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Oxidative stress has been considered to be closely related to spaceflight-induced bone loss; however, mechanism is elusive and there are no effective countermeasures. Using cultured rat calvarial osteoblasts exposed to microgravity simulated by a random positioning machine, this study addressed the hypotheses that microgravity-induced shortening of primary cilia leads to oxidative stress and that primary cilium protection prevents oxidative stress and osteogenesis loss. Microgravity was found to induce oxidative stress (as represented by increased levels of reactive oxygen species (ROS) and malondialdehyde production, and decreased activities of antioxidant enzymes), which was perfectly replicated in osteoblasts growing in NG with abrogated primary cilia (created by transfection of an interfering RNA), suggesting the possibility that shortening of primary cilia leads to oxidative stress. Oxidative stress was accompanied by mitochondrial dysfunction (represented by increased mitochondrial ROS and decreased mitochondrial membrane potential) and intracellular Ca 2+ overload, and the latter was found to be caused by increased activity of Ca 2+ channel transient receptor potential vanilloid 4 (TRPV4), as also evidenced by TRPV4 agonist GSK1016790A-elicited Ca 2+ influx. Supplementation of HC-067047, a specific antagonist of TRPV4, attenuated microgravity-induced mitochondrial dysfunction, oxidative stress, and osteogenesis loss. Although TRPV4 was found localized in primary cilia and expressed at low levels in NG, microgravity-induced shortening of primary cilia led to increased TRPV4 levels and Ca 2+ influx. When primary cilia were protected by miR-129-3p overexpression or supplementation with a natural flavonoid moslosooflavone, microgravity-induced increased TRPV4 expression, mitochondrial dysfunction, oxidative stress, and osteogenesis loss were all prevented. Our data revealed a new mechanism that primary cilia function as a controller for TRPV4 expression. Microgravity-induced injury on primary cilia leads to increased expression and overactive channel of TRPV4, causing intracellular Ca 2+ overload and oxidative stress, and primary cilium protection could be an effective countermeasure against microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts., (© 2023 Wiley Periodicals LLC.)

Electromagnetic fields (EMFs) have emerged as a versatile means for osteoporosis treatment and prevention. However, its optimal application parameters are still elusive. Here, we optimized the frequency parameter first by cell culture screening and then by animal experiment validation. Osteoblasts isolated from newborn rats (ROBs) were exposed 90 min/day to 1.8 mT SEMFs at different frequencies (ranging from 10 to 100 Hz, interval of 10 Hz). SEMFs of 1.8 mT inhibited ROB proliferation at 30, 40, 50, 60 Hz, but increased proliferation at 10, 70, 80 Hz. SEMFs of 10, 50, and 70 Hz promoted ROB osteogenic differentiation and mineralization as shown by alkaline phosphatase (ALP) activity, calcium content, and osteogenesis-related molecule expression analyses, with 50 Hz showing greater effects than 10 and 70 Hz. Treatment of young rats with 1.8 mT SEMFs at 10, 50, or 100 Hz for 2 months significantly increased whole-body bone mineral density (BMD) and femur microarchitecture, with the 50 Hz group showing the greatest effect. Furthermore, 1.8 mT SEMFs extended primary cilia lengths of ROBs and increased protein kinase A (PKA) activation also in a frequency-dependent manner, again with 50 Hz SEMFs showing the greatest effect. Pretreatment of ROBs with the PKA inhibitor KT5720 abolished the effects of SEMFs to increase primary cilia length and promote osteogenic differentiation/mineralization. These results indicate that 1.8 mT SEMFs have a frequency window effect in promoting osteogenic differentiation/mineralization in ROBs and bone formation in growing rats, which involve osteoblast primary cilia length extension and PKA activation., (© 2021 International Federation for Cell Biology.)

Extremely low-frequency electromagnetic fields have been considered a potential candidate for the prevention and treatment of osteoporosis; however, their action mechanism and optimal magnetic flux density (intensity) parameter are still elusive. The present study found that 50-Hz sinusoidal electromagnetic fields (SEMFs) at 1.8 mT increased the peak bone mass of young rats by increasing bone formation. Gene array expression studies with femoral bone samples showed that SEMFs increased the expression levels of collagen-1α1 and Wnt10b, a critical ligand of the osteogenic Wnt/β-catenin pathway. Consistently, SEMFs promoted osteogenic differentiation and maturation of rat calvarial osteoblasts (ROBs) in vitro through activating the Wnt10b/β-catenin pathway. This osteogenesis-promoting effect of SEMFs via Wnt10b/β-catenin signaling was found to depend on the functional integrity of primary cilia in osteoblasts. When the primary cilia were abrogated by small interfering RNA (siRNA) targeting IFT88, the ability of SEMFs to promote the osteogenic differentiation of ROBs through activating Wnt10b/β-catenin signaling was blocked. Although the knockdown of Wnt10b expression with RNA interference had no effect on primary cilia, it significantly suppressed the promoting effect of SEMFs on osteoblastic differentiation/maturation. Wnt10b was normally localized at the bases of primary cilia, but it disappeared (or was released) from the cilia upon SEMF treatment. Interestingly, primary cilia were elongated to different degrees by different intensities of 50-Hz SEMFs, with the window effect observed at 1.8 mT, and the expression level of Wnt10b increased in accord with the lengths of primary cilia. These results indicate that 50-Hz 1.8-mT SEMFs increase the peak bone mass of growing rats by promoting osteogenic differentiation/maturation of osteoblasts, which is mediated, at least in part, by Wnt10b at the primary cilia and the subsequent activation of Wnt/β-catenin signaling. © 2019 American Society for Bone and Mineral Research., (© 2019 American Society for Bone and Mineral Research.)

