Analysis of collagen types synthesized by rabbit ear cartilage chondrocytes in vivo and in vitro

Abstract

This study compares the collagen types present in rabbit ear cartilage with those synthesized by dissociated chondrocytes in cell culture. The cartilage was first extracted with 4M-guanidinium chloride to remove proteoglycans. This step also extracted type I collagen. After pepsin solubilization of the residue, three additional, genetically distinct collagen types could be separated by fractional salt precipitation. On SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis they were identified as type II collagen, (1 alpha, 2 alpha, 3 alpha) collagen and M-collagen fragments, a collagen pattern identical with that found in hyaline cartilage. Types I, II, (1 alpha, 2 alpha, 3 alpha) and M-collagen fragments represent 20, 75, 3.5, and 1% respectively of the total collagen. In frozen sections of ear cartilage, type II collagen was located by immunofluorescence staining in the extracellular matrix, whereas type I collagen was closely associated with the chondrocytes. Within 24h after release from elastic cartilage by enzymic digestion, auricular chondrocytes began to synthesize type III collagen, in addition to the above-mentioned collagens. This was shown after labelling of freshly dissociated chondrocytes with [3H]proline 1 day after plating, fractionation of the pepsin-treated collagens from medium and cell layer by NaCl precipitation, and analysis of the fractions by CM(carboxymethyl)-cellulose chromatography and SDS/polyacrylamide-gel electrophoresis. The 0.8 M-NaCl precipitate of cell-layer extracts consisted predominantly of type II collagen. The 0.8 M-NaCl precipitate obtained from the medium contained type I, II, and III collagen. In the supernatant of the 0.8 M-NaCl precipitation remained, both in the cell extract and medium, predominantly 1 alpha-, 2 alpha-, and 3 alpha-chains and M-collagen fragments. These results indicate that auricular chondrocytes are similar to chondrocytes from hyaline cartilage in that they produce, with the exception of type I collagen, the same collagen types in vivo, but change their cellular phenotype more rapidly after transfer to monolayer culture, as indicated by the prompt onset of type III collagen synthesis.

Development of Scaffold-Free Elastic Cartilaginous Constructs with Structural Similarities to Auricular Cartilage

“…Although the development of perichondrium-like regions in neocartilage constructs have been observed in relatively few previous studies-most notably by Yanaga and coworkers who also showed a spontaneous recapitulation of a perichondrium-like region after implantation, 46 the majority of elastic cartilage tissue engineering approaches tend to only utilize cells isolated from the cartilaginous region. 10,11,[47][48][49][50][51] The creation of engineered constructs possessing both cartilaginous and perichondrium regions, similar to that observed in native auricular cartilage, is of benefit toward the development of functional engineered constructs suitable for implantation. 2,10,44 The cartilaginous portion of the engineered constructs also appeared to be similar to that of native auricular cartilage with the presence of elastin, collagen types II and X, and sulfated proteoglycans.…”

Structural and biomechanical characteristics of the diaphragmatic tendon in infancy and childhood: an initial analysis

“…During the GAG extraction process, acid-soluble collagen (mainly type I) was simultaneously extracted [9]. Samples were then digested with 1% (wt/vol) pepsin (Sigma) in 0.5 mol/L acetic acid (Fisher Scientific, Pittsburgh, PA) on an orbital shaker at 4°C for 24 hours.…”

The burned ear; possibilities and challenges in framework reconstruction and coverage

“…Unfortunately, adequate proliferation of chondrocytes whilst maintaining their chondrogenic capacity has proven to be a challenge, as cells tend to dedifferentiate upon culturing [49]. Apart from growth factors and other soluble mediators, the biomechanical environment has proven to play an import role in cell behavior [50][51][52]. Different approaches to maintain cartilage phenotype have been proposed including growth factor mediation [53], coculturing [54] and 3D matrix culture environments [55].…”