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Objective: To initially investigate the function of neuronal pentraxin 1 ( NPTX1 ) gene on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs)., Methods: hBMSCs were induced to undergo osteogenic differentiation, and then RNA was collected at different time points, namely 0, 3, 7, 10 and 14 d. The mRNA expression levels of key genes related with osteogenic differentiation, including runt-related transcription factor 2 ( RUNX2 ), alkaline phosphatase ( ALP ), osteocalcin ( OCN ), and NPTX1 , were detected on the basis of quantitative real-time polymerase chain reaction (qPCR) technology. In order to establish a stable NPTX1 -knockdown hBMSCs cell line, NPTX1 shRNA lentivirus was constructed and used to infect hBMSCs. ALP staining, alizarin red (AR) staining, and qPCR were employed to assess the impact of NPTX1 -knockdown on the osteogenic differentiation ability of hBMSCs., Results: The results showed that during the osteogenic differentiation of hBMSCs in vitro , the mRNA expression levels of osteogenic genes RUNX2 , ALP and OCN significantly increased compared with 0 d, while NPTX1 expression decreased markedly ( P < 0.01) as the osteogenic induction period exten-ded. At 72 h post-infection with lentivirus, the result of qPCR indicated that the knockdown efficiency of NPTX1 was over 60%. After knocking down NPTX1 in hBMSCs, RNA was extracted from both the NPTX1 -knockdown group (sh NPTX1 group) and the control group (shNC group) cultured in regular proliferation medium. The results of qPCR showed that the expression levels of osteogenic-related genes RUNX2 and osterix ( OSX ) were significantly higher in the sh NPTX1 group compared with the shNC group ( P < 0.01). ALP staining revealed a significantly deeper coloration in the sh NPTX1 group than in the shNC group at the end of 7 d of osteogenic induction. AR staining demonstrated a marked increase in mineralized nodules in the sh NPTX1 group compared with the shNC group at the end of 14 d of osteogenic induction., Conclusion: NPTX1 exerts a modulatory role in the osteogenic differentiation of hBMSCs, and its knockdown has been found to enhance the osteogenic differentiation of hBMSCs. This finding implies that NPTX1 could potentially serve as a therapeutic target for the treatment of osteogenic abnormalities, including osteoporosis.

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., Competing Interests: 利益冲突　所有作者均声明不存在利益冲突, (© 2024《四川大学学报（医学版）》编辑部 版权所有Copyright ©2024 Editorial Board of Journal of Sichuan University (Medical Sciences).)

Objective: To explore the effect of ubiquitin-specific protease 42 (USP42) on osteogenic differentiation of human adipose-derived stem cells (hASCs) in vivo and in vitro ., Methods: A combination of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy. Alkaline phosphatase (ALP) staining and quantification, alizarin red S (ARS) staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group (knockdown group and overexpression group) and the control group. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of osteogenesis related genes in the experimental group and control group, and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group. Nude mice ectopic implantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo ., Results: The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group, and those in overexpression group were significantly higher than those in control group. After 7 days of osteogenic induction, the ALP activity in the knockdown group was significantly higher than that in the control group, and ALP activity in overexpression group was significantly lower than that in control group. After 14 days of osteogenic induction, ARS staining was significantly deeper in the knockdown group than in the control group, and significantly lighter in overexpression group than in the control group. The results of qRT-PCR showed that the mRNA expression levels of ALP, osterix (OSX) and collagen type Ⅰ (COLⅠ) in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction, and those in overexpression group were significantly lower than those in control group. The results of Western blotting showed that the expression levels of runt-related transcription factor 2 (RUNX2), OSX and COLⅠ in the knockout group were significantly higher than those in the control group at 14 days after osteogenic induction, while the expression levels of RUNX2, OSX and COLⅠ in the overexpression group were significantly lower than those in the control group. Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group., Conclusion: Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vivo , and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs, and USP42 can provide a potential therapeutic target for bone tissue engineering.

Purpose: Bioactive magnesium ions were successfully incorporated into the nanoporous titanium base coating by micro-arc oxidation(MAO), and its physical properties and osteogenic effects were explored., Methods: Non-magnesium-containing and magnesium-containing titanium porous titanium coatings(MAO, MAO-mg) were prepared by changing the composition of MAO electrolyte and controlling the doping of magnesium in porous titanium coatings. The samples were characterized by scanning electron microscope (SEM), roughness, contact angle and energy dispersive X-ray spectrometer (EDS). Mg 2+ release ability of magnesium-doped nanoporous titanium coatings was determined by inductively coupled plasma/optical emission spectrometer(ICP-OES). The structure of the cytoskeleton was determined by live/dead double staining, CCK-8 detection of material proliferation-toxicity, and staining of β-actin using FITC-phalloidin. The effects of the coating on osteogenic differentiation in vitro were determined by alizarin red (ARS), alkaline phosphatase (ALP) staining and real-time polymerase chain reaction (qRT-PCR). SPSS 25.0 software package was used for statistical analysis., Results: The MAO electrolyte with magnesium ions did not change the surface characteristics of the porous titanium coating. Each group prepared by MAO had similar microporous structure(P＞0.05). There was no significant difference in surface roughness and contact angle between MAO treatment group (MAO, MAO-mg)(P＞0.05), but significantly higher than that of Ti group (P＜0.05). With the passage of cell culture time, MAO-mg group promoted cell proliferation (P＜0.05). MAO-mg group was significantly higher than other groups in ALP and ARS staining. The expression of Runx2 mRNA (P＜0.05), ALP(P＜0.05) and osteocalcin OCN(P＜0.05) in MAO-mg group was significantly higher than that in Ti and MAO groups., Conclusions: MAO successfully prepared magnesium-containing nanoporous titanium coating, and showed a significant role in promoting osteogenic differentiation.

Purpose: To clarify the effect of genistein(GEN) on osteogenic differentiation and explore the effect of GEN loaded by platelet-rich fibrin (PRF) on the repair process of bone defects in obese mice., Methods: In in vitro experiments, the effect of GEN(0, 0.1, 1, 10, 50 μmol/L) on the proliferation of mouse embryonic osteoblast precursor cells (MC3T3-E1) was determined by CCK 8. Alkaline phosphatase(ALP) staining and quantitative detection of ALP activity were performed to determine the changes of ALP activity in cells; RNA and protein expression levels of ALP, osteopontin (OPN) and osteocalcin (OCN) were detected by quantitative real-time PCR(qRT-PCR) and Western blot. Alizarin red staining was used to define the effect of GEN on mineralization of MC3T3-E1. To verify the feasibility of the PRF drug loading, the ultrastructure of PRF was subsequently observed under SEM. In in vivo experiments, obese C57 mouse models were established by high-fat diet feeding. On this basis, skull defect models with a diameter of 2.8 mm were established, and the prepared GEN/PRF complexes were placed into the bone defect area. The effects of GEN on skull defect repair in obese mice were evaluated by Micro-CT scanning and hematoxylin-eosin(H-E) staining. Statistical analysis was performed with GraphPad Prism 5.0 software package., Results: CCK 8 results showed that 0.1, 1 μmol/L GEN promoted cell proliferation within 7 days(P＜0.05); 10 μmol/L GEN had no significant effect on the process of cell proliferation. From the second day, 50 μmol/L GEN significantly inhibited cell growth and showed cytotoxicity(P＜0.05). These two concentrations had similar effects in promoting cellular osteogenic differentiation. SEM results showed that PRF presented a 3-dimensional network structure, providing space for loading drug molecules. In in vivo experiments, the body weight of mice in the high-fat diet (HFD) group was 27.7% greater than that in the normal diet group(P＜0.05) and had abnormal glucose tolerance (P＜0.05). Micro-CT showed that compared with the normal diet group, the number of bone trabeculae in the femur of obese mice was decreased(P＜0.05), the distance between bone trabeculae was widened(P＜0.05), and the bone density was decreased (P＜0.05). In addition, GEN (0.1, 1.0 μmol/L) loaded by PRF increased bone volume fraction in the skull of obese mice (P＜0.05). H-E results showed that GEN/PRF promoted the healing of the bone defects., Conclusions: GEN promotes osteogenic differentiation of MC3T3-E1, and it can effectively accelerate the healing of cranial bone defects after loading with PRF in obese mice.

Purpose: To explore how bone morphogenetic protein 9(BMP9) promotes the osteogenic differentiation of periodontal ligament stem cells(PDLSCs) via ERK5/KLF4 signaling pathway under an inflammatory environment., Methods: Recombinant adenovirus to overexpress BMP9 in PDLSCs stimulated with TNF-α was used and the expression level of osteogenic-related genes and proteins in BMP9-treated cells was examined using RT-PCR and Western blot. Then, KLF4 was overexpressed in PDLSCs via transfection and subjected to inflammatory stimulation to observe its effects on osteogenic differentiation and the expression of osteogenic-related genes. overexpression of BMP9 under the condition of KLF4 knockdown was conducted and its effects on osteogenic differentiation and the expression of osteogenic-related genes was analyzed. ERK5 inhibitor BIX02189 was used to explore the role of ERK5 in BMP9-induced osteogenic differentiation. Statistical analysis was performed with SPSS 20.0 software package., Results: BMP9 could promote the osteogenic differentiation of PDLSCs and the expression of KLF4 in an inflammatory environment. Moreover, the overexpression of KLF4 further promoted the osteogenic differentiation of PDLSCs. However, when KLF4 was knocked down, the osteogenic differentiation promotion effect of BMP9 on PDLSCs was weakened. After BMP9 treatment, the phosphorylation level of ERK5 significantly increased. However, after adding ERK5 inhibitor, the promoting effect of BMP9 on osteogenic differentiation was significantly reduced., Conclusions: BMP9 can promote the osteogenic differentiation of PDLSCs via ERK5/KLF4 signaling pathway in an inflammatory environment.

