The Experts below are selected from a list of 1242 Experts worldwide ranked by ideXlab platform
Andrew Hopkinson - One of the best experts on this subject based on the ideXlab platform.
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Corneal Keratocyte transition to mesenchymal stem cell phenotype and reversal using serum free medium supplemented with fibroblast growth factor 2 transforming growth factor β3 and retinoic acid
Journal of Tissue Engineering and Regenerative Medicine, 2018Co-Authors: Laura E Sidney, Andrew HopkinsonAbstract:Keratocytes of the Corneal limbal stroma can derive populations of mesenchymal stem cells (MSC) when expanded in vitro. However, once a Corneal MSC (cMSC) phenotype is achieved, regaining the Keratocyte phenotype can be challenging, and there is no standardised differentiation medium. Here, we investigated the transition of Keratocytes to cMSC and compared different supplements in their ability to return cMSC to a Keratocyte phenotype. Immunofluorescence and quantitative reverse transcription polymerase chain reaction demonstrated in vivo Keratocyte expression of aldehyde dehydrogenase 3A1, CD34 and keratocan, but not any of the typical MSC markers (CD73, CD90, CD105). As the Keratocytes were expanded in vitro, the phenotypic profile reversed and the cells expressed MSC markers but not Keratocyte markers. Differentiating the cMSC back to a Keratocyte phenotype using nonsupplemented, serum-free medium restored Keratocyte markers but did not maintain cell viability or support Corneal extracellular matrix production. Supplementing the differentiation medium with combinations of fibroblast growth factor-2, transforming growth factor-β3 and retinoic acid maintained viability, restored expression of CD34, aldehyde dehydrogenase 3A1 and keratocan, and facilitated production of abundant extracellular matrix as shown by immunofluorescent staining for collagen-I and lumican, alongside quantitative assays for collagen and glycosaminoglycan production. However, no differentiation medium was able to downregulate the expression of MSC markers in the 21-day culture period. This study shows that the Keratocyte to MSC transition can be partially reversed using serum-free media and supplementation with retinoic acid, fibroblast growth factor-2 and transforming growth factor-β3 and can enhance this effect. This is relevant for development of Corneal regenerative strategies that require the production of a Keratocyte phenotype. Copyright © 2016 John Wiley & Sons, Ltd.
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Corneal Keratocyte transition to mesenchymal stem cell phenotype and reversal using serum-free medium supplemented with FGF-2, TGF-ß3 and retinoic acid
Journal of Tissue Engineering and Regenerative Medicine, 2017Co-Authors: Laura E Sidney, Andrew HopkinsonAbstract:Keratocytes of the Corneal limbal stroma can derive populations of mesenchymal stem cells (MSC) when expanded in vitro. However, once a Corneal MSC (cMSC) phenotype is achieved, regaining the Keratocyte phenotype can be challenging, and there is no standardised differentiation medium. Here, we investigated the transition of Keratocytes to cMSC and compared different supplements in their ability to return cMSC to a Keratocyte phenotype. Immunofluorescence and RT-qPCR demonstrated in vivo Keratocyte expression of ALDH3A1, CD34 and keratocan, but not any of the typical MSC markers (CD73, CD90, CD105). As the Keratocytes were expanded in vitro, the phenotypic profile reversed and the cells expressed MSC markers but not Keratocyte markers. Differentiating the cMSC back to a Keratocyte phenotype using non-supplemented, serum-free medium restored Keratocyte markers but did not maintain cell viability or support Corneal extracellular matrix (ECM) production. Supplementing the differentiation medium with combinations of fibroblast growth factor-2 (FGF-2), transforming growth factor-β3 (TGF-β3) and retinoic acid (RA) maintained viability, restored expression of CD34, ALDH3A1 and keratocan, and facilitated production of abundant ECM as shown by immunofluorescent staining for collagen-I and lumican, alongside quantitative assays for collagen and glycosaminoglycan production. However, no differentiation medium was able to downregulate the expression of MSC markers in the 21-day culture period. This study shows that the Keratocyte to MSC transition can be partially reversed using serum-free media and supplementation with RA, FGF-2 and TGF-β3 can enhance this effect. This is relevant for development of Corneal regenerative strategies that require the production of a Keratocyte phenotype.
W. Matthew Petroll - One of the best experts on this subject based on the ideXlab platform.
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Effect of HDAC Inhibitors on Corneal Keratocyte Mechanical Phenotypes in 3-D Collagen Matrices.
