Construction Of Tissue Engineering Intervertebral Disc Using Allograft Intervertebral Disc With The Cannie Nucleus Pulposus Cell Expressing Bone Morphogenetic Protein-7

Human intervertebral disc degeneration is mainly reason for low back pain and neck-should pain. The ratio of degenerative disc disease step up obviously with lengthening average longevity of human and increasing social work stress. Moreover, the expectant treatment and surgical treatment couldn't thoroughly solve clinical problem at present. It has been proved that the frozen allogenic intervertebral discs could survive in animal study and clinical trail. Furthermore, the allogenic intervertebral disc could completely relieve clinical symptoms in clinical trail. The motion and stability of the spinal unit is preserved after transplantation of frozen allogenic intervertebral discs. But signs of mild disc degeneration were observed in allogenic intervertebral disc transplantation. This mild disc degeneration will cause clinical symptoms emergence once more. The present study results showed that the change of intervertebral disc degeneration could be prevent, even repair, by increasing the active of nucleus pulposus cell. So in this study, tissue engineering intervertebral disc were constructed by using allograft intervertebral disc with the cannie nucleus pulposus cell expressing bone morphogenetic protein-7. Furthermore, the bioactive and function were also investigate in this study.1. Effects of different storage temperatures and times on cell viability of cryopreserved intervertebral discObjectives. To investigate effects of different storage temperatures and times on cell viability of cryopreserved intervertebral disc and obtain the optimal storage condition.Methods. The 52 canine intervertebral discs were divided into the control group, liquid nitrogen group and -80℃group. The intervertebral discs in liquid nitrogen group and -80℃group were immerged into the freezing preservation medium and storage in liquid nitrogen and -80℃low temperature refrigerator. At the time-points of 2w,1m,2m,4m,6m and 12m, the ratio of different position cell survival were detected by EB/FDA dying method, the PG content of NP tissue were detected by DMMB method, and the collagen content were detected by hydroxyproline method.Results. The ratio of cell survival in outer layer annulus fibrosus is higher than that in inner layer annulus fibrosus and nucleus pulposus in both groups (p<0.05). The ratio of cell survival is not significantly different in inner layer annulus fibrosus and nucleus pulposus. The ratio of cell survival in inner layer annulus fibrosus in liquid nitrogen group is higher that in -80℃group in the 2nd storage week (p<0.05). The ratio of cell survival in the both groups was not different from 1st to 12th storage month (p>0.05). The PG content reduced gradually with storage duration. From 1st to 12th storage month, the PG content in liquid nitrogen is higher than that in -80℃group. The collagen content in both groups reduced gradually in storage duration, and was not different(p>0.05). Conclusion:Both the ratio of cell survival and PG content of intervertebral disc storaged in liquid nitrogen is higher than that of in -80℃. In 1 year storage duration, intervertebral disc storaged in liquid nitrogen maintains the stable ratio of cell survival and PG content, is able to suitable for the basic requirement to allogenic transplantation, is able to be the scaffold of tissue engineering.2. Construction and identification of recombination adeno-associated virus type-2 vector carrying human bone morphogenetic protein-7Objectives: To construct and identification of the recombinant adeno-associated virus type-2 vector carrying human bone morphogenetic protein ( rAAV2-hBMP7).Methods: The recombinant AAV2 packaging plasmid pSNAV2.0 and full-length hBMP7 cDNA was respectively obtained by the simultaneous digestion of plasmid pDC316-BMP7-IRES-EGFP with AgeIase and NheIase. The full-length hBMP7 cDNA and recombinant AAV2 packaging plasmid pSNAV2.0 were then integrated using T4 DNA ligase. The integrated product was used to transfect the competence Bacillus coli DH-5α. The transfected bacillus coli DH-5αwas cultured in agarose culture medium containing ampicil. Monoclonal colonies were selected and inoculated into Luria Broth (LB) culture medium containing ampicil. After 12 hours in culture, plasmid pSNAV2.0-hBMP7 was extracted and the base sequence was analyzed. After constructed and identified plasmid pSNAV2.0-hBMP7, rAAV2-hBMP7 vector were constructed, amplifying and purifying by AAVMaxTM package and purification system. The optimal multiplicity of infection (MOI) for hNP cell were detected by rAAV vector carrying enhanced green fluorescent protein r(AAV2-EGFP).Results: Plasmid pSNAV2.0-hBMP7 was successfully constructed by integrating the full-length hBMP-7 cDNA and recombinant AAV2 packaging plasmid pSNAV2.0 using T4 DNA ligase. Plasmid pSNAV2.0-hBMP7 base sequence was correct by PCR detection and assay