Repairing Cartilage Defect On Rabbit Knee Joints With Tissue Engineered Cartilage Constructed By Allogeneic Chondrocyte Through Cryopreservation

ObjectiveTo observe the repairing of cartilage defect on rabbit knee joint with tissue engineered cartilage constructed by allogeneic chondrocyte through cryopreservation in the way of morphology and ultrastructure. Thus, the feasibility and effectiveness are explored which the allogeneic chondrocyte is used as the seed cells for cartilage engineering to repair the cartilage defects on rabbit knee joints, providing a theoretical basis for clinical application.MethodsExperiment in vitro: To use the double knee cartilage of the healthy adult rabbits which is stored in the way of slow access cooling method as the chondrocyte and rewarm them in 37℃water for 2 minutes. Take a certain amount of sodium alginate suspension sterilizatized by membrane and add accurate ammount of chondrocyte, blow them fully and mix well with the planting density of 4×107/m1. Then make the cell plate in the silica gel plate, add the Cacl2 liquid with the quality fraction of 1.06%, and gel for 10min. Put 20% FBS to DMEM/F12 serum-free medium into the 24-well culture plate to culture for 2 weeks. To respectively three whole cell plates on the 1,2,4,6,8,10,12,14 day, and take the average figure to draw the growth curve of chondrocytes. Draw in 2 weeks and stain with HE, and observe the distribution and forms of cartilage cells.Experiment In vivo: To select 27 adult New Zealand white rabbits, and use an electric drill to make a model of full-thickness cartilage defects with the diameter of 3.0mm and depth of 3.0mm in the center of patellofemoral joint of the femur. To randomly implant chondrocyte-alginate sodium plate cultured for 2 weeks with the experiment in vivo (group A) and cell-free alginate carrier (group B) and the not treated group (group C). Kill the animal with air embolism and harvest them after the 4, 8, and 12 week respectively, and conduct the general observation, HE, toluidine blue staining and immunohistochemical analysis of observation. To assess with Wakitani histological scores and observe the vivo effect of articular cartilage defects.Data will be presented by mean±standard deviation ( x±s) and be analyzed by statistical package of SAS8.1, using two-factor variant analysis to test the experiment effect and time effect, and multiply compare the results of different groups by LSD-t test and the result of P <0.05 will be considered statistically significant.Results1. Observation and detection of cartilage in cell plate: the sell cells are proliferated in scaffold of sodium alginate after 4 days. In 2 weeks, it continues to grow and form tissue engineered cartilage at length.2. General observation at transplantation: After operation, three groups were observed. There were no hyperemia, edema, or joint fluid abnormalities, and the joint capsule was without adhesion in intra-articular synovial. 12 weeks after the operation, repair defects organization and cartilage tissue is difficult to distinguish from the normal surroundings in group A. There were no obvious boundaries compared with the normal surrounding tissue, with similar color, smooth surface and consistent elastic. However, not complete defect repair is found in the other two groups; there were sag and slight elastic. Cartilage tissue was not found and no obvious difference was observed between cartilage tissue and surrounding tissue.3. Histological observation: After 12 weeks of the operation, there was mainly transparent cartilage repairing in group A. Positive cartilage cells were found by immunohistochemical, the synthesis and secretion of collagen matrix were so exuberant by toluidine blue staining. There were mainly smoothiness in the defected parts. The repaired tissue cartilage was much integrated with surrounding tissues with not much difference. During the whole observation period, invasion of blood-vessel and lymphatic were not found. In the other two groups, fibrous filling is found in the tissue defect area with slight sag, and the full repairing is not seen. Through toluidine blue staining and immunohistochemical, little cartilage cells expression is found in group B and no cartilage cells expression in group C, and extracellular matrix and type II collagen were not expressed as well.4. Wakitani histological score and statistical analysis show that there are significant differences between repair effects of group A and the other two groups (P <0.05).Conclusion 1. The persevered chondrocyte by gradient cooling can be used as sell cells of tissue engineered cartilage.2. Sodium alginate was regarded as an ideal scaffold material, the chondrocyte-sodium alginate's complex in vitro can generate tissue engineered cartilage.3. The tissue engineered cartilage can be used to repair the cartilage defect of rabbit knee joint.