Interaction between bone and immune cells: Implications for postmenopausal osteoporosis.

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. Th17 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.

Role of Mast-Cell-Derived RANKL in Ovariectomy-Induced Bone Loss in Mice.

Mast cells may contribute to osteoporosis development, because patients with age-related or post-menopausal osteoporosis exhibit more mast cells in the bone marrow, and mastocytosis patients frequently suffer from osteopenia. We previously showed that mast cells crucially regulated osteoclastogenesis and bone loss in ovariectomized, estrogen-depleted mice in a preclinical model for post-menopausal osteoporosis and found that granular mast cell mediators were responsible for these estrogen-dependent effects. However, the role of the key regulator of osteoclastogenesis, namely, receptor activator of NFκB ligand (RANKL), which is secreted by mast cells, in osteoporosis development has, to date, not been defined. Here, we investigated whether mast-cell-derived RANKL participates in ovariectomy (OVX)-induced bone loss by using female mice with a conditional Rankl deletion. We found that this deletion in mast cells did not influence physiological bone turnover and failed to protect against OVX-induced bone resorption in vivo, although we demonstrated that RANKL secretion was significantly reduced in estrogen-treated mast cell cultures. Furthermore, Rankl deletion in mast cells did not influence the immune phenotype in non-ovariectomized or ovariectomized mice. Therefore, other osteoclastogenic factors released by mast cells might be responsible for the onset of OVX-induced bone loss.

Chronic psychosocial stress compromises the immune response and endochondral ossification during bone fracture healing via ß-AR signaling.

Chronic psychosocial stress/trauma represents an increasing burden in our modern society and a risk factor for the development of mental disorders, including posttraumatic stress disorder (PTSD). PTSD, in turn, is highly comorbid with a plethora of inflammatory disorders and has been associated with increased bone fracture risk. Since a balanced inflammatory response after fracture is crucial for successful bone healing, we hypothesize that stress/trauma alters the inflammatory response after fracture and, consequently, compromises fracture healing. Here we show, employing the chronic subordinate colony housing (CSC) paradigm as a clinically relevant mouse model for PTSD, that mice subjected to CSC displayed increased numbers of neutrophils in the early fracture hematoma, whereas T lymphocytes and markers for cartilage-to-bone transition and angiogenesis were reduced. At late stages of fracture healing, CSC mice were characterized by decreased bending stiffness and bony bridging of the fracture callus. Strikingly, a single systemic administration of the ß-adrenoreceptor (AR) blocker propranolol before femur osteotomy prevented bone marrow mobilization of neutrophils and invasion of neutrophils into the fracture hematoma, both seen in the early phase after fracture, as well as a compromised fracture healing in CSC mice. We conclude that chronic psychosocial stress leads to an imbalanced immune response after fracture via ß-AR signaling, accompanied by disturbed fracture healing. These findings offer possibilities for clinical translation in patients suffering from PTSD and fracture.

Reduced Terminal Complement Complex Formation in Mice Manifests in Low Bone Mass and Impaired Fracture Healing.

The terminal complement complex (TCC) is formed on activation of the complement system, a crucial arm of innate immunity. TCC formation on cell membranes results in a transmembrane pore leading to cell lysis. In addition, sublytic TCC concentrations can modulate various cellular functions. TCC-induced effects may play a role in the pathomechanisms of inflammatory disorders of the bone, including rheumatoid arthritis and osteoarthritis. In this study, we investigated the effect of the TCC on bone turnover and repair. Mice deficient for complement component 6 (C6), an essential component for TCC assembly, and mice with a knockout of CD59, which is a negative regulator of TCC formation, were used in this study. The bone phenotype was analyzed in vivo, and bone cell behavior was analyzed ex vivo. In addition, the mice were subjected to a femur osteotomy. Under homeostatic conditions, C6-deficient mice displayed a reduced bone mass, mainly because of increased osteoclast activity. After femur fracture, the inflammatory response was altered and bone formation was disturbed, which negatively affected the healing outcome. By contrast, CD59-knockout mice only displayed minor skeletal alterations and uneventful bone healing, although the early inflammatory reaction to femur fracture was marginally enhanced. These results demonstrate that TCC-mediated effects regulate bone turnover and promote an adequate response to fracture, contributing to an uneventful healing outcome.

