Targeting EZH2 attenuates the ferroptosis-mediated osteoblast-osteoclast imbalance in rheumatoid arthritis.

OBJECTIVE: The enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) can regulate osteogenesis and osteoclastogenesis. This study aimed to further explore the effects of EZH2 modification on ferroptosis and the osteoblast-osteoclast balance in rheumatoid arthritis (RA) in vitro and in vivo. METHODS: Bone marrow mesenchymal stromal cells were transfected with EZH2 overexpression (oeEZH2) and EZH2 shRNA (shEZH2) plasmids with or without ferrostatin-1 (Fer-1) treatment and subjected to an osteoblast differentiation assay. The cells were then cocultured with bone marrow-derived macrophages and subjected to an osteoclast differentiation assay. Collagen-induced arthritis (CIA) mice were generated and injected with shEZH2 adeno-associated virus (AAV). RESULTS: OeEZH2 repressed osteoblast differentiation, as reflected by decreased ALP and Alizarin Red S staining and increased OPN, RUNX2, OPG and RANKL; however, shEZH2 had the opposite effects. Besides, oeEZH2 promoted osteoblast ferroptosis, as suggested by increased MDA, Fe2+, ROS, and PTGS2 but decreased GPX4 and SLC7A11; these effects could be attenuated by Fer-1 treatment. In contrast, shEZH2 ameliorated osteoblast ferroptosis. OeEZH2 subsequently increased osteoclast differentiation, as indicated by increased TRAP+ multinucleated cells, NFATC1, CTSK, and c-FOS; however, shEZH2 had the opposite effect, except that it did not regulate CTSK. In CIA mice, shEZH2 AAV decreased the clinical symptom score, histological score of cartilage, and systemic inflammation (TNF-α and IL-6) and repressed bone ferroptosis and restored the osteoblast-osteoclast balance to some extent, as reflected by immunohistochemical staining of related markers. CONCLUSION: Targeting EZH2 attenuates the ferroptosis-mediated osteoblast-osteoclast imbalance in RA, revealing its potential as a treatment target.

Wnt3a-induced LRP6 phosphorylation enhances osteoblast differentiation to alleviate osteoporosis through activation of mTORC1/ß-catenin signaling.

OBJECTIVE: Osteoporosis (OP) is a common cause of morbidity and mortality in older individuals. The importance of Wnt3a in osteogenic activity and bone tissue homeostasis is well known. Here, we explored the possible molecular mechanism by which Wnt3a mediates the LRP6/mTORC1/ß-catenin axis to regulate osteoblast differentiation in OP. METHODS: OP-related key genes were identified through a bioinformatics analysis. A ROS17/2.8 cell differentiation system for rat osteogenic progenitors and a rat model of senile OP were constructed for in vitro and in vivo mechanism verification. RESULTS: Bioinformatics analysis revealed that LRP6 was poorly expressed in OP and may play a key role in the occurrence of OP by affecting osteoblast differentiation. LRP6 knockdown inhibited osteoblast differentiation in an in vitro model. In addition, Wnt3a promoted osteoblast differentiation by inducing LRP6 phosphorylation. Moreover, LRP6 promoted mTORC1 expression, which indirectly promoted ß-catenin expression, thus promoting osteoblast differentiation. Finally, an in vivo assay revealed that LRP6 inhibition improved OP. CONCLUSION: Our study provides evidence that Wnt3a induces phosphorylation of LRP6 to activate the mTORC1/ß-catenin axis, thus promoting osteoblast differentiation and ultimately improving OP in aged rats.

BMSCs-derived exosomes carrying miR-668-3p promote progression of osteoblasts in osteonecrosis of the femoral head: Expression of proteins CD63 and CD9.

