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In an effort to bolster our understanding of regulation of bone formation in the context of osteoporosis, we screened out differentially expressed genes in osteoporosis patients with high and low bone mineral density by bioinformatics analysis. PIK3R1 is increasingly being nominated as a pivotal mediator in the differentiation of osteoblasts and osteoclasts that is closely related to bone formation. However, the specific mechanisms underlying the way that PIK3R1 affects bone metabolism are not fully elucidated. We intended to examine the potential mechanism by which PIK3R1 regulates osteoblast differentiation. Enrichment analysis was therefore carried out for differentially expressed genes. We noted that the estrogen signaling pathway, TNF signaling pathway, and osteoclast differentiation were markedly associated with ossification, and they displayed enrichment in PIK3R1. Based on western blot, qRT-PCR, and differentiation analysis in vitro , we found that upregulation of PIK3R1 enhanced osteoblastic differentiation, as evidenced by increased levels of investigated osteoblast-related genes as well as activities of ALP and ARS, while it notably decreased levels of investigated osteoclast-related genes. On the contrary, downregulation of PIK3R1 decreased levels of osteoblast-related genes and increased levels of osteoclast-related genes. Besides, in vitro experiments revealed that PIK3R1 facilitated proliferation and repressed apoptosis of osteoblasts but had an opposite impact on osteoclasts. In summary, PIK3R1 exhibits an osteoprotective effect via regulating osteoblast differentiation, which can be represented as a promising therapeutic target for osteoporosis., Competing Interests: All authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2021 Haitao Zhu et al.)

Currently, there are no medications available to treat aseptic loosening of orthopedic implants. Using osteoprotegerin fusion protein (OPG-Fc), we previously blocked instability-induced osteoclast differentiation and peri-prosthetic osteolysis. Wnt/β-catenin signaling, which regulates OPG secretion from osteoblasts, also modulates the bone tissue response to mechanical loading. We hypothesized that activating Wnt/β-catenin signaling by inhibiting glycogen synthase kinase-3β (GSK-3β) would reduce instability-induced bone loss through regulation of both osteoblast and osteoclast differentiation. We examined effects of GSK-3β inhibition on regulation of RANKL and OPG in a rat model of mechanical instability-induced peri-implant osteolysis. The rats were treated daily with a GSK-3β inhibitor, AR28 (20 mg/kg bw), for up to 5 days. Bone tissue and blood serum were assessed by qRT-PCR, immunohistochemistry, and ELISA on days 3 and 5, and by micro-CT on day 5. After 3 days of treatment with AR28, mRNA levels of β-catenin, Runx2, Osterix, Col1α1, and ALP were increased leading to higher osteoblast numbers compared to vehicle-treated animals. BMP-2 and Wnt16 mRNA levels were downregulated by mechanical instability and this was rescued by GSK-3β inhibition. Osteoclast numbers were decreased significantly after 3 days of GSK-3β inhibition, which correlated with enhanced OPG mRNA expression. This was accompanied by decreased serum levels of TRAP5b on days 3 and 5. Treatment with AR28 upregulated osteoblast differentiation, while osteoclastogenesis was blunted, leading to increased bone mass by day 5. These data suggest that GSK-3β inactivation suppresses osteolysis through regulating both osteoblast and osteoclast differentiation in a rat model of instability-induced osteolysis., (© 2017 Wiley Periodicals, Inc.)

The roles of prolyl hydroxylase domain proteins (PHDs) in bone are incompletely understood. Here we deleted the expression of genes encoding PHD1, PHD2, and PHD3 in osteoblasts in mice by breeding the floxed Phd1-3 mice with Col1a1-Cre transgenic mice. Results showed that mice lacking PHD1-3 in osteoblasts (Phd1-3 ob-/- ) had increased bone mass. Bone parameters such as bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were increased, while trabecular spacing (Tb.Sp) was decreased in Phd1-3 ob-/- relative to wild-type (WT) femurs. In contrast, loss of PHD1-3 in osteoblasts did not alter cortical thickness (Cort.Th). The mineralization apposition rate (MAR) was increased in Phd1-3 ob-/- bone compared to that of wild-type (WT) bone, demonstrating an enhancement of osteoblast function. Loss of PHD1-3 increased the number of osteoblast progenitors (CFU-OBs) in bone marrow cultures. Interestingly, deleting Phd1-3 genes in osteoblasts increased osteoclast formation in vitro and in bone., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Due to the reduction in bone mass and deterioration in bone microarchitecture, osteoporosis is an important risk factor for impairing implant osseointegration. Recently, low-magnitude, high-frequency (LMHF) vibration (LM: <1×g; HF: 20-90Hz) has been shown to exhibit anabolic, but anti-resorptive effects on skeletal homeostasis. Therefore, we hypothesized that LMHF loading, in terms of whole body vibration (WBV), may improve implant fixation under osteoporotic status. In the in vivo study, WBV treatment (magnitude: 0.3g, frequency: 40Hz, time: 30min/12h, 5days/week) was applied after hydroxyapatite-coated titanium implants were inserted in the bilateral tibiae of ovariectomized rats. The bone mass and the osteospecific gene expressions were measured at 12weeks post implantation. In the in vitro study, the cellular and molecular mechanisms underlying osteoblastic and osteoclastic activities were fully investigated using various experimental assays. Micro-CT examination showed that WBV could enhance osseointegration by improving microstructure parameters surrounding implants. WBV-regulated gene levels in favor of bone formation over resorption may be the reason for the favorable adaptive bone remolding on bone-implant surface. The in vitro study showed that vibration (magnitude: 0.3g, frequency: 40Hz, time: 30min/12h) up-regulated osteoblast differentiation, matrix synthesis and mineralization. However, mechanically regulated osteoclastic activity was mainly through the effect on osteoblastic cells producing osteoclastogenesis-associated key soluble factors, including RANKL and M-CSF. Osteoblasts were therefore the direct target cells during the mechanotransduction process. The ERK1/2 pathway was demonstrated to play an essential role in vibration-induced enhancement of bone formation and decreased bone resorption. Our data suggests that WBV was a helpful non-pharmacological intervention for improving osseointegration under osteoporosis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

While it has been well-documented that excessive fluoride exposure caused the skeletal disease and osteoblasts played a critical role in the advanced skeletal fluorosis, the underlying mechanism that mediated these effects remain poorly understood. The present study was undertaken to examine the effect of fluoride on bone of rats and MC3T3-E1 cells in vitro. Herein we found pathological features of high bone turnover in fluoride-treated rats, which was supported by an increase of osteogenic and osteoclastogenic genes expression in different stages of fluoride exposure. The skeletal toxicity of fluoride was accompanied by activation of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR). A novel finding of this study was that expression of PKR-like endoplasmic reticulum kinase (PERK) was the same trend with receptor activator for nuclear factor-κ B ligand (RANKL), and NF-E2 p45-related factor 2 (Nrf2) was the same trend with Runt-related transcription factor 2 (Runx2) in bones of rats exposed to varied fluoride condition. Based on these data, we hypothesized that up-regulation of PERK probably played a role in mediating bone turnover induced by fluoride. Action of fluoride on MC3T3-E1 cells differentiation was demonstrated through analysis of alkaline phosphatase (ALP) activity and mineralized nodules formation. Meantime, an increase of binding immunoglobulin protein (BiP) expression indicated the active ER stress in cells exposed to various dose of fluoride. Blocking PERK expression using siRNA showed the obvious decrease of osteogenic and osteoclastogenic factors expression in MC3T3-E1 cells exposed to certain dose of fluoride that could positively stimulate osteoblastic viability. In conclusion these findings underscore the importance of PERK in modulating fluoride induced bone formation and bone resorption. Understanding the link between PERK and bone turnover could probe into the mechanism underlying different bone lesion of skeletal fluorosis., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Summary: The basic mechanisms by which strontium ranelate acts on bone are still unclear. We show that an important action of strontium salts is to block calcification in cultures of osteoblasts, the bone-forming cells. These results suggest that strontium treatment could have previously overlooked effects on bone., Introduction: The basic mechanisms of action of strontium ranelate (SrR) on bone have remained unclear. We studied the direct actions of Sr(2+) salts in functional cultures of osteoblasts and osteoclasts., Methods: Cultures of primary osteoblasts from rat calvariae and osteoclast-forming mouse marrow cells were treated continuously with either SrR or strontium chloride (SrCl2)., Results: Abundant, discretely mineralised 'trabecular' bone structures formed in control osteoblast cultures after 14 days. SrR at 0.01, 0.1 and 1 mM inhibited mineralisation to 59, 98 and 100 % (all p < 0.001) of control values, respectively. SrCl2 at the same concentrations caused similar inhibitions. Osteoblast cell numbers and alkaline phosphatase activity were unaltered. SrR dose-dependently reduced the formation of multinucleated osteoclasts from marrow mononuclear cells cultured on dentine for 8 days in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL), with a 50 % inhibition occurring at 1 mM; SrCl2 was slightly less effective, eliciting a maximal 30 % inhibition. Corresponding decreases in total resorption pit formation were observed, suggesting Sr(2+) salts affect osteoclast formation rather than resorptive activity., Conclusion: Our findings are consistent with the documented physicochemical inhibitory action of Sr(2+) on mineralisation but contrast with reports that Sr(2+) increases osteoblast activity and number in vitro. Our results suggest that rather than acting as an agent that 'uncouples' bone formation and resorption, Sr(2+) acts as a global inhibitor of bone cell function, with particularly marked effects on mineralisation. The potential effects of long-term Sr(2+) on secondary mineralisation in bone deserve investigation.

