Dataset Information

Induction of Tenogenic Differentiation Mediated by Extracellular Tendon Matrix and Short-Term Cyclic Stretching.

ABSTRACT: Tendon and ligament pathologies are still a therapeutic challenge, due to the difficulty in restoring the complex extracellular matrix architecture and biomechanical strength. While progress is being made in cell-based therapies and tissue engineering approaches, comprehensive understanding of the fate of progenitor cells in tendon healing is still lacking. The aim of this study was to investigate the effect of decellularized tendon matrix and moderate cyclic stretching as natural stimuli which could potentially direct tenogenic fate. Equine adipose-derived mesenchymal stromal cells (MSC) were seeded on decellularized tendon matrix scaffolds. Mechanical stimulation was applied in a custom-made cyclic strain bioreactor. Assessment was performed 4?h, 8?h, and 24?h following mechanical stimulation. Scaffold culture induced cell alignment and changes in expression of tendon-related genes, although cell viability was decreased compared to monolayer culture. Short mechanical stimulation periods enhanced most of the scaffold-induced effects. Collagen 1A2 expression levels were decreased, while collagen 3A1 and decorin levels were increased. Tenascin-C and scleraxis expression showed an initial decrease but had increased 24?h after stimulation. The results obtained suggest that decellularized tendon matrix, supported by cyclic stretching, can induce tenogenic differentiation and the synthesis of tendon components important for matrix remodeling.

SUBMITTER: Burk J

PROVIDER: S-EPMC5007347 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Induction of Tenogenic Differentiation Mediated by Extracellular Tendon Matrix and Short-Term Cyclic Stretching.

Burk Janina J Plenge Amelie A Brehm Walter W Heller Sandra S Pfeiffer Bastian B Kasper Cornelia C

Stem cells international 20160818

Tendon and ligament pathologies are still a therapeutic challenge, due to the difficulty in restoring the complex extracellular matrix architecture and biomechanical strength. While progress is being made in cell-based therapies and tissue engineering approaches, comprehensive understanding of the fate of progenitor cells in tendon healing is still lacking. The aim of this study was to investigate the effect of decellularized tendon matrix and moderate cyclic stretching as natural stimuli which ...[more]

PMID: 27630718

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Project description:BackgroundTendon healing is clinically challenging largely due to its inferior regenerative capacity. We have previously prepared a soluble, DNA-free, urea-extracted bovine tendon-derived extracellular matrix (tECM) that exhibits strong pro-tenogenic bioactivity on human adipose-derived stem cells (hASCs). In this study, we aimed to elucidate the mechanism of tECM bioactivity via characterization of tECM protein composition and comparison of transcriptomic profiles of hASC cultures treated with tECM versus collagen type I (Col1) as a control ECM component.MethodsThe protein composition of tECM was characterized by SDS-PAGE, hydroxyproline assay, and proteomics analysis. To investigate tECM pro-tenogenic bioactivity and mechanism of action, differentiation of tECM-treated hASC cultures was compared to serum control medium or Col1-treated groups, as assessed via immunofluorescence for tenogenic markers and RNA Sequencing (RNA-Seq).ResultsUrea-extracted tECM yielded consistent protein composition, including collagens (20% w/w) and at least 17 non-collagenous proteins (< 100 kDa) based on MS analysis. Compared to current literature, tECM included key tendon ECM components that are functionally involved in tendon regeneration, as well as those that are involved in similar principal Gene Ontology (GO) functions (ECM-receptor interaction and collagen formation) and signaling pathways (ECM-receptor interaction and focal adhesion). When used as a cell culture supplement, tECM enhanced hASC proliferation and tenogenic differentiation compared to the Col1 and FBS treatment groups based on immunostaining of tenogenesis-associated markers. Furthermore, RNA-Seq analysis revealed a total of 584 genes differentially expressed among the three culture groups. Specifically, Col1-treated hASCs predominantly exhibited expression of genes and pathways related to ECM-associated processes, while tECM-treated hASCs expressed a mixture of ECM- and cell activity-associated processes, which may explain in part the enhanced proliferation and tenogenic differentiation of tECM-treated hASCs.ConclusionsOur findings showed that urea-extracted tECM contained 20% w/w collagens and is significantly enriched with other non-collagenous tendon ECM components. Compared to Col1 treatment, tECM supplementation enhanced hASC proliferation and tenogenic differentiation as well as induced distinct gene expression profiles. These findings provide insights into the potential mechanism of the pro-tenogenic bioactivity of tECM and support the development of future tECM-based approaches for tendon repair.

