RANKL Expression Research Articles

Paracrine activity of Smurf1-silenced mesenchymal stem cells enhances bone regeneration and reduces bone loss in postmenopausal osteoporosis

BackgroundOsteoporosis (OP), characterized by reduced bone mass and mineral density, is a global metabolic disorder that severely impacts the quality of life in affected individuals. Although current pharmacological treatments are effective, their long-term use is often associated with adverse effects, highlighting the need for safer, more sustainable therapeutic strategies. This study investigates the pro-osteogenic and anti-resorptive potential of the secretome from Smurf1-silenced mesenchymal stem cells (MSCs) as a promising cell-free therapy for bone regeneration.MethodsConditioned media (CM) from Smurf1-silenced rat (rCM-Smur1) and human MSCs (hCM-Smurf1) was collected and analyzed. Pro-osteogenic potential was assessed by measuring in vitro mineralization in human and rat MSCs cultures. In vivo, studies were conducted using a rat ectopic bone formation model and a post-menopausal osteoporotic mouse model. Additionally, primary human osteoporotic MSCs were preconditioned with hCM-Smurf1, and their osteogenic capacity was compared to that induced by BMP2 treatment. Ex vivo, human bone explants were treated with hCM-Smurf1 to assess anti-resorptive effects. Proteomic analysis of the soluble and vesicular CM fractions identified key proteins involved in bone regeneration.ResultsCM from Smurf1-silenced MSCs significantly enhanced mineralization in vitro and bone formation in vivo. Preconditioning human osteoporotic MSCs with hCM-Smurf1 significantly increases in vitro mineralization, with levels comparable to those achieved with BMP2 treatment. Additionally, in ex vivo human bone cultures, treatment with hCM-Smurf1 significantly reduced RANKL expression without affecting OPG levels, indicating an anti-resorptive effect. In vivo, CM from Smurf1-silenced MSCs significantly increased bone formation in a rat ectopic model, and its local administration reduced trabecular bone loss by 50% in a post-menopausal osteoporotic mouse model after a single administration within just four weeks. Proteomic analysis revealed both soluble and vesicular fractions of hCM-Smurf1 were enriched with proteins essential for ossification and extracellular matrix organization, enhancing osteogenic differentiation.ConclusionsThe Smurf1-silenced MSCs' secretome shows potent osteogenic and anti-resorptive effects, significantly enhancing bone formation and reducing bone loss. This study provides compelling evidence for the therapeutic potential of Smurf1-silenced MSC-derived secretome as a non-toxic and targeted treatment for osteoporosis. These findings warrant further in vivo studies and clinical trials to validate its therapeutic efficacy and safety.

Tideglusib enhances ALP activity and upregulates RANKL expression in Osteoblast-macrophage Co-cultures within a 3D collagen scaffold.

Tideglusib (Tx) is known for its osteogenic potential, yet its effects on the interplay between osteoblasts and M1 macrophages remain underexplored. This in vitro study aimed to isolate and evaluate both the individual and combined roles of M1 macrophages and osteoblasts in macrophage differentiation and osteoblast function, specifically focusing on how these interactions influence protein expression of osteogenesis and osteoclastogenesis in the presence or absence of Tx. Osteoblast and macrophage cells were co-cultured in direct contact for 24 and 48 h, with or without the presence of Tx. ALP activity, the expression of inflammatory-related genes using RT-qPCR, and histological analyses were performed. Co-culturing osteoblasts and M1 macrophages with Tx increased alkaline phosphatase production, indicative of enhanced osteoblast activity. Histological assessments revealed that Tx treatment contributed to the stability and maintenance of cell morphology within the scaffold, suggesting a supportive environment for cell viability and function. Tx significantly reduced the expression of pro-inflammatory markers, such as TNF-α and IL-1β, in the co-culture at both 24 and 48 h Tx also effectively inhibited osteoclastogenic differentiation in macrophages, thereby diminishing their pro-inflammatory phenotype. Tx increased ALP activity and produced a significant up-regulation of RANKL expression, indicating enhanced osteoblast differentiation and osteoclast activation. Tx mitigates macrophage-driven inflammation. Tx may enhance bone regeneration by modulating inflammatory responses and preserving cell integrity.

Role of Cathepsin K in bone invasion of pituitary adenomas: A dual mechanism involving cell proliferation and osteoclastogenesis.

