Effect Of AMSCs Compound Chitosan/Gp/collagen Ⅰ Transplantation On TNF-α ,IL-1β Content In Early Degenerated Intervertebral Disc Of Rabbit

Objective:To observe the effect of AMSCs (adipose-derived mesenchymal stem cells) compound injective thermo-sensitive chitosan/Gp/ collagenⅠscaffold transplantation on content of TNF-α,IL-1βin early degenerated lumbar intervertebral disc of rabit, and analyze the significances. To investigate the bromodeoxyuridine(BrdU) for labeling of rabit AMSCs in vitro and in vivo and explore the feasibility of using BrdU as a tracer to label stem cells.Methods:AMSCs were isolated from rabbit fat tissue by type I collagenase digestion and puri-fied by differential adhesion. A total of 24 white New Zealand rabbits (no limit of male or female) are randomly and equally divided into four groups. A. Degeneration model group: nucleus aspiration. B. AMSCs compound Chitosan/Gp/collagenⅠTransplantation group. C. Cell-free chitosan Transplantation group. D. Blank control group: only explore the target intervertebral disc. L2-3,L3-4,L4-5,L5-6 was chosed as target intervertebral disc. When aspirate pulposus with 21G needle,inject AMSCs-scaffold complex and chitosan scaffold respectively. The samples of L2-3,L3-4,L4-5,L5-6 intervertebral disc were obtained from two rabit at 2,4,8 week in each group after surgery. Elisa was adopted to inspect the contents of TNF-α,IL-1β, and differences among groups were compared.Result:25μmol/L Brdu cultivate with DMEM didn't affect the growth with AMSCs compared to normal cultured AMSCs, as the MTT test showed (P> 0.05). The immunofluorescence staining of target disc injected with AMSCs had showed BrdU lembled AMSCs survived at 2,4,8 week after surgry. All animals survived after the surgery. Compare with the Blank control group, the contents of TNF-α,IL-1βin degenerated model group both were increased signicantly(P<0.05). AMSCs compound chitosan Transplantation group and Cell-free chitosan Transplantation group compared to Degeneration model group , TNF-α,IL-1βdecreased dramatically, and the differences were significantly(P<0.05). When AMSCs compound chitosan Transplantation group compared to Cell-free chitosan Transplantation group, IL-1βdecreased significantly (P<0.05) at 8 week after surgery.Conclusion:1.AMSCs compound injective thermo-sensitive chitosan scaffold Transplantation in early degenerated lumbar intervertebral disc could decrease the content of TNF-α,IL-1β, and they may regulate the inflammatory response.2. The BrdU-labeling method achieves a high labeling rate, and has little impact on the growth of AMSCs, and it could be used in vivo study of intervertebral disc tissue engineering.