EVALUATION OF A 3-D ALGINATE SCAFFOLD FOR INTERVERTEBRAL DISC ANNULUS FIBROSUS CELLS
TISSUE ENGINEERING
*Shao, XX; +*Hunter, CJ
+*University of Calgary, Calgary, Alberta, Canada
[email protected]
INTRODUCTION
Degeneration of the intervertebral discs cause discogenic low back
pain and limited mobility. Current surgical treatments include spine
fusion, which eliminates pain but does not restore disc function, or
implantation of synthetic disc prostheses. However, these materials face
challenges in fixation, mechanical strength and loosing of the implant
[1]. Tissue engineering strategies may produce new promising
treatments for intervertebral disc disease, using functional living tissues
constructed from cells and scaffolds. Unlike articular cartilage, the
intervertebral disc has two distinct regions, the annulus fibrosus (AF)
and the nucleus pulposus (NP), each containing different cell type and
extracellular matrix. The aim of the present study was to develop a
nonwoven three-dimensional alginate felt for supporting annulus
fibrosus cell growth.
METHODS
Scaffold: Alginate felts were synthesized by extruding a solution of
1.