Objective： To investigate the effect of miR-34a on osteogenic differentiation of hOBMSCs. Methods： Real-time quantitative RT-PCR was used to detect the expression of miR-34a/miR-34b/miR-34c in hOBMSCs during osteogenic induction. The effect of miR-34a on osteogenic differentiation of hOBMSCs were detected by alkaline phosphatase staining and alizarin red staining. The regulation role of miR-34a on the expression of osteogenic markers (RUNX2, OSX, OPN, OCN, COL1A1) during osteogenic induction and the effect of miR-34a on DKK1 protein expression under normal cell culture conditions were detected by westernblot. The target relationship of miR-34a and DKK1 was measured by dual luciferase reporter assay. The effect of miR-34a on osteogenesis of hOBMSCs was examined by subcutaneous osteogenesis in nude mice. Results： MiR-34a was demonstrated to be upregulated during the osteogenic differentiation of hOBMSCs. Overexpression of miR-34a significantly increased alkaline phosphatase activity, mineralization capacity, and the expression of osteogenesis-associated genes in hOBMSCs in vitro. Further investigations revealed that miR-34a inhibited the expression of DKK1 and reduced the luciferase activity of reporter gene construct comprising putative miR-34a binding sites in the 3′-UTR of DKK1. In vivo experiments also confirmed that miR-34a can promote osteogenic differentiation of hOBMSCs. Conclusions： MiR-34a promoted hOBNSCs osteogenic differentiation via down-regulating the expression of DKK1.

Objective： To explore the effects of SB431542, a TGF-β signaling pathway inhibitor, on the growing status and osteogenic differentiation of human gingival mesenchymal stem cells(hGMSCs) in vitro . Methods：The hGMSCs were isolated and cultured from clinically discarded gingival tissues. The surface markers of hGMSCs at passage 3 were by flow cytometry. Then the hGMSCs were treated with various concentrations of SB435142 (0, 0.1, 1, and 10 μmol/L) for the following detections. The proliferation and apoptosis of cells were analyzed using Cell Counting Kit-8 assay and Apoptosis Detection Kit, respectively. The osteogenic differentiation of hGMSCs was investigated using Alizarin Red staining after induced with osteogenic induction medium for 21 days. In addition, osteoblastic differentiation-related protein and downstream molecules of TGF-β signaling pathway in hGMSCs were measured by Western Blot analysis during osteogenic differentiation at the presence of SB431542(1μmol/L) or not. Results：The hGMSCs at passage 3 were positive for the typical MSC marker proteins CD105（99.8%）、CD90（100%）、and CD73（98.1%）,but negative for hematopoietic stem cell markers or macrophage and B-cells makers CD14（0.3%）、CD34（0.4%）、CD19（0.7%）、CD45（0.2%）、and HLA-DR（1.4%）. Apoptosis rates of the groups with SB431542 treatment (0.1,1, and 10μmol/L) were not statistically different with the control group（P＞0.05）.But the CCK-8 results showed that the proliferation of hGMSCs was inhibited in 10μmol/L SB431542 group, compared with control group at day 7(P < 0.5). After osteogenic induction for 21 days, 1μmol/L SB431542 treatment dramatically accelerated the calcified nodule formation compared to the control group（P < 0.5）.The collagen type I (COL-1), alkaline phosphatase（ALP）and runt-related transcription factor 2 (Runx2) protein expressions were remarkedly higher in SB431542 treatment group than that in control group after induced by osteogenic medium for 11 days（P < 0.5）. In addition, SB431542 treatment markedly inhibited the Smad3 phosphorylation in hGMSCs induced by osteogenic differentiation medium at 30 and 60 minutes（P < 0.5）. Conclusions：Treatment with 1μmol/L SB431542 could induce a robust osteogenic differentiation in hGMSCs in vitro, which may be regulated by inhibiting the phosphorylation level of Smad3 during osteogenic differentiation, indicating it is a great potential approach for repair and reconstruction of bone defect in future clinical application.

