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).

Bone is a mechanosensitive tissue for which mechanical stimuli are crucial in maintaining its structure and function. Bone cells react to their biomechanical environment by activating molecular signaling pathways, which regulate their proliferation, differentiation, and matrix production. Bone implants influence the mechanical conditions in the adjacent bone tissue. Optimizing their mechanical properties can support bone regeneration. Furthermore, external biomechanical stimulation can be applied to improve implant osseointegration and accelerate bone regeneration. One promising anabolic therapy is vertical whole-body low-magnitude high-frequency vibration (LMHFV). This form of vibration is currently extensively investigated to serve as an easy-to-apply, cost-effective, and efficient treatment for bone disorders and regeneration. This review aims to provide an overview of LMHFV effects on bone cells in vitro and on implant integration and bone fracture healing in vivo. In particular, we review the current knowledge on cellular signaling pathways which are influenced by LMHFV within bone tissue. Most of the in vitro experiments showed that LMHFV is able to enhance mesenchymal stem cell (MSC) and osteoblast proliferation. Furthermore, osteogenic differentiation of MSCs and osteoblasts was shown to be accelerated by LMHFV, whereas osteoclastogenic differentiation was inhibited. Furthermore, LMHFV increased bone regeneration during osteoporotic fracture healing and osseointegration of orthopedic implants. Important mechanosensitive pathways mediating the effects of LMHFV might be the Wnt/beta-catenin signaling pathway, the estrogen receptor (ER) signaling pathway, and cytoskeletal remodeling.

In the adult skeleton, bone remodeling is required to replace damaged bone and functionally adapt bone mass and structure according to the mechanical requirements. It is regulated by multiple endocrine and paracrine factors, including hormones and growth factors, which interact in a coordinated manner. Because the response of bone to mechanical signals is dependent on functional estrogen receptor (ER) and Wnt/β-catenin signaling and is impaired in postmenopausal osteoporosis by estrogen deficiency, it is of paramount importance to elucidate the underlying mechanisms as a basis for the development of new strategies in the treatment of osteoporosis. The present study aimed to investigate the effectiveness of the activation of the ligand-dependent ER and the Wnt/β-catenin signal transduction pathways on mechanically induced bone formation using ovariectomized mice as a model of postmenopausal bone loss. We demonstrated that both pathways interact in the regulation of bone mass adaption in response to mechanical loading and that the activation of Wnt/β-catenin signaling considerably increased mechanically induced bone formation, whereas the effects of estrogen treatment strictly depended on the estrogen status in the mice.

Clinical and experimental studies demonstrate the potential of low-magnitude high-frequency vibration (LMHFV) to enhance bone formation in the intact skeleton and during fracture healing. Moreover, it was shown that the effects of vibration therapy during fracture healing are highly dependent on the estrogen status of the vibrated individual and that estrogen receptor (ER) α signaling plays a major role in mechanotransduction of LMHFV. Because it is known that LMHFV can directly act on osteogenic cells, we hypothesize that the differential effects of LMHFV in the presence and absence of estrogen are mediated by ERα signaling in osteoblasts. To prove this hypothesis, we subjected preosteoblastic MC3T3-E1 cells and primary osteoblasts to LMHFV in vitro. We found increased Cox2 gene expression, cell metabolic activity and cell proliferation after LMHFV in the absence of estrogen, whereas the effects were contrary in the presence of estrogen. Blocking of ERα signaling by Esr1-siRNA knockdown or adding the selective ERα antagonist MPP dihydrochloride abolished the effects of LMHFV on osteoblast proliferation and Cox2 expression. Furthermore, primary osteoblasts isolated from ERα-knockout mice did not show a response towards LMHFV in the presence of estrogen. Additionally, blocking of actin cytoskeletal remodeling by adding the p160ROCK inhibitor Y-27632 abolished the effects of LMHFV. In contrast, expression of primary cilium was not necessary for mechanotransduction of LMHFV. These results suggest that direct effects of LMHFV on osteoblasts are dependent on ERα signaling and cytoskeletal remodeling.