Distinct Effects of IL-6 Classic and Trans-Signaling in Bone Fracture Healing.

Bone healing is a complex process with closely linked phases of inflammation, regeneration, and remodeling. IL-6 may crucially regulate this process; however, the underlying mechanisms are unclear. IL-6 signals are transmitted via the transmembrane glycoprotein 130 by two distinct mechanisms: classic signaling using the membrane-anchored IL-6 receptor and trans-signaling using its soluble form. Herein, we investigated the hypothesis that IL-6 classic and trans-signaling have different functions during bone healing. To investigate fracture healing, 12-week-old C57BL/6J mice underwent a femur osteotomy. To study the function of IL-6 during the inflammatory phase, either an anti-IL-6 antibody, which inhibits IL-6 classic and trans-signaling, or soluble glycoprotein 130 fusion protein, which selectively blocks trans-signaling, was injected after 30 minutes and 48 hours. To analyze IL-6 effects in the repair phase, compounds were injected from day 7 onwards. Global IL-6 inhibition in the early phase after fracture reduced systemic inflammation, the recruitment of immune cells, and bone regeneration, resulting in delayed fracture healing. Global IL-6 inhibition during the repair phase disturbed bone formation and remodeling. In contrast, inhibition of IL-6 trans-signaling exerted minor effects on the immune response and did not influence bone repair, suggesting that the classic pathway accounts for most of the effects observed after global IL-6 inhibition. Our results reveal that IL-6 classic signaling, but not IL-6 trans-signaling, is essential for bone repair.

Loss of p53 compensates osteopenia in murine Mysm1 deficiency.

Histone modifications critically contribute to the epigenetic orchestration of bone homeostasis-in part, by modifying the access of transcription factors to specific genes involved in the osteogenic differentiation process of bone marrow mesenchymal stem cells (MSCs) and osteoblasts. Based on our previous finding that histone H2A deubiquitinase 2A-DUB/Mysm1 interacts with the p53 axis in hematopoiesis and tissue development, we analyzed the molecular basis of the skeletal phenotype of Mysm1-deficient mice and dissected the underlying p53-dependent and -independent mechanisms. Visible morphologic, skeletal deformations of young Mysm1-deficient mice-including a kinked and truncated tail and shortened long bones-were associated with osteopenia of long bones. On the cellular level, Mysm1-deficient primary osteoblasts displayed reduced potential to differentiate into mature osteoblasts, as indicated by decreased expression of osteogenic markers. Reduced osteogenic differentiation capacity of Mysm1-deficient osteoblasts was accompanied by an impaired induction of osteogenic transcription factor Runx2. Osteogenic differentiation of Mysm1-/- MSCs, however, was not compromised in vitro. In line with defective hematopoietic development of Mysm1-deficient mice, Mysm1-/- osteoclasts had reduced resorption activity and were more prone to apoptosis in TUNEL assays. Skeletal alterations and osteopenia of Mysm1-deficient mice were phenotypically completely rescued by simultaneous ablation of p53 in p53-/-Mysm1-/- double-deficient mice-although p53 deficiency did not restore Runx2 expression in Mysm1-/- osteoblasts on the molecular level but, instead, enhanced proliferation and osteogenic differentiation of MSCs. In summary, our results demonstrate novel roles for Mysm1 in osteoblast differentiation and osteoclast formation, resulting in osteopenia in Mysm1-deficient mice that could be abrogated by the loss of p53 from increased osteogenic differentiation of Mysm1-/-p53-/- MSCs.-Haffner-Luntzer, M., Kovtun, A., Fischer, V., Prystaz, K., Hainzl, A., Kroeger, C. M., Krikki, I., Brinker, T. J., Ignatius, A., Gatzka, M. Loss of p53 compensates osteopenia in murine Mysm1 deficiency.

Influence of Menopause on Inflammatory Cytokines during Murine and Human Bone Fracture Healing.

