Effects Of Concentrated Growth Factors On Proliferation And Osteogenic Differentiation In Beagle Adipose-derived Stem Cells

Objective:Autologous concentrated growth factors(CGF)is the third generation of autologous blood extract after platelet-rich plasma(PRP)and platelet-rich fibrin(PRF).Compared to PRP and PRF,CGF has stronger promoting tissue regeneration ability due to its higher internal fibrin or growth factor polymerization degree and content.Adipose-derived stromal cells(ADSCs)are mesenchymal stem cells isolated from adopts tissue and have regenerating capacity.ADSCs have many advantages such as abundant reserves,simple preparation and strong regeneration ability,compared with stem cells from other sources,and gradually become the important seed cells of tissue engineering.This experiment with beagle adipose stem cells as the object of the research,adopts the method of observing the effect of CGF in vitro proliferation and osteogenic differentiation of ADSCs in dogs to explore the feasibility of combined application of the two in bone regeneration.Method:1.Isolation and culture of ADSCs in beaglesA healthy beagle aged 12months(10-12kg)was selected for making unilateral groin fat tissue under aseptic conditions.The third generation cells were selected for subsequent experiments by enzyme digestion method and adherent method to isolate and culture ADSCs.2.Identification of polymorphic differentiation of ADSCsAfter the ADSCs were adhered to the wall,the normal medium was replaced with the osteoblasts,lipids and chondrogenic medium to induce the cells respectively.After 21 d inducement,the culture medium was removed and fixed,and the osteogenic group was stained with alizarin red,the lipids group was stained with oil red O,the cartilage group was stained with all new blue.3.Preparation and experimental grouping of CGF.10 mL of venous blood of the forelimb of the beagle dog was placed in the negative pressure centrifuge tube and placed in the Medifuge CGF centrifuge.After the preparation,CGF was prepared according to the procedure,and the condition medium was added after shearing.The cells were grouped according to the CGF in the medium.One is the normal medium + CGF group(CGF group)the other is the normal medium group(control group).4.The effect of CGF on proliferation of ADSCs was detected by CCK-8 method.5.The effect of CGF on the activity of alkaline phosphatase in osteoinductive ADSCs was determined by alkaline phosphatase staining.6.Rt-PCR was used to detect the expression levels of osteogenic related genes of I-type collagen,osteocalcin,runx-2,osteopontin and other osteogenic related genes.7.Statistical processingAll data were analyzed using SPSS 22.0 software and independent sample t test was used for comparison between groups,and P<0.05 was considered statistically significant.Results:1.The ADSCs,isolated and cultured in vitro by enzymatic digestion and adherent method,were observed to be long fusiform and arranged in bundles or vortices and were able to proliferate and propagate in vitro.2.Alizarin red staining of ADSCs was conducted after osteogenic induction of 21 d.Lipid-induced cells were stained by oil red O staining.The cell volume increased under the microscope,and bright red lipid droplets were seen in the cytoplasm.A new alli blue stain was carried out after 21 d induced by cartilage.3.The results of CCK-8 showed that the proliferation activity of fat cells in the two groups was normal and the growth curve was “S" and the proliferation tendency of CGF group was significantly higher than the control group in the 3-7 days.4.Alkaline phosphatase staining results showed that the cytoplasm and extracellular matrix were observed in the cytoplasm and extracellular matrix after osteosynthesis induced 7d.However,the depth of staining of CGF group and the density of mineralized particles were significantly greater than that in the control group.5.The results of RT-PCR showed that the mRNA levels of ALP,OCN,OPN and Runx2 in CGF group were slightly higher than that in the control group after osteosynthesis induced 4d,and there was no statistically significant difference between the groups(P>0.05).However,the mRNA levels of ALP,Col-?,OCN,OPN and Runx2 in CGF group were significantly higher than that in the control group(P<0.05)after osteosynthesis induced 7d.Conclusion:1.This experiment successfully isolated ADSCs from Beagles' fat tissue and cultured in vitro.A variety of staining methods were used to identify the triaxial differentiation ability and the cells were obtained as mesenchymal stem cells.2.CGF can significantly enhance the proliferation ability of big fat stem cells in vitro.3.CGF can enhance the induction effect of osteogenic culture and can effectively promote the osteogenic differentiation of adipose stem cells.