Sclerostin inhibits interleukin-1ß-induced late stage chondrogenic differentiation through downregulation of Wnt/ß-catenin signaling pathway.

It is known that Wnt/ß-catenin signaling induces endochondral ossification and plays a significant role in the pathophysiology of osteoarthritis (OA). Sclerostin is a potent inhibitor of the Wnt/ß-catenin signaling pathway. This study investigated the role of sclerostin in the endochondral differentiation under an OA-like condition induced by proinflammatory cytokines. ATDC5 cells were used to investigate chondrogenic differentiation and terminal calcification, and 10 ng/ml IL-1ß and/or 200 ng/ml sclerostin were added to the culture medium. IL-1ß impaired early chondrogenesis from undifferentiated state into proliferative chondrocytes, and it was not altered by sclerostin. IL-1ß induced progression of chondrogenic differentiation in the late stage and promoted terminal calcification. These processes were inhibited by sclerostin and chondrogenic phenotype was restored. In addition, sclerostin restored IL-1ß-induced upregulation of Wnt/ß-catenin signaling in the late stage. This study provides insights into the possible role of sclerostin in the chondrogenic differentiation under the IL-1ß-induced OA-like environment. Suppression of Wnt signaling by an antagonist may play a key role in the maintenance of articular homeostasis and has a potential to prevent the progression of OA. Thus, sclerostin is a candidate treatment option for OA.

Platelet-Rich Plasma Promotes Migration, Proliferation, and the Gene Expression of Scleraxis and Vascular Endothelial Growth Factor in Paratenon-Derived Cells In Vitro.

BACKGROUND:: Platelet-rich plasma (PRP) is a treatment option for tendon injury because of its effective tendon-healing properties. At the early stage of tendon repair, paratenon-derived cells (PDCs) are thought to play a more important role than tendon proper-derived cells (TDCs). However, there has been no study investigating the effects of PRP on PDCs. HYPOTHESIS:: PRP promotes the migration, proliferation, and differentiation of PDCs in vitro. STUDY DESIGN:: Controlled laboratory study. METHODS:: TDCs and PDCs were isolated from the tendon proper and paratenon of rat Achilles tendons and were cultured to the third passage. PRP was prepared from the rats using the double-spin method. Third-passage TDCs and PDCs were cultured in Dulbecco's modified Eagle medium with 2% fetal bovine serum (control group) or 2% fetal bovine serum plus 5% PRP (PRP group), and cell migration, proliferation, and differentiation were evaluated. The relative mRNA expression levels of scleraxis (Scx), tenomodulin (Tnmd), collagen type I alpha 1 (Col1a1), collagen type III alpha 1 (Col3a1), and vascular endothelial growth factor A (VEGF) were examined by quantitative real-time reverse transcription polymerase chain reaction. RESULTS:: The cell migration rate was significantly higher in the PDCs of the PRP group than in the control group (1.4-fold increase; P = 0.02). Cell proliferation was significantly higher in the PDCs of the PRP group (2.2-fold increase; P < 0.01). In the PDCs, the gene expression levels of Scx, Col1a1, and VEGF were significantly increased by PRP (Scx: 2.0-fold increase, P = 0.01; Col1a1: 5.3-fold increase, P = 0.01; VEGF: 7.8-fold increase, P = 0.01), but the gene expression level of Tnmd, a factor for tendon maturation, was significantly reduced by PRP (0.11-fold decrease; P = 0.02). CONCLUSION:: In vitro PRP promoted migration, proliferation, and tenogenic differentiation with the upregulation of Scx in PDCs. PRP also upregulated the expression of the angiogenic marker VEGF. CLINICAL RELEVANCE:: Our results suggest that PRP treatment in vitro may enhance the tendon-healing properties of PDCs at the initial stage of tendon repair.

Sclerostin is upregulated in the early stage of chondrogenic differentiation, but not required in endochondral ossification in vitro.

Sclerostin is a potent inhibitor of the canonical Wnt signaling pathway. Wnt signaling pathways have multiple roles in the regulation of cartilage development, growth, and maintenance. This study focused on the role of sclerostin in the process of chondrogenic differentiation. We hypothesized that sclerostin is essential to induce chondrogenic differentiation and regulate endochondral ossification. ATDC5 cells were used to investigate chondrogenic differentiation and terminal calcification. During chondrogenic differentiation, intrinsic sclerostin was upregulated in the early stage, but downregulated in the late stage. Addition of sclerostin elevated expressions of Sox9 and Col2a1 (P<0.05) and reduced expressions of Runx2, Col10a1, MMP-3, MMP-13, and ADAMTS5 (P<0.05) through inhibition of the Wnt-ß-catenin signaling pathway (P<0.05). Terminal calcification was significantly inhibited by sclerostin (P<0.05). In contrast, deletion of sclerostin decreased expressions of Sox9 and Col2a1 (P<0.05), increased expressions of Runx2, Col10a1, MMP-3, and MMP-13 (P<0.05), and promoted terminal calcification (P<0.05). This study provides insights into the possible role of sclerostin in the regulation of chondrogenic differentiation. Sclerostin is upregulated in the early stage of chondrogenic differentiation, but is not required in endochondral ossification. Sclerostin is a candidate modulator for chondrogenic differentiation.

Effect of Low-Intensity Pulsed Ultrasound (LIPUS) on Endochondral Ossification via the Wnt Signaling Pathway.

OBJECTIVE: We have previously demonstrated that LIPUS stimulates endochondral ossification with decreased expression of sclerostin. The Wnt signaling pathway was examined in vitro to further address the effect of LIPUS on endochondral ossification. MATERIALS AND METHODS: ATDC5 cells were plated at an initial density of 6 × 10 cells/well in a 6-multiwell plate and cultured in the presence of 5% FBS plus ITS. The bottom of the culture plate was treated every day with LIPUS for 20 minutes. The level of calcification and the expression of Wnt signaling were investigated. RESULTS: The area of calcified nodules in the LIPUS-treated group was significantly greater than in the control group. The expression of Wnt was significantly elevated by LIPUS exposure. Markers associated with endochondral ossification were increased in the LIPUS-treated group. When sclerostin was added to the culture media, calcified nodule formation and the expression of Wnt were inhibited in both the LIPUS-treated group and the control group. DISCUSSION: This study suggests that endochondral ossification was stimulated by LIPUS via the Wnt signaling pathway.