Glial Fibrillary Acidic Protein as Biomarker Indicates Purity and Property of Auricular Chondrocytes

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.

Microscopic anatomy of the subcommissural organ in the brain of the adult greater cane rat (Rodentia: Thryonomyidae)

“…The tissue was prepared for immunostaining according the method described by Gilbert et al., (2020). In order to determine different types of cells in the SCO of adult GCR, we made use of anti‐Glial fibrillary acid protein (GFAP) for fibrillary protein localization (Nishizawa et al., 2020), Iba1 for calcium binding protein localization (Ito et al., 1998) and NeuN for expression of neurons (Mullen et al., 1992). Paraffinized brain sections of 5 μm thickness were mounted on well‐labelled adhesive glass slides and then baked in an oven regulated for 1 h 30 min at 60°C to melt the paraffin wax.…”

Cadherin‐11 promotes the mechanical strength of engineered elastic cartilage by enhancing extracellular matrix synthesis and microstructure

“…Auricular cartilage defects, including congenital microtia and auricle injury are common in surgery (Nishizawa et al, 2020;Zhang et al, 2019). Conventionally, autologous costal cartilage graft is the most effective clinical treatment.…”