Bone cells, including osteoblasts, osteoclasts, and osteocytes, have the ability to develop and maintain bone architecture. Although improved experimental testing approaches are increasing our understanding of the complex structures and functions of bone cells and bone, computational models, particularly finite element analyses, are being used to extend this knowledge and to develop a more theoretical understanding of bone cell behaviors. There are many challenges to developing an accurate and validated computational model due to the complex structure and biomechanical behaviors of the bone cells and bone tissue. A better understanding of the geometry and material properties of bone cells and bone will improve our understanding of the bone's biomechanical behaviors. In this review, we summarize and discuss the different geometric representations and material properties that have been used to model the bone cells. The current status of computational models, a comprehensive overview of the modeling methods for the bone cells, and the challenges for validating the models are presented.

Faulty bony repair causes dysrepair of injured growth plate cartilage and bone growth defects in children; however, the underlying mechanisms are unclear. Recently, we observed the prominent induction of neurotrophin‑3 (NT-3) and its important roles as an osteogenic and angiogenic factor promoting the bony repair. The current study investigated its roles in regulating injury site remodelling. In a rat tibial growth plate drill-hole injury repair model, NT-3 was expressed prominently in osteoblasts at the injury site. Recombinant NT-3 (rhNT-3) systemic treatment enhanced, but NT-3 immunoneutralization attenuated, expression of cartilage-removal proteases (MMP-9 and MMP-13), presence of bone-resorbing osteoclasts and expression of osteoclast protease cathepsin K, and remodelling at the injury site. NT-3 was also highly induced in cultured mineralizing rat bone marrow stromal cells, and the conditioned medium augmented osteoclast formation and resorptive activity, an ability that was blocked by presence of anti-NT-3 antibody. Moreover, NT-3 and receptor TrkC were induced during osteoclastogenesis, and rhNT-3 treatment activated TrkC downstream kinase Erk1/2 in differentiating osteoclasts although rhNT-3 alone did not affect activation of osteoclastogenic transcription factors NF-κB or NFAT in RAW 264.7 osteoclast precursor cells. Furthermore, rhNT-3 treatment increased, but NT-3 neutralization reduced, expression of osteoclastogenic cytokines (RANKL, TNF-α, and IL-1) in mineralizing osteoblasts and in growth plate injury site, and rhNT-3 augmented the induction of these cytokines caused by RANKL treatment in RAW 264.7 cells. Thus, injury site osteoblast-derived NT-3 is important in promoting growth plate injury site remodelling, as it induces cartilage proteases for cartilage removal and augments osteoclastogenesis and resorption both directly (involving activing Erk1/2 and substantiating RANKL-induced increased expression of osteoclastogenic signals in differentiating osteoclasts) and indirectly (inducing osteoclastogenic signals in osteoblasts)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Osteoblast differentiation is a multistep process delicately regulated by many factors, including cytoskeletal dynamics and signaling pathways. Microtubule actin crosslinking factor 1 (MACF1), a key cytoskeletal linker, has been shown to play key roles in signal transduction and in diverse cellular processes; however, its role in regulating osteoblast differentiation is still needed to be elucidated. To further uncover the functions and mechanisms of action of MACF1 in osteoblast differentiation, we examined effects of MACF1 knockdown (MACF1-KD) in MC3T3-E1 osteoblastic cells on their osteoblast differentiation and associated molecular mechanisms. The results showed that knockdown of MACF1 significantly suppressed mineralization of MC3T3-E1 cells, down-regulated the expression of key osteogenic genes alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and type I collagen α1 (Col Iα1). Knockdown of MACF1 dramatically reduced the nuclear translocation of β-catenin, decreased the transcriptional activation of T cell factor 1 (TCF1), and down-regulated the expression of TCF1, lymphoid enhancer-binding factor 1 (LEF1), and Runx2, a target gene of β-catenin/TCF1. In addition, MACF1-KD increased the active level of glycogen synthase kinase-3β (GSK-3β), which is a key regulator for β-catenin signal transduction. Moreover, the reduction of nuclear β-catenin amount and decreased expression of TCF1 and Runx2 were significantly reversed in MACF1-KD cells when treated with lithium chloride, an agonist for β-catenin by inhibiting GSK-3β activity. Taken together, these findings suggest that knockdown of MACF1 in osteoblastic cells inhibits osteoblast differentiation through suppressing the β-catenin/TCF1-Runx2 axis. Thus, a novel role of MACF1 in and a new mechanistic insight of osteoblast differentiation are uncovered., (© 2017 Wiley Periodicals, Inc.)

Leptin, an adipocyte-derived cytokine associated with bone metabolism, is believed to play a critical role in the pathogenesis of heterotopic ossification (HO). The effect and underlying action mechanism of leptin were investigated on osteogenic differentiation of tendon-derived stem cells (TDSCs) in vitro and the HO formation in rat tendons. Isolated rat TDSCs were treated with various concentrations of leptin in the presence or absence of mTORC1 signaling specific inhibitor rapamycin in vitro. A rat model with Achilles tenotomy was employed to evaluate the effect of leptin on HO formation together with or without rapamycin treatment. In vitro studies with TDSCs showed that leptin increased the expression of osteogenic biomarkers (alkaline phosphatase, runt-related transcription factor 2, osterix, osteocalcin) and enhanced mineralization of TDSCs via activating the mTORC1 signal pathway (as indicated by phosphorylation of p70 ribosomal S6 kinase 1 and p70 ribosomal S6). However, mTORC1 signaling blockade with rapamycin treatment suppressed leptin-induced osteogenic differentiation and mineralization. In vivo studies showed that leptin promoted HO formation in the Achilles tendon after tenotomy, and rapamycin treatment blocked leptin-induced HO formation. In conclusion, leptin can promote TDSC osteogenic differentiation and heterotopic bone formation via mTORC1 signaling in both vitro and vivo model, which provides a new potential therapeutic target for HO prevention., (© 2017 Wiley Periodicals, Inc.)

