Calcium Phosphate-coated Plates Research Articles

Suppression of bone resorption in ovariectomized mice using estrogen-immobilized polyphosphodiesters

Estrogens play a crucial role in bone remodeling. In this study, we synthesized two types of bone-targeted polyphosphodiesters (PPDEs): poly(diethyl sodium phosphate) (PEP•Na) and β-estradiol 17-valerate terminated PEP•Na (E2V-PEP•Na), and determined the inhibitory effect of the terminal estrogen on ovariectomy-induced bone resorption. We first assessed the impact of these polymers on the differentiation of osteoblasts and osteoclasts in vitro. Both PPDEs effectively promoted the differentiation of mouse osteoblastic MC3T3-E1 cells, with E2V-PEP•Na significantly enhancing the mineralization of these cells. Conversely, the density of osteoclasts formed from bone marrow mononuclear cells (BMNCs) significantly decreased when exposed to E2V-PEP•Na. Additionally, the formation of resorption pits on calcium phosphate-coated plates was reduced after treating BMNCs with both PPDEs. We then investigated the role of these polymers in treating osteoporosis using ovariectomized (OVX) mice. PEP•Na or E2V-PEP•Na was separately injected into the tail vein of the mice three times at 2-week intervals, starting 1 day after OVX treatment. Two weeks after the final injection, we observed that E2V-PEP•Na was distributed in bone tissue in a dose-dependent manner. The injection of E2V-PEP•Na significantly reduced bone resorption, and all bone parameters of OVX mice treated with E2V-PEP•Na were superior to those of sham mice. In contrast, PEP•Na did not affect bone mineral metabolism. In conclusion, our study is the first to demonstrate that E2V-PEP•Na can inhibit bone resorption in vivo, offering a promising new polymeric drug for preventing osteoporosis.

2E-Decene-4,6-diyn-1-ol-acetate inhibits osteoclastogenesis through mitogen-activated protein kinase-c-Fos-NFATc1 signalling pathways.

An imbalance of osteoclasts and osteoblasts can result in a variety of bone-related diseases, including osteoporosis. Thus, decreasing the activity of osteoclastic bone resorption is the main therapeutic method for treating osteoporosis. 2E-Decene-4, 6-diyn-1-ol-acetate (DDA) is a natural bioactive compound with anti-inflammatory and anti-cancer properties. However, its effects on osteoclastogenesis are unknown. Murine bone marrow-derived macrophages (BMMs) or RAW264.7 cells were treated with DDA, followed by evaluation of cell viability, RANKL-induced osteoclast differentiation, and pit formation assay. Effects of DDA on RANKL-induced phosphorylation of MAPKs were assayed by western blot analysis. Expression of osteoclast-specific genes was examined with reverse transcription-PCR (RT-PCR) and western blot analysis. In this study, DDA significantly inhibited RANKL-induced osteoclast differentiation in RAW264.7 cells as well as in BMMs without cytotoxicity. DDA also strongly blocked the resorbing capacity of BMM on calcium phosphate-coated plates. DDA inhibited RANKL-induced phosphorylation of ERK, JNK and p38MAPKs, as well as expression of c-Fos and NFATc1, which are essential transcription factors for osteoclastogenesis. In addition, DDA decreased expression levels of osteoclastogenesis-specific genes, including matrix metalloproteinase-9 (MMP-9), tartrate-resistant acid phosphatase (TRAP), and receptor activator of NF-κB (RANK) in RANKL-induced RAW264.7 cells. Collectively, these findings indicated that DDA attenuates RANKL-induced osteoclast formation by suppressing the MAPKs-c-Fos-NFATc1signalling pathway and osteoclast-specific genes. These results indicate that DDA may be a potential candidate for bone diseases associated with abnormal osteoclast formation and function.

Increase in the Number of Bone Marrow Osteoclast Precursors at Different Skeletal Sites, Particularly in Long Bone and Jaw Marrow in Mice Lacking IL-1RA.

