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Sample records for 3d collagen gels

  1. Generation of 3D Collagen Gels with Controlled Diverse Architectures.

    PubMed

    Doyle, Andrew D

    2016-01-01

    Rat tail collagen solutions have been used as polymerizable in vitro three dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. Factors such as ECM concentration, pH, ionic concentration, and temperature can alter collagen polymerization and ECM architecture. This unit describes how to generate 3D collagen gels that have distinct architectures ranging from a highly reticular meshwork of short thin fibrils with small pores to a loose matrix consisting of stiff, parallel-bundled long fibrils by changing collagen polymerization temperature. This permits analysis of 3D cell migration in different ECM architectures found in vivo while maintaining a similar ECM concentration. Also included are collagen labeling techniques helpful for ECM visualization during live fluorescence imaging. © 2016 by John Wiley & Sons, Inc. PMID:27580704

  2. Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration.

    PubMed

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

    2015-01-01

    Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled. The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The "static bioreactor" provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues. PMID:26132527

  3. Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

    PubMed Central

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

    2015-01-01

    Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled. The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The “static bioreactor” provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues. PMID:26132527

  4. Micro-structured materials and mechanical cues in 3D collagen gels.

    PubMed

    Phillips, James B; Brown, Robert

    2011-01-01

    Collagen gels provide a versatile and widely used substrate for three-dimensional (3D) cell culture. Here we describe how cell-seeded Type-I collagen gels can be adapted to provide powerful 3D models to support a wide range of research applications where cell/substrate alignment, density, stiffness/compliance, and strain are critical factors. In their fully hydrated form, rectangular collagen gels can be tethered such that endogenous forces generated as resident cells attach to and remodel the fibrillar collagen network can align the substrate in a controllable, predictable, and quantifiable manner. By removing water from collagen gels (plastic compression), their density increases towards that of body tissues, facilitating the engineering of a range of biomimetic constructs with controllable mechanical properties. This dense collagen can be used in combination with other components to achieve a range of functional properties from controlled perfusion, or tensile/compressive strength to new micro-structures. Detailed methodology is provided for the assembly of a range of 3D collagen materials including tethered aligned hydrogels and plastic compressed constructs. A range of techniques for analysing cell behaviour within these models, including microscopy and molecular analyses are described. These systems therefore provide a highly controllable mechanical and chemical micro-environment for investigating a wide range of cellular responses. PMID:21042973

  5. Microfluidic Generation of Haptotactic Gradients through 3D Collagen Gels for Enhanced Neurite Growth

    PubMed Central

    Sundararaghavan, Harini G.; Masand, Shirley N.

    2011-01-01

    Abstract We adapted a microfluidic system used previously to generate durotactic gradients of stiffness in a 3D collagen gel, to produce haptotactic gradients of adhesive ligands through the collagen gel. Oligopeptide sequences that included bioactive peptide sequences from laminin, YIGSR, or IKVAV, were grafted separately onto type I collagen using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Solutions of peptide-grafted collagen and untreated collagen were then used as source and sink input solutions, respectively, in an H-shaped microfluidic network fabricated using traditional soft lithography. One-dimensional gradients of the peptide-grafted collagen solution were generated in the channel that connected the source and sink channels, and these gradients became immobilized upon self-assembly of the collagen into a 3D fibrillar gel. The slope and average concentration of the gradients were adjusted by changing the concentration of the source solutions and by changing the length of the cross-channel. A separate, underlying channel in the microfluidic construct allowed the introduction of a chick embryo dorsal root ganglion into the network. Neurites from these explants grew significantly longer up steep gradients of YIGSR, but shallow gradients of IKVAV in comparison to untreated collagen controls. When these two gradients were presented in combination, the bias in growth acceleration was the largest and most consistent. No differences were observed in the number of neurites choosing to grow up or down the gradients in any condition. These results suggest that the incorporation of distinct gradients of multiple bioactive ligands can improve directional acceleration of regenerating axons. PMID:21473683

  6. Toward single cell traction microscopy within 3D collagen matrices

    SciTech Connect

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-10-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels.

  7. Noncanonical roles of membranous lysyl-tRNA synthetase in transducing cell-substrate signaling for invasive dissemination of colon cancer spheroids in 3D collagen I gels

    PubMed Central

    Nam, Seo Hee; Kim, Doyeun; Lee, Mi-Sook; Lee, Doohyung; Kwak, Tae Kyoung; Kang, Minkyung; Ryu, Jihye; Kim, Hye-Jin; Song, Haeng Eun; Choi, Jungeun; Lee, Gyu-Ho; Kim, Sang-Yeob; Park, Song Hwa; Kim, Dae Gyu; Kwon, Nam Hoon; Kim, Tai Young; Thiery, Jean Paul; Kim, Sunghoon; Lee, Jung Weon

    2015-01-01

    The adhesion properties of cells are involved in tumor metastasis. Although KRS at the plasma membrane is shown important for cancer metastasis, additionally to canonical roles of cytosolic KRS in protein translation, how KRS and its downstream effectors promote the metastatic migration remains unexplored. Disseminative behaviors (an earlier metastatic process) of colon cancer cell spheroids embedded in 3D collagen gels were studied with regards to cell adhesion properties, and relevance in KRS−/+ knocked-down animal and clinical colon cancer tissues. Time-lapse imaging revealed KRS-dependent cell dissemination from the spheroids, whereas KRS-suppressed spheroids remained static due to the absence of outbound movements supported by cell-extracellular matrix (ECM) adhesion. While keeping E-cadherin at the outward disseminative cells, KRS caused integrin-involved intracellular signaling for ERK/c-Jun, paxillin, and cell-ECM adhesion-mediated signaling to modulate traction force for crawling movement. KRS-suppressed spheroids became disseminative following ERK or paxillin re-expression. The KRS-dependent intracellular signaling activities correlated with the invasiveness in clinical colon tumor tissues and in KRS−/+ knocked-down mice tissues. Collectively, these observations indicate that KRS at the plasma membrane plays new roles in metastatic migration as a signaling inducer, and causes intracellular signaling for cancer dissemination, involving cell-cell and cell-ECM adhesion, during KRS-mediated metastasis. PMID:26091349

  8. Towards Single Cell Traction Microscopy within 3D Collagen Matrices

    PubMed Central

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-01-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell-ECM and cell-cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cells migration within collagen gels. PMID:23806281

  9. Single Cell Traction Microscopy within 3D Collagen Matrices

    NASA Astrophysics Data System (ADS)

    Wu, Mingming

    2014-03-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion and migration. Cells require the three dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, our current understanding of cell-ECM and cell-cell mechanical interactions is largely derived from 2D traction force microscopy, in which cells are cultured on a flat substrate. It is now clear that what we learn about cellular behavior on a 2D substrate does not always apply to cells embedded within a 3D biomatrix. 3D traction microscopy is emerging for mapping traction fields of single cells embedded in 3D gel, but current methods cannot account for the fibrous and nonlinear properties of collagen gel. In this talk, I will present a forward computation algorithm that we have developed for 3D cell traction measurements within collagen gels. The application of this technology to understanding cancer migration and invasion will be discussed. This work is supported by the National Center for Research Resources (5R21RR025801-03, NIH) and the National Institute of General Medical Sciences (8 R21 GM103388-03,NIH), and the Cornell Center on the Microenvironment & Metastasis.

  10. Tuning 3D Collagen Matrix Stiffness Independently of Collagen Concentration Modulates Endothelial Cell Behavior

    PubMed Central

    Mason, Brooke N.; Starchenko, Alina; Williams, Rebecca M.; Bonassar, Lawrence J.; Reinhart-King, Cynthia A.

    2012-01-01

    Numerous studies have described the effects of matrix stiffening on cell behavior using two dimensional (2D) synthetic surfaces; however less is known about the effects of matrix stiffening on cells embedded in three dimensional (3D) in vivo-like matrices. A primary limitation in investigating the effects of matrix stiffness in 3D is the lack of materials that can be tuned to control stiffness independently of matrix density. Here, we use collagen-based scaffolds where the mechanical properties are tuned using non-enzymatic glycation of the collagen in solution, prior to polymerization. Collagen solutions glycated prior to polymerization result in collagen gels with a 3-fold increase in compressive modulus without significant changes to the collagen architecture. Using these scaffolds, we show that endothelial cell spreading increases with matrix stiffness, as does the number and length of angiogenic sprouts and the overall spheroid outgrowth. Differences in sprout length are maintained even when the receptor for advanced glycation endproducts is inhibited. Our results demonstrate the ability to de-couple matrix stiffness from matrix density and structure in collagen gels, and that increased matrix stiffness results in increased sprouting and outgrowth. PMID:22902816

  11. Uncertainty in 3D gel dosimetry

    NASA Astrophysics Data System (ADS)

    De Deene, Yves; Jirasek, Andrew

    2015-01-01

    Three-dimensional (3D) gel dosimetry has a unique role to play in safeguarding conformal radiotherapy treatments as the technique can cover the full treatment chain and provides the radiation oncologist with the integrated dose distribution in 3D. It can also be applied to benchmark new treatment strategies such as image guided and tracking radiotherapy techniques. A major obstacle that has hindered the wider dissemination of gel dosimetry in radiotherapy centres is a lack of confidence in the reliability of the measured dose distribution. Uncertainties in 3D dosimeters are attributed to both dosimeter properties and scanning performance. In polymer gel dosimetry with MRI readout, discrepancies in dose response of large polymer gel dosimeters versus small calibration phantoms have been reported which can lead to significant inaccuracies in the dose maps. The sources of error in polymer gel dosimetry with MRI readout are well understood and it has been demonstrated that with a carefully designed scanning protocol, the overall uncertainty in absolute dose that can currently be obtained falls within 5% on an individual voxel basis, for a minimum voxel size of 5 mm3. However, several research groups have chosen to use polymer gel dosimetry in a relative manner by normalizing the dose distribution towards an internal reference dose within the gel dosimeter phantom. 3D dosimetry with optical scanning has also been mostly applied in a relative way, although in principle absolute calibration is possible. As the optical absorption in 3D dosimeters is less dependent on temperature it can be expected that the achievable accuracy is higher with optical CT. The precision in optical scanning of 3D dosimeters depends to a large extend on the performance of the detector. 3D dosimetry with X-ray CT readout is a low contrast imaging modality for polymer gel dosimetry. Sources of error in x-ray CT polymer gel dosimetry (XCT) are currently under investigation and include inherent

  12. 3D dosimetry fundamentals: gels and plastics

    NASA Astrophysics Data System (ADS)

    Lepage, M.; Jordan, K.

    2010-11-01

    Many different materials have been developed for 3D radiation dosimetry since the Fricke gel dosimeter was first proposed in 1984. This paper is intended as an entry point into these materials where we provide an overview of the basic principles for the most explored materials. References to appropriate sources are provided such that the reader interested in more details can quickly find relevant information.

  13. FIBROBLAST MECHANICS IN 3D COLLAGEN MATRICES

    PubMed Central

    Rhee, Sangmyung; Grinnell, Frederick

    2007-01-01

    Connective tissues provide mechanical support and frameworks for the other tissues of the body. Type 1 collagen is the major protein component of ordinary connective tissue, and fibroblasts are the cell type primarily responsible for its biosynthesis and remodeling. Research on fibroblasts interacting with collagen matrices explores all four quadrants of cell mechanics: pro-migratory vs. pro-contractile growth factor environments on one axis; high tension vs. low tension cell-matrix interactions on the other. The dendritic fibroblast – probably equivalent to the resting tissue fibroblast – can be observed only in the low tension quadrant and generally has not been appreciated from research on cells incubated with planar culture surfaces. Fibroblasts in the low tension quadrant require microtubules for formation of dendritic extensions, whereas fibroblasts in the high tension quadrant require microtubules for polarization but not for spreading. Ruffling of dendritic extensions rather than their overall protrusion or retraction provides the mechanism for remodeling of floating collagen matrices, and floating matrix remodeling likely reflects a model of tissue mechanical homeostasis. PMID:17825456

  14. Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

    PubMed

    Thievessen, Ingo; Fakhri, Nikta; Steinwachs, Julian; Kraus, Viola; McIsaac, R Scott; Gao, Liang; Chen, Bi-Chang; Baird, Michelle A; Davidson, Michael W; Betzig, Eric; Oldenbourg, Rudolf; Waterman, Clare M; Fabry, Ben

    2015-11-01

    Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation. PMID:26195589

  15. 3D gel printing for soft-matter systems innovation

    NASA Astrophysics Data System (ADS)

    Furukawa, Hidemitsu; Kawakami, Masaru; Gong, Jin; Makino, Masato; Kabir, M. Hasnat; Saito, Azusa

    2015-04-01

    In the past decade, several high-strength gels have been developed, especially from Japan. These gels are expected to use as a kind of new engineering materials in the fields of industry and medical as substitutes to polyester fibers, which are materials of artificial blood vessels. We consider if various gel materials including such high-strength gels are 3D-printable, many new soft and wet systems will be developed since the most intricate shape gels can be printed regardless of the quite softness and brittleness of gels. Recently we have tried to develop an optical 3D gel printer to realize the free-form formation of gel materials. We named this apparatus Easy Realizer of Soft and Wet Industrial Materials (SWIM-ER). The SWIM-ER will be applied to print bespoke artificial organs, including artificial blood vessels, which will be possibly used for both surgery trainings and actual surgery. The SWIM-ER can print one of the world strongest gels, called Double-Network (DN) gels, by using UV irradiation through an optical fiber. Now we also are developing another type of 3D gel printer for foods, named E-Chef. We believe these new 3D gel printers will broaden the applications of soft-matter gels.

  16. Microscale 3D collagen cell culture assays in conventional flat-bottom 384-well plates.

    PubMed

    Leung, Brendan M; Moraes, Christopher; Cavnar, Stephen P; Luker, Kathryn E; Luker, Gary D; Takayama, Shuichi

    2015-04-01

    Three-dimensional (3D) culture systems such as cell-laden hydrogels are superior to standard two-dimensional (2D) monolayer cultures for many drug-screening applications. However, their adoption into high-throughput screening (HTS) has been lagging, in part because of the difficulty of incorporating these culture formats into existing robotic liquid handling and imaging infrastructures. Dispensing cell-laden prepolymer solutions into 2D well plates is a potential solution but typically requires large volumes of reagents to avoid evaporation during polymerization, which (1) increases costs, (2) makes drug penetration variable and (3) complicates imaging. Here we describe a technique to efficiently produce 3D microgels using automated liquid-handling systems and standard, nonpatterned, flat-bottomed, 384-well plates. Sub-millimeter-diameter, cell-laden collagen gels are deposited on the bottom of a ~2.5 mm diameter microwell with no concerns about evaporation or meniscus effects at the edges of wells, using aqueous two-phase system patterning. The microscale cell-laden collagen-gel constructs are readily imaged and readily penetrated by drugs. The cytotoxicity of chemotherapeutics was monitored by bioluminescence and demonstrated that 3D cultures confer chemoresistance as compared with similar 2D cultures. Hence, these data demonstrate the importance of culturing cells in 3D to obtain realistic cellular responses. Overall, this system provides a simple and inexpensive method for integrating 3D culture capability into existing HTS infrastructure. PMID:25510473

  17. Microscale 3-D collagen cell culture assays in conventional flat-bottom 384-well plates

    PubMed Central

    Leung, Brendan M.; Moraes, Christopher; Cavnar, Stephen; Luker, Kathryn E.; Luker, Gary D.; Takayama, Shuichi

    2015-01-01

    Three-dimensional culture systems such as cell-laden hydrogels are superior to standard 2-D monolayer cultures for many drug-screening applications. However, their adoption in high throughput screening (HTS) have been lagging, in part due to the difficulty of incorporating these culture formats into existing robotic liquid handling and imaging infrastructures. Dispensing cell-laden pre-polymer solutions into 2-D well-plates is a potential solution, but typically requires large volumes of reagents to avoid evaporation during polymerization, which increases cost, makes drug penetration variable and imaging complex. Here we describe a technique to efficiently produce 3-D ‘microgels’ using automated liquid handling systems and standard, non-patterned, flat-bottomed, 384-well plates. Sub-millimeter-diameter, cell-laden collagen gels are deposited on the bottom of ~2.5 mm-diameter microwell with no concerns over evaporation and meniscus effects at the edges of wells, using aqueous two-phase system patterning. The microscale cell-laden collagen-gel constructs are readily imaged and readily penetrated by drugs. Cytotoxicity of chemotherapeutics were monitored by bioluminescence and demonstrates that 3-D cultures confer chemoresistance, as compared to similar 2-D culture. This data hence, demonstrates the importance of culturing cells in 3-D to obtain realistic cellular responses. Overall, this system provided a simple and inexpensive method for integrating 3-D culture capability into existing HTS infrastructure. PMID:25510473

  18. Boundary Stiffness Regulates Fibroblast Behavior in Collagen Gels

    PubMed Central

    John, Jeffrey; Quinlan, Angela Throm; Silvestri, Chiara; Billiar, Kristen

    2010-01-01

    Recent studies have illustrated the profound dependence of cellular behavior on the stiffness of 2D culture substrates. The goal of this study was to develop a method to alter the stiffness cells experience in a standard 3D collagen gel model without affecting the physiochemical properties of the extracellular matrix. A device was developed utilizing compliant anchors (0.048–0.64 N m−1) to tune the boundary stiffness of suspended collagen gels in between the commonly utilized free and fixed conditions (zero and infinite stiffness boundary stiffness). We demonstrate the principle of operation with finite element analyses and a wide range of experimental studies. In all cases, boundary stiffness has a strong influence on cell behavior, most notably eliciting higher basal tension and activated force (in response to KCl) and more pronounced remodeling of the collagen matrix at higher boundary stiffness levels. Measured equibiaxial forces for gels seeded with 3 million human foreskin fibroblasts range from 0.05 to 1 mN increasing monotonically with boundary stiffness. Estimated force per cell ranges from 17 to 100 nN utilizing representative volume element analysis. This device provides a valuable tool to independently study the effect of the mechanical environment of the cell in a 3D collagen matrix. PMID:20012205

  19. 3D jet printer of edible gels for food creation

    NASA Astrophysics Data System (ADS)

    Serizawa, Ryo; Shitara, Mariko; Gong, Jin; Makino, Masato; Kabir, M. Hasnat; Furukawa, Hidemitsu

    2014-03-01

    In recent years, aging is progressing in Japan. Elderly people can't swallow the food well. So, the need of soft food is increasing greatly with the aging of the population. There are so few satisfying foods for the elderly to enjoy a meal. An equipment of printing soft food gives the elderly a big dream and is promising. In this study, we aim at developing a 3D edible gel printer in order to make soft food for the elderly. We made a prototype of the 3D edible gel printer. The printer consists of syringe pump and dispenser. The syringe pump extrudes the solution. The dispenser allows to model threedimensional objects. We use agar solution as the ink to carry out the printing. Agar's gelation deeply depends on temperature. Therefore temperature control of the solution is important to mold optimal shapes because the physical crosslinking network of agar's solution is instable. We succeeded in making the gels and plate-shape gel using the 3D edible gel printer. Further more, in order to increase the gelation speed agar's solution, we changed the dispenser and the printing test is being done now. 4 kinds of soft food prepared from agar and gelatin were printed by the 3D edible gel printer. The compression tests of the printed soft food samples were done and their hardness is measured because the hardness is one of very important factors which influence the food texture greatly. In the future, the viscosity of the agar solution or other food ink should be adjusted to suitable for printing.

  20. Dose rate dependency of micelle leucodye 3D gel dosimeters

    NASA Astrophysics Data System (ADS)

    Vandecasteele, J.; Ghysel, S.; De Deene, Y.

    2010-11-01

    Recently a novel 3D radiochromic gel dosimeter was introduced which uses micelles to dissolve a leucodye in a gelatin matrix. Experimental results show that this 3D micelle gel dosimeter was found to be dose rate dependent. A maximum difference in optical dose sensitivity of 70% was found for dose rates between 50 cGy min-1 and 400 cGy min-1. A novel composition of 3D radiochromic dosimeter is proposed composed of gelatin, sodium dodecyl sulphate, chloroform, trichloroacetic acid and leucomalachite green. The novel gel dosimeter formulation exhibits comparable radio-physical properties in respect to the composition previously proposed. Nevertheless, the novel formulation was found to be still dose rate dependent. A maximum difference of 33% was found for dose rates between 50 cGy min-1 and 400 cGy min-1. On the basis of these experimental results it is concluded that the leucodye micelle gel dosimeter is still unsatisfactory for clinical radiation therapy dose verifications. Some insights in the physico-chemical mechanisms were obtained and are discussed.

  1. Fibronectin- and Collagen-Mimetic Ligands Regulate BMSC Chondrogenesis in 3D Hydrogels

    PubMed Central

    Connelly, J.T.; Petrie, T.A.; García, A.J.; Levenston, M.E.

    2016-01-01

    Modification of tissue engineering scaffolds with bioactive molecules is a potential strategy for modulating cell behavior and guiding tissue regeneration. While adhesion to RGD peptides has been shown to inhibit in vitro chondrogenesis, the effects of extracellular matrix (ECM)-mimetic ligands with complex secondary and tertiary structures are unknown. This study aimed to determine whether collagen- and fibronectin-mimetic ligands would retain biologic functionality in three-dimensional (3D) hydrogels, whether different ECM-mimetic ligands differentially influence in vitro chondrogenesis, and if effects of ligands on differentiation depend on soluble biochemical stimuli. A linear RGD peptide, a recombinant fibronectin fragment containing the seven to 10 Type III repeats (FnIII7-10) and a triple helical, collagen mimetic peptide with the GFOGER motif were covalently coupled to agarose gels using the sulfo-SANPAH crosslinker, and bone marrow stromal cells (BMSCs) were cultured within the 3D hydrogels.. The ligands retained biologic functionality within the agarose gels and promoted density-dependent BMSC spreading. Interactions with all adhesive ligands inhibited stimulation by chondrogenic factors of collagen Type II and aggrecanmRNA levels and deposition of sulfated glycosaminoglycans. In medium containing fetal bovine serum, interactions with the GFOGER peptide enhanced mRNA expression of the osteogenic gene osteocalcin whereas FnIII7-10 inhibited osteocalcin expression. In conclusion, modification of agarose hydrogels with ECM-mimetic ligands can influence the differentiation of BMSCs in a manner that depends strongly on the presence and nature of soluble biochemical stimuli. PMID:21932193

  2. GEM printer: 3D gel printer for free shaping of functional gel engineering materials

    NASA Astrophysics Data System (ADS)

    Furukawa, Hidemitsu; Muroi, Hisato; Yamamoto, Kouki; Serizawa, Ryo; Gong, Jin

    2013-04-01

    In the past decade, several high-strength gels have been developed. These gels are expected to use as a kind of new engineering materials in the fields of industry and medical as substitutes to polyester fibers, which are materials of artificial blood vessels. The gels have both low surface friction and well permeability due to a large amount of water absorbed in the gels, which are superiority of the gels compering to the polyester fibers. It is, however, difficult for gels to be forked structure or cavity structure by using cutting or mold. Consequently, it is necessary to develop the additive manufacturing device to synthesize and mode freely gels at the same time. Here we try to develop an optical 3D gel printer that enables gels to be shaped precisely and freely. For the free forming of high-strength gels, the 1st gels are ground to particles and mixed with 2nd pregel solution, and the mixed solution is gelled by the irradiation of UV laser beam through an optical fiber. The use of the optical fiber makes one-point UV irradiation possible. Since the optical fiber is controlled by 3D-CAD, the precise and free molding in XYZ directions is easily realized. We successfully synthesized tough gels using the gel printer.

  3. Free forming of the gel by 3D gel printer SWIM-ER

    NASA Astrophysics Data System (ADS)

    Okada, Koji; Tase, Taishi; Saito, Azusa; Makino, Masato; Gong, Jin; Kawakami, Masaru; Furukawa, Hidemitsu

    2015-04-01

    Gels, soft and wet materials, have unique properties such as material permeability, biocompatibility and low friction, which are hardly found in hard and dry materials. These superior characteristics of hydrogels promise to expand the medical applications. In recent years, the optical 3D gel printer named SWIM-ER (Soft and Wet Industrial - Easy Realizer) was developed by our team in order to fabricate tough gels with free form. We are aiming to create artificial blood vessel of the gel material by 3D gel printer. Artificial blood vessel is expected to be used for vascular surgery practice. The artificial blood vessel made by 3D gel printer can be create to free form on the basis of the biological data of the patient. Therefore, we believe it is possible to contribute to increasing the success rate and safety of vascular surgery by creating artificial blood vessel with 3D gel printer. The modeling method of SWIM-ER is as follow. Pregel solution is polymerized by one-point UV irradiation with optical fiber. The irradiation area is controlled by computer program, so that exact 3D free forming is realized. In this study, synthesis conditions are re-examined in order to improve the degree of freedom of fabrication. The dimensional accuracy in height direction is improved by increasing the cross linker concentration. We examined the relationship of resolution to the pitch and UV irradiation time in order to improve the modeling accuracy.

  4. 3D Gel Map of Arabidopsis Complex I

    PubMed Central

    Peters, Katrin; Belt, Katharina; Braun, Hans-Peter

    2013-01-01

    Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves, and roots. Subunits of complex I were resolved by 3D blue-native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, seven of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of subunits, 40 of which represent homologs of bovine complex I. The nine other subunits represent special proteins absent in the animal linage of eukaryotes, most prominently a group of subunits related to bacterial gamma-type carbonic anhydrases. A GelMap http://www.gelmap.de/arabidopsis-3d-complex-i/ is presented for promoting future complex I research in Arabidopsis thaliana. PMID:23761796

  5. Microfluidic vascular channels in gels using commercial 3D printers

    NASA Astrophysics Data System (ADS)

    Selvaganapathy, P. Ravi; Attalla, Rana

    2016-03-01

    This paper details the development of a three dimensional (3D) printing system with a modified microfluidic printhead used for the generation of complex vascular tissue scaffolds. The print-head features an integrated coaxial nozzle that allows the fabrication of hollow, calcium-polymerized alginate tubes that can easily be patterned using 3Dbioprinting techniques. This microfluidic design allows the incorporation of a wide range of scaffold materials as well as biological constituents such as cells, growth factors, and ECM material. With this setup, gel constructs with embedded arrays of hollow channels can be created and used as a potential substitute for blood vessel networks.

  6. A Novel 3D Fibril Force Assay Implicates Src in Tumor Cell Force Generation in Collagen Networks

    PubMed Central

    Polackwich, Robert J.; Koch, Daniel; Arevalo, Richard; Miermont, Anne M.; Jee, Kathleen J.; Lazar, John; Urbach, Jeffrey; Mueller, Susette C.; McAllister, Ryan G.

    2013-01-01

    New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM) environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction and motility in 3D fibrillar environments. The assay consists of a controlled-density fibrillar collagen gel atop a controlled-stiffness polyacrylamide (PAA) surface. Forces generated by living cells and their migration in the 3D collagen gel were measured with the 3D motion of tracer beads within the PAA layer. Here, this 3D fibril force assay is used to study the role of the invasion-associated protein kinase Src in mechanotransduction and motility. Src expression and activation are linked with proliferation, invasion, and metastasis, and have been shown to be required in 2D for invadopodia membranes to direct and mediate invasion. Breast cancer cell line MDA-MD-231 was stably transfected with GFP-tagged constitutively active Src or wild-type Src. In 3D fibrillar collagen matrices we found that, relative to wild-type Src, constitutively active Src: 1) increased the strength of cell-induced forces on the ECM, 2) did not significantly change migration speed, and 3) increased both the duration and the length, but not the number, of long membrane protrusions. Taken together, these results support the hypothesis that Src controls invasion by controlling the ability of the cell to form long lasting cellular protrusions to enable penetration through tissue barriers, in addition to its role in promoting invadopodia matrix-degrading activity. PMID:23536784

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

    SciTech Connect

    Chen, Chih-Hao; Kuo, Shyh Ming; Liu, Guei-Sheung; Chen, Wan-Nan U.; Chuang, Chin-Wen; Liu, Li-Feng

    2012-11-09

    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 porous 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.

  8. Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model

    PubMed Central

    Terryn, Christine; Garnotel, Roselyne; Jeannesson, Pierre; Sockalingum, Ganesh D.; Manfait, Michel; Perraut, François; Dinten, Jean-Marc; Koenig, Anne; Piot, Olivier

    2016-01-01

    During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models. PMID:26885896

  9. Cell Proliferation on Macro/Nano Surface Structure and Collagen Immobilization of 3D Polycaprolactone Scaffolds.

    PubMed

    Park, Young-Ouk; Myung, Sung-Woon; Kook, Min-Suk; Jung, Sang-Chul; Kim, Byung-Hoon

    2016-02-01

    In this study, 3D polycaprolactone (PCL) scaffolds were fabricated by 3D printing technique. The macro/nano morphology of, 3D PCL scaffolds surface was etched with oxygen plasma. Acrylic acid (AA) plasma-polymerization was performed to functionalize the macro/nano surface with carboxyl groups and then collagen was immobilized with plasma-polymerized 3D PCL scaffolds. After O2 plasma and AA plasma-polymerization, contact angles were decreased. The FE-SEM and AFM results showed that O2 plasma is increased the surface roughness. The MTT assay results showed that proliferation of the M3CT3-E1 cells increased on the oxygen plasma treated and collagen immobilized 3D PCL scaffolds. PMID:27433597

  10. Quantitative Assessment of Local Collagen Matrix Remodeling in 3-D Culture: The Role of Rho Kinase

    PubMed Central

    Kim, Areum; Lakshman, Neema; Petroll, W.Matthew

    2007-01-01

    The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100 μm thick fibrillar collagen matrices and cultured for 24 hours in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p < 0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p<0.01). Overall, the data suggests that Rho kinase dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction. PMID:16978606

  11. Characterization of Fibrin and Collagen Gels for Engineering Wound Healing Models

    PubMed Central

    Moreno-Arotzena, Oihana; Meier, Johann G.; del Amo, Cristina; García-Aznar, José Manuel

    2015-01-01

    Hydrogels are used for 3D in vitro assays and tissue engineering and regeneration purposes. For a thorough interpretation of this technology, an integral biomechanical characterization of the materials is required. In this work, we characterize the mechanical and functional behavior of two specific hydrogels that play critical roles in wound healing, collagen and fibrin. A coherent and complementary characterization was performed using a generalized and standard composition of each hydrogel and a combination of techniques. Microstructural analysis was performed by scanning electron microscopy and confocal reflection imaging. Permeability was measured using a microfluidic-based experimental set-up, and mechanical responses were analyzed by rheology. We measured a pore size of 2.84 and 1.69 μm for collagen and fibrin, respectively. Correspondingly, the permeability of the gels was 1.00·10−12 and 5.73·10−13 m2. The shear modulus in the linear viscoelastic regime was 15 Pa for collagen and 300 Pa for fibrin. The gels exhibited strain-hardening behavior at ca. 10% and 50% strain for fibrin and collagen, respectively. This consistent biomechanical characterization provides a detailed and robust starting point for different 3D in vitro bioapplications, such as collagen and/or fibrin gels. These features may have major implications for 3D cellular behavior by inducing divergent microenvironmental cues. PMID:26290683

  12. Primary human osteoblast culture on 3D porous collagen-hydroxyapatite scaffolds.

    PubMed

    Jones, Gemma L; Walton, Robin; Czernuszka, Jan; Griffiths, Sarah L; El Haj, Alicia J; Cartmell, Sarah H

    2010-09-15

    There is a need in tissue-engineering for 3D scaffolds that mimic the natural extracellular matrix of bone to enhance cell adhesion, proliferation, and differentiation. The scaffold is also required to be degradable. A highly porous scaffold has been developed to incorporate two of the extracellular components found in bone-collagen and hydroxyapatite (HA). The scaffold's collagen component is an afibrillar monomeric type I atelocollagen extracted from foetal calf's skin. This provided a novel environment for the inclusion of HA powder. Five hundred thousand primary human osteoblasts were seeded onto 4 mm cubed scaffolds that varied in ratio of HA to collagen. Weight ratios of 1:99, 25:75, 50:50, and 75:25 hydroxyapatite:collagen (HA:Collagen) were analysed. The scaffolds plus cells were cultured for 21 days. DNA assays and live/dead viability staining demonstrated that all of the scaffolds supported cell proliferation and viability. An alkaline phosphatase assay showed similar osteoblast phenotype maintenance on all of the 3D scaffolds analysed at 21 days. MicroCT analysis demonstrated an increase in total sample volume (correlating to increase in unmineralised matrix production). An even distribution of HA throughout the collagen matrix was observed using this technique. Also at 3 weeks, reductions in the percentage of the mineralised phase of the constructs were seen. These results indicate that each of the ratios of HA/collagen scaffolds have great potential for bone tissue engineering. PMID:20694991

  13. Wound healing properties of a 3-D scaffold comprising soluble silkworm gland hydrolysate and human collagen.

    PubMed

    Kim, Kyu-Oh; Lee, Youngjun; Hwang, Jung-Wook; Kim, Hojin; Kim, Sun Mi; Chang, Sung Woon; Lee, Heui Sam; Choi, Yong-Soo

    2014-04-01

    Biomaterials that serve as scaffolds for cell proliferation and differentiation are increasingly being used in wound repair. In this study, the potential regenerative properties of a 3-D scaffold containing soluble silkworm gland hydrolysate (SSGH) and human collagen were evaluated. The scaffold was generated by solid-liquid phase separation and a freeze-drying method using a homogeneous aqueous solution. The porosity, swelling behavior, protein release, cytotoxicity, and antioxidative properties of scaffolds containing various ratios of SSGH and collagen were evaluated. SSGH/collagen scaffolds had a high porosity of 61-81% and swelling behavior studies demonstrated a 50-75% increase in swelling, along with complete protein release in the presence of phosphate-buffered saline. Cytocompatibility of the SSGH/collagen scaffold was demonstrated using mesenchymal stem cells from human umbilical cord. Furthermore, SSGH/collagen efficiently attenuated oxidative stress-induced cell damage. In an in vivo mouse model of wound healing, the SSGH/collagen scaffold accelerated wound re-epithelialization over a 15-day period. Overall, the microporous SSGH/collagen 3-D scaffold maintained optimal hydration of the exposed tissues and decreased wound healing time. These results contribute to the generation of advanced wound healing materials and may have future therapeutic implications. PMID:24503353

  14. Collagen telopeptides (cross-linking sites) play a role in collagen gel lattice contraction

    NASA Technical Reports Server (NTRS)

    Woodley, D. T.; Yamauchi, M.; Wynn, K. C.; Mechanic, G.; Briggaman, R. A.

    1991-01-01

    Solubilized interstitial collagens will form a fibrillar, gel-like lattice when brought to physiologic conditions. In the presence of human dermal fibroblasts the collagen lattice will contract. The rate of contraction can be determined by computer-assisted planemetry. The mechanisms involved in contraction are as yet unknown. Using this system it was found that the rate of contraction was markedly decreased when collagen lacking telopeptides was substituted for native collagen. Histidinohydroxylysinonorleucine (HHL) is a major stable trifunctional collagen cross-link in mature skin that involves a carboxyl terminal, telopeptide site 16c, the sixteenth amino acid residue from the carboxy terminal of the telopeptide region of alpha 1 (I) in type I collagen. Little, if any, HHL was present in native, purified, reconstituted, soluble collagen fibrils from 1% acetic acid-extracted 2-year-old bovine skin. In contrast, HHL cross-links were present (0.22 moles of cross-link per mole of collagen) in lattices of the same collagen contracted by fibroblasts. However, rat tail tendon does not contain HHL cross-links, and collagen lattices made of rat tail tendon collagen are capable of contraction. This suggests that telopeptide sites, and not mature HHL cross-links per se, are essential for fibroblasts to contract collagen lattices. Beta-aminopropionitrile fumarate (BAPN), a potent lathyrogen that perturbs collagen cross-linking by inhibition of lysyl oxidase, also inhibited the rate of lattice cell contraction in lattices composed of native collagen. However, the concentrations of BAPN that were necessary to inhibit the contraction of collagen lattices also inhibited fibroblast growth suggestive of cellular toxicity. In accordance with other studies, we found no inhibition of the rate of lattice contraction when fibronectin-depleted serum was used. Electron microscopy of contracted gels revealed typical collagen fibers with a characteristic axial periodicity. The data

  15. 3D Printing of Composite Calcium Phosphate and Collagen Scaffolds for Bone Regeneration

    PubMed Central

    Inzana, Jason A.; Olvera, Diana; Fuller, Seth M.; Kelly, James P.; Graeve, Olivia A.; Schwarz, Edward M.; Kates, Stephen L.; Awad, Hani A.

    2014-01-01

    Low temperature 3D printing of calcium phosphate scaffolds holds great promise for fabricating synthetic bone graft substitutes with enhanced performance over traditional techniques. Many design parameters, such as the binder solution properties, have yet to be optimized to ensure maximal biocompatibility and osteoconductivity with sufficient mechanical properties. This study tailored the phosphoric acid-based binder solution concentration to 8.75 wt% to maximize cytocompatibility and mechanical strength, with a supplementation of Tween 80 to improve printing. To further enhance the formulation, collagen was dissolved into the binder solution to fabricate collagen-calcium phosphate composites. Reducing the viscosity and surface tension through a physiologic heat treatment and Tween 80, respectively, enabled reliable thermal inkjet printing of the collagen solutions. Supplementing the binder solution with 1–2 wt% collagen significantly improved maximum flexural strength and cell viability. To assess the bone healing performance, we implanted 3D printed scaffolds into a critically sized murine femoral defect for 9 weeks. The implants were confirmed to be osteoconductive, with new bone growth incorporating the degrading scaffold materials. In conclusion, this study demonstrates optimization of material parameters for 3D printed calcium phosphate scaffolds and enhancement of material properties by volumetric collagen incorporation via inkjet printing. PMID:24529628

  16. Characterizing matrix remodeling in collagen gels using optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Levitz, David; Hinds, Monica T.; Hanson, Stephen R.; Jacques, Steven L.

    2010-02-01

    Optical coherence tomography (OCT) has shown promise at non-destructively characterizing engineered tissues such as collagen gels. However, as the collagen gels develop, the OCT images lose contrast of structures as the gels develop, making visual assessment difficult. Our group proposed quantitatively characterizing these gels by fitting the optical properties from the OCT signals. In this paper, we imaged collagen gels seeded with smooth muscle cells (SMCs) over a 5-day period and used the data to measure their optical properties. Our results showed that over time, the reflectivity of the samples increased 10-fold, corresponding to a decrease in anisotropy factor g, without much change in the scattering coefficient μs. Overall, the optical properties appeared to be dominated by scattering from the collagen matrix, not the cells. However, SMCs remodeled the collagen matrix, and this collagen remodeling by the cells is what causes the observed changes in optical properties. Moreover, the data showed that the optical properties were sensitive to the activity of matrix metalloproteinases (MMPs), enzymes that break down local collagen fibrils into smaller fragments. Blocking MMPs in the SMC gels greatly impeded both the remodeling process and change in optical properties at day 5. Treating day 1 acellular gels with MMP-8 for 3 hr managed to partially reproduce the remodeling observed in SMC gels at day 5. Altogether, we conclude that matrix remodeling in general, and MMPs specifically, greatly affect the local optical properties of the sample, and OCT is a unique tool that can assess MMP activity in collagen gels both non-destructively and label free.

  17. Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes.

    PubMed

    Yates, Karen E; Allemann, Florin; Glowacki, Julie

    2005-01-01

    Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS(+3)) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering. PMID:15735900

  18. Effects of Decorin Proteoglycan on Fibrillogenesis, Ultrastructure, and Mechanics of Type I Collagen Gels

    PubMed Central

    Reese, Shawn P.; Underwood, Clayton J.; Weiss, Jeffrey A.