Icariin, a prenylated flavonol glycoside isolated from the herb Epimedium , has been considered as a potential alternative therapy for osteoporosis. Previous research has shown that, unlike other flavonoids, icariin is unlikely to act via the estrogen receptor, but its exact mechanism of action is unknown. In this study, using rat calvarial osteoblast culture and rat bone growth models, we demonstrated that icariin promotes bone formation by activating the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway requiring functional primary cilia of osteoblasts. We found that icariin increases the peak bone mass attained by young rats and promotes the maturation and mineralization of rat calvarial osteoblasts. Icariin activated cAMP/PKA/CREB signaling of the osteoblasts by increasing intracellular cAMP levels and facilitating phosphorylation of both PKA and CREB. Blocking cAMP/PKA/CREB signaling with inhibitors of the cAMP-synthesizing adenylyl cyclase (AC) and PKA inhibitors significantly inhibited the osteogenic effect of icariin in the osteoblasts. Icariin-activated cAMP/PKA/CREB signaling was localized to primary cilia, as indicated by localization of soluble AC and phosphorylated PKA. Furthermore, blocking ciliogenesis via siRNA knockdown of a cilium assembly protein, IFT88, inhibited icariin-induced PKA and CREB phosphorylation and also abolished icariin's osteogenic effect. Finally, several of these outcomes were validated in icariin-treated rats. Together, these results provide new insights into icariin function and its mechanisms of action and strengthen existing ties between cAMP-mediated signaling and osteogenesis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

It is well documented that microgravity in space environment leads to bone loss in astronauts. These physiological changes have also been validated by human and animal studies and modeled in cell-based analogs. However, the underlying mechanisms are elusive. In the current study, we identified a novel phenomenon that primary cilia (key sensors and functioning organelles) of rat calvarial osteoblasts (ROBs) gradually shrank and disappeared almost completely after exposure to simulated microgravity generated by a random positioning machine (RPM). Along with the abrogation of primary cilia, the differentiation, maturation and mineralization of ROBs were inhibited. We also found that the disappearance of primary cilia was prevented by treating ROBs with cytochalasin D, but not with LiCl or dynein light chain Tctex-type 1 (Dynlt1) siRNA. The repression of the differentiation, maturation and mineralization of ROBs was effectively offset by cytochalasin D treatment in microgravity conditions. Blocking ciliogenesis using intraflagellar transport protein 88 (IFT88) siRNA knockdown inhibited the ability of cytochalasin D to counteract this reduction of osteogenesis. These results indicate that the abrogation of primary cilia may be responsible for the microgravity's inhibition on osteogenesis. Reconstruction of primary cilia may become a potential strategy against bone loss induced by microgravity.

Pulsed electromagnetic fields (PEMFs) have been considered as a potential candidate for the prevention and treatment of osteoporosis, however, the mechanism of its action is still elusive. We have previously reported that 50Hz 0.6mT PEMFs stimulate osteoblastic differentiation and mineralization in a primary cilium- dependent manner, but did not know the reason. In the current study, we found that the PEMFs promoted osteogenic differentiation and maturation of rat calvarial osteoblasts (ROBs) by activating bone morphogenetic protein BMP-Smad1/5/8 signaling on the condition that primary cilia were normal. Further studies revealed that BMPRII, the primary binding receptor of BMP ligand, was readily and strongly upregulated by PEMF treatment and localized at the bases of primary cilia. Abrogation of primary cilia with small interfering RNA sequence targeting IFT88 abolished the PEMF-induced upregulation of BMPRII and its ciliary localization. Knockdown of BMPRII expression level with RNA interference had no effects on primary cilia but significantly decreased the promoting effect of PEMFs on osteoblastic differentiation and maturation. These results indicated that PEMFs stimulate osteogenic differentiation and maturation of osteoblast by primary cilium-mediated upregulation of BMPRII expression and subsequently activation of BMP-Smad1/5/8 signaling, and that BMPRII is the key component linking primary cilium and BMP-Smad1/5/8 pathway. This study has thus revealed the molecular mechanism for the osteogenic effect of PEMFs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

BACKGROUND Although it has been reported that hypoxic exposure can attenuate hypertension, heart disease, diabetes, and some other diseases, effects of hypoxia on osteoporosis are still unknown. MATERIAL AND METHODS The current study investigated whether short-term hypoxic exposure (in comparison with normoxic conditions) affects bone metabolism in normal or ovariectomized (OVX) adult female rats in an vivo study. Micro-computed tomography bone volume/structural analyses, histological examination, and serum bone turnover biochemical assays were used. In addition, the expressions of some associated major regulatory molecules were measured in osteoblastic cultures. RESULTS While the 14-day hypoxic exposure did not change the bone-remodeling process in normal adult female rats, it decreased bone volume, osteoclast density, and serum bone formation marker (alkaline phosphatase) level, but increased osteoclast density and serum bone resorption marker (C-telopeptide of collagen) level in OVX rats. The bone marrow adipocyte number and serum fatty acid binding protein-4 level were increased in OVX-hypoxic rats compared with OVX-normoxic rats. Consistently, in human MG-63 osteoblastic cultures, the hypoxic condition suppressed protein expression of osteogenic transcriptional factors Runx2 and osterix, elevated protein expression of osteoclastogenic cytokine receptor activator of nuclear factor kappa-B ligand, but reduced that of osteoclastogenic inhibitor osteoprotegerin. CONCLUSIONS Our results suggest that, although no change occurred in the bone-remodeling process in normal adult female rats after hypoxic exposure, under the estrogen-deficient osteoporotic condition, the hypoxic condition can alter the bone microenvironment so that it may further impair osteoblastic differentiation and enhance osteoclastic formation, and thus reduce bone formation, enhance bone resorption, and accelerate bone loss.

