[Changes of nucleus pulposus after culture of whole intervertebral discs].
OBJECTIVE
To explore a practical method of culturing discs organ system by observing the changes of the nucleus pulposus after the whole intervertebral discs (including cartilage end-plate, nucleus pulposus, and annulus fibrous) were cultivated.


METHODS
A total of 335 intervertebral discs were taken out completely from 60 healthy SD rats (about 150 g) aged 5-6 weeks of clear grade and rinsed by high osmotic saline solution containing heparin, then put to the culture plate after being divided into 5 groups randomly. The whole intervertebral discs were cultured with high osmotic (410 mOsmol/kg) culture medium and changed the medium once every day, then the cell viability (n=15), HE staining (n=15), Safranin O staining (n=15), and immunohistochemistry staining (n=2) were observed at 0, 3, 7, 14, and 21 days; RT-PCR result (n=5) was observed at 0, 3, 7, and 14 days.


RESULTS
The cell viability was not changed significantly within 14 days (P > 0.05) and was significantly lower at 21 days than at other time points (P < 0.01). The immunohistochemistry staining results for collagen type II were positive in nucleus pulposus cells at every time point. HE staining showed that the tissue integrity and morphology of the whole intervertebral discs were not changed within 14 days. Safranin O staining showed no significant difference in the matrix grey scale within 14 days (P > 0.05) and significant differences between 21 days and 0-14 days (P < 0.05). RT-PCR results showed that the mRNA expression of collagen type I increased with time, but the expressions of collagen type II, aggrecan, and decorin decreased, showing significant differences in the mRNA expressions of the matrix protein at each time point (P < 0.05).


CONCLUSIONS
High osmotic saline solution containing heparin could be used to cultivate the whole intervertebral discs, it is an ideal model for further studies on physiology and pathology of intervertebral discs.
Li
Gang
G
Department of Orthopaedics, Sir Run Run Shaw Hospital Affiliated School of Medicine, Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou Zhejiang, 310016, P.R. China.
Fan
Shunwu
S
Zhao
Fengdong
F
Zhang
Congming
C
He
Rongli
R
D000818:Animals; D002470:Cell Survival; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism*; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley
D024042:Collagen Type I; D024043:Collagen Type II