenerative medicine. Passive biomaterials may have non-linear elasticity and losses, but engineered muscle tissue also exhibits time- and force-dependent contractions. Historically, mathematical muscle models include series-elastic, parallel-elastic, contractile, and viscous elements. While hearts-on-a-chip can demonstrate in vitro the contractile properties of engineered cardiac constructs and their response to drugs, most of these use cellular monolayers that cannot be readily probed with controlled forces. The I-Wire platform described in the preceding paper by Sidorov et al. addresses these limitations with three-dimensional tissue constructs to which controlled forces can be applied. In this companion paper, we show how to characterize I-Wire constructs using a non-linear, active Hill model, which should be useful for qualifying cells prior to their use in cardiac regenerative medicine. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

State-of-the-Art Review of 3D Bioprinting for Cardiovascular Tissue Engineering.

PubMed

Duan, Bin

2017-01-01

3D bioprinting is a group of rapidly growing techniques that allows building engineered tissue constructs with complex and hierarchical structures, mechanical and biological heterogeneity. It enables implementation of various bioinks through different printing mechanisms and precise deposition of cell and/or biomolecule laden biomaterials in predefined locations. This review briefly summarizes applicable bioink materials and various bioprinting techniques, and presents the recent advances in bioprinting of cardiovascular tissues, with focusing on vascularized constructs, myocardium and heart valve conduits. Current challenges and further perspectives are also discussed to help guide the bioink and bioprinter development, improve bioprinting strategies and direct future organ bioprinting and translational applications.

Mechanical Testing of Hydrogels in Cartilage Tissue Engineering: Beyond the Compressive Modulus

PubMed Central

Xiao, Yinghua; Friis, Elizabeth A.; Gehrke, Stevin H.

2013-01-01

Injuries to articular cartilage result in significant pain to patients and high medical costs. Unfortunately, cartilage repair strategies have been notoriously unreliable and/or complex. Biomaterial-based tissue-engineering strategies offer great promise, including the use of hydrogels to regenerate articular cartilage. Mechanical integrity is arguably the most important functional outcome of engineered cartilage, although mechanical testing of hydrogel-based constructs to date has focused primarily on deformation rather than failure properties. In addition to deformation testing, as the field of cartilage tissue engineering matures, this community will benefit from the addition of mechanical failure testing to outcome analyses, given the crucial clinical importance of the success of engineered constructs. However, there is a tremendous disparity in the methods used to evaluate mechanical failure of hydrogels and articular cartilage. In an effort to bridge the gap in mechanical testing methods of articular cartilage and hydrogels in cartilage regeneration, this review classifies the different toughness measurements for each. The urgency for identifying the common ground between these two disparate fields is high, as mechanical failure is ready to stand alongside stiffness as a functional design requirement. In comparing toughness measurement methods between hydrogels and cartilage, we recommend that the best option for evaluating mechanical failure of hydrogel-based constructs for cartilage tissue engineering may be tensile testing based on the single edge notch test, in part because specimen preparation is more straightforward and a related American Society for Testing and Materials (ASTM) standard can be adopted in a fracture mechanics context. PMID:23448091

Bioinspired coupled helical coils for soft tissue engineering of tubular structures - Improved mechanical behavior of tubular collagen type I templates.

PubMed

Janke, H P; Bohlin, J; Lomme, R M L M; Mihaila, S M; Hilborn, J; Feitz, W F J; Oosterwijk, E

2017-09-01

The design of constructs for tubular tissue engineering is challenging. Most biomaterials need to be reinforced with supporting structures such as knittings, meshes or electrospun material to comply with the mechanical demands of native tissues. In this study, coupled helical coils (CHCs) were manufactured to mimic collagen fiber orientation as found in nature. Monofilaments of different commercially available biodegradable polymers were wound and subsequently fused, resulting in right-handed and left-handed polymer helices fused together in joints where the filaments cross. CHCs of different polymer composition were tested to determine the tensile strength, strain recovery, hysteresis, compressive strength and degradation of CHCs of different composition. Subsequently, seamless and stable hybrid constructs consisting of PDSII® USP 2-0 CHCs embedded in porous collagen type I were produced. Compared to collagen alone, this hybrid showed superior strain recovery (93.5±0.9% vs 71.1±12.6% in longitudinal direction; 87.1±6.6% vs 57.2±4.6% in circumferential direction) and hysteresis (18.9±2.7% vs 51.1±12.0% in longitudinal direction; 11.5±4.6% vs 46.3±6.3% in circumferential direction). Furthermore, this hybrid construct showed an improved Young's modulus in both longitudinal (0.5±0.1MPavs 0.2±0.1MPa; 2.5-fold) and circumferential (1.65±0.07MPavs (2.9±0.3)×10 -2 MPa; 57-fold) direction, respectively, compared to templates created from collagen alone. Moreover, hybrid template characteristics could be modified by changing the CHC composition and CHCs were produced showing a mechanical behavior similar to the native ureter. CHC-enforced templates, which are easily tunable to meet different demands may be promising for tubular tissue engineering. Most tubular constructs lack sufficient strength and tunability to comply with the mechanical demands of native tissues. Therefore, we embedded coupled helical coils (CHCs) produced from biodegradable polymers - to mimic collagen fiber orientation as found in nature - in collagen type I sponges. We show that the mechanical behavior of CHCs is very similar to native tissue and strengths structurally weak tubular constructs. The production procedure is relatively easy, reproducible and mechanical features can be controlled to meet different mechanical demands. This is promising in template manufacture, hence offering new opportunities in tissue engineering of tubular organs and preventing graft failure. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Ultrasound Technologies for the Spatial Patterning of Cells and Extracellular Matrix Proteins and the Vascularization of Engineered Tissue

NASA Astrophysics Data System (ADS)

Garvin, Kelley A.

Technological advancements in the field of tissue engineering could save the lives of thousands of organ transplant patients who die each year while waiting for donor organs. Currently, two of the primary challenges preventing tissue engineers from developing functional replacement tissues and organs are the need to recreate complex cell and extracellular microenvironments and to vascularize the tissue to maintain cell viability and function. Ultrasound is a form of mechanical energy that can noninvasively and nondestructively interact with tissues at the cell and protein level. In this thesis, novel ultrasound-based technologies were developed for the spatial patterning of cells and extracellular matrix proteins and the vascularization of three-dimensional engineered tissue constructs. Acoustic radiation forces associated with ultrasound standing wave fields were utilized to noninvasively control the spatial organization of cells and cell-bound extracellular matrix proteins within collagen-based engineered tissue. Additionally, ultrasound induced thermal mechanisms were exploited to site-specifically pattern various extracellular matrix collagen microstructures within a single engineered tissue construct. Finally, ultrasound standing wave field technology was used to promote the rapid and extensive vascularization of three-dimensional tissue constructs. As such, the ultrasound technologies developed in these studies have the potential to provide the field of tissue engineering with novel strategies to spatially pattern cells and extracellular matrix components and to vascularize engineered tissue, and thus, could advance the fabrication of functional replacement tissues and organs in the field of tissue engineering.

Bioprinting Using Mechanically Robust Core-Shell Cell-Laden Hydrogel Strands.

PubMed

Mistry, Pritesh; Aied, Ahmed; Alexander, Morgan; Shakesheff, Kevin; Bennett, Andrew; Yang, Jing

2017-06-01

The strand material in extrusion-based bioprinting determines the microenvironments of the embedded cells and the initial mechanical properties of the constructs. One unmet challenge is the combination of optimal biological and mechanical properties in bioprinted constructs. Here, a novel bioprinting method that utilizes core-shell cell-laden strands with a mechanically robust shell and an extracellular matrix-like core has been developed. Cells encapsulated in the strands demonstrate high cell viability and tissue-like functions during cultivation. This process of bioprinting using core-shell strands with optimal biochemical and biomechanical properties represents a new strategy for fabricating functional human tissues and organs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Challenges in engineering osteochondral tissue grafts with hierarchical structures Ivana Gadjanski, Gordana Vunjak Novakovic

PubMed Central

Gadjanski, Ivana; Vunjak-Novakovic, Gordana

2015-01-01

Introduction A major hurdle in treating osteochondral (OC) defects are the different healing abilities of two types of tissues involved - articular cartilage and subchondral bone. Biomimetic approaches to OC-construct-engineering, based on recapitulation of biological principles of tissue development and regeneration, have potential for providing new treatments and advancing fundamental studies of OC tissue repair. Areas covered This review on state of the art in hierarchical OC tissue graft engineering is focused on tissue engineering approaches designed to recapitulate the native milieu of cartilage and bone development. These biomimetic systems are discussed with relevance to bioreactor cultivation of clinically sized, anatomically shaped human cartilage/bone constructs with physiologic stratification and mechanical properties. The utility of engineered OC tissue constructs is evaluated for their use as grafts in regenerative medicine, and as high-fidelity models in biological research. Expert opinion A major challenge in engineering OC tissues is to generate a functionally integrated stratified cartilage-bone structure starting from one single population of mesenchymal cells, while incorporating perfusable vasculature into the bone, and in bone-cartilage interface. To this end, new generations of advanced scaffolds and bioreactors, implementation of mechanical loading regimens, and harnessing of inflammatory responses of the host will likely drive the further progress. PMID:26195329

Composition-function relations of cartilaginous tissues engineered from chondrocytes and mesenchymal stem cells isolated from bone marrow and infrapatellar fat pad.

PubMed

Vinardell, T; Buckley, C T; Thorpe, S D; Kelly, D J

2011-10-01

The objective of this study was to determine the functional properties of cartilaginous tissues generated by porcine MSCs isolated from different tissue sources, and to compare these properties to those derived from chondrocytes (CCs). MSCs were isolated from bone marrow (BM) and infrapatellar fat pad (FP), while CCs were harvested from the articular surface of the femoro-patellar joint. Culture-expanded CCs and MSCs were encapsulated in agarose hydrogels and cultured in the presence of TGFβ3. Samples were analysed biomechanically, biochemically and histologically at days 0, 21 and 42. After 42 days in free swelling culture, mean GAG content was 1.50% w/w in CC-seeded constructs, compared to 0.95% w/w in FP- and 0.43% w/w in BM-seeded constructs. Total collagen accumulation was highest in FP constructs. DNA content increased with time for all the groups. The mechanical functionality of cartilaginous tissues engineered using CCs was superior to that generated from either source of MSCs. Differences were also observed in the spatial distribution of matrix components in tissues engineered using CCs and MSCs, which appears to have a strong influence on the apparent mechanical properties of the constructs. Therefore, while functional cartilaginous tissues can be engineered using MSCs isolated from different sources, the spatial composition of these tissues is unlike that generated using chondrocytes, suggesting that MSCs and chondrocytes respond differently to the regulatory factors present within developing cartilaginous constructs. Copyright © 2010 John Wiley & Sons, Ltd.

Nanocellulose reinforced gellan-gum hydrogels as potential biological substitutes for annulus fibrosus tissue regeneration.

PubMed

Pereira, Diana R; Silva-Correia, Joana; Oliveira, Joaquim M; Reis, Rui L; Pandit, Abhay; Biggs, Manus J

2018-04-01

Intervertebral disc (IVD) degeneration is associated with both structural damage and aging related degeneration. Annulus fibrosus (AF) defects such as annular tears, herniation and discectomy require novel tissue engineering strategies to functionally repair AF tissue. An ideal construct will repair the AF by providing physical and biological support, facilitating regeneration. The presented strategy herein proposes a gellan gum-based construct reinforced with cellulose nanocrystals (nCell) as a biological self-gelling AF substitute. Nanocomposite hydrogels were fabricated and characterized with respect to hydrogel swelling capacity, degradation rate in vitro and mechanical properties. Rheological evaluation on the nanocomposites demonstrated the GGMA reinforcement with nCell promoted matrix entanglement with higher scaffold stiffness observed upon ionic crosslinking. Compressive mechanical tests demonstrated compressive modulus values close to those of the human AF tissue. Furthermore, cell culture studies with encapsulated bovine AF cells indicated that nanocomposite constructs promoted cell viability and a physiologically relevant cell morphology for up to fourteen days in vitro. Copyright © 2017 Elsevier Inc. All rights reserved.

Engineering zonal cartilaginous tissue by modulating oxygen levels and mechanical cues through the depth of infrapatellar fat pad stem cell laden hydrogels.

PubMed

Luo, Lu; O'Reilly, Adam R; Thorpe, Stephen D; Buckley, Conor T; Kelly, Daniel J

2017-09-01

Engineering tissues with a structure and spatial composition mimicking those of native articular cartilage (AC) remains a challenge. This study examined if infrapatellar fat pad-derived stem cells (FPSCs) can be used to engineer cartilage grafts with a bulk composition and a spatial distribution of matrix similar to the native tissue. In an attempt to mimic the oxygen gradients and mechanical environment within AC, FPSC-laden hydrogels (either 2 mm or 4 mm in height) were confined to half of their thickness and/or subjected to dynamic compression (DC). Confining FPSC-laden hydrogels was predicted to accentuate the gradient in oxygen tension through the depth of the constructs (higher in the top and lower in the bottom), leading to enhanced glycosaminoglycan (GAG) and collagen synthesis in 2 mm high tissues. When subjected to DC alone, both GAG and collagen accumulation increased within 2 mm high unconfined constructs. Furthermore, the dynamic modulus of constructs increased from 0.96 MPa to 1.45 MPa following the application of DC. There was no synergistic benefit of coupling confinement and DC on overall levels of matrix accumulation; however in all constructs, irrespective of their height, the combination of these boundary conditions led to the development of engineered tissues that spatially best resembled native AC. The superficial region of these constructs mimicked that of native tissue, staining weakly for GAG, strongly for type II collagen, and in 4 mm high tissues more intensely for proteoglycan 4 (lubricin). This study demonstrated that FPSCs respond to joint-like environmental conditions by producing cartilage tissues mimicking native AC. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Design and Validation of a Compressive Tissue Stimulator with High-Throughput Capacity and Real-Time Modulus Measurement Capability

PubMed Central

Salvetti, David J.; Pino, Christopher J.; Manuel, Steven G.; Dallmeyer, Ian; Rangarajan, Sanjeet V.; Meyer, Tobias; Kotov, Misha

2012-01-01

Mechanical stimulation has been shown to impact the properties of engineered hyaline cartilage constructs and is relevant for engineering of cartilage and osteochondral tissues. Most mechanical stimulators developed to date emphasize precision over adaptability to standard tissue culture equipment and protocols. The realization of mechanical characteristics in engineered constructs approaching native cartilage requires the optimization of complex variables (type of stimulus, regimen, and bimolecular signals). We have proposed and validated a stimulator design that focuses on high construct capacity, compatibility with tissue culture plastic ware, and regimen adaptability to maximize throughput. This design utilizes thin force sensors in lieu of a load cell and a linear encoder to verify position. The implementation of an individual force sensor for each sample enables the measurement of Young's modulus while stimulating the sample. Removable and interchangeable Teflon plungers mounted using neodymium magnets contact each sample. Variations in plunger height and design can vary the strain and force type on individual samples. This allows for the evaluation of a myriad of culture conditions and regimens simultaneously. The system was validated using contact accuracy, and Young's modulus measurements range as key parameters. Contact accuracy for the system was excellent within 1.16% error of the construct height in comparison to measurements made with a micrometer. Biomaterials ranging from bioceramics (cancellous bone, 123 MPa) to soft gels (1% agarose, 20 KPa) can be measured without any modification to the device. The accuracy of measurements in conjunction with the wide range of moduli tested demonstrate the unique characteristics of the device and the feasibility of using this device in mapping real-time changes to Young's modulus of tissue constructs (cartilage, bone) through the developmental phases in ex vivo culture conditions. PMID:21988089

In vitro physical stimulation of tissue-engineered and native cartilage.

PubMed

Li, Kelvin W; Klein, Travis J; Chawla, Kanika; Nugent, Gayle E; Bae, Won C; Sah, Robert L

2004-01-01

Because of the limited availability of donor cartilage for resurfacing defects in articular surfaces, there is tremendous interest in the in vitro bioengineering of cartilage replacements for clinical applications. However, attaining mechanical properties in engineered cartilaginous constructs that approach those of native cartilage has not been previously achieved when constructs are cultured under free-swelling conditions. One approach toward stimulating the development of constructs that are mechanically more robust is to expose them to physical environments that are similar, in certain ways, to those encountered by native cartilage. This is a strategy motivated by observations in numerous short-term experiments that certain mechanical signals are potent stimulators of cartilage metabolism. On the other hand, excess mechanical loading can have a deleterious effect on cartilage. Culture conditions that include a physical stimulation component are made possible by the use of specialized bioreactors. This chapter addresses some of the issues involved in using bioreactors as integral components of cartilage tissue engineering and in studying the physical regulation of cartilage. We first consider the generation of cartilaginous constructs in vitro. Next we describe the rationale and design of bioreactors that can impart either mechanical deformation or fluid-induced mechanical signals.

Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

PubMed

Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana

2013-10-01

Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

Construction Strategy and Progress of Whole Intervertebral Disc Tissue Engineering.

PubMed

Yang, Qiang; Xu, Hai-wei; Hurday, Sookesh; Xu, Bao-shan

2016-02-01

Degenerative disc disease (DDD) is the major cause of low back pain, which usually leads to work absenteeism, medical visits and hospitalization. Because the current conservative procedures and surgical approaches to treatment of DDD only aim to relieve the symptoms of disease but not to regenerate the diseased disc, their long-term efficiency is limited. With the rapid developments in medical science, tissue engineering techniques have progressed markedly in recent years, providing a novel regenerative strategy for managing intervertebral disc disease. However, there are as yet no ideal methods for constructing tissue-engineered intervertebral discs. This paper reviews published reports pertaining to intervertebral disc tissue engineering and summarizes data concerning the seed cells and scaffold materials for tissue-engineered intervertebral discs, construction of tissue-engineered whole intervertebral discs, relevant animal experiments and effects of mechanics on the construction of tissue-engineered intervertebral disc and outlines the existing problems and future directions. Although the perfect regenerative strategy for treating DDD has not yet been developed, great progress has been achieved in the construction of tissue-engineered intervertebral discs. It is believed that ongoing research on intervertebral disc tissue engineering will result in revolutionary progress in the treatment of DDD. © 2016 Chinese Orthopaedic Association and John Wiley & Sons Australia, Ltd.

Application of new optical coherence elastography to monitor the mineralization processing in bone tissue engineering constructs

NASA Astrophysics Data System (ADS)

Guan, Guangying; Song, Shaozhen; Huang, Zhihong; Yang, Ying

2015-03-01

Generation of functional tissue in vitro through tissue engineering technique is a promising direction to repair and replace malfunctioned organ and tissue in the modern medicine for various diseases which could not been treated well by conventional therapy. Similar to the embryo development, the generation of tissue in vitro is a highly dynamic processing. Obtaining the feedback of the processing real time is highly demanded. In this study, a new methodology has been explored aiming to monitor the morphological and mechanical property alteration of bone tissue engineering constructs simultaneously. Optical coherence elastography (OCE) equipped with a LDS V201 permanent magnet shaker and a modulated acoustic radiation force (ARF) to provide a vibration signal, has been used for the real time and non-destructive monitoring. A phantom construct system has been used to optimize the measurement conditions in which agar hydrogel with concentration from 0, 0.75 to 2% with/without hydroxyappatite particles have been injected to 3D porous poly (lactic acid) scaffolds to simulate the collagenous extracellular matrix (ECM) and mineralized ECM. The structural and elastography images of the constructs have clearly demonstrated the linear relation with the increased mechanical property versus the increase of agar concentration within the pores of the scaffolds. The MG63 bone cells seeded in the scaffolds and cultured for 4 weeks have been monitored by the established protocol exhibiting the increased mechanical strength in the pore wall where the ECM or mineralized ECM was assumed to be formed in comparison to empty pores. This study confirms that OCE-ARF could become a valuable tool in regenerative medicine to assess the biological events during in vitro culture and conditioning.

Mechanical stimulation enhances integration in an in vitro model of cartilage repair.

PubMed

Theodoropoulos, John S; DeCroos, Amritha J N; Petrera, Massimo; Park, Sam; Kandel, Rita A

2016-06-01

(1) To characterize the effects of mechanical stimulation on the integration of a tissue-engineered construct in terms of histology, biochemistry and biomechanical properties; (2) to identify whether cells of the implant or host tissue were critical to implant integration; and (3) to study cells believed to be involved in lateral integration of tissue-engineered cartilage to host cartilage. We hypothesized that mechanical stimulation would enhance the integration of the repair implant with host cartilage in an in vitro integration model. Articular cartilage was harvested from 6- to 9-month-old bovine metacarpal-phalangeal joints. Constructs composed of tissue-engineered cartilage implanted into host cartilage were placed in spinner bioreactors and maintained on a magnetic stir plate at either 0 (static control) or 90 (experimental) rotations per minute (RPM). The constructs from both the static and spinner bioreactors were harvested after either 2 or 4 weeks of culture and evaluated histologically, biochemically, biomechanically and for gene expression. The extent and strength of integration between tissue-engineered cartilage and native cartilage improved significantly with both time and mechanical stimulation. Integration did not occur if the implant was not viable. The presence of stimulation led to a significant increase in collagen content in the integration zone between host and implant at 2 weeks. The gene profile of cells in the integration zone differs from host cartilage demonstrating an increase in the expression of membrane type 1 matrix metalloproteinase (MT1-MMP), aggrecan and type II collagen. This study shows that the integration of in vitro tissue-engineered implants with host tissue improves with mechanical stimulation. The findings of this study suggests that consideration should be given to implementing early loading (mechanical stimulation) into future in vivo studies investigating the long-term viability and integration of tissue-engineered cartilage for the treatment of cartilage injuries. This could simply be done through the use of continuous passive motion (CPM) in the post-operative period or through a more complex and structured rehabilitation program with a gradual increase in forces across the joint over time.

A puzzle assembly strategy for fabrication of large engineered cartilage tissue constructs.

PubMed

Nover, Adam B; Jones, Brian K; Yu, William T; Donovan, Daniel S; Podolnick, Jeremy D; Cook, James L; Ateshian, Gerard A; Hung, Clark T

2016-03-21

Engineering of large articular cartilage tissue constructs remains a challenge as tissue growth is limited by nutrient diffusion. Here, a novel strategy is investigated, generating large constructs through the assembly of individually cultured, interlocking, smaller puzzle-shaped subunits. These constructs can be engineered consistently with more desirable mechanical and biochemical properties than larger constructs (~4-fold greater Young׳s modulus). A failure testing technique was developed to evaluate the physiologic functionality of constructs, which were cultured as individual subunits for 28 days, then assembled and cultured for an additional 21-35 days. Assembled puzzle constructs withstood large deformations (40-50% compressive strain) prior to failure. Their ability to withstand physiologic loads may be enhanced by increases in subunit strength and assembled culture time. A nude mouse model was utilized to show biocompatibility and fusion of assembled puzzle pieces in vivo. Overall, the technique offers a novel, effective approach to scaling up engineered tissues and may be combined with other techniques and/or applied to the engineering of other tissues. Future studies will aim to optimize this system in an effort to engineer and integrate robust subunits to fill large defects. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Puzzle Assembly Strategy for Fabrication of Large Engineered Cartilage Tissue Constructs

PubMed Central

Nover, Adam B.; Jones, Brian K.; Yu, William T.; Donovan, Daniel S.; Podolnick, Jeremy D.; Cook, James L.; Ateshian, Gerard A.; Hung, Clark T.

2016-01-01

Engineering of large articular cartilage tissue constructs remains a challenge as tissue growth is limited by nutrient diffusion. Here, a novel strategy is investigated, generating large constructs through the assembly of individually cultured, interlocking, smaller puzzle-shaped subunits. These constructs can be engineered consistently with more desirable mechanical and biochemical properties than larger constructs (~4-fold greater Young's modulus). A failure testing technique was developed to evaluate the physiologic functionality of constructs, which were cultured as individual subunits for 28 days, then assembled and cultured for an additional 21-35 days. Assembled puzzle constructs withstood large deformations (40-50% compressive strain) prior to failure. Their ability to withstand physiologic loads may be enhanced by increases in subunit strength and assembled culture time. A nude mouse model was utilized to show biocompatibility and fusion of assembled puzzle pieces in vivo. Overall, the technique offers a novel, effective approach to scaling up engineered tissues and may be combined with other techniques and/or applied to the engineering of other tissues. Future studies will aim to optimize this system in an effort to engineer and integrate robust subunits to fill large defects. PMID:26895780

Bioactive glass 13-93 as a subchondral substrate for tissue-engineered osteochondral constructs: a pilot study.

PubMed

Jayabalan, Prakash; Tan, Andrea R; Rahaman, Mohammed N; Bal, B Sonny; Hung, Clark T; Cook, James L

2011-10-01

Replacement of diseased areas of the joint with tissue-engineered osteochondral grafts has shown potential in the treatment of osteoarthritis. Bioactive glasses are candidates for the osseous analog of these grafts. (1) Does Bioactive Glass 13-93 (BG 13-93) as a subchondral substrate improve collagen and glycosaminoglycan production in a tissue-engineered cartilage layer? (2) Does BG 13-93 as a culture medium supplement increase the collagen and glycosaminoglycan production and improve the mechanical properties in a tissue-engineered cartilage layer? In Study 1, bioactive glass samples (n = 4) were attached to a chondrocyte-seeded agarose layer to form an osteochondral construct, cultured for 6 weeks, and compared to controls. In Study 2, bioactive glass samples (n = 5) were cocultured with cell-seeded agarose for 6 weeks. The cell-seeded agarose layer was exposed to BG 13-93 either continuously or for the first or last 2 weeks in culture or had no exposure. Osteochondral constructs with a BG 13-93 base had improved glycosaminoglycan deposition but less collagen II content. Agarose scaffolds that had a temporal exposure to BG 13-93 within the culture medium had improved mechanical and biochemical properties compared to continuous or no exposure. When used as a subchondral substrate, BG 13-93 did not improve biochemical properties compared to controls. However, as a culture medium supplement, BG 13-93 improved the biochemical and mechanical properties of a tissue-engineered cartilage layer. BG 13-93 may not be suitable in osteochondral constructs but could have potential as a medium supplement for neocartilage formation.

Biomimetic microenvironment complexity to redress the balance between biodegradation and de novo matrix synthesis during early phase of vascular tissue engineering.

PubMed

Vatankhah, Elham; Prabhakaran, Molamma P; Ramakrishna, Seeram

2017-12-01

Physiological functionality of a tissue engineered vascular construct depends on the phenotype of smooth muscle cells (SMCs) cultured into the scaffold and mechanical robust of the construct relies on two simultaneous mechanisms including scaffold biodegradation and de novo matrix synthesis by SMCs which both can be influenced by scaffold properties and culture condition. Our focus in this study was to provide an appropriate environmental condition within tissue engineering context to meet foregoing requisites for a successful vascular regeneration. To this end, SMCs seeded onto electrospun Tecophilic/gelatin (TP(70)/gel(30)) scaffolds were subjected to orbital shear stress. Given the improvement in mechanical properties of dynamically stimulated cell-seeded constructs after a span of 10days, effect of fluctuating shear stress on scaffold biodegradation and SMC behavior was investigated. Compared to static condition, SMCs proliferated more rapidly and concomitantly built up greater collagen content in response to dynamic culture, suggesting a reasonable balance between scaffold biodegradation and matrix turnover for maintaining the structural integrity and mechanical support to seeded cells during early phase of vascular tissue engineering. Despite higher proliferation of SMCs under dynamic condition, cells preserved nearly spindle like morphology and contractile protein expression likely thanks to composition of the scaffold. Copyright © 2017 Elsevier B.V. All rights reserved.

Tissue Engineering Strategies for Myocardial Regeneration: Acellular Versus Cellular Scaffolds?

PubMed

Domenech, Maribella; Polo-Corrales, Lilliana; Ramirez-Vick, Jaime E; Freytes, Donald O

2016-12-01

Heart disease remains one of the leading causes of death in industrialized nations with myocardial infarction (MI) contributing to at least one fifth of the reported deaths. The hypoxic environment eventually leads to cellular death and scar tissue formation. The scar tissue that forms is not mechanically functional and often leads to myocardial remodeling and eventual heart failure. Tissue engineering and regenerative medicine principles provide an alternative approach to restoring myocardial function by designing constructs that will restore the mechanical function of the heart. In this review, we will describe the cellular events that take place after an MI and describe current treatments. We will also describe how biomaterials, alone or in combination with a cellular component, have been used to engineer suitable myocardium replacement constructs and how new advanced culture systems will be required to achieve clinical success.

Fabrication of Cardiac Patch with Decellularized Porcine Myocardial Scaffold and Bone Marrow Mononuclear Cells

PubMed Central

Wang, Bo; Borazjani, Ali; Tahai, Mina; de Jongh Curry, Amy L.; Simionescu, Dan T.; Guan, Jianjun; To, Filip; Elder, Steve H.; Liao, Jun

2010-01-01

Tissue engineered cardiac grafts are a promising therapeutic mode for ventricular wall reconstruction. Recently, it has been found that acellular tissue scaffolds provide natural ultrastructural, mechanical, and compositional cues for recellularization and tissue remodeling. We thus assess the potential of decellularized porcine myocardium as a scaffold for thick cardiac patch tissue engineering. Myocardial sections with 2 mm thickness were decellularized using 0.1% sodium dodecyl sulfate (SDS), and then reseeded with differentiated bone marrow mononuclear cells. We found that thorough decellularization could be achieved after 2.5 weeks treatment. Reseeded cells were found to infiltrate and proliferate in the tissue constructs. Immunohistological staining studies showed that the reseeded cells maintained cardiomyocyte-like phenotype and possible endothelialization was found in locations close to vasculature channels, indicating angiogenesis potential. Both biaxial and uniaxial mechanical testing showed a stiffer mechanical response of the acellular myocardial scaffolds; however, tissue extensibility and tensile modulus were found to recover in the constructs along with the culture time, as expected from increased cellular content. The cardiac patch that we envision for clinical application will benefit from the natural architecture of myocardial extracellular matrix, which has the potential to promote stem cell differentiation, cardiac regeneration, and angiogenesis. PMID:20694977

Development of large engineered cartilage constructs from a small population of cells.

PubMed

Brenner, Jillian M; Kunz, Manuela; Tse, Man Yat; Winterborn, Andrew; Bardana, Davide D; Pang, Stephen C; Waldman, Stephen D

2013-01-01

Confronted with articular cartilage's limited capacity for self-repair, joint resurfacing techniques offer an attractive treatment for damaged or diseased tissue. Although tissue engineered cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for the repair of large defects. As routine cell expansion methods tend to elicit negative effects on chondrocyte function, we have developed an approach to generate phenotypically stable, large-sized engineered constructs (≥3 cm(2) ) directly from a small amount of donor tissue or cells (as little as 20,000 cells to generate a 3 cm(2) tissue construct). Using rabbit donor tissue, the bioreactor-cultivated constructs were hyaline-like in appearance and possessed a biochemical composition similar to native articular cartilage. Longer bioreactor cultivation times resulted in increased matrix deposition and improved mechanical properties determined over a 4 week period. Additionally, as the anatomy of the joint will need to be taken in account to effectively resurface large affected areas, we have also explored the possibility of generating constructs matched to the shape and surface geometry of a defect site through the use of rapid-prototyped defect tissue culture molds. Similar hyaline-like tissue constructs were developed that also possessed a high degree of shape correlation to the original defect mold. Future studies will be aimed at determining the effectiveness of this approach to the repair of cartilage defects in an animal model and the creation of large-sized osteochondral constructs. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Application of Detergents or High Hydrostatic Pressure as Decellularization Processes in Uterine Tissues and Their Subsequent Effects on In Vivo Uterine Regeneration in Murine Models

PubMed Central

Hirota, Yasushi; Aizawa, Masanori; Yoshino, Osamu; Kishida, Akio; Osuga, Yutaka; Saito, Shigeru; Ushida, Takashi; Furukawa, Katsuko S.

2014-01-01

Infertility caused by ovarian or tubal problems can be treated using In Vitro Fertilization and Embryo Transfer (IVF-ET); however, this is not possible for women with uterine loss and malformations that require uterine reconstruction for the treatment of their infertility. In this study, we are the first to report the usefulness of decellularized matrices as a scaffold for uterine reconstruction. Uterine tissues were extracted from Sprague Dawley (SD) rats and decellularized using either sodium dodecyl sulfate (SDS) or high hydrostatic pressure (HHP) at optimized conditions. Histological staining and quantitative analysis showed that both SDS and HHP methods effectively removed cells from the tissues with, specifically, a significant reduction of DNA contents for HHP constructs. HHP constructs highly retained the collagen content, the main component of extracellular matrices in uterine tissue, compared to SDS constructs and had similar content levels of collagen to the native tissue. The mechanical strength of the HHP constructs was similar to that of the native tissue, while that of the SDS constructs was significantly elevated. Transmission electron microscopy (TEM) revealed no apparent denaturation of collagen fibers in the HHP constructs compared to the SDS constructs. Transplantation of the decellularized tissues into rat uteri revealed the successful regeneration of the uterine tissues with a 3-layer structure 30 days after the transplantation. Moreover, a lot of epithelial gland tissue and Ki67 positive cells were detected. Immunohistochemical analyses showed that the regenerated tissues have a normal response to ovarian hormone for pregnancy. The subsequent pregnancy test after 30 days transplantation revealed successful pregnancy for both the SDS and HHP groups. These findings indicate that the decellularized matrix from the uterine tissue can be a potential scaffold for uterine regeneration. PMID:25057942

Dental pulp stem cells express tendon markers under mechanical loading and are a potential cell source for tissue engineering of tendon-like tissue.

PubMed

Chen, Yu-Ying; He, Sheng-Teng; Yan, Fu-Hua; Zhou, Peng-Fei; Luo, Kai; Zhang, Yan-Ding; Xiao, Yin; Lin, Min-Kui

2016-12-16

Postnatal mesenchymal stem cells have the capacity to differentiate into multiple cell lineages. This study explored the possibility of dental pulp stem cells (DPSCs) for potential application in tendon tissue engineering. The expression of tendon-related markers such as scleraxis, tenascin-C, tenomodulin, eye absent homologue 2, collagens I and VI was detected in dental pulp tissue. Interestingly, under mechanical stimulation, these tendon-related markers were significantly enhanced when DPSCs were seeded in aligned polyglycolic acid (PGA) fibre scaffolds. Furthermore, mature tendon-like tissue was formed after transplantation of DPSC-PGA constructs under mechanical loading conditions in a mouse model. This study demonstrates that DPSCs could be a potential stem cell source for tissue engineering of tendon-like tissue.

Fibre-reinforced hydrogels for tissue engineering

NASA Astrophysics Data System (ADS)

Waters, Sarah; Byrne, Helen; Chen, Mike; Dias Castilho, Miguel; Kimpton, Laura; Please, Colin; Whiteley, Jonathan

2017-11-01

Tissue engineers aim to grow replacement tissues in vitro to replace those in the body that have been damaged through age, trauma or disease. One approach is to seed cells within a scaffold consisting of an interconnected 3D-printed lattice of polymer fibres, cast in a hydrogel, and subject the construct (cell-seeded scaffold) to an applied load in a bioreactor. A key question is to understand how this applied load is distributed throughout the construct to the mechanosensitive cells. To address this, we exploit the disparate length scales (small inter-fibre spacing compared with construct dimensions). The fibres are treated as a linear elastic material and the hydrogel as a poroelastic material. We employ homogenisation theory to derive equations governing the material properties of a periodic, elastic-poroelastic composite. To validate the mobel, model solutions are compared to experimental data describing the unconfined compression of the fibre-reinforced hydrogels. The model is used to derive the bulk mechanical properties of a cylindrical construct of the composite material for a range of fibre spacings, and the local mechanical environment experienced by cells embedded within the construct is determined. Funded by the European Union Seventh Framework Programme (FP7/2007-2013).

Three-dimensional bioprinting is not only about cell-laden structures.

PubMed

Zhang, Hong-Bo; Xing, Tian-Long; Yin, Rui-Xue; Shi, Yong; Yang, Shi-Mo; Zhang, Wen-Jun

2016-08-01

In this review, we focused on a few obstacles that hinder three-dimensional (3D) bioprinting process in tissue engineering. One of the obstacles is the bioinks used to deliver cells. Hydrogels are the most widely used bioink materials; however, they aremechanically weak in nature and cannot meet the requirements for supporting structures, especially when the tissues, such as cartilage, require extracellular matrix to be mechanically strong. Secondly and more importantly, tissue regeneration is not only about building all the components in a way that mimics the structures of living tissues, but also about how to make the constructs function normally in the long term. One of the key issues is sufficient nutrient and oxygen supply to the engineered living constructs. The other is to coordinate the interplays between cells, bioactive agents and extracellular matrix in a natural way. This article reviews the approaches to improve the mechanical strength of hydrogels and their suitability for 3D bioprinting; moreover, the key issues of multiple cell lines coprinting with multiple growth factors, vascularization within engineered living constructs etc. were also reviewed.

A Dual-Mode Bioreactor System for Tissue Engineered Vascular Models.

PubMed

Bono, N; Meghezi, S; Soncini, M; Piola, M; Mantovani, D; Fiore, Gianfranco Beniamino

2017-06-01

In the past decades, vascular tissue engineering has made great strides towards bringing engineered vascular tissues to the clinics and, in parallel, obtaining in-lab tools for basic research. Herein, we propose the design of a novel dual-mode bioreactor, useful for the fabrication (construct mode) and in vitro stimulation (culture mode) of collagen-based tubular constructs. Collagen-based gels laden with smooth muscle cells (SMCs) were molded directly within the bioreactor culture chamber. Based on a systematic characterization of the bioreactor culture mode, constructs were subjected to 10% cyclic strain at 0.5 Hz for 5 days. The effects of cyclic stimulation on matrix re-arrangement and biomechanical/viscoelastic properties were examined and compared vs. statically cultured constructs. A thorough comparison of cell response in terms of cell localization and expression of contractile phenotypic markers was carried out as well. We found that cyclic stimulation promoted cell-driven collagen matrix bi-axial compaction, enhancing the mechanical strength of strained samples with respect to static controls. Moreover, cyclic strain positively affected SMC behavior: cells maintained their contractile phenotype and spread uniformly throughout the whole wall thickness. Conversely, static culture induced a noticeable polarization of cell distribution to the outer rim of the constructs and a sharp reduction in total cell density. Overall, coupling the use of a novel dual-mode bioreactor with engineered collagen-gel-based tubular constructs demonstrated to be an interesting technology to investigate the modulation of cell and tissue behavior under controlled mechanically conditioned in vitro maturation.

Engineered Three-Dimensional Cardiac Fibrotic Tissue to Study Fibrotic Remodeling

PubMed Central

Sadeghi, Amir Hossein; Shin, Su Ryon; Deddens, Janine C.; Fratta, Giuseppe; Mandla, Serena; Yazdi, Iman K.; Prakash, Gyan; Antona, Silvia; Demarchi, Danilo; Buijsrogge, Marc P.; Sluijter, Joost P.G.; Hjortnaes, Jesper

2017-01-01

Activation of cardiac fibroblasts (CF) into myofibroblasts is considered to play an essential role in cardiac remodeling and fibrosis. A limiting factor in studying this process is the spontaneous activation of CFs when cultured on two-dimensional (2D) culture plates. Here, a simplified 3D hydrogel platform of contractile cardiac tissue, stimulated by transforming growth factor-β1 (TGF-β1), is presented to recapitulate a fibrogenic micro-environment. It was hypothesized that the quiescent state of CFs can be maintained by mimicking the mechanical stiffness of native heart tissue. To test this hypothesis, a 3D cell culture model consisting of cardiomyocytes and CFs encapsulated within mechanically engineered gelatin methacryloyl (GelMA) hydrogel, was developed. The study shows that CFs maintain their quiescent phenotype in mechanically tuned hydrogels. Additionally, treatment with a beta-adrenergic agonist increased beating frequency, demonstrating physiologic-like behavior of the heart constructs. Subsequently, quiescent CFs within the constructs were activated by the exogenous addition of TGF-β1. The expression of fibrotic protein markers (and the functional changes in mechanical stiffness) in the fibrotic-like tissues were analyzed to validate the model. Overall, this 3D engineered culture model of contractile cardiac tissue enabled controlled activation of CFs, demonstrating the usability of this platform to study fibrotic remodeling. PMID:28498548

Computed tomography-guided tissue engineering of upper airway cartilage.

PubMed

Brown, Bryan N; Siebenlist, Nicholas J; Cheetham, Jonathan; Ducharme, Norm G; Rawlinson, Jeremy J; Bonassar, Lawrence J

2014-06-01

Normal laryngeal function has a large impact on quality of life, and dysfunction can be life threatening. In general, airway obstructions arise from a reduction in neuromuscular function or a decrease in mechanical stiffness of the structures of the upper airway. These reductions decrease the ability of the airway to resist inspiratory or expiratory pressures, causing laryngeal collapse. We propose to restore airway patency through methods that replace damaged tissue and improve the stiffness of airway structures. A number of recent studies have utilized image-guided approaches to create cell-seeded constructs that reproduce the shape and size of the tissue of interest with high geometric fidelity. The objective of the present study was to establish a tissue engineering approach to the creation of viable constructs that approximate the shape and size of equine airway structures, in particular the epiglottis. Computed tomography images were used to create three-dimensional computer models of the cartilaginous structures of the larynx. Anatomically shaped injection molds were created from the three-dimensional models and were seeded with bovine auricular chondrocytes that were suspended within alginate before static culture. Constructs were then cultured for approximately 4 weeks post-seeding and evaluated for biochemical content, biomechanical properties, and histologic architecture. Results showed that the three-dimensional molded constructs had the approximate size and shape of the equine epiglottis and that it is possible to seed such constructs while maintaining 75%+ cell viability. Extracellular matrix content was observed to increase with time in culture and was accompanied by an increase in the mechanical stiffness of the construct. If successful, such an approach may represent a significant improvement on the currently available treatments for damaged airway cartilage and may provide clinical options for replacement of damaged tissue during treatment of obstructive airway disease.

Ultrasound Elastography for Estimation of Regional Strain of Multilayered Hydrogels and Tissue-Engineered Cartilage

PubMed Central

Chung, Chen-Yuan; Heebner, Joseph; Baskaran, Harihara; Welter, Jean F.; Mansour, Joseph M.

2015-01-01

Tissue-engineered (TE) cartilage constructs tend to develop inhomogeneously, thus, to predict the mechanical performance of the tissue, conventional biomechanical testing, which yields average material properties, is of limited value. Rather, techniques for evaluating regional and depth-dependent properties of TE cartilage, preferably non-destructively, are required. The purpose of this study was to build upon our previous results and to investigate the feasibility of using ultrasound elastography to non-destructively assess the depth-dependent biomechanical characteristics of TE cartilage while in a sterile bioreactor. As a proof-of-concept, and to standardize an assessment protocol, a well-characterized three-layered hydrogel construct was used as a surrogate for TE cartilage, and was studied under controlled incremental compressions. The strain field of the construct predicted by elastography was then validated by comparison with a poroelastic finite-element analysis (FEA). On average, the differences between the strains predicted by elastography and the FEA were within 10%. Subsequently engineered cartilage tissue was evaluated in the same test fixture. Results from these examinations showed internal regions where the local strain was 1–2 orders of magnitude greater than that near the surface. These studies document the feasibility of using ultrasound to evaluate the mechanical behaviors of maturing TE constructs in a sterile environment. PMID:26077987

Mechanical evaluation of a tissue-engineered zone of calcification in a bone–hydrogel osteochondral construct

PubMed Central

Hollenstein, Jérôme; Terrier, Alexandre; Cory, Esther; Chen, Albert C.; Sah, Robert L.; Pioletti, Dominique P.

2016-01-01

The objective of this study was to test the hypothesis that mechanical properties of artificial osteochondral constructs can be improved by a tissue-engineered zone of calcification (teZCC) at the bone–hydrogel interface. Experimental push-off tests were performed on osteochondral constructs with or without a teZCC. In parallel, a numerical model of the osteochondral defect treatment was developed and validated against experimental results. Experimental results showed that the shear strength at the bone–hydrogel interface increased by 100% with the teZCC. Numerical predictions of the osteochondral defect treatment showed that the shear stress at the bone–hydrogel interface was reduced with the teZCC. We conclude that a teZCC in osteochondral constructs can provide two improvements. First, it increases the strength of the bone–hydrogel interface and second, it reduces the stress at this interface. PMID:23706035

Physiologically Distributed Loading Patterns Drive the Formation of Zonally Organized Collagen Structures in Tissue-Engineered Meniscus.

PubMed

Puetzer, Jennifer L; Bonassar, Lawrence J

2016-07-01

The meniscus is a dense fibrocartilage tissue that withstands the complex loads of the knee via a unique organization of collagen fibers. Attempts to condition engineered menisci with compression or tensile loading alone have failed to reproduce complex structure on the microscale or anatomic scale. Here we show that axial loading of anatomically shaped tissue-engineered meniscus constructs produced spatial distributions of local strain similar to those seen in the meniscus when the knee is loaded at full extension. Such loading drove formation of tissue with large organized collagen fibers, levels of mechanical anisotropy, and compressive moduli that match native tissue. Loading accelerated the development of native-sized and aligned circumferential and radial collagen fibers. These loading patterns contained both tensile and compressive components that enhanced the major biochemical and functional properties of the meniscus, with loading significantly improved glycosaminoglycan (GAG) accumulation 200-250%, collagen accumulation 40-55%, equilibrium modulus 1000-1800%, and tensile moduli 500-1200% (radial and circumferential). Furthermore, this study demonstrates local changes in mechanical environment drive heterogeneous tissue development and organization within individual constructs, highlighting the importance of recapitulating native loading environments. Loaded menisci developed cartilage-like tissue with rounded cells, a dense collagen matrix, and increased GAG accumulation in the more compressively loaded horns, and fibrous collagen-rich tissue in the more tensile loaded outer 2/3, similar to native menisci. Loaded constructs reached a level of organization not seen in any previous engineered menisci and demonstrate great promise as meniscal replacements.

Recent Advances in Bioink Design for 3D Bioprinting of Tissues and Organs.

PubMed

Ji, Shen; Guvendiren, Murat

2017-01-01

There is a growing demand for alternative fabrication approaches to develop tissues and organs as conventional techniques are not capable of fabricating constructs with required structural, mechanical, and biological complexity. 3D bioprinting offers great potential to fabricate highly complex constructs with precise control of structure, mechanics, and biological matter [i.e., cells and extracellular matrix (ECM) components]. 3D bioprinting is an additive manufacturing approach that utilizes a "bioink" to fabricate devices and scaffolds in a layer-by-layer manner. 3D bioprinting allows printing of a cell suspension into a tissue construct with or without a scaffold support. The most common bioinks are cell-laden hydrogels, decellulerized ECM-based solutions, and cell suspensions. In this mini review, a brief description and comparison of the bioprinting methods, including extrusion-based, droplet-based, and laser-based bioprinting, with particular focus on bioink design requirements are presented. We also present the current state of the art in bioink design including the challenges and future directions.

Recent Advances in Bioink Design for 3D Bioprinting of Tissues and Organs

PubMed Central

Ji, Shen; Guvendiren, Murat

2017-01-01

There is a growing demand for alternative fabrication approaches to develop tissues and organs as conventional techniques are not capable of fabricating constructs with required structural, mechanical, and biological complexity. 3D bioprinting offers great potential to fabricate highly complex constructs with precise control of structure, mechanics, and biological matter [i.e., cells and extracellular matrix (ECM) components]. 3D bioprinting is an additive manufacturing approach that utilizes a “bioink” to fabricate devices and scaffolds in a layer-by-layer manner. 3D bioprinting allows printing of a cell suspension into a tissue construct with or without a scaffold support. The most common bioinks are cell-laden hydrogels, decellulerized ECM-based solutions, and cell suspensions. In this mini review, a brief description and comparison of the bioprinting methods, including extrusion-based, droplet-based, and laser-based bioprinting, with particular focus on bioink design requirements are presented. We also present the current state of the art in bioink design including the challenges and future directions. PMID:28424770

LARGE STRAIN STIMULATION PROMOTES EXTRACELLULAR MATRIX PRODUCTION AND STIFFNESS IN AN ELASTOMERIC SCAFFOLD MODEL

PubMed Central

D’more, Antonio; Soares, Joao; Stella, John A.; Zhang, Will; Amoroso, Nicholas J.; Mayer, John E.; Wagner, William R.; Sacks, Michael S.

2016-01-01

Mechanical conditioning of engineered tissue constructs is widely recognized as one of the most relevant methods to enhance tissue accretion and microstructure, leading to improved mechanical behaviors. The understanding of the underlying mechanisms remains rather limited, restricting the development of in silico models of these phenomena, and the translation of engineered tissues into clinical application. In the present study, we examined the role of large strip-biaxial strains (up to 50%) on ECM synthesis by vascular smooth muscle cells (VSMCs) micro-integrated into electrospun polyester urethane urea (PEUU) constructs over the course of 3 weeks. Experimental results indicated that VSMC biosynthetic behavior was quite sensitive to tissue strain maximum level, and that collagen was the primary ECM component synthesized. Moreover, we found that while a 30% peak strain level achieved maximum ECM synthesis rate, further increases in strain level lead to a reduction in ECM biosynthesis. Subsequent mechanical analysis of the formed collagen fiber network was performed by removing the scaffold mechanical responses using a strain-energy based approach, showing that the de-novo collagen also demonstrated mechanical behaviors substantially better than previously obtained with small strain training and comparable to mature collagenous tissues. We conclude that the application of large deformations can play a critical role not only in the quantity of ECM synthesis (i.e. the rate of mass production), but also on the modulation of the stiffness of the newly formed ECM constituents. The improved understanding of the process of growth and development of ECM in these mechano-sensitive cell-scaffold systems will lead to more rational design and manufacturing of engineered tissues operating under highly demanding mechanical environments. PMID:27344402

Biphasic Finite Element Modeling Reconciles Mechanical Properties of Tissue-Engineered Cartilage Constructs Across Testing Platforms.

PubMed

Meloni, Gregory R; Fisher, Matthew B; Stoeckl, Brendan D; Dodge, George R; Mauck, Robert L

2017-07-01

Cartilage tissue engineering is emerging as a promising treatment for osteoarthritis, and the field has progressed toward utilizing large animal models for proof of concept and preclinical studies. Mechanical testing of the regenerative tissue is an essential outcome for functional evaluation. However, testing modalities and constitutive frameworks used to evaluate in vitro grown samples differ substantially from those used to evaluate in vivo derived samples. To address this, we developed finite element (FE) models (using FEBio) of unconfined compression and indentation testing, modalities commonly used for such samples. We determined the model sensitivity to tissue radius and subchondral bone modulus, as well as its ability to estimate material parameters using the built-in parameter optimization tool in FEBio. We then sequentially tested agarose gels of 4%, 6%, 8%, and 10% weight/weight using a custom indentation platform, followed by unconfined compression. Similarly, we evaluated the ability of the model to generate material parameters for living constructs by evaluating engineered cartilage. Juvenile bovine mesenchymal stem cells were seeded (2 × 10 7 cells/mL) in 1% weight/volume hyaluronic acid hydrogels and cultured in a chondrogenic medium for 3, 6, and 9 weeks. Samples were planed and tested sequentially in indentation and unconfined compression. The model successfully completed parameter optimization routines for each testing modality for both acellular and cell-based constructs. Traditional outcome measures and the FE-derived outcomes showed significant changes in material properties during the maturation of engineered cartilage tissue, capturing dynamic changes in functional tissue mechanics. These outcomes were significantly correlated with one another, establishing this FE modeling approach as a singular method for the evaluation of functional engineered and native tissue regeneration, both in vitro and in vivo.

Mechanical stretching for tissue engineering: two-dimensional and three-dimensional constructs.

PubMed

Riehl, Brandon D; Park, Jae-Hong; Kwon, Il Keun; Lim, Jung Yul

2012-08-01

Mechanical cell stretching may be an attractive strategy for the tissue engineering of mechanically functional tissues. It has been demonstrated that cell growth and differentiation can be guided by cell stretch with minimal help from soluble factors and engineered tissues that are mechanically stretched in bioreactors may have superior organization, functionality, and strength compared with unstretched counterparts. This review explores recent studies on cell stretching in both two-dimensional (2D) and three-dimensional (3D) setups focusing on the applications of stretch stimulation as a tool for controlling cell orientation, growth, gene expression, lineage commitment, and differentiation and for achieving successful tissue engineering of mechanically functional tissues, including cardiac, muscle, vasculature, ligament, tendon, bone, and so on. Custom stretching devices and lab-specific mechanical bioreactors are described with a discussion on capabilities and limitations. While stretch mechanotransduction pathways have been examined using 2D stretch, studying such pathways in physiologically relevant 3D environments may be required to understand how cells direct tissue development under stretch. Cell stretch study using 3D milieus may also help to develop tissue-specific stretch regimens optimized with biochemical feedback, which once developed will provide optimal tissue engineering protocols.

Mechanical Stretching for Tissue Engineering: Two-Dimensional and Three-Dimensional Constructs

PubMed Central

Riehl, Brandon D.; Park, Jae-Hong; Kwon, Il Keun

2012-01-01

Mechanical cell stretching may be an attractive strategy for the tissue engineering of mechanically functional tissues. It has been demonstrated that cell growth and differentiation can be guided by cell stretch with minimal help from soluble factors and engineered tissues that are mechanically stretched in bioreactors may have superior organization, functionality, and strength compared with unstretched counterparts. This review explores recent studies on cell stretching in both two-dimensional (2D) and three-dimensional (3D) setups focusing on the applications of stretch stimulation as a tool for controlling cell orientation, growth, gene expression, lineage commitment, and differentiation and for achieving successful tissue engineering of mechanically functional tissues, including cardiac, muscle, vasculature, ligament, tendon, bone, and so on. Custom stretching devices and lab-specific mechanical bioreactors are described with a discussion on capabilities and limitations. While stretch mechanotransduction pathways have been examined using 2D stretch, studying such pathways in physiologically relevant 3D environments may be required to understand how cells direct tissue development under stretch. Cell stretch study using 3D milieus may also help to develop tissue-specific stretch regimens optimized with biochemical feedback, which once developed will provide optimal tissue engineering protocols. PMID:22335794

Evaluation of nanostructural, mechanical, and biological properties of collagen-nanotube composites.

PubMed

Tan, Wei; Twomey, John; Guo, Dongjie; Madhavan, Krishna; Li, Min

2010-06-01

Collagen I is an essential structural and mechanical building block of various tissues, and it is often used as tissue-engineering scaffolds. However, collagen-based constructs reconstituted in vitro often lacks robust fiber structure, mechanical stability, and molecule binding capability. To enhance these performances, the present study developed 3-D collagen-nanotube composite constructs with two types of functionalized carbon nanotubes, carboxylated nanotubes and covalently functionalized nanotubes (CFNTs). The influences of nanotube functionalization and loading concentration on the collagen fiber structure, mechanical property, biocompatibility, and molecule binding were examined. Results revealed that surface modification and loading concentration of nanotubes determined the interactions between nanotubes and collagen fibrils, thus altering the structure and property of nanotube-collagen composites. Scanning electron microscopy and confocal microscopy revealed that the incorporation of CFNT in collagen-based constructs was an effective means of restructuring collagen fibrils because CFNT strongly bound to collagen molecules inducing the formation of larger fibril bundles. However, increased nanotube loading concentration caused the formation of denser fibril network and larger aggregates. Static stress-strain tests under compression showed that the addition of nanotube into collagen-based constructs did not significantly increase static compressive moduli. Creep/recovery testing under compression revealed that CFNT-collagen constructs showed improved mechanical stability under continuous loading. Testing with endothelial cells showed that biocompatibility was highly dependent on nanotube loading concentration. At a low loading level, CFNT-collagen showed higher endothelial coverage than the other tested constructs or materials. Additionally, CFNT-collagen showed capability of binding to other biomolecules to enhance the construct functionality. In conclusion, functionalized nanotube-collagen composites, particularly CFNT-collagen composites, could be promising materials, which provide structural support showing bundled fibril structure, biocompatibility, multifunctionality, and mechanical stability, but rigorous control over chemical modification, loading concentration, and nanotube dispersion are needed.

Application of Extrusion-Based Hydrogel Bioprinting for Cartilage Tissue Engineering.

PubMed

You, Fu; Eames, B Frank; Chen, Xiongbiao

2017-07-23

Extrusion-based bioprinting (EBB) is a rapidly developing technique that has made substantial progress in the fabrication of constructs for cartilage tissue engineering (CTE) over the past decade. With this technique, cell-laden hydrogels or bio-inks have been extruded onto printing stages, layer-by-layer, to form three-dimensional (3D) constructs with varying sizes, shapes, and resolutions. This paper reviews the cell sources and hydrogels that can be used for bio-ink formulations in CTE application. Additionally, this paper discusses the important properties of bio-inks to be applied in the EBB technique, including biocompatibility, printability, as well as mechanical properties. The printability of a bio-ink is associated with the formation of first layer, ink rheological properties, and crosslinking mechanisms. Further, this paper discusses two bioprinting approaches to build up cartilage constructs, i.e., self-supporting hydrogel bioprinting and hybrid bioprinting, along with their applications in fabricating chondral, osteochondral, and zonally organized cartilage regenerative constructs. Lastly, current limitations and future opportunities of EBB in printing cartilage regenerative constructs are reviewed.

Helicoidal multi-lamellar features of RGD-functionalized silk biomaterials for corneal tissue engineering.

PubMed

Gil, Eun Seok; Mandal, Biman B; Park, Sang-Hyug; Marchant, Jeffrey K; Omenetto, Fiorenzo G; Kaplan, David L

2010-12-01

RGD-coupled silk protein-biomaterial lamellar systems were prepared and studied with human cornea fibroblasts (hCFs) to match functional requirements. A strategy for corneal tissue engineering was pursued to replicate the structural hierarchy of human corneal stroma within thin stacks of lamellae-like tissues, in this case constructed from scaffolds constructed with RGD-coupled, patterned, porous, mechanically robust and transparent silk films. The influence of RGD-coupling on the orientation, proliferation, ECM organization, and gene expression of hCFs was assessed. RGD surface modification enhanced cell attachment, proliferation, alignment and expression of both collagens (type I and V) and proteoglycans (decorin and biglycan). Confocal and histological images of the lamellar systems revealed that the bio-functionalized silk human cornea 3D constructs exhibited integrated corneal stroma tissue with helicoidal multi-lamellar alignment of collagen-rich and proteoglycan-rich extracellular matrix, with transparency of the construct. This biomimetic approach to replicate corneal stromal tissue structural hierarchy and architecture demonstrates a useful strategy for engineering human cornea. Further, this approach can be exploited for other tissue systems due to the pervasive nature of such helicoids in most human tissues. Copyright © 2010 Elsevier Ltd. All rights reserved.

Energy dissipation in quasi-linear viscoelastic tissues, cells, and extracellular matrix.

PubMed

Babaei, Behzad; Velasquez-Mao, A J; Pryse, Kenneth M; McConnaughey, William B; Elson, Elliot L; Genin, Guy M

2018-05-21

Characterizing how a tissue's constituents give rise to its viscoelasticity is important for uncovering how hidden timescales underlie multiscale biomechanics. These constituents are viscoelastic in nature, and their mechanics must typically be assessed from the uniaxial behavior of a tissue. Confounding the challenge is that tissue viscoelasticity is typically associated with nonlinear elastic responses. Here, we experimentally assessed how fibroblasts and extracellular matrix (ECM) within engineered tissue constructs give rise to the nonlinear viscoelastic responses of a tissue. We applied a constant strain rate, "triangular-wave" loading and interpreted responses using the Fung quasi-linear viscoelastic (QLV) material model. Although the Fung QLV model has several well-known weaknesses, it was well suited to the behaviors of the tissue constructs, cells, and ECM tested. Cells showed relatively high damping over certain loading frequency ranges. Analysis revealed that, even in cases where the Fung QLV model provided an excellent fit to data, the the time constant derived from the model was not in general a material parameter. Results have implications for design of protocols for the mechanical characterization of biological materials, and for the mechanobiology of cells within viscoelastic tissues. Copyright © 2018. Published by Elsevier Ltd.

Differences in time-dependent mechanical properties between extruded and molded hydrogels

PubMed Central

Ersumo, N; Witherel, CE; Spiller, KL

2016-01-01

The mechanical properties of hydrogels used in biomaterials and tissue engineering applications are critical determinants of their functionality. Despite the recent rise of additive manufacturing, and specifically extrusion-based bioprinting, as a prominent biofabrication method, comprehensive studies investigating the mechanical behavior of extruded constructs remain lacking. To address this gap in knowledge, we compared the mechanical properties and swelling properties of crosslinked gelatin-based hydrogels prepared by conventional molding techniques or by 3D bioprinting using a BioBots Beta pneumatic extruder. A preliminary characterization of the impact of bioprinting parameters on construct properties revealed that both Young's modulus and optimal extruding pressure increased with polymer content, and that printing resolution increased with both printing speed and nozzle gauge. High viability (>95%) of encapsulated NIH 3T3 fibroblasts confirmed the cytocompatibility of the construct preparation process. Interestingly, the Young's moduli of extruded and molded constructs were not different, but extruded constructs did show increases in both the rate and extent of time-dependent mechanical behavior observed in creep. Despite similar polymer densities, extruded hydrogels showed greater swelling over time compared to molded hydrogels, suggesting that differences in creep behavior derived from differences in microstructure and fluid flow. Because of the crucial roles of time-dependent mechanical properties, fluid flow, and swelling properties on tissue and cell behavior, these findings highlight the need for greater consideration of the effects of the extrusion process on hydrogel properties. PMID:27550945

Towards a unified theory for morphomechanics

PubMed Central

Taber, Larry A.

2009-01-01

Mechanical forces are closely involved in the construction of an embryo. Experiments have suggested that mechanical feedback plays a role in regulating these forces, but the nature of this feedback is poorly understood. Here, we propose a general principle for the mechanics of morphogenesis, as governed by a pair of evolution equations based on feedback from tissue stress. In one equation, the rate of growth (or contraction) depends on the difference between the current tissue stress and a target (homeostatic) stress. In the other equation, the target stress changes at a rate that depends on the same stress difference. The parameters in these morphomechanical laws are assumed to depend on stress rate. Computational models are used to illustrate how these equations can capture a relatively wide range of behaviours observed in developing embryos, as well as show the limitations of this theory. Specific applications include growth of pressure vessels (e.g. the heart, arteries and brain), wound healing and sea urchin gastrulation. Understanding the fundamental principles of tissue construction can help engineers design new strategies for creating replacement tissues and organs in vitro. PMID:19657011

A novel device to stretch multiple tissue samples with variable patterns: application for mRNA regulation in tissue-engineered constructs.

PubMed

Imsirovic, Jasmin; Derricks, Kelsey; Buczek-Thomas, Jo Ann; Rich, Celeste B; Nugent, Matthew A; Suki, Béla

2013-01-01

A broad range of cells are subjected to irregular time varying mechanical stimuli within the body, particularly in the respiratory and circulatory systems. Mechanical stretch is an important factor in determining cell function; however, the effects of variable stretch remain unexplored. In order to investigate the effects of variable stretch, we designed, built and tested a uniaxial stretching device that can stretch three-dimensional tissue constructs while varying the strain amplitude from cycle to cycle. The device is the first to apply variable stretching signals to cells in tissues or three dimensional tissue constructs. Following device validation, we applied 20% uniaxial strain to Gelfoam samples seeded with neonatal rat lung fibroblasts with different levels of variability (0%, 25%, 50% and 75%). RT-PCR was then performed to measure the effects of variable stretch on key molecules involved in cell-matrix interactions including: collagen 1α, lysyl oxidase, α-actin, β1 integrin, β3 integrin, syndecan-4, and vascular endothelial growth factor-A. Adding variability to the stretching signal upregulated, downregulated or had no effect on mRNA production depending on the molecule and the amount of variability. In particular, syndecan-4 showed a statistically significant peak at 25% variability, suggesting that an optimal variability of strain may exist for production of this molecule. We conclude that cycle-by-cycle variability in strain influences the expression of molecules related to cell-matrix interactions and hence may be used to selectively tune the composition of tissue constructs.

An electromechanical based deformable model for soft tissue simulation.

PubMed

Zhong, Yongmin; Shirinzadeh, Bijan; Smith, Julian; Gu, Chengfan

2009-11-01

Soft tissue deformation is of great importance to surgery simulation. Although a significant amount of research efforts have been dedicated to simulating the behaviours of soft tissues, modelling of soft tissue deformation is still a challenging problem. This paper presents a new deformable model for simulation of soft tissue deformation from the electromechanical viewpoint of soft tissues. Soft tissue deformation is formulated as a reaction-diffusion process coupled with a mechanical load. The mechanical load applied to a soft tissue to cause a deformation is incorporated into the reaction-diffusion system, and consequently distributed among mass points of the soft tissue. Reaction-diffusion of mechanical load and non-rigid mechanics of motion are combined to govern the simulation dynamics of soft tissue deformation. An improved reaction-diffusion model is developed to describe the distribution of the mechanical load in soft tissues. A three-layer artificial cellular neural network is constructed to solve the reaction-diffusion model for real-time simulation of soft tissue deformation. A gradient based method is established to derive internal forces from the distribution of the mechanical load. Integration with a haptic device has also been achieved to simulate soft tissue deformation with haptic feedback. The proposed methodology does not only predict the typical behaviours of living tissues, but it also accepts both local and large-range deformations. It also accommodates isotropic, anisotropic and inhomogeneous deformations by simple modification of diffusion coefficients.

Oxygen tension differentially regulates the functional properties of cartilaginous tissues engineered from infrapatellar fat pad derived MSCs and articular chondrocytes.

PubMed

Buckley, C T; Vinardell, T; Kelly, D J

2010-10-01

For current tissue engineering or regenerative medicine strategies, chondrocyte (CC)- or mesenchymal stem cell (MSC)-seeded constructs are typically cultured in normoxic conditions (20% oxygen). However, within the knee joint capsule a lower oxygen tension exists. The objective of this study was to investigate how CCs and infrapatellar fad pad derived MSCs will respond to a low oxygen (5%) environment in 3D agarose culture. Our hypothesis was that culture in a low oxygen environment (5%) will enhance the functional properties of cartilaginous tissues engineered using both cell sources. Cell-encapsulated agarose hydrogel constructs (seeded with CCs or infrapatellar fat pad (IFP) derived MSCs) were prepared and cultured in a chemically defined serum-free medium in the presence (CCs and MSCs) or absence (CCs only) of transforming growth factor-beta3 (TGF-β3) in normoxic (20%) or low oxygen (5%) conditions for 42 days. Constructs were assessed at days 0, 21 and 42 in terms of mechanical properties, biochemical content and histologically. Low oxygen tension (5%) was observed to promote extracellular matrix (ECM) production by CCs cultured in the absence of TGF-β3, but was inhibitory in the presence of TGF-β3. In contrast, a low oxygen tension enhanced chondrogenesis of IFP constructs in the presence of TGF-β3, leading to superior mechanical functionality compared to CCs cultured in identical conditions. Extrapolating the results of this study to the in vivo setting, it would appear that joint fat pad derived MSCs may possess a superior potential to generate a functional repair tissue in low oxygen tensions. However, in the context of in vitro cartilage tissue engineering, CCs maintained in normoxic conditions in the presence of TGF-β3 generate the most mechanically functional tissue. Copyright © 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration.

PubMed

Meghezi, Sébastien; Seifu, Dawit G; Bono, Nina; Unsworth, Larry; Mequanint, Kibret; Mantovani, Diego

2015-06-16

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled. The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The "static bioreactor" provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

Advances in Application of Mechanical Stimuli in Bioreactors for Cartilage Tissue Engineering.

PubMed

Li, Ke; Zhang, Chunqiu; Qiu, Lulu; Gao, Lilan; Zhang, Xizheng

2017-08-01

Articular cartilage (AC) is the weight-bearing tissue in diarthroses. It lacks the capacity for self-healing once there are injuries or diseases due to its avascularity. With the development of tissue engineering, repairing cartilage defects through transplantation of engineered cartilage that closely matches properties of native cartilage has become a new option for curing cartilage diseases. The main hurdle for clinical application of engineered cartilage is how to develop functional cartilage constructs for mass production in a credible way. Recently, impressive hyaline cartilage that may have the potential to provide capabilities for treating large cartilage lesions in the future has been produced in laboratories. The key to functional cartilage construction in vitro is to identify appropriate mechanical stimuli. First, they should ensure the function of metabolism because mechanical stimuli play the role of blood vessels in the metabolism of AC, for example, acquiring nutrition and removing wastes. Second, they should mimic the movement of synovial joints and produce phenotypically correct tissues to achieve the adaptive development between the micro- and macrostructure and function. In this article, we divide mechanical stimuli into three types according to forces transmitted by different media in bioreactors, namely forces transmitted through the liquid medium, solid medium, or other media, then we review and summarize the research status of bioreactors for cartilage tissue engineering (CTE), mainly focusing on the effects of diverse mechanical stimuli on engineered cartilage. Based on current researches, there are several motion patterns in knee joints; but compression, tension, shear, fluid shear, or hydrostatic pressure each only partially reflects the mechanical condition in vivo. In this study, we propose that rolling-sliding-compression load consists of various stimuli that will represent better mechanical environment in CTE. In addition, engineers often ignore the importance of biochemical factors to the growth and development of engineered cartilage. In our point of view, only by fully considering synergistic effects of mechanical and biochemical factors can we find appropriate culture conditions for functional cartilage constructs. Once again, rolling-sliding-compression load under appropriate biochemical conditions may be conductive to realize the adaptive development between the structure and function of engineered cartilage in vitro.

The design and development of a high-throughput magneto-mechanostimulation device for cartilage tissue engineering.

PubMed

Brady, Mariea A; Vaze, Reva; Amin, Harsh D; Overby, Darryl R; Ethier, C Ross

2014-02-01

To recapitulate the in vivo environment and create neo-organoids that replace lost or damaged tissue requires the engineering of devices, which provide appropriate biophysical cues. To date, bioreactors for cartilage tissue engineering have focused primarily on biomechanical stimulation. There is a significant need for improved devices for articular cartilage tissue engineering capable of simultaneously applying multiple biophysical (electrokinetic and mechanical) stimuli. We have developed a novel high-throughput magneto-mechanostimulation bioreactor, capable of applying static and time-varying magnetic fields, as well as multiple and independently adjustable mechanical loading regimens. The device consists of an array of 18 individual stations, each of which uses contactless magnetic actuation and has an integrated Hall Effect sensing system, enabling the real-time measurements of applied field, force, and construct thickness, and hence, the indirect measurement of construct mechanical properties. Validation tests showed precise measurements of thickness, within 14 μm of gold standard calliper measurements; further, applied force was measured to be within 0.04 N of desired force over a half hour dynamic loading, which was repeatable over a 3-week test period. Finally, construct material properties measured using the bioreactor were not significantly different (p=0.97) from those measured using a standard materials testing machine. We present a new method for articular cartilage-specific bioreactor design, integrating combinatorial magneto-mechanostimulation, which is very attractive from functional and cost viewpoints.

Regulation of decellularized tissue remodeling via scaffold-mediated lentiviral delivery in anatomically-shaped osteochondral constructs.

PubMed

Rowland, Christopher R; Glass, Katherine A; Ettyreddy, Adarsh R; Gloss, Catherine C; Matthews, Jared R L; Huynh, Nguyen P T; Guilak, Farshid

2018-05-30

Cartilage-derived matrix (CDM) has emerged as a promising scaffold material for tissue engineering of cartilage and bone due to its native chondroinductive capacity and its ability to support endochondral ossification. Because it consists of native tissue, CDM can undergo cellular remodeling, which can promote integration with host tissue and enables it to be degraded and replaced by neotissue over time. However, enzymatic degradation of decellularized tissues can occur unpredictably and may not allow sufficient time for mechanically competent tissue to form, especially in the harsh inflammatory environment of a diseased joint. The goal of the current study was to engineer cartilage and bone constructs with the ability to inhibit aberrant inflammatory processes caused by the cytokine interleukin-1 (IL-1), through scaffold-mediated delivery of lentiviral particles containing a doxycycline-inducible IL-1 receptor antagonist (IL-1Ra) transgene on anatomically-shaped CDM constructs. Additionally, scaffold-mediated lentiviral gene delivery was used to facilitate spatial organization of simultaneous chondrogenic and osteogenic differentiation via site-specific transduction of a single mesenchymal stem cell (MSC) population to overexpress either chondrogenic, transforming growth factor-beta 3 (TGF-β3), or osteogenic, bone morphogenetic protein-2 (BMP-2), transgenes. Controlled induction of IL-1Ra expression protected CDM hemispheres from inflammation-mediated degradation, and supported robust bone and cartilage tissue formation even in the presence of IL-1. In the absence of inflammatory stimuli, controlled cellular remodeling was exploited as a mechanism for fusing concentric CDM hemispheres overexpressing BMP-2 and TGF-β3 into a single bi-layered osteochondral construct. Our findings demonstrate that site-specific delivery of inducible and tunable transgenes confers spatial and temporal control over both CDM scaffold remodeling and neotissue composition. Furthermore, these constructs provide a microphysiological in vitro joint organoid model with site-specific, tunable, and inducible protein delivery systems for examining the spatiotemporal response to pro-anabolic and/or inflammatory signaling across the osteochondral interface. Copyright © 2018 Elsevier Ltd. All rights reserved.

Differential osteogenic activity of osteoprogenitor cells on HA and TCP/HA scaffold of tissue engineered bone.

PubMed

Ng, Angela M H; Tan, K K; Phang, M Y; Aziyati, O; Tan, G H; Isa, M R; Aminuddin, B S; Naseem, M; Fauziah, O; Ruszymah, B H I

2008-05-01

Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering. Copyright 2007 Wiley Periodicals, Inc.

The influence of construct scale on the composition and functional properties of cartilaginous tissues engineered using bone marrow-derived mesenchymal stem cells.

PubMed

Buckley, Conor T; Meyer, Eric G; Kelly, Daniel J

2012-02-01

Engineering cartilaginous tissue of a scale necessary to treat defects observed clinically is a well-documented challenge in the field of cartilage tissue engineering. The objective of this study was to determine how the composition and mechanical properties of cartilaginous tissues that are engineered by using bone marrow-derived mesenchymal stem cells (MSCs) depend on the scale of the construct. Porcine bone marrow-derived MSCs were encapsulated in agarose hydrogels, and constructs of different cylindrical geometries (Ø4×1.5 mm; Ø5×3 mm; Ø6×4.5 mm; Ø8×4.5 mm) were fabricated and maintained in a chemically defined serum-free medium supplemented with transforming growth factor-β3 for 42 days. Total sulfated glycosaminoglycan (sGAG) accumulation by day 42 increased from 0.14% w/w to 0.88% w/w as the construct geometry increased from Ø4×1.5 to Ø8×4.5 mm, with collagen accumulation increasing from 0.31% w/w to 1.62% w/w. This led to an increase in the dynamic modulus from 90.81 to 327.51 kPa as the engineered tissue increased in scale from Ø4×1.5 to Ø8×4.5 mm. By decreasing the external oxygen tension from 20% to 5%, it was possible to achieve these higher levels of mechanical functionality in the smaller engineered tissues. Constructs were then sectioned into smaller subregions to quantify the spatial accumulation of extracellular matrix components, and a model of oxygen diffusion and consumption was used to predict spatial gradients in oxygen concentration throughout the construct. sGAG accumulation was always highest in regions where oxygen concentration was predicted to be lowest. In addition, as the size of the engineered construct increased, different regions of the construct preferentially supported either sGAG or collagen accumulation, thus suggesting that gradients in regulatory factors other than oxygen were playing a role in determining levels of collagen synthesis. The identification of such factors and the means to control their spatial concentration within developing tissues represents a central challenge in engineering large cartilaginous grafts.

Growth Factor Stimulation Improves the Structure and Properties of Scaffold-Free Engineered Auricular Cartilage Constructs

PubMed Central

Rosa, Renata G.; Joazeiro, Paulo P.; Bianco, Juares; Kunz, Manuela; Weber, Joanna F.; Waldman, Stephen D.

2014-01-01

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation. PMID:25126941

Tunability of collagen matrix mechanical properties via multiple modes of mineralization

PubMed Central

Smith, Lester J.; Deymier, Alix C.; Boyle, John J.; Li, Zhen; Linderman, Stephen W.; Pasteris, Jill D.; Xia, Younan; Genin, Guy M.; Thomopoulos, Stavros

2016-01-01

Functionally graded, mineralized collagen tissues exist at soft-to-hard material attachments throughout the body. However, the details of how collagen and hydroxyapatite mineral (HA) interact are not fully understood, hampering efforts to develop tissue-engineered constructs that can assist with repair of injuries at the attachments of soft tissues to bone. In this study, spatial control of mineralization was achieved in collagen matrices using simulated body fluids (SBFs). Based upon previous observations of poor bonding between reconstituted collagen and HA deposited using SBF, we hypothesized that mineralizing collagen in the presence of fetuin (which inhibits surface mineralization) would lead to more mineral deposition within the scaffold and therefore a greater increase in stiffness and toughness compared with collagen mineralized without fetuin. We tested this hypothesis through integrated synthesis, mechanical testing and modelling of graded, mineralized reconstituted collagen constructs. Results supported the hypothesis, and further suggested that mineralization on the interior of reconstituted collagen constructs, as promoted by fetuin, led to superior bonding between HA and collagen. The results provide us guidance for the development of mineralized collagen scaffolds, with implications for bone and tendon-to-bone tissue engineering. PMID:26855755

Bioink properties before, during and after 3D bioprinting.

PubMed

Hölzl, Katja; Lin, Shengmao; Tytgat, Liesbeth; Van Vlierberghe, Sandra; Gu, Linxia; Ovsianikov, Aleksandr

2016-09-23

Bioprinting is a process based on additive manufacturing from materials containing living cells. These materials, often referred to as bioink, are based on cytocompatible hydrogel precursor formulations, which gel in a manner compatible with different bioprinting approaches. The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural resolution, shape fidelity and cell survival. However, it is the final properties of the matured bioprinted tissue construct that are crucial for the end application. During tissue formation these properties are influenced by the amount of cells present in the construct, their proliferation, migration and interaction with the material. A calibrated computational framework is able to predict the tissue development and maturation and to optimize the bioprinting input parameters such as the starting material, the initial cell loading and the construct geometry. In this contribution relevant bioink properties are reviewed and discussed on the example of most popular bioprinting approaches. The effect of cells on hydrogel processing and vice versa is highlighted. Furthermore, numerical approaches were reviewed and implemented for depicting the cellular mechanics within the hydrogel as well as for prediction of mechanical properties to achieve the desired hydrogel construct considering cell density, distribution and material-cell interaction.

Bioactive scaffold for bone tissue engineering: An in vivo study

NASA Astrophysics Data System (ADS)

Livingston, Treena Lynne

Massive bone loss of the proximal femur is a common problem in revision cases of total hip implants. Allograft is typically used to reconstruct the site for insertion of the new prosthesis. However, for long term fixation and function, it is desirable that the allograft becomes fully replaced by bone tissue and aids in the regeneration of bone to that site. However, allograft use is typically associated with delayed incorporation and poor remodeling. Due to these profound limitations, alternative approaches are needed. Tissue engineering is an attractive approach to designing improved graft materials. By combining osteogenic activity with a resorbable scaffold, bone formation can be stimulated while providing structure and stability to the limb during incorporation and remodeling of the scaffold. Porous, surface modified bioactive ceramic scaffolds (pSMC) have been developed which stimulate the expression of the osteoblastic phenotype and production of bone-like tissue in vitro. The scaffold and two tissue-engineered constructs, osteoprogenitor cells seeded onto scaffolds or cells expanded in culture to form bone tissue on the scaffolds prior to implantation, were investigated in a long bone defect model. The rate of incorporation was assessed. Both tissue-engineered constructs stimulated bone formation and comparable repair at 2 weeks. In a rat femoral window defect model, bone formation increased over time for all groups in concert with scaffold resorption, leading to a 40% increase in bone and 40% reduction of the scaffold in the defect by 12 weeks. Both tissue-engineered constructs enhanced the rate of mechanical repair of long bones due to better bony union with the host cortex. Long bones treated with tissue engineered constructs demonstrated a return in normal torsional properties by 4 weeks as compared to 12 weeks for long bones treated with pSMC. Culture expansion of cells to produce bone tissue in vitro did not accelerate incorporation over the treatment with cells seeded at the time of surgery. Porous, surface modified bioactive ceramic is a promising scaffold material for tissue-engineered bone repair. Bone formation and scaffold resorption act in concert for maintenance and improvement of the structural properties of the long bones over time. As determined histomorphometrically and mechanically, the rate of incorporation of the scaffold was enhanced with the tissue-engineered constructs.

Construction of stable capillary networks using a microfluidic device.

PubMed

Sudo, Ryo

2015-01-01

Construction of stable capillary networks is required to provide sufficient oxygen and nutrients to the deep region of thick tissues, which is important in the context of 3D tissue engineering. Although conventional in vitro culture models have been used to investigate the mechanism of capillary formation, recent advances in microfluidics technologies allowed us to control biophysical and biochemical culture environments more precisely, which led to the construction of functional and stable capillary networks. In this study, endothelial cells and mesenchymal stem cells were co-cultured in microfluidic devices to construct stable capillary networks, which resulted in the construction of luminal structures covered by pericytes. Interactions between endothelial cells and mesenchymal stem cells are also discussed in the context of capillary formation.

Engineered disc-like angle-ply structures for intervertebral disc replacement.

