Preliminary Research Of Repairing Articular Cartilage Defects By Transgeneic Allogenic Tissue-engineered Cartilage In Rabbits

Objective:Exogenous hTERT gene will be transferred into articular chondrocytes for immortalization with retroviral vectors, and the characteristics of immortalized cells will be researched. Immortalized chondrocytes will be regarded as seed cells, and combined with BMG (Bone Matrix Gelatin, BMG) to build tissue-engineered cartilage. We will further evaluate its ability of repairing rabbit articular cartilage defects. So as to explore hTERT transfected immortalized chondrocytes as seed cells for cartilage tissue engineering feasibility and BMG as cartilage tissue engineering scaffold material superiority.Method:1. Rabbit articular chondrocytes were isolated and cultured, and then their characteristic phenotype was identified. pLEGFP-C1-hTERT plasmid and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by cationic liposome, and virus supernatants were collected and concentrated within 72 hours after transfection. The 2nd generation of isolated articular chondrocytes was reinfected, and we observed the expression of green fluorescent protein GFP in GP2-293 cells and chondrocytes. The original virus and the concentrated virus for virus titer were determined with the NIH3T3 cells, and the two virus titers were compared. The infected articular chondrocytes were screened by G418, and the resulting clone was expanded and cultured. Screening chondrocytes were detected by RT-PCR for the expression of hTERT gene. Cartilage cells which were transduced the hTERT gene were observed the morphology and growth under inverted microscope. Drawing cell growth curve and calculating the doubling time in order to observe the effect on the proliferation of chondrocytes after being transduced the hTERT gene. In order to evaluate the impact on phenotype of chondrocytes through transducing hTERT gene and adding cytokine BMP-4, we used RT-PCR method and immunohistochemical staining. Finally, we evaluated the impact on cell's tendency of transformation by transferring hTERT gene through the formation of flat clone experimental observation.2. Rabbit bone matrix gelatin BMG need be prepared firstly. The BMG was freeze-dried after trimmed, sterilized by ethylene oxide and placed in the environment of-80℃. Immortalized chondrocytes grow in BMG, as cell-carrier complex. RT-PCR method and immunohistochemical staining were conducted to detect the expression of collagen typeⅡafter immortalized chondrocytes grow with BMG 3 days. Eight male New Zealand white rabbits were randomly divided into A, B groups, and established the model of bilateral femoral trochlear articular cartilage defect. Each joint had two knee cartilage defects, including experimental and control groups. A group: experimental group, immortalized chondrocytes and BMG (A1), control group, blank BMG (A2); B group:experimental group, immortalized chondrocytes and BMG (B1), control group, the 2nd generation chondrocytes and BMG (B2). Articular cartilage of each group was harvested for gross observation and Moran gross score after 6 weeks and 12 weeks.Result:1.The morphous of rabbit articular cartilage cells is polygonal and triangular, rich in cytoplasm, has a good refraction. Toluidine blue staining and typeⅡcollagen immunocytochemical staining results showed positive. pLEGFP-C1-hTERT plasmid and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by cationic liposome, GFP can be expressed in GP2-293 cells, the virus supernatant through concentrated virus titer markedly improved. yellow-green fluorescent protein can be expressed intensely in the cytoplasm of cells after rabbit articular chondrocytes were infected for 72 hours. The final clones were obtained by G418 selected for 4 weeks, and then clones were amplified. Clones have spread to 25 consecutive generations, initially established immortalized chondrocytes line. But chondrocytes without being transfected all died after G418 screened for 1 week. The expression of hTERT gene in mRNA level can be detected by the method of RT-PCR. In vitro continuous culture, immortalized cartilage cells proliferation actively increased, the growth of cell accelerated and doubling time was reduced. But the shape of such cells decreased and gradually elongated, spindle or radial alignment. Collagen typeⅡimmunocytochemical staining was negative. The expression of collagenⅡnearly can not be detected by the method of RT-PCR, standardized gray value was significantly lower than the group of the 2nd normal generation of chondrocytes and the group of immortalized chondrocytes added BMP-4 (P<0.05). Immortalized chondrocytes in vitro have the phenomenon of contact inhibition; the rate of colony formation was 5.8%, far below the rate of Hela cells. Therefore, chondrocytes do not have the tendency to malignant transformation.2.BMG was loose, porous sponge-like structure, with a certain toughness and strength, easily cut into desired shape. When the dedifferentiation of immortalized chondrocytes growing with BMG in vitro for 3 days, collagenⅡcan be translated and expressed again. At 6th week and 12th week, A1, B1, B2 were repaired by the cartilage-like tissue after surgery, articular surfaces were in their integrity, the cartilage-like tissue to repair integrated with the surrounding normal cartilage perfectly. However, for A2, cartilage defects sag, there were white fill transparent-like substance inside the defects, the surrounding boundaries were clear. The result of Moran's gross score showed that in the two time points experimental group (A1, B1), normal positive control group (B2), the results were significantly better than the blank BMG group (A2), the difference was significant(P<0.05). Experimental group (A1, B1) and normal positive control group (B2), scores were different but not statistically significant at 6th week (P>0.05). However, after 12 weeks the experimental group (A1, B1) is better than the normal positive control group (B2), there was significant (P<0.05). After 12 weeks each group's scores are higher than each group's scores after 6 weeks(P<0.05).Conclusion:1. We established a cartilaginous immortalized cell lines by transducing exogenous hTERT gene, so the life of articular chondrocytes can be significantly extended in vitro culture.2. The phenotype of immortalized chondrocytes gradually lost in vitro monolayer culture, cytokines BMP-4 can make phenotype of immortalized chondrocytes maintained.3. BMG can be used as a good carrier of the seed cells, and can make the dedifferentiation of immortalized chondrocytes redifferentiate.4. Immortalized chondrocytes constructed by hTERT have feasibility as seed cells for cartilage tissue engineering.