This study aimed to investigate the regulation and underlying mechanism of Cathepsin K (CTSK) in bone-invasive pituitary adenomas (BIPAs). A total of 1437 patients with pituitary adenomas were included and followed up. RNA sequencing, immunohistochemistry, and qRT-PCR were used to analyze CTSK expression. The effects of CTSK on cellular proliferation, bone matrix degradation, and osteoclast differentiation were determined by gain/loss of function experiments in vitro and in vivo. The exploration of signaling pathways was determined through molecular biology experiments. Here, we reported a significant fraction (∼10%) of pituitary adenoma patients developed bone invasion, which was correlated with tumor recurrence. Patients with BIPAs had shorter recurrence-free survival. CTSK expression was increased in BIPA patients and was strongly associated with a worse prognosis. Increased CTSK expression enhanced pituitary adenoma cell proliferation through the activation of the mammalian target of rapamycin (mTOR) signaling pathway and promoted bone invasion by increasing osteoclast differentiation both in vitro and in vivo. Treatment with the CTSK inhibitor odanacatib effectively inhibited pituitary adenoma cell proliferation and bone invasion in these models. Additionally, CTSK facilitated osteoclast differentiation by promoting RANKL expression in MC3T3-E1 cells via interaction with TLR4. Based on these findings, we conclude that CTSK has the potential to become a novel predictive biomarker and therapeutic target for BIPAs.

Adjunctive Treatment Effect of Non-Thermal Atmospheric Pressure Plasma in Periodontitis-Induced Rats

Background/Objectives: As non-thermal atmospheric pressure plasma (NTP) is known to have advantages in application in the medical field, we consider its applicability to periodontitis, a representative chronic inflammatory disease. The purpose of this study was to evaluate the effect of NTP in inhibiting the progression of periodontitis in a rat model when additionally used in scaling and root planing (SRP). Methods: To induce experimental periodontitis in 20 rats, ligatures were placed in the maxillary second molar and lipopolysaccharide from Porphyromonas gingivalis was injected around the teeth. Then, NTP treatment was performed for 2 or 5 min, together with scaling and root planing (SRP). To evaluate alveolar bone loss, micro-computed tomography (micro-CT) analysis and hematoxylin–eosin (H-E) staining were performed. Tartrate-resistant acid phosphatase (TRAP) analysis was performed to compare the number of osteoclasts, while immunohistochemistry (IHC) analysis was performed to determine the expression levels of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG). Enzyme-linked immunosorbent assay (ELISA) analysis was performed for the detection of cytokines (TNF-α, IL-1β, and IL-10) in tissues and sera. Results: When SRP was combined with NTP, alveolar bone loss was decreased, the number of osteoclasts and RANKL expression were decreased, OPG expression was increased, and pro-inflammatory cytokine (TNF-α and IL-1β) levels were significantly decreased. Compared with the NTP treatment for 2 min, when treated for 5 min, less alveolar bone loss, fewer osteoclasts, a lower RANKL expression level, and a higher OPG expression level were observed. Conclusions: This study evaluated the adjunctive treatment effect of NTP in periodontitis-induced rats. Based on the results of this study, we suggest that supplemental NTP treatment may be a good option for non-surgical periodontal treatment; however, further studies are needed to elucidate the mechanism through which NTP suppresses periodontal inflammation.

Molecular Profiling of Odontoclasts during Physiological Tooth Replacement.

The odontoclast is a rarely studied cell type that is overly active in many dental pathologies, leading to tooth loss. It is difficult to find diphyodont mammals in which either physiological or pathological root resorption can be studied. Here we use the adult leopard gecko, which has repeated cycles of physiological tooth resorption and shedding. RNA-seq was carried out to compare gene expression profiles of functional teeth to developing teeth. Genes more highly expressed in bell-stage developing teeth were related to morphogenesis (PTHLH, SFRP2, SHH, EDAR). Some genes expressed in osteoclasts (ACP5, CTSK, CSF1R) were relatively more abundant in functional teeth compared with developing teeth. There was, however, no differential expression of RANK and RANKL in the 2 tooth types. In addition, functional teeth expressed proteolysis genes not found in osteoclasts (ADAMTS2, 3, 4, 14; CTSA, CTSH, CTSS). We used tartrate acid resistant phosphatase and cathepsin K (CTSK) staining to identify odontoclasts in and around the gecko dentition. There were 3 populations of CTSK cells: (1) large, functional multinucleated odontoclasts in the crown of the tooth with a ruffled border inside resorption pits; (2) smaller, precursor cells in the pulp with fewer nuclei; and (3) flattened external precursor cells next to the root and bone of attachment. We found a positive relationship between developing teeth and the population of CTSK+ cells on the root surface. We tested a candidate signal that may be involved in CTSK+ cell presence. An antagonist of CSF1R was delivered to developing teeth in vivo, which resulted in a significant decrease in CTSK and CSF1R compared with DMSO controls. Thus, the CSF1 signaling pathway is upstream of CTSK in teeth. This is the first work to detail the molecular characteristics of odontoclasts during physiological tooth shedding and to demonstrate that in vivo, local drug delivery is possible in the gecko model.

