Osteoclasts In Rats Research Articles

Effect and mechanism of aqueous extract of Strychni Semen on bone destruction in rats with type Ⅱ collagen-induced rheumatoid arthritis

To investigate the mechanism of action of aqueous extract of Strychni Semen(SA) on bone destruction in rats with type Ⅱ collagen-induced arthritis(CIA), the SD rats were randomly divided into normal group, model group, low, medium, and high dose(2.85, 5.70, and 11.40 mg·kg~(-1)) groups of SA, and methotrexate group. Except for the normal group, the CIA model was prepared for the other groups. After the second immunization, different doses of SA were given to the low, medium, and high dose groups of SA once a day, and the methotrexate group was given once every three days. 0.3% sodium hydroxymethylcellulose(CMC-Na) was given once a day to the normal and model groups for 28 d. The clinical score of arthritis was evaluated every three days. Micro computed tomography(Micro-CT) method was used to evaluate the degree of bone destruction. Histopathological changes in the joint tissue and the number of osteoclasts in CIA rats were evaluated by hematoxylin-eosin(HE) staining and tartrate-resistant acid phosphatase(TRAP) staining. The expression of interleukin-1β(IL-1β) in the joint tissue of rats was detected by immunohistochemistry. Western blot was used to detect key protein expression in mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) signaling pathways in the joint tissue of rats. The results showed that different doses of SA were able to improve the red and swollen inflammatory joint and joint deformity in CIA rats to varying degrees, reduce the clinical score, inhibit synovial inflammation, vascular opacification, cartilage erosion, and bone destruction, and reduce the number of TRAP-positive cells in bone tissue. Micro-CT results showed that the SA was able to increase bone mineral density, bone volume fraction, trabecular reduce, and trabecular number and reduce bone surface/bone volume and trabecular separation/spacing. Different doses of SA could down-regulate the protein expression of IL-1β, p-JNK, p-ERK, p-p38, PI3K, and p-Akt to varying degrees. In conclusion, SA can improve disease severity, attenuate histopathological and imaging changes in joints, and have osteoprotective effects in CIA rats, and its mechanism of action may be related to the inhibition of the overactivation of MAPK and PI3K/Akt signaling pathways.

ED-71 Improves Bone Mass in Ovariectomized Rats by Inhibiting Osteoclastogenesis Through EphrinB2-EphB4-RANKL/OPG Axis.

Estrogen deficiency is the main reason of postmenopausal osteoporosis. Eldecalcitol (ED-71) is a new active vitamin D analogue clinically used in the treatment of postmenopausal osteoporosis. We aimed to investigate whether EphrinB2-EphB4 and RANKL/RANK/OPG signaling cooperate in mediating the process of osteoporosis by ED-71. In vivo, the ovariectomized (OVX) rats were administered orally with 30 ng/kg ED-71 once a day for 8 weeks. HE staining, Masson staining and Immunofluorescence staining were used to evaluate bone mass, bone formation, osteoclastogenesis associated factors and the expression of EphrinB2, EphB4, RANKL and OPG. In vitro, H2O2 stimulation was used to simulate the cell environment in osteoporosis. Immunofluorescence, quantitative real time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western Blot were applied to detect the expression of EphrinB2, EphB4, RANKL and OPG. In osteoblasts, EphB4 was knocked down by EphB4 small-interfering RNA (siRNA) transfection. LY294002 (PI3K inhibitor) or ARQ092 (AKT inhibitor) was used to block PI3K/AKT pathway. An indirect co-culture system of osteoblasts and osteoclasts was established. The mRNA and protein expression of osteoclastogenes is associated factors were tested by qRT-PCR and Western Blot. ED-71 increased bone mass and decreased the number of osteoclasts in OVX rats. Moreover, ED-71 promoted the expression of EphrinB2, EphB4, and decreased the RANKL/OPG ratio in osteoblasts. Osteoclastogenesis was restrained when osteoclasts were indirectly co-cultured with ED-71-treated osteoblasts. After silencing of EphB4 expression in osteoblasts, ED-71 inhibited the expression of P-PI3K and P-AKT and increased the ratio of RANKL/OPG. This reversed the inhibitory effect of ED-71 on osteoclastogenes. Therefore, in ED-71-inhibited osteoclastogenes, EphB4 is a key factor affecting the secretion of RANKL and OPG by osteoblasts. EphB4 suppressed the RANKL/OPG ratio through activating PI3K/AKT signaling in osteoblasts. ED-71 inhibits osteoclastogenesis through EphrinB2-EphB4-RANKL/OPG axis, improving bone mass in ovariectomized rats. PI3K/AKT pathway is involved this process.

