Number Of TRAP-positive Cells Research Articles

Pleurotus sajor-caju (Fr.) Singer β-1,3-Glucanoligosaccharide (Ps-GOS) Suppresses RANKL-Induced Osteoclast Differentiation and Function in Pre-Osteoclastic RAW 264.7 Cells by Inhibiting the RANK/NFκB/cFOS/NFATc1 Signalling Pathway.

Edible grey oyster mushroom, Pleurotus sajor-caju, β (1,3), (1,6) glucan possesses a wide range of biological activities, including anti-inflammation, anti-microorganism and antioxidant. However, its biological activity is limited by low water solubility resulting from its high molecular weight. Our previous study demonstrated that enzymatic hydrolysis of grey oyster mushroom β-glucan using Hevea β-1,3-glucanase isozymes obtains a lower molecular weight and higher water solubility, Pleurotus sajor-caju glucanoligosaccharide (Ps-GOS). Additionally, Ps-GOS potentially reduces osteoporosis by enhancing osteoblast-bone formation, whereas its effect on osteoclast-bone resorption remains unknown. Therefore, our study investigated the modulatory activities and underlying mechanism of Ps-GOS on Receptor activator of nuclear factor kappa-Β ligand (RANKL) -induced osteoclastogenesis in pre-osteoclastic RAW 264.7 cells. Cell cytotoxicity of Ps-GOS on RAW 264.7 cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and its effect on osteoclast differentiation was determined by tartrate-resistant acid phosphatase (TRAP) staining. Additionally, its effect on osteoclast bone-resorptive ability was detected by pit formation assay. The osteoclastogenic-related factors were assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), Western blot and immunofluorescence. The results revealed that Ps-GOS was non-toxic and significantly suppressed the formation of mature osteoclast multinucleated cells and their resorption activity by reducing the number of TRAP-positive cells and pit formation areas in a dose-dependent manner. Additionally, Ps-GOS attenuated the nuclear factor kappa light chain-enhancer of activated B cells' P65 (NFκB-P65) expression and their subsequent master osteoclast modulators, including nuclear factor of activated T cell c1 (NFATc1) and Fos proto-oncogene (cFOS) via the NF-κB pathway. Furthermore, Ps-GOS markedly inhibited RANK expression, which serves as an initial transmitter of many osteoclastogenesis-related cascades and inhibited proteolytic enzymes, including TRAP, matrix metallopeptidase 9 (MMP-9) and cathepsin K (CTK). These findings indicate that Ps-GOS could potentially be beneficial as an effective natural agent for bone metabolic disease.

In Vitro Effects of Weissella cibaria CMU and CMS1 on Receptor Activator of NF-κB Ligand (RANKL)-Induced Osteoclast Differentiation.

Excessive osteoclast activity can promote periodontitis-associated bone destruction. The inhibitory mechanisms of Weissella cibaria strains CMU and CMS1 against periodontitis have not yet been fully elucidated. In this study, we aimed to investigate whether heat-killed (HK) W. cibaria CMU and CMS1 or their respective cell-free supernatants (CFSs) inhibit osteoclast differentiation and bone resorption in response to receptor activator of nuclear factor kappa-B ligand (RANKL)-treated RAW 264.7 cells. TRAP (tartrate-resistant acid phosphatase) staining and bone resorption assays revealed that both HK bacteria and CFSs significantly suppressed the number of TRAP-positive cells, TRAP activity, and bone pit formation compared to the RANKL-treated control (p < 0.05). HK bacteria dose-dependently inhibited osteoclastogenesis while selectively regulating certain genes in CFSs (p < 0.05). We found that disrupting the direct interaction between HK bacteria and RAW 264.7 cells abolished the inhibitory effect of HK bacteria on the expression of osteoclastogenesis-associated proteins (c-Fos, nuclear factor of activated T cells c1 (NFATc1), and cathepsin K). These results suggest that dead bacteria suppress osteoclast differentiation more effectively than the metabolites and may serve as beneficial agents in preventing periodontitis by inhibiting osteoclast differentiation via direct interaction with cells.

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.

Application of mesenchymal stromal cell sheets to prevent medication-related osteonecrosis of the jaw with titanium implants in rats.

