*2.6.4. CD8 + TEMRA cells*

the prominent macrophage phenotype during the initial phase is M1. Upon attenuating of the proinflammatory phase, the macrophage phenotype changes towards the M2 phenotype [77]. In a proof of concept study in mice, we were able to show that an induction of the M2 phenotype

The T cell population is highly divers and probably pleiotropic as well as interchangeable. Among the T cells, there seem to be subpopulations supporting the fracture healing process and also other subpopulations, which have negative effects on the healing process. CD4+ and CD8+ T cell subsets have been addressed in this context. CD4+ T cells have been shown to increase osteogenic differentiation in human mesenchymal stem cell cultures in *in vitro* assays using their conditioned medium, whereas this effect was missing when observing CD8+ T cells [115]. The osteogenic effect of CD4+ T cells was further supported through their positive effects during wound healing [116], however without a more specific determination of the responsible CD4+ T cell subset. In later studies, regulatory T cells came more and more into the focus as a CD4+ T cell subset with positive effects on bone healing. Mice with an increased percentage of regulatory T cells showed higher bone mass and decreased bone resorption when compared to wild type mice [117, 118]. Regulatory T cells support osteoblast differentiation and have a negative impact on osteoclast differentiation and function [119]. In a skull defect model in mice, it was possible to enhance bone healing through the addition of regulatory T cells in combi‐ nation with applied autologous bone graft [120]. Currently under investigation is the possi‐ bility of a direct interaction of regulatory T cells and bone-forming cells or their progenitor cells, the mesenchymal stromal/stem cells. This interaction is supported by the fact that mesenchymal stromal/stem cells, as osteoblast precursors, and regulatory T cells use similar suppression mechanisms for an immune response [121]. The direct interaction between regulatory T cells and bone-forming cells as well as mesenchymal stromal/stem cells could proceed through coordination of the CD39-CD73-(adenosine)-ADOR pathway. This puriner‐ gic signaling would potentiate the differentiation of mesenchymal stromal/stem cells and thus facilitate bone regeneration [122]. Another direct interaction between osteoblasts and regula‐ tory T cells could be the induction of IDO (indoleamine 2,3-dioxygenase) and HO-1 (heme oxygenase-1) by regulatory T cells [123] or the fact that regulatory T cells can inhibit CD40L and thus regulating the RANKL-OPG balance in favor of osteoblast differentiation [124].

The lead cytokine expressed by Th17 (T helper 17) is IL-17. The dual effect of IL-17 on osteoclasts and osteoblasts has been mentioned before. However, these cells are of interest as novel therapeutics targeting IL-12, IL-23, IL-17, and IL-17 receptor and which are now used to successfully treat psoriasis by either repressing Th17 differentiation (IL-12/IL-23) or by directly targeting IL-17. Psoriasis has two manifestations, one in skin (psoriasis vulgaris) and one in bone (psoriasis arthritis), and the immune modulatory treatment shows positive results in both [125]. Th-17 cell differentiation is induced by IL-1β, IL-6 and TGF-β [126, 127], with TGF-β

early in the fracture healing cascade can enhance bone healing [77].

*2.6.2. Regulatory T cells*

182 Advanced Techniques in Bone Regeneration

*2.6.3. T helper 17 cells*

A direct crosstalk between activated T cells and bone-forming cells can be assumed during the healing process. Among these T cells, CD8+ TEMRA cells were confirmed to have a negative effect on the bone regenerative process. High expression levels of TNF-α and interferon-γ (IFN-γ) of CD8+ T cells decreased the osteogenic differentiation capacity *in vitro* [91]. CD8+ TEMRA cells can be triggered to express these cytokines without antigen-presenting cells and do not necessarily need costimulatory molecules like CD80/86-CD28 but are activated by bystander responsiveness [131–133]. These cells accumulate in the fracture hematoma due to their tissue homing qualities and they occur in higher numbers in patients experiencing a delayed healing [91]. In the clinical setting, the recognition of a delayed or missing bone healing is so far only possible when these healing disturbances become visible in X-ray or computed tomography evaluations of the fractured bone. An early identification of patients at risk of a delayed or disturbed fracture healing is still missing. CD8+ TEMRA cells could proof to be a marker for delayed healing risk in patients, since these cells also show elevated values in peripheral blood. Predicting patients with an extended need for special fracture treatment could thus just be done by analyzing the CD8+ TEMRA percentage in peripheral blood early on in the healing process.

#### *2.6.5. Outlook*

Not only the interaction of the skeletal and immune system in fracture healing is not well understood so far, the immune reaction in itself is also still not unraveled. Aside from the complexity of the cytokine pattern guiding the regenerative process, the plasticity of the immune cells is still a vast challenge: M1 macrophage phenotype changing towards M2, Th1 changing towards Th2 response, regulatory T cells changing into Th17 cells and vice versa, to mention only a few aspects that still have to be understood. First approaches have been successful in influencing the fracture treatment through immune modulation (NSAIDs or IL-23 neutralization antibodies) but the possibilities are far from being exploited. A stratification of patients can help to decide, which treatment is optimal for which patient, especially with respect to the current immune status of these patients. With the numbers of delayed healing fracture patients still vastly unknown and possibly massively underestimated, and the demographic prognostic of a substantial increase in the elderly population during the next years, the need for further treatment options is rising together with the necessity of enhanced basic research in the field of osteoimmunology.
