| There has been no satisfactorily effective method for the clinical treatment of articular cartilage degradation and functional incapacitation due to traumas and cartilage lesions up till now. Tissue engineering, which has been being developed in the past decade, provides a possible and hopeful approach to the solution of the above-mentioned problem. In addition, cartilage tissue engineering, one of the fast developed branches in tissue engineering, may bring about complete solution to the medical problem. However, development of cartilaginous tissue engineering is obsessed by another unsolved problem which is how to obtain a lot of seed cells (chondrocyte) possessing normal phenotype.With the development of cloning and transgenic technology, many researches had been conducted about this question and some progress has been made. One of the most important study fields is how to induce stem cells, including ES cells and MSC cells, to differentiate into chondrocytes directly or elongate the life span of chondrocytes in vitro.In this study, we dealt with human telomerase reverse transcriptase (hTERT) gene introduced into mandibular condylar chondrocytes (MCC) of goat by eukaryotic vector and Lipofectin transgenic method. The biological characteristics of the positive clones selected with G418 were detected. Engineered cartilage is fabricated with the complexes immortalized by chondrocyte-loaded b-tricalcium phosphate (b -TCP) and implanted into goat humeral cartilage with defects. The cartilage defect repair was evaluated. The brief results are presented as follows:After the identification of double enzyme digestion and electrophoresis, the eukaryotic vector pCI-hTERT was massively extracted and purified. Then human telomerase reverse transcriptase (hTERT) gene was introduced into mandibular condylar chondrocytes (MCC) of goat by eukaryotic vector and Lipofectin transgenic method. Positive clones were selected with G418 or limited dilution method for culture and subculture. The majority of MCCs were in polygonal shape in the primary culture, but more fusiform and spindle-shaped cells were found after 6-8 passages cultured in vitro. With slow down speed of proliferation, the chondrocytes presented the senescent appearance and stoppedgrowing. On the contrary, 13 out of the 20 positive strains of transduced cells, cultured more than 40 passages, have maintained for more than 150 passages in nearly one year and still display a vigorous proliferative capacity without any signs of senescence, which were termed as immortalized mandibular condylar chondrocytes (IMCCs) with the similar polygonal shape during the course of culture to that of primary MCCs, but the size of IMCCs is smaller than that of MCCs.The growth curve revealed that the population doubling time of IMCCs and MCCs was 22.4 h and 65.2 h, respectively. IMCCs and MCCs stopped proliferating in DMEM without serum, but the proliferating rate of IMCCs was significantly higher than that of MCCs with the same serum concentration.Tissue and morphological assays found that 92% MCCs of the fourth passage had 60 chromosomes, but 83% and 75% normal karyotype were found in 40th and 80th IMCCs passage respectively. IMCCs could grow in soft agar and its clone formation efficiency was 6.2% compared to 16% clone formation efficiency on tissue culture dishes. No tumor was found 6 months later after IMCCs were injected subcutaneously in 6-weel-old BALB/c nu-/nu- mice.TRAP (telomeric repeat amplification protocol) assay proved that positive expression of telomerase was found in IMCCs but negative in MCCs. IMCCs showed positive staining for collagen type II mRNA in situ hybridization and maintained the normal phenotype. Alkaline Phosphatase (ALP) activity of IMCCs was lower than that of MCCs and osteoblast. The result of Von Kossa staining for IMCCs was negative.The assay of cartilage defect repair demonstrated that engineered cartilage based on IMCCs as seed cells had a better ability for the repair of cartilage defect, which formed a lot of cartilaginou...
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