Perpetual phenotype of self-assembled tissue-engineered cartilages transferred with lentiviral-mediated C-1-1

Abstract

Objective To observe the effect of lentiviral-mediated C-1-1 on maintenance of perpetual phenotype of self-assembled engineered cartilages.Methods The lentiviral expression vector carrying C-1-1 gene was constructed and transfected into cultured human bone marrow mesenchymal stem cells (hMSCs),and then resistance clones were acquired by puromycin screening.The expression of C-1-1 gene was detected by using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Westem blotting.hMSCs at passage 3 were induced with chondrogenic medium containing 200 μg/L growth differentiation factor 5 (GDF-5) for 3 weeks.Three weeks later,the cells were suspended and then inoculated into each well of 2％ agarose-coated 24-well plates at a density of 5 × 106/mL.Another 3 weeks later,the differentiating effect was identified by histological staining.Results The successful construction of the lentiviral expression vector carrying C-1-1 gene was identified by sequencing.After the lentiviral expression vector was transfected into the hMSCs,the expression of C-1-1 mRNA and protein was enhanced.After self-assembly culture for 3 weeks,toluidine blue staining was positive.The mean absorbance (A) values of Collagen Ⅱ in C-1-1 gene transfection group was (0.3754 ± 0.0255),which was not higher than that in the control group (P ＞0.05).The mean A values of Collagen X in C-1-1 gene transfection group was (0.0115 ±0.0062),which was lower than that in the control group (P ＜0.001 ).Conclusion After C-1-1 gene was transfected through lentiviruses into the hMSCs,the phenotype of self-assembled engineered cartilages was more stable while their maturation and hypertrophy was inhibited. Key words: Cartilages; Gene transfection; Growth differentiation factor 5