Cucumber seed polypeptides regulate RANKL-induced osteoclastogenesis through OPG/RANKL/RANK and NF-κB

Postmenopausal osteoporosis (PMOP) is a common disease that endangers the health of elderly women. Cucumber seeds have shown excellent therapeutic effects on PMOP, but the mechanism of cucumber seed peptide (CSP) remains unclear. The expression levels of NF-κB and osteoclast-related genes were detected by RT-qPCR. The levels of apoptosis-related proteins were detected by Western blotting. Nuclear translocation of NF-κB p65 and osteoclast formation were detected by immunofluorescence and tartrate-resistant acid phosphatase (TRAP) staining, respectively. ELISA was used to detect the expression levels of OPG, M-CSF, and RANKL. Hematoxylin–eosin (H&E) and TRAP staining were used to observe the effects of CSP on bone formation. In RAW264.7 cells, CSP (0.4 mg/L, 4 mg/L, and 40 mg/L) effectively inhibited the expression of osteoclast-related genes (Cathepsin-K, MT1-MMP, MMP-9, and TRAP). TRAP-positive multinucleated giant cells gradually decreased. Furthermore, NF-κB pathway activation downstream of RANK was inhibited. In bone marrow stromal cells (BMSCs), the expression levels of M-CSF and RANKL gradually decreased, and OPG gradually increased with increasing CSP concentrations. Treatment of RAW264.7 cells with pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB) prevented the formation of osteoclasts. Treatment with different concentrations of CSP effectively decreased the levels of RANKL and M-CSF in rat serum and increased the expression of OPG in the oophorectomy (OVX) rat model. Furthermore, different concentrations of CSP could ameliorate the loss of bone structure and inhibit the formation of osteoclasts in rats. CSP inhibits osteoclastogenesis by regulating the OPG/RANKL/RANK pathway and inhibiting the NF-kB pathway.

The combined treatment of insulin and naringin improves bone properties in rats with type 1 diabetes mellitus.

We have studied the effects of individual and combined treatment of insulin (I) and naringin (NAR) on the bone structure and biomechanical properties of femurs from streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were divided into five groups: (1) controls, (2) STZ-induced diabetic rats, (3) STZ-induced diabetic rats treated with I, (4) STZ-induced diabetic rats treated with NAR, and (5) STZ-induced diabetic rats treated with I+NAR. Bone mineral density (BMD), bone histomorphometry, biomechanical testing, and bone biomarker expressions were accomplished in femur of all animals, as well as serum biochemical analyses. The combined treatment of I+NAR increased the body weight and the femur BMD from STZ-induced diabetic rats. The bone biomechanical properties and the bone morphology of the femurs from STZ-induced diabetic rats were also improved by the combined treatment. The increased number of osteoclasts in STZ-induced diabetic rats was partially prevented by I, NAR, or I+NAR. NAR or I+NAR completely blocked the decrease in the number of osteocalcin (+) cells in the femur from STZ-induced diabetic rats. RUNX family transcription factor 2 immunostaining was much lower in STZ-induced diabetic rats than in control animals; the combination of I+NAR totally blocked this effect. The combined treatment not only ameliorated bone quality and function, but also normalized the variables related to glucose metabolism. Therefore, the combination of I+NAR might be a better therapeutic strategy than the individual I or NAR administration to reduce bone complications in diabetic patients.

Single-cell RNA sequencing reveals classical monocytes are the major precursors of rat osteoclasts

To dissect which subset of bone marrow monocyte is the major precursor of osteoclast, 3-month-old rat bone marrow was obtained for single-cell RNA sequencing. A total of 6091 cells were acquired for detailed analysis, with a median number of 1206 genes detected per cell and 17,959 genes detected in total. A total of 19 cell clusters were recognized, with the main lineages identified as B cells, Granulocytes, Monocytes, T cells, Erythrocytes and Macrophages. Monocytes were further divided into classical monocytes and non-classical monocytes. Compared with non-classical monocytes, classical monocytes highly expressed osteoclast differentiation related genes Mitf, Spi1, Fos and Csf1r. Additionally, biological processes of classical monocytes were related to osteoclast differentiation. qPCR revealed differentially expressed genes of classical monocytes played a role in osteoclast differentiation. In conclusion, classical monocytes were identified as the main precursors of osteoclasts in rats, and may contribute to osteoclast differentiation by regulating S100a4, S100a6, S100a10, Fn1, Vcan and Bcl2a1. The results of this study contribute to the understanding of the origin of osteoclasts and may provide potential biomarkers for early diagnosis of bone metabolic diseases, as well as molecular and cellular targets for clinical intervention in bone metabolic diseases.