Purpose: To explore the effect of sodium alginate-g-deferoxamine/chitosan (SA-g-DFO/CS) microspheres on proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs)., Methods: A kind of SA-g-DFO/CS microsphere was developed through electrostatic interaction between porous chitosan microspheres and sodium alginate chemically grafted on the surface of DFO. Its morphology, porosity rate, pore size and sustained release of DFO in vitro were examined. Rat BMSCs were isolated and co-cultured with microspheres in osteogenic differentiation medium. MTT assay was used to study the influence of cell proliferation, and Calcein-AM/PI staining was used to observe the cell viability. Alkaline phosphatase (ALP) activity assay was conducted. PCR was used to detect the expression of genes related to angiogenesis and osteogenesis. Statistical analysis was performed using SPSS 22.0 software package., Results: The SA-g-DFO/CS porous microspheres were successfully prepared with a sustained re6lease of DFO. Compared with SA/CS microspheres, the SA-g-DFO/CS microspheres were conducive to cell proliferation and differentiation, with the increases in expression level of ALP, related angiogenesis genes HIF-1α, VEGF and osteogenesis genes COLI, OCN., Conclusions: The SA-g-DFO/CS porous microspheres can provide a new choice for the development of alveolar bone regeneration.

It has been well documented that exercise can improve bone metabolism, promote bone growth and development, and alleviate bone loss. MicroRNAs (miRNAs) are widely involved in the proliferation and differentiation of bone marrow mesenchymal stem cells, osteoblasts, osteoclasts and other bone tissue cells, and regulation of balance between bone formation and bone resorption by targeting osteogenic factors or bone resorption factors. Thus miRNAs play an important role in the regulation of bone metabolism. Recently, regulation of miRNAs are shown to be one of the ways by which exercise or mechanical stress promotes the positive balance of bone metabolism. Exercise induces changes of miRNAs expression in bone tissue and regulates the expression of related osteogenic factors or bone resorption factors, to further strengthen the osteogenic effect of exercise. This review summarizes relevant studies on the mechanism whereby exercise regulates bone metabolism via miRNAs, providing a theoretical basis for osteoporosis prevention and treatment with exercise.

Objective: To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1)., Methods: The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture., Results: The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P <0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P <0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P >0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P <0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P <0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P >0.05)., Conclusion: Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.

Purpose: To investigate the possible role of circRASA2 in periodontitis and its potential regulatory mechanism., Methods: Periodontitis cell model was established by lipopolysaccharide(LPS)-induced periodontal ligament cells(PDLCs). Cell proliferation activity was detected by CCK-8 assay, cell migration ability was detected by Transwell chamber assay, and the expression of osteogenic differentiation-related proteins in cells was detected by Western blot. The target miRNA of circRASA2 and its downstream target genes were predicted using the databases circinteractome and starBase, respectively, and the targeting relationship between the target genes was verified by dual-luciferase reporter gene experiment. GraphPad Prism 8.0 software package was used to analyze the data., Results: circRASA2 was highly expressed in LPS-treated PDLCs cells. LPS-induced PDLCs cell proliferation activity, migration ability and osteogenic differentiation ability decreased, while knockdown of circRASA2 promoted proliferation, migration and osteogenic differentiation ability of PDLCs under LPS treatment. circRASA2 targeted and negatively regulated the expression of miR-543, and overexpression of miR-543 promoted proliferation, migration and osteogenic differentiation of PDLCs under LPS treatment. TRAF6 was a downstream target gene of miR-543, knockdown of circRASA2 down-regulated the expression of TRAF6 through the sponge action of miR-543. Overexpression of TRAF6 reversed the promotion of circRASA2 knockdown on proliferation, migration and osteogenic differentiation of PDLCs., Conclusions: circRASA2 accelerated the pathological process of periodontitis in vitro through miR-543/TRAF6 axis, and might improve periodontitis by targeting down the expression of circRASA2.

Objective: To observe the effect of miR-335-5p derived from human bone marrow mesenchymal stem cell (hBMMSCs) exosomes on osteogenic differentiation of human periodontal ligament stem cell (PDLSCs) model of periodontitis and explore its mechanism., Methods: The exosomes extracted from hBMMSCs were identified by transmission electron microscopy, Western blotting and PKH67 labeling. The human PDLSC model of TNF-α-induced periodontitis were co-cultured with the extracted exosomes, and qRT-PCR was performed to detect the changes in the expressions of miR-335-5p and the mRNA levels of pro-inflammatory cytokines (IL-1β, IL-6, and IL-8) and the osteogenic marker genes (RunX2, OCN and BMP-2). Alizarin red staining and ALP staining were used to detect the formation of calcium nodules in the treated cells, and the expression level of DKK1 protein was detected with Western blotting. Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-335-5p and DKK1., Results: High expressions of CD9 and CD81 were detected in the extracted hBMMSC exosomes ( P < 0.05). In TNF-α-induced hPDLSCs, treatment with the extracted exosomes significantly reduced the mRNA expressions of IL-1β, IL-6 and IL-8, enhanced the mRNA expressions of RunX2, OCN, and BMP-2, and promoted the formation of calcium nodules. MiR-335-5p was highly expressed in hBMMSC-derived exosomes, and overexpression of miR-335-5p significantly downregulated DKK1 protein expression, inhibited the mRNA expressions of IL-1β, IL-6 and IL-8, and promoted the mRNA expressions of osteogenic markers and the formation of calcium nodules in hPDLSCs., Conclusion: HBMMSC exosome-derived miR-335-5p promotes osteogenic differentiation of hPDLSCs and inhibits the development of periodontitis by downregulating DKK1.

Purpose: To study the role of microRNA (miR)-497-5p in the differentiation and mineralization of pre-osteoblasts MC3T3-E1, and to explore the related mechanisms., Methods: The third generation MC3T3-E1 cells were transfected into the miR-497-5p overexpression plasmid miR-497-5p mimics, the low expression plasmid miR-497-5p inhibitor, and the negative control plasmid miR-497-5p NC. They were set up as the miR-497-5p mimics group, miR-497-5p inhibitor group, and miR-497-5p NC group. The cells untreated was set up as the blank group. Fourteen days after osteogenic induction, alkaline phosphatase (ALP) activity was detected. The expression of osteocalcin (OCN) and type I collagen (COL-I) proteins related to osteogenic differentiation were detected by Western blotting. Mineralization was observed by alizarin red staining method. The expression of Smad ubiquitination regulatory factor 2 (Smurf2) protein was detected by Western blotting. The targeting relationship between miR-497-5p and Smurf2 was verified by dual luciferase experiment. Statistical analysis was performed by SPSS 25.0 software package., Results: Compared with the blank group and miR-497-5p NC group, ALP activity of the miR-497-5p mimics group was enhanced, the expression of OCN, COL-I protein and the ratio of the area of mineralized nodules was increased, and the expression of Smurf2 protein was decreased(P＜0.05). ALP activity of the miR-497-5p inhibitor group was weakened, the expression of OCN, COL-I protein and the ratio of the area of mineralized nodules was decreased, and the expression of Smurf2 protein was increased(P＜0.05). Compared with Smurf2 3'-UTR-WT+miR-497-5p NC group, Smurf2 3'-UTR-MT+miR-497-5p mimics group, Smurf2 3'-UTR-MT+miR-497-5p NC group, the activity of dual luciferase in the WT+miR-497-5p mimics group was decreased (P＜0.05)., Conclusions: Overexpression of miR-497-5p can promote the differentiation and mineralization of pre-osteoblasts MC3T3-E1, and its mechanism may be related to the negatively targeted regulation of Smurf2 protein expression.

Objective: To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM., Methods: Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ , DLK1 , DGAT1 , FABP4 , FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor., Results: The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ , DLK1 , DGAT1 , FABP4 and FASN , but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells., Conclusion: The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.

Purpose: To explore the effect and molecular mechanism of long non-coding RNA(lncRNA) AWPPH on proliferation and osteogenic differentiation of human periodontal ligament cells by regulating the Notch signaling pathway., Methods: Human periodontal ligament cells were cultured in vitro, and osteogenic differentiation was induced. Quantitative real-time polymerase chain reaction (qRT-PCR) experiment were used to detect the AWPPH expression level of cells at 0, 3, 7, and 14 days. Human periodontal ligament cells were divided into blank control group (NC), empty vector group (vector), AWPPH overexpression group (AWPPH), and overexpression AWPPH+ pathway inhibitor group (AWPPH+DAPT). qRT-PCR experiment was used to detect the expression level of AWPPH; thiazole blue (MTT), cloning experiment was used to detect cell proliferation. Western blot was performed to detect the protein expression of alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), Notch1 and Hes1. SPSS 21.0 software package was used for statistical analysis., Results: The AWPPH expression level in periodontal ligament cells decreased after 0, 3, 7, and 14 days of osteogenic differentiation. Overexpression of AWPPH increased the A value of periodontal ligament cells, the number of cloned cells, and up-regulated the protein expression of ALP, OPN, OCN, Notch1, and Hes1. After adding the pathway inhibitor DAPT, the A value and the number of cloned cells decreased, and the protein expression of Notch1, Hes1, ALP, OPN, and OCN decreased., Conclusions: Overexpression of AWPPH may inhibit the proliferation and osteogenic differentiation of periodontal ligament cells by reducing the expression of related proteins in the Notch signaling pathway.

Objective: To investigate the expression of pyruvate kinase M2 (PKM2) in bone marrow mesenchymal stem cells (BMSCs) in myeloma bone disease (MBD) and its effect on osteogenic and adipogenic differentiation of BMSCs., Methods: BMSCs were isolated from bone marrow of five patients with multiple myeloma (MM) (MM group) and five with iron deficiency anemia (control group) for culture and identification. The expression of PKM2 protein were compared between the two groups. The differences between osteogenic and adipogenic differentiation of BMSCs were assessed by using alkaline phosphatase (ALP) and oil red O staining, and detecting marker genes of osteogenesis and adipogenesis. The effect of MM cell line (RPMI-8226) and BMSCs co-culture on the expression of PKM2 was explored. Functional analysis was performed to investigate the correlations of PKM2 expression of MM-derived BMSCs with osteogenic and adipogenic differentiation by employing PKM2 activator and inhibitor. The role of orlistat was explored in regulating PKM2 expression, osteogenic and adipogenic differentiation of MM-derived BMSCs., Results: Compared with control, MM-originated BMSCs possessed the ability of increased adipogenic and decreased osteogenic differentiation, and higher level of PKM2 protein. Co-culture of MM cells with BMSCs markedly up-regulated the expression of PKM2 of BMSCs. Up-regulation of PKM2 expression could promote adipogenic differentiation and inhibit osteogenic differentiation of MM-derived BMSCs, while down-regulation of PKM2 showed opposite effect. Orlistat significantly promoted osteogenic differentiation in MM-derived BMSCs via inhibiting the expression of PKM2., Conclusion: The overexpression of PKM2 can induce the inhibition of osteogenic differentiation of BMSCs in MBD. Orlistat can promote the osteogenic differentiation of BMSCs via inhibiting the expression of PKM2, indicating a potential novel agent of anti-MBD therapy.

