Use Of Bmp Type I Alk-3 Knockout Mice To Develop Novel Treatment Strategies For Trauma-induced Heterotopic Ossification
Benjamin Levi, MD, Jonathan Peterson, MD, Shailesh Agarwal, MD, Eboda Oluwatobi, MD, Kavitha Ranganathan, MD, Yuji Mishina, PhD, Steven R. Buchman, MD, Stewart C. Wang, MD, PhD, Paul S. Cederna, MD.
University of Michigan, Ann Arbor, MI, USA.
Heterotopic ossification (HO) is a debilitating process characterized by the formation of ectopic bone following trauma, burn injury, and amputations. HO formation in trauma and burn patients is poorly understood at a mechanistic level, contributing to the lack of therapeutic options. In this study, we demonstrate the role of BMP type I receptor Alk3 in HO development and target downstream Alk3 signaling with a novel small molecule (LDN-193189) to prevent HO.
Conditional Alk3 knockout (KO) mice were engineered using a C57BL/6 background; these mice are no longer able to express Alk3 upon exposure to the AdCre adenovirus. Mesenchymal stem cells
(MSCs) from bone marrow and adipose tissue were harvested from these mice (N=4/group). These MSCs were then exposed to AdCre or AdLac (control virus) with subsequent assessment of
osteogenic differentiation using qRT-PCR. A separate set of wild type and conditional Alk3 KO mice underwent Achilles tenotomy with 30% total body surface area partial thickness burn (N=4/group).
Mice underwent injection of AdCre or AdLac (control) at the tenotomy site. An additional set of wild type mice underwent injection of the small molecule LDN-193189, a known Alk3 pathway inhibitor.
Serial micro-CT scans were completed 1-9 weeks post injury and HO volume was assessed. Functional impairment was assessed by ankle range of motion (ROM).
AdCre-mediated knockout of Alk3 resulted in significantly decreased osteogenic capacity of MSCs as demonstrated by reduced alkaline phosphatase (ALP) stain, ALP enzymatic activity, and a three fold decrease in osteoid deposition by alizarin red stain (p<0.05). In vivo ectopic bone formation was significantly reduced after 9 weeks in ALK-3 KO mice which received the AdCre injection at the tenotomy site by μCT scan (Fig. 1) (n=4, p<0.05). Ankle range of motion was similarly significantly enhanced in mice with introduction of AdCre in ALK-3 KO mice (Fig. 2; p<0.05). Small molecule
inhibition of downstream Alk-3 signaling with a novel small molecule LDN-193189 resulted in a dramatic decrease in osteogenic differentiation of MSCs in vitro and abrogated HO formation and ROM in vivo (n=5; p<0.05).
Alk3a plays a vital role in the osteogenic response to burn injury with respect to MSC osteogenic differentiation and HO development. Introduction of AdCre in conditional KO Alk3 mice reduces HO and joint contractures. Novel small molecule inhibitors such as LDN-193189 may be used to inhibit Alk3 signaling and prevent the formation of HO in patients in the future.
Back to Program