Experimental Research On Effect Of Biological Behavior Of Platelet-Rich Plasma On Humann Osteosarcoma Cell Lines MG63, HumanAdipose-Derived Mesenchymal Stem Cells And Human Dermal Fibroblasts

OBJECTIVE: Platelet-rich plasma (PRP) is widely used to promote tissue healing. However, there is no concrete evidence for the biological effects of PRP. This study evaluated the biological effects of PRP on the proliferation, differentiation and the ability of bone formation of human osteosarcoma cell lines MG63, human adipose-derived mesenchymal stem cells (hADMSc) and human dermal fibroblasts (hDFb), while, the aim of the study also discuss the effects of PRP on the rehabilitate of crandial bone defect of rabbit and illustrate the biofuntion of PRP and clean some questions in the application of PRP through cytology and molecular biology.METHODS: the experiments in the study1. Study the methods of preparation of the platelet-rich plasma.PRP was prepared from freshly drawn human venous blood containing a large number of platelets. We discussed the attention when prepare PRP and compared growth factor contents of platelet-derived growth factor (PDGF), transforming growth factor (TGF-β) and vascular endothelial growth factor (VEGF) in three different centrifugation techniques by ELISA. The methods are PCCS PRP system, Curasan PRP kit system and the third method, which blood was centrifuged at 1 600 r/min for 10 min and then centrifuged at 5 000 r/min for 5 min. The studies also evaluate the content of PDGF, TGF-βand VEGF in PRP by different active methods. The study also discussed the effects of the activation percent of platelet in the PRP by four different centrifugation techniques. The study also explored the manner of infliction on PRP in cell culture.2. Study the effect of PRP on human osteoblast-like cells MG63 of biological behavior in vitro.A phase contrast microscope was used to observe the MG63 cells biology behavior. The differentiation of cells was detected by alkaline phosphatase (ALP) activity. Propidium iodide staining (PI) fluorescent coloration was used to observe the morphology of cells using by a phase contrast microscope. Immunocytochemistry was used to evaluate the expression of TGF-βof MG63 in different concentration PRP. The morphology of cells, which adhere to titanium dish and the artificial calcium phosphate bone material was observed by scanning electron microscopy. The proliferation was evaluated by using CCK-8 proliferation assay. The cell cycle assay was performed to study the effect of PRP on MG63 in different times.The expression of PDGF, VEGF, collagen type I (COL-1)and bone morphogenetic protein (BMP-4) mRNA was measured using semiquantitative reverse transcriptase-polymerase chain reaction and Real Time-PCR reaction. The protein expression of c-fos, c-myc, p27 and vinculin also determined by western blotting.3. Study the Effect of PRP on human adipose-derived mesenchymal stem cells of biological behavior in vitro.In the osteogenesis inducement experiment the morphology of cells was studied with a phase contrast microscope. To evaluate the growth differentiation, alkaline phosphatase activity was assessed. The CCK-8 assay was used to examine the effects of PRP on cells viability with and without osteogenic induction culture medium. A phase contrast microscope was used to observe the ability of mineralization detected by using calcium-cobalt staining method. The expression of PDGF, VEGF, COL-1and bone morphogenetic protein(BMP-4) mRNA was measured using semiquantitative reverse transcriptase-polymerase chain reaction and Real Time-PCR reaction.4. Study the effect of PRP on human dermal fibroblasts of biological behavior in vitro.A phase contrast microscope was used to observe the alizarin reds staining for the formation of calcium node. The activity of alkaline phosphatase was detected by using calcium-cobalt staining method. Fluorescent coloration was used to observe the cell proliferation, which adhered to the titanium material. Immunocytochemistry was used to evaluate the expression of PDGF, TGF-βand VEGF in different concentration PRP. Cell cycle of hDFbs was detected by flow cytometry. Evaluation of cell proliferation by CCK-8 assay in osteogenic induction condition and without osteogenic induction. The expression of PDGF and VEGF mRNA was measured using semiquantitative reverse transcriptase- polymerase chain reaction and Real Time-PCR reaction.5. Study the effect of PRP on rabbit calvaria defects rehabilitation.X-ray was useds to urvey the bone density and histologic analyses was used to determin newly formed bone in defects.RESULTS:The results of ELISA demonstrate that the level of PDGF achieved by the third method was higher than others, TGF-βby the PCCS was higher than other kits, the concentrations of VEGF in the Curasan PRP kits was most high. Only use calcium chloride and calcium chloride added thrombin were more effective activation than control group, but the two activation methods no statistics signification. The third method for preparing PRP has the lowest the activation percent of platelet than that of other groups, but there was a marked increased for PCCS PRP system in room temperature of activated platelet. The microscope investigation shows that centrifugation could clean out the RBC in the PRP this will be advantageous for observing cells.These data suggest that PRP has a positive influence on MG63 proliferation, transference. PRP improved MG63 attached on the material. Semiquantitative reverse- transcription polymerase chain reaction and RT-PCR analysis showed that PRP enhanced the levels of VEGF, Collagen type Iand BMP-4mRNA but PDGF levels descended. Western blotting res result suggested that the levels of protein expression of c-fos,c-myc,p27gene were enhanced when added PRP but vinculin adhesion plaques gene was restrained.A suitable concentration improved the proliferation of hADSCs. PRP added osteogenic induction culture medium had a more strong stimulate in cell proliferation than without osteogenesis inducement. PRP enhanced the levels of PDGF, VEGF, Collagen type I and BMP-4mRNA.A suitable concentration improved the proliferation of hDFb. The differentiation was stimulated when PRP in osteogenic induction condition than without PRP. The notability increase in PDGF was observed when added higher concentration PRP, but TGF-βand VEGF need a suitable concentration to improve expression in cells. The levels of PDGF ascended when only in osteogenesis inducement but decended when PRP was added in osteogenic induction culture medium. The reactions of VEGF to PRP were mostly ascending.Compared with the defects were filled by artificial bone material only, the platelet-rich plasma added with material group had a statistically greater amount of bone formation than control group. The bone density also improved by PRP. PRP accelerate the amalgamation of bone material and autologous bone.CONCLUSIONS:Within the limits of this study, it can be concluded thatThird method which blood was centrifuged at 1 600 r/min for 10 min and then centrifuged at 5 000 r/min for 5 min can decrease the the activation percent of platelet in the PRP. Low temperature is necessary for preparing PRP. Freezen preservation can't affect the biofunction of PRP. Only CaCl₂ can active the PRP without thrombin. The most important function of PRP was to promote the proliferation of cells, which were provided in tissue rehabilitation. The different function of the cells were endowed by other factors, which secreted by the cells in different local tissues.The cells coming from different species and different systems, staying at different biology stats and different inside environment will behave different reaction in the PRP.The reason of PRP improving the migration of MG63 may be due to depress the the levels of protein expression of vinculin adhesion plaques gene.The experiments in vitro and in vivo all approve the favorable effection of suitable concentration PRP markedly enhanced cells proliferation, bone-related gene and collagen production,These experiments established the groundwork of futher study, afforded theory evidences for clinic application, provided pathway for producing large numbers of mesenchymal stem cells in tissue engineering research.