Tartrate-resistant Acid Phosphatase-positive Cells Research Articles

Caffeine induces alveolar bone loss in rats submitted to orthodontic movement via activation of receptor activator of nuclear factor ҡB, receptor activator of nuclear factor ҡB ligand, and osteoprotegerin pathway

Caffeine is a widely consumed substance with several effects on bone metabolism. This study aimed to investigate the effect of caffeine on the bone tissue of rats submitted to orthodontic movement. Twenty-five male Wistar rats underwent orthodontic movement (21 days) of the first permanent maxillary molars on the left side. The experimental group (caffeine; n= 13) and control group (n= 12) received caffeine and water, respectively, by gavage. Microcomputed tomography was performed to analyze orthodontic movement. Histologic analysis of the inflammatory infiltrate and osteoclast count by tartrate-resistant acid phosphatase were conducted. Maxilla tissue was evaluated for receptor activator of nuclear factor ҡB (RANK), RANK ligand (RANKL), and osteoprotegerin by immunohistochemistry. Caffeine exhibited a lower bone volume/tissue volume ratio (78.09% ± 5.83%) than the control (86.84% ± 4.89%; P<0.05). Inflammatory infiltrate was increased in the caffeine group compared with the control group (P<0.05). A higher number of tartrate-resistant acid phosphatase-positive cells was observed in the caffeine (9.67 ± 1.73) than in the control group (2.66 ± 0.76; P<0.01). Immunoexpression of RANK and RANKL in the caffeine group was greater than the control (P<0.05). The use of caffeine thermogenic induces alveolar bone loss in rats submitted to orthodontic movement via activation of RANK, RANKL, and osteoprotegerin signaling pathways.

Senolytics ameliorate the failure of bone regeneration through the cell senescence-related inflammatory signalling pathway

Stress-induced premature senescent (SIPS) cells induced by various stresses deteriorate cell functions. Dasatinib and quercetin senolytics (DQ) can alleviate several diseases by eliminating senescent cells. α-tricalcium phosphate (α-TCP) is a widely used therapeutic approach for bone restoration but induces bone formation for a comparatively long time. Furthermore, bone infection exacerbates the detrimental prognosis of bone formation during material implant surgery due to oral cavity bacteria and unintentional contamination. It is essential to mitigate the inhibitory effects on bone formation during surgical procedures. Little is known that DQ improves bone formation in Lipopolysaccharide (LPS)-contaminated implants and its intrinsic mechanisms in the study of maxillofacial bone defects. This study aims to investigate whether the administration of DQ ameliorates the impairments on bone repair inflammation and contamination by eliminating SIPS cells. α-TCP and LPS-contaminated α-TCP were implanted into Sprague-Dawley rat calvaria bone defects. Simultaneously, bone formation in the bone defects was investigated with or without the oral administration of DQ. Micro-computed tomography and hematoxylin-eosin staining showed that senolytics significantly enhanced bone formation at the defect site. Histology and immunofluorescence staining revealed that the levels of p21- and p16-positive senescent cells, inflammation, macrophages, reactive oxygen species, and tartrate-resistant acid phosphatase-positive cells declined after administering DQ. DQ could partially alleviate the production of senescent markers and senescence-associated secretory phenotypes in vitro. This study indicates that LPS-contaminated α-TCP-based biomaterials can induce cellular senescence and hamper bone regeneration. Senolytics have significant therapeutic potential in reducing the adverse osteogenic effects of biomaterial-related infections and improving bone formation capacity.

Biomodulation of Induced Tooth Movement by Three Methods, Corticopuncture, Photobiomodulation, and Their Combination: An Animal Study.

