Original Article
Tenascin-C promotes the repair of cartilage defects in mice

https://doi.org/10.1016/j.jos.2019.03.013Get rights and content

Abstract

Background

The effects of tenascin-C (TNC) on cartilage repair were examined in cartilage defect model mice. An in vitro study was also performed to determine the mechanism of cartilage repair with TNC.

Methods

Full-thickness osteochondral defects were filled with TNC (group A: 100 μg/ml, group B: 10 μg/ml, group C: empty). Mice were sacrificed at 1, 2, 3, and 6 weeks postoperatively. Cartilage repair was histologically evaluated using the modified WAKITANI score. Chondrocytes were isolated and cultured, and they were treated with TNC. The expressions of various mRNAs including TNC, inflammatory cytokines, and anabolic and catabolic factors for cartilage were compared by real-time polymerase chain reaction.

Results

The defects in group A were covered with hyaline-like cartilage after 3 weeks. Average modified WAKITANI scores were significantly better in group A than in groups B and C at 3 and 6 weeks. TNC upregulated the expressions of endogenous TNC, inflammatory cytokines, and anabolic and catabolic factors for cartilage. TNC downregulated the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5.

Conclusions

Intra-articular injection of full-length TNC repaired cartilage in murine models of full-thickness osteochondral defects. TNC upregulated the expression of ADAMTS4, but downregulated the expression of ADAMTS5 that contributed to cartilage degradation.

Introduction

Articular cartilage is known to show a limited capacity for self-repair. Osteoarthritis (OA) is one of the leading causes of disability in elderly people. For many years, scientists have been searching for ways to intervene in the disease process and retard or prevent progression of joint damage [1].

Tenascin-C (TNC) is an extracellular matrix glycoprotein that is expressed transiently during embryonic development, but it is absent in most normal adult tissue. This glycoprotein is re-expressed under pathological conditions such as inflammation, tumorigenesis, wound healing, and regeneration. In articular cartilage, TNC expression is also associated with development of cartilage, but it is finally almost abolished in adult articular cartilage [2]. TNC is increased in the synovial fluid in patients with OA and rheumatoid arthritis (RA) [3]. Using an enzyme-linked immunosorbent assay, the TNC level of joint fluids was found to increase in parallel with the radiographic progression of OA [4]. A previous study demonstrated that full-length TNC promotes chondrocyte proliferation and increases proteoglycan content in vitro [5]. In addition, full-length TNC was found to promote the repair of full-thickness osteochondral defects in rabbits [6], and TNC prevented articular cartilage degeneration in murine models of OA for 6 weeks [7]. In contrast, another report showed that the FBG domain of TNC enhanced joint inflammation in murine models of RA [8].

We hypothesized that TNC could be an important molecule for repair of articular cartilage damage. Thus, whether intra-articular injections of full-length TNC could repair the cartilage in the full-thickness osteochondral defects model without exacerbating synovitis was investigated. In addition, to determine the mechanism of prevention of cartilage degeneration and repair of cartilage using full-length TNC, the role of TNC in chondrocyte production of various cytokines was evaluated.

Section snippets

Purification of TNC

TNC was purified from culture supernatant of the U-251MG glioma cell line by ammonium sulfate precipitation, Sephacryl S-500 gel filtration, Mono Q ion exchange chromatography, and using a hydroxyapatite column, as previously described [7], [9].

Animals

A total of 60 male 8-week-old BALB/c strain mice were used according to guidelines approved by our institution. This study protocol was approved by the Institutional Ethics Review Board.

Full-thickness osteochondral defects model

A total of 60 mice were anesthetized with an intramuscular injection

Microscopic analysis of the full-thickness osteochondral defects model

Histological sections of each group that were stained with H&E and Saf-O, immunolabeled for type II collagen, and stained with picrosirius red were examined.

Cartilage: At 1 and 2 weeks, the defects were not filled with repaired tissue in all groups on Saf-O staining. At 3 and 6 weeks, the defects in group A were covered with hyaline-like cartilage, but Saf-O was not uniformly distributed in the upper part of cartilage. In comparison with group A, cartilage repair was not found in groups B and C

Discussion

In the previous study, injected TNC was maintained in the cartilage and synovial cells for at least 2 weeks after injection using labeled TNC [7]. In this in vivo study, the injection of 100 μg/ml of full-length TNC into the knee joint could result in partial repair with hyaline-like cartilage and subchondral bone formation in murine full-thickness osteochondral defects models. In addition, full-length TNC did not exacerbate the synovitis in the full-thickness osteochondral defects model. These

Funding

This work was supported by KAKENHI (Grants-in-Aid for Scientific Research) Grant Number JP25462367.

Conflict of interest

T.Y. receives a royalty from IBL. Other authors received no financial support for the research, authorship, and/or publication of this article.

Acknowledgments

We are grateful to Ms. K. Chiba for technical assistance.

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