Chitosan oligosaccharide inhibits skull resorption induced by lipopolysaccharides in mice.

BACKGROUND: Low-molecular-weight chitosan oligosaccharide (LMCOS), a chitosan degradation product, is water-soluble and easily absorbable, rendering it a popular biomaterial to study. However, its effect on bone remodelling remains unknown. Therefore, we evaluated the effect of LMCOS on lipopolysaccharide (LPS)-induced bone resorption in mice. METHODS: Six-week-old male C57BL/6 mice (n = five per group) were randomly divided into five groups: PBS, LPS, LPS + 0.005% LMCOS, LPS + 0.05% LMCOS, and LPS + 0.5% LMCOS. Then, the corresponding reagents (300 µL) were injected into the skull of the mice. To induce bone resorption, LPS was administered at 10 mg/kg per injection. The mice were injected three times a week with PBS alone or LPS without or with LMCOS and sacrificed 2 weeks later. The skull was removed for micro-computed tomography, haematoxylin-eosin staining, and tartrate-resistant acid phosphatase staining. The area of bone damage and osteoclast formation were evaluated and recorded. RESULTS: LMCOS treatment during LPS-induced skull resorption led to a notable reduction in the area of bone destruction; we observed a dose-dependent decrease in the area of bone destruction and number of osteoclasts with increasing LMCOS concentration. CONCLUSIONS: Our findings showed that LMCOS could inhibit skull bone damage induced by LPS in mice, further research to investigate its therapeutic potential for treating osteolytic diseases is required.

Synthesis of some novel orcinol based coumarin triazole hybrids with capabilities to inhibit RANKL-induced osteoclastogenesis through NF-κB signaling pathway.

A total of twenty-two novel coumarin triazole hybrids (4a-4k and 6a-6k) were synthesized from orcinol in good to excellent yields of 70-94%. The structures of all the synthesized compounds were elucidated by spectroscopic techniques such as 1H NMR, 13C NMR, and HRMS. The anti-inflammatory potential of synthesized compounds was investigated against the proinflammatory cytokine, TNF-α on U937 cell line and compounds 4d, 4j, and 6j were found to exhibit promising anti-inflammatory activity. These three compounds were further screened against TNF-α on LPS-stimulated RAW 264.7 cells, which confirm their anti-inflammatory potential. Furthermore, the above said active compounds were tested for their inhibitory effect on RANKL-induced osteoclastogenesis in RAW 264.7 cells by using tartrate resistant acid phosphatase (TRAP) staining assay at 10 µM. Molecular mechanism studies demonstrated that compound 4d exhibited dose dependent inhibition of RANKL-induced osteoclastogenesis by suppression of the NF-kB pathway. Thus, compound 4d is a promising candidate for further optimization to develop as a potent anti-osteoporotic agent.

Neural crest-mediated bone resorption is a determinant of species-specific jaw length.

Precise control of jaw length during development is crucial for proper form and function. Previously we have shown that in birds, neural crest mesenchyme (NCM) confers species-specific size and shape to the beak by regulating molecular and histological programs for the induction and deposition of cartilage and bone. Here we reveal that a hitherto unrecognized but similarly essential mechanism for establishing jaw length is the ability of NCM to mediate bone resorption. Osteoclasts are considered the predominant cells that resorb bone, although osteocytes have also been shown to participate in this process. In adults, bone resorption is tightly coupled to bone deposition as a means to maintain skeletal homeostasis. Yet, the role and regulation of bone resorption during growth of the embryonic skeleton have remained relatively unexplored. We compare jaw development in short-beaked quail versus long-billed duck and find that quail have substantially higher levels of enzymes expressed by bone-resorbing cells including tartrate-resistant acid phosphatase (TRAP), Matrix metalloproteinase 13 (Mmp13), and Mmp9. Then, we transplant NCM destined to form the jaw skeleton from quail to duck and generate chimeras in which osteocytes arise from quail donor NCM and osteoclasts come exclusively from the duck host. Chimeras develop quail-like jaw skeletons coincident with dramatically elevated expression of TRAP, Mmp13, and Mmp9. To test for a link between bone resorption and jaw length, we block resorption using a bisphosphonate, osteoprotegerin protein, or an MMP13 inhibitor, and this significantly lengthens the jaw. Conversely, activating resorption with RANKL protein shortens the jaw. Finally, we find that higher resorption in quail presages their relatively lower adult jaw bone mineral density (BMD) and that BMD is also NCM-mediated. Thus, our experiments suggest that NCM not only controls bone resorption by its own derivatives but also modulates the activity of mesoderm-derived osteoclasts, and in so doing enlists bone resorption as a key patterning mechanism underlying the functional morphology and evolution of the jaw.