PubMed

Nerurkar, Nandan L; Sen, Sounok; Huang, Alice H; Elliott, Dawn M; Mauck, Robert L

2010-04-15

To develop a construction algorithm in which electrospun nanofibrous scaffolds are coupled with a biocompatible hydrogel to engineer a mesenchymal stem cell (MSC)-based disc replacement. To engineer a disc-like angle-ply structure (DAPS) that replicates the multiscale architecture of the intervertebral disc. Successful engineering of a replacement for the intervertebral disc requires replication of its mechanical function and anatomic form. Despite many attempts to engineer a replacement for ailing and degenerated discs, no prior study has replicated the multiscale hierarchical architecture of the native disc, and very few have assessed the mechanical function of formed neo-tissues. A new algorithm for the construction of a disc analogue was developed, using agarose to form a central nucleus pulposus (NP) and oriented electrospun nanofibrous scaffolds to form the anulus fibrosus region (AF). Bovine MSCs were seeded into both regions and biochemical, histologic, and mechanical maturation were evaluated with in vitro culture. We show that mechanical testing in compression and torsion, loading methods commonly used to assess disc mechanics, reveal equilibrium and time-dependent behaviors that are qualitatively similar to native tissue, although lesser in magnitude. Further, we demonstrate that cells seeded into both AF and NP regions adopt distinct morphologies that mirror those seen in native tissue, and that, in the AF region, this ordered community of cells deposit matrix that is organized in an angle-ply configuration. Finally, constructs demonstrate functional development with long-term in vitro culture. These findings provide a new approach for disc tissue engineering that replicates multi-scale form and function of the intervertebral disc, providing a foundation from which to build a multi-scale, biologic, anatomically and hierarchically relevant composite disc analogue for eventual disc replacement.

Thick Acellular Heart Extracellular Matrix with Inherent Vasculature: A Potential Platform for Myocardial Tissue Regeneration

PubMed Central

Sarig, Udi; Au-Yeung, Gigi C.T.; Wang, Yao; Bronshtein, Tomer; Dahan, Nitsan; Boey, Freddy Y.C.; Venkatraman, Subbu S.

2012-01-01

The decellularization of porcine heart tissue offers many opportunities for the production of physiologically relevant myocardial mimetic scaffolds. Earlier, we reported the successful isolation of a thin porcine cardiac extracellular matrix (pcECM) exhibiting relevant bio-mechanical properties for myocardial tissue engineering. Nevertheless, since native cardiac tissue is much thicker, such thin scaffolds may offer limited regeneration capacity. However, generation of thicker myocardial mimetic tissue constructs is hindered by diffusion limitations (∼100 μm), and the lack of a proper vascular-like network within these constructs. In our present work, we focused on optimizing the decellularization procedure for thicker tissue slabs (10–15 mm), while retaining their inherent vasculature, and on characterizing the resulting pcECM. The trypsin/Triton-based perfusion procedure that resulted in a nonimmunogenic and cell-supportive pcECM was found to be more effective in cell removal and in the preservation of fiber morphology and structural characteristics than stirring, sonication, or sodium dodecyl sulfate/Triton-based procedures. Mass spectroscopy revealed that the pcECM is mainly composed of ECM proteins with no apparent cellular protein remains. Mechanical testing indicated that the obtained pcECM is viscoelastic in nature and possesses the typical stress-strain profile of biological materials. It is stiffer than native tissue yet exhibits matched mechanical properties in terms of energy dissipation, toughness, and ultimate stress behavior. Vascular network functionality was maintained to the first three–four branches from the main coronary vessels. Taken together, these results reaffirm the efficiency of the decellularization procedure reported herein for yielding thick nonimmunogenic cell-supportive pcECM scaffolds, preserving both native tissue ultra-structural properties and an inherent vascular network. When reseeded with the appropriate progenitor cells, these scaffolds can potentially serve as ex vivo screening platforms for new therapeutics, as models for human cardiac ECM, or as biomedical constructs for patch or transmural transplantation strategies. PMID:22663095

Engineering Lubrication in Articular Cartilage

PubMed Central

McNary, Sean M.; Athanasiou, Kyriacos A.

2012-01-01

Despite continuous progress toward tissue engineering of functional articular cartilage, significant challenges still remain. Advances in morphogens, stem cells, and scaffolds have resulted in enhancement of the bulk mechanical properties of engineered constructs, but little attention has been paid to the surface mechanical properties. In the near future, engineered tissues will be able to withstand and support the physiological compressive and tensile forces in weight-bearing synovial joints such as the knee. However, there is an increasing realization that these tissue-engineered cartilage constructs will fail without the optimal frictional and wear properties present in native articular cartilage. These characteristics are critical to smooth, pain-free joint articulation and a long-lasting, durable cartilage surface. To achieve optimal tribological properties, engineered cartilage therapies will need to incorporate approaches and methods for functional lubrication. Steady progress in cartilage lubrication in native tissues has pushed the pendulum and warranted a shift in the articular cartilage tissue-engineering paradigm. Engineered tissues should be designed and developed to possess both tribological and mechanical properties mirroring natural cartilage. In this article, an overview of the biology and engineering of articular cartilage structure and cartilage lubrication will be presented. Salient progress in lubrication treatments such as tribosupplementation, pharmacological, and cell-based therapies will be covered. Finally, frictional assays such as the pin-on-disk tribometer will be addressed. Knowledge related to the elements of cartilage lubrication has progressed and, thus, an opportune moment is provided to leverage these advances at a critical step in the development of mechanically and tribologically robust, biomimetic tissue-engineered cartilage. This article is intended to serve as the first stepping stone toward future studies in functional tissue engineering of articular cartilage that begins to explore and incorporate methods of lubrication. PMID:21955119

Hydrogels for lung tissue engineering: Biomechanical properties of thin collagen-elastin constructs.

PubMed

Dunphy, Siobhán E; Bratt, Jessica A J; Akram, Khondoker M; Forsyth, Nicholas R; El Haj, Alicia J

2014-10-01

In this study, collagen-elastin constructs were prepared with the aim of producing a material capable of mimicking the mechanical properties of a single alveolar wall. Collagen has been used in a wide range of tissue engineering applications; however, due to its low mechanical properties its use is limited to non load-bearing applications without further manipulation using methods such as cross-linking or mechanical compression. Here, it was hypothesised that the addition of soluble elastin to a collagen hydrogel could improve its mechanical properties. Hydrogels made from collagen only and collagen plus varying amounts elastin were prepared. Young׳s modulus of each membrane was measured using the combination of a non-destructive indentation and a theoretical model previously described. An increase in Young׳s modulus was observed with increasing concentration of elastin. The use of non-destructive indentation allowed for online monitoring of the elastic moduli of cell-seeded constructs over 8 days. The addition of lung fibroblasts into the membrane increased the stiffness of the hydrogels further and cell-seeded collagen hydrogels were found to have a stiffness equal to the theoretical value for a single alveolar wall (≈5kPa). Through provision of some of the native extracellular matrix components of the lung parenchyma these scaffolds may be able to provide an initial building block toward the regeneration of new functional lung tissue. Copyright © 2014 Elsevier Ltd. All rights reserved.

The local matrix distribution and the functional development of tissue engineered cartilage, a finite element study.

PubMed

Sengers, B G; Van Donkelaar, C C; Oomens, C W J; Baaijens, F P T

2004-12-01

Assessment of the functionality of tissue engineered cartilage constructs is hampered by the lack of correlation between global measurements of extra cellular matrix constituents and the global mechanical properties. Based on patterns of matrix deposition around individual cells, it has been hypothesized previously, that mechanical functionality arises when contact occurs between zones of matrix associated with individual cells. The objective of this study is to determine whether the local distribution of newly synthesized extracellular matrix components contributes to the evolution of the mechanical properties of tissue engineered cartilage constructs. A computational homogenization approach was adopted, based on the concept of a periodic representative volume element. Local transport and immobilization of newly synthesized matrix components were described. Mechanical properties were taken dependent on the local matrix concentration and subsequently the global aggregate modulus and hydraulic permeability were derived. The transport parameters were varied to assess the effect of the evolving matrix distribution during culture. The results indicate that the overall stiffness and permeability are to a large extent insensitive to differences in local matrix distribution. This emphasizes the need for caution in the visual interpretation of tissue functionality from histology and underlines the importance of complementary measurements of the matrix's intrinsic molecular organization.

Creating homogenous strain distribution within 3D cell-encapsulated constructs using a simple and cost-effective uniaxial tensile bioreactor: Design and validation study.

PubMed

Subramanian, Gayathri; Elsaadany, Mostafa; Bialorucki, Callan; Yildirim-Ayan, Eda

2017-08-01

Mechanical loading bioreactors capable of applying uniaxial tensile strains are emerging to be a valuable tool to investigate physiologically relevant cellular signaling pathways and biochemical expression. In this study, we have introduced a simple and cost-effective uniaxial tensile strain bioreactor for the application of precise and homogenous uniaxial strains to 3D cell-encapsulated collagen constructs at physiological loading strains (0-12%) and frequencies (0.01-1 Hz). The bioreactor employs silicone-based loading chambers specifically designed to stretch constructs without direct gripping to minimize stress concentration at the ends of the construct and preserve its integrity. The loading chambers are driven by a versatile stepper motor ball-screw actuation system to produce stretching of the constructs. Mechanical characterization of the bioreactor performed through Finite Element Analysis demonstrated that the constructs experienced predominantly uniaxial tensile strain in the longitudinal direction. The strains produced were found to be homogenous over a 15 × 4 × 2 mm region of the construct equivalent to around 60% of the effective region of characterization. The strain values were also shown to be consistent and reproducible during cyclic loading regimes. Biological characterization confirmed the ability of the bioreactor to promote cell viability, proliferation, and matrix organization of cell-encapsulated collagen constructs. This easy-to-use uniaxial tensile strain bioreactor can be employed for studying morphological, structural, and functional responses of cell-embedded matrix systems in response to physiological loading of musculoskeletal tissues. It also holds promise for tissue-engineered strategies that involve delivery of mechanically stimulated cells at the site of injury through a biological carrier to develop a clinically useful therapy for tissue healing. Biotechnol. Bioeng. 2017;114: 1878-1887. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A nano-sandwich construct built with graphene nanosheets and carbon nanotubes enhances mechanical properties of hydroxyapatite-polyetheretherketone scaffolds.

PubMed

Feng, Pei; Peng, Shuping; Wu, Ping; Gao, Chengde; Huang, Wei; Deng, Youwen; Xiao, Tao; Shuai, Cijun

2016-01-01

A nano-sandwich construct was built by combining two-dimensional graphene nanosheets (GNSs) and one-dimensional carbon nanotubes (CNTs) to improve the mechanical properties of hydroxyapatite-polyetheretherketone (HAP-PEEK) scaffolds for bone tissue engineering. In this nano-sandwich construct, the long tubular CNTs penetrated the interlayers of graphene and prevented their aggregation, increasing the effective contact area between the construct and matrix. The combination of GNSs and CNTs in a weight ratio of 2:8 facilitated the dispersion of each other and provided a synergetic effect in enhancing the mechanical properties. The compressive strength and modulus of the scaffolds were increased by 63.58% and 56.54% at this time compared with those of HAP-PEEK scaffolds, respectively. The carbon-based fillers, pulling out and bridging, were also clearly observed in the matrix. Moreover, the dangling of CNTs and their entangling with GNSs further reinforced the mechanical properties. Furthermore, apatite layer formed on the scaffold surface after immersing in simulated body fluid, and the cells attached and spread well on the surface of the scaffolds and displayed good viability, proliferation, and differentiation. These evidence indicate that the HAP-PEEK scaffolds enhanced by GNSs and CNTs are a promising alternative for bone tissue engineering.

A nano-sandwich construct built with graphene nanosheets and carbon nanotubes enhances mechanical properties of hydroxyapatite–polyetheretherketone scaffolds

PubMed Central

Feng, Pei; Peng, Shuping; Wu, Ping; Gao, Chengde; Huang, Wei; Deng, Youwen; Xiao, Tao; Shuai, Cijun

2016-01-01

A nano-sandwich construct was built by combining two-dimensional graphene nanosheets (GNSs) and one-dimensional carbon nanotubes (CNTs) to improve the mechanical properties of hydroxyapatite–polyetheretherketone (HAP–PEEK) scaffolds for bone tissue engineering. In this nano-sandwich construct, the long tubular CNTs penetrated the interlayers of graphene and prevented their aggregation, increasing the effective contact area between the construct and matrix. The combination of GNSs and CNTs in a weight ratio of 2:8 facilitated the dispersion of each other and provided a synergetic effect in enhancing the mechanical properties. The compressive strength and modulus of the scaffolds were increased by 63.58% and 56.54% at this time compared with those of HAP–PEEK scaffolds, respectively. The carbon-based fillers, pulling out and bridging, were also clearly observed in the matrix. Moreover, the dangling of CNTs and their entangling with GNSs further reinforced the mechanical properties. Furthermore, apatite layer formed on the scaffold surface after immersing in simulated body fluid, and the cells attached and spread well on the surface of the scaffolds and displayed good viability, proliferation, and differentiation. These evidence indicate that the HAP–PEEK scaffolds enhanced by GNSs and CNTs are a promising alternative for bone tissue engineering. PMID:27555770

Identifying and exploiting trait-relevant tissues with multiple functional annotations in genome-wide association studies

PubMed Central

Zhang, Shujun

2018-01-01

Genome-wide association studies (GWASs) have identified many disease associated loci, the majority of which have unknown biological functions. Understanding the mechanism underlying trait associations requires identifying trait-relevant tissues and investigating associations in a trait-specific fashion. Here, we extend the widely used linear mixed model to incorporate multiple SNP functional annotations from omics studies with GWAS summary statistics to facilitate the identification of trait-relevant tissues, with which to further construct powerful association tests. Specifically, we rely on a generalized estimating equation based algorithm for parameter inference, a mixture modeling framework for trait-tissue relevance classification, and a weighted sequence kernel association test constructed based on the identified trait-relevant tissues for powerful association analysis. We refer to our analytic procedure as the Scalable Multiple Annotation integration for trait-Relevant Tissue identification and usage (SMART). With extensive simulations, we show how our method can make use of multiple complementary annotations to improve the accuracy for identifying trait-relevant tissues. In addition, our procedure allows us to make use of the inferred trait-relevant tissues, for the first time, to construct more powerful SNP set tests. We apply our method for an in-depth analysis of 43 traits from 28 GWASs using tissue-specific annotations in 105 tissues derived from ENCODE and Roadmap. Our results reveal new trait-tissue relevance, pinpoint important annotations that are informative of trait-tissue relationship, and illustrate how we can use the inferred trait-relevant tissues to construct more powerful association tests in the Wellcome trust case control consortium study. PMID:29377896

Application of Extrusion-Based Hydrogel Bioprinting for Cartilage Tissue Engineering

PubMed Central

You, Fu; Eames, B. Frank; Chen, Xiongbiao

2017-01-01

Extrusion-based bioprinting (EBB) is a rapidly developing technique that has made substantial progress in the fabrication of constructs for cartilage tissue engineering (CTE) over the past decade. With this technique, cell-laden hydrogels or bio-inks have been extruded onto printing stages, layer-by-layer, to form three-dimensional (3D) constructs with varying sizes, shapes, and resolutions. This paper reviews the cell sources and hydrogels that can be used for bio-ink formulations in CTE application. Additionally, this paper discusses the important properties of bio-inks to be applied in the EBB technique, including biocompatibility, printability, as well as mechanical properties. The printability of a bio-ink is associated with the formation of first layer, ink rheological properties, and crosslinking mechanisms. Further, this paper discusses two bioprinting approaches to build up cartilage constructs, i.e., self-supporting hydrogel bioprinting and hybrid bioprinting, along with their applications in fabricating chondral, osteochondral, and zonally organized cartilage regenerative constructs. Lastly, current limitations and future opportunities of EBB in printing cartilage regenerative constructs are reviewed. PMID:28737701

Engineering on the straight and narrow: the mechanics of nanofibrous assemblies for fiber-reinforced tissue regeneration.

PubMed

Mauck, Robert L; Baker, Brendon M; Nerurkar, Nandan L; Burdick, Jason A; Li, Wan-Ju; Tuan, Rocky S; Elliott, Dawn M

2009-06-01

Tissue engineering of fibrous tissues of the musculoskeletal system represents a considerable challenge because of the complex architecture and mechanical properties of the component structures. Natural healing processes in these dense tissues are limited as a result of the mechanically challenging environment of the damaged tissue and the hypocellularity and avascular nature of the extracellular matrix. When healing does occur, the ordered structure of the native tissue is replaced with a disorganized fibrous scar with inferior mechanical properties, engendering sites that are prone to re-injury. To address the engineering of such tissues, we and others have adopted a structurally motivated approach based on organized nanofibrous assemblies. These scaffolds are composed of ultrafine polymeric fibers that can be fabricated in such a way to recreate the structural anisotropy typical of fiber-reinforced tissues. This straight-and-narrow topography not only provides tailored mechanical properties, but also serves as a 3D biomimetic micropattern for directed tissue formation. This review describes the underlying technology of nanofiber production and focuses specifically on the mechanical evaluation and theoretical modeling of these structures as it relates to native tissue structure and function. Applying the same mechanical framework for understanding native and engineered fiber-reinforced tissues provides a functional method for evaluating the utility and maturation of these unique engineered constructs. We further describe several case examples where these principles have been put to test, and discuss the remaining challenges and opportunities in forwarding this technology toward clinical implementation.

Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-Reinforced Tissue Regeneration

PubMed Central

Baker, Brendon M.; Nerurkar, Nandan L.; Burdick, Jason A.; Li, Wan-Ju; Tuan, Rocky S.; Elliott, Dawn M.

2009-01-01

Tissue engineering of fibrous tissues of the musculoskeletal system represents a considerable challenge because of the complex architecture and mechanical properties of the component structures. Natural healing processes in these dense tissues are limited as a result of the mechanically challenging environment of the damaged tissue and the hypocellularity and avascular nature of the extracellular matrix. When healing does occur, the ordered structure of the native tissue is replaced with a disorganized fibrous scar with inferior mechanical properties, engendering sites that are prone to re-injury. To address the engineering of such tissues, we and others have adopted a structurally motivated approach based on organized nanofibrous assemblies. These scaffolds are composed of ultrafine polymeric fibers that can be fabricated in such a way to recreate the structural anisotropy typical of fiber-reinforced tissues. This straight-and-narrow topography not only provides tailored mechanical properties, but also serves as a 3D biomimetic micropattern for directed tissue formation. This review describes the underlying technology of nanofiber production and focuses specifically on the mechanical evaluation and theoretical modeling of these structures as it relates to native tissue structure and function. Applying the same mechanical framework for understanding native and engineered fiber-reinforced tissues provides a functional method for evaluating the utility and maturation of these unique engineered constructs. We further describe several case examples where these principles have been put to test, and discuss the remaining challenges and opportunities in forwarding this technology toward clinical implementation. PMID:19207040

Automated 3D bioassembly of micro-tissues for biofabrication of hybrid tissue engineered constructs.

PubMed

Mekhileri, N V; Lim, K S; Brown, G C J; Mutreja, I; Schon, B S; Hooper, G J; Woodfield, T B F

2018-01-12

Bottom-up biofabrication approaches combining micro-tissue fabrication techniques with extrusion-based 3D printing of thermoplastic polymer scaffolds are emerging strategies in tissue engineering. These biofabrication strategies support native self-assembly mechanisms observed in developmental stages of tissue or organoid growth as well as promoting cell-cell interactions and cell differentiation capacity. Few technologies have been developed to automate the precise assembly of micro-tissues or tissue modules into structural scaffolds. We describe an automated 3D bioassembly platform capable of fabricating simple hybrid constructs via a two-step bottom-up bioassembly strategy, as well as complex hybrid hierarchical constructs via a multistep bottom-up bioassembly strategy. The bioassembly system consisted of a fluidic-based singularisation and injection module incorporated into a commercial 3D bioprinter. The singularisation module delivers individual micro-tissues to an injection module, for insertion into precise locations within a 3D plotted scaffold. To demonstrate applicability for cartilage tissue engineering, human chondrocytes were isolated and micro-tissues of 1 mm diameter were generated utilising a high throughput 96-well plate format. Micro-tissues were singularised with an efficiency of 96.0 ± 5.1%. There was no significant difference in size, shape or viability of micro-tissues before and after automated singularisation and injection. A layer-by-layer approach or aforementioned bottom-up bioassembly strategy was employed to fabricate a bilayered construct by alternatively 3D plotting a thermoplastic (PEGT/PBT) polymer scaffold and inserting pre-differentiated chondrogenic micro-tissues or cell-laden gelatin-based (GelMA) hydrogel micro-spheres, both formed via high-throughput fabrication techniques. No significant difference in viability between the construct assembled utilising the automated bioassembly system and manually assembled construct was observed. Bioassembly of pre-differentiated micro-tissues as well as chondrocyte-laden hydrogel micro-spheres demonstrated the flexibility of the platform while supporting tissue fusion, long-term cell viability, and deposition of cartilage-specific extracellular matrix proteins. This technology provides an automated and scalable pathway for bioassembly of both simple and complex 3D tissue constructs of clinically relevant shape and size, with demonstrated capability to facilitate direct spatial organisation and hierarchical 3D assembly of micro-tissue modules, ranging from biomaterial free cell pellets to cell-laden hydrogel formulations.

Development and Translation of a Tissue-Engineered Disc in a Preclinical Rodent Model

DTIC Science & Technology

2014-02-01

maturation of MSC - laden DAPS at a length scale of the full intact tissue. This is the first study to our knowledge 5 showing beneficial effects of...was also published in the Journal of the Mechanical Behavior of Biomedical Materials in 2012. To carry out this study , MSCs were isolated from...in our preliminary studies . Cylindrical MSC -based NP constructs (Ø4 × 2.25mm) were cultured in a chemically defined medium (1mL/construct

Bioreactor System Using Noninvasive Imaging and Mechanical Stretch for Biomaterial Screening

PubMed Central

Kluge, Jonathan A.; Leisk, Gary G.; Cardwell, Robyn S.; Fernandes, Alexander P.; House, Michael; Ward, Andrew; Dorfmann, A. Luis; Kaplan, David L.

2012-01-01

Screening biomaterial and tissue systems in vitro, for guidance of performance in vivo, remains a major requirement in the field of tissue engineering. It is critical to understand how culture stimulation affects both tissue construct maturation and function, with the goal of eliminating resource-intensive trial-and-error screening and better matching specifications for various in vivo needs. We present a multifunctional and robust bioreactor design that addresses this need. The design enables a range of mechanical inputs, durations, and frequencies to be applied in coordination with noninvasive optical assessments. A variety of biomaterial systems, including micro- and nano-fiber and porous sponge biomaterials, as well as cell-laden tissue engineering constructs were used in validation studies in order to demonstrate the versatility and utility of this new bioreactor design. The silk-based biomaterials highlighted in these studies offered several unique optical signatures for use in label-free nondestructive imaging that allowed for sequential profiling. Both short- and long-term culture studies were conducted to evaluate several practical scenarios of usage: on a short-term basis, we demonstrate that construct cellularity can be monitored by usage of nonpermanent dyes; on a more long-term basis, we show that cell ingrowth can be monitored by GFP-labeling and construct integrity probed with concurrent load/displacement data. The ability to nondestructively track cells, biomaterials, and new matrix formation without harvesting designated samples at each time point will lead to less resource-intensive studies and should enhance our understanding and the discovery of biomaterial designs related to functional tissue engineering. PMID:21298345

Mechanical cues in orofacial tissue engineering and regenerative medicine.

PubMed

Brouwer, Katrien M; Lundvig, Ditte M S; Middelkoop, Esther; Wagener, Frank A D T G; Von den Hoff, Johannes W

2015-01-01

Cleft lip and palate patients suffer from functional, aesthetical, and psychosocial problems due to suboptimal regeneration of skin, mucosa, and skeletal muscle after restorative cleft surgery. The field of tissue engineering and regenerative medicine (TE/RM) aims to restore the normal physiology of tissues and organs in conditions such as birth defects or after injury. A crucial factor in cell differentiation, tissue formation, and tissue function is mechanical strain. Regardless of this, mechanical cues are not yet widely used in TE/RM. The effects of mechanical stimulation on cells are not straight-forward in vitro as cellular responses may differ with cell type and loading regime, complicating the translation to a therapeutic protocol. We here give an overview of the different types of mechanical strain that act on cells and tissues and discuss the effects on muscle, and skin and mucosa. We conclude that presently, sufficient knowledge is lacking to reproducibly implement external mechanical loading in TE/RM approaches. Mechanical cues can be applied in TE/RM by fine-tuning the stiffness and architecture of the constructs to guide the differentiation of the seeded cells or the invading surrounding cells. This may already improve the treatment of orofacial clefts and other disorders affecting soft tissues. © 2015 by the Wound Healing Society.

Nondestructive mechanical characterization of developing biological tissues using inflation testing.

PubMed

Oomen, P J A; van Kelle, M A J; Oomens, C W J; Bouten, C V C; Loerakker, S

2017-10-01

One of the hallmarks of biological soft tissues is their capacity to grow and remodel in response to changes in their environment. Although it is well-accepted that these processes occur at least partly to maintain a mechanical homeostasis, it remains unclear which mechanical constituent(s) determine(s) mechanical homeostasis. In the current study a nondestructive mechanical test and a two-step inverse analysis method were developed and validated to nondestructively estimate the mechanical properties of biological tissue during tissue culture. Nondestructive mechanical testing was achieved by performing an inflation test on tissues that were cultured inside a bioreactor, while the tissue displacement and thickness were nondestructively measured using ultrasound. The material parameters were estimated by an inverse finite element scheme, which was preceded by an analytical estimation step to rapidly obtain an initial estimate that already approximated the final solution. The efficiency and accuracy of the two-step inverse method was demonstrated on virtual experiments of several material types with known parameters. PDMS samples were used to demonstrate the method's feasibility, where it was shown that the proposed method yielded similar results to tensile testing. Finally, the method was applied to estimate the material properties of tissue-engineered constructs. Via this method, the evolution of mechanical properties during tissue growth and remodeling can now be monitored in a well-controlled system. The outcomes can be used to determine various mechanical constituents and to assess their contribution to mechanical homeostasis. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro fabrication of a tissue engineered human cardiovascular patch for future use in cardiovascular surgery.

PubMed

Yang, Chao; Sodian, Ralf; Fu, Ping; Lüders, Cora; Lemke, Thees; Du, Jing; Hübler, Michael; Weng, Yuguo; Meyer, Rudolf; Hetzer, Roland

2006-01-01

One approach to tissue engineering has been the development of in vitro conditions for the fabrication of functional cardiovascular structures intended for implantation. In this experiment, we developed a pulsatile flow system that provides biochemical and biomechanical signals in order to regulate autologous, human patch-tissue development in vitro. We constructed a biodegradable patch scaffold from porous poly-4-hydroxy-butyrate (P4HB; pore size 80 to 150 microm). The scaffold was seeded with pediatric aortic cells. The cell-seeded patch constructs were placed in a self-developed bioreactor for 7 days to observe potential tissue formation under dynamic cell culture conditions. As a control, cell-seeded scaffolds were not conditioned in the bioreactor system. After maturation in vitro, the analysis of the tissue engineered constructs included biochemical, biomechanical, morphologic, and immunohistochemical examination. Macroscopically, all tissue engineered constructs were covered by cells. After conditioning in the bioreactor, the cells were mostly viable, had grown into the pores, and had formed tissue on the patch construct. Electron microscopy showed confluent smooth surfaces. Additionally, we demonstrated the capacity to generate collagen and elastin under in vitro pulsatile flow conditions in biochemical examination. Biomechanical testing showed mechanical properties of the tissue engineered human patch tissue without any statistical differences in strength or resistance to stretch between the static controls and the conditioned patches. Immunohistochemical examination stained positive for alpha smooth muscle actin, collagen type I, and fibronectin. There was minor tissue formation in the nonconditioned control samples. Porous P4HB may be used to fabricate a biodegradable patch scaffold. Human vascular cells attached themselves to the polymeric scaffold, and extracellular matrix formation was induced under controlled biomechanical and biodynamic stimuli in a self-developed pulsatile bioreactor system.

Silk: a potential medium for tissue engineering.

PubMed

Sobajo, Cassandra; Behzad, Farhad; Yuan, Xue-Feng; Bayat, Ardeshir

2008-01-01

Human skin is a complex bilayered organ that serves as a protective barrier against the environment. The loss of integrity of skin by traumatic experiences such as burns and ulcers may result in considerable disability or ultimately death. Therefore, in skin injuries, adequate dermal substitutes are among primary care targets, aimed at replacing the structural and functional properties of native skin. To date, there are very few single application tissue-engineered dermal constructs fulfilling this criterion. Silk produced by the domestic silkworm, Bombyx mori, has a long history of use in medicine. It has recently been increasingly investigated as a promising biomaterial for dermal constructs. Silk contains 2 fibrous proteins, sericin and fibroin. Each one exhibits unique mechanical and biological properties. Comprehensive review of randomized-controlled trials investigating current dermal constructs and the structures and properties of silk-based constructs on wound healing. This review revealed that silk-fibroin is regarded as the most promising biomaterial, providing options for the construction of tissue-engineered skin. The research available indicates that silk fibroin is a suitable biomaterial scaffold for the provision of adequate dermal constructs.

Optimization and translation of MSC-based hyaluronic acid hydrogels for cartilage repair

NASA Astrophysics Data System (ADS)

Erickson, Isaac E.

2011-12-01

Traumatic injury and disease disrupt the ability of cartilage to carry joint stresses and, without an innate regenerative response, often lead to degenerative changes towards the premature development of osteoarthritis. Surgical interventions have yet to restore long-term mechanical function. Towards this end, tissue engineering has been explored for the de novo formation of engineered cartilage as a biologic approach to cartilage repair. Research utilizing autologous chondrocytes has been promising, but clinical limitations in their yield have motivated research into the potential of mesenchymal stem cells (MSCs) as an alternative cell source. MSCs are multipotent cells that can differentiate towards a chondrocyte phenotype in a number of biomaterials, but no combination has successfully recapitulated the native mechanical function of healthy articular cartilage. The broad objective of this thesis was to establish an MSC-based tissue engineering approach worthy of clinical translation. Hydrogels are a common class of biomaterial used for cartilage tissue engineering and our initial work demonstrated the potential of a photo-polymerizable hyaluronic acid (HA) hydrogel to promote MSC chondrogenesis and improved construct maturation by optimizing macromer and MSC seeding density. The beneficial effects of dynamic compressive loading, high MSC density, and continuous mixing (orbital shaker) resulted in equilibrium modulus values over 1 MPa, well in range of native tissue. While compressive properties are crucial, clinical translation also demands that constructs stably integrate within a defect. We utilized a push-out testing modality to assess the in vitro integration of HA constructs within artificial cartilage defects. We established the necessity for in vitro pre-maturation of constructs before repair to achieve greater integration strength and compressive properties in situ. Combining high MSC density and gentle mixing resulted in integration strength over 500 kPa, nearly 10-fold greater than previous reports of integration with MSC-based constructs. Furthermore, we demonstrated the durability of this repair system by applying dynamic loading and showed its functional contribution to the distribution of compressive loads across the repair space. Overall, the studies contained within this thesis offer the first MSC-based tissue engineering strategy that successfully recapitulates native mechanical function while also demonstrating the potential for complete functional cartilage repair.

Chondroprotective supplementation promotes the mechanical properties of injectable scaffold for human nucleus pulposus tissue engineering.

PubMed

Foss, Berit L; Maxwell, Thomas W; Deng, Ying

2014-01-01

A result of intervertebral disc (IVD) degeneration, the nucleus pulposus (NP) is no longer able to withstand applied load leading to pain and disability. The objective of this study is to fabricate a tissue-engineered injectable scaffold with chondroprotective supplementation in vitro to improve the mechanical properties of a degenerative NP. Tissue-engineered scaffolds were fabricated using different concentrations of alginate and calcium chloride and mechanically evaluated. Fabrication conditions were based on structural and mechanical resemblance to the native NP. Chondroprotective supplementation, glucosamine (GCSN) and chondroitin sulfate (CS), were added to scaffolds at concentrations of 0:0µg/mL (0:0-S), 125:100µg/mL (125:100-S), 250:200µg/mL (250:200-S), and 500:400µg/mL (500:400-S), GCSN and CS, respectively. Scaffolds were used to fabricate tissue-engineered constructs through encapsulation of human nucleus pulposus cells (HNPCs). The tissue-engineered constructs were collected at days 1, 14, and 28 for biochemical and biomechanical evaluations. Confocal microscopy showed HNPC viability and rounded morphology over the 28 day period. MTT analysis resulted in significant increases in cell proliferation for each group. Collagen type II ELISA quantification and compressive aggregate moduli (HA) showed increasing trends for both 250:200-S and the 500:400-S groups on Day 28 with significantly greater HA compared to 0:0-S group. Glycosaminoglycan and water content decreased for all groups. Results indicate the increased mechanical properties of the 250:200-S and the 500:400-S was due to production of a functional matrix. This study demonstrated potential for a chondroprotective supplemented injectable scaffold to restore biomechanical function of a degenerative disc through the production of a mechanically functional matrix. Copyright © 2013 Elsevier Ltd. All rights reserved.

Bioinks for 3D bioprinting: an overview.

PubMed

Gungor-Ozkerim, P Selcan; Inci, Ilyas; Zhang, Yu Shrike; Khademhosseini, Ali; Dokmeci, Mehmet Remzi

2018-05-01

Bioprinting is an emerging technology with various applications in making functional tissue constructs to replace injured or diseased tissues. It is a relatively new approach that provides high reproducibility and precise control over the fabricated constructs in an automated manner, potentially enabling high-throughput production. During the bioprinting process, a solution of a biomaterial or a mixture of several biomaterials in the hydrogel form, usually encapsulating the desired cell types, termed the bioink, is used for creating tissue constructs. This bioink can be cross-linked or stabilized during or immediately after bioprinting to generate the final shape, structure, and architecture of the designed construct. Bioinks may be made from natural or synthetic biomaterials alone, or a combination of the two as hybrid materials. In certain cases, cell aggregates without any additional biomaterials can also be adopted for use as a bioink for bioprinting processes. An ideal bioink should possess proper mechanical, rheological, and biological properties of the target tissues, which are essential to ensure correct functionality of the bioprinted tissues and organs. In this review, we provide an in-depth discussion of the different bioinks currently employed for bioprinting, and outline some future perspectives in their further development.

Ultrasound Imaging Techniques for Spatiotemporal Characterization of Composition, Microstructure, and Mechanical Properties in Tissue Engineering.

PubMed

Deng, Cheri X; Hong, Xiaowei; Stegemann, Jan P

2016-08-01

Ultrasound techniques are increasingly being used to quantitatively characterize both native and engineered tissues. This review provides an overview and selected examples of the main techniques used in these applications. Grayscale imaging has been used to characterize extracellular matrix deposition, and quantitative ultrasound imaging based on the integrated backscatter coefficient has been applied to estimating cell concentrations and matrix morphology in tissue engineering. Spectral analysis has been employed to characterize the concentration and spatial distribution of mineral particles in a construct, as well as to monitor mineral deposition by cells over time. Ultrasound techniques have also been used to measure the mechanical properties of native and engineered tissues. Conventional ultrasound elasticity imaging and acoustic radiation force imaging have been applied to detect regions of altered stiffness within tissues. Sonorheometry and monitoring of steady-state excitation and recovery have been used to characterize viscoelastic properties of tissue using a single transducer to both deform and image the sample. Dual-mode ultrasound elastography uses separate ultrasound transducers to produce a more potent deformation force to microscale characterization of viscoelasticity of hydrogel constructs. These ultrasound-based techniques have high potential to impact the field of tissue engineering as they are further developed and their range of applications expands.

Long-Term Morphological and Microarchitectural Stability of Tissue-Engineered, Patient-Specific Auricles In Vivo

PubMed Central

Cohen, Benjamin Peter; Hooper, Rachel C.; Puetzer, Jennifer L.; Nordberg, Rachel; Asanbe, Ope; Hernandez, Karina A.; Spector, Jason A.

2016-01-01

Current techniques for autologous auricular reconstruction produce substandard ear morphologies with high levels of donor-site morbidity, whereas alloplastic implants demonstrate poor biocompatibility. Tissue engineering, in combination with noninvasive digital photogrammetry and computer-assisted design/computer-aided manufacturing technology, offers an alternative method of auricular reconstruction. Using this method, patient-specific ears composed of collagen scaffolds and auricular chondrocytes have generated auricular cartilage with great fidelity following 3 months of subcutaneous implantation, however, this short time frame may not portend long-term tissue stability. We hypothesized that constructs developed using this technique would undergo continued auricular cartilage maturation without degradation during long-term (6 month) implantation. Full-sized, juvenile human ear constructs were injection molded from high-density collagen hydrogels encapsulating juvenile bovine auricular chondrocytes and implanted subcutaneously on the backs of nude rats for 6 months. Upon explantation, constructs retained overall patient morphology and displayed no evidence of tissue necrosis. Limited contraction occurred in vivo, however, no significant change in size was observed beyond 1 month. Constructs at 6 months showed distinct auricular cartilage microstructure, featuring a self-assembled perichondrial layer, a proteoglycan-rich bulk, and rounded cellular lacunae. Verhoeff's staining also revealed a developing elastin network comparable to native tissue. Biochemical measurements for DNA, glycosaminoglycan, and hydroxyproline content and mechanical properties of aggregate modulus and hydraulic permeability showed engineered tissue to be similar to native cartilage at 6 months. Patient-specific auricular constructs demonstrated long-term stability and increased cartilage tissue development during extended implantation, and offer a potential tissue-engineered solution for the future of auricular reconstructions. PMID:26847742

Geometric modeling of space-optimal unit-cell-based tissue engineering scaffolds

NASA Astrophysics Data System (ADS)

Rajagopalan, Srinivasan; Lu, Lichun; Yaszemski, Michael J.; Robb, Richard A.

2005-04-01

Tissue engineering involves regenerating damaged or malfunctioning organs using cells, biomolecules, and synthetic or natural scaffolds. Based on their intended roles, scaffolds can be injected as space-fillers or be preformed and implanted to provide mechanical support. Preformed scaffolds are biomimetic "trellis-like" structures which, on implantation and integration, act as tissue/organ surrogates. Customized, computer controlled, and reproducible preformed scaffolds can be fabricated using Computer Aided Design (CAD) techniques and rapid prototyping devices. A curved, monolithic construct with minimal surface area constitutes an efficient substrate geometry that promotes cell attachment, migration and proliferation. However, current CAD approaches do not provide such a biomorphic construct. We address this critical issue by presenting one of the very first physical realizations of minimal surfaces towards the construction of efficient unit-cell based tissue engineering scaffolds. Mask programmability, and optimal packing density of triply periodic minimal surfaces are used to construct the optimal pore geometry. Budgeted polygonization, and progressive minimal surface refinement facilitate the machinability of these surfaces. The efficient stress distributions, as deduced from the Finite Element simulations, favor the use of these scaffolds for orthopedic applications.

Fabrication of Poly(ε-caprolactone) Scaffolds Reinforced with Cellulose Nanofibers, with and without the Addition of Hydroxyapatite Nanoparticles.

PubMed

Morouço, Pedro; Biscaia, Sara; Viana, Tânia; Franco, Margarida; Malça, Cândida; Mateus, Artur; Moura, Carla; Ferreira, Frederico C; Mitchell, Geoffrey; Alves, Nuno M

2016-01-01

Biomaterial properties and controlled architecture of scaffolds are essential features to provide an adequate biological and mechanical support for tissue regeneration, mimicking the ingrowth tissues. In this study, a bioextrusion system was used to produce 3D biodegradable scaffolds with controlled architecture, comprising three types of constructs: (i) poly( ε -caprolactone) (PCL) matrix as reference; (ii) PCL-based matrix reinforced with cellulose nanofibers (CNF); and (iii) PCL-based matrix reinforced with CNF and hydroxyapatite nanoparticles (HANP). The effect of the addition and/or combination of CNF and HANP into the polymeric matrix of PCL was investigated, with the effects of the biomaterial composition on the constructs (morphological, thermal, and mechanical performances) being analysed. Scaffolds were produced using a single lay-down pattern of 0/90°, with the same processing parameters among all constructs being assured. The performed morphological analyses showed a satisfactory distribution of CNF within the polymer matrix and high reliability was obtained among the produced scaffolds. Significant effects on surface wettability and thermal properties were observed, among scaffolds. Regarding the mechanical properties, higher scaffold stiffness in the reinforced scaffolds was obtained. Results from the cytotoxicity assay suggest that all the composite scaffolds presented good biocompatibility. The results of this first study on cellulose and hydroxyapatite reinforced constructs with controlled architecture clearly demonstrate the potential of these 3D composite constructs for cell cultivation with enhanced mechanical properties.

A comparative study of skin cell activities in collagen and fibrin constructs.

PubMed

Law, Jia Xian; Musa, Faiza; Ruszymah, Bt Hj Idrus; El Haj, Alicia J; Yang, Ying

2016-09-01

Collagen and fibrin are widely used in tissue engineering due to their excellent biocompatibility and bioactivities that support in vivo tissue formation. These two hydrogels naturally present in different wound healing stages with different regulatory effects on cells, and both of them are mechanically weak in the reconstructed hydrogels. We conducted a comparative study by the growth of rat dermal fibroblasts or dermal fibroblasts and epidermal keratinocytes together in collagen and fibrin constructs respectively with and without the reinforcement of electrospun poly(lactic acid) nanofiber mesh. Cell proliferation, gel contraction and elastic modulus of the constructs were measured on the same gels at multiple time points during the 22 day culturing period using multiple non-destructive techniques. The results demonstrated considerably different cellular activities within the two types of constructs. Co-culturing keratinocytes with fibroblasts in the collagen constructs reduced the fibroblast proliferation, collagen contraction and mechanical strength at late culture point regardless of the presence of nanofibers. Co-culturing keratinocytes with fibroblasts in the fibrin constructs promoted fibroblast proliferation but exerted no influence on fibrin contraction and mechanical strength. The presence of nanofibers in the collagen and fibrin constructs played a favorable role on the fibroblast proliferation when keratinocytes were absent. Thus, this study exhibited new evidence of the strong cross-talk between keratinocytes and fibroblasts, which can be used to control fibroblast proliferation and construct contraction. This cross-talk activity is extracellular matrix-dependent in terms of the fibrous network morphology, density and strength. Copyright © 2016 IPEM. Published by Elsevier Ltd. All rights reserved.

The effects of intermittent hydrostatic pressure on self-assembled articular cartilage constructs.

PubMed

Hu, Jerry C; Athanasiou, Kyriacos A

2006-05-01

To date, static culture for the tissue engineering of articular cartilage has shown to be inadequate in conferring functionality to constructs. Various forms of mechanical stimuli accompany articular cartilage development in vivo, and one of these is hydrostatic pressure. This study used histology, biochemistry, and biomechanics to examine the effects of intermittent hydrostatic pressure, applied at 10 MPa and 1 Hz for 4 h per day for 5 days per week for up to 8 weeks on self-assembled chondrocyte constructs. The self-assembling process is a novel approach that allows engineering of articular cartilage constructs without the use of exogenous scaffolds. The self-assembled constructs were found to be capable of enduring this loading regimen. Significant increases in collagen production were only observed in pressurized samples. Intermittent hydrostatic pressure prevented a significant decrease in total GAG, which was significant in controls. Aside from the beneficial effects intermittent hydrostatic pressure may have on ECM synthesis, its effects on mechanical properties may require longer culture periods to manifest. This study demonstrates the successful use of the self-assembling process to produce articular cartilage constructs. It also shows for the first time that long-term culture of tissue-engineered articular cartilage construct benefits from intermittent hydrostatic pressure.

Hydrogel tissue construct-based high-content compound screening.

PubMed

Lam, Vy; Wakatsuki, Tetsuro

2011-01-01

Current pharmaceutical compound screening systems rely on cell-based assays to identify therapeutic candidates and potential toxicities. However, cells grown on 2D substrata or in suspension do not exhibit the mechanical or physiological properties of cells in vivo. To address this limitation, the authors developed an in vitro, high-throughput, 3D hydrogel tissue construct (HTC)-based assay system to quantify cell and tissue mechanical properties and multiple parameters of physiology. HTC mechanics was quantified using an automated device, and physiological status was assessed using spectroscopy-based indicators that were read on microplate readers. To demonstrate the application of this system, the authors screened 4 test compounds--rotenone (ROT), cytochalasin D (CD), 2,4-dinitrophenol (DNP), and Rho kinase inhibitor (H-1152)--for their ability to modulate HTC contractility without affecting actin integrity, mitochondrial membrane potential (MMP), or viability. All 4 compounds dose-dependently reduced HTC contractility. However, ROT was toxic, DNP dissipated MMP, and CD reduced both intracellular F-actin and viability. H-1152 was found to be the best candidate compound since it reduced HTC contractility with minimal side effects. The authors propose that their HTC-based assay system can be used to screen for compounds that modulate HTC contractility and assess the underlying physiological mechanism(s) of compound activity and toxicity.

Sol gel-derived hydroxyapatite films over porous calcium polyphosphate substrates for improved tissue engineering of osteochondral-like constructs.

PubMed

Lee, Whitaik David; Gawri, Rahul; Pilliar, Robert M; Stanford, William L; Kandel, Rita A

2017-10-15

Integration of in vitro-formed cartilage on a suitable substrate to form tissue-engineered implants for osteochondral defect repair is a considerable challenge. In healthy cartilage, a zone of calcified cartilage (ZCC) acts as an intermediary for mechanical force transfer from soft to hard tissue, as well as an effective interlocking structure to better resist interfacial shear forces. We have developed biphasic constructs that consist of scaffold-free cartilage tissue grown in vitro on, and interdigitated with, porous calcium polyphosphate (CPP) substrates. However, as CPP degrades, it releases inorganic polyphosphates (polyP) that can inhibit local mineralization, thereby preventing the formation of a ZCC at the interface. Thus, we hypothesize that coating CPP substrate with a layer of hydroxyapatite (HA) might prevent or limit this polyP release. To investigate this we tested both inorganic or organic sol-gel processing methods, asa barrier coating on CPP substrate to inhibit polyP release. Both types of coating supported the formation of ZCC in direct contact with the substrate, however the ZCC appeared more continuous in the tissue formed on the organic HA sol gel coated CPP. Tissues formed on coated substrates accumulated comparable quantities of extracellular matrix and mineral, but tissues formed on organic sol-gel (OSG)-coated substrates accumulated less polyP than tissues formed on inorganic sol-gel (ISG)-coated substrates. Constructs formed with OSG-coated CPP substrates had greater interfacial shear strength than those formed with ISG-coated and non-coated substrates. These results suggest that the OSG coating method can modify the location and distribution of ZCC and can be used to improve the mechanical integrity of tissue-engineered constructs formed on porous CPP substrates. Articular cartilage interfaces with bone through a zone of calcified cartilage. This study describes a method to generate an "osteochondral-like" implant that mimics this organization using isolated deep zone cartilage cells and a sol-gel hydroxyapatite coated bone substitute material composed of calcium polyphosphate (CPP). Developing a layer of calcified cartilage at the interface should contribute to enhancing the success of this "osteochondral-like" construct following implantation to repair cartilage defects. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Channeled Scaffolds for Engineering Myocardium with Mechanical Stimulation

PubMed Central

Zhang, Ting; Wan, Leo Q.; Xiong, Zhuo; Marsano, Anna; Maidhof, Robert; Park, Miri; Yan, Yongnian; Vunjak-Novakovic, Gordana

2011-01-01

The characteristics of the matrix (composition, structure, mechanical properties) and external culture environment (pulsatile perfusion, physical stimulation) are critically important for engineering functional myocardial tissue. We report the development of chitosan-collagen scaffolds with micro-pores and an array of parallel channels (~200 μm in diameter) that were specifically designed for cardiac tissue engineering with mechanical stimulation. The scaffolds were designed to have the structural and mechanical properties similar to those of the native human heart matrix. Scaffolds were seeded with neonatal rat heart cells and subjected to dynamic tensile stretch using a custom-designed bioreactor. The channels enhanced oxygen transport and facilitated the establishment of cell connections within the construct. The myocardial patches (14 mm in diameter, 1–2 mm thick) consisted of metabolically active cells and started to contract synchronously after 3 days of culture. Mechanical stimulation with high tensile stresses promoted cell alignment, elongation, and the expression of connexin-43 (Cx-43). This study confirms the importance of scaffold design and mechanical stimulation for the formation of contractile cardiac constructs. PMID:22081518

Channelled scaffolds for engineering myocardium with mechanical stimulation.

PubMed

Zhang, Ting; Wan, Leo Q; Xiong, Zhuo; Marsano, Anna; Maidhof, Robert; Park, Miri; Yan, Yongnian; Vunjak-Novakovic, Gordana

2012-10-01

The characteristics of the matrix (composition, structure, mechanical properties) and external culture environment (pulsatile perfusion, physical stimulation) of the heart are important characteristics in the engineering of functional myocardial tissue. This study reports on the development of chitosan-collagen scaffolds with micropores and an array of parallel channels (~ 200 µm in diameter) that were specifically designed for cardiac tissue engineering using mechanical stimulation. The scaffolds were designed to have similar structural and mechanical properties of those of native heart matrix. Scaffolds were seeded with neonatal rat heart cells and subjected to dynamic tensile stretch using a custom designed bioreactor. The channels enhanced oxygen transport and facilitated the establishment of cell connections within the construct. The myocardial patches (14 mm in diameter, 1-2 mm thick) consisted of metabolically active cells that began to contract synchronously after 3 days of culture. Mechanical stimulation with high tensile stress promoted cell alignment, elongation, and expression of connexin-43 (Cx-43). This study confirms the importance of scaffold design and mechanical stimulation for the formation of contractile cardiac constructs. Copyright © 2011 John Wiley & Sons, Ltd.

Development of a thermosensitive HAMA-containing bio-ink for the fabrication of composite cartilage repair constructs.

PubMed

Mouser, V H M; Abbadessa, A; Levato, R; Hennink, W E; Vermonden, T; Gawlitta, D; Malda, J

2017-03-23

Fine-tuning of bio-ink composition and material processing parameters is crucial for the development of biomechanically relevant cartilage constructs. This study aims to design and develop cartilage constructs with tunable internal architectures and relevant mechanical properties. More specifically, the potential of methacrylated hyaluronic acid (HAMA) added to thermosensitive hydrogels composed of methacrylated poly[N-(2-hydroxypropyl)methacrylamide mono/dilactate] (pHPMA-lac)/polyethylene glycol (PEG) triblock copolymers, to optimize cartilage-like tissue formation by embedded chondrocytes, and enhance printability was explored. Additionally, co-printing with polycaprolactone (PCL) was performed for mechanical reinforcement. Chondrocyte-laden hydrogels composed of pHPMA-lac-PEG and different concentrations of HAMA (0%-1% w/w) were cultured for 28 d in vitro and subsequently evaluated for the presence of cartilage-like matrix. Young's moduli were determined for hydrogels with the different HAMA concentrations. Additionally, hydrogel/PCL constructs with different internal architectures were co-printed and analyzed for their mechanical properties. The results of this study demonstrated a dose-dependent effect of HAMA concentration on cartilage matrix synthesis by chondrocytes. Glycosaminoglycan (GAG) and collagen type II content increased with intermediate HAMA concentrations (0.25%-0.5%) compared to HAMA-free controls, while a relatively high HAMA concentration (1%) resulted in increased fibrocartilage formation. Young's moduli of generated hydrogel constructs ranged from 14 to 31 kPa and increased with increasing HAMA concentration. The pHPMA-lac-PEG hydrogels with 0.5% HAMA were found to be optimal for cartilage-like tissue formation. Therefore, this hydrogel system was co-printed with PCL to generate porous or solid constructs with different mesh sizes. Young's moduli of these composite constructs were in the range of native cartilage (3.5-4.6 MPa). Interestingly, the co-printing procedure influenced the mechanical properties of the final constructs. These findings are relevant for future bio-ink development, as they demonstrate the importance of selecting proper HAMA concentrations, as well as appropriate print settings and construct designs for optimal cartilage matrix deposition and final mechanical properties of constructs, respectively.

3D bioprinted functional and contractile cardiac tissue constructs.

PubMed

Wang, Zhan; Lee, Sang Jin; Cheng, Heng-Jie; Yoo, James J; Atala, Anthony

2018-04-01

Bioengineering of a functional cardiac tissue composed of primary cardiomyocytes has great potential for myocardial regeneration and in vitro tissue modeling. However, its applications remain limited because the cardiac tissue is a highly organized structure with unique physiologic, biomechanical, and electrical properties. In this study, we undertook a proof-of-concept study to develop a contractile cardiac tissue with cellular organization, uniformity, and scalability by using three-dimensional (3D) bioprinting strategy. Primary cardiomyocytes were isolated from infant rat hearts and suspended in a fibrin-based bioink to determine the priting capability for cardiac tissue engineering. This cell-laden hydrogel was sequentially printed with a sacrificial hydrogel and a supporting polymeric frame through a 300-µm nozzle by pressured air. Bioprinted cardiac tissue constructs had a spontaneous synchronous contraction in culture, implying in vitro cardiac tissue development and maturation. Progressive cardiac tissue development was confirmed by immunostaining for α-actinin and connexin 43, indicating that cardiac tissues were formed with uniformly aligned, dense, and electromechanically coupled cardiac cells. These constructs exhibited physiologic responses to known cardiac drugs regarding beating frequency and contraction forces. In addition, Notch signaling blockade significantly accelerated development and maturation of bioprinted cardiac tissues. Our results demonstrated the feasibility of bioprinting functional cardiac tissues that could be used for tissue engineering applications and pharmaceutical purposes. Cardiovascular disease remains a leading cause of death in the United States and a major health-care burden. Myocardial infarction (MI) is a main cause of death in cardiovascular diseases. MI occurs as a consequence of sudden blocking of blood vessels supplying the heart. When occlusions in the coronary arteries occur, an immediate decrease in nutrient and oxygen supply to the cardiac muscle, resulting in permanent cardiac cell death. Eventually, scar tissue formed in the damaged cardiac muscle that cannot conduct electrical or mechanical stimuli thus leading to a reduction in the pumping efficiency of the heart. The therapeutic options available for end-stage heart failure is to undergo heart transplantation or the use of mechanical ventricular assist devices (VADs). However, many patients die while being on a waiting list, due to the organ shortage and limitation of VADs, such as surgical complications, infection, thrombogenesis, and failure of the electrical motor and hemolysis. Ultimately, 3D bioprinting strategy aims to create clinically applicable tissue constructs that can be immediately implanted in the body. To date, the focus on replicating complex and heterogeneous tissue constructs continues to increase as 3D bioprinting technologies advance. In this study, we demonstrated the feasibility of 3D bioprinting strategy to bioengineer the functional cardiac tissue that possesses a highly organized structure with unique physiological and biomechanical properties similar to native cardiac tissue. This bioprinting strategy has great potential to precisely generate functional cardiac tissues for use in pharmaceutical and regenerative medicine applications. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Hyaluronan supplementation as a mechanical regulator of cartilage tissue development under joint-kinematic-mimicking loading.

PubMed

Wu, Yabin; Stoddart, Martin J; Wuertz-Kozak, Karin; Grad, Sibylle; Alini, Mauro; Ferguson, Stephen J

2017-08-01

Articular cartilage plays an essential role in joint lubrication and impact absorption. Through this, the mechanical signals are coupled to the tissue's physiological response. Healthy synovial fluid has been shown to reduce and homogenize the shear stress acting on the cartilage surfaces due to its unique shear-thinning viscosity. As cartilage tissues are sensitive to mechanical changes in articulation, it was hypothesized that replacing the traditional culture medium with a healthy non-Newtonian lubricant could enhance tissue development in a cartilage engineering model, where joint-kinematic-mimicking mechanical loading is applied. Different amounts of hyaluronic acid were added to the culture medium to replicate the viscosities of synovial fluid at different health states. Hyaluronic acid supplementation, especially at a physiologically healthy concentration (2.0 mg ml -1 ), promoted a better preservation of chondrocyte phenotype. The ratio of collagen II to collagen I mRNA was 4.5 times that of the control group, implying better tissue development (however, with no significant difference of measured collagen II content), with a good retention of collagen II and proteoglycan in the mechanically active region. Simulating synovial fluid properties by hyaluronic acid supplementation created a favourable mechanical environment for mechanically loaded constructs. These findings may help in understanding the influence of joint articulation on tissue homeostasis, and moreover, improve methods for functional cartilage tissue engineering. © 2017 The Author(s).

Learning from real and tissue-engineered jellyfish: How to design and build a muscle-powered pump at intermediate Reynolds numbers

NASA Astrophysics Data System (ADS)

Nawroth, Janna; Lee, Hyungsuk; Feinberg, Adam; Ripplinger, Crystal; McCain, Megan; Grosberg, Anna; Dabiri, John; Parker, Kit

2012-11-01

Tissue-engineered devices promise to advance medical implants, aquatic robots and experimental platforms for tissue-fluid interactions. The design, fabrication and systematic improvement of tissue constructs, however, is challenging because of the complex interactions of living cell, synthetic materials and their fluid environments. In a proof of concept study we have tissue-engineered a construct that mimics the swimming of a juvenile jellyfish, a simple model system for muscle-powered pumps at intermediate Reynolds numbers with quantifiable fluid dynamics and morphological properties. Optimally designed constructs achieved jellyfish-like swimming and generated biomimetic propulsion and feeding currents. Focusing on the fluid interactions, we discuss failed and successful designs and the lessons learned in the process. The main challenges were (1) to derive a body shape and deformation suitable for effective fluid transport under physiological fluid conditions, (2) to understand the mechanical properties of muscle and bell matrix and device a design capable of the desired deformation, (3) to establish adequate 3D kinematics of power and recovery stroke, and (4) to evaluate the performance of the design.

Growth factor effects on costal chondrocytes for tissue engineering fibrocartilage

PubMed Central

Johns, D.E.; Athanasiou, K.A.

2010-01-01

Tissue engineered fibrocartilage could become a feasible option for replacing tissues like the knee meniscus or temporomandibular joint disc. This study employed five growth factors insulin-like growth factor-I, transforming growth factor-β1, epidermal growth factor, platelet-derived growth factor-BB, and basic fibroblast growth factor in a scaffoldless approach with costal chondrocytes, attempting to improve biochemical and mechanical properties of engineered constructs. Samples were quantitatively assessed for total collagen, glycosaminoglycans, collagen type I, collagen type II, cells, compressive properties, and tensile properties at two time points. Most treated constructs were worse than the no growth factor control, suggesting a detrimental effect, but the IGF treatment tended to improve the constructs. Additionally, the 6wk time point was consistently better than 3wks, with total collagen, glycosaminoglycans, and aggregate modulus doubling during this time. Further optimization of the time in culture and exogenous stimuli will be important in making a more functional replacement tissue. PMID:18597118

Development of a 3D cell printed construct considering angiogenesis for liver tissue engineering.

PubMed

Lee, Jin Woo; Choi, Yeong-Jin; Yong, Woon-Jae; Pati, Falguni; Shim, Jin-Hyung; Kang, Kyung Shin; Kang, In-Hye; Park, Jaesung; Cho, Dong-Woo

2016-01-12

Several studies have focused on the regeneration of liver tissue in a two-dimensional (2D) planar environment, whereas actual liver tissue is three-dimensional (3D). Cell printing technology has been successfully utilized for building 3D structures; however, the poor mechanical properties of cell-laden hydrogels are a major concern. Here, we demonstrate the printing of a 3D cell-laden construct and its application to liver tissue engineering using 3D cell printing technology through a multi-head tissue/organ building system. Polycaprolactone (PCL) was used as a framework material because of its excellent mechanical properties. Collagen bioink containing three different types of cells-hepatocytes (HCs), human umbilical vein endothelial cells , and human lung fibroblasts--was infused into the canals of a PCL framework to induce the formation of capillary--like networks and liver cell growth. A co-cultured 3D microenvironment of the three types of cells was successfully established and maintained. The vascular formation and functional abilities of HCs (i.e., albumin secretion and urea synthesis) demonstrated that the heterotypic interaction among HCs and nonparenchymal cells increased the survivability and functionality of HCs within the collagen gel. Therefore, our results demonstrate the prospect of using cell printing technology for the creation of heterotypic cellular interaction within a structure for liver tissue engineering.

Multiscale strain analysis of tissue equivalents using a custom-designed biaxial testing device.

PubMed

Bell, B J; Nauman, E; Voytik-Harbin, S L

2012-03-21

Mechanical signals transferred between a cell and its extracellular matrix play an important role in regulating fundamental cell behavior. To further define the complex mechanical interactions between cells and matrix from a multiscale perspective, a biaxial testing device was designed and built. Finite element analysis was used to optimize the cruciform specimen geometry so that stresses within the central region were concentrated and homogenous while minimizing shear and grip effects. This system was used to apply an equibiaxial loading and unloading regimen to fibroblast-seeded tissue equivalents. Digital image correlation and spot tracking were used to calculate three-dimensional strains and associated strain transfer ratios at macro (construct), meso, matrix (collagen fibril), cell (mitochondria), and nuclear levels. At meso and matrix levels, strains in the 1- and 2-direction were statistically similar throughout the loading-unloading cycle. Interestingly, a significant amplification of cellular and nuclear strains was observed in the direction perpendicular to the cell axis. Findings indicate that strain transfer is dependent upon local anisotropies generated by the cell-matrix force balance. Such multiscale approaches to tissue mechanics will assist in advancement of modern biomechanical theories as well as development and optimization of preconditioning regimens for functional engineered tissue constructs. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model.

PubMed

Lee-Barthel, Ann; Baar, Keith; West, Daniel W D

2017-06-11

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

PubMed Central

Lee-Barthel, Ann; Baar, Keith; West, Daniel W. D.

2017-01-01

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration. PMID:28654031

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints.

PubMed

Shahin, Kifah; Doran, Pauline M

2012-04-01

The effect of dynamic mechanical shear and compression on the synthesis of human tissue-engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA-alginate scaffolds were precultured in shaking T-flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak-to-peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T-flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3- and 10-fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4-fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue-engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II. Copyright © 2011 Wiley Periodicals, Inc.

A mathematical model for the determination of forming tissue moduli in needled-nonwoven scaffolds.

PubMed

Soares, João S; Zhang, Will; Sacks, Michael S

2017-03-15

Formation of engineering tissues (ET) remains an important scientific area of investigation for both clinical translational and mechanobiological studies. Needled-nonwoven (NNW) scaffolds represent one of the most ubiquitous biomaterials based on their well-documented capacity to sustain tissue formation and the unique property of substantial construct stiffness amplification, the latter allowing for very sensitive determination of forming tissue modulus. Yet, their use in more fundamental studies is hampered by the lack of: (1) substantial understanding of the mechanics of the NNW scaffold itself under finite deformations and means to model the complex mechanical interactions between scaffold fibers, cells, and de novo tissue; and (2) rational models with reliable predictive capabilities describing their evolving mechanical properties and their response to mechanical stimulation. Our objective is to quantify the mechanical properties of the forming ET phase in constructs that utilize NNW scaffolds. We present herein a novel mathematical model to quantify their stiffness based on explicit considerations of the modulation of NNW scaffold fiber-fiber interactions and effective fiber stiffness by surrounding de novo ECM. Specifically, fibers in NNW scaffolds are effectively stiffer than if acting alone due to extensive fiber-fiber cross-over points that impart changes in fiber geometry, particularly crimp wavelength and amplitude. Fiber-fiber interactions in NNW scaffolds also play significant role in the bulk anisotropy of the material, mainly due to fiber buckling and large translational out-of-plane displacements occurring to fibers undergoing contraction. To calibrate the model parameters, we mechanically tested impregnated NNW scaffolds with polyacrylamide (PAM) gels with a wide range of moduli with values chosen to mimic the effects of surrounding tissues on the scaffold fiber network. Results indicated a high degree of model fidelity over a wide range of planar strains. Lastly, we illustrated the impact of our modeling approach quantifying the stiffness of engineered ECM after in vitro incubation and early stages of in vivo implantation obtained in a concurrent study of engineered tissue pulmonary valves in an ovine model. Regenerative medicine has the potential to fully restore diseased tissues or entire organs with engineered tissues. Needled-nonwoven scaffolds can be employed to serve as the support for their growth. However, there is a lack of understanding of the mechanics of these materials and their interactions with the forming tissues. We developed a mathematical model for these scaffold-tissue composites to quantify the mechanical properties of the forming tissues. Firstly, these measurements are pivotal to achieve functional requirements for tissue engineering implants; however, the theoretical development yielded critical insight into particular mechanisms and behaviors of these scaffolds that were not possible to conjecture without the insight given by modeling, let alone describe or foresee a priori. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Growing Tissues in Real and Simulated Microgravity: New Methods for Tissue Engineering

PubMed Central

Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Ulbrich, Claudia; Magnusson, Nils E.; Infanger, Manfred; Bauer, Johann

2014-01-01

Tissue engineering in simulated (s-) and real microgravity (r-μg) is currently a topic in Space medicine contributing to biomedical sciences and their applications on Earth. The principal aim of this review is to highlight the advances and accomplishments in the field of tissue engineering that could be achieved by culturing cells in Space or by devices created to simulate microgravity on Earth. Understanding the biology of three-dimensional (3D) multicellular structures is very important for a more complete appreciation of in vivo tissue function and advancing in vitro tissue engineering efforts. Various cells exposed to r-μg in Space or to s-μg created by a random positioning machine, a 2D-clinostat, or a rotating wall vessel bioreactor grew in the form of 3D tissues. Hence, these methods represent a new strategy for tissue engineering of a variety of tissues, such as regenerated cartilage, artificial vessel constructs, and other organ tissues as well as multicellular cancer spheroids. These aggregates are used to study molecular mechanisms involved in angiogenesis, cancer development, and biology and for pharmacological testing of, for example, chemotherapeutic drugs or inhibitors of neoangiogenesis. Moreover, they are useful for studying multicellular responses in toxicology and radiation biology, or for performing coculture experiments. The future will show whether these tissue-engineered constructs can be used for medical transplantations. Unveiling the mechanisms of microgravity-dependent molecular and cellular changes is an up-to-date requirement for improving Space medicine and developing new treatment strategies that can be translated to in vivo models while reducing the use of laboratory animals. PMID:24597549

Growing tissues in real and simulated microgravity: new methods for tissue engineering.

PubMed

Grimm, Daniela; Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Ulbrich, Claudia; Magnusson, Nils E; Infanger, Manfred; Bauer, Johann

2014-12-01

Tissue engineering in simulated (s-) and real microgravity (r-μg) is currently a topic in Space medicine contributing to biomedical sciences and their applications on Earth. The principal aim of this review is to highlight the advances and accomplishments in the field of tissue engineering that could be achieved by culturing cells in Space or by devices created to simulate microgravity on Earth. Understanding the biology of three-dimensional (3D) multicellular structures is very important for a more complete appreciation of in vivo tissue function and advancing in vitro tissue engineering efforts. Various cells exposed to r-μg in Space or to s-μg created by a random positioning machine, a 2D-clinostat, or a rotating wall vessel bioreactor grew in the form of 3D tissues. Hence, these methods represent a new strategy for tissue engineering of a variety of tissues, such as regenerated cartilage, artificial vessel constructs, and other organ tissues as well as multicellular cancer spheroids. These aggregates are used to study molecular mechanisms involved in angiogenesis, cancer development, and biology and for pharmacological testing of, for example, chemotherapeutic drugs or inhibitors of neoangiogenesis. Moreover, they are useful for studying multicellular responses in toxicology and radiation biology, or for performing coculture experiments. The future will show whether these tissue-engineered constructs can be used for medical transplantations. Unveiling the mechanisms of microgravity-dependent molecular and cellular changes is an up-to-date requirement for improving Space medicine and developing new treatment strategies that can be translated to in vivo models while reducing the use of laboratory animals.

Detection of abnormalities in the superficial zone of cartilage repaired using a tissue engineered construct derived from synovial stem cells.

PubMed

Ando, Wataru; Fujie, Hiromichi; Moriguchi, Yu; Nansai, Ryosuke; Shimomura, Kazunori; Hart, David A; Yoshikawa, Hideki; Nakamura, Norimasa

2012-09-28

The present study investigated the surface structure and mechanical properties of repair cartilage generated from a tissue engineered construct (TEC) derived from synovial mesenchymal stem cells at six months post-implantation compared to those of uninjured cartilage. TEC-mediated repair tissue was cartilaginous with Safranin O staining, and had comparable macro-scale compressive properties with uninjured cartilage. However, morphological assessments revealed that the superficial zone of TEC-mediated tissue was more fibrocartilage-like, in contrast to the middle or deep zones that were more hyaline cartilage-like with Safranin O staining. Histological scoring of the TEC-mediated tissue was significantly lower in the superficial zone than in the middle and deep zones. Scanning electron microscopy showed a thick tangential bundle of collagen fibres at the most superficial layer of uninjured cartilage, while no corresponding structure was detected at the surface of TEC-mediated tissue. Immunohistochemical analysis revealed that PRG4 was localised in the superficial area of uninjured cartilage, as well as the TEC-mediated tissue. Friction testing showed that the lubrication properties of the two tissues was similar, however, micro-indentation analysis revealed that the surface stiffness of the TEC-repair tissue was significantly lower than that of uninjured cartilage. Permeability testing indicated that the TEC-mediated tissue exhibited lower water retaining capacity than did uninjured cartilage, specifically at the superficial zone. Thus, TEC-mediated tissue exhibited compromised mechanical properties at the superficial zone, properties which need improvement in the future for maintenance of long term repair cartilage integrity.

Bioreactors as engineering support to treat cardiac muscle and vascular disease.

PubMed

Massai, Diana; Cerino, Giulia; Gallo, Diego; Pennella, Francesco; Deriu, Marco A; Rodriguez, Andres; Montevecchi, Franco M; Bignardi, Cristina; Audenino, Alberto; Morbiducci, Umberto

2013-01-01

Cardiovascular disease is the leading cause of morbidity and mortality in the Western World. The inability of fully differentiated, load-bearing cardiovascular tissues to in vivo regenerate and the limitations of the current treatment therapies greatly motivate the efforts of cardiovascular tissue engineering to become an effective clinical strategy for injured heart and vessels. For the effective production of organized and functional cardiovascular engineered constructs in vitro, a suitable dynamic environment is essential, and can be achieved and maintained within bioreactors. Bioreactors are technological devices that, while monitoring and controlling the culture environment and stimulating the construct, attempt to mimic the physiological milieu. In this study, a review of the current state of the art of bioreactor solutions for cardiovascular tissue engineering is presented, with emphasis on bioreactors and biophysical stimuli adopted for investigating the mechanisms influencing cardiovascular tissue development, and for eventually generating suitable cardiovascular tissue replacements.

Cell-matrix mechanical interaction in electrospun polymeric scaffolds for tissue engineering: Implications for scaffold design and performance.

PubMed

Kennedy, Kelsey M; Bhaw-Luximon, Archana; Jhurry, Dhanjay

2017-03-01

Engineered scaffolds produced by electrospinning of biodegradable polymers offer a 3D, nanofibrous environment with controllable structural, chemical, and mechanical properties that mimic the extracellular matrix of native tissues and have shown promise for a number of tissue engineering applications. The microscale mechanical interactions between cells and electrospun matrices drive cell behaviors including migration and differentiation that are critical to promote tissue regeneration. Recent developments in understanding these mechanical interactions in electrospun environments are reviewed, with emphasis on how fiber geometry and polymer structure impact on the local mechanical properties of scaffolds, how altering the micromechanics cues cell behaviors, and how, in turn, cellular and extrinsic forces exerted on the matrix mechanically remodel an electrospun scaffold throughout tissue development. Techniques used to measure and visualize these mechanical interactions are described. We provide a critical outlook on technological gaps that must be overcome to advance the ability to design, assess, and manipulate the mechanical environment in electrospun scaffolds toward constructs that may be successfully applied in tissue engineering and regenerative medicine. Tissue engineering requires design of scaffolds that interact with cells to promote tissue development. Electrospinning is a promising technique for fabricating fibrous, biomimetic scaffolds. Effects of electrospun matrix microstructure and biochemical properties on cell behavior have been extensively reviewed previously; here, we consider cell-matrix interaction from a mechanical perspective. Micromechanical properties as a driver of cell behavior has been well established in planar substrates, but more recently, many studies have provided new insights into mechanical interaction in fibrillar, electrospun environments. This review provides readers with an overview of how electrospun scaffold mechanics and cell behavior work in a dynamic feedback loop to drive tissue development, and discusses opportunities for improved design of mechanical environments that are conducive to tissue development. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

3D Printing of Tissue Engineered Constructs for In Vitro Modeling of Disease Progression and Drug Screening.

PubMed

Vanderburgh, Joseph; Sterling, Julie A; Guelcher, Scott A

2017-01-01

2D cell culture and preclinical animal models have traditionally been implemented for investigating the underlying cellular mechanisms of human disease progression. However, the increasing significance of 3D vs. 2D cell culture has initiated a new era in cell culture research in which 3D in vitro models are emerging as a bridge between traditional 2D cell culture and in vivo animal models. Additive manufacturing (AM, also known as 3D printing), defined as the layer-by-layer fabrication of parts directed by digital information from a 3D computer-aided design file, offers the advantages of simultaneous rapid prototyping and biofunctionalization as well as the precise placement of cells and extracellular matrix with high resolution. In this review, we highlight recent advances in 3D printing of tissue engineered constructs that recapitulate the physical and cellular properties of the tissue microenvironment for investigating mechanisms of disease progression and for screening drugs.

3D Printing of Tissue Engineered Constructs for in vitro Modeling of Disease Progression and Drug Screening

PubMed Central

Vanderburgh, Joseph; Sterling, Julie A.

2016-01-01

2D cell culture and preclinical animal models have traditionally been implemented for investigating the underlying cellular mechanisms of human disease progression. However, the increasing significance of 3D versus 2D cell culture has initiated a new era in cell culture research in which 3D in vitro models are emerging as a bridge between traditional 2D cell culture and in vivo animal models. Additive manufacturing (AM, also known as 3D printing), defined as the layer-by-layer fabrication of parts directed by digital information from a 3D computer-aided design (CAD) file, offers the advantages of simultaneous rapid prototyping and biofunctionalization as well as the precise placement of cells and extracellular matrix with high resolution. In this review, we highlight recent advances in 3D printing of tissue engineered constructs (TECs) that recapitulate the physical and cellular properties of the tissue microenvironment for investigating mechanisms of disease progression and for screening drugs. PMID:27169894

A convex optimization approach for identification of human tissue-specific interactomes.

PubMed

Mohammadi, Shahin; Grama, Ananth

2016-06-15

Analysis of organism-specific interactomes has yielded novel insights into cellular function and coordination, understanding of pathology, and identification of markers and drug targets. Genes, however, can exhibit varying levels of cell type specificity in their expression, and their coordinated expression manifests in tissue-specific function and pathology. Tissue-specific/tissue-selective interaction mechanisms have significant applications in drug discovery, as they are more likely to reveal drug targets. Furthermore, tissue-specific transcription factors (tsTFs) are significantly implicated in human disease, including cancers. Finally, disease genes and protein complexes have the tendency to be differentially expressed in tissues in which defects cause pathology. These observations motivate the construction of refined tissue-specific interactomes from organism-specific interactomes. We present a novel technique for constructing human tissue-specific interactomes. Using a variety of validation tests (Edge Set Enrichment Analysis, Gene Ontology Enrichment, Disease-Gene Subnetwork Compactness), we show that our proposed approach significantly outperforms state-of-the-art techniques. Finally, using case studies of Alzheimer's and Parkinson's diseases, we show that tissue-specific interactomes derived from our study can be used to construct pathways implicated in pathology and demonstrate the use of these pathways in identifying novel targets. http://www.cs.purdue.edu/homes/mohammas/projects/ActPro.html mohammadi@purdue.edu. © The Author 2016. Published by Oxford University Press.

Tissue grown in space in NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens of cartilage tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Constructs grown on Mir (A) tended to become more spherical, whereas those grown on Earth (B) maintained their initial disc shape. These findings might be related to differences in cultivation conditions, i.e., videotapes showed that constructs floated freely in microgravity but settled and collided with the rotating vessel wall at 1g (Earth's gravity). In particular, on Mir the constructs were exposed to uniform shear and mass transfer at all surfaces such that the tissue grew equally in all directions, whereas on Earth the settling of discoid constructs tended to align their flat circular areas perpendicular to the direction of motion, increasing shear and mass transfer circumferentially such that the tissue grew preferentially in the radial direction. A and B are full cross sections of constructs from Mir and Earth groups shown at 10-power. C and D are representative areas at the construct surfaces enlarged to 200-power. They are stained red with safranin-O. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Photo credit: Proceedings of the National Academy of Sciences.

Improved properties of bone and cartilage tissue from 3D inkjet-bioprinted human mesenchymal stem cells by simultaneous deposition and photocrosslinking in PEG-GelMA.

PubMed

Gao, Guifang; Schilling, Arndt F; Hubbell, Karen; Yonezawa, Tomo; Truong, Danh; Hong, Yi; Dai, Guohao; Cui, Xiaofeng

2015-11-01

Bioprinting of bone and cartilage suffers from low mechanical properties. Here we have developed a unique inkjet bioprinting approach of creating mechanically strong bone and cartilage tissue constructs using poly(ethylene glycol) dimethacrylate, gelatin methacrylate, and human MSCs. The printed hMSCs were evenly distributed in the polymerized PEG-GelMA scaffold during layer-by-layer assembly. The procedure showed a good biocompatibility with >80% of the cells surviving the printing process and the resulting constructs provided strong mechanical support to the embedded cells. The printed mesenchymal stem cells showed an excellent osteogenic and chondrogenic differentiation capacity. Both osteogenic and chondrogenic differentiation as determined by specific gene and protein expression analysis (RUNX2, SP7, DLX5, ALPL, Col1A1, IBSP, BGLAP, SPP1, Col10A1, MMP13, SOX9, Col2A1, ACAN) was improved by PEG-GelMA in comparison to PEG alone. These observations were consistent with the histological evaluation. Inkjet bioprinted-hMSCs in simultaneously photocrosslinked PEG-GelMA hydrogel scaffolds demonstrated an improvement of mechanical properties and osteogenic and chondrogenic differentiation, suggesting its promising potential for usage in bone and cartilage tissue engineering.

A multiphase model for tissue construct growth in a perfusion bioreactor.

PubMed

O'Dea, R D; Waters, S L; Byrne, H M

2010-06-01

The growth of a cell population within a rigid porous scaffold in a perfusion bioreactor is studied, using a three-phase continuum model of the type presented by Lemon et al. (2006, Multiphase modelling of tissue growth using the theory of mixtures. J. Math. Biol., 52, 571-594) to represent the cell population (and attendant extracellular matrix), culture medium and porous scaffold. The bioreactor system is modelled as a 2D channel containing the cell-seeded rigid porous scaffold (tissue construct) which is perfused with culture medium. The study concentrates on (i) the cell-cell and cell-scaffold interactions and (ii) the impact of mechanotransduction mechanisms on construct composition. A numerical and analytical analysis of the model equations is presented and, depending upon the relative importance of cell aggregation and repulsion, markedly different cell movement is revealed. Additionally, mechanotransduction effects due to cell density, pressure and shear stress-mediated tissue growth are shown to generate qualitative differences in the composition of the resulting construct. The results of our simulations indicate that this model formulation (in conjunction with appropriate experimental data) has the potential to provide a means of identifying the dominant regulatory stimuli in a cell population.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Thao D.; Grazier, John Mark; Boyce, Brad Lee

Biological tissues are uniquely structured materials with technologically appealing properties. Soft tissues such as skin, are constructed from a composite of strong fibrils and fluid-like matrix components. This was the first coordinated experimental/modeling project at Sandia or in the open literature to consider the mechanics of micromechanically-based anisotropy and viscoelasticity of soft biological tissues. We have exploited and applied Sandia's expertise in experimentation and mechanics modeling to better elucidate the behavior of collagen fibril-reinforced soft tissues. The purpose of this project was to provide a detailed understanding of the deformation of ocular tissues, specifically the highly structured skin-like tissue inmore » the cornea. This discovery improved our knowledge of soft/complex materials testing and modeling. It also provided insight into the way that cornea tissue is bio-engineered such that under physiologically-relevant conditions it has a unique set of properties which enhance functionality. These results also provide insight into how non-physiologic loading conditions, such as corrective surgeries, may push the cornea outside of its natural design window, resulting in unexpected non-linear responses. Furthermore, this project created a clearer understanding of the mechanics of soft tissues that could lead to bio-inspired materials, such as highly supple and impact resistant body armor, and improve our design of human-machine interfaces, such as micro-electrical-mechanical (MEMS) based prosthetics.« less

Stem Cells and Scaffolds for Vascularizing Engineered Tissue Constructs

NASA Astrophysics Data System (ADS)

Luong, E.; Gerecht, S.

The clinical impact of tissue engineering depends upon our ability to direct cells to form tissues with characteristic structural and mechanical properties from the molecular level up to organized tissue. Induction and creation of functional vascular networks has been one of the main goals of tissue engineering either in vitro, for the transplantation of prevascularized constructs, or in vivo, for cellular organization within the implantation site. In most cases, tissue engineering attempts to recapitulate certain aspects of normal development in order to stimulate cell differentiation and functional tissue assembly. The induction of tissue growth generally involves the use of biodegradable and bioactive materials designed, ideally, to provide a mechanical, physical, and biochemical template for tissue regeneration. Human embryonic stem cells (hESCs), derived from the inner cell mass of a developing blastocyst, are capable of differentiating into all cell types of the body. Specifically, hESCs have the capability to differentiate and form blood vessels de novo in a process called vasculogenesis. Human ESC-derived endothelial progenitor cells (EPCs) and endothelial cells have substantial potential for microvessel formation, in vitro and in vivo. Human adult EPCs are being isolated to understand the fundamental biology of how these cells are regulated as a population and to explore whether these cells can be differentiated and reimplanted as a cellular therapy in order to arrest or even reverse damaged vasculature. This chapter focuses on advances made toward the generation and engineering of functional vascular tissue, focusing on both the scaffolds - the synthetic and biopolymer materials - and the cell sources - hESCs and hEPCs.

