RANKL Expression Research Articles

Curcumin Co-Encapsulation Potentiates Anti-Arthritic Efficacy of Meloxicam Biodegradable Nanoparticles in Adjuvant-Induced Arthritis Animal Model.

This study aimed to evaluate the anti-arthritic activity of curcumin and meloxicam co-loaded PLGA nanoparticles in adjuvant-induced arthritic rats. PLGA nanoparticles encapsulating curcumin (nCur) and meloxicam (nMlx) alone and in combination (nCur/Mlx) were used to characterize zeta size and potential, polydispersity index, encapsulation efficiency (%), compound-polymer interactions (FT-IR analysis), and surface morphology (SEM imaging). In vivo, Complete Freund's adjuvant-induced arthritic rats were intraperitoneally (i.p.) administered with curcumin, meloxicam, curcumin plus meloxicam, nCur, nMlx, and nCur/Mlx for 28 consecutive days. Results showed that nCur, nMlx, and nCur/Mlx significantly (p ≤ 0.05) reduced paw swelling and arthritic score, restored body weight and the immune organ index (thymus and spleen), as well as attenuated serum inflammatory markers (RF, CRP, and PGE2) and oxidative stress parameters (MDA, SOD, and CAT) in adjuvant-induced arthritic rats compared to free compounds. In addition, mono- and dual-compound-loaded nanoparticles significantly (p ≤ 0.05) down-regulated pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), up-regulated anti-inflammatory cytokines (IL-4, IL-10, and IFN-γ), and modulated OPG and RANKL expressions in paw tissue. The aforementioned results were further confirmed through radiological and histopathological examinations. Furthermore, the anti-arthritic effect of nCur/Mlx was notably (p ≤ 0.05) enhanced compared to nCur or nMlx alone. In conclusion, the co-nanoencapsulation of curcumin could potentiate the anti-arthritic activity of meloxicam and could provide a novel therapeutic approach for the formulation of nanocarrier pharmaceutical products for the management of arthritis.

IL-21/23 axis modulates inflammatory cytokines and RANKL expression in RA CD4+ T cells via p-Akt1 signaling.

CD4+ T cells are critically involved in the pathogenesis of Rheumatoid Arthritis; an autoimmune disorder characterized by joint inflammation and bone degeneration. In this study, we focused on the critical role of cytokines, IL-21 and IL-23 in facilitating the aberrant status of RA Th17-like cells and report their significant contribution(s) in modulating the expression of inflammatory cytokines and RANKL. Blood and synovial fluid collected from a total of 167 RA patients and 25 healthy volunteers were assessed for various inflammatory markers and RANKL expression in plasma and CD4+ T cells. Subsequent ex vivo studies examined the role of specific cytokines, IL-21 and IL-23 in mediating inflammation and RANKL upregulation by blocking their expression with neutralizing antibodies in RA CD4+ T cells and terminally differentiated human Th17 cells. Further, the role of p-Akt1 as a signalling target downstream of IL-21 and IL-23 was evinced with IL-21 and IL-23 inhibition and phospho Akt-1/2 kinase inhibitor. Our observations highlighted the augmented inflammatory cytokine levels in plasma and an aberrant CD4+ T cell phenotype expressing exaggerated inflammatory cytokines and membrane RANKL expression in RA as opposed to healthy controls. Neutralization of either IL-21 or IL-23 (p19 and p40) or both, resulted in downregulation of the cytokines, TNF-α, IFN-γ and IL-17 and RANKL expression in these cells, signifying the critical role of IL-21/23 axis in modulating inflammation and RANKL. Subsequent dissection of the signaling pathway found p-Akt1 as the key phosphoprotein downstream of both IL-21 and IL-23, capable of increasing inflammatory cytokines and RANKL production. Our findings unequivocally identify IL-21/23 axis in RA CD4+ T cells as a key regulator dictating two critical processes i.e. exaggerated inflammation and higher RANKL expression and provide critical targets in their downstream signalling for therapeutic approaches.

Jaw Bone Invasion of Oral Squamous Cell Carcinoma Is Associated with Osteoclast Count and Expression of Its Regulating Proteins in Patients and Organoids.