Bone cells are deformed according to mechanical stimulation they receive and their mechanical characteristics. However, how osteoblasts are affected by mechanical vibration frequency and acceleration amplitude remains unclear. By developing 3D osteoblast finite element (FE) models, this study investigated the effect of cell shapes on vibration characteristics and effect of acceleration (vibration intensity) on vibrational responses of cultured osteoblasts. Firstly, the developed FE models predicted natural frequencies of osteoblasts within 6.85-48.69 Hz. Then, three different levels of acceleration of base excitation were selected (0.5, 1, and 2 g) to simulate vibrational responses, and acceleration of base excitation was found to have no influence on natural frequencies of osteoblasts. However, vibration response values of displacement, stress, and strain increased with the increase of acceleration. Finally, stress and stress distributions of osteoblast models under 0.5 g acceleration in Z -direction were investigated further. It was revealed that resonance frequencies can be a monotonic function of cell height or bottom area when cell volumes and material properties were assumed as constants. These findings will be useful in understanding how forces are transferred and influence osteoblast mechanical responses during vibrations and in providing guidance for cell culture and external vibration loading in experimental and clinical osteogenesis studies., Competing Interests: The authors declare that they have no competing interests.

Iron pyrite, an important component of traditional Chinese medicine, has a poor solubility, bioavailability, and patient compliance due to a high dose required and associated side effects, all of which have limited its clinical applications and experimental studies on its action mechanisms in improving fracture healing. This study investigated Acidithiobacillus ferrooxidans (A.f)-bioleaching of two kinds of pyrites and examined bioactivities of the derived solutions in viability and osteogenic differentiation in rat calvarial osteoblasts. A.f bioleaching improved element contents (Fe, Mn, Zn, Cu, and Se) in the derived solutions and the solutions concentration-dependently affected osteoblast viability and differentiation. While the solutions had no effects at low concentrations and inhibited the osteoblast alkaline phosphatase (ALP) activity at high concentrations, they improved ALP activity at their optimal concentrations. The improved osteoblast differentiation and osteogenic function at optimal concentrations were also revealed by levels of ALP cytochemical staining, calcium deposition, numbers and areas of mineralized nodules formed, mRNA and protein expression levels of osteogenesis-related genes (osteocalcin, Bmp-2, Runx-2, and IGF-1), and Runx-2 nuclear translocation. Data from this study will be useful in offering new strategies for improving pyrite bioavailability and providing a mechanistic explanation for the beneficial effects of pyrite in improving bone healing.

Although pulsed electromagnetic fields (PEMFs) have been approved as a therapy for osteoporosis, action mechanisms and optimal parameters are elusive. To determine the optimal intensity, exposure effects of 50 Hz PEMFs of 0.6-3.6 mT (0.6 interval at 90 min/day) were investigated on proliferation and osteogenic differentiation of cultured calvarial osteoblasts. All intensity groups stimulated proliferation significantly with the highest effect at 0.6 mT. The 0.6 mT group also obtained the optimal osteogenic effect as demonstrated by the highest ALP activity, ALP(+) CFU-f colony formation, nodule mineralization, and expression of COL-1 and BMP-2. To verify our hypothesis that the primary cilia are the cellular sensors for PEMFs, osteoblasts were also transfected with IFT88 siRNA or scrambled control, and osteogenesis-promoting effects of 0.6 mT PEMFs were found abrogated when primary cilia were inhibited by IFT88 siRNA. Thus primary cilia of osteoblasts play an indispensable role in mediating PEMF osteogenic effect in vitro., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Noninvasive electromagnetic fields (EMFs) have been known to be able to improve bone health; however, their optimal application parameters and action mechanisms remain unclear. This study compared the effects of different forms of EMFs (sinusoidal, triangular, square, and serrated, all set at 50 Hz frequency and 1.8 mT intensity) on proliferation, differentiation and mineralization of rat calvarial osteoblasts. Square EMFs stimulated osteoblast proliferation but sinusoidal EMFs inhibited it. Sinusoidal and triangular EMFs produced significantly greater alkaline phosphatase (ALP) activity, ALP staining areas, calcium deposition, mineralized nodule areas, and mRNA expression of Runx-2, osteoprotegerin and insulin-like growth factor-I than square and serrated EMFs (P < 0.01). Triangular EMFs had a greater effect than sinusoidal EMFs on every indices except for Runx-2 mRNA expression (P < 0.05). These results indicated that while square EMFs promoted proliferation and had no effect on the differentiation of osteoblasts, sinusoidal EMFs inhibited proliferation but enhanced osteogenic differentiation. Triangular EMFs did not affect cell proliferation but induced the strongest osteogenic activity among the four waveforms of EMFs. Thus, the effects of EMFs on proliferation and differentiation of osteoblasts in vitro were dependent on their waveforms., (Copyright © 2013 Wiley Periodicals, Inc.)

An effective method for preventing bone loss is by promoting osteoblast differentiation and bone formation. While dexamethasone has been routinely used as a classical inducer for osteoblast differentiation, limitations have been observed with its usage, including its varied effects on expression of osteoblast genes in different species and its potentials in suppressing osteoblastic differentiation and mineralization. In this study, we assessed the ability of flavonoid icariin in enhancing differentiation and mineralization of cultured rat primary osteoblasts in the absence of dexamethasone. It was found that, compared to the non-stimulated control, icariin at 10(-5) M produced a higher alkaline phosphatase activity, more and larger areas of alkaline phosphatase-positive colonies (CFU-FALP) and mineralized nodules, more osteocalcin secretion and calcium deposition, higher levels of mRNA expression of alkaline phosphatase, osteoblastic transcription factors osterix and runt-related transcription factor 2, and collagen 1α, higher levels of protein expression of collagen 1α, alkaline phosphatese, osterix, and runt-related transcription factor 2. In addition, icariin at 10(-5) M was always more potent than dexamethasone at its optimal concentration of 10(-8) M on the above osteoblast differentiation and mineralization markers. Taken together, our studies demonstrated that icariin has a pronounced ability in promoting osteoblast differentiation in vitro in the absence of dexamethasone., (Georg Thieme Verlag KG Stuttgart · New York.)