Recently, it was shown that interleukin-1β (IL-1β) has diverse stimulatory effects on different murine long bone marrow osteoclast precursors (OCPs) in vitro. In this study, interleukin-1 receptor antagonist deficient (Il1rn−/−) and wild-type (WT) mice were compared to investigate the effects of enhanced IL-1 signaling on the composition of OCPs in long bone, calvaria, vertebra, and jaw. Bone marrow cells were isolated from these sites and the percentage of early blast (CD31hi Ly-6C−), myeloid blast (CD31+ Ly-6C+), and monocyte (CD31− Ly-6Chi) OCPs was assessed by flow cytometry. At the time-point of cell isolation, Il1rn−/− mice showed no inflammation or bone destruction yet as determined by histology and microcomputed tomography. However, Il1rn−/− mice had an approximately two-fold higher percentage of OCPs in long bone and jaw marrow compared to WT. Conversely, vertebrae and calvaria marrow contained a similar composition of OCPs in both strains. Bone marrow cells were cultured with macrophage colony stimulating factor (M-CSF) and receptor of NfκB ligand (RANKL) on bone slices to assess osteoclastogenesis and on calcium phosphate-coated plates to analyze mineral dissolution. Deletion of Il1rn increased osteoclastogenesis from long bone, calvaria, and jaw marrows, and all Il1rn−/− cultures showed increased mineral dissolution compared to WT. However, osteoclast markers increased exclusively in Il1rn−/− osteoclasts from long bone and jaw. Collectively, these findings indicate that a lack of IL-1RA increases the numbers of OCPs in vivo, particularly in long bone and jaw, where rheumatoid arthritis and periodontitis develop. Thus, increased bone loss at these sites may be triggered by a larger pool of OCPs due to the disruption of IL-1 inhibitors.

Thymol inhibits RANKL-induced osteoclastogenesis in RAW264.7 and BMM cells and LPS-induced bone loss in mice

Thymol was identified as one of key compounds contributing to the aroma of thyme leaves. We investigated the effects of thymol on receptor activator NF-κB ligand (RANKL)-induced osteoclastogenesis in murine macrophage RAW264.7 cells and bone marrow derived macrophage (BMMs) cells and lipopolysaccharide (LPS)-induced bone loss in vivo. Thymol markedly reduced RANKL-stimulated osteoclast formation and differentiation in RAW264.7 cells and BMMs cells without any cytotoxic effects. The in vitro and in vivo osteoclastogenesis inhibitory effect of thymol was assessed by calculating the quantity of TRAP (+) multinucleated cells and its inhibitory effects on the resorbing capacity were examined on calcium phosphate-coated plates. Moreover, the inhibitory effects of thymol resulted in a reduction of RANK, cathepsin K, matrix metalloproteinase-9 (MMP-9), dendritic cell-specific transmembrane protein (DC-STAMP), c-terminal myc kinase (C-MYC), C-terminal Src kinase (C-SRC), GRB2-associated-binding protein 2 (GAB2), microphthalmia-associated transcription factor (MITF), and carbonic anhydrase II genes. Similarly, activities of ERK, JNK and AKT and protein expressions of NFATc1, C-FOS, MMP-9 and cathepsin K were downregulated by thymol. More importantly, the application of thymol significantly reduced LPS-induced inflammatory bone loss in mice. In conclusion, these findings identified that thymol could be a useful therapeutic agent for the prevention of bone destructive diseases.

The Inhibitory Effects of Forsythia Koreana Extracts on the Metastatic Ability of Breast Cancer Cells and Bone Resorption by Osteoclasts.

