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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.

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.

Sanguisorba officinalis L. (Rosaceae) is a perennial herbaceous plant and its roots have been used as an important traditional medicine for over 2000 years. Ziyuglycoside I (Ziyu), an active compound isolated from the roots of S. officinalis L., has shown biological effects such as anti-oxidant, antiviral, and antiwrinkle activities. This study aimed to elucidate the underlying mechanisms of action of Ziyu on cytotoxicity, migration, and differentiation of pre-osteoblasts. Herein, at concentrations ranging from 1 to 100 $\mu$M, Ziyu was not cytotoxic against pre-osteoblasts. Alkaline phosphatase activity assay and staining, and migration assay showed that Ziyu increased cell migration and promoted early osteoblast differentiation, followed by the enhancement of mineralized nodule formation in a dose-dependent manner, as indicated by Alizarin Red S staining. In addition, Ziyu increased the protein levels of runt-related transcription factor 2 (RUNX2) during osteoblast differentiation, whereas it did not affect the phosphorylation of Smad1/5/8 and GSK3b and expression of $\beta$-catenin. Ziyu also activated ERK1/2 and mitogen-activated protein kinase during osteoblast differentiation, and ERK1/2 inhibitor attenuated Ziyu-mediated RUNX2 expression and nuclear accumulation. Furthermore, Ziyu-mediated early and late osteoblast differentiation was significantly suppressed by the inhibition of ERK1/2, which was accompanied by attenuation in the mRNA levels of osteoblast-related genes including bone sialoprotein, osteopontin, and osteocalcin. Taken together, the findings of this study provide evidence that Ziyu promotes cell migration, osteoblast differentiation, and bone mineralization and suggest a potential role for Ziyu in the treatment of bone diseases.

Titanium, zirconia and HAp were known as good biocompatible materials for tissue engineering. Osteblastic cell response is influence by the surface topography of material and its chemical composition as well. To evaluate the influence of different chemical compositions on osteoblast-like cells the specimens were polished until they have almost identical surface roughness. The commercially pure titanium, zirconia/alumina composite and nano-sized hydroxyapatite (HAp) specimens synthesized by hydrothermal method were used to evaluate the cell attachment, proliferation and differentiation. Confocal laser microscopy was used measurement of surface roughness, and flourescence microscopy and SEM were used to evaluate initial cell attachment and morphology after 3 hours. MTS assay was performed for cell proliferation after 1, 3, 7 days and ALP assay was used for cell differentiation after 7, 10, 14 days of cell culture period. Surface topography of nano-HAp specimen was almost identical compared with those of titanium and zirconia specimen. Under this condition, proliferation and differentiation of MC3T3-E1 cells was not significantly different with those on titanium and zirconia specimen. However, cells on Nano-HAp specimen showed quicker and more active cellular reaction for attachment when measured by the expression of adhesion proteins through confocal laser microscopy. The results suggested that the new nano-sized HAp can be applied as a suitable material for skeletal tissue engineering.

A SiC coating for carbon/carbon (C/C) composites was produced by pack cementation to use as a bonding and buffer layer between C/C composites and bioactive ceramic for application in orthopaedic implants. The microstructure and MG63 osteoblast-like cell responses of the coating were investigated. The results confirmed that the SiC coating displayed a dense and uniform microstructure. MG63 cells attached and spread favorably on SiC coating, and cell proliferation was better on SiC coating than on uncoated C/C composites surface. The SiC coated C/C composites have the potential to be used in artificial implants.

Osteoblast-osteoclast interaction plays an important role in the bone remodeling. During long duration space flight, astronauts undergo serious bone loss mainly due to the disruption of equivalence between bone formation and bone resorption. Osteoclast precursors often operate under the control of osteoblasts. However, here we show that the osteoclast precursors could in turn influence osteoblasts. RAW264.7 cells, the murine osteoclast precursors, were treated in the simulated weightlessness produced by a Random Positioning Machine (RPM). After 72 h, conditioned mediums (CM) by the RAW264.7 cells from RPM (RCM) or static control (CCM) were collected and were used to culture osteoblastic-like MC3T3-E1 cells. The results showed that the RCM culture inhibited cell viability and slightly altered cell cycle, but the morphology of the MC3T3-E1 cells was not changed by RCM compared to that of CCM. Furthermore, the intracellular ALP level, NO release and expression of osteoblastic marker genes were all down-regulated by RCM culture. These results suggest that osteoclast precursors subjected to RPM exert negative regulation on osteoblasts.