    2013-01-01

    The proteoglycan decorin is known to affect both the fibrillogenesis and the resulting ultrastructure of in vitro polymerized collagen gels. However, little is known about its effects on mechanical properties. In this study, 3D collagen gels were polymerized into tensile test specimens in the presence of decorin proteoglycan, decorin core protein, or dermatan sulfate (DS). Collagen fibrillogenesis, ultrastructure, and mechanical properties were then quantified using a turbidity assay, 2 forms of microscopy (SEM and confocal), and tensile testing. The presence of decorin proteoglycan or core protein decreased the rate and ultimate turbidity during fibrillogenesis and decreased the number of fibril aggregates (fibers) compared to control gels. The addition of decorin and core protein increased the linear modulus by a factor of 2 compared to controls, while the addition of DS reduced the linear modulus by a factor of 3. Adding decorin after fibrillogenesis had no effect, suggesting that decorin must be present during fibrillogenesis to increase the mechanical properties of the resulting gels. These results show that the inclusion of decorin proteoglycan during fibrillogenesis of Type I collagen increases the modulus and tensile strength of resulting collagen gels. The increase in mechanical properties when polymerization occurs in the presence of the decorin proteoglycan is due to a reduction in the aggregation of fibrils into larger order structures such as fibers and fiber bundles. PMID:23608680

  19. MMP Regulation of Corneal Keratocyte Motility and Mechanics in 3-D Collagen Matrices

    PubMed Central

    Zhou, Chengxin; Petroll, W. Matthew

    2014-01-01

    Previous studies have shown that platelet derived growth factor (PDGF) can stimulate corneal keratocyte spreading and migration within 3-D collagen matrices, without inducing transformation to a contractile, fibroblastic phenotype. The goal of this study was to investigate the role of matrix metalloproteinases (MMPs) in regulating PDGF-induced changes in keratocyte motility and mechanical differentiation. Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-) to maintain their quiescent phenotype. A nested collagen matrix construct was used to assess 3-D cell migration, and a standard collagen matrix model was used to assess cell morphology and cell-mediated matrix contraction. In both cases constructs were cultured in S- supplemented with PDGF, with or without the broad spectrum MMP inhibitors GM6001 or BB-94. After 4 days, f-actin, nuclei and collagen fibrils were imaged using confocal microscopy. To assess sub-cellular mechanical activity (extension and retraction of cell processes), time-lapse DIC imaging was also performed. MT1-MMP expression and MMP-mediated collagen degradation by were also examined. Results demonstrated that neither GM6001 nor BB-94 affected corneal keratocyte viability or proliferation in 3-D culture. PDGF stimulated elongation and migration of corneal keratocytes within type I collagen matrices, without causing a loss of their dendritic morphology or inducing formation of intracellular stress fibers. Treatment with GM6001 and BB-94 inhibited PDGF-induced keratocyte spreading and migration. Relatively low levels of keratocyte-induced matrix contraction were also maintained in PDGF, and the amount of PDGF-induced collagen degradation was similar to that observed in S- controls. The collagen degradation pattern was consistent with membrane-associated MMP activity, and keratocytes showed positive staining for MT1-MMP, albeit weak. Both matrix contraction and collagen degradation were reduced by MMP inhibition. For most

  20. Heterogeneity in dermatosparaxis is shown by contraction of collagen gels.

    PubMed

    Ramshaw, J A; Mitrangas, K; Bateman, J F

    1991-01-01

    Dermal fibroblasts from sheep exhibiting a mild form of dermatosparaxis were able to contract reconstituted, fibrillar collagen gels at the same rate as control dermal fibroblasts, indicating a normal interaction between the cells and a collagenous matrix. An extract from dermatosparactic skin was shown, after partial purification, to have N-proteinase activity, although the level of activity was much lower than found in normal skin. These data show that dermatosparaxis is a heterogeneous disease, since in the severe forms of the disease the defect has been characterized as an absence of N-proteinase and an inability of the cells to interact with and contract collagen gels. PMID:2060304

  1. Microfabricated collagen tracks facilitate single cell metastatic invasion in 3D.

    PubMed

    Kraning-Rush, Casey M; Carey, Shawn P; Lampi, Marsha C; Reinhart-King, Cynthia A

    2013-03-01

    While the mechanisms employed by metastatic cancer cells to migrate remain poorly understood, it has been widely accepted that metastatic cancer cells can invade the tumor stroma by degrading the extracellular matrix (ECM) with matrix metalloproteinases (MMPs). Although MMP inhibitors showed early promise in preventing metastasis in animal models, they have largely failed clinically. Recently, studies have shown that some cancer cells can use proteolysis to mechanically rearrange their ECM to form tube-like "microtracks" which other cells can follow without using MMPs themselves. We speculate that this mode of migration in the secondary cells may be one example of migration which can occur without endogenous protease activity in the secondary cells. Here we present a technique to study this migration in a 3D, collagen-based environment which mimics the size and topography of the tracks produced by proteolytically active cancer cells. Using time-lapse phase-contrast microscopy, we find that these microtracks permit the rapid and persistent migration of noninvasive MCF10A mammary epithelial cells, which are unable to otherwise migrate in 3D collagen. Additionally, while highly metastatic MDAMB231 breast cancer cells are able to invade a 3D collagen matrix, seeding within the patterned microtracks induced significantly increased cell migration speed, which was not decreased by pharmacological MMP inhibition. Together, these data suggest that microtracks within a 3D ECM may facilitate the migration of cells in an MMP-independent fashion, and may reveal novel insight into the clinical challenges facing MMP inhibitors. PMID:23388698

  2. Rapid Quantification of 3D Collagen Fiber Alignment and Fiber Intersection Correlations with High Sensitivity

    PubMed Central

    Sun, Meng; Bloom, Alexander B.; Zaman, Muhammad H.

    2015-01-01

    Metastatic cancers aggressively reorganize collagen in their microenvironment. For example, radially orientated collagen fibers have been observed surrounding tumor cell clusters in vivo. The degree of fiber alignment, as a consequence of this remodeling, has often been difficult to quantify. In this paper, we present an easy to implement algorithm for accurate detection of collagen fiber orientation in a rapid pixel-wise manner. This algorithm quantifies the alignment of both computer generated and actual collagen fiber networks of varying degrees of alignment within 5°°. We also present an alternative easy method to calculate the alignment index directly from the standard deviation of fiber orientation. Using this quantitative method for determining collagen alignment, we demonstrate that the number of collagen fiber intersections has a negative correlation with the degree of fiber alignment. This decrease in intersections of aligned fibers could explain why cells move more rapidly along aligned fibers than unaligned fibers, as previously reported. Overall, our paper provides an easier, more quantitative and quicker way to quantify fiber orientation and alignment, and presents a platform in studying effects of matrix and cellular properties on fiber alignment in complex 3D environments. PMID:26158674

  3. 3D Raman mapping of the collagen fibril orientation in human osteonal lamellae.

    PubMed

    Schrof, Susanne; Varga, Peter; Galvis, Leonardo; Raum, Kay; Masic, Admir

    2014-09-01

    Chemical composition and fibrillar organization are the major determinants of osteonal bone mechanics. However, prominent methodologies commonly applied to investigate mechanical properties of bone on the micro scale are usually not able to concurrently describe both factors. In this study, we used polarized Raman spectroscopy (PRS) to simultaneously analyze structural and chemical information of collagen fibrils in human osteonal bone in a single experiment. Specifically, the three-dimensional arrangement of collagen fibrils in osteonal lamellae was assessed. By analyzing the anisotropic intensity of the amide I Raman band of collagen as a function of the orientation of the incident laser polarization, different parameters related to the orientation of the collagen fibrils and the degree of alignment of the fibrils were derived. Based on the analysis of several osteons, two major fibrillar organization patterns were identified, one with a monotonic and another with a periodically changing twist direction. These results confirm earlier reported twisted and oscillating plywood arrangements, respectively. Furthermore, indicators of the degree of alignment suggested the presence of disordered collagen within the lamellar organization of the osteon. The results show the versatility of the analytical PRS approach and demonstrate its capability in providing not only compositional, but also 3D structural information in a complex hierarchically structured biological material. The concurrent assessment of chemical and structural features may contribute to a comprehensive characterization of the microstructure of bone and other collagen-based tissues. PMID:25025981

  4. Rapid Quantification of 3D Collagen Fiber Alignment and Fiber Intersection Correlations with High Sensitivity.

    PubMed

    Sun, Meng; Bloom, Alexander B; Zaman, Muhammad H

    2015-01-01

    Metastatic cancers aggressively reorganize collagen in their microenvironment. For example, radially orientated collagen fibers have been observed surrounding tumor cell clusters in vivo. The degree of fiber alignment, as a consequence of this remodeling, has often been difficult to quantify. In this paper, we present an easy to implement algorithm for accurate detection of collagen fiber orientation in a rapid pixel-wise manner. This algorithm quantifies the alignment of both computer generated and actual collagen fiber networks of varying degrees of alignment within 5°°. We also present an alternative easy method to calculate the alignment index directly from the standard deviation of fiber orientation. Using this quantitative method for determining collagen alignment, we demonstrate that the number of collagen fiber intersections has a negative correlation with the degree of fiber alignment. This decrease in intersections of aligned fibers could explain why cells move more rapidly along aligned fibers than unaligned fibers, as previously reported. Overall, our paper provides an easier, more quantitative and quicker way to quantify fiber orientation and alignment, and presents a platform in studying effects of matrix and cellular properties on fiber alignment in complex 3D environments. PMID:26158674

  5. Characterization of collagen gel solutions and collagen matrices for cell culture.

    PubMed

    Sheu, M T; Huang, J C; Yeh, G C; Ho, H O

    2001-07-01

    The influence of glutaraldehyde as a crosslinking agent to increase the strength of collagen matrices for cell culture was examined in this study. Collagen solutions of 1% were treated with different concentrations (0-0.2%) of glutaraldehyde for 24 h. The viscoelasticity of the resulting collagen gel solution was measured using dynamic mechanical analysis (DMA), which demonstrated that all collagen gel solutions examined followed the same model pattern. The creep compliance model of Voigt-Kelvin satisfactorily described the change of viscoelasticity expressed by these collagen gel solutions. These crosslinked collagen gel solutions were freeze-dried to form a matrix with a thickness of about 0.2-0.3 mm. The break modulus of these collagen matrices measured by DMA revealed that the higher the degree of crosslinking. the higher the break modulus. The compatibility of fibroblasts isolated from nude mouse skin with these collagen matrices was found to be acceptable at a cell density of 3 x 10(5) cells/cm2 with no contraction, even when using a concentration of glutaraldehyde of up to 0.2%. PMID:11396874

  6. Texture analysis of the 3D collagen network and automatic classification of the physiology of articular cartilage.

    PubMed

    Duan, Xiaojuan; Wu, Jianping; Swift, Benjamin; Kirk, Thomas Brett

    2015-07-01

    A close relationship has been found between the 3D collagen structure and physiological condition of articular cartilage (AC). Studying the 3D collagen network in AC offers a way to determine the condition of the cartilage. However, traditional qualitative studies are time consuming and subjective. This study aims to develop a computer vision-based classifier to automatically determine the condition of AC tissue based on the structural characteristics of the collagen network. Texture analysis was applied to quantitatively characterise the 3D collagen structure in normal (International Cartilage Repair Society, ICRS, grade 0), aged (ICRS grade 1) and osteoarthritic cartilages (ICRS grade 2). Principle component techniques and linear discriminant analysis were then used to classify the microstructural characteristics of the 3D collagen meshwork and the condition of the AC. The 3D collagen meshwork in the three physiological condition groups displayed distinctive characteristics. Texture analysis indicated a significant difference in the mean texture parameters of the 3D collagen network between groups. The principle component and linear discriminant analysis of the texture data allowed for the development of a classifier for identifying the physiological status of the AC with an expected prediction error of 4.23%. An automatic image analysis classifier has been developed to predict the physiological condition of AC (from ICRS grade 0 to 2) based on texture data from the 3D collagen network in the tissue. PMID:24428581

  7. Strain-enhanced stress relaxation impacts nonlinear elasticity in collagen gels.

    PubMed

    Nam, Sungmin; Hu, Kenneth H; Butte, Manish J; Chaudhuri, Ovijit

    2016-05-17

    The extracellular matrix (ECM) is a complex assembly of structural proteins that provides physical support and biochemical signaling to cells in tissues. The mechanical properties of the ECM have been found to play a key role in regulating cell behaviors such as differentiation and malignancy. Gels formed from ECM protein biopolymers such as collagen or fibrin are commonly used for 3D cell culture models of tissue. One of the most striking features of these gels is that they exhibit nonlinear elasticity, undergoing strain stiffening. However, these gels are also viscoelastic and exhibit stress relaxation, with the resistance of the gel to a deformation relaxing over time. Recent studies have suggested that cells sense and respond to both nonlinear elasticity and viscoelasticity of ECM, yet little is known about the connection between nonlinear elasticity and viscoelasticity. Here, we report that, as strain is increased, not only do biopolymer gels stiffen but they also exhibit faster stress relaxation, reducing the timescale over which elastic energy is dissipated. This effect is not universal to all biological gels and is mediated through weak cross-links. Mechanistically, computational modeling and atomic force microscopy (AFM) indicate that strain-enhanced stress relaxation of collagen gels arises from force-dependent unbinding of weak bonds between collagen fibers. The broader effect of strain-enhanced stress relaxation is to rapidly diminish strain stiffening over time. These results reveal the interplay between nonlinear elasticity and viscoelasticity in collagen gels, and highlight the complexity of the ECM mechanics that are likely sensed through cellular mechanotransduction. PMID:27140623

  8. Effect of HDAC Inhibitors on Corneal Keratocyte Mechanical Phenotypes in 3-D Collagen Matrices

    PubMed Central

    Koppaka, Vindhya; Lakshman, Neema

    2015-01-01

    Purpose: Histone deacetylase inhibitors (HDAC) have been shown to inhibit the TGFβ-induced myofibroblast transformation of corneal fibroblasts in 2-D culture. However, the effect of HDAC inhibitors on keratocyte spreading, contraction, and matrix remodeling in 3-D culture has not been directly assessed. The goal of this study was to investigate the effects of the HDAC inhibitors Trichostatin A (TSA) and Vorinostat (SAHA) on corneal keratocyte mechanical phenotypes in 3-D culture using defined serum-free culture conditions. Methods: Rabbit corneal keratocytes were plated within standard rat tail type I collagen matrices (2.5 mg/ml) or compressed collagen matrices (~100 mg/ml) and cultured for up to 4 days in serum-free media, PDGF BB, TGFβ1, and either 50 nM TSA, 10 μM SAHA, or vehicle (DMSO). F-actin, α-SM-actin, and collagen fibrils were imaged using confocal microscopy. Cell morphology and global matrix contraction were quantified digitally. The expression of α-SM-actin was assessed using western blotting. Results: Corneal keratocytes in 3-D matrices had a quiescent mechanical phenotype, as indicated by a dendritic morphology, a lack of stress fibers, and minimal cell-induced matrix remodeling. This phenotype was generally maintained following the addition of TSA or SAHA. TGFβ1 induced a contractile phenotype, as indicated by a loss of dendritic cell processes, the development of stress fibers, and significant matrix compaction. In contrast, cells cultured in TGFβ1 plus TSA or SAHA remained dendritic and did not form stress fibers or induce ECM compaction. Western blotting showed that the expression of α-SM actin after treatment with TGFβ1 was inhibited by TSA and SAHA. PDGF BB stimulated the elongation of keratocytes and the extension of dendritic processes within 3-D matrices without inducing stress fiber formation or collagen reorganization. This spreading response was maintained in the presence of TSA or SAHA. Conclusions: Overall, HDAC inhibitors

  9. Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix.

    PubMed

    Stout, David A; Toyjanova, Jennet; Franck, Christian

    2015-01-01

    The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis. PMID:26131645

  10. Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

    PubMed Central

    Chwalek, Karolina; Sood, Disha; Cantley, William L.; White, James D.; Tang-Schomer, Min; Kaplan, David L.

    2015-01-01

    Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions. PMID:26555926

  11. An Experimental Model for Assessing Fibroblast Migration in 3-D Collagen Matrices

    PubMed Central

    Karamichos, Dimitris; Lakshman, Neema; Petroll, W. Matthew

    2009-01-01

    The purpose of this study was to develop and test a novel culture model for studying fibroblast migration in 3-D collagen matrices. Human corneal fibroblasts were seeded within dense, randomly oriented compressed collagen matrices. A 6 mm diameter button of this cell-seeded matrix was placed in the middle of an acellular, less dense outer collagen matrix. These constructs were cultured for 1, 3, 5 or 7 days in serum-free media, 10% fetal bovine serum (FBS), or 50 ng/ml PDGF. Constructs were then fixed and labeled with AlexaFluor 546 phalloidin (for f-actin) and TOTO-3 (for nuclei). Cell-matrix interactions were assessed using a combination of fluorescent and reflected light confocal imaging. Human corneal fibroblasts in serum-free media showed minimal migration into the outer (non-compressed) matrix. In contrast, culture in serum or PDGF stimulated cell migration. Cell-induced collagen matrix reorganization in the outer matrix could be directly visualized using reflected light imaging, and was highest following culture in 10% FBS. Cellular contraction in 10% FBS often led to alignment of cells parallel to the outer edge of the inner matrix, similar to the pattern observed during corneal wound healing following incisional surgery. Overall, this 3-D model allows the effects of different culture conditions on cell migration and matrix remodeling to be assessed simultaneously. In addition, the design allows for ECM density, geometry and mechanical constraints to be varied in a controlled fashion. These initial results demonstrate differences in cell and matrix patterning during migration in response to serum and PDGF. PMID:19061246

  12. Development of bioartificial myocardium by electrostimulation of 3D collagen scaffolds seeded with stem cells

    PubMed Central

    Haneef, Kanwal; Lila, Nermine; Benadda, Samira; Legrand, Fabien; Carpentier, Alain; Chachques, Juan C.

    2012-01-01

    Electrostimulation (ES) can be defined as a safe physical method to induce stem cell differentiation. The aim of this study is to evaluate the effectiveness of ES on bone marrow mesenchymal stem cells (BMSCs) seeded in collagen scaffolds in terms of proliferation and differentiation into cardiomyocytes. BMSCs were isolated from Wistar rats and seeded into 3D collagen type 1 templates measuring 25 × 25 × 6 mm. Bipolar in vitro ES was performed during 21 days. Electrical impedance and cell proliferation were measured. Expression of cardiac markers was assessed by immunocytochemistry. Viscoelasticity of collagen matrix was evaluated. Electrical impedance assessments showed a low resistance of 234±41 Ohms which indicates good electrical conductivity of collagen matrix. Cell proliferation at 570 nm as significantly increased in ES groups after seven day (ES 0.129±0.03 vs non-stimulated control matrix 0.06±0.01, P=0.002) and after 21 days, (ES 0.22±0.04 vs control 0.13±0.01, P=0.01). Immunocytoche mistry of BMSCs after 21 days ES showed positive staining of cardiac markers, troponin I, connexin 43, sarcomeric alpha-actinin, slow myosin, fast myosin and desmin. Staining for BMSCs marker CD29 after 21 days was negative. Electrostimulation of cell-seeded collagen matrix changed stem cell morphology and biochemical characteristics, increasing the expression of cardiac markers. Thus, MSC-derived differentiated cells by electrostimulation grafted in biological scaffolds might result in a convenient tissue engineering source for myocardial diseases. PMID:23185681

  13. Fully automated, quantitative, noninvasive assessment of collagen fiber content and organization in thick collagen gels

    NASA Astrophysics Data System (ADS)

    Bayan, Christopher; Levitt, Jonathan M.; Miller, Eric; Kaplan, David; Georgakoudi, Irene

    2009-05-01

    Collagen is the most prominent protein of human tissues. Its content and organization define to a large extent the mechanical properties of tissue as well as its function. Methods that have been used traditionally to visualize and analyze collagen are invasive, provide only qualitative or indirect information, and have limited use in studies that aim to understand the dynamic nature of collagen remodeling and its interactions with the surrounding cells and other matrix components. Second harmonic generation (SHG) imaging emerged as a promising noninvasive modality for providing high-resolution images of collagen fibers within thick specimens, such as tissues. In this article, we present a fully automated procedure to acquire quantitative information on the content, orientation, and organization of collagen fibers. We use this procedure to monitor the dynamic remodeling of collagen gels in the absence or presence of fibroblasts over periods of 12 or 14 days. We find that an adaptive thresholding and stretching approach provides great insight to the content of collagen fibers within SHG images without the need for user input. An additional feature-erosion and feature-dilation step is useful for preserving structure and noise removal in images with low signal. To quantitatively assess the orientation of collagen fibers, we extract the orientation index (OI), a parameter based on the power distribution of the spatial-frequency-averaged, two-dimensional Fourier transform of the SHG images. To measure the local organization of the collagen fibers, we access the Hough transform of small tiles of the image and compute the entropy distribution, which represents the probability of finding the direction of fibers along a dominant direction. Using these methods we observed that the presence and number of fibroblasts within the collagen gel significantly affects the remodeling of the collagen matrix. In the absence of fibroblasts, gels contract, especially during the first few

  14. Fibroblast cluster formation on 3D collagen matrices requires cell contraction dependent fibronectin matrix organization.

    PubMed

    da Rocha-Azevedo, Bruno; Ho, Chin-Han; Grinnell, Frederick

    2013-02-15

    Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin (FN) fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy. PMID:23117111

  15. Microfluidics 3D gel-island chip for single cell isolation and lineage-dependent drug responses study.

    PubMed

    Zhang, Zhixiong; Chen, Yu-Chih; Cheng, Yu-Heng; Luan, Yi; Yoon, Euisik

    2016-07-01

    3D cell culture in the extracellular matrix (ECM), which not only provides structural support to cellular constituents, but also initiates regulatory biochemical cues for a variety of important cell functions in tissue, has become more and more important in understanding cancer pathology and drug testing. Although the ECM-gel has been used in cell culture both in bulk and on-chip, previous studies focused on collective cell behavior rather than single-cell heterogeneity. To track the behavior of each individual cell, we have developed a gel-island chip, which can form thousands of islands containing single cells encapsulated by the desired ECM. Optimized by Poisson's distribution, the device can attain 34% single cell capture efficiency of the exact number of single cells per island. A good culture media exchange rate and high cell viability can be achieved in the gel-islands. The cells in the islands can be automatically counted for high-throughput analysis. As a proof of concept, we monitored the proliferation and differentiation of single Notch+ (stem-like) T47D breast cancer cells. The 3D collagen gel environment was found to be favorable for the stem-like phenotype through better self-renewal and de-differentiation (Notch- to Notch+ transition). More interestingly, we found that the Notch- de-differentiated cells were more resistant to doxorubicin and cisplatin than the Notch+ cells. Combining the 3D ECM culture and single cell resolution, the presented platform can automatically analyze the individual cell behaviors of hundreds of cells using a small amount of drug and reagents. PMID:27270563

  16. 3D composites based on the blends of chitosan and collagen with the addition of hyaluronic acid.

    PubMed

    Sionkowska, Alina; Kaczmarek, Beata; Lewandowska, Katarzyna; Grabska, Sylwia; Pokrywczyńska, Marta; Kloskowski, Tomasz; Drewa, Tomasz

    2016-08-01

    3D porous composites based on blends of chitosan, collagen and hyaluronic acid were obtained through the lyophilization process. Mechanical properties, swelling behavior and thermal stability of the blends were studied. Moreover, SEM images were taken and the structure of the blends was studied. Biological properties of the materials obtained were investigated by analyzing of proliferation rate of fibroblast cells incubated with biomaterial extract using MTT assay (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide). The results showed that the properties of 3D composites based on the blends of chitosan and collagen were altered after the addition 1%, 2% and 5% of hyaluronic acid. Mechanical properties and thermal stability of chitosan/collagen blends were improved in the presence of hyaluronic acid in the composite. New 3D materials based on the blends of chitosan, collagen and hyaluronic acid were non-toxic and did not significantly affect cell morphology. PMID:27151670

  17. True 3D chemical dosimetry (gels, plastics): Development and clinical role

    NASA Astrophysics Data System (ADS)

    Schreiner, L. J.

    2015-01-01

    Since the introduction of volumetric chemical dosimetry with Fricke gel dosimeters in the 1980s, three-dimensional (3D) dosimetry has been a promising technique for the clinic, since it provides a unique methodology for 3D dose measurement of the complex conformal dose distributions achieved by modern techniques such as Intensity Modulated and Volumetric Arc Radiation Therapy. In the last decade, the potential for improved clinical applicability has been advanced by the development of improved 3D dosimeters such as normoxic polymer gel systems, radiochromic plastics (such as PRESAGE) and, recently, newer radiochromic gel dosimeters. Some of these new 3D dosimetry systems were enabled by the availability of optical computed tomography imaging systems for fast dose readout. However, despite its promise, true 3D dosimetry is still not widely practiced in the community. Its use has been confined primarily to select centres of expertise and to specialised quality assurance or commissioning roles where other dosimetry techniques are difficult to implement. In this paper I review some of the current 3D chemical dosimeters available, discuss the requirements for their use and briefly review the roles that these systems can provide to complement the other dose delivery validation approaches available in the clinic. I conclude by describing two roles that may be uniquely served by 3D chemical dosimetry in end-to-end process testing and validation in the complex environment coming into play with the development of Image Guided Adaptive Radiation Therapy.

  18. 3D scanning of internal structure in gel engineering materials with visual scanning microscopic light scattering

    NASA Astrophysics Data System (ADS)

    Watanabe, Yosuke; Gong, Jing; Masato, Makino; Kabir, M. Hasnat; Furukawa, Hidemitsu

    2014-04-01

    The 3D printing technology, causing much attention from the beginning of 2013, will be possibly an alternative method to fabricate the biological soft tissues. Recently our group of Yamagata University has developed the world-first 3D Gel Printer to fabricate the complicated gel-materials with high-strength and biocompatibility. However, there are no 3D scanners that collect the data from the internal structure of complicated gel objects such as eye lens. It means that a new system for scanning the internal structure is needed now. In this study, firstly, we have tried to investigate the gel network of synthetic and biological gel with scanning microscopic light scattering (SMILS). We calculated the Young's modulus of synthetic gels with the SMILS and with the tensile test, and precisely compared the results between them. The temperature dependences of the inside structure and the transparency are observed in the pig crystalline lens. The quantitative analysis indicates the importance of the internal structure of real object. Secondary, we show the new system named Gel-scanner that can provide the 2-dimentional data of the internal structure. From examining our findings, the scanning of internal structure will enable us to expect physical properties of the real object. We convince that the gelscanner will play major role in the various fields.

  19. Control of vascular network location in millimeter-sized 3D-tissues by micrometer-sized collagen coated cells.

    PubMed

    Liu, Chun-Yen; Matsusaki, Michiya; Akashi, Mitsuru

    2016-03-25

    Engineering three-dimensional (3D) vascularized constructs remains a central challenge because capillary network structures are important for sufficient oxygen and nutrient exchange to sustain the viability of engineered constructs. However, construction of 3D-tissues at single cell level has yet to be reported. Previously, we established a collagen coating method for fabricating a micrometer-sized collagen matrix on cell surfaces to control cell distance or cell densities inside tissues. In this study, a simple fabrication method is presented for constructing vascular networks in 3D-tissues over micrometer-sized or even millimeter-sized with controlled cell densities. From the results, well vascularized 3D network structures can be observed with a fluorescence label method mixing collagen coated cells and endothelia cells, indicating that constructed ECM rich tissues have the potential for vascularization, which opens up the possibility for various applications in pharmaceutical or tissue engineering fields. PMID:26920051

  20. 3D Printed PEG-Based Hybrid Nanocomposites Obtained by Sol-Gel Technique.

    PubMed

    Chiappone, Annalisa; Fantino, Erika; Roppolo, Ignazio; Lorusso, Massimo; Manfredi, Diego; Fino, Paolo; Pirri, Candido Fabrizio; Calignano, Flaviana

    2016-03-01

    In this work, three-dimensional (3D) structured hybrid materials were fabricated combining 3D printing technology with in situ generation of inorganic nanoparticles by sol-gel technique. Those materials, consisting of silica nanodomains covalently interconnected with organic polymers, were 3D printed in complex multilayered architectures, incorporating liquid silica precursors into a photocurable oligomer in the presence of suitable photoinitiators and exposing them to a digital light system. A post sol-gel treatment in acidic vapors allowed the in situ generation of the inorganic phase in a dedicated step. This method allows to build hybrid structures operating with a full liquid formulation without meeting the drawbacks of incorporating inorganic powders into 3D printable formulations. The influence of the generated silica nanoparticle on the printed objects was deeply investigated at macro- and nanoscale; the resulting light hybrid structures show improved mechanical properties and, thus, have a huge potential for applications in a variety of advanced technologies. PMID:26871993

  1. Topographical guidance of 3D tumor cell migration at an interface of collagen densities

    NASA Astrophysics Data System (ADS)

    Bordeleau, Francois; Tang, Lauren N.; Reinhart-King, Cynthia A.

    2013-12-01

    During cancer progression, metastatic cells leave the primary tumor and invade into the fibrous extracellular matrix (ECM) within the surrounding stroma. This ECM network is highly heterogeneous, and interest in understanding how this network can affect cell behavior has increased in the past several decades. However, replicating this heterogeneity has proven challenging. Here, we designed and utilized a method to create a well-defined interface between two distinct regions of high- and low-density collagen gels to mimic the heterogeneities in density found in the tumor stroma. We show that cells will invade preferentially from the high-density side into the low-density side. We also demonstrate that the net cell migration is a function of the density of the collagen in which the cells are embedded, and the difference in density between the two regions has minimal effect on cell net displacement and distance travelled. Our data further indicate that a low-to-high density interface promotes directional migration and induces formation of focal adhesion on the interface surface. Together, the current results demonstrate how ECM heterogeneities, in the form of interfacial boundaries, can affect cell migration.

  2. Bioactive fish collagen/polycaprolactone composite nanofibrous scaffolds fabricated by electrospinning for 3D cell culture.

    PubMed

    Choi, Da Jeong; Choi, Seung Mi; Kang, Hae Yeong; Min, Hye-Jin; Lee, Rira; Ikram, Muhammad; Subhan, Fazli; Jin, Song Wan; Jeong, Young Hun; Kwak, Jong-Young; Yoon, Sik

    2015-07-10

    One of the most challenging objectives of 3D cell culture is the development of scaffolding materials with outstanding biocompatibility and favorable mechanical strength. In this study, we fabricated a novel nanofibrous scaffold composed of fish collagen (FC) and polycaprolactone (PCL) blends by using the electrospinning method. Nanofibrous scaffolds were characterized using a scanning electron microscope (SEM), and it was revealed that the diameter of nanofibers decreased as FC content was increased in the FC/PCL composite nanofibers. The cytocompatibility of the FC/PCL scaffolds was evaluated by SEM, WST-1 assay, confocal microscopy, western blot, and RT-PCR. It was found that the scaffolds not only facilitated the adhesion, spreading, protrusions, and proliferation of thymic epithelial cells (TECs), but also stimulated the expression of genes and proteins involved in cell adhesion and T-cell development. Thus, these results suggest that the FC/PCL composite nanofibrous scaffolds will be a useful model of 3D cell culture for TECs and may have wide applicability in the future for engineering tissues or organs. PMID:25617682

  3. 3D bioprinting of BMSC-laden methacrylamide gelatin scaffolds with CBD-BMP2-collagen microfibers.

    PubMed

    Du, Mingchun; Chen, Bing; Meng, Qingyuan; Liu, Sumei; Zheng, Xiongfei; Zhang, Cheng; Wang, Heran; Li, Hongyi; Wang, Nuo; Dai, Jianwu

    2015-01-01

    Three-dimensional (3D) bioprinting combines biomaterials, cells and functional components into complex living tissues. Herein, we assembled function-control modules into cell-laden scaffolds using 3D bioprinting. A customized 3D printer was able to tune the microstructure of printed bone mesenchymal stem cell (BMSC)-laden methacrylamide gelatin scaffolds at the micrometer scale. For example, the pore size was adjusted to 282 ± 32 μm and 363 ± 60 μm. To match the requirements of the printing nozzle, collagen microfibers with a length of 22 ± 13 μm were prepared with a high-speed crusher. Collagen microfibers bound bone morphogenetic protein 2 (BMP2) with a collagen binding domain (CBD) as differentiation-control module, from which BMP2 was able to be controllably released. The differentiation behaviors of BMSCs in the printed scaffolds were compared in three microenvironments: samples without CBD-BMP2-collagen microfibers in the growth medium, samples without microfibers in the osteogenic medium and samples with microfibers in the growth medium. The results indicated that BMSCs showed high cell viability (>90%) during printing; CBD-BMP2-collagen microfibers induced BMSC differentiation into osteocytes within 14 days more efficiently than the osteogenic medium. Our studies suggest that these function-control modules are attractive biomaterials and have potential applications in 3D bioprinting. PMID:26684899

  4. Fabrication of 3D tissue equivalent: an in vitro platform for understanding collagen evolution in healthy and diseased models

    NASA Astrophysics Data System (ADS)

    Urciuolo, F.; Imparato, G.; Casale, C.; Scamardella, S.; Netti, P.

    2013-04-01

    In this study we realized a three-dimensional human dermis equivalent (3D-HDE) and, by exploiting multi-photon microscopy (MPM) we validated its use as an in vitro model to study collagen network re-arrangement under simulated solar exposure. The realization of 3D-HDE has been pursed by means of a bottom-up tissue engineering strategy that comprises firstly the fabrication of micron sized tissue building blocks and then their assembly in a 3D tissue construct. The building blocks injected in a maturation chamber, and cultured under optimized culture condition, were able to fuse due to the establishment of cell-cell and cell-extra cellular matrix (ECM) interactions that induced a biological sintering process resulting in 3D-HDE production. The final 3D tissue was made-up by fibroblasts embedded in their own ECM rich in endogenous collagen type I, resembling the composition and the architecture of native human dermis. Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (TPEF) imaging have been exploited to assess modification in collagen assembly before and after UV irradiation. Textural features and SHG to TPFE ratio of the endogenous ECM within 3D-HDE have been shown to vary after UVA irradiation, proving the hypothesis that the 3DHDE realized can be used as biological platform in vitro to study ECM modifications induced by photo-damage.

  5. Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

    SciTech Connect

    Ko, Jae Hyung; Kim, Yang Hee; Jeong, Seong Hee; Lee, Song; Park, Si-Nae; Shim, In Kyong; Kim, Song Cheol

    2015-08-07

    Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3 dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release.

  6. Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture.

    PubMed

    Lee, Yeong-Bae; Polio, Samuel; Lee, Wonhye; Dai, Guohao; Menon, Lata; Carroll, Rona S; Yoo, Seung-Schik

    2010-06-01

    Time-released delivery of soluble growth factors (GFs) in engineered hydrogel tissue constructs promotes the migration and proliferation of embedded cells, which is an important factor for designing scaffolds that ultimately aim for neural tissue regeneration. We report a tissue engineering technique to print murine neural stem cells (C17.2), collagen hydrogel, and GF (vascular endothelial growth factor: VEGF)-releasing fibrin gel to construct an artificial neural tissue. We examined the morphological changes of the printed C17.2 cells embedded in the collagen and its migration toward the fibrin gel. The cells showed high viability (92.89+/-2.32%) after printing, which was equivalent to that of manually-plated cells. C17.2 cells printed within 1mm from the border of VEGF-releasing fibrin gel showed GF-induced changes in their morphology. The cells printed in this range also migrated toward the fibrin gel, with the total migration distance of 102.4+/-76.1microm over 3days. The cells in the control samples (fibrin without the VEGF or VEGF printed directly in collagen) neither proliferated nor migrated. The results demonstrated that bio-printing of VEGF-containing fibrin gel supported sustained release of the GF in the collagen scaffold. The presented method can be gainfully used in the development of three-dimensional (3D) artificial tissue assays and neural tissue regeneration applications. PMID:20211178

  7. Electrical and Neurotrophin Enhancement of Neurite Outgrowth within a 3D Collagen Scaffold

    PubMed Central

    Adams, Robert D.; Rendell, Sara R.; Counts, Lauren R.; Papke, Jason B.; Willits, Rebecca K.; Harkins, Amy B.

    2016-01-01

    Electrical and chemical stimulation have been studied as potent mechanisms of enhancing nerve regeneration and wound healing. However, it remains unclear how electrical stimuli affect nerve growth, particularly in the presence of neurotrophic factors. The objective of this study was to explore (1) the effect of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) supplementation to support neurite outgrowth in a 3D scaffold, and (2) the effect of brief, low voltage, electrical stimulation (ES) on neurite outgrowth prior to neurotrophin supplementation. Dissociated E11 chick dorsal root ganglia (DRG) were seeded within a 1.5 mg/mL type-I collagen scaffold. For neurotrophin treatments, scaffolds were incubated for 24 hrs in culture media containing nerve growth factor (NGF, 10 ng/mL) or BDNF (200 ng/mL), or both. For ES groups, scaffolds containing neurons were stimulated for 10 min at 8–10 V/m DC, then incubated for 24 hrs with neurotrophin. Fixed and labeled neurons were imaged to measure neurite growth and directionality. BDNF supplementation was not as effective as NGF at supporting DRG neurite outgrowth. ES prior to NGF supplementation improved DRG neurite outgrowth compared to NGF alone. This combination of brief ES with NGF treatment was the most effective treatment compared to NGF or BDNF alone. Brief ES had no impact on neurite directionality in the 3D scaffolds. These results demonstrate that ES improves neurite outgrowth in the presence of neurotrophins, and could provide a potential therapeutic approach to improve nerve regeneration when coupled with neurotrophin treatment. PMID:24710795

  8. Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

    SciTech Connect

    Wang, Hong-Ju; He, Wen-Qi; Chen, Ling; Liu, Wei-Wei; Xu, Qian; Xia, Ming-Yu; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2015-02-20

    Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. - Highlights: • Collagen gel culture changed the morphology of L929 cells. • L929 cell cultured on collagen gel were resistant to TNFα-induced cell death. • Collagen gel culture inhibited TNFα-induced autophagy in L929 cells.

  9. Strategies for Directing the Structure and Function of 3D Collagen Biomaterials across Length Scales

    PubMed Central

    Walters, Brandan D.; Stegemann, Jan P.

    2013-01-01

    Collagen type I is a widely used natural biomaterial that has found utility in a variety of biological and medical applications. Its well characterized structure and role as an extracellular matrix protein make it a highly relevant material for controlling cell function and mimicking tissue properties. Collagen type I is abundant in a number of tissues, and can be isolated as a purified protein. This review focuses on hydrogel biomaterials made by reconstituting collagen type I from a solubilized form, with an emphasis on in vitro studies in which collagen structure can be controlled. The hierarchical structure of collagen from the nanoscale to the macroscale is described, with an emphasis on how structure is related to function across scales. Methods of reconstituting collagen into hydrogel materials are presented, including molding of macroscopic constructs, creation of microscale modules, and electrospinning of nanoscale fibers. The modification of collagen biomaterials to achieve desired structures and functions is also addressed, with particular emphasis on mechanical control of collagen structure, creation of collagen composite materials, and crosslinking of collagenous matrices. Biomaterials scientists have made remarkable progress in rationally designing collagen-based biomaterials and in applying them to both the study of biology and for therapeutic benefit. This broad review illustrates recent examples of techniques used to control collagen structure, and to thereby direct its biological and mechanical functions. PMID:24012608

  10. Flexible Fabrication of Shape-Controlled Collagen Building Blocks for Self-Assembly of 3D Microtissues.

    PubMed

    Zhang, Xu; Meng, Zhaoxu; Ma, Jingyun; Shi, Yang; Xu, Hui; Lykkemark, Simon; Qin, Jianhua

    2015-08-12

    Creating artificial tissue-like structures that possess the functionality, specificity, and architecture of native tissues remains a big challenge. A new and straightforward strategy for generating shape-controlled collagen building blocks with a well-defined architecture is presented, which can be used for self-assembly of complex 3D microtissues. Collagen blocks with tunable geometries are controllably produced and released via a membrane-templated microdevice. The formation of functional microtissues by embedding tissue-specific cells into collagen blocks with expression of specific proteins is described. The spontaneous self-assembly of cell-laden collagen blocks into organized tissue constructs with predetermined configurations is demonstrated, which are largely driven by the synergistic effects of cell-cell and cell-matrix interactions. This new strategy would open up new avenues for the study of tissue/organ morphogenesis, and tissue engineering applications. PMID:25920010

  11. Rapid 3D Printing of Multifunctional Calcium Alginate Gel Pipes using Coaxial Jet Extruder

    NASA Astrophysics Data System (ADS)

    Rykaczewski, Konrad; Damle, Viraj

    2014-11-01

    Calcium alginate (CA) forms when solution containing sodium alginate (SA) comes in contact with a CaCl2 solution. The resulting gel is biocompatible as well as edible and is used in production of bio-scaffolds, artificial plant seeds, and edible substances. In the latter application, referred to in the culinary world as ``spherification,'' flavored liquids are mixed with the SA and dripped into CaCl2 solution to form gel encapsulated flavored ``marbles.'' Previously, crude 3D printing of CA structures has been achieved by stacking of such flavored liquid filled marbles. In turn, solid CA rods have been fabricated by properly mixing flow of the two solutions using a microfluidic device. Here we show that by using two circular cross-section coaxial nozzles to produce coaxial jets of the SA and CaCl2 solutions, liquid filled CA micro-to-mili scale gel pipes can be produced at speeds around ~ 150 mm/s. Such extrusion rate is compatible with most commercially available 3D printers, facilitating adoption of the CA pipe coaxial jet extruder. Here, the impact of inner and outer liquid properties and flow speeds on the gel pipe extrusion process is discussed. KR acknowledges startup funding from ASU.