Project description:Age-related degenerative changes in tendon tissue represent a common cause for acute tendon pathologies. Although the regenerative potential of multipotent mesenchymal stromal cells (MSC) was reported to restore functionality in injured tendon tissue, cellular mechanisms of action remain partly unclear. Potential tenogenic differentiation of applied MSC is affected by various intrinsic and extrinsic factors. The current study presents an in vitro model to evaluate the combined extrinsic effects of decellularized equine tendon matrix, transforming growth factor beta 3 (TGFβ3) and bone morphogenetic protein 12 (BMP12) on the tenogenic fate of equine adipose tissue-derived MSC. Monolayer MSC cultures supplemented with TGFβ3 and BMP12 as well as MSC cultured on tendon matrix scaffolds preloaded with the growth factors were incubated for 3 and 5 days. Histological evaluation and real time reverse transcription polymerase chain reaction (RT-PCR) revealed that growth factor-mediated tenogenic induction of MSC was modified by the conditions of the surrounding microenvironment. While the gene expression pattern in monolayer cultures supplemented with TGFβ3 or TGFβ3 and BMP12 revealed an upregulation for collagen 1A2, collagen 3A1, tenascin c, scleraxis and mohawk ( p < 0.05 ), the presence of tendon matrix led to an upregulation of decorin and osteopontin as well as to a downregulation of smad8 ( p < 0.05). Preloading of scaffolds with either TGFβ3, or with TGFβ3 and BMP12 promoted a tenocyte-like phenotype and improved cell alignment. Furthermore, gene expression in scaffold culture was modulated by TGFβ3 and/or BMP12, with downregulation of collagen 1A2, collagen 3A1, decorin, scleraxis, smad8 and osteopontin, whereas gene expression of tenascin c was increased. This study shows that growth factor-induced tenogenic differentiation of equine MSC is markedly altered by topographical constraints of decellularized tendon tissue in vitro. While TGFβ3 represents an effective mediator for tenogenic induction, the role of BMP12 in tenogenesis may be of modulatory character and needs further evaluation.

Project description:Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim we investigated the in vitro tenogenic differentiation potential of ASCs using a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL) suitable for cell therapy and both supplemented with CTGF, TGFβ-3, BMP-12 and ascorbic acid (AA) soluble factors. Human ASCs were isolated from 4 healthy donors and they were inducted to differentiate until 14 days in both hPL and SF tenogenic media (hPL-TENO and SF-TENO). Cell viability and immunophenotype profile were analysed to evaluate mesenchymal stem cell (MSC) characteristics in both xenogenic-free media. Moreover, the expression of stemness and tendon-related markers upon cell differentiation by RT-PCR, protein staining and cytofluorimetric analysis were also performed. Our results showed the two xenogenic-free media well support cell viability of ASCs and maintain their MSC nature as demonstrated by their typical immunophenototype profile and by the expression of NANOG, OCT4 and Ki67 genes. Moreover, both hPL-TENO and SF-TENO expressed significant high levels of the tendon-related genes SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 already at early time points in comparison to the respective controls. Significant up-regulations in scleraxis, collagen and tenomodulin proteins were also demonstrated at in both differentiated SF and hPL ASCs. In conclusion, we demonstrated firstly the feasibility of both serum and xenogenic-free media tested to culture ASCs moving forward the GMP-compliant approaches for clinical scale expansion of human MSCs needed for therapeutical application of stem cells. Moreover, a combination of CTGF, BMP-12, TGFβ3 and AA factors strongly and rapidly induce human ASCs to differentiate into tenocyte-like cells.

Project description:Tendon injury is a common but tough medical problem. Unsatisfactory clinical results have been reported in tendon repair using mesenchymal stem cell (MSC) therapy, creating a need for a better strategy to induce MSCs to tenogenic differentiation. This study was designed to examine the effect of hypoxia on the tenogenic differentiation of different MSCs and their tenogenic differentiation capacities under hypoxia condition in vitro and to investigate the in vivo inductility of hypoxia in tenogenesis. Adipose tissue-derived MSCs (AMSCs) and bone marrow-derived MSCs (BMSCs) were isolated and characterized. The expression of hypoxia-induced factor-1 alpha (Hif-1?) was examined to confirm the establishment of hypoxia condition. qRT-PCR, western blot, and immunofluorescence staining were used to evaluate the expression of tendon-associated marker Col-1a1, Col-3a1, Dcn, and Tnmd in AMSCs and BMSCs under hypoxia condition, compared with Tgf-?1 induction. In vivo, a patellar tendon injury model was established. Normoxic and hypoxic BMSCs were cultured and implanted. Histological, biomechanical, and transmission electron microscopy analyses were performed to assess the improved healing effect of hypoxic BMSCs on tendon injury. Our in vitro results showed that hypoxia remarkably increased the expression of Hif-1? and that hypoxia not only promoted a significant increase in tenogenic markers in both AMSCs and BMSCs compared with the normoxia group but also showed higher inductility compared with Tgf-?1. In addition, hypoxic BMSCs exhibited higher potential of tenogenic differentiation than hypoxic AMSCs. Our in vivo results demonstrated that hypoxic BMSCs possessed better histological and biomechanical properties than normoxic BMSCs, as evidenced by histological scores, patellar tendon biomechanical parameters, and the range and average of collagen fibril diameters. These findings suggested that hypoxia may be a practical and reliable strategy to induce tenogenic differentiation of BMSCs for tendon repair and could enhance the effectiveness of MSCs therapy in treating tendon injury.

Dataset Information

Induction of Tenogenic Differentiation Mediated by Extracellular Tendon Matrix and Short-Term Cyclic Stretching.

Publications

Induction of Tenogenic Differentiation Mediated by Extracellular Tendon Matrix and Short-Term Cyclic Stretching.

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