| Objective:To demenstrate the regenerative effects of transplanting with injectable tissue engineering nucleus pulposus to the degenerated intervertebral disc, and to provide a new approach for the treatment of the degenerative intervertebral disc disease.Methods:1 The culture,identification and choose of the Seed cells1.1 In vitro culture and identification of the Rabbit NP cells2-week-old healthy rabbit nucleus pulposus cells were cultured in DMEM / F12 medium with 15 % fetal bovine serum. Cell biological features were researched by inverted phase contrast microscope,light microscope,electron microscope,cell vitality assay,cell growth curve and cells staining.1.2 In vitro culture and identification of the Rabbit MSCsThe bone marrow was extracted from the tib of the Juvenile rabbit under anesthesia and sterile conditions. mesenchymal stem cells were obtained by density gradient centrifugation and inoculated to 100ml culture flask. The changes of their shapes, anchoring rate and ultrastructure were observed. The cell surface markers of mesenchymal stem cells were detected by flowcytometry, and induce it differentiate into osteoblast.1.3 Compared the NP cells with MSCs, and choose one for the seed cells to continue next expriment.2 The consturction of injectable tissue engineering nucleus pulposus and its identification.Isolated and cultured rabbit bone mesenchymal stem cells in vitro, cultured the third generation bone mesenchymal stem cells in chitosan hydrogels. We use the cells-hydrogels Complex to slice for HE staining,safranin O staining and type II collagen immunohistochemical stain to identify its differentiation.3 Establishment of intervertebral disc degenerative model Needle puncture injury disc applying microendoscopic discetomy-like technology to establish intervertebral disc degenerative model4 The transplantation of injectable tissue engineering nucleus pulposusInjected the tissue engineering nucleus pulposus into the degenerative disc.After raised for 4 weeks and 8 weeks,sacrificed the animals, isolated the injected disc and observed by histological stain and gene express essay,and respectively compared the results with untreated degenerative disc and normal disc to identify whether the complex could repair the degenerative disc.Result:1 The culture,identification and choose of the Seed cells1.1 Rabbit NP cells: 1,Observing by inverted phase contrast microscope showed that the primary NPCs adherenced in 5 days, and growed exponentially in 6～8 days, finally subcultured in 17 days. With the times of passage increased, however, the phenotype of the NPCs changed from polygon to long fusiform; 2,The vitality assay showed that there was about 95%～97% NPCs survived just before isolated from intervertebral disc, and during the cultured in primary,fisrt generation and second generation, there were respectively about 98%～100%,100% and 75%～80%; 3,The toluidine blue stain of the NPCs was strongly positive,and HE stain showed clearly the cell nucleus and cytoplasm. The type I collagen immunocytochemical stain showed negative in primay and first generation NPCs, but type II collagen immunocytochemical stain and safranin O stain showed positive.However, The type I collagen immunocytochemical stain showed positive in second generation NPCs, and type II collagen immunocytochemical stain and safranin O stain showed all weakly positive; 4,cell growth curve showed the same to the growth course of cell cultured in vitro; 5,It was observed by transmission electron microscope that there were many glycogen particles and less chondrosomes, with the times of passage increased, the glycogen particles decreased and the chondrosomes increased, and cell organ became swell.1.2 Rabbit MSCs: 1,The mesenchymal stem cells began to be anchored after culture for 2 hours, and more than 90% mesenchymal stem cells were fused on 15 days;2,By observing with transmission electron microscopy, the cultured cells showed the typical ultrastructure of stem cells in which there are so many microvillis on the one side of the cells, the nucleus of the cells is irregular and there are lots of cell organs;3,The cell surface markers of mesenchymal stem cells analysis indicated positive expression of CD44,CD90 and negtive expression of CD45. Cell growth cycle essay showed almost (about 97% in all cells essayed) mesenchymal stem cells were stayed in stage G1 and stage G2;4,After cultured 3 weeks by induced osteoblast, the mesenchymal stem cells gradually produced small calcium salts crystal, and the stainning of alkaline phosphatase showed positive, the stainning of Von Kossa showed black reaction places in which there are so many calcium salts in the cell clone.1.3 Because its resource is widespread and easy to culture and amplification, the MSCs is the most suitable seed cell for constructing the tissue engineering nucleus pulposus.2 The consturction of injectable tissue engineering nucleus pulposus and its identification.MSCs can survive and proliferation in hydrogels, and we also find it can differentiate to nucleus pulposus-like cells which synthesis type II collagen and aggrecan.3 Establishment of intervertebral disc degenerative model3.1 results of HE stainning4 weeks after operation,there are so many leakages and chondrocyte metaplasia in the region of nucleus pulposus , the height of intervertebral disc decreased , Nishimura classify:2-3;8 weeks after operation,the height of intervertebral disc decrease mostly,nucleus pulposus cells disappeared, Nishimura classify:4-5.3.2 results of IHC stainning4 weeks after operation,the positive sign of degenerative disc is weaker than normal disc,structure of disc is abnormal;8 weeks after operation,the result of degenerative disc IHC staining is negtive。4 The transplantation of injectable tissue engineering nucleus pulposus1. the HE stainning showed that the control and the treat teams can remain the integrity of IVD,but the degenerated team showed that the constructure of IVD is loss; 2. Aggrecan safarine O stainning and II-collagen IHC showed that they could differentiate to nucleus pulposus-like cells which synthesis and secrete II-collagen and aggrecan,the complex can remain the biological characteristic of nucleus pulposus.In contrast,the untreated discs showed that their nucleus pulposus collapse and fibrosis which in return the synthesis of the II-collagen and aggrecan decreased dramatically. 3. Aggrecan and II-collagen RT-PCR shows that the gene express between treat team and normal team is no significant variance,but which has significant variance with the untreat team. (p<0.05)Conclusion:1. The MSCs which its resource is widespread and is easy to be isolated are the most suitable resource for the tissue engineering seed cells;2. chitocan has favourable histocompatibility and can induces MSCs to differentiated to NP cell-like cells;3. The tissue engineering nucleus pulposus can repair the degeneration of intervertebral disc.
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