Fibroblast-populated Collagen Matrix Research Articles

Treatment of Aged Wound Healing Models with FGF2 and ABT-737 Reduces the Senescent Cell Population and Increases Wound Closure Rate.

Delayed tissue repair in the aged presents a major socio-economic and clinical problem. Age-associated delay in wound healing can be attributed to multiple factors, including an increased presence of senescent cells persisting in the wound. Although the transient presence of senescent cells is physiologic during the resolution phase of normal healing, increased senescent cell accumulation with age can negatively impact tissue repair. The objective of the study was to test interventional strategies that could mitigate the negative effect of senescent cell accumulation and possibly improve the age-associated delay in wound healing. We utilized a 3D in vitro senescent fibroblast populated collagen matrix (FPCM) to study cellular events associated with senescence and delayed healing. Senescent fibroblasts showed an increase in anti-apoptotic B-cell lymphoma 2 (BCL-2) family proteins. We hypothesized that reducing the senescent cell population and promoting non-senescent cell functionality would mitigate the negative effect of senescence and improve healing kinetics. BCL-2 inhibition and mitogen stimulation (FGF2) improved healing in the in vitro senescent models. These results were confirmed with an ex vivo human skin biopsy model. These data suggested that modulation of the senescent cell population with soluble factors improved the healing outcome in our in vitro and ex vivo healing models. This article is protected by copyright. All rights reserved.

Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in an In Vitro Hydrogel Model.

Current cellular hydrogel-based skin grafts composed of human dermal fibroblasts and a hydrogel scaffold tend to minimize contraction of full-thickness skin wounds and support skin regeneration. However, there has been no comparison between the sources of the dermal fibroblast used. Products using human adult or neonatal foreskin dermal fibroblasts are often expanded in vitro and used after multiple passages without a clear understanding of the effects of this initial production step on the quality and reproducibility of the cellular behavior. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may influence cellular properties and contractile behavior in a fibroblast-populated collagen matrix. Using porcine skin as a model based on its similarity to human skin in structure and wound healing properties, we isolated porcine dermal fibroblasts of three different donor ages for use in a 2D proliferation assay and in a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1 in vitro. However, after five passages in culture, there was no difference in contractility between ages. These results show how cellular responses in a hydrogel scaffold differ based on donor age and time in culture in vitro, and suggest that consistency in the cellular component of bioengineered skin products could be beneficial in the biomanufacturing of consistent, reliable skin grafts and graft in vivo models. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding. Impact statement Little is known about the impact of donor cell age and time in culture on the contraction of cellular, hydrogel-based skin grafts. These results show how cellular phenotypes of porcine fibroblasts differ based on donor age and time in culture. This information is beneficial when addressing important inconsistencies in biomanufacturing of bioengineered skin grafts and in vitro models. These findings are relevant to research and therapies using bioengineered skin graft models and the results can be used to increase reproducibility and consistency during the production of bioengineered skin constructs. The information from this study can be extrapolated to future in vivo studies using human dermal fibroblasts in an in vivo model to help determine the best donor age and time in culture for optimal wound healing outcomes or more reproducible in vitro testing constructs.

Novel PEGylated Lipid Nanoparticles Have a High Encapsulation Efficiency and Effectively Deliver MRTF-B siRNA in Conjunctival Fibroblasts.

The master regulator of the fibrosis cascade is the myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway, making it a key target for anti-fibrotic therapeutics. In the past, inhibitors and small interfering RNAs (siRNAs) targeting the MRTF-B gene have been deployed to counter fibrosis in the eye, with the latter showing promising results. However, the biggest challenge in implementing siRNA therapeutics is the method of delivery. In this study, we utilised the novel, pH-sensitive, cationic lipid CL4H6, which has previously demonstrated potent targeting of hepatocytes and endosomal escape, to safely and efficiently deliver an MRTF-B siRNA into human conjunctival fibroblasts. We prepared two lipid nanoparticle (LNP) formulations, incorporating targeting cleavable peptide cY in one of them, and measured their physicochemical properties and silencing effect in human conjunctival fibroblasts. Both proved to be non-cytotoxic at a concentration of 50 nM and effectively silenced the MRTF-B gene in vitro, with the targeting cleavable peptide not affecting the silencing efficiency [LNP with cY: 62.1% and 81.5% versus LNP without cY: 77.7% and 80.2%, at siRNA concentrations of 50 nM (p = 0.06) and 100 nM (p = 0.09), respectively]. On the other hand, the addition of the targeting cleavable peptide significantly increased the encapsulation efficiency of the LNPs from 92.5% to 99.3% (p = 0.0005). In a 3D fibroblast-populated collagen matrix model, both LNP formulations significantly decreased fibroblast contraction after a single transfection. We conclude that the novel PEGylated CL4H6-MRTF-B siRNA-loaded LNPs represent a promising therapeutic approach to prevent conjunctival fibrosis after glaucoma filtration surgery.

