Construction of Tissue-engineered Cartilage In Vivo from Microtia Chondrocytes After Transfection with Human VEGF165 Genes Mediated by a Recombinant Adeno-Associated Viral Vector

Objective To evaluate the transfection efficiency of cultured chondrocytes from individuals with microtia (microtia chondrocytes) with the recombinant adenoassociated virus vector rAAV2-hVEGF(165) -IRES-EGFP and hVEGF(165) in vitro. To test whether VEGF(165) gene-modified microtia chondrocytes can enhance the survival and quality of tissue-engineered cartilage. Method The recombinant plasmid rAAV2-hVEGF165-IRES-EGFP was inserted into rAAV2 virus vectors to construct rAAV2-hVEGF165-IRES-EGFP using the AATMaxTM system. The second-passage microtia chondrocytes were divided into 3 groups in vitro: the Ctr group (without transfection), Exp1 group (transfected with rAAV2-IRES-EGFP), and Exp2 group (transfected with rAAV2-hVEGF165-IRES-EGFP). At 24 h, 48 h, 72 h and 7 d after transfection, cell viability was measured by MTT staining. Transfection efficiency was determined by the rate of fluorescence-positive cells. The mRNA expression of hVEG165 was detected by RT-PCR (reverse transcription PCR) and agarose gel electrophoresis, and the VEGF165 protein levels in the supernatant fluids were measured by ELISAs. The second passage microtia chondrocytes with (Exp) and without (Ctr) transfection of VEGF(165) genes were mixed with 0.5 ml 30% Pluronic F-127 at 4 degrees C and then injected subcutaneously into the opposing side of the back of nude mice. Eight weeks after injection, the cartilage-like tissues of nude mice were harvested for morphological and histologic examination. Results Chondrocyte viability increased in a time-dependent manner but did not differ among the 3 groups at the same time point. The mRNA and protein levels of VEGF increased in a time-dependent manner in the 3 groups. The mRNA and protein levels of VEGF165 were much higher in the Exp 2 group than in the Ctr and Exp 1 groups at the same time point, but the levels were not significantly different between the Exp 1 and Exp 2 groups. Both the Ctr group and the Exp1 group formed mature cartilage with mature lacunar structures, metachromatic matrices, collagen, and elastic fibers, and the structure of neonatal cartilage was not significantly different between the 2 groups. However, the wet weight of the neonatal cartilage was much larger in the Exp group (127.4 +/- 12.4 mg) than in the Ctr group (58.5 +/- 12.2 mg, p < 0.05). VEGF protein staining also showed a higher level in the Exp group. Conclusion The HVEGF(165) gene was transfected efficiently into microtia chondrocytes using the recombinant adeno-associated virus vector rAAV2-hVEGF(165) -IRES-EGFP. After transfection, the mRNA and protein levels of hVEGF(165 )increased in a time-dependent manner. VEGF(165) gene-modified microtia chondrocytes showed enhanced survival in vivo but did not improve the texture of tissue-engineered cartilage. No Level Assigned This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.corn/00266.

Automated summary

Transfection of HVEGF(165) gene can enhance the survival of microtia chondrocytes, but does not improve the texture of tissue-engineered cartilage.