Development of 1-Amino-4-(phenylamino)anthraquinone-2-sulfonate Sodium Derivatives as a New Class of Inhibitors of RANKL-Induced Osteoclastogenesis.

A series of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium derivatives was synthesized and evaluated for osteoclast inhibition using a TRAP-staining assay. Among them, two compounds, LCCY-13 and LCCY-15, dose-dependently suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. Moreover, the cytotoxicity assay on RAW264.7 cells suggested that the inhibition of osteoclastic bone resorption by these compounds was not a result of their cytotoxicity. Further, the inhibitory activities of compounds LCCY-13 and LCCY-15 were further confirmed by including specific inhibition of NFATc1 expression levels in nuclei using an immunofluorescent analysis. In addition, LCCY-13 and LCCY-15 also significantly attenuated the bone resorption activity of osteoclasts according to a pit formation assay. Thus, a new class of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium compounds might be considered as an essential lead structure for the further development of anti-resorptive agents.

Novel inhibitors of RANKL-induced osteoclastogenesis: Design, synthesis, and biological evaluation of 6-(2,4-difluorophenyl)-3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-diones.

A series of novel 6-(2,4-difluorophenyl)-3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives were synthesized and evaluated for their inhibitory effects on osteoclast activities by using TRAP-staining assay. Among the tested compounds, 3d and 3h exhibited more potent osteoclast-inhibitory activities than the lead compound NDMC503 (a ring-fused structure of NDMC101), as reported in our previous study. Both 3d and 3h exhibited two-fold increase in activity compared to NDMC503. In addition, our biological results indicated that 3d and 3h could suppress RANKL-induced osteoclastogenesis-related marker genes, such as NFATc1, c-fos, TRAP, and cathepsin K. Notably, 3d could significantly attenuate the bone-resorbing activity of osteoclasts in the pit formation assay. Thus, this study might provide a new class of lead structures that warrant further development as potential anti-resorptive agents.

Surface Pre-Reacted Glass-Ionomer Eluate Suppresses Osteoclastogenesis through Downregulation of the MAPK Signaling Pathway.

Surface pre-reacted glass-ionomer (S-PRG) is a new bioactive filler utilized for the restoration of decayed teeth by its ability to release six bioactive ions that prevent the adhesion of dental plaque to the tooth surface. Since ionic liquids are reported to facilitate transepithelial penetration, we reasoned that S-PRG applied to root caries could impact the osteoclasts (OCs) in the proximal alveolar bone. Therefore, this study aimed to investigate the effect of S-PRG eluate solution on RANKL-induced OC-genesis and mineral dissolution in vitro. Using RAW264.7 cells as OC precursor cells (OPCs), TRAP staining and pit formation assays were conducted to monitor OC-genesis and mineral dissolution, respectively, while OC-genesis-associated gene expression was measured using quantitative real-time PCR (qPCR). Expression of NFATc1, a master regulator of OC differentiation, and the phosphorylation of MAPK signaling molecules were measured using Western blotting. S-PRG eluate dilutions at 1/200 and 1/400 showed no cytotoxicity to RAW264.7 cells but did significantly suppress both OC-genesis and mineral dissolution. The same concentrations of S-PRG eluate downregulated the RANKL-mediated induction of OCSTAMP and CATK mRNAs, as well as the expression of NFATc1 protein and the phosphorylation of ERK, JNK, and p38. These results demonstrate that S-PRG eluate can downregulate RANKL-induced OC-genesis and mineral dissolution, suggesting that its application to root caries might prevent alveolar bone resorption.

Effect of phosphodiester composition in polyphosphoesters on the inhibition of osteoclastic differentiation of murine bone marrow mononuclear cells.

Osteoporosis is a common bone disorder characterized by reduced bone density and increased risk of fractures. The modulation of bone cell functions, particularly the inhibition of osteoclastic differentiation, plays a crucial role in osteoporosis treatment. Polyphosphoesters (PPEs) have shown the potential in reducing the function of osteoclast cells, but the effect of their chemical structure on osteoclastic differentiation remains largely unexplored. In this study, we evaluated the effect of PPE's chemical structure on the inhibition of osteoclastic differentiation of murine bone marrow mononuclear cells (BMNCs). PPEs containing phosphotriester and phosphodiester units at varying compositions were synthesized. Cytotoxicity testing confirmed the biocompatibility of the copolymers at concentrations below 0.5 mg/mL. Isolated from long bones, BMNCs were cultured in a differentiation medium supplemented with different PPE concentrations. Osteoclast formation was assessed through tartrate-resistant acid phosphatase and phalloidin staining. A significant decrease in the size of osteoclast cells formed upon BMNC contact with PPEs was observed, with a more pronounced effect observed at higher PPE concentrations. In addition, an increased composition of phosphodiester units in the PPEs yielded a decreased density of differentiated osteoclasts. Furthermore, real-time PCR analysis of major osteoclastic markers provided gene expression data that correlated with microscopic observations, confirming the effect of phosphodiester units in suppressing osteoclast differentiation of BMNCs from the early stages. These findings highlight the potential of PPEs as polymers are capable of modulating bone cell functions through their chemical structures.

