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Due to the chemical composition and structure of the target tissue, autologous bone grafting remains the gold standard for orthopedic applications worldwide. However, ongoing advancements in alternative grafting materials show that 3D-printed synthetic biomaterials offer many advantages. For instance, they provide high availability, have low clinical limitations, and can be designed with a chemical composition and structure comparable to the target tissue. This study aimed to compare the influences of particle size and sintering temperature on the mechanical properties and biocompatibility of calcium phosphate (CaP) gyroid scaffolds. CaP gyroid scaffolds were fabricated by 3D printing using powders with the same chemical composition but different particle sizes and sintering temperatures. The physicochemical characterization of the scaffolds was performed using X-ray diffractometry, scanning electron microscopy, and microtomography analyses. The immortalized human mesenchymal stem cell line SCP-1 (osteoblast-like cells) and osteoclast-like cells (THP-1 cells) were seeded on the scaffolds as mono- or co-cultures. Bone cell attachment, number of live cells, and functionality were assessed at different time points over a period of 21 days. Improvements in mechanical properties were observed for scaffolds fabricated with narrow-particle-size-distribution powder. The physicochemical analysis showed that the microstructure varied with sintering temperature and that narrow particle size distribution resulted in smaller micropores and a smoother surface. Viable osteoblast- and osteoclast-like cells were observed for all scaffolds tested, but scaffolds produced with a smaller particle size distribution showed less attachment of osteoblast-like cells. Interestingly, low attachment of osteoclast-like cells was observed for all scaffolds regardless of surface roughness. Although bone cell adhesion was lower in scaffolds made with powder containing smaller particle sizes, the long-term function of osteoblast-like and osteoclast-like cells was superior in scaffolds with improved mechanical properties. [ABSTRACT FROM AUTHOR]

Due to the chemical composition and structure of the target tissue, autologous bone grafting remains the gold standard for orthopedic applications worldwide. However, ongoing advancements in alternative grafting materials show that 3D-printed synthetic biomaterials offer many advantages. For instance, they provide high availability, have low clinical limitations, and can be designed with a chemical composition and structure comparable to the target tissue. This study aimed to compare the influences of particle size and sintering temperature on the mechanical properties and biocompatibility of calcium phosphate (CaP) gyroid scaffolds. CaP gyroid scaffolds were fabricated by 3D printing using powders with the same chemical composition but different particle sizes and sintering temperatures. The physicochemical characterization of the scaffolds was performed using X-ray diffractometry, scanning electron microscopy, and microtomography analyses. The immortalized human mesenchymal stem cell line SCP-1 (osteoblast-like cells) and osteoclast-like cells (THP-1 cells) were seeded on the scaffolds as mono- or co-cultures. Bone cell attachment, number of live cells, and functionality were assessed at different time points over a period of 21 days. Improvements in mechanical properties were observed for scaffolds fabricated with narrow-particle-size-distribution powder. The physicochemical analysis showed that the microstructure varied with sintering temperature and that narrow particle size distribution resulted in smaller micropores and a smoother surface. Viable osteoblast- and osteoclast-like cells were observed for all scaffolds tested, but scaffolds produced with a smaller particle size distribution showed less attachment of osteoblast-like cells. Interestingly, low attachment of osteoclast-like cells was observed for all scaffolds regardless of surface roughness. Although bone cell adhesion was lower in scaffolds made with powder containing smaller particle sizes, the long-term function of osteoblast-like and osteoclast-like cells was superior in scaffolds with improved mechanical properties.

With the advent of nanotechnology, the use of nanoparticles as drug delivery system (DDS) has attracted great interest. We aimed to apply carbon nanohorns (CNHs) as DDS in the development of new treatments for bone diseases. We evaluated the in vitro and in vivo cellular responses of CNHs in bone-related cells compared with carbon blacks (CBs), which are similar in particle size but differ in surface and structural morphologies. Although in vitro experiments revealed that both CNHs and CBs were incorporated into the lysosomes of RAW264-induced osteoclast-like cells (OCs) and MC3T3-E1 osteoblast-like cells (OBs), no severe cytotoxicity was observed. CNHs reduced the tartrate-resistant acid phosphatase activity and expression of the differentiation marker genes in OCs at noncytotoxic concentrations, whereas the alkaline phosphatase activity and differentiation of OBs increased. Under calcification of OBs, CNHs increased the number of calcified nodules and were intra- and extracellularly incorporated into calcified vesicles to form crystal nuclei. The in vivo experiments showed significant promotion of bone regeneration in the CNH group alone, with localized CNHs being found in the bone matrix and lacunae. The suppression of OCs and promotion of OBs suggested that CNHs may be effective against bone diseases and could be applied as DDS. [ABSTRACT FROM AUTHOR]

Arterial calcification refers to the abnormal deposition of calcium salts in the arterial wall, which results in vessel lumen stenosis and vascular remodeling. Studies increasingly show that arterial calcification is a cell mediated, reversible and active regulated process similar to physiological bone mineralization. The osteoblasts and chondrocytes-like cells are present in large numbers in the calcified lesions, and express osteogenic transcription factor and bone matrix proteins that are known to initiate and promote arterial calcification. In addition, osteoclast-like cells have also been detected in calcified arterial walls wherein they possibly inhibit vascular calcification, similar to the catabolic process of bone mineral resorption. Therefore, tilting the balance between osteoblast-like and osteoclast-like cells to the latter maybe a promising therapeutic strategy against vascular calcification. In this review, we have summarized the current findings on the origin and functions of osteoblast-like and osteoclast-like cells in the development and progression of vascular progression, and explored novel therapeutic possibilities.