  12. Collagen-poly(dialdehyde) guar gum based porous 3D scaffolds immobilized with growth factor for tissue engineering applications.

    PubMed

    Ragothaman, Murali; Palanisamy, Thanikaivelan; Kalirajan, Cheirmadurai

    2014-12-19

    Here we report the preparation of collagen-poly(dialdehyde) guar gum based hybrid functionalized scaffolds covalently immobilized with platelet derived growth factor - BB for tissue engineering applications. Poly(dialdehyde) guar gum was synthesized from selective oxidation of guar gum using sodium periodate. The synthesized poly(dialdehyde) guar gum not only promotes crosslinking of collagen but also immobilizes the platelet derived growth factor through imine bonds. The covalent crosslinking formed in collagen improves thermal, swelling and biodegradation properties of the hybrid scaffolds. The prepared hybrid scaffolds show 3D interconnected honeycomb porous structure when viewed under a microscope. The release of immobilized platelet derived growth factor was seen up to 13th day of incubation thereby proving its sustained delivery. The developed hybrid scaffold leads to a quantum increase in NIH 3T3 fibroblast cell density and proliferation thereby demonstrating its potential for tissue engineering applications. PMID:25263907

  13. Focusing optics of a parallel beam CCD optical tomography apparatus for 3D radiation gel dosimetry.

    PubMed

    Krstajić, Nikola; Doran, Simon J

    2006-04-21

    Optical tomography of gel dosimeters is a promising and cost-effective avenue for quality control of radiotherapy treatments such as intensity-modulated radiotherapy (IMRT). Systems based on a laser coupled to a photodiode have so far shown the best results within the context of optical scanning of radiosensitive gels, but are very slow ( approximately 9 min per slice) and poorly suited to measurements that require many slices. Here, we describe a fast, three-dimensional (3D) optical computed tomography (optical-CT) apparatus, based on a broad, collimated beam, obtained from a high power LED and detected by a charged coupled detector (CCD). The main advantages of such a system are (i) an acquisition speed approximately two orders of magnitude higher than a laser-based system when 3D data are required, and (ii) a greater simplicity of design. This paper advances our previous work by introducing a new design of focusing optics, which take information from a suitably positioned focal plane and project an image onto the CCD. An analysis of the ray optics is presented, which explains the roles of telecentricity, focusing, acceptance angle and depth-of-field (DOF) in the formation of projections. A discussion of the approximation involved in measuring the line integrals required for filtered backprojection reconstruction is given. Experimental results demonstrate (i) the effect on projections of changing the position of the focal plane of the apparatus, (ii) how to measure the acceptance angle of the optics, and (iii) the ability of the new scanner to image both absorbing and scattering gel phantoms. The quality of reconstructed images is very promising and suggests that the new apparatus may be useful in a clinical setting for fast and accurate 3D dosimetry. PMID:16585845

  14. 3D Assembly of All-Inorganic Colloidal Nanocrystals into Gels and Aerogels.

    PubMed

    Sayevich, Vladimir; Cai, Bin; Benad, Albrecht; Haubold, Danny; Sonntag, Luisa; Gaponik, Nikolai; Lesnyak, Vladimir; Eychmüller, Alexander

    2016-05-17

    We report an efficient approach to assemble a variety of electrostatically stabilized all-inorganic semiconductor nanocrystals (NCs) by their linking with appropriate ions into multibranched gel networks. These all-inorganic non-ordered 3D assemblies benefit from strong interparticle coupling, which facilitates charge transport between the NCs with diverse morphologies, compositions, sizes, and functional capping ligands. Moreover, the resulting dry gels (aerogels) are highly porous monolithic structures, which preserve the quantum confinement of their building blocks. The inorganic semiconductor aerogel made of 4.5 nm CdSe colloidal NCs capped with I(-) ions and bridged with Cd(2+) ions had a large surface area of 146 m(2)  g(-1) . PMID:27100131

  15. Preparation and characterization of cross-linked collagen-phospholipid polymer hybrid gels.

    PubMed

    Nam, Kwangwoo; Kimura, Tsuyoshi; Kishida, Akio

    2007-01-01

    2-methacryloyloxyethyl phosphorylcholine (MPC)-immobilized collagen gel was developed. Using 1-ethyl-3-(3-dimethyl aminopropyl)-1-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), we cross-linked a collagen film in 2-morpholinoethane sulfonic acid (MES) buffer (EN gel). EN gel was prepared under both pH 4.5 and pH 9.0 in order to observe changes in cross-linking ability. To cross-link MPC to collagen gel, poly(MPC-co-methacrylic acid) (PMA) having a carboxyl group side chain was chosen. E/N gel was added to the MES buffer having pre-NHS activated PMA to make MPC-immobilized collagen gel (MiC gel). MiC gel was prepared under both acidic and alkaline conditions to observe the changes in the cross-linking ability of PMA. X-ray photoelectron spectroscopy showed that the PMA was cross-linked with collagen under both acidic and alkaline conditions. Differential scanning calorimetry (DSC) results showed that the shrinkage temperature increased for the MiC gels and that the increase would be greater for the MiC gel prepared under alkaline conditions. The data showed that swelling would be less when the MiC gel was prepared under alkaline conditions. The biodegradation caused by collagenase was suppressed for the MiC gel prepared under alkaline conditions due to stable inter- and intrahelical networks. PMID:16959313

  16. Best fit refractive index of matching liquid for 3D NIPAM gel dosimeters using optical CT

    NASA Astrophysics Data System (ADS)

    Chen, Chin-Hsing; Wu, Jay; Hsieh, Bor-Tsung; Chen, De-Shiou; Wang, Tzu-Hwei; Chien, Sou-Hsin; Chang, Yuan-Jen

    2014-11-01

    The accuracy of an optical computed tomography (CT)-based dosimeter is significantly affected by the refractive index (RI) of the matching liquid. Mismatched RI induces reflection and refraction as the laser beam passes through the gel phantom. Moreover, the unwanted light rays collected by the photodetector produce image artifacts after image reconstruction from the collected data. To obtain the best image quality, this study investigates the best-fit RI of the matching liquid for a 3D NIPAM gel dosimeter. The three recipes of NIPAM polymer gel used in this study consisted of 5% gelatin, 5% NIPAM and 3% N,N'-methylene bisacrylamide, which were combined with three compositions (5, 10, and 20 mM) of Tetrakis (hydroxymethyl) phosphonium chloride. Results were evaluated using a quantitative evaluation method of the gamma evaluation technique. Results showed that the best-fit RI for the non-irradiated NIPAM gel ranges from 1.340 to 1.346 for various NIPAM recipes with sensitivities ranging from 0.0113 to 0.0227. The greatest pass rate of 88.00% is achieved using best-fit RI=1.346 of the matching liquid. The adoption of mismatching RI decreases the gamma pass rate by 2.63% to 16.75% for all three recipes of NIPAM gel dosimeters. In addition, the maximum average deviation is less than 0.1% for the red and transparent matching liquids. Thus, the color of the matching liquid does not affect the measurement accuracy of the NIPAM gel dosimeter, as measured by optical CT.

  17. Fabrication of Collagen Gel Hollow Fibers by Covalent Cross-Linking for Construction of Bioengineering Renal Tubules.

    PubMed

    Shen, Chong; Zhang, Guoliang; Wang, Qichen; Meng, Qin

    2015-09-01

    Collagen, the most used natural biomacromolecule, has been extensively utilized to make scaffolds for cell cultures in tissue engineering, but has never been fabricated into the configuration of a hollow fiber (HF) for cell culture due to its poor mechanical properties. In this study, renal tubular cell-laden collagen hollow fiber (Col HF) was fabricated by dissolving sacrificial Ca-alginate cores from collagen shells strengthened by carbodiimide cross-linking. The inner/outer diameters of the Col HF were precisely controlled by the flow rates of core alginate/shell collagen solution in the microfluidic device. As found, the renal tubular cells self-assembled into renal tubules with diameters of 50-200 μm post to the culture in Col HF for 10 days. According to the 3D reconstructed confocal images or HE staining, the renal cells appeared as a tight tubular monolayer on the Col HF inner surface, sustaining more 3D cell morphology than the cell layer on the 2D flat collagen gel surface. Moreover, compared with the cultures in either a Transwell or polymer HF membrane, the renal tubules in Col HF exhibited at least 1-fold higher activity on brush border enzymes of alkaline phosphatase and γ-glutamyltransferase, consistent with their gene expressions. The enhancement occurred similarly on multidrug resistance protein 2 and glucose uptake. Such bioengineered renal tubules in Col HF will present great potential as alternatives to synthetic HF in both clinical use and pharmaceutical investigation. PMID:26280545

  18. Improved MAGIC gel for higher sensitivity and elemental tissue equivalent 3D dosimetry

    SciTech Connect

    Zhu Xuping; Reese, Timothy G.; Crowley, Elizabeth M.; El Fakhri, Georges

    2010-01-15

    Purpose: Polymer-based gel dosimeter (MAGIC type) is a preferable phantom material for PET range verification of proton beam therapy. However, improvement in elemental tissue equivalency (specifically O/C ratio) is very desirable to ensure realistic time-activity measurements. Methods: Glucose and urea was added to the original MAGIC formulation to adjust the O/C ratio. The dose responses of the new formulations were tested with MRI transverse relaxation rate (R2) measurements. Results: The new ingredients improved not only the elemental composition but also the sensitivity of the MAGIC gel. The O/C ratios of our new gels agree with that of soft tissue within 1%. The slopes of dose response curves were 1.6-2.7 times larger with glucose. The melting point also increased by 5 deg. C. Further addition of urea resulted in a similar slope but with an increased intercept and a decreased melting point. Conclusions: Our improved MAGIC gel formulations have higher sensitivity and better elemental tissue equivalency for 3D dosimetry applications involving nuclear reactions.

  19. Improved MAGIC gel for higher sensitivity and elemental tissue equivalent 3D dosimetry

    PubMed Central

    Zhu, Xuping; Reese, Timothy G.; Crowley, Elizabeth M.; El Fakhri, Georges

    2010-01-01

    Purpose: Polymer-based gel dosimeter (MAGIC type) is a preferable phantom material for PET range verification of proton beam therapy. However, improvement in elemental tissue equivalency (specifically O∕C ratio) is very desirable to ensure realistic time-activity measurements. Methods: Glucose and urea was added to the original MAGIC formulation to adjust the O∕C ratio. The dose responses of the new formulations were tested with MRI transverse relaxation rate (R2) measurements. Results: The new ingredients improved not only the elemental composition but also the sensitivity of the MAGIC gel. The O∕C ratios of our new gels agree with that of soft tissue within 1%. The slopes of dose response curves were 1.6–2.7 times larger with glucose. The melting point also increased by 5 °C. Further addition of urea resulted in a similar slope but with an increased intercept and a decreased melting point. Conclusions: Our improved MAGIC gel formulations have higher sensitivity and better elemental tissue equivalency for 3D dosimetry applications involving nuclear reactions. PMID:20175480

  20. Study of a non-diffusing radiochromic gel dosimeter for 3D radiation dose imaging

    NASA Astrophysics Data System (ADS)

    Marsden, Craig Michael

    2000-12-01

    This thesis investigates the potential of a new radiation gel dosimeter, based on nitro-blue tetrazolium (NBTZ) suspended in a gelatin mold. Unlike all Fricke based gel dosimeters this dosimeter does not suffer from diffusive loss of image stability. Images are obtained by an optical tomography method. Nitro blue tetrazolium is a common biological indicator that when irradiated in an aqueous medium undergoes reduction to a highly colored formazan, which has an absorbance maximum at 525nm. Tetrazolium is water soluble while the formazan product is insoluble. The formazan product sticks to the gelatin matrix and the dose image is maintained for three months. Methods to maximize the sensitivity of the system were evaluated. It was found that a chemical detergent, Triton X-100, in combination with sodium formate, increased the dosimeter sensitivity significantly. An initial G-value of formazan production for a dosimeter composed of 1mM NBTZ, gelatin, and water was on the order of 0.2. The addition of Triton and formate produced a G-value in excess of 5.0. The effects of NBTZ, triton, formate, and gel concentration were all investigated. All the gels provided linear dose vs. absorbance plots for doses from 0 to >100 Gy. It was determined that gel concentration had minimal if any effect on sensitivity. Sensitivity increased slightly with increasing NBTZ concentration. Triton and formate individually and together provided moderate to large increases in dosimeter sensitivity. The dosimeter described in this work can provide stable 3D radiation dose images for all modalities of radiation therapy equipment. Methods to increase sensitivity are developed and discussed.

  1. Sol-gel assisted fabrication of collagen hydrolysate composite scaffold: a novel therapeutic alternative to the traditional collagen scaffold.

    PubMed

    Ramadass, Satiesh Kumar; Perumal, Sathiamurthi; Gopinath, Arun; Nisal, Anuya; Subramanian, Saravanan; Madhan, Balaraman

    2014-09-10

    Collagen is one of the most widely used biomaterial for various biomedical applications. In this Research Article, we present a novel approach of using collagen hydrolysate, smaller fragments of collagen, as an alternative to traditionally used collagen scaffold. Collagen hydrolysate composite scaffold (CHCS) was fabricated with sol-gel transition procedure using tetraethoxysilane as the silica precursor. CHCS exhibits porous morphology with pore sizes varying between 380 and 780 μm. Incorporation of silica conferred CHCS with controlled biodegradation and better water uptake capacity. Notably, 3T3 fibroblast proliferation was seen to be significantly better under CHCS treatment when compared to treatment with collagen scaffold. Additionally, CHCS showed excellent antimicrobial activity against the wound pathogens Staphylococcus aureus, Bacillus subtilis, and Escherichia coli due to the inherited antimicrobial activity of collagen hydrolysate. In vivo wound healing experiments with full thickness excision wounds in rat model demonstrated that wounds treated with CHCS showed accelerated healing when compared to wounds treated with collagen scaffold. These findings indicate that the CHCS scaffold from collagen fragments would be an effective and affordable alternative to the traditionally used collagen structural biomaterials. PMID:25105509

  2. Doxorubicin-conjugated dendrimer/collagen hybrid gels for metastasis-associated drug delivery systems.

    PubMed

    Kojima, Chie; Suehiro, Tomoyuki; Watanabe, Kenji; Ogawa, Mikako; Fukuhara, Ayano; Nishisaka, Eiko; Harada, Atsushi; Kono, Kenji; Inui, Takashi; Magata, Yasuhiro

    2013-03-01

    Metastasis is a characteristic property of cancer cells, which degrade extracellular matrix containing collagen. We prepared a polymer prodrug-embedded collagen gel for metastasis-associated drug delivery. A collagen peptide-modified dendrimer that attached doxorubicin (Dox) via a pH-degradable linkage was synthesized as a polymer prodrug. Compared with free Dox, the diffusion of the dendrimer prodrug from the collagen gel was suppressed. Highly invasive MDA-MB-231 cells were more sensitive to the prodrug-hybrid collagen gel than poorly invasive MCF-7 cells, even though the cytotoxicity of the dendrimer prodrug by itself against these cells was almost identical. The cytotoxicity against MDA-MB-231 cells decreased in the presence of a matrix metalloproteinase (MMP) inhibitor, suggesting that the dendrimer prodrug/collagen hybrid gel was affected by MMP activity. The dendrimer prodrug/collagen hybrid gel not only suppressed tumor growth but also attenuated metastatic activity in vivo. Therefore, the dendrimer prodrug-embedded collagen gel is useful for cancer chemotherapy. PMID:23164946

  3. Cutaneous Wound Healing After Treatment with Plant-Derived Human Recombinant Collagen Flowable Gel

    PubMed Central

    Roth, Sigal; Amzel, Tal; Harel-Adar, Tamar; Tamir, Eran; Grynspan, Frida; Shoseyov, Oded

    2013-01-01

    Chronic wounds, particularly diabetic ulcers, represent a main public health concern with significant costs. Ulcers often harbor an additional obstacle in the form of tunneled or undermined wounds, requiring treatments that can reach the entire wound tunnel, because bioengineered grafts are typically available only in a sheet form. While collagen is considered a suitable biodegradable scaffold material, it is usually extracted from animal and human cadaveric sources, and accompanied by potential allergic and infectious risks. The purpose of this study was to test the performance of a flowable gel made of human recombinant type I collagen (rhCollagen) produced in transgenic tobacco plants, indicated for the treatment of acute, chronic, and tunneled wounds. The performance of the rhCollagen flowable gel was tested in an acute full-thickness cutaneous wound-healing rat model and compared to saline treatment and two commercial flowable gel control products made of bovine collagen and cadaver human skin collagen. When compared to the three control groups, the rhCollagen-based gel accelerated wound closure and triggered a significant jumpstart to the healing process, accompanied by enhanced re-epithelialization. In a cutaneous full-thickness wound pig model, the rhCollagen-based flowable gel induced accelerated wound healing compared to a commercial product made of bovine tendon collagen. By day 21 post-treatment, 95% wound closure was observed with the rhCollagen product compared to 68% closure in wounds treated with the reference product. Moreover, rhCollagen treatment induced an early angiogenic response and induced a significantly lower inflammatory response than in the control group. In summary, rhCollagen flowable gel proved to be efficacious in animal wound models and is expected to be capable of reducing the healing time of human wounds. PMID:23259631

  4. Cutaneous wound healing after treatment with plant-derived human recombinant collagen flowable gel.

    PubMed

    Shilo, Shani; Roth, Sigal; Amzel, Tal; Harel-Adar, Tamar; Tamir, Eran; Grynspan, Frida; Shoseyov, Oded

    2013-07-01

    Chronic wounds, particularly diabetic ulcers, represent a main public health concern with significant costs. Ulcers often harbor an additional obstacle in the form of tunneled or undermined wounds, requiring treatments that can reach the entire wound tunnel, because bioengineered grafts are typically available only in a sheet form. While collagen is considered a suitable biodegradable scaffold material, it is usually extracted from animal and human cadaveric sources, and accompanied by potential allergic and infectious risks. The purpose of this study was to test the performance of a flowable gel made of human recombinant type I collagen (rhCollagen) produced in transgenic tobacco plants, indicated for the treatment of acute, chronic, and tunneled wounds. The performance of the rhCollagen flowable gel was tested in an acute full-thickness cutaneous wound-healing rat model and compared to saline treatment and two commercial flowable gel control products made of bovine collagen and cadaver human skin collagen. When compared to the three control groups, the rhCollagen-based gel accelerated wound closure and triggered a significant jumpstart to the healing process, accompanied by enhanced re-epithelialization. In a cutaneous full-thickness wound pig model, the rhCollagen-based flowable gel induced accelerated wound healing compared to a commercial product made of bovine tendon collagen. By day 21 post-treatment, 95% wound closure was observed with the rhCollagen product compared to 68% closure in wounds treated with the reference product. Moreover, rhCollagen treatment induced an early angiogenic response and induced a significantly lower inflammatory response than in the control group. In summary, rhCollagen flowable gel proved to be efficacious in animal wound models and is expected to be capable of reducing the healing time of human wounds. PMID:23259631

  5. On the validity of 3D polymer gel dosimetry: III. MRI-related error sources

    NASA Astrophysics Data System (ADS)

    Vandecasteele, Jan; De Deene, Yves

    2013-01-01

    In MRI (PAGAT) polymer gel dosimetry, there exists some controversy on the validity of 3D dose verifications of clinical treatments. The relative contribution of important sources of uncertainty in MR scanning to the overall accuracy and precision of 3D MRI polymer gel dosimetry is quantified in this study. The performance in terms of signal-to-noise and imaging artefacts was evaluated on three different MR scanners (two 1.5 T and a 3 T scanner). These include: (1) B0-field inhomogeneity, (2) B1-field inhomogeneity, (3) dielectric effects (losses and standing waves) and (4) temperature inhomogeneity during scanning. B0-field inhomogeneities that amount to maximum 5 ppm result in dose deviations of up to 4.3% and deformations of up to 5 pixels. Compensation methods are proposed. B1-field inhomogeneities were found to induce R2 variations in large anthropomorphic phantoms both at 1.5 and 3 T. At 1.5 T these effects are mainly caused by the coil geometry resulting in dose deviations of up to 25%. After the correction of the R2 maps using a heuristic flip angle-R2 relation, these dose deviations are reduced to 2.4%. At 3 T, the dielectric properties of the gel phantoms are shown to strongly influence B1-field homogeneity, hence R2 homogeneity, especially of large anthropomorphic phantoms. The low electrical conductivity of polymer gel dosimeters induces standing wave patterns resulting in dose deviations up to 50%. Increasing the conductivity of the gel by adding NaCl reduces the dose deviation to 25% after which the post-processing is successful in reducing the remaining inhomogeneities caused by the coil geometry to within 2.4%. The measurements are supported by computational modelling of the B1-field. Finally, temperature fluctuations of 1 °C frequently encountered in clinical MRI scanners result in dose deviations up to 15%. It is illustrated that with adequate temperature stabilization, the dose uncertainty is reduced to within 2.58%. Both authors contributed

  6. On the validity of 3D polymer gel dosimetry: III. MRI-related error sources.

    PubMed

    Vandecasteele, Jan; De Deene, Yves

    2013-01-01

    In MRI (PAGAT) polymer gel dosimetry, there exists some controversy on the validity of 3D dose verifications of clinical treatments. The relative contribution of important sources of uncertainty in MR scanning to the overall accuracy and precision of 3D MRI polymer gel dosimetry is quantified in this study. The performance in terms of signal-to-noise and imaging artefacts was evaluated on three different MR scanners (two 1.5 T and a 3 T scanner). These include: (1) B₀-field inhomogeneity, (2) B₁-field inhomogeneity, (3) dielectric effects (losses and standing waves) and (4) temperature inhomogeneity during scanning. B₀-field inhomogeneities that amount to maximum 5 ppm result in dose deviations of up to 4.3% and deformations of up to 5 pixels. Compensation methods are proposed. B₁-field inhomogeneities were found to induce R₂ variations in large anthropomorphic phantoms both at 1.5 and 3 T. At 1.5 T these effects are mainly caused by the coil geometry resulting in dose deviations of up to 25%. After the correction of the R₂ maps using a heuristic flip angle-R₂ relation, these dose deviations are reduced to 2.4%. At 3 T, the dielectric properties of the gel phantoms are shown to strongly influence B₁-field homogeneity, hence R₂ homogeneity, especially of large anthropomorphic phantoms. The low electrical conductivity of polymer gel dosimeters induces standing wave patterns resulting in dose deviations up to 50%. Increasing the conductivity of the gel by adding NaCl reduces the dose deviation to 25% after which the post-processing is successful in reducing the remaining inhomogeneities caused by the coil geometry to within 2.4%. The measurements are supported by computational modelling of the B₁-field. Finally, temperature fluctuations of 1 °C frequently encountered in clinical MRI scanners result in dose deviations up to 15%. It is illustrated that with adequate temperature stabilization, the dose uncertainty is reduced to within 2.58%. PMID

  7. Modulating the Rigidity and Mineralization of Collagen Gels Using Poly(Lactic-Co-Glycolic Acid) Microparticles

    PubMed Central

    DeVolder, Ross J.; Kim, Il Won; Kim, Eun-Suk

    2012-01-01

    Extensive efforts have been made to prepare osteoconductive collagen gels for the regeneration of normal bone and the pathological examination of diseased bone; however, collagen gels are often plagued by limited controllability of their rigidity and mineral deposition. This study reports a simple but efficient strategy that tunes the mechanical properties of, and apatite formation in, collagen gels by incorporating hydrolyzable poly(lactic-co-glycolic acid) (PLGA) microparticles within the gels. The PLGA microparticles are associated with the collagen fibrils and increased both the gel's elasticity and rigidity while minimally influencing its permeability. As compared with pure collagen gels, the PLGA microparticle-filled collagen gels, termed PLGA-Col hydrogels, significantly enhanced the deposition of apatite-like minerals within the gels when incubated in simulated body fluid or encapsulated with mesenchymal stem cells (MSCs) undergoing osteogenic differentiation. Finally, PLGA-Col hydrogels mineralized by differentiated MSCs led to an enhanced formation of bone-like tissues within the hydrogels. Overall, the PLGA-Col hydrogel system developed in this study will serve to improve the quality of osteoconductive matrices for both fundamental and clinical studies that are relevant to bone repair, regeneration, and pathogenesis. PMID:22480235

  8. Effect of papain-based gel on type I collagen - spectroscopy applied for microstructural analysis

    PubMed Central

    Júnior, Zenildo Santos Silva; Botta, Sergio Brossi; Ana, Patricia Aparecida; França, Cristiane Miranda; Fernandes, Kristianne Porta Santos; Mesquita-Ferrari, Raquel Agnelli; Deana, Alessandro; Bussadori, Sandra Kalil

    2015-01-01

    Considering the improvement of biomaterials that facilitate atraumatic restorative techniques in dentistry, a papain-based gel can be used in the chemomechanical removal of decayed dental tissue. However, there is no information regarding the influence of this gel on the structure of sound collagen. The aim of the present study was to investigate the adsorption of a papain-based gel (PapacarieTM) to collagen and determine collagen integrity after treatment. A pilot study was first performed with 10 samples of type I collagen membrane obtained from bovine Achilles deep tendon to compare the influence of hydration (Milli-Q water) on infrared bands of collagen. In a further experiment, 10 samples of type I collagen membrane were used to evaluate the effects of PapacarieTM on the collagen microstructure. All analyses were performed using the attenuated total reflectance technique of Fourier transform infrared (ATR-FTIR). The results demonstrated that the application of PapacarieTM does not lead to the degradation of collagen and this product can be safely used in minimally invasive dentistry. As the integrity of sound collagen is preserved after the application of the papain-based gel, this product is indicated for the selective removal of infected dentin, leaving the affected dentin intact and capable of re-mineralization. PMID:26101184

  9. Functional properties of human thyroid follicles cultured within collagen gel.

    PubMed

    Massart, C; Hody, B; Condé, D; Leclech, G; Edan, G; Nicol, M

    1988-04-01

    Cultures of human thyroid follicles embedded in collagen gel were performed to investigate certain functional properties under bovine thyrotropin (TSH) stimulation. Follicles obtained from normal glands responded to increasing concentrations of TSH administered on day 4 in culture and for 3 days by increased amounts of cyclic AMP (cAMP), thyroglobulin (Tg) and triiodothyronine (T3) and by decreased levels of thyroxine (T4). Effect was maximal at 2000 microU/ml TSH (cAMP) or 200 microU/ml (Tg, T3, T4). When methimazole or propylthiouracil (PTU) were added, the T3 levels decreased. Follicle lumens contained a periodic acid-Schiff substance which was identified by immunoreaction as Tg. Thyroid follicles obtained from Graves' disease glands gave modified results with an earlier and intensified T3 response and no increase in Tg. These data show that (1) Tg and T3 are secretory products of functional follicles giving a cAMP-mediated response to TSH. (2) The detected T3 also derives from T4 5'-deiodination inhibited by PTU. (3) Intensified T3 response in Graves' follicles is probably due to enhanced conversion of T4 to T3. PMID:2836248

  10. Growth of Normal Mouse Vaginal Epithelial Cells in and on Collagen Gels

    NASA Astrophysics Data System (ADS)

    Iguchi, Taisen; Uchima, Francis-Dean A.; Ostrander, Patricia L.; Bern, Howard A.

    1983-06-01

    Sustained growth in primary culture of vaginal epithelial cells from ovariectomized adult BALB/cCrg1 mice embedded within or seeded on collagen gel matrix was achieved in a serum-free medium composed of Ham's F-12 medium/Dulbecco's modified Eagle's medium, 1:1 (vol/vol), supplemented with insulin, bovine serum albumin fraction V, epidermal growth factor, cholera toxin, and transferrin. Three-dimensional growth of vaginal epithelial cells occurred inside the collagen gel matrix. Cell numbers increased 4- to 8-fold in collagen gel and about 4-fold on collagen gel after 9-10 days in culture. The effect of 17β -estradiol (0.00018-180 nM in gel or 0.018-180 nM on gel) and diethylstilbestrol (DES; 0.0186-186 nM in gel) on the growth of vaginal epithelial cells was examined. The addition of estrogen did not enhance the growth of vaginal epithelial cells during this time period either in the complete medium or in a suboptimal medium. Cultures on floating collagen gels in the serum-free medium are composed of 1-3 cell layers with superficial cornification. Estrogen does not appear to be a direct mitogen for vaginal epithelial cells, at least in this system.

  11. Diffusion and convection in collagen gels: implications for transport in the tumor interstitium.

    PubMed Central

    Ramanujan, Saroja; Pluen, Alain; McKee, Trevor D; Brown, Edward B; Boucher, Yves; Jain, Rakesh K

    2002-01-01

    Diffusion coefficients of tracer molecules in collagen type I gels prepared from 0-4.5% w/v solutions were measured by fluorescence recovery after photobleaching. When adjusted to account for in vivo tortuosity, diffusion coefficients in gels matched previous measurements in four human tumor xenografts with equivalent collagen concentrations. In contrast, hyaluronan solutions hindered diffusion to a lesser extent when prepared at concentrations equivalent to those reported in these tumors. Collagen permeability, determined from flow through gels under hydrostatic pressure, was compared with predictions obtained from application of the Brinkman effective medium model to diffusion data. Permeability predictions matched experimental results at low concentrations, but underestimated measured values at high concentrations. Permeability measurements in gels did not match previous measurements in tumors. Visualization of gels by transmission electron microscopy and light microscopy revealed networks of long collagen fibers at lower concentrations along with shorter fibers at high concentrations. Negligible assembly was detected in collagen solutions pregelation. However, diffusion was similarly hindered in pre and postgelation samples. Comparison of diffusion and convection data in these gels and tumors suggests that collagen may obstruct diffusion more than convection in tumors. These findings have significant implications for drug delivery in tumors and for tissue engineering applications. PMID:12202388

  12. Effect of gravity and diffusion interface proximity on the morphology of collagen gels.

    PubMed

    Roedersheimer, M T; Bateman, T A; Simske, S J

    1997-11-01

    Collagen solutions (0.25% w/v) were polymerized in microgravity (STS-77, 10 days) along with simultaneous ground controls. Assembly conditions were achieved by the passage of buffer ions across a dialysis membrane into a reaction chamber containing the dissolved collagen. The gels were analyzed macroscopically and microscopically to assess the influence of gravity and the oriented diffusion of buffer ions on the resulting product. Double-blind rankings based on visual observation of the gels established that all of the flight gels (n = 8) were more uniform in appearance than all of the ground gels (n = 6). Photography using side illumination of the gels revealed the more granular appearance of the ground gels relative to the highly uniform appearance of the flight gels. Scanning electron microscopy established this difference at the microscopic level. Proximity to the dialysis interface and the presence or absence of gravity were both found to control the porosity and uniformity of the matrix. PMID:9358322

  13. Aligned 3D human aortic smooth muscle tissue via layer by layer technique inside microchannels with novel combination of collagen and oxidized alginate hydrogel.

    PubMed

    Rayatpisheh, Shahrzad; Poon, Yin Fun; Cao, Ye; Feng, Jie; Chan, Vincent; Chan-Park, Mary B

    2011-08-01

    Tissue engineering of the small diameter blood vessel medial layer has been challenging. Recreation of the circumferentially aligned multilayer smooth muscle tissue has been one of the major technical difficulties. Some research has utilized cyclic stress to align smooth muscle cells (SMCs) but due to the long time conditioning needed, it was not possible to use primary human cells because of expeditious senescence occurred . We demonstrate rapid buildup of a homogeneous relatively thick (30-40 μm) aligned smooth muscle tissue via layer by layer (LBL) technique within microchannels and a soft cell-adhesive hydrogel. Using a microchannelled scaffold with gapped microwalls, two layers of primary human SMCs separated by an interlayer hydrogel were cultured to confluence within the microchannels. The SMCs aligned along the microchannels because of the physically constraining microwalls. A novel double layered gel consisting of a mixture of pristine and oxidized alginate hydrogel coated with collagen was designed to place between each layer of cells, leading to a thicker tissue in a shorter time. The SMCs penetrated the soft thin interlayer hydrogel within 6 days of seeding of the 2nd cell layer so that the entire construct became more or less homogeneously populated by the SMCs. The unique LBL technique applied within the micropatterned scaffold using a soft cell-adhesive gel interlayer allows rapid growth and confluence of SMCs on 2D surface but at the same time aligns the cells and builds up multiple layers into a 3D tissue. This pseudo-3D buildup method avoids the typical steric resistance of hydrogel embedding. PMID:21548018

  14. Thickness sensing of hMSCs on collagen gel directs stem cell fate

    SciTech Connect

    Leong, Wen Shing; Tay, Chor Yong; Yu, Haiyang; Li, Ang; Wu, Shu Cheng; Duc, Duong-Hong; Lim, Chwee Teck; Tan, Lay Poh

    2010-10-15

    Research highlights: {yields} hMSCs appeared to sense thin collagen gel (130 {mu}m) with higher effective modulus as compared to thick gel (1440 {mu}m). {yields} Control of collagen gel thickness can modulate cellular behavior, even stem cell fate (neuronal vs. Quiescent). {yields} Distinct cellular behavior of hMSCs on thin and thick collagen gel suggests long range interaction of hMSCs with collagen gel. -- Abstract: Mechanically compliant substrate provides crucial biomechanical cues for multipotent stem cells to regulate cellular fates such as differentiation, proliferation and maintenance of their phenotype. Effective modulus of which cells sense is not only determined by intrinsic mechanical properties of the substrate, but also the thickness of substrate. From our study, it was found that interference from underlying rigid support at hundreds of microns away could induce significant cellular response. Human mesenchymal stem cells (hMSCs) were cultured on compliant biological gel, collagen type I, of different thickness but identical ECM composition and local stiffness. The cells sensed the thin gel (130 {mu}m) as having a higher effective modulus than the thick gel (1440 {mu}m) and this was reflected in their changes in morphology, actin fibers structure, proliferation and tissue specific gene expression. Commitment into neuronal lineage was observed on the thin gel only. Conversely, the thick gel (1440 {mu}m) was found to act like a substrate with lower effective modulus that inhibited actin fiber polymerization. Stem cells on the thick substrate did not express tissue specific genes and remained at their quiescent state. This study highlighted the need to consider not only the local modulus but also the thickness of biopolymer gel coating during modulation of cellular responses.

  15. Collagen-gel-induced resistance of overlying keratinocyte cultures to photosensitization

    NASA Astrophysics Data System (ADS)

    Katsantonis, John C.; Georgiou, Savas K.; Providaki, Mary G.; Vlahonikolis, John G.; Tosca, Andronicki D.

    1997-12-01

    In the present study it was found that human keratinocytes grown on collagen substrate, exhibited increased resistance to the hematoprophyrin-mediated photodynamic treatment, in comparison to keratinocytes grown on Petri dishes without collagen. Interestingly, no protection was afforded by the collagen gel to the cells in the corresponding control ('hematoprophyrin only' and 'light only') experiments. This observation was found to be independent of light dose and drug concentration, and the relative degree of resistance was the same for both normal and malignant cells. The degree of keratinocyte resistance was found to be closely related to the duration of cell attachment on the collagen substrate. These results are indicative of an active interference of collagen gel with the cellular evolution of the photodynamic phenomenon and they are also suggestive of variation in the photodynamic treatment efficacy according to the cellular environment.

  16. Microencapsulation of Neuroblastoma Cells and Mesenchymal Stromal Cells in Collagen Microspheres: A 3D Model for Cancer Cell Niche Study

    PubMed Central

    Yeung, Pan; Sin, Hoi Shun; Chan, Shing; Chan, Godfrey Chi Fung; Chan, Barbara Pui

    2015-01-01

    There is a growing trend for researchers to use in vitro 3D models in cancer studies, as they can better recapitulate the complex in vivo situation. And the fact that the progression and development of tumor are closely associated to its stromal microenvironment has been increasingly recognized. The establishment of such tumor supportive niche is vital in understanding tumor progress and metastasis. The mesenchymal origin of many cells residing in the cancer niche provides the rationale to include MSCs in mimicking the niche in neuroblastoma. Here we co-encapsulate and co-culture NBCs and MSCs in a 3D in vitro model and investigate the morphology, growth kinetics and matrix remodeling in the reconstituted stromal environment. Results showed that the incorporation of MSCs in the model lead to accelerated growth of cancer cells as well as recapitulation of at least partially the tumor microenvironment in vivo. The current study therefore demonstrates the feasibility for the collagen microsphere to act as a 3D in vitro cancer model for various topics in cancer studies. PMID:26657086

  17. Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique

    PubMed Central

    Wu, Jian P; Kirk, Thomas B; Zheng, Ming H

    2008-01-01

    Introduction The collagen structure in the superficial zone of articular cartilage is critical to the tissue's durability. Early osteoarthritis is often characterized with fissures on the articular surface. This is closely related to the disruption of the collagen network. However, the traditional histology can not offer visualization of the collagen structure in articular cartilage because it uses conventional optical microscopy that does not have insufficient imaging resolution to resolve collagen from proteoglycans in hyaline articular cartilage. This study examines the 3D collagen network of articular cartilage scored from 0 to 2 in the scoring system of International Cartilage Repair Society, and aims to develop a 3D histology for assessing early osteoarthritis. Methods Articular cartilage was visually classified into five physiological groups: normal cartilage, aged cartilage, cartilage with artificial and natural surface disruption, and fibrillated. The 3D collagen matrix of the cartilage was acquired using a 3D imaging technique developed previously. Traditional histology was followed to grade the physiological status of the cartilage in the scoring system of International Cartilage Repair Society. Results Normal articular cartilage contains interwoven collagen bundles near the articular surface, approximately within the lamina splendens. However, its collagen fibres in the superficial zone orient predominantly in a direction spatially oblique to the articular surface. With age and disruption of the articular surface, the interwoven collagen bundles are gradually disappeared, and obliquely oriented collagen fibres change to align predominantly in a direction spatially perpendicular to the articular surface. Disruption of the articular surface is well related to the disappearance of the interwoven collagen bundles. Conclusion A 3D histology has been developed to supplement the traditional histology and study the subtle changes in the collagen network in the

  18. Compression loading-induced stress responses in intervertebral disc cells encapsulated in 3D collagen constructs

    PubMed Central

    Chooi, Wai Hon; Chan, Barbara Pui

    2016-01-01

    Cells protect themselves from stresses through a cellular stress response. In the interverebral disc, such response was also demonstrated to be induced by various environmental stresses. However, whether compression loading will cause cellular stress response in the nucleus pulposus cells (NPCs) is not well studied. By using an in vitro collagen microencapsulation model, we investigated the effect of compression loading on the stress response of NPCs. Cell viability tests, and gene and protein expression experiments were conducted, with primers for the heat shock response (HSR: HSP70, HSF1, HSP27 and HSP90), and unfolded protein response (UPR: GRP78, GRP94, ATF4 and CHOP) genes and an antibody to HSP72. Different gene expression patterns occurred due to loading type throughout experiments. Increasing the loading strain for a short duration did not increase the stress response genes significantly, but over longer durations, HSP70 and HSP27 were upregulated. Longer loading durations also resulted in a continuous upregulation of HSR genes and downregulation of UPR genes, even after load removal. The rate of apoptosis did not increase significantly after loading, suggesting that stress response genes might play a role in cell survival following mechanical stress. These results demonstrate how mechanical stress might induce and control the expression of HSR and UPR genes in NPCs. PMID:27197886

  19. Compression loading-induced stress responses in intervertebral disc cells encapsulated in 3D collagen constructs.

    PubMed

    Chooi, Wai Hon; Chan, Barbara Pui

    2016-01-01

    Cells protect themselves from stresses through a cellular stress response. In the interverebral disc, such response was also demonstrated to be induced by various environmental stresses. However, whether compression loading will cause cellular stress response in the nucleus pulposus cells (NPCs) is not well studied. By using an in vitro collagen microencapsulation model, we investigated the effect of compression loading on the stress response of NPCs. Cell viability tests, and gene and protein expression experiments were conducted, with primers for the heat shock response (HSR: HSP70, HSF1, HSP27 and HSP90), and unfolded protein response (UPR: GRP78, GRP94, ATF4 and CHOP) genes and an antibody to HSP72. Different gene expression patterns occurred due to loading type throughout experiments. Increasing the loading strain for a short duration did not increase the stress response genes significantly, but over longer durations, HSP70 and HSP27 were upregulated. Longer loading durations also resulted in a continuous upregulation of HSR genes and downregulation of UPR genes, even after load removal. The rate of apoptosis did not increase significantly after loading, suggesting that stress response genes might play a role in cell survival following mechanical stress. These results demonstrate how mechanical stress might induce and control the expression of HSR and UPR genes in NPCs. PMID:27197886

  20. Automatic and quantitative measurement of collagen gel contraction using model-guided segmentation

    NASA Astrophysics Data System (ADS)

    Chen, Hsin-Chen; Yang, Tai-Hua; Thoreson, Andrew R.; Zhao, Chunfeng; Amadio, Peter C.; Sun, Yung-Nien; Su, Fong-Chin; An, Kai-Nan

    2013-08-01

    Quantitative measurement of collagen gel contraction plays a critical role in the field of tissue engineering because it provides spatial-temporal assessment (e.g., changes of gel area and diameter during the contraction process) reflecting the cell behavior and tissue material properties. So far the assessment of collagen gels relies on manual segmentation, which is time-consuming and suffers from serious intra- and inter-observer variability. In this study, we propose an automatic method combining various image processing techniques to resolve these problems. The proposed method first detects the maximal feasible contraction range of circular references (e.g., culture dish) and avoids the interference of irrelevant objects in the given image. Then, a three-step color conversion strategy is applied to normalize and enhance the contrast between the gel and background. We subsequently introduce a deformable circular model which utilizes regional intensity contrast and circular shape constraint to locate the gel boundary. An adaptive weighting scheme was employed to coordinate the model behavior, so that the proposed system can overcome variations of gel boundary appearances at different contraction stages. Two measurements of collagen gels (i.e., area and diameter) can readily be obtained based on the segmentation results. Experimental results, including 120 gel images for accuracy validation, showed high agreement between the proposed method and manual segmentation with an average dice similarity coefficient larger than 0.95. The results also demonstrated obvious improvement in gel contours obtained by the proposed method over two popular, generic segmentation methods.