Monocrotophos, an organophosphorus insecticide, induces cortical and trabecular bone loss in Swiss albino mice.

Till now monocrotophos (MCP) has been addressed as a neurotoxic stressor. Limited studies investigate its aftermath on bone pathologies. Given the fact that MCP is a propensely used insecticide in developing countries, this study investigates its potential to mirror osteoporotic features and bone loss incurred in a rodent model. Briefly, Swiss albino mice were orally gavaged daily with varying doses of MCP for 8 weeks. Musculoskeletal changes were analyzed through micro-computed tomography and histology. A series of in vitro and ex vivo cell culture experiments were performed on MC3T3E-1 and primary osteoclast cultures. Results highlight that oral gavaging with MCP causes bone loss from the cortico-trabecular interface by decreasing the osteoblast and increasing the osteoclast number. Results from in vitro studies establish that MCP treatment increases the TRAP-positive multinucleated cell number during osteoclast differentiation. Ex-vivo experiments with MCP-treated animal sera further substantiate the in vivo claims with significant decreases seen in cell viability, proliferation, mineralization and differentiation studies. In conclusion MCP induces osteoclastogenesis (bone loss) on direct stimulation and alters the circulating factors in MCP-treated serum. Holistically, this work would be of potential significance to patients suffering from pesticide induced osteoporosis.

Osteoclast Differentiation Assay.

Osteoclasts are highly specialized multinucleated cells derived from the monocyte/macrophage hematopoietic lineage that are uniquely capable of adhering to bone matrix and resorbing bone. The tartrate-resistant acid phosphatase (TRAP) assay is the most common method to detect osteoclasts population in vitro. Here we described a general protocol of inducing osteoclast differentiation from the murine macrophage cell line, RAW264.7, and identification of osteoclasts with the classical TRAP assay.

Generation and Analysis of Human and Murine Osteoclasts.

Osteoclasts are the only bone-resorbing cells in the body. Together with bone-forming osteoblasts, they are responsible for bone homeostasis and constant bone remodeling. Aberrant activation of osteoclasts leads to bone loss, as seen in postmenopausal osteoporosis or in autoimmune diseases like rheumatoid arthritis. Although much research has been performed to understand and prevent osteoclast-mediated bone loss, the mechanisms of osteoclast hyperactivation are not completely understood. This unit describes several protocols for ex vivo generation of murine and human osteoclasts, allowing study of the effects of specific cells, cytokines, or chemical substances on osteoclast formation and activity without the need for expensive and time-consuming animal experiments. In addition, we provide protocols for specific staining of osteoclasts and for analysis of resorption activity using calcium phosphate-coated surfaces or bone slices. © 2019 by John Wiley & Sons, Inc.

In vitro Osteoclastogenesis Assays Using Primary Mouse Bone Marrow Cells.

Osteoclasts are a group of bone-absorbing cells to degenerate bone matrix and play pivotal roles in bone growth and homeostasis. The unbalanced induction of osteoclast differentiation (osteoclastogenesis) in pathological conditions, such as osteoporosis, arthritis and skeleton metastasis of cancer, causes great pain, bone fracture, hypercalcemia or even death to patients. In vitro osteoclastogenesis analysis is useful to better understand osteoclast formation in physiological and pathological conditions. Here we summarized an easy-to-follow osteoclastogenesis protocol, which is suitable to evaluate the effect of different factors (cytokines, small molecular chemicals and conditioned medium from cell culture) on osteoclast differentiation using primary murine bone marrow cells.

Characterization of the F-box Proteins FBXW2 and FBXL14 in the Initiation of Bone Regeneration in Transplants given to Nude Mice.

