Production of a natural disc scaffold for intervertebral disc tissue engineering

Abstract

INTRODUCTION: The physiological properties of the intervertebral disc (IVD) are highly linked to the composition of the extracellular matrix (ECM), of which current synthetic scaffolds lack a natural niche for cell growth. Decellularization helps to removecellular contents from a tissue whilst minimizing adverse effects on the ECM. This study aims to develop a natural disc scaffold with preserved ECM for disc cell culture by decellularization. METHODS: Bovine caudal discs (18‐23 mm in diameter) were harvested. Discs were incubated with 25 mL of phosphate buffer solution containing 0.1% sodium dodecyl sulphate (SDS) and protease inhibitor with constant agitation. Optimum conditions were identified using 4 different protocols which tested the effects of the duration and temperature of washing, frequency of solution replacement and number of snap‐freezing cycles during IVD decellularization. Live/Dead staining and the Alamar Blue assay were used for analysing the number of remaining cells and their metabolic activity after treatment. RESULTS: The most effective protocol enabled removal of 69% and 73% cell content from the annulus fibrosus and nucleus pulposus, respectively. Data from Alamar Blue assays suggested that the majority of the cell activity was eliminated. DISCUSSION: Increase of the duration of washing, frequency of solution replacement, and number of snap‐freezing cycles, decreased cell viability and facilitated removal of cellular contents. In this study, decellularization was achieved by physical treatment (snap‐freezing cycles) and chemical washing (SDS). Our findings imply that formation of intracellular ice crystals during the cycles facilitated the disruption of the cell membrane which allowed disc cell contents to be washed out. SDS buffer has a role in solubilizing cytoplasmic and nuclear membranes which aided in the clearance of cell remnants. Overall, this study shows that varying the frequency of solution change and duration of washing can effectively eliminate the majority of disc cells from the IVD. © 2010 Lippincott Williams & Wilkins, Inc.