The periprosthetic osteolysis induced by wear particles contributes to aseptic loosening after joint arthroplasty. The molecular mechanism underlying osteolysis remains to be described. In this study, cultured MC3T3-E1 cells were incubated with titanium particles. We investigated the role of p38 mitogen-activated protein kinase in the expression of MMP-2 in response to wear particles. Our results demonstrated MC3T3-E1 cells exposed to titanium particles had significantly increased levels of MMP-2 and MT1-MMP mRNA, whereas the TIMP-2 mRNA level was unchanged. In MC3T3-E1 cells, the protein expression of MMP-2, MT1-MMP, and active p38 was also elevated after titanium particle exposure, as detected by Western blot and Biotrak activity analyses. Inhibition studies showed that the specific p38 inhibitor SB203580 completely abrogated the increase in MMP-2 and MT1-MMP production induced by the titanium particles. Moreover, our results revealed that conditioned media-stimulated osteoclast formation was related to the MMP-2 activity of osteoblasts that were challenged with Ti particles. This study demonstrated that p38 signaling is required for MMP-2 activity in osteoblasts under wear particle-induced conditions. MMP-2 could act as a catabolic element or a proinflammatory factor contributing to periprosthetic osteolysis. Therefore, the p38 pathway and MMP-2 may play a critical role in the development of aseptic loosening., (© 2013 Wiley Periodicals, Inc.)

In recent years a paradigm shift in understanding of human bone formation has occurred that starts to change current concepts in tissue engineering of bone and cartilage. New discoveries revealed that fundamental steps in biomineralization are enzyme driven, not only during hydroxyapatite deposition, but also during initial bioseed formation, involving the transient deposition and subsequent transformation of calcium carbonate to calcium phosphate mineral. The principal enzymes mediating these reactions, carbonic anhydrase and alkaline phosphatase, open novel targets for pharmacological intervention of bone diseases like osteoporosis, by applying compounds acting as potential activators of these enzymes. It is expected that these new findings will give an innovation boost for the development of scaffolds for bone repair and reconstruction, which began with the use of bioinert materials, followed by bioactive materials and now leading to functional regenerative tissue units. These new developments have become possible with the discovery of the morphogenic activity of bioinorganic polymers, biocalcit, bio-polyphosphate and biosilica that are formed by a biogenic, enzymatic mechanism, a driving force along with the development of novel rapid-prototyping three-dimensional (3D) printing methods and bioprinting (3D cell printing) techniques that may allow a fabrication of customized implants for patients suffering in bone diseases in the future.

Bone remodeling, a physiological process characterized by bone formation by osteoblasts (OBs) and resorption of preexisting bone matrix by osteoclasts (OCs), is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature, osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence, anabolic therapeutics that stimulate bone accrual is in high clinical demand. Here we identify Ca²⁺/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics because its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones, along with significantly more OBs and fewer multinuclear OCs. In vitro, although Camkk2⁻/⁻ mesenchymal stem cells (MSCs) yield significantly higher numbers of OBs, bone marrow cells from Camkk2⁻/⁻ mice produce fewer multinuclear OCs. Acute inhibition of CaMKK2 by its selective, cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further, we find phospho-protein kinase A (PKA) and Ser¹³³ phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand, genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target, nuclear factor of activated T cells, cytoplasmic (NFATc1). Moreover, in vivo administration of STO-609 results in increased OBs and diminished OCs, conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall, our findings reveal a novel function for CaMKK2 in bone remodeling and highlight the potential for its therapeutic inhibition as a valuable bone anabolic strategy that also inhibits OC differentiation in the treatment of osteoporosis., (Copyright © 2013 American Society for Bone and Mineral Research.)

Although previous studies have demonstrated that hydrogen sulfide (H(2)S) stimulated or inhibited osteoclastic differentiation, little is known about the effects of H(2)S on the differentiation of osteoblasts and osteoclasts. To determine the possible bioactivities of H(2)S on bone metabolism, we investigated the in vitro effects of H(2)S on cytotoxicity, osteoblastic, and osteoclastic differentiation as well as the underlying mechanism in lipopolysaccharide (LPS) and nicotine-stimulated human periodontal ligament cells (hPDLCs). The H(2)S donor, NaHS, protected hPDLCs from nicotine and LPS-induced cytotoxicity and recovered nicotine- and LPS-downregulated osteoblastic differentiation, such as alkaline phosphatase (ALP) activity, mRNA expression of osteoblasts, including ALP, osteopontin (OPN), and osteocalcin (OCN), and mineralized nodule formation. Concomitantly, NaHS inhibited the differentiation of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in mouse bone marrow cells and blocked nicotine- and LPS-induced osteoclastogenesis regulatory molecules, such as RANKL, OPG, M-CSF, MMP-9, TRAP, and cathepsin K mRNA. NaHS blocked nicotine and LPS-induced activation of p38, ERK, MKP-1, PI3K, PKC, and PKC isoenzymes, and NF-κB. The effects of H(2)S on nicotine- and LPS-induced osteoblastic and osteoclastic differentiation were remarkably reversed by MKP-1 enzyme inhibitor (vanadate) and expression inhibitor (triptolide). Taken together, we report for the first time that H(2)S inhibited cytotoxicity and osteoclastic differentiation and recovered osteoblastic differentiation in a nicotine- and periodontopathogen-stimulated hPDLCs model, which has potential therapeutic value for treatment of periodontal and inflammatory bone diseases., (Copyright © 2012 Wiley Periodicals, Inc.)

The aim of this study was to investigate the effect of alendronate released from chitosan scaffolds on enhancement of osteoblast functions and inhibition of osteoclast differentiation in vitro. The surface and cell morphologies of chitosan scaffolds and alendronate-loaded chitosan scaffolds were characterized by variable pressure field emission scanning electron microscope (VP-FE-SEM). Alendronate was released in a sustained manner. For evaluating osteoblast functions in MG-63 cells, we investigated cell proliferation, alkaline phosphatase (ALP) activity, and calcium deposition. Furthermore, for evaluating inhibition of osteoclast differentiation in RAW 264.7 cells, we investigated tartrate-resistant acid phosphatase (TRAP) activity, TRAP staining, and gene expressions. The in vitro studies revealed that osteoblasts grown on alendronate-loaded chitosan scaffold showed a significant increment in cell proliferation, ALP activity, and calcium deposition as compared to those grown on chitosan scaffolds. In addition, the in vitro study showed that osteoclast differentiation in RAW 264.7 cells cultured on alendronate-loaded chitosan scaffolds was greatly inhibited as compared to those cultured on chitosan scaffolds by the results of TRAP activity, TRAP staining, and gene expressions. Taken together, alendronate-loaded chitosan scaffolds could achieve the dual functions of improvement in osteoblast functions and inhibition of osteoclast differentiation. Thus, alendronate-eluting chitosan substrates are promising materials for enhancing osteoblast functions and inhibiting osteoclast differentiation in orthopedic and dental fields.

