Mkx mediates tenogenic differentiation but incompletely inhibits the proliferation of hypoxic MSCs

Abstract

Background: Hypoxia has been shown to be able to induce tenogenic differentiation and proliferation of mesenchymal stem cells (MSCs) which lead hypoxia-induced MSCs to be a potential treatment for tendon injury. However, little is known about the mechanism underlying the tenogenic differentiation and proliferation process of hypoxic MSCs, which limited the application of differentiation-inducing therapies in tendon repair. This study was designed to investigate the role of Mohawk homeobox (Mkx) in tenogenic differentiation and proliferation of hypoxic MSCs. Methods: qRT-PCR, western blot, and immunofluorescence staining were performed to evaluate the expression of Mkx and other tendon-associated markers in adipose-derived MSCs (AMSCs) and bone marrow-derived MSCs (BMSCs) under hypoxia condition. Small interfering RNA technique was applied to observe the effect of Mkx levels on the expression of tendon-associated markers in normoxic and hypoxic BMSCs. Hypoxic BMSCs infected with Mkx-specific short hair RNA (shRNA) or scramble were implanted into the wound gaps of injured patellar tendons to assess the effect of Mkx levels on tendon repair. In addition, cell counting kit-8 assay, colony formation unit assay, cell cycle analysis, and EdU assay were adopted to determine the proliferation capacity of normoxic or hypoxic BMSCs infected with or without Mkx-specific shRNA. Results: Our data showed that the expression of Mkx significantly increased in hypoxic AMSCs and increased much higher in hypoxic BMSCs. Our results also detected that the expression of tenogenic differentiation markers after downregulation of Mkx were significantly decreased not only in normoxic BMSCs, but also in hypoxic BMSCs which paralleled the inferior histological evidences, worse biomechanical properties, and smaller diameters of collagen fibrils in vivo. In addition, our in vitro data demonstrated that the optical density values, the clone numbers, the percentage of cells in S phage, and cell proliferation potential of both normoxic and hypoxic BMSCs were all significantly increased after knockdown of Mkx and were also significantly enhanced in both AMSCs and BMSCs in hypoxia condition under which the expression of Mkx was upregulated. Conclusions: These findings strongly suggested that Mkx mediated hypoxia-induced tenogenic differentiation of MSCs but could not completely repress the proliferation of hypoxic MSCs.