Oral squamous cell carcinoma (OSCC) frequently invades the jaw. The exact mechanism of bone invasion remains unclear. This study investigates (premature) osteoclasts and the expression of its differentiation regulating proteins RANKL, OPG and RANK in patients with OSCC. Resection specimens from OSCC patients were divided into NI group (No Invasion), E group (Erosion) or I group (bone Invasion). Tissue sections were stained with Cathepsin K (osteoclast-counting), RANKL, OPG and RANK. The staining intensity was scored on different regions of the tumor: front, center, back and normal mucosa. Immunohistochemistry and qPCR for RANKL/OPG/RANK were performed on five head and neck squamous cell carcinoma (HNSCC) organoids. The mean number of osteoclasts (I group) and premature osteoclasts (E group) was significantly higher compared to the NI group (p = 0.003, p = 0.036). RANKL expression was significantly higher in the tumor front and tumor center compared to normal mucosa (all groups). In the I group, RANKL and RANK expression was significantly higher in the tumor front compared to the tumor back and there was a trend of higher RANKL expression in the tumor front compared to the E group and NI group. qPCR showed a 20-43 times higher RANKL mRNA expression in three out of five tumor organoids compared to a normal squamous cell organoid line. There was no correlation between protein and mRNA expression in the HNSCC organoids. These findings suggest that OSCCs induce bone invasion by stimulating osteoclast activation by regulating the production of RANKL and RANK proteins.

Azilsartan inhibits inflammation-triggered bone resorption and osteoclastogenesis in vivo via suppression of TNF-α expression in macrophages.

Hypertension is a major risk factor for cardiovascular disease (CVD) and is associated with increased bone loss due to excessive activity of the local renin-angiotensin system (RAS). Angiotensinogen/Angiotensin (ANG) II/Angiotensin II type 1 receptor (AT1R) axis is considered as the core axis regulating RAS activity. Azilsartan is an FDA-approved selective AT1R antagonist that is used to treat hypertension. This study aimed to determine whether azilsartan affects formation of osteoclast, resorption of bone, and the expression of cytokines linked with osteoclastogenesis during lipopolysaccharide (LPS)-triggered inflammation in vivo. In vivo, following a 5-day supracalvarial injection of LPS or tumor necrosis factor-alpha (TNF-α) with or without azilsartan, the proportion of bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, which are identified as osteoclasts on mice calvariae were counted. The mRNA expression levels of TRAP, cathepsin K, receptor activator of NF-κB ligand (RANKL), and TNF-α were also evaluated. In vitro, the effect of azilsartan (0, 0.01, 0.1, 1, and 10 μM) on RANKL and TNF-α-triggered osteoclastogenesis were investigated. Also, whether azilsartan restrains LPS-triggered TNF-α mRNA and protein expression in macrophages and RANKL expression in osteoblasts were assessed. Furthermore, western blotting for analysis of mitogen-activated protein kinases (MAPKs) signaling was conducted. Azilsartan-treated calvariae exhibited significantly lower bone resorption and osteoclastogenesis than those treated with LPS alone. In vivo, LPS with azilsartan administration resulted in lower levels of receptor activator of RANKL and TNF-α mRNA expression than LPS administration alone. Nevertheless, azilsartan did not show inhibitory effect on RANKL- and TNF-α-triggered osteoclastogenesis in vitro. Compared to macrophages treated with LPS, TNF-α mRNA and protein levels were lower in macrophages treated by LPS with azilsartan. In contrast, RANKL mRNA and protein expression levels in osteoblasts were the same in cells co-treated with azilsartan and LPS and those exposed to LPS only. Furthermore, azilsartan suppressed LPS-triggered MAPKs signaling pathway in macrophages. After 5-day supracalvarial injection, there is no difference between TNF-α injection group and TNF-α with azilsartan injection group. These findings imply that azilsartan prevents LPS-triggered TNF-α production in macrophages, which in turn prevents LPS-Triggered osteoclast formation and bone resorption in vivo.

Panicum miliaceum L. and Bone Healing Properties in Rat Model of Femur Fracture by Activating Phosphate Stimulating Macrophages via BMP2 and RANK Signaling Pathway

Objectives The aim of this study was to investigate the effect of Panicum miliaceum involved in the repair of femur bone fracture in rats. Materials and Methods In this study, 40 Wistar rats in five groups were studied as healthy, rats with lesion induced by surgery in femur (FX), FX rats treated with P. miliaceum extract (FX +100 and 200 P. miliaceum) and FX rats treated with Osteocare syrup (FX + OC). After 40 days, the serum levels of blood calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP) along with activity of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) enzymes, and nitric oxide (NO) level were measured. The serum level of cytokines (IL-6, C-reactive protein [CRP], IL-10, IL-1β, and tumor necrosis factor-α [TNF-α]) was also measured by ELISA. The expression of OPG, RANK, RANKL, and BMP-2 genes and proteins were evaluated by real-time PCR and Western blotting techniques, respectively. Results The results showed that P. miliaceum was able to decrease the activity of ALP enzyme in addition to increasing serum Ca and P levels and GPx, SOD, and CAT activity. P. miliaceum reduces serum IL-6, IL-1β, and TNF-α, and NO levels. Also, the extract of this plant could increase the expression of OPG/RANK/BMP-2 pathway genes and proteins. Conclusion P. miliaceum can be used as a prodrug in traditional medicine to repair bone fractures and osteoporosis.

