N. K. Patel1, A. K. Huber1, C. Pagani1, D. Matera1, C. Hwang1, S. Loder2, N. D. Visser1, K. Vasquez1, J. Li1, Y. Mishina1, S. Agarwal3, B. Baker1, B. Levi1 1University Of Michigan,Ann Arbor, MI, USA 2University Of Pittsburg,Pittsburgh, PA, USA 3Massachusetts General Hospital,Boston, MA, USA
Introduction: Traumatic heterotopic ossification (HO) is a debilitating condition where aberrant bone is formed outside the skeleton due to a fate switch of tissue resident mesenchymal/progenitor cells (MSCs). HO can occur after extremity trauma, burns, and extremity surgeries including amputations and joint replacements. No effective preventive strategies exist as the underlying mechanisms have not been elucidated. Though HO forms at sites of mechanical stress such as joints, the role of mobilization on HO has not been clearly defined. We hypothesize that movement is central to HO formation through mechanotransductive signaling and can provide a basis for improving post-trauma guidelines.
Methods: HO was induced in mice via a dorsal partial thickness burn with concomitant Achilles tenotomy (B/T). Mice were randomly assigned to move ad lib (mobile) or their injured extremity was immobilized. Hindlimb bone volume was assessed at 9 weeks post-B/T by MicroCT (uCT). Immunofluorescent (IF) labeling for PDGFRα and SOX9, was done on 1 week cross sections to assess MSC chondrogenic signaling. Oil Red O (ORO) stain on adjacent sections was done to evaluate for adipogenic differentiation. Atomic Force Microscopy (AFM) was used to obtain elastic modulus of the tissue at site of HO formation. To visualize collagen alignment, second harmonic generation (SHG) microscopy was performed. Anisotropy was calculated for quantification of collagen alignment.
Results: Immobilization of the injured extremity substantially decreased HO by uCT after B/T (Fig A). HO progenitor cells exhibited decreased signaling of chondrogenic transcription factor (SOX9) in immobilized mice by IF, indicating decreased endochondral ossification; circular regions lacking fluorescence in the immobilized samples suggested adipocytes (Fig B). ORO staining confirmed significantly more adipocytes in the immobilized group near the peritenon space (Fig. C), indicating an MSC fate switch. Given the known effect of tissue stiffness on MSC fate, injury site elastic modulus was analyzed by AFM. We found that HO forming regions in immobilized mice were stiffer compared to mobile control (Fig. D) which would normally drive osteogenesis. Immobilized mice demonstrated an increase in collagen anisotropy (Fig. E), which has been shown to drive MSC adipogenic fate.
Conclusion: Immobilization plays a significant role in our model of HO formation by diverting differentiation of MSCs from chondrogenesis to adipogenesis. Future studies will elucidate the mechanisms underlying immobilization resulting in alteration in ECM alignment and cell differentiation.