Retraction Statement.

Retraction Statement.

Ac-SDKP Attenuates Activation of Lung Macrophages and Bone Osteoclasts in Rats Exposed to Silica by Inhibition of TLR4 and RANKL Signaling Pathways.

BackgroundSilica-induced inflammatory activation is associated with silicosis and various non-respiratory conditions. The present study was designed to examine the anti-inflammatory effects of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on lung macrophages and bone osteoclasts after silica inhalation in rats.MethodsWistar rats and NR8383 and RAW 264.7 cell lines were used in the present study. The receptor activator of nuclear factor kappa-B ligand (RANKL) and toll-like receptor 4 (TLR4) signaling pathways was measured in the lung tissue of rats or NR8383/RAW 264.7 cells exposed to silica. The microarchitecture of the trabecular bone in the tibia and femur was evaluated in silicotic rats. Furthermore, the roles of Ac-SDKP on silicotic rats, silica-treated NR8383/RAW 264.7 cells, and RANKL-induced osteoclast differentiation were studied.ResultsThe data indicated that silica inhalation might activate the RANKL and TLR4 signaling pathways in lung macrophages, thus inducing the lung inflammatory and proteolytic phenotype of macrophages and osteoclasts in lung and bone. Ac-SDKP maintained the lung elastin level by inhibiting lung inflammation and macrophage activation via the RANKL and TLR4 signaling pathways. Ac-SDKP also attenuated the reduction in femoral bone mineral density in silicotic rats by inhibiting osteoclast differentiation via the RANKL signaling pathway.ConclusionOur findings support the hypothesis that inhalation of crystalline silica induces activation of lung macrophages and bone osteoclasts via the RANKL and TLR4 signaling pathways. Ac-SDKP has the potential to stabilize lung homeostasis and bone metabolism.

Phagocytic osteoclasts in the alveolar bone of diabetic rats with periodontitis

Periodontitis is a bacteria-induced inflammatory disease associated with alveolar bone loss. Osteoclast is a macrophage-lineage cell that exhibits phagocytic activity; however, osteoclast phagocytic activity has not been demonstrated under pathological conditions. Diabetes is a pathological condition that exacerbates alveolar bone loss via periodontitis; therefore, we examined phagocytic osteoclasts in diabetic rats that had periodontitis. The rats were divided into the control (C), periodontitis (P), and diabetes with periodontitis (DP) groups. Diabetes and periodontitis were induced by streptozotocin injection and ligature of the mandibular first molars, respectively. On days 3 and 20 after the ligature, the rats were sacrificed, and osteoclasts containing inclusions were quantified by tartrate-resistant acid phosphatase staining. On day 3, there were more osteoclasts containing inclusions in the DP group than in the C group. Among inclusions, osteocyte-like cells and dense bodies were more frequently observed in the DP group than in the C group. Cytoplasm-like structures were elevated more in the DP group than in the C and P groups. However, no differences were observed on day 20. Interestingly, some osteoclasts were in contact with the osteocytes within the exposed lacunae and contained several inclusions within a large vacuole. Thus, the elevation of phagocytic osteoclasts in rats with diabetes and periodontitis provides insight into the role of osteoclast phagocytic activity under pathological conditions.

Calcitonin Induces Bone Formation by Increasing Expression of Wnt10b in Osteoclasts in Ovariectomy-Induced Osteoporotic Rats.

