RESUMEN
OBJECTIVES: Progressive bone resorption and destruction is one of the most critical clinical features of middle ear cholesteatoma, potentially leading to various intracranial and extracranial complications. However, the mechanisms underlying bone destruction in middle ear cholesteatoma remain unclear. This study aims to explore the role of parathyroid hormone-related protein (PTHrP) in bone destruction associated with middle ear cholesteatoma. METHODS: A total of 25 cholesteatoma specimens and 13 normal external auditory canal skin specimens were collected from patients with acquired middle ear cholesteatoma. Immunohistochemical staining was used to detect the expressions of PTHrP, receptor activator for nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in cholesteatoma and normal tissues. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the presence of TRAP positive multi-nucleated macrophages in cholesteatoma and normal tissues. Mono-nuclear macrophage RAW264.7 cells were subjected to interventions, divided into a RANKL intervention group and a PTHrP+ RANKL co-intervention group. TRAP staining was used to detect osteoclast formation in the 2 groups. The mRNA expression levels of osteoclast-related genes, including TRAP, cathepsin K (CTSK), and nuclear factor of activated T cell cytoplasmic 1 (NFATc1), were measured using real-time polymerase chain reaction (real-time PCR) after the interventions. Bone resorption function of osteoclasts was assessed using a bone resorption pit analysis. RESULTS: Immunohistochemical staining showed significantly increased expression of PTHrP and RANKL and decreased expression of OPG in cholesteatoma tissues (all P<0.05). PTHrP expression was significantly positively correlated with RANKL, the RANKL/OPG ratio, and negatively correlated with OPG expression (r=0.385, r=0.417, r=-0.316, all P<0.05). Additionally, the expression levels of PTHrP and RANKL were significantly positively correlated with the degree of bone destruction in cholesteatoma (r=0.413, r=0.505, both P<0.05). TRAP staining revealed a large number of TRAP-positive cells, including multi-nucleated osteoclasts with three or more nuclei, in the stroma surrounding the cholesteatoma epithelium. After 5 days of RANKL or PTHrP+RANKL co-intervention, the number of osteoclasts was significantly greater in the PTHrP+RANKL co-intervention group than that in the RANKL group (P<0.05), with increased mRNA expression levels of TRAP, CTSK, and NFATc1 (all P<0.05). Scanning electron microscopy of bone resorption pits showed that the number (P<0.05) and size of bone resorption pits on bone slices were significantly greater in the PTHrP+RANKL co-intervention group compared with the RANKL group. CONCLUSIONS: PTHrP may promote the differentiation of macrophages in the surrounding stroma of cholesteatoma into osteoclasts through RANKL induction, contributing to bone destruction in middle ear cholesteatoma.
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Resorción Ósea , Diferenciación Celular , Colesteatoma del Oído Medio , Macrófagos , Osteoclastos , Osteoprotegerina , Proteína Relacionada con la Hormona Paratiroidea , Ligando RANK , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Ligando RANK/genética , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Humanos , Animales , Ratones , Osteoprotegerina/metabolismo , Colesteatoma del Oído Medio/metabolismo , Colesteatoma del Oído Medio/patología , Resorción Ósea/metabolismo , Macrófagos/metabolismo , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/genética , Células RAW 264.7 , MasculinoRESUMEN
BACKGROUND: Orthodontic tooth movement (OTM) is a dynamic equilibrium of bone remodeling, involving the osteogenesis of new bone and the osteoclastogenesis of old bone, which is mediated by mechanical force. Periodontal ligament stem cells (PDLCSs) in the periodontal ligament (PDL) space can transmit mechanical signals and regulate osteoclastogenesis during OTM. KAT6A is a histone acetyltransferase that plays a part in the differentiation of stem cells. However, whether KAT6A is involved in the regulation of osteoclastogenesis by PDLSCs remains unclear. RESULTS: In this study, we used the force-induced OTM model and observed that KAT6A was increased on the compression side of PDL during OTM, and also increased in PDLSCs under compression force in vitro. Repression of KAT6A by WM1119, a KAT6A inhibitor, markedly decreased the distance of OTM. Knockdown of KAT6A in PDLSCs decreased the RANKL/OPG ratio and osteoclastogenesis of THP-1. Mechanistically, KAT6A promoted osteoclastogenesis by binding and acetylating YAP, simultaneously regulating the YAP/TEAD axis and increasing the RANKL/OPG ratio in PDLSCs. TED-347, a YAP-TEAD4 interaction inhibitor, partly attenuated the elevation of the RANKL/OPG ratio induced by mechanical force. CONCLUSION: Our study showed that the PDLSCs modulated osteoclastogenesis and increased the RANKL/OPG ratio under mechanical force through the KAT6A/YAP/TEAD4 pathway. KAT6A might be a novel target to accelerate OTM.