Extracellular vesicles derived from M1 macrophages enhance rat midpalatal suture expansion by promoting initial bone turnover and inflammation

BackgroundMidpalatal suture (MPS) expansion can be affected by many factors, and researchers have attempted to regulate the initial inflammatory stage of expansion to optimize clinical outcomes and their underlying mechanisms. This study aimed to investigate the potential effects and mechanisms of M1 macrophage small extracellular vesicles during rat MPS expansion.Materials and methodsRAW264.7 cells were induced to M1 or M2 polarization and, small extracellular vesicles were isolated from the polarized macrophages. Male Sprague–Dawley rats (6–7 weeks) were administered 70 ± 5 g expansion force devices for 7 days. Rats with expanders without force served as controls. M1/M2 small extracellular vesicles were injected into the MPS region (50 µg/day) in the M1 and M2 small extracellular vesicle-assisted groups, while 0.9% saline was injected into the expansion-only group. Suture width, bone mass, and morphological changes in the region of interest (ROI) were examined.ResultsThe M1 small extracellular vesicle-assisted group showed a significantly increased MPS suture width in vivo (P < 0.001), and less bone mass was observed in the ROI (P < 0.05). Histological examination showed that the M1 small extracellular vesicle-assisted group exhibited a wider palatal area and obvious fibrous tissue rearrangement. The expression of RANKL and the number of osteoclasts were increased (P < 0.01) in the bony edges, and the p65 protein expression was significantly higher (P < 0.001).ConclusionsM1 macrophage-derived small extracellular vesicles have a positive effect in MPS expansion and increase p65 protein content and RANKL expression, thus promoting bone turnover. This study may contribute to the clinical application of small extracellular vesicles in the expansion of the palatal suture.

OPG gene-modified adipose-derived stem cells improve bone formation around implants in osteoporotic rat maxillae

BackgroundOsteoporosis is a significant barrier to the use of dental implants in the elderly for the treatment of tooth defects. Adipose derived stem cells (ADSCs) have demonstrated extensive potential for tissue repair and regeneration. The present study aimed to investigate the effectiveness of ADSCs engineered to express high levels of osteoprotegerin (OPG) for the treatment of bone loss in implant dentistry caused by estrogen deficiency. MethodsA rat model of osteoporosis was established through double oophorectomy, and the rats were treated by gene modified cells Adv-OPG-ADSCs. The effects of the treatment on maxilla tissue changes were evaluated using HE staining and micro-CT. Additionally, ALP and TRAP staining were used to assess osteoblast and osteoclast alterations. Finally, the changes in related osteoblast and osteoclast indicators were measured by RT-qPCR, Western blot, and ELISA. ResultsThe successfully generated high-OPG-expressing ADSCs led to increase of cell viability, proliferation, and osteoblast differentiation. Treatment with Adv-OPG-ADSCs significantly ameliorated maxillary morphology, trabecular volume reduction, and bone mineral density decline in the model of estrogen-deficient maxillary implant dentistry. Furthermore, the treatment was beneficial to promoting the generation of osteoblasts and inhibiting the generation of osteoclast. Adv-OPG-ADSCs increased OPG, ALP, OCN, and Runx-2 expressions in the maxilla while suppressing RANKL expression, and also increased the concentration of COL I and PINP, as well as decreased the concentration of CTX-1. ConclusionAdv-OPG-ADSCs promote the formation of osteoblasts and inhibit the generation of osteoclasts, thereby inhibiting bone absorption, facilitating bone formation, and promoting the repair of maxillary bone after dental implantation in the presence of osteoporosis-related complications, especially in the setting of estrogen deficiency, providing scientific basis for the application of Adv-OPG-ADSCs in the treatment of implant related osteoporosis.