Objective: The aim of this study was to assess the effects of accelerated tooth movement (ATM) methods: corticopuncture (CP), photobiomodulation (PBM), and their combination (CP + PBM) by evaluating tooth displacement, alveolar bone changes, and molecular and cellular response compared with conventional induced tooth movement. Materials and methods: Tooth movement and bone changes were evaluated on days 1, 3, and 7 (9 animals per time point) using microtomography, histological, and immunohistochemical evaluation, at compression and tension sites. CP groups received two perforations in the palate and one mesial to the molars. PBM was performed using GaAlAs diode laser applied every other day for 7 days (λ = 808 nm, 100 mW) in two points for 15 sec/point and total energy of 3 J. Results: Tooth movement was significantly increased in all three ATM groups after 7 days compared with the control group (mean 0.24 mm) by 27% PBM (0.31 mm), 45% CP (0.35 mm), and 57% CP + PBM (0.38 mm) (p < 0.05). At the compression side, all ATM groups showed significant decrease in bone density on day 3 (p < 0.05) and significant less bone volume on day 7 compared with Control (p < 0.05). At the tension side, PBM group showed a significant increase in bone density and volume on day 3 (p < 0.05). Immunohistochemistry analysis showed that at the compression side, tartrate-resistant acid phosphatase-positive cells, RANKL, and tumor necrosis factor-alpha expression were highly marked of the PBM and the combined method groups (p < 0.05). PBM and CP + PBM groups showed a significant increase in expression Runt-related transcription factor 2 and osterix (p < 0.05) at the tension side. Conclusions: All ATM groups showed increase on tooth displacement with CP + PBM group showing greatest tooth displacement. CP method appears to stimulate bone catabolism, PBM has more effect on bone formation, and the combined method showing a synergistic effect on bone remodeling.

TMBIM6 deficiency leads to bone loss by accelerating osteoclastogenesis

TMBIM6 is an endoplasmic reticulum (ER) protein that modulates various physiological and pathological processes, including metabolism and cancer. However, its involvement in bone remodeling has not been investigated. In this study, we demonstrate that TMBIM6 serves as a crucial negative regulator of osteoclast differentiation, a process essential for bone remodeling. Our investigation of Tmbim6-knockout mice revealed an osteoporotic phenotype, and knockdown of Tmbim6 inhibited the formation of multinucleated tartrate-resistant acid phosphatase-positive cells, which are characteristic of osteoclasts. Transcriptome and immunoblot analyses uncovered that TMBIM6 exerts its inhibitory effect on osteoclastogenesis by scavenging reactive oxygen species and preventing p65 nuclear localization. Additionally, TMBIM6 depletion was found to promote p65 localization to osteoclast-related gene promoters. Notably, treatment with N-acetyl cysteine, an antioxidant, impeded the osteoclastogenesis induced by TMBIM6-depleted cells, supporting the role of TMBIM6 in redox regulation. Furthermore, we discovered that TMBIM6 controls redox regulation via NRF2 signaling pathways. Our findings establish TMBIM6 as a critical regulator of osteoclastogenesis and suggest its potential as a therapeutic target for the treatment of osteoporosis.

Effects of green tea extract epigallocatechin-3-gallate (EGCG) on orthodontic tooth movement and root resorption in rats

ObjectiveTo study the effect of EGCG on tooth movement and root resorption during orthodontic treatment in rats. MethodsA total of thirty six male Wistar rats were randomly and equally divided into three groups: control, 50 mg/kg EGCG, and 100 mg/kg EGCG. During the experiment, the subjects were submitted to an orthodontic tooth movement (OTM) model, rats in the experimental groups were given the corresponding dose of EGCG, while rats in the control group were administrated with an equal volume of normal saline solution by gavage. After 14 days of OTM, the rats were sacrificed by transcardial perfusion. Micro-CT of rat maxillaes was taken to analyze OTM distance and root resorption. The maxillary samples were prepared as histological sections for H&E staining, tartrate-resistant acid phosphatase (TRAP) staining and immunohistochemical (IHC) staining to be observed and analyzed. ResultsThe OTM distance and root resorption of rats in the dosed group decreased, and the number of TRAP positive cells in their periodontium decreased significantly. The expression level of RANKL was decreased in the EGCG group compared to the control group, while that of OPG, OCN and Runx2 was increased. Effects were more pronounced in 100 mg/kg group than in 50 mg/kg group. ConclusionEGCG reduces OTM and orthodontic induced root resorption (OIRR) in rats, and is able to attenuate osteoclastogenesis on the pressure side and promote osteogenesis on the tension side.

