Objective:To study the effects of IGF-1 on the proliferation and differentiation of Hertwig’s epithelial root sheath(HERS) in vitro.Methods:HERS cells were isolated and cultured in vitro, and were treated with IGF-1. Proliferation of HERS cells was evaluated by CCK8 assay. Immunofluorescence staining detected the expression of CK14, E-cadherin, Vimentin of HERS cells. Real-time PCR detected the expression of E-cadherin, N-cadherin, Vimentin, Bsp,Col1, Runx2, Alp. And osteogenic differentiation of HERS cells was determined by Alizarin red staining.Results:HERS cells could have the expression of the marker gene of epithelia and mesenchyme.IGF-1 treatment significantly decreased the expression of epithelial marker gene and increased the expression of mesenchymal marker gene and enhanced the cell proliferation. HERS cells culturedin mineralization-induction medium showed significantly upregulated the expression of genes related to the cementoblastic differentiation such as Bsp,Col1, Runx2, Alp on day 7 and day 14(P＜0.05). IGF-1 treatment also significantly enhanced the mineralization nodule formation of HERS cells. Conclusions:IGF-1 regulated the proliferation, cementoblastic differentiation and EMT of HERS cells.

Objective: To investigate the effects of polyacrylamide (PA) hydrogel substrates with different stiffness on the growth and differentiation of cell line of osteoblastic (MG63). Methods: PA hydrogels with different stiffness was prepared and its properties such as water absorption, wettability and elastic modulus were tested. Then we inoculated MG63 cells on different PA hydrogels coated with Sulfo-SANPAH and collagen I, and we observed the cell growth by light microscope and scanning electron microscope, then the real-time quantitative RT-PCR and cell immunofluorescence staining were used to detect the expression of genes and proteins about osteogenesis and stemness of MG63. Results: PA hydrogels (PA1 and PA2) with stiffness of (31.32±1.04) kPa and (147.62±1.30) kPa were prepared with good hydrophilic properties. The cells grew in clumps on PA hydrogels. Compared with traditional culture dishes, the expression of sex-determining region Y-box 2 (SOX2), octamer-binding transcription factor 4 (OCT4) genes and protein in cells cultured on PA hydrogels were significantly increased (P＜0.05), and the expression of runt related transcription factor 2 (RUNX2), osteocalcin (OCN) genes and protein were significantly decreased (P＜0.05). There was no significant difference in the expression of osteogenic genes in the cells growing on PA1 and PA2 (P＞0.05), while the expression of OCT4 and SOX2 genes in the cells of the PA2 group was higher than that of PA1 (P＜0.05). Conclusions: The stiffer PA hydrogel is beneficial to the growth and maintenance of the stemness of osteoblast.

Objective: To investigate the role of age-related proteoglycans in fracture healing by establishing a model of femur fracture in mice. Methods: We established right femur fracture models on 2 months and 9 months C57BL/6J, they were sacrificed at 7 days post surgery. The fracture healing was observed by histological stain, the expression of chondrogenic genes and proteoglycan was detected by Real-time PCR. Results: The mouse femur fracture model was successfully constructed. Histological staining showed that the callus of 9 months mice was smaller than that of 2 months mice after fracture. Real-time PCR showed the expression of cartilage-related genes such as Col10, Sox9, Mmp9 and proteoglycan-related genes such as Dmp1, Vcan, Dcn, Bgn during fracture healing of 9 months mice were lower than that of 2 months mice (P<0.05). Conclusion: In the process of fracture healing, the expression of proteoglycans in aged mice is decreased, suggesting that proteoglycans may be related to the poor fracture healing in aged mice.

Objective: To investigate the role of major vault protein (MVP) in periodontitis by establishing an experimentalmodel of periodontitison mice and osteoclast induction of bone marrow mesenchymal stem cells (BMMSCs) in vitro. Methods:A model of experimental periodontitis was established by local injection of lipopolysaccharide (LPS) into wild-type mice (WT) and MVP knockout (MVP-/-) mice.The severity of periodontitis was detected by micro-CT and TRAP staining. In vitro, the role of MVP in osteoclast differentiation was verified by TRAP staining and Wheat Germ Agglutinin (WGA) staining after cultured with RANKL and M-CSF. Results:In experimental periodontitis induced by LPS, MVP-/- group developed more bone resorption and more osteoclasts could be seen in the injection area. In vitro experiments showed that knockout MVP promoted osteoclast differentiation and bone resorption.Conclusions:MVP plays a protective role in periodontitis by inhibiting osteoclast differentiation.