The effects of dynamic compression on the development of cartilage grafts engineered using bone marrow and infrapatellar fat pad derived stem cells.

PubMed

Luo, Lu; Thorpe, Stephen D; Buckley, Conor T; Kelly, Daniel J

2015-09-21

Bioreactors that subject cell seeded scaffolds or hydrogels to biophysical stimulation have been used to improve the functionality of tissue engineered cartilage and to explore how such constructs might respond to the application of joint specific mechanical loading. Whether a particular cell type responds appropriately to physiological levels of biophysical stimulation could be considered a key determinant of its suitability for cartilage tissue engineering applications. The objective of this study was to determine the effects of dynamic compression on chondrogenesis of stem cells isolated from different tissue sources. Porcine bone marrow (BM) and infrapatellar fat pad (FP) derived stem cells were encapsulated in agarose hydrogels and cultured in a chondrogenic medium in free swelling (FS) conditions for 21 d, after which samples were subjected to dynamic compression (DC) of 10% strain (1 Hz, 1 h d(-1)) for a further 21 d. Both BM derived stem cells (BMSCs) and FP derived stem cells (FPSCs) were capable of generating cartilaginous tissues with near native levels of sulfated glycosaminoglycan (sGAG) content, although the spatial development of the engineered grafts strongly depended on the stem cell source. The mechanical properties of cartilage grafts generated from both stem cell sources also approached that observed in skeletally immature animals. Depending on the stem cell source and the donor, the application of DC either enhanced or had no significant effect on the functional development of cartilaginous grafts engineered using either BMSCs or FPSCs. BMSC seeded constructs subjected to DC stained less intensely for collagen type I. Furthermore, histological and micro-computed tomography analysis showed mineral deposition within BMSC seeded constructs was suppressed by the application of DC. Therefore, while the application of DC in vitro may only lead to modest improvements in the mechanical functionality of cartilaginous grafts, it may play an important role in the development of phenotypically stable constructs.

A chondroitinase-ABC and TGF-β1 treatment regimen for enhancing the mechanical properties of tissue-engineered fibrocartilage.

PubMed

MacBarb, Regina F; Makris, Eleftherios A; Hu, Jerry C; Athanasiou, Kyriacos A

2013-01-01

The development of functionally equivalent fibrocartilage remains elusive despite efforts to engineer tissues such as knee meniscus, intervertebral disc and temporomandibular joint disc. Attempts to engineer these structures often fail to create tissues with mechanical properties on a par with native tissue, resulting in constructs unsuitable for clinical applications. The objective of this study was to engineer a spectrum of biomimetic fibrocartilages representative of the distinct functional properties found in native tissues. Using the self-assembly process, different co-cultures of meniscus cells and articular chondrocytes were seeded into agarose wells and treated with the catabolic agent chondroitinase-ABC (C-ABC) and the anabolic agent transforming growth factor-β1 (TGF-β1) via a two-factor (cell ratio and bioactive treatment), full factorial study design. Application of both C-ABC and TGF-β1 resulted in a beneficial or positive increase in the collagen content of treated constructs compared to controls. Significant increases in both the collagen density and fiber diameter were also seen with this treatment, increasing these values by 32 and 15%, respectively, over control values. Mechanical testing found the combined bioactive treatment to synergistically increase the Young's modulus and ultimate tensile strength of the engineered fibrocartilages compared to controls, with values reaching the lower spectrum of those found in native tissues. Together, these data demonstrate that C-ABC and TGF-β1 interact to develop a denser collagen matrix better able to withstand tensile loading. This study highlights a way to optimize the tensile properties of engineered fibrocartilage using a biochemical and a biophysical agent together to create distinct fibrocartilages with functional properties mimicking those of native tissue. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A chondroitinase-ABC and TGF-β1 treatment regimen for enhancing the mechanical properties of tissue engineered fibrocartilage

PubMed Central

MacBarb, Regina F.; Makris, Eleftherios A.; Hu, Jerry C.; Athanasiou, Kyriacos A.

2012-01-01

The development of functionally equivalent fibrocartilage remains elusive despite efforts to engineer tissues such as the knee menisci, intervertebral disc, and TMJ disc. Attempts to engineer these structures often fail to create tissues with mechanical properties on par with native tissue, resulting in constructs unsuitable for clinical applications. The objective of this study was to engineer a spectrum of biomimetic fibrocartilages representative of the distinct functional properties found in native tissues. Using the self-assembly process, different co-cultures of meniscus cells (MCs) and articular chondrocytes (ACs) were seeded into agarose wells and treated with the catabolic agent chondroitinase-ABC (C-ABC) and the anabolic agent transforming growth factor-β1 (TGF-β1) via a two-factor (cell ratio and bioactive treatment), full factorial study design. Application of both C-ABC and TGF-β1 resulted in a beneficial or positive increase in the collagen content of treated constructs compared to controls. Significant increases in both the collagen density and fiber diameter were also seen with this treatment, increasing these values 32% and 15%, respectively, over control values. Mechanical testing found the combined bioactive treatment to synergistically increase the Young’s modulus and ultimate tensile strength of the engineered fibrocartilages compared to controls, with values reaching the lower spectrum of those found in native tissues. Together, these data demonstrate that C-ABC and TGF-β1 interact to develop a denser collagen matrix better able to withstand tensile loading. This study highlights a way to optimize the tensile properties of engineered fibrocartilage using a biochemical and biophysical agent together to create distinct fibrocartilages with functional properties mimicking those of native tissue. PMID:23041782

Image-guided tissue engineering of anatomically shaped implants via MRI and micro-CT using injection molding.

PubMed

Ballyns, Jeffery J; Gleghorn, Jason P; Niebrzydowski, Vicki; Rawlinson, Jeremy J; Potter, Hollis G; Maher, Suzanne A; Wright, Timothy M; Bonassar, Lawrence J

2008-07-01

This study demonstrates for the first time the development of engineered tissues based on anatomic geometries derived from widely used medical imaging modalities such as computed tomography (CT) and magnetic resonance imaging (MRI). Computer-aided design and tissue injection molding techniques have demonstrated the ability to generate living implants of complex geometry. Due to its complex geometry, the meniscus of the knee was used as an example of this technique's capabilities. MRI and microcomputed tomography (microCT) were used to design custom-printed molds that enabled the generation of anatomically shaped constructs that retained shape throughout 8 weeks of culture. Engineered constructs showed progressive tissue formation indicated by increases in extracellular matrix content and mechanical properties. The paradigm of interfacing tissue injection molding technology can be applied to other medical imaging techniques that render 3D models of anatomy, demonstrating the potential to apply the current technique to engineering of many tissues and organs.

A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone

PubMed Central

Thibaudeau, Laure; Taubenberger, Anna V.; Holzapfel, Boris M.; Quent, Verena M.; Fuehrmann, Tobias; Hesami, Parisa; Brown, Toby D.; Dalton, Paul D.; Power, Carl A.; Hollier, Brett G.; Hutmacher, Dietmar W.

2014-01-01

ABSTRACT The skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact ‘organ’ bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo. PMID:24713276

Mussel-inspired tough hydrogels with self-repairing and tissue adhesion

NASA Astrophysics Data System (ADS)

Gao, Zijian; Duan, Lijie; Yang, Yongqi; Hu, Wei; Gao, Guanghui

2018-01-01

The mussel-inspired polymeric hydrogels have been attractively explored owing to their self-repairing or adhesive property when the catechol groups of dopamine could chelate metal ions. However, it was a challenge for self-repairing hydrogels owning high mechanical properties. Herein, a synergistic strategy was proposed by combining catechol-Fe3+ complexes and hydrophobic association. The resulting hydrogels exhibited seamless self-repairing behavior, tissue adhesion and high mechanical property. Moreover, the pH-dependent stoichiometry of catechol-Fe3+ and temperature-sensitive hydrophobic association endue hydrogels with pH/thermo responsive characteristics. Subsequently, the self-repairing rate and mechanical property of hydrogels were investigated at different pH and temperature. This bio-inspired strategy would build an avenue for designing and constructing a new generation of self-repairing, tissue-adhesive and tough hydrogel.

Fiber Angle and Aspect Ratio Influence the Shear Mechanics of Oriented Electrospun Nanofibrous Scaffolds

PubMed Central

Driscoll, Tristan P.; Nerurkar, Nandan L.; Jacobs, Nathan T.; Elliott, Dawn M.; Mauck, Robert L.

2011-01-01

Fibrocartilages, including the knee meniscus and the annulus fibrosus (AF) of the intervertebral disc, play critical mechanical roles in load transmission across joints and their function is dependent upon well-defined structural hierarchies, organization, and composition. All, however, are compromised in the pathologic transformations associated with tissue degeneration. Tissue engineering strategies that address these key features, for example, aligned nanofibrous scaffolds seeded with mesenchymal stem cells (MSCs), represent a promising approach for the regeneration of these fibrous structures. While such engineered constructs can replicate native tissue structure and uniaxial tensile properties, the multidirectional loading encountered by these tissues in vivo necessitates that they function adequately in other loading modalities as well, including shear. As previous findings have shown that native tissue tensile and shear properties are dependent on fiber angle and sample aspect ratio, respectively, the objective of the present study was to evaluate the effects of a changing fiber angle and sample aspect ratio on the shear properties of aligned electrospun poly(ε-caprolactone) (PCL) scaffolds, and to determine how extracellular matrix deposition by resident MSCs modulates the measured shear response. Results show that fiber orientation and sample aspect ratio significantly influence the response of scaffolds in shear, and that measured shear strains can be predicted by finite element models. Furthermore, acellular PCL scaffolds possessed a relatively high shear modulus, 2–4 fold greater than native tissue, independent of fiber angle and aspect ratio. It was further noted that under testing conditions that engendered significant fiber stretch, the aggregate resistance to shear was higher, indicating a role for fiber stretch in the overall shear response. Finally, with time in culture, the shear modulus of MSC laden constructs increased, suggesting that deposited ECM contributes to the construct shear properties. Collectively, these findings show that aligned electrospun PCL scaffolds are a promising tool for engineering fibrocartilage tissues, and that the shear properties of both acellular and cell-seeded formulations can match or exceed native tissue benchmarks. PMID:22098865

Two-photon induced collagen cross-linking in bioartificial cardiac tissue

NASA Astrophysics Data System (ADS)

Kuetemeyer, Kai; Kensah, George; Heidrich, Marko; Meyer, Heiko; Martin, Ulrich; Gruh, Ina; Heisterkamp, Alexander

2011-08-01

Cardiac tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs for transplantation. Besides contractile function, the stiffness of tissue engineered constructs is crucial to generate transplantable tissue surrogates with sufficient mechanical stability to withstand the high pressure present in the heart. Although several collagen cross-linking techniques have proven to be efficient in stabilizing biomaterials, they cannot be applied to cardiac tissue engineering, as cell death occurs in the treated area. Here, we present a novel method using femtosecond (fs) laser pulses to increase the stiffness of collagen-based tissue constructs without impairing cell viability. Raster scanning of the fs laser beam over riboflavin-treated tissue induced collagen cross-linking by two-photon photosensitized singlet oxygen production. One day post-irradiation, stress-strain measurements revealed increased tissue stiffness by around 40% being dependent on the fibroblast content in the tissue. At the same time, cells remained viable and fully functional as demonstrated by fluorescence imaging of cardiomyocyte mitochondrial activity and preservation of active contraction force. Our results indicate that two-photon induced collagen cross-linking has great potential for studying and improving artificially engineered tissue for regenerative therapies.

High Throughput and Mechano-Active Platforms to Promote Cartilage Regeneration and Repair

NASA Astrophysics Data System (ADS)

Mohanraj, Bhavana

Traumatic joint injuries initiate acute degenerative changes in articular cartilage that can lead to progressive loss of load-bearing function. As a result, patients often develop post-traumatic osteoarthritis (PTOA), a condition for which there currently exists no biologic interventions. To address this need, tissue engineering aims to mimic the structure and function of healthy, native counterparts. These constructs can be used to not only replace degenerated tissue, but also build in vitro, pre-clinical models of disease. Towards this latter goal, this thesis focuses on the design of a high throughput system to screen new therapeutics in a micro-engineered model of PTOA, and the development of a mechanically-responsive drug delivery system to augment tissue-engineered approaches for cartilage repair. High throughput screening is a powerful tool for drug discovery that can be adapted to include 3D tissue constructs. To facilitate this process for cartilage repair, we built a high throughput mechanical injury platform to create an engineered cartilage model of PTOA. Compressive injury of functionally mature constructs increased cell death and proteoglycan loss, two hallmarks of injury observed in vivo. Comparison of this response to that of native cartilage explants, and evaluation of putative therapeutics, validated this model for subsequent use in small molecule screens. A primary screen of 118 compounds identified a number of 'hits' and relevant pathways that may modulate pathologic signaling post-injury. To complement this process of therapeutic discovery, a stimuli-responsive delivery system was designed that used mechanical inputs as the 'trigger' mechanism for controlled release. The failure thresholds of these mechanically-activated microcapsules (MAMCs) were influenced by physical properties and composition, as well as matrix mechanical properties in 3D environments. TGF-beta released from the system upon mechano-activation stimulated stem cell chondrogenesis, demonstrating the potential of MAMCs to actively deliver therapeutics within demanding mechanical environments. Taken together, this work advances our capacity to identify and deliver new compounds of clinical relevance to modulate disease progression following traumatic injury using state-of-the-art micro-engineered screening tools and a novel mechanically-activated delivery system. These platforms advance strategies for cartilage repair and regeneration in PTOA and provide new options for the treatment of this debilitating condition.

Photo-cross-linkable methacrylated gelatin and hydroxyapatite hybrid hydrogel for modularly engineering biomimetic osteon.

PubMed

Zuo, Yicong; Liu, Xiaolu; Wei, Dan; Sun, Jing; Xiao, Wenqian; Zhao, Huan; Guo, Likun; Wei, Qingrong; Fan, Hongsong; Zhang, Xingdong

2015-05-20

Modular tissue engineering holds great potential in regenerating natural complex tissues by engineering three-dimensional modular scaffolds with predefined geometry and biological characters. In modular tissue-like construction, a scaffold with an appropriate mechanical rigidity for assembling fabrication and high biocompatibility for cell survival is the key to the successful bioconstruction. In this work, a series of composite hydrogels (GH0, GH1, GH2, and GH3) based on a combination of methacrylated gelatin (GelMA) and hydroxyapatite (HA) was exploited to enhance hydrogel mechanical rigidity and promote cell functional expression for osteon biofabrication. These composite hydrogels presented a lower swelling ratio, higher mechanical moduli, and better biocompatibility when compared to the pure GelMA hydrogel. Furthermore, on the basis of the composite hydrogel and photolithograph technology, we successfully constructed an osteon-like concentric double-ring structure in which the inner ring encapsulating human umbilical vascular endothelial cells (HUVECs) was designed to imitate blood vessel tubule while the outer ring encapsulating human osteoblast-like cells (MG63s) acts as part of bone. During the coculture period, MG63s and HUVECs exhibited not only satisfying growth status but also the enhanced genic expression of osteogenesis-related and angiogenesis-related differentiations. These results demonstrate this GelMA-HA composite hydrogel system is promising for modular tissue engineering.

Fabrication of injectable high strength hydrogel based on 4-arm star PEG for cartilage tissue engineering.

PubMed

Wang, Jianqi; Zhang, Fengjie; Tsang, Wing Pui; Wan, Chao; Wu, Chi

2017-03-01

Hydrogels prepared from poly(ethylene glycol) (PEG) are widely applied in tissue engineering, especially those derived from a combination of functional multi-arm star PEG and linear crosslinker, with an expectation to form a structurally ideal network. However, the poor mechanical strength still renders their further applications. Here we examined the relationship between the dynamics of the pre-gel solution and the mechanical property of the resultant hydrogel in a system consisting of 4-arm star PEG functionalized with vinyl sulfone and short dithiol crosslinker. A method to prepare mechanically strong hydrogel for cartilage tissue engineering is proposed. It is found that when gelation takes place at the overlap concentration, at which a slow relaxation mode just appears in dynamic light scattering (DLS), the resultant hydrogel has a local maximum compressive strength ∼20 MPa, while still keeps ultralow mass concentration and Young's modulus. Chondrocyte-laden hydrogel constructed under this condition was transplanted into the subcutaneous pocket and an osteochondral defect model in SCID mice. The in vivo results show that chondrocytes can proliferate and maintain their phenotypes in the hydrogel, with the production of abundant extracellular matrix (ECM) components, formation of typical chondrocyte lacunae structure and increase in Young's modulus over 12 weeks, as indicated by histological, immunohistochemistry, gene expression analyses and mechanical test. Moreover, newly formed hyaline cartilage was observed to be integrated with the host articular cartilage tissue in the defects injected with chondrocytes/hydrogel constructs. The results suggest that this hydrogel is a promising candidate scaffold for cartilage tissue engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bio-printing cell-laden Matrigel–agarose constructs

PubMed Central

Fan, Rong; Piou, Marine; Darling, Evan; Cormier, Denis; Sun, Jun; Wan, Jiandi

2017-01-01

3D printing of biological architectures that mimic the structural and functional features of in vivo tissues is of great interest in tissue engineering and the development of transplantable organ constructs. Printable bio-inks that are compatible with cellular activities play critical roles in the process of 3D bio-printing. Although a variety of hydrogels have been used as bio-inks for 3D bio-printing, they inherit poor mechanical properties and/or the lack of essential protein components that compromise their performance. Here, a hybrid Matrigel–agarose hydrogel system has been demonstrated that possesses both desired rheological properties for bio-printing and biocompatibility for long-term (11 days) cell culture. The agarose component in the hybrid hydrogel system enables the maintenance of 3D-printed structures, whereas Matrigel provides essential microenvironments for cell growth. When human intestinal epithelial HCT116 cells are encapsulated in the printed Matrigel–agarose constructs, high cell viability and proper cell spreading morphology are observed. Given that Matrigel is used extensively for 3D cell culturing, the developed 3D-printable Matrigel–agarose system will open a new way to construct Matrigel-based 3D constructs for cell culture and tissue engineering. PMID:27638155

Role of chondroitin sulphate tethered silk scaffold in cartilaginous disc tissue regeneration.

PubMed

Bhattacharjee, Maumita; Chawla, Shikha; Chameettachal, Shibu; Murab, Sumit; Bhavesh, Neel Sarovar; Ghosh, Sourabh

2016-04-12

Strategies for tissue engineering focus on scaffolds with tunable structure and morphology as well as optimum surface chemistry to simulate the anatomy and functionality of the target tissue. Silk fibroin has demonstrated its potential in supporting cartilaginous tissue formation both in vitro and in vivo. In this study, we investigate the role of controlled lamellar organization and chemical composition of biofunctionalized silk scaffolds in replicating the structural properties of the annulus region of an intervertebral disc using articular chondrocytes. Covalent attachment of chondroitin sulfate (CS) to silk is characterized. CS-conjugated silk constructs demonstrate enhanced cellular metabolic activity and chondrogenic redifferentiation potential with significantly improved mechanical properties over silk-only constructs. A matrix-assisted laser desorption ionization-time of flight analysis and protein-protein interaction studies help to generate insights into how CS conjugation can facilitate the production of disc associated matrix proteins, compared to a silk-only based construct. An in-depth understanding of the interplay between such extra cellular matrix associated proteins should help in designing more rational scaffolds for cartilaginous disc regeneration needs.

Tough and flexible CNT-polymeric hybrid scaffolds for engineering cardiac constructs.

PubMed

Kharaziha, Mahshid; Shin, Su Ryon; Nikkhah, Mehdi; Topkaya, Seda Nur; Masoumi, Nafiseh; Annabi, Nasim; Dokmeci, Mehmet R; Khademhosseini, Ali

2014-08-01

In the past few years, a considerable amount of effort has been devoted toward the development of biomimetic scaffolds for cardiac tissue engineering. However, most of the previous scaffolds have been electrically insulating or lacked the structural and mechanical robustness to engineer cardiac tissue constructs with suitable electrophysiological functions. Here, we developed tough and flexible hybrid scaffolds with enhanced electrical properties composed of carbon nanotubes (CNTs) embedded aligned poly(glycerol sebacate):gelatin (PG) electrospun nanofibers. Incorporation of varying concentrations of CNTs from 0 to 1.5% within the PG nanofibrous scaffolds (CNT-PG scaffolds) notably enhanced fiber alignment and improved the electrical conductivity and toughness of the scaffolds while maintaining the viability, retention, alignment, and contractile activities of cardiomyocytes (CMs) seeded on the scaffolds. The resulting CNT-PG scaffolds resulted in stronger spontaneous and synchronous beating behavior (3.5-fold lower excitation threshold and 2.8-fold higher maximum capture rate) compared to those cultured on PG scaffold. Overall, our findings demonstrated that aligned CNT-PG scaffold exhibited superior mechanical properties with enhanced CM beating properties. It is envisioned that the proposed hybrid scaffolds can be useful for generating cardiac tissue constructs with improved organization and maturation. Copyright © 2014 Elsevier Ltd. All rights reserved.

ISSLS prize winner: integrating theoretical and experimental methods for functional tissue engineering of the annulus fibrosus.

PubMed

Nerurkar, Nandan L; Mauck, Robert L; Elliott, Dawn M

2008-12-01

Integrating theoretical and experimental approaches for annulus fibrosus (AF) functional tissue engineering. Apply a hyperelastic constitutive model to characterize the evolution of engineered AF via scalar model parameters. Validate the model and predict the response of engineered constructs to physiologic loading scenarios. There is need for a tissue engineered replacement for degenerate AF. When evaluating engineered replacements for load-bearing tissues, it is necessary to evaluate mechanical function with respect to the native tissue, including nonlinearity and anisotropy. Aligned nanofibrous poly-epsilon-caprolactone scaffolds with prescribed fiber angles were seeded with bovine AF cells and analyzed over 8 weeks, using experimental (mechanical testing, biochemistry, histology) and theoretical methods (a hyperelastic fiber-reinforced constitutive model). The linear region modulus for phi = 0 degrees constructs increased by approximately 25 MPa, and for phi = 90 degrees by approximately 2 MPa from 1 day to 8 weeks in culture. Infiltration and proliferation of AF cells into the scaffold and abundant deposition of s-GAG and aligned collagen was observed. The constitutive model had excellent fits to experimental data to yield matrix and fiber parameters that increased with time in culture. Correlations were observed between biochemical measures and model parameters. The model was successfully validated and used to simulate time-varying responses of engineered AF under shear and biaxial loading. AF cells seeded on nanofibrous scaffolds elaborated an organized, anisotropic AF-like extracellular matrix, resulting in improved mechanical properties. A hyperelastic fiber-reinforced constitutive model characterized the functional evolution of engineered AF constructs, and was used to simulate physiologically relevant loading configurations. Model predictions demonstrated that fibers resist shear even when the shearing direction does not coincide with the fiber direction. Further, the model suggested that the native AF fiber architecture is uniquely designed to support shear stresses encountered under multiple loading configurations.

Global De Novo Protein-Protein Interactome Elucidates Interactions of Drought-Responsive Proteins in Horse Gram (Macrotyloma uniflorum).

PubMed

Bhardwaj, Jyoti; Gangwar, Indu; Panzade, Ganesh; Shankar, Ravi; Yadav, Sudesh Kumar

2016-06-03

Inspired by the availability of de novo transcriptome of horse gram (Macrotyloma uniflorum) and recent developments in systems biology studies, the first ever global protein-protein interactome (PPI) map was constructed for this highly drought-tolerant legume. Large-scale studies of PPIs and the constructed database would provide rationale behind the interplay at cascading translational levels for drought stress-adaptive mechanisms in horse gram. Using a bidirectional approach (interolog and domain-based), a high-confidence interactome map and database for horse gram was constructed. Available transcriptomic information for shoot and root tissues of a sensitive (M-191; genotype 1) and a drought-tolerant (M-249; genotype 2) genotype of horse gram was utilized to draw comparative PPI subnetworks under drought stress. High-confidence 6804 interactions were predicted among 1812 proteins covering about one-fourth of the horse gram proteome. The highest number of interactions (33.86%) in horse gram interactome matched with Arabidopsis PPI data. The top five hub nodes mostly included ubiquitin and heat-shock-related proteins. Higher numbers of PPIs were found to be responsive in shoot tissue (416) and root tissue (2228) of genotype 2 compared with shoot tissue (136) and root tissue (579) of genotype 1. Characterization of PPIs using gene ontology analysis revealed that kinase and transferase activities involved in signal transduction, cellular processes, nucleocytoplasmic transport, protein ubiquitination, and localization of molecules were most responsive to drought stress. Hence, these could be framed in stress adaptive mechanisms of horse gram. Being the first legume global PPI map, it would provide new insights into gene and protein regulatory networks for drought stress tolerance mechanisms in horse gram. Information compiled in the form of database (MauPIR) will provide the much needed high-confidence systems biology information for horse gram genes, proteins, and involved processes. This information would ease the effort and increase the efficacy for similar studies on other legumes. Public access is available at http://14.139.59.221/MauPIR/ .

Tendon and ligament as novel cell sources for engineering the knee meniscus.

PubMed

Hadidi, P; Paschos, N K; Huang, B J; Aryaei, A; Hu, J C; Athanasiou, K A

2016-12-01

The application of cell-based therapies in regenerative medicine is hindered by the difficulty of acquiring adequate numbers of competent cells. For the knee meniscus in particular, this may be solved by harvesting tissue from neighboring tendons and ligaments. In this study, we have investigated the potential of cells from tendon and ligament, as compared to meniscus cells, to engineer scaffold-free self-assembling fibrocartilage. Self-assembling meniscus-shaped constructs engineered from a co-culture of articular chondrocytes and either meniscus, tendon, or ligament cells were cultured for 4 weeks with TGF-β1 in serum-free media. After culture, constructs were assessed for their mechanical properties, histological staining, gross appearance, and biochemical composition including cross-link content. Correlations were performed to evaluate relationships between biochemical content and mechanical properties. In terms of mechanical properties as well as biochemical content, constructs engineered using tenocytes and ligament fibrocytes were found to be equivalent or superior to constructs engineered using meniscus cells. Furthermore, cross-link content was found to be correlated with engineered tissue tensile properties. Tenocytes and ligament fibrocytes represent viable cell sources for engineering meniscus fibrocartilage using the self-assembling process. Due to greater cross-link content, fibrocartilage engineered with tenocytes and ligament fibrocytes may maintain greater tensile properties than fibrocartilage engineered with meniscus cells. Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Tendon and ligament as novel cell sources for engineering the knee meniscus

PubMed Central

Hadidi, Pasha; Paschos, Nikolaos K.; Huang, Brian J.; Aryaei, Ashkan; Hu, Jerry C.; Athanasiou, Kyriacos A.

2016-01-01

Objective The application of cell-based therapies in regenerative medicine is hindered by the difficulty of acquiring adequate numbers of competent cells. For the knee meniscus in particular, this may be solved by harvesting tissue from neighboring tendons and ligaments. In this study, we have investigated the potential of cells from tendon and ligament, as compared to meniscus cells, to engineer scaffold-free self-assembling fibrocartilage. Method Self-assembling meniscus-shaped constructs engineered from a co-culture of articular chondrocytes and either meniscus, tendon, or ligament cells were cultured for 4 weeks with TGF-β1 in serum-free media. After culture, constructs were assessed for their mechanical properties, histological staining, gross appearance, and biochemical composition including cross-link content. Correlations were performed to evaluate relationships between biochemical content and mechanical properties. Results In terms of mechanical properties as well as biochemical content, constructs engineered using tenocytes and ligament fibrocytes were found to be equivalent or superior to constructs engineered using meniscus cells. Furthermore, cross-link content was found to be correlated with engineered tissue tensile properties. Conclusion Tenocytes and ligament fibrocytes represent viable cell sources for engineering meniscus fibrocartilage using the self-assembling process. Due to greater cross-link content, fibrocartilage engineered with tenocytes and ligament fibrocytes may maintain greater tensile properties than fibrocartilage engineered with meniscus cells. PMID:27473559

High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties.

PubMed

Cigan, Alexander D; Roach, Brendan L; Nims, Robert J; Tan, Andrea R; Albro, Michael B; Stoker, Aaron M; Cook, James L; Vunjak-Novakovic, Gordana; Hung, Clark T; Ateshian, Gerard A

2016-06-14

Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young's moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microfluidic perfusion culture system for multilayer artery tissue models.

PubMed

Yamagishi, Yuka; Masuda, Taisuke; Matsusaki, Michiya; Akashi, Mitsuru; Yokoyama, Utako; Arai, Fumihito

2014-11-01

We described an assembly technique and perfusion culture system for constructing artery tissue models. This technique differed from previous studies in that it does not require a solid biodegradable scaffold; therefore, using sheet-like tissues, this technique allowed the facile fabrication of tubular tissues can be used as model. The fabricated artery tissue models had a multilayer structure. The assembly technique and perfusion culture system were applicable to many different sizes of fabricated arteries. The shape of the fabricated artery tissue models was maintained by the perfusion culture system; furthermore, the system reproduced the in vivo environment and allowed mechanical stimulation of the arteries. The multilayer structure of the artery tissue model was observed using fluorescent dyes. The equivalent Young's modulus was measured by applying internal pressure to the multilayer tubular tissues. The aim of this study was to determine whether fabricated artery tissue models maintained their mechanical properties with developing. We demonstrated both the rapid fabrication of multilayer tubular tissues that can be used as model arteries and the measurement of their equivalent Young's modulus in a suitable perfusion culture environment.

Wound ballistics of firearm-related injuries--part 1: missile characteristics and mechanisms of soft tissue wounding.

PubMed

Stefanopoulos, P K; Filippakis, K; Soupiou, O T; Pazarakiotis, V C

2014-12-01

Firearm-related injuries are caused by a wide variety of weapons and projectiles. The kinetic energy of the penetrating projectile defines its ability to disrupt and displace tissue, whereas the actual tissue damage is determined by the mode of energy release during the projectile-tissue interaction and the particular characteristics of the tissues and organs involved. Certain projectile factors, namely shape, construction, and stability, greatly influence the rate of energy transfer to the tissues along the wound track. Two zones of tissue damage can be identified, the permanent cavity created by the passage of the bullet and a potential area of contused tissue surrounding it, produced mainly by temporary cavitation which is a manifestation of effective high-energy transfer to tissue. Due to the complex nature of these injuries, wound assessment and the type and extent of treatment required should be based on an understanding of the various mechanisms contributing to tissue damage. Copyright © 2014 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Proteomic Profiling of Tissue-Engineered Blood Vessel Walls Constructed by Adipose-Derived Stem Cells

PubMed Central

Wang, Chen; Guo, Fangfang; Zhou, Heng; Zhang, Yun; Xiao, Zhigang

2013-01-01

Adipose-derived stem cells (ASCs) can differentiate into smooth muscle cells and have been engineered into elastic small diameter blood vessel walls in vitro. However, the mechanisms involved in the development of three-dimensional (3D) vascular tissue remain poorly understood. The present study analyzed protein expression profiles of engineered blood vessel walls constructed by human ASCs using methods of two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). These results were compared to normal arterial walls. A total of 1701±15 and 1265±26 protein spots from normal and engineered blood vessel wall extractions were detected by 2DE, respectively. A total of 20 spots with at least 2.0-fold changes in expression were identified, and 38 differently expressed proteins were identified by 2D electrophoresis and ion trap MS. These proteins were classified into seven functional categories: cellular organization, energy, signaling pathway, enzyme, anchored protein, cell apoptosis/defense, and others. These results demonstrated that 2DE, followed by ion trap MS, could be successfully utilized to characterize the proteome of vascular tissue, including tissue-engineered vessels. The method could also be employed to achieve a better understanding of differentiated smooth muscle protein expression in vitro. These results provide a basis for comparative studies of protein expression in vascular smooth muscles of different origin and could provide a better understanding of the mechanisms of action needed for constructing blood vessels that exhibit properties consistent with normal blood vessels. PMID:22963350

Proteomic profiling of tissue-engineered blood vessel walls constructed by adipose-derived stem cells.

PubMed

Wang, Chen; Guo, Fangfang; Zhou, Heng; Zhang, Yun; Xiao, Zhigang; Cui, Lei

2013-02-01

Adipose-derived stem cells (ASCs) can differentiate into smooth muscle cells and have been engineered into elastic small diameter blood vessel walls in vitro. However, the mechanisms involved in the development of three-dimensional (3D) vascular tissue remain poorly understood. The present study analyzed protein expression profiles of engineered blood vessel walls constructed by human ASCs using methods of two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). These results were compared to normal arterial walls. A total of 1701±15 and 1265±26 protein spots from normal and engineered blood vessel wall extractions were detected by 2DE, respectively. A total of 20 spots with at least 2.0-fold changes in expression were identified, and 38 differently expressed proteins were identified by 2D electrophoresis and ion trap MS. These proteins were classified into seven functional categories: cellular organization, energy, signaling pathway, enzyme, anchored protein, cell apoptosis/defense, and others. These results demonstrated that 2DE, followed by ion trap MS, could be successfully utilized to characterize the proteome of vascular tissue, including tissue-engineered vessels. The method could also be employed to achieve a better understanding of differentiated smooth muscle protein expression in vitro. These results provide a basis for comparative studies of protein expression in vascular smooth muscles of different origin and could provide a better understanding of the mechanisms of action needed for constructing blood vessels that exhibit properties consistent with normal blood vessels.

Nondestructive measurement of esophageal biaxial mechanical properties utilizing sonometry

NASA Astrophysics Data System (ADS)

Aho, Johnathon M.; Qiang, Bo; Wigle, Dennis A.; Tschumperlin, Daniel J.; Urban, Matthew W.

2016-07-01

Malignant esophageal pathology typically requires resection of the esophagus and reconstruction to restore foregut continuity. Reconstruction options are limited and morbid. The esophagus represents a useful target for tissue engineering strategies based on relative simplicity in comparison to other organs. The ideal tissue engineered conduit would have sufficient and ideally matched mechanical tolerances to native esophageal tissue. Current methods for mechanical testing of esophageal tissues both in vivo and ex vivo are typically destructive, alter tissue conformation, ignore anisotropy, or are not able to be performed in fluid media. The aim of this study was to investigate biomechanical properties of swine esophageal tissues through nondestructive testing utilizing sonometry ex vivo. This method allows for biomechanical determination of tissue properties, particularly longitudinal and circumferential moduli and strain energy functions. The relative contribution of mucosal-submucosal layers and muscular layers are compared to composite esophagi. Swine thoracic esophageal tissues (n  =  15) were tested by pressure loading using a continuous pressure pump system to generate stress. Preconditioning of tissue was performed by pressure loading with the pump system and pre-straining the tissue to in vivo length before data was recorded. Sonometry using piezocrystals was utilized to determine longitudinal and circumferential strain on five composite esophagi. Similarly, five mucosa-submucosal and five muscular layers from thoracic esophagi were tested independently. This work on esophageal tissues is consistent with reported uniaxial and biaxial mechanical testing and reported results using strain energy theory and also provides high resolution displacements, preserves native architectural structure and allows assessment of biomechanical properties in fluid media. This method may be of use to characterize mechanical properties of tissue engineered esophageal constructs.

Roles of Diffusion Dynamics in Stem Cell Signaling and Three-Dimensional Tissue Development.

PubMed

McMurtrey, Richard J

2017-09-15

Recent advancements in the ability to construct three-dimensional (3D) tissues and organoids from stem cells and biomaterials have not only opened abundant new research avenues in disease modeling and regenerative medicine but also have ignited investigation into important aspects of molecular diffusion in 3D cellular architectures. This article describes fundamental mechanics of diffusion with equations for modeling these dynamic processes under a variety of scenarios in 3D cellular tissue constructs. The effects of these diffusion processes and resultant concentration gradients are described in the context of the major molecular signaling pathways in stem cells that both mediate and are influenced by gas and nutrient concentrations, including how diffusion phenomena can affect stem cell state, cell differentiation, and metabolic states of the cell. The application of these diffusion models and pathways is of vital importance for future studies of developmental processes, disease modeling, and tissue regeneration.

Biologically and mechanically driven design of an RGD-mimetic macroporous foam for adipose tissue engineering applications.

PubMed

Rossi, Eleonora; Gerges, Irini; Tocchio, Alessandro; Tamplenizza, Margherita; Aprile, Paola; Recordati, Camilla; Martello, Federico; Martin, Ivan; Milani, Paolo; Lenardi, Cristina

2016-10-01

Despite clinical treatments for adipose tissue defects, in particular breast tissue reconstruction, have certain grades of efficacy, many drawbacks are still affecting the long-term survival of new formed fat tissue. To overcome this problem, in the last decades, several scaffolding materials have been investigated in the field of adipose tissue engineering. However, a strategy able to recapitulate a suitable environment for adipose tissue reconstruction and maintenance is still missing. To address this need, we adopted a biologically and mechanically driven design to fabricate an RGD-mimetic poly(amidoamine) oligomer macroporous foam (OPAAF) for adipose tissue reconstruction. The scaffold was designed to fulfil three fundamental criteria: capability to induce cell adhesion and proliferation, support of in vivo vascularization and match of native tissue mechanical properties. Poly(amidoamine) oligomers were formed into soft scaffolds with hierarchical porosity through a combined free radical polymerization and foaming reaction. OPAAF is characterized by a high water uptake capacity, progressive degradation kinetics and ideal mechanical properties for adipose tissue reconstruction. OPAAF's ability to support cell adhesion, proliferation and adipogenesis was assessed in vitro using epithelial, fibroblast and endothelial cells (MDCK, 3T3L1 and HUVEC respectively). In addition, in vivo subcutaneous implantation in murine model highlighted OPAAF potential to support both adipogenesis and vessels infiltration. Overall, the reported results support the use of OPAAF as a scaffold for engineered adipose tissue construct. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microgravity

NASA Image and Video Library

1998-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens of cartilage tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Constructs grown on Mir (A) tended to become more spherical, whereas those grown on Earth (B) maintained their initial disc shape. These findings might be related to differences in cultivation conditions, i.e., videotapes showed that constructs floated freely in microgravity but settled and collided with the rotating vessel wall at 1g (Earth's gravity). In particular, on Mir the constructs were exposed to uniform shear and mass transfer at all surfaces such that the tissue grew equally in all directions, whereas on Earth the settling of discoid constructs tended to align their flat circular areas perpendicular to the direction of motion, increasing shear and mass transfer circumferentially such that the tissue grew preferentially in the radial direction. A and B are full cross sections of constructs from Mir and Earth groups shown at 10-power. C and D are representative areas at the construct surfaces enlarged to 200-power. They are stained red with safranin-O. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Photo credit: Proceedings of the National Academy of Sciences.

NUTRIENT CHANNELS AND STIRRING ENHANCED THE COMPOSITION AND STIFFNESS OF LARGE CARTILAGE CONSTRUCTS

PubMed Central

Cigan, Alexander D.; Nims, Robert J.; Albro, Michael B.; Vunjak-Novakovic, Gordana; Hung, Clark T.; Ateshian, Gerard A.

2014-01-01

A significant challenge in cartilage tissue engineering is to successfully culture functional tissues that are sufficiently large to treat osteoarthritic joints. Transport limitations due to nutrient consumption by peripheral cells produce heterogeneous constructs with matrix-deficient centers. Incorporation of nutrient channels into large constructs is a promising technique for alleviating transport limitations, in conjunction with simple yet effective methods for enhancing media flow through channels. Cultivation of cylindrical channeled constructs flat in culture dishes, with or without orbital shaking, produced asymmetric constructs with poor tissue properties. We therefore explored a method for exposing the entire construct surface to the culture media, while promoting flow through the channels. To this end, chondrocyte-seeded agarose constructs (Ø10 mm, 2.34 mm thick), with zero or three nutrient channels (Ø1 mm), were suspended on their sides in custom culture racks and subjected to three media stirring modes for 56 days: uniaxial rocking, orbital shaking, or static control. Orbital shaking led to the highest construct EY, glycosaminoglycan (GAG), and collagen contents, whereas rocking had detrimental effects on GAG and collagen versus static control. Nutrient channels increased EY as well as GAG homogeneity, and the beneficial effects of channels were most marked in orbitally shaken samples. Under these conditions, the constructs developed symmetrically and reached or exceeded native levels of EY (~400 kPa) and glycosaminoglycans (GAG; ~9%/ww). These results suggest that the cultivation of channeled constructs in culture racks with orbital shaking is a promising method for engineering mechanically competent large cartilage constructs. PMID:25458579

Nutrient channels and stirring enhanced the composition and stiffness of large cartilage constructs.

PubMed

Cigan, Alexander D; Nims, Robert J; Albro, Michael B; Vunjak-Novakovic, Gordana; Hung, Clark T; Ateshian, Gerard A

2014-12-18

A significant challenge in cartilage tissue engineering is to successfully culture functional tissues that are sufficiently large to treat osteoarthritic joints. Transport limitations due to nutrient consumption by peripheral cells produce heterogeneous constructs with matrix-deficient centers. Incorporation of nutrient channels into large constructs is a promising technique for alleviating transport limitations, in conjunction with simple yet effective methods for enhancing media flow through channels. Cultivation of cylindrical channeled constructs flat in culture dishes, with or without orbital shaking, produced asymmetric constructs with poor tissue properties. We therefore explored a method for exposing the entire construct surface to the culture media, while promoting flow through the channels. To this end, chondrocyte-seeded agarose constructs (∅10mm, 2.34mm thick), with zero or three nutrient channels (∅1mm), were suspended on their sides in custom culture racks and subjected to three media stirring modes for 56 days: uniaxial rocking, orbital shaking, or static control. Orbital shaking led to the highest construct EY, sulfated glycosaminoglycan (sGAG), and collagen contents, whereas rocking had detrimental effects on sGAG and collagen versus static control. Nutrient channels increased EY as well as sGAG homogeneity, and the beneficial effects of channels were most marked in orbitally shaken samples. Under these conditions, the constructs developed symmetrically and reached or exceeded native levels of EY (~400kPa) and sGAG (~9%/ww). These results suggest that the cultivation of channeled constructs in culture racks with orbital shaking is a promising method for engineering mechanically competent large cartilage constructs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Three-dimensional Macroscopic Scaffolds With a Gradient in Stiffness for Functional Regeneration of Interfacial Tissues

PubMed Central

Singh, Milind; Dormer, Nathan; Salash, Jean R.; Christian, Jordan M.; Moore, David S.; Berkland, Cory; Detamore, Michael S.

2010-01-01

A novel approach has been demonstrated to construct biocompatible, macroporous 3-D tissue engineering scaffolds containing a continuous macroscopic gradient in composition that yields a stiffness gradient along the axis of the scaffold. Polymeric microspheres, made of poly(d,l-lactic-co-glycolic acid) (PLGA), and composite microspheres encapsulating a higher stiffness nano-phase material (PLGA encapsulating CaCO3 or TiO2 nanoparticles) were used for the construction of microsphere-based scaffolds. Using controlled infusion of polymeric and composite microspheres, gradient scaffolds displaying an anisotropic macroscopic distribution of CaCO3/TiO2 were fabricated via an ethanol sintering technique. The controllable mechanical characteristics and biocompatible nature of these scaffolds warrants further investigation for interfacial tissue engineering applications. PMID:20336753

In vitro culture increases mechanical stability of human tissue engineered cartilage constructs by prevention of microscale scaffold buckling.

PubMed

Middendorf, Jill M; Shortkroff, Sonya; Dugopolski, Caroline; Kennedy, Stephen; Siemiatkoski, Joseph; Bartell, Lena R; Cohen, Itai; Bonassar, Lawrence J

2017-11-07

Many studies have measured the global compressive properties of tissue engineered (TE) cartilage grown on porous scaffolds. Such scaffolds are known to exhibit strain softening due to local buckling under loading. As matrix is deposited onto these scaffolds, the global compressive properties increase. However the relationship between the amount and distribution of matrix in the scaffold and local buckling is unknown. To address this knowledge gap, we studied how local strain and construct buckling in human TE constructs changes over culture times and GAG content. Confocal elastography techniques and digital image correlation (DIC) were used to measure and record buckling modes and local strains. Receiver operating characteristic (ROC) curves were used to quantify construct buckling. The results from the ROC analysis were placed into Kaplan-Meier survival function curves to establish the probability that any point in a construct buckled. These analysis techniques revealed the presence of buckling at early time points, but bending at later time points. An inverse correlation was observed between the probability of buckling and the total GAG content of each construct. This data suggests that increased GAG content prevents the onset of construct buckling and improves the microscale compressive tissue properties. This increase in GAG deposition leads to enhanced global compressive properties by prevention of microscale buckling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Engineering anisotropic biomimetic fibrocartilage microenvironment by bioprinting mesenchymal stem cells in nanoliter gel droplets.

PubMed

Gurkan, Umut A; El Assal, Rami; Yildiz, Simin E; Sung, Yuree; Trachtenberg, Alexander J; Kuo, Winston P; Demirci, Utkan

2014-07-07

Over the past decade, bioprinting has emerged as a promising patterning strategy to organize cells and extracellular components both in two and three dimensions (2D and 3D) to engineer functional tissue mimicking constructs. So far, tissue printing has neither been used for 3D patterning of mesenchymal stem cells (MSCs) in multiphase growth factor embedded 3D hydrogels nor been investigated phenotypically in terms of simultaneous differentiation into different cell types within the same micropatterned 3D tissue constructs. Accordingly, we demonstrated a biochemical gradient by bioprinting nanoliter droplets encapsulating human MSCs, bone morphogenetic protein 2 (BMP-2), and transforming growth factor β1 (TGF- β1), engineering an anisotropic biomimetic fibrocartilage microenvironment. Assessment of the model tissue construct displayed multiphasic anisotropy of the incorporated biochemical factors after patterning. Quantitative real time polymerase chain reaction (qRT-PCR) results suggested genomic expression patterns leading to simultaneous differentiation of MSC populations into osteogenic and chondrogenic phenotype within the multiphasic construct, evidenced by upregulation of osteogenesis and condrogenesis related genes during in vitro culture. Comprehensive phenotypic network and pathway analysis results, which were based on genomic expression data, indicated activation of differentiation related mechanisms, via signaling pathways, including TGF, BMP, and vascular endothelial growth factor.

Engineering Anisotropic Biomimetic Fibrocartilage Microenvironment by Bioprinting Mesenchymal Stem Cells in Nanoliter Gel Droplets

PubMed Central

2015-01-01

Over the past decade, bioprinting has emerged as a promising patterning strategy to organize cells and extracellular components both in two and three dimensions (2D and 3D) to engineer functional tissue mimicking constructs. So far, tissue printing has neither been used for 3D patterning of mesenchymal stem cells (MSCs) in multiphase growth factor embedded 3D hydrogels nor been investigated phenotypically in terms of simultaneous differentiation into different cell types within the same micropatterned 3D tissue constructs. Accordingly, we demonstrated a biochemical gradient by bioprinting nanoliter droplets encapsulating human MSCs, bone morphogenetic protein 2 (BMP-2), and transforming growth factor β1 (TGF- β1), engineering an anisotropic biomimetic fibrocartilage microenvironment. Assessment of the model tissue construct displayed multiphasic anisotropy of the incorporated biochemical factors after patterning. Quantitative real time polymerase chain reaction (qRT-PCR) results suggested genomic expression patterns leading to simultaneous differentiation of MSC populations into osteogenic and chondrogenic phenotype within the multiphasic construct, evidenced by upregulation of osteogenesis and condrogenesis related genes during in vitro culture. Comprehensive phenotypic network and pathway analysis results, which were based on genomic expression data, indicated activation of differentiation related mechanisms, via signaling pathways, including TGF, BMP, and vascular endothelial growth factor. PMID:24495169

Prediction of water loss and viscoelastic deformation of apple tissue using a multiscale model.

PubMed

Aregawi, Wondwosen A; Abera, Metadel K; Fanta, Solomon W; Verboven, Pieter; Nicolai, Bart

2014-11-19

A two-dimensional multiscale water transport and mechanical model was developed to predict the water loss and deformation of apple tissue (Malus × domestica Borkh. cv. 'Jonagold') during dehydration. At the macroscopic level, a continuum approach was used to construct a coupled water transport and mechanical model. Water transport in the tissue was simulated using a phenomenological approach using Fick's second law of diffusion. Mechanical deformation due to shrinkage was based on a structural mechanics model consisting of two parts: Yeoh strain energy functions to account for non-linearity and Maxwell's rheological model of visco-elasticity. Apparent parameters of the macroscale model were computed from a microscale model. The latter accounted for water exchange between different microscopic structures of the tissue (intercellular space, the cell wall network and cytoplasm) using transport laws with the water potential as the driving force for water exchange between different compartments of tissue. The microscale deformation mechanics were computed using a model where the cells were represented as a closed thin walled structure. The predicted apparent water transport properties of apple cortex tissue from the microscale model showed good agreement with the experimentally measured values. Deviations between calculated and measured mechanical properties of apple tissue were observed at strains larger than 3%, and were attributed to differences in water transport behavior between the experimental compression tests and the simulated dehydration-deformation behavior. Tissue dehydration and deformation in the high relative humidity range ( > 97% RH) could, however, be accurately predicted by the multiscale model. The multiscale model helped to understand the dynamics of the dehydration process and the importance of the different microstructural compartments (intercellular space, cell wall, membrane and cytoplasm) for water transport and mechanical deformation.

Use of magnetic micro-cantilevers to study the dynamics of 3D engineered smooth muscle constructs

NASA Astrophysics Data System (ADS)

Liu, Alan; Zhao, Ruogang; Copeland, Craig; Chen, Christopher; Reich, Daniel

2013-03-01

The normal and pathological response of arterial tissue to mechanical stimulus sheds important light on such conditions as atherosclerosis and hypertension. While most previous methods of determining the biomechanical properties of arteries have relied on excised tissue, we have devised a system that enables the growth and in situ application of forces to arrays of stable suspended microtissues consisting of arterial smooth muscle cells (SMCs). Briefly, this magnetic microtissue tester system consists of arrays of pairs of elastomeric magnetically actuated micro-cantilevers between which SMC-infused 3D collagen gels self-assemble and remodel into aligned microtissue constructs. These devices allow us to simultaneously apply force and track stress-strain relationships of multiple microtissues per substrate. We have studied the dilatory capacity and subsequent response of the tissues and find that the resulting stress-strain curves show viscoelastic behavior as well as a linear dynamic recovery. These results provide a foundation for elucidating the mechanical behavior of this novel model system as well as further experiments that simulate pathological conditions. Supported in part by NIH grant HL090747.

Bioengineering vascularized tissue constructs using an injectable cell-laden enzymatically crosslinked collagen hydrogel derived from dermal extracellular matrix.

PubMed

Kuo, Kuan-Chih; Lin, Ruei-Zeng; Tien, Han-Wen; Wu, Pei-Yun; Li, Yen-Cheng; Melero-Martin, Juan M; Chen, Ying-Chieh

2015-11-01

Tissue engineering promises to restore or replace diseased or damaged tissue by creating functional and transplantable artificial tissues. The development of artificial tissues with large dimensions that exceed the diffusion limitation will require nutrients and oxygen to be delivered via perfusion instead of diffusion alone over a short time period. One approach to perfusion is to vascularize engineered tissues, creating a de novo three-dimensional (3D) microvascular network within the tissue construct. This significantly shortens the time of in vivo anastomosis, perfusion and graft integration with the host. In this study, we aimed to develop injectable allogeneic collagen-phenolic hydroxyl (collagen-Ph) hydrogels that are capable of controlling a wide range of physicochemical properties, including stiffness, water absorption and degradability. We tested whether collagen-Ph hydrogels could support the formation of vascularized engineered tissue graft by human blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSC) in vivo. First, we studied the growth of adherent ECFCs and MSCs on or in the hydrogels. To examine the potential formation of functional vascular networks in vivo, a liquid pre-polymer solution of collagen-Ph containing human ECFCs and MSCs, horseradish peroxidase and hydrogen peroxide was injected into the subcutaneous space or abdominal muscle defect of an immunodeficient mouse before gelation, to form a 3D cell-laden polymerized construct. These results showed that extensive human ECFC-lined vascular networks can be generated within 7 days, the engineered vascular density inside collagen-Ph hydrogel constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with the existing vasculature to further support the survival of host muscle tissues. Finally, optimized conditions of the cell-laden collagen-Ph hydrogel resulted in not only improving the long-term differentiation of transplanted MSCs into mineralized osteoblasts, but the collagen-Ph hydrogel also improved an increased of adipocytes within the vascularized bioengineered tissue in a mouse after 1 month of implantation. We reported a method for preparing autologous extracellular matrix scaffolds, murine collagen-Ph hydrogels, and demonstrated its suitability for use in supporting human progenitor cell-based formation of 3D vascular networks in vitro and in vivo. Results showed extensive human vascular networks can be generated within 7 days, engineered vascular density inside collagen-Ph constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with existing vasculature to further support the survival of host muscle tissues. Moreover, optimized conditions of cell-laden collagen-Ph hydrogel resulted in not only improving the long-term differentiation of transplanted MSCs into mineralized osteoblasts, but the collagen-Ph hydrogel also improved an increased of adipocytes within the vascularized bioengineered tissue in a mouse. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Beta-lactamase targeted enzyme activatable photosensitizers for antimicrobial PDT

NASA Astrophysics Data System (ADS)

Zheng, Xiang; Verma, Sarika; Sallum, Ulysses W.; Hasan, Tayyaba

2009-06-01

Photodynamic therapy (PDT) as a treatment modality for infectious disease has shown promise. However, most of the antimicrobial photosensitizers (PS) non-preferentially accumulate in both bacteria and host tissues, causing host tissue phototoxicity during treatment. We have developed a new antimicrobial PDT strategy which exploits beta-lactam resistance mechanism, one of the major drug-resistance bacteria evolved, to achieve enhanced target specificity with limited host damage. Our strategy comprises a prodrug construct with a PS and a quencher linked by beta-lactam ring, resulting in a diminished phototoxicity. This construct, beta-lactamase enzyme-activated-photosensitizer (beta-LEAP), can only be activated in the presence of both light and bacteria, and remains inactive elsewhere such as mammalian tissue. Beta-LEAP construct had shown specific cleavage by purified beta-lactamase and by beta-lactamase over-expressing methicillin resistant Staphylococcus aureus (MRSA). Specific photodynamic toxicity was observed towards MRSA, while dark and light toxicity were equivalent to reference strains. The prodrug design, synthesis and photophysical properties will be discussed.

Magnetically actuated tissue engineered scaffold: insights into mechanism of physical stimulation

NASA Astrophysics Data System (ADS)

Sapir-Lekhovitser, Yulia; Rotenberg, Menahem Y.; Jopp, Juergen; Friedman, Gary; Polyak, Boris; Cohen, Smadar

2016-02-01

Providing the right stimulatory conditions resulting in efficient tissue promoting microenvironment in vitro and in vivo is one of the ultimate goals in tissue development for regenerative medicine. It has been shown that in addition to molecular signals (e.g. growth factors) physical cues are also required for generation of functional cell constructs. These cues are particularly relevant to engineering of biological tissues, within which mechanical stress activates mechano-sensitive receptors, initiating biochemical pathways which lead to the production of functionally mature tissue. Uniform magnetic fields coupled with magnetizable nanoparticles embedded within three dimensional (3D) scaffold structures remotely create transient physical forces that can be transferrable to cells present in close proximity to the nanoparticles. This study investigated the hypothesis that magnetically responsive alginate scaffold can undergo reversible shape deformation due to alignment of scaffold's walls in a uniform magnetic field. Using custom made Helmholtz coil setup adapted to an Atomic Force Microscope we monitored changes in matrix dimensions in situ as a function of applied magnetic field, concentration of magnetic particles within the scaffold wall structure and rigidity of the matrix. Our results show that magnetically responsive scaffolds exposed to an externally applied time-varying uniform magnetic field undergo a reversible shape deformation. This indicates on possibility of generating bending/stretching forces that may exert a mechanical effect on cells due to alternating pattern of scaffold wall alignment and relaxation. We suggest that the matrix structure deformation is produced by immobilized magnetic nanoparticles within the matrix walls resulting in a collective alignment of scaffold walls upon magnetization. The estimated mechanical force that can be imparted on cells grown on the scaffold wall at experimental conditions is in the order of 1 pN, which correlates well with reported threshold to induce mechanotransduction effects on cellular level. This work is our next step in understanding of how to accurately create proper stimulatory microenvironment for promotion of cellular organization to form mature tissue engineered constructs.

Magnetically actuated tissue engineered scaffold: insights into mechanism of physical stimulation

PubMed Central

Sapir-Lekhovitser, Yulia; Rotenberg, Menahem Y.; Jopp, Juergen; Friedman, Gary; Polyak, Boris; Cohen, Smadar

2016-01-01

Providing the right stimulatory conditions resulting in efficient tissue promoting microenvironment in vitro and in vivo is one of the ultimate goals in tissue development for regenerative medicine. It has been shown that in addition to molecular signals (e.g. growth factors) physical cues are also required for generation of functional cell constructs. These cues are particularly relevant to engineering of biological tissues, within which mechanical stress activates mechano-sensitive receptors, initiating biochemical pathways which lead to the production of functionally mature tissue. Uniform magnetic fields coupled with magnetizable nanoparticles embedded within three dimensional (3D) scaffold structures remotely create transient physical forces that can be transferrable to cells present in close proximity to the nanoparticles. This study investigated the hypothesis that magnetically responsive alginate scaffold can undergo reversible shape deformation due to alignment of scaffold’s walls in a uniform magnetic field. Using custom made Helmholtz coil setup adapted to an Atomic Force Microscope we monitored changes in matrix dimensions in situ as a function of applied magnetic field, concentration of magnetic particles within the scaffold wall structure and rigidity of the matrix. Our results show that magnetically responsive scaffolds exposed to an externally applied time-varying uniform magnetic field undergo a reversible shape deformation. This indicates on possibility of generating bending/stretching forces that may exert a mechanical effect on cells due to alternating pattern of scaffold wall alignment and relaxation. We suggest that the matrix structure deformation is produced by immobilized magnetic nanoparticles within the matrix walls resulting in a collective alignment of scaffold walls upon magnetization. The estimated mechanical force that can be imparted on cells grown on the scaffold wall at experimental conditions is in the order of 1 pN, which correlates well with reported threshold to induce mechanotransduction effects on cellular level. This work is our next step in understanding of how to accurately create proper stimulatory microenvironment for promotion of cellular organization to form mature tissue engineered constructs. PMID:26790538

Comparative assessment of intrinsic mechanical stimuli on knee cartilage and compressed agarose constructs.

PubMed

Completo, A; Bandeiras, C; Fonseca, F

2017-06-01

A well-established cue for improving the properties of tissue-engineered cartilage is mechanical stimulation. However, the explicit ranges of mechanical stimuli that correspond to favorable metabolic outcomes are elusive. Usually, these outcomes have only been associated with the applied strain and frequency, an oversimplification that can hide the fundamental relationship between the intrinsic mechanical stimuli and the metabolic outcomes. This highlights two important key issues: the firstly is related to the evaluation of the intrinsic mechanical stimuli of native cartilage; the second, assuming that the intrinsic mechanical stimuli will be important, deals with the ability to replicate them on the tissue-engineered constructs. This study quantifies and compares the volume of cartilage and agarose subjected to a given magnitude range of each intrinsic mechanical stimulus, through a numerical simulation of a patient-specific knee model coupled with experimental data of contact during the stance phase of gait, and agarose constructs under direct-dynamic compression. The results suggest that direct compression loading needs to be parameterized with time-dependence during the initial culture period in order to better reproduce each one of the intrinsic mechanical stimuli developed in the patient-specific cartilage. A loading regime which combines time periods of low compressive strain (5%) and frequency (0.5Hz), in order to approach the maximal principal strain and fluid velocity stimulus of the patient-specific cartilage, with time periods of high compressive strain (20%) and frequency (3Hz), in order to approach the pore pressure values, may be advantageous relatively to a single loading regime throughout the full culture period. Copyright © 2017 IPEM. Published by Elsevier Ltd. All rights reserved.

Design and evaluation of a novel subatmospheric pressure bioreactor for the preconditioning of tissue-engineered vascular constructs.

PubMed

Coakley, Daniel N; Shaikh, Faisal M; O'Sullivan, Kathleen; Kavanagh, Eamon G; Grace, Pierce A; McGloughlin, Tim M

2016-02-01

The pre-conditioning of tissue-engineered vascular scaffolds with mechanical stimuli is being recognised as an essential step in producing a functional vascular construct. In this study we design and evaluate a novel bioreactor, which exerts a mechanical strain on developing vascular scaffolds via subatmospheric pressure. We design and construct a bioreactor, which exerts subatmospheric pressure via a vacuum assisted closure unit. Vascular scaffolds seeded with human umbilical endothelial cells were evaluated for structural integrity, microbial contamination, cellular viability, von Willebrand factor (VWF) production, cell proliferation and morphology under a range of subatmospheric pressures (75-200mmHg). The bioreactor produced sustained subatmospheric pressures, which exerted a mechanical strain on the vascular scaffold. No microbial contamination was found during the study. The structural integrity of the vascular construct was maintained. There was no difference in cellular viability between control or subatmospheric pressure groups (p = 0.817). Cells continued to produce VWF under a range of subatmospheric pressures. Cells subjected to subatmospheric pressures of 125mmHg and 200mmHg exhibited higher levels of growth than cells in atmospheric pressure at 24 (p≤0.016) and 48 hour (p≤0.001). Negative pressure affected cellular morphology, which were more organised, elongated and expanded when exposed to subatmospheric pressure. We have constructed and validated a novel subatmospheric bioreactor. The bioreactor maintained a continuous subatmospheric pressure to the vascular scaffolds in a stable, sterile and constant environment. The bioreactor exerted a strain on the vascular sheets, which was shown to alter cellular morphology and enhance cellular proliferation.

Textile Technologies and Tissue Engineering: A Path Towards Organ Weaving

PubMed Central

Akbari, Mohsen; Tamayol, Ali; Bagherifard, Sara; Serex, Ludovic; Mostafalu, Pooria; Faramarzi, Negar; Mohammadi, Mohammad Hossein

2016-01-01

Textile technologies have recently attracted great attention as potential biofabrication tools for engineering tissue constructs. Using current textile technologies, fibrous structures can be designed and engineered to attain the required properties that are demanded by different tissue engineering applications. Several key parameters such as physiochemical characteristics of fibers, pore size and mechanical properties of the fabrics play important role in the effective use of textile technologies in tissue engineering. This review summarizes the current advances in the manufacturing of biofunctional fibers. Different textile methods such as knitting, weaving, and braiding are discussed and their current applications in tissue engineering are highlighted. PMID:26924450

A computational modeling approach for the characterization of mechanical properties of 3D alginate tissue scaffolds.

PubMed

Nair, K; Yan, K C; Sun, W

2008-01-01

Scaffold guided tissue engineering is an innovative approach wherein cells are seeded onto biocompatible and biodegradable materials to form 3-dimensional (3D) constructs that, when implanted in the body facilitate the regeneration of tissue. Tissue scaffolds act as artificial extracellular matrix providing the environment conducive for tissue growth. Characterization of scaffold properties is necessary to understand better the underlying processes involved in controlling cell behavior and formation of functional tissue. We report a computational modeling approach to characterize mechanical properties of 3D gellike biomaterial, specifically, 3D alginate scaffold encapsulated with cells. Alginate inherent nonlinearity and variations arising from minute changes in its concentration and viscosity make experimental evaluation of its mechanical properties a challenging and time consuming task. We developed an in silico model to determine the stress-strain relationship of alginate based scaffolds from experimental data. In particular, we compared the Ogden hyperelastic model to other hyperelastic material models and determined that this model was the most suitable to characterize the nonlinear behavior of alginate. We further propose a mathematical model that represents the alginate material constants in Ogden model as a function of concentrations and viscosity. This study demonstrates the model capability to predict mechanical properties of 3D alginate scaffolds.

In vitro characterization of 3D printed scaffolds aimed at bone tissue regeneration.

PubMed

Boga, João C; Miguel, Sónia P; de Melo-Diogo, Duarte; Mendonça, António G; Louro, Ricardo O; Correia, Ilídio J

2018-05-01

The incidence of fractures and bone-related diseases like osteoporosis has been increasing due to aging of the world's population. Up to now, grafts and titanium implants have been the principal therapeutic approaches used for bone repair/regeneration. However, these types of treatment have several shortcomings, like limited availability, risk of donor-to-recipient infection and tissue morbidity. To overcome these handicaps, new 3D templates, capable of replicating the features of the native tissue, are currently being developed by researchers from the area of tissue engineering. These 3D constructs are able to provide a temporary matrix on which host cells can adhere, proliferate and differentiate. Herein, 3D cylindrical scaffolds were designed to mimic the natural architecture of hollow bones, and to allow nutrient exchange and bone neovascularization. 3D scaffolds were produced with tricalcium phosphate (TCP)/alginic acid (AA) using a Fab@home 3D printer. Furthermore, graphene oxide (GO) was incorporated into the structure of some scaffolds to further enhance their mechanical properties. The results revealed that the scaffolds incorporating GO displayed greater porosity, without impairing their mechanical properties. These scaffolds also presented a controlled swelling profile, enhanced biomineralization capacity and were able to increase the Alkaline Phosphatase (ALP) activity. Such characteristics make TCP/AA scaffolds functionalized with GO promising 3D constructs for bone tissue engineering applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Microfabrication of a platform to measure and manipulate the mechanics of engineered microtissues.

PubMed

Ramade, Alexandre; Legant, Wesley R; Picart, Catherine; Chen, Christopher S; Boudou, Thomas

2014-01-01

Engineered tissues can be used to understand fundamental features of biology, develop organotypic in vitro model systems, and as engineered tissue constructs for replacing damaged tissue in vivo. However, a key limitation is an inability to test the wide range of parameters that might impact the engineered tissue in a high-throughput manner and in an environment that mimics the three-dimensional (3D) native architecture. We developed a microfabricated platform to generate arrays of microtissues embedded within 3D micropatterned matrices. Microcantilevers simultaneously constrain microtissue formation and report forces generated by the microtissues in real time, opening the possibility to use high-throughput, low-volume screening for studies on engineered tissues. Thanks to the micrometer scale of the microtissues, this platform is also suitable for high-throughput monitoring of drug-induced effect on architecture and contractility in engineered tissues. Moreover, independent variations of the mechanical stiffness of the cantilevers and collagen matrix allow the measurement and manipulation of the mechanics of the microtissues. Thus, our approach will likely provide valuable opportunities to elucidate how biomechanical, electrical, biochemical, and genetic/epigenetic cues modulate the formation and maturation of 3D engineered tissues. In this chapter, we describe the microfabrication, preparation, and experimental use of such microfabricated tissue gauges. Copyright © 2014 Elsevier Inc. All rights reserved.

Preconditioning of skeletal myoblast-based engineered tissue constructs enables functional coupling to myocardium in vivo

PubMed Central

Treskes, Philipp; Neef, Klaus; Srinivasan, Sureshkumar Perumal; Halbach, Marcel; Stamm, Christof; Cowan, Douglas; Scherner, Maximilian; Madershahian, Navid; Wittwer, Thorsten; Hescheler, Jürgen; Wahlers, Thorsten; Choi, Yeong-Hoon

2015-01-01

Objective Skeletal myoblasts fuse to form functional syncytial myotubes as an integral part of the skeletal muscle. During this differentiation process, expression of proteins for mechanical and electrical integration is seized, which is a major drawback for the application of skeletal myoblasts in cardiac regenerative cell therapy, because global heart function depends on intercellular communication. Methods Mechanically preconditioned engineered tissue constructs containing neonatal mouse skeletal myoblasts were transplanted epicardially. A Y-chromosomal specific polymerase chain reaction (PCR) was undertaken up to 10 weeks after transplantation to confirm the presence of grafted cells. Histologic and electrophysiologic analyses were carried out 1 week after transplantation. Results Cells within the grafted construct expressed connexin 43 at the interface to the host myocardium, indicating electrical coupling, confirmed by sharp electrode recordings. Analyses of the maximum stimulation frequency (5.65 ± 0.37 Hz), conduction velocity (0.087 ± 0.011 m/s) and sensitivity for pharmacologic conduction block (0.736 ± 0.080 mM 1-heptanol) revealed effective electrophysiologic coupling between graft and host cells, although significantly less robust than in native myocardial tissue (maximum stimulation frequency, 11.616 ± 0.238 Hz, P<.001; conduction velocity, 0.300 ± 0.057 m/s, P<.01; conduction block, 1.983 ± 0.077 mM 1-heptanol, P<.001). Conclusions Although untreated skeletal myoblasts cannot couple to cardiomyocytes, we confirm that mechanical preconditioning enables transplanted skeletal myoblasts to functionally interact with cardio-myocytes in vivo and, thus, reinvigorate the concept of skeletal myoblast-based cardiac cell therapy. PMID:25439779

Tibial Plateau Fractures in Elderly Patients

PubMed Central

Vemulapalli, Krishna C.; Gary, Joshua L.; Donegan, Derek J.

2016-01-01

Tibial plateau fractures are common in the elderly population following a low-energy mechanism. Initial evaluation includes an assessment of the soft tissues and surrounding ligaments. Most fractures involve articular depression leading to joint incongruity. Treatment of these fractures may be complicated by osteoporosis, osteoarthritis, and medical comorbidities. Optimal reconstruction should restore the mechanical axis, provide a stable construct for mobilization, and reestablish articular congruity. This is accomplished through a variety of internal or external fixation techniques or with acute arthroplasty. Regardless of the treatment modality, particular focus on preservation and maintenance of the soft tissue envelope is paramount. PMID:27551570

A biomimetic three-dimensional woven composite scaffold for functional tissue engineering of cartilage

NASA Astrophysics Data System (ADS)

Moutos, Franklin T.; Freed, Lisa E.; Guilak, Farshid

2007-02-01

Tissue engineering seeks to repair or regenerate tissues through combinations of implanted cells, biomaterial scaffolds and biologically active molecules. The rapid restoration of tissue biomechanical function remains an important challenge, emphasizing the need to replicate structural and mechanical properties using novel scaffold designs. Here we present a microscale 3D weaving technique to generate anisotropic 3D woven structures as the basis for novel composite scaffolds that are consolidated with a chondrocyte-hydrogel mixture into cartilage tissue constructs. Composite scaffolds show mechanical properties of the same order of magnitude as values for native articular cartilage, as measured by compressive, tensile and shear testing. Moreover, our findings showed that porous composite scaffolds could be engineered with initial properties that reproduce the anisotropy, viscoelasticity and tension-compression nonlinearity of native articular cartilage. Such scaffolds uniquely combine the potential for load-bearing immediately after implantation in vivo with biological support for cell-based tissue regeneration without requiring cultivation in vitro.

Contractile recovery of microtissues after giant shear events

NASA Astrophysics Data System (ADS)

Morley, Cameron; Bhattacharjee, Tapomoy; Ellison, Sarah; Sawyer, W.; Angelini, Thomas

Cells are often dispersed in extracellular matrix (ECM) gels like collagen and Matrigel as minimal tissue models. Generally, large-scale contraction of these constructs is observed, in which the degree of contraction of the entire system correlates with cell density and ECM concentration. The freedom to perform diverse mechanical experiments on these contracting constructs is limited by the challenges of handling and supporting these delicate samples. Here, we present a method to create simple cell-ECM constructs that can be manipulated with significantly reduced experimental limitations. We 3D print mixtures of MCF10A cells and ECM (collagen-I and Matrigel) into a 3D growth medium made from jammed microgels. With this approach, we are able to apply shear stresses to the cell constructs times after printing and observe the collective response. Preliminary results reveal that, following shear deformations that exceed 300% and dramatically smear cells and matrix in space, the cells actively re-contract the construct toward the un-sheared construct. These results suggest that new principles of collective recovery can be employed for tissue engineering applications using jammed microgels as a re-configurable support medium.

Engineering tissue constructs to mimic native aortic and pulmonary valve leaflets' structures and mechanics

NASA Astrophysics Data System (ADS)

Masoumi, Nafiseh

There are several disadvantages correlated with current heart valve replacement, including anticoagulation therapy for patients with mechanical valves and the low durability of bioprosthetic valves. The non-viable nature of such devices is a critical drawback especially for pediatric cases due to the inability of the graft to grow in vivo with the patients. A tissue engineered heart valve (TEHV) with remodeling and growth ability, is conceptually appealing to use in the surgical repair and could serve as a permanent replacements when operating for pediatric valvular lesions. It is critical that scaffolds for functional heart valve tissue engineering, be capable of mimicking the native leaflet's structure and mechanical properties at the time of implantation. Meanwhile, the scaffolds should be able to support cellular proliferation and native-like tissue formation as the TEHV remodels toward a scaffold-free state. Our overall hypothesis is that an "ideal" engineered construct, designed based on native leaflet's structure and mechanics, will complement a native heart valve leaflet in providing benchmarks for use in the design of clinically-applicable TEHV. This hypothesis was addressed through several experiments conducted in the present study. To establish a functional biomimetic TEHV, we developed scaffolds capable of matching the anisotropic stiffness of native leaflet while promoting native-like cell and collagen content and supporting the ECM generation. Scaffolds with various polymer contents (e.g., poly (glycerol sebacate) (PGS) and poly (epsilon-caprolactone) (PCL)) and structural designs (e.g., microfabricated and microfibrous scaffolds), were fabricated based on native leaflet's structure and mechanics. It was found that the tri-layered scaffold, designed with assembly of microfabricated PGS and microfibrous PGS/PCL was a functional leaflet capable of promoting tissue formation. Furthermore, to investigate the effect of cyclic stress and flexure individually on the TEHV development, we designed a simple and novel stretch-flexure bioreactor in which samples were subjected to well-defined stimulations with a controlled strain-rate. The stretch and flexure was found to accelerate and increase tissue formation on the microfabricated PGS scaffolds cultivated in the bioreactors.

Construction of biocompatible porous tissue scaffold from the decellularized umbilical artery.

PubMed

Xin, Yi; Wu, Guanghui; Wu, Man; Zhang, Xiaoxia; Velot, Emilie; Decot, Véronique; Cui, Wei; Huang, Yimin; Stoltz, Jean-Francois; Du, Jie; Li, Na

2015-01-01

The scaffolds prepared from the tissue decellularization conserve the porous 3-D structure and provide an optimal matrix for the tissue regeneration. Since decade, the enzymatic digestion, chemical reagent treatment and mechanical actions such as eversion and abrasion have been used to remove the cells from the intact matrix. In this study, we optimized an enzymatic method to decellularize the umbilical artery to construct a 3-D porous scaffold which is suitable for the culture of mesenchymal stem cells (MSCs). The scaffold maintained the interconnected porous structure. It remained the similar high water content 95.3 ± 1% compared to 94.9 ± 0.6% in the intact umbilical artery (p>0.05). The decellularization process decreased the stress from 0.24 ± 0.05 mPa to 0.15 ± 0.06 mPa (p<0.05). However the decellularization did not change the strain of the artery (45 ± 15% vs. 53 ± 10%, p>0.05). When the scaffold was transplanted to the subcutaneous tissue in the wild type mice, there were less T cells appeared in the surrounding tissue which meant the decreased the immunogenicity by decellularization. This scaffold also supported the adhesion and proliferation of the MSCs. In this study, we constructed a biological compatible porous scaffold from the decellularized umbilical artery which may provide a suitable scaffold for cell-matrix interaction studies and for tissue engineering.

A Tissue Engineered Model of Aging: Interdependence and Cooperative Effects in Failing Tissues.

PubMed

Acun, A; Vural, D C; Zorlutuna, P

2017-07-11

Aging remains a fundamental open problem in modern biology. Although there exist a number of theories on aging on the cellular scale, nearly nothing is known about how microscopic failures cascade to macroscopic failures of tissues, organs and ultimately the organism. The goal of this work is to bridge microscopic cell failure to macroscopic manifestations of aging. We use tissue engineered constructs to control the cellular-level damage and cell-cell distance in individual tissues to establish the role of complex interdependence and interactions between cells in aging tissues. We found that while microscopic mechanisms drive aging, the interdependency between cells plays a major role in tissue death, providing evidence on how cellular aging is connected to its higher systemic consequences.

Direct 3D bioprinting of perfusable vascular constructs using a blend bioink.

PubMed

Jia, Weitao; Gungor-Ozkerim, P Selcan; Zhang, Yu Shrike; Yue, Kan; Zhu, Kai; Liu, Wanjun; Pi, Qingment; Byambaa, Batzaya; Dokmeci, Mehmet Remzi; Shin, Su Ryon; Khademhosseini, Ali

2016-11-01

Despite the significant technological advancement in tissue engineering, challenges still exist towards the development of complex and fully functional tissue constructs that mimic their natural counterparts. To address these challenges, bioprinting has emerged as an enabling technology to create highly organized three-dimensional (3D) vascular networks within engineered tissue constructs to promote the transport of oxygen, nutrients, and waste products, which can hardly be realized using conventional microfabrication techniques. Here, we report the development of a versatile 3D bioprinting strategy that employs biomimetic biomaterials and an advanced extrusion system to deposit perfusable vascular structures with highly ordered arrangements in a single-step process. In particular, a specially designed cell-responsive bioink consisting of gelatin methacryloyl (GelMA), sodium alginate, and 4-arm poly(ethylene glycol)-tetra-acrylate (PEGTA) was used in combination with a multilayered coaxial extrusion system to achieve direct 3D bioprinting. This blend bioink could be first ionically crosslinked by calcium ions followed by covalent photocrosslinking of GelMA and PEGTA to form stable constructs. The rheological properties of the bioink and the mechanical strengths of the resulting constructs were tuned by the introduction of PEGTA, which facilitated the precise deposition of complex multilayered 3D perfusable hollow tubes. This blend bioink also displayed favorable biological characteristics that supported the spreading and proliferation of encapsulated endothelial and stem cells in the bioprinted constructs, leading to the formation of biologically relevant, highly organized, perfusable vessels. These characteristics make this novel 3D bioprinting technique superior to conventional microfabrication or sacrificial templating approaches for fabrication of the perfusable vasculature. We envision that our advanced bioprinting technology and bioink formulation may also have significant potentials in engineering large-scale vascularized tissue constructs towards applications in organ transplantation and repair. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hydroxypropyl cellulose methacrylate as a photo-patternable and biodegradable hybrid paper substrate for cell culture and other bioapplications.

PubMed

Qi, Aisha; Hoo, Siew Pei; Friend, James; Yeo, Leslie; Yue, Zhilian; Chan, Peggy P Y

2014-04-01

In addition to the choice of appropriate material properties of the tissue construct to be used, such as its biocompatibility, biodegradability, cytocompatibility, and mechanical rigidity, the ability to incorporate microarchitectural patterns in the construct to mimic that found in the cellular microenvironment is an important consideration in tissue engineering and regenerative medicine. Both these issues are addressed by demonstrating a method for preparing biodegradable and photo-patternable constructs, where modified cellulose is cross-linked to form an insoluble structure in an aqueous environment. Specifically, hydroxypropyl cellulose (HPC) is rendered photocrosslinkable by grafting with methylacrylic anhydride, whose linkages also render the cross-linked construct hydrolytically degradable. The HPC is then cross-linked via a photolithography-based fabrication process. The feasibility of functionalizing these HPC structures with biochemical cues is verified post-fabrication, and shown to facilitate the adhesion of mesenchymal progenitor cells. The HPC constructs are shown to be biocompatible and hydrolytically degradable, thus enabling cell proliferation and cell migration, and therefore constituting an ideal candidate for long-term cell culture and implantable tissue scaffold applications. In addition, the potential of the HPC structure is demonstrated as an alternative substrate to paper microfluidic diagnostic devices for protein and cell assays. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Carbon nanotube-incorporated collagen hydrogels improve cell alignment and the performance of cardiac constructs

PubMed Central

Sun, Hongyu; Zhou, Jing; Huang, Zhu; Qu, Linlin; Lin, Ning; Liang, Chengxiao; Dai, Ruiwu; Tang, Lijun; Tian, Fuzhou

2017-01-01

Carbon nanotubes (CNTs) provide an essential 2-D microenvironment for cardiomyocyte growth and function. However, it remains to be elucidated whether CNT nanostructures can promote cell–cell integrity and facilitate the formation of functional tissues in 3-D hydrogels. Here, single-walled CNTs were incorporated into collagen hydrogels to fabricate (CNT/Col) hydrogels, which improved mechanical and electrical properties. The incorporation of CNTs (up to 1 wt%) exhibited no toxicity to cardiomyocytes and enhanced cell adhesion and elongation. Through the use of immunohistochemical staining, transmission electron microscopy, and intracellular calcium-transient measurement, the incorporation of CNTs was found to improve cell alignment and assembly remarkably, which led to the formation of engineered cardiac tissues with stronger contraction potential. Importantly, cardiac tissues based on CNT/Col hydrogels were noted to have better functionality. Collectively, the incorporation of CNTs into the Col hydrogels improved cell alignment and the performance of cardiac constructs. Our study suggests that CNT/Col hydrogels offer a promising tissue scaffold for cardiac constructs, and might serve as injectable biomaterials to deliver cell or drug molecules for cardiac regeneration following myocardial infarction in the near future. PMID:28450785

Generating favorable growth factor and protease release profiles to enable extracellular matrix accumulation within an in vitro tissue engineering environment.

