Use ADSCs And PRF On The Periodontal Healing Of The Reimpanted Teeth

Objective:Traumatic dislocation teeth,defined as the complete displacement of the tooth from the alveolar socket with Injury of periodontal membrane and pulp,is one of the most serious dental trauma about 16% of permanent tooth trauma.The periodontal membrane was destroyed when the tooth was dislocated,and the cells gradually dehydrated and necrosis with the growth of the drying environment.When the teeth are replanted,the dead cells cannot function properly.Adipose-derived stem cells(ADSCs),as the new generation of seed cells,have many advantages in vitro and in culture compared with others.Study had shown that ADSCs have multiple differentiation abilities,which can differentiate into fat,bone,cartilage,muscle and nerve cells.Because of these advantages,ADSCs are used as seed cells in tissue engineering in many fields.Platelet-rich fibrin(PRF)as a new generation of rich platelet concentrate,has the characteristics of simple preparation,pure autogenous blood and no artificial preparation,so as to avoid the ethical controversies and immune rejection and the risk of cross infection of blood.The PRF has been proved to be beneficial to the proliferation and differentiation of ADSCs.Therefore,it is possible to the periodontal regeneration.In our study,ADSCs as seed cells,and preparation into the cell membrane fragment,at the same time the PRF as the source of the scaffolds and growth factor,construct the ADSCs/PRF composite grafts to promote the regeneration of the periodontal tissue.This transplant method may provide new opportunities for the clinical application of periodontal healing.Method:Part ?:The effects of PRF on biological characteristics of canine ADSCs in vitro1.Platelet-rich fibrin preparation and detection:5ml forearm vein blood of a dog was collected and immediately centrifuged at 400 g for 10 min.The fibrin clots were obtained and gently compressed into a membrane.After treatment,HE staining and scanning electron microscopy were used to observe the histological morphology of PRF.2.Isolation,cultivation and identification of canine ADSCs: 5ml inguinal fat tissue were harvested from dogs and ADSCs were successfully obtained,the cultured canine ADSCs were detected the proliferative activity by cck-8 and their osteogenic/dentinogenesis and adipogenic differentiation potential were detected.3.The effects of PRF on osteogenic capability of the ADSCs: The P3 ADSCs were induced by different osteoblast medium.The expression of ALP and Runx2 were detected by Real time PCR.After 7 days of induction,ALP staining was conducted,and 14 days after induction,alizarin red staining was performed.Part? : The combined use of ADSCs cell sheets and PRF for periodontal healing of reimplantation of avulsed tooth1.The preparation and detection of ADSCs cell sheet: The suspension of P3 ADSCs Inoculated into a petri dish and film-forming induction when cell adhesion.After two weeks cell culture,the cell sheets were matured and the micro-and ultra-structure of membrane were observed by HE staining and scan electron microscope.The PRF and cell sheet were cultured together and the ultrastructure of different constructs were observed by SEM.2.In vivo study of auto-ADSCs and PRF promote periodontal healing of reimplantation of avulsed tooth: Use animal model to evaluate the effects of the ADSCs/PRF on the periodontal healing of immediate and delayed reimplanted teeth.Result:Part?:1.PRF was Three-dimensional fibrin network structure,The upper end of the PRF was a fibrous collagen structure and the lower end was embedded in fibrin by white blood cells containing a small amount of red blood cells and some blue-stained nuclei.2.Adipose-derived stem cells maintain good cellular morphology and proliferation.After alizarin red staining,visible mineralized nodules were formed,and there was a single or fusion of red lipid droplets in the cells of different sizes in the oil red O staining3.ADSCs can be induced to further differentiate into the osteogenic direction by PRF,and in the early stages of osteogenesis,the expression of osteogenic genes becomes more and more obvious with the increase of induction time.Part?:1.In the ADSCs cell sheet,the cell are arranged,and the extracellular matrix is closely connected with the cells,and the cells are closely aligned.The membrane complex has a very good combination.2.In the immediate replantation groups,the new bone formation in ADSCs/PRF group was higher than that in the ADSCs group and the PRF group significantly,and the control group had no obvious new bone formation.Periodontal fibrin-liked tissue has been seen in periodontal cavity and little replacement resorption occurs on the surface of the teeth.In the delayed replantation group,new bone formation was still present in the alveolar bone,but there was less formation.ADSCs/PRF group have little fiber tissue form,ADSCs and PRF had a serious replacement resorption,and the osteoclast and resorption in the root surface in control group were obvious than that in the other group.Conclusion:1.ADSCs have the potential to proliferate and differentiate into fat,bone and cartilage.PRF can obviously promote cell proliferation and differentiation of bone.2.The application of autologous PRF in the replantation of avulsed tooth can obviously promote the periodontal healing and reduce its pathological absorption.However,the repair effect was negatively correlated with the dislocation time,and the immediate replantation of PRF group was significantly different from that of delayed replantation of PRF.3.ADSCs/PRF group has stronger ability to form periodontal tissue in comparison with the ADSCs group and PRF group.At the same time,the periodontal healing ability of the immediate replantation group was significantly higher than that of the corresponding delayed replantation group.