HBP/O-GlcNAcylation Metabolic Axis Regulates Bone Resorption Outcome

Osteoclasts play a key role in the regulation of bone mass and are highly active metabolically. Here we show that a metabolic reprogramming toward the hexosamine biosynthetic pathway (HBP) is required not only for osteoclast differentiation but also to determine the bone resorption mode during physiological and pathological bone remodeling. We found that pharmacological inhibition of O-GlcNAc transferase (OGT) significantly reduced protein O-GlcNAcylation and osteoclast differentiation. Accordingly, genetic deletion of OGT also inhibited osteoclast formation and downregulated critical markers related to osteoclasts differentiation and function (NFATc1, αvintegrin, cathepsin K). Indeed, cells treated with OSMI-1, an OGT inhibitor, also reduced nuclear translocation of NFATc1. Furthermore, the addition of exogenous N-acetylglucosamine (GlcNAc) strongly increased osteoclast formation and demineralization ability. Strikingly, our data show for the first time that O-GlcNAcylation facilitates an aggressive trench resorption mode in human cells. The incubation of osteoclasts with exogenous GlcNAc increases the percentage of erosion by trench while having no effect on pit resorption mode. Through time-lapse recording, we documented that osteoclasts making trenches moving across the bone surface are sensitive to GlcNAcylation. Finally, osteoclast-specific Ogt-deficient mice show increased bone density and reduced inflammation-induced bone loss during apical periodontitis model. We show that osteoclast-specific Ogt-deficient mice are less susceptible to develop bacterial-induced periapical lesion. Consistent with this, Ogt-deleted mice showed a decreased number of tartrate-resistant acid phosphatase-positive cells lining the apical periodontitis site. In summary, here we describe a hitherto undiscovered role of the HBP/O-GlcNAcylation axis tuning resorption mode and dictating bone resorption outcome.

The effects of seeding density and osteoclastic supplement concentration on osteoclastic differentiation and resorption

The bone resorbing osteoclasts are a complex type of cell essential for in vivo bone remodeling. There is no consensus on medium composition and seeding density for in vitro osteoclastogenesis, despite the importance thereof on osteoclastic differentiation and activity. The aim of this study was to investigate the relative effect of monocyte or peripheral blood mononuclear cell (PBMC) seeding density, osteoclastic supplement concentration and priming on the in vitro generation of functional osteoclasts, and to explore and evaluate the usefulness of commonly used markers for osteoclast cultures. Morphology and osteoclast formation were analyzed with fluorescence imaging for tartrate resistant acid phosphatase (TRAP) and integrin β3 (Iβ3). TRAP release was analyzed from supernatant samples, and resorption was analyzed from culture on Corning® Osteo Assay plates. In this study, we have shown that common non-standardized culturing conditions of monocyte or PBMCs had a significant effect on the in vitro generation of functional osteoclasts. We showed how increased osteoclastic supplement concentrations supported osteoclastic differentiation and resorption but not TRAP release, while priming resulted in increased TRAP release as well. Increased monocyte seeding densities resulted in more and large TRAP positive bi-nuclear cells, but not directly in more multinucleated osteoclasts, resorption or TRAP release. Increasing PBMC seeding densities resulted in more and larger osteoclasts and more resorption, although resorption was disproportionally low compared to the monocyte seeding density experiment. Exploration of commonly used markers for osteoclast cultures demonstrated that Iβ3 staining was an excellent and specific osteoclast marker in addition to TRAP staining, while supernatant TRAP measurements could not accurately predict osteoclastic resorptive activity. With improved understanding of the effect of seeding density and osteoclastic supplement concentration on osteoclasts, experiments yielding higher numbers of functional osteoclasts can ultimately improve our knowledge of osteoclasts, osteoclastogenesis, bone remodeling and bone diseases.

The effect of resolvin D1 on bone regeneration in a rat calvarial defect model.