PubMed

Zhang, Xiaoqing; Battiston, Kyle G; Labow, Rosalind S; Simmons, Craig A; Santerre, J Paul

2017-05-01

Tissue engineering (particularly for the case of load-bearing cardiovascular and connective tissues) requires the ability to promote the production and accumulation of extracellular matrix (ECM) components (e.g., collagen, glycosaminoglycan and elastin). Although different approaches have been attempted in order to enhance ECM accumulation in tissue engineered constructs, studies of underlying signalling mechanisms that influence ECM deposition and degradation during tissue remodelling and regeneration in multi-cellular culture systems have been limited. The current study investigated vascular smooth muscle cell (VSMC)-monocyte co-culture systems using different VSMC:monocyte ratios, within a degradable polyurethane scaffold, to assess their influence on ECM generation and degradation processes, and to elucidate relevant signalling molecules involved in this in vitro vascular tissue engineering system. It was found that a desired release profile of growth factors (e.g. insulin growth factor-1 (IGF-1)) and hydrolytic proteases (e.g. matrix-metalloproteinases 2, 9, 13 and 14 (MMP2, MMP9, MMP13 and MMP14)), could be achieved in co-culture systems, yielding an accumulation of ECM (specifically for 2:1 and 4:1 VSMC:monocyte culture systems). This study has significant implications for the tissue engineering field (including vascular tissue engineering), not only because it identified important cytokines and proteases that control ECM accumulation/degradation within synthetic tissue engineering scaffolds, but also because the established culture systems could be applied to improve the development of different types of tissue constructs. Sufficient extracellular matrix accumulation within cardiovascular and connective tissue engineered constructs is a prerequisite for their appropriate function in vivo. This study established co-culture systems with tissue specific cells (vascular smooth muscle cells (VSMCs)) and defined ratios of immune cells (monocytes) to investigate extracellular matrix (ECM) generation and degradation processes, revealing important mechanisms underlying ECM turnover during vascular tissue regeneration/remodelling. A specific growth factor (IGF-1), as well as hydrolytic proteases (e.g. MMP2, MMP9, MMP13 and MMP14), were identified as playing important roles in these processes. ECM accumulation was found to be dependent on achieving a desired release profile of these ECM-promoting and ECM-degrading factors within the multi-cellular microenvironment. The findings enhance our understanding of ECM deposition and degradation during in vitro tissue engineering and would be applicable to the repair or regeneration of a variety of tissues. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Frozen tissue preparation for high-resolution multiplex histological analyses of human brain specimens.

PubMed

Shao, Fangjie; Jiang, Wenhong; Gao, Qingqing; Li, Baizhou; Sun, Chongran; Wang, Qiyuan; Chen, Qin; Sun, Bing; Shen, Hong; Zhu, Keqing; Zhang, Jianmin; Liu, Chong

2017-10-01

The availability of a comprehensive tissue library is essential for elucidating the function and pathology of human brains. Considering the irreplaceable status of the formalin-fixation-paraffin-embedding (FFPE) preparation in routine pathology and the advantage of ultra-low temperature to preserve nucleic acids and proteins for multi-omics studies, these methods have become major modalities for the construction of brain tissue libraries. Nevertheless, the use of FFPE and snap-frozen samples is limited in high-resolution histological analyses because the preparation destroys tissue integrity and/or many important cellular markers. To overcome these limitations, we detailed a protocol to prepare and analyze frozen human brain samples that is particularly suitable for high-resolution multiplex immunohistological studies. As an alternative, we offered an optimized procedure to rescue snap-frozen tissues for the same purpose. Importantly, we provided a guideline to construct libraries of frozen tissue with minimal effort, cost and space. Taking advantage of this new tissue preparation modality to nicely preserve the cellular information that was otherwise damaged using conventional methods and to effectively remove tissue autofluorescence, we described the high-resolution landscape of the cellular composition in both lower-grade gliomas and glioblastoma multiforme samples. Our work showcases the great value of fixed frozen tissue in understanding the cellular mechanisms of CNS functions and abnormalities.

It's all in the timing: modeling isovolumic contraction through development and disease with a dynamic dual electromechanical bioreactor system.

PubMed

Morgan, Kathy Ye; Black, Lauren Deems

2014-01-01

This commentary discusses the rationale behind our recently reported work entitled "Mimicking isovolumic contraction with combined electromechanical stimulation improves the development of engineered cardiac constructs," introduces new data supporting our hypothesis, and discusses future applications of our bioreactor system. The ability to stimulate engineered cardiac tissue in a bioreactor system that combines both electrical and mechanical stimulation offers a unique opportunity to simulate the appropriate dynamics between stretch and contraction and model isovolumic contraction in vitro. Our previous study demonstrated that combined electromechanical stimulation that simulated the timing of isovolumic contraction in healthy tissue improved force generation via increased contractile and calcium handling protein expression and improved hypertrophic pathway activation. In new data presented here, we further demonstrate that modification of the timing between electrical and mechanical stimulation to mimic a non-physiological process negatively impacts the functionality of the engineered constructs. We close by exploring the various disease states that have altered timing between the electrical and mechanical stimulation signals as potential future directions for the use of this system.

Passaged adult chondrocytes can form engineered cartilage with functional mechanical properties: a canine model.

PubMed

Ng, Kenneth W; Lima, Eric G; Bian, Liming; O'Conor, Christopher J; Jayabalan, Prakash S; Stoker, Aaron M; Kuroki, Keiichi; Cook, Cristi R; Ateshian, Gerard A; Cook, James L; Hung, Clark T

2010-03-01

It was hypothesized that previously optimized serum-free culture conditions for juvenile bovine chondrocytes could be adapted to generate engineered cartilage with physiologic mechanical properties in a preclinical, adult canine model. Primary or passaged (using growth factors) adult chondrocytes from three adult dogs were encapsulated in agarose, and cultured in serum-free media with transforming growth factor-beta3. After 28 days in culture, engineered cartilage formed by primary chondrocytes exhibited only small increases in glycosaminoglycan content. However, all passaged chondrocytes on day 28 elaborated a cartilage matrix with compressive properties and glycosaminoglycan content in the range of native adult canine cartilage values. A preliminary biocompatibility study utilizing chondral and osteochondral constructs showed no gross or histological signs of rejection, with all implanted constructs showing excellent integration with surrounding cartilage and subchondral bone. This study demonstrates that adult canine chondrocytes can form a mechanically functional, biocompatible engineered cartilage tissue under optimized culture conditions. The encouraging findings of this work highlight the potential for tissue engineering strategies using adult chondrocytes in the clinical treatment of cartilage defects.

Monitoring Cartilage Tissue Engineering Using Magnetic Resonance Spectroscopy, Imaging, and Elastography

PubMed Central

Klatt, Dieter; Magin, Richard L.

2013-01-01

A key technical challenge in cartilage tissue engineering is the development of a noninvasive method for monitoring the composition, structure, and function of the tissue at different growth stages. Due to its noninvasive, three-dimensional imaging capabilities and the breadth of available contrast mechanisms, magnetic resonance imaging (MRI) techniques can be expected to play a leading role in assessing engineered cartilage. In this review, we describe the new MR-based tools (spectroscopy, imaging, and elastography) that can provide quantitative biomarkers for cartilage tissue development both in vitro and in vivo. Magnetic resonance spectroscopy can identify the changing molecular structure and alternations in the conformation of major macromolecules (collagen and proteoglycans) using parameters such as chemical shift, relaxation rates, and magnetic spin couplings. MRI provides high-resolution images whose contrast reflects developing tissue microstructure and porosity through changes in local relaxation times and the apparent diffusion coefficient. Magnetic resonance elastography uses low-frequency mechanical vibrations in conjunction with MRI to measure soft tissue mechanical properties (shear modulus and viscosity). When combined, these three techniques provide a noninvasive, multiscale window for characterizing cartilage tissue growth at all stages of tissue development, from the initial cell seeding of scaffolds to the development of the extracellular matrix during construct incubation, and finally, to the postimplantation assessment of tissue integration in animals and patients. PMID:23574498

Rationally designed synthetic protein hydrogels with predictable mechanical properties.

PubMed

Wu, Junhua; Li, Pengfei; Dong, Chenling; Jiang, Heting; Bin Xue; Gao, Xiang; Qin, Meng; Wang, Wei; Bin Chen; Cao, Yi

2018-02-12

Designing synthetic protein hydrogels with tailored mechanical properties similar to naturally occurring tissues is an eternal pursuit in tissue engineering and stem cell and cancer research. However, it remains challenging to correlate the mechanical properties of protein hydrogels with the nanomechanics of individual building blocks. Here we use single-molecule force spectroscopy, protein engineering and theoretical modeling to prove that the mechanical properties of protein hydrogels are predictable based on the mechanical hierarchy of the cross-linkers and the load-bearing modules at the molecular level. These findings provide a framework for rationally designing protein hydrogels with independently tunable elasticity, extensibility, toughness and self-healing. Using this principle, we demonstrate the engineering of self-healable muscle-mimicking hydrogels that can significantly dissipate energy through protein unfolding. We expect that this principle can be generalized for the construction of protein hydrogels with customized mechanical properties for biomedical applications.

Fluid-Structure Analysis of Opening Phenomena in a Collapsible Airway

NASA Astrophysics Data System (ADS)

Ghadiali, Samir N.; Banks, Julie; Swarts, J. Douglas

2003-11-01

Several physiological functions require the opening of collapsed respiratory airways. For example, the Eustachian tube (ET), which connects the nasopharynx with the middle ear (ME), must be periodically opened to maintain ambient ME pressures. These openings normally occur during swallowing when muscle contraction deforms the surrounding soft tissue. The inability to open the ET results in the most common and costly ear disease in children, Otitis Media. Although tissue-based treatments have been purposed, the influence of the various tissue mechanical properties on flow phenomena has not been investigated. A computational model of ET opening was developed using in-vivo structural data to investigate these fluid-structure interactions. This model accounts for both tissue deformation and the resulting airflow in a non-circular conduit. Results indicate that ET opening is more sensitive to the applied muscle forces than elastic tissue properties. These models have therefore identified how different tissue elements alter ET opening phenomena, which elements should be targeted for treatment and the optimal mechanical properties of these tissue constructs. Research supported by NIH grant DC005345.

Bioengineering pediatric scaffold-free auricular cartilaginous constructs.

PubMed

Akbari, Pedram; Waldman, Stephen D; Cushing, Sharon L; Papsin, Blake C; Propst, Evan J; Weber, Joanna F; Yeger, Herman; Farhat, Walid A

2017-05-01

The use of exogenous materials as scaffolds in cartilage tissue engineering has limited the clinical application of resultant constructs due to the risk of postoperative complications. In an effort to minimize such complications, we aim to generate human, scaffold-free auricular cartilaginous constructs. Laboratory study using pediatric auricular cartilage. Remnant, normal pediatric auricular cartilage samples that would have otherwise been discarded were collected and digested to free cells. Harvested cells were cultured and expanded in vitro for two passages and plated as micromass cultures. The culture medium was replaced with a chemically defined chondrogenic medium, and cellular monolayers surrounding micromass cultures were continuously scraped off. Constructs were allowed to mature for a period of 8 weeks. Micromass constructs showed mechanical stability and structurally resembled native auricular tissue, with a perichondrium-like layer of cells surrounding the inner cartilaginous zone. Constructs accumulated equivalent sulphated glycosaminoglycan and 50% of collagen content compared to native auricular cartilage by mass, while displaying 156% more cellularity. High-density micromass cultures of pediatric auricular chondrocytes can generate stable cartilaginous constructs following prolonged chondrogenic inductions in vitro. This technique is an essential step toward the development of three-dimensional constructs to recreate clinically applicable auricular cartilaginous constructs. NA. Laryngoscope, 127:E153-E158, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Textile Technologies and Tissue Engineering: A Path Toward Organ Weaving.

PubMed

Akbari, Mohsen; Tamayol, Ali; Bagherifard, Sara; Serex, Ludovic; Mostafalu, Pooria; Faramarzi, Negar; Mohammadi, Mohammad Hossein; Khademhosseini, Ali

2016-04-06

Textile technologies have recently attracted great attention as potential biofabrication tools for engineering tissue constructs. Using current textile technologies, fibrous structures can be designed and engineered to attain the required properties that are demanded by different tissue engineering applications. Several key parameters such as physiochemical characteristics of fibers, microarchitecture, and mechanical properties of the fabrics play important roles in the effective use of textile technologies in tissue engineering. This review summarizes the current advances in the manufacturing of biofunctional fibers. Different textile methods such as knitting, weaving, and braiding are discussed and their current applications in tissue engineering are highlighted. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluating 3D-printed biomaterials as scaffolds for vascularized bone tissue engineering.

PubMed

Wang, Martha O; Vorwald, Charlotte E; Dreher, Maureen L; Mott, Eric J; Cheng, Ming-Huei; Cinar, Ali; Mehdizadeh, Hamidreza; Somo, Sami; Dean, David; Brey, Eric M; Fisher, John P

2015-01-07

There is an unmet need for a consistent set of tools for the evaluation of 3D-printed constructs. A toolbox developed to design, characterize, and evaluate 3D-printed poly(propylene fumarate) scaffolds is proposed for vascularized engineered tissues. This toolbox combines modular design and non-destructive fabricated design evaluation, evaluates biocompatibility and mechanical properties, and models angiogenesis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and Translation of a Tissue-Engineered Disc in a Preclinical Rodent Model

DTIC Science & Technology

2014-12-01

annulus fibrosus tissue into full 3D Disc-like Angle Ply Structures (DAPS), inclusive of a hyaluronic acid hydrogel seeded with adult stem cells, that...AF constructs surrounding an engineered nucleus pulposus (NP) composed of a hyaluronic acid (HA) hydrogel. Measure the disc structural mechanics in...exposure to TGF-ß3 improves the functional properties of MSC-seeded photocrosslinked hyaluronic acid hydrogels by authors Minwook Kim, Isaac E

A review of evolution of electrospun tissue engineering scaffold: From two dimensions to three dimensions.

PubMed

Ngadiman, Nor Hasrul Akhmal; Noordin, M Y; Idris, Ani; Kurniawan, Denni

2017-07-01

The potential of electrospinning process to fabricate ultrafine fibers as building blocks for tissue engineering scaffolds is well recognized. The scaffold construct produced by electrospinning process depends on the quality of the fibers. In electrospinning, material selection and parameter setting are among many factors that contribute to the quality of the ultrafine fibers, which eventually determine the performance of the tissue engineering scaffolds. The major challenge of conventional electrospun scaffolds is the nature of electrospinning process which can only produce two-dimensional electrospun mats, hence limiting their applications. Researchers have started to focus on overcoming this limitation by combining electrospinning with other techniques to fabricate three-dimensional scaffold constructs. This article reviews various polymeric materials and their composites/blends that have been successfully electrospun for tissue engineering scaffolds, their mechanical properties, and the various parameters settings that influence the fiber morphology. This review also highlights the secondary processes to electrospinning that have been used to develop three-dimensional tissue engineering scaffolds as well as the steps undertaken to overcome electrospinning limitations.

Deformation micromechanisms of collagen fibrils under uniaxial tension

PubMed Central

Tang, Yuye; Ballarini, Roberto; Buehler, Markus J.; Eppell, Steven J.

2010-01-01

Collagen, an essential building block of connective tissues, possesses useful mechanical properties due to its hierarchical structure. However, little is known about the mechanical properties of collagen fibril, an intermediate structure between the collagen molecule and connective tissue. Here, we report the results of systematic molecular dynamics simulations to probe the mechanical response of initially unflawed finite size collagen fibrils subjected to uniaxial tension. The observed deformation mechanisms, associated with rupture and sliding of tropocollagen molecules, are strongly influenced by fibril length, width and cross-linking density. Fibrils containing more than approximately 10 molecules along their length and across their width behave as representative volume elements and exhibit brittle fracture. Shorter fibrils experience a more graceful ductile-like failure. An analytical model is constructed and the results of the molecular modelling are used to find curve-fitted expressions for yield stress, yield strain and fracture strain as functions of fibril structural parameters. Our results for the first time elucidate the size dependence of mechanical failure properties of collagen fibrils. The associated molecular deformation mechanisms allow the full power of traditional material and structural engineering theory to be applied to our understanding of the normal and pathological mechanical behaviours of collagenous tissues under load. PMID:19897533

Preparation and features of polycaprolactone vascular grafts with the incorporated vascular endothelial growth factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sevostyanova, V. V., E-mail: sevostyanova.victoria@gmail.com; Khodyrevskaya, Y. I.; Glushkova, T. V.

The development of tissue-engineered small-diameter vascular grafts is an urgent issue in cardiovascular surgery. In this study, we assessed how the incorporation of the vascular endothelial growth factor (VEGF) affects morphological and mechanical properties of polycaprolactone (PCL) vascular grafts along with its release kinetics. Vascular grafts were prepared using two-phase electrospinning. In pursuing our aims, we performed scanning electron microscopy, mechanical testing, and enzyme-linked immunosorbent assay. Our results demonstrated the preservation of a highly porous structure and improvement of PCL/VEGF scaffold mechanical properties as compared to PCL grafts. A prolonged VEGF release testifies the use of this construct as amore » scaffold for tissue-engineered vascular grafts.« less

Mechanical properties of ceramic structures based on Triply Periodic Minimal Surface (TPMS) processed by 3D printing

NASA Astrophysics Data System (ADS)

Restrepo, S.; Ocampo, S.; Ramírez, J. A.; Paucar, C.; García, C.

2017-12-01

Repairing tissues and organs has been the main goal of surgical procedures. Since the 1990s, the main goal of tissue engineering has been reparation, using porous scaffolds that serve as a three-dimensional template for the initial fixation of cells and subsequent tissue formation both in vitro and in vivo. A scaffold must have specific characteristics of porosity, interconnectivity, surface area, pore volume, surface tortuosity, permeability and mechanical properties, which makes its design, manufacturing and characterization a complex process. Inspired by nature, triply periodic minimal surfaces (TPMS) have emerged as an alternative for the manufacture of porous pieces with design requirements, such as scaffolds for tissue repair. In the present work, we used the technique of 3D printing to obtain ceramic structures with Gyroid, Schwarz Primitive and Schwarz Diamond Surfaces shapes, three TPMS that fulfil the geometric requirements of a bone tissue scaffold. The main objective of this work is to compare the mechanical properties of ceramic pieces of three different forms of TPMS printed in 3D using a commercial ceramic paste. In this way it will be possible to clarify which is the TPMS with appropriate characteristics to construct scaffolds of ceramic materials for bone repair. A dependence of the mechanical properties with the geometry was found being the Primitive Surface which shows the highest mechanical properties.

Tissue architecture, cell traction, deformable scaffolds, and the forces that shape the embryo during morphogenesis.

NASA Astrophysics Data System (ADS)

Davidson, Lance

2005-03-01

Morphogenesis is the process of constucting form and shape. Morphogenesis during early development of the embryo involves orchestrated movements of cells and tissues. These morphogenetic movements establish the body plan and organs of the early embryo. The rates and trajectories of these movements depend on three physical features of the early embryo: 1) the forces generated by cells, 2) the mechanical properties of the tissues, and 3) the architecture of the tissues. These three mechanical features of the embryo are some of the earliest phenotypic features generated by the genome. We are taking an interdisciplinary approach combining biophysical, cell biological, and classical embryological techniques to understand the mechanics of morphogenesis. Using nanoNewton-sensitive force transducers we can apply forces and measure time dependent elastic modulii of tissue fragments 100 micrometers across. Using traction-force microscopy we can measure forces generated by cells on their environment. We use drugs and chimeric proteins to investigate the localization and function of molecular complexes responsible for force generation and the modulus. We use microsurgery to take-apart and construct novel tissues to investigate the role of geometry and architecture in the mechanics of morphogenesis. Together with simulation techniques these quantitative approaches will provide us with a practical nuts-and-bolts understanding of how the genome encodes the shapes and forms of life.

Use of hydrodynamic forces to engineer cartilaginous tissues resembling the non-uniform structure and function of meniscus.

PubMed

Marsano, Anna; Wendt, David; Raiteri, Roberto; Gottardi, Riccardo; Stolz, Martin; Wirz, Dieter; Daniels, Alma U; Salter, Donald; Jakob, Marcel; Quinn, Thomas M; Martin, Ivan

2006-12-01

The aim of this study was to demonstrate that differences in the local composition of bi-zonal fibrocartilaginous tissues result in different local biomechanical properties in compression and tension. Bovine articular chondrocytes were loaded into hyaluronan-based meshes (HYAFF-11) and cultured for 4 weeks in mixed flask, a rotary Cell Culture System (RCCS), or statically. Resulting tissues were assessed histologically, immunohistochemically, by scanning electron microscopy and mechanically in different regions. Local mechanical analyses in compression and tension were performed by indentation-type scanning force microscopy and by tensile tests on punched out concentric rings, respectively. Tissues cultured in mixed flask or RCCS displayed an outer region positively stained for versican and type I collagen, and an inner region positively stained for glycosaminoglycans and types I and II collagen. The outer fibrocartilaginous capsule included bundles (up to 2 microm diameter) of collagen fibers and was stiffer in tension (up to 3.6-fold higher elastic modulus), whereas the inner region was stiffer in compression (up to 3.8-fold higher elastic modulus). Instead, molecule distribution and mechanical properties were similar in the outer and inner regions of statically grown tissues. In conclusion, exposure of articular chondrocyte-based constructs to hydrodynamic flow generated tissues with locally different composition and mechanical properties, resembling some aspects of the complex structure and function of the outer and inner zones of native meniscus.

Fluid mechanics of spinner-flask bioreactors

NASA Astrophysics Data System (ADS)

Sucosky, Philippe; Neitzel, G. Paul

2000-11-01

The dynamic environment within bioreactors used for in vitro tissue growth has been observed to affect the development of mammalian cells. Many studies have shown that moderate mechanical stress enhances growth of some tissues whereas high shear levels and turbulence seem to damage cells. In order to optimize the design and the operating conditions of bioreactors, it is important to understand the fluid-dynamic characteristics and to control the stress levels within these devices. The present research focuses on the characterization of the flow field within a spinner-flask bioreactor. The dynamic properties of the flow are investigated experimentally using particle-image velocimetry with a refractive-index-matched model. Phase-locked ensemble-averaging is employed to provide some information on the turbulence characteristics of the model culture medium in the vicinity of a model tissue construct.

Engineered skeletal muscle tissue for soft robotics: fabrication strategies, current applications, and future challenges.

PubMed

Duffy, Rebecca M; Feinberg, Adam W

2014-01-01

Skeletal muscle is a scalable actuator system used throughout nature from the millimeter to meter length scales and over a wide range of frequencies and force regimes. This adaptability has spurred interest in using engineered skeletal muscle to power soft robotics devices and in biotechnology and medical applications. However, the challenges to doing this are similar to those facing the tissue engineering and regenerative medicine fields; specifically, how do we translate our understanding of myogenesis in vivo to the engineering of muscle constructs in vitro to achieve functional integration with devices. To do this researchers are developing a number of ways to engineer the cellular microenvironment to guide skeletal muscle tissue formation. This includes understanding the role of substrate stiffness and the mechanical environment, engineering the spatial organization of biochemical and physical cues to guide muscle alignment, and developing bioreactors for mechanical and electrical conditioning. Examples of engineered skeletal muscle that can potentially be used in soft robotics include 2D cantilever-based skeletal muscle actuators and 3D skeletal muscle tissues engineered using scaffolds or directed self-organization. Integration into devices has led to basic muscle-powered devices such as grippers and pumps as well as more sophisticated muscle-powered soft robots that walk and swim. Looking forward, current, and future challenges include identifying the best source of muscle precursor cells to expand and differentiate into myotubes, replacing cardiomyocytes with skeletal muscle tissue as the bio-actuator of choice for soft robots, and vascularization and innervation to enable control and nourishment of larger muscle tissue constructs. © 2013 Wiley Periodicals, Inc.

A bioreactor test system to mimic the biological and mechanical environment of oral soft tissues and to evaluate substitutes for connective tissue grafts.

PubMed

Mathes, Stephanie H; Wohlwend, Lorenz; Uebersax, Lorenz; von Mentlen, Roger; Thoma, Daniel S; Jung, Ronald E; Görlach, Christoph; Graf-Hausner, Ursula

2010-12-15

Gingival cells of the oral connective tissue are exposed to complex mechanical forces during mastication, speech, tooth movement and orthodontic treatments. Especially during wound healing following surgical procedures, internal and external forces may occur, creating pressure upon the newly formed tissue. This clinical situation has to be considered when developing biomaterials to augment soft tissue in the oral cavity. In order to pre-evaluate a collagen sponge intended to serve as a substitute for autogenous connective tissue grafts (CTGs), a dynamic bioreactor system was developed. Pressure and shear forces can be applied in this bioreactor in addition to a constant medium perfusion to cell-material constructs. Three-dimensional volume changes and stiffness of the matrices were analyzed. In addition, cell responses such as cell vitality and extracellular matrix (ECM) production were investigated. The number of metabolic active cells constantly increased under fully dynamic culture conditions. The sponges remained elastic even after mechanical forces were applied for 14 days. Analysis of collagen type I and fibronectin revealed a statistically significant accumulation of these ECM molecules (P < 0.05-0.001) when compared to static cultures. An increased expression of tenascin-c, indicating tissue remodeling processes, was observed under dynamic conditions only. The results indicate that the tested in vitro cell culture system was able to mimic both the biological and mechanical environments of the clinical situation in a healing wound. © 2010 Wiley Periodicals, Inc.

Towards an ideal polymer scaffold for tendon/ligament tissue engineering

NASA Astrophysics Data System (ADS)

Sahoo, Sambit; Ouyang, Hong Wei; Goh, James Cho-Hong; Tay, Tong-Earn; Toh, Siew Lok

2005-04-01

Tissue engineering holds promise in treating injured tendons and ligaments by replacing the injured tissues with "engineered tissues" with identical mechanical and functional characteristics. A biocompatible, biodegradable, porous scaffold with optimized architecture, sufficient surface area for cell attachment, growth and proliferation, faborable mechanical properties, and suitable degradation rate is a pre-requisite to achieve success with this aproach. Knitted poly(lactide-co-glycolide) (PLGA) scaffolds comprising of microfibers of 25 micron diameter were coated with PLGA nanofibers on their surfaces by electrospinning technique. A cell suspension of pig bone marrow stromal cells (BMSC) was seeded on the scaffolds by pipetting, and the cell-scaffold constructs were cultured in a CO2 incubator, at 37°C for 1-2 weeks. The "engineered tissues" were then assessed for cell attachment and proliferation, tissue formation, and mechanical properties. Nanofibers, of diameter 300-900 nm, were spread randomly over the knitted scaffold. The reduction in pore-size from about 1 mm (in the knitted scaffold) to a few micrometers (in the nano-microscaffold) allowed cell seeding by direct pipetting, and eliminated the need of a cell-delivery system like fibrin gel. BMSCs were seen to attach and proliferate well on the nano-microscaffold, producing abundant extracellular matrix. Mechanical testing revealed that the cell-seeded nano-microscaffolds possessed slightly higher values of failure load, elastic-region stiffness and toe-region stiffness, than the unseeded scaffolds. The combination of superior mechanical strength and integrity of knitted microfibers, with the large surface area and improved hydrophilicity of the electrospun nanofibers facilitated cell attachment and new tissue formation. This holds promise in tissue engineering of tendon/ligament.

Allogeneic versus autologous derived cell sources for use in engineered bone-ligament-bone grafts in sheep anterior cruciate ligament repair.

PubMed

Mahalingam, Vasudevan D; Behbahani-Nejad, Nilofar; Horine, Storm V; Olsen, Tyler J; Smietana, Michael J; Wojtys, Edward M; Wellik, Deneen M; Arruda, Ellen M; Larkin, Lisa M

2015-03-01

The use of autografts versus allografts for anterior cruciate ligament (ACL) reconstruction is controversial. The current popular options for ACL reconstruction are patellar tendon or hamstring autografts, yet advances in allograft technologies have made allogeneic grafts a favorable option for repair tissue. Despite this, the mismatched biomechanical properties and risk of osteoarthritis resulting from the current graft technologies have prompted the investigation of new tissue sources for ACL reconstruction. Previous work by our lab has demonstrated that tissue-engineered bone-ligament-bone (BLB) constructs generated from an allogeneic cell source develop structural and functional properties similar to those of native ACL and vascular and neural structures that exceed those of autologous patellar tendon grafts. In this study, we investigated the effectiveness of our tissue-engineered ligament constructs fabricated from autologous versus allogeneic cell sources. Our preliminary results demonstrate that 6 months postimplantation, our tissue-engineered auto- and allogeneic BLB grafts show similar histological and mechanical outcomes indicating that the autologous grafts are a viable option for ACL reconstruction. These data indicate that our tissue-engineered autologous ligament graft could be used in clinical situations where immune rejection and disease transmission may preclude allograft use.

Porous titanium bases for osteochondral tissue engineering

PubMed Central

Nover, Adam B.; Lee, Stephanie L.; Georgescu, Maria S.; Howard, Daniel R.; Saunders, Reuben A.; Yu, William T.; Klein, Robert W.; Napolitano, Anthony P.; Ateshian, Gerard A.

2015-01-01

Tissue engineering of osteochondral grafts may offer a cell-based alternative to native allografts, which are in short supply. Previous studies promote the fabrication of grafts consisting of a viable cell-seeded hydrogel integrated atop a porous, bone-like metal. Advantages of the manufacturing process have led to the evaluation of porous titanium as the bone-like base material. Here, porous titanium was shown to support the growth of cartilage to produce native levels of Young’s modulus, using a clinically relevant cell source. Mechanical and biochemical properties were similar or higher for the osteochondral constructs compared to chondral-only controls. Further investigation into the mechanical influence of the base on the composite material suggests that underlying pores may decrease interstitial fluid pressurization and applied strains, which may be overcome by alterations to the base structure. Future studies aim to optimize titanium-based tissue engineered osteochondral constructs to best match the structural architecture and strength of native grafts. Statement of Significance The studies described in this manuscript follow up on previous studies from our lab pertaining to the fabrication of osteochondral grafts that consist of a bone-like porous metal and a chondrocyte-seeded hydrogel. Here, tissue engineered osteochondral grafts were cultured to native stiffness using adult chondrocytes, a clinically relevant cell source, and a porous titanium base, a material currently used in clinical implants. This porous titanium is manufactured via selective laser melting, offering the advantages of precise control over shape, pore size, and orientation. Additionally, this manuscript describes the mechanical influence of the porous base, which may have applicability to porous bases derived from other materials. PMID:26320541

Porous titanium bases for osteochondral tissue engineering.

PubMed

Nover, Adam B; Lee, Stephanie L; Georgescu, Maria S; Howard, Daniel R; Saunders, Reuben A; Yu, William T; Klein, Robert W; Napolitano, Anthony P; Ateshian, Gerard A; Hung, Clark T

2015-11-01

Tissue engineering of osteochondral grafts may offer a cell-based alternative to native allografts, which are in short supply. Previous studies promote the fabrication of grafts consisting of a viable cell-seeded hydrogel integrated atop a porous, bone-like metal. Advantages of the manufacturing process have led to the evaluation of porous titanium as the bone-like base material. Here, porous titanium was shown to support the growth of cartilage to produce native levels of Young's modulus, using a clinically relevant cell source. Mechanical and biochemical properties were similar or higher for the osteochondral constructs compared to chondral-only controls. Further investigation into the mechanical influence of the base on the composite material suggests that underlying pores may decrease interstitial fluid pressurization and applied strains, which may be overcome by alterations to the base structure. Future studies aim to optimize titanium-based tissue engineered osteochondral constructs to best match the structural architecture and strength of native grafts. The studies described in this manuscript follow up on previous studies from our lab pertaining to the fabrication of osteochondral grafts that consist of a bone-like porous metal and a chondrocyte-seeded hydrogel. Here, tissue engineered osteochondral grafts were cultured to native stiffness using adult chondrocytes, a clinically relevant cell source, and a porous titanium base, a material currently used in clinical implants. This porous titanium is manufactured via selective laser melting, offering the advantages of precise control over shape, pore size, and orientation. Additionally, this manuscript describes the mechanical influence of the porous base, which may have applicability to porous bases derived from other materials. Copyright © 2015. Published by Elsevier Ltd.

A comparison of the functionality and in vivo phenotypic stability of cartilaginous tissues engineered from different stem cell sources.

PubMed

Vinardell, Tatiana; Sheehy, Eamon J; Buckley, Conor T; Kelly, Daniel J

2012-06-01

Joint-derived stem cells are a promising alternative cell source for cartilage repair therapies that may overcome many of the problems associated with the use of primary chondrocytes (CCs). The objective of this study was to compare the in vitro functionality and in vivo phenotypic stability of cartilaginous tissues engineered using bone marrow-derived stem cells (BMSCs) and joint tissue-derived stem cells following encapsulation in agarose hydrogels. Culture-expanded BMSCs, fat pad-derived stem cells (FPSCs), and synovial membrane-derived stem cells (SDSCs) were encapsulated in agarose and maintained in a chondrogenic medium supplemented with transforming growth factor-β3. After 21 days of culture, constructs were either implanted subcutaneously into the back of nude mice for an additional 28 days or maintained for a similar period in vitro in either chondrogenic or hypertrophic media formulations. After 49 days of in vitro culture in chondrogenic media, SDSC constructs accumulated the highest levels of sulfated glycosaminoglycan (sGAG) (∼2.8% w/w) and collagen (∼1.8% w/w) and were mechanically stiffer than constructs engineered using other cell types. After subcutaneous implantation in nude mice, sGAG content significantly decreased for all stem cell-seeded constructs, while no significant change was observed in the control constructs engineered using primary CCs, indicating that the in vitro chondrocyte-like phenotype generated in all stem cell-seeded agarose constructs was transient. FPSCs and SDSCs appeared to undergo fibrous dedifferentiation or resorption, as evident from increased collagen type I staining and a dramatic loss in sGAG content. BMSCs followed a more endochondral pathway with increased type X collagen expression and mineralization of the engineered tissue. In conclusion, while joint tissue-derived stem cells possess a strong intrinsic chondrogenic capacity, further studies are needed to identify the factors that will lead to the generation of a more stable chondrogenic phenotype.

In situ monitoring of localized shear stress and fluid flow within developing tissue constructs by Doppler optical coherence tomography

NASA Astrophysics Data System (ADS)

Jia, Yali; Bagnaninchi, Pierre O.; Wang, Ruikang K.

2008-02-01

Mechanical stimuli can be introduced to three dimensional (3D) cell cultures by use of perfusion bioreactor. Especially in musculoskeletal tissues, shear stress caused by fluid flow generally increase extra-cellular matrix (ECM) production and cell proliferation. The relationship between the shear stress and the tissue development in situ is complicated because of the non-uniform pore distribution within the cell-seeded scaffold. In this study, we firstly demonstrated that Doppler optical coherence tomography (DOCT) is capable of monitoring localized fluid flow and shear stress in the complex porous scaffold by examining their variation trends at perfusion rate of 5, 8, 10 and 12 ml/hr. Then, we developed the 3D porous cellular constructs, cell-seeded chitosan scaffolds monitored during several days by DOCT. The fiber based fourier domain DOCT employed a 1300 nm superluminescent diode with a bandwidth of 52 nm and a xyz resolution of 20×20×15 μm in free space. This setup allowed us not only to assess the cell growth and ECM deposition by observing their different scattering behaviors but also to further investigate how the cell attachment and ECM production has the effect on the flow shear stress and the relationship between flow rate and shear stress in the developing tissue construct. The possibility to monitor continuously the constructs under perfusion will easily indicate the effect of flow rate or shear stress on the cell viability and cell proliferation, and then discriminate the perfusion parameters affecting the pre-tissue formation rate growth.

Evaluation of articular cartilage repair using biodegradable nanofibrous scaffolds in a swine model: a pilot study.

PubMed

Li, Wan-Ju; Chiang, Hongsen; Kuo, Tzong-Fu; Lee, Hsuan-Shu; Jiang, Ching-Chuan; Tuan, Rocky S

2009-01-01

The aim of this study was to evaluate a cell-seeded nanofibrous scaffold for cartilage repair in vivo. We used a biodegradable poly(epsilon-caprolactone) (PCL) nanofibrous scaffold seeded with allogeneic chondrocytes or xenogeneic human mesenchymal stem cells (MSCs), or acellular PCL scaffolds, with no implant as a control to repair iatrogenic, 7 mm full-thickness cartilage defects in a swine model. Six months after implantation, MSC-seeded constructs showed the most complete repair in the defects compared to other groups. Macroscopically, the MSC-seeded constructs regenerated hyaline cartilage-like tissue and restored a smooth cartilage surface, while the chondrocyte-seeded constructs produced mostly fibrocartilage-like tissue with a discontinuous superficial cartilage contour. Incomplete repair containing fibrocartilage or fibrous tissue was found in the acellular constructs and the no-implant control group. Quantitative histological evaluation showed overall higher scores for the chondrocyte- and MSC-seeded constructs than the acellular construct and the no-implant groups. Mechanical testing showed the highest equilibrium compressive stress of 1.5 MPa in the regenerated cartilage produced by the MSC-seeded constructs, compared to 1.2 MPa in the chondrocyte-seeded constructs, 1.0 MPa in the acellular constructs and 0.2 MPa in the no-implant group. No evidence of immune reaction to the allogeneically- and xenogeneically-derived regenerated cartilage was observed, possibly related to the immunosuppressive activities of MSCs, suggesting the feasibility of allogeneic or xenogeneic transplantation of MSCs for cell-based therapy. Taken together, our results showed that biodegradable nanofibrous scaffolds seeded with MSCs effectively repair cartilage defects in vivo, and that the current approach is promising for cartilage repair. 2008 John Wiley & Sons, Ltd

Evaluation of articular cartilage repair using biodegradable nanofibrous scaffolds in a swine model: a pilot study

PubMed Central

Li, Wan-Ju; Chiang, Hongsen; Kuo, Tzong-Fu; Lee, Hsuan-Shu; Jiang, Ching-Chuan; Tuan, Rocky S.

2013-01-01

The aim of this study was to evaluate a cell-seeded nanofibrous scaffold for cartilage repair in vivo. We used a biodegradable poly(ε-caprolactone) (PCL) nanofibrous scaffold seeded with allogeneic chondrocytes or xenogeneic human mesenchymal stem cells (MSCs), or acellular PCL scaffolds, with no implant as a control to repair iatrogenic, 7 mm full-thickness cartilage defects in a swine model. Six months after implantation, MSC-seeded constructs showed the most complete repair in the defects compared to other groups. Macroscopically, the MSC-seeded constructs regenerated hyaline cartilage-like tissue and restored a smooth cartilage surface, while the chondrocyte-seeded constructs produced mostly fibrocartilage-like tissue with a discontinuous superficial cartilage contour. Incomplete repair containing fibrocartilage or fibrous tissue was found in the acellular constructs and the no-implant control group. Quantitative histological evaluation showed overall higher scores for the chondrocyte- and MSC-seeded constructs than the acellular construct and the no-implant groups. Mechanical testing showed the highest equilibrium compressive stress of 1.5 MPa in the regenerated cartilage produced by the MSC-seeded constructs, compared to 1.2 MPa in the chondrocyte-seeded constructs, 1.0 MPa in the acellular constructs and 0.2 MPa in the no-implant group. No evidence of immune reaction to the allogeneically- and xenogeneically-derived regenerated cartilage was observed, possibly related to the immunosuppressive activities of MSCs, suggesting the feasibility of allogeneic or xenogeneic transplantation of MSCs for cell-based therapy. Taken together, our results showed that biodegradable nanofibrous scaffolds seeded with MSCs effectively repair cartilage defects in vivo, and that the current approach is promising for cartilage repair. PMID:19004029

Biomimetic 3D tissue printing for soft tissue regeneration.

PubMed

Pati, Falguni; Ha, Dong-Heon; Jang, Jinah; Han, Hyun Ho; Rhie, Jong-Won; Cho, Dong-Woo

2015-09-01

Engineered adipose tissue constructs that are capable of reconstructing soft tissue with adequate volume would be worthwhile in plastic and reconstructive surgery. Tissue printing offers the possibility of fabricating anatomically relevant tissue constructs by delivering suitable matrix materials and living cells. Here, we devise a biomimetic approach for printing adipose tissue constructs employing decellularized adipose tissue (DAT) matrix bioink encapsulating human adipose tissue-derived mesenchymal stem cells (hASCs). We designed and printed precisely-defined and flexible dome-shaped structures with engineered porosity using DAT bioink that facilitated high cell viability over 2 weeks and induced expression of standard adipogenic genes without any supplemented adipogenic factors. The printed DAT constructs expressed adipogenic genes more intensely than did non-printed DAT gel. To evaluate the efficacy of our printed tissue constructs for adipose tissue regeneration, we implanted them subcutaneously in mice. The constructs did not induce chronic inflammation or cytotoxicity postimplantation, but supported positive tissue infiltration, constructive tissue remodeling, and adipose tissue formation. This study demonstrates that direct printing of spatially on-demand customized tissue analogs is a promising approach to soft tissue regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Critical seeding density improves properties and translatability of self-assembling anatomically shaped knee menisci

PubMed Central

Hadidi, Pasha; Yeh, Timothy C.; Hu, Jerry C.; Athanasiou, Kyriacos A.

2014-01-01

A recent development in the field of tissue engineering is the rise of all-biologic, scaffold-free engineered tissues. Since these biomaterials rely primarily upon cells, investigation of initial seeding densities constitutes a particularly relevant aim for tissue engineers. In this study, a scaffold-free method was used to create fibrocartilage in the shape of the rabbit knee meniscus. The objectives of this study were: (i) to determine the minimum seeding density, normalized by an area of 44 mm2, necessary for the self-assembling process of fibrocartilage to occur, (ii) examine relevant biomechanical properties of engineered fibrocartilage, such as tensile and compressive stiffness and strength, and their relationship to seeding density, and (iii) identify a reduced, or optimal, number of cells needed to produce this biomaterial. It was found that a decreased initial seeding density, normalized by the area of the construct, produced superior mechanical and biochemical properties. Collagen per wet weight, glycosaminoglycans per wet weight, tensile properties, and compressive properties were all significantly greater in the 5 million cells per construct group as compared to the historical 20 million cells per construct group. Scanning electron microscopy demonstrated that a lower seeding density results in a denser tissue. Additionally, the translational potential of the self-assembling process for tissue engineering was improved though this investigation, as fewer cells may be used in the future. The results of this study underscore the potential for critical seeding densities to be investigated when researching scaffold-free engineered tissues. PMID:25234157

Cell–scaffold interaction within engineered tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Haiping; Liu, Yuanyuan, E-mail: Yuanyuan_liu@shu.edu.cn; Jiang, Zhenglong

The structure of a tissue engineering scaffold plays an important role in modulating tissue growth. A novel gelatin–chitosan (Gel–Cs) scaffold with a unique structure produced by three-dimensional printing (3DP) technology combining with vacuum freeze-drying has been developed for tissue-engineering applications. The scaffold composed of overall construction, micro-pore, surface morphology, and effective mechanical property. Such a structure meets the essential design criteria of an ideal engineered scaffold. The favorable cell–matrix interaction supports the active biocompatibility of the structure. The structure is capable of supporting cell attachment and proliferation. Cells seeded into this structure tend to maintain phenotypic shape and secreted largemore » amounts of extracellular matrix (ECM) and the cell growth decreased the mechanical properties of scaffold. This novel biodegradable scaffold has potential applications for tissue engineering based upon its unique structure, which acts to support cell growth. - Highlights: • The scaffold is not only for providing a surface for cell residence but also for determining cell phenotype and retaining structural integrity. • The mechanical property of scaffold can be affected by activities of cell. • The scaffold provides a microenvironment for cell attachment, growth, and migration.« less

Continuum theory of fibrous tissue damage mechanics using bond kinetics: application to cartilage tissue engineering.

PubMed

Nims, Robert J; Durney, Krista M; Cigan, Alexander D; Dusséaux, Antoine; Hung, Clark T; Ateshian, Gerard A

2016-02-06

This study presents a damage mechanics framework that employs observable state variables to describe damage in isotropic or anisotropic fibrous tissues. In this mixture theory framework, damage is tracked by the mass fraction of bonds that have broken. Anisotropic damage is subsumed in the assumption that multiple bond species may coexist in a material, each having its own damage behaviour. This approach recovers the classical damage mechanics formulation for isotropic materials, but does not appeal to a tensorial damage measure for anisotropic materials. In contrast with the classical approach, the use of observable state variables for damage allows direct comparison of model predictions to experimental damage measures, such as biochemical assays or Raman spectroscopy. Investigations of damage in discrete fibre distributions demonstrate that the resilience to damage increases with the number of fibre bundles; idealizing fibrous tissues using continuous fibre distribution models precludes the modelling of damage. This damage framework was used to test and validate the hypothesis that growth of cartilage constructs can lead to damage of the synthesized collagen matrix due to excessive swelling caused by synthesized glycosaminoglycans. Therefore, alternative strategies must be implemented in tissue engineering studies to prevent collagen damage during the growth process.

Continuum theory of fibrous tissue damage mechanics using bond kinetics: application to cartilage tissue engineering

PubMed Central

Nims, Robert J.; Durney, Krista M.; Cigan, Alexander D.; Hung, Clark T.; Ateshian, Gerard A.

2016-01-01

This study presents a damage mechanics framework that employs observable state variables to describe damage in isotropic or anisotropic fibrous tissues. In this mixture theory framework, damage is tracked by the mass fraction of bonds that have broken. Anisotropic damage is subsumed in the assumption that multiple bond species may coexist in a material, each having its own damage behaviour. This approach recovers the classical damage mechanics formulation for isotropic materials, but does not appeal to a tensorial damage measure for anisotropic materials. In contrast with the classical approach, the use of observable state variables for damage allows direct comparison of model predictions to experimental damage measures, such as biochemical assays or Raman spectroscopy. Investigations of damage in discrete fibre distributions demonstrate that the resilience to damage increases with the number of fibre bundles; idealizing fibrous tissues using continuous fibre distribution models precludes the modelling of damage. This damage framework was used to test and validate the hypothesis that growth of cartilage constructs can lead to damage of the synthesized collagen matrix due to excessive swelling caused by synthesized glycosaminoglycans. Therefore, alternative strategies must be implemented in tissue engineering studies to prevent collagen damage during the growth process. PMID:26855751

Pulsatile perfusion bioreactor for cardiac tissue engineering.

PubMed

Brown, Melissa A; Iyer, Rohin K; Radisic, Milica

2008-01-01

Cardiovascular disease is the number one cause of mortality in North America. Cardiac tissue engineering aims to engineer a contractile patch of physiological thickness to use in surgical repair of diseased heart tissue. We previously reported that perfusion of engineered cardiac constructs resulted in improved tissue assembly. Because heart tissues respond to mechanical stimuli in vitro and experience rhythmic mechanical forces during contraction in vivo, we hypothesized that provision of pulsatile interstitial medium flow to an engineered cardiac patch would result in enhanced tissue assembly by way of mechanical conditioning and improved mass transport. Thus, we constructed a novel perfusion bioreactor capable of providing pulsatile fluid flow at physiologically relevant shear stresses and flow rates. Pulsatile perfusion (PP) was achieved by incorporation of a normally closed solenoid pinch valve into the perfusion loop and was carried out at a frequency of 1 Hz and a flow rate of 1.50 mL/min (PP) or 0.32 mL/min (PP-LF). Nonpulsatile flow at 1.50 mL/min (NP) or 0.32 mL/min (NP-LF) served as controls. Static controls were cultivated in well plates. The main experimental groups were seeded with cells enriched for cardiomyocytes by one preplating step (64% cardiac Troponin I+, 34% prolyl-4-hydroxylase+), whereas pure cardiac fibroblasts and cells enriched for cardiomyocytes by two preplating steps (81% cardiac Troponin I+, 16% prolyl-4-hydroxylase+) served as controls. Cultivation under pulsatile flow had beneficial effects on contractile properties. Specifically, the excitation threshold was significantly lower in the PP condition (pulsatile perfusion at 1.50 mL/min) than in the Static control, and the contraction amplitude was the highest; whereas high maximum capture rate was observed for the PP-LF conditions (pulsatile perfusion at 0.32 mL/min). The enhanced hypertrophy index observed for the PP-LF group was consistent with the highest cellular length and diameter in this group. Within the same cultivation groups (Static, NP-LF, PP-LF, PP, and NP) there were no significant differences in the diameter between fibroblasts and cardiomyocytes, although cardiomyocytes were significantly more elongated than fibroblasts under PP-LF conditions. Cultivation of control cell populations resulted in noncontractile constructs when cardiac fibroblasts were used (as expected) and no overall improvement in functional properties when two steps of preplating were used to enrich for cardiomyocytes in comparison with only one step of preplating.

Effect of Substrate Mechanics on Cardiomyocyte Maturation and Growth

PubMed Central

Tallawi, Marwa; Rai, Ranjana; Boccaccini, Aldo. R.

2015-01-01

Cardiac tissue engineering constructs are a promising therapeutic treatment for myocardial infarction, which is one of the leading causes of death. In order to further advance the development and regeneration of engineered cardiac tissues using biomaterial platforms, it is important to have a complete overview of the effects that substrates have on cardiomyocyte (CM) morphology and function. This article summarizes recent studies that investigate the effect of mechanical cues on the CM differentiation, maturation, and growth. In these studies, CMs derived from embryos, neonates, and mesenchymal stem cells were seeded on different substrates of various elastic modulus. Measuring the contractile function by force production, work output, and calcium handling, it was seen that cell behavior on substrates was optimized when the substrate stiffness mimicked that of the native tissue. The contractile function reflected changes in the sarcomeric protein confirmation and organization that promoted the contractile ability. The analysis of the literature also revealed that, in addition to matrix stiffness, mechanical stimulation, such as stretching the substrate during cell seeding, also played an important role during cell maturation and tissue development. PMID:25148904

Bioengineering vascularized tissue constructs using an injectable cell-laden enzymatically crosslinked collagen hydrogel derived from dermal extracellular matrix

PubMed Central

Kuo, Kuan-Chih; Lin, Ruei-Zeng; Tien, Han-Wen; Wu, Pei-Yun; Li, Yen-Cheng; Melero-Martin, Juan M.; Chen, Ying-Chieh

2015-01-01

Tissue engineering promises to restore or replace diseased or damaged tissue by creating functional and transplantable artificial tissues. The development of artificial tissues with large dimensions that exceed the diffusion limitation will require nutrients and oxygen to be delivered via perfusion instead of diffusion alone over a short time period. One approach to perfusion is to vascularize engineered tissues, creating a de novo three-dimensional (3D) microvascular network within the tissue construct. This significantly shortens the time of in vivo anastomosis, perfusion and graft integration with the host. In this study, we aimed to develop injectable allogeneic collagen-phenolic hydroxyl (collagen-Ph) hydrogels that are capable of controlling a wide range of physicochemical properties, including stiffness, water absorption and degradability. We tested whether collagen-Ph hydrogels could support the formation of vascularized engineered tissue graft by human blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSC) in vivo. First, we studied the growth of adherent ECFCs and MSCs on or in the hydrogels. To examine the potential formation of functional vascular networks in vivo, a liquid pre-polymer solution of collagen-Ph containing human ECFCs and MSCs, horseradish peroxidase and hydrogen peroxide was injected into the subcutaneous space or abdominal muscle defect of an immunodeficient mouse before gelation, to form a 3D cell-laden polymerized construct. These results showed that extensive human ECFC-lined vascular networks can be generated within 7 days, the engineered vascular density inside collagen-Ph hydrogel constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with the existing vasculature to further support the survival of host muscle tissues. Finally, optimized conditions of the cell-laden collagen-Ph hydrogel resulted in not only improving the long-term differentiation of transplanted MSCs into mineralized osteoblasts, but the collagen-Ph hydrogel also improved an increased of adipocytes within the vascularized bioengineered tissue in a mouse after 1 month of implantation. PMID:26348142

Evaluation of the Growth Environment of a Hydrostatic Force Bioreactor for Preconditioning of Tissue-Engineered Constructs

PubMed Central

Reinwald, Yvonne; Leonard, Katherine H.L.; Henstock, James R.; Whiteley, Jonathan P.; Osborne, James M.; Waters, Sarah L.; Levesque, Philippe

2015-01-01

Bioreactors have been widely acknowledged as valuable tools to provide a growth environment for engineering tissues and to investigate the effect of physical forces on cells and cell-scaffold constructs. However, evaluation of the bioreactor environment during culture is critical to defining outcomes. In this study, the performance of a hydrostatic force bioreactor was examined by experimental measurements of changes in dissolved oxygen (O2), carbon dioxide (CO2), and pH after mechanical stimulation and the determination of physical forces (pressure and stress) in the bioreactor through mathematical modeling and numerical simulation. To determine the effect of hydrostatic pressure on bone formation, chick femur skeletal cell-seeded hydrogels were subjected to cyclic hydrostatic pressure at 0–270 kPa and 1 Hz for 1 h daily (5 days per week) over a period of 14 days. At the start of mechanical stimulation, dissolved O2 and CO2 in the medium increased and the pH of the medium decreased, but remained within human physiological ranges. Changes in physiological parameters (O2, CO2, and pH) were reversible when medium samples were placed in a standard cell culture incubator. In addition, computational modeling showed that the distribution and magnitude of physical forces depends on the shape and position of the cell-hydrogel constructs in the tissue culture format. Finally, hydrostatic pressure was seen to enhance mineralization of chick femur skeletal cell-seeded hydrogels. PMID:24967717

Evaluation of the growth environment of a hydrostatic force bioreactor for preconditioning of tissue-engineered constructs.

PubMed

Reinwald, Yvonne; Leonard, Katherine H L; Henstock, James R; Whiteley, Jonathan P; Osborne, James M; Waters, Sarah L; Levesque, Philippe; El Haj, Alicia J

2015-01-01

Bioreactors have been widely acknowledged as valuable tools to provide a growth environment for engineering tissues and to investigate the effect of physical forces on cells and cell-scaffold constructs. However, evaluation of the bioreactor environment during culture is critical to defining outcomes. In this study, the performance of a hydrostatic force bioreactor was examined by experimental measurements of changes in dissolved oxygen (O2), carbon dioxide (CO2), and pH after mechanical stimulation and the determination of physical forces (pressure and stress) in the bioreactor through mathematical modeling and numerical simulation. To determine the effect of hydrostatic pressure on bone formation, chick femur skeletal cell-seeded hydrogels were subjected to cyclic hydrostatic pressure at 0-270 kPa and 1 Hz for 1 h daily (5 days per week) over a period of 14 days. At the start of mechanical stimulation, dissolved O2 and CO2 in the medium increased and the pH of the medium decreased, but remained within human physiological ranges. Changes in physiological parameters (O2, CO2, and pH) were reversible when medium samples were placed in a standard cell culture incubator. In addition, computational modeling showed that the distribution and magnitude of physical forces depends on the shape and position of the cell-hydrogel constructs in the tissue culture format. Finally, hydrostatic pressure was seen to enhance mineralization of chick femur skeletal cell-seeded hydrogels.

Construction of human induced pluripotent stem cell-derived oriented bone matrix microstructure by using in vitro engineered anisotropic culture model.

PubMed

Ozasa, Ryosuke; Matsugaki, Aira; Isobe, Yoshihiro; Saku, Taro; Yun, Hui-Suk; Nakano, Takayoshi

2018-02-01

Bone tissue has anisotropic microstructure based on collagen/biological apatite orientation, which plays essential roles in the mechanical and biological functions of bone. However, obtaining an appropriate anisotropic microstructure during the bone regeneration process remains a great challenging. A powerful strategy for the control of both differentiation and structural development of newly-formed bone is required in bone tissue engineering, in order to realize functional bone tissue regeneration. In this study, we developed a novel anisotropic culture model by combining human induced pluripotent stem cells (hiPSCs) and artificially-controlled oriented collagen scaffold. The oriented collagen scaffold allowed hiPSCs-derived osteoblast alignment and further construction of anisotropic bone matrix which mimics the bone tissue microstructure. To the best of our knowledge, this is the first report showing the construction of bone mimetic anisotropic bone matrix microstructure from hiPSCs. Moreover, we demonstrated for the first time that the hiPSCs-derived osteoblasts possess a high level of intact functionality to regulate cell alignment. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 360-369, 2018. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc.

Hierarchical Design of Tissue Regenerative Constructs.

PubMed

Rose, Jonas C; De Laporte, Laura

2018-03-01

The worldwide shortage of organs fosters significant advancements in regenerative therapies. Tissue engineering and regeneration aim to supply or repair organs or tissues by combining material scaffolds, biochemical signals, and cells. The greatest challenge entails the creation of a suitable implantable or injectable 3D macroenvironment and microenvironment to allow for ex vivo or in vivo cell-induced tissue formation. This review gives an overview of the essential components of tissue regenerating scaffolds, ranging from the molecular to the macroscopic scale in a hierarchical manner. Further, this review elaborates about recent pivotal technologies, such as photopatterning, electrospinning, 3D bioprinting, or the assembly of micrometer-scale building blocks, which enable the incorporation of local heterogeneities, similar to most native extracellular matrices. These methods are applied to mimic a vast number of different tissues, including cartilage, bone, nerves, muscle, heart, and blood vessels. Despite the tremendous progress that has been made in the last decade, it remains a hurdle to build biomaterial constructs in vitro or in vivo with a native-like structure and architecture, including spatiotemporal control of biofunctional domains and mechanical properties. New chemistries and assembly methods in water will be crucial to develop therapies that are clinically translatable and can evolve into organized and functional tissues. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of collagenase-sensitive polyureas for ligament tissue engineering.

PubMed

Benhardt, Hugh; Sears, Nick; Touchet, Tyler; Cosgriff-Hernandez, Elizabeth

2011-08-11

Recently, poly(ester urethanes) were investigated for use as ligament grafts due to their exceptional mechanical properties and highly tunable structure; however, these grafts are susceptible to hydrolytic degradation that occurs independent of tissue regeneration. To address this limitation, polyureas containing collagen-derived peptides were synthesized which enable cellular release of proteases to dictate degradation rate. It is hypothesized that this cell-responsive design will facilitate load transfer from the biodegradable scaffold to neotissue at a rate that promotes proper tissue orientation and function while maintaining construct integrity. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

From single fiber to macro-level mechanics: A structural finite-element model for elastomeric fibrous biomaterials

PubMed Central

D'Amore, Antonio; Amoroso, Nicholas; Gottardi, Riccardo; Hobson, Christopher; Carruthers, Christopher; Watkins, Simon; Wagner, William R.; Sacks, Michael S.

2014-01-01

In the present work, we demonstrate that the mesoscopic in-plane mechanical behavior of membrane elastomeric scaffolds can be simulated by replication of actual quantified fibrous geometries. Elastomeric electrospun polyurethane (ES-PEUU) scaffolds, with and without particulate inclusions, were utilized. Simulations were developed from experimentally-derived fiber network geometries, based on a range of scaffold isotropic and anisotropic behaviors. These were chosen to evaluate the effects on macro-mechanics based on measurable geometric parameters such as fiber intersections, connectivity, orientation, and diameter. Simulations were conducted with only the fiber material model parameters adjusted to match the macro-level mechanical test data. Fiber model validation was performed at the microscopic level by individual fiber mechanical tests using AFM. Results demonstrated very good agreement to the experimental data, and revealed the formation of extended preferential fiber orientations spanning the entire model space. We speculate that these emergent structures may be responsible for the tissue-like macroscale behaviors observed in electrospun scaffolds. To conclude, the modeling approach has implications for (1) gaining insight on the intricate relationship between fabrication variables, structure, and mechanics to manufacture more functional devices/materials, (2) elucidating the effects of cell or particulate inclusions on global construct mechanics, and (3) fabricating better performing tissue surrogates that could recapitulate native tissue mechanics. PMID:25128869

Fabrication of a Neotrachea Using Engineered Cartilage

PubMed Central

Weidenbecher, Mark; Tucker, Harvey M.; Awadallah, Amad; Dennis, James E.

2008-01-01

Objectives Surgical management of long-segment tracheal stenosis is an ongoing problem. Many types of tracheal prostheses have been tried but with limited success because of immune rejection, graft ischemia, or restenosis. Tissue engineered cartilage may offer a solution to this problem, although scaffolds, which are currently often used for support, can lead to biocompatibility problems. This study investigated the feasibility of scaffold-free cartilage to tissue engineer a vascularized neotrachea in rabbits. Study Design Animal study. Methods Autologous neotracheal constructs were implanted in the abdomen of six New Zealand white rabbits. Auricular chondrocytes were used to engineer scaffold-free cartilage sheets. A muscle flap raised from the external abdominal oblique muscle and the engineered cartilage were wrapped around a silicone stent to fabricate a vascularized neotrachea in vivo. In two of the six rabbits, a full thickness skin graft was used to create an epithelial lining. The constructs were harvested after either 6 or 10 weeks. Results All neotracheal constructs were healthy with well-vascularized and integrated layers. The implanted engineered cartilage underwent a remodeling process, forming a solid tracheal framework. Constructs harvested after 10 weeks proved to have significantly better mechanical properties than after 6 weeks and were comparable with the rabbit's native trachea. Conclusion Scaffold-free engineered cartilage can successfully fabricate a well-vascularized, autologous neotrachea with excellent mechanical properties. The results suggest that this approach can be used to reconstruct tracheal defects in rabbits. PMID:18197138

Wound ballistics 101: the mechanisms of soft tissue wounding by bullets.

PubMed

Stefanopoulos, P K; Pinialidis, D E; Hadjigeorgiou, G F; Filippakis, K N

2017-10-01

The mechanisms of soft tissue injury by bullets are reviewed, in the belief that the current incidence of firearm injuries in many urban areas necessitates an understanding of wound ballistics on the part of trauma surgeons who may not be familiar with the wounding factors involved. Review of the literature, with technical information obtained from appropriate non-medical texts. Despite numerous publications concerning the treatment of gunshot wounds, relatively few papers contain details on the mechanisms of ballistic trauma, with the main body of evidence derived from previous laboratory and animal studies which have only recently been systematically appraised. These studies have shown that in rifle injuries the main wound tract is surrounded by an area of damaged tissue as a result of the temporary cavitation induced once the bullet becomes destabilized or deformed. On the other hand, the more commonly encountered non-deforming handgun bullets cause damage limited to the bullet's path, mainly as a result of localized crush injury. The bullet's construction and ballistic behavior within tissue determine to what extent the previously overestimated velocity factor may influence wound severity. The damage produced from temporary cavitation depends on the tensile properties of the tissues involved, and in high-energy injuries may lead to progressive muscle tissue necrosis. Therefore, the term "high-energy" should be reserved for those injuries with substantial tissue damage extending beyond the visible wound tract.

Tissue material properties and computational modelling of the human tibiofemoral joint: a critical review

PubMed Central

Akhtar, Riaz; Comerford, Eithne J.; Bates, Karl T.

2018-01-01

Understanding how structural and functional alterations of individual tissues impact on whole-joint function is challenging, particularly in humans where direct invasive experimentation is difficult. Finite element (FE) computational models produce quantitative predictions of the mechanical and physiological behaviour of multiple tissues simultaneously, thereby providing a means to study changes that occur through healthy ageing and disease such as osteoarthritis (OA). As a result, significant research investment has been placed in developing such models of the human knee. Previous work has highlighted that model predictions are highly sensitive to the various inputs used to build them, particularly the mathematical definition of material properties of biological tissues. The goal of this systematic review is two-fold. First, we provide a comprehensive summation and evaluation of existing linear elastic material property data for human tibiofemoral joint tissues, tabulating numerical values as a reference resource for future studies. Second, we review efforts to model tibiofemoral joint mechanical behaviour through FE modelling with particular focus on how studies have sourced tissue material properties. The last decade has seen a renaissance in material testing fuelled by development of a variety of new engineering techniques that allow the mechanical behaviour of both soft and hard tissues to be characterised at a spectrum of scales from nano- to bulk tissue level. As a result, there now exists an extremely broad range of published values for human tibiofemoral joint tissues. However, our systematic review highlights gaps and ambiguities that mean quantitative understanding of how tissue material properties alter with age and OA is limited. It is therefore currently challenging to construct FE models of the knee that are truly representative of a specific age or disease-state. Consequently, recent tibiofemoral joint FE models have been highly generic in terms of material properties even relying on non-human data from multiple species. We highlight this by critically evaluating current ability to quantitatively compare and model (1) young and old and (2) healthy and OA human tibiofemoral joints. We suggest that future research into both healthy and diseased knee function will benefit greatly from a subject- or cohort-specific approach in which FE models are constructed using material properties, medical imagery and loading data from cohorts with consistent demographics and/or disease states. PMID:29379690

Tissue material properties and computational modelling of the human tibiofemoral joint: a critical review.

PubMed

Peters, Abby E; Akhtar, Riaz; Comerford, Eithne J; Bates, Karl T

2018-01-01

Understanding how structural and functional alterations of individual tissues impact on whole-joint function is challenging, particularly in humans where direct invasive experimentation is difficult. Finite element (FE) computational models produce quantitative predictions of the mechanical and physiological behaviour of multiple tissues simultaneously, thereby providing a means to study changes that occur through healthy ageing and disease such as osteoarthritis (OA). As a result, significant research investment has been placed in developing such models of the human knee. Previous work has highlighted that model predictions are highly sensitive to the various inputs used to build them, particularly the mathematical definition of material properties of biological tissues. The goal of this systematic review is two-fold. First, we provide a comprehensive summation and evaluation of existing linear elastic material property data for human tibiofemoral joint tissues, tabulating numerical values as a reference resource for future studies. Second, we review efforts to model tibiofemoral joint mechanical behaviour through FE modelling with particular focus on how studies have sourced tissue material properties. The last decade has seen a renaissance in material testing fuelled by development of a variety of new engineering techniques that allow the mechanical behaviour of both soft and hard tissues to be characterised at a spectrum of scales from nano- to bulk tissue level. As a result, there now exists an extremely broad range of published values for human tibiofemoral joint tissues. However, our systematic review highlights gaps and ambiguities that mean quantitative understanding of how tissue material properties alter with age and OA is limited. It is therefore currently challenging to construct FE models of the knee that are truly representative of a specific age or disease-state. Consequently, recent tibiofemoral joint FE models have been highly generic in terms of material properties even relying on non-human data from multiple species. We highlight this by critically evaluating current ability to quantitatively compare and model (1) young and old and (2) healthy and OA human tibiofemoral joints. We suggest that future research into both healthy and diseased knee function will benefit greatly from a subject- or cohort-specific approach in which FE models are constructed using material properties, medical imagery and loading data from cohorts with consistent demographics and/or disease states.

Tissue-Engineered Fibrin-Based Heart Valve with a Tubular Leaflet Design

PubMed Central

Weber, Miriam; Heta, Eriona; Moreira, Ricardo; Gesche, Valentine N.; Schermer, Thomas; Frese, Julia

2014-01-01

The general approach in heart valve tissue engineering is to mimic the shape of the native valve in the attempt to recreate the natural haemodynamics. In this article, we report the fabrication of the first tissue-engineered heart valve (TEHV) based on a tubular leaflet design, where the function of the leaflets of semilunar heart valves is performed by a simple tubular construct sutured along a circumferential line at the root and at three single points at the sinotubular junction. The tubular design is a recent development in pericardial (nonviable) bioprostheses, which has attracted interest because of the simplicity of the construction and the reliability of the implantation technique. Here we push the potential of the concept further from the fabrication and material point of view to realize the tube-in-tube valve: an autologous, living HV with remodelling and growing capability, physiological haemocompatibility, simple to construct and fast to implant. We developed two different fabrication/conditioning procedures and produced fibrin-based constructs embedding cells from the ovine umbilical cord artery according to the two different approaches. Tissue formation was confirmed by histology and immunohistology. The design of the tube-in-tube foresees the possibility of using a textile coscaffold (here demonstrated with a warp-knitted mesh) to achieve enhanced mechanical properties in vision of implantation in the aortic position. The tube-in-tube represents an attractive alternative to the conventional design of TEHVs aiming at reproducing the valvular geometry. PMID:23829551

Implantation of In Vitro Tissue Engineered Muscle Repair Constructs and Bladder Acellular Matrices Partially Restore In Vivo Skeletal Muscle Function in a Rat Model of Volumetric Muscle Loss Injury

PubMed Central

Corona, Benjamin T.; Ward, Catherine L.; Baker, Hannah B.; Walters, Thomas J.

2014-01-01

The frank loss of a large volume of skeletal muscle (i.e., volumetric muscle loss [VML]) can lead to functional debilitation and presents a significant problem to civilian and military medicine. Current clinical treatment for VML involves the use of free muscle flaps and physical rehabilitation; however, neither are effective in promoting regeneration of skeletal muscle to replace the tissue that was lost. Toward this end, skeletal muscle tissue engineering therapies have recently shown great promise in offering an unprecedented treatment option for VML. In the current study, we further extend our recent progress (Machingal et al., 2011, Tissue Eng; Corona et al., 2012, Tissue Eng) in the development of tissue engineered muscle repair (TEMR) constructs (i.e., muscle-derived cells [MDCs] seeded on a bladder acellular matrix (BAM) preconditioned with uniaxial mechanical strain) for the treatment of VML. TEMR constructs were implanted into a VML defect in a tibialis anterior (TA) muscle of Lewis rats and observed up to 12 weeks postinjury. The salient findings of the study were (1) TEMR constructs exhibited a highly variable capacity to restore in vivo function of injured TA muscles, wherein TEMR-positive responders (n=6) promoted an ≈61% improvement, but negative responders (n=7) resulted in no improvement compared to nonrepaired controls, (2) TEMR-positive and -negative responders exhibited differential immune responses that may underlie these variant responses, (3) BAM scaffolds (n=7) without cells promoted an ≈26% functional improvement compared to uninjured muscles, (4) TEMR-positive responders promoted muscle fiber regeneration within the initial defect area, while BAM scaffolds did so only sparingly. These findings indicate that TEMR constructs can improve the in vivo functional capacity of the injured musculature at least, in part, by promoting generation of functional skeletal muscle fibers. In short, the degree of functional recovery observed following TEMR implantation (BAM+MDCs) was 2.3×-fold greater than that observed following implantation of BAM alone. As such, this finding further underscores the potential benefits of including a cellular component in the tissue engineering strategy for VML injury. PMID:24066899

Anatomically shaped tissue-engineered cartilage with tunable and inducible anticytokine delivery for biological joint resurfacing

PubMed Central

Moutos, Franklin T.; Glass, Katherine A.; Compton, Sarah A.; Ross, Alison K.; Gersbach, Charles A.; Estes, Bradley T.

2016-01-01

Biological resurfacing of entire articular surfaces represents an important but challenging strategy for treatment of cartilage degeneration that occurs in osteoarthritis. Not only does this approach require anatomically sized and functional engineered cartilage, but the inflammatory environment within an arthritic joint may also inhibit chondrogenesis and induce degradation of native and engineered cartilage. The goal of this study was to use adult stem cells to engineer anatomically shaped, functional cartilage constructs capable of tunable and inducible expression of antiinflammatory molecules, specifically IL-1 receptor antagonist (IL-1Ra). Large (22-mm-diameter) hemispherical scaffolds were fabricated from 3D woven poly(ε-caprolactone) (PCL) fibers into two different configurations and seeded with human adipose-derived stem cells (ASCs). Doxycycline (dox)-inducible lentiviral vectors containing eGFP or IL-1Ra transgenes were immobilized to the PCL to transduce ASCs upon seeding, and constructs were cultured in chondrogenic conditions for 28 d. Constructs showed biomimetic cartilage properties and uniform tissue growth while maintaining their anatomic shape throughout culture. IL-1Ra–expressing constructs produced nearly 1 µg/mL of IL-1Ra upon controlled induction with dox. Treatment with IL-1 significantly increased matrix metalloprotease activity in the conditioned media of eGFP-expressing constructs but not in IL-1Ra–expressing constructs. Our findings show that advanced textile manufacturing combined with scaffold-mediated gene delivery can be used to tissue engineer large anatomically shaped cartilage constructs that possess controlled delivery of anticytokine therapy. Importantly, these cartilage constructs have the potential to provide mechanical functionality immediately upon implantation, as they will need to replace a majority, if not the entire joint surface to restore function. PMID:27432980

Reverse engineering the mechanical and molecular pathways in stem cell morphogenesis.

PubMed

Lu, Kai; Gordon, Richard; Cao, Tong

2015-03-01

The formation of relevant biological structures poses a challenge for regenerative medicine. During embryogenesis, embryonic cells differentiate into somatic tissues and undergo morphogenesis to produce three-dimensional organs. Using stem cells, we can recapitulate this process and create biological constructs for therapeutic transplantation. However, imperfect imitation of nature sometimes results in in vitro artifacts that fail to recapitulate the function of native organs. It has been hypothesized that developing cells may self-organize into tissue-specific structures given a correct in vitro environment. This proposition is supported by the generation of neo-organoids from stem cells. We suggest that morphogenesis may be reverse engineered to uncover its interacting mechanical pathway and molecular circuitry. By harnessing the latent architecture of stem cells, novel tissue-engineering strategies may be conceptualized for generating self-organizing transplants. Copyright © 2013 John Wiley & Sons, Ltd.

Characterization of Mechanical Properties of Tissue Scaffolds by Phase Contrast Imaging and Finite Element Modeling.

PubMed

Bawolin, Nahshon K; Dolovich, Allan T; Chen, Daniel X B; Zhang, Chris W J

2015-08-01

In tissue engineering, the cell and scaffold approach has shown promise as a treatment to regenerate diseased and/or damaged tissue. In this treatment, an artificial construct (scaffold) is seeded with cells, which organize and proliferate into new tissue. The scaffold itself biodegrades with time, leaving behind only newly formed tissue. The degradation qualities of the scaffold are critical during the treatment period, since the change in the mechanical properties of the scaffold with time can influence cell behavior. To observe in time the scaffold's mechanical properties, a straightforward method is to deform the scaffold and then characterize scaffold deflection accordingly. However, experimentally observing the scaffold deflection is challenging. This paper presents a novel study on characterization of mechanical properties of scaffolds by phase contrast imaging and finite element modeling, which specifically includes scaffold fabrication, scaffold imaging, image analysis, and finite elements (FEs) modeling of the scaffold mechanical properties. The innovation of the work rests on the use of in-line phase contrast X-ray imaging at 20 KeV to characterize tissue scaffold deformation caused by ultrasound radiation forces and the use of the Fourier transform to identify movement. Once deformation has been determined experimentally, it is then compared with the predictions given by the forward solution of a finite element model. A consideration of the number of separate loading conditions necessary to uniquely identify the material properties of transversely isotropic and fully orthotropic scaffolds is also presented, along with the use of an FE as a form of regularization.

Organ Printing

NASA Astrophysics Data System (ADS)

Cho, Dong-Woo; Lee, Jung-Seob; Jang, Jinah; Jung, Jin Woo; Park, Jeong Hun; Pati, Falguni

2015-10-01

This book introduces various 3D printing systems, biomaterials, and cells for organ printing. In view of the latest applications of several 3D printing systems, their advantages and disadvantages are also discussed. A basic understanding of the entire spectrum of organ printing provides pragmatic insight into the mechanisms, methods, and applications of this discipline. Organ printing is being applied in the tissue engineering field with the purpose of developing tissue/organ constructs for the regeneration of both hard (bone, cartilage, osteochondral) and soft tissues (heart). There are other potential application areas including tissue/organ models, disease/cancer models, and models for physiology and pathology, where in vitro 3D multicellular structures developed by organ printing are valuable.

Electro-mechanical dynamics of spiral waves in a discrete 2D model of human atrial tissue.

PubMed

Brocklehurst, Paul; Ni, Haibo; Zhang, Henggui; Ye, Jianqiao

2017-01-01

We investigate the effect of mechano-electrical feedback and atrial fibrillation induced electrical remodelling (AFER) of cellular ion channel properties on the dynamics of spiral waves in a discrete 2D model of human atrial tissue. The tissue electro-mechanics are modelled using the discrete element method (DEM). Millions of bonded DEM particles form a network of coupled atrial cells representing 2D cardiac tissue, allowing simulations of the dynamic behaviour of electrical excitation waves and mechanical contraction in the tissue. In the tissue model, each cell is modelled by nine particles, accounting for the features of individual cellular geometry; and discrete inter-cellular spatial arrangement of cells is also considered. The electro-mechanical model of a human atrial single-cell was constructed by strongly coupling the electrophysiological model of Colman et al. to the mechanical myofilament model of Rice et al., with parameters modified based on experimental data. A stretch-activated channel was incorporated into the model to simulate the mechano-electrical feedback. In order to investigate the effect of mechano-electrical feedback on the dynamics of spiral waves, simulations of spiral waves were conducted in both the electromechanical model and the electrical-only model in normal and AFER conditions, to allow direct comparison of the results between the models. Dynamics of spiral waves were characterized by tracing their tip trajectories, stability, excitation frequencies and meandering range of tip trajectories. It was shown that the developed DEM method provides a stable and efficient model of human atrial tissue with considerations of the intrinsically discrete and anisotropic properties of the atrial tissue, which are challenges to handle in traditional continuum mechanics models. This study provides mechanistic insights into the complex behaviours of spiral waves and the genesis of atrial fibrillation by showing an important role of the mechano-electrical feedback in facilitating and promoting atrial fibrillation.

Electro-mechanical dynamics of spiral waves in a discrete 2D model of human atrial tissue

PubMed Central

Zhang, Henggui

2017-01-01

We investigate the effect of mechano-electrical feedback and atrial fibrillation induced electrical remodelling (AFER) of cellular ion channel properties on the dynamics of spiral waves in a discrete 2D model of human atrial tissue. The tissue electro-mechanics are modelled using the discrete element method (DEM). Millions of bonded DEM particles form a network of coupled atrial cells representing 2D cardiac tissue, allowing simulations of the dynamic behaviour of electrical excitation waves and mechanical contraction in the tissue. In the tissue model, each cell is modelled by nine particles, accounting for the features of individual cellular geometry; and discrete inter-cellular spatial arrangement of cells is also considered. The electro-mechanical model of a human atrial single-cell was constructed by strongly coupling the electrophysiological model of Colman et al. to the mechanical myofilament model of Rice et al., with parameters modified based on experimental data. A stretch-activated channel was incorporated into the model to simulate the mechano-electrical feedback. In order to investigate the effect of mechano-electrical feedback on the dynamics of spiral waves, simulations of spiral waves were conducted in both the electromechanical model and the electrical-only model in normal and AFER conditions, to allow direct comparison of the results between the models. Dynamics of spiral waves were characterized by tracing their tip trajectories, stability, excitation frequencies and meandering range of tip trajectories. It was shown that the developed DEM method provides a stable and efficient model of human atrial tissue with considerations of the intrinsically discrete and anisotropic properties of the atrial tissue, which are challenges to handle in traditional continuum mechanics models. This study provides mechanistic insights into the complex behaviours of spiral waves and the genesis of atrial fibrillation by showing an important role of the mechano-electrical feedback in facilitating and promoting atrial fibrillation. PMID:28510575

Tissue engineering therapy for cardiovascular disease.

PubMed

Nugent, Helen M; Edelman, Elazer R

2003-05-30

The present treatments for the loss or failure of cardiovascular function include organ transplantation, surgical reconstruction, mechanical or synthetic devices, or the administration of metabolic products. Although routinely used, these treatments are not without constraints and complications. The emerging and interdisciplinary field of tissue engineering has evolved to provide solutions to tissue creation and repair. Tissue engineering applies the principles of engineering, material science, and biology toward the development of biological substitutes that restore, maintain, or improve tissue function. Progress has been made in engineering the various components of the cardiovascular system, including blood vessels, heart valves, and cardiac muscle. Many pivotal studies have been performed in recent years that may support the move toward the widespread application of tissue-engineered therapy for cardiovascular diseases. The studies discussed include endothelial cell seeding of vascular grafts, tissue-engineered vascular conduits, generation of heart valve leaflets, cardiomyoplasty, genetic manipulation, and in vitro conditions for optimizing tissue-engineered cardiovascular constructs.

A microfabricated platform with hydrogel arrays for 3D mechanical stimulation of cells.

PubMed

Liu, Haijiao; Usprech, Jenna; Sun, Yu; Simmons, Craig A

2016-04-01

Cellular microenvironments present cells with multiple stimuli, including not only soluble biochemical and insoluble matrix cues but also mechanical factors. Biomaterial array platforms have been used to combinatorially and efficiently probe and define two-dimensional (2D) and 3D microenvironmental cues to guide cell functions for tissue engineering applications. However, there are few examples of array platforms that include dynamic mechanical forces, particularly to enable stretching of 3D cell-seeded biomaterials, which is relevant to engineering connective and cardiovascular tissues. Here we present a deformable membrane platform that enables 3D dynamic mechanical stretch of arrayed biomaterial constructs. Cell-seeded polyethylene glycol norbornene (PEG-NB) hydrogels were bound to miniaturized deformable membranes via a thiol-ene reaction with off-stoichiometry thiol-ene based polydimethylsiloxane (OSTE-PDMS) as the membrane material. Bonding to OSTE-PDMS enabled the 3D hydrogel microconstructs to be cyclically deformed and stretched by the membrane. As a first demonstration, human mesenchymal stromal cells (MSCs) embedded in PEG-NB were stretched for several days. They were found to be viable, spread in the 3D hydrogels, and exhibited a contractile myofibroblast phenotype when exposed to dynamic 3D mechanical deformation. This platform, which is readily scalable to larger arrays, enables systematic interrogation of the relationships between combinations of 3D mechanobiological cues and cellular responses, and thus has the potential to identify strategies to predictably control the construction of functional engineered tissues. Current high-throughput biomaterial screening approaches fail to consider the effects of dynamic mechanical stimulation, despite its importance in a wide variety of regenerative medicine applications. To meet this need, we developed a deformable membrane platform that enables 3D dynamic stretch of arrayed biomaterial constructs. Our approach combines microtechnologies fabricated with off-stoichiometry thiol-ene based polydimethylsiloxane membranes that can covalently bond cell-seeded polyethylene glycol norbornene 3D hydrogels, a model biomaterial with tunable adhesive, elastic and degradation characteristics. As a first demonstration, we show that human mesenchymal stromal cells embedded in hydrogels and subjected to dynamic mechanical stimulation undergo myofibroblast differentiation. This system is readily scaled up to larger arrays, and will enable systematic and efficient screening of combinations of 3D mechanobiological and biomaterial cues on cell fate and function. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Morphological changes in paraurethral area after introduction of tissue engineering construct on the basis of adipose tissue stromal cells.