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Histona Acetiltransferasas , Osteogénesis , Osteoprotegerina , Ligamento Periodontal , Ligando RANK , Técnicas de Movimiento Dental , Factores de Transcripción , Técnicas de Movimiento Dental/métodos , Ligando RANK/metabolismo , Ligamento Periodontal/citología , Ligamento Periodontal/metabolismo , Factores de Transcripción/metabolismo , Osteogénesis/fisiología , Humanos , Histona Acetiltransferasas/metabolismo , Osteoprotegerina/metabolismo , Proteínas de Unión al ADN/metabolismo , Osteoclastos/metabolismo , Células Madre , Transducción de Señal/fisiología , AnimalesRESUMEN
A thorough comprehension of age-related variances in orthodontic tooth movement (OTM) and bone remodeling response to mechanical force holds significant implications for enhancing orthodontic treatment. Mitophagy plays a crucial role in bone metabolism and various age-related diseases. However, the impact of mitophagy on the bone remodeling process during OTM remains elusive. Using adolescent (6 weeks old) and adult (12 months old) rats, we established OTM models and observed that orthodontic force increased the expression of the mitophagy proteins PTEN-induced putative kinase 1 (PINK1) and Parkin, as well as the number of tartrate-resistant acid phosphatase-positive osteoclasts and osteocalcin-positive osteoblasts. These biological changes were found to be age-related. In vitro, compression force loading promoted PINK1/Parkin-dependent mitophagy in periodontal ligament stem cells (PDLSCs) derived from adolescents (12-16 years old) and adults (25-35 years old). Furthermore, adult PDLSCs exhibited lower levels of mitophagy, impaired mitochondrial function, and a decreased ratio of RANKL/OPG compared to young PDLSCs after compression. Transfection of siRNA confirmed that inhibition of mitophagy in PDLSC resulted in decreased mitochondrial function and reduced RANKL/OPG ratio. Application of mitophagy inducer Urolithin A enhanced bone remodeling and accelerated OTM in rats, while the mitophagy inhibitor Mdivi-1 had the opposite effect. These findings indicate that force-stimulated PDLSC mitophagy contributes to alveolar bone remodeling during OTM, and age-related impairment of mitophagy negatively impacts the PDLSC response to mechanical stimulus. Our findings enhance the understanding of mitochondrial mechanotransduction and offer new targets to tackle current clinical challenges in orthodontic therapy.
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Mitocondrias , Mitofagia , Osteoprotegerina , Ligamento Periodontal , Ligando RANK , Técnicas de Movimiento Dental , Animales , Mitofagia/fisiología , Ratas , Ligando RANK/metabolismo , Ligamento Periodontal/metabolismo , Osteoprotegerina/metabolismo , Mitocondrias/metabolismo , Masculino , Proteínas Quinasas/metabolismo , Ratas Sprague-Dawley , Adolescente , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Células Madre/metabolismo , Remodelación Ósea/fisiología , Células CultivadasRESUMEN
BACKGROUND: Palatal expansion is a common way of treating maxillary transverse deficiency. Under mechanical force, the midpalatal suture is expanded, causing local immune responses. This study aimed to determine whether macrophages participate in bone remodeling of the midpalatal suture during palatal expansion and the effects on bone remodeling. METHODS: Palatal expansion model and macrophage depletion model were established. Micro-CT, histological staining, and immunohistochemical staining were used to investigate the changes in the number and phenotype of macrophages during palatal expansion as well as the effects on bone remodeling of the midpalatal suture. Additionally, the effect of mechanically induced M2 macrophages on palatal osteoblasts was also elucidated in vitro. RESULTS: The number of macrophages increased significantly and polarized toward M2 phenotype with the increase of the expansion time, which was consistent with the trend of bone remodeling. After macrophage depletion, the function of osteoblasts and bone formation at the midpalatal suture were impaired during palatal expansion. In vitro, conditioned medium derived from M2 macrophages facilitated osteogenic differentiation of osteoblasts and decreased the RANKL/OPG ratio. CONCLUSIONS: Macrophages through polarizing toward M2 phenotype participated in midpalatal suture bone remodeling during palatal expansion, which may provide a new idea for promoting bone remodeling from the perspective of regulating macrophage polarization.