Introduction: Sympathetic hyperinnervation plays an important role in modulating the vascular smooth muscle cell (VSMC) phenotype and vascular diseases, but its role in abdominal aortic aneurysm (AAA) is still unknown., Objectives: This study aimed to investigate the role of sympathetic hyperinnervation in promoting AAA development and the underlying mechanism involved., Methods: Western blotting and immunochemical staining were used to detect sympathetic hyperinnervation. We performed sympathetic denervation through coeliac ganglionectomy (CGX) and 6-OHDA administration to understand the role of sympathetic hyperinnervation in AAA and investigated the underlying mechanisms through transcriptome and functional studies. Sema4D knockout (Sema4D -/- ) mice were utilized to determine the involvement of Sema4D in inducing sympathetic hyperinnervation and AAA development., Results: We observed sympathetic hyperinnervation, the most important form of sympathetic neural remodeling, in both mouse AAA models and AAA patients. Elimination of sympathetic hyperinnervation by CGX or 6-OHDA significantly inhibited AAA development and progression. We further revealed that sympathetic hyperinnervation promoted VSMC phenotypic switching in AAA by releasing extracellular ATP (eATP) and activating eATP-P2rx4-p38 signaling. Moreover, single-cell RNA sequencing revealed that Sema4D secreted by osteoclast-like cells induces sympathetic nerve diffusion and hyperinnervation through binding to Plxnb1. We consistently observed that AAA progression was significantly ameliorated in Sema4D-deficient mice., Conclusions: Sympathetic hyperinnervation driven by osteoclast-like cell-derived Sema4D promotes VSMC phenotypic switching and accelerates pathological aneurysm progression by activating the eATP/P2rx4/p38 pathway. Inhibition of sympathetic hyperinnervation emerges as a potential novel therapeutic strategy for preventing and treating AAA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

The receptor activator of nuclear factor κB ligand (RANKL) is an important factor for osteoclastogenesis and contributes to the pathology of rheumatoid arthritis (RA); thus, the anti-RANKL antibody (Ab) has been expected to protect joint destruction in RA patients. IL-8 also has osteoclastogenic activity; however, the role of IL-8 in the bone pathology of RA as well as the relation between IL-8 and RANKL remain unclear. In the present study, clinical observation revealed serum IL-8 levels of 611 pg ml−1 in RA patients with anti-RANKL Ab and 266 pg ml−1 in the same patients without anti-RANKL Ab. In vitro assay showed that anti-RANKL Ab induced production of IL-8 from pre-osteoclast-like cells (OCLs), and IL-8 promoted the formation of OCLs from peripheral monocytes even without RANKL activity. We further showed that treatment with FK506 (tacrolimus) possibly inhibits the increase in IL-8 levels in RA patients with anti-RANKL Ab, and in vitro assay confirmed that FK506 suppressed IL-8 production in pre-OCLs. These results suggest that inhibition of RANKL induces the change in osteoclastogenesis-promoting factor from RANKL to IL-8, and FK506 may be a valuable combination drug to support the use of anti-RANKL Ab in treatment of RA. [ABSTRACT FROM AUTHOR]

Osteoclast-like cells are known to inhibit arterial calcification. Receptor activator of NF-κB ligand (RANKL) is likely to act as an inducer of osteoclast-like cell differentiation. However, several studies have shown that RANKL promotes arterial calcification rather than inhibiting arterial calcification. The present study was conducted in order to investigate and elucidate this paradox. Firstly, RANKL was added into the media, and the monocyte precursor cells were cultured. Morphological observation and Tartrate resistant acid phosphatase (TRAP) staining were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells. During arterial calcification, in vivo and in vitro expression of RANKL and its inhibitor, osteoprotegerin (OPG), was detected by real-time PCR. The extent of osteoclast-like cell differentiation was also assessed. It was found RANKL could induce osteoclast-like cell differentiation. There was no in vivo or in vitro expression of osteoclast-like cells in the early stage of calcification. At that time, the ratio of RANKL to OPG was very low. In the late stage of calcification, a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG. According to the results, the ratio of RANKL to OPG was very low during most of the arterial calcification period. This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation. This likely explains why RANKL had the ability to induce osteoclast-like cell differentiation but acted as a promoter of calcification instead. [ABSTRACT FROM AUTHOR]

Background and aims: Pulsed electromagnetic fields (PEMF) have proven to be an effective noninvasive method in the prevention and treatment of osteoporosis. This study evaluated the effects of PEMF on the expression of the NFATc1, CAII and RANK genes in mouse osteoclast-like cells. Methods: Bone marrow from bilateral tibiae and femurs was cultured in differentiation medium in the presence of soluble macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). After 5 days, the osteoclast-like cells were confirmed by both tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assays. The osteoclast-like cells were divided into five groups and exposed to the following treatments for 3 days: M-CSF; M-CSF + RANKL; M-CSF + RANKL + osteoprotegerin (OPG), M-CSF + RANKL + premarin (E); and M-CSF + RANKL + PEMF. The numbers of multinucleated, TRAP-positive osteoclast-like cells and resorption pits formed were determined. The expression of NFATc1, CAII and RANK mRNA was determined with real-time fluorescent quantitative polymerase chain reaction. Results: PEMF substantially reduced the number of osteoclast-like cells in the culture with M-CSF + RANKL. The level of NFATc1, CAII, and RANK mRNA expression was decreased in the M-CSF + RANKL + PEMF group compared to the M-CSF + RANKL group ( p = 0.007, p = 0.039, p = 0.001, respectively). The mRNA expression of NFATc1, CAII, and RANK was not higher in the M-CSF + RANKL + OPG group compared to the M-CSF + RANKL + PEMF group ( p = 0.682, p = 0.200, p = 0.924, respectively). In addition, there was no difference in the expression of mRNA from NFATc1, CAII, and RANK between the M-CSF + RANKL + PEMF group and the M-CSF + RANKL + E group ( p = 0.853, p = 0.509, p = 0.664, respectively). Conclusions: These data suggest that PEMF might modulate the process of osteoclastogenesis and subsequent bone resorption, at least partially, through NFATc1, CAII and RANK. [ABSTRACT FROM AUTHOR]