Glucocorticoids on bone remodeling in systemic lupus erythematosus mice.

Systemic lupus erythematosus requires glucocorticoids for management. This study investigates how glucocorticoids influence bone in a SLE mouse model, focusing on bone mineral density (BMD), microstructure, and remodeling markers. MRL/lpr and C57BL/6 mice were administered dexamethasone or saline as a control for 4-weeks. Bone assessments included analyses of BMD, bone structure, and serum levels of RANKL and OPG. Dexamethasone decreased BMD and altered cortical and trabecular bone thickness in both MRL/lpr and C57BL/6 mice. In C57BL/6 mice, cortical bone exhibited increased catabolism while trabecular bone showed signs of increased anabolism, whereas MRL/lpr mice did not show significant changes in bone turnover. Both strains experienced weight loss, with a significant decrease in femur length observed only in C57BL/6 mice. Dexamethasone exacerbated BMD reduction in MRL/lpr mice and halted its increase in C57BL/6 mice. C57BL/6 mice exhibited notable changes in cortical and trabecular bone structure, while MRL/lpr mice didn't. After receiving dexamethasone, both strains showed higher serum RANKL levels, especially in C57BL/6 mice. OPG decreased in both strains. Both glucocorticoids and SLE contribute to abnormal bone remodeling through RANKL/OPG pathway. Glucocorticoid (GC) treatment in a mouse model of systemic lupus erythematosus (SLE) leads to significant changes in bone parameters, including decreased bone mineral density (BMD) and alterations in bone structure. Those change are associated with the modulation of RANKL and OPG expression. Both GC and inflammation in SLE contribute to BMD reduction, and GC may have a certain protective effect on bone in the early stage of chronic inflammation. GC can upregulate RANKL expression and downregulate OPG expression in vivo. During a state of chronic inflammation, RANKL expression increases. However, OPG may not exert a significant influence on inflammatory stimulation.

Antiosteoporosis and Bone Protective Effect of Phyllanthin Against Glucocorticoid-induced Osteoporosis in Rats via Alteration of HO-1/Nrf2 and RANK/RANKL/OPG Pathway.

: SD rats were used in this study and subcutaneous administration of DEX (3 mg/kg) was used for the induction of osteoporosis and rats were treated with phyllanthin and alendronate for 12 weeks. The body weight, femur mass, length, hormones, nutrients, antioxidant, cytokines and bone parameters were estimated. The mRNA expression of HO-1, Nrf2, RANK, RANKL and OPG were estimated. : Phyllanthin treatment significantly (p < 0.001) improved the body weight, femur mass and femur length. Phyllanthin significantly (p < 0.001) altered the level of hormones estrodiol, PTH; nutrients such as calcium, phosphorus, magnesium; Bone mineral content (BMC) and bone mineral density (BMD); Bone formation marker like ALP, TRAP, osteocalcin, β-CTX, BGP, cathepsin K, DPD; Bone parameters viz., Tb.N, BV/TV, Tb.sp, BS/BV, Tb.Th; Bone structure analysis includes maximum load, energy, stiffness, maximum stress, young's modules; oxidative stress parameters such as TBARS, CAT, GPx, GSH, GR; cytokines such as TNF-α, IL-1β, IL-6, IL-10 and antioxidant marker such as HO-1 and Nrf2. Phyllanthin significantly (P < 0.001) altered the mRNA expression of HO-1, Nrf2, RANK, RANKL and OPG. : On the basis of result, we can say that phyllanthin exhibited the antiosteoporosis effect against glucocorticoid-induced osteoporosis in rats via alteration of HO-1/Nrf2 and RANK/RANKL/OPG pathway.

Gossypin Ameliorates Collagen-Induced Arthritic Rats by Inhibiting Angiogenesis, Inflammation and Oxidative Stress.

Rheumatoid arthritis (RA) is a long-term inflammatory autoimmune disease that damages cartilage and synovial membranes while also affecting bones and joints. The aim of the current study was to investigate the antiarthritic effect of gossypin against collagen-induced arthritis (CIA) in rats. Intraperitoneal administration of Type II collagen (2 mg/mL) was used to induce arthritis in the rats, followed by oral administration of gossypin (5, 10 and 15 mg/kg) for 28 days. Various parameters were assessed, including body weight, paw swelling, arthritis score, organ weights, RF, haematological parameters, inflammatory markers, MMP levels, antioxidants, cytokines and nonhepatic and hepatic liver parameters. Additionally, the expression of various mRNAs was analysed. Gossypin significantly (p < 0.001) altered the body weight, paw swelling, arthritis score and organ weights along with RF level. Gossypin treatment significantly (p < 0.001) altered the level of antioxidant and hepatic parameters; inflammatory cytokines; and inflammatory parameters such as PGE2, COX-2, NF-κB and VEGF, respectively. Gossypin also enhanced the level of HO-1 and Nrf2 and suppressed the level of MMP parameters such as MMP-2, MMP-3 and MMP-9. Gossypin also modified the mRNA expression of MMP-3, MMP-9, TRAP, RANK, Ctsk and RANKL, respectively. Gossypin demonstrated antiarthritic effects against CIA in rats.