PubMed

Makarov, A V; Arutyunyan, I V; Bol'shakova, G B; Volkov, A V; Gol'dshtein, D V

2009-10-01

We studied morphological changes in the paraurethral area of Wistar rats after introduction of tissue engineering constructs on the basis of multipotent mesenchymal stem cells and gelatin sponge. The tissue engineering construct containing autologous culture of the stromal fraction of the adipose tissue was most effective. After introduction of this construct we observed more rapid degradation of the construct matrix and more intensive formation of collagen fibers.

Biocompatibility and calcification of bovine pericardium employed for the construction of cardiac bioprostheses treated with different chemical crosslink methods.

PubMed

Jorge-Herrero, Eduardo; Fonseca, Carlos; Barge, Alexandra P; Turnay, Javier; Olmo, Nieves; Fernández, Pilar; Lizarbe, María A; García Páez, José M

2010-05-01

The use of biological materials in the construction of bioprostheses requires the application of different chemical procedures to improve the durability of the material without producing any undesirable effects. A number of crosslinking methods have been tested in biological tissues composed mainly of collagen. The aim of this study was to evaluate the in vitro biocompatibility, the mechanical properties, and in vivo calcification of chemically modified bovine pericardium using glutaraldehyde acetals (GAAs) in comparison with glutaraldehyde (GA) treatment. Homsy's tests showed that the most cytotoxic treatment is GA whereas GAA treatments showed lower cytotoxicity. Regarding the mechanical properties of the modified materials, no significant differences in stress at rupture were detected among the different treatments. Zeta-Potential showed higher negative values for GA treatment (-4.9 +/- 0.6 mV) compared with GAA-0.625% (-2.2 +/- 0.5 mV) and GAA-1% (-2.2 +/- 0.4 mV), which presented values similar to native tissue. Similar results were obtained for calcium permeability coefficients which showed the highest values for GA treatment (0.12 +/- 0.02 mm(2)/min), being significantly lower for GAA treatments or non-crosslinked pericardium. These results confirmed the higher propensity of the GA-treated tissues for attraction of calcium cations and were in good agreement with the calcification degree obtained after 60 days implantation into young rats, which was significantly higher for the GA group (22.70 +/- 20.80 mg/g dry tissue) compared with GAA-0.625% and GAA-1% groups (0.49 +/- 0.28 mg/g dry tissue and 3.51 +/- 3.27 mg/g dry tissue, respectively; P < 0.001). In conclusion, GAA treatments can be considered a promising alternative to GA treatment.

3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture.

PubMed

Ghezzi, Chiara E; Marelli, Benedetto; Omenetto, Fiorenzo G; Funderburgh, James L; Kaplan, David L

2017-01-01

The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear silk protein films, in combination with human corneal stromal stem cells (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison to previously developed 2D approaches. Silk film contact guidance was used to control the alignment and distribution of hCSSCs on RGD-treated single porous silk films, which were then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These systems were compared similar systems generated with human corneal fibroblasts (hCFs). Both cell types were viable and preferentially aligned along the biomaterial patterns for up to 9 weeks in culture. H&E histological sections showed that the systems seeded with the hCSSCs displayed ECM production throughout the entire thickness of the constructs. In addition, the ECM proteins tested positive for keratocyte-specific tissue markers, including keratan sulfate, lumican, and keratocan. The quantification of hCSSC gene expression of keratocyte-tissue markers, including keratocan, lumican, human aldehyde dehydrogenase 3A1 (ALDH3A1), prostaglandin D2 synthase (PTDGS), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), within the 3D tissue systems demonstrated upregulation when compared to 2D single silk films and to the systems generated with the hCFs. Furthermore, the production of ECM from the hCSSC seeded systems and subsequent remodeling of the initial matrix significantly improved cohesiveness and mechanical performance of the constructs, while maintaining transparency after 9 weeks.

A high throughput mechanical screening device for cartilage tissue engineering.

PubMed

Mohanraj, Bhavana; Hou, Chieh; Meloni, Gregory R; Cosgrove, Brian D; Dodge, George R; Mauck, Robert L

2014-06-27

Articular cartilage enables efficient and near-frictionless load transmission, but suffers from poor inherent healing capacity. As such, cartilage tissue engineering strategies have focused on mimicking both compositional and mechanical properties of native tissue in order to provide effective repair materials for the treatment of damaged or degenerated joint surfaces. However, given the large number design parameters available (e.g. cell sources, scaffold designs, and growth factors), it is difficult to conduct combinatorial experiments of engineered cartilage. This is particularly exacerbated when mechanical properties are a primary outcome, given the long time required for testing of individual samples. High throughput screening is utilized widely in the pharmaceutical industry to rapidly and cost-effectively assess the effects of thousands of compounds for therapeutic discovery. Here we adapted this approach to develop a high throughput mechanical screening (HTMS) system capable of measuring the mechanical properties of up to 48 materials simultaneously. The HTMS device was validated by testing various biomaterials and engineered cartilage constructs and by comparing the HTMS results to those derived from conventional single sample compression tests. Further evaluation showed that the HTMS system was capable of distinguishing and identifying 'hits', or factors that influence the degree of tissue maturation. Future iterations of this device will focus on reducing data variability, increasing force sensitivity and range, as well as scaling-up to even larger (96-well) formats. This HTMS device provides a novel tool for cartilage tissue engineering, freeing experimental design from the limitations of mechanical testing throughput. © 2013 Published by Elsevier Ltd.

Decoupling the effect of shear stress and stretch on tissue growth & remodeling in a vascular graft.

PubMed

van Haaften, Eline E; Wissing, Tamar B; Rutten, Marcel; Bulsink, Jurgen A; Gashi, Kujtim; van Kelle, Mathieu A J; Smits, Anthal; Bouten, Carlijn; Kurniawan, Nicholas A

2018-06-07

The success of cardiovascular tissue engineering strategies largely depends on the mechanical environment in which cells develop a neo-tissue via growth and remodeling processes. This mechanical environment is defined by the local scaffold architecture to which cells adhere, i.e., the micro-environment, and by external mechanical cues to which cells respond, i.e., hemodynamic loading. The hemodynamic environment of early-developing blood vessels consists of both shear stress (due to blood flow) and circumferential stretch (due to blood pressure). Experimental platforms that recapitulate this mechanical environment in a controlled and tunable manner are thus critical for investigating cardiovascular tissue engineering. In traditional perfusion bioreactors, however, shear stress and stretch are coupled, hampering a clear delineation of their effects on cell and tissue response. Here, we uniquely designed a bioreactor that independently combines these two types of mechanical cues in eight parallel vascular grafts. The system is computationally and experimentally validated, through finite element analysis and culture of tissue constructs respectively, to distinguish various levels of shear stress (up to 5 Pa) and cyclic stretch (up to 1.10). To illustrate the usefulness of the system, we investigated the relative contribution of cyclic stretch (1.05 at 0.5 Hz) and shear stress (1 Pa) to tissue development. Both types of hemodynamic loading contributed to cell alignment, but the contribution of shear stress overruled stretch-induced cell proliferation and matrix (i.e., collagen and glycosaminoglycan) production. At a macroscopic level, cyclic stretching led to the most linear stress-stretch response, which was not related to the presence of shear stress. In conclusion, we have developed a bioreactor that is particularly suited to further unravel the interplay between hemodynamics and in situ tissue engineering processes. Using the new system, the present work highlights the importance of hemodynamic loading to the study of developing vascular tissues.

Hybrid Aorta Constructs via In Situ Crosslinking of Poly(glycerol-sebacate) Elastomer Within a Decellularized Matrix.

PubMed

Guler, Selcan; Hosseinian, Pezhman; Aydin, Halil Murat

2017-01-01

Decellularization of tissues and organs has high potential to obtain unique conformation and composition as native tissue structure but may result in weakened tissue mechanical strength. In this study, poly(glycerol-sebacate) (PGS) elastomers were combined with decellularized aorta fragments to investigate the changes in mechanical properties. PGS prepolymer was synthesized via microwave irradiation and then in situ crosslinked within the decellularized aorta extracellular matrix (ECM). Tensile strength (σ) values were found comparable as 0.44 ± 0.10 MPa and 0.57 ± 0.18 MPa for native and hybrid aorta samples, respectively, while elongation at break (ɛ) values were 261% ± 17%, 7.5% ± 0.57%, and 22.18% ± 2.48% for wet control (native), decellularized dried aortae, and hybrid matrices, showing elastic contribution. Young's modulus data indicate that there was a threefold decrease in stiffness compared to decellularized samples once PGS is introduced into the ECM structure. Scanning electron microscopy (SEM) analysis of hybrid grafts revealed that the construct preserves porosity in medial layer of the vessel. Biocompatibility analyses showed no cytotoxic effects on human abdominal aorta smooth muscle cells. Cell studies showed 98% activity in hybrid graft extracts. As a control, collagen coating of the hybrid grafts was performed in the recellularization stage. SEM analysis of recellularized hybrid grafts revealed that cells were attached to the surface of the hybrid graft and proliferated during the 14 days of culture in both groups. This study shows that introducing an elastomer into the native ECM structure following decellularization process can be a useful approach for the preparation of mechanically enhanced composites for soft tissues.

Optimized adipose tissue engineering strategy based on a neo-mechanical processing method.

PubMed

He, Yunfan; Lin, Maohui; Wang, Xuecen; Guan, Jingyan; Dong, Ziqing; Feng, Lu; Xing, Malcolm; Feng, Chuanbo; Li, Xiaojian

2018-05-26

Decellularized adipose tissue (DAT) represents a promising scaffold for adipose tissue engineering. However, the unique and prolonged lipid removal process required for adipose tissue can damage extracellular matrix (ECM) constituents. Moreover, inadequate vascularization limits the recellularization of DAT in vivo. We proposed a neo-mechanical protocol for rapidly breaking adipocytes and removing lipid content from adipose tissue. The lipid-depleted adipose tissue was then subjected to a fast and mild decellularization to fabricate high-quality DAT (M-DAT). Adipose liquid extract (ALE) derived from this mechanical process was collected and incorporated into M-DAT to further optimize in vivo recellularization. Ordinary DAT was fabricated and served as a control. This developed strategy was evaluated based on decellularization efficiency, ECM quality, and recellularization efficiency. Angiogenic factor components and angiogenic potential of ALE were evaluated in vivo and in vitro. M-DAT achieved the same decellularization efficiency, but exhibited better retention of ECM components and recellularization, compared to those with ordinary DAT. Protein quantification revealed considerable levels of angiogenic factors (basic fibroblast growth factor, epidermal growth factor, transforming growth factor-β1, and vascular endothelial growth factor) in ALE. ALE promoted tube formation in vitro and induced intense angiogenesis in M-DAT in vivo; furthermore, higher expression of the adipogenic factor PPARγ and greater numbers of adipocytes were evident following ALE treatment, compared to those in the M-DAT group. Mechanical processing of adipose tissue led to the production of high-quality M-DAT and angiogenic factor-enriched ALE. The combination of ALE and M-DAT could be a promising strategy for engineered adipose tissue construction. This article is protected by copyright. All rights reserved. © 2018 by the Wound Healing Society.

Quantitative analysis of tissue deformation dynamics reveals three characteristic growth modes and globally aligned anisotropic tissue deformation during chick limb development.

PubMed

Morishita, Yoshihiro; Kuroiwa, Atsushi; Suzuki, Takayuki

2015-05-01

Tissue-level characterization of deformation dynamics is crucial for understanding organ morphogenetic mechanisms, especially the interhierarchical links among molecular activities, cellular behaviors and tissue/organ morphogenetic processes. Limb development is a well-studied topic in vertebrate organogenesis. Nevertheless, there is still little understanding of tissue-level deformation relative to molecular and cellular dynamics. This is mainly because live recording of detailed cell behaviors in whole tissues is technically difficult. To overcome this limitation, by applying a recently developed Bayesian approach, we here constructed tissue deformation maps for chick limb development with high precision, based on snapshot lineage tracing using dye injection. The precision of the constructed maps was validated with a clear statistical criterion. From the geometrical analysis of the map, we identified three characteristic tissue growth modes in the limb and showed that they are consistent with local growth factor activity and cell cycle length. In particular, we report that SHH signaling activity changes dynamically with developmental stage and strongly correlates with the dynamic shift in the tissue growth mode. We also found anisotropic tissue deformation along the proximal-distal axis. Morphogenetic simulation and experimental studies suggested that this directional tissue elongation, and not local growth, has the greatest impact on limb shaping. This result was supported by the novel finding that anisotropic tissue elongation along the proximal-distal axis occurs independently of cell proliferation. Our study marks a pivotal point for multi-scale system understanding in vertebrate development. © 2015. Published by The Company of Biologists Ltd.

Remote Determination of Time-Dependent Stiffness of Surface-Degrading-Polymer Scaffolds Via Synchrotron-Based Imaging.

PubMed

Bawolin, N K; Chen, X B

2017-04-01

Surface-degrading polymers have been widely used to fabricate scaffolds with the mechanical properties appropriate for tissue regeneration/repair. During their surface degradation, the material properties of polymers remain approximately unchanged, but the scaffold geometry and thus mechanical properties vary with time. This paper presents a novel method to determine the time-dependent mechanical properties, particularly stiffness, of scaffolds from the geometric changes captured by synchrotron-based imaging, with the help of finite element analysis (FEA). Three-dimensional (3D) tissue scaffolds were fabricated from surface-degrading polymers, and during their degradation, the tissue scaffolds were imaged via the synchrotron-based imaging to characterize their changing geometry. On this basis, the stiffness behavior of scaffolds was estimated from the FEA, and the results obtained were compared to the direct measurements of scaffold stiffness from the load-displacement material testing. The comparison illustrates that the Young's moduli estimated from the FEA and characterized geometry are in agreement with the ones of direct measurements. The developed method of estimating the mechanical behavior was also demonstrated effective with a nondegrading scaffold that displays the nonlinear stress-strain behavior. The in vivo monitoring of Young's modulus by morphology characterization also suggests the feasibility of characterizing experimentally the difference between in vivo and in vitro surface degradation of tissue engineering constructs.

Nanostructured 3D constructs based on chitosan and chondroitin sulphate multilayers for cartilage tissue engineering.

PubMed

Silva, Joana M; Georgi, Nicole; Costa, Rui; Sher, Praveen; Reis, Rui L; Van Blitterswijk, Clemens A; Karperien, Marcel; Mano, João F

2013-01-01

Nanostructured three-dimensional constructs combining layer-by-layer technology (LbL) and template leaching were processed and evaluated as possible support structures for cartilage tissue engineering. Multilayered constructs were formed by depositing the polyelectrolytes chitosan (CHT) and chondroitin sulphate (CS) on either bidimensional glass surfaces or 3D packet of paraffin spheres. 2D CHT/CS multi-layered constructs proved to support the attachment and proliferation of bovine chondrocytes (BCH). The technology was transposed to 3D level and CHT/CS multi-layered hierarchical scaffolds were retrieved after paraffin leaching. The obtained nanostructured 3D constructs had a high porosity and water uptake capacity of about 300%. Dynamical mechanical analysis (DMA) showed the viscoelastic nature of the scaffolds. Cellular tests were performed with the culture of BCH and multipotent bone marrow derived stromal cells (hMSCs) up to 21 days in chondrogenic differentiation media. Together with scanning electronic microscopy analysis, viability tests and DNA quantification, our results clearly showed that cells attached, proliferated and were metabolically active over the entire scaffold. Cartilaginous extracellular matrix (ECM) formation was further assessed and results showed that GAG secretion occurred indicating the maintenance of the chondrogenic phenotype and the chondrogenic differentiation of hMSCs.

Construction and Characterization of a Novel Vocal Fold Bioreactor

PubMed Central

Zerdoum, Aidan B.; Tong, Zhixiang; Bachman, Brendan; Jia, Xinqiao

2014-01-01

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 µm. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues. PMID:25145349

Construction and characterization of a novel vocal fold bioreactor.

PubMed

Zerdoum, Aidan B; Tong, Zhixiang; Bachman, Brendan; Jia, Xinqiao

2014-08-01

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 µm. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

Cell-laden hydrogels for osteochondral and cartilage tissue engineering.

PubMed

Yang, Jingzhou; Zhang, Yu Shrike; Yue, Kan; Khademhosseini, Ali

2017-07-15

Despite tremendous advances in the field of regenerative medicine, it still remains challenging to repair the osteochondral interface and full-thickness articular cartilage defects. This inefficiency largely originates from the lack of appropriate tissue-engineered artificial matrices that can replace the damaged regions and promote tissue regeneration. Hydrogels are emerging as a promising class of biomaterials for both soft and hard tissue regeneration. Many critical properties of hydrogels, such as mechanical stiffness, elasticity, water content, bioactivity, and degradation, can be rationally designed and conveniently tuned by proper selection of the material and chemistry. Particularly, advances in the development of cell-laden hydrogels have opened up new possibilities for cell therapy. In this article, we describe the problems encountered in this field and review recent progress in designing cell-hydrogel hybrid constructs for promoting the reestablishment of osteochondral/cartilage tissues. Our focus centers on the effects of hydrogel type, cell type, and growth factor delivery on achieving efficient chondrogenesis and osteogenesis. We give our perspective on developing next-generation matrices with improved physical and biological properties for osteochondral/cartilage tissue engineering. We also highlight recent advances in biomanufacturing technologies (e.g. molding, bioprinting, and assembly) for fabrication of hydrogel-based osteochondral and cartilage constructs with complex compositions and microarchitectures to mimic their native counterparts. Despite tremendous advances in the field of regenerative medicine, it still remains challenging to repair the osteochondral interface and full-thickness articular cartilage defects. This inefficiency largely originates from the lack of appropriate tissue-engineered biomaterials that replace the damaged regions and promote tissue regeneration. Cell-laden hydrogel systems have emerged as a promising tissue-engineering platform to address this issue. In this article, we describe the fundamental problems encountered in this field and review recent progress in designing cell-hydrogel constructs for promoting the reestablishment of osteochondral/cartilage tissues. Our focus centers on the effects of hydrogel composition, cell type, and growth factor delivery on achieving efficient chondrogenesis and osteogenesis. We give our perspective on developing next-generation hydrogel/inorganic particle/stem cell hybrid composites with improved physical and biological properties for osteochondral/cartilage tissue engineering. We also highlight recent advances in biomanufacturing and bioengineering technologies (e.g. 3D bioprinting) for fabrication of hydrogel-based osteochondral and cartilage constructs. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A Review of Three-Dimensional Printing in Tissue Engineering.

PubMed

Sears, Nick A; Seshadri, Dhruv R; Dhavalikar, Prachi S; Cosgriff-Hernandez, Elizabeth

2016-08-01

Recent advances in three-dimensional (3D) printing technologies have led to a rapid expansion of applications from the creation of anatomical training models for complex surgical procedures to the printing of tissue engineering constructs. In addition to achieving the macroscale geometry of organs and tissues, a print layer thickness as small as 20 μm allows for reproduction of the microarchitectures of bone and other tissues. Techniques with even higher precision are currently being investigated to enable reproduction of smaller tissue features such as hepatic lobules. Current research in tissue engineering focuses on the development of compatible methods (printers) and materials (bioinks) that are capable of producing biomimetic scaffolds. In this review, an overview of current 3D printing techniques used in tissue engineering is provided with an emphasis on the printing mechanism and the resultant scaffold characteristics. Current practical challenges and technical limitations are emphasized and future trends of bioprinting are discussed.

Potential of Bioactive Glasses for Cardiac and Pulmonary Tissue Engineering

PubMed Central

Hamzehlou, Sepideh

2017-01-01

Repair and regeneration of disorders affecting cardiac and pulmonary tissues through tissue-engineering-based approaches is currently of particular interest. On this matter, different families of bioactive glasses (BGs) have recently been given much consideration with respect to treating refractory diseases of these tissues, such as myocardial infarction. The inherent properties of BGs, including their ability to bond to hard and soft tissues, to stimulate angiogenesis, and to elicit antimicrobial effects, along with their excellent biocompatibility, support these newly proposed strategies. Moreover, BGs can also act as a bioactive reinforcing phase to finely tune the mechanical properties of polymer-based constructs used to repair the damaged cardiac and pulmonary tissues. In the present study, we evaluated the potential of different forms of BGs, alone or in combination with other materials (e.g., polymers), in regards to repair and regenerate injured tissues of cardiac and pulmonary systems. PMID:29244726

Faster tissue interface analysis from Raman microscopy images using compressed factorisation

NASA Astrophysics Data System (ADS)

Palmer, Andrew D.; Bannerman, Alistair; Grover, Liam; Styles, Iain B.

2013-06-01

The structure of an artificial ligament was examined using Raman microscopy in combination with novel data analysis. Basis approximation and compressed principal component analysis are shown to provide efficient compression of confocal Raman microscopy images, alongside powerful methods for unsupervised analysis. This scheme allows the acceleration of data mining, such as principal component analysis, as they can be performed on the compressed data representation, providing a decrease in the factorisation time of a single image from five minutes to under a second. Using this workflow the interface region between a chemically engineered ligament construct and a bone-mimic anchor was examined. Natural ligament contains a striated interface between the bone and tissue that provides improved mechanical load tolerance, a similar interface was found in the ligament construct.

Transplantation of Tissue-Engineered Cartilage in an Animal Model (Xenograft and Autograft): Construct Validation.

PubMed

Nemoto, Hitoshi; Watson, Deborah; Masuda, Koichi

2015-01-01

Tissue engineering holds great promise for cartilage repair with minimal donor-site morbidity. The in vivo maturation of a tissue-engineered construct can be tested in the subcutaneous tissues of the same species for autografts or of immunocompromised animals for allografts or xenografts. This section describes detailed protocols for the surgical transplantation of a tissue-engineered construct into an animal model to assess construct validity.

Dynamic soft tissue deformation estimation based on energy analysis

NASA Astrophysics Data System (ADS)

Gao, Dedong; Lei, Yong; Yao, Bin

2016-10-01

The needle placement accuracy of millimeters is required in many needle-based surgeries. The tissue deformation, especially that occurring on the surface of organ tissue, affects the needle-targeting accuracy of both manual and robotic needle insertions. It is necessary to understand the mechanism of tissue deformation during needle insertion into soft tissue. In this paper, soft tissue surface deformation is investigated on the basis of continuum mechanics, where a geometry model is presented to quantitatively approximate the volume of tissue deformation. The energy-based method is presented to the dynamic process of needle insertion into soft tissue based on continuum mechanics, and the volume of the cone is exploited to quantitatively approximate the deformation on the surface of soft tissue. The external work is converted into potential, kinetic, dissipated, and strain energies during the dynamic rigid needle-tissue interactive process. The needle insertion experimental setup, consisting of a linear actuator, force sensor, needle, tissue container, and a light, is constructed while an image-based method for measuring the depth and radius of the soft tissue surface deformations is introduced to obtain the experimental data. The relationship between the changed volume of tissue deformation and the insertion parameters is created based on the law of conservation of energy, with the volume of tissue deformation having been obtained using image-based measurements. The experiments are performed on phantom specimens, and an energy-based analytical fitted model is presented to estimate the volume of tissue deformation. The experimental results show that the energy-based analytical fitted model can predict the volume of soft tissue deformation, and the root mean squared errors of the fitting model and experimental data are 0.61 and 0.25 at the velocities 2.50 mm/s and 5.00 mm/s. The estimating parameters of the soft tissue surface deformations are proven to be useful for compensating the needle-targeting error in the rigid needle insertion procedure, especially for percutaneous needle insertion into organs.

Biomimetic heterogenous elastic tissue development.

PubMed

Tsai, Kai Jen; Dixon, Simon; Hale, Luke Richard; Darbyshire, Arnold; Martin, Daniel; de Mel, Achala

2017-01-01

There is an unmet need for artificial tissue to address current limitations with donor organs and problems with donor site morbidity. Despite the success with sophisticated tissue engineering endeavours, which employ cells as building blocks, they are limited to dedicated labs suitable for cell culture, with associated high costs and long tissue maturation times before available for clinical use. Direct 3D printing presents rapid, bespoke, acellular solutions for skull and bone repair or replacement, and can potentially address the need for elastic tissue, which is a major constituent of smooth muscle, cartilage, ligaments and connective tissue that support organs. Thermoplastic polyurethanes are one of the most versatile elastomeric polymers. Their segmented block copolymeric nature, comprising of hard and soft segments allows for an almost limitless potential to control physical properties and mechanical behaviour. Here we show direct 3D printing of biocompatible thermoplastic polyurethanes with Fused Deposition Modelling, with a view to presenting cell independent in-situ tissue substitutes. This method can expeditiously and economically produce heterogenous, biomimetic elastic tissue substitutes with controlled porosity to potentially facilitate vascularisation. The flexibility of this application is shown here with tubular constructs as exemplars. We demonstrate how these 3D printed constructs can be post-processed to incorporate bioactive molecules. This efficacious strategy, when combined with the privileges of digital healthcare, can be used to produce bespoke elastic tissue substitutes in-situ, independent of extensive cell culture and may be developed as a point-of-care therapy approach.

Osteograft, plastic material for regenerative medicine

NASA Astrophysics Data System (ADS)

Zaidman, A. M.; Korel, A. V.; Shevchenko, A. I.; Shchelkunova, E. I.; Sherman, K. M.; Predein, Yu. A.; Kosareva, O. S.

2016-08-01

Creating tissue-engineering constructs based on the mechanism of cartilage-bone evolution is promising for traumatology and orthopedics. Such a graft was obtained from a chondrograft by transdifferentiation. The hondrograft placed in osteogenic medium is undergoing osteogenic differentiation for 14-30 days. Tissue specificity of the osteograft was studied by morphology, immunohistochemistry, electron microscopy, and the expression of the corresponding genes was estimated. The expression of osteonectin, fibronectin, collagen of type I, izolektin and CD 44 is determined. Alkaline phosphatase and matrix vesicles are determined in osteoblasts. Calcificates are observed in the matrix. Chondrogenic proteins expression is absent. These findings evidence the tissue specificity of the developed osteograft.

Electrospinning of gelatin and SMPU with carbon nanotubes for tissue engineering scaffolds.

PubMed

Mejia, Monica A; Hoyos, Lina M; Zapata, Jenniffer; Restrepo, Luz M; Moneada, Maria E

2016-08-01

The nanofibres created by electrospinning technique are currently used for a variety of applications in tissue engineering; and Gelatin and Polyurethane Shape-Memory (SMPU) have important results in biomedicine. Similarly, carbon nanotubes combined with other biomaterials change important properties, opening new opportunities for biomedical applications. In this work, we constructed scaffold using electrospinning technique based in bovine-hide gelatin, SMPU and both materials hybrid with carbon nanotube. Morphology and cytotoxicity were evaluated and mechanical properties for two materials were obtained in scaffold building. Morphological, mechanical and citotoxic properties of the electrospun fibers were found to be dependent of alteration in materials concentration, electrospinning conditions and MWCNT concentration. According to morphological, cytotoxic and mechanical analysis, SMPU more MWCNT were the best material, with nanofibers of 451 nm, tensile strength of 1.912 MPa, and a high ratio surface volume.

Physiologically relevant organs on chips

PubMed Central

Yum, Kyungsuk; Hong, Soon Gweon; Lee, Luke P.

2015-01-01

Recent advances in integrating microengineering and tissue engineering have generated promising microengineered physiological models for experimental medicine and pharmaceutical research. Here we review the recent development of microengineered physiological systems, or organs on chips, that reconstitute the physiologically critical features of specific human tissues and organs and their interactions. This technology uses microengineering approaches to construct organ-specific microenvironments, reconstituting tissue structures, tissue–tissue interactions and interfaces, and dynamic mechanical and biochemical stimuli found in specific organs, to direct cells to assemble into functional tissues. We first discuss microengineering approaches to reproduce the key elements of physiologically important, dynamic mechanical microenvironments, biochemical microenvironments, and microarchitectures of specific tissues and organs in microfluidic cell culture systems. This is followed by examples of microengineered individual organ models that incorporate the key elements of physiological microenvironments into single microfluidic cell culture systems to reproduce organ-level functions. Finally, microengineered multiple organ systems that simulate multiple organ interactions to better represent human physiology, including human responses to drugs, is covered in this review. This emerging organs-on-chips technology has the potential to become an alternative to 2D and 3D cell culture and animal models for experimental medicine, human disease modeling, drug development, and toxicology. PMID:24357624

Multifunctional Bioreactor System for Human Intestine Tissues

PubMed Central

2017-01-01

The three-dimensional (3D) cultivation of intestinal cells and tissues in dynamic bioreactor systems to represent in vivo intestinal microenvironments is essential for developing regenerative medicine treatments for intestinal diseases. We have previously developed in vitro human intestinal tissue systems using a 3D porous silk scaffold system with intestinal architectures and topographical features for the adhesion, growth, and differentiation of intestinal cells under static culture conditions. In this study, we designed and fabricated a multifunctional bioreactor system that incorporates pre-epithelialized 3D silk scaffolds in a dynamic culture environment for in vitro engineering of human intestine tissues. The bioreactor system allows for control of oxygen levels in perfusion fluids (aerobic simulated intestinal fluid (SIF), microaerobic SIF, and anaerobic SIF), while ensuring control over the mechanical and chemical microenvironments present in native human intestines. The bioreactor system also enables 3D cell culture with spatial separation and cultivation of cocultured epithelial and stromal cells. Preliminary functional analysis of tissues housed in the bioreactor demonstrated that the 3D tissue constructs survived and maintained typical phenotypes of intestinal epithelium, including epithelial tight junction formation, intestinal biomarker expression, microvilli formation, and mucus secretion. The unique combination of a dynamic bioreactor and 3D intestinal constructs offers utility for engineering human intestinal tissues for the study of intestinal diseases and discovery options for new treatments. PMID:29333491

From single fiber to macro-level mechanics: A structural finite-element model for elastomeric fibrous biomaterials.

PubMed

D'Amore, Antonio; Amoroso, Nicholas; Gottardi, Riccardo; Hobson, Christopher; Carruthers, Christopher; Watkins, Simon; Wagner, William R; Sacks, Michael S

2014-11-01

In the present work, we demonstrate that the mesoscopic in-plane mechanical behavior of membrane elastomeric scaffolds can be simulated by replication of actual quantified fibrous geometries. Elastomeric electrospun polyurethane (ES-PEUU) scaffolds, with and without particulate inclusions, were utilized. Simulations were developed from experimentally-derived fiber network geometries, based on a range of scaffold isotropic and anisotropic behaviors. These were chosen to evaluate the effects on macro-mechanics based on measurable geometric parameters such as fiber intersections, connectivity, orientation, and diameter. Simulations were conducted with only the fiber material model parameters adjusted to match the macro-level mechanical test data. Fiber model validation was performed at the microscopic level by individual fiber mechanical tests using AFM. Results demonstrated very good agreement to the experimental data, and revealed the formation of extended preferential fiber orientations spanning the entire model space. We speculate that these emergent structures may be responsible for the tissue-like macroscale behaviors observed in electrospun scaffolds. To conclude, the modeling approach has implications for (1) gaining insight on the intricate relationship between fabrication variables, structure, and mechanics to manufacture more functional devices/materials, (2) elucidating the effects of cell or particulate inclusions on global construct mechanics, and (3) fabricating better performing tissue surrogates that could recapitulate native tissue mechanics. Copyright © 2014 Elsevier Ltd. All rights reserved.

A three-dimensional bioprinting system for use with a hydrogel-based biomaterial and printing parameter characterization.

PubMed

Song, Seung-Joon; Choi, Jaesoon; Park, Yong-Doo; Lee, Jung-Joo; Hong, So Young; Sun, Kyung

2010-11-01

Bioprinting is an emerging technology for constructing tissue or bioartificial organs with complex three-dimensional (3D) structures. It provides high-precision spatial shape forming ability on a larger scale than conventional tissue engineering methods, and simultaneous multiple components composition ability. Bioprinting utilizes a computer-controlled 3D printer mechanism for 3D biological structure construction. To implement minimal pattern width in a hydrogel-based bioprinting system, a study on printing characteristics was performed by varying printer control parameters. The experimental results showed that printing pattern width depends on associated printer control parameters such as printing flow rate, nozzle diameter, and nozzle velocity. The system under development showed acceptable feasibility of potential use for accurate printing pattern implementation in tissue engineering applications and is another example of novel techniques for regenerative medicine based on computer-aided biofabrication system. © 2010, Copyright the Authors. Artificial Organs © 2010, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Bioinspired porous membranes containing polymer nanoparticles for wound healing.

PubMed

Ferreira, Ana M; Mattu, Clara; Ranzato, Elia; Ciardelli, Gianluca

2014-12-01

Skin damages covering a surface larger than 4 cm(2) require a regenerative strategy based on the use of appropriate wound dressing supports to facilitate the rapid tissue replacement and efficient self-healing of the lost or damaged tissue. In the present work, A novel biomimetic approach is proposed for the design of a therapeutic porous construct made of poly(L-lactic acid) (PLLA) fabricated by thermally induced phase separation (TIPS). Biomimicry of ECM was achieved by immobilization of type I collagen through a two-step plasma treatment for wound healing. Anti-inflammatory (indomethacin)-containing polymeric nanoparticles (nps) were loaded within the porous membranes in order to minimize undesired cell response caused by post-operative inflammation. The biological response to the scaffold was analyzed by using human keratinocytes cell cultures. In this work, a promising biomimetic construct for wound healing and soft tissue regeneration with drug-release properties was fabricated since it shows (i) proper porosity, pore size, and mechanical properties, (ii) biomimicry of ECM, and (iii) therapeutic potential. © 2014 Wiley Periodicals, Inc.

Mechanobiologic Research in a Microgravity Environment Bioreactor

NASA Astrophysics Data System (ADS)

Guidi, A.; Dubini, G.; Tominetti, F.; Raimondi, M.

A current problem in tissue culturing technology is the unavailability of an effective Bioreactor for the in vitro cultivation of cells and explants. It has, in fact, proved extremely difficult to promote the high-density three-dimensional in vitro growth of human tissues that have been removed from the body and deprived of their normal in vivo vascular sources of nutrients and gas exchange. A variety of tissue explants can be maintained for a short period of time on a supportive collagen matrix surrounded by culture medium. But this system provides only limited mass transfer of nutrients and wastes through the tissue, and gravity-induced sedimentation prevents complete three- dimensional cell-cell and cell-matrix interactions. Several devices presently on the market have been used with only limited success since each has limitations, which restrict usefulness and versatility. Further, no Bioreactor or culture vessel is known that will allow for unimpeded growth of three dimensional cellular aggregates or tissue. Extensive research on the effect of mechanical stimuli on cell metabolism suggests that tissues may respond to mechanical stimulation via loading-induced flow of the interstitial fluids. During the culture, cells are subject to a flow of culture medium. Flow properties such as flow field, flow regime (e.g. turbulent or laminar), flow pattern (e.g. circular), entity and distribution of the shear stress acting on the cells greatly influence fundamental aspects of cell function, such as regulation and gene expression. This has been demonstrated for endothelial cells and significant research efforts are underway to elucidate these mechanisms in various other biological systems. Local fluid dynamics is also responsible of the mass transfer of nutrients and catabolites as well as oxygenation through the tissue. Most of the attempts to culture tissue-engineered constructs in vitro have utilized either stationary cultures or systems generating relatively small mechanical forces. For example, cartilage constructs have been cultured in spinner flasks under mixed or unmixed conditions, in simulated and in real microgravity. In these mixing studies, however, it is difficult to definitively quantify the effects of mixing-induced mechanical forces from those of convection-enhanced transport of nutrients to and of catabolites away from the cells. At the state of the art, the presence of a more controlled mechanical environment may be the condition required in order to study the biochemical and mechanical response of these biological systems. Such a controlled environment could lead to an advanced fluid dynamic design of the culture chamber that could both enhance the local mass transfer phenomena and match the needs of specific macroscopic mechanical effects in tissue development. The bioreactor is an excellent example of how the skills and resources of two distinctly different fields can complement each other. Microgravity can be used to enhance the formation of tissue like aggregates in specially designed bioreactors. Theoretical and experimental projects are under way to improve cell culture techniques using microgravity conditions experienced during space flights. Bioreactors usable under space flight conditions impose constructional principles which are different from those intended solely for ground applications. The Columbus Laboratory as part of the International Space Station (ISS) will be an evolving facility in low Earth orbit. Its mission is to support scientific, technological, and commercial activities in space. A goal of this research is to design a unique bioreactor for use sequentially from ground research to space research. One of the particularities of the simulated microgravity obtained through time averaging of the weight vector is that by varying the rotational velocity the same results can be obtained with a different value of g. One of the first applications of this technique in space biology was in fact the Rotating Wall Vessel developed by NASA, and originally designed to protect cell culture from the high shear forces generated during the launch and the landing of the Space Shuttle. A Bioreactor that is used both for ground and flight experiments provides the additional benefit of isolating dependent variable of gravity. This continuity will provide a means to compare results to a control experiment.

Fibrous composite material for textile heart valve design: in vitro assessment.

PubMed

Amri, Amna; Laroche, Gaetan; Chakfe, Nabil; Heim, Frederic

2018-04-17

With over 150,000 implantations performed over the world, transcatheter aortic valve replacement (TAVR) has become a surgical technique, which largely competes with open surgery valve replacement for an increasing number of patients. The success of the procedure favors the research toward synthetic valve leaflet materials as an alternative to biological tissues, whose durability remains unknown. In particular, fibrous constructions have recently proven to be durable in vivo over a 6-month period of time in animal sheep models. Exaggerated fibrotic tissue formation remains, however, a critical issue to be addressed. This work investigates the design of a composite fibrous construction combining a woven polyethylene terephthalate (PET) layer and a non-woven PET mat, which are expected to provide, respectively, strength and appropriate topography toward limited fibrotic tissue ingrowth. For this purpose, a specific equipment has been developed to produce non-woven PET mats made from fibers with small diameter. These mats were assembled with woven PET substrates using various assembling techniques in order to obtain hybrid fibrous constructions. The physical and mechanical properties of the obtained materials were assessed and valve samples were manufactured to be tested in vitro for hydrodynamic performances. The results show that the composite fibrous construction is characterized by properties suitable for the valve leaflet function, but the durability of the assembling is however limited under accelerated cyclic loading.

Effect of Implanting a Soft Tissue Autograft in a Central-Third Patellar Tendon Defect: Biomechanical and Histological Comparisons

PubMed Central

Kinneberg, Kirsten R. C.; Galloway, Marc T.; Butler, David L.; Shearn, Jason T.

2011-01-01

Previous studies by our laboratory have demonstrated that implanting a stiffer tissue engineered construct at surgery is positively correlated with repair tissue stiffness at 12 weeks. The objective of this study was to test this correlation by implanting a construct that matches normal tissue biomechanical properties. To do this, we utilized a soft tissue patellar tendon autograft to repair a central-third patellar tendon defect. Patellar tendon auto-graft repairs were contrasted against an unfilled defect repaired by natural healing (NH). We hypothesized that after 12 weeks, patellar tendon autograft repairs would have biomechanical properties superior to NH. Bilateral defects were established in the central-third patellar tendon of skeletally mature (one year old), female New Zealand White rabbits (n = 10). In one limb, the excised tissue, the patellar tendon autograft, was sutured into the defect site. In the contralateral limb, the defect was left empty (natural healing). After 12 weeks of recovery, the animals were euthanized and their limbs were dedicated to bio-mechanical (n = 7) or histological (n = 3) evaluations. Only stiffness was improved by treatment with patellar tendon autograft relative to natural healing (p = 0.009). Additionally, neither the patellar tendon autograft nor natural healing repairs regenerated a normal zonal insertion site between the tendon and bone. Immunohistochemical staining for collagen type II demonstrated that fibrocartilage-like tissue was regenerated at the tendon-bone interface for both repairs. However, the tissue was disorganized. Insufficient tissue integration at the tendon-to-bone junction led to repair tissue failure at the insertion site during testing. It is important to re-establish the tendon-to-bone insertion site because it provides joint stability and enables force transmission from muscle to tendon and subsequent loading of the tendon. Without loading, tendon mechanical properties deteriorate. Future studies by our laboratory will investigate potential strategies to improve patellar tendon autograft integration into bone using this model. [DOI: 10.1115/1.4004948] PMID:22010737

Extracellular matrix fragmentation in young, healthy cartilaginous tissues.

PubMed

Craddock, R J; Hodson, N W; Ozols, M; Shearer, T; Hoyland, J A; Sherratt, M J

2018-02-09

Although the composition and structure of cartilaginous tissues is complex, collagen II fibrils and aggrecan are the most abundant assemblies in both articular cartilage (AC) and the nucleus pulposus (NP) of the intervertebral disc (IVD). Whilst structural heterogeneity of intact aggrecan ( containing three globular domains) is well characterised, the extent of aggrecan fragmentation in healthy tissues is poorly defined. Using young, yet skeletally mature (18-30 months), bovine AC and NP tissues, it was shown that, whilst the ultrastructure of intact aggrecan was tissue-dependent, most molecules (AC: 95 %; NP: 99.5 %) were fragmented (lacking one or more globular domains). Fragments were significantly smaller and more structurally heterogeneous in the NP compared with the AC (molecular area; AC: 8543 nm2; NP: 4625 nm2; p < 0.0001). In contrast, fibrillar collagen appeared structurally intact and tissue-invariant. Molecular fragmentation is considered indicative of a pathology; however, these young, skeletally mature tissues were histologically and mechanically (reduced modulus: AC: ≈ 500 kPa; NP: ≈ 80 kPa) comparable to healthy tissues and devoid of notable gelatinase activity (compared with rat dermis). As aggrecan fragmentation was prevalent in neonatal bovine AC (99.5 % fragmented, molecular area: 5137 nm2) as compared with mature AC (95.0 % fragmented, molecular area: 8667 nm2), it was hypothesised that targeted proteolysis might be an adaptive process that modified aggrecan packing (as simulated computationally) and, hence, tissue charge density, mechanical properties and porosity. These observations provided a baseline against which pathological and/or age-related fragmentation of aggrecan could be assessed and suggested that new strategies might be required to engineer constructs that mimic the mechanical properties of native cartilaginous tissues.

Measurement and Comparison of Mechanical Properties of Nitinol Stents

NASA Astrophysics Data System (ADS)

Hanus, Josef; Zahora, Jiri

2005-01-01

The self expandable Nitinol stents or stentgrafts are typically used for miniinvasive treatment of stenosis and aneurysms in the cardiovascular system. The minimal traumatisation of the patient, shorter time of hospitalization are typical advantages of these methods. More than ten years of experience has yielded also important information about the performance of stents in interaction with biological system and the possible problems related with it. The leakage or the shift of stent are some typical disadvantages, that can be related among other in the construction of the stent. The problem is that the mechanical properties, dimensions and the dynamical properties of the stent do not exactly correspond to the properties of the vessel or generally of tissue where this stent is introduced. The measurement, the description and the comparison of the relations between the mechanical properties of stents and tissues can be one of the possible ways to minimize these disadvantages. The developed original computer controlled measuring system allows the measurement of mechanical properties of stents, the measurement of strain-stress curves or simulation of interaction of the stent and vessel for exactly defined hemodynamic conditions. We measured and compared the mechanical parameters of different selfexpandable Nitinol stents, which differed in geometry (radius and length), in the type of construction (number of branches and rising of winding) and in the diameter of used wire. The results of measurements confirmed the theoretical assumptions that just the diameter of the Nitinol wire significantly influences the rigidity and the level of compressibility of the stent as well. A compromise must be found between the required rigidity of the stent and the minimal size of the delivery system. The exact description of the relation between the mechanical properties and geometry and construction of the stents enables to design the stent to fit the patient and it is expected that this access improves the efficiency of treatment. The results of measurement are also necessary for the design and identification of the parameters of the models of the stents.

From Microscale Devices to 3D Printing: Advances in Fabrication of 3D Cardiovascular Tissues

PubMed Central

Borovjagin, Anton V.; Ogle, Brenda; Berry, Joel; Zhang, Jianyi

2016-01-01

Current strategies for engineering cardiovascular cells and tissues have yielded a variety of sophisticated tools for studying disease mechanisms, for development of drug therapies, and for fabrication of tissue equivalents that may have application in future clinical use. These efforts are motivated by the need to extend traditional two-dimensional (2D) cell culture systems into 3D to more accurately replicate in vivo cell and tissue function of cardiovascular structures. Developments in microscale devices and bioprinted 3D tissues are beginning to supplant traditional 2D cell cultures and pre-clinical animal studies that have historically been the standard for drug and tissue development. These new approaches lend themselves to patient-specific diagnostics, therapeutics, and tissue regeneration. The emergence of these technologies also carries technical challenges to be met before traditional cell culture and animal testing become obsolete. Successful development and validation of 3D human tissue constructs will provide powerful new paradigms for more cost effective and timely translation of cardiovascular tissue equivalents. PMID:28057791

Polypropylene Surgical Mesh Coated with Extracellular Matrix Mitigates the Host Foreign Body Response

PubMed Central

Wolf, Matthew T.; Carruthers, Christopher A.; Dearth, Christopher L.; Crapo, Peter M.; Huber, Alexander; Burnsed, Olivia A.; Londono, Ricardo; Johnson, Scott A.; Daly, Kerry A.; Stahl, Elizabeth C.; Freund, John M.; Medberry, Christopher J.; Carey, Lisa E.; Nieponice, Alejandro; Amoroso, Nicholas J.; Badylak, Stephen F.

2013-01-01

Surgical mesh devices composed of synthetic materials are commonly used for ventral hernia repair. These materials provide robust mechanical strength and are quickly incorporated into host tissue; factors which contribute to reduced hernia recurrence rates. However, such mesh devices cause a foreign body response with the associated complications of fibrosis and patient discomfort. In contrast, surgical mesh devices composed of naturally occurring extracellular matrix (ECM) are associated with constructive tissue remodeling, but lack the mechanical strength of synthetic materials. A method for applying a porcine dermal ECM hydrogel coating to a polypropylene mesh is described herein with the associated effects upon the host tissue response and biaxial mechanical behavior. Uncoated and ECM coated heavy-weight BARD™ Mesh were compared to the light-weight ULTRAPRO™ and BARD™ Soft Mesh devices in a rat partial thickness abdominal defect overlay model. The ECM coated mesh attenuated the pro-inflammatory response compared to all other devices, with a reduced cell accumulation and fewer foreign body giant cells. The ECM coating degraded by 35 days, and was replaced with loose connective tissue compared to the dense collagenous tissue associated with the uncoated polypropylene mesh device. Biaxial mechanical characterization showed that all of the mesh devices were of similar isotropic stiffness. Upon explantation, the light-weight mesh devices were more compliant than the coated or uncoated heavy-weight devices. The present study shows that an ECM coating alters the default host response to a polypropylene mesh, but not the mechanical properties in an acute in vivo abdominal repair model. PMID:23873846

Enhanced mechanical properties of thermosensitive chitosan hydrogel by silk fibers for cartilage tissue engineering.

PubMed

Mirahmadi, Fereshteh; Tafazzoli-Shadpour, Mohammad; Shokrgozar, Mohammad Ali; Bonakdar, Shahin

2013-12-01

Articular cartilage has limited repair capability following traumatic injuries and current methods of treatment remain inefficient. Reconstructing cartilage provides a new way for cartilage repair and natural polymers are often used as scaffold because of their biocompatibility and biofunctionality. In this study, we added degummed chopped silk fibers and electrospun silk fibers to the thermosensitive chitosan/glycerophosphate hydrogels to reinforce two hydrogel constructs which were used as scaffold for hyaline cartilage regeneration. The gelation temperature and gelation time of hydrogel were analyzed by the rheometer and vial tilting method. Mechanical characterization was measured by uniaxial compression, indentation and dynamic mechanical analysis assay. Chondrocytes were then harvested from the knee joint of the New Zealand white rabbits and cultured in constructs. The cell proliferation, viability, production of glycosaminoglycans and collagen type II were assessed. The results showed that mechanical properties of the hydrogel were significantly enhanced when a hybrid with two layers of electrospun silk fibers was made. The results of GAG and collagen type II in cell-seeded scaffolds indicate support of the chondrogenic phenotype for chondrocytes with a significant increase in degummed silk fiber-hydrogel composite for GAG content and in two-layer electrospun fiber-hydrogel composite for Col II. It was concluded that these two modified scaffolds could be employed for cartilage tissue engineering. © 2013.

Hybrid Carbon-Based Scaffolds for Applications in Soft Tissue Reconstruction

PubMed Central

Lafdi, Khalid; Joseph, Robert M.; Tsonis, Panagiotis A.

2012-01-01

Current biomedical scaffolds utilized in surgery to repair soft tissues commonly fail to meet the optimal combination of biomechanical and tissue regenerative properties. Carbon is a scaffold alternative that potentially optimizes the balance between mechanical strength, durability, and function as a cell and biologics delivery vehicle that is necessary to restore tissue function while promoting tissue repair. The goals of this study were to investigate the feasibility of fabricating hybrid fibrous carbon scaffolds modified with biopolymer, polycaprolactone and to analyze their mechanical properties and ability to support cell growth and proliferation. Environmental scanning electron microscopy, micro-computed tomography, and cell adhesion and cell proliferation studies were utilized to test scaffold suitability as a cell delivery vehicle. Mechanical properties were tested to examine load failure and elastic modulus. Results were compared to an acellular dermal matrix scaffold control (GraftJacket® [GJ] Matrix), selected for its common use in surgery for the repair of soft tissues. Results indicated that carbon scaffolds exhibited similar mechanical maximums and capacity to support fibroblast adhesion and proliferation in comparison with GJ. Fibroblast adhesion and proliferation was collinear with carbon fiber orientation in regions of sparsely distributed fibers and occurred in clusters in regions of higher fiber density and low porosity. Overall, fibroblast adhesion and proliferation was greatest in lower porosity carbon scaffolds with highly aligned fibers. Stepwise multivariate regression showed that the variability in maximum load of carbon scaffolds and controls were dependent on unique and separate sets of parameters. These finding suggested that there were significant differences in the functional implications of scaffold design and material properties between carbon and dermis derived scaffolds that affect scaffold utility as a tissue replacement construct. PMID:22092333

Assessing the functional mechanical properties of bioengineered organs with emphasis on the lung.

PubMed

Suki, Béla

2014-09-01

Recently, an exciting new approach has emerged in regenerative medicine pushing the forefront of tissue engineering to create bioartificial organs. The basic idea is to create biological scaffolds made of extracellular matrix (ECM) that preserves the three-dimensional architecture of an entire organ. These scaffolds are then used as templates for functional tissue and organ reconstruction after re-seeding the structure with stem cells or appropriately differentiated cells. In order to make sure that these bioartificial organs will be able to function in the mechanical environment of the native tissue, it is imperative to fully characterize their mechanical properties and match them with those of the normal native organs. This mini-review briefly summarizes modern measurement techniques of mechanical function characterized mostly by the material or volumetric stiffness. Micro-scale and macro-scale techniques such as atomic force microscopy and the tissue strip stress-strain approach are discussed with emphasis on those that combine mechanical measurements with structural visualization. Proper micro-scale stiffness helps attachment and differentiation of cells in the bioartificial organ whereas macro-scale functionality is provided by the overall mechanical properties of the construct. Several approaches including failure mechanics are also described, which specifically probe the contributions of the main ECM components including collagen, elastin, and proteoglycans to organ level ECM function. Advantages, drawbacks, and possible pitfalls as well as interpretation of the data are given throughout. Finally, specific techniques to assess the functionality of the ECM of bioartificial lungs are separately discussed. © 2014 Wiley Periodicals, Inc.

Fourier Transform Infrared Imaging Microspectroscopy and Tissue-Level Mechanical Testing Reveal Intraspecies Variation in Mouse Bone Mineral and Matrix Composition

PubMed Central

Courtland, Hayden-William; Nasser, Philip; Goldstone, Andrew B.; Spevak, Lyudmila; Boskey, Adele L.; Jepsen, Karl J.

2009-01-01

Fracture susceptibility is heritable and dependent upon bone morphology and quality. However, studies of bone quality are typically overshadowed by emphasis on bone geometry and bone mineral density. Given that differences in mineral and matrix composition exist in a variety of species, we hypothesized that genetic variation in bone quality and tissue-level mechanical properties would also exist within species. Sixteen-week-old female A/J, C57BL/6J (B6), and C3H/HeJ (C3H) inbred mouse femora were analyzed using Fourier transform infrared imaging and tissue-level mechanical testing for variation in mineral composition, mineral maturity, collagen cross-link ratio, and tissue-level mechanical properties. A/J femora had an increased mineral-to-matrix ratio compared to B6. The C3H mineral-to-matrix ratio was intermediate of A/J and B6. C3H femora had reduced acid phosphate and carbonate levels and an increased collagen cross-link ratio compared to A/J and B6. Modulus values paralleled mineral-to-matrix values, with A/J femora being the most stiff, B6 being the least stiff, and C3H having intermediate stiffness. In addition, work-to-failure varied among the strains, with the highly mineralized and brittle A/J femora performing the least amount of work-to-failure. Inbred mice are therefore able to differentially modulate the composition of their bone mineral and the maturity of their bone matrix in conjunction with tissue-level mechanical properties. These results suggest that specific combinations of bone quality and morphological traits are genetically regulated such that mechanically functional bones can be constructed in different ways. PMID:18855037

Fourier transform infrared imaging microspectroscopy and tissue-level mechanical testing reveal intraspecies variation in mouse bone mineral and matrix composition.

PubMed

Courtland, Hayden-William; Nasser, Philip; Goldstone, Andrew B; Spevak, Lyudmila; Boskey, Adele L; Jepsen, Karl J

2008-11-01

Fracture susceptibility is heritable and dependent upon bone morphology and quality. However, studies of bone quality are typically overshadowed by emphasis on bone geometry and bone mineral density. Given that differences in mineral and matrix composition exist in a variety of species, we hypothesized that genetic variation in bone quality and tissue-level mechanical properties would also exist within species. Sixteen-week-old female A/J, C57BL/6J (B6), and C3H/HeJ (C3H) inbred mouse femora were analyzed using Fourier transform infrared imaging and tissue-level mechanical testing for variation in mineral composition, mineral maturity, collagen cross-link ratio, and tissue-level mechanical properties. A/J femora had an increased mineral-to-matrix ratio compared to B6. The C3H mineral-to-matrix ratio was intermediate of A/J and B6. C3H femora had reduced acid phosphate and carbonate levels and an increased collagen cross-link ratio compared to A/J and B6. Modulus values paralleled mineral-to-matrix values, with A/J femora being the most stiff, B6 being the least stiff, and C3H having intermediate stiffness. In addition, work-to-failure varied among the strains, with the highly mineralized and brittle A/J femora performing the least amount of work-to-failure. Inbred mice are therefore able to differentially modulate the composition of their bone mineral and the maturity of their bone matrix in conjunction with tissue-level mechanical properties. These results suggest that specific combinations of bone quality and morphological traits are genetically regulated such that mechanically functional bones can be constructed in different ways.

Physiologically relevant organs on chips.

PubMed

Yum, Kyungsuk; Hong, Soon Gweon; Healy, Kevin E; Lee, Luke P

2014-01-01

Recent advances in integrating microengineering and tissue engineering have generated promising microengineered physiological models for experimental medicine and pharmaceutical research. Here we review the recent development of microengineered physiological systems, or also known as "ogans-on-chips", that reconstitute the physiologically critical features of specific human tissues and organs and their interactions. This technology uses microengineering approaches to construct organ-specific microenvironments, reconstituting tissue structures, tissue-tissue interactions and interfaces, and dynamic mechanical and biochemical stimuli found in specific organs, to direct cells to assemble into functional tissues. We first discuss microengineering approaches to reproduce the key elements of physiologically important, dynamic mechanical microenvironments, biochemical microenvironments, and microarchitectures of specific tissues and organs in microfluidic cell culture systems. This is followed by examples of microengineered individual organ models that incorporate the key elements of physiological microenvironments into single microfluidic cell culture systems to reproduce organ-level functions. Finally, microengineered multiple organ systems that simulate multiple organ interactions to better represent human physiology, including human responses to drugs, is covered in this review. This emerging organs-on-chips technology has the potential to become an alternative to 2D and 3D cell culture and animal models for experimental medicine, human disease modeling, drug development, and toxicology. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human cartilage tissue fabrication using three-dimensional inkjet printing technology.

PubMed

Cui, Xiaofeng; Gao, Guifang; Yonezawa, Tomo; Dai, Guohao

2014-06-10

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

Homologous structure-function relationships between native fibrocartilage and tissue engineered from MSC-seeded nanofibrous scaffolds.

PubMed

Nerurkar, Nandan L; Han, Woojin; Mauck, Robert L; Elliott, Dawn M

2011-01-01

Understanding the interplay of composition, organization and mechanical function in load-bearing tissues is a prerequisite in the successful engineering of tissues to replace diseased ones. Mesenchymal stem cells (MSCs) seeded on electrospun scaffolds have been successfully used to generate organized tissues that mimic fibrocartilages such as the knee meniscus and the annulus fibrosus of the intervertebral disc. While matrix deposition has been observed in parallel with improved mechanical properties, how composition, organization, and mechanical function are related is not known. Moreover, how this relationship compares to that of native fibrocartilage is unclear. Therefore, in the present work, functional fibrocartilage constructs were formed from MSC-seeded nanofibrous scaffolds, and the roles of collagen and glycosaminoglycan (GAG) in compressive and tensile properties were determined. MSCs deposited abundant collagen and GAG over 120 days of culture, and these extracellular molecules were organized in such a way that they performed similar mechanical functions to their native roles: collagen dominated the tensile response while GAG was important for compressive properties. GAG removal resulted in significant stiffening in tension. A similar stiffening response was observed when GAG was removed from native inner annulus fibrosus, suggesting an interaction between collagen fibers and their surrounding extrafibrillar matrix that is shared by both engineered and native fibrocartilages. These findings strongly support the use of electrospun scaffolds and MSCs for fibrocartilage tissue engineering, and provide insight on the structure-function relations of both engineered and native biomaterials. Copyright © 2010 Elsevier Ltd. All rights reserved.

HOMOLOGOUS STRUCTURE-FUNCTION RELATIONSHIPS BETWEEN NATIVE FIBROCARTILAGE AND TISSUE ENGINEERED FROM MSC-SEEDED NANOFIBROUS SCAFFOLDS

PubMed Central

Nerurkar, Nandan L.; Han, Woojin; Mauck, Robert L.; Elliott, Dawn M.

2010-01-01

Understanding the interplay of composition, organization and mechanical function in load-bearing tissues is a prerequisite in the successful engineering of replacement tissues for diseased ones. Mesenchymal stem cells (MSCs) seeded on electrospun scaffolds have been successfully used to generate organized tissues that mimic fibrocartilages such as the knee meniscus and the annulus fibrosus of the intervertebral disc. While matrix deposition has been observed in parallel with improved mechanical properties, how composition, organization, and mechanical function are related is not known. Moreover, how this relationship compares to that of native fibrocartilage is unclear. Therefore, in the present work, functional fibrocartilage constructs were formed from MSC-seeded nanofibrous scaffolds, and the roles of collagen and glycosaminoglycan (GAG) in compressive and tensile properties were determined. MSCs deposited abundant collagen and GAG over 120 days of culture, and these extracellular molecules were organized in such a way that they performed similar mechanical functions to their native roles: collagen dominated the tensile response while GAG was important for compressive properties. GAG removal resulted in significant stiffening in tension. A similar stiffening response was observed when GAG was removed from native inner annulus fibrosus, suggesting an interaction between collagen fibers and their surrounding extrafibrillar matrix that is shared by both engineered and native fibrocartilages. These findings strongly support the use of electrospun scaffolds and MSCs for fibrocartilage tissue engineering, and provide insight on the structure-function relations of both engineered and native biomaterials. PMID:20880577

Monitoring sinew contraction during formation of tissue-engineered fibrin-based ligament constructs.

PubMed

Paxton, Jennifer Z; Wudebwe, Uchena N G; Wang, Anqi; Woods, Daniel; Grover, Liam M

2012-08-01

The ability to study the gross morphological changes occurring during tissue formation is vital to producing tissue-engineered structures of clinically relevant dimensions in vitro. Here, we have used nondestructive methods of digital imaging and optical coherence tomography to monitor the early-stage formation and subsequent maturation of fibrin-based tissue-engineered ligament constructs. In addition, the effect of supplementation with essential promoters of collagen synthesis, ascorbic acid (AA) and proline (P), has been assessed. Contraction of the cell-seeded fibrin gel occurs unevenly within the first 5 days of culture around two fixed anchor points before forming a longitudinal ligament-like construct. AA+P supplementation accelerates gel contraction in the maturation phase of development, producing ligament-like constructs with a higher collagen content and distinct morphology to that of unsupplemented constructs. These studies highlight the importance of being able to control the methods of tissue formation and maturation in vitro to enable the production of tissue-engineered constructs with suitable replacement tissue characteristics for repair of clinical soft-tissue injuries.

Functional reconstruction of critical-sized load-bearing bone defects using a Sclerostin-targeting miR-210-3p-based construct to enhance osteogenic activity.

PubMed

Hu, Bin; Li, Yan; Wang, Mohan; Zhu, Youming; Zhou, Yong; Sui, Baiyan; Tan, Yu; Ning, Yujie; Wang, Jie; He, Jiacai; Yang, Chi; Zou, Duohong

2018-06-10

A considerable amount of research has focused on improving regenerative therapy strategies for repairing defects in load-bearing bones. The enhancement of tissue regeneration with microRNAs (miRNAs) is being developed because miRNAs can simultaneously regulate multiple signaling pathways in an endogenous manner. In this study, we developed a miR-210-based bone repair strategy. We identified a miRNA (miR-210-3p) that can simultaneously up-regulate the expression of multiple key osteogenic genes in vitro. This process resulted in enhanced bone formation in a subcutaneous mouse model with a miR-210-3p/poly-L-lactic acid (PLLA)/bone marrow-derived stem cell (BMSC) construct. Furthermore, we constructed a model of critical-sized load-bearing bone defects and implanted a miR-210-3p/β-tricalcium phosphate (β-TCP)/bone mesenchymal stem cell (BMSC) construct into the defect. We found that the load-bearing defect was almost fully repaired using the miR-210-3p construct. We also identified a new mechanism by which miR-210-3p regulates Sclerostin protein levels. This miRNA-based strategy may yield novel therapeutic methods for the treatment of regenerative defects in vital load-bearing bones by utilizing miRNA therapy for tissue engineering. The destroyed maxillofacial bone reconstruction is still a real challenge for maxillofacial surgeon, due to that functional bone reconstruction involved load-bearing. Base on the above problem, this paper developed a novel miR-210-3p/β-tricalcium phosphate (TCP)/bone marrow-derived stem cell (BMSC) construct (miR-210-3p/β-TCP/BMSCs), which lead to functional reconstruction of critical-size mandible bone defect. We found that the load-bearing defect was almost fully repaired using the miR-210-3p construct. In addition, we also found the mechanism of how the delivered microRNA activated the signaling pathways of endogenous stem cells, leading to the defect regeneration. This miRNA-based strategy can be used to regenerate defects in vital load-bearing bones, thus addressing a critical challenge in regenerative medicine by utilizing miRNA therapy for tissue engineering. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Skeletal muscle derived stem cells microintegrated into a biodegradable elastomer for reconstruction of the abdominal wall.

PubMed

Takanari, Keisuke; Hashizume, Ryotaro; Hong, Yi; Amoroso, Nicholas J; Yoshizumi, Tomo; Gharaibeh, Burhan; Yoshida, Osamu; Nonaka, Kazuhiro; Sato, Hideyoshi; Huard, Johnny; Wagner, William R

2017-01-01

A variety of techniques have been applied to generate tissue engineered constructs, where cells are combined with degradable scaffolds followed by a period of in vitro culture or direct implantation. In the current study, a cellularized scaffold was generated by concurrent deposition of electrospun biodegradable elastomer (poly(ester urethane)urea, PEUU) and electrosprayed culture medium + skeletal muscle-derived stem cells (MDSCs) or electrosprayed culture medium alone as a control. MDSCs were obtained from green fluorescent protein (GFP) transgenic rats. The created scaffolds were implanted into allogenic strain-matched rats to replace a full thickness abdominal wall defect. Both control and MDSC-integrated scaffolds showed extensive cellular infiltration at 4 and 8 wk. The number of blood vessels was higher, the area of residual scaffold was lower, number of multinucleated giant cells was lower and area of connective tissue was lower in MDSC-integrated scaffolds (p < 0.05). GFP + cells co-stained positive for VEGF. Bi-axial mechanical properties of the MDSC-microintegrated constructs better approximated the anisotropic behavior of the native abdominal wall. GFP + cells were observed throughout the scaffold at ∼5% of the cell population at 4 and 8 wk. RNA expression at 4 wk showed higher expression of early myogenic marker Pax7, and b-FGF in the MDSC group. Also, higher expression of myogenin and VEGF were seen in the MDSC group at both 4 and 8 wk time points. The paracrine effect of donor cells on host cells likely contributed to the differences found in vivo between the groups. This approach for the rapid creation of highly-cellularized constructs with soft tissue like mechanics offers an attractive methodology to impart cell-derived bioactivity into scaffolds providing mechanical support during the healing process and might find application in a variety of settings. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tissue-mimicking bladder wall phantoms for evaluating acoustic radiation force-optical coherence elastography systems.

PubMed

Ejofodomi, O'tega A; Zderic, Vesna; Zara, Jason M

2010-04-01

Acoustic radiation force-optical coherence elastography (ARF-OCE) systems are novel imaging systems that have the potential to simultaneously quantify and characterize the optical and mechanical properties of in vivo tissues. This article presents the construction of bladder wall phantoms for use in ARF-OCE systems. Mechanical, acoustic, and optical properties are reported and compared to published values for the urinary bladder. The phantom consisted of 0.2000 +/- 0.0089 and 6.0000 +/- 0.2830 microm polystyrene microspheres (Polysciences Inc., Warrington, PA, Catalog Nos. 07304 and 07312), 7.5 +/- 1.5 microm copolymer microspheres composed of acrylonitrile and vinylidene chloride, (Expancel, Duluth, GA, Catalog No. 461 DU 20), and bovine serum albumin within a gelatin matrix. Young's modulus was measured by successive compression of the phantom and obtaining the slope of the resulting force-displacement data. Acoustic measurements were performed using the transmission method. The phantoms were submerged in a water bath and placed between transmitting and receiving 13 mm diameter unfocused transducers operating at a frequency of 3.5 MHz. A MATLAB algorithm to extract the optical scattering coefficient from optical coherence tomography (OCT) images of the phantom was used. The phantoms possess a Young's modulus of 17.12 +/- 2.72 kPa, a mass density of 1.05 +/- 0.02 g/cm3, an acoustic attenuation coefficient of 0.66 +/- 0.08 dB/cm/MHz, a speed of sound of 1591 +/- 8.76 m/s, and an optical scattering coefficient of 1.80 +/- 0.23 mm(-1). Ultrasound and OCT images of the bladder wall phantom are presented. A material that mimics the mechanical, optical, and acoustic properties of healthy bladder wall has been developed. This tissue-mimicking bladder wall phantom was developed as a control tool to investigate the feasibility of using ARF-OCE to detect the mechanical and optical changes that may be indicative of the onset or development of cancer in the urinary bladder. By following the methods used in this article, phantoms matching the optical, acoustic, and mechanical properties of other biological tissues can also be constructed.

A silk-based scaffold platform with tunable architecture for engineering critically-sized tissue constructs.

PubMed

Wray, Lindsay S; Rnjak-Kovacina, Jelena; Mandal, Biman B; Schmidt, Daniel F; Gil, Eun Seok; Kaplan, David L

2012-12-01

In the field of tissue engineering and regenerative medicine there is significant unmet need for critically-sized, fully degradable biomaterial scaffold systems with tunable properties for optimizing tissue formation in vitro and tissue regeneration in vivo. To address this need, we have developed a silk-based scaffold platform that has tunable material properties, including localized and bioactive functionalization, degradation rate, and mechanical properties and that provides arrays of linear hollow channels for delivery of oxygen and nutrients throughout the scaffold bulk. The scaffolds can be assembled with dimensions that range from millimeters to centimeters, addressing the need for a critically-sized platform for tissue formation. We demonstrate that the hollow channel arrays support localized and confluent endothelialization. This new platform offers a unique and versatile tool for engineering 'tailored' scaffolds for a range of tissue engineering and regenerative medicine needs. Copyright © 2012 Elsevier Ltd. All rights reserved.

Biomechanical regulation of in vitro cardiogenesis for tissue-engineered heart repair.

PubMed

Zimmermann, Wolfram-Hubertus

2013-01-01

The heart is a continuously pumping organ with an average lifespan of eight decades. It develops from the onset of embryonic cardiogenesis under biomechanical load, performs optimally within a defined range of hemodynamic load, and fails if acutely or chronically overloaded. Unloading of the heart leads to defective cardiogenesis in utero, but can also lead to a desired therapeutic outcome (for example, in patients with heart failure under left ventricular assist device therapy). In light of the well-documented relevance of mechanical loading for cardiac physiology and pathology, it is plausible that tissue engineers have integrated mechanical stimulation regimens into protocols for heart muscle construction. To achieve optimal results, physiological principles of beat-to-beat myocardial loading and unloading should be simulated. In addition, heart muscle engineering, in particular if based on pluripotent stem cell-derived cardiomyocytes, may benefit from staggered tonic loading protocols to simulate viscoelastic properties of the prenatal and postnatal myocardial stroma. This review will provide an overview of heart muscle mechanics, summarize observations on the role of mechanical loading for heart development and postnatal performance, and discuss how physiological loading regimens can be exploited to advance myocardial tissue engineering towards a therapeutic application.

Biomechanical regulation of in vitro cardiogenesis for tissue-engineered heart repair

PubMed Central

2013-01-01

The heart is a continuously pumping organ with an average lifespan of eight decades. It develops from the onset of embryonic cardiogenesis under biomechanical load, performs optimally within a defined range of hemodynamic load, and fails if acutely or chronically overloaded. Unloading of the heart leads to defective cardiogenesis in utero, but can also lead to a desired therapeutic outcome (for example, in patients with heart failure under left ventricular assist device therapy). In light of the well-documented relevance of mechanical loading for cardiac physiology and pathology, it is plausible that tissue engineers have integrated mechanical stimulation regimens into protocols for heart muscle construction. To achieve optimal results, physiological principles of beat-to-beat myocardial loading and unloading should be simulated. In addition, heart muscle engineering, in particular if based on pluripotent stem cell-derived cardiomyocytes, may benefit from staggered tonic loading protocols to simulate viscoelastic properties of the prenatal and postnatal myocardial stroma. This review will provide an overview of heart muscle mechanics, summarize observations on the role of mechanical loading for heart development and postnatal performance, and discuss how physiological loading regimens can be exploited to advance myocardial tissue engineering towards a therapeutic application. PMID:24229468

Macro- to microscale strain transfer in fibrous tissues is heterogeneous and tissue-specific.

PubMed

Han, Woojin M; Heo, Su-Jin; Driscoll, Tristan P; Smith, Lachlan J; Mauck, Robert L; Elliott, Dawn M

2013-08-06

Mechanical deformation applied at the joint or tissue level is transmitted through the macroscale extracellular matrix to the microscale local matrix, where it is transduced to cells within these tissues and modulates tissue growth, maintenance, and repair. The objective of this study was to investigate how applied tissue strain is transferred through the local matrix to the cell and nucleus in meniscus, tendon, and the annulus fibrosus, as well as in stem cell-seeded scaffolds engineered to reproduce the organized microstructure of these native tissues. To carry out this study, we developed a custom confocal microscope-mounted tensile testing device and simultaneously monitored strain across multiple length scales. Results showed that mean strain was heterogeneous and significantly attenuated, but coordinated, at the local matrix level in native tissues (35-70% strain attenuation). Conversely, freshly seeded scaffolds exhibited very direct and uniform strain transfer from the tissue to the local matrix level (15-25% strain attenuation). In addition, strain transfer from local matrix to cells and nuclei was dependent on fiber orientation and tissue type. Histological analysis suggested that different domains exist within these fibrous tissues, with most of the tissue being fibrous, characterized by an aligned collagen structure and elongated cells, and other regions being proteoglycan (PG)-rich, characterized by a dense accumulation of PGs and rounder cells. In meniscus, the observed heterogeneity in strain transfer correlated strongly with cellular morphology, where rounder cells located in PG-rich microdomains were shielded from deformation, while elongated cells in fibrous microdomains deformed readily. Collectively, these findings suggest that different tissues utilize distinct strain-attenuating mechanisms according to their unique structure and cellular phenotype, and these differences likely alter the local biologic response of such tissues and constructs in response to mechanical perturbation. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Growth factor delivery for oral and periodontal tissue engineering

PubMed Central

Kaigler, Darnell; Cirelli, Joni A; Giannobile, William V

2008-01-01

The treatment of oral and periodontal diseases and associated anomalies accounts for a significant proportion of the healthcare burden, with the manifestations of these conditions being functionally and psychologically debilitating. Growth factors are critical to the development, maturation, maintenance and repair of craniofacial tissues, as they establish an extracellular environment that is conducive to cell and tissue growth. Tissue-engineering principles aim to exploit these properties in the development of biomimetic materials that can provide an appropriate microenvironment for tissue development. These materials have been constructed into devices that can be used as vehicles for delivery of cells, growth factors and DNA. In this review, different mechanisms of drug delivery are addressed in the context of novel approaches to reconstruct and engineer oral- and tooth-supporting structures, namely the periodontium and alveolar bone. PMID:16948560

Bioprinting technology and its applications.

PubMed

Seol, Young-Joon; Kang, Hyun-Wook; Lee, Sang Jin; Atala, Anthony; Yoo, James J

2014-09-01

Bioprinting technology has emerged as a powerful tool for building tissue and organ structures in the field of tissue engineering. This technology allows precise placement of cells, biomaterials and biomolecules in spatially predefined locations within confined three-dimensional (3D) structures. Various bioprinting technologies have been developed and utilized for applications in life sciences, ranging from studying cellular mechanisms to constructing tissues and organs for implantation, including heart valve, myocardial tissue, trachea and blood vessels. In this article, we introduce the general principles and limitations of the most widely used bioprinting technologies, including jetting- and extrusion-based systems. Application-based research focused on tissue regeneration is presented, as well as the current challenges that hamper clinical utility of bioprinting technology. © The Author 2014. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Atomic force microscopy reveals the mechanical design of a modular protein

PubMed Central

Li, Hongbin; Oberhauser, Andres F.; Fowler, Susan B.; Clarke, Jane; Fernandez, Julio M.

2000-01-01

Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanical strength, we can generate proteins with novel elastic properties. Our experiments reveal the simple mechanical design of modular proteins and open the way for the engineering of elastic proteins with defined mechanical properties, which can be used in tissue and fiber engineering. PMID:10823913

Atomic force microscopy reveals the mechanical design of a modular protein.

PubMed

Li, H; Oberhauser, A F; Fowler, S B; Clarke, J; Fernandez, J M

2000-06-06

Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanical strength, we can generate proteins with novel elastic properties. Our experiments reveal the simple mechanical design of modular proteins and open the way for the engineering of elastic proteins with defined mechanical properties, which can be used in tissue and fiber engineering.

Assessment of post-implantation integration of engineered tissues using fluorescence lifetime spectroscopy

NASA Astrophysics Data System (ADS)

Elahi, Sakib F.; Lee, Seung Y.; Lloyd, William R.; Chen, Leng-Chun; Kuo, Shiuhyang; Zhou, Ying; Kim, Hyungjin M.; Kennedy, Robert; Marcelo, Cynthia; Feinberg, Stephen E.; Mycek, Mary-Ann

2018-02-01

Clinical translation of engineered tissue constructs requires noninvasive methods to assess construct health and viability after implantation in patients. However, current practices to monitor post-implantation construct integration are either qualitative (visual assessment) or destructive (tissue histology). As label-free fluorescence lifetime sensing can noninvasively characterize pre-implantation construct viability, we employed a handheld fluorescence lifetime spectroscopy probe to quantitatively and noninvasively assess tissue constructs that were implanted in a murine model. We designed the system to be suitable for intravital measurements: portability, localization with precise maneuverability, and rapid data acquisition. Our model tissue constructs were manufactured from primary human cells to simulate patient variability and were stressed to create a range of health states. Secreted amounts of three cytokines that relate to cellular viability were measured in vitro to assess pre-implantation construct health. In vivo optical sensing assessed tissue integration of constructs at one-week and three-weeks post-implantation. At one-week post-implantation, optical parameters correlated with in vitro pre-implantation secretion levels of all three cytokines (p < 0.05). This relationship was no longer seen at three-weeks post-implantation, suggesting comparable tissue integration independent of preimplantation health. Histology confirmed re-epithelialization of these constructs independent of pre-implantation health state, supporting the lack of a correlation. These results suggest that clinical optical diagnostic tools based on label-free fluorescence lifetime sensing of endogenous tissue fluorophores could noninvasively monitor post-implantation integration of engineered tissues.

Influence of intermittent pressure, fluid flow, and mixing on the regenerative properties of articular chondrocytes.

PubMed

Carver, S E; Heath, C A

1999-11-05

Equine articular chondrocytes, embedded within a polyglycolic acid nonwoven mesh, were cultured with various combinations of intermittent pressure, fluid flow, and mixing to examine the effects of different physical stimuli on neochondrogenesis from young cells. The cell/polymer constructs were cultured first in 125 ml spinner flasks for 1, 2, or 4 weeks and then in a perfusion system with intermittent pressure for a total of up to 6 weeks. Additional constructs were either cultured for all 6 weeks in the spinner flasks or for 1 week in spinners followed by 5 weeks in the perfusion system without intermittent pressure. Tissue constructs cultivated for 2 or 4 weeks in spinner flasks followed by perfusion with intermittent pressure had significantly higher concentrations of both sulfated glycosaminoglycan and collagen than constructs cultured entirely in spinners or almost entirely in the pressure/perfusion system. Initial cultivation in the spinner flasks, with turbulent mixing, enhanced both cell attachment and early development of the extracellular matrix. Subsequent culture with perfusion and intermittent pressure appeared to accelerate matrix formation. While the correlation was much stronger in the pressurized constructs, the compressive modulus was directly proportional to the concentration of sulfated glycosaminoglycan in all physically stressed constructs. Constructs that were not stressed beyond the 1-week seeding period lost mechanical integrity upon harvest, suggesting that physical stimulation, particularly with intermittent pressure, of immature tissue constructs during their development may contribute to their ultimate biomechanical functionality. Copyright 1999 John Wiley & Sons, Inc.

Three types of dermal grafts in rats: the importance of mechanical property and structural design.

PubMed

You, Chuangang; Wang, Xingang; Zheng, Yurong; Han, Chunmao

2013-12-04

To determine how the mechanical property and micro structure affect tissue regeneration and angiogenesis, three types of scaffolds were studied. Acellular dermal matrices (ADM), produced from human skin by removing the epidermis and cells, has been widely used in wound healing because of its high mechanical strength. Collagen scaffolds (CS) incorporated with poly(glycolide-co-L-lactide) (PLGA) mesh forms a well-supported hybrid dermal equivalent poly(glycolide-co-L-lactide) mesh/collagen scaffolds (PMCS). We designed this scaffold to enhance the CS mechanical property. These three different dermal substitutes-ADM, CS and PMCSs are different in the tensile properties and microstructure. Several basic physical characteristics of dermal substitutes were investigated in vitro. To characterize the angiogenesis and tissue regeneration, the materials were embedded subcutaneously in Sprague-Dawley (SD) rats. At weeks 1, 2, 4 and 8 post-surgery, the tissue specimens were harvested for histology, immunohistochemistry and real-time quantitative PCR (RT-qPCR). In vitro studies demonstrated ADM had a higher Young's modulus (6.94 MPa) rather than CS (0.19 MPa) and PMCS (3.33 MPa) groups in the wet state. Compared with ADMs and CSs, PMCSs with three-dimensional porous structures resembling skin and moderate mechanical properties can promote tissue ingrowth more quickly after implantation. In addition, the vascularization of the PMCS group is more obvious than that of the other two groups. The incorporation of a PLGA knitted mesh in CSs can improve the mechanical properties with little influence on the three-dimensional porous microstructure. After implantation, PMCSs can resist the contraction and promote cell infiltration, neotissue formation and blood vessel ingrowth, especially from the mesh side. Although ADM has high mechanical strength, its vascularization is poor because the pore size is too small. In conclusion, the mechanical properties of scaffolds are important for maintaining the three-dimensional microarchitecture of constructs used to induce tissue regeneration and vascularization. The results illustrated that tissue regeneration requires the proper pore size and an appropriate mechanical property like PMCS which could satisfy these conditions to sustain growth.