  1. Tetrapeptide-coumarin conjugate 3D networks based on hydrogen-bonded charge transfer complexes: gel formation and dye release.

    PubMed

    Guo, Zongxia; Gong, Ruiying; Jiang, Yi; Wan, Xiaobo

    2015-08-14

    Oligopeptide-based derivatives are important synthons for bio-based functional materials. In this article, a Gly-(L-Val)-Gly-(L-Val)-coumarin (GVGV-Cou) conjugate was synthesized, which forms 3D networks in ethanol. The gel nanostructures were characterized by UV-vis spectroscopy, FT-IR spectroscopy, X-ray diffraction (XRD), SEM and TEM. It is suggested that the formation of charge transfer (CT) complexes between the coumarin moieties is the main driving force for the gel formation. The capability of the gel to encapsulate and release dyes was explored. Both Congo Red (CR) and Methylene Blue (MB) can be trapped in the CT gel matrix and released over time. The present gel might be used as a functional soft material for guest encapsulation and release. PMID:26138931

  2. Disulfide-Based Diblock Copolymer Worm Gels: A Wholly-Synthetic Thermoreversible 3D Matrix for Sheet-Based Cultures.

    PubMed

    Simon, Karen A; Warren, Nicholas J; Mosadegh, Bobak; Mohammady, Marym R; Whitesides, George M; Armes, Steven P

    2015-12-14

    It is well-known that 3D in vitro cell cultures provide a much better model than 2D cell cultures for understanding the in vivo microenvironment of cells. However, significant technical challenges in handling and analyzing 3D cell cultures remain, which currently limits their widespread application. Herein, we demonstrate the application of wholly synthetic thermoresponsive block copolymer worms in sheet-based 3D cell culture. These worms form a soft, free-standing gel reversibly at 20-37 °C, which can be rapidly converted into a free-flowing dispersion of spheres on cooling to 5 °C. Functionalization of the worms with disulfide groups was found to be essential for ensuring sufficient mechanical stability of these hydrogels to enable long-term cell culture. These disulfide groups are conveniently introduced via statistical copolymerization of a disulfide-based dimethacrylate under conditions that favor intramolecular cyclization and subsequent thiol/disulfide exchange leads to the formation of reversible covalent bonds between adjacent worms within the gel. This new approach enables cells to be embedded within micrometer-thick slabs of gel with good viability, permits cell culture for at least 12 days, and facilitates recovery of viable cells from the gel simply by incubating the culture in buffer at 4 °C (thus, avoiding the enzymatic degradation required for cell harvesting when using commercial protein-based gels, such as Matrigel). PMID:26509930

  3. 3D conformal MRI-guided transurethral ultrasound therapy: results of gel phantom experiments

    NASA Astrophysics Data System (ADS)

    N'Djin, W. A.; Burtnyk, M.; McCormick, S.; Bronskill, M.; Chopra, R.

    2011-09-01

    MRI-guided transurethral ultrasound therapy shows promise for minimally invasive treatment of localized prostate cancer. Previous in-vivo studies demonstrated the feasibility of performing conservative treatments using real-time temperature feedback to control accurately the establishment of coagulative lesions within circumscribed prostate regions. This in-vitro study tested device configuration and control options for achieving full prostate treatments. A multi-channel MRI compatible ultrasound therapy system was evaluated in gel phantoms using 3 canine prostate models. Prostate profiles were 5 mm-step-segmented from T2-weighted MR images performed during previous in-vivo experiments. During ultrasound exposures, each ultrasound element was controlled independently by the 3D controller. Decisions on acoustic power, frequency, and device rotation rate were made in real time based on MR thermometry feedback and prostate radii. Low and high power treatment approaches using maximum acoustic powers of 10 or 20 W.cm-2 were tested as well as single and dual-frequency strategies (4.05/13.10 MHz). The dual-frequency strategy used either the fundamental frequency or the 3rd harmonic component, depending on the prostate radius. The 20 W.cm-2 dual frequency approach was the most efficient configuration in achieving full prostate treatments. Treatment times were about half the duration of those performed with 10 W.cm-2 configurations. Full prostate coagulations were performed in 16.3±6.1 min at a rate of 1.8±0.2 cm3.min-1, and resulted in very little undertreated tissue (<3%). Surrounding organs positioned beyond a safety distance of 1.4±1.0 mm from prostate boundaries were not damaged, particularly rectal wall tissues. In this study, a 3D, MR-thermometry-guided transurethral ultrasound therapy was validated in vitro in a tissue-mimicking phantom for performing full prostate treatment. A dual-frequency configuration with 20 W.cm-2 ultrasound intensity exposure showed good

  4. Fiber based optical tweezers for simultaneous in situ force exertion and measurements in a 3D polyacrylamide gel compartment

    PubMed Central

    Ti, Chaoyang; Thomas, Gawain M; Ren, Yundong; Zhang, Rui; Wen, Qi; Liu, Yuxiang

    2015-01-01

    Optical tweezers play an important role in biological applications. However, it is difficult for traditional optical tweezers based on objective lenses to work in a three-dimensional (3D) solid far away from the substrate. In this work, we develop a fiber based optical trapping system, namely inclined dual fiber optical tweezers, that can simultaneously apply and measure forces both in water and in a 3D polyacrylamide gel matrix. In addition, we demonstrate in situ, non-invasive characterization of local mechanical properties of polyacrylamide gel by measurements on an embedded bead. The fiber optical tweezers measurements agree well with those of atomic force microscopy (AFM). The inclined dual fiber optical tweezers provide a promising and versatile tool for cell mechanics study in 3D environments. PMID:26203364

  5. Cyclic mechanical strain induces TGFβ1-signalling in dermal fibroblasts embedded in a 3D collagen lattice.

    PubMed

    Peters, Andreas S; Brunner, Georg; Krieg, Thomas; Eckes, Beate

    2015-03-01

    Many tissues are constantly exposed to mechanical stress, e.g. shear stress in vascular endothelium, compression forces in cartilage or tensile strain in the skin. Dermal fibroblasts can differentiate into contractile myofibroblasts in a process requiring the presence of TGFβ1 in addition to mechanical load. We aimed at investigating the effect of cyclic mechanical strain on dermal fibroblasts grown in a three-dimensional environment. Therefore, murine dermal fibroblasts were cultured in collagen gels and subjected to cyclic tension at a frequency of 0.1 Hz (6 cycles/min) with a maximal increase in surface area of 10 % for 24 h. This treatment resulted in a significant increase in active TGFβ1 levels, leaving the amount of total TGFβ1 unaffected. TGFβ1 activation led to pSMAD2-mediated transcriptional elevation of downstream mediators, such as CTGF, and an auto-induction of TGFβ1, respectively. PMID:25348252

  6. Tissue in Cube: In Vitro 3D Culturing Platform with Hybrid Gel Cubes for Multidirectional Observations.

    PubMed

    Hagiwara, Masaya; Kawahara, Tomohiro; Nobata, Rina

    2016-07-01

    An in vitro 3D culturing platform enabling multidirectional observations of 3D biosamples is presented. The 3D structure of biosamples can be recognized without fluorescence. The cubic platform employs two types of hydrogels that are compatible with conventional culture dishes or well plates, facilitating growth in culture, ease of handling, and viewing at multiple angles. PMID:27128576

  7. Optical CT scanner for in-air readout of gels for external radiation beam 3D dosimetry.

    PubMed

    Ramm, Daniel; Rutten, Thomas P; Shepherd, Justin; Bezak, Eva

    2012-06-21

    Optical CT scanners for a 3D readout of externally irradiated radiosensitive hydrogels currently require the use of a refractive index (RI) matching liquid bath to obtain suitable optical ray paths through the gel sample to the detector. The requirement for a RI matching liquid bath has been negated by the design of a plastic cylindrical gel container that provides parallel beam geometry through the gel sample for the majority of the projection. The design method can be used for various hydrogels. Preliminary test results for the prototype laser beam scanner with ferrous xylenol-orange gel show geometric distortion of 0.2 mm maximum, spatial resolution limited to beam spot size of about 0.4 mm and 0.8% noise (1 SD) for a uniform irradiation. Reconstruction of a star pattern irradiated through the cylinder walls demonstrates the suitability for external beam applications. The extremely simple and cost-effective construction of this optical CT scanner, together with the simplicity of scanning gel samples without RI matching fluid increases the feasibility of using 3D gel dosimetry for clinical external beam dose verifications. PMID:22644104

  8. Developing a Procedure for the Characterization of Mechanical Properties of Collagen Gels

    NASA Astrophysics Data System (ADS)

    Chambers, Christopher; Lovelady, Heather; Matthews, Garrett

    2011-03-01

    The characterization of bulk mechanical properties of type I collagen gels is critical to understanding the role of collagen in the extracellular matrix (ECM), and developing biocompatible devices for use in the human body. Understanding the mechanical properties of the gel state of collagen can lead to the ability to adjust these properties for multiple uses. Here, we examined the Young's modulus of the synthesized gels. This project used a microrheological approach to discover these properties. Gels were first formed using a known process and magnetic microspheres were embedded in the gel prior to formation. An optical microscope was fitted with a magnetic chamber used to drive the embedded beads in two modes, an oscillatory motion and a pulse motion. Tracking software was modified and used to analyze the motion of the beads recorded with a CCD camera on the microscope. These techniques should be sufficient to obtain a reliable value for the Young's modulus of collagen gels, as well as other similar materials. This work was supported by NSF REU program (award No DMR-1004873).

  9. Editorial on the original article entitled “3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration” published in the Biomaterials on February 14, 2014

    PubMed Central

    Li, Lan

    2015-01-01

    The paper entitled “3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration” published in the Biomaterials recently illuminated the way to make particular scaffolds with calcium phosphate (CaP) powder, phosphoric acid, type I collagen and Tween 80 in low temperature. After the optimal concentration of each component was determined, the scaffolds were evaluated in a critically sized murine femoral defect model and exhibited good material properties. We made some related introduction of materials applied in 3D printing for bone tissue engineering based on this article to demonstrate the current progress in this field of study. PMID:26046065

  10. 1alpha,25-dihydroxyvitamin D3 rapidly inhibits fibroblast-induced collagen gel contraction.

    PubMed

    Greiling, D; Thieroff-Ekerdt, R

    1996-06-01

    1alpha,25-Dihydroxyvitamin D3 (1,25-D3) inhibits the proliferation of fibroblasts in vitro in monolayer culture. We investigated the effect of 1,25-D3 on normal murine and human fibroblasts cultured in collagen type I gels, which more closely resembles the in vivo situation in the dermis. In this culture system 1,25-D3 had no effect on fibroblast proliferation; however, the fibroblast-induced collagen gel contraction was inhibited in a time- and concentration-dependent manner in the nanomolar concentration range. 25-Hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were inactive. 1,25-D3 had no effect in fibroblasts lacking a functional vitamin D receptor. Pretreatment of fibroblasts in monolayer culture for 5 min was sufficient to trigger the inhibition of collagen gel contraction. Nifedipine increased collagen gel contraction and counteracted the effect of 1,25-D3. The inhibition of collagen gel contraction by 1,25-D3 is supposed to be mediated by the vitamin D receptor because a functional vitamin D receptor is required, and vitamin D metabolites with low affinity to the vitamin D receptor were inactive. Brief pretreatment of fibroblasts was sufficient to trigger the inhibitory effect of 1,25-D3, suggesting a nongenomic effect. A genomic mode of action could not be ruled out, however, because the inhibition was first measured after 24 h. The antagonism of the calcium channel antagonist nifedipine probably represents the sum of two opposite effects rather than supporting evidence for a nongenomic mode of action of 1,25-D3. In conclusion, 1,25-D3 has a specific and rapidly triggered inhibitory effect on fibroblast-induced collagen gel contraction. PMID:8752663

  11. Injectable gels of anionic collagen:rhamsan composites for plastic correction: preparation, characterization, and rheological properties.

    PubMed

    de Paula, Márcio; Goissis, Gilberto; Martins, Virgínia C A; da Silva Trindade, José Carlos

    2005-11-01

    The present article describes the preparation and characterization of anionic collagen gels obtained from porcine intestinal submucosa after 72 h of alkaline treatment and in the form of rhamsan composites to develop injectable biomaterials for plastic reconstruction. All materials were characterized by SDS/polyacrylamide gel electrophoresis, infrared spectroscopy, thermal stability, potentiometric titration, rheological properties, and fluidity tests. Biocompatibility was appraised after the injection of anionic collagen: rhamsan composites at 2.5% in 60 North Folk rabbits. Independently of processing, the collagen's secondary structure was preserved in all cases, and after 72 h of hydrolysis the collagen was characterized by a carboxyl group content of 346+/-9, which, at physiological pH, corresponds to an increase of 106+/-17 negative charges, in comparison to native collagen, due to the selective hydrolysis of asparagine and glutamine carboxyamide side chain. Rheological studies of composites at pH 7.4 in concentrations of 2, 4, and 6% (in proportions of 75:1 and 50:1) showed a viscoelastic behavior dependent on the frequency, which is independent of concentration and proportion. In both, the concentration of the storage modulus always predominated over the loss modulus (G'>G'' and delta<45 degrees ). The results from creep experiments confirmed this behavior and showed that anionic collagen:rhamsan composites at pH 7.4 in the proportion of 50:1 are less elastic and more susceptible to deformation in comparison to gels in the proportion of 75:1, independent of concentration. This was further confirmed by flow experiments, indicating that the necessary force for the extrusion of anionic collagen:rhamsan composites, in comparison to anionic collagen, was significantly smaller and with a smooth flow. Biocompatibility studies showed that the tissue reaction of anionic collagen:rhamsan composites at 2.5% in the proportion of 75:1 was compatible with the application

  12. Fibrin binds to collagen and provides a bridge for αVβ3 integrin-dependent contraction of collagen gels

    PubMed Central

    Reyhani, Vahid; Seddigh, Pegah; Guss, Bengt; Gustafsson, Renata; Rask, Lars; Rubin, Kristofer

    2014-01-01

    The functional significance of fibrin deposits typically seen in inflammatory lesions, carcinomas and in healing wounds is not fully understood. In the present study, we demonstrate that fibrinogen/fibrin specifically bound to native Col I (collagen type I) and used the Col I fibre network as a base to provide a functional interface matrix that connects cells to the Col I fibres through αVβ3 integrins. This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via αVβ3 integrin. We show that fibrinogen specifically bound to immobilized native Col I at the site known to bind matrix metalloproteinase-1, discoidin domain receptor-2 and fibronectin, and that binding had no effect on Col I fibrillation. A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels. Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects αVβ3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures. PMID:24840544

  13. Comparison of the Expression of Hepatic Genes by Human Wharton’s Jelly Mesenchymal Stem Cells Cultured in 2D and 3D Collagen Culture Systems

    PubMed Central

    Khodabandeh, Zahra; Vojdani, Zahra; Talaei-Khozani, Tahereh; Jaberipour, Mansoureh; Hosseini, Ahmad; Bahmanpour, Soghra

    2016-01-01

    Background: Human Wharton’s jelly mesenchymal stem cells (HWJMSCs) express liver-specific markers such as albumin, alpha-fetoprotein, cytokeratin-19, cytokeratin-18, and glucose-6-phosphatase. Therefore, they can be considered as a good source for cell replacement therapy for liver diseases. This study aimed to evaluate the effects of various culture systems on the hepatocyte-specific gene expression pattern of naïve HWJMSCs. Methods: HWJMSCs were characterized as MSCs by detecting the surface CD markers and capability to differentiate toward osteoblast and adipocyte. HWJMSCs were cultured in 2D collagen films and 3D collagen scaffolds for 21 days and were compared to control cultures. Real time RT-PCR was used to evaluate the expression of liver-specific genes. Results: The HWJMSCs which were grown on non-coated culture plates expressed cytokeratin-18 and -19, alpha-fetoprotein, albumin, glucose-6-phosphatase, and claudin. The expression of the hepatic nuclear factor 4 (HNF4) was very low. The cells showed a significant increase in caludin expression when they cultured in 3D collagen scaffolds compared to the conventional monolayer culture and 2D collagen scaffold. Conclusion: Various culture systems did not influence on hepatocyte specific marker expression by HWJMSCs, except for claudin. The expression of claudin showed that 3D collagen scaffold provided the extracellular matrix for induction of the cells to interconnect with each other. PMID:26722142

  14. Functional 3D Neural Mini-Tissues from Printed Gel-Based Bioink and Human Neural Stem Cells.

    PubMed

    Gu, Qi; Tomaskovic-Crook, Eva; Lozano, Rodrigo; Chen, Yu; Kapsa, Robert M; Zhou, Qi; Wallace, Gordon G; Crook, Jeremy M

    2016-06-01

    Direct-write printing of stem cells within biomaterials presents an opportunity to engineer tissue for in vitro modeling and regenerative medicine. Here, a first example of constructing neural tissue by printing human neural stem cells that are differentiated in situ to functional neurons and supporting neuroglia is reported. The supporting biomaterial incorporates a novel clinically relevant polysaccharide-based bioink comprising alginate, carboxymethyl-chitosan, and agarose. The printed bioink rapidly gels by stable cross-linking to form a porous 3D scaffold encapsulating stem cells for in situ expansion and differentiation. Differentiated neurons form synaptic contacts, establish networks, are spontaneously active, show a bicuculline-induced increased calcium response, and are predominantly gamma-aminobutyric acid expressing. The 3D tissues will facilitate investigation of human neural development, function, and disease, and may be adaptable for engineering other 3D tissues from different stem cell types. PMID:27028356

  15. Integration of 3D Printed and Micropatterned Polycaprolactone Scaffolds for Guidance of Oriented Collagenous Tissue Formation In Vivo.

    PubMed

    Pilipchuk, Sophia P; Monje, Alberto; Jiao, Yizu; Hao, Jie; Kruger, Laura; Flanagan, Colleen L; Hollister, Scott J; Giannobile, William V

    2016-03-01

    Scaffold design incorporating multiscale cues for clinically relevant, aligned tissue regeneration has potential to improve structural and functional integrity of multitissue interfaces. The objective of this preclinical study is to develop poly(ε-caprolactone) (PCL) scaffolds with mesoscale and microscale architectural cues specific to human ligament progenitor cells and assess their ability to form aligned bone-ligament-cementum complexes in vivo. PCL scaffolds are designed to integrate a 3D printed bone region with a micropatterned PCL thin film consisting of grooved pillars. The patterned film region is seeded with human ligament cells, fibroblasts transduced with bone morphogenetic protein-7 genes seeded within the bone region, and a tooth dentin segment positioned on the ligament region prior to subcutaneous implantation into a murine model. Results indicate increased tissue alignment in vivo using micropatterned PCL films, compared to random-porous PCL. At week 6, 30 μm groove depth significantly enhances oriented collagen fiber thickness, overall cell alignment, and nuclear elongation relative to 10 μm groove depth. This study demonstrates for the first time that scaffolds with combined hierarchical mesoscale and microscale features can align cells in vivo for oral tissue repair with potential for improving the regenerative response of other bone-ligament complexes. PMID:26820240

  16. Smad signal pathway regulates angiogenesis via endothelial cell in an adipose-derived stromal cell/endothelial cell co-culture, 3D gel model.

    PubMed

    Lin, Shiyu; Xie, Jing; Gong, Tao; Shi, Sirong; Zhang, Tao; Fu, Na; Lin, Yunfeng

    2016-01-01

    Co-implantation of adipose-derived stromal cells (ASCs) and endothelial cells (ECs) can markedly expedite the formation of functional microvascular beds and provides possible methods for cell-based revascularization therapies to treat various diseases. Furthermore, we investigated the role of TGFβ/Smad signaling pathway for angiogenesis in a three-dimensional (3D) collagen gel model established in vitro with co-culture between ASCs and ECs. We found that angiogenesis was attenuated in the co-culture gels after inhibition of ALK5/Smad2/3 with SB431542. Genes coding for VEGF-A, VEGF-B, VE-ca, FGF-1, PDGF, BMP-4, and BMP-7 were significantly reduced in both mono-cultured and co-cultured ECs. Furthermore, the decrease in co-cultured ECs was prominent relative to mono-cultured ECs. Taken together, these findings suggest that in the co-culture between ASCs and ECs, TGFβ/Smad signal pathway regulates angiogenesis via ECs; moreover, the findings that the co-cultured ECs were regulated more significantly than mono-cultured ECs suggest that suppression of Smad signal pathway may regulate the paracrine secretion of ASCs to further modulate angiogenesis of ECs. PMID:26694166

  17. Mathematical Modeling of Uniaxial Mechanical Properties of Collagen Gel Scaffolds for Vascular Tissue Engineering

    PubMed Central

    Irastorza, Ramiro M.; Drouin, Bernard; Blangino, Eugenia; Mantovani, Diego

    2015-01-01

    Small diameter tissue-engineered arteries improve their mechanical and functional properties when they are mechanically stimulated. Applying a suitable stress and/or strain with or without a cycle to the scaffolds and cells during the culturing process resides in our ability to generate a suitable mechanical model. Collagen gel is one of the most used scaffolds in vascular tissue engineering, mainly because it is the principal constituent of the extracellular matrix for vascular cells in human. The mechanical modeling of such a material is not a trivial task, mainly for its viscoelastic nature. Computational and experimental methods for developing a suitable model for collagen gels are of primary importance for the field. In this research, we focused on mechanical properties of collagen gels under unconfined compression. First, mechanical viscoelastic models are discussed and framed in the control system theory. Second, models are fitted using system identification. Several models are evaluated and two nonlinear models are proposed: Mooney-Rivlin inspired and Hammerstein models. The results suggest that Mooney-Rivlin and Hammerstein models succeed in describing the mechanical behavior of collagen gels for cyclic tests on scaffolds (with best fitting parameters 58.3% and 75.8%, resp.). When Akaike criterion is used, the best is the Mooney-Rivlin inspired model. PMID:25834840

  18. Mathematical modeling of uniaxial mechanical properties of collagen gel scaffolds for vascular tissue engineering.

    PubMed

    Irastorza, Ramiro M; Drouin, Bernard; Blangino, Eugenia; Mantovani, Diego

    2015-01-01

    Small diameter tissue-engineered arteries improve their mechanical and functional properties when they are mechanically stimulated. Applying a suitable stress and/or strain with or without a cycle to the scaffolds and cells during the culturing process resides in our ability to generate a suitable mechanical model. Collagen gel is one of the most used scaffolds in vascular tissue engineering, mainly because it is the principal constituent of the extracellular matrix for vascular cells in human. The mechanical modeling of such a material is not a trivial task, mainly for its viscoelastic nature. Computational and experimental methods for developing a suitable model for collagen gels are of primary importance for the field. In this research, we focused on mechanical properties of collagen gels under unconfined compression. First, mechanical viscoelastic models are discussed and framed in the control system theory. Second, models are fitted using system identification. Several models are evaluated and two nonlinear models are proposed: Mooney-Rivlin inspired and Hammerstein models. The results suggest that Mooney-Rivlin and Hammerstein models succeed in describing the mechanical behavior of collagen gels for cyclic tests on scaffolds (with best fitting parameters 58.3% and 75.8%, resp.). When Akaike criterion is used, the best is the Mooney-Rivlin inspired model. PMID:25834840

  19. Automatic and Quantitative Measurement of Collagen Gel Contraction Using Model-Guided Segmentation.

    PubMed

    Chen, Hsin-Chen; Yang, Tai-Hua; Thoreson, Andrew R; Zhao, Chunfeng; Amadio, Peter C; Sun, Yung-Nien; Su, Fong-Chin; An, Kai-Nan

    2013-08-01

    Quantitative measurement of collagen gel contraction plays a critical role in the field of tissue engineering because it provides spatial-temporal assessment (e.g., changes of gel area and diameter during the contraction process) reflecting the cell behaviors and tissue material properties. So far the assessment of collagen gels relies on manual segmentation, which is time-consuming and suffers from serious intra- and inter-observer variability. In this study, we propose an automatic method combining various image processing techniques to resolve these problems. The proposed method first detects the maximal feasible contraction range of circular references (e.g., culture dish) and avoids the interference of irrelevant objects in the given image. Then, a three-step color conversion strategy is applied to normalize and enhance the contrast between the gel and background. We subsequently introduce a deformable circular model (DCM) which utilizes regional intensity contrast and circular shape constraint to locate the gel boundary. An adaptive weighting scheme was employed to coordinate the model behavior, so that the proposed system can overcome variations of gel boundary appearances at different contraction stages. Two measurements of collagen gels (i.e., area and diameter) can readily be obtained based on the segmentation results. Experimental results, including 120 gel images for accuracy validation, showed high agreement between the proposed method and manual segmentation with an average dice similarity coefficient larger than 0.95. The results also demonstrated obvious improvement in gel contours obtained by the proposed method over two popular, generic segmentation methods. PMID:24092954

  20. Integrin {beta}1-dependent invasive migration of irradiation-tolerant human lung adenocarcinoma cells in 3D collagen matrix

    SciTech Connect

    Ishihara, Seiichiro; Haga, Hisashi; Yasuda, Motoaki; Mizutani, Takeomi; Kawabata, Kazushige; Shirato, Hiroki; Nishioka, Takeshi

    2010-06-04

    Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin {beta}1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin {beta}1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin {beta}1-dependent phenotype, and integrin {beta}1 might be a potentially effective therapeutic target in combination with radiotherapy.

  1. New radiochromic gel for 3D dosimetry based on Turnbull blue: basic properties.

    PubMed

    Solc, Jaroslav; Spevácek, Václav

    2009-09-01

    The recently developed new radiochromic gel dosimeter based on Turnbull blue dye formed by irradiation (the TBG dosimeter) does not exhibit dose pattern degradation due to diffusion effects as observed in the Fricke-gel dosimeter with xylenol orange incorporated into the gel matrix (the FXG dosimeter). The TBG dosimeter can be easily prepared and its optical properties enable evaluation of the gel's response using the cone-beam optical computed tomography technique. The preparation procedure is described in the paper along with the basic characteristics of the gel, including dose response, dose sensitivity, ageing under different storage conditions, diffusion rates of Turnbull blue and gel density. The measurement of diffusion is described in more detail. The same method was applied to the FXG dosimeter for direct comparison. It was found that the diffusion coefficient of the TBG dosimeter stored at 24 degrees C is less than 4 x 10(-3) mm(2) h(-1) (1sigma confidence level), compared to the value of 7.3 x 10(-1) mm(2) h(-1) (1sigma) of the FXG dosimeter measured at the same temperature. Although the TBG dosimeter is less sensitive than the FXG dosimeter, its diffusion coefficient is practically negligible and, therefore, it offers large potential as a three-dimensional dosimeter for applications encompassing sharp dose gradients such as high-dose-rate brachytherapy. PMID:19652291

  2. Fabrication and characterization of gels with integrated channels using 3D printing with microfluidic nozzle for tissue engineering applications.

    PubMed

    Attalla, R; Ling, C; Selvaganapathy, P

    2016-02-01

    The lack of a simple and effective method to integrate vascular network with engineered scaffolds and tissue constructs remains one of the biggest challenges in true 3D tissue engineering. Here, we detail the use of a commercially available, low-cost, open-source 3D printer modified with a microfluidic print-head in order to develop a method for the generation of instantly perfusable vascular network integrated with gel scaffolds seeded with cells. The print-head features an integrated coaxial nozzle that allows the fabrication of hollow, calcium-polymerized alginate tubes that can be easily patterned using 3D printing techniques. The diameter of the hollow channel can be precisely controlled and varied between 500 μm - 2 mm by changing applied flow rates or print-head speed. These channels are integrated into gel layers with a thickness of 800 μm - 2.5 mm. The structural rigidity of these constructs allows the fabrication of multi-layered structures without causing the collapse of hollow channels in lower layers. The 3D printing method was fully characterized at a range of operating speeds (0-40 m/min) and corresponding flow rates (1-30 mL/min) were identified to produce precise definition. This microfluidic design also allows the incorporation of a wide range of scaffold materials as well as biological constituents such as cells, growth factors, and ECM material. Media perfusion of the channels causes a significant viability increase in the bulk of cell-laden structures over the long-term. With this setup, gel constructs with embedded arrays of hollow channels can be created and used as a potential substitute for blood vessel networks. PMID:26842949

  3. Microstructural modeling of collagen network mechanics and interactions with the proteoglycan gel in articular cartilage.

    PubMed

    Quinn, T M; Morel, V

    2007-01-01

    Cartilage matrix mechanical function is largely determined by interactions between the collagen fibrillar network and the proteoglycan gel. Although the molecular physics of these matrix constituents have been characterized and modern imaging methods are capable of localized measurement of molecular densities and orientation distributions, theoretical tools for using this information for prediction of cartilage mechanical behavior are lacking. We introduce a means to model collagen network contributions to cartilage mechanics based upon accessible microstructural information (fibril density and orientation distributions) and which self-consistently follows changes in microstructural geometry with matrix deformations. The interplay between the molecular physics of the collagen network and the proteoglycan gel is scaled up to determine matrix material properties, with features such as collagen fibril pre-stress in free-swelling cartilage emerging naturally and without introduction of ad hoc parameters. Methods are developed for theoretical treatment of the collagen network as a continuum-like distribution of fibrils, such that mechanical analysis of the network may be simplified by consideration of the spherical harmonic components of functions of the fibril orientation, strain, and stress distributions. Expressions for the collagen network contributions to matrix stress and stiffness tensors are derived, illustrating that only spherical harmonic components of orders 0 and 2 contribute to the stress, while orders 0, 2, and 4 contribute to the stiffness. Depth- and compression-dependent equilibrium mechanical properties of cartilage matrix are modeled, and advantages of the approach are illustrated by exploration of orientation and strain distributions of collagen fibrils in compressed cartilage. Results highlight collagen-proteoglycan interactions, especially for very small physiological strains where experimental data are relatively sparse. These methods for

  4. Second-harmonic generation scattering directionality predicts tumor cell motility in collagen gels

    NASA Astrophysics Data System (ADS)

    Burke, Kathleen A.; Dawes, Ryan P.; Cheema, Mehar K.; Van Hove, Amy; Benoit, Danielle S. W.; Perry, Seth W.; Brown, Edward

    2015-05-01

    Second-harmonic generation (SHG) allows for the analysis of tumor collagen structural changes throughout metastatic progression. SHG directionality, measured through the ratio of the forward-propagating to backward-propagating signal (F/B ratio), is affected by collagen fibril diameter, spacing, and disorder of fibril packing within a fiber. As tumors progress, these parameters evolve, producing concurrent changes in F/B. It has been recently shown that the F/B of highly metastatic invasive ductal carcinoma (IDC) breast tumors is significantly different from less metastatic tumors. This suggests a possible relationship between the microstructure of collagen, as measured by the F/B, and the ability of tumor cells to locomote through that collagen. Utilizing in vitro collagen gels of different F/B ratios, we explored the relationship between collagen microstructure and motility of tumor cells in a "clean" environment, free of the myriad cells, and signals found in in vivo. We found a significant relationship between F/B and the total distance traveled by the tumor cell, as well as both the average and maximum velocities of the cells. Consequently, one possible mechanism underlying the observed relationship between tumor F/B and metastatic output in IDC patient samples is a direct influence of collagen structure on tumor cell motility.

  5. Second-harmonic generation scattering directionality predicts tumor cell motility in collagen gels.

    PubMed

    Burke, Kathleen A; Dawes, Ryan P; Cheema, Mehar K; Van Hove, Amy; Benoit, Danielle S W; Perry, Seth W; Brown, Edward

    2015-05-01

    Second-harmonic generation (SHG) allows for the analysis of tumor collagen structural changes throughout metastatic progression. SHG directionality, measured through the ratio of the forward-propagating to backward-propagating signal (F/B ratio), is affected by collagen fibril diameter, spacing, and disorder of fibril packing within a fiber. As tumors progress, these parameters evolve, producing concurrent changes in F/B. It has been recently shown that the F/B of highly metastatic invasive ductal carcinoma (IDC) breast tumors is significantly different from less metastatic tumors. This suggests a possible relationship between the microstructure of collagen, as measured by the F/B, and the ability of tumor cells to locomote through that collagen. Utilizing in vitro collagen gels of different F/B ratios, we explored the relationship between collagen microstructure and motility of tumor cells in a “clean” environment, free of the myriad cells, and signals found in in vivo. We found a significant relationship between F/B and the total distance traveled by the tumor cell, as well as both the average and maximum velocities of the cells. Consequently, one possible mechanism underlying the observed relationship between tumor F/B and metastatic output in IDC patient samples is a direct influence of collagen structure on tumor cell motility. PMID:25625899

  6. Small-Field Measurements of 3D Polymer Gel Dosimeters through Optical Computed Tomography

    PubMed Central

    Shih, Cheng-Ting; Lee, Yao-Ting; Wu, Shin-Hua; Yao, Chun-Hsu; Hsieh, Bor-Tsung

    2016-01-01

    With advances in therapeutic instruments and techniques, three-dimensional dose delivery has been widely used in radiotherapy. The verification of dose distribution in a small field becomes critical because of the obvious dose gradient within the field. The study investigates the dose distributions of various field sizes by using NIPAM polymer gel dosimeter. The dosimeter consists of 5% gelatin, 5% monomers, 3% cross linkers, and 5 mM THPC. After irradiation, a 24 to 96 hour delay was applied, and the gel dosimeters were read by a cone beam optical computed tomography (optical CT) scanner. The dose distributions measured by the NIPAM gel dosimeter were compared to the outputs of the treatment planning system using gamma evaluation. For the criteria of 3%/3 mm, the pass rates for 5 × 5, 3 × 3, 2 × 2, 1 × 1, and 0.5 × 0.5 cm2 were as high as 91.7%, 90.7%, 88.2%, 74.8%, and 37.3%, respectively. For the criteria of 5%/5 mm, the gamma pass rates of the 5 × 5, 3 × 3, and 2 × 2 cm2 fields were over 99%. The NIPAM gel dosimeter provides high chemical stability. With cone-beam optical CT readouts, the NIPAM polymer gel dosimeter has potential for clinical dose verification of small-field irradiation. PMID:26974434

  7. How do monomeric components of a polymer gel dosimeter respond to ionising radiation: A steady-state radiolysis towards preparation of a 3D polymer gel dosimeter

    NASA Astrophysics Data System (ADS)

    Kozicki, Marek

    2011-12-01

    Ionising radiation-induced reactions of aqueous single monomer solutions and mixtures of poly(ethylene glycol) diacrylate (PEGDA) and N, N'-methylenebisacrylamide (Bis) in a steady-state condition are presented below and above gelation doses in order to highlight reactions in irradiated 3D polymer gel dosimeters, which are assigned for radiotherapy dosimetry. Both monomers are shown to undergo radical polymerisation and cross-linking, which result in the measured increase in molecular weight and radius of gyration of the formed polydisperse polymer coils. The formation of nanogels was also observed for Bis solutions at a low concentration. In the case of PEGDA-Bis mixtures, co-polymerisation is suggested as well. At a sufficiently high radiation dose, the formation of a polymer network was observed for both monomers and their mixture. For this reason a sol-gel analysis for PEGDA and Bis was performed gravimetrically and a proposition of an alternative to this method employing a nuclear magnetic resonance technique is made. The two monomers were used for preparation of 3D polymer gel dosimeters having the acronyms PABIG and PABIG nx. The latter is presented for the first time in this work and is a type of the formerly established PABIG polymer gel dosimeter. The elementary characteristics of the new composition are presented, underlining the ease of its preparation, low dose threshold, and slightly increased sensitivity but lower quasi-linear range of dose response in comparison to PABIG.

