MECHANORESPONSIVE PERIOSTEAL STEM CELLS IN DISTRACTION OSTEOGENESIS

Recent studies have identified periosteal stem cells (PSCs) as the main contributors to bone repair; however, the mechanisms by which PSCs respond to mechanical stimulation in distraction osteogenesis (DO) remain unclear. This study aimed to elucidate how PSCs respond to tensile strain and promote bone regeneration, providing new insights into the role of mechanical stimulation in osteogenesis. To identify the contribution of different progenitor cells in DO, we compared the therapeutic effect of local injection of PSCs or bone marrow-derived mesenchymal stem cells (BMSCs) in the bone lengthening model with periosteum defect at the regenerate sites. We performed an RNA-seq assay followed by qRT-PCR using the DO callus isolated from the affected femur, or PSCs and BMSCs from contralateral intact femur. In addition, single-cell RNA sequencing of the DO callus and bone defect models was employed to explore the mechanoresponses of PSCs. To validate these findings, an ex vivo mechanical stretching was conducted to assess the mechanoresponse of PSCs in DO. We further investigated the role of PI3K-AKT in bone healing outcomes in both bone lengthening model and bone transport model through applying PI3K activator or inhibitor at the regenerate sites. We identified PSCs played a predominant role in DO callus formation compared to BMSCs, with a significant enrichment of the mechanotransductive PI3K/AKT pathway. Inhibition of Akt1 resulted in delayed union, whereas PI3K activation enhanced bone consolidation and increased the expression of PSC markers. Mechanistic studies further revealed an embryonic-like skeletal development phenomenon at the regenerative site. We successfully identified a subgroup of PSCs in DO highly expressing genes related to embryonic skeletal development. In vitro studies confirmed the upregulation of stemness markers, such as Grem-1, along with activation of the Wnt signaling pathway in PSCs under tensile stress stimulation. PSC has been identified as a mechanoresponsive cell type indispensable for callus formation in DO. Activating the PI3K pathway in PSCs has emerged as a promising therapeutic strategy to enhance bone regeneration and mitigate nonunion. Notably, the DO regenerate regressed to an embryonic skeletal growth state, with a specific subgroup of PSCs acquiring an embryonic skeletal development signature. In conclusion, these findings provide novel insights into PSCs as the primary mechanoresponders in DO, elucidating the cellular mechanisms by which mechanical stimulation promotes osteogenesis and offering cues for reprogramming PSCs through external mechanical stimulation. Acknowledgements: This work was supported by the RGC HK (14119124 & 14113723).