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.

Altered early immune response after fracture and traumatic brain injury.

Introduction: Clinical and preclinical data suggest accelerated bone fracture healing in subjects with an additional traumatic brain injury (TBI). Mechanistically, altered metabolism and neuro-endocrine regulations have been shown to influence bone formation after combined fracture and TBI, thereby increasing the bone content in the fracture callus. However, the early inflammatory response towards fracture and TBI has not been investigated in detail so far. This is of great importance, since the early inflammatory phase of fracture healing is known to be essential for the initiation of downstream regenerative processes for adequate fracture repair. Methods: Therefore, we analyzed systemic and local inflammatory mediators and immune cells in mice which were exposed to fracture only or fracture + TBI 6h and 24h after injury. Results: We found a dysregulated systemic immune response and significantly fewer neutrophils and mast cells locally in the fracture hematoma. Further, local CXCL10 expression was significantly decreased in the animals with combined trauma, which correlated significantly with the reduced mast cell numbers. Discussion: Since mast cells and mast cell-derived CXCL10 have been shown to increase osteoclastogenesis, the reduced mast cell numbers might contribute to higher bone content in the fracture callus of fracture + TBI mice due to decreased callus remodeling.

Wnt1 Boosts Fracture Healing by Enhancing Bone Formation in the Fracture Callus.

Despite considerable improvement in fracture care, 5%-10% of all fractures still heal poorly or result in nonunion formation. Therefore, there is an urgent need to identify new molecules that can be used to improve bone fracture healing. One activator of the Wnt-signaling cascade, Wnt1, has recently gained attention for its intense osteoanabolic effect on the intact skeleton. The aim of the present study was to investigate whether Wnt1 might be a promising molecule to accelerate fracture healing both in skeletally healthy and osteoporotic mice that display a diminished healing capacity. Transgenic mice for a temporary induction of Wnt1 specifically in osteoblasts (Wnt1-tg) were subjected to femur osteotomy. Non-ovariectomized and ovariectomized Wnt1-tg mice displayed significantly accelerated fracture healing based on a strong increase in bone formation in the fracture callus. Transcriptome profiling revealed that Hippo/yes1-associated transcriptional regulator (YAP)-signaling and bone morphogenetic protein (BMP) signaling pathways were highly enriched in the fracture callus of Wnt1-tg animals. Immunohistochemical staining confirmed increased activation of YAP1 and expression of BMP2 in osteoblasts in the fracture callus. Therefore, our data indicate that Wnt1 boosts bone formation during fracture healing via YAP/BMP signaling both under healthy and osteoporotic conditions. To further test a potential translational application of Wnt1, we applied recombinant Wnt1 embedded into a collagen gel during critical-size bone-defect repair. Mice treated with Wnt1 displayed increased bone regeneration compared to control mice accompanied by increased YAP1/BMP2 expression in the defect area. These findings are of high clinical relevance because they indicate that Wnt1 could be used as a new therapeutic agent to treat orthopedic complications in the clinic. © 2023 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 Drive Systemic Inflammation and Compromised Bone Repair After Trauma.

There is evidence that mast cells contribute to inflammation induced by hemorrhagic shock, severe tissue injury or sepsis. Mast cells are highly responsive to alarm signals generated after trauma, and release many inflammatory mediators including interleukin-6, a key mediator of posttraumatic inflammation. An overwhelming posttraumatic inflammation causes compromised bone healing; however, the underlying cellular and molecular mechanisms are poorly understood. Recently, we found that mast cells trigger local and systemic inflammation after isolated fracture leading to uneventful bone repair. Here, we investigated whether mast cells critically contribute to trauma-induced compromised bone healing. Male Mcpt5-Cre+ R-DTA mice, which lack connective tissue type mast cells, and their mast cell-competent Cre- littermates underwent a femur fracture with/without thoracic trauma. Posttraumatic systemic and local inflammation and bone repair were assessed 3 h and 21 d post injury. Both, the systemic and pulmonary inflammation was significantly increased in mast cell-competent mice upon combined trauma compared to isolated fracture. In mast cell-deficient mice, the increase of inflammatory mediators in the circulation induced by the severe trauma was abolished. In the bronchoalveolar lavage fluid, the trauma-induced increase of inflammatory cytokines was not reduced, but the neutrophil invasion into the lungs was significantly diminished in the absence of mast cells. Locally in the fracture hematoma, mast cell-competent mice displayed reduced inflammatory mediator concentrations after combined trauma compared to isolated fracture, which was abolished in mast cell-deficient mice. Notably, while combined trauma resulted in compromised bone repair in mast cell-competent mice, indicated by significantly reduced bone and increased cartilage fracture callus contents, this was abolished in Mcpt5-Cre+ R-DTA mice. Therefore, mast cells contribute to trauma-induced compromised bone repair and could be a potential target for new treatment options to improve fracture healing in multiply injured patients.

