The Effect Of Leukocyte-removed Optimized Platelet-rich Plasma On The Biology Of Stem Cells Derived From Nucleus Pulposus In Early Degeneration Of Intervertebral Discs

Part ? Characterization of NPSCs from early degenerated intervertebral discs ObjectiveTo characterize NPSCs from early degenerated intervertebral discs.Methods1)Rabbit NPSCs from early degenerated discs induced by needle puncture were isolated and cultured.2)The isolated NPSCs were then characterized by Polymerase chain reaction(PCR)for stem cell markers and induced differentiation potential.Results1)The morphology of cells in the colonies also varied,with some of them being cobblestone-like and others being spindle-like.2)The NPSCs isolated from early degenerated discs(D-NPSCs)and healthy discs(H-NPSCs)had strong expression of CD29,CD44,and CD166;Meanwhile,they had negligible expression of CD4,CD8,and CD14.3)The isolated NPSCs were effective in the induced differentiation processes(osteogenesis,adipogenesis,and chondrogenesis).Compared to D-NPSCs,H-NPSCs formed more colonies and proliferated faster.ConclusionNPSCs can be isolated from rabbit early degenerated and healthy discs,which are effective in colony forming,MSCs markers' expression and induced multi-differentiation.Compared to D-NPSCs,H-NPSCs formed more colonies and proliferated faster.Part ? Effect of P-PRP and L-PRP on the proliferation and differentiation of NPSCs from early degenerated intervertebral discsObjectiveTo evaluate the effect of P-PRP and L-PRP on the proliferation and differentiation of NPSCs from early degenerated intervertebral discs.Methods1)NPSCs were seeded at the density of 1 × 104 in a 24-transwell system and cultured in DMEM-LG contain-ing P-PRP or L-PRP at different concentrations: 0%,5%,10%,15%,and 20%(vol/vol).2)To determine the effect of P-PRP and L-PRP on the gene expression of NPSCs,quantitative real-time poly-merase chain reaction(q RT-PCR)was used to analyze the following genes :stem cell-related gene(Oct-4,Nango),NP-related genes(Col II and AGC).3)To determine the effects of P-PRP and L-PRP on the protein expression of NPSCs,Western blot analysis and Immunostaining was used to analyze the following proteins :AGC and Col II.Results1)In the presence of P-PRP or L-PRP,cell proliferation rate increased in a PRP dose-dependent manner.At each time point,10% PRP concentration induced significantly higher cell proliferation rate compared with other groups.Compared to L-PRP,P-PRP showed similar effect in induced cell proliferation.2)Two markers of active NP cells,including AGC and Col II increased significantly compared to the control group,and P-PRP yielded the highest gene expression(P<0.05);Both P-PRP and L-PRP decreased the stemness of NPSCs.3)Western blot and Immunostaining of the active NP cell proteins,AGC,and Col II,were confirmed in highest intensity P-PRP treatment(P<0.05).ConclusionCell proliferation induced by P-PRP and L-PRP is in a dose-dependent manner with maximum proliferation at 10% dose;The exclusion of leukocytes in PRP exhibited no superiority in induced cell proliferation;P-PRP and L-PRP specifically induces NPSCs into active NP cells,and P-PRP can be more effective.Part ? Effect of P-PRP and L-PRP on the inflammation and catabolism of NPSCs from early degenerated intervertebral discsObjectiveTo evaluate the effect of P-PRP and L-PRP on the inflammation and catabolism of NPSCs from early degenerated intervertebral discs.Methods1)To determine the effects of P-PRP and L-PRP on the gene expression of NPSCs,quantitative real-time poly-merase chain reaction(q RT-PCR)was used to analyze the following genes: catabolic genes(MMP-1,MMP-13)and inflammatory marker genes(IL-1?,IL-6,IL-8,and TNF-?).2)To determine the effects of P-PRP and L-PRP on the inflammation of NPSCs,ELISA was used to analyze the following proteins: TNF-??IL-1?.Results1)P-PRP and L-PRP both decreased the inflammatory and catobolic gene expression.The inflammatory and catobolic gene expression was significantly lower in P-PRP group(P<0.05).2)The level of inflammatory cytokines(IL-1?,TNF-?)in the cell supernatants was consistent with the above gene expression data.The inflammatory and catobolic proteins was significantly lower in P-PRP group(P<0.05).ConclusionP-PRP activates less inflammatory and catabolic effects on NPSCs from early degenerated intervertebral discs.