Pangasius djambal gelatin increases osteoprotegerin, decreases RANK and RANKL after tooth extraction in vivo

Background: Osteoprotegerin (OPG) plays a role in preventing osteoclast formation and osteoclastic bone resorption by inhibiting the receptor activator nuclear factor-β and its ligand (RANK–RANKL) interaction that causes bone resorption. Pangasius djambal gelatin contains essential amino acids that may stimulate bone remodeling. Purpose: This study aimed to investigate the expressions of OPG, RANKL, and RANK following tooth extraction to ascertain whether OPG is associated with the RANKL and RANK ratios following gelatin induction. Methods: This study was a true experimental study with a simple, randomized post-test only control group design. Twenty-four albino rats (Rattus norvegicus) were divided into two groups (n = 4). The treatment group was treated with P. djambal gelatin following tooth extraction, while the control group was left untreated. Animal models undergoing tooth extraction were anesthetized intraperitoneally with ketamine (1,000 mg/10 ml up to 0.2 ml). The tooth was then extracted slowly so as not to break it. P. djambal gelatin was dripped (0.1 ml) into the socket after the extraction procedure. The association of OPG with RANKL and RANK immunohistochemistry expressions was evaluated after three, five, and seven days. Results: There was a significant increase in OPG expression on day 7 (P &lt; 0.05) and a significant decrease in the expression of RANKL and RANK (P &lt; 0.05) in the treatment group. Conclusion: The application of P. djambal gelatin could increase OPG expression, decrease RANKL and RANK expression in the socket following tooth extraction, and might be associated with the inhibition of bone resorption.

Early gene expression profiles of anabolic and catabolic molecules in murine bone after a single PTH injection

The current study examined the gene expression profiles of anabolic and catabolic molecules after a single parathyroid hormone (PTH) injection in mice. No significant changes were observed in alkaline phosphatase area/tissue volume, tartrate-resistant acid phosphatase–positive osteoclasts, or static bone histomorphometry parameters. However, a sudden and significant decrease in Runx2 expression occurred at 1.5 h post-injection followed by immediate elevation, while sclerostin level was initially downregulated but gradually recovered. Meanwhile, Rankl expression initially increased and then returned to baseline. The prolonged elevation of anabolic molecules and transient increase in catabolic molecules may contribute to the anabolic effect of PTH treatment.

Mechanism Underlying the Alveolar Bone Destruction by Prevotella intermedia: Systematic Review

Introduction: Periodontitis is an inflammatory disorder caused by oral microorganisms, including Prevotella intermedia, that destroy the periodontal ligament and alveolar bone. The detailed mechanism of P. intermedia-inducing bone damage is essential to establish a novel approach to controlling P. intermedia that prevents alveolar bone loss. Purpose: To elucidate the mechanism of P. intermedia's role in alveolar bone destruction by systematically reviewing various related publications. Methods: From September 2020 through February 2021, this systematic review was performed by selecting publications for relevant material from two electronic databases, PubMed and Scopus. The literature must be in English, published within the last ten years, be available in full-text form, and be a research article to meet the criteria for inclusion. Results: Three final articles passed the eligibility evaluation stage and met the inclusion requirements. They all discussed how P. intermedia lipopolysaccharide (LPS) affects target cells to destroy bone, including human dental follicle stem cells (hDFSCs), human periodontal ligament fibroblasts (hPDLs), and macrophages. Tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-8, and prostaglandin E2 (PGE2) are a few of the inflammatory mediators that LPS P. intermedia cause a rise in target cells, leading to bone damage. These inflammatory mediators stimulate nuclear factor-kappa ligand receptor (RANKL) expression, resulting in osteoclast activation and differentiation, leading to bone loss. Conclusion: P. intermedia significantly contributes to alveolar bone degradation by enhancing inflammatory mediators. As observed in periodontitis, these mediators stimulate RANKL expression and osteoclast activation, resulting in alveolar bone damage.

Identification of an intronic enhancer regulating RANKL expression in osteocytic cells

The bony skeleton is continuously renewed throughout adult life by the bone remodeling process, in which old or damaged bone is removed by osteoclasts via largely unknown mechanisms. Osteocytes regulate bone remodeling by producing the osteoclast differentiation factor RANKL (encoded by the TNFSF11 gene). However, the precise mechanisms underlying RANKL expression in osteocytes are still elusive. Here, we explored the epigenomic landscape of osteocytic cells and identified a hitherto-undescribed osteocytic cell-specific intronic enhancer in the TNFSF11 gene locus. Bioinformatics analyses showed that transcription factors involved in cell death and senescence act on this intronic enhancer region. Single-cell transcriptomic data analysis demonstrated that cell death signaling increased RANKL expression in osteocytic cells. Genetic deletion of the intronic enhancer led to a high-bone-mass phenotype with decreased levels of RANKL in osteocytic cells and osteoclastogenesis in the adult stage, while RANKL expression was not affected in osteoblasts or lymphocytes. These data suggest that osteocytes may utilize a specialized regulatory element to facilitate osteoclast formation at the bone surface to be resorbed by linking signals from cellular senescence/death and RANKL expression.