Effect of vortioxetine and fluoxetine on immunohistochemical expression of Caspase-8, RANKL, and IL-6 in testicular tissue in an experimental depression model.

The main symptoms of depression, a chronic mental illness, include sadness, low self-esteem, and a diminished sense of enjoyment in life. Many factors have been suggested to be associated with depression, one of which is low testosterone in men. The serotonin reuptake inhibitor fluoxetine (FLU), used to treat depression, has been reported to potentially have detrimental effects on spermatogenesis in rats after long-term use. The multimodal antidepressant vortioxetine (VTX) offers new promise for the treatment of depression. The chronic unpredictable mild stress (CUMS) model is widely known as an experimental paradigm used to study depression-like behaviors in rodents. Stress leads to various neurochemical and immune changes, affecting multiple organs. Our study aims to examine the histopathological findings in testicular tissue induced by CUMS and the immunohistochemical expression of Cas-8, IL-6, and RANKL using a depression model in rats. Rats were split into 4 groups of 7 animals each at random. Group 1 (control) did not experience any stress. Group 2 (CUMS) was exposed to chronic, unpredictable mild stress using a specific procedure. Group 3 (CUMS+VTX) and Group 4 (CUMS+FLU) underwent CUMS and received intraperitoneal drug treatment at a dose of 10 mg/kg during the final three weeks of the study. The rat testicles collected during necropsy were evaluated histopathologically and immunohistochemically for Cas-8, IL-6, and RANKL expressions using a light microscope. In Group 1, histological analysis showed normal tissue architecture in the testicles and epididymis. In Group 2, there was significant depletion of spermatozoa in the seminiferous tubules and empty tubules in the epididymis. In Groups 3 and 4, FLU and VTX treatment led to improvements in the testicles. Cas-8, RANKL, and IL-6 immunohistochemistry revealed increased expression in Group 2, primarily in interstitial cells. In Groups 1, 3, and 4, no or very slight expression of these markers was observed. The results of this study showed that sperm production in the testes is negatively affected in CUMS-induced depression and that Cas-8, IL-6, and RANKL expression is increased, particularly in interstitial cells. VTX and FLU, used in the treatment of depression, suggest potential for mitigating the adverse effects of CUMS on the testes.

Implications of Mineralization Biomarkers in Breast Cancer Outcomes Beyond Calcifications.

The aim of this work was to explore the biomarkers associated with epithelial to mesenchymal transition (EMT) and mineralization processes as new prognostic factors across different breast cancer phenotypes. To this end, 133 breast biopsies, including benign and malignant lesions, with or without microcalcifications, were retrospectively collected. Immunohistochemical analysis was performed to evaluate the expression of vimentin, BMP-2, BMP-4, RANKL, Runx2, OPN, PTX3, and SDF-1, while Kaplan-Meier plots were used to assess their prognostic impact on overall survival in a dataset of 2976 breast cancer patients. The expression of vimentin, BMP-2, BMP-4, and SDF-1 was significantly higher in malignant lesions compared to benign ones, regardless of the presence of microcalcifications. Notably, these markers showed no correlation with traditional prognostic factors, such as tumor grade or hormone receptor status. The bioinformatics analysis provided valuable insights into the possible prognostic and therapeutic significance of BMP-2, BMP-4, SDF-1, and vimentin in breast cancer. In fact, all these biomarkers impact on the overall survival in specific molecular breast cancer types. In addition, high expression of SDF-1 and vimentin is able to predict the response to chemotherapy. The findings here reported suggest that vimentin, BMP-2, BMP-4, and SDF-1 could be independent prognostic biomarkers in breast cancer, providing insights beyond traditional clinical factors.

