194. Mesenchymal Progenitor Cell Polyamine Metabolism Promotes Trauma-induced Heterotopic Ossification

PURPOSE: Alterations in metabolic flux dictate cellular differentiation in development and disease. However, little is known about post-injury metabolic shifts and their impact on aberrant wound healing, such as heterotopic ossification (HO). HO is the formation of ectopic bone in muscle and tendon following traumatic injury. Using HO as a model, we aimed to understand how cellular metabolism drives mesenchymal cell differentiation after injury. METHODS: We utilized a reproducible HO model in which mice are subjected to a dorsal burn and concurrent Achilles’ tenotomy. To assess how cellular metabolism influences differentiation, we performed untargeted metabolomics, analyzed single-cell RNA-sequencing (scRNA) from published datasets, conducted immunofluorescence histology, traced metabolites in vitro, and used micro-computed tomography to measure bone volume (p*<0.05,p**<0.01). RESULTS: Pathway analysis of untargeted metabolomics highlighted that “Spermidine and Spermine Metabolism” was significantly upregulated in day seven post-injury tissue compared to uninjured tissue (A, n=5/group, ranked 2nd by p-value and pathway impact score). This pathway, involving the polyamines putrescine, spermidine, and spermine, plays roles in autophagy, DNA replication, and differentiation. However, its mechanism in HO remains unclear, prompting further investigation. Previous studies by our group have shown that mesenchymal progenitor cells (MPCs), which differentiate into chondrocytes and osteoblasts after HO-forming injury, upregulate key polyamine enzymes ornithine decarboxylase (Odc1) and spermidine synthase (Srm) in HO. To determine whether this also occurs in development, we analyzed scRNA-seq data from human and mouse embryonic limb development (Wellcome Sanger Institute), which revealed polyamine enzyme expression in MPCs, osteoblasts, and chondrocytes (not shown). We confirmed ODC and SRM expression in chondrocytes and osteoblasts in post-natal day 2 limb histology, indicating that polyamine flux may be crucial to limb development (not shown). To determine if polyamine metabolism occurs in MPCs, we treated MPCs with arginine containing heavy nitrogen in osteogenic or control media. We detected labeled nitrogen in putrescine, with a higher trend in osteogenic conditions, suggesting that MPCs increase polyamine flux during osteogenesis. Lastly, we assessed polyamine biosynthesis during HO formation in vivo. Using inhibitors of polyamine synthesis enzymes, we observed decreased HO volumes compared to vehicle controls. Conversely, supplementation of spermidine in drinking water increased HO formation and rescued the HO phenotype in mice treated with a polyamine inhibitor. Additionally, genetic deletion of Odc1 via adenovirus-cre injection to the injury site in Odc1 flox mice reduced HO formation. These findings identify polyamine metabolism as essential to HO formation. CONCLUSION: This study reveals a previously uncharacterized metabolic pathway in HO and suggests that polyamine metabolism plays a critical role in bone and limb development. Targeting this pathway may prevent HO and have implications for other bone-related conditions, such as fracture healing or osteoporosis. Given that polyamines are also present in the diet, therapeutic dietary interventions could improve healing after injury or surgery.