Three types of dermal grafts in rats: the importance of mechanical property and structural design

PubMed Central

2013-01-01

Background To determine how the mechanical property and micro structure affect tissue regeneration and angiogenesis, three types of scaffolds were studied. Acellular dermal matrices (ADM), produced from human skin by removing the epidermis and cells, has been widely used in wound healing because of its high mechanical strength. Collagen scaffolds (CS) incorporated with poly(glycolide-co-L-lactide) (PLGA) mesh forms a well-supported hybrid dermal equivalent poly(glycolide-co-L-lactide) mesh/collagen scaffolds (PMCS). We designed this scaffold to enhance the CS mechanical property. These three different dermal substitutes—ADM, CS and PMCSs are different in the tensile properties and microstructure. Methods Several basic physical characteristics of dermal substitutes were investigated in vitro. To characterize the angiogenesis and tissue regeneration, the materials were embedded subcutaneously in Sprague–Dawley (SD) rats. At weeks 1, 2, 4 and 8 post-surgery, the tissue specimens were harvested for histology, immunohistochemistry and real-time quantitative PCR (RT-qPCR). Results In vitro studies demonstrated ADM had a higher Young’s modulus (6.94 MPa) rather than CS (0.19 MPa) and PMCS (3.33 MPa) groups in the wet state. Compared with ADMs and CSs, PMCSs with three-dimensional porous structures resembling skin and moderate mechanical properties can promote tissue ingrowth more quickly after implantation. In addition, the vascularization of the PMCS group is more obvious than that of the other two groups. The incorporation of a PLGA knitted mesh in CSs can improve the mechanical properties with little influence on the three-dimensional porous microstructure. After implantation, PMCSs can resist the contraction and promote cell infiltration, neotissue formation and blood vessel ingrowth, especially from the mesh side. Although ADM has high mechanical strength, its vascularization is poor because the pore size is too small. In conclusion, the mechanical properties of scaffolds are important for maintaining the three-dimensional microarchitecture of constructs used to induce tissue regeneration and vascularization. Conclusion The results illustrated that tissue regeneration requires the proper pore size and an appropriate mechanical property like PMCS which could satisfy these conditions to sustain growth. PMID:24304500

Biological and mechanical evaluation of a Bio-Hybrid scaffold for autologous valve tissue engineering.

PubMed

Jahnavi, S; Saravanan, U; Arthi, N; Bhuvaneshwar, G S; Kumary, T V; Rajan, S; Verma, R S

2017-04-01

Major challenge in heart valve tissue engineering for paediatric patients is the development of an autologous valve with regenerative capacity. Hybrid tissue engineering approach is recently gaining popularity to design scaffolds with desired biological and mechanical properties that can remodel post implantation. In this study, we fabricated aligned nanofibrous Bio-Hybrid scaffold made of decellularized bovine pericardium: polycaprolactone-chitosan with optimized polymer thickness to yield the desired biological and mechanical properties. CD44 + , αSMA + , Vimentin + and CD105 - human valve interstitial cells were isolated and seeded on these Bio-Hybrid scaffolds. Subsequent biological evaluation revealed interstitial cell proliferation with dense extra cellular matrix deposition that indicated the viability for growth and proliferation of seeded cells on the scaffolds. Uniaxial mechanical tests along axial direction showed that the Bio-Hybrid scaffolds has at least 20 times the strength of the native valves and its stiffness is nearly 3 times more than that of native valves. Biaxial and uniaxial mechanical studies on valve interstitial cells cultured Bio-Hybrid scaffolds revealed that the response along the axial and circumferential direction was different, similar to native valves. Overall, our findings suggest that Bio-Hybrid scaffold is a promising material for future development of regenerative heart valve constructs in children. Copyright © 2016 Elsevier B.V. All rights reserved.

A Cost-Minimization Analysis of Tissue-Engineered Constructs for Corneal Endothelial Transplantation

PubMed Central

Tan, Tien-En; Peh, Gary S. L.; George, Benjamin L.; Cajucom-Uy, Howard Y.; Dong, Di; Finkelstein, Eric A.; Mehta, Jodhbir S.

2014-01-01

Corneal endothelial transplantation or endothelial keratoplasty has become the preferred choice of transplantation for patients with corneal blindness due to endothelial dysfunction. Currently, there is a worldwide shortage of transplantable tissue, and demand is expected to increase further with aging populations. Tissue-engineered alternatives are being developed, and are likely to be available soon. However, the cost of these constructs may impair their widespread use. A cost-minimization analysis comparing tissue-engineered constructs to donor tissue procured from eye banks for endothelial keratoplasty was performed. Both initial investment costs and recurring costs were considered in the analysis to arrive at a final tissue cost per transplant. The clinical outcomes of endothelial keratoplasty with tissue-engineered constructs and with donor tissue procured from eye banks were assumed to be equivalent. One-way and probabilistic sensitivity analyses were performed to simulate various possible scenarios, and to determine the robustness of the results. A tissue engineering strategy was cheaper in both investment cost and recurring cost. Tissue-engineered constructs for endothelial keratoplasty could be produced at a cost of US$880 per transplant. In contrast, utilizing donor tissue procured from eye banks for endothelial keratoplasty required US$3,710 per transplant. Sensitivity analyses performed further support the results of this cost-minimization analysis across a wide range of possible scenarios. The use of tissue-engineered constructs for endothelial keratoplasty could potentially increase the supply of transplantable tissue and bring the costs of corneal endothelial transplantation down, making this intervention accessible to a larger group of patients. Tissue-engineering strategies for corneal epithelial constructs or other tissue types, such as pancreatic islet cells, should also be subject to similar pharmacoeconomic analyses. PMID:24949869

A cost-minimization analysis of tissue-engineered constructs for corneal endothelial transplantation.

PubMed

Tan, Tien-En; Peh, Gary S L; George, Benjamin L; Cajucom-Uy, Howard Y; Dong, Di; Finkelstein, Eric A; Mehta, Jodhbir S

2014-01-01

Corneal endothelial transplantation or endothelial keratoplasty has become the preferred choice of transplantation for patients with corneal blindness due to endothelial dysfunction. Currently, there is a worldwide shortage of transplantable tissue, and demand is expected to increase further with aging populations. Tissue-engineered alternatives are being developed, and are likely to be available soon. However, the cost of these constructs may impair their widespread use. A cost-minimization analysis comparing tissue-engineered constructs to donor tissue procured from eye banks for endothelial keratoplasty was performed. Both initial investment costs and recurring costs were considered in the analysis to arrive at a final tissue cost per transplant. The clinical outcomes of endothelial keratoplasty with tissue-engineered constructs and with donor tissue procured from eye banks were assumed to be equivalent. One-way and probabilistic sensitivity analyses were performed to simulate various possible scenarios, and to determine the robustness of the results. A tissue engineering strategy was cheaper in both investment cost and recurring cost. Tissue-engineered constructs for endothelial keratoplasty could be produced at a cost of US$880 per transplant. In contrast, utilizing donor tissue procured from eye banks for endothelial keratoplasty required US$3,710 per transplant. Sensitivity analyses performed further support the results of this cost-minimization analysis across a wide range of possible scenarios. The use of tissue-engineered constructs for endothelial keratoplasty could potentially increase the supply of transplantable tissue and bring the costs of corneal endothelial transplantation down, making this intervention accessible to a larger group of patients. Tissue-engineering strategies for corneal epithelial constructs or other tissue types, such as pancreatic islet cells, should also be subject to similar pharmacoeconomic analyses.

Microcomputed tomography characterization of neovascularization in bone tissue engineering applications.

PubMed

Young, Simon; Kretlow, James D; Nguyen, Charles; Bashoura, Alex G; Baggett, L Scott; Jansen, John A; Wong, Mark; Mikos, Antonios G

2008-09-01

Vasculogenesis and angiogenesis have been studied for decades using numerous in vitro and in vivo systems, fulfilling the need to elucidate the mechanisms involved in these processes and to test potential therapeutic agents that inhibit or promote neovascularization. Bone tissue engineering in particular has benefited from the application of proangiogenic strategies, considering the need for an adequate vascular supply during healing and the challenges associated with the vascularization of scaffolds implanted in vivo. Conventional methods of assessing the in vivo angiogenic response to tissue-engineered constructs tend to rely on a two-dimensional assessment of microvessel density within representative histological sections without elaboration of the true vascular tree. The introduction of microcomputed tomography (micro-CT) has recently allowed investigators to obtain a diverse range of high-resolution, three-dimensional characterization of structures, including renal, coronary, and hepatic vascular networks, as well as bone formation within healing defects. To date, few studies have utilized micro-CT to study the vascular response to an implanted tissue engineering scaffold. In this paper, conventional in vitro and in vivo models for studying angiogenesis will be discussed, followed by recent developments in the use of micro-CT for vessel imaging in bone tissue engineering research. A new study demonstrating the potential of contrast-enhanced micro-CT for the evaluation of in vivo neovascularization in bony defects is described, which offers significant potential in the evaluation of bone tissue engineering constructs.

Biocompatible magnetic core-shell nanocomposites for engineered magnetic tissues

NASA Astrophysics Data System (ADS)

Rodriguez-Arco, Laura; Rodriguez, Ismael A.; Carriel, Victor; Bonhome-Espinosa, Ana B.; Campos, Fernando; Kuzhir, Pavel; Duran, Juan D. G.; Lopez-Lopez, Modesto T.

2016-04-01

The inclusion of magnetic nanoparticles into biopolymer matrixes enables the preparation of magnetic field-responsive engineered tissues. Here we describe a synthetic route to prepare biocompatible core-shell nanostructures consisting of a polymeric core and a magnetic shell, which are used for this purpose. We show that using a core-shell architecture is doubly advantageous. First, gravitational settling for core-shell nanocomposites is slower because of the reduction of the composite average density connected to the light polymer core. Second, the magnetic response of core-shell nanocomposites can be tuned by changing the thickness of the magnetic layer. The incorporation of the composites into biopolymer hydrogels containing cells results in magnetic field-responsive engineered tissues whose mechanical properties can be controlled by external magnetic forces. Indeed, we obtain a significant increase of the viscoelastic moduli of the engineered tissues when exposed to an external magnetic field. Because the composites are functionalized with polyethylene glycol, the prepared bio-artificial tissue-like constructs also display excellent ex vivo cell viability and proliferation. When implanted in vivo, the engineered tissues show good biocompatibility and outstanding interaction with the host tissue. Actually, they only cause a localized transitory inflammatory reaction at the implantation site, without any effect on other organs. Altogether, our results suggest that the inclusion of magnetic core-shell nanocomposites into biomaterials would enable tissue engineering of artificial substitutes whose mechanical properties could be tuned to match those of the potential target tissue. In a wider perspective, the good biocompatibility and magnetic behavior of the composites could be beneficial for many other applications.The inclusion of magnetic nanoparticles into biopolymer matrixes enables the preparation of magnetic field-responsive engineered tissues. Here we describe a synthetic route to prepare biocompatible core-shell nanostructures consisting of a polymeric core and a magnetic shell, which are used for this purpose. We show that using a core-shell architecture is doubly advantageous. First, gravitational settling for core-shell nanocomposites is slower because of the reduction of the composite average density connected to the light polymer core. Second, the magnetic response of core-shell nanocomposites can be tuned by changing the thickness of the magnetic layer. The incorporation of the composites into biopolymer hydrogels containing cells results in magnetic field-responsive engineered tissues whose mechanical properties can be controlled by external magnetic forces. Indeed, we obtain a significant increase of the viscoelastic moduli of the engineered tissues when exposed to an external magnetic field. Because the composites are functionalized with polyethylene glycol, the prepared bio-artificial tissue-like constructs also display excellent ex vivo cell viability and proliferation. When implanted in vivo, the engineered tissues show good biocompatibility and outstanding interaction with the host tissue. Actually, they only cause a localized transitory inflammatory reaction at the implantation site, without any effect on other organs. Altogether, our results suggest that the inclusion of magnetic core-shell nanocomposites into biomaterials would enable tissue engineering of artificial substitutes whose mechanical properties could be tuned to match those of the potential target tissue. In a wider perspective, the good biocompatibility and magnetic behavior of the composites could be beneficial for many other applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr00224b

Computational model-informed design and bioprinting of cell-patterned constructs for bone tissue engineering.

PubMed

Carlier, Aurélie; Skvortsov, Gözde Akdeniz; Hafezi, Forough; Ferraris, Eleonora; Patterson, Jennifer; Koç, Bahattin; Van Oosterwyck, Hans

2016-05-17

Three-dimensional (3D) bioprinting is a rapidly advancing tissue engineering technology that holds great promise for the regeneration of several tissues, including bone. However, to generate a successful 3D bone tissue engineering construct, additional complexities should be taken into account such as nutrient and oxygen delivery, which is often insufficient after implantation in large bone defects. We propose that a well-designed tissue engineering construct, that is, an implant with a specific spatial pattern of cells in a matrix, will improve the healing outcome. By using a computational model of bone regeneration we show that particular cell patterns in tissue engineering constructs are able to enhance bone regeneration compared to uniform ones. We successfully bioprinted one of the most promising cell-gradient patterns by using cell-laden hydrogels with varying cell densities and observed a high cell viability for three days following the bioprinting process. In summary, we present a novel strategy for the biofabrication of bone tissue engineering constructs by designing cell-gradient patterns based on a computational model of bone regeneration, and successfully bioprinting the chosen design. This integrated approach may increase the success rate of implanted tissue engineering constructs for critical size bone defects and also can find a wider application in the biofabrication of other types of tissue engineering constructs.

Mueller-matrix mapping of biological tissues in differential diagnosis of optical anisotropy mechanisms of protein networks

NASA Astrophysics Data System (ADS)

Ushenko, V. A.; Sidor, M. I.; Marchuk, Yu F.; Pashkovskaya, N. V.; Andreichuk, D. R.

2015-03-01

We report a model of Mueller-matrix description of optical anisotropy of protein networks in biological tissues with allowance for the linear birefringence and dichroism. The model is used to construct the reconstruction algorithms of coordinate distributions of phase shifts and the linear dichroism coefficient. In the statistical analysis of such distributions, we have found the objective criteria of differentiation between benign and malignant tissues of the female reproductive system. From the standpoint of evidence-based medicine, we have determined the operating characteristics (sensitivity, specificity and accuracy) of the Mueller-matrix reconstruction method of optical anisotropy parameters and demonstrated its effectiveness in the differentiation of benign and malignant tumours.

Structural and rheological characterization of hyaluronic acid-based scaffolds for adipose tissue engineering.

PubMed

Borzacchiello, Assunta; Mayol, Laura; Ramires, Piera A; Pastorello, Andrea; Di Bartolo, Chiara; Ambrosio, Luigi; Milella, Evelina

2007-10-01

In this study the attention has been focused on the ester derivative of hyaluronic acid (HA), HYAFF11, as a potential three-dimensional scaffold in adipose tissue engineering. Different HYAFF11 sponges having different pore sizes, coated or not coated with HA, have been studied from a rheological and morphological point of view in order to correlate their structure to the macroscopic and degradation properties both in vitro and in vivo, using rat model. The in vitro results indicate that the HYAFF11 sponges possess proper structural and mechanical properties to be used as scaffolds for adipose tissue engineering and, among all the analysed samples, uncoated HYAFF11 large-pore sponges showed a longer lasting mechanical stability. From the in vivo results, it was observed that the elastic modulus of scaffolds seeded with preadipocytes, the biohybrid constructs, and explanted after 3 months of implantation in autologous rat model are over one order of magnitude higher than the corresponding values for the native tissue. These results could suggest that the implanted scaffolds can be invaded and populated by different cells, not only adipocytes, that can produce new matrix having different properties from that of adipose tissue.

3D bioprinting of urethra with PCL/PLCL blend and dual autologous cells in fibrin hydrogel: An in vitro evaluation of biomimetic mechanical property and cell growth environment.

PubMed

Zhang, Kaile; Fu, Qiang; Yoo, James; Chen, Xiangxian; Chandra, Prafulla; Mo, Xiumei; Song, Lujie; Atala, Anthony; Zhao, Weixin

2017-03-01

Urethral stricture is a common condition seen after urethral injury. The currently available treatments are inadequate and there is a scarcity of substitute materials used for treatment of urethral stricture. The traditional tissue engineering of urethra involves scaffold design, fabrication and processing of multiple cell types. In this study, we have used 3D bioprinting technology to fabricate cell-laden urethra in vitro with different polymer types and structural characteristics. We hypothesized that use of PCL and PLCL polymers with a spiral scaffold design could mimic the structure and mechanical properties of natural urethra of rabbits, and cell-laden fibrin hydrogel could give a better microenvironment for cell growth. With using an integrated bioprinting system, tubular scaffold was formed with the biomaterials; meanwhile, urothelial cells (UCs) and smooth muscle cells (SMCs) were delivered evenly into inner and outer layers of the scaffold separately within the cell-laden hydrogel. The PCL/PLCL (50:50) spiral scaffold demonstrated mechanical properties equivalent to the native urethra in rabbit. Evaluation of the cell bioactivity in the bioprinted urethra revealed that UCs and SMCs maintained more than 80% viability even at 7days after printing. Both cell types also showed active proliferation and maintained the specific biomarkers in the cell-laden hydrogel. These results provided a foundation for further studies in 3D bioprinting of urethral constructs that mimic the natural urethral tissue in mechanical properties and cell bioactivity, as well a possibility of using the bioprinted construct for in vivo study of urethral implantation in animal model. The 3D bioprinting is a new technique to replace traditional tissue engineering. The present study is the first demonstration that it is feasible to create a urethral construct. Two kinds of biomaterials were used and achieved mechanical properties equivalent to that of native rabbit urethra. Bladder epithelial cells and smooth muscle cells were loaded in hydrogel and maintained sufficient viability and proliferation in the hydrogel. The highly porous scaffold could mimic a natural urethral base-membrane, and facilitate contacts between the printed epithelial cells and smooth muscle cells on both sides of the scaffold. These results provided a strong foundation for future studies on 3D bioprinted urethra. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Alternating air-medium exposure in rotating bioreactors optimizes cell metabolism in 3D novel tubular scaffold polyurethane foams.

PubMed

Tresoldi, Claudia; Stefani, Ilaria; Ferracci, Gaia; Bertoldi, Serena; Pellegata, Alessandro F; Farè, Silvia; Mantero, Sara

2017-04-26

In vitro dynamic culture conditions play a pivotal role in developing engineered tissue grafts, where the supply of oxygen and nutrients, and waste removal must be permitted within construct thickness. For tubular scaffolds, mass transfer is enhanced by introducing a convective flow through rotating bioreactors with positive effects on cell proliferation, scaffold colonization and extracellular matrix deposition. We characterized a novel polyurethane-based tubular scaffold and investigated the impact of 3 different culture configurations over cell behavior: dynamic (i) single-phase (medium) rotation and (ii) double-phase exposure (medium-air) rotation; static (iii) single-phase static culture as control. A new mixture of polyol was tested to create polyurethane foams (PUFs) as 3D scaffold for tissue engineering. The structure obtained was morphologically and mechanically analyzed tested. Murine fibroblasts were externally seeded on the novel porous PUF scaffold, and cultured under different dynamic conditions. Viability assay, DNA quantification, SEM and histological analyses were performed at different time points. The PUF scaffold presented interesting mechanical properties and morphology adequate to promote cell adhesion, highlighting its potential for tissue engineering purposes. Results showed that constructs under dynamic conditions contain enhanced viability and cell number, exponentially increased for double-phase rotation; under this last configuration, cells uniformly covered both the external surface and the lumen. The developed 3D structure combined with the alternated exposure to air and medium provided the optimal in vitro biochemical conditioning with adequate nutrient supply for cells. The results highlight a valuable combination of material and dynamic culture for tissue engineering applications.

Tissue-engineered cartilaginous constructs for the treatment of caprine cartilage defects, including distribution of laminin and type IV collagen.

PubMed

Jeng, Lily; Hsu, Hu-Ping; Spector, Myron

2013-10-01

The purpose of this study was the immunohistochemical evaluation of (1) cartilage tissue-engineered constructs; and (2) the tissue filling cartilage defects in a goat model into which the constructs were implanted, particularly for the presence of the basement membrane molecules, laminin and type IV collagen. Basement membrane molecules are localized to the pericellular matrix in normal adult articular cartilage, but have not been examined in tissue-engineered constructs cultured in vitro or in tissue filling cartilage defects into which the constructs were implanted. Cartilaginous constructs were engineered in vitro using caprine chondrocyte-seeded type II collagen scaffolds. Autologous constructs were implanted into 4-mm-diameter defects created to the tidemark in the trochlear groove in the knee joints of skeletally mature goats. Eight weeks after implantation, the animals were sacrificed. Constructs underwent immunohistochemical and histomorphometric evaluation. Widespread staining for the two basement membrane molecules was observed throughout the extracellular matrix of in vitro and in vivo samples in a distribution unlike that previously reported for cartilage. At sacrifice, 70% of the defect site was filled with reparative tissue, which consisted largely of fibrous tissue and some fibrocartilage, with over 70% of the reparative tissue bonded to the adjacent host tissue. A novel finding of this study was the observation of laminin and type IV collagen in in vitro engineered cartilaginous constructs and in vivo cartilage repair samples from defects into which the constructs were implanted, as well as in normal caprine articular cartilage. Future work is needed to elucidate the role of basement membrane molecules during cartilage repair and regeneration.

Tissue-Engineered Cartilaginous Constructs for the Treatment of Caprine Cartilage Defects, Including Distribution of Laminin and Type IV Collagen

PubMed Central

Jeng, Lily; Hsu, Hu-Ping

2013-01-01

The purpose of this study was the immunohistochemical evaluation of (1) cartilage tissue-engineered constructs; and (2) the tissue filling cartilage defects in a goat model into which the constructs were implanted, particularly for the presence of the basement membrane molecules, laminin and type IV collagen. Basement membrane molecules are localized to the pericellular matrix in normal adult articular cartilage, but have not been examined in tissue-engineered constructs cultured in vitro or in tissue filling cartilage defects into which the constructs were implanted. Cartilaginous constructs were engineered in vitro using caprine chondrocyte-seeded type II collagen scaffolds. Autologous constructs were implanted into 4-mm-diameter defects created to the tidemark in the trochlear groove in the knee joints of skeletally mature goats. Eight weeks after implantation, the animals were sacrificed. Constructs underwent immunohistochemical and histomorphometric evaluation. Widespread staining for the two basement membrane molecules was observed throughout the extracellular matrix of in vitro and in vivo samples in a distribution unlike that previously reported for cartilage. At sacrifice, 70% of the defect site was filled with reparative tissue, which consisted largely of fibrous tissue and some fibrocartilage, with over 70% of the reparative tissue bonded to the adjacent host tissue. A novel finding of this study was the observation of laminin and type IV collagen in in vitro engineered cartilaginous constructs and in vivo cartilage repair samples from defects into which the constructs were implanted, as well as in normal caprine articular cartilage. Future work is needed to elucidate the role of basement membrane molecules during cartilage repair and regeneration. PMID:23672504

A Comparison of Bone Marrow and Cord Blood Mesenchymal Stem Cells for Cartilage Self-Assembly.

PubMed

White, Jamie L; Walker, Naomi J; Hu, Jerry C; Borjesson, Dori L; Athanasiou, Kyriacos A

2018-04-02

Joint injury is a common cause of premature retirement for the human and equine athlete alike. Implantation of engineered cartilage offers the potential to increase the success rate of surgical intervention and hasten recovery times. Mesenchymal stem cells (MSCs) are a particularly attractive cell source for cartilage engineering. While bone marrow-derived MSCs (BM-MSCs) have been most extensively characterized for musculoskeletal tissue engineering, studies suggest that cord blood MSCs (CB-MSCs) may elicit a more robust chondrogenic phenotype. The objective of this study was to determine a superior equine MSC source for cartilage engineering. MSCs derived from bone marrow or cord blood were stimulated to undergo chondrogenesis through aggregate redifferentiation and used to generate cartilage through the self-assembling process. The resulting neocartilage produced from either BM-MSCs or CB-MSCs was compared by measuring mechanical, biochemical, and histological properties. We found that while BM constructs possessed higher tensile properties and collagen content, CB constructs had superior compressive properties comparable to that of native tissue and higher GAG content. Moreover, CB constructs had alkaline phosphatase activity, collagen type X, and collagen type II on par with native tissue suggesting a more hyaline cartilage-like phenotype. In conclusion, while both BM-MSCs and CB-MSCs were able to form neocartilage, CB-MSCs resulted in tissue more closely resembling native equine articular cartilage as determined by a quantitative functionality index. Therefore, CB-MSCs are deemed a superior source for the purpose of articular cartilage self-assembly.

Remodeling by fibroblasts alters the rate-dependent mechanical properties of collagen.

PubMed

Babaei, Behzad; Davarian, Ali; Lee, Sheng-Lin; Pryse, Kenneth M; McConnaughey, William B; Elson, Elliot L; Genin, Guy M

2016-06-01

The ways that fibroblasts remodel their environment are central to wound healing, development of musculoskeletal tissues, and progression of pathologies such as fibrosis. However, the changes that fibroblasts make to the material around them and the mechanical consequences of these changes have proven difficult to quantify, especially in realistic, viscoelastic three-dimensional culture environments, leaving a critical need for quantitative data. Here, we observed the mechanisms and quantified the mechanical effects of fibroblast remodeling in engineered tissue constructs (ETCs) comprised of reconstituted rat tail (type I) collagen and human fibroblast cells. To study the effects of remodeling on tissue mechanics, stress-relaxation tests were performed on ETCs cultured for 24, 48, and 72h. ETCs were treated with deoxycholate and tested again to assess the ECM response. Viscoelastic relaxation spectra were obtained using the generalized Maxwell model. Cells exhibited viscoelastic damping at two finite time constants over which the ECM showed little damping, approximately 0.2s and 10-30s. Different finite time constants in the range of 1-7000s were attributed to ECM relaxation. Cells remodeled the ECM to produce a relaxation time constant on the order of 7000s, and to merge relaxation finite time constants in the 0.5-2s range into a single time content in the 1s range. Results shed light on hierarchical deformation mechanisms in tissues, and on pathologies related to collagen relaxation such as diastolic dysfunction. As fibroblasts proliferate within and remodel a tissue, they change the tissue mechanically. Quantifying these changes is critical for understanding wound healing and the development of pathologies such as cardiac fibrosis. Here, we characterize for the first time the spectrum of viscoelastic (rate-dependent) changes arising from the remodeling of reconstituted collagen by fibroblasts. The method also provides estimates of the viscoelastic spectra of fibroblasts within a three-dimensional culture environment. Results are of particular interest because of the ways that fibroblasts alter the mechanical response of collagen at loading frequencies associated with cardiac contraction in humans. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Microvascular Guidance: A Challenge to Support the Development of Vascularised Tissue Engineering Construct

PubMed Central

Sukmana, Irza

2012-01-01

The guidance of endothelial cell organization into a capillary network has been a long-standing challenge in tissue engineering. Some research efforts have been made to develop methods to promote capillary networks inside engineered tissue constructs. Capillary and vascular networks that would mimic blood microvessel function can be used to subsequently facilitate oxygen and nutrient transfer as well as waste removal. Vascularization of engineering tissue construct is one of the most favorable strategies to overpass nutrient and oxygen supply limitation, which is often the major hurdle in developing thick and complex tissue and artificial organ. This paper addresses recent advances and future challenges in developing three-dimensional culture systems to promote tissue construct vascularization allowing mimicking blood microvessel development and function encountered in vivo. Bioreactors systems that have been used to create fully vascularized functional tissue constructs will also be outlined. PMID:22623881

Platform technology for scalable assembly of instantaneously functional mosaic tissues

PubMed Central

Zhang, Boyang; Montgomery, Miles; Davenport-Huyer, Locke; Korolj, Anastasia; Radisic, Milica

2015-01-01

Engineering mature tissues requires a guided assembly of cells into organized three-dimensional (3D) structures with multiple cell types. Guidance is usually achieved by microtopographical scaffold cues or by cell-gel compaction. The assembly of individual units into functional 3D tissues is often time-consuming, relying on cell ingrowth and matrix remodeling, whereas disassembly requires an invasive method that includes either matrix dissolution or mechanical cutting. We invented Tissue-Velcro, a bio-scaffold with a microfabricated hook and loop system. The assembly of Tissue-Velcro preserved the guided cell alignment realized by the topographical features in the 2D scaffold mesh and allowed for the instant establishment of coculture conditions by spatially defined stacking of cardiac cell layers or through endothelial cell coating. The assembled cardiac 3D tissue constructs were immediately functional as measured by their ability to contract in response to electrical field stimulation. Facile, on-demand tissue disassembly was demonstrated while preserving the structure, physical integrity, and beating function of individual layers. PMID:26601234

Three-Dimensional Printing and Cell Therapy for Wound Repair.

PubMed

van Kogelenberg, Sylvia; Yue, Zhilian; Dinoro, Jeremy N; Baker, Christopher S; Wallace, Gordon G

2018-05-01

Significance: Skin tissue damage is a major challenge and a burden on healthcare systems, from burns and other trauma to diabetes and vascular disease. Although the biological complexities are relatively well understood, appropriate repair mechanisms are scarce. Three-dimensional bioprinting is a layer-based approach to regenerative medicine, whereby cells and cell-based materials can be dispensed in fine spatial arrangements to mimic native tissue. Recent Advances: Various bioprinting techniques have been employed in wound repair-based skin tissue engineering, from laser-induced forward transfer to extrusion-based methods, and with the investigation of the benefits and shortcomings of each, with emphasis on biological compatibility and cell proliferation, migration, and vitality. Critical issues: Development of appropriate biological inks and the vascularization of newly developed tissues remain a challenge within the field of skin tissue engineering. Future Directions: Progress within bioprinting requires close interactions between material scientists, tissue engineers, and clinicians. Microvascularization, integration of multiple cell types, and skin appendages will be essential for creation of complex skin tissue constructs.

Enhancing Post-Expansion Chondrogenic Potential of Costochondral Cells in Self-Assembled Neocartilage

PubMed Central

Murphy, Meghan K.; Huey, Daniel J.; Reimer, Andrew J.; Hu, Jerry C.; Athanasiou, Kyriacos A.

2013-01-01

The insufficient healing capacity of articular cartilage necessitates mechanically functional biologic tissue replacements. Using cells to form biomimetic cartilage implants is met with the challenges of cell scarcity and donor site morbidity, requiring expanded cells that possess the ability to generate robust neocartilage. To address this, this study assesses the effects of expansion medium supplementation (bFGF, TFP, FBS) and self-assembled construct seeding density (2, 3, 4 million cells/5 mm dia. construct) on the ability of costochondral cells to generate biochemically and biomechanically robust neocartilage. Results show TFP (1 ng/mL TGF-β1, 5 ng/mL bFGF, 10 ng/mL PDGF) supplementation of serum-free chondrogenic expansion medium enhances the post-expansion chondrogenic potential of costochondral cells, evidenced by increased glycosaminoglycan content, decreased type I/II collagen ratio, and enhanced compressive properties. Low density (2 million cells/construct) enhances matrix synthesis and tensile and compressive mechanical properties. Combined, TFP and Low density interact to further enhance construct properties. That is, with TFP, Low density increases type II collagen content by over 100%, tensile stiffness by over 300%, and compressive moduli by over 140%, compared with High density. In conclusion, the interaction of TFP and Low density seeding enhances construct material properties, allowing for a mechanically functional, biomimetic cartilage to be formed using clinically relevant costochondral cells. PMID:23437288

Tissue Equivalents Based on Cell-Seeded Biodegradable Microfluidic Constructs

PubMed Central

Borenstein, Jeffrey T.; Megley, Katie; Wall, Kimberly; Pritchard, Eleanor M.; Truong, David; Kaplan, David L.; Tao, Sarah L.; Herman, Ira M.

2010-01-01

One of the principal challenges in the field of tissue engineering and regenerative medicine is the formation of functional microvascular networks capable of sustaining tissue constructs. Complex tissues and vital organs require a means to support oxygen and nutrient transport during the development of constructs both prior to and after host integration, and current approaches have not demonstrated robust solutions to this challenge. Here, we present a technology platform encompassing the design, construction, cell seeding and functional evaluation of tissue equivalents for wound healing and other clinical applications. These tissue equivalents are comprised of biodegradable microfluidic scaffolds lined with microvascular cells and designed to replicate microenvironmental cues necessary to generate and sustain cell populations to replace dermal and/or epidermal tissues lost due to trauma or disease. Initial results demonstrate that these biodegradable microfluidic devices promote cell adherence and support basic cell functions. These systems represent a promising pathway towards highly integrated three-dimensional engineered tissue constructs for a wide range of clinical applications.

3D surface reconstruction and visualization of the Drosophila wing imaginal disc at cellular resolution

NASA Astrophysics Data System (ADS)

Bai, Linge; Widmann, Thomas; Jülicher, Frank; Dahmann, Christian; Breen, David

2013-01-01

Quantifying and visualizing the shape of developing biological tissues provide information about the morphogenetic processes in multicellular organisms. The size and shape of biological tissues depend on the number, size, shape, and arrangement of the constituting cells. To better understand the mechanisms that guide tissues into their final shape, it is important to investigate the cellular arrangement within tissues. Here we present a data processing pipeline to generate 3D volumetric surface models of epithelial tissues, as well as geometric descriptions of the tissues' apical cell cross-sections. The data processing pipeline includes image acquisition, editing, processing and analysis, 2D cell mesh generation, 3D contourbased surface reconstruction, cell mesh projection, followed by geometric calculations and color-based visualization of morphological parameters. In their first utilization we have applied these procedures to construct a 3D volumetric surface model at cellular resolution of the wing imaginal disc of Drosophila melanogaster. The ultimate goal of the reported effort is to produce tools for the creation of detailed 3D geometric models of the individual cells in epithelial tissues. To date, 3D volumetric surface models of the whole wing imaginal disc have been created, and the apicolateral cell boundaries have been identified, allowing for the calculation and visualization of cell parameters, e.g. apical cross-sectional area of cells. The calculation and visualization of morphological parameters show position-dependent patterns of cell shape in the wing imaginal disc. Our procedures should offer a general data processing pipeline for the construction of 3D volumetric surface models of a wide variety of epithelial tissues.

Analytic Models of Oxygen and Nutrient Diffusion, Metabolism Dynamics, and Architecture Optimization in Three-Dimensional Tissue Constructs with Applications and Insights in Cerebral Organoids

PubMed Central

2016-01-01

Diffusion models are important in tissue engineering as they enable an understanding of gas, nutrient, and signaling molecule delivery to cells in cell cultures and tissue constructs. As three-dimensional (3D) tissue constructs become larger, more intricate, and more clinically applicable, it will be essential to understand internal dynamics and signaling molecule concentrations throughout the tissue and whether cells are receiving appropriate nutrient delivery. Diffusion characteristics present a significant limitation in many engineered tissues, particularly for avascular tissues and for cells whose viability, differentiation, or function are affected by concentrations of oxygen and nutrients. This article seeks to provide novel analytic solutions for certain cases of steady-state and nonsteady-state diffusion and metabolism in basic 3D construct designs (planar, cylindrical, and spherical forms), solutions that would otherwise require mathematical approximations achieved through numerical methods. This model is applied to cerebral organoids, where it is shown that limitations in diffusion and organoid size can be partially overcome by localizing metabolically active cells to an outer layer in a sphere, a regionalization process that is known to occur through neuroglial precursor migration both in organoids and in early brain development. The given prototypical solutions include a review of metabolic information for many cell types and can be broadly applied to many forms of tissue constructs. This work enables researchers to model oxygen and nutrient delivery to cells, predict cell viability, study dynamics of mass transport in 3D tissue constructs, design constructs with improved diffusion capabilities, and accurately control molecular concentrations in tissue constructs that may be used in studying models of development and disease or for conditioning cells to enhance survival after insults like ischemia or implantation into the body, thereby providing a framework for better understanding and exploring the characteristics and behaviors of engineered tissue constructs. PMID:26650970

In vitro 3D corneal tissue model with epithelium, stroma, and innervation.

PubMed

Wang, Siran; Ghezzi, Chiara E; Gomes, Rachel; Pollard, Rachel E; Funderburgh, James L; Kaplan, David L

2017-01-01

The interactions between corneal nerve, epithelium, and stroma are essential for maintaining a healthy cornea. Thus, corneal tissue models that more fully mimic the anatomy, mechanical properties and cellular components of corneal tissue would provide useful systems to study cellular interactions, corneal diseases and provide options for improved drug screening. Here a corneal tissue model was constructed to include the stroma, epithelium, and innervation. Thin silk protein film stacks served as the scaffolding to support the corneal epithelial and stromal layers, while a surrounding silk porous sponge supported neuronal growth. The neurons innervated the stromal and epithelial layers and improved function and viability of the tissues. An air-liquid interface environment of the corneal tissue was also mimicked in vitro, resulting in a positive impact on epithelial maturity. The inclusion of three cell types in co-culture at an air-liquid interface provides an important advance for the field of in vitro corneal tissue engineering, to permit improvements in the study of innervation and corneal tissue development, corneal disease, and tissue responses to environmental factors. Copyright © 2016 Elsevier Ltd. All rights reserved.

From Microscale Devices to 3D Printing: Advances in Fabrication of 3D Cardiovascular Tissues.

PubMed

Borovjagin, Anton V; Ogle, Brenda M; Berry, Joel L; Zhang, Jianyi

2017-01-06

Current strategies for engineering cardiovascular cells and tissues have yielded a variety of sophisticated tools for studying disease mechanisms, for development of drug therapies, and for fabrication of tissue equivalents that may have application in future clinical use. These efforts are motivated by the need to extend traditional 2-dimensional (2D) cell culture systems into 3D to more accurately replicate in vivo cell and tissue function of cardiovascular structures. Developments in microscale devices and bioprinted 3D tissues are beginning to supplant traditional 2D cell cultures and preclinical animal studies that have historically been the standard for drug and tissue development. These new approaches lend themselves to patient-specific diagnostics, therapeutics, and tissue regeneration. The emergence of these technologies also carries technical challenges to be met before traditional cell culture and animal testing become obsolete. Successful development and validation of 3D human tissue constructs will provide powerful new paradigms for more cost effective and timely translation of cardiovascular tissue equivalents. © 2017 American Heart Association, Inc.

Regenerating Articular Tissue by Converging Technologies

PubMed Central

Paoluzzi, Luca; Pieper, Jeroen; de Wijn, Joost R.; van Blitterswijk, Clemens A.

2008-01-01

Scaffolds for osteochondral tissue engineering should provide mechanical stability, while offering specific signals for chondral and bone regeneration with a completely interconnected porous network for cell migration, attachment, and proliferation. Composites of polymers and ceramics are often considered to satisfy these requirements. As such methods largely rely on interfacial bonding between the ceramic and polymer phase, they may often compromise the use of the interface as an instrument to direct cell fate. Alternatively, here, we have designed hybrid 3D scaffolds using a novel concept based on biomaterial assembly, thereby omitting the drawbacks of interfacial bonding. Rapid prototyped ceramic particles were integrated into the pores of polymeric 3D fiber-deposited (3DF) matrices and infused with demineralized bone matrix (DBM) to obtain constructs that display the mechanical robustness of ceramics and the flexibility of polymers, mimicking bone tissue properties. Ostechondral scaffolds were then fabricated by directly depositing a 3DF structure optimized for cartilage regeneration adjacent to the bone scaffold. Stem cell seeded scaffolds regenerated both cartilage and bone in vivo. PMID:18716660

[Design and biological evaluation of poly-lactic-co-glycolic acid (PLGA) mesh/collagen-chitosan hybrid scaffold (CCS) as a dermal substitute].

PubMed

Wang, Xin-Gang; You, Chuan-Gang; Sun, Hua-Feng; Hu, Xin-Lei; Han, Chun-Mao; Zhang, Li-Ping; Zheng, Yu-Rong; Li, Qi-Yin

2011-02-01

To design and construct a kind of dermal regeneration template with mesh, and to preliminarily evaluate its biological characteristics. PLGA mesh was integrated into CCS with freeze-drying method for constructing PLGA mesh/CCS composite (PCCS). The micromorphologies and mechanical properties among PLGA mesh, CCS, and PCCS were compared. PCCS and CCS was respectively implanted into subcutaneous tissue of SD rats (PCCS and CCS groups, 9 rats in each group). The tissue samples were collected at post operation week (POW) 1, 2, and 4 for histopathological and immunohistochemical observation. Protein levels of CD68, MPO, IL-1beta, IL-10 were examined by Western blot, with expression of gray value. Data were processed with one-way analysis of variance and t test. Three-dimensional porous structure of PCCS was similar to that of CCS. Mechanical property of PLGA mesh and PCCS was respectively (3.07 +/- 0.10), (3.26 +/- 0.15) MPa, and they were higher than that of CCS [(0.42 +/- 0.21) MPa, F = 592.3, P < 0.0001)]. The scaffolds were filled with newly formed tissue in PCCS group at POW 2, while those in CCS group were observed at POW 4. A large accumulation of macrophages was observed in both groups, especially at POW 2, and more macrophage infiltration was observed in CCS group. The protein level of IL-10 in PCCS group at POW 2 was obviously higher than that in CCS group, while the protein levels of CD68, MPO, IL-1beta were significantly decreased as compared with those in CCS group (with t value from -4.06 to 2.89, P < 0.05 or P < 0.01). PCCS has excellent mechanical property with appropriate three-dimensional porous structure. Meanwhile, it can rapidly induce formation of new tissue and vascularization, and it has a prospect of serving as a dermal substitute.

[Construction of forward and reverse subtracted cDNA libraries between muscle tissue of Meishan and Landrace pigs].

PubMed

Xu, De-Quan; Zhang, Yi-Bing; Xiong, Yuan-Zhu; Gui, Jian-Fang; Jiang, Si-Wen; Su, Yu-Hong

2003-07-01

Using suppression subtractive hybridization (SSH) technique, forward and reverse subtracted cDNA libraries were constructed between Longissimus muscles from Meishan and Landrace pigs. A housekeeping gene, G3PDH, was used to estimate the efficiency of subtractive cDNA. In two cDNA libraries, G3PDH was subtracted very efficiently at appropriate 2(10) and 2(5) folds, respectively, indicating that some differentially expressed genes were also enriched at the same folds and the two subtractive cDNA libraries were very successful. A total of 709 and 673 positive clones were isolated from forward and reverse subtracted cDNA libraries, respectively. Analysis of PCR showed that most of all plasmids in the clones contained 150-750 bp inserts. The construction of subtractive cDNA libraries between muscle tissue from different pig breeds laid solid foundations for isolating and identifying the genes determining muscle growth and meat quality, which will be important to understand the mechanism of muscle growth, determination of meat quality and practice of molecular breeding.

* Constrained Cage Culture Improves Engineered Cartilage Functional Properties by Enhancing Collagen Network Stability.

PubMed

Nims, Robert J; Cigan, Alexander D; Durney, Krista M; Jones, Brian K; O'Neill, John D; Law, Wing-Sum A; Vunjak-Novakovic, Gordana; Hung, Clark T; Ateshian, Gerard A

2017-08-01

When cultured with sufficient nutrient supply, engineered cartilage synthesizes proteoglycans rapidly, producing an osmotic swelling pressure that destabilizes immature collagen and prevents the development of a robust collagen framework, a hallmark of native cartilage. We hypothesized that mechanically constraining the proteoglycan-induced tissue swelling would enhance construct functional properties through the development of a more stable collagen framework. To test this hypothesis, we developed a novel "cage" growth system to mechanically prevent tissue constructs from swelling while ensuring adequate nutrient supply to the growing construct. The effectiveness of constrained culture was examined by testing constructs embedded within two different scaffolds: agarose and cartilage-derived matrix hydrogel (CDMH). Constructs were seeded with immature bovine chondrocytes and cultured under free swelling (FS) conditions for 14 days with transforming growth factor-β before being placed into a constraining cage for the remainder of culture. Controls were cultured under FS conditions throughout. Agarose constructs cultured in cages did not expand after the day 14 caging while FS constructs expanded to 8 × their day 0 weight after 112 days of culture. In addition to the physical differences in growth, by day 56, caged constructs had higher equilibrium (agarose: 639 ± 179 kPa and CDMH: 608 ± 257 kPa) and dynamic compressive moduli (agarose: 3.4 ± 1.0 MPa and CDMH 2.8 ± 1.0 MPa) than FS constructs (agarose: 193 ± 74 kPa and 1.1 ± 0.5 MPa and CDMH: 317 ± 93 kPa and 1.8 ± 1.0 MPa for equilibrium and dynamic properties, respectively). Interestingly, when normalized to final day wet weight, cage and FS constructs did not exhibit differences in proteoglycan or collagen content. However, caged culture enhanced collagen maturation through the increased formation of pyridinoline crosslinks and improved collagen matrix stability as measured by α-chymotrypsin solubility. These findings demonstrate that physically constrained culture of engineered cartilage constructs improves functional properties through improved collagen network maturity and stability. We anticipate that constrained culture may benefit other reported engineered cartilage systems that exhibit a mismatch in proteoglycan and collagen synthesis.

Additive manufacturing: From implants to organs.

PubMed

Douglas, Tania S

2014-05-12

Additive manufacturing (AM) constructs 3D objects layer by layer under computer control from 3D models. 3D printing is one example of this kind of technology. AM offers geometric flexibility in its products and therefore allows customisation to suit individual needs. Clinical success has been shown with models for surgical planning, implants, assistive devices and scaffold-based tissue engineering. The use of AM to print tissues and organs that mimic nature in structure and function remains an elusive goal, but has the potential to transform personalised medicine, drug development and scientific understanding of the mechanisms of disease. 

Evaluating the Use of Monocytes with a Degradable Polyurethane for Vascular Tissue Regeneration

NASA Astrophysics Data System (ADS)

Battiston, Kyle Giovanni

Monocytes are one of the first cell types present following the implantation of a biomaterial or tissue engineered construct. Depending on the monocyte activation state supported by the biomaterial, monocytes and their derived macrophages (MDMs) can act as positive contributors to tissue regeneration and wound healing, or conversely promote a chronic inflammatory response that leads to fibrous encapsulation and implant rejection. A degradable polar hydrophobic iconic polyurethane (D-PHI) has been shown to reduce pro-inflammatory monocyte/macrophage response compared to tissue culture polystyrene (TCPS), a substrate routinely used for in vitro culture of cells, as well as poly(lactide- co-glycolide) (PLGA), a standard synthetic biodegradable biomaterial in the tissue engineering field. D-PHI has also shown properties suitable for use in a vascular tissue engineering context. In order to understand the mechanism through which D-PHI attenuates pro-inflammatory monocyte response, this thesis investigated the ability of D-PHI to modulate interactions with adsorbed serum proteins and the properties of D-PHI that were important for this activity. D-PHI was shown to regulate protein adsorption in a manner that produced divergent monocyte responses compared to TCPS and PLGA when coated with the serum proteins alpha2-macroglobulin or immunoglobulin G (IgG). In the case of IgG, D-PHI was shown to reduce pro-inflammatory binding site exposure as a function of the material's polar, hydrophobic, and ionic character. Due to the favourable monocyte activation state supported by D-PHI, and the importance of monocytes/macrophages in regulating the response of tissue-specific cell types in vivo, the ability of a D-PHI-stimulated monocyte/macrophage activation state to contribute to modulating the response of vascular smooth muscle cells (VSMCs) in a vascular tissue engineering context was investigated. D-PHI- stimulated monocytes promoted VSMC growth and migration through biomolecule release. Coupling monocyte-VSMC co-culture with biomechanical strain further enhanced these effects, while also promoting extracellular matrix deposition (collagen I, collagen III, and elastin) and enhancing the mechanical properties of VSMC-monocyte seeded tissue constructs. This thesis identifies the use of biomaterials with immunomodulatory capacity to harness the stimulatory potential of MDMs and contribute to tissue engineering strategies in vitro. This latter work in turn has contributed to identifying aspects of biomaterial design that can contribute to supporting desirable monocyte-biomaterial interactions that can facilitate this process.

An antibody based approach for multi-coloring osteogenic and chondrogenic proteins in tissue engineered constructs.

PubMed

Leferink, Anne M; Reis, Diogo Santos; van Blitterswijk, Clemens A; Moroni, Lorenzo

2018-04-11

When tissue engineering strategies rely on the combination of three-dimensional (3D) polymeric or ceramic scaffolds with cells to culture implantable tissue constructs in vitro, it is desirable to monitor tissue growth and cell fate to be able to more rationally predict the quality and success of the construct upon implantation. Such a 3D construct is often referred to as a 'black-box' since the properties of the scaffolds material limit the applicability of most imaging modalities to assess important construct parameters. These parameters include the number of cells, the amount and type of tissue formed and the distribution of cells and tissue throughout the construct. Immunolabeling enables the spatial and temporal identification of multiple tissue types within one scaffold without the need to sacrifice the construct. In this report, we concisely review the applicability of antibodies (Abs) and their conjugation chemistries in tissue engineered constructs. With some preliminary experiments, we show an efficient conjugation strategy to couple extracellular matrix Abs to fluorophores. The conjugated probes proved to be effective in determining the presence of collagen type I and type II on electrospun and additive manufactured 3D scaffolds seeded with adult human bone marrow derived mesenchymal stromal cells. The conjugation chemistry applied in our proof of concept study is expected to be applicable in the coupling of any other fluorophore or particle to the Abs. This could ultimately lead to a library of probes to permit high-contrast imaging by several imaging modalities.

Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues.

PubMed

Abraham, N G; da Silva, J L; Lavrovsky, Y; Stoltz, R A; Kappas, A; Dunn, M W; Schwartzman, M L

1995-10-01

Heme oxygenase-1 (HO-1) is a stress protein induced up to 100-fold within a few hours after exposure to oxidative stress, and it has been shown to counteract oxidative injury induced by ultraviolet light or free radicals. The current study was undertaken to determine whether the HO-1 gene can be introduced into adult rabbit ocular tissues by microinjection of a recombinant replication-deficient adenovirus human HO-1 cDNA (Adv-HHO). Human HO-1 gene was used for transfection studies to differentiate endogenous from transfected HO. The purified Adv-HHO construct (10(8) pfu/ml) was mixed with lipofectamine and microinjected into the anterior chamber, vitreous cavity, and subretinal space of New Zealand rabbit eyes. After 2 weeks, total RNA was extracted from different ocular tissues, reverse transcription-polymerase chain reaction was performed using specific human HO-1 primers, and amplification products were subjected to Southern hybridization. Transfection with the Adv-HHO construct into rabbit corneal epithelial cells in culture resulted in a functional expression of the human HO-1 gene; the human HO-1 mRNA was detected, and enzyme activity increased threefold. Human HO-1 mRNA was detected in the retina after microinjection of the Adv-HHO construct into the subretinal space. Microinjection into the vitreous resulted in HO-1 mRNA expression in the corneal endothelium, iris, lens, and retina; after intracameral injection of the Adv-HHO construct, human HO-1 mRNA was detected in corneal epithelium and endothelium, ciliary body, lens, and iris. Regardless of the injection site, transfected human HO-1 mRNA was undetectable in tissues outside the eye, that is, brain, liver, and kidney. These results demonstrated a tissue-selective functional transfer of the human HO-1 gene into rabbit ocular tissues in vivo. This technique may be a promising means for delivering HO-1 gene in vivo as a protective mechanism against oxidative stress that contributes to the pathogenesis of ocular diseases such as cataract, light-induced injury, age-related macular degeneration, and diabetic retinopathy.

A construction of novel iron-foam-based calcium phosphate/chitosan coating biodegradable scaffold material.

PubMed

Wen, Zhaohui; Zhang, Liming; Chen, Chao; Liu, Yibo; Wu, Changjun; Dai, Changsong

2013-04-01

Slow corrosion rate and poor bioactivity restrict iron-based implants in biomedical application. In this study, we design a new iron-foam-based calcium phosphate/chitosan coating biodegradable composites offering a priority mechanical and bioactive property for bone tissue engineering through electrophoretic deposition (EPD) followed by a conversion process into a phosphate buffer solution (PBS). Tensile test results showed that the mechanical property of iron foam could be regulated through altering the construction of polyurethane foam. The priority coatings were deposited from 40% nano hydroxyapatite (nHA)/ethanol suspension mixed with 60% nHA/chitosan-acetic acid aqueous solution. In vitro immersion test showed that oxidation-iron foam as the matrix decreased the amount of iron implanted and had not influence on the bioactivity of this implant, obviously. So, this method could also be a promising method for the preparation of a new calcium phosphate/chitosan coating on foam construction. Copyright © 2012. Published by Elsevier B.V.

Endochondral Priming: A Developmental Engineering Strategy for Bone Tissue Regeneration.

PubMed

Freeman, Fiona E; McNamara, Laoise M

2017-04-01

Tissue engineering and regenerative medicine have significant potential to treat bone pathologies by exploiting the capacity for bone progenitors to grow and produce tissue constituents under specific biochemical and physical conditions. However, conventional tissue engineering approaches, which combine stem cells with biomaterial scaffolds, are limited as the constructs often degrade, due to a lack of vascularization, and lack the mechanical integrity to fulfill load bearing functions, and as such are not yet widely used for clinical treatment of large bone defects. Recent studies have proposed that in vitro tissue engineering approaches should strive to simulate in vivo bone developmental processes and, thereby, imitate natural factors governing cell differentiation and matrix production, following the paradigm recently defined as "developmental engineering." Although developmental engineering strategies have been recently developed that mimic specific aspects of the endochondral ossification bone formation process, these findings are not widely understood. Moreover, a critical comparison of these approaches to standard biomaterial-based bone tissue engineering has not yet been undertaken. For that reason, this article presents noteworthy experimental findings from researchers focusing on developing an endochondral-based developmental engineering strategy for bone tissue regeneration. These studies have established that in vitro approaches, which mimic certain aspects of the endochondral ossification process, namely the formation of the cartilage template and the vascularization of the cartilage template, can promote mineralization and vascularization to a certain extent both in vitro and in vivo. Finally, this article outlines specific experimental challenges that must be overcome to further exploit the biology of endochondral ossification and provide a tissue engineering construct for clinical treatment of large bone/nonunion defects and obviate the need for bone tissue graft.

A pancreas specificity results from the combination of polyomavirus and Moloney murine leukemia virus enhancer.

PubMed Central

Rochford, R; Campbell, B A; Villarreal, L P

1987-01-01

An infectious recombinant polyomavirus was constructed in which a regulatory region of its genome, the B enhancer region (nucleotides 5128-5265) has been replaced with the 72- or 73-base-pair repeat enhancer from the Moloney murine leukemia virus genome. We show that this recombinant polyomavirus displays a strong tissue specificity for the pancreas of mice. This organ was not permissive for either the parental polyomavirus, which is predominantly kidney and salivary gland specific, or the Moloney murine leukemia virus, which is lymphotropic. This result indicated that tissue specificity can be achieved by a combination of apparently modular elements. Some of the implications of a modular mechanism of tissue specificity are considered. Images PMID:3025873

Recent advances in computational mechanics of the human knee joint.

PubMed

Kazemi, M; Dabiri, Y; Li, L P

2013-01-01

Computational mechanics has been advanced in every area of orthopedic biomechanics. The objective of this paper is to provide a general review of the computational models used in the analysis of the mechanical function of the knee joint in different loading and pathological conditions. Major review articles published in related areas are summarized first. The constitutive models for soft tissues of the knee are briefly discussed to facilitate understanding the joint modeling. A detailed review of the tibiofemoral joint models is presented thereafter. The geometry reconstruction procedures as well as some critical issues in finite element modeling are also discussed. Computational modeling can be a reliable and effective method for the study of mechanical behavior of the knee joint, if the model is constructed correctly. Single-phase material models have been used to predict the instantaneous load response for the healthy knees and repaired joints, such as total and partial meniscectomies, ACL and PCL reconstructions, and joint replacements. Recently, poromechanical models accounting for fluid pressurization in soft tissues have been proposed to study the viscoelastic response of the healthy and impaired knee joints. While the constitutive modeling has been considerably advanced at the tissue level, many challenges still exist in applying a good material model to three-dimensional joint simulations. A complete model validation at the joint level seems impossible presently, because only simple data can be obtained experimentally. Therefore, model validation may be concentrated on the constitutive laws using multiple mechanical tests of the tissues. Extensive model verifications at the joint level are still crucial for the accuracy of the modeling.

Recent Advances in Computational Mechanics of the Human Knee Joint

PubMed Central

Kazemi, M.; Dabiri, Y.; Li, L. P.

2013-01-01

Computational mechanics has been advanced in every area of orthopedic biomechanics. The objective of this paper is to provide a general review of the computational models used in the analysis of the mechanical function of the knee joint in different loading and pathological conditions. Major review articles published in related areas are summarized first. The constitutive models for soft tissues of the knee are briefly discussed to facilitate understanding the joint modeling. A detailed review of the tibiofemoral joint models is presented thereafter. The geometry reconstruction procedures as well as some critical issues in finite element modeling are also discussed. Computational modeling can be a reliable and effective method for the study of mechanical behavior of the knee joint, if the model is constructed correctly. Single-phase material models have been used to predict the instantaneous load response for the healthy knees and repaired joints, such as total and partial meniscectomies, ACL and PCL reconstructions, and joint replacements. Recently, poromechanical models accounting for fluid pressurization in soft tissues have been proposed to study the viscoelastic response of the healthy and impaired knee joints. While the constitutive modeling has been considerably advanced at the tissue level, many challenges still exist in applying a good material model to three-dimensional joint simulations. A complete model validation at the joint level seems impossible presently, because only simple data can be obtained experimentally. Therefore, model validation may be concentrated on the constitutive laws using multiple mechanical tests of the tissues. Extensive model verifications at the joint level are still crucial for the accuracy of the modeling. PMID:23509602

Nano/micro hybrid scaffold of PCL or P3HB nanofibers combined with silk fibroin for tendon and ligament tissue engineering.

PubMed

Naghashzargar, Elham; Farè, Silvia; Catto, Valentina; Bertoldi, Serena; Semnani, Dariush; Karbasi, Saeed; Tanzi, Maria Cristina

2015-07-04

A novel biodegradable nano/micro hybrid structure was obtained by electrospinning P3HB or PCL nanofibers onto a twisted silk fibroin (SF) structure, with the aim of fabricating a suitable scaffold for tendon and ligament tissue engineering. The electrospinning (ES) processing parameters for P3HB and PCL were optimized on 2D samples, and applied to produce two different nano/micro hybrid constructs (SF/ES-PCL and SF/ES-P3HB).Morphological, chemico-physical and mechanical properties of the novel hybrid scaffolds were evaluated by SEM, ATR FT-IR, DSC, tensile and thermodynamic mechanical tests. The results demonstrated that the nanofibers were tightly wrapped around the silk filaments, and the crystallinity of the SF twisted yarns was not influenced by the presence of the electrospun polymers. The slightly higher mechanical properties of the hybrid constructs confirmed an increase of internal forces due to the interaction between nano and micro components. Cell culture tests with L929 fibroblasts, in the presence of the sample eluates or in direct contact with the hybrid structures, showed no cytotoxic effects and a good level of cytocompatibility of the nano/micro hybrid structures in term of cell viability, particularly at day 1. Cell viability onto the nano/micro hybrid structures decreased from the first to the third day of culture when compared with the control culture plastic, but appeared to be higher when compared with the uncoated SF yarns. Although additional in vitro and in vivo tests are needed, the original fabrication method here described appears promising for scaffolds suitable for tendon and ligament tissue engineering.

Development of Scaffold-Free Elastic Cartilaginous Constructs with Structural Similarities to Auricular Cartilage

PubMed Central

Giardini-Rosa, Renata; Joazeiro, Paulo P.; Thomas, Kathryn; Collavino, Kristina; Weber, Joanna

2014-01-01

External ear reconstruction with autologous cartilage still remains one of the most difficult problems in the fields of plastic and reconstructive surgery. As the absence of tissue vascularization limits the ability to stimulate new tissue growth, relatively few surgical approaches are currently available (alloplastic implants or sculpted autologous cartilage grafts) to repair or reconstruct the auricle (or pinna) as a result of traumatic loss or congenital absence (e.g., microtia). Alternatively, tissue engineering can offer the potential to grow autogenous cartilage suitable for implantation. While tissue-engineered auricle cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for reconstruction. Similarly, as routine cell expansion can elicit negative effects on chondrocyte function, we have developed an approach to generate large-sized engineered auricle constructs (≥3 cm2) directly from a small population of donor cells (20,000–40,000 cells/construct). Using rabbit donor cells, the developed bioreactor-cultivated constructs adopted structural-like characteristics similar to native auricular cartilage, including the development of distinct cartilaginous and perichondrium-like regions. Both alterations in media composition and seeding density had profound effects on the formation of engineered elastic tissue constructs in terms of cellularity, extracellular matrix accumulation, and tissue structure. Higher seeding densities and media containing sodium bicarbonate produced tissue constructs that were closer to the native tissue in terms of structure and composition. Future studies will be aimed at improving the accumulation of specific tissue constituents and determining the clinical effectiveness of this approach using a reconstructive animal model. PMID:24124666

Development of scaffold-free elastic cartilaginous constructs with structural similarities to auricular cartilage.

PubMed

Giardini-Rosa, Renata; Joazeiro, Paulo P; Thomas, Kathryn; Collavino, Kristina; Weber, Joanna; Waldman, Stephen D

2014-03-01

External ear reconstruction with autologous cartilage still remains one of the most difficult problems in the fields of plastic and reconstructive surgery. As the absence of tissue vascularization limits the ability to stimulate new tissue growth, relatively few surgical approaches are currently available (alloplastic implants or sculpted autologous cartilage grafts) to repair or reconstruct the auricle (or pinna) as a result of traumatic loss or congenital absence (e.g., microtia). Alternatively, tissue engineering can offer the potential to grow autogenous cartilage suitable for implantation. While tissue-engineered auricle cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for reconstruction. Similarly, as routine cell expansion can elicit negative effects on chondrocyte function, we have developed an approach to generate large-sized engineered auricle constructs (≥3 cm(2)) directly from a small population of donor cells (20,000-40,000 cells/construct). Using rabbit donor cells, the developed bioreactor-cultivated constructs adopted structural-like characteristics similar to native auricular cartilage, including the development of distinct cartilaginous and perichondrium-like regions. Both alterations in media composition and seeding density had profound effects on the formation of engineered elastic tissue constructs in terms of cellularity, extracellular matrix accumulation, and tissue structure. Higher seeding densities and media containing sodium bicarbonate produced tissue constructs that were closer to the native tissue in terms of structure and composition. Future studies will be aimed at improving the accumulation of specific tissue constituents and determining the clinical effectiveness of this approach using a reconstructive animal model.

Printing of Three-Dimensional Tissue Analogs for Regenerative Medicine

PubMed Central

Lee, Vivian K.; Dai, Guohao

2016-01-01

3-D cell printing, which can accurately deposit cells, biomaterial scaffolds and growth factors in precisely defined spatial patterns to form biomimetic tissue structures, has emerged as a powerful enabling technology to create live tissue and organ structures for drug discovery and tissue engineering applications. Unlike traditional 3-D printing that uses metals, plastics and polymers as the printing materials, cell printing has to be compatible with living cells and biological matrix. It is also required that the printing process preserves the biological functions of the cells and extracellular matrix, and to mimic the cell-matrix architectures and mechanical properties of the native tissues. Therefore, there are significant challenges in order to translate the technologies of traditional 3-D printing to cell printing, and ultimately achieve functional outcomes in the printed tissues. So it is essential to develop new technologies specially designed for cell printing and in-depth basic research in the bioprinted tissues, such as developing novel biomaterials specifically for cell printing applications, understanding the complex cell-matrix remodeling for the desired mechanical properties and functional outcomes, establishing proper vascular perfusion in bioprinted tissues, etc. In recent years, many exciting research progresses have been made in the 3-D cell printing technology and its application in engineering live tissue constructs. This review paper summarized the current development in 3-D cell printing technologies; focus on the outcomes of the live printed tissues and their potential applications in drug discovery and regenerative medicine. Current challenges and limitations are highlighted, and future directions of 3-D cell printing technology are also discussed. PMID:27066784

The application of multiple biophysical cues to engineer functional neocartilage for treatment of osteoarthritis. Part I: cellular response.

PubMed

Brady, Mariea A; Waldman, Stephen D; Ethier, C Ross

2015-02-01

Osteoarthritis (OA) is a complex disease of the joint for which current treatments are unsatisfactory, thus motivating development of tissue engineering (TE)-based therapies. To date, TE strategies have had some success, developing replacement tissue constructs with biochemical properties approaching that of native cartilage. However, poor biomechanical properties and limited postimplantation integration with surrounding tissue are major shortcomings that need to be addressed. Functional tissue engineering strategies that apply physiologically relevant biophysical cues provide a platform to improve TE constructs before implantation. In the previous decade, new experimental and theoretical findings in cartilage biomechanics and electromechanics have emerged, resulting in an increased understanding of the complex interplay of multiple biophysical cues in the extracellular matrix of the tissue. The effect of biophysical stimulation on cartilage, and the resulting chondrocyte-mediated biosynthesis, remodeling, degradation, and repair, has, therefore, been extensively explored by the TE community. This article compares and contrasts the cellular response of chondrocytes to multiple biophysical stimuli, and may be read in conjunction with its companion paper that compares and contrasts the subsequent intracellular signal transduction cascades. Mechanical, magnetic, and electrical stimuli promote proliferation, differentiation, and maturation of chondrocytes within established dose parameters or "biological windows." This knowledge will provide a framework for ongoing studies incorporating multiple biophysical cues in TE functional neocartilage for treatment of OA.

Analysis of molecular pathways in pancreatic ductal adenocarcinomas with a bioinformatics approach.

PubMed

Wang, Yan; Li, Yan

2015-01-01

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death worldwide. Our study aimed to reveal molecular mechanisms. Microarray data of GSE15471 (including 39 matching pairs of pancreatic tumor tissues and patient-matched normal tissues) was downloaded from Gene Expression Omnibus (GEO) database. We identified differentially expressed genes (DEGs) in PDAC tissues compared with normal tissues by limma package in R language. Then GO and KEGG pathway enrichment analyses were conducted with online DAVID. In addition, principal component analysis was performed and a protein-protein interaction network was constructed to study relationships between the DEGs through database STRING. A total of 532 DEGs were identified in the 38 PDAC tissues compared with 33 normal tissues. The results of principal component analysis of the top 20 DEGs could differentiate the PDAC tissues from normal tissues directly. In the PPI network, 8 of the 20 DEGs were all key genes of the collagen family. Additionally, FN1 (fibronectin 1) was also a hub node in the network. The genes of the collagen family as well as FN1 were significantly enriched in complement and coagulation cascades, ECM-receptor interaction and focal adhesion pathways. Our results suggest that genes of collagen family and FN1 may play an important role in PDAC progression. Meanwhile, these DEGs and enriched pathways, such as complement and coagulation cascades, ECM-receptor interaction and focal adhesion may be important molecular mechanisms involved in the development and progression of PDAC.

FABRICA: A Bioreactor Platform for Printing, Perfusing, Observing, & Stimulating 3D Tissues.

PubMed

Smith, Lester J; Li, Ping; Holland, Mark R; Ekser, Burcin

2018-05-15

We are introducing the FABRICA, a bioprinter-agnostic 3D-printed bioreactor platform designed for 3D-bioprinted tissue construct culture, perfusion, observation, and analysis. The computer-designed FABRICA was 3D-printed with biocompatible material and used for two studies: (1) Flow Profile Study: perfused 5 different media through a synthetic 3D-bioprinted construct and ultrasonically analyzed the flow profile at increasing volumetric flow rates (VFR); (2) Construct Perfusion Study: perfused a 3D-bioprinted tissue construct for a week and compared histologically with a non-perfused control. For the flow profile study, construct VFR increased with increasing pump VFR. Water and other media increased VFR significantly while human and pig blood showed shallow increases. For the construct perfusion study, we confirmed more viable cells in perfused 3D-bioprinted tissue compared to control. The FABRICA can be used to visualize constructs during 3D-bioprinting, incubation, and to control and ultrasonically analyze perfusion, aseptically in real-time, making the FABRICA tunable for different tissues.

A review of rapid prototyping techniques for tissue engineering purposes.

PubMed

Peltola, Sanna M; Melchels, Ferry P W; Grijpma, Dirk W; Kellomäki, Minna

2008-01-01

Rapid prototyping (RP) is a common name for several techniques, which read in data from computer-aided design (CAD) drawings and manufacture automatically three-dimensional objects layer-by-layer according to the virtual design. The utilization of RP in tissue engineering enables the production of three-dimensional scaffolds with complex geometries and very fine structures. Adding micro- and nanometer details into the scaffolds improves the mechanical properties of the scaffold and ensures better cell adhesion to the scaffold surface. Thus, tissue engineering constructs can be customized according to the data acquired from the medical scans to match the each patient's individual needs. In addition RP enables the control of the scaffold porosity making it possible to fabricate applications with desired structural integrity. Unfortunately, every RP process has its own unique disadvantages in building tissue engineering scaffolds. Hence, the future research should be focused on the development of RP machines designed specifically for fabrication of tissue engineering scaffolds, although RP methods already can serve as a link between tissue and engineering.

Unravelling the effects of mechanical physiological conditioning on cardiac adipose tissue-derived progenitor cells in vitro and in silico.

PubMed

Llucià-Valldeperas, Aida; Bragós, Ramon; Soler-Botija, Carolina; Roura, Santiago; Gálvez-Montón, Carolina; Prat-Vidal, Cristina; Perea-Gil, Isaac; Bayes-Genis, Antoni

2018-01-11

Mechanical conditioning is incompletely characterized for stimulating therapeutic cells within the physiological range. We sought to unravel the mechanism of action underlying mechanical conditioning of adipose tissue-derived progenitor cells (ATDPCs), both in vitro and in silico. Cardiac ATDPCs, grown on 3 different patterned surfaces, were mechanically stretched for 7 days at 1 Hz. A custom-designed, magnet-based, mechanical stimulator device was developed to apply ~10% mechanical stretching to monolayer cell cultures. Gene and protein analyses were performed for each cell type and condition. Cell supernatants were also collected to analyze secreted proteins and construct an artificial neural network. Gene and protein modulations were different for each surface pattern. After mechanostimulation, cardiac ATDPCs increased the expression of structural genes and there was a rising trend on cardiac transcription factors. Finally, secretome analyses revealed upregulation of proteins associated with both myocardial infarction and cardiac regeneration, such as regulators of the immune response, angiogenesis or cell adhesion. To conclude, mechanical conditioning of cardiac ATDPCs enhanced the expression of early and late cardiac genes in vitro. Additionally, in silico analyses of secreted proteins showed that mechanical stimulation of cardiac ATDPCs was highly associated with myocardial infarction and repair.

Nanofiber Scaffold-Based Tissue-Engineered Retinal Pigment Epithelium to Treat Degenerative Eye Diseases

PubMed Central

Khristov, Vladimir; Wan, Qin; Sharma, Ruchi; Jha, Balendu Shekhar; Lotfi, Mostafa; Maminishkis, Arvydas; Simon, Carl G.

2016-01-01

Abstract Clinical-grade manufacturing of a functional retinal pigment epithelium (RPE) monolayer requires reproducing, as closely as possible, the natural environment in which RPE grows. In vitro, this can be achieved by a tissue engineering approach, in which the RPE is grown on a nanofibrous biological or synthetic scaffold. Recent research has shown that nanofiber scaffolds perform better for cell growth and transplantability compared with their membrane counterparts and that the success of the scaffold in promoting cell growth/function is not heavily material dependent. With these strides, the field has advanced enough to begin to consider implementation of one, or a combination, of the tissue engineering strategies discussed herein. In this study, we review the current state of tissue engineering research for in vitro culture of RPE/scaffolds and the parameters for optimal scaffold design that have been uncovered during this research. Next, we discuss production methods and manufacturers that are capable of producing the nanofiber scaffolds in such a way that would be biologically, regulatory, clinically, and commercially viable. Then, a discussion of how the scaffolds could be characterized, both morphologically and mechanically, to develop a testing process that is viable for regulatory screening is performed. Finally, an example of a tissue-engineered RPE/scaffold construct is given to provide the reader a framework for understanding how these pieces could fit together to develop a tissue-engineered RPE/scaffold construct that could pass regulatory scrutiny and can be commercially successful. PMID:27110730

Microcomputed Tomography Characterization of Neovascularization in Bone Tissue Engineering Applications

PubMed Central

Young, Simon; Kretlow, James D.; Nguyen, Charles; Bashoura, Alex G.; Baggett, L. Scott; Jansen, John A.; Wong, Mark

2008-01-01

Abstract Vasculogenesis and angiogenesis have been studied for decades using numerous in vitro and in vivo systems, fulfilling the need to elucidate the mechanisms involved in these processes and to test potential therapeutic agents that inhibit or promote neovascularization. Bone tissue engineering in particular has benefited from the application of proangiogenic strategies, considering the need for an adequate vascular supply during healing and the challenges associated with the vascularization of scaffolds implanted in vivo. Conventional methods of assessing the in vivo angiogenic response to tissue-engineered constructs tend to rely on a two-dimensional assessment of microvessel density within representative histological sections without elaboration of the true vascular tree. The introduction of microcomputed tomography (micro-CT) has recently allowed investigators to obtain a diverse range of high-resolution, three-dimensional characterization of structures, including renal, coronary, and hepatic vascular networks, as well as bone formation within healing defects. To date, few studies have utilized micro-CT to study the vascular response to an implanted tissue engineering scaffold. In this paper, conventional in vitro and in vivo models for studying angiogenesis will be discussed, followed by recent developments in the use of micro-CT for vessel imaging in bone tissue engineering research. A new study demonstrating the potential of contrast-enhanced micro-CT for the evaluation of in vivo neovascularization in bony defects is described, which offers significant potential in the evaluation of bone tissue engineering constructs. PMID:18657028

Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells.

PubMed

Rohringer, Sabrina; Hofbauer, Pablo; Schneider, Karl H; Husa, Anna-Maria; Feichtinger, Georg; Peterbauer-Scherb, Anja; Redl, Heinz; Holnthoner, Wolfgang

2014-10-01

Vascularization of tissue-engineered constructs is essential to provide sufficient nutrient supply and hemostasis after implantation into target sites. Co-cultures of adipose-derived stem cells (ASC) with outgrowth endothelial cells (OEC) in fibrin gels were shown to provide an effective possibility to induce vasculogenesis in vitro. However, the mechanisms of the interaction between these two cell types remain unclear so far. The aim of this study was to evaluate differences of direct and indirect stimulation of ASC-induced vasculogenesis, the influence of ASC on network stabilization and molecular mechanisms involved in vascular structure formation. Endothelial cells (EC) were embedded in fibrin gels either containing non-coated or ASC-coated microcarrier beads as well as ASC alone. Moreover, EC-seeded constructs incubated with ASC-conditioned medium were used in addition to constructs with ASC seeded on top. Vascular network formation was visualized by green fluorescent protein expressing cells or immunostaining for CD31 and quantified. RT-qPCR of cells derived from co-cultures in fibrin was performed to evaluate changes in the expression of EC marker genes during the first week of culture. Moreover, angiogenesis-related protein levels were measured by performing angiogenesis proteome profiler arrays. The results demonstrate that proximity of endothelial cells and ASC is required for network formation and ASC stabilize EC networks by developing pericyte characteristics. We further showed that ASC induce controlled vessel growth by secreting pro-angiogenic and regulatory proteins. This study reveals angiogenic protein profiles involved in EC/ASC interactions in fibrin matrices and confirms the usability of OEC/ASC co-cultures for autologous vascular tissue engineering.

Braided nanofibrous scaffold for tendon and ligament tissue engineering.

PubMed

Barber, John G; Handorf, Andrew M; Allee, Tyler J; Li, Wan-Ju

2013-06-01

Tendon and ligament (T/L) injuries present an important clinical challenge due to their intrinsically poor healing capacity. Natural healing typically leads to the formation of scar-like tissue possessing inferior mechanical properties. Therefore, tissue engineering has gained considerable attention as a promising alternative for T/L repair. In this study, we fabricated braided nanofibrous scaffolds (BNFSs) as a potential construct for T/L tissue engineering. Scaffolds were fabricated by braiding 3, 4, or 5 aligned bundles of electrospun poly(L-lactic acid) nanofibers, thus introducing an additional degree of flexibility to alter the mechanical properties of individual scaffolds. We observed that the Young's modulus, yield stress, and ultimate stress were all increased in the 3-bundle compared to the 4- and 5-bundle BNFSs. Interestingly, acellular BNFSs mimicked the normal tri-phasic mechanical behavior of native tendon and ligament (T/L) during loading. When cultured on the BNFSs, human mesenchymal stem cells (hMSCs) adhered, aligned parallel to the length of the nanofibers, and displayed a concomitant realignment of the actin cytoskeleton. In addition, the BNFSs supported hMSC proliferation and induced an upregulation in the expression of key pluripotency genes. When cultured on BNFSs in the presence of tenogenic growth factors and stimulated with cyclic tensile strain, hMSCs differentiated into the tenogenic lineage, evidenced most notably by the significant upregulation of Scleraxis gene expression. These results demonstrate that BNFSs provide a versatile scaffold capable of supporting both stem cell expansion and differentiation for T/L tissue engineering applications.

Moldable elastomeric polyester-carbon nanotube scaffolds for cardiac tissue engineering.

PubMed

Ahadian, Samad; Davenport Huyer, Locke; Estili, Mehdi; Yee, Bess; Smith, Nathaniel; Xu, Zhensong; Sun, Yu; Radisic, Milica

2017-04-01

Polymer biomaterials are used to construct scaffolds in tissue engineering applications to assist in mechanical support, organization, and maturation of tissues. Given the flexibility, electrical conductance, and contractility of native cardiac tissues, it is desirable that polymeric scaffolds for cardiac tissue regeneration exhibit elasticity and high electrical conductivity. Herein, we developed a facile approach to introduce carbon nanotubes (CNTs) into poly(octamethylene maleate (anhydride) 1,2,4-butanetricarboxylate) (124 polymer), and developed an elastomeric scaffold for cardiac tissue engineering that provides electrical conductivity and structural integrity to 124 polymer. 124 polymer-CNT materials were developed by first dispersing CNTs in poly(ethylene glycol) dimethyl ether porogen and mixing with 124 prepolymer for molding into shapes and crosslinking under ultraviolet light. 124 polymers with 0.5% and 0.1% CNT content (wt) exhibited improved conductivity against pristine 124 polymer. With increasing the CNT content, surface moduli of hybrid polymers were increased, while their bulk moduli were decreased. Furthermore, increased swelling of hybrid 124 polymer-CNT materials was observed, suggesting their improved structural support in an aqueous environment. Finally, functional characterization of engineered cardiac tissues using the 124 polymer-CNT scaffolds demonstrated improved excitation threshold in materials with 0.5% CNT content (3.6±0.8V/cm) compared to materials with 0% (5.1±0.8V/cm) and 0.1% (5.0±0.7V/cm), suggesting greater tissue maturity. 124 polymer-CNT materials build on the advantages of 124 polymer elastomer to give a versatile biomaterial for cardiac tissue engineering applications. Achieving a high elasticity and a high conductivity in a single cardiac tissue engineering material remains a challenge. We report the use of CNTs in making electrically conductive and mechanically strong polymeric scaffolds in cardiac tissue regeneration. CNTs were incorporated in elastomeric polymers in a facile and reproducible approach. Polymer-CNT materials were able to construct complicated scaffold structures by injecting the prepolymer into a mold and crosslinking the prepolymer under ultraviolet light. CNTs enhanced electrical conductivity and structural support of elastomeric polymers. Hybrid polymeric scaffolds containing 0.5wt% CNTs increased the maturation of cardiac tissues fabricated on them compared to pure polymeric scaffolds. The cardiac tissues on hybrid polymer-CNT scaffolds showed earlier beating than those on pure polymer scaffolds. In the future, fabricated polymer-CNT scaffolds could also be used to fabricate other electro-active tissues, such neural and skeletal muscle tissues. In the future, fabricated polymer-CNT scaffolds could also be used to fabricate other electro-active tissues, such as neural and skeletal muscle tissues. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Effects of matrix composition, microstructure, and viscoelasticity on the behaviors of vocal fold fibroblasts cultured in three-dimensional hydrogel networks.

PubMed

Farran, Alexandra J E; Teller, Sean S; Jha, Amit K; Jiao, Tong; Hule, Rohan A; Clifton, Rodney J; Pochan, Darrin P; Duncan, Randall L; Jia, Xinqiao

2010-04-01

Vocal fold diseases and disorders are difficult to treat surgically or therapeutically. Tissue engineering offers an alternative strategy for the restoration of functional vocal folds. As a first step toward vocal fold tissue engineering, we investigated the responses of primary vocal fold fibroblasts (PVFFs) to two types of collagen and hyaluronic acid (HA)-based hydrogels that are compositionally similar, but structurally variable and mechanically different. Type A hydrogels were composed of mature collagen fibers reinforced by oxidized HA, whereas type B hydrogels contained immature collagen fibrils interpenetrated in an amorphous, covalently cross-linked HA matrix. PVFFs encapsulated in either matrix adopted a fibroblastic morphology and expressed genes related to important extracellular matrix proteins. DNA analysis indicated a linear growth profile for cells encapsulated in type B gels from day 0 to 21, in contrast to an initial dormant, nonproliferative period from day 0 to 3 experienced by cells in type A gels. At the end of the culture, similar DNA content was detected in both types of constructs. A reduction in collagen content was observed for both types of constructs after 28 days of culture, with type A constructs generally retaining higher amounts of collagen than type B constructs. The HA content in the constructs decreased steadily throughout the culture, with type A constructs consistently exhibiting less HA than type B constructs. Using the torsional wave analysis, we found that the elastic moduli for type A constructs decreased sharply during the first week of culture, followed by 2 weeks of matrix stabilization without significant changes in matrix stiffness. Conversely, the elastic modulus for type B constructs increased moderately over time. It is postulated that PVFFs residing in gels alter the matrix organization, chemical compositions, and viscoelasticity through cell-mediated remodeling processes.