The Effect of Chitosan Derivatives on the Compaction and Tension Generation of the Fibroblast-populated Collagen Matrix.

Fibrotic diseases, such as Dupuytren’s contracture (DC), involve excess scar tissue formation. The differentiation of fibroblasts into myofibroblasts is a significant mechanism in DC, as it generates tissue contraction in areas without wound openings, leading to the deposition of scar tissue, and eventually flexing one or more fingers in a restrictive fashion. Additionally, DC has a high recurrence rate. Previously, we showed that N-dihydrogalactochitosan (GC), an immunostimulant, inhibited myofibroblast differentiation in a DC fibroblast culture. Our goal of this study was to expand our previous study to include other DC and normal cell lines and other chitosan derivatives (GC and single-walled carbon nanotube-conjugated GC) to determine the specific mechanism of inhibition. Derivative-incorporated and vehicle control (water) anchored fibroblast-populated collagen matrices (aFPCM) were used to monitor compaction (anchored matrix height reduction) using microscopy and optical coherence tomography (OCT) for six days. Fibroblasts were unable to compact chitosan derivative aFPCM to the same extent as vehicle control aFPCM in repeated experiments. Similarly, chitosan derivative aFPCM contracted less than control aFPCM when released from anchorage. Proliferative myofibroblasts were identified by the presence of alpha smooth muscle actin via myofibroblast proliferative assay. In all tested conditions, a small percentage of myofibroblasts and proliferative cells were present. However, when aFPCM were treated with transforming growth factor-beta 1 (TGF-β1), all tested samples demonstrated increased myofibroblasts, proliferation, compaction, and contraction. Although compaction and contraction were reduced, there was sufficient tension present in the chitosan derivative aFPCM to allow exogenous stimulation of the myofibroblast phenotype.

Predictable fibroblast tension generation by measuring compaction of anchored collagen matrices using microscopy and optical coherence tomography

ABSTRACTThe anchored fibroblast-populated collagen matrix (aFPCM) is an appropriate model to study fibrocontractive disease mechanisms. Our goal was to determine if aFPCM height reduction (compaction) during development is sufficient to predict tension generation. Compaction was quantified daily by both traditional light microscopy and an optical coherence tomography (OCT) system. Contraction in aFPCM was revealed by releasing them from anchorage. We found that aFPCM contraction increase was correlated to the compaction increase. Cytochalasin D treatment reversibly inhibited compaction. Therefore, we demonstrated that aFPCM height reduction efficiently measures compaction, contraction, and relative maturity of the collagen matrix during development or treatment. In addition, we showed that OCT is suitable for effectively imaging the cross-sectional morphology of the aFPCM in culture. This study will pave the way for more efficient studies on the mechanisms of (and treatments that target) migration and contraction in wound healing and Dupuytren’s contracture in a tissue environment.

Effects of Geometry on the Mechanics and Alignment of Three-Dimensional Engineered Microtissues.

