The Mechanism, Regulatory Effect of Wnt5α/Ca2+ Signaling Pathway on hMSCs Osteogenic Differentiation Under Infection Conditions

Abstract

Background: The paper aimed to reveal the role and possible regulatory mechanisms of Wnt5 gene and its non-canonical Beta-catenin signaling pathway in osteogenic differentiation and bone repairment of hMSCs under infection environment. Material and Methods : CCK-8 method was employed to detect the effect of SPA on the proliferation of different hMSCs cells. The histological staining of alizarin red was used to observe the differentiation of cells into osteoblasts. The semi-quantitative detection of osteoblast alkaline phosphatase (ALP) was performed. Fluo-2 assay was employed to measure the intracellular calcium concentration of different hMSCs before and after osteogenic differentiation stimulated by SPA. RT-PCR was applied to detect the specific gene fluorescence of osteoblasts differentiation. Western Blotting was employed to determine the differentiation of hMSCs into osteoblasts. Results: Different hMSCs were able to proliferate in vitro under the stimulation of 100 ng/mL SPA. CCK-8 kit was used to detect lentivirus-infected hMSCs and non-transduced hMSCs in vitro. The results showed that all the cells gradually proliferate with time and an increasing rate. The hMSCs treated with different Wnt5 genes were stained with alizarin red after osteogenic differentiation, and the activities of AMSCs ALP increased. The intracellular free calcium concentration increased with the induction time and peaked in the Wnt5 overexpression group. PCR results showed that ALP, Runx-2, Collagen I, OC, Osterix and BSP genes were all increased in contrast with the non-induction Control group. Conclusion: Under the inflammatory condition of SPA virulence factors, non-canonical Wnt signaling pathway is associated with the classical Wnt/ β-catenin signaling pathway in the process of osteogenic differentiation of hMSCs in vitro, which has a certain synergistic effect.