[Generation and validation of inducible osteoblast-specific Stat3 knockout mice].

PURPOSE: Based on the Cre-Loxp gene knockout system, this study intended to construct tamoxifen-inducible STAT3 conditional knockout mice and verify the knockout efficiency. METHODS: The inducible osteoblasts-specific Stat3 knockout mice Stat3Col1ERT2 were obtained by hybridization through C57 mice of Stat3fl/fl and Col1 creERT2. Bone mesenchymal stem cells(BMSCs) of these mice were isolated and cultured with or without 4-hydroxytamoxin(4-OTH), to verify the effect of Stat3 knockout in vitro by real-time quantitative PCR and Western blotting in the level of mRNA and protein. Meanwhile, wild type and Stat3Col1ERT2 mice were both intraperitoneally injected with tamoxifen, the expression of STAT3 in the maxillary alveolar bone was observed by immunofluorescent staining to confirm the knockout effect in vivo. Statistical analysis was conducted with SPSS 24.0 software package. RESULTS: Real-time quantitative PCR and Western blotting results demonstrated that mRNA(P<0.05) and protein levels of STAT3 were significantly decreased (P<0.05) in BMSCs derived from Stat3Col1ERT2 mice by 4-OHT induced knockout in vitro. Immunofluorescent staining indicated that STAT3 expression was significantly reduced(P<0.05) in osteoblasts of the maxillary alveolar bone in Stat3Col1ERT2 mice. CONCLUSIONS: This study successfully constructed the inducible osteoblasts-specific Stat3 gene knockout mice, which helped investigators control the time and space of gene knockout, therefore providing new insights and guidance for research fields of orthodontic tooth movement, distraction osteogenesis and jaw fractures in the future.

[Expression of STAT3 in alveolar bone during orthodontic tooth movement in rats].

PURPOSE: To study the changes of expression of signal transducer and activator of transcription 3 (STAT3) during orthodontic tooth movement (OTM). METHODS: Twenty six-week-old SD rats were selected, the upper left first molars were moved by coil spring and lasted for 7 days. The maxillary tissues were obtained at 0, 1, 3, 7 d and subjected to histological study. Statistical analysis was performed using SPSS 16.0 software package. RESULTS: The main changes in alveolar bone during orthodontic tooth movement included significant increase in osteoblast number and bone formation in the tension area, and bone resorption in the pressure area. The positive cells of osteocalcin in the tension area increased during OTM. The expression of STAT3 increased in the tension area at 3 d and 7 d in comparison with that at 0 d. CONCLUSIONS: Orthodontic force can stimulate alveolar bone remolding. The expression of STAT3 in the tension area may have effects on alveolar bone remolding during orthodontic tooth movement.

[The effect of estrogen on adipogenic differentiation of rat bone mesenchymal stem cells].

PURPOSE: In this study, 10⁻9 mol/L 17 ß-estradiol (E2) was applied in the adipogenic differentiation of rat bone mesenchymal stem cells (rBMSCs) and the effect of E2 was explored. METHODS: Rat BMSCs were obtained from the femurs and tibias of SD rats. 10⁻9 mol/L E2 was involved in the adipogenic differentiation of rBMSCs. Oil red staining, real time PCR and Western blot were carried out to detect the effect of 10⁻9 mol/L E2 on adipogenic differentiation of rBMSCs. The data was statistically analyzed using SPSS 19.0 software package. RESULTS: The use of 10⁻9 mol/L E2 decreased the amount of lipid droplets in rBMSCs and weakened the expression of adipogenic related genes and proteins like C/EBP α, C/EBP ß, PPAR γ, aP2, and ARDP, which were significantly lower than the adipogenic induced group. CONCLUSIONS: The use of 10⁻9 mol/L E2 inhibited adipogenic differentiation of rBMSCs significantly in vitro.

[Changes of the microarchitecture of alveolar bone due to different duration of ovariectomy: a Micro-CT study in rats].

PURPOSE: To study the changes of microarchitecture of alveolar bone due to different duration of ovariectomy in rats. METHODS: Twenty-four virgin Sprague-Dawley rats were randomly assigned to ovariectomy group (OVX) or sham-ovariectomy group (sham). OVX rats were subjected to bilateral ovariectomy and sham-ovariectomy was conducted in sham rats. Six rats of each group were sacrificed respectively 3 months and 6 months after surgery. The right semi-maxilla of all rats were scanned by Micro-CT, and the inter-radicular alveolar bone of the maxillary first molar was analyzed. Statistical analysis was carried out with SPSS 16.0 software package. RESULTS: Two-dimensional and three-dimensional reconstructed images of the alveolar bone showed porotic changes in rats both 3 months and 6 months after ovariectomy, including thinner, looser trabeculae and expanded bone marrow. When compared with corresponding sham rats, BMD, BV/TV and Tb.Th of alveolar bone significantly decreased in OVX rats both 3 months and 6 months after ovariectomy (P<0.05). Tb.N and Tb.Sp significantly increased in both OVX groups (P<0.05). When compared with 3 months after ovariectomy, the rats 6 months after ovaroectomy shared deceased BMD, BV/TV and Tb.Th (P<0.05) and increased Tb.N (P<0.05). CONCLUSIONS: Bone loss and deterioration of trabeculae of alveolar bone aggravates with the extended duration of ovariectomy in OVX rats.

[The effect of estrogen on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells].

PURPOSE: Different concentrations of 17 ß-estradiol (E2) were applied in the osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs), and the proliferation and apoptosis of BMSCs were explored. METHODS: BMSCs were obtained from the femurs and tibias of SD rats. The proliferation curve was conducted to rBMSCs in culture medium containing 0, 10(-9), and 10(-7) mol/L 17 ß-estradiol by CCK-8 for 7 days. Annexin V and PI for flow cytometry were applied to detect the impact of E2 on apoptosis of rBMSCs. After 1, 7, 11 and 14 days of osteogenic induction, the activity of alkaline phosphatase (ALP) was assayed; ALP staining was performed on day 7 and day 14; Alizarin red staining for calcium deposits was carried out on day 21. Concentrations of 0, 10(-9), and 10(-7) mol/L 17 ß-estradiol were administrated to rBMSCs for real-time PCR of osteogenic related genes on day 1, 3, 5, 7, 14, and day 21. The data was statistically analyzed using SPSS 19.0 software package. RESULTS: The effect of 17 ß-estradiol on proliferation and apoptosis of rBMSCs was not obvious. However, after osteogenic induction, the ALP activity and Alizarin red staining were significantly stronger in the groups containing 17 ß-estradiol. Especially, the use of 17 ß-estradiol with the concentration of 10(-9) mol/L enhanced the expression of osteogenic related genes like RUNX2, ALP, COL I, and OCN, which was significantly higher than other groups. CONCLUSIONS: 17 ß-estradiol promotes osteogenic differentiation of BMSCs in a dose-dependent pattern in vitro.