INTRODUCTION Neurogenic heterotopic ossifications (NHO) are abnormal development of heterotopic bones in periarticular muscles after spinal cord injury (SCI), traumatic brain injury (TBI), or cerebral stroke. NHO causes joint ankylosis and compromises the quality of life for these patients. Retrospective studies in SCI and TBI patients suggest that infections are a significant risk factor of developing NHO. Our initial experiments show that mesenchymal fibro-adipoprogenitors in the muscles are the cells-of-origin of NHO. We have previously developed a mouse model in which NHO develops in injured hamstrings after SCI. In this model, we investigated whether pathogen-associated molecular patterns (PAMPs) from microbes, have the potential to exacerbate NHO.
METHODS Through osteogenic assays, purified PAMPs were added in cultures of sorted muscle (FAPs), to test the potential of PAMPs to enhance mineralisation, both directly and indirectly via macrophages, which are essential drivers of NHO. After 10-14 days, calcium deposition was measured with alizarin red staining. For in vivo experiments, C57BL/6 mice underwent SCI, and PAMPs were co-administered with cardiotoxin (CDTX) in hamstring muscles. NHO volumes in injured muscles were measured via µCT at day 7-21 post-injury.
RESULTS In vitro, PAMPs such as Pam2CSK4, Pam3CSK4 and Zymosan induced a dose-dependent increase in FAP mineralisation. We also observed that all PAMPs increased the mineralisation potential of FAPs indirectly. Supernatants from bone marrow-derived macrophages cultured in the presence PAMPs could promote FAP mineralisation in vitro. In vivo, co-administration of lipopolysaccharides (LPS) or lipoteichoic acid (LTA) with cardiotoxin significantly increased NHO volumes. Interestingly, PAMPs such as zymosan and gardiquimod significantly decreased NHO formation.
CONCLUSION We have established that PAMPs could increase the mineralization capacity of FAPs. Some bacterial PAMPs exacerbated NHO when injected in the injured muscles. Future investigations are warranted to determine whether intramuscular injection of zymosan and gardiquimod deplete/compromise macrophages in injured muscles.