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Remodelación Ósea , Macrófagos , Osteoblastos , Técnica de Expansión Palatina , Microtomografía por Rayos X , Remodelación Ósea/fisiología , Animales , Hueso Paladar , Ligando RANK , Suturas Craneales , Osteogénesis/fisiología , Diferenciación Celular , Ratones , Osteoprotegerina , Masculino , Estrés Mecánico , FenotipoRESUMEN
Biomaterial wound dressings, such as hydrogels, interact with host cells to regulate tissue repair. This study investigates how crosslinking of gelatin-based hydrogels influences immune and stromal cell behavior and wound healing in female mice. We observe that softer, lightly crosslinked hydrogels promote greater cellular infiltration and result in smaller scars compared to stiffer, heavily crosslinked hydrogels. Using single-cell RNA sequencing, we further show that heavily crosslinked hydrogels increase inflammation and lead to the formation of a distinct macrophage subpopulation exhibiting signs of oxidative activity and cell fusion. Conversely, lightly crosslinked hydrogels are more readily taken up by macrophages and integrated within the tissue. The physical properties differentially affect macrophage and fibroblast interactions, with heavily crosslinked hydrogels promoting pro-fibrotic fibroblast activity that drives macrophage fusion through RANKL signaling. These findings suggest that tuning the physical properties of hydrogels can guide cellular responses and improve healing, offering insights for designing better biomaterials for wound treatment.
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Fibroblastos , Hidrogeles , Macrófagos , Cicatrización de Heridas , Animales , Hidrogeles/química , Cicatrización de Heridas/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Femenino , Comunicación Celular/efectos de los fármacos , Materiales Biocompatibles/química , Ligando RANK/metabolismo , Ratones Endogámicos C57BL , Reactivos de Enlaces Cruzados/química , Gelatina/química , Inflamación/metabolismo , Inflamación/patologíaRESUMEN
OBJECTIVES: To evaluate the effects of coenzyme Q10 (CoQ10) on alveolar bone remodeling and orthodontic tooth movement (OTM). MATERIALS AND METHODS: An orthodontic appliance was placed in 42 female SpragueâDawley rats were divided into two groups: the orthodontic force (OF) group (n = 21) and the OF + CoQ10 (CoQ10) treatment group (n = 21). Each group was divided into 3 subgroups, and the rats were sacrificed on days 3, 7 and 14. The rats in CoQ10 and OF groups were administered 100 mg/kg b.w./day CoQ10 (in 1 mL/b.w. soybean oil) and 1 mL b.w./day soybean oil, respectively, by orogastric gavage. The OTM was measured at the end of the experiment. The osteoclast, osteoblast and capillary numbers; vascular endothelial growth factor (VEGF), receptor activator nuclear kappa B ligand (RANKL) and osteoprotegrin (OPG) levels in tissue; and total antioxidant status (TAS) and total oxidant status (TOS) in blood were determined. RESULTS: Compared with the OF group, the CoQ10 treatment group exhibited decreased orthodontic tooth movement and osteoclast and capillary numbers. Indeed, the levels of VEGF and RANKL decreased, while the levels of OPG increased except on day 7. Additionally, the CoQ10 treatment group exhibited lower TOS and higher TAS on days 7 and 14 (p < 0.05). Histological findings showed that the morphology of osteoblasts changed in the CoQ10 group; however, there was no significant difference in the number of osteoblasts between the groups (p > 0.05). CONCLUSION: Due to its effect on oxidative stress and inflammation, CoQ10 regulates bone remodeling by inhibiting osteoclast differentiation, promoting osteoblast differentiation and reducing the amount of OTM. CLINICAL RELEVANCE: Considering that OTM may be slowed with the use of CoQ10, topics such as orthodontic treatment duration, orthodontic force activation and appointment frequency should be considered in treatment planning. It is predicted that the use of CoQ10 will support the effectiveness of treatment in clinical applications such as preventing relapse in orthodontic treatment by regulating bone modulation and anchorage methods that suppress/optimize unwanted tooth movement.
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Remodelación Ósea , Ratas Sprague-Dawley , Técnicas de Movimiento Dental , Ubiquinona , Ubiquinona/análogos & derivados , Ubiquinona/farmacología , Animales , Ratas , Femenino , Remodelación Ósea/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ligando RANK/metabolismo , Proceso Alveolar/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Antioxidantes/farmacologíaRESUMEN
Periapical lesions are common pathologies affecting the alveolar bone, often initiated by intraradicular lesions resulting from microbial exposure to dental pulp. These microorganisms trigger inflammatory and immune responses. When endodontic treatment fails to eliminate the infection, periapical lesions persist, leading to bone loss. The RANK/RANKL/OPG pathway plays a crucial role in both the formation and the destruction of the bone. In this study, the objective was to inhibit the RANK/RANKL pathway in vitro within exposed Thp-1 macrophages to endodontic microorganisms, specifically Enterococcus faecalis, which was isolated from root canals of 20 patients with endodontic secondary/persistent infection, symptomatic and asymptomatic, and utilizing an α-IRAK-4 inhibitor, we introduced endodontic microorganisms and/or lipoteichoic acid from Streptococcus spp. to cellular cultures in a culture plate, containing thp-1 cells and/or PBMC from patients with apical periodontitis. Subsequently, we assessed the percentages of RANK+, RANKL+, and OPG+ cells through flow cytometry and measured the levels of several inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) in the cellular culture supernatant through a CBA kit and performed analysis by flow cytometry. A significant difference was observed in the percentages of RANK+RANKL+, OPG+ RANKL+ cells in thp-1 cells and PBMCs from patients with apical periodontitis. The findings revealed significant differences in the percentages of the evaluated cells, highlighting the novel role of the IRAK-4 inhibitor in addressing this oral pathology, apical periodontitis, where bone destruction is observed.
