Purpose: The author developed and clinically applied a new culture method with which a sufficiently large volume of chondrocytes for the reconstruction of normal cartilage in human body is developed from a small piece of auricular cartilage. This report presents overall results of the basic studies and clinical applications.

Methods: Approximately one cm² of cartilage was collected from the posterior part of the auricle, and then treated with collagenase, and chondrocytes were obtained. The chondrocytes were subcultured, and the cells at the second and/or third passage were used for grafting. The culture medium was a blend of F-12 medium and DME medium in an equal volume supplemented with 10% autoserum, penicillin G (50 unit/ml) and streptomycin sulfate (0.1 mg/ml). Finally, the chondrocytes obtained by the subculture were seeded at a density of 1x10⁴ cells/cm² 2~3 times over the previously seeded cells and gel-like chondrocytes were obtained. In the culture, increase in cell numbers were counted, and the levels of hyaluronic acid and alkaline phosphatase (ALP) were measured. It took approximately 4 weeks from the primary culture to grafting to the patient. The grafting volumes range was 2.5 to, and the number of chondrocytes per 1cc was 0.5~5 x10⁸cells. They were then aspirated into a syringe and inject-grafted into the subcutaneous pocket that was produced by the surgical dissection of the deformed area. After the grafting, the graft area was tightly sutured and taped for the compression and modeling. Histology of the newly formed cartilage was examined in 10 patients. To date, this methodology has been applied to 60 patients, i.e., nasal augmentation (n = 47), cranio-facial deformity (n = 5), mentoplasty (n = 4) and microtia (n = 4). The longest follow-up period is 6 years.

Results: A sufficiently large volume of chondrocytes was prepared with this culture method. The differentiated chondrocytes produced the differentiation marker ALP as well as Type II collagen and hyaluronic acid that are the necessary substances for the production of cell matrix in vitro. In clinical application, cartilage formation was confirmed 3 weeks after the grafting. Histologically, toluidine blue staining revealed the presence of metachromasie in newly formed cartilage, and the presence of cartilage-specific human Type II collagen was confirmed. EVG staining revealed the presence of elastic fibers that are a feature of auricular cartilage. In all the 60 patients, the shape and contour of the grafted areas were excellent, and good aesthetic results were obtained. Frequency was 0/60 for complication, 0/60 for total absorption of the grafted chondrocytes and 5/60 for partial absorption of the chondrocytes.

Conclusion: Successful expansion of cultured auricular chondrocytes is the prerequisite for autologous cell transplantation. With this method, the chondrocytes take a gel form in the culture, but after grafting they form a matrix by getting natural scaffolds and regenerate cartilage tissue. The grafted chondrocytes formed elastic caltilage derived from their origin. These culture method and grafting technique will be a highly useful new treatment method with less invasion. This method of cultured chondrocyte grafting could be applicable as a cell therapy to a wide range of cranio-facial treatment.