Medication-related osteonecrosis of the jaw (MRONJ) is an intractable adverse event. Dental implants are one of the triggering factors of MRONJ, and implant therapy with low MRONJ risk is required. This study aimed to investigate a rat model of MRONJ induced by extraoral placement of titanium materials and the use of mesenchymal stromal cell (MSCs) sheets to prevent MRONJ. Eight-week-old male rats were administered zoledronate and dexamethasone thrice weekly until killing. A week after drug initiation, a titanium screw and a plate were placed on the left buccal side of the mandible. Allogeneic bone marrow-derived MSC sheets were co-grafted with the titanium plates in the MSC sheet ( +) group. Six weeks after titanium placement, the rats were killed, and their excised mandibular bones were subjected to micro-computed tomography (CT) analysis. Histological analysis was performed after the titanium implants were removed. Empty lacunae visualized on hematoxylin and eosin staining were used as evidence of bone necrosis. Bone necrosis was reduced in the MSC sheet ( +) group. Tartrate-resistant acid phosphatase (TRAP) staining revealed a decreased number of TRAP-positive cells in areas with a large number of empty lacunae in the MSC sheet (-) group. Micro-CT analyses demonstrated that the bone volume fraction (BV/TV) was not significantly different between the MSC sheet (-) and ( +) groups. We conclude that MRONJ can be triggered by a titanium placement in rats, and grafting of allogeneic MSC sheets has the potential to prevent MRONJ.

Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways.

Coenzyme Q10 (CoQ10) is a potent antioxidant that is implicated in the inhibition of osteoclastogenesis, but the underlying mechanism has not been determined. We explored the underlying molecular mechanisms involved in this process. RAW264.7 cells received receptor activator of NF-κB ligand (RANKL) and CoQ10, after which the differentiation and viability of osteoclasts were assessed. After the cells were treated with CoQ10 and/or H2O2 and RANKL, the levels of reactive oxygen species (ROS) and proteins involved in the PI3K/AKT/mTOR and MAPK pathways and autophagy were tested. Moreover, after the cells were pretreated with or without inhibitors of the two pathways or with the mitophagy agonist, the levels of autophagy-related proteins and osteoclast markers were measured. CoQ10 significantly decreased the number of TRAP-positive cells and the level of ROS but had no significant impact on cell viability. The relative phosphorylation levels of PI3K, AKT, mTOR, ERK, and p38 were significantly reduced, but the levels of FOXO3/LC3/Beclin1 were significantly augmented. Moreover, the levels of FOXO3/LC3/Beclin1 were significantly increased by the inhibitors and mitophagy agonist, while the levels of osteoclast markers showed the opposite results. Our data showed that CoQ10 prevented RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways in RAW264.7 cells.

The inhibition of Wnt signaling attenuates RANKL-induced osteoclastogenic macrophage activation.

Abdominal aortic aneurysms (AAAs) have been linked to the activation of osteoclastogenic macrophages. Reports have suggested that Wnt signaling has a dual effect of proliferation and differentiation during osteoclastogenesis. The Wnt/β-Catenin pathway is a critical regulator of cell pluripotency, cell survival, and cell fate decisions. It regulates cell proliferation and differentiation through transcriptional co-activators, CBP, and p300, respectively. The inhibition of β-catenin suppresses proliferation but induces differentiation of osteoclast precursor cells. This study aimed to examine the effect of ICG-001, a β-catenin/CBP-specific Wnt signaling inhibitor, on osteoclastogenesis by inhibiting proliferation without inducing differentiation. To induce osteoclastogenesis, RAW 264.7 macrophages were stimulated with a soluble receptor activator of NF-κB ligand (RANKL). The effect of Wnt signaling inhibition was examined by treating macrophages with or without ICG-001 during RANKL stimulation. The activation and differentiation of macrophages were examined through western blotting, quantitative PCR, and tartrate-resistant acid phosphate (TRAP) staining in vitro. The relative expression level of the nuclear factor of activated T-cells cytoplasmic 1 protein was significantly suppressed by ICG-001 treatment. The relative expression levels of mRNA of TRAP, cathepsin K, and matrix metalloproteinase-9 were significantly lower in the ICG-001-treated group. The number of TRAP-positive cells decreased in the ICG-001-treated group relative to the non-treated group. The inhibition of Wnt signaling pathway via ICG-001 suppressed osteoclastogenic macrophage activation. Our previous studies have shown the importance of osteoclastogenic macrophage activation in AAA. Further research to examine the therapeutic potential of ICG-001 on AAA is warranted.