Objective: To investigate the effects of tolerance induced by Porphyromonas gingivalis (P.gingivalis) on bone resorption related cytokines, including IL-1β, osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL), and alveolar bone resorption in mice. Methods: Tolerance in murine periodontal tissues was induced by orally inoculation with 1×109CFU P.gingivalis for 5 days, washing with PBS and inoculation with P.gingivalis again for additional 2 days. After 10 days, fresh gingivae were collected and the expression levels of IL-1β, OPG and RANKL were detected by western bolt. 6 weeks later, maxillae were collected and the distances from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) were evaluated by methylene blue staining. Results: The expression levels of IL-1β in gingivae from P.gingivalis-tolerant mice were lower than those from non-tolerant group and the ratio of OPG/RANKL expression levels was higher than that of non-tolerant group. Less bone absorption was observed in tolerant group compared with that in non-tolerant mice (P<0.05). Conclusion:Tolerance induced by P.gingivalis could suppress

Objective: To explore the effects of periodontal inflammation on the formation of macrophage extracellular traps (METs) in gingivae. Methods: 29 patients with periodontitis who finished periodontal non-surgical treatment and needed flap surgery and 20 periodontal healthy patients who needed crown lengthening surgery were recruited. Before surgeries, clinical periodontal parameters, including plaque index (PI), gingival index (GI), probing depth (PD) and clinical attachment loss (CAL), were recorded and gingival crevicular fluid (GCF) was collected. In patients with periodontitis, gingival samples from the most serious sites were collected during flap surgeries, while healthy gingivea were collected during crowning lengthening surgeries. METs were observed by laser confocal microscopy and the levels of TNF-α and IL-9 in GCF were measured by ELISA. Correlations between the scores of METs formation and periodontal clinical parameters, the levels of TNF-α, IL-9 were analyzed. Results: Clinical periodontal parameters, including PLI, GI, PD and CAL, the scores of METs formation and the levels of TNF-α and IL-9 in periodontitis group were higher than those in control group (P<0.05). The scores of METs formation were positively correlated with PD and CAL (P<0.05). Conclusions: Inflammatory destruction in periodontal tissues might contribute to the formation of METs.

Objective: To investigate the expression of TEA domain transcription factor 4 (TEAD4) in oral squamous cell carcinoma (OSCC) and its correlation with clinical pathological parameters and prognosis value, and explore the biological function in OSCC invasion and migration ability. Methods: Immunohistochemistry was assessed to detect TEAD4 expression in 79 primary OSCC sample and 21 normal oral mucosa. The associations between TEAD4 and multiple clinicopathological parameters and prognosis were analyzed. In vivo assay, Western Blot, q-PCR, wound healing assay and Transwell invasion assay were assessed to detect the invasion and migration ability when TEAD4 silencing. Results: Immunohistochemistry data showed that the expression of TEAD4 in OSCC was significantly higher than that in normal oral mucosa (P＜0.05). And its expression in OSCC was significantly correlated with cervical lymph node metastasis, clinical stage, and overall survival rate (P＜0.05). The invasion and migration ability of Cal27 was impaired when TEAD4 silencing in vivo assay. Conclusions: Our findings reveal that overexpression of TEAD4 correlate with OSCC malignant phenotype of, the prognosis of patients and promoting OSCC cell migration and invasion.

Objective: To explore the potential role of myeloid-derived suppressor cells (MDSC) in the pathogenesis of this disease by establishing a mouse model of Bisphosphonate-related of osteonecrosis of the jaw (BRONJ). Methods: BRONJ-like disease was induced in C57BL/6 mice by intravenous injection of zoledronate to observe gingival healing and alveolar bone repair. In addition, flow cytometry was used to detect the changes in the proportion of MDSC and their subgroups in peripheral blood of BRONJ mice. Results: The extraction cavity of BRONJ mice did not completely heal, showing the empty alveolar fossa. Micro-CT manifested that the extraction socket had scattered high-density images. HE staining showed the absorption of trabecular bone surrounded by multinucleated giant cells. The proportion of MDSC and their subgroups in the peripheral blood of mice was significantly reduced. Conclusions: MDSC differentiation in BRONJ mice model was inhibited which was used to further confirm the mechanism of immune dysfunction in BRONJ.

