Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications

PubMed Central

Fortunato, Tiago M.; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A.; Pula, Giordano

2016-01-01

Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs. PMID:27141997

Engineering micropatterned surfaces to modulate the function of vascular stem cells.

PubMed

Li, Jennifer; Wu, Michelle; Chu, Julia; Sochol, Ryan; Patel, Shyam

2014-02-21

Multipotent vascular stem cells have been implicated in vascular disease and in tissue remodeling post therapeutic intervention. Hyper-proliferation and calcified extracellular matrix deposition of VSC cause blood vessel narrowing and plaque hardening thereby increasing the risk of myocardial infarct. In this study, to optimize the surface design of vascular implants, we determined whether micropatterned polymer surfaces can modulate VSC differentiation and calcified matrix deposition. Undifferentiated rat VSC were cultured on microgrooved surfaces of varied groove widths, and on micropost surfaces. 10μm microgrooved surfaces elongated VSC and decreased cell proliferation. However, microgrooved surfaces did not attenuate calcified extracellular matrix deposition by VSC cultured in osteogenic media conditions. In contrast, VSC cultured on micropost surfaces assumed a dendritic morphology, were significantly less proliferative, and deposited minimal calcified extracellular matrix. These results have significant implications for optimizing the design of cardiovascular implant surfaces. Copyright © 2014 Elsevier Inc. All rights reserved.

Detergent-enzymatic decellularization of swine blood vessels: insight on mechanical properties for vascular tissue engineering.

PubMed

Pellegata, Alessandro F; Asnaghi, M Adelaide; Stefani, Ilaria; Maestroni, Anna; Maestroni, Silvia; Dominioni, Tommaso; Zonta, Sandro; Zerbini, Gianpaolo; Mantero, Sara

2013-01-01

Small caliber vessels substitutes still remain an unmet clinical need; few autologous substitutes are available, while synthetic grafts show insufficient patency in the long term. Decellularization is the complete removal of all cellular and nuclear matters from a tissue while leaving a preserved extracellular matrix representing a promising tool for the generation of acellular scaffolds for tissue engineering, already used for various tissues with positive outcomes. The aim of this work is to investigate the effect of a detergent-enzymatic decellularization protocol on swine arteries in terms of cell removal, extracellular matrix preservation, and mechanical properties. Furthermore, the effect of storage at -80°C on the mechanical properties of the tissue is evaluated. Swine arteries were harvested, frozen, and decellularized; histological analysis revealed complete cell removal and preserved extracellular matrix. Furthermore, the residual DNA content in decellularized tissues was far low compared to native one. Mechanical testings were performed on native, defrozen, and decellularized tissues; no statistically significant differences were reported for Young's modulus, ultimate stress, compliance, burst pressure, and suture retention strength, while ultimate strain and stress relaxation of decellularized vessels were significantly different from the native ones. Considering the overall results, the process was confirmed to be suitable for the generation of acellular scaffolds for vascular tissue engineering.

Neoproteoglycans in tissue engineering.

PubMed

Weyers, Amanda; Linhardt, Robert J

2013-05-01

Proteoglycans, comprised of a core protein to which glycosaminoglycan chains are covalently linked, are an important structural and functional family of macromolecules found in the extracellular matrix. Advances in our understanding of biological interactions have lead to a greater appreciation for the need to design tissue engineering scaffolds that incorporate mimetics of key extracellular matrix components. A variety of synthetic and semisynthetic molecules and polymers have been examined by tissue engineers that serve as structural, chemical and biological replacements for proteoglycans. These proteoglycan mimetics have been referred to as neoproteoglycans and serve as functional and therapeutic replacements for natural proteoglycans that are often unavailable for tissue engineering studies. Although neoproteoglycans have important limitations, such as limited signaling ability and biocompatibility, they have shown promise in replacing the natural activity of proteoglycans through cell and protein binding interactions. This review focuses on the recent in vivo and in vitro tissue engineering applications of three basic types of neoproteoglycan structures, protein-glycosaminoglycan conjugates, nano-glycosaminoglycan composites and polymer-glycosaminoglycan complexes. © 2013 The Authors Journal compilation © 2013 FEBS.

Neoproteoglycans in tissue engineering

PubMed Central

Weyers, Amanda; Linhardt, Robert J.

2014-01-01

Proteoglycans, comprised of a core protein to which glycosaminoglycan chains are covalently linked, are an important structural and functional family of macromolecules found in the extracellular matrix. Advances in our understanding of biological interactions have lead to a greater appreciation for the need to design tissue engineering scaffolds that incorporate mimetics of key extracellular matrix components. A variety of synthetic and semisynthetic molecules and polymers have been examined by tissue engineers that serve as structural, chemical and biological replacements for proteoglycans. These proteoglycan mimetics have been referred to as neoproteoglycans and serve as functional and therapeutic replacements for natural proteoglycans that are often unavailable for tissue engineering studies. Although neoproteoglycans have important limitations, such as limited signaling ability and biocompatibility, they have shown promise in replacing the natural activity of proteoglycans through cell and protein binding interactions. This review focuses on the recent in vivo and in vitro tissue engineering applications of three basic types of neoproteoglycan structures, protein–glycosaminoglycan conjugates, nano-glycosaminoglycan composites and polymer–glycosaminoglycan complexes. PMID:23399318

Tissue engineering on the nanoscale: lessons from the heart.

PubMed

Fleischer, Sharon; Dvir, Tal

2013-08-01

Recognizing the limitations of biomaterials for engineering complex tissues and the desire for closer recapitulation of the natural matrix have led tissue engineers to seek new technologies for fabricating 3-dimensional (3D) cellular microenvironments. In this review, through examples from cardiac tissue engineering, we describe the nanoscale hallmarks of the extracellular matrix that tissue engineers strive to mimic. Furthermore, we discuss the use of inorganic nanoparticles and nanodevices for improving and monitoring the performance of engineered tissues. Finally, we offer our opinion on the main challenges and prospects of applying nanotechnology in tissue engineering. Copyright © 2012 Elsevier Ltd. All rights reserved.

Influence of culture conditions and extracellular matrix alignment on human mesenchymal stem cells invasion into decellularized engineered tissues.

PubMed

Weidenhamer, Nathan K; Moore, Dusty L; Lobo, Fluvio L; Klair, Nathaniel T; Tranquillo, Robert T

2015-05-01

The variables that influence the in vitro recellularization potential of decellularized engineered tissues, such as cell culture conditions and scaffold alignment, have yet to be explored. The goal of this work was to explore the influence of insulin and ascorbic acid and extracellular matrix (ECM) alignment on the recellularization of decellularized engineered tissue by human mesenchymal stem cells (hMSCs). Aligned and non-aligned tissues were created by specifying the geometry and associated mechanical constraints to fibroblast-mediated fibrin gel contraction and remodelling using circular and C-shaped moulds. Decellularized tissues (matrices) of the same alignment were created by decellularization with detergents. Ascorbic acid promoted the invasion of hMSCs into the matrices due to a stimulated increase in motility and proliferation. Invasion correlated with hyaluronic acid secretion, α-smooth muscle actin expression and decreased matrix thickness. Furthermore, hMSCs invasion into aligned and non-aligned matrices was not different, although there was a difference in cell orientation. Finally, we show that hMSCs on the matrix surface appear to differentiate toward a smooth muscle cell or myofibroblast phenotype with ascorbic acid treatment. These results inform the strategy of recellularizing decellularized engineered tissue with hMSCs. Copyright © 2014 John Wiley & Sons, Ltd.

Comparative analysis of poly-glycolic acid-based hybrid polymer starter matrices for in vitro tissue engineering.

PubMed

Generali, Melanie; Kehl, Debora; Capulli, Andrew K; Parker, Kevin K; Hoerstrup, Simon P; Weber, Benedikt

2017-10-01

Biodegradable scaffold matrixes form the basis of any in vitro tissue engineering approach by acting as a temporary matrix for cell proliferation and extracellular matrix deposition until the scaffold is replaced by neo-tissue. In this context several synthetic polymers have been investigated, however a concise systematic comparative analyses is missing. Therefore, the present study systematically compares three frequently used polymers for the in vitro engineering of extracellular matrix based on poly-glycolic acid (PGA) under static as well as dynamic conditions. Ultra-structural analysis was used to examine the polymers structure. For tissue engineering (TE) three human fibroblast cell lines were seeded on either PGA-poly-4-hydroxybutyrate (P4HB), PGA-poly-lactic acid (PLA) or PGA-poly-caprolactone (PCL) patches. These patches were analyzed after 21days of culture qualitative by histology and quantitative by determining the amount of DNA, glycosaminoglycan and hydroxyproline. We found that PGA-P4HB and PGA-PLA scaffolds enhance tissue formation significantly higher than PGA-PCL scaffolds (p<0.05). Polymer remnants were visualized by polarization microscopy. In addition, biomechanical properties of the tissue engineered patches were determined in comparison to native tissue. This study may allow future studies to specifically select certain polymer starter matrices aiming at specific tissue properties of the bioengineered constructs in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

Design properties of hydrogel tissue-engineering scaffolds

PubMed Central

Zhu, Junmin; Marchant, Roger E

2011-01-01

This article summarizes the recent progress in the design and synthesis of hydrogels as tissue-engineering scaffolds. Hydrogels are attractive scaffolding materials owing to their highly swollen network structure, ability to encapsulate cells and bioactive molecules, and efficient mass transfer. Various polymers, including natural, synthetic and natural/synthetic hybrid polymers, have been used to make hydrogels via chemical or physical crosslinking. Recently, bioactive synthetic hydrogels have emerged as promising scaffolds because they can provide molecularly tailored biofunctions and adjustable mechanical properties, as well as an extracellular matrix-like microenvironment for cell growth and tissue formation. This article addresses various strategies that have been explored to design synthetic hydrogels with extracellular matrix-mimetic bioactive properties, such as cell adhesion, proteolytic degradation and growth factor-binding. PMID:22026626

Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment.

PubMed

Palchesko, Rachelle N; Szymanski, John M; Sahu, Amrita; Feinberg, Adam W

2014-09-01

Cell-matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo . Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell-matrix adhesions and cell behavior in 2D and 3D microenvironments.

Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment

PubMed Central

Palchesko, Rachelle N.; Szymanski, John M.; Sahu, Amrita; Feinberg, Adam W.

2014-01-01

Cell-matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo. Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell-matrix adhesions and cell behavior in 2D and 3D microenvironments. PMID:25530816

Comparison of four decontamination treatments on porcine renal decellularized extracellular matrix structure, composition, and support of human renal cortical tubular epithelium cells.

PubMed

Poornejad, Nafiseh; Nielsen, Jeffery J; Morris, Ryan J; Gassman, Jason R; Reynolds, Paul R; Roeder, Beverly L; Cook, Alonzo D

2016-03-01

Engineering whole organs from porcine decellularized extracellular matrix and human cells may lead to a plentiful source of implantable organs. Decontaminating the porcine decellularized extracellular matrix scaffolds is an essential step prior to introducing human cells. However, decontamination of whole porcine kidneys is a major challenge because the decontamination agent or irradiation needs to diffuse deep into the structure to eliminate all microbial contamination while minimizing damage to the structure and composition of the decellularized extracellular matrix. In this study, we compared four decontamination treatments that could be applicable to whole porcine kidneys: 70% ethanol, 0.2% peracetic acid in 1 M NaCl, 0.2% peracetic acid in 4% ethanol, and gamma (γ)-irradiation. Porcine kidneys were decellularized by perfusion of 0.5% (w/v) aqueous solution of sodium dodecyl sulfate and the four decontamination treatments were optimized using segments (n = 60) of renal tissue to ensure a consistent comparison. Although all four methods were successful in decontamination, γ-irradiation was very damaging to collagen fibers and glycosaminoglycans, leading to less proliferation of human renal cortical tubular epithelium cells within the porcine decellularized extracellular matrix. The effectiveness of the other three optimized solution treatments were then all confirmed using whole decellularized porcine kidneys (n = 3). An aqueous solution of 0.2% peracetic acid in 1 M NaCl was determined to be the best method for decontamination of porcine decellularized extracellular matrix. © The Author(s) 2015.

Functional Characterization of Detergent-Decellularized Equine Tendon Extracellular Matrix for Tissue Engineering Applications

PubMed Central

Youngstrom, Daniel W.; Barrett, Jennifer G.; Jose, Rod R.; Kaplan, David L.

2013-01-01

Natural extracellular matrix provides a number of distinct advantages for engineering replacement orthopedic tissue due to its intrinsic functional properties. The goal of this study was to optimize a biologically derived scaffold for tendon tissue engineering using equine flexor digitorum superficialis tendons. We investigated changes in scaffold composition and ultrastructure in response to several mechanical, detergent and enzymatic decellularization protocols using microscopic techniques and a panel of biochemical assays to evaluate total protein, collagen, glycosaminoglycan, and deoxyribonucleic acid content. Biocompatibility was also assessed with static mesenchymal stem cell (MSC) culture. Implementation of a combination of freeze/thaw cycles, incubation in 2% sodium dodecyl sulfate (SDS), trypsinization, treatment with DNase-I, and ethanol sterilization produced a non-cytotoxic biomaterial free of appreciable residual cellular debris with no significant modification of biomechanical properties. These decellularized tendon scaffolds (DTS) are suitable for complex tissue engineering applications, as they provide a clean slate for cell culture while maintaining native three-dimensional architecture. PMID:23724028

Engineering hydrogels as extracellular matrix mimics

PubMed Central

Geckil, Hikmet; Xu, Feng; Zhang, Xiaohui; Moon, SangJun

2010-01-01

Extracellular matrix (ECM) is a complex cellular environment consisting of proteins, proteoglycans, and other soluble molecules. ECM provides structural support to mammalian cells and a regulatory milieu with a variety of important cell functions, including assembling cells into various tissues and organs, regulating growth and cell–cell communication. Developing a tailored in vitro cell culture environment that mimics the intricate and organized nanoscale meshwork of native ECM is desirable. Recent studies have shown the potential of hydrogels to mimic native ECM. Such an engineered native-like ECM is more likely to provide cells with rational cues for diagnostic and therapeutic studies. The research for novel biomaterials has led to an extension of the scope and techniques used to fabricate biomimetic hydrogel scaffolds for tissue engineering and regenerative medicine applications. In this article, we detail the progress of the current state-of-the-art engineering methods to create cell-encapsulating hydrogel tissue constructs as well as their applications in in vitro models in biomedicine. PMID:20394538

Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine

DOE PAGES

Martino, Mikael M.; Brkic, Sime; Bovo, Emmanuela; ...

2015-04-01

In this study, blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF) is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular,more » the spatial localization of angiogenic signals in the extracellular matrix (ECM) is crucial to ensure the proper assembly and maturation of new vascular structures. Here, we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis on VEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of ECM to optimize the signaling microenvironment of vascular growth factors.« less

Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine

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

Martino, Mikael M.; Brkic, Sime; Bovo, Emmanuela

In this study, blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF) is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular,more » the spatial localization of angiogenic signals in the extracellular matrix (ECM) is crucial to ensure the proper assembly and maturation of new vascular structures. Here, we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis on VEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of ECM to optimize the signaling microenvironment of vascular growth factors.« less

Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine.

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

Martino, Mikael M.; Brkic, Sime; Bovo, Emmanuela

Blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF) is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular, the spatial localizationmore » of angiogenic signals in the extracellular matrix (ECM) is crucial to ensure the proper assembly and maturation of new vascular structures. Here, we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis on VEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of ECM to optimize the signaling microenvironment of vascular growth factors.« less

Mitochondrial function in engineered cardiac tissues is regulated by extracellular matrix elasticity and tissue alignment.

PubMed

Lyra-Leite, Davi M; Andres, Allen M; Petersen, Andrew P; Ariyasinghe, Nethika R; Cho, Nathan; Lee, Jezell A; Gottlieb, Roberta A; McCain, Megan L

2017-10-01

Mitochondria in cardiac myocytes are critical for generating ATP to meet the high metabolic demands associated with sarcomere shortening. Distinct remodeling of mitochondrial structure and function occur in cardiac myocytes in both developmental and pathological settings. However, the factors that underlie these changes are poorly understood. Because remodeling of tissue architecture and extracellular matrix (ECM) elasticity are also hallmarks of ventricular development and disease, we hypothesize that these environmental factors regulate mitochondrial function in cardiac myocytes. To test this, we developed a new procedure to transfer tunable polydimethylsiloxane disks microcontact-printed with fibronectin into cell culture microplates. We cultured Sprague-Dawley neonatal rat ventricular myocytes within the wells, which consistently formed tissues following the printed fibronectin, and measured oxygen consumption rate using a Seahorse extracellular flux analyzer. Our data indicate that parameters associated with baseline metabolism are predominantly regulated by ECM elasticity, whereas the ability of tissues to adapt to metabolic stress is regulated by both ECM elasticity and tissue alignment. Furthermore, bioenergetic health index, which reflects both the positive and negative aspects of oxygen consumption, was highest in aligned tissues on the most rigid substrate, suggesting that overall mitochondrial function is regulated by both ECM elasticity and tissue alignment. Our results demonstrate that mitochondrial function is regulated by both ECM elasticity and myofibril architecture in cardiac myocytes. This provides novel insight into how extracellular cues impact mitochondrial function in the context of cardiac development and disease. NEW & NOTEWORTHY A new methodology has been developed to measure O 2 consumption rates in engineered cardiac tissues with independent control over tissue alignment and matrix elasticity. This led to the findings that matrix elasticity regulates basal mitochondrial function, whereas both matrix elasticity and tissue alignment regulate mitochondrial stress responses. Copyright © 2017 the American Physiological Society.

Engineering a Biocompatible Scaffold with Either Micrometre or Nanometre Scale Surface Topography for Promoting Protein Adsorption and Cellular Response

PubMed Central

Le, Xuan; Poinern, Gérrard Eddy Jai; Ali, Nurshahidah; Berry, Cassandra M.; Fawcett, Derek

2013-01-01

Surface topographical features on biomaterials, both at the submicrometre and nanometre scales, are known to influence the physicochemical interactions between biological processes involving proteins and cells. The nanometre-structured surface features tend to resemble the extracellular matrix, the natural environment in which cells live, communicate, and work together. It is believed that by engineering a well-defined nanometre scale surface topography, it should be possible to induce appropriate surface signals that can be used to manipulate cell function in a similar manner to the extracellular matrix. Therefore, there is a need to investigate, understand, and ultimately have the ability to produce tailor-made nanometre scale surface topographies with suitable surface chemistry to promote favourable biological interactions similar to those of the extracellular matrix. Recent advances in nanoscience and nanotechnology have produced many new nanomaterials and numerous manufacturing techniques that have the potential to significantly improve several fields such as biological sensing, cell culture technology, surgical implants, and medical devices. For these fields to progress, there is a definite need to develop a detailed understanding of the interaction between biological systems and fabricated surface structures at both the micrometre and nanometre scales. PMID:23533416

Matrix elasticity regulates the optimal cardiac myocyte shape for contractility

PubMed Central

McCain, Megan L.; Yuan, Hongyan; Pasqualini, Francesco S.; Campbell, Patrick H.

2014-01-01

Concentric hypertrophy is characterized by ventricular wall thickening, fibrosis, and decreased myocyte length-to-width aspect ratio. Ventricular thickening is considered compensatory because it reduces wall stress, but the functional consequences of cell shape remodeling in this pathological setting are unknown. We hypothesized that decreases in myocyte aspect ratio allow myocytes to maximize contractility when the extracellular matrix becomes stiffer due to conditions such as fibrosis. To test this, we engineered neonatal rat ventricular myocytes into rectangles mimicking the 2-D profiles of healthy and hypertrophied myocytes on hydrogels with moderate (13 kPa) and high (90 kPa) elastic moduli. Actin alignment was unaffected by matrix elasticity, but sarcomere content was typically higher on stiff gels. Microtubule polymerization was higher on stiff gels, implying increased intracellular elastic modulus. On moderate gels, myocytes with moderate aspect ratios (∼7:1) generated the most peak systolic work compared with other cell shapes. However, on stiffer gels, low aspect ratios (∼2:1) generated the most peak systolic work. To compare the relative contributions of intracellular vs. extracellular elasticity to contractility, we developed an analytical model and used our experimental data to fit unknown parameters. Our model predicted that matrix elasticity dominates over intracellular elasticity, suggesting that the extracellular matrix may potentially be a more effective therapeutic target than microtubules. Our data and model suggest that myocytes with lower aspect ratios have a functional advantage when the elasticity of the extracellular matrix decreases due to conditions such as fibrosis, highlighting the role of the extracellular matrix in cardiac disease. PMID:24682394

Engineering nanoscale stem cell niche: direct stem cell behavior at cell-matrix interface.

PubMed

Zhang, Yan; Gordon, Andrew; Qian, Weiyi; Chen, Weiqiang

2015-09-16

Biophysical cues on the extracellular matrix (ECM) have proven to be significant regulators of stem cell behavior and evolution. Understanding the interplay of these cells and their extracellular microenvironment is critical to future tissue engineering and regenerative medicine, both of which require a means of controlled differentiation. Research suggests that nanotopography, which mimics the local, nanoscale, topographic cues within the stem cell niche, could be a way to achieve large-scale proliferation and control of stem cells in vitro. This Progress Report reviews the history and contemporary advancements of this technology, and pays special attention to nanotopographic fabrication methods and the effect of different nanoscale patterns on stem cell response. Finally, it outlines potential intracellular mechanisms behind this response. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling interlamellar interactions in angle-ply biologic laminates for annulus fibrosus tissue engineering

PubMed Central

Nerurkar, Nandan L.; Mauck, Robert L.

2012-01-01

Mechanical function of the annulus fibrosus of the intervertebral disc is dictated by the composition and microstructure of its highly ordered extracellular matrix. Recent work on engineered angle-ply laminates formed from mesenchymal stem cell (MSC)-seeded nanofibrous scaffolds indicates that the organization of collagen fibers into planes of alternating alignment may play an important role in annulus fibrosus tissue function. Specifically, these engineered tissues can resist tensile deformation through shearing of the interlamellar matrix as layers of collagen differentially reorient under load. In the present work, a hyperelastic constitutive model was developed to describe the role of interlamellar shearing in reinforcing the tensile response of biologic laminates, and was applied to experimental results from engineered annulus constructs formed from MSC-seeded nanofibrous scaffolds. By applying the constitutive model to uniaxial tensile stress–strain data for bilayers with three different fiber orientations, material parameters were generated that characterize the contributions of extrafibrillar matrix, fibers, and interlamellar shearing interactions. By 10 weeks of in vitro culture, interlamellar shearing accounted for nearly 50% of the total stress associated with uniaxial extension in the anatomic range of ply angle. The model successfully captured changes in function with extracellular matrix deposition through variations in the magnitude of model parameters with culture duration. This work illustrates the value of engineered tissues as tools to further our understanding of structure–function relations in native tissues and as a test-bed for the development of constitutive models to describe them. PMID:21287395

Modeling interlamellar interactions in angle-ply biologic laminates for annulus fibrosus tissue engineering.

PubMed

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

2011-12-01

Mechanical function of the annulus fibrosus of the intervertebral disc is dictated by the composition and microstructure of its highly ordered extracellular matrix. Recent work on engineered angle-ply laminates formed from mesenchymal stem cell (MSC)-seeded nanofibrous scaffolds indicates that the organization of collagen fibers into planes of alternating alignment may play an important role in annulus fibrosus tissue function. Specifically, these engineered tissues can resist tensile deformation through shearing of the interlamellar matrix as layers of collagen differentially reorient under load. In the present work, a hyperelastic constitutive model was developed to describe the role of interlamellar shearing in reinforcing the tensile response of biologic laminates, and was applied to experimental results from engineered annulus constructs formed from MSC-seeded nanofibrous scaffolds. By applying the constitutive model to uniaxial tensile stress-strain data for bilayers with three different fiber orientations, material parameters were generated that characterize the contributions of extrafibrillar matrix, fibers, and interlamellar shearing interactions. By 10 weeks of in vitro culture, interlamellar shearing accounted for nearly 50% of the total stress associated with uniaxial extension in the anatomic range of ply angle. The model successfully captured changes in function with extracellular matrix deposition through variations in the magnitude of model parameters with culture duration. This work illustrates the value of engineered tissues as tools to further our understanding of structure-function relations in native tissues and as a test-bed for the development of constitutive models to describe them.

Engineering micropatterned surfaces to modulate the function of vascular stem cells

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

Li, Jennifer; Wu, Michelle; Chu, Julia

2014-02-21

Highlights: • We examine vascular stem cell function on microgrooved and micropost patterned polymer substrates. • 10 μm microgrooved surfaces significantly lower VSC proliferation but do not modulate calcified matrix deposition. • Micropost surfaces significantly lower VSC proliferation and decrease calcified matrix deposition. - Abstract: Multipotent vascular stem cells have been implicated in vascular disease and in tissue remodeling post therapeutic intervention. Hyper-proliferation and calcified extracellular matrix deposition of VSC cause blood vessel narrowing and plaque hardening thereby increasing the risk of myocardial infarct. In this study, to optimize the surface design of vascular implants, we determined whether micropatterned polymermore » surfaces can modulate VSC differentiation and calcified matrix deposition. Undifferentiated rat VSC were cultured on microgrooved surfaces of varied groove widths, and on micropost surfaces. 10 μm microgrooved surfaces elongated VSC and decreased cell proliferation. However, microgrooved surfaces did not attenuate calcified extracellular matrix deposition by VSC cultured in osteogenic media conditions. In contrast, VSC cultured on micropost surfaces assumed a dendritic morphology, were significantly less proliferative, and deposited minimal calcified extracellular matrix. These results have significant implications for optimizing the design of cardiovascular implant surfaces.« less

Feasibility of autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold for cartilage tissue engineering.

PubMed

Tang, Cheng; Xu, Yan; Jin, Chengzhe; Min, Byoung-Hyun; Li, Zhiyong; Pei, Xuan; Wang, Liming

2013-12-01

Extracellular matrix (ECM) materials are widely used in cartilage tissue engineering. However, the current ECM materials are unsatisfactory for clinical practice as most of them are derived from allogenous or xenogenous tissue. This study was designed to develop a novel autologous ECM scaffold for cartilage tissue engineering. The autologous bone marrow mesenchymal stem cell-derived ECM (aBMSC-dECM) membrane was collected and fabricated into a three-dimensional porous scaffold via cross-linking and freeze-drying techniques. Articular chondrocytes were seeded into the aBMSC-dECM scaffold and atelocollagen scaffold, respectively. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The current results showed that the aBMSC-dECM scaffold had a good microstructure and biocompatibility. After 4 weeks in vitro culture, the engineered cartilage in the aBMSC-dECM scaffold group formed thicker cartilage tissue with more homogeneous structure and higher expressions of cartilaginous gene and protein compared with the atelocollagen scaffold group. Furthermore, the engineered cartilage based on the aBMSC-dECM scaffold showed better cartilage formation in terms of volume and homogeneity, cartilage matrix content, and compressive modulus after 3 weeks in vivo implantation. These results indicated that the aBMSC-dECM scaffold could be a successful novel candidate scaffold for cartilage tissue engineering. © 2013 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation.

Cartilaginous extracellular matrix-modified chitosan hydrogels for cartilage tissue engineering.

PubMed

Choi, Bogyu; Kim, Soyon; Lin, Brian; Wu, Benjamin M; Lee, Min

2014-11-26

Cartilaginous extracellular matrix (ECM) components such as type-II collagen (Col II) and chondroitin sulfate (CS) play a crucial role in chondrogenesis. However, direct clinical use of natural Col II or CS as scaffolds for cartilage tissue engineering is limited by their instability and rapid enzymatic degradation. Here, we investigate the incorporation of Col II and CS into injectable chitosan hydrogels designed to gel upon initiation by exposure to visible blue light (VBL) in the presence of riboflavin. Unmodified chitosan hydrogel supported proliferation and deposition of cartilaginous ECM by encapsulated chondrocytes and mesenchymal stem cells. The incorporation of native Col II or CS into chitosan hydrogels further increased chondrogenesis. The incorporation of Col II, in particular, was found to be responsible for the enhanced cellular condensation and chondrogenesis observed in modified hydrogels. This was mediated by integrin α10 binding to Col II, increasing cell-matrix adhesion. These findings demonstrate the potential of cartilage ECM-modified chitosan hydrogels as biomaterials to promote cartilage regeneration.

Design of biomimetic cellular scaffolds for co-culture system and their application

PubMed Central

Kook, Yun-Min; Jeong, Yoon; Lee, Kangwon; Koh, Won-Gun

2017-01-01

The extracellular matrix of most natural tissues comprises various types of cells, including fibroblasts, stem cells, and endothelial cells, which communicate with each other directly or indirectly to regulate matrix production and cell functionality. To engineer multicellular interactions in vitro, co-culture systems have achieved tremendous success achieving a more realistic microenvironment of in vivo metabolism than monoculture system in the past several decades. Recently, the fields of tissue engineering and regenerative medicine have primarily focused on three-dimensional co-culture systems using cellular scaffolds, because of their physical and biological relevance to the extracellular matrix of actual tissues. This review discusses several materials and methods to create co-culture systems, including hydrogels, electrospun fibers, microfluidic devices, and patterning for biomimetic co-culture system and their applications for specific tissue regeneration. Consequently, we believe that culture systems with appropriate physical and biochemical properties should be developed, and direct or indirect cell–cell interactions in the remodeled tissue must be considered to obtain an optimal tissue-specific microenvironment. PMID:29081966

Design of biomimetic cellular scaffolds for co-culture system and their application.

PubMed

Kook, Yun-Min; Jeong, Yoon; Lee, Kangwon; Koh, Won-Gun

2017-01-01

The extracellular matrix of most natural tissues comprises various types of cells, including fibroblasts, stem cells, and endothelial cells, which communicate with each other directly or indirectly to regulate matrix production and cell functionality. To engineer multicellular interactions in vitro, co-culture systems have achieved tremendous success achieving a more realistic microenvironment of in vivo metabolism than monoculture system in the past several decades. Recently, the fields of tissue engineering and regenerative medicine have primarily focused on three-dimensional co-culture systems using cellular scaffolds, because of their physical and biological relevance to the extracellular matrix of actual tissues. This review discusses several materials and methods to create co-culture systems, including hydrogels, electrospun fibers, microfluidic devices, and patterning for biomimetic co-culture system and their applications for specific tissue regeneration. Consequently, we believe that culture systems with appropriate physical and biochemical properties should be developed, and direct or indirect cell-cell interactions in the remodeled tissue must be considered to obtain an optimal tissue-specific microenvironment.

Cell Based Meniscal Repair Using an Aligned Bioactive Nanofibrous Sheath

DTIC Science & Technology

2017-07-01

to rapid joint degeneration (i.e., osteoarthritis). Tissue engineering approaches, including the combination of cells, scaffolds, and bioactive...nano/microfibers comprising engineered scaffolds can mimic the ultrastructure of the native meniscal extracellular matrix (ECM); when seeded with adult...explant and in vivo goat model. 2. KEYWORDS: Provide a brief list of keywords (limit to 20 words). Meniscus tissue engineering , electrospun

A fast and mild decellularization protocol for obtaining extracellular matrix.

PubMed

Mirzarafie, Ariana; Grainger, Rhian K; Thomas, Ben; Bains, William; Ustok, Fatma I; Lowe, Chris R

2014-04-01

Degradation of extracellular matrix (ECM) function with age is a major cause of loss of tissue function with age that we would wish to reverse. Tissue engineering to provide replacement tissue requires an ECM-mimicking scaffold for cell organization. The standard protocols for achieving this take 10 days and include steps that may change the protein structure of the ECM. Here we describe a much shorter protocol for decellularizing chicken muscle, skin, and tendon samples that achieves the same efficiency as the original protocol without protein cross-link interference. Our protocol can be completed in 72 hr.

A gold nanoparticle coated porcine cholecyst-derived bioscaffold for cardiac tissue engineering.

PubMed

Nair, Reshma S; Ameer, Jimna Mohamed; Alison, Malcolm R; Anilkumar, Thapasimuthu V

2017-09-01

Extracellular matrices of xenogeneic origin have been extensively used for biomedical applications, despite the possibility of heterogeneity in structure. Surface modification of biologically derived biomaterials using nanoparticles is an emerging strategy for improving topographical homogeneity when employing these scaffolds for sophisticated tissue engineering applications. Recently, as a tissue engineering scaffold, cholecyst derived extracellular matrix (C-ECM) has been shown to have several advantages over extracellular matrices derived from other organs such as jejunum and urinary bladder. This study explored the possibility of adding gold nanoparticles, which have a large surface area to volume ratio on C-ECM for achieving homogeneity in surface architecture, a requirement for cardiac tissue engineering. In the current study, gold nanoparticles (AuNPs) were synthesized and functionalised for conjugating with a porcine cholecystic extracellular matrix scaffold. The conjugation of nanoparticles to C-ECM was achieved by 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide/N-hydroxysuccinimide chemistry and further characterized by Fourier transform infrared spectroscopy, environmental scanning electron microscopy, energy dispersive X-ray spectroscopy and thermogravimetric analysis. The physical properties of the modified scaffold were similar to the original C-ECM. Biological properties were evaluated by using H9c2 cells, a cardiomyoblast cell line commonly used for cellular and molecular studies of cardiac cells. The modified scaffold was found to be a suitable substrate for the growth and proliferation of the cardiomyoblasts. Further, the non-cytotoxic nature of the modified scaffold was established by direct contact cytotoxicity testing and live/dead staining. Thus, the modified C-ECM appears to be a potential biomaterial for cardiac tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Cardiac tissue engineering: from matrix design to the engineering of bionic hearts.

PubMed

Fleischer, Sharon; Feiner, Ron; Dvir, Tal

2017-04-01

The field of cardiac tissue engineering aims at replacing the scar tissue created after a patient has suffered from a myocardial infarction. Various technologies have been developed toward fabricating a functional engineered tissue that closely resembles that of the native heart. While the field continues to grow and techniques for better tissue fabrication continue to emerge, several hurdles still remain to be overcome. In this review we will focus on several key advances and recent technologies developed in the field, including biomimicking the natural extracellular matrix structure and enhancing the transfer of the electrical signal. We will also discuss recent developments in the engineering of bionic cardiac tissues which integrate the fields of tissue engineering and electronics to monitor and control tissue performance.

Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: a requisite for tissue engineering of intervertebral disc.

PubMed

Mwale, F; Roughley, P; Antoniou, J

2004-12-15

Tissue engineering of intervertebral discs (IVD) using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. However, since there is no unique marker characteristic of discs and since hyaline cartilage and immature nucleus pulposus (NP) possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. This study was performed to compare the proteoglycan to collagen ratio (measured as GAG to hydroxyproline ratio) in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals and test the hypothesis that this ratio can be used for in vivo studies to distinguish between a normal NP and hyaline cartilage phenotype. Whole human lumbar spine specimens from fresh cadavers, ranging in age from 12 weeks to 79 years, were used to harvest the IVDs and adjacent endplates. The GAG to hydroxyproline ratio within the NP of young adults is approximately 27:1, whereas the ratio within the hyaline cartilage endplate of the same aged individuals is about 2:1. The production of an extracellular matrix with a high proteoglycan to collagen ratio can be used in vivo to distinguish NP cells from chondrocytes, and could help in identifying a NP-like phenotype in vivo as opposed to a chondrocyte when MSCs are induced to differentiate for tissue engineering of a disc.

Topographical Control of Ocular Cell Types for Tissue Engineering

PubMed Central

McHugh, Kevin J.; Saint-Geniez, Magali; Tao, Sarah L.

2014-01-01

Visual impairment affects over 285 million people worldwide and has a major impact on an individual’s quality of life. Tissue engineering has the potential to increase quality of life for many of these patients by preventing vision loss or restoring vision using cell-based therapies. However, these strategies will require an understanding of the microenvironmental factors that influence cell behavior. The eye is a well-organized organ whose structural complexity is essential for proper function. Interactions between ocular cells and their highly ordered extracellular matrix are necessary for maintaining key tissue properties including corneal transparency and retinal lamination. Therefore, it is not surprising that culturing these cells in vitro on traditional flat substrates result in irregular morphology. Instead, topographically patterned biomaterials better mimic native extracellular matrix and have been shown to elicit in vivo-like morphology and gene expression which is essential for tissue engineering. Herein we review multiple methods for producing well-controlled topography and discuss optimal biomaterial scaffold design for cells of the cornea, retina, and lens. PMID:23744715

Extracellular matrix regenerative graft attenuates the negative impact of polypropylene prolapse mesh on vagina in rhesus macaque.

PubMed

Liang, Rui; Knight, Katrina; Barone, William; Powers, Robert W; Nolfi, Alexis; Palcsey, Stacy; Abramowitch, Steven; Moalli, Pamela A

2017-02-01

The use of wide pore lightweight polypropylene mesh to improve anatomical outcomes in the surgical repair of prolapse has been hampered by mesh complications. One of the prototype prolapse meshes has been found to negatively impact the vagina by inducing a decrease in smooth muscle volume and contractility and the degradation of key structural proteins (collagen and elastin), resulting in vaginal degeneration. Recently, bioscaffolds derived from extracellular matrix have been used to mediate tissue regeneration and have been widely adopted in tissue engineering applications. Here we aimed to: (1) define whether augmentation of a polypropylene prolapse mesh with an extracellular matrix regenerative graft in a primate sacrocolpopexy model could mitigate the degenerative changes; and (2) determine the impact of the extracellular matrix graft on vagina when implanted alone. A polypropylene-extracellular matrix composite graft (n = 9) and a 6-layered extracellular matrix graft alone (n = 8) were implanted in 17 middle-aged parous rhesus macaques via sacrocolpopexy and compared to historical data obtained from sham (n = 12) and the polypropylene mesh (n = 12) implanted by the same method. Vaginal function was measured in passive (ball-burst test) and active (smooth muscle contractility) mechanical tests. Vaginal histomorphologic/biochemical assessments included hematoxylin-eosin and trichrome staining, immunofluorescent labeling of α-smooth muscle actin and apoptotic cells, measurement of total collagen, collagen subtypes (ratio III/I), mature elastin, and sulfated glycosaminoglycans. Statistical analyses included 1-way analysis of variance, Kruskal-Wallis, and appropriate post-hoc tests. The host inflammatory response in the composite mesh-implanted vagina was reduced compared to that following implantation with the polypropylene mesh alone. The increase in apoptotic cells observed with the polypropylene mesh was blunted in the composite (overall P < .001). Passive mechanical testing showed inferior parameters for both polypropylene mesh alone and the composite compared to sham whereas the contractility and thickness of smooth muscle layer in the composite were improved with a value similar to sham, which was distinct from the decreases observed with polypropylene mesh alone. Biochemically, the composite had similar mature elastin content, sulfated glycosaminoglycan content, and collagen subtype III/I ratio but lower total collagen content when compared to sham (P = .011). Multilayered extracellular matrix graft alone showed overall comparable values to sham in aspects of the biomechanical, histomorphologic, or biochemical endpoints of the vagina. The increased collagen subtype ratio III/I with the extracellular matrix graft alone (P = .033 compared to sham) is consistent with an ongoing active remodeling response. Mesh augmentation with a regenerative extracellular matrix graft attenuated the negative impact of polypropylene mesh on the vagina. Application of the extracellular matrix graft alone had no measurable negative effects suggesting that the benefits of this extracellular matrix graft occur when used without a permanent material. Future studies will focus on understanding mechanisms. Copyright © 2016. Published by Elsevier Inc.

Extracellular matrix regenerative graft attenuates the negative impact of polypropylene prolapse mesh on vagina in rhesus macaque

PubMed Central

Liang, Rui; Knight, Katrina; Barone, William; Powers, Robert W.; Nolfi, Alexis; Palcsey, Stacy; Abramowitch, Steven; Moalli, Pamela A.

2016-01-01

BACKGROUND The use of wide pore lightweight polypropylene mesh to improve anatomical outcomes in the surgical repair of prolapse has been hampered by mesh complications. One of the prototype prolapse meshes has been found to negatively impact the vagina by inducing a decrease in smooth muscle volume and contractility and the degradation of key structural proteins (collagen and elastin), resulting in vaginal degeneration. Recently, bioscaffolds derived from extracellular matrix have been used to mediate tissue regeneration and have been widely adopted in tissue engineering applications. OBJECTIVE Here we aimed to: (1) define whether augmentation of a polypropylene prolapse mesh with an extracellular matrix regenerative graft in a primate sacrocolpopexy model could mitigate the degenerative changes; and (2) determine the impact of the extracellular matrix graft on vagina when implanted alone. STUDY DESIGN A polypropylene-extracellular matrix composite graft (n = 9) and a 6-layered extracellular matrix graft alone (n = 8) were implanted in 17 middle-aged parous rhesus macaques via sacrocolpopexy and compared to historical data obtained from sham (n = 12) and the polypropylene mesh (n = 12) implanted by the same method. Vaginal function was measured in passive (ball-burst test) and active (smooth muscle contractility) mechanical tests. Vaginal histomorphologic/ biochemical assessments included hematoxylin-eosin and trichrome staining, immunofluorescent labeling of α-smooth muscle actin and apoptotic cells, measurement of total collagen, collagen subtypes (ratio III/ I), mature elastin, and sulfated glycosaminoglycans. Statistical analyses included 1-way analysis of variance, Kruskal-Wallis, and appropriate posthoc tests. RESULTS The host inflammatory response in the composite mesh-implanted vagina was reduced compared to that following implantation with the polypropylene mesh alone. The increase in apoptotic cells observed with the polypropylene mesh was blunted in the composite (overall P < .001). Passive mechanical testing showed inferior parameters for both polypropylene mesh alone and the composite compared to sham whereas the contractility and thickness of smooth muscle layer in the composite were improved with a value similar to sham, which was distinct from the decreases observed with polypropylene mesh alone. Biochemically, the composite had similar mature elastin content, sulfated glycosaminoglycan content, and collagen subtype III/I ratio but lower total collagen content when compared to sham (P = .011). Multilayered extracellular matrix graft alone showed overall comparable values to sham in aspects of the biomechanical, histomorphologic, or biochemical end-points of the vagina. The increased collagen subtype ratio III/I with the extracellular matrix graft alone (P = .033 compared to sham) is consistent with an ongoing active remodeling response. CONCLUSION Mesh augmentation with a regenerative extracellular matrix graft attenuated the negative impact of polypropylene mesh on the vagina. Application of the extracellular matrix graft alone had no measurable negative effects suggesting that the benefits of this extra-cellular matrix graft occur when used without a permanent material. Future studies will focus on understanding mechanisms. PMID:27615441

Ultrasonic and electromagnetic enhancement of a culture of human SAOS-2 osteoblasts seeded onto a titanium plasma-spray surface.

PubMed

Fassina, Lorenzo; Saino, Enrica; Sbarra, Maria Sonia; Visai, Livia; Cusella De Angelis, Maria Gabriella; Mazzini, Giuliano; Benazzo, Francesco; Magenes, Giovanni

2009-06-01

Several studies suggest that the surface coating of titanium could play an important role in bone tissue engineering. In the present study, we have followed a particular biomimetic strategy where ultrasonically or electromagnetically stimulated SAOS-2 human osteoblasts proliferated and built their extracellular matrix on a titanium plasma-spray surface. In comparison with control conditions, the ultrasonic stimulation (average power, 149 mW; frequency, 1.5 MHz) and the electromagnetic stimulation (magnetic field intensity, 2 mT; frequency, 75 Hz) caused higher cell proliferation, and increased surface coating with decorin, osteocalcin, osteopontin, and type I collagen together with higher incorporation of calcium and phosphorus inside the extracellular matrix. The immunofluorescence related to the preceding bone matrix proteins showed their colocalization in the cell-rich areas. The use of the two physical stimulations aimed at obtaining the coating of the rough titanium plasma-spray surface in terms of cell colonization and deposition of extracellular matrix. The superficially cultured biomaterial could be theoretically used, in clinical applications, as an implant for bone repair.

Lysophosphatidic acid enhances collagen deposition and matrix thickening in engineered tissue.

PubMed

Chabaud, Stéphane; Marcoux, Thomas-Louis; Deschênes-Rompré, Marie-Pier; Rousseau, Alexandre; Morissette, Amélie; Bouhout, Sara; Bernard, Geneviève; Bolduc, Stéphane

2015-11-01

The time needed to produce engineered tissue is critical. A self-assembly approach provided excellent results regarding biological functions and cell differentiation because it closely respected the microenvironment of cells. Nevertheless, the technique was time consuming for producing tissue equivalents with enough extracellular matrix to allow manipulations. Unlike L-arginine supplementation that only increased accumulation of collagen in cell culture supernatant in our model, addition of lysophosphatidic acid, a natural bioactive lipid, did not modify the amount of accumulated collagen in the cell culture supernatant; however, it enhanced the matrix deposition rate without inducing fibroblast hyperproliferation and tissue fibrosis. Copyright © 2013 John Wiley & Sons, Ltd.

Scaffolds for peripheral nerve repair and reconstruction.

PubMed

Yi, Sheng; Xu, Lai; Gu, Xiaosong

2018-06-02

Trauma-associated peripheral nerve defect is a widespread clinical problem. Autologous nerve grafting, the current gold standard technique for the treatment of peripheral nerve injury, has many internal disadvantages. Emerging studies showed that tissue engineered nerve graft is an effective substitute to autologous nerves. Tissue engineered nerve graft is generally composed of neural scaffolds and incorporating cells and molecules. A variety of biomaterials have been used to construct neural scaffolds, the main component of tissue engineered nerve graft. Synthetic polymers (e.g. silicone, polyglycolic acid, and poly(lactic-co-glycolic acid)) and natural materials (e.g. chitosan, silk fibroin, and extracellular matrix components) are commonly used along or together to build neural scaffolds. Many other materials, including the extracellular matrix, glass fabrics, ceramics, and metallic materials, have also been used to construct neural scaffolds. These biomaterials are fabricated to create specific structures and surface features. Seeding supporting cells and/or incorporating neurotrophic factors to neural scaffolds further improve restoration effects. Preliminary studies demonstrate that clinical applications of these neural scaffolds achieve satisfactory functional recovery. Therefore, tissue engineered nerve graft provides a good alternative to autologous nerve graft and represents a promising frontier in neural tissue engineering. Copyright © 2018 Elsevier Inc. All rights reserved.

Unphysiologically high magnesium concentrations support chondrocyte proliferation and redifferentiation.

PubMed

Feyerabend, Frank; Witte, Frank; Kammal, Michael; Willumeit, Regine

2006-12-01

The effect of unphysiologically high extracellular magnesium concentrations on chondrocytes, induced by the supplementation of magnesium sulfate, was studied using a 3-phase tissue engineering model. The experiments showed that chondrocyte proliferation and redifferentiation, on the gene and protein expression level, are enhanced. A negative influence was found during chondrogenesis where an inhibition of extracellular matrix formation was observed. In addition, a direct impact on chondrocyte metabolism, elevated magnesium concentrations also affected growth factor effectiveness by consecutive influences during chondrogenesis. All observations were dosage dependent. The results of this study indicate that magnesium may be a useful tool for cartilage tissue engineering.

Cauda equina-derived extracellular matrix for fabrication of nanostructured hybrid scaffolds applied to neural tissue engineering.

PubMed

Wen, Xiaoxiao; Wang, Yu; Guo, Zhiyuan; Meng, Haoye; Huang, Jingxiang; Zhang, Li; Zhao, Bin; Zhao, Qing; Zheng, Yudong; Peng, Jiang

2015-03-01

Extracellular matrix (ECM) components have become important candidate materials for use as neural scaffolds for neural tissue engineering. In the current study, we prepared cauda equina-derived ECM materials for the production of scaffolds. Natural porcine cauda equina was decellularized using Triton X-100 and sodium deoxycholate, shattered physically, and made into a suspension by differential centrifugation. The decellularization procedure resulted in the removal of >94% of the nuclear material and preserved the extracellular collagen and sulfated glycosaminoglycan. Immunofluorescent staining confirmed the presence of collagen type I, laminin, and fibronectin in the ECM. The cauda equine-derived ECM was blended with poly(l-lactide-co-glycolide) (PLGA) to fabricate nanostructured scaffolds using electrospinning. The incorporation of the ECM increased the hydrophilicity of the scaffolds. Fourier transform infrared spectroscopy and multiphoton-induced autofluorescence images showed the presence of the ECM in the scaffolds. ECM/PLGA scaffolds were beneficial for the survival of Schwann cells compared with scaffolds consisting of PLGA alone, and the aligned fibers could regulate cell morphologic features by modulating cellular orientation. Axons in the dorsal root ganglia explants extended to a greater extent along ECM/PLGA compared with PLGA-alone fibers. The cauda equina ECM might be a promising material for forming scaffolds for use in neural tissue engineering.

Stretching the boundaries of extracellular matrix research.

PubMed

Hynes, Richard O

2014-12-01

Extracellular matrix (ECM) proteins constitute >1% of the proteome and interact with many modifiers and growth factors to affect most aspects of cellular behaviour during development and normal physiology, as well as in diseases such as fibroses, cancer and many genetic disorders. In addition to biochemical signals provided to cells by ECM proteins, important cell–ECM interactions involve bidirectional mechanotransduction influences, which are dependent on the physical structure and organization of the ECM. These are beginning to be understood using twenty-first-century approaches, including biophysics, nanotechnology, biological engineering and modern microscopy. Articles in this issue of Nature Reviews Molecular Cell Biology review progress in our understanding of the ECM.

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix.

PubMed

Piotrowski-Daspit, Alexandra S; Nelson, Celeste M

2016-07-10

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

Programmable biofilm-based materials from engineered curli nanofibres.

PubMed

Nguyen, Peter Q; Botyanszki, Zsofia; Tay, Pei Kun R; Joshi, Neel S

2014-09-17

The significant role of biofilms in pathogenicity has spurred research into preventing their formation and promoting their disruption, resulting in overlooked opportunities to develop biofilms as a synthetic biological platform for self-assembling functional materials. Here we present Biofilm-Integrated Nanofiber Display (BIND) as a strategy for the molecular programming of the bacterial extracellular matrix material by genetically appending peptide domains to the amyloid protein CsgA, the dominant proteinaceous component in Escherichia coli biofilms. These engineered CsgA fusion proteins are successfully secreted and extracellularly self-assemble into amyloid nanofibre networks that retain the functions of the displayed peptide domains. We show the use of BIND to confer diverse artificial functions to the biofilm matrix, such as nanoparticle biotemplating, substrate adhesion, covalent immobilization of proteins or a combination thereof. BIND is a versatile nanobiotechnological platform for developing robust materials with programmable functions, demonstrating the potential of utilizing biofilms as large-scale designable biomaterials.

Rapid Engineering of Three-Dimensional, Multicellular Tissues With Polymeric Scaffolds

NASA Technical Reports Server (NTRS)

Gonda, Steve R.; Jordan, Jacqueline; Fraga, Denise N.

2007-01-01

A process has been developed for the rapid tissue engineering of multicellular-tissue-equivalent assemblies by the controlled enzymatic degradation of polymeric beads in a low-fluid-shear bioreactor. In this process, the porous polymeric beads serve as temporary scaffolds to support the assemblies of cells in a tissuelike 3D configuration during the critical initial growth phases of attachment of anchorage-dependent cells, aggregation of the cells, and formation of a 3D extracellular matrix. Once the cells are assembled into a 3D array and enmeshed in a structural supportive 3D extracellular matrix (ECM), the polymeric scaffolds can be degraded in the low-fluid-shear environment of the NASA-designed bioreactor. The natural 3D tissuelike assembly, devoid of any artificial support structure, is maintained in the low-shear bioreactor environment by the newly formed natural cellular/ECM. The elimination of the artificial scaffold allows normal tissue structure and function.

Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering

PubMed Central

Singelyn, Jennifer M.; DeQuach, Jessica A.; Seif-Naraghi, Sonya B.; Littlefield, Robert B.; Schup-Magoffin, Pamela J.; Christman, Karen L.

2009-01-01

Myocardial tissue lacks the ability to significantly regenerate itself following a myocardial infarction, thus tissue engineering strategies are required for repair. Several injectable materials have been examined for cardiac tissue engineering; however, none have been designed specifically to mimic the myocardium. The goal of this study was to investigate the in vitro properties and in vivo potential of an injectable myocardial matrix designed to mimic the natural myocardial extracellular environment. Porcine myocardial tissue was decellularized and processed to form a myocardial matrix with the ability to gel in vitro at 37°C and in vivo upon injection into rat myocardium. The resulting myocardial matrix maintained a complex composition, including glycosaminoglycan content, and was able to self-assemble to form a nanofibrous structure. Endothelial cells and smooth muscle cells were shown to migrate towards the myocardial matrix both in vitro and in vivo, with a significant increase in arteriole formation at 11 days post-injection. The matrix was also successfully pushed through a clinically used catheter, demonstrating its potential for minimally invasive therapy. Thus, we have demonstrated the initial feasibility and potential of a naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering. PMID:19608268

Development and characterization of decellularized human nasoseptal cartilage matrix for use in tissue engineering.

PubMed

Graham, M Elise; Gratzer, Paul F; Bezuhly, Michael; Hong, Paul

2016-10-01

Reconstruction of cartilage defects in the head and neck can require harvesting of autologous cartilage grafts, which can be associated with donor site morbidity. To overcome this limitation, tissue-engineering approaches may be used to generate cartilage grafts. The objective of this study was to decellularize and characterize human nasoseptal cartilage with the aim of generating a biological scaffold for cartilage tissue engineering. Laboratory study using nasoseptal cartilage. Remnant human nasoseptal cartilage specimens were collected and subjected to a novel decellularization treatment. The decellularization process involved several cycles of enzymatic detergent treatments. For characterization, decellularized and fresh (control) specimens underwent histological, biochemical, and mechanical analyses. Scanning electron microscopy and biocompatibility assay were also performed. The decellularization process had minimal effect on glycosaminoglycan content of the cartilage extracellular matrix. Deoxyribonucleic acid (DNA) analysis revealed the near-complete removal of genomic DNA from decellularized tissues. The effectiveness of the decellularization process was also confirmed on histological and scanning electron microscopic analyses. Mechanical testing results showed that the structural integrity of the decellularized tissue was maintained, and biocompatibility was confirmed. Overall, the current decellularization treatment resulted in significant reduction of genetic/cellular material with preservation of the underlying extracellular matrix structure. This decellularized material may serve as a potential scaffold for cartilage tissue engineering. N/A. Laryngoscope, 126:2226-2231, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Extracellular Matrix-Inspired Growth Factor Delivery Systems for Skin Wound Healing

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

Briquez, Priscilla S.; Hubbell, Jeffrey A.; Martino, Mikaël M.

2015-08-01

Blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF) is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular, the spatial localizationmore » of angiogenic signals in the extracellular matrix (ECM) is crucial to ensure the proper assembly and maturation of new vascular structures. Here, we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis on VEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of ECM to optimize the signaling microenvironment of vascular growth factors.« less

A novel bioactive membrane by cell electrospinning.

PubMed

Chen, Haiping; Liu, Yuanyuan; Hu, Qingxi

2015-11-01

Electrospinning permits fabrication of biodegradable matrices that can resemble the both scale and mechanical behavior of the native extracellular matrix. However, achieving high-cellular density and infiltration of cells within matrices with traditional technique remain challenging and time consuming. The cell electrospinning technique presented in this paper can mitigate the problems associated with these limitations. Cells encapsulated by the material in the cell electrospinning technique survived well and distributed homogenously within the nanofibrous membrane, and their vitality was improved to 133% after being cultured for 28 days. The electrospun nanofibrous membrane has a certain degradation property and favorable cell-membrane interaction that supports the active biocompatibility of the membrane. Its properties are helpful for supporting cell attachment and growth, maintaining phenotypic shape, and secreting an ample amount of extracellular matrix (ECM). This novel membrane may be a potential application within the field of tissue engineering. The ability of cell electrospinning to microintegrate cells into a biodegradable fibrous matrix embodies a novel tissue engineering approach that could be applied to fabricate a high cell density elastic tissue mimetic. Copyright © 2015 Elsevier Inc. All rights reserved.

Extracellular matrix components direct porcine muscle stem cell behavior

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

Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl

2010-02-01

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatinmore » and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.« less

[Self-assembly tissue engineering fibrocartilage model of goat temporomandibular joint disc].

PubMed

Kang, Hong; Li, Zhen-Qiang; Bi, Yan-Da

2011-06-01

To construct self-assembly fibrocartilage model of goat temporomandibular joint disc and observe the biological characteristics of the self-assembled fibrocartilage constructs, further to provide a basis for tissue engineering of the temporomandibular joint disc and other fibrocartilage. Cells from temporomandibular joint discs of goats were harvested and cultured. 5.5 x 10(6) cells were seeded in each agarose well with diameter 5 mm x depth 10 mm, daily replace of medium, cultured for 2 weeks. One day after seeding, goat temporomandibular joint disc cells in agarose wells were gathered and began to self-assemble into a disc-shaped base, then gradually turned into a round shape. When cultured for 2 weeks, hematoxylin-eosin staining was conducted and observed that cells were round and wrapped around by the matrix. Positive Safranin-O/fast green staining for glycosaminoglycans was observed throughout the entire constructs, and picro-sirius red staining was examined and distribution of numerous type I collagen was found. Immunohistochemistry staining demonstrated brown yellow particles in cytoplasm and around extracellular matrix, which showed self-assembly construct can produce type I collagen as native temporomandibular joint disc tissue. Production of extracellular matrix in self-assembly construct as native temporomandibular joint disc tissue indicates that the use of agarose wells to construct engineered temporomandibular joint disc will be possible and practicable.

Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration

PubMed Central

2009-01-01

Neural tissue repair and regeneration strategies have received a great deal of attention because it directly affects the quality of the patient's life. There are many scientific challenges to regenerate nerve while using conventional autologous nerve grafts and from the newly developed therapeutic strategies for the reconstruction of damaged nerves. Recent advancements in nerve regeneration have involved the application of tissue engineering principles and this has evolved a new perspective to neural therapy. The success of neural tissue engineering is mainly based on the regulation of cell behavior and tissue progression through the development of a synthetic scaffold that is analogous to the natural extracellular matrix and can support three-dimensional cell cultures. As the natural extracellular matrix provides an ideal environment for topographical, electrical and chemical cues to the adhesion and proliferation of neural cells, there exists a need to develop a synthetic scaffold that would be biocompatible, immunologically inert, conducting, biodegradable, and infection-resistant biomaterial to support neurite outgrowth. This review outlines the rationale for effective neural tissue engineering through the use of suitable biomaterials and scaffolding techniques for fabrication of a construct that would allow the neurons to adhere, proliferate and eventually form nerves. PMID:19939265

On the role of hydrogel structure and degradation in controlling the transport of cell-secreted matrix molecules for engineered cartilage.

PubMed

Dhote, Valentin; Skaalure, Stacey; Akalp, Umut; Roberts, Justine; Bryant, Stephanie J; Vernerey, Franck J

2013-03-01

Damage to cartilage caused by injury or disease can lead to pain and loss of mobility, diminishing one's quality of life. Because cartilage has a limited capacity for self-repair, tissue engineering strategies, such as cells encapsulated in synthetic hydrogels, are being investigated as a means to restore the damaged cartilage. However, strategies to date are suboptimal in part because designing degradable hydrogels is complicated by structural and temporal complexities of the gel and evolving tissue along multiple length scales. To address this problem, this study proposes a multi-scale mechanical model using a triphasic formulation (solid, fluid, unbound matrix molecules) based on a single chondrocyte releasing extracellular matrix molecules within a degrading hydrogel. This model describes the key players (cells, proteoglycans, collagen) of the biological system within the hydrogel encompassing different length scales. Two mechanisms are included: temporal changes of bulk properties due to hydrogel degradation, and matrix transport. Numerical results demonstrate that the temporal change of bulk properties is a decisive factor in the diffusion of unbound matrix molecules through the hydrogel. Transport of matrix molecules in the hydrogel contributes both to the development of the pericellular matrix and the extracellular matrix and is dependent on the relative size of matrix molecules and the hydrogel mesh. The numerical results also demonstrate that osmotic pressure, which leads to changes in mesh size, is a key parameter for achieving a larger diffusivity for matrix molecules in the hydrogel. The numerical model is confirmed with experimental results of matrix synthesis by chondrocytes in biodegradable poly(ethylene glycol)-based hydrogels. This model may ultimately be used to predict key hydrogel design parameters towards achieving optimal cartilage growth. Copyright © 2012 Elsevier Ltd. All rights reserved.

On the role of hydrogel structure and degradation in controlling the transport of cell-secreted matrix molecules for engineered cartilage

PubMed Central

Dhote, Valentin; Skaalure, Stacey; Akalp, Umut; Roberts, Justine; Bryant, Stephanie J.; Vernerey, Franck J.

2012-01-01

Damage to cartilage caused by injury or disease can lead to pain and loss of mobility, diminishing one’s quality of life. Because cartilage has a limited capacity for self-repair, tissue engineering strategies, such as cells encapsulated in synthetic hydrogels, are being investigated as a means to restore the damaged cartilage. However, strategies to date are suboptimal in part because designing degradable hydrogels is complicated by structural and temporal complexities of the gel and evolving tissue along multiple length scales. To address this problem, this study proposes a multi-scale mechanical model using a triphasic formulation (solid, fluid, unbound matrix molecules) based on a single chondrocyte releasing extracellular matrix molecules within a degrading hydrogel. This model describes the key players (cells, proteoglycans, collagen) of the biological system within the hydrogel encompassing different length scales. Two mechanisms are included: temporal changes of bulk properties due to hydrogel degradation, and matrix transport. Numerical results demonstrate that the temporal change of bulk properties is a decisive factor in the diffusion of unbound matrix molecules through the hydrogel. Transport of matrix molecules in the hydrogel contributes both to the development of the pericellular matrix and the extracellular matrix and is dependent on the relative size of matrix molecules and the hydrogel mesh. The numerical results also demonstrate that osmotic pressure, which leads to changes in mesh size, is a key parameter for achieving a larger diffusivity for matrix molecules in the hydrogel. The numerical model is confirmed with experimental results of matrix synthesis by chondrocytes in biodegradable poly(ethylene glycol)-based hydrogels. This model may ultimately be used to predict key hydrogel design parameters towards achieving optimal cartilage growth. PMID:23276516

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.

Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering.

PubMed

El-Amin, S F; Lu, H H; Khan, Y; Burems, J; Mitchell, J; Tuan, R S; Laurencin, C T

2003-03-01

The nature of the extracellular matrix (ECM) is crucial in regulating cell functions via cell-matrix interactions, cytoskeletal organization, and integrin-mediated signaling. In bone, the ECM is composed of proteins such as collagen (CO), fibronectin (FN), laminin (LM), vitronectin (VN), osteopontin (OP) and osteonectin (ON). For bone tissue engineering, the ECM should also be considered in terms of its function in mediating cell adhesion to biomaterials. This study examined ECM production, cytoskeletal organization, and adhesion of primary human osteoblastic cells on biodegradable matrices applicable for tissue engineering, namely polylactic-co-glycolic acid 50:50 (PLAGA) and polylactic acid (PLA). We hypothesized that the osteocompatible, biodegradable polymer surfaces promote the production of bone-specific ECM proteins in a manner dependent on polymer composition. We first examined whether the PLAGA and PLA matrices could support human osteoblastic cell growth by measuring cell adhesion at 3, 6 and 12h post-plating. Adhesion on PLAGA was consistently higher than on PLA throughout the duration of the experiment, and comparable to tissue culture polystyrene (TCPS). ECM components, including CO, FN, LM, ON, OP and VN, produced on the surface of the polymers were quantified by ELISA and localized by immunofluorescence staining. All of these proteins were present at significantly higher levels on PLAGA compared to PLA or TCPS surfaces. On PLAGA, OP and ON were the most abundant ECM components, followed by CO, FN, VN and LN. Immunofluorescence revealed an extracellular distribution for CO and FN, whereas OP and ON were found both intracellularly as well as extracellularly on the polymer. In addition, the actin cytoskeletal network was more extensive in osteoblasts cultured on PLAGA than on PLA or TCPS. In summary, we found that osteoblasts plated on PLAGA adhered better to the substrate, produced higher levels of ECM molecules, and showed greater cytoskeletal organization than on PLA and TCPS. We propose that this difference in ECM composition is functionally related to the enhanced cell adhesion observed on PLAGA. There is initial evidence that specific composition of the PLAGA polymer favors the ECM. Future studies will seek to optimize ECM production on these matrices for bone tissue engineering applications.

A multi-scale controlled tissue engineering scaffold prepared by 3D printing and NFES technology

NASA Astrophysics Data System (ADS)

Yan, Feifei; Liu, Yuanyuan; Chen, Haiping; Zhang, Fuhua; Zheng, Lulu; Hu, Qingxi

2014-03-01

The current focus in the field of life science is the use of tissue engineering scaffolds to repair human organs, which has shown great potential in clinical applications. Extracellular matrix morphology and the performance and internal structure of natural organs are required to meet certain requirements. Therefore, integrating multiple processes can effectively overcome the limitations of the individual processes and can take into account the needs of scaffolds for the material, structure, mechanical properties and many other aspects. This study combined the biological 3D printing technology and the near-field electro-spinning (NFES) process to prepare a multi-scale controlled tissue engineering scaffold. While using 3D printing technology to directly prepare the macro-scaffold, the compositing NFES process to build tissue micro-morphology ultimately formed a tissue engineering scaffold which has the specific extracellular matrix structure. This scaffold not only takes into account the material, structure, performance and many other requirements, but also focuses on resolving the controllability problems in macro- and micro-forming which further aim to induce cell directed differentiation, reproduction and, ultimately, the formation of target tissue organs. It has in-depth immeasurable significance to build ideal scaffolds and further promote the application of tissue engineering.

Tissue Culture in Microgravity

NASA Technical Reports Server (NTRS)

Pellis, Neal R.; Duray, Paul H.; Hatfill, Steven J.

1997-01-01

Attempts to simulate normal tissue micro-environments in vitro have been thwarted by the complexity and plasticity of the extracellular matrix, which is important in regulating cytoskeletal and nuclear matrix proteins. Gravity is one of the problems, tending to separate components that should be kept together. For space shuttle experiments, NASA engineers devised a double-walled rotating bioreactor, which is proving to be a useful tissue culture device on earth as well as in space.

A novel culture device for the evaluation of three-dimensional extracellular matrix materials.

PubMed

Akhyari, Payam; Ziegler, Heiko; Gwanmesia, Patricia; Barth, Mareike; Schilp, Soeren; Huelsmann, Joern; Hoffmann, Stefanie; Bosch, Julia; Kögler, Gesine; Lichtenberg, Artur

2014-09-01

Cell-matrix interactions in a three-dimensional (3D) extracellular matrix (ECM) are of fundamental importance in living tissue, and their in vitro reconstruction in bioartificial structures represents a core target of contemporary tissue engineering concepts. For a detailed analysis of cell-matrix interaction under highly controlled conditions, we developed a novel ECM evaluation culture device (EECD) that allows for a precisely defined surface-seeding of 3D ECM scaffolds, irrespective of their natural geometry. The effectiveness of EECD was evaluated in the context of heart valve tissue engineering. Detergent decellularized pulmonary cusps were mounted in EECD and seeded with endothelial cells (ECs) to study EC adhesion, morphology and function on a 3D ECM after 3, 24, 48 and 96 h. Standard EC monolayers served as controls. Exclusive top-surface-seeding of 3D ECM by viable ECs was demonstrated by laser scanning microscopy (LSM), resulting in a confluent re-endothelialization of the ECM after 96 h. Cell viability and protein expression, as demonstrated by MTS assay and western blot analysis (endothelial nitric oxide synthase, von Willebrand factor), were preserved at maintained levels over time. In conclusion, EECD proves as a highly effective system for a controlled repopulation and in vitro analysis of cell-ECM interactions in 3D ECM. Copyright © 2012 John Wiley & Sons, Ltd.

Electrospun conductive nanofibrous scaffolds for engineering cardiac tissue and 3D bioactuators.

PubMed

Wang, Ling; Wu, Yaobin; Hu, Tianli; Guo, Baolin; Ma, Peter X

2017-09-01

Mimicking the nanofibrous structure similar to extracellular matrix and conductivity for electrical propagation of native myocardium would be highly beneficial for cardiac tissue engineering and cardiomyocytes-based bioactuators. Herein, we developed conductive nanofibrous sheets with electrical conductivity and nanofibrous structure composed of poly(l-lactic acid) (PLA) blending with polyaniline (PANI) for cardiac tissue engineering and cardiomyocytes-based 3D bioactuators. Incorporating of varying contents of PANI from 0wt% to 3wt% into the PLA polymer, the electrospun nanofibrous sheets showed enhanced conductivity while maintaining the same fiber diameter. These PLA/PANI conductive nanofibrous sheets exhibited good cell viability and promoting effect on differentiation of H9c2 cardiomyoblasts in terms of maturation index and fusion index. Moreover, PLA/PANI nanofibrous sheets enhanced the cell-cell interaction, maturation and spontaneous beating of primary cardiomyocytes. Furthermore, the cardiomyocytes-laden PLA/PANI conductive nanofibrous sheets can form 3D bioactuators with tubular and folding shapes, and spontaneously beat with much higher frequency and displacement than that on cardiomyocytes-laden PLA nanofibrous sheets. Therefore, these PLA/PANI conductive nanofibrous sheets with conductivity and extracellular matrix like nanostructure demonstrated promising potential in cardiac tissue engineering and cardiomyocytes-based 3D bioactuators. Cardiomyocytes-based bioactuators have been paid more attention due to their spontaneous motion by integrating cardiomyocytes into polymer structures, but developing suitable scaffolds for bioactuators remains challenging. Electrospun nanofibrous scaffolds have been widely used in cardiac tissue engineering because they can mimic the extracellular matrix of myocardium. Developing conductive nanofibrous scaffolds by electrospinning would be beneficial for cardiomyocytes-based bioactuators, but such scaffolds have been rarely reported. This work presented a conductive nanofibrous sheet based on polylactide and polyaniline via electrospinning with tunable conductivity. These conductive nanofibrous sheets performed the ability to enhance cardiomyocytes maturation and spontaneous beating, and further formed cardiomyocytes-based 3D bioactuators with tubular and folding shapes, which indicated their great potential in cardiac tissue engineering and bioactuators applications. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

In vitro synthesis of tensioned synoviocyte bioscaffolds for meniscal fibrocartilage tissue engineering.