Mast Cells Trigger Disturbed Bone Healing in Osteoporotic Mice.

Mast cells are important tissue-resident sensor and effector immune cells but also play a major role in osteoporosis development. Mast cells are increased in numbers in the bone marrow of postmenopausal osteoporotic patients, and mast cell-deficient mice are protected from ovariectomy (OVX)-induced bone loss. In this study, we showed that mast cell-deficient Mcpt5-Cre R-DTA mice were protected from OVX-induced disturbed fracture healing, indicating a critical role for mast cells in the pathomechanisms of impaired bone repair under estrogen-deficient conditions. We revealed that mast cells trigger the fracture-induced inflammatory response by releasing inflammatory mediators, including interleukin-6, midkine (Mdk), and C-X-C motif chemokine ligand 10 (CXCL10), and promote neutrophil infiltration into the fracture site in OVX mice. Furthermore, mast cells were responsible for reduced osteoblast and increased osteoclast activities in OVX mice callus, as well as increased receptor activator of NF-κB ligand serum levels in OVX mice. Additional in vitro studies with human cells showed that mast cells stimulate osteoclastogenesis by releasing the osteoclastogenic mediators Mdk and CXCL10 in an estrogen-dependent manner, which was mediated via the estrogen receptor alpha on mast cells. In conclusion, mast cells negatively affect the healing of bone fractures under estrogen-deficient conditions. Hence, targeting mast cells might provide a therapeutic strategy to improve disturbed bone repair in postmenopausal osteoporosis. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Complement receptor C5aR1 on osteoblasts regulates osteoclastogenesis in experimental postmenopausal osteoporosis.

In recent years, evidence has accumulated that the complement system, an integral part of innate immunity, may be involved in the regulation of bone homeostasis as well as inflammatory bone loss, for example, in rheumatoid arthritis and periodontitis. Complement may also contribute to osteoporosis development, but investigation of the mechanism is limited. Using mice with a conditional deletion of the complement anaphylatoxin receptor C5aR1, we here demonstrated that C5aR1 in osteoblasts (C5aR1 Runx2-Cre mice) or osteoclasts (C5aR1 LysM-Cre mice) did not affect physiological bone turnover or age-related bone loss in either sex, as confirmed by micro-computed tomography, histomorphometry, and biomechanical analyses of the bone and by the measurement of bone turnover markers in the blood serum. When female mice were subjected to ovariectomy (OVX), a common model for postmenopausal osteoporosis, significant bone loss was induced in C5aR1 fl/fl and C5aR1 LysM-Cre mice, as demonstrated by a significantly reduced bone volume fraction, trabecular number and thickness as well as an increased trabecular separation in the trabecular bone compartment. Confirming this, the osteoclast number and the receptor activator of nuclear factor k-B (RANK) ligand (RANKL) serum level were significantly elevated in these mouse lines. By contrast, C5aR1 Runx2-Cre mice were protected from bone loss after OVX and the serum RANKL concentration was not increased after OVX. These data suggested that bone cell-specific C5aR1 may be redundant in bone homeostasis regulation under physiological conditions. However, C5aR1 on osteoblasts was crucial for the induction of bone resorption under osteoporotic conditions by stimulating RANKL release, whereas C5aR1 on osteoclasts did not regulate OVX-induced bone loss. Therefore, our results implicate C5aR1 on osteoblasts as a potential target for treating postmenopausal osteoporosis.

