Complement C3 plays a critical role in periodontitis. However, its source, role and underlying mechanisms remain unclear. In our study, by analyzing single-cell sequencing data from mouse model of periodontitis, we identified that C3 is primarily derived from periodontal fibroblasts. Subsequently, we demonstrated that C3a has a detrimental effect in ligature-induced periodontitis. C3ar^-/- mice exhibited significantly less destruction of periodontal support tissues compared to wild-type mice, characterized by mild gingival tissue damage and reduced alveolar bone loss. This reduction was associated with decreased production of pro-inflammatory mediators and reduced osteoclast infiltration in the periodontal tissues. Mechanistic studies suggested that C3a could promote macrophage polarization and osteoclast differentiation. Finally, by analyzing single-cell sequencing data from the periodontal tissues of patients with periodontitis, we found that the results observed in mice were consistent with human data. Therefore, our findings clearly demonstrate the destructive role of fibroblast-derived C3 in ligature-induced periodontitis, driven by macrophage M1 polarization and osteoclast differentiation. These data strongly support the feasibility of C3a-targeted interventions for the treatment of human periodontitis.
Animals ; Osteoclasts/cytology* ; Periodontitis/metabolism* ; Cell Differentiation ; Mice ; Fibroblasts/metabolism* ; Macrophages ; Disease Models, Animal ; Complement C3/metabolism* ; Humans ; Disease Progression ; Mice, Inbred C57BL ; Male ; Mice, Knockout

This study investigated the regulatory potential of salidroside (SAL), a primary active compound in Rhodiola rosea L., on osteoclast differentiation by modulating the hypoxia-inducible factor 1-alpha (HIF-1a) pathway in osteoblasts. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were employed to validate whether the receptor activator of nuclear factor-?B ligand (RANKL) is the downstream target gene of HIF-1a in osteoblasts. The study also utilized lipopolysaccharide (LPS)-induced mouse osteolysis to examine the impact of SAL on osteolysis in vivo. Furthermore, conditioned medium (CM) from SAL-pretreated osteoblasts was used to investigate the paracrine effects on osteoclastogenesis through the HIF-1a pathway. Hypoxic condition-induced overexpression of HIF-1a upregulated RANKL levels by binding to the RANKL promoter and enhancing transcription in osteoblastic cells. In vivo, SAL significantly alleviated bone tissue hypoxia and decreased the expression of HIF-1a by downregulating the expression of RANKL, vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), and angiopoietin-like 4 (ANGPTL4). In the paracrine experiment, conditioned media from SAL-pretreated osteoblasts inhibited differentiation through the HIF-1a/RANKL, VEGF, IL-6, and ANGPTL4 pathways. RANKL emerges as the downstream target gene regulated by HIF-1a in osteoblasts. SAL significantly alleviates bone tissue hypoxia and bone loss in LPS-induced osteolysis through the HIF-1a/RANKL, VEGF, IL-6, and ANGPTL4 pathways. SAL inhibits osteoclast differentiation by regulating osteoblast paracrine secretion.
Animals ; Osteoblasts/cytology* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Glucosides/administration & dosage* ; Cell Differentiation/drug effects* ; Phenols/administration & dosage* ; Mice ; Osteoclasts/metabolism* ; RANK Ligand/genetics* ; Rhodiola/chemistry* ; Osteogenesis/drug effects* ; Signal Transduction/drug effects* ; Interleukin-6/genetics* ; Male ; RAW 264.7 Cells ; Osteolysis/genetics* ; Humans ; Mice, Inbred C57BL

OBJECTIVETo establish osteoblast-osteoclast cell co-culture system in a Transwell chamber, and detect cell viability of osteoblasts and osteoclasts in system.