Objective To investigate the osteogenic differentiation of bone marrow stromal cells (BMSC) with Notch signaling activation in vitro. Methods The BMSC derived from Notch1-NICD flox/flox mice were infected with recombinant adenovirus expressing Cre or GFP respectively, and designated as Ad-Cre group and Ad-GFP group. The expression of Notch1 was evaluated by Western blot analysis. Alkaline phosphatase (ALP) activity was determined by ALP staining and biochemical quantification, and the calcium deposition was analyzed with Alizarin red S staining. Real-time fluorescence quantitative PCR (qPCR) was used to quantify the expression of Notch target genes(Hes1, Hey1, Hey2, HeyL), osteogenic differentiation-related genes(ALP, RUNX2, osterix, osteocalcin), and angiogenesis factors(VEGF, HIF-1α). Results Notch signaling in BMSC of Notch1-NICDflox/flox mice was activated successfully by Ad-Cre, as was evidenced by the significantly elevated expression of Notch1 and Notch target genes. Compared with the Ad-GFP group, ALP activity and the late calcium deposition were significantly increased upon treatment with Ad-Cre. qPCR results demonstrated that the expression of ALP, RUNX2, osterix, osteocalcin, VEGF, HIF-1α in the Ad-Cre group were significantly upregulated. Conclusion Activation of Notch signaling in BMSC in vitro significantly promotes osteogenic differentiation.

Purpose: To evaluate the effect of microRNA (miR)-124 on osteogenic differentiation of dental pulp mesenchymal stem cells (DPSCs) and to explore the possible mechanism., Methods: Logarithmic DPSCs were collected and divided into blank group, no-load group, miR-124 inhibitor group, miR-124 inhibitor combined with N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-ph (DAPT, Notch signaling pathway inhibitor) group. The blank group was not treated, the empty group was transfected with negative control vector inhibitor-NC, the miR-124 inhibitor group was transfected with miR-124 inhibitor, the miR-124 inhibitor combined with DAPT group was transfected with miR-124 inhibitor, and DAPT was added to make the final concentration of 5 μmol/L. The proliferation ability was tested by CCK-8 method 48 h after transfection. Alkaline phosphatase (ALP) activity was tested by p-nitrophenyl phosphate (P-NPP) method after 2 weeks of induction. The area of calcified nodules was tested by alizarin red staining method. The protein expression of hair-like division-related enhancer 1 (HEY1), hair-like division-related enhancer 2 (HEY2), and cyclin D1 gene (CCND1) were tested by Western blot. The data was analyzed by SPSS 19.0 software package., Results: Compared with the blank group and no-load group, the A450 value at 24, 48, 72 h detected by CCK-8 experiment, A450 value of ALP activity, the area composition ratio of calcified nodules, and expression of HEY1, HEY2, and CCND1 in the miR-124 inhibitor group were increased (P<0.05). Compared with miR-124 inhibitor group, the A450 value at 24, 48, 72 h detected by CCK-8 experiment, A450 value of ALP activity, the area composition ratio of calcified nodules, and the expression of HEY1, HEY2, and CCND1 in the miR-124 inhibitor combined with DAPT group were significantly decreased(P<0.05)., Conclusions: Down-regulation of miR-124 can promote osteogenic differentiation of DPSCs. It is speculated that the mechanism of action is related to the activation of Notch signaling pathway.

Objective: To explore whether the effect of low-frequency pulsed electromagnetic fields (PEMFs) in promoting osteoblast mineralization and maturation is related to the primary cilia, polycystin2 (PC2) and sAC/PKA/CREB signaling pathway., Methods: We detected the expression levels of PC2, sAC, PKA, CREB and their phosphorylated proteins in primary rat calvarial osteoblasts exposed to 50 Hz 0.6 mT PEMFs for 0, 5, 15, 30, 60, 90, and 120 min. We blocked PC2 function with amiloride hydrochloride and detected the changes in the activity of sAC/PKA/CREB signal pathway and the mineralization and maturation of the osteoblasts. These examinations were repeated in the osteoblasts after specific knockdown of PC2 via RNA interference and were the co-localization of PC2, sAC, PKA, CREB and their phosphorylated proteins with the primary cilia were using immunofluorescence staining. The expressions of PC2 and the signaling proteins of sAC/PKA/CREB pathway were detected after inhibition of primary ciliation by RNA interference., Results: The expression levels of PC2, sAC, p-PKA and p- CREB were significantly increased in the osteoblasts after exposure to PEMFs for different time lengths ( P < 0.01). Blocking PC2 function or PC2 knockdown in the osteoblasts resulted in failure of sAC/PKA/CREB signaling pathway activation and arrest of osteoblast mineralization and maturation. PC2, sAC, p-PKA and p-CREB were localized to the entire primary cilia or its roots, but PKA and CREB were not detected in the primary cilia. After interference of the primary cilia, PEMFs exposure no longer caused increase of PC2 expression and failed to activate the sAC/PKA/CREB signaling pathway or promote osteoblast mineralization and maturation., Conclusion: PC2, located on the surface of the primary cilia of osteoblasts, can perceive and transmit the physical signals from PEMFs and promote the mineralization and maturation of osteoblasts by activating the PC2/ sAC/PKA/CREB signaling pathway.

Purpose: To investigate the effects of inflammatory microenvironment on proliferation and osteogenic differentiation of periodontal ligament cells(PDLCs) in vitro., Methods: Human PDLCs were isolated and characterized. MTT was used to investigate the proliferation rate of PDLCs under different concentration of lipopolysaccharide（LPS）. The PDLCs' osteogenic differentiation was investigated using real-time PCR and Western blot. The date were statistically analyzed with SPSS 13.0 software package., Results: Treatment with 0.1 μg/mL LPS increased proliferation of PDLCs and enhanced the expression of osteogenic gene and protein. The proliferation of PDLCs and expression of alkaline phosphatase(ALP), RUNX2, Collagen-I, BMP2 were significantly decreased by 10 μg/mL LPS., Conclusions: The inflammatory microenvironment (10 μg/mL LPS) inhibits the proliferation and osteogenic differentiation of human PDLCs.

Objective: To investigate the effect of RUNX2 gene overexpression vector modified exosomes derived from bone marrow mesenchymal stem cells (BMSCs) combined with calcium carbonate scaffold system in bone defect., Methods: Rabbit BMSCs were used as the research object, and BMSCs were identified by flow cytometry. Construct RUNX2 gene overexpression vector, transfect BMSCs with lentivirus, and collect exosomes by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscope, the expression of exosome marker CD63 was detected by Western blot, and the calcium carbonate scaffold was constructed by three chamber parallel automatic temperature control reaction system. According to whether the RUNX2 gene overexpression vector was transfected or not, the complex of BMSCs and calcium carbonate scaffold was divided into three groups, namely BMSCs group, RUNX2 overexpression group and exosome group. The osteogenic differentiation of BMSCs was detected by oil red O staining and RT-PCR. There were 9 clean adult healthy male New Zealand white rabbits, aged (12.97±1.21) months, with a body weight of (19.3±3.6) kg, with 3 rabbits in each group. The animal model of skull defect was constructed by surgical method, and the repair of bone defect was evaluated by imaging, he staining and Masson staining., Results: The results of flow cytometry showed that the expression of CD29 protein, CD44 protein, CD11b protein and CD45 protein on the surface of BMSCs were 99.5%, 100%, 0.1% and 0.1%, respectively. Transmission electron microscopy showed that the exosomes were bilayer vesicles with a diameter of 50 to 150 nm. Western blot showed that the molecular marker CD63 of exosomes was positive. Oil red O staining showed that the osteogenic differentiation of BMSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group ( P <0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group ( P <0.05). MSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group( P <0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group( P <0.05)., Conclusion: Compared with RUNX2 gene overexpression vector transfection, extraction of exosomes directly can promote the differentiation of BMSCs into osteoblasts more efficiently, and the combination with calcium carbonate scaffold can better promote the healing of bone defects. So as to provide new ideas and methods for the clinical treatment of bone defects.

Objective: To summarize the mechanism of long non-coding RNA (lncRNA) in signal pathways related to osteogenic differentiation., Methods: Relevant domestic and foreign researches in recent years were consulted. The characteristics and biological functions of lncRNA were introduced, and the specific mechanism of lncRNA regulating related signal pathways in osteogenic differentiation was elaborated., Results: The exertion and maintenance of normal function of bone requires the closed coordination of transcription networks and signal pathways. However, most of these signal pathways or networks are dysregulated under pathological conditions that affect bone homeostasis. lncRNA can regulate the differentiation of various bone cells by activating or inhibiting signal pathways to achieve the balance of bone homeostasis, thereby reversing the pathological state of bones and achieving the purpose of treating bone metabolic diseases., Conclusion: At present, the research on the mechanism of lncRNA regulating various osteogenic differentiation pathways is still in the early stage. Its in-depth regulator mechanism, especially the cross-talk of complex signal pathways needs to be further studied. And how to apply these molecular targets to clinical treatment is also a big challenge.