Calcitonin is a small peptide hormone secreted from the parafollicular cells of the thyroid gland in response to an increase in serum calcium. The inhibition of osteoclastic resorption is the main mechanism by which calcitonin quickly decreases circulating calcium levels. Although calcitonin pharmacologically acts on osteoclasts to prevent bone resorption, the results of studies on genetically modified animals have shown that the physiological effect of calcitonin is in the inhibition of osteoblastic bone formation. Because the calcitonin receptor is only expressed in osteoclasts, the effect of calcitonin on osteoblasts maybe indirect and mediated via osteoclasts. Wnt ligands are involved in various aspects of skeletal biology, including bone remodeling and endochondral bone formation. Wnt10b has recently been recognized as a clastokine, and is potentially a therapeutic target for treating bone disorders. However, the extent to which Wnt signaling is involved in bone physiology and disease is not yet fully understood. We hypothesize that calcitonin indirectly increases osteoblastic bone formation by inducing Wnt10b expression in osteoclasts. Micro-CT analysis revealed reduced bone loss in calcitonin-treated ovariectomized rats. The serum of animals treated with calcitonin had decreased TRAP5b and CTX-1 but increased osteocalcin, P1NP, and Wnt10b. Immunohistochemistry staining showed that the level of Wnt10b in the femur was increased in calcitonin-treated groups as compared with control groups. Hematopoietic mononuclear cells were separated from rat femur and tibia bone marrow, and were induced into osteoclasts following treatment with M-CSF and RANKL. In these cells, immunoconfocal microscopy and Western blot analysis showed that calcitonin induced an increase in Wnt10b expression. In a culture of osteoblasts isolated from neonatal rat calvariae, the calcitonin-treated osteoclast supernatant showed an increase in mineralization, as indicated by ALP and alizarin red staining. Taken together, these results indicate that calcitonin induces bone formation by increasing the expression of Wnt10b in osteoclasts in ovariectomy-induced osteoporotic rats. The present study provides in-depth information about the effects of calcitonin on bone remodeling and will thus help in the development of future potential therapeutic strategies for postmenopausal osteoporosis.

Postorthodontic Relapse Prevention by Administration of Grape Seed (Vitis vinifera) Extract Containing Cyanidine in Rats.

Objective The aim of this study was to analyze the effect of grape seed extract containing cyanidin on osteoclastogenesis (by means of receptor activator of nuclear factor-κ B ligand [RANKL] and osteoprotegerin [OPG] levels) and the number of osteoclasts during orthodontic relapse in Wistar rats. Materials and Methods This study is an in vivo quasi experimental research. A total of 32 male Wistar rats were used in the study, which were randomly split equally into two groups, grape seed (GS) and control group (CG). All rats were given an orthodontic force of 35 cN using a stainless steel 3-spin coil spring that was activated for 7 days and then conditioned to be passive. During this phase, the GS group was administered grape seed extract containing cyanidin once per day. Orthodontic appliances were removed from both groups afterward, and then the alveolar bone tissue was isolated consecutively according to observation days (days 1, 3, 7, and 14), while OPG and RANKL levels were analyzed in their gingival crevicular fluid using a specific enzyme-linked immunosorbent assay (ELISA). Tissues were then stained with hematoxylin–eosin (H&E) and observed under a light microscope to count the number of osteoclast cells. Data were analyzed statistically using an independent t -test ( p < 0.05). Results The number of osteoclasts in the GS group was significantly lower than that in the CG group on all experiment days ( p = 0.021; p = 0.001; p = 0.024; p = 0.001; p < 0.05). ELISA results showed that the RANKL level of the GS group was significantly lower on days 3 and 7 ( p = 0.025; p = 0.039; p < 0.05), while the OPG level was significantly higher on days 1 and 3 in the GS group than in the CG group ( p = 0.039; p = 0.021; p < 0.05). Conclusion Grape seed extract can prevent postorthodontic relapse movement by inhibiting osteoclastogenesis and reducing the number of osteoclasts in Wistar rats.

Alveolar Bone Osteoclast Profile in the Periodontitis Wistar Rats Model with the Snail Slime (Achatina Fulica) Application.

BACKGROUND:Bone damage is a result of periodontal disease that occurs due to changes in osteoclast and osteoblast activity in response to local inflammation. The bacteria Aggregatibacter actinomycetemcomitans produces Lipopolysaccharide (LPS), which can increase osteoclast activity.AIM:This study aimed to analyse the decrease in alveolar bone osteoclasts in periodontitis rats’ model with the application of snail slime.METHODS:Wistar rats (27) with periodontitis divided into three groups, namely the control group (debridement), P1 group (debridement and application of oral snail slime) 300 Mg/Kg Body weight, P2 group (debridement, application of topical snail slime) 0.1 Mg. Osteoclast profile analysis was carried out by HE staining procedure to determine the histological feature of osteoclasts. The statistical significance was determined using the Shapiro-Wilk Test, One Way ANOVA, and Post Hoc test (p < 0.05).RESULTS:Osteoclast profile in rats with periodontitis applied with snail slime significantly decreased the number of osteoclasts with both oral and topical administration, there were significant differences in the number of osteoclasts between groups (one way ANOVA, p < 0.05) and there were no significant differences between groups P1 and P2 (Post Hoc, p > 0.05).CONCLUSION:In this study, there was a decrease in the number of osteoclasts which were slipped by snail slime in Wistar rats with periodontitis; this indicates a periodontitis healing process.