Cbl ubiquitin ligases are important molecules that control the process of ubiquitination and degradation of proteins by the proteasome. Because this process regulates several intracellular mechanisms, alterations in Cbl activity lead to several pathologies including cancer. In bone, the c-Cbl ubiquitin ligase is known to control osteoclast activity. Our studies indicate that c-Cbl also regulates osteoblast proliferation, differentiation and survival. We recently showed that inhibition of c-Cbl activity using a c-Cbl mutant leads to promote osteoblast differentiation in mesenchymal stromal cells as a consequence of increased receptor tyrosine kinase expression. Conversely, we found that overexpression of c-Cbl leads to inhibit osteosarcoma cell proliferation and tumorigenesis through downregulation of these receptors. Thus, the use of pharmacological agents capable of modulating c-Cbl activity may be of therapeutic interest for promoting bone formation in normal bone, or to reduce tumorigenesis in primary bone cancer., (© 2012 médecine/sciences – Inserm / SRMS.)

Objective: To investigate the effect of osteoclast bone resorption supernatants on the osteogenic activity of mouse MC3T3-E1 cell line., Methods: Mouse RAW264.7 cell line was induced to osteoclast which was identified with tartrate resistant acid phosphatase (TRAP) staining and osteoclast specific gene detection. The differentiated RAW264.7 osteoclast was co-cultured with bovine milling bone specimen followed by toluidine blue staining. Then mouse MC3T3-E1 cell was cultured with supernatant from the osteoclast bone absorbent model. Methyl thiazolyl tetrazolium (MTT) method, alizarin red S staining, enzyme-linked immunosorbent assay detection of osteocalcin, and reverse transcriptase polymerase chain reaction detection were adopted to investigate the proliferation, calcification and osteogenic activity of MC3T3-E1 cells., Results: TRAP staining, osteoclast specific gene detection and toluidine blue staining all indicated that RAW264.7 cell could be differentiated into functioning osteoclast. The supernatant from the osteoclast bone absorbent model could inhibit the proliferation of MC3T3-E1 cells, with the A value between 0.062 ± 0.004 and 0.405 ± 0.033 (P < 0.05). It could also increase the formation of calcification nods, promote the osteocalcin level which peaked with the tenth day's supernatant at a level of (2.965 ± 0.047) µg/L, as well as enhance the transcription of the alkaline phosphatase and Runt related transcription factor 2 gene., Conclusions: RAW264.7 osteoclast bone absorbent supernatant might influence the osteogenic activity of osteoblast-like cell by inhibiting proliferation, promoting differentiation and calcification.

Farnesyl pyrophosphate synthase (FPPS), an enzyme in the mevalonate pathway, is the inhibition target of alendronate, a potent FDA-approved nitrogen-containing bisphosphonate (N-BP) drug, at the molecular level. Alendronate not only inhibits osteoclasts but also has been reported to positively affect osteoblasts. This study assesses the knockdown effects of siRNA targeting FPPS compared with alendronate in both osteoclast and osteoblast cultures. Primary murine bone marrow cell-induced osteoclasts and the preosteoblast MC3T3-E1 cell line were used to assess effects of anti-FPPS siRNA compared with alendronate. Results show that both FPPS mRNA message and protein knockdown in serum-based culture is correlated with reduced osteoclast viability. FPPS siRNA is more potent than 10 μM alendronate, but less potent than 50 μM alendronate on reducing osteoclast viability. Despite FPPS knockdown, no significant changes were observed in osteoblast proliferation. FPPS knockdown promotes osteoblast differentiation significantly but not cell mineral deposition. However, compared with 50 μM alendronate dosing, FPPS siRNA does not exhibit cytotoxic effects on osteoblasts while producing significant effects on ostoblast differentiation. Both siRNA and alendronate at tested concentrations do not have significant effects on cultured osteoblast mineralization. Overall, results indicate that siRNA against FPPS could be useful for selectively inhibiting osteoclast-mediated bone resorption and improving bone mass maintenance by influencing both osteoclasts and osteoblasts in distinct ways.

High maternal blood glucose levels caused by diabetes mellitus can irreversibly lead to maldevelopment of the growing fetus with specific effects on the skeleton. To date, it remains controversial at which stage embryonic development is affected. Specifically during embryonic bone development, it is unclear whether diminished bone mineral density is caused by reduced osteoblast or rather enhanced osteoclast function. Therefore, the aim of this study was to characterize the growth as well as the skeletal differentiation capability of pluripotent embryonic stem cells (ESCs), which may serve as an in vitro model for all stages of embryonic development, when cultured in diabetic levels of D-glucose (4.5 g/L) versus physiological levels (1.0 g/L). Results showed that cells cultivated in physiological glucose gave rise to a higher number of colonies with an undifferentiated character as compared to cells grown in diabetic glucose concentrations. In contrast, these cultures were characterized by slightly decreased expression of proteins associated with the stem cell state. Furthermore, differentiation of ESCs into osteoblasts and osteoclasts was favored in physiological glucose concentrations, demonstrated by an increased matrix calcification, enhanced expression of cell-type-specific mRNAs, as well as activity of the cell-type-specific enzymes, alkaline, and tartrate resistant acidic phosphatase. In fact, this pattern was noted in murine as well as in primate ESCs. Our study suggests that an interplay between both the osteoblast and the osteoclast lineage is needed for proper skeletal development to occur, which seems impaired in hyperglycemic conditions.

The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that couples ATP hydrolysis to proton pumping across membranes. Recently, there is increasing evidence that V-ATPase may contribute to the pathogenesis of bone resorption disorders due to it is predominantly expressed in osteoclasts also function in bone resorption making it a good candidate in a therapeutic target for osteoporosis. Osteoclasts are capable of generating an acidic microenvironment necessary for bone resorption by utilizing V-ATPases to pump protons into the resorption lacuna. In addition, it has been shown that therapeutic interventions have been proposed that specifically target inhibition of the osteoclast proton pump. Modulation of osteoclastic V-ATPase activity has been considered to be a suitable therapy for the treatment of osteoporosis. All theses findings suggest that V-ATPase have important biological effects in bone resorption that might be a promising therapeutic target for osteoporosis. In this review, we will briefly discuss the biological features of osteoporosis and summarize recent advances on the role of V-ATPase in the pathogenesis and treatment of osteoporosis.

Bioactive glasses (BG) which contain strontium have the potential to combine the known bone regenerative properties of BG with the anabolic and anti-catabolic effects of strontium cations. Here we created a BG series (SiO(2)-P(2)O(5)-Na(2)O-CaO) in which 0-100% of the calcium was substituted by strontium and tested their effects on osteoblasts and osteoclasts in vitro. We show that ions released from strontium-substituted BG enhance metabolic activity in osteoblasts. They also inhibit osteoclast activity by both reducing tartrate resistant acid phosphatase activity and inhibiting resorption of calcium phosphate films in a dose-dependent manner. Additionally, osteoblasts cultured in contact with BG show increased proliferation and alkaline phosphatase activity with increasing strontium substitution, while osteoclasts adopt typical resorption morphologies. These results suggest that similarly to the osteoporosis drug strontium ranelate, strontium-substituted BG may promote an anabolic effect on osteoblasts and an anti-catabolic effect on osteoclasts. These effects, when combined with the advantages of BG such as controlled ion release and delivery versatility, may make strontium-substituted BG an effective biomaterial choice for a range of bone regeneration therapies., (Copyright 2010 Elsevier Ltd. All rights reserved.)

We investigated the interplay between parathyroid hormone (PTH) and phosphodiesterases (PDEs) in osteoblasts. PDE4 negatively regulated PTH-induced cAMP accumulation. PDE4 inhibitor enhanced PTH-induced osteoclast formation and RANKL mRNA expression, which is partially mediated by COX-2 mRNA expression. Two CRE sites in the COX-2 promoter were required for the increase in COX-2 transcription by PDE4 inhibitor, and the expression of a dominant-negative form of CREB abolished COX-2 mRNA expression in response to PDE4 inhibitor or PTH in osteoblasts. Taken together, our data indicate that PDE4 inhibitor promotes PTH-induced osteoclast formation partially via CRE-mediated COX-2 mRNA expression.