  8. Assessment of angiogenesis in osseointegration of a silica-collagen biomaterial using 3D-nano-CT.

    PubMed

    Alt, Volker; Kögelmaier, Daniela Vera; Lips, Katrin S; Witt, Vera; Pacholke, Sabine; Heiss, Christian; Kampschulte, Marian; Heinemann, Sascha; Hanke, Thomas; Thormann, Ulrich; Schnettler, Reinhard; Langheinrich, Alexander C

    2011-10-01

    Bony integration of biomaterials is a complex process in which angiogenesis plays a crucial role. We evaluated micro- and nano-CT imaging to demonstrate and quantify neovascularization in bony integration of a biomaterial and to give an image based estimation for the needed resolution for imaging angiogenesis in an animal model of femora defect healing. In 8 rats 5mm full-size defects were created at the left femur that was filled with silica-collagen bone substitute material and internally fixed with plate osteosynthesis. After 6 weeks the femora were infused in situ with Microfil, harvested and scanned for micro-CT (9 μm)(3) and nano-CT (3 μm)(3) imaging. Using those 3D images, the newly formed blood vessels in the area of the biomaterial were assessed and the total vascular volume fraction, the volume of the bone substitute material and the volume of the bone defect were quantitatively characterized. Results were complemented by histology. Differences were statistically assessed using (ANOVA). High-resolution nano-CT demonstrated new blood vessel formation surrounding the biomaterial in all animals at capillary level. Immunohistochemistry confirmed the newly formed blood vessels surrounding the bone substitute material. The mean vascular volume fraction (VVF) around the implant was calculated to be 3.01 ± 0.4%. The VVF was inversely correlated with the volume of the bone substitute material (r=0.8) but not with the dimension of the fracture zone (r=0.3). Nano-CT imaging is feasible for quantitative analysis of angiogenesis during bony integration of biomaterials and a promising tool in this context for the future. PMID:21723963

  9. Injectable, high-density collagen gels for annulus fibrosus repair: An in vitro rat tail model.

    PubMed

    Borde, Brandon; Grunert, Peter; Härtl, Roger; Bonassar, Lawrence J

    2015-08-01

    A herniated intervertebral disc often causes back pain when disc tissue is displaced through a damaged annulus fibrosus. Currently, the only methods available for annulus fibrosus repair involve mechanical closure of defect, which does little to address biological healing in the damaged tissue. Collagen hydrogels are injectable and have been used to repair annulus defects in vivo. In this study, high-density collagen hydrogels at 5, 10, and 15 mg/mL were used to repair defects made to intact rat caudal intervertebral discs in vitro. A group of gels at 15 mg/mL were also cross-linked with riboflavin at 0.03 mM, 0.07 mM, or 0.10 mM. These cross-linked, high-density collagen gels maintained their presence in the defect under loading and contributed positively to the mechanical response of damaged discs. Discs exhibited increases to 95% of undamaged effective equilibrium and instantaneous moduli as well as up to fourfold decreases in effective hydraulic permeability from the damaged discs. These data suggest that high-density collagen gels may be effective at restoring mechanical function of injured discs as well as potential vehicles for the delivery of biological agents such as cells or growth factors that may aid in the repair of the annulus fibrosus. PMID:25504661

  10. Gamma Knife 3-D dose distribution near the area of tissue inhomogeneities by normoxic gel dosimetry

    SciTech Connect

    Isbakan, Fatih; Uelgen, Yekta; Bilge, Hatice; Ozen, Zeynep; Agus, Onur; Buyuksarac, Bora

    2007-05-15

    The accuracy of the Leksell GammaPlan registered , the dose planning system of the Gamma Knife Model-B, was evaluated near tissue inhomogeneities, using the gel dosimetry method. The lack of electronic equilibrium around the small-diameter gamma beams can cause dose calculation errors in the neighborhood of an air-tissue interface. An experiment was designed to investigate the effects of inhomogeneity near the paranosal sinuses cavities. The homogeneous phantom was a spherical glass balloon of 16 cm diameter, filled with MAGIC gel; i.e., the normoxic polymer gel. Two hollow PVC balls of 2 cm radius, filled with N{sub 2} gas, represented the air cavities inside the inhomogeneous phantom. For dose calibration purposes, 100 ml gel-containing vials were irradiated at predefined doses, and then scanned in a MR unit. Linearity was observed between the delivered dose and the reciprocal of the T2 relaxation time constant of the gel. Dose distributions are the results of a single shot of irradiation, obtained by collimating all 201 cobalt sources to a known target in the phantom. Both phantoms were irradiated at the same dose level at the same coordinates. Stereotactic frames and fiducial markers were attached to the phantoms prior to MR scanning. The dose distribution predicted by the Gamma Knife planning system was compared with that of the gel dosimetry. As expected, for the homogeneous phantom the isodose diameters measured by the gel dosimetry and the GammaPlan registered differed by 5% at most. However, with the inhomogeneous phantom, the dose maps in the axial, coronal and sagittal planes were spatially different. The diameters of the 50% isodose curves differed 43% in the X axis and 32% in the Y axis for the Z=90 mm axial plane; by 44% in the X axis and 24% in the Z axis for the Y=90 mm coronal plane; and by 32% in the Z axis and 42% in the Y axis for the X=92 mm sagittal plane. The lack of ability of the GammaPlan registered to predict the rapid dose fall off, due

  11. Gamma Knife 3-D dose distribution near the area of tissue inhomogeneities by normoxic gel dosimetry.

    PubMed

    Isbakan, Fatih; Ulgen, Yekta; Bilge, Hatice; Ozen, Zeynep; Agus, Onur; Buyuksarac, Bora

    2007-05-01

    The accuracy of the Leksell GammaPlan, the dose planning system of the Gamma Knife Model-B, was evaluated near tissue inhomogeneities, using the gel dosimetry method. The lack of electronic equilibrium around the small-diameter gamma beams can cause dose calculation errors in the neighborhood of an air-tissue interface. An experiment was designed to investigate the effects of inhomogeneity near the paranosal sinuses cavities. The homogeneous phantom was a spherical glass balloon of 16 cm diameter, filled with MAGIC gel; i.e., the normoxic polymer gel. Two hollow PVC balls of 2 cm radius, filled with N2 gas, represented the air cavities inside the inhomogeneous phantom. For dose calibration purposes, 100 ml gel-containing vials were irradiated at predefined doses, and then scanned in a MR unit. Linearity was observed between the delivered dose and the reciprocal of the T2 relaxation time constant of the gel. Dose distributions are the results of a single shot of irradiation, obtained by collimating all 201 cobalt sources to a known target in the phantom. Both phantoms were irradiated at the same dose level at the same coordinates. Stereotactic frames and fiducial markers were attached to the phantoms prior to MR scanning. The dose distribution predicted by the Gamma Knife planning system was compared with that of the gel dosimetry. As expected, for the homogeneous phantom the isodose diameters measured by the gel dosimetry and the GammaPlan differed by 5% at most. However, with the inhomogeneous phantom, the dose maps in the axial, coronal and sagittal planes were spatially different. The diameters of the 50% isodose curves differed 43% in the X axis and 32% in the Y axis for the Z =90 mm axial plane; by 44% in the X axis and 24% in the Z axis for the Y=90 mm coronal plane; and by 32% in the Z axis and 42% in the Y axis for the X=92 mm sagittal plane. The lack of ability of the GammaPlan to predict the rapid dose fall off, due to the air cavities behind or near the

  12. Fabrication of Compositionally and Topographically Complex Robust Tissue Forms by 3D-Electrochemical Compaction of Collagen

    PubMed Central

    Younesi, Mousa; Islam, Anowarul; Kishore, Vipuil; Panit, Stefi; Akkus, Ozan

    2015-01-01

    Collagen solutions are phase-transformed to mechanically robust shell structures with curviplanar topographies using electrochemically induced pH gradients. The process enables rapid layer-by-layer deposition of collagen-rich mixtures over the entire field simultaneously to obtain compositionally diverse multilayered structures. In-plane tensile strength and modulus of the electrocompacted collagen sheet samples were 5200 -fold and 2300 -fold greater than that of uncompacted collagen samples. Out of plane compression tests showed 27 -fold and fold increase in compressive stress and 46 -fold increase in compressive modulus compared to uncompacted collagen sheets. Cells proliferated 4.9 times faster, and cellular area spread was 2.7 times greater on compacted collagen sheets. Electrocompaction also resulted in 2.9 times greater focal adhesion area than on regular collagen hydrogel. The reported improvements in the cell-matrix interactions with electrocompaction would serve to expedite the population of electrocompacted collagen scaffolds by cells. The capacity of the method to fabricate nonlinear curved topographies with compositional heterogeneous layers is demonstrated by sequential deposition of collagenhydroxyapatite layer over a collagen layer. The complex curved topography of the nasal structure is replicated by the electrochemical compaction method. The presented electrochemical compaction process is an enabling modality which holds significant promise for reconstruction of a wide spectrum of topographically complex systems such as joint surfaces, craniofacial defects, ears, nose or urogenital forms. PMID:26069162

  13. 3D Tissue Culturing: Tissue in Cube: In Vitro 3D Culturing Platform with Hybrid Gel Cubes for Multidirectional Observations (Adv. Healthcare Mater. 13/2016).

    PubMed

    Hagiwara, Masaya; Kawahara, Tomohiro; Nobata, Rina

    2016-07-01

    An in vitro 3D culturing platform enabling multidirectional observations of 3D biosamples is presented by M. Hagiwara and co-workers on page 1566. 3D recognition of a sample structure can be achieved by facilitating multi-directional views using a standard microscope without a laser system. The cubic platform has the potential to promote 3D culture studies, offering easy handling and compatibility with commercial culture plates at a low price tag. PMID:27384934

  14. Sustained Delivery of Bioactive GDNF from Collagen and Alginate-Based Cell-Encapsulating Gel Promoted Photoreceptor Survival in an Inherited Retinal Degeneration Model

    PubMed Central

    Chan, Barbara P.; Lo, Amy C. Y.

    2016-01-01

    Encapsulated-cell therapy (ECT) is an attractive approach for continuously delivering freshly synthesized therapeutics to treat sight-threatening posterior eye diseases, circumventing repeated invasive intravitreal injections and improving local drug availability clinically. Composite collagen-alginate (CAC) scaffold contains an interpenetrating network that integrates the physical and biological merits of its constituents, including biocompatibility, mild gelling properties and availability. However, CAC ECT properties and performance in the eye are not well-understood. Previously, we reported a cultured 3D CAC system that supported the growth of GDNF-secreting HEK293 cells with sustainable GDNF delivery. Here, the system was further developed into an intravitreally injectable gel with 1x104 or 2x105 cells encapsulated in 2mg/ml type I collagen and 1% alginate. Gels with lower alginate concentration yielded higher initial cell viability but faster spheroid formation while increasing initial cell density encouraged cell growth. Continuous GDNF delivery was detected in culture and in healthy rat eyes for at least 14 days. The gels were well-tolerated with no host tissue attachment and contained living cell colonies. Most importantly, gel-implanted in dystrophic Royal College of Surgeons rat eyes for 28 days retained photoreceptors while those containing higher initial cell number yielded better photoreceptor survival. CAC ECT gels offers flexible system design and is a potential treatment option for posterior eye diseases. PMID:27441692

  15. Sustained Delivery of Bioactive GDNF from Collagen and Alginate-Based Cell-Encapsulating Gel Promoted Photoreceptor Survival in an Inherited Retinal Degeneration Model.

    PubMed

    Wong, Francisca S Y; Wong, Calvin C H; Chan, Barbara P; Lo, Amy C Y

    2016-01-01

    Encapsulated-cell therapy (ECT) is an attractive approach for continuously delivering freshly synthesized therapeutics to treat sight-threatening posterior eye diseases, circumventing repeated invasive intravitreal injections and improving local drug availability clinically. Composite collagen-alginate (CAC) scaffold contains an interpenetrating network that integrates the physical and biological merits of its constituents, including biocompatibility, mild gelling properties and availability. However, CAC ECT properties and performance in the eye are not well-understood. Previously, we reported a cultured 3D CAC system that supported the growth of GDNF-secreting HEK293 cells with sustainable GDNF delivery. Here, the system was further developed into an intravitreally injectable gel with 1x104 or 2x105 cells encapsulated in 2mg/ml type I collagen and 1% alginate. Gels with lower alginate concentration yielded higher initial cell viability but faster spheroid formation while increasing initial cell density encouraged cell growth. Continuous GDNF delivery was detected in culture and in healthy rat eyes for at least 14 days. The gels were well-tolerated with no host tissue attachment and contained living cell colonies. Most importantly, gel-implanted in dystrophic Royal College of Surgeons rat eyes for 28 days retained photoreceptors while those containing higher initial cell number yielded better photoreceptor survival. CAC ECT gels offers flexible system design and is a potential treatment option for posterior eye diseases. PMID:27441692

  16. Crack Propagation Versus Fiber Alignment in Collagen Gels: Experiments and Multiscale Simulation.

    PubMed

    Vanderheiden, Sarah M; Hadi, Mohammad F; Barocas, V H

    2015-12-01

    It is well known that the organization of the fibers constituting a collagenous tissue can affect its failure behavior. Less clear is how that effect can be described computationally so as to predict the failure of a native or engineered tissue under the complex loading conditions that can occur in vivo. Toward the goal of a general predictive strategy, we applied our multiscale model of collagen gel mechanics to the failure of a double-notched gel under tension, comparing the results for aligned and isotropic samples. In both computational and laboratory experiments, we found that the aligned gels were more likely to fail by connecting the two notches than the isotropic gels. For example, when the initial notches were 30% of the sample width (normalized tip-to-edge distance = 0.7), the normalized tip-to-tip distance at which the transition occurred from between-notch failure to across-sample failure shifted from 0.6 to 1.0. When the model predictions for the type of failure event (between the two notches versus across the sample width) were compared to the experimental results, the two were found to be strongly covariant by Fisher's exact test (p < 0.05) for both the aligned and isotropic gels with no fitting parameters. Although the double-notch system is idealized, and the collagen gel system is simpler than a true tissue, it presents a simple model system for studying failure of anisotropic tissues in a controlled setting. The success of the computational model suggests that the multiscale approach, in which the structural complexity is incorporated via changes in the model networks rather than via changes to a constitutive equation, has the potential to predict tissue failure under a wide range of conditions. PMID:26355475

  17. Physics of soft hyaluronic acid-collagen type II double network gels

    NASA Astrophysics Data System (ADS)

    Morozova, Svetlana; Muthukumar, Murugappan

    2015-03-01

    Many biological hydrogels are made up of multiple interpenetrating, charged components. We study the swelling, elastic diffusion, mechanical, and optical behaviors of 100 mol% ionizable hyaluronic acid (HA) and collagen type II fiber networks. Dilute, 0.05-0.5 wt% hyaluronic acid networks are extremely sensitive to solution salt concentration, but are stable at pH above 2. When swelled in 0.1M NaCl, single-network hyaluronic acid gels follow scaling laws relevant to high salt semidilute solutions; the elastic shear modulus G' and diffusion constant D scale with the volume fraction ϕ as G' ~ϕ 9 / 4 and D ~ϕ 3 / 4 , respectively. With the addition of a collagen fiber network, we find that the hyaluronic acid network swells to suspend the rigid collagen fibers, providing extra strength to the hydrogel. Results on swelling equilibria, elasticity, and collective diffusion on these double network hydrogels will be presented.

  18. Mechanical Restrictions on Biological Responses by Adherent Cells within Collagen Gels

    PubMed Central

    Simon, D.D.; Horgan, C.O.; Humphrey, J.D.

    2012-01-01

    Cell-seeded collagen and fibrin gels represent excellent assays for studying interactions between adherent interstitial cells and the three-dimensional extracellular matrix in which they reside. Over one hundred papers have employed the free-floating collagen gel assay alone since its introduction in 1979 and much has been learned about mechanobiological responses of diverse types of cells. Yet, given that mechanobiology is the study of biological responses by cells to mechanical stimuli that must respect the basic laws of mechanics, we must quantify better the mechanical conditions that are imposed on or arise in cell-seeded gels. In this paper, we suggest that cell responses and associated changes in matrix organization within the classical free-floating gel assay are highly restricted by the mechanics. In particular, many salient but heretofore unexplained or misinterpreted observations in free-floating gels can be understood in terms of apparent cell-mediated residual stress fields that satisfy quasi-static equilibria and continuity of tractions. There is a continuing need, therefore, to bring together the allied fields of mechanobiology and biomechanics as we continue to elucidate cellular function within both native connective tissues and tissue equivalents that are used in basic scientific investigations or regenerative medicine. PMID:23022259

  19. Radio-physical properties of micelle leucodye 3D integrating gel dosimeters

    NASA Astrophysics Data System (ADS)

    Vandecasteele, J.; Ghysel, S.; Baete, S. H.; De Deene, Y.

    2011-02-01

    Recently, novel radiochromic leucodye micelle hydrogel dosimeters were introduced in the literature. In these studies, gel measured electron depth dose profiles were compared with ion chamber depth dose data, from which it was concluded that leucocrystal violet-type dosimeters were independent of dose rate. Similar conclusions were drawn for leucomalachite green-type dosimeters, only after pre-irradiating the samples to a homogeneous radiation dose. However, in our extensive study of the radio-physical properties of leucocrystal violet- and leucomalachite green-type dosimeters, a significant dose rate dependence was found. For a dose rate variation between 50 and 400 cGy\\,min^{-1}, a maximum difference of 75% was found in optical dose sensitivity for the leucomalachite green-type dosimeter. Furthermore, the measured optical dose sensitivity of the leucomalachite green-type dosimeter was four times lower than the value previously reported in the literature. For the leucocrystal violet-type dosimeter, a maximum difference in optical dose sensitivity of 55% was found between 50 and 400 cGy\\,min^{-1}. A modified composition of the leucomalachite green-type dosimeter is proposed. This dosimeter is composed of gelatin, sodium dodecyl sulfate, chloroform, trichloroacetic acid and leucomalachite green. The optical dose sensitivity amounted to 4.375 \\times 10^{-5} \\,cm^{-1}\\; cGy^{-1} (dose rate 400 cGy\\,min^{-1}). No energy dependence for photon energies between 6 and 18 MV was found. No temperature dependence during readout was found notwithstanding a temperature dependence during irradiation of 1.90 cGy °C-1 increase on a total dose of 100 cGy. The novel gel dosimeter formulation exhibits an improved spatial stability (2.45 \\times 10^{-7} \\,cm^{2}\\; s^{-1} (= 0.088 mm^2 \\; h^{-1})) and good water/soft tissue equivalence. Nevertheless, the novel formulation was also found to have a significant, albeit reduced, dose rate dependence, as a maximum difference of 33

  20. Probing the 3D structure of cornea-like collagen liquid crystals with polarization-resolved SHG microscopy.

    PubMed

    Teulon, Claire; Tidu, Aurélien; Portier, François; Mosser, Gervaise; Schanne-Klein, Marie-Claire

    2016-07-11

    This work aims at characterizing the three-dimensional organization of liquid crystals composed of collagen, in order to determine the physico-chemical conditions leading to highly organized structures found in biological tissues such as cornea. To that end, we use second-harmonic generation (SHG) microscopy, since aligned collagen structures have been shown to exhibit intrinsic SHG signals. We combine polarization-resolved SHG experiments (P-SHG) with the theoretical derivation of the SHG signal of collagen molecules tilted with respect to the focal plane. Our P-SHG images exhibit striated patterns with variable contrast, as expected from our analytical and numerical calculations for plywood-like nematic structures similar to the ones found in the cornea. This study demonstrates the benefits of P-SHG microscopy for in situ characterization of highly organized biopolymers at micrometer scale, and the unique sensitivity of this nonlinear optical technique to the orientation of collagen molecules. PMID:27410876

  1. The feasibility assessment of radiation dose of movement 3D NIPAM gel by magnetic resonance imaging

    NASA Astrophysics Data System (ADS)

    Hsieh, Chih-Ming; Leung, Joseph Hang; Ng, Yu-Bun; Cheng, Chih-Wu; Sun, Jung-Chang; Lin, Ping-Chin; Hsieh, Bor-Tsung

    2015-11-01

    NIPAM dosimeter is widely accepted and recommended for its 3D distribution and accuracy in dose absorption. Up to the moment, most research works on dose measurement are based on a fixed irradiation target without the consideration of the effect from physiological motion. We present a study to construct a respiratory motion simulating patient anatomical and dosimetry model for the study of dosimetic effect of organ motion. The dose on fixed and motion targets was measured by MRI after a dose adminstration of 1, 2, 5, 8, and 10 Gy from linear accelerator. Comparison of two situations is made. The average sensitivity of fixed NIPAM was 0.1356 s-1/Gy with linearity R2=0.998. The average sensitivity of movement NIPAM was 0.1366 s-1/Gy with linearity R2=0.998 both having only 0.001 of the sensitivity difference. The difference between the two based on dose rate dependency, position and depth was not significant. There was thus no apparent impact on NIPAM dosimeter from physiological motion. The high sensitivity, linearity and stability of NIPAM dosimeter proved to be an ideal apparatus in the dose measurement in these circumstances.

  2. Treatment with Y-27632, a ROCK Inhibitor, Increases the Proinvasive Nature of SW620 Cells on 3D Collagen Type 1 Matrix

    PubMed Central

    Vishnubhotla, Ramana; Bharadwaj, Shruthi; Sun, Shan; Metlushko, Vitali; Glover, Sarah C.

    2012-01-01

    The concept of using tissue density as a mechanism to diagnose a tumor has been around for centuries. However, this concept has not been sufficiently explored in a laboratory setting. Therefore, in this paper, we observed the effects of cell density and extracellular matrix (ECM) density on colon cancer invasion and proliferation using SW620 cells. We also attempted to inhibit ROCK-I to determine its effect on cell invasion and proliferation using standard molecular biology techniques and advanced imaging. Increasing cell seeding density resulted in a 2-fold increase in cell invasion as well as cell proliferation independent of treatment with Y-27632. Increasing collagen I scaffold density resulted in a 2.5-fold increase in cell proliferation while treatment with Y-27632 attenuated this effect although 1.5 fold increase in cell invasion was observed in ROCK inhibited samples. Intriguingly, ROCK inhibition also resulted in a 3.5-fold increase in cell invasion within 3D collagen scaffolds for cells seeded at lower densities. We show in this paper that ROCK-I inhibition leads to increased invasion within 3D collagen I microenvironments. This data suggests that although ROCK inhibitors have been used clinically to treat several medical conditions, its effect largely depends on the surrounding microenvironment. PMID:22690219

  3. Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres

    PubMed Central

    Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

    2016-01-01

    Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering. PMID:26760956

  4. Controlling coupling reaction of EDC and NHS for preparation of collagen gels using ethanol/water co-solvents.

    PubMed

    Nam, Kwangwoo; Kimura, Tsuyoshi; Kishida, Akio

    2008-01-01

    To control the crosslinking rate of the collagen gel, ethanol/water co-solvent was adopted for the reaction solvent for the collagen microfibril crosslinking. Collagen gel was prepared by using EDC and NHS as coupling agents. Ethanol did not denaturate the helical structure of the collagen and prevented the hydrolysis of EDC, but showed the protonation of carboxylate anions. In order to control the intra- and interhelical crosslink of the collagen triple helix, variations of the mole ratio of carboxyl group/EDC/NHS, and of the ethanol mole concentration were investigated. Increase in the EDC ratio against the carboxyl group increased the crosslinking rate. Furthermore, an increase in the ethanol mole concentration resulted in an increase of the crosslinking rate until ethanol mole concentration was 0.12, but showed gradual decrease as the ethanol mole concentration was further increased. This is because the adsorption of solvent by the collagen gel, protonation of carboxylate anion, and hydrolysis of EDC is at its most optimum condition for the coupling reaction when the ethanol mole concentration is 0.12. The re-crosslinking of the collagen gel showed an increase in the crosslinking rate, but did not show further increase when the coupling reaction was executed for the third time. This implied that the highest possible crosslinking rate for the intra- and interhelical is approximately 60% when EDC/NHS is used. PMID:18023082

  5. Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions

    PubMed Central

    Rodrigues, Juliany C.F.; Viana, Nathan B.; Pontes, Bruno; Pereira, Camila F.A.; Silva-Filho, Fernando C.

    2014-01-01

    Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM) of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D) environment mainly composed of Collagen I (COL I). This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited “freeze and run” migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular “home”—macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model. PMID:24765565

  6. The endogenous fluorescence of fibroblast in collagen gels as indicator of stiffness of the extracellular matrix

    NASA Astrophysics Data System (ADS)

    Padilla-Martinez, J. P.; Ortega-Martinez, A.; Franco, W.

    2016-03-01

    The stiffness or rigidity of the extracellular matrix (ECM) regulates cell response. Established mechanical tests to measure stiffness, such as indentation and tensile tests, are invasive and destructive to the sample. Endogenous or native molecules to cells and ECM components, like tryptophan and cross-links of collagen, display fluorescence upon irradiation with ultraviolet light. Most likely, the concentration of these endogenous fluorophores changes as the stiffness of the ECM changes. In this work we investigate the endogenous fluorescence of collagen gels containing fibroblasts as a non-invasive non-destructive method to measure stiffness of the ECM. Human fibroblast cells were cultured in three-dimensional gels of type I collagen (50,000 cells/ml). This construct is a simple model of tissue contraction. During contraction, changes in the excitation-emission matrix (a fluorescence map in the 240-520/290-530 nm range) of constructs were measured with a spectrofluoremeter, and changes in stiffness were measured with a standard indentation test over 16 days. Results show that a progressive increase in fluorescence of the 290/340 nm excitation-emission pair correlates with a progressive increase in stiffness (r=0.9, α=0.5). The fluorescence of this excitation-emission pair is ascribed to tryptophan and variations in the fluorescence of this pair correlate with cellular proliferation. In this tissue model, the endogenous functional fluorescence of proliferating fibroblast cells is a biomechanical marker of stiffness of the ECM.

  7. Tooth periodontal ligament: Direct 3D microCT visualization of the collagen network and how the network changes when the tooth is loaded.

    PubMed

    Naveh, Gili R S; Brumfeld, Vlad; Shahar, Ron; Weiner, Steve

    2013-02-01

    The periodontal ligament (PDL), a soft tissue connecting the tooth and the bone, is essential for tooth movement, bone remodeling and force dissipation. A collagenous network that connects the tooth root surface to the alveolar jaw bone is one of the major components of the PDL. The organization of the collagenous component and how it changes under load is still poorly understood. Here using a state-of-the-art custom-made loading apparatus and a humidified environment inside a microCT, we visualize the PDL collagenous network of a fresh rat molar in 3D at 1 μm voxel size without any fixation or contrasting agents. We demonstrate that the PDL collagen network is organized in sheets. The spaces between sheets vary thus creating dense and sparse networks. Upon vertical loading, the sheets in both networks are stretched into well aligned arrays. The sparse network is located mainly in areas which undergo compressive loading as the tooth moves towards the bone, whereas the dense network functions mostly in tension as the tooth moves further from the bone. This new visualization method can be used to study other non-mineralized or partially mineralized tissues, and in particular those that are subjected to mechanical loads. The method will also be valuable for characterizing diseased tissues, as well as better understanding the phenotypic expressions of genetic mutants. PMID:23110851

  8. Synthesis and studies of Zn-doped LaMnO3+d nanoperovskite by sol-gel processing

    NASA Astrophysics Data System (ADS)

    Alemi, A. A.; Karimpour, E.; Shokri, H.

    Nanoparticles with high surface area of LaMn1-xZnxO3+d (LMZ) (0.0≤;x≤;0.38) were synthesized by the Pechini-type polymerizable complex technique based on in situ polyesterification between citric acid (CA) and ethylene glycol (EG). Heating at 90 °C of a mixed solution of CA, EG, H2O, and nitrates of La, Mn, and Zn, gave a polymeric resin. The polymeric resin or citrate-derived gel was preheated at 450 °C for 2 h to give a powdered precursor of LMZ. The final heat treatment of the powdered precursor was done in static air at a temperature range 700-800 °C for 6 h. After calcinations of the dried gels, the products were characterized by the X-ray powder diffraction technique, Fourier transform infrared analysis, and scanning electron microscopy. The products are of considerable interest both for utilization as catalysts and for their exceptional magnetotransport properties.

  9. Multiphoton microscopy of engineered dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast contraction

    NASA Astrophysics Data System (ADS)

    Pena, Ana-Maria; Fagot, Dominique; Olive, Christian; Michelet, Jean-François; Galey, Jean-Baptiste; Leroy, Frédéric; Beaurepaire, Emmanuel; Martin, Jean-Louis; Colonna, Anne; Schanne-Klein, Marie-Claire

    2010-09-01

    Dermal fibroblasts are responsible for the generation of mechanical forces within their surrounding extracellular matrix and can be potentially targeted by anti-aging ingredients. Investigation of the modulation of fibroblast contraction by these ingredients requires the implementation of three-dimensional in situ imaging methodologies. We use multiphoton microscopy to visualize unstained engineered dermal tissue by combining second-harmonic generation that reveals specifically fibrillar collagen and two-photon excited fluorescence from endogenous cellular chromophores. We study the fibroblast-induced reorganization of the collagen matrix and quantitatively evaluate the effect of Y-27632, a RhoA-kinase inhibitor, on dermal substitute contraction. We observe that collagen fibrils rearrange around fibroblasts with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA-kinase inhibitor. Moreover, we show that the inhibitory effects are reversible. Our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the extracellular matrix induced by fibroblast contraction or other processes.

  10. The 3-D collagen structure of equine articular cartilage, characterized using variable-angle-of-incidence polarization-sensitive optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Ugryumova, Nadya; Gangnus, Sergei V.; Matcher, Stephen J.

    2005-08-01

    Polarization-sensitive optical coherence tomography has been used to spatially map the birefringence of equine articular cartilage. Images obtained in the vicinity of visible osteoarthritic lesions display a characteristic disruption of the regular birefringence bands shown by normal cartilage. We also note that significant (e.g. ×2) variations in the apparent birefringence of samples taken from young (18 month) animals that otherwise appear visually homogeneous are found over spatial scales of a few millimeters. We suggest that whilst some of this variation may be due to changes in the intrinsic birefringence of the tissue, the 3-D orientation of the collagen fibers relative to the plane of the joint surface should also be taken into account. We propose a method based on multiple angles of illumination to determine the polar angle of the collagen fibers.

  11. A modified collagen gel dressing promotes angiogenesis in a preclinical swine model of chronic ischemic wounds.

    PubMed

    Elgharably, Haytham; Ganesh, Kasturi; Dickerson, Jennifer; Khanna, Savita; Abas, Motaz; Ghatak, Piya Das; Dixit, Sriteja; Bergdall, Valerie; Roy, Sashwati; Sen, Chandan K

    2014-01-01

    We recently performed proteomic characterization of a modified collagen gel (MCG) dressing and reported promising effects of the gel in healing full-thickness excisional wounds. In this work, we test the translational relevance of our aforesaid findings by testing the dressing in a swine model of chronic ischemic wounds recently reported by our laboratory. Full-thickness excisional wounds were established in the center of bipedicle ischemic skin flaps on the backs of animals. Ischemia was verified by laser Doppler imaging, and MCG was applied to the test group of wounds. Seven days post wounding, macrophage recruitment to the wound was significantly higher in MCG-treated ischemic wounds. In vitro, MCG up-regulated expression of Mrc-1 (a reparative M2 macrophage marker) and induced the expression of anti-inflammatory cytokine interleukin (IL)-10 and of fibroblast growth factor-basic (β-FGF). An increased expression of CCR2, an M2 macrophage marker, was noted in the macrophages from MCG treated wounds. Furthermore, analyses of wound tissues 7 days post wounding showed up-regulation of transforming growth factor-β, vascular endothelial growth factor, von Willebrand's factor, and collagen type I expression in MCG-treated ischemic wounds. At 21 days post wounding, MCG-treated ischemic wounds displayed higher abundance of proliferating endothelial cells that formed mature vascular structures and increased blood flow to the wound. Fibroblast count was markedly higher in MCG-treated ischemic wound-edge tissue. In addition, MCG-treated wound-edge tissues displayed higher abundance of mature collagen with increased collagen type I : III deposition. Taken together, MCG helped mount a more robust inflammatory response that resolved in a timely manner, followed by an enhanced proliferative phase, angiogenic outcome, and postwound tissue remodeling. Findings of the current study warrant clinical testing of MCG in a setting of ischemic chronic wounds. PMID:25224310

  12. Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry.

    PubMed

    Davidenko, Natalia; Schuster, Carlos F; Bax, Daniel V; Farndale, Richard W; Hamaia, Samir; Best, Serena M; Cameron, Ruth E

    2016-10-01

    Studies of cell attachment to collagen-based materials often ignore details of the binding mechanisms-be they integrin-mediated or non-specific. In this work, we have used collagen and gelatin-based substrates with different dimensional characteristics (monolayers, thin films and porous scaffolds) in order to establish the influence of composition, crosslinking (using carbodiimide) treatment and 2D or 3D architecture on integrin-mediated cell adhesion. By varying receptor expression, using cells with collagen-binding integrins (HT1080 and C2C12 L3 cell lines, expressing α2β1, and Rugli expressing α1β1) and a parent cell line C2C12 with gelatin-binding receptors (αvβ3 and α5β1), the nature of integrin binding sites was studied in order to explain the bioactivity of different protein formulations. We have shown that alteration of the chemical identity, conformation and availability of free binding motifs (GxOGER and RGD), resulting from addition of gelatin to collagen and crosslinking, have a profound effect on the ability of cells to adhere to these formulations. Carbodiimide crosslinking ablates integrin-dependent cell activity on both two-dimensional and three-dimensional architectures while the three-dimensional scaffold structure also leads to a high level of non-specific interactions remaining on three-dimensional samples even after a rigorous washing regime. This phenomenon, promoted by crosslinking, and attributed to cell entrapment, should be considered in any assessment of the biological activity of three-dimensional substrates. Spreading data confirm the importance of integrin-mediated cell engagement for further cell activity on collagen-based compositions. In this work, we provide a simple, but effective, means of deconvoluting the effects of chemistry and dimensional characteristics of a substrate, on the cell activity of protein-derived materials, which should assist in tailoring their biological properties for specific tissue engineering

  13. Corneal Stromal Cells use both High- and Low-Contractility Migration Mechanisms in 3-D Collagen Matrices

    PubMed Central

    Kim, Areum; Zhou, Chengxin; Lakshman, Neema; Petroll, W. Matthew

    2012-01-01

    Corneal keratocyte migration can impact both corneal clarity and refractive outcome following injury or refractive surgery. In this study, we investigated how culture conditions, ECM properties, and Rho kinase activity regulate the mechanics of keratocyte migration, using a nested collagen matrix model. Time-lapse imaging demonstrated that both serum and PDGF stimulate keratocyte migration into the outer matrix. Although the velocity of cell migration was similar, cells in serum were bipolar and induced significant matrix deformation during migration, whereas PDGF induced extension of branching dendritic processes with smaller, more localized force generation. These differences in cell-induced matrix reorganization were verified with a global matrix contraction assay and confocal reflection imaging, using both bovine and rat tail collagen. When constructs were detached from the substrate to lower the effective stiffness, migration was significantly reduced in serum; but was unchanged in PDGF. These differences in migration mechanics were mediated, in part, by Rho kinase. Overall, corneal keratocytes can effectively migrate through collagen matrices using varying degrees of cellular force generation. Low-contractility migration may facilitate keratocyte repopulation of the stroma following surgery or injury, without altering the structural and mechanical properties that are critical to maintaining corneal transparency. PMID:22233682

  14. The 3D structure of the collagen fibril network in human trabecular bone: relation to trabecular organization.

    PubMed

    Reznikov, Natalie; Chase, Hila; Brumfeld, Vlad; Shahar, Ron; Weiner, Steve

    2015-02-01

    Trabecular bone is morphologically and functionally different from compact bone at the tissue level, but both are composed of lamellae at the micrometer-scale level. We present a three-dimensional study of the collagenous network of human trabecular lamellar bone from the proximal femur using the FIB-SEM serial surface view method. The results are compared to human compact lamellar bone of the femoral shaft, studied by the same method. Both demineralized trabecular and compact lamellar bone display the same overall structural organization, namely the presence of ordered and disordered materials and the confinement of the canalicular network to the disordered material. However, in trabecular bone lamellae a significant proportion of the ordered collagen fibril arrays is aligned with the long axis of the trabecula and, unlike in compact bone, is not related to the anatomical axis of the whole femur. The remaining ordered collagen fibrils are offset from the axis of a trabecula either by about 30° or 70°. Interestingly, at the tissue scale of millimeters, the most abundant angles between any two connected trabeculae - the inter-trabecular angles - center around 30° and 70°. This implies that within a framework of interconnected trabeculae the same lamellar structure will always have a significant component of the fibrils aligned with the long axes of connected trabeculae. This structural complementarity at different hierarchical levels presumably reflects an adaptation of trabecular bone to function. PMID:25445457

  15. A gel aspiration-ejection system for the controlled production and delivery of injectable dense collagen scaffolds.

    PubMed

    Kamranpour, Neysan O; Miri, Amir K; James-Bhasin, Mark; Nazhat, Showan N

    2016-03-01

    A gel aspiration-ejection (GAE) system has been developed for the advanced production and delivery of injectable dense collagen (I-DC) gels of unique collagen fibrillar densities (CFDs). Through the creation of negative pressure, GAE aspirates prefabricated highly hydrated collagen gels into a needle, simultaneously inducing compaction and meso-scale anisotropy (i.e., fibrillar alignment) on the gels, and by subsequent reversal of the pressure, I-DC gels can be controllably ejected. The system generates I-DC gels with CFDs ranging from 5 to 32 wt%, controlling the initial scaffold microstructure, anisotropy, hydraulic permeability, and mechanical properties. These features could potentially enable the minimally invasive delivery of more stable hydrogels. The viability, metabolic activity, and differentiation of seeded mesenchymal stem cells (MSCs) was investigated in the I-DC gels of distinct CFDs and extents of anisotropy produced through two different gauge needles. MSC osteoblastic differentiation was found to be relatively accelerated in I-DC gels that combined physiologically relevant CFDs and increased fibrillar alignment. The ability to not only support homogenous cell seeding, but also to direct and accelerate their differentiation through tissue-equivalent anisotropy, creates numerous opportunities in regenerative medicine. PMID:27003606

  16. Two-way regulation between cells and aligned collagen fibrils: local 3D matrix formation and accelerated neural differentiation of human decidua parietalis placental stem cells.

    PubMed

    Li, Wen; Zhu, Bofan; Strakova, Zuzana; Wang, Rong

    2014-08-01

    It has been well established that an aligned matrix provides structural and signaling cues to guide cell polarization and cell fate decision. However, the modulation role of cells in matrix remodeling and the feedforward effect on stem cell differentiation have not been studied extensively. In this study, we report on the concerted changes of human decidua parietalis placental stem cells (hdpPSCs) and the highly ordered collagen fibril matrix in response to cell-matrix interaction. With high-resolution imaging, we found the hdpPSCs interacted with the matrix by deforming the cell shape, harvesting the nearby collagen fibrils, and reorganizing the fibrils around the cell body to transform a 2D matrix to a localized 3D matrix. Such a unique 3D matrix prompted high expression of β-1 integrin around the cell body that mediates and facilitates the stem cell differentiation toward neural cells. The study offers insights into the coordinated, dynamic changes at the cell-matrix interface and elucidates cell modulation of its matrix to establish structural and biochemical cues for effective cell growth and differentiation. PMID:25003322

  17. Longitudinal, 3D Imaging of Collagen Remodeling in Murine Hypertrophic Scars In Vivo using Polarization-sensitive Optical Frequency Domain Imaging

    PubMed Central

    Lo, William C. Y.; Villiger, Martin; Golberg, Alexander; Broelsch, G. Felix; Khan, Saiqa; Lian, Christine G.; Austen, William G.; Yarmush, Martin; Bouma, Brett E.

    2016-01-01

    Hypertrophic scars (HTS), frequently seen after traumatic injuries and surgery, remain a major clinical challenge due to the limited success of existing therapies. A significant obstacle to understanding HTS etiology is the lack of tools to monitor scar remodeling longitudinally and non-invasively. We present an in vivo, label-free technique using polarization-sensitive optical frequency domain imaging (PS-OFDI) for the 3D, longitudinal assessment of collagen remodeling in murine HTS. In this study, HTS was induced with a mechanical tension device for 4 to 10 days on incisional wounds and imaged up to one month after device removal; an excisional HTS model was also imaged at 6 months after injury to investigate deeper and more mature scars. We showed that local retardation (LR) and degree of polarization (DOP) provide a robust signature for HTS. Compared to normal skin with heterogeneous LR and low DOP, HTS was characterized by an initially low LR, which increased as collagen fibers remodeled, and a persistently high DOP. This study demonstrates that PS-OFDI offers a powerful tool to gain significant biological insights into HTS remodeling by enabling longitudinal assessment of collagen in vivo, which is critical to elucidating HTS etiology and developing more effective HTS therapies. PMID:26763427

  18. Longitudinal, 3D Imaging of Collagen Remodeling in Murine Hypertrophic Scars In Vivo Using Polarization-Sensitive Optical Frequency Domain Imaging.

    PubMed

    Lo, William C Y; Villiger, Martin; Golberg, Alexander; Broelsch, G Felix; Khan, Saiqa; Lian, Christine G; Austen, William G; Yarmush, Martin; Bouma, Brett E

    2016-01-01

    Hypertrophic scars (HTS), frequently seen after traumatic injuries and surgery, remain a major clinical challenge because of the limited success of existing therapies. A significant obstacle to understanding HTS etiology is the lack of tools to monitor scar remodeling longitudinally and noninvasively. We present an in vivo, label-free technique using polarization-sensitive optical frequency domain imaging for the 3D, longitudinal assessment of collagen remodeling in murine HTS. In this study, HTS was induced with a mechanical tension device for 4-10 days on incisional wounds and imaged up to 1 month after device removal; an excisional HTS model was also imaged at 6 months after injury to investigate deeper and more mature scars. We showed that local retardation and degree of polarization provide a robust signature for HTS. Compared with normal skin with heterogeneous local retardation and low degree of polarization, HTS was characterized by an initially low local retardation, which increased as collagen fibers remodeled, and a persistently high degree of polarization. This study demonstrates that polarization-sensitive optical frequency domain imaging offers a powerful tool to gain significant biological insights into HTS remodeling by enabling longitudinal assessment of collagen in vivo, which is critical to elucidating HTS etiology and developing more effective HTS therapies. PMID:26763427

  19. Ectopic bone formation in rapidly fabricated acellular injectable dense collagen-Bioglass hybrid scaffolds via gel aspiration-ejection.

    PubMed

    Miri, Amir K; Muja, Naser; Kamranpour, Neysan O; Lepry, William C; Boccaccini, Aldo R; Clarke, Susan A; Nazhat, Showan N

    2016-04-01

    Gel aspiration-ejection (GAE) has recently been introduced as an effective technique for the rapid production of injectable dense collagen (IDC) gel scaffolds with tunable collagen fibrillar densities (CFDs) and microstructures. Herein, a GAE system was applied for the advanced production and delivery of IDC and IDC-Bioglass(®) (IDC-BG) hybrid gel scaffolds for potential bone tissue engineering applications. The efficacy of GAE in generating mineralizable IDC-BG gels (from an initial 75-25 collagen-BG ratio) produced through needle gauge numbers 8G (3.4 mm diameter and 6 wt% CFD) and 14G (1.6 mm diameter and 14 wt% CFD) was investigated. Second harmonic generation (SHG) imaging of as-made gels revealed an increase in collagen fibril alignment with needle gauge number. In vitro mineralization of IDC-BG gels was confirmed where carbonated hydroxyapatite was detected as early as day 1 in simulated body fluid, which progressively increased up to day 14. In vivo mineralization of, and host response to, acellular IDC and IDC-BG gel scaffolds were further investigated following subcutaneous injection in adult rats. Mineralization, neovascularization and cell infiltration into the scaffolds was enhanced by the addition of BG and at day 21 post injection, there was evidence of remodelling of granulation tissue into woven bone-like tissue in IDC-BG. SHG imaging of explanted scaffolds indicated collagen fibril remodelling through cell infiltration and mineralization over time. In sum, the results suggest that IDC-BG hybrid gels have osteoinductive properties and potentially offer a novel therapeutic approach for procedures requiring the injectable delivery of a malleable and dynamic bone graft that mineralizes under physiological conditions. PMID:26871889

  20. Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells.

