gms | German Medical Science

Objectives: The goal of regenerative therapy approaches is to fully restore structural integrity to the pre-injury state. Bone is one of the few organs capable of scar-free regeneration. However, the source of this regenerative ability remains elusive. This study aimed to elucidate the regenerative directionality using a murine bone graft transplantation model. Fluorochrome cell marker staining was used to evaluate cell migration, vascularization, collagen organization, and bone marrow remodeling. By using graft transplantation, this research allowed for the simultaneous analysis of two spatially distinct bone healing processes, one proximal and one distal, within the same animal.

Methods: Black 6 mice expressing either ubiquitous red fluorescent protein (RFP) or yellow fluorescent protein (YFP) were matched so that each chimeric mouse could serve as either donor or recipient. Here, RFP-labeled 2 mm pieces of bone shaft were cut with a wire saw and then transplanted into YFP mice. This strategy allowed simultaneous tracking and analysis of RFP-labeled cells during bone regeneration at both the proximal and distal osteotomy sites. A distraction system was used to stabilize both the osteotomies and the bone graft. Healing was evaluated at 3, 7, and 14 days. Flushed bone shaft pieces without intact marrow graft served as controls. Cryosections were stained with CD146 to identify microvascular structures, and second harmonic generation (SHG) imaging was used to identify thick collagen bundles at each time point.

Results and conclusion: After transplantation, host cells from the bone marrow migrated into the bone graft from the proximal areas at both osteotomy sites, proximal and distal. By day 7, these host cells had infiltrated approximately half of the graft, and by day 14, they had completely infiltrated the entire graft, predominantly from the proximal direction. Notably, there was a reduction in bone marrow cell density anterior to the invasion zone, suggesting apoptosis of resident bone marrow graft cells and subsequent bone marrow remodeling. Vascular sprouts originated from the proximal host bone marrow and infiltrated the graft, resulting in complete vascularization by day 14. In the control group with flushed bone marrow, vascularization within the transplanted bone was increased compared to the non-flushed group. Our study elucidates the distinct proximal-to-distal invasion pattern of regenerating cells and vasculature in a scar-free bone regeneration model. These findings provide insights that may improve strategies for efficient and targeted scar-free bone regeneration and shed light on endogenous healing mechanisms that hold promise for clinical applications.