Narrow Results

In this experiment, we investigate the biochemical effects of traditional Chinese medicines via an in vitro bone cell culture. Ten different Chinese medicines were used in this study. The rat osteoblast-osteoclast co-culture system was used as the experimental model. After the cells grew to 80% confluence, various tested materials were added. The mitochondria activity of the bone cells after exposure to various preparations of Chinese medicines was determined by colorimetric assay. Biochemical markers such as protein content, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and acid phosphatase (ACP) titer were analyzed to evaluate the bone cell activity. When cultured with various Chinese medicines for 24 hours, only four of these ten Chinese medicines had potential beneficial effects on the bone cell culture; and only Drynaria fortunei (Kunze) J. Sm. had a universal beneficial effect on bone cell metabolism. The major beneficial effect of Drynaria fortunei (Kunze) J. Sm. on bone cells is probably mediated by the induction of apoptosis of the osteoclast cell population. Continued study of alterations in gene expression of bone cells caused by Chinese medicines will improve our understanding of bone cell responses to various pharmacological interventions.

Dang-Gui-Ji-Hwang Yeum (DGJHY; an Oriental prescription), has been successfully used for the management of osteoporotic disorders in China, Japan and Korea. In this study, we have characterized the effect of DGJHY on osteoporosis-associated phenomena in ovariectomized (OVX) rats by measuring body weights and bone histomorphometries in sham, OVX and DGJHY-administered OVX rats. Light microscopic analyses showed a porous or an eroded appearance on the tibial trabecular bone surface in OVX rats, whereas those of the sham and DGJHY-administered OVX rats were composed of fine particles. The trabecular bone area and the trabecular thickness in OVX rats were significantly lower than those of sham rats. Moreover, these reductions in OVX rats were significantly reversed by the administration of DGJHY for 7 weeks, and osteoclast numbers were also significantly reduced. Although no differences were observed between OVX and DGJHY-administered OVX rats and the sham animals at the T₃ level, we have found significant differences between these two groups at the T₄ level. However, serum phosphorus, calcium, mechanical strength and the surface appearance of osteoblasts in the DGJHY-administered OVX rats were similar to those of OVX rats. These results suggest that DGJHY is effective at preventing bone loss in OVX rats.

This study was carried out to investigate the effect of Spatholobus suberectus Dunn (SS) on the protection of chondral defect and inhibition of osteoclastogenesis. To examine these effects, we measured the matrix metalloproteinase (MMP) levels in SW1353 chondrosarcoma cells and performed tartrate-resistant acid phosphatase (TRAP) staining in bone marrow macrophage (BMM)-derived osteoclasts. To investigate the anti-osteoarthritis (OA) effects, we assessed TNF-α-induced MMP-1, -3, -9 and tissue inhibitors of matrix metalloproteinase (TIMP) expression levels in SW1353 cells. We observed that SS extract significantly inhibited MMP and TIMP expression in SW1353 cells. Also, SS extract inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. These results suggest that SS extract may have a potential in the treatment of bone loss and chondral defect by suppressing osteoclast differentiation and decreasing the expression of OA factors. Therefore, clarification of the mechanism of the action of SS extract and its active components is needed.

Angelica tenuissima has been traditionally used in oriental medicine for its therapeutic effects in headache, toothache, and flu symptoms. It also exerts anti-inflammatory activity via the inhibition of the expression of cyclooxygenase-2 (COX-2). However, the effect of Angelica tenuissima on osteoclast differentiation has not been identified until recently. In this study, we first confirmed that Angelica tenuissima water extract (ATWE) significantly interrupted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) in a dose-dependent manner without any cytotoxicity. Next, we clarified the underlying mechanisms linking the suppression effects of ATWE on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. At the molecular level, ATWE induced the dephosphorylation of c-Jun N-terminal kinase (JNK) and Akt and decreased the degradation of IκB in RANKL-dependent early signaling pathways. Subsequently, ATWE caused impaired activation of the protein and mRNA levels of c-Fos and nuclear factor of activated T cell c1 (NFATc1). Moreover, the disassembly of filamentous actin (F-actin) ring and anti-resorptive activity of mature osteoclasts were triggered by ATWE treatment. Although ATWE did not enhance osteogenesis in primary osteoblasts, our results showed that ATWE is a potential candidate for anti-resorptive agent in osteoporosis, a common metabolic bone disorder.