[Abstract]Objective: Polycaprolactone/type I collagen/fluoroapatite composite scaffolds were prepared for periodontal tissue regeneration and biocompatibility and osteogenic induction were tested. Methods: Polycaprolactone/type I collagen electrospun nanofiber membrane was prepared, and the fluoroapatite mineralized coating was synthesized on the surface. The morphology was observed by scanning electron microscopy. At the same time, human periodontal ligament cells were isolated and cultured on the surface of the scaffold, and the cell proliferation was detected by CCK-8 method. The effects of composite scaffold on osteogenic differentiation of human periodontal ligament cells were observed by alkaline phosphatase staining, alizarin red staining and Von Kossa staining.Results: The polycaprolactone/type I collagen/fluoroapatite composite scaffold showed a three-dimensional network structure, and the fluoroapatite crystals were distributed on the surface of the scaffold. The CCK-8 results showed that the human periodontal ligament cells proliferated well on the composite scaffold. Alkaline phosphatase staining, alizarin red staining and Von Kossa staining results suggest that the composite scaffold can induce osteogenic differentiation of human periodontal ligament cells. Conclusion: The polycaprolactone/type I collagen/fluoroapatite composite scaffold has good biocompatibility and osteogenic induction potential, and it is expected to be used as a scaffold material for periodontal tissue engineering.

[Abstract] Objective：This study aimed to prepare monoclonal antibodies against human amelogenin and characterize their immunological properties. Methods：BALB/c mice were immunized with the recombinant full-length human amelogenin expressed in prokaryotic expression system. The spleen cells of immunized mice were fused with mouse myeloma SP2/0 cells, and hybridoma cell lines capable of stably secreting monoclonal antibodies were screened out by the limited dilution method and indirect enzyme-linked immunosorbent assay (ELISA) method. Ascites were produced in mice, and then purified by saturated ammonium sulfate precipitation and protein G affinity chromatography. Results：Three hybridoma cell lines stably secreting monoclonal antibodies against human amelogenin were screened and named as H10G1, H3G1 and G5H3. The ascites titers were 1:243000, 1:729000, 1:729000, and the titers of monoclonal antibody after purification was 1:81000、1:243000、1:729000, respectively. The results of Western blotting showed that all of 3 monoclonal antibodies recognize the 45-149 amino acid of human amelogenin. Conclusions：The specific monoclonal antibody against human amelogenin was successfully prepared for further study on the amelogenin in root development and periodontal regeneration.

Abstract: Objective: To evaluate the antibacterial activity of ozonated oil on Porphyromonas gingivalis (P. gingivalis). Methods: Agar diffusion assay was used to investigate the sensitivity of P. gingivalis to ozonated oil. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution method. The effects of ozonated oil on bacterial viability, biofilm structure and cell morphology were observed by Laser confocal microscopy (CLSM) and scanning electron microscopy (SEM). Results: Ozonated oil significantly inhibited the growth of P. gingivalis with the inhibition diameter of 68.13 mm. And the MIC and MBC of ozonated oil on P. gingivalis were 0.04 mg/mL. CLSM and SEM images confirmed that after ozonated oil treatment, no dense biofilm structure was observed, the viability and density of biofilm decreased, the intact cell shape was damaged and the cells shrink and lysed. Conclusions: Ozonated oil had a strong inhibitory effect on planktonic and biofilm forms of P. gingivalis.

Objective：To investigate the effect of D-leucine on biofilm formation by Streptococcus mutans.Methods：The Spectrophotometer was used to evaluate the growth curve of Streptococcus mutans exposed to 20.0 mmol/LD-leucine. Construct Streptococcus mutans biofilm model in vitro. Biofilms were formed with 2.5, 5.0, 10.0 and 20.0 mmol/LD-leucine, MTT assay method was used to compare the biofilm formation quantificationally. Confocal laser scanning microscope (CLSM) was applied to observe the biofilm structure after treated with different concentrations of D-leucine. Results：D-leucinedid not affect the growth curves of planktonic S. mutanswithin 24 h. The growth curves attained exponential phase after 3 h andreached stationary phase at 12 h. The quantity of biofilm formed by Streptococcus mutanssignificantlydecreased (P<0.05) and the structure of the biofilm showed by CLSM became thinner with the increase of D-leucine concentration.Conclusions：D-leucinecan inhibit the biofilm formation of Streptococcus mutans.