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Macrófagos , Periodontitis Periapical , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Transducción de Señal , Humanos , Ligando RANK/metabolismo , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Células THP-1 , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Periodontitis Periapical/metabolismo , Periodontitis Periapical/microbiología , Periodontitis Periapical/patología , Citocinas/metabolismo , Enterococcus faecalis , Lipopolisacáridos , Cavidad Pulpar/microbiología , Cavidad Pulpar/metabolismo , Masculino , Osteoprotegerina/metabolismo , Adulto , Ácidos Teicoicos/farmacologíaRESUMEN
Background: Periprosthetic osteolysis is a prevalent complication following total ankle arthroplasty (TAA), implicating various cytokines in osteoclastogenesis as pivotal in this process. This study aimed to evaluate the relationship between osteolysis and the concentrations of osteoclastogenesis-related cytokines in synovial fluid and investigate its clinical value following TAA. Methods: Synovial fluid samples from 23 ankles that underwent revision surgery for osteolysis following TAA were analyzed as the osteolysis group. As a control group, we included synovial fluid samples obtained from 23 ankles during primary TAA for osteoarthritis. The receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) ratio in these samples was quantified using sandwich enzyme-linked immunosorbent assay techniques, and a bead-based multiplex immunoassay facilitated the detection of specific osteoclastogenesis-related cytokines. Results: RANKL levels averaged 487.9 pg/mL in 14 of 23 patients in the osteolysis group, with no detection in the control group's synovial fluid. Conversely, a significant reduction in OPG levels was observed in the osteolysis group (p = 0.002), resulting in a markedly higher mean RANKL/OPG ratio (0.23) relative to controls (p = 0.020). Moreover, the osteolysis group had increased concentrations of various osteoclastogenesis-related cytokines (tumor necrosis factor-α, interleukin [IL]-1ß, IL-6, IL-8, IP-10, and monocyte chemotactic protein-1) in the synovial fluid relative to the control group. Conclusions: Our results demonstrated that periprosthetic osteolysis was associated with osteoclastogenesis activation through an elevated RANKL/OPG ratio following TAA. We assume that RANKL and other osteoclastogenesis-related cytokines in the synovial fluid have clinical value as a potential marker for the development and progression of osteolysis following TAA.
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Artroplastia de Reemplazo de Tobillo , Biomarcadores , Osteólisis , Osteoprotegerina , Ligando RANK , Líquido Sinovial , Humanos , Líquido Sinovial/metabolismo , Líquido Sinovial/química , Osteólisis/metabolismo , Osteólisis/etiología , Masculino , Femenino , Ligando RANK/metabolismo , Anciano , Persona de Mediana Edad , Artroplastia de Reemplazo de Tobillo/efectos adversos , Osteoprotegerina/metabolismo , Osteoprotegerina/análisis , Biomarcadores/metabolismo , Biomarcadores/análisis , Anciano de 80 o más Años , Citocinas/metabolismo , Citocinas/análisis , ReoperaciónRESUMEN
As a member of the tumor necrosis factor receptor family, osteoprotegerin (OPG) is highly expressed in adults in the lung, heart, kidney, liver, spleen, thymus, prostate, ovary, small intestines, thyroid gland, lymph nodes, trachea, adrenal gland, the testis, and bone marrow. Together with the receptor activator of nuclear factor-κB (RANK) and the receptor activator of nuclear factor-κB ligand (RANKL), it forms the RANK/RANKL/OPG pathway, which plays an important role in the molecular mechanism of the development of various diseases. MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs performing regulatory functions in eukaryotes, with a size of about 20-25 nucleotides. miRNA genes are transcribed into primary transcripts by RNA polymerase, bind to RNA-induced silencing complexes, identify target mRNAs through complementary base pairing, with a single miRNA being capable of targeting hundreds of mRNAs, and influence the expression of many genes through pathways involved in functional interactions. In recent years, a large number of studies have been done to explore the mechanism of action of miRNA in diseases through miRNA isolation, miRNA quantification, miRNA spectrum analysis, miRNA target detection, in vitro and in vivo regulation of miRNA levels, and other technologies. It was found that miRNA can play a key role in the pathogenesis of osteoporosis, rheumatoid arthritis, and other diseases by targeting OPG. The purpose of this review is to explore the interaction between miRNA and OPG in various diseases, and to propose new ideas for studying the mechanism of action of OPG in diseases.
