Experimental Study On The Repairment For Articular Cartilage Defects Of Rabbit With The Novel Tissue-engineered Cartilage

Objective: To evaluate the treatment for the articular cartilage defects bytissue-engineered cartilage which constructed with a novel chitosanhydrogel and costal chondrocytes.Methods:（1）The costal cartilage tissue harvested from24-weeks-oldrabbit was digested and cultured to get chondroeytes in vitro.（2）Chondroeytes were subcultured and proliferated in vitro, then the morphologywas observed and the growth curve of cell was drawn. The second generationcells were identified by safranin-O, fast green, toluidine blue, aggrecan andtype II collagen immunohistochemical staining. The extracellular matrixsecretion ability of different generation chondroeytes was observed by typeⅡcollagen immunohistochemical staining.（3）The chitosan hydrogel mixedwith the second generation chondrocytes to construct tissue-engineeredcartilage and cultured in the completely DMEM medium of20%fetalbovine serum for1week.Then it was frozen at-20℃and5μm sections wereprepared for normal H&E staining, toluidine blue staining and type Ⅱcollagen immunochemical staining to observe the growth of chondroeytes（.4）The rabbit model of knee cartilage defect was made and divided into3groups.In the experimental group: Tissue-engineered cartilage was implanted intothe defects. In control group: The defects were treated by simple chitosanhydrogels. In blank group: the defects without anything treatment. Then theknee joints were harvested at4,8and12weeks, the repairments wereanalyzed through histologically and immunohistochemically.And the repairments were scored through the ICRS`standard.Results:（1）Massive high-purity chondrocytes could be got by culturedin vitro. It was confirmed that cultured chondrocytes within the thirdgeneration could maintain its stable phenotype, and the chondrocytes after thethird generation proliferated slowly and differentiated.（2） Thetissue-engineered cartilage was sliced and dying by HE staining, and theresults shows that chondrocytes were scattered in scaffold’s pores and grewwell. The results of toluidine blue and type II collagen immunohistochemicalstains were positive, which suggested that chondrocytes grew well on thescaffold（.3）The defects were obviously repaired in experimental group at4thweek after implantion. After8weeks, the repairment was better and thereexisted hyaline-like cartilage in the regenerate tissue through thehistochemical staining. And the defects were repaired completely on grossmorphology after12weeks. The regenerate tissue closely connects withsubchondral bone and the boundary with normal tissue was fuzzy.Thecartilage lacuna in the regenerate tissue was similar with normal cartilagelacuna. It was confirmed that the regenerate tissue was mainly hyaline-likecartilage. Compared with the experimental group, the defects in control grouphad been repaird partly, while the repairment in in blank group was worse.After12weeks, the defects was still obviously and the regenerate tissue wasmainly fibrocartilage. The results of ICRS gross and histological gradingshowed there were significant difference among the three groups（P<0.05）.Conclusions:（1）Massive high-purity chondrocytes could be got invitro by digested with trypsin and collagenase.（2）Chondrocytes within thethird generation could maintain their phenotypic features and stable secretionof the extracellular matrix, which were suitable for research（.3）Chondrocytes implanted in the scaffold could adhere, proliferate and secrete theextracellular matrix.（4）The novel tissue-engineered cartilage constructedin our research could repair the articular cartilage defects completely instructure.