BACKGROUND: Cultured bovine-periosteum-derived cells can form three-dimensional structures on tissue culture dishes without artificial scaffolding material, can induce bone regeneration in vivo. The utility of cultured bovine-periosteum-derived cells for bone tissue regeneration after their transplantation into nude mice has been reported, the precise F-box molecular mechanism was unclear. OBJECTIVE: The aim of this study was to investigate the specific F-box proteins required for bone regeneration by cultured bovine-periosteum-derived cells in vitro. METHODS: In the present study, periosteum tissue and cultured periosteum-derived cells were cultured for 5 weeks in vitro and then embedded in collagen gel with a green tissue-marking dye. Electrophoresis and immunohistochemistry were used to identify the specific F-box proteins required for tissue bone regeneration. RESULTS: The bovine-periosteum-derived cells were observed to form bone shortly after the expression of F-box proteins. After the initial phase of bone formation, the expression of the F-box proteins ceased. FBXW2 was shown to be expressed in the periosteum, but not in cultured periosteum-derived cells. Furthermore, FBXL14 disappeared during bone formation. CONCLUSIONS: Bone regeneration requires progenitor cells, such as bovine-periosteum-derived cells and the activation of the F-box Proteins FBXW2 and FBXL14, over time the expression of these proteins ceases. Further scientific and clinical trials are needed to investigate how the F-box Proteins can be used therapeutically to treat osteoporosis and osteonecrosis.

Effect of low level laser on osteoclasts and collagen fiber remodeling during the process of tooth retention after tooth movement in rats / 口腔疾病防治

Objective@#To investigate the effect of low level laser on osteoclast and collagen fiber remodeling during the process of tooth retention after tooth movement in rats and to provide the experimental basis for clinical application.@*Methods @# In total, 20 eight-week-old Wistar rats were selected to establish a mesial movement model of the maxillary first molar and then randomly divided into four groups after the appliance was removed. In total, 5 rats were included in each group, including baseline group (without force as blank control), control group (without any intervention after removing the force appliance), retention group (teeth were wrapped with orthodontic ligature wires that were screwed into hemp flower as fixed retention to maintain the space between the first molar and incisor after appliances were removed) and retention and low energy laser irradiation group (teeth were wrapped with the orthodontic ligature wires that were screwed into hemp flower as fixed retention and low energy laser irradiation was applied on days 0, 3, 6, 9 and 12 after appliance removal). Two weeks later, all the rats were sacrificed and the first molar tissue blocks of each group were collected. The distribution of osteoclasts and collagen fiber were studied by HE staining, TRAP staining and Masson staining to illustrate the process of alveolar bone and collagen fiber remodeling.@*Results @# Two weeks after appliances were removed, collagen fibers were deposited on both sides of the root in the baseline group, but no osteoclasts were observed in the distal side of the root. In the control group, collagen fibers on the two sides of the root were not obvious and osteoclasts were active on the distal side. In the retention group, collagen fibers were obvious on the two sides of the root and the osteoclasts on the distal side were less active than the control group. Regarding the retention and low energy laser irradiation group, collagen fibers were significantly obvious and osteoclasts were not seen. The difference was statistically significant between the retention and low energy laser irradiation group and the other three groups (P＜0.05). @*Conclusion@#These results suggest that fixed retention with simultaneous low level laser can effectively promote the synthesis of collagen fibers and inhibit the activity of osteoclasts during the process of tooth retention after movement, thus reducing the possibility of molar recurrence.

Effects of Tim-3 on osteoclast-like cell formation and bone resorption induced by peripheral blood mononuclear cells / 临床检验杂志

Objective@#To investigate the effects of Tim-3 on osteoclast-like cell (OLC) formation and bone resorption induced by peripheral blood mononuclear cells (PBMCs). @*Methods@#The expression levels of Tim-3 in of rheumatoid arthritis (RA) patients and healthy controls were detected by flow cytometry. The OLCs were induced by human PBMCs in vitro. The expression levels of tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK) and matrix metalloproteinase 9 (MMP9) mRNAs in the formation of OLCs were detected by real-time quantitative PCR. The morphology of OLCs was observed by Wright′s staining and G-actin staining, and the number of OLCs was counted by TRAP staining. The number and area of bone resorption pits in OLCs were detected by the Corning Osteo Assay Surface. @*Results@#The expression levels of Tim-3 in PBMCs of RA patients ([77.31±10.66]%) were significantly higher than that of healthy controls ([51.72±16.69]%, t=7.593, P＜0.01). When PBMCs with different Tim-3 levels were induced into OLCs, the area of bone resorption pits in the high Tim-3 level group ([1.054±0.085] S/mm 2 ) were significantly lower than those in the intermediate Tim-3 level group ([1.889±0.053] S/mm 2 ) and the low Tim-3 level group ([2.763±0.066] S/mm 2 , F=9.318, P＜0.05). @*Conclusion@#Tim-3 may negatively regulate the bone resorption of OLCs.