3D bioprinting for vascularized tissue fabrication

PubMed Central

Richards, Dylan; Jia, Jia; Yost, Michael; Markwald, Roger; Mei, Ying

2016-01-01

3D bioprinting holds remarkable promise for rapid fabrication of 3D tissue engineering constructs. Given its scalability, reproducibility, and precise multi-dimensional control that traditional fabrication methods do not provide, 3D bioprinting provides a powerful means to address one of the major challenges in tissue engineering: vascularization. Moderate success of current tissue engineering strategies have been attributed to the current inability to fabricate thick tissue engineering constructs that contain endogenous, engineered vasculature or nutrient channels that can integrate with the host tissue. Successful fabrication of a vascularized tissue construct requires synergy between high throughput, high-resolution bioprinting of larger perfusable channels and instructive bioink that promotes angiogenic sprouting and neovascularization. This review aims to cover the recent progress in the field of 3D bioprinting of vascularized tissues. It will cover the methods of bioprinting vascularized constructs, bioink for vascularization, and perspectives on recent innovations in 3D printing and biomaterials for the next generation of 3D bioprinting for vascularized tissue fabrication. PMID:27230253

Non-invasive and Non-destructive Characterization of Tissue Engineered Constructs Using Ultrasound Imaging Technologies: A Review.

PubMed

Kim, Kang; Wagner, William R

2016-03-01

With the rapid expansion of biomaterial development and coupled efforts to translate such advances toward the clinic, non-invasive and non-destructive imaging tools to evaluate implants in situ in a timely manner are critically needed. The required multi-level information is comprehensive, including structural, mechanical, and biological changes such as scaffold degradation, mechanical strength, cell infiltration, extracellular matrix formation and vascularization to name a few. With its inherent advantages of non-invasiveness and non-destructiveness, ultrasound imaging can be an ideal tool for both preclinical and clinical uses. In this review, currently available ultrasound imaging technologies that have been applied in vitro and in vivo for tissue engineering and regenerative medicine are discussed and some new emerging ultrasound technologies and multi-modality approaches utilizing ultrasound are introduced.

Non-invasive and non-destructive characterization of tissue engineered constructs using ultrasound imaging technologies: a review

PubMed Central

Kim, Kang; Wagner, William R.

2015-01-01

With the rapid expansion of biomaterial development and coupled efforts to translate such advances toward the clinic, non-invasive and non-destructive imaging tools to evaluate implants in situ in a timely manner are critically needed. The required multilevel information is comprehensive, including structural, mechanical, and biological changes such as scaffold degradation, mechanical strength, cell infiltration, extracellular matrix formation and vascularization to name a few. With its inherent advantages of non-invasiveness and non-destructiveness, ultrasound imaging can be an ideal tool for both preclinical and clinical uses. In this review, currently available ultrasound imaging technologies that have been applied in vitro and in vivo for tissue engineering and regenerative medicine are discussed and some new emerging ultrasound technologies and multi-modality approaches utilizing ultrasound are introduced. PMID:26518412

Design and characterization of a biodegradable double-layer scaffold aimed at periodontal tissue-engineering applications.

PubMed

Requicha, João F; Viegas, Carlos A; Hede, Shantesh; Leonor, Isabel B; Reis, Rui L; Gomes, Manuela E

2016-05-01

The inefficacy of the currently used therapies in achieving the regeneration ad integrum of the periodontium stimulates the search for alternative approaches, such as tissue-engineering strategies. Therefore, the core objective of this study was to develop a biodegradable double-layer scaffold for periodontal tissue engineering. The design philosophy was based on a double-layered construct obtained from a blend of starch and poly-ε-caprolactone (30:70 wt%; SPCL). A SPCL fibre mesh functionalized with silanol groups to promote osteogenesis was combined with a SPCL solvent casting membrane aiming at acting as a barrier against the migration of gingival epithelium into the periodontal defect. Each layer of the double-layer scaffolds was characterized in terms of morphology, surface chemical composition, degradation behaviour and mechanical properties. Moreover, the behaviour of seeded/cultured canine adipose-derived stem cells (cASCs) was assessed. In general, the developed double-layered scaffolds demonstrated adequate degradation and mechanical behaviour for the target application. Furthermore, the biological assays revealed that both layers of the scaffold allow adhesion and proliferation of the seeded undifferentiated cASCs, and the incorporation of silanol groups into the fibre-mesh layer enhance the expression of a typical osteogenic marker. This study allowed an innovative construct to be developed, combining a three-dimensional (3D) scaffold with osteoconductive properties and with potential to assist periodontal regeneration, carrying new possible solutions to current clinical needs. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

Embryonic rather than extraembryonic tissues have more impact on the development of placental hyperplasia in cloned mice.

PubMed

Miki, H; Wakisaka, N; Inoue, K; Ogonuki, N; Mori, M; Kim, J-M; Ohta, A; Ogura, A

2009-06-01

Somatic cell cloning by nuclear transfer (NT) in mice is associated with hyperplastic placentas at term. To dissect the effects of embryonic and extraembryonic tissues on this clone-associated phenotype, we constructed diploid (2n) fused with (<-->) tetraploid (4n) chimeras from NT- and fertilization-derived (FD) embryos. Generally, the 4n cells contributed efficiently to all the extraembryonic tissues but not to the embryo itself. Embryos constructed by 2n NT<-->4n FD aggregation developed hyperplastic placentas (0.33+/-0.22 g) with a predominant contribution by NT-derived cells. Even when the population of FD-derived cells in placentas was increased using multiple FD embryos (up to four) for aggregation, most placentas remained hyperplastic (0.36+/-0.13 g). By contrast, placentas of the reciprocal combination, 2n FD<-->4n NT, were less hyperplastic (0.15+/-0.02 g). These nearly normal-looking placentas had a large proportion of NT-derived cells. Thus, embryonic rather than extraembryonic tissues had more impact on the onset of placental hyperplasia, and that the abnormal placentation in clones occurs in a noncell-autonomous manner. These findings suggest that for improvement of cloning efficiency we should understand the mechanisms regulating placentation, especially those of embryonic origin that might control the proliferation of trophoblastic lineage cells.

Naturally Engineered Maturation of Cardiomyocytes

PubMed Central

Scuderi, Gaetano J.; Butcher, Jonathan

2017-01-01

Ischemic heart disease remains one of the most prominent causes of mortalities worldwide with heart transplantation being the gold-standard treatment option. However, due to the major limitations associated with heart transplants, such as an inadequate supply and heart rejection, there remains a significant clinical need for a viable cardiac regenerative therapy to restore native myocardial function. Over the course of the previous several decades, researchers have made prominent advances in the field of cardiac regeneration with the creation of in vitro human pluripotent stem cell-derived cardiomyocyte tissue engineered constructs. However, these engineered constructs exhibit a functionally immature, disorganized, fetal-like phenotype that is not equivalent physiologically to native adult cardiac tissue. Due to this major limitation, many recent studies have investigated approaches to improve pluripotent stem cell-derived cardiomyocyte maturation to close this large functionality gap between engineered and native cardiac tissue. This review integrates the natural developmental mechanisms of cardiomyocyte structural and functional maturation. The variety of ways researchers have attempted to improve cardiomyocyte maturation in vitro by mimicking natural development, known as natural engineering, is readily discussed. The main focus of this review involves the synergistic role of electrical and mechanical stimulation, extracellular matrix interactions, and non-cardiomyocyte interactions in facilitating cardiomyocyte maturation. Overall, even with these current natural engineering approaches, pluripotent stem cell-derived cardiomyocytes within three-dimensional engineered heart tissue still remain mostly within the early to late fetal stages of cardiomyocyte maturity. Therefore, although the end goal is to achieve adult phenotypic maturity, more emphasis must be placed on elucidating how the in vivo fetal microenvironment drives cardiomyocyte maturation. This information can then be utilized to develop natural engineering approaches that can emulate this fetal microenvironment and thus make prominent progress in pluripotent stem cell-derived maturity toward a more clinically relevant model for cardiac regeneration. PMID:28529939

Biomechanical Comparison of Glutaraldehyde-Crosslinked Gelatin Fibrinogen Electrospun Scaffolds to Porcine Coronary Arteries

PubMed Central

Tamimi, E.; Ardila, D. C.; Haskett, D. G.; Doetschman, T.; Slepian, M. J.; Kellar, R. S.; Vande Geest, J. P.

2016-01-01

Cardiovascular disease (CVD) is the leading cause of death for Americans. As coronary artery bypass graft surgery (CABG) remains a mainstay of therapy for CVD and native vein grafts are limited by issues of supply and lifespan, an effective readily available tissue-engineered vascular graft (TEVG) for use in CABG would provide drastic improvements in patient care. Biomechanical mismatch between vascular grafts and native vasculature has been shown to be the major cause of graft failure, and therefore, there is need for compliance-matched biocompatible TEVGs for clinical implantation. The current study investigates the biaxial mechanical characterization of acellular electrospun glutaraldehyde (GLUT) vapor-crosslinked gelatin/fibrinogen cylindrical constructs, using a custom-made microbiaxial optomechanical device (MOD). Constructs crosslinked for 2, 8, and 24 hrs are compared to mechanically characterized porcine left anterior descending coronary (LADC) artery. The mechanical response data were used for constitutive modeling using a modified Fung strain energy equation. The results showed that constructs crosslinked for 2 and 8 hrs exhibited circumferential and axial tangential moduli (ATM) similar to that of the LADC. Furthermore, the 8-hrs experimental group was the only one to compliance-match the LADC, with compliance values of 0.0006±0.00018 mm Hg−1 and 0.00071±0.00027 mm Hg−1, respectively. The results of this study show the feasibility of meeting mechanical specifications expected of native arteries through manipulating GLUT vapor crosslinking time. The comprehensive mechanical characterization of cylindrical biopolymer constructs in this study is an important first step to successfully develop a biopolymer compliance-matched TEVG. PMID:26501189

Chondrocyte and Mesenchymal Stem Cell Derived Engineered Cartilage Exhibits Differential Sensitivity to Pro-Inflammatory Cytokines.

PubMed

Mohanraj, Bhavana; Huang, Alice H; Yeger-McKeever, Meira J; Schmidt, Megan J; Dodge, George R; Mauck, Robert L

2018-05-29

Tissue engineering is a promising approach for the repair of articular cartilage defects, with engineered constructs emerging that match native tissue properties. However, the inflammatory environment of the damaged joint might compromise outcomes, and this may be impacted by the choice of cell source in terms of their ability to operate anabolically in an inflamed environment. Here, we compared the response of engineered cartilage derived from native chondrocytes and mesenchymal stem cells (MSCs) to challenge by TNFα and IL-1β in order to determine if either cell type possessed an inherent advantage. Compositional (extracellular matrix) and functional (mechanical) characteristics, as well as the release of catabolic mediators (matrix metalloproteinases (MMPs), nitric oxide (NO)) were assessed to determine cell- and tissue- level changes following exposure to IL-1β or TNF-α. Results demonstrated that MSC-derived constructs were more sensitive to inflammatory mediators than chondrocyte-derived constructs, exhibiting a greater loss of proteoglycans and functional properties at lower cytokine concentrations. While MSCs and chondrocytes both have the capacity to form functional engineered cartilage in vitro, this study suggests that the presence of an inflammatory environment is more likely to impair the in vivo success of MSC-derived cartilage repair. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Repair of Avascular Meniscus Tears with Electrospun Collagen Scaffolds Seeded with Human Cells

PubMed Central

Baek, Jihye; Sovani, Sujata; Glembotski, Nicholas E.; Du, Jiang; Jin, Sungho; Grogan, Shawn P.

2016-01-01

The self-healing capacity of an injured meniscus is limited to the vascularized regions and is especially challenging in the inner avascular regions. As such, we investigated the use of human meniscus cell-seeded electrospun (ES) collagen type I scaffolds to produce meniscal tissue and explored whether these cell-seeded scaffolds can be implanted to repair defects created in meniscal avascular tissue explants. Human meniscal cells (derived from vascular and avascular meniscal tissue) were seeded on ES scaffolds and cultured. Constructs were evaluated for cell viability, gene expression, and mechanical properties. To determine potential for repair of meniscal defects, human meniscus avascular cells were seeded and cultured on aligned ES collagen scaffolds for 4 weeks before implantation. Surgical defects resembling “longitudinal tears” were created in the avascular zone of bovine meniscus and implanted with cell-seeded collagen scaffolds and cultured for 3 weeks. Tissue regeneration and integration were evaluated by histology, immunohistochemistry, mechanical testing, and magentic resonance imaging. Ex vivo implantation with cell-seeded collagen scaffolds resulted in neotissue that was significantly better integrated with the native tissue than acellular collagen scaffolds or untreated defects. Human meniscal cell-seeded ES collagen scaffolds may therefore be useful in facilitating meniscal repair of avascular meniscus tears. PMID:26842062

Prediction of oxygen distribution in aortic valve leaflet considering diffusion and convection.

PubMed

Wang, Ling; Korossis, Sotirios; Fisher, John; Ingham, Eileen; Jin, Zhongmin

2011-07-01

Oxygen supply and transport is an important consideration in the development of tissue engineered constructs. Previous studies from our group have focused on the effect of tissue thickness on the oxygen diffusion within a three-dimensional aortic valve leaflet model, and highlighted the necessity for additional transport mechanisms such as oxygen convection. The aims of this study were to investigate the effect of interstitial fluid flow within the aortic valve leaflet, induced by the cyclic loading of the leaflet, on oxygen transport. Indentation testing and finite element modelings were employed to derive the biphasic properties of the leaflet tissue. The biphasic properties were subsequently used in the computational modeling of oxygen convection in the leaflet, which was based on the effective interstitial fluid velocity and the tissue deformation. Subsequently, the oxygen profile was predicted within the valve leaflet model by solving the diffusion and convection equation simultaneously utilizing the finite difference method. The compression modulus (E) and hydraulic permeability were determined by adapting a finite element model to the experimental indentation test on valvular tissue, E = 0.05MPa, and k =2.0 mm4/Ns. Finite element model of oxygen convection in valvular tissue incorporating the predicted biphasic properties was developed and the interstitial fluid flow rate was calculated falling in range of 0.025-0.25 mm/s depending on the tissue depth. Oxygen distribution within valvular tissue was predicted using one-dimensional oxygen diffusion model taking into consider the interstitial fluid effect. It was found that convection did enhance the oxygen transport in valvular tissue by up to 68% increase in the minimum oxygen tension within the tissue, depending on the strain level of the tissue as reaction of the magnitude and frequencies of the cardiac loading. The effective interstitial fluid velocity was found to play an important role in enhancing the oxygen transport within the valve leaflet. Such an understanding is important in the development of valvular tissue engineered constructs.

Photothermal optical coherence tomography of epidermal growth factor receptor in live cells using immunotargeted gold nanospheres

NASA Astrophysics Data System (ADS)

Skala, Melissa C.; Crow, Matthew J.; Wax, Adam; Izatt, Joseph A.

2009-02-01

Molecular imaging is a powerful tool for investigating disease processes and potential therapies in both in vivo and in vitro systems. However, high resolution molecular imaging has been limited to relatively shallow penetration depths that can be accessed with microscopy. Optical coherence tomography (OCT) is an optical analogue to ultrasound with relatively good penetration depth (1-2 mm) and resolution (~1-10 μm). We have developed and characterized photothermal OCT as a molecular contrast mechanism that allows for high resolution molecular imaging at deeper penetration depths than microscopy. Our photothermal system consists of an amplitude-modulated heating beam that spatially overlaps with the focused spot of the sample arm of a spectral-domain OCT microscope. Validation experiments in tissue-like phantoms containing gold nanospheres that absorb at 532 nm revealed a sensitivity of 14 parts per million nanospheres (weight/weight) in a tissue-like environment. The nanospheres were then conjugated to anti-EGFR, and molecular targeting was confirmed in cells that over-express EGFR (MDA-MB-468) and cells that express low levels of EGFR (MDA-MB-435). Molecular imaging in three-dimensional tissue constructs was confirmed with a significantly lower photothermal signal (p<0.0001) from the constructs composed of cells that express low levels of EGFR compared to the over-expressing cell constructs (300% signal increase). This technique could potentially augment confocal and multiphoton microscopy as a method for deep-tissue, depth-resolved molecular imaging with relatively high resolution and target sensitivity, without photobleaching or cytotoxicity.

Fabrication of triple-layered bifurcated vascular scaffold with a certain degree of three-dimensional structure

NASA Astrophysics Data System (ADS)

Liu, Yuanyuan; Jiang, Weijian; Yang, Yang; Pu, Huayan; Peng, Yan; Xin, Liming; Zhang, Yi; Sun, Yu

2018-01-01

Constructing vascular scaffolds is important in tissue engineering. However, scaffolds with characteristics such as multiple layers and a certain degree of spatial morphology still cannot be readily constructed by current vascular scaffolds fabrication techniques. This paper presents a three-layered bifurcated vascular scaffold with a curved structure. The technique combines 3D printed molds and casting hydrogel and fugitive ink to create vessel-mimicking constructs with customizable structural parameters. Compared with other fabrication methods, the technique can create more native-like 3D geometries. The diameter and wall thickness of the fabricated constructs can be independently controlled, providing a feasible approach for vascular scaffold construction. Enzymatically-crosslinked gelatin was used as the scaffold material. The morphology and mechanical properties were evaluated. Human umbilical cord derived endothelial cells (HUVECs) were seeded on the scaffolds and cultured for 72 h. Cell viability and morphology were assessed. The results showed that the proposed process had good application potentials, and will hopefully provide a feasible approach for constructing vascular scaffolds.

Osteogenic Treatment Initiating a Tissue-Engineered Cartilage Template Hypertrophic Transition.

PubMed

Fu, J Y; Lim, S Y; He, P F; Fan, C J; Wang, D A

2016-10-01

Hypertrophic chondrocytes play a critical role in endochondral bone formation as well as the progress of osteoarthritis (OA). An in vitro cartilage hypertrophy model can be used as a platform to study complex molecular mechanisms involved in these processes and screen new drugs for OA. To develop an in vitro cartilage hypertrophy model, we treated a tissue-engineered cartilage template, living hyaline cartilaginous graft (LhCG), with osteogenic medium for hypertrophic induction. In addition, endothelial progenitor cells (EPCs) were seeded onto LhCG constructs to mimic vascular invasion. The results showed that osteogenic treatment significantly inhibited the synthesis of endostatin in LhCG constructs and enhanced expression of hypertrophic marker-collagen type X (Col X) and osteogenic markers, as well as calcium deposition in vitro. Upon subcutaneous implantation, osteogenic medium-treated LhCG constructs all stained positive for Col X and showed significant calcium deposition and blood vessel invasion. Col X staining and calcium deposition were most obvious in osteogenic medium-treated only group, while there was no difference between EPC-seeded and non-seeded group. These results demonstrated that osteogenic treatment was of the primary factor to induce hypertrophic transition of LhCG constructs and this model may contribute to the establishment of an in vitro cartilage hypertrophy model.

Requirement for serum in medium supplemented with insulin-transferrin-selenium for hydrodynamic cultivation of engineered cartilage.

PubMed

Yang, Yueh-Hsun; Barabino, Gilda A

2011-08-01

Achievement of viable engineered tissues through in vitro cultivation in bioreactor systems requires a thorough understanding of the complex interplay between hydrodynamic forces and biochemical cues such as serum. To this end, chondrocyte-seeded constructs were cultured under continuous fluid-induced shear forces with reduced serum content (0%-2%, v/v), which was partially or completely replaced by a potential substitute, insulin-transferrin-selenium, to minimize deleterious effects associated with the use of culture media containing high levels of serum (10%-20%). Low-serum cultures yielded constructs with similar biochemical properties to those cultivated with high-serum supplements, whereas the serum-free constructs exhibited poor cell proliferation, insufficient extracellular matrix production, and rapid degradation of and/or shear-induced damage to polyglycolic acid scaffolds. A fibrous outer capsule typically observed in hydrodynamic cultures and characterized by increased cell density and decreased (virtually none) glycosaminoglycan deposition was eliminated when serum concentration was equal to or <0.2% in the presence of hydrodynamic stimuli. Our findings suggest that serum is a requirement in insulin-transferrin-selenium-supplemented cultures in order for constructs to exhibit improved properties in response to hydrodynamic forces, and that mechanical and biochemical stimuli may synergistically modulate tissue properties and morphology through shear-responsive signals.

A poroelastic model describing nutrient transport and cell stresses within a cyclically strained collagen hydrogel.

PubMed

Vaughan, Benjamin L; Galie, Peter A; Stegemann, Jan P; Grotberg, James B

2013-11-05

In the creation of engineered tissue constructs, the successful transport of nutrients and oxygen to the contained cells is a significant challenge. In highly porous scaffolds subject to cyclic strain, the mechanical deformations can induce substantial fluid pressure gradients, which affect the transport of solutes. In this article, we describe a poroelastic model to predict the solid and fluid mechanics of a highly porous hydrogel subject to cyclic strain. The model was validated by matching the predicted penetration of a bead into the hydrogel from the model with experimental observations and provides insight into nutrient transport. Additionally, the model provides estimates of the wall-shear stresses experienced by the cells embedded within the scaffold. These results provide insight into the mechanics of and convective nutrient transport within a cyclically strained hydrogel, which could lead to the improved design of engineered tissues. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Layer by Layer Three-dimensional Tissue Epitaxy by Cell-Laden Hydrogel Droplets

PubMed Central

Moon, SangJun; Hasan, Syed K.; Song, Young S.; Xu, Feng; Keles, Hasan Onur; Manzur, Fahim; Mikkilineni, Sohan; Hong, Jong Wook; Nagatomi, Jiro; Haeggstrom, Edward; Khademhosseini, Ali

2010-01-01

The ability to bioengineer three-dimensional (3D) tissues is a potentially powerful approach to treat diverse diseases such as cancer, loss of tissue function, or organ failure. Traditional tissue engineering methods, however, face challenges in fabricating 3D tissue constructs that resemble the native tissue microvasculature and microarchitectures. We have developed a bioprinter that can be used to print 3D patches of smooth muscle cells (5 mm × 5 mm × 81 μm) encapsulated within collagen. Current inkjet printing systems suffer from loss of cell viability and clogging. To overcome these limitations, we developed a system that uses mechanical valves to print high viscosity hydrogel precursors containing cells. The bioprinting platform that we developed enables (i) printing of multilayered 3D cell-laden hydrogel structures (16.2 μm thick per layer) with controlled spatial resolution (proximal axis: 18.0 ± 7.0 μm and distal axis: 0.5 ± 4.9 μm), (ii) high-throughput droplet generation (1 s per layer, 160 droplets/s), (iii) cell seeding uniformity (26 ± 2 cells/mm2 at 1 million cells/mL, 122 ± 20 cells/mm2 at 5 million cells/mL, and 216 ± 38 cells/mm2 at 10 million cells/mL), and (iv) long-term viability in culture (>90%, 14 days). This platform to print 3D tissue constructs may be beneficial for regenerative medicine applications by enabling the fabrication of printed replacement tissues. PMID:19586367

Micromechanical analysis of volumetric growth in the context of open systems thermodynamics and configurational mechanics. Application to tumor growth

NASA Astrophysics Data System (ADS)

Ganghoffer, J. F.; Boubaker, M. B.

2017-03-01

We adopt in this paper the physically and micromechanically motivated point of view that growth (resp. resorption) occurs as the expansion (resp. contraction) of initially small tissue elements distributed within a host surrounding matrix, due to the interfacial motion of their boundary. The interface motion is controlled by the availability of nutrients and mechanical driving forces resulting from the internal stresses that built in during the growth. A general extremum principle of the zero potential for open systems witnessing a change of their mass due to the diffusion of nutrients is constructed, considering the framework of open systems thermodynamics. We postulate that the shape of the tissue element evolves in such a way as to minimize the zero potential among all possible admissible shapes of the growing tissue elements. The resulting driving force for the motion of the interface sets a surface growth models at the scale of the growing tissue elements, and is conjugated to a driving force identified as the interfacial jump of the normal component of an energy momentum tensor, in line with Hadamard's structure theorem. The balance laws associated with volumetric growth at the mesoscopic level result as the averaging of surface growth mechanisms occurring at the microscopic scale of the growing tissue elements. The average kinematics has been formulated in terms of the effective growth velocity gradient and elastic rate of deformation tensor, both functions of time. This formalism is exemplified by the simulation of the avascular growth of multicell spheroids in the presence of diffusion of nutrients, showing the respective influence of mechanical and chemical driving forces in relation to generation of internal stresses.

Nondestructive evaluation of a new hydrolytically degradable and photo-clickable PEG hydrogel for cartilage tissue engineering.

PubMed

Neumann, Alexander J; Quinn, Timothy; Bryant, Stephanie J

2016-07-15

Photopolymerizable and hydrolytically labile poly(ethylene glycol) (PEG) hydrogels formed from photo-clickable reactions were investigated as cell delivery platforms for cartilage tissue engineering (TE). PEG hydrogels were formed from thiol-norbornene PEG macromers whereby the crosslinks contained caprolactone segments with hydrolytically labile ester linkages. Juvenile bovine chondrocytes encapsulated in the hydrogels were cultured for up to four weeks and assessed biochemically and histologically, using standard destructive assays, and for mechanical and ultrasound properties, as nondestructive assays. Bulk degradation of acellular hydrogels was confirmed by a decrease in compressive modulus and an increase in mass swelling ratio over time. Chondrocytes deposited increasing amounts of sulfated glycosaminoglycans and collagens in the hydrogels with time. Spatially, collagen type II and aggrecan were present in the neotissue with formation of a territorial matrix beginning at day 21. Nondestructive measurements revealed an 8-fold increase in compressive modulus from days 7 to 28, which correlated with total collagen content. Ultrasound measurements revealed changes in the constructs over time, which differed from the mechanical properties, and appeared to correlate with ECM structure and organization shown by immunohistochemical analysis. Overall, non-destructive and destructive measurements show that this new hydrolytically degradable PEG hydrogel is promising for cartilage TE. Designing synthetic hydrogels whose degradation matches tissue growth is critical to maintaining mechanical integrity as the hydrogel degrades and new tissue forms, but is challenging due to the nature of the hydrogel crosslinks that inhibit diffusion of tissue matrix molecules. This study details a promising, new, photo-clickable and synthetic hydrogel whose degradation supports cartilaginous tissue matrix growth leading to the formation of a territorial matrix, concomitant with an increase in mechanical properties. Nondestructive assays based on mechanical and ultrasonic properties were also investigated using a novel instrument and found to correlate with matrix deposition and evolution. In sum, this study presents a new hydrogel platform combined with nondestructive assessments, which together have potential for in vitro cartilage tissue engineering. Copyright © 2016 Acta Materialia Inc. All rights reserved.

Designing injectable beta-hairpin peptide hydrogels for cartilage tissue engineering application

NASA Astrophysics Data System (ADS)

Sinthuvanich, Chomdao

In this work, it was demonstrated that peptide-based gels having different electrostatic network character but similar mechanical properties can be designed by modulating the primary sequence of the peptides used for self-assembly. As a result, HLT2 and HET1 peptides, having formal charge states of +5 per monomer, were designed using MAX8, a peptide with a charge state of +7 per monomer, as a template. Using gels prepared from all three peptides (MAX8, HLT2, and HET1), it was shown that the electropositive character of the network influences chondrocyte behavior. Specifically, the less electropositive gel (HLT2) is able to maintain chondrocyte viability and phenotype. In contrast, chondrocytes encapsulated in the more positively charged gel (MAX8) are more prone to dedifferentiation, resulting in tissue constructs with inferior mechanical properties. Gels prepared from peptides having the same net charge but differing only in their primary sequences (HLT2 and HET1) were also shown to influence cell behavior, but only during the early period of culturing. If constructs derived from these two different peptide gels are allowed to culture for extended times, their mechanical properties become similar. This suggests that the amino acid composition and sequence of the peptides used to make the gels also influences cell behavior, but perhaps not to the extent that network electrostatics plays. Supplementation of bioactive factors in the culturing media, as opposed to being encapsulated directly in the network, was shown to adversely affect the cellular response resulting in tissue constructs where extracellular matrix (ECM) components are non-uniformly distributed. When bioactive factors were encapsulated and co-delivered with cells, positive results were observed, particularly when cells were co-encapsulated with the growth factor, TGF-β1. The effect of TGF-β1 on cellular response and the mechanical properties of the tissue-engineered constructs is largely governed by the ability of the growth factor to be retained within the hydrogels and made available to the cells, which in turn, dictate the quality of the engineered tissue. Rational peptide design was also employed to generate negatively charged peptides capable of folding and self-assembling under physiological conditions to afford electronegative gel. Initial designs resulted in peptides that undergo gelation in response to a change in environmental pH and temperature. Modification of these initially designed peptides led to the design of VE3 and VEQ1, two negatively charged peptides that can be used to directly encapsulate chondrocytes providing gel-cell constructs with homogeneous cellular distribution. Finally, the positively charged peptide gel (HET1) and negatively charged peptide gel (VE3) were compared to investigate the influence of vastly different network electrostatics on the response of encapsulated primary chondrocytes. In these gels, a majority of cells were able to retain their chondrocyte phenotype within the scaffold regardless of which gel was used for encapsulation and delivery. However, the positively charge hydrogel is better at supporting cell proliferation and ECM accumulation. On the other hand, the cells encapsulated in the negatively charged hydrogel were less proliferative and the negatively charged hydrogel had a limited ability to retain ECM produced by the cells. In contrast, when culturing in the presence of TGF-β1, constructs derived from the negatively charged gel showed greater compressive moduli than those derived from the positively charged hydrogel. This difference is largely due to the amount of TGF-β1 made available to the encapsulated cells as a function of time, which was found to be governed by the electrostatic character of the hydrogel network. This work indicates that network electrostatics influence the response of encapsulated chondrocytes, retention of secreted ECM, and the diffusion of bioactive factors necessary for the generation of engineered cartilage. During the course of these studies, I have a serendipitous discovery that a derivative of one of the material forming β-hairpin peptides displays anticancer activity. Chapter 8 describes this peptide, SVS-1, and its mechanism of action. (Abstract shortened by UMI.).

Dynamic reciprocity in cell-scaffold interactions.

PubMed

Mauney, Joshua R; Adam, Rosalyn M

2015-03-01

Tissue engineering in urology has shown considerable promise. However, there is still much to understand, particularly regarding the interactions between scaffolds and their host environment, how these interactions regulate regeneration and how they may be enhanced for optimal tissue repair. In this review, we discuss the concept of dynamic reciprocity as applied to tissue engineering, i.e. how bi-directional signaling between implanted scaffolds and host tissues such as the bladder drives the process of constructive remodeling to ensure successful graft integration and tissue repair. The impact of scaffold content and configuration, the contribution of endogenous and exogenous bioactive factors, the influence of the host immune response and the functional interaction with mechanical stimulation are all considered. In addition, the temporal relationships of host tissue ingrowth, bioactive factor mobilization, scaffold degradation and immune cell infiltration, as well as the reciprocal signaling between discrete cell types and scaffolds are discussed. Improved understanding of these aspects of tissue repair will identify opportunities for optimization of repair that could be exploited to enhance regenerative medicine strategies for urology in future studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Induction of functional tissue-engineered skeletal muscle constructs by defined electrical stimulation.

PubMed

Ito, Akira; Yamamoto, Yasunori; Sato, Masanori; Ikeda, Kazushi; Yamamoto, Masahiro; Fujita, Hideaki; Nagamori, Eiji; Kawabe, Yoshinori; Kamihira, Masamichi

2014-04-24

Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3 V/mm amplitude, 4 ms width, and 1 Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50-60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs.

Multizone Paper Platform for 3D Cell Cultures

PubMed Central

Derda, Ratmir; Hong, Estrella; Mwangi, Martin; Mammoto, Akiko; Ingber, Donald E.; Whitesides, George M.

2011-01-01

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (“cells-in-gels-in-paper” or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, “sections” all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures. PMID:21573103

Regeneration of the anterior cruciate ligament: Current strategies in tissue engineering

PubMed Central

Nau, Thomas; Teuschl, Andreas

2015-01-01

Recent advancements in the field of musculoskeletal tissue engineering have raised an increasing interest in the regeneration of the anterior cruciate ligament (ACL). It is the aim of this article to review the current research efforts and highlight promising tissue engineering strategies. The four main components of tissue engineering also apply in several ACL regeneration research efforts. Scaffolds from biological materials, biodegradable polymers and composite materials are used. The main cell sources are mesenchymal stem cells and ACL fibroblasts. In addition, growth factors and mechanical stimuli are applied. So far, the regenerated ACL constructs have been tested in a few animal studies and the results are encouraging. The different strategies, from in vitro ACL regeneration in bioreactor systems to bio-enhanced repair and regeneration, are under constant development. We expect considerable progress in the near future that will result in a realistic option for ACL surgery soon. PMID:25621217

Cellulose/poly-(m-phenylene isophthalamide) porous film as a tissue-engineered skin bioconstruct

NASA Astrophysics Data System (ADS)

Lee, Jae Woong; Han, Sung Soo; Zo, Sum Mi; Choi, Soon Mo

2018-06-01

Regarding the porous structure, coagulated cellulose may not provide sufficient voids for cell proliferation, resulting in tissue growth. For this reason, it was blended with poly(m-phenylene isophthalamide) (PMIA), which could produce a porous structure in the resulting construct. The aim of this study was to confirm the potential of a novel cellulose/PMIA porous film as a tissue-engineered bioconstruct for impaired skin. The films were fabricated by a coagulation process added with a peel-off method, and the structural, mechanical properties were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, and capillary flow porometry. CRL-2310 human keratinocytes were used to determine the biocompatibility of the prepared films. The attachment and proliferation of cells were investigated by scanning electron microscopy, DAPI staining, and a cell viability assay. The results show that cellulose/PMIA porous films have potential use as wound matrices for skin tissue genesis.

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer.

PubMed

Dennis, Sarah Grace; Trusk, Thomas; Richards, Dylan; Jia, Jia; Tan, Yu; Mei, Ying; Fann, Stephen; Markwald, Roger; Yost, Michael

2015-09-22

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

Engineering tubular bone using mesenchymal stem cell sheets and coral particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geng, Wenxin; Ma, Dongyang; Yan, Xingrong

Highlights: • We developed a novel engineering strategy to solve the limitations of bone grafts. • We fabricated tubular constructs using cell sheets and coral particles. • The composite constructs showed high radiological density and compressive strength. • These characteristics were similar to those of native bone. -- Abstract: The development of bone tissue engineering has provided new solutions for bone defects. However, the cell-scaffold-based approaches currently in use have several limitations, including low cell seeding rates and poor bone formation capacity. In the present study, we developed a novel strategy to engineer bone grafts using mesenchymal stem cell sheetsmore » and coral particles. Rabbit bone marrow mesenchymal stem cells were continuously cultured to form a cell sheet with osteogenic potential and coral particles were integrated into the sheet. The composite sheet was then wrapped around a cylindrical mandrel to fabricate a tubular construct. The resultant tubular construct was cultured in a spinner-flask bioreactor and subsequently implanted into a subcutaneous pocket in a nude mouse for assessment of its histological characteristics, radiological density and mechanical property. A similar construct assembled from a cell sheet alone acted as a control. In vitro observations demonstrated that the composite construct maintained its tubular shape, and exhibited higher radiological density, compressive strength and greater extracellular matrix deposition than did the control construct. In vivo experiments further revealed that new bone formed ectopically on the composite constructs, so that the 8-week explants of the composite sheets displayed radiological density similar to that of native bone. These results indicate that the strategy of using a combination of a cell sheet and coral particles has great potential for bone tissue engineering and repairing bone defects.« less

Mesoderm Lineage 3D Tissue Constructs Are Produced at Large-Scale in a 3D Stem Cell Bioprocess.

PubMed

Cha, Jae Min; Mantalaris, Athanasios; Jung, Sunyoung; Ji, Yurim; Bang, Oh Young; Bae, Hojae

2017-09-01

Various studies have presented different approaches to direct pluripotent stem cell differentiation such as applying defined sets of exogenous biochemical signals and genetic/epigenetic modifications. Although differentiation to target lineages can be successfully regulated, such conventional methods are often complicated, laborious, and not cost-effective to be employed to the large-scale production of 3D stem cell-based tissue constructs. A 3D-culture platform that could realize the large-scale production of mesoderm lineage tissue constructs from embryonic stem cells (ESCs) is developed. ESCs are cultured using our previously established 3D-bioprocess platform which is amenable to mass-production of 3D ESC-based tissue constructs. Hepatocarcinoma cell line conditioned medium is introduced to the large-scale 3D culture to provide a specific biomolecular microenvironment to mimic in vivo mesoderm formation process. After 5 days of spontaneous differentiation period, the resulting 3D tissue constructs are composed of multipotent mesodermal progenitor cells verified by gene and molecular expression profiles. Subsequently the optimal time points to trigger terminal differentiation towards cardiomyogenesis or osteogenesis from the mesodermal tissue constructs is found. A simple and affordable 3D ESC-bioprocess that can reach the scalable production of mesoderm origin tissues with significantly improved correspondent tissue properties is demonstrated. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Airway and tissue loading in postinterrupter response of the respiratory system - an identification algorithm construction.

PubMed

Jablonski, Ireneusz; Mroczka, Janusz

2010-01-01

The paper offers an enhancement of the classical interrupter technique algorithm dedicated to respiratory mechanics measurements. Idea consists in exploitation of information contained in postocclusional transient states during indirect measurement of parameter characteristics by model identification. It needs the adequacy of an inverse analogue to general behavior of the real system and a reliable algorithm of parameter estimation. The second one was a subject of reported works, which finally showed the potential of the approach to separation of airway and tissue response in a case of short-term excitation by interrupter valve operation. Investigations were conducted in a regime of forward-inverse computer experiment.

Factors affecting the structure and maturation of human tissue engineered skeletal muscle.

PubMed

Martin, Neil R W; Passey, Samantha L; Player, Darren J; Khodabukus, Alastair; Ferguson, Richard A; Sharples, Adam P; Mudera, Vivek; Baar, Keith; Lewis, Mark P

2013-07-01

Tissue engineered skeletal muscle has great utility in experimental studies of physiology, clinical testing and its potential for transplantation to replace damaged tissue. Despite recent work in rodent tissue or cell lines, there is a paucity of literature concerned with the culture of human muscle derived cells (MDCs) in engineered constructs. Here we aimed to tissue engineer for the first time in the literature human skeletal muscle in self-assembling fibrin hydrogels and determine the effect of MDC seeding density and myogenic proportion on the structure and maturation of the constructs. Constructs seeded with 4 × 10(5) MDCs assembled to a greater extent than those at 1 × 10(5) or 2 × 10(5), and immunostaining revealed a higher fusion index and a higher density of myotubes within the constructs, showing greater structural semblance to in vivo tissue. These constructs primarily expressed perinatal and slow type I myosin heavy chain mRNA after 21 days in culture. In subsequent experiments MACS(®) technology was used to separate myogenic and non-myogenic cells from their heterogeneous parent population and these cells were seeded at varying myogenic (desmin +) proportions in fibrin based constructs. Only in the constructs seeded with 75% desmin + cells was there evidence of striations when immunostained for slow myosin heavy chain compared with constructs seeded with 10 or 50% desmin + cells. Overall, this work reveals the importance of cell number and myogenic proportions in tissue engineering human skeletal muscle with structural resemblance to in vivo tissue. Copyright © 2013 Elsevier Ltd. All rights reserved.

The effect of cyclic hydrostatic pressure on the functional development of cartilaginous tissues engineered using bone marrow derived mesenchymal stem cells.

PubMed

Meyer, E G; Buckley, C T; Steward, A J; Kelly, D J

2011-10-01

Mechanical signals can play a key role in regulating the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to determine if the long-term application of cyclic hydrostatic pressure could be used to improve the functional properties of cartilaginous tissues engineered using bone marrow derived MSCs. MSCs were isolated from the femora of two porcine donors, expanded separately under identical conditions, and then suspended in cylindrical agarose hydrogels. Constructs from both donors were maintained in a chemically defined media supplemented with TGF-β3 for 42 days. TGF-β3 was removed from a subset of constructs from day 21 to 42. Loaded groups were subjected to 10 MPa of cyclic hydrostatic pressurisation at 1 Hz for one hour/day, five days/week. Loading consisted either of continuous hydrostatic pressure (CHP) initiated at day 0, or delayed hydrostatic pressure (DHP) initiated at day 21. Free swelling (FS) constructs were cultured in parallel as controls. Constructs were assessed at days 0, 21 and 42. MSCs isolated from both donors were morphologically similar, demonstrated comparable colony forming unit-fibroblast (CFU-F) numbers, and accumulated near identical levels of collagen and GAG following 42 days of free swelling culture. Somewhat unexpectedly the two donors displayed a differential response to hydrostatic pressure. For one donor the application of CHP resulted in increased collagen and GAG accumulation by day 42, resulting in an increased dynamic modulus compared to FS controls. In contrast, CHP had no effect on matrix accumulation for the other donor. The application of DHP had no effect on either matrix accumulation or construct mechanical properties for both donors. Variability in the response to hydrostatic pressure was also observed for three further donors. In conclusion, this study demonstrates that the application of long-term hydrostatic pressure can be used to improve the functional properties of cartilaginous tissues engineered using bone marrow derived MSCs by enhancing collagen and GAG accumulation. The response to such loading however is donor dependent, which has implications for the clinical utilisation of such a stimulus when engineering cartilaginous grafts using autologous MSCs. Copyright © 2011 Elsevier Ltd. All rights reserved.

Hydrogel Bioprinted Microchannel Networks for Vascularization of Tissue Engineering Constructs

PubMed Central

Bertassoni, Luiz E.; Cecconi, Martina; Manoharan, Vijayan; Nikkhah, Mehdi; Hjortnaes, Jesper; Cristino, Ana Luiza; Barabaschi, Giada; Demarchi, Danilo; Dokmeci, Mehmet R.; Yang, Yunzhi; Khademhosseini, Ali

2014-01-01

Vascularization remains a critical challenge in tissue engineering. The development of vascular networks within densely populated and metabolically functional tissues facilitate transport of nutrients and removal of waste products, thus preserving cellular viability over a long period of time. Despite tremendous progress in fabricating complex tissue constructs in the past few years, approaches for controlled vascularization within hydrogel based engineered tissue constructs have remained limited. Here, we report a three dimensional (3D) micromolding technique utilizing bioprinted agarose template fibers to fabricate microchannel networks with various architectural features within photo cross linkable hydrogel constructs. Using the proposed approach, we were able to successfully embed functional and perfusable microchannels inside methacrylated gelatin (GelMA), star poly (ethylene glycol-co-lactide) acrylate (SPELA), poly (ethylene glycol) dimethacrylate (PEGDMA) and poly (ethylene glycol) diacrylate (PEGDA) hydrogels at different concentrations. In particular, GelMA hydrogels were used as a model to demonstrate the functionality of the fabricated vascular networks in improving mass transport, cellular viability and differentiation within the cell-laden tissue constructs. In addition, successful formation of endothelial monolayers within the fabricated channels was confirmed. Overall, our proposed strategy represents an effective technique for vascularization of hydrogel constructs with useful applications in tissue engineering and organs on a chip. PMID:24860845

Ballistics reviews: mechanisms of bullet wound trauma.

PubMed

Maiden, Nicholas

2009-01-01

The location of an entrance wound (bullet placement) and the projectile path are the most important factors in causing significant injury or death following a shooting. The head followed by the torso are the most vulnerable areas, with incapacitation resulting from central nervous system (brain or cord) disruption, or massive organ destruction with hemorrhage. Tissue and organ trauma result from the permanent wound cavity caused by direct destruction by the bullet, and also from radial stretching of surrounding tissues causing a temporary wound cavity. The extent of tissue damage is influenced by the type of bullet, its velocity and mass, as well as the physical characteristics of the tissues. The latter includes resistance to strain, physical dimensions of an organ, and the presence or absence of surrounding anatomical constraints. Bullet shape and construction will also affect tissue damage and bullets which display greater yaw will be associated with increased temporary cavitation. Military bullet designs do not include bullets that will expand or flatten as these cause greater wound trauma and are regulated by convention.

Potential applications of three-dimensional structure of silk fibroin/poly(ester-urethane) urea nanofibrous scaffold in heart valve tissue engineering

NASA Astrophysics Data System (ADS)

Du, Juan; Zhu, Tonghe; Yu, Haiyan; Zhu, Jingjing; Sun, Changbing; Wang, Jincheng; Chen, Sihao; Wang, Jihu; Guo, Xuran

2018-07-01

Tissue engineering heart valves (TEHV) are thought to have many advantages in low immunogenicity, good histocompatibility, excellent mechanical properties. In this paper, we reported the fabrication and characterization of a novel composite nanofibrous scaffold consisting of silk fibroin (SF) and poly(ester-urethane) urea (LDI-PEUU) by using electrospinning. Chemical and physical properties of scaffolds were evaluated using scanning electron microscopy, attenuated total reflectance Fourier transform infrared, X-ray diffraction, contact angle measurement, thermogravimetric analysis, biodegradation test and tensile strength analysis. We determined that the composite scaffolds supported the growth of human umbilical vein endothelial cell (HUVEC). The results of cell proliferation and cell morphology indicate that SF/LDI-PEUU nanofibers promoted cell viability, which supporting the application in tissue engineering. All results clarified that SF/LDI-PEUU (40:60) nanofibrous scaffolds meet the required specifications for tissue engineering and could be used as a promising construct for heart valve tissue engineering.

Multilayered tissue mimicking skin and vessel phantoms with tunable mechanical, optical, and acoustic properties

PubMed Central

Chen, Alvin I.; Balter, Max L.; Chen, Melanie I.; Gross, Daniel; Alam, Sheikh K.; Maguire, Timothy J.; Yarmush, Martin L.

2016-01-01

Purpose: This paper describes the design, fabrication, and characterization of multilayered tissue mimicking skin and vessel phantoms with tunable mechanical, optical, and acoustic properties. The phantoms comprise epidermis, dermis, and hypodermis skin layers, blood vessels, and blood mimicking fluid. Each tissue component may be individually tailored to a range of physiological and demographic conditions. Methods: The skin layers were constructed from varying concentrations of gelatin and agar. Synthetic melanin, India ink, absorbing dyes, and Intralipid were added to provide optical absorption and scattering in the skin layers. Bovine serum albumin was used to increase acoustic attenuation, and 40 μm diameter silica microspheres were used to induce acoustic backscatter. Phantom vessels consisting of thin-walled polydimethylsiloxane tubing were embedded at depths of 2–6 mm beneath the skin, and blood mimicking fluid was passed through the vessels. The phantoms were characterized through uniaxial compression and tension experiments, rheological frequency sweep studies, diffuse reflectance spectroscopy, and ultrasonic pulse-echo measurements. Results were then compared to in vivo and ex vivo literature data. Results: The elastic and dynamic shear behavior of the phantom skin layers and vessel wall closely approximated the behavior of porcine skin tissues and human vessels. Similarly, the optical properties of the phantom tissue components in the wavelength range of 400–1100 nm, as well as the acoustic properties in the frequency range of 2–9 MHz, were comparable to human tissue data. Normalized root mean square percent errors between the phantom results and the literature reference values ranged from 1.06% to 9.82%, which for many measurements were less than the sample variability. Finally, the mechanical and imaging characteristics of the phantoms were found to remain stable after 30 days of storage at 21 °C. Conclusions: The phantoms described in this work simulate the mechanical, optical, and acoustic properties of human skin tissues, vessel tissue, and blood. In this way, the phantoms are uniquely suited to serve as test models for multimodal imaging techniques and image-guided interventions. PMID:27277058

Murine tissue-engineered stomach demonstrates epithelial differentiation.

PubMed

Speer, Allison L; Sala, Frederic G; Matthews, Jamil A; Grikscheit, Tracy C

2011-11-01

Gastric cancer remains the second largest cause of cancer-related mortality worldwide. Postgastrectomy morbidity is considerable and quality of life is poor. Tissue-engineered stomach is a potential replacement solution to restore adequate food reservoir and gastric physiology. In this study, we performed a detailed investigation of the development of tissue-engineered stomach in a mouse model, specifically evaluating epithelial differentiation, proliferation, and the presence of putative stem cell markers. Organoid units were isolated from <3 wk-old mouse glandular stomach and seeded onto biodegradable scaffolds. The constructs were implanted into the omentum of adult mice. Implants were harvested at designated time points and analyzed with histology and immunohistochemistry. Tissue-engineered stomach grows as an expanding sphere with a simple columnar epithelium organized into gastric glands and an adjacent muscularis. The regenerated gastric epithelium demonstrates differentiation of all four cell types: mucous, enteroendocrine, chief, and parietal cells. Tissue-engineered stomach epithelium proliferates at a rate comparable to native glandular stomach and expresses two putative stem cell markers: DCAMKL-1 and Lgr5. This study demonstrates the successful generation of tissue-engineered stomach in a mouse model for the first time. Regenerated gastric epithelium is able to appropriately proliferate and differentiate. The generation of murine tissue-engineered stomach is a necessary advance as it provides the transgenic tools required to investigate the molecular and cellular mechanisms of this regenerative process. Delineating the mechanism of how tissue-engineered stomach develops in vivo is an important precursor to its use as a human stomach replacement therapy. Copyright © 2011 Elsevier Inc. All rights reserved.

Knitting for heart valve tissue engineering

PubMed Central

Ayad, Nadia; Wojciechowska, Dorota; Zielińska, Dorota; Struszczyk, Marcin H.; Latif, Najma; Yacoub, Magdi

Knitting is a versatile technology which offers a large portfolio of products and solutions of interest in heart valve (HV) tissue engineering (TE). One of the main advantages of knitting is its ability to construct complex shapes and structures by precisely assembling the yarns in the desired position. With this in mind, knitting could be employed to construct a HV scaffold that closely resembles the authentic valve. This has the potential to reproduce the anisotropic structure that is characteristic of the heart valve with the yarns, in particular the 3-layered architecture of the leaflets. These yarns can provide oriented growth of cells lengthwise and consequently enable the deposition of extracellular matrix (ECM) proteins in an oriented manner. This technique, therefore, has a potential to provide a functional knitted scaffold, but to achieve that textile engineers need to gain a basic understanding of structural and mechanical aspects of the heart valve and in addition, tissue engineers must acquire the knowledge of tools and capacities that are essential in knitting technology. The aim of this review is to provide a platform to consolidate these two fields as well as to enable an efficient communication and cooperation among these two research areas. PMID:29043276

Endothelial Progenitor Cells as a Sole Source for Ex Vivo Seeding of Tissue-Engineered Heart Valves

PubMed Central

Mettler, Bret A.; Engelmayr, George C.; Aikawa, Elena; Bischoff, Joyce; Martin, David P.; Exarhopoulos, Alexis; Moses, Marsha A.; Schoen, Frederick J.; Sacks, Michael S.

2010-01-01

Purposes: We investigated whether circulating endothelial progenitor cells (EPCs) can be used as a cell source for the creation of a tissue-engineered heart valve (TEHV). Methods: Trileaflet valved conduits were fabricated using nonwoven polyglycolic acid/poly-4-hydroxybutyrate polymer. Ovine peripheral blood EPCs were dynamically seeded onto a valved conduit and incubated for 7, 14, and 21 days. Results: Before seeding, EPCs were shown to express CD31+, eNOS+, and VE-Cadherin+ but not α-smooth muscle actin. Histological analysis demonstrated relatively homogenous cellular ingrowth throughout the valved conduit. TEHV constructs revealed the presence of endothelial cell (EC) markers and α-smooth muscle actin+ cells comparable with native valves. Protein levels were comparable with native valves and exceeded those in unseeded controls. EPC-TEHV demonstrated a temporal pattern of matrix metalloproteinases-2/9 expression and tissue inhibitors of metalloproteinase activities comparable to that of native valves. Mechanical properties of EPC-TEHV demonstrated significantly greater stiffness than that of the unseeded scaffolds and native valves. Conclusions: Circulating EPC appears to have the potential to provide both interstitial and endothelial functions and could potentially serve as a single-cell source for construction of autologous heart valves. PMID:19698056

Engineering of In Vitro 3D Capillary Beds by Self-Directed Angiogenic Sprouting

PubMed Central

Chan, Juliana M.; Zervantonakis, Ioannis K.; Rimchala, Tharathorn; Polacheck, William J.; Whisler, Jordan; Kamm, Roger D.

2012-01-01

In recent years, microfluidic systems have been used to study fundamental aspects of angiogenesis through the patterning of single-layered, linear or geometric vascular channels. In vivo, however, capillaries exist in complex, three-dimensional (3D) networks, and angiogenic sprouting occurs with a degree of unpredictability in all x,y,z planes. The ability to generate capillary beds in vitro that can support thick, biological tissues remains a key challenge to the regeneration of vital organs. Here, we report the engineering of 3D capillary beds in an in vitro microfluidic platform that is comprised of a biocompatible collagen I gel supported by a mechanical framework of alginate beads. The engineered vessels have patent lumens, form robust ∼1.5 mm capillary networks across the devices, and support the perfusion of 1 µm fluorescent beads through them. In addition, the alginate beads offer a modular method to encapsulate and co-culture cells that either promote angiogenesis or require perfusion for cell viability in engineered tissue constructs. This laboratory-constructed vascular supply may be clinically significant for the engineering of capillary beds and higher order biological tissues in a scalable and modular manner. PMID:23226527

Development of a Cyclic Strain Bioreactor for Mechanical Enhancement and Assessment of Bioengineered Myocardial Constructs

PubMed Central

Salazar, Betsy H.; Cashion, Avery T.; Dennis, Robert G.; Birla, Ravi K.

2015-01-01

Purpose The purpose of this study was to develop enabling bioreactor technologies using a novel voice coil actuator system for investigating the effects of periodic strain on cardiac patches fabricated with rat cardiomyocytes. Methods The bioengineered muscle constructs used in this study were formed by culturing rat neonatal primary cardiac cells on a fibrin gel. The physical design of the bioreactor was initially conceived using Solidworks to test clearances and perform structural strain analysis. Once the software design phase was completed the bioreactor was assembled using a combination of commercially available, custom machined, and 3-D printed parts. We utilized the bioreactor to evaluate the effect of a 4-hour stretch protocol on the contractile properties of the tissue after which immunohistological assessment of the tissue was also performed. Results An increase in contractile force was observed after the strain protocol of 10% stretch at 1Hz, with no significant increase observed in the control group. Additionally, an increase in cardiac myofibril alignment, connexin 43 expression, and collagen type I distribution were noted. Conclusion In this study we demonstrated the effectiveness of a new bioreactor design to improve contractility of engineered cardiac muscle tissue. PMID:26577484

Development of a Cyclic Strain Bioreactor for Mechanical Enhancement and Assessment of Bioengineered Myocardial Constructs.

PubMed

Salazar, Betsy H; Cashion, Avery T; Dennis, Robert G; Birla, Ravi K

2015-12-01

The purpose of this study was to develop enabling bioreactor technologies using a novel voice coil actuator system for investigating the effects of periodic strain on cardiac patches fabricated with rat cardiomyocytes. The bioengineered muscle constructs used in this study were formed by culturing rat neonatal primary cardiac cells on a fibrin gel. The physical design of the bioreactor was initially conceived using Solidworks to test clearances and perform structural strain analysis. Once the software design phase was completed the bioreactor was assembled using a combination of commercially available, custom machined, and 3-D printed parts. We utilized the bioreactor to evaluate the effect of a 4-h stretch protocol on the contractile properties of the tissue after which immunohistological assessment of the tissue was also performed. An increase in contractile force was observed after the strain protocol of 10% stretch at 1 Hz, with no significant increase observed in the control group. Additionally, an increase in cardiac myofibril alignment, connexin 43 expression, and collagen type I distribution were noted. In this study we demonstrated the effectiveness of a new bioreactor design to improve contractility of engineered cardiac muscle tissue.

Biofabricated constructs as tissue models: a short review.

PubMed

Costa, Pedro F

2015-04-01

Biofabrication is currently able to provide reliable models for studying the development of cells and tissues into multiple environments. As the complexity of biofabricated constructs is becoming increasingly higher their ability to closely mimic native tissues and organs is also increasing. Various biofabrication technologies currently allow to precisely build cell/tissue constructs at multiple dimension ranges with great accuracy. Such technologies are also able to assemble together multiple types of cells and/or materials and generate constructs closely mimicking various types of tissues. Furthermore, the high degree of automation involved in these technologies enables the study of large arrays of testing conditions within increasingly smaller and automated devices both in vitro and in vivo. Despite not yet being able to generate constructs similar to complex tissues and organs, biofabrication is rapidly evolving in that direction. One major hurdle to be overcome in order for such level of complex detail to be achieved is the ability to generate complex vascular structures within biofabricated constructs. This review describes several of the most relevant technologies and methodologies currently utilized within biofabrication and provides as well a brief overview of their current and future potential applications.

A tissue adaptation model based on strain-dependent collagen degradation and contact-guided cell traction.

PubMed

Heck, T A M; Wilson, W; Foolen, J; Cilingir, A C; Ito, K; van Donkelaar, C C

2015-03-18

Soft biological tissues adapt their collagen network to the mechanical environment. Collagen remodeling and cell traction are both involved in this process. The present study presents a collagen adaptation model which includes strain-dependent collagen degradation and contact-guided cell traction. Cell traction is determined by the prevailing collagen structure and is assumed to strive for tensional homeostasis. In addition, collagen is assumed to mechanically fail if it is over-strained. Care is taken to use principally measurable and physiologically meaningful relationships. This model is implemented in a fibril-reinforced biphasic finite element model for soft hydrated tissues. The versatility and limitations of the model are demonstrated by corroborating the predicted transient and equilibrium collagen adaptation under distinct mechanical constraints against experimental observations from the literature. These experiments include overloading of pericardium explants until failure, static uniaxial and biaxial loading of cell-seeded gels in vitro and shortening of periosteum explants. In addition, remodeling under hypothetical conditions is explored to demonstrate how collagen might adapt to small differences in constraints. Typical aspects of all essentially different experimental conditions are captured quantitatively or qualitatively. Differences between predictions and experiments as well as new insights that emerge from the present simulations are discussed. This model is anticipated to evolve into a mechanistic description of collagen adaptation, which may assist in developing load-regimes for functional tissue engineered constructs, or may be employed to improve our understanding of the mechanisms behind physiological and pathological collagen remodeling. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of a novel bioreactor system for 3D cellular mechanobiology studies.

PubMed

Cook, Colin A; Huri, Pinar Y; Ginn, Brian P; Gilbert-Honick, Jordana; Somers, Sarah M; Temple, Joshua P; Mao, Hai-Quan; Grayson, Warren L

2016-08-01

In vitro engineering systems can be powerful tools for studying tissue development in response to biophysical stimuli as well as for evaluating the functionality of engineered tissue grafts. It has been challenging, however, to develop systems that adequately integrate the application of biomimetic mechanical strain to engineered tissue with the ability to assess functional outcomes in real time. The aim of this study was to design a bioreactor system capable of real-time conditioning (dynamic, uniaxial strain, and electrical stimulation) of centimeter-long 3D tissue engineered constructs simultaneously with the capacity to monitor local strains. The system addresses key limitations of uniform sample loading and real-time imaging capabilities. Our system features an electrospun fibrin scaffold, which exhibits physiologically relevant stiffness and uniaxial alignment that facilitates cell adhesion, alignment, and proliferation. We have demonstrated the capacity for directly incorporating human adipose-derived stromal/stem cells into the fibers during the electrospinning process and subsequent culture of the cell-seeded constructs in the bioreactor. The bioreactor facilitates accurate pre-straining of the 3D constructs as well as the application of dynamic and static uniaxial strains while monitoring bulk construct tensions. The incorporation of fluorescent nanoparticles throughout the scaffolds enables in situ monitoring of local strain fields using fluorescent digital image correlation techniques, since the bioreactor is imaging compatible, and allows the assessment of local sample stiffness and stresses when coupled with force sensor measurements. In addition, the system is capable of measuring the electromechanical coupling of skeletal muscle explants by applying an electrical stimulus and simultaneously measuring the force of contraction. The packaging of these technologies, biomaterials, and analytical methods into a single bioreactor system has produced a powerful tool that will enable improved engineering of functional 3D ligaments, tendons, and skeletal muscles. Biotechnol. Bioeng. 2016;113: 1825-1837. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Knitted polylactide 96/4 L/D structures and scaffolds for tissue engineering: Shelf life, in vitro and in vivo studies

PubMed Central

Ellä, Ville; Annala, Tuija; Länsman, Satu; Nurminen, Manu; Kellomäki, Minna

2011-01-01

This study covers the whole production cycle, from biodegradable polymer processing to an in vivo tissue engineered construct. Six different biodegradable polylactide 96/4 L/D single jersey knits were manufactured using either four or eight multifilament fiber batches. The properties of those were studied in vitro for 42 weeks and in 0- to 3-year shelf life studies. Three types (Ø 12, 15 and 19 mm) of cylindrical scaffolds were manufactured from the knit, and the properties of those were studied in vitro for 48 weeks. For the Ø 15 mm scaffold type, mechanical properties were also studied in a one-year in vivo experiment. The scaffolds were implanted in the rat subcutis. All the scaffolds were g-irradiated prior to the studies. In vitro, all the knits lost 99% of their mechanical strength in 30 weeks. In the three-year follow up of shelf life properties, there was no decrease in the mechanical properties due to the storage time and only a 12% decrease in molecular weight. The in vitro and in vivo scaffolds lost their mechanical properties after 1 week. In the case of the in vivo samples, the mechanical properties were restored again, stepwise, by the presence of growing/maturing tissue between weeks 3 and 12. Faster degradation was observed with in vitro scaffolds compared to in vivo scaffolds during the one-year follow up. PMID:23507732

Nanotechnology in bone tissue engineering.

PubMed

Walmsley, Graham G; McArdle, Adrian; Tevlin, Ruth; Momeni, Arash; Atashroo, David; Hu, Michael S; Feroze, Abdullah H; Wong, Victor W; Lorenz, Peter H; Longaker, Michael T; Wan, Derrick C

2015-07-01

Nanotechnology represents a major frontier with potential to significantly advance the field of bone tissue engineering. Current limitations in regenerative strategies include impaired cellular proliferation and differentiation, insufficient mechanical strength of scaffolds, and inadequate production of extrinsic factors necessary for efficient osteogenesis. Here we review several major areas of research in nanotechnology with potential implications in bone regeneration: 1) nanoparticle-based methods for delivery of bioactive molecules, growth factors, and genetic material, 2) nanoparticle-mediated cell labeling and targeting, and 3) nano-based scaffold construction and modification to enhance physicochemical interactions, biocompatibility, mechanical stability, and cellular attachment/survival. As these technologies continue to evolve, ultimate translation to the clinical environment may allow for improved therapeutic outcomes in patients with large bone deficits and osteodegenerative diseases. Traditionally, the reconstruction of bony defects has relied on the use of bone grafts. With advances in nanotechnology, there has been significant development of synthetic biomaterials. In this article, the authors provided a comprehensive review on current research in nanoparticle-based therapies for bone tissue engineering, which should be useful reading for clinicians as well as researchers in this field. Copyright © 2015 Elsevier Inc. All rights reserved.

The biological response to orthopedic implants for joint replacement. II: Polyethylene, ceramics, PMMA, and the foreign body reaction

PubMed Central

Gibon, Emmanuel; Córdova, Luis A.; Lu, Laura; Lin, Tzu-Hua; Yao, Zhenyu; Hamadouche, Moussa; Goodman, Stuart B.

2017-01-01

Novel evidence-based prosthetic designs and biomaterials facilitate the performance of highly successful joint replacement (JR) procedures. To achieve this goal, constructs must be durable, biomechanically sound, and avoid adverse local tissue reactions. Different biomaterials such as metals and their alloys, polymers, ceramics, and composites are currently used for JR implants. This review focuses on (1) the biological response to the different biomaterials used for TJR and (2) the chronic inflammatory and foreign-body response induced by byproducts of these biomaterials. A homeostatic state of bone and surrounding soft tissue with current biomaterials for JR can be achieved with mechanically stable, infection free and intact (as opposed to the release of particulate or ionic byproducts) implants. Adverse local tissue reactions (an acute/chronic inflammatory reaction, periprosthetic osteolysis, loosening and subsequent mechanical failure) may evolve when the latter conditions are not met. This article (Part 2 of 2) summarizes the biological response to the non-metallic materials commonly used for joint replacement including polyethylene, ceramics, and polymethylmethacrylate (PMMA), as well as the foreign body reaction to byproducts of these materials. PMID:27080740

Mechanical behavior of a cellulose-reinforced scaffold in vascular tissue engineering.

PubMed

Pooyan, Parisa; Tannenbaum, Rina; Garmestani, Hamid

2012-03-01

Scaffolds constitute an essential structural component in tissue engineering of a vascular substitute for small grafts by playing a significant role in integrating the overall tissue constructs. The microstructure and mechanical properties of such scaffolds are important parameters to promote further cellular activities and neo-tissue development. Cellulose nanowhiskers (CNWs), an abundant, biocompatible material, could potentially constitute an acceptable candidate in scaffolding of a tissue-engineered vessel. Inspired by the advantages of cellulose and its derivatives, we have designed a biomaterial comprising CNWs embedded in a matrix of cellulose acetate propionate to fabricate a fully bio-based scaffold. To ensure uniform distribution, CNWs were delicately extracted from a multi-stage process and dispersed in an acetone suspension prior to the composite fabrication. Comparable to carbon nanotubes or kevlar, CNWs impart significant strength and directional rigidity even at 0.2 wt% and almost double that at only 3.0 wt%. To ensure the accuracy of our experimental data and to predict the unusual reinforcing effect of CNWs in a cellulose-based composite, homogenization schemes such as the mean field approach and the percolation technique were also investigated. Based on these comparisons, the tendency of CNWs to interconnect with one another through strong hydrogen bonding confirmed the formation of a three-dimensional rigid percolating network, fact which imparted an excellent mechanical stability to the entire structure at such low filler contents. Hence, our fibrous porous microstructure with improved mechanical properties could introduce a potential scaffold to withstand the physiological pressure and to mimic the profile features of native extracellular matrix in a human vessel. We believe that our nanohybrid design not only could expand the biomedical applications of renewable cellulose-based materials but also could provide a potential scaffold candidate in tissue engineering of small diameter grafts. Copyright © 2011 Elsevier Ltd. All rights reserved.

The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability.

PubMed

Billiet, Thomas; Gevaert, Elien; De Schryver, Thomas; Cornelissen, Maria; Dubruel, Peter

2014-01-01

In the present study, we report on the combined efforts of material chemistry, engineering and biology as a systemic approach for the fabrication of high viability 3D printed macroporous gelatin methacrylamide constructs. First, we propose the use and optimization of VA-086 as a photo-initiator with enhanced biocompatibility compared to the conventional Irgacure 2959. Second, a parametric study on the printing of gelatins was performed in order to characterize and compare construct architectures. Hereby, the influence of the hydrogel building block concentration, the printing temperature, the printing pressure, the printing speed, and the cell density were analyzed in depth. As a result, scaffolds could be designed having a 100% interconnected pore network in the gelatin concentration range of 10-20 w/v%. In the last part, the fabrication of cell-laden scaffolds was studied, whereby the application for tissue engineering was tested by encapsulation of the hepatocarcinoma cell line (HepG2). Printing pressure and needle shape was revealed to impact the overall cell viability. Mechanically stable cell-laden gelatin methacrylamide scaffolds with high cell viability (>97%) could be printed. Copyright © 2013 Elsevier Ltd. All rights reserved.

Quantifying the correlation between spatially defined oxygen gradients and cell fate in an engineered three-dimensional culture model.

PubMed

Ardakani, Amir G; Cheema, Umber; Brown, Robert A; Shipley, Rebecca J

2014-09-06

A challenge in three-dimensional tissue culture remains the lack of quantitative information linking nutrient delivery and cellular distribution. Both in vivo and in vitro, oxygen is delivered by diffusion from its source (blood vessel or the construct margins). The oxygen level at a defined distance from its source depends critically on the balance of diffusion and cellular metabolism. Cells may respond to this oxygen environment through proliferation, death and chemotaxis, resulting in spatially resolved gradients in cellular density. This study extracts novel spatially resolved and simultaneous data on tissue oxygenation, cellular proliferation, viability and chemotaxis in three-dimensional spiralled, cellular collagen constructs. Oxygen concentration gradients drove preferential cellular proliferation rates and viability in the higher oxygen zones and induced chemotaxis along the spiral of the collagen construct; an oxygen gradient of 1.03 mmHg mm(-1) in the spiral direction induced a mean migratory speed of 1015 μm day(-1). Although this movement was modest, it was effective in balancing the system to a stable cell density distribution, and provided insights into the natural cell mechanism for adapting cell number and activity to a prevailing oxygen regime.

Noninvasive Assessment of Glycosaminoglycan Production in Injectable Tissue-Engineered Cartilage Constructs Using Magnetic Resonance Imaging

PubMed Central

Ramaswamy, Sharan; Uluer, Mehmet C.; Leen, Stephanie; Bajaj, Preeti; Fishbein, Kenneth W.

2008-01-01

Abstract The glycosaminoglycan (GAG) content of engineered cartilage is a determinant of biochemical and mechanical quality. The ability to measure the degree to which GAG content is maintained or increases in an implant is therefore of importance in cartilage repair procedures. The gadolinium exclusion magnetic resonance imaging (MRI) method for estimating matrix fixed charge density (FCD) is ideally suited to this. One promising approach to cartilage repair is use of seeded injectable hydrogels. Accordingly, we assess the reliability of measuring GAG content in such a system ex vivo using MRI. Samples of the photo-polymerizable hydrogel, poly(ethylene oxide) diacrylate, were seeded with bovine chondrocytes (∼2.4 million cells/sample). The FCD of the constructs was determined using MRI after 9, 16, 29, 36, 43, and 50 days of incubation. Values were correlated with the results of biochemical determination of GAG from the same samples. FCD and GAG were found to be statistically significantly correlated (R2 = 0.91, p <0.01). We conclude that MRI-derived FCD measurements of FCD in injectable hydrogels reflect tissue GAG content and that this methodology therefore has potential for in vivo monitoring of such constructs. PMID:18620483

In Vivo Investigation of the Effectiveness of a Hyper-viscoelastic Model in Simulating Brain Retraction

NASA Astrophysics Data System (ADS)

Li, Ping; Wang, Weiwei; Zhang, Chenxi; An, Yong; Song, Zhijian

2016-07-01

Intraoperative brain retraction leads to a misalignment between the intraoperative positions of the brain structures and their previous positions, as determined from preoperative images. In vitro swine brain sample uniaxial tests showed that the mechanical response of brain tissue to compression and extension could be described by the hyper-viscoelasticity theory. The brain retraction caused by the mechanical process is a combination of brain tissue compression and extension. In this paper, we first constructed a hyper-viscoelastic framework based on the extended finite element method (XFEM) to simulate intraoperative brain retraction. To explore its effectiveness, we then applied this framework to an in vivo brain retraction simulation. The simulation strictly followed the clinical scenario, in which seven swine were subjected to brain retraction. Our experimental results showed that the hyper-viscoelastic XFEM framework is capable of simulating intraoperative brain retraction and improving the navigation accuracy of an image-guided neurosurgery system (IGNS).

Injuring mechanisms of gunshot wounds.

PubMed

White, K M

1989-03-01

The severity of injury in a gunshot wound is dependent on many factors, including the type of firearm; the velocity, mass, and construction of the bullet; and the structural properties of the tissues that are wounded. Knowledge of ballistics and an appreciation of the wounding potential of certain firearms and their ammunition can help clinicians anticipate the severity of a wound and raise the index of suspicion for occult but severe internal trauma.

Collagen-alginate as bioink for three-dimensional (3D) cell printing based cartilage tissue engineering.

PubMed

Yang, Xingchen; Lu, Zhenhui; Wu, Huayu; Li, Wei; Zheng, Li; Zhao, Jinmin

2018-02-01

Articular cartilage repair is still a huge challenge for researchers and clinicians. 3D bioprinting could be an innovative technology for cartilage tissue engineering. In this study, we used collagen type I (COL) or agarose (AG) mixed with sodium alginate (SA) to serve as 3D bioprinting bioinks and incorporated chondrocytes to construct in vitro 3D printed cartilage tissue. Swelling ratio, mechanical properties, scanning electron microscopy (SEM), cell viability and cytoskeleton, biochemistry analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to investigate the function of different bioinks in 3D printing cartilage tissue engineering applications. The results showed that the mechanical strength was improved in both SA/COL and SA/AG groups compared to SA alone. Besides, the addition of COL or AG has little impact on gelling behavior, demonstrating the advantage as bioinks for 3D printing. Among the three scaffolds, SA/COL could distinctly facilitated cell adhesion, accelerated cell proliferation and enhanced the expression of cartilage specific genes such as Acan, Col2al and Sox9 than the other two groups. Lower expression of Col1a1, the fibrocartilage marker, was present in SA/COL group than that in both of SA and SA/AG groups. The results indicated that SA/COL effectively suppressed dedifferentiation of chondrocytes and preserved the phenotype. In summary, 3D bioprinted SA/COL with favorable mechanical strength and biological functionality is promising in cartilage tissue engineering. Copyright © 2017. Published by Elsevier B.V.

Biaxial mechanics and inter-lamellar shearing of stem-cell seeded electrospun angle-ply laminates for annulus fibrosus tissue engineering.

PubMed

Driscoll, Tristan P; Nakasone, Ryan H; Szczesny, Spencer E; Elliott, Dawn M; Mauck, Robert L

2013-06-01

The annulus fibrosus (AF) of the intervertebral disk plays a critical role in vertebral load transmission that is heavily dependent on the microscale structure and composition of the tissue. With degeneration, both structure and composition are compromised, resulting in a loss of AF mechanical function. Numerous tissue engineering strategies have addressed the issue of AF degeneration, but few have focused on recapitulation of AF microstructure and function. One approach that allows for generation of engineered AF with appropriate (+/-)30° lamellar microstructure is the use of aligned electrospun scaffolds seeded with mesenchymal stem cells (MSCs) and assembled into angle-ply laminates (APL). Previous work indicates that opposing lamellar orientation is necessary for development of near native uniaxial tensile properties. However, most native AF tensile loads are applied biaxially, as the disk is subjected to multi-axial loads and is constrained by its attachments to the vertebral bodies. Thus, the objective of this study was to evaluate the biaxial mechanical response of engineered AF bilayers, and to determine the importance of opposing lamellar structure under this loading regime. Opposing bilayers, which replicate native AF structure, showed a significantly higher modulus in both testing directions compared to parallel bilayers, and reached ∼60% of native AF biaxial properties. Associated with this increase in biaxial properties, significantly less shear, and significantly higher stretch in the fiber direction, was observed. These results provide additional insight into native tissue structure-function relationships, as well as new benchmarks for engineering functional AF tissue constructs. Copyright © 2013 Orthopaedic Research Society.

Biomaterials in co-culture systems: towards optimizing tissue integration and cell signaling within scaffolds.

PubMed

Battiston, Kyle G; Cheung, Jane W C; Jain, Devika; Santerre, J Paul

2014-05-01

Most natural tissues consist of multi-cellular systems made up of two or more cell types. However, some of these tissues may not regenerate themselves following tissue injury or disease without some form of intervention, such as from the use of tissue engineered constructs. Recent studies have increasingly used co-cultures in tissue engineering applications as these systems better model the natural tissues, both physically and biologically. This review aims to identify the challenges of using co-culture systems and to highlight different approaches with respect to the use of biomaterials in the use of such systems. The application of co-culture systems to stimulate a desired biological response and examples of studies within particular tissue engineering disciplines are summarized. A description of different analytical co-culture systems is also discussed and the role of biomaterials in the future of co-culture research are elaborated on. Understanding the complex cell-cell and cell-biomaterial interactions involved in co-culture systems will ultimately lead the field towards biomaterial concepts and designs with specific biochemical, electrical, and mechanical characteristics that are tailored towards the needs of distinct co-culture systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

Living nanofiber yarn-based woven biotextiles for tendon tissue engineering using cell tri-culture and mechanical stimulation.

PubMed

Wu, Shaohua; Wang, Ying; Streubel, Philipp N; Duan, Bin

2017-10-15

Non-woven nanofibrous scaffolds have been developed for tendon graft application by using electrospinning strategies. However, electrospun nanofibrous scaffolds face some obstacles and limitations, including suboptimal scaffold structure, weak tensile and suture-retention strengths, and compact structure for cell infiltration. In this work, a novel nanofibrous, woven biotextile, fabricated based on electrospun nanofiber yarns, was implemented as a tissue engineered tendon scaffold. Based on our modified electrospinning setup, polycaprolactone (PCL) nanofiber yarns were fabricated with reproducible quality, and were further processed into plain-weaving fabrics interlaced with polylactic acid (PLA) multifilaments. Nonwoven nanofibrous PCL meshes with random or aligned fiber structures were generated using typical electrospinning as comparative counterparts. The woven fabrics contained 3D aligned microstructures with significantly larger pore size and obviously enhanced tensile mechanical properties than their nonwoven counterparts. The biological results revealed that cell proliferation and infiltration, along with the expression of tendon-specific genes by human adipose derived mesenchymal stem cells (HADMSC) and human tenocytes (HT), were significantly enhanced on the woven fabrics compared with those on randomly-oriented or aligned nanofiber meshes. Co-cultures of HADMSC with HT or human umbilical vein endothelial cells (HUVEC) on woven fabrics significantly upregulated the functional expression of most tenogenic markers. HADMSC/HT/HUVEC tri-culture on woven fabrics showed the highest upregulation of most tendon-associated markers than all the other mono- and co-culture groups. Furthermore, we conditioned the tri-cultured constructs with dynamic conditioning and demonstrated that dynamic stretch promoted total collagen secretion and tenogenic differentiation. Our nanofiber yarn-based biotextiles have significant potential to be used as engineered scaffolds to synergize the multiple cell interaction and mechanical stimulation for promoting tendon regeneration. Tendon grafts are essential for the treatment of various tendon-related conditions due to the inherently poor healing capacity of native tendon tissues. In this study, we combined electrospun nanofiber yarns with textile manufacturing strategies to fabricate nanofibrous woven biotextiles with hierarchical features, aligned fibrous topography, and sufficient mechanical properties as tendon tissue engineered scaffolds. Comparing to traditional electrospun random or aligned meshes, our novel nanofibrous woven fabrics possess strong tensile and suture-retention strengths and larger pore size. We also demonstrated that the incorporation of tendon cells and vascular cells promoted the tenogenic differentiation of the engineered tendon constructs, especially under dynamic stretch. This study not only presents a novel tissue engineered tendon scaffold fabrication technique but also provides a useful strategy to promote tendon differentiation and regeneration. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Organ-On-A-Chip Platforms: A Convergence of Advanced Materials, Cells, and Microscale Technologies.

PubMed

Ahadian, Samad; Civitarese, Robert; Bannerman, Dawn; Mohammadi, Mohammad Hossein; Lu, Rick; Wang, Erika; Davenport-Huyer, Locke; Lai, Ben; Zhang, Boyang; Zhao, Yimu; Mandla, Serena; Korolj, Anastasia; Radisic, Milica

2018-01-01

Significant advances in biomaterials, stem cell biology, and microscale technologies have enabled the fabrication of biologically relevant tissues and organs. Such tissues and organs, referred to as organ-on-a-chip (OOC) platforms, have emerged as a powerful tool in tissue analysis and disease modeling for biological and pharmacological applications. A variety of biomaterials are used in tissue fabrication providing multiple biological, structural, and mechanical cues in the regulation of cell behavior and tissue morphogenesis. Cells derived from humans enable the fabrication of personalized OOC platforms. Microscale technologies are specifically helpful in providing physiological microenvironments for tissues and organs. In this review, biomaterials, cells, and microscale technologies are described as essential components to construct OOC platforms. The latest developments in OOC platforms (e.g., liver, skeletal muscle, cardiac, cancer, lung, skin, bone, and brain) are then discussed as functional tools in simulating human physiology and metabolism. Future perspectives and major challenges in the development of OOC platforms toward accelerating clinical studies of drug discovery are finally highlighted. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photoactivated methods for enabling cartilage-to-cartilage tissue fixation

NASA Astrophysics Data System (ADS)

Sitterle, Valerie B.; Roberts, David W.

2003-06-01

The present study investigates whether photoactivated attachment of cartilage can provide a viable method for more effective repair of damaged articular surfaces by providing an alternative to sutures, barbs, or fibrin glues for initial fixation. Unlike artificial materials, biological constructs do not possess the initial strength for press-fitting and are instead sutured or pinned in place, typically inducing even more tissue trauma. A possible alternative involves the application of a photosensitive material, which is then photoactivated with a laser source to attach the implant and host tissues together in either a photothermal or photochemical process. The photothermal version of this method shows potential, but has been almost entirely applied to vascularized tissues. Cartilage, however, exhibits several characteristics that produce appreciable differences between applying and refining these techniques when compared to previous efforts involving vascularized tissues. Preliminary investigations involving photochemical photosensitizers based on singlet oxygen and electron transfer mechanisms are discussed, and characterization of the photodynamic effects on bulk collagen gels as a simplified model system using FTIR is performed. Previous efforts using photothermal welding applied to cartilaginous tissues are reviewed.

