Objective: To investigate the effect of calcium hydroxide paste extract on the osteogenic differentiation of rat BMMSCs, and to provide theoretical basis for the clinical use of calcium hydroxide paste. Methods: Rat BMMSCs were isolated and cultured by whole bone marrow adherent method, and identified by flow cytometry. After induced by different conditions, Alizarin red staining, oil red O staining and alcian blue staining were used to identify the osteogenic, adipogenic and chondrogenic differentiation ability of stem cells. Calcium hydroxide paste was dried and ground into powder, then calcium hydroxide paste extract and calcium hydroxide paste conditioned medium (CH) were prepared. After being cultured with different concentrations of CH (0, 0.02, 0.2, 1, 1.5, 2 g/L)， ALP activity was measured to screen the best concentration for subsequent experiments. ALP staining, ALP activity assay and alizarin red staining were used to detect the changes in ALP activity and mineralization ability after CH induction. Western blot was used to detect the expression levels of COL1A1, ALP, RUNX2, and OSX. Results: Rat BMMSCs were successfully isolated and the cells had the characteristics of mesenchymal stem cells such as osteogenic, adipogenic and chondrogenic differentiation. 0.2 g/L was the optimal concentration of CH in inducing ALP activity of rat BMMSCs（P＜0.01）. The expressions of COL1A1, ALP, RUNX2 and OSX were up-regulated after the cells were treated with 0.2 g/L CH（P＜0.01）. Conclusion: Calcium hydroxide paste extract can potentially promote the osteogenic capacity of rat BMMSCs.

Objective: To explore the differences in inflammatory responses between oral polymorphonuclear neutrophil (oPMN) and circulating polymorphonuclear neutrophil (cPMN), and the roles of oPMN in maintaining periodontal health. Methods：cPMN was obtained from peripheral venous blood of healthy individuals by density gradient centrifugation, while oPMN was collected from oral rinses of healthy subjects by centrifugal filtration. Both of PMNs were stimulated with 1μg/ml Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) and Escherichia coli (E. coli) LPS was employed as a positive control. Apoptosis levels and phagocytosis of P. gingivalis or E. coli by two PMNs were assessed by flow cytometry. Moreover, after IL-8 stimulation, chemotactic responses of the cells were determined by a transwell migration assay. Production of interleukin (IL)-1β, IL-8 and IL-10 were analyzed by ELISA. Formation of neutrophil extracellular traps (NETs) was observed by immunofluorescence staining and levels of extracellular DNA were quantified by Sytox Green fluorescence staining. Furthermore, phosphorylation of mitogen?activated protein kinase1/2 (MEK1/2), extracellular regulated protein kinases1/2 (ERK1/2) and nuclear factor kappa-B (NF-κB) were measured by Western blot. Results：After P. gingivalis/E. coli LPS stimulation, higher levels of apoptosis in oPMN were disclosed compared with cPMN (p<0.05). No significant difference was confirmed in chemotactic ability of the two PMNs after IL-8 stimulation (p>0.05). Expression levels of IL-1β, IL-8 and IL-10 in oPMN were higher compared with cPMN (p<0.05) and more quantities of P. gingivalis or E. coli were phagocytized by oPMN than cPMN (p<0.05). Moreover, more quantities of NETs, higher levels of extracellular DNA, and greater phosphorylation levels of MEK1/2, ERK1/2 and NF-κB P65 were revealed in oPMN compared with cPMN (p<0.05). Conclusion：oPMN might be an overactive phenotype of neutrophil with increased capacities of phagocytosis, NET formation and cytokine secretion compared to cPMN, which might be associated with the increased phosphorylation of MEK1/2, ERK1/2 and NF-κB.

Objective:?To investigate the effect of inhibiting miR-145 on inflammatory response in experimental peri-implantitis in mice. Methods:?Forty male C57BL/6J mice were randomly divided into four groups: normal control group, peri-implantitis control group, peri-implantitis miR-NC group, and peri-implantitis miR-inhibitor group. All groups received dental implant insertion in maxillae, then the three peri-implantitis groups received ligation around implants to induce peri-implantitis. Afterwards, miR-145 inhibitor was injected into peri-implant gingiva in peri-implantitis miR-inhibitor group, and its negative control reagent was injected in peri-implantitis miR-NC group. Peri-implant bone loss in each group was detected using micro-computer tomography, and infiltration of inflammatory cells around the implants was examined using hematoxylin and eosin staining. Expression levels of miR-145, C-C motif chemokine ligand 2 (CCL2), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 10 (IL-10) and matrix metalloproteinase 8 (MMP8) in peri-implant gingival tissues were detected using real-time quantitative PCR. Results:?Compared with normal control group, bone loss and inflammatory cell infiltration in all peri-implantitis groups were greater (P<0.05), and the expressions of inflammation-related factors CCL2, TNF-α, IL-1β, IL-10 and MMP8 were increased (P<0.05). Upon miR-145 inhibition, the bone loss and inflammatory cell infiltration were ameliorated, and the expression of inflammation-related factors CCL2, TNF-α, IL-1β and MMP8 was decreased, while the expression of IL-10 enhanced (P<0.05). Conclusions:?Inhibition of miR-145 expression could suppress peri-implantitis, which may be associated with the regulationof inflammation-related factor expression in peri-implant gingival tissues.