Background:Breast cancer is the most common malignant disease in women. The patients with advanced breast cancer develop metastasis to bone. Bone metastasis and skeletal-related events by breast cancer are frequently associated with the invasiveness of breast cancer cells and osteoclasts-mediated bone resorption. Forsythia koreana is used in oriental traditional medicine to treat asthma, atopy, and allergic diseases. The aim of this study was to evaluate the inhibitory effects of F. koreana extracts on the invasion of breast cancer cells and bone resorption by osteoclasts.Methods:Cell viability was measured by an MTT assay and the migration and invasion of MDA-MB-231 cells were detected by a Boyden chamber assay. The formation of osteoclasts and pit was detected using tartrate-resistant acid phosphatase staining and calcium phosphate-coated plates, respectively. The activities of matrix metalloproteinases (MMPs) and cathepsin K were evaluated by gelatin zymography and a cathepsin K detection kit.Results:The fruit and leaf extracts of F. koreana significantly inhibited the invasion of MDA-MB-231 cells at noncytotoxic concentrations. The fruit extract of F. koreana reduced the transforming growth factor β1-induced migration, invasion and MMPs activities of MDA-MB-231 cells. In addition, the fruit, branch, and leaf extracts of F. koreana also inhibited the receptor activator of nuclear factor kappa-B ligand-induced osteoclast formation and osteoclast-mediated bone-resorbing activity by reducing the activities of MMPs and cathepsin K.Conclusions:The extracts of F. koreana may possess the potential to inhibit the breast cancer-induced bone destruction through blocking invasion of breast cancer cells, osteoclastogenesis, and the activity of mature osteoclasts.

Surface vacuolar ATPase in ameloblastoma contributes to tumor invasion of the jaw bone.

Ameloblastoma is the most common benign odontogenic tumor in Japan. It is believed that it expands in the jaw bone through peritumoral activation of osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) released from the ameloblastoma, as in bone metastases of cancer cells. However, the clinical features of ameloblastoma, including its growth rate and patterns of invasion, are quite different from those of bone metastasis of cancer cells, suggesting that different underlying mechanisms are involved. Therefore, in the present study, we examined the possible mechanisms underlying the invasive expansion of ameloblastoma in the jaw bone. Expression levels of RANKL assessed by western blotting were markedly lower in ameloblastoma (AM-1) cells than in highly metastatic oral squamous cell carcinoma (HSC-3) cells. Experiments coculturing mouse macrophages (RAW264.7) with AM-1 demonstrated low osteoclastogenic activity, as assessed by tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cell formation, probably because of low release of RANKL, whereas cocultures of RAW264.7 with HSC-3 cells exhibited very high osteoclastogenic activity. Thus, RANKL release from AM-1 appeared to be too low to generate osteoclasts. However, AM-1 cultured directly on calcium phosphate-coated plates formed resorption pits, and this was inhibited by application of bafilomycin A1. Furthermore, vacuolar-type H+-ATPase (V-ATPase) and H+/Cl- exchange transporter 7 (CLC-7) were detected on the surface of AM-1 cells by plasma membrane biotinylation and immunofluorescence analysis. Immunohistochemical analysis of clinical samples of ameloblastoma also showed plasma membrane-localized V-ATPase and CLC-7 in the epithelium of plexiform, follicular and basal cell types. The demineralization activity of AM-1 was only 1.7% of osteoclasts demineralization activity, and the growth rate was 20% of human normal skin keratinocytes and HSC-3 cells. These results suggest that the slow expansion of several typical types of ameloblastomas in jaw bone is attributable to its slow growth and low demineralization ability.

Bromo-honaucin A inhibits osteoclastogenic differentiation in RAW 264.7 cells via Akt and ERK signaling pathways

Osteoclasts are unique bone remodeling cells derived from multinucleated myeloid progenitor cells. They play homeostatic vital roles in skeletal modeling and remodeling but also destroy bone masses in many pathological conditions such as osteoporosis and rheumatoid arthritis. Receptor activation of NF-κB ligand (RANKL) is essential to osteoclastogenesis. In this study, we investigated the effects of bromo-honaucin A (Br-H A) isolated from Leptolyngbya crossbyana (cyanobacterium). To investigate the mechanism of the inhibitory effect of Br-H A on osteoclastogenesis, we employed Br-H Ain RANKL-treated murine monocyte/macrophage RAW 264.7 cells for osteoclastic differentiation in-vitro. The inhibitory effects on in-vitro osteoclastogenesis was evaluated by counting the number of Tartarate resistant acid phospatase (TRAP) positive multinucleated cells and by measuring the expression level of osteoclast-specific genes like matrix metalloproteinase 9 (MMP9), cathepsin K (CATH K), GRB2-associated-binding protein 2 (GAB2), c-terminal myc kinase (C-MYC), C-terminal Src kinase (C-SRC) and Microphthalmia-associated transcription factor (MITF). Moreover, Br-H A blocked the resorbing capacity of RAW 264.7 cells on calcium phosphate-coated plates. Finally, Br-H A clearly decreased the expression of Akt and also decreased the activation of ERK. Thus, the study identifies Br-H A as potent inhibitor potentialin the treatment of diseases involving abnormal bone lysis such as osteoporosis, rheumatoid arthritis, and periodontal bone degradation.

Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVβ3 and Inhibits Osteoclast Differentiation.

Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVβ3, and inhibited the direct binding of OA to integrin αVβ3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVβ3 and HSPG.

Agelasine D suppresses RANKL-induced osteoclastogenesis via down-regulation of c-Fos, NFATc1 and NF-κB.

In the present study, we investigated the effect of agelasine D (AD) on osteoclastogenesis. Treatment of bone marrow macrophages (BMMs) with receptor activator of nuclear factor κB ligand (RANKL) resulted in a differentiation of BMMs into osteoclasts as evidenced by generation of tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated cells and formation of pits in calcium phosphate-coated plates. However, RANKL-induced osteoclastogenesis was significantly suppressed by AD treatment. We also confirmed the increased mRNA and protein expression of osteoclastic markers, such as TRAP, cathepsin K and matrix metalloproteinase-9, during RANKL-induced osteoclast differentiation and this was down-regulated by AD treatment. Moreover, AD treatment significantly suppressed RANKL-induced mRNA expression of DC-STAMP and OC-STAMP and cell fusion of TRAP-positive mononuclear osteoclast precursors. In addition, AD suppressed RANKL-induced expression of transcription factors, c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important transcription factors involved in differentiation of BMMs into osteoclasts. Furthermore, RANKL-induced phosphorylation of extracellular signal-related kinase (ERK) and activation of NF-κB were also inhibited by AD treatment. Collectively, these results suggest that AD inhibits RANKL-induced osteoclastogenesis by down-regulation of multiple signaling pathways involving c-Fos, NFATc1, NF-κB and ERK. Our results also suggest that AD might be a potential therapeutic agent for prevention and treatment of osteoporosis.

Targeting CD26 with Humanized Monoclonal Antibody, As a Novel Approach to Inhibit Human Osteoclast Differentiation and Subsequent Bone Resorption