Purpose: To investigate the expression change of microRNA (miR) - 199a in MC3T3-E1 cells stimulated by mechanical stretch and its mechanism of osteogenic differentiation., Methods: MC3T3-E1 cells cultured in vitro were loaded with 12% stretch for 0, 3, 6, 12 and 24 hours, alkaline phosphatase (ALP) activity was detected by ALP activity test kit, the expressions of osteocalcin (OCN), osteoblast specific transcription factor osterix (OSX), Runt related transcription factor 2 (Runx2) mRNA and miR-199a were detected by real-time fluorescence quantitative PCR. MC3T3-E1 cells were divided into control group, stretch group, stretch + miR-NC group and stretch + miR-199a group, and the expressions of miR-199a, OCN, OSX, Runx2 mRNA and protein and ALP activity were observed after 12% stretch and transfection of miR-199a. Alizarin red S (ARS) staining was used to observe calcium nodule formation ability. The target relationship between miR-199a and insulin like growth factor-1 (IGF1) was detected by double luciferase reporter gene assay; in addition, the effect of miR-199a mimic on IGF1 mRNA and protein expression was detected by real-time fluorescent quantitative PCR and Western blot. SPSS 24.0 software package was used to analyze the data., Results: Compared with those at the time point of 0 h, ALP activity and expression level of OCN, OSX and Runx2 mRNA of MC3T3-E1 cells at 3, 6, 12 and 24 hours after mechanical stretch stimulation were significantly higher, while the expression level of miR-199a was significantly lower(P＜0.05), and the change was most significant at 12 h. Compared with those in the control group, the expression level of miR-199a was significantly lower in the stretch group, while ALP activity, the expression level of OCN, OSX and Runx2 mRNA and protein, calcium nodule formation level were significantly high in the stretch group(P＜0.05); there was no significant difference in the above indexes between the stretch group and stretch + miR-NC group(P＞0.05). Compared with stretch + miR-NC group, the expression level of miR-199a in stretch + miR-199a group was significantly higher; while ALP activity, OCN, OSX, Runx2 mRNA and protein expression level, calcium nodule formation level were significantly lower(P＜0.05). miR-199a could targetedly bind to IGF1, and the expression level of IGF1 mRNA and protein in MC3T3-E1 cells was significantly reduced by miR-199a mimic(P＜0.05)., Conclusions: MiR-199a can inhibit the osteogenic differentiation of MC3T3-E1 cells induced by mechanical stretch stimulation, and its mechanism may be related to the targeted regulation of IGF1 expression.

It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro . However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.

Objective To investigate the role of miR-100-5p in the pathogenesis of non-traumatic osteonecrosis of the femoral head (NONFH). Methods The miRNA expression in patients with NONFH was detected by real-time quantitative PCR, the high expression of miR-100-5p in femoral head tissues of the patients determined. Rat bone marrow mesenchymal stem cells (rBMSCs) were cultured and divided into 5 groups: blank control group, dexamethasone treatment group (treated with dexamethasone for 3 days), miR-NC group (transfected with miR-NC), agomiR-100-5p group (overexpression of miR-100-5p), and antagomiR-100-5p group (transfected with miR-100-5p antagonist). The mRNA expression levels of miR-100-5p, alkaline phosphatase (ALP), Runt-associated transcription factor 2 (RUNX2), and collagen type I (Col1) were detected by real-time quantitative PCR. The protein expressions of ALP, RUNX2, Col1, and bone morphogenetic protein receptor 2 (BMPR2) were detected by Western blotting. The effect of miR-100-5p on the migration ability of rBMSCs was evaluated by scratch healing assay. And the effect of miR-100-5p on osteogenic differentiation ability of rBMSCs was investigated by alizarin red staining. Results miR-100-5p was significantly upregulated in the femoral head bone tissue of NONFH patients compared with normal femoral head bone tissue. Compared with those in the normal rBMSCs, the expression of miR-100-5p in rBMSCs treated with 20 μmol/L of dexamethasone was up-regulated. The upregulation of miR-100-5p in rBMSCs reduced the expressions of ALP, RUNX2, Col1, and BMPR2, and inhibited the osteogenic differentiation and migration abilities of rBMSCs. Conclusion The expression of miR-100-5p is elevated in bone tissues of NONFH patients and in rBMSCs treated with 20 μmol/L of dexamethasone. The up-regulated miR-100-5p may be involved in the pathogenesis of NONFH by inhibiting the migration and osteogenic differentiation of rBMSCs.

Objective: To explore the effects of siRNA hsa-circ-0000885 modified bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation, cell proliferation and apoptosis in order to provide new ideas and methods for the clinical treatment of osteoporosis (OP)., Methods: From September 2018 to February 2020, 13 patients with osteoporosis admitted to our hospital were selected as the research objects, including 11 females and 2 males, with an age of (65.45±10.77) years old. After obtaining the informed consent of patients, peripheral blood tissues were extracted. Then the expression level of cir-cRNA in peripheral blood mononuclear cells(PBMC) was detected by circ RNA chip. The expression of circ RNA was silenced by siRNA technology. The BMSCs were transfected with lentivirus. According to the siRNA interference plasmid hsa-circ-0000885, the cells were divided into the blank group, the empty vector group and the siRNA interference group. After 72 hours of treatment, the cell cycle was detected by flow cytometry, the apoptosis level was detected by AV-PI kit, and the osteogenic differentiation ability of BMSCs was detected by ALP staining., Results: The expression of hsa-circ-0000885 in PBMC of patients with osteoporosis was significantly higher than that of healthy controls ( t =2.119, P <0.05). ALP staining showed that siR-NA hsa-circ-0000885 could promote the osteogenic differentiation of BMSCs, which was obviously too much in the blank group and blank plasmid group ( F =9.132, q =2.995, 2.897; P =0.009, 0.012<0.05). The results of CCK-8 showed that siRNA hsa-circ-0000885 could promote the proliferation of BMSCs, which was significantly higher than that of the blank group and blank plasmid group ( F =9.881, q =2.457, 2.904; P =0.032, 0.016<0.05). The results of AV-PI showed that the apoptosis rate of siRNA interference group was significantly lower than that of blank group and blank plasmid group( F =10.208; q =2.885, 3.001; P =0.019, 0.011<0.05)., Conclusion: The lentivirus mediated siRNA hsa-circ-0000885 plasmid transfected into BMSCs and osteoclast co culture system can promote cell proliferation, inhibit apoptosis and promote osteogenic differentiation of BMSCs, which can be used as a potential therapeutic target for OP patients.

Purpose: To explore the effect of enamel matrix proteins(EMPs) on osteogenesis and adipogenesis of stem cells from human exfoliated deciduous teeth SHED), and explore its molecular mechanism., Methods: SHEDs were used to detect the expression of its surface antigens CD73, CD146, CD34 and CD45 by flow cytometry. SHED was induced by OB osteogenic induction liquid, and then the osteogenic differentiation ability was measured by alizarin red staining. SHEDs were divided into 4 groups, NC group had invalid sequence shRNA interfered with SHED, EMPs group had invalid sequence shRNA interfered with SHED. Then 100 μg/L EMPs was used to interfere with SHED. In miR-32 inhibitor group, miR-32 shRNA plasmid was used to interfere with SHED; while in EMPs+miR-32 inhibitor group, 100 μg/L EMP was used to intervene SHED after silencing miR-32. QPCR was used to detect the expression of miR-32, dentin sialophosphoprotein (DSPP), dentin matrix protein 1, DMP-1, peroxisome proliferators-activated receptor γ (PPARγ) and CCAAT enhancer binding protein α (C/EBPα) gene expression; Western blot was used to detect the expression of DSPP, DMP-1, PPARγ and C/EBPα protein expression; Alizarin red staining was used to detect SHED osteogenic capacity; Oil red O staining was used to detect adipogenetic capacity of SHED., Results: The results of flow cytometry showed that SHED had positive expression of CD146 and CD73, and negative expression of CD34 and CD45, which was consistent with the characteristics of stem cell surface markers. Alizarin red staining and oil red O staining showed mineralized nodules and oil droplets increased significantly, consistent with the multi-directional differentiation characteristics of stem cells. Compared with NC group, the expression of miR-32 gene in EMPs group was significantly increased(P<0.05), and the expression of miR-32 in miR-32 inhibitor group and EMPs+miR-32 inhibitor group was significantly decreased(P<0.05). Compared with NC group, the expression of DSPP and DMP-1, the number of mineralized nodules in EMPs group were significantly increased(P<0.05), the expression of PPARγ and C/EBPa and the number of lipid droplets were significantly decreased (P<0.05), while the result of miR-32 inhibitor group was the opposite (P<0.05). Compared with miR-32 inhibitor group, there was no significant difference in the expression of DSPP, DMP-1, PPARγ and C/EBPα, number of mineralized nodules and oil droplets in EMPs+miR-32 inhibitor group(P>0.05). Compared with EMPs group, the expression of DSPP and DMP-1 and the number of mineralized nodules in EMPs+miR-32 inhibitor group were significantly reduced(P<0.05), while the expression of PPARγ and C/EBPα and the number of lipid droplets were significantly increased(P<0.05)., Conclusions: EMPs can regulate osteogenic and adipogenic differentiation of SHED by promoting the expression of miR-32.