The structure and stiffness of the extracellular matrix (ECM) in living tissues play a significant role in facilitating cellular functions and maintaining tissue homeostasis. However, the wide variation and complexity in tissue composition across different tissue types make comparative study of the impact of matrix architecture and alignment on tissue mechanics difficult. Here we present a microtissue-based system capable of controlling the degree of ECM alignment in 3D self-assembled fibroblast-populated collagen matrix, anchored around multiple elastic micropillars. The pillars provide structural constraints, control matrix alignment, enable measurement of the microtissues' contractile forces, and provide the ability to apply tensile strain using magnetic particles. Utilizing finite element models (FEMs) to parametrize results of mechanical measurements, spatial variations in the microtissues' Young's moduli across different regions were shown to be correlated with the degree of ECM fiber alignment. The aligned regions were up to six times stiffer than the unaligned regions. The results were not affected by suppression of cellular contractile forces in matured microtissues. However, comparison to a distributed fiber anisotropic model shows that variations in fiber alignment alone cannot account for the variations in the observed moduli, indicating that fiber density and tissue geometry also play important roles in the microtissues' properties. These results suggest a complex interplay between mechanical boundary constraints, ECM alignment, density, and mechanics and offer an approach combining engineered microtissues and computational modeling to elucidate these relationships.

A novel engineered dermis for in vitro photodamage research.

The realization of biologically relevant human tissue equivalents as an in vitro model to investigate human diseases, as well as to test the efficacy or toxicity of novel compounds, is emerging as a new challenge in tissue engineering. Currently, the in vitro three-dimensional (3D) dermis model mainly involves the use of cells embedded in exogenous non-human matrices. However, such models feature biological and functional disparities with native dermis, therefore limiting their relevance to the in vivo situation. The purpose of this study was to provide a reliable endogenous human dermal equivalent (HDE) able to recapitulate the extracellular matrix (ECM) remodelling of the native dermis occurring after external damage. To this end, UVA irradiation was used to induce photodamage to both the HDE and to a fibroblast-populated collagen matrix. The photodamage was investigated at the cellular and ECM level and the results showed that, although a cellular response was detected in both systems, no ECM reorganization characteristic of the in vivo photo-aged dermis could be detected in the fibroblast-populated collagen matrix. In contrast in the HDE, the neosynthesized ECM recapitulated the characteristic ageing behaviour of the dermis found in vivo, in terms of collagen and hyaluronic acid synthesis as well as collagen organization remodelling. This study therefore demonstrates the role of the endogenous ECM in recapitulating in vitro the functionality of the human dermis and the proposed HDE as a novel tool for photoprotection trials. Copyright © 2016 John Wiley & Sons, Ltd.

MCPIP1 Regulates Fibroblast Migration in 3-D Collagen Matrices Downstream of MAP Kinases and NF-κB

The fibroblast-populated 3D collagen matrix has been used to model matrix contraction, cell motility, and general fibroblast biology. MCPIP1 (monocyte chemotactic protein-induced protein 1) has been shown to regulate inflammation, angiogenesis, and cellular motility. In the present study, we demonstrated induction of MCPIP1 in human fibroblasts embedded in the stress-released 3D collagen matrix, which occurred through activation of mitogen-activated protein kinases, phosphoinositide 3-kinase, and NF-κB. Furthermore, MCPIP1 induction was associated with inhibition of fibroblast migration out of the nested collagen matrix. MCPIP1 induction or ectopic expression also upregulated p53. RNA interference of p53 prevented the inhibition of migration produced by induction or ectopic expression of MCPIP1. Our findings suggest a new role for MCPIP1 as a molecular switch that regulates fibroblast migration in the nested collagen matrix model.

Disruption of Nuclear Factor–kappa-B Signaling in the Fibroblast-Populated Collagen Matrix

Disruption of Nuclear Factor–kappa-B Signaling in the Fibroblast-Populated Collagen Matrix

Expression of green fluorescent protein in human foreskin fibroblasts for use in 2D and 3D culture models

The availability of fibroblasts that express green fluorescent protein (GFP) would be of interest for the monitoring of cell growth, migration, contraction, and other processes within the fibroblast-populated collagen matrix and other culture systems. A plasmid lentiviral vector-GFP (pLV-GFP) was utilized for gene delivery to produce primary human foreskin fibroblasts (HFFs) that stably express GFP. Cell morphology, cell migration, and collagen contraction were compared between nontransduced HFFs and transduced GFP-HFFs; no differences were observed. Immunocytochemical staining showed no differences in cell morphology between nontransduced and GFP-HFFs in both two-dimensional and three-dimensional culture systems. Furthermore, there was no significant difference in cellular population growth within the collagen matrix populated with nontransduced vs. GFP-HFFs. Within the limits of our assays, we conclude that transduction of GFP into HFFs did not alter the observed properties of HFFs compared with nontransduced fibroblasts. The GFP-HFFs may represent a new tool for the convenient monitoring of living primary fibroblast processes in two-dimensional or three-dimensional culture.

