PGA-associated heterotopic chondrocyte cocultures: implications of nasoseptal and auricular chondrocytes in articular cartilage repair

Abstract

The availability of autologous articular chondrocytes remains a limiting issue in matrix assisted autologous chondrocyte transplantation. Non-articular heterotopic chondrocytes could be an alternative autologous cell source. The aims of this study were to establish heterotopic chondrocyte cocultures to analyze cell-cell compatibilities and to characterize the chondrogenic potential of nasoseptal chondrocytes compared to articular chondrocytes. Primary porcine and human nasoseptal and articular chondrocytes were investigated for extracellular cartilage matrix (ECM) expression in a monolayer culture. 3D polyglycolic acid-(PGA) associated porcine heterotopic mono-and cocultures were assessed for cell vitality, types II, I, and total collagen-, and proteoglycan content. The type II collagen, lubricin, and Sox9 gene expressions were significantly higher in articular compared with nasoseptal monolayer chondrocytes, while type IX collagen expression was lower in articular chondrocytes. Only b1-integrin gene expression was significantly inferior in humans but not in porcine nasoseptal compared with articular chondrocytes, indicating species-dependent differences. Heterotopic chondrocytes in PGA cultures revealed high vitality with proteoglycan-rich hyaline-like ECM production. Similar amounts of type II collagen deposition and type II/I collagen ratios were found in heterotopic chondrocytes cultured on PGA compared to articular chondrocytes. Quantitative analyses revealed a time-dependent increase in total collagen and proteoglycan content, whereby the differences between heterotopic and articular chondrocyte cultures were not significant. Nasoseptal and auricular chondrocytes monocultured in PGA or cocultured with articular chondrocytes revealed a comparable high chondrogenic potential in a tissue engineering setting, which created the opportunity to test them in vivo for articular cartilage repair.Gene expression was determined using RTD-PCR. Porcine (passage 1) and human (passage 0) articular and nasoseptal chondrocytes were cultured for 5 days until 70% confluence. 62 K. El Sayed et al. Hydroxyproline assay for the assessment of total collagen contentDry weight of the probes was measured after dehydration in an exsiccator filled with phosphorus pentoxide as a desiccant. Next, samples were hydrolyzed with 6 M HCl at 105 C for 24 h. The hydrolyzate was buffered by adding citrate acetate buffer containing 0.24 M citric acid monohydrate, 0.88 M sodium acetate trihydrate, 0.85 M sodium hydroxide, and 1.2% acetic acid volume at pH 6.0. Next, chloramine-T (Merk, Darmstadt, Germany) solved in ≥ 99.5 % methyl cellosolve (Carl-Roth) was added and 64 K. El Sayed et al.

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