Zinc Chloride Treatment in ATDC5 cells Induces Chondrocyte Maturation

Abstract

Objectives: Chondrogenesis is an integral part of endochondral ossification during bone healing. Insulin and insulin mimetic compounds like ZnCl2 have been shown to improve bone healing in both normal and diabetic bone healing models. Although the effects of ZnCl2 in processes like osteoblast proliferation and osteoclastogenesis have been evaluated, its role in chondrogenesis is unclear [1-4]. In this study, we determine if ZnCl2 treatment can induce chondrogenic differentiation in the murine chondrogenic ATDC5 cell line in the absence of insulin. Methods: ATDC5 cells were treated with 10ug/ml insulin (IDM), 0µM, 100µM, or 1000µM of ZnCl2 every other day and harvested at the following time points 2hrs, 4hrs, 1d, 2d, 4d, 7d, 10d, 14d, 17d, 21d, 24d, and 28d. ATDC5 chondrogenic differentiation was assayed via Alizarin Red S (ARS) staining, Alcian blue staining, immunoblotting, and QPCR. Results: Our results demonstrate that ZnCl2 treatment can induce proteoglycan and calcium deposition as per Alcian blue and ARS staining in ATDC5 chondrocyte cells. ZnCl2 treatment also increased transcriptional expression of Col2a1 and increased phospho-Akt protein levels over time. Conclusions: The experimental results indicate that ZnCl2 can be promote ATDC5 chondrocytes to differentiate in vitro. Our results coincide with other studies that show that Zn+2 can stimulation collagen production or enhance bone healing outcomes in animal models [4, 5]. Our data indicate that ZnCl2 may be a useful adjuvant for promoting chondrogenesis during endochondral ossification, enhancing cartilage repair, or improving cartilage tissue engineering applications.