Objective: To study the regulatory effect of YTH domain-containing protein 2 (YTHDC2), a member of N 6 -methyladenosine (m 6 A) readers, on the osteogenic or adipogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs)., Methods: YTHDC 2 expression was knocked down by small interfering RNA (siRNA) in vitro . Osteogenic differentiation and adipogenic differentiation of hBMSCs were induced after YTHDC 2 knockdown in order to study the changes in the differentiation phenotype of hBMSCs. Alkaline phosphatase staining (ALP staining) and alizarin red S staining were performed to examine osteogenic activity and calcium-nodular formation. Nile red staining was performed to examine lipid-droplet formation. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to assess the expression of osteogenesis and adipogenesis-related genes. RNA-sequencing was performed to identify the transcriptome changes after YTHDC 2 knockdown and to explore the potential regulatory mechanism by which YTHDC2 regulated the diferentiation of hBMSCs., Results: In this study, we found that siRNA-induced YTHDC 2 knockdown resulted in increased ALP activity and calcium-nodular formation of hBMSCs during osteogenic differentiation, and significantly upregulated the expression of osteogenesis-related genes. In addition, the lipid-droplet formation capacity of hBMSCs was decreased during adipogenic differentiation. The expression of adipogenesis-related genes was significantly down-regulated. Gene-set enrichmen analysis of RNA-seq data showed that YTHDC2 was significantly correlated with ribosome function and mRNA-translation-related signaling pathways., Conclusion: The findings indicate that YTHDC2 knockdown can promote the osteogenic differentiation of hBMSCs and inhibit the adipogenic differentiation. YTHDC2 knockdown may cause changes in ribosome function., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

The proliferation and multi-directional differentiation potential of mesenchymal stem cells (MSCs) enabled its wide use in the development of new therapies for bone and cartilage repair. Although preliminary work has been done to verify the gene expression profile of MSCs osteogenic and chondrogenic differentiation, it is still unclear what key factors initiate the differentiation of MSCs, resulting in its limited application in bone and cartilage tissue engineering. The epigenetic mechanism mediated by histone demethylases (lysine [K]-specific histone demethylases, KDMs) is the key link in regulating MSCs lineage differentiation. The lysine-specific histone demethylase (LSD) family containing Tower domain and the histone demethylase family containing Jumonji C (JmjC) domain regulate the expression of various osteogenic-related genes, including Runt-related transcription factor 2 ( RUNX 2), osterix ( OSX ), osteocalcin ( OCN ), to mediate MSCs osteogenic differentiation. The KDM2/4/6 subfamilies regulate the chondrogenic differentiation of MSCs through multiple pathways centered on SRY-related high-mobility-group-box gene 9 ( SOX 9). In addition, nanotopology, mircoRNAs, etc. regulate the expression of a variety of osteogenic and chondrogenic transcription factors through up- and down-regulation of KDMs. In summary, the role of histone demethylase in the osteogenic and chondrogenic differentiation of mesenchymal stem cells will help us better understand the pathogenesis of bone and cartilage damage diseases, and establish the foundation of future clinical applications for bone and cartilage tissue engineering., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Purpose: To investigate the biological characteristics of human periodontal stem cells (hPDLSCs) modified with platelet derived growth factor BB(PDGFBB) gene, and to explore its influence on proliferation, migration and osteogenic induction of hPDLSCs., Methods: hPDLSCs were isolated and amplified, and immunofluorescence staining was performed to identify cell surface markers and osteogenic differentiation ability. hPDLSCs were transfected with PDGFBB gene by lentivirus vector, and the effects on cell proliferation and migration were detected by CCK-8 and scratch test after transfection. Real-time PCR was performed to analyze the mRNA expression levels of osteogenic and angiogenic genes in hPDLSCs cells transfected with PDGFBB gene. Statistical analysis was performed using SPSS 22.0 software package., Results: hPDLSCs were successfully obtained by tissue mass culture and finite dilution method. Compared with the blank virus group and non-transfected group, the proliferation and migration ability of the cells in the transfection group were significantly increased, and the mRNA expression levels of OPN, COL-1 and VEGF were significantly up-regulated(P<0.05)., Conclusions: Lentiviral vector can transfer PDGFBB gene into hPDLSCs in vitro and obtain continuous and stable expression. PDGFBB can promote proliferation and migration of hPDLSCs cells and up-regulate expression of osteogenic and angiogenic genes.

Purpose: To investigate the role of P2X7 receptor (P2X7r) in osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs)., Methods: hPDLSCs were isolated from the premolars collected in the First Affiliated Hospital of Guiyang University of Traditional Chinese Medicine, and divided into four groups. Group A was cultured in conventional medium, group B was cultured in osteogenic induction medium, group C was cultured in osteogenic induction medium + 100 nmol/L adenosine triphosphate (ATP) solution, and group D was cultured in osteogenic induction medium + 100 nmol/L P2X7 receptor specific antagonist KN-62. After 7 days, alizarin red staining was used to observe the osteogenic effect of hPDLSCs in each group. The mRNA expression of osteocalcin (OCN), RUNX2 and P2X7r in hPDLSCs was detected by real-time PCR reaction (RT-PCR). The data were processed by SPSS 22.0 software package., Results: Alisarin red staining showed that the morphology of hPDLSCs cells in group B and group C was significantly changed. The pale calcified nodules in group C were significantly more than those in group B, while very few calcified nodules were found in group A and group D. The mRNA expression of OCN, RUNX2 and P2X7r in hPDLSCs were the highest in group C, followed by group B(P<0.05), and no difference was found between group A and group D(P>0.05)., Conclusions: P2X7 receptor can promote osteogenic differentiation of human periodontal ligament stem cells after being activated by ATP, which may provide a new direction for clinical treatment of periodontitis.

Purspose: To investigate the role of P2X7 receptor in osteogenic differentiation of human periodontal ligament stem cells., Methods: Human periodontal ligament stem cells obtained from primary culture were divided into 4 groups: control group, adenosine triphosphate group, osteogenic induction group, adenosine triphosphate + osteogenic induction group. The differences of RUNX2, OCN gene expression and P2X7 receptor mRNA expression between the four groups were compared. Statistical analysis was performed with SPSS 16.0 software package., Results: One week after osteogenic formation and two weeks after osteogenic formation, the expression of RUNX2 and OCN mRNA in the adenosine triphosphate + osteogenic induction group was significantly higher than that in the osteogenic induction group (P<0.05). The expression of RUNX2 and OCN mRNA in the 1 week after adenosine triphosphate + osteogenic induction fluid was significantly higher than that 2 weeks after osteogenic formation, and the difference was statistically significant (P<0.05). The expression of P2X7 receptor mRNA in the adenosine triphosphate group and the adenosine triphosphate + osteogenic induction group was significantly higher than that in the control group and the osteogenic induction group 1 week after osteogenesis and 2 weeks after osteogenesis (P<0.05). The expression of P2X7 receptor mRNA in the adenosine triphosphate group was significantly higher than that in the adenosine triphosphate + osteogenic induction group 2 weeks after osteogenesis(P<0.05). The expression of P2X7 receptor mRNA was significantly higher than that of osteogenic induction 1 week after adenosine triphosphate composition(P<0.05)., Conclusions: P2X7 receptor can significantly improve the osteogenesis of periodontal ligament stem cells, and adenosine triphosphate can activate the expression of P2X7 receptor.

Objective: To investigate the effect of micro RNA (miR)-335-5p regulating bone morphogenetic protein 2 (BMP-2) on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs)., Methods: hBMSCs were cultured in vitro and randomly divided into control group (group A), miR-335-5p mimics group (group B), miR-335-5p mimics negative control group (group C), miR-335-5p inhibitor group (group D), and miR-335-5p inhibitor negative control group (group E). After grouping treatment and induction of osteogenic differentiation, the osteogenic differentiation of cells in each group was detected by alkaline phosphatase (ALP) and alizarin red staining; the expressions of miR-335-5p and BMP-2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) mRNAs were detected by real-time fluorescence quantitative PCR analysis; the expressions of Runx2, OPN, OCN, and BMP-2 proteins were detected by Western blot., Results: Compared with group A, the relative proportion of ALP positive cells and the relative content of mineralized nodules, the relative expressions of BMP-2, miR-335-5p, OPN, OCN, Runx2 mRNAs, the relative expressions of Runx2, OPN, OCN, and BMP-2 proteins in group B were significantly increased ( P <0.05); the above indexes in group D were significantly decreased ( P <0.05); the above indexes between groups C, E and group A were not significantly different ( P >0.05)., Conclusion: miR-335-5p can up-regulate BMP-2 expression and promote osteogenic differentiation of hBMSCs.

Objective: To investigate the correlation between down-regulation of miR-381-3p and inhibition of osteogenic differentiation of mouse embryonic palatal mesenchymal (MEPM) cells in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cleft palate of fetal mice., Methods: Thirty-two pregnant mice were randomly divided into TCDD group and control group, 16 in each group. On embryonic day 10.5 (E10.5), the pregnant mice in TCDD group were orally administrated with TCDD at dosage of 28 μg/kg, while the pregnant mice in control group received equivalent corn oil. The pregnant mice in each group were sacrificed on E13.5 and E14.5, fetal palates were collected for analysis. The expression of miR-381-3p was detected by real-time fluorescent quantitative PCR and the protein expressions of runt- related transcription factor 2 (RUNX2) and osteopontin (OPN) were detected by Western blot. MEPM cells were extracted from fetal palates on E14.5 in control group and passaged. The 3rd passage cells were cultured with TCDD at dosage of 10 nmol/L for 0, 0.5, 1, 2, and 3 days. The expression of miR-381-3p was detected after 0, 0.5, 1, 2, and 3 days and the protein expressions of RUNX2 and OPN were detected after 0, 1, 2, and 3 days. Then, the 3rd passage cells were divided into 4 groups. The MEPM cells were transfected with miR-381-3p inhibitor (inhibitor group), NC inhibitor (NC inhibitor group) and miR-381-3p mimics (mimics group), NC mimics (NC mimics group) for 48 hours, respectively. And the expressions of miR-381-3p and the protein expressions of RUNX2 and OPN were detected., Results: On E13.5 and E14.5, 96 fetal mice in control group and 92 in TCDD group were obtained. The bilateral palates contacted in control group on E14.5, and a gap between the bilateral palates existed in TCDD group. On E13.5 and E14.5, the relative expressions of miR-381-3p and RUNX2 and OPN proteins were significant lower in TCDD group than in control group ( P <0.05). The relative expression of miR-381-3p at 0.5 and 1 day after TCDD treatment of MEPM cells were significantly lower than that at 0 day ( P <0.05); then, the relative expressions at 2 and 3 days significantly increased, showing no significant difference when compared with that at 0 day ( P >0.05). The relative expressions of RUNX2 and OPN proteins at 1, 2, and 3 days were significantly lower than that at 0 day ( P <0.05). The relative expressions of miR-381-3p and RUNX2 and OPN proteins significantly lower in inhibitor group than in NC inhibitor group ( P <0.05) and higher in mimics group than in NC mimics group ( P <0.05)., Conclusion: Down-regulation of miR-381-3p expression may be associated with inhibition of osteogenic differentiation of MEPM cells in TCDD-induced cleft palate of fetal mice.