Osteoblast lineage Sod2 deficiency leads to an osteoporosis-like phenotype in mice.

Osteoporosis is a systemic metabolic skeletal disease characterized by low bone mass and strength associated with fragility fractures. Oxidative stress, which results from elevated intracellular reactive oxygen species (ROS) and arises in the aging organism, is considered one of the critical factors contributing to osteoporosis. Mitochondrial (mt)ROS, as the superoxide anion (O2-) generated during mitochondrial respiration, are eliminated in the young organism by antioxidant defense mechanisms, including superoxide dismutase 2 (SOD2), the expression and activity of which are decreased in aging mesenchymal progenitor cells, accompanied by increased mtROS production. Using a mouse model of osteoblast lineage cells with Sod2 deficiency, we observed significant bone loss in trabecular and cortical bones accompanied by decreased osteoblast activity, increased adipocyte accumulation in the bone marrow and augmented osteoclast activity, suggestive of altered mesenchymal progenitor cell differentiation and osteoclastogenesis. Furthermore, osteoblast senescence was increased. To date, there are only a few studies suggesting a causal association between mtROS and cellular senescence in tissue in vivo. Targeting SOD2 to improve redox homeostasis could represent a potential therapeutic strategy for maintaining bone health during aging.

Differences in Fracture Healing Between Female and Male C57BL/6J Mice.

BACKGROUND: Mice are increasingly used in fracture healing research because of the opportunity to use transgenic animals. While both, male and female mice are employed, there is no consensus in the literature whether fracture healing differs between both sexes. Therefore, the aim of the present study was to analyze diaphyseal fracture healing in female and male C57BL/6J mice, a commonly used mouse strain in bone research. METHODS: For that purpose, 12-week-old Female (17-20 g) and Male mice (22-26 g) received a standardized femur midshaft osteotomy stabilized by an external fixator. Mice were euthanized 10 and 21 days after fracture and bone healing was analyzed by biomechanical testing, µCT, histology, immunohistochemistry and qPCR. RESULTS: Ten days after fracture, Male mice displayed significantly more cartilage but less fibrous tissue in the fracture callus compared to Female mice, whereas the amount of bone did not differ. At day 21, Male mice showed a significantly larger fracture callus compared to Female mice. The relative amount of bone in the fracture callus did not significantly differ between both sexes, whereas its tissue mineral density was significantly higher in Male mice on day 21, indicating more mature bone and slightly more rapid fracture healing. These results were confirmed by a significantly greater absolute bending stiffness of the fractured femurs of Male mice on day 21. On the molecular level, Male mice displayed increased active ß-catenin expression in the fracture callus, whereas estrogen receptor α (ERα) expression was lower. CONCLUSION: These results suggest that Male mice display more rapid fracture healing with more prominent cartilaginous callus formation. This might be due to the higher weight of Male mice, resulting in increased mechanical loading of the fracture. Furthermore, Male mice displayed significantly greater activation of osteoanabolic Wnt/ß-catenin signaling, which might also contribute to more rapid bone regeneration.

Estrogen Receptor α Signaling in Osteoblasts is Required for Mechanotransduction in Bone Fracture Healing.