Molecular vision, 2015Co-Authors: Vindhya Koppaka, Neema Lakshman, W. Matthew PetrollAbstract: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 appear to mitigate the effects of TGFβ1 on the transformation of Corneal Keratocytes to a contractile, myofibroblast phenotype in both compliant and rigid 3-D matrices while preserving normal cell spreading and their ability to respond to the pro-migratory growth factor PDGF.
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MMP regulation of Corneal Keratocyte motility and mechanics in 3-D collagen matrices
Experimental eye research, 2014Co-Authors: Chengxin Zhou, W. Matthew PetrollAbstract: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 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 outcome measures, the inhibitory effect of BB-94 was significantly greater than that of GM6001. Overall, the data demonstrate for the first time that even under conditions in which low levels of contractility and extracellular matrix proteolysis are maintained, MMPs still play an important role in mediating cell spreading and migration within 3-D collagen matrices. This appears to be mediated at least in part by membrane-tethered MMPs, such as MT1-MMP.
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Growth factor regulation of Corneal Keratocyte mechanical phenotypes in 3-D collagen matrices.
Investigative ophthalmology & visual science, 2012Co-Authors: Neema Lakshman, W. Matthew PetrollAbstract:Stromal Keratocytes play a central role in mediating the Corneal response to lacerating injury or refractive surgery.1 During wound healing, quiescent Corneal Keratocytes surrounding the area of injury generally become activated, and transform into a fibroblastic repair phenotype.2,3 These activated fibroblasts proliferate, migrate into the provisional matrix, and generate the forces required for wound closure. In certain wound types, fibroblasts further differentiate into myofibroblasts, which generate even stronger forces and synthesize a fibrotic extracellular matrix (ECM) associated with scar formation.4,5 These wound healing responses can cause a permanent reduction in Corneal clarity, and can also decrease the refractive effect of vision correction surgeries such as photorefractive keratectomy (PRK).6,7 Peptide growth factors present in the cornea and tear film, such as insulin-like growth factor (IGF), platelet derived growth factor (PDGF), FGF, IL-1α and TGFβ,8–11 are postulated to play an important role in modulating the Keratocyte phenotype during Corneal wound healing. In cell culture, these growth factors differentially regulate Keratocyte proliferation, cytoskeletal organization, and ECM synthesis. Keratocytes cultured under serum-free conditions maintain the quiescent, dendritic phenotype normally observed in vivo.12,13 IGF stimulates Keratocyte proliferation without altering cell morphology, cytoskeletal organization, or downregulating keratocan sulfate proteoglycan (KSPG) synthesis.14,15 In contrast, exposure to serum results in fibroblast differentiation, as indicated by the assumption of a bipolar morphology, formation of intracellular stress fibers, and downregulation of keratin sulfate proteoglycan expression.13,16–18 PDGF and basic FGF (FGF2) have also been shown to activate Corneal Keratocytes in vitro, but have distinct effects on cell morphology, cytoskeletal organization, and proteoglycan synthesis.14,15,19,20 TGFβ induces myofibroblast differentiation, as indicated by expression of stress fibers containing α-smooth muscle actin (α-SMA),21 and production of abnormal, fibrotic ECM.15,20,22 PDGF participates in TGFβ-induced myofibroblast differentiation through an autocrine feedback loop,23 whereas FGF2 has been shown to reduce expression of α-SMA.24 Most previous studies investigating the effect of these growth factors on Corneal Keratocyte differentiation have been performed using rigid, two-dimensional (2-D) substrates. However, Keratocytes reside within a complex 3-D extracellular matrix in vivo, and significant differences in cell morphology, adhesion organization, and mechanical behavior have been identified in 2-D versus 3-D culture models.25–27 Furthermore, unlike rigid 2-D substrates, 3-D models also allow assessment of cellular force generation and cell-induced matrix reorganization; biomechanical activities that are critically involved in the migratory, contractile, and remodeling phases of wound healing. Recent studies also suggest a potential linkage between increased Keratocyte contractility and altered ECM synthesis, further highlighting the potential importance of cell mechanics in the wound repair process.20,28 We have recently established 3-D culture models which support Keratocyte differentiation in serum-free media, and allow changes in cell morphology, mechanical activity, and matrix reorganization to be measured using quantitative imaging techniques.29,30 In this study, we use these models to assess the effects of PDGF BB, FGF2, IGF, TGFβ1, and TGFβ2 on the Keratocyte mechanical phenotype in 3-D collagen matrices, and compare these responses with those observed on a rigid 2-D substrate.