Mechanostimulation by low-magnitude high frequency vibration (LMHFV) has been shown to provoke anabolic effects on the intact skeleton in both mice and humans. However, experimental studies revealed that, during bone fracture healing, the effect of whole-body vibration is profoundly influenced by the estrogen status. LMHFV significantly improved fracture healing in ovariectomized (OVX) mice being estrogen deficient, whereas bone regeneration was significantly reduced in non-OVX, estrogen-competent mice. Furthermore, estrogen receptors a (ER alpha) and beta (ER beta) were differentially expressed in the fracture callus after whole-body vibration, depending on the estrogen status. Based on these data, we hypothesized that ERs may mediate vibration-induced effects on fracture healing. To prove this hypothesis, we investigated the effects of LMHFV on bone healing in mice lacking ER alpha or ER beta. To study the influence of the ER ligand estrogen, both non-OVX and OVX mice were used. All mice received a femur osteotomy stabilized by an external fixator. Half of the mice were sham-operated or subjected to OVX 4 weeks before osteotomy. Half of each group received LMHFV with 03 g and 45 Hz for 20 min per day, 5 days per week. After 21 days, fracture healing was evaluated by biomechanical testing, mu CT analysis, histomorphometry and immunohistochemistry. Absence of ER alpha or ER beta did not affect fracture healing in sham-treated mice. Wildtype (Wt) and ER beta-knockout mice similarly displayed impaired bone regeneration after OVX, whereas ER alpha-knockout mice did not. Confirming previous data, in WT mice, LMHFV negatively affected bone repair in non-OVX mice, whereas OVX-induced compromised healing was significantly improved by vibration. In contrast, vibrated ER alpha-knockout mice did not display significant differences in fracture healing compared to non-vibrated animals, both in non-OVX and OVX mice. Fracture healing in ER beta-knockout mice was similarly affected by LMHFV as in WT mice. These results suggest that ER alpha-signaling may be crucial for vibration-induced effects on fracture healing, whereas ER beta-signaling may play a minor role. (C) 2018 Elsevier Inc. All rights reserved.

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.

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.

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

The anaphylatoxin receptor C5aR1 plays an important role not only in innate immune responses, but also in bone metabolism and fracture healing, being highly expressed on immune and bone cells, including osteoblasts and osteoclasts. C5aR1 induces osteoblast migration, cytokine generation and osteoclastogenesis, however, the exact role of C5aR1-mediated signaling in osteoblasts is not entirely known. Therefore, we hypothesized that osteoblasts are essential target cells for C5a and that fracture healing should be disturbed in mice with an osteoblast-specific C5aR1 overexpression (Col1a1-C5aR1). Osteoblast activity in vitro, bone phenotype and fracture healing after isolated osteotomy and after combined osteotomy with additional thoracic trauma were analyzed. The systemic and local inflammatory reactions were analyzed by determining C5a and IL-6 concentrations in blood, bronchoalveolar lavage fluid and fracture callus and the recruitment of immune cells. In vitro, osteoblast proliferation and differentiation were similar to wildtype cells, and phosphorylation of p38 and expression of IL-6 and RANKL were increased in osteoblasts derived from Col1a1-C5aR1 mice. Bone phenotype and the inflammatory reaction were unaffected in Col1a1-C5aR1 mice. Fracture healing was significantly impaired as demonstrated by significantly reduced bone content, bone mineral density and flexural rigidity, possibly due to significantly increased osteoclast numbers. C5aR1 signaling in osteoblasts might possibly affect RANKL/OPG balance, leading to increased bone resorption. Additional trauma significantly impaired fracture healing, particularly in Col1a1-C5aR1 mice. In conclusion, the data indicate that C5aR1 signaling in osteoblasts plays a detrimental role in bone regeneration after fracture.

Background: 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.Methods: 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.Results: 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.Conclusions: These data indicate that oestrogen-deficiency influences the early inflammatory phase after fracture. This may contribute to delayed fracture healing after oestrogen depletion.

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

The heparin-binding growth and differentiation factor midkine (Mdk) is proposed to negatively regulate osteoblast activity and bone formation in the adult skeleton. As Mdk-deficient mice were protected from ovariectomy (OVX)-induced bone loss, this factor may also play a role in the pathogenesis of postmenopausal osteoporosis. We have previously demonstrated that Mdk negatively influences bone regeneration during fracture healing. Here, we investigated whether the inhibition of Mdk using an Mdk-antibody (Mdk-Ab) improves compromised bone healing in osteoporotic OVX-mice. Using a standardized femur osteotomy model, we demonstrated that Mdk serum levels were significantly enhanced after fracture in both non-OVX and OVX-mice, however, the increase was considerably greater in osteoporotic mice. Systemic treatment with the Mdk-Ab significantly improved bone healing in osteoporotic mice by increasing bone formation in the fracture callus. On the molecular level, we demonstrated that the OVX-induced reduction of the osteoanabolic beta-catenin signaling in the bony callus was abolished by Mdk-Ab treatment. Furthermore, the injection of the Mdk-Ab increased trabecular bone mass in the skeleton of the osteoporotic mice. These results implicate that antagonizing Mdk may be useful for the therapy of osteoporosis and osteoporotic fracture-healing complications.