Resolvin D1 (RvD1) is a pro-resolving lipid mediator of inflammation, endogenously synthesized from omega-3 docosahexaenoic acid. The purpose of this study was to investigate the effect of RvD1 on bone regeneration using a rat calvarial defect model. Collagen 3D nanopore scaffold (COL) and Pluronic F127 hydrogel (F127) incorporated with RvD1 (RvD1-COL-F127 group) or COL and F127 (COL-F127 group) were implanted in symmetrical calvarial defects. After implantation, RvD1 was administrated subcutaneously every 7days for 4weeks. The rats were sacrificed at weeks 1 and 8 post-implantation. Tissue samples were analyzed by real-time reverse transcriptase-polymerase chain reaction and histology at week 1. Radiographical and histological analyses were done at week 8. At week 1, calvarial defects treated with RvD1 exhibited decreased numbers of inflammatory cells and tartrate-resistant acid phosphatase (TRAP) positive cells, greater numbers of newly formed blood vessels, upregulated gene expression of vascular endothelial growth factor and alkaline phosphatase, and downregulated gene expression of receptor activator of nuclear factor-κB ligand, interleukin-1β and tumor necrosis factor-α. At week 8, the radiographical results showed that osteoid area fraction of the RvD1-COL-F127 group was higher than that of the COL-F127 group, and histological examination exhibited enhanced osteoid formation and newly formed blood vessels in the RvD1-COL-F127 group. In conclusion, this study showed that RvD1 enhanced bone formation and vascularization in rat calvarial defects.

Inflammasome Activation in the Hip Synovium of Rapidly Destructive Coxopathy Patients and Its Relationship with the Development of Synovitis and Bone Loss

Rapidly destructive coxopathy (RDC), a rare disease of unknown etiology, is characterized by the rapid destruction of the hip joint. In the current study, the potential involvement of inflammasome signaling in the progression of RDC was investigated. Histopathologic changes and the gene expression of inflammasome activation markers in hip synovial tissues collected from patients with RDC were evaluated and compared with those of osteoarthritis and osteonecrosis of the femoral head patients. The synovial tissues of patients with RDC exhibited remarkable increases in the number of infiltrated macrophages and osteoclasts, and the expression of inflammasome activation markers was also increased compared with those of osteoarthritis and osteonecrosis of the femoral head patients. To further understand the histopathologic changes in the joint, a co-culture model of macrophages and synoviocytes that mimicked the joint environment was developed. Remarkably, the gene expression levels of NLRP3, GSDMD, IL1B, TNFA, ADMTS4, ADMTS5, MMP3, MMP9, and RANKL were significantly elevated in the synoviocytes that were co-cultured with activated THP-1 macrophages, suggesting the association between synovitis and inflammasome activation. Consistent with these findings, osteoclast precursor cells that were co-cultured with stimulated synoviocytes exhibited an increased number of tartrate-resistant acid phosphatase-positive cells, compared with cells that were co-cultured with non-stimulated synoviocytes. These findings suggest that the activation of inflammasome signaling in the synovium results in an increase in local inflammation and osteoclastogenesis, thus leading to the rapid bone destruction in RDC.

Therapeutic potential of PPARα agonist in ligature-induced experimental periodontitis.

Inflammation-related immune responses and bone metabolism lead to extensive tooth loss in periodontitis.Objective:This study aims to investigate the effect of peroxisome proliferator-activated receptor (PPAR) alpha agonist anti-inflammatory treatment in vitro and in ligature-induced experimental periodontitis in vivo .Methodology:Splenocytes were isolated from C57BL/6J mice and cultured for 48 hours under the following conditions: control, P. gingivalis lipopolysaccharide (LPS) (1 µg/ml); experimental, LPS (1 µg/ml) + PPARα agonist (fenofibrate) at 1, 10, 50, 100 µM. MRNA and secreted protein levels of TNF-α expression were detected by RT-qPCR and ELISA, respectively. Silk ligatures (7-0) were tied around maxillary second molars of C57BL/6J mice for two weeks. Optimized doses of fenofibrate (50 µM) and vehicle control were injected into the contralateral side of the palatal gingiva on days three, six, and nine. At day 14, bone resorption, osteoclastogenesis, and gingival mRNA expression levels of TNF-α, IL-1β, IL-6, and RANKL/OPG were measured by micro-computed tomography, Tartrate-resistant acid phosphatase (TRAP) staining, and Real-time quantitative PCR, respectively.Results:TNF-α expression in cultured spleen cells were significantly increased in the presence of LPS, when compared with the control group, and significantly reduced by fenofibrate treatment in a dose-dependent manner from 1-100 µM (p<0.05). Gingival mRNA levels of TNF-α, IL-1β, IL-6, and the ratio of RANKL/OPG, were significantly decreased after injection of fenofibrate, when compared to the control side (p<0.05). Periodontal bone loss and TRAP positive cell formation were significantly decreased on the side with an injection of fenofibrate, as compared to the control side (p<0.05).Conclusions:An anti-inflammatory treatment, PPARα agonist, inhibited inflammation and periodontal bone loss in ligature-induced experimental periodontitis.