PubMed

Warnock, Jennifer J; Baker, Lindsay; Ballard, George A; Ott, Jesse

2013-12-03

Meniscal injury is a common cause of lameness in the dog. Tissue engineered bioscaffolds may be a treatment option for meniscal incompetency, and ideally would possess meniscus- like extracellular matrix (ECM) and withstand meniscal tensile hoop strains. Synovium may be a useful cell source for meniscal tissue engineering because of its natural role in meniscal deficiency and its in vitro chondrogenic potential. The objective of this study is to compare meniscal -like extracellular matrix content of hyperconfluent synoviocyte cell sheets ("HCS") and hyperconfluent synoviocyte sheets which have been tensioned over wire hoops (tensioned synoviocyte bioscaffolds, "TSB") and cultured for 1 month. Long term culture with tension resulted in higher GAG concentration, higher chondrogenic index, higher collagen concentration, and type II collagen immunoreactivity in TSB versus HCS. Both HCS and TSB were immunoreactive for type I collagen, however, HCS had mild, patchy intracellular immunoreactivity while TSB had diffuse moderate immunoreactivity over the entire bisocaffold. The tissue architecture was markedly different between TSB and HCS, with TSB containing collagen organized in bands and sheets. Both HCS and TSB expressed alpha smooth muscle actin and displayed active contractile behavior. Double stranded DNA content was not different between TSB and HCS, while cell viability decreased in TSB. Long term culture of synoviocytes with tension improved meniscal- like extra cellular matrix components, specifically, the total collagen content, including type I and II collagen, and increased GAG content relative to HCS. Future research is warranted to investigate the potential of TSB for meniscal tissue engineering.

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.

Engineering the extracellular matrix for clinical applications: endoderm, mesoderm, and ectoderm.

PubMed

Williams, Miguel L; Bhatia, Sujata K

2014-03-01

Tissue engineering is rapidly progressing from a research-based discipline to clinical applications. Emerging technologies could be utilized to develop therapeutics for a wide range of diseases, but many are contingent on a cell scaffold that can produce proper tissue ultrastructure. The extracellular matrix, which a cell scaffold simulates, is not merely a foundation for tissue growth but a dynamic participant in cellular crosstalk and organ homeostasis. Cells change their growth rates, recruitment, and differentiation in response to the composition, modulus, and patterning of the substrate on which they reside. Cell scaffolds can regulate these factors through precision design, functionalization, and application. The ideal therapy would utilize highly specialized cell scaffolds to best mimic the tissue of interest. This paper discusses advantages and challenges of optimized cell scaffold design in the endoderm, mesoderm, and ectoderm for clinical applications in tracheal transplant, cardiac regeneration, and skin grafts, respectively. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Colonization of bone matrices by cellular components

NASA Astrophysics Data System (ADS)

Shchelkunova, E. I.; Voropaeva, A. A.; Korel, A. V.; Mayer, D. A.; Podorognaya, V. T.; Kirilova, I. A.

2017-09-01

Practical surgery, traumatology, orthopedics, and oncology require bioengineered constructs suitable for replacement of large-area bone defects. Only rigid/elastic matrix containing recipient's bone cells capable of mitosis, differentiation, and synthesizing extracellular matrix that supports cell viability can comply with these requirements. Therefore, the development of the techniques to produce structural and functional substitutes, whose three-dimensional structure corresponds to the recipient's damaged tissues, is the main objective of tissue engineering. This is achieved by developing tissue-engineering constructs represented by cells placed on the matrices. Low effectiveness of carrier matrix colonization with cells and their uneven distribution is one of the major problems in cell culture on various matrixes. In vitro studies of the interactions between cells and material, as well as the development of new techniques for scaffold colonization by cellular components are required to solve this problem.

Receptor control in mesenchymal stem cell engineering

NASA Astrophysics Data System (ADS)

Dalby, Matthew J.; García, Andrés J.; Salmeron-Sanchez, Manuel

2018-03-01

Materials science offers a powerful tool to control mesenchymal stem cell (MSC) growth and differentiation into functional phenotypes. A complex interplay between the extracellular matrix and growth factors guides MSC phenotypes in vivo. In this Review, we discuss materials-based bioengineering approaches to direct MSC fate in vitro and in vivo, mimicking cell-matrix-growth factor crosstalk. We first scrutinize MSC-matrix interactions and how the properties of a material can be tailored to support MSC growth and differentiation in vitro, with an emphasis on MSC self-renewal mechanisms. We then highlight important growth factor signalling pathways and investigate various materials-based strategies for growth factor presentation and delivery. Integrin-growth factor crosstalk in the context of MSC engineering is introduced, and bioinspired material designs with the potential to control the MSC niche phenotype are considered. Finally, we summarize important milestones on the road to MSC engineering for regenerative medicine.

Capturing extracellular matrix properties in vitro: Microengineering materials to decipher cell and tissue level processes.

PubMed

Abdeen, Amr A; Lee, Junmin; Kilian, Kristopher A

2016-05-01

Rapid advances in biology have led to the establishment of new fields with tremendous translational potential including regenerative medicine and immunoengineering. One commonality to these fields is the need to extract cells for manipulation in vitro; however, results obtained in laboratory cell culture will often differ widely from observations made in vivo. To more closely emulate native cell biology in the laboratory, designer engineered environments have proved a successful methodology to decipher the properties of the extracellular matrix that govern cellular decision making. Here, we present an overview of matrix properties that affect cell behavior, strategies for recapitulating important parameters in vitro, and examples of how these properties can affect cell and tissue level processes, with emphasis on leveraging these tools for immunoengineering. © 2016 by the Society for Experimental Biology and Medicine.

Matrix-directed differentiation of human adipose-derived mesenchymal stem cells to dermal-like fibroblasts that produce extracellular matrix.

PubMed

Sivan, Unnikrishnan; Jayakumar, K; Krishnan, Lissy K

2016-10-01

Commercially available skin substitutes lack essential non-immune cells for adequate tissue regeneration of non-healing wounds. A tissue-engineered, patient-specific, dermal substitute could be an attractive option for regenerating chronic wounds, for which adipose-derived mesenchymal stem cells (ADMSCs) could become an autologous source. However, ADMSCs are multipotent in nature and may differentiate into adipocytes, osteocytes and chondrocytes in vitro, and may develop into undesirable tissues upon transplantation. Therefore, ADMSCs committed to the fibroblast lineage could be a better option for in vitro or in vivo skin tissue engineering. The objective of this study was to standardize in vitro culture conditions for ADMSCs differentiation into dermal-like fibroblasts which can synthesize extracellular matrix (ECM) proteins. Biomimetic matrix composite, deposited on tissue culture polystyrene (TCPS), and differentiation medium (DM), supplemented with fibroblast-conditioned medium and growth factors, were used as a fibroblast-specific niche (FSN) for cell culture. For controls, ADMSCs were cultured on bare TCPS with either DM or basal medium (BM). Culture of ADMSCs on FSN upregulated the expression of differentiation markers such as fibroblast-specific protein-1 (FSP-1) and a panel of ECM molecules specific to the dermis, such as fibrillin-1, collagen I, collagen IV and elastin. Immunostaining showed the deposition of dermal-specific ECM, which was significantly higher in FSN compared to control. Fibroblasts derived from ADMSCs can synthesize elastin, which is an added advantage for successful skin tissue engineering as compared to fibroblasts from skin biopsy. To obtain rapid differentiation of ADMSCs to dermal-like fibroblasts for regenerative medicine, a matrix-directed differentiation strategy may be employed. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

Biologically active chitosan systems for tissue engineering and regenerative medicine.

PubMed

Jiang, Tao; Kumbar, Sangamesh G; Nair, Lakshmi S; Laurencin, Cato T

2008-01-01

Biodegradable polymeric scaffolds are widely used as a temporary extracellular matrix in tissue engineering and regenerative medicine. By physical adsorption of biomolecules on scaffold surface, physical entrapment of biomolecules in polymer microspheres or hydrogels, and chemical immobilization of oligopeptides or proteins on biomaterials, biologically active biomaterials and scaffolds can be derived. These bioactive systems show great potential in tissue engineering in rendering bioactivity and/or specificity to scaffolds. This review highlights some of the biologically active chitosan systems for tissue engineering application and the associated strategies to develop such bioactive chitosan systems.

Preparation and Characterization of a Chitosan/Gelatin/Extracellular Matrix Scaffold and Its Application in Tissue Engineering.

PubMed

Wang, Xiaoyan; Yu, Tailong; Chen, Guanghua; Zou, Jilong; Li, Jianzhong; Yan, Jinglong

2017-03-01

Previous studies have demonstrated that extracellular matrix (ECM) can be used in tissue engineering due to its bioactivity. However, adipose-derived ECM (A-dECM) has never been applied in bone tissue engineering, and it is unknown whether it would be beneficial to the growth of bone marrow mesenchymal stem cells (BMSCs). In this study, we produced chitosan/gelatin/A-dECM (C/G/A-dECM) scaffolds via lyophilization and crosslinking; chitosan/gelatin (C/G) scaffolds were used as controls. For the C/G/A-dECM scaffolds, the average pore size was 285.93 ± 85.39 μm; the average porosity was 90.62 ± 3.65%; the average compressive modulus was 0.87 ± 0.05 kPa; and the average water uptake ratio was 13.73 ± 1.16. In vitro, A-dECM scaffolds could promote the attachment and proliferation of BMSCs. In the same osteogenic-inducing reagent, better osteogenic differentiation could be observed for the C/G/A-dECM scaffolds than for the C/G scaffolds. Thus, we conclude that A-dECM is a promising material and that C/G/A-dECM scaffolds are a candidate for bone tissue engineering.

Targeting extracellular matrix remodeling in disease: Could resveratrol be a potential candidate?

PubMed

Agarwal, Renu; Agarwal, Puneet

2017-02-01

Disturbances of extracellular matrix homeostasis are associated with a number of pathological conditions. The ability of extracellular matrix to provide contextual information and hence control the individual or collective cellular behavior is increasingly being recognized. Hence, newer therapeutic approaches targeting extracellular matrix remodeling are widely investigated. We reviewed the current literature showing the effects of resveratrol on various aspects of extracellular matrix remodeling. This review presents a summary of the effects of resveratrol on extracellular matrix deposition and breakdown. Mechanisms of action of resveratrol in extracellular matrix deposition involving growth factors and their signaling pathways are discussed. Involvement of phosphoinositol-3-kinase/Akt and mitogen-activated protein kinase pathways and role of transcription factors and sirtuins on the effects of resveratrol on extracellular matrix homeostasis are summarized. It is evident from the literature presented in this review that resveratrol has significant effects on both the synthesis and breakdown of extracellular matrix. The major molecular targets of the action of resveratrol are growth factors and their signaling pathways, phosphoinositol-3-kinase/Akt and mitogen-activated protein kinase pathways, transcription factors, and SIRT-1. The effects of resveratrol on extracellular matrix and the molecular targets appear to be related to experimental models, experimental environment as well as the doses.

Targeting extracellular matrix remodeling in disease: Could resveratrol be a potential candidate?

PubMed Central

Agarwal, Puneet

2016-01-01

Disturbances of extracellular matrix homeostasis are associated with a number of pathological conditions. The ability of extracellular matrix to provide contextual information and hence control the individual or collective cellular behavior is increasingly being recognized. Hence, newer therapeutic approaches targeting extracellular matrix remodeling are widely investigated. We reviewed the current literature showing the effects of resveratrol on various aspects of extracellular matrix remodeling. This review presents a summary of the effects of resveratrol on extracellular matrix deposition and breakdown. Mechanisms of action of resveratrol in extracellular matrix deposition involving growth factors and their signaling pathways are discussed. Involvement of phosphoinositol-3-kinase/Akt and mitogen-activated protein kinase pathways and role of transcription factors and sirtuins on the effects of resveratrol on extracellular matrix homeostasis are summarized. It is evident from the literature presented in this review that resveratrol has significant effects on both the synthesis and breakdown of extracellular matrix. The major molecular targets of the action of resveratrol are growth factors and their signaling pathways, phosphoinositol-3-kinase/Akt and mitogen-activated protein kinase pathways, transcription factors, and SIRT-1. The effects of resveratrol on extracellular matrix and the molecular targets appear to be related to experimental models, experimental environment as well as the doses. PMID:27798117

Regional variations in the distribution and colocalization of extracellular matrix proteins in the juvenile bovine meniscus

PubMed Central

Vanderploeg, Eric J; Wilson, Christopher G; Imler, Stacy M; Ling, Carrie Hang-Yin; Levenston, Marc E

2012-01-01

A deeper understanding of the composition and organization of extracellular matrix molecules in native, healthy meniscus tissue is required to fully appreciate the degeneration that occurs in joint disease and the intricate environment in which an engineered meniscal graft would need to function. In this study, regional variations in the tissue-level and pericellular distributions of collagen types I, II and VI and the proteoglycans aggrecan, biglycan and decorin were examined in the juvenile bovine meniscus. The collagen networks were extensively, but not completely, colocalized, with tissue-level organization that varied with radial position across the meniscus. Type VI collagen exhibited close association with large bundles composed of type I and II collagen and, in contrast to type I and II collagen, was further concentrated in the pericellular matrix. Aggrecan was detected throughout the inner region of the meniscus but was restricted to the pericellular matrix and sheaths of collagen bundles in the middle and outer regions. The small proteoglycans biglycan and decorin exhibited regional variations in staining intensity but were consistently localized in the intra- and/or peri-cellular compartments. These results provide insight into the complex hierarchy of extracellular matrix organization in the meniscus and provide a framework for better understanding meniscal degeneration and disease progression and evaluating potential repair and regeneration strategies. PMID:22703476

Extracellular-Matrix-Based and Arg-Gly-Asp–Modified Photopolymerizing Hydrogels for Cartilage Tissue Engineering

PubMed Central

Kim, Hwan D.; Heo, Jiseung; Hwang, Yongsung; Kwak, Seon-Yeong; Park, Ok Kyu; Kim, Hyunbum; Varghese, Shyni

2015-01-01

Articular cartilage damage is a persistent and increasing problem with the aging population. Strategies to achieve complete repair or functional restoration remain a challenge. Photopolymerizing-based hydrogels have long received an attention in the cartilage tissue engineering, due to their unique bioactivities, flexible method of synthesis, range of constituents, and desirable physical characteristics. In the present study, we have introduced unique bioactivity within the photopolymerizing-based hydrogels by copolymerizing polyethylene glycol (PEG) macromers with methacrylated extracellular matrix (ECM) molecules (hyaluronic acid and chondroitin sulfate [CS]) and integrin binding peptides (RGD peptide). Results indicate that cellular morphology, as observed by the actin cytoskeleton structures, was strongly dependent on the type of ECM component as well as the presence of integrin binding moieties. Further, CS-based hydrogel with integrin binding RGD moieties increased the lubricin (or known as superficial zone protein [SZP]) gene expression of the encapsulated chondrocytes. Additionally, CS-based hydrogel displayed cell-responsive degradation and resulted in increased DNA, GAG, and collagen accumulation compared with other hydrogels. This study demonstrates that integrin-mediated interactions within CS microenvironment provide an optimal hydrogel scaffold for cartilage tissue engineering application. PMID:25266634

Regulation of skeletal myotube formation and alignment by nanotopographically controlled cell-secreted extracellular matrix.

PubMed

Jiao, Alex; Moerk, Charles T; Penland, Nisa; Perla, Mikael; Kim, Jinsung; Smith, Alec S T; Murry, Charles E; Kim, Deok-Ho

2018-06-01

Skeletal muscle has a well-organized tissue structure comprised of aligned myofibers and an encasing extracellular matrix (ECM) sheath or lamina, within which reside satellite cells. We hypothesize that the organization of skeletal muscle tissues in culture can affect both the structure of the deposited ECM and the differentiation potential of developing myotubes. Furthermore, we posit that cellular and ECM cues can be a strong determinant of myoblast fusion and morphology in 3D tissue culture environments. To test these, we utilized a thermoresponsive nanofabricated substratum to engineer anisotropic sheets of myoblasts which could then be transferred and stacked into multilayered tissues. Within such engineered tissues, we found that myoblasts rapidly sense topography and deposit structurally organized ECM proteins. Furthermore, the initial tissue structure was found to exert significant control over myoblast fusion and eventual myotube organization. These results highlight the importance of ECM structure on myoblast fusion and organization, and provide insights into substrate-mediated control of myotube formation in the development of novel, more effective, engineered skeletal muscle tissues. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1543-1551, 2018. © 2018 Wiley Periodicals, Inc.

Laminated electrospun nHA/PHB-composite scaffolds mimicking bone extracellular matrix for bone tissue engineering.

PubMed

Chen, Zhuoyue; Song, Yue; Zhang, Jing; Liu, Wei; Cui, Jihong; Li, Hongmin; Chen, Fulin

2017-03-01

Electrospinning is an effective means to generate nano- to micro-scale polymer fibers resembling native extracellular matrix for tissue engineering. However, a major problem of electrospun materials is that limited pore size and porosity may prevent adequate cellular infiltration and tissue ingrowth. In this study, we first prepared thin layers of hydroxyapatite nanoparticle (nHA)/poly-hydroxybutyrate (PHB) via electrospinning. We then laminated the nHA/PHB thin layers to obtain a scaffold for cell seeding and bone tissue engineering. The results demonstrated that the laminated scaffold possessed optimized cell-loading capacity. Bone marrow mesenchymal stem cells (MSCs) exhibited better adherence, proliferation and osteogenic phenotypes on nHA/PHB scaffolds than on PHB scaffolds. Thereafter, we seeded MSCs onto nHA/PHB scaffolds to fabricate bone grafts. Histological observation showed osteoid tissue formation throughout the scaffold, with most of the scaffold absorbed in the specimens 2months after implantation, and blood vessels ingrowth into the graft could be observed in the graft. We concluded that electrospun and laminated nanoscaled biocomposite scaffolds hold great therapeutic potential for bone regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

Small intestinal submucosa extracellular matrix (CorMatrix®) in cardiovascular surgery: a systematic review

PubMed Central

Mosala Nezhad, Zahra; Poncelet, Alain; de Kerchove, Laurent; Gianello, Pierre; Fervaille, Caroline; El Khoury, Gebrine

2016-01-01

Extracellular matrix (ECM) derived from small intestinal submucosa (SIS) is widely used in clinical applications as a scaffold for tissue repair. Recently, CorMatrix® porcine SIS-ECM (CorMatrix Cardiovascular, Inc., Roswell, GA, USA) has gained popularity for ‘next-generation’ cardiovascular tissue engineering due to its ease of use, remodelling properties, lack of immunogenicity, absorbability and potential to promote native tissue growth. Here, we provide an overview of the biology of porcine SIS-ECM and systematically review the preclinical and clinical literature on its use in cardiovascular surgery. CorMatrix® has been used in a variety of cardiovascular surgical applications, and since it is the most widely used SIS-ECM, this material is the focus of this review. Since CorMatrix® is a relatively new product for cardiovascular surgery, some clinical and preclinical studies published lack systematic reporting of functional and pathological findings in sufficient numbers of subjects. There are also emerging reports to suggest that, contrary to expectations, an undesirable inflammatory response may occur in CorMatrix® implants in humans and longer-term outcomes at particular sites, such as the heart valves, may be suboptimal. Large-scale clinical studies are needed driven by robust protocols that aim to quantify the pathological process of tissue repair. PMID:26912574

Knee Ligament Injury and the Clinical Application of Tissue Engineering Techniques: A Systematic Review.

PubMed

Riley, Thomas C; Mafi, Reza; Mafi, Pouya; Khan, Wasim S

2018-02-23

The incidence of knee ligament injury is increasing and represents a significant cost to healthcare providers. Current interventions include tissue grafts, suture repair and non-surgical management. These techniques have demonstrated good patient outcomes but have been associated graft rejection, infection, long term immobilization and reduced joint function. The limitations of traditional management strategies have prompted research into tissue engineering of knee ligaments. This paper aims to evaluate whether tissue engineering of knee ligaments offers a viable alternative in the clinical management of knee ligament injuries. A search of existing literature was performed using OVID Medline, Embase, AMED, PubMed and Google Scholar, and a manual review of citations identified within these papers. Silk, polymer and extracellular matrix based scaffolds can all improve graft healing and collagen production. Fibroblasts and stem cells demonstrate compatibility with scaffolds, and have been shown to increase organized collagen production. These effects can be augmented using growth factors and extracellular matrix derivatives. Animal studies have shown tissue engineered ligaments can provide the biomechanical characteristics required for effective treatment of knee ligament injuries. There is a growing clinical demand for a tissue engineered alternative to traditional management strategies. Currently, there is limited consensus regarding material selection for use in tissue engineered ligaments. Further research is required to optimize tissue engineered ligament production before clinical application. Controlled clinical trials comparing the use of tissue engineered ligaments and traditional management in patients with knee ligament injury could determine whether they can provide a cost-effective alternative. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Continuum-level modelling of cellular adhesion and matrix production in aggregates.

PubMed

Geris, Liesbet; Ashbourn, Joanna M A; Clarke, Tim

2011-05-01

Key regulators in tissue-engineering processes such as cell culture and cellular organisation are the cell-cell and cell-matrix interactions. As mathematical models are increasingly applied to investigate biological phenomena in the biomedical field, it is important, for some applications, that these models incorporate an adequate description of cell adhesion. This study describes the development of a continuum model that represents a cell-in-gel culture system used in bone-tissue engineering, namely that of a cell aggregate embedded in a hydrogel. Cell adhesion is modelled through the use of non-local (integral) terms in the partial differential equations. The simulation results demonstrate that the effects of cell-cell and cell-matrix adhesion are particularly important for the survival and growth of the cell population and the production of extracellular matrix by the cells, concurring with experimental observations in the literature.

Alignment hierarchies: engineering architecture from the nanometre to the micrometre scale.

PubMed

Kureshi, Alvena; Cheema, Umber; Alekseeva, Tijna; Cambrey, Alison; Brown, Robert

2010-12-06

Natural tissues are built of metabolites, soluble proteins and solid extracellular matrix components (largely fibrils) together with cells. These are configured in highly organized hierarchies of structure across length scales from nanometre to millimetre, with alignments that are dominated by anisotropies in their fibrillar matrix. If we are to successfully engineer tissues, these hierarchies need to be mimicked with an understanding of the interaction between them. In particular, the movement of different elements of the tissue (e.g. molecules, cells and bulk fluids) is controlled by matrix structures at distinct scales. We present three novel systems to introduce alignment of collagen fibrils, cells and growth factor gradients within a three-dimensional collagen scaffold using fluid flow, embossing and layering of construct. Importantly, these can be seen as different parts of the same hierarchy of three-dimensional structure, as they are all formed into dense collagen gels. Fluid flow aligns collagen fibrils at the nanoscale, embossed topographical features provide alignment cues at the microscale and introducing layered configuration to three-dimensional collagen scaffolds provides microscale- and mesoscale-aligned pathways for protein factor delivery as well as barriers to confine protein diffusion to specific spatial directions. These seemingly separate methods can be employed to increase complexity of simple extracellular matrix scaffolds, providing insight into new approaches to directly fabricate complex physical and chemical cues at different hierarchical scales, similar to those in natural tissues.

Reconstruction of structure and function in tissue engineering of solid organs: Toward simulation of natural development based on decellularization.

PubMed

Zheng, Chen-Xi; Sui, Bing-Dong; Hu, Cheng-Hu; Qiu, Xin-Yu; Zhao, Pan; Jin, Yan

2018-04-27

Failure of solid organs, such as the heart, liver, and kidney, remains a major cause of the world's mortality due to critical shortage of donor organs. Tissue engineering, which uses elements including cells, scaffolds, and growth factors to fabricate functional organs in vitro, is a promising strategy to mitigate the scarcity of transplantable organs. Within recent years, different construction strategies that guide the combination of tissue engineering elements have been applied in solid organ tissue engineering and have achieved much progress. Most attractively, construction strategy based on whole-organ decellularization has become a popular and promising approach, because the overall structure of extracellular matrix can be well preserved. However, despite the preservation of whole structure, the current constructs derived from decellularization-based strategy still perform partial functions of solid organs, due to several challenges, including preservation of functional extracellular matrix structure, implementation of functional recellularization, formation of functional vascular network, and realization of long-term functional integration. This review overviews the status quo of solid organ tissue engineering, including both advances and challenges. We have also put forward a few techniques with potential to solve the challenges, mainly focusing on decellularization-based construction strategy. We propose that the primary concept for constructing tissue-engineered solid organs is fabricating functional organs based on intact structure via simulating the natural development and regeneration processes. Copyright © 2018 John Wiley & Sons, Ltd.

Decellularized skin/adipose tissue flap matrix for engineering vascularized composite soft tissue flaps.

PubMed

Zhang, Qixu; Johnson, Joshua A; Dunne, Lina W; Chen, Youbai; Iyyanki, Tejaswi; Wu, Yewen; Chang, Edward I; Branch-Brooks, Cynthia D; Robb, Geoffrey L; Butler, Charles E

2016-04-15

Using a perfusion decellularization protocol, we developed a decellularized skin/adipose tissue flap (DSAF) comprising extracellular matrix (ECM) and intact vasculature. Our DSAF had a dominant vascular pedicle, microcirculatory vascularity, and a sensory nerve network and retained three-dimensional (3D) nanofibrous structures well. DSAF, which was composed of collagen and laminin with well-preserved growth factors (e.g., vascular endothelial growth factor, basic fibroblast growth factor), was successfully repopulated with human adipose-derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs), which integrated with DSAF and formed 3D aggregates and vessel-like structures in vitro. We used microsurgery techniques to re-anastomose the recellularized DSAF into nude rats. In vivo, the engineered flap construct underwent neovascularization and constructive remodeling, which was characterized by the predominant infiltration of M2 macrophages and significant adipose tissue formation at 3months postoperatively. Our results indicate that DSAF co-cultured with hASCs and HUVECs is a promising platform for vascularized soft tissue flap engineering. This platform is not limited by the flap size, as the entire construct can be immediately perfused by the recellularized vascular network following simple re-integration into the host using conventional microsurgical techniques. Significant soft tissue loss resulting from traumatic injury or tumor resection often requires surgical reconstruction using autologous soft tissue flaps. However, the limited availability of qualitative autologous flaps as well as the donor site morbidity significantly limits this approach. Engineered soft tissue flap grafts may offer a clinically relevant alternative to the autologous flap tissue. In this study, we engineered vascularized soft tissue free flap by using skin/adipose flap extracellular matrix scaffold (DSAF) in combination with multiple types of human cells. Following vascular reanastomosis in the recipient site, the engineered products successful regenerated large-scale fat tissue in vivo. This approach may provide a translatable platform for composite soft tissue free flap engineering for microsurgical reconstruction. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Cellular and nerve regeneration within a biosynthetic extracellular matrix for corneal transplantation

NASA Astrophysics Data System (ADS)

Li, Fengfu; Carlsson, David; Lohmann, Chris; Suuronen, Erik; Vascotto, Sandy; Kobuch, Karin; Sheardown, Heather; Munger, Rejean; Nakamura, Masatsugu; Griffith, May

2003-12-01

Our objective was to determine whether key properties of extracellular matrix (ECM) macromolecules can be replicated within tissue-engineered biosynthetic matrices to influence cellular properties and behavior. To achieve this, hydrated collagen and N-isopropylacrylamide copolymer-based ECMs were fabricated and tested on a corneal model. The structural and immunological simplicity of the cornea and importance of its extensive innervation for optimal functioning makes it an ideal test model. In addition, corneal failure is a clinically significant problem. Matrices were therefore designed to have the optical clarity and the proper dimensions, curvature, and biomechanical properties for use as corneal tissue replacements in transplantation. In vitro studies demonstrated that grafting of the laminin adhesion pentapeptide motif, YIGSR, to the hydrogels promoted epithelial stratification and neurite in-growth. Implants into pigs' corneas demonstrated successful in vivo regeneration of host corneal epithelium, stroma, and nerves. In particular, functional nerves were observed to rapidly regenerate in implants. By comparison, nerve regeneration in allograft controls was too slow to be observed during the experimental period, consistent with the behavior of human cornea transplants. Other corneal substitutes have been produced and tested, but here we report an implantable matrix that performs as a physiologically functional tissue substitute and not simply as a prosthetic device. These biosynthetic ECM replacements should have applicability to many areas of tissue engineering and regenerative medicine, especially where nerve function is required. regenerative medicine | tissue engineering | cornea | implantation | innervation

Modulating bacterial and gut mucosal interactions with engineered biofilm matrix proteins.

PubMed

Duraj-Thatte, Anna M; Praveschotinunt, Pichet; Nash, Trevor R; Ward, Frederick R; Joshi, Neel S

2018-02-22

Extracellular appendages play a significant role in mediating communication between bacteria and their host. Curli fibers are a class of bacterial fimbria that is highly amenable to engineering. We demonstrate the use of engineered curli fibers to rationally program interactions between bacteria and components of the mucosal epithelium. Commensal E. coli strains were engineered to produce recombinant curli fibers fused to the trefoil family of human cytokines. Biofilms formed from these strains bound more mucins than those producing wild-type curli fibers, and modulated mucin rheology as well. When treated with bacteria producing the curli-trefoil fusions mammalian cells behaved identically in terms of their migration behavior as when they were treated with the corresponding soluble trefoil factors. Overall, this demonstrates the potential utility of curli fibers as a scaffold for the display of bioactive domains and an untapped approach to rationally modulating host-microbe interactions using bacterial matrix proteins.

Tissue Extracellular Matrix Nanoparticle Presentation in Electrospun Nanofibers

PubMed Central

Gibson, Matt; Mao, Hai-Quan; Elisseeff, Jennifer

2014-01-01

Biomaterials derived from the decellularization of mature tissues retain biological and architectural features that profoundly influence cellular activity. However, the clinical utility of such materials remains limited as the shape and physical properties are difficult to control. In contrast, scaffolds based on synthetic polymers can be engineered to exhibit specific physical properties, yet often suffer from limited biological functionality. This study characterizes composite materials that present decellularized extracellular matrix (DECM) particles in combination with synthetic nanofibers and examines the ability of these materials to influence stem cell differentiation. Mechanical processing of decellularized tissues yielded particles with diameters ranging from 71 to 334 nm. Nanofiber scaffolds containing up to 10% DECM particles (wt/wt) derived from six different tissues were engineered and evaluated to confirm DECM particle incorporation and to measure bioactivity. Scaffolds containing bone, cartilage, and fat promoted osteogenesis at 1 and 3 weeks compared to controls. In contrast, spleen and lung DECM significantly reduced osteogenic outcomes compared to controls. These findings highlight the potential to incorporate appropriate source DECM nanoparticles within nanofiber composites to design a scaffold with bioactivity targeted to specific applications. PMID:24971329

Targeting Heparin to Collagen within Extracellular Matrix Significantly Reduces Thrombogenicity and Improves Endothelialization of Decellularized Tissues.

PubMed

Jiang, Bin; Suen, Rachel; Wertheim, Jason A; Ameer, Guillermo A

2016-12-12

Thrombosis within small-diameter vascular grafts limits the development of bioartificial, engineered vascular conduits, especially those derived from extracellular matrix (ECM). Here we describe an easy-to-implement strategy to chemically modify vascular ECM by covalently linking a collagen binding peptide (CBP) to heparin to form a heparin derivative (CBP-heparin) that selectively binds a subset of collagens. Modification of ECM with CBP-heparin leads to increased deposition of functional heparin (by ∼7.2-fold measured by glycosaminoglycan composition) and a corresponding reduction in platelet binding (>70%) and whole blood clotting (>80%) onto the ECM. Furthermore, addition of CBP-heparin to the ECM stabilizes long-term endothelial cell attachment to the lumen of ECM-derived vascular conduits, potentially through recruitment of heparin-binding growth factors that ultimately improve the durability of endothelialization in vitro. Overall, our findings provide a simple yet effective method to increase deposition of functional heparin on the surface of ECM-based vascular grafts and thereby minimize thrombogenicity of decellularized tissue, overcoming a significant challenge in tissue engineering of bioartificial vessels and vascularized organs.

Artificial extracellular matrix for biomedical applications: biocompatible and biodegradable poly (tetramethylene ether) glycol/poly (ε-caprolactone diol)-based polyurethanes.

PubMed

Shahrousvand, Mohsen; Mir Mohamad Sadeghi, Gity; Salimi, Ali

2016-12-01

The cells as a tissue component need to viscoelastic, biocompatible, biodegradable, and wettable extracellular matrix for their biological activity. In this study, in order to prepare biomedical polyurethane elastomers with good mechanical behavior and biodegradability, a series of novel polyester-polyether- based polyurethanes (PUs) were synthesized using a two-step bulk reaction by melting pre-polymer method, taking 1,4-Butanediol (BDO) as chain extender, hexamethylene diisocyanate as the hard segment, and poly (tetramethylene ether) glycol (PTMEG) and poly (ε-caprolactone diol) (PCL-Diol) as the soft segment without a catalyst. The soft to the hard segment ratio was kept constant in all samples. Polyurethane characteristics such as thermal and mechanical properties, wettability and water adsorption, biodegradability, and cellular behavior were changed by changing the ratio of polyether diol to polyester diol composition in the soft segment. Our present work provides a new procedure for the preparation of engineered polyurethanes in surface properties and biodegradability, which could be a good candidate for bone, cartilage, and skin tissue engineering.

Biomaterials for Tissue Engineering

PubMed Central

Lee, Esther J.; Kasper, F. Kurtis; Mikos, Antonios G.

2013-01-01

Biomaterials serve as an integral component of tissue engineering. They are designed to provide architectural framework reminiscent of native extracellular matrix in order to encourage cell growth and eventual tissue regeneration. Bone and cartilage represent two distinct tissues with varying compositional and mechanical properties. Despite these differences, both meet at the osteochondral interface. This article presents an overview of current biomaterials employed in bone and cartilage applications, discusses some design considerations, and alludes to future prospects within this field of research. PMID:23820768

Hyaluronic acid-based scaffolds for tissue engineering.

PubMed

Chircov, Cristina; Grumezescu, Alexandru Mihai; Bejenaru, Ludovic Everard

2018-01-01

Hyaluronic acid (HA) is a natural glycosaminoglycan found in the extracellular matrix of most connective tissues. Due to its chemical structure, HA is a hydrophilic polymer and it is characterized by a fast degradation rate. HA-based scaffolds for tissue engineering are intensively studied due to their increased biocompatibility, biodegradability and chemical modification. Depending on the processing technique, scaffolds can be prepared in the form of hydrogels, sponges, cryogels, and injectable hydrogels, all discussed in this review.

The emerging role of skeletal muscle extracellular matrix remodelling in obesity and exercise.

PubMed

Martinez-Huenchullan, S; McLennan, S V; Verhoeven, A; Twigg, S M; Tam, C S

2017-07-01

Skeletal muscle extracellular matrix remodelling has been proposed as a new feature associated with obesity and metabolic dysfunction. Exercise training improves muscle function in obesity, which may be mediated by regulatory effects on the muscle extracellular matrix. This review examined available literature on skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise. A non-systematic literature review was performed on PubMed of publications from 1970 to 2015. A total of 37 studies from humans and animals were retained. Studies reported overall increases in gene and protein expression of different types of collagen, growth factors and enzymatic regulators of the skeletal muscle extracellular matrix in obesity. Only two studies investigated the effects of exercise on skeletal muscle extracellular matrix during obesity, with both suggesting a regulatory effect of exercise. The effects of exercise on muscle extracellular matrix seem to be influenced by the duration and type of exercise training with variable effects from a single session compared with a longer duration of exercise. More studies are needed to elucidate the mechanisms behind skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise. © 2017 World Obesity Federation.

Hydrocortisone and triiodothyronine regulate hyaluronate synthesis in a tissue-engineered human dermal equivalent through independent pathways.

PubMed

Deshpande, Madhura; Papp, Suzanne; Schaffer, Lana; Pouyani, Tara

2015-02-01

Hydrocortisone (HC) and triiodothyronine (T3) have both been shown to be capable of independently inhibiting hyaluronate (HA, hyaluronic acid) synthesis in a self-assembled human dermal equivalent (human dermal matrix). We sought to investigate the action of these two hormones in concert on extracellular matrix formation and HA inhibition in the tissue engineered human dermal matrix. To this end, neonatal human dermal fibroblasts were cultured in defined serum-free medium for 21 days in the presence of each hormone alone, or in combination, in varying concentrations. Through a process of self-assembly, a substantial dermal extracellular matrix formed that was characterized. The results of these studies demonstrate that combinations of the hormones T3 and hydrocortisone showed significantly higher levels of hyaluronate inhibition as compared to each hormone alone in the human dermal matrix. In order to gain preliminary insight into the genes regulating HA synthesis in this system, a differential gene array analysis was conducted in which the construct prepared in the presence of 200 μg/mL HC and 0.2 nM T3 was compared to the normal construct (0.4 μg/mL HC and 20 pM T3). Using a GLYCOv4 gene chip containing approximately 1260 human genes, we observed differential expression of 131 genes. These data suggest that when these two hormones are used in concert a different mechanism of inhibition prevails and a combination of degradation and inhibition of HA synthesis may be responsible for HA regulation in the human dermal matrix. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Cytocompatibility and biologic characteristics of synthetic scaffold materials of rabbit acellular vascular matrix combining with human-like collagen I.

PubMed

Liu, Xuqian; Wang, Jie; Dong, Fusheng; Song, Peng; Tian, Songbo; Li, Hexiang; Hou, Yali

2017-10-01

Scaffold material provides a three-dimensional growing environment for seed cells in the research field of tissue engineering. In the present study, rabbit arterial blood vessel cells were chemically removed with trypsin and Triton X-100 to prepare rabbit acellular vascular matrix scaffold material. Observation by He&Masson staining revealed that no cellular components or nuclei existed in the vascular intima and media after decellularization. Human-like collagen I was combined with acellular vascular matrix by freeze-drying to prepare an acellular vascular matrix-0.25% human-like collagen I scaffold to compensate for the extracellular matrix loss during the decellularization process. We next performed a series of experiments to test the water absorbing quality, biomechanics, pressure resistance, cytotoxicity, and ultra-micro structure of the acellular vascular matrix composite material and natural rabbit artery and found that the acellular vascular matrix-0.25% human-like collagen I material behaved similarly to natural rabbit artery. In conclusion, the acellular vascular matrix-0.25% human-like collagen I composite material provides a new approach and lays the foundation for novel scaffold material research into tissue engineering of blood vessels.

Challenges and Opportunities to Harnessing the (Hematopoietic) Stem Cell Niche

PubMed Central

Choi, Ji Sun; Harley, Brendan A. C.

2016-01-01

In our body, stem cells reside in a microenvironment termed the niche. While the exact composition and therefore the level of complexity of a stem cell niche can vary significantly tissue-to-tissue, the stem cell niche microenvironment is dynamic, typically containing spatial and temporal variations in both cellular, extracellular matrix, and biomolecular components. This complex flow of secreted or bound biomolecules, cytokines, extracellular matrix components, and cellular constituents all contribute to the regulation of stem cell fate specification events, making engineering approaches at the nano- and micro-scale of particular interest for creating an artificial niche environment in vitro. Recent advances in fabrication approaches have enabled biomedical researchers to capture and recreate the complexity of stem cell niche microenvironments in vitro. Such engineered platforms show promise as a means to enhance our understanding of the mechanisms underlying niche-mediated stem cell regulation as well as offer opportunities to precisely control stem cell expansion and differentiation events for clinical applications. While these principles generally apply to all adult stem cells and niches, in this review, we focus on recent developments in engineering synthetic niche microenvironments for one of the best-characterized stem cell populations, hematopoietic stem cells (HSC). Specifically, we highlight recent advances in platforms designed to facilitate the extrinsic control of HSC fate decisions. PMID:27134819

Fluorescent Labeling of Collagen Production by Cells for Noninvasive Imaging of Extracellular Matrix Deposition.

PubMed

Bardsley, Katie; Yang, Ying; El Haj, Alicia J

2017-04-01

Extracellular matrix (ECM) is an essential component of tissues and provides both integrity and biological cues for cells. Collagen is one of the major proteins found within the ECM and therefore is an essential component of all engineered tissues. Therefore, in this article, we present a method for the online real-time monitoring of collagen deposition in three-dimensional engineered constructs. This method revolves around modification of collagen through the addition of azide-L-proline to cell culture media. The incorporation of azide-L-proline into the neocollagen produced by cells can then be detected by reaction with 10 mM of a Click-IT Alexa Fluor 488 DIBO Alkyne. The reaction was shown as being specific to the collagen as little background staining was observed in cultures, which did not contain the modified proline, and the staining was also depleted after treatment with collagenase and colocalization of collagen type I staining by immunochemistry assay. Real-time online staining of collagen deposition was observed under different culture conditions without affecting proliferation. Collagen deposition was observed to be increased under mechanical stimulation; however, the localization varied across stimulation regimes. This is a new technique for real-time monitoring of cell-produced collagen and will be a valuable addition to the tissue engineering field.

Multilayered Electrospun Scaffolds for Tendon Tissue Engineering

PubMed Central

Chainani, Abby; Hippensteel, Kirk J.; Kishan, Alysha; Garrigues, N. William; Ruch, David S.; Guilak, Farshid

2013-01-01

Full-thickness rotator cuff tears are one of the most common causes of shoulder pain in people over the age of 65. High retear rates and poor functional outcomes are common after surgical repair, and currently available extracellular matrix scaffold patches have limited abilities to enhance new tendon formation. In this regard, tissue-engineered scaffolds may provide a means to improve repair of rotator cuff tears. Electrospinning provides a versatile method for creating nanofibrous scaffolds with controlled architectures, but several challenges remain in its application to tissue engineering, such as cell infiltration through the full thickness of the scaffold as well as control of cell growth and differentiation. Previous studies have shown that ligament-derived extracellular matrix may enhance differentiation toward a tendon or ligament phenotype by human adipose stem cells (hASCs). In this study, we investigated the use of tendon-derived extracellular matrix (TDM)-coated electrospun multilayered scaffolds compared to fibronectin (FN) or phosphate-buffered saline (PBS) coating for use in rotator cuff tendon tissue engineering. Multilayered poly(ɛ-caprolactone) scaffolds were prepared by sequentially collecting electrospun layers onto the surface of a grounded saline solution into a single scaffold. Scaffolds were then coated with TDM, FN, or PBS and seeded with hASCs. Scaffolds were maintained without exogenous growth factors for 28 days in culture and evaluated for protein content (by immunofluorescence and biochemical assay), markers of tendon differentiation, and tensile mechanical properties. The collagen content was greatest by day 28 in TDM-scaffolds. Gene expression of type I collagen, decorin, and tenascin C increased over time, with no effect of scaffold coating. Sulfated glycosaminoglycan and dsDNA contents increased over time in culture, but there was no effect of scaffold coating. The Young's modulus did not change over time, but yield strain increased with time in culture. Histology demonstrated cell infiltration through the full thickness of all scaffolds and immunofluorescence demonstrated greater expression of type I, but not type III collagen through the full thickness of the scaffold in TDM-scaffolds compared to other treatment groups. Together, these data suggest that nonaligned multilayered electrospun scaffolds permit tenogenic differentiation by hASCs and that TDM may promote some aspects of this differentiation. PMID:23808760

Aggrecan-Like Biomimetic Proteoglycans (BPGs) Composed of Natural Chondroitin Sulfate Bristles Grafted onto Poly(acrylic acid) Core for Molecular Engineering of the Extracellular Matrix.

PubMed

Prudnikova, K; Lightfoot Vidal, S E; Sarkar, S; Yu, T; Yucha, R W; Ganesh, N; Penn, L S; Han, L; Schauer, C L; Vresilovic, E J; Marcolongo, M S

2018-05-10

Biomimetic proteoglycans (BPGs) were designed to mimic the three-dimensional (3D) bottlebrush architecture of natural extracellular matrix (ECM) proteoglycans, such as aggrecan. BPGs were synthesized by grafting native chondroitin sulfate bristles onto a synthetic poly(acrylic acid) core to form BPGs at a molecular weight of approximately ∼1.6 MDa. The aggrecan mimics were characterized chemically, physically, and structurally, confirming the 3D bottlebrush architecture as well as a level of water uptake, which is greater than that of the natural proteoglycan, aggrecan. Aggrecan mimics were cytocompatible at physiological concentrations. Fluorescently labeled BPGs were injected into the nucleus pulposus of the intervertebral disc ex vivo and were retained in tissue before and after static loading and equilibrium conditioning. BPGs infiltrated the tissue, distributed and integrated with the ECM on a molecular scale, in the absence of a bolus, thus demonstrating a new molecular approach to tissue repair: molecular matrix engineering. Molecular matrix engineering may compliment or offer an acellular alternative to current regenerative medicine strategies. Aggrecan is a natural biomolecule that is essential for connective tissue hydration and mechanics. Aggrecan is composed of negatively charged chondroitin sulfate bristles attached to a protein core in a bottlebrush configuration. With age and degeneration, enzymatic degradation of aggrecan outpaces cellular synthesis resulting in a loss of this important molecule. We demonstrate a novel biomimetic molecule composed of natural chondroitin sulfate bristles grafted onto an enzymatically-resistant synthetic core. Our molecule mimics a 3D architecture and charge density of the natural aggrecan, can be delivered via a simple injection and is retained in tissue after equilibrium conditioning and loading. This novel material can serve as a platform for molecular repair, drug delivery and tissue engineering in regenerative medicine approaches. Copyright © 2018. Published by Elsevier Ltd.

Integrative Utilization of Microenvironments, Biomaterials and Computational Techniques for Advanced Tissue Engineering.

PubMed

Shamloo, Amir; Mohammadaliha, Negar; Mohseni, Mina

2015-10-20

This review aims to propose the integrative implementation of microfluidic devices, biomaterials, and computational methods that can lead to a significant progress in tissue engineering and regenerative medicine researches. Simultaneous implementation of multiple techniques can be very helpful in addressing biological processes. Providing controllable biochemical and biomechanical cues within artificial extracellular matrix similar to in vivo conditions is crucial in tissue engineering and regenerative medicine researches. Microfluidic devices provide precise spatial and temporal control over cell microenvironment. Moreover, generation of accurate and controllable spatial and temporal gradients of biochemical factors is attainable inside microdevices. Since biomaterials with tunable properties are a worthwhile option to construct artificial extracellular matrix, in vitro platforms that simultaneously utilize natural, synthetic, or engineered biomaterials inside microfluidic devices are phenomenally advantageous to experimental studies in the field of tissue engineering. Additionally, collaboration between experimental and computational methods is a useful way to predict and understand mechanisms responsible for complex biological phenomena. Computational results can be verified by using experimental platforms. Computational methods can also broaden the understanding of the mechanisms behind the biological phenomena observed during experiments. Furthermore, computational methods are powerful tools to optimize the fabrication of microfluidic devices and biomaterials with specific features. Here we present a succinct review of the benefits of microfluidic devices, biomaterial, and computational methods in the case of tissue engineering and regeneration medicine. Furthermore, some breakthroughs in biological phenomena including the neuronal axon development, cancerous cell migration and blood vessel formation via angiogenesis by virtue of the aforementioned approaches are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Nano-biomimetics for nano/micro tissue regeneration.

PubMed

Singh, Dolly; Singh, Deepti; Zo, Sunmi; Han, Sung Soo

2014-10-01

Nanostructured biomimetics have recently shown great promise in the field of tissue engineering. They can be used as nanoscaffolds and tailored at the molecular level. The scaffold topography closely resembles the native extracellular matrix in terms of framing, porosity and bio-functionality. This review covers the approaches used for biomimetic fabrication, including soft lithography, the plasmonic nanohybrid matrix method and multilayer self-assembly scaffolds for tissue regeneration. It brings together knowledge from different arenas about the synthesis, characterization and functionalization of matrices to accelerate the tissue regeneration process. Every tissue in the body presents different challenges and requires a specific fabrication process designed to identify and mirror the particular organ. For example, microfluidics systems aim to mimic the extracellular matrix of vascular and cartilage tissue, and these systems have different parts with completely different mechanical strength, cellular adhesion and interplay between matrix and cells. A fully functional nanomatrix designed by a self-assembling methodology for use as a vascular tissue engineering scaffold needs to have intrinsic microvessels that facilitate the transportation of metabolites and nutrients. Similarly, in the case of peripheral nerve regeneration, a scaffold needs to have sufficient mechanical strength to protect the regenerating tissue, yet be biodegradable enough to avoid a possible second surgery. To enhance the functionality of scaffolds, increasing focus has been placed on in vitro and in vivo research to achieve optimal scaffold design. Nanobiomimetics unarguably offer the most suitable physicochemical scaffold properties for tissue regeneration.

Putting Electrospun Nanofibers to Work for Biomedical Research

PubMed Central

Xie, Jingwei; Li, Xiaoran; Xia, Younan

2009-01-01

Electrospinning has been exploited for almost one century to process polymers and related materials into nanofibers with controllable compositions, diameters, porosities, and porous structures for a variety of applications. Owing to its high porosity and large surface area, a non-woven mat of electrospun nanofibers can serve as an ideal scaffold to mimic the extracellular matrix for cell attachment and nutrient transportation. The nanofiber itself can also be functionalized through encapsulation or attachment of bioactive species such as extracellular matrix proteins, enzymes, and growth factors. In addition, the nanofibers can be further assembled into a variety of arrays or architectures by manipulating their alignment, stacking, or folding. All these attributes make electrospinning a powerful tool for generating nanostructured materials for a range of biomedical applications that include controlled release, drug delivery, and tissue engineering. PMID:20011452

Biophysical Stimuli: A Review of Electrical and Mechanical Stimulation in Hyaline Cartilage.

PubMed

Vaca-González, Juan J; Guevara, Johana M; Moncayo, Miguel A; Castro-Abril, Hector; Hata, Yoshie; Garzón-Alvarado, Diego A

2017-09-01

Objective Hyaline cartilage degenerative pathologies induce morphologic and biomechanical changes resulting in cartilage tissue damage. In pursuit of therapeutic options, electrical and mechanical stimulation have been proposed for improving tissue engineering approaches for cartilage repair. The purpose of this review was to highlight the effect of electrical stimulation and mechanical stimuli in chondrocyte behavior. Design Different information sources and the MEDLINE database were systematically revised to summarize the different contributions for the past 40 years. Results It has been shown that electric stimulation may increase cell proliferation and stimulate the synthesis of molecules associated with the extracellular matrix of the articular cartilage, such as collagen type II, aggrecan and glycosaminoglycans, while mechanical loads trigger anabolic and catabolic responses in chondrocytes. Conclusion The biophysical stimuli can increase cell proliferation and stimulate molecules associated with hyaline cartilage extracellular matrix maintenance.

Extracellular Matrix Degradation and Remodeling in Development and Disease

PubMed Central

Lu, Pengfei; Takai, Ken; Weaver, Valerie M.; Werb, Zena

2011-01-01

The extracellular matrix (ECM) serves diverse functions and is a major component of the cellular microenvironment. The ECM is a highly dynamic structure, constantly undergoing a remodeling process where ECM components are deposited, degraded, or otherwise modified. ECM dynamics are indispensible during restructuring of tissue architecture. ECM remodeling is an important mechanism whereby cell differentiation can be regulated, including processes such as the establishment and maintenance of stem cell niches, branching morphogenesis, angiogenesis, bone remodeling, and wound repair. In contrast, abnormal ECM dynamics lead to deregulated cell proliferation and invasion, failure of cell death, and loss of cell differentiation, resulting in congenital defects and pathological processes including tissue fibrosis and cancer. Understanding the mechanisms of ECM remodeling and its regulation, therefore, is essential for developing new therapeutic interventions for diseases and novel strategies for tissue engineering and regenerative medicine. PMID:21917992

Tissue-engineered cartilage with inducible and tunable immunomodulatory properties

PubMed Central

Glass, Katherine A.; Link, Jarrett M.; Brunger, Jonathan M.; Moutos, Franklin T.; Gersbach, Charles A.; Guilak, Farshid

2014-01-01

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (MSC) chondrogenesis. In this study, we combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in MSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce MSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis. PMID:24767790

Tissue engineering in endodontics.

PubMed

Saber, Shehab El-Din M

2009-12-01

Tissue engineering is the science of design and manufacture of new tissues to replace impaired or damaged ones. The key ingredients for tissue engineering are stem cells, the morphogens or growth factors that regulate their differentiation, and a scaffold of extracellular matrix that constitutes the microenvironment for their growth. Recently, there has been increasing interest in applying the concept of tissue engineering to endodontics. The aim of this study was to review the body of knowledge related to dental pulp stem cells, the most common growth factors, and the scaffolds used to control their differentiation, and a clinical technique for the management of immature non-vital teeth based on this novel concept.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components.

PubMed

Qing, Chang; Wei-ding, Cui; Wei-min, Fan

2011-04-01

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Chondrogenic properties of collagen type XI, a component of cartilage extracellular matrix.

PubMed

Li, Ang; Wei, Yiyong; Hung, Clark; Vunjak-Novakovic, Gordana

2018-08-01

Cartilage extracellular matrix (ECM) has been used for promoting tissue engineering. However, the exact effects of ECM on chondrogenesis and the acting mechanisms are not well understood. In this study, we investigated the chondrogenic effects of cartilage ECM on human mesenchymal stem cells (MSCs) and identified the contributing molecular components. To this end, a preparation of articular cartilage ECM was supplemented to pellets of chondrogenically differentiating MSCs, pellets of human chondrocytes, and bovine articular cartilage explants to evaluate the effects on cell proliferation and the production of cartilaginous matrix. Selective enzymatic digestion and screening of ECM components were conducted to identify matrix molecules with chondrogenic properties. Cartilage ECM promoted MSC proliferation, production of cartilaginous matrix, and maturity of chondrogenic differentiation, and inhibited the hypertrophic differentiation of MSC-derived chondrocytes. Selective digestion of ECM components revealed a contributory role of collagens in promoting chondrogenesis. The screening of various collagen subtypes revealed strong chondrogenic effect of collagen type XI. Finally, collagen XI was found to promote production and inhibit degradation of cartilage matrix in human articular chondrocyte pellets and bovine articular cartilage explants. Our results indicate that cartilage ECM promotes chondrogenesis and inhibits hypertrophic differentiation in MSCs. Collagen type XI is the ECM component that has the strongest effects on enhancing the production and inhibiting the degradation of cartilage matrix. Copyright © 2018 Elsevier Ltd. All rights reserved.

Glycopolymer functionalization of engineered spider silk protein-based materials for improved cell adhesion.

PubMed

Hardy, John G; Pfaff, André; Leal-Egaña, Aldo; Müller, Axel H E; Scheibel, Thomas R

2014-07-01

Silk protein-based materials are promising biomaterials for application as tissue scaffolds, due to their processability, biocompatibility, and biodegradability. The preparation of films composed of an engineered spider silk protein (eADF4(C16)) and their functionalization with glycopolymers are described. The glycopolymers bind proteins found in the extracellular matrix, providing a biomimetic coating on the films that improves cell adhesion to the surfaces of engineered spider silk films. Such silk-based materials have potential as coatings for degradable implantable devices. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cell–material interactions on biphasic polyurethane matrix

PubMed Central

Dicesare, Patrick; Fox, Wade M.; Hill, Michael J.; Krishnan, G. Rajesh; Yang, Shuying; Sarkar, Debanjan

2013-01-01

Cell–matrix interaction is a key regulator for controlling stem cell fate in regenerative tissue engineering. These interactions are induced and controlled by the nanoscale features of extracellular matrix and are mimicked on synthetic matrices to control cell structure and functions. Recent studies have shown that nanostructured matrices can modulate stem cell behavior and exert specific role in tissue regeneration. In this study, we have demonstrated that nanostructured phase morphology of synthetic matrix can control adhesion, proliferation, organization and migration of human mesenchymal stem cells (MSCs). Nanostructured biodegradable polyurethanes (PU) with segmental composition exhibit biphasic morphology at nanoscale dimensions and can control cellular features of MSCs. Biodegradable PU with polyester soft segment and hard segment composed of aliphatic diisocyanates and dipeptide chain extender were designed to examine the effect polyurethane phase morphology. By altering the polyurethane composition, morphological architecture of PU was modulated and its effect was examined on MSC. Results show that MSCs can sense the nanoscale morphology of biphasic polyurethane matrix to exhibit distinct cellular features and, thus, signifies the relevance of matrix phase morphology. The role of nanostructured phases of a synthetic matrix in controlling cell–matrix interaction provides important insights for regulation of cell behavior on synthetic matrix and, therefore, is an important tool for engineering tissue regeneration. PMID:23255285

Injectable hydrogels for cartilage and bone tissue engineering

PubMed Central

Liu, Mei; Zeng, Xin; Ma, Chao; Yi, Huan; Ali, Zeeshan; Mou, Xianbo; Li, Song; Deng, Yan; He, Nongyue

2017-01-01

Tissue engineering has become a promising strategy for repairing damaged cartilage and bone tissue. Among the scaffolds for tissue-engineering applications, injectable hydrogels have demonstrated great potential for use as three-dimensional cell culture scaffolds in cartilage and bone tissue engineering, owing to their high water content, similarity to the natural extracellular matrix (ECM), porous framework for cell transplantation and proliferation, minimal invasive properties, and ability to match irregular defects. In this review, we describe the selection of appropriate biomaterials and fabrication methods to prepare novel injectable hydrogels for cartilage and bone tissue engineering. In addition, the biology of cartilage and the bony ECM is also summarized. Finally, future perspectives for injectable hydrogels in cartilage and bone tissue engineering are discussed. PMID:28584674

A Structurally and Functionally Biomimetic Biphasic Scaffold for Intervertebral Disc Tissue Engineering

PubMed Central

Choy, Andrew Tsz Hang; Chan, Barbara Pui

2015-01-01

Tissue engineering offers high hopes for the treatment of intervertebral disc (IVD) degeneration. Whereas scaffolds of the disc nucleus and annulus have been extensively studied, a truly biomimetic and mechanically functional biphasic scaffold using naturally occurring extracellular matrix is yet to be developed. Here, a biphasic scaffold was fabricated with collagen and glycosaminoglycans (GAGs), two of the most abundant extracellular matrix components in the IVD. Following fabrication, the scaffold was characterized and benchmarked against native disc. The biphasic scaffold was composed of a collagen-GAG co-precipitate making up the nucleus pulposus-like core, and this was encapsulated in multiple lamellae of photochemically crosslinked collagen membranes comprising the annulus fibrosus-like lamellae. On mechanical testing, the height of our engineered disc recovered by ~82-89% in an annulus-independent manner, when compared with the 99% recovery exhibited by native disc. The annulus-independent nature of disc height recovery suggests that the fluid replacement function of the engineered nucleus pulposus core might mimic this hitherto unique feature of native disc. Biphasic scaffolds comprised of 10 annulus fibrosus-like lamellae had the best overall mechanical performance among the various designs owing to their similarity to native disc in most aspects, including elastic compliance during creep and recovery, and viscous compliance during recovery. However, the dynamic mechanical performance (including dynamic stiffness and damping factor) of all the biphasic scaffolds was similar to that of the native discs. This study contributes to the rationalized design and development of a biomimetic and mechanically viable biphasic scaffold for IVD tissue engineering. PMID:26115332

Nanomaterials for Craniofacial and Dental Tissue Engineering.

PubMed

Li, G; Zhou, T; Lin, S; Shi, S; Lin, Y

2017-07-01

Tissue engineering shows great potential as a future treatment for the craniofacial and dental defects caused by trauma, tumor, and other diseases. Due to the biomimetic features and excellent physiochemical properties, nanomaterials are of vital importance in promoting cell growth and stimulating tissue regeneration in tissue engineering. For craniofacial and dental tissue engineering, the frequently used nanomaterials include nanoparticles, nanofibers, nanotubes, and nanosheets. Nanofibers are attractive for cell invasion and proliferation because of their resemblance to extracellular matrix and the presence of large pores, and they have been used as scaffolds in bone, cartilage, and tooth regeneration. Nanotubes and nanoparticles improve the mechanical and chemical properties of scaffold, increase cell attachment and migration, and facilitate tissue regeneration. In addition, nanofibers and nanoparticles are also used as a delivery system to carry the bioactive agent in bone and tooth regeneration, have better control of the release speed of agent upon degradation of the matrix, and promote tissue regeneration. Although applications of nanomaterials in tissue engineering remain in their infancy with numerous challenges to face, the current results indicate that nanomaterials have massive potential in craniofacial and dental tissue engineering.

Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments

NASA Astrophysics Data System (ADS)

Baker, Brendon M.; Trappmann, Britta; Wang, William Y.; Sakar, Mahmut S.; Kim, Iris L.; Shenoy, Vivek B.; Burdick, Jason A.; Chen, Christopher S.

2015-12-01

To investigate how cells sense stiffness in settings structurally similar to native extracellular matrices, we designed a synthetic fibrous material with tunable mechanics and user-defined architecture. In contrast to flat hydrogel surfaces, these fibrous materials recapitulated cell-matrix interactions observed with collagen matrices including stellate cell morphologies, cell-mediated realignment of fibres, and bulk contraction of the material. Increasing the stiffness of flat hydrogel surfaces induced mesenchymal stem cell spreading and proliferation; however, increasing fibre stiffness instead suppressed spreading and proliferation for certain network architectures. Lower fibre stiffness permitted active cellular forces to recruit nearby fibres, dynamically increasing ligand density at the cell surface and promoting the formation of focal adhesions and related signalling. These studies demonstrate a departure from the well-described relationship between material stiffness and spreading established with hydrogel surfaces, and introduce fibre recruitment as a previously undescribed mechanism by which cells probe and respond to mechanics in fibrillar matrices.

Extracellular Matrix Scaffold Technology for Bioartificial Pancreas Engineering

PubMed Central

Salvatori, Marcus; Katari, Ravi; Patel, Timil; Peloso, Andrea; Mugweru, Jon; Owusu, Kofi

2014-01-01

Emergent technologies in regenerative medicine may soon overcome the limitations of conventional diabetes therapies. Collaborative efforts across the subfields of stem cell technology, islet encapsulation, and biomaterial carriers seek to produce a bioengineered pancreas capable of restoring endocrine function in patients with insulin-dependent diabetes. These technologies rely on a robust understanding of the extracellular matrix (ECM), the supportive 3-dimensional network of proteins necessary for cellular attachment, proliferation, and differentiation. Although these functions can be partially approximated by biosynthetic carriers, novel decellularization protocols have allowed researchers to discover the advantages afforded by the native pancreatic ECM. The native ECM has proven to be an optimal platform for recellularization and whole-organ pancreas bioengineering, an exciting new field with the potential to resolve the dire shortage of transplantable organs. This review seeks to contextualize recent findings, discuss current research goals, and identify future challenges of regenerative medicine as it applies to diabetes management. PMID:24876552

Cartilage extracellular matrix as a biomaterial for cartilage regeneration.

PubMed

Kiyotake, Emi A; Beck, Emily C; Detamore, Michael S

2016-11-01

The extracellular matrix (ECM) of various tissues possesses the model characteristics that biomaterials for tissue engineering strive to mimic; however, owing to the intricate hierarchical nature of the ECM, it has yet to be fully characterized and synthetically fabricated. Cartilage repair remains a challenge because the intrinsic properties that enable its durability and long-lasting function also impede regeneration. In the last decade, cartilage ECM has emerged as a promising biomaterial for regenerating cartilage, partly because of its potentially chondroinductive nature. As this research area of cartilage matrix-based biomaterials emerged, investigators facing similar challenges consequently developed convergent solutions in constructing robust and bioactive scaffolds. This review discusses the challenges, emerging trends, and future directions of cartilage ECM scaffolds, including a comparison between two different forms of cartilage matrix: decellularized cartilage (DCC) and devitalized cartilage (DVC). To overcome the low permeability of cartilage matrix, physical fragmentation greatly enhances decellularization, although the process itself may reduce the chondroinductivity of fabricated scaffolds. The less complex processing of a scaffold composed of DVC, which has not been decellularized, appears to have translational advantages and potential chondroinductive and mechanical advantages over DCC, without detrimental immunogenicity, to ultimately enhance cartilage repair in a clinically relevant way. © 2016 New York Academy of Sciences.

Tissue Engineering Using Transfected Growth-Factor Genes

NASA Technical Reports Server (NTRS)

Madry, Henning; Langer, Robert S.; Freed, Lisa E.; Trippel, Stephen; Vunjak-Novakovic, Gordana

2005-01-01

A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

Mesoscopic Rigid Body Modelling of the Extracellular Matrix Self-Assembly.

PubMed

Wong, Hua; Prévoteau-Jonquet, Jessica; Baud, Stéphanie; Dauchez, Manuel; Belloy, Nicolas

2018-06-11

The extracellular matrix (ECM) plays an important role in supporting tissues and organs. It even has a functional role in morphogenesis and differentiation by acting as a source of active molecules (matrikines). Many diseases are linked to dysfunction of ECM components and fragments or changes in their structures. As such it is a prime target for drugs. Because of technological limitations for observations at mesoscopic scales, the precise structural organisation of the ECM is not well-known, with sparse or fuzzy experimental observables. Based on the Unity3D game and physics engines, along with rigid body dynamics, we propose a virtual sandbox to model large biological molecules as dynamic chains of rigid bodies interacting together to gain insight into ECM components behaviour in the mesoscopic range. We have preliminary results showing how parameters such as fibre flexibility or the nature and number of interactions between molecules can induce different structures in the basement membrane. Using the Unity3D game engine and virtual reality headset coupled with haptic controllers, we immerse the user inside the corresponding simulation. Untrained users are able to navigate a complex virtual sandbox crowded with large biomolecules models in a matter of seconds.

New advances in probing cell–extracellular matrix interactions

PubMed Central

2017-01-01

The extracellular matrix (ECM) provides structural and biochemical support to cells within tissues. An emerging body of evidence has established that the ECM plays a key role in cell mechanotransduction – the study of coupling between mechanical inputs and cellular phenotype – through either mediating transmission of forces to the cells, or presenting mechanical cues that guide cellular behaviors. Recent progress in cell mechanotransduction research has been facilitated by advances of experimental tools, particularly microtechnologies, engineered biomaterials, and imaging and analytical methods. Microtechnologies have enabled the design and fabrication of controlled physical microenvironments for the study and measurement of cell–ECM interactions. Advances in engineered biomaterials have allowed researchers to develop synthetic ECMs that mimic tissue microenvironments and investigate the impact of altered physicochemical properties on various cellular processes. Finally, advanced imaging and spectroscopy techniques have facilitated the visualization of the complex interaction between cells and ECM in vitro and in living tissues. This review will highlight the application of recent innovations in these areas to probing cell–ECM interactions. We believe cross-disciplinary approaches, combining aspects of the different technologies reviewed here, will inspire innovative ideas to further elucidate the secrets of ECM-mediated cell control. PMID:28352896

The role of environmental factors in regulating the development of cartilaginous grafts engineered using osteoarthritic human infrapatellar fat pad-derived stem cells.

PubMed

Liu, Yurong; Buckley, Conor T; Downey, Richard; Mulhall, Kevin J; Kelly, Daniel J

2012-08-01

Engineering functional cartilaginous grafts using stem cells isolated from osteoarthritic human tissue is of fundamental importance if autologous tissue engineering strategies are to be used in the treatment of diseased articular cartilage. It has previously been demonstrated that human infrapatellar fat pad (IFP)-derived stem cells undergo chondrogenesis in pellet culture; however, the ability of such cells to generate functional cartilaginous grafts has not been adequately addressed. The objective of this study was to explore how environmental conditions regulate the functional development of cartilaginous constructs engineered using diseased human IFP-derived stem cells (FPSCs). FPSCs were observed to display a diminished chondrogenic potential upon encapsulation in a three-dimensional hydrogel compared with pellet culture, synthesizing significantly lower levels of glycosaminoglycan and collagen on a per cell basis. To engineer more functional cartilaginous grafts, we next explored whether additional biochemical and biophysical stimulations would enhance chondrogenesis within the hydrogels. Serum stimulation was observed to partially recover the diminished chondrogenic potential within hydrogel culture. Over 42 days, stem cells that had first been expanded in a low-oxygen environment proliferated extensively on the outer surface of the hydrogel in response to serum stimulation, assembling a dense type II collagen-positive cartilaginous tissue resembling that formed in pellet culture. The application of hydrostatic pressure did not further enhance extracellular matrix synthesis within the hydrogels, but did appear to alter the spatial accumulation of extracellular matrix leading to the formation of a more compact tissue with superior mechanically functionality. Further work is required in order to recapitulate the environmental conditions present during pellet culture within scaffolds or hydrogels in order to engineer more functional cartilaginous grafts using human osteoarthritic FPSCs.

Engineered matrices for bone regeneration

NASA Astrophysics Data System (ADS)

Winn, Shelley R.; Hu, Yunhua; Pugh, Amy; Brown, Leanna; Nguyen, Jesse T.; Hollinger, Jeffrey O.

