| Part Ⅰ In vitro study:Comparison of the effects of autologous Lp-PRP and Lr-PRP on human degenerated intervertebral disc nucleus pulposus cellsObjective:To compare the effects of autologous Lp-PRP and Lr-PRP on human degenerated intervertebral disc nucleus pulposus cells.Methods:Eligible patients were included according to the inclusion criteria.Their blood was collected for preparing both Lp-PRP and Lr-PRP,blood count was detected and TGF-β1,PDGF-BB,TNFα and IL-1β was measured by ELISA.Then the degenerated nucleus pulposus tissues was collected from the same patients from surgery for primary cell culture.The nucleus pulposus cells were then treated with autologous Lp-PRP,autologous Lr-PRP or PBS.Cell proliferation was detected by CCK8 and Ed U staining experiment,cell migration was detected by Transwell experiment,and apoptosis was detected through Annexin V/PI double staining method.Expression level of anabolic genes(Col Ⅱ、Aggrecan)and catabolic genes(MMP-1、3、13)were detected by q PCR.Col Ⅱ and Aggrecan protein synthesis were detected by immunofluorescence,Western blot,and Alcin blue staining.Results:1.There was no significant difference in platelet counts between the prepared autologous Lp-PRP and Lr-PRP(P>0.05),and also no significant difference in PDGF-BB and TGF-β1 level between the two kinds of PRP(P>0.05,respectively).The leucocytes in Lr-PRP were significantly higher than those in Lp-PRP(P<0.05),and also the level of TNFα and IL-1β were significantly higher in Lr-PRP(P<0.05,respectively).2.Compared to the control group,both kinds of the autologous PRP can promote the proliferation and migration of human nucleus pulposus cell,and promote both gene and protein expression of Col Ⅱ and Aggrecan(P<0.05,respectively).3.The above promoting effects were greater with Lp-PRP when compared to Lr-PRP(P<0.05,respectively),and Lp-PRP can also inhibit the apoptosis of human nucleus pulposus cells when compared to both control and Lr-PRP group(P<0.05,respectively),while Lr-PRP promoted the expression of MMP-1,3 and 13(P<0.05,respectively).Conclusion:1.Lp-PRP and Lr-PRP both contain a high concentration of platelets and growth factors such as TGF-β1 and PDGF-BB,while Lr-PRP contains more leucocytes and inflammatory cytokines such as TNFα and IL-1β.2.Through the experiments of human degenerated nucleus pulposus cells,we confirmed that both autologous Lp-PRP and Lr-PRP have the effect to repair degenerated intervertebral disc,and autologous Lp-PRP was better than Lr-PRP.The reason why Lr-PRP was less effective than Lp-PRP may due to the antagonism and catabolism induced by inflammatory cytokines such as TNFα and IL-1β in Lr-PRP.Part Ⅱ In vivo study:Comparison of autologous Lp-PRP and Lr-PRP intradiscal injection to treat rabbit disc degenerationObjective:To compare the effects of autologous Lp-PRP and Lr-PRP intradiscal injection to treat rabbit disc degeneration.Methods:24 rabbits were randomly divided into three groups(Lp-PRP group,Lr-PRP group and control group),blood was collected and Lp-PRP or Lr-PRP was prepared according to the grouping situation.Then the rabbit disc degeneration model was induced by annulus puncture at L4/5 and L5/6 level.4 weeks later,MRI was used to confirm that the degeneration model was created.Then intradiscal injection of autologous Lp-PRP,autologous Lr-PRP or Saline was conducted according to their grouping.X-ray and MRI were examined before injection,4 and 8 weeks after injection.X ray was used to measure the disc height and MRI quantitative T2 mapping technique was used to measure the nucleus pulposus T2 value.Disc samples were collected 8 weeks after injection,then HE staining and Col Ⅱ immunohistochemistry were examined.Results:The rabbit degeneration models were created and all confirmed by MRI 4 weeks after surgery.The X-ray measurement showed there was no significant difference between the three groups in disc height before injection(P>0.05,respectively),however,the disc height was shown as Lp-PRP group>Lr-PRP group>control group at 4 weeks after injection(P<0.05,respectively),and this trend maintained until 8 weeks after injection(P<0.05,respectively).Longitudinally,disc height in the control group gradually decreased,while it gradually recovered in both autologous PRP group.The MRI results showed there was no significant difference between the three groups in T2 value before injection(P>0.05,respectively),however,the T2 value was shown as Lp-PRP group>Lr-PRP group>control group at 4 weeks after injection(P<0.05,respectively),and this trend maintained until 8 weeks after injection(P<0.05,respectively).Also T2 value in the control group gradually decreased,while it gradually recovered in both autologous PRP group.The degree of degeneration was shown as Lp-PRP<Lr-PRP<control group in HE staining.And the Col Ⅱ content was shown as Lp-PRP group>Lr-PRP group>control group according to the immunohistochemistry(P<0.05,respectively).Conclusion:In vivo,we further verified that both autologous Lp-PRP and Lr-PRP intradiscal injection can repair disc degeneration in rabbit model,and autologous Lp-PRP has a better effect than Lr-PRP.Part Ⅲ Preliminary clinical trial:Safety and short-term efficacy of autologous Lp-PRP intradiscal injection to treat lumbar discogenic painObjective:To investigate the safety and short-term efficacy of autologous Lp-PRP intradiscal injection to treat lumbar discogenic pain.Methods:A prospective cohort study was designed.Eligible patients were included according to the inclusion criteria and their baseline data were collected.The autologous LpPRP was harvested through apheresis method and then was injected into the painful disc.The patients were followed up 1 and 3 month after injection.The symptoms were evaluated by VAS and ODI scores and the complications were recorded.MRI was performed to evaluate disc degeneration at 3 month follow-up.Results:A total of 8 patients were included.Platelets concentration was 1492.9±266.2×10~9/L in the harvested Lp-PRP and it was 6.5 times of the whole blood.There was no leucocytes in the Lp-PRP.All patients acquired 3 months follow-up.The effective rate of LpPRP treatment was 62.5%.The average VAS score was 3.8±1.3,4.4±1.2 at 1 and 3 month follow-up,and that were significantly improved compared to 6.6±1.1 at the baseline(P<0.05,respectively).The average ODI score was 21.1±5.8,23.9±5.8 at 1 and 3 month follow-up,and that also were significantly improved compared to 37.8±8 at the baseline(P<0.05,respectively).No complications or adverse effects were reported.Conclusion:Autologous Lp-PRP intradiscal injection for the treatment of lumbar discogenic pain has good safety and short-term efficacy. |
