HISTOLOGY AND HISTOPATHOLOGY

Cellular and Molecular Biology

 

Development and phenotypic characterization of a high density in vitro model of auricular chondrocytes with applications in reconstructive plastic surgery

A. Haisch1, U. Marzahn1, A. Mobasheri2, G. Schulze-Tanzil3 and M. Shakibaei3,4

1Charité Medicine University Berlin, Campus Benjamin Franklin, Department of Otorhinolaryngology, Head and Neck Surgery, Berlin, Germany, 2Connective Tissue and Molecular Pathogenesis Research Groups, Faculty of Veterinary Science, University of Liverpool, Liverpool, United Kingdom, 3Charité Medicine University Berlin, Campus Benjamin Franklin, Institute of Anatomy, Department of Cell and Neurobiology, Berlin, Germany, 4Musculoskeletal Research Group, Institute of Anatomy, Ludwig-Maximilian-University Munich, Munich, Germany

Offprint requests to: Dr Andreas Haisch, Charité Medicine University Berlin, Campus Benjamin Franklin, Department of Otorhinolaryngology Head and Neck Surgery, Berlin, Germany. e-mail: andreas.haisch@charite.de


Summary. Cultivation of phenotypically stable auricular chondrocytes will have applications in autologous chondrocyte transplantation and reconstructive surgery of cartilage. Chondrocytes grown in monolayer culture rapidly dedifferentiate assuming a fibroblast-like morphology and lose their cartilage-specific pattern of gene expression. Three-dimensional high-density culture models mimic more closely the in vivo conditions of cartilage. Therefore, this study was undertaken to test whether the high-density cultures might serve as a suitable model system to acquire phenotypically and functionally differentiated auricular chondrocytes from porcine cartilage.
Freshly isolated porcine auricular chondrocytes were cultured for 7 passages in monolayer culture. From each passage (passage 0 and 1-7) cells were introduced to high-density cultures and examined by transmission electron microscopy. Western blotting was used to analyse the expression of cartilage-specific markers, such as collagen type II and cartilage specific proteo-glycan, fibronectin, cell adhesion and signal transduction receptor ß1-integrin, matrix metalloproteinases (MMP-9, MMP-13), cyclo-oxygenase (COX)-2 and the apoptosis commitment marker, activated caspase-3.
When dedifferentiated auricular chondrocytes from monolayer passages 0-4 were cultured in high-density culture, they recovered their chondrocytic phenotype and formed cartilage nodules surrounded by fibroblast-like cells and synthesised collagen type II, proteoglycans, fibronectin and ß1-integrins. However, chondrocytes from monolayer passages 5-7 did not redifferentiate to chondrocytes even when transferred to high-density culture, and did not synthesize a chondrocyte-specific extracellular matrix. Instead, they produced increasing amounts of MMP-9, MMP-13, COX-2, activated caspase-3 and underwent apoptosis.
Three-dimensional high-density cultures may therefore be used to obtain sufficient quantities of fully differentiated auricular chondrocytes for autologous chondrocyte transplantation and reconstructive plastic surgery. Histol Histopathol 21, 467-476 (2006)

Key words: Auricular chondrocytes, High-density culture, Redifferentiation, Dedifferentiation, Apoptosis, Reconstructive surgery

DOI: 10.14670/HH-21.467