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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]

To present knowledge, macrophages are found in all tissues of the human body. They are a cell population with high plasticity which come with a multitude of functions which appear to be adapted to the respective tissue niche and micro-environment in which they reside. Bone harbors multiple macrophage subpopulations, with the osteoclasts as classical representative of a bone resorbing cells and osteomacs as a bone tissue resident macrophage first described by the expression of F4/80. Both subtypes are found throughout all phases in bone healing. In vivo data on bone regeneration have demonstrated their essential role in initiating the healing cascade (inflammatory phase) but also of the later phases of healing (e.g. endochondral and intramembranous bone formation). To participate in such diverse processes macrophages have to be highly plastic in their functionality. Thus, the widely used M1/M2 paradigm to distinguish macrophage subpopulations may not mirror the comprehensive role of the dynamics of macrophage plasticity. From a clinical perspective it is especially relevant to distinguish what drives macrophages in impaired healing scenarios, implant loosening or infections, where their specific role of a misbalanced inflammatory setting is so far only partially known. With this review we aim at illustrating current knowledge and gaps of knowledge on macrophage plasticity and function during the cascades of regeneration and reconstitution of bone tissue. We propose aspects of the known biological mechanisms of macrophages and their specific subsets that might serve as targets to control their function in impaired healing and eventually support a scar-free regeneration. Macrophages are essential for successful regeneration. In scar-free healing such as in bone, a complete failure of healing was shown if macrophages were depleted; the M1/M2 switch appears to be key to the progression from pro-inflammation to regeneration. However, experimental data illustrate that the classical M1/M2 paradigm does not completely mirror the complexity of observed macrophage functions during bone healing and thus demands a broader perspective. Within this review we discuss the high degree of plasticity of macrophages and the relevant contribution of the different and more specific M2 subtypes (M2a-M2f) during (bone) regeneration. It summarizes the versatile roles of macrophages in skeletal regeneration and thereby highlights potential target points for immunomodulatory approaches to enable or even foster bone repair. [Display omitted] [ABSTRACT FROM AUTHOR]

The interaction of hematopoietic cells and the bone microenvironment to maintain bone homeostasis is increasingly appreciated. We hypothesized that the transfer of allogeneic T lymphocytes has extensive effects on bone biology and investigated trabecular and cortical bone structures, the osteoblast reconstitution, and the bone vasculature in experimental hematopoietic stem cell transplantations (HSCT). Allogeneic or syngeneic hematopoietic stem cells (HSC) and allogeneic T lymphocytes were isolated and transferred in a murine model. After 20, 40, and 60 days, bone structures were visualized using microCT and histology. Immune cells were monitored using flow cytometry and bone vessels, bone cells and immune cells were fluorescently stained and visualized. Remodeling of the bone substance, the bone vasculature and bone cell subsets were found to occur as early as day +20 after allogeneic HSCT (including allogeneic T lymphocytes) but not after syngeneic HSCT. We discovered that allogeneic HSCT (including allogeneic T lymphocytes) results in a transient increase of trabecular bone number and bone vessel density. This was paralleled by a cortical thinning as well as disruptive osteoblast lining and loss of B lymphocytes. In summary, our data demonstrate that the adoptive transfer of allogeneic HSCs and allogeneic T lymphocytes can induce profound structural and spatial changes of bone tissue homeostasis as well as bone marrow cell composition, underlining the importance of the adaptive immune system for maintaining a balanced bone biology. [ABSTRACT FROM AUTHOR]