The Neuropeptide VIP Limits Human Osteoclastogenesis: Clinical Associations with Bone Metabolism Markers in Patients with Early Arthritis.

We aimed to evaluate the direct action of VIP on crucial molecules involved in human osteoclast differentiation and function. We also investigated the relationship between VIP serum levels and bone remodeling mediators in early arthritis patients. The expression of VIP receptors and osteoclast gene markers in monocytes and in vitro differentiated osteoclasts was studied by real-time PCR. NFATc1 activity was measured using a TransAM® kit. Osteoclastogenesis was confirmed by quantification of tartrate-resistant acid phosphatase positive multinucleated cells. OsteoAssay® Surface Multiple Well Plate was used to evaluate bone-resorbing activity. The ring-shaped actin cytoskeleton and the VPAC1 and VPAC2 expression were analyzed by immunofluorescence. We described the presence of VIP receptors in monocytes and mature osteoclasts. Osteoclasts that formed in the presence of VIP showed a decreased expression of osteoclast differentiation gene markers and proteolytic enzymes involved in bone resorption. VIP reduced the resorption activity and decreased both β3 integrin expression and actin ring formation. Elevated serum VIP levels in early arthritis patients were associated with lower BMD loss and higher serum OPG concentration. These results demonstrate that VIP exerts an anti-osteoclastogenic action impairing both differentiation and resorption activity mainly through the negative regulation of NFATc1, evidencing its bone-protective effects in humans.

Orthodontic force and extracorporeal shock wave therapy: Assessment of orthodontic tooth movement and bone morphometry in a rat model

ObjectiveThe objective was to investigate the effect of extracorporeal shock wave therapy (ESWT) on the magnitude of orthodontic tooth movement, in a rat model, based on a previously established treatment protocol. DesignIn conjunction with orthodontic force commencement, rats underwent ESWT. The amount of tooth movement along with different microarchitectural parameters were measured after three weeks by means of microcomputed tomography. In addition, the percentage of cells expressing vascular endothelial growth factor, the number of tartrate-resistant acid phosphatase (TRAP) positive cells/area and blood vessel density were evaluated both for the pressure and tension sides. ResultsThe addition of ESWT to the orthodontic force after three weeks more than doubled the average tooth movement. The addition of ESWT on the pressure side induced a significant decrease in volumetric bone mineral density. Blood vessel density and the number of TRAP positive cells were higher after the application of ESWT. ConclusionThe induction of ESWT during orthodontic tooth movement in a rat model increases the rate of tooth movement by accelerating bone resorption on the pressure side and possibly enhances bone formation on the tension side.

Enhancement of Bone-Forming Ability on Beta-Tricalcium Phosphate by Modulating Cellular Senescence Mechanisms Using Senolytics.

Various stresses latently induce cellular senescence that occasionally deteriorates the functioning of surrounding tissues. Nevertheless, little is known about the appearance and function of senescent cells, caused by the implantation of beta-tricalcium phosphate (β-TCP)—used widely in dentistry and orthopedics for treating bone diseases. In this study, two varying sizes of β-TCP granules (<300 μm and 300–500 μm) were implanted, and using histological and immunofluorescent staining, appearances of senescent-like cells in critical-sized bone defects in the calvaria of Sprague Dawley rats were evaluated. Parallelly, bone formation in defects was investigated with or without the oral administration of senolytics (a cocktail of dasatinib and quercetin). A week after the implantation, the number of senescence-associated beta-galactosidase, p21-, p19-, and tartrate-resistant acid phosphatase-positive cells increased and then decreased upon administrating senolytics. This administration of senolytics also attenuated 4-hydroxy-2-nonenal staining, representing reactive oxygen species. Combining senolytic administration with β-TCP implantation significantly enhanced the bone formation in defects as revealed by micro-computed tomography analysis and hematoxylin-eosin staining. This study demonstrates that β-TCP granules latently induce senescent-like cells, and senolytic administration may improve the bone-forming ability of β-TCP by inhibiting senescence-associated mechanisms.