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MicroARNs , Osteoprotegerina , Receptor Activador del Factor Nuclear kappa-B , Osteoprotegerina/metabolismo , Osteoprotegerina/genética , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Receptor Activador del Factor Nuclear kappa-B/genética , Ligando RANK/metabolismo , Ligando RANK/genética , Neoplasias/genética , Neoplasias/metabolismo , Animales , Transducción de Señal , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismoRESUMEN
BACKGROUND: Skeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation. METHODS: To dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl-/-) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs). RESULTS: We found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl-/- mice as compared to wild-type. Rankl-/- mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl-/- SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures. CONCLUSIONS: We provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance.
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Diferenciación Celular , Osteogénesis , Ligando RANK , Animales , Ligando RANK/metabolismo , Ligando RANK/genética , Ratones , Osteogénesis/genética , Humanos , Células Madre/metabolismo , Células Madre/citología , Ratones Noqueados , Denosumab/farmacología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Células Cultivadas , Ratones Endogámicos C57BLRESUMEN
Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.
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Resorción Ósea , Diferenciación Celular , N-Metiltransferasa de Histona-Lisina , Factores de Transcripción NFATC , Osteoclastos , Osteogénesis , Animales , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Ratones , Osteoclastos/metabolismo , Resorción Ósea/metabolismo , Osteogénesis/fisiología , Ratones Noqueados , Ligando RANK/metabolismo , Ratones Endogámicos C57BL , Células CultivadasRESUMEN
The aim of this study was to evaluate the influence of implant macrodesign and surface hydrophilicity on osteoclast (OC) differentiation, activation, and survival in vitro. Titanium disks were produced with a sandblasted, dual acid-etched surface, with or without additional chemical modification for increasing hydrophilicity (SAE-HD and SAE, respectively) and different macrodesign comprising trapezoidal (HLX) or triangular threads (TMX). This study evaluated 7 groups in total, 4 of which were experimental: HLX/SAE-HD, HLX-SAE, TMX/SAE-HD, and TMX/SAE; and 3 control groups comprising OC differentiated on polystyrene plates (CCPC): a positive CCPC (+), a negative CCPC (-), and a lipopolysaccharide-stimulated assay positive control group, CCPC-LPS. Murine macrophage RAW264.7 cells were seeded on the disks, differentiated to OC (RAW-OC) by receptor activator of nuclear factor-κB ligand (RANKL) treatment and cultured for 5 days. Osteoclast differentiation and cell viability were respectively assessed by specific enzymatic Tartrate-Resistant Acid Phosphatase (TRAP) activity and MTT assays. Expression levels of various OC-related genes were measured at the mRNA level by quantitative polymerase chain reaction (qPCR). HLX/SAE-HD, TMX/SAE-HD, and HLX/SAE significantly suppressed OC differentiation when compared to CCPC (+). Cell viability was significantly increased in TMX/SAE and reduced in HLX/SAE-HD. In addition, the expression of Interleukin (IL)-6 and Tumour Necrosis Factor (TNF)-α was upregulated in TMX/SAE-HD compared to CCPC (+). Hydrophilic surfaces negatively modulate macrophage/osteoclast viability. Specifically, SAE-HD with double triangular threads increases the cellular pro-inflammatory status, while surface hydrophilicity and macrodesign do not seem to have a distinct impact on osteoclast differentiation, activation, or survival.