METHODSOsteoblast MC3T3-E1 and mouse monocytes RAW264.7 were cultivated in vitro. RANKL-induced mouse RAW264.7 monocytes differentiated into mature osteoclasts, osteoblast-osteoclast cell co-culture system was established in Transwell chamber. Cell activity of osteoblasts and osteoclasts were detected by CCK-8 experimenting, Alizarin Red staining, TRAP staining. The expression of OPG, ALP, RANKL, TGF-b1 gene and RANKL protein in osteoblast MC3T3-E1 were detected by PCR, Western-Blot methods. Also, the expression of RANK, NF-κB in gene and protein level in osteoclast were measured through the same method respectively.

RESULTSThe co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell were established in Transwell chamber. Co-culture system affected cell division activities of osteoblasts and osteoclasts. Differentiation of osteoblasts were increased, while differentiation of osteoclast division were slight decreased under microscope observation. OPG (0.65±0.08) and ALP (0.16±0.01) gene expression of co-culture system were less than single culture OPG(1.00±0.08) and ALP (1.01±0.16); TGF-b1(4.42±0.21) and RANKL(4.12±1.04) of osteoblasts in co-culture system were higher than TGF-b1(1.00±0.10) and RANKL(1.00±0.09) under single culture. However, gene expression of RANK(0.63±0.06) and NF-κB(0.64±0.08) in co-culture system were decreased than RANK(1.00±0.08) and NF-κB(1.00±0.09), in single culture, and had significant differences. Similarly, protein expression of OPG(0.43±0.05) and NF-κB(0.59±0.05) of co-culture system were less than OPG(0.84±0.06) and NF-κB(1.13±0.03) of single culture. While RANKL protein expression (0.54±0.03)of co-culture system was more than single culture RANKL(0.31±0.03), and had statistically differences, which was in agreement of the trend of gene expression change.

CONCLUSIONSCo-culture system of mouse MC3T3-E1 cells and RAW264.7 cell was viable in Transwell chamber, and the activity of osteoblasts is higher than osteoclasts in co-culture system.

3T3 Cells ; Animals ; Cell Differentiation ; Coculture Techniques ; Mice ; NF-kappa B ; metabolism ; Osteoblasts ; cytology ; Osteoclasts ; cytology ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism ; RAW 264.7 Cells ; Receptor Activator of Nuclear Factor-kappa B ; metabolism ; Transforming Growth Factor beta1 ; metabolism

As a dioxygenase, Ten-Eleven Translocation 2 (TET2) catalyzes subsequent steps of 5-methylcytosine (5mC) oxidation. TET2 plays a critical role in the self-renewal, proliferation, and differentiation of hematopoietic stem cells, but its impact on mature hematopoietic cells is not well-characterized. Here we show that Tet2 plays an essential role in osteoclastogenesis. Deletion of Tet2 impairs the differentiation of osteoclast precursor cells (macrophages) and their maturation into bone-resorbing osteoclasts in vitro. Furthermore, Tet2 mice exhibit mild osteopetrosis, accompanied by decreased number of osteoclasts in vivo. Tet2 loss in macrophages results in the altered expression of a set of genes implicated in osteoclast differentiation, such as Cebpa, Mafb, and Nfkbiz. Tet2 deletion also leads to a genome-wide alteration in the level of 5-hydroxymethylcytosine (5hmC) and altered expression of a specific subset of macrophage genes associated with osteoclast differentiation. Furthermore, Tet2 interacts with Runx1 and negatively modulates its transcriptional activity. Our studies demonstrate a novel molecular mechanism controlling osteoclast differentiation and function by Tet2, that is, through interactions with Runx1 and the maintenance of genomic 5hmC. Targeting Tet2 and its pathway could be a potential therapeutic strategy for the prevention and treatment of abnormal bone mass caused by the deregulation of osteoclast activities.
5-Methylcytosine ; analogs & derivatives ; chemistry ; metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; DNA-Binding Proteins ; physiology ; Genome ; Genomics ; Mice ; Mice, Knockout ; Osteoclasts ; cytology ; metabolism ; Proto-Oncogene Proteins ; physiology

OBJECTIVETo compare the osteogenic differentiation potential and osteoclast capacity between stem cells from human exfoliated deciduous teeth (SHED) in the physiological root resorption period and dental pulp stem cells (DPSCs).