Objective: To examine the growth activity and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) in rats with Type 2 diabetes mellitus (T2DM) as well as the expression level of Dickkopf-1 (DKK-1) in bone marrow, and to explore the relationship between the osteogenic activity of BMSCs and the expression of DKK-1.
 Methods: The BMSCs were isolated from T2DM rats and were cultured in vitro. The BMSCs were divided into a T2DM group and a control group. The proliferation of BMSCs was detected by cell counting kit-8 (CCK8). Apoptosis rate was detected by annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining. In the osteogenic induction phase, the expression level of alkaline phosphatase (ALP) in BMSCs was detected by ALP staining and ALP activity assay kit. The osteogenic differentiation of BMSCs was analyzed by alizarin red staining and mineralized nodule quantification. In addition, the expression of Runx2 and DKK-1 in BMSCs was detected by qRT-PCR.
 Results: Compared with the control group, the proliferation of BMSCs was decreased and the apoptosis was increased in the T2DM group (both P<0.01). In the osteogenic induction process of BMSCs, the expression of ALP significantly decreased, the formation of calcium nodules reduced, and the expression of osteoblast transcription factor Runx2 was down-regulated in the T2DM group compared with those in the control group (all P<0.01). The levels of DKK-1 protein and mRNA were up-regulated in the T2DM group, which were higher than those in the control group (both P<0.01). The levels of DKK-1 protein and mRNA were related to the increase of Runx2 (both P<0.01).
 Conclusion: The growth activity of BMSCs and the potential of osteogenic differentiation are attenuated in the T2DM rats, which may be related to the increase of DKK-1 expression in BMSCs.

Objective: To investigate the role of miR-144-3p in regulating osteogenic differentiation of bone marrow mesenchymal stem cells and predict its target genes., Methods: Rat bone marrow mesenchymal stem cells (BMSCs) with induced osteogenic differentiation were examined for the expressions of Runx2, OCN and miR-144-3p. The effects of transfection with a miR-144-3p mimic or a miR-144-3p inhibitor were tested on the osteogenic differentiation of the BMSCs. The changes in the expressions of the predicted target of miR-144-3p in the BMSCs during induced osteogenic differentiation were examined using Western blotting and qRT-PCR., Results: Rat BMSCs with induced differentiation into osteoblasts exhibited a progressive increase in the expressions of Runx2 and OCN (two markers of osteogenic differentiation), while the expression of miR-144-3p gradually decreased during the differentiation till reaching the lowest level at 21 days of induction. In rat BMSCs, transfection with the miR-144-3p mimic significantly decreased ALP activity ( P &lt; 0.05) wile transfection with the miR-144-3p inhibitor significantly increased ALP activity ( P &lt; 0.05) in rat BMSCs. Analysis based on miRanda, microRNA.org database and TargetScan suggested that Smad4 was the most likely target gene of miR-144-3p. The results of qRT-PCR showed no significant differences in expression levels of Smad4 among the cells with different treatments ( P &gt; 0.05), while Western blotting revealed a significantly decreased expression of Smad4 in the cells transfected with miR-144-3p mimics and an increased Smad4 expression in the cells transfected with the miR-144-3p inhibitor as compared with the control cells ( P &lt; 0.05)., Conclusions: miR-144-3p participates in the regulation of osteogenic differentiation of rat BMSCs, and its inhibitory effect on osteogenic differentiation is achieved probably by decreasing the expression level of Smad4.

Objective: To determine whether quercetin inhibits oxidized low-density lipoprotein (Ox-LDL)-induced osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) and understand the underlying mechanism., Methods: The calcification of human VSMCs following Ox-LDL treatment was assessed using alizarin red staining and by detecting ALP activity. The mRNA expressions of the bone-related genes including Msx2, BMP2 and Osterix, and the contractile proteins including SMA and SM22a were analyzed using qPCR. The effects of quercetin were investigated on OxLDL-induced VSMC calcification and changes in ALP activity, expressions of Msx2, BMP2, Osterix, SMA and SM22a, ROS levels and SOD activity. The effect of Toll like receptor 4 (TLR4) silencing mediated by siRNA transfection on cell calcification, ALP activity, gene expressions and ROS levels were investigated., Results: Ox-LDL treatment promoted VSMC calcification and up-regulated TLR4 expression. Quercetin treatment significantly attenuated Ox-LDL-induced VSMC calcification, reduced ALP activity, down-regulated the expression levels of Msx2, BMP2 and Osterix, and up-regulated the expressions of vascular smooth muscle contractile proteins SMA and SM22a. In addition, Quercetin treatment markedly increased SOD activity, reduced ROS levels and TLR4 expression in VSMCs. Silencing TLR4 expression using TLR4 siRNA also significantly decreased calcification of the VSMCs., Conclusions: Quercetin inhibits Ox-LDL-induced VSMC calcification in VSMCs possibly by targeting the ROS/TLR4 signaling pathway.

Objective: To explore the effect and mechanism of miR-21 down-regulated which was induced by H 2 O 2 on osteogenic differentiation of MC3T3-E1 cells., Methods: MC3T3-E1 cells were cultured and passaged, and the 7th generation cells were harvested to use in experiment. The MC3T3-E1 cells were treated with different concentrations (0, 40, 80, 160, and 320 μmol/L) of H 2 O 2 . The expression of miR-21 was detected by real-time quantitative PCR (RT-PCR) and the cell viability was determined by MTS. Then the appropriate concentration of H 2 O 2 was obtained. To analyze the effect of H 2 O 2 on osteogenic differentiation of MC3T3-E1 cells, the MC3T3-E1 cells were divided into blank control group (group A), H 2 O 2 group (group B), osteogenic induction group (group C), and H 2 O 2 +osteogenic induction group (group D). The expression of miR-21 and the osteogenesis related genes expressions of Runx2, osteopontin (OPN), and collagen type Ⅰ alpha 1 (Col1a1) were detected by RT-PCR. The expression of phosphatase and tensin homolog (PTEN) was detected by Western blot. The extracellular calcium deposition was detected by alizarin red staining. To analyze the effect on osteogenic differentiation of MC3T3-E1 cells after the transfection of miR-21 inhibitor and siRNA-PTEN, the MC3T3-E1 cells were divided into H 2 O 2 group (group A1), H 2 O 2 +osteogenic induction group (group B1), H 2 O 2 +osteogenic induction+miR-21 inhibitor group (group C1), and H 2 O 2 +osteogenic induction+miR-21 inhibitor negative control group (group D1); and H 2 O 2 group (group A2), H 2 O 2 +osteogenic induction group (group B2), H 2 O 2 +osteogenic induction+siRNA-PTEN negative control group (group C2), and H 2 O 2 +osteogenic induction+siRNA-PTEN group (group D2). The osteogenesis related genes were detected by RT-PCR and the extracellular calcium deposition was detected by alizarin red staining., Results: The results of MTS and RT-PCR showed that the appropriate concentration of H 2 O 2 was 160 μmol/L. The expression of miR-21 was significantly lower in group B than in group A at 1 and 2 weeks ( P <0.05). The expression of miR-21 was significantly lower in group D than in group C at 1 and 2 weeks ( P <0.05). The expression of PTEN protein was significantly lower in group C than in groups A and D ( P <0.05). The mRNA expressions of Runx2, OPN, and Col1a1 were significantly lower in group D than in group C at 1 and 2 weeks ( P <0.05). The extracellular calcium deposition in group D was obviously less than that in group C. The expression of PTEN protein was significantly higher in group C1 than in group D1 ( P <0.05). The mRNA expressions of Runx2 and OPN were significantly lower in group C1 than in groups B1 and D1 at 1 and 2 weeks ( P <0.05). The mRNA expression of Col1a1 was significantly lower in group C1 than in groups B1 and D1 at 2 weeks ( P <0.05). The extracellular calcium deposition in group C1 was obviously less than those in groups B1 and D1. The mRNA expressions of OPN and Col1a1 were significantly higher in group D2 than in groups B2 and C2 at 1 week ( P <0.05). The extracellular calcium deposition in group D2 was obviously more than those in groups B2 and C2., Conclusion: H 2 O 2 inhibits the osteogenic differentiation of MC3T3-E1 cells, which may be induced by down-regulating the expression of miR-21.

Objective: To investigate the effect of concentrated growth factor (CGF) on proliferation and differentiation in Beagle adipose-derived stem cells (ADSCs).
 Methods: ADSCs were isolated from adipose tissue of healthy Beagles and cultured. The multi-directional differentiation potential of ADSCs was identified. The ADSCs were assigned to a CGF group and a control group. The rate of proliferation was analyzed by CCK-8 assay. The osteogenic differentiation capability was detected by ALP staining after the osteoinduction. Bone formation-related gene expression was detected by RT-PCR.
 Results: CGF promoted the proliferation of ADSCs in vitro. ADSCs in the CGF group showed higher level of ALP activity than that in the control group (P<0.05). CGF stimulated the expression of the genes associated with osteogenesis, such as Col-I and Runx2. 
 Conclusion: CGF can promote the proliferation and osteogenic differentiation in ADSCs in vitro.

Objective: To investigate the effect of Wnt3a on osteogenic differentiation of human dental pulp stem cells (DPSC). Methods: DPSCs were subjected to different concentrations of Wnt3a (0, 5, 20, 50 and 100 μg/L) and at seven days after culture the alkaline phosphatase (ALP) activity was tested. Mineralized nodule formation was examined by alizarin red staining. Osteogenic-related gene expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type Ⅰ (COL-Ⅰ), Runt-related transcription factor-2 (RUNX2) was examined by quantitative real-time PCR (qPCR). Results: After seven days of induction by DPSC, Wnt3a protein could inhibit the ALP activity (concentration 0: 1.076±0.203, 5 μg/L: 0.828±0.118, 20 μg/L: 0.505±0.044, 50 μg/L: 0.499±0.038, 100 μg/L: 0.483±0.060). The expression of OCN in 5 μg/L Wnt3a group (0.092±0.005) was lower than that in culture medium (0.858±0.190)( P <0.05). Alizarin red staining showed that 5 μg/L Wnt3a had no mineralization induction effect on DPSC. Conclusions: Wnt3a could inhibit osteogenic differentiation of dental pulp stem cells.