With the aging of the population and a correlated increase in the incidence of osteoarticular damage, great attention is focused on regenerative nanomedicine solutions to restore durable articular function and comfort. A durable cartilage repair is not effective without regeneration of an intact subchondral bed along with the surface chondral regeneration. Our expected outcomes are the development of clinical applications in the field of tissue engineering and nanomedicine, and more particularly in bone-cartilage unit regeneration. Here we report for the first time the nanomechanical analysis of the retrieved active implant after subchondral bone regeneration in vivo, which is much more efficient and long lasting solution to osteochondral defects than the existing ones. We believe that our results make a significant contribution to the area of regenerative nanomedicine. The concepts discovered here may serve to design sophisticated implants for placement into a broad variety of tissues.

Modification of Ti–6Al–4V alloys with silver-loaded TiO₂ nanotubes was investigated. In this study, TiO₂ nanotube (TiNT) was grown on the surface of Ti–6Al–4V plates by means of anodization in an electrolyte solution containing glycerol, water and 0.5wt.% of NH₄F. Silver particles were deposited on TiNT using a Photo-Assisted Deposition (PAD) method. Formation of crystalline phase of TiO₂ on the surface was confirmed by means of XRD while its superficial morphology was observed using FESEM/EDS. Hydrophilicity was assessed by means of contact angle measurement. As-synthesized silver-loaded TiNT on osteoblast ATCC growth in vitro was also investigated in terms of its capacity in supporting osseointegration. The cell viability was determined by MTT (3-[4,5-dimethylthiazol-2yl]-2,5diphenyl-2H-tetrazolium bromide) assay and its differentiation activity was measured by alkaline phosphatase (ALP) assay. The results showed that desposition of silver on TiNT increased cell viability after 14 days culture while improving the hydrophilicity feature. Silver-loaded TiNT on Ti–6Al–4V alloy with Ag precursor concentration of 0.10M showed the optimum viability of osteoblast growth, with 14% improvement in comparison to its unmodified counterpart. The MTT assay showed that no cytotoxicity in vitro was observed on this material. This study provides corroborating evidences that the modification of Ti–6Al–4V alloy may enhance the cell viability and its prominence as dental implant materials.

Synthetic bioactive and bioresorbable composite materials are becoming increasingly important as scaffolds for bone tissue engineering. Next generation biomaterials should combine bioactive and bioresorbable properties to activate in vivo mechanisms of tissue regeneration, stimulating the body to heal itself and leading to replacement of the scaffold by the regenerating tissue. In the present chapter composite materials based on smart combinations of biodegradable polymers and bioactive ceramics, including hydroxyapatite and bioactive glasses, are discussed as suitable materials for scaffold fabrication. These composites exhibit tailored physical, biological and mechanical properties as well as predictable degradation behaviour. The appropriate selection of a particular composite for a given application requires a detailed understanding of relevant cells and/or tissue response. Knowledge concerning interactions between cells and their immediate local environment in composite scaffolds has deeply improved in the last years. An overview of these findings is presented highlighting the influence of material processing methods, scaffold microstructure as well as the importance of the nature and amount of the bioactive ceramic particulate included in specific polymer matrices. The chapter also emphasises the response diversity according to the cell type used in vitro or the chosen in vivo models (species and location), suggesting the utility of standardisation in this field of biomaterials science. Bioactive composites discussed in this chapter, enhanced by microstructural optimisation and surface engineering, are suggested as the materials of choice for development of optimal bone tissue engineering scaffolds.

Four batches of carbonate-substituted hydroxyapatite discs, designated CA1, CA2, CA3 and CA4, were prepared with carbonate contents of 5.4, 6.1, 8.2 and 11.3 wt%, respectively. The biological response to these materials was compared with the response to stoichiometric hydroxyapatite (HA) by biochemical evaluation of human osteoblast-like cells (HOBs) incubated both in direct contact with the discs and in culture medium containing solutes from the five substrates. Assessment of the cellular response to solutes from the five substrates demonstrated enhanced activity in those cells exposed to CA3 discs. Biochemical and morphological assessment of HOBS in direct contact with the disc surfaces supported this finding with enhanced proliferation being achieved in cells incubated on CA3 discs as compared to HA. However, while the response to CA4 was found to be comparable to the HA, proliferation was suppressed in cells incubated on CAl and CA2 discs, as confirmed by the poor morphological appearance of cells on these discs, apparently due to increased ceramic solubility leading to a degradation in the cell/substrate interface. This indicates that HOB cell metabolism is highly sensitive to the precise level of carbonate ion substitution, which in low carbonate HA reflects the solubility of the material. Moreover, the optimum level of carbonate substitution has been identified as similar to that found in bone mineral.

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.

Osteopontin(OPN), one of the major non-collagenous bone proteins, has an important role in bone remodeling. OPN not only has a role in triggering the osteoblast early differentiation, but activates osteoclast resorption function. On this point of view, we proposed a model for bone remodeling via integrin-OPN binding, by which OPN could act as a signaling molecule between osteoblasts and osteoclasts in the remodeling procedure. Moreover, OPN proved to induce the in vivo osteogenic potential of bone marrow derived osteoblasts/hydroxyapatitc(HA) construct.