2000-06-01

Traditional therapies of autografts and allogeneic banked bone can promote reasonable clinical outcome to repair damaged bone. However, under certain conditions the success of these traditional approaches plummets, providing the incentive for researchers to develop clinical alternatives. The evolving field of tissue engineering in the musculoskeletal system attempts to mimic many of the components from the intact, healthy subject. Those components consist of a biologic scaffold, cells, extracellular matrix, and signaling molecules. The bone biomimetic, i.e., an engineered matrix, provides a porous structural architecture for the regeneration and ingrowth of osseous tissue at the site of injury. To further enhance the regenerative cascade, our strategy has involved porous biodegradable scaffolds containing and releasing signaling molecules and providing a suitable environment for cell attachment, growth and differentiation. In addition, the inclusion of genetically modified osteogenic precursor cells has brought the technology closer to developing a tissue-engineered equivalent. The presentation will describe various formulations and the methods utilized to evaluate the clinical utility of these biomimetics.

Student Award for Outstanding Research Winner in the Ph.D. Category for the 9th World Biomaterials Congress, Chengdu, China, June 1-5, 2012: The interplay of bone-like extracellular matrix and TNF-α signaling on in vitro osteogenic differentiation of mesenchymal stem cells.

PubMed

Mountziaris, Paschalia M; Tzouanas, Stephanie N; Mikos, Antonios G

2012-05-01

As an initial step in the development of a bone tissue engineering strategy to rationally control inflammation, we investigated the interplay of bone-like extracellular matrix (ECM) and varying doses of the inflammatory cytokine tumor necrosis factor alpha (TNF-α) on osteogenically differentiating mesenchymal stem cells (MSCs) cultured in vitro on 3D poly(ε-caprolactone) (PCL) microfiber scaffolds containing pregenerated bone-like ECM. To generate the ECM, PCL scaffolds were seeded with MSCs and cultured in medium containing the typically required osteogenic supplement dexamethasone. However, since dexamethasone antagonizes TNF-α, the interplay of ECM and TNF-α was investigated by culturing naïve MSCs on the decellularized scaffolds in the absence of dexamethasone. MSCs cultured on ECM-coated scaffolds continued to deposit mineralized matrix, a late stage marker of osteogenic differentiation. Mineralized matrix deposition was not adversely affected by exposure to TNF-α for 4-8 days, but was significantly reduced after continuous exposure to TNF-α over 16 days, which simulates the in vivo response, where brief TNF-α signaling stimulates bone regeneration, while prolonged exposure has damaging effects. This underscores the exciting potential of PCL/ECM constructs as a more clinically realistic in vitro culture model to facilitate the design of new bone tissue engineering strategies that rationally control inflammation to promote regeneration. Copyright © 2012 Wiley Periodicals, Inc.

Modeling extracellular matrix degradation balance with proteinase/transglutaminase cycle.

PubMed

Larreta-Garde, Veronique; Berry, Hugues

2002-07-07

Extracellular matrix mass balance is implied in many physiological and pathological events, such as metastasis dissemination. Widely studied, its destructive part is mainly catalysed by extracellular proteinases. Conversely, the properties of the constructive part are less obvious, cellular neo-synthesis being usually considered as its only element. In this paper, we introduce the action of transglutaminase in a mathematical model for extracellular matrix remodeling. This extracellular enzyme, catalysing intermolecular protein cross-linking, is considered here as a reverse proteinase as far as the extracellular matrix physical state is concerned. The model is based on a proteinase/transglutaminase cycle interconverting insoluble matrix and soluble proteolysis fragments, with regulation of cellular proteinase expression by the fragments. Under "closed" (batch) conditions, i.e. neglecting matrix influx and fragment efflux from the system, the model is bistable, with reversible hysteresis. Extracellular matrix proteins concentration abruptly switches from low to high levels when transglutaminase activity exceeds a threshold value. Proteinase concentration usually follows the reverse complementary kinetics, but can become apparently uncoupled from extracellular matrix concentration for some parameter values. When matrix production by the cells and fragment degradation are taken into account, the dynamics change to sustained oscillations because of the emergence of a stable limit cycle. Transitions out of and into oscillation areas are controlled by the model parameters. Biological interpretation indicates that these oscillations could represent the normal homeostatic situation, whereas the other exhibited dynamics can be related to pathologies such as tumor invasion or fibrosis. These results allow to discuss the insights that the model could contribute to the comprehension of these complex biological events.

Evaluation of Small Intestine Grafts Decellularization Methods for Corneal Tissue Engineering

PubMed Central

Oliveira, Ana Celeste; Garzón, Ingrid; Ionescu, Ana Maria; Carriel, Victor; Cardona, Juan de la Cruz; González-Andrades, Miguel; Pérez, María del Mar; Alaminos, Miguel; Campos, Antonio

2013-01-01

Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications. PMID:23799114

Integrating valve-inspired design features into poly(ethylene glycol) hydrogel scaffolds for heart valve tissue engineering.

PubMed

Zhang, Xing; Xu, Bin; Puperi, Daniel S; Yonezawa, Aline L; Wu, Yan; Tseng, Hubert; Cuchiara, Maude L; West, Jennifer L; Grande-Allen, K Jane

2015-03-01

The development of advanced scaffolds that recapitulate the anisotropic mechanical behavior and biological functions of the extracellular matrix in leaflets would be transformative for heart valve tissue engineering. In this study, anisotropic mechanical properties were established in poly(ethylene glycol) (PEG) hydrogels by crosslinking stripes of 3.4 kDa PEG diacrylate (PEGDA) within 20 kDa PEGDA base hydrogels using a photolithographic patterning method. Varying the stripe width and spacing resulted in a tensile elastic modulus parallel to the stripes that was 4.1-6.8 times greater than that in the perpendicular direction, comparable to the degree of anisotropy between the circumferential and radial orientations in native valve leaflets. Biomimetic PEG-peptide hydrogels were prepared by tethering the cell-adhesive peptide RGDS and incorporating the collagenase-degradable peptide PQ (GGGPQG↓IWGQGK) into the polymer network. The specific amounts of RGDS and PEG-PQ within the resulting hydrogels influenced the elongation, de novo extracellular matrix deposition and hydrogel degradation behavior of encapsulated valvular interstitial cells (VICs). In addition, the morphology and activation of VICs grown atop PEG hydrogels could be modulated by controlling the concentration or micro-patterning profile of PEG-RGDS. These results are promising for the fabrication of PEG-based hydrogels using anatomically and biologically inspired scaffold design features for heart valve tissue engineering. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

MAGP1, the extracellular matrix, and metabolism

PubMed Central

Craft, Clarissa S

2014-01-01

Adipose tissue and the extracellular matrix were once considered passive players in regulating physiological processes. Now, both entities are acknowledged for their capacity to engage signal transduction pathways, and for their involvement in maintaining normal tissue homeostasis. We recently published a series of studies that identified a novel mechanism whereby an extracellular matrix molecule, MAGP1 (microfibril associated glycoprotein 1), can regulate energy metabolism in adipose tissue. MAGP1 is a component of extracellular microfibrils and plays a supportive role in maintaining thermoregulation by indirectly regulating expression of the thermogenic uncoupling proteins (UCPs). The focus of this commentary is to draw attention to the role of the extracellular matrix in regulating the bioavailability of signaling molecules, like transforming growth factor β (TGFβ), and exemplify that a better understanding of the extracellular matrix's biological properties could unveil a new source of therapeutic targets for metabolic diseases. PMID:26167404

MAGP1, the extracellular matrix, and metabolism.

PubMed

Craft, Clarissa S

2015-01-01

Adipose tissue and the extracellular matrix were once considered passive players in regulating physiological processes. Now, both entities are acknowledged for their capacity to engage signal transduction pathways, and for their involvement in maintaining normal tissue homeostasis. We recently published a series of studies that identified a novel mechanism whereby an extracellular matrix molecule, MAGP1 (microfibril associated glycoprotein 1), can regulate energy metabolism in adipose tissue. MAGP1 is a component of extracellular microfibrils and plays a supportive role in maintaining thermoregulation by indirectly regulating expression of the thermogenic uncoupling proteins (UCPs). The focus of this commentary is to draw attention to the role of the extracellular matrix in regulating the bioavailability of signaling molecules, like transforming growth factor β (TGFβ), and exemplify that a better understanding of the extracellular matrix's biological properties could unveil a new source of therapeutic targets for metabolic diseases.

Designing ECM-mimetic Materials Using Protein Engineering

PubMed Central

Cai, Lei; Heilshorn, Sarah C.

2014-01-01

The natural extracellular matrix (ECM), with its multitude of evolved cell-instructive and cell-responsive properties, provides inspiration and guidelines for the design of engineered biomaterials. One strategy to create ECM-mimetic materials is the modular design of protein-based engineered ECM (eECM) scaffolds. This modular design strategy involves combining multiple protein domains with different functionalities into a single, modular polymer sequence, resulting in a multifunctional matrix with independent tunability of the individual domain functions. These eECMs often enable decoupled control over multiple material properties for fundamental studies of cell-matrix interactions. In addition, since the eECMs are frequently composed entirely of bioresorbable amino acids, these matrices have immense clinical potential for a variety of regenerative medicine applications. This brief review demonstrates how fundamental knowledge gained from structure-function studies of native proteins can be exploited in the design of novel protein-engineered biomaterials. While the field of protein-engineered biomaterials has existed for over 20 years, the community is only now beginning to fully explore the diversity of functional peptide modules that can be incorporated into these materials. We have chosen to highlight recent examples that either (1) demonstrate exemplary use as matrices with cell-instructive and cell-responsive properties or (2) demonstrate outstanding creativity in terms of novel molecular-level design and macro-level functionality. PMID:24365704

Microfluidic hydrogels for tissue engineering.

PubMed

Huang, Guo You; Zhou, Li Hong; Zhang, Qian Cheng; Chen, Yong Mei; Sun, Wei; Xu, Feng; Lu, Tian Jian

2011-03-01

With advanced properties similar to the native extracellular matrix, hydrogels have found widespread applications in tissue engineering. Hydrogel-based cellular constructs have been successfully developed to engineer different tissues such as skin, cartilage and bladder. Whilst significant advances have been made, it is still challenging to fabricate large and complex functional tissues due mainly to the limited diffusion capability of hydrogels. The integration of microfluidic networks and hydrogels can greatly enhance mass transport in hydrogels and spatiotemporally control the chemical microenvironment of cells, mimicking the function of native microvessels. In this review, we present and discuss recent advances in the fabrication of microfluidic hydrogels from the viewpoint of tissue engineering. Further development of new hydrogels and microengineering technologies will have a great impact on tissue engineering.

Polymeric Nanofibers in Tissue Engineering

PubMed Central

Dahlin, Rebecca L.; Kasper, F. Kurtis

2011-01-01

Polymeric nanofibers can be produced using methods such as electrospinning, phase separation, and self-assembly, and the fiber composition, diameter, alignment, degradation, and mechanical properties can be tailored to the intended application. Nanofibers possess unique advantages for tissue engineering. The small diameter closely matches that of extracellular matrix fibers, and the relatively large surface area is beneficial for cell attachment and bioactive factor loading. This review will update the reader on the aspects of nanofiber fabrication and characterization important to tissue engineering, including control of porous structure, cell infiltration, and fiber degradation. Bioactive factor loading will be discussed with specific relevance to tissue engineering. Finally, applications of polymeric nanofibers in the fields of bone, cartilage, ligament and tendon, cardiovascular, and neural tissue engineering will be reviewed. PMID:21699434

Using Polymeric Materials to Control Stem Cell Behavior for Tissue Regeneration

PubMed Central

Zhang, Nianli; Kohn, David H.

2017-01-01

Patients with organ failure often suffer from increased morbidity and decreased quality of life. Current strategies of treating organ failure have limitations, including shortage of donor organs, low efficiency of grafts, and immunological problems. Tissue engineering emerged about two decades ago as a strategy to restore organ function with a living, functional engineered substitute. However, the ability to engineer a functional organ substitute is limited by a limited understanding of the interactions between materials and cells that are required to yield functional tissue equivalents. Polymeric materials are one of the most promising classes of materials for use in tissue engineering due to their biodegradability, flexibility in processing and property design, and the potential to use polymer properties to control cell function. Stem cells offer potential in tissue engineering because of their unique capacity to self renew and differentiate into neurogenic, osteogenic, chondrogenic, myogenic lineages under appropriate stimuli from extracellular components. This review examines recent advances in stem cell-polymer interactions for tissue regeneration, specifically highlighting control of polymer properties to direct adhesion, proliferation, and differentiation of stem cells, and how biomaterials can be designed to provide some of the stimuli to cells that the natural extracellular matrix does. PMID:22457178

Next Generation Tissue Engineering of Orthopedic Soft Tissue-to-Bone Interfaces.

PubMed

Boys, Alexander J; McCorry, Mary Clare; Rodeo, Scott; Bonassar, Lawrence J; Estroff, Lara A

2017-09-01

Soft tissue-to-bone interfaces are complex structures that consist of gradients of extracellular matrix materials, cell phenotypes, and biochemical signals. These interfaces, called entheses for ligaments, tendons, and the meniscus, are crucial to joint function, transferring mechanical loads and stabilizing orthopedic joints. When injuries occur to connected soft tissue, the enthesis must be re-established to restore function, but due to structural complexity, repair has proven challenging. Tissue engineering offers a promising solution for regenerating these tissues. This prospective review discusses methodologies for tissue engineering the enthesis, outlined in three key design inputs: materials processing methods, cellular contributions, and biochemical factors.

Next Generation Tissue Engineering of Orthopedic Soft Tissue-to-Bone Interfaces

PubMed Central

Boys, Alexander J.; McCorry, Mary Clare; Rodeo, Scott; Bonassar, Lawrence J.; Estroff, Lara A.

2017-01-01

Soft tissue-to-bone interfaces are complex structures that consist of gradients of extracellular matrix materials, cell phenotypes, and biochemical signals. These interfaces, called entheses for ligaments, tendons, and the meniscus, are crucial to joint function, transferring mechanical loads and stabilizing orthopedic joints. When injuries occur to connected soft tissue, the enthesis must be re-established to restore function, but due to structural complexity, repair has proven challenging. Tissue engineering offers a promising solution for regenerating these tissues. This prospective review discusses methodologies for tissue engineering the enthesis, outlined in three key design inputs: materials processing methods, cellular contributions, and biochemical factors. PMID:29333332

Tissue-engineered cartilage with inducible and tunable immunomodulatory properties.

PubMed

Glass, Katherine A; Link, Jarrett M; Brunger, Jonathan M; Moutos, Franklin T; Gersbach, Charles A; Guilak, Farshid

2014-07-01

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (MSC) chondrogenesis. In this study, we combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in MSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce MSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis. Copyright © 2014 Elsevier Ltd. All rights reserved.

In vivo engineering of the vocal fold extracellular matrix with injectable hyaluronic acid hydrogels: early effects on tissue repair and biomechanics in a rabbit model.

PubMed

Hansen, Jennifer K; Thibeault, Susan L; Walsh, Jennifer F; Shu, Xiao Zheng; Prestwich, Glenn D

2005-09-01

A prospective, controlled animal study was performed to determine whether the use of injectable, chemically modified hyaluronic acid (HA) derivatives at the time of intentional vocal fold resection might facilitate wound repair and preserve the unique viscoelastic properties of the vocal fold extracellular matrix. We performed bilateral vocal fold biopsies on 33 rabbits. Two groups of rabbits were unilaterally treated with 2 different HA derivatives--Carbylan-SX and HA-DTPH-PEGDA--at the time of resection. Saline was injected as a control into the contralateral fold. The animals were painlessly sacrificed 3 weeks after biopsy and injection. The outcomes measured included histologic fibrosis level, tissue HA level, and tissue viscosity and elasticity. The Carbylan-SX-treated vocal folds were found to have significantly less fibrosis than the saline-treated controls. The levels of HA in the treated vocal folds were not significantly different from those in the controls at 3 weeks as measured by enzyme-linked immunosorbent assay. The Carbylan-SX-treated vocal folds had significantly improved biomechanical properties of elasticity and viscosity. The HA-DTPH-PEGDA injections yielded significantly improved viscosity, but not elasticity. Prophylactic in vivo manipulation of the extracellular matrix with an injectable Carbylan-SX hydrogel appears to induce vocal fold tissue regeneration to yield optimal tissue composition and biomechanical properties favorable for phonation.

Solubilized liver extracellular matrix maintains primary rat hepatocyte phenotype in-vitro.

PubMed

Loneker, Abigail E; Faulk, Denver M; Hussey, George S; D'Amore, Antonio; Badylak, Stephen F

2016-04-01

Whole organ engineering and cell-based regenerative medicine approaches are being investigated as potential therapeutic options for end-stage liver failure. However, a major challenge of these strategies is the loss of hepatic specific function after hepatocytes are removed from their native microenvironment. The objective of the present study was to determine if solubilized liver extracellular matrix (ECM), when used as a media supplement, can better maintain hepatocyte phenotype compared to type I collagen alone or solubilized ECM harvested from a non-liver tissue source. Liver extracellular matrix (LECM) from four different species was isolated via liver tissue decellularization, solubilized, and then used as a media supplement for primary rat hepatocytes (PRH). The four species of LECM investigated were human, porcine, canine and rat. Cell morphology, albumin secretion, and ammonia metabolism were used to assess maintenance of hepatocyte phenotype. Biochemical and mechanical characterization of each LECM were also conducted. Results showed that PRH's supplemented with canine and porcine LECM maintained their phenotype to a greater extent compared to all other groups. PRH's supplemented with canine and porcine LECM showed increased bile production, increased albumin production, and the formation of multinucleate cells. The findings of the present study suggest that solubilized liver ECM can support in-vitro hepatocyte culture and should be considered for therapeutic and diagnostic techniques that utilize hepatocytes. © 2016 Wiley Periodicals, Inc.

Organ reconstruction: Dream or reality for the future.

PubMed

Stoltz, J-F; Zhang, L; Ye, J S; De Isla, N

2017-01-01

The relevance of research on reconstructed organs is justified by the lack of organs available for transplant and the growing needs for the ageing population. The development of a reconstructed organ involves two parallel complementary steps: de-cellularization of the organ with the need to maintain the structural integrity of the extracellular matrix and vascular network and re-cellularization of the scaffold with stem cells or resident cells.Whole organ engineering for liver, heart, lung or kidneys, is particularly difficult because of the structural complexity of organs and heterogeneity of cells. Rodent, porcine and rhesus monkey organs have been de-cellularized to obtain a scaffold with preserved extracellular matrix and vascular network. As concern the cells for re-cellularization, embryonic, foetal, adult, progenitor stem cells and also iPS have been proposed.Heart construction could be an alternative option for the treatment of cardiac insufficiency. It is based on the use of an extra-cellular matrix coming from an animal's heart and seeded with cells likely to reconstruct a normal cardiac function. Though de-cellularization techniques now seem controlled, the issues posed by the selection of cells capable of generating the various components of cardiac tissue are not settled yet. In addition, the recolonisation of the matrix does not only depend on the phenotype of cells that are used, but it is also impacted by the nature of biochemical signals emitted.Recent researches have shown that it is possible to use decellularized whole liver treated by detergents as scaffold, which keeps the entire network of blood vessels and the integrated extracellular matrix (ECM). Beside of decellularized whole organ scaffold seeding cells selected to repopulate a decellularized liver scaffold are critical for the function of the bioengineered liver. At present, potential cell sources are hepatocyte, and mesenchymal stem cells.Pulmonary regeneration using engineering approaches is complex. In fact, several types of local progenitor cells that contribute to cell repair have been described at different levels of the respiratory tract. Moving towards the alveoles, one finds bronchioalveolar stem cells as well as epithelial cells and pneumocytes. A promising option to increase the donor organ pool is to use allogeneic or xenogeneic decellularized lungs as a scaffold to engineer functional lung tissue ex vivo.The kidney is certainly one of the most difficult organs to reconstruct due to its complex nature and the heterogeneous nature of the cells. There is relatively little research on auto-construction, and experiments have been performed on rats, pigs and monkeys.Nevertheless, before these therapeutic approaches can be applied in clinical practice, many researches are necessary to understand and in particular the behaviour of cells on the decellularized organs as well as the mechanisms of their interaction with the microenvironment. Current knowledges allow optimism for the future but definitive answers can only be given after long term animal studies and controlled clinical studies.

Emerging interactions between matrix components during biofilm development.

PubMed

Payne, David E; Boles, Blaise R

2016-02-01

Bacterial cells are most often found in the form of multicellular aggregates commonly referred to as biofilms. Biofilms offer their member cells several benefits, such as resistance to killing by antimicrobials and predation. During biofilm formation there is a production of extracellular substances that, upon assembly, constitute an extracellular matrix. The ability to generate a matrix encasing the microbial cells is a common feature of biofilms, but there is diversity in matrix composition and in interaction between matrix components. The different components of bacterial biofilm extracellular matrixes, known as matrix interactions, and resulting implications are discussed in this review.

Xenogeneic Acellular Conjunctiva Matrix as a Scaffold of Tissue-Engineered Corneal Epithelium

PubMed Central

Zhao, Haifeng; Qu, Mingli; Wang, Yao; Wang, Zhenyu; Shi, Weiyun

2014-01-01

Amniotic membrane-based tissue-engineered corneal epithelium has been widely used in the reconstruction of the ocular surface. However, it often degrades too early to ensure the success of the transplanted corneal epithelium when treating patients with severe ocular surface disorders. In the present study, we investigated the preparation of xenogeneic acellular conjunctiva matrix (aCM) and evaluated its efficacy and safety as a scaffold of tissue-engineered corneal epithelium. Native porcine conjunctiva was decellularized with 0.1% sodium dodecyl sulfate (SDS) for 12 h at 37°C and sterilized via γ-irradiation. Compared with native conjunctiva, more than 92% of the DNA was removed, and more than 90% of the extracellular matrix components (glycosaminoglycan and collagen) remained after the decellularization treatment. Compared with denuded amniotic membrane (dAM), the aCM possessed favorable optical transmittance, tensile strength, stability and biocompatibility as well as stronger resistance to degradation both in vitro and in vivo. The corneal epithelial cells seeded on aCM formed a multilayered epithelial structure and endured longer than did those on dAM. The aCM-based tissue-engineered corneal epithelium was more effective in the reconstruction of the ocular surface in rabbits with limbal stem cell deficiency. These findings support the application of xenogeneic acellular conjunctiva matrix as a scaffold for reconstructing the ocular surface. PMID:25375996

Fiber Development and Matrix Production in Tissue Engineered Menisci using Bovine Mesenchymal Stem Cells and Fibrochondrocytes

PubMed Central

McCorry, Mary Clare; Bonassar, Lawrence J.

2017-01-01

Mesenchymal stem cells (MSCs) have been investigated with promising results for meniscus healing and tissue engineering. While MSCs are known to contribute to ECM production, less is known about how MSCs produce and align large organized fibers for application to tissue engineering the meniscus. The goal of this study was to investigate the capability of MSCs to produce and organize extracellular matrix molecules compared to meniscal fibrochondrocytes (FCCs). Bovine FCCs and MSCs were encapsulated in an anatomically accurate collagen meniscus using mono-culture and co-culture of each cell type. Each meniscus was mechanically anchored at the horns to mimic the physiological fixation by the meniscal entheses. Mechanical fixation generates a static mechanical boundary condition previously shown to induce formation of oriented fiber by FCCs. Samples were cultured for 4 weeks and then evaluated for biochemical composition and fiber development. MSCs increased the GAG and collagen production in both co-culture and mono-culture groups compared to FCC mono-culture. Collagen organization was greatest in the FCC mono-culture group. While MSCs had increased matrix production they lacked the fiber organization capabilities of FCCs. This study suggests that GAG production and fiber formation are linked. Co-culture can be used as a means of balancing the synthetic properties of MSCs and the matrix remodeling capabilities of FCCs for tissue engineering applications. PMID:27925474

Chondrogenic potential of physically treated bovine cartilage matrix derived porous scaffolds on human dermal fibroblast cells.

PubMed

Moradi, Ali; Ataollahi, Forough; Sayar, Katayoun; Pramanik, Sumit; Chong, Pan-Pan; Khalil, Alizan Abdul; Kamarul, Tunku; Pingguan-Murphy, Belinda

2016-01-01

Extracellular matrices have drawn attention in tissue engineering as potential biomaterials for scaffold fabrication because of their bioactive components. Noninvasive techniques of scaffold fabrication and cross-linking treatments are believed to maintain the integrity of bioactive molecules while providing proper architectural and mechanical properties. Cartilage matrix derived scaffolds are designed to support the maintenance of chondrocytes and provide proper signals for differentiation of chondroinducible cells. Chondroinductive potential of bovine articular cartilage matrix derived porous scaffolds on human dermal fibroblasts and the effect of scaffold shrinkage on chondrogenesis were investigated. An increase in sulfated glycosaminoglycans production along with upregulation of chondrogenic genes confirmed that physically treated cartilage matrix derived scaffolds have chondrogenic potential on human dermal fibroblasts. © 2015 Wiley Periodicals, Inc.

Tissue-Engineered Nanofibrous Nerve Grafts for Enhancing the Rate of Nerve Regeneration

DTIC Science & Technology

2015-10-01

structured nanofibrous biodegradable nerve graft system that present ECM protein, neurotrophic factor, and pre-seeded with bone marrow stromal cells in...nanofibrous biodegradable nerve graft system that present extracellular matrix (ECM) protein, nerve growth factor, and pre-seeded with bone marrow stromal...proposed novel structured nanofibrous biodegradable grafts will provide the micro environment, bioactivity, transport features and mechanics ideal for

Bridging the gap: from 2D cell culture to 3D microengineered extracellular matrices

PubMed Central

Li, Yanfen

2016-01-01

Historically the culture of mammalian cells in the laboratory has been performed on planar substrates with media cocktails that are optimized to maintain phenotype. However, it is becoming increasingly clear that much of biology discerned from 2D studies does not translate well to the 3D microenvironment. Over the last several decades, 2D and 3D microengineering approaches have been developed that better recapitulate the complex architecture and properties of in vivo tissue. Inspired by the infrastructure of the microelectronics industry, lithographic patterning approaches have taken center stage because of the ease in which cell-sized features can be engineered on surfaces and within a broad range of biocompatible materials. Patterning and templating techniques enable precise control over extracellular matrix properties including: composition, mechanics, geometry, cell-cell contact, and diffusion. In this review article we will explore how the field of engineered extracellular matrices has evolved with the development of new hydrogel chemistry and the maturation of micro- and nano- fabrication. Guided by the spatiotemporal regulation of cell state in developing tissues, we will review the maturation of micropatterning in 2D, pseudo-3D systems, and patterning within 3D hydrogels in the context of translating the information gained from 2D systems to synthetic engineered 3D tissues. PMID:26592366

Six Years Experience with Porcine Extracellular Matrix: A New Paradigm for Pelvic Floor Repair

DTIC Science & Technology

2017-05-06

MDW/SGVU SUBJECT: Profess ional Presentation Approval 20 APR 20 17 1. Your paper, entitled Six Years Experience with Porcine Extracellular Matrix: A...NIA 6. TITLE OF MATERIAL TO BE PUBLISHED OR PRESENTED: Six Years Experience with Porcine Extracellular Matrix: A New Paradigm for Pelvic Floor Repair

Clinical applications of decellularized extracellular matrices for tissue engineering and regenerative medicine.

PubMed

Parmaksiz, Mahmut; Dogan, Arin; Odabas, Sedat; Elçin, A Eser; Elçin, Y Murat

2016-03-17

Decellularization is the process of removing the cellular components from tissues or organs. It is a promising technology for obtaining a biomaterial with a highly preserved extracellular matrix (ECM), which may also act as a biological scaffold for tissue engineering and regenerative therapies. Decellularized products are gaining clinical importance and market space due to their ease of standardized production, constant availability for grafting and mechanical or biochemical superiority against competing clinical options, yielding clinical results ahead of the ones with autografts in some applications. Current drawbacks and limitations of traditional treatments and clinical applications can be overcome by using decellularized or acellular matrices. Several companies are leading the market with versatile acellular products designed for diverse use in the reconstruction of tissues and organs. This review describes ECM-based decellularized and acellular products that are currently in use for different branches of clinic.

Evaluation of cultured human dermal- and dermo-epidermal substitutes focusing on extracellular matrix components: Comparison of protein and RNA analysis.

PubMed

Oostendorp, Corien; Meyer, Sarah; Sobrio, Monia; van Arendonk, Joyce; Reichmann, Ernst; Daamen, Willeke F; van Kuppevelt, Toin H

2017-05-01

Treatment of full-thickness skin defects with split-thickness skin grafts is generally associated with contraction and scar formation and cellular skin substitutes have been developed to improve skin regeneration. The evaluation of cultured skin substitutes is generally based on qualitative parameters focusing on histology. In this study we focused on quantitative evaluation to provide a template for comparison of human bio-engineered skin substitutes between clinical and/or research centers, and to supplement histological data. We focused on extracellular matrix proteins since these components play an important role in skin regeneration. As a model we analyzed the human dermal substitute denovoDerm and the dermo-epidermal skin substitute denovoSkin. The quantification of the extracellular matrix proteins type III collagen and laminin 5 in tissue homogenates using western blotting analysis and ELISA was not successful. The same was true for assaying lysyl oxidase, an enzyme involved in crosslinking of matrix molecules. As an alternative, gene expression levels were measured using qPCR. Various RNA isolation procedures were probed. The gene expression profile for specific dermal and epidermal genes could be measured reliably and reproducibly. Differences caused by changes in the cell culture conditions could easily be detected. The number of cells in the skin substitutes was measured using the PicoGreen dsDNA assay, which was found highly quantitative and reproducible. The (dis) advantages of assays used for quantitative evaluation of skin substitutes are discussed. Copyright © 2016 Elsevier Ltd and ISBI. All rights reserved.

Matrix modulation and heart failure: new concepts question old beliefs.

PubMed

Deschamps, Anne M; Spinale, Francis G

2005-05-01

Myocardial remodeling is a complex process involving several molecular and cellular factors. Extracellular matrix has been implicated in the remodeling process. Historically, the myocardial extracellular matrix was thought to serve solely as a means to align cells and provide structure to the tissue. Although this is one of its important functions, evidence suggests that the extracellular matrix plays a complex and divergent role in influencing cell behavior. This paper characterizes some of the notable studies on this dynamic entity and on adverse myocardial remodeling that have been published over the past year, which further question the belief that the extracellular matrix is a static structure. Progress has been made in understanding how the extracellular matrix is operative in the three major conditions (myocardial infarction, left ventricular hypertrophy due to overload, and dilated cardiomyopathy) that involve myocardial remodeling. Several studies have examined plasma profiles of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases following myocardial infarction and during left ventricular hypertrophy as surrogate markers of remodeling/remodeled myocardium. It has been demonstrated that bioactive signaling molecules and growth factors, proteases, and structural proteins influence cell-matrix interactions in the context of left ventricular hypertrophy. Finally, studies that either removed or added tissue inhibitor of metalloproteinases species in the myocardium demonstrated the importance of this regulatory protein in the remodeling process. Understanding the cellular and molecular triggers that in turn give rise to changes in the extracellular matrix could provide opportunities to modify the remodeling process.

Processed xenogenic cartilage as innovative biomatrix for cartilage tissue engineering: effects on chondrocyte differentiation and function.

PubMed

Schwarz, Silke; Elsaesser, Alexander F; Koerber, Ludwig; Goldberg-Bockhorn, Eva; Seitz, Andreas M; Bermueller, Christian; Dürselen, Lutz; Ignatius, Anita; Breiter, Roman; Rotter, Nicole

2015-12-01

One key point in the development of new bioimplant matrices for the reconstruction and replacement of cartilage defects is to provide an adequate microenvironment to ensure chondrocyte migration and de novo synthesis of cartilage-specific extracellular matrix (ECM). A recently developed decellularization and sterilization process maintains the three-dimensional (3D) collagen structure of native septal cartilage while increasing matrix porosity, which is considered to be crucial for cartilage tissue engineering. Human primary nasal septal chondrocytes were amplified in monolayer culture and 3D-cultured on processed porcine nasal septal cartilage scaffolds. The influence of chondrogenic growth factors on neosynthesis of ECM proteins was examined at the protein and gene expression levels. Seeding experiments demonstrated that processed xenogenic cartilage matrices provide excellent environmental properties for human nasal septal chondrocytes with respect to cell adhesion, migration into the matrix and neosynthesis of cartilage-specific ECM proteins, such as collagen type II and aggrecan. Matrix biomechanical stability indicated that the constructs retrieve full stability and function during 3D culture for up to 42 days, proportional to collagen type II and GAG production. Thus, processed xenogenic cartilage offers a suitable environment for human nasal chondrocytes and has promising potential for cartilage tissue engineering in the head and neck region. Copyright © 2012 John Wiley & Sons, Ltd.

Microfabrication of Cell-Laden Hydrogels for Engineering Mineralized and Load Bearing Tissues.

PubMed

Li, Chia-Cheng; Kharaziha, Mahshid; Min, Christine; Maas, Richard; Nikkhah, Mehdi

2015-01-01

Microengineering technologies and advanced biomaterials have extensive applications in the field of regenerative medicine. In this chapter, we review the integration of microfabrication techniques and hydrogel-based biomaterials in the field of dental, bone, and cartilage tissue engineering. We primarily discuss the major features that make hydrogels attractive candidates to mimic extracellular matrix (ECM), and we consider the benefits of three-dimensional (3D) culture systems for tissue engineering applications. We then focus on the fundamental principles of microfabrication techniques including photolithography, soft lithography and bioprinting approaches. Lastly, we summarize recent research on microengineering cell-laden hydrogel constructs for dental, bone and cartilage regeneration, and discuss future applications of microfabrication techniques for load-bearing tissue engineering.

Synthetic biodegradable functional polymers for tissue engineering: a brief review.

PubMed

BaoLin, Guo; Ma, Peter X

2014-04-01

Scaffolds play a crucial role in tissue engineering. Biodegradable polymers with great processing flexibility are the predominant scaffolding materials. Synthetic biodegradable polymers with well-defined structure and without immunological concerns associated with naturally derived polymers are widely used in tissue engineering. The synthetic biodegradable polymers that are widely used in tissue engineering, including polyesters, polyanhydrides, polyphosphazenes, polyurethane, and poly (glycerol sebacate) are summarized in this article. New developments in conducting polymers, photoresponsive polymers, amino-acid-based polymers, enzymatically degradable polymers, and peptide-activated polymers are also discussed. In addition to chemical functionalization, the scaffold designs that mimic the nano and micro features of the extracellular matrix (ECM) are presented as well, and composite and nanocomposite scaffolds are also reviewed.

Cell adhesion molecules, the extracellular matrix and oral squamous carcinoma.

PubMed

Lyons, A J; Jones, J

2007-08-01

Carcinomas are characterized by invasion of malignant cells into the underlying connective tissue and migration of malignant cells to form metastases at distant sites. These processes require alterations in cell-cell and cell-extracellular matrix interactions. As cell adhesion molecules play a role in cell-cell and cell-extracellular matrix adhesion and interactions they are involved in the process of tumour invasion and metastases. In epithelial tissues, receptors of the integrin family mediate adhesion to the adjacent matrix whereas cadherins largely mediate intercellular adhesion. These and other cell adhesion molecules such as intercellular adhesion molecule-1, CD44, dystroglycans and selectins, are involved and undergo changes in carcinomas, which provide possible targets for anti-cancer drug treatments. In the extracellular matrix that is associated with tumours, laminin 5, oncofetal fibronectin and tenascin C appear. The degree of expression of some of these moieties indicates prognosis in oral cancer and offer targets for antibody-directed radiotherapy. Metalloproteases which degrade the extracellular matrix are increased in carcinomas, and their activity is necessary for tumour angiogenesis and consequent invasion and metastases. Metalloprotease inhibitors have begun to produce decreases in mortality in clinical trials. This report provides a brief overview of our current understanding of cell adhesion molecules, the extracellular matrix, tumour invasion and metastasis.

Extracellular matrix directions estimation of the heart on micro-focus x-ray CT volumes

NASA Astrophysics Data System (ADS)

Oda, Hirohisa; Oda, Masahiro; Kitasaka, Takayuki; Akita, Toshiaki; Mori, Kensaku

2017-03-01

In this paper we propose an estimation method of extracellular matrix directions of the heart. Myofiber are surrounded by the myocardial cell sheets whose directions have strong correspondence between heart failure. Estimation of the myocardial cell sheet directions is difficult since they are very thin. Therefore, we estimate the extracellular matrices which are touching to the sheets as if piled up. First, we perform a segmentation of the extracellular matrices by using the Hessian analysis. Each extracellular matrix region has sheet-like shape. We estimate the direction of each extracellular matrix region by the principal component analysis (PCA). In our experiments, mean inclination angles of two normal canine hearts were 50.6 and 46.2 degrees, while the angle of a failing canine heart was 57.4 degrees. This results well fit the anatomical knowledge that failing hearts tend to have vertical myocardical cell sheets.

Development of a cellularly degradable PEG hydrogel to promote articular cartilage extracellular matrix deposition.

PubMed

Sridhar, Balaji V; Brock, John L; Silver, Jason S; Leight, Jennifer L; Randolph, Mark A; Anseth, Kristi S

2015-04-02

Healing articular cartilage remains a significant clinical challenge because of its limited self-healing capacity. While delivery of autologous chondrocytes to cartilage defects has received growing interest, combining cell-based therapies with scaffolds that capture aspects of native tissue and promote cell-mediated remodeling could improve outcomes. Currently, scaffold-based therapies with encapsulated chondrocytes permit matrix production; however, resorption of the scaffold does not match the rate of production by cells leading to generally low extracellular matrix outputs. Here, a poly (ethylene glycol) (PEG) norbornene hydrogel is functionalized with thiolated transforming growth factor (TGF-β1) and cross-linked by an MMP-degradable peptide. Chondrocytes are co-encapsulated with a smaller population of mesenchymal stem cells, with the goal of stimulating matrix production and increasing bulk mechanical properties of the scaffold. The co-encapsulated cells cleave the MMP-degradable target sequence more readily than either cell population alone. Relative to non-degradable gels, cellularly degraded materials show significantly increased glycosaminoglycan and collagen deposition over just 14 d of culture, while maintaining high levels of viability and producing a more widely-distributed matrix. These results indicate the potential of an enzymatically degradable, peptide-functionalized PEG hydrogel to locally influence and promote cartilage matrix production over a short period. Scaffolds that permit cell-mediated remodeling may be useful in designing treatment options for cartilage tissue engineering applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

What Makes the Optimal Wound Healing Material? A Review of Current Science and Introduction of a Synthetic Nanofabricated Wound Care Scaffold.

PubMed

MacEwan, Matthew R; MacEwan, Sarah; Kovacs, Tamas R; Batts, Joel

2017-10-02

Wound matrix materials are used to improve the regeneration of dermal and epidermal layers in both acute and chronic wounds. Contemporary wound matrices are primarily composed of biologic materials such as processed xenogeneic and allogeneic tissues. Unfortunately, existing biologic wound matrices possess multiple limitations including poor longevity, durability, strength, and enzymatic resistance required for persistent support for new tissue formation. A fully-synthetic, resorbable electrospun material (Restrata Wound Matrix, Acera, St.Louis, Missouri ) that exhibits structural similarities to the native extracellular matrix offers a new approach to the treatment of acute and chronic wounds. This novel matrix is the first product to combine the advantages of synthetic construction (e.g. resistance to enzymatic degradation, excellent biocompatibility, strength/durability and controlled degradation) with the positive attributes of biologic materials (e.g. biomimetic architecture similar to human extracellular matrix (ECM), fibrous architecture optimized to support cellular migration and proliferation, engineered porosity to encourage tissue ingrowth and vascularization). These features allow RWM to achieve rapid and complete healing of full-thickness wounds that, in preclinical studies, is comparable to Integra Bilayer Wound Matrix (Integra LifeSciences, Plainsboro, New Jersey), a gold standard biologic material with diverse clinical indications in the wound care. Together, this review suggests that the RWM offers a unique fully-synthetic alternative to existing biologic matrices that is effective, widely available, easy to store, simple to apply and low cost.

Advances in Tissue Regeneration

DTIC Science & Technology

2010-01-01

Conference Hand Transplants Face transplants Skin Graft Stretching 33 www.afirm.mil Josh Maloney 1st AFIRM Hand Transplant 2010 MHS Conference...34www.afirm.mil Cell spraying in place of skin grafting for burn AFIRM: clinical trials scheduled for FY 10 2010 MHS Conference Before After patients...ReCell) Using Extracellular matrix to regrow lost muscle tissue. Weeks 6 & 7 Week 9 Week 8 Week 10 Week 11 Autologous engineered skin grafts Not

Role of nanoparticle size in self-assemble processes of collagen for tissue engineering application.

PubMed

Vedhanayagam, Mohan; Nidhin, Marimuthu; Duraipandy, Natarajan; Naresh, Niranjan Dhanasekar; Jaganathan, Ganesh; Ranganathan, Mohan; Kiran, Manikantan Syamala; Narayan, Shoba; Nair, Balachandran Unni; Sreeram, Kalarical Janardhanan

2017-06-01

Nanoparticle mediated extracellular matrix may offer new and improved biomaterial to wound healing and tissue engineering applications. However, influence of nanoparticle size in extracellular matrix is still unclear. In this work, we synthesized different size of silver nanoparticles (AgNPs) comprising of 10nm, 35nm and 55nm using nutraceuticals (pectin) as reducing as well as stabilization agents through microwave irradiation method. Synthesized Ag-pectin nanoparticles were assimilated in the self-assemble process of collagen leading to fabricated collagen-Ag-pectin nanoparticle based scaffolds. Physico-chemical properties and biocompatibility of scaffolds were analyzed through FT-IR, SEM, DSC, mechanical strength analyzer, antibacterial activity and MTT assay. Our results suggested that 10nm sized Ag-pectin nanoparticles significantly increased the denaturation temperature (57.83°C) and mechanical strength (0.045MPa) in comparison with native collagen (50.29°C and 0.011MPa). The in vitro biocompatibility assay reveals that, collagen-Ag-pectin nanoparticle based scaffold provided higher antibacterial activity against to Gram positive and Gram negative as well as enhanced cell viability toward keratinocytes. This work opens up a possibility of employing the pectin caged silver nanoparticles to develop collagen-based nanoconstructs for biomedical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

N-Isopropylacrylamide-co-glycidylmethacrylate as a Thermoresponsive Substrate for Corneal Endothelial Cell Sheet Engineering

PubMed Central

Madathil, Bernadette K.; Anil Kumar, Pallickaveedu RajanAsari; Kumary, Thrikkovil Variyath

2014-01-01

Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate) polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty. PMID:25003113

Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials.

PubMed

Briquez, Priscilla S; Lorentz, Kristen M; Larsson, Hans M; Frey, Peter; Hubbell, Jeffrey A

2017-08-01

Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a human-derived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-β A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis in vitro, outperforming aprotinin. In vivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Chitosan/silk fibroin-based, Schwann cell-derived extracellular matrix-modified scaffolds for bridging rat sciatic nerve gaps.

PubMed

Gu, Yun; Zhu, Jianbin; Xue, Chengbin; Li, Zhenmeiyu; Ding, Fei; Yang, Yumin; Gu, Xiaosong

2014-02-01

Extracellular matrix (ECM) plays a prominent role in establishing and maintaining an ideal microenvironment for tissue regeneration, and ECM scaffolds are used as a feasible alternative to cellular and molecular therapy in the fields of tissue engineering. Because of their advantages over tissue-derived ECM scaffolds, cultured cell-derived ECM scaffolds are beginning to attract attention, but they have been scarcely studied for peripheral nerve repair. Here we aimed to develop a tissue engineered nerve scaffold by reconstituting nerve cell-derived ECM with natural biomaterials. A protocol was adopted to prepare and characterize the cultured Schwann cell (SC)-derived ECM. A chitosan conduit and silk fibroin (SF) fibers were prepared, cultured with SCs for ECM deposition, and subjected to decellularization, followed by assembly into a chitosan/SF-based, SC-derived ECM-modified scaffold, which was used to bridge a 10 mm rat sciatic nerve gap. The results from morphological analysis as well as electrophysiological examination indicated that regenerative outcomes achieved by our developed scaffold were similar to those by an acellular nerve graft (namely a nerve tissue-derived ECM scaffold), but superior to those by a plain chitosan/SF scaffold. Moreover, blood and histopathological parameters confirmed the safety of scaffold modification by SC-derived ECM. Therefore, a hybrid scaffold based on joint use of acellular and classical biomaterials represents a promising approach to nerve tissue engineering. Copyright © 2013 Elsevier Ltd. All rights reserved.

Guiding tissue regeneration with ultrasound in vitro and in vivo

NASA Astrophysics Data System (ADS)

Dalecki, Diane; Comeau, Eric S.; Raeman, Carol H.; Child, Sally Z.; Hobbs, Laura; Hocking, Denise C.

2015-05-01

Developing new technologies that enable the repair or replacement of injured or diseased tissues is a major focus of regenerative medicine. This paper will discuss three ultrasound technologies under development in our laboratories to guide tissue regeneration both in vitro and in vivo. A critical obstacle in tissue engineering is the need for rapid and effective tissue vascularization strategies. To address this challenge, we are developing acoustic patterning techniques for microvascular tissue engineering. Acoustic radiation forces associated with ultrasound standing wave fields provide a rapid, non-invasive approach to spatially pattern cells in three dimensions without affecting cell viability. Acoustic patterning of endothelial cells leads to the rapid formation of microvascular networks throughout the volumes of three-dimensional hydrogels, and the morphology of the resultant microvessel networks can be controlled by design of the ultrasound field. A second technology under development uses ultrasound to noninvasively control the microstructure of collagen fibers within engineered tissues. The microstructure of extracellular matrix proteins provides signals that direct cell functions critical to tissue regeneration. Thus, controlling collagen microfiber structure with ultrasound provides a noninvasive approach to regulate the mechanical properties of biomaterials and control cellular responses. The third technology employs therapeutic ultrasound to enhance the healing of chronic wounds. Recent studies demonstrate increased granulation tissue thickness and collagen deposition in murine dermal wounds exposed to pulsed ultrasound. In summary, ultrasound technologies offer noninvasive approaches to control cell behaviors and extracellular matrix organization and thus hold great promise to advance tissue regeneration in vitro and in vivo.

Laser surface modification of decellularized extracellular cartilage matrix for cartilage tissue engineering.

PubMed

Goldberg-Bockhorn, Eva; Schwarz, Silke; Subedi, Rachana; Elsässer, Alexander; Riepl, Ricarda; Walther, Paul; Körber, Ludwig; Breiter, Roman; Stock, Karl; Rotter, Nicole

2018-02-01

The implantation of autologous cartilage as the gold standard operative procedure for the reconstruction of cartilage defects in the head and neck region unfortunately implicates a variety of negative effects at the donor site. Tissue-engineered cartilage appears to be a promising alternative. However, due to the complex requirements, the optimal material is yet to be determined. As demonstrated previously, decellularized porcine cartilage (DECM) might be a good option to engineer vital cartilage. As the dense structure of DECM limits cellular infiltration, we investigated surface modifications of the scaffolds by carbon dioxide (CO 2 ) and Er:YAG laser application to facilitate the migration of chondrocytes inside the scaffold. After laser treatment, the scaffolds were seeded with human nasal septal chondrocytes and analyzed with respect to cell migration and formation of new extracellular matrix proteins. Histology, immunohistochemistry, SEM, and TEM examination revealed an increase of the scaffolds' surface area with proliferation of cell numbers on the scaffolds for both laser types. The lack of cytotoxic effects was demonstrated by standard cytotoxicity testing. However, a thermal denaturation area seemed to hinder the migration of the chondrocytes inside the scaffolds, even more so after CO 2 laser treatment. Therefore, the Er:YAG laser seemed to be better suitable. Further modifications of the laser adjustments or the use of alternative laser systems might be advantageous for surface enlargement and to facilitate migration of chondrocytes into the scaffold in one step.

Culture of human anulus fibrosus cells on polyamide nanofibers: extracellular matrix production.

PubMed

Gruber, Helen E; Hoelscher, Gretchen; Ingram, Jane A; Hanley, Edward N

2009-01-01

Studies were approved by the authors' Human Subjects Institutional Review Board. Human anulus cells were tested for growth and extracellular matrix (ECM) production in vitro. To investigate cell attachment, cell proliferation, and ECM production of human intervertebral disc anulus cells seeded onto randomly oriented electrospun polyamide nanofibers. Because nanofibrillar matrices have the potential to promote microenvironments, which may mimic in vivo conditions and resemble connective tissue, their utilization opens new avenues for cell-based tissue engineering applications for disc cells. Anulus cells were isolated from 4 cervical spine surgical disc specimens, expanded, and seeded into either routine plastic culture (control) or a nanofiber surface of randomly oriented electrospun polyamide nanofibers (Ultra-Web-coated culture dish, Corning) with a positive charge or without a charge. Cells were cultured for 9 days, digital images captured, cells harvested, embedded in paraffin, and examined for production of extracellular matrix (ECM). Additional anulus cultures were tested to quantitatively assess total proteoglycan production and cell proliferation under control or nanofiber cultures. Cells attached well and exhibited cell extensions within the nanofiber layers; cells on the charged nanofiber surface deposited greater amounts of chondroitin sulfate than of type II collagen than cells cultured on the uncharged nanofiber surface. Results showed that culture of anulus cells on nanofibers was permissive for secretion and assembly of type II collagen and chondroitin sulfate. Significantly greater total proteoglycan formation was present after culture on the nanofiber with added charge conditions {control, 0.6116 microg/mL +/- 0.186 [4] [mean +/- sem(n)] vs. 1.201 +/- 0.2509 [4], P < 0.05}. Cell proliferation, however, did not differ among treatment groups. Culture of anulus cells on nanofibers was found to be permissive for secretion and assembly of type II collagen and chondroitin sulfate, and culture on nanofibers with added charge significantly increased total proteoglycan production. These novel findings point to the need for further examination of nanofibrillar 3D culture of anulus cells for tissue engineering applications.

Pulmonary immunity and extracellular matrix interactions.

PubMed

O'Dwyer, David N; Gurczynski, Stephen J; Moore, Bethany B

2018-04-09

The lung harbors a complex immune system composed of both innate and adaptive immune cells. Recognition of infection and injury by receptors on lung innate immune cells is crucial for generation of antigen-specific responses by adaptive immune cells. The extracellular matrix of the lung, comprising the interstitium and basement membrane, plays a key role in the regulation of these immune systems. The matrix consists of several hundred assembled proteins that interact to form a bioactive scaffold. This template, modified by enzymes, acts to facilitate cell function and differentiation and changes dynamically with age and lung disease. Herein, we explore relationships between innate and adaptive immunity and the lung extracellular matrix. We discuss the interactions between extracellular matrix proteins, including glycosaminoglycans, with prominent effects on innate immune signaling effectors such as toll-like receptors. We describe the relationship of extracellular matrix proteins with adaptive immunity and leukocyte migration to sites of injury within the lung. Further study of these interactions will lead to greater knowledge of the role of matrix biology in lung immunity. The development of novel therapies for acute and chronic lung disease is dependent on a comprehensive understanding of these complex matrix-immunity interactions. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Controlling the Porosity and Microarchitecture of Hydrogels for Tissue Engineering

PubMed Central

Annabi, Nasim; Nichol, Jason W.; Zhong, Xia; Ji, Chengdong; Koshy, Sandeep; Khademhosseini, Ali

2010-01-01

Tissue engineering holds great promise for regeneration and repair of diseased tissues, making the development of tissue engineering scaffolds a topic of great interest in biomedical research. Because of their biocompatibility and similarities to native extracellular matrix, hydrogels have emerged as leading candidates for engineered tissue scaffolds. However, precise control of hydrogel properties, such as porosity, remains a challenge. Traditional techniques for creating bulk porosity in polymers have demonstrated success in hydrogels for tissue engineering; however, often the conditions are incompatible with direct cell encapsulation. Emerging technologies have demonstrated the ability to control porosity and the microarchitectural features in hydrogels, creating engineered tissues with structure and function similar to native tissues. In this review, we explore the various technologies for controlling the porosity and microarchitecture within hydrogels, and demonstrate successful applications of combining these techniques. PMID:20121414

The ECM moves during primitive streak formation--computation of ECM versus cellular motion.

PubMed

Zamir, Evan A; Rongish, Brenda J; Little, Charles D

2008-10-14

Galileo described the concept of motion relativity--motion with respect to a reference frame--in 1632. He noted that a person below deck would be unable to discern whether the boat was moving. Embryologists, while recognizing that embryonic tissues undergo large-scale deformations, have failed to account for relative motion when analyzing cell motility data. A century of scientific articles has advanced the concept that embryonic cells move ("migrate") in an autonomous fashion such that, as time progresses, the cells and their progeny assemble an embryo. In sharp contrast, the motion of the surrounding extracellular matrix scaffold has been largely ignored/overlooked. We developed computational/optical methods that measure the extent embryonic cells move relative to the extracellular matrix. Our time-lapse data show that epiblastic cells largely move in concert with a sub-epiblastic extracellular matrix during stages 2 and 3 in primitive streak quail embryos. In other words, there is little cellular motion relative to the extracellular matrix scaffold--both components move together as a tissue. The extracellular matrix displacements exhibit bilateral vortical motion, convergence to the midline, and extension along the presumptive vertebral axis--all patterns previously attributed solely to cellular "migration." Our time-resolved data pose new challenges for understanding how extracellular chemical (morphogen) gradients, widely hypothesized to guide cellular trajectories at early gastrulation stages, are maintained in this dynamic extracellular environment. We conclude that models describing primitive streak cellular guidance mechanisms must be able to account for sub-epiblastic extracellular matrix displacements.

Electrospun Fibrous Scaffolds for Tissue Engineering: Viewpoints on Architecture and Fabrication.

PubMed

Jun, Indong; Han, Hyung-Seop; Edwards, James R; Jeon, Hojeong

2018-03-06

Electrospinning has been used for the fabrication of extracellular matrix (ECM)-mimicking fibrous scaffolds for several decades. Electrospun fibrous scaffolds provide nanoscale/microscale fibrous structures with interconnecting pores, resembling natural ECM in tissues, and showing a high potential to facilitate the formation of artificial functional tissues. In this review, we summarize the fundamental principles of electrospinning processes for generating complex fibrous scaffold geometries that are similar in structural complexity to the ECM of living tissues. Moreover, several approaches for the formation of three-dimensional fibrous scaffolds arranged in hierarchical structures for tissue engineering are also presented.

A Review of Cellularization Strategies for Tissue Engineering of Whole Organs

PubMed Central

Scarritt, Michelle E.; Pashos, Nicholas C.; Bunnell, Bruce A.

2015-01-01

With the advent of whole organ decellularization, extracellular matrix scaffolds suitable for organ engineering were generated from numerous tissues, including the heart, lung, liver, kidney, and pancreas, for use as alternatives to traditional organ transplantation. Biomedical researchers now face the challenge of adequately and efficiently recellularizing these organ scaffolds. Herein, an overview of whole organ decellularization and a thorough review of the current literature for whole organ recellularization are presented. The cell types, delivery methods, and bioreactors employed for recellularization are discussed along with commercial and clinical considerations, such as immunogenicity, biocompatibility, and Food and Drug Administartion regulation. PMID:25870857

Creating biomaterials with spatially organized functionality.

PubMed

Chow, Lesley W; Fischer, Jacob F

2016-05-01

Biomaterials for tissue engineering provide scaffolds to support cells and guide tissue regeneration. Despite significant advances in biomaterials design and fabrication techniques, engineered tissue constructs remain functionally inferior to native tissues. This is largely due to the inability to recreate the complex and dynamic hierarchical organization of the extracellular matrix components, which is intimately linked to a tissue's biological function. This review discusses current state-of-the-art strategies to control the spatial presentation of physical and biochemical cues within a biomaterial to recapitulate native tissue organization and function. © 2016 by the Society for Experimental Biology and Medicine.

Biophysical properties of dermal building-blocks affects extra cellular matrix assembly in 3D endogenous macrotissue.

PubMed

Urciuolo, F; Garziano, A; Imparato, G; Panzetta, V; Fusco, S; Casale, C; Netti, P A

2016-01-29

The fabrication of functional tissue units is one of the major challenges in tissue engineering due to their in vitro use in tissue-on-chip systems, as well as in modular tissue engineering for the construction of macrotissue analogs. In this work, we aim to engineer dermal tissue micromodules obtained by culturing human dermal fibroblasts into porous gelatine microscaffold. We proved that such stromal cells coupled with gelatine microscaffolds are able to synthesize and to assemble an endogenous extracellular matrix (ECM) resulting in tissue micromodules, which evolve their biophysical features over the time. In particular, we found a time-dependent variation of oxygen consumption kinetic parameters, of newly formed ECM stiffness and of micromodules self-aggregation properties. As consequence when used as building blocks to fabricate larger tissues, the initial tissue micromodules state strongly affects the ECM organization and maturation in the final macrotissue. Such results highlight the role of the micromodules properties in controlling the formation of three-dimensional macrotissue in vitro, defining an innovative design criterion for selecting tissue-building blocks for modular tissue engineering.

Extraction and Assembly of Tissue-Derived Gels for Cell Culture and Tissue Engineering

PubMed Central

Uriel, Shiri; Labay, Edwardine; Francis-Sedlak, Megan; Moya, Monica L.; Weichselbaum, Ralph R.; Ervin, Natalia; Cankova, Zdravka

2009-01-01

Interactions with the extracellular matrix (ECM) play an important role in regulating cell function. Cells cultured in, or on, three-dimensional ECM recapitulate similar features to those found in vivo that are not present in traditional two-dimensional culture. In addition, both natural and synthetic materials containing ECM components have shown promise in a number of tissue engineering applications. Current materials available for cell culture and tissue engineering do not adequately reflect the diversity of ECM composition between tissues. In this paper, a method is presented for extracting solutions of proteins and glycoproteins from soft tissues and inducing assembly of these proteins into gels. The extracts contain ECM proteins specific to the tissue source with low levels of intracellular molecules. Gels formed from the tissue-derived extracts have nanostructure similar to ECM in vivo and can be used to culture cells as both a thin substrate coating and a thick gel. This technique could be used to assemble hydrogels with varying composition depending upon the tissue source, hydrogels for three-dimensional culture, as scaffolds for tissue engineering therapies, and to study cell–matrix interactions. PMID:19115821

“Engineering Substrate Micro- and Nanotopography to Control Cell Function”

PubMed Central

Bettinger, Christopher J; Langer, Robert; Borenstein, Jeffrey T

2010-01-01

Lead-In The interaction of mammalian cells with nanoscale topography has proven to be an important signaling modality in controlling cell function. Naturally occurring nanotopographic structures within the extracellular matrix present surrounding cells with mechanotransductive cues that influence local migration, cell polarization, and other functions. Synthetically nanofabricated topography can also influence cell morphology, alignment, adhesion, migration, proliferation, and cytoskeleton organization. Here we review the use of in vitro synthetic cell-nanotopography interactions to control cell behavior and influence complex cellular processes including stem cell differentiation and tissue organization. Future challenges and opportunities in cell-nanotopography engineering will also be discussed including the elucidation of mechanisms and applications in tissue engineering. PMID:19492373

Design and preparation of polymeric scaffolds for tissue engineering.

PubMed

Weigel, Thomas; Schinkel, Gregor; Lendlein, Andreas

2006-11-01

Polymeric scaffolds for tissue engineering can be prepared with a multitude of different techniques. Many diverse approaches have recently been under development. The adaptation of conventional preparation methods, such as electrospinning, induced phase separation of polymer solutions or porogen leaching, which were developed originally for other research areas, are described. In addition, the utilization of novel fabrication techniques, such as rapid prototyping or solid free-form procedures, with their many different methods to generate or to embody scaffold structures or the usage of self-assembly systems that mimic the properties of the extracellular matrix are also described. These methods are reviewed and evaluated with specific regard to their utility in the area of tissue engineering.

Agarose-based biomaterials for tissue engineering.

PubMed

Zarrintaj, Payam; Manouchehri, Saeed; Ahmadi, Zahed; Saeb, Mohammad Reza; Urbanska, Aleksandra M; Kaplan, David L; Mozafari, Masoud

2018-05-01

Agarose is a natural polysaccharide polymer having unique characteristics that give reason to consider it for tissue engineering applications. Special characteristics of agarose such as its excellent biocompatibility, thermo-reversible gelation behavior and physiochemical features support its use as a biomaterial for cell growth and/or controlled/localized drug delivery. The resemblance of this natural carbohydrate polymer to the extracellular matrix results in attractive features that bring about a strong interest in its usage in the field. The scope of this review is to summarize the extensive researches addressing agarose-based biomaterials in order to provide an in-depth understanding of its tissue engineering-related applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Three-dimensional bioprinting of thick vascularized tissues

NASA Astrophysics Data System (ADS)

Kolesky, David B.; Homan, Kimberly A.; Skylar-Scott, Mark A.; Lewis, Jennifer A.

2016-03-01

The advancement of tissue and, ultimately, organ engineering requires the ability to pattern human tissues composed of cells, extracellular matrix, and vasculature with controlled microenvironments that can be sustained over prolonged time periods. To date, bioprinting methods have yielded thin tissues that only survive for short durations. To improve their physiological relevance, we report a method for bioprinting 3D cell-laden, vascularized tissues that exceed 1 cm in thickness and can be perfused on chip for long time periods (>6 wk). Specifically, we integrate parenchyma, stroma, and endothelium into a single thick tissue by coprinting multiple inks composed of human mesenchymal stem cells (hMSCs) and human neonatal dermal fibroblasts (hNDFs) within a customized extracellular matrix alongside embedded vasculature, which is subsequently lined with human umbilical vein endothelial cells (HUVECs). These thick vascularized tissues are actively perfused with growth factors to differentiate hMSCs toward an osteogenic lineage in situ. This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.

Extracellular Matrix Scaffold Technology for Bioartificial Pancreas Engineering: State of the Art and Future Challenges.

PubMed

Salvatori, Marcus; Katari, Ravi; Patel, Timil; Peloso, Andrea; Mugweru, Jon; Owusu, Kofi; Orlando, Giuseppe

2014-01-01

Emergent technologies in regenerative medicine may soon overcome the limitations of conventional diabetes therapies. Collaborative efforts across the subfields of stem cell technology, islet encapsulation, and biomaterial carriers seek to produce a bioengineered pancreas capable of restoring endocrine function in patients with insulin-dependent diabetes. These technologies rely on a robust understanding of the extracellular matrix (ECM), the supportive 3-dimensional network of proteins necessary for cellular attachment, proliferation, and differentiation. Although these functions can be partially approximated by biosynthetic carriers, novel decellularization protocols have allowed researchers to discover the advantages afforded by the native pancreatic ECM. The native ECM has proven to be an optimal platform for recellularization and whole-organ pancreas bioengineering, an exciting new field with the potential to resolve the dire shortage of transplantable organs. This review seeks to contextualize recent findings, discuss current research goals, and identify future challenges of regenerative medicine as it applies to diabetes management. © 2014 Diabetes Technology Society.

Structural and biomechanical characterizations of porcine myocardial extracellular matrix

PubMed Central

Wang, Bo; Tedder, Mary E.; Perez, Clara E.; Wang, Guangjun; de Jongh Curry, Amy L.; To, Filip; Elder, Steven H.; Williams, Lakiesha N.; Simionescu, Dan T.; Liao, Jun

2012-01-01

Extracellular matrix (ECM) of myocardium plays an important role to maintain a multilayered helical architecture of cardiomyocytes. In this study, we have characterized the structural and biomechanical properties of porcine myocardial ECM. Fresh myocardium were decellularized in a rotating bioreactor using 0.1 % sodium dodecyl sulfate solution. Masson’s trichrome staining and SEM demonstrated the removal of cells and preservation of the interconnected 3D cardiomyocyte lacunae. Movat’s pentachrome staining showed the preservation of cardiac elastin ultrastructure and vascular elastin distribution/alignment. DNA assay result confirmed a 98.59 % reduction in DNA content; the acellular myocardial scaffolds were found completely lack of staining for the porcine α-Gal antigen; and the accelerating enzymatic degradation assessment showed a constant degradation rate. Tensile and shear properties of the acellular myocardial scaffolds were also evaluated. Our observations showed that the acellular myocardial ECM possessed important traits of biodegradable scaffolds, indicating the potentials in cardiac regeneration and whole heart tissue engineering. PMID:22584822

Visualizing cell extracellular matrix (ECM) deposited by cells cultured on aligned bacteriophage M13 thin films.

PubMed

Wu, Laying; Lee, L Andrew; Niu, Zhongwei; Ghoshroy, Soumitra; Wang, Qian

2011-08-02

Topographical features ranging from micro- to nanometers can affect cell orientation and migratory pathways, which are important factors in tissue engineering and tumor migration. In our previous study, a convective assembly of bacteriophage M13 resulted in thin films which could be used to control the alignment of cells. However, several questions regarding its underlying reasons to dictate cell alignment remained unanswered. Here, we further study the nanometer topographical features generated by the bacteriophage M13 crystalline film, which results in the alignment of the cells and extracellular matrix (ECM) proteins. Sequential imaging analyses at micro- and nanoscale levels of aligned cells and fibrillar matrix proteins were documented using scanning electron microscopy and immunofluorescence microscopy. As a result, we observed baby hamster kidney cells with higher degree of alignment on the ordered M13 substrates than NIH-3T3 fibroblasts, a difference which could be attributed to the intrinsic nature of the cells' production of ECM proteins. The results from this study provide a crucial insight into the topographical features of a biological thin film, which can be utilized to control the orientation of cells and surrounding ECM proteins.

XanoMatrix surfaces as scaffolds for mesenchymal stem cell culture and growth

PubMed Central

Bhardwaj, Garima; Webster, Thomas J

2016-01-01

Stem cells are being widely investigated for a wide variety of applications in tissue engineering due to their ability to differentiate into a number of cells such as neurons, osteoblasts, and fibroblasts. This ability of stem cells to differentiate into different types of cells is greatly based on mechanical and chemical cues received from their three-dimensional environments. All organs are formed by a number of cells linked together via an extracellular matrix (ECM). The ECM is a complex network of proteins and carbohydrates, which occupies intercellular spaces and regulates cellular activity by controlling cell adhesion, migration, proliferation, and differentiation. The ECM is composed of two main types of macromolecules, namely, polysaccharide glycosaminoglycans, which are covalently attached to proteins in the form of proteoglycans and fibrous proteins belonging to two functional groups, structural (collagen and elastin) and adhesive (fibronectin, laminin, vitronectin, etc). Tissue engineering is a multidisciplinary field that aims to develop biomimetic scaffolds that emulate properties of the ECM to help repair or regenerate diseased or damaged tissue. This study introduces one of these matrices, XanoMatrix, as an optimal scaffold for tissue engineering applications, in particular, for stem cell research, based on its composition, nanofibrous structure, and porosity. Results of this study suggest that XanoMatrix scaffolds are promising for stem cell tissue engineering applications and as improved cell culture inserts for studying stem cell functions (compared to traditional Corning and Falcon cell culture plates) and, thus, should be further studied. PMID:27354795

Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions

PubMed Central

Kean, Thomas J.; Dennis, James E.

2015-01-01

Background Current tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential. Methods Porcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically. Results Human chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions. Conclusions Synoviocyte-derived matrix supports enhanced expansion of human chondrocytes such that the chondrocytes are maintained in a state from which they can re-differentiate into a cartilage phenotype after significantly more population doublings. Also, low oxygen tension supports GAG, but not collagen, accumulation. These findings are a step towards the production of a more functional, tissue engineered cartilage. PMID:26075742

Development of hydrogels for regenerative engineering.

PubMed

Guan, Xiaofei; Avci-Adali, Meltem; Alarçin, Emine; Cheng, Hao; Kashaf, Sara Saheb; Li, Yuxiao; Chawla, Aditya; Jang, Hae Lin; Khademhosseini, Ali

2017-05-01

The aim of regenerative engineering is to restore complex tissues and biological systems through convergence in the fields of advanced biomaterials, stem cell science, and developmental biology. Hydrogels are one of the most attractive biomaterials for regenerative engineering, since they can be engineered into tissue mimetic 3D scaffolds to support cell growth due to their similarity to native extracellular matrix. Advanced nano- and micro-technologies have dramatically increased the ability to control properties and functionalities of hydrogel materials by facilitating biomimetic fabrication of more sophisticated compositions and architectures, thus extending our understanding of cell-matrix interactions at the nanoscale. With this perspective, this review discusses the most commonly used hydrogel materials and their fabrication strategies for regenerative engineering. We highlight the physical, chemical, and functional modulation of hydrogels to design and engineer biomimetic tissues based on recent achievements in nano- and micro-technologies. In addition, current hydrogel-based regenerative engineering strategies for treating multiple tissues, such as musculoskeletal, nervous and cardiac tissue, are also covered in this review. The interaction of multiple disciplines including materials science, cell biology, and chemistry, will further play an important role in the design of functional hydrogels for the regeneration of complex tissues. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of freezing-induced cell-fluid-matrix interactions on the cells and extracellular matrix of engineered tissues.

