Abstracts Posters SICOT-SOF meeting Gothenburg 2010 _2_

Poster
Topic: Spine
Abstract number: 25328
EFFECTS OF DETERGENTS ON BOVINE DISC DECELLULARIZATION FOR THE
GENERATION OF NATURAL DISC SCAFFOLDS FOR INTERVERTEBRAL DISC
TISSUE ENGINEERING
Kelvin YEUNG 1 , Kit Ying CHAN 2 , Victor YL LEUNG 2 , Vivian TAM 2 , William LU 2 , K.Y
SZE 2 , Kenneth CHEUNG 2
1 The University of Hong Kong, Hong Kong (HONG KONG)
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.
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