Effect of long non-coding RNA LINC00311 on proliferation of osteoclasts in osteoporotic rats

Objective To explore the effect of long non-coding RNA (lncRNA) LINC00311 on the proliferation of osteoclasts in osteoporotic rats. Methods Osteoclasts were isolated from ovariectomized rat models of ovariectomy. These osteoclasts were then transfected into the LINC00331 vector (observation group), short hairpin RNA (shRNA)-LINC00331 (control group), and not transfected (blank group). Finally, Western blotting was used to detect the expression of Delta-like 3 (DLL3), Notch1, Notch2, Jagged1, hairy and enhancer of split-1 (Hes-1) and tartrate resistant acid phosphatase (TRAP). The cell relative survival rate was detected by methyl thiazol tetrazolium (MTT) method, and the cell apoptosis and cell cycle were detected by flow cytometry. Results The expression of Notch2 and TRAP in the observation group (302.23±45.23, 324.32±42.12) was significantly higher than that in the blank group, the expression level of DLL3, Notch1, Jagged1 and Hes-1, and the apoptosis rate was significantly lower than that of the blank group [(52.33±12.36)%, (72.32±5.36)%, (175.23±23.25)%, (72.36±21.02)%, (3.87±0.91)%]. The cell related indexes of the control group were opposite to those of the observation group (0.96±0.11). The expression of cell Notch2 and TRAP, the relative survival rate of cell survival in the control group were (36.63±12.12, 112.02±32.01, 0.31±0.05) significantly lower than that of the blank group [(198.35±25.41)%, (259.63±35.26)%, (298.36±45.56)%, (198.69±32.25)%, (17.95±2.04)%], and the expression level of DLL3, Notch1, Jagged1 and Hes-1 of the control group were significantly higher than that of the blank group . Conclusion LncRNA LINC00311 can inhibit osteoclast apoptosis and increase the proliferation ability of osteoclasts by down regulating Notch signaling pathway. Key words: Long non-coding RNA; Osteoporosis; Osteoclasts; Proliferation; Differentiation

Anti-inflammatory role of papaya seed extracts in inhibiting osteoclastogenesis of rats with Periodontitis

Some previous studies mentioned that papaya seed extract has anti-inflammatory activity because it contains polyphenols including phenolic and flavonoid acids. Polyphenols of the papaya seed extracts are known to inhibit the severity of periodontitis. It can prevent the release of excessive osteoclastogenesis which may promote bone resorption aggravating periodontitis. Hence, this study aims to determine the role of papaya seed extract in reducing the number of osteoclasts in rats with periodontitis. Twenty-eight male Wistar rats were divided into 4 groups of induced periodontitis using lipopolisakarida (LPS) Porphyromonas gingivalis (P. Gingivalis) and followed by wire ligature in “8” placed around the mandible mandibular incisor. The first group was given standard food without any papaya extract, while each rat in three other groups with periodontitis (P2, P3, P4) was given with Papaya seed extracts of 200 mg/kgBW, 300 mg/kgBW, and 400 mg/kgBW. Seven rats in one group were not induced by periodontitis and were fed with standard food as a control group. On the 30th day, the rat was sacrificed, and its transverse pieces of alveolar bone around the mandibular incisor were stained with Hematoxylin-Eosin. The data analyzed by one way ANOVA indicated a significant result. Thus, it is conclusive that extract of papaya seed ethanol inhibited osteoclastogenesis in Wistar rats induced with periodontitis.

Beneficial effects of Cuscuta chinensis extract on glucocorticoid-induced osteoporosis through modulation of RANKL/OPG signals.

