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Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells

ABSTRACT: Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation in vivo. The transforming growth factor (TGF)-β, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-β3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-β3-induced tenogenic differentiation. The MSC were incubated with and without TGF-β3 (10 ng/ml), Y-27632 (10 μM), or both. On day 1 and day 3, the signalling pathway of TGF-β and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-β3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species (p < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-β3 and ROCK inhibition were combined (p < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-β3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-β3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.

SUBMITTER: Melzer M

PROVIDER: S-EPMC8519677 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHigh post-surgical failure rates following tendon injury generate high medical costs and poor patient recovery. Cell-based tendon tissue engineering has the potential to produce fully functional replacement tissue and provide new strategies to restore tendon function and healing. In this endeavour, the application of mesenchymal stromal cells (MSCs) encapsulated in biomaterial scaffolds has shown great promise. However, a consensus on optimal promotion of tenogenic differentiation of MSCs has yet to be reached, although growth factors and mechanical cues are generally acknowledged as important factors.MethodsIn this study, we prepared a hydrogel cell culture system consisting of methacrylated poly(d,l-lactic acid-ethylene glycol-d,l-lactic acid) (P(LA-EG-LA)) and gelatin methacrylate (GelMA) to encapsulate human bone marrow-derived MSCs (hBMSCs). We further systematically investigated the influence of static and intermittent cyclic uniaxial strain mechanical stimulation, in combination with transforming growth factor-β3 (TGF-β3) supplementation, on tenogenic differentiation of hBMSCs.ResultsIncreased TGF-β3 concentration upregulated the tenogenic genes Scleraxis (SCX) and collagen type I (COL1A1) but showed no effects on tenascin-c (TNC) and collagen type III (COL3A1) expression. Mechanical stimulation had no observable effect on gene expression, but intermittent cyclic uniaxial strain stimulation improved matrix deposition. Together, these data provide new insights into how TGF-β3 and mechanical stimulation regulate MSC tenogenesis, with TGF-β3 promoting the expression of key tenogenic genes whilst mechanical stimulation aided matrix deposition in the engineered tissue. Furthermore, intermittent cyclic uniaxial strain at 3% elongation and 0.33 Hz for 1 h/day showed improved matrix effects compared to static strain.ConclusionTogether, the most promising result for tenogenic differentiation of hBMSCs was identified as treatment with 5 ng/ml TGF-β3 under intermittent cyclic uniaxial strain (3% strain; 0.33 Hz; 1 h/day). This knowledge is of importance for the development of an improved protocol for tenogenic differentiation of MSCs and thereby for tendon tissue engineering.The translational potential of this articleTissue-engineered strategies for tendon repair require a consensus on the differentiation of mesenchymal stromal cells to tenocytes, which is currently lacking. This article provides a systematic investigation of two main tenogenic differentiation conditions to further development of a tenogenic differentiation protocol.

Project description:To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.

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Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells

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