Effect Of Concentrated Growth Factors On The Adhesion,Proliferation And Osteogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells On The Surface Of Mineralized Collagen Materials

Objective: This experiment was to investigate the adhesion,proliferation,and osteogenic differentiation of concentrated growth factors(CGF)on human bone marrow mesenchymal stem cells(hBMSCs)on the surface of mineralized collagen(MC)material so as to provide a theoretical basis for the clinical application of concentrated growth factors in the regeneration of bone defects and stomatology.Methods:1.With the consent of the ethics committee of the Stomatological Hospital Affiliated to China Medical University and informed consents of the patients,venous blood from healthy volunteers was extracted and prepared into a CGF gel by a high-speed centrifuge.The the CGF gel was then cut,ground,frozen,stirred,and filtered to prepare concentrated growth factor extracts(CGFe).2.Material acquisition: The materials were provided by Beijing Aojing Company which are mineralized collagen discs with a diameter of 10 mm and a thickness of 2mm.3.Scanning Electron Microscopye(SEM)was used to observe the surface characteristics of MC materials.4.Obtain hBMSCs cell lines and observe the morphology of hBMSCs using an inverted phase contrast microscope.5.The extracted CGFe was added to the culture medium to prepare four groups of culture medium,and hBMSCs were cultured separately.Group A: ?-MEM medium containing2% CGFe(10% FBS),group B:?-MEM medium containing 5% CGFe(10% FBS),group C: ?-MEM medium(10% FBS),control group D: CGFe-free-?-MEM medium(10%FBS).6.SEM was used to observe the effects of different concentrations of CGFe medium on the adhesion and growth of hBMSCs inoculated on MC material.7.The CCK-8 method was used to detect the effects of different concentrations of CGFe medium on the proliferation of hBMSCs inoculated on MC material.8.hBMSCs cells were inducted into osteogenesis direction,with four osteoinduction induction media containing CGFe with different concentrations(includingdexamethasone,?-glyceride,vitamin C,10% FBS,1% double antibody)prepared according to concentration gradients.Grouping is the same as above,and the cells were induced separately.9.Alkaline phosphatase(ALP)activity was used to detect the effects of different concentrations of CGFe osteogenesis induction medium on the ALP activity of hBMSCs inoculated on MC material.10.Western blot was used to detect the effect of different concentrations of CGFe osteogenesis induction medium on the expression of osteopontin(OPN)of hBMSCs inoculated on MC material.Results:1.Surface characteristics of MC material: The surface of MC material is loose and porous,and it shows three-dimensional porous structure with different pore sizes under SEM.The size of pores ranges from 50 to 500?m,and the porosity is greater than 70%.2.hBMSCs cell morphology: hBMSCs were round and polygonal in the initial stage of adherence.With the increase of culture time,the number of cells increased,and the morphology gradually became long spindle-shaped and grew in a spiral direction.3.CGF gel: A three-layer separation structure obtained by venous blood centrifugation.The topmost layer is a pale yellow liquid.The bottommost layer is a jelly-like substance with loose texture.The middle layer of the centrifuge tube was a fibrin layer,and the translucent light yellow gel is CGF.The surface is smooth,the texture is flexible and elastic,and various biological factors are enriched at the junction of the middle and bottommost layers.4.SEM observation of cell extension and adhesion growth: After hBMSCs was inoculated with MC material for 24 h,the cells adhered to the surface and pores of MC material.The cells were expanded to varying degrees under the culture of each group of media,showing a polygonal,long shuttle shape spread.The CGFe-free control group had well spread cells with shorter and fewer pseudopods.The experimental group with different concentrations of CGFe extended the length of the pseudopod compared to the control group.The cells,as well as the ends of the pseudofoot grew into the material pores.5.Cell proliferation detected by CCK-8: With the increase of culture time,the number ofhBMSCs in each group gradually increased,and the growth curve showed an S-type.The proliferation ability of cultured cells containing different concentrations of CGFe was greater than that of the control group without CGFe.Among them,at each time point,the absorbed value of the 10% CGFe group was the highest(p<0.05).6.Detection of ALP activity: ALP activity of each group increased with time.At each time point,the ALP activities of cells cultured in media containing different concentrations of CGFe were higher than the control group without CGFe.The differences among groups at 7d and 14 d were statistically significant(p<0.05).7.Western blot detection of OPN protein content: OPN protein was expressed in each group.The levels of OPN protein in the 2%,5% and 10% groups were quite higher than that of the control group.As the CGFe content increased,the OPN protein level also increased.The differences among those groups were statistically significant.Conclusions:1.hBMSCs grow well on MC materials,and MC materials are of good biocompatibility.2.CGF can effectively promote the adhesion,proliferation and osteogenic differentiation of hBMSCs.3.Under the conditions set in this experiment,the CGF concentration is positively correlated with its concentration,with 10% CGF being the most significant.