Postmenopausal osteoporosis is a systemic disease characterized by the loss of bone mass and increased bone fracture risk largely resulting from significantly reduced levels of the hormone estrogen after menopause. Besides the direct negative effects of estrogen-deficiency on bone, indirect effects of altered immune status in postmenopausal women might contribute to ongoing bone destruction, as postmenopausal women often display a chronic low-grade inflammatory phenotype with altered cytokine expression and immune cell profile. In this context, it was previously shown that various immune cells interact with osteoblasts and osteoclasts either via direct cell-cell contact, or more likely via paracrine mechanisms. For example, specific subtypes of T lymphocytes express TNFα, which was shown to increase osteoblast apoptosis and to indirectly stimulate osteoclastogenesis via B cell-produced receptor-activator of NF-κB ligand (RANKL), thereby triggering bone loss during postmenopausal osteoporosis. T_h17 cells release interleukin-17 (IL-17), which directs mesenchymal stem cell differentiation towards the osteogenic lineage, but also indirectly increases osteoclast differentiation. B lymphocytes are a major regulator of osteoclast formation via granulocyte colony-stimulating factor secretion and the RANKL/osteoprotegerin system under estrogen-deficient conditions. Macrophages might act differently on bone cells dependent on their polarization profile and their secreted paracrine factors, which might have implications for the development of postmenopausal osteoporosis, because macrophage polarization is altered during disease progression. Likewise, neutrophils play an important role during bone homeostasis, but their over-activation under estrogen-deficient conditions contributes to osteoblast apoptosis via the release of reactive oxygen species and increased osteoclastogenesis via RANKL signaling. Furthermore, mast cells might be involved in the development of postmenopausal osteoporosis, because they store high levels of osteoclastic mediators, including IL-6 and RANKL, in their granules and their numbers are greatly increased in osteoporotic bone. Additionally, bone fracture healing is altered under estrogen-deficient conditions with the increased presence of pro-inflammatory cytokines, including IL-6 and Midkine, which might contribute to healing disturbances. Consequently, in addition to the direct negative influence of estrogen-deficiency on bone, immune cell alterations contribute to the pathogenesis of postmenopausal osteoporosis.

The skeleton is a dynamic tissue continuously adapting to mechanical stimuli. Although matrix‐embedded osteocytes are considered as the key mechanoresponsive bone cells, all other skeletal cell types are principally exposed to macroenvironmental and microenvironmental mechanical influences that could potentially affect their activities. It was recently reported that Piezo1, one of the two mechanically activated ion channels of the Piezo family, functions as a mechanosensor in osteoblasts and osteocytes. Here we show that Piezo1 additionally plays a critical role in the process of endochondral bone formation. More specifically, by targeted deletion of Piezo1 or Piezo2 in either osteoblast (Runx2Cre) or osteoclast lineage cells (Lyz2Cre), we observed severe osteoporosis with numerous spontaneous fractures specifically in Piezo1^Runx2Cre mice. This phenotype developed at an early postnatal stage and primarily affected the formation of the secondary spongiosa. The presumptive Piezo1^Runx2Cre osteoblasts in this region displayed an unusual flattened appearance and were positive for type X collagen. Moreover, transcriptome analyses of primary osteoblasts identified an unexpected induction of chondrocyte‐related genes in Piezo1^Runx2Cre cultures. Because Runx2 is not only expressed in osteoblast progenitor cells, but also in prehypertrophic chondrocytes, these data suggested that Piezo1 functions in growth plate chondrocytes to ensure trabecular bone formation in the process of endochondral ossification. To confirm this hypothesis, we generated mice with Piezo1 deletion in chondrocytes (Col2a1Cre). These mice essentially recapitulated the phenotype of Piezo1^Runx2Cre animals, because they displayed early‐onset osteoporosis with multiple fractures, as well as impaired formation of the secondary spongiosa with abnormal osteoblast morphology. Our data identify a previously unrecognized key function of Piezo1 in endochondral ossification, which, together with its role in bone remodeling, suggests that Piezo1 represents an attractive target for the treatment of skeletal disorders. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Mast cells (MCs) are important sensor and effector cells of the immune system that are involved in many physiological and pathological conditions. Increasing evidence suggests that they also play an important role in bone metabolism and bone disorders. MCs are located in the bone marrow and secrete a wide spectrum of mediators, which can be rapidly released upon activation of mature MCs following their differentiation in mucosal or connective tissues. Many of these mediators can exert osteocatabolic effects by promoting osteoclast formation [e.g., histamine, tumor necrosis factor (TNF), interleukin-6 (IL-6)] and/or by inhibiting osteoblast activity (e.g., IL-1, TNF). By contrast, MCs could potentially act in an osteoprotective manner by stimulating osteoblasts (e.g., transforming growth factor-β) or reducing osteoclastogenesis (e.g., IL-12, interferon-γ). Experimental studies investigating MC functions in physiological bone turnover using MC-deficient mouse lines give contradictory results, reporting delayed or increased bone turnover or no influence depending on the mouse model used. By contrast, the involvement of MCs in various pathological conditions affecting bone is evident. MCs may contribute to the pathogenesis of primary and secondary osteoporosis as well as inflammatory disorders, including rheumatoid arthritis and osteoarthritis, because increased numbers of MCs were found in patients suffering from these diseases. The clinical observations could be largely confirmed in experimental studies using MC-deficient mouse models, which also provide mechanistic insights. MCs also regulate bone healing after fracture by influencing the inflammatory response toward the fracture, vascularization, bone formation, and callus remodeling by osteoclasts. This review summarizes the current view and understanding of the role of MCs on bone in both physiological and pathological conditions.