Abstract 1348Bone disease is a hallmark of malignancy with osteolytic bone metastasis, including multiple myeloma (MM) and targeting osteoclasts (OCs) to alleviate bone destruction is a component of the standard care for MM. CD26 is a 110-kDa multifunctional membrane-bound glycoprotein, with dipeptidyl peptidase IV (DPPIV) enzyme activity in its extracellular domain and is critical in T-cell activation and several tumor developments, including malignant lymphoma. However, little is known about the role of CD26 in regulating bone remodeling. In this study, we show that CD26 is expressed on normal human osteoclasts and moreover, intensely expressed on activated human osteoclasts with osteolytic bone metastasis, including MM. We explore the function of CD26 in osteoclastgenesis (OCG) and investigate the effects of humanized anti-CD26 monoclonal antibody (huCD26mAb), which has shown promising clinical activity in T-cell lymphoma, on human OC differentiation, maturation and function. We further define the molecular targets of CD26 signaling cascade in OCG and explore the therapeutic potential of huCD26mAb for treating osteolytic bone metastasis. Human bone marrow mononuclear cells (BMMs) were cultured with human M-CSF (25ng/ml) plus sRANKL (50ng/ml) in the absence or presence of huCD26mAb for the indicated times. Then, M-CSF and sRANKL stimulate CD26 expressions during OCG, in a dose-dependent manner. The expression of CD26 up-regulates mitogen-activated protein kinase14 (p38MAPK) phosphorylation. P38MAPK phosphorylation also occurs downstream of RANK signaling in OCs and stimulates its downstream activation of microphthalmia-associated transcription factor (mi/Mitf), which plays an important role in OC function. Importantly, huCD26mAb decreased the number of multinucleated OCs (>3 nuclei) by tartrate-resistant phosphates (TRAP)/CD26 staining and the secretion of TRAP-5b and type 1 collagen; specific mature OC markers. It decreased the size of OCs and the number of nuclei per OC, with significantly defective bone resorption activity, as evidenced by diminished pit formation on fluoresceinated calcium phosphate-coated plates. In contrast, huCD26mAb added after 4- or 7- days’ BMM cultures with M-CSF plus sRANKL did not have significant effects on mature osteoclast formation and function. Given these dual roles of CD26 in OCG, we next examined the effects of huCD26mAb on the phosphorylation of p38MAPK in OC precursor cells and mature OCs. At first, in the absence of huCD26mAb, similar amounts of p38MAPK and MKK3/6 (a molecule that is upstream of p38MAPK) were present in OC precursor cells and OCs. In response to RANKL, MKK3/6-p38MAPK was phosphorylated within 15 minutes in OC precursor cells and reached a maximal level within 30 minutes, and was maintained up to 60 minutes. Moreover, mi/Mitf was subsequently rapidly activated and persisted for 24hours. In the presence of huCD26mAb, when huCD26mAb bound to CD26 on OC precursor cells, only the MKK3/6-p38MAPK pathway was specifically rapidly inactivated, as shown by the persistent decrease in the phosphorylation of p38MAPK, together with MKK3/6, starting within 15 minutes of RANKL stimulation. Subsequent mi/Mitf phosphorylation was also persistently inhibited. In contrast, MKK3/6-p38MAPK was not phosphorylated at all in mature OCs after RANKL stimulation, regardless of the absence or presence of huCD26mAb. These results suggest that huCD26mAb suppressed RANKL induced p38MAPK phosphorylation in OC precursor cells, but not in OCs. The activation of other MAPKs including ERK and SAPK/JNK, or NFκB were rapidly induced in response to RANKL both in OC precursor cells and OCs, regardless of the absence or presence of huCD26mAb. Moreover, p38MAPK inhibitor also strongly inhibited OC formation and function through the suppression of p38 MAPK phosphorylation and subsequent mi/Mitf activation in OC precursor cells, but not in OCs. In conclusion, these data demonstrate that targeting CD26 on OC precursor cells with huCD26mAb suppressed human osteoclast differentiation, via the inhibition of MKK3/6-p38MAPK-mi/Mitf phosphorylation pathway and impaired subsequent mature osteoclast formation and function. Our results strongly suggest that targeting OCs with huCD26mAb has a promising alternative therapeutic potential for the treatment of osteolytic bone metastasis, including MM, to reduce the occurrence of total skeletal-related events. Disclosures:No relevant conflicts of interest to declare.

The response of dental pulp-derived cells to zoledronate depends on the experimental model

To investigate whether zoledronate (ZOL) can cause a cytotoxic response in dental pulp-derived cells (DPCs) in vitro. Cell activity was assessed utilizing MTT tests, (3) [H]thymidine, and (3) [H]leucine incorporation assays in human DPCs in response to ZOL. Cell activity assays were also preformed on calcium phosphate-coated plates. Cell death was analysed with annexin V/propidium iodide, trypan blue staining and Western blot analysis. Micromolar concentrations of ZOL were required to decrease the activity of DPCs. The decreased activity of DPCs was associated with the occurrence of apoptosis and necrosis. No adverse effects were observed when DPCs were cultured on calcium phosphate-coated plates with ZOL. High concentrations of soluble ZOL were required to cause adverse effects in vitro. These adverse effects are abolished when the bisphosphonate was bound to a mineralized surface. However, the clinical relevance of these results remains to be determined.

