[Construction of injectable tissue engineered nucleus pulposus in vitro].
OBJECTIVE
To investigate the feasibility of using thermo-sensitive chitosan hydrogen as a scaffold to construct tissue engineered injectable nucleus pulposus (NP).


METHODS
Three-month-old neonatal New Zealand rabbits (male or female) weighing 150-200 g were selected to isolate and culture NP cells. The thermo-sensitive chitosan hydrogel scaffold was made of chitosan, disodium beta-glycerophosphate and hydroxyethyl cellulose. Its physical properties and gross condition were observed. The tissue engineered NP was constructed by compounding the scaffold and rabbit NP cells. Then, the viability of NP cells in the chitosan hydrogel was observed 2 days after compound culture and the growth condition of NP cells on the scaffold was observed by SEM 7 days after compound culture. NP cells went through histology and immunohistochemistry detection and their secretion of aggrecan and expression of Col II mRNA were analyzed by RT-PCR 21 days after compound culture.


RESULTS
The thermo-sensitive chitosan hydrogel was liquid at room temperature and solidified into gel at 37 degrees C (15 minutes) due to crosslinking reaction. Acridine orange-propidium iodide staining showed that the viability rate of NP cells in chitosan hydrogel was above 90%. Scanning electron microscope observation demonstrated that the NP cells were distributed in the reticulate scaffold, with ECM on their surfaces. The results of HE, toluidine blue, safranin O and histology and immunohistochemistry staining confirmed that the NP cells in chitosan hydrogel were capable of producing ECM. RT-PCR results showed that the secretion of Col II and aggrecan mRNA in NP cells cultured three-dimensionally by chitosan hydrogen scaffold were 0.631 +/- 0.064 and 0.832 +/- 0.052, respectively, showing more strengths of producing matrix than that of monolayer culture (0.528 +/- 0.039, 0.773 +/- 0.046) with a significant difference (P < 0.05).


CONCLUSIONS
With good cellular compatibilities, the thermo-sensitive chitosan hydrogel makes it possible for NP cells to maintain their normal morphology and secretion after compound culture, and may be a potential NP cells carrier for tissue engineered NP.
Tian
Huake
H
Department of Orthopedics, Xinqiao Hospital, the Third Military Medical University, Chongqing, 400037, P.R. China.
Wang
Jian
J
Chen
Chao
C
Liu
Jie
J
Zhou
Yue
Y
D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry; D001854:Bone Marrow Cells / Q000166:cytology*; D018929:Cell Culture Techniques; D048271:Chitosan; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D007403:Intervertebral Disc / Q000166:cytology; D011817:Rabbits; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds
D001672:Biocompatible Materials; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D048271:Chitosan