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. 2023 Feb 14;10(1):15.
doi: 10.1186/s40634-023-00581-4.

Degenerative tendon matrix induces tenogenic differentiation of mesenchymal stem cells

Joongwon Hwang #  1   2 , Seung Yeon Lee #  2 , Chris Hyunchul Jo  3   4
Affiliations

Degenerative tendon matrix induces tenogenic differentiation of mesenchymal stem cells

Joongwon Hwang et al. J Exp Orthop. .

Abstract

Purpose: Mesenchymal stem cells (MSCs) react dynamically with the surrounding microenvironment to promote tissue-specific differentiation and hence increase targeted regenerative capacity. Extracellular matrix (ECM) would be the first microenvironment to interact with MSCs injected into the tissue lesion. However, degenerative tissues would have different characteristics of ECM in comparison with healthy tissues. Therefore, the influence of degenerative ECM on tissue-specific differentiation of MSCs and the formation of matrix composition need to be considered for the sophisticated therapeutic application of stem cells for tissue regeneration.

Methods: Human degenerative tendon tissues were obtained from patients undergoing rotator cuff repair and finely minced into 2 ~ 3 mm fragments. Different amounts of tendon matrix (0.005 g, 0.01 g, 0.025 g, 0.05 g, 0.1 g, 0.25 g, 0.5 g, 1 g, and 2 g) were co-cultured with bone marrow MSCs (BM MSCs) for 7 days. Six tendon-related markers, scleraxis, tenomodulin, collagen type I and III, decorin, and tenascin-C, osteogenic marker, alkaline phosphatase (ALP), and chondrogenic marker, aggrecan (ACAN), were analyzed by qRT-PCR. Cell viability and senescence-associated beta-galactosidase assays were performed. The connective tissue growth factor was used as a positive control.

Results: The expressions of six tendon-related markers were significantly upregulated until the amount of tendon matrix exceeded 0.5 g, the point where the mRNA expressions of all six genes analyzed started to decrease. The tendon matrix exerted an inhibitory effect on ACAN expression but had a negligible effect on ALP expression. Cell viability did not change significantly over the culture period. The amount of tendon matrix exceeding 0.01 g significantly increased the SA-βgal activity of BM MSCs.

Conclusion: This study successfully demonstrated tendon ECM-stimulated tenogenesis of BM MSCs through an indirect co-culture system without the use of exogenous growth factors and the alteration of cellular viability. In contrast to the initial hypothesis, the tenogenesis of BM MSCs induced with the degenerative tendon matrix accompanied cellular senescence.

Keywords: Degenerative tendon matrix; Extracellular matrix; Mesenchymal stem cells; Senescence; Tenogenic differentiation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Phenotypic characterization of BM MSCs. A Morphology of human BM MSCs (original magnification: 100 × and 200x, scale bar = 100 μm). B CFU-F assay of human BM MSCs with two different initial seeding cell densities and its semi-quantification. C Immunophenotype expression analysis of human BM MSCs using flow cytometry
Fig. 2
Fig. 2
Effects of human degenerative tendon matrix on gene expression of six tendon-related markers. Relative mRNA expression of (A) two tenogenic markers, including SCX and TNMD, and (B) four matrix molecules, including Col I, Col III, DCN, and TNC, was detected by qRT-PCR. *p < 0.05, **p < 0.01, and ***p < 0.001
Fig. 3
Fig. 3
Effects of human degenerative tendon matrix on gene expressions of osteogenic and chondrogenic markers of human BM MSCs. Relative mRNA expression of (A) osteogenic marker, ALP, and (B) chondrogenic marker, ACAN, was detected by qRT-PCR. ***p < 0.001
Fig. 4
Fig. 4
Effects of human degenerative tendon matrix on viability and senescence of human BM MSCs. A Measurement of cell viability using EZ-Cytox cell viability assay kit. B Measurement of cellular senescence using senescence β-galactosidase staining kit. C Morphology of human BM MSCs after β-Galactosidase Staining (original magnification: 100x, scale bar = 200 μm). ***p < 0.001
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