PubMed

Teo, Ka Yaw; DeHoyos, Tenok O; Dutton, J Craig; Grinnell, Frederick; Han, Bumsoo

2011-08-01

The two most significant challenges for successful cryopreservation of engineered tissues (ETs) are preserving tissue functionality and controlling highly tissue-type dependent preservation outcomes. In order to address these challenges, freezing-induced cell-fluid-matrix interactions should be understood, which determine the post-thaw cell viability and extracellular matrix (ECM) microstructure. However, the current understanding of this tissue-level biophysical interaction is still limited. In this study, freezing-induced cell-fluid-matrix interactions and their impact on the cells and ECM microstructure of ETs were investigated using dermal equivalents as a model ET. The dermal equivalents were constructed by seeding human dermal fibroblasts in type I collagen matrices with varying cell seeding density and collagen concentration. While these dermal equivalents underwent an identical freeze/thaw condition, their spatiotemporal deformation during freezing, post-thaw ECM microstructure, and cellular level cryoresponse were characterized. The results showed that the extent and characteristics of freezing-induced deformation were significantly different among the experimental groups, and the ETs with denser ECM microstructure experienced a larger deformation. The magnitude of the deformation was well correlated to the post-thaw ECM structure, suggesting that the freezing-induced deformation is a good indicator of post-thaw ECM structure. A significant difference in the extent of cellular injury was also noted among the experimental groups, and it depended on the extent of freezing-induced deformation of the ETs and the initial cytoskeleton organization. These results suggest that the cells have been subjected to mechanical insult due to the freezing-induced deformation as well as thermal insult. These findings provide insight on tissue-type dependent cryopreservation outcomes, and can help to design and modify cryopreservation protocols for new types of tissues from a pre-developed cryopreservation protocol. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mesenchymal stem cell-derived extracellular matrix enhances chondrogenic phenotype of and cartilage formation by encapsulated chondrocytes in vitro and in vivo.

PubMed

Yang, Yuanheng; Lin, Hang; Shen, He; Wang, Bing; Lei, Guanghua; Tuan, Rocky S

2018-03-15

Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21 days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14 days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo. Current cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for xenogenic scaffolds, and suboptimal cartilage formation. We present here a novel technique that utilizes adult stem cell-derived extracellular matrix, as a culture substrate and/or encapsulation scaffold for human adult chondrocytes, for the repair of cartilage defects. Chondrocytes cultured in stem cell-derived matrix showed higher proliferation, better chondrocytic phenotype, and improved redifferentiation ability upon in vitro culture expansion. Most importantly, 3-dimensional constructs formed from chondrocytes folded within stem cell matrix manifested excellent cartilage formation both in vitro and in vivo. These findings demonstrate the suitability of stem cell-derived extracellular matrix as a culture substrate for chondrocyte expansion as well as a candidate bioactive matrix for cartilage regeneration. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

Bridging the Gap: From 2D Cell Culture to 3D Microengineered Extracellular Matrices.

PubMed

Li, Yanfen; Kilian, Kristopher A

2015-12-30

Historically the culture of mammalian cells in the laboratory has been performed on planar substrates with media cocktails that are optimized to maintain phenotype. However, it is becoming increasingly clear that much of biology discerned from 2D studies does not translate well to the 3D microenvironment. Over the last several decades, 2D and 3D microengineering approaches have been developed that better recapitulate the complex architecture and properties of in vivo tissue. Inspired by the infrastructure of the microelectronics industry, lithographic patterning approaches have taken center stage because of the ease in which cell-sized features can be engineered on surfaces and within a broad range of biocompatible materials. Patterning and templating techniques enable precise control over extracellular matrix properties including: composition, mechanics, geometry, cell-cell contact, and diffusion. In this review article we explore how the field of engineered extracellular matrices has evolved with the development of new hydrogel chemistry and the maturation of micro- and nano- fabrication. Guided by the spatiotemporal regulation of cell state in developing tissues, techniques for micropatterning in 2D, pseudo-3D systems, and patterning within 3D hydrogels will be discussed in the context of translating the information gained from 2D systems to synthetic engineered 3D tissues. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acellular Mouse Kidney ECM can be Used as a Three-Dimensional Substrate to Test the Differentiation Potential of Embryonic Stem Cell Derived Renal Progenitors.

PubMed

Sambi, Manpreet; Chow, Theresa; Whiteley, Jennifer; Li, Mira; Chua, Shawn; Raileanu, Vanessa; Rogers, Ian M

2017-08-01

The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.

The emergence of extracellular matrix mechanics and cell traction forces as important regulators of cellular self-organization.

PubMed

Checa, Sara; Rausch, Manuel K; Petersen, Ansgar; Kuhl, Ellen; Duda, Georg N

2015-01-01

Physical cues play a fundamental role in a wide range of biological processes, such as embryogenesis, wound healing, tumour invasion and connective tissue morphogenesis. Although it is well known that during these processes, cells continuously interact with the local extracellular matrix (ECM) through cell traction forces, the role of these mechanical interactions on large scale cellular and matrix organization remains largely unknown. In this study, we use a simple theoretical model to investigate cellular and matrix organization as a result of mechanical feedback signals between cells and the surrounding ECM. The model includes bi-directional coupling through cellular traction forces to deform the ECM and through matrix deformation to trigger cellular migration. In addition, we incorporate the mechanical contribution of matrix fibres and their reorganization by the cells. We show that a group of contractile cells will self-polarize at a large scale, even in homogeneous environments. In addition, our simulations mimic the experimentally observed alignment of cells in the direction of maximum stiffness and the building up of tension as a consequence of cell and fibre reorganization. Moreover, we demonstrate that cellular organization is tightly linked to the mechanical feedback loop between cells and matrix. Cells with a preference for stiff environments have a tendency to form chains, while cells with a tendency for soft environments tend to form clusters. The model presented here illustrates the potential of simple physical cues and their impact on cellular self-organization. It can be used in applications where cell-matrix interactions play a key role, such as in the design of tissue engineering scaffolds and to gain a basic understanding of pattern formation in organogenesis or tissue regeneration.

Similar Properties of Chondrocytes from Osteoarthritis Joints and Mesenchymal Stem Cells from Healthy Donors for Tissue Engineering of Articular Cartilage

PubMed Central

Fernandes, Amilton M.; Herlofsen, Sarah R.; Karlsen, Tommy A.; Küchler, Axel M.; Fløisand, Yngvar; Brinchmann, Jan E.

2013-01-01

Lesions of hyaline cartilage do not heal spontaneously, and represent a therapeutic challenge. In vitro engineering of articular cartilage using cells and biomaterials may prove to be the best solution. Patients with osteoarthritis (OA) may require tissue engineered cartilage therapy. Chondrocytes obtained from OA joints are thought to be involved in the disease process, and thus to be of insufficient quality to be used for repair strategies. Bone marrow (BM) derived mesenchymal stem cells (MSCs) from healthy donors may represent an alternative cell source. We have isolated chondrocytes from OA joints, performed cell culture expansion and tissue engineering of cartilage using a disc-shaped alginate scaffold and chondrogenic differentiation medium. We performed real-time reverse transcriptase quantitative PCR and fluorescence immunohistochemistry to evaluate mRNA and protein expression for a range of molecules involved in chondrogenesis and OA pathogenesis. Results were compared with those obtained by using BM-MSCs in an identical tissue engineering strategy. Finally the two populations were compared using genome-wide mRNA arrays. At three weeks of chondrogenic differentiation we found high and similar levels of hyaline cartilage-specific type II collagen and fibrocartilage-specific type I collagen mRNA and protein in discs containing OA and BM-MSC derived chondrocytes. Aggrecan, the dominant proteoglycan in hyaline cartilage, was more abundantly distributed in the OA chondrocyte extracellular matrix. OA chondrocytes expressed higher mRNA levels also of other hyaline extracellular matrix components. Surprisingly BM-MSC derived chondrocytes expressed higher mRNA levels of OA markers such as COL10A1, SSP1 (osteopontin), ALPL, BMP2, VEGFA, PTGES, IHH, and WNT genes, but lower levels of MMP3 and S100A4. Based on the results presented here, OA chondrocytes may be suitable for tissue engineering of articular cartilage. PMID:23671648

Methods of Manufacturing Bioactive Gels from Extracellular Matrix Material

NASA Technical Reports Server (NTRS)

Janis, Abram D. (Inventor); Kentner, Kimberly A. (Inventor); Stuart, Katherine A. (Inventor)

2014-01-01

The present invention is directed to methods of manufacturing bioactive gels from ECM material, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. The manufacturing methods take advantage of a new recognition that bioactive gels from ECM material can be created by digesting particularized ECM material in an alkaline environment and neutralizing to provide bioactive gels.

Methods of Manufacturing Bioactive Gels from Extracellular Matrix Material

NASA Technical Reports Server (NTRS)

Kentner, Kimberly A. (Inventor); Stuart, Katherine A. (Inventor); Janis, Abram D. (Inventor)

2015-01-01

The present invention is directed to methods of manufacturing bioactive gels from ECM material, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. The manufacturing methods take advantage of a new recognition that bioactive gels from ECM material can be created by digesting particularized ECM material in an alkaline environment and neutralizing to provide bioactive gels.

Methods of Manufacturing Bioactive Gels from Extracellular Matrix Material

NASA Technical Reports Server (NTRS)

Kentner, Kimberly A. (Inventor); Stuart, Katherine A. (Inventor); Janis, Abram D. (Inventor)

2016-01-01

The present invention is directed to methods of manufacturing bioactive gels from ECM material, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. The manufacturing methods take advantage of a new recognition that bioactive gels from ECM material can be created by digesting particularized ECM material in an alkaline environment and neutralizing to provide bioactive gels.

Methods of Manufacturing Bioactive Gels from Extracellular Matrix Material

NASA Technical Reports Server (NTRS)

Kentner, Kimberly (Inventor); Janis, Abram D. (Inventor); Stuart, Katherine A. (Inventor)

2017-01-01

The present invention is directed to methods of manufacturing bioactive gels from ECM material, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. The manufacturing methods take advantage of a new recognition that bioactive gels from ECM material can be created by digesting particularized ECM material in an alkaline environment and neutralizing to provide bioactive gels.

Right ventricular function after repair of tetralogy of Fallot: a comparison between bovine pericardium and porcine small intestinal extracellular matrix.

PubMed

Naik, Ronak; Johnson, Jason; Kumar, T K S; Philip, Ranjit; Boston, Umar; Knott-Craig, Christopher J

2017-05-29

The porcine small intestinal extracellular matrix reportedly has the potential to differentiate into viable myocardial cells. When used in tetralogy of Fallot repair, it may improve right ventricular function. We evaluated right ventricular function after repair of tetralogy of Fallot with extracellular matrix versus bovine pericardium. Subjects with non-transannular repair of tetralogy of Fallot with at least 1 year of follow-up were selected. The extracellular matrix and bovine pericardium groups were compared. We used three-dimensional right ventricular ejection fraction, right ventricle global longitudinal strain, and tricuspid annular plane systolic excursion to assess right ventricular function. The extracellular matrix group had 11 patients, whereas the bovine pericardium group had 10 patients. No differences between the groups were found regarding sex ratio, age at surgery, and cardiopulmonary bypass time. The follow-up period was 28±12.6 months in the extracellular matrix group and 50.05±17.6 months in the bovine pericardium group (p=0.001). The mean three-dimensional right ventricular ejection fraction (55.7±5.0% versus 55.3±5.2%, p=0.73), right ventricular global longitudinal strain (-18.5±3.0% versus -18.0±2.2%, p=0.44), and tricuspid annular plane systolic excursions (1.59±0.16 versus 1.59±0.2, p=0.93) were similar in the extracellular matrix group and in the bovine pericardium group, respectively. Right ventricular global longitudinal strain in healthy children is reported at -29±3% in literature. In a small cohort of the patients undergoing non-transannular repair of tetralogy of Fallot, there was no significant difference in right ventricular function between groups having extracellular matrix versus bovine pericardium patches followed-up for more than 1 year. Lower right ventricular longitudinal strain noted in both the groups compared to healthy children.

Elasticity-mediated nematiclike bacterial organization in model extracellular DNA matrix.

PubMed

Smalyukh, Ivan I; Butler, John; Shrout, Joshua D; Parsek, Matthew R; Wong, Gerard C L

2008-09-01

DNA is a common extracellular matrix component of bacterial biofilms. We find that bacteria can spontaneously order in a matrix of aligned concentrated DNA, in which rod-shaped cells of Pseudomonas aeruginosa follow the orientation of extended DNA chains. The alignment of bacteria is ensured by elasticity and liquid crystalline properties of the DNA matrix. These findings show how behavior of planktonic bacteria may be modified in extracellular polymeric substances of biofilms and illustrate the potential of using complex fluids to manipulate embedded nanosized and microsized active particles.

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.

Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.

PubMed

Ott, Harald C; Matthiesen, Thomas S; Goh, Saik-Kia; Black, Lauren D; Kren, Stefan M; Netoff, Theoden I; Taylor, Doris A

2008-02-01

About 3,000 individuals in the United States are awaiting a donor heart; worldwide, 22 million individuals are living with heart failure. A bioartificial heart is a theoretical alternative to transplantation or mechanical left ventricular support. Generating a bioartificial heart requires engineering of cardiac architecture, appropriate cellular constituents and pump function. We decellularized hearts by coronary perfusion with detergents, preserved the underlying extracellular matrix, and produced an acellular, perfusable vascular architecture, competent acellular valves and intact chamber geometry. To mimic cardiac cell composition, we reseeded these constructs with cardiac or endothelial cells. To establish function, we maintained eight constructs for up to 28 d by coronary perfusion in a bioreactor that simulated cardiac physiology. By day 4, we observed macroscopic contractions. By day 8, under physiological load and electrical stimulation, constructs could generate pump function (equivalent to about 2% of adult or 25% of 16-week fetal heart function) in a modified working heart preparation.

Advantages and challenges offered by biofunctional core-shell fiber systems for tissue engineering and drug delivery.

PubMed

Sperling, Laura E; Reis, Karina P; Pranke, Patricia; Wendorff, Joachim H

2016-08-01

Whereas highly porous scaffolds composed of electrospun nanofibers can mimick major features of the extracellular matrix in tissue engineering, they lack the ability to incorporate and release biocompounds (drugs, growth factors) safely in a controlled way. Here, electrospun core-shell fibers (core made from water and aqueous solutions of hydrophilic polymers and the shell from materials with well-defined release mechanisms) offer unique advantages in comparison with those that have helped make porous nanofibrillar scaffolds highly successful in tissue engineering. This review considers the preparation and biofunctionalization of such core-shell fibers as well as applications in various areas, including neural, vascular, cardiac, cartilage and bone tissue engineering, and touches on the topic of clinical trials. Copyright © 2016 Elsevier Ltd. All rights reserved.

De novo biosynthesis of glycosaminoglycans in the extracellular matrix of skin studied by matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Böhme, Julia; Anderegg, Ulf; Nimptsch, Ariane; Nimptsch, Kathrin; Hacker, Michael; Schulz-Siegmund, Michaela; Huster, Daniel; Schiller, Jürgen

2012-02-15

The self-healing capacity of skin is limited, and medical intervention is often unavoidable. Skin may be generated ex vivo from cultured fibroblasts. Because the molecular composition of de novo formed skin (mostly collagen and glycosaminoglycans [GAGs]) is crucial, analytical methods are required for the quality control of tissue-engineered products. Here, we show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of fibroblast cultures subsequent to digestion with chondroitinase ABC is a reliable and fast method to monitor the GAG content of native and bioengineered skin. Furthermore, the supplementation of the fibroblast medium with ¹³C-labeled glucose provides insights into the biosynthesis of GAGs. Copyright © 2011 Elsevier Inc. All rights reserved.

Mechanical influence of tissue culture plates and extracellular matrix on mesenchymal stem cell behavior: A topical review.

PubMed

Tatullo, Marco; Marrelli, Massimo; Falisi, Giovanni; Rastelli, Claudio; Palmieri, Francesca; Gargari, Marco; Zavan, Barbara; Paduano, Francesco; Benagiano, Vincenzo

2016-03-01

Tissue engineering applications need a continuous development of new biomaterials able to generate an ideal cell-extracellular matrix interaction. The stem cell fate is regulated by several factors, such as growth factors or transcription factors. The most recent literature has reported several publications able to demonstrate that environmental factors also contribute to the regulation of stem cell behavior, leading to the opinion that the environment plays the major role in the cell differentiation.The interaction between mesenchymal stem cells (MSCs) and extracellular environment has been widely described, and it has a crucial role in regulating the cell phenotype. In our laboratory (Tecnologica Research Institute, Crotone, Italy), we have recently studied how several physical factors influence the distribution and the morphology of MSCs isolated from dental pulp, and how they are able to regulate stem cell differentiation. Mechanical and geometrical factors are only a small part of the environmental factors able to influence stem cell behavior, however, this influence should be properly known: in fact, this assumption must be clearly considered during those studies involving MSCs; furthermore, these interactions should be considered as an important bias that involves an high number of studies on the MSCs, since in worldwide laboratories the scientists mostly use tissue culture plates for their experiments. © The Author(s) 2015.

Hydrogel scaffolds for tissue engineering: Progress and challenges

PubMed Central

El-Sherbiny, Ibrahim M.; Yacoub, Magdi H.

2013-01-01

Designing of biologically active scaffolds with optimal characteristics is one of the key factors for successful tissue engineering. Recently, hydrogels have received a considerable interest as leading candidates for engineered tissue scaffolds due to their unique compositional and structural similarities to the natural extracellular matrix, in addition to their desirable framework for cellular proliferation and survival. More recently, the ability to control the shape, porosity, surface morphology, and size of hydrogel scaffolds has created new opportunities to overcome various challenges in tissue engineering such as vascularization, tissue architecture and simultaneous seeding of multiple cells. This review provides an overview of the different types of hydrogels, the approaches that can be used to fabricate hydrogel matrices with specific features and the recent applications of hydrogels in tissue engineering. Special attention was given to the various design considerations for an efficient hydrogel scaffold in tissue engineering. Also, the challenges associated with the use of hydrogel scaffolds were described. PMID:24689032

Mesenchymal Stem Cells Sense Three Dimensional Type I Collagen through Discoidin Domain Receptor 1.

PubMed

Lund, A W; Stegemann, J P; Plopper, G E

2009-01-01

The extracellular matrix provides structural and organizational cues for tissue development and defines and maintains cellular phenotype during cell fate determination. Multipotent mesenchymal stem cells use this matrix to tightly regulate the balance between their differentiation potential and self-renewal in the native niche. When understood, the mechanisms that govern cell-matrix crosstalk during differentiation will allow for efficient engineering of natural and synthetic matrices to specifically direct and maintain stem cell phenotype. This work identifies the discoidin domain receptor 1 (DDR1), a collagen activated receptor tyrosine kinase, as a potential link through which stem cells sense and respond to the 3D organization of their extracellular matrix microenvironment. DDR1 is dependent upon both the structure and proteolytic state of its collagen ligand and is specifically expressed and localized in three dimensional type I collagen culture. Inhibition of DDR1 expression results in decreased osteogenic potential, increased cell spreading, stress fiber formation and ERK1/2 phosphorylation. Additionally, loss of DDR1 activity alters the cell-mediated organization of the naïve type I collagen matrix. Taken together, these results demonstrate a role for DDR1 in the stem cell response to and interaction with three dimensional type I collagen. Dynamic changes in cell shape in 3D culture and the tuning of the local ECM microstructure, directs crosstalk between DDR1 and two dimensional mechanisms of osteogenesis that can alter their traditional roles.

A human in vitro model of Duchenne muscular dystrophy muscle formation and contractility.

PubMed

Nesmith, Alexander P; Wagner, Matthew A; Pasqualini, Francesco S; O'Connor, Blakely B; Pincus, Mark J; August, Paul R; Parker, Kevin Kit

2016-10-10

Tongue weakness, like all weakness in Duchenne muscular dystrophy (DMD), occurs as a result of contraction-induced muscle damage and deficient muscular repair. Although membrane fragility is known to potentiate injury in DMD, whether muscle stem cells are implicated in deficient muscular repair remains unclear. We hypothesized that DMD myoblasts are less sensitive to cues in the extracellular matrix designed to potentiate structure-function relationships of healthy muscle. To test this hypothesis, we drew inspiration from the tongue and engineered contractile human muscle tissues on thin films. On this platform, DMD myoblasts formed fewer and smaller myotubes and exhibited impaired polarization of the cell nucleus and contractile cytoskeleton when compared with healthy cells. These structural aberrations were reflected in their functional behavior, as engineered tongues from DMD myoblasts failed to achieve the same contractile strength as healthy tongue structures. These data suggest that dystrophic muscle may fail to organize with respect to extracellular cues necessary to potentiate adaptive growth and remodeling. © 2016 Nesmith et al.

Decellularized Tissue and Cell-Derived Extracellular Matrices as Scaffolds for Orthopaedic Tissue Engineering

PubMed Central

Cheng, Christina W.; Solorio, Loran D.; Alsberg, Eben

2014-01-01

The reconstruction of musculoskeletal defects is a constant challenge for orthopaedic surgeons. Musculoskeletal injuries such as fractures, chondral lesions, infections and tumor debulking can often lead to large tissue voids requiring reconstruction with tissue grafts. Autografts are currently the gold standard in orthopaedic tissue reconstruction; however, there is a limit to the amount of tissue that can be harvested before compromising the donor site. Tissue engineering strategies using allogeneic or xenogeneic decellularized bone, cartilage, skeletal muscle, tendon and ligament have emerged as promising potential alternative treatment. The extracellular matrix provides a natural scaffold for cell attachment, proliferation and differentiation. Decellularization of in vitro cell-derived matrices can also enable the generation of autologous constructs from tissue specific cells or progenitor cells. Although decellularized bone tissue is widely used clinically in orthopaedic applications, the exciting potential of decellularized cartilage, skeletal muscle, tendon and ligament cell-derived matrices has only recently begun to be explored for ultimate translation to the orthopaedic clinic. PMID:24417915

Patterning methods for polymers in cell and tissue engineering.

PubMed

Kim, Hong Nam; Kang, Do-Hyun; Kim, Min Sung; Jiao, Alex; Kim, Deok-Ho; Suh, Kahp-Yang

2012-06-01

Polymers provide a versatile platform for mimicking various aspects of physiological extracellular matrix properties such as chemical composition, rigidity, and topography for use in cell and tissue engineering applications. In this review, we provide a brief overview of patterning methods of various polymers with a particular focus on biocompatibility and processability. The materials highlighted here are widely used polymers including thermally curable polydimethyl siloxane, ultraviolet-curable polyurethane acrylate and polyethylene glycol, thermo-sensitive poly(N-isopropylacrylamide) and thermoplastic and conductive polymers. We also discuss how micro- and nanofabricated polymeric substrates of tunable elastic modulus can be used to engineer cell and tissue structure and function. Such synergistic effect of topography and rigidity of polymers may be able to contribute to constructing more physiologically relevant microenvironment.

SHIP, a novel factor to ameliorate extracellular matrix accumulation via suppressing PI3K/Akt/CTGF signaling in diabetic kidney disease.

PubMed

Li, Fan; Li, Lisha; Cheng, Meijuan; Wang, Xiumin; Hao, Jun; Liu, Shuxia; Duan, Huijun

2017-01-22

Tubular interstitial extracellular matrix accumulation, which plays a key role in the pathogenesis and progression of diabetic kidney disease (DKD), is believed to be mediated by activation of PI3K/Akt signal pathway. However, it is still not clear whether SH2 domain-containing inositol 5'-phosphatase (SHIP), known as a negative regulator of PI3K/Akt pathway is also involved in extracellular matrix metabolism of diabetic kidney. In the present study, decreased SHIP and increased phospho-Akt (Ser 473, Thr 308) were found in renal tubular cells of diabetic mice accompanied by overexpression of connective tissue growth factor (CTGF) and extracellular matrix deposition versus normal mice. Again, high glucose attenuated SHIP expression in a time-dependent manner, concomitant with activation of PI3K/Akt signaling and extracellular matrix production in human renal proximal tubular epithelial cells (HK2) cultured in vitro, which was significantly prevented by transfection of M90-SHIP vector. Furthermore, in vivo delivery of rAd-INPP5D vector (SHIP expression vector) via intraperitoneal injection in diabetic mice increased SHIP expression by 3.36 times followed by 65.26%, 70.38% and 46.71% decreases of phospho-Akt (Ser 473), phospho-Akt (Thr 308) and CTGF expression versus diabetic mice receiving rAd-EGFP vector. Meanwhile, increased renal extracellular matrix accumulation of diabetic mice was also inhibited with intraperitoneal injection of rAd-INPP5D vector. These above data suggested that overexpression of SHIP might be a potent method to lessen renal extracellular matrix accumulation via inactivation of PI3K/Akt pathway and suppression of CTGF expression in DKD. Copyright © 2016 Elsevier Inc. All rights reserved.

Bio-inspired 3D microenvironments: a new dimension in tissue engineering.

PubMed

Magin, Chelsea M; Alge, Daniel L; Anseth, Kristi S

2016-03-04

Biomaterial scaffolds have been a foundational element of the tissue engineering paradigm since the inception of the field. Over the years there has been a progressive move toward the rational design and fabrication of bio-inspired materials that mimic the composition as well as the architecture and 3D structure of tissues. In this review, we chronicle advances in the field that address key challenges in tissue engineering as well as some emerging applications. Specifically, a summary of the materials and chemistries used to engineer bio-inspired 3D matrices that mimic numerous aspects of the extracellular matrix is provided, along with an overview of bioprinting, an additive manufacturing approach, for the fabrication of engineered tissues with precisely controlled 3D structures and architectures. To emphasize the potential clinical impact of the bio-inspired paradigm in biomaterials engineering, some applications of bio-inspired matrices are discussed in the context of translational tissue engineering. However, focus is also given to recent advances in the use of engineered 3D cellular microenvironments for fundamental studies in cell biology, including photoresponsive systems that are shedding new light on how matrix properties influence cell phenotype and function. In an outlook for future work, the need for high-throughput methods both for screening and fabrication is highlighted. Finally, microscale organ-on-a-chip technologies are highlighted as a promising area for future investment in the application of bio-inspired microenvironments.

Nanofabricated Collagen-Inspired Synthetic Elastomers for Primary Rat Hepatocyte Culture

PubMed Central

Bettinger, Christopher J.; Kulig, Katherine M.; Vacanti, Joseph P.

2009-01-01

Synthetic substrates that mimic the properties of extracellular matrix proteins hold significant promise for use in systems designed for tissue engineering applications. In this report, we designed a synthetic polymeric substrate that is intended to mimic chemical, mechanical, and topological characteristics of collagen. We found that elastomeric poly(ester amide) substrates modified with replica-molded nanotopographic features enhanced initial attachment, spreading, and adhesion of primary rat hepatocytes. Further, hepatocytes cultured on nanotopographic substrates also demonstrated reduced albumin secretion and urea synthesis, which is indicative of strongly adherent hepatocytes. These results suggest that these engineered substrates can function as synthetic collagen analogs for in vitro cell culture. PMID:18847357

Albumin fiber scaffolds for engineering functional cardiac tissues.

PubMed

Fleischer, Sharon; Shapira, Assaf; Regev, Omri; Nseir, Nora; Zussman, Eyal; Dvir, Tal

2014-06-01

In recent years attempts to engineer contracting cardiac patches were focused on recapitulation of the myocardium extracellular microenvironment. We report here on our work, where for the first time, a three-dimensional cardiac patch was fabricated from albumin fibers. We hypothesized that since albumin fibers' mechanical properties resemble those of cardiac tissue extracellular matrix (ECM) and their biochemical character enables their use as protein carriers, they can support the assembly of cardiac tissues capable of generating strong contraction forces. Here, we have fabricated aligned and randomly oriented electrospun albumin fibers and investigated their structure, mechanical properties, and chemical nature. Our measurements showed that the scaffolds have improved elasticity as compared to synthetic electrospun PCL fibers, and that they are capable of adsorbing serum proteins, such as laminin leading to strong cell-matrix interactions. Moreover, due to the functional groups on their backbone, the fibers can be chemically modified with essential biomolecules. When seeded with rat neonatal cardiac cells the engineered scaffolds induced the assembly of aligned cardiac tissues with high aspect ratio cardiomyocytes and massive actinin striation. Compared to synthetic fibrous scaffolds, cardiac cells cultured within aligned or randomly oriented scaffolds formed functional tissues, exhibiting significantly improved function already on Day 3, including higher beating rate (P = 0.0002 and P < 0.0001, respectively), and higher contraction amplitude (P = 0.009 and P = 0.003, respectively). Collectively, our results suggest that albumin electrospun scaffolds can play a key role in contributing to the ex vivo formation of a contracting cardiac muscle tissue. © 2014 Wiley Periodicals, Inc.

Strategies for Enhancing the Accumulation and Retention of Extracellular Matrix in Tissue-Engineered Cartilage Cultured in Bioreactors

PubMed Central

Shahin, Kifah; Doran, Pauline M.

2011-01-01

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min−1) and gradually increasing (0.075–0.2 mL min−1) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0–4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8–5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min−1. GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention. PMID:21858004

Freezing-induced deformation of biomaterials in cryomedicine

NASA Astrophysics Data System (ADS)

Ozcelikkale, Altug

Cryomedicine utilizes low temperature treatments of biological proteins, cells and tissues for cryopreservation, materials processing and cryotherapy. Lack of proper understanding of cryodamage that occurs during these applications remains to be the primary bottleneck for development of successful tissue cryopreservation and cryosurgery procedures. An engineering approach based on a view of biological systems as functional biomaterials can help identify, predict and control the primary cryodamage mechanisms by developing an understanding of underlying freezing-induced biophysical processes. In particular, freezing constitutes the main structural/mechanical origin of cryodamage and results in significant deformation of biomaterials at multiple length scales. Understanding of these freezing-induced deformation processes and their effects on post-thaw biomaterial functionality is currently lacking but will be critical to engineer improved cryomedicine procedures. This dissertation addresses this problem by presenting three separate but related studies of freezing-induced deformation at multiple length scales including nanometer-scale protein fibrils, single cells and whole tissues. A combination of rigorous experimentation and computational modeling is used to characterize post-thaw biomaterial structure and properties, predict biomaterial behavior and assess its post-thaw biological functionality. Firstly, freezing-induced damage on hierarchical extracellular matrix structure of collagen is investigated at molecular, fibril and matrix levels. Results indicate to a specific kind of fibril damage due to freezing-induced expansion of intrafibrillar fluid. This is followed by a study of freezing-induced cell and tissue deformation coupled to osmotically driven cellular water transport. Computational and semi empirical modeling of these processes indicate that intracellular deformation of the cell during freezing is heterogeneous and can interfere with cellular water transport, thereby leading to previously unconsidered mechanisms of cell freezing response. In addition, cellular water transport is identified as the critical limiting factor on the amount of freezing-induced tissue deformation, particularly in native tissues with high cell densities. Finally, effects of cryopreservation on post-thaw biological functionality of collagen engineered tissue constructs is investigated where cell-matrix interactions during fibroblast migration are considered as the functional response. Simultaneous cell migration and extracellular matrix deformation are characterized. Results show diminished cell-matrix coupling by freeze/thaw accompanied by a subtle decrease in cell migration. A connection between these results and freezing-induced collagen fibril damage is also suggested. Overall, this dissertation provides new fundamental knowledge on cryodamage mechanisms and a collection of novel multi-purpose engineering tools that will open the way for rational design of cryomedicine technologies.

Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms.

PubMed

Klein, Marlise I; Hwang, Geelsu; Santos, Paulo H S; Campanella, Osvaldo H; Koo, Hyun

2015-01-01

Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS), eDNA, and lipoteichoic acid (LTA). EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan) synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases.

Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms

PubMed Central

Klein, Marlise I.; Hwang, Geelsu; Santos, Paulo H. S.; Campanella, Osvaldo H.; Koo, Hyun

2015-01-01

Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS), eDNA, and lipoteichoic acid (LTA). EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan) synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases. PMID:25763359

Modifying alginate with early embryonic extracellular matrix, laminin, and hyaluronic acid for adipose tissue engineering.

PubMed

Chen, Yo-Shen; Chen, Yen-Yu; Hsueh, Yu-Sheng; Tai, Hao-Chih; Lin, Feng-Huei

2016-03-01

Extracellular matrix provides both mechanistic and chemical cues that can influence cellular behaviors such as adhesion, migration, proliferation, and differentiation. In this study, a new material, HA-L-Alg, was synthesized by linking developmentally essential ECM constituents hyaluronic acid (HA) and laminin(L) to alginate (Alg). The fabrication of HA-L-Alg was confirmed by FTIR spectroscopy, and it was used to form 3D cell-carrying beads. HA-L-Alg beads had a steady rate of degradation and retained 63.25% of mass after 9 weeks. HA-L-Alg beads showed biocompatibility comparable to beads formed by Alg-only with no obvious cytotoxic effect on the embedded 3T3-L1 preadipocytes. HA-L-Alg encapsulated 3T3-L1 cells were found to have a higher proliferation rate over those in Alg-only beads. These cells also showed better differentiation capacity after 2 weeks of adipogenic induction within HA-L-Alg beads. These results support that HA-L-Alg facilitated cell survival and proliferation, as well as stimulated and maintained cell differentiation. Our results suggest that HA-L-Alg has a great clinical potential to be used as stem cell carrier for adipose tissue engineering. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 669-677, 2016. © 2015 Wiley Periodicals, Inc.

Effects of electromagnetic field frequencies on chondrocytes in 3D cell-printed composite constructs.

PubMed

Yi, Hee-Gyeong; Kang, Kyung Shin; Hong, Jung Min; Jang, Jinah; Park, Moon Nyeo; Jeong, Young Hun; Cho, Dong-Woo

2016-07-01

In cartilage tissue engineering, electromagnetic field (EMF) therapy has been reported to have a modest effect on promoting cartilage regeneration. However, these studies were conducted using different frequencies of EMF to stimulate chondrocytes. Thus, it is necessary to investigate the effect of EMF frequency on cartilage formation. In addition to the stimulation, a scaffold is required to satisfy the characteristics of cartilage such as its hydrated and dense extracellular matrix, and a mechanical resilience to applied loads. Therefore, we 3D-printed a composite construct composed of a polymeric framework and a chondrocyte-laden hydrogel. Here, we observed frequency-dependent positive and negative effects on chondrogenesis using a 3D cell-printed cartilage tissue. We found that a frequency of 45 Hz promoted gene expression and secretion of extracellular matrix molecules of chondrocytes. In contrast, a frequency of 7.5 Hz suppressed chondrogenic differentiation in vitro. Additionally, the EMF-treated composite constructs prior to implantation showed consistent results with those of in vitro, suggesting that in vitro pre-treatment with different EMF frequencies provides different capabilities for the enhancement of cartilage formation in vivo. This correlation between EMF frequency and 3D-printed chondrocytes suggests the necessity for optimization of EMF parameters when this physical stimulus is applied to engineered cartilage. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1797-1804, 2016. © 2016 Wiley Periodicals, Inc.

Human fibroblast-derived extracellular matrix constructs for bone tissue engineering applications.

PubMed

Tour, Gregory; Wendel, Mikael; Tcacencu, Ion

2013-10-01

We exploited the biomimetic approach to generate constructs composed of synthetic biphasic calcium phosphate ceramic and extracellular matrix (SBC-ECM) derived from adult human dermal fibroblasts in complete xeno-free culture conditions. The construct morphology and composition were assessed by scanning electron microscopy, histology, immunohistochemistry, Western blot, glycosaminoglycan, and hydroxyproline assays. Residual DNA quantification, endotoxin testing, and local inflammatory response after implantation in a rat critical-sized calvarial defect were used to access the construct biocompatibility. Moreover, in vitro interaction of human mesenchymal stem cells (hMSCs) with the constructs was studied. The bone marrow- and adipose tissue-derived mesenchymal stem cells were characterized by flow cytometry and tested for osteogenic differentiation capacity prior seeding onto SBC-ECM, followed by alkaline phosphatase, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and real-time quantitative polymerase chain reaction to assess the osteogenic differentiation of hMSCs after seeding onto the constructs at different time intervals. The SBC-ECM constructs enhanced osteogenic differentiation of hMSCs in vitro and exhibited excellent handling properties and high biocompatibility in vivo. Our results highlight the ability to generate in vitro fibroblast-derived ECM constructs in complete xeno-free conditions as a step toward clinical translation, and the potential use of SBC-ECM in craniofacial bone tissue engineering applications. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Production and characterization of bacterial cellulose membranes with hyaluronic acid from chicken comb.

PubMed

de Oliveira, Sabrina Alves; da Silva, Bruno Campos; Riegel-Vidotti, Izabel Cristina; Urbano, Alexandre; de Sousa Faria-Tischer, Paula Cristina; Tischer, Cesar Augusto

2017-04-01

The bacterial cellulose (BC), from Gluconacetobacter hansenii, is a biofilm with a high degree of crystallinity that can be used for therapeutic purposes and as a candidate for healing wounds. Hyaluronic acid (HA) is a constitutive polysaccharide found in the extracellular matrix and is a material used in tissue engineering and scaffolding for tissue regeneration. In this study, polymeric composites were produced in presence of hyaluronic acid isolated from chicken comb on different days of fermentation, specifically on the first (BCHA-SABT0) and third day (BCHA-SABT3) of fermentation. The structural characteristics, thermal stability and molar mass of hyaluronic acid from chicken comb were evaluated. Native membrane and polymeric composites were characterized with respect to their morphology and crystallinity. The optimized process of extraction and purification of hyaluronic acid resulted in low molar mass hyaluronic acid with structural characteristics similar to the standard commercial hyaluronic acid. The results demonstrate that the polymeric composites (BC/HA-SAB) can be produced in situ. The membranes produced on the third day presented better incorporation of HA-SAB between cellulose microfiber, resulting in membranes with higher thermal stability, higher roughness and lower crystallinity. The biocompatiblily of bacterial cellulose and the importance of hyaluronic acid as a component of extracellular matrix qualify the polymeric composites as promising biomaterials for tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

The extracellular matrix of Staphylococcus aureus biofilms comprises cytoplasmic proteins that associate with the cell surface in response to decreasing pH.

PubMed

Foulston, Lucy; Elsholz, Alexander K W; DeFrancesco, Alicia S; Losick, Richard

2014-09-02

Biofilm formation by Staphylococcus aureus involves the formation of an extracellular matrix, but the composition of this matrix has been uncertain. Here we report that the matrix is largely composed of cytoplasmic proteins that reversibly associate with the cell surface in a manner that depends on pH. We propose a model for biofilm formation in which cytoplasmic proteins are released from cells in stationary phase. These proteins associate with the cell surface in response to decreasing pH during biofilm formation. Rather than utilizing a dedicated matrix protein, S. aureus appears to recycle cytoplasmic proteins that moonlight as components of the extracellular matrix. Staphylococcus aureus is a leading cause of multiantibiotic-resistant nosocomial infections and is often found growing as a biofilm in catheters and chronic wounds. Biofilm formation is an important pathogenicity strategy that enhances resistance to antimicrobials, thereby limiting treatment options and ultimately contributing to increased morbidity and mortality. Cells in a biofilm are held together by an extracellular matrix that consists in whole or in part of protein, but the nature of the proteins in the S. aureus matrix is not well understood. Here we postulate that S. aureus recycles proteins from the cytoplasm to form the extracellular matrix. This strategy, of cytoplasmic proteins moonlighting as matrix proteins, could allow enhanced flexibility and adaptability for S. aureus in forming biofilms under infection conditions and could promote the formation of mixed-species biofilms in chronic wounds. Copyright © 2014 Foulston et al.

Teaching the Extracellular Matrix and Introducing Online Databases within a Multidisciplinary Course with i-Cell-MATRIX: A Student-Centered Approach

ERIC Educational Resources Information Center

Sousa, Joao Carlos; Costa, Manuel Joao; Palha, Joana Almeida

2010-01-01

The biochemistry and molecular biology of the extracellular matrix (ECM) is difficult to convey to students in a classroom setting in ways that capture their interest. The understanding of the matrix's roles in physiological and pathological conditions study will presumably be hampered by insufficient knowledge of its molecular structure.…

Effects of chitosan-coated fibers as a scaffold for three-dimensional cultures of rabbit fibroblasts for ligament tissue engineering.

PubMed

Sarukawa, Junichiro; Takahashi, Masaaki; Abe, Masashi; Suzuki, Daisuke; Tokura, Seiichi; Furuike, Tetsuya; Tamura, Hiroshi

2011-01-01

Material selection in tissue-engineering scaffolds is one of the primary factors defining cellular response and matrix formation. In this study, we fabricated chitosan-coated poly(lactic acid) (PLA) fiber scaffolds to test our hypothesis that PLA fibers coated with chitosan highly promoted cell supporting properties compared to those without chitosan. Both PLA fibers (PLA group) and chitosan-coated PLA fibers (PLA-chitosan group) were fabricated for this study. Anterior cruciate ligament (ACL) fibroblasts were isolated from Japanese white rabbits and cultured on scaffolds consisting of each type of fiber. The effects of cell adhesivity, proliferation, and synthesis of the extracellular matrix (ECM) for each fiber were analyzed by cell counting, hydroxyproline assay, scanning electron microscopy and quantitative RT-PCR. Cell adhesivity, proliferation, hydroxyproline content and the expression of type-I collagen mRNA were significantly higher in the PLA-chitosan group than in the PLA group. Scanning electron microscopic observation showed that fibroblasts proliferated with a high level of ECM synthesis around the cells. Chitosan coating improved ACL fibroblast adhesion and proliferation, and had a positive effect on matrix production. Thus, the advantages of chitosan-coated PLA fibers show them to be a suitable biomaterial for ACL tissue-engineering scaffolds.

The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

PubMed

Johnson, Chad J; Cabezas-Olcoz, Jonathan; Kernien, John F; Wang, Steven X; Beebe, David J; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E

2016-09-01

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps

PubMed Central

Cabezas-Olcoz, Jonathan; Wang, Steven X.; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E.

2016-01-01

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix. PMID:27622514

Dynamic Manipulation of Hydrogels to Control Cell Behavior: A Review

PubMed Central

Vats, Kanika

2013-01-01

For many tissue engineering applications and studies to understand how materials fundamentally affect cellular functions, it is important to have the ability to synthesize biomaterials that can mimic elements of native cell–extracellular matrix interactions. Hydrogels possess many properties that are desirable for studying cell behavior. For example, hydrogels are biocompatible and can be biochemically and mechanically altered by exploiting the presentation of cell adhesive epitopes or by changing hydrogel crosslinking density. To establish physical and biochemical tunability, hydrogels can be engineered to alter their properties upon interaction with external driving forces such as pH, temperature, electric current, as well as exposure to cytocompatible irradiation. Additionally, hydrogels can be engineered to respond to enzymes secreted by cells, such as matrix metalloproteinases and hyaluronidases. This review details different strategies and mechanisms by which biomaterials, specifically hydrogels, can be manipulated dynamically to affect cell behavior. By employing the appropriate combination of stimuli and hydrogel composition and architecture, cell behavior such as adhesion, migration, proliferation, and differentiation can be controlled in real time. This three-dimensional control in cell behavior can help create programmable cell niches that can be useful for fundamental cell studies and in a variety of tissue engineering applications. PMID:23541134

A Drug-Induced Hybrid Electrospun Poly-Capro-Lactone: Cell-Derived Extracellular Matrix Scaffold for Liver Tissue Engineering.

PubMed

Grant, Rhiannon; Hay, David C; Callanan, Anthony

2017-07-01

Liver transplant is the only treatment option for patients with end-stage liver failure, however, there are too few donor livers available for transplant. Whole organ tissue engineering presents a potential solution to the problem of rapidly escalating donor liver shortages worldwide. A major challenge for liver tissue engineers is the creation of a hepatocyte microenvironment; a niche in which liver cells can survive and function optimally. While polymers and decellularized tissues pose an attractive option for scaffold manufacturing, neither alone has thus far proved sufficient. This study exploited cell's native extracellular matrix (ECM) producing capabilities using two different histone deacetylase inhibitors, and combined these with the customizability and reproducibility of electrospun polymer scaffolds to produce a "best of both worlds" niche microenvironment for hepatocytes. The resulting hybrid poly-capro-lactone (PCL)-ECM scaffolds were validated using HepG2 hepatocytes. The hybrid PCL-ECM scaffolds maintained hepatocyte growth and function, as evidenced by metabolic activity and DNA quantitation. Mechanical testing revealed little significant difference between scaffolds, indicating that cells were responding to a biochemical and topographical profile rather than mechanical changes. Immunohistochemistry showed that the biochemical profile of the drug-derived and nondrug-derived ECMs differed in ratio of Collagen I, Laminin, and Fibronectin. Furthermore, the hybrid PCL-ECM scaffolds influence the gene expression profile of the HepG2s drastically; with expression of Albumin, Cytochrome P450 Family 1 Subfamily A Polypeptide 1, Cytochrome P450 Family 1 Subfamily A Polypeptide 2, Cytochrome P450 Family 3 Subfamily A Polypeptide 4, Fibronectin, Collagen I, and Collagen IV undergoing significant changes. Our results demonstrate that drug-induced hybrid PCL-ECM scaffolds provide a viable, translatable platform for creating a niche microenvironment for hepatocytes, supporting in vivo phenotype and function. These scaffolds offer great potential for tissue engineering and regenerative medicine strategies for whole organ tissue engineering.

Genetic engineering for skeletal regenerative medicine.

PubMed

Gersbach, Charles A; Phillips, Jennifer E; García, Andrés J

2007-01-01

The clinical challenges of skeletal regenerative medicine have motivated significant advances in cellular and tissue engineering in recent years. In particular, advances in molecular biology have provided the tools necessary for the design of gene-based strategies for skeletal tissue repair. Consequently, genetic engineering has emerged as a promising method to address the need for sustained and robust cellular differentiation and extracellular matrix production. As a result, gene therapy has been established as a conventional approach to enhance cellular activities for skeletal tissue repair. Recent literature clearly demonstrates that genetic engineering is a principal factor in constructing effective methods for tissue engineering approaches to bone, cartilage, and connective tissue regeneration. This review highlights this literature, including advances in the development of efficacious gene carriers, novel cell sources, successful delivery strategies, and optimal target genes. The current status of the field and the challenges impeding the clinical realization of these approaches are also discussed.

Nanotechnology in the Regeneration of Complex Tissues

PubMed Central

Cassidy, John W.

2015-01-01

Modern medicine faces a growing crisis as demand for organ transplantations continues to far outstrip supply. By stimulating the body’s own repair mechanisms, regenerative medicine aims to reduce demand for organs, while the closely related field of tissue engineering promises to deliver “off-the-self” organs grown from patients’ own stem cells to improve supply. To deliver on these promises, we must have reliable means of generating complex tissues. Thus far, the majority of successful tissue engineering approaches have relied on macroporous scaffolds to provide cells with both mechanical support and differentiative cues. In order to engineer complex tissues, greater attention must be paid to nanoscale cues present in a cell’s microenvironment. As the extracellular matrix is capable of driving complexity during development, it must be understood and reproduced in order to recapitulate complexity in engineered tissues. This review will summarize current progress in engineering complex tissue through the integration of nanocomposites and biomimetic scaffolds. PMID:26097381

Optical Spectroscopy and Imaging for the Noninvasive Evaluation of Engineered Tissues

PubMed Central

Rice, William L.; Hronik-Tupaj, Marie; Kaplan, David L.

2008-01-01

Optical spectroscopy and imaging approaches offer the potential to noninvasively assess different aspects of the cellular, extracellular matrix, and scaffold components of engineered tissues. In addition, the combination of multiple imaging modalities within a single instrument is highly feasible, allowing acquisition of complementary information related to the structure, organization, biochemistry, and physiology of the sample. The ability to characterize and monitor the dynamic interactions that take place as engineered tissues develop promises to enhance our understanding of the interdependence of processes that ultimately leads to functional tissue outcomes. It is expected that this information will impact significantly upon our abilities to optimize the design of biomaterial scaffolds, bioreactors, and cell systems. Here, we review the principles and performance characteristics of the main methodologies that have been exploited thus far, and we present examples of corresponding tissue engineering studies. PMID:18844604

Heparin Stimulates Elastogenesis: Application to Silk-Based Vascular Grafts

PubMed Central

Baughman, Cassandra; Kaplan, David L.; Castellot, John J.

2013-01-01

With over 500,000 coronary artery bypass grafts (CABG) performed annually in the United States alone, there is a significant clinical need for a small diameter tissue engineered vascular graft. A principle goal in tissue engineering is to develop materials and growth conditions that encourage appropriate re-cellularization and extracellular matrix formation in vivo. A particular challenge in vascular tissue engineering results from the inability of adult cells to produce elastin, as its expression is developmentally limited. We investigated factors to stimulate elastogenesis in vitro, and found that heparin treatment of adult human vascular smooth muscle cells promoted the formation of elastic fibers. This effect was heparin-specific, and dependent on cell density and growth state. We then applied this information to a silk-based construct, and found that immobilized heparin showed essentially identical biological effects to that of soluble heparin. These findings indicate that heparinized vascular grafts may promote elastin formation and regulate restenosis, in addition to heparin’s well-established antithrombotic properties. Given the increase in elastin mRNA level and the increase in extracellular elastin present, our data suggests that there may be multiple levels of elastin regulation that are mediated by heparin treatment. PMID:21600981

Low‑dose halofuginone inhibits the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes.

PubMed

Li, Zeng; Fei, Hao; Wang, Zhen; Zhu, Tianyi

2017-09-01

Full‑thickness and large area defects of articular cartilage are unable to completely repair themselves and require surgical intervention, including microfracture, autologous or allogeneic osteochondral grafts, and autologous chondrocyte implantation. A large proportion of regenerative cartilage exists as fibrocartilage, which is unable to withstand impacts in the same way as native hyaline cartilage, owing to excess synthesis of type I collagen in the matrix. The present study demonstrated that low‑dose halofuginone (HF), a plant alkaloid isolated from Dichroa febrifuga, may inhibit the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes. In addition, HF was revealed to inhibit the phosphorylation of mothers against decapentaplegic homolog (Smad)2/3 and promoted Smad7 expression, as well as decrease the synthesis of type I collagen synthesis. Results from the present study indicated that HF treatment suppressed the synthesis of type I collagen by inhibiting the transforming growth factor‑β signaling pathway in chondrocytes. These results may provide an alternative solution to the problems associated with fibrocartilage, and convert fibrocartilage into hyaline cartilage at the mid‑early stages of cartilage regeneration. HF may additionally be used to improve monolayer expansion or 3D cultures of seed cells for the tissue engineering of cartilage.

Raman spectroscopy in biomedicine – non-invasive in vitro analysis of cells and extracellular matrix components in tissues

PubMed Central

Brauchle, Eva; Schenke-Layland, Katja

2013-01-01

Raman spectroscopy is an established laser-based technology for the quality assurance of pharmaceutical products. Over the past few years, Raman spectroscopy has become a powerful diagnostic tool in the life sciences. Raman spectra allow assessment of the overall molecular constitution of biological samples, based on specific signals from proteins, nucleic acids, lipids, carbohydrates, and inorganic crystals. Measurements are non-invasive and do not require sample processing, making Raman spectroscopy a reliable and robust method with numerous applications in biomedicine. Moreover, Raman spectroscopy allows the highly sensitive discrimination of bacteria. Rama spectra retain information on continuous metabolic processes and kinetics such as lipid storage and recombinant protein production. Raman spectra are specific for each cell type and provide additional information on cell viability, differentiation status, and tumorigenicity. In tissues, Raman spectroscopy can detect major extracellular matrix components and their secondary structures. Furthermore, the non-invasive characterization of healthy and pathological tissues as well as quality control and process monitoring of in vitro-engineered matrix is possible. This review provides comprehensive insight to the current progress in expanding the applicability of Raman spectroscopy for the characterization of living cells and tissues, and serves as a good reference point for those starting in the field. PMID:23161832

Protein- mediated enamel mineralization

PubMed Central

Moradian-Oldak, Janet

2012-01-01

Enamel is a hard nanocomposite bioceramic with significant resilience that protects the mammalian tooth from external physical and chemical damages. The remarkable mechanical properties of enamel are associated with its hierarchical structural organization and its thorough connection with underlying dentin. This dynamic mineralizing system offers scientists a wealth of information that allows the study of basic principals of organic matrix-mediated biomineralization and can potentially be utilized in the fields of material science and engineering for development and design of biomimetic materials. This chapter will provide a brief overview of enamel hierarchical structure and properties as well as the process and stages of amelogenesis. Particular emphasis is given to current knowledge of extracellular matrix protein and proteinases, and the structural chemistry of the matrix components and their putative functions. The chapter will conclude by discussing the potential of enamel for regrowth. PMID:22652761

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.

From Nano to Macro: Studying the Hierarchical Structure of the Corneal Extracellular Matrix

PubMed Central

Quantock, Andrew J.; Winkler, Moritz; Parfitt, Geraint J.; Young, Robert D.; Brown, Donald J.; Boote, Craig; Jester, James V.

2014-01-01

In this review, we discuss current methods for studying ocular extracellular matrix (ECM) assembly from the ‘nano’ to the ‘macro’ levels of hierarchical organization. Since collagen is the major structural protein in the eye, providing mechanical strength and controlling ocular shape, the methods presented focus on understanding the molecular assembly of collagen at the nanometer level using x-ray scattering through to the millimeter to centimeter level using nonlinear optical (NLO) imaging of second harmonic generated (SHG) signals. Three-dimensional analysis of ECM structure is also discussed, including electron tomography, serial block face scanning electron microscopy (SBF-SEM) and digital image reconstruction. Techniques to detect non-collagenous structural components of the ECM are also presented, and these include immunoelectron microscopy and staining with cationic dyes. Together, these various approaches are providing new insights into the structural blueprint of the ocular ECM, and in particular that of the cornea, which impacts upon our current understanding of the control of corneal shape, pathogenic mechanisms underlying ectatic disorders of the cornea and the potential for corneal tissue engineering. PMID:25819457

The Histochemistry and Cell Biology omnium-gatherum: the year 2015 in review.

PubMed

Taatjes, Douglas J; Roth, Jürgen

2016-03-01

We provide here our annual review/synopsis of all of the articles published in Histochemistry and Cell Biology (HCB) for the preceding year. In 2015, HCB published 102 articles, representing a wide variety of topics and methodologies. For ease of access to these differing topics, we have created categories, as determined by the types of articles presented to provide a quick index representing the general areas covered. This year, these categories include: (1) advances in methodologies; (2) molecules in health and disease; (3) organelles, subcellular structures, and compartments; (4) the nucleus; (5) stem cells and tissue engineering; (6) cell cultures: properties and capabilities; (7) connective tissues and extracellular matrix; (8) developmental biology; (9) nervous system; (10) musculoskeletal system; (11) respiratory and cardiovascular system; (12) liver and gastrointestinal tract; and (13) male and female reproductive systems. Of note, the categories proceed from methods development, to molecules, intracellular compartments, stem cells and cell culture, extracellular matrix, developmental biology, and finishing with various organ systems, hopefully presenting a logical journey from methods to organismal molecules, cells, and whole tissue systems.

Role of substrate biomechanics in controlling (stem) cell fate: Implications in regenerative medicine.

PubMed

Macri-Pellizzeri, Laura; De-Juan-Pardo, Elena M; Prosper, Felipe; Pelacho, Beatriz

2018-04-01

Tissue-specific stem cells reside in a specialized environment known as niche. The niche plays a central role in the regulation of cell behaviour and, through the concerted action of soluble molecules, supportive somatic cells, and extracellular matrix components, directs stem cells to proliferate, differentiate, or remain quiescent. Great efforts have been done to decompose and separately analyse the contribution of these cues in the in vivo environment. Specifically, the mechanical properties of the extracellular matrix influence many aspects of cell behaviour, including self-renewal and differentiation. Deciphering the role of biomechanics could thereby provide important insights to control the stem cells responses in a more effective way with the aim to promote their therapeutic potential. In this review, we provide a wide overview of the effect that the microenvironment stiffness exerts on the control of cell behaviour with a particular focus on the induction of stem cells differentiation. We also describe the process of mechanotransduction and the molecular effectors involved. Finally, we critically discuss the potential involvement of tissue biomechanics in the design of novel tissue engineering strategies. Copyright © 2017 John Wiley & Sons, Ltd.

Effect of Urea and Thiourea on Generation of Xenogeneic Extracellular Matrix Scaffolds for Tissue Engineering

PubMed Central

Wong, Maelene L.; Wong, Janelle L.; Horn, Rebecca M.; Sannajust, Kimberley C.; Rice, Dawn A.

2016-01-01

Effective solubilization of proteins by chaotropes in proteomic applications motivates their use in solubilization-based antigen removal/decellularization strategies. A high urea concentration has previously been reported to significantly reduce lipophilic antigen content of bovine pericardium (BP); however, structure and function of the resultant extracellular matrix (ECM) scaffold were compromised. It has been recently demonstrated that in vivo ECM scaffold fate is determined by two primary outcome measures as follows: (1) sufficient reduction in antigen content to avoid graft-specific adaptive immune responses and (2) maintenance of native ECM structural proteins to avoid graft-specific innate responses. In this work, we assessed residual antigenicity, ECM architecture, ECM content, thermal stability, and tensile properties of BP subjected to a gradient of urea concentrations to determine whether an intermediate concentration exists at which both antigenicity and structure–function primary outcome measures for successful in vivo scaffold outcome can simultaneously be achieved. Alteration in tissue structure–function properties at various urea concentrations with decreased effectiveness for antigen removal makes use of urea-mediated antigen removal unlikely to be suitable for functional scaffold generation. PMID:27230226

Linearized texture of three-dimensional extracellular matrix is mandatory for bladder cancer cell invasion.

PubMed

Alfano, Massimo; Nebuloni, Manuela; Allevi, Raffaele; Zerbi, Pietro; Longhi, Erika; Lucianò, Roberta; Locatelli, Irene; Pecoraro, Angela; Indrieri, Marco; Speziali, Chantal; Doglioni, Claudio; Milani, Paolo; Montorsi, Francesco; Salonia, Andrea

2016-10-25

In the fields of biomaterials and tissue engineering simulating the native microenvironment is of utmost importance. As a major component of the microenvironment, the extracellular matrix (ECM) contributes to tissue homeostasis, whereas modifications of native features are associated with pathological conditions. Furthermore, three-dimensional (3D) geometry is an important feature of synthetic scaffolds favoring cell stemness, maintenance and differentiation. We analyzed the 3D structure, geometrical measurements and anisotropy of the ECM isolated from (i) human bladder mucosa (basal lamina and lamina propria) and muscularis propria; and, (ii) bladder carcinoma (BC). Next, binding and invasion of bladder metastatic cell line was observed on synthetic scaffold recapitulating anisotropy of tumoral ECM, but not on scaffold with disorganized texture typical of non-neoplastic lamina propria. This study provided information regarding the ultrastructure and geometry of healthy human bladder and BC ECMs. Likewise, using synthetic scaffolds we identified linearization of the texture as a mandatory feature for BC cell invasion. Integrating microstructure and geometry with biochemical and mechanical factors could support the development of an innovative synthetic bladder substitute or a tumoral scaffold predictive of chemotherapy outcomes.

Three-dimensional finite element modeling of pericellular matrix and cell mechanics in the nucleus pulposus of the intervertebral disk based on in situ morphology.

PubMed

Cao, Li; Guilak, Farshid; Setton, Lori A

2011-02-01

Nucleus pulposus (NP) cells of the intervertebral disk (IVD) have unique morphological characteristics and biologic responses to mechanical stimuli that may regulate maintenance and health of the IVD. NP cells reside as single cell, paired or multiple cells in a contiguous pericellular matrix (PCM), whose structure and properties may significantly influence cell and extracellular matrix mechanics. In this study, a computational model was developed to predict the stress-strain, fluid pressure and flow fields for cells and their surrounding PCM in the NP using three-dimensional (3D) finite element models based on the in situ morphology of cell-PCM regions of the mature rat NP, measured using confocal microscopy. Three-dimensional geometries of the extracellular matrix and representative cell-matrix units were used to construct 3D finite element models of the structures as isotropic and biphasic materials. In response to compressive strain of the extracellular matrix, NP cells and PCM regions were predicted to experience volumetric strains that were 1.9-3.7 and 1.4-2.1 times greater than the extracellular matrix, respectively. Volumetric and deviatoric strain concentrations were generally found at the cell/PCM interface, while von Mises stress concentrations were associated with the PCM/extracellular matrix interface. Cell-matrix units containing greater cell numbers were associated with higher peak cell strains and lower rates of fluid pressurization upon loading. These studies provide new model predictions for micromechanics of NP cells that can contribute to an understanding of mechanotransduction in the IVD and its changes with aging and degeneration.

Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones

PubMed Central

Hansen, R K; Bissell, M J

2010-01-01

The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellular matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function. PMID:10903527

Rapid enzyme regeneration results in the striking catalytic longevity of an engineered, single species, biocatalytic biofilm.

PubMed

Tong, Xiaoxue; Barberi, Tania Triscari; Botting, Catherine H; Sharma, Sunil V; Simmons, Mark J H; Overton, Tim W; Goss, Rebecca J M

2016-10-21

Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis. The catalytic longevity of the engineered biofilm is striking, and we had postulated that this was likely to largely result from protection conferred to recombinant enzymes by biofilm's extracellular matrix. SILAC (stable isotopic labelled amino acids in cell cultures), and in particular dynamic SILAC, in which pulses of different isotopically labelled amino acids are administered to cells over a time course, has been used to follow the fate of proteins. To explore within our spin coated biofilm, whether the recombinant enzyme's longevity might be in part due to its regeneration, we introduced pulses of isotopically labelled lysine and phenylalanine into medium overlaying the biofilm and followed their incorporation over the course of biofilm development. Through SILAC analysis, we reveal that constant and complete regeneration of recombinant enzymes occurs within spin coated biofilms. The striking catalytic longevity within the biofilm results from more than just simple protection of active enzyme by the biofilm and its associated extracellular matrix. The replenishment of recombinant enzyme is likely to contribute significantly to the catalytic longevity observed for the engineered biofilm system. Here we provide the first evidence of a recombinant enzyme's regeneration in an engineered biofilm. The recombinant enzyme was constantly replenished over time as evidenced by dynamic SILAC, which suggests that the engineered E. coli biofilms are highly metabolically active, having a not inconsiderable energetic demand. The constant renewal of recombinant enzyme highlights the attractive possibility of utilising this biofilm system as a dynamic platform into which enzymes of interest can be introduced in a "plug-and-play" fashion and potentially be controlled through promoter switching for production of a series of desired fine chemicals.

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.

Limitation of Cell Adhesion by the Elasticity of the Extracellular Matrix

PubMed Central

Nicolas, Alice; Safran, Samuel. A.

2006-01-01

Cell/matrix adhesions are modulated by cytoskeletal or external stresses and adapt to the mechanical properties of the extracellular matrix. We propose that this mechanosensitivity arises from the activation of a mechanosensor located within the adhesion itself. We show that this mechanism accounts for the observed directional growth of focal adhesions and the reduction or even cessation of their growth when cells adhere to a soft extracellular matrix. We predict quantitatively that both the elasticity and the thickness of the matrix play a key role in the dynamics of focal adhesions. Two different types of dynamics are expected depending on whether the thickness of the matrix is of order of or much larger than the adhesion size. In the latter situation, we predict that the adhesion region reaches a saturation size that can be tuned by the mechanical properties of the matrix. PMID:16581840

3D extracellular matrix interactions modulate tumour cell growth, invasion and angiogenesis in engineered tumour microenvironments.

PubMed

Taubenberger, Anna V; Bray, Laura J; Haller, Barbara; Shaposhnykov, Artem; Binner, Marcus; Freudenberg, Uwe; Guck, Jochen; Werner, Carsten

2016-05-01

Interactions between tumour cells and extracellular matrix proteins of the tumour microenvironment play crucial roles in cancer progression. So far, however, there are only a few experimental platforms available that allow us to study these interactions systematically in a mechanically defined three-dimensional (3D) context. Here, we have studied the effect of integrin binding motifs found within common extracellular matrix (ECM) proteins on 3D breast (MCF-7) and prostate (PC-3, LNCaP) cancer cell cultures, and co-cultures with endothelial and mesenchymal stromal cells. For this purpose, matrix metalloproteinase-degradable biohybrid poly(ethylene) glycol-heparin hydrogels were decorated with the peptide motifs RGD, GFOGER (collagen I), or IKVAV (laminin-111). Over 14days, cancer spheroids of 100-200μm formed. While the morphology of poorly invasive MCF-7 and LNCaP cells was not modulated by any of the peptide motifs, the aggressive PC-3 cells exhibited an invasive morphology when cultured in hydrogels comprising IKVAV and GFOGER motifs compared to RGD motifs or nonfunctionalised controls. PC-3 (but not MCF-7 and LNCaP) cell growth and endothelial cell infiltration were also significantly enhanced in IKVAV and GFOGER presenting gels. Taken together, we have established a 3D culture model that allows for dissecting the effect of biochemical cues on processes relevant to early cancer progression. These findings provide a basis for more mechanistic studies that may further advance our understanding of how ECM modulates cancer cell invasion and how to ultimately interfere with this process. Threedimensional in vitro cancer models have generated great interest over the past decade. However, most models are not suitable to systematically study the effects of environmental cues on cancer development and progression. To overcome this limitation, we have developed an innovative hydrogel platform to study the interactions between breast and prostate cancer cells and extracellular matrix ligands relevant to the tumour microenvironment. Our results show that hydrogels with laminin- and collagen-derived adhesive peptides induce a malignant phenotype in a cell-line specific manner. Thus, we have identified a method to control the incorporation of biochemical cues within a three dimensional culture model and anticipate that it will help us in better understanding the effects of the tumour microenvironment on cancer progression. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Imaging articular cartilage using second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Mansfield, Jessica C.; Winlove, C. Peter; Knapp, Karen; Matcher, Stephen J.

2006-02-01

Sub cellular resolution images of equine articular cartilage have been obtained using both second harmonic generation microscopy (SHGM) and two-photon fluorescence microscopy (TPFM). The SHGM images clearly map the distribution of the collagen II fibers within the extracellular matrix while the TPFM images show the distribution of endogenous two-photon fluorophores in both the cells and the extracellular matrix, highlighting especially the pericellular matrix and bright 2-3μm diameter features within the cells. To investigate the source of TPF in the extracellular matrix experiments have been carried out to see if it may originate from the proteoglycans. Pure solutions of the following proteoglycans hyaluronan, chondroitin sulfate and aggrecan have been imaged, only the aggrecan produced any TPF and here the intensity was not great enough to account for the TPF in the extracellular matrix. Also cartilage samples were subjected to a process to remove proteoglycans and cellular components. After this process the TPF from the samples had decreased by a factor of two, with respect to the SHG intensity.

Defining the hierarchical organisation of collagen VI microfibrils at nanometre to micrometre length scales.

PubMed

Godwin, Alan R F; Starborg, Tobias; Sherratt, Michael J; Roseman, Alan M; Baldock, Clair

2017-04-01

Extracellular matrix microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the matrix allowing for transduction of biochemical and mechanical signals. It is not understood how collagen VI is arranged into microfibrils or how these microfibrils are arranged into tissues. Therefore we have characterised the hierarchical organisation of collagen VI across multiple length scales. The frozen hydrated nanostructure of purified collagen VI microfibrils was reconstructed using cryo-TEM. The bead region has a compact hollow head and flexible tail regions linked by the collagenous interbead region. Serial block face SEM imaging coupled with electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM has a meshwork-like organisation formed from globular densities ∼30nm in diameter. These approaches can characterise structures spanning nanometer to millimeter length scales to define the nanostructure of individual collagen VI microfibrils and the micro-structural organisation of these fibrils within tissues to help in the future design of better mimetics for tissue engineering. Cartilage is a connective tissue rich in extracellular matrix molecules and is tough and compressive to cushion the bones of joints. However, in adults cartilage is poorly repaired after injury and so this is an important target for tissue engineering. Many connective tissues contain collagen VI, which forms microfibrils and networks but we understand very little about these assemblies or the tissue structures they form. Therefore, we have use complementary imaging techniques to image collagen VI microfibrils from the nano-scale to the micro-scale in order to understand the structure and the assemblies it forms. These findings will help to inform the future design of scaffolds to mimic connective tissues in regenerative medicine applications. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Reversible non-stick behaviour of a bacterial protein polymer provides a tuneable molecular mimic for cell and tissue engineering.

PubMed

Roque, Ana I; Soliakov, Andrei; Birch, Mark A; Philips, Sion R; Shah, Deepan S H; Lakey, Jeremy H

2014-05-01

Yersina pestis, the bubonic plague bacterium, is coated with a polymeric protein hydrogel for protection from host defences. The protein, which is robust and non-stick, resembles structures found in many eukaryotic extracellular-matrix proteins. Cells grown on the natural polymer cannot adhere and grow poorly; however, when cell-adhesion motifs are inserted into the protein, the cells proliferate. © The Authors, 2014. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Naturally derived and synthetic scaffolds for skeletal muscle reconstruction☆

PubMed Central

Wolf, Matthew T.; Dearth, Christopher L.; Sonnenberg, Sonya B.; Loboa, Elizabeth G.; Badylak, Stephen F.