Inhibitory Effects of Gold and Silver Nanoparticles on the Differentiation into Osteoclasts In Vitro.

Gold nanoparticles (GNPs) have been widely studied to inhibit differentiation into osteoclasts. However, reports of the inhibitory effects of silver nanoparticles (SNPs) during the process of differentiation into osteoclasts are rare. We compared the inhibitory effect of GNPs and SNPs during the process of differentiation into osteoclasts. Bone marrow-derived cells were differentiated into osteoclasts by the receptor activator of the nuclear factor-kappa-Β ligand (RANKL). The inhibitory effect of GNPs or SNPs during the process of differentiation into osteoclasts was investigated using tartrate-resistant acid phosphatase (TRAP) and actin ring staining. The formation of TRAP positive (+) multinuclear cells (MNCs) with the actin ring structure was most inhibited in the SNP group. In addition, the expression of specific genes related to the differentiation into osteoclasts, such as c-Fos, the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), TRAP, and Cathepsin K (CTSK) were also inhibited in the SNP groups. As a result, the levels related to differentiation into osteoclasts were consistently lower in the SNP groups than in the GNP groups. Our study suggests that SNPs can be a useful material for inhibiting differentiation into osteoclasts and they can be applied to treatments for osteoporosis patients.

Inhibition of osteoclastogenesis and inflammation by phosvitin and phosvitin hydrolysate via NF-κB and MAPK pathways in RAW 264.7 cells

Phosvitin (PV) is an egg protein. Our recent study showed both phosvitin and phosvitin hydrolysate (PVH) could promote osteoblast differentiation in osteoblast cells. The objective of the study was to investigate the effects of PV and PVH on osteoclastogenesis and possible signalling pathways in RAW264.7 cells. Both PV and PVH inhibited osteoclastogenesis (fewer tartrate-resistant acid phosphatase (TRAP) positive cells and lower TRAP activity), reduced levels of transcription factors, c-Fos and NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1), and suppressed inflammatory biomarkers TNF-α (tumor necrosis factor alpha), MCP-1 (monocyte chemoattractant protein 1), RANTES (regulated on activation, normal T cell expressed and secreted), and inducible nitric oxide synthase. The inhibitory effects of PV and PVH on RAW264.7 cells differentiation were likely mediated through p38, c-Jun N-terminal kinases (JNK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. These results indicated that PV and PVH might inhibit bone resorption activities.

In vivo study of conductive 3D printed PCL/MWCNTs scaffolds with electrical stimulation for bone tissue engineering

Critical bone defects are considered one of the major clinical challenges in reconstructive bone surgery. The combination of 3D printed conductive scaffolds and exogenous electrical stimulation (ES) is a potential favorable approach for bone tissue repair. In this study, 3D conductive scaffolds made with biocompatible and biodegradable polycaprolactone (PCL) and multi-walled carbon nanotubes (MWCNTs) were produced using the extrusion-based additive manufacturing to treat large calvary bone defects in rats. Histology results show that the use of PCL/MWCNTs scaffolds and ES contributes to thicker and increased bone tissue formation within the bone defect. Angiogenesis and mineralization are also significantly promoted using high concentration of MWCNTs (3 wt%) and ES. Moreover, scaffolds favor the tartrate-resistant acid phosphatase (TRAP) positive cell formation, while the addition of MWCNTs seems to inhibit the osteoclastogenesis but present limited effects on the osteoclast functionalities (receptor activator of nuclear factor κβ ligand (RANKL) and osteoprotegerin (OPG) expressions). The use of ES promotes the osteoclastogenesis and RANKL expressions, showing a dominant effect in the bone remodeling process. These results indicate that the combination of 3D printed conductive PCL/MWCNTs scaffold and ES is a promising strategy to treat critical bone defects and provide a cue to establish an optimal protocol to use conductive scaffolds and ES for bone tissue engineering.