It is increasingly evident that micronutrient environment experienced before birth and in infancy is important for achieving optimal bone mass by adolescence and maintaining bone health. This study determined whether maternal supplementation with ω3-polyunsaturated fatty acids (n3FA) improved offspring bone growth and adult bone mass. Female rats were fed a diet containing 0.1% (control, n = 10) or 1% (n3FA, n = 11) docosahexanoic acid (DHA) during pregnancy and lactation. Offspring were weaned onto a control rat chow diet. Tibial growth plate and metaphysis structure, osteoblast/osteoclast density and differentiation, and gene expression were assessed in offspring at 3 wk (weaning), 6 wk (adolescent), and 3 months (adult). Maternal n3FA supplementation elevated offspring plasma n3FA levels at 3 and 6 wk. Although total growth plate heights were unaffected at any age, the resting zone thickness was increased in both male and female offspring at 3 wk. In n3FA males, but not females, bone trabecular number and thickness were increased at 3 wk but not other ages. The wk 3 n3FA males also exhibited an increased bone volume, an increased osteoblast but decreased osteoclast density, and lower expression of osteoclastogenic cytokines receptor activator of nuclear factor-κB ligand, TNF-α, and IL-6. No effects were seen at 6 wk or 3 months in either sex. Thus, perinatal n3FA supplementation is associated with increased bone formation, decreased resorption, and a higher bone mass in males, but not in females, at weaning; these effects do not persist into adolescence and adulthood and are unlikely to produce lasting improvements in bone health.

Electromagnetic fields (EMFs) have been used clinically to slow down osteoporosis and promote fracture healing for many years. However, the underlying action mechanisms and optimal parameters of the EMF applications are unclear. In this study, we investigated the effects of treatment for different durations with 50 Hz sinusoidal electromagnetic fields (SEMFs) at different intensities on proliferation, differentiation and mineralization potentials of rat osteoblasts. Osteoblasts isolated from neonatal rats were treated with SEMFs (50 Hz at 0.9 mT-4.8 mT, 0.3 mT interval, 30 min/day up to 15 days). Compared to untreated control, SEMFs inhibited osteoblast proliferation (after 3 days' treatment) but increased alkaline phosphatase (ALP) activity (after treatment for 9 days) from 0.9 mT to 1.8 mT, declined from 1.8 mT until 3.0 mT, and then increased again from 3.0 mT to 3.6 mT and decreased once again from 3.6 mT to 4.8 mT. Numbers of colonies stained positive for ALP after 8 days and mineralized nodules stained by Alizarin red after 10 days showed the same bimodal tendency as with the ALP activity, with two peaks at 1.8 mT and 3.6 mT. SEMFs also bimodally increased Runx-2, Col1α2 and Bmp-2 mRNA expression levels in osteoblasts at 12, 24 and 96 h after exposure. The results indicated that while exposure to 50 Hz SEMFs inhibits the osteoblast proliferation, it significantly promotes differentiation and mineralization potentials of osteoblasts in an intensity-dependent manner with peak activity at 1.8 mT and 3.6 mT., (Copyright © 2011 Elsevier Inc. All rights reserved.)

There has been a strong interest in searching for natural therapies for osteoporosis. Genistein, an isoflavone abundant in soy, and icariin, a prenylated flavonol glycoside isolated from Epimedium Herb, have both been identified to exert beneficial effects in preventing postmenopausal bone loss. However, the relative potency in osteogenesis between the individual phytoestrogen flavonoids remains unknown. The present study compared ability of genistein and icariin in enhancing differentiation and mineralization of cultured rat calvarial osteoblasts in vitro. Dose-dependent studies in osteoblast differentiation measuring alkaline phosphatase (ALP) activity revealed optimal concentrations of genistein and icarrin for stimulating osteogenesis to be both at 10(-5) M. Time course studies comparing the two compounds both at 10(-5) M demonstrated that icariin treatment always produced higher ALP activity, more and larger areas of CFU-F(ALP) colonies and mineralized nodules, more osteocalcin secretion, and calcium deposition, and a higher level of mRNA expression of osteogenesis-related genes COL1α2, BMP-2, OSX, and RUNX-2. However, they inhibited the proliferation of osteoblasts to a similar degree. In conclusion, although future in vivo studies are required to investigate whether icariin is more efficient in improving bone mass and/or preventing bone loss, our in vitro studies have demonstrated that icariin has a stronger osteogenic activity than genistein. In addition, while the prenyl group on C-8 of icariin could be the active group that takes part in osteoblastic differentiation and explains its greater potency in osteogenesis, mechanisms of action, and reasons for the relative potency of icariin versus genistein need to be further studied., (Copyright © 2010 Wiley-Liss, Inc.)

