The Effects Of Concentrated Growth Factors On The Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells

ObjectiveThe aim of the study to bring the original training or periodontal membrane stem cells in vitro inoculation in fibrin membranes,CGF for wide clinical application of the sea the biofilm by contrast,in vitro to observe the periodontal ligament stem cells in the proliferation and osteogenesis differentiation two kinds of membrane surface,and apply the beagle in vivo animal model was established,in CGF site preservation effects of the application of operation after tooth extraction,detection of osteogenesis ability to change.For CGF fibrous protein membrane provides the theory basis for further clinical application.Methods1.The isolation and culture and identification of human periodontal ligament stem cellsFirstly,the method of enzymatic digestion tissue block and cloning rings were employed to isolate and culture human periodontal ligament stem cells;by flow cytometry instrument,we detected the expression of surface markers on mesenchymal stem cells;then we induced periodontal ligament stem cells into osteoblast,lipoblast and chondroblast,to test its multi-directional differentiation potential;last,collected the third generation periodontal ligament stem cells and used in the subsequent experiment.2.The Preparation of CGF:Venous blood in 9 ml sterile Vacuette tubes was obtained from the patients,placing in Medifuge centrifuge machine,then set the CGF preparation program.The sample was divided into tree layers after centrifuging.We discarded the upper layer,and cut from the junction of the middle and bottom layer,pressing CGF into membrane.HE method was used to detect its organizational structure and ELISA was used to analyze the release characteristics of internal growth factor.3.The experimental groups and cells inoculation(1)The experimental groups included CGF membrane group,biological membrane group and blank group.(2)Cells inoculation:The third generation of periodontal ligament stem cells were dispensed into single cell suspension with the special density.Then the suspension were inoculated on CGF membrane surface,the loose layer of the membrane surface,and the blank control.Set them in 37 0C and 5%CO2 culture saturated humidity incubator and transfer the culture liquid in a 3 day.4.Adherence test:Culture for five days,light-microscopy was used to observe the morphology of periodontal ligament stem cells on two different membrane.5.CCK-8 assay:After 1,3,5,7 days culture,CCK-8 assay were used to detect periodontal ligament stem cell proliferation of three different groups and drawn cell growth curve.6.Real-time quantitative PCR and Western-Blot was adopted to assay osteogenesis mRNA and osteogenesis protein expression of periodontal ligament stem cells of three different groups after 7 days osteogenic induction.Results1.We isolated and cultured periodontal ligament stem cells with enzyme digestion method in vitro successfully.The selected cells,who were long spindle mononuclear,conformed to the characteristic of fiber cells.Under induction condition,cells could differentiate in the direction of osteoblast,lipoblast and chondroblast,fitting the characteristics of stem cells.Flow cytometry instrument tests showed that surface markers of human mesenchymal stem cells(CD73,CD90,CD105 and CD44)presented in high expression,negative markers(CD11b,CD 19,CD34,CD45 and HLA-DR)were absent expression on PDLPCs surface.2.After centrifugation venous blood was divided into three layers,the middle layer was CGF.HE results showed that CGF was composed of fibrin grid,a large number of white blood cells and platelets,and a thin red blood cells at the bottom.ELISA test demonstrated that the releasing regularity of TGF beta-1,PDGF-BB and IGF-1 is different,but all can long-acting release.3.Adherence test results displayed that the periodontal ligament stem cells climbed out of the CGF membrane,and their axis were always perpendicular to CGF surface,presenting radioactive growth for the center with membrane.So that CGF could not drift with the medium.However cells surrounding with HEAL-All membrane,who were spindle,grew disorderly and sparsely,therefore,there was no attachment between cells and membrane.4.The CCK 8 results illuminated that CGF group was much higher than control and blank group on the all detected day(P<0.05).But the absorbance value of control and blank group had no significant difference on the first day and third day(P>0.05);the absorbance value of control group was much higher than blank group on the fifth day;while the absorbance value of blank group was higher than control group on the seventh days(P<0.05).5.After 7 days,the expression of osteogenesis related genes of ALP,Col-I,OPN,RUNX-2 in the cells of CGF group were higher than other two groups under the induce condition(P<0.05),then control group,and blank group was the least.6.Western Blot results was accordance with PT-PCR,the expression of osteogenesis related protein of ALP,Col-I,OPN,RUNX-2 in the cells of CGF group were higher than other two groups under the induce condition,then control group,and blank group was the least.Conclusion1.hPDLSCs has strong proliferation ability,with multi-directional differentiation potential under the induced condition,and can be used as a primary source of seed cells in tissue engineering.2.CGF can be as a good potential scaffold in the field of tissue engineering,which is able to provide good support for seed cells and microenvironment of rich in growth factors.3.CGF has obvious promoting effect in the proliferation,and it also can significantly enhance the ability of osteogenesis differentiation of hPDLSCs,in the joint application with osteogenesis induced condition.