Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Defining The Role Of The Vasculogenic Niche In Trauma-induced Heterotopic Ossification Using Novel Imaging And Lineage-tracing Mice
Shailesh Agarwal, MD1, Shawn Loder, BS1, Cameron Brownley, BS1, Jonathan Peterson, BS1, Eboda Oluwatobi, BS1, Ronald Tompkins, MD2, David Herndon, MD3, Hsiao Sung, DDs, MD1, Wenzhao Zeng, PhD4, Dolrudee Jumlongras, PhD5, Bjorn Olsen, PhD5, Paul S. Cederna, MD1, Steven R. Buchman, MD1, Benjamin Levi, MD1.
1University of Michigan, Ann Arbor, MI, USA, 2Massachusetts General Hospital, Boston, MA, USA, 3University of Texas Medical Branch, Galveston, TX, USA, 4Stanford University, Stanford, CA, USA, 5Harvard Dental School, Boston, MA, USA.

PURPOSE:Heterotopic ossification (HO) is extra-skeletal lamellar bone in soft tissues following trauma. Prior to HO deposition, patients develop inflammation and hypervascularization. Endothelial progenitor cells contribute to neo-angiogenesis by differentiating into mature endothelium. We hypothesized that angiogenesis establishes the niche necessary for HO, and that endothelial cells contribute to HO thereby providing a target for future inhibition.
METHODS:Debrided tissue from 244 patients with >20% TBSA burn within 96 hours of burn was compared to 34 non-burn controls for vasculogenic gene expression. Separately, C57BL/6 mice underwent our proven HO model (Achilles’ tenotomy + 30% total body surface area burn) or control tenotomy no burn injury (n=6 per group). The vasculogenic niche was assessed using flow cytometry, lineage tracing mice, in vivo confocal microscopy, histology, bioluminescensce, microCT, microcapture mRNA quantification, and protein quantification. Next, Cdh5+/+-Cre/RFP+/+ mice were used to visualize the presence of cells (expressing red fluorescent protein) of endothelial progenitor lineage at the HO site (3 and 6 weeks) after trauma. Laser capture microdissection (LCM) was performed to extract tissues of interest and RNA capture was performed with subsequent qRT-PCR to evaluate for gene expression. Protein was collected from the hind limb soft tissue and immunoblotted for VEGF.
RESULTS:Tissue from burn patients demonstrated significant up-regulation of vasculogenic genes (VEGF/HIF-1alpha/VE-cadherin/CD31) compared to unburned patients. In our mouse model, bioluminescent imaging demonstrated increased vascularity at the site of future HO when compared with the contralateral limb (1A). Consistent with these findings, burned mice developed more HO at the site of trauma when compared to unburned mice (1B). In vivo confocal microscopy using Cdh5+/0/RFP+/+ mice confirmed the presence of robust vascularity at the site of HO when compared with the contralateral limb at 6 weeks (1C). Flow cytometry of cells from the HO site showed 20% enrichment of mature endothelial cells (Tie2+/CD31+) and 100% enrichment of endothelial progenitor cells (CD34+/CD133+) compared with the control uninjured leg at three weeks (2A; p <0.05). 3wks after trauma, Cdh5+/0-Cre/RFP+/+ lineage-tracing mice also demonstrated enrichment of endothelial-lineage cells at the HO site compared to the contralateral control limb (2B). However, HO osteoblasts did not express RFP, indicating they are not of endothelial-lineage (2B). LCM sections showed more angiogenic RNA transcript (vegfa) and protein (VEGFA) in the burned mice compared with unburned mice. Western blot of protein from hind limb confirmed these findings (2C).
CONCLUSION:We demonstrate that the development of heterotopic ossification is dependent on the presence of a vasculogenic niche. Mice with robust HO demonstrate an enrichment of mature endothelial and endothelial precursor cells. Although cells of endothelial progenitor origin play a role in chondrogenesis, they do not contribute to HO osteoblasts after endochondral ossification. Targeting angiogenesis may serve as a therapeutic opportunity to prevent HO.


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