A multilayered scaffold for regeneration of smooth muscle and connective tissue layers.
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ABSTRACT:
SUBMITTER: Garrison CM
PROVIDER: S-EPMC7855544 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A major hurdle in engineering thick and laminated tissues such as skin is how to vascularize the tissue. This study introduces a promising strategy for generating multi-layering engineered tissue sheets consisting of fibroblasts and endothelial cells co-seeded on highly micro-fibrous, biodegradable polycaprolactone membrane. Analysis of the conditions for induction of the vessels in vivo showed that addition of endothelial cell sheets into the laminated structure increases the number of incorporated cells and promotes primitive endothelial vessel growth. In vivo analysis of 11-layered constructs showed that seeding a high number of endothelial cells resulted in better cell survival and vascularization 4 weeks after implantation. Within one week after implantation in vivo, red blood cells were detected in the middle section of three-layered engineered tissue sheets composed of polycaprolactone/collagen membranes. Our engineered tissue sheets have several advantages, such as easy handling for cell seeding, manipulation by stacking each layer, a flexible number of cells for next-step applications and versatile tissue regeneration, and automated thick tissue generation with proper vascularization.
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Project description:BACKGROUND:Connective tissue growth factor (CTGF) is a potent profibrotic factor, which is implicated in fibroblast proliferation, angiogenesis and extracellular matrix (ECM) synthesis. It is a downstream mediator of some of the effects of transforming growth factor beta (TGFbeta) and is potentially induced by hyperglycemia in human renal mesangial cells. However, whether high glucose could induce the CTGF expression in vascular smooth muscle cells (VSMCs) remains unknown. Therefore, this study was designed to test whether high glucose could regulate CTGF expression in human VSMC. The effect of modulating CTGF expression on VSMC proliferation and migration was further investigated. RESULTS:Expression of CTGF mRNA was up-regulated as early as 6 hours in cultured human VSMCs after exposed to high glucose condition, followed by ECM components (collagen type I and fibronectin) accumulation. The upregulation of CTGF mRNA appears to be TGFbeta-dependent since anti-TGFbeta antibody blocks the effect of high glucose on CTGF gene expression. A small interference RNA (siRNA) targeting CTGF mRNA (CTGF-siRNA) effectively suppressed CTGF up-regulation stimulated by high glucose up to 79% inhibition. As a consequence of decreased expression of CTGF gene, the deposition of ECM proteins in the VSMC was also declined. Moreover, CTGF-siRNA expressing vector partially inhibited the high glucose-induced VSMC proliferation and migration. CONCLUSION:Our data suggest that in the development of macrovascular complications in diabetes, CTGF might be an important factor involved in the patho-physiological responses to high glucose in human VSMCs. In addition, the modulatory effects of CTGF-siRNA during this process suggest that specific targeting CTGF by RNA interference could be useful in preventing intimal hyperplasia in diabetic macrovascular complications.
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Project description:Neovascularization, increased basal membrane thickness and increased airway smooth muscle (ASM) bulk are hallmarks of airway remodelling in asthma. In this study, we examined connective tissue growth factor (CTGF) dysregulation in human lung tissue and animal models of allergic airway disease. Immunohistochemistry revealed that ASM cells from patients with severe asthma (A) exhibited high expression of CTGF, compared to mild and non-asthmatic (NA) tissues. This finding was replicated in a sheep model of allergic airways disease. In vitro, transforming growth factor (TGF)-? increased CTGF expression both in NA- and A-ASM cells but the expression was higher in A-ASM at both the mRNA and protein level as assessed by PCR and Western blot. Transfection of CTGF promoter-luciferase reporter constructs into NA- and A-ASM cells indicated that no region of the CTGF promoter (-1500 to +200 bp) displayed enhanced activity in the presence of TGF-?. However, in silico analysis of the CTGF promoter suggested that distant transcription factor binding sites may influence CTGF promoter activation by TGF-? in ASM cells. The discord between promoter activity and mRNA expression was also explained, in part, by differential post-transcriptional regulation in A-ASM cells due to enhanced mRNA stability for CTGF. In patients, higher CTGF gene expression in bronchial biopsies was correlated with increased basement membrane thickness indicating that the enhanced CTGF expression in A-ASM may contribute to airway remodelling in asthma.
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Project description:Smooth muscle cell containing organs (bladder, heart, blood vessels) are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs) can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF) suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain) as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.