SummaryBackground: Osteoblast- and osteoclast-like cells are responsible for coordinated bone maintenance, illustrated by a balanced formation and resorption. Both parameters appear to be influenced by mechanical constrains acting on each of these cell types individually. We hypothesized that the interactions between both cell types are also influenced by mechanical stimulation. Methods: Co-cultures of osteoblast- and osteoclast-like cells were stimulated with 1,100 µstrain, 0.1 or 0.3 Hz for 1-5 min/day over 5 days. Two different setups depending on the differentiation of the osteoclast-like cells were used: i) differentiation assay for the fusion of pre-osteoclasts to osteoclasts, ii) resorption assay to determine the activity level of osteoclast-like cells. Results: In the differentiation assay (co-culture of osteoblasts with unfused osteoclast precursor cells) the mechanical stimulation resulted in a significant decrease of collagen-1 and osteocalcin produced by osteoblast-like cells. Significantly more TRAP-iso5b was measured after stimulation for 3 min with 0.1 Hz, indicating enhanced osteoclastogenesis. In the resorption assay (co-culture of osteoblasts with fused osteoclasts) the stimulation for 3 min with 0.3 Hz significantly increased the resorption activity of osteoclasts measured by the pit formation and the collagen resorption. The same mechanical stimulation resulted in an increased collagen-1 production by the osteoblast-like cells. The ratio of RANKL/OPG was not different between the groups. Conclusion: These findings demonstrate that already small changes in duration or frequency of mechanical stimulation had significant consequences for the behavior of osteoblast- and osteoclast-like cells in co-culture, which partially depend on the differentiation status of the osteoclast-like cells. © 2014 S. Karger GmbH, Freiburg [ABSTRACT FROM AUTHOR]

- Background: Hank's balanced salt solution (HBSS) and milk have gained wide acceptance as storage media for avulsed tooth. However, the effect of the media and storage time on the periodontal ligament (PDL) cells involvement in the development of root resorption is still unclear. The purpose of this study was to evaluate whether precultured PDL cells in HBSS, milk, or modified Eagle's medium alpha (α-MEM) would affect osteoclastogenesis. Materials and methods: PDL cells were precultured in HBSS, milk, or α-MEM for 1 h or 6 h before being co-cultured with RAW 264.7 cells for an additional 3 days for mRNA analysis and 11 days for osteoclastogenesis assay. Results: Cyclooxygenase-2 (COX-2) mRNA was detected immediately in PDL cells precultured in the three storage media. The expression was up-regulated markedly in all co-cultures when compared with RAW cells alone. As a result of the co-culture, interleukin-1β (IL-1β) expression was detectable in both PDL and RAW cells. TRAP+ multinucleated, osteoclast-like cells developed in all co-cultures; the number of TRAP+ cells was highest ( P < 0.05) in the co-cultures that PDL cells precultured in milk for 6 h. The mRNA level of receptor activator of nuclear factor-kappa B ligand (RANKL) was not detected in PDL cells. Osteoprotegerin (OPG) mRNA expression reduced with increased preculture time, but the difference was not significant ( P > 0.05). Conclusions: PDL cells kept in the three storage media led to TRAP+ multinucleated, osteoclast-like cells formation via RANKL-independent signaling. The ability to induce osteoclastogenesis may be considered as one of the factors to evaluate the ability of storage medium to maintain PDL viability after tooth avulsion. [ABSTRACT FROM AUTHOR]

Background: Arterial medial calcification (AMC) is frequent prevalence in patients with end stage renal disease. Evidence about hyperphosphatemia induced anabolic crosstalk between osteoblast and osteoclast in AMC of uremia is rare. Lanthanum carbonate as an orally administered phosphate-binding agent to reduce phosphate load and ameliorate AMC, but direct evidence is missing.Methods: Detailed time-course studies were conducted of Sprague-Dawley rats fed with adenine and high phosphate diet to imitate the onset and progression of AMC of uremia. Calcification in great arteries was evaluated by VonKossa's and Masson's trichrome staining. Osteoblast (Runx2, Osteocalcin) and osteoclast (RANKL, Cathepsin K, TRAP) related genes were analyzed by Immunohistochemistry and qRT-PCR. Serum PTH, RANKL and OPG levels were detected by ELISA kit.Results: Serum phosphate was markedly increased in CRF group (6.94 ± 0.97 mmol/L) and 2%La group (5.12 ± 0.84 mmol/L) at week 4, while the latter group diminished significantly (2.92 ± 0.73 mmol/L vs CRF Group 3.48 ± 0.69, p < 0.01) at week 10. The rats that did not receive 2%La treatment had extensive von kossa staining for medial calcification in CRF group. In contrast, the rats in 2%La group just exhibit mild medial calcification. Inhibitory effect on progression of AMC was reflected by down regulated osteogenic genes and altered osteoclast-like genes. RANKL/OPG ratio in local calcification area was declined in 2%La group (vs CRF group, p <0.01), whereas marginal difference in serum among the three groups. In contrast to the robust expression of cathepsinK in calcified area, TRAP expression was not found.Conclusions: Abnormal phosphate homeostasis, induction of osteogenic conversion and osteoclast suppression were contributed to the current mechanisms of uremia associated arterial medial calcification based on our studies. Beneficial effects of Lanthanum carbonate could be mainly due to the decreased phosphate retention and cross-talk between osteoblast and osteoclast-like cell, both of which can be the therapeutic target for uremia associated with AMC. [ABSTRACT FROM AUTHOR]