Objective: To explore repairing results of VEGF165 gene modified adipose-derived stem cells for diabetic rats with bone defect., Methods: Seventy-eight male Wistar rats weighted 180 to 220 g were selected, 72 rats were established diabetic animal models by streptozotocin inducement method, blood glucose level was more than 16.7 mmol/L. Experimental animals were randomly divided into 5 groups, 6 rats in normal group and each 18 rats in other groups. VEGF165 gene modified adipose-derived stem cells were implanted into normal group with bone defect; single diabetic rats with bone defect were named as diabetic group;vascular endothelial growth factor implanted into single diabetic rats with bone defect named as growth factor group; adipose-derived stem cells implanted into diabetic rats with bone defect names as stem cell group; VEGF165 gene modified adipose-derived stem cells implanted diabetic rats with bone defect named as experimental group. After combination of VEGF165-ADSCs (5×106) cells combined with gel sponge, implanted into diabetic rats with bone defect. On the forth week, general form of defect repairing tissue were observed by optical microscopy;local density of micro-vessel were detected by immunohistochemistry method; content of Ca, P and ALP of repairing callus were detected by IRIS Intrepid XSP inductively coupled plasma emission spectrometer. Efficacy of the VEGF165-ADSCs repairing function was evaluated by SPSS statistic software., Results: Fluorescent staining results showed that expression of VEGF165 located on cytoplasm of ADSCs, expression percentage was more than 87%; general histology results showed that callus formation and quality was near to normal group, repairing results in diabetes group, growth factor group and stem cell group were poor. On the Forth week after implantation, content of Ca, P and ALP of repairing callus in experimental group were higher than those in growth group and stem cell group, and without significant differences compared with normal group; blood vessel density in experimental group was lower than normal group, but higher than other groups., Conclusions: VEGF165 gene modified adipose-derived stem cells for repairing diabetic rats with bone defect has advantages of osteogenesis and angiogenesis, and should be one of the effective method for repairing diabetic rats with bone defect., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Objective: To build scaffold materials with different concentrations of alginate and collagen, and to observe the effects of alginate/collagen ratio on the proliferation of human adipose-derived mesenchymal stem cells (hAMSC) and osteogenic differentiation. The optimal concentration of alginate/collagen will be chosen for constructing hydrogel that will be used for bone tissue engineering. Methods: Soluble hydrogel scaffold materials containing alginate/collagen were prepared, and the following groups were established based on different alginate/collagen ratio: 4∶1 (group A), 2∶1 (group B), and 1∶1 (group C). Cell proliferation on the material surface was observed using the cell counting kit-8 (CCK-8) assay, while cell viability in each material group were observed using live/dead staining. Quantitative real-time PCR(qPCR) was used to measure the differential expression of osteogenesis-related genes on and in the materials. Immunofluorescence staining was used to measure the differential gene expression of osteogenesis-related proteins in each group. Results: The results from the CCK-8 assay showed increasing cell proliferation rate on the lyophilized hydrogel material surface as the collagen concentration increased, and the highest cell proliferation was observed in group C. Live/dead staining assay indicated that cells were able to proliferate in all three types of hydrogel materials, and the highest cell viability was found in material from group B ([87.50±2.65]%). qPCR showed that the expression of osteogenesis-related genes in group C was the highest, among the three groups, while the expression of osteocalcin in group B was significantly higher than those in the other two groups ( P< 0.05). Immunofluorescence staining was carried out for osteocalcin on and in the hydrogel material and the results were consistent with that of qPCR. Conclusions: The alginate/collagen scaffold materials did not show adverse effects on the cell proliferation of hAMSC and osteogenenic differentiation. Bone tissue engineering can use 10% hydrogel material, and when the sodium alginate and collagen have a ratio of 2∶1, the hydrogel can be conducive to cell differentiation and proliferation.

Objective: To evaluate the adhesion, proliferation, alkaline phosphatase (ALP) activity and the expression of osteogenesis-related genes and osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) of osteoblast-like cells on a type of near β-type titanium alloys (Ti-5Zr-3Sn-5Mo-15Nb, TLM) surfaces modified by the double glow plasma nitriding technology, and to investigate the effect of the modified surfaces on the initial functions of osteoblast-like cells. Methods: The surfaces of TLM were modified by the double glow plasma nitriding technology. TLM surfaces without modification were used as control. Cell morphology was observed with scanning electron microscopy (SEM). Methyl thiazolyl tetrazolium (MTT) method was used to measure cell proliferation. Cell ALP activity was evaluated by using reagent kits. The mRNA expression of Runt-related transcription factor-2 (RUNX2), typeⅠcollagen alpha 1 chain (COLⅠ α1) and OPG/RANKL were examined by quantitative real-time PCR(qRT-PCR). Results: Four hour following cell alture, cells on modified surfaces extend filopodia and intercellular junction was tight. Three days later, cell proliferation (0.277±0.007) was significantly higher than that in control group (0.249±0.004) ( P< 0.01). After two weeks, ALP activity on TLM modified layer (173.6±1.89) was significantly higher than that on unmodified TLM (162.6±2.4) ( P< 0.01). The mRNA expression of osteoblast marker RUNX2, COLⅠα1 were stronger than that in control group ( P< 0.05). The expression of OPG mRNA was higher than that in control group ( P< 0.01), and RANKL mRNA expression was significantly lower than that in control group ( P< 0.05). Conclusions: The TLM surface modified by the double glow plasma nitriding technology has a positive effect on osteoblasts initial adhesion, proliferation and differentiation, and it can also improve expression of OPG mRNA and has an inhibitory effect on RANKL mRNA expression of osteoblasts.

Objective: To investigate whether mechanical stretch stimulation affects the expression of the immediate early gene c-fos mRNA in rat Achilles-derived tendon stem cells (TSCs) in vitro ., Methods: TSCs were isolated from the Achilles tendons of 8 weeks old male Sprague Dawley rats by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated by a uniaxial cyclic stretching loading system under the condition of 1 Hz, respectively with 4% or 8% stretch intensity for 0, 5, 15, 30, 60, and 120 minutes. At each time point, TSCs were collected to detect c-fos mRNA expressions and to find the best time-point T max by real-time fluorescence quantitative PCR. Then, TSCs were simulated with 2%, 4%, 6%, 8%, or 12% stretch intensity for T max to observe the relative expressions of c-fos mRNA under different stretch intensities. Next, TSCs were stretched for 0, 5, or 15 minutes respectively and followed by incubation at relax status up to T max to observe the changes of c-fos mRNA expressions after short period stimulation. Finally, TSCs were stimulated with 4% or 8% stretch intensity respectively for 0, T max , or 120 minutes to detect the expressions of the tenogenic differentiation related genes [collagen type I, tenomodulin (TNMD)], the osteogenic differentiation related genes [runt related transcription factor 2 (Runx2), distal-less homeobox 5 (Dlx5)], and the adipogenic differentiation related gene [fatty acid binding protein 4 (FABP4)]., Results: Under 4% or 8% stretch intensity, the relative expressions of c-fos mRNA significantly increased at 15 minutes ( P <0.05), reached the maximum at 30 minutes ( P <0.05), and returned to baseline at 60 minutes ( P >0.05) when compared with expression at 0 minute. Therefore, T max was 30 minutes. The stretch intensity of 2% was enough to cause the expression of c-fos mRNA at 30 minutes, and the expression was significantly higher under the stretch intensity of 6%, 8%, and 12% than 2% and 4% ( P <0.05). Even for a short period stimulation of 5 minutes, c-fos mRNA expression could still significantly increase at 30 minutes ( P <0.05). The relative expressions of differentiation related genes at 30 and 120 minutes showed no significant difference when compared with the expression at 0 minute under 4% stretch intensity ( P >0.05); but the relative expression of Runx2 gene significantly increased at 30 minutes, and the relative expressions of collagen type I, TNMD, Dlx5, and Runx2 increased at 120 minutes under 8% stretch intensity ( P <0.05)., Conclusion: Mechanical stretch stimulation can affect the relative expression of the immediate early gene c-fos mRNA of rat Achilles-derived tendon stem cells in vitro , and there is time- and intensity-dependence. It is suggested that the mechanical stimulation with different time or intensity may affect the differentiation of TSCs at early stage. This study is meaningful for the further study on TSCs intracellular mechanical signal transfer mechanism.

Objective To construct a recombinant adenovirus expressing siRNA targeting human sclerostin (SOST) gene, and test the function of MG-63 cells while co-cultured with MDA-MB-231 cells infected by Ad-siSOST. MethodsAccording to the RNA sequence of SOST gene, two pairs of primers which contained 3 siRNA sequences were designed, and a pB2B plasmid was taken as template to amplify 2 DNA sequences. Both of the 2 DNA sequences were ligated to pAdTrace-OK by Gibson DNA Assembly way. After homologous recombination between recombinant shuttle plasmid and adenovirus vector plasmid, the adenovirus was packaged in HEK-293 cells. PCR and ELISA were used to test the expression of SOST in MDA-MB-231 cells which were infected with Ad-siSOST. In the co-culture system of MG-63 cells and MDA-MB-231 cells infected Ad-siSOST, osteoprotegerin (OPG), osteocalcin (OCN), integrin binding sialoprotein (IBSP) and receptor activator of nuclear factor-κB ligand (RANKL) were tested by quantitative real-time PCR. Results Recombinant shuttle plasmid which contained 3 interfering fragments was constructed successfully, and Ad-siSOST was obtained after homologous recombination and packaging. SOST expression in MDA-MB-231 cells was downregulated significantly after infeceted with Ad-siSOST. The mRNA levels of OPG, OCN, IBSP in MG-63 cells increased significantly, while the level of RANKL mRNA decreased significantly, and all 4 gene expressions were reversed after the infection of Ad-siSOST. Conclusion Knockdown of SOST in MG-63 cells increases osteogenesis and ratio of OPG/RANKL in vitro.

