Intervertebral disc degeneration is a chronic and complicate procedure, but its mechanism is still unclear and difficult for self-repairs. Recent studies addressing stem cell transplantation in the treatment of disc degeneration disease are still limited to the laboratory study. Research on biological properties of nucleus pulposus cells can provide theoretical basis for studying the mechanism underlying intervertebral disc degeneration, construction of the intervertebral disc by tissue engineering, and gene therapy.OBJECTIVE:
To investigate the characteristics of different generations of rabbit nucleus pulposus cells, searching for the best suitable seed cells to treat degenerative disc diseases.METHODS:
Nucleus pulposus cells from New Zealand white rabbits were separated, cultured and then passaged. The morphological changes of primary, passages 3 and 4 nucleus pulposus cells were observed by hematoxylin-eosin staining under an inverted microscope. The biological properties of rabbit nucleus pulposus cells were observed. Aggrecan and type II collagen expressions were detected by toluidine blue and immunocytochemistry staining, respectively. Type II collagen and glycosaminoglycan mRNA expressions in the nucleus pulposus cells were detected by reverse transcription-PCR.RESULTS AND CONCLUSION:
Rabbit nucleus pulposus cells were successfully cultured and passaged in vitro. Primary nucleus pulposus cells were round or polygonal, and the average adherence time was 7 days. The first and third generations of nucleus pulposus cells were round or polygonal, and have strong vitality. Hematoxylin-eosin staining showed that nuclei were in a uniform blue-black, and cytoplasm showed light pink. The cytoplasm of nucleus pulposus cells was sky blue stained for toluidine blue staining, and type II collagen immunohistochemical staining showed the cytoplasm of nucleus pulposus cells displayed yellowish-brown. Passage 4 nucleus pulposus cells appeared with degeneration, and type II collagen and glycosaminoglycan mRNA expression was significantly decreased compared with previous generations. The first three generations of nucleus pulposus cells were exuberant in metabolism and showed consistent phenotypes and normal expression of aggrecan and type II collagen. Passage 4 nucleus pulposus cells began to age and degenerate.