| Myostatin (GDF-8), a member of the transforming growth factor beta superfamily, is a negative regulator of skeletal muscle growth and development. Loss of myostatin leads to an increase in fracture callus volume. Although myostatin is immediately expressed during the early phases of fracture healing, little is known about its source after injury, the time course of its expression, and its mechanism of action. We analyzed the temporal & spatial expression patterns of myostatin & its primary receptor (ActRIIB) using a fibular osteotomy in wildtype mice in a time-course immunohistochemical study. We showed that myostatin is highly expressed by the injured skeletal muscle fibers surrounding the site of bone fracture during the first 48 hours following osteotomy. Myostatin was also highly expressed by soft callus chondrocytes, cells that express ActRIIB and play a key role in fracture healing. These data reveal an autocrine mode of action of myostatin by chondrocytes, in addition to the muscle-derived paracrine source. Next, we examined the effect of myostatin on proliferation & chondrogenic differentiation potential of bone marrow-derived mesenchymal stem cells (BMSCs) as key steps in fracture healing. BMSCs were isolated from a GFP-Col2 reporter mouse using immunomagnetic beads, and purity was analyzed by high-speed flowcytometry. A high mass aggregate culture was treated in the presence or absence of recombinant myostatin in chondrogenic medium. Results showed that myostatin suppresses the proliferation, & chondrogenic differentiation of BMSCs by decreasing the production of Col2 (major cartilage collagen) and the expression of Sox5, and Sox9 (key chondrogenic transcription factors). We also showed that the negative effects of myostatin on BMSCs chondrogenic differentiation were associated with unique changes in the Wnt/beta-catenin signaling pathway, in particular down regulation of Wnt-5a. And finally, we assessed the direct role of myostatin in fracture healing using fibular osteotomy in wild type mice after receiving increasing doses of recombinant myostatin loaded in a biodegradable hydrogel injected directly into the open fracture site. MicroCT imaging and histological sections showed a dose-dependent decrease in fracture callus total bone volume, cartilage formation, and muscle regeneration with myostatin treatment. Together, these findings suggest that myostatin can regulate the initial differentiation of chondrogenic progenitors, and can represent a novel therapeutic target for management of orthopedic trauma where bone and muscle are damaged. |
