The Experimental Studies Of Construction Tissue Engineered Cartilage In Vivo From Microtia Chondrocytes After Transfection With Human VEGF₁₆₅ Genes Mediated By Recombinant Adeno-associated Viral Vector

Partâ… Construction and Identification of the Recombinant Adeno-associatedVirus Vectors Containing hVEGF₁₆₅ Genes and EGFP GenesObjective To construct the recombinant adeno-associated virus vectors whichcontain human vascular endothelial growth factor 165 (hVEGF₁₆₅) genes andenhanced green fluorescent protein (EGFP) genes.Methods The pSNAV-hVEGF₁₆₅-IRES-EGFP recombinant plasmid wasconstructed by recombination of the eukaryotic expression plasmidpDC316-IRES-EGFP which contains the reporter gene and pSNAV-hVEGF₁₆₅ whichcontains the target gene. The recombinant plasmid was identified by PCR and DNAsequence analysis, and then was packed into rAAV₂ virus vectors to constructrAAV2-hVEGF₁₆₅-IRES-EGFP using the AATMax^TM systems.Results The recombinant plasmids pSNAV-hVEGF₁₆₅-IRES-EGFP wereconfirmed by PCR amplification, and DNA sequence analysis. Moreover, the virusvectors rAAV2-hVEGF₁₆₅-IRES-EGFP were successfully obtained.Conclusion The recombinant adeno-associated virus vectorsrAAV2-hVEGF₁₆₅-IRES-EGFP are constructed successfully, which are ready totransfect the seed cells-chondrocytes- in the following engineered cartilage studies. Partâ…¡Transfection of Microtia Chondrocytes with Human VEGF₁₆₅ GenesMediated by Recombinant Adeno-associated Virus Vectors in VitroObjective To transfect cultured microtia chondrocytes with recombinantadeno-associated virus vector rAAV2-hVEGF₁₆₅-IRES-EGFP, which was constructedin the first part of our studies, and transfection efficiency and hVEGF₁₆₅ genesexpression were examined.Methods The microtia chondrocytes were dissociated from microtia residualcartilages and cultured in monolayer. The chondrocytes at second passage weredivided into 3 groups: Ctr goroup, without transfection; Exp1 group, transfected withrAAV2-IRES-EGFP; Exp2 group, transfected with rAAV2-hVEGF₁₆₅-1RES-EGFP.At 24 h, 48 h, 72 h and 7 days after transfection, cell viability was measured by MTTstaining. The green flurescent light which mirrors EGFP was observed with thereverse fluorescent microscope; the transfection efficiency was determined by the rateof positive cells with fluorescent lights. The mRNA expression of hVEG₁₆₅ wasdetected by RT-PCR and the VEGF protein levels in the supernatant fluids weremeasured by ELISA.Results The cell viability increased time-dependently, but did not differ amongthe 3 groups at the same time point. The green fluorescent light was seen in Exp 2 at24 h after transfection. The intensity of the green fluorescent light and transfectionefficiency increased gradually during 7 days after transfection, the period we observed.The levels of the mRMA and the protein of VEGF increased in a time-dependantmanner in 3 groups. The levels of the mRMA and the protein of VEGF were muchhigher in Exp 2 than those in Ctr group and Exp1 group at the same time piont, butthe levels were not significantly different between Exp 1 and Exp2.Conclusion The hVEGF₁₆₅ gene is transfected safely and efficiently to microtiachondrocytes using the recombinant adeno-associated virus vectorsrAAV2-hVEGF₁₆₅-IRES-EGFP. After transfection, the levels of the mRNA and theprotein of hVEGF₁₆₅ enhanced and increased time-dependantly. Partâ…¢Construction of Tissue Engineered Cartilage from Microtia ChondrocytesTransfected with Human VEGF₁₆₅ Genes in VivoObjective To test the hypothesis that VEGF₁₆₅ genes modified microtiachondrocytes can enhance the survival of chondrocytes after combined with PluronicF-127 injected into nude mice and improve the quality of tissue engineered cartilage.Methods The second passage of microtia chondrocytes with (Exp.) or without(Ctr.) transfection of VEGF₁₆₅ genes were combined with 0.5 ml 30% Pluronic F-127at 4℃, and then were injected subcutaneously into opposing side of the backs of nudemice. Eight weeks after injection, the neonatal tissues in nude mice were taken outfor morphological examination, Haematoxylin and Eosin (HE) staining, Verhoeffstaining of elastin, Toluidine Blue staining for glycosaminoglycan, Masson staining ofcollagen, and VEGF immunohistochemical staining.Results Both the Ctr. group and Exp.1group formed mature cartilages withmature lacuna structures, metachromatic matrices, collagen, and elastic fibers. Thestructure of neonatal cartilages was not significantly different between the 2 groups.However, the wet weight of the neonatal cartilages was much larger in Exp. group(127.4±12.4 mg) than in Ctr. group (58.5±12.2 mg, p＜0.05)). VEGF protein stainingshowed a higher level of VEGF in Exp group too.Conclusion VEGF₁₆₅ gene modified microtia chondrocytes can enhance the cellsurvival after seeded in vivo but cannot improve the texture of tissue engineeredcartilage.