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

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The Impact of the Adaptive Immune System On Ectopic Bone Formation
Kavitha Ranganathan, MD, Shuli Li, PhD, Shawn Loder, BS, Cameron Brownley, BS, Shailesh Agarwal, MD, Casey Kraft, BS, Oluwatobi Eboda, BS, Jonathan Peterson, BS, Paul S. Cederna, MD, Steven R. Buchman, MD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.

PURPOSE: Heterotopic ossification (HO) is the abnormal formation of bone in soft tissue structures that commonly occurs after extremity trauma, burn injury, or joint arthroplasties. Although the exact etiology of HO remains unclear, inflammation appears to catalyze the initial formation of HO. The goal of this study is to determine the role of the adaptive immune system in the development of heterotopic bone, and specifically identify the role of B and T cells in this process.
METHODS: An Achilles tenotomy and 30% total body surface area dorsal burn model was used in immunocompromised (C57BL/RagT1Mom-T-cell and B-cell deficient) and immunocompetent (C57BL) mice to study the in vivo and in vitro relationship between the adaptive immune system and heterotopic ossification. Micro CT imaging analysis was used to quantify the extent of HO formation at the tenotomy site. Further histological analysis was performed to assess immune signaling and osteogenic signaling. Flow cytometry of the region of heterotopic bone formation was performed to assess enrichment of immune cells at the site of future bone formation. Given the role of mesenchymal cells in HO formation, adipose-derived mesenchymal cells from immunocompromised and immunocompetent mice sustaining burn injuries were harvested and cultured in osteogenic differentiation medium. The osteogenic potential of these cells was quantified using alkaline phosphatase activity, alizarin red staining, and RNA and protein levels.
RESULTS: Mice in the immunocompromised group formed 60% less bone compared to the immunocompetent mice (4.67 ± 1.5 mm3 vs. 7.76 ± .65 mm3 p=.001). This bone was thinner and had a less developed stroma. Remote burn injury led to significant enrichment of inflammatory cells consisting of endothelial (CD146+/CD45+) and B and T cells (CD45+) at the site of future HO formation as measured by flow cytometry (Figure 1). Mesenchymal cells derived from immunocompromised mice demonstrated significantly decreased osteogenic potential as compared to immunocompetent mice by alkaline phosophatase and alizarin red staining and quantification (29.1 ± .54 vs. 12.1 ± .14; p<0.001).
CONCLUSIONS: We demonstrate that the adaptive immune system plays a prominent role in the inflammatory cascade leading to ectopic bone formation. Furthermore, we demonstrate that a remote burn injury increases inflammatory cell migration to the site of trauma. Inhibition of the adaptive immune system led to a significant decrease in the extent of HO formation. Additional studies are being performed to isolate and track the pathway of individual B and T cells to the tenotomy site itself. This information will enable the use of currently available immunomodulating agents for treatment of heterotopic ossification and bone tissue engineering in the future.



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