    PubMed

    Greenburg, G; Hay, E D

    1982-10-01

    This study of epithelial-mesenchymal transformation and epithelial cell polarity in vitro reveals that environmental conditions can have a profound effect on the epithelial phenotype, cell shape, and polarity as expressed by the presence of apical and basal surfaces. A number of different adult and embryonic epithelia were suspended within native collagen gels. Under these conditions, cells elongate, detach from the explants, and migrate as individual cells within the three-dimensional lattice, a previously unknown property of well-differentiated epithelia. Epithelial cells from adult and embryonic anterior lens were studied in detail. Elongated cells derived from the apical surface develop pseudopodia and filopodia characteristic of migratory cells and acquire a morphology and ultrastructure virtually indistinguishable from that of mesenchymal cells in vivo. It is concluded from these experiments that the three-dimensional collagen gel can promote dissociation, migration, and acquisition of secretory organelles by differentiated epithelial cells, and can abolish the apical-basal cell polarity characteristic of the original epithelium. PMID:7142291

  1. 3D polymer gel dosimetry and Geant4 Monte Carlo characterization of novel needle based X-ray source

    NASA Astrophysics Data System (ADS)

    Liu, Y.; Sozontov, E.; Safronov, V.; Gutman, G.; Strumban, E.; Jiang, Q.; Li, S.

    2010-11-01

    In the recent years, there have been a few attempts to develop a low energy x-ray radiation sources alternative to conventional radioisotopes used in brachytherapy. So far, all efforts have been centered around the intent to design an interstitial miniaturized x-ray tube. Though direct irradiation of tumors looks very promising, the known insertable miniature x-ray tubes have many limitations: (a) difficulties with focusing and steering the electron beam to the target; (b)necessity to cool the target to increase x-ray production efficiency; (c)impracticability to reduce the diameter of the miniaturized x-ray tube below 4mm (the requirement to decrease the diameter of the x-ray tube and the need to have a cooling system for the target have are mutually exclusive); (c) significant limitations in changing shape and energy of the emitted radiation. The specific aim of this study is to demonstrate the feasibility of a new concept for an insertable low-energy needle x-ray device based on simulation with Geant4 Monte Carlo code and to measure the dose rate distribution for low energy (17.5 keV) x-ray radiation with the 3D polymer gel dosimetry.

  2. Polarity reversal of inside-out thyroid follicles cultured within collagen gel: reexpression of specific functions.

    PubMed

    Chambard, M; Verrier, B; Gabrion, J; Mauchamp, J

    1984-01-01

    Isolated porcine thyroid cells cultured in suspension in Eagle Minimum Essential Medium supplemented with calf serum (5-20%) reorganize to form vesicles, i.e. closed structures in which all cells have an inverted polarity as compared to that found in follicles: the apical membranes are bathed by the culture medium. Under these conditions, cells neither concentrate iodide nor respond to acute thyrotropin (TSH) stimulation. When embedded in collagen gel, these vesicles undergo polarity reversal to form follicles. We describe here the change in the orientation of cell polarity and the subsequent reappearance of specific thyroid functions. Six hr after embedding, membrane areas in contact with collagen fibers show basal characteristics. At this time, cells begin to concentrate iodide and to respond to acute TSH stimulation (iodide efflux and increased cAMP levels). Most cells form follicles 24 hr after embedding, but 48 hr are required for the transformation of all vesicles into follicles. This occurs without opening of the tight junctions. Iodide organification is detected 24 hr after embedding, when periodic acid-Schiff positive material, identified as thyroglobulin by immunofluorescence, accumulates in the lumen. Iodide concentration and organification, as well as response to TSH stimulation reach maximal levels after 3 days in the collagen matrix. After a 5-day culture in the collagen matrix in the absence of TSH, cell activity can be stimulated by chronic treatment with low hormone concentrations (10-100 microU/ml). As shown with thyroid cells grown in monolayer on permeable substrates (Chambard M., et al., 1983, J. Cell Biol. 96, 1172-1177), iodide uptake and cAMP-mediated TSH responses are expressed when the halogen and the hormone have direct access to the basal membrane. Organification, on the contrary, requires a closed apical compartment. PMID:6098327

  3. Matrix forming characteristics of inner and outer human meniscus cells on 3D collagen scaffolds under normal and low oxygen tensions

    PubMed Central

    2013-01-01

    toward chondrogenic phenotype and ECM production. Oxygen tension played a key role in modulating the redifferentiation of meniscal fibrochondrocytes on a 3D collagen scaffold in vitro. PMID:24330551

  4. The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets.

    PubMed

    Nagamoto, Yasuhito; Tashiro, Katsuhisa; Takayama, Kazuo; Ohashi, Kazuo; Kawabata, Kenji; Sakurai, Fuminori; Tachibana, Masashi; Hayakawa, Takao; Furue, Miho Kusuda; Mizuguchi, Hiroyuki

    2012-06-01

    Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are known to be a useful cell source for drug screening. We recently developed an efficient hepatic differentiation method from hESCs and hiPSCs by sequential transduction of FOXA2 and HNF1α. It is known that the combination of three-dimensional (3D) culture and co-culture, namely 3D co-culture, can maintain the functions of primary hepatocytes. However, hepatic maturation of hESC- or hiPSC-derived hepatocyte-like cells (hEHs or hiPHs, respectively) by 3D co-culture systems has not been examined. Therefore, we utilized a cell sheet engineering technology to promote hepatic maturation. The gene expression levels of hepatocyte-related markers (such as cytochrome P450 enzymes and conjugating enzymes) and the amount of albumin secretion in the hEHs or hiPHs, which were 3D co-cultured with the Swiss 3T3 cell sheet, were significantly up-regulated in comparison with those in the hEHs or hiPHs cultured in a monolayer. Furthermore, we found that type I collagen synthesized in Swiss 3T3 cells plays an important role in hepatic maturation. The hEHs or hiPHs that were 3D co-cultured with the Swiss 3T3 cell sheet would be powerful tools for medical applications, such as drug screening. PMID:22445253

  5. Optical laser scanning of a leucodye micelle gel: preliminary results of a 3D dose verification of an IMRT treatment for a brain tumor

    NASA Astrophysics Data System (ADS)

    Vandecasteele, J.; De Deene, Y.

    2013-06-01

    In the present study an in-house developed leucodye micelle gel was used in combination with an in-house developed optical laser scanner for the 3D dose verification of an IMRT treatment of a pituitary adenoma. In an initial prospective study, a gel measured depth dose distribution of a square 6 MV photon beam was compared with an ion chamber measurement. In a second experiment, the gel and scanner were used to verify a clinical dose distribution on a recently installed linear accelerator. The calibration procedure is identified as the major source of dose deviations.

  6. Intervention with Formulated Collagen Gel for Chronic Heel Pressure Ulcers in Older Adults with Diabetes.

    PubMed

    Agosti, Jennifer K; Chandler, Lois A

    2015-11-01

    Chronic pressure ulcers (PrUs), ulcers that fail to progress through the expected phases of wound healing in a timely fashion, are not only a concern for the patients afflicted with them, but are also a significant burden for the long-term-care facilities in which patients reside. The heel is the second most common location for PrUs. Morbidity and mortality rates for heel PrUs, particularly in the diabetic population, are alarming. Therefore, a consistently effective, cost-conscious, and user-friendly topical treatment for heel ulcers would be welcomed by patients and clinicians. This article describes a marked and rapid improvement in wound granulation in 3 older adult patients following weekly treatment for 8 weeks of chronic (≥1-year duration) heel ulcers with an easy-to-use, cost-effective, topical, formulated collagen gel. PMID:26479694

  7. Cyclic traction machine for long-term culture of fibroblast-populated collagen gels.

    PubMed

    Langelier, E; Rancourt, D; Bouchard, S; Lord, C; Stevens, P P; Germain, L; Auger, F A

    1999-01-01

    Our research group has been investigating the effect of cyclic deformations on the evolution of fibroblast populated collagen gels (FPCG). Since existing traction machines are not designed for such an application, we had to design a cyclic traction machine adapted to tissue culture inside an incubator over an extended period of time. Biocompatible materials were used for fabrication to allow for easy sterilization and to prevent any adverse reaction from the tissue. The traction machine is based on a computer-controlled stepping motor system for easy adjustment of the deformation amplitude and frequency. The maximum stretching speed achieved is around 1 mm/s. The traction machine can measure FPCG mechanical properties and perform rupture tests to determine its ultimate strength. Several FPCGs have been successfully cultured with the machine for up to four weeks without any adverse reaction. PMID:9916762

  8. Interstitial flow promotes vascular fibroblast, myofibroblast, and smooth muscle cell motility in 3-D collagen I via upregulation of MMP-1

    PubMed Central

    Shi, Zhong-Dong; Ji, Xin-Ying; Qazi, Henry

    2009-01-01

    Neointima formation often occurs in regions where the endothelium has been damaged and the transmural interstitial flow is elevated. Vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) contribute to intimal thickening by migrating from the media and adventitia into the site of injury. In this study, for the first time, the direct effects of interstitial flow on SMC and FB/MFB migration were investigated in an in vitro three-dimensional system. Collagen I gels were used to mimic three-dimensional extracellular matrix (ECM) for rat aortic SMCs and FBs/MFBs. Exposure to interstitial flow induced by 1 cmH2O pressure differential (shear stress, ∼0.05 dyn/cm2; flow velocity, ∼0.5 μm/s; and Darcy permeability, ∼10−11 cm2) substantially enhanced cell motility. Matrix metalloproteinase (MMP) inhibitor (GM-6001) abolished flow-induced migration augmentation, which suggested that the enhanced motility was MMP dependent. The upregulation of MMP-1 played a critical role for the flow-enhanced motility, which was further confirmed by silencing MMP-1 gene expression. Longer exposures to higher flows suppressed the number of migrated cells, although MMP-1 gene expression remained high. This suppression was a result of both flow-induced tissue inhibitor of metalloproteinase-1 upregulation and increased apoptotic and necrotic cell death. Interstitial flow did not affect MMP-2 gene expression or activity in the collagen I gel for any cell type. Our findings shed light on the mechanism by which vascular SMCs and FBs/MFBs contribute to intimal thickening in regions of vascular injury where interstitial flow is elevated. PMID:19465549

  9. Photo-crosslinkable hydrogel-based 3D microfluidic culture device.

    PubMed

    Lee, Youlee; Lee, Jong Min; Bae, Pan-Kee; Chung, Il Yup; Chung, Bong Hyun; Chung, Bong Geun

    2015-04-01

    We developed the photo-crosslinkable hydrogel-based 3D microfluidic device to culture neural stem cells (NSCs) and tumors. The photo-crosslinkable gelatin methacrylate (GelMA) polymer was used as a physical barrier in the microfluidic device and collagen type I gel was employed to culture NSCs in a 3D manner. We demonstrated that the pore size was inversely proportional to concentrations of GelMA hydrogels, showing the pore sizes of 5 and 25 w/v% GelMA hydrogels were 34 and 4 μm, respectively. It also revealed that the morphology of pores in 5 w/v% GelMA hydrogels was elliptical shape, whereas we observed circular-shaped pores in 25 w/v% GelMA hydrogels. To culture NSCs and tumors in the 3D microfluidic device, we investigated the molecular diffusion properties across GelMA hydrogels, indicating that 25 w/v% GelMA hydrogels inhibited the molecular diffusion for 6 days in the 3D microfluidic device. In contrast, the chemicals were diffused in 5 w/v% GelMA hydrogels. Finally, we cultured NSCs and tumors in the hydrogel-based 3D microfluidic device, showing that 53-75% NSCs differentiated into neurons, while tumors were cultured in the collagen gels. Therefore, this photo-crosslinkable hydrogel-based 3D microfluidic culture device could be a potentially powerful tool for regenerative tissue engineering applications. PMID:25641332

  10. SU-E-T-105: Development of 3D Dose Verification System for Volumetric Modulated Arc Therapy Using Improved Polyacrylamide-Based Gel Dosimeter

    SciTech Connect

    Ono, K; Fujimoto, S; Akagi, Y; Hirokawa, Y; Hayashi, S; Miyazawa, M

    2014-06-01

    Purpose: The aim of this dosimetric study was to develop 3D dose verification system for volumetric modulated arc therapy (VMAT) using polyacrylamide-based gel (PAGAT) dosimeter improved the sensitivity by magnesium chloride (MgCl{sub 2}). Methods: PAGAT gel containing MgCl{sub 2} as a sensitizer was prepared in this study. Methacrylic-acid-based gel (MAGAT) was also prepared to compare the dosimetric characteristics with PAGAT gel. The cylindrical glass vials (4 cm diameter, 12 cm length) filled with each polymer gel were irradiated with 6 MV photon beam using Novalis Tx linear accelerator (Varian/BrainLAB). The irradiated polymer gel dosimeters were scanned with Signa 1.5 T MRI system (GE), and dose calibration curves were obtained using T{sub 2} relaxation rate (R{sub 2} = 1/T{sub 2}). Dose rate (100-600 MU min{sup −1}) and fractionation (1-8 fractions) were varied. In addition, a cubic acrylic phantom (10 × 10 × 10 cm{sup 3}) filled with improved PAGAT gel inserted into the IMRT phantom (IBA) was irradiated with VMAT (RapidArc). C-shape structure was used for the VMAT planning by the Varian Eclipse treatment planning system (TPS). The dose comparison of TPS and measurements with the polymer gel dosimeter was accomplished by the gamma index analysis, overlaying the dose profiles for a set of data on selected planes using in-house developed software. Results: Dose rate and fractionation dependence of improved PAGAT gel were smaller than MAGAT gel. A high similarity was found by overlaying the dose profiles measured with improved PAGAT gel dosimeter and the TPS dose, and the mean pass rate of the gamma index analysis using 3%/3 mm criteria was achieved 90% on orthogonal planes for VMAT using improved PAGAT gel dosimeter. Conclusion: In-house developed 3D dose verification system using improved polyacrylamide-based gel dosimeter had a potential as an effective tool for VMAT QA.

  11. Epidermal growth factor promotes a mesenchymal over an amoeboid motility of MDA-MB-231 cells embedded within a 3D collagen matrix

    NASA Astrophysics Data System (ADS)

    Geum, Dongil T.; Kim, Beum Jun; Chang, Audrey E.; Hall, Matthew S.; Wu, Mingming

    2016-01-01

    The receptor of epidermal growth factor (EGFR) critically regulates tumor cell invasion and is a potent therapeutic target for treatment of many types of cancers, including carcinomas and glioblastomas. It is known that EGF regulates cell motility when tumor cells are embedded within a 3D biomatrix. However, roles of EGF in modulating tumor cell motility phenotype are largely unknown. In this article, we report that EGF promotes a mesenchymal over an amoeboid motility phenotype using a malignant breast tumor cell line, MDA-MB-231, embedded within a 3D collagen matrix. Amoeboid cells are rounded in shape, while mesenchymal cells are elongated, and their migrations are governed by a distinctly different set of biomolecules. Using single cell tracking analysis, we also show that EGF promotes cell dissemination through a significant increase in cell persistence along with a moderate increase of speed. The increase of persistence is correlated with the increase of the percentage of the mesenchymal cells within the population. Our work reveals a novel role of microenvironmental cue, EGF, in modulating heterogeneity and plasticity of tumor cell motility phenotype. In addition, it suggests a potential visual cue for diagnosing invasive states of breast cancer cells. This work can be easily extended beyond breast cancer cells.

  12. Sol-gel processed mupirocin silica microspheres loaded collagen scaffold: a synergistic bio-composite for wound healing.

    PubMed

    Perumal, Sathiamurthi; Ramadass, Satiesh kumar; Madhan, Balaraman

    2014-02-14

    Development of a bio-composite using synergistic combination is a promising strategy to address various pathological manifestations of acute and chronic wounds. In the present work, we have combined three materials viz., mupirocin as an antimicrobial drug, sol-gel processed silica microsphere as drug carrier for sustained delivery of drug and collagen, an established wound healer as scaffold. The mupirocin-loaded silica microspheres (Mu-SM) and Mu-SM loaded collagen scaffold were characterized for surface morphology, entrapment efficiency and distribution homogeneity, in vitro drug release, water uptake capacity, cell proliferation and antibacterial activity. In vivo wound healing efficacy of the bio-composite was experimented using full thickness excision wound model in Wistar albino rats. The Mu-SM incorporated collagen scaffold showed good in vitro characteristics in terms of better water uptake, sustained drug availability and antimicrobial activity. The wound closure analysis revealed that the complete epithelialisation was observed at 14.2 ± 0.44 days for Mu-SM loaded collagen, whereas this was 17.4 ± 0.44 days and 20.6 ± 0.54 days for collagen and control groups, respectively. Consequently, the synergistic strategy of combining mupirocin-loaded silica microspheres and collagen as a Mu-SM loaded collagen dressing material would be an ideal biomaterial for the treatment of surface wounds, burns and foot ulcers. PMID:24514452

  13. Hybrid Macro-Porous Titanium Ornamented by Degradable 3D Gel/nHA Micro-Scaffolds for Bone Tissue Regeneration

    PubMed Central

    Yin, Bo; Ma, Pei; Chen, Jun; Wang, Hai; Wu, Gui; Li, Bo; Li, Qiang; Huang, Zhifeng; Qiu, Guixing; Wu, Zhihong

    2016-01-01

    Porous titanium is a kind of promising material for bone substitution, while its bio-inert property results in demand of modifications to improve the osteointegration capacity. In this study, gelatin (Gel) and nano-hydroxyapatite (nHA) were used to construct 3D micro-scaffolds in the pores of porous titanium in the ratios of Gel:nHA = 1:0, Gel:nHA = 1:1, and Gel:nHA = 1:3, respectively. Cell attachment and proliferation, and gene and protein expression levels of osteogenic markers were evaluated in MC3T3-E1 cells, followed by bone regeneration assessment in a rabbit radius defect model. All hybrid scaffolds with different composition ratio were found to have significant promotional effects in cell adhesion, proliferation and differentiation, in which the group with Gel:nHA = 1:1 showed the best performance in vitro, as well as the most bone regeneration volume in vivo. This 3D micro-scaffolds modification may be an innovative method for porous titanium ornamentation and shows potential application values in clinic. PMID:27092492

  14. Complementary roles of microtubules and microfilaments in the lung fibroblast-mediated contraction of collagen gels: Dynamics and the influence of cell density.

    PubMed

    Redden, Robert A; Doolin, Edward J

    2006-01-01

    Fibroblasts are important cellular components in wound healing, scar formation, and fibrotic disorders; and the fibroblast-populated collagen-gel (FPCG) model allows examination of fibroblast behavior in an in vitro three-dimensional environment similar to that in vivo. Contraction of free-floating FPCGs depends on an active and dynamic cytoskeleton, and the contraction dynamics are highly influenced by cell density. We investigated mechanistic differences between high- and low-cell density FPCG contraction by evaluating contraction dynamics in detail, using specific cytoskeletal disruptors. Collagen gels were seeded with human lung fibroblasts at either high (HD) or low (LD) density, and incubated with or without cytoskeletal disruptors colchicine (microtubules) or cytochalasin D (microfilaments). Gel area was measured daily. FPCG contraction curves were essentially sigmoidal, featuring an initial period of no contraction (lag phase), followed by a period of rapid contraction (log phase). Contraction curves of HD-FPCGs were distinct from those of LD-FPCGs. For example, HD-FPCGs had a negligible lag phase (compared with 3 d for LD-FPCGs) and exhibited a higher rate of log-phase contraction. Both colchicine and cytochalasin dose-dependently inhibited contraction but specifically affected different phases of contraction in HD- and LD-FPCGs; and colchicine inhibited LD-FPCGs much more than HD-FPCGs. The data indicate that LD- and HD-FPCGs contract through different primary mechanisms. Microtubules and microfilaments are both complementarily and dynamically involved in the contraction of FPCGs, and cell density influences primary cytoskeletal mechanisms. These results provide valuable information about fibroblast behavior in healing and fibrosis, and may suggest novel treatment options. PMID:16759151

  15. 3D printing of soft and wet systems benefit from hard-to-soft transition of transparent shape memory gels (presentation video)

    NASA Astrophysics Data System (ADS)

    Furukawa, Hidemitsu; Gong, Jin; Makino, Masato; Kabir, Md. Hasnat

    2014-04-01

    Recently we successfully developed novel transparent shape memory gels. The SMG memorize their original shapes during the gelation process. In the room temperature, the SMG are elastic and show plasticity (yielding) under deformation. However when heated above about 50˚C, the SMG induce hard-to-soft transition and go back to their original shapes automatically. We focus on new soft and wet systems made of the SMG by 3-D printing technology.

  16. Formulated collagen gel accelerates healing rate immediately after application in patients with diabetic neuropathic foot ulcers

    PubMed Central

    Blume, Peter; Driver, Vickie R; Tallis, Arthur J; Kirsner, Robert S; Kroeker, Roy; Payne, Wyatt G; Wali, Soma; Marston, William; Dove, Cyaandi; Engler, Robert L; Chandler, Lois A; Sosnowski, Barbara K

    2011-01-01

    We assessed the safety and efficacy of Formulated Collagen Gel (FCG) alone and with Ad5PDGF-B (GAM501) compared with Standard of Care (SOC) in patients with 1.5–10.0 cm2 chronic diabetic neuropathic foot ulcers that healed <30% during Run-in. Wound size was assessed by planimetry of acetate tracings and photographs in 124 patients. Comparison of data sets revealed that acetate tracings frequently overestimated areas at some sites. For per-protocol analysis, 113 patients qualified using acetate tracings but only 82 qualified using photographs. Prior animal studies suggested that collagen alone would have little effect on healing and would serve as a negative control. Surprisingly trends for increased incidence of complete closure were observed for both GAM501 (41%) and FCG (45%) vs. Standard of Care (31%). By photographic data, Standard of Care had no significant effect on change in wound radius (mm/week) from during Run-in to Week 1 (−0.06±0.32 to 0.78±1.53, p=ns) but both FCG (−0.08±0.61 to 1.97±1.77, p<0.002) and GAM501 (−0.02±0.58 to 1.46±1.37, p<0.002) significantly increased healing rates that gradually declined over subsequent weeks. Both GAM501 and FCG appeared to be safe and well tolerated, and alternate dosing schedules hold promise to improve overall complete wound closure in adequately powered trials. PMID:21371164

  17. Deformation simulation of cells seeded on a collagen-GAG scaffold in a flow perfusion bioreactor using a sequential 3D CFD-elastostatics model.

    PubMed

    Jungreuthmayer, C; Jaasma, M J; Al-Munajjed, A A; Zanghellini, J; Kelly, D J; O'Brien, F J

    2009-05-01

    Tissue-engineered bone shows promise in meeting the huge demand for bone grafts caused by up to 4 million bone replacement procedures per year, worldwide. State-of-the-art bone tissue engineering strategies use flow perfusion bioreactors to apply biophysical stimuli to cells seeded on scaffolds and to grow tissue suitable for implantation into the patient's body. The aim of this study was to quantify the deformation of cells seeded on a collagen-GAG scaffold which was perfused by culture medium inside a flow perfusion bioreactor. Using a microCT scan of an unseeded collagen-GAG scaffold, a sequential 3D CFD-deformation model was developed. The wall shear stress and the hydrostatic wall pressure acting on the cells were computed through the use of a CFD simulation and fed into a linear elastostatics model in order to calculate the deformation of the cells. The model used numerically seeded cells of two common morphologies where cells are either attached flatly on the scaffold wall or bridging two struts of the scaffold. Our study showed that the displacement of the cells is primarily determined by the cell morphology. Although cells of both attachment profiles were subjected to the same mechanical load, cells bridging two struts experienced a deformation up to 500 times higher than cells only attached to one strut. As the scaffold's pore size determines both the mechanical load and the type of attachment, the design of an optimal scaffold must take into account the interplay of these two features and requires a design process that optimizes both parameters at the same time. PMID:19109048

  18. Comparison of Uncultured Marrow Mononuclear Cells and Culture-Expanded Mesenchymal Stem Cells in 3D Collagen-Chitosan Microbeads for Orthopedic Tissue Engineering

    PubMed Central

    Wise, Joel K.; Alford, Andrea I.; Goldstein, Steven A.

    2014-01-01

    Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25×106 cells/mL, containing an estimated 5×104 MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2×105 cells/mL) were added to a 65–35 wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead

  19. Construction of a 3D rGO-collagen hybrid scaffold for enhancement of the neural differentiation of mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Guo, Weibo; Wang, Shu; Yu, Xin; Qiu, Jichuan; Li, Jianhua; Tang, Wei; Li, Zhou; Mou, Xiaoning; Liu, Hong; Wang, Zhonglin

    2016-01-01

    The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold because the surface of the scaffold can determine the fate of stem cells. A conductive surface is required for a scaffold to direct stem cells toward neural differentiation. However, most conductive polymers are toxic and not amenable to biological degradation, which restricts the design of neural tissue engineering scaffolds. In this study, we used a bioactive three-dimensional (3D) porcine acellular dermal matrix (PADM), which is mainly composed of type I collagen, as a basic material and successfully assembled a layer of reduced graphene oxide (rGO) nanosheets on the surface of the PADM channels to obtain a porous 3D, biodegradable, conductive and biocompatible PADM-rGO hybrid neural tissue engineering scaffold. Compared with the PADM scaffold, assembling the rGO into the scaffold did not induce a significant change in the microstructure but endowed the PADM-rGO hybrid scaffold with good conductivity. A comparison of the neural differentiation of rat bone-marrow-derived mesenchymal stem cells (MSCs) was performed by culturing the MSCs on PADM and PADM-rGO scaffolds in neuronal culture medium, followed by the determination of gene expression and immunofluorescence staining. The results of both the gene expression and protein level assessments suggest that the rGO-assembled PADM scaffold may promote the differentiation of MSCs into neuronal cells with higher protein and gene expression levels after 7 days under neural differentiation conditions. This study demonstrated that the PADM-rGO hybrid scaffold is a promising scaffold for neural tissue engineering; this scaffold can not only support the growth of MSCs at a high proliferation rate but also enhance the differentiation of MSCs into neural cells.The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold

  20. Construction of a 3D rGO-collagen hybrid scaffold for enhancement of the neural differentiation of mesenchymal stem cells.

    PubMed

    Guo, Weibo; Wang, Shu; Yu, Xin; Qiu, Jichuan; Li, Jianhua; Tang, Wei; Li, Zhou; Mou, Xiaoning; Liu, Hong; Wang, Zhonglin

    2016-01-28

    The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold because the surface of the scaffold can determine the fate of stem cells. A conductive surface is required for a scaffold to direct stem cells toward neural differentiation. However, most conductive polymers are toxic and not amenable to biological degradation, which restricts the design of neural tissue engineering scaffolds. In this study, we used a bioactive three-dimensional (3D) porcine acellular dermal matrix (PADM), which is mainly composed of type I collagen, as a basic material and successfully assembled a layer of reduced graphene oxide (rGO) nanosheets on the surface of the PADM channels to obtain a porous 3D, biodegradable, conductive and biocompatible PADM-rGO hybrid neural tissue engineering scaffold. Compared with the PADM scaffold, assembling the rGO into the scaffold did not induce a significant change in the microstructure but endowed the PADM-rGO hybrid scaffold with good conductivity. A comparison of the neural differentiation of rat bone-marrow-derived mesenchymal stem cells (MSCs) was performed by culturing the MSCs on PADM and PADM-rGO scaffolds in neuronal culture medium, followed by the determination of gene expression and immunofluorescence staining. The results of both the gene expression and protein level assessments suggest that the rGO-assembled PADM scaffold may promote the differentiation of MSCs into neuronal cells with higher protein and gene expression levels after 7 days under neural differentiation conditions. This study demonstrated that the PADM-rGO hybrid scaffold is a promising scaffold for neural tissue engineering; this scaffold can not only support the growth of MSCs at a high proliferation rate but also enhance the differentiation of MSCs into neural cells. PMID:26750302

  1. TU-C-BRE-04: 3D Gel Dosimetry Using ViewRay On-Board MR Scanner: A Feasibility Study

    SciTech Connect

    Zhang, L; Du, D; Green, O; Rodriguez, V; Wooten, H; Xiao, Z; Yang, D; Hu, Y; Li, H

    2014-06-15

    Purpose: MR based 3D gel has been proposed for radiation therapy dosimetry. However, access to MR scanner has been one of the limiting factors for its wide acceptance. Recent commercialization of an on-board MR-IGRT device (ViewRay) may render the availability issue less of a concern. This work reports our attempts to simulate MR based dose measurement accuracy on ViewRay using three different gels. Methods: A spherical BANG gel dosimeter was purchased from MGS Research. Cylindrical MAGIC gel and Fricke gel were fabricated in-house according to published recipes. After irradiation, BANG and MAGIC were imaged using a dual-echo spin echo sequence for T2 measurement on a Philips 1.5T MR scanner, while Fricke gel was imaged using multiple spin echo sequences. Difference between MR measured and TPS calculated dose was defined as noise. The noise power spectrum was calculated and then simulated for the 0.35 T magnetic field associated with ViewRay. The estimated noise was then added to TG-119 test cases to simulate measured dose distributions. Simulated measurements were evaluated against TPS calculated doses using gamma analysis. Results: Given same gel, sequence and coil setup, with a FOV of 180×90×90 mm3, resolution of 3×3×3 mm3, and scanning time of 30 minutes, the simulated measured dose distribution using BANG would have a gamma passing rate greater than 90% (3%/3mm and absolute). With a FOV 180×90×90 mm3, resolution of 4×4×5 mm3, and scanning time of 45 minutes, the simulated measuremened dose distribution would have a gamma passing rate greater than 97%. MAGIC exhibited similar performance while Fricke gel was inferior due to much higher noise. Conclusions: The simulation results demonstrated that it may be feasible to use MAGIC and BANG gels for 3D dose verification using ViewRay low-field on-board MRI scanner.

  2. Morphology of capillary-like structures in a three-dimensional aorta/collagen gel culture.

    PubMed

    Akita, M; Murata, E; Merker, H J; Kaneko, K

    1997-04-01

    The morphology of capillary-like tubes was investigated by electron microscopy (TEM and SEM) using an in vitro model of capillarogenesis (aorta/collagen type I gel). This model allowed morphological comparisons with in vivo capillaries and an evaluation of the functional maturity of the endothelium to be made. The lumina developing in vitro were demarcated by endothelial cells of varying thickness (0.1-2 microns). Pericytes were resting on the outside. The endothelial cells were characterized by contacts of varying length with tight and gap junctions and occasional indentations. The inner surface exhibited areas both with pronounced and without any endocytotic activity. In addition to a large Golgi apparatus, a varying number of cell organelles occurred depending on the thickness of the endothelium. Bundles consisting of microfilaments were often located underneath the outer cell membrane and in the vicinity of contact areas. A lamina densa was in the process of formation. The capillaries grown in vitro closely resembled those in vivo and showed a high degree of differentiation. Hence, this in vitro model allows the study of a number of functions of endothelial cells. PMID:9134083

  3. Role of xenogenous bovine platelet gel embedded within collagen implant on tendon healing: an in vitro and in vivo study.

    PubMed

    Oryan, Ahmad; Moshiri, Ali; Meimandi-Parizi, Abdolhamid; Maffulli, Nicola

    2015-02-01

    Surgical reconstruction of large Achilles tendon defects is demanding. Platelet concentrates may be useful to favor healing in such conditions. The characteristics of bovine platelet-gel embedded within a collagen-implant were determined in vitro, and its healing efficacy was examined in a large Achilles tendon defect in rabbits. Two cm of the left Achilles tendon of 60 rabbits were excised, and the animals were randomly assigned to control (no implant), collagen-implant, or bovine-platelet-gel-collagen-implant groups. The tendon edges were maintained aligned using a Kessler suture. No implant was inserted in the control group. In the two other groups, a collagen-implant or bovine-platelet-gel-collagen-implant was inserted in the defect. The bioelectricity and serum platelet-derived growth factor levels were measured weekly and at 60 days post injury, respectively. After euthanasia at 60 days post injury, the tendons were tested at macroscopic, microscopic, and ultrastructural levels, and their dry matter and biomechanical performances were also assessed. Another 60 rabbits were assigned to receive no implant, a collagen-implant, or a bovine-platelet-gel-collagen-implant, euthanized at 10, 20, 30, and 40 days post injury, and their tendons were evaluated grossly and histologically to determine host-graft interactions. Compared to the control and collagen-implant, treatment with bovine-platelet-gel-collagen-implant improved tissue bioelectricity and serum platelet-derived growth factor levels, and increased cell proliferation, differentiation, and maturation. It also increased number, diameter, and density of the collagen fibrils, alignment and maturation of the collagen fibrils and fibers, biomechanical properties and dry matter content of the injured tendons at 60 days post injury. The bovine-platelet-gel-collagen-implant also increased biodegradability, biocompatibility, and tissue incorporation behavior of the implant compared to the collagen-implant alone

  4. An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions.

    PubMed

    Ishihara, Seiichiro; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

    2016-01-01

    Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagen-containing gels interfered with detection of the p65 protein (one of the subunits in the NF-κB family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serum-free culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel. PMID:25005915

  5. Strong and fast-recovery organic/inorganic hybrid AuNPs-supramolecular gels based on loofah-like 3D networks.

    PubMed

    He, Huiwen; Chen, Si; Tong, Xiaoqian; Chen, Yining; Wu, Bozhen; Ma, Meng; Wang, Xiaosong; Wang, Xu

    2016-01-21

    Super strong and fast-recovery organic/inorganic hybrid gold nanoparticle (AuNPs)-supramolecular gels based on a three-dimensional loofah-like nanoscale network self-assembled by polyhedral oligomeric silsesquioxane (POSS) core supramolecular gelators are reported for the first time. Two series of POSS core organic/inorganic hybrid gelators, POSS-BOC-l-Homophenylalanine (POSS-Hpy) and POSS-Boc-Cys(Bzl)-OH (POSS-Cys), with two types of peripherals having different abilities for driving the self-assembly of AuNPs in gels were designed and synthesized, both of which self-assembled into three-dimensional loofah-like nanoscale gel networks producing hybrid physical gels with fast-recovery behaviors. The mechanical properties of the resultant hybrid gels were dramatically increased by as much as 100 times in the system of sulfur containing POSS-Cys gelators without destroying the fast-recovery behaviors, with the addition of AuNPs, which had direct interaction with AuNPs to give S-Au non-covalent driving force to lead AuNPs self-assemble onto the 3D loofah-like network nanofibres in the supramolecular hybrid gel system. However, in the POSS-Hpy gelator system without sulfur, no strong interaction with AuNPs existed and the POSS-Hpy nanocomposites showed no clear changes in morphology, thermal stability or rheological properties, confirmed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), tube-inversion and rotational rheometer measurements. This indicated that the organic/inorganic hybrid gelator POSS-Cys could be applied to the formation of soft materials in which AuNPs were self-assembled and closely arranged into three-dimensional nanoscale networks. This hybrid material has great potential for applications in self-recovery, nano- and micron-scale electronic devices, because it has both a large mechanical strength and a fast-recovery capability. PMID:26568047

  6. Comparison between Monte Carlo simulation and measurement with a 3D polymer gel dosimeter for dose distributions in biological samples

    NASA Astrophysics Data System (ADS)

    Furuta, T.; Maeyama, T.; Ishikawa, K. L.; Fukunishi, N.; Fukasaku, K.; Takagi, S.; Noda, S.; Himeno, R.; Hayashi, S.

    2015-08-01

    In this research, we used a 135 MeV/nucleon carbon-ion beam to irradiate a biological sample composed of fresh chicken meat and bones, which was placed in front of a PAGAT gel dosimeter, and compared the measured and simulated transverse-relaxation-rate (R2) distributions in the gel dosimeter. We experimentally measured the three-dimensional R2 distribution, which records the dose induced by particles penetrating the sample, by using magnetic resonance imaging. The obtained R2 distribution reflected the heterogeneity of the biological sample. We also conducted Monte Carlo simulations using the PHITS code by reconstructing the elemental composition of the biological sample from its computed tomography images while taking into account the dependence of the gel response on the linear energy transfer. The simulation reproduced the experimental distal edge structure of the R2 distribution with an accuracy under about 2 mm, which is approximately the same as the voxel size currently used in treatment planning.

  7. Comparison between Monte Carlo simulation and measurement with a 3D polymer gel dosimeter for dose distributions in biological samples.

    PubMed

    Furuta, T; Maeyama, T; Ishikawa, K L; Fukunishi, N; Fukasaku, K; Takagi, S; Noda, S; Himeno, R; Hayashi, S

    2015-08-21

    In this research, we used a 135 MeV/nucleon carbon-ion beam to irradiate a biological sample composed of fresh chicken meat and bones, which was placed in front of a PAGAT gel dosimeter, and compared the measured and simulated transverse-relaxation-rate (R2) distributions in the gel dosimeter. We experimentally measured the three-dimensional R2 distribution, which records the dose induced by particles penetrating the sample, by using magnetic resonance imaging. The obtained R2 distribution reflected the heterogeneity of the biological sample. We also conducted Monte Carlo simulations using the PHITS code by reconstructing the elemental composition of the biological sample from its computed tomography images while taking into account the dependence of the gel response on the linear energy transfer. The simulation reproduced the experimental distal edge structure of the R2 distribution with an accuracy under about 2 mm, which is approximately the same as the voxel size currently used in treatment planning. PMID:26266894

  8. Experimental validation of an extended Jones matrix calculus model to study the 3D structural orientation of the collagen fibers in articular cartilage using polarization-sensitive optical coherence tomography

    PubMed Central

    Kasaragod, Deepa K.; Lu, Zenghai; Jacobs, James; Matcher, Stephen J.

    2012-01-01

    We report results to verify a theoretical framework to analyze the 3D depth-wise structural organization of collagen fibers in articular cartilage using polarization-sensitive optical coherence tomography. Apparent birefringence data obtained from multi-angle measurements using a time domain polarization-sensitive optical coherence tomography system has been compared with simulated data based on the extended Jones matrix calculus. Experimental data has been shown to agree with the lamellar model previously proposed for the cartilage microstructure based on scanning electron microscopy data. This tool could have potential application in mapping the collagen structural orientation information of cartilage non-invasively during arthroscopy. PMID:22435087

  9. Strategies for the crystallization of viruses: using phase diagrams and gels to produce 3D crystals of Grapevine fanleaf virus.

