Abstract
Instead of the silicone implants previously used for repair and reconstruction of the auricle and nose lost due to accidents and disease, a new treatment method using tissue-engineered cartilage has been attracting attention. The quality of cultured cells is important in this method because it affects treatment outcomes. However, a marker of chondrocytes, particularly auricular chondrocytes, has not yet been established. The objective of this study was to establish an optimal marker to evaluate the quality of cultured auricular chondrocytes as a cell source of regenerative cartilage tissue. Gene expression levels were comprehensively compared using the microarray method between human undifferentiated and dedifferentiated auricular chondrocytes to investigate a candidate quality control index with an expression level that is high in differentiated cells, but markedly decreases in dedifferentiated cells. We identified glial fibrillary acidic protein (GFAP) as a marker that decreased with serial passages in auricular chondrocytes. GFAP was not detected in articular chondrocytes, costal chondrocytes, or fibroblasts, which need to be distinguished from auricular chondrocytes in cell cultures. GFAP mRNA expression was observed in cultured auricular chondrocytes, and GFAP protein levels were also measured in the cell lysates and culture supernatants of these cells. However, GFAP levels detected from mRNA and protein in cell lysates were significantly decreased by increases in the incubation period. In contrast, the amount of protein in the cell supernatant was not affected by the incubation period. Furthermore, the protein level of GFAP in the supernatants of cultured cells correlated with the in vitro and in vivo production of the cartilage matrix by these cells. The productivity of the cartilage matrix in cultured auricular chondrocytes may be predicted by measuring GFAP protein levels in the culture supernatants of these cells. Thus, GFAP is regarded as a marker of the purity and properties of cultured auricular chondrocytes.
Highlights
The external nose and external ear are organs maintaining their shape with cartilage, and repair and reconstruction are performed when these are deformed or lost due to congenital anomalies or trauma, in which a frame prepared with silicone, costal cartilage, and other biomaterials or transplants is transplanted to the affected region
To confirm the decrease in glial fibrillary acidic protein (GFAP) expression at P8 from that at passage 3 (P3), we examined changes in mRNA expression in a long-term culture of human auricular chondrocytes with repeated passages, and the results obtained revealed the marked downregulation of GFAP expression (Fig. 1B)
GFAP expression was downregulated with serial passages, the mRNA expression of vimentin (VIM), a typical intermediate filament of the mesenchymal cell lineage, actin beta (ACTB), a component of the microfilaments and mediator of internal cell motility, and tubulin (TUBA1A), the basic structural unit of microtubules, was not downregulated, implying that a decrease in mRNA expression according to the dedifferentiation of auricular chondrocytes was not common in the cytoskeleton, but was specific to GFAP
Summary
The external nose and external ear are organs maintaining their shape with cartilage, and repair and reconstruction are performed when these are deformed or lost due to congenital anomalies or trauma, in which a frame prepared with silicone, costal cartilage, and other biomaterials or transplants is transplanted to the affected region. Since silicone is an artificial material, it may have a negative influence after transplantation. Costal cartilage is a superior onlay graft, but its collection is very invasive and it has a risk of development of pneumothorax and thoracic deformity. A frame made of costal cartilage may be deformed after transplantation.[1,2] A new treatment strategy applying tissue engineering has been attracting attention as a method to overcome these problems.
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