METHODSSHED and DPSCs were isolated, purified and cultured in vitro. The two stem cells were examined with ALP staining at 14 days and with alizarin red staining at 21 days of osteogenic induction, and the expressions of the genes associated with osteogenesis and osteoclastogenesis were detected using real-time PCR.

RESULTSThe isolated SHED and DPSCs both showed an elongate spindle-shaped morphology. After osteogenic induction of the cells, Alizarin red staining visualized a greater number of mineralized nodules in SHED than in DPSCs (P<0.05), and SHED also exhibited a stronger ALP activity than DPSCs (P<0.05). RT-PCR test results showed that the two stem cells expressed RANKL,OCN, ALP, OPG and Runx2 mRNA after osteogenic induction, but the expression levels of Runx2, OCN and ALP were lower in DPSCs than in SHED (P<0.05), and the ratio of RANKL/OPG was significantly higher in SHED (P<0.05).

CONCLUSIONSCompared with DPSCs, SHED has not only the ability of osteogenic differentiation but also an osteoclast capacity, which sheds light on the regulatory role of SHED in physiological root resorption bone remodeling.

Alkaline Phosphatase ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Dental Pulp ; cytology ; Humans ; Osteoclasts ; cytology ; Osteogenesis ; Osteopontin ; metabolism ; RANK Ligand ; metabolism ; Real-Time Polymerase Chain Reaction ; Stem Cells ; cytology ; Tooth, Deciduous ; cytology

OBJECTIVETo explore the role of epithelial sodium channel (ENaC) in regulating the functional activity of osteoclasts.

METHODSMultinucleated osteoclasts were obtained by inducing the differentiation of rat bone marrow cells with macrophage colony-stimulating factor (M-CSF) and RANKL. The osteoclasts were exposed to different concentrations of the ENaC inhibitor amiloride, and the expression of ENaC on osteoclasts was examined using immunofluorescence technique. The osteoclasts were identified with tartrate-resistant acid phosphatase (TRAP) staining, and the positive cells were incubated with fresh bovine femoral bone slices and the number of bone absorption pits was counted by computer-aided image processing. RT-PCR was performed to analyze the expression of cathepsin K in the osteoclasts.

RESULTSs Exposure to different concentrations of amiloride significantly inhibited the expression of ENaC and reduced the number of TRAP-positive osteoclasts. Exposure of the osteoclasts to amiloride also reduced the number of bone resorption pits on bone slices and the expression of osteoclast-specific gene cathepsin K.

CONCLUSIONs ENaC may participate in the regulation of osteoclast differentiation and bone resorption, suggesting its role in functional regulation of the osteoclasts and a possibly new signaling pathway related with ENaC regulation for modulating bone metabolism.

Animals ; Bone Marrow Cells ; cytology ; Bone Resorption ; Cathepsin K ; metabolism ; Cattle ; Cell Differentiation ; Epithelial Sodium Channels ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Osteoclasts ; cytology ; RANK Ligand ; metabolism ; Rats ; Signal Transduction

PURPOSETo investigate the influence of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro.

METHODSPrimary osteoblasts, bone marrow-derived mesenchymal stem cells (BMSCs, cultured in osteoinductive medium) and RAW264.7 cells cultured in osteoclast inductive medium were all subjected to a 1000 μstrain (μs) at 1 Hz cyclic mechanical stretch for 30 min (twice a day).

RESULTSAfter mechanical stimulation, the alkaline phosphatase (ALP) activity, osteocalcin protein level of the osteoblasts and BMSCs were all enhanced, and the mRNA levels of ALP and collagen type I increased. Additionally, extracellular-deposited calcium of both osteoblasts and BMSCs increased. At the same time, the activity of secreted tartrate-resistant acid phosphatase, the number of tartrate-resistant acid phosphatase-positive multinucleated cells, matrix metalloproteinase-9 protein levels of RAW264.7 cells and the extracellular calcium solvency all decreased.

