Tissue-engineered Allograft Intervertebral Disc Transplantation For The Treatment Of Degenerative Disc Disease: An In Vitro And In Vivo Study

Degenerative Disc Disease (DDD) is one of the most popular disease whichusually leads to substantial pain, functional disorder and declining ofperformance on tasks. Nucleotomy and spine fusion has relieved the suffering oflots of patients. However, removal of the entire disc without furtherreconstruction of the defect alters the normal alignment and stability of the spineand usually leads to high incidence segmental instability and adjacent segmentdisease. The broad objectives of any treatments for disc degeneration should beboth to alleviate painful symptoms and to restore mechanical function. TissueEngineering treatment strategies, when appropriately targeted, have the potentialto effectively satisfy both objectives. Disc transplantation might be a promisingtreatment option for degerative disc diseaase. However, the degenerative changesof the transplanted disc might limit the long term clinical outcome of the disctransplantation. Thus, a modified disc regeneration techniche based on the totaldisc transplantation was needed to ensure the disc survival and the long termfunction preservation.In this study, drawing on the experience of tissue engineering idea, wemanage to optimize the effect of allograft total disc transplantation by directinjecting hTERT activated NP cells into the allograft disc. Furthermore, wedesign to testify whether the intervention of hTERT gene transfected NP cells canprevent the degeneration process after allograft total disc transplantation in abeagle dog model in vivo. 1. Construction of Tissue-Engineered Allograft Intervertebral Disc And InVitro ObservationObjectives: To investigate the NP cells activity and phenotype preservation afterrAAV2-hTERT transfection and to testify the feasibility to construct atissue-engineered allograft intervertebral disc by hTERT-NPc intervention.Methods: Nucleus pulposus cells were transfected with rAAV2-hTERT atmultiplicities of infection of1×105genomes per cell. The expression of hTERTmRNA and protein were assessed by RT-PCR and Western-blot at5,10,15,30and60days post-transfection; Real-time PCR and Elisa were used to testproteoglycan and type Ⅱcollagen secreted by nucleus pulposus cells, comparisonanalysis were performed between the test group and the control. The hTERT-NPcwere injected into the cryopreserved disc to construct a “Tissue-engineered”allograft disc (group A). EGFP-NPcs were used for negative control (group B)and untreated allografts were used for vacuity contrast (group C). A total of30discs received the injection procedure. Proteoglycan content within the NP areawas analyzed at10,20and30days post cell injection. PKH-26tracer technologywas used to determine the survival of the injected cells. In vitro cultivation of thetissue-engineered allograft intervertebral disc showed that the overallmorphology and structure of the disc was well preserved.Results: NP cells could be effectively transfected by rAAV2, positive hTERTmRNA and protein expression could be detected at5,10,15,30and60days posttransfection. No positive hTERT mRNA and protein expression was detected atthe control group. The hTERT gene expression reached a peak at10days posttransfection and dropped slowly with increasing culturing time. RAAV-hTERTtransfection effectively upregulated the proteoglycan and type Ⅱcollagenexpression which was significantly higher than the control. As to the expression of type Ⅰ collagen, however, no significant difference was detected between thetest and control. PKH-26positive cells were identified within the allograft discsin group A and EGFP positive cells in group B at30days. The proteoglycancontent in the NP displayed increasing trend to a different extent in the3groupsof disc, significant difference was identified between group C and group A/B.Conclusion：RAAV-hTERT could effectively transfect NP cells and upregulatethe proliferative capacity and ECM expression of NP cells cultured in vitro.Exogenous NP cells could survive within the allograft intervertebral disc for atleast30days and promote proteoglycan secretion. The function of the allograftintervertebral disc could be effectively optimized by this cell interventiontechnique.2. Tissue-engineered allograft intervertebral disc transplantation: an in vivostudy in beagle modelObjectives: To observe the outcome of the allograft intervertebral disc aftertransplantation and to testify whether the intervention of NP cells or hTERT genetransfected NP cells can prevent the degeneration process after allograft total disctransplantation in a beagle dog model in vivo.Methods:18beagle dogs were randomly divided into3groups and respectivelyhTERT-NPc allograft intervertebral disc implantation (Group A), NPc allograftintervertebral disc implantation (Group B, positive control) and allograftintervertebral disc implantation (Group C, blank control). MRI and X-rayexamination were performed at4,8and12weeks post implantation to observethe outcome of the implanted allograft intervertebral disc and to assess thedegeneration degree by analysis the disc height and the MRI T2signal intensityof the NP. At12weeks after operation, all dogs were sacrificed and the lumbarspines were harvested for the biomechanical analysis and then the intervertebral disc allografts were applied for histological analysis, ectogenic NP cell tracingand hTERT mRNA expressing analysis.Results: The implantation procedure could be successfully accomplished througha lateral frontal approach. Few animals appeared surgical complications such asincision hernia, temporal partial paralysis, graft falling off and suppurativeinfection. Except two cases of graft falling off, fine bone fusion betweenintervertebral disc allograft and adjacent intervertebral body were observed in therest animals. The disc height preservation in group A and B was better than groupC. MRI scan showed the typical disc degenerative changes in group C, the T2signal intensity of group A and B were higher than group C at all time points. At12weeks post operation, the normalized grayscale of the transplanted disc ofgroup A was significant higher than that of group B and C. Biomechanicalshowed that the ROM of flexion-extension and left-right rotation in group C wassignificant bigger than that of group A and group B, which indicated a poorstability preservation in group C. PKH-26positive cells were identified withinthe allograft discs in group A at12weeks, providing evidence of cell survival.Histological analysis showed the NP cell phenotype, cell number and distributionof the allograft discs was better preserved in group A and B compared with groupC at12weeks follow up.Conclusion: The allograft intervertebral disc could successfully survive in thehost and accomplish fine bone fusion with the host vertebral body. Exogenouscells could survive within the allograft intervertebral disc in the host for at least12weeks. NP cells or hTERT loaded NP cells intervention could effectively resistthe degeneration of the allogenic transplanted intervertebral discs in a beaglemodel. The hTERT loaded NP cells had a better anti-degeneration effect on thetransplanted disc than NP cells. Despite the need of a long time in vivo experiment to further validate our preliminary conclusion, this modified discregeneration technique through cell intervention has the potential to ensure thelong term function preservation of allograft disc transplantation.