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Corneal stromal cells use both high- and low-contractility migration mechanisms in 3-D collagen matrices.
Experimental cell research, 2012Co-Authors: Areum Kim, Chengxin Zhou, Neema Lakshman, W. Matthew PetrollAbstract: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.
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Characterization of Corneal Keratocyte Morphology and Mechanical Activity within 3-D Collagen Matrices
Experimental eye research, 2009Co-Authors: Neema Lakshman, Areum Kim, W. Matthew PetrollAbstract:Abstract The purpose of this study was to assess quantitatively the differences in morphology, cytoskeletal organization and mechanical behavior between quiescent Corneal Keratocytes and activated fibroblasts in a 3-D culture model. Primary cultures of rabbit Corneal Keratocytes and fibroblasts were plated inside type I collagen matrices in serum-free media or 10% FBS, and allowed to spread for 1–5 days. Following F-actin labeling using phalloidin, and immunolabeling of tubulin, α-smooth muscle actin or connexin 43, fluorescent and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. In other experiments, dynamic imaging was performed using differential interference contrast microscopy, and finite element modeling was used to map ECM deformations. Corneal Keratocytes developed a stellate morphology with numerous cell processes that ran a tortuous path between and along collagen fibrils without any apparent impact on their alignment. Fibroblasts on the other hand, had a more bipolar morphology with pseudopodial processes (P ≤ 0.001). Time-lapse imaging of Keratocytes revealed occasional extension and retraction of dendritic processes with only transient displacements of collagen fibrils, whereas fibroblasts exerted stronger myosin II-dependent contractile forces (P
Laura E Sidney - One of the best experts on this subject based on the ideXlab platform.
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Corneal Keratocyte transition to mesenchymal stem cell phenotype and reversal using serum free medium supplemented with fibroblast growth factor 2 transforming growth factor β3 and retinoic acid
Journal of Tissue Engineering and Regenerative Medicine, 2018Co-Authors: Laura E Sidney, Andrew HopkinsonAbstract:Keratocytes of the Corneal limbal stroma can derive populations of mesenchymal stem cells (MSC) when expanded in vitro. However, once a Corneal MSC (cMSC) phenotype is achieved, regaining the Keratocyte phenotype can be challenging, and there is no standardised differentiation medium. Here, we investigated the transition of Keratocytes to cMSC and compared different supplements in their ability to return cMSC to a Keratocyte phenotype. Immunofluorescence and quantitative reverse transcription polymerase chain reaction demonstrated in vivo Keratocyte expression of aldehyde dehydrogenase 3A1, CD34 and keratocan, but not any of the typical MSC markers (CD73, CD90, CD105). As the Keratocytes were expanded in vitro, the phenotypic profile reversed and the cells expressed MSC markers but not Keratocyte markers. Differentiating the cMSC back to a Keratocyte phenotype using nonsupplemented, serum-free medium restored Keratocyte markers but did not maintain cell viability or support Corneal extracellular matrix production. Supplementing the differentiation medium with combinations of fibroblast growth factor-2, transforming growth factor-β3 and retinoic acid maintained viability, restored expression of CD34, aldehyde dehydrogenase 3A1 and keratocan, and facilitated production of abundant extracellular matrix as shown by immunofluorescent staining for collagen-I and lumican, alongside quantitative assays for collagen and glycosaminoglycan production. However, no differentiation medium was able to downregulate the expression of MSC markers in the 21-day culture period. This study shows that the Keratocyte to MSC transition can be partially reversed using serum-free media and supplementation with retinoic acid, fibroblast growth factor-2 and transforming growth factor-β3 and can enhance this effect. This is relevant for development of Corneal regenerative strategies that require the production of a Keratocyte phenotype. Copyright © 2016 John Wiley & Sons, Ltd.
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Corneal Keratocyte transition to mesenchymal stem cell phenotype and reversal using serum-free medium supplemented with FGF-2, TGF-ß3 and retinoic acid
Journal of Tissue Engineering and Regenerative Medicine, 2017Co-Authors: Laura E Sidney, Andrew HopkinsonAbstract:Keratocytes of the Corneal limbal stroma can derive populations of mesenchymal stem cells (MSC) when expanded in vitro. However, once a Corneal MSC (cMSC) phenotype is achieved, regaining the Keratocyte phenotype can be challenging, and there is no standardised differentiation medium. Here, we investigated the transition of Keratocytes to cMSC and compared different supplements in their ability to return cMSC to a Keratocyte phenotype. Immunofluorescence and RT-qPCR demonstrated in vivo Keratocyte expression of ALDH3A1, CD34 and keratocan, but not any of the typical MSC markers (CD73, CD90, CD105). As the Keratocytes were expanded in vitro, the phenotypic profile reversed and the cells expressed MSC markers but not Keratocyte markers. Differentiating the cMSC back to a Keratocyte phenotype using non-supplemented, serum-free medium restored Keratocyte markers but did not maintain cell viability or support Corneal extracellular matrix (ECM) production. Supplementing the differentiation medium with combinations of fibroblast growth factor-2 (FGF-2), transforming growth factor-β3 (TGF-β3) and retinoic acid (RA) maintained viability, restored expression of CD34, ALDH3A1 and keratocan, and facilitated production of abundant ECM as shown by immunofluorescent staining for collagen-I and lumican, alongside quantitative assays for collagen and glycosaminoglycan production. However, no differentiation medium was able to downregulate the expression of MSC markers in the 21-day culture period. This study shows that the Keratocyte to MSC transition can be partially reversed using serum-free media and supplementation with RA, FGF-2 and TGF-β3 can enhance this effect. This is relevant for development of Corneal regenerative strategies that require the production of a Keratocyte phenotype.
Juiyang Lai - One of the best experts on this subject based on the ideXlab platform.
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dependence of Corneal Keratocyte adhesion spreading and integrin β1 expression on deacetylated chitosan coating
Materials Science and Engineering: C, 2016Co-Authors: Shihfeng Chou, Juiyang Lai, Chinghsie Cho, Chihhung LeeAbstract:This study reports, for the first time, the regulation of Corneal Keratocyte adhesion, spreading, morphology, and integrin gene expression on chitosan coating due to the effects of deacetylation. The degree of deacetylation (DD) in chitosan materials was confirmed by elemental analysis, gel permeation chromatography, and Fourier transform infrared spectroscopy. In this study, chitosan samples with the same molecular weight level but varying DD (74.1 ± 0.5%, 84.4 ± 0.7%, and 94.2 ± 0.5%) were obtained by heat-alkaline treatment under a nitrogen atmosphere. For higher DD groups, the biopolymer carried abundant amino groups since the deacetylation process removed larger amount of acetyl groups from the chitosan molecules. Results showed that the mechanical stability and crystallinity of the chitosan coatings significantly increased with increasing DD value. Fibronectin adsorption, Keratocyte adhesion, and cell spreading exhibited a positive correlation with DD due to the chemical functionality of polysaccharides (bearing acetyl and amino groups) and increase of substrate stiffness and crystallinity. In particular, when adhered to chitosan coatings with a DD value of 74.1%, the Keratocytes appeared to be fibroblastic, elongated, and spindle shape, indicating a loss of their characteristic dendritic morphology. Furthermore, the gene expression of integrin β1 (i.e., a cell-matrix adhesion molecule) was significantly up-regulated on the chitosan coatings with higher DD, which supports favorable attachment of Corneal Keratocytes. Our findings suggest that DD-mediated physicochemical properties of chitosan coatings greatly affect cell-substrate crosstalk during Corneal Keratocyte cultivation.
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relationships between surface roughness stiffness of chitosan coatings and fabrication of Corneal Keratocyte spheroids effect of degree of deacetylation
Colloids and Surfaces B: Biointerfaces, 2016Co-Authors: Shihfeng Chou, Juiyang Lai, Ching-hsien Cho, Chihhung LeeAbstract:Fabrication of the cell spheroids from Corneal Keratocytes has important implications to the advance in tissue engineering while stimulation from the interface of a biopolymer coating has the ability to modulate this event. This study aims to investigate the dependence of Keratocyte migration, proliferation, and differentiation on the surface roughness/stiffness of the chitosan coatings through modifications by degree of deacetylation (DD). After a series of deacetylation process, chitosan coatings with increasing DD exhibited significantly decreased surface roughness and increased surface stiffness. Relationships between the behaviors of rabbit Corneal Keratocytes (RCKs) and biopolymer coatings with varying DDs (between 75% and 96%) were also found during in vitro cultivation. Both the surface roughness increase and stiffness decrease could lead to enhanced cell migration, which is the main driving force for the early stage spheroid formation on chitosan substrates (e.g., within 8h). With these stimulations from the substrate interfaces, the size and morphology of RCK spheroids were greatly affected by the DD of chitosan. When fabricated on a lowered DD of chitosan material, the spheroids had a larger size with abundant extracellular matrix produced around the cells. At a later stage of spheroid cultivation (e.g., 5 days), significantly higher amount of RCKs on chitosan coatings was noted with increasing DD, indicating the substrate interface effects on cell proliferation. The keratocan expression of RCK spheroids grown on a lowered DD of chitosan was up-regulated, suggesting that both the surface roughness increase and stiffness decrease may facilitate the microenvironment for preservation of cellular phenotype. Overall, our work contributes to the scientific understanding of the Keratocyte behaviors and spheroid fabrications in response to DD-mediated surface roughness/stiffness of chitosan coatings.