Plum-Derived Exosome-like Nanovesicles Induce Differentiation of Osteoblasts and Reduction of Osteoclast Activation

Osteoblasts and osteoclasts play crucial roles in bone formation and bone resorption. We found that plum-derived exosome-like nanovesicles (PENVs) suppressed osteoclast activation and modulated osteoblast differentiation. PENVs increased the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells and osteoblasts from mouse bone marrow cultures. Notably, PENVs elevated the expression of osteoblastic transcription factors and osteoblast differentiation marker proteins in MC3T3-E1 cells. Higher levels of phosphorylated BMP-2, p38, JNK, and smad1 proteins were detected in PENV-treated MC3T3-E1 cells. Additionally, the number of TRAP-positive cells was significantly decreased in PENV-treated osteoclasts isolated from osteoblasts from mouse bone marrow cultures. Importantly, osteoclastogenesis of marker proteins such as PPAR-gamma, NFATc1, and c-Fos were suppressed by treatment with PENVs (50 μg/mL). Taken together, these results demonstrate that PENVs can be used as therapeutic targets for treating bone-related diseases by improving osteoblast differentiation and inhibiting osteoclast activation for the first time.

Resolvin D1 improves allograft osteointegration and directly enhances osteoblasts differentiation.

Allografts are the most common bone grafts for repairing osseous defects. However, their use is associated with an increased risk for infections, donor disease transmission and osteointegration deficiency. Resolvin D1 (RvD1) is an endogenous lipid with a scientifically proven pivotal role in inflammation resolution and osteoclastogenesis inhibition. Yet, its biological relevance as a potential bone regenerative drug has been scarcely studied. Here, we aim to investigate the RvD1 effect on allograft osteointegration in the alveolar bone regeneration (ABR) murine model. ABR model consisted of osseous defects that were generated by the extraction of the maxillary first molar in C57BL/6 mice. The sockets were filled with allograft and analyzed via RNA sequencing. Then they were locally injected with either RvD1 or saline via single or repeated administrations. The mice were sacrificed 2W after the procedure, and regenerated sites were analyzed using µCT and histology. First, MC3T3-E1 preosteoblasts were plated with IL-17 pro-inflammatory medium, and RANKL/OPG ratio was measured. Secondly, the MC3T3-E1 were cultured w/o RvD1, for 3W. Osteoblasts' markers were evaluated in different days, using qRT-PCR and Alizarin Red staining for calcified matrix. In vivo, neither allograft alone nor single RvD1 administration promote bone regeneration in comparison to the control of spontaneous healing and even triggered an elevation in NR1D1 and IL1RL1 expression, markers associated with inflammation and inhibition of bone cell differentiation. However, repeated RvD1 treatment increased bone content by 135.92% ± 45.98% compared to its specific control, repeated sham, and by 39.12% ± 26.3% when compared to the spontaneous healing control group (n=7/group). Histologically, repeated RvD1 reduced the number of TRAP-positive cells, and enhanced allograft osteointegration with new bone formation. In vitro, RvD1 rescued OPG expression and decreased RANKL/OPG ratio in IL-17 pro-inflammatory conditions. Furthermore, RvD1 increased the expression of RUNX2, OSX, BSP and OC/BGLAP2 and the mineralized extracellular matrix during MC3T3-E1 osteoblasts differentiation. Repeated administrations of RvD1 promote bone regeneration via a dual mechanism: directly, via enhancement of osteoblasts' differentiation and indirectly, through reduction of osteoclastogenesis and RANKL/OPG ratio. This suggests that RvD1 may be a potential therapeutic bioagent for osseous regeneration following allograft implantation.

Changes in the Subchondral Bone Affect Pain in the Natural Course of Traumatic Articular Cartilage Defects.