Alpinia officinarum rhizome has been used as a traditional herbal remedy to treat inflammatory and internal diseases. Based on the previously observed inhibitory effect of A. officinarum rhizome in an arthritis model, we evaluated whether a water extract of A. officinarum rhizome (WEAO) would enhance in vitro osteoblast mineralization using calvarial osteoblast precursor cells or would inhibit in vitro osteoclast differentiation and bone resorption using bone marrow derived macrophages. In osteoblasts, WEAO enhanced the mRNA levels of transcription factor (runt-related transcription factor 2, smad1, smad5, and junB) and marker (bone morphogenetic protein-2, collagen type 1alpha1, and osteocalcin) genes related to osteoblast mineralization, consistent with increased alizarin red S staining intensity. WEAO markedly inhibited osteoclast differentiation by suppressing the receptor activator for nuclear factor-$\kappa$B ligand-induced downregulation of inhibitor of DNA binding 2 and V-maf musculoaponeurotic fibrosarcoma oncogene homolog B and the phosphorylation of c-Jun N-terminal kinase, p38, nuclear factor-$\kappa$B, c-Src, and Bruton’s tyrosine kinase to induce nuclear factor of activated T cells cytoplasmic 1 expression. WEAO also suppressed the resorbing activity of mature osteoclasts by altering actin ring formation. Therefore, the results of this study demonstrate that WEAO stimulates osteoblast mineralization and inhibits osteoclast differentiation. Thus, WEAO may be a promising herbal candidate to treat or prevent pathological bone diseases by regulating the balance between osteoclast and osteoblast activity.

Artemisia capillaris has been used to treat jaundice and relieve high liver-heat in traditional medicine. In this study, we found that the administration of a water extract from A. capillaris (WEAC) to the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced bone loss model significantly prevents osteoporotic bone loss, increasing bone volume/trabecular volume by 22% and trabecular number by 24%, and decreasing trabecular separation by 29%. WEAC stimulated invitro osteoblast mineralization from primary osteoblasts in association with increasing expression of osterix, nuclear factor of activated T cells cytoplasmic 1, and activator protein-1, as well as phosphorylation of extracellular signal-regulated kinase. In contrast to the anabolic effect of WEAC, WEAC significantly suppressed invitro osteoclast formation from bone marrow macrophages by inhibiting the RANKL signaling pathways and bone resorption by downregulating the expression of resorption markers. Therefore, this study demonstrated that WEAC has a beneficial effect on bone loss through the regulation of osteoblast mineralization, as well as osteoclast formation and bone resorption. These results suggest that A. capillaris may be a promising herbal candidate for therapeutic agents to treat or prevent osteoporotic bone diseases.

Puerariae radix, the dried root of Pueraria lobate Ohwi, is known to prevent bone loss in ovariectomized mice; however, the precise molecular mechanisms are not understood. In this study, we investigated the effects and underlying mechanisms of action of Puerariae radix extract (PRE) on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. PRE dose-dependently inhibited osteoclast differentiation and formation, decreased the bone-resorbing activity of osteoclasts, and downregulated the expression of osteoclast differentiation marker genes. The expression of osteoclastogenic factors produced by PRE-treated osteoblasts such as RANKL, macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) was comparable to that of untreated (control) cells. However, the formation of osteoclasts via bone marrow cell and calvaria-derived osteoblast co-cultures was suppressed by PRE treatment. Therefore, the inhibitory effects of PRE on osteoclastogenesis clearly targeted osteoclasts, but not osteoblasts. PRE treatment considerably reduced RANKL-induced mitogen-activated protein kinases (MAPKs) activity, especially c-Jun N-terminal kinase, in osteoclast precursor cells. In addition, PRE markedly suppressed cAMP response element-binding protein (CREB) activation and the induction of peroxisome proliferator-activated receptor gamma coactivator 1β (PGC1β), which stimulate osteoclastogenesis — an effect that was not observed for puerarin and 17-β estradiol. Finally, PRE treatment significantly repressed the expression of c-Fos and the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), which is a master transcription factor for osteoclastogenesis in vitro and in vivo. Overall, these results strongly suggest that PRE is an effective inhibitor of RANKL-induced osteoclastogenesis and may be a potent therapeutic agent for bone-related diseases such as osteoporosis, rheumatoid arthritis, and periodontitis.