The combination of a bone graft material with bisphosphonates (BPs) might be advantageous for an optimal balance of material resorption and stimulation of bone formation. This study investigated the effect of β-tricalcium phosphate (β-TCP) bone grafts coated with zoledronic acid (ZOL) on osteoblast-like cells and osteoclast-like cells (OLC). As a drug carrier, the polymer poly(D,L-lactide) was used and three different concentrations of ZOL were tested. β-TCP coated with ZOL stimulated the production of osteocalcin (OC), osteoprotegerin, and sRANKL in osteoblast-like cells. The polymer coating alone caused a significant increase in collagen type 1 and OC production. OLC viability was inhibited and the tartrate-resistant acidic phosphatase isoform-5b was significantly decreased after cultivation on polymer-coated β-TCP for 12 days. The three different concentrations of ZOL decreased cell viability and no TRAPiso-5b was detectable, indicating a strong reduction of the TRAPiso-5b after 12 days in culture. After 21 days in culture, only the higher ZOL concentrations significantly reduced cell viability and TRAPiso-5b. The results of this study show that coating of β-TCP with ZOL has stimulating effects on osteoblast-like cells. Additionally, an inhibition of osteoclasts was seen. The combination of this bone grafting material with BPs might, therefore, be effective in the treatment of large bone defects. [ABSTRACT FROM AUTHOR]

Bone substitute materials are required to support the remodeling process, which consists of osteoclastic resorption and osteoblastic synthesis. Osteoclasts, the bone-resorbing cells, generate from differentiation of hemopoietic mononuclear cells. In the present study, we have evaluated the effects of 1.0 wt % strontium (Sr) and 1.0 wt % magnesium (Mg) doping in beta-tricalcium phosphate (β-TCP) on the differentiation of mononuclear cells into osteoclast-like cells and its resorptive activity. In vitro osteoclast-like cell formation, adhesion, and resorption were studied using osteoclast precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κβ ligand (RANKL). Osteoclast-like cell formation was noticed on pure and Sr-doped β-TCP samples at day 8, which was absent on Mg-doped β-TCP samples indicating decrease in initial osteoclast differentiation due to Mg doping. After 21 days of culture, osteoclast-like cell formation was evident on all samples with osteoclastic markers such as actin ring, multiple nuclei, and presence of vitronectin receptor αvβ3 integrin. After osteoclast differentiation, all substrates showed osteoclast-like cell-mediated degradation, however, significantly restricted for Mg-doped β-TCP samples. Our present results indicated that substrate chemistry controlled osteoclast differentiation and resorptive activity, which can be used in designing TCP-based resorbable bone substitutes with controlled degradation properties. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A: 2450-2461, 2012. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Degenerative aortic valve disease is characterized by some of the histological features of atherosclerotic lesions. Gamma-glutamyltransferase (GGT) has been recently implicated in pathogenesis of atherosclerosis, as well as in modulation of cells involved in calcium metabolism. We aimed to evaluate the possible implication of this enzyme activity in aortic valve disease. Methods: GGT immunohistochemistry was performed on valve leaflets of 64 patients with aortic valve stenosis undergoing valve replacement. Fractional GGT activity in plasma and tissue was analysed in a subgroup of cases by molecular exclusion chromatography. Results: A close association was found between tissue extracellular GGT staining and lipid deposits (p <0.0001). GGT was expressed by CD68-positive cells around neovessels, as well as by MMP-9- and TRAP-positive multinucleated cells in the vicinity of bone metaplasia areas. Total plasma GGT levels were associated with low HDL-c (p =0.028) and high triglycerides (p =0.017). Total GGT activity in tissue was negatively correlated with the extent of valves calcification (p =0.03). Both serum and tissue GGT levels were negatively associated with severity of valve stenosis, as judged by peak transvalvular pressure gradients (p <0.0003 and p <0.002, respectively). Conclusions: Accumulation of GGT activity inside the lipid component of valves leaflets suggests a common mechanism of lesion shaping underlying both atherosclerosis and degenerative aortic valve disease. Moreover, the finding of GGT expression in cells with an osteoclast-like phenotype, and its negative correlation with both valves calcification and degree of valvular stenosis lend additional support to the recently envisaged involvement of GGT in the homeostasis of calcified tissues. [ABSTRACT FROM AUTHOR]

Abstract: In this study the tissue reaction to five different β-tricalcium phosphate (β-TCP)-based bone substitute materials differing only in size, shape and porosity was analyzed over 60days, at 3, 10, 15, 30 and 60days after implantation. Using the subcutaneous implantation model in Wistar rats both the inflammatory response within the implantation bed and the resulting vascularization of the biomaterials were qualitatively and quantitatively assessed by means of standard and special histological staining methods. The data from this study showed that all investigated β-TCP bone substitutes induced the formation of multinucleated giant cells. Changes in size, shape and porosity influenced the integration of the biomaterials within the implantation bed and the formation of tartrate-resistant acid phosphatase (TRAP)-positive and TRAP-negative multinucleated giant cells, as well as the rate of vascularization. While a high porosity generally allowed cell and fiber in-growth within the center of the bone substitute, a lower porosity resulted in a mosaic-like integration of the materials, with the granules serving as place holders. The number of multinucleated giant cells located in the implantation bed positively correlated with the vascularization rate. These data emphasize that all biomaterials investigated were capable of inducing the formation of TRAP-positive multinucleated giant cells as a sign of biomaterial stability. Furthermore, these cells directly influenced vascularization by secretion of vascular endothelial growth factor (VEGF), as well as other chemokines. Based on these findings, the role of multinucleated giant cells in the foreign body reaction to biomaterials might need to be reconsidered. This study demonstrates that variations in the physical properties of a bone substitute material clearly influence the (extent of the) inflammatory reaction and its consequences. [ABSTRACT FROM AUTHOR]

