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Background: The healing of a bone injury is a highly complex process involving a multitude of different tissue and cell types, including immune cells, which play a major role in the initiation and progression of bone regeneration. Methods: We histologically analyzed the spatio-temporal occurrence of cells of the innate immune system (macrophages), the adaptive immune system (B and T lymphocytes), and bone cells (osteoblasts and osteoclasts) in the fracture area of a femoral osteotomy over the healing time. This study was performed in a bone osteotomy gap mouse model. We also investigated two key challenges of successful bone regeneration: hypoxia and revascularization. Results: Macrophages were present in and around the fracture gap throughout the entire healing period. The switch from initially pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype coincided with the revascularization as well as the appearance of osteoblasts in the fracture area. This indicates that M2 macrophages are necessary for the restoration of vessels and that they also play an orchestrating role in osteoblastogenesis during bone healing. The presence of adaptive immune cells throughout the healing process emphasizes their essential role for regenerative processes that exceeds a mere pathogen defense. B and T cells co-localize consistently with bone cells throughout the healing process, consolidating their crucial role in guiding bone formation. These histological data provide, for the first time, comprehensive information about the complex interrelationships of the cellular network during the entire bone healing process in one standardized set up. With this, an overall picture of the spatio-temporal interplay of cellular key players in a bone healing scenario has been created. Conclusions: A spatio-temporal distribution of immune cells, bone cells, and factors driving bone healing at time points that are decisive for this process—especially during the initial steps of inflammation and revascularization, as well as the soft and hard callus phases—has been visualized. The results show that the bone healing cascade does not consist of five distinct, consecutive phases but is a rather complex interrelated and continuous process of events, especially at the onset of healing. [ABSTRACT FROM AUTHOR]

Despite significant advances in surgical techniques, treatment options for impaired bone healing are still limited. Inadequate bone regeneration is not only associated with pain, prolonged immobilization and often multiple revision surgeries, but also with high socioeconomic costs, underlining the importance of a detailed understanding of the bone healing process. In this regard, we previously showed that mice lacking the calcitonin receptor (CTR) display increased bone formation mediated through the increased osteoclastic secretion of sphingosine-1-phosphate (S1P), an osteoanabolic molecule promoting osteoblast function. Although strong evidence is now available for the crucial role of osteoclast-to-osteoblast coupling in normal bone hemostasis, the relevance of this paracrine crosstalk during bone regeneration is unknown. Therefore, our study was designed to test whether increased osteoclast-to-osteoblast coupling, as observed in CTR-deficient mice, may positively affect bone repair. In a standardized femoral osteotomy model, global CTR-deficient mice displayed no alteration in radiologic callus parameters. Likewise, static histomorphometry demonstrated moderate impairment of callus microstructure and normal osseous bridging of osteotomy ends. In conclusion, bone regeneration is not accelerated in CTR-deficient mice, and contrary to its osteoanabolic action in normal bone turnover, osteoclast-to-osteoblast coupling specifically involving the CTR-S1P axis, may only be of minor relevance during bone healing. [ABSTRACT FROM AUTHOR]