Cuscuta chinensis Lam. (Convolvulaceae) is an important herbal medicine widely used to improve sexual function, treat osteoporosis, and prevent aging, and has been reported to exhibit anti-osteoporotic effects in vitro. However, the activity of Cuscuta chinensis Lam. on glucocorticoid-induced osteoporosis still remains unclear. The present study aimed to assess the protective effect and the underlying mechanism of action of Cuscuta chinensis extract (CCE) against glucocorticoid-induced osteoporosis in vivo. Sprague-Dawley rats were randomly divided into four groups as follows: control group, osteoporosis group, and 2 CCE-treated osteoporosis groups (100 mg·kg-1·day-1). Blood samples and femur bones were collected for immunohistochemistry, biochemical, mRNA expression, and western blot analysis. HPLC analysis revealed that chlorogenic acid, quercetin, and hyperin were the major constituents of CCE. The results indicated that CCE increased bone length, bone weight, and bone mineral density and suppressed dexamethasone (DEX)-induced reduction in body weight. In addition, TRAP staining indicated that CCE reduced osteoclasts in DEX-induced osteoporosis rats. Mechanistically, CCE treatment alleviated the increase of bone resorption markers and the decline of osteogenic markers, which might be partially mediated by regulation of RANKL/OPG and RunX2 pathways. These results suggest that CCE showed promising effects in the protection against glucocorticoid-induced osteoporosis through protecting osteoblasts and suppressing osteoclastogenesis.

Tetrandrine attenuates the bone erosion in collagen-induced arthritis rats by inhibiting osteoclastogenesis via spleen tyrosine kinase.

Tetrandrine, a bisbenzylisoquinoline alkaloid, was previously demonstrated to attenuate inflammation and cartilage destruction in the ankles of mice with collagen-induced arthritis (CIA). Here, we explored the underlying mechanism by which tetrandrine prevented arthritis-induced bone erosion by focusing on the differentiation and function of osteoclasts. We found that daily administration of tetrandrine (30 mg/kg) markedly reduced the bone damage and decreased the number of osteoclasts in CIA rats. In vitro, tetrandrine inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis at the early stage and reduced the expressions of osteoclast-related marker genes. In bone marrow-derived macrophages and RAW264.7 cells, tetrandrine inhibited RANKL-induced translocation of NF-κB-p65 and nuclear factor of activated T cell 1 (NFATc1) through suppressing spleen tyrosine kinase (Syk)-Bruton's tyrosine kinase-PLCγ2-Ca2+ signaling. Of interest, tetrandrine did not affect the phosphorylation of immunoreceptor tyrosine-based activation motifs, the conventional upstream of Syk, but it inhibited the activity of Syk by enhancing its ubiquitination and degradation. The anti-osteoclastogenesis effect of tetrandrine nearly disappeared when it was used in combination with the Syk inhibitor piceatannol or in constitutively activated Syk-overexpressing cells. Taken together, tetrandrine attenuated CIA-induced bone destruction by inhibiting osteoclastogenesis through hindering the translocation of NF-κB-p65 and NFATc1 via reducing the activation of Syk.-Jia, Y., Miao, Y., Yue, M., Shu, M., Wei, Z., Dai, Y. Tetrandrine attenuates the bone erosion in collagen-induced arthritis rats by inhibiting osteoclastogenesis via spleen tyrosine kinase.

LncRNA LINC00311 Promotes the Proliferation and Differentiation of Osteoclasts in Osteoporotic Rats Through the Notch Signaling Pathway by Targeting DLL3

Osteoporosis is a commonly occurring condition marked by a loss of bone density. Previous evidence has highlighted the roles played by microRNAs as potential treatment tools for the disease. At present, the influence of long non-coding RNAs (lncRNAs) on the progression of osteoporosis remains largely unclear. Thus, an investigation was conducted into the target relationship between LINC00311, which has been reported to be highly expressed in osteoporosis, and delta-like 3 (DLL3), which is involved in the Notch signaling pathway, in connection with a series of bioinformatic methods. An osteoporotic rat model was established by means of ovariectomy (OVX) to evaluate the influence exerted by DLL3-binding LINC00311 on osteoclasts through the Notch signaling pathway. Osteoclasts were extracted from osteoporotic rats and transfected with the LINC00311-vector, shRNA-LINC00311, Notch activator, or a combination of the Notch activator and LINC00311-vector. Western blotting and RT-qPCR techniques were applied to determine the expression levels of LINC00311, DLL3, Notch1, Notch2, Jagged1, Hes-1 and TRAP in tissues and cells, while cell activity was detected by MTT assay. The cell cycle as well as the rate of apoptosis was detected by flow cytometry. The successfully established osteoporotic rats were designated into the OVX-siRNA, OVX-LINC00311 and OVX-control groups to observe the effects of LINC00311 on the proliferation and differentiation of osteoclasts. Cells transfected with the LINC00311-vector exhibited increased expression levels of Notch2 and TRPA as well as increased cell activity, while decreased expression levels of DLL3, Notch1, Jagged1 and Hes-1, along with a decreased cell apoptosis rate, were observed. The opposite tendencies of these parameters were observed in the cells treated with shRNA-LINC00311. A key observation was made when the Notch signaling pathway was activated, in that the cell activity was decreased while the rate of apoptosis increased. In comparison with the OVX-control group, the expression levels of LINC00311, Notch2 and TRAP as well as the positive expression rate of TRAP all exhibited reductions, while those of DLL3, Jagged1 and Notch1 were elevated in the OVX-siRNA group. Compared with those in the sham group, in the OVX-control and OVX-LINC00311 groups, LINC00311 and the expression levels of Notch2 and TRAP were increased; however, decreased levels of DLL3, Jagged1 and Notch1 were noted. Taken together, the key findings of the present study suggest that LINC00311 induces proliferation and inhibits apoptosis of osteoclasts via the regulation of the Notch signaling pathway by inhibiting DLL3 expression, ultimately demonstrating that LINC00311 and its target gene DLL3 may serve as independent factors in cases of osteoporosis.

