Background: Bone marrow reticular stromal cells (RSCs), which act as adult skeletal stem cells, are indispensable for injury-responsive bone regeneration and marrow homeostasis. RSCs directly differentiate into regenerative osteoblasts in response to acute injury, contributing to bone repair and subsequently reoccupying the bone marrow as RSCs. However, whether the healed bone marrow environment fully recapitulates the original state and whether RSCs can indefinitely retain their stem cell potential after injury remains unclear.
Methods: We subjected Lepr-Cre;R26RtdTomato and Cxcl12-CreER;R26RtdTomato mice to either a single femoral injury at 13 weeks or two injuries at 9 and 13 weeks. We quantified Col1a1+ osteoblasts, perilipin+ adipocytes, and p16INK4a+ senescent cells by immunohistochemistry. Single-cell RNA sequencing (scRNA-seq) of Lepr+ RSCs and their progeny at various post-injury stages delineated transcriptional changes. Finally, we performed conditional deletion of β-catenin in Lepr+ cells to evaluate Wnt signaling's role in maintaining lineage commitment of RSCs after injury.
Results: Single injury drove robust Col1a1+ osteoblast formation, whereas repeated injuries sharply reduced osteogenesis and increased perilipin+ adipocytes and p16INK4a+ senescent cells. scRNA-seq of Lepr+ cells after repeated injury revealed upregulation of adipogenic (Fabp4, Adipor2), senescence (Cdkn1b, Sesn3), and inflammatory (Nfkbia, Tnfa) genes alongside downregulation of Wnt/β-catenin components (Ctnnb1). Conditional β-catenin deletion in Lepr+ RSCs mimicked exhaustion—further impairing osteogenesis and promoting adipogenesis and senescence even after a single injury.
Conclusions: Recurrent marrow injury drives RSC exhaustion, impairs osteoblast formation, and skews differentiation toward adipocytes and senescent cells. Wnt/β-catenin signaling is crucial for sustaining RSC lineage commitment. Therapeutic modulation of this pathway may rejuvenate RSC function and enhance bone repair, with implications for treating osteoporosis and fracture nonunion.