The molecular mechanism for the microgravity‐induced decrease in bone formation remains unclear and there is a lack of effective specific preventative therapies. We recently reported that primary cilia of osteoblasts became shorter and even disappeared when the cells were exposed to random positioning machine (RPM)‐simulated microgravity and that the microgravity‐induced loss of osteogenic potential of osteoblasts could be attenuated when the resorption of primary cilia was prevented by treatment with 0.1 μM cytochalasin D. In the current study, it was further found that the loss of the osteogenic capacity of rat calvarial osteoblasts (ROBs) was associated with the inhibition of the BMP‐2/Smad1/5/8 signalling pathway, of which most of the signalling proteins including BMP‐2, BMPRII, Smad1/5/8 and p‐Smad1/5/8 were found localized to primary cilia. Accompanying the resorption of primary cilia following the cells being exposed to simulated microgravity, the expression levels of these signalling proteins were reduced significantly. Furthermore, the expression of miRNA‐129‐3p, a microRNA previously reported to control cilium biogenesis, was found to be reduced quickly and changed in a similar tendency with the length of primary cilia. Moreover, overexpression of miRNA‐129‐3p in ROBs significantly attenuated microgravity‐induced inhibition of BMP‐2 signalling and loss of osteogenic differentiation and mineralization. These results indicated the important role of miRNA‐129‐3p in microgravity‐induced resorption of primary cilia of osteoblasts and the potential of replenishing the miRNA‐129‐3p as an effective countermeasure against microgravity‐induced loss of primary cilia and impairment of osteoblast function. [ABSTRACT FROM AUTHOR]

Methotrexate (MTX) is a commonly used antimetabolite in cancer treatment. Its intensive use is linked with skeletal adverse effects such as reduced bone formation and bone loss, and yet little information is available on molecular mechanisms underlying MTX‐induced impaired bone formation. This study investigated the effects of MTX treatment at a clinical chemotherapy relevant dose on osteogenic differentiation in MC3T3E1 osteoblastic cells. To investigate the potential mechanisms, the expression of 87 genes regulating osteoblast differentiation and bone homeostasis was screened in MTX‐treated versus untreated cells by polymerase chain reaction (PCR) arrays and results illustrated significant upregulation of Notch2 and Notch target genes at both early and late stages of MC3T3E1 differentiation following MTX treatment. To confirm the roles of Notch2 pathway and its potential action mechanisms, MC3T3E1 cells were treated with MTX with an anti‐Notch2 neutralizing antibody or control IgG and effects were examined on osteogenesis and activation of the Wnt/β‐catenin pathway. Our results demonstrated that induction of Notch2 activity is associated with MTX adverse effects on osteogenic differentiation and blocking Notch2 rescues osteoblast differentiation by preserving activation of the Wnt/β‐catenin pathway. [ABSTRACT FROM AUTHOR]

The application of pulsed electromagnetic fields (PEMFs) in the prevention and treatment of osteoporosis has long been an area of interest. However, the clinical application of PEMFs remains limited because of the poor understanding of the PEMF action mechanism. Here, we report that PEMFs promote bone formation by activating soluble adenylyl cyclase (sAC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element‐binding protein (CREB) signaling pathways. First, it was found that 50 Hz 0.6 millitesla (mT) PEMFs promoted osteogenic differentiation of rat calvarial osteoblasts (ROBs), and that PEMFs activated cAMP–PKA–CREB signaling by increasing intracellular cAMP levels, facilitating phosphorylation of PKA and CREB, and inducing nuclear translocation of phosphorylated (p)‐CREB. Blocking the signaling by adenylate cyclase (AC) and PKA inhibitors both abolished the osteogenic effect of PEMFs. Second, expression of sAC isoform was found to be increased significantly by PEMF treatment. Blocking sAC using sAC‐specific inhibitor KH7 dramatically inhibited the osteogenic differentiation of ROBs. Finally, the peak bone mass of growing rats was significantly increased after 2 months of PEMF treatment with 90 min/day. The serum cAMP content, p‐PKA, and p‐CREB as well as the sAC protein expression levels were all increased significantly in femurs of treated rats. The current study indicated that PEMFs promote bone formation in vitro and in vivo by activating sAC–cAMP–PKA–CREB signaling pathway of osteoblasts directly or indirectly. Our results indicated that 50 Hz 0.6 millitesla pulsed electromagnetic fields (PEMFs) promote osteogenic differentiation and mineralization of osteoblasts and augment peak bone mass attainment in young rats, and that PEMFs stimulate osteogenic differentiation and mineralization of osteoblasts by activating soluble adenylyl cyclase–cyclic adenosine monophosphate–protein kinase A–cAMP response element‐binding protein signaling pathway. [ABSTRACT FROM AUTHOR]

Neurotrophins and their receptors are key molecules that are known to be critical in regulating nervous system development and maintenance and have been recognized to be also involved in regulating tissue formation and healing in skeletal tissues. Studies have shown that neurotrophins and their receptors are widely expressed in skeletal tissues, implicated in chondrogenesis, osteoblastogenesis, and osteoclastogenesis, and are also involved in regulating tissue formation and healing events in skeletal tissue. Increased mRNA expression for neurotrophins NGF, BDNF, NT-3, and NT-4, and their Trk receptors has been observed in injured bone tissues, and NT-3 and its receptor, TrkC, have been identified to have the highest induction at the injury site in a drill-hole injury repair model in both bone and the growth plate. In addition, NT-3 has also recently been shown to be both an osteogenic and angiogenic factor, and this neurotrophin can also enhance expression of the key osteogenic factor, BMP-2, as well as the major angiogenic factor, VEGF, to promote bone formation, vascularization, and healing of the injury site. Further studies, however, are needed to investigate if different neurotrophins have differential roles in skeletal repair, and if NT-3 can be a potential target of intervention for promoting bone fracture healing. [ABSTRACT FROM AUTHOR]

Intensive cancer chemotherapy is known to cause bone defects, which currently lack treatments. This study investigated the effects of polyphenol resveratrol (RES) in preventing bone defects in rats caused by methotrexate (MTX), a commonly used antimetabolite in childhood oncology. Young rats received five daily MTX injections at 0.75 mg/kg/day. RES was orally gavaged daily for seven days prior to, and during, five-day MTX administration. MTX reduced growth plate thickness, primary spongiosa height, trabecular bone volume, increased marrow adipocyte density, and increased mRNA expression of the osteogenic, adipogenic, and osteoclastogenic factors in the tibial bone. RES at 10 mg/kg was found not to affect bone health in normal rats, but to aggravate the bone damage in MTX-treated rats. However, RES supplementation at 1 mg/kg preserved the growth plate, primary spongiosa, bone volume, and lowered the adipocyte density. It maintained expression of genes involved in osteogenesis and decreased expression of adipogenic and osteoclastogenic factors. RES suppressed osteoclast formation ex vivo of bone marrow cells from the treated rats. These data suggest that MTX can enhance osteoclast and adipocyte formation and cause bone loss, and that RES supplementation at 1 mg/kg may potentially prevent these bone defects. [ABSTRACT FROM AUTHOR]

Mechanotransduction is an important process that influences bone remodeling and maintains viability of bone cells. To understand the effect of the vibrational mechanical stimulation on biomechanic responses of bone cells, a viscoelastic osteoblast finite element (FE) model was developed. Firstly, the mode shapes and natural frequencies of a spreading osteoblast were assessed using the FE modal analysis. The osteoblast FE model predicted the natural frequencies of osteoblasts (within the range about 19.99-34.48 Hz). Then, the effect of acceleration on the vibrational responses of in-vitro cultured osteoblasts was investigated. Three different accelerations of base excitation were selected (0.15 g , 0.3 g and 0.5 g , where g = 9.8 m/s2) and the vibrational responses (displacement, strain and stress) of osteoblasts were simulated. It was found that values of displacement, strain and stress increase with the increase of base excitation acceleration. In addition, the response values in Z-direction are much higher than those in the other directions (X, Y-direction) for the same base excitation acceleration. These findings will provide useful information to understand how vibrational mechanical stimulus influences bone cells and provide guidance for in vitro cell culture and experimental research and ultimately clinical treatment using the external vibrating loading. [ABSTRACT FROM AUTHOR]

Osteocytes, the major type of bone cells which reside in their lacunar and canalicular system within the bone matrix, function as biomechanosensors and biomechanotransducers of the bone. Although biomechanical behaviour of the osteocyte-lacunar-canalicular system has been investigated in previous studies mostly using computational 2-dimensional (2D) geometric models, only a few studies have used the 3-dimensional (3D) finite element (FE) model. In the current study, a 3D FE model was used to predict the responses of strain distributions of osteocyte-lacunar-canalicular system analyzed under static and cyclic loads. The strain amplification factor was calculated for all simulations. Effects on the strain of the osteocyte system were investigated under 500, 1500, 2000, and 3000 microstrain loading magnitudes and 1, 5, 10, 40, and 100 Hz loading frequencies. The maximum strain was found to change with loading magnitude and frequency. It was observed that maximum strain under 3000-microstrain loading was higher than those under 500, 1500, and 2000 microstrains. When the loading strain reached the maximum magnitude, the strain amplification factor of 100 Hz was higher than those of the other frequencies. Data from this 3D FE model study suggests that the strain amplification factor of the osteocyte-lacunar-canalicular system increases with loading frequency and loading strain increasing. [ABSTRACT FROM AUTHOR]

Abstract: Methotrexate (MTX) is a commonly used anti-metabolite in childhood oncology and is known to cause bone growth arrest and osteoporosis; yet the underlying mechanisms for MTX-induced bone growth defects remain largely unclear. This study characterized damaging effects in young rats of acute chemotherapy with 5 once-daily doses of MTX (0.75 mg/kg) on the cellular activities in the growth plate in producing calcified cartilage and trabecular bone and on activities of osteoblastic cells in the metaphysis. MTX treatment significantly induced chondrocyte apoptosis. MTX also suppressed chondrocyte proliferation and reduced collagen-II mRNA expression and total thickness of the growth plate, with the damage being most obvious on day 9 after the first injection, and with the growth plate histological structure returning normal on day 14. In the adjacent metaphyseal bone, mirroring the decrease in the width of the growth plate, production of primary spongiosa bone was markedly reduced and bone volume of the secondary spongiosa was decreased. Furthermore, MTX treatment significantly induced osteocyte apoptosis in the primary spongiosa and reduced proliferation of osteoblasts and preosteoblasts particularly in the secondary spongiosa. These observations suggest that methotrexate chemotherapy may cause bone growth defects by arresting cellular activities in the growth plate in producing calcified cartilage and primary trabecular bone and by decreasing pools of metaphyseal osteoblastic cells. However, this short-term MTX treatment only caused transit suppressions on growth plate cartilage and trabecular bone, as most cellular and histological parameters had recovered by day 14 or 21. [Copyright &y& Elsevier]

Abstract: Introduction: Injured growth plate cartilage is often repaired by bony tissue, causing bone growth defects in children. Currently, mechanisms for the undesirable repair remain unclear and there are no biological treatments available to prevent the associated bone growth defects. Osterix is known as a vital transcription factor for osteoblast differentiation which is critical for normal bone formation and bone repair, and osterix is known to be regulated by protein kinase-D; however it is unknown whether protein kinase-D–osterix signalling plays any roles in the bony repair of injured growth plate. Methods: Using a rat model, this study investigated potential roles of protein kinase-D (PKD) in regulating expression of osteogenic transcription factor osterix and the growth plate bony repair. 4 days post injury at the proximal tibial growth plate, rats received four once-daily injections of vehicle or 2.35mg/kg gö6976 (a PKD inhibitor), and growth plate tissues collected at day 10 were examined histologically and molecularly. In addition, effects of PKD inhibition on osteogenic and chondrogenic differentiation were examined in vitro using rat bone marrow mesenchymal stromal cells. Results: Compared to vehicle control, PKD inhibition caused a decrease in bone volume (p <0.05), an increase in % of mesenchymal tissue (p <0.01), and an increase in cartilaginous tissue within the injury site. Consistently, gö6976 treatment tended to decrease expression of bone-related genes (osterix, osteocalcin) and increase levels of cartilage-related genes (Sox9, collagen-2a, collagen-10a1). In support, in vitro experiments showed that gö6976 presence in the primary rat marrow stromal cell culture resulted in a decrease of alkaline phosphatase+ CFU-f colonies formed (p <0.05) and an increase in collagen-2a expression in chondrogenic pellet culture (p <0.05). Conclusion: These studies suggest that PKD is important for growth plate bony repair and its inhibition after growth plate injury may result in less bone formation and potentially more cartilage repair. [Copyright &y& Elsevier]