2017-01-01

Skeletal muscle tissue has an inherent capacity for regeneration following injury. However, severe trauma, such as volumetric muscle loss, overwhelms these natural muscle repair mechanisms prompting the search for a tissue engineering/regenerative medicine approach to promote functional skeletal muscle restoration. A desirable approach involves a bioscaffold that simultaneously acts as an inductive microenvironment and as a cell/drug delivery vehicle to encourage muscle ingrowth. Both biologically active, naturally derived materials (such as extracellular matrix) and carefully engineered synthetic polymers have been developed to provide such a muscle regenerative environment. Next generation naturally derived/synthetic “hybrid materials” would combine the advantageous properties of these materials to create an optimal platform for cell/drug delivery and possess inherent bioactive properties. Advances in scaffolds using muscle tissue engineering are reviewed herein. PMID:25174309

Patterning Methods for Polymers in Cell and Tissue Engineering

PubMed Central

Kim, Hong Nam; Kang, Do-Hyun; Kim, Min Sung; Jiao, Alex; Kim, Deok-Ho; Suh, Kahp-Yang

2017-01-01

Polymers provide a versatile platform for mimicking various aspects of physiological extracellular matrix properties such as chemical composition, rigidity, and topography for use in cell and tissue engineering applications. In this review, we provide a brief overview of patterning methods of various polymers with a particular focus on biocompatibility and processability. The materials highlighted here are widely used polymers including thermally curable polydimethyl siloxane, ultraviolet-curable polyurethane acrylate and polyethylene glycol, thermo-sensitive poly(N-isopropylacrylamide) and thermoplastic and conductive polymers. We also discuss how micro- and nanofabricated polymeric substrates of tunable elastic modulus can be used to engineer cell and tissue structure and function. Such synergistic effect of topography and rigidity of polymers may be able to contribute to constructing more physiologically relevant microenvironment. PMID:22258887

Rheological Properties of Cross-Linked Hyaluronan–Gelatin Hydrogels for Tissue Engineering

PubMed Central

Vanderhooft, Janssen L.; Alcoutlabi, Mataz; Magda, Jules J.; Prestwich, Glenn D.

2009-01-01

Hydrogels that mimic the natural extracellular matrix (ECM) are used in three-dimensional cell culture, cell therapy, and tissue engineering. A semi-synthetic ECM based on cross-linked hyaluronana offers experimental control of both composition and gel stiffness. The mechanical properties of the ECM in part determine the ultimate cell phenotype. We now describe a rheological study of synthetic ECM hydrogels with storage shear moduli that span three orders of magnitude, from 11 to 3 500 Pa, a range important for engineering of soft tissues. The concentration of the chemically modified HA and the cross-linking density were the main determinants of gel stiffness. Increase in the ratio of thiol-modified gelatin reduced gel stiffness by diluting the effective concentration of the HA component. PMID:18839402

Recent advancements in electrospinning design for tissue engineering applications: A review.

PubMed

Kishan, Alysha P; Cosgriff-Hernandez, Elizabeth M

2017-10-01

Electrospinning, a technique used to fabricate fibrous scaffolds, has gained popularity in recent years as a method to produce tissue engineered grafts with architectural similarities to the extracellular matrix. Beyond its versatility in material selection, electrospinning also provides many tools to tune the fiber morphology and scaffold geometry. Recent efforts have focused on extending the capabilities of electrospinning to produce scaffolds that better recapitulate tissue properties and enhance regeneration. This review highlights these advancements by providing an overview of the processing variables and setups used to modulate scaffold architecture, discussing strategies to improve cellular infiltration and guide cell behavior, and providing a summary of electrospinning applications in tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2892-2905, 2017. © 2017 Wiley Periodicals, Inc.

[NEW PROGRESS OF ACELLULAR FISH SKIN AS NOVEL TISSUE ENGINEERED SCAFFOLD].

PubMed

Wei, Xiaojuan; Wang, Nanping; He, Lan; Guo, Xiuyu; Gu, Qisheng

2016-11-08

To review the recent research progress of acellular fish skin as a tissue engineered scaffold, and to analyze the feasibility and risk management in clinical application. The research and development, application status of acellular fish skin as a tissue engineered scaffold were comprehensively analyzed, and then several key points were put forward. Acellular fish skin has a huge potential in clinical practice as novel acellular extracellular matrix, but there have been no related research reports up to now in China. As an emerging point of translational medicine, investigation of acellular fish skin is mainly focused on artificial skin, surgical patch, and wound dressings. Development of acellular fish skin-based new products is concerned to be clinical feasible and necessary, but a lot of applied basic researches should be carried out.

Mutations in extracellular matrix genes NID1 and LAMC1 cause autosomal dominant Dandy-Walker malformation and occipital cephaloceles

PubMed Central

Darbro, Benjamin W.; Mahajan, Vinit B.; Gakhar, Lokesh; Skeie, Jessica M.; Campbell, Elizabeth; Wu, Shu; Bing, Xinyu; Millen, Kathleen J.; Dobyns, William B.; Kessler, John A.; Jalali, Ali; Cremer, James; Segre, Alberto; Manak, J. Robert; Aldinger, Kimerbly A.; Suzuki, Satoshi; Natsume, Nagato; Ono, Maya; Hai, Huynh Dai; Viet, Le Thi; Loddo, Sara; Valente, Enza M.; Bernardini, Laura; Ghonge, Nitin; Ferguson, Polly J.; Bassuk, Alexander G.

2013-01-01

We performed whole-exome sequencing of a family with autosomal dominant Dandy-Walker malformation and occipital cephaloceles (ADDWOC) and detected a mutation in the extracellular matrix protein encoding gene NID1. In a second family, protein interaction network analysis identified a mutation in LAMC1, which encodes a NID1 binding partner. Structural modeling the NID1-LAMC1 complex demonstrated that each mutation disrupts the interaction. These findings implicate the extracellular matrix in the pathogenesis of Dandy-Walker spectrum disorders. PMID:23674478

Physical, Spatial, and Molecular Aspects of Extracellular Matrix of In Vivo Niches and Artificial Scaffolds Relevant to Stem Cells Research

PubMed Central

Akhmanova, Maria; Osidak, Egor; Domogatsky, Sergey; Rodin, Sergey; Domogatskaya, Anna

2015-01-01

Extracellular matrix can influence stem cell choices, such as self-renewal, quiescence, migration, proliferation, phenotype maintenance, differentiation, or apoptosis. Three aspects of extracellular matrix were extensively studied during the last decade: physical properties, spatial presentation of adhesive epitopes, and molecular complexity. Over 15 different parameters have been shown to influence stem cell choices. Physical aspects include stiffness (or elasticity), viscoelasticity, pore size, porosity, amplitude and frequency of static and dynamic deformations applied to the matrix. Spatial aspects include scaffold dimensionality (2D or 3D) and thickness; cell polarity; area, shape, and microscale topography of cell adhesion surface; epitope concentration, epitope clustering characteristics (number of epitopes per cluster, spacing between epitopes within cluster, spacing between separate clusters, cluster patterns, and level of disorder in epitope arrangement), and nanotopography. Biochemical characteristics of natural extracellular matrix molecules regard diversity and structural complexity of matrix molecules, affinity and specificity of epitope interaction with cell receptors, role of non-affinity domains, complexity of supramolecular organization, and co-signaling by growth factors or matrix epitopes. Synergy between several matrix aspects enables stem cells to retain their function in vivo and may be a key to generation of long-term, robust, and effective in vitro stem cell culture systems. PMID:26351461

Characterization of the Vibrio cholerae extracellular matrix: a top-down solid-state NMR approach.

PubMed

Reichhardt, Courtney; Fong, Jiunn C N; Yildiz, Fitnat; Cegelski, Lynette

2015-01-01

Bacterial biofilms are communities of bacterial cells surrounded by a self-secreted extracellular matrix. Biofilm formation by Vibrio cholerae, the human pathogen responsible for cholera, contributes to its environmental survival and infectivity. Important genetic and molecular requirements have been identified for V. cholerae biofilm formation, yet a compositional accounting of these parts in the intact biofilm or extracellular matrix has not been described. As insoluble and non-crystalline assemblies, determinations of biofilm composition pose a challenge to conventional biochemical and biophysical analyses. The V. cholerae extracellular matrix composition is particularly complex with several proteins, complex polysaccharides, and other biomolecules having been identified as matrix parts. We developed a new top-down solid-state NMR approach to spectroscopically assign and quantify the carbon pools of the intact V. cholerae extracellular matrix using ¹³C CPMAS and ¹³C{(¹⁵N}, ¹⁵N{³¹P}, and ¹³C{³¹P}REDOR. General sugar, lipid, and amino acid pools were first profiled and then further annotated and quantified as specific carbon types, including carbonyls, amides, glycyl carbons, and anomerics. In addition, ¹⁵N profiling revealed a large amine pool relative to amide contributions, reflecting the prevalence of molecular modifications with free amine groups. Our top-down approach could be implemented immediately to examine the extracellular matrix from mutant strains that might alter polysaccharide production or lipid release beyond the cell surface; or to monitor changes that may accompany environmental variations and stressors such as altered nutrient composition, oxidative stress or antibiotics. More generally, our analysis has demonstrated that solid-state NMR is a valuable tool to characterize complex biofilm systems. Copyright © 2014. Published by Elsevier B.V.

A major protein component of the Bacillus subtilis biofilm matrix.

PubMed

Branda, Steven S; Chu, Frances; Kearns, Daniel B; Losick, Richard; Kolter, Roberto

2006-02-01

Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

Modeling the formation of cell-matrix adhesions on a single 3D matrix fiber.

PubMed

Escribano, J; Sánchez, M T; García-Aznar, J M

2015-11-07

Cell-matrix adhesions are crucial in different biological processes like tissue morphogenesis, cell motility, and extracellular matrix remodeling. These interactions that link cell cytoskeleton and matrix fibers are built through protein clutches, generally known as adhesion complexes. The adhesion formation process has been deeply studied in two-dimensional (2D) cases; however, the knowledge is limited for three-dimensional (3D) cases. In this work, we simulate different local extracellular matrix properties in order to unravel the fundamental mechanisms that regulate the formation of cell-matrix adhesions in 3D. We aim to study the mechanical interaction of these biological structures through a three dimensional discrete approach, reproducing the transmission pattern force between the cytoskeleton and a single extracellular matrix fiber. This numerical model provides a discrete analysis of the proteins involved including spatial distribution, interaction between them, and study of the different phenomena, such as protein clutches unbinding or protein unfolding. Copyright © 2015 Elsevier Ltd. All rights reserved.

Micrometer scale guidance of mesenchymal stem cells to form structurally oriented large-scale tissue engineered cartilage.

PubMed

Chou, Chih-Ling; Rivera, Alexander L; Williams, Valencia; Welter, Jean F; Mansour, Joseph M; Drazba, Judith A; Sakai, Takao; Baskaran, Harihara

2017-09-15

Current clinical methods to treat articular cartilage lesions provide temporary relief of the symptoms but fail to permanently restore the damaged tissue. Tissue engineering, using mesenchymal stem cells (MSCs) combined with scaffolds and bioactive factors, is viewed as a promising method for repairing cartilage injuries. However, current tissue engineered constructs display inferior mechanical properties compared to native articular cartilage, which could be attributed to the lack of structural organization of the extracellular matrix (ECM) of these engineered constructs in comparison to the highly oriented structure of articular cartilage ECM. We previously showed that we can guide MSCs undergoing chondrogenesis to align using microscale guidance channels on the surface of a two-dimensional (2-D) collagen scaffold, which resulted in the deposition of aligned ECM within the channels and enhanced mechanical properties of the constructs. In this study, we developed a technique to roll 2-D collagen scaffolds containing MSCs within guidance channels in order to produce a large-scale, three-dimensional (3-D) tissue engineered cartilage constructs with enhanced mechanical properties compared to current constructs. After rolling the MSC-scaffold constructs into a 3-D cylindrical structure, the constructs were cultured for 21days under chondrogenic culture conditions. The microstructure architecture and mechanical properties of the constructs were evaluated using imaging and compressive testing. Histology and immunohistochemistry of the constructs showed extensive glycosaminoglycan (GAG) and collagen type II deposition. Second harmonic generation imaging and Picrosirius red staining indicated alignment of neo-collagen fibers within the guidance channels of the constructs. Mechanical testing indicated that constructs containing the guidance channels displayed enhanced compressive properties compared to control constructs without these channels. In conclusion, using a novel roll-up method, we have developed large scale MSC based tissue-engineered cartilage that shows microscale structural organization and enhanced compressive properties compared to current tissue engineered constructs. Tissue engineered cartilage constructs made with human mesenchymal stem cells (hMSCs), scaffolds and bioactive factors are a promising solution to treat cartilage defects. A major disadvantage of these constructs is their inferior mechanical properties compared to the native tissue, which is likely due to the lack of structural organization of the extracellular matrix of the engineered constructs. In this study, we developed three-dimensional (3-D) cartilage constructs from rectangular scaffold sheets containing hMSCs in micro-guidance channels and characterized their mechanical properties and metabolic requirements. The work led to a novel roll-up method to embed 2-D microscale structures in 3-D constructs. Further, micro-guidance channels incorporated within the 3-D cartilage constructs led to the production of aligned cell-produced matrix and enhanced mechanical function. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Presynaptic neurones may contribute a unique glycoprotein to the extracellular matrix at the synapse

NASA Astrophysics Data System (ADS)

Caroni, Pico; Carlson, Steven S.; Schweitzer, Erik; Kelly, Regis B.

1985-04-01

As the extracellular matrix at the original site of a neuromuscular junction seems to play a major part in the specificity of synaptic regeneration1-5, considerable attention has been paid to unique molecules localized to this region6-11. Here we describe an extracellular matrix glycoprotein of the elasmobranch electric organ that is localized near the nerve endings. By immunological criteria, it is synthesized in the cell bodies, transported down the axons and is related to a glycoprotein in the synaptic vesicles of the neurones that innervate the electric organ. It is apparently specific for these neurones, as it cannot be detected elsewhere in the nervous system of the fish. Therefore, neurones seem to contribute unique extracellular matrix glycoproteins to the synaptic region. Synaptic vesicles could be involved in transporting these glycoproteins to or from the nerve terminal surface.

A Novel Biodegradable Polyurethane Matrix for Auricular Cartilage Repair: An In Vitro and In Vivo Study.

PubMed

Iyer, Kartik; Dearman, Bronwyn L; Wagstaff, Marcus J D; Greenwood, John E

2016-01-01

Auricular reconstruction poses a challenge for reconstructive and burns surgeons. Techniques involving cartilage tissue engineering have shown potential in recent years. A biodegradable polyurethane matrix developed for dermal reconstruction offers an alternative to autologous, allogeneic, or xenogeneic biologicals for cartilage reconstruction. This study assesses such a polyurethane matrix for this indication in vivo and in vitro. To evaluate intrinsic cartilage repair, three pigs underwent auricular surgery to create excisional cartilage ± perichondrial defects, measuring 2 × 3 cm in each ear, into which acellular polyurethane matrices were implanted. Biopsies were taken at day 28 for histological assessment. Porcine chondrocytes ± perichondrocytes were cultured and seeded in vitro onto 1 × 1 cm polyurethane scaffolds. The total culture period was 42 days; confocal, histological, and immunohistochemical analyses of scaffold cultures were performed on days 14, 28, and 42. In vivo, the polyurethane matrices integrated with granulation tissue filling all biopsy samples. Minimal neocartilage invasion was observed marginally on some samples. Tissue composition was identical between ears whether perichondrium was left intact, or not. In vitro, the polyurethane matrix was biocompatible with chondrocytes ± perichondrocytes and supported production of extracellular matrix and Type II collagen. No difference was observed between chondrocyte culture alone and chondrocyte/perichondrocyte scaffold coculture. The polyurethane matrix successfully integrated into the auricular defect and was a suitable scaffold in vitro for cartilage tissue engineering, demonstrating its potential application in auricular reconstruction.

Engineering-derived approaches for iPSC preparation, expansion, differentiation and applications.

PubMed

Li, Yang; Li, Ling; Chen, Zhi-Nan; Gao, Ge; Yao, Rui; Sun, Wei

2017-07-31

Remarkable achievements have been made since induced pluripotent stem cells (iPSCs) were first introduced in 2006. Compared with non-pluripotent stem cells, iPSC research faces several additional complexities, such as the choice of extracellular matrix proteins, growth and differentiation factors, as well as technical challenges related to self-renewal and directed differentiation. Overcoming these challenges requires the integration of knowledge and technologies from multiple fields including cell biology, biomaterial science, engineering, physics and medicine. Here, engineering-derived iPSC approaches are reviewed according to three aspects of iPSC studies: preparation, expansion, differentiation and applications. Engineering strategies, such as 3D systems establishment, cell-matrix mechanics and the regulation of biophysical and biochemical cues, together with engineering techniques, such as 3D scaffolds, cell microspheres and bioreactors, have been applied to iPSC studies and have generated insightful results and even mini-organs such as retinas, livers and intestines. Specific results are given to demonstrate how these approaches impact iPSC behavior, and related mechanisms are discussed. In addition, cell printing technologies are presented as an advanced engineering-derived approach since they have been applied in both iPSC studies and the construction of diverse tissues and organs. Further development and possible innovations of cell printing technologies are presented in terms of creating complex and functional iPSC-derived living tissues and organs.

Optimization and critical evaluation of decellularization strategies to develop renal extracellular matrix scaffolds as biological templates for organ engineering and transplantation.

PubMed

Caralt, M; Uzarski, J S; Iacob, S; Obergfell, K P; Berg, N; Bijonowski, B M; Kiefer, K M; Ward, H H; Wandinger-Ness, A; Miller, W M; Zhang, Z J; Abecassis, M M; Wertheim, J A

2015-01-01

The ability to generate patient-specific cells through induced pluripotent stem cell (iPSC) technology has encouraged development of three-dimensional extracellular matrix (ECM) scaffolds as bioactive substrates for cell differentiation with the long-range goal of bioengineering organs for transplantation. Perfusion decellularization uses the vasculature to remove resident cells, leaving an intact ECM template wherein new cells grow; however, a rigorous evaluative framework assessing ECM structural and biochemical quality is lacking. To address this, we developed histologic scoring systems to quantify fundamental characteristics of decellularized rodent kidneys: ECM structure (tubules, vessels, glomeruli) and cell removal. We also assessed growth factor retention--indicating matrix biofunctionality. These scoring systems evaluated three strategies developed to decellularize kidneys (1% Triton X-100, 1% Triton X-100/0.1% sodium dodecyl sulfate (SDS) and 0.02% Trypsin-0.05% EGTA/1% Triton X-100). Triton and Triton/SDS preserved renal microarchitecture and retained matrix-bound basic fibroblast growth factor and vascular endothelial growth factor. Trypsin caused structural deterioration and growth factor loss. Triton/SDS-decellularized scaffolds maintained 3 h of leak-free blood flow in a rodent transplantation model and supported repopulation with human iPSC-derived endothelial cells and tubular epithelial cells ex vivo. Taken together, we identify an optimal Triton/SDS-based decellularization strategy that produces a biomatrix that may ultimately serve as a rodent model for kidney bioengineering. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

The role of nanotechnology in induced pluripotent and embryonic stem cells research.

PubMed

Chen, Lukui; Qiu, Rong; Li, Lushen

2014-12-01

This paper reviews the recent studies on development of nanotechnology in the field of induced pluripotent and embryonic stem cells. Stem cell therapy is a promising therapy that can improve the quality of life for patients with refractory diseases. However, this option is limited by the scarcity of tissues, ethical problem, and tumorigenicity. Nanotechnology is another promising therapy that can be used to mimic the extracellular matrix, label the implanted cells, and also can be applied in the tissue engineering. In this review, we briefly introduce implementation of nanotechnology in induced pluripotent and embryonic stem cells research. Finally, the potential application of nanotechnology in tissue engineering and regenerative medicine is also discussed.

Suspended, Shrinkage-Free, Electrospun PLGA Nanofibrous Scaffold for Skin Tissue Engineering.

PubMed

Ru, Changhai; Wang, Feilong; Pang, Ming; Sun, Lining; Chen, Ruihua; Sun, Yu

2015-05-27

Electrospinning is a technique for creating continuous nanofibrous networks that can architecturally be similar to the structure of extracellular matrix (ECM). However, the shrinkage of electrospun mats is unfavorable for the triggering of cell adhesion and further growth. In this work, electrospun PLGA nanofiber assemblies are utilized to create a scaffold. Aided by a polypropylene auxiliary supporter, the scaffold is able to maintain long-term integrity without dimensional shrinkage. This scaffold is also able to suspend in cell culture medium; hence, keratinocyte cells seeded on the scaffold are exposed to air as required in skin tissue engineering. Experiments also show that human skin keratinocytes can proliferate on the scaffold and infiltrate into the scaffold.

Effects of radial compression on a novel simulated intervertebral disc-like assembly using bone marrow-derived mesenchymal stem cell cell-sheets for annulus fibrosus regeneration.

PubMed

See, Eugene Yong-Shun; Toh, Siew Lok; Goh, James Cho-Hong

2011-10-01

The aim of this study was to develop a tissue engineering approach in regenerating the annulus fibrosus (AF) as part of an overall strategy to produce a tissue-engineered intervertebral disc (IVD) replacement. To determine whether a rehabilitative simulation regime on bone marrow–derived mesenchymal stem cell cell-sheet is able to aid the regeneration of the AF. No previous study has used bone marrow–derived mesenchymal stem cell cell-sheets simulated by a rehabilitative regime to regenerate the AF. The approach was to use bone marrow–derived stem cells to form cell-sheets and incorporating them onto silk scaffolds to simulate the native lamellae of the AF. The in vitro experimental model used to study the efficacy of such a system was made up of the tissue engineering AF construct wrapped around a silicone disc to form a simulated IVD-like assembly. The assembly was cultured within a custom-designed bioreactor that provided a compressive mechanical stimulation onto the silicone disc. The silicone nucleus pulposus would bulge radially and compress the simulated AF to mimic the physiological conditions. The simulated IVD-like assembly was compressed using a rehabilitative regime that lasted for 4 weeks at 0.25 Hz, for 15 minutes each day. With the rehabilitative regime, the cell-sheets remained viable but showed a decrease in cell numbers and viability. Gene expression analysis showed significant upregulation of IVD-related genes and there was an increased ratio of collagen type II to collagen type I found within the extracellular matrix. The results suggested that a rehabilitative regime caused extensive remodeling to take place within the simulated IVD-like assembly, producing extracellular matrix similar to that found in the inner AF.

A collagen-based scaffold delivering exogenous microrna-29B to modulate extracellular matrix remodeling.

PubMed

Monaghan, Michael; Browne, Shane; Schenke-Layland, Katja; Pandit, Abhay

2014-04-01

Directing appropriate extracellular matrix remodeling is a key aim of regenerative medicine strategies. Thus, antifibrotic interfering RNA (RNAi) therapy with exogenous microRNA (miR)-29B was proposed as a method to modulate extracellular matrix remodeling following cutaneous injury. It was hypothesized that delivery of miR-29B from a collagen scaffold will efficiently modulate the extracellular matrix remodeling response and reduce maladaptive remodeling such as aggressive deposition of collagen type I after injury. The release of RNA from the scaffold was assessed and its ability to silence collagen type I and collagen type III expression was evaluated in vitro. When primary fibroblasts were cultured with scaffolds doped with miR-29B, reduced levels of collagen type I and collagen type III mRNA expression were observed for up to 2 weeks of culture. When the scaffolds were applied to full thickness wounds in vivo, reduced wound contraction, improved collagen type III/I ratios and a significantly higher matrix metalloproteinase (MMP)-8: tissue inhibitor of metalloproteinase (TIMP)-1 ratio were detected when the scaffolds were functionalized with miR-29B. Furthermore, these effects were significantly influenced by the dose of miR-29B in the collagen scaffold (0.5 versus 5 μg). This study shows a potential of combining exogenous miRs with collagen scaffolds to improve extracellular matrix remodeling following injury.

Detection of extracellular matrix modification in cancer models with inverse spectroscopic optical coherence tomography

NASA Astrophysics Data System (ADS)

Spicer, Graham L. C.; Azarin, Samira M.; Yi, Ji; Young, Scott T.; Ellis, Ronald; Bauer, Greta M.; Shea, Lonnie D.; Backman, Vadim

2016-10-01

In cancer biology, there has been a recent effort to understand tumor formation in the context of the tissue microenvironment. In particular, recent progress has explored the mechanisms behind how changes in the cell-extracellular matrix ensemble influence progression of the disease. The extensive use of in vitro tissue culture models in simulant matrix has proven effective at studying such interactions, but modalities for non-invasively quantifying aspects of these systems are scant. We present the novel application of an imaging technique, Inverse Spectroscopic Optical Coherence Tomography, for the non-destructive measurement of in vitro biological samples during matrix remodeling. Our findings indicate that the nanoscale-sensitive mass density correlation shape factor D of cancer cells increases in response to a more crosslinked matrix. We present a facile technique for the non-invasive, quantitative study of the micro- and nano-scale structure of the extracellular matrix and its host cells.

Manipulation of in vitro collagen matrix architecture for scaffolds of improved physiological relevance

NASA Astrophysics Data System (ADS)

Hapach, Lauren A.; VanderBurgh, Jacob A.; Miller, Joseph P.; Reinhart-King, Cynthia A.

2015-12-01

Type I collagen is a versatile biomaterial that is widely used in medical applications due to its weak antigenicity, robust biocompatibility, and its ability to be modified for a wide array of applications. As such, collagen has become a major component of many tissue engineering scaffolds, drug delivery platforms, and substrates for in vitro cell culture. In these applications, collagen constructs are fabricated to recapitulate a diverse set of conditions. Collagen fibrils can be aligned during or post-fabrication, cross-linked via numerous techniques, polymerized to create various fibril sizes and densities, and copolymerized into a wide array of composite scaffolds. Here, we review approaches that have been used to tune collagen to better recapitulate physiological environments for use in tissue engineering applications and studies of basic cell behavior. We discuss techniques to control fibril alignment, methods for cross-linking collagen constructs to modulate stiffness, and composite collagen constructs to better mimic physiological extracellular matrix.

Non-Fouling Biodegradable Poly(ϵ-caprolactone) Nanofibers for Tissue Engineering.

PubMed

Kostina, Nina Yu; Pop-Georgievski, Ognen; Bachmann, Michael; Neykova, Neda; Bruns, Michael; Michálek, Jiří; Bastmeyer, Martin; Rodriguez-Emmenegger, Cesar

2016-01-01

Poly(ϵ-caprolactone) (PCL) nanofibers are very attractive materials for tissue engineering (TE) due to their degradability and structural similarity to the extracellular matrix (ECM). However, upon exposure to biological media, their surface is rapidly fouled by proteins and cells, which may lead to inflammation and foreign body reaction. In this study, an approach for the modification of PCL nanofibers to prevent protein fouling from biological fluids and subsequent cell adhesion is introduced. A biomimetic polydopamine (PDA) layer was deposited on the surface of the PCL nanofibers and four types of antifouling polymer brushes were grown by surface-initiated atom transfer radical polymerization (SI-ATRP) from initiator moieties covalently attached to the PDA layer. Cell adhesion was assessed with mouse embryonic fibroblasts (MEFs). MEFs rapidly adhered and formed cell-matrix adhesions (CMAs) with PCL and PCL-PDA nanofibers. Importantly, the nanofibers modified with antifouling polymer brushes were able to suppress non-specific protein adsorption and thereby cell adhesion. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biomechanical cell regulatory networks as complex adaptive systems in relation to cancer.

PubMed

Feller, Liviu; Khammissa, Razia Abdool Gafaar; Lemmer, Johan

2017-01-01

Physiological structure and function of cells are maintained by ongoing complex dynamic adaptive processes in the intracellular molecular pathways controlling the overall profile of gene expression, and by genes in cellular gene regulatory circuits. Cytogenetic mutations and non-genetic factors such as chronic inflammation or repetitive trauma, intrinsic mechanical stresses within extracellular matrix may induce redirection of gene regulatory circuits with abnormal reactivation of embryonic developmental programmes which can now drive cell transformation and cancer initiation, and later cancer progression and metastasis. Some of the non-genetic factors that may also favour cancerization are dysregulation in epithelial-mesenchymal interactions, in cell-to-cell communication, in extracellular matrix turnover, in extracellular matrix-to-cell interactions and in mechanotransduction pathways. Persistent increase in extracellular matrix stiffness, for whatever reason, has been shown to play an important role in cell transformation, and later in cancer cell invasion. In this article we review certain cell regulatory networks driving carcinogenesis, focussing on the role of mechanical stresses modulating structure and function of cells and their extracellular matrices.

Periodontal Bioengineering: A Discourse in Surface Topographies, Progenitor Cells and Molecular Profiles

NASA Astrophysics Data System (ADS)

Dangaria, Smit J.

2011-12-01

Stem/progenitor cells are a population of cells capable of providing replacement cells for a given differentiated cell type. We have applied progenitor cell-based technologies to generate novel tissue-engineered implants that use biomimetic strategies with the ultimate goal of achieving full regeneration of lost periodontal tissues. Mesenchymal periodontal tissues such as cementum, alveolar bone (AB), and periodontal ligament (PDL) are neural crest-derived entities that emerge from the dental follicle (DF) at the onset of tooth root formation. Using a systems biology approach we have identified key differences between these periodontal progenitors on the basis of global gene expression profiles, gene cohort expression levels, and epigenetic modifications, in addition to differences in cellular morphologies. On an epigenetic level, DF progenitors featured high levels of the euchromatin marker H3K4me3, whereas PDL cells, AB osteoblasts, and cementoblasts contained high levels of the transcriptional repressor H3K9me3. Secondly, we have tested the influence of natural extracellular hydroxyapatite matrices on periodontal progenitor differentiation. Dimension and structure of extracellular matrix surfaces have powerful influences on cell shape, adhesion, and gene expression. Here we show that natural tooth root topographies induce integrin-mediated extracellular matrix signaling cascades in tandem with cell elongation and polarization to generate physiological periodontium-like tissues. In this study we replanted surface topography instructed periodontal ligament progenitors (PDLPs) into rat alveolar bone sockets for 8 and 16 weeks, resulting in complete attachment of tooth roots to the surrounding alveolar bone with a periodontal ligament fiber apparatus closely matching physiological controls along the entire root surface. Displacement studies and biochemical analyses confirmed that progenitor-based engineered periodontal tissues were similar to control teeth and uniquely derived from pre-implantation green fluorescent protein (GFP)-labeled progenitors. Together, these studies illustrate the capacity of natural extracellular surface topographies to instruct PDLPs to fully regenerate complex cellular and structural morphologies of tissues once lost to disease. We suggest that our strategy could be used for the replantation of teeth lost due to trauma or as a novel approach for tooth replacement using tooth-shaped replicas.

Use of Mueller matrix polarimetry and optical coherence tomography in the characterization of cervical collagen anisotropy.

PubMed

Chue-Sang, Joseph; Bai, Yuqiang; Stoff, Susan; Gonzalez, Mariacarla; Holness, Nola; Gomes, Jefferson; Jung, Ranu; Gandjbakhche, Amir; Chernomordik, Viktor V; Ramella-Roman, Jessica C

2017-08-01

Preterm birth (PTB) presents a serious medical health concern throughout the world. There is a high incidence of PTB in both developed and developing countries ranging from 11% to 15%, respectively. Recent research has shown that cervical collagen orientation and distribution changes during pregnancy may be useful in predicting PTB. Polarization imaging is an effective means to measure optical anisotropy in birefringent materials, such as the cervix's extracellular matrix. Noninvasive, full-field Mueller matrix polarimetry (MMP) imaging methodologies, and optical coherence tomography (OCT) imaging were used to assess cervical collagen content and structure in nonpregnant porcine cervices. We demonstrate that the highly ordered structure of the nonpregnant porcine cervix can be observed with MMP. Furthermore, when utilized ex vivo, OCT and MMP yield very similar results with a mean error of 3.46% between the two modalities. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Injectable Hydrogel Scaffold from Decellularized Human Lipoaspirate

PubMed Central

Young, D. Adam; Ibrahim, Dina O.; Hu, Diane; Christman, Karen L.

2010-01-01

Soft tissue fillers are rapidly gaining popularity for aesthetic improvements or repair of adipose tissue deficits. Several injectable biopolymers have been investigated for this purpose but often face rapid resorption or limited adipogenesis, and do not mimic the native adipose extracellular matrix (ECM). We have generated an injectable adipose matrix scaffold by efficiently removing both the cellular and lipid contents of human lipoaspirate. The decellularized material retained a complex composition of peptides and glycosaminoglycans found in native adipose ECM. This matrix can be further processed by solubilizing the extracted ECM to generate a thermally-responsive hydrogel that self-assembles upon subcutaneous injection. This hydrogel also supports the growth and survival of patient matched adipose - derived stem cells in vitro. The development of an injectable hydrogel from human lipoaspirate represents a minimally-invasive option for adipose tissue engineering in terms of both the collection of source material and delivery of the scaffold. PMID:20932943

Enhancement of matrix production and cell proliferation in human annulus cells under bioreactor culture.

PubMed

Yang, Xinlin; Wang, Daidong; Hao, Jianrong; Gong, Meiqing; Arlet, Vincent; Balian, Gary; Shen, Francis H; Li, Xudong Joshua

2011-06-01

Tissue engineering is a promising approach for treatment of disc degeneration. Herein, we evaluated effects of rotating bioreactor culture on the extracellular matrix production and proliferation of human annulus fibrosus (AF) cells. AF cells were embedded into alginate beads, and then cultured up to 3 weeks in a rotating wall vessel bioreactor or a static vessel. By real-time reverse transcription-polymerase chain reaction, expression of aggrecan, collagen type I and type II, and collagen prolyl 4-hydroxylase II was remarkably elevated, whereas expression of matrix metalloproteinase 3 and a disintegrin and metalloproteinase with thrombospondin motifs 5 was significantly decreased under bioreactor. Biochemical analysis revealed that the levels of the whole cell-associated proteoglycan and collagen were approximately five- and twofolds in rotating bioreactor, respectively, compared to those in static culture. Moreover, AF cell proliferation was augmented in rotating bioreactor. DNA contents were threefolds higher in rotating bioreactor than that in static culture. Expression of the proliferating cell nuclear antigen was robustly enhanced in rotating bioreactor as early as 1 week. Our findings suggested that rotating bioreactor culture would be an effective technique for expansion of human annulus cells for tissue engineering driven treatment of disc degeneration.

Design of nano- and microfiber combined scaffolds by electrospinning of collagen onto starch-based fiber meshes: a man-made equivalent of natural extracellular matrix.

PubMed

Tuzlakoglu, Kadriye; Santos, Marina I; Neves, Nuno; Reis, Rui L

2011-02-01

Mimicking the structural organization and biologic function of natural extracellular matrix has been one of the main goals of tissue engineering. Nevertheless, the majority of scaffolding materials for bone regeneration highlights biochemical functionality in detriment of mechanical properties. In this work we present a rather innovative construct that combines in the same structure electrospun type I collagen nanofibers with starch-based microfibers. These combined structures were obtained by a two-step methodology and structurally consist in a type I collagen nano-network incorporated on a macro starch-based support. The morphology of the developed structures was assessed by several microscopy techniques and the collagenous nature of the nano-network was confirmed by immunohistochemistry. In addition, and especially regarding the requirements of large bone defects, we also successfully introduced the concept of layer by layer, as a way to produce thicker structures. In an attempt to recreate bone microenvironment, the design and biochemical composition of the combined structures also envisioned bone-forming cells and endothelial cells (ECs). The inclusion of a type I collagen nano-network induced a stretched morphology and improved the metabolic activity of osteoblasts. Regarding ECs, the presence of type I collagen on the combined structures provided adhesive support and obviated the need of precoating with fibronectin. It was also importantly observed that ECs on the nano-network organized into circular structures, a three-dimensional arrangement distinct from that observed for osteoblasts and resembling the microcappillary-like organizations formed during angiogenesis. By providing simultaneously physical and chemical cues for cells, the herein-proposed combined structures hold a great potential in bone regeneration as a man-made equivalent of extracellular matrix.

Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds.

PubMed

Butler, Colin R; Hynds, Robert E; Crowley, Claire; Gowers, Kate H C; Partington, Leanne; Hamilton, Nicholas J; Carvalho, Carla; Platé, Manuela; Samuel, Edward R; Burns, Alan J; Urbani, Luca; Birchall, Martin A; Lowdell, Mark W; De Coppi, Paolo; Janes, Sam M

2017-04-01

Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3-8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Development and Characterization of Acellular Extracellular Matrix Scaffolds from Porcine Menisci for Use in Cartilage Tissue Engineering

PubMed Central

Chen, Ying-Chen; Chen, Ray-Neng; Jhan, Hua-Jing; Liu, Der-Zen; Ho, Hsiu-O; Mao, Yong; Kohn, Joachim

2015-01-01

Given the growing number of arthritis patients and the limitations of current treatments, there is great urgency to explore cartilage substitutes by tissue engineering. In this study, we developed a novel decellularization method for menisci to prepare acellular extracellular matrix (ECM) scaffolds with minimal adverse effects on the ECM. Among all the acid treatments, formic acid treatment removed most of the cellular contents and preserved the highest ECM contents in the decellularized porcine menisci. Compared with fresh porcine menisci, the content of DNA decreased to 4.10%±0.03%, and there was no significant damage to glycosaminoglycan (GAG) or collagen. Histological staining also confirmed the presence of ECM and the absence of cellularity. In addition, a highly hydrophilic scaffold with three-dimensional interconnected porous structure was fabricated from decellularized menisci tissue. Human chondrocytes showed enhanced cell proliferation and synthesis of chondrocyte ECM including type II collagen and GAG when cultured in this acellular scaffold. Moreover, the scaffold effectively supported chondrogenesis of human bone marrow-derived mesenchymal stem cells. Finally, in vivo implantation was conducted in rats to assess the biocompatibility of the scaffolds. No significant inflammatory response was observed. The acellular ECM scaffold provided a native environment for cells with diverse physiological functions to promote cell proliferation and new tissue formation. This study reported a novel way to prepare decellularized meniscus tissue and demonstrated the potential as scaffolds to support cartilage repair. PMID:25919905

Synthetic design of growth factor sequestering extracellular matrix mimetic hydrogel for promoting in vivo bone formation.

PubMed

Yan, Hong Ji; Casalini, Tommaso; Hulsart-Billström, Gry; Wang, Shujiang; Oommen, Oommen P; Salvalaglio, Matteo; Larsson, Sune; Hilborn, Jöns; Varghese, Oommen P

2018-04-01

Synthetic scaffolds that possess an intrinsic capability to protect and sequester sensitive growth factors is a primary requisite for developing successful tissue engineering strategies. Growth factors such as recombinant human bone morphogenetic protein-2 (rhBMP-2) is highly susceptible to premature degradation and to provide a meaningful clinical outcome require high doses that can cause serious side effects. We discovered a unique strategy to stabilize and sequester rhBMP-2 by enhancing its molecular interactions with hyaluronic acid (HA), an extracellular matrix (ECM) component. We found that by tuning the initial protonation state of carboxylic acid residues of HA in a covalently crosslinked hydrogel modulate BMP-2 release at physiological pH by minimizing the electrostatic repulsion and maximizing the Van der Waals interactions. At neutral pH, BMP-2 release is primarily governed by Fickian diffusion, whereas at acidic pH both diffusion and electrostatic interactions between HA and BMP-2 become important as confirmed by molecular dynamics simulations. Our results were also validated in an in vivo rat ectopic model with rhBMP-2 loaded hydrogels, which demonstrated superior bone formation with acidic hydrogel as compared to the neutral counterpart. We believe this study provides new insight on growth factor stabilization and highlights the therapeutic potential of engineered matrices for rhBMP-2 delivery and may help to curtail the adverse side effects associated with the high dose of the growth factor. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds

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

Chen, Chih-Hao; Neurosurgery, Department of Surgery, Kaohsiung Veterans General Hospital, Taiwan, ROC; Department of Biomedical Engineering, I-Shou University, Taiwan, ROC

Highlights: Black-Right-Pointing-Pointer Neuron cancer stem cells (NCSCs) behave high multiply of growth on collagen scaffold. Black-Right-Pointing-Pointer Enhancement of NCSCs neurite outgrowth on porous collagen scaffold. Black-Right-Pointing-Pointer 3-D collagen culture of NCSCs shows an advance differentiation than 2-D culture. -- Abstract: Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porousmore » collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 {mu}m porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.« less

Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: a pragmatic approach.

PubMed

Prestwich, Glenn D

2007-08-15

The common technique of growing cells on tissue culture plastic (TCP) is gradually being supplanted by methods for culturing cells in two-dimensions (2-D) on matrices with more appropriate physical and biological properties or by encapsulation of cells in three-dimensions (3-D). The universal acceptance of the new 3-D paradigm is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. In this Prospect, I argue that the standard for 3-D cell culture should be bio-inspired, biomimetic materials that can be used "as is" in drug discovery, toxicology, cell banking, and ultimately in medicine. Such biomaterials must therefore be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end-users-physicians and patients-must dictate the key design criteria. Herein I describe the development of one such material that meets these requirements: a covalently crosslinked, biodegradable, simplified mimic of the extracellular matrix (ECM) that permits 3-D culture of cells in vitro and enables tissue formation in vivo. In contrast to materials that were designed for in vitro cell culture and then found unsuitable for clinical use, these semi-synthetic hyaluronan-derived materials were developed for in vivo tissue repair, and are now being re-engineered for in vitro applications in research.

Deposition of tropoelastin into the extracellular matrix requires a competent elastic fiber scaffold but not live cells.

PubMed

Kozel, Beth A; Ciliberto, Christopher H; Mecham, Robert P

2004-04-01

The initial steps of elastic fiber assembly were investigated using an in vitro assembly model in which purified recombinant tropoelastin (rbTE) was added to cultures of live or dead cells. The ability of tropoelastin to associate with preexisting elastic fibers or microfibrils in the extracellular matrix was then assessed by immunofluorescence microscopy using species-specific tropoelastin antibodies. Results show that rbTE can associate with elastic fiber components in the absence of live cells through a process that does not depend on crosslink formation. Time course studies show a transformation of the deposited protein from an initial globular appearance early in culture to a more fibrous structure as the matrix matures. Deposition required the C-terminal region of tropoelastin and correlated with the presence of preexisting elastic fibers or microfibrils. Association of exogenously added tropoelastin to the cellular extracellular matrix was inhibited by the addition of heparan sulfate but not chondroitin sulfate sugars. Together, these results suggest that the matrix elaborated by the cell is sufficient for the initial deposition of tropoelastin in the extracellular space and that elastin assembly may be influenced by the composition of sulfated proteoglycans in the matrix.

Tissue constructs: platforms for basic research and drug discovery.

PubMed

Elson, Elliot L; Genin, Guy M

2016-02-06

The functions, form and mechanical properties of cells are inextricably linked to their extracellular environment. Cells from solid tissues change fundamentally when, isolated from this environment, they are cultured on rigid two-dimensional substrata. These changes limit the significance of mechanical measurements on cells in two-dimensional culture and motivate the development of constructs with cells embedded in three-dimensional matrices that mimic the natural tissue. While measurements of cell mechanics are difficult in natural tissues, they have proven effective in engineered tissue constructs, especially constructs that emphasize specific cell types and their functions, e.g. engineered heart tissues. Tissue constructs developed as models of disease also have been useful as platforms for drug discovery. Underlying the use of tissue constructs as platforms for basic research and drug discovery is integration of multiscale biomaterials measurement and computational modelling to dissect the distinguishable mechanical responses separately of cells and extracellular matrix from measurements on tissue constructs and to quantify the effects of drug treatment on these responses. These methods and their application are the main subjects of this review.

Tissue constructs: platforms for basic research and drug discovery

PubMed Central

Elson, Elliot L.; Genin, Guy M.

2016-01-01

The functions, form and mechanical properties of cells are inextricably linked to their extracellular environment. Cells from solid tissues change fundamentally when, isolated from this environment, they are cultured on rigid two-dimensional substrata. These changes limit the significance of mechanical measurements on cells in two-dimensional culture and motivate the development of constructs with cells embedded in three-dimensional matrices that mimic the natural tissue. While measurements of cell mechanics are difficult in natural tissues, they have proven effective in engineered tissue constructs, especially constructs that emphasize specific cell types and their functions, e.g. engineered heart tissues. Tissue constructs developed as models of disease also have been useful as platforms for drug discovery. Underlying the use of tissue constructs as platforms for basic research and drug discovery is integration of multiscale biomaterials measurement and computational modelling to dissect the distinguishable mechanical responses separately of cells and extracellular matrix from measurements on tissue constructs and to quantify the effects of drug treatment on these responses. These methods and their application are the main subjects of this review. PMID:26855763

Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture

NASA Astrophysics Data System (ADS)

Abbah, Sunny A.; Thomas, Dilip; Browne, Shane; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

2016-02-01

Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.

Assembly and Development of the Pseudomonas aeruginosa Biofilm Matrix

PubMed Central

Ma, Luyan; Conover, Matthew; Lu, Haiping; Parsek, Matthew R.; Bayles, Kenneth; Wozniak, Daniel J.

2009-01-01

Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell–cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications. PMID:19325879

Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

PubMed

Ma, Luyan; Conover, Matthew; Lu, Haiping; Parsek, Matthew R; Bayles, Kenneth; Wozniak, Daniel J

2009-03-01

Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell-cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

Cell-Extracellular Matrix Mechanobiology: Forceful Tools and Emerging Needs for Basic and Translational Research.

PubMed

Holle, Andrew W; Young, Jennifer L; Van Vliet, Krystyn J; Kamm, Roger D; Discher, Dennis; Janmey, Paul; Spatz, Joachim P; Saif, Taher

2018-01-10

Extracellular biophysical cues have a profound influence on a wide range of cell behaviors, including growth, motility, differentiation, apoptosis, gene expression, adhesion, and signal transduction. Cells not only respond to definitively mechanical cues from the extracellular matrix (ECM) but can also sometimes alter the mechanical properties of the matrix and hence influence subsequent matrix-based cues in both physiological and pathological processes. Interactions between cells and materials in vitro can modify cell phenotype and ECM structure, whether intentionally or inadvertently. Interactions between cell and matrix mechanics in vivo are of particular importance in a wide variety of disorders, including cancer, central nervous system injury, fibrotic diseases, and myocardial infarction. Both the in vitro and in vivo effects of this coupling between mechanics and biology hold important implications for clinical applications.

Cellulose biosynthesis by the beta-proteobacterium, Chromobacterium violaceum.

PubMed

Recouvreux, Derce O S; Carminatti, Claudimir A; Pitlovanciv, Ana K; Rambo, Carlos R; Porto, Luismar M; Antônio, Regina V

2008-11-01

The Chromobacterium violaceum ATCC 12472 genome was sequenced by The Brazilian National Genome Project Consortium. Previous annotation reported the presence of cellulose biosynthesis genes in that genome. Analysis of these genes showed that, as observed in other bacteria, they are organized in two operons. In the present work, experimental evidences of the presence of cellulose in the extracellular matrix of the biofilm produced by C. violaceum in static cultures are shown. Biofilm samples were enzymatically digested by cellulase, releasing glucose units, suggesting the presence of cellulose as an extracellular matrix component. Fluorescence microscopy observations showed that C. violaceum produces a cellulase-sensitive extracellular matrix composed of fibers able to bind calcofluor. C. violaceum grows on medium containing Congo red, forming brown-red colonies. Together, these results suggest that cellulase-susceptible matrix material is cellulose. Scanning electronic microscopy analysis showed that the extracellular matrix exhibited a network of microfibrils, typical of bacterial cellulose. Although cellulose production is widely distributed between several bacterial species, including at least the groups of Gram-negative proteobacteria alpha and gamma, we give for the first time experimental evidence for cellulose production in beta-proteobacteria.

Effect of spaceflight on the extracellular matrix of skeletal muscle after a crush injury

NASA Technical Reports Server (NTRS)

Stauber, W. T.; Fritz, V. K.; Burkovskaia, T. E.; Il'ina-Kakueva, E. I.

1992-01-01

The organization and composition of the extracellular matrix were studied in the crush-injured gastrocnemius muscle of rats subjected to 0 G. After 14 days of flight on Cosmos 2044, the gastrocnemius muscle was removed and evaluated by histochemical and immunohistochemical techniques from the five injured flight rodents and various earth-based treatment groups. In general, the repair process was similar in all injured muscle samples with regard to the organization of the extracellular matrix and myofibers. Small and large myofibers were present within an expanded extracellular matrix, indicative of myogenesis and muscle regeneration. In the tail-suspended animals, a more complete repair was observed with nonenlarged area of nonmuscle cells or matrix material visible. In contrast, the muscle samples from the flight animals were less well organized and contained more macrophages and blood vessels in the repair region, indicative of a delayed repair process, but did not demonstrate any chronic inflammation. Myofiber repair did vary in muscles from the different groups, being slowest in the flight animals and most complete in the tail-suspended ones.

Calreticulin--an endoplasmic reticulum protein with calcium-binding activity is also found in the extracellular matrix.

PubMed

Somogyi, Eszter; Petersson, Ulrika; Hultenby, Kjell; Wendel, Mikael

2003-04-01

Previous studies have reported that calreticulin (CRT), a calcium-binding and chaperoning protein, is expressed only in the endoplasmatic reticulum, nucleus and at the cell surface. In this study we clearly show that odontoblasts and predentin matrix contain CRT. To our knowledge, this is the first time CRT has been described in the extracellular matrix. The expression of CRT was studied by immunohistochemistry, ultrastructural immunocytochemistry and in situ hybridization in developing rat teeth. CRT was detected as a 59-kDa protein in rat pulp cell culture medium and dentin extracellular matrix extract by Western blotting. The presence of the protein was shown in rat odontoblasts and predentin with immunohistochemistry. At the ultrastructural level, the labeling was distributed in the rat odontoblasts, ameloblasts and predentin. Northern blotting showed the presence of CRT mRNA in rat molars, which was confirmed by in situ hybridization in odontoblasts and ameloblasts. We now present the first convincing evidence that CRT is found in extracellular matrix where it may play an important role in mineralization.

Engineered extracellular microenvironment with a tunable mechanical property for controlling cell behavior and cardiomyogenic fate of cardiac stem cells.

PubMed

Choi, Min-Young; Kim, Jong-Tae; Lee, Won-Jin; Lee, Yunki; Park, Kyung Min; Yang, Young-Il; Park, Ki Dong

2017-03-01

Endogenous cardiac stem cells (CSCs) are known to play a certain role in the myocardial homeostasis of the adult heart. The extracellular matrix (ECM) surrounding CSCs provides mechanical signals to regulate a variety of cell behaviors, yet the impact in the adult heart of these mechanical properties of ECM on CSC renewal and fate decisions is mostly unknown. To elucidate CSC mechanoresponses at the individual cell and myocardial level, we used the sol-to-gel transitional gelatin-poly(ethylene glycol)-tyramine (GPT) hydrogel with a tunable mechanical property to construct a three-dimensional (3D) matrix for culturing native myocardium and CSCs. The elastic modulus of the GPT hydrogel was controlled by adjusting cross-linking density using hydrogen peroxide. The GPT hydrogel showed an ability to transduce integrin-mediated signals into the myocardium and to permit myocardial homeostatic processes in vitro, including CSC migration and proliferation into the hydrogel from the myocardium. Decreasing the elastic modulus of the hydrogel resulted in upregulation of phosphorylated integrin-mediated signaling molecules in CSCs, which were associated with significant increases in cell spreading, migration, and proliferation of CSCs in a modulus-dependent manner. However, increasing the elastic modulus of hydrogel induced the arrest of cell growth but led to upregulation of cardiomyocyte-associated mRNAs in CSCs. This work demonstrates that tunable 3D-engineered microenvironments created by GPT hydrogel are able to control CSC behavior and to direct cardiomyogenic fate. Our system may also be appropriate for studying the mechanoresponse of CSCs in a 3D context as well as for developing therapeutic strategies for in situ myocardial regeneration. The extracellular matrix (ECM) provides a physical framework of myocardial niches in which endogenous cardiac stem cells (CSCs) reside, renew, differentiate, and replace cardiac cells. Interactions between ECM and CSCs might be critical for the maintenance of myocardial homeostasis in the adult heart. Yet most studies done so far have used irrelevant cell types and have been performed at the individual cell level, none able to reflect the in vivo situation. By the use of a chemically defined hydrogel to create a tunable 3D microenvironment, we succeeded in controlling CSC behavior at the myocardial and individual cell level and directing the cardiomyogenic fate. Our work may provide insight into the design of biomaterials for in situ myocardial regeneration as well as for tissue engineering. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Extracellular matrix scaffold as a tubular graft for ascending aorta aneurysm repair.

PubMed

Abu Saleh, Walid K; Al Jabbari, Odeaa; Grande-Allen, Jane; Ramchandani, Mahesh

2015-08-01

Although extracellular xenograft repair has produced encouraging results when applied to cardiac, valvular, and specific aortic defects, its employment as a tube graft to replace the ascending aorta has not been reported. We describe a patient who underwent resection and replacement of an infected ascending aortic graft with an extracellular matrix conduit. The patient did well, but 14 months later developed a pseudoaneurysm from the staple line used to construct the extracellular matrix conduit. The patient underwent a repeat sternotomy and removal of the graft. Because of the increased risk of graft failure, a homograft was felt to be more appropriate in this setting. Ultimately, we were unable to implant the homograft because it was too small for the aortic root; therefore we decided to construct a tubular graft from Cormatrix extracellular matrix (CorMatrix, Roswell, GA, USA). Fourteen months later, he presented with shortness of breath. Computed tomography scan revealed a 3.5 cm pseudoaneurysm of the ascending aorta. It appeared as if there was a disruption of the staple line in the extra cellular matrix graft. The plan was to replace it with a Dacron graft. The Cormatrix graft material was removed and sent for culture and histological analysis. A 28-mm Gel weave graft (Terumo Cardiovascular Systems, Ann Arbor, MI, USA) was implanted. The patient tolerated the procedure well with good hemodynamics. Our experience suggests that the superior strength, handling characteristics, and resistance to infection make extra cellular matrix scaffold a possible alternative conduit to cryopreserved homografts. Applicability as an aortic conduit merits further investigation to better understand behavior of extra cellular matrix in this situation. © 2015 Wiley Periodicals, Inc.

Regulation of extracellular matrix vesicles via rapid responses to steroid hormones during endochondral bone formation.

PubMed

Asmussen, Niels; Lin, Zhao; McClure, Michael J; Schwartz, Zvi; Boyan, Barbara D

2017-12-09

Endochondral bone formation is a precise and highly ordered process whose exact regulatory framework is still being elucidated. Multiple regulatory pathways are known to be involved. In some cases, regulation impacts gene expression, resulting in changes in chondrocyte phenotypic expression and extracellular matrix synthesis. Rapid regulatory mechanisms are also involved, resulting in release of enzymes, factors and micro RNAs stored in extracellular matrisomes called matrix vesicles. Vitamin D metabolites modulate endochondral development via both genomic and rapid membrane-associated signaling pathways. 1α,25-dihydroxyvitamin D3 [1α,25(OH) 2 D 3 ] acts through the vitamin D receptor (VDR) and a membrane associated receptor, protein disulfide isomerase A3 (PDIA3). 24R,25-dihydroxyvitamin D3 [24R,25(OH) 2 D 3 ] affects primarily chondrocytes in the resting zone (RC) of the growth plate, whereas 1α,25(OH) 2 D 3 affects cells in the prehypertrophic and upper hypertrophic cell zones (GC). This includes genomically directing the cells to produce matrix vesicles with zone specific characteristics. In addition, vitamin D metabolites produced by the cells interact directly with the matrix vesicle membrane via rapid signal transduction pathways, modulating their activity in the matrix. The matrix vesicle payload is able to rapidly impact the extracellular matrix via matrix processing enzymes as well as providing a feedback mechanism to the cells themselves via the contained micro RNAs. Copyright © 2017. Published by Elsevier Inc.

Carbon nanotube interaction with extracellular matrix proteins producing scaffolds for tissue engineering

PubMed Central

Tonelli, Fernanda MP; Santos, Anderson K; Gomes, Katia N; Lorençon, Eudes; Guatimosim, Silvia; Ladeira, Luiz O; Resende, Rodrigo R

2012-01-01

In recent years, significant progress has been made in organ transplantation, surgical reconstruction, and the use of artificial prostheses to treat the loss or failure of an organ or bone tissue. In recent years, considerable attention has been given to carbon nanotubes and collagen composite materials and their applications in the field of tissue engineering due to their minimal foreign-body reactions, an intrinsic antibacterial nature, biocompatibility, biodegradability, and the ability to be molded into various geometries and forms such as porous structures, suitable for cell ingrowth, proliferation, and differentiation. Recently, grafted collagen and some other natural and synthetic polymers with carbon nanotubes have been incorporated to increase the mechanical strength of these composites. Carbon nanotube composites are thus emerging as potential materials for artificial bone and bone regeneration in tissue engineering. PMID:22923989

Electrospun nanofibrous 3D scaffold for bone tissue engineering.

PubMed

Eap, Sandy; Ferrand, Alice; Palomares, Carlos Mendoza; Hébraud, Anne; Stoltz, Jean-François; Mainard, Didier; Schlatter, Guy; Benkirane-Jessel, Nadia

2012-01-01

Tissue engineering aims at developing functional substitutes for damaged tissues by mimicking natural tissues. In particular, tissue engineering for bone regeneration enables healing of some bone diseases. Thus, several methods have been developed in order to produce implantable biomaterial structures that imitate the constitution of bone. Electrospinning is one of these methods. This technique produces nonwoven scaffolds made of nanofibers which size and organization match those of the extracellular matrix. Until now, seldom electrospun scaffolds were produced with thickness exceeding one millimeter. This article introduces a new kind of electrospun membrane called 3D scaffold of thickness easily exceeding one centimeter. The manufacturing involves a solution of poly(ε-caprolactone) in DMF/DCM system. The aim is to establish parameters for electrospinning in order to characterize these 3D scaffolds and, establish whether such scaffolds are potentially interesting for bone regeneration.

Tissue engineering for human urethral reconstruction: systematic review of recent literature.

PubMed

de Kemp, Vincent; de Graaf, Petra; Fledderus, Joost O; Ruud Bosch, J L H; de Kort, Laetitia M O

2015-01-01

Techniques to treat urethral stricture and hypospadias are restricted, as substitution of the unhealthy urethra with tissue from other origins (skin, bladder or buccal mucosa) has some limitations. Therefore, alternative sources of tissue for use in urethral reconstructions are considered, such as ex vivo engineered constructs. To review recent literature on tissue engineering for human urethral reconstruction. A search was made in the PubMed and Embase databases restricted to the last 25 years and the English language. A total of 45 articles were selected describing the use of tissue engineering in urethral reconstruction. The results are discussed in four groups: autologous cell cultures, matrices/scaffolds, cell-seeded scaffolds, and clinical results of urethral reconstructions using these materials. Different progenitor cells were used, isolated from either urine or adipose tissue, but slightly better results were obtained with in vitro expansion of urothelial cells from bladder washings, tissue biopsies from the bladder (urothelium) or the oral cavity (buccal mucosa). Compared with a synthetic scaffold, a biological scaffold has the advantage of bioactive extracellular matrix proteins on its surface. When applied clinically, a non-seeded matrix only seems suited for use as an onlay graft. When a tubularized substitution is the aim, a cell-seeded construct seems more beneficial. Considerable experience is available with tissue engineering of urethral tissue in vitro, produced with cells of different origin. Clinical and in vivo experiments show promising results.

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.

Advances in vascular tissue engineering.

PubMed

Thomas, Anita C; Campbell, Gordon R; Campbell, Julie H

2003-01-01

Coronary and peripheral artery bypass grafting is commonly used to relieve the symptoms of vascular deficiencies, but the supply of autologous artery or vein may not be sufficient or suitable for multiple bypass or repeat procedures, necessitating the use of other materials. Synthetic materials are suitable for large bore arteries but often thrombose when used in smaller arteries. Suitable replacement grafts must have appropriate characteristics, including resistance to infection, low immunogenicity and good biocompatability and thromboresistance, with appropriate mechanical and physiological properties and cheap and fast manufacture. Current avenues of graft development include coating synthetic grafts with either biological chemicals or cells with anticoagulatory properties. Matrix templates or acellular tubes of extracellular matrix (such as collagen) may be coated or infiltrated with cultured cells. Once placed into the artery, these grafts may become colonised by host cells and gain many of the properties of normal artery. "Tissue-engineered blood vessels" may also be formed from layers of human vascular cells grown in culture. These engineered vessels have many of the characteristics of arteries formed in vivo. "Artificial arteries" may be also be derived from peritoneal granulation tissue in body "bioreactors" by adapting the body's natural wound healing response to produce a hollow tube.

Unusual glycosaminoglycans from a deep sea hydrothermal bacterium improve fibrillar collagen structuring and fibroblast activities in engineered connective tissues.

PubMed

Senni, Karim; Gueniche, Farida; Changotade, Sylvie; Septier, Dominique; Sinquin, Corinne; Ratiskol, Jacqueline; Lutomski, Didier; Godeau, Gaston; Guezennec, Jean; Colliec-Jouault, Sylvia

2013-04-23

Biopolymers produced by marine organisms can offer useful tools for regenerative medicine. Particularly, HE800 exopolysaccharide (HE800 EPS) secreted by a deep-sea hydrothermal bacterium displays an interesting glycosaminoglycan-like feature resembling hyaluronan. Previous studies demonstrated its effectiveness to enhance in vivo bone regeneration and to support osteoblastic cell metabolism in culture. Thus, in order to assess the usefulness of this high-molecular weight polymer in tissue engineering and tissue repair, in vitro reconstructed connective tissues containing HE800 EPS were performed. We showed that this polysaccharide promotes both collagen structuring and extracellular matrix settle by dermal fibroblasts. Furthermore, from the native HE800 EPS, a low-molecular weight sulfated derivative (HE800 DROS) displaying chemical analogy with heparan-sulfate, was designed. Thus, it was demonstrated that HE800 DROS mimics some properties of heparan-sulfate, such as promotion of fibroblast proliferation and inhibition of matrix metalloproteinase (MMP) secretion. Therefore, we suggest that the HE800EPS family can be considered as an innovative biotechnological source of glycosaminoglycan-like compounds useful to design biomaterials and drugs for tissue engineering and repair.

Unusual Glycosaminoglycans from a Deep Sea Hydrothermal Bacterium Improve Fibrillar Collagen Structuring and Fibroblast Activities in Engineered Connective Tissues

PubMed Central

Senni, Karim; Gueniche, Farida; Changotade, Sylvie; Septier, Dominique; Sinquin, Corinne; Ratiskol, Jacqueline; Lutomski, Didier; Godeau, Gaston; Guezennec, Jean; Colliec-Jouault, Sylvia

2013-01-01

Biopolymers produced by marine organisms can offer useful tools for regenerative medicine. Particularly, HE800 exopolysaccharide (HE800 EPS) secreted by a deep-sea hydrothermal bacterium displays an interesting glycosaminoglycan-like feature resembling hyaluronan. Previous studies demonstrated its effectiveness to enhance in vivo bone regeneration and to support osteoblastic cell metabolism in culture. Thus, in order to assess the usefulness of this high-molecular weight polymer in tissue engineering and tissue repair, in vitro reconstructed connective tissues containing HE800 EPS were performed. We showed that this polysaccharide promotes both collagen structuring and extracellular matrix settle by dermal fibroblasts. Furthermore, from the native HE800 EPS, a low-molecular weight sulfated derivative (HE800 DROS) displaying chemical analogy with heparan-sulfate, was designed. Thus, it was demonstrated that HE800 DROS mimics some properties of heparan-sulfate, such as promotion of fibroblast proliferation and inhibition of matrix metalloproteinase (MMP) secretion. Therefore, we suggest that the HE800EPS family can be considered as an innovative biotechnological source of glycosaminoglycan-like compounds useful to design biomaterials and drugs for tissue engineering and repair. PMID:23612369

Monitoring tissue formation and organization of engineered tendon by optical coherence tomography

NASA Astrophysics Data System (ADS)

Bagnaninchi, P. O.; Yang, Y.; Maffulli, N.; Wang, R. K.; El Haj, A.

2006-02-01

The uniaxial orientation and bundle formation of collagen fibres determine the mechanical properties of tendons. Thus the particular challenge of tendon tissue engineering is to build the tissue with a highly organized structure of collagen fibres. Ultimately the engineered construct will be used as autologous grafts in tendon surgery, withstanding physiological loading. We grew pig tenocytes in porous chitosan scaffolds with multiple microchannels of 250-500 μm. The cell proliferation and production of extra-cellular matrix (ECM) within the scaffolds have been successfully monitored by Optical Coherence Tomography (OCT), a bench-top OCT system equipped with a broadband light source centred at 1300 nm. Under sterile condition, the measurements were performed on-line and in a non-destructive manner. In addition, a novel method based on OCT imaging, which calculates the occupation ratio of the microchannel derived from the scattered intensity has been developed. It is confirmed that the occupation ratio is correlated to cell proliferation and ECM production in the scaffolds. Thus this method has been utilised to assess the effect of different culture conditions on the tissue formation. The use of a perfusion bioreactor has resulted in a significantly (p<1e -3) higher cell proliferation and matrix production.

Escherichia coli Biofilms Have an Organized and Complex Extracellular Matrix Structure

PubMed Central

Hung, Chia; Zhou, Yizhou; Pinkner, Jerome S.; Dodson, Karen W.; Crowley, Jan R.; Heuser, John; Chapman, Matthew R.; Hadjifrangiskou, Maria; Henderson, Jeffrey P.; Hultgren, Scott J.

2013-01-01

ABSTRACT Bacterial biofilms are ubiquitous in nature, and their resilience is derived in part from a complex extracellular matrix that can be tailored to meet environmental demands. Although common developmental stages leading to biofilm formation have been described, how the extracellular components are organized to allow three-dimensional biofilm development is not well understood. Here we show that uropathogenic Escherichia coli (UPEC) strains produce a biofilm with a highly ordered and complex extracellular matrix (ECM). We used electron microscopy (EM) techniques to image floating biofilms (pellicles) formed by UPEC. EM revealed intricately constructed substructures within the ECM that encase individual, spatially segregated bacteria with a distinctive morphology. Mutational and biochemical analyses of these biofilms confirmed curli as a major matrix component and revealed important roles for cellulose, flagella, and type 1 pili in pellicle integrity and ECM infrastructure. Collectively, the findings of this study elucidated that UPEC pellicles have a highly organized ultrastructure that varies spatially across the multicellular community. PMID:24023384

Thermomechanical analysis of freezing-induced cell-fluid-matrix interactions in engineered tissues

PubMed Central

Han, Bumsoo; Teo, Ka Yaw; Ghosh, Soham; Dutton, J. Craig; Grinnell, Frederick

2012-01-01

Successful cryopreservation of functional engineered tissues (ETs) is significant to tissue engineering and regenerative medicine, but it is extremely challenging to develop a successful protocol because the effects of cryopreservation parameters on the post-thaw functionality of ETs are not well understood. Particularly, the effects on the microstructure of their extracellular matrix (ECM) have not been well studied, which determines many functional properties of the ETs. In this study, we investigated the effects of two key cryopreservation parameters – i) freezing temperature and corresponding cooling rate; and ii) the concentration of cryoprotective agent (CPA) on the ECM microstructure as well as the cellular viability. Using dermal equivalent as a model ET and DMSO as a model CPA, freezing-induced spatiotemporal deformation and post-thaw ECM microstructure of ETs was characterized while varying the freezing temperature and DMSO concentrations. The spatial distribution of cellular viability and the cellular actin cytoskeleton was also examined. The results showed that the tissue dilatation increased significantly with reduced freezing temperature (i.e., rapid freezing). A maximum limit of tissue deformation was observed for preservation of ECM microstructure, cell viability and cell-matrix adhesion. The dilatation decreased with the use of DMSO, and a freezing temperature dependent threshold concentration of DMSO was observed. The threshold DMSO concentration increased with lowering freezing temperature. In addition, an analysis was performed to delineate thermodynamic and mechanical components of freezing-induced tissue deformation. The results are discussed to establish a mechanistic understanding of freezing-induced cell-fluid-matrix interaction and phase change behavior within ETs in order to improve cryopreservation of ETs. PMID:23246556

Incorporation of Biomaterials in Multicellular Aggregates Modulates Pluripotent Stem Cell Differentiation

PubMed Central

Bratt-Leal, Andrés M.; Carpenedo, Richard L.; Ungrin, Mark; Zandstra, Peter W.; McDevitt, Todd C.

2010-01-01

Biomaterials are increasingly being used to engineer the biochemical and biophysical properties of the extracellular stem cell microenvironment in order to tailor niche characteristics and direct cell phenotype. To date, stem cell-biomaterial interactions have largely been studied by introducing stem cells into artificial environments, such as 2D cell culture on biomaterial surfaces, encapsulation of cell suspensions within hydrogel materials, or cell seeding on 3D polymeric scaffolds. In this study, microparticles fabricated from different materials, such as agarose, PLGA and gelatin, were stably integrated, in a dose-dependent manner, within aggregates of pluripotent stem cells (PSCs) prior to differentiation as a means to directly examine stem cell-biomaterial interactions in 3D. Interestingly, the presence of the materials within the stem cell aggregates differentially modulated the gene and protein expression patterns of several differentiation markers without adversely affecting cell viability. Microparticle incorporation within 3D stem cell aggregates can control the spatial presentation of extracellular environmental cues (i.e. soluble factors, extracellular matrix and intercellular adhesion molecules) as a means to direct the differentiation of stem cells for tissue engineering and regenerative medicine applications. In addition, these results suggest that the physical presence of microparticles within stem cell aggregates does not compromise PSC differentiation, but in fact the choice of biomaterials can impact the propensity of stem cells to adopt particular differentiated cell phenotypes. PMID:20864164

A Comparative Analysis of the In Vitro Effects of Pulsed Electromagnetic Field Treatment on Osteogenic Differentiation of Two Different Mesenchymal Cell Lineages

PubMed Central

Ceccarelli, Gabriele; Bloise, Nora; Mantelli, Melissa; Gastaldi, Giulia; Fassina, Lorenzo; De Angelis, Maria Gabriella Cusella; Ferrari, Davide; Imbriani, Marcello

2013-01-01

Abstract Human mesenchymal stem cells (MSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications. Exposure of MSCs to physical stimuli favors early and rapid activation of the tissue repair process. In this study we investigated the in vitro effects of pulsed electromagnetic field (PEMF) treatment on the proliferation and osteogenic differentiation of bone marrow MSCs (BM-MSCs) and adipose-tissue MSCs (ASCs), to assess if both types of MSCs could be indifferently used in combination with PEMF exposure for bone tissue healing. We compared the cell viability, cell matrix distribution, and calcified matrix production in unstimulated and PEMF-stimulated (magnetic field: 2 mT, amplitude: 5 mV) mesenchymal cell lineages. After PEMF exposure, in comparison with ASCs, BM-MSCs showed an increase in cell proliferation (p<0.05) and an enhanced deposition of extracellular matrix components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type-I and -III collagens (p<0.05). Calcium deposition was 1.5-fold greater in BM-MSC–derived osteoblasts (p<0.05). The immunofluorescence related to the deposition of bone matrix proteins and calcium showed their colocalization to the cell-rich areas for both types of MSC-derived osteoblast. Alkaline phosphatase activity increased nearly 2-fold (p<0.001) and its protein content was 1.2-fold higher in osteoblasts derived from BM-MSCs. The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis revealed up-regulated transcription specific for bone sialoprotein, osteopontin, osteonectin, and Runx2, but at a higher level for cells differentiated from BM-MSCs. All together these results suggest that PEMF promotion of bone extracellular matrix deposition is more efficient in osteoblasts differentiated from BM-MSCs. PMID:23914335

Engineering of arteries in vitro

PubMed Central

Huang, Angela H.; Niklason, Laura E.