Objective: To develop a colloidal gold-based immunochromatographic test strip for the rapid detection of IL-1β levels in the gingival crevicular fluid (GCF), expecting to provide experimental evidence for the accurate diagnosis of chronic periodontitis. Methods: The colloidal gold was prepared by chloroauric acid and trisodium citrate. After optimizing the reaction conditions, the colloidal gold-antibody complex was prepared. Then, with the assembled colloidal gold chromatography test strips, the standard curve was tested and the clinical samples were detected, whose results were analyzed by Spearman correlation with those of ELISA. Results: The colloidal gold immunochromatographic test strips were successfully prepared, and the detection sensitivity could reach 5 ng/mL. The clinical samples detection showed that the Spearman correlation coefficient between the test strips and ELISA was 0.992 for detecting IL-1β levels in GCF in periodontitis patients. Conclusion: The developed colloidal gold-based immunochromatographic test strips can perform reliable detections, which provide experimental evidence for rapid detection of IL-1β levels in GCF in periodontitis patients.

Objective: To evaluate changes and correlations of facial soft and hard tissues in skeletal Class III malocclusion patients after a combined orthodontic and orthognathic surgery treatment. Method: In this study, 32 patients with skeletal class III malocclusion received a combined orthodontic and surgical treatment. Cone-beam computed tomography (CBCT) was performed pre-(T1) and post-(T2) treatment. Lateral cephalometric radiographs were obtained from CBCT scans. Landmarks on the sagittal plane were selected to measure the angle, thickness and distance of the profile. Paired t-test was used to compare the angles and thicknesses, and Pearson linear correlation analysis was conducted to examine the correlation between soft and hard tissue variations. Results: There were significant angle differences in NLA, MLA, MP-SN and L1-SN (P< 0.05) with the exception of U1-SN (P >0.05). Soft tissue thickness TLL', TBs significantly increased (P < 0.05). However, TSn, UL', TUL' and TPos did not significantly change (P > 0.05). The soft and hard tissue variation ratios of Sn/A, UL’/UI, LL’/LI, Bs/B, Pos/Po, Gns/Gn were 0.69, 0.59,0.73, 0.85, 0.90 and 0.97, respectively. Conclusion: Profile improvement was remarkable after orthodontic-orthognathic treatment in skeletal class III malocclusion patients. Pearson’s correlation analysis showed that the position predictability was more favorable for the chin compared to the lip. Additionally, the position predictability of the mandible outdid the maxilla.

Objective: To establish and explore a predictive model for hand wrist bone age calculation based on two-dimensional (2D) and three-dimensional (3D) morphological measurements of cervical vertebra in cone beam computerized tomography (CBCT) images. Methods: Hand-wrist X-ray images and craniofacial CBCT images of 150 Eastern Chinese female adolescents (aged from 8 to 16 years) were collected, and 2D and 3D morphological variables from cervical vertebras were measured. According to Fishman's Skeletal Maturity Index, the stages of bone age were determined. Multiple stepwise regression analysis was used to develop two different bone age predictive formulas, excluding and including 3D measurements. The paired t-test was applied to detect the prediction reliability of the models. The adjusted R-squared and accuracy of the models excluding and including 3D measurements were analyzed and compared. Results: Age and quantitative variables of the second, third and fourth cervical vertebrae were positively correlated with bone age. The paired t-test showed that there was no significant difference between the predicted results of the two bone age formulas and the gold standard (P >0.05). The adjusted R-squared and accuracy of the formula including 3D measurements were improved compared to the formula which 3D measurements were excluded. Conclusions: The adjusted R-squared and accuracy of the hand wrist bone age predictive model formulated were improved by conjunctive using of 2D and 3D morphological measurements, which can provide a reliable index for quantitative craniomaxillofacial growth studies in eastern Chinese female adolescents, and also a reference for individual bone maturity.