Recently, exosomes that are derived from bone marrow mesenchymal stem cells (BMSCs) have garnered considerable interest due to their significant roles in the processes of bone regeneration and repair. Among the various molecular components present within these exosomes, miR-668-3p has emerged as a pivotal microRNA that may be instrumental in modulating the function and proliferation of osteoblasts, the cells responsible for bone formation. The primary objective of this research was to examine the enhancing effects of BMSC-derived exosomes that are enriched with miR-668-3p on the advancement of osteoblasts in the context of osteonecrosis of the femoral head. Furthermore, the study aimed to analyze how the expression of specific exosomal proteins, namely CD63 and CD9, influences this biological process. To conduct the investigation, BMSCs were isolated from healthy rat models, followed by the extraction of their secreted exosomes. The subsequent phase of the study involved assessing the proliferation and differentiation of osteoblasts by introducing the exosomes enriched with miR-668-3p into an experimental setup representing osteonecrosis of the femoral head. The findings revealed that exosomes derived from BMSCs, which contained miR-668-3p, significantly enhanced the proliferation of osteoblasts as well as the expression of key osteogenic marker genes. Notably, the levels of CD63 and CD9 proteins were markedly increased in the treated groups, indicating that the mechanisms underlying this promotion might involve cell adhesion and the endocytic uptake of exosomes.

microRNA-324 mediates bone homeostasis and the regulation of osteoblast and osteoclast differentiation and activity.

MicroRNAs (miRNAs) modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. Defects in bone homeostasis result in the common age-related diseases including osteoporosis. Serum level of miR-324-3p positively correlate with several features of bone maintenance. In contrast here, using in vivo micro-computed tomography and histology, global miR-324-null mice demonstrated increased bone mineral density and both trabecular and cortical thickness, with effect magnitudes increasing with age. The bone marrow of miR-324-null mice had reduced lipid content while TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation. Ex vivo assays showed that the high bone mass phenotype of miR-324-null mice resulted from both increased osteoblast activity and decreased osteoclastogenesis. RNA-seq analysis of osteoblasts, osteoclasts and bone marrow macrophages and target validation assays identified that the osteoclast fusion regulator Pin1 and the master osteogenic regulator Runx2 were targets of miR-324-5p in osteoclast lineage cells and osteoblasts, respectively. Indeed, in vitro Runx2 overexpression recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo by the loss of miR-324. Overall, these data demonstrate the importance of miR-324 in bone homeostasis by regulating aspects of both bone formation and remodelling. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis.

Tissue-Engineered Bone Regeneration for Medium-to-Large Osteonecrosis of the Femoral Head in the Weight-Bearing Portion: An Observational Study.

Background: Stem cell therapy for the treatment of osteonecrosis of the femoral head (ONFH) showed promising outcomes. However, ONFH with a large lesion in the weight-bearing portion is a poor prognostic factor and still challenging issue to be solved. We aimed to evaluate the effect of tissue-engineered bone regeneration for this challenging condition to preserve the femoral head. Methods: A total of 7 patients (9 hips) with ONFH who received osteoblasts expanded ex vivo from bone marrow-derived mesenchymal stem cells (BMdMSCs) and calcium metaphosphate (CMP) as scaffolds from March 2002 to March 2004 were retrospectively reviewed. The median age was 27.0 years (interquartile range [IQR], 23.0-34.0 years), and the median follow-up period was 20.0 years (IQR, 11.0-20.0 years). After culture and expansion of stem cells, we performed core decompression with BMdMSC implantation at a median number of 10.1 ×107 (IQR, 9.9-10.9 ×107). To evaluate radiographic outcomes, the Association Research Circulation Osseous (ARCO) classifications, the Japanese Investigation Committee (JIC) classification, and modified Kerboul combined necrotic angle (mKCNA) were evaluated preoperatively and during follow-up. Clinical outcomes were evaluated by a visual analog scale (VAS) and Harris Hip Score (HHS). Results: The preoperative stage of ONFH was ARCO 2 in 5 hips and ARCO 3a in 4 hips. The ARCO staging was maintained in 3 hips of ARCO 2 and 4 hips of ARCO 3a. Two hips of ARCO 2 with radiographic progression underwent total hip arthroplasty. According to mKCNA, 2 hips showed medium lesions, and 7 hips showed large lesions. The size of necrotic lesion was decreased in 4 hips (2 were ARCO 2 and 2 were ARCO 3a). There were no significant changes in JIC classification in all hips (type C1: 3 hips and type C2: 6 hips) (p = 0.655). Clinically, there were no significant changes in the VAS and HHS between preoperative and last follow-up (p = 0.072 and p = 0.635, respectively). Conclusions: Tissue engineering technique using osteoblasts expanded ex vivo from BMdMSC and CMP showed promising outcomes for the treatment of pre-collapsed and early-collapsed stage ONFH with medium-to-large size, mainly located in weight-bearing areas.

Effect of recombinant irisin on recombinant human bone morphogenetic protein-2 induced osteogenesis and osteoblast differentiation.

Osteoporotic fragility fractures substantially impact aging societies, necessitating long-term care and increasing healthcare costs. Myokine irisin, secreted by skeletal muscle, influences bone metabolism; however, a comprehensive understanding of the mechanisms by which irisin affects bone metabolism is still lacking. Therefore, this study aimed to explore the effects of irisin on osteogenesis and osteoblast differentiation triggered by bone morphogenetic protein-2 (BMP-2). We used 4-week-old male ICR mice and implanted polyethylene glycol pellets containing recombinant human BMP-2 (rh-BMP-2) into the left dorsal muscle pouch. Mice received weekly intraperitoneal injections of either phosphate-buffered saline or recombinant irisin (re-irisin). Ectopic bone formation was evaluated 3 weeks post-surgery using micro-computed tomography (µ-CT) and histological analysis. In vitro experiments, C2C12 cells were treated with or without rh-BMP-2 and re-irisin, and we assessed osteoblast differentiation markers, e.g., runt-related transcription factor 2, alkaline phosphatase, osteocalcin, and osteopontin, using real-time reverse transcription-polymerase chain reaction. The µ-CT analyses showed that re-irisin significantly increased bone mineral content and bone volume of ectopic bones newly formed by rh-BMP-2. The gene expressions of the osteoblast markers were significantly increased by rh-BMP-2 and further upregulated by re-irisin. The treatment of cyclic AMP response element-binding protein (CREB) small interfering RNA attenuated these effects, suggesting that CREB signaling pathway was involved in rh-BMP-2/re-irisin-induced osteoblastic differentiation. This study demonstrates the potential of irisin to enhance osteogenesis through BMP signaling, offering insights for osteoporosis treatment and highlighting irisin as a promising therapeutic target for improving bone health and extending a healthy lifespan.

Synthesis and Evaluation of a Novel Series of Diphenylamine and Diphenylether Derivatives with Osteoblastogenic and Osteogenic Effects via CDK8 Inhibition.

Osteoporosis is induced by an imbalance between osteogenesis and bone resorption, and is treated with osteogenic drugs and/or resorption inhibitors. Resorption inhibitors, such as bisphosphonates, are orally used; however, orally active small molecules with osteogenic activity are not clinically available. We synthesized various types of small molecules and identified a series of diphenylamine and diphenylether derivatives that promoted osteoblast differentiation. Among them, diphenylether derivatives 13a, 13g, and 13h potently promoted osteoblast differentiation (EC200 for increasing alkaline phosphatase activity = 11.3, 31.1, and 12.3 nM, respectively) and inhibited cyclin-dependent kinase 8 (CDK8) activity (IC50 = 2.5, 7.8, and 3.9 nM, respectively), suggesting that their osteoblastgenic effects are mediated by the inhibition of CDK8. The ratio of the maximal plasma concentration after oral administration at 10 mg/kg in female rats and EC200 for osteoblastogenesis was 148.1 for compound 13a, 53.4 for 13g, and 101.8 for 13h, indicating possible in vivo osteoblastogenic and osteogenic effects. In ovariectomized female rats, 13g and 13h at 10 mg/kg/d for 8 weeks increased plasma bone-type alkaline phosphatase activity, indicating enhanced in vivo osteoblastogenesis. Furthermore, micro-computed tomography (micro-CT) showed that both compounds increased femoral cortical bone volume and mineral contents, which were unaffected by ovariectomy, while having negligible effects on trabecular bone volume and mineral contents, which were markedly reduced by ovariectomy. In conclusion, diphenylamine and diphenylether structures are novel scaffolds for osteoblastogenesis enhancers via the inhibition of CDK8. Among them, 13g and 13h are candidates for anti-osteoporotic drugs with cortical bone-selective osteogenic effects.

[Calcifications and phosphocalcic metabolism]. / Calcifications et métabolisme phosphocalcique.

Extraosseous calcifications correspond to ubiquitous deposits of intra-tissue calcium salts leading to dysfunction of the affected tissue or organ. There are two types: metastatic calcifications and dystrophic calcifications. Their formation mechanism is by mimicking the physiological mineralization process with an "osteoblast-like" cell. The cause of extra-osseous calcification is variable and depends on risk factors. If the subject is young, you will have to think about a genetic syndrome!

Europium-Containing Nanospheres for Treating Ovariectomy-Induced Osteoporosis: Targeted Bone Remodeling and Macrophage Polarization Modulation.

Purpose: Osteoporosis, characterized by reduced bone mass and structural deterioration, poses a significant healthcare challenge. Traditional treatments, while effective in reducing fracture risks, are often limited by side effects. This study introduces a novel nanocomplex, europium (Eu) ions-doped superparamagnetic iron oxide (SPIO) nanocrystals encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanospheres, abbreviated as SPIO:Eu@PLGA nanospheres, as a potential therapeutic agent for osteoporosis by modulating macrophage polarization, enhancing osteoblast differentiation and inhibiting osteoclastogenesis. Methods: SPIO and SPIO:Eu nanocrystals were synthesized through pyrolysis and encapsulated in PLGA using an emulsification method. To evaluate the impact of SPIO:Eu@PLGA nanospheres on macrophage reprogramming and reactive oxygen species (ROS) production, flow cytometry analysis was conducted. Furthermore, an ovariectomized (OVX) rat model was employed to assess the therapeutic efficacy of SPIO:Eu@PLGA nanospheres in preventing the deterioration of osteoporosis. Results: In vitro, SPIO:Eu@PLGA nanospheres significantly attenuated M1 macrophage activation induced by lipopolysaccharides, promoting a shift towards the M2 phenotype. This action is linked to the modulation of ROS and the NF-κB pathway. Unlike free Eu ions, which do not achieve similar results when not incorporated into the SPIO nanocrystals. SPIO:Eu@PLGA nanospheres enhanced osteoblast differentiation and matrix mineralization while inhibiting RANKL-induced osteoclastogenesis. In vivo studies demonstrated that SPIO:Eu@PLGA nanospheres effectively targeted trabecular bone surfaces in OVX rats under magnetic guidance, preserving their structure and repairing trabecular bone loss by modulating macrophage polarization, thus restoring bone remodeling homeostasis. The study underscores the critical role of Eu doping in boosting the anti-osteoporotic effects of SPIO:Eu@PLGA nanospheres, evident at both cellular and tissue levels in vitro and in vivo. Conclusion: The inclusion of Eu into SPIO matrix suggests a novel approach for developing more effective osteoporosis treatments, particularly for conditions induced by OVX. This research provides essential insights into SPIO:Eu@PLGA nanospheres as an innovative osteoporosis treatment, addressing the limitations of conventional therapies through targeted delivery and macrophage polarization modulation.

Natural Compounds for Bone Remodeling: Targeting osteoblasts and relevant signaling pathways.

In the process of bone metabolism and bone remodeling, bone marrow mesenchymal stem cells (BM-MSCs) differentiate into osteoblasts (OBs) under certain conditions to enable the formation of new bone, and normal bone reconstruction and pathological bone alteration are closely related to the differentiation and proliferation functions of OBs. Osteogenic differentiation of BM-MSCs involves multiple signaling pathways, which function individually but interconnect intricately to form a complex signaling regulatory network. Natural compounds have fewer adverse effects than chemically synthesized drugs, optimize bone health, and are more suitable for long-term use. In this paper, we focus on OBs, summarize the current research progress of signaling pathways related to OBs differentiation, and review the molecular mechanisms by which chemically synthesized drugs with potential anti-osteoporosis properties regulate OBs-mediated bone formation.

sEV-mediated Lipid Droplets Transferred from Bone Marrow Adipocytes promote ferroptosis and impair Osteoblast Function.

Osteoporosis is linked to increased bone marrow adipocyte (BMAd) proliferation, which displaces bone-forming cells and alters the local environment. The impact of BMAd lipid droplets on bone health and osteoblast function remains unclear. This study investigates the interplay between BMAd-derived lipid droplets and osteoblast functionality, focusing on ferroptosis pathways. Osteoblast cultures were treated with conditioned media from adipocytes to simulate in vivo conditions. High-throughput mRNA sequencing and Western blot analysis were used to profile changes in gene expression and protein levels related to ferroptosis, oxidative phosphorylation, and osteogenic markers. Cellular assays assessed the direct impact of lipid droplets on osteoblast activity. Results showed that osteoblasts exposed to adipocyte-conditioned media had increased intracellular lipid droplet accumulation, upregulation of ferroptosis-related genes and proteins, and downregulation of oxidative phosphorylation and osteoblast differentiation markers. Treatment with ferroptosis inhibitors reversed the detrimental effects on osteoblasts, indicating the functional relevance of this pathway in osteoporosis. BMAd-derived lipid droplets contribute to osteoblast dysfunction through ferroptosis induction. Inhibiting ferroptosis could preserve osteoblast function and combat osteoporosis-related bone issues, suggesting that modulating lipid metabolism and redox balance in bone cells may be promising for future treatments.

Role of oxytocin in bone.

Oxytocin (OT) is a posterior pituitary hormone that, in addition to its role in regulating childbirth and lactation, also exerts direct regulatory effects on the skeleton through peripheral OT and oxytocin receptor (OTR). Bone marrow mesenchymal stem cells (BMSCs), osteoblasts (OB), osteoclasts (OC), chondrocytes, and adipocytes all express OT and OTR. OT upregulates RUNX2, BMP2, ALP, and OCN, thereby enhancing the activity of BMSCs and promoting their differentiation towards OB rather than adipocytes. OT also directly regulates OPG/RANKL to inhibit adipocyte generation, increase the expression of SOX9 and COMP, and enhance chondrocyte differentiation. OB can secrete OT, exerting influence on the surrounding environment through autocrine and paracrine mechanisms. OT directly increases OC formation through the NκB/MAP kinase signaling pathway, inhibits osteoclast proliferation by triggering cytoplasmic Ca2+ release and nitric oxide synthesis, and has a dual regulatory effect on OCs. Under the stimulation of estrogen, OB synthesizes OT, amplifying the biological effects of estrogen and OT. Mediated by estrogen, the OT/OTR forms a feedforward loop with OB. Apart from estrogen, OT also interacts with arginine vasopressin (AVP), prostaglandins (PGE2), leptin, and adiponectin to regulate bone metabolism. This review summarizes recent research on the regulation of bone metabolism by OT and OTR, aiming to provide insights into their clinical applications and further research.

Low-molecular-weight estrogenic phytoprotein suppresses osteoporosis development through positive modulation of skeletal estrogen receptors.

Age-related osteoporosis is a metabolic skeletal disorder caused by estrogen deficiency in postmenopausal women. Prolonged use of anti-osteoporotic drugs such as bisphosphonates and FDA-approved anti-resorptive selective estrogen receptor modulators (SERMs) has been associated with various clinical drawbacks. We recently discovered a low-molecular-weight biocompatible and osteoanabolic phytoprotein, called HKUOT-S2 protein (32 kDa), from Dioscorea opposita Thunb that can accelerate bone defect healing. Here, we demonstrated that the HKUOT-S2 protein treatment can enhance osteoblasts-induced ossification and suppress osteoporosis development by upregulating skeletal estrogen receptors (ERs) ERα, ERß, and GPR30 expressions in vivo. Also, HKUOT-S2 protein estrogenic activities promoted hMSCs-osteoblasts differentiation and functions by increasing osteogenic markers, ALP, and RUNX2 expressions, ALP activity, and osteoblast biomineralization in vitro. Fulvestrant treatment impaired the HKUOT-S2 protein-induced ERs expressions, osteoblasts differentiation, and functions. Finally, we demonstrated that the HKUOT-S2 protein could bind to ERs to exert osteogenic and osteoanabolic properties. Our results showed that the biocompatible HKUOT-S2 protein can exert estrogenic and osteoanabolic properties by positively modulating skeletal estrogen receptor signaling to promote ossification and suppress osteoporosis. Currently, there is no or limited data if any, on osteoanabolic SERMs. The HKUOT-S2 protein can be applied as a new osteoanabolic SERM for osteoporosis treatment.

Cyclophilin D, regulator of the mitochondrial permeability transition, impacts bone development and fracture repair.

Mitochondrial Permeability Transition Pore (MPTP) and its key positive regulator, Cyclophilin D (CypD), control activity of cell oxidative metabolism important for differentiation of stem cells of various lineages including osteogenic lineage. Our previous work (Sautchuk et al., 2022) showed that CypD gene, Ppif, is transcriptionally repressed during osteogenic differentiation by regulatory Smad transcription factors in BMP canonical pathway, a major driver of osteoblast (OB) differentiation. Such a repression favors closure of the MPTP, priming OBs to higher usage of mitochondrial oxidative metabolism. The physiological role of CypD/MPTP regulation was demonstrated by its inverse correlation with BMP signaling in aging and bone fracture healing in addition to the negative effect of CypD gain-of-function (GOF) on bone maintenance. Here we show evidence that CypD GOF also negatively affects bone development and growth as well as fracture healing in adult mice. Developing craniofacial and long bones presented with delayed ossification and decreased growth rate, respectively, whereas in fracture, bony callus volume was diminished. Given that Genome Wide Association Studies showed that PPIF locus is associated with both body height and bone mineral density, our new data provide functional evidence for the role of PPIF gene product, CypD, and thus MPTP in bone growth and repair.

Examining the level of inflammatory cytokines TNF-α and IL-8 produced by osteoblasts differentiated from dental pulp stem cells.

BACKGROUND: The use of dental pulp stem cells (DPSCs) in clinical applications instead of bone marrow stem cells is a very promising method capable of significantly changing the future of medical treatment. If further studies prove that DPSCs and the cells differentiated from them do not stimulate the immune system, these cells can be used more reliably in treatment of autoimmune diseases. METHODS: In this research, we examined the isolated DPSCs and differentiated osteoblasts from them in medium without inflammatory stimulants in terms of TLR3 and TLR4 gene expression and inflammatory cytokines, including TNF-α and IL-8 using qRT-PCR, and measured the concentration of inflammatory cytokines IL-8 and TNF-α produced by these two types of cells through ELISA. RESULTS: The obtained results showed that the expression level of inflammatory cytokines IL-8 and TNF-α in differentiated osteoblasts is significantly different as compared with DPSCs. However, no significant difference was observed in TLR-4 expression between two groups. An increase in TNF-α expression level was found to directly correlate with an increase in the expression of IL-8. The concentration of cytokine TNF-α in osteoblasts was significantly higher than that of IL-8 in DPSCs. CONCLUSION: In comparison to DPSCs, osteoblast cells first lead to inflammatory responses. These responses reduce overtime. However, DPSCs retain their immunomodulatory properties and do not show inflammatory responses.

Hdac3 deficiency limits periosteal reaction associated with Western diet feeding in female mice.

Diet-induced obesity is associated with enhanced systemic inflammation that limits bone regeneration. HDAC inhibitors are currently being explored as anti-inflammatory agents. Prior reports show that myeloid progenitor-directed Hdac3 ablation enhances intramembranous bone healing in female mice. In this study, we determined if Hdac3 ablation increased intramembranous bone regeneration in mice fed a high-fat/high-sugar (HFD) diet. Micro-CT analyses demonstrated that HFD-feeding enhanced the formation of periosteal reaction tissue of control littermates, reflective of suboptimal bone healing. We confirmed enhanced bone volume within the defect of Hdac3-ablated females and showed that Hdac3 ablation reduced the amount of periosteal reaction tissue following HFD feeding. Osteoblasts cultured in a conditioned medium derived from Hdac3-ablated cells exhibited a four-fold increase in mineralization and enhanced osteogenic gene expression. We found that Hdac3 ablation elevated the secretion of several chemokines, including CCL2. We then confirmed that Hdac3 deficiency increased the expression of Ccl2. Lastly, we show that the proportion of CCL2-positve cells within bone defects was significantly higher in Hdac3-deficient mice and was further enhanced by HFD. Overall, our studies demonstrate that Hdac3 deletion enhances intramembranous bone healing in a setting of diet-induced obesity, possibly through increased production of CCL2 by macrophages within the defect.

Gallein but not fluorescein enhances the PGD2-stimulated synthesis of osteoprotegerin and interleukin-6 in osteoblasts: Amplification of osteoprotegerin/interleukin-6 by gallein.

Gallein, a small molecule related to fluorescein, is established as an inhibitor of Gßγ subunits to inhibit G protein (Gs) signaling. This agent is providing a potential therapeutic strategy to ameliorate organ dysfunctions especially involved in inflammation, however; the effects on bone metabolism have not yet been clarified. Prostaglandins (PGs) play important roles as autacoids including osteoblasts, and d-type prostanoid (DP) receptor, a member of G protein-coupled receptor specific to PGD2, is expressed on osteoblasts. We previously reported that prostaglandin D2 (PGD2) induces the syntheses of osteoprotegerin (OPG) and interleukin-6 (IL-6), essential factors in bone remodelling process, and p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/p42 MAPK are involved in the signal transduction of PGD2 in osteoblast-like MC3T3-E1 cells. Thus, we investigated in this study that the effect and the underlying mechanism of gallein, an inhibitor Gßɤ subunits, on the syntheses of OPG and IL-6 induced by PGD2 in these cells. The cultured cells were treated with gallein or fluorescein, a structurally related compound inactive to Gßɤ subunits, and subsequently stimulated with PGD2. Not fluorescein but gallein amplified the PGD2-stimulated releases of OPG and IL-6. Gallein enhanced the PGD2-upregulated mRNA expression levels of OPG and IL-6. Regarding the signaling mechanism, gallein did not affect the PGD2-induced phosphorylation of p38 MAPK, JNK, or p42 MAPK. In conclusion, gallein upregulates the PGD2-stimulated syntheses of OPG and IL-6 by the specific effect to inhibit Gßγ subunits in osteoblasts, but the effect is not exerted at the upstream of p38 MAPK, JNK, or p44/p42 MAPK activation.

Regulation of Osteogenic and Angiogenic Markers in Alkali Treated Titanium for Hard Tissue Engineering Applications.