Protective effect of low-dose risedronate against osteocyte apoptosis and bone loss in ovariectomized rats

Osteocyte apoptosis is the first reaction to estrogen depletion, thereby stimulating osteoclastic bone resorption resulting in bone loss. We investigated the effects of two different risedronate (RIS) doses (high and low) on osteocyte apoptosis, osteoclast activity and bone loss in ovariectomized rats. Forty rats with ovariectomy (OVX) and sham ovariectomy (SHAM) were divided into 4 groups: 1) SHAM rats treated with saline (SHAM); 2) OVX rats treated with saline (OVX); 3) OVX rats treated with low-dose RIS (OVX-LR, 0.08 μg/kg/day); 4) OVX rats treated with high-dose RIS (OVX-HR, 0.8 μg/kg/day). All animals were sacrificed 90 days after surgery for the examinations of osteocyte apoptosis by caspase-3 staining, osteoclast activity by TRAP staining and bone volume by micro-CT scanning in lumbar vertebral cancellous bone. Both low and high dose RIS significantly reduced caspase-3 positive osteocytes, empty lacunae and TRAP positive osteoclasts in OVX rats. Although the difference in caspase-3 positive osteocytes was not significant between the OVX-LR and OVX-HR groups, numerically these cells were significantly more prevalent in OVX-HR (not OVX-LR) group than in SHAM group. TRAP positive osteoclasts were significantly higher in OVX-LR group than in SHAM or OVX-HR group. There was no significant difference in bone volume among the OVX-LR, OVX-HR and SHAM groups, but lower in OVX group alone. However, significant increase in trabecular thickness only occurred in OVX-LR group. We conclude that both low and high dose RIS significantly inhibit osteocyte apoptosis and osteoclast activity in OVX rats, but the low-dose RIS has weaker effect on osteoclast activity. However, low-dose RIS preserves cancellous bone mass and microarchitecture as well as high-dose RIS after estrogen depletion.

Age-related effects on osteoclastic activities after orthodontic tooth movement.

ObjectivesTo elucidate the effects of age on the expression levels of the receptor activator of the nuclear factor-κB ligand (RANKL) and osteoclasts in the periodontal ligament during orthodontic mechanical loading and post-orthodontic retention.Materials and MethodsThe study included 20 male Sprague-Dawley rats, ten in the young group (aged four to five weeks) and ten in the adult group (aged 18 to 20 weeks). In each rat, the upper-left first molar was subjected to a seven-day orthodontic force loading followed by a seven-day retention period. The upper-right first molar served as a control. The amount of orthodontic tooth movement was measured after seven-day force application and seven-day post-orthodontic retention. The expression levels of RANKL and the tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were evaluated on day 7 (end of mechanical force loading) and day 14 (after seven days of post-orthodontic retention). Statistical analysis was performed using the t-test, and significance was set at p < 0.05.ResultsThere was no significant difference between the amount of tooth movement in the young group (0.96, standard deviation (sd) 0.30mm) and that in the adult group (0.80mm, sd 0.28) (p > 0.05) after the seven-day force application. On the compression side, the expression of RANKL and TRAP-positive osteoclasts in both the young and the adult groups increased after the application of force for seven days, and then decreased at the end of the seven-day retention period. However, by the end of the period, the expression of RANKL on the compression side dropped to the control level in the young group (p > 0.05), while it was still higher than that on the control side in the adult group (p < 0.05). The expression of RANKL on the compression side did not show significant difference between the young and the adult groups after seven-day force application (p > 0.05), but it was significantly higher in the adult group than that in the young group after seven-day post-orthodontic retention (p < 0.05). Similarly, the decreasing trend of TRAP-positive osteoclasts during the retention period in the adult group was less obvious than that in the young group.ConclusionsThe bone-resorptive activity in the young rats was more dynamic than that in the adult rats. The expression of RANKL and the number of osteoclasts in adult rats did not drop to the control level during the post-orthodontic retention period while RANKL expression and the number of osteoclasts in young rats had returned to the baseline.Cite this article: X. Li, M. Li, J. Lu, Y. Hu, L. Cui, D. Zhang, Y. Yang. Age-related effects on osteoclastic activities after orthodontic tooth movement. Bone Joint Res 2016;5:492–499. DOI: 10.1302/2046-3758.510.BJR-2016-0004.R2.

