Heterotopic ossification (HO) is a debilitating condition defined by the de novo formation of bone within non-osseous soft tissues, such as skeletal muscle. Despite continued efforts to identify the cellular and molecular events leading to HO, the mechanisms of pathogenesis remain elusive. This study utilises a novel in vitro model of skeletal muscle to better understand the pathology of HO. A muscle model was developed by seeding C2C12 myoblasts within a type-I collagen gel. Uniaxial tension was produced by anchoring the gel at opposing ends of a well plate using polyethylene flotation bars. The model was cultured with high glucose DMEM and 20% FBS for 4 days, after which the FBS was replaced with 2% horse serum to facilitate myoblast fusion along the axis of tension. After myotube formation the model was exposed to inflammatory factors both independently and in combination, and the results compared with untreated and 2D controls. Cells were released from the model by digestion with 0.1% collagenase, plated at 10x104 cells/cm2 and cultured in growth or osteogenic medium. The osteogenic potential of the cells was determined using Alizarin Red (AR) staining. The results obtained are novel and show that a population of cells released from the collagen constructs remained unfused and did not contribute to myogenesis. Culture of these unfused cells in osteogenic medium led to ossification, as determined by AR staining. No ossification was observed in 2D controls. Our data demonstrated for the first time that exposure to TGF-β1 and PDGF led to the formation of mineralised nodules, independent of osteogenic medium. This study identified the presence of an osteogenic/non-myogenic population of C2C12 myoblasts when cultured in a 3D collagen model. We also showed that exposure to inflammatory factors identified at the site of tissue damage can lead to ossification, and may contribute to HO.
Disclosure: The authors declared no competing interests. This work was generously supported by a grant from the Defense science and technology laboratory (Dstl).