Signaling through phosphatidylinositol-3 kinases (PI3K) regulates fundamental cellular processes such as survival and growth, and these lipid kinases are currently being investigated as therapeutic targets in several contexts. In skeletal tissue, experiments using pan-specific PI3K inhibitors have suggested that PI3K signaling influences both osteoclast and osteoblast function, but the contributions of specific PI3K isoforms to these effects have not been examined. In the current work, we assessed the effects of pharmacological inhibitors of the class Ia PI3Ks, alpha, beta, and delta, on bone cell growth, differentiation and function in vitro. Each of the class Ia PI3K isoforms is expressed and functionally active in bone cells. No consistent effects of inhibitors of p110-beta or p110-delta on bone cells were observed. Inhibitors of p110-alpha decreased osteoclastogenesis by 60-80% (p<0.001 vs control) by direct actions on osteoclast precursors, and decreased the resorptive activity of mature osteoclasts by 60% (p<0.01 vs control). The p110-alpha inhibitors also decreased the growth of osteoblastic and stromal cells (p<0.001 vs control), and decreased differentiated osteoblast function by 30% (p<0.05 vs control). These data suggest that signaling through the p110-alpha isoform of class Ia PI3Ks positively regulates the development and function of both osteoblasts and osteoclasts. Therapeutic agents that target this enzyme have the potential to significantly affect bone homeostasis, and evaluation of skeletal endpoints in clinical trials of such agents is warranted., (Copyright 2009 Elsevier Inc. All rights reserved.)

Osteoblasts and chondrocytes arise from common osteo-chondroprogenitor cells. We show here that inactivation of ERK1 and ERK2 in osteo-chondroprogenitor cells causes a block in osteoblast differentiation and leads to ectopic chondrogenic differentiation in the bone-forming region in the perichondrium. Furthermore, increased mitogen-activated protein kinase signaling in mesenchymal cells enhances osteoblast differentiation and inhibits chondrocyte differentiation. These observations indicate that extracellular signal-regulated kinase 1 (ERK1) and ERK2 play essential roles in the lineage specification of mesenchymal cells. The inactivation of ERK1 and ERK2 resulted in reduced beta-catenin expression, suggesting a role for canonical Wnt signaling in ERK1 and ERK2 regulation of skeletal lineage specification. Furthermore, inactivation of ERK1 and ERK2 significantly reduced RANKL expression, accounting for a delay in osteoclast formation. Thus, our results indicate that ERK1 and ERK2 not only play essential roles in the lineage specification of osteo-chondroprogenitor cells but also support osteoclast formation in vivo.

Background and Objective: Low-Level Laser Therapy (LLLT) has been suggested to improve bone tissue healing. The cellular and molecular mechanisms involved in this effect are still unclear but bone cell proliferation and differentiation alteration have been proposed. The aim of the present study was to investigate, in vitro, the effect of LLLT on bone cell proliferation, osteoblastic and osteoclastic differentiation, both involved in bone remodeling and regeneration., Study Design/materials and Methods: Murine bone marrow cells, which contain both osteoblast and osteoclast progenitors, were cultured and induced to differentiate in the absence or in the presence of LLLT. Laser exposition parameters were determined using a powermeter and consisted in an 808 nm infrared wavelength laser light in continuous mode, with an energy density of 4 J/cm(2) administered three times a week. Cell proliferation and differentiation were assessed after specific staining and microscopic analysis of the cultures after various times, as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteoclast markers after nucleic acid extraction., Results: The use of a powermeter revealed that the power emitted by the optical fiber of the laser device was markedly reduced compared to the displayed power. This allowed to adjust the LLLT parameters to a final energy density exposure of 4 J/cm(2). In these conditions, proliferation of bone marrow mesenchymal stem cells as well as osteoclast or osteoblast differentiation of the corresponding progenitors were found similar in control and LLLT conditions., Conclusion: Using the present experimental protocol, we concluded that an 808 nm wavelength infrared LLLT does not alter murine bone progenitor cell proliferation and differentiation. Moreover our results confirm the necessary use of a powermeter to fix LLLT protocol parameters.

Glutamate, an important central excitatory neurotransmitter, is also secreted by osteoblasts and may be involved in the regulation of bone metabolism. Glutamate receptors for N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) are demonstrated in bone cells. Here we investigated the in vivo effects of glutamate by local injection of AMPA, NMDA, and their antagonists into tibia as well as their in vitro effects on the maturation of osteoblasts and formation of osteoclasts. AMPA receptor antagonist CNQX and NMDA receptor antagonist MK-801 significantly inhibited the maturation and mineralization of osteoblasts in high-glutamate alpha-MEM. On the other hand, AMPA and NMDA up-regulated the mineralized deposition and osteocalcin mRNA expression of primary osteoblasts cultured in glutamate-free DMEM. AMPA and NMDA induced the phosphorylation of extracellular signal-related kinases (ERK) in osteoblasts within 15 min. In addition, NMDA but not AMPA up-regulated the number of osteoclasts while MK801 antagonized this potentiating effect. To explore the action of glutamate agonists on bone formation in animal model, AMPA was locally injected into tibia and it was found that the bone volume in secondary spongiosa significantly increased and co-treatment of CNQX antagonized the enhancing effect of AMPA. These results suggest that glutamate may play a physiological role in regulating the maturation of osteoblasts and osteoclastogenesis. Activation of both AMPA and NMDA receptors regulates the maturation of osteoblasts. NMDA but not AMPA affects receptor for activation of NF-kappaB ligand (RANKL)-induced osteoclastogenesis.

Enhanced and deficient immune responses are associated with abnormal bone homeostasis. A new study by Shinohara et al. (2008) shows that protein phosphorylation by the tyrosine kinases Bruton and Tec links immunity and bone as well as two signaling pathways in precursors of osteoclasts, the cells that degrade bone.

Unlabelled: Several lines of evidence suggest that imatinib may affect skeletal tissue. We show that inhibition by imatinib of PDGFR signaling in osteoblasts activates osteoblast differentiation and inhibits osteoblast proliferation and that imatinib inhibits osteoclastogenesis by both stromal cell-dependent and direct effects on osteoclast precursors., Introduction: Imatinib mesylate, an orally active inhibitor of the c-abl, c-kit, and platelet-derived growth factor receptor (PDGFR) tyrosine kinases, is in clinical use for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal cell tumors. Interruption of both c-kit and c-abl signaling in mice induces osteopenia, suggesting that imatinib might have adverse effects on the skeleton. However, biochemical markers of bone formation increase in patients with CML starting imatinib therapy, whereas bone resorption is unchanged, despite secondary hyperparathyroidism. We assessed the actions of imatinib on bone cells in vitro to study the cellular and molecular mechanism(s) underlying the skeletal effects we observed in imatinib-treated patients., Materials and Methods: Osteoblast differentiation was assessed using a mineralization assay, proliferation by [(3)H]thymidine incorporation, and apoptosis by a TUNEL assay. Osteoclastogenesis was assessed using murine bone marrow cultures and RAW 264.7 cells. RT and multiplex PCR were performed on RNA prepared from human bone marrow samples, osteoblastic cells, and murine bone marrow cultures. Osteoprotegerin was measured by ELISA., Results: The molecular targets of imatinib are expressed in bone cells. In vitro, imatinib increases osteoblast differentiation and prevents PDGF-induced inhibition of this process. Imatinib inhibits proliferation of osteoblast-like cells induced by serum and PDGF. In murine bone marrow cultures, imatinib inhibits osteoclastogenesis stimulated by 1,25-dihydroxyvitamin D(3) and partially inhibits osteoclastogenesis induced by RANKL and macrophage-colony stimulating factor. Imatinib partially inhibited osteoclastogenesis in RANKL-stimulated RAW-264.7 cells. Treatment with imatinib increases the expression of osteoprotegerin in bone marrow from patients with CML and osteoblastic cells., Conclusions: Taken together with recent in vivo data, these results suggest a role for the molecular targets of imatinib in bone cell function, that inhibition by imatinib of PDGFR signaling in osteoblasts activates bone formation, and that the antiresorptive actions of imatinib are mediated by both stromal cell-dependent and direct effects on osteoclast precursors.