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Relationships between surface roughness/stiffness of chitosan coatings and fabrication of Corneal Keratocyte spheroids: Effect of degree of deacetylation
Colloids and surfaces. B Biointerfaces, 2016Co-Authors: Shihfeng Chou, Juiyang Lai, Ching-hsien Cho, Chihhung LeeAbstract:Fabrication of the cell spheroids from Corneal Keratocytes has important implications to the advance in tissue engineering while stimulation from the interface of a biopolymer coating has the ability to modulate this event. This study aims to investigate the dependence of Keratocyte migration, proliferation, and differentiation on the surface roughness/stiffness of the chitosan coatings through modifications by degree of deacetylation (DD). After a series of deacetylation process, chitosan coatings with increasing DD exhibited significantly decreased surface roughness and increased surface stiffness. Relationships between the behaviors of rabbit Corneal Keratocytes (RCKs) and biopolymer coatings with varying DDs (between 75% and 96%) were also found during in vitro cultivation. Both the surface roughness increase and stiffness decrease could lead to enhanced cell migration, which is the main driving force for the early stage spheroid formation on chitosan substrates (e.g., within 8h). With these stimulations from the substrate interfaces, the size and morphology of RCK spheroids were greatly affected by the DD of chitosan. When fabricated on a lowered DD of chitosan material, the spheroids had a larger size with abundant extracellular matrix produced around the cells. At a later stage of spheroid cultivation (e.g., 5 days), significantly higher amount of RCKs on chitosan coatings was noted with increasing DD, indicating the substrate interface effects on cell proliferation. The keratocan expression of RCK spheroids grown on a lowered DD of chitosan was up-regulated, suggesting that both the surface roughness increase and stiffness decrease may facilitate the microenvironment for preservation of cellular phenotype. Overall, our work contributes to the scientific understanding of the Keratocyte behaviors and spheroid fabrications in response to DD-mediated surface roughness/stiffness of chitosan coatings.
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Biofunctionalization of gelatin microcarrier with oxidized hyaluronic acid for Corneal Keratocyte cultivation
Colloids and surfaces. B Biointerfaces, 2014Co-Authors: Juiyang LaiAbstract:Abstract Development of microcarriers as alternative architecture models to traditional three-dimensional porous scaffolds can potentially eliminate the concern of extensive geographic necrosis during Keratocyte cultivation. For the first time, this work was performed to evaluate the in vitro expansion of rabbit Corneal Keratocytes (RCKs) on the gelatin microcarrier (GMC) modified with hyaluronic acid (HA). In order to confirm the successful synthesis, the oxidized HA (oHA) samples following treatment of HA with sodium periodate were investigated by chemical characterization, including Fourier transform infrared and nuclear magnetic resonance spectroscopy. Biofunctionalization of GMC with oHA was then achieved by the formation of aldimine linkage between free amino groups in protein and aldehyde groups in oxidized polysaccharide. The grafting yield of oHA onto GMC determined by alcian blue staining assay was 26.9 ± 0.1%. Tissue culture polystyrene (TCPS) plates and the microcarriers from either the GMC or GMC-oHA group were used for suspension culture of RCKs. Results of WST-1 assays, scanning electron microscopy, extracellular matrix production assays, and quantitative real-time reverse transcription polymerase chain reaction and Western blot analysis showed that among all the groups studied, the GMC-oHA samples could most effectively support a large scale growth of RCKs while enhancing their total collagen and glycosaminoglycan contents (i.e., indicative of biosynthetic capacity) and keratocan and ALDH1 gene and protein expressions (i.e., indicative of cell phenotype and function). It is concluded that the aforementioned Keratocyte behaviors are found to be strongly dependent on the type of culture substrate (i.e., TCPS plate versus biopolymer microcarrier) and on the biomaterial functionalization (i.e., modification of gelatin with oHA).