Osteoblast harvest from rat bone and bone marrow by mechanical isolation and induction culture methods, respectively, is described in this chapter. Osteoclasts act as a cell model for pharmaceutical evaluation on anti-bone resorption drug screening by providing a technical platform to study the regulation of bone resorption and to evaluate the agents developed for prevention and treatment of metabolic bone disorders.

The interplay between neoplastic cells and multinucleate osteoclast-like giant cells found in giant cell tumor has been considered as a model of the cellular interactions that occur during bone resorption in both primary and metastatic neoplasms. This chapter describes the tissue culture techniques of giant cell tumor of bone. The main proliferating cells maintained in culture are the spindle-shaped stromal-like mononuclear cells, which represent the neoplastic component of this tumor.

The following sections are included:

• Introduction
• Different types of RhoGEF inhibitors
• An example of preclinical application of a RhoGEF inhibitor: Dock5 and osteolytic diseases
• Acknowledgments
• References

Cbfal is a transcription factor, which belongs to the runt-domain gene family. Cbfal is expressed in osteoblasts and chondrocytes, and its expression is increased according to their maturation. Cbfal has two isotypes with different N-terminal domains, and the two isotypes show different activities in the regulation of their target genes. Cbfal-deficient mice showed a complete lack of bone formation in the absence of mature osteoblasts, and forced expression of Cbfal in mesenchymal cells induced osteoblastic markers, indicating that Cbfal is an essential factor for osteoblast differentiation. Chondrocyte maturation and osteoclast differentiation was also blocked in mutant mice. RANKL was barely detectable in Cbfal-deficient mice, suggesting that Cbfal induces osteoclast differentiation by the regulation of RANKL in osteoblastic cells. These data demonstrate that Cbfal plays an essential role in bone formation.

Microgravity in space has been reported to enhance bone resorption in vitro and in vivo, however, the gravity effect on bone remodeling is not well understood. In particular, there are only a few reports about gravity effect on osteoclasts. In the present study, we examined the hypergravity effect on mRNA expressions of osteoclast marker enzymes such as TRAP (tartrate-resistant acid phosphatase) and cathepsin K. Osteoclasts were exposed to 30 xg for 2 hr or 18 hr. Results suggested that hypergravity enhanced the mRNA expression of both enzymes with different manner; the expression of the TRAP showed a slight increase, that of the cathepsin K showed a non-monotonous time course with maximum hypergravity effect for short time (2 hr) incubation.

The mechanism of osteoclasts attachment to the bone surface has not been well understood. In particular sealing zone is a very specific structure of active osteoclast on bone. The molecular basis on its formation has recently attracted much attention in biologists. In this study, we have performed differential display (DD) PCR with the template RNAs extracted from osteoclasts cultured on ivory or collagen type I to identify genes specifically expressed in osteoclasts attached to ivory. Highly enriched osteoclasts were harvested from 10-day-old rabbit according to the method of Kakudo et al. and cultured on each surface. DDPCR was performed using their total RNAs, then PCR products were cloned into pCR-TRAP vector, followed by the DNA sequencing. 22 cDNA fragment clones were found in osteoclast attached to ivory. Among them, 11 clones which located at 3’terminal of related genes were sequenced. Two of them were found as known genes, i. e. PTX3 and DRAK 1. We selected 6 clones and examined tissue distribution by slot blot. The data showed that 6 clones expressed in various pattern. These result indicated that different pattern of gene expression was revealed when osteoclast attached to different matrix. It also suggested osteoclasts could form the effective sealing zone with the participation of multiple genes.

Hydroxyapatite (HA) has been used successfully for anchorage of endoprostheses. Data regarding the cellular resorption of HA coatings are varying. Until now it is not clear, why some HA materials were degraded by osteoclasts and others were not. In these cases foreign body giant cells (FBGC) were observed. In this study three different HA materials with different crystal sizes were tested. Only microcrystalline HA (crystal size in the range of human bone crystals) was resorbed actively by osteoclasts. Typical lacunae were observed in the surface of the material. If the crystal size of HA was higher (0.1-0.3 μm or 1-3 μm) lacunae were not observed any more. In such cases FBGC were detected on implant surfaces. In all three implant types tested bone bonding was observed in a similar amount. The data obtained are important with regard to the design of bioactive materials to special needs, e.g. coatings of endoprostheses.