Characterization of chondrocyte sheets prepared using a co-culture method with temperature-responsive culture inserts

Abstract

Conventional culture methods using temperature-responsive culture dishes require 4-5 weeks to prepare layered chondrocyte sheets that can be used in articular cartilage repair and regeneration. This study investigated whether the use of synovial tissue obtained from the same joint as the chondrocyte nutritive supply source could more quickly facilitate the preparation of chondrocyte sheets. After culturing derived synoviocytes and chondrocytes together (i.e. combined culture or co-culture) on temperature-responsive inserts, chondrocyte growth was assessed and a molecular analysis of the chondrocyte sheets was performed. Transplantable tissue could be obtained more quickly using this method (average 10.5 days). Real-time polymerase chain reaction and immunostaining of the three-layer chondrocyte sheets confirmed the significant expression of genes critical to cartilage maintenance, including type II collagen (COL2), aggrecan-1 and tissue metallopeptidase inhibitor 1. However, the expression of COL1, matrix metalloproteinase 3 (MMP3), MMP13 and A-disintegrin and metalloproteinase with thrombospondin motifs 5 was suppressed. The adhesive factor fibronectin-1 (FN1) was observed in all sheet layers, whereas in sheets generated using conventional preparation methods positive FN1 immunostaining was observed only on the surface of the sheets. The results indicate that synoviocyte co-cultures provide an optimal environment for the preparation of chondrocyte sheets for tissue transplantation and are particularly beneficial for shortening the required culture period. Copyright © 2013 John Wiley & Sons, Ltd.

Annulus fibrosus cell sheets limit disc degeneration in a rat annulus fibrosus injury model

“…Previous investigations on chondrocytes revealed a strong increase in both SOX9 or COL2 expression in layered chondrocyte sheets compared to a standard monolayer culture . Not only was chondrocyte phenotype maintained but the gene expression of cell‐adhesion molecules, such as fibronectin or integrin α10, significantly increased and could be detected throughout chondrocyte sheets contributing to the high tissue‐adhesive properties of the constructs . Moreover, investigators found that it is possible to maintain the ECM deposited by the cells while harvesting and transplanting the cell sheets .…”

Rabbit xenogeneic transplantation model for evaluating human chondrocyte sheets used in articular cartilage repair

“…Under the approval and guidance of the Tokai University Ethics Committee and with patients' informed consent, cartilage and synovial tissues were obtained from five patients (age 61–80 years; average age 71.4 years) who underwent TKA at Tokai University Hospital. Chondrocytes and synovial cells were enzymatically isolated, and chondrocyte sheets made from cells obtained from TKA (TKA sheets) were prepared separately from each donor using the coculture method as previously described (Kokubo et al, ). Briefly, cartilage tissue and synovial tissue were separately minced and digested with 5 mg/ml collagenase Type I (Worthington Biochemical Corp., Lakewood, NJ, USA) in Dulbecco's modified Eagle's medium–Nutrient Mixture F‐12 (DMEM/F12; Gibco, Waltham, MA, USA) supplemented with 20% fetal bovine serum (FBS; Ausgenex, Molendinar, Australia) and 1% antibiotic–antimycotic solution (AA; Gibco).…”

Intra-articular administration of EP2 enhances the articular cartilage repair in a rabbit model

“…There are structural and temporal differences in the formation of chondrocyte sheets between animal species (Kokubo et al, ). New treatments that can be used in combination with chondrocyte sheet transplantation are needed to provide stable results from a very early stage after chondrocyte sheet transplantation.…”