Preliminary Study On The Prepareation Growth Factors From Mixed Human Platelet Rich Concentration

As we all know that platelet plays an important role in promoting angiogenesis and blood coagulation. As research continues, people found that platelet also play an important role in promoting wound healing and tissue repairing.Nowadays,Clinical blood platelet preparations have been used in orthopaedic, dermatology, chronic care and other clinical fields, but they are all autografted. This research discusses the possibility of mixed human platelet growth factor preparations that has been inactivated by virus and complement, used in cells culture and tissue repair, by comparing the differencein growth factor content and the effect on cells proliferation of platelets mixed people blood platelet preparations with monomer platelet preparation. Furthermore, this paper also exploreswhat are the key materials in platelet related preparationthat can promote cell proliferation.Objective:Find outcell’s maximum tolerated concentration for the S/D reagent concentrations. To explore the feasibility of platelet growth factors preparated from mixed human platelets used in cell culture. Preliminary explore whether the growth factor is the main content of blood products in promoting cell proliferation.Methods:Add the S/D reagents, in a certain gradient concentration, to the culture medium containing 10% fetal bovine serum and the culture medium containing 10% platelet growth factors solution, then use them to culture cells separately, and compare the difference of their cell proliferate. Compare the difference of growth factor contents by repeated freezing and thawing methods and the effects on promoting cell proliferation of several bags platelet concentrate and their blend separately. Divide the growth factor rich solution into two parts,one of themdoes complement inactivated processing(56℃, 30min), the other has no special treatment. Contrast their differences in growth factor content and promoting mice fibroblast cell and human fibroblast cell proliferation. Get platelet growth factors by S/D method, and,at the same time, achieve the role of virus inactivating, purificate them through column chromatography, then test the growth factor’s recovery ratio andthe effect on promoting cell proliferation in the whole process. Use saline to wash fresh platelet concentrate, to get platelet-poor plasma, washing platelet, physiological saline supernatant and untreated platelet concentrate. Make growth factors in the above-mentioned four kinds of solutions by repeated freezing and thawing method,divide all sample solutions into two parts, one of them does complement inactivated processing(56℃,30min), the other has no special treatment. Test their difference in promoting mice fibroblast cell proliferation and human fibroblast cell proliferation.Results:Higb concentration of S/D can inhabit cells proliferation obviously.By the observation of polarizing microscope, it appears cytomorphosis and cells rupturing. When the concentration of S/D in culture medium, which contains 10% platelet growth factors,falls below TNBP 5ppm, it has no effect on cell proliferation.While,the maximum tolerated concentrationof cells in culture medium containing 10% fetal bovine serum is TNBP 10ppm. By ELASA test, we find that there is no differencein growth factors’ contents when the Severalbags platelets and their blends been through the repeated freezing and thawing process. Also, there is no statistical discrepancy in growth factors’ contents before and after the Solution rich in platelet growth factors been complements inactivated. S/D stimulatesplatelet to release growth factors while it inactivated virus at the same time. Samples are purificated by column chomatography, test the changes in content of growth factors before and after the process. We find that the recovery rate is below 70% and S/D residue concentration is higher than stipulated in the National Pharmacopoeia Safety. And use the samples to culture cells, but results in the death of cells.Use Saline to wash platelet rich concentrate to get several components, then make up growth factors through repeated freezing and thawing.But it shows that there’s no statistical difference in the content before and aftercomplement inactivation.In promoting cell proliferation, for human epithelial fibroblasts, there is no significantstatistically difference in promoting cell proliferation before and after the complement inactivation. Comparingthe various components, there is no statistically significant differences of promoting cell proliferation between platelet rich plasma/platelet concentrate, poor platelets and suspension platelet group component, but their effect on promoting proliferation are all greater than the physiological saline group. For mice fibroblast proliferation, platelet rich plasma/plateletconcentrate andpoor platelet plasma group samples inhibit the proliferation of cells before been complement inactivation, butpromote proliferation of cells afterbeen complementinactivated. While washed plateletpromote proliferation of cells either they havebeen complementinactivated or not.Comparison between each component, influence on cell proliferation is different significantly.Conclusions:There is no obviousdifference neither in quantity level nor promoting cells proliferation between growth factors rich solution prepared from monomer and mixed platelet. S/D can effectively stimulate platelet to release growth factors and inactive virus at the same time. The main materials to promote cell proliferation in platelet related products are more than growth factors, it’s a combination of multiple factors, and there may be differences among species.