Osteoclastogenesis markers in craniofacial bone defects after demineralized dentin material membrane implantation as guided bone regeneration.

this article aims to analyze the expression of TNF-α, RANKL, and osteoclast cells count after application of DDMM as GBR in mandibular bone defects. this is an experimental study with a post-test only control group design, which began with the randomization of 120 rats into five groups: K(-), without membrane implantation; K(+), PPCM; P1, DDMM; P2, DDMM + bone graft; P3, PPCM + bone graft. The expression of TNF-α, RANKL, and osteoclast cells count were observed, followed by analysis using a one-way ANOVA and post hoc Tukey HSD comparison test. there were significant differences in the expression of TNF-α, RANKL, and osteoclast cells count in all study groups (p=0.000). TNF-α showed a decreasing difference with the highest expression in the K(-) group on day 3 of 12.00±2.16. RANKL expression increased on day 14 and decreased on day 21 in all groups. The osteoclast cells count generally showed a critical period with the highest increase in the K(-) group on day 14 of 73.00±0.00. DDMM has the potential to be a superior membrane substitute compared to PPCM as GBR in alternative treatment for craniofacial bone defects reconstruction.

Transcriptional regulation of Rankl by Txnip-Ecd in aging and diabetic related osteoporosis.

Bone homeostasis between osteoclast bone resorption and osteoblastic bone formation is tightly regulated by a series of factors such as the receptor activator of nuclear factor-κB ligand (RANKL). Denosumab that neutralizes RANKL is effective and widely applied in the treatment of postmenopausal osteoporosis. However, factors that participated in the RANKL-related bone remodeling process in primary and secondary osteoporosis are less known. Revealing the novel transcriptional regulatory mechanism of RANKL is of great significance for the treatment of osteoporosis. After differential expression genes (DEGs) intersection and screening, we generated Thioredoxin-interacting protein (Txnip) bone marrow-derived mesenchymal stromal cells (BMSCs) genetic knockout mice and performed bone histomorphometry and histological analysis. RNA-Sequencing, Western blotting and immunofluorescence staining verified Rankl downregulation. Co-immunoprecipitation and immunofluorescence staining were used for Rankl regulation mechanism exploration. A specific inhibitor was selected for treatment effect verification. Txnip knockout in BMSCs impaired its osteogenic differentiation, suppressed Rankl expression and subsequent osteoclast formation and thus led to increased bone mass. The regulatory function of Txnip on Rankl expression was revealed for the first time through the novel transcription-related Ecdysoneless (Ecd)-P300 axis. Pharmacological inhibition of Txnip can effectively prevent bilateral ovariectomy (OVX)-induced osteoporosis. Inhibition of Txnip is an alternative way to suppress Rankl-mediated osteoblast and osteoclast crosstalk. This interesting finding rendered Txnip an ideal therapeutic target for the treatment of both ovariectomy-induced and diabetes-induced osteoporosis.

Suppressing anti-citrullinated protein antibody-induced osteoclastogenesis in rheumatoid arthritis using anti-CD64 and PAD-2 inhibitors.

To evaluate the role of Fcγ receptors (FcγR) and peptidyl arginine deiminase (PAD) in anti-citrullinated protein antibody (ACPA)-induced fibroblast-like synoviocytes (FLSs)-mediated osteoclastogenesis in patients with rheumatoid arthritis (RA). FLSs and peripheral blood mononuclear cells were collected from patients with RA. We stimulated RA-FLS with ACPA (100 ng/ml) with and without anti-cluster of differentiation (CD)32a/CD64 (FcγRIIA/FcγRI) antibody and PAD-2/4 inhibitors. Flow cytometry and enzyme-linked immunosorbent assay were also performed. CD14+ monocytes were cultured with receptor activator of nuclear factor kappa beta (RANKL) and macrophage colony-stimulating factor, and ACPA-stimulated RA-FLSs were added. These cells were cultured for 14 days, and osteoclastogenesis was quantified using tartrate-resistant acid phosphatase (TRAP) staining. ACPA increased RANKL+ and tumour necrotic factor-alpha (TNF-α+) FLS, which decreased dose-dependently by adding 5 and 10 ug/mL anti-CD64 antibody rather than anti-CD32a antibody. In PAD inhibitor experiments, the proportion of RANKL+ and TNF-α+ FLS decreased in 50 μM condition containing PAD-2 inhibitor rather than PAD-4 inhibitor. The co-culture of ACPA-stimulated RA-FLSs and osteoclast precursors increased the TRAP+ multinucleated osteoclast count, which was decreased by anti-CD64 antibody and PAD2 inhibitor. The present study showed that ACPA increased RANKL and pro-inflammatory cytokine expression in RA-FLSs, and ACPA-activated RA-FLSs could augment osteoclastogenesis. These processes were inhibited by treatment with anti-CD64 antibody and PAD-2 inhibitors. These results show that CD64 and PAD-2-induced pathways may be involved in ACPA-induced FLS activation and osteoclastogenesis in patients with RA. Therefore, regulating the CD64 and PAD-2 pathways may improve RA treatment.

Efficacy and Mechanism of Highly Active Umbilical Cord Mesenchymal Stem Cells in the Treatment of Osteoporosis in Rats.

