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Bone is a unique organ able to regenerate itself after injuries. This regeneration requires the local interplay between different biological systems such as inflammation and matrix formation. Structural reconstitution is initiated by an inflammatory response orchestrated by the host immune system. However, the individual role of T cells and B cells in regeneration and their relationship to bone tissue reconstitution remain unknown. Comparing bone and fracture healing in animals with and without mature T and B cells revealed the essential role of these immune cells in determining the tissue mineralization and thus the bone quality. Bone without mature T and B cells is stiffer when compared to wild-type bone thus lacking the elasticity that helps to absorb forces, thus preventing fractures. In-depth analysis showed dysregulations in collagen deposition and osteoblast distribution upon lack of mature T and B cells. These changes in matrix deposition have been correlated with T cells rather than B cells within this study. This work presents, for the first time, a direct link between immune cells and matrix formation during bone healing after fracture. It illustrates specifically the role of T cells in the collagen organization process and the lack thereof in the absence of T cells.

Background and objectivesThe critical interdependency between the skeletal and immune system has been demonstrated for the fracture healing process. However, the current state of the art is contradictory. On one hand, lymphopenic mice display alleged better healing after injury. On the other hand, several studies attribute either a positive or negative role to T and B cells in bone biology, autoimmunity and fracture healing. The objective of this study is to elucidate the specific role of T and B cells in fracture repair toward early callus mineralization, bone quality, migration of osteoblast precursors and Collagen I deposition.Materials and methodsUnilateral closed femoral fractures were produced in RAG1-/-, TCR βδ chain -/-, JHT -/- and C57BL/6 wild-type mice. Bone healing was assessed by histology, biomechanical testing and fluorochrome deposition. Deposition and structural properties of CollagenI were studied by second harmonic and confocal microscopy. Callus gene expression was determined by microarray analysis. Osteoblast precursors, T and B cell migration in the callus were visualised by confocal microscopy.ResultsFractured bones of RAG -/- mice mineralized faster, but were much less capable to withstand deformation. Similarly, RAG -/- mice deposited bone quicker during the earliest phases of healing, whereas at later stages underperformed relative to controls. RAG -/- mice also failed to deposit a cross gap layer of Collagen I which correlated with diminished immigration of osteoblast precursors in the peripheral callus. The remaining network of Collagen I fibres in these mice was found to be highly disordered. Interestingly, no alteration in osteoblast precursor migration was observed when only either T or B cells were  absent. However, Collagen I deposition was strongly impaired in T cell but not in B cell deficient mice. There, Collagen I fibres failed to organise across the gap in the peripheral callus displaying a highly disordered pattern of deposition.ConclusionsOverall our data show that lack of an adaptive immune system does not lead to better, but rather deregulated and suboptimal bone healing. These features were recapitulated in mice that specifically lacked T and not B cells.

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Background and objectives Fracture healing is a regenerative process and is one of the two processes in the human body in which an organ is restored without scar tissue formation. The healing cascade initiates with a cycle of inflammation, cell migration, proliferation and differentiation. Immune cells invade the fracture site immediately upon bone damage and contribute to the initial phase of the healing process and appear to recruit accessory cells to the injury site. However, little is known about the role of the immune system in the later stages of fracture repair, in particular, whether lymphocytes participate in soft and hard callus formation. Materials and methodsIn order to answer this question, we analysed femoral fracture healing in mice by confocal microscopy, flow cytometry and molecular biology. Results Upon fracture, we could detect generalised increased frequencies of T cells in the bone marrow of injured mice which persisted throughout healing. By closely analysing the fractured bone, we found that surprisingly, after the initial inflammatory phase, when soft callus developed, T and B cells withdrew from the fracture site. Thereafter lymphocytes massively infiltrated the callus region (around day 14 after injury), during callus mineralisation. Interestingly, lymphocytes were not found within cartilaginous areas of the callus but only nearby newly forming bone. During healing, B cell numbers seemed to exceed those of T cells and B cells progressively underwent effector maturation associated with high OPG production capacity. During bone regeneration, both osteoblasts and osteoclasts were found to undergo direct cell-cell contact with lymphocytes. The latter were also found to secrete OPG in the callus and their appearance also correlated with a progressive increase in the ratio of OPG vs. RANKL gene expression during fracture repair. Conclusions Our data strongly suggests a regulatory role of T and B cells not only in the initial phase of healing but specifically also in the later stages of fracture remodelling.

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