Objective: To study the effect of nano hydroxyapatite on human adipose-derived mesenchymal stem cells(hASCs) mixture 3D bio-printing for cells' proliferation and osteogenesis., Methods: P5 hASCs were used as seed cells, 10 g/L nano hydroxyapatite was added into the cell-sodium alginate-gelatin mixture (concentration: 20 g/L sodium alginate, 80 g/L gelatin; cell density: 1×10 6 /mL), then the mixture was printed by 3D bio-printer as the experimental group. And the cell-sodium alginate-gelatin mixture without nano hydroxyapatite was printed as the control group. Respectively, both the experimental and control groups were detected by microscope, CCK-8, Western blot and PCR at certain time pointsafter being printed, whose cells' proliferation and osteogenic differentiation were analyzed., Results: The microscopic observation and CCK-8 results showed that the cells of the experimental group and the control group both had a good proliferation 24 h and 7 d after being printed. The Western blot results showed that 14 d after printing, the expression of Runt-related transcription factor 2 (RUNX2) had no statistical difference between the experimental group and control group. The PCR results showed that 14 d after printing, the expression of osteogenesis-related genes (RUNX2, osterix, and osteocalcin) was significantly higher in the experimental group than in the control group., Conclusion: Nano hydroxyapatite can increase osteogenic differentiation of the hASCs mixture after bio-printing, in which the cells still have a good proliferation.

Objective: To explore the effect of H 2 O 2 -actived RAW264.7 macrophages on the migration, proliferation, and osteogenesis gene expression of MC3T3-E1 in mice., Methods: MC3T3-E1 cells and RAW264.7 cells were cultured to the 7th generation. RAW264.7 macrophages were stimulated with 0, 25, 50 and 100 μmol/L H 2 O 2 , the cell proliferation rate was detected by MTS at 1, 3, and 6 hours after stimulated, and superoxide dismutase (SOD) content by SOD assay kit at 1 hour after stimulated. The appropriate concentration and action time of H 2 O 2 -actived RAW264.7 were obtained. The supernatant of RAW264.7 macrophages stimulated by H 2 O 2 or not was collected at 24 hours. Then, the supernatant was used to culture MC3T3-E1 cells in groups B (not stimulated by H 2 O 2 ) and C (stimulated by H 2 O 2 ), and DMEM was used as a control in group A. The migration of MC3T3-E1 cells was detected at 12 and 24 hours by cell scratch test, the proliferation of MC3T3-E1 cells at 24, 48, and 72 hours by MTS assay. MC3T3-E1 cells were cultured with only complete medium in blank control group, with complete medium containing 50 μg/mL vitamin C + 10 nmol/L β sodium glycerophosphate in positive group, normal control group (adding the supernatant not stimulated by H 2 O 2 ), and experimental group (adding the supernatant stimulated by H 2 O 2 ). At 3, 7, and 14 days, RT-PCR was used to determine the osteogenesis related mRNA expressions of alkaline phosphatase (ALP), Runx2, osteopontin (OPN), osteocalcin (OC), bone sialoprotein (BSP), and collagen type I (COL-I)., Results: The results of MTS and SOD assay showed that the appropriate concentration and action time of H 2 O 2 -actived RAW264.7 macrophages were 25 μmol/L and 1 hour, respectively. MTS assay showed that the proliferation rate of MC3T3-E1 cells was significant higher in groups B and C than group A ( P <0.05), in group B than group C, and significant difference was shown between groups at 2 and 3 days ( P <0.05). The cell scratch test indicated that the migration of MC3T3-E1 cells was significantly faster in groups B and C than group A, and in group C than group B at 12 hours ( P <0.05); many migrated cells were observed in all scratch sites of groups B and C at 24 hours. When compared with positive control group, the mRNA expressions of ALP, Runx2, OC and BSP in experimental group were significantly down-regulated at 7 and 14 days ( P <0.05). When compared blank control group, the mRNA expressions of OPN and COL-I in experimental group were significantly down-regulated at 7 and 14 days ( P <0.05)., Conclusions: The appropriate concentration and action time of H 2 O 2 -actived RAW264.7 macrophages are 25 μmol/L and 1 hour. The H 2 O 2 -actived RAW264.7 cells can promote MC3T3-E1 cells migration, and suppress MC3T3-E1 cells proliferation and expressions of osteogenesis related genes.

Objective: To investigate the effect of miR-705 on osteogenic differentiation of mouse embryo osteoblast precursor (MC3T3-E1) cells. Methods: miR-705 mimics, inhibitors and negative control were transfected into MC3T3-E1 cells. Alkaline phosphates (ALP) staining were performed and quantified after 7 days of osteogenic medium induction. The mRNA and protein expression levels of runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected by real-time RT-PCR and Western blot after 14 days of osteogenic induction. Alizarin red staining was performed and quantified in MC3T3-E1 cells after 21 days of osteogenic induction. Results: After 7 days of osteogenic induction, ALP staining showed that overexpression of miR-705 significantly reduced ALP activity, whereas knockdown of miR-705 increased ALP activity (all P <0.05). Consistently, after 14 days of osteogenic induction, mRNA and protein expressions of Runx2 and OCN were suppressed by overexpression of miR-705, whereas they were promoted by knockdown of miR-705 (all P <0.05). After 21 days of osteogenic induction, alizarin red staining showed that overexpression of miR-705 significantly reduced the formation of mineralized node, the opposite results were found in miR-705 knockdown group (all P <0.05). Conclusion: miR-705 can inhibit the osteogenic differentiation of MC3T3-E1 cells.

Objective: To study the genic and non-genic regulation of 50 Hz 0.6 mT pulsed electromagnenic fields (PEMF) on rat calvarial osteoblasts (ROB) differentiation. Methods: ROBs were achieved by enzyme digestion, and treated with 50 Hz 0.6 mT PEMFs for 1.5 hours after subculture. The alkaline phosphatase (ALP) activity, mRNA transcription of ALP, Runx2 and OSX and protein expression of Runx2 and OSX were detected at 0, 3, 6, 9 and 12 hours after PEMF treatment. Results: The ALP activity at 3 hours after treatment was significantly higher than that in the control( P <0.01), while the mRNA transcription of ALP began to increase at 6 hours after treatment. The mRNA transcription of Runx2 increased immediately after treatment and regressed at 6 hours, then increased again. The protein expression of it corresponded but with a little lag. The mRNA transcription of OSX also raised instantly after treatment, then returned to the level of control at 6 hours, and lower than control at 12 hours significantly. The protein expression of it also corresponded but with a bit delay. Conclusions: There are genic regulation for the protein expression of Runx2 and OSX, and non-genic regulation for the ALP activity on the process of 50 Hz 0.6 mT PEMFs prompts ROBs differentiation.

Objective: To compare the expression of histone deacetylase(HDAC)1-11 of human periodontal ligament stem cells(PDLSC)in normal and inflammatory microenvironments, and to investigate the effect of histone deacetylase inhibitor trichostatin A(TSA)on the osteogenic differentiation potential of PDLSC in inflammatory microenvironment induced by tumor necrosis factor-α(TNF-α)stimulation., Methods: PDLSC were isolated from periodontal ligament tissues obtained from the surgically extracted human teeth and cultured by single-colony selection. The expression of HDAC1-11 in cells with or without TNF-α(10 μg/L)stimulation was evaluated by quantitative real time-PCR(RT-PCR). The effect of TSA on cell proliferation was investigated by methyl thiazolyl tetrazolium(MTT)assay. The influence of TSA on osteogenic differentiation of PDLSC in inflammatory microenvironment with TNF-α stimulation was assessed by alizarin red staining, quantitative RT-PCR and Western blotting, respectively., Results: The expression of HDAC in PDLSC with TNF-α stimulation was significantly higher than that in normal PDLSC(P<0.05)(except HDAC7, P=0.243). TSA had no significant effect on PDLSC proliferation at the concentration of 50 nmol/L(P=0.232). The alizarin red staining showed that PDLSC in TNF-α group generated less mineralized nodule than the control group, while the cell matrix mineralization in TSA group was improved obviously. TNF-α had an inhibitory effect on the expression of osteogenesis related genes, runt-related transcription factor-2(RUNX2)and alkaline phosphatase(ALP), with relative gene expression ratio(experimental/control)decreased to 0.17 ± 0.02 and 0.32 ± 0.03, while TSA could significantly increase the genes' expression to 0.67±0.03 and 0.89±0.02(P<0.01). Western blotting test showed that in TNF-α group the expression of osteogenesis related proteins was obviously reduced, and compared with the TNF-α group, TSA could significantly promote the expression of proteinsin inflammatory microenvironment., Conclusions: PDLSC in inflammatory microenvironment by TNF-α stimulation had a higher expression of HDAC than that in normal conditions. TSA, as a histone deacetylase inhibitor, could significantly promote the osteogenic differentiation potential of PDLSC in inflammatory microenvironment by suppressing HDAC.

Objective: This work investigated mesenchymal stem cells (MSCs) modified with Runt-related transcription factor 2 (Runx2) therapy for bone regeneration in rabbit mandibular distraction osteogenesis., Methods: Forty-eight New Zealand mature white rabbits were randomly divided into three groups after the rabbit model of mandibular distraction osteogenesis was established: reconstruction plasmid modified with Runx2 (group A), plasmid without Runx2 (group B), and the same dose of saline as control (group C). At the fifth day of distraction phase, MSCs with reconstruction plasmid modified with adv-hRunx2-gfp were injected into the distraction gap of group A. MSCs with reconstruction plasmid modified with adv-gfp was injected into the distraction gap of group B, whereas group C was injected with the same dose of saline. At 8 weeks after injection, all animals were sacrificed, and the distracted mandibles were harvested. The general imaging histological observation and three-point bending test were used for evaluation., Results: CT plain scan and histological analysis confirmed that the amount of new bone forming in the distraction gap of group A was significantly higher than those in groups B and C. Dual-energy X ray and three-point bending test results also showed that the bone mineral density, bone mineral content, and maximum load of the distraction gap of group A were significantly higher than those of groups B and C (P<0.01)., Conclusion: Runx2-ex vivo gene therapy based on MSCs can effectively promote the bone regeneration in rabbit mandibular distraction osteogenesis and shorten the stationary phase. Therefore, reconstruction of craniofacial fracture would be a valuable strategy