| Objective: The aim of this study is to explore a convenient technology for repairing articular cartilage defects with tissue-engineered cartilage which constructed with the chitosan hydrogel and adipose-derived stem cells.Methods:(1) In vitro culture and identification of adipose derived stem cells. The adipose tissues were harvest from the 24-week old New Zealand rabbits’ groin or back, then the tissue were digested by collagenase and cultured in vitro. The differentiation ability was identified by directed differentiation into chondrocytes.(2) Preparation of Chitosan Hydrogel. After 1,6-adipic acid isocyanate and polyethylene glycol-400 formed crosslinker, the chitosan hydrogel was prepared by 2%(wt%) chitosan solution and the cross-linking agent.(3) Construction of tissue engineering cartilage in vitro. After the freeze-dried chitosan scaffolds were sterilized by ultraviolet radiation, the adipose-derived stem cells were implanted into the scaffold and cultured in 20% FBS DMEM culture medium for 1 week.(4) Evaluation of articular cartilage defects. The articular cartilage defects animal model was induced by electric drilled in the patellar groove. Rabbits were randomly separated into three groups: the control group which underwent no implantation(A group); the chitosan hydrogel group(B group); and the experiment group which was implanted with tissue-engineered cartilage(C group). The samples were grossly examined, histologically analysis, immunohisto-chemical staining and ICRS scoring system at 4, 8 and 12 weeks, respectively.Results:(1) Using the method of collagenase digestion can obtain abundant adipose derived mesenchymal stem cells with the ability of self-renewing and multi differentiation, which can be used as seed cells to construct tissue engineering cartilage.(2) Gross observation showed that the cartilage defects were repaired with cartilage-like tissues in experimental group. The new organization is closely integrated with the normal tissue, which structure and secretion of extracellular matrix is similar to normal tissue. Yet those of the control group and the chitosan hydrogel group were repaired incompletely.(3) Histological observation and immunohistochemical staining confirmed that the repairing tissues were hyaline cartilage-like tissues in the experimental group, which structure and extracellular matrix secretion was similar with the normal cartilage tissue. Compared with the experiment group, the degradation rate of the hydrogel in the chitosan hydrogel group was faster than the formation rate of the regeneration tissue, so there was notable hole in the regeneration tissue after 12 weeks post operation. The repair in the control group was the worst in all of the three groups. The cartilage defect remained obviously in the control group. ICRS gross and histological scores for repairing tissues showed that the experimental group was significantly better than the other two groups(P<0.05).Conclusions: Adipose derived stem cells could be used as seed cells for tissue engineering cartilage construction. The tissue-engineered cartilage constructed by adipose derived stem cells and chitosan hydrogel could repair the defect of articular cartilage. |
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