Objective: To explore suitable concentration of recombinant human transforming growth factor β1 (rhTGF-β1) usage and study the effect of rhTGF-β1 on differentiation of dental pulp stem cells (DPSCs).Methods: DPSCs were isolated from the undiseased third molars of people aged 18-25 years and cultured according to instructions in vitro.Different concentrations (1 , 6 , 10 μg/L) of rhTGF-β1 were added to the culture medium to examine DPSCs proliferation by CCK-8 (cell counting kit-8) assay.The suitable concentration was then selected.For differentiation, the DPSCs were incubated for 7 or 14 days with rhTGF-β1 supplemented with osteo/odontoblastic induction medium containing 10 nmol/L dexamethasone, 10 mmol/L b-glycerophosphate, 50 g/L ascorbate phosphate, 10 nmol/L 1,25-dihydroxyvitamin D3 and 10% fetal bovine serum.The cells were then washed 3 times with phosphate-buffered saline and sonicated with 1%Triton X-100 for 30 minutes on ice.Cellular alkaline phosphatase (ALP) activity was assayed with p-nitrophenyl phosphate as the substrate.The enzyme activity was expressed as p-nitrophenyl produced per milligram of protein [bicinchoninic acid (BCA) protein assay kit].To examine mineral nodule formation, the cultured cells were fixed in 4% paraformaldehyde and washed in water, and the mineralization of the extracellular matrix was assayed by 1% alizarin red S staining and elution of staining was examined as optical density (D) under microplate reader.The mean difference was considered significant at 0.05 and 95% confidence interval.Results: The DPSCs had ty-pical fibroblast morphology and could form mineral nodules after being cultured with osteo/odontoblstic induction medium for 14 days.6 μg/L rhTGF-β1 significantly promoted the DPSCs proliferation on the 3rd and 5th days.After the incubation of osteo/odontoblastic induction medium, the DPSCs with the 6 μg/L rhTGF-β1 increased ALP activities compared with the control;D values in the 6 μg/L rhTGF-β1 group was 0.31±0.03, while the control group was 0.02±0.01(P<0.05).The total protein content in the 6 μg/L rhTGF-β1 group was (2 775.46±83.54) mg/L, and the control group was (1 432.20±110.83) mg/L (P<0.05).To eliminate the cells proliferation influence, relative ALP activities, which was defined as the total ALP divided by the total protein content, the 6μg/L rhTGF-β1 group was 6 times higher than the control group.Alizarin red S staining showed increased mineral nodule formation in the rhTGF-β1 group.The elution of staining under microplate reader also showed more optical density in the 6 μg/L rhTGF-β1-treated cells (0.83±0.02) than that in the control groups (0.55±0.05, P<0.05).Conclusion: 6 μg/L rhTGF-β1 could significantly promote DPSCs proliferation and odontoblastic differentiation in vitro.

Objective:To explore the feasibility and clinical efficacy of stratified suture of lateral pharyngeal wall(SSLPW) combined with soft palate radiofrequency coblation in the treatment of the patients with severe obstructive sleep apnea hypopnea syndrome(OSAHS).Methods:The clinical data of 21 severe OSAHS patients underwent lateral pharyngoplasty(LP) combined with soft palate radiofrequency coblation (LP group) and 39 severe OSAHS patients underwent SSLPW combined with soft palate radiofrequency coblation (SSLPW group) were selected.The apnea hypopnea index(AHI),lowest SaO2(LSaO2),Epworth sleepiness scale(ESS) scores,and related postoperative complications of the patients in two groups were analyzed before and after operation.The successful rates of operation of the patients in two were compared.Results:In LP group,1 patient was cured (5%),19 patients were improved markedly (90%),1 patient was effective (5%),and no patient was invalid;the successful rate was 95.2%(20/21).In SSLPW group,2 patients were cured (5.1%),33 patients were improved markedly (84.6%),4 patients were effective (10.3%),and no patients was invalid;the successful rate was 89.7%(35/39);there was no statistical difference in the successful rate of operation of the patients between two groups (P>0.05).There was statistically significant improvement of the subjective symptoms.All the patients returned to normal subjective swallowing functions without nasal pharyngeal reflux and dysphagia in one month follow-up after operation.Compared with before operation,the AHI and ESS scores of the patients in two groups after operation were decreased(P<0.05);the LSaO2 scores were increased(P<0.05).Conclusion:The AHI,LSaO2 and ESS scores are significantly improved in the OSAHS patients after treated with SSLPW combined with soft palate radiofrequency coblation.The method is a valid option for the severe OSAHS patients.