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Diferenciación Celular , Supervivencia Celular , Interacciones Hidrofóbicas e Hidrofílicas , Osteoclastos , Propiedades de Superficie , Titanio , Titanio/química , Osteoclastos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Ratones , Factores de Tiempo , Grabado Ácido Dental , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Ensayo de Materiales , Reproducibilidad de los Resultados , Fosfatasa Ácida Tartratorresistente/análisis , Análisis de Varianza , Ligando RANK/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Células RAW 264.7 , Valores de Referencia , Macrófagos/efectos de los fármacosRESUMEN
Seven new formononetin derivatives (1-7) were designed and prepared from formononetin (phase II phytoestrogen). The derivatives 9-butyl-3-(4-methoxyphenyl)-9,10-dihydro-4H,8H-chromeno[8,7-e][1,3]oxazin-4-one (2) and 9-(furan-3-ylmethyl)-3-(4-methoxyphenyl)-9,10-dihydro-4H,8H-chromeno[8,7-e][1,3]oxazin-4-one (7) promoted significant osteoblast formation by modulating the BMP/Smad pathway. Compound 7 exhibited potent antiosteoclastogenesis activity in RANKL-induced RAW264.7 cells and ovariectomy (OVX)-induced osteoporosis in mice by regulation of the RANK/RANKL/OPG pathway. Compound 7 regulated osteoblast and osteoclast simultaneously and showed better effect than the well-known drug ipriflavone in vivo, suggesting 7 as a patented antiosteoporosis candidate.
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Isoflavonas , Osteoblastos , Osteoclastos , Osteoporosis , Ligando RANK , Isoflavonas/farmacología , Isoflavonas/química , Animales , Osteoblastos/efectos de los fármacos , Ratones , Osteoporosis/tratamiento farmacológico , Osteoclastos/efectos de los fármacos , Células RAW 264.7 , Ligando RANK/metabolismo , Ligando RANK/efectos de los fármacos , Femenino , Estructura Molecular , Ovariectomía , OsteoprotegerinaRESUMEN
Although bone dehiscence may occur during orthodontic tooth movement into the narrow alveolar ridge, a non-invasive prevention method is yet to be fully established. We show for the first time prevention of bone dehiscence associated with orthodontic tooth movement by prophylactic injection of bone anabolic agents in mice. In this study, we established a bone dehiscence mouse model by applying force application and used the granular type of scaffold materials encapsulated with bone morphogenetic protein (BMP)-2 and OP3-4, the receptor activator of NF-κB ligand (RANKL)-binding peptide, for the prophylactic injection to the alveolar bone. In vivo micro-computed tomography revealed bone dehiscence with decreased buccal alveolar bone thickness and height after force application, whereas no bone dehiscence was observed with the prophylactic injection after force application, and alveolar bone thickness and height were kept at similar levels as those in the control group. Bone histomorphometry analyses revealed that both bone formation and resorption parameters were significantly higher in the injection with force application group than in the force application without the prophylactic injection group. These findings suggest that the prophylactic local delivery of bone anabolic reagents can prevent bone dehiscence with increased bone remodelling activity.
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Anabolizantes , Proteína Morfogenética Ósea 2 , Técnicas de Movimiento Dental , Microtomografía por Rayos X , Animales , Ratones , Técnicas de Movimiento Dental/efectos adversos , Anabolizantes/farmacología , Anabolizantes/administración & dosificación , Masculino , Osteogénesis/efectos de los fármacos , Remodelación Ósea/efectos de los fármacos , Ligando RANK/metabolismo , Proceso Alveolar/efectos de los fármacos , Proceso Alveolar/diagnóstico por imagen , Proceso Alveolar/patología , Modelos Animales de EnfermedadRESUMEN
The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored mechanisms of silica-induced pulmonary fibrosis in human lung samples collected from patients with occupational exposure to silica and in a longitudinal mouse model of silicosis using multiple modalities including whole-lung single-cell RNA sequencing and histological, biochemical, and physiologic assessments. In addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor κΒ ligand (RANKL) in pulmonary lymphocytes, and alveolar type II cells. Anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated pulmonary fibrosis. We conclude that silica induces differentiation of pulmonary osteoclast-like cells leading to progressive lung injury, likely due to sustained elaboration of bone-resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.
Asunto(s)
Diferenciación Celular , Osteoclastos , Fibrosis Pulmonar , Dióxido de Silicio , Silicosis , Dióxido de Silicio/toxicidad , Animales , Humanos , Osteoclastos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/metabolismo , Ratones , Silicosis/patología , Silicosis/metabolismo , Silicosis/etiología , Diferenciación Celular/efectos de los fármacos , Ligando RANK/metabolismo , Modelos Animales de Enfermedad , Masculino , Pulmón/patología , Pulmón/metabolismo , Pulmón/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Macrófagos Alveolares/efectos de los fármacos , FemeninoRESUMEN
Bone-modifying agents are a class of drugs that alleviate a series of bone-related events such as pain, pathologic fracture, spinal cord compression, and hypercalcemia caused by bone metastases, and currently include bisphosphonates and RANKL inhibitors. Due to the widespread use of bone-modifying agents, the adverse effects of them are gradually increasing and affecting patients' quality of life. The Breast Cancer Group, Chinese Medical Doctor Association, and the International Medical Society, Chinese Anti-Cancer Association have organized relevant experts to focus on the treatment of bone metastases of advanced malignant tumors based on evidence-based medicine, discuss the management of adverse reactions to bone-modifying agents and form the consensus. Based on the first Expert Consensus on Safety Management of Bone-modifying Agents in China, this consensus added the definition of osteonecrosis of the jaw related to bone-modifying agents, the occurrence of adverse reactions of bone-modifying drugs reported in the literature, and summarized the clinical experience of clinicians in the management of adverse reactions in practice in recent years, and ultimately, the expert group members discussed and proposed reasonable suggestions to guide clinicians in the safety management of bone-modifying agents.