Abstract: Nanocrystalline calcium phosphates containing carbonate have a high similarity to bone mineral. The reactions of bone cells (primary osteoblasts and osteoclast-like cells) on these materials as well as on sintered β-tricalcium phosphate and hydroxyapatite (HA) confirmed a good biocompatibility of the nanocrystalline samples. However, osteoclastic differentiation was constrained on the carbonate-rich samples, leading to a small number of osteoclast-like cells on the materials and few resorption pits. The grain size of the calcium phosphate ceramics (nano vs. micro) was less important than expected from to physico-chemical considerations. When comparing the nanocrystalline samples, the highest resorption rate was found for nano-HA with a low carbonate content, which strongly stimulated the differentiation of osteoclast-like cells on its surface. [Copyright &y& Elsevier]

Bone-forming osteoblasts and bone-resorbing osteoclasts play an important role during maintenance, adaptation and healing of bone, and both cell types are influenced by physical activity. The aim of the present study was to investigate the effect of a narrow mechanical stimulation window on osteoblast- and osteoclast-like cells. Primary human cells were cultured on a bone-like structure (dentine) and three-point bending with approximately 1,100 microstrain was applied to the dentine at varying frequencies (0.1 and 0.3 Hz) and duration (1, 3 and 5 min daily over 5 days) resulting in different patterns of mechanical stimulation of osteoblast- and osteoclast-like cells. The longest stimulation (5 min at 0.1 Hz) induced a significant increase in osteoblast alkaline phosphatase activity and a significant decrease in osteoprotegerin (OPG) production, and resulted in a significant increase in the soluble receptor activator of NF-κB ligand (sRANKL)/OPG ratio towards sRANKL in comparison to the unstimulated osteoblast-like cells. All stimulations caused a significant decrease in collagen type 1 synthesis. Stimulation for 1 min at 0.3 Hz decreased the fusion and resorption activity of the osteoclast-like cells. These results demonstrate a direct effect of mechanical stimuli on osteoblast-like cells as well as on osteoclast formation and activity in vitro. The change in the sRANKL/OPG ratio towards the stimulation of osteoclastogenesis stresses the necessity to investigate the effect of the same stimulation parameter on the co-culture of both cell types. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The development of composites has been recognized as a promising strategy to fulfil the complex requirements of biomaterials. The present study reports on the modification of a novel silica–collagen composite material by varying the inorganic/organic mass ratio and introducing calcium phosphate cement (CPC) as a third component. The sol–gel technique is used for processing, followed by xerogel formation under specific temperature and relative humidity conditions. Cylindrical monolithic samples up to 400mm3 were obtained without any sintering processes. Various hierarchical phases of the organic component were applied, ranging from tropocollagen and collagen fibrils up to collagen fibers, each characterized by atomic force microscopy. Focusing on the application of fibrils, various inorganic/organic mass ratios were used: 100/0, 85/15 and 70/30; their influence on the structure of the composite material was demonstrated by scanning electron microscopy. The composition was extended by the addition of 25wt.% CPC which led to increased bioactivity by accelerating the formation of bone apatite layers in simulated body fluid. Synchrotron microcomputed tomography demonstrated the homogeneous distribution of the cement particles in the silica–collagen matrix. Compressive strength tests showed that the mechanical properties of the brittle pure silica gel are changed significantly due to collagen addition. The highest ultimate strength of about 115MPa at about 18% total strain was registered for the 70/30 silica–collagen composite xerogels. Incorporation of CPC lowered the gel’s strength. By demonstrating differentiation of human monocytes into osteoclast-like cells, an important feature of the composite material regarding successful bone remodeling is fulfilled. [Copyright &y& Elsevier]