    PubMed

    Schellenberger, Pascale; Demangeat, Gérard; Lemaire, Olivier; Ritzenthaler, Christophe; Bergdoll, Marc; Oliéric, Vincent; Sauter, Claude; Lorber, Bernard

    2011-05-01

    The small icosahedral plant RNA nepovirus Grapevine fanleaf virus (GFLV) is specifically transmitted by a nematode and causes major damage to vineyards worldwide. To elucidate the molecular mechanisms underlying the recognition between the surface of its protein capsid and cellular components of its vector, host and viral proteins synthesized upon infection, the wild type GFLV strain F13 and a natural mutant (GFLV-TD) carrying a Gly₂₉₇Asp mutation were purified, characterized and crystallized. Subsequently, the geometry and volume of their crystals was optimized by establishing phase diagrams. GFLV-TD was twice as soluble as the parent virus in the crystallization solution and its crystals diffracted X-rays to a resolution of 2.7 Å. The diffraction limit of GFLV-F13 crystals was extended from 5.5 to 3 Å by growth in agarose gel. Preliminary crystallographic analyses indicate that both types of crystals are suitable for structure determination. Keys for the successful production of GFLV crystals include the rigorous quality control of virus preparations, crystal quality improvement using phase diagrams, and crystal lattice reinforcement by growth in agarose gel. These strategies are applicable to the production of well-diffracting crystals of other viruses and macromolecular assemblies. PMID:21352920

  10. Preparation of collagen modified photopolymers: a new type of biodegradable gel for cell growth.

    PubMed

    Bayramoğlu, Gülay; Kayaman-Apohan, Nilhan; Akçakaya, Handan; Vezir Kahraman, Memet; Erdem Kuruca, Serap; Güngör, Atilla

    2010-02-01

    In this study a new branched methacrylated poly(propylene glycol-co-lactic acid) (PPG-PLA-IEM) and methacrylated cellulose acetate butyrate resin (CAB-IEM) were synthesized. Hydrogels with various amounts of PPG-PLA-IEM and CAB-IEM (25, 50 and 75 wt% IEM modified) were prepared by photopolymerization. Collagen tethered PEG-monoacrylate (PEGMA-collagen) was prepared and introduced as a bioactive moiety to modify the hydrogel in order to enhance cell affinity. In vitro attachment and growth of 3T3 mouse fibroblasts and human umbilical vein endothelial cells (HUVEC) on the hydrogels with and without collagen were also investigated. It was observed that, the collagen improves the cell adhesion onto the hydrogel surface. With the increasing amount of collagen, cell viability increased by 28% for ECV304 (P < 0.05) and 30% for 3T3 (P < 0.05). PMID:19936889

  11. Comparison of 3D dose distributions for HDR {sup 192}Ir brachytherapy sources with normoxic polymer gel dosimetry and treatment planning system

    SciTech Connect

    Senkesen, Oznur; Tezcanli, Evrim; Buyuksarac, Bora; Ozbay, Ismail

    2014-10-01

    Radiation fluence changes caused by the dosimeter itself and poor spatial resolution may lead to lack of 3-dimensional (3D) information depending on the features of the dosimeter and quality assurance of dose distributions for high–dose rate (HDR) iridium-192 ({sup 192}Ir) brachytherapy sources is challenging and experimental dosimetry methods used for brachytherapy sources are limited. In this study, we investigated 3D dose distributions of {sup 192}Ir brachytherapy sources for irradiation with single and multiple dwell positions using a normoxic gel dosimeter and compared them with treatment planning system (TPS) calculations. For dose calibration purposes, 100-mL gel-containing vials were irradiated at predefined doses and then scanned in an magnetic resonance (MR) imaging unit. Gel phantoms prepared in 2 spherical glasses were irradiated with {sup 192}Ir for the calculated dwell positions, and MR scans of the phantoms were obtained. The images were analyzed with MATLAB software. Dose distributions and profiles derived with 1-mm resolution were compared with TPS calculations. Linearity was observed between the delivered dose and the reciprocal of the T2 relaxation time constant of the gel. The x-, y-, and z-axes were defined as the sagittal, coronal, and axial planes, respectively, the sagittal and axial planes were defined parallel to the long axis of the source while the coronal plane was defined horizontally to the long axis of the source. The differences between measured and calculated profile widths of 3-cm source length and point source for 70%, 50%, and 30% isodose lines were evaluated at 3 dose levels using 18 profiles of comparison. The calculations for 3-cm source length revealed a difference of > 3 mm in 1 coordinate at 50% profile width on the sagittal plane and 3 coordinates at 70% profile width and 2 coordinates at 50% and 30% profile widths on the axial plane. Calculations on the coronal plane for 3-cm source length showed > 3-mm difference in 1

  12. Comparison of 3D dose distributions for HDR 192Ir brachytherapy sources with normoxic polymer gel dosimetry and treatment planning system.

    PubMed

    Senkesen, Oznur; Tezcanli, Evrim; Buyuksarac, Bora; Ozbay, Ismail

    2014-01-01

    Radiation fluence changes caused by the dosimeter itself and poor spatial resolution may lead to lack of 3-dimensional (3D) information depending on the features of the dosimeter and quality assurance of dose distributions for high-dose rate (HDR) iridium-192 ((192)Ir) brachytherapy sources is challenging and experimental dosimetry methods used for brachytherapy sources are limited. In this study, we investigated 3D dose distributions of (192)Ir brachytherapy sources for irradiation with single and multiple dwell positions using a normoxic gel dosimeter and compared them with treatment planning system (TPS) calculations. For dose calibration purposes, 100-mL gel-containing vials were irradiated at predefined doses and then scanned in an magnetic resonance (MR) imaging unit. Gel phantoms prepared in 2 spherical glasses were irradiated with (192)Ir for the calculated dwell positions, and MR scans of the phantoms were obtained. The images were analyzed with MATLAB software. Dose distributions and profiles derived with 1-mm resolution were compared with TPS calculations. Linearity was observed between the delivered dose and the reciprocal of the T2 relaxation time constant of the gel. The x-, y-, and z-axes were defined as the sagittal, coronal, and axial planes, respectively, the sagittal and axial planes were defined parallel to the long axis of the source while the coronal plane was defined horizontally to the long axis of the source. The differences between measured and calculated profile widths of 3-cm source length and point source for 70%, 50%, and 30% isodose lines were evaluated at 3 dose levels using 18 profiles of comparison. The calculations for 3-cm source length revealed a difference of > 3mm in 1 coordinate at 50% profile width on the sagittal plane and 3 coordinates at 70% profile width and 2 coordinates at 50% and 30% profile widths on the axial plane. Calculations on the coronal plane for 3-cm source length showed > 3-mm difference in 1 coordinate at

  13. Fast and mild strategy, using superhydrophobic surfaces, to produce collagen/platelet lysate gel beads for skin regeneration.

    PubMed

    Lima, Ana Catarina; Mano, João F; Concheiro, Angel; Alvarez-Lorenzo, Carmen

    2015-02-01

    Platelet lysate (PL) was encapsulated in collagen (Coll) millimetric gel beads, on biomimetic superhydrophobic surfaces, under mild conditions, with the aim of obtaining easy-to-handle formulations able to provide sustained release of multiple growth factors for skin ulcers treatment. The gel particles were prepared with various concentrations of PL incorporating or not stem cells, and tested as freshly prepared or after being freeze-dried or cryopreserved. Coll + PL particles were evaluated regarding degradation in collagenase-rich environment (simulating the aggressive environment of the chronic ulcers), sustained release of total protein, PDGF-BB and VEGF, cell proliferation (using particles as the only source of growth factors), scratch wound recovery and angiogenic capability. Compared to Coll solely particles, incorporation of PL notably enhanced cell proliferation (inside and outside gels) and favored scratch wound recovery and angiogenesis. Moreover, cell-laden gel particles containing PL notably improved cell proliferation and even migration of cells from one particle towards a neighbor one, which led to cell-cell contacts and the spontaneous formation of tissue layers in which the spherical gels were interconnected by the stem cells. PMID:25120225

  14. Maintenance of a bone collagen phenotype by osteoblast-like cells in 3D periodic porous titanium (Ti-6Al-4 V) structures fabricated by selective electron beam melting

    PubMed Central

    Hrabe, Nikolas W.; Heinl, Peter; Bordia, Rajendra K.; Körner, Carolin; Fernandes, Russell J.

    2013-01-01

    Regular 3D periodic porous Ti-6Al-4 V structures were fabricated by the selective electron beam melting method (EBM) over a range of relative densities (0.17–0.40) and pore sizes (500–1500 μm). Structures were seeded with human osteoblast-like cells (SAOS-2) and cultured for four weeks. Cells multiplied within these structures and extracellular matrix collagen content increased. Type I and type V collagens typically synthesized by osteoblasts were deposited in the newly formed matrix with time in culture. High magnification scanning electron microscopy revealed cells attached to surfaces on the interior of the structures with an increasingly fibrous matrix. The in-vitro results demonstrate that the novel EBM-processed porous structures, designed to address the effect of stress-shielding, are conducive to osteoblast attachment, proliferation and deposition of a collagenous matrix characteristic of bone. PMID:23869614

  15. Collagen Gel Contraction by Fibroblasts: The Role of Myosin 2 and Gravity Effects

    NASA Technical Reports Server (NTRS)

    Johnson-Wint, Barbara P.; Malouvier, Alexandre; Holton, Emily

    1996-01-01

    Several lines of evidence suggest that collagen organization by connective tissue cells is sensitive to force. For instance, in flight experiments on rats the collagen fibrils which were produced under weightlessness and which were immediately next to the tendon fibroblasts were shown to be oriented randomly around the cells while the older fibrils right next to these and which were produced under 1 G, were highly organized.

  16. Collagen gel contraction as a measure of fibroblast function in an animal model of subsynovial connective tissue fibrosis.

    PubMed

    Yang, Tai-Hua; Thoreson, Andrew R; Gingery, Anne; Larson, Dirk R; Passe, Sandra M; An, Kai-Nan; Zhao, Chunfeng; Amadio, Peter C

    2015-05-01

    Carpal tunnel syndrome (CTS) is a peripheral neuropathy characterized by non-inflammatory fibrosis of the subsynovial connective tissues (SSCT). A rabbit model of CTS was developed to test the hypothesis that SSCT fibrosis causes the neuropathy. We used a cell-seeded collagen-gel contraction model to characterize the fibrosis in this model in terms of cellular mechanics, specifically to compare the ability of SSCT cells from the rabbit model and normal rabbits to contract the gel, and to assess the effect of transforming growth factor-β1,which is upregulated in CTS, on these cells. SSCT fibrosis was induced in six retired breeder female rabbits which were sacrificed at 6 weeks (N = 3) and 12 weeks (n = 3). An additional two rabbits served as controls. SSCT was harvested according to a standard protocol. Gels seeded with SSCT cells from rabbits sacrificed at 6 weeks had significantly higher tensile strength (p < 0.001) and Young's modulus (p < 0.001) than gels seeded with cells from rabbits sacrificed at 12 weeks or control animals. TGF-β1 significantly increased the decay time constant (p < 0.001), tensile strength (p < 0.001), and Young's modulus (p < 0.001) regardless of the cell source. This model may be useful in screening therapeutic agents that may block SSCT fibrosis, identifying possible candidates for CTS treatment. PMID:25626430

  17. 3D-printed polylactic acid supports for enhanced ionization efficiency in desorption electrospray mass spectrometry analysis of liquid and gel samples.

    PubMed

    Elviri, Lisa; Foresti, Ruben; Bianchera, Annalisa; Silvestri, Marco; Bettini, Ruggero

    2016-08-01

    The potential of 3D printing technology was here exploited to prepare tailored polylactic acid (PLA) supports for desorption electrospray ionization (DESI) experiments. PLA rough solid supports presenting wells of different shape (i.e. cylindrical, cubic and hemispherical cavities) were designed to accommodate samples of different physical state. The potentials of such supports in terms of sample loading capacity, sensitivity, signal stability were tested by analysing a peptide (i.e. insulin) and an aminoglycoside antibiotic (i.e. gentamicin sulphate) from solution and a chitosan-based gel. The results obtained were compared with those obtained by using a traditional polytetrafluoroethylene (PTFE) support and discussed. By using PLA support on the flat side, signal intensity improved almost twice with respect to PTFE support, whereas with spherical wells a five times improved signal sensitivity and good stability (RSD<6%) were obtained for the analysis of two model molecules. Limits of detection were in the 3-10nM range and linearity was demonstrated for both analytes in the 0.05-0.5μM range for semi-quantitative or quantitative purposes. The use of a well and the set-up of optimal source parameters allowed the analysis of samples in a gel state with good precision (RSD<10%) and accuracy (86±6-102±9%), otherwise difficult to analyse on a flat smooth surface. These findings are of great interest and stimulus to exploit the advantages of 3D printing technology for the development of devices for a DESI source, presenting different shapes or configuration as a function of the sample types. PMID:27216689

  18. Hyaluronan in aged collagen matrix increases prostate epithelial cell proliferation

    PubMed Central

    Damodarasamy, Mamatha; Vernon, Robert B.; Chan, Christina K.; Plymate, Stephen R.; Wight, Thomas N.

    2015-01-01

    The extracellular matrix (ECM) of the prostate, which is comprised primarily of collagen, becomes increasingly disorganized with age, a property that may influence the development of hyperplasia and cancer. Collageous ECM extracted from the tails of aged mice exhibits many characteristics of collagen in aged tissues, including the prostate. When polymerized into a 3-dimensional (3D) gel, these collagen extracts can serve as models for the study of specific cell-ECM interactions. In the present study, we examined the behaviors of human prostatic epithelial cell lines representing normal prostate epithelial cells (PEC), benign prostatic hyperplasia (BPH-1), and adenocarcinoma (LNCaP) cultured in contact with 3D gels made from collagen extracts of young and aged mice. We found that proliferation of PEC, BPH-1, and LNCaP cells were all increased by culture on aged collagen gels relative to young collagen gels. In examining age-associated differences in the composition of the collagen extracts, we found that aged and young collagen had a similar amount of several collagen-associated ECM components, but aged collagen had a much greater content of the glycosaminoglycan hyaluronan (HA) than young collagen. The addition of HA (of similar size and concentration to that found in aged collagen extracts) to cells placed in young collagen elicited significantly increased proliferation in BPH-1 cells, but not in PEC or LNCaP cells, relative to controls not exposed to HA. Of note, histochemical analyses of human prostatic tissues showed significantly higher expression of HA in BPH and prostate cancer stroma relative to stroma of normal prostate. Collectively, these results suggest that changes in ECM involving increased levels of HA contribute to the growth of prostatic epithelium with aging. PMID:25124870

  19. Adipose-derived mesenchymal stem cells from the sand rat: transforming growth factor beta and 3D co-culture with human disc cells stimulate proteoglycan and collagen type I rich extracellular matrix

    PubMed Central

    Tapp, Hazel; Deepe, Ray; Ingram, Jane A; Kuremsky, Marshall; Hanley, Edward N; Gruber, Helen E

    2008-01-01

    Introduction Adult mesenchymal stem cell therapy has a potential application in the biological treatment of disc degeneration. Our objectives were: to direct adipose-derived mesenchymal stem cells (AD-MSC) from the sand rat to produce a proteoglycan and collagen type I extracellular matrix (ECM) rich in known ECM components of the annulus fibrosis of disc; and to stimulate proteoglycan production by co-culture of human annulus cells with AD-MSC. Methods AD-MSC were isolated and characterised by adherence to plastic, appropriate expression of cluster of differentiation (CD) markers, and differentiation to osteoblasts and chondrocytes in vitro. AD-MSC were grown in three-dimensional (3D) culture and treated with or without transforming growth factor beta (TGFβ) to direct them to produce annulus-like ECM as determined by proteoglycan content and collagen expression. AD-MSC were co-cultured with human annulus cells and grown in 3D culture. Results AD-MSC produced a proteoglycan and collagen type I rich ECM after treatment with TGFβ in 3D culture as confirmed by a 48% increase in proteoglycan content assayed by 1,9-dimethylmethylene blue (DMB), and by immunohistochemical identification of ECM components. Co-culture of human annulus and sand rat AD-MSC in 3D culture resulted in a 20% increase in proteoglycan production compared with the predicted value of the sum of the individual cultures. Conclusion Results support the hypothesis that AD-MSC have potential in cell-based therapy for disc degeneration. PMID:18691412

  20. Gelatin-methacrylamide gel loaded with microspheres to deliver GDNF in bilayer collagen conduit promoting sciatic nerve growth.

    PubMed

    Zhuang, Hai; Bu, Shoushan; Hua, Lei; Darabi, Mohammad A; Cao, Xiaojian; Xing, Malcolm

    2016-01-01

    In this study, we fabricated glial cell-line derived neurotrophic factor (GDNF)-loaded microspheres, then seeded the microspheres in gelatin-methacrylamide hydrogel, which was finally integrated with the commercial bilayer collagen membrane (Bio-Gide(®)). The novel composite of nerve conduit was employed to bridge a 10 mm long sciatic nerve defect in a rat. GDNF-loaded gelatin microspheres had a smooth surface with an average diameter of 3.9±1.8 μm. Scanning electron microscopy showed that microspheres were uniformly distributed in both the GelMA gel and the layered structure. Using enzyme-linked immunosorbent assay, in vitro release studies (pH 7.4) of GDNF from microspheres exhibited an initial burst release during the first 3 days (18.0%±1.3%), and then, a prolonged-release profile extended to 32 days. However, in an acidic condition (pH 2.5), the initial release percentage of GDNF was up to 91.2%±0.9% within 4 hours and the cumulative release percentage of GDNF was 99.2%±0.2% at 48 hours. Then the composite conduct was implanted in a 10 mm critical defect gap of sciatic nerve in a rat. We found that the nerve was regenerated in both conduit and autograft (AG) groups. A combination of electrophysiological assessment and histomorphometry analysis of regenerated nerves showed that axonal regeneration and functional recovery in collagen tube filled with GDNF-loaded microspheres (GM + CT) group were similar to AG group (P>0.05). Most myelinated nerves were matured and arranged densely with a uniform structure of myelin in a neat pattern along the long axis in the AG and GM + CT groups, however, regenerated nerve was absent in the BLANK group, left the 10 mm gap empty after resection, and the nerve fiber exhibited a disordered arrangement in the collagen tube group. These results indicated that the hybrid system of bilayer collagen conduit and GDNF-loaded gelatin microspheres combined with gelatin-methacrylamide hydrogels could serve as a new biodegradable

  1. Gelatin-methacrylamide gel loaded with microspheres to deliver GDNF in bilayer collagen conduit promoting sciatic nerve growth

    PubMed Central

    Zhuang, Hai; Bu, Shoushan; Hua, Lei; Darabi, Mohammad A; Cao, Xiaojian; Xing, Malcolm

    2016-01-01

    In this study, we fabricated glial cell-line derived neurotrophic factor (GDNF)-loaded microspheres, then seeded the microspheres in gelatin-methacrylamide hydrogel, which was finally integrated with the commercial bilayer collagen membrane (Bio-Gide®). The novel composite of nerve conduit was employed to bridge a 10 mm long sciatic nerve defect in a rat. GDNF-loaded gelatin microspheres had a smooth surface with an average diameter of 3.9±1.8 μm. Scanning electron microscopy showed that microspheres were uniformly distributed in both the GelMA gel and the layered structure. Using enzyme-linked immunosorbent assay, in vitro release studies (pH 7.4) of GDNF from microspheres exhibited an initial burst release during the first 3 days (18.0%±1.3%), and then, a prolonged-release profile extended to 32 days. However, in an acidic condition (pH 2.5), the initial release percentage of GDNF was up to 91.2%±0.9% within 4 hours and the cumulative release percentage of GDNF was 99.2%±0.2% at 48 hours. Then the composite conduct was implanted in a 10 mm critical defect gap of sciatic nerve in a rat. We found that the nerve was regenerated in both conduit and autograft (AG) groups. A combination of electrophysiological assessment and histomorphometry analysis of regenerated nerves showed that axonal regeneration and functional recovery in collagen tube filled with GDNF-loaded microspheres (GM + CT) group were similar to AG group (P>0.05). Most myelinated nerves were matured and arranged densely with a uniform structure of myelin in a neat pattern along the long axis in the AG and GM + CT groups, however, regenerated nerve was absent in the BLANK group, left the 10 mm gap empty after resection, and the nerve fiber exhibited a disordered arrangement in the collagen tube group. These results indicated that the hybrid system of bilayer collagen conduit and GDNF-loaded gelatin microspheres combined with gelatin-methacrylamide hydrogels could serve as a new biodegradable

  2. Introducing gel dosimetry in a clinical environment: Customization of polymer gel composition and magnetic resonance imaging parameters used for 3D dose verifications in radiosurgery and intensity modulated radiotherapy

    SciTech Connect

    Crescenti, Remo A.; Scheib, Stefan G.; Schneider, Uwe; Gianolini, Stefano

    2007-04-15

    calculated and measured relative 3D dose distributions performed for radiosurgery and IMRT showed an acceptable overall correlation. The gamma criterion for the radiosurgery verification with a voxel size of 1.5x1.5x1.5 mm{sup 3} was passed by 96.8% of the voxels (1.5 mm distance, 8% in dose). For the IMRT verification using a voxel size of 1.25x1.25x5 mm{sup 3} the gamma criterion was passed by 50.3% of the voxels (3 mm distance, 3% dose uncertainty). Using dedicated data analysis and visualization software, MR based normoxic gel dosimetry was found to be a valuable tool for clinically based dose verification, provided that customized gel compositions and MR imaging parameters are used. While high dose precision was achieved, further work is required to achieve clinically acceptable dose accuracy.

  3. Introducing gel dosimetry in a clinical environment: customization of polymer gel composition and magnetic resonance imaging parameters used for 3D dose verifications in radiosurgery and intensity modulated radiotherapy.

    PubMed

    Crescenti, Remo A; Scheib, Stefan G; Schneider, Uwe; Gianolini, Stefano

    2007-04-01

    Radiation sensitive gels have been used as dosimeters for clinical dose verification of different radiation therapy modalities. However, the use of gels is not widespread, because careful techniques are required to achieve the dose precision and accuracy aimed for in clinical dose verification. Here, the introduction of gel dosimetry in a clinical environment is described, including the whole chain of customizations and preparations required to introduce magnetic resonance (MR) based gel dosimetry into clinical routine. In order to standardize gel dosimetry in dose verifications for radiosurgery and intensity modulated radiotherapy (IMRT), we focused on both the customization of the gel composition and of the MR imaging parameters to increase its precision. The relative amount of the components of the normoxic, methacrylic acid based gel (MAGIC) was changed to obtain linear and steep dose response relationships. MR imaging parameters were customized for the different dose ranges used in order to lower the relative standard deviation of the measured transversal relaxation rate (R2). An optimization parameter was introduced to quantify the change in the relative standard deviation of R2 (sigma(R2,rel)) taking the increase in MR time into account. A 9% methacrylic acid gel customized for radiosurgery was found to give a linear dose response up to 40 Gy with a slope of 0.94 Gy(-1) s(-1), while a 6% methacrylic acid gel customized for IMRT had a linear range up to 3 Gy with a slope of 1.86 Gy(-1) s(-1). With the help of an introduced optimization parameter, the mean sigma(R2,rel) was improved by 13% for high doses and by 55% for low doses, without increasing MR time to unacceptable values. A mean dose resolution of less than 0.13 Gy has been achieved with the gel and imaging parameters customized for IMRT and a dose resolution from 0.97 Gy (at 5 Gy) to 2.15 Gy (at 40 Gy) for the radiosurgery dose range. The comparisons of calculated and measured relative 3D dose

  4. Chemical and biological evaluation of Egyptian Nile Tilapia (Oreochromis niloticas) fish scale collagen.

    PubMed

    El-Rashidy, Aiah A; Gad, Ahmed; Abu-Hussein, Abd El-Hay G; Habib, Shaymaa I; Badr, Nadia A; Hashem, Azza A

    2015-08-01

    Collagen is considered to be one of the most useful biomaterials with different medical applications. However, collagen properties differ from one source to another. The aim of this study was to extract, purify, characterize and perform preliminary biological evaluation of type I collagen from scales of Egyptian Nile Tilapia. Pepsin-solubilized collagen (PSC) was successfully prepared from Nile Tilapia fish scale waste. Lyophilized collagen was dissolved in dilute HCl to form acidic collagen solutions (ACS) which was neutralized to form gel. To confirm the biocompatibility of the produced gel, baby hamster kidney (BHK-21) fibroblast cells were seeded onto a 3D collagen gel (0.3% and 0.5%, w/v). The results of an SDS-PAGE test showed that the extracted collagens were type I collagen, with α chain composition of (α1)2α2. Thermal analysis showed that the denaturation temperature was 32 °C. X-ray diffraction (XRD) analysis and Fourier-transform infrared spectra (FTIR) showed that the extracted collagen had a triple helix structure. Active proliferation of BHK-21 cells with no signs of toxicity was evident with both collagen gel concentrations tested. The results show that Nile Tilapia scales can be an effective source of collagen extraction that could be used as a potential biomaterial in biomedical applications. PMID:26026980

  5. Noninvasive Quantitative Imaging of Collagen Microstructure in Three-Dimensional Hydrogels Using High-Frequency Ultrasound

    PubMed Central

    Mercado, Karla P.; Helguera, María; Hocking, Denise C.

    2015-01-01

    Collagen I is widely used as a natural component of biomaterials for both tissue engineering and regenerative medicine applications. The physical and biological properties of fibrillar collagens are strongly tied to variations in collagen fiber microstructure. The goal of this study was to develop the use of high-frequency quantitative ultrasound to assess collagen microstructure within three-dimensional (3D) hydrogels noninvasively and nondestructively. The integrated backscatter coefficient (IBC) was employed as a quantitative ultrasound parameter to detect, image, and quantify spatial variations in collagen fiber density and diameter. Collagen fiber microstructure was varied by fabricating hydrogels with different collagen concentrations or polymerization temperatures. IBC values were computed from measurements of the backscattered radio-frequency ultrasound signals collected using a single-element transducer (38-MHz center frequency, 13–47 MHz bandwidth). The IBC increased linearly with increasing collagen concentration and decreasing polymerization temperature. Parametric 3D images of the IBC were generated to visualize and quantify regional variations in collagen microstructure throughout the volume of hydrogels fabricated in standard tissue culture plates. IBC parametric images of corresponding cell-embedded collagen gels showed cell accumulation within regions having elevated collagen IBC values. The capability of this ultrasound technique to noninvasively detect and quantify spatial differences in collagen microstructure offers a valuable tool to monitor the structural properties of collagen scaffolds during fabrication, to detect functional differences in collagen microstructure, and to guide fundamental research on the interactions of cells and collagen matrices. PMID:25517512

  6. Variable angle-of-incidence polarization-sensitive optical coherence tomography: its use to study the 3D collagen structure of equine articular cartilage

    NASA Astrophysics Data System (ADS)

    Ugryumova, Nadya; Gangnus, Sergei V.; Matcher, Stephen J.

    2006-02-01

    Polarization-sensitive optical coherence tomography has been used to spatially map the birefringence of equine articular cartilage. The polar orientation of the collagen fibers relative to the plane of the joint surface must be taken into account if a quantitative measurement of true birefringence is required. Using a series of images taken at different angles of illumination, we determine the fiber polar angle and true birefringence at one site on a sample of equine cartilage, on the assumption that the fibers lie within the plane of imaging. We propose a more general method based on the extended Jones matrix formalism to determine both the polar and azimuthal orientation of the collagen fibers as well as the true birefringence as functions of depth.

  7. Synthesis, development, characterization and effectiveness of bovine pure platelet gel-collagen-polydioxanone bioactive graft on tendon healing.

    PubMed

    Moshiri, Ali; Oryan, Ahmad; Meimandi-Parizi, Abdolhamid

    2015-06-01

    Bovine platelet gel (BPG) is an accessible and cost-effective source of growth factors which may have a value in tendon regenerative medicine. We produced a collagen implant (CI) as a tendon proper, covered it with polydioxanone (PDS) sheath to simulate paratenon and finally embedded the BPG as an active source of growth factor within the bioimplant to test whether BPG would be able to accelerate and enhance tendon regeneration and repair. After in vitro characterization of the bioactive grafts, the grafts were implanted in rabbit large tendon defect model. Untreated tendons and tendons treated with either CI or CI-PDS were served as controls for the CI-PDS-BPG. The animals were investigated clinically, ultrasonographically and haematologically for 120 days. After euthanasia, dry matter content, water uptake and delivery characteristics and also gross morphological, histopathological and scanning electron microscopic features of the healing tendons were assessed. In vitro, the activated platelets in the scaffold, released their growth factors significantly more than the controls. BPG also increased cell viability, and enhanced cellular differentiation, maturation and proliferation inside the CI-PDS compared with the controls. In vivo, the BPG modulated inflammation, increased quality and rate of fibroplasia and produced a remodelled tendon that had significantly higher collagen content and superior collagen fibril and fibre differentiation than controls. Treatment also significantly improved tendon water uptake and delivery characteristics, animals' serum PDGF level, CI-PDS biocompatibility and biodegradability and reduced peritendinous adhesions, muscle fibrosis and atrophy. BPG was effective on tendon healing and CI-PDS-BPG may be a valuable bioscaffold in tendon reconstructive surgery. PMID:25702535

  8. Synthesis, development, characterization and effectiveness of bovine pure platelet gel-collagen-polydioxanone bioactive graft on tendon healing

    PubMed Central

    Moshiri, Ali; Oryan, Ahmad; Meimandi-Parizi, Abdolhamid

    2015-01-01

    Bovine platelet gel (BPG) is an accessible and cost-effective source of growth factors which may have a value in tendon regenerative medicine. We produced a collagen implant (CI) as a tendon proper, covered it with polydioxanone (PDS) sheath to simulate paratenon and finally embedded the BPG as an active source of growth factor within the bioimplant to test whether BPG would be able to accelerate and enhance tendon regeneration and repair. After in vitro characterization of the bioactive grafts, the grafts were implanted in rabbit large tendon defect model. Untreated tendons and tendons treated with either CI or CI-PDS were served as controls for the CI-PDS-BPG. The animals were investigated clinically, ultrasonographically and haematologically for 120 days. After euthanasia, dry matter content, water uptake and delivery characteristics and also gross morphological, histopathological and scanning electron microscopic features of the healing tendons were assessed. In vitro, the activated platelets in the scaffold, released their growth factors significantly more than the controls. BPG also increased cell viability, and enhanced cellular differentiation, maturation and proliferation inside the CI-PDS compared with the controls. In vivo, the BPG modulated inflammation, increased quality and rate of fibroplasia and produced a remodelled tendon that had significantly higher collagen content and superior collagen fibril and fibre differentiation than controls. Treatment also significantly improved tendon water uptake and delivery characteristics, animals’ serum PDGF level, CI-PDS biocompatibility and biodegradability and reduced peritendinous adhesions, muscle fibrosis and atrophy. BPG was effective on tendon healing and CI-PDS-BPG may be a valuable bioscaffold in tendon reconstructive surgery. PMID:25702535

  9. Self-assembled smooth muscle cell tissue rings exhibit greater tensile strength than cell-seeded fibrin or collagen gel rings

    PubMed Central

    Adebayo, Olufunmilayo; Gwyther, Tracy A.; Hu, Jason Z.; Billiar, Kristen L.; Rolle, Marsha W.

    2012-01-01

    In this study, we created self-assembled smooth muscle cell (SMC) tissue rings (comprised entirely of cells and cell-derived matrix; CDM) and compared their structure and material properties with tissue rings created from SMC-seeded fibrin or collagen gels. All tissue rings were cultured statically for 7 days in supplemented growth medium (with ε-amino caproic acid, ascorbic acid, and insulin-transferrin-selenium), prior to uniaxial tensile testing and histology. Self-assembled CDM rings exhibited ultimate tensile strength and stiffness values that were two-fold higher than fibrin gel and collagen gel rings. Tensile testing of CDM, fibrin gel and collagen gel rings treated with deionized water to lyse cells showed little to no change in mechanical properties relative to untreated ring samples, indicating that the ECM dominates the measured ring mechanics. In addition, CDM rings cultured in supplemented growth medium were significantly stronger than CDM rings cultured in standard, unsupplemented growth medium. These results illustrate the potential utility of self-assembled cell rings as model CDM constructs for tissue engineering and biomechanical analysis of ECM material properties. PMID:22865465

  10. Fabrication of injectable, cellular, anisotropic collagen tissue equivalents with modular fibrillar densities.

    PubMed

    Marelli, Benedetto; Ghezzi, Chiara E; James-Bhasin, Mark; Nazhat, Showan N

    2015-01-01

    Technological improvements in collagen gel fabrication are highly desirable as they may enable significant advances in the formation of tissue-equivalent biomaterials for regenerative medicine, three-dimensional (3D) in vitro tissue models, and injectable scaffolds for cell and drug delivery applications. Thus, strategies to modulate collagen gel fibrillar density and organization in the mesostructure have been pursued to fabricate collagenous matrices with extracellular matrix-like features. Herein, we introduce a robust and simple method, namely gel aspiration-ejection (GAE), to engineer 3D, anisotropic, cell seeded, injectable dense collagen (I-DC) gels with controllable fibrillar densities, without the use of crosslinking. GAE allows for the hybridization of collagen gels with bioactive agents for increased functionality and supports highly aligned homogenous cell seeding, thus providing I-DC gels with distinct properties when compared to isotropic DC gels of random fibrillar orientation. The hybridization of I-DC with anionic fibroin derived polypeptides resulted in the nucleation of carbonated hydroxyapatite within the aligned nanofibrillar network upon exposure to simulated body fluid, yielding a 3D, anisotropic, mineralized collagen matrix. In addition, I-DC gels accelerated the osteoblastic differentiation of seeded murine mesenchymal stem cells (m-MSCs) when exposed to osteogenic supplements, which resulted in the cell-mediated, bulk mineralization of the osteoid-like gels. In addition, and upon exposure to neuronal transdifferentiation medium, I-DC gels supported and accelerated the differentiation of m-MSCs toward neuronal cells. In conclusion, collagen GAE presents interesting opportunities in a number of fields spanning tissue engineering and regenerative medicine to drug and cell delivery. PMID:25453949

  11. Effectiveness factor and diffusion limitations in collagen gel modules containing HepG2 cells

    PubMed Central

    Corstorphine, Lindsay; Sefton, Michael V.

    2010-01-01

    A major obstacle in tissue engineering is overcoming hypoxia in thick, three-dimensional engineered tissues, which is caused by the diffusional limitations of oxygen and lack of internal vasculature to facilitate mass transfer. Modular tissue engineering is a bio-mimetic strategy that forms scalable, vascularized and uniform three-dimensional constructs by assembling small (sub-mm), cell-containing modules. It was previously assumed that mass transfer resistance within the individual modules was negligible, due to their small size. In the present work, this assumption was tested using theoretical analysis of oxygen transport within the module (effectiveness factor) and experimental studies. Small (400μm diameter, post contraction) and large (700μm diameter, post-contraction) HepG2-collagen modules were made for a range of seeding densities (2 × 106 – 1 × 107 cells/ml collagen). Cell density, distribution and morphology within the modules showed that the small modules were capable of sustaining high cell densities (8.0 × 107 ± 4.4 × 107 cells/ cm3) with negligible mass transfer inhibition. Conversely, large modules developed a necrotic core and had significantly (p < 0.05) reduced cell densities (1.5 × 107 ± 9.2 × 106 cells/cm3). It was also observed that the embedded cells responded quickly to the oxygen availability, by proliferating or dying to reach a sustainable density of approximately 8000 cells/module. Furthermore, a simple effectiveness factor calculation was successful in estimating the maximum cell density per module. The results gathered in this study confirm the previous assumption that the small diameter modules avoid the internal mass transfer limitations that are often observed in larger constructs. PMID:20653045

  12. Fabrication of type I collagen microcarrier using a microfluidic 3D T-junction device and its application for the quantitative analysis of cell-ECM interactions.

    PubMed

    Yoon, Junghyo; Kim, Jaehoon; Jeong, Hyo Eun; Sudo, Ryo; Park, Myung-Jin; Chung, Seok

    2016-01-01

    We presented a new quantitative analysis for cell and extracellular matrix (ECM) interactions, using cell-coated ECM hydrogel microbeads (hydrobeads) made of type I collagen. The hydrobeads can carry cells as three-dimensional spheroidal forms with an ECM inside, facilitating a direct interaction between the cells and ECM. The cells on hydrobeads do not have a hypoxic core, which opens the possibility for using as a cell microcarrier for bottom-up tissue reconstitution. This technique can utilize various types of cells, even MDA-MB-231 cells, which have weak cell-cell interactions and do not form spheroids in conventional spheroid culture methods. Morphological indices of the cell-coated hydrobead visually present cell-ECM interactions in a quantitative manner. PMID:27563029

  13. Use of collagen gel as an alternative extracellular matrix for the in vitro and in vivo growth of murine small intestinal epithelium.

    PubMed

    Jabaji, Ziyad; Sears, Connie M; Brinkley, Garrett J; Lei, Nan Ye; Joshi, Vaidehi S; Wang, Jiafang; Lewis, Michael; Stelzner, Matthias; Martín, Martín G; Dunn, James C Y

    2013-12-01

    Methods for the in vitro culture of primary small intestinal epithelium have improved greatly in recent years. A critical barrier for the translation of this methodology to the patient's bedside is the ability to grow intestinal stem cells using a well-defined extracellular matrix. Current methods rely on the use of Matrigel(™), a proprietary basement membrane-enriched extracellular matrix gel produced in mice that is not approved for clinical use. We demonstrate for the first time the capacity to support the long-term in vitro growth of murine intestinal epithelium in monoculture, using type I collagen. We further demonstrate successful in vivo engraftment of enteroids co-cultured with intestinal subepithelial myofibroblasts in collagen gel. Small intestinal crypts were isolated from 6 to 10 week old transgenic enhanced green fluorescent protein (eGFP+) mice and suspended within either Matrigel or collagen gel; cultures were supported using previously reported media and growth factors. After 1 week, cultures were either lysed for DNA or RNA extraction or were implanted subcutaneously in syngeneic host mice. Quantitative real-time polymerase chain reaction (qPCR) was performed to determine expansion of the transgenic eGFP-DNA and to determine the mRNA gene expression profile. Immunohistochemistry was performed on in vitro cultures and recovered in vivo explants. Small intestinal crypts reliably expanded to form enteroids in either Matrigel or collagen in both mono- and co-cultures as confirmed by microscopy and eGFP-DNA qPCR quantification. Collagen-based cultures yielded a distinct morphology with smooth enteroids and epithelial monolayer growth at the gel surface; both enteroid and monolayer cells demonstrated reactivity to Cdx2, E-cadherin, CD10, Periodic Acid-Schiff, and lysozyme. Collagen-based enteroids were successfully subcultured in vitro, whereas pure monolayer epithelial sheets did not survive passaging. Reverse transcriptase-polymerase chain reaction

  14. Agent-based modeling traction force mediated compaction of cell-populated collagen gels using physically realistic fibril mechanics.

    PubMed

    Reinhardt, James W; Gooch, Keith J

    2014-02-01

    Agent-based modeling was used to model collagen fibrils, composed of a string of nodes serially connected by links that act as Hookean springs. Bending mechanics are implemented as torsional springs that act upon each set of three serially connected nodes as a linear function of angular deflection about the central node. These fibrils were evaluated under conditions that simulated axial extension, simple three-point bending and an end-loaded cantilever. The deformation of fibrils under axial loading varied <0.001% from the analytical solution for linearly elastic fibrils. For fibrils between 100 μm and 200 μm in length experiencing small deflections, differences between simulated deflections and their analytical solutions were <1% for fibrils experiencing three-point bending and <7% for fibrils experiencing cantilever bending. When these new rules for fibril mechanics were introduced into a model that allowed for cross-linking of fibrils to form a network and the application of cell traction force, the fibrous network underwent macroscopic compaction and aligned between cells. Further, fibril density increased between cells to a greater extent than that observed macroscopically and appeared similar to matrical tracks that have been observed experimentally in cell-populated collagen gels. This behavior is consistent with observations in previous versions of the model that did not allow for the physically realistic simulation of fibril mechanics. The significance of the torsional spring constant value was then explored to determine its impact on remodeling of the simulated fibrous network. Although a stronger torsional spring constant reduced the degree of quantitative remodeling that occurred, the inclusion of torsional springs in the model was not necessary for the model to reproduce key qualitative aspects of remodeling, indicating that the presence of Hookean springs is essential for this behavior. These results suggest that traction force mediated matrix

  15. Significant Type I and Type III Collagen Production from Human Periodontal Ligament Fibroblasts in 3D Peptide Scaffolds without Extra Growth Factors

    PubMed Central

    Kumada, Yoshiyuki; Zhang, Shuguang

    2010-01-01

    We here report the development of two peptide scaffolds designed for periodontal ligament fibroblasts. The scaffolds consist of one of the pure self-assembling peptide scaffolds RADA16 through direct coupling to short biologically active motifs. The motifs are 2-unit RGD binding sequence PRG (PRGDSGYRGDS) and laminin cell adhesion motif PDS (PDSGR). RGD and laminin have been previously shown to promote specific biological activities including periodontal ligament fibroblasts adhesion, proliferation and protein production. Compared to the pure RADA16 peptide scaffold, we here show that these designer peptide scaffolds significantly promote human periodontal ligament fibroblasts to proliferate and migrate into the scaffolds (for ∼300 µm/two weeks). Moreover these peptide scaffolds significantly stimulated periodontal ligament fibroblasts to produce extracellular matrix proteins without using extra additional growth factors. Immunofluorescent images clearly demonstrated that the peptide scaffolds were almost completely covered with type I and type III collagens which were main protein components of periodontal ligament. Our results suggest that these designer self-assembling peptide nanofiber scaffolds may be useful for promoting wound healing and especially periodontal ligament tissue regeneration. PMID:20421985

  16. Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis.

