J. Drake1, S. Agarwal1, K. Shigemori1, S. Loder1, C. Hwang1, S. Li1, Y. Mishina1, S. Wang1, B. Levi1 1University Of Michigan,Ann Arbor, MI, USA
Introduction: Large burns and high-energy trauma can lead to heterotopic ossification (HO), a process by which pathologic, ectopic bone forms within soft tissue. Management of HO is limited by efficacy of available treatments, difficulty identifying at-risk patients, and high recurrence rates following surgical excision. The cellular and molecular basis of HO is unknown. Here we investigate whether the cells themselves or their environment drive HO formation. We demonstrate that human and mouse cells isolated from sites of HO retain increased osteogenic capacity when cultured outside of an inflammatory environment.
Methods: Human cells were cultured from HO and surrounding normal bone. Mouse cells were obtained from two models including trauma-induced and genetic HO. Cells from the trauma induced model were isolated from the tendon transection site of mice which had undergone a dorsal burn with tendon transection (burn/tenotomy) at 1, 2, and 3 weeks after injury, a model that reliably produces HO. In the genetic HO model (Nfatc1-cre/caACVR1fl/wt) normal and HO-derived osteoblasts were isolated from 1, 2, and 3 week old mice. Osteogenic differentiation was assessed for by alkaline phosphatase production, alizarin red stain for mineral deposition, RNA expression, and protein expression. Cell proliferation was also assessed.
Results: Human HO cells showed increased osteogenic signaling compared to human osteoblasts from non-HO bone (Fig. 1). Cells isolated from the burn/tenotomy mice 2 and 3 weeks after injury demonstrated significantly increased cell proliferation, alkaline phosphatase, alizarin red stain, and pSmad 1/5 expression when compared with controls. Similarly, HO-derived cells from our genetic HO model in 2 or 3 week old mice exhibited increased cell proliferation, alkaline phosphatase, alizarin red stain, and pSmad 1/5 expression when compared with non-HO osteoblasts from the same mice (Fig. 2). Finally, targeting these cells with inhibitors of smad5 phosphorylation (LDN-193189) decreased osteogenic capacity by alkaline phosphatase and alizarin red quantification (p<0.05), consistent with its effect on HO formation in our trauma model.
Conclusion: In vitro analysis demonstrates significant differences in cellular behavior with regard to proliferation and osteogenic differentiation in HO models when cells are cultured separate from their in vivo environment. This data suggest that changes in cell behavior drive the process of HO as cell characteristics are preserved after they are removed from their environment. Furthermore, the striking differences in these HO-derived cells from normal cells suggests that they may be used for in vitro assays to study potential therapies targeting HO development.