Postmenopausal females display a chronic inflammatory phenotype with higher levels of circulating pro-inflammatory cytokines. Furthermore, the inflammatory response to injury may be altered under estrogen-deficiency, because it was shown previously that estrogen-deficient mice displayed increased levels of the inflammatory cytokines Midkine (Mdk) and Interleukin-6 (IL-6) in the early fracture hematoma. Because a balanced immune response to fracture is required for successful bone regeneration, this might contribute to the delayed fracture healing frequently observed in osteoporotic, postmenopausal fracture patients. In this study, we aimed to investigate whether further cytokines in addition to Mdk and IL-6 might be affected by estrogen-deficiency after fracture in mice and whether these cytokines are also relevant during human fracture healing. Additionally, we aimed to investigate whether serum from male vs. female fracture patients affects osteogenic differentiation of human mesenchymal stem cells (MSCs). To address these questions, female mice were either sham-operated or ovariectomized (OVX) and subjected to standardized femur osteotomy. A broad panel of pro- and anti-inflammatory cytokines was determined systemically and locally in the fracture hematoma. In a translational approach, serum was collected from healthy controls and patients with an isolated fracture. Mdk and IL-6 serum levels were determined at day 0, day 14 and day 42 after fracture. Subgroup analysis was performed to investigate differences between male and female fracture patients after menopause. In an in vitro approach, human MSCs were cultured with the collected patient serum and osteogenic differentiation was assessed by qPCR and alkaline-phosphatase staining. Our results suggest an important role for the pro-inflammatory cytokines Mdk and IL-6 in the response to fracture in estrogen-deficient mice among all of the measured inflammatory mediators. Notably, both cytokines were also significantly increased in the serum of patients after fracture. However, only Mdk serum levels differed significantly between male and female fracture patients after menopause. MSCs cultivated with serum from female fracture patients displayed significantly reduced osteogenic differentiation, which was attenuated by Mdk-antibody treatment. In conclusion, our study demonstrated increased Mdk levels after fracture in OVX mice and female fracture patients after menopause. Because Mdk is a negative regulator of bone formation, this might contribute to impaired osteoporotic fracture healing.

Fracture healing is known to be delayed in postmenopausal, osteoporotic females under oestrogen-deficient conditions. Confirming this, experimental studies demonstrated impaired callus formation in ovariectomised animals. Oestrogen-deficiency is known to affect the immune system and the inflammatory response during wound healing. Because a balanced immune response is required for proper bone healing, we were interested to ascertain whether the early immune response after facture is affected by oestrogen depletion. To address the above question, female mice received either a bilateral ovariectomy (OVX) or were sham-operated, and femur osteotomy was performed 8 weeks after OVX/sham operation. The effects of OVX on the presence of immune cells and pro-inflammatory cytokines were evaluated by flow cytometry and immunohistochemistry of the fracture calli on days 1 and 3 after fracture. One day after fracture, immune cell numbers and populations in the fracture haematoma did not differ between OVX- and sham-mice. However, on day 3 after fracture, OVX-mice displayed significantly greater numbers of neutrophils. Local expression of the oestrogen-responsive and pro-inflammatory cytokine midkine (Mdk) and interleukin-6 (IL-6) expression in the fracture callus were increased in OVX-mice on day 3 after fracture compared with sham-mice, indicating that both factors might be involved in the increased presence of neutrophils. Confirming this, Mdk-antibody treatment decreased the number of neutrophils in the fracture callus and reduced local IL-6 expression in OVX-mice. These data indicate that oestrogen-deficiency influences the early inflammatory phase after fracture. This may contribute to delayed fracture healing after oestrogen depletion.

Calcium and vitamin-D (Ca/VitD) deficiency is a major risk factor for osteoporosis. It may also contribute to the compromised bone healing frequently observed in osteoporotic patients, since calcium is essential for fracture-callus mineralization. Additionally, clinical data suggest systemic bone loss following fracture, which may aggravate osteoporosis and thus increase the risk for fragility fractures in osteoporotic patients further. However, the role of Ca/VitD in fracture healing and posttraumatic bone turnover has to date been poorly investigated. Here, we studied bone regeneration and posttraumatic bone turnover in C57BL/6 J mice with ovariectomy-induced osteoporosis. Mice were fed a standard or a Ca/VitD-deficient diet. Notably, fracture healing was only marginally disturbed in Ca/VitD-deficient mice. However, deficient mice displayed significantly increased serum parathyroid hormone levels and osteoclast activity, as well as reduced bone mass in the intact skeleton post-fracture, suggesting considerably enhanced calcium mobilization from the intact skeleton during bone regeneration. Ca/VitD supplementation initiated post-fracture prevented posttraumatic bone loss by reducing bone resorption and furthermore improved bone repair. These results imply that adequate Ca/VitD supply post-fracture is essential to provide sufficient calcium for callus-mineralization in order to prevent posttraumatic bone loss and to reduce the risk for secondary fractures in osteoporotic patients with Ca/VitD deficiency.