Objective:To study the inhibitory effect of berberine (BBR) on Porphyromonas gingivali (Pg) and explore its mechanism. Methods:The minimum inhibitory concentration (MIC) of BBR on the inhibition of standard strains Pg ATCC33277 and hypervirulent strains Pg W83 was detected by concentration gradient dilution method. The minimum bactericidal concentration (MBC) of BBR on Pg ATCC33277 and Pg W83 was detected by blood plate experiment. The effect of BBR on bacterial activity of Pg ATCC33277 was studied by MTT. The effect of BBR on the cell walls of Pg ATCC33277 was determined by alkaline phosphatase activity. The effect of BBR on the morphology of Pg ATCC33277 was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Results:The MIC and MBC of BBR for Pg ATCC33277 and Pg W83 were 15.625 μg/mL. At this concentration, BBR can significantly reduce the bacterial activity of Pg ATCC33277 (P<0.05), reduce the activity of alkaline phosphatase after 12 hours (P<0.05). Pg ATCC33277 cells were deformed and wrinkled after BBR action and lost the normal bacterial morphology under SEM and TEM. Conclusions:BBR has a significant inhibitory effect on Pg ATCC33277 and Pg W83. It may reduce bacterial viability by disrupting cell walls and cell membranes.

Objective:To clarify the effects of feather keratin-montmorillonite hydrogels on the biological behavior and osteogenic properties of BMSCs. Methods:Keratins were extracted from chicken feathers by the modified Shindai method. The feather keratin-montmorillonite hydrogel was prepared by mixing the montmorillonite and feather keratin by mass ratio（0%, 1%, 3%). The feather keratin was identified and confirmed as coomassie brilliant blue staining and Fourier transform infrared spectroscopy. The morphology of feather keratin-montmorillonite hydrogels was shown by SEM. BMSCs were cultured at the leach liquor of feather keratin-montmorillonite hydrogels in the medium. The cytotoxicity was measured by CCK-8 assay. The BMSCs of ALP activity was also quantified using an ALP assay kit. Mineralization of the extracellular matrix of BMSCs is investigated by alizarin red staining. The expressions of related proteins in BMP/SMAD signaling pathway were detected by Western blot. Results:The results of coomassie brilliant blue staining and Fourier transform infrared spectroscopy demonstrate that we successfully extracted feather keratin from chicken feather. The pores present in feather keratin hydrogels were well-formed and interconnected. The addition of morillonite changes their structure. When 3%MMT was added, the pore size of feather keratin-montmorillonite hydrogel was significantly reduced. The results of the CCK-8 proliferation experiment showed that the leach liquors of the feather keratin+3%MMT group had inhibitory effects on the growth of the cells. The feather keratin+1%MMT group expressed the highest level of ALP activity, ALP staining, and alizarin red staining, and the data indicate that the leach liquors of the feather keratin+1%MMT group may promote the osteogenic differentiation of BMSCs. Protein expression of BMP-2, Wnt3a, p-SMAD1/5/8, RUNX2, OCN, and COL-1 was significantly higher in feather keratin+1%MMT group（P＜0.01）. Conclusions:The feather keratin-montmorillonite hydrogels can potentially promote osteogenic differentiation through BMP/SMAD signaling pathway.

Objective: To evaluate the effects of the thickness of tooth preparation, abutment color, and resin cement shade on the color of digital-guided glass full-crown restoration for incisor with different thickness of crown. Methods: The all-ceramic crown restorations were randomly divided into 18 groups according to three factors of the thickness of tooth preparation (1.0 mm and 1.5 mm), abutment color (IPS-ND2, ND5, ND8) and resin cement shade (Translucent-TR, B0.5 opaque-B0.5, White opaque-WO), with 10 specimens in each group. The monothetic glass ceramic crown was fabricated by CAD/CAM. The abutments were made using IPS nature die resin according to different colors as designed. The all-ceramic crowns in each group were temporally fixed on the resin abutment with the try-in paste for the color assessment using VITA Easyshade spectrophotometer and the results were recorded by L*a*b* system. Results: The thickness of tooth preparation, abutment color and resin cement shade and their interaction all showed a significant effect on the color of digital-manufactured monothetic glass ceramic crown for incisor (P<0.001). Conclusions: In case of restorationwith all ceramic crown that fabricated by CAD/CAM technique in anterior teeth, clinicians should make an optimal design of the tooth preparation and the thickness of the crown, according to the different abutment color. Furthermore, a resin cement with proper color should be selected for cementation in order to achieve an aesthetic and durable restoration.assessment using VITA Easyshade spectrophotometer and the results were recorded by L*a*b* system. Results: The thickness of tooth preparation, abutment color and resin cement shade and their interaction all showed a significant effect on the color of digital- manufactured monothetic glass ceramic crown (P<0.001). Conclusion: In case of restoration with all ceramic crown that fabricated by CAD/CAM technique in anterior teeth, clinicians should make an optimal design of the tooth preparation and the thickness of the crown, according to the different abutment color. Furthermore, a resin cement with proper color should be selected for cementation in order to achieve an aesthetic and durable restoration.