The Experiment On Microtia Chondrocytes As A Donor Source For Tissue-Engineering Cartilage

Part I Biological Characteristics of the Human Microtia Chondrocytes Cultured In VitroObjective: To investigate the culturing methods of human microtia chondrocytes and to observe the biological characteristics.Methods: The human microtia chondrocytes were isolated and cultured to observe morphological changes. The origin were identified by type II collagen antibody by immunohistochemistry. Meanwhile, the expressions of collagen II and Aggrecan were detected by RT-PCR.Results: The human microtia chondrocytes were transformed from polygon or triangle into the shape of the fibroblast-like cell with the passage increasing. Most cells of the sixth passage turned into fusiform cell. The cells expressed type II collagen were identified as chondrocytes by immunohistochemistry. The expression of collagen II and Aggrecan mRNA were high before the third passage and decreased with the passage increasing in the mRNA level and that of the fifth passage was negative.Conclusion: The microtia chondrocytes from the first passage to the second passage seem suitable for tissue-engineering cartilage.Part II Age Dependence of Biological Characteristics of the Human Microtia Chondrocytes Cultured In VitroObjective: To investigate the effects of donor age on biological characteristics of human microtia chondrocytes cultured in vitro.Methods: The human microtia chondrocytes from 30 patients ranging in age from 6 to 30 years were isolated and cultured. The origin were identified by type II collagen antibody by immunohistochemistry. The comparison was done in morphology, population doubling time and the expression levels of collagen II and Aggrecan mRNA by RT-PCR.Results: The human microtia chondrocytes isolated from different age show the similar morphology, population doubling time and the expression levels of collagen II and Aggrecan mRNA. The human microtia chondrocytes were transformed from polygon or triangle into the shape of the fibroblast-like cell with the passage increasing. The second passage cells of every age group were identified as chondrocytes by immunohistochemistry.The population doubling time showed that the proliferation of the first to the third passage was powerful. The expression of collagen II and Aggrecan mRNA were high in the first and second passage, decreased in the third passage distinctly in the mRNA level and was negative after the fourth passage.Conclusion: The biological characteristics of human microtia chondrocytes seem to be independence of donor age for the span from 6 to 30 years. The human microtia chondrocytes of the first and the second passage may be suitable for tissue-engineering cartilage. Part III Effect of b-FGF on Human Microtia Chondrocytes in Vitro CultureObjective: To investigate the effect of basic fibroblast growth factor (b-FGF) on proliferation and chondrogenetic potency of human microtia chondrocytes cultured in vitro.Methods: Cells were harvested from human microtia chondrocytes. Viability and quantification of the cells was determined. Cells were isolated and cultured at equal concentration.The effect of b-FGF on cell growth and chondrogenetic potency was studied in the following groups: Group 1 (control group) receiving Ham's F-12 with supplements but no b-FGF; Group 2 receiving 5 ng/ml b-FGF; Group 3 receiving 10 ng/ml b-FGF; Group 4 receiving 25 ng/ml b-FGF; Group 5 receiving 50 ng/ml b-FGF respectively. The effect of on microtia chondrocytes was detected by MTT and that of the expression levels of collagen II and Aggrecan were detected by RT-PCR. Results: Chondrocytes displayed morphology similar to fibroblasts in the media containing b-FGF. When the b-FGF concentration was 10～50 ng/ml, the numbers of cultured microtia chondrocytes increased markedly. The expression levels of collagen II and Aggrecan in the experimental group with concentration of 10 ng/ml was higher than that in the control. This difference denoted statistical significance (P<0.05).Conclusion: The using of b-FGF stimulates the proliferation of the human microtia chondrocytes and has the positive role on maintaining the chondroitic phenotype. The optimal concentration is at the level of 10 ng/ml.Part IV Microtia Chondrocytes as a Donor Source for Tissue Engineering CartilageObjective: To evaluate the biological potential of chondrocytes isolated from microtia cartilage as a source of tissue-engineering cartilage. Methods: Cartilage specimens from 18 microtia patients were obtained. The chondrocytes were isolated and cultured in vitro, and chondrocyte count was increased by passaging. The chondrocytes were isolated and suspended into a hydrogel (Pluronic F-127) at a cell concentration of 50×10~6/ml and were implanted in nude mice to generate tissue-engineering cartilage. Eight weeks after implantation the specimens were dissected and removed.Results: An initial mean cell count of 150,000 cells increased to an average cell count of 130 million cells in microtia group at the end of the second passage. Histologically, chondrocytes from microtia and normal auricular all generated tissue engineering cartilage.Conclusion: The study demonstrated the potential of cells isolated from microtia cartilage to generate tissue-engineering cartilage. Microtia cartilage represents an important additional donor source for the possible generation of a human tissue-engineering auricle.