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Conservadores de la Densidad Ósea , Neoplasias Óseas , Consenso , Difosfonatos , Humanos , Neoplasias Óseas/secundario , Neoplasias Óseas/tratamiento farmacológico , Conservadores de la Densidad Ósea/efectos adversos , Conservadores de la Densidad Ósea/uso terapéutico , Difosfonatos/efectos adversos , Difosfonatos/uso terapéutico , Ligando RANK/antagonistas & inhibidores , China , Calidad de Vida , Osteonecrosis de los Maxilares Asociada a Difosfonatos/etiología , Osteonecrosis de los Maxilares Asociada a Difosfonatos/prevención & controlRESUMEN
Tumour necrosis factor (TNF) superfamily member 11 (TNFSF11), also known as RANKL, plays a crucial role in regulating several physiological and pathological activities. Additionally, it is a vital factor in bone physiology, and the sex hormone progesterone regulates the expansion of stem cells and the proliferation of mammary epithelial cells. It is essential for animal growth and reproductive physiological processes. This study aimed to evaluate the tissue-specific expression characteristics and promoter activity of the TNFSF11 gene in pigs. As a result, the study examined the presence of TNFSF11 expression in the tissues of Xiangsu pigs at 0.6 and 12 months of age. Moreover, the core promoter region of TNFSF11 was also identified by utilizing a combination of bioinformatic prediction and dual-luciferase activity tests. Finally, the effect of transcription factors on the transcriptional activity of the core promoter region was determined using site-directed mutagenesis. TNFSF11 was uniformly expressed in all tissues; however, its expression in muscles was comparatively low. The core promoter region of TNFSF11 was located in the -555 to -1 region. The prediction of the transcription start site of TNFSF11 gene-2000 â¼ + 500bp showed that there was a CpG site in 17 â¼ + 487bp. Analysis of mutations in the transcription factor binding sites revealed that mutations in the Stat5b, Myog, Trl, and EN1 binding sites had significant effects on the transcriptional activity of the TNFSF11 gene, particularly following the EN1 binding site mutation (P < 0.001). This study provides insights into both the tissue-specific expression patterns of TNFSF11 in the tissues of Xiangsu pigs and the potential regulatory effects of transcription factors on its promoter activity. These results may be helpful for future research aimed at clarifying the expression and role of the porcine TNFSF11 gene.
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Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Ligando RANK , Animales , Porcinos/genética , Ligando RANK/genética , Ligando RANK/metabolismoRESUMEN
The Receptor Activator Nuclear of κB Ligand (RANKL) plays an important function in immune responses, activating osteoclast cells and unchanged bone resorption, which in turn leads to bone erosion and inflammation. Genetic variants in the promoter region of the RANKL gene could lead to a higher risk of rheumatoid arthritis (RA). OBJECTIVE: To assess the association of rs9533155 (-693C>G) and rs9533156 (-643T>C) genetic variants with RA risk. METHODS: A case-control study was carried out. A total of 94 patients with RA (RA group) and 134 subjects without any rheumatologic disease (control group) were included. Genetic DNA was extracted from peripheral white blood cells (leukocytes). Genetic variant rs9533155 (-693C>G) was screened by an approach based on Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP), while rs9533156 (-643T>C) was screened using quantitative polymerase chain reaction (qPCR) with TaqMan probes. RANKL serum levels were measured by ELISA. RESULTS: For rs9533155 (-693C>G), the polymorphic homozygous genotype frequencies (CC) were higher in the RA group (p = 0.006). Individuals carrying the risk genotype presented higher levels of serum RANKL. Carriers of the polymorphic homozygous genotype in the dominant model (CC vs. CG + GG) had an increased risk of developing RA (OR: 1.8, 95% CI 1.04 to 3.1). No association between rs9533156 (-643T>C) and the haplotypes with RA risk was observed. CONCLUSION: The rs9533155 (-693C>G) genetic variant exhibits a potential role in RA risk. The studied population had no association with the rs9533156 (-643T>C) genetic variant.