Articular cartilage defect causes joint pain and finally progresses to osteoarthritis. Although the subchondral bone condition affects clinical outcomes of cartilage defects, the natural course of changes in subchondral bone and associated pain in full-thickness cartilage defects remain unknown. Therefore, we investigated the natural course of histological changes in subchondral bone and joint pain in cartilage defects using a rat model. Full-thickness cartilage defects were created at the medial femoral condyle of 10-week-old male Sprague-Dawley rats. Rats were sacrificed at 3, 7, 14, 28, and 56 days postoperatively, and histological including immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining and micro-computed tomography (μCT) analyses of their knees were performed. Pain was evaluated using behavioral analysis and immunofluorescence staining of the dorsal root ganglion (DRG). The contour of the subchondral bone plate was maintained until day 3, but it was absorbed just under the cartilage defect from day 7 to 14. Starting on day 28, sclerotic changes surrounding the bone absorption area were detected. In the subchondral bone, the number of TRAP-positive cells peaked on day 14. Osteocalcin-positive cells were observed at 7 days, and their number gradually increased till day 56. Behavioral analysis showed that the total distance and the number of getting up by hind legs decreased on day 14. The number of calcitonin gene-related peptide-positive fibers in the DRG increased and was the highest on day 14. The subchondral bone condition under cartilage defects dynamically changes from bone resorption to sclerosis and is related to pain level.

Use of Photobiomodulation Combined with Fibrin Sealant and Bone Substitute Improving the Bone Repair of Critical Defects.

In this preclinical protocol, an adjunct method is used in an attempt to overcome the limitations of conventional therapeutic approaches applied to bone repair of large bone defects filled with scaffolds. Thus, we evaluate the effects of photobiomodulation therapy (PBMT) on the bone repair process on defects filled with demineralized bovine bone (B) and fibrin sealant (T). The groups were BC (blood clot), BT (B + T), BCP (BC + PBMT), and BTP (B + T + PBMT). Microtomographically, BC and BCP presented a hypodense cavity with hyperdense regions adjacent to the border of the wound, with a slight increase at 42 days. BT and BTP presented discrete hyperdensing areas at the border and around the B particles. Quantitatively, BCP and BTP (16.96 ± 4.38; 17.37 ± 4.38) showed higher mean bone density volume in relation to BC and BT (14.42 ± 3.66; 13.44 ± 3.88). Histologically, BC and BCP presented deposition of immature bone at the periphery and at 42 days new bone tissue became lamellar with organized total collagen fibers. BT and BTP showed inflammatory infiltrate along the particles, but at 42 days, it was resolved, mainly in BTP. In the birefringence analysis, BT and BTP, the percentage of red birefringence increased (9.14% to 20.98% and 7.21% to 27.57%, respectively), but green birefringence was similar in relation to 14 days (3.3% to 3.5% and 3.5% to 4.2%, respectively). The number of osteocytes in the neoformed bone matrix proportionally reduced in all evaluated groups. Immunostaining of bone morphogenetic protein (BMP—2/4), osteocalcin (OCN), and vascular endothelial growth factor (VEGF) were higher in BCP and BTP when compared to the BC and BT groups (p < 0.05). An increased number of TRAP positive cells (tartrate resistant acid phosphatase) was observed in BT and BTP. We conclude that PBMT positively influenced the repair of bone defects filled with B and T.

CpG Oligodeoxynucleotides Inhibit RANKL-Induced Osteoclast Formation by Upregulating A20 Deubiquitinase in RAW 264.7 Cells.

Objective Activation of toll-like receptor 9 (TLR9) has been proposed to play an inhibitory role in RANKL-induced osteoclastogenesis. A20 deubiquitinase has been found to be related to bone loss. This study investigated the role of CpG oligodeoxynucleotides (CpG-ODNs) through regulation of A20 deubiquitinase in RANKL-induced osteoclast formation. Methods RAW 264.7 cells, a murine monocyte-macrophage cell line, were incubated with or without CpG-ODN in the presence of RANKL. Osteoclast-specific genes and their related signaling molecules were measured by real-time quantitative polymerase chain reaction and Western blot assay. Morphological assessment for osteoclast formation was performed using tartrate-resistant acid phosphatase (TRAP) staining and F-actin ring formation staining. Results RANKL-induced osteoclast-related genes and proteins, c-Fos, NFATc1, TRAP, cathepsin K, and carbonic anhydrase II were significantly inhibited in RAW 264.7 cells stimulated with CpG-ODN. CpG-ODN attenuated TNF receptor-associated factor 6 (TRAF6), p-IκBα, and p-NF-κB expression in RAW 264 cells mediated by RANKL. CpG-ODN increased A20 gene and proteins in time-dependent manner. A20 expression under costimulation with CpG-ODN and RANKL was more decreased than under stimulation with RANKL alone. Cells transfected with A20 siRNA augmented expression of osteoclast-related genes and proteins. Number of TRAP-positive cells transfected with A20 siRNA was higher than those transfected with NC siRNA. A20 expression in cells transfected with IL-1β siRNA in the presence of both RANKL and CpG-ODN was more decreased than those with NC siRNA. Conclusion This study showed that CpG-ODN suppressed RANKL-induced osteoclast formation through regulation of the A20-TRAF6 axis in RAW 264.7 cells.

