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

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Optimizing timing and targeting of Bone Morphogenetic Protein Receptor One to Treat Heterotopic Ossification with novel kinase inhibitors and ligand traps
John Li, MD1, Shailesh Agarwal, MD1, Shawn Loder, BS1, Cameron Brownley, BS1, Jonathan Peterson, BS1, Kavitha Ranganathan, MD1, Paul S. Cederna, MD1, Eboda Oluwatobi, BS1, Agustin Mohedas, PhD2, Paul Yu, MD2, Benjamin Levi, MD1.
1University of Michigan, Ann Arbor, MI, USA, 2Brigham and Womens Hospital, Boston, MA, USA.

PURPOSE:Aberrant formation of ectopic bone in soft tissues is a sequela of major burns, pressure ulcers and blast injuries leading to disability and impaired quality of life. This process, called heterotopic ossification (HO), is a challenge in the clinical setting with no definitive medical or surgical options for prevention or treatment. Post-traumatic heterotopic ossification relies on bone morphogenetic protein (BMP)-SMAD1/5 signaling. Small molecule inhibitors have been developed to target either Alk2/3 nonspecifically (LDN-193189) or to specifically inhibit Alk2 (LDN-212854). LDN 19 has been shown to decrease HO formation when given continuously, however, such non-specific treatments have a significant toxicity profile. We set out to determine if pulsed dosing of Alk2 specific inhibitor (LDN-21) would mitigate HO formation.
METHODS: Eight week-old male C57BL/6J mice received a dorsal 30% total body surface area (TBSA) partial thickness burn with Achilles tenotomy. Mice then were treated with daily intraperitoneal injection of either LDN-19, LDN-21, or saline in the following groups: LDN-19 (weeks 0-9), LDN-21 (weeks 0-9), LDN-21 pulse weeks 0-2, LDN-21 weeks 2-4, LDN-21 weeks 4-6, and control saline injection (n=6 per group). µCT and histologic analysis were performed 5 and 9 weeks post injury. Histologic analysis included pentachrome, TRAP (osteoclastogenesis) and immunohistochemistry for BMP signaling (smad 5; psmad 1/5). HO volume and thickness was quantified by µCT. Separately, mouse and human HO-derived osteoblasts and control normal osteoblasts from the same patient and non-HO control patients were cultured in vitro with osteogenic differentiation medium (ODM) with either a) no LDN; b) LDN (19 or 21) treatment for days 1-3; or c) LDN (19 or 21) treatment for days 4-6. Alkaline phosphatase and alizarin red staining and quantification were performed to assess osteogenic differentiation.
RESULTS:In-vivo µCT at 9 weeks post-trauma demonstrated that mean HO volume was significantly less when LDN-21 was administered between 2-4 or 4-6 weeks compared to those treated 0-2 weeks or untreated controls (p<0.05; Fig. 1). Compared to untreated controls 4-6 weeks treatment groups had increased osteoclastogenesis. In vitro, osteogenic differentiation studies demonstrate that pulsed dosing of LDN-19 or LDN-21 at either 1-3 or 4-6 days post initiation of differentiation demonstrated decreased mineralization versus untreated controls in human and mouse derived HO osteoblasts by alkaline phosphatase and alizarin red staining and quantification (p<0.05).
CONCLUSION:
Targeted Alk2 inhibition with LDN-21 administered for a brief pulse interval significantly mitigates HO formation in vivo. Delayed pulse dosing of LDN-21 between 2-4 and 4-6 weeks demonstrates equivalent effect versus continuous dosing. Interestingly, delayed dosing led to resorption of the HO. Immediate pulse dosing between 0-2 weeks is insufficient to prevent HO at 5- or 9- weeks. LDN-19 and LDN-21 pulse treatments also decreases osteogenic differentiation of human and mouse heterotopic ossification derived osteoblasts. The efficacy of this novel LDN-21 compound in our mouse HO model and on human cells demonstrates its potential translation to treat patients at risk for HO.


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