Bone regeneration is influenced by systemic factors like aging and by local mechanical conditions such as fixation stability. Aging disrupts adaptive immunity, often impairing healing and causing delayed or non-union fractures. While previous research has examined age-related changes in T cells and their effect on bone healing, the role of aging on B cell function in this context remains poorly characterized. Similarly, although mechanical stability is known to be critical during the early inflammatory phase, its influence on the later mineralization stage of bone healing is still unclear. In the first part of this study, we investigated how immune aging affects B cell function in early bone regeneration. To investigate how immune experience affects B cell behavior during bone healing, we compared young, non-immune-experienced (NE), and immune-experienced (IE) mice. Through single-cell proteo-genomic profiling, we examined thousands of transcriptomes derived from the hematoma and neighboring bone marrow regions following osteotomy. In both young and NE mice, the proportion of B cells within the hematoma remained largely unchanged between days 2 and 5 post-osteotomy. In contrast, IE mice showed a pronounced reduction in B cell proportion and a concurrent decrease of B cell–associated genes, suggesting a selective impairment in B cell function. Trajectory analysis further revealed that B cell differentiation, particularly the shift from immature to mature B cell states, was notably impaired in IE animals compared with NE and young mice. These findings suggest that immune experience—and, by extension, immune aging—disrupts effective B cell-mediated responses during the early stages of bone healing, thereby contributing to the delayed healing outcomes observed in IE mice. In the second part, we examined the impact of mechanical fixation on bone matrix mineralization. Using a 2D–3D image registration approach, we integrated histological (collagen and Osterix signals) and µCT data to visualize spatial relationships between collagen deposition and hydroxyapatite mineralization. We compared bone healing under rigid versus semi-rigid fixation. Under a mechanical, more stable, and rigid fixation (which leads to fast healing), mineralization occurred closer to collagen bundles, indicating more efficient matrix mineralization. In contrast, semi-rigid fixation showed delayed mineralization and greater spatial separation between collagen and mineral. This suggests that mechanical stability directly influences the efficiency and spatial coordination of bone matrix mineralization. Together, these findings highlight two drivers of impaired bone healing: B cell dysfunction related to immune experience during early inflammation, and diminished collagen mineralization under suboptimal fixation. Addressing each mechanism could help improve strategies for promoting effective bone regeneration.
Mireille Ngokingha Tchouto (Thu,) studied this question.
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