Abstract
Introduction
Collagen is the predominant component of extracellular matrix in various connective tissues and makes up to 25% to 35% of the whole protein content in animal bodies. Type II collagen was first introduced from chicken sternal cartilage and presents supportive function in cartilaginous tissue. Since type II collagen is the major component of cartilage in joint, this study is aiming to determine an optimal type II collagen material for the development of medical devices for articular cartilage regeneration. In order to make more effective use of underutilized food waste, type II collagens from mammalian tissue sources (porcine tracheal cartilage; auricular cartilage; articular cartilage) and marine tissue sources (cuckoo ray, blonde ray, thorn back ray, lesser spotted dogfish) were isolated through acid-pepsin digestion under 4°C and characterized by various biological, biochemical and biophysical analysis. Pepsin cleaves the telopeptide region of the collagen molecule and pepsin treated collagen extraction ensures higher collagen yield. Telopeptide-free collagen reveals cytocompatibility, biodegradability and lower toxicity. The number and size of collagen chains were revealed by SDS-polyacrylamide gel electrophoresis. Intermolecular crosslinking density was quantified by Ninhydrin assay. Thermal stability was tested by differential scanning calorimetry (DSC) and enzymatic degradation was assessed by collagenase assay. Human chondrocytes were seeded on to collagen sponges at a density of 30,000 cells per sponge. Cell morphology (DAPI/ Rhodamine Phalloidin), viability(LIVE/DEAD®), proliferation(PicoGreen®) and metabolic activity (alamarBlue®) were analysed. Quantitative morphometric analysis was carried out using ImageJ software.
Conclusion
Porcine articular cartilage and cartilaginous fishes yield high purity type II collagen. Type II collagen isolated from cartilaginous fishes exhibited similar crosslinking density and thermal stability. Among various porcine cartilaginous tissues, articular cartilage was the most resistant to enzymatic degradation and female trachea exhibited the highest cross-linking density. The biological, biochemical and thermal properties of type II collagen are dependent on the tissue and gender from which the collagen was extracted.