Effect of Periodontal Ligament Stem Cells-Derived Conditioned Medium on Gene Expression and Differentiation of Tumor Necrosis Factor-α-Challenged Osteoblasts.

Tumor necrosis factor-α (TNF-α) causes bone resorption in periodontitis. It induces the production of receptor activator of NF-κB ligand (RANKL) from osteoblasts, leading to the disturbance of bone homeostasis through RANKL, RANK, and osteoprotegerin (OPG) axis. This study aimed to explore the effect of periodontal ligament stem cells-derived conditioned medium (PDLSCs-CM) on gene expression related to bone homeostasis and the differentiation of TNF-α-challenged osteoblasts. Human osteoblasts were cultured with 50 ng/mL of TNF-α and 0, 1, 10, and 100 µg/ mL of PDLSCs-CM. Osteoblasts cultured without TNF-α and PDLSCs-CM were served as control. Gene expression of RANKL, OPG, and interleukin-1β (IL-1β) was evaluated by reverse transcription quantitative polymerase chain reaction at 48 hours. The early-stage and late-stage differentiation of TNF-α-challenged osteoblasts without or with PDLSCs-CM was explored by alkaline phosphatase (ALP) activity and alizarin red staining, respectively, at day 1, 3, 6, 9, and 12. Mann-Whitney U test was used to analyze the differences in gene expression of TNF-α-challenged osteoblasts at 24 and 48 hours, and Kruskal-Wallis test was used to analyze the effect of PDLSCs-CM on gene expression and ALP activity among all experimental groups using SPSS software version 21.0. Statistical significance was considered with p-value less than 0.05. Expression of RANKL, OPG and IL-1β was significantly upregulated in TNF-α-challenged osteoblasts compared to the untreated control. The PDLSCs-CM at 1 and 10 μg/mL downregulated gene expression of TNF-α-challenged osteoblasts compared to the group without PDLSCs-CM, but the difference did not reach statistical significance. The ALP activity was decreased in TNF-α-challenged osteoblasts. The addition of PDLSCs-CM did not alter ALP activity of TNF-α-challenged osteoblasts. Alizarin red staining was comparable in the TNF-α-challenged osteoblasts cultured without or with PDLSCs-CM. The PDLSCs-CM did not alter gene expression involved in bone homeostasis and differentiation of TNF-α-challenged osteoblasts.

Effect of periostin on bone metabolic and autophagy factors during tooth eruption in mice.

This study aimed to investigate the effect of periostin (PN) on the expression of receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), microtubule-associated protein1 light chain 3B (LC3B), and Beclin1 in mouse alveolar bone specimens and cultured osteoblasts in vitro, to preliminarily explore the role of PN and autophagy in remodeling bone metabolism during tooth eruption. Mice at 5 days of age were injected with 75 ng/mL recombinant PN protein under the periosteum for 3 consecutive days according to the standard of 1 mL/100 g/day. Then, their mandibles were removed, and the expression of bone metabolic and autophagy factors was detected by immunohistochemistry. Mouse osteoblast-like cells cultured in vitro were treated with recombinant PN at a concentration of 75 ng/mL. The changes in the aforementioned indicators were compared again by immunofluorescence and western blotting 72 h after dosing. The results of the mouse samples showed that the protein expression of RANKL, LC3B, and Beclin1 decreased, accompanied by the decrease in RANKL/OPG ratio. However, OPG protein expression increased in the dosing group. Immunofluorescence and western blotting results of osteoblasts cultured in vitro showed that the protein expression of RANKL, LC3B, Beclin1, and the RANKL/OPG ratio in the experimental group decreased, but OPG expression increased. PN may regulate alveolar bone metabolism during tooth eruption by inhibiting the RANKL/OPG ratio and autophagy, which will provide a new research perspective for further exploration of the mechanisms during tooth eruption.

Sustained release of a highly specific GSK3β inhibitor SB216763 in the PCL scaffold creates an osteogenic niche for osteogenesis, anti-adipogenesis, and potential angiogenesis.