Biomechanical stimulation by whole-body low-magnitude high-frequency vibration (LMHFV) has demonstrated to provoke anabolic effects on bone metabolism in both non-osteoporotic and osteoporotic animals and humans. However, preclinical studies reported that vibration improved fracture healing and bone formation in osteoporotic, ovariectomized (OVX) mice representing an estrogen-deficient hormonal status, but impaired bone regeneration in skeletally healthy non-OVX mice. These effects were abolished in general estrogen receptor α (ERα)-knockout (KO) mice. However, it remains to be elucidated which cell types in the fracture callus are targeted by LMHFV during bone healing. To answer this question, we generated osteoblast lineage-specific ERα-KO mice that were subjected to ovariectomy, femur osteotomy and subsequent vibration. We found that the ERα specifically on osteoblastic lineage cells facilitated the vibration-induced effects on fracture healing, because in osteoblast lineage-specific ERα-KO (ERαfl/fl; Runx2Cre) mice the negative effects in non-OVX mice were abolished, whereas the positive effects of vibration in OVX mice were reversed. To gain greater mechanistic insights, the influence of vibration on murine and human osteogenic cells was investigated in vitro by whole genome array analysis and qPCR. The results suggested that particularly canonical WNT and Cox2/PGE2 signaling is involved in the mechanotransduction of LMHFV under estrogen-deficient conditions. In conclusion, our study demonstrates a critical role of the osteoblast lineage-specific ERα in LMHFV-induced effects on fracture healing and provides further insights into the molecular mechanism behind these effects.

Effects of immune cells on mesenchymal stem cells during fracture healing.

In vertebrates, bone is considered an osteoimmune system which encompasses functions of a locomotive organ, a mineral reservoir, a hormonal organ, a stem cell pool and a cradle for immune cells. This osteoimmune system is based on cooperatively acting bone and immune cells, cohabitating within the bone marrow. They are highly interdependent, a fact that is confounded by shared progenitors, mediators, and signaling pathways. Successful fracture healing requires the participation of all the precursors, immune and bone cells found in the osteoimmune system. Recent evidence demonstrated that changes of the immune cell composition and function may negatively influence bone healing. In this review, first the interplay between different immune cell types and osteoprogenitor cells will be elaborated more closely. The separate paragraphs focus on the specific cell types, starting with the cells of the innate immune response followed by cells of the adaptive immune response, and the complement system as mediator between them. Finally, a brief overview on the challenges of preclinical testing of immune-based therapeutic strategies to support fracture healing will be given.

Piezo1 Inactivation in Chondrocytes Impairs Trabecular Bone Formation.

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 Piezo1Runx2Cre mice. This phenotype developed at an early postnatal stage and primarily affected the formation of the secondary spongiosa. The presumptive Piezo1Runx2Cre 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 Piezo1Runx2Cre 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 Piezo1Runx2Cre 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).

The Role of Mast Cells in Bone Metabolism and Bone Disorders.

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-ß) 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.

A novel mouse model to study fracture healing of the proximal femur.

The majority of fractures, especially in elderly and osteoporotic patients, occurs in metaphyseal bone. However, only a few experimental models exist to study metaphyseal bone healing in mice. Currently used mouse models of metaphyseal fracture healing are either based on drill hole defects, lacking adequate biomechanical stimulation at the site of fracture and therefore endochondral ossification in the fracture callus, or are introduced into the distal part of the mouse femur stabilized by a locking plate, which is challenging due to the small specimen size. Therefore, the aim of the current study was to develop a new mouse model to study metaphyseal fracture healing of the proximal femur. We chose a combination between an open osteotomy and a closed intramedullary stabilization. A 24 G needle was inserted into the femur in a closed manner, then an osteotomy was made with a 0.4-mm Gigli wire saw between the third and the lesser trochanter of the femur using an open approach. Fractured femurs were analyzed using microcomputed tomography and histology at days 14 and 21 after surgery. No animals were lost due to surgery or anesthesia. All animals displayed normal limb loading and a physiological gait pattern within the first three days after fracture. We found robust endochondral ossification during the fracture healing process with high expression of late chondrocyte and early osteogenic markers at day 14 (d14). By day 21 (d21), all fractures had a bony bridging score of 3 or more, indicating successful healing. Callus volume significantly decreased from d14 to d21, whereas high numbers of osteoclasts appeared at the fracture callus until d21, indicating that callus remodeling had already started at d21. In conclusion, we successfully developed a novel mouse model to study endochondral fracture healing of the proximal femur. This model might be useful for future studies using transgenic animals to unravel molecular mechanisms of osteoporotic metaphyseal fracture healing.

Publisher Correction: Calcium and vitamin-D deficiency marginally impairs fracture healing but aggravates posttraumatic bone loss in osteoporotic mice.

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