[BMSCs-chitosan hydrogel complex transplantation for treating intervertebral disc degeneration].

OBJECTIVE: To investigate the therapeutic effect of BMSCs-chitosan hydrogel complex transplantation on intervertebral disc degeneration and to provide experimental basis for its clinical application.

METHODS: Two milliliter of bone marrow from 6 healthy one-month-old New Zealand rabbits were selected to isolate and culture BMSCs. Then, BMSCs at passage 3 were labeled by 5-BrdU and mixed with chitosan hydrogel to prepare BMSCs-chitosan hydrogel complex. Six rabbits were selected to establish the model of intervertebral disc degeneration and randomized into 3 groups (n=2 per group): control group in which intervertebral disc was separated and exposed but without further processing; transplantation group in which 30 microL of autogenous BMSCs-chitosan hydrogel complex was injected into the center of defected intervertebral disc; degeneration group in which only 30 microL of 0.01 mol/L PBS solution was injected. Animals were killed 4 weeks later and the repaired discs were obtained. Then cell 5-BrdU labeling detection, HE staining, aggrecan safranin O staining, Col II immunohistochemical staining and gray value detection were conducted.

RESULTS: Cell labeling detection showed that autogenous BMSCs survived and proliferated after transplantation, forming cell clone. HE staining showed that in the control and transplantation groups, the intervertebral disc had a clear structure, a distinct boundary between the central nucleus pulposus and the outer anulus fibrosus, and the obviously stained cell nuclear and cytochylema; while the intervertebral disc in the degeneration group had a deranged structure and an indistinct division between the nucleus pulposus and the outer anulus fibrosus. Aggrecan safarine O staining notified that intervertebral disc in the control and transplantation groups were stained obviously, with a clear structure; while the intervertebral disc in the degeneration group demonstrated a deranged structure with an indistinct division between the nucleus pulposus and the anulus fibrosus. Col II immunohistochemical staining showed that the tawny-stained region in the control group was located primarily in the central nucleus pulposus with a clear structure of intervertebral disc, the central nucleus pulposus in the transplantation group was positive with obvious tawny-stained intercellular substances and a complete gross structure, while the stained color in the degeneration group was lighter than that of other two groups, with a indistinct structure. Gray value assay of Col II immunohistochemical staining section showed that the gray value of the control, the transplantation and the degeneration group was 223.84 +/- 3.93, 221.03 +/- 3.53 and 172.50 +/- 3.13, respectively, indicating there was no significant difference between the control and the transplantation group (P > 0.05), but a significant difference between the control and transplantation groups and the degeneration group (P < 0.05).

CONCLUSION: The rabbit BMSCs-chitosan hydrogel complex can repair intervertebral disc degeneration, providing an experimental foundation for the clinical application of injectable tissue engineered nucleus pulposus complex to treat intervertebral disc degeneration.