Inhibition of osteoclastogenic differentiation by Ikarisoside A in RAW 264.7 cells via JNK and NF-κB signaling pathways

Osteoclasts are specialized bone-resorbing cells derived from multipotent myeloid progenitor cells. They play a crucial homeostatic role in skeletal modeling and remodeling and destroy bone in many pathologic conditions. Receptor activator of NF-kappaB ligand (RANKL) is essential to osteoclastogenesis. In this study, we investigated the effects of Ikarisoside A, isolated from Epimedium koreanum (Berberidaceae), on osteoclastogenesis in RANKL-treated murine monocyte/macrophage RAW 264.7 cells. The results indicate that Ikarisoside A is a potent inhibitor of osteoclastogenesis in RANKL-stimulated RAW 264.7 cells as well as in bone marrow-derived macrophages. The inhibitory effect of Ikarisoside A resulted in decrease of osteoclast-specific genes like matrix metalloproteinase 9 (MMP9), tartrate-resistant acid phosphatase (TRAP), receptor activator of NF-kappaB (RANK), and cathepsin K. Moreover, Ikarisoside A blocked the resorbing capacity of RAW 264.7 cells on calcium phosphate-coated plates. Ikarisoside A also has inhibitory effects on the RANKL-mediated activation of NF-kappaB, JNK, and Akt. Finally, Ikarisoside A clearly decreased the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1) as well as the transcriptional activity of NFATc1, the master regulator of osteoclast differentiation. The data indicate that Ikarisoside A has potential for use in treatment of diseases involving abnormal bone lysis such as osteoporosis, rheumatoid arthritis, and periodontal bone erosion.

Bone tissue engineering on calcium phosphate-coated titanium plates utilizing cultured rat bone marrow cells: a preliminary study.

The use of osteoinductive in vitro tissue-coated implants in orthopaedic and dental surgery (e.g. revision hip arthroplasty), could result in a better fixation of these implants. However, this tissue engineering technology has only proved to be effective in porous materials and not on flat implant surfaces. In this study we have demonstrated that calcium phosphate-coated titanium plates with a layer of cultured osteogenic cells and their extracellular matrix can initiate bone formation in vivo. Both primary and subcultured rat bone marrow cells were grown on to biomimetic calcium phosphate-coated titanium plates. After 7 d of culture, in the presence or absence of dexamethasone, the implants were subcutaneously implanted in nude mice for 4 wk. Control samples, which consisted of calcium phosphate-coated plates without cultured cells and porous calcium phosphate particles with or without cultured cells, were also implanted subcutaneously. At autopsy, no bone formation could be detected on any of the control samples without cells and samples with subcultured cells, which were primary cultured in medium without dexamethasone. In contrast, clear de novo bone formation could be observed on the calcium phosphate-coated plates and in the porous calcium phosphate particles with primary or subcultured cells, which had been continuously cultured in medium with dexamethasone. These results indicate that this hybrid technology offers great potential for the fixation of flat bone replacement implants (e.g. artificial hips) in inferior bone in the future.

Osteoblastic cells are prerequisite for inducing not only osteoclast differentiation but also function

Osteoclasts are unique bone remodeling cells derived from multinucleated myeloid progenitor cells. They play homeostatic vital roles in skeletal modeling and remodeling but also destroy bone masses in many pathological conditions such as osteoporosis and rheumatoid arthritis. Receptor activation of NF-κB ligand (RANKL) is essential to osteoclastogenesis. In this study, we investigated the effects of bromo-honaucin A (Br-H A) isolated from Leptolyngbya crossbyana (cyanobacterium). To investigate the mechanism of the inhibitory effect of Br-H A on osteoclastogenesis, we employed Br-H Ain RANKL-treated murine monocyte/macrophage RAW 264.7 cells for osteoclastic differentiation in-vitro. The inhibitory effects on in-vitro osteoclastogenesis was evaluated by counting the number of Tartarate resistant acid phospatase (TRAP) positive multinucleated cells and by measuring the expression level of osteoclast-specific genes like matrix metalloproteinase 9 (MMP9), cathepsin K (CATH K), GRB2-associated-binding protein 2 (GAB2), c-terminal myc kinase (C-MYC), C-terminal Src kinase (C-SRC) and Microphthalmia-associated transcription factor (MITF). Moreover, Br-H A blocked the resorbing capacity of RAW 264.7 cells on calcium phosphate-coated plates. Finally, Br-H A clearly decreased the expression of Akt and also decreased the activation of ERK. Thus, the study identifies Br-H A as potent inhibitor potentialin the treatment of diseases involving abnormal bone lysis such as osteoporosis, rheumatoid arthritis, and periodontal bone degradation.