Immuno-Regulatory Function of Indoleamine 2,3 Dioxygenase through Modulation of Innate Immune Responses

Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO–expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11±1.47 vs. 70.5±7.57 cells/HPF), T-cells (8.75±1.03 vs. 75.75±5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.

Attachment-regulated signaling networks in the fibroblast-populated 3D collagen matrix

Fibroblasts in the attached collagen matrix are in a pro-survival, pro-proliferative state relative to fibroblasts in the released collagen matrix, such that matrix cell number increases in the former over time. Gene array data from attached vs. released matrices were analyzed for putative networks that regulated matrix cell number. Select networks then underwent augmentation and/or inhibition in order to determine their biologic relevance. Matrix stress-release was associated with modulation of signaling networks that involved IL6, IL8, NF-κB, TGF-β1, p53, interferon-γ, and other entities as central participants. Perturbation of select networks in multiple fibroblast strains suggested that IL6 and IL8 secretion may have been involved in preservation of matrix cell population in the released matrix, though there was variability in testing results among the strains. NF-κB activation may have contributed to the induction of population regression after matrix release.

Embedding Islet in a Liquid Scaffold Increases Islet Viability and Function

IntroductionIslet transplantation is a promising strategy to restore efficient insulin regulation in type 1 diabetes mellitus patients. However, shortage of islet donors, poor islet survival and toxicity of immunosuppressants often reduce the graft functional lifetime. MethodsWe previously showed that a fibroblast populated-collagen matrix (CM) significantly improved engrafted islet viability/function. However, this composite was prone to gradual biodegradation and contraction. Moreover, to avoid use of systemic immunosuppressants, we proposed the use of a local immunosuppressive enzyme, indoleamine-2,3-dioxygenase (IDO). We developed a novel bioengineered crosslinked CM (CCM) to provide optimal matrix biomimetic. Viability and insulin secretory function of islets embedded within fibroblast populated CCM (FP-CCM) was evaluated in vitro and in vivo. IDO expression was transduced in fibroblasts by a lentiviral vector carrying IDO gene and islet viability was evaluated in the presence and absence of IDO producing cells. ResultsIslet survival/function markedly improved within FP-CCM. Furthermore, our data shows that local lentiviral induction of IDO delivered by FP-CCM is nontoxic to the embedded islets. ConclusionsThis promising finding offers a new approach to improving islet transplant outcome.

Effect of An NF-kB Inhibitor on the Cell Population in the Fibroblast-Populated Collagen Matrix

Effect of An NF-kB Inhibitor on the Cell Population in the Fibroblast-Populated Collagen Matrix

Improving the Pancreatic Islet Graft Structure and Function: Application of a Novel Bioengineered Composite Scaffold