Exaggerated inflammatory environment decreases BMP-2/ACS-induced ectopic bone mass in a rat model: implications for clinical use of BMP-2

Numerous recent reports have observed a low osteoinductive efficacy property of bone morphogenetic protein-2 (BMP-2) and disappointing long-term outcomes in clinical cases. An alternative hypothesis, that these observations are caused by an exaggerated inflammatory environment, needs experimental evidence. Thirty-seven Sprague Dawley (SD) rats were administrated with Lipopolysaccharide (LPS) injections and BMP-2/absorbable collagen sponge (ACS) implantation to respectively mimic pre-operative and post-operative inflammatory responses. Blood samples and BMP-2/ACS implants were analyzed by enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (PCR), micro-computed tomography (μCT) and histological examination. LPS injections and BMP-2/ACS implantation provoked a significant elevation of inflammatory cytokines in serum and an obvious infiltration of inflammatory cells around BMP-2/ACS implants. The bone volume, mineral content and mineral density of the BMP-2/ACS implants from LPS-injected rats were significantly decreased, indicating that attenuated BMP-2-induced bone mass might be associated with down-regulated bone formation activity and up-regulated bone resorption activity. Furthermore, histological examination of the rhBMP-2/ACS implants showed a decreased expression of osteocalcin (OCN) and an increased number of osteoclasts in LPS-injected rats at 8 weeks; the expression level of bone turnover markers in serum and BMP-2/ACS implants revealed inhibited osteoblastogenesis activity and activated osteoclastogenesis activity in LPS-injected rats. Among the top three elevated pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) showed a suppressive effect on BMP-2-induced osteoblastic differentiation invitro. These data indicatethat an exaggerated inflammatory environment may decrease BMP-2/ACS-induced bone mass invivo by suppressing BMP-2-induced osteoblastic differentiation and by increasing the number or activity of osteoclasts. The negative role of exaggerated inflammation deserves consideration for future clinical use of BMP-2 in inducing bone regeneration.

Structural and functional changes in the alveolar bone osteoclasts of estrogen‐treated rats

This study investigated structural and functional features of apoptotic alveolar bone osteoclasts in estrogen-treated rats. For this purpose, 15 female rats 22 days old were divided into three groups: Estrogen (EG), Sham (SG) and Control (CG). The rats of EG received daily intramuscular injection of estrogen for 7 days. The SG received only the oil vehicle. Maxillary fragments containing alveolar bone were removed and processed for light and transmission electron microscopy. Area (OcA) and number of nuclei (OcN) and bone resorption surface per TRAP-positive osteoclasts (BS/OC) were obtained. Vimentin, caspase-3 and MMP-9 immunoreactions, TUNEL/TRAP and MMP-9/TUNEL combined reactions were performed. In EG, the OcA, OcN and BS/Oc were reduced. Moreover, osteoclasts showed cytoplasm immunolabelled by caspase-3 and a different pattern of vimentin expression in comparison with CG and SG. MMP-9 expression was not affected by estrogen and the TUNEL-positive osteoclasts were MMP-9-immunolabelled. In EG, ultrastructural images showed that apoptotic osteoclasts did not exhibit ruffled borders or clear zones and were shedding mononucleated portions. TRAP-positive structures containing irregular and dense chromatin were partially surrounded by fibroblast-like cells. In conclusion, the reduction in the BS/Oc may be due to reduction in OcA and OcN; these effects seem to be related to vimentin disarrangement rather than to an interference of estrogen with osteoclast MMP-9 expression. Osteoclast apoptosis involves caspase-3 activity and vimentin degradation; these cells release portions containing one apoptotic nucleus and, subsequently, undergo fragmentation, giving rise to apoptotic bodies.