Directed vascularization in bone regeneration requires bone marrow reconstitution

Bone has the unique ability to regenerate without scarring, yet the cellular dynamics and directional organization underlying this process, and how they are linked to marrow reconstitution, remain incompletely understood. To investigate these mechanisms, we developed a novel double osteotomy model in mice, involving transplantation of a 2-mm bone graft between genetically distinct fluorescent reporter lines (yellow fluorescent protein and red fluorescent protein). This approach enabled precise tracking of cellular migration and vascularization over 3, 7, and 14 d post-transplantation. Our findings revealed directed migration of host-derived proximal bone marrow cells into the graft, starting at day 3 and leading to complete host cell infiltration by day 14. CD146-positive blood vessels, mainly originating from the proximal host marrow, invaded the graft coinciding with graft marrow remodeling. Marrow disintegration within the graft occurred prior to vascular and cellular invasion, with subsequent reconstitution progressing from the proximal side. Flushing the graft marrow cavity prior to transplantation resulted in more extensive marrow niche formation by day 14 suggesting that marrow reconstitution can proceed more rapidly without the need for prior remodeling. This study introduces a new model to dissect the spatial and temporal coordination of cellular migration and vessel invasion during bone regeneration. Our results uncover the directional nature of healing and underscore the critical role of marrow reconstitution in guiding regenerative processes-insights that may inform surgical and biomaterial strategies to enhance bone repair.