Osteoporosis increases bone brittleness and the risk of fracture. Umbilical cord mesenchymal stem cell (UCMSC) treatment is effective, but how to improve the biological activity and clinical efficacy of UCMSCs has not been determined. A rat model of osteoporosis was induced with dexamethasone sodium phosphate. Highly active umbilical cord mesenchymal stem cells (HA-UCMSCs) and UCMSCs were isolated, cultured, identified, and infused intravenously once at a dose of 2.29 × 106 cells/kg. In the 4th week of treatment, bone mineral density (BMD) was evaluated via cross-micro-CT, tibial structure was observed via HE staining, osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) was examined via alizarin red staining, and carboxy-terminal cross-linked telopeptide (CTX), nuclear factor-κβ ligand (RANKL), procollagen type 1 N-terminal propeptide (PINP) and osteoprotegerin (OPG) levels were investigated via enzyme-linked immunosorbent assays (ELISAs). BMMSCs were treated with 10-6 mol/L dexamethasone and cocultured with HA-UCMSCs and UCMSCs in transwells. The osteogenic and adipogenic differentiation of BMMSCs was subsequently examined through directional induction culture. The protein expression levels of WNT, β-catenin, RUNX2, IFN-γ and IL-17 in the bone tissue were measured via Western blotting. The BMD in the healthy group was higher than that in the model group. Both UCMSCs and HA-UCMSCs exhibited a fusiform morphology; swirling growth; high expression of CD73, CD90 and CD105; and low expression of CD34 and CD45 and could differentiate into adipocytes, osteoblasts and chondrocytes, while HA-UCMSCs were smaller in size; had a higher nuclear percentage; and higher differentiation efficiency. Compared with those in the model group, the BMD increased, the bone structure improved, the trabecular area, number, and perimeter increased, the osteogenic differentiation of BMMSCs increased, RANKL expression decreased, and PINP expression increased after UCMSC and HA-UCMSC treatment for 4 weeks. Furthermore, the BMD, trabecular area, number and perimeter, calcareous nodule counts, and OPG/RANKL ratio were higher in the HA-UCMSC treatment group than in the UCMSC treatment group. The osteogenic and adipogenic differentiation of dexamethasone-treated BMMSCs was enhanced after the coculture of UCMSCs and HA-UCMSCs, and the HA-UCMSC group exhibited better effects than the UCMSC coculture group. The protein expression of WNT, β-catenin, and runx2 was upregulated, and IFN-γ and IL-17 expression was downregulated after UCMSC and HA-UCMSC treatment. HA-UCMSCs have a stronger therapeutic effect on osteoporosis compared with that of UCMSCs. These effects include an improved bone structure, increased BMD, an increased number and perimeter of trabeculae, and enhanced osteogenic differentiation of BMMSCs via activation of the WNT/β-catenin pathway and inhibition of inflammation.

The Application of Resolvin D1-Loaded Gelatin Methacrylate in a Rat Periodontitis Model.

Objective: To evaluate the drug release, cytocompatibility with periodontal ligament cells (PDLCs), and therapeutic efficacy of GelMA hydrogel loaded with resolvin D1 (RvD1) in treating rat periodontal inflammation and alveolar bone damage. Methods: An RvD1 complexed with GelMA was prepared, and its release kinetics and compatibility with PDLCs were assessed. Rats with induced periodontitis were treated weekly with topical applications of vehicle, GelMA, RvD1, or RvD1 complexed with GelMA for four weeks. Periodontal inflammation and tissue regeneration were evaluated using quantitative PCR (qPCR) and histochemical staining, while alveolar bone repair and regeneration were analyzed through micro-CT. Results: The RvD1 complexed with GelMA effectively released RvD1 and enhanced the proliferation and viability of PDLCs. Compared to RvD1 alone, treatment with RvD1 complexed with GelMA significantly reduced inflammatory cell infiltration, TNF-α and RANKL expression, and osteoclast formation in periodontal tissues. Additionally, it promoted the expression of specific anti-inflammatory and regenerative markers. Micro-CT analysis confirmed significant new bone formation in the RvD1 complexed with GelMA-treated group. Conclusions: RvD1 complexed with GelMA provides sustained drug release and biocompatibility, effectively resolves periodontal inflammation, and promotes tissue regeneration in periodontitis.

Roles of osteoclasts in pathological conditions.