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Nanoscale modification of porous gelatin scaffolds with chondroitin sulfate for Corneal stromal tissue engineering.
International journal of nanomedicine, 2012Co-Authors: Juiyang Lai, Ching-hsien ChoAbstract:Recent studies reflect the importance of using naturally occurring biopolymers as three-dimensional Corneal Keratocyte scaffolds and suggest that the porous structure of gelatin materials may play an important role in controlling nutrient uptake. In the current study, the authors further consider the application of carbodiimide cross-linked porous gelatin as an alternative to collagen for Corneal stromal tissue engineering. The authors developed Corneal Keratocyte scaffolds by nanoscale modification of porous gelatin materials with chondroitin sulfate (CS) using carbodiimide chemistry. Scanning electron microscopy/energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy showed that the amount of covalently incorporated polysaccharide was significantly increased when the CS concentration was increased from 0% to 1.25% (w/v). In addition, as demonstrated by dimethylmethylene blue assays, the CS content in these samples was in the range of 0.078-0.149 nmol per 10 mg scaffold. When compared with their counterparts without CS treatment, various CS-modified porous gelatin membranes exhibited higher levels of water content, light transmittance, and amount of permeated nutrients but possessed lower Young's modulus and resistance against protease digestion. The hydrophilic and mechanical properties of scaffolds modified with 0.25% CS were comparable with those of native corneas. The samples from this group were biocompatible with the rabbit Corneal Keratocytes and showed enhanced proliferative and biosynthetic capacity of cultured cells. In summary, the authors found that the nanoscale-level modification has influence on the characteristics and cell-material interactions of CS-containing gelatin hydrogels. Porous membranes with a CS content of 0.112 ± 0.003 nmol per 10 mg scaffold may hold potential for use in Corneal stromal tissue engineering.
Joseph A. Bonanno - One of the best experts on this subject based on the ideXlab platform.
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Hypoxia reduces TGFβ1-induced Corneal Keratocyte myofibroblast transformation
Molecular vision, 2009Co-Authors: D. Xing, Joseph A. BonannoAbstract:PURPOSE The purpose of this study was to determine whether transient hypoxia had an effect on transforming growth factor beta1 (TGFbeta1)-induced rabbit Corneal Keratocyte myofibroblast transformation. METHODS Primary isolated rabbit Corneal Keratocytes were cultured in a serum-free medium. The effect of transient hypoxia treatment (1% oxygen, 4 h/day) on TGFbeta1 (5 ng/ml)-induced alpha-smooth muscle actin (alpha-SM actin) expression was examined by immunofluorescence, flow cytometry, and immunocytochemistry 72 h after treatment. We found that hypoxia treatment significantly reduced the myofibroblast phenotype and alpha-SM actin expression that was induced by TGFbeta1. To explore the possible mechanism for this effect, we screened for the effects of hypoxia on several early TGFbeta-dependent signaling events including activated pSmad3, CREB (cAMP response element binding) binding protein (CBP), MAPKs (Mitogen-activated protein kinase), and RhoA by co-immunoprecipitation and western blotting. RESULTS Hypoxia alone increased alpha-SM actin expression and the association of pSmad3 to CBP, but it did not induce the myofibroblast phenotype. The levels of pERK (the extracellular signal-regulated protein kinase) and pSmad3 or the extent of the interaction between pSmad3 and CBP induced by TGFbeta1 were not affected by hypoxia whereas the activation of RhoA induced by TGFbeta1 was significantly reduced. CONCLUSIONS We conclude that hypoxia can inhibit TGFbeta1-induced Corneal myofibroblast transformation and alpha-SM actin expression. Our data show that this inhibition does not occur by altering Smads or MAPK signaling but possibly by reducing the early activation of RhoA.
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Effect of cAMP on TGFβ1-induced Corneal Keratocyte-myofibroblast transformation.
Investigative ophthalmology & visual science, 2008Co-Authors: D. Xing, Joseph A. BonannoAbstract:Purpose TGFβ is the major mediator to induce myofibroblast differentiation in the Corneal wound-healing process. Elevated cAMP can reduce TGFβ-induced fibrosis in other tissues. This study was conducted to determine whether elevated cAMP can inhibit TGFβ1-induced rabbit Corneal Keratocyte–myofibroblast transformation.