Effects Of PRP And LyPRP On Osteogenic Differentiation Of Mesenchymal Stem And Its Composite Bone Repair Scaffold

Objective:Platelet-rich plasma(PRP)is rich in a variety of growth factors and plays an important role in the proliferation and differentiation of mesenchymal stem cells.Preparation of lyophilizer platelet-rich plasma(LyPRP)can preserve platelets for long periods of time.Our study compared the osteogenic induction of growth factors to mesenchymal stem cells in PRP and LyPRP.Finally,collagen was used as a carrier to investigate the preliminary study of collagen and PRP,LyPRP composite bone repair materials.Method:The first part,the platelet-rich plasma(PRP)and lyophilizer platelet-rich plasma platelet-rich plasma(LyPRP)were preparation,Analysis the content of platelets,white blood cells,and red blood cells in PRP,LyPRP,and whole blood by animal blood cell analyzer.The platelets distributed on the PRP and LyPRP gels and the size of internal of pore was observed by scanning electron microscopy after PRP and LyPRP were activated.Enzyme-linked immunosorbent assay(ELISA)was used to detect the release of growth factors after activated PRP and LyPRP,such as transforming growth factor(TGF-?),vascular endothelial growth factor(VEGF),and platelet-derived growth factor(PDGF).In the second part,PRP and LyPRP stimulated rabbit bone marrow mesenchymal stem cells to explore the osteogenic differentiation.Standardized extraction of bone marrow mesenchymal stem cells from newborn rabbit and cultured third generation in vitro.The concentrations of PRP and LyPRP were 5%,10%and 20%respectively.Experimental group were mesenchymal stem cells cultured with PRP and LyPRP conditioned medium.Control group:mesenchymal stem cells cultured with osteogenic induction medium.CCK-8 was used to detect the proliferation of mesenchymal stem cells cultured with different concentrations of PRP and LyPRP culture medium.FDA/PI staining was used to analyze cell viability at 1 day and 3 days to determine whether different concentrations of PRP and LyPRP inhibited cell viability.Stimulating cells for 1,3,6,and 9 days under different concentrations of PRP and LyPRP conditioned medium,Alizarin red staining was performed to verify the presence of calcium nodules in the cells.The quantification of alizarin red was used to verify the accuracy of calcium salt content in the calcium nodules secretion of the experiment.Alkaline phosphatase staining was another important characterization of osteogenic induction,the cells were observed the alkaline phosphatase secreted at 9 days to determine whether the cells induced successfully.The alkaline phosphatase activity assay corresponded to alkaline phosphatase staining to further verify the experimental results.The osteogenic genes expression level of ALP,BMP-2 and OCN to detection from bone marrow mesenchymal stimulated with different concentrations culture medium at 1 days,3 days,6 days and 9 days.In the third part,collagen as a carrier was combined with PRP and LyPRP respectively for the preliminary study of bone repair scaffolds.The experimental groups were PRP(5%,10%,20%)+type ? collagen;LyPRP(5%,10%,20%)+type ? collagen.Control group was type ? collagen.Enzyme-linked immunosorbent assay(ELISA)was used to detect the release of growth factors such as TGF-?,VEGF,and PDGF in collagen combined with PRP and LyPRP.Scanning electron microscopy was used to observe the distribution of platelets on the collagen surface.The DMA mechanical tester was used to verify the mechanical properties of type ? collagen changed after adding different concentrations of PRP and LyPRP.Then the degradation experiments of the composite PRP and LyPRP hydrogels were carried out to calculate the degradation rate and find the optimal group with slow degradation.Bone marrow mesenchymal stem cells from newborn rabbits were wrapped into composite hydrogel(combined with different concentrations of PRP and LyPRP collagen),by culturing the encapsulated cell hydrogel for 1 day,3 days,7 days,and 14 days,it was macroscopically judged whether the contraction of collagen was effectively inhibited by the addition of PRP and LyPRP.FDA/PI staining was used to observe the activity of bone marrow mesenchymal cells in hydrogels.CCK-8 assay determines the proliferation of bone marrow mesenchymal stem cells in composite hydrogel.Results:Successfully prepared PRP and LyPRP,the content of platelets in PRP and LyPRP exceeds the content of platelets in whole blood.Platelets were observed on the surface of PRP and LyPRP gel fibers by scanning electron microscopy.In the enzyme-linked immunosorbent assay,the levels of VEGF and PDGF were higher than those of TGF-?.In the CCK-8 experiment,20%PRP and 10%LyPRP can effectively promote cell proliferation.In bone marrow mesenchymal stem cells cultured in 20%PRP and 10%LyPRP conditioned medium,obvious calcium nodules appeared at 9 days,and the alizarin red quantitative test also verified that the calcium salt content was the highest at this time.Alkaline phosphatase was secreted in 20%PRP and 10%LyPRP groups at 9 days.In both groups,on day 9,the activity of alkaline phosphatase was higher than other experimental groups.In the expression of related osteogenic genes,the expression of ALP,BMP-2 and OCN was significantly increased in 10%PRP and 5%LyPRP at 9 days.In 20%PRP and 10%LyPRP,cells proliferate too fast,and cell membranes grow.The metabolism of cells and the matrix secreted by cells will affect the regulation of related genes to some extent.The 10%PRP and 5%LyPRP were both reduced by one gradient from the previous concentration.The proliferation of cells is in a steady state environment,and the secreted matrix and the like are insufficient to cause interference with genes.In the hydrogel of PRP and LyPRP complex type ? collagen,the content of TGF-?is higher than the content of VEGF and PDGF.Platelets were uniformly distributed on the collagen surface by scanning electron microscopy.And the higher the concentration of PRP and LyPRP,the more platelets are loaded on the collagen surface.In the determination of growth factor content.The mechanical strength of the 10%PRP and 10%LyPRP composite collagen in DMA mechanical testing is the largest.In the degradation experiments,10%PRP and 10%LyPRP hydrogels were slowly degraded in PBS.Wrap the bone marrow mesenchymal stem cells into a type ? collagen gel rich in PRP and LyPRP,the cell gel block was cultured for 14 days,we found that collagen contraction was inhibited after the addition of PRP and LyPRP.FDA/PI staining also found that cell viability was best in 10%PRP and 5%LyPRP groups.It was found by CCK-8 that cell proliferation was significantly increased in 10%PRP and 5%LyPRP composite collagen.Conclusion:Both PRP and LyPRP can effectively promote the proliferation and induce osteogenic differentiation of mesenchymal stem cells.Compared with the osteogenic induction effect of PRP and LyPRP,PRP is superior to LyPRP.The reason is related to the lyophilization environment of LyPRP,and the performance of platelets is affected during the lyophilization process.The release of growth factors are slowed and the nutrients and differentiation functions provided by the cells are reduced.After adding PRP and LyPRP to collagen,its mechanical properties are improved.Wrap the cells into the complex collagen,inhibiting collagen shrinkage,material has good biocompatibility.Our results will provide a better donor for bone repair.