[Experimental study on biological feature of rabbit intervertebral disc nucleus pulposus in vitro].
OBJECTIVE
To research the biological feature of intervertebral disc nucleus pulposus cells (NPCs) by observing cell amorphous, phenotype and ultramicrostructure.


METHODS
The NPCs from 2-week-old healthy rabbit were cultured in DMEM/F12 medium with 15% FBS. The cell biological features were observed by inverted phase contrast microscope, light microscope, electron microscope, cell vitality assay, cell growth curve and cells staining after harvest and during the periods of culturing the primary, the 1st passage and 2nd passage.


RESULTS
The results of inverted phase contrast microscope showed that the primary passage adhered at 5 days, grew exponentially at 6-8 days, and were subcultured after covering the bottom at 17 days. The phenotype of the NPCs changed from polygon to long fusiform with passage increased; the vitality assay showed that there was about 95%-97%, 98%-100%, 100% and 75%-80% NPCs survived just after isolation from intervertebral disc, during the period of culturing the primary, the 1st passage and the 2nd passage, respectively. The toluidine blue staining of the NPCs was strongly positive, and HE staining showed clear cell nucleus and cytoplasm. The I collagen immunohistochemical staining showed negative results in the 1st passage, but II collagen immunohistochemical staining and safranin O staining showed positive results. However, the I collagen immunohistochemical staining showed positive result in the 2nd passage, and II collagen immunohistochemical staining and safranin O staining showed weakly positive results. The cell growth curve showed the same as the growth course of cell cultured in vitro. The results of TEM showed that there were many glycogen particles and less chondriosomes in the primary passage. With the increased passage, the glycogen particles decreased and the chondriosomes increased, and cell organ became swell.


CONCLUSIONS
This study clarifies the biological feature of NPCs in vitro, providing the experimental basis for the seed cell research of the nucleus pulposus tissue.
Liu
Jie
J
Department of Orthopaedics, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, P.R. China.
Wang
Jian
J
Zhou
Yue
Y
D000818:Animals; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism*; D015388:Organelles / Q000473:pathology; D011817:Rabbits; D023822:Tissue Engineering
D024042:Collagen Type I; D024043:Collagen Type II