Heterotopic ossification (HO) is the abnormal formation of bone in structures such as muscle or joints. This devastating complication occurs following major trauma, burns, spinal cord injury/traumatic brain injury, and orthopedic surgery procedures. Patients with HO develop chronic pain, open wounds, restricted joint motion, and nerve entrapment resulting in a significantly diminished quality of life. Patients with HO often require surgical excision, although recurrence and residual deformities are common. Commonly trialed drugs such as bisphosphonates are not tolerated by all patients, have limited efficacy, and do not target the mechanism responsible for HO. Therefore, there is a need to identify solutions to prevent HO in patients. Based on our clinical experience, HO is often preceded by a significant inflammatory insult. We have developed a mouse model of hindlimb tendon transection and 30% total body surface area burn over the dorsum, which consistently produces HO at the tenotomy site. Our evaluation of the tenotomy site revealed substantial macrophage infiltration apparent by 5 days after injury and present in proximity to developing HO even 2 weeks later. Our preliminary findings also show enrichment of a population of osteoprogenitor cells at the injury site present 3 weeks prior to radiographically evident HO. Finally, targeted inhibition of the bone morphogenetic protein (BMP) receptor pathway using small molecule inhibitors significantly decreases HO burden in our mouse model. Through this proposal, we plan to improve prevention strategies for injuries with high HO risk by targeting 1) macrophages or 2) BMP signaling to prevent HO, and the enrichment of osteoprogenitor cells which precedes HO.
In Aim 1, we will characterize the contribution of macrophage accumulation to HO using knockout mice incapable of recruiting macrophages to injury sites (Ccr2-null), or by treating mice locally or systemically with inhibitors of COX-1/-2 or TNF?, both of which are involved in macrophage development. We will examine macrophage infiltration using histology, overall HO development using imaging, osteoprogenitor cell presence at the tenotomy site, and osteoprogenitor cell differentiation capacity in vitro.
In Aim 2, we will target ALK2; a bone morphogenetic protein (BMP) receptor which we hypothesize is involved in trauma-induced HO. We will use transgenic mice or small molecule inhibitors to target ALK2 locally or globally and examine the effect on HO. We will additionally study the effect on osteoprogenitor cell quantity in vivo and osteogenic differentiation based on in vitro assays. Human tissue in Aim 2 will also be examined for osteoprogenitor cell quantity and in vitro differentiation. Upon completion of this study, we will elucidate how macrophages and/or ALK2 signaling mediate HO formation and whether strategies targeting either of these mechanisms can inhibit HO following trauma.

Public Health Relevance

Heterotopic ossification (HO), the abnormal formation of bone in structures such as muscle or joints, occurs in patients following major trauma, burns, spinal cord injury/traumatic brain injury, and orthopedic surgery procedures. The overall goal of this proposal is to study how inflammation and BMP cellular signaling can be targeted to prevent HO and the progenitor cells responsible for its development. These findings will provide us with several avenues for the prevention of heterotopic ossification.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F15-P (20)L)
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Chen, Faye H
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
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Loder, Shawn J; Agarwal, Shailesh; Chung, Michael T et al. (2018) Characterizing the Circulating Cell Populations in Traumatic Heterotopic Ossification. Am J Pathol 188:2464-2473
Kraft, Casey; Millet, John D; Agarwal, Shailesh et al. (2017) SPECT/CT in the Evaluation of Frostbite. J Burn Care Res 38:e227-e234
Strong, Amy L; Agarwal, Shailesh; Cederna, Paul S et al. (2017) Peripheral Neuropathy and Nerve Compression Syndromes in Burns. Clin Plast Surg 44:793-803
Dey, Devaveena; Wheatley, Benjamin M; Cholok, David et al. (2017) The traumatic bone: trauma-induced heterotopic ossification. Transl Res 186:95-111
Agarwal, Shailesh; Loder, Shawn J; Breuler, Christopher et al. (2017) Strategic Targeting of Multiple BMP Receptors Prevents Trauma-Induced Heterotopic Ossification. Mol Ther 25:1974-1987
Agarwal, Shailesh; Loder, Shawn; Levi, Benjamin (2017) Heterotopic Ossification Following Upper Extremity Injury. Hand Clin 33:363-373
Agarwal, Shailesh; Lloyd, William R; Loder, Shawn J et al. (2017) Combined reflectance and Raman spectroscopy to assess degree of in vivo angiogenesis after tissue injury. J Surg Res 209:174-177
Agarwal, Shailesh; Loder, Shawn J; Cholok, David et al. (2017) Scleraxis-Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon. Stem Cells 35:705-710
Sorkin, Michael; Agarwal, Shailesh; Ranganathan, Kavitha et al. (2017) Hair follicle specific ACVR1/ALK2 critically affects skin morphogenesis and attenuates wound healing. Wound Repair Regen 25:521-525
Agarwal, Shailesh; Loder, Shawn; Cholok, David et al. (2017) Surgical Excision of Heterotopic Ossification Leads to Re-Emergence of Mesenchymal Stem Cell Populations Responsible for Recurrence. Stem Cells Transl Med 6:799-806

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