Interferon-gamma inducible protein 16 and type I interferon receptors expression in experimental apical periodontitis induced in wild-type mice.

The aim of this study was to evaluate the IFI16 and IFN-α/β receptors expression during the genesis and development of experimental apical periodontitis (AP) in mice teeth. Apical periodontitis was induced in the lower first molars of 40 C57BL/6 mice. They were divided according to the experimental periods 2, 7, 14, 21 and 42 days (n=8 per group). Five animals were used as a control group (without AP). Specimens were submitted to histological processing for description of the inflammatory process, immunostaining for the presence/absence and localization of IFI16 and IFN-α/β receptors (qualitative and semi-quantitative analysis) and tartrate-resistant acid phosphatase (TRAP) histoenzimology. The results showed a gradual development of AP over the experimental times. The expression of IFI16 was noticeably more exacerbated in the experimental early period (day 2) whilst the lowest expression was observed in the control group (p=.02). For IFN-α/β receptors, a higher intensity staining was observed 42 days after AP induction, that was statistically different from the control group (p=.02). In addition, the number of TRAP-positive cells was higher on the later periods (days 21 and 42; p < .001). IFI16 protein expression was highest during the early periods after AP induction in mice teeth, whilst IFN-α/β receptor expression was highest after AP became established.

Inhibition of osteoclastogenesis by interleukin-33 administration in the periodontal ligament under mechanical loading.

The molecular mechanisms mediating external root resorption are poorly understood. Interleukin-33 (IL-33) expression increased remarkably in the periodontal ligament (PDL) under orthodontic loading. The IL-33-driven responses are delicately cell type- and tissue context-dependent. It is unknown how IL-33 act on osteoclastogenesis in the context of root surface. This study aimed to investigate the effect of IL-33 on osteoclastogenesis in the PDL under mechanical loading. C57BL/6J mice were treated with injections of phosphate buffer saline (PBS) or recombinant mouse IL-33 (rmIL-33, 6μl, 30 μg/ml), and subjected to models of orthodontic tooth movement. Tartrated resistant acid phosphates (TRAP)-positive cells and IL-33 expressions were examined in the PDL. IL-33 release from human PDL cells (hPDLCs) was detected by ELISA. Cementoblast-like (OCCM-30) cells were cultured in the presence of rmIL-33 to examine the release of osteoclast-regulatory proteins. The effects of rmIL-33 on osteoclastogenesis were examined in vitro in cultures of bone marrow macrophages (BMMs) and in BMMs-OCCM-30 cocultures. Expressions of osteoclast-specific or -related genes and proteins were investigated in BMMs-OCCM-30 cocultures treated with or without rmIL-33, in the presence or absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) neutralizing antibody. Interleukin-33 expressions were upregulated in the PDL under orthodontic loading. Static compressive force enhanced expression and release of IL-33 from hPDLCs. Administration of rmIL-33 resulted in reduced number of TRAP-positive cells in the PDL, and inhibited osteoclast differentiation from BMMs in vitro. OCCM-30 cells had varied osteoprotegerin (OPG) / receptor activator for nuclear factor-κB ligand (RANKL) secretion and increased release of GM-CSF under rmIL-33 stimulation. Treatment with rmIL-33 in BMMs-OCCM-30 cocultures resulted in inhibited differentiation and decreased activity of osteoclasts, and these effects were partially reversed by GM-CSF neutralizing antibody. Interleukin-33 inhibits osteoclastogenesis in the PDL under orthodontic loading. The anti-osteoclastogenic effects were mediated partly by directly affecting osteoclast precursors and partly by cementoblast-mediated release of GM-CSF.

Semaphorin 3A protects against alveolar bone loss during orthodontic tooth movement in mice with periodontitis.

This study investigated the effect of local semaphorin 3A (Sema3A) administration on alveolar bone loss during OTM in a mouse model of periodontitis. Orthodontic tooth movement (OTM) for patients with periodontal disease is known to increase the risk of exacerbating alveolar bone loss due to inflammation of the periodontal tissue. However, its mechanism of action and prevention remains unclear. Mice (male 7-8 weeks old, C57BL/6J, n= 12) were divided into six groups: untreated group (control), without OTM and recovered from induced periodontitis (RP), with OTM and administered PBS or Sema3A to the gingiva after induced periodontitis (VehPO, SemaPO), with OTM and administered PBS or Sema3A to the gingiva without periodontitis induction (VehNO, SemaNO). Samples were collected on 14 days, and bone loss, histological analysis, cytokine production level, and tooth movement were assessed. Cultured human periodontal ligament (hPDL) cells were stimulated with lipopolysaccharide (LPS) and compressive force (CF), and mRNA expression levels of Sema3A and its receptors were analyzed. The bone loss was significantly lower in the SemaPO group than in the VehPO group. The number of TRAP-positive cells in the SemaPO group was significantly lower than that in the VehPO group and was at the same level as that in the control group. The receptor activator of nuclear factor (NF)-kB-ligand/osteoprotegerin (RANKL/OPG) ratio and the levels of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-17, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, in the gingival tissues were significantly lower in the SemaPO group than in the VehPO group. Additionally, Sema3A mRNA expression in hPDL cells was significantly decreased by co-stimulation with LPS and CF compared with that in the control group. Finally, the distance moved (dist.) and the mesial tipping angle (θ) was significantly smaller in the SemaPO group than in the VehPO group and was not significantly different from that of VehNO. Pathological alveolar bone loss exacerbated by OTM in periodontitis might be prevented by local administration of Sema3A without inhibiting OTM.

Inhibitory effect of artesunate on bone destruction in rheumatoid arthritis: an exploration based on AhR/ARNT/NQO1 signaling pathway

This study aimed to explore the effect of artesunate(ARS) on bone destruction in rheumatoid arthritis(RA) based on the aryl hydrocarbon receptor(AhR)/AhR nucleart ranslocator(ARNT)/NAD(P)H quinone dehydrogenase 1(NQO1) signaling pathway. Macrophage-colony stimulating factor(M-CSF) and receptor activator of nuclear factor-κB(RANKL) were used to induce the differentiation of primary bone marrow-derived mouse macrophages into osteoclasts. After intervention with ARS(0.2, 0.4, and 0.8 μmol·L~(-1)), the formation and differentiation of osteoclasts were observed by tartrate-resistant acid phosphatase(TRAP) and F-actin staining. The protein expression levels of AhR and NQO1 were detected by Western blot, and their distribution in osteoclasts was observed by immunofluorescence localization. Simultaneously, the collagen induced arthritis(CIA) rat model was established using type Ⅱ bovine collagen emulsion and then treated with ARS(7.5, 15, and 30 mg·kg~(-1)) by gavage for 30 days. Following the observation of spinal cord and bone destruction in CIA rats by Masson staining, the expression of AhR and ARNT in rat knee joint tissue was measured by immunohistochemistry and the NQO1 protein expression in the knee joint tissue by Western blot. The results showed that a large number of TRAP-positive cells were present in RANKL-induced rats. Compared with the RANKL-induced group, ARS(0.2, 0.4, and 0.8 μmol·L~(-1)) inhibited the number of TRAP-positive cells in a dose-dependent manner. F-actin staining results showed that the inhibition of F-actin formation was enhanced with the increase in ARS dose. As revealed by Western blot and immunofluorescence assay, ARS significantly promoted the expression of AhR and its transfer to the nucleus, thereby activating the protein expression of downstream ARNT and antioxidant enzyme NQO1. At the same time, the CIA rat model was successfully established. Masson staining revealed serious joint destruction in the model group, manifested by the failed staining of surface cartilage, disordered arrangement of collagen fibers, and unclear boundaries of cartilage and bone. The positive drug and ARS at different doses all improved cartilage and bone destruction to varying degrees, with the best efficacy detected in the high-dose ARS group. According to immunohistochemistry, ARS promoted AhR and ARNT protein expression in knee cartilage and bone of CIA rats and also NQO1 protein expression in rat knee and ankle joint tissues. In conclusion, ARS inhibited osteoclast differentiation by activating the AhR/ARNT/NQO1 signaling pathway, thus alleviating RA.

Effects of ultraviolet irradiation on beta-tricalcium phosphate as a bone graft substitute.

Surface modification of various materials using ultraviolet (UV) irradiation improves their wettability. The purpose of this study was to investigate the wettability of a β-tricalcium phosphate (TCP) surface and the composition changes and bioactivity of β-TCP after UV irradiation. We applied 172nm UV treatment to a β-TCP surface and measured the contact angle before and after UV irradiation. Energy-dispersive X-ray and Fourier transform infrared spectroscopy examinations were performed on the β-TCP disk with or without UV treatment. In an adhesion test of bone marrow cells using β-TCP disks with and without UV irradiation, cell attachment was measured 10, 30, 50, and 70h after β-TCP insertion. UV-irradiated β-TCP osteogenesis and absorption of bone substitutes were evaluated using hematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP) staining in a rabbit sinus model. The contact angle on the TCP surface decreased from 70° to 10° owing to UV irradiation. Conversely, UV irradiation did not change the composition of carbon, oxygen, and phosphorus. In the cell adhesion test, UV-irradiated β-TCP significantly increased cell adhesion compared with UV-unirradiated β-TCP after 10 to 30h of culture. In the rabbit sinus model, TRAP staining showed that UV-irradiated β-TCP significantly increased the number of TRAP-positive cells compared with unirradiated β-TCP granules in the central part of β-TCP. Our results indicate that the UV irradiation of β-TCP improves its clinical utility for surgical bone augmentation in the oral and maxillofacial region.

Effects of anti-mouse RANKL antibody on orthodontic tooth movement in mice

Background/purposeOrthodontic tooth movement is achieved by alveolar bone remodeling, and therefore the balance of bone resorption and formation is important. Receptor activator of nuclear factor-κB ligand (RANKL) plays a crucial role in bone resorption. We previously reported that tumor necrosis factor-α (TNF-α) is also important in bone resorption during tooth movement. In this study, we focused on bone and root resorption during orthodontic tooth movement in mice using anti-mouse RANKL antibody (anti-mRANKL ab). Materials and methodsAnti-mRANKL ab was administered intraperitoneally to mice that subsequently underwent orthodontic tooth movement. After 10 days, tissues around the moved teeth were histologically evaluated. To confirm the effects of anti-mRANKL ab on TNF-α induced bone resorption, TNF-α was administered with and without anti-mRANKL ab into the supracalvaria and the sutures of the calvaria were histologically evaluated. ResultsOrthodontic tooth movement was suppressed in mice treated with anti-mRANKL ab. Root resorption was observed after orthodontic tooth movement, but not in mice treated with anti-mRANKL ab. In the calvarial experiment, the number of TRAP-positive cells in the calvarial sutures was lower in mice administered TNF-α with anti-mRANKL ab than in mice administered TNF-α alone. ConclusionOur findings suggest that anti-mRANKL ab suppressed orthodontic tooth movement. This needs to be considered when orthodontic tooth movement is required in patients using anti-RANKL antibody.

Condylar Resorption Following Compressive Mechanical Stress in Rabbit Model - Association of Matrix Metalloproteinases.

Idiopathic condylar resorption (ICR) is a morphological change of the condylar head that occurs following orthodontic treatment or orthognathic surgery. This complication is serious, as it can cause relapse after mandible treatment. The aim of this experimental study was to evaluate the mechanism of influence of condylar resorption on compressive mechanical stress in temporomandibular joint following a change in occlusal position by mandible advancement. An osteotomy procedure at the midline of mandible was performed in 15 rabbits, with the left side moved forward by 3.5 mm. Advancement of the left side of the mandible resulted in compressive mechanical stress on condylar head on the left side. Samples were subjected to micro-computed tomography, histological staining and immunohistochemistry. The area and depth of anterior condylar resorption at two weeks were significantly different as compared to those at one week (p<0.05). TRAP staining confirmed the significantly largest number of TRAP-positive cells after two weeks (p=0.02), compared to one week. MMP-3 and MMP-13 immunostaining of the anterior condylar head at two weeks revealed high levels of both proteins from the surface to the deep layer of cartilage. Compressive mechanical stress following mandible advancement results in load on the anterior surface of the condylar head, which leads to bone resorption there, and induces MMP-3 and MMP-13 related to degradation of condylar head cartilage.

Influence of the treatment with the antineoplastic agents 5-fluorouracil and cisplatin on the severity of experimental periodontitis in rats.

The determination on how antineoplastic agents interfere on the progression of periodontitis is critical for improvement and even development of novel therapeutic approaches for periodontal management. This study evaluated the influence of chemotherapy with 5-fluorouracil (5-FU) or cisplatin (CIS) on healthy periodontal tissues and on the progression of experimental periodontitis (EP). One hundred forty-four male rats were divided into six groups (n = 24). Each group was treated with physiological saline solution (PSS) 0.9%, 5-FU, or CIS. Experimental periodontitis (EP) was induced by ligature placement. Animals were euthanized at 7, 15, and 30days after treatment. Data were statistically analyzed (p ≤ 0.05). The groups with EP and treated with 5-FU or CIS showed lower percentage of bone volume in the furcation region and higher percentage of alveolar bone loss, higher number of TRAP-positive cells, and lower number of PCNA-positive cells when compared group with EP and treated with PSS (p ≤ 0.05). Groups with EP and treated with 5-FU or CIS showed high immunolabelling pattern of RANKL, TNF-α, and IL-1β, moderate of BAX, and low of HIF-1α. Histological analysis showed severe tissue breakdown in the groups with EP and treated with 5-FU or CIS. Chemotherapy with antineoplastic agents 5-FU and CIS increased the intensity and duration of the inflammation and compromised tissue repair by reduction in cellular and vascular turnover. The more severe periodontal breakdown was caused by 5-FU.

Effects of I-125 seeds combined with anlotinib on tumor growth and bone metabolism in A549 tumor-bearing mice

Purpose This study aimed to investigate the therapeutic potential of tumor suppression and mechanism for different implantation modes of iodine-125 (I-125) seeds irradiation in a mice xenograft model, and its skeletal complications. Materials and methods A total of 24 mice carrying A549 lung tumor-derived xenografts were randomly assigned to four groups, including non-radioactive (sham) seeds implantation, I-125 seeds fractional implantation, I-125 seeds single implantation and I-125 seeds single implantation combined with anlotinib. Ki67 immunohistochemistry, TUNEL immunofluorescence and CD31 morphometric analysis were used to determine the proliferation index, rate of apoptotic cells and microvessel density, respectively. Additionally, the side effects on the skeletal system in mice treated with I-125 seeds implantation were evaluated by histomorphometric staining with tartrate-resistant acid phosphate (TRAP) and alkaline phosphatase (ALP) expression in femur, tartrate-resistant acid phosphatase 5b (TRACP-5b) and procollagen type I N-terminal propeptide (PINP) levels in serum were evaluated by enzyme-linked immunosorbent assay (ELISA). Results The I-125 seeds single and fractionated implantation had similar therapeutic effects and complications when the total number of I-125 seeds was the same. A single implantation of I-125 seeds with or without anlotinib could analogously inhibit the tumor growth in xenografts mice, while the single implantation combined with anlotinib had more effective in tumor inhibition. The results of Ki67, TUNEL and CD31 staining confirmed an evident reduction in tumor cell proliferation and angiogenesis, as well as an increase in apoptosis. A relatively integrated bone metabolism was indicated after I-125 seeds single implantation with or without anlotinib, and the results were similar in I-125 seeds fractional implantation, including a reduction in the number of TRAP-positive cells and an increase in ALP expression level. Additionally, the serum TRACP-5b activity was decreased and the serum PINP concentration was increased following I-125 seeds implantation. Conclusions Single and fractionated implantation pattern of I-125 radioactive seeds had similar therapeutic efficacy against tumor growth, while brachytherapy with I-125 seeds implantation may be an effective and safe treatment strategy for its potential protection against cancer treatment-induced bone loss.