Bone is a unique organ crucial for locomotion, mineral metabolism, and hematopoiesis. It maintains homeostasis through a balance between bone formation by osteoblasts and bone resorption by osteoclasts, which is regulated by the basic multicellular unit (BMU). Abnormal bone metabolism arises from an imbalance in the BMU. Osteoclasts, derived from the monocyte-macrophage lineage, are regulated by the RANKL-RANK-OPG system, which is a key factor in osteoclast differentiation. RANKL activates osteoclasts through its receptor RANK, while OPG acts as a decoy receptor that inhibits RANKL. In trabecular bone, high turnover involves rapid bone formation and resorption, influenced by conditions such as malignancy and inflammatory cytokines that increase RANKL expression. Cortical bone remodeling, regulated by aged osteocytes expressing RANKL, is less understood, despite ongoing research into how Rett syndrome, characterized by MeCP2 abnormalities, affects RANKL expression. Balancing trabecular and cortical bone involves mechanisms that preserve cortical bone, despite overall bone mass reduction due to aging or oxidative stress. Research into genes like sFRP4, which modulates bone mass, highlights the complex regulation by BMUs. The roles of the RANKL-RANK-OPG system extend beyond bone, affecting processes such as aortic valve formation and temperature regulation, which highlight the interconnected nature of biological research.

Effects of Semaglutide and Tirzepatide on Bone Metabolism in Type 2 Diabetic Mice.

Background/Objectives: Type 2 diabetes and weight loss are associated with detrimental skeletal health. Incretin-based therapies (GLP-1 receptor agonists, and dual GIP/GLP-1 receptor agonists) are used clinically to treat diabetes and obesity. The potential effects of semaglutide and tirzepatide on bone metabolism in type 2 diabetic mice remain uncertain. Methods: Combined streptozotocin and high fat feeding were employed in female C57BL/6J mice to promote hyperglycemia. Mice were administered for 4 weeks with a saline vehicle (sc., once-daily), semaglutide (40 μg/kg/d, sc., every three days), or tirzepatide (10 nmol/kg, sc., once-daily). Bone strength was assessed by three-point bending. Femur microarchitecture was determined by micro-CT, and bone formation and resorption parameters were measured by histomorphometric analysis. Serum was collected to measure bone resorption (C-telopeptide fragments of type I collagen, CTX) and formation (procollagen type 1 N-terminal propeptide, P1NP) biomarkers, respectively. The expression of bone metabolism-related genes was evaluated in the bone using RT-PCR. Results: Glucose levels significantly reduced after 4 weeks of semaglutide and tirzepatide treatment (both p < 0.05) compared with vehicle treatment. Tirzepatide led to more weight loss than semaglutide. Compared to saline-treated diabetic mice, the mean femur length was shorter in the tirzepatide group. After treatment with tirzepatide or semaglutide, cortical bone and trabecular bone parameters did not change significantly compared to saline-treated diabetic mice, except that cortical thickness was lower in the semaglutide group compared to the saline group (p = 0.032). Though CTX and P1NP levels decreased, however, the change in CTX and P1NP levels did not differ among the four groups during the 4 weeks of treatment (all p > 0.05). Semaglutide affected RANKL and OPG mRNA expression and increased the ratio of OPG/RANKL. No significant difference was found in the quantity of Col1a1, RANKL, OPG, and RUNX2 between tirzepatide- and saline-treated diabetic mice. Conclusions: The 4-week treatment with semaglutide and tirzepatide had a neutral effect on bone mass compared with the controls, and most of the bone microarchitecture parameters were also comparable between groups in diabetic mice. A better understanding of incretin-based therapies on bone metabolism in patients with diabetes requires further evaluation in large clinical trials.

The role of FOXM1 in acetylcysteine improving diabetic periodontitis.