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Artritis Reumatoide , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Ligando RANK , Humanos , Artritis Reumatoide/genética , Femenino , Masculino , México , Persona de Mediana Edad , Estudios de Casos y Controles , Ligando RANK/genética , Ligando RANK/sangre , Adulto , Frecuencia de los Genes , AncianoRESUMEN
Staphylococcus aureus osteomyelitis leads to extensive bone destruction. Osteoclasts are bone resorbing cells that are often increased in bone infected with S. aureus. The cytokine RANKL is essential for osteoclast formation under physiological conditions but in vitro evidence suggests that inflammatory cytokines may by-pass the requirement for RANKL. The goal of this study was to determine whether RANKL-dependent osteoclast formation is essential for the bone loss that occurs in a murine model of S. aureus osteomyelitis. To this end, humanized-RANKL mice were infected by direct inoculation of S. aureus into a unicortical defect in the femur. Mice were treated with vehicle or denosumab, a human monoclonal antibody that inhibits RANKL, both before and during a 14-day infection period. The severe cortical bone destruction caused by infection was completely prevented by denosumab administration even though the bacterial burden in the femur was not affected. Osteoclasts were abundant near the inoculation site in vehicle-treated mice but absent in denosumab-treated mice. In situ hybridization demonstrated that S. aureus infection potently stimulated RANKL expression in bone marrow stromal cells. The extensive reactive bone formation that occurs in this osteomyelitis model was also reduced by denosumab administration. Lastly, there was a notable lack of osteoblasts near the infection site suggesting that the normal coupling of bone formation to bone resorption was disrupted by S. aureus infection. These results demonstrate that RANKL-mediated osteoclast formation is required for the bone loss that occurs in S. aureus infection and suggest that disruption of the coupling of bone formation to bone resorption may also contribute to bone loss in this condition.
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Resorción Ósea , Denosumab , Modelos Animales de Enfermedad , Osteoclastos , Osteomielitis , Ligando RANK , Infecciones Estafilocócicas , Staphylococcus aureus , Animales , Osteomielitis/microbiología , Osteomielitis/patología , Osteomielitis/metabolismo , Ligando RANK/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patología , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , Ratones , Resorción Ósea/patología , Resorción Ósea/microbiología , Resorción Ósea/metabolismo , Denosumab/farmacología , Humanos , Fémur/patología , Fémur/microbiología , Anticuerpos Monoclonales Humanizados/farmacologíaRESUMEN
BACKGROUND: Reactive Oxygen Species (ROS) is a key factor in the pathogenesis of osteoporosis (OP) primarily characterized by excessive osteoclast activity. Active fraction of Polyrhachis vicina Rogers (AFPR) exerts antioxidant effects and possesses extensive promising therapeutic effects in various conditions, however, its function in osteoclastogenesis and OP is unknown. PURPOSE: The aim of this study is to elucidate the cellular and molecular mechanisms of AFPR in OP. STUDY DESIGN AND METHODS: CCK8 assay was used to evaluate the cell viability under AFPR treatment. TRAcP staining, podosome belts staining and bone resorption were used to test the effect of AFPR on osteoclastogenesis. Immunofluorescence staining was used to observe the effect of AFPR on ROS production. si-RNA transfection, coimmunoprecipitation and Western-blot were used to clarify the underlying mechanisms. Further, an ovariectomy (OVX) -induced OP mice model was used to identify the effect of AFPR on bone loss using Micro-CT scanning and histological examination. RESULTS: In the present study, AFPR inhibited osteoclast differentiation and bone resorption induced by nuclear factor-κB receptor activator (NF-κB) ligand (RANKL) in dose-/ time-dependent with no cytotoxicity. Meanwhile, AFPR decreased RANKL-mediated ROS levels and enhanced ROS scavenging enzymes. Mechanistically, AFPR promoted proteasomal degradation of TRAF6 by significantly upregulating its K48-linked ubiquitination, subsequently inhibiting NFATc1 activity. We further observed that tripartite motif protein 38 (TRIM38) could mediate the ubiquitination of TRAF6 in response to RANKL. Moreover, TRIM38 could negatively regulate the RANKL pathway by binding to TRAF6 and promoting K48-linked polyubiquitination. In addition, TRIM38 deficiency rescued the inhibition of AFPR on ROS and NFATc1 activity and osteoclastogenesis. In line with these results, AFPR reduced OP caused by OVX through ameliorating osteoclastogenesis. CONCLUSION: AFPR alleviates ovariectomized-induced bone loss via suppressing ROS and NFATc1 by targeting Trim38 mediated proteasomal degradation of TRAF6. The research offers innovative perspectives on AFPR's suppressive impact in vivo OVX mouse model and in vitro, and clarifies the fundamental mechanism.