Comparative Effects Of Pure Platelet-Rich Plasma And Leukocyte-and Platelet-Rich Plasma On The Biological Characteristics Of Nucleus Pulposus-Derived Mesenchymal Stem Cells

Objective:This study is to comparetively evaluate effects of pure platelet-rich plasma(P-PRP)and leukocyte-and platelet-rich plasma(L-PRP)on proliferation,differentiation,expression of extracellular matrix and activation of NF-?B signaling pathway in rabbit nucleus pulposus-derived mesenchymal stem cells(NPMSCs).Methods:A total of 24 adult New Zealand White rabbits(Female,6–8months old,3.0–4.0 kg)were randomly divided into three groups: control,P-PRP and L-PRP groups.NPMSCs isolated from NP tissues at P2 were treated with different concentrations(0%,5%,10%and 20% Vol/Vol)of L-PRP or P-PRP,and the control was treated with basic medium(DMEM +2%FBS)only.The cell proliferation and the expression of stem cell markers(Oct4,Nanog),pro-inflammatory cytokines(TNF-?,IL-?)production of extracellular matrix(Collagen II,Aggrecan)and nuclear NF-?B p65 protein were validated by CCK-8assay,quantitative real-time polymerase chain reaction(qRT-PCR),enzymelinked immunosorbent assay,immunostaining and westernblot respectively.Results:Both P-PRP and L-PRP promoted cell proliferation in a dose-dependent manner with maximum proliferation at a 10 % PRP dose.The cells morphology of experimental groups gradually changed from cobblestone-like cells to elongated spindle-shaped nucleus pulposus-like cells after two weeks.Furthermore,the results of qRT-PCR suggested that the expression of stem cell marker gene(Oct-4,Nanog)in experimental groups decreased 30% compared with controls.In contrast,the expression of nucleus pulposus cell-related genes(Collagen?,Aggrecan)in the experimental groups was 3 to 4 holds higher than that in control group L-PRP had significantly higher concentrations of leukocytes and pro-inflammatory cytokines(TNF-?,IL-1?)compared with P-PRP(P<0.05).Meanwhile,L-PRP induced predominantly inflammatory changes in differentiated NPMSCs by up-regulating the gene expression of pro-inflammatory cytokines(IL-1?,TNF-?)and the corresponding protein expression.Furthermore,L-PRP enhanced activation of the NF-?B pathway in differentiated NPMSCs and increased the expression of catabolic marker,including matrix metalloproteinase-1(MMP-1,MMP-13).In contrast,P-PRP promoted more extracellular matrix production of differentiated NPMSCs.Conclusion:Both P-PRP and L-PRP can induce the proliferation and NP-differetiation of NPMSCs;Compared to L-PRP,P-PRP can prohibit the activation of the NF-?B pathway and reduce the inflammatory and catabolic responses.