The effects of vasoactive intestinal peptide on RANKL-induced osteoclast formation.

BackgroundCongenital pseudarthrosis of the tibia is a rare disease characterized by an imbalance in bone remodeling. Vasoactive intestinal peptide (VIP) has been proven to modulate bone resorption and the formation of osteoclasts. This study aimed to explore the effects of VIP on the homeostasis of bone metabolism in diverse in vitro systems.MethodsBone marrow-derived macrophages (BMMs) were differentiated into tartrate-resistant acid phosphatase-positive cells through incubation with receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). In vitro resorption pit detection was carried out to assess the effects of VIP on osteoclastic activity. Rat osteosarcoma cell line ROS 17/2.8 was cultured alone or co-cultured with rat BMMs in the presence or absence of VIP at various concentrations. The expression levels of RANKL, RANK, OPG, NF-κB, IL-6, ERK, CAII, and GAPDH were determined by qRT-PCR and WB assay.ResultsVIP was observed to repress osteoclast differentiation without affecting the number of osteoclast precursor cells. Furthermore, the modulation of the RANKL/osteoprotegerin (OPG), nuclear factor-κB (NF-κB), and extracellular signal-regulated kinase (ERK) signaling pathways were involved in the inhibitive influence of VIP upon bone erosion. Additionally, VIP affected the expression levels of osteoclastic factors including RANKL, OPG, and interleukin-6 in osteoblast cells. Furthermore, the expression levels of RANKL and RANK were increased, while OPG expression was reduced after treatment with VIP in the co-culture of ROS 17/2.8 and rat BMMs. ERK and NF-κB signal pathways were demonstrated to be involved in the effect of VIP in the co-culture system.ConclusionsVIP plays a critical role in bone remodeling and might serve as a potential target in the development of treatments for congenital pseudarthrosis of the tibia.

Bone Marrow Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Promote Periodontal Regeneration.

Bone marrow mesenchymal stem cell-derived small extracellular vesicles (BMSC-sEVs) can be used as a potential cell-free strategy for periodontal tissue regeneration, and we aim to investigate the effect and possible mechanism of BMSC-sEV in periodontal tissue regeneration in this study. The BMSC-sEV was isolated by the Exosome Isolation™ reagent and identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. The human periodontal ligament cells (hPDLCs) were cocultured with BMSC-sEV in vitro to detect the effects of BMSC-sEV on hPDLC migration, proliferation, and differentiation. The BMSC-sEV loaded by hydrogel was injected into experimental periodontitis rats to verify the therapeutic effect and possible mechanism. The results showed that BMSC-sEVs were 30-150 nm vesicles and expressed the exosome protein CD63 and tumor susceptibility 101 (TSG101), which could promote the migration, proliferation, osteogenic differentiation of hPDLCs. The BMSC-sEV-hydrogel had a therapeutic effect on periodontitis rats. After administration for 4-8 weeks, microcomputed tomography and histological analysis showed that alveolar bone loss, inflammatory infiltration, and collagen destruction in the BMSC-sEV-hydrogel group were less than that in the phosphate-buffered saline (PBS)-hydrogel group and periodontitis group. Further immunohistochemical and immunofluorescent staining revealed that the number of tartrate-resistant acid phosphatase-positive cells and the expression ratio of osteoprotegerin (OPG) and receptor-activator of nuclear factor kappa beta ligand (RANKL) in the BMSC-sEV-hydrogel group were lower than that in the PBS-hydrogel group and periodontitis group, while the expression of transforming growth factor-beta 1 (TGF-β1) and the ratio of macrophage type 2 and macrophage type 1 (M2/M1) were opposite. Therefore, BMSC-sEV can promote the regeneration of periodontal tissues, and that may be partly due to BMSC-sEV involvement in the OPG-RANKL-RANK signaling pathway to regulate the function of osteoclasts and affect the macrophage polarization and TGF-β1 expression to modulate the inflammatory immune response, thereby inhibiting the development of periodontitis and immune damage of periodontal tissue. Impact statement Bone marrow mesenchymal stem cell-derived small extracellular vesicles (BMSCs-sEVs) have been proven to have similar functions to BMSCs, such as promoting the regeneration of heart, liver, kidney, and bone tissue and regulating immune responses. BMSCs are candidate seed cells of periodontal regeneration, but it is unclear about the role of BMSC-sEV on periodontal regeneration. In this study, we explored the effects and possible mechanism of BMSC-sEV on periodontal regeneration. The results of this study provide the evidence of BMSC-sEV as a cell-free strategy for periodontal regeneration.