CONCLUSIONThe results demonstrated that 1000 μs cyclic mechanical loading enhanced osteoblasts activity, promoted osteoblastic differentiation of BMSCs and restrained osteoclastogenesis of RAW264.7 cells in vitro.

Animals ; Biomechanical Phenomena ; Cell Differentiation ; Cells, Cultured ; Mice ; Mice, Inbred C57BL ; Osteoblasts ; cytology ; Osteoclasts ; physiology ; Osteogenesis ; physiology ; Tartrate-Resistant Acid Phosphatase ; metabolism

Osteoclast-like cells are known to inhibit arterial calcification. Receptor activator of NF-κB ligand (RANKL) is likely to act as an inducer of osteoclast-like cell differentiation. However, several studies have shown that RANKL promotes arterial calcification rather than inhibiting arterial calcification. The present study was conducted in order to investigate and elucidate this paradox. Firstly, RANKL was added into the media, and the monocyte precursor cells were cultured. Morphological observation and Tartrate resistant acid phosphatase (TRAP) staining were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells. During arterial calcification, in vivo and in vitro expression of RANKL and its inhibitor, osteoprotegerin (OPG), was detected by real-time PCR. The extent of osteoclast-like cell differentiation was also assessed. It was found RANKL could induce osteoclast-like cell differentiation. There was no in vivo or in vitro expression of osteoclast-like cells in the early stage of calcification. At that time, the ratio of RANKL to OPG was very low. In the late stage of calcification, a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG. According to the results, the ratio of RANKL to OPG was very low during most of the arterial calcification period. This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation. This likely explains why RANKL had the ability to induce osteoclast-like cell differentiation but acted as a promoter of calcification instead.
Acid Phosphatase ; genetics ; metabolism ; Animals ; Aorta ; drug effects ; metabolism ; pathology ; Cell Differentiation ; Coculture Techniques ; Gene Expression Regulation ; Isoenzymes ; genetics ; metabolism ; Male ; Monocytes ; cytology ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; pathology ; Osteoclasts ; drug effects ; metabolism ; pathology ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tartrate-Resistant Acid Phosphatase ; Vascular Calcification ; genetics ; metabolism ; pathology

Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.
Animals ; Anti-Inflammatory Agents/pharmacology/*therapeutic use ; Arthritis, Experimental/chemically induced/*drug therapy ; Arthritis, Rheumatoid/drug therapy/pathology ; Cell Differentiation/*drug effects ; Cells, Cultured ; Collagen Type II ; Cytokines/biosynthesis ; Disease Models, Animal ; Drugs, Chinese Herbal/therapeutic use ; Female ; Flavonoids/pharmacology/*therapeutic use ; Humans ; Inflammation/drug therapy/immunology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Lymph Nodes/cytology ; Mice ; Mice, Inbred DBA ; Monocytes/cytology ; Osteoclasts/*cytology ; Plant Extracts/pharmacology ; RNA, Messenger/biosynthesis ; Synovial Membrane/cytology ; T-Lymphocytes, Regulatory/cytology/immunology ; Tumor Necrosis Factor-alpha/pharmacology

Osteoclasts are multinucleated giant cell, which derived from mononuclear myeloid hematopoietic stem cells with the function of bone absorption. Osteoclasts plays a key role in bone metabolism, therefore the body is very strict to regulation of osteoclastogenesis. Mobilization and differentiation of osteoclast maturation is a complex and sophisticated multi-level regulatory processes. In the relevant regulatory mechanisms, OPG/RANKL/RANK system plays a pivotal role in the process of osteoclast differentiation and maturation. Recent studies revealed that immune cells and osteoclasts were closely connect with each other in the field of bone metabolism, also provide a new therapeutic target for the treatment of bone diseases. The apoptosis of osteoclasts in bone metabolism have been payed more attention,while its mechanism is still not clear, which need further research.
Animals ; Cell Differentiation ; Gene Expression Regulation ; Humans ; Osteoclasts ; cytology ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; metabolism ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism