Porphyromonas gingivalis LPS-stimulated BMSC-derived exosome promotes osteoclastogenesis via miR-151-3p/PAFAH1B1.

OBJECTIVES: Porphyromonas gingivalis-LPS regulated bone metabolism by triggering dysfunction of osteoblasts directly, and affecting activity of osteoclasts through intracellular communication. Exosome, as the mediator of intercellular communication, was important vesicle to regulate osteogenesis and osteoclastogenesis. This research was designed for investigating the mechanism of BMSCs-EXO in modulating osteoclastic activity under the P. gingivalis-LPS. MATERIALS AND METHODS: The cytotoxicity and osteogenic effects of P. gingivalis-LPS on BMSCs was evaluated, and then osteoclastic activity of RAW264.7 co-cultured with exosomes was detected. Besides, Affymetrix miRNA array and luciferase reporter assay were used to identify the target exosomal miRNA signal pathway. RESULTS: BMSCs' osteogenic differentiation and proliferation were decreased under 1 and 10 µg/mL P. gingivalis-LPS. Osteoclastic-related genes and proteins levels were promoted by P. gingivalis-LPS-stimulated BMSCs-EXO. Based on the miRNA microarray analysis, exosomal miR-151-3p was lessened in BMExo-LPS group, which facilitated osteoclastic differentiation through miR-151-3p/PAFAH1B1. CONCLUSIONS: Porphyromonas gingivalis-LPS could regulated bone metabolism by inhibiting proliferation and osteogenesis of BMSCs directly. Also, P. gingivalis-LPS-stimulated BMSCs-EXO promoted osteoclastogenesis via activating miR-151-3p/PAFAH1B1 signal pathway.

[Research Progress in the Regulatory Role of circRNA-miRNA Network in Bone Remodeling]. / circRNA-miRNAç½‘ç»œè°ƒæŽ§éª¨é‡å¡‘çš„ç ”ç©¶è¿›å±•.

The dynamic balance between bone formation and bone resorption is a critical process of bone remodeling. The imbalance of bone formation and bone resorption is closely associated with the occurrence and development of various bone-related diseases. Under both physiological and pathological conditions, non-coding RNAs (ncRNAs) play a crucial regulatory role in protein expression through either inhibiting mRNAs translation or promoting mRNAs degradation. Circular RNAs (circRNAs) are a type of non-linear ncRNAs that can resist the degradation of RNA exonucleases. There is accumulating evidence suggesting that circRNAs and microRNAs (miRNAs) serve as critical regulators of bone remodeling through their direct or indirect regulation of the expression of osteogenesis-related genes. Additionally, recent studies have revealed the involvement of the circRNAs-miRNAs regulatory network in the process by which mesenchymal stem cells (MSCs) differentiate towards the osteoblasts (OB) lineage and the process by which bone marrow-derived macrophages (BMDM) differentiate towards osteoclasts (OC). The circRNA-miRNA network plays an important regulatory role in the osteoblastic-osteoclastic balance of bone remodeling. Therefore, a thorough understanding of the circRNA-miRNA regulatory mechanisms will contribute to a better understanding of the regulatory mechanisms of the balance between osteoblastic and osteoclastic activities in the process of bone remodeling and the diagnosis and treatment of related diseases. Herein, we reviewed the functions of circRNA and microRNA. We also reviewed their roles in and the mechanisms of the circRNA-miRNA regulatory network in the process of bone remodeling. This review provides references and ideas for further research on the regulation of bone remodeling and the prevention and treatment of bone-related diseases.

Mechanism of miRNA Intervention in Osteoporosis and Intervention Effect of Traditional Chinese Medicine： A Review / 中国实验方剂学杂志

Osteoporosis （OP） is a skeletal metabolic disease characterized by bone loss and destruction of bone microstructure. Changes in estrogen levels are not the only pathogenic factors for the occurrence and development of OP. MicroRNA （miRNA） plays an important regulatory role in cells. The complementary sequences of miRNA and targeted mRNA combine to inhibit the expression of targeted mRNA through post-transcriptional regulation， forming a complex regulatory network. Research suggests that miRNA is closely related to the occurrence and development of various diseases， including inflammatory diseases， metabolic diseases， and cancer. Targeted mRNA participates in post-transcriptional gene expression regulation in OP， mainly regulating the balance among bone construction， bone resorption， and osteoblast differentiation. Therefore， miRNA-based gene therapy is a rapidly developing disease treatment strategy. Traditional Chinese medicine can improve bone metabolism by intervening in miRNA differential expression to target and regulate osteogenic/osteoclast differentiation. This article summarized the targeting effects of miRNAs in physiological and developmental processes such as bone cell proliferation， differentiation， survival， and apoptosis， reviewed and classified their mechanisms of action and targets， and sorted out the current treatment methods of traditional Chinese medicine for preventing and treating OP and drugs that exert bone protective functions through miRNAs. This review is expected to provide theoretical reference and research guidance for future research on OP treatment by regulating miRNA.

Effect of graphene-oxide-modified osteon-like concentric microgrooved surface on the osteoclastic differentiation of macrophages.

OBJECTIVES: This study aimed to investigate the effect of new biomimetic micro/nano surfaces on the osteoclastic differentiation of RAW264.7 macrophages by simulating natural osteons for the design of concentric circular structures and modifying graphene oxide (GO). METHODS: The groups were divided into smooth titanium surface group (SS), concentric microgrooved titanium surface group (CMS), and microgroove modified with GO group (GO-CMS). The physicochemical properties of the material surfaces were studied using scanning electron microscopy (SEM), contact-angle measurement, atomic force microscopy, X-ray photoelectron spectroscopy analysis, and Raman spectroscopy. The effect of the modified material surface on the cell biological behavior of RAW264.7 was investigated by cell-activity assay, SEM, and laser confocal microscopy. The effect on the osteoclastic differentiation of macrophages was investiga-ted by tartrate-resistant acid phosphatase (TRAP) immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) experiments. RESULTS: Macrophages were arranged in concentric circles along the microgrooves, and after modification with GO, the oxygen-containing groups on the surface of the material increased and hydrophilicity increased. Osteoclasts in the GO-CMS group were small in size and number and had the lowest TRAP expression. Although it promoted the proliferation of macrophages in the GO-CMS group, the expression of osteoclastic differentiation-related genes was lower than that in the SS group, and the difference was statistically significant (P<0.05). CONCLUSIONS: Concentric circular microgrooves restricted the fusion of osteoclasts and the formation of sealing zones. Osteomimetic concentric microgrooves modified with GO inhibited the osteoclastic differentiation of RAW 264.7 macrophages.

Reduction of Osteoclastic Differentiation of Raw 264.7 Cells by EMF Exposure through TRPV4 and p-CREB Pathway.

In this study, we investigated the effect of EMF exposure on the regulation of RANKL-induced osteoclast differentiation in Raw 264.7 cells. In the EMF-exposed group, the cell volume did not increase despite RANKL treatment, and the expression levels of Caspase-3 remained much lower than those in the RANKL-treated group. TRAP and F-actin staining revealed smaller actin rings in cells exposed to EMF during RANKL-induced differentiation, indicating that EMF inhibited osteoclast differentiation. EMF-irradiated cells exhibited reduced mRNA levels of osteoclastic differentiation markers cathepsin K (CTSK), tartrate-resistant acid phosphatase (TRAP), and matrix metalloproteinase 9 (MMP-9). Furthermore, as measured by RT-qPCR and Western blot, EMF induced no changes in the levels of p-ERK and p-38; however, it reduced the levels of TRPV4 and p-CREB. Overall, our findings indicate that EMF irradiation inhibits osteoclast differentiation through the TRPV4 and p-CREB pathway.

Effect of graphene-oxide-modified osteon-like concentric microgrooved surface on the osteoclastic differentiation of macrophages / 华西口腔医学杂志

OBJECTIVES@#This study aimed to investigate the effect of new biomimetic micro/nano surfaces on the osteoclastic differentiation of RAW264.7 macrophages by simulating natural osteons for the design of concentric circular structures and modifying graphene oxide (GO).@*METHODS@#The groups were divided into smooth titanium surface group (SS), concentric microgrooved titanium surface group (CMS), and microgroove modified with GO group (GO-CMS). The physicochemical properties of the material surfaces were studied using scanning electron microscopy (SEM), contact-angle measurement, atomic force microscopy, X-ray photoelectron spectroscopy analysis, and Raman spectroscopy. The effect of the modified material surface on the cell biological behavior of RAW264.7 was investigated by cell-activity assay, SEM, and laser confocal microscopy. The effect on the osteoclastic differentiation of macrophages was investiga-ted by tartrate-resistant acid phosphatase (TRAP) immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) experiments.@*RESULTS@#Macrophages were arranged in concentric circles along the microgrooves, and after modification with GO, the oxygen-containing groups on the surface of the material increased and hydrophilicity increased. Osteoclasts in the GO-CMS group were small in size and number and had the lowest TRAP expression. Although it promoted the proliferation of macrophages in the GO-CMS group, the expression of osteoclastic differentiation-related genes was lower than that in the SS group, and the difference was statistically significant (P<0.05).@*CONCLUSIONS@#Concentric circular microgrooves restricted the fusion of osteoclasts and the formation of sealing zones. Osteomimetic concentric microgrooves modified with GO inhibited the osteoclastic differentiation of RAW 264.7 macrophages.

Mechanical strain regulates osteoclastogenesis via modulating the PTEN/PI3K/Akt signal pathway through miR-21.

Mechanical strain regulated osteoclastic differentiation and angiogenesis are crucial for bone modeling and remodeling, and previous data indicate that high-magnitude strain within physiological load regulates osteoclastic differentiation. However, the underlying mechanisms are still not fully understood. In the present study, the RAW264.7 mouse monocyte/macrophage was used as an osteoclast precursor, and the bone marrow-derived macrophages (BMMs) were isolated and cultured in vitro. The above cells were subjected to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) for the induction of osteoclast differentiation. Subsequently, the above cells were stretched by differential strain magnitudes to simulate the mechanical stimuli in the physiological conditions, and we found that low-magnitude strain (100 µÎµ) increased the expression levels of Acp5, Clcn7, MMP9 and Ctsk to promote osteoclastogenesis, while high-magnitude strain (3000 µÎµ) had opposite effects. In addition, we noticed that high-magnitude strain upregulated PTEN to inactivate the PI3K/Akt signaling pathway, and silencing of PTEN abrogated the suppressing effects of high-magnitude strain on osteoclastic differentiation. Next, we screened out that high-magnitude strain downregulated miR-21 to promote PTEN expressions in a competing endogenous RNA (ceRNA)-dependent manner. Finally, upregulation of miR-21 recovered osteoclastic differentiation in RAW264.7 and BMMs cells stimulated with high-magnitude strain. Collectively, our findings suggested that high-magnitude mechanical strain affected osteoclastic differentiation through modulating the miR-21/PTEN/PI3K/Akt signaling cascade, which provided potential strategies for the treatment of bone-related diseases. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00507-x.

A novel hierarchical biofunctionalized 3D-printed porous Ti6Al4V scaffold with enhanced osteoporotic osseointegration through osteoimmunomodulation.

BACKGROUND: Femoral stem of titanium alloy has been widely used for hip arthroplasty with considerable efficacy; however, the application of this implant in patients with osteoporosis is limited due to excessive bone resorption. Macrophages participate in the regulation of inflammatory response and have been a topic of increasing research interest in implant field. However, few study has explored the link between macrophage polarization and osteogenic-osteoclastic differentiation. The present study aims to develop a novel hierarchical biofunctionalized 3D-printed porous Ti6Al4V scaffold with enhanced osteoporotic osseointegration through immunotherapy. METHOD: To improve the osteointegration under osteoporosis, we developed a hierarchical biofunctionalized 3D-printed porous Ti6Al4V scaffold (PT). Biomimetic extracellular matrix (ECM) was constructed inside the interconnected pores of PT in micro-scale. And in nano-scale, a drug cargo icariin@Mg-MOF-74 (ICA@MOF) was wrapped in ECM-like structure that can control release of icariin and Mg2+. RESULTS: In this novel hierarchical biofunctionalized 3D-printed porous Ti6Al4V scaffold, the macroporous structure provides mechanical support, the microporous structure facilitates cell adhesion and enhances biocompatibility, and the nanostructure plays a biological effect. We also demonstrate the formation of abundant new bone at peripheral and internal sites after intramedullary implantation of the biofunctionalized PT into the distal femur in osteoporotic rats. We further find that the controlled-release of icariin and Mg2+ from the biofunctionalized PT can significantly improve the polarization of M0 macrophages to M2-type by inhibiting notch1 signaling pathway and induce the secretion of anti-inflammatory cytokines; thus, it significantly ameliorates bone metabolism, which contributes to improving the osseointegration between the PT and osteoporotic bone. CONCLUSION: The therapeutic potential of hierarchical PT implants containing controlled release system are effective in geriatric orthopaedic osseointegration.

LncRNA metastasis-associated lung adenocarcinoma transcript-1 promotes osteogenic differentiation of bone marrow stem cells and inhibits osteoclastic differentiation of Mø in osteoporosis via the miR-124-3p/IGF2BP1/Wnt/ß-catenin axis.

Osteoporosis is defined as a skeletal disorder characterized by impairment in bone strength. The potential application of lncRNAs as therapeutic targets for osteoporosis has been unveiled. This study investigated the regulatory mechanism of lncRNA MALAT1 in the differentiation of bone marrow stem cells (BMSCs) and macrophages (Mø) in osteoporosis. MALAT1 expression in peripheral blood of elderly osteoporosis patients and healthy volunteers was detected. BMSCs and mononuclear Mø were isolated and cultured. Osteogenic differentiation of BMSCs and osteoclastic differentiation of Mø were induced. BMSCs and Mø were transfected with si-MALAT1, miR-124-3p mimics, miR-124-3p inhibitor, or pcDNA IGF2BP1, followed by detection of cell differentiation. The target microRNAs (miRs) and downstream genes and signaling pathways of MALAT1 were examined. The ovariectomy-induced mouse model of osteoporosis was established, and the mice were injected with pcDNA-MALAT1. MALAT1 was downregulated in osteoporosis patients, increased in BMSCs after osteogenic differentiation, and diminished in Mø after osteoclastic differentiation. Downregulation of MALAT1 repressed osteogenic differentiation of BMSCs and facilitated osteoclastic differentiation of Mø. MALAT1 upregulated IGF2BP1 expression by competitively binding to miR-124-3p. miR-124-3p silencing reversed the effect of si-MALAT1 on BMSCs and Mø differentiation, and IGF2BP1 upregulation averted the effect of overexpressed-miR-124-3p by activating the Wnt/ß-catenin pathway. Upregulation of MALAT1 activated the Wnt/ß-catenin pathway and attenuated bone injury in mice. In conclusion, lncRNA MALAT1 promoted the osteogenic differentiation of BMSCs and inhibited osteoclastic differentiation of Mø in osteoporosis via the miR-124-3p/IGF2BP1/Wnt/ß-catenin axis.

Combined effects of fluoride exposure and low nutrition on osteogenesis and osteoclastic differentiation in rats / 中华地方病学杂志

Objective:To investigate the combined effect of fluoride exposure and low nutrition on osteogenesis and osteoclastic differentiation in rats.Methods:SD rats were divided into four groups by the method of random number table, namely normal nutrition group, low nutrition treatment group, fluoride exposure group and co-treatment of fluoride and low nutrition group according to 2 × 2 factorial experimental design, 8 rats in each group, half male and half female. Five months after the experiment, immunohistochemistry was used to test the expression levels of femoral alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL). Analysis of variance of factorial design was used to determine the interaction between fluoride exposure and low nutrition on osteogenesis and osteoclastic differentiation.Results:The immunohistochemical results of bone tissue showed that there were significant differences in the expression levels of osteogenesis differentiation markers ALP and Runx2 between different groups ( F = 25.98, 17.77, P < 0.001). Compared with normal nutrition group (0.005 2 ± 0.002 7, 0.003 1 ± 0.001 4), the expression levels of ALP and Runx2 in fluoride exposure group were higher (0.019 5 ± 0.005 0, 0.014 4 ± 0.004 4, P < 0.05). There was no significant difference between low nutrition treatment group (0.002 6 ± 0.001 8, 0.004 4 ± 0.003 2) and co-treatment of fluoride and low nutrition group (0.003 6 ± 0.000 7, 0.002 9 ± 0.000 8, P > 0.05). The expression levels of ALP and Runx2 in co-treatment of fluoride and low nutrition group were lower than those of fluoride exposure group ( P < 0.05). There were significant differences in the expression level osteoclastic differentiation marker of RANKL and the ratio of RANKL/OPG ( F = 10.50, 31.05, P < 0.001). Among them, the RANKL/OPG ratio (0.115 3 ± 0.039 5) in fluoride exposure group was lower than that in normal nutrition group (1.426 3 ± 0.777 2), and the RANKL expression level and RANKL/OPG ratio (0.019 5 ± 0.007 7, 7.258 7 ± 3.674 3) in co-treatment of fluoride and low nutrition group were higher than those in normal nutrition group (0.004 4 ± 0.002 5, 1.426 3 ± 0.777 2, P < 0.05). However, there was no significant difference in the RANKL expression level and RANKL/OPG ratio (0.004 0 ± 0.001 9, 2.022 3 ± 0.753 7) in low nutrition treatment group ( P > 0.05). The expression level of RANKL and the ratio of RANKL/OPG in the co-treatment of fluoride and low nutrition group were higher than those in low nutrition treatment group and fluoride exposure group ( P < 0.05). The 2 × 2 analysis of variance of factorial design showed that fluoride exposure and low nutrition had interaction on ALP, Runx2, RANKL expression levels and RANKL/OPG ratio ( F = 4.38, 19.39, 22.12, 108.00, P < 0.05), antagonistic effect on ALP and Runx2 expression, synergistic effect on RANKL expression and RANKL/OPG ratio. Conclusions:In rat bone tissue, fluoride exposure promotes osteogenesis differentiation, inhibits osteoclastic differentiation dominated by active osteogenic function. The interaction between fluoride and low nutrition on osteogenesis and osteoclastic differentiation is antagonistic osteogenesis differentiation and synergistic promotion of osteoclastic differentiation. Normal nutrition conditions are material basis of osteogenesis differentiation, and low nutrition is the inducement of enhanced osteoclastic differentiation.

SETD2-mediated epigenetic regulation of noncanonical Wnt5A during osteoclastogenesis.

To define the role of SETD2 in the WNT5a signaling in the context of osteoclastogenesis, we exploited two different models: in vitro osteoclast differentiation, and K/BxN serum-induced arthritis model. We found that SETD2 and WNT5a were upregulated during osteoclast differentiation and after induction of arthritis. Using gain- and loss-of-function approaches in the myeloid cell, we confirmed that SETD2 regulated the osteoclast markers, and WNT5a via modulating active histone marks by enriching H3K36me3, and by reducing repressive H3K27me3 mark. Additionally, during osteoclastic differentiation, the transcription of Wnt5a was also associated with the active histone H3K9 and H4K8 acetylations. Mechanistically, SETD2 directed induction of NF-κß expression facilitated the recruitment of H3K9Ac and H4K8Ac around the TSS region of the Wnt5a gene, thereby, assisting osteoclast differentiation. Together these findings for the first time revealed that SETD2 mediated epigenetic regulation of Wnt5a plays a critical role in osteoclastogenesis and induced arthritis. Model for the Role of SETD2 dependent regulation of osteoclastic differentiation. A In monocyte cells SETD2-dependent H3K36 trimethylation help to create open chromatin region along with active enhancer mark, H3K27Ac. This chromatin state facilitated the loss of a suppressive H3K27me3 mark. B Additionally, SETD2 mediated induction of NF-κß expression leads to the recruitment of histone acetyl transferases, P300/PCAF, to the Wnt5a gene and establish H3K9Ac and H4K8Ac marks. Along with other activation marks, these acetylation marks help in Wnt5a transcription which leads to osteoclastogenesis.

Rosehip Extract-Functionalized Magnesium Hydroxide Nanoparticles and Its Effect on Osteoblastic and Osteoclastic Cells.

Considering the role of magnesium in bone metabolism and the increasing relevance of plant-mediated green-synthesis, this work compares the bone cytocompatibility of magnesium hydroxide nanoparticles (NPs) produced by using pure water, Mg(OH)2, or a rosehip (RH) aqueous extract, Mg(OH)2RH. The NPs were evaluated for dose- and time-dependent effects on human osteoblastic and osteoclastic response, due to the direct involvement of the two cell types in bone metabolism. Mg(OH)2 NPs presented nanoplatelet-like morphology (mean diameter ~90 nm) and a crystalline structure (XRD analysis); the RH-mediated synthesis yielded smaller rounded particles (mean diameter <10 nm) with decreased crystallinity. On the ATR-FTIR spectra, both NPs presented the characteristic Mg-OH peaks; Mg(OH)2RH exhibited additional vibration bands associated with the presence of phytochemicals. On osteoblastic cells, NPs did not affect cell growth and morphology but significantly increased alkaline phosphatase (ALP) activity; on osteoclastic cells, particles had little effect in protein content, tartrate-resistant acid phosphatase (TRAP) activity, percentage of multinucleated cells, and cell area. However, compared with Mg(OH)2, Mg(OH)2RH increased osteoblastic differentiation by inducing ALP activity and promoting the expression of Runx2, SP7, Col1a1, and ALP, and had a negative effect on the expression of the osteoclastic genes NFATC1, CA2, and CTSK. These observations suggest the potential usefulness of Mg(OH)2RH NPs in bone regeneration.

Zinc Sulfate Stimulates Osteogenic Phenotypes in Periosteum-Derived Cells and Co-Cultures of Periosteum-Derived Cells and THP-1 Cells.

Coupling between osteoblast-mediated bone formation and osteoclast-mediated bone resorption maintains both mechanical integrity and mineral homeostasis. Zinc is required for the formation, mineralization, growth, and maintenance of bones. We examined the effects of zinc sulfate on osteoblastic differentiation of human periosteum-derived cells (hPDCs) and osteoclastic differentiation of THP-1 cells. Zinc sulfate enhanced the osteoblastic differentiation of hPDCs; however, it did not affect the osteoclastic differentiation of THP-1 cells. The levels of extracellular signaling-related kinase (ERK) were strongly increased during osteoblastic differentiation in zinc sulfate-treated hPDCs, compared with other mitogen-activated protein kinases (MAPKs). Zinc sulfate also promoted osteogenesis in hPDCs and THP-1 cells co-cultured with the ratio of one osteoclast to one osteoblast, as indicated by alkaline phosphatase levels, mineralization, and cellular calcium contents. In addition, the receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio was decreased in the zinc sulfate-treated co-cultures. Our results suggest that zinc sulfate enhances osteogenesis directly by promoting osteoblastic differentiation and osteogenic activities in osteoblasts and indirectly by inhibiting osteoclastic bone resorption through a reduced RANKL/OPG ratio in co-cultured osteoblasts and osteoclasts.

[Research progress of nanomaterials in osteomyelitis treatment].

OBJECTIVE: To review the related studies on the application of nanomaterials in the treatment of osteomyelitis, and to provide new ideas for the research and clinical treatment of osteomyelitis. METHODS: The literature about the treatment of osteomyelitis with nanomaterials at home and abroad in recent years was reviewed and analyzed. RESULTS: At present, surgical treatment and antibiotic application are the main treatment options for osteomyelitis. But there are many defects such as antibiotic resistance, residual bone defect, and low effective concentration of local drugs. The application of nanomaterials can make up for the above defects. In recent years, nanomaterials play an important role in the treatment of osteomyelitis by filling bone defects, establishing local drug delivery system, and self-antibacterial properties. CONCLUSION: It will provide a new idea and an important research direction for the treatment of osteomyelitis to fully study the related characteristics of nanomaterials and select beneficial materials to make drug delivery system or substitute drugs.

Rutaecarpine exerted anti-osteroporosis / 药学学报

Osteoprotegerin (OPG), secreted by osteoblasts, is a marker of bone turnover. OPG can inhibit osteoclastic differentiation by binding receptor activator of nuclear factor-κB ligand (RANKL). In this study, we found that rutaecarpine (RUT) had the up-regulating OPG activity, and it could significantly increase OPG protein levels in both mouse embryonic osteogenic precursor MC3T3-E1 and human osteosarcoma U-2OS cells. Osteoblastogenic differentiation calcified nodules staining results showed that RUT significantly promoted the osteogenic differentiation of MC3T3-E1 cells. Osteoclastic differentiation tartrate resistant acid phosphatase (TRAP) staining results showed that RUT obviously inhibited the osteoclast differentiation of mouse macrophages RAW264.7 induced by RANKL. In vivo studies showed that low-dose RUT group (5 mg·kg-1·day-1) and high-dose RUT group (45 mg·kg-1·day-1) treatments for 3 months significantly increased bone density in ovariectomized (OVX) rats; calcein double labeling experiment and toluidine blue staining results indicated that low-dose RUT group promoted bone formation and decreased bone loss in vivo; immunohistochemistry results showed that low-dose RUT group increased the expression of OPG in rat femur. All animal procedures were performed in accordance with the regulations of the Institutional Animal Care and Use Committee of Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. In summary, this study demonstrated that RUT could up-regulate OPG expression and had promoting osteoblastic differentiation and inhibiting osteoclastic differentiation effects in vitro and in vivo.

N-Acetyl-l-Leucine-Polyethyleneimine-Mediated Delivery of CpG Oligodeoxynucleotides 2006 Inhibits RAW264.7 Cell Osteoclastogenesis.

INTRODUCTION: CpG oligodeoxynucleotides (CpG ODN) play important roles in resisting inflammation and bone resorption. However, the inherent instability and rapid degradation hinder their wider application. This study aimed to evaluate whether N-acetyl-L-leucine-modified polyethyleneimine (N-Ac-L-Leu-PEI) could effectively deliver CpG ODN 2006 to RAW264.7 cells and and if it can regulate osteoclastogenesis in vitro. MATERIALS AND METHODS: Gel retardation assay was conducted to evaluate whether N- Ac-L-Leu-PEI and CpG ODN could form a stable complex. RAW264.7 cells were divided into four groups of control group, ODN group, phosphorothioate ODN group and N-Ac-L-Leu-PEI/ODN group. Fluorescence assay was conducted to evaluate the transfection rate of ODNs in different groups. Cell viability was determined by MTT assay. Cell apoptosis was determined by live-dead cell staining and flow cytometry assay. Relative expression levels of osteoclastic differentiation factors, including Nfatc, c-fos, receptor activator of nuclear factor κB (RANK), and matrix metalloproteinase 9 (MMP9), were determined by real-time PCR and Western blot. RESULTS: N-Ac-L-Leu-PEI and CpG ODN could form a stable complex at a mass ratio of 1:1 (w:w). MTT assay showed that the cell viability of N-Ac-L-Leu-PEI was relatively high even at a mass ratio of 8 µg/mL. The transfection rate of N-Ac-L-Leu-PEI-ODN complex was higher than 90%. The cell proliferation and apoptosis was significantly enhanced in N-Ac-L-Leu-PEI- CpG ODN group when compared to those in phosphorothioate CpG ODN. The expression levels of Nfatc, c-fos, RANK, and MMP9 were significantly decreased in N-Ac-L-Leu-PEI/ODN complex group. DISCUSSION: N-Ac-L-Leu-PEI could be a potential gene vehicle for the prevention of periodontitis-mediated bone resorption.

Asiatic Acid Attenuates Bone Loss by Regulating Osteoclastic Differentiation.

Anti-resorptive agents like bisphosphonates have been widely used for the treatment of postmenopausal osteoporosis. However, their long-term safety and efficacy are still controversial. This study is to examine the effect of Asiatic acid (AA) in osteoclastic differentiation, and further to investigate its effect on bone quality in animals. Effect of AA on osteoclastic differentiation was measured by Tartrate-resistant acid phosphatase stain, bone resorption pit assays, and quantitative real-time polymerase chain reaction. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transforming growth factor-ß (TGF-ß) signaling were measured by western blot before and after AA treatment. Ovariectomized (OVX) wild-type or Smad7 partially knock out mice were used to evaluate the effects of AA on bone quality by micro-computed tomography, mechanical test, and histomorphometry. Results revealed a dose-dependent inhibitory effect of AA on osteoclastic differentiation. After AA treatment, Smad7 was upregulated, while NF-κB and TGF-ß signaling were inhibited during osteoclastic differentiation. Results from animal study revealed that AA prevented bone from further loss caused by OVX and increased the mechanical properties of femur in wild-type animals. AA also prevented bone loss in the Smad7-deficient animals. When combining with OVX in the Smad7-deficient mice, AA could only partially preserve their bone mass. Taken together, we found that AA effectively inhibited osteoclastic differentiation and attenuated osteoporosis, which effects may be through TGF-ß and NF-κB pathways. This study reveals that AA may be a potential anti-resorptive agent for postmenopausal osteoporosis.

Endothelial Progenitor Cells Enhance the Migration and Osteoclastic Differentiation of Bone Marrow-Derived Macrophages in vitro and in a Mouse Femur Fracture Model through Talin-1.

BACKGROUND/AIMS: Bone resorption mediated by osteoclasts plays an important role in bone healing. Endothelial progenitor cells (EPCs) promote bone repair by stimulating neovascularization and osteogenesis. However, the role of EPCs in osteoclast formation and function is not well defined. The aim of this study was to elucidate mechanisms of EPCs in osteoclast formation and function. METHODS: In this study, we examined the effects of EPCs on the proliferation, migration and osteoclastic differentiation of primary mouse bone marrow-derived macrophages (BMMs) in a co-culture system in vitro. We also evaluated the effects of EPC co-transplantation on the homing and osteoclastic differentiation of transplanted BMMs in a mouse bone fracture model in vivo. The technology of immunofluorescence, immunohistochemical, western blot, Rt-PCR, cell co-culture and Transwell were used in this study. RESULTS: EPCs secreted TGF-ß1 in the EPC-BMM co-culture medium and increased Talin-1 expression in the co-cultured BMMs. Treatment with a TGF-ß1 neutralizing antibody or Talin-1 silencing in BMMs completely inhibited BMM osteoclastic differentiation in the co-culture system. These results indicated that the osteoclastogenic effects of EPCs were mediated by TGF-ß1-mediated Talin-1 expression in BMMs. In the femur fracture model, BMMs co-transplanted with EPCs exhibited enhanced engraftment into the fracture site and osteoclastic differentiation compared with those transplanted alone. Mice treated with EPC-BMM co-transplantation exhibited increased neovascularization at the fracture site and accelerated fracture healing compared with those treated with BMMs alone. CONCLUSION: Taken together, the results suggest that EPCs can promote bone repair by enhancing recruitment and differentiation of osteoclast precursors.

Bone mesenchymal stem cell secretion of sRANKL/OPG/M-CSF in response to macrophage-mediated inflammatory response influences osteogenesis on nanostructured Ti surfaces.

Although it has been well established that osteogenic differentiation of bone mesenchymal stem cells (bMSCs) as well as osteoclastic differentiation of macrophages can be manipulated by the nanostructure of biomaterial surfaces, the interactions among the effects of the surface on immune cells and bMSCs remained unknown. Therefore, in this study, the osteogenic behaviors and secretion of osteoclastogenesis-related cytokines of human bMSCs on TiO2 nanotubular (NT) surfaces in conditioned medium (CM) generated by macrophages cultured on the respective NT surfaces (NT-CM) were analyzed. Although bMSCs showed consistent osteogenic behaviors on the NT5 and NT20 surfaces in both standard culture medium and both types of NT-CM, collagen synthesis and extracellular matrix mineralization were partially impeded on the NT20 surface in NT20-CM and bMSC cytokine secretions on the NT20 surface in NT20-CM elicited remarkable multinuclear giant cell and osteoclast formation compared with that observed on the NT5 surface in NT5-CM. After implantation in vivo, mineralized bone formation was significantly delayed around the NT20 implant compared with the NT5 implant, but both surfaces contributed to good bone formation after 12 weeks. The results obtained in this study advance our understanding of the confounding influence of the implant surface nanostructure, macrophage inflammatory response, and osteogenic differentiation of bMSCs as well as the retro-regulative effects of bMSCs on the osteoclastic differentiation of macrophages, and the culture system based on different NT surfaces and CM generated on the respective surfaces may provide a systematic research model for evaluating the performance of endosseous implants as well as a prospective approach for improving implant osseointegration via immune-regulation.

Effects of Osteogenic-Conditioned Medium from Human Periosteum-Derived Cells on Osteoclast Differentiation.

Stem/progenitor cell-based regenerative medicine using the osteoblast differentiation of mesenchymal stem cells (MSCs) is regarded as a promising approach for the therapeutic treatment of various bone defects. The effects of the osteogenic differentiation of stem/progenitor cells on osteoclast differentiation may have important implications for use in therapy. However, there is little data regarding the expression of osteoclastogenic proteins during osteoblastic differentiation of human periosteum-derived cells (hPDCs) and whether factors expressed during this process can modulate osteoclastogenesis. In the present study, we measured expression of RANKL in hPDCs undergoing osteoblastic differentiation and found that expression of RANKL mRNA was markedly increased in these cells in a time-dependent manner. RANKL protein expression was also significantly enhanced in osteogenic-conditioned media from hPDCs undergoing osteoblastic differentiation. We then isolated and cultured CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood (UCB) mononuclear cells (MNCs) and found that these cells were well differentiated into several hematopoietic lineages. Finally, we co-cultured human trabecular bone osteoblasts (hOBs) with CD34+ HSCs and used the conditioned medium, collected from hPDCs during osteoblastic differentiation, to investigate whether factors produced during osteoblast maturation can affect osteoclast differentiation. Specifically, we measured the effect of this osteogenic-conditioned media on expression of osteoclastogenic markers and osteoclast cell number. We found that osteoclastic marker gene expression was highest in co-cultures incubated with the conditioned medium collected from hPDCs with the greatest level of osteogenic maturation. Although further study will be needed to clarify the precise mechanisms that underlie osteogenic-conditioned medium-regulated osteoclastogenesis, our results suggest that the osteogenic maturation of hPDCs could promote osteoclastic potential.