Bone Formation During Correction of Vertebral Rounding Deformity in a Rat Model of Pediatric Spondylolisthesis.

A study using rat spondylolisthesis models. The aim of this study was to elucidate the mechanism for correction of vertebral rounding deformity. Vertebral rounding deformity is the strongest risk factor for high-grade slippage associated with spondylolisthesis in adolescents. We previously reported that inadequate endochondral ossification of the anterior upper corner of the vertebral growth plate in response to mechanical stress could be the pathological mechanism of vertebral rounding deformity. We created a model of spondylolisthesis using 4-week-old rats. They were divided into a tail suspension group that underwent tail suspension to decrease mechanical stress starting at 2 weeks postoperatively and a ground control group with no intervention. Radiographs and microcomputed tomography scans were obtained once weekly for 6 weeks postoperatively. The lumbar spines were then harvested for histological analysis. Immunohistochemical studies detected types I, II, and X collagen in the growth plate cartilage. Bone histomorphometrical analysis was also performed. Radiological and histological evidence in the ground control group showed progress the rounding deformity with time as previously reported. Formation of normal cancellous bone was observed radiologically over time in the tail suspension group, indicating correction of rounding deformity. Histologically, the site showing radiological evidence of correction was derived from cartilage tissue. After starting tail suspension, the growth plate stained positive for type X collagen and the corrected site stained for types II and X collagen in a mosaic pattern. Chondrocytes expressing types I and II collagen and tartrate-resistant acid phosphatase-positive cells were also present at the corrected site. Histomorphometrically, more endochondral bone was detected at the corrected site than in the posterior aspect of the normal growth plate. Correction of vertebral rounding deformity was associated with improvement of chondrocyte differentiation; furthermore, there is possible involvement of a third mechanism, namely transchondroid bone ossification.Level of Evidence: N/A.

Quercetin Decreased Alveolar Bone Loss and Apoptosis in Experimentally Induced Periodontitis Model in Wistar Rats

Quercetin is a flavonoid which has potent anti-inflammatory, antibacterial, and antioxidant effect. Purpose of this study was to evaluate effects of quercetin on alveolar bone loss and histopathological changes in ligature-induced periodontitis in rats. Wistar rats were divided into four experimental groups: non-ligated control (C, n=8) group; periodontitis (P, n=8) group; ligature and low dose quercetin group (75 mg/kg/day quercetin, Q75 group, n=8); ligature and high dose quercetin group (150 mg/kg/day quercetin, Q150 group, n=8). Silk ligatures were placed at gingival margin of lower first molars of mandibular right quadrant. Study duration was 15 days, and animals were sacrificed end of this period. Changes in alveolar bone levels were clinically measured and tissues were immunohistochemically examined, matrix metalloproteinase 8 (MMP 8), inducible nitric oxide synthase (iNOS), tissue inhibitor of metalloproteinase 1 (TIMP 1), Cysteine-aspartic proteases 3 (Caspase 3), and tartrate-resistant acid phosphatase (TRAP) positive osteoclast cells, osteoblast, and neutrophil counts were also determined. Alveolar bone loss was highest in P group, and differences among P, Q75, and Q150 groups were significant. Both doses of quercetin decreased TRAP+ osteoclast cells and increased osteoblast cells. Inflammation in P group was also higher than those of C, Q75, and Q150 groups indicating anti-inflammatory effect of quercetin. iNOS, MMP-8, and caspase-3 levels were highest, and TIMP-1 expression was lowest in P group; differences were statistically significant. Within limits of this study, it can be suggested that quercetin administration may reduce alveolar bone loss by increasing osteoblastic activity, decreasing osteoclastic activity, apoptosis, and inflammation in an experimental model of periodontitis.