Design, construction and performance evaluation of the target tissue thickness measurement system in intraoperative radiotherapy for breast cancer

NASA Astrophysics Data System (ADS)

Yazdani, Mohammad Reza; Setayeshi, Saeed; Arabalibeik, Hossein; Akbari, Mohammad Esmaeil

2017-05-01

Intraoperative electron radiation therapy (IOERT), which uses electron beams for irradiating the target directly during the surgery, has the advantage of delivering a homogeneous dose to a controlled layer of tissue. Since the dose falls off quickly below the target thickness, the underlying normal tissues are spared. In selecting the appropriate electron energy, the accuracy of the target tissue thickness measurement is critical. In contrast to other procedures applied in IOERT, the routine measurement method is considered to be completely traditional and approximate. In this work, a novel mechanism is proposed for measuring the target tissue thickness with an acceptable level of accuracy. An electronic system has been designed and manufactured with the capability of measuring the tissue thickness based on the recorded electron density under the target. The results indicated the possibility of thickness measurement with a maximum error of 2 mm for 91.35% of data. Aside from system limitation in estimating the thickness of 5 mm phantom, for 88.94% of data, maximum error is 1 mm.

Direct plasma interaction with living tissue

NASA Astrophysics Data System (ADS)

Fridman, Gregory

For some time, plasma has been used in medicine to cauterize or cut tissue using heat and mechanical energy. In the recent decade, some researchers around the world have started to investigate how gas jets that pass through thermal plasma can be employed in medicine. This thesis presents the first investigation of biomedical uses of non-thermal plasma discharge which comes in direct contact with living tissue. It is demonstrated that the direct application of non-thermal plasma in air can cause rapid deactivation of bacteria on surfaces of tissues without causing any visible tissue damage. Medical need for such a device is discussed. Construction and operation of various types of non-thermal plasma power supplies and many types of treatment electrodes are presented as well. Application of this plasma to living organisms is shown to be safe from both the electrical perspective and from the biological perspective. Biological safety is revealed through a series of differential skin toxicity trials on human cadaver tissue, live hairless mouse skin tissue, live pig skin tissue, and finally in an open wound model on pigs. Direct non-thermal plasma in air is shown to deactivate bacteria about 100 times faster than indirect application using jets. A series of experiments reveal that this effectiveness is due to the ability of direct discharge to bring charges to tissue surfaces. It is demonstrated that neither ultraviolet (UV) radiation nor neutral active species such as hydroxyl radicals or ozone produced in plasma are responsible for the main effect on bacteria. Although much additional work remains on establishing detailed mechanism by which charges from plasma achieve this effect, the work carried out in this thesis clearly demonstrates that direct application of non-thermal plasma in air can be a very useful tool in medicine.

Development of novel force-limiting grasping forceps with a simple mechanism.

PubMed

Sakaguchi, Yasuto; Sato, Toshihiko; Yutaka, Yojiro; Muranishi, Yusuke; Komatsu, Teruya; Yoshizawa, Akihiko; Nakajima, Naoki; Nakamura, Tatsuo; Date, Hiroshi

2018-06-06

In endoscopic surgery, fragile tissues may be damaged by the application of excessive force. Thus, we developed novel endoscopic forceps with a simple force-limiting mechanism. The novel forceps were constructed with a leaf spring, and the spring thickness determines grasping pressure. We established an evaluation system (maximum score is 11 points) for lung tissue damage leading to complications. We tested the conventional forceps (186.8 kPa) and 3 novel spring forceps with the following thicknesses: 1.3 mm (53.0 kPa), 2.2 mm (187.7 kPa) and 2.8 mm (369.2 kPa). After grasping, peripheral canine lung tissues were microscopically examined for acute- and late-phase damages. In the acute phase (20 sites), the novel forceps caused capillary congestion and haemorrhage in the subpleural tissue, whereas the conventional forceps caused deep tissue and pleural damages. In the late phase (30 sites), both forceps caused fibroblast formation and interstitial thickening, which progressed to the deeper tissues as grasping pressure increased. In the acute phase, the median scores were 2.0 and 6.0 for the novel and conventional forceps, respectively (P = 0.003). In the late phase, the median scores were 2.0, 2.5 and 5.0 for 1.3-, 2.2- and 2.8-mm thick forceps, respectively, and 5.0 for the conventional forceps (P < 0.001). In both phases, the novel forceps with grasping pressure set below 187.7 kPa (2.2 mm) caused significantly less lung tissue damage than the conventional forceps. The novel endoscopic forceps are able to regulate the tissue-grasping pressure and induce less damage in lung tissues than conventional forceps.

Multiscale Inorganic Hierarchically Materials: Towards an Improved Orthopaedic Regenerative Medicine.

PubMed

Ruso, Juan M; Sartuqui, Javier; Messina, Paula V

2015-01-01

Bone is a biologically and structurally sophisticated multifunctional tissue. It dynamically responds to biochemical, mechanical and electrical clues by remodelling itself and accordingly the maximum strength and toughness are along the lines of the greatest applied stress. The challenge is to develop an orthopaedic biomaterial that imitates the micro- and nano-structural elements and compositions of bone to locally match the properties of the host tissue resulting in a biologically fixed implant. Looking for the ideal implant, the convergence of life and materials sciences occurs. Researchers in many different fields apply their expertise to improve implantable devices and regenerative medicine. Materials of all kinds, but especially hierarchical nano-materials, are being exploited. The application of nano-materials with hierarchical design to calcified tissue reconstructive medicine involve intricate systems including scaffolds with multifaceted shapes that provides temporary mechanical function; materials with nano-topography modifications that guarantee their integration to tissues and that possesses functionalized surfaces to transport biologic factors to stimulate tissue growth in a controlled, safe, and rapid manner. Furthermore materials that should degrade on a timeline coordinated to the time that takes the tissues regrow, are prepared. These implantable devices are multifunctional and for its construction they involve the use of precise strategically techniques together with specific material manufacturing processes that can be integrated to achieve in the design, the required multifunctionality. For such reasons, even though the idea of displacement from synthetic implants and tissue grafts to regenerative-medicine-based tissue reconstruction has been guaranteed for well over a decade, the reality has yet to emerge. In this paper, we examine the recent approaches to create enhanced bioactive materials. Their design and manufacturing procedures as well as the experiments to integrate them into engineer hierarchical inorganic materials for their practical application in calcified tissue reparation are evaluated.

Quasi-static elastography comparison of hyaline cartilage structures

NASA Astrophysics Data System (ADS)

McCredie, A. J.; Stride, E.; Saffari, N.

2009-11-01

Joint cartilage, a load bearing structure in mammals, has only limited ability for regeneration after damage. For tissue engineers to design functional constructs, better understanding of the properties of healthy tissue is required. Joint cartilage is a specialised structure of hyaline cartilage; a poroviscoelastic solid containing fibril matrix reinforcements. Healthy joint cartilage is layered, which is thought to be important for correct tissue function. However, the behaviour of each layer during loading is poorly understood. Ultrasound elastography provides access to depth-dependent information in real-time for a sample during loading. A 15 MHz focussed transducer provided details from scatterers within a small fixed region in each sample. Quasi-static loading was applied to cartilage samples while ultrasonic signals before and during compressions were recorded. Ultrasonic signals were processed to provide time-shift profiles using a sum-squared difference method and cross-correlation. Two structures of hyaline cartilage have been tested ultrasonically and mechanically to determine method suitability for monitoring internal deformation differences under load and the effect of the layers on the global mechanical material behaviour. Results show differences in both the global mechanical properties and the ultrasonically tested strain distributions between the two structures tested. It was concluded that these differences are caused primarily by the fibril orientations.

Tissue grown in space in NASA Bioreactor

NASA Technical Reports Server (NTRS)

2001-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Final samples from Mir and Earth appeared histologically cartilaginous throughout their entire cross sections (5-8 mm thick), with the exception of fibrous outer capsules. Constructs grown on Earth (A) appeared to have a more organized extracellular matrix with more uniform collagen orientation as compared with constructs grown on Mir (B), but the average collagen fiber diameter was similar in the two groups (22 +- 2 nm) and comparable to that previously reported for developing articular cartilage. Randomly oriented collagen in Mir samples would be consistent with previous reports that microgravity disrupts fibrillogenesis. These are transmission electron micrographs of constructs from Mir (A) and Earth (B) groups at magnifications of x3,500 and x120,000 (Inset). The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Credit: Proceedings of the National Academy of Sciences.

Fabrication of a Highly Aligned Neural Scaffold via a Table Top Stereolithography 3D Printing and Electrospinning^.

PubMed

Lee, Se-Jun; Nowicki, Margaret; Harris, Brent; Zhang, Lijie Grace

2017-06-01

Three-dimensional (3D) bioprinting is a rapidly emerging technique in the field of tissue engineering to fabricate extremely intricate and complex biomimetic scaffolds in the range of micrometers. Such customized 3D printed constructs can be used for the regeneration of complex tissues such as cartilage, vessels, and nerves. However, the 3D printing techniques often offer limited control over the resolution and compromised mechanical properties due to short selection of printable inks. To address these limitations, we combined stereolithography and electrospinning techniques to fabricate a novel 3D biomimetic neural scaffold with a tunable porous structure and embedded aligned fibers. By employing two different types of biofabrication methods, we successfully utilized both synthetic and natural materials with varying chemical composition as bioink to enhance biocompatibilities and mechanical properties of the scaffold. The resulting microfibers composed of polycaprolactone (PCL) polymer and PCL mixed with gelatin were embedded in 3D printed hydrogel scaffold. Our results showed that 3D printed scaffolds with electrospun fibers significantly improve neural stem cell adhesion when compared to those without the fibers. Furthermore, 3D scaffolds embedded with aligned fibers showed an enhancement in cell proliferation relative to bare control scaffolds. More importantly, confocal microscopy images illustrated that the scaffold with PCL/gelatin fibers greatly increased the average neurite length and directed neurite extension of primary cortical neurons along the fiber. The results of this study demonstrate the potential to create unique 3D neural tissue constructs by combining 3D bioprinting and electrospinning techniques.

Characterization of local fluid flow in 3D porous construct characterized by Fourier domain Doppler optical coherence tomography

NASA Astrophysics Data System (ADS)

Bagnaninchi, P. O.; Yang, Y.; El Haj, A.; Hinds, M. T.; Wang, R. K.

2007-02-01

In order to achieve functional tissue with the correct biomechanical properties it is critical to stimulate mechanically the cells. Perfusion bioreactor induces fluid shear stress that has been well characterized for two-dimensional culture where both simulation and experimental data are available. However these results can't be directly translated to tissue engineering that makes use of complex three-dimensional porous scaffold. Moreover, stimulated cells produce extensive extra-cellular matrix (ECM) that alter dramatically the micro-architecture of the constructs, changing the local flow dynamic. In this study a Fourier domain Doppler optical coherent tomography (FD-DOCT) system working at 1300nm with a bandwidth of 50nm has been used to determine the local flow rate inside different types of porous scaffolds used in tissue engineering. Local flow rates can then be linearly related, for Newtonian fluid, to the fluid shear stress occurring on the pores wall. Porous chitosan scaffolds (\\fgr 1.5mm x 3mm) with and without a central 250 μm microchannel have been produced by a freeze-drying technique. This techniques allow us to determine the actual shear stress applied to the cells and to optimise the input flow rate consequently, but also to relate the change of the flow distribution to the amount of ECM production allowing the monitoring of tissue formation.

Directing collagen fibers using counter-rotating cone extrusion.

PubMed

Hoogenkamp, Henk R; Bakker, Gert-Jan; Wolf, Louis; Suurs, Patricia; Dunnewind, Bertus; Barbut, Shai; Friedl, Peter; van Kuppevelt, Toin H; Daamen, Willeke F

2015-01-01

The bio-inspired engineering of tissue equivalents should take into account anisotropic morphology and the mechanical properties of the extracellular matrix. This especially applies to collagen fibrils, which have various, but highly defined, orientations throughout tissues and organs. There are several methods available to control the alignment of soluble collagen monomers, but the options to direct native insoluble collagen fibers are limited. Here we apply a controlled counter-rotating cone extrusion technology to engineer tubular collagen constructs with defined anisotropy. Driven by diverging inner and outer cone rotation speeds, collagen fibrils from bovine skin were extruded and precipitated onto mandrels as tubes with oriented fibers and bundles, as examined by second harmonic generation microscopy and quantitative image analysis. A clear correlation was found whereby the direction and extent of collagen fiber alignment during extrusion were a function of the shear forces caused by a combination of the cone rotation and flow direction. A gradual change in the fiber direction, spanning +50 to -40°, was observed throughout the sections of the sample, with an average decrease ranging from 2.3 to 2.6° every 10μm. By varying the cone speeds, the collagen constructs showed differences in elasticity and toughness, spanning 900-2000kPa and 19-35mJ, respectively. Rotational extrusion presents an enabling technology to create and control the (an)isotropic architecture of collagen constructs for application in tissue engineering and regenerative medicine. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Electrical Stimulation of Artificial Heart Muscle: A Look Into the Electrophysiologic and Genetic Implications.

PubMed

Mohamed, Mohamed A; Islas, Jose F; Schwartz, Robert J; Birla, Ravi K

Development of tissue-engineered hearts for treatment of myocardial infarction or biologic pacemakers has been hindered by the production of mostly arrhythmic or in-synergistic constructs. Electrical stimulation (ES) of these constructs has been shown to produce tissues with greater twitch force and better adrenergic response. To further our understanding of the mechanisms underlying the effect of ES, we fabricated a bioreactor capable of delivering continuous or intermittent waveforms of various types to multiple constructs simultaneously. In this study, we examined the effect of an intermittent biphasic square wave on our artificial heart muscle (AHM) composed of neonatal rat cardiac cells and fibrin gel. Twitch forces, spontaneous contraction rates, biopotentials, gene expression profiles, and histologic observations were examined for the ES protocol over a 12 day culture period. We demonstrate improved consistency between samples for twitch force and contraction rate, and higher normalized twitch force amplitudes for electrically stimulated AHMs. Improvements in electrophysiology within the AHM were noted by higher conduction velocities and lower latency in electrical response for electrically stimulated AHMs. Genes expressing key electrophysiologic and structural markers peaked at days 6 and 8 of culture, only a few days after the initiation of ES. These results may be used for optimization strategies to establish protocols for producing AHMs capable of replacing damaged heart tissue in either a contractile or electrophysiologic capacity. Optimized AHMs can lead to alternative treatments to heart failure and alleviate the limited donor supply crisis.

Electrical Stimulation of Artificial Heart Muscle: a look into the electrophysiological and genetic implications

PubMed Central

Mohamed, Mohamed A; Islas, Jose F; Schwartz, Robert J; Birla, Ravi K

2016-01-01

Development of tissue-engineered hearts for treatment of myocardial infarction or biological pacemakers has been hindered by the production of mostly arrhythmic or in-synergistic constructs. Electrical stimulation (ES) of these constructs has been shown to produce tissues with greater twitch force and better adrenergic response. In order to further our understanding of the mechanisms underlying the effect of ES, we fabricated a bioreactor capable of delivering continuous or intermittent waveforms of various types to multiple constructs simultaneously. In this study, we examined the effect of an intermittent biphasic square wave on our artificial heart muscle (AHM) composed of neonatal rat cardiac cells and fibrin gel. Twitch forces, spontaneous contraction rates, biopotentials, gene expression profiles, and histological observations were examined for the ES protocol over a 12 day culture period. We demonstrate improved consistency between samples for twitch force and contraction rate, and higher normalized twitch force amplitudes for electrically stimulated AHM. Improvements in electrophysiology within the AHM was noted by higher conduction velocities and lower latency in electrical response for electrically stimulated AHM. Genes expressing key electrophysiological and structural markers peaked at days 6 and 8 of culture, only a few days after the initiation of ES. These results may be used for optimization strategies to establish protocols for producing AHM capable of replacing damaged heart tissue in either a contractile or electrophysiological capacity. Optimized AHM can lead to alternative treatments to heart failure and alleviate the limited donor supply crisis. PMID:28459744

Heterogeneous engineered cartilage growth results from gradients of media-supplemented active TGF-β and is ameliorated by the alternative supplementation of latent TGF-β.

PubMed

Albro, Michael B; Nims, Robert J; Durney, Krista M; Cigan, Alexander D; Shim, Jay J; Vunjak-Novakovic, Gordana; Hung, Clark T; Ateshian, Gerard A

2016-01-01

Transforming growth factor beta (TGF-β) has become one of the most widely utilized mediators of engineered cartilage growth. It is typically exogenously supplemented in the culture medium in its active form, with the expectation that it will readily transport into tissue constructs through passive diffusion and influence cellular biosynthesis uniformly. The results of this investigation advance three novel concepts regarding the role of TGF-β in cartilage tissue engineering that have important implications for tissue development. First, through the experimental and computational analysis of TGF-β concentration distributions, we demonstrate that, contrary to conventional expectations, media-supplemented exogenous active TGF-β exhibits a pronounced concentration gradient in tissue constructs, resulting from a combination of high-affinity binding interactions and a high cellular internalization rate. These gradients are sustained throughout the entire culture duration, leading to highly heterogeneous tissue growth; biochemical and histological measurements support that while biochemical content is enhanced up to 4-fold at the construct periphery, enhancements are entirely absent beyond 1 mm from the construct surface. Second, construct-encapsulated chondrocytes continuously secrete large amounts of endogenous TGF-β in its latent form, a portion of which undergoes cell-mediated activation and enhances biosynthesis uniformly throughout the tissue. Finally, motivated by these prior insights, we demonstrate that the alternative supplementation of additional exogenous latent TGF-β enhances biosynthesis uniformly throughout tissue constructs, leading to enhanced but homogeneous tissue growth. This novel demonstration suggests that latent TGF-β supplementation may be utilized as an important tool for the translational engineering of large cartilage constructs that will be required to repair the large osteoarthritic defects observed clinically. Copyright © 2015. Published by Elsevier Ltd.

Heterogeneous engineered cartilage growth results from gradients of media-supplemented active TGF-β and is ameliorated by the alternative supplementation of latent TGF-β

PubMed Central

Durney, Krista M.; Cigan, Alexander D.; Shim, Jay J.; Vunjak-Novakovic, Gordana; Hung, Clark T.; Ateshian, Gerard A.

2016-01-01

Transforming growth factor beta (TGF-β) has become one of the most widely utilized mediators of engineered cartilage growth. It is typically exogenously supplemented in the culture medium in its active form, with the expectation that it will readily transport into tissue constructs through passive diffusion and influence cellular biosynthesis uniformly. The results of this investigation advance three novel concepts regarding the role of TGF-β in cartilage tissue engineering that have important implications for tissue development. First, through the experimental and computational analysis of TGF-β concentration distributions, we demonstrate that, contrary to conventional expectations, media-supplemented exogenous active TGF-β exhibits a pronounced concentration gradient in tissue constructs, resulting from a combination of high-affinity binding interactions and a high cellular internalization rate. These gradients are sustained throughout the entire culture duration, leading to highly heterogeneous tissue growth; biochemical and histological measurements support that while biochemical content is enhanced up to 4-fold at the construct periphery, enhancements are entirely absent beyond 1 mm from the construct surface. Second, construct-encapsulated chondrocytes continuously secrete large amounts of endogenous TGF-β in its latent form, a portion of which undergoes cell-mediated activation and enhances biosynthesis uniformly throughout the tissue. Finally, motivated by these prior insights, we demonstrate that the alternative supplementation of additional exogenous latent TGF-β enhances biosynthesis uniformly throughout tissue constructs, leading to enhanced but homogeneous tissue growth. This novel demonstration suggests that latent TGF-β supplementation may be utilized as an important tool for the translational engineering of large cartilage constructs that will be required to repair the large osteoarthritic defects observed clinically. PMID:26599624

Calculation of dose distribution in compressible breast tissues using finite element modeling, Monte Carlo simulation and thermoluminescence dosimeters

NASA Astrophysics Data System (ADS)

Mohammadyari, Parvin; Faghihi, Reza; Mosleh-Shirazi, Mohammad Amin; Lotfi, Mehrzad; Rahim Hematiyan, Mohammad; Koontz, Craig; Meigooni, Ali S.

2015-12-01

Compression is a technique to immobilize the target or improve the dose distribution within the treatment volume during different irradiation techniques such as AccuBoost® brachytherapy. However, there is no systematic method for determination of dose distribution for uncompressed tissue after irradiation under compression. In this study, the mechanical behavior of breast tissue between compressed and uncompressed states was investigated. With that, a novel method was developed to determine the dose distribution in uncompressed tissue after irradiation of compressed breast tissue. Dosimetry was performed using two different methods, namely, Monte Carlo simulations using the MCNP5 code and measurements using thermoluminescent dosimeters (TLD). The displacement of the breast elements was simulated using a finite element model and calculated using ABAQUS software. From these results, the 3D dose distribution in uncompressed tissue was determined. The geometry of the model was constructed from magnetic resonance images of six different women volunteers. The mechanical properties were modeled by using the Mooney-Rivlin hyperelastic material model. Experimental dosimetry was performed by placing the TLD chips into the polyvinyl alcohol breast equivalent phantom. The results determined that the nodal displacements, due to the gravitational force and the 60 Newton compression forces (with 43% contraction in the loading direction and 37% expansion in the orthogonal direction) were determined. Finally, a comparison of the experimental data and the simulated data showed agreement within 11.5%  ±  5.9%.

Calculation of dose distribution in compressible breast tissues using finite element modeling, Monte Carlo simulation and thermoluminescence dosimeters.

PubMed

Mohammadyari, Parvin; Faghihi, Reza; Mosleh-Shirazi, Mohammad Amin; Lotfi, Mehrzad; Hematiyan, Mohammad Rahim; Koontz, Craig; Meigooni, Ali S

2015-12-07

Compression is a technique to immobilize the target or improve the dose distribution within the treatment volume during different irradiation techniques such as AccuBoost(®) brachytherapy. However, there is no systematic method for determination of dose distribution for uncompressed tissue after irradiation under compression. In this study, the mechanical behavior of breast tissue between compressed and uncompressed states was investigated. With that, a novel method was developed to determine the dose distribution in uncompressed tissue after irradiation of compressed breast tissue. Dosimetry was performed using two different methods, namely, Monte Carlo simulations using the MCNP5 code and measurements using thermoluminescent dosimeters (TLD). The displacement of the breast elements was simulated using a finite element model and calculated using ABAQUS software. From these results, the 3D dose distribution in uncompressed tissue was determined. The geometry of the model was constructed from magnetic resonance images of six different women volunteers. The mechanical properties were modeled by using the Mooney-Rivlin hyperelastic material model. Experimental dosimetry was performed by placing the TLD chips into the polyvinyl alcohol breast equivalent phantom. The results determined that the nodal displacements, due to the gravitational force and the 60 Newton compression forces (with 43% contraction in the loading direction and 37% expansion in the orthogonal direction) were determined. Finally, a comparison of the experimental data and the simulated data showed agreement within 11.5%  ±  5.9%.

Negating Tissue Contracture Improves Volume Maintenance and Longevity of In Vivo Engineered Tissues.

PubMed

Lytle, Ian F; Kozlow, Jeffrey H; Zhang, Wen X; Buffington, Deborah A; Humes, H David; Brown, David L

2015-10-01

Engineering large, complex tissues in vivo requires robust vascularization to optimize survival, growth, and function. Previously, the authors used a "chamber" model that promotes intense angiogenesis in vivo as a platform for functional three-dimensional muscle and renal engineering. A silicone membrane used to define the structure and to contain the constructs is successful in the short term. However, over time, generated tissues contract and decrease in size in a manner similar to capsular contracture seen around many commonly used surgical implants. The authors hypothesized that modification of the chamber structure or internal surface would promote tissue adherence and maintain construct volume. Three chamber configurations were tested against volume maintenance. Previously studied, smooth silicone surfaces were compared to chambers modified for improved tissue adherence, with multiple transmembrane perforations or lined with a commercially available textured surface. Tissues were allowed to mature long term in a rat model, before analysis. On explantation, average tissue masses were 49, 102, and 122 mg; average volumes were 74, 158 and 176 μl; and average cross-sectional areas were 1.6, 6.7, and 8.7 mm for the smooth, perforated, and textured groups, respectively. Both perforated and textured designs demonstrated significantly greater measures than the smooth-surfaced constructs in all respects. By modifying the design of chambers supporting vascularized, three-dimensional, in vivo tissue engineering constructs, generated tissue mass, volume, and area can be maintained over a long time course. Successful progress in the scale-up of construct size should follow, leading to improved potential for development of increasingly complex engineered tissues.

Bone tissue engineering scaffolding: computer-aided scaffolding techniques.

PubMed

Thavornyutikarn, Boonlom; Chantarapanich, Nattapon; Sitthiseripratip, Kriskrai; Thouas, George A; Chen, Qizhi

Tissue engineering is essentially a technique for imitating nature. Natural tissues consist of three components: cells, signalling systems (e.g. growth factors) and extracellular matrix (ECM). The ECM forms a scaffold for its cells. Hence, the engineered tissue construct is an artificial scaffold populated with living cells and signalling molecules. A huge effort has been invested in bone tissue engineering, in which a highly porous scaffold plays a critical role in guiding bone and vascular tissue growth and regeneration in three dimensions. In the last two decades, numerous scaffolding techniques have been developed to fabricate highly interconnective, porous scaffolds for bone tissue engineering applications. This review provides an update on the progress of foaming technology of biomaterials, with a special attention being focused on computer-aided manufacturing (Andrade et al. 2002) techniques. This article starts with a brief introduction of tissue engineering (Bone tissue engineering and scaffolds) and scaffolding materials (Biomaterials used in bone tissue engineering). After a brief reviews on conventional scaffolding techniques (Conventional scaffolding techniques), a number of CAM techniques are reviewed in great detail. For each technique, the structure and mechanical integrity of fabricated scaffolds are discussed in detail. Finally, the advantaged and disadvantage of these techniques are compared (Comparison of scaffolding techniques) and summarised (Summary).

Bioprinting for vascular and vascularized tissue biofabrication.

PubMed

Datta, Pallab; Ayan, Bugra; Ozbolat, Ibrahim T

2017-03-15

Bioprinting is a promising technology to fabricate design-specific tissue constructs due to its ability to create complex, heterocellular structures with anatomical precision. Bioprinting enables the deposition of various biologics including growth factors, cells, genes, neo-tissues and extra-cellular matrix-like hydrogels. Benefits of bioprinting have started to make a mark in the fields of tissue engineering, regenerative medicine and pharmaceutics. Specifically, in the field of tissue engineering, the creation of vascularized tissue constructs has remained a principal challenge till date. However, given the myriad advantages over other biofabrication methods, it becomes organic to expect that bioprinting can provide a viable solution for the vascularization problem, and facilitate the clinical translation of tissue engineered constructs. This article provides a comprehensive account of bioprinting of vascular and vascularized tissue constructs. The review is structured as introducing the scope of bioprinting in tissue engineering applications, key vascular anatomical features and then a thorough coverage of 3D bioprinting using extrusion-, droplet- and laser-based bioprinting for fabrication of vascular tissue constructs. The review then provides the reader with the use of bioprinting for obtaining thick vascularized tissues using sacrificial bioink materials. Current challenges are discussed, a comparative evaluation of different bioprinting modalities is presented and future prospects are provided to the reader. Biofabrication of living tissues and organs at the clinically-relevant volumes vitally depends on the integration of vascular network. Despite the great progress in traditional biofabrication approaches, building perfusable hierarchical vascular network is a major challenge. Bioprinting is an emerging technology to fabricate design-specific tissue constructs due to its ability to create complex, heterocellular structures with anatomical precision, which holds a great promise in fabrication of vascular or vascularized tissues for transplantation use. Although a great progress has recently been made on building perfusable tissues and branched vascular network, a comprehensive review on the state-of-the-art in vascular and vascularized tissue bioprinting has not reported so far. This contribution is thus significant because it discusses the use of three major bioprinting modalities in vascular tissue biofabrication for the first time in the literature and compares their strengths and limitations in details. Moreover, the use of scaffold-based and scaffold-free bioprinting is expounded within the domain of vascular tissue fabrication. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Additive Manufacturing of Vascular Grafts and Vascularized Tissue Constructs.

PubMed

Elomaa, Laura; Yang, Yunzhi Peter

2017-10-01

There is a great need for engineered vascular grafts among patients with cardiovascular diseases who are in need of bypass therapy and lack autologous healthy blood vessels. In addition, because of the severe worldwide shortage of organ donors, there is an increasing need for engineered vascularized tissue constructs as an alternative to organ transplants. Additive manufacturing (AM) offers great advantages and flexibility of fabrication of cell-laden, multimaterial, and anatomically shaped vascular grafts and vascularized tissue constructs. Various inkjet-, extrusion-, and photocrosslinking-based AM techniques have been applied to the fabrication of both self-standing vascular grafts and porous, vascularized tissue constructs. This review discusses the state-of-the-art research on the use of AM for vascular applications and the key criteria for biomaterials in the AM of both acellular and cellular constructs. We envision that new smart printing materials that can adapt to their environment and encourage rapid endothelialization and remodeling will be the key factor in the future for the successful AM of personalized and dynamic vascular tissue applications.

Multi-scale Multi-mechanism Design of Tough Hydrogels: Building Dissipation into Stretchy Networks

PubMed Central

Zhao, Xuanhe

2014-01-01

As swollen polymer networks in water, hydrogels are usually brittle. However, hydrogels with high toughness play critical roles in many plant and animal tissues as well as in diverse engineering applications. Here we review the intrinsic mechanisms of a wide variety of tough hydrogels developed over past few decades. We show that tough hydrogels generally possess mechanisms to dissipate substantial mechanical energy but still maintain high elasticity under deformation. The integrations and interactions of different mechanisms for dissipating energy and maintaining elasticity are essential to the design of tough hydrogels. A matrix that combines various mechanisms is constructed for the first time to guide the design of next-generation tough hydrogels. We further highlight that a particularly promising strategy for the design is to implement multiple mechanisms across multiple length scales into nano-, micro-, meso-, and macro-structures of hydrogels. PMID:24834901

Precise stacking of decellularized extracellular matrix based 3D cell-laden constructs by a 3D cell printing system equipped with heating modules.

PubMed

Ahn, Geunseon; Min, Kyung-Hyun; Kim, Changhwan; Lee, Jeong-Seok; Kang, Donggu; Won, Joo-Yun; Cho, Dong-Woo; Kim, Jun-Young; Jin, Songwan; Yun, Won-Soo; Shim, Jin-Hyung

2017-08-17

Three-dimensional (3D) cell printing systems allow the controlled and precise deposition of multiple cells in 3D constructs. Hydrogel materials have been used extensively as printable bioinks owing to their ability to safely encapsulate living cells. However, hydrogel-based bioinks have drawbacks for cell printing, e.g. inappropriate crosslinking and liquid-like rheological properties, which hinder precise 3D shaping. Therefore, in this study, we investigated the influence of various factors (e.g. bioink concentration, viscosity, and extent of crosslinking) on cell printing and established a new 3D cell printing system equipped with heating modules for the precise stacking of decellularized extracellular matrix (dECM)-based 3D cell-laden constructs. Because the pH-adjusted bioink isolated from native tissue is safely gelled at 37 °C, our heating system facilitated the precise stacking of dECM bioinks by enabling simultaneous gelation during printing. We observed greater printability compared with that of a non-heating system. These results were confirmed by mechanical testing and 3D construct stacking analyses. We also confirmed that our heating system did not elicit negative effects, such as cell death, in the printed cells. Conclusively, these results hold promise for the application of 3D bioprinting to tissue engineering and drug development.

Anatomic Mesenchymal Stem Cell-Based Engineered Cartilage Constructs for Biologic Total Joint Replacement

PubMed Central

Saxena, Vishal; Kim, Minwook; Keah, Niobra M.; Neuwirth, Alexander L.; Stoeckl, Brendan D.; Bickard, Kevin; Restle, David J.; Salowe, Rebecca; Wang, Margaret Ye; Steinberg, David R.

2016-01-01

Cartilage has a poor healing response, and few viable options exist for repair of extensive damage. Hyaluronic acid (HA) hydrogels seeded with mesenchymal stem cells (MSCs) polymerized through UV crosslinking can generate functional tissue, but this crosslinking is not compatible with indirect rapid prototyping utilizing opaque anatomic molds. Methacrylate-modified polymers can also be chemically crosslinked in a cytocompatible manner using ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED). The objectives of this study were to (1) compare APS/TEMED crosslinking with UV crosslinking in terms of functional maturation of MSC-seeded HA hydrogels; (2) generate an anatomic mold of a complex joint surface through rapid prototyping; and (3) grow anatomic MSC-seeded HA hydrogel constructs using this alternative crosslinking method. Juvenile bovine MSCs were suspended in methacrylated HA (MeHA) and crosslinked either through UV polymerization or chemically with APS/TEMED to generate cylindrical constructs. Minipig porcine femoral heads were imaged using microCT, and anatomic negative molds were generated by three-dimensional printing using fused deposition modeling. Molded HA constructs were produced using the APS/TEMED method. All constructs were cultured for up to 12 weeks in a chemically defined medium supplemented with TGF-β3 and characterized by mechanical testing, biochemical assays, and histologic analysis. Both UV- and APS/TEMED-polymerized constructs showed increasing mechanical properties and robust proteoglycan and collagen deposition over time. At 12 weeks, APS/TEMED-polymerized constructs had higher equilibrium and dynamic moduli than UV-polymerized constructs, with no differences in proteoglycan or collagen content. Molded HA constructs retained their hemispherical shape in culture and demonstrated increasing mechanical properties and proteoglycan and collagen deposition, especially at the edges compared to the center of these larger constructs. Immunohistochemistry showed abundant collagen type II staining and little collagen type I staining. APS/TEMED crosslinking can be used to produce MSC-seeded HA-based neocartilage and can be used in combination with rapid prototyping techniques to generate anatomic MSC-seeded HA constructs for use in filling large and anatomically complex chondral defects or for biologic joint replacement. PMID:26871863

Use of Interim Scaffolding and Neotissue Development to Produce a Scaffold-Free Living Hyaline Cartilage Graft.

PubMed

Lau, Ting Ting; Leong, Wenyan; Peck, Yvonne; Su, Kai; Wang, Dong-An

2015-01-01

The fabrication of three-dimensional (3D) constructs relies heavily on the use of biomaterial-based scaffolds. These are required as mechanical supports as well as to translate two-dimensional cultures to 3D cultures for clinical applications. Regardless of the choice of scaffold, timely degradation of scaffolds is difficult to achieve and undegraded scaffold material can lead to interference in further tissue development or morphogenesis. In cartilage tissue engineering, hydrogel is the highly preferred scaffold material as it shares many similar characteristics with native cartilaginous matrix. Hence, we employed gelatin microspheres as porogens to create a microcavitary alginate hydrogel as an interim scaffold to facilitate initial chondrocyte 3D culture and to establish a final scaffold-free living hyaline cartilaginous graft (LhCG) for cartilage tissue engineering.

Bioprinting of a functional vascularized mouse thyroid gland construct.

PubMed

Bulanova, Elena A; Koudan, Elizaveta V; Degosserie, Jonathan; Heymans, Charlotte; Pereira, Frederico DAS; Parfenov, Vladislav A; Sun, Yi; Wang, Qi; Akhmedova, Suraya A; Sviridova, Irina K; Sergeeva, Natalia S; Frank, Georgy A; Khesuani, Yusef D; Pierreux, Christophe E; Mironov, Vladimir A

2017-08-18

Bioprinting can be defined as additive biofabrication of three-dimensional (3D) tissues and organ constructs using tissue spheroids, capable of self-assembly, as building blocks. The thyroid gland, a relatively simple endocrine organ, is suitable for testing the proposed bioprinting technology. Here we report the bioprinting of a functional vascularized mouse thyroid gland construct from embryonic tissue spheroids as a proof of concept. Based on the self-assembly principle, we generated thyroid tissue starting from thyroid spheroids (TS) and allantoic spheroids (AS) as a source of thyrocytes and endothelial cells (EC), respectively. Inspired by mathematical modeling of spheroid fusion, we used an original 3D bioprinter to print TS in close association with AS within a collagen hydrogel. During the culture, closely placed embryonic tissue spheroids fused into a single integral construct, EC from AS invaded and vascularized TS, and epithelial cells from the TS progressively formed follicles. In this experimental setting, we observed formation of a capillary network around follicular cells, as observed during in utero thyroid development when thyroid epithelium controls the recruitment, invasion and expansion of EC around follicles. To prove that EC from AS are responsible for vascularization of the thyroid gland construct, we depleted endogenous EC from TS before bioprinting. EC from AS completely revascularized depleted thyroid tissue. The cultured bioprinted construct was functional as it could normalize blood thyroxine levels and body temperature after grafting under the kidney capsule of hypothyroid mice. Bioprinting of functional vascularized mouse thyroid gland construct represents a further advance in bioprinting technology, exploring the self-assembling properties of tissue spheroids.

[Differential gene expression profile in ischemic myocardium of Wistar rats with acute myocardial infarction: the study on gene construction, identification and function].

PubMed

Guo, Chun Yu; Yin, Hui Jun; Jiang, Yue Rong; Xue, Mei; Zhang, Lu; Shi, Da Zhuo

2008-06-18

To construct the differential genes expressed profile in the ischemic myocardium tissue reduced from acute myocardial infarction(AMI), and determine the biological functions of target genes. AMI model was generated by ligation of the left anterior descending coronary artery in Wistar rats. Total RNA was extracted from the normal and the ischemic heart tissues under the ligation point 7 days after the operation. Differential gene expression profiles of the two samples were constructed using Long Serial Analysis of Gene Expression(LongSAGE). Real time fluorescence quantitative PCR was used to verify gene expression profile and to identify the expression of 2 functional genes. The activities of enzymes from functional genes were determined by histochemistry. A total of 15,966 tags were screened from the normal and the ischemic LongSAGE maps. The similarities of the sequences were compared using the BLAST algebra in NCBI and 7,665 novel tags were found. In the ischemic tissue 142 genes were significantly changed compared with those in the normal tissue (P<0.05). These differentially expressed genes represented the proteins which might play important roles in the pathways of oxidation and phosphorylation, ATP synthesis and glycolysis. The partial genes identified by LongSAGE were confirmed using real time fluorescence quantitative PCR. Two genes related to energy metabolism, COX5a and ATP5e, were screened and quantified. Expression of two functional genes down-regulated at their mRNA levels and the activities of correlative functional enzymes decreased compared with those in the normal tissue. AMI causes a series of changes in gene expression, in which the abnormal expression of genes related to energy metabolism could be one of the molecular mechanisms of AMI. The intervention of the expressions of COX5a and ATP5e may be a new target for AMI therapy.

Design and Fabrication of Anatomical Bioreactor Systems Containing Alginate Scaffolds for Cartilage Tissue Engineering

PubMed Central

Gharravi, Anneh Mohammad; Orazizadeh, Mahmoud; Ansari-Asl, Karim; Banoni, Salem; Izadi, Sina; Hashemitabar, Mahmoud

2012-01-01

The aim of the present study was to develop a tissue-engineering approach through alginate gel molding to mimic cartilage tissue in a three-dimensional culture system. The perfusion biomimetic bioreactor was designed to mimic natural joint. The shear stresses exerting on the bioreactor chamber were calculated by Computational Fluid Dynamic (CFD). Several alginate/bovine chondrocyte constructs were prepared, and were cultured in the bioreactor. Histochemical and immunohistochemical staining methods for the presence of glycosaminoglycan(GAG), overall matrix production and type II collagen protein were performed, respectively. The dynamic mechanical device applied a linear mechanical displacement of 2 mm to 10 mm. The CFD modeling indicated peak velocity and maximum wall shear stress were 1.706×10−3 m/s and 0.02407 dyne/cm 2, respectively. Histochemical and immunohistochemical analysis revealed evidence of cartilage-like tissue with lacunas similar to those of natural cartilage and the production of sulfated GAG of matrix by the chondrons, metachromatic territorial matrix-surrounded cells and accumulation of type II collagen around the cells. The present study indicated that when chondrocytes were seeded in alginate hydrogel and cultured in biomimetic cell culture system, cells survived well and secreted newly synthesized matrix led to improvement of chondrogenesis. PMID:23408660

Improving Joint Function Using Photochemical Hydrogels for Articular Surface Repair

DTIC Science & Technology

2017-02-01

dilution groups , tdeg increased with increasing concentration of EDC/NHS. Mechanical testing Values for storage modulus in spontaneous control gels (25.86...red) in 48-well nontreated tissue culture plates. As a positive control , a subset group of gels without tethered growth factor was exposed to 0.3 nM...in a cartilage ring and capped with fibrin and collagen gel. A control group consisted of chondrocytes encapsulated in fibrin gel. Constructs were

Epithelial Patterning, Morphogenesis, and Evolution: Drosophila Eggshell as a Model.

PubMed

Osterfield, Miriam; Berg, Celeste A; Shvartsman, Stanislav Y

2017-05-22

Understanding the mechanisms driving tissue and organ formation requires knowledge across scales. How do signaling pathways specify distinct tissue types? How does the patterning system control morphogenesis? How do these processes evolve? The Drosophila egg chamber, where EGF and BMP signaling intersect to specify unique cell types that construct epithelial tubes for specialized eggshell structures, has provided a tractable system to ask these questions. Work there has elucidated connections between scales of development, including across evolutionary scales, and fostered the development of quantitative modeling tools. These tools and general principles can be applied to the understanding of other developmental processes across organisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Osteograft, plastic material for regenerative medicine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zaidman, A. M., E-mail: AZaydman@niito.ru; Korel, A. V., E-mail: AKorel@niito.ru; Shchelkunova, E. I., E-mail: EShelkunova@niito.ru

Creating tissue-engineering constructs based on the mechanism of cartilage-bone evolution is promising for traumatology and orthopedics. Such a graft was obtained from a chondrograft by transdifferentiation. The hondrograft placed in osteogenic medium is undergoing osteogenic differentiation for 14–30 days. Tissue specificity of the osteograft was studied by morphology, immunohistochemistry, electron microscopy, and the expression of the corresponding genes was estimated. The expression of osteonectin, fibronectin, collagen of type I, izolektin and CD 44 is determined. Alkaline phosphatase and matrix vesicles are determined in osteoblasts. Calcificates are observed in the matrix. Chondrogenic proteins expression is absent. These findings evidence the tissuemore » specificity of the developed osteograft.« less

Antibiotic-Releasing Porous Polymethylmethacrylate/Gelatin/Antibiotic Constructs for Craniofacial Tissue Engineering

PubMed Central

Shi, Meng; Kretlow, James D.; Spicer, Patrick P.; Tabata, Yasuhiko; Demian, Nagi; Wong, Mark E.; Kasper, F. Kurtis; Mikos, Antonios G.

2011-01-01

An antibiotic-releasing porous polymethylmethacrylate (PMMA) construct was developed to maintain the bony space and prime the wound site in the initial step of a two-stage regenerative medicine approach toward reconstructing significant bony or composite craniofacial tissue defects. Porous polymethylmethacrylate (PMMA) constructs incorporating gelatin microparticles (GMPs) were fabricated by the sequential assembly of GMPs, the antibiotic colistin, and a clinically used bone cement formulation of PMMA powder and methylmethacrylate liquid. PMMA/gelatin/antibiotic constructs with varying gelatin incorporation and drug content were investigated to elucidate the relationship between material composition and construct properties (porosity and drug release kinetics). The porosity of PMMA/gelatin/antibiotic constructs ranged between 7.6±1.8–38.4±1.4% depending on the amount of gelatin incorporated and the drug solution added for gelatin swelling. The constructs released colistin over 10 or 14 days with an average release rate per day above 10 µg/ml. The porosity and in vitro colistin release kinetics of PMMA/gelatin/antibiotic constructs were tuned by varying the material composition and fabrication parameters. This study demonstrates the potential of gelatin-incorporating PMMA constructs as a functional space maintainer for both promoting tissue healing/coverage and addressing local infections, enabling better long-term success of the definitive regenerated tissue construct. PMID:21295086

[Development of computer aided forming techniques in manufacturing scaffolds for bone tissue engineering].

PubMed

Wei, Xuelei; Dong, Fuhui

2011-12-01

To review recent advance in the research and application of computer aided forming techniques for constructing bone tissue engineering scaffolds. The literature concerning computer aided forming techniques for constructing bone tissue engineering scaffolds in recent years was reviewed extensively and summarized. Several studies over last decade have focused on computer aided forming techniques for bone scaffold construction using various scaffold materials, which is based on computer aided design (CAD) and bone scaffold rapid prototyping (RP). CAD include medical CAD, STL, and reverse design. Reverse design can fully simulate normal bone tissue and could be very useful for the CAD. RP techniques include fused deposition modeling, three dimensional printing, selected laser sintering, three dimensional bioplotting, and low-temperature deposition manufacturing. These techniques provide a new way to construct bone tissue engineering scaffolds with complex internal structures. With rapid development of molding and forming techniques, computer aided forming techniques are expected to provide ideal bone tissue engineering scaffolds.

Application of Hydrogels in Heart Valve Tissue Engineering

PubMed Central

Zhang, Xing; Xu, Bin; Puperi, Daniel S.; Wu, Yan; West, Jennifer L.; Grande-Allen, K. Jane

2015-01-01

With an increasing number of patients requiring valve replacement, there is heightened interest in advancing heart valve tissue engineering (HVTE) to provide solutions to the many limitations of current surgical treatments. A variety of materials have been developed as scaffolds for HVTE including natural polymers, synthetic polymers, and decellularized valvular matrices. Among them, biocompatible hydrogels are generating growing interest. Natural hydrogels, such as collagen and fibrin, generally show good bioactivity, but poor mechanical durability. Synthetic hydrogels, on the other hand, have tunable mechanical properties; however, appropriate cell-matrix interactions are difficult to obtain. Moreover, hydrogels can be used as cell carriers when the cellular component is seeded into the polymer meshes or decellularized valve scaffolds. In this review, we discuss current research strategies for HVTE with an emphasis on hydrogel applications. The physicochemical properties and fabrication methods of these hydrogels, as well as their mechanical properties and bioactivities are described. Performance of some hydrogels including in vitro evaluation using bioreactors and in vivo tests in different animal models are also discussed. For future HVTE, it will be compelling to examine how hydrogels can be constructed from composite materials to replicate mechanical properties and mimic biological functions of the native heart valve. PMID:25955010

Balancing mechanical strength with bioactivity in chitosan-calcium phosphate 3D microsphere scaffolds for bone tissue engineering: air- vs. freeze-drying processes.

PubMed

Nguyen, D T; McCanless, J D; Mecwan, M M; Noblett, A P; Haggard, W O; Smith, R A; Bumgardner, J D

2013-01-01

The objective of this study was to evaluate the potential benefit of 3D composite scaffolds composed of chitosan and calcium phosphate for bone tissue engineering. Additionally, incorporation of mechanically weak lyophilized microspheres within those air-dried (AD) was considered for enhanced bioactivity. AD microsphere, alone, and air- and freeze-dried microsphere (FDAD) 3D scaffolds were evaluated in vitro using a 28-day osteogenic culture model with the Saos-2 cell line. Mechanical testing, quantitative microscopy, and lysozyme-driven enzymatic degradation of the scaffolds were also studied. FDAD scaffold showed a higher concentration (p < 0.01) in cells per scaffold mass vs. AD constructs. Collagen was ∼31% greater (p < 0.01) on FDAD compared to AD scaffolds not evident in microscopy of microsphere surfaces. Alternatively, AD scaffolds demonstrated a superior threefold increase in compressive strength over FDAD (12 vs. 4 MPa) with minimal degradation. Inclusion of FD spheres within the FDAD scaffolds allowed increased cellular activity through improved seeding, proliferation, and extracellular matrix production (as collagen), although mechanical strength was sacrificed through introduction of the less stiff, porous FD spheres.

Towards the design of 3D multiscale instructive tissue engineering constructs: Current approaches and trends.

PubMed

Oliveira, Sara M; Reis, Rui L; Mano, João F

2015-11-01

The design of 3D constructs with adequate properties to instruct and guide cells both in vitro and in vivo is one of the major focuses of tissue engineering. Successful tissue regeneration depends on the favorable crosstalk between the supporting structure, the cells and the host tissue so that a balanced matrix production and degradation are achieved. Herein, the major occurring events and players in normal and regenerative tissue are overviewed. These have been inspiring the selection or synthesis of instructive cues to include into the 3D constructs. We further highlight the importance of a multiscale perception of the range of features that can be included on the biomimetic structures. Lastly, we focus on the current and developing tissue-engineering approaches for the preparation of such 3D constructs: top-down, bottom-up and integrative. Bottom-up and integrative approaches present a higher potential for the design of tissue engineering devices with multiscale features and higher biochemical control than top-down strategies, and are the main focus of this review. Copyright © 2015 Elsevier Inc. All rights reserved.

Three-dimensional bioprinting in tissue engineering and regenerative medicine.

PubMed

Gao, Guifang; Cui, Xiaofeng

2016-02-01

With the advances of stem cell research, development of intelligent biomaterials and three-dimensional biofabrication strategies, highly mimicked tissue or organs can be engineered. Among all the biofabrication approaches, bioprinting based on inkjet printing technology has the promises to deliver and create biomimicked tissue with high throughput, digital control, and the capacity of single cell manipulation. Therefore, this enabling technology has great potential in regenerative medicine and translational applications. The most current advances in organ and tissue bioprinting based on the thermal inkjet printing technology are described in this review, including vasculature, muscle, cartilage, and bone. In addition, the benign side effect of bioprinting to the printed mammalian cells can be utilized for gene or drug delivery, which can be achieved conveniently during precise cell placement for tissue construction. With layer-by-layer assembly, three-dimensional tissues with complex structures can be printed using converted medical images. Therefore, bioprinting based on thermal inkjet is so far the most optimal solution to engineer vascular system to the thick and complex tissues. Collectively, bioprinting has great potential and broad applications in tissue engineering and regenerative medicine. The future advances of bioprinting include the integration of different printing mechanisms to engineer biphasic or triphasic tissues with optimized scaffolds and further understanding of stem cell biology.

Anisotropic Shape-Memory Alginate Scaffolds Functionalized with Either Type I or Type II Collagen for Cartilage Tissue Engineering.

PubMed

Almeida, Henrique V; Sathy, Binulal N; Dudurych, Ivan; Buckley, Conor T; O'Brien, Fergal J; Kelly, Daniel J

2017-01-01

Regenerating articular cartilage and fibrocartilaginous tissue such as the meniscus is still a challenge in orthopedic medicine. While a range of different scaffolds have been developed for joint repair, none have facilitated the development of a tissue that mimics the complexity of soft tissues such as articular cartilage. Furthermore, many of these scaffolds are not designed to function in mechanically challenging joint environments. The overall goal of this study was to develop a porous, biomimetic, shape-memory alginate scaffold for directing cartilage regeneration. To this end, a scaffold was designed with architectural cues to guide cellular and neo-tissue alignment, which was additionally functionalized with a range of extracellular matrix cues to direct stem cell differentiation toward the chondrogenic lineage. Shape-memory properties were introduced by covalent cross-linking alginate using carbodiimide chemistry, while the architecture of the scaffold was modified using a directional freezing technique. Introducing such an aligned pore structure was found to improve the mechanical properties of the scaffold, and promoted higher levels of sulfated glycosaminoglycans (sGAG) and collagen deposition compared to an isotropic (nonaligned) pore geometry when seeded with adult human stem cells. Functionalization with collagen improved stem cell recruitment into the scaffold and facilitated more homogenous cartilage tissue deposition throughout the construct. Incorporating type II collagen into the scaffolds led to greater cell proliferation, higher sGAG and collagen accumulation, and the development of a stiffer tissue compared to scaffolds functionalized with type I collagen. The results of this study demonstrate how both scaffold architecture and composition can be tailored in a shape-memory alginate scaffold to direct stem cell differentiation and support the development of complex cartilaginous tissues.

Combined chemical and structural signals of biomaterials synergistically activate cell-cell communications for improving tissue regeneration.

PubMed

Xu, Yachen; Peng, Jinliang; Dong, Xin; Xu, Yuhong; Li, Haiyan; Chang, Jiang

2017-06-01

Biomaterials are only used as carriers of cells in the conventional tissue engineering. Considering the multi-cell environment and active cell-biomaterial interactions in tissue regeneration process, in this study, structural signals of aligned electrospun nanofibers and chemical signals of bioglass (BG) ionic products in cell culture medium are simultaneously applied to activate fibroblast-endothelial co-cultured cells in order to obtain an improved skin tissue engineering construct. Results demonstrate that the combined biomaterial signals synergistically activate fibroblast-endothelial co-culture skin tissue engineering constructs through promotion of paracrine effects and stimulation of gap junctional communication between cells, which results in enhanced vascularization and extracellular matrix protein synthesis in the constructs. Structural signals of aligned electrospun nanofibers play an important role in stimulating both of paracrine and gap junctional communication while chemical signals of BG ionic products mainly enhance paracrine effects. In vivo experiments reveal that the activated skin tissue engineering constructs significantly enhance wound healing as compared to control. This study indicates the advantages of synergistic effects between different bioactive signals of biomaterials can be taken to activate communication between different types of cells for obtaining tissue engineering constructs with improved functions. Tissue engineering can regenerate or replace tissue or organs through combining cells, biomaterials and growth factors. Normally, for repairing a specific tissue, only one type of cells, one kind of biomaterials, and specific growth factors are used to support cell growth. In this study, we proposed a novel tissue engineering approach by simply using co-cultured cells and combined biomaterial signals. Using a skin tissue engineering model, we successfully proved that the combined biomaterial signals such as surface nanostructures and bioactive ions could synergistically stimulate the cell-cell communication in co-culture system through paracrine effects and gap junction activation, and regulated expression of growth factors and extracellular matrix proteins, resulting in an activated tissue engineering constructs that significantly enhanced skin regeneration. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Biomimetic and synthetic esophageal tissue engineering.

PubMed

Jensen, Todd; Blanchette, Alex; Vadasz, Stephanie; Dave, Apeksha; Canfarotta, Michael; Sayej, Wael N; Finck, Christine

2015-07-01

A tissue-engineered esophagus offers an alternative for the treatment of pediatric patients suffering from severe esophageal malformations, caustic injury, and cancer. Additionally, adult patients suffering from carcinoma or trauma would benefit. Donor rat esophageal tissue was physically and enzymatically digested to isolate epithelial and smooth muscle cells, which were cultured in epithelial cell medium or smooth muscle cell medium and characterized by immunofluorescence. Isolated cells were also seeded onto electrospun synthetic PLGA and PCL/PLGA scaffolds in a physiologic hollow organ bioreactor. After 2 weeks of in vitro culture, tissue-engineered constructs were orthotopically transplanted. Isolated cells were shown to give rise to epithelial, smooth muscle, and glial cell types. After 14 days in culture, scaffolds supported epithelial, smooth muscle and glial cell phenotypes. Transplanted constructs integrated into the host's native tissue and recipients of the engineered tissue demonstrated normal feeding habits. Characterization after 14 days of implantation revealed that all three cellular phenotypes were present in varying degrees in seeded and unseeded scaffolds. We demonstrate that isolated cells from native esophagus can be cultured and seeded onto electrospun scaffolds to create esophageal constructs. These constructs have potential translatable application for tissue engineering of human esophageal tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biomaterial-mesenchymal stem cell constructs for immunomodulation in composite tissue engineering.

PubMed

Hanson, Summer; D'Souza, Rena N; Hematti, Peiman

2014-08-01

Cell-based treatments are being developed as a novel approach for the treatment of many diseases in an effort to repair injured tissues and regenerate lost tissues. Interest in the potential use of multipotent progenitor or stem cells has grown significantly in recent years, specifically the use of mesenchymal stem cells (MSCs), for tissue engineering in combination with extracellular matrix-based scaffolds. An area that warrants further attention is the local or systemic host responses toward the implanted cell-biomaterial constructs. Such immunological responses could play a major role in determining the clinical efficacy of the therapeutic device or biomaterials used. MSCs, due to their unique immunomodulatory properties, hold great promise in tissue engineering as they not only directly participate in tissue repair and regeneration but also modulate the host foreign body response toward the engineered constructs. The purpose of this review was to summarize the current state of knowledge and applications of MSC-biomaterial constructs as a potential immunoregulatory tool in tissue engineering. Better understanding of the interactions between biomaterials and cells could translate to the development of clinically relevant and novel cell-based therapeutics for tissue reconstruction and regenerative medicine.

In situ patterned micro 3D liver constructs for parallel toxicology testing in a fluidic device

PubMed Central

Skardal, Aleksander; Devarasetty, Mahesh; Soker, Shay; Hall, Adam R

2017-01-01

3D tissue models are increasingly being implemented for drug and toxicology testing. However, the creation of tissue-engineered constructs for this purpose often relies on complex biofabrication techniques that are time consuming, expensive, and difficult to scale up. Here, we describe a strategy for realizing multiple tissue constructs in a parallel microfluidic platform using an approach that is simple and can be easily scaled for high-throughput formats. Liver cells mixed with a UV-crosslinkable hydrogel solution are introduced into parallel channels of a sealed microfluidic device and photopatterned to produce stable tissue constructs in situ. The remaining uncrosslinked material is washed away, leaving the structures in place. By using a hydrogel that specifically mimics the properties of the natural extracellular matrix, we closely emulate native tissue, resulting in constructs that remain stable and functional in the device during a 7-day culture time course under recirculating media flow. As proof of principle for toxicology analysis, we expose the constructs to ethyl alcohol (0–500 mM) and show that the cell viability and the secretion of urea and albumin decrease with increasing alcohol exposure, while markers for cell damage increase. PMID:26355538

In situ patterned micro 3D liver constructs for parallel toxicology testing in a fluidic device.

PubMed

Skardal, Aleksander; Devarasetty, Mahesh; Soker, Shay; Hall, Adam R

2015-09-10

3D tissue models are increasingly being implemented for drug and toxicology testing. However, the creation of tissue-engineered constructs for this purpose often relies on complex biofabrication techniques that are time consuming, expensive, and difficult to scale up. Here, we describe a strategy for realizing multiple tissue constructs in a parallel microfluidic platform using an approach that is simple and can be easily scaled for high-throughput formats. Liver cells mixed with a UV-crosslinkable hydrogel solution are introduced into parallel channels of a sealed microfluidic device and photopatterned to produce stable tissue constructs in situ. The remaining uncrosslinked material is washed away, leaving the structures in place. By using a hydrogel that specifically mimics the properties of the natural extracellular matrix, we closely emulate native tissue, resulting in constructs that remain stable and functional in the device during a 7-day culture time course under recirculating media flow. As proof of principle for toxicology analysis, we expose the constructs to ethyl alcohol (0-500 mM) and show that the cell viability and the secretion of urea and albumin decrease with increasing alcohol exposure, while markers for cell damage increase.

Relationship between micro-porosity, water permeability and mechanical behavior in scaffolds for cartilage engineering.

PubMed

Vikingsson, L; Claessens, B; Gómez-Tejedor, J A; Gallego Ferrer, G; Gómez Ribelles, J L

2015-08-01

In tissue engineering the design and optimization of biodegradable polymeric scaffolds with a 3D-structure is an important field. The porous scaffold provide the cells with an adequate biomechanical environment that allows mechanotransduction signals for cell differentiation and the scaffolds also protect the cells from initial compressive loading. The scaffold have interconnected macro-pores that host the cells and newly formed tissue, while the pore walls should be micro-porous to transport nutrients and waste products. Polycaprolactone (PCL) scaffolds with a double micro- and macro-pore architecture have been proposed for cartilage regeneration. This work explores the influence of the micro-porosity of the pore walls on water permeability and scaffold compliance. A Poly(Vinyl Alcohol) with tailored mechanical properties has been used to simulate the growing cartilage tissue inside the scaffold pores. Unconfined and confined compression tests were performed to characterize both the water permeability and the mechanical response of scaffolds with varying size of micro-porosity while volume fraction of the macro-pores remains constant. The stress relaxation tests show that the stress response of the scaffold/hydrogel construct is a synergic effect determined by the performance of the both components. This is interesting since it suggests that the in vivo outcome of the scaffold is not only dependent upon the material architecture but also the growing tissue inside the scaffold׳s pores. On the other hand, confined compression results show that compliance of the scaffold is mainly controlled by the micro-porosity of the scaffold and less by hydrogel density in the scaffold pores. These conclusions bring together valuable information for customizing the optimal scaffold and to predict the in vivo mechanical behavior. Copyright © 2015 Elsevier Ltd. All rights reserved.

* Three-Dimensional Bioprinting of Polycaprolactone Reinforced Gene Activated Bioinks for Bone Tissue Engineering.

PubMed

Cunniffe, Gráinne M; Gonzalez-Fernandez, Tomas; Daly, Andrew; Sathy, Binulal N; Jeon, Oju; Alsberg, Eben; Kelly, Daniel J

2017-09-01

Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-γ-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bone marrow-derived mesenchymal stem cells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization and mineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs.

3D Bioprinting of Artificial Tissues: Construction of Biomimetic Microstructures.

PubMed

Luo, Yongxiang; Lin, Xin; Huang, Peng

2018-04-24

It is promising that artificial tissues/organs for clinical application can be produced via 3D bioprinting of living cells and biomaterials. The construction of microstructures biomimicking native tissues is crucially important to create artificial tissues with biological functions. For instance, the fabrication of vessel-like networks to supply cells with initial nutrient and oxygen, and the arrangement of multiple types of cells for creating lamellar/complex tissues through 3D bioprinting are widely reported. The current advances in 3D bioprinting of artificial tissues from the view of construction of biomimetic microstructures, especially the fabrication of lamellar, vascular, and complex structures are summarized. In the end, the conclusion and perspective of 3D bioprinting for clinical applications are elaborated. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Design and Fabrication of an MRI-Compatible, Autonomous Incubation System.

PubMed

Khalilzad-Sharghi, Vahid; Xu, Huihui

2015-10-01

Tissue engineers have long sought access to an autonomous, imaging-compatible tissue incubation system that, with minimum operator handling, can provide real-time visualization and quantification of cells, tissue constructs, and organs. This type of screening system, capable of operating noninvasively to validate tissue, can overcome current limitations like temperature shock, unsustainable cellular environments, sample contamination, and handling/stress. However, this type of system has been a major challenge, until now. Here, we describe the design, fabrication, and characterization of an innovative, autonomous incubation system that is compatible with a 9.4 T magnetic resonance imaging (MRI) scanner. Termed the e-incubator (patent pending; application number: 13/953,984), this microcontroller-based system is integrated into an MRI scanner and noninvasively screens cells and tissue cultures in an environment where temperature, pH, and media/gas handling are regulated. The 4-week study discussed herein details the continuous operation of the e-incubator for a tissue-engineered osteogenic construct, validated by LIVE/DEAD(®) cell assays and histology. The evolving MR quantitative parameters of the osteogenic construct were used as biomarkers for bone tissue engineering and to further validate the quality of the product noninvasively before harvesting. Importantly, the e-incubator reliably facilitates culturing cells and tissue constructs to create engineered tissues and/or investigate disease therapies.

"Deep-media culture condition" promoted lumen formation of endothelial cells within engineered three-dimensional tissues in vitro.

PubMed

Sekiya, Sachiko; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2011-03-01

In the field of tissue engineering, the induction of microvessels into tissues is an important task because of the need to overcome diffusion limitations of oxygen and nutrients within tissues. Powerful methods to create vessels in engineered tissues are needed for creating real living tissues. In this study, we utilized three-dimensional (3D) highly cell dense tissues fabricated by cell sheet technology. The 3D tissue constructs are close to living-cell dense tissue in vivo. Additionally, creating an endothelial cell (EC) network within tissues promoted neovascularization promptly within the tissue after transplantation in vivo. Compared to the conditions in vivo, however, common in vitro cell culture conditions provide a poor environment for creating lumens within 3D tissue constructs. Therefore, for determining adequate conditions for vascularizing engineered tissue in vitro, our 3D tissue constructs were cultured under a "deep-media culture conditions." Compared to the control conditions, the morphology of ECs showed a visibly strained cytoskeleton, and the density of lumen formation within tissues increased under hydrostatic pressure conditions. Moreover, the increasing expression of vascular endothelial cadherin in the lumens suggested that the vessels were stabilized in the stimulated tissues compared with the control. These findings suggested that deep-media culture conditions improved lumen formation in engineered tissues in vitro.

Ontogenetic tissue modification in Malus fruit peduncles: the role of sclereids

PubMed Central

Horbens, Melanie; Feldner, Alexander; Höfer, Monika; Neinhuis, Christoph

2014-01-01

Background and Aims Apple (Malus) fruit peduncles are highly modified stems with limited secondary growth because fruit ripening lasts only one season. They must reliably connect rather heavy fruits to the branch and cope with increasing fruit weight, which induces dynamic stresses under oscillating wind loads. This study focuses on tissue modification of these small, exposed structures during fruit development. Methods A combination of microscopic, static and dynamic mechanical tests, as well as Raman spectroscopy, was used to study structure–function relationships in peduncles of one cultivar and 12 wild species, representatively chosen from all sections of the genus Malus. Tissue differentiation and ontogenetic changes in mechanical properties of Malus peduncles were observed throughout one growing season and after successive removal of tissues. Key Results Unlike in regular stems, the vascular cambium produces mainly phloem during secondary growth. Hence, in addition to a reduced xylem, all species developed a centrally arranged sclerenchyma ring composed of fibres and brachysclereids. Based on differences in cell-wall thickness, and proportions and arrangement of sclereids, two types of peduncle construction could be distinguished. Fibres provide an increased maximum tensile strength and contribute most to the overall axial rigidity of the peduncles. Sclereids contribute insignificantly to peduncle strength; however, despite being shown to have a lower elastic modulus than fibres, they are the most effective tissue in stiffening peduncles against bending. Conclusions The experimental data revealed that sclereids originating from cortical parenchyma act as ‘accessory’ cells to enhance proportions of sclerenchyma during secondary growth in peduncles. The mechanism can be interpreted as an adaptation to continuously increasing fruit loads. Under oscillating longitudinal stresses, sclereids may be regarded as regulating elements between maintenance of stiffness and viscous damping, the latter property being attributed to the cortical parenchyma. PMID:24287811

Human embryonic stem cell-derived mesodermal progenitors display substantially increased tissue formation compared to human mesenchymal stem cells under dynamic culture conditions in a packed bed/column bioreactor.

PubMed

de Peppo, Giuseppe Maria; Sladkova, Martina; Sjövall, Peter; Palmquist, Anders; Oudina, Karim; Hyllner, Johan; Thomsen, Peter; Petite, Hervé; Karlsson, Camilla

2013-01-01

Bone tissue engineering represents a promising strategy to obviate bone deficiencies, allowing the ex vivo construction of bone substitutes with unprecedented potential in the clinical practice. Considering that in the human body cells are constantly stimulated by chemical and mechanical stimuli, the use of bioreactor is emerging as an essential factor for providing the proper environment for the reproducible and large-scale production of the engineered substitutes. Human mesenchymal stem cells (hMSCs) are experimentally relevant cells but, regardless the encouraging results reported after culture under dynamic conditions in bioreactors, show important limitations for tissue engineering applications, especially considering their limited proliferative potential, loss of functionality following protracted expansion, and decline in cellular fitness associated with aging. On the other hand, we previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold great potential to provide a homogenous and unlimited source of cells for bone engineering applications. Based on prior scientific evidence using different types of stem cells, in the present study we hypothesized that dynamic culture of hES-MPs in a packed bed/column bioreactor had the potential to affect proliferation, expression of genes involved in osteogenic differentiation, and matrix mineralization, therefore resulting in increased bone-like tissue formation. The reported findings suggest that hES-MPs constitute a suitable alternative cell source to hMSCs and hold great potential for the construction of bone substitutes for tissue engineering applications in clinical settings.

In vitro osteogenesis of human stem cells by using a three-dimensional perfusion bioreactor culture system: a review.

PubMed

Ceccarelli, Gabriele; Bloise, Nora; Vercellino, Marco; Battaglia, Rosalia; Morgante, Lucia; De Angelis, Maria Gabriella Cusella; Imbriani, Marcello; Visai, Livia

2013-04-01

Tissue engineering (by culturing cells on appropriate scaffolds, and using bioreactors to drive the correct bone structure formation) is an attractive alternative to bone grafting or implantation of bone substitutes. Osteogenesis is a biological process that involves many molecular intracellular pathways organized to optimize bone modeling. The use of bioreactor systems and especially the perfusion bioreactor, provides both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues. In this mini-review all the characteristics for the production of an appropriate bone construct are analyzed: the stem cell source, scaffolds useful for the seeding of pre-osteoblastic cells and the effects of fluid flow on differentiation and proliferation of bone precursor cells. By automating and standardizing tissue manufacture in controlled closed systems, engineered tissues may reduce the gap between the process of bone formation in vitro and subsequent graft of bone substitutes in vivo.

Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels.

PubMed

Bertassoni, Luiz E; Cardoso, Juliana C; Manoharan, Vijayan; Cristino, Ana L; Bhise, Nupura S; Araujo, Wesleyan A; Zorlutuna, Pinar; Vrana, Nihal E; Ghaemmaghami, Amir M; Dokmeci, Mehmet R; Khademhosseini, Ali

2014-06-01

Fabrication of three dimensional (3D) organoids with controlled microarchitectures has been shown to enhance tissue functionality. Bioprinting can be used to precisely position cells and cell-laden materials to generate controlled tissue architecture. Therefore, it represents an exciting alternative for organ fabrication. Despite the rapid progress in the field, the development of printing processes that can be used to fabricate macroscale tissue constructs from ECM-derived hydrogels has remained a challenge. Here we report a strategy for bioprinting of photolabile cell-laden methacrylated gelatin (GelMA) hydrogels. We bioprinted cell-laden GelMA at concentrations ranging from 7 to 15% with varying cell densities and found a direct correlation between printability and the hydrogel mechanical properties. Furthermore, encapsulated HepG2 cells preserved cell viability for at least eight days following the bioprinting process. In summary, this work presents a strategy for direct-write bioprinting of a cell-laden photolabile ECM-derived hydrogel, which may find widespread application for tissue engineering, organ printing and the development of 3D drug discovery platforms.

The Crosstalk between Tissue Engineering and Pharmaceutical Biotechnology: Recent Advances and Future Directions.

PubMed

Pacheco, Daniela P; Reis, Rui L; Correlo, Vítor M; Marques, Alexandra P

2015-01-01

Tissue-engineered constructs made of biotechnology-derived materials have been preferred due to their chemical and physical composition, which offers both high versatility and a support to enclose/ incorporate relevant signaling molecules and/or genes known to therapeutically induce tissue repair. Herein, a critical overview of the impact of different biotechnology-derived materials, scaffolds, and recombinant signaling molecules over the behavior of cells, another element of tissue engineered constructs, as well its regulatory role in tissue regeneration and disease progression is given. Additionally, these tissue-engineered constructs evolved to three-dimensional (3D) tissue-like models that, as an advancement of two-dimensional standard culture methods, are expected to be a valuable tool in the field of drug discovery and pharmaceutical research. Despite the improved design and conception of current proposed 3D tissue-like models, advanced control systems to enable and accelerate streamlining and automation of the numerous labor-intensive steps intrinsic to the development of tissue-engineered constructs are still to be achieved. In this sense, this review intends to present the biotechnology- derived materials that are being explored in the field of tissue engineering to generate 3D tissue-analogues and briefly highlight their foremost breakthroughs in tissue regeneration and drug discovery. It also aims to reinforce that the crosstalk between tissue engineering and pharmaceutical biotechnology has been fostering the outcomes of tissue engineering approaches through the use of biotechnology-derived signaling molecules. Gene delivery/therapy is also discussed as a forefront area that represents another cross point between tissue engineering and pharmaceutical biotechnology, in which nucleic acids can be considered a "super pharmaceutical" to drive biological responses, including tissue regeneration.

Synchronous Measuring Techniques in Parallel to MRE: Study of Pressure, Pre-Tension, and Surface Dynamics

NASA Astrophysics Data System (ADS)

Brinker, Spencer Thomas

The contents of this dissertation include investigations in Magnetic Resonance Elastography (MRE) using a preclinical 9.4 Tesla small animal Magnetic Resonance Imaging (MRI) system along with synthetic materials that mimic the mechanical properties of soft human tissue. MRE is used for studying the mechanical behavior of soft tissue particularly applicable to medical applications. Wave motion induced by a mechanical driver is measured with MRI to acquire internal displacement fields over time and space within a material media. Complex shear modulus of the media is calculated from the response of mechanical wave transmission through the material. Changes in soft tissue stiffness is associated with disease progression and thus, is why assessing tissue mechanical properties with MRE has powerful diagnostic potential due to the noninvasive procedure of MRI. The experiments performed in this dissertation used elastic phantoms and specimens to observe the influence of pre-stress on MRE derived mechanical properties while additional mechanical measurements from other related material testing methods were synchronously collected alongside MRI scanning. An organ simulating phantom was used to explore changes in MRE stiffness in response to gas and liquid cyclic pressure loading. MRE stiffness increased with pressure and hysteresis was observed in cyclic pressure loading. The results suggest MRE is applicable to pressure related disease assessment. In addition, an interconnected porosity pressure phantom was constructed for future porous media investigations. A custom system was also built to demonstrate concurrent tensile testing during MRE for investigating homogeneous soft material media undergoing pre-tension. Stiffness increased with uniaxial tensile stress and strain. The tension and stiffness relationship explored can be related to the stress analysis of voluntary muscle. The results also offer prospective experimental strategies for community wide standards on MRE calibration methods. Lastly, a novel platform was developed for synchronous acquisition of Scanning Laser Doppler Vibrometry (SLDV) and MRE for examining surface wave dynamics related to internal media wave propagation in soft material experiencing sinusoidal mechanical excitation. The results indicate that optical displacement measurements of media on the surface are similar in nature to internal displacement measured from MRE. It is concluded that optical and MRI based elastography yield similar values of complex shear modulus.

Tissue Modeling and Analyzing with Finite Element Method: A Review for Cranium Brain Imaging

PubMed Central

Yue, Xianfang; Wang, Li; Wang, Ruonan

2013-01-01

For the structure mechanics of human body, it is almost impossible to conduct mechanical experiments. Then the finite element model to simulate mechanical experiments has become an effective tool. By introducing several common methods for constructing a 3D model of cranial cavity, this paper carries out systematically the research on the influence law of cranial cavity deformation. By introducing the new concepts and theory to develop the 3D cranial cavity model with the finite-element method, the cranial cavity deformation process with the changing ICP can be made the proper description and reasonable explanation. It can provide reference for getting cranium biomechanical model quickly and efficiently and lay the foundation for further biomechanical experiments and clinical applications. PMID:23476630

In Vitro Engineering of Vascularized Tissue Surrogates

PubMed Central

Sakaguchi, Katsuhisa; Shimizu, Tatsuya; Horaguchi, Shigeto; Sekine, Hidekazu; Yamato, Masayuki; Umezu, Mitsuo; Okano, Teruo

2013-01-01

In vitro scaling up of bioengineered tissues is known to be limited by diffusion issues, specifically a lack of vasculature. Here, we report a new strategy for preserving cell viability in three-dimensional tissues using cell sheet technology and a perfusion bioreactor having collagen-based microchannels. When triple-layer cardiac cell sheets are incubated within this bioreactor, endothelial cells in the cell sheets migrate to vascularize in the collagen gel, and finally connect with the microchannels. Medium readily flows into the cell sheets through the microchannels and the newly developed capillaries, while the cardiac construct shows simultaneous beating. When additional triple-layer cell sheets are repeatedly layered, new multi-layer construct spontaneously integrates and the resulting construct becomes a vascularized thick tissue. These results confirmed our method to fabricate in vitro vascularized tissue surrogates that overcomes engineered-tissue thickness limitations. The surrogates promise new therapies for damaged organs as well as new in vitro tissue models. PMID:23419835

Repair of Segmental Bone Defect Using Totally Vitalized Tissue Engineered Bone Graft by a Combined Perfusion Seeding and Culture System

PubMed Central

Feng, Ya-Fei; Li, Xiang; Hu, Yun-Yu; Wang, Zhen; Ma, Zhen-Sheng; Lei, Wei

2014-01-01

Background The basic strategy to construct tissue engineered bone graft (TEBG) is to combine osteoblastic cells with three dimensional (3D) scaffold. Based on this strategy, we proposed the “Totally Vitalized TEBG” (TV-TEBG) which was characterized by abundant and homogenously distributed cells with enhanced cell proliferation and differentiation and further investigated its biological performance in repairing segmental bone defect. Methods In this study, we constructed the TV-TEBG with the combination of customized flow perfusion seeding/culture system and β-tricalcium phosphate (β-TCP) scaffold fabricated by Rapid Prototyping (RP) technique. We systemically compared three kinds of TEBG constructed by perfusion seeding and perfusion culture (PSPC) method, static seeding and perfusion culture (SSPC) method, and static seeding and static culture (SSSC) method for their in vitro performance and bone defect healing efficacy with a rabbit model. Results Our study has demonstrated that TEBG constructed by PSPC method exhibited better biological properties with higher daily D-glucose consumption, increased cell proliferation and differentiation, and better cell distribution, indicating the successful construction of TV-TEBG. After implanted into rabbit radius defects for 12 weeks, PSPC group exerted higher X-ray score close to autograft, much greater mechanical property evidenced by the biomechanical testing and significantly higher new bone formation as shown by histological analysis compared with the other two groups, and eventually obtained favorable healing efficacy of the segmental bone defect that was the closest to autograft transplantation. Conclusion This study demonstrated the feasibility of TV-TEBG construction with combination of perfusion seeding, perfusion culture and RP technique which exerted excellent biological properties. The application of TV-TEBG may become a preferred candidate for segmental bone defect repair in orthopedic and maxillofacial fields. PMID:24728277

Subcritical CO2 Sintering of Microspheres of Different Polymeric Materials to Fabricate Scaffolds for Tissue Engineering

PubMed Central

Bhamidipati, Manjari; Sridharan, BanuPriya; Scurto, Aaron M; Detamore, Michael S.

2013-01-01

The aim of this study was to use CO2 at sub-critical pressures as a tool to sinter 3D, macroporous, microsphere-based scaffolds for bone and cartilage Tissue Engineering Porous scaffolds composed of ~200 µm microspheres of either poly(lactic-co-glycolic acid) (PLGA) or polycaprolactone (PCL) were prepared using dense phase CO2 sintering, which were seeded with rat bone marrow mesenchymal stromal cells (rBMSCs), and exposed to either osteogenic (PLGA, PCL) or chondrogenic (PLGA) conditions for 6 weeks. Under osteogenic conditions, the PLGA constructs produced over an order of magnitude more calcium than the PCL constructs, whereas the PCL constructs had far superior mechanical and structural integrity (125 times stiffer than PLGA constructs) at week 6, along with twice the cell content of the PLGA constructs. Chondrogenic cell performance was limited in PLGA constructs, perhaps as a result of the polymer degradation rate being too high. The current study represents the first long-term culture of CO2-sintered microsphere-based scaffolds, and has established important thermodynamic differences in sintering between the selected formulations of PLGA and PCL, with the former requiring adjustment of pressure only, and the latter requiring the adjustment of both pressure and temperature. Based on more straightforward sintering conditions and more favorable cell performance, PLGA may be the material of choice for microspheres in a CO2 sintering application, although a different PLGA formulation with the encapsulation of growth factors, extracellular matrix-derived nanoparticles, and/or buffers in the microspheres may be advantageous for achieving a more superior cell performance than observed here. PMID:24094202

Application of stem cells for cardiovascular grafts tissue engineering.

PubMed

Wu, Kaihong; Liu, Ying Long; Cui, Bin; Han, Zhongchao

2006-06-01

Congenital and acquired heart diseases are leading causes of morbidity and mortality world-wide. Currently, the synthetic materials or bioprosthetic replacement devices for cardiovascular surgery are imperfect and subject patients to one or more ongoing risks including thrombosis, limited durability and need for reoperations due to lack of growth in children and young adults. Suitable replacement grafts should have appropriate characteristics, including resistance to infection, low immunogenicity, good biocompatability and thromboresistance, with appropriate mechanical and physiological properties. Tissue engineering is a new scientific field aiming at fabrication of living, autologous grafts having structure or function properties that can be used to restore, maintain or improve tissue function. The use of autologous stem cells in cardiovascular tissue engineering is quite promising due to their capacity of self-renewal, high proliferation, and differentiation into specialized progeny. Progress has been made in engineering the various components of the cardiovascular system, including myocardial constructs, heart valves, and vascular patches or conduits with autologous stem cells. This paper will review the current achievements in stem cell-based cardiovascular grafts tissue engineering, with an emphasis on its clinical or possible clinical use in cardiovascular surgery.

Numerical analysis of stress distribution in the upper arm tissues under an inflatable cuff: Implications for noninvasive blood pressure measurement

NASA Astrophysics Data System (ADS)

Deng, Zhipeng; Liang, Fuyou

2016-10-01

An inflatable cuff wrapped around the upper arm is widely used in noninvasive blood pressure measurement. However, the mechanical interaction between cuff and arm tissues, a factor that potentially affects the accuracy of noninvasive blood pressure measurement, remains rarely addressed. In the present study, finite element (FE) models were constructed to quantify intra-arm stresses generated by cuff compression, aiming to provide some theoretical evidence for identifying factors of importance for blood pressure measurement or explaining clinical observations. Obtained results showed that the simulated tissue stresses were highly sensitive to the distribution of cuff pressure on the arm surface and the contact condition between muscle and bone. In contrast, the magnitude of cuff pressure and small variations in elastic properties of arm soft tissues had little influence on the efficiency of pressure transmission in arm tissues. In particular, it was found that a thickened subcutaneous fat layer in obese subjects significantly reduced the effective pressure transmitted to the brachial artery, which may explain why blood pressure overestimation occurs more frequently in obese subjects in noninvasive blood pressure measurement.