Introduction: Transplantation of the insulin-producing islet cells of the pancreas has been a promising strategy for management of type 1 diabetes. However, shortage of islet donors, and poor survival of the islets after transplantation are some of the main challenges of this procedure. Enzymatic digestion of the pancreas in the process of islet isolation results in the loss of peri-insular extracellular matrix (ECM) and peri-vascular basement membrane of the islets that can significantly disrupt the ECM-mediated interactions and result in apoptosis. Naturally a collagen matrix provides for a viable environment in which both stromal and islet cells can survive. However this matrix is mainly biodegradable and subject to gradual disintegration and contraction after transplantation. We have previously shown that a fibroblast populated collagen matrix (FPCM) significantly improves islet cell viability and glucose responsiveness and reduces the marginal islet mass required for hyperglycemia reversal in diabetic mice. Fibroblasts were incorporated to provide a favorable support for islets by producing various growth and angiogenic factors, while maintaining the integrity of the collagen matrix. Although this composite was able to promote the islet graft survival and function, it was still prone to gradual biodegradation. For this reason, we designed a novel bioengineered crosslinked-interpenetrating network of glycosaminoglycan (GAG) and type I collagen (Collagen-GAG matrix, CGM). When populated with fibroblasts (FP-CGM), it would provide an optimal matrix biomimetic with reduced contraction and enhanced mechanical strength for the transplanted islets. Methods: Pancreatic islets and dermal fibroblasts were obtained from Balb/c mice. Islets (50 islets per well in 48-well plates) were cultured in medium (2D), acellular collagen matrix (CM), FPCM, acellular CGM and FP-CGM composites. In order to prepare the FPCM and FP-CGM composites, 200,000 fibroblasts per 100μl of the gel were used. The viability of the islets within composites was assessed by a Live/Dead assay kit on days 1, 15 and 30 post culture. Islets were retrieved on days 1, 15 and 30 post culture and paraffin-embedded sections were stained for insulin, glucagon, caspase-3 and Ki67 and the β-cell proliferation rate was calculated. Retrieved islets were evaluated for glucose-stimulated insulin secretion. Finally, the expression of the islet key specific genes including insulin, PDX1, GLUT2, Pax4, and Pax6 were examined before and after culture at different conditions using reverse transcriptase-PCR analysis. Results: Our preliminary findings have demonstrated that our matrix is non-toxic to both fibroblasts and islets and able to withstand contraction in vitro: Live and Dead assay showed that islets perfectly survive within the new composite matrix. Histology revealed reductions in graft contraction and fibroblast cellularity, increased insulin and glucagon production, and decreased apoptosis rate compared to naked islets in 2D system. Islet morphology remained intact within the composite FP-CGM. PCR analysis showed high expression of islets key genes when islets are embedded within the proposed matrix. Conclusions: We have identified that the use of a crosslinked collagen-GAG graft has the potential to improve graft survival, improving the post-surgical outcome of the transplanted islets.

Three-Dimensional Scaffolds Reduce Islet Amyloid Formation and Enhance Survival and Function of Cultured Human Islets

Islet transplantation provides a promising approach for treatment of type 1 diabetes mellitus. Amyloid formation and loss of extracellular matrix are two nonimmune factors contributing to death of isolated human islets. We tested the effects of two types of three-dimensional scaffolds, collagen matrix (CM) and fibroblast-populated collagen matrix (FPCM), on amyloid formation, viability, and function of isolated islets. Islets from cadaveric donors were cultured in FPCM, CM, or two-dimensional plate (2D) for 7 days. After 7 days, compared with the 2D culture condition, CM and FPCM markedly reduced amyloid formation of cultured islets and decreased apoptotic β-cell rate by ∼75%. IL-1β and Fas levels were also reduced in scaffold-embedded islets. Furthermore, β/α cell ratios were increased by ∼18% and ∼36% in CM- and FPCM-embedded islets, respectively. Insulin content and insulin response to elevated glucose were also enhanced by both three-dimensional scaffolds. Moreover, culture in CM and FPCM (but not 2D) preserved insulin, GLUT-2, and PDX-1 mRNA expression. FPCM-embedded islets had significantly higher insulin response and lower amyloid formation than CM-embedded islets. These findings suggest that three-dimensional scaffolds reduce amyloid formation and improve viability and function of human islets in vitro, and that CM and fibroblasts have additive effects in enhancing islet function and reducing amyloid formation. Using this strategy is likely to improve outcome in human islet transplantation.

Human Skin Model Containing Melanocytes: Essential Role of Keratinocyte Growth Factor for Constitutive Pigmentation—Functional Response to α-Melanocyte Stimulating Hormone and Forskolin

To study human skin pigmentation in a physiological in vitro model, we developed a pigmented reconstructed skin reproducing the three-dimensional architecture of the melanocyte environment and the interactions of melanocyte with its cellular partners, keratinocytes, and fibroblasts. Co-seeding melanocytes and keratinocytes onto a fibroblast-populated collagen matrix led to a correct integration of melanocytes within the epidermal basal layer, but melanocytes remained amelanotic even after supplementation with promelanogenic factors. Interestingly, normalization of keratinocyte differentiation using keratinocyte growth factor instead of epidermal growth factor finally allowed an active pigmentary system to develop, as shown by the expression of key melanogenic markers, the production, and transfer of melanosome-containing melanin into keratinocytes. Various degrees of constitutive pigmentation were reproduced using melanocytes from different skin phenotypes. Furthermore, induction of pigmentation was achieved by treatment with known propigmenting molecules, αMSH and forskolin, thus demonstrating the functionality of the pigmentary system. This pigmented full-thickness skin model therefore represents a highly relevant tool to study the role of cell-cell, cell-matrix, and mesenchymal-epithelial interactions in the control of skin pigmentation.

