Effects of detergents on bovine disc decullularization for the generation of natural disc scaffolds for intervertebral disc tissue engineering

Abstract

Study of intervertebral disc (IVD) cells in a 3D formation within a niche resembling native extracellular matrix (ECM) is important for researchers to understand the actual cell behaviour and etiology of disc degeneration/regeneration. Removal of resident cells and cellular contents whilst minimizing adverse effects on ECM from IVD is necessary in making a natural scaffold. We hypothesize that bovine discs may be effectively decellularised using a detergent to produce a scaffold resembling native IVD for disc cell investigation. Bovine caudal discs (18-20mm diameter) were harvested and incubated with either 25 mL of PBS containing 0.1% SDS or 1% Triton X-100 (TX-100) in Tris-HCL solution with protease inhibitor. Live/Dead staining and Alamar Blue assay were used to determine numbers of resident cells and their metabolic activity after treatment, respectively.0.1% SDS solution enabled removal of 69% and 73% cell contents from annulus fibrosus and nucleus pulposus respectively, whereas 1% TX-100 removed 54% from both tissues. SDS was the most efficient in removing resident cells from IVD. Being an ionic reagent, SDS has a role in solubilising cytoplasmic and nuclear membranes, which caused the disruption of protein-protein interactions and aided in the clearance of cells. The successful development of a natural scaffold will allow IVD cells to be studied in a physiological 3D microenvironment without the use of in vivo animal models. This methodology also has the potential to provide a significant source of readily available tissue for IVD degeneration/regeneration studies.