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Central Role Of Early Vascularization In Burn and Trauma-Induced Heterotopic Ossification
Shailesh Agarwal, MD, Jonathan Peterson, BS, Sara De La Rosa, BS, Kavitha Ranganathan, MD, Alexis Donneys, MD, Stewart Wang, MD, PhD, Paul S. Cederna, MD, Steven R. Buchman, MD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.
Introduction: Heterotopic ossification (HO) is a condition in which bone forms within non-osseous tissues after severe trauma, burn and orthopedic surgery. Previous studies have demonstrated that normal bone development and repair depends on vasculogenic and angiogenic signaling. However, the roles of key mediators of angiogenesis have not been fully evaluated in the pathogenesis of burn-induced HO. We hypothesize that burn injury increases early vasculogenic signaling and vessel formation resulting in increased heterotopic bone formation.
Methods:C57/BL6 male mice underwent Achilles tenotomy with 1) 30% total body surface area burn or 2) no burn injury (n=4/group). HO formation was evaluated weekly (1-9 weeks) using micro-CT and at 9 weeks with nano-CT. At 5 weeks postoperatively, the vascular density in the region of the HO was assessed by Microfil injection followed by micro and nano-CT. Immunohistochemistry and immunofluorescent staining with anti-CD31 and anti-HIF1A antibodies was performed to assess angiogenesis. Separately, adipose-derived mesenchymal stem cells (MSCs) were isolated after 2 hours or five days from mice with or without 30% TBSA burn injury and cultured in osteogenic differentiation medium for 3 days. The robustness of vascular signaling was evaluated using qRT-PCR to quantify Vegfa gene transcription and Western blotting to quantify protein expression (VEGFA). Furthermore, an in vivo Matrigel plug assay (1 million MSCs seeded in 20ul of matrigel and implanted subcutaneously) was used to assess the effect of burn injury on angiogenesis.
Results: Mice with Achilles’ tenotomy and burn injury developed HO with intertwined blood vessels as demonstrated by nano-CT at 9 weeks. Microfil quantification showed that mice with Achilles tenotomy and burn developed greater vascular density at HO sites than mice with Achilles tenotomy alone (Fig. 1) (n=6*p<0.05). We also noted significantly more IHC staining for CD31 and HIF1A in burn mice at the site of HO (Fig. 2). MSCs harvested from mice with a burn injury had 30% more Vegfa gene transcript and over 65% more VEGFA protein expression. Finally, in vivo tubule assays demonstrated increased angiogenic signaling in those mice with a burn injury compared to control.
Conclusions: These data suggest that early angiogenesis plays a role in HO formation. Furthermore, burn injury enhances the in vitro angiogenic capacity of MSCs and stimulates an increase in in vivo vessel formation at the Achilles tenotomy site. Increased presence of HIF-alpha and VEGF-A after a burn injury may create a permissive niche for HO to form. This is the first study demonstrating the role of angiogenesis in the formation of burn- induced HO. Localized regulation of vessel formation may serve as a viable target to treat or prevent HO in trauma and burn patients.
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