    PubMed

    Feraud, O; Cao, Y; Vittet, D

    2001-12-01

    The formation of new blood vessels proceeds by both vasculogenesis and angiogenesis. The development of models, which fully recapitulate spatio-temporal events involved during these processes, are crucial to fully understand their mechanisms of regulation. In vitro differentiation of murine embryonic stem (ES) cells has been shown to be a useful tool to investigate factors and genes potentially involved in vasculogenesis (Hirashima et al, 1999; Risau et al, 1988; Vittet et al, 1996; Wang et al, 1992; Wartenberg et al, 1998). We asked here whether this model system can also recapitulate angiogenesis, which may offer new means to study mechanisms involved in this process. ES-derived embryoid bodies (EBs) obtained after 11 days of differentiation, in which a primitive vascular network had formed, were then subcultured into a type I collagen matrix. In the presence of angiogenic growth factors, EBs rapidly developed branching pseudopods. Whole mount immunostainings with a PECAM antibody revealed that more than 75% EBs displayed, within a few days, a large number of endothelial outgrowths that can give tube-like structures with concomitant differentiation of alpha-smooth muscle actin positive cells, thus evoking sprouting angiogenesis. High expression levels of flk1 (VEGFR2), flt1 (VEGFR1), tie-1, and tie-2 are also found, indicating that budding endothelial cells displayed an angiogenic phenotype. The endothelial sprouting response was specifically induced by angiogenic factors with a major contribution of vascular endothelial growth factor (VEGF). Known angiostatic agents, such as platelet factor 4 (PF4), angiostatin, and endostatin inhibited the formation of endothelial sprouts induced by angiogenic factors. Moreover, consistent with the in vivo phenotype, VE-cadherin deficient EBs failed to develop angiogenesis in this model. ES cell differentiation can then recapitulate, in addition to vasculogenesis, the early stages of sprouting angiogenesis. This model system

  17. 3D dose verification with polymer gel detectors of brain-spine match line for proton pencil beam cranio-spinal: A preliminary study

    NASA Astrophysics Data System (ADS)

    Avery, S.; Cardin, A.; Lin, L.; Kirk, M.; Kassaee, A.; Maryanski, M. J.

    2015-01-01

    This paper is intended as a preliminary study to demonstrate the quality assurance benefits from polymer gel detectors for proton pencil beam cranio-spinal treatments. A stable gel type was selected for protons to suppress the LET dependence at the end of the Bragg peak. The depth dose distributions in the gels were examined with regard of its dose dependences and compared to baseline measurements. The preliminary experimental results indicate polymer gel detectors may be able to verify dose in three dimensions along match line for proton therapy treatments.

  18. Gel electrophoretic studies of photochemical cross-linking of type I collagen with brominated 1,8-naphthalimide dyes and visible light

    NASA Astrophysics Data System (ADS)

    Judy, Millard M.; Fuh, L.; Matthews, James Lester; Lewis, David E.; Utecht, Ronald E.

    1994-09-01

    Insoluble Type I collagen from bovine Achilles tendon (Sigma C9879) was suspended in a 3 mM solution of the dye diEd66Br dissolved in Cremophor ELR (BASF) to give a molecular concentration ratio. Fifty-microliter aliquots in 5-mm-diameter wells were exposed to 458 J/cm2 (225 mW/cm2, 1800 sec) of 457.9-nm light from an argon ion laser; similar aliquots with and without dye were kept in the dark to serve as controls. Following pelleting of the collagen by centrifugation and 3x washing in phosphate-buffered saline, aliquots of light-treated and control sample pellets were (1) digested in collagenase (Sigma C9891) or (2) extracted in 0.5 M acetic acid, followed by centrifugative ultrafiltration (10-kd cutoff) in 0.01 M acetic acid. Aliquots of the supernatant of the acid-extracted collagen also were digested in pepsin. Electrophoretic protein migration in 8% to 25% gradient polyacrylamide gels following SDS solubilization disclosed numerous, densely packed, essentially contiguous protein bands. These studies indicate that the dye and light treatment of insoluble Type I collagen (1) results in cross-linking of collagen molecules and (2) does not denature the trimer conformation sufficiently to enable significant digestion by pepsin.

  19. Collagen matrix as a tool in studying fibroblastic cell behavior.

    PubMed

    Kanta, Jiří

    2015-01-01

    Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes. PMID:25734486

  20. Collagen matrix as a tool in studying fibroblastic cell behavior

    PubMed Central

    Kanta, Jiří

    2015-01-01

    Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes. PMID:25734486

  1. The influence of particle size and static magnetic fields on the uptake of magnetic nanoparticles into three dimensional cell-seeded collagen gel cultures.

    PubMed

    Lewis, Emily E L; Child, Hannah W; Hursthouse, Andrew; Stirling, David; McCully, Mark; Paterson, David; Mullin, Margaret; Berry, Catherine C

    2015-08-01

    Over recent decades there has been and continues to be major advances in the imaging, diagnosis and potential treatment of medical conditions, by the use of magnetic nanoparticles. However, to date the majority of cell delivery studies employ a traditional 2D monolayer culture. This article aims to determine the ability of various sized magnetic nanoparticles to penetrate and travel through a cell seeded collagen gel model, in the presence or absence of a magnetic field. Three different sized (100, 200, and 500 nm) nanoparticles were employed in the study. The results showed cell viability was unaffected by the presence of nanoparticles over a 24-h test period. The initial uptake of the 100 nm nanoparticle into the collagen gel structure was superior compared to the larger sized nanoparticles under the influence of a magnetic field and incubated for 24 h. Interestingly, it was the 200 nm nanoparticles, which proved to penetrate the gel furthest, under the influence of a magnetic field, during the initial culture stage after 1-h incubation. PMID:25358626

  2. Validation of high-resolution 3D patient QA for proton PBS and IMPT using laser CT of improved polymer gel dosimeters

    NASA Astrophysics Data System (ADS)

    Cardin, A.; Ding, X.; Kassaee, A.; Lin, L.; Maryanski, M. J.; Avery, S.

    2015-01-01

    Laser CT scanning of LET-independent BANG3-Pro2® polymer gel dosimeters has recently shown potential in proton dosimetry. However, raw materials' impurities impart some variability. This study aimed to validate a new method of compensating for this variability, and to validate the suitability of the improved dosimeter for patient-specific QA in pencil beam scanning (PBS) and IMPT. Six modifications of the BANG3-Pro2® gel dosimeter were analysed for their sensitivity to proton dose and to LET. One formulation was selected for a clinical QA feasibility study, in which one composite IMPT plan, two single-field IMPT plans, and one SFUD plan were delivered to identical gel phantoms. New commercial VOLQATM software (beta version) was used for data analysis. Both validations were successful.

  3. Fibrin Gels Exhibit Improved Biological, Structural, and Mechanical Properties Compared with Collagen Gels in Cell-Based Tendon Tissue-Engineered Constructs

    PubMed Central

    Dyment, Nathaniel A.; Lu, Yinhui; Rao, Marepalli; Shearn, Jason T.; Rowe, David W.; Kadler, Karl E.; Butler, David L.

    2015-01-01

    The prevalence of tendon and ligament injuries and inadequacies of current treatments is driving the need for alternative strategies such as tissue engineering. Fibrin and collagen biopolymers have been popular materials for creating tissue-engineered constructs (TECs), as they exhibit advantages of biocompatibility and flexibility in construct design. Unfortunately, a few studies have directly compared these materials for tendon and ligament applications. Therefore, this study aims at determining how collagen versus fibrin hydrogels affect the biological, structural, and mechanical properties of TECs during formation in vitro. Our findings show that tendon and ligament progenitor cells seeded in fibrin constructs exhibit improved tenogenic gene expression patterns compared with their collagen-based counterparts for approximately 14 days in culture. Fibrin-based constructs also exhibit improved cell-derived collagen alignment, increased linear modulus (2.2-fold greater) compared with collagen-based constructs. Cyclic tensile loading, which promotes the maturation of tendon constructs in a previous work, exhibits a material-dependent effect in this study. Fibrin constructs show trending reductions in mechanical, biological, and structural properties, whereas collagen constructs only show improved tenogenic expression in the presence of mechanical stimulation. These findings highlight that components of the mechanical stimulus (e.g., strain amplitude or time of initiation) need to be tailored to the material and cell type. Given the improvements in tenogenic expression, extracellular matrix organization, and material properties during static culture, in vitro findings presented here suggest that fibrin-based constructs may be a more suitable alternative to collagen-based constructs for tissue-engineered tendon/ligament repair. PMID:25266738

  4. Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization.

    PubMed

    McCarty, William J; Prodanov, Ljupcho; Bale, Shyam Sundhar; Bhushan, Abhinav; Jindal, Rohit; Yarmush, Martin L; Usta, O Berk

    2015-01-01

    Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices. PMID:26485274

  5. Stem Cell Bioprinting: Functional 3D Neural Mini-Tissues from Printed Gel-Based Bioink and Human Neural Stem Cells (Adv. Healthcare Mater. 12/2016).

    PubMed

    Gu, Qi; Tomaskovic-Crook, Eva; Lozano, Rodrigo; Chen, Yu; Kapsa, Robert M; Zhou, Qi; Wallace, Gordon G; Crook, Jeremy M

    2016-06-01

    On page 1429 G. G. Wallace, J. M. Crook, and co-workers report the first example of fabricating neural tissue by 3D bioprinting human neural stem cells. A novel polysaccharide based bioink preserves stem cell viability and function within the printed construct, enabling self-renewal and differentiation to neurons and supporting neuroglia. Neurons are predominantly GABAergic, establish networks, are spontaneously active, and show a bicuculline induced increased calcium response. PMID:27333401

  6. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

    PubMed Central

    Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

    2015-01-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils. PMID:26548801

  7. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions.

    PubMed

    Doyle, Andrew D; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M

    2015-01-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils. PMID:26548801

  8. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

    NASA Astrophysics Data System (ADS)

    Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

    2015-11-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

  9. Sol-gel synthesis, phase composition, morphological and structural characterization of Ca10(PO4)6(OH)2: XRD, FTIR, SEM, 3D SEM and solid-state NMR studies

    NASA Astrophysics Data System (ADS)

    Kareiva, Simonas; Klimavicius, Vytautas; Momot, Aleksandr; Kausteklis, Jonas; Prichodko, Aleksandra; Dagys, Laurynas; Ivanauskas, Feliksas; Sakirzanovas, Simas; Balevicius, Vytautas; Kareiva, Aivaras

    2016-09-01

    Aqueous sol-gel chemistry route based on ammonium-hydrogen phosphate as the phosphorus precursor, calcium acetate monohydrate as source of calcium ions, and 1,2-ethylendiaminetetraacetic acid (EDTA), or 1,2-diaminocyclohexanetetracetic acid (DCTA), or tartaric acid (TA), or ethylene glycol (EG), or glycerol (GL) as complexing agents have been used to prepare calcium hydroxyapatite (Ca10(PO4)6(OH)2, CHAp). The phase transformations, composition, and structural changes in the polycrystalline samples were studied by infrared spectroscopy (FTIR), X-ray powder diffraction analysis (XRD), and scanning electron microscopy (SEM). The local short-range (nano- and mezo-) scale effects in CHAp were studied using solid-state NMR spectroscopy. The spatial 3D data from the SEM images of CHAp samples obtained by TA, EG and GL sol-gel routes were recovered for the first time to our knowledge.

  10. Controlling sensitivity and stability of ferrous-xylenol orange-gelatin 3D gel dosimeters by doping with phenanthroline-type ligands and glyoxal

    NASA Astrophysics Data System (ADS)

    Penev, Kalin I.; Mequanint, Kibret

    2013-03-01

    The ferrous-xylenol orange-gelatin (FXG) dosimeter is widely used for three-dimensional ionizing radiation field mapping through optical scanning. Upon irradiation, the ferrous iron (Fe2+) is oxidized to ferric iron (Fe3+), which forms an intensely coloured complex with xylenol orange (XO). XO also acts as a diffusion-limiting additive; however, its presence may cause rapid auto-oxidation of Fe2+ during storage and low stability of the dose response. In this work, phenanthroline-type ligands were added to FXG system in a bid to bind the ferrous iron in a stable complex and minimize the rate of the auto-oxidation, whereas glyoxal was used as a chemical cross-linker, aiming to minimize the ferric iron diffusion. It was found that addition of either 1,10-phenanthroline or 5-nitro-1,10-phenanthroline can improve the auto-oxidation behaviour of the gels. However, the initial background absorbance was slightly increased, and the sensitivity of the dosimeters was decreased. Doping with glyoxal led to a moderate decrease of the diffusion only in those gels that also contained a phenanthroline-type ligand, and did not affect the initial dose response. Glyoxal also afforded an extended period of stable background absorbance level after an initial period of bleaching of the gel. Following re-irradiation, most glyoxal-containing dosimeters showed an excellent linearity of the dose response, albeit at a decreased sensitivity. We recommend further testing of FXG dosimeters, doped with phenanthroline-type ligands and glyoxal as a means for controlling the dose response and improving the long-term storage properties of the gels and the potential for dose fractionation.

  11. Hot embossing for fabrication of a microfluidic 3D cell culture platform

    PubMed Central

    Jeon, Jessie S.; Chung, Seok; Kamm, Roger D.; Charest, Joseph L.

    2011-01-01

    Clinically relevant studies of cell function in vitro require a physiologically-representative microenvironment possessing aspects such as a 3D extracellular matrix (ECM) and controlled biochemical and biophysical parameters. A polydimethylsiloxane (PDMS) microfluidic system with a 3D collagen gel has previously served for analysis of factors inducing different responses of cells in a 3D microenvironment under controlled biochemical and biophysical parameters. In the present study, applying the known commercially-viable manufacturing methods to a cyclic olefin copolymer (COC) material resulted in a microfluidic device with enhanced 3D gel capabilities, controlled surface properties, and improved potential to serve high-volume applications. Hot embossing and roller lamination molded and sealed the microfluidic device. A combination of oxygen plasma and thermal treatments enhanced the sealing, ensured proper placement of the 3D gel, and created controlled and stable surface properties within the device. Culture of cells in the new device indicated no adverse effects of the COC material or processing as compared to previous PDMS devices. The results demonstrate a methodology to transition microfludic devices for 3D cell culture from scientific research to high-volume applications with broad clinical impact. PMID:21113663

  12. 3D Extracellular Matrix from Sectioned Human Tissues

    PubMed Central

    Campbell, Catherine B; Cukierman, Edna; Artym, Vira V

    2014-01-01

    Three-dimensional (3D) matrices have significant advantages compared to conventional two-dimensional (2D) matrices for studying cell adhesion, migration, and tissue organization. Cellular behavior is dependent on the surrounding matrix environment for signaling and induction of biological responses (Carletti, et al., 2011; Pampaloni, et al., 2007; Vlodavsky, 1999). 2D cultures induce an artificial polarity in cultured cells between upper and lower surfaces not present normally in the in vivo environment. No longer nonpolar, many aspects of cellular behavior are altered (Beacham, et al., 2007; Grinnell and Petroll, 2010; Yamada and Cukierman, 2007). In addition, 2D models lack the physical properties of 3D matrix, such as topography, stiffness, and dimensionality. To begin to mimic the 3D environment of in vivo connective tissue extracellular matrix (ECM), collagen gels have been used widely (see Unit 10.3). Culture of cells in collagen gels results in a bipolar fibroblast morphology that resembles the in vivo phenotype (Friedl and Brocker, 2000; Even-Ram and Yamada, 2005; Grinnell and Petroll, 2010). Although more physiological, 3D collagen gels lack the complex biochemical and physical microenvironment present in an in vivo ECM that regulates cellular physiological properties (Beacham, et al., 2007). A variety of methods to create a more in vivo-like ECM have been published (Yamada and Cukierman, 2007). Adding critical ECM components to 3D collagen matrices, including fibronectin, hyaluronan, link protein and glycosaminoglycans, can more accurately mimic the structural microenvironment of the native ECM (Friedl and Brocker, 2000). Other ECM models use cultured cell lines, such as fibroblasts, to derive an ECM lattice through secretion of an organized ECM (Beacham, et al., 2007). Different cell lines have been chosen to generate a specific microenvironment for study of particularly types of cellular behavior (Kutys and Yamada, 2013). For example, cultured bovine

  13. A novel injectable thermoresponsive and cytocompatible gel of poly(N-isopropylacrylamide) with layered double hydroxides facilitates siRNA delivery into chondrocytes in 3D culture.

    PubMed

    Yang, Hsiao-yin; van Ee, Renz J; Timmer, Klaas; Craenmehr, Eric G M; Huang, Julie H; Öner, F Cumhur; Dhert, Wouter J A; Kragten, Angela H M; Willems, Nicole; Grinwis, Guy C M; Tryfonidou, Marianna A; Papen-Botterhuis, Nicole E; Creemers, Laura B

    2015-09-01

    Hybrid hydrogels composed of poly(N-isopropylacrylamide) (pNIPAAM) and layered double hydroxides (LDHs) are presented in this study as novel injectable and thermoresponsive materials for siRNA delivery, which could specifically target several negative regulators of tissue homeostasis in cartilaginous tissues. Effectiveness of siRNA transfection using pNIPAAM formulated with either MgAl-LDH or MgFe-LDH platelets was investigated using osteoarthritic chondrocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing. No significant adverse effects of pNIPAAM/LDH hydrogels on cell viability were noticed. Cellular uptake of fluorescently labeled siRNA was greatly enhanced (>75%) in pNIPAAM/LDH hydrogel constructs compared to alginate, hyaluronan and fibrin gels, and was absent in pNIPAAM hydrogel without LDH platelets. When using siRNA against GAPDH, 82-98% reduction of gene expression was found in both types of pNIPAAM/LDH hydrogel constructs after 6 days of culturing. In the pNIPAAM/MgAl-LDH hybrid hydrogel, 80-95% of GAPDH enzyme activity was reduced in parallel with gene. Our findings show that the combination of a cytocompatible hydrogel and therapeutic RNA oligonucleotides is feasible. Thus it might hold promise in treating degeneration of cartilaginous tissues by providing supporting scaffolds for cells and interference with locally produced degenerative factors. PMID:26022968

  14. Role of Cell-Matrix Interactions on VIC Phenotype and Tissue Deposition in 3D PEG Hydrogels

    PubMed Central

    Gould, Sarah T.; Anseth, Kristi S.

    2014-01-01

    Valvular interstitial cells (VICs) respond to 3D matrix interactions in a complex manner, but better understanding these effects on VIC function is important for applications ranging from valve tissue engineering to studying valve disease. Here, we encapsulated VICs in poly(ethylene glycol) (PEG) hydrogels modified with three different adhesive ligands derived from fibronectin (RGDS), elastin (VGVAPG), and collagen-1 (P15). By day 14, VICs became significantly more elongated in RGDS containing gels compared to VGVAPG or P15. This difference in cell morphology appeared to correlate with global matrix metalloproteinase (MMP) activity, as VICs encapsulated in RGDS-functionalized hydrogels secreted higher levels of active MMP at day 2. VIC activation to a myofibroblast phenotype was also characterized by staining for α-smooth muscle actin (αSMA) at day 14. The percentage of αSMA+ VICs in the VGVAPG gels was the highest (56%) compared to RGDS (33%) or P15 (38%) gels. Matrix deposition and composition were also characterized at day 14 and 42 and found to depend on the initial hydrogel composition. All gel formulations had similar levels of collagen, elastin, and chondroitin sulfate deposited as the porcine aortic valve. However, the composition of collagen deposited by VICs in VGVAPG functionalized gels had a significantly higher collagen-X to collagen-1 ratio, which is associated with stenotic valves. Taken together, these data suggest that peptide functionalized PEG hydrogels are a useful system to culture VICs in 3D, and with the ability to systematically alter biochemical and biophysical properties, this platform may prove useful in manipulating VIC function for valve regeneration. PMID:24130082

  15. Cigarette smoke extract inhibits chemotaxis and collagen gel contraction mediated by human bone marrow osteoprogenitor cells and osteoblast-like cells.

    PubMed

    Liu, Xiangde; Kohyama, Tadashi; Kobayashi, Tetsu; Abe, Shinji; Kim, Hui Jung; Reed, Elizabeth C; Rennard, Stephen I

    2003-05-01

    Cell migration and matrix remodeling are key events in tissue repair and restructuring. Osteoblasts are responsible for the production of new bone matrix during bone remodeling. The activity of these cells can be modulated by a number of factors. The current study evaluated the hypothesis that cigarette smoke extract can alter repair and remodeling responses of human osteoprogenitor cells and osteoblast-like cells and, therefore, could explain one mechanism by which cigarette smoking leads to osteoporosis. Human osteoprogenitor cells were isolated from normal human bone marrow and maintained in culture under either control conditions or conditions that induced differentiation into osteoblast-like cells. Both cell types migrated toward fibronectin and PDGF-BB as chemoattractants. Neither responded to TGF-beta1. The osteoprogenitor cells were more active in their chemotactic response. The chemotactic response of both cell types was inhibited by cigarette smoke extract in a concentration-dependent manner. Both cell types, when cultured in three-dimensional native collagen gels maintained in floating culture, induced contraction of their surrounding matrices. Contraction was augmented by serum, PDGF-BB, and TGF-beta1. Osteoprogenitor cells were less active in inducing contraction than were osteoblast-like cells. Contraction of both cell types was inhibited by cigarette smoke extract. Cigarette smoke extract also inhibited the production of fibronectin by both cell types maintained in three-dimensional culture. Addition of exogenous fibronectin partially restored the ability of the cells to contract three-dimensional collagen gels. The current study demonstrates that cigarette smoke can interfere with the ability of bone cells to participate in repair and remodeling events. Such an effect may be one mechanism leading to the development of osteoporosis. PMID:12730796

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

    PubMed

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

    2016-03-01

    Several studies have focused on the regeneration of liver tissue in a two-dimensional (2D) planar environment, whereas actual liver tissue is three-dimensional (3D). Cell printing technology has been successfully utilized for building 3D structures; however, the poor mechanical properties of cell-laden hydrogels are a major concern. Here, we demonstrate the printing of a 3D cell-laden construct and its application to liver tissue engineering using 3D cell printing technology through a multi-head tissue/organ building system. Polycaprolactone (PCL) was used as a framework material because of its excellent mechanical properties. Collagen bioink containing three different types of cells-hepatocytes (HCs), human umbilical vein endothelial cells , and human lung fibroblasts--was infused into the canals of a PCL framework to induce the formation of capillary--like networks and liver cell growth. A co-cultured 3D microenvironment of the three types of cells was successfully established and maintained. The vascular formation and functional abilities of HCs (i.e., albumin secretion and urea synthesis) demonstrated that the heterotypic interaction among HCs and nonparenchymal cells increased the survivability and functionality of HCs within the collagen gel. Therefore, our results demonstrate the prospect of using cell printing technology for the creation of heterotypic cellular interaction within a structure for liver tissue engineering. PMID:26756962

  17. Effects of Matrix Alignment and Mechanical Constraints on Cellular Behavior in 3D Engineered Microtissues

    NASA Astrophysics Data System (ADS)

    Bose, Prasenjit; Eyckmans, Jeroen; Chen, Christopher; Reich, Daniel

    The adhesion of cells to the extracellular matrix (ECM) plays a crucial role in a variety of cellular functions. The main building blocks of the ECM are 3D networks of fibrous proteins whose structure and alignments varies with tissue type. However, the impact of ECM alignment on cellular behaviors such as cell adhesion, spreading, extension and mechanics remains poorly understood. We present results on the development of a microtissue-based system that enables control of the structure, orientation, and degree of fibrillar alignment in 3D fibroblast-populated collagen gels. The tissues self-assemble from cell-laden collagen gels placed in micro-fabricated wells containing sets of elastic pillars. The contractile action of the cells leads to controlled alignment of the fibrous collagen, depending on the number and location of the pillars in each well. The pillars are elastic, and are utilized to measure the contractile forces of the microtissues, and by incorporating magnetic material in selected pillars, time-varying forces can be applied to the tissues for dynamic stimulation and measurement of mechanical properties. Results on the effects of varying pillar shape, spacing, location, and stiffness on microtissue organization and contractility will be presented. This work is supported by NSF CMMI-1463011.

  18. Using a 3D Culture System to Differentiate Visceral Adipocytes In Vitro.

    PubMed

    Emont, Margo P; Yu, Hui; Jun, Heejin; Hong, Xiaowei; Maganti, Nenita; Stegemann, Jan P; Wu, Jun

    2015-12-01

    It has long been recognized that body fat distribution and regional adiposity play a major role in the control of metabolic homeostasis. However, the ability to study and compare the cell autonomous regulation and response of adipocytes from different fat depots has been hampered by the difficulty of inducing preadipocytes isolated from the visceral depot to differentiate into mature adipocytes in culture. Here, we present an easily created 3-dimensional (3D) culture system that can be used to differentiate preadipocytes from the visceral depot as robustly as those from the sc depot. The cells differentiated in these 3D collagen gels are mature adipocytes that retain depot-specific characteristics, as determined by imaging, gene expression, and functional assays. This 3D culture system therefore allows for study of the development and function of adipocytes from both depots in vitro and may ultimately lead to a greater understanding of site-specific functional differences of adipose tissues to metabolic dysregulation. PMID:26425808

  19. Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices.

    PubMed

    Sung, Kyung Eun; Su, Gui; Pehlke, Carolyn; Trier, Steven M; Eliceiri, Kevin W; Keely, Patricia J; Friedl, Andreas; Beebe, David J

    2009-09-01

    Interest in constructing a reliable 3-dimensional (3D) collagen culture platform in microfabricated systems is increasing as researchers strive to investigate reciprocal interaction between extracellular matrix (ECM) and cells under various conditions. However, in comparison to conventional 2-dimensional (2D) cell culture research, relatively little work has been reported about the polymerization of collagen type I matrix in microsystems. We, thus, present a study of 3D collagen polymerization to achieve reproducible 3D cell culture in microfluidic devices. Array-based microchannels are employed to efficiently examine various polymerization conditions, providing more replicates with less sample volume than conventional means. Collagen fibers assembled in microchannels were almost two-times thinner than those in conventional gels prepared under similar conditions, and the fiber thickness difference influenced viability and morphology of embedded human mammary fibroblast (HMF) cells. HMF cells contained more actin stress fibers and showed increased viability in 3D collagen matrix composed of thicker collagen fibers. Relatively low pH of the collagen solution within a physiological pH range (6.5-8.5) and pre-incubation at low temperature (approximately 4 degrees C) before polymerization at 37 degrees C allow sufficient time for molecular assembly, generating thicker collagen fibers and enhancing HMF cell viability. The results provide the basis for improved process control and reproducibility of 3D collagen matrix culture in microchannels, allowing predictable modifications to provide optimum conditions for specific cell types. In addition, the presented method lays the foundation for high throughput 3D cellular screening. PMID:19540580

  20. Tenogenic Induction of Human MSCs by Anisotropically Aligned Collagen Biotextiles

    PubMed Central

    Younesi, Mousa; Islam, Anowarul; Kishore, Vipuil; Anderson, James M.; Akkus, Ozan

    2015-01-01

    A novel biofabrication modality, electrophoretic compaction with macromolecular alignment, was utilized to make collagen threads that mimic the native tendon’s structure and mechanical properties. A device with kinematic electrodes was designed to fabricate collagen threads in continuous length. For the first time, a 3D-biotextile was woven purely from collagen. Mechanical properties and load-displacement behavior of the biotextile mimicked those of the native tendon while presenting a porosity of 80%. The open pore network facilitated cell seeding across the continuum of the bioscaffold. Mesenchymal stem cells (MSCs) seeded in the woven scaffold underwent tenogenic differentiation in the absence of growth factors and synthesized a matrix that was positive for tenomodulin, COMP and type I collagen. Up-regulation of tenomodulin, a tendon specific marker, was 11.6 ± 3.5 fold, COMP was up-regulated 16.7 ± 5.5 fold, and Col I was up-regulated 6.9 ± 2.7 fold greater on ELAC threads when compared to randomly oriented collagen gels. These results demonstrate that a bioscaffold woven by using collagen threads with densely compacted and anisotropically aligned substrate texture stimulates tenogenesis topographically, rendering the electrochemically aligned collagen as a promising candidate for functional repair of tendons and ligaments. PMID:25750610

  1. Europeana and 3D

    NASA Astrophysics Data System (ADS)

    Pletinckx, D.

    2011-09-01

    The current 3D hype creates a lot of interest in 3D. People go to 3D movies, but are we ready to use 3D in our homes, in our offices, in our communication? Are we ready to deliver real 3D to a general public and use interactive 3D in a meaningful way to enjoy, learn, communicate? The CARARE project is realising this for the moment in the domain of monuments and archaeology, so that real 3D of archaeological sites and European monuments will be available to the general public by 2012. There are several aspects to this endeavour. First of all is the technical aspect of flawlessly delivering 3D content over all platforms and operating systems, without installing software. We have currently a working solution in PDF, but HTML5 will probably be the future. Secondly, there is still little knowledge on how to create 3D learning objects, 3D tourist information or 3D scholarly communication. We are still in a prototype phase when it comes to integrate 3D objects in physical or virtual museums. Nevertheless, Europeana has a tremendous potential as a multi-facetted virtual museum. Finally, 3D has a large potential to act as a hub of information, linking to related 2D imagery, texts, video, sound. We describe how to create such rich, explorable 3D objects that can be used intuitively by the generic Europeana user and what metadata is needed to support the semantic linking.

  2. Role of cell-matrix interactions on VIC phenotype and tissue deposition in 3D PEG hydrogels.

    PubMed

    Gould, Sarah T; Anseth, Kristi S

    2013-10-16

    Valvular interstitial cells (VICs) respond to 3D matrix interactions in a complex manner, but understanding these effects on VIC function better is important for applications ranging from valve tissue engineering to studying valve disease. Here, we encapsulated VICs in poly(ethylene glycol) (PEG) hydrogels modified with three different adhesive ligands, derived from fibronectin (RGDS), elastin (VGVAPG) and collagen-1 (P15). By day 14, VICs became significantly more elongated in RGDS-containing gels compared to VGVAPG or P15. This difference in cell morphology appeared to correlate with global matrix metalloproteinase (MMP) activity, as VICs encapsulated in RGDS-functionalized hydrogels secreted higher levels of active MMP at day 2. VIC activation to a myofibroblast phenotype was also characterized by staining for α-smooth muscle actin (αSMA) at day 14. The percentage of αSMA(+) VICs in the VGVAPG gels was the highest (56%) compared to RGDS (33%) or P15 (38%) gels. Matrix deposition and composition were also characterized at days 14 and 42 and found to depend on the initial hydrogel composition. All gel formulations had similar levels of collagen, elastin and chondroitin sulphate deposited as the porcine aortic valve. However, the composition of collagen deposited by VICs in VGVAPG-functionalized gels had a significantly higher collagen-X:collagen-1 ratio, which is associated with stenotic valves. Taken together, these data suggest that peptide-functionalized PEG hydrogels are a useful system for culturing VICs three-dimensionally and, with the ability to systematically alter biochemical and biophysical properties, this platform may prove useful in manipulating VIC function for valve regeneration. Copyright © 2013 John Wiley & Sons, Ltd. PMID:24130082

  3. Clinical applications of 3-D dosimeters

    NASA Astrophysics Data System (ADS)

    Wuu, Cheng-Shie

    2015-01-01

    Both 3-D gels and radiochromic plastic dosimeters, in conjunction with dose image readout systems (MRI or optical-CT), have been employed to measure 3-D dose distributions in many clinical applications. The 3-D dose maps obtained from these systems can provide a useful tool for clinical dose verification for complex treatment techniques such as IMRT, SRS/SBRT, brachytherapy, and proton beam therapy. These complex treatments present high dose gradient regions in the boundaries between the target and surrounding critical organs. Dose accuracy in these areas can be critical, and may affect treatment outcome. In this review, applications of 3-D gels and PRESAGE dosimeter are reviewed and evaluated in terms of their performance in providing information on clinical dose verification as well as commissioning of various treatment modalities. Future interests and clinical needs on studies of 3-D dosimetry are also discussed.

  4. Microfabricated tissue gauges to measure and manipulate forces from 3D microtissues

    PubMed Central

    Legant, Wesley R.; Pathak, Amit; Yang, Michael T.; Deshpande, Vikram S.; McMeeking, Robert M.; Chen, Christopher S.

    2009-01-01

    Physical forces generated by cells drive morphologic changes during development and can feedback to regulate cellular phenotypes. Because these phenomena typically occur within a 3-dimensional (3D) matrix in vivo, we used microelectromechanical systems (MEMS) technology to generate arrays of microtissues consisting of cells encapsulated within 3D micropatterned matrices. Microcantilevers were used to simultaneously constrain the remodeling of a collagen gel and to report forces generated during this process. By concurrently measuring forces and observing matrix remodeling at cellular length scales, we report an initial correlation and later decoupling between cellular contractile forces and changes in tissue morphology. Independently varying the mechanical stiffness of the cantilevers and collagen matrix revealed that cellular forces increased with boundary or matrix rigidity whereas levels of cytoskeletal and extracellular matrix (ECM) proteins correlated with levels of mechanical stress. By mapping these relationships between cellular and matrix mechanics, cellular forces, and protein expression onto a bio-chemo-mechanical model of microtissue contractility, we demonstrate how intratissue gradients of mechanical stress can emerge from collective cellular contractility and finally, how such gradients can be used to engineer protein composition and organization within a 3D tissue. Together, these findings highlight a complex and dynamic relationship between cellular forces, ECM remodeling, and cellular phenotype and describe a system to study and apply this relationship within engineered 3D microtissues. PMID:19541627

  5. Increasing 3D Matrix Rigidity Strengthens Proliferation and Spheroid Development of Human Liver Cells in a Constant Growth Factor Environment.

    PubMed

    Bomo, Jérémy; Ezan, Frédéric; Tiaho, François; Bellamri, Medjda; Langouët, Sophie; Theret, Nathalie; Baffet, Georges

    2016-03-01

    Mechanical forces influence the growth and shape of virtually all tissues and organs. Recent studies show that increased cell contractibility, growth and differentiation might be normalized by modulating cell tensions. Particularly, the role of these tensions applied by the extracellular matrix during liver fibrosis could influence the hepatocarcinogenesis process. The objective of this study is to determine if 3D stiffness could influence growth and phenotype of normal and transformed hepatocytes and to integrate extracellular matrix (ECM) stiffness to tensional homeostasis. We have developed an appropriate 3D culture model: hepatic cells within three-dimensional collagen matrices with varying rigidity. Our results demonstrate that the rigidity influenced the cell phenotype and induced spheroid clusters development whereas in soft matrices, Huh7 transformed cells were less proliferative, well-spread and flattened. We confirmed that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas ERK2 mainly controlled proliferation. As compared to 2D culture, 3D cultures are associated with epithelial markers expression. Interestingly, proliferation of normal hepatocytes was also induced in rigid gels. Furthermore, biotransformation activities are increased in 3D gels, where CYP1A2 enzyme can be highly induced/activated in primary culture of human hepatocytes embedded in the matrix. In conclusion, we demonstrated that increasing 3D rigidity could promote proliferation and spheroid developments of liver cells demonstrating that 3D collagen gels are an attractive tool for studying rigidity-dependent homeostasis of the liver cells embedded in the matrix and should be privileged for both chronic toxicological and pharmacological drug screening. PMID:26331987

  6. Chemotherapeutic efficiency of drugs in vitro: Comparison of doxorubicin exposure in 3D and 2D culture matrices.

    PubMed

    Casey, A; Gargotti, M; Bonnier, F; Byrne, H J

    2016-06-01

    The interest in the use of 3D matrices for in vitro analysis, with a view to increasing the relevance of in vitro studies and reducing the dependence on in vivo studies, has been growing in recent years. Cells grown in a 3D in vitro matrix environment have been reported to exhibit significantly different properties to those in a conventional 2D culture environment. However, comparison of 2D and 3D cell culture models have recently been noted to result in differing responses of cytotoxic assays, without any associated change in viability. The effect was attributed to differing conversion rates and effective concentrations of the resazurin assay in 2D and 3D environments, rather than differences in cellular metabolism. In this study, the efficacy of a chemotherapeutic agent, doxorubicin, is monitored and compared in conventional 2D and 3D collagen gel exposures of immortalized human cervical cells. Viability was monitored with the aid of the Alamar Blue assay and drug internalisation was verified using confocal microscopy. Drug uptake and retention within the collagen matrix was monitored by absorption spectroscopy. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to a 3D environment causing alterations to dye resazurin uptake and conversion rates. The use of 3D culture matrices has widely been interpreted to result in "reduced" toxicity or cellular "resistance" to the chemotherapeutic agent. The results of this study show that the reduced efficiency of the drug to cells grown in the 3D environment can be accounted for by a sequential reduction of the effective concentration of the test compound and assay. This is due to absorption within the collagen gel inducing a higher uptake of both drug and assay thereby influencing the toxic impact of the drug and conversion rate of resazurin, and. The increased effective surface area of the cell exposed to the drug

  7. Biocompatible 3D Matrix with Antimicrobial Properties.

    PubMed

    Ion, Alberto; Andronescu, Ecaterina; Rădulescu, Dragoș; Rădulescu, Marius; Iordache, Florin; Vasile, Bogdan Ștefan; Surdu, Adrian Vasile; Albu, Madalina Georgiana; Maniu, Horia; Chifiriuc, Mariana Carmen; Grumezescu, Alexandru Mihai; Holban, Alina Maria

    2016-01-01

    The aim of this study was to develop, characterize and assess the biological activity of a new regenerative 3D matrix with antimicrobial properties, based on collagen (COLL), hydroxyapatite (HAp), β-cyclodextrin (β-CD) and usnic acid (UA). The prepared 3D matrix was characterized by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Microscopy (FT-IRM), Transmission Electron Microscopy (TEM), and X-ray Diffraction (XRD). In vitro qualitative and quantitative analyses performed on cultured diploid cells demonstrated that the 3D matrix is biocompatible, allowing the normal development and growth of MG-63 osteoblast-like cells and exhibited an antimicrobial effect, especially on the Staphylococcus aureus strain, explained by the particular higher inhibitory activity of usnic acid (UA) against Gram positive bacterial strains. Our data strongly recommend the obtained 3D matrix to be used as a successful alternative for the fabrication of three dimensional (3D) anti-infective regeneration matrix for bone tissue engineering. PMID:26805790

  8. Screening for Stromal and Matrix Effects in 3D Microenvironments of Breast Cancer Cells

    NASA Astrophysics Data System (ADS)

    Montanez-Sauri, Sara I.

    Breast cancer progression ensures through the acquisition of genetic mutations, the uncontrollable growth of cells, and their progression to invasion. Studies have shown that the surrounding three-dimensional (3D) microenvironment can also influence breast cancer cell progression by controlling the morphology, differentiation, proliferation, and migration of cells. However, most of the currently available in vitro screening platforms are based on the two-dimensional (2D) culture of cells, and do not provide cells with the complex 3D microenvironment that exists in vivo. Therefore, there is a need for more biologically relevant in vitro platforms to help decipher the complexity of the microenvironment and its influence in breast cancer. In this dissertation we present an automated microfluidic platform that allows to efficiently screen for the effect of multiple matrix and stromal microenvironment in 3D cultures of breast cancer cells. Several extracellular matrix (ECM) compositions and stromal cells are included in the 3D microenvironments to examine their influence on breast cancer cell behavior. The screening results suggest that collagen gels with fibronectin might be influencing paracrine signals between breast cancer cells and stromal cells. The ability of the platform to culture and treat cells in 3D microenvironments offers a powerful screening tool for the identification of compounds and interactions using more in vivo-like 3D microenvironments. The identification of these mechanisms will increase our current understanding of breast cancer, and will aid in the identification of potential therapeutics.

  9. Software for 3D radiotherapy dosimetry. Validation

    NASA Astrophysics Data System (ADS)

    Kozicki, Marek; Maras, Piotr; Karwowski, Andrzej C.

    2014-08-01

    The subject of this work is polyGeVero® software (GeVero Co., Poland), which has been developed to fill the requirements of fast calculations of 3D dosimetry data with the emphasis on polymer gel dosimetry for radiotherapy. This software comprises four workspaces that have been prepared for: (i) calculating calibration curves and calibration equations, (ii) storing the calibration characteristics of the 3D dosimeters, (iii) calculating 3D dose distributions in irradiated 3D dosimeters, and (iv) comparing 3D dose distributions obtained from measurements with the aid of 3D dosimeters and calculated with the aid of treatment planning systems (TPSs). The main features and functions of the software are described in this work. Moreover, the core algorithms were validated and the results are presented. The validation was performed using the data of the new PABIGnx polymer gel dosimeter. The polyGeVero® software simplifies and greatly accelerates the calculations of raw 3D dosimetry data. It is an effective tool for fast verification of TPS-generated plans for tumor irradiation when combined with a 3D dosimeter. Consequently, the software may facilitate calculations by the 3D dosimetry community. In this work, the calibration characteristics of the PABIGnx obtained through four calibration methods: multi vial, cross beam, depth dose, and brachytherapy, are discussed as well.

  10. 3d-3d correspondence revisited

    NASA Astrophysics Data System (ADS)

    Chung, Hee-Joong; Dimofte, Tudor; Gukov, Sergei; Sułkowski, Piotr

    2016-04-01

    In fivebrane compactifications on 3-manifolds, we point out the importance of all flat connections in the proper definition of the effective 3d {N}=2 theory. The Lagrangians of some theories with the desired properties can be constructed with the help of homological knot invariants that categorify colored Jones polynomials. Higgsing the full 3d theories constructed this way recovers theories found previously by Dimofte-Gaiotto-Gukov. We also consider the cutting and gluing of 3-manifolds along smooth boundaries and the role played by all flat connections in this operation.