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Project description:Endothelial progenitor cells (EPCs) play a vital role in endothelial repair following vascular injury by maintaining the integrity of endothelium. As EPCs home to endothelial injury sites, they may communicate with exposed vascular smooth muscle cells (VSMCs), which are subjected to cyclic stretch generated by blood flow. In this study, the synergistic effect of cyclic stretch and communication with neighboring VSMCs on EPC function during vascular repair was investigated. In vivo study revealed that EPCs adhered to the injury site and were contacted to VSMCs in the Sprague-Dawley (SD) rat carotid artery injury model. In vitro, EPCs were cocultured with VSMCs, which were exposed to cyclic stretch at a magnitude of 5% (which mimics physiological stretch) and a constant frequency of 1.25 Hz for 12 h. The results indicated that stretched VSMCs modulated EPC differentiation into mature endothelial cells (ECs) and promoted angiogenesis. Meanwhile, cyclic stretch upregulated the mRNA expression and secretion level of connective tissue growth factor (CTGF) in VSMCs. Recombinant CTGF (r-CTGF) treatment promoted endothelial differentiation of EPCs and angiogenesis, and increased their protein levels of FZD8 and β-catenin. CTGF knockdown in VSMCs inhibited cyclic stretch-induced EPC differentiation into ECs and attenuated EPC tube formation via modulation of the FZD8/β-catenin signaling pathway. FZD8 knockdown repressed endothelial differentiation of EPCs and their angiogenic activity. Wnt signaling inhibitor decreased the endothelial differentiation and angiogenetic ability of EPCs cocultured with stretched VSMCs. Consistently, an in vivo Matrigel plug assay demonstrated that r-CTGF-treated EPCs exhibited enhanced angiogenesis; similarly, stretched VSMCs also induced cocultured EPC differentiation toward ECs. In a rat vascular injury model, r-CTGF improved EPC reendothelialization capacity. The present results indicate that cyclic stretch induces VSMC-derived CTGF secretion, which, in turn, activates FZD8 and β-catenin to promote both differentiation of cocultured EPCs into the EC lineage and angiogenesis, suggesting that CTGF acts as a key intercellular mediator and a potential therapeutic target for vascular repair.
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Project description:Functionally contracting smooth muscle is an essential part of the engineered intestine that has not been replicated in vitro. The purpose of this study is to produce contracting smooth muscle in culture by maintaining the native smooth muscle organization. We employed intact smooth muscle strips and compared them to dissociated smooth muscle cells in culture for 14 days. Cells isolated by enzymatic digestion quickly lost maturity markers for smooth muscle cells and contained few enteric neural and glial cells. Cultured smooth muscle strips exhibited periodic contraction and maintained neural and glial markers. Smooth muscle strips cultured for 14 days also exhibited regular fluctuation of intracellular calcium, whereas cultured smooth muscle cells did not. After implantation in omentum for 14 days on polycaprolactone scaffolds, smooth muscle strip constructs expressed high levels of smooth muscle maturity markers as well as enteric neural and glial cells. Intact smooth muscle strips may be a useful component for engineered intestinal smooth muscle.
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Project description:Extracellular matrix (ECM) structures within skeletal muscle play an important, but under-appreciated, role in muscle development, function and adaptation. Each individual muscle is surrounded by epimysial connective tissue and within the muscle there are two distinct extracellular matrix (ECM) structures, the perimysium and endomysium. Together, these three ECM structures make up the intramuscular connective tissue (IMCT). There are large variations in the amount and composition of IMCT between functionally different muscles. Although IMCT acts as a scaffold for muscle fiber development and growth and acts as a carrier for blood vessels and nerves to the muscle cells, the variability in IMCT between different muscles points to a role in the variations in active and passive mechanical properties of muscles. Some traditional measures of the contribution of endomysial IMCT to passive muscle elasticity relied upon tensile measurements on single fiber preparations. These types of measurements may now be thought to be missing the important point that endomysial IMCT networks within a muscle fascicle coordinate forces and displacements between adjacent muscle cells by shear and that active contractile forces can be transmitted by this route (myofascial force transmission). The amount and geometry of the perimysial ECM network separating muscle fascicles varies more between different muscle than does the amount of endomysium. While there is some evidence for myofascial force transmission between fascicles via the perimysium, the variations in this ECM network appears to be linked to the amount of shear displacements between fascicles that must necessarily occur when the whole muscle contracts and changes shape. Fast growth of muscle by fiber hypertrophy is not always associated with a high turnover of ECM components, but slower rates of growth and muscle wasting may be associated with IMCT remodeling. A hypothesis arising from this observation is that the level of cell signaling via shear between integrin and dystroglycan linkages on the surface of the muscle cells and the overlying endomysium may be the controlling factor for IMCT turnover, although this idea is yet to be tested.
| S-EPMC7248366 | biostudies-literature
Project description:PURPOSE:To assess the impact of dietary protein ingestion on intramuscular connective tissue protein synthesis rates during overnight recovery from a single bout of resistance exercise. METHODS:Thirty-six healthy, young males were randomly assigned to one of three treatments. One group ingested 30 g intrinsically L-[1-C]-phenylalanine-labeled casein protein prior to sleep (PRO, n=12). The other two groups performed a bout of resistance exercise in the evening and ingested either a placebo (EX, n=12) or 30 g intrinsically L-[1-C]-phenylalanine-labeled casein protein prior to sleep (EX+PRO, n=12). Continuous intravenous infusions of L-[ring-H5]-phenylalanine and L-[1-C]-leucine were applied and blood and muscle tissue samples were collected to assess connective tissue protein synthesis rates and dietary protein-derived amino acid incorporation in the connective tissue protein fraction. RESULTS:Resistance exercise resulted in higher connective tissue protein synthesis rates when compared to rest (0.086±0.017 (EX) and 0.080±0.019 (EX+PRO) vs 0.059±0.016 (PRO) %·h; P<0.05). Post-exercise casein protein ingestion did not result in higher connective tissue protein synthesis rates when compared to post-exercise placebo ingestion (P=1.00). Dietary protein-derived amino acids were incorporated into the connective tissue protein fraction at rest, and to a greater extent during recovery from exercise (P=0.002). CONCLUSION:Resistance exercise increases intramuscular connective tissue protein synthesis rates during overnight sleep, with no further impact of post-exercise protein ingestion. However, dietary protein-derived amino acids are being utilized as precursors to support de novo connective tissue protein synthesis.
| S-EPMC7431152 | biostudies-literature