2014-01-01

This review will focus on two elements that are essential for functional arterial regeneration in vitro: the mechanical environment and the bioreactors used for tissue growth. The importance of the mechanical environment to embryological development, vascular functionality, and vascular graft regeneration will be discussed. Bioreactors generate mechanical stimuli to simulate the biomechanical environment of the arterial system. This system has been used to reconstruct arterial grafts with appropriate mechanical strength for implantation by controlling the chemical and mechanical environments in which the grafts are grown. Bioreactors are powerful tools to study the effect of mechanical stimuli on extracellular matrix (ECM) architecture and the mechanical properties of engineered vessels. Hence biomimetic systems enable us to optimize chemo-biomechanical culture conditions to regenerate engineered vessels with physiological properties similar to those of native arterial vessels. In addition, this review will introduce and examine various approaches and techniques that have been used to engineer biologically-based vascular grafts, including collagen-based grafts, fibrin-gel grafts, cell sheet engineering, biodegradable polymers, and decellularization of native vessels. PMID:24399290

Silk fibroin in tissue engineering.

PubMed

Kasoju, Naresh; Bora, Utpal

2012-07-01

Tissue engineering (TE) is a multidisciplinary field that aims at the in vitro engineering of tissues and organs by integrating science and technology of cells, materials and biochemical factors. Mimicking the natural extracellular matrix is one of the critical and challenging technological barriers, for which scaffold engineering has become a prime focus of research within the field of TE. Amongst the variety of materials tested, silk fibroin (SF) is increasingly being recognized as a promising material for scaffold fabrication. Ease of processing, excellent biocompatibility, remarkable mechanical properties and tailorable degradability of SF has been explored for fabrication of various articles such as films, porous matrices, hydrogels, nonwoven mats, etc., and has been investigated for use in various TE applications, including bone, tendon, ligament, cartilage, skin, liver, trachea, nerve, cornea, eardrum, dental, bladder, etc. The current review extensively covers the progress made in the SF-based in vitro engineering and regeneration of various human tissues and identifies opportunities for further development of this field. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

PubMed Central

Pati, Falguni; Jang, Jinah; Ha, Dong-Heon; Won Kim, Sung; Rhie, Jong-Won; Shim, Jin-Hyung; Kim, Deok-Ho; Cho, Dong-Woo

2014-01-01

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method. PMID:24887553

Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

NASA Astrophysics Data System (ADS)

Pati, Falguni; Jang, Jinah; Ha, Dong-Heon; Won Kim, Sung; Rhie, Jong-Won; Shim, Jin-Hyung; Kim, Deok-Ho; Cho, Dong-Woo

2014-06-01

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.

Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV.

PubMed

López-Jiménez, Alberto J; Basak, Trayambak; Vanacore, Roberto M

2017-10-13

Lysyl oxidase-like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Depressed immune surveillance against cancer: role of deficient T cell: extracellular matrix interactions.

PubMed

Górski, A; Castronovo, V; Stepień-Sopniewska, B; Grieb, P; Ryba, M; Mrowiec, T; Korczak-Kowalska, G; Wierzbicki, P; Matysiak, W; Dybowska, B

1994-07-01

Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.

Swarm chondrosarcoma: a continued resource for chondroblastic-like extracellular matrix and chondrosarcoma biology research.

PubMed

Stevens, Jeff W

2013-01-01

Since its first description over four decades ago, the Swarm chondrosarcoma (Swarm rat chondrosarcoma, SRC) remains a valuable tool for studies of chondroblastic-like extracellular matrix (ECM) biology and as an animal model of human chondrosarcoma of histological grades I-III. Moreover, articular joints and skeletal anomalies such as arthritis as well as cartilage regeneration, skeletal development, tissue engineering, hard tissue tumorigenesis and space flight physiology are advanced through studies in hyaline cartilage-like models. With more than 500 articles published since the first report on the characteristics of mucopolysaccharides (glycosaminoglycans) of the tumor in 1971, several transplantable tumor and cell lines have been developed by multiple laboratories worldwide. This review describes the characterization of SRC tumors and cell lines, including the use of SRC lines as a resource for isolation and characterization of several ECM elements that have become vital for the advancement of our understanding of cartilage biology. Also presented is the importance of pertubation of ECM components and the influence of the tumor microenvironment on disease progression. Therapeutic failure and currently pursued avenues of intervention utilizing the SRC lines in treatment of chondrosarcoma are also discussed.

Influence of Extracellular Matrix Proteins and Substratum Topography on Corneal Epithelial Cell Alignment and Migration

PubMed Central

Raghunathan, VijayKrishna; McKee, Clayton; Cheung, Wai; Naik, Rachel; Nealey, Paul F.; Russell, Paul

2013-01-01

The basement membrane (BM) of the corneal epithelium presents biophysical cues in the form of topography and compliance that can impact the phenotype and behaviors of cells and their nuclei through modulation of cytoskeletal dynamics. In addition, it is also well known that the intrinsic biochemical attributes of BMs can modulate cell behaviors. In this study, the influence of the combination of exogenous coating of extracellular matrix proteins (ECM) (fibronectin-collagen [FNC]) with substratum topography was investigated on cytoskeletal architecture as well as alignment and migration of immortalized corneal epithelial cells. In the absence of FNC coating, a significantly greater percentage of cells aligned parallel with the long axis of the underlying anisotropically ordered topographic features; however, their ability to migrate was impaired. Additionally, changes in the surface area, elongation, and orientation of cytoskeletal elements were differentially influenced by the presence or absence of FNC. These results suggest that the effects of topographic cues on cells are modulated by the presence of surface-associated ECM proteins. These findings have relevance to experiments using cell cultureware with biomimetic biophysical attributes as well as the integration of biophysical cues in tissue-engineering strategies and the development of improved prosthetics. PMID:23488816

Macromolecular crowding meets oxygen tension in human mesenchymal stem cell culture - A step closer to physiologically relevant in vitro organogenesis

NASA Astrophysics Data System (ADS)

Cigognini, Daniela; Gaspar, Diana; Kumar, Pramod; Satyam, Abhigyan; Alagesan, Senthilkumar; Sanz-Nogués, Clara; Griffin, Matthew; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

2016-08-01

Modular tissue engineering is based on the cells’ innate ability to create bottom-up supramolecular assemblies with efficiency and efficacy still unmatched by man-made devices. Although the regenerative potential of such tissue substitutes has been documented in preclinical and clinical setting, the prolonged culture time required to develop an implantable device is associated with phenotypic drift and/or cell senescence. Herein, we demonstrate that macromolecular crowding significantly enhances extracellular matrix deposition in human bone marrow mesenchymal stem cell culture at both 20% and 2% oxygen tension. Although hypoxia inducible factor - 1α was activated at 2% oxygen tension, increased extracellular matrix synthesis was not observed. The expression of surface markers and transcription factors was not affected as a function of oxygen tension and macromolecular crowding. The multilineage potential was also maintained, albeit adipogenic differentiation was significantly reduced in low oxygen tension cultures, chondrogenic differentiation was significantly increased in macromolecularly crowded cultures and osteogenic differentiation was not affected as a function of oxygen tension and macromolecular crowding. Collectively, these data pave the way for the development of bottom-up tissue equivalents based on physiologically relevant developmental processes.

Macromolecular crowding meets oxygen tension in human mesenchymal stem cell culture - A step closer to physiologically relevant in vitro organogenesis

PubMed Central

Cigognini, Daniela; Gaspar, Diana; Kumar, Pramod; Satyam, Abhigyan; Alagesan, Senthilkumar; Sanz-Nogués, Clara; Griffin, Matthew; O’Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

2016-01-01

Modular tissue engineering is based on the cells’ innate ability to create bottom-up supramolecular assemblies with efficiency and efficacy still unmatched by man-made devices. Although the regenerative potential of such tissue substitutes has been documented in preclinical and clinical setting, the prolonged culture time required to develop an implantable device is associated with phenotypic drift and/or cell senescence. Herein, we demonstrate that macromolecular crowding significantly enhances extracellular matrix deposition in human bone marrow mesenchymal stem cell culture at both 20% and 2% oxygen tension. Although hypoxia inducible factor - 1α was activated at 2% oxygen tension, increased extracellular matrix synthesis was not observed. The expression of surface markers and transcription factors was not affected as a function of oxygen tension and macromolecular crowding. The multilineage potential was also maintained, albeit adipogenic differentiation was significantly reduced in low oxygen tension cultures, chondrogenic differentiation was significantly increased in macromolecularly crowded cultures and osteogenic differentiation was not affected as a function of oxygen tension and macromolecular crowding. Collectively, these data pave the way for the development of bottom-up tissue equivalents based on physiologically relevant developmental processes. PMID:27478033

The use of three-dimensional nanostructures to instruct cells to produce extracellular matrix for regenerative medicine strategies

PubMed Central

Schenke-Layland, Katja; Rofail, Fady; Heydarkhan, Sanaz; Gluck, Jessica M.; Ingle, Nilesh P.; Angelis, Ekaterini; Choi, Chang-Hwan; MacLellan, W Robb; Beygui, Ramin E; Shemin, Richard J; Heydarkhan-Hagvall, Sepideh

2009-01-01

Synthetic polymers or naturally-derived extracellular matrix (ECM) proteins have been used to create tissue engineering scaffolds; however, the need for surface modification in order to achieve polymer biocompatibility and the lack of biomechanical strength of constructs built using proteins alone remain major limitations. To overcome these obstacles, we developed novel hybrid constructs composed of both strong biosynthetic materials and natural human ECM proteins. Taking advantage of the ability of cells to produce their own ECM, human foreskin fibroblasts were grown on silicon-based nanostructures exhibiting various surface topographies that significantly enhanced ECM protein production. After 4 weeks, cell-derived sheets were harvested and histology, immunochemistry, biochemistry and multiphoton imaging revealed the presence of collagens, tropoelastin, fibronectin and glycosaminoglycans. Following decellularization, purified sheet-derived ECM proteins were mixed with poly(ε-caprolactone) to create fibrous scaffolds using electrospinning. These hybrid scaffolds exhibited excellent biomechanical properties with fiber and pore sizes that allowed attachment and migration of adipose tissue-derived stem cells. Our study represents an innovative approach to generate strong, non-cytotoxic scaffolds that could have broad applications in tissue regeneration strategies. PMID:19524289

Glycol chitosan/oxidized hyaluronic acid hydrogels functionalized with cartilage extracellular matrix particles and incorporating BMSCs for cartilage repair.

PubMed

Liu, Chun; Liu, Deshuai; Wang, Yingying; Li, Yun; Li, Tao; Zhou, Zhiyou; Yang, Zhijian; Wang, Jianhua; Zhang, Qiqing

2018-02-05

In this article, we fabricated a bioactive hydrogel composed of glycol chitosan (G-CS) and oxidized hyaluronic acid (OHA) via Schiff base reaction. Cartilage extracellular matrix (ECM) particles with different concentrations were used to functionalize G-CS/OHA (S1) hydrogel. The results demonstrated that S3 (G-CS/OHA/ECM 2% w/v) hydrogel exhibited the most suitable compression strength and provided the optimal environment for proliferation of bone marrow mesenchymal stem cells (BMSCs). To assess the chondroinductivity of ECM in vitro, we compared the chondrogenesis of BMSCs in S1 (G-CS/OHA) and S3 (G-CS/OHA/ECM 2% w/v) hydrogels. The results confirmed that the higher levels of glycosaminoglycans (GAGs) and collagen type II (COL II) were accumulated in S3 hydrogel. In vivo, cartilage defects of rats generated most mature tissue within BMSCs-laden S3 hydrogel (S3/BMSCs group). The tissues were more integrative and contained higher levels of COL II and GAGs compared to the other groups. All these results suggested that the G-CS/OHA hydrogel functionalized with ECM particles is a good candidate biomaterial to be applied in cartilage tissue engineering.

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.

Differential effect of extracellular matrix derived from papillary and reticular fibroblasts on epidermal development in vitro.

PubMed

Janson, David; Rietveld, Marion; Mahé, Christian; Saintigny, Gaëlle; El Ghalbzouri, Abdoelwaheb

2017-06-01

Papillary and reticular fibroblasts have different effects on keratinocyte proliferation and differentiation. The aim of this study was to investigate whether these effects are caused by differential secretion of soluble factors or by differential generation of extracellular matrix from papillary and reticular fibroblasts. To study the effect of soluble factors, keratinocyte monolayer cultures were grown in papillary or reticular fibroblast-conditioned medium. To study the effect of extracellular matrix, keratinocytes were grown on papillary or reticular-derived matrix. Conditioned medium from papillary or reticular fibroblasts did not differentially affect keratinocyte viability or epidermal development. However, keratinocyte viability was increased when grown on matrix derived from papillary, compared with reticular, fibroblasts. In addition, the longevity of the epidermis was increased when cultured on papillary fibroblast-derived matrix skin equivalents compared with reticular-derived matrix skin equivalents. The findings indicate that the matrix secreted by papillary and reticular fibroblasts is the main causal factor to account for the differences in keratinocyte growth and viability observed in our study. Differences in response to soluble factors between both populations were less significant. Matrix components specific to the papillary dermis may account for the preferential growth of keratinocytes on papillary dermis.

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

PubMed Central

Glass, RH; Aggeler, J; Spindle, A; Pederson, RA; Werb, Z

1983-01-01

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0 PMID:6339525

Extracellular-matrix-mediated osmotic pressure drives Vibrio cholerae biofilm expansion and cheater exclusion.

PubMed

Yan, Jing; Nadell, Carey D; Stone, Howard A; Wingreen, Ned S; Bassler, Bonnie L

2017-08-23

Biofilms, surface-attached communities of bacteria encased in an extracellular matrix, are a major mode of bacterial life. How the material properties of the matrix contribute to biofilm growth and robustness is largely unexplored, in particular in response to environmental perturbations such as changes in osmotic pressure. Here, using Vibrio cholerae as our model organism, we show that during active cell growth, matrix production enables biofilm-dwelling bacterial cells to establish an osmotic pressure difference between the biofilm and the external environment. This pressure difference promotes biofilm expansion on nutritious surfaces by physically swelling the colony, which enhances nutrient uptake, and enables matrix-producing cells to outcompete non-matrix-producing cheaters via physical exclusion. Osmotic pressure together with crosslinking of the matrix also controls the growth of submerged biofilms and their susceptibility to invasion by planktonic cells. As the basic physicochemical principles of matrix crosslinking and osmotic swelling are universal, our findings may have implications for other biofilm-forming bacterial species.Most bacteria live in biofilms, surface-attached communities encased in an extracellular matrix. Here, Yan et al. show that matrix production in Vibrio cholerae increases the osmotic pressure within the biofilm, promoting biofilm expansion and physical exclusion of non-matrix producing cheaters.

The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

PubMed

Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

2006-05-01

Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

Tendon Functional Extracellular Matrix

PubMed Central

Screen, H.R.C.; Birk, D.E.; Kadler, K.E.; Ramirez, F; Young, M.F.

2015-01-01

This article is one of a series, summarising views expressed at the Orthopaedic Research Society New Frontiers in Tendon Research Conference. This particular article reviews the three workshops held under the “Functional Extracellular Matrix” stream. The workshops focused on the roles of the tendon extracellular matrix, such as performing the mechanical functions of tendon, creating the local cell environment and providing cellular cues. Tendon is a complex network of matrix and cells, and its biological functions are influenced by widely-varying extrinsic and intrinsic factors such as age, nutrition, exercise levels and biomechanics. Consequently, tendon adapts dynamically during development, ageing and injury. The workshop discussions identified research directions associated with understanding cell-matrix interactions to be of prime importance for developing novel strategies to target tendon healing or repair. PMID:25640030

Sterilization of Lung Matrices by Supercritical Carbon Dioxide

PubMed Central

Balestrini, Jenna L.; Liu, Angela; Gard, Ashley L.; Huie, Janet; Blatt, Kelly M.S.; Schwan, Jonas; Zhao, Liping; Broekelmann, Tom J.; Mecham, Robert P.; Wilcox, Elise C.

2016-01-01

Lung engineering is a potential alternative to transplantation for patients with end-stage pulmonary failure. Two challenges critical to the successful development of an engineered lung developed from a decellularized scaffold include (i) the suppression of resident infectious bioburden in the lung matrix, and (ii) the ability to sterilize decellularized tissues while preserving the essential biological and mechanical features intact. To date, the majority of lungs are sterilized using high concentrations of peracetic acid (PAA) resulting in extracellular matrix (ECM) depletion. These mechanically altered tissues have little to no storage potential. In this study, we report a sterilizing technique using supercritical carbon dioxide (ScCO2) that can achieve a sterility assurance level 10−6 in decellularized lung matrix. The effects of ScCO2 treatment on the histological, mechanical, and biochemical properties of the sterile decellularized lung were evaluated and compared with those of freshly decellularized lung matrix and with PAA-treated acellular lung. Exposure of the decellularized tissue to ScCO2 did not significantly alter tissue architecture, ECM content or organization (glycosaminoglycans, elastin, collagen, and laminin), observations of cell engraftment, or mechanical integrity of the tissue. Furthermore, these attributes of lung matrix did not change after 6 months in sterile buffer following sterilization with ScCO2, indicating that ScCO2 produces a matrix that is stable during storage. The current study's results indicate that ScCO2 can be used to sterilize acellular lung tissue while simultaneously preserving key biological components required for the function of the scaffold for regenerative medicine purposes. PMID:26697757

Advances in biomimetic regeneration of elastic matrix structures

PubMed Central

Sivaraman, Balakrishnan; Bashur, Chris A.

2012-01-01

Elastin is a vital component of the extracellular matrix, providing soft connective tissues with the property of elastic recoil following deformation and regulating the cellular response via biomechanical transduction to maintain tissue homeostasis. The limited ability of most adult cells to synthesize elastin precursors and assemble them into mature crosslinked structures has hindered the development of functional tissue-engineered constructs that exhibit the structure and biomechanics of normal native elastic tissues in the body. In diseased tissues, the chronic overexpression of proteolytic enzymes can cause significant matrix degradation, to further limit the accumulation and quality (e.g., fiber formation) of newly deposited elastic matrix. This review provides an overview of the role and importance of elastin and elastic matrix in soft tissues, the challenges to elastic matrix generation in vitro and to regenerative elastic matrix repair in vivo, current biomolecular strategies to enhance elastin deposition and matrix assembly, and the need to concurrently inhibit proteolytic matrix disruption for improving the quantity and quality of elastogenesis. The review further presents biomaterial-based options using scaffolds and nanocarriers for spatio-temporal control over the presentation and release of these biomolecules, to enable biomimetic assembly of clinically relevant native elastic matrix-like superstructures. Finally, this review provides an overview of recent advances and prospects for the application of these strategies to regenerating tissue-type specific elastic matrix structures and superstructures. PMID:23355960

Mineralization of the Sea Urchin Skeleton

NASA Astrophysics Data System (ADS)

Wilt, F.

2001-12-01

The sea urchin possess a calcareous skeleton composed of over 99% magnesian calcite,an enveloping extracellular matrix, and an occluded protein matrix. The most intensively studied skeletal element is the spicule of the embryo. At the 32 cell stage of development a cohort of 4 cells becomes irrevocably dedicated to spicule formation. At the early gastrula stage the descendants of these founder cells form the primary mesenchyme (PMC). The PMCs fuse to form a multinucleated syncytium connected by cytoplasmic cables, and the calcitic skeleton is formed within these cables. Our primary concern is with the cellular and molecular mechanisms that support the formation of the mineralized spicules. The import of calcium into the PMCs results in appearance of intracellular vesicles containing precipitated calcium, which is neither very stable nor birefringent, and could be amorphous. The precipitated calcium is vectorially secreted into an extracellular space. This space is almost completely enclosed by cytoplasmic strands, and the mineral is encased in an extracellular matrix. Proteins destined for the extracellular matrix, and for inclusion in the spicule, are present in the Golgi membranes and in small intracellular vesicles. These vesicles apparently deliver the matrix proteins to the growing spicule. Our current view is that the matrix molecules are much more than a passive armature, but are actively involved in precipitation, secretion, and organization of the mineral phase.

The effects of scaffold architecture and fibrin gel addition on tendon cell phenotype.

PubMed

Pawelec, K M; Wardale, R J; Best, S M; Cameron, R E

2015-01-01

Development of tissue engineering scaffolds relies on careful selection of pore architecture and chemistry of the cellular environment. Repair of skeletal soft tissue, such as tendon, is particularly challenging, since these tissues have a relatively poor healing response. When removed from their native environment, tendon cells (tenocytes) lose their characteristic morphology and the expression of phenotypic markers. To stimulate tendon cells to recreate a healthy extracellular matrix, both architectural cues and fibrin gels have been used in the past, however, their relative effects have not been studied systematically. Within this study, a combination of collagen scaffold architecture, axial and isotropic, and fibrin gel addition was assessed, using ovine tendon-derived cells to determine the optimal strategy for controlling the proliferation and protein expression. Scaffold architecture and fibrin gel addition influenced tendon cell behavior independently in vitro. Addition of fibrin gel within a scaffold doubled cell number and increased matrix production for all architectures studied. However, scaffold architecture dictated the type of matrix produced by cells, regardless of fibrin addition. Axial scaffolds, mimicking native tendon, promoted a mature matrix, with increased tenomodulin, a marker for mature tendon cells, and decreased scleraxis, an early transcription factor for connective tissue. This study demonstrated that both architectural cues and fibrin gel addition alter cell behavior and that the combination of these signals could improve clinical performance of current tissue engineering constructs.

Cryopreservation of tissue engineered constructs for bone.

PubMed

Kofron, Michelle D; Opsitnick, Natalie C; Attawia, Mohamed A; Laurencin, Cato T

2003-11-01

The large-scale clinical use of tissue engineered constructs will require provisions for its mass availability and accessibility. Therefore, it is imperative to understand the effects of low temperature (-196 degrees C) on the tissue engineered biological system. Initial studies used samples of the osteoblast-like cell line (SaOS-2) adhered to a two-dimensional poly(lactide-co-glycolide) thin film (2D-PLAGA) or a three-dimensional poly(lactide-co-glycolide) sintered microsphere matrix (3D-PLAGA) designed for bone tissue engineering. Experimental samples were tested for their ability to maintain cell viability, following low temperature banking for one week, in solutions of the penetrating cryoprotective agents, dimethylsulfoxide (DMSO), ethylene glycol, and glycerol. Results indicated the DMSO solution yielded the greatest percent cell survival for SaOS-2 cells adhered to both the 2D- and 3D-PLAGA scaffolds; therefore, DMSO was used to cryopreserve mineralizing primary rabbit osteoblasts cells adhered to 2D-PLAGA matrices for 35 days. Results indicated retention of the extracellular matrix architecture as no statistically significant difference in the pre- and post-thaw mineralized structures was measured. Percent cell viability of the mineralized constructs following low temperature storage was approximately 50%. These are the first studies to address the issue of preservation techniques for tissue engineered constructs. The ability to successfully cryopreserve mineralized tissue engineered matrices for bone may offer an unlimited and readily available source of bone-like materials for orthopaedic applications.

Extracellular matrix biomimicry for the creation of investigational and therapeutic devices.

PubMed

Pellowe, Amanda S; Gonzalez, Anjelica L

2016-01-01

The extracellular matrix (ECM) is a web of fibrous proteins that serves as a scaffold for tissues and organs, and is important for maintaining homeostasis and facilitating cellular adhesion. Integrin transmembrane receptors are the primary adhesion molecules that anchor cells to the ECM, thus integrating cells with their microenvironments. Integrins play a critical role in facilitating cell-matrix interactions and promoting signal transduction, both from the cell to the ECM and vice versa, ultimately mediating cell behavior. For this reason, many advanced biomaterials employ biomimicry by replicating the form and function of fibrous ECM proteins. The ECM also acts as a reservoir for small molecules and growth factors, wherein fibrous proteins directly bind and present these bioactive moieties that facilitate cell activity. Therefore biomimicry can be enhanced by incorporating small molecules into ECM-like substrates. Biomimetic ECM materials have served as invaluable research tools for studying interactions between cells and the surrounding ECM, revealing that cell-matrix signaling is driven by mechanical forces, integrin engagement, and small molecules. Mimicking pathological ECMs has also elucidated disease specific cell behaviors. For example, biomimetic tumor microenvironments have been used to induce metastatic cell behaviors, and have thereby shown promise for in vitro cancer drug testing and targeting. Further, ECM-like substrates have been successfully employed for autologous cell recolonization for tissue engineering and wound healing. As we continue to learn more about the mechanical and biochemical characteristics of the ECM, these properties can be harnessed to develop new biomaterials, biomedical devices, and therapeutics. © 2015 Wiley Periodicals, Inc.

The impact of quercetin on wound healing relates to changes in αV and β1 integrin expression.

PubMed

Doersch, Karen M; Newell-Rogers, M Karen

2017-08-01

Overly fibrotic wound healing can lead to excess scar formation, causing functional impairment and undesirable cosmetic results. However, there are few successful treatments available to prevent or remediate scars. This study sought to explore the molecular mechanisms by which quercetin, a naturally-occurring antifibrotic agent, diminishes scar formation. Using both mice and fibroblast cells, we examined quercetin's impact on fibrosis and the wound healing rate, and potential molecular mechanisms underlying the quercetin-mediated reduction of fibrosis. While cultured fibroblasts demonstrated normal growth in response to quercetin, quercetin increased surface αV integrin and decreased β1 integrin. These changes in surface integrin expression may impact factors that contribute to fibrosis including cell migration, proliferation, and extracellular matrix production. In both quercetin-treated and control mice, wounds healed in about 14 days. Masson's trichrome stain revealed diminished fibrosis at the wound site in quercetin-treated animals despite the normal healing rate, indicating the potential for better cosmetic results without delaying healing. An in vitro scratch wound model using cells plated on an artificial extracellular matrix demonstrated delayed closure following quercetin treatment. The extracellular matrix also ameliorated quercetin's effect on αV integrin. Thus, αV integrin recruitment in response to quercetin treatment may promote the quercetin-mediated decrease extracellular matrix because cells require less extracellular matrix to migrate into a wound. With added extracellular matrix, β1 integrin remained diminished in response to quercetin, indicating that quercetin's effect on β1 integrin expression is independent of extracellular matrix -mediated signaling and is likely driven by inhibition of the intracellular mechanisms driving β1 expression. These findings suggest that quercetin could alter the cells' interactions with the extracellular matrix through the regulation of integrin expression to promote a decrease in fibrosis. Furthermore, this work demonstrates that this naturally occurring and commercially available supplement could be used to improve wound healing by impacting integrin expression, leading to a lower extracellular matrix requirement to achieve healing. Impact statement Scar formation during wound healing can be problematic for patients but there are limited therapies available to treat or prevent excess fibrosis at wound sites. This work examines the impact of quercetin, a flavonoid that decreases fibrosis, on wound healing, and relates quercetin's effects to changes in integrin expression on the surface of fibroblast cells. To our knowledge, this is the first report that quercetin alters integrin expression or that this impact may be part of the mechanism by which quercetin prevents fibrosis. This work demonstrates that quercetin can be used to modulate integrin expression and that this effect may in turn reduce fibrosis during wound healing. Furthermore, this paper identifies the modulation of integrin expression as a possible therapeutic target in preventing scars. This information could be used to improve therapeutics to aid in the cosmetic and functional results following wound healing.

HTRA1, an age-related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1.

PubMed

Lin, Michael K; Yang, Jin; Hsu, Chun Wei; Gore, Anuradha; Bassuk, Alexander G; Brown, Lewis M; Colligan, Ryan; Sengillo, Jesse D; Mahajan, Vinit B; Tsang, Stephen H

2018-05-05

High-temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age-related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high-risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high-risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP-1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Endochondral Ossification for Enhancing Bone Regeneration: Converging Native Extracellular Matrix Biomaterials and Developmental Engineering In Vivo

PubMed Central

Dennis, S. Connor; Berkland, Cory J.; Bonewald, Lynda F.

2015-01-01

Autologous bone grafting (ABG) remains entrenched as the gold standard of treatment in bone regenerative surgery. Consequently, many marginally successful bone tissue engineering strategies have focused on mimicking portions of ABG's “ideal” osteoconductive, osteoinductive, and osteogenic composition resembling the late reparative stage extracellular matrix (ECM) in bone fracture repair, also known as the “hard” or “bony” callus. An alternative, less common approach that has emerged in the last decade harnesses endochondral (EC) ossification through developmental engineering principles, which acknowledges that the molecular and cellular mechanisms involved in developmental skeletogenesis, specifically EC ossification, are closely paralleled during native bone healing. EC ossification naturally occurs during the majority of bone fractures and, thus, can potentially be utilized to enhance bone regeneration for nearly any orthopedic indication, especially in avascular critical-sized defects where hypoxic conditions favor initial chondrogenesis instead of direct intramembranous ossification. The body's native EC ossification response, however, is not capable of regenerating critical-sized defects without intervention. We propose that an underexplored potential exists to regenerate bone through the native EC ossification response by utilizing strategies which mimic the initial inflammatory or fibrocartilaginous ECM (i.e., “pro-” or “soft” callus) observed in the early reparative stage of bone fracture repair. To date, the majority of strategies utilizing this approach rely on clinically burdensome in vitro cell expansion protocols. This review will focus on the confluence of two evolving areas, (1) native ECM biomaterials and (2) developmental engineering, which will attempt to overcome the technical, business, and regulatory challenges that persist in the area of bone regeneration. Significant attention will be given to native “raw” materials and ECM-based designs that provide necessary osteo- and chondro-conductive and inductive features for enhancing EC ossification. In addition, critical perspectives on existing stem cell-based therapeutic strategies will be discussed with a focus on their use as an extension of the acellular ECM-based designs for specific clinical indications. Within this framework, a novel realm of unexplored design strategies for bone tissue engineering will be introduced into the collective consciousness of the regenerative medicine field. PMID:25336144

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.

Disorganized collagen scaffold interferes with fibroblast mediated deposition of organized extracellular matrix in vitro.

PubMed

Saeidi, Nima; Guo, Xiaoqing; Hutcheon, Audrey E K; Sander, Edward A; Bale, Shyam Sundar; Melotti, Suzanna A; Zieske, James D; Trinkaus-Randall, Vickery; Ruberti, Jeffrey W

2012-10-01

Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold-based and scaffold-free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS--five donors: average age 34.4) or on a bare polycarbonate membrane (five donors: average age 32.4 controls). The organization and morphology of the two culture systems were compared over the long-term at 4, 8, and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick-freeze/deep-etch electron microscopy. Both the scaffold-free and the collagen-based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell-derived matrix in the scaffold-based constructs was significantly lower than that produced by scaffold-free constructs (controls). We also observed significant variability in the remodeling of RCS scaffold by PHCFCs. PHCFCs which penetrated the RCS scaffold did exert robust local control over secreted collagen but did not appear to globally reorganize the scaffold effectively in the time period of the study. Consistent with our hypothesis, the results demonstrate that the presence of the scaffold appears to interfere with the global organization of the cell-derived matrix. The production of highly organized local matrix by fibroblasts which penetrated the scaffold suggests that there is a mechanism which operates close to the cell membrane capable of controlling fibril organization. Nonetheless, the local control of the collagen alignment produced by cells within the scaffold was not continuous and did not result in overall global organization of the construct. Using a disorganized scaffold as a guide to produce highly organized tissue has the potential to delay the production of useful matrix or prevent uniform remodeling. The results of this study may shed light on the recent attempts to use disorganized collagenous matrix as a temporary corneal replacement in vivo which led to a variable remodeling response. Copyright © 2012 Wiley Periodicals, Inc.

Disorganized collagen scaffold interferes with fibroblast mediated deposition of organized extracellular matrix in vitro

PubMed Central

Saeidi, Nima; Guo, Xiaoqing; Hutcheon, Audrey E. K.; Sander, Edward A.; Bale, Shyam Sundar; Melotti, Suzanna A.; Zieske, James D.; Trinkaus-Randall, Vickery; Ruberti, Jeffrey W.

2013-01-01

Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold-based and scaffold-free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS - 5 donors: average age 34.4) or on a bare polycarbonate membrane (5 donors: average age 32.4-controls). The organization and morphology of the two culture systems were compared over the long-term at 4, 8 and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick-freeze/deep-etch electron microscopy. Both the scaffold-free and the collagen-based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell-derived matrix in the scaffold-based constructs was significantly lower than that produced by scaffold-free constructs (controls). We also observed significant variability in the remodeling of RCS scaffold by PHCFCs. PHCFCs which penetrated the RCS scaffold did exert robust local control over secreted collagen but did not appear to globally reorganize the scaffold effectively in the time period of the study. Consistent with our hypothesis, the results demonstrate that the presence of the scaffold appears to interfere with the global organization of the cell-derived matrix. The production of highly-organized local matrix by fibroblasts which penetrated the scaffold suggests that there is a mechanism which operates close to the cell membrane capable of control fibril organization. Nonetheless, the local control of the collagen alignment produced by cells within the scaffold was not continuous and did not result in overall global organization of the construct. Using a disorganized scaffold as a guide to produce highly-organized tissue has the potential to delay the production of useful matrix or prevent uniform remodeling. The results of this study may shed light on the recent attempts to use disorganized collagenous matrix as a temporary corneal replacement in vivo which led to a variable remodeling response. PMID:22528405

Tissue-engineered trachea regeneration using decellularized trachea matrix treated with laser micropore technique.

PubMed

Xu, Yong; Li, Dan; Yin, Zongqi; He, Aijuan; Lin, Miaomiao; Jiang, Gening; Song, Xiao; Hu, Xuefei; Liu, Yi; Wang, Jinpeng; Wang, Xiaoyun; Duan, Liang; Zhou, Guangdong

2017-08-01

Tissue-engineered trachea provides a promising approach for reconstruction of long segmental tracheal defects. However, a lack of ideal biodegradable scaffolds greatly restricts its clinical translation. Decellularized trachea matrix (DTM) is considered a proper scaffold for trachea cartilage regeneration owing to natural tubular structure, cartilage matrix components, and biodegradability. However, cell residual and low porosity of DTM easily result in immunogenicity and incomplete cartilage regeneration. To address these problems, a laser micropore technique (LMT) was applied in the current study to modify trachea sample porosity to facilitate decellular treatment and cell ingrowth. Decellularization processing demonstrated that cells in LMT treated samples were more easily removed compared with untreated native trachea. Furthermore, after optimizing the protocols of LMT and decellular treatments, the LMT-treated DTM (LDTM) could retain their original tubular shape with only mild extracellular matrix damage. After seeding with chondrocytes and culture in vitro for 8 weeks, the cell-LDTM constructs formed tubular cartilage with relatively homogenous cell distribution in both micropores and bilateral surfaces. In vivo results further confirmed that the constructs could form mature tubular cartilage with increased DNA and cartilage matrix contents, as well as enhanced mechanical strength, compared with native trachea. Collectively, these results indicate that LDTM is an ideal scaffold for tubular cartilage regeneration and, thus, provides a promising strategy for functional reconstruction of trachea cartilage. Lacking ideal biodegradable scaffolds greatly restricts development of tissue-engineered trachea. Decellularized trachea matrix (DTM) is considered a proper scaffold for trachea cartilage regeneration. However, cell residual and low porosity of DTM easily result in immunogenicity and incomplete cartilage regeneration. By laser micropore technique (LMT), the current study efficiently enhanced the porosity and decellularized efficacy of DTM. The LMT-treated DTM basically retained the original tubular shape with mild matrix damage. After chondrocyte seeding followed by in vitro culture and in vivo implantation, the constructs formed mature tubular cartilage with matrix content and mechanical strength similar to native trachea. The current study provides an ideal scaffold and a promising strategy for cartilage regeneration and functional reconstruction of trachea. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Supra-organization and optical anisotropies of the extracellular matrix in the amniotic membrane and limbal stroma before and after explant culture

PubMed Central

Valdetaro, Gisele P.; Aldrovani, Marcela; Padua, Ivan R. M.; Cristovam, Priscila C.; Gomes, José A. P.; Laus, José L.

2016-01-01

In this research we evaluated the supramolecular organizations and the optical anisotropical properties of the de-epithelialized human amniotic membrane and rabbit limbal stroma, before and after explant culture. Birefringence, monochromatic light spectral absorption and linear dichroism of the main extracellular matrix biopolymers, that is, the fibrillar collagens and proteoglycans, were investigated by polarized light microscopy combined with image analysis. Our results demonstrated that the culture procedure–induced stimuli altered the supra-organizational characteristics (in terms of collagens/proteoglycans spatial orientation and ordered-aggregational state) of the amniotic and limbal extracellular matrix, which led to changes in optical anisotropical properties. PMID:28018719

Angiogenic Type I Collagen Extracellular Matrix Integrated with Recombinant Bacteriophages Displaying Vascular Endothelial Growth Factors.

PubMed

Yoon, Junghyo; Korkmaz Zirpel, Nuriye; Park, Hyun-Ji; Han, Sewoon; Hwang, Kyung Hoon; Shin, Jisoo; Cho, Seung-Woo; Nam, Chang-Hoon; Chung, Seok

2016-01-21

Here, a growth-factor-integrated natural extracellular matrix of type I collagen is presented that induces angiogenesis. The developed matrix adapts type I collagen nanofibers integrated with synthetic colloidal particles of recombinant bacteriophages that display vascular endothelial growth factor (VEGF). The integration is achieved during or after gelation of the type I collagen and the matrix enables spatial delivery of VEGF into a desired region. Endothelial cells that contact the VEGF are found to invade into the matrix to form tube-like structures both in vitro and in vivo, proving the angiogenic potential of the matrix. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms.

PubMed

Kornmuller, Anna; Brown, Cody F C; Yu, Claire; Flynn, Lauren E

2017-04-11

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

Guiding the orientation of smooth muscle cells on random and aligned polyurethane/collagen nanofibers.

PubMed

Jia, Lin; Prabhakaran, Molamma P; Qin, Xiaohong; Ramakrishna, Seeram

2014-09-01

Fabricating scaffolds that can simulate the architecture and functionality of native extracellular matrix is a huge challenge in vascular tissue engineering. Various kinds of materials are engineered via nano-technological approaches to meet the current challenges in vascular tissue regeneration. During this study, nanofibers from pure polyurethane and hybrid polyurethane/collagen in two different morphologies (random and aligned) and in three different ratios of polyurethane:collagen (75:25; 50:50; 25:75) are fabricated by electrospinning. The fiber diameters of the nanofibrous scaffolds are in the range of 174-453 nm and 145-419 for random and aligned fibers, respectively, where they closely mimic the nanoscale dimensions of native extracellular matrix. The aligned polyurethane/collagen nanofibers expressed anisotropic wettability with mechanical properties which is suitable for regeneration of the artery. After 12 days of human aortic smooth muscle cells culture on different scaffolds, the proliferation of smooth muscle cells on hybrid polyurethane/collagen (3:1) nanofibers was 173% and 212% higher than on pure polyurethane scaffolds for random and aligned scaffolds, respectively. The results of cell morphology and protein staining showed that the aligned polyurethane/collagen (3:1) scaffold promote smooth muscle cells alignment through contact guidance, while the random polyurethane/collagen (3:1) also guided cell orientation most probably due to the inherent biochemical composition. Our studies demonstrate the potential of aligned and random polyurethane/collagen (3:1) as promising substrates for vascular tissue regeneration. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Superelastic, superabsorbent and 3D nanofiber-assembled scaffold for tissue engineering.

PubMed

Chen, Weiming; Ma, Jun; Zhu, Lei; Morsi, Yosry; -Ei-Hamshary, Hany; Al-Deyab, Salem S; Mo, Xiumei

2016-06-01

Fabrication of 3D scaffold to mimic the nanofibrous structure of the nature extracellular matrix (ECM) with appropriate mechanical properties and excellent biocompatibility, remain an important technical challenge in tissue engineering. The present study reports the strategy to fabricate a 3D nanofibrous scaffold with similar structure to collagen in ECM by combining electrospinning and freeze-drying technique. With the technique reported here, a nanofibrous structure scaffold with hydrophilic and superabsorbent properties can be readily prepared by Gelatin and Polylactic acid (PLA). In wet state the scaffold also shows a super-elastic property, which could bear a compressive strain as high as 80% and recovers its original shape afterwards. Moreover, after 6 days of culture, L-929 cells grow, proliferate and infiltrated into the scaffold. The results suggest that this 3D nanofibrous scaffold would be promising for varied field of tissue engineering application. Copyright © 2016 Elsevier B.V. All rights reserved.

Making microenvironments: A look into incorporating macromolecular crowding into in vitro experiments, to generate biomimetic microenvironments which are capable of directing cell function for tissue engineering applications.

PubMed

Benny, Paula; Raghunath, Michael

2017-01-01

Biomimetic microenvironments are key components to successful cell culture and tissue engineering in vitro. One of the most accurate biomimetic microenvironments is that made by the cells themselves. Cell-made microenvironments are most similar to the in vivo state as they are cell-specific and produced by the actual cells which reside in that specific microenvironment. However, cell-made microenvironments have been challenging to re-create in vitro due to the lack of extracellular matrix composition, volume and complexity which are required. By applying macromolecular crowding to current cell culture protocols, cell-made microenvironments, or cell-derived matrices, can be generated at significant rates in vitro. In this review, we will examine the causes and effects of macromolecular crowding and how it has been applied in several in vitro systems including tissue engineering.

Recent Applications of Coaxial and Emulsion Electrospinning Methods in the Field of Tissue Engineering

PubMed Central

McClellan, Phillip; Landis, William J.

2016-01-01

Abstract Electrospinning has emerged as an effective method of producing nanoscale fibers for use in multiple fields of study. One area of significant interest is nanofiber utilization for tissue engineering because the nanofibrous mats can mimic the native extracellular matrix of biological tissues. A logical next step is the inclusion of certain molecules and compounds to accelerate or increase the efficacy of tissue regeneration. Two methods are under scrutiny for their capability to encapsulate therapeutic compounds within electrospun nanofibers: emulsion and coaxial electrospinning. Both have advantages and disadvantages, which need to be taken into careful consideration when deciding to use them in a specific application. Several examples are provided here to highlight the vast potential of multilayered nanofibers as well as the emergence of new techniques to produce three-dimensional scaffolds of nanofibers for use in the field of tissue engineering. PMID:27610268

[Application of electrostatic spinning technology in nano-structured polymer scaffold].

PubMed

Chen, Denglong; Li, Min; Fang, Qian

2007-04-01

To review the latest development in the research on the application of the electrostatic spinning technology in preparation of the nanometer high polymer scaffold. The related articles published at home and abroad during the recent years were extensively reviewed and comprehensively analyzed. Micro/nano-structure and space topology on the surfaces of the scaffold materials, especially the weaving structure, were considered to have an important effect on the cell adhesion, proliferation, directional growth, and biological activation. The electrospun scaffold was reported to have a resemblance to the structure of the extracellular matrix and could be used as a promising scaffold for the tissue engineering application. The electrospun scaffolds were applied to the cartilage, bone, blood vessel, heart, and nerve tissue engineering fields. The nano-structured polymer scaffold can support the cell adhesion, proliferation, location, and differentiation, and this kind of scaffold has a considerable value in the tissue engineering field.

TOPICAL REVIEW: Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

NASA Astrophysics Data System (ADS)

Amranul Haque, Md; Nagaoka, Masato; Hexig, Bayar; Akaike, Toshihiro

2010-02-01

Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.

Prediction of Metastasis Using Second Harmonic Generation

DTIC Science & Technology

2016-07-01

extracellular matrix through which metastasizing cells must travel. We and others have demonstrated that tumor collagen structure, as measured with the...algorithm using separate training and validation sets, etc. Keywords: metastasis, overtreatment, extracellular matrix , collagen , second harmonic...optical process called second harmonic generation (SHG), influences tumor metastasis. This suggests that collagen structure may provide prognostic

Leg ulcer treatment outcomes with new ovine collagen extracellular matrix dressing: a retrospective case series.

PubMed

Bohn, Gregory A; Gass, Kimberly

2014-10-01

The purpose of this study was to describe the rate of closure observed in venous leg ulcers during treatment with ovine collagen extracellular matrix dressings and compression. Fourteen patients with 23 wounds were retrospectively evaluated with respect to healing rates, time to closure, and weekly facility charge fees.

The effect of scaffold pore size in cartilage tissue engineering.

PubMed

Nava, Michele M; Draghi, Lorenza; Giordano, Carmen; Pietrabissa, Riccardo

2016-07-26

The effect of scaffold pore size and interconnectivity is undoubtedly a crucial factor for most tissue engineering applications. The aim of this study was to examine the effect of pore size and porosity on cartilage construct development in different scaffolds seeded with articular chondrocytes. We fabricated poly-L-lactide-co-trimethylene carbonate scaffolds with different pore sizes, using a solvent-casting/particulate-leaching technique. We seeded primary bovine articular chondrocytes on these scaffolds, cultured the constructs for 2 weeks and examined cell proliferation, viability and cell-specific production of cartilaginous extracellular matrix proteins, including GAG and collagen. Cell density significantly increased up to 50% with scaffold pore size and porosity, likely facilitated by cell spreading on the internal surface of bigger pores, and by increased mass transport of gases and nutrients to cells, and catabolite removal from cells, allowed by lower diffusion barriers in scaffolds with a higher porosity. However, both the cell metabolic activity and the synthesis of cartilaginous matrix proteins significantly decreased by up to 40% with pore size. We propose that the association of smaller pore diameters, causing 3-dimensional cell aggregation, to a lower oxygenation caused by a lower porosity, could have been the condition that increased the cell-specific synthesis of cartilaginous matrix proteins in the scaffold with the smallest pores and the lowest porosity among those tested. In the initial steps of in vitro cartilage engineering, the combination of small scaffold pores and low porosity is an effective strategy with regard to the promotion of chondrogenesis.

Laser-assisted patch clamping: a methodology

NASA Technical Reports Server (NTRS)

Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1997-01-01

Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

Effect of ECM2 expression on bovine skeletal muscle-derived satellite cell differentiation.

PubMed

Liu, Chang; Tong, Huili; Li, Shufeng; Yan, Yunqin

2018-05-01

Extracellular matrix components have important regulatory functions during cell proliferation and differentiation. In recent study, extracellular matrix were shown to have a strong effect on skeletal muscle differentiation. Here, we aimed to elucidate the effects of extracellular matrix protein 2 (ECM2), an extracellular matrix component, on the differentiation of bovine skeletal muscle-derived satellite cells (MDSCs). Western blot and immunofluorescence analyses were used to elucidate the ECM2 expression pattern in bovine MDSCs during differentiation in vitro. CRISPR/Cas9 technology was used to activate or inhibit ECM2 expression to study its effects on the in vitro differentiation of bovine MDSCs. ECM2 expression was shown to increase gradually during bovine MDSC differentiation, and the levels of this protein were higher in more highly differentiated myotubes. ECM2 activation promoted MDSC differentiation, whereas its suppression inhibited the differentiation of these cells. Here, for the first time, we demonstrated the importance of ECM2 expression during bovine MDSC differentiation; these results could lead to treatments that help to increase beef cattle muscularity. © 2018 International Federation for Cell Biology.

Effects of boron derivatives on extracellular matrix formation.

PubMed

Benderdour, M; Van Bui, T; Hess, K; Dicko, A; Belleville, F; Dousset, B

2000-10-01

Boric acid solution (3%) dramatically improves wound healing through action on the extracellular matrix, a finding that has been obtained in vitro. Consequently, investigations are presently underway to produce boronated compounds having a therapeutical effectiveness similar to that of boric acid. On the basis of experimental results obtained with boric acid, we examined the effects of boron derivatives on extracellular matrix formation and degradation and analyzed their potential toxicity by using two biological models (chick embryo cartilage and human fibroblasts). The four boron derivatives tested in this study (triethanolamine borate; N-diethyl-phosphoramidate-propylboronique acid; 2,2 dimethylhexyl-1,3-propanediol-aminopropylboronate and 1,2 propanediol-aminopropylboronate) mimicked the effects of boric acid. They induced a decrease of intracellular concentrations in extracellular matrix macromolecules (proteoglycans, proteins)-associated with an increase of their release in culture medium and stimulated the activity of intra- and extracellular proteases. Similarly to boric acid, these actions occurred after exposure of the cells to concentrations of all boron derivatives without apparent toxic effects. The compounds were found to be more toxic than boric acid itself when concentrations were calculated according to their molecular weight. Nevertheless, these in vitro preliminary results demonstrate effects of boron derivatives that may be of therapeutic benefit in wound repair.

Bottom-Up and Top-Down Solid-State NMR Approaches for Bacterial Biofilm Matrix Composition

PubMed Central

Cegelski, Lynette

2015-01-01

The genomics and proteomics revolutions have been enormously successful in providing crucial “parts lists” for biological systems. Yet, formidable challenges exist in generating complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell assemblies. Bacterial biofilms are complex multicellular bacterial communities protected by a slime-like extracellular matrix that confers protection to environmental stress and enhances resistance to antibiotics and host defenses. As a non-crystalline, insoluble, heterogeneous assembly, the biofilm extracellular matrix poses a challenge to compositional analysis by conventional methods. In this Perspective, bottom-up and top-down solid-state NMR approaches are described for defining chemical composition in complex macrosystems. The “sum-of-theparts” bottom-up approach was introduced to examine the amyloid-integrated biofilms formed by E. coli and permitted the first determination of the composition of the intact extracellular matrix from a bacterial biofilm. An alternative top-down approach was developed to define composition in V. cholerae biofilms and relied on an extensive panel of NMR measurements to tease out specific carbon pools from a single sample of the intact extracellular matrix. These two approaches are widely applicable to other heterogeneous assemblies. For bacterial biofilms, quantitative parameters of matrix composition are needed to understand how biofilms are assembled, to improve the development of biofilm inhibitors, and to dissect inhibitor modes of action. Solid-state NMR approaches will also be invaluable in obtaining parameters of matrix architecture. PMID:25797008

Bottom-up and top-down solid-state NMR approaches for bacterial biofilm matrix composition.

PubMed

Cegelski, Lynette

2015-04-01

The genomics and proteomics revolutions have been enormously successful in providing crucial "parts lists" for biological systems. Yet, formidable challenges exist in generating complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell assemblies. Bacterial biofilms are complex multicellular bacterial communities protected by a slime-like extracellular matrix that confers protection to environmental stress and enhances resistance to antibiotics and host defenses. As a non-crystalline, insoluble, heterogeneous assembly, the biofilm extracellular matrix poses a challenge to compositional analysis by conventional methods. In this perspective, bottom-up and top-down solid-state NMR approaches are described for defining chemical composition in complex macrosystems. The "sum-of-the-parts" bottom-up approach was introduced to examine the amyloid-integrated biofilms formed by Escherichia coli and permitted the first determination of the composition of the intact extracellular matrix from a bacterial biofilm. An alternative top-down approach was developed to define composition in Vibrio cholerae biofilms and relied on an extensive panel of NMR measurements to tease out specific carbon pools from a single sample of the intact extracellular matrix. These two approaches are widely applicable to other heterogeneous assemblies. For bacterial biofilms, quantitative parameters of matrix composition are needed to understand how biofilms are assembled, to improve the development of biofilm inhibitors, and to dissect inhibitor modes of action. Solid-state NMR approaches will also be invaluable in obtaining parameters of matrix architecture. Copyright © 2015 Elsevier Inc. All rights reserved.

Bottom-up and top-down solid-state NMR approaches for bacterial biofilm matrix composition

NASA Astrophysics Data System (ADS)

Cegelski, Lynette

2015-04-01

The genomics and proteomics revolutions have been enormously successful in providing crucial "parts lists" for biological systems. Yet, formidable challenges exist in generating complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell assemblies. Bacterial biofilms are complex multicellular bacterial communities protected by a slime-like extracellular matrix that confers protection to environmental stress and enhances resistance to antibiotics and host defenses. As a non-crystalline, insoluble, heterogeneous assembly, the biofilm extracellular matrix poses a challenge to compositional analysis by conventional methods. In this perspective, bottom-up and top-down solid-state NMR approaches are described for defining chemical composition in complex macrosystems. The "sum-of-the-parts" bottom-up approach was introduced to examine the amyloid-integrated biofilms formed by Escherichia coli and permitted the first determination of the composition of the intact extracellular matrix from a bacterial biofilm. An alternative top-down approach was developed to define composition in Vibrio cholerae biofilms and relied on an extensive panel of NMR measurements to tease out specific carbon pools from a single sample of the intact extracellular matrix. These two approaches are widely applicable to other heterogeneous assemblies. For bacterial biofilms, quantitative parameters of matrix composition are needed to understand how biofilms are assembled, to improve the development of biofilm inhibitors, and to dissect inhibitor modes of action. Solid-state NMR approaches will also be invaluable in obtaining parameters of matrix architecture.

Preliminary results of recurrent cubital tunnel syndrome treated with neurolysis and porcine extracellular matrix nerve wrap.

PubMed

Papatheodorou, Loukia K; Williams, Benjamin G; Sotereanos, Dean G

2015-05-01

To evaluate the clinical results of revision neurolysis and wrapping with porcine extracellular matrix (AxoGuard Nerve Protector, AxoGen Inc., Alachua, FL) for cubital tunnel syndrome after one previous surgical decompression. Twelve patients with recurrent cubital tunnel syndrome were treated with decompression, porcine extracellular matrix nerve wrap, and minimal medial epicondylectomy (if not previously performed). The average follow-up period was 41 months (range, 24-61 mo). All patients had recurrent symptoms after having previously undergone one surgical decompression. The mean patient age was 45 years (range, 30-58 y). All patients were evaluated subjectively and objectively (pain, satisfaction, static 2-point discrimination, grip strength, and pinch strength). A significant improvement was demonstrated in postoperative pain levels (from 8.5 to 1.7), grip strength (from 41% to 86% of the unaffected side), and pinch strength (from 64% to 83% of the unaffected side). Static 2-point discrimination improved from an average 10.4 mm preoperatively to 7.6 mm postoperatively. Eleven of 12 patients demonstrated 2 mm or more improvement in 2-point discrimination postoperatively. There were no complications related to the use of the porcine extracellular matrix for nerve wrapping. This study found that secondary decompression combined with porcine extracellular matrix nerve wrapping was an effective and safe treatment for patients with recurrent cubital tunnel syndrome. Therapeutic IV. Copyright © 2015 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.

Regulation of Osteoblast Survival by the Extracellular Matrix and Gravity

NASA Technical Reports Server (NTRS)

Globus. Ruth K.; Almeida, Eduardo A. C.; Searby, Nancy D.; Bowley, Susan M. (Technical Monitor)

2000-01-01

Spaceflight adversely affects the skeleton, posing a substantial risk to astronaut's health during long duration missions. The reduced bone mass observed in growing animals following spaceflight is due at least in part to inadequate bone formation by osteoblasts. Thus, it is of central importance to identify basic cellular mechanisms underlying normal bone formation. The fundamental ideas underlying our research are that interactions between extracellular matrix proteins, integrin adhesion receptors, cytoplasmic signaling and cytoskeletal proteins are key ingredients for the proper functioning of osteoblasts, and that gravity impacts these interactions. As an in vitro model system we used primary fetal rat calvarial cells which faithfully recapitulate osteoblast differentiation characteristically observed in vivo. We showed that specific integrin receptors ((alpha)3(beta)1), ((alpha)5(beta)1), ((alpha)8(betal)1) and extracellular matrix proteins (fibronectin, laminin) were needed for the differentiation of immature osteoblasts. In the course of maturation, cultured osteoblasts switched from depending on fibronectin and laminin for differentiation to depending on these proteins for their very survival. Furthermore, we found that manipulating the gravity vector using ground-based models resulted in activation of key intracellular survival signals generated by integrin/extracellular matrix interactions. We are currently testing the in vivo relevance of some of these observations using targeted transgenic technology. In conclusion, mechanical factors including gravity may participate in regulating survival via cellular interactions with the extracellular matrix. This leads us to speculate that microgravity adversely affects the survival of osteoblasts and contributes to spaceflight-induced osteoporosis.

Molecularly Imprinted Intelligent Scaffolds for Tissue Engineering Applications.

PubMed

Neves, Mariana I; Wechsler, Marissa E; Gomes, Manuela E; Reis, Rui L; Granja, Pedro L; Peppas, Nicholas A

2017-02-01

The development of molecularly imprinted polymers (MIPs) using biocompatible production methods enables the possibility to further exploit this technology for biomedical applications. Tissue engineering (TE) approaches use the knowledge of the wound healing process to design scaffolds capable of modulating cell behavior and promote tissue regeneration. Biomacromolecules bear great interest for TE, together with the established recognition of the extracellular matrix, as an important source of signals to cells, both promoting cell-cell and cell-matrix interactions during the healing process. This review focuses on exploring the potential of protein molecular imprinting to create bioactive scaffolds with molecular recognition for TE applications based on the most recent approaches in the field of molecular imprinting of macromolecules. Considerations regarding essential components of molecular imprinting technology will be addressed for TE purposes. Molecular imprinting of biocompatible hydrogels, namely based on natural polymers, is also reviewed here. Hydrogel scaffolds with molecular memory show great promise for regenerative therapies. The first molecular imprinting studies analyzing cell adhesion report promising results with potential applications for cell culture systems, or biomaterials for implantation with the capability for cell recruitment by selectively adsorbing desired molecules.

Accelerated Development of Supramolecular Corneal Stromal-Like Assemblies from Corneal Fibroblasts in the Presence of Macromolecular Crowders.

PubMed

Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Rochev, Yury; Rodriguez, Brian J; Gorelov, Alexander; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I

2015-07-01

Tissue engineering by self-assembly uses the cells' secretome as a regeneration template and biological factory of trophic factors. Despite the several advantages that have been witnessed in preclinical and clinical settings, the major obstacle for wide acceptance of this technology remains the tardy extracellular matrix formation. In this study, we assessed the influence of macromolecular crowding (MMC)/excluding volume effect, a biophysical phenomenon that accelerates thermodynamic activities and biological processes by several orders of magnitude, in human corneal fibroblast (HCF) culture. Our data indicate that the addition of negatively charged galactose derivative (carrageenan) in HCF culture, even at 0.5% serum, increases by 12-fold tissue-specific matrix deposition, while maintaining physiological cell morphology and protein/gene expression. Gene analysis indicates that a glucose derivative (dextran sulfate) may drive corneal fibroblasts toward a myofibroblast lineage. Collectively, these results indicate that MMC may be suitable not only for clinical translation and commercialization of tissue engineering by self-assembly therapies, but also for the development of in vitro pathophysiology models.

Skeletal biology: Where matrix meets mineral

PubMed Central

Young, Marian F.

2017-01-01

The skeleton is unique from all other tissues in the body because of its ability to mineralize. The incorporation of mineral into bones and teeth is essential to give them strength and structure for body support and function. For years, researchers have wondered how mineralized tissues form and repair. A major focus in this context has been on the role of the extracellular matrix, which harbors key regulators of the mineralization process. In this introductory minireview, we will review some key concepts of matrix biology as it related to mineralized tissues. Concurrently, we will highlight the subject of this special issue covering many aspects of mineralized tissues, including bones and teeth and their associated structures cartilage and tendon. Areas of emphasis are on the generation and analysis of new animal models with permutations of matrix components as well as the development of new approaches for tissue engineering for repair of damaged hard tissue. In assembling key topics on mineralized tissues written by leaders in our field, we hope the reader will get a broad view of the topic and all of its fascinating complexities. PMID:27131884

Chitosan microspheres with an extracellular matrix-mimicking nanofibrous structure as cell-carrier building blocks for bottom-up cartilage tissue engineering

NASA Astrophysics Data System (ADS)

Zhou, Yong; Gao, Huai-Ling; Shen, Li-Li; Pan, Zhao; Mao, Li-Bo; Wu, Tao; He, Jia-Cai; Zou, Duo-Hong; Zhang, Zhi-Yuan; Yu, Shu-Hong

2015-12-01

Scaffolds for tissue engineering (TE) which closely mimic the physicochemical properties of the natural extracellular matrix (ECM) have been proven to advantageously favor cell attachment, proliferation, migration and new tissue formation. Recently, as a valuable alternative, a bottom-up TE approach utilizing cell-loaded micrometer-scale modular components as building blocks to reconstruct a new tissue in vitro or in vivo has been proved to demonstrate a number of desirable advantages compared with the traditional bulk scaffold based top-down TE approach. Nevertheless, micro-components with an ECM-mimicking nanofibrous structure are still very scarce and highly desirable. Chitosan (CS), an accessible natural polymer, has demonstrated appealing intrinsic properties and promising application potential for TE, especially the cartilage tissue regeneration. According to this background, we report here the fabrication of chitosan microspheres with an ECM-mimicking nanofibrous structure for the first time based on a physical gelation process. By combining this physical fabrication procedure with microfluidic technology, uniform CS microspheres (CMS) with controlled nanofibrous microstructure and tunable sizes can be facilely obtained. Especially, no potentially toxic or denaturizing chemical crosslinking agent was introduced into the products. Notably, in vitro chondrocyte culture tests revealed that enhanced cell attachment and proliferation were realized, and a macroscopic 3D geometrically shaped cartilage-like composite can be easily constructed with the nanofibrous CMS (NCMS) and chondrocytes, which demonstrate significant application potential of NCMS as the bottom-up cell-carrier components for cartilage tissue engineering.Scaffolds for tissue engineering (TE) which closely mimic the physicochemical properties of the natural extracellular matrix (ECM) have been proven to advantageously favor cell attachment, proliferation, migration and new tissue formation. Recently, as a valuable alternative, a bottom-up TE approach utilizing cell-loaded micrometer-scale modular components as building blocks to reconstruct a new tissue in vitro or in vivo has been proved to demonstrate a number of desirable advantages compared with the traditional bulk scaffold based top-down TE approach. Nevertheless, micro-components with an ECM-mimicking nanofibrous structure are still very scarce and highly desirable. Chitosan (CS), an accessible natural polymer, has demonstrated appealing intrinsic properties and promising application potential for TE, especially the cartilage tissue regeneration. According to this background, we report here the fabrication of chitosan microspheres with an ECM-mimicking nanofibrous structure for the first time based on a physical gelation process. By combining this physical fabrication procedure with microfluidic technology, uniform CS microspheres (CMS) with controlled nanofibrous microstructure and tunable sizes can be facilely obtained. Especially, no potentially toxic or denaturizing chemical crosslinking agent was introduced into the products. Notably, in vitro chondrocyte culture tests revealed that enhanced cell attachment and proliferation were realized, and a macroscopic 3D geometrically shaped cartilage-like composite can be easily constructed with the nanofibrous CMS (NCMS) and chondrocytes, which demonstrate significant application potential of NCMS as the bottom-up cell-carrier components for cartilage tissue engineering. Electronic supplementary information (ESI) available: Additional figures and table. See DOI: 10.1039/c5nr06876b

Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

PubMed

Müller, Stephan A; van der Smissen, Anja; von Feilitzsch, Margarete; Anderegg, Ulf; Kalkhof, Stefan; von Bergen, Martin

2012-12-01

Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

Nanostructured Biomaterials for Tissue Engineered Bone Tissue Reconstruction

PubMed Central

Chiara, Gardin; Letizia, Ferroni; Lorenzo, Favero; Edoardo, Stellini; Diego, Stomaci; Stefano, Sivolella; Eriberto, Bressan; Barbara, Zavan

2012-01-01

Bone tissue engineering strategies are emerging as attractive alternatives to autografts and allografts in bone tissue reconstruction, in particular thanks to their association with nanotechnologies. Nanostructured biomaterials, indeed, mimic the extracellular matrix (ECM) of the natural bone, creating an artificial microenvironment that promotes cell adhesion, proliferation and differentiation. At the same time, the possibility to easily isolate mesenchymal stem cells (MSCs) from different adult tissues together with their multi-lineage differentiation potential makes them an interesting tool in the field of bone tissue engineering. This review gives an overview of the most promising nanostructured biomaterials, used alone or in combination with MSCs, which could in future be employed as bone substitutes. Recent works indicate that composite scaffolds made of ceramics/metals or ceramics/polymers are undoubtedly more effective than the single counterparts in terms of osteoconductivity, osteogenicity and osteoinductivity. A better understanding of the interactions between MSCs and nanostructured biomaterials will surely contribute to the progress of bone tissue engineering. PMID:22312283

Advances in Tissue Engineering Techniques for Articular Cartilage Repair

PubMed Central

Haleem, AM; Chu, CR

2010-01-01

The limited repair potential of human articular cartilage contributes to development of debilitating osteoarthritis and remains a great clinical challenge. This has led to evolution of cartilage treatment strategies from palliative to either reconstructive or reparative methods in an attempt to delay or “bridge the gap” to joint replacement. Further development of tissue engineering-based cartilage repair methods have been pursued to provide a more functional biological tissue. Currently, tissue engineering of articular cartilage has three cornerstones; a cell population capable of proliferation and differentiation into mature chondrocytes, a scaffold that can host these cells, provide a suitable environment for cellular functioning and serve as a sustained-release delivery vehicle of chondrogenic growth factors and thirdly, signaling molecules and growth factors that stimulate the cellular response and the production of a hyaline extracellular matrix (ECM). The aim of this review is to summarize advances in each of these three fields of tissue engineering with specific relevance to surgical techniques and technical notes. PMID:29430164

Bioprinting Cartilage Tissue from Mesenchymal Stem Cells and PEG Hydrogel.

PubMed

Gao, Guifang; Hubbell, Karen; Schilling, Arndt F; Dai, Guohao; Cui, Xiaofeng

2017-01-01

Bioprinting based on thermal inkjet printing is one of the most attractive enabling technologies for tissue engineering and regeneration. During the printing process, cells, scaffolds , and growth factors are rapidly deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations. Ideally, the bioprinted tissues are able to mimic the native anatomic structures in order to restore the biological functions. In this study, a bioprinting platform for 3D cartilage tissue engineering was developed using a commercially available thermal inkjet printer with simultaneous photopolymerization . The engineered cartilage demonstrated native zonal organization, ideal extracellular matrix (ECM ) composition, and proper mechanical properties. Compared to the conventional tissue fabrication approach, which requires extended 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 profile. Therefore, this platform is ideal for anatomic tissue engineering with accurate cell distribution and arrangement.

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.

Biomaterials and cells for neural tissue engineering: Current choices.

PubMed

Sensharma, Prerana; Madhumathi, G; Jayant, Rahul D; Jaiswal, Amit K

2017-08-01

The treatment of nerve injuries has taken a new dimension with the development of tissue engineering techniques. Prior to tissue engineering, suturing and surgery were the only options for effective treatment. With the advent of tissue engineering, it is now possible to design a scaffold that matches the exact biological and mechanical properties of the tissue. This has led to substantial reduction in the complications posed by surgeries and suturing to the patients. New synthetic and natural polymers are being applied to test their efficiency in generating an ideal scaffold. Along with these, cells and growth factors are also being incorporated to increase the efficiency of a scaffold. Efforts are being made to devise a scaffold that is biodegradable, biocompatible, conducting and immunologically inert. The ultimate goal is to exactly mimic the extracellular matrix in our body, and to elicit a combination of biochemical, topographical and electrical cues via various polymers, cells and growth factors, using which nerve regeneration can efficiently occur. Copyright © 2017 Elsevier B.V. All rights reserved.

Using Acellular Bioactive Extracellular Matrix Scaffolds to Enhance Endogenous Cardiac Repair

PubMed Central

Svystonyuk, Daniyil A.; Mewhort, Holly E. M.; Fedak, Paul W. M.

2018-01-01

An inability to recover lost cardiac muscle following acute ischemic injury remains the biggest shortcoming of current therapies to prevent heart failure. As compared to standard medical and surgical treatments, tissue engineering strategies offer the promise of improved heart function by inducing regeneration of functional heart muscle. Tissue engineering approaches that use stem cells and genetic manipulation have shown promise in preclinical studies but have also been challenged by numerous critical barriers preventing effective clinical translational. We believe that surgical intervention using acellular bioactive ECM scaffolds may yield similar therapeutic benefits with minimal translational hurdles. In this review, we outline the limitations of cellular-based tissue engineering strategies and the advantages of using acellular biomaterials with bioinductive properties. We highlight key anatomic targets enriched with cellular niches that can be uniquely activated using bioactive scaffold therapy. Finally, we review the evolving cardiovascular tissue engineering landscape and provide critical insights into the potential therapeutic benefits of acellular scaffold therapy. PMID:29696148

Human collagen produced in plants: more than just another molecule.

