| Insufficient residual bone height is the main disadvantage of posterior maxilla after tooth loss,which often makes it difficult for implant placement.Maxillary sinus floor elevation(MSFE)is the common bone augmentation technique to overcome this problem.The principle of MSFE is to surgically separate the maxillary sinus membrane from the sinus floor and raise it,and place bone graft material between the membrane and the bone wall of the sinus floor.Bone formation pattern in the maxillary sinus is controversial,some scholars believed that bone formation in maxillary sinus is in the direction from the bottom wall of the maxillary sinus to Schneiderian membrane,but some scholars thought that Schneiderian membrane also has osteogenesis potential.Bone formation could happen from Schneiderian membrane to maxillary bone wall.The reason is that after teeth extraction or maxillary sinus cyst extraction,newborn bone could be observed near the Schneiderian membrane;In maxillary sinus floor elevation,although no bone graft material was used,new bone formation was still observed at the apex of implant during postoperative follow-up.Therefore,the study on the osteogenic potential of Schneiderian membrane is helpful for understanding the bone formation pattern in maxillary sinus and can provide basic theory for new maxillary sinus floor elevation procedure.Schneiderian membrane can be used as a source of mesenchymal stem cells(MSCs)due to abundant capillaries and connective tissues in its lamina propria.MSCs have the ability of self-renew and multidirectional differentiation,and are widely used as seed cells in tissue engineering.In certain conditions,MSCs can be induced to differentiate into osteoblasts and eventually form bone in a certain microenvironment.Platelet-rich fibrin(PRF)is rich in a large number of growth factors that promote bone tissue regeneration and healing,and its dense three-dimensional fiber network provides a scaffold structure for cell growth.However,so far,there have been several reports on isolating,culturing and identifying MSCs from Schneiderian membrane,and inducing their osteogenic differentiation with PRF stimulation.Therefore,this study intends to isolate,culture and identify MSCs derived from Schneiderian membrane(SMMSCs),and induce their osteogenic differentiation with PRF stimulation,so as to further explore the osteogenic mechanism,and then apply the research results to clinical practice,in order to provide new ideas and methods for clinical work.The specific content of this research is divided into four parts:Experiment 1: Isolation,culture and identification of SMMSCsRabbit SMMSCs were isolated and cultured by enzyme digestion.The surface markers were identified by flow cytometry.Alkaline phosphatase staining and activity test,alizarin red staining to evaluate the ability of osteogenic differentiation;The ability of adipogenic differentiation was judged by oil red O staining;The ability of chondroblast differentiation was judged by alicin blue staining after 3D suspension culture.Experiment 2: Effects of PRF on biological behavior of SMMSCsThe ultrastructure of PRF was observed by scanning electron microscopy(SEM)and the release of growth factors was analyzed.CCK-8,RTCA,and cell fluorescence staining were used to evaluate the effect of PRF on SMMSCs proliferation.Cell scratch assay and Transwell assay were used to evaluate the effect of PRF on SMMSCs migration.Alkaline phosphatase staining and activity test,alizarin red staining,and q PCR were used to evaluate the effect of PRF on osteogenic differentiation of SMMSCs.Western Blot was used to evaluate the regulatory effect of PRF on the ERK 1/2 signaling pathway in inhibited and non-inhibited states,as well as the expression of RUNX2,a key osteogenic factor downstream.Experiment 3: PRF promotes osteogenesis in maxillary sinus in vivoA rabbit model of maxillary sinus floor elevation was constructed,and different bone graft materials were placed into the maxillary sinus.Samples were respectively obtained at 8 and 12 weeks after surgery.Micro-CT imaging evaluation was performed to determine the amount of new bone,bone volume fraction,number,thickness and separation of bone trabeculae.Double fluorescence labeling,HE staining,masson staining and toluidine blue staining were used to evaluate the osteogenic effect of different experimental groups.Experiment 4: The clinical trial of MSFE with PRF grafting and simultaneous implant placement.A prospective clinical trial was conducted to evaluate the clinical effect,success rate,endo-sinus bone production,marginal bone loss and bone density of MSFE with PRF grafting and simultaneous implant placement.Through the above 4 parts experiments,the following results were obtained:1.SMMSCs were successfully isolated and cultured.The cell surface markers were positive for expression of mesenchymal stem cell markers CD90,CD105,and negative for expression of CD34,CD45.SMMSCs had the ability of osteogenic,adipogenic and chondrogenic differentiation and could be defined as mesenchymal stem cells.2.PRF had a dense three-dimensional network structure,with few cell components near the serum side,and extremely rich platelets and leukocytes near the erythrocyte side.PRF sustained released growth factors such as platelet-derived growth factor(PDGF),transforming growth factor-β(TGF-β),vascular endothelial growth factor(VEGF)up to 14 days.Growth factors released from PRF could stimulate proliferation,migration and osteogenic differentiation of SMMSCs.PRF could promote osteogenic differentiation of SMMSCs by up-regulating the ERK 1/2 signaling pathway.3.PRF could accelerate the formation of new bone in maxillary sinus and increase the amount of new bone formation.The expression of osteogenic marker factor in bone graft area was significantly increased.4.During the 1-year follow-up,considerable endo-sinus bone augmentation was obtained by “PESS”,implant survival rate was high,marginal bone loss was low,and peri-implant bone density increased gradually over time.“PESS” is a minimally invasive,safe,and effective clinical approach to promote new bone formation in the sinus,shorten the duration of treatment,and extend the indications for maxillary sinus floor elevation through transcrestal approach. |
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