The safe and effective use of Wnt signaling is a hot topic in developing osteogenic drugs. SB216763 (S33) is a widely used highly specific GSK3β inhibitor. Here, we show that S33 initiates canonical Wnt signaling by inhibiting GSK3β activity in the bone marrow stromal cell line ST2 and increases osteoblast marker alkaline phosphatase activity, osteoblast marker gene expression including Alpl, Col1α1, and Runx2, promoting osteogenic differentiation and mineralization of ST2 cells. In addition, S33 suppressed the expression of adipogenic transcription factors Pparg and Cebpa in ST2 cells to suppress adipogenesis. ICRT-14, a specific transcriptional inhibitor of Wnt signaling, reversed the effects of S33 on the differentiation of ST2 cells. S33 also increased the expression of osteoclast cytokines RANKL and Opg but decreased the RANKL/Opg ratio and had the potential to inhibit osteoclast differentiation. In addition, we printed the PSCI3D (polycaprolactone, S33, cell-integrated 3D) scaffolds using a newly established integrated 3D printing system for hard materials and cells. S33 sustained release in the hydrogel of the scaffold with 25.4% release on day 1% and 81.7% release over 7days. Cells in the scaffolds had good cell viability. The ratio of live/dead cells remained above 94% for 7days, while the cells in the scaffolds proliferated linearly, and the proliferative activity of the PSCI3D scaffold group increased 1.4-fold and 1.7-fold on days 4 and 7, respectively. Similarly, in PSCI3D scaffolds, osteogenic differentiation of st2 cells was increased. The alkaline phosphatase activity increased 1.4- and 4.0-fold on days 7 and 14, respectively, and mineralization increased 1.7-fold at 21days. In addition, PSCI3D conditioned medium promoted migration and tubulogenesis of HUVECs, and S33 upregulated the expression of Vegfa, a key factor in angiogenesis. In conclusion, our study suggests that S33 functions in osteogenesis, anti-adipogenesis, and potential inhibition of osteoclast differentiation. And the sustained release of S33 in PSCI3D scaffolds creates a safe osteogenic niche, which promotes cell proliferation, osteogenesis, and angiogenesis and has application prospects.

Eicosapentaenoic acid supplementation modulates the osteoblast/osteoclast balance in inflammatory environments and protects against estrogen deficiency-induced bone loss in mice

An imbalance of osteoblasts (OBs) and osteoclasts (OCs) in a chronic inflammatory microenvironment is an important pathological factor leading to osteoporosis. Eicosapentaenoic acid (EPA) has been shown to suppress inflammation in macrophages and adipocytes. However, the effect of EPA on OBs and OCs has yet to be fully elucidated. We explored the roles of EPA in the differentiation of OBs and OCs, as well as the coupling between OBs and OCs in an inflammatory microenvironment. The effects of EPA on estrogen deficiency-induced osteoporosis were also evaluated. Mouse bone marrow mesenchymal stem cells (mBMSCs) and mouse bone marrow-derived macrophages (mBMMs) were used for invitro OBs and OCs differentiation. TNF-α was used to create an inflammatory microenvironment. We examined the effects of EPA on osteoblastogenesis in the absence or presence of TNF-α and collect OBs' culture medium as the conditioned medium (CM). Then we examined the effects of EPA and CM on RANKL-induced osteoclastogenesis. The invivo effects of EPA were determined using an ovariectomized (OVX) mouse model treated with EPA or vehicle. High-dose EPA was shown to promote osteoblastogenesis in an inflammatory environment invitro, as well as upregulate expression of OBs-specific proteins and genes. ARS and ALP staining also showed that high-dose EPA-treated groups restored mBMSCs' impaired osteogenic capacity caused by TNFa. Mechanistically, EPA suppressed the NF-κB pathway activated by TNF-α in mBMSCs and rescued TNF-α-mediated inhibition of osteoblastogenesis. EPA was also shown to inhibit expression of RANKL and decrease the RANKL/OPG ratio in OBs in an inflammatory environment. CM from TNF-α-stimulated OBs promoted osteoclastogenesis of mBMMs; EPA-treated CM prevented this. In the OVX mouse model, EPA supplementation prevented bone loss in an estrogen deficiency-induced inflammatory environment. EPA was demonstrated for the first time to restore mBMSCs' impaired osteogenic capacity caused by TNFa-induced inflammation and rescue the OBs/OCs balance via regulation of RANKL and OPG expression in OBs. EPA showed a remarkable ability to prevent bone loss in OVX mice, suggesting a potential application of EPA in postmenopausal osteoporosis.

