Transfection Of Lentivirus-bone Morphogenetic Protein 2 And Lentivirus-inhibitor Of Differentiation 1 Into Nucleus Pulposus For Delaying Intervertebral Disc Degeneration In An In Vivo Rabbit Model

Objective: To investigate if the course of intervertebral disc degeneration(IDD)is delayed by injecting lentivirus(Lv)vector carrying bone morphogenetic protein 2(BMP-2)and inhibitor of differentiation 1(Id1)genes directly into the nucleus pulposus.Methods:(1)The annular puncture model of IVD degeneration in rabbits established via the transabdominal approach was validated radiologically and histologically;(2)Thirty-two New Zealand white rabbits,2.0-2.5 kg in weight and 3 months in age,were used to establish the IDD models at L3/4、L4/5 and L5/6 discs with annular puncture via transabdominal approach.Thirty rabbits with successful modeling were randomly divided into 5 groups,6 rabbits every group.At 4 weeks after modeling,rabbits were injected with Lv-BMP-2(group A),with Lv-BMP-2 and Lv-Id1(group B),with Lv-Id1(group C),with Lv-green fluorescent protein(group D),and with PBS(group E).At 2,4,and 8 weeks after injection,T2-mapping MRI was performed on 2 rabbits each group to obtain the T2 values,and then subsequently the lumbar disc tissues were harvested to test the m RNA expressions and contents of collagen type Ⅱ and proteoglycan by real-time fluorescent quantitative PCR and ELISA methods.Results:(1)The operations had no significant effects on the rabbit body weight,and partial mild intra-abdominal adhesions were found in only 1 rabbit.The punctured discs were confirmed to be those of interest post-surgery and displayed progressive degeneration in disc height index(%)and MRI signal.A histological analysis at 4 weeks after puncture revealed notochordal cell loss from the nucleus pulposus,fibrocartilage filling the nucleus pulposus space,and annulus fibrosus disorganization.(2)T2-mapping MRI demonstrated that there was no significant difference in the T2 value between different groups at immediate and 2 weeks after injection(P>0.05).The T2 value of groups A and B was significantly higher than that of groups C,D,and E at 4 weeks after injection(P<0.05),but no significant difference was observed between group A and group B(P>0.05).The T2 value of group B was significantly higher than that of the other groups at 8 weeks after injection(P<0.05).The real-time fluorescent quantitative PCR and ELISA showed that the expressions and contents of collagen type Ⅱ and proteoglycan in group B were significantly higher than those in the other groups at 2,4,and 8 weeks after injection(P<0.05).Conclusion:(1)The annular needle puncture model established through a transabdominal approach,which demonstrates better visualization,exact identification,consistent degeneration degrees and minimal complications,is radiologically and histologically consistent with human IVD degeneration.(2)Combined application of Lv-BMP-2 and Lv-Id1 can delay IDD changes in rabbit IDD models.