Diabetic periodontitis (DP) stems from hyperglycemia-driven oxidative stress amplification and chronic inflammation, leading to periodontal tissue breakdown. Misregulated forkhead box protein M1 (FOXM1) play key roles in this process, exacerbating both inflammation and oxidative stress. In light of N-Acetylcysteine (NAC)'s potent anti-oxidative capacity and anti-inflammatory potential, understanding how it modulates these central pathways to alleviate DP holds high scientific and clinical importance. An animal model of diabetic mice periodontitis was established, and the model mice were injected with FOXM 1 adenovirus to enrich FOXM 1, and the periodontal pathological histology of each group was evaluated by HE staining. Western blotting and RT-PCR evaluated the expression levels of factors involved in bone destruction. ELISA evaluated the amount of inflammatory factors in mice serum. FOXM 1 over-expression and NAC were treated in murine macrophages, and the intracellular reactive oxygen species(ROS) levels in macrophages were measured using a DCFH-DA probe. Receptor activator of NF-κB ligand (RANKL) and lipopolysaccharide (LPS) were used to establish the macrophage osteoclast differentiation model and test the expression level of osteoclast differentiation factors after giving NAC. Hydrogen peroxide was used to establish a peroxidation environment, the plasmid silenced C-JUN, and the DNA binding activity of activating protein-1(AP1) was detected by EMSA. The effect of peroxidation on the osteoclast differentiation level was determined by WB. Mice with DP model had epithelial damage and inflammatory infiltration in periodontal tissues, and in the FOXM1 enriched group, the periodontal epithelial damage was repaired and inflammation was alleviated. FOXM1 enrichment resulted in DP model lower expression of RANKL (P < 0.01), macrophage colony-stimulating factor (M-CSF) (P < 0.01) and elevated expression of osteoprotegerin (OPG) (P < 0.001). Serum levels of pro-inflammatory factors interleukin (IL)-1β, tumor necrosis factor (TNF-α), and inducible nitric oxide synthase (iNOS) were elevated in DP mice (P < 0.001), and anti-inflammatory factor IL-10 was reduced(P < 0.001),, and FOXM1 enrichment significantly reversed inflammatory factor levels (P < 0.01). Overexpression of FOXM1 reduced ROS content in macrophages (P < 0.001), and NAC was performed to further reduce ROS content (P < 0.01). Silencing of FOXM1 elevated the expression of osteoclast-specific genes NFATc1, TRAP and OSCAR (P < 0.01), and the addition of NAC on top of silencing of FOXM1 markedly suppressed the expression level of osteoclast-specific genes (P < 0.01). ROS increased the transcriptional activity of AP1 (P < 0.001), which promoted osteoclast-specific gene expression (P < 0.001), and osteoclast-specific gene expression was decreased after silencing C-JUN (P < 0.01). FOXM1 relieve diabetic periodontitis inflammation and promote bone formation, regulates ROS production and ROS increases the transcriptional activity of AP1 and affects the osteoclastic differentiation of macrophages, which plays a positive role in bone protection in diabetic periodontitis.

TMCO1-deficient mice exhibit a high incidence of otitis media associated with impaired bone homeostasis in the middle ear.

Craniofacial dysmorphism, skeletal anomalies and impaired intellectual development syndrome" (CFSMR1; OMIM#213980) is characterized by craniofacial dysmorphism, skeletal anomalies, and mental retardation. However, reports of hearing issues have been limited. To investigate hearing-related aspects of CFSMR1, Tmco1 knockout mice (Tmco1-/-) exhibiting similar symptoms to human patients were utilized in this study. Otitis media was discovered in approximately 80% of Tmco1-/- mice, which led to moderate conductive hearing loss at 3 months old and further progressed to deafness two months later. Pathology studies of Tmco1-/- mice revealed a thickened middle ear (ME) epithelium and pronounced inflammatory infiltrates in the ME cavity (MEC) and Eustachian tube (ET) of Tmco1-/- OM mice. Micro-CT scan of 5-month-old Tmco1-/- OM mice showed significantly reduced ME volume and ME malformation. Tartrate-resistant acid phosphatase (TRAP) and RUNX2, RANKL expression in ME revealed increased osteoclast activity and significantly decreased bone formation, suggesting potential causes of ME malformation. This study represents the first report of the audiological characteristics and the elucidation of potential mechanisms in Tmco1-/- mice. It enriches our understanding of the phenotypes associated with CFSMR1 in the field of otology and provides a promising model for chronic OM with conductive hearing loss.

TGF-β3 Restrains Osteoclastic Resorption Through Autophagy.

While TGF-β3 promoted defect healing in a primate baboon skull defect model and patients, it remains unclear whether TGF-β3 affects the formation of osteoclasts and bone resorption between osteogenesis and osteolysis. Analysis of the full transcriptome of hPDLSCs (human periodontal ligament stem cells) revealed that the expression of RANKL was significantly up-regulated after TGF-β3 treatment during osteogenesis, which suggests its involvement in clock-controlled autophagy in bone metabolism. TRAP staining and bone resorption lacunae were used to assess the osteoclasts formed from RANKL-induced differentiated BMMs. During osteoclast differentiation, the characteristics of autophagy regulated by TGF-β3 were observed in BMMs through MDC staining, transmission electron microscopy, and LC3 immunofluorescence. The expression of related genes and proteins were detected on the sixth day in mCherry-EGFP-LC3B lentivirus-transfected BMMs using RT-qPCR and WB. Finally, a trans-well co-culture system was used to evaluate the effects of osteogenic differentiated hPDLSCs treated with TGF-β3 on the osteoclastic differentiation of BMMs. The results showed 10 ng/mL of TGF-β3 significantly suppressed osteoclastic differentiation and bone resorption in BMMs (p < 0.05 vs. RANKL). In particular, TGF-β3 augments the expression of LC3-II to stimulate autophagy, consequently restraining osteoclastic resorption. These findings provide a molecular basis and are beneficial to illustrate the potential druggability of TGF-β3 in osteoporotic diseases.