Gene Networks Involved With Mechanoregulation of Cell Population in the Fibroblast-Populated 3D Collagen Matrix

Introduction: The 3D collagen matrix has been used to study mechanical states in wound healing and tissue engineering. Human foreskin fibroblasts (HFFs) in a mechanically-stressed matrix (MS; attached to culture plate) increase their population, while HFFs in a stress-released matrix (SR; detached, floating in medium) decrease their population. Our intention was to use gene array data to uncover relevant gene networks which might mediate the decrease in matrix cell number associated with the MS-SR transition.

Fibroblast populated collagen matrix promotes islet survival and reduces the number of islets required for diabetes reversal

Islet transplantation represents a viable treatment for type 1 diabetes. However, due to loss of substantial mass of islets early after transplantation, islets from two or more donors are required to achieve insulin independence. Islet-extracellular matrix disengagement, which occurs during islet isolation process, leads to subsequent islet cell apoptosis and is an important contributing factor to early islet loss. In this study, we developed a fibroblast populated collagen matrix (FPCM) as a novel scaffold to improve islet cell viability and function post-transplantation. FPCM was developed by embedding fibroblasts within type-I collagen and used as scaffold for islet grafts. Viability and insulin secretory function of islets embedded within FPCM was evaluated in vitro and in a syngeneic murine islet transplantation model. Islets embedded within acellular matrix or naked islets were used as control. Islet cell survival and function was markedly improved particularly after embedding within FPCM. The composite scaffold significantly promoted islet isograft survival and reduced the critical islet mass required for diabetes reversal by half (from 200 to 100 islets per recipient). Fibroblast embedded within FPCM produced fibronectin and growth factors and induced islet cell proliferation. No evidence of fibroblast over-growth within composite grafts was noticed. These results confirm that FPCM significantly promotes islet viability and functionality, enhances engraftment of islet grafts and decreases the critical islet mass needed to reverse hyperglycemia. This promising finding offers a new approach to reducing the number of islet donors per recipient and improving islet transplant outcome.

Platelet-derived growth factor acts via both the Rho-kinase and p38 signaling enzymes to stimulate contraction in an in vitro model of equine wound healing

Horses are more prone to complications in the wound healing process than other species, and problems such as chronic inflammation, delayed epithelialization, poor wound contraction, and exuberant granulation tissue are commonly seen, particularly in wounds on the distal limbs. In comparison, wounds of the oral mucosa heal rapidly in a scarless fashion with a high degree of wound contraction. The effect of platelet-derived growth factor BB (PDGF), insulin-like growth factor (IGF)-1, and transforming growth factor β1 (TGFβ1) on the contraction of a fibroblast-populated collagen matrix (FPCM) as a model of equine wound contraction was investigated using equine oral fibroblasts. The fibroblasts were embedded into floating FPCM and treated with PDGF, IGF-1, and TGFβ1. The surface areas of the FPCM were determined daily for 5 d. Platelet-derived growth factor significantly stimulated the contraction of the FPCM at an optimal concentration of 10 ng/mL ( P = 0.025). Insulin-like growth factor-1 and TGFβ1 did not significantly affect the contraction of the FPCM relative to the control. To elucidate the mechanisms by which PDGF stimulated contraction of FPCM, the Rho-kinase and p38 cell signaling pathways were blocked, resulting in a significant inhibition ( P < 0.001) of PDGF-stimulated contraction. Platelet-derived growth factor BB is a potent stimulator of fibroblast migration, and hence the FPCM contraction generated here is probably a result of its effects on cell migration. The results of the present experiment suggest that this effect is stimulated via both the Rho-kinase and p38 signaling pathways in equine oral fibroblasts.