Bone can adapt to changing load demands by mechanically regulated bone remodeling. Osteocytes, osteoblasts, and mesenchymal stem cells are mechanosensitive and respond to mechanical signals through the activation of specific molecular signaling pathways. The process of bone regeneration after fracture is similarly and highly regulated by the biomechanical environment at the fracture site. Depending on the tissue strains, mesenchymal cells differentiate into fibroblasts, chondrocytes, or osteoblasts, determining the course and the success of healing. In the aged organism, mechanotransduction in both intact and fractured bones may be altered due to changed hormone levels and expression of growth factors and other signaling molecules. It is proposed that altered mechanotransduction may contribute to disturbed healing in aged patients. This review explains the basic principles of mechanotransduction in the bone and the fracture callus and summarizes the current knowledge on aging-induced changes in mechanobiology. Furthermore, the methods for external biomechanical stimulation of intact and fractured bones are discussed with respect to a possible application in the elderly patient.

Efficient calcium absorption is essential for skeletal health. Patients with impaired gastric acidification display low bone mass and increased fracture risk because calcium absorption is dependent on gastric pH. We investigated fracture healing and post-traumatic bone turnover in mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells. Cckbr-/- mice display hypochlorhydria, calcium malabsorption, and osteopenia. Cckbr-/- and wildtype (WT) mice received a femur osteotomy and were fed either a standard or calcium-enriched diet. Healed and intact bones were assessed by biomechanical testing, histomorphometry, micro-computed tomography, and quantitative backscattering. Parathyroid hormone (PTH) serum levels were determined by enzyme-linked immunosorbent assay. Fracture healing was unaffected in Cckbr-/- mice. However, Cckbr-/- mice displayed increased calcium mobilization from the intact skeleton during bone healing, confirmed by significantly elevated PTH levels and osteoclast numbers compared to WT mice. Calcium supplementation significantly reduced secondary hyperparathyroidism and bone resorption in the intact skeleton in both genotypes, but more efficiently in WT mice. Furthermore, calcium administration improved bone healing in WT mice, indicated by significantly increased mechanical properties and bone mineral density of the fracture callus, whereas it had no significant effect in Cckbr-/- mice. Therefore, under conditions of hypochlorhydria-induced calcium malabsorption, calcium, which is essential for callus mineralization, appears to be increasingly mobilized from the intact skeleton in favor of fracture healing. Calcium supplementation during fracture healing prevented systemic calcium mobilization, thereby maintaining bone mass and improving fracture healing in healthy individuals whereas the effect was limited by gastric hypochlorhydria. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1914-1921, 2016.

Bone can adapt to changing load demands by mechanically regulated bone remodeling. Osteocytes, osteoblasts, and mesenchymal stem cells are mechanosensitive and respond to mechanical signals through the activation of specific molecular signaling pathways. The process of bone regeneration after fracture is similarly and highly regulated by the biomechanical environment at the fracture site. Depending on the tissue strains, mesenchymal cells differentiate into fibroblasts, chondrocytes, or osteoblasts, determining the course and the success of healing. In the aged organism, mechanotransduction in both intact and fractured bones may be altered due to changed hormone levels and expression of growth factors and other signaling molecules. It is proposed that altered mechanotransduction may contribute to disturbed healing in aged patients. This review explains the basic principles of mechanotransduction in the bone and the fracture callus and summarizes the current knowledge on aging-induced changes in mechanobiology. Furthermore, the methods for external biomechanical stimulation of intact and fractured bones are discussed with respect to a possible application in the elderly patient.

Previous findings suggest that the growth and differentiation factor midkine (Mdk) is a negative regulator of osteoblast activity and bone formation, thereby raising the possibility that a specific Mdk antagonist might improve bone formation during fracture healing.

Micro‐environmental clues are critical to cell behavior. One of the key elements of migration is the generation and response to forces. Up to now there is no definitive concept on how the generation and responses to cellular forces influence cell behavior. Here, we show that phosphorylation of paxillin is a crucial event in the response to exogenous forces. Application of force induced growth of adhesion sites and this phenomenon was accompanied by a downregulation of Src family kinase activity, which in turn led to a decrease in the phosphorylation of paxillin at the tyrosine residues Y31 and Y118. The force‐dependent growth of adhesion sites is mediated by a decrease in the turnover‐rate of paxillin in focal contacts. This turnover critically depended on the phosphorylation state of paxillin at Y31/118. Paxillin is an important regulator in the control of the aggregate state of the whole adhesion site since the turnover of other adhesion site proteins such as vinculin is influenced by the phosphorylation state of paxillin as well. Taken together these data suggest that SFK dependent phosphorylation of paxillin is a crucial event in the regulation of adhesion site function in response to force. © 2015 Wiley Periodicals, Inc.

Mechanical stimuli are known to significantly influence bone metabolism and fracture healing. Various studies have demonstrated the involvement of complex molecular mechanotransduction pathways, such as the Wnt/beta-catenin, bone morphogenetic protein (BMP) and estrogen receptor signaling pathways in mechanotransduction. Mechanotransduction is influenced by aging and the comorbidities of the patient. Pharmacological modulation of signal transduction influences bone formation and the mechanosensitivity of skeletal tissue. The combination of pharmacological and biomechanical therapies may be useful for the treatment of fractures with impaired healing.