PubMed

Shoseyov, Oded; Posen, Yehudit; Grynspan, Frida

2014-01-01

Consequential to its essential role as a mechanical support and affinity regulator in extracellular matrices, collagen constitutes a highly sought after scaffolding material for regeneration and healing applications. However, substantiated concerns have been raised with regard to quality and safety of animal tissue-extracted collagen, particularly in relation to its immunogenicity, risk of disease transmission and overall quality and consistency. In parallel, contamination with undesirable cellular factors can significantly impair its bioactivity, vis-a-vis its impact on cell recruitment, proliferation and differentiation. High-scale production of recombinant human collagen Type I (rhCOL1) in the tobacco plant provides a source of an homogenic, heterotrimeric, thermally stable "virgin" collagen which self assembles to fine homogenous fibrils displaying intact binding sites and has been applied to form numerous functional scaffolds for tissue engineering and regenerative medicine. In addition, rhCOL1 can form liquid crystal structures, yielding a well-organized and mechanically strong membrane, two properties indispensable to extracellular matrix (ECM) mimicry. Overall, the shortcomings of animal- and cadaver-derived collagens arising from their source diversity and recycled nature are fully overcome in the plant setting, constituting a collagen source ideal for tissue engineering and regenerative medicine applications.

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

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

Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi

2008-10-20

In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for {beta}-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of {beta}-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissuesmore » in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of {beta}-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.« less

Comparative Biology of Decellularized Lung Matrix: Implications of Species Mismatch in Regenerative Medicine

PubMed Central

Balestrini, Jenna L.; Gard, Ashley L.; Gerhold, Kristin A.; Wilcox, Elise C.; Liu, Angela; Schwan, Jonas; Le, Andrew V.; Baevova, Pavlina; Dimitrievska, Sashka; Zhao, Liping; Sundaram, Sumati; Sun, Huanxing; Rittié, Laure; Dyal, Rachel; Broekelmann, Tom J.; Mecham, Robert P.; Schwartz, Martin A.; Niklason, Laura E.; White, Eric S.

2016-01-01

Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration. PMID:27344365

Influence of bone morphogenetic protein-2 on the extracellular matrix, material properties, and gene expression of long-term articular chondrocyte cultures: loss of chondrocyte stability.

PubMed

Krawczak, David A; Westendorf, Jennifer J; Carlson, Cathy S; Lewis, Jack L

2009-06-01

The aim of this study was to determine the effects of bone morphogenetic protein-2 (BMP-2) on articular chondrocyte tissues grown as monolayers in vitro for up to 8 weeks. Articular chondrocytes were isolated from New Zealand White rabbits and plated in monolayer cultures. The cultures were supplemented with 100 ng/mL of BMP-2 for up to 8 weeks and the extracellular matrix (ECM) composition, material properties, and messenger RNA (mRNA) expression were analyzed. mRNA expression of cartilage-specific genes, type II collagen, and aggrecan showed that BMP-2 enhanced chondrocyte stability for up to 3 weeks. After 3 weeks in culture, there was substantially more type I collagen expression and more osteopontin and runt-related transcription factor 2 expression in 5- and 8-week cultures treated with BMP-2 than in controls. Additionally, matrix metalloproteinase-13 and ADAMTS-5 (A disintegrin-like and metalloproteinase with thrombospondin 5) were upregulated in 5- and 8-week cultures treated with BMP-2, coinciding with a loss of ECM density, collagen, and proteoglycan. Eight-week tissue stimulated with BMP-2 was more fragile and tore more easily when removed from the culture dish as compared to controls, suggesting temporal limitations to the effectiveness of BMP-2 in monolayer systems and perhaps other models to enhance the generation of a cartilage-like tissue for tissue engineering purposes.

Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro.

PubMed

Miyanishi, Keita; Trindade, Michael C D; Lindsey, Derek P; Beaupré, Gary S; Carter, Dennis R; Goodman, Stuart B; Schurman, David J; Smith, R Lane

2006-06-01

This study examined the effects of intermittent hydrostatic pressure (IHP) and transforming growth factor-beta 3 on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. Chondrogenic gene expression was determined by quantifying mRNA signal levels for SOX9, a transcription factor critical for cartilage development and the cartilage matrix proteins, aggrecan and type II collagen. Extracellular matrix production was determined by weight and histology. IHP was applied to hMSCs in pellet culture at a level of 10 MPa and a frequency of 1 Hz for 4 h per day for periods of 3, 7, and 14 days. hMSCs responded to addition of TGF-beta 3 (10 ng/mL) with a greater than 10-fold increase (p < 0.01) in mRNA levels for each, SOX9, type II collagen, and aggrecan during a 14-day culture period. Applying IHP in the presence of TGF-beta 3 further increased the mRNA levels for these proteins by 1.9-, 3.3-, and 1.6-fold, respectively, by day 14. Chondrogenic mRNA levels were increased with just exposure to IHP. Extracellular matrix deposition of type II collagen and aggrecan increased in the pellets as a function of treatment conditions and time of culture. This study demonstrated adjunctive effects of IHP on TGF-beta 3-induced chondrogenesis and suggests that mechanical loading can facilitate articular cartilage tissue engineering.

Biomaterials and Culture Technologies for Regenerative Therapy of Liver Tissue.

PubMed

Perez, Roman A; Jung, Cho-Rok; Kim, Hae-Won

2017-01-01

Regenerative approach has emerged to substitute the current extracorporeal technologies for the treatment of diseased and damaged liver tissue. This is based on the use of biomaterials that modulate the responses of hepatic cells through the unique matrix properties tuned to recapitulate regenerative functions. Cells in liver preserve their phenotype or differentiate through the interactions with extracellular matrix molecules. Therefore, the intrinsic properties of the engineered biomaterials, such as stiffness and surface topography, need to be tailored to induce appropriate cellular functions. The matrix physical stimuli can be combined with biochemical cues, such as immobilized functional groups or the delivered actions of signaling molecules. Furthermore, the external modulation of cells, through cocultures with nonparenchymal cells (e.g., endothelial cells) that can signal bioactive molecules, is another promising avenue to regenerate liver tissue. This review disseminates the recent approaches of regenerating liver tissue, with a focus on the development of biomaterials and the related culture technologies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Hypoxic Regulation of Functional Extracellular Matrix Elaboration by Nucleus Pulposus Cells in Long-Term Agarose Culture

PubMed Central

Gorth, Deborah J; Lothstein, Katherine E; Chiaro, Joseph A; Farrell, Megan J; Dodge, George R; Elliott, Dawn M; Malhotra, Neil R; Mauck, Robert L; Smith, Lachlan J

2015-01-01

Degeneration of the intervertebral discs is strongly implicated as a cause of low back pain. Since current treatments for discogenic low back pain show poor long-term efficacy, a number of new, biological strategies are being pursued. For such therapies to succeed, it is critical that they be validated in conditions that mimic the unique biochemical microenvironment of the nucleus pulposus (NP), which include low oxygen tension. Therefore, the objective of this study was to investigate the effects of oxygen tension on NP cell functional extracellular matrix elaboration in 3D culture. Bovine NP cells were encapsulated in agarose constructs and cultured for 14 or 42 days in either 20% or 2% oxygen in defined media containing transforming growth factor beta-3. At each time point, extracellular matrix composition, biomechanics and mRNA expression of key phenotypic markers were evaluated. Results showed that while bulk mechanics and composition were largely independent of oxygen level, low oxygen promoted improved restoration of the NP phenotype, higher mRNA expression of extracellular matrix and NP specific markers, and more uniform matrix elaboration. These findings indicate that culture under physiological oxygen levels is an important consideration for successful development of cell and growth factor-based regenerative strategies for the disc. PMID:25640328

Large-scale production and isolation of Candida biofilm extracellular matrix.

PubMed

Zarnowski, Robert; Sanchez, Hiram; Andes, David R

2016-12-01

The extracellular matrix of biofilm is unique to the biofilm lifestyle, and it has key roles in community survival. A complete understanding of the biochemical nature of the matrix is integral to the understanding of the roles of matrix components. This knowledge is a first step toward the development of novel therapeutics and diagnostics to address persistent biofilm infections. Many of the assay methods needed for refined matrix composition analysis require milligram amounts of material that is separated from the cellular components of these complex communities. The protocol described here explains the large-scale production and isolation of the Candida biofilm extracellular matrix. To our knowledge, the proposed procedure is the only currently available approach in the field that yields milligram amounts of biofilm matrix. This procedure first requires biofilms to be seeded in large-surface-area roller bottles, followed by cell adhesion and biofilm maturation during continuous movement of the medium across the surface of the rotating bottle. The formed matrix is then separated from the entire biomass using sonication, which efficiently removes the matrix without perturbing the fungal cell wall. Subsequent filtration, dialysis and lyophilization steps result in a purified matrix product sufficient for biochemical, structural and functional assays. The overall protocol takes ∼11 d to complete. This protocol has been used for Candida species, but, using the troubleshooting guide provided, it could be adapted for other fungi or bacteria.

Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones

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

Hansen, R K; Bissell, M J

The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellularmore » matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function.« less

Material properties of biofilms – key methods for understanding permeability and mechanics

PubMed Central

Billings, Nicole; Birjiniuk, Alona; Samad, Tahoura S.; Doyle, Patrick S.; Ribbeck, Katharina

2015-01-01

Microorganisms can form biofilms, which are multicellular communities surrounded by a hydrated extracellular matrix of polymers. Central properties of the biofilm are governed by this extracellular matrix, which provides mechanical stability to the three-dimensional biofilm structure, regulates the ability of the biofilm to adhere to surfaces, and determines the ability of the biofilm to adsorb gasses, solutes, and foreign cells. Despite their critical relevance for understanding and eliminating of biofilms, the materials properties of the extracellular matrix are understudied. Here, we offer the reader a guide to current technologies that can be utilized to specifically assess the permeability and mechanical properties of the biofilm matrix and its interacting components. In particular, we highlight technological advances in instrumentation and interactions between multiple disciplines that have broadened the spectrum of methods available to conduct these studies. We review pioneering work that furthers our understanding of the material properties of biofilms. PMID:25719969

Material properties of biofilms—a review of methods for understanding permeability and mechanics

NASA Astrophysics Data System (ADS)

Billings, Nicole; Birjiniuk, Alona; Samad, Tahoura S.; Doyle, Patrick S.; Ribbeck, Katharina

2015-02-01

Microorganisms can form biofilms, which are multicellular communities surrounded by a hydrated extracellular matrix of polymers. Central properties of the biofilm are governed by this extracellular matrix, which provides mechanical stability to the 3D biofilm structure, regulates the ability of the biofilm to adhere to surfaces, and determines the ability of the biofilm to adsorb gases, solutes, and foreign cells. Despite their critical relevance for understanding and eliminating of biofilms, the materials properties of the extracellular matrix are understudied. Here, we offer the reader a guide to current technologies that can be utilized to specifically assess the permeability and mechanical properties of the biofilm matrix and its interacting components. In particular, we highlight technological advances in instrumentation and interactions between multiple disciplines that have broadened the spectrum of methods available to conduct these studies. We review pioneering work that furthers our understanding of the material properties of biofilms.

Specialisation of extracellular matrix for function in tendons and ligaments

PubMed Central

Birch, Helen L.; Thorpe, Chavaunne T.; Rumian, Adam P.

2013-01-01

Summary Tendons and ligaments are similar structures in terms of their composition, organisation and mechanical properties. The distinction between them stems from their anatomical location; tendons form a link between muscle and bone while ligaments link bones to bones. A range of overlapping functions can be assigned to tendon and ligaments and each structure has specific mechanical properties which appear to be suited for particular in vivo function. The extracellular matrix in tendon and ligament varies in accordance with function, providing appropriate mechanical properties. The most useful framework in which to consider extracellular matrix differences therefore is that of function rather than anatomical location. In this review we discuss what is known about the relationship between functional requirements, structural properties from molecular to gross level, cellular gene expression and matrix turnover. The relevance of this information is considered by reviewing clinical aspects of tendon and ligament repair and reconstructive procedures. PMID:23885341

Scaffolds for Controlled Release of Cartilage Growth Factors.

PubMed

Morille, Marie; Venier-Julienne, Marie-Claire; Montero-Menei, Claudia N

2015-01-01

In recent years, cell-based therapies using adult stem cells have attracted considerable interest in regenerative medicine. A tissue-engineered construct for cartilage repair should provide a support for the cell and allow sustained in situ delivery of bioactive factors capable of inducing cell differentiation into chondrocytes. Pharmacologically active microcarriers (PAMs), made of biodegradable and biocompatible poly (D,L-lactide-co-glycolide acid) (PLGA), are a unique system which combines these properties in an adaptable and simple microdevice. This device relies on nanoprecipitation of proteins encapsulated in polymeric microspheres with a solid in oil in water emulsion-solvent evaporation process, and their subsequent coating with extracellular matrix protein molecules. Here, we describe their preparation process, and some of their characterization methods for an application in cartilage tissue engineering.

The Extracellular Protease Matrix Metalloproteinase-9 Is Activated by Inhibitory Avoidance Learning and Required for Long-Term Memory

ERIC Educational Resources Information Center

Nagy, Vanja; Bozdagi, Ozlem; Huntley, George W.

2007-01-01

Matrix metalloproteinases (MMPs) are a family of extracellularly acting proteolytic enzymes with well-recognized roles in plasticity and remodeling of synaptic circuits during brain development and following brain injury. However, it is now becoming increasingly apparent that MMPs also function in normal, nonpathological synaptic plasticity of the…

Altered Liver Proteoglycan/Glycosaminoglycan Structure as a Manifestation of Extracellular Matrix Remodeling upon BCG-induced Granulomatosis in Mice.

PubMed

Kim, L B; Shkurupy, V A; Putyatina, A N

2017-01-01

Experimental BCG-induced granulomatosis in mice was used to study changes in the dynamics of individual liver proteoglycan components reflecting phasic extracellular matrix remodeling, determined by the host-parasite interaction and associated with granuloma development. In the early BCG-granulomatosis period, the increase in individual proteoglycan components promotes granuloma formation, providing conditions for mycobacteria adhesion to host cells, migration of phagocytic cells from circulation, and cell-cell interaction leading to granuloma development and fibrosis. Later, reduced reserve capacity of the extracellular matrix, development of interstitial fibrosis and granuloma fibrosis can lead to trophic shortage for cells within the granulomas, migration of macrophages out of them, and development of spontaneous necrosis and apoptosis typical of tuberculosis.

3D culture models of tissues under tension.

PubMed

Eyckmans, Jeroen; Chen, Christopher S

2017-01-01

Cells dynamically assemble and organize into complex tissues during development, and the resulting three-dimensional (3D) arrangement of cells and their surrounding extracellular matrix in turn feeds back to regulate cell and tissue function. Recent advances in engineered cultures of cells to model 3D tissues or organoids have begun to capture this dynamic reciprocity between form and function. Here, we describe the underlying principles that have advanced the field, focusing in particular on recent progress in using mechanical constraints to recapitulate the structure and function of musculoskeletal tissues. © 2017. Published by The Company of Biologists Ltd.

Fabrication of porous scaffolds with decellularized cartilage matrix for tissue engineering application.

PubMed

Nasiri, Bita; Mashayekhan, Shohreh

2017-07-01

Due to the avascular nature of articular cartilage, damaged tissue has little capacity for spontaneous healing. Three-dimensional scaffolds have potential for use in tissue engineering approach for cartilage repair. In this study, bovine cartilage tissue was decellularized and chemically crosslinked hybrid chitosan/extracellular matrix (ECM) scaffolds were fabricated with different ECM weight ratios by simple freeze drying method. Various properties of chitosan/ECM scaffolds such as microstructure, mechanical strength, swelling ratio, and biodegradability rate were investigated to confirm improved structural and biological characteristics of chitosan scaffolds in the presence of ECM. The results indicated that by introducing ECM to chitosan, pore sizes in scaffolds with 1% and 2% ECM decreased and thus the mechanical properties were improved. The presence of ECM in the same scaffolds also improved the swelling ratio and biodegradation rate in the hybrid scaffolds. MTT cytotoxicity assays performed on chondrocyte cells cultured on chitosan/ECM scaffolds having various amounts of ECM showed that the greatest cell attachment belongs to the sample with intermediate ECM content (2% ECM). Overall, it can be concluded from all obtained results that the prepared scaffold with intermediate concentration of ECM could be a proper candidate for use in cartilage tissue engineering. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Engineering a clinically-useful matrix for cell therapy.

PubMed

Prestwich, Glenn D

2008-01-01

The design criteria for matrices for encapsulation of cells for cell therapy include chemical, biological, engineering, marketing, regulatory, and financial constraints. What is required is a biocompatible material for culture of cells in three-dimensions (3-D) that offers ease of use, experimental flexibility to alter composition and compliance, and a composition that would permit a seamless transition from in vitro to in vivo use. The challenge is to replicate the complexity of the native extracellular matrix (ECM) environment with the minimum number of components necessary to allow cells to rebuild a given tissue. Our approach is to deconstruct the ECM to a few modular components that can be reassembled into biomimetic materials that meet these criteria. These semi-synthetic ECMs (sECMs) employ thiol-modified derivatives of hyaluronic acid (HA) that can form covalently crosslinked, biodegradable hydrogels. These sECMs are "living" biopolymers, meaning that they can be crosslinked in the presence of cells or tissues to enable cell therapy and tissue engineering. Moreover, the sECMs allow inclusion of the appropriate biological cues needed to simulate the complexity of the ECM of a given tissue. Taken together, the sECM technology offers a manufacturable, highly reproducible, flexible, FDA-approvable, and affordable vehicle for cell expansion and differentiation in 3-D.

Advanced techniques for in situ analysis of the biofilm matrix (structure, composition, dynamics) by means of laser scanning microscopy.

PubMed

Neu, Thomas R; Lawrence, John R

2014-01-01

The extracellular constituents in bioaggregates and biofilms can be imaged four dimensionally by using laser scanning microscopy. In this protocol we provide guidance on how to examine the various extracellular compartments in between microbial cells and communities associated with interfaces. The current options for fluorescence staining of matrix compounds and extracellular microhabitats are presented. Furthermore, practical aspects are discussed and useful notes are added. The chapter ends with a brief introduction to other approaches for EPS analysis and an outlook for future needs.

3D cell printing of in vitro stabilized skin model and in vivo pre-vascularized skin patch using tissue-specific extracellular matrix bioink: A step towards advanced skin tissue engineering.

PubMed

Kim, Byoung Soo; Kwon, Yang Woo; Kong, Jeong-Sik; Park, Gyu Tae; Gao, Ge; Han, Wonil; Kim, Moon-Bum; Lee, Hyungseok; Kim, Jae Ho; Cho, Dong-Woo

2018-06-01

3D cell-printing technique has been under spotlight as an appealing biofabrication platform due to its ability to precisely pattern living cells in pre-defined spatial locations. In skin tissue engineering, a major remaining challenge is to seek for a suitable source of bioink capable of supporting and stimulating printed cells for tissue development. However, current bioinks for skin printing rely on homogeneous biomaterials, which has several shortcomings such as insufficient mechanical properties and recapitulation of microenvironment. In this study, we investigated the capability of skin-derived extracellular matrix (S-dECM) bioink for 3D cell printing-based skin tissue engineering. S-dECM was for the first time formulated as a printable material and retained the major ECM compositions of skin as well as favorable growth factors and cytokines. This bioink was used to print a full thickness 3D human skin model. The matured 3D cell-printed skin tissue using S-dECM bioink was stabilized with minimal shrinkage, whereas the collagen-based skin tissue was significantly contracted during in vitro tissue culture. This physical stabilization and the tissue-specific microenvironment from our bioink improved epidermal organization, dermal ECM secretion, and barrier function. We further used this bioink to print 3D pre-vascularized skin patch able to promote in vivo wound healing. In vivo results revealed that endothelial progenitor cells (EPCs)-laden 3D-printed skin patch together with adipose-derived stem cells (ASCs) accelerates wound closure, re-epithelization, and neovascularization as well as blood flow. We envision that the results of this paper can provide an insightful step towards the next generation source for bioink manufacturing. Copyright © 2018 Elsevier Ltd. All rights reserved.

Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation.

PubMed

Li, Bojun; Lin, Zhe; Mitsi, Maria; Zhang, Yang; Vogel, Viola

2015-01-01

An increasing body of evidence suggests important roles of extracellular matrix (ECM) in regulating stem cell fate. This knowledge can be exploited in tissue engineering applications for the design of ECM scaffolds appropriate to direct stem cell differentiation. By probing the conformation of fibronectin (Fn) using fluorescence resonance energy transfer (FRET), we show here that heparin treatment of the fibroblast-derived ECM scaffolds resulted in more extended conformations of fibrillar Fn in ECM. Since heparin is a highly negatively charged molecule while fibronectin contains segments of positively charged modules, including FnIII13, electrostatic interactions between Fn and heparin might interfere with residual quaternary structure in relaxed fibronectin fibers thereby opening up buried sites. The conformation of modules FnIII12-14 in particular, which contain one of the heparin binding sites as well as binding sites for many growth factors, may be activated by heparin, resulting in alterations in growth factor binding to Fn. Indeed, upregulated osteogenic differentiation was observed when hMSCs were seeded on ECM scaffolds that had been treated with heparin and were subsequently chemically fixed. In contrast, either rigidifying relaxed fibers by fixation alone, or heparin treatment without fixation had no effect. We hypothesize that fibronectin's conformations within the ECM are activated by heparin such as to coordinate with other factors to upregulate hMSC osteogenic differentiation. Thus, the conformational changes of fibronectin within the ECM could serve as a 'converter' to tune hMSC differentiation in extracellular matrices. This knowledge could also be exploited to promote osteogenic stem cell differentiation on biomedical surfaces.

Tissue-engineered lymphatic graft for the treatment of lymphedema

PubMed Central

Kanapathy, Muholan; Patel, Nikhil M.; Kalaskar, Deepak M.; Mosahebi, Afshin; Mehrara, Babak J.; Seifalian, Alexander M.

2015-01-01

Background Lymphedema is a chronic debilitating condition and curative treatment is yet to be found. Tissue engineering approach, which combines cellular components, scaffold, and molecular signals hold great potential in the treatment of secondary lymphedema with the advent of lymphatic graft to reconstruct damaged collecting lymphatic vessel. This review highlights the ideal characteristics of lymphatic graft, the limitation and challenges faced, and the approaches in developing tissue-engineered lymphatic graft. Methods Literature on tissue engineering of lymphatic system and lymphatic tissue biology was reviewed. Results The prime challenge in the design and manufacturing of this graft is producing endothelialized conduit with intraluminal valves. Suitable scaffold material is needed to ensure stability and functionality of the construct. Endothelialization of the construct can be enhanced via biofunctionalization and nanotopography, which mimics extracellular matrix. Nanocomposite polymers with improved performance over existing biomaterials are likely to benefit the development of lymphatic graft. Conclusions With the in-depth understanding of tissue engineering, nanotechnology, and improved knowledge on the biology of lymphatic regeneration, the aspiration to develop successful lymphatic graft is well achievable. PMID:25248852

Osteochondral Interface Tissue Engineering Using Macroscopic Gradients of Bioactive Signals

PubMed Central

Dormer, Nathan H.; Singh, Milind; Wang, Limin; Berkland, Cory J.; Detamore, Michael S.

2013-01-01

Continuous gradients exist at osteochondral interfaces, which may be engineered by applying spatially patterned gradients of biological cues. In the present study, a protein-loaded microsphere-based scaffold fabrication strategy was applied to achieve spatially and temporally controlled delivery of bioactive signals in three-dimensional (3D) tissue engineering scaffolds. Bone morphogenetic protein-2 and transforming growth factor-β1-loaded poly(d,llactic- co-glycolic acid) microspheres were utilized with a gradient scaffold fabrication technology to produce microsphere-based scaffolds containing opposing gradients of these signals. Constructs were then seeded with human bone marrow stromal cells (hBMSCs) or human umbilical cord mesenchymal stromal cells (hUCMSCs), and osteochondral tissue regeneration was assessed in gradient scaffolds and compared to multiple control groups. Following a 6-week cell culture, the gradient scaffolds produced regionalized extracellular matrix, and outperformed the blank control scaffolds in cell number, glycosaminoglycan production, collagen content, alkaline phosphatase activity, and in some instances, gene expression of major osteogenic and chondrogenic markers. These results suggest that engineered signal gradients may be beneficial for osteochondral tissue engineering. PMID:20379780

Effects of cell-attachment and extracellular matrix on bone formation in vivo in collagen-hydroxyapatite scaffolds.

PubMed

Villa, Max M; Wang, Liping; Rowe, David W; Wei, Mei

2014-01-01

Cell-based tissue engineering can be used to replace missing or damaged bone, but the optimal methods for delivering therapeutic cells to a bony defect have not yet been established. Using transgenic reporter cells as a donor source, two different collagen-hydroxyapatite (HA) scaffolds, and a critical-size calvarial defect model, we investigated the effect of a cell-attachment period prior to implantation, with or without an extracellular matrix-based seeding suspension, on cell engraftment and osteogenesis. When quantitatively compared, the in-house scaffold implanted immediately had a higher mean radiopacity than in-house scaffolds incubated overnight. Both scaffold types implanted immediately had significantly higher area fractions of donor cells, while the in-house collagen-HA scaffolds implanted immediately had higher area fractions of the mineralization label compared with groups incubated overnight. When the cell loading was compared in vitro for each delivery method using the in-house scaffold, immediate loading led to higher numbers of delivered cells. Immediate loading may be preferable in order to ensure robust bone formation in vivo. The use of a secondary ECM carrier improved the distribution of donor cells only when a pre-attachment period was applied. These results have improved our understanding of cell delivery to bony defects in the context of in vivo outcomes.

Influence of residual composition on the structure and properties of extracellular matrix derived hydrogels.

PubMed

Claudio-Rizo, Jesús A; Rangel-Argote, Magdalena; Castellano, Laura E; Delgado, Jorge; Mata-Mata, José L; Mendoza-Novelo, Birzabith

2017-10-01

In this work, hydrolysates of extracellular matrix (hECM) were obtained from rat tail tendon (TR), bovine Achilles tendon (TAB), porcine small intestinal submucosa (SIS) and bovine pericardium (PB), and they were polymerized to generate ECM hydrogels. The composition of hECM was evaluated by quantifying the content of sulphated glycosaminoglycans (sGAG), fibronectin and laminin. The polymerization process, structure, physicochemical properties, in vitro degradation and biocompatibility were studied and related to their composition. The results indicated that the hECM derived from SIS and PB were significantly richer in sGAG, fibronectin and laminin, than those derived from TAB and TR. These differences in hECM composition influenced the polymerization and the structural characteristics of the fibrillar gel network. Consequently, the swelling, mechanics and degradation of the hydrogels showed a direct relationship with the remaining composition. Moreover, the cytocompatibility and the secretion of transforming growth factor beta-1 (TGF-β1) by macrophages were enhanced in hydrogels with the highest residual content of ECM biomolecules. The results of this work evidenced the role of the ECM molecules remaining after both decellularization and hydrolysis steps to produce tissue derived hydrogels with structure and properties tailored to enhance their performance in tissue engineering and regenerative medicine applications. Copyright © 2017 Elsevier B.V. All rights reserved.

In Vivo Engineering of the Vocal Fold ECM with Injectable HA Hydrogels -- Late Effects on Tissue Repair and Biomechanics in a Rabbit Model

PubMed Central

Klemuk, Sarah A.; Chen, Xia; Quinchia Johnson, Beatriz H.

2009-01-01

Objectives To determine if the utilization of injectable chemically-modified hyaluronan (HA) derivative at the time of intentional vocal fold resection may facilitate wound repair and preserve the unique viscoelastic properties of the extracellular matrix and lamina propria 6 months after treatment. Study Design Prospective, controlled animal study. Methods Twelve rabbit vocal folds were biopsied bilaterally, and the left side of vocal fold was treated with Extracel, an injectable, chemically-modified HA derivative, and the right side of vocal fold was injected with saline as control at the time of resection. Animals were sacrificed six months after biopsy and injection. Outcomes measured include transcription levels for procollagen, fibronectin, fibromodulin, TGF-β1, hyaluronan synthase and hyaluronidase and tissue biomechanics -- viscosity and elasticity. Results Extracel treated vocal folds were found to have significantly less fibrosis than saline treated controls. Extracel treated vocal folds had significantly improved biomechanical properties of elasticity and viscosity. Significantly decreased levels of fibronectin, fibromodulin, TGF-β1, procollagen I and hyaluronan synthase were measured. Conclusions Prophylactic in vivo manipulation of the extracellular matrix with an injectable HA hydrogel appears to induce vocal fold tissue regeneration to yield improved tissue composition and biomechanical properties at 6 months. PMID:20456912

Programmable Control in Extracellular Matrix-mimicking Polymer Hydrogels.

PubMed

Hof, Kevin S; Bastings, Maartje M C

2017-06-28

The extracellular matrix (ECM) and cells have a reciprocal relationship, one shapes the other and vice versa. One of the main challenges of synthetic material systems for developmental cell culturing, organoid and stem cell work includes the implementation of this reciprocal nature. The largest hurdle to achieve true cell-instructive materials in biomaterials engineering is a lack of spatial and temporal control over material properties and the display of bioactive signals compared to the natural cell environment. ECM-mimicking hydrogels have been developed using a wide range of polymers, assembly and cross-linking strategies. While our synthetic toolbox is larger than nature, often our systems underperform when compared to ECM systems with natural components like Matrigel. Material properties and three-dimensional structure ill-represent the three-dimensional ECM reciprocal nature and ligand presentation is an oversimplified version of the complexity found in nature. We hypothesize that the lack of programmable control in properties and ligand presentation forms the basis of this mismatch in performance and analyze the presence of control in current state of the art ECM-mimicking systems based on covalent, supramolecular and recombinant polymers. We conclude that through combining the dynamics of supramolecular materials, robustness from covalent systems and the programmable spatial control of bio-activation in recombinant ECM materials, the optimal synthetic artificial ECM could be assembled.

Vascularized tissue-engineered chambers promote survival and function of transplanted islets and improve glycemic control.

PubMed

Knight, K R; Uda, Y; Findlay, M W; Brown, D L; Cronin, K J; Jamieson, E; Tai, T; Keramidaris, E; Penington, A J; Rophael, J; Harrison, L C; Morrison, W A

2006-03-01

We have developed a chamber model of islet engraftment that optimizes islet survival by rapidly restoring islet-extracellular matrix relationships and vascularization. Our aim was to assess the ability of syngeneic adult islets seeded into blood vessel-containing chambers to correct streptozotocin-induced diabetes in mice. Approximately 350 syngeneic islets suspended in Matrigel extracellular matrix were inserted into chambers based on either the splenic or groin (epigastric) vascular beds, or, in the standard approach, injected under the renal capsule. Blood glucose was monitored weekly for 7 weeks, and an intraperitoneal glucose tolerance test performed at 6 weeks in the presence of the islet grafts. Relative to untreated diabetic animals, glycemic control significantly improved in all islet transplant groups, strongly correlating with islet counts in the graft (P<0.01), and with best results in the splenic chamber group. Glycemic control deteriorated after chambers were surgically removed at week 8. Immunohistochemistry revealed islets with abundant insulin content in grafts from all groups, but with significantly more islets in splenic chamber grafts than the other treatment groups (P<0.05). It is concluded that hyperglycemia in experimental type 1 diabetes can be effectively treated by islets seeded into a vascularized chamber functioning as a "pancreatic organoid."

Effects of transforming growth factor-beta1 on cell motility, collagen gel contraction, myofibroblastic differentiation, and extracellular matrix expression of human adipose-derived stem cell.

PubMed

Kakudo, Natsuko; Kushida, Satoshi; Suzuki, Kenji; Ogura, Tsunetaka; Notodihardjo, Priscilla Valentin; Hara, Tomoya; Kusumoto, Kenji

2012-12-01

Human adipose-derived stem cells (ASCs) are adult pluripotent stem cells, and their usefulness in plastic surgery has garnered attention in recent years. Although, there have been expectations that ASCs might function in wound repair and regeneration, no studies to date have examined the role of ASCs in the mechanism that promotes wound-healing. Transforming growth factor-beta1 (TGF-β1) is a strong candidate cytokine for the triggering of mesenchymal stem cell migration, construction of extracellular matrices, and differentiation of ASCs into myofibroblasts. Cell proliferation, motility, and differentiation, as well as extracellular matrix production, play an important role in wound-healing. We have evaluated the capacity of ASCs to proliferate and their potential to differentiate into phenotypic myofibroblasts, as well as their cell motility and collagen gel contraction ability, when cultured with TGF-β1. Cell motility was analyzed using a wound-healing assay. ASCs that differentiated into myofibroblasts expressed the gene for alpha-smooth muscle actin, and its protein expression was detected immunohistochemically. The extracellular matrix expression in ASCs was evaluated using real-time RT-PCR. Based on the results, we conclude that human ASCs have the potential for cell motility, extracellular matrix gene expression, gel contraction, and differentiation into myofibroblasts and, therefore, may play an important role in the wound-healing process.

Different properties of skin of different body sites: The root of keloid formation?

PubMed

Butzelaar, Liselotte; Niessen, Frank B; Talhout, Wendy; Schooneman, Dennis P M; Ulrich, Magda M; Beelen, Robert H J; Mink van der Molen, Aebele B

2017-09-01

The purpose of this study was to examine extracellular matrix composition, vascularization, and immune cell population of skin sites prone to keloid formation. Keloids remain a complex problem, posing esthetical as well as functional difficulties for those affected. These scars tend to develop at anatomic sites of preference. Mechanical properties of skin vary with anatomic location and depend largely on extracellular matrix composition. These differences in extracellular matrix composition, but also vascularization and resident immune cell populations might play a role in the mechanism of keloid formation. To examine this hypothesis, skin samples of several anatomic locations were taken from 24 human donors within zero to 36 hours after they had deceased. Collagen content and cross-links were determined through high-performance liquid chromatography. The expression of several genes, involved in extracellular matrix production and degradation, was measured by means of real-time PCR. (Immuno)histochemistry was performed to detect fibroblasts, collagen, elastin, blood vessels, Langerhans cells, and macrophages. Properties of skin of keloid predilections sites were compared to properties of skin from other locations (nonpredilection sites [NPS]). The results indicated that there are site specific variations in extracellular matrix properties (collagen and cross-links) as well as macrophage numbers. Moreover, predilection sites (PS) for keloid formation contain larger amounts of collagen compared to NPS, but decreased numbers of macrophages, in particular classically activated CD40 positive macrophages. In conclusion, the altered (histological, protein, and genetic) properties of skin of keloid PS may cause a predisposition for and contribute to keloid formation. © 2017 by the Wound Healing Society.

Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

NASA Technical Reports Server (NTRS)

Ehrhart, E. J.; Gillette, E. L.; Barcellos-Hoff, M. H.; Chaterjee, A. (Principal Investigator)

1996-01-01

High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by gamma irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin immunoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid-remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation.

Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

PubMed

Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

2011-12-01

The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

Tissue-engineered vascular grafts for use in the treatment of congenital heart disease: from the bench to the clinic and back again.

PubMed

Patterson, Joseph T; Gilliland, Thomas; Maxfield, Mark W; Church, Spencer; Naito, Yuji; Shinoka, Toshiharu; Breuer, Christopher K

2012-05-01

Since the first tissue-engineered vascular graft (TEVG) was implanted in a child over a decade ago, growth in the field of vascular tissue engineering has been driven by clinical demand for improved vascular prostheses with performance and durability similar to an autologous blood vessel. Great strides were made in pediatric congenital heart surgery using the classical tissue engineering paradigm, and cell seeding of scaffolds in vitro remained the cornerstone of neotissue formation. Our second-generation bone marrow cell-seeded TEVG diverged from tissue engineering dogma with a design that induces the recipient to regenerate vascular tissue in situ. New insights suggest that neovessel development is guided by cell signals derived from both seeded cells and host inflammatory cells that infiltrate the graft. The identification of these signals and the regulatory interactions that influence cell migration, phenotype and extracellular matrix deposition during TEVG remodeling are yielding a next-generation TEVG engineered to guide neotissue regeneration without the use of seeded cells. These developments represent steady progress towards our goal of an off-the-shelf tissue-engineered vascular conduit for pediatric congenital heart surgery.

[Three-dimensional parallel collagen scaffold promotes tendon extracellular matrix formation].

PubMed

Zheng, Zefeng; Shen, Weiliang; Le, Huihui; Dai, Xuesong; Ouyang, Hongwei; Chen, Weishan

2016-03-01

To investigate the effects of three-dimensional parallel collagen scaffold on the cell shape, arrangement and extracellular matrix formation of tendon stem cells. Parallel collagen scaffold was fabricated by unidirectional freezing technique, while random collagen scaffold was fabricated by freeze-drying technique. The effects of two scaffolds on cell shape and extracellular matrix formation were investigated in vitro by seeding tendon stem/progenitor cells and in vivo by ectopic implantation. Parallel and random collagen scaffolds were produced successfully. Parallel collagen scaffold was more akin to tendon than random collagen scaffold. Tendon stem/progenitor cells were spindle-shaped and unified orientated in parallel collagen scaffold, while cells on random collagen scaffold had disorder orientation. Two weeks after ectopic implantation, cells had nearly the same orientation with the collagen substance. In parallel collagen scaffold, cells had parallel arrangement, and more spindly cells were observed. By contrast, cells in random collagen scaffold were disorder. Parallel collagen scaffold can induce cells to be in spindly and parallel arrangement, and promote parallel extracellular matrix formation; while random collagen scaffold can induce cells in random arrangement. The results indicate that parallel collagen scaffold is an ideal structure to promote tendon repairing.

Interaction between the extracellular matrix and lymphatics - consequences for lymphangiogenesis and lymphatic function

PubMed Central

Wiig, Helge; Keskin, Doruk; Kalluri, Raghu

2014-01-01

The lymphatic system is important for body fluid balance as well as immunological surveillance. Due to the identification of new molecular markers during the last decade, there has been a recent dramatic increase in our knowledge on the molecular mechanisms involved in lymphatic vessel growth (lymphangiogenesis) and lymphatic function. Here we review data showing that although it is often overlooked, the extracellular matrix plays an important role in the generation of new lymphatic vessels as a response to physiological and pathological stimuli. Extracellular matrix-lymphatic interactions as well as biophysical characteristics of the stroma have consequences for tumor formation, growth and metastasis. During the recent years, anti-lymphangiogenesis has emerged as an additional therapeutic modality to the clinically applied anti-angiogenesis strategy. Oppositely, enhancement of lymphangiogenesis in situations of lymph accumulation is seen as a promising strategy to a set of conditions where few therapeutic avenues are available. Knowledge on the interaction between the extracellular matrix and the lymphatics may enhance our understanding of the underlying mechanisms and may ultimately lead to better therapies for conditions where reduced or increased lymphatic function is the therapeutic target PMID:20727409

Paracrine signaling in a bacterium.

PubMed

López, Daniel; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2009-07-15

Cellular differentiation is triggered by extracellular signals that cause target cells to adopt a particular fate. Differentiation in bacteria typically involves autocrine signaling in which all cells in the population produce and respond to the same signal. Here we present evidence for paracrine signaling in bacterial populations-some cells produce a signal to which only certain target cells respond. Biofilm formation in Bacillus involves two centrally important signaling molecules, ComX and surfactin. ComX triggers the production of surfactin. In turn, surfactin causes a subpopulation of cells to produce an extracellular matrix. Cells that produced surfactin were themselves unable to respond to it. Likewise, once surfactin-responsive cells commenced matrix production, they no longer responded to ComX and could not become surfactin producers. Insensitivity to ComX was the consequence of the extracellular matrix as mutant cells unable to make matrix responded to both ComX and surfactin. Our results demonstrate that extracellular signaling was unidirectional, with one subpopulation producing a signal and a different subpopulation responding to it. Paracrine signaling in a bacterial population ensures the maintenance, over generations, of particular cell types even in the presence of molecules that would otherwise cause those cells to differentiate into other cell types.

An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus.

PubMed

Dengler, Vanina; Foulston, Lucy; DeFrancesco, Alicia S; Losick, Richard

2015-12-01

Staphylococcus aureus is an important human pathogen that can form biofilms on various surfaces. These cell communities are protected from the environment by a self-produced extracellular matrix composed of proteins, DNA, and polysaccharide. The exact compositions and roles of the different components are not fully understood. In this study, we investigated the role of extracellular DNA (eDNA) and its interaction with the recently identified cytoplasmic proteins that have a moonlighting role in the biofilm matrix. These matrix proteins associate with the cell surface upon the drop in pH that naturally occurs during biofilm formation, and we found here that this association is independent of eDNA. Conversely, the association of eDNA with the matrix was dependent on matrix proteins. Both proteinase and DNase treatments severely reduced clumping of resuspended biofilms; highlighting the importance of both proteins and eDNA in connecting cells together. By adding an excess of exogenous DNA to DNase-treated biofilm, clumping was partially restored, confirming the crucial role of eDNA in the interconnection of cells. On the basis of our results, we propose that eDNA acts as an electrostatic net, interconnecting cells surrounded by positively charged matrix proteins at a low pH. Extracellular DNA (eDNA) is an important component of the biofilm matrix of diverse bacteria, but its role in biofilm formation is not well understood. Here we report that in Staphylococcus aureus, eDNA associates with cells in a manner that depends on matrix proteins and that eDNA is required to link cells together in the biofilm. These results confirm previous studies that showed that eDNA is an important component of the S. aureus biofilm matrix and also suggest that eDNA acts as an electrostatic net that tethers cells together via the proteinaceous layer of the biofilm matrix. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Substance P up-regulates matrix metalloproteinase-1 and down-regulates collagen in human lung fibroblast.

PubMed

Ramos, Carlos; Montaño, Martha; Cisneros, Jose; Sommer, Bettina; Delgado, Javier; Gonzalez-Avila, Georgina

2007-01-01

Substance P is involved in inflammatory processes, but its effect on extracellular matrix metabolism has not been studied; therefore, the authors evaluated its effect on collagen synthesis and degradation, expression of pro-alpha1(I) collagen, matrix metalloproteinase-1 and -2, and tissue inhibitor of metalloproteinase-1 and -2 in normal human lung fibroblast strains. Substance P induced a decrease in collagen biosynthesis, concomitant to a down-regulation of pro-alpha1(I) collagen mRNA. In contrast, an increase in collagen degradation was observed, accompanied with an up-regulation of matrix metalloproteinase-1. Substance P did not influence tissue inhibitor of metalloproteinase-1 and -2 or matrix metalloproteinase-2 expression. The results suggest that substance P participates in extracellular matrix metabolism.

Cell Microenvironment Engineering and Monitoring for Tissue Engineering and Regenerative Medicine: The Recent Advances

PubMed Central

Barthes, Julien; Özçelik, Hayriye; Hindié, Mathilde; Ndreu-Halili, Albana; Hasan, Anwarul

2014-01-01

In tissue engineering and regenerative medicine, the conditions in the immediate vicinity of the cells have a direct effect on cells' behaviour and subsequently on clinical outcomes. Physical, chemical, and biological control of cell microenvironment are of crucial importance for the ability to direct and control cell behaviour in 3-dimensional tissue engineering scaffolds spatially and temporally. In this review, we will focus on the different aspects of cell microenvironment such as surface micro-, nanotopography, extracellular matrix composition and distribution, controlled release of soluble factors, and mechanical stress/strain conditions and how these aspects and their interactions can be used to achieve a higher degree of control over cellular activities. The effect of these parameters on the cellular behaviour within tissue engineering context is discussed and how these parameters are used to develop engineered tissues is elaborated. Also, recent techniques developed for the monitoring of the cell microenvironment in vitro and in vivo are reviewed, together with recent tissue engineering applications where the control of cell microenvironment has been exploited. Cell microenvironment engineering and monitoring are crucial parts of tissue engineering efforts and systems which utilize different components of the cell microenvironment simultaneously can provide more functional engineered tissues in the near future. PMID:25143954

Cell microenvironment engineering and monitoring for tissue engineering and regenerative medicine: the recent advances.

PubMed

Barthes, Julien; Özçelik, Hayriye; Hindié, Mathilde; Ndreu-Halili, Albana; Hasan, Anwarul; Vrana, Nihal Engin

2014-01-01

In tissue engineering and regenerative medicine, the conditions in the immediate vicinity of the cells have a direct effect on cells' behaviour and subsequently on clinical outcomes. Physical, chemical, and biological control of cell microenvironment are of crucial importance for the ability to direct and control cell behaviour in 3-dimensional tissue engineering scaffolds spatially and temporally. In this review, we will focus on the different aspects of cell microenvironment such as surface micro-, nanotopography, extracellular matrix composition and distribution, controlled release of soluble factors, and mechanical stress/strain conditions and how these aspects and their interactions can be used to achieve a higher degree of control over cellular activities. The effect of these parameters on the cellular behaviour within tissue engineering context is discussed and how these parameters are used to develop engineered tissues is elaborated. Also, recent techniques developed for the monitoring of the cell microenvironment in vitro and in vivo are reviewed, together with recent tissue engineering applications where the control of cell microenvironment has been exploited. Cell microenvironment engineering and monitoring are crucial parts of tissue engineering efforts and systems which utilize different components of the cell microenvironment simultaneously can provide more functional engineered tissues in the near future.

Serum, liver, and lung levels of the major extracellular matrix components at the early stage of BCG-induced granulomatosis depending on the infection route.

PubMed

Kim, L B; Shkurupy, V A; Putyatina, A N

2015-01-01

Experiments on the model of mouse BCG-induced granulomatous showed that the content of glycosaminoglycans and proteoglycans in the extracellular matrix of the liver and lungs are changed at the early stages of inflammation (days 3 and 30 postinfection) before cell destruction in the organs begins. This is related to degradation of extracellular matrix structures. Their high content in the blood and interstitium probably contributes to the formation of granulomas, fibroblast proliferation and organ fibrosis. These processes depend on the infection route that determines different conditions for generalization of the inflammation process. Intravenous method of vaccine injection is preferable to use when designing the experiments simulating tuberculosis granulomatosis, especially for the analysis of its early stages.

Microfluidic vascularized bone tissue model with hydroxyapatite-incorporated extracellular matrix.

PubMed

Jusoh, Norhana; Oh, Soojung; Kim, Sudong; Kim, Jangho; Jeon, Noo Li

2015-10-21

Current in vitro systems mimicking bone tissues fail to fully integrate the three-dimensional (3D) microvasculature and bone tissue microenvironments, decreasing their similarity to in vivo conditions. Here, we propose 3D microvascular networks in a hydroxyapatite (HA)-incorporated extracellular matrix (ECM) for designing and manipulating a vascularized bone tissue model in a microfluidic device. Incorporation of HA of various concentrations resulted in ECM with varying mechanical properties. Sprouting angiogenesis was affected by mechanically modulated HA-extracellular matrix interactions, generating a model of vascularized bone microenvironment. Using this platform, we observed that hydroxyapatite enhanced angiogenic properties such as sprout length, sprouting speed, sprout number, and lumen diameter. This new platform integrates fibrin ECM with the synthetic bone mineral HA to provide in vivo-like microenvironments for bone vessel sprouting.

The Molecular Basis of Memory

PubMed Central

2012-01-01

We propose a tripartite biochemical mechanism for memory. Three physiologic components are involved, namely, the neuron (individual and circuit), the surrounding neural extracellular matrix, and the various trace metals distributed within the matrix. The binding of a metal cation affects a corresponding nanostructure (shrinking, twisting, expansion) and dielectric sensibility of the chelating node (address) within the matrix lattice, sensed by the neuron. The neural extracellular matrix serves as an electro-elastic lattice, wherein neurons manipulate multiple trace metals (n > 10) to encode, store, and decode coginive information. The proposed mechanism explains brains low energy requirements and high rates of storage capacity described in multiples of Avogadro number (NA = 6 × 1023). Supportive evidence correlates memory loss to trace metal toxicity or deficiency, or breakdown in the delivery/transport of metals to the matrix, or its degradation. Inherited diseases revolving around dysfunctional trace metal metabolism and memory dysfunction, include Alzheimer's disease (Al, Zn, Fe), Wilson’s disease (Cu), thalassemia (Fe), and autism (metallothionein). The tripartite mechanism points to the electro-elastic interactions of neurons with trace metals distributed within the neural extracellular matrix, as the molecular underpinning of “synaptic plasticity” affecting short-term memory, long-term memory, and forgetting. PMID:23050060

New intracellular activities of matrix metalloproteinases shine in the moonlight.

PubMed

Jobin, Parker G; Butler, Georgina S; Overall, Christopher M

2017-11-01

Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis.

PubMed

Al Shammari, Basim; Shiomi, Takayuki; Tezera, Liku; Bielecka, Magdalena K; Workman, Victoria; Sathyamoorthy, Tarangini; Mauri, Francesco; Jayasinghe, Suwan N; Robertson, Brian D; D'Armiento, Jeanine; Friedland, Jon S; Elkington, Paul T

2015-08-01

A central tenet of tuberculosis pathogenesis is that caseous necrosis leads to extracellular matrix destruction and bacterial transmission. We reconsider the underlying mechanism of tuberculosis pathology and demonstrate that collagen destruction may be a critical initial event, causing caseous necrosis as opposed to resulting from it. In human tuberculosis granulomas, regions of extracellular matrix destruction map to areas of caseous necrosis. In mice, transgenic expression of human matrix metalloproteinase 1 causes caseous necrosis, the pathological hallmark of human tuberculosis. Collagen destruction is the principal pathological difference between humanised mice and wild-type mice with tuberculosis, whereas the release of proinflammatory cytokines does not differ, demonstrating that collagen breakdown may lead to cell death and caseation. To investigate this hypothesis, we developed a 3-dimensional cell culture model of tuberculosis granuloma formation, using bioelectrospray technology. Collagen improved survival of Mycobacterium tuberculosis-infected cells analyzed on the basis of a lactate dehydrogenase release assay, propidium iodide staining, and measurement of the total number of viable cells. Taken together, these findings suggest that collagen destruction is an initial event in tuberculosis immunopathology, leading to caseous necrosis and compromising the immune response, revealing a previously unappreciated role for the extracellular matrix in regulating the host-pathogen interaction. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Aggrecan-based extracellular matrix shows unique cortical features and conserved subcortical principles of mammalian brain organization in the Madagascan lesser hedgehog tenrec (Echinops telfairi Martin, 1838).

PubMed

Morawski, M; Brückner, G; Jäger, C; Seeger, G; Künzle, H; Arendt, T

2010-02-03

The Madagascan tenrecs (Afrotheria), an ancient mammalian clade, are characterized by unique brain anatomy. Striking features are an expanded paleocortex but a small and poorly differentiated neocortex devoid of a distinct granular layer IV. To investigate the organization of cortical areas we analyzed extracellular matrix components in perineuronal nets (PNs) using antibodies to aggrecan, lectin staining and hyaluronan-binding protein. Selected subcortical regions were studied to correlate the cortical patterns with features in evolutionary conserved systems. In the neocortex, paleocortex and hippocampus PNs were associated with nonpyramidal neurons. Quantitative analysis in the cerebral cortex revealed area-specific proportions and laminar distribution patterns of neurons ensheathed by PNs. Cortical PNs showed divergent structural phenotypes. Diffuse PNs forming a cotton wool-like perisomatic rim were characteristic of the paleocortex. These PNs were associated with a dense pericellular plexus of calretinin-immunoreactive fibres. Clearly contoured PNs were devoid of a calretinin-positive plexus and predominated in the neocortex and hippocampus. The organization of the extracellular matrix in subcortical nuclei followed the widely distributed mammalian type. We conclude that molecular properties of the aggrecan-based extracellular matrix are conserved during evolution of mammals; however, the matrix scaffold is adapted to specific wiring patterns of cortical and subcortical neuronal networks. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Effect of chondrocyte-derived early extracellular matrix on chondrogenesis of placenta-derived mesenchymal stem cells.

PubMed

Park, Yong-Beom; Seo, Sinji; Kim, Jin-A; Heo, Jin-Chul; Lim, Young-Cheol; Ha, Chul-Won

2015-06-24

The extracellular matrix (ECM) surrounding cells contains a variety of proteins that provide structural support and regulate cellular functions. Previous studies have shown that decellularized ECM isolated from tissues or cultured cells can be used to improve cell differentiation in tissue engineering applications. In this study we evaluated the effect of decellularized chondrocyte-derived ECM (CDECM) on the chondrogenesis of human placenta-derived mesenchymal stem cells (hPDMSCs) in a pellet culture system. After incubation with or without chondrocyte-derived ECM in chondrogenic medium for 1 or 3 weeks, the sizes and wet masses of the cell pellets were compared with untreated controls (hPDMSCs incubated in chondrogenic medium without chondrocyte-derived ECM). In addition, histologic analysis of the cell pellets (Safranin O and collagen type II staining) and quantitative reverse transcription-PCR analysis of chondrogenic markers (aggrecan, collagen type II, and SOX9) were carried out. Our results showed that the sizes and masses of hPDMSC pellets incubated with chondrocyte-derived ECM were significantly higher than those of untreated controls. Differentiation of hPDMSCs (both with and without chondrocyte-derived ECM) was confirmed by Safranin O and collagen type II staining. Chondrogenic marker expression and glycosaminoglycan (GAG) levels were significantly higher in hPDMSC pellets incubated with chondrocyte-derived ECM compared with untreated controls, especially in cells precultured with chondrocyte-derived ECM for 7 d. Taken together, these results demonstrate that chondrocyte-derived ECM enhances the chondrogenesis of hPDMSCs, and this effect is further increased by preculture with chondrocyte-derived ECM. This preculture method for hPDMSC chondrogenesis represents a promising approach for cartilage tissue engineering.

Comparison of three methods for the derivation of a biologic scaffold composed of adipose tissue extracellular matrix.

PubMed

Brown, Bryan N; Freund, John M; Han, Li; Rubin, J Peter; Reing, Janet E; Jeffries, Eric M; Wolf, Mathew T; Tottey, Stephen; Barnes, Christopher A; Ratner, Buddy D; Badylak, Stephen F

2011-04-01

Extracellular matrix (ECM)-based scaffold materials have been used successfully in both preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. Results of numerous studies have shown that ECM scaffolds are capable of supporting the growth and differentiation of multiple cell types in vitro and of acting as inductive templates for constructive tissue remodeling after implantation in vivo. Adipose tissue represents a potentially abundant source of ECM and may represent an ideal substrate for the growth and adipogenic differentiation of stem cells harvested from this tissue. Numerous studies have shown that the methods by which ECM scaffold materials are prepared have a dramatic effect upon both the biochemical and structural properties of the resultant ECM scaffold material as well as the ability of the material to support a positive tissue remodeling outcome after implantation. The objective of the present study was to characterize the adipose ECM material resulting from three methods of decellularization to determine the most effective method for the derivation of an adipose tissue ECM scaffold that was largely free of potentially immunogenic cellular content while retaining tissue-specific structural and functional components as well as the ability to support the growth and adipogenic differentiation of adipose-derived stem cells. The results show that each of the decellularization methods produced an adipose ECM scaffold that was distinct from both a structural and biochemical perspective, emphasizing the importance of the decellularization protocol used to produce adipose ECM scaffolds. Further, the results suggest that the adipose ECM scaffolds produced using the methods described herein are capable of supporting the maintenance and adipogenic differentiation of adipose-derived stem cells and may represent effective substrates for use in tissue engineering and regenerative medicine approaches to soft tissue reconstruction.

3D cancer cell migration in a confined matrix

NASA Astrophysics Data System (ADS)

Alobaidi, Amani; Sun, Bo

Cancer cell migration is widely studied in 2D motion, which does not mimic the invasion processes in vivo. More recently, 3D cell migration studies have been performed. The ability of cancer cells to migrate within the extracellular matrix depends on the physical and biochemical features of the extracellular matrix. We present a model of cell motility in confined matrix geometry. The aim of the study is to study cancer migration in collagen matrix, as a soft tissue, to investigate their motility within the confined and surrounding collagen environment. Different collagen concentrations have been used to show the ability of these cancer cells to move through such a complex structure by measuring Cancer cell migration velocity as well as the displacement. Graduate student physics department.

Tissue engineering a human phalanx.

PubMed

Landis, W J; Chubinskaya, S; Tokui, T; Wada, Y; Isogai, N; Jacquet, R

2017-08-01

A principal purpose of tissue engineering is the augmentation, repair or replacement of diseased or injured human tissue. This study was undertaken to determine whether human biopsies as a cell source could be utilized for successful engineering of human phalanges consisting of both bone and cartilage. This paper reports the use of cadaveric human chondrocytes and periosteum as a model for the development of phalanx constructs. Two factors, osteogenic protein-1 [OP-1/bone morphogenetic protein-7 (BMP7)], alone or combined with insulin-like growth factor (IGF-1), were examined for their potential enhancement of chondrocytes and their secreted extracellular matrices. Design of the study included culture of chondrocytes and periosteum on biodegradable polyglycolic acid (PGA) and poly-l-lactic acid (PLLA)-poly-ε-caprolactone (PCL) scaffolds and subsequent implantation in athymic nu/nu (nude) mice for 5, 20, 40 and 60 weeks. Engineered constructs retrieved from mice were characterized with regard to genotype and phenotype as a function of developmental (implantation) time. Assessments included gross observation, X-ray radiography or microcomputed tomography, histology and gene expression. The resulting data showed that human cell-scaffold constructs could be successfully developed over 60 weeks, despite variability in donor age. Cartilage formation of the distal phalanx models enhanced with both OP-1 and IGF-1 yielded more cells and extracellular matrix (collagen and proteoglycans) than control chondrocytes without added factors. Summary data demonstrated that human distal phalanx models utilizing cadaveric chondrocytes and periosteum were successfully fabricated and OP-1 and OP-1/IGF-1 accelerated construct development and mineralization. The results suggest that similar engineering and transplantation of human autologous tissues in patients are clinically feasible. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Regulation of Extracellular Matrix Synthesis by Shell Extracts from the Marine Bivalve Pecten maximus in Human Articular Chondrocytes- Application for Cartilage Engineering.

PubMed

Bouyoucef, Mouloud; Rakic, Rodolphe; Gómez-Leduc, Tangni; Latire, Thomas; Marin, Frédéric; Leclercq, Sylvain; Carreiras, Franck; Serpentini, Antoine; Lebel, Jean-Marc; Galéra, Philippe; Legendre, Florence

2018-04-07

The shells of the bivalve mollusks are organo-mineral structures predominantly composed of calcium carbonate, but also of a minor organic matrix, a mixture of proteins, glycoproteins, and polysaccharides. These proteins are involved in mineral deposition and, more generally, in the spatial organization of the shell crystallites in well-defined microstructures. In this work, we extracted different organic shell extracts (acid-soluble matrix, acid-insoluble matrix, water-soluble matrix, guanidine HCl/EDTA-extracted matrix, referred as ASM, AIM, WSM, and EDTAM, respectively) from the shell of the scallop Pecten maximus and studied their biological activities on human articular chondrocytes (HACs). We found that these extracts differentially modulate the biological activities of HACs, depending on the type of extraction and the concentration used. Furthermore, we showed that, unlike ASM and AIM, WSM promotes maintenance of the chondrocyte phenotype in monolayer culture. WSM increased the expression of chondrocyte-specific markers (aggrecan and type II collagen), without enhancing that of the main chondrocyte dedifferentiation marker (type I collagen). We also demonstrated that WSM could favor redifferentiation of chondrocyte in collagen sponge scaffold in hypoxia. Thus, this study suggests that the organic matrix of Pecten maximus, particularly WSM, may contain interesting molecules with chondrogenic effects. Our research emphasizes the potential use of WSM of Pecten maximus for cell therapy of cartilage.

Effect of transforming growth factor-beta and growth differentiation factor-5 on proliferation and matrix production by human bone marrow stromal cells cultured on braided poly lactic-co-glycolic acid scaffolds for ligament tissue engineering.

PubMed

Jenner, J M G Th; van Eijk, F; Saris, D B F; Willems, W J; Dhert, W J A; Creemers, Laura B

2007-07-01

Tissue engineering of ligaments based on biomechanically suitable biomaterials combined with autologous cells may provide a solution for the drawbacks associated with conventional graft material. The aim of the present study was to investigate the contribution of recombinant human transforming growth factor beta 1 (rhTGF-beta1) and growth differentiation factor (GDF)-5, known for their role in connective tissue regeneration, to proliferation and matrix production by human bone marrow stromal cells (BMSCs) cultured onto woven, bioabsorbable, 3-dimensional (3D) poly(lactic-co-glycolic acid) scaffolds. Cells were cultured for 12 days in the presence or absence of these growth factors at different concentrations. Human BMSCs attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I and collagen III. No differentiation was demonstrated toward cartilage or bone tissue. The addition of rhTGF-beta1 (1-10 ng/mL) and GDF-5 (10-100 ng/mL) increased cell content (p < 0.05), but only TGF-beta1 also increased total collagen production (p < 0.05) and collagen production per cell, which is a parameter indicating differentiation. In conclusion, stimulation with rhTGF-beta1, and to a lesser extent with GDF-5, can modulate human BMSCs toward collagenous soft tissue when applied to a 3D hybrid construct. The use of growth factors could play an important role in the improvement of ligament tissue engineering.

Bone as an ion exchange system: evidence for a link between mechanotransduction and metabolic needs.

PubMed

Rubinacci, A; Covini, M; Bisogni, C; Villa, I; Galli, M; Palumbo, C; Ferretti, M; Muglia, M A; Marotti, G

2002-04-01

To detect whether the mutual interaction occurring between the osteocytes-bone lining cells system (OBLCS) and the bone extracellular fluid (BECF) is affected by load through a modification of the BECF-extracellular fluid (ECF; systemic extracellular fluid) gradient, mice metatarsal bones immersed in ECF were subjected ex vivo to a 2-min cyclic axial load of different amplitudes and frequencies. The electric (ionic) currents at the bone surface were measured by a vibrating probe after having exposed BECF to ECF through a transcortical hole. The application of different loads and different frequencies increased the ionic current in a dose-dependent manner. The postload current density subsequently decayed following an exponential pattern. Postload increment's amplitude and decay were dependent on bone viability. Dummy and static loads did not induce current density modifications. Because BECF is perturbed by loading, it is conceivable that OBLCS tends to restore BECF preload conditions by controlling ion fluxes at the bone-plasma interface to fulfill metabolic needs. Because the electric current reflects the integrated activity of OBLCS, its evaluation in transgenic mice engineered to possess genetic lesions in channels or matrix constituents could be helpful in the characterization of the mechanical and metabolic functions of bone.

Novel valve replacement with an extracellular matrix scaffold in an infant with single ventricle physiology.

PubMed

Guariento, Alvise; Burke, Redmond; Fedrigo, Marny; Angelini, Annalisa; Maschietto, Nicola; Vida, Vladimiro; Thiene, Gaetano; Stellin, Giovanni; Padalino, Massimo

2016-01-01

Valve replacement in children with functionally univentricular hearts remains challenging. The absence of small prostheses, the lack of growth, and the need for anticoagulation limit these procedures. We describe a 1-year follow-up of an extracellular matrix scaffold tube used as systemic atrio-ventricular valve in an infant. Copyright © 2015 Elsevier Inc. All rights reserved.

Development of collagen/polydopamine complexed matrix as mechanically enhanced and highly biocompatible semi-natural tissue engineering scaffold.

PubMed

Hu, Yang; Dan, Weihua; Xiong, Shanbai; Kang, Yang; Dhinakar, Arvind; Wu, Jun; Gu, Zhipeng

2017-01-01

To improve the mechanical properties and biocompatibility of collagen I matrix, a novel and facile strategy was developed to modify porcine acellular dermal matrix (PADM) via dopamine self-polymerization followed by collagen immobilization to enhance the biological, mechanical and physicochemical properties of PADM. Mechanism study indicated that the polymerization of dopamine onto PADM surface could be regulated by controlling the amount of hydrogen bonds forming between phenol hydroxyl (COH) and nitrogen atom (NCO) within collagen fibers of PADM. The investigations of surface interactions between PDA and PADM illustrated that PDA-PADM system yielded better mechanical properties, thermal stability, surface hydrophilicity and the structural integrity of PADM was maintained after dopamine coating. Furthermore, collagen (COL) was immobilized onto the fresh PDA-PADM to fabricate the collagen-PDA-PADM (COL-PDA-PADM) complexed scaffold. The MTT assay and CLSM observation showed that COL-PDA-PADM had better biocompatibility and higher cellular attachment than pure PADM and COL-PADM without dopamine coating, thus demonstrating the efficacy of PDA as the intermediate layer. Meanwhile, the expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) of COL-PDA-PADM were investigated by an in vivo study. The results revealed that COL-PDA-PADM could effectively promote bFGF and VEGF expression, possibly leading to enhancing the dura repairing process. Overall, this work contributed a new insight into the development of a semi-natural tissue engineering scaffold with high biocompatibility and good mechanical properties. Obtaining scaffolds with high biocompatibility and good mechanical properties is still one of the most challenging issues in tissue engineering. To have excellent in vitro and in vivo performance, scaffolds are desired to have similar mechanical and biological properties as the natural extracellular matrix, such as collagen based matrix. Utilizing the surface self-crosslinking and coating strategy, we successfully obtained a novel semi-natural platform with excellent biological and mechanical properties from porcine acellular dermal matrix (PADM), polydopamine and collagen. The results confirmed that this scaffold platform has very excellent cellular performance and very little toxicity/side effects in vivo. Therefore, this semi-natural scaffold may be an appropriate platform for tissue engineering and this strategy would further help to develop more robust scaffolds. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation

PubMed Central

Seper, Andrea; Fengler, Vera H I; Roier, Sandro; Wolinski, Heimo; Kohlwein, Sepp D; Bishop, Anne L; Camilli, Andrew; Reidl, Joachim; Schild, Stefan

2011-01-01

Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae. PMID:22032623

Collagenous Extracellular Matrix Biomaterials for Tissue Engineering: Lessons from the Common Sea Urchin Tissue.

PubMed

Goh, Kheng Lim; Holmes, David F

2017-04-25

Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM) of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs). The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action-the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre reinforced composites to a wide variety of collagen reinforcing (non-mutable) connective tissue, has allowed us to draw general conclusions concerning the mechanical response of the MCT at specific mechanical states, namely the stiff and complaint states. The intent of this review is to provide the latest insights, as well as identify technical challenges and opportunities, that may be useful for developing methods for effective mechanical support when adapting decellularised connective tissues from the sea urchin for tissue engineering or for the design of a synthetic analogue.

Collagenous Extracellular Matrix Biomaterials for Tissue Engineering: Lessons from the Common Sea Urchin Tissue

PubMed Central

Goh, Kheng Lim; Holmes, David F.

2017-01-01

Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM) of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs). The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action—the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre reinforced composites to a wide variety of collagen reinforcing (non-mutable) connective tissue, has allowed us to draw general conclusions concerning the mechanical response of the MCT at specific mechanical states, namely the stiff and complaint states. The intent of this review is to provide the latest insights, as well as identify technical challenges and opportunities, that may be useful for developing methods for effective mechanical support when adapting decellularised connective tissues from the sea urchin for tissue engineering or for the design of a synthetic analogue. PMID:28441344

Enhanced differentiation of dental pulp cells cultured on microtubular polymer scaffolds in vitro.

PubMed

Haeri, Morteza; Sagomonyants, Karen; Mina, Mina; Kuhn, Liisa T; Goldberg, A Jon

2017-06-01

Dental caries (tooth decay) is the most common chronic disease. Dental tissue engineering is a promising alternative approach to alleviate the shortcomings of the currently available restorative materials. Mimicking the natural extracellular matrix (ECM) could enhance the performance of tissue engineering scaffolds. In this study, we developed microtubular (~20 μm diameter) polymethyl methacrylate (PMMA) scaffolds resembling the tubular (~2.5 μm diameter) structure of dentin, the collagen-based mineralized tissue that forms the major portion of teeth, to study the effect of scaffold architecture on differentiation of mouse dental pulp cells in vitro . Flat (control), plasma-treated solid and microtubular PMMA scaffolds with densities of 240±15, 459±51 and 480±116 tubules/mm 2 were first characterized using scanning electron microscopy and contact angle measurements. Dental pulp cells were cultured on the surface of the scaffolds for up to 21 days and examined using various assays. Cell proliferation and mineralization were examined using Alamar Blue and Xylenol Orange (XO) staining assays, respectively. The differentiation of pulp cells into odontoblasts was examined by immunostaining for Nestin and by quantitative PCR analysis for dentin matrix protein 1 ( Dmp1 ), dentin sialophosphoprotein ( Dspp ) and osteocalcin ( Ocn ). Our results showed that the highest tubular density scaffolds significantly (p<0.05) enhanced differentiation of pulp cells into odontoblasts as compared to control flat scaffolds, as evidenced by increased expression of Nestin (5.4x). However, mineralization was suppressed on all surfaces, possibly due to low cell density. These results suggest that the microtubular architecture may be a desirable feature of scaffolds developed for clinical applications. Regenerative engineering of diseased or traumatized tooth structure could avoid the deficiencies of traditional dental restorative (filling) materials. Cells in the dental pulp have the potential to differentiate to dentin-producing odontoblast cells. Furthermore, cell-supporting scaffolds that mimic a natural extracellular matrix (ECM) are known to influence behavior of progenitor cells. Accordingly, we hypothesized that a dentin-like microtubular scaffold would enhance differentiation of dental pulp cells. The hypothesis was proven true and differentiation to odontoblasts increased with increasing density of the microtubules. However, mineralization was suppressed, possibly due to a low density of cells. The results demonstrate the potential benefits of a microtubular scaffold design to promote odontoblast cells for regeneration of dentin.