Titanium (Ti) and Ti alloys are of great interest in bone and dental tissue engineering applications due to their biocompatibility, corrosion resistance, and close mechanical properties to natural bone. However, the formation of fibrous tissue prevents osteointegration and results in implant loosening. Thus, physical and chemical methods are used to improve the surface properties of Ti. This study aimed to understand the role of alkali treatment conditions, including alkali medium concentration, temperature, rotation speed, and post-heat treatment. Our results showed that alkali treatment using 5 and 10 molar sodium hydroxide solution allows the formation of web-like microstructure. However, a higher concentration of 15 molar resulted in cracks along the surface. Interaction between the human fetal osteoblast cells (hfOBs) and Ti samples showed that heat treatment is necessary for increased cellular proliferation, which was not significantly different at later time points compared to the polished Ti. Alkali heat treatment did not induce inflammatory reactions at later time points. It showed an increase in vascular endothelial growth factor, osteoprotegerin/nuclear factor kappa-В ligand ratio, and osteocalcin expression, which is evidence for accelerated osteoblast cell maturation and bone remodeling in surface-modified samples. Together, these data show that alkali treatment using 5 or 10 molar of NaOH followed by heat treatment may have therapeutic effect and assist with bone tissue integration with Ti implant.

Levels of B-ALP and TRAP-5b in a patient with short root anomaly: a case report.

The purpose of this study was to observe the changes in bone-specific alkaline phosphatase (B-ALP) and tartrate-resistant acid phosphatase-5b (TRAP-5b) in a patient diagnosed with short root anomaly (SRA). The detailed clinical data and history of related clinical symptoms of the SRA patient were retrieved. Oral examination showed that the shape and color of the tooth crown were normal. Tooth 11 and 12 were missing, and the mobility degree of other teeth was II-III. Panoramic radiograph examination showed that the root length only reached the neck of the tooth. Laboratory results showed that blood spectrum, chromosome and trace elements were normal. Endocrinological evaluation indicated that hormone levels were within normal limits; however, both B-ALP and TRAP-5b were higher than the normal range. The present case shows that SRA may be related to an imbalance in osteoblast/osteoclast metabolism, which provides a new direction for the etiological research of this disease.

ED-71 Ameliorates Bone Loss in Type 2 Diabetes Mellitus by Enhancing Osteogenesis Through Upregulation of the Circadian Rhythm Coregulator BMAL1.

Purpose: Bone loss is a common complication of type 2 diabetes mellitus (T2DM). Circadian rhythms play a significant role in T2DM and bone remodeling. Eldecalcitol (ED-71), a novel active vitamin D analog, has shown promise in ameliorating T2DM. We aimed to investigate whether the circadian rhythm coregulator BMAL1 mediates the anti-osteoporotic effect of ED-71 in T2DM and its associated mechanisms. Methods: A T2DM mouse model was established using high-fat diet (HDF) and streptozotocin (STZ) injection, and blood glucose levels were monitored weekly. HE staining, Masson staining, and Micro-CT were performed to assess the changes in bone mass. IHC staining and IF staining were used to detect osteoblast status and BMAL1 expression and RT-qPCR was applied to detect the change of oxidative stress factors. In vitro, high glucose (HG) stimulation was used to simulate the cell environment in T2DM. RT-qPCR, Western blot, IF, ALP staining and AR staining were used to detect osteogenic differentiation and SIRT1/GSK3ß signaling pathway. DCFH-DA staining was used to detect reactive oxygen species (ROS) levels. Results: ED-71 increased bone mass and promoted osteogenesis in T2DM mice. Moreover, ED-71 inhibited oxidative stress and promoted BMAL1 expression in osteoblasts The addition of STL1267, an agonist of the BMAL1 transcriptional repressor protein REV-ERB, reversed the inhibitory effect of ED-71 on oxidative stress and the promotional effect on osteogenic differentiation. In addition, ED-71 facilitated SIRT1 expression and reduced GSK3ß activity. The inhibition of SIRT1 with EX527 partially attenuated ED-71's effects, whereas the GSK3ß inhibitor LiCl further enhanced ED-71's positive effects on BMAL1 expression. Conclusion: ED-71 ameliorates bone loss in T2DM by upregulating the circadian rhythm coregulator BMAL1 and promoting osteogenesis through inhibition of oxidative stress. The SIRT1/GSK3ß signaling pathway is involved in the regulation of BMAL1.