Bone mass and turnover are maintained by the coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts, under regulation of many systemic and local factors. Phosphoinositide-dependent serine-threonine protein kinase Akt is one of the key players in the signaling of potent bone anabolic factors. This study initially showed that the disruption of Akt1, a major Akt in osteoblasts and osteoclasts, in mice led to low-turnover osteopenia through dysfunctions of both cells. Ex vivo cell culture analyses revealed that the osteoblast dysfunction was traced to the increased susceptibility to the mitochondria-dependent apoptosis and the decreased transcriptional activity of runt-related transcription factor 2 (Runx2), a master regulator of osteoblast differentiation. Notably, our findings revealed a novel role of Akt1/forkhead box class O (FoxO) 3a/Bim axis in the apoptosis of osteoblasts: Akt1 phosphorylates the transcription factor FoxO3a to prevent its nuclear localization, leading to impaired transactivation of its target gene Bim which was also shown to be a potent proapoptotic molecule in osteoblasts. The osteoclast dysfunction was attributed to the cell autonomous defects of differentiation and survival in osteoclasts and the decreased expression of receptor activator of nuclear factor-kappaB ligand (RANKL), a major determinant of osteoclastogenesis, in osteoblasts. Akt1 was established as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover. The molecular network found in this study will provide a basis for rational therapeutic targets for bone disorders.

The 'vanishing bone' or inherited osteolysis/arthritis syndromes represent a heterogeneous group of skeletal disorders characterized by mineralization defects of affected bones and joints. Differing in anatomical distribution, severity and associated syndromic features, gene identification in each 'vanishing bone' disorder should provide unique insights into genetic/molecular pathways contributing to the overall control of skeletal growth and development. We previously described and then demonstrated that the novel autosomal recessive osteolysis/arthritis syndrome, multicentric osteolysis with arthritis (MOA) (MIM #605156), was caused by inactivating mutations in the MMP2 gene [Al Aqeel, A., Al Sewairi, W., Edress, B., Gorlin, R.J., Desnick, R.J. and Martignetti, J.A. (2000) Inherited multicentric osteolysis with arthritis: A variant resembling Torg syndrome in a Saudi family. Am. J. Med. Genet., 93, 11-18.]. These in vivo results were counterintuitive and unexpected since previous in vitro studies suggested that MMP-2 overexpression and increased activity, not deficiency, would result in the bone and joint features of MOA. The apparent lack of a murine model [Itoh, T., Ikeda, T., Gomi, H., Nakao, S., Suzuki, T. and Itohara, S. (1997) Unaltered secretion of beta-amyloid precursor protein in gelatinase A (matrix metalloproteinase 2)-deficient mice. J. Biol. Chem., 272, 22389-22392.] has hindered studies on disease pathogenesis and, more fundamentally, in addressing the paradox of how functional loss of a single proteolytic enzyme results in an apparent increase in bone loss. Here, we report that Mmp2-/- mice display attenuated features of human MOA including progressive loss of bone mineral density, articular cartilage destruction and abnormal long bone and craniofacial development. Moreover, these changes are associated with markedly and developmentally restricted decreases in osteoblast and osteoclast numbers in vivo. Mmp2-/- mice have approximately 50% fewer osteoblasts and osteoclasts than control littermates at 4 days of life but these differences have nearly resolved by 4 weeks of age. In addition, despite normal cell numbers in vivo at 8 weeks of life, Mmp2-/- bone marrow cells are unable to effectively support osteoblast and osteoclast growth and differentiation in culture. Targeted inhibition of MMP-2 using siRNA in human SaOS2 and murine MC3T3 osteoblast cell lines resulted in decreased cell proliferation rates. Taken together, our findings suggest that MMP-2 plays a direct role in early skeletal development and bone cell growth and proliferation. Thus, Mmp2-/- mice provide a valuable biological resource for studying the pathophysiological mechanisms underlying the human disease and defining the in vivo physiological role of MMP-2.

The biostimulatory effects of laser irradiation focus not only in the field of soft tissue but also bone formation. Studies have shown that the light of a nanosecond pulsed laser which has a high peak power can produce stress waves in tissue. We have hypothesized that nanosecond pulsed laser irradiation stimulates bone formation. Our aim was to clarify the mechanism of increased bone volume by nanosecond pulsed laser irradiation. Rat femur was irradiated with a Q-switched Nd:YAG laser, which has a wavelength of 1064 nm. The quantification of trabecular architecture using three-dimensional morphometric analysis and measurement of bone mineral density (BMD) using pQCT was performed on day 1, day 3, day 5, and day 7 after laser irradiation. The laser effects on bone cells were also investigated using histological and immunohistochemical analysis. On day 1 after laser irradiation, bone volume (BV/TV), trabecular thickness (Tb.Th), and other parameters of the irradiated group did not significantly differ from the non-irradiation group (control). However, the mean BV/TV, Tb.Th, mineral apposition rate, and BMD of the laser group on day 7 after laser irradiation were significantly greater than those of the control. On histological analysis, the number of TRAP-positive osteoclasts was lower on day 3 after laser irradiation. Osteoblasts with activated clearance were seen in the laser irradiated group on day 1 and day 3. These data reveal that the increased bone volume by nanosecond pulsed laser irradiation causes an increase in osteoblast activity and a decrease in osteoclast number.

Vitamin D is an important regulator of mineral homeostasis and bone metabolism. 1Alpha-hydroxylation of 25-(OH)D3 to form the bioactive vitamin D hormone, 1alpha,25-(OH)2D3, is classically considered to take place in the kidney. However, 1alpha-hydroxylase has been reported at extrarenal sites. Whether bone is a 1alpha,25-(OH)2D3 synthesizing tissue is not univocal. The aim of this study was to investigate an autocrine/paracrine function for 1alpha,25-(OH)2D3 in bone. We show that 1alpha-hydroxylase is expressed in human osteoblasts, as well as the vitamin D binding protein receptors megalin and cubilin. Functional analyses demonstrate that after incubation with the 1alpha-hydroxylase substrate 25-(OH)D3, the osteoblasts can produce sufficient 1alpha,25-(OH)2D3 to modulate osteoblast activity, resulting in induced alkaline phosphatase (ALP) activity, osteocalcin (OC) and CYP24 mRNA expression, and mineralization. The classical renal regulators of 1alpha-hydroxylase, parathyroid hormone, and ambient calcium do not regulate 1alpha-hydroxylase in osteoblasts. In contrast, interleukin (IL)-1beta strongly induces 1alpha-hydroxylase. Besides the bone-forming cells, we demonstrate 1alpha-hydroxylase activity in the bone resorbing cells, the osteoclasts. This is strongly dependent on osteoclast inducer RANKL. This study showing expression, activity, and functionality of 1alpha-hydroxylase unequivocally demonstrates that vitamin D can act in an auto/paracrine manner in bone.

Osteogenesis in the teleost was morphologically observed using regenerating scales of goldfish. Histological observations indicated that osteoblasts around the regenerating scales on days 7 to 10 were greater in size and number than those at other stages. Therefore, further experiments were carried out to examine the activity of osteoblasts in the regenerating period. To quantify their osteoblastic activities, scales on the left side of the body were taken, and the regenerating scales were then used to measure the activities of alkaline phosphatase (ALP), a marker of osteoblasts, on days 7, 10, and 15. The ontogenic scales on the right side of the body were also collected and used to measure ALP activity on the same days. Osteoblasts at all stages of regenerating scales were more active than those in the remaining ontogenic scales. The regenerating scales on day 10 had the highest activity. Furthermore, we found that estrogen receptor (ER) mRNA was expressed in the regenerating scales because estrogen participates in osteoblastic growth and differentiation in mammals. Therefore, using a scale culture system reported previously, the estrogenic response was examined in the ontogenic and regenerating scales on day 10. The reactivity was much higher in regenerating scales, although estrogen treatment significantly activated the osteoblastic activities in both scales. We are the first to demonstrate that ER is expressed in regenerating scales and that estrogen participates in osteogenesis as it does in mammalian bone. Our findings strongly suggest that regenerating scales can be used as a model of osteogenesis in vertebrates.

Our previous study showed that tooth germs at late embryonic stage [later than embryonic day 17.5 (E17.5)] and neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are compressed or penetrated by the surrounding alveolar bone tissue. In vivo and in vitro studies have shown that the development of the tooth germ proper is not disturbed, but insufficient alveolar bone resorption, due to the decreased number and hypofunction of osteoclasts, is the main cause of this abnormality. In addition to the insufficient alveolar bone resorption, progressive bone formation toward tooth germs was observed in homozygous mice, suggesting that accelerated bone formation also contributes to this abnormality. To further investigate this, homozygous mice at E14.0 and E15.5, when alveolar bone is forming, were used for histochemical and bone histomorphometric analyses. In contrast to the late embryonic stage, the alveolar bone did not yet compress developing tooth germs in homozygous mice on E14.0, but a larger amount of bone tissue was seen compared to wild-type littermates. Histomorphometric analysis of bone at E14.0 revealed that the osteoblast numbers and surfaces in the mandibles and in the bone collar of femora of homozygous mice were significantly higher than those of wild-type mice. However, unlike our previous study showing the osteoclast surface on E18.5 in homozygous mice to be significantly lower than that of wild-type mice, this study at E14.0 showed no significant difference between the two genotypes. To evaluate the amount of calcification around tooth germs, 3D images of mandibles were reconstructed from the calcein-labeled sections of the wild-type and mutant mice. Labeling was performed at E14.0, and the mice were sacrificed 1 h after the calcein injection to minimize the effect of bone resorption. Comparison of the 3D images revealed that the labeled surface was larger around developing tooth germs in homozygous mouse than in wild-type mouse. On day E15.5, osteoblasts approached the enamel organ of homozygous mice but this was not observed in wild-type mice. In this study, we report a systemic increase in osteoblast number and accelerated bone formation in homozygous PTHrP-knockout mice, both of which contribute to the abnormal tooth development.

Protein tyrosine phosphorylation is a major regulator of bone metabolism. Tyrosine phosphatases participate in regulating phosphorylation, but roles of specific phosphatases in bone metabolism are largely unknown. We demonstrate that young (<12 weeks) female mice lacking tyrosine phosphatase epsilon (PTPepsilon) exhibit increased trabecular bone mass due to cell-specific defects in osteoclast function. These defects are manifested in vivo as reduced association of osteoclasts with bone and as reduced serum concentration of C-terminal collagen telopeptides, specific products of osteoclast-mediated bone degradation. Osteoclast-like cells are generated readily from PTPepsilon-deficient bone-marrow precursors. However, cultures of these cells contain few mature, polarized cells and perform poorly in bone resorption assays in vitro. Podosomes, structures by which osteoclasts adhere to matrix, are disorganized and tend to form large clusters in these cells, suggesting that lack of PTPepsilon adversely affects podosomal arrangement in the final stages of osteoclast polarization. The gender and age specificities of the bone phenotype suggest that it is modulated by hormonal status, despite normal serum levels of estrogen and progesterone in affected mice. Stimulation of bone resorption by RANKL and, surprisingly, Src activity and Pyk2 phosphorylation are normal in PTPepsilon-deficient osteoclasts, indicating that loss of PTPepsilon does not cause widespread disruption of these signaling pathways. These results establish PTPepsilon as a phosphatase required for optimal structure, subcellular organization, and function of osteoclasts in vivo and in vitro.

Although type 5 TRACP is recognized as a histochemical and biochemical marker of osteoclasts, there is evidence that bone forming cells, osteoblasts, and osteocytes also express a type 5 TRACP. Accordingly, an osteoblastic type 5 TRACP has been purified from human osteoblasts and from bovine cortical bone matrices. Comparison of biochemical properties of osteoblastic type 5 TRACP with those of osteoclastic type 5 TRACP suggests that osteoblastic type 5 TRACP is a different isoenzyme from osteoclastic type 5 TRACP. Two properties of osteoblastic type 5 TRACP may be relevant to its physiological functions: (1) it acts as a protein-tyrosine phosphatase (protein tyrosine phosphorylation) under physiologically relevant conditions, and (2) it is sensitive to inhibition by clinically relevant concentrations of fluoride. Because fluoride is a stimulator of osteoblastic proliferation and differentiation and a potent osteogenic agent and because protein tyrosine phosphorylation plays an important regulatory role in cell proliferation and differentiation, these unique properties and other evidence summarized in this review led to the proposal that the osteogenic action of fluoride is mediated, at least in part, by the fluoride-mediated inhibition of osteoblastic type 5 TRACP/protein tyrosine phosphorylation, which leads to a stimulation of osteoblast proliferation and differentiation, and subsequently, an increase in bone formation.

The direct effect of bisphenol A on osteoclasts and osteoblasts was examined using a culture system of goldfish scales. Tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were used as markers of osteoclasts and osteoblasts, respectively. Bisphenol A (10(5) M) significantly suppressed both TRAP and ALP activities. These data were reproduced in an in vivo experiment. From an analysis of a reverse transcription-polymerase chain reaction in the in vitro-cultured scales, it was demonstrated that the insulin-like growth factor (IGF)-1 mRNA expression decreased by a bisphenol A treatment. On the other hand, 17beta-estradiol (E(2)) stimulated both TRAP and ALP activities and did not change IGF-1 mRNA expression, suggesting that bisphenol A has a different effect from E(2) on bone metabolism. This study is the first to demonstrate that bisphenol A functions to suppress directly osteoblasts and osteoclasts among vertebrates, which strongly suggests that this scale in an in vitro assay system can be utilized for the evaluation of the effects of endocrine disrupters on bone cells.

It is well accepted that mechanical loading inhibits bone resorption and increases in vivo bone formation. It is also known that cyclic mechanical loading, in particular, can enhance bone formation significantly. These findings suggest a significant role for mechanical stimuli in bone remodelling mediated by various local growth factors including insulin-like growth factor-I (IGF-I). Earlier studies showed that the nasal bone length and premaxillary bone width were significantly greater in mice fed a solid diet rather than a granulated diet, and that these dimensions increased significantly in a solid-diet group treated with IGF-I. The present study sought to examine the effect of IGF-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture subjected to different masticatory loadings. For the solid-diet groups, the numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells and osteoblasts were significantly greater in the group injected with IGF-I than in the animals injected with physiological saline. In the groups fed a granulated diet, no significant differences in the numbers of TRAP-positive osteoclastic cells and osteoblasts were found over the entire experimental period between mice injected with either IGF-I or physiological saline. It is shown that IGF-I significantly induces the expression of osteoclasts and osteoblasts and the subsequent bone remodelling, and that the effect may be additive as compared to that of mechanical masticatory loading, which seems to be more important in bone remodelling in terms of the numbers of osteoclasts and osteoblasts.

Net activity of tissue-nonspecific alkaline phosphatase (TNAP) and acid phosphatase (ACP) remains to be determined since enzyme histochemistry has adopted biochemically determined optimal pH, which is not likely to represent local pH in vivo. The present study aimed to evaluate TNAP and ACP activities associated with bone cells at physiological pH. At the physiological pH of tissue fluid, intense phosphatase reactions were demonstrable in osteoblasts and osteoclasts as well as the bone matrix associated with osteoclasts. In fresh-frozen and freeze-substituted specimens, intense phosphatase reactions appeared at both alkaline and neutral pH along the entire surface of osteoblasts including the osteoidal surface, where TNAP was shown to be absent by immunohistochemistry. Combined specificity tests suggested that TNAP and ACP in bone cells can serve as phosphohydrolases at pH 7.3 and that reactions along the osteoidal surface of osteoblasts differ from that of TNAP and represent novel enzyme.

Osteoblasts, osteocytes and osteoclasts are specialised cells of bone that play crucial roles in the formation, maintenance and resorption of bone matrix. Bone formation and resorption critically depend on optimal intracellular calcium and phosphate homeostasis and on the expression and activity of plasma membrane transport systems in all three cell types. Osteotropic agents, mechanical stimulation and intracellular pH are important parameters that determine the fate of bone matrix and influence the activity, expression, regulation and cell surface abundance of plasma membrane transport systems. In this paper the role of ATPase pumps is reviewed in the context of their expression in bone cells, their contribution to ion homeostasis and their relation to other transport systems regulating bone turnover.

There is accumulating evidence that interleukin-1 (IL-1) levels are increased locally at the site of active bone resorption in a variety of diseases including osteoporosis, periodontal disease and rheumatoid arthritis. However, the pathogenic role of IL-1 in bone loss remains to be fully elucidated. We present here additional evidence that IL-1beta enhances endothelial activation and thereby stimulates mobilization of peripheral blood mononuclear cells (PBMCs) from luminal to abluminal spaces across the endothelium. Furthermore, IL-1beta stimulates the differentiation of PBMCs into osteoclast-like cells with bone-resorbing activity in the presence of human osteoblastic SaOS-2 cells without systemic hormones. These findings provide circumstantial evidence for the hypothesis that IL-1beta generated in the bone microenviroment plays a stimulatory role in PBMC mobilization from the peripheral circulation and their subsequent differentiation into osteoclast-like cells in the bone tissue. In addition, the present study supports the notion that osteoclast progenitor cells might be derived from the peripheral circulating blood mononuclear cells in human.

A review of histochemical and immunohistochemical studies on alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) in osteoblasts leads to the following conclusions: 1) AP is a typical (non-specific) marker of osteoblasts, and TRAP is a typical (non-specific) marker of osteoclasts; 2) both enzymes may be used to identify the precursors of these cells and are found in young osteocytes; 3) both are released into the extracellular space; 4) both are expressed by metaphyseal osteoblast-like cells whose ultrastructural characteristics are similar to those of post-osteoclastic cells of the basic multicellular unit (BMU) and are also exhibited by osteoblasts and macrophages; 5) the increased numbers of these cells in hypocalcemic animals suggests that the local calcium ion concentration may trigger the transition of the reversal into the formation phase of the BMU.

Tissue nonspecific alkaline phosphatase (TNAP) knockout (ko) mice manifest defects in bone mineralization that mimic the phenotypic abnormalities of infantile hypophosphatasia. In this article, we have searched for phenotypic differences between calvarial osteoblasts and osteoclasts in wild-type (wt), heterozygous and homozygous TNAP null mice. In vitro release of 45Ca from calvarial bones, with and without stimulation with parathyroid hormone (PTH), revealed no functional difference between osteoclasts from the three TNAP genotypes. Studies of primary cultures of TNAP+/+, TNAP+/-, and TNAP-/- calvarial osteoblasts revealed no differences in the rate of protein synthesis or in the expression levels of messenger RNAs (mRNAs) for osteopontin (OP), osteocalcin (OC), collagen type I, core binding factor alpha1 (Cbfa 1), N-cadherin, Smad 5, and Smad 7. Release of interleukin-6 (IL-6) from calvarial osteoblasts under basal conditions and after stimulation with PTH, tumor necrosis factor alpha (TNF-alpha) or IL-1beta was similar in all genotypes. The amount of cyclic adenosine monophosphate (cAMP) accumulation also was comparable. However, although cultures of primary TNAP-/- osteoblasts were able to form cellular nodules as well as TNAP positive osteoblasts do, they lacked the ability to mineralize these nodules in vitro. Mineralization also was delayed in TNAP+/- osteoblast cultures compared with cultures of wt osteoblasts. Incubation with media supplemented with recombinant TNAP, but not with enzymatically inactive TNAP, restored mineralization in ko osteoblast cultures. Our data provide evidence that osteoblasts in TNAP null mice differentiate normally but are unable to initiate mineralization in vitro. The fact that even heterozygous osteoblasts show delayed mineralization provides a rationale for the presence of bone disease in carriers of hypophosphatasia.

The development of the potential of osteoblasts to support bone resorption by osteoclasts in response to roughness on bone slices was examined in the co-incubation cell system of immature osteoclasts and osteoblastic cells. The immature osteoclasts, which need alkaline phosphatase (ALP)-positive osteoblastic cells for bone resorption, were generated in mouse spleen cultures with 1, 25-dihydroxyvitamin D(3) and prostaglandin E(2). ALP-negative osteoblastic cells from mouse calvaria were incubated on rough surfaced bone slices for 3 days. The number of ALP-positive cells increased greatly on the rough surface, but little on the smooth surface. When immature osteoclasts were added and incubated for 1 more day, the resorption pit number and the total pit areas on the smooth surface were not much different from those before incubation but were approximately four times higher on the rough surface.

This study aimed to observe the influence of the new compound-XW630 on the proliferation of osteoblast, ALP action, and the forming of osteoclast. The MTT method and the alkaline phosphatase method were adopted to investigate the influence of XW630 on ALP action and the proliferation of osteoblast cultured from grown rat's skull. The Tartrate-resistant acid phosphatase dyeing method was used to observe the influence of XW630 on forming of cultured osteoclast in vitro. The result showed that, when XW630 concentration was 10(-6)-10(-8) mol/L, it obviously enhanced the proliferation of osteoblast and improved the activity of ALP, and it evidently provented PTH from stimulating the forming of osteoclast. It is concluded that XW630 is obviously effective for stimulating the proliferation of cultivated osteoblast in vitro and for inhibiting the forming of osteoclast.

Three distinct osteoblastic cell lines (KS418, KS460, and KS483) were subcloned from the mouse osteoblastic KS-4 cells, which possessed the abilities not only to differentiate into mature osteoblasts, but also to support osteoclast differentiation in coculture with spleen cells. The order of the magnitude of the basal alkaline phosphatase (ALP) activity was KS483 > KS418 > KS460. KS483 cells were also more differentiated than KS418 and KS460 in terms of ALP activity and osteocalcin production, when cultured in growth medium containing 10% fetal bovine serum. In long-term culture, KS418 and KS483 apparently differentiated into mature osteoblasts and formed calcified nodules without addition of beta-glycerophosphate. Electron microscopic analysis demonstrated that calcification occurring in the nodules was initiated in the matrix vesicles as observed in bone formation in vivo. Nodule formation and mineral deposition occurred simultaneously in the presence of beta-glycerophosphate, but the former always preceded the latter without addition of beta-glycerophosphate. In contrast, KS460 cells did not show time-dependent increases of ALP activity, type I collagen expression and osteocalcin production, which were induced by treatment with recombinant osteogenic protein-1 (OP-1). The three cell lines similarly supported osteoclast differentiation in coculture with spleen cells in response to 1,25-dihydroxyvitamin D3. These results indicate that the three cell lines subcloned from the original KS-4 cells represent phenotypically distinct osteoblasts during osteoblast differentiation, but are equipped similarly with the capacity to support osteoclast differentiation. The subcloned cells of the KS-4 series may provide useful systems in which to study osteoblast differentiation and function.

Studies in some animal species have demonstrated the production of metalloproteinases by bone cells, suggesting that they may play a role in bone modeling and remodeling. The aim of the present study was to investigate the expression of collagenase in human bone in situ, using heterotopic and osteophytic bone. Immunohistochemistry was performed on chilled sections of bone, using well characterized polyclonal antibodies to human collagenase. The heterotopic and osteophytic bone exhibited high turnover and both bone modeling and remodeling were evident. Collagenase expression by osteoblasts was demonstrated in cells synthesising matrix and in lining cells; the strongest signal was seen in areas of de novo matrix formation, where bridges of woven bone were being formed between areas of mineralized bone. Collagenase was also present in some osteoclasts associated with eroded bone surfaces and in some mononuclear cells that were present in resorption cavities and in the bone marrow. Our results provide the first demonstration, in situ, of collagenase in human bone and suggest that it may play a role in human bone modeling and remodeling. Production of collagenase by active osteoblasts and lining cells suggest that it may be involved both in matrix formation and activation of bone remodeling. The presence of collagenase in osteoclasts provides further evidence that metalloproteinases may play a role in bone resorption.

Objective: We have recently shown that administration of long-term, low-dose methotrexate (MTX) causes severe osteopenia in female rats. This osteopenia is characterized both by decreased osteoblast function without a decrease in osteoblast numbers, and by increased bone resorption that is believed to represent a physiologic remodeling response by osteoclasts. The present study investigates the effects of varying doses of MTX on mouse bone cells in culture., Methods: Cells were obtained by sequential digestion of neonatal mouse calvariae, and cultured with fetal calf serum (10% for osteoblast-like cells and 2% for osteoclast-like cells). After 1 week, MTX was added to each culture in concentrations of 0.6 microM, 0.4 microM, 0.2 microM, 0.1 microM, 1 nM, and 0.5 nM. All experiments were done on 24 wells for each MTX concentration and for the controls. The effect on osteoblastic cells was assessed, at 7 days, by cell counts and by measurement of lysate alkaline phosphatase and supernatant osteocalcin levels, and, at 21 days, by analysis of the calcified matrix production, which was cultured with ascorbic acid and beta-glycerophosphate. For osteoclastic cells, cell count and lysate acid phosphatase levels were determined., Results: Levels of osteoblastic cells and lysate alkaline phosphatase were not changed by any of the concentrations of MTX. Matrix calcification and supernatant osteocalcin levels were diminished by MTX in a dose-responsive manner. Osteoclast-like cell numbers and acid phosphatase levels were not significantly affected by MTX., Conclusion: These results suggest that diminished mouse osteoblastic cell function occurs with very low mean concentrations of MTX, in a dose-responsive manner. The mechanism seems to be inability of the cell to synthesize and calcify matrix, possibly through defective osteocalcin production. Thus, low-dose MTX may have an important impact on bone density by slowing osteoblastic matrix production.

Prostaglandin E2 (PGE2) is an important local regulator in bone. The present study was performed to investigate the effect of PGE2 on osteoclast-like cell formation and bone-resorbing activity of mature osteoclasts in the presence or absence of osteoblasts, PGE2 (10(-8) to 10(-6) M) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although it did not affect osteoclast-like cell formation from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in osteoblast-free mouse spleen cell cultures. The conditioned medium from osteoblastic UMR-106 cells pretreated with PGE2 (10(-8) and 10(-6) M) significantly stimulated osteoclast-like cell formation from hemopoietic blast cells. PGE2 also significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures. In contrast, PGE2 significantly inhibited the bone-resorbing activity and osteopontin mRNA expression in isolated rabbit osteoclasts. Rp-cAMPS, a direct protein kinase (PKA) antagonist, significantly inhibited PGE2-stimulated osteoclast-like cell formation and the bone-resorbing activity of mature osteoclasts, although protein kinase C inhibitors, dantrolene (an inhibitor of calcium release from the intracellular calcium pool) and voltage-dependent calcium channel blockers did not affect PGE2-stimulated osteoclast-like cell formation. In conclusion, PGE2 stimulated osteoclast-like cell formation and bone-resorbing activity in mouse bone cell cultures presumably through osteoblasts. The activation of PKA is linked to PGE2-stimulated osteoclast-like cell formation and bone-resorbing activity.

Activation of osteoclasts by parathyroid hormone (PTH) is mediated by PTH stimulation of osteoblasts, and is dependent on a PTH-induced rise in protein kinase C activity. Physiological levels of insulin reduce the ability of PTH to activate protein kinase C in osteoblasts, suggesting that insulin may be a physiological antagonist of bone resorption. In addition, insulin is known to promote collagen production by osteoblasts. These findings imply that efficient insulin activity may exert an anabolic effect on bone, and rationalize the many clinical studies demonstrating reduced bone density in Type I diabetes. Recently, the insulin-sensitizing nutrient chromium picolinate has been found to reduce urinary excretion of hydroxyproline and calcium in postmenopausal women, presumably indicative of a reduced rate of bone resorption. This nutrient also raised serum levels of dehydroepiandrosterone-sulfate, which may play a physiological role in the preservation of postmenopausal bone density. The impact of chromium picolinate (alone or in conjunction with calcium and other micronutrients) on bone metabolism and bone density, merits further evaluation in controlled studies.

When bone is cultured in acidic medium produced by a reduced bicarbonate concentration ([HCO(3-)]), a model of metabolic acidosis, there is greater net calcium efflux than when the same decrement in pH is produced by an increased partial pressure of carbon dioxide (PCO2), a model of respiratory acidosis. To determine the effects of metabolic and respiratory acidosis on bone cell function we cultured neonatal mouse calvariae for 48 h under control conditions (pH approximately 7.40, PCO2 approximately 41 mmHg, [HCO(3-)] approximately 25 meq/l) or under isohydric acidic conditions simulating metabolic (pH approximately 7.09, [HCO(3-)] approximately 12) or respiratory (pH approximately 7.10, PCO2 approximately 86) acidosis and measured osteoblastic collagen synthesis and alkaline phosphatase activity and osteoclastic beta-glucuronidase activity. Collagen synthesis was inhibited by metabolic (23.2 +/- 1.3 vs. 30.3 +/- 1.0% in control) but was not altered by respiratory (32.3 +/- 0.6) acidosis. Alkaline phosphatase activity was inhibited by metabolic (402 +/- 16 vs. 471 +/- 15 nmol P.min-1.mg protein-1 in control) but not altered by respiratory (437 +/- 25) acidosis. beta-Glucuronidase activity was stimulated by metabolic (1.02 +/- 0.06 vs. 0.78 +/- 0.05 micrograms phenolphthalein released.bone-1.h-1 in control) but not altered by respiratory (0.73 +/- 0.06) acidosis. Net calcium efflux in control was increased by metabolic (783 +/- 57 vs. 20 +/- 57 nmol.bone-1.48 h-1 in control) and by respiratory (213 +/- 45) acidosis; however, calcium efflux with metabolic was greater than with respiratory acidosis.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was performed to clarify the role of high calcium concentration and the appearance of mononuclear cells at the resorptive site in bone remodeling. Our recent study revealed that the high concentration of extracellular calcium ([Ca2+]e) stimulated DNA synthesis in osteoblastic MC3T3-E1 cells not only directly but also indirectly via monocytes. Human monocyte-conditioned medium (CM) significantly stimulated DNA synthesis and inhibited alkaline phosphatase (ALP) activity. In contrast, when monocytes were cultured at high [Ca2+]e concentrations (more than 3 mM), CM from these monocytes significantly stimulated ALP activity in MC3T3-E1 cells. Such stimulatory effect of CM was not observed at a high magnesium concentration (Mg2+, 5 mM). Treatment of monocytes with the calcium ionophore A23187 did not affect the CM-induced effect on DNA synthesis and ALP activity in these cells. To determine the migration potency of MC3T3-E1 cells and monocytes toward the high [Ca2+]e, chemotaxis assay was performed. The increasing [Ca2+]e (more than 3 mM) induced a chemotactic response of MC3T3-E1 cells as well as monocytes, but the high concentration of Mg2+ (5 mM) did not induce it. On the other hand, treatment with high [Ca2+]e (more than 3 mM) or CM significantly inhibited the 1,25-(OH)2D3-induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) from their precursors derived from mouse spleen cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Sodium butyrate increased alkaline phosphatase (ALP) activity of cloned osteoblastic cell line MC3T3-E1 by the stimulation of de novo enzyme synthesis. 2. Sodium butyrate did not affect mature osteoblastic cells but affected preosteoblastic cells. 3. Sodium butyrate decreased tartrate resistant acid phosphatase (TRACP)-positive multinucleated cells (MNC) formation from bone marrow cells. This related to the cytotoxicity of sodium butyrate on bone marrow cells.

Bone alkaline and tartrate-resistant acid phosphatase activities were studied in paraffin sections obtained from experimentally induced periapical lesions of rats. These enzyme activities were demonstrated despite the use of high temperatures during embedding. In the formation phase, numerous osteoblasts and periodontal ligament cells showed strong alkaline phosphatase activity. On the other hand, a number of osteoclasts and preosteoclasts showed tartrate-resistant acid phosphatase activity in the activated resorption sites of periapical lesions. It is suggested that enzyme histochemical applications to paraffin sections are a useful means for clarifying the behavior of bone cells and that bone remodeling has occurred in the periapical lesions.