Kindler syndrome.

PubMed

Ashton, G H S

2004-03-01

Kindler syndrome is a rare, autosomal recessive skin fragility disorder characterized by blistering in infancy, followed by photosensitivity and progressive poikiloderma. Ultrastructural examination reveals marked basement membrane reduplication and variable levels of cleavage at the dermal-epidermal junction. The molecular pathology underlying Kindler syndrome has recently been shown to involve loss-of-function mutations in a novel gene, KIND1, encoding kindlin-1. Immunofluorescence, gene expression and cell biology studies have shown that kindlin-1 is expressed mainly in basal keratinocytes and plays a role in the attachment of the actin cytoskeleton via focal contacts to the extracellular matrix. Thus, Kindler syndrome is the first genodermatosis caused by a defect in actin-extracellular matrix linkage rather than the classic keratin-extracellular matrix linkage underlying the pathology of other inherited skin fragility disorders such as epidermolysis bullosa. This article reviews the clinical features as well as the molecular and cellular pathology of Kindler syndrome and highlights the importance of the new protein, kindlin-1, in cell-matrix adhesion and its intriguing link to photosensitivity.

Regulation of Extracellular Matrix Remodeling Proteins by Osteoblasts in Titanium Nanoparticle-Induced Aseptic Loosening Model.

PubMed

Xie, Jing; Hou, Yanhua; Fu, Na; Cai, Xiaoxiao; Li, Guo; Peng, Qiang; Lin, Yunfeng

2015-10-01

Titanium (Ti)-wear particles, formed at the bone-implant interface, are responsible for aseptic loosening, which is a main cause of total joint replacement failure. There have been many studies on Ti particle-induced function changes in mono-cultured osteoblasts and synovial cells. However, little is known on extracellular matrix remodeling displayed by osteoblasts when in coexistence with Synovial cells. To further mimic the bone-implant interface environment, we firstly established a nanoscaled-Ti particle-induced aseptic loosening system by co-culturing osteoblasts and Synovial cells. We then explored the impact of the Synovial cells on Ti particle-engulfed osteoblasts in the mimicked flamed niche. The matrix metalloproteinases and lysyl oxidases expression levels, two protein families which are critical in osseointegration, were examined under induction by tumor necrosis factor-alpha. It was found that the co-culture between the osteoblasts and Synovial cells markedly increased the migration and proliferation of the osteoblasts, even in the Ti-particle engulfed osteoblasts. Importantly, the Ti-particle engulfed osteoblasts, induced by TNF-alpha after the co-culture, enhanced the release of the matrix metalloproteinases and reduced the expressions of lysyl oxidases. The regulation of extracellular matrix remodeling at the protein level was further assessed by investigations on gene expression of the matrix metalloproteinases and lysyl oxidases, which also suggested that the regulation started at the genetic level. Our research work has therefore revealed the critical role of multi cell-type interactions in the extracellular matrix remodeling within the peri-prosthetic tissues, which provides new insights on aseptic loosening and brings new clues about incomplete osseointegration between the implantation materials and their surrounding bones.

The Extracellular Matrix of Fungal Biofilms.

PubMed

Mitchell, Kaitlin F; Zarnowski, Robert; Andes, David R

A key feature of biofilms is their production of an extracellular matrix. This material covers the biofilm cells, providing a protective barrier to the surrounding environment. During an infection setting, this can include such offenses as host cells and products of the immune system as well as drugs used for treatment. Studies over the past two decades have revealed the matrix from different biofilm species to be as diverse as the microbes themselves. This chapter will review the composition and roles of matrix from fungal biofilms, with primary focus on Candida species, Saccharomyces cerevisiae, Aspergillus fumigatus, and Cryptococcus neoformans. Additional coverage will be provided on the antifungal resistance proffered by the Candida albicans matrix, which has been studied in the most depth. A brief section on the matrix produced by bacterial biofilms will be provided for comparison. Current tools for studying the matrix will also be discussed, as well as suggestions for areas of future study in this field.

Synthesis of Macroporous Poly(dimethylsiloxane) Scaffolds for Tissue Engineering Applications

PubMed Central

Pedraza, Eileen; Brady, Ann-Christina; Fraker, Christopher A.

2015-01-01

Macroporous, biostable scaffolds with controlled porous architecture were prepared from poly(dimethylsiloxane) (PDMS) using sodium chloride particles (NaCl) and a solvent casting and particulate leaching (SCPL) technique. The effect of particulate size range and overall porosity on the resulting structure was evaluated. Results found 90% v/v scaffolds and particulate ranges above 100 µm to have the most optimal open framework and porosity. Resulting hydrophobic PDMS scaffolds were coated with fibronectin and evaluated as a platform for adherent cell culture using human mesenchymal stem cells. Biocompatibility of PDMS scaffolds was also evaluated in a rodent model, where implants were found to be highly biocompatibile and biostable, with positive extracellular matrix deposition throughout the scaffold. These results demonstrate the suitability of macroporous PDMS scaffolds for tissue engineering applications where strong integration with the host is desired. PMID:23683037

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

Fabrication of Biopolymer Nanofibers of Hyaluronic Acid via Electrospinning

NASA Astrophysics Data System (ADS)

Young, Denice; Queen, Hailey; Krause, Wendy

2006-03-01

Electrospinning is a novel technology that uses an electric field to form fibrous materials from a polymer solution. Unlike traditional spinning techniques, electrospinning can produce fibers on the order of 100 nm that can be utilized in applications where nanoscale fibers are necessary for successful implementation, including tissue engineering. Hyaluronic acid (HA) is a widely used biopolymer found in the extracellular matrix and currently marketed in medical applications for joint lubrications and tissue engineering. The high viscosity and surface tension of HA make it an unlikely candidate for electrospinning processes as viscosity is an important parameter in successful electrospinning. To promote HA fiber formation by electrospinning, the effects of salt (NaCl), which is used to reduce the viscosity of aqueous HA solutions; molecular weight of the HA; and an additional biocompatible polymer (e.g., PEO) are under investigation.

Engineering a biospecific communication pathway between cells and electrodes

NASA Astrophysics Data System (ADS)

Collier, Joel H.; Mrksich, Milan

2006-02-01

Methods for transducing the cellular activities of mammalian cells into measurable electronic signals are important in many biotechnical applications, including biosensors, cell arrays, and other cell-based devices. This manuscript describes an approach for functionally integrating cellular activities and electrical processes in an underlying substrate. The cells are engineered with a cell-surface chimeric receptor that presents the nonmammalian enzyme cutinase. Action of this cell-surface cutinase on enzyme substrate self-assembled monolayers switches a nonelectroactive hydroxyphenyl ester to an electroactive hydroquinone, providing an electrical activity that can be identified with cyclic voltammetry. In this way, cell-surface enzymatic activity is transduced into electronic signals. The development of strategies to directly interface the activities of cells with materials will be important to enabling a broad class of hybrid microsystems that combine living and nonliving components. biomaterial | extracellular matrix | signal transduction

Demineralized bone matrix fibers formable as general and custom 3D printed mold-based implants for promoting bone regeneration.

PubMed

Rodriguez, Rudy U; Kemper, Nathan; Breathwaite, Erick; Dutta, Sucharita M; Hsu, Erin L; Hsu, Wellington K; Francis, Michael P

2016-07-26

Bone repair frequently requires time-consuming implant construction, particularly when using un-formed implants with poor handling properties. We therefore developed osteoinductive, micro-fibrous surface patterned demineralized bone matrix (DBM) fibers for engineering both defect-matched and general three-dimensional implants. Implant molds were filled with demineralized human cortical bone fibers there were compressed and lyophilized, forming mechanically strong shaped DBM scaffolds. Enzyme linked immunosorbent assays and mass spectrometry confirmed that DBM fibers contained abundant osteogenic growth factors (bone morphogenetic proteins, insulin-like growth factor-I) and extracellular matrix proteins. Mercury porosimetry and mechanical testing showed interconnected pores within the mechanically stable, custom DBM fiber scaffolds. Mesenchymal stem cells readily attached to the DBM and showed increasing metabolic activity over time. DBM fibers further increased alkaline phosphatase activity in C2C12 cells. In vivo, DBM implants elicited osteoinductive potential in a mouse muscle pouch, and also promoted spine fusion in a rat arthrodesis model. DBM fibers can be engineered into custom-shaped, osteoinductive and osteoconductive implants with potential for repairing osseous defects with precise fitment, potentially reducing operating time. By providing pre-formed and custom implants, this regenerative allograft may improve patient outcomes following surgical bone repair, while further advancing personalized orthopedic and craniomaxillofacial medicine using three-dimensional-printed tissue molds.

The role of laminins in cartilaginous tissues: from development to regeneration.

PubMed

Sun, Y; Wang, T L; Toh, W S; Pei, M

2017-07-21

As a key molecule of the extracellular matrix, laminin provides a delicate microenvironment for cell functions. Recent findings suggest that laminins expressed by cartilage-forming cells (chondrocytes, progenitor cells and stem cells) could promote chondrogenesis. However, few papers outline the effect of laminins on providing a favorable matrix microenvironment for cartilage regeneration. In this review, we delineated the expression of laminins in hyaline cartilage, fibrocartilage and cartilage-like tissue (nucleus pulposus) throughout several developmental stages. We also examined the effect of laminins on the biological activities of chondrocytes, including adhesion, migration and survival. Furthermore, we scrutinized the potential influence of various laminin isoforms on cartilage-forming cells' proliferation and chondrogenic differentiation. With this information, we hope to facilitate the understanding of the spatial and temporal interactions between cartilage-forming cells and laminin microenvironment to eventually advance cell-based cartilage engineering and regeneration.

Fibrillar films obtained from sodium soap fibers and polyelectrolyte multilayers.

PubMed

Zawko, Scott A; Schmidt, Christine E

2011-08-01

An objective of tissue engineering is to create synthetic polymer scaffolds with a fibrillar microstructure similar to the extracellular matrix. Here, we present a novel method for creating polymer fibers using the layer-by-layer method and sacrificial templates composed of sodium soap fibers. Soap fibers were prepared from neutralized fatty acids using a sodium chloride crystal dissolution method. Polyelectrolyte multilayers (PEMs) of polystyrene sulfonate and polyallylamine hydrochloride were deposited onto the soap fibers, crosslinked with glutaraldehyde, and then the soap fibers were leached with warm water and ethanol. The morphology of the resulting PEM structures was a dense network of fibers surrounded by a nonfibrillar matrix. Microscopy revealed that the PEM fibers were solid structures, presumably composed of polyelectrolytes complexed with residual fatty acids. These fibrillar PEM films were found to support the attachment of human dermal fibroblasts. Copyright © 2011 Wiley Periodicals, Inc.

Endosteal-like extracellular matrix expression on melt electrospun written scaffolds.

PubMed

Muerza-Cascante, Maria Lourdes; Shokoohmand, Ali; Khosrotehrani, Kiarash; Haylock, David; Dalton, Paul D; Hutmacher, Dietmar W; Loessner, Daniela

2017-04-01

Tissue engineering technology platforms constitute a unique opportunity to integrate cells and extracellular matrix (ECM) proteins into scaffolds and matrices that mimic the natural microenvironment in vitro. The development of tissue-engineered 3D models that mimic the endosteal microenvironment enables researchers to discover the causes and improve treatments for blood and immune-related diseases. The aim of this study was to establish a physiologically relevant in vitro model using 3D printed scaffolds to assess the contribution of human cells to the formation of a construct that mimics human endosteum. Melt electrospun written scaffolds were used to compare the suitability of primary human osteoblasts (hOBs) and placenta-derived mesenchymal stem cells (plMSCs) in (non-)osteogenic conditions and with different surface treatments. Using osteogenic conditions, hOBs secreted a dense ECM with enhanced deposition of endosteal proteins, such as fibronectin and vitronectin, and osteogenic markers, such as osteopontin and alkaline phosphatase, compared to plMSCs. The expression patterns of these proteins were reproducibly identified in hOBs derived from three individual donors. Calcium phosphate-coated scaffolds induced the expression of osteocalcin by hOBs when maintained in osteogenic conditions. The tissue-engineered endosteal microenvironment supported the growth and migration of primary human haematopoietic stem cells (HSCs) when compared to HSCs maintained using tissue culture plastic. This 3D testing platform represents an endosteal bone-like tissue and warrants future investigation for the maintenance and expansion of human HSCs. This work is motivated by the recent interest in melt electrospinning writing, a 3D printing technique used to produce porous scaffolds for biomedical applications in regenerative medicine. Our team has been among the pioneers in building a new class of melt electrospinning devices for scaffold-based tissue engineering. These scaffolds allow structural support for various cell types to invade and deposit their own ECM, mimicking a characteristic 3D microenvironment for experimental studies. We used melt electrospun written polycaprolactone scaffolds to develop an endosteal bone-like tissue that promotes the growth of HSCs. We combine tissue engineering concepts with cell biology and stem cell research to design a physiologically relevant niche that is of prime interest to the scientific community. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Study of the relationship between mononuclear inflammatory infiltrate and Ki-67 and basement membrane and extracellular matrix protein expression in radicular cysts.

PubMed

Mourão, R V C; Júnior, E C Pinheiro; Barros Silva, P G; Turatti, E; Mota, M R L; Alves, A P N N

2016-05-01

To evaluate the relationship between mononuclear inflammatory infiltrate and the expression of a proliferative immunomarker (Ki-67) as well as to evaluate basement membrane and extracellular matrix proteins (laminin and collagen type IV) in radicular cysts and dentigerous cysts (DC). Immunohistochemical analyses were performed in heavily inflamed radicular cysts (HIRC), slightly inflamed radicular cysts (SIRC) and DC (n = 20) using Ki-67 (Dako(®) , 1 : 50), anticollagen type IV (DBS(®) , 1 : 40) and antilaminin (DBS(®) , 1 : 20). The data were analysed using anova/Tukey's test (Ki-67) and Kruskal-Wallis/Dunn's test (collagen type IV and laminin) (P < 0.05). The immunoexpression of Ki-67 was significantly greater in the SIRC group compared with the HIRC and DC (P = 0.0040). Likewise, the immunoexpression of collagen type IV in the basement membrane of the SIRC group was significantly more continuous (P = 0.0475) than in the HIRC group. DC had significantly less collagen type IV in extracellular matrix immunoexpression than HIRC and SIRC (P = 0.0246). Laminin was absent in the basement membrane in the SIRC and DC groups, and the extracellular matrix of the HIRC was weak and punctate. The presence of inflammatory factors in the radicular cyst wall modified the expression of proliferation factors in the epithelial lining and the expression of collagen type IV and laminin in the basement membrane, but did not modify extracellular matrix behaviour in radicular cysts. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

The extracellular matrix of rat pacinian corpuscles: an analysis of its fine structure.

PubMed

Dubový, P; Bednárová, J

1999-12-01

The Pacinian corpuscle consists of a sensory axon terminal that is enveloped by two different structures, the inner core and the capsule. Since proteoglycans are extremely water soluble and are extracted by conventional methods for electron microscopy, the current picture of the structural composition of the extracellular matrix in the inner core and the capsule of the Pacinian corpuscle is incomplete. To study the structural composition of the extracellular matrix of the Pacinian corpuscles, cationic dyes (ruthenium red, alcian blue, acridine orange) and tannic acid were applied simultaneously with the aldehyde fixation. The interosseal Pacinian corpuscles of the rat were fixed either in 2% formaldehyde and 1.5% glutaraldehyde, with the addition of one of these cationic dyes or, in Zamboni's fixative, with tannic acid added. The cationic dyes and tannic acid revealed a different structural pattern of proteoglycans in the extracellular matrix in the inner core and in the capsule of the rat Pacinian corpuscles. The inner core surrounding the sensory axon terminal is a compartment containing proteoglycans that were distributed not only in the extracellular matrix but also in the cytoplasm of the lamellae. In addition, this excitable domain was separated from the capsular fluid by a thick layer of proteoglycans on its surface. An enlarged interlamellar space of the capsule contained large amounts of proteoglycans that were removed by digestion with chondroitinase-ABC. Ruthenium red and alcian blue provided only electron dense granules, probably corresponding to collapsed monomeric proteoglycan molecules. Acridine orange and tannic acid preserved proteoglycans very well and made it possible to visualize them as "bottlebrush" structures in the electron microscope. These results show that the inner core and the capsule of rat Pacinian corpuscles have different structural patterns of proteoglycans, which are probably involved in different functions.

Inhibitory effect of OPC-15161, a component of fungus Thielavia minor, on proliferation and extracellular matrix production of rat cultured hepatic stellate cells.

PubMed

Sugawara, H; Ueno, T; Torimura, T; Inuzuka, S; Tanikawa, K

1998-03-01

A component of fungus Thielavia minor, OPC-15161, has been shown to inhibit the proliferation and extracellular matrix production of extracellular matrix-producing mesangial cells in the kidney in vivo. In this study, we examined the effects of OPC-15161 on the proliferation and extracellular matrix production of rat cultured hepatic stellate cells (HSCs). To determine the effect of OPC-15161 on proliferation of HSCs, the cell number and the uptake of [3H]thymidine were investigated in the presence and absence of interleukin-1beta (IL-1beta). IL-1beta significantly increased the uptake of [3H]thymidine in the HSCs, and the addition of OPC-15161 inhibited the uptake in a dose-dependent manner. The cell number of HSCs was also increased by IL-1beta, which was inhibited by OPC-15161. Production of extracellular matrix by OPC-15161 was studied by the production of [3H]-hydroxyproline in the presence and absence of transforming growth factor-beta1 (TGF-beta1). TGF-beta1 significantly increased the production of [3H]-hydroxyproline in the cells, whereas the addition of OPC-15161 inhibited this effect dose dependently. We also investigated the effects of OPC-15161 on Ca2+ mobilization and measured D-myo-inositol 1,4,5-triphosphate (IP3) in the HSCs. IL-1beta induced the increase of intracellular Ca2+ and IP3 concentrations in the HSCs, which were decreased by OPC-15161. Based on these results, we conclude that OPC-1 5161 inhibited the proliferation and production of hydroxyproline in cultured rat HSCs, and thus, it may have a role in prevention of liver fibrosis in vivo.

Escherichia coli biofilms have an organized and complex extracellular matrix structure.

PubMed

Hung, Chia; Zhou, Yizhou; Pinkner, Jerome S; Dodson, Karen W; Crowley, Jan R; Heuser, John; Chapman, Matthew R; Hadjifrangiskou, Maria; Henderson, Jeffrey P; Hultgren, Scott J

2013-09-10

Bacterial biofilms are ubiquitous in nature, and their resilience is derived in part from a complex extracellular matrix that can be tailored to meet environmental demands. Although common developmental stages leading to biofilm formation have been described, how the extracellular components are organized to allow three-dimensional biofilm development is not well understood. Here we show that uropathogenic Escherichia coli (UPEC) strains produce a biofilm with a highly ordered and complex extracellular matrix (ECM). We used electron microscopy (EM) techniques to image floating biofilms (pellicles) formed by UPEC. EM revealed intricately constructed substructures within the ECM that encase individual, spatially segregated bacteria with a distinctive morphology. Mutational and biochemical analyses of these biofilms confirmed curli as a major matrix component and revealed important roles for cellulose, flagella, and type 1 pili in pellicle integrity and ECM infrastructure. Collectively, the findings of this study elucidated that UPEC pellicles have a highly organized ultrastructure that varies spatially across the multicellular community. Bacteria can form biofilms in diverse niches, including abiotic surfaces, living cells, and at the air-liquid interface of liquid media. Encasing these cellular communities is a self-produced extracellular matrix (ECM) that can be composed of proteins, polysaccharides, and nucleic acids. The ECM protects biofilm bacteria from environmental insults and also makes the dissolution of biofilms very challenging. As a result, formation of biofilms within humans (during infection) or on industrial material (such as water pipes) has detrimental and costly effects. In order to combat bacterial biofilms, a better understanding of components required for biofilm formation and the ECM is required. This study defined the ECM composition and architecture of floating pellicle biofilms formed by Escherichia coli.

Raloxifene microsphere-embedded collagen/chitosan/β-tricalcium phosphate scaffold for effective bone tissue engineering.

PubMed

Zhang, Ming-Lei; Cheng, Ji; Xiao, Ye-Chen; Yin, Ruo-Feng; Feng, Xu

2017-02-25

Engineering novel scaffolds that can mimic the functional extracellular matrix (ECM) would be a great achievement in bone tissue engineering. This paper reports the fabrication of novel collagen/chitosan/β-tricalcium phosphate (CCTP) based tissue engineering scaffold. In order to improve the regeneration ability of scaffold, we have embedded raloxifene (RLX)-loaded PLGA microsphere in the CCTP scaffold. The average pore of scaffold was in the range of 150-200μm with ideal mechanical strength and swelling/degradation characteristics. The release rate of RLX from the microsphere (MS) embedded scaffold was gradual and controlled. Also a significantly enhanced cell proliferation was observed in RLX-MS exposed cell group suggesting that microsphere/scaffold could be an ideal biomaterial for bone tissue engineering. Specifically, RLX-MS showed a significantly higher Alizarin red staining indicating the higher mineralization capacity of this group. Furthermore, a high alkaline phosphatase (ALP) activity for RLX-MS exposed group after 15days incubation indicates the bone regeneration capacity of MC3T3-E1 cells. Overall, present study showed that RLX-loaded microsphere embedded scaffold has the promising potential for bone tissue engineering applications. Copyright © 2016. Published by Elsevier B.V.

Research progress on reconstruction of meniscus in tissue engineering.

PubMed

Zhang, Yu; Li, Pengsong; Wang, Hai; Wang, Yiwei; Song, Kedong; Li, Tianqing

2017-05-01

Meniscus damages are most common in sports injuries and aged knees. One third of meniscus lesions are known as white-white zone or nonvascular zones, which are composed of chondrocyte and extracellular matrix composition only. Due to low vascularization the ability of regeneration in such zones is inherently limited, leading to impossible self-regeneration post damage. Meniscus tissue engineering is known for emerging techniques for treating meniscus damage, but there are questions that need to be answered, including an optimal and suitable cell source, the usability of growth factor, the selectivity of optimal biomaterial scaffolds as well as the technology for improving partial reconstruction of meniscus tears. This review focuses on current research on the in vitro reconstruction of the meniscus using tissue engineering methods with the expectation to develop a series of tissue engineering meniscus products for the benefit of sports injuries. With rapid growth of clinical demand, the key breakthrough of meniscus tissue engineering research foundation is enlarged to a great extent. This review discusses aspects of meniscus tissue engineering, which is relative to the clinical treatment of meniscus injuries for further support and establishment of fundamental and clinical studies.

Decellularized material as scaffolds for tissue engineering studies in long gap esophageal atresia.

PubMed

Lee, Esmond; Milan, Anna; Urbani, Luca; De Coppi, Paolo; Lowdell, Mark W

2017-05-01

Esophageal atresia refers to an anomaly in foetal development in which the esophagus terminates in a blind end. Whilst surgical correction is achievable in most patients, when a long gap is present it still represents a major challenge associated with higher morbidity and mortality. In this context, tissue engineering could represent a successful alternative to restore oesophageal function and structure. Naturally derived biomaterials made of decellularized tissues retain native extracellular matrix architecture and composition, providing a suitable bed for the anchorage and growth of relevant cell types. Areas covered: This review outlines the various strategies and challenges in esophageal tissue engineering, highlighting the evolution of ideas in the development of decellularized scaffolds for clinical use. It explores the interplay between clinical needs, ethical dilemmas, and manufacturing challenges in the development of a tissue engineered decellularized scaffold for oesophageal atresia. Expert opinion: Current progress on oesophageal tissue engineering has enabled effective repair of patch defects, whilst the development of a full circumferential construct remains a challenge. Despite the different approaches available and the improvements achieved, a gold standard for fully functional tissue engineered oesophageal constructs has not been defined yet.

Characterization of cell surface and extracellular matrix remodeling of Azospirillum brasilense chemotaxis-like 1 signal transduction pathway mutants by atomic force microscopy.

PubMed

Edwards, Amanda Nicole; Siuti, Piro; Bible, Amber N; Alexandre, Gladys; Retterer, Scott T; Doktycz, Mitchel J; Morrell-Falvey, Jennifer L

2011-01-01

To compete in complex microbial communities, bacteria must sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the regulation of multiple behaviors in response to changes in the environment, including motility patterns, exopolysaccharide production, and cell-to-cell interactions. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting flocculation. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Whereas the wild-type strain produces a smooth mucosal extracellular matrix after 24 h, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition, lectin-binding assays, and comparison of lipopolysaccharides profiles suggest that the extracellular matrix differs between the cheA1 and the cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition. FEMS Microbiology Letters © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original US government works.

Printing of Patterned, Engineered E. coli Biofilms with a Low-Cost 3D Printer.

PubMed

Schmieden, Dominik T; Basalo Vázquez, Samantha J; Sangüesa, Héctor; van der Does, Marit; Idema, Timon; Meyer, Anne S

2018-05-18

Biofilms can grow on virtually any surface available, with impacts ranging from endangering the lives of patients to degrading unwanted water contaminants. Biofilm research is challenging due to the high degree of biofilm heterogeneity. A method for the production of standardized, reproducible, and patterned biofilm-inspired materials could be a boon for biofilm research and allow for completely new engineering applications. Here, we present such a method, combining 3D printing with genetic engineering. We prototyped a low-cost 3D printer that prints bioink, a suspension of bacteria in a solution of alginate that solidifies on a calcium-containing substrate. We 3D-printed Escherichia coli in different shapes and in discrete layers, after which the cells survived in the printing matrix for at least 1 week. When printed bacteria were induced to form curli fibers, the major proteinaceous extracellular component of E. coli biofilms, they remained adherent to the printing substrate and stably spatially patterned even after treatment with a matrix-dissolving agent, indicating that a biofilm-mimicking structure had formed. This work is the first demonstration of patterned, biofilm-inspired living materials that are produced by genetic control over curli formation in combination with spatial control by 3D printing. These materials could be used as living, functional materials in applications such as water filtration, metal ion sequestration, or civil engineering, and potentially as standardizable models for certain curli-containing biofilms.

Regulation of Corneal Stroma Extracellular Matrix Assembly

PubMed Central

Chen, Shoujun; Mienaltowski, Michael J.; Birk, David E.

2014-01-01

The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus. PMID:25819456

Decellularized Cartilage May Be a Chondroinductive Material for Osteochondral Tissue Engineering

PubMed Central

Sutherland, Amanda J.; Beck, Emily C.; Dennis, S. Connor; Converse, Gabriel L.; Hopkins, Richard A.; Berkland, Cory J.; Detamore, Michael S.

2015-01-01

Extracellular matrix (ECM)-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were cultured in cell pellets containing cells only (control), chondrogenic differentiation medium (TGF-β), chemically decellularized cartilage particles (DCC), or physically devitalized cartilage particles (DVC). The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG) within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-β in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the ‘raw material’ building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration. PMID:25965981

Biomechanical regulation of cell orientation and fate

PubMed Central

Lopez, JI; Mouw, JK; Weaver, VM

2009-01-01

Biomechanical regulation of tumor phenotypes have been noted for several decades, yet the function of mechanics in the co-evolution of the tumor epithelium and altered cancer extracellular matrix has not been appreciated until fairly recently. In this review, we examine the dynamic interaction between the developing epithelia and the extracellular matrix, and discuss how similar interactions are exploited by the genetically modified epithelium during tumor progression. We emphasize the process of mechanoreciprocity, which is a phenomenon observed during epithelial transformation, in which tension generated within the extracellular microenvironment induce and cooperate with opposing reactive forces within transformed epithelium to drive tumor progression and metastasis. We highlight the importance of matrix remodeling, and present a new, emerging paradigm that underscores the importance of tissue morphology as a key regulator of epithelial cell invasion and metastasis. PMID:19029939

The Interplay of Dental Pulp Stem Cells and Endothelial Cells in an Injectable Peptide Hydrogel on Angiogenesis and Pulp Regeneration In Vivo

PubMed Central

Dissanayaka, Waruna Lakmal; Hargreaves, Kenneth M.; Jin, Lijian; Samaranayake, Lakshman P.

2015-01-01

Securing an adequate blood supply for the survival of cell transplants is critical for a successful outcome in tissue engineering. Interactions between endothelial and progenitor/stem cells are important for vascularization of regenerating tissue. Recently, self-assembling peptide nanofibers were described as a promising environment for pulp regeneration due to their synthetic nature and controlled physicochemical properties. In this study, the peptide hydrogel PuraMatrix™ was used as a scaffold system to investigate the role of dental pulp stem cells (DPSCs) in triggering angiogenesis and the potential for regenerating vascularized pulp in vivo. Human umbilical vein endothelial cells (HUVECs), DPSCs, or cocultures of both cell types were encapsulated in three-dimensional PuraMatrix. The peptide nanofiber microenvironment supported cell survival, cell migration, and capillary network formation in the absence of exogenous growth factors. DPSCs increased early vascular network formation by facilitating the migration of HUVECs and by increasing vascular endothelial growth factor (VEGF) expression. Both the DPSC-monoculture and coculture groups exhibited vascularized pulp-like tissue with patches of osteodentin after transplantation in mice. The cocultured groups exhibited more extracellular matrix, vascularization, and mineralization than the DPSC-monocultures in vivo. The DPSCs play a critical role in initial angiogenesis, whereas coordinated efforts by the HUVECs and DPSCs are required to achieve a balance between extracellular matrix deposition and mineralization. The findings of this study also highlighted the importance of a microenvironment that supports cell–cell interactions and cell migration, which contribute to successful dental pulp regeneration. PMID:25203774

Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms.

PubMed

Sivakumar, Krishnakumar; Mukherjee, Manisha; Cheng, Hsin-I; Zhang, Yingdan; Ji, Lianghui; Cao, Bin

2015-03-01

Biofilms are the most ubiquitous and resilient form of microbial life on earth. One most important feature of a biofilm is the presence of a self-produced matrix, which creates highly heterogeneous and dynamic microenvironments within biofilms. Redox status in biofilm microenvironments plays a critical role in biofilm development and function. However, there is a lack of non-intrusive tools to quantify extracellular redox status of microenvironments within a biofilm matrix. In this study, using Shewanella oneidensis as a model organism, we demonstrated a novel approach to monitor extracellular redox status in biofilm microenvironments. Specifically, we displayed a redox sensitive fluorescence protein roGFP onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed roGFP as a sensor to quantify the extracellular redox status in the matrix of S. oneidensis biofilms. The fusion of roGFP into BpfA has no negative impacts on cell growth and biofilm formation. Upon exposure to oxidizing agents such as H2 O2 , Ag(+) , and SeO3 (2-) , S. oneidensis BpfA-roGFP cells exhibited a characteristic fluorescence of roGFP. Proteinase treatment assay and super-resolution structured illumination microscopy confirmed the surface localization of BpfA-roGFP. We further used the surface displayed roGFP monitored the extracellular redox status in the matrix at different depths of a biofilm exposed to H2 O2 . This study provides a novel approach to non-invasively monitor extracellular redox status in microenvironments within biofilms, which can be used to understand redox responses of biofilms to environmental perturbations. © 2014 Wiley Periodicals, Inc.

Quantification of focal adhesion dynamics of cell movement based on cell-induced collagen matrix deformation using second-harmonic generation microscopy.

PubMed

Kang, Yong Guk; Jang, Hwanseok; Yang, Taeseok Daniel; Notbohm, Jacob; Choi, Youngwoon; Park, Yongdoo; Kim, Beop-Min

2018-06-01

Mechanical interactions of living cells with the surrounding environment via focal adhesion (FA) in three dimensions (3-D) play a key role in dynamic biological events, such as tissue regeneration, wound healing, and cancer invasion. Recently, several methods for observing 3-D cell-extracellular matrix (ECM) interactions have been reported, lacking solid and quantitative analysis on the dynamics of the physical interaction between the cell and the ECM. We measured the submicron displacements of ECM deformation in 3-D due to protrusion-retraction dynamics during cell migration, using second-harmonic generation without labeling the matrix structures. We then quantitatively analyzed the mechanical deformation between the ECM and the cells based on spatiotemporal volumetric correlations. The greatest deformations within the collagen matrix were found to occur at sites of colocalization of the FA site-related proteins vinculin and actin, which confirms that FA sites play a critical role in living cells within the ECM as a point for adhesion, traction, and migration. We believe that this modality can be used in studies of cell-ECM interaction during angiogenesis, wound healing, and metastasis. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development

PubMed Central

Pandya, Mirali; Lin, Tiffani; Li, Leo; Allen, Michael J.; Jin, Tianquan; Luan, Xianghong; Diekwisch, Thomas G. H.

2017-01-01

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8–16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit. PMID:29089900

Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development.

PubMed

Pandya, Mirali; Lin, Tiffani; Li, Leo; Allen, Michael J; Jin, Tianquan; Luan, Xianghong; Diekwisch, Thomas G H

2017-01-01

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8-16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit.

The Effects of Simulated Micro-gravity on Cultured Chicken Embryonic Chondrocytes

NASA Astrophysics Data System (ADS)

Li, X.; Zhang, X.; Yang, S.; Li, S.; Peidong, J.; Lin, Z.

T he effects of simulated microgravity on the microtubular system, extracellular matrix, alkaline phosphatase activity, intracellular free calcium concentration, mitochondrial ATP synthase activity and oligomycin inhibition rate of cultured chicken embryonic chondrocytes were studied with a clinostat. The microtubular content decreased. The extracellualr matrix decreased significantly after rotating, and the fibers in the extracellular matrix were more tiny and disorderly. There was a time course decrease in alkaline phosphatase activity of chondrocytes, a marker of matrix mineralization. Meanwhile a significant drop in the intracellular calcium concentration happened at the beginning of rotation. No significant changes happened in the mitochondrial ATP synthase activity and oligomycin inhibition rate. The possible mechanisms about them were discussed.

[Preparation of acellular matrix from antler cartilage and its biological compatibility].

PubMed

Fu, Jing; Zhang, Wei; Zhang, Aiwu; Ma, Lijuan; Chu, Wenhui; Li, Chunyi

2017-06-01

To study the feasibility of acellular matrix materials prepared from deer antler cartilage and its biological compatibility so as to search for a new member of the extracellular matrix family for cartilage regeneration. The deer antler mesenchymal (M) layer tissue was harvested and treated through decellular process to prepare M layer acellular matrix; histologic observation and detection of M layer acellular matrix DNA content were carried out. The antler stem cells [antlerogenic periosteum (AP) cells] at 2nd passage were labelled by fluorescent stains and by PKH26. Subsequently, the M layer acellular matrix and the AP cells at 2nd passage were co-cultured for 7 days; then the samples were transplanted into nude mice to study the tissue compatibility of M layer acellular matrix in the living animals. HE and DAPI staining confirmed that the M layer acellular matrix did not contain nucleus; the DNA content of the M layer acellular matrix was (19.367±5.254) ng/mg, which was significantly lower than that of the normal M layer tissue [(3 805.500±519.119) ng/mg]( t =12.630, P =0.000). In vitro co-culture experiments showed that AP cells could adhere to or even embedded in the M layer acellular matrix. Nude mice transplantation experiments showed that the introduced AP cells could proliferate and induce angiogenesis in the M layer acellular matrix. The deer antler cartilage acellular matrix is successfully prepared. The M layer acellular matrix is suitable for adhesion and proliferation of AP cells in vitro and in vivo , and it has the function of stimulating angiogenesis. This model for deer antler cartilage acellular matrix can be applied in cartilage tissue engineering in the future.

Basic components of connective tissues and extracellular matrix: elastin, fibrillin, fibulins, fibrinogen, fibronectin, laminin, tenascins and thrombospondins.

PubMed

Halper, Jaroslava; Kjaer, Michael

2014-01-01

Collagens are the most abundant components of the extracellular matrix and many types of soft tissues. Elastin is another major component of certain soft tissues, such as arterial walls and ligaments. Many other molecules, though lower in quantity, function as essential components of the extracellular matrix in soft tissues. Some of these are reviewed in this chapter. Besides their basic structure, biochemistry and physiology, their roles in disorders of soft tissues are discussed only briefly as most chapters in this volume deal with relevant individual compounds. Fibronectin with its muldomain structure plays a role of "master organizer" in matrix assembly as it forms a bridge between cell surface receptors, e.g., integrins, and compounds such collagen, proteoglycans and other focal adhesion molecules. It also plays an essential role in the assembly of fibrillin-1 into a structured network. Laminins contribute to the structure of the extracellular matrix (ECM) and modulate cellular functions such as adhesion, differentiation, migration, stability of phenotype, and resistance towards apoptosis. Though the primary role of fibrinogen is in clot formation, after conversion to fibrin by thrombin, it also binds to a variety of compounds, particularly to various growth factors, and as such fibrinogen is a player in cardiovascular and extracellular matrix physiology. Elastin, an insoluble polymer of the monomeric soluble precursor tropoelastin, is the main component of elastic fibers in matrix tissue where it provides elastic recoil and resilience to a variety of connective tissues, e.g., aorta and ligaments. Elastic fibers regulate activity of TGFβs through their association with fibrillin microfibrils. Elastin also plays a role in cell adhesion, cell migration, and has the ability to participate in cell signaling. Mutations in the elastin gene lead to cutis laxa. Fibrillins represent the predominant core of the microfibrils in elastic as well as non-elastic extracellular matrixes, and interact closely with tropoelastin and integrins. Not only do microfibrils provide structural integrity of specific organ systems, but they also provide a scaffold for elastogenesis in elastic tissues. Fibrillin is important for the assembly of elastin into elastic fibers. Mutations in the fibrillin-1 gene are closely associated with Marfan syndrome. Fibulins are tightly connected with basement membranes, elastic fibers and other components of extracellular matrix and participate in formation of elastic fibers. Tenascins are ECM polymorphic glycoproteins found in many connective tissues in the body. Their expression is regulated by mechanical stress both during development and in adulthood. Tenascins mediate both inflammatory and fibrotic processes to enable effective tissue repair and play roles in pathogenesis of Ehlers-Danlos, heart disease, and regeneration and recovery of musculo-tendinous tissue. One of the roles of thrombospondin 1 is activation of TGFβ. Increased expression of thrombospondin and TGFβ activity was observed in fibrotic skin disorders such as keloids and scleroderma. Cartilage oligomeric matrix protein (COMP) or thrombospondin-5 is primarily present in the cartilage. High levels of COMP are present in fibrotic scars and systemic sclerosis of the skin, and in tendon, especially with physical activity, loading and post-injury. It plays a role in vascular wall remodeling and has been found in atherosclerotic plaques as well.

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.

Red light accelerates the formation of a human dermal equivalent.

PubMed

Oliveira, Anna Cb; Morais, Thayz Fl; Bernal, Claudia; Martins, Virginia Ca; Plepis, Ana Mg; Menezes, Priscila Fc; Perussi, Janice R

2018-04-01

Development of biomaterials' substitutes and/or equivalents to mimic normal tissue is a current challenge in tissue engineering. Thus, three-dimensional cell culture using type I collagen as a polymeric matrix cell support designed to promote cell proliferation and differentiation was employed to create a dermal equivalent in vitro, as well to evaluate the photobiomodulation using red light. Polymeric matrix cell support was prepared from porcine serous collagen (1.1%) hydrolyzed for 96 h. The biomaterial exhibited porosity of 95%, a median pore of 44 µm and channels with an average distance between the walls of 78 ± 14 µm. The absorption of culture medium was 95%, and the sponge showed no cytotoxicity to Vero cells, a non-tumor cell line. Additionally, it was observed that irradiation with light at 630 nm (fluency 30 J cm -2 ) leads to the cellular photobiomodulation in both monolayer and human dermal equivalent (three-dimensional cell culture system). It was also verified that the cells cultured in the presence of the polymeric matrix cell support, allows differentiation and extracellular matrix secretion. Therefore, the results showed that the collagen sponge used as polymeric matrix cell support and the photobiomodulation at 630 nm are efficient for the production of a reconstructed human dermal equivalent in vitro.

Effects of geometry and cell-matrix interactions on the mechanics of 3D engineered microtissues

NASA Astrophysics Data System (ADS)

Bose, Prasenjit; Eyckmans, Jeroen; Chen, Christopher; Reich, Daniel

Approaches to measure and control cell-extracellular matrix (ECM) interactions in a dynamic mechanical environment are important both for studies of mechanobiology and for tissue design for bioengineering applications. We have developed a microtissue-based platform capable of controlling the ECM alignment of 3D engineered microtissues while simultaneously permitting measurement of cellular contractile forces and the tissues' mechanical properties. The tissues self-assemble from cell-laden collagen gels placed in micro-fabricated wells containing sets of flexible elastic pillars. Tissue geometry and ECM alignment are controlled by the pillars' number, shape and location. Optical tracking of the pillars provides readout of the tissues' contractile forces. Magnetic materials bound to selected pillars allow quasi-static or dynamic stretching of the tissue, and together with simultaneous measurements of the tissues' local dynamic strain field, enable characterization of the mechanical properties of the system, including their degree of anisotropy. Results on the effects of symmetry and degree of ECM alignment and organization on the role of cell-ECM interactions in determining tissue mechanical properties will be discussed. This work is supported by NSF CMMI-1463011 and CMMI-1462710.

Laser direct writing of combinatorial libraries of idealized cellular constructs: Biomedical applications

NASA Astrophysics Data System (ADS)

Schiele, Nathan R.; Koppes, Ryan A.; Corr, David T.; Ellison, Karen S.; Thompson, Deanna M.; Ligon, Lee A.; Lippert, Thomas K. M.; Chrisey, Douglas B.

2009-03-01

The ability to control cell placement and to produce idealized cellular constructs is essential for understanding and controlling intercellular processes and ultimately for producing engineered tissue replacements. We have utilized a novel intra-cavity variable aperture excimer laser operated at 193 nm to reproducibly direct write mammalian cells with micrometer resolution to form a combinatorial array of idealized cellular constructs. We deposited patterns of human dermal fibroblasts, mouse myoblasts, rat neural stem cells, human breast cancer cells, and bovine pulmonary artery endothelial cells to study aspects of collagen network formation, breast cancer progression, and neural stem cell proliferation, respectively. Mammalian cells were deposited by matrix assisted pulsed laser evaporation direct write from ribbons comprised of a UV transparent quartz coated with either a thin layer of extracellular matrix or triazene as a dynamic release layer using CAD/CAM control. We demonstrate that through optical imaging and incorporation of a machine vision algorithm, specific cells on the ribbon can be laser deposited in spatial coherence with respect to geometrical arrays and existing cells on the receiving substrate. Having the ability to direct write cells into idealized cellular constructs can help to answer many biomedical questions and advance tissue engineering and cancer research.

Transient inter-cellular polymeric linker.

PubMed

Ong, Siew-Min; He, Lijuan; Thuy Linh, Nguyen Thi; Tee, Yee-Han; Arooz, Talha; Tang, Guping; Tan, Choon-Hong; Yu, Hanry

2007-09-01

Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell-cell and cell-matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell-cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell-cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell-cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications.

SXR, A Novel Target for Breast Cancer Therapeutics

DTIC Science & Technology

2007-04-01

similar, fat-soluble, 2-methyl-1,4-naphthoqui- nones, including phylloquinone (K1), menaquinones (K2), and menadi- one ( K3 ). Vitamin K1 and vitamin K2...xenobiotic receptor SXR mediates vitamin K2- activated transcription of extracellular matrix-related genes and collagen accumulation in osteoblastic cells...2003. 89: p. 1-34. 10 Steroid and Xenobiotic Receptor SXR Mediates Vitamin K2- activated Transcription of Extracellular Matrix-related Genes and

Tenascin-C mimetic Peptide nanofibers direct stem cell differentiation to osteogenic lineage.

PubMed

Sever, Melike; Mammadov, Busra; Guler, Mustafa O; Tekinay, Ayse B

2014-12-08

Extracellular matrix contains various signals for cell surface receptors that regulate cell fate through modulation of cellular activities such as proliferation and differentiation. Cues from extracellular matrix components can be used for development of new materials to control the stem cell fate. In this study, we achieved control of stem cell fate toward osteogenic commitment by using a single extracellular matrix element despite the contradictory effect of mechanical stiffness. For this purpose, we mimicked bone extracellular matrix by incorporating functional sequence of fibronectin type III domain from native tenascin-C on self-assembled peptide nanofibers. When rat mesenchymal stem cells (rMSCs) were cultured on these peptide nanofibers, alkaline phosphatase (ALP) activity and alizarin red staining indicated osteogenic differentiation even in the absence of osteogenic supplements. Moreover, expression levels of osteogenic marker genes were significantly enhanced revealed by quantitative real-time polymerase chain reaction (qRT-PCR), which showed the remarkable bioactive role of this nanofiber system on osteogenic differentiation. Overall, these results showed that tenascin-C mimetic peptides significantly enhanced the attachment, proliferation, and osteogenic differentiation of rMSCs even in the absence of any external bioactive factors and regardless of the suitable stiff mechanical properties normally required for osteogenic differentiation. Thus, these peptide nanofibers provide a promising new platform for bone regeneration.

Hyaluronidase and Collagenase Increase the Transfection Efficiency of Gene Electrotransfer in Various Murine Tumors

PubMed Central

Golzio, Muriel; Sersa, Gregor; Escoffre, Jean-Michel; Coer, Andrej; Vidic, Suzana; Teissie, Justin

2012-01-01

Abstract One of the applications of electroporation/electropulsation in biomedicine is gene electrotransfer, the wider use of which is hindered by low transfection efficiency in vivo compared with viral vectors. The aim of our study was to determine whether modulation of the extracellular matrix in solid tumors, using collagenase and hyaluronidase, could increase the transfection efficiency of gene electrotransfer in histologically different solid subcutaneous tumors in mice. Tumors were treated with enzymes before electrotransfer of plasmid DNA encoding either green fluorescent protein or luciferase. Transfection efficiency was determined 3, 9, and 15 days posttransfection. We demonstrated that pretreatment of tumors with a combination of enzymes significantly increased the transfection efficiency of electrotransfer in tumors with a high extracellular matrix area (LPB fibrosarcoma). In tumors with a smaller extracellular matrix area and less organized collagen lattice, the increase was not so pronounced (SA-1 fibrosarcoma and EAT carcinoma), whereas in B16 melanoma, in which only traces of collagen are present, pretreatment of tumors with hyaluronidase alone was more efficient than pretreatment with both enzymes. In conclusion, our results suggest that modification of the extracellular matrix could improve distribution of plasmid DNA in solid subcutaneous tumors, demonstrated by an increase in transfection efficiency, and thus have important clinical implications for electrogene therapy. PMID:21797718

Aberrant Epicardial Adipose Tissue Extracellular Matrix Remodeling in Patients with Severe Ischemic Cardiomyopathy: Insight from Comparative Quantitative Proteomics.

PubMed

Jiang, Ding-Sheng; Zeng, Hao-Long; Li, Rui; Huo, Bo; Su, Yun-Shu; Fang, Jing; Yang, Qing; Liu, Li-Gang; Hu, Min; Cheng, Cai; Zhu, Xue-Hai; Yi, Xin; Wei, Xiang

2017-03-03

There is ample evidence indicating that epicardial adipose tissue (EAT) volume and thickness is positively associated with coronary artery disease (CAD). However, the exact pathological changes in the human EAT after myocardial ischemia remains largely unclear. In the current study, we applied a comparative quantitative proteomics to elucidate the altered biological processes in the EAT of ischemic cardiomyopathy (ICM) patients. A total of 1649 proteins were successfully quantified in our study, among which 165 proteins were significantly changed (ratio <0.8 or >1.2 fold and p < 0.05 in both repetitions) in EAT of ICM individuals. Gene ontology (GO) enrichment analysis revealed that cardiac structure and cellular metabolism were over-represented among these regulated proteins. The hypertrophic cardiomyopathy, adrenergic signaling in cardiomyocytes, extracellular matrix (ECM)-receptor interaction, phagosome, Glycolysis/Gluconeogenesis, and PPAR signaling pathway were highlighted by the KEGG PATHWAY analysis. More importantly, we found that the proteins responsible for extracellular matrix organization were dramatically increased in EAT of ICM patients. In addition, the picrosirius red (PSR) staining results showed that the collagen fiber content was prominently increased, which indicated the EAT of ICM individuals underwent extracellular matrix remodeling and ERK1/2 activation maybe responsible for these pathological changes partially.

Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice.

PubMed

Kyriakides, T R; Zhu, Y H; Yang, Z; Huynh, G; Bornstein, P

2001-10-01

The matricellular angiogenesis inhibitor, thrombospondin (TSP) 2, has been shown to be an important modulator of wound healing and the foreign body response. Specifically, TSP2-null mice display improved healing with minimal scarring and form well-vascularized foreign body capsules. In this study we performed subcutaneous implantation of sponges and investigated the resulting angiogenic and fibrogenic responses. Histological and immunohistochemical analysis of sponges, excised at 7, 14, and 21 days after implantation, revealed significant differences between TSP2-null and wild-type mice. Most notably, TSP2-null mice exhibited increased angiogenesis and fibrotic encapsulation of the sponge. However, invasion of dense tissue was compromised, even though its overall density was increased. Furthermore, histomorphometry and biochemical assays demonstrated a significant increase in the extracellular distribution of matrix metalloproteinase (MMP) 2, but no change in the levels of active transforming growth factor-beta(1). The alterations in neovascularization, dense tissue invasion, and MMP2 in TSP2-null mice coincided with the deposition of TSP2 in the extracellular matrix of wild-type animals. These observations support the proposed role of TSP2 as a modulator of angiogenesis and matrix remodeling during tissue repair. In addition, they provide in vivo evidence for a newly proposed function of TSP2 as a modulator of extracellular MMP2 levels.

Investigation of Aspergillus fumigatus biofilm formation by various “omics” approaches

PubMed Central

Muszkieta, Laetitia; Beauvais, Anne; Pähtz, Vera; Gibbons, John G.; Anton Leberre, Véronique; Beau, Rémi; Shibuya, Kazutoshi; Rokas, Antonis; Francois, Jean M.; Kniemeyer, Olaf; Brakhage, Axel A.; Latgé, Jean P.

2013-01-01

In the lung, Aspergillus fumigatus usually forms a dense colony of filaments embedded in a polymeric extracellular matrix called biofilm (BF). This extracellular matrix embeds and glues hyphae together and protects the fungus from an outside hostile environment. This extracellular matrix is absent in fungal colonies grown under classical liquid shake conditions (PL), which were historically used to understand A. fumigatus pathobiology. Recent works have shown that the fungus in this aerial grown BF-like state exhibits reduced susceptibility to antifungal drugs and undergoes major metabolic changes that are thought to be associated to virulence. These differences in pathological and physiological characteristics between BF and liquid shake conditions suggest that the PL condition is a poor in vitro disease model. In the laboratory, A. fumigatus mycelium embedded by the extracellular matrix can be produced in vitro in aerial condition using an agar-based medium. To provide a global and accurate understanding of A. fumigatus in vitro BF growth, we utilized microarray, RNA-sequencing, and proteomic analysis to compare the global gene and protein expression profiles of A. fumigatus grown under BF and PL conditions. In this review, we will present the different signatures obtained with these three “omics” methods. We will discuss the advantages and limitations of each method and their complementarity. PMID:23407341

Targeting the extracellular matrix of ovarian cancer using functionalized, drug loaded lyophilisomes.

PubMed

van der Steen, Sophieke C H A; Raavé, René; Langerak, Sjoerd; van Houdt, Laurens; van Duijnhoven, Sander M J; van Lith, Sanne A M; Massuger, Leon F A G; Daamen, Willeke F; Leenders, William P; van Kuppevelt, Toin H

2017-04-01

Epithelial ovarian cancer is characterized by a high mortality rate and is in need for novel therapeutic avenues to improve patient outcome. The tumor's extracellular matrix ("stroma") offers new possibilities for targeted drug-delivery. Recently we identified highly sulfated chondroitin sulfate (CS-E) as a component abundantly present in the ovarian cancer extracellular matrix, and as a novel target for anti-cancer therapy. Here, we report on the functionalization of drug-loaded lyophilisomes (albumin-based biocapsules) to specifically target the stroma of ovarian carcinomas with the potential to eliminate cancer cells. To achieve specific targeting, we conjugated single chain antibodies reactive with CS-E to lyophilisomes using a two-step approach comprising sortase-mediated ligation and bioorthogonal click chemistry. Antibody-functionalized lyophilisomes specifically targeted the ovarian cancer stroma through CS-E. In a CS-E rich micro-environment in vitro lyophilisomes induced cell death by extracellular release of doxorubicin which localized to the nucleus. Immunohistochemistry identified CS-E rich stroma in a variety of solid tumors other than ovarian cancer, including breast, lung and colon cancer indicating the potential versatility of matrix therapy and the use of highly sulfated chondroitin sulfates in cancer stroma as a micro-environmental hook for targeted drug delivery. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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).

Applications and Emerging Trends of Hyaluronic Acid in Tissue Engineering, as a Dermal Filler, and in Osteoarthritis Treatment

PubMed Central

Fakhari, Amir; Berkland, Cory

2013-01-01

Hyaluronic acid (HA) is a naturally occurring biodegradable polymer with a variety of applications in medicine including scaffolding for tissue engineering, dermatological fillers, and viscosupplementation for osteoarthritis treatment. HA is available in most connective tissues in body fluids such as synovial fluid and the vitreous humor of the eye. HA is responsible for several structural properties of tissues as a component of extracellular matrix (ECM) and is involved in cellular signaling. Degradation of HA is a step-wise process that can occur via enzymatic or non-enzymatic reactions. A reduction in HA mass or molecular weight via degradation or slowing of synthesis affects physical and chemical properties such as tissue volume, viscosity, and elasticity. This review addresses the distribution, turnover, and tissue-specific properties of HA. This information is used as context for considering recent products and strategies for modifying the viscoelastic properties of HA in tissue engineering, as a dermal filler, and in osteoarthritis treatment. PMID:23507088

Matrigel immobilization on the shish-kebab structured poly(ɛ-caprolactone) nanofibers for skin tissue engineering

NASA Astrophysics Data System (ADS)

Jing, Xin; Mi, Hao-Yang; Peng, Xiang-Fang; Turng, Lih-Sheng

2016-03-01

Surface properties of tissue engineering scaffolds such as topography, hydrophilicity, and functional groups play a vital role in cell adhesion, migration, proliferation, and apoptosis. First, poly(ɛ-caprolactone) (PCL) shish-kebab scaffolds (PCL-SK), which feature a three-dimensional structure comprised of electrospun PCL nanofibers covered by periodic, self-induced PCL crystal lamellae on the surface, was created to mimic the nanotopography of native collagen fibrils in the extracellular matrix (ECM). Second, matrigel was covalently immobilized on the surface of alkaline hydrolyzed PCL-SK scaffolds to enhance their hydrophilicity. This combined approach not only mimics the nanotopography of native collagen fibrils, but also simulates the surface features of collagen fibrils for cell growth. To investigate the viability of such scaffolds, HEF1 fibroblast cell assays were conducted and the results revealed that the nanotopography of the PCL-SK scaffolds facilitated cell adhesion and proliferation. The matrigel functionalization on PCL-SK scaffolds further enhanced cellular response, which suggested elevated biocompatibility and greater potential for skin tissue engineering applications.

Chitosan-collagen scaffolds with nano/microfibrous architecture for skin tissue engineering.

PubMed

Sarkar, Soumi Dey; Farrugia, Brooke L; Dargaville, Tim R; Dhara, Santanu

2013-12-01

In this study, a hierarchical nano/microfibrous chitosan/collagen scaffold that approximates structural and functional attributes of native extracellular matrix has been developed for applicability in skin tissue engineering. Scaffolds were produced by electrospinning of chitosan followed by imbibing of collagen solution, freeze-drying, and subsequent cross-linking of two polymers. Scanning electron microscopy showed formation of layered scaffolds with nano/microfibrous architechture. Physicochemical properties of scaffolds including tensile strength, swelling behavior, and biodegradability were found satisfactory for intended application. 3T3 fibroblasts and HaCaT keratinocytes showed good in vitro cellular response on scaffolds thereby indicating the matrices, cytocompatible nature. Scaffolds tested in an ex vivo human skin equivalent wound model, as a preliminary alternative to animal testing, showed keratinocyte migration and wound re-epithelization-a prerequisite for healing and regeneration. Taken together, the herein proposed chitosan/collagen scaffold, shows good potential for skin tissue engineering. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Fabrication of porous chitosan/poly(vinyl alcohol) reinforced single-walled carbon nanotube nanocomposites for neural tissue engineering.

PubMed

Shokrgozar, Mohammad Ali; Mottaghitalab, Fatemeh; Mottaghitalab, Vahid; Farokhi, Mehdi

2011-04-01

With the ability to form a nano-sized fibrous structure with large pore sizes mimicking the extracellular matrix (ECM), electrospinning was used to fabricate chitosan/poly(vinyl alcohol) nanofibers reinforced by single-walled carbon nanotube (SWNT-CS/PVA) for potential use in neural tissue engineering. Moreover, ultrasonication was performed to fabricate highly dispersed SWNT/CS solution with 7%, 12%, and 17% SWNT content prior to electrospinning process. In the present study, a number of properties of CS/PVA reinforced SWNTs nanocomposites were evaluated. The in vitro biocompatibility of the electrospun fiber mats was also assessed using human brain-derived cells and U373 cell lines. The results have shown that SWNTs as reinforcing phase can augment the morphology, porosity, and structural properties of CS/PVA nanofiber composites and thus benefit the proliferation rate of both cell types. In addition, the cells exhibit their normal morphology while integrating with surrounding fibers. The results confirmed the potential of SWNT-CS/PVA nanocomposites as scaffold for neural tissue engineering.

Smart Polymeric Hydrogels for Cartilage Tissue Engineering: A Review on the Chemistry and Biological Functions.

PubMed

Eslahi, Niloofar; Abdorahim, Marjan; Simchi, Abdolreza

2016-11-14

Stimuli responsive hydrogels (SRHs) are attractive bioscaffolds for tissue engineering. The structural similarity of SRHs to the extracellular matrix (ECM) of many tissues offers great advantages for a minimally invasive tissue repair. Among various potential applications of SRHs, cartilage regeneration has attracted significant attention. The repair of cartilage damage is challenging in orthopedics owing to its low repair capacity. Recent advances include development of injectable hydrogels to minimize invasive surgery with nanostructured features and rapid stimuli-responsive characteristics. Nanostructured SRHs with more structural similarity to natural ECM up-regulate cell-material interactions for faster tissue repair and more controlled stimuli-response to environmental changes. This review highlights most recent advances in the development of nanostructured or smart hydrogels for cartilage tissue engineering. Different types of stimuli-responsive hydrogels are introduced and their fabrication processes through physicochemical procedures are reported. The applications and characteristics of natural and synthetic polymers used in SRHs are also reviewed with an outline on clinical considerations and challenges.

Biomimetic and bioactive nanofibrous scaffolds from electrospun composite nanofibers

PubMed Central

Zhang, YZ; Su, B; Venugopal, J; Ramakrishna, S; Lim, CT

2007-01-01

Electrospinning is an enabling technology that can architecturally (in terms of geometry, morphology or topography) and biochemically fabricate engineered cellular scaffolds that mimic the native extracellular matrix (ECM). This is especially important and forms one of the essential paradigms in the area of tissue engineering. While biomimesis of the physical dimensions of native ECM’s major constituents (eg, collagen) is no longer a fabrication-related challenge in tissue engineering research, conveying bioactivity to electrospun nanofibrous structures will determine the efficiency of utilizing electrospun nanofibers for regenerating biologically functional tissues. This can certainly be achieved through developing composite nanofibers. This article gives a brief overview on the current development and application status of employing electrospun composite nanofibers for constructing biomimetic and bioactive tissue scaffolds. Considering that composites consist of at least two material components and phases, this review details three different configurations of nanofibrous composite structures by using hybridizing basic binary material systems as example. These are components blended composite nanofiber, core-shell structured composite nanofiber, and nanofibrous mingled structure. PMID:18203429

Engineering growth factors for regenerative medicine applications.

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

Mitchell, Aaron C.; Briquez, Priscilla S.; Hubbell, Jeffrey A.

Growth factors are important morphogenetic proteins that instruct cell behavior and guide tissue repair and renewal. Although their therapeutic potential holds great promise in regenerative medicine applications, translation of growth factors into clinical treatments has been hindered by limitations including poor protein stability, low recombinant expression yield, and suboptimal efficacy. This review highlights current tools, technologies, and approaches to design integrated and effective growth factor-based therapies for regenerative medicine applications. The first section describes rational and combinatorial protein engineering approaches that have been utilized to improve growth factor stability, expression yield, biodistribution, and serum half-life, or alter their cell traffickingmore » behavior or receptor binding affinity. The second section highlights elegant biomaterial-based systems, inspired by the natural extracellular matrix milieu, that have been developed for effective spatial and temporal delivery of growth factors to cell surface receptors. Although appearing distinct, these two approaches are highly complementary and involve principles of molecular design and engineering to be considered in parallel when developing optimal materials for clinical applications.« less

Introduction of N-cadherin-binding motif to alginate hydrogels for controlled stem cell differentiation.

PubMed

Lee, Jae Won; An, Hyoseok; Lee, Kuen Yong

2017-07-01

Control of stem cell fate and phenotype using biomimetic synthetic extracellular matrices (ECMs) is an important tissue engineering approach. Many studies have focused on improving cell-matrix interactions. However, proper control of cell-cell interactions using synthetic ECMs could be critical for tissue engineering, especially with undifferentiated stem cells. In this study, alginate hydrogels were modified with a peptide derived from the low-density lipoprotein receptor-related protein 5 (LRP5), which is known to bind to N-cadherin, as a cell-cell interaction motif. In vitro changes in the morphology and differentiation of mouse bone marrow stromal cells (D1 stem cells) cultured in LRP5-alginate hydrogels were investigated. LRP5-alginate gels successfully induced stem cell aggregation and enhanced chondrogenic differentiation of D1 stem cells, compared to RGD-alginate gels, at low cell density. This approach to tailoring synthetic biomimetic ECMs using cell-cell interaction motifs may be critical in tissue engineering approaches using stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro comparison of human fibroblasts from intact and ruptured ACL for use in tissue engineering.

PubMed

Brune, T; Borel, A; Gilbert, T W; Franceschi, J P; Badylak, S F; Sommer, P

2007-12-17

The present study compares fibroblasts extracted from intact and ruptured human anterior cruciate ligaments (ACL) for creation of a tissue engineered ACL-construct, made of porcine small intestinal submucosal extracellular matrix (SIS-ECM) seeded with these ACL cells. The comparison is based on histological, immunohistochemical and RT-PCR analyses. Differences were observed between cells in a ruptured ACL (rACL) and cells in an intact ACL (iACL), particularly with regard to the expression of integrin subunits and smooth muscle actin (SMA). Despite these differences in the cell source, both cell populations behaved similarly when seeded on an SIS-ECM scaffold, with similar cell morphology, connective tissue organization and composition, SMA and integrin expression. This study shows the usefulness of naturally occurring scaffolds such as SIS-ECM for the study of cell behaviour in vitro, and illustrates the possibility to use autologous cells extracted from ruptured ACL biopsies as a source for tissue engineered ACL constructs.

The extracellular matrix in myocardial injury, repair, and remodeling

PubMed Central

2017-01-01

The cardiac extracellular matrix (ECM) not only provides mechanical support, but also transduces essential molecular signals in health and disease. Following myocardial infarction, dynamic ECM changes drive inflammation and repair. Early generation of bioactive matrix fragments activates proinflammatory signaling. The formation of a highly plastic provisional matrix facilitates leukocyte infiltration and activates infarct myofibroblasts. Deposition of matricellular proteins modulates growth factor signaling and contributes to the spatial and temporal regulation of the reparative response. Mechanical stress due to pressure and volume overload and metabolic dysfunction also induce profound changes in ECM composition that contribute to the pathogenesis of heart failure. This manuscript reviews the role of the ECM in cardiac repair and remodeling and discusses matrix-based therapies that may attenuate remodeling while promoting repair and regeneration. PMID:28459429

Ultrastructure and biological function of matrix vesicles in bone mineralization.

PubMed

Hasegawa, Tomoka

2018-04-01

Bone mineralization is initiated by matrix vesicles, small extracellular vesicles secreted by osteoblasts, inducing the nucleation and subsequent growth of calcium phosphate crystals inside. Although calcium ions (Ca 2+ ) are abundant throughout the tissue fluid close to the matrix vesicles, the influx of phosphate ions (PO4 3- ) into matrix vesicles is a critical process mediated by several enzymes and transporters such as ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), ankylosis (ANK), and tissue nonspecific alkaline phosphatase (TNSALP). The catalytic activity of ENPP1 in osteoblasts generates inorganic pyrophosphate (PPi) intracellularly and extracellularly, and ANK may allow the intracellular PPi to pass through the plasma membrane to the outside of the osteoblasts. Although the extracellular PPi binds to growing hydroxyapatite crystals to prevent crystal overgrowth, TNSALP on the osteoblasts and matrix vesicles hydrolyzes PPi into PO4 3- monomers: the prevention of crystal growth is blocked, and PO4 3- monomers are supplied to matrix vesicles. In addition, PHOSPHO1 is thought to function inside matrix vesicles to catalyze phosphocoline, a constituent of the plasma membrane, consequently increasing PO4 3- in the vesicles. Accumulation of Ca 2+ and PO4 3- inside the matrix vesicles then initiates crystalline nucleation associated with the inner leaflet of the matrix vesicles. Calcium phosphate crystals elongate radially, penetrate the matrix vesicle's membrane, and finally grow out of the vesicles to form calcifying nodules, globular assemblies of needle-shaped mineral crystals retaining some of those transporters and enzymes. The subsequent growth of calcifying nodules appears to be regulated by surrounding organic compounds, finally leading to collagen mineralization.

Novel image analysis methods for quantification of in situ 3-D tendon cell and matrix strain.

PubMed

Fung, Ashley K; Paredes, J J; Andarawis-Puri, Nelly

2018-01-23

Macroscopic tendon loads modulate the cellular microenvironment leading to biological outcomes such as degeneration or repair. Previous studies have shown that damage accumulation and the phases of tendon healing are marked by significant changes in the extracellular matrix, but it remains unknown how mechanical forces of the extracellular matrix are translated to mechanotransduction pathways that ultimately drive the biological response. Our overarching hypothesis is that the unique relationship between extracellular matrix strain and cell deformation will dictate biological outcomes, prompting the need for quantitative methods to characterize the local strain environment. While 2-D methods have successfully calculated matrix strain and cell deformation, 3-D methods are necessary to capture the increased complexity that can arise due to high levels of anisotropy and out-of-plane motion, particularly in the disorganized, highly cellular, injured state. In this study, we validated the use of digital volume correlation methods to quantify 3-D matrix strain using images of naïve tendon cells, the collagen fiber matrix, and injured tendon cells. Additionally, naïve tendon cell images were used to develop novel methods for 3-D cell deformation and 3-D cell-matrix strain, which is defined as a quantitative measure of the relationship between matrix strain and cell deformation. The results support that these methods can be used to detect strains with high accuracy and can be further extended to an in vivo setting for observing temporal changes in cell and matrix mechanics during degeneration and healing. Copyright © 2017. Published by Elsevier Ltd.

Cell delivery in regenerative medicine: the cell sheet engineering approach.

PubMed

Yang, Joseph; Yamato, Masayuki; Nishida, Kohji; Ohki, Takeshi; Kanzaki, Masato; Sekine, Hidekazu; Shimizu, Tatsuya; Okano, Teruo

2006-11-28

Recently, cell-based therapies have developed as a foundation for regenerative medicine. General approaches for cell delivery have thus far involved the use of direct injection of single cell suspensions into the target tissues. Additionally, tissue engineering with the general paradigm of seeding cells into biodegradable scaffolds has also evolved as a method for the reconstruction of various tissues and organs. With success in clinical trials, regenerative therapies using these approaches have therefore garnered significant interest and attention. As a novel alternative, we have developed cell sheet engineering using temperature-responsive culture dishes, which allows for the non-invasive harvest of cultured cells as intact sheets along with their deposited extracellular matrix. Using this approach, cell sheets can be directly transplanted to host tissues without the use of scaffolding or carrier materials, or used to create in vitro tissue constructs via the layering of individual cell sheets. In addition to simple transplantation, cell sheet engineered constructs have also been applied for alternative therapies such as endoscopic transplantation, combinatorial tissue reconstruction, and polysurgery to overcome limitations of regenerative therapies and cell delivery using conventional approaches.

Natural Origin Materials for Osteochondral Tissue Engineering.

PubMed

Bonani, Walter; Singhatanadgige, Weerasak; Pornanong, Aramwit; Motta, Antonella

2018-01-01

Materials selection is a critical aspect for the production of scaffolds for osteochondral tissue engineering. Synthetic materials are the result of man-made operations and have been investigated for a variety of tissue engineering applications. Instead, the products of physiological processes and the metabolic activity of living organisms are identified as natural materials. Over the recent decades, a number of natural materials, namely, biopolymers and bioceramics, have been proposed as the main constituent of osteochondral scaffolds, but also as cell carriers and signaling molecules. Overall, natural materials have been investigated both in the bone and in the cartilage compartment, sometimes alone, but often in combination with other biopolymers or synthetic materials. Biopolymers and bioceramics possess unique advantages over their synthetic counterparts due similarity with natural extracellular matrix, the presence of cell recognition sites and tunable chemistry. However, the characteristics of natural origin materials can vary considerably depending on the specific source and extraction process. A deeper understanding of the relationship between material variability and biological activity and the definition of standardized manufacturing procedures will be crucial for the future of natural materials in tissue engineering.