Structured Abstract Authors – Fujita S, Yamaguchi M, Utsunomiya T, Yamamoto H, Kasai K Objective – Recent studies have demonstrated that low-energy laser irradiation stimulates bone formation in vitro and in vivo. However, very little is known about the effects of laser irradiation on osteoclastogenesis. The receptor activator of the nuclear factor- kB (RANK) / RANK ligand (RANKL) / osteoprotegerin (OPG) system is essential and sufficient for osteoclastogenesis. The present study was designed to examine the effects of low-energy laser irradiation on expressions of RANK, RANKL, and OPG during experimental tooth movement. Design – To induce experimental tooth movement in rats, 10 g of orthodontic force was applied to the molars. Next, a Ga–Al–As diode laser was used to irradiate the area around the moved tooth and the amount of tooth movement was measured for 7 days. Immunohistochemical staining with RANK, RANKL, and OPG was performed. Real time PCR was also performed to elucidate the expression of RANK in irradiated rat osteoclast precursor cells in vitro. Results – In the irradiation group, the amount of tooth movement was significantly greater than in the non-irradiation group by the end of the experimental period. Cells that showed positive immunoreactions to the primary antibodies of RANKL and RANK were significantly increased in the irradiation group on day 2 and 3, compared with the non-irradiation group. In contrast, the expression of OPG was not changed. Further, RANK expression in osteoclast precursor cells was detected at an early stage (day 2 and 3) in the irradiation group. Conclusion – These findings suggest that low-energy laser irradiation stimulates the velocity of tooth movement via induction of RANK and RANKL. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have demonstrated that low-energy laser irradiation stimulates bone formation in vitro and in vivo. However, very little is known about the effects of laser irradiation on osteoclastogenesis. Macrophage colony-stimulating factor (M-CSF) is essential and sufficient for osteoclastogenesis. The present study was designed to examine the effects of low-energy laser irradiation on expressions of M-CSF and c-fms during experimental tooth movement. A total of 10g of orthodontic force was applied to rat molars to cause experimental tooth movement. A Ga-Al-As diode laser was used to irradiate the area around the moved tooth and the amount of tooth movement was measured for 7 days. Immunohistochemical staining with M-CSF and c-fms was performed. RT-PCR was also performed to elucidate the expression of c-fms from irradiated rat osteoclast precursor cells. In the irradiation group, the amount of tooth movement was significantly greater than that of the non-irradiation group at the end of the experiment. Cells positively stained with M-CSF and c-fms were found to be significantly increased in the irradiation group on days 2 and 3 as compared with the non-irradiation group. Further, c-fms expression in osteoclast precursor cells was detected at an early stage (days 2 and 3) in the irradiation group. These findings suggest that low-energy laser irradiation stimulates the velocity of tooth movement via the expressions of M-CSF and c-fms. [Copyright &y& Elsevier]

Abstract: Objective: Increasing research suggested that Cimicifugae rhizoma might be protective against osteoporosis. In this study, we investigated the effects of three cycloartane-type triterpenoids isolated from Cimicifugae rhizoma, cimicidol-3-O-β-d-xyloside (1), cimicidanol-3-O-β-d-xyloside (2) and acetylacteol-3-O-β-d-xyloside (3) on bone resorption in vitro and bone loss in ovariectomized (OVX) mice. Methods: The activities of the tested compounds on bone resorption were evaluated using three assays, neonatal mouse parietal bone organ culture, osteoclast-like cells (OCLs) formation and pit formation. The effects on bone mineral density (BMD) and uterine weight were examined using OVX mice. Using LC–MS/MS method, the serum concentrations of the triterpenoids were measured in mice serum collected at 0.5, 1, 3, 6 and 12h following its oral administration. Results: All of the tested compounds exerted the inhibitory effects on bone resorption in bone organ culture, suppressed both of the formation and the resorbing activity of OCLs. Furthermore, a synergistic effect was observed among those compounds. In vivo studies revealed that compounds 1–3 and the mixture of compounds 1–3 prevented the bone loss in OVX mice without affecting uterine weight, and each compound was detected in the mice serum after single oral administration. Conclusions: The triterpenoids exerted the inhibitory effects on osteoclastic bone resorption through the suppression of both OCLs formation and the resorbing activity of OCLs, and also showed a significant protective effect on BMD in OVX mice. The present results might provide a new pharmacological potential for the treatment of osteoporosis. [Copyright &y& Elsevier]

Some practitioners of the Nishino Breathing Method (NBM) were found to have a higher bone density than the average values of age- and gender-matched non-practitioners. Using bone cell culture models, we investigated a possible mechanism behind this observation. For the study of bone mineralization, we performed the following two experiments using cultured osteoblastic MC3T3-E1 cells: (i) Kozo Nishino, a Japanese Ki expert, sent Ki-energy to the cells once for 5 or 10min after they were seeded in culture dishes in the presence of 10% fetal bovine serum (FBS). They were incubated for 72h and the cells were counted. The number in the dish with 10-min Ki-exposure was significantly greater than that in the control (P<0.01 with n=8). We performed a reverse transcription-polymerase chain reaction (RT PCR) study using these cells, but the mRNA expressions did not change significantly. (ii) After cells were incubated for 72 h without Ki-exposure (in the presence of FBS), they were further cultured for 48 h (in the absence of FBS) to promote differentiation. At the beginning of the second culture stage, Ki was applied once for 10 min. Alter 48 h, RT PCR was performed. The mRNA expressions which are related to bone mineralization, such as Runx2, α1(I) collagen, alkaline phosphatase and osteocalcin, increased significantly (P<0.05 and n=4 for all). For the bone resorption study, we used mouse marrow cultures, which can form osteoclast-like cells in the presence of (1 34) parathyroid hormone (PTH), and stimulate resorption. We exposed these cells to Ki-energy twice for the duration of 5 or 10min on day 0 and day 4. On day 7, the cells were counted. The number of osteoclast-like cells in dishes with Ki exposure was significantly smaller than those in control dishes (P<0.05 with n = 5). The difference between 5-min exposure and 10-min exposure was not statistically significant. All of our data suggest that the Ki-effect on osteoporosis should be further explored. [ABSTRACT FROM AUTHOR]

The addition of 10% deep-sea water (DW) from a depth of 620 m, from which NaCl was eliminated, to the medium induced the best cellular growth and the highest activities of alkaline phosphatase (ALP) in MC3T3-E1 osteoblasts and tartrate-resistant acid phosphatase (TRAP) in osteoclast-like cells compared with those induced by the control medium. The 45CaCl2 uptake in MC3T3-E1 cells was enhanced by media containing 10% DW (126%) compared with those induced by the control medium, whereas 45Ca uptake in osteoclast-like cells was suppressed. The results from the cell culture study indicated that DW stimulates both osteoblastogenesis and osteoclastogenesis, i.e., bone turnover. The administration of DW to SAMP6 and SAMR1 for 6 months improved the biomechanical properties of SAM mouse skeleton on three-point bending tests. [Copyright &y& Elsevier]