Objective To explore the application of UW solution in the replantation of the rat limb,and to explore a simple method for the ideal preservation of the amputated limbs.Methods From September,2017 to Janurary,2018,one hundred and twenty healthy adult male SD rats,weight 320-350 g,were randomly divided into 8 groups:A,B,C,D,E,F,G,H group.The isolated limbs of the control group A,C,E,G were cryopreserved in refrigerator at 4 ℃,while those of the experiment group B,D,F,H were preserved under the same condition after being perfused with UW solution.All the severed limbs were replanted back to the rats after different time length of cry opreservation:A,B 12 hours,C,D 20 hours,E,F 28 hours and G,H 36 hours.During the operations the femoral artery and venous patency were detected immediately after anastomosis.And after the operations the survival rates of limb replantation were observed.On the third day after the limb replantation,the tibial posterior muscle tissue was examined for pathology.The cell areas in pathological photographs were compared with Image analysis software Image-pro Plus 6.0 and the ultra structural changes of skeletal muscle tissue were observed.Results The femoral artery and venous patency in experiment group A,C,E and control group B,D,F were all 100 percent.However,in the group G,H the severed limb vessels changed obviously,the patency dropped markedly and the survival rates of limb replantation turned out to be 0.The experiment groups perfused with UW solutions,had relatively higher survival rates compared with the control groups (12 h:93.33％ vs.53.33％,20 h:80.00％ vs.40.00％,28 h:53.33％ vs.13.33％) and the skeletal muscles edema was lighter than that of the control group,with a larger proportion of cell areas(12 h:69.49±8.57 vs.44.75±6.34,20 h:56.34±7.73 vs.36.69±5.74,28 h:45.76±8.23 vs.31.49±7.72).There was a significantly difference (P＜0.05).Electron microscope observation showed that the experiment group bore a lighter damage of myofibril,interstitial tissue and mitochondria compared with the control group.Conclusion The combination of UW solution and cryopreservation of organs is an ideal and proper way to preserve the amputation limb,to extend the time limit for replantation,and to achieve a better recovery effect of postoperative limb function.Besides,UW solution is easy to be promoted in the treatment of isolated limb in clinical work.

Objective: To evaluate the effect of exogenous stem cells from apical papillae (SCAP) in the pulp revascularization treatment for the immature permanent tooth with periapical periodontitis in animal model. Methods: After the SCAP were isolated and cultured from the Beagle dogs, stem cell properties of these cells were characterized by analyzing their colony-forming ability, the expression of mesenchymal stem cell markers and the multidifferentiation characteristics including osteogenic, adipogenic, and chondrogenic potentials. Models of young permanent tooth with periapical periodontitis were established in dogs and the infection in each of the model tooth was eliminated by root canal irrigation and intracanal medication. After that, all of the model teeth were randomly divided into 4 groups: Group 1: normal developing teeth with no treatment applied;Group 2: teeth that periapical tissues were irritated to induce blood flowing into the root canals;Group 3: teeth that peripheral blood was delivered into the root canals;Group 4: teeth that SCAP were resuspended in peripheral blood and delivered into the root canals. In Group 2-4, firm coronal seal was performed after revascularization procedure and radiographs were taken periodically in order to observe the development of roots. After a 12-week-period, alveolar samples were collected and observed histologically. Results: The isolated SCAP showed clonogenic ability and multilineage differentiation ability including osteogenic, adipogenic and chondrogenic potentials. These cells also expressed the mesenchymal stem cell markers such as STRO-1 and CD146, while no cytokeratin was detected. The thickening of canal wall was observed radiographically 12 weeks after procedures of infection control and revascularization. Histologically, the newly formed tissues on the inner canal wall were found bone lacuna like structure in Group 2 and 3, and the new tissue formed in the Group 3 seemed easy to separate from the canal wall. The newly formed tissues in Group 4 were much thicker compare to those in the Group 2 and 3, and the dentine tubule like structure instead of bone lacuna was noticed although the orientation of these tubules were various. Conclusions SCAP seem to play an important role in the tissue regeneration procedure when infection is well controlled in young permanent teeth with periapical periodontitis. It is difficult to achieve real tissue regeneration due to the lack of endogenous SCAP in apical area, therefore delivering adequate exogenous SCAP isolated and cultured in vitro could be a promising approach to overcome the challenge.