[Abstract] Objective: The aim of this study was to compare the efficacy of different irrigation techniques on the removal of intracanal medicaments.Methods: 90 extrated single-rooted premolars were decoronated to obtain a standardized root length of 13 mm.The roots were split longitudinally into two halves and an artificial groove was prepared in the apical part of one segment.The triple antibiotic paste and calcium hydroxide were placed into the grooves.Two weeks later,the root canals were then irrigated with 10ml 1% NaOCl by three irrigation techiniques（ conventional syringe irrigation ,vibringe sonic irrigation and passive ultrasonic irrigation）.The amounts of intrcanal medicaments remaining in the grooves were scored with a stereomicroscope ,and the data were evaluated statistically.Results:Vibringe sonic irrigation and passive ultrasonic irrigation with 1% NaOCl removed significantly more triple antibiotic paste than the syringe irrigation(p<0.01).The syringe irrigation was associated with significantly more remaining calcium hydroxide than passive ultrasonic irrigation(p<0.01). There were no significant differences between vibringe sonic irrigation and the other two methods(vibringe sonic irrigation and syringe irrigation) on removing the calcium hydroxide.The groups of two medicaments with the same irrigation technique did not have significant differences.Conclusions:All the irrigation techniques cannot completely remove the intracanal medicaments.The use of vibringe sonic root canal irrigator and ultrasonic system may improve the efficacy of irrigation on the removal of intracanal medicaments.

Objective：To study the difference of cement residual and adhesion in the conventional coating method and extra-oral cementation method for single zirconia crown supported by dental implants with different thickness of cement. Methods: The Ankylos standard type B 6mm implant abutment analog was scanned by the Sinora CEREC system and the 3D digital model of zirconia single crown with the thickness of 50m, 80m and 110m of the cement layer and 1mm of the thickness was designed. The zirconia single crown was cut and sintered by CAD/CAM of Sinora CEREC system. The zirconia crowns with the different thickness of cement layer was cemented on the Ankylos standard type B 6mm implant abutment analogs by traditional coating method and extra-oral cementation method with the resin reinforced glass ionic cement. The residual of cement in the artificial gingiva was observed in each group, and the adhesion of two methods in each group was detected respectively. Results: There was significant difference in the adhesion between the group with the cement layer thickness of 50 m and the other groups. However, there was no significant difference in the adhesion between the group with the cement layer thickness of 80 m and 110 m. The adhesion of the cement layer thickness of 110 m group of adhesion is significantly less than the other groups by the conventional coating method. There is no significant difference in adhesion between the cement layer thickness of 80 m and 50 m groups. The residual cement around the implant and in the artificial gingiva of the three kinds of zirconia single crowns with three different cement thickness layers cemented by conventional coating method were more than that by the extra-oral cementation method. Conclusion: The zirconia single crown was cemented by extra-oral method with the resin reinforced glass ionic cement was better to be with 50~80 m cement thickness, while by conventional coating method was better to be with 80~110 m cement thickness .

Non-syndromic cleft lip and palate is a common congenital malformation. With the development of genome-wide association research, the genetic research on non-syndromic cleft lip and palate has made a lot of achievements, and a large number of candidate mutation sites have been found. However, most of these mutations are located in the non-coding region of susceptible genes and only have statistical significance. Further functional studies are needed to verify the true pathogenicity of these mutations. At present, the research method of coding region variation of cleft lip and palate has been more mature, but because of the difference of pathogenic mechanism, this method will not be fully applicable to study the variation of non-coding region. Therefore, this paper will introduce the following functional studies of non-coding region mutation from the aspects of big data utilization, software function prediction and functional experiment in vivo and in vitro, and provide reference for more studies on non-coding region mutation of cleft lip and palate in the future.

Wnt signaling pathway regulates growth and development, which is closely related to the development of various tissues and organs. Moreover, it is also related to the development of inflammation, wound healing and tissue regeneration. This article reviews the composition of Wnt signaling pathway and its roles in the development of periodontal tissues and periodontal diseases.

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) is a stable remineralization system of calcium-phosphorus derived from milk, which can be used in the minimally invasive treatment of early enamel caries. CPP-ACP can be utilized with fluoride. However, the synergy of CPP-ACP and fluoride is still controversial.

Adenoid cystic carcinoma is a very rare malignant tumor, accounting for 1% of head and neck malignancies, and approximately 10% of salivary tumors are adenoid cystic carcinoma. Adenoid cystic carcinoma has a slow growth process, but a high propensity for perineural invasion. Surgery is regarded as the main treatment for the disease. However, due to the nature of perineural invasion, it is difficult to obtain a clear surgical margin, resulting in postoperative recurrence and unfavorable prognosis. Therefore, it is particularly urgent to study the mechanism of perineural invasion systematically and comprehensively. In recent years, many researches on salivary adenoid cystic carcinoma have focused on the molecular mechanism of perineural invasion. This paper reviews the research progresses of neurotrophic factors, such as nerve growth factors, brain derived neurotrophic factor, glial cell derived neurotrophic factors and neurotrophic factor-3.