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Stem cells international2021; 2021; 8284690; doi: 10.1155/2021/8284690

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 . The transforming growth factor (TGF)-, 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-3/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-3-induced tenogenic differentiation. The MSC were incubated with and without TGF-3 (10 ng/ml), Y-27632 (10 M), or both. On day 1 and day 3, the signalling pathway of TGF- 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-3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species ( < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-3 and ROCK inhibition were combined ( < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.
Copyright © 2021 Michaela Melzer et al.
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Publication Date: 2021-10-08 PubMed ID: 34659420PubMed Central: PMC8519677DOI: 10.1155/2021/8284690Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research looked at enhancing tendon regeneration by accelerating the transformation of Mesenchymal Stromal Cells (MSC) into tendon cells through manipulating Rho/ROCK and TGF-β3/Smad signaling. The study found that inhibiting Rho/ROCK signaling while stimulating TGF-β3/Smad signaling effectively transformed MSC into tendon cells, a finding that potentially improves MSC-based therapies for tendon injuries.

Study Rationale and Objective

  • The focus of the research is on two important biological processes for tendon regeneration — the process of MSC transforming into tendon cells (tenogenic differentiation) and the interplay between two signaling pathways, Rho/ROCK and TGF-β3/Smad, which regulate this transformation.
  • The goal is to optimize this process to make MSC-based tendon therapies more efficacious.

Methodology

  • MSC from human and equine were isolated and cultured under different conditions: no treatment, TGF-β3 treatment to stimulate tenogenic differentiation, Rho/ROCK inhibitor Y-27632 treatment to suppress Rho/ROCK signaling, and both TGF-β3 and Y-27632 treatments.
  • Both monolayer culture and culture on tendon-derived decellularized scaffolds were used to mimic realities of tissue construction and cell transplantation.
  • Gene expression of signaling molecules and tendon markers and the actin cytoskeleton were evaluated on days 1 and 3 to assess and compare the effectiveness of different treatments.

Findings

  • Rho/ROCK inhibition by Y-27632 was confirmed through the disruption of the actin cytoskeleton.
  • Activation of Smad 2/3 with nuclear translocation, a key process in TGF-β-mediated tenogenic differentiation, was observed when TGF-β3 was applied, and this effect was even more pronounced with additional ROCK inhibition.
  • The expression of scleraxis, a marker for tendon cells, increased the most when TGF-β3 and ROCK inhibition were combined, indicating the highest rate of tenogenic differentiation under this condition.
  • Differences were observed between human and equine MSC in the regulation pattern of tendon extracellular matrix components and the signaling molecules TGF-β3 and Smad 8.

Implications

  • The results suggest that combined Rho/ROCK inhibition and TGF-β3/Smad stimulation could enhance tenogenic differentiation in MSC, which could optimize MSC-based therapies for tendon injuries.
  • The observed species differences in response to treatments warrant further investigation to reach more widely applicable conclusions.

Cite This Article

APA
Melzer M, Schubert S, Müller SF, Geyer J, Hagen A, Niebert S, Burk J. (2021). Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells. Stem Cells Int, 2021, 8284690. https://doi.org/10.1155/2021/8284690

Publication

Stem cells international
ISSN: 1687-966X
NlmUniqueID: 101535822
Country: United States
Language: English
Volume: 2021
Pages: 8284690
PII: 8284690

Researcher Affiliations

Melzer, Michaela
  • Equine Clinic (Surgery, Orthopedics), Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Schubert, Susanna
  • Saxon Incubator for Clinical Translation, University of Leipzig, 04103 Leipzig, Germany.
  • Institute of Human Genetics, University of Leipzig Hospitals and Clinics, 04103 Leipzig, Germany.
Müller, Simon Franz
  • Biomedical Research Center Seltersberg (BFS), Institute of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Geyer, Joachim
  • Biomedical Research Center Seltersberg (BFS), Institute of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Hagen, Alina
  • Equine Clinic (Surgery, Orthopedics), Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Niebert, Sabine
  • Equine Clinic (Surgery, Orthopedics), Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Burk, Janina
  • Equine Clinic (Surgery, Orthopedics), Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.

Conflict of Interest Statement

The authors declare that there is no conflict of interest regarding the publication of this paper.

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Citations

This article has been cited 12 times.
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