of base seguence. The rAAV2-hBMP7 vector was packaged by pSNAV2.0-hBMP7 plasmid using the standard calcium phosphate precipitation method. The titer was determined using quantitative DNA dot blots and purity was examined using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Titers averaged approximately 5.1×1011 vg/ml and purity was >97%.Conclusion:The rAAV2-hBMP7 vector can be successfully constructed by plasmid pSNAV2.0 package system. The titers and purity of the rAAV2-hBMP7 vector is suitable for next experiments.3.Effects of adeno-associated virus-2 mediated human BMP-7 gene transfection on the chondrocytic phenotype of nucleus pulposus cellsObjectives: To investigate the effects of adeno-associated virus-2 expressing human bone morphogenetic protein-7 (rAAV2-hBMP7) on canine nucleus pulposus cells.Methods: Following infection with rAAV-BMP7 vector at multiplicities of infection of 1×105 genomes per cell and subsequent culture, nucleus pulposus cells transferred with hBMP7 gene were assessed semi-qualitatively for BMP7 expression with real-time PCR. At 7 days post-transfection, proliferative ability of nucleus pulposus cells were comparative in the transfection and no-transfection cells. Aggrecan, typeⅠand typeⅡcollagen secreted by nucleus pulposus cells were qualitatively accessed at 4, 7 and 14 days post-transfection in the transfection and control groups.Results: The adeno-associated virus can successfully transfer human BMP7 into the canine NP cell. The NP cell transfected by rAAV-hBMP7 vector express human BMP7 at least 14 days. The expressed human BMP7 promote remarkably accumulation of proteoglycans 42% and 77% (p<0.05) higher than no-transfection cells at 7 and 14 days post-transfection, and typeⅡcollagen 63% and 94% (p<0.05).Conclusion: The rAAV-based gene delivery approach is capable of promoting the expression of proteoglycans and typeⅡcollagen of nucleus pulposus. This approach may be applied for the treatment of degenerative disc disease in the future.4. Construction of Tissue Engineering Intervertebral Disc Using allograft Intervertebral Disc with the Cannie Nucleus Pulposus Cell Expressing Bone morphogenetic protein-7Objective: To construct the tissue engineering intervertebral disc using allograft intervertebral disc with the cannie nucleus pulposus cell expressing hBMP7Methods: The 24 intervertebral discs storage in liquid nitrogen for 2 months were divided into EGFP, 1×104,1×105 and 1×106 groups. EGFP group: 20ul cell suspension including 1×105 nucleus pulposus cells expressing EGFP were injected into intervertebral disc. 1×104 group: 20ul cell suspension including 1×104 nucleus pulposus cells expressing hBMP7 by PKH-26 dying were injected into intervertebral disc. 1×105 group: 20ul cell suspension including 1×105 nucleus pulposus cells expressing hBMP7 by PKH-26 dying were injected into intervertebral disc. 1×106 group: 20ul cell suspension including 1×106 nucleus pulposus cells expressing hBMP7 by PKH-26 dying were injected into intervertebral disc. Intervertebral discs injected were immerged into 30 ml culture medium. The morphous, cell survival, cell fluorescence intensity, PG and collagen content of intervertebral discs in every group were assessed at 4, 7, 14 culture days.Results: The morphous of intervertebral discs in every group didn't change at 4 and 7 culture days. However, the calcium content in the lamina terminalis of intervertebral disc lost at 14 culture days. In EGFP group, nucleus pulposus cells expressing hBMP7 could be observed at any time points. Results showed that cell fluorescence intensity in 1×105 group was higher than that of 1×106 and 1×104 groups. Moreover, the PG and collagen contents in 1×105group were higher than that of 1×106 and 1×104 groups.Conclusion: The ideal tissue engineering intervertebral disc could be constructed using allograft intervertebral disc storaged in liquid combined with the cannie nucleus pulposus cell expressing hBMP7, and then be cultured for 7days in vitro.Summary:1. Both the ratio of cell survival and PG content of intervertebral disc storaged in liquid nitrogen is higher than that of in -80℃. In 1 year storage duration, intervertebral disc storaged in liquid nitrogen maintains the stable ratio of cell survival and PG content, is able to suitable for the basic requirement to allogenic transplantation, is able to be the scaffold of tissue engineering.2. The rAAV2-hBMP7 vector can be successfully constructed by Plasmid pSNAV2.0 package system. The titers and purity of the rAAV2-hBMP7 vector is suitable for next experiments.3. The rAAV-based gene delivery approach is capable of promoting the expression of proteoglycans and typeⅡcollagen of nucleus pulposus. This approach may be applied for the treatment of degenerative disc disease in the future. 4. The ideal tissue engineering intervertebral disc could be constructed using allograft intervertebral disc storaged in liquid combined with the cannie nucleus pulposus cell expressing hBMP7, and then be cultured for 7days in vitro.