[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).


CONCLUSIONS
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.
Liu
Jie
J
Department of Orthopedics, Xinqiao Hospital, the Third Military Medical University, Chongqing, 400037, P.R. China.
Wang
Jian
J
Zhou
Yue
Y
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D017690:Cell Transplantation; D048271:Chitosan / Q000627:therapeutic use*; D004195:Disease Models, Animal; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D007403:Intervertebral Disc / Q000473:pathology; D007405:Intervertebral Disc Displacement / Q000601:surgery*; D011817:Rabbits; D023822:Tissue Engineering; D014184:Transplantation, Homologous
D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D048271:Chitosan