TNF-α–TNFR1 Signaling Mediates Inflammation and Bone Resorption in Apical Periodontitis

IntroductionThe aim of this study was to investigate the role of the proinflammatory axis TNF-α–TNFR1 in experimentally induced periapical inflammation and bone resorption in mice. MethodsAfter receiving Ethics Committee Approval (2019.1.139.58.0), experimental apical periodontitis was induced by means of inoculating oral microorganisms into the root canals of molars of mice. Genetically deficient tumor necrosis factor-α receptor-1 mice (TNFR1-/-; n = 50) response was compared with that of C57Bl6 wild-type mice (wild-type; n = 50) after 7, 14, 28, and 42 days. The analyses performed were micro–computed tomographic, histopathologic, histomicrobiological, and histometric evaluation, tartrate-resistant acid phosphatase staining, immunohistochemistry, and quantitative reverse transcriptase polymerase chain reaction. Data were analyzed by using one-way analysis of variance, followed by Tukey or Bonferroni tests (α = 5%). ResultsTNFR1-/- mice exhibited lower recruitment of neutrophils at 14, 28, and 42 days (P < .05), which resulted in reduced area and volume of apical periodontitis at 42 days (P < .05). The number of osteoclasts was also lower in TNFR1-/- animals at 14 and 42 days (P < .01), along with reduced synthesis of CTSK, MMP-9, and COX-2. Expression of RANKL, but not OPG, was reduced at 14 and 42 days (P < .001). The highest RANKL expression over OPG (ratio > 1) was found in wild-type animals at 7 (P < .0001) and 42 days (P < .001). ConclusionsPeriapical inflammation and bone resorption were exacerbated in wild-type animals compared with TNFR1-/- mice, demonstrating that the TNF-α–TNFR1 signaling pathway mediated catabolic events in bone after root canal contamination.

Osteoclast-derived IGF1 induces RANKL production in osteocytes and contributes to pagetic lesion formation.

We previously reported that measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) of patients with Paget disease (PD) or targeted to the OCL lineage in MVNP-transgenic mice (MVNP mice) increases IGF1 production in osteoclasts (OCL-IGF1) and leads to development of PD OCLs and pagetic bone lesions (PDLs). Conditional deletion of Igf1 in OCLs of MVNP mice fully blocked development of PDLs. In this study, we examined whether osteocytes (OCys), key regulators of normal bone remodeling, contribute to PD. OCys in PDLs of patients and of MVNP mice expressed less sclerostin, and had increased RANKL expression compared with OCys in bones from WT mice or normal patients. To test whether increased OCL-IGF1 is sufficient to induce PDLs and PD phenotypes, we generated TRAP-Igf1 (T-Igf1) transgenic mice to determine whether increased IGF1 expression in the absence of MVNP in OCLs is sufficient to induce PDLs and pagetic OCLs. We found that T-Igf1 mice at 16 months of age developed PD OCLs, PDLs, and OCys, with decreased sclerostin and increased RANKL, similar to MVNP mice. Thus, pagetic phenotypes could be induced by OCLs expressing increased IGF1. OCL-IGF1 in turn increased RANKL production in OCys to induce PD OCLs and PDLs.

Effect of amoxicillin and clindamycin on the gene expression of markers involved in osteoblast physiology

Background/purposeAmoxicillin and clindamycin are the most effective decontaminants for intraoral bone grafts before their application in bone regeneration without cytotoxic effects on osteoblasts, but their effects on the gene expression of markers involved in osteoblast growth and differentiation remain unclear. The study objective was to determine the effects of amoxicillin and clindamycin on the gene expression of markers involved in osteoblast growth and differentiation. Materials and methodsReal-time polymerase chain reaction (RT-PCR) was performed to explore the effect of 150 μg/mL clindamycin or 400 μg/mL amoxicillin on the gene expression by primary human osteoblasts (HOBs) of runt-related transcription factor 2 (Runx-2), osterix (OSX), alkaline phosphatase (ALP), osteocalcin (OSC), osteoprotegerin (OPG), receptor activator for nuclear factor κ B ligand (RANKL), type I collagen (Col-I), bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), TGF-β1 and TGF-β receptors (TGF-βR1, TGF-βR2, and TGF-βR3), and vascular endothelial growth factor (VEGF). ResultsTreatment with 150 μg/mL clindamycin significantly increased the gene expression of TFG-β1, TGF-βR1, TGF-βR2, TGF-βR3, RUNX-2, Col-1, OSX, OSC, BMP-2, BMP-7, ALP, VEGF, and RANKL by HOBs. Treatment with 400 μg/mL amoxicillin significantly increased the gene expression of TGF-β R1, Col-I, OSC, RANKL, and OPG alone. ConclusionThese findings suggest that 150 μg/mL clindamycin is the decontaminant of choice to treat intraoral bone grafts before their application in bone regeneration. The osteogenic and antibacterial properties of clindamycin can favor and accelerate the integration of bone grafts in the oral cavity.

