Isolation and characterization of pepsin solubilized type Ⅱ collagen from porcine cartilage and cartilagious fish for intervertebral disc (IVD) regeneration

摘要

Introduction: Collagen is the main component of ECM with 29 types identified. Type Ⅱ collagen presents supportive function in cartilaginous tissues. Since type Ⅱ collagen is the major component of nucleus pulposus in intervertebral disc, the potential of injectable type Ⅱ collagen hydrogel as a cell carrier for intervertebral disc regeneration is being investigated in this project. At this stage, we isolated and characterised type Ⅱ collagen from porcine cartilage nad cartilaginous fish. Type Ⅱ collagen was cross-linked with 4arm and 8arm StarPEG. We hypothesise that pure type Ⅱ collagen can be obtained from cartilaginous tissue and 8arm StarPEG crosslinking will increase the mechanical, biological and biomedical properties of collagen hydrogel. Materials and Methods: Tyep Ⅱ collagen was extracted from porcine trachea, ear, articular cartilage and cartilaginous fish through acid-pepsin digestion at 4°C. SDS-PAGE was used to reveal the number and size of collagen chains. Thermal stability was tested by differential scanning calorimetry (DSC). Enzymatic degradation was assessed by collagenase assay. TNBSA assay will be carried out to test the free amine group in cross-linked collagen hydrogel. Human chondrocyte cell phenotype maintenance will be assessed with proteina and gene assays. Results: Conclusions: Pure type Ⅱ collagen can be extracted from porcine cartilage and cartilaginous fish. Further biological assays will be carried out to characterise collagen enzymatic degradation and thermal stability.