| Objective:To explore the effects of PDGF-BB on the proliferation,migration and osteogenesis of BMSCs which was pretreated by ALN.To study the preventive effect of PDGF-BB on the occurrence of bisphosphonate-associated osteonecrosis after tooth extraction in SD rats.Method:1.BMSCs were treated with ALN at different concentrations(10-4M,10-5M,10-6M,10-7M),and the proliferation capacity of cck-8（cell counting kit-8）was detected at different point-in-times（1d,3d,5d,7d）.According to the results of the proliferation experiment,10-5M ALN was selected for subsequent exper-iment.After BMSCs were treated with 10-5ALN,the ALP was quantitatively determined by p NPP when it was ossified for 7d and 14d.2.BMSCs were treated with 10-5M ALN for 72h and then replaced with PDGF-BB medium for 48h.The proliferation ability of BMSCs was detected by cck-8.The ability to migrate of BMSCs was detected by Transwell cell compartment.After osteogenesis induction for 14 days,the calcium nodule formation expression of BMSCs was qualitatively detected by alizarin red staining.The ALP expression of BMSCs was quantitatively detected by p NPP method.The expression of BMSCs osteogenic proteins was detected by WB.3.12 6-month-old female SD rats were subjected to bilateral oophorectomy,after 3 months,ALN was injected subcutaneously for 60 days.All rats were randomly divided into the prevention group and the control group,at the same time,their first and second molar of the left mandibular were extracted.For the prevention group,their tooth extraction fossas of rats were inserted into the absorbable gelatin sponge loaded with PDGF-BB and then sutured,while the control group were inserted into the normal absorbable gelatin sponge and sutured tightly.8 weeks after tooth extraction,various tests were performed by gross observation,HE section staining pathology examination,and Micro-CT imaging;ELISA was used to determine the expression of c-terminal telopeptides of type I collagen（CTX I）in serum of rats.Result:1.When the concentration of ALN was less than or equal to 10-7M,cck-8 results showed a time-dependent promotion effect on BMSCs proliferation（P<0.05）which was treated with ALN for 3 days.When the concentration of ALN was greater than or equal to 10-5M,it was obvious that the proliferation of cells could be inhibited after treated with ALN for 3days（P<0.05）,and the dead cells could be observed under the microscope.The proliferation of BMSCs was inhibited by ALN,and the effect increased with time.After 3 days of ALN treatment,there was no significant difference in the expression of ALP in BMSCs at 7d of osteogenic induction（P>0.05）,but the expression of ALP was significantly less than the control group at 14d（P<0.05）.2.The results of cck-8 and Transwell showed that PDGF-BB could promote the proliferation of BMSCs who pretreated with 10-5M ALN（P<0.05）.However,there was no significant change in its inhibited migration ability.In further quantitative analysis of ALP and WB experiments,we found that the expression of osteogenic proteins（ALP,RUNX2,OCN）of BMSCs was inhibited under the effect of ALN,but their expression was restored in the PDGF-BB treatment group.3.In the prevention group of SD rats with PDGF-BB absorbable sponge loaded with tooth extraction fossa packing,the incidence rate of BRONJ was33.3%（2/6）,and 83.3%（5/6）in the control group,and the incidence rate of BRONJ in the prevention group was significantly reduced.The results of tissue sections showed that the bone tissue lacunae and bone cell loss of the prevention group were significantly less than those in the control group（P<0.05）,while the new bone deposition increased and the structure was more dense and uniform.Micro-CT results showed that the bone mass increased significantly in the prevention group,the number of bone trabecula increased,the pore wall thickened,and the newly formed bone structure became more dense and continuous.There was no significant difference in CTX I between the control group and the prevention group（P>0.05）.Conclusion:In vitro experiments,ALN could inhibit the proliferation and osteogenic differentiation of BMSCs,while PDGF-BB could reverse the inhibition.PDGF-BB could make an excellent effect on BRONJ prevention after tooth extraction in experimental animals. |
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