Absence of melatonin during development impairs craniofacial and dental onset in rats.

Herein, we evaluated pinealectomy-induced melatonin absence to determine its effects on craniofacial and dental development in the offspring. Female Wistar rats in three groups, i.e., intact pregnant rats, pinealectomized pregnant rats (PINX), and pinealectomized pregnant rats subjected to oral melatonin replacement therapy, were crossed 30days after surgery. The heads of 7-day-old pups were harvested for cephalometric and histological analyses, and maxillae and incisors were collected for mRNA expression analysis. The PINX pups exhibited a reduction in neurocranial and facial parameters such as a decrease in alveolar bone area, incisor size and proliferation, and an increase in odontoblasts and the dentin layer. Based on incisor mRNA expression analysis, we found that Dmp1 expression was upregulated, whereas Col1a1 expression was downregulated. Maxillary mRNA expression revealed that Rankl expression was upregulated, whereas that of Opn and Osx was downregulated. Our results demonstrated that the absence of maternal melatonin during early life could affect dental and maxillary development in offspring, as well as delay odontogenesis and osteogenesis in maxillary tissues. Our findings suggest that disruptions or a lack of melatonin during pregnancy may cause changes in craniofacial and dental development, at least in animal experiments; however, in humans, these feedings are still poorly understood, and thus careful evaluations of melatonin levels in humans need to be investigated in craniofacial alterations.

Radiation Induces Bone Microenvironment Disruption by Activating the STING-TBK1 Pathway.

Background and Objectives: Damage to normal bone tissue following therapeutic irradiation (IR) represents a significant concern, as IR-induced bone microenvironment disruption can cause bone loss and create a more favorable environment for tumor metastases. The aim of the present study was to explore the cellular regulatory mechanism of IR-induced bone microenvironment disruption to effectively prevent radiotherapy-associated adverse effects in the future. Materials and Methods: In this study, a mouse model of local IR was established via local irradiation of the left hind limb of BALB/c mice with 12 Gy X-rays, and an in vitro osteocyte (OCY) model was established by exposing osteocyte-like MLO-Y4 cells to 2, 4, and 8 Gy irradiation to analyze multicellular biological injuries and cellular senescence. Small interfering RNA (siRNA) transfection at the cellular level and a selective antagonist intervention C-176 at the animal level were used to explore the potential role of the stimulator of interferon genes (STING) on IR-induced bone microenvironment disruption. Results: The results showed that 12 Gy local IR induces multicellular dysfunction, manifested as ascension of OCYs exfoliation, activation of osteoclastogenesis, degeneration of osteogenesis and fate conversion of adipogenesis, as well as cellular senescence and altered senescence-associated secretory phenotype (SASP) secretion. Furthermore, the expression of STING was significantly elevated, both in the primary OCYs harvested from locally irradiated mice and in vitro irradiated MLO-Y4 cells, accompanied by the markedly upregulated levels of phosphorylated TANK-binding kinase 1 (P-TBK1), RANKL and sclerostin (SOST). STING-siRNA transfection in vitro restored IR-induced upregulated protein expression of P-TBK1 and RANKL, as well as the mRNA expression levels of inflammatory cytokines, such as IL-1α, IL-6 and NF-κB, accompanied by the alleviation of excessive osteoclastogenesis. Finally, administration of the STING inhibitor C-176 mitigated IR-induced activation of osteoclastogenesis and restraint of osteogenesis, ameliorating the IR-induced biological damage of OCYs, consistent with the inhibition of P-TBK1, RANKL and SOST. Conclusions: The STING-P-TBK1 signaling pathway plays a crucial role in the regulation of the secretion of inflammatory cytokines and osteoclastogenesis potential in IR-induced bone microenvironment disruption. The selective STING antagonist can be used to intervene to block the STING pathway and, thereby, repair IR-induced multicellular biological damage and mitigate the imbalance between osteoclastogenesis and osteoblastgenesis.