Experimental Studies On Temperature-responsive Chitosan Hydrogel Of The Injectable Engineered Nucleus Pulposus

Objective: To investigate the feasibility of using thermo-responsive chitosan hydrogen as a scaffold to construct tissue engineered injectable nucleus pulposus (NP).Methods: The temperature-responsive chitosan hydrogel scaffold was made of chitosan,disodiumβ-glycerophosphate and hydroxyethyl cellulose. Its physical properties and general condition were observed and its characters of the toxicity and histocompatibility were evaluated. The tissue engineered NP was constructed by compounding the scaffold and rabbit NP cells. Then, the viabil ity 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 scanning electron microscopy 7 days after compound culture. NP cells went through histology and immunohistochemistry detection and their secretion of aggrecan and expression of collagen II mRNA were analyzed by RT-PCR 21 days after compound culture. Models of degenerative intervertebral disc were established in eighteen New Zealand rabbits and the animals were randomly divided into three groups: in treatment group, The tissue engineered NP were transplanted into the degenerative disc; in control group, only chitosan hydrogel were transplanted; in blank group, nothing was transplanted. The changes in intervertebral disc height were detected. Gross histological observation was performed at 12 weeks postoperatively. TypeⅡcollagen content was detected by immunohistochemistry, and glycosaminoglycan was quantitated by spectrophotometer to observe the repair effect.Results: The chitosan hydrogel was temperature-responsive : it was liquid when be put on room temperature , and became solid hydrogel within 10 - 15 minutes when be put on 37℃. The toxic and histocompatibility test suggested that this material has good biocompatibility. Acridine orange-propidiumiodide staining showed that the viability rate of NP cells 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, 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 type II collagen and aggrecan mRNA in NP cells cultured three-dimensionally by chitosan hydrogen scaffold was 0.631±0.064and 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). the changes in disc height in postoperative 12 weeks were significantly different compared with the blank group and control group (P < 0.01). In gross observation, the nucleus pulposus and annulus fibrosus of intervertebral disc in blank and control groups were not clear, and the nucleus pulposus in central region were replaced by fibrous tissues. While in treatment group, the degeneration was relieved, and the tissue structure was still clear. By spectrophotometer and immunohistochemistry, the content of glycosaminoglycan and typeⅡcollagen in treatment was significantly higher (P < 0.05) than that in blank and control group in the twelfth postoperative week.Conclusion: With good biocompatibility, the temperature-responsive chitosan hydrogel makes it possible for NP cells to maintain their normal morphology and secretion in the compound culture. The tissue engineered NP can repair the degenerative disc in animal study. the temperature-responsive chitosan hydrogel can be used as scaffold in the tissue engineered study on the repair of degenerative disc.