The microstructure of biomaterials influences the cellular and biological responses in the bone. Octacalcium phosphate (OCP) exhibits higher biodegradability and osteoconductivity than hydroxyapatite (HA) during the conversion process from OCP to HA. However, the effect of the microstructure of OCP crystals on long tubular bones has not been clarified. In this study, two types of OCPs with different microstructures, fine-OCP (F-OCP) and coarse-OCP (C-OCP), were implanted in rat tibia for 4 weeks. F-OCP promoted cortical bone regeneration compared with C-OCP. The osteoclasts appearance was significantly higher in the C-OCP group than in the control group (defect only) at 1-week post-implantation. To investigate whether the solubility equilibrium depends on the different particle sizes of OCPs, Nano-OCP, which consisted of nanometer-sized OCPs, was prepared. The degree of supersaturation (DS) tended to decrease modestly in the order of C-OCP, F-OCP, and Nano-OCP with respect to HA and OCP in Tris-HCl buffer. F-OCP showed a higher phosphate ion concentration and lower calcium ion concentration after immersion in the buffer than C-OCP. The crystal structures of both OCPs tended to be converted to HA by rat abdominal implantation. These results suggest that differences in the microstructure of OCPs may affect osteoclastogenesis and result in osteoconductivity of this material in long tubular bone by altering dissolution behavior. [ABSTRACT FROM AUTHOR]

Salicylates are reported to have an inhibitory effect on bone resorption in vivo and in vitro. The present study examined the effect of sodium salicylate on the formation of osteoclast-like cells in vitro. When mouse bone marrow cells were cultured for 8 days with 10(-8) M 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25(OH)2D3), numerous clusters of mononuclear and multinucleated cells (MNCs) formed, which stained positive for tartrate-resistant acid phosphatase (TRAP-positive). In similar cultures using sodium salicylate, the number of both TRAP-positive mononuclear and TRAP-positive MNCs were found to diminish in proportion to the concentration of sodium salicylate. A time-course experimental model showed that the number of TRAP-positive MNCs decreased slightly when sodium salicylate was given early in the culture period, and decreased markedly when the drugs were given later in the culture period. Pit formation and bone-resorption area on the bone slices were also inhibited by adding sodium salicylate continuously with 1 alpha, 25(OH)2D3. The sodium salicylate showed no cytotoxic effect because the total number of adherent cells, including both TRAP-positive and TRAP-negative cells, was independent of the presence of sodium salicylate. These results suggest that sodium salicylate has an inhibitory effect on the recruitment of osteoclast-like MNCs and that this inhibition is greater during the later stage of mouse bone marrow culture. [ABSTRACT FROM AUTHOR]

Inflammatory reactions in rheumatoid arthritis (RA) often cause severe joint destruction. However, the mechanism of bone destruction is still a matter of controversy. To determine whether multinuclear cells found in the rheumatoid synovium can resorb bone, isolated synovial cells were assessed for tartrate-resistant acid phosphatase (TRAP) staining and the ability to resorb bone in a dentine resorption assay. TRAP-positive multinuclear cells were found in six out of 10 samples. These six samples showed resorption pit formation on dentine slices. The other four samples did not form resorption pits. The results of this study demonstrate that TRAP-positive multinuclear cells isolated from the rheumatoid synovium form resorption pits on dentine slices. Our results suggest that inflamed synovial cells in rheumatoid joints might participate in bone destruction. [ABSTRACT FROM PUBLISHER]

Osteoporosis is associated with vessel diseases attributed to hyperlipidemia, and bone resorption by multinucleated osteoclasts is related to lipid metabolism. In this study, we generated low-density lipoprotein receptor (LDLR)/lectin-like oxidized LDL receptor-1 (LOX-1, also known as Olr1) double knockout (dKO) mice. We found that, like LDLR single KO (sKO), LDLR/LOX-1 dKO impaired cell-cell fusion of osteoclast-like cells (OCLs). LDLR/LOX-1 dKO and LDLR sKO preosteoclasts exhibited decreased uptake of LDL. The cell surface cholesterol levels of both LDLR/LOX-1 dKO and LDLR sKO osteoclasts were lower than the levels of wild-type OCLs. Additionally, the amount of phosphatidylethanolamine (PE) on the cell surface was attenuated in LDLR/LOX-1 dKO and LDLR sKO preosteoclasts, whereas the PE distribution in wild-type OCLs was concentrated on the filopodia in contact with neighboring cells. Abrogation of the ATP binding cassette G1 (ABCG1) transporter, which transfers PE to the cell surface, caused decreased PE translocation to the cell surface and subsequent cell-cell fusion. The findings of this study indicate the involvement of a novel cascade (LDLR~ABCG1~PE translocation to cell surface~cell-cell fusion) in multinucleation of OCLs. [ABSTRACT FROM AUTHOR]

Abdominal aortic aneurysms (AAA) are a major cause of death. Currently, the mainstay of treatment for AAA is surgical repair and there are no FDA approved medical therapies for AAA. Much research is in progress to discover new medical therapies for AAA. The pathophysiology of AAA is understood to be a complex interplay of inflammatory and proteolytic processes that degenerate the aneurysm wall. Arterial calcification, which is observed in AAA but to a lesser extent than in arterial occlusive disease, occurs in a highly regulated manner in a similar process as mineral deposition in bone. Osteoblasts-like cells are responsible for mineral deposition in atherosclerotic plaques. Recently, osteoclast-like cells - the catabolic counterpart to osteoblasts - were discovered in atherosclerotic plaques. Additionally, osteoclast-like cells are present in the wall of AAA but not in healthy aortas. Osteoclast-like cells secrete matrix metalloproteinases (MMP) - proteases implicated in arterial aneurysm wall degeneration - and may contribute to the degredation of the aneurysm wall. Inhibiting osteoclast-like cells may prevent aneurysm progression by reducing tissue levels of MMPs. In this review, we discuss the pathophysiology of AAA formation and the current role of medical therapy in treatment of AAA. Furthermore, we highlight the emerging hypothesis that osteoclasts play a key role in the development of AAA and discuss therapies to inhibit osteoclastogenesis in AAA., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