Microtubule actin crosslinking factor 1 (MACF1) is a large spectraplakin protein known to have crucial roles in regulating cytoskeletal dynamics, cell migration, growth, and differentiation. However, its role and action mechanism in bone remain unclear. The present study investigated optimal conditions for effective transfection of the large plasmid PEGFP-C1A-ACF7 (∼21 kbp) containing full-length human MACF1 cDNA, as well as the potential role of MACF1 in bone formation. To enhance MACF1 expression, the plasmid was transfected into osteogenic cells by electroporation in vitro and into mouse calvaria with nanoparticles. Then, transfection efficiency, osteogenic marker expression, calvarial thickness, and bone formation were analyzed. Notably, MACF1 overexpression triggered a drastic increase in osteogenic gene expression, alkaline phosphatase activity, and matrix mineralization in vitro. Mouse calvarial thickness, mineral apposition rate, and osteogenic marker protein expression were significantly enhanced by local transfection. In addition, MACF1 overexpression promoted β-catenin expression and signaling. In conclusion, MACF1 overexpression by transfecting the large plasmid containing full-length MACF1 cDNA promotes osteoblast differentiation and bone formation via β-catenin signaling. Current data will provide useful experimental parameters for the transfection of large plasmids and a novel strategy based on promoting bone formation for prevention and therapy of bone disorders. [ABSTRACT FROM AUTHOR]

Icariin, a prenylated flavonol glycoside isolated from the herb Epimedium, has been considered as a potential alternative therapy for osteoporosis. Previous research has shown that, unlike other flavonoids, icariin is unlikely to act via the estrogen receptor, but its exact mechanism of action is unknown. In this study, using rat calvarial osteoblast culture and rat bone growth models, we demonstrated that icariin promotes bone formation by activating the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway requiring functional primary cilia of osteoblasts. We found that icariin increases the peak bone mass attained by young rats and promotes the maturation and mineralization of rat calvarial osteoblasts. Icariin activated cAMP/PKA/CREB signaling of the osteoblasts by increasing intracellular cAMP levels and facilitating phosphorylation of both PKA and CREB. Blocking cAMP/PKA/CREB signaling with inhibitors of the cAMP-synthesizing adenylyl cyclase (AC) and PKA inhibitors significantly inhibited the osteogenic effect of icariin in the osteoblasts. Icariin-activated cAMP/PKA/CREB signaling was localized to primary cilia, as indicated by localization of soluble AC and phosphorylated PKA. Furthermore, blocking ciliogenesis via siRNA knockdown of a cilium assembly protein, IFT88, inhibited icariin-induced PKA and CREB phosphorylation and also abolished icariin's osteogenic effect. Finally, several of these outcomes were validated in icariintreated rats. Together, these results provide new insights into icariin function and its mechanisms of action and strengthen existing ties between cAMP-mediated signaling and osteogenesis. [ABSTRACT FROM AUTHOR]

Abstract: From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities, which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of overexpression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis, and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how downregulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis. [Copyright &y& Elsevier]

Abstract: Growth plate cartilage is responsible for longitudinal growth of the long bone in children, and its injury is often repaired by bony tissue, which can cause limb length discrepancy and/or bone angulation deformities. Whilst earlier studies with a rat growth plate injury repair model have identified inflammatory, mesenchymal infiltration, osteogenesis and remodeling responses, the molecular mechanisms involved in the bony repair remain unknown. Since our recent microarray study has strongly suggested involvement of Wnt–β-catenin signalling pathway in regulating the growth plate repair and the pathway is known to play a crucial role in the osteogenic differentiation of mesenchymal progenitor cells, the current study investigated the potential roles of Wnt–β-catenin signalling pathway in the bony repair of injured tibial growth plate in rats. Immunohistochemical analysis of the growth plate injury site revealed β-catenin immunopositive cells within the growth plate injury site. Treatment of the injured rats with the β-catenin inhibitor ICG-001 (oral gavage at 200mg/kg/day for 8days, commenced at day 2 post injury) enhanced COL2A1 gene expression (by qRT-PCR) and increased proportion of cartilage tissue (by histological analysis), but decreased level of osterix expression and amount of bone tissue, at the injury site by day 10 post-injury (n =8, P <0.01 compared to vehicle controls). Consistently, in vitro studies with bone marrow stromal cells from normal rats showed that β-catenin inhibitor ICG-001 dose dependently inhibited expression of Wnt target genes Cyclin D1 and survivin (P <0.01). At 25mM, ICG-001 suppressed osteogenic (by CFU-f-ALP assay) but enhanced chondrogenic (by pellet culture) differentiation. These results suggest that Wnt/β-catenin signalling pathway is involved in regulating growth plate injury repair by promoting osteoblastogenesis, and that intervention of this signalling could represent a potential approach in enhancing cartilage repair after growth plate injury. [Copyright &y& Elsevier]