1α, 25-dihydroxyvitamin D3 (1α, 25(OH)2D3) acts on the osteoblasts to enhance the expressions of receptor activator of nuclear factor κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) and induce the formation of osteoclasts. However, the mechanism in osteoblasts by which 1α, 25(OH)2D3 promotes osteoclastogenesis has not yet been completely understood. This study aimed to select the first generation of murine osteoblasts to explore the underlying mechanism of 1α, 25(OH)2D3-induced osteoclastic formation from bone marrow mononuclear cells (BMMNCs). We discovered the activation of osteoblastic NF-κB pathway under 10(-8) mol/L 1α, 25(OH)2D3 treatment, as evidenced by the transfer of NF-κB p65 from cytoplasm to nuclei. Then, the NF-κB p65-siRNA was designed, constructed, and transfected into osteoblastic cells. Immunofluorescence assay confirmed the successfully silenced NF-κB p65 gene in osteoblasts. In the co-culture system of osteoblasts and BMMNCs with 1α, 25(OH)2D3 added, the multinucleated osteoclast-like cells containing 2-3 nuclei were observed in BMMNCs co-cultured with non-transfection osteoblasts, conversely, silencing osteoblastic NF-κB p65 resulted in failed differentiation of BMMNCs along with substantial vacuolar degeneration in cytoplasm. In addition, the expressions of RANKL and M-CSF were notably decreased in NF-κB p65-silenced osteoblasts. Taken together, our data indicated that osteoblastic NF-κB pathway was involved in 1α, 25(OH)2D3-induced osteoclast-like cells formation from BMMNCs through regulating the expression of RANKL and M-CSF. Therefore, our findings further identified the mechanism of 1α, 25(OH)2D3-induced osteoclastogenesis on the basis of prior studies.

Objective: Neuropeptide FF (NPFF) has been implicated in many physiological processes but not osteoclastogenesis. We previously demonstrated that NPFF modulates the viability and nitric oxide (NO) production of RAW264.7 macrophages. This study was designed to investigate the effect of NPFF on receptor activator of nuclear factor κB ligand (RANKL)-mediated differentiation of RAW264.7 cells into osteoclast-like cells., Design: RAW264.7 cells were cultured in 96-stripwell plates or in Corning Osteo Assay Surface 96-well plates in the presence of various concentrations of NPFF with or without RANKL for 3 or 6 d. The differentiation of osteoclast-like cells was analyzed by tartrate-resistant acid phosphatase (TRAP) stain, TRAP activity and bone resorption capacity, respectively. The mRNA expression of NPFF2 receptor (NPFFR2) and osteoclast genes was evaluated by using real-time quantitative PCR which includes TRAP, RANK (receptor activator of NF-κB), Cathepsin K, MMP-9 (matrix metallopeptidase 9), Intβ3 (integrin β3) and NFATc1 (nuclear factor of activated T cells cytoplasmic 1). In addition, the influence of NPFF on the cell viability and NO release of RAW264.7 cells was measured by MTT assay and Griess method, respectively., Results: NPFF dose-dependently inhibited RANKL-induced osteoclast-like cells differentiation including TRAP-positive cell formation, TRAP activity and bone resorption capacity. Moreover, NO release and osteoclast gene expression of osteoclast-like cells were downregulated by NPFF. In addition, NPFFR2 gene expression in osteoclast-like cells was augmented in response to NPFF treatment., Conclusion: Our findings showed that NPFF could attenuate osteoclast-like cells differentiation in an in vitro osteoclastogenesis model., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Bone replacement materials must be able to regulate both osteoblastic synthesis of new bone and osteoclastic resorption process in order to maintain the balance of bone remodeling. Osteoclasts generate from differentiation of mononuclear cells. In the present study, we have studied the osteoclast-like-cells responses (differentiation from mononuclear cells and resorption) to beta tricalcium phosphate (β-TCP) doped with zinc (Zn) and strontium (Sr). Osteoclast-like-cells differentiation and resorption was studied in vitro using osteoclast-like-cells precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κβ ligand (RANKL). Morphological and immunohistochemical analysis confirmed successful differentiation of osteoclast-like-cells on the doped and undoped β-TCP substrates after 8 days of culture. Cells on the substrate surface expressed specific osteoclast markers such as; actin ring, multiple nucleus, tartrate-resistant acid phosphatase (TRAP) synthesis, and vitronectin receptor. However, quantitative TRAP assay indicated the inhibiting effect of Zn on osteoclast differentiation. Although, Zn doped β-TCP restricted osteoclast-like-cells differentiation, the samples were resorbed much faster. An increased resorption pit volume was noticed on Zn doped β-TCP samples after 28 days of culture compared to pure and Sr doped β-TCP. In this work, we demonstrated that β-TCP bone substitute materials can be successfully resorbed by osteoclast-like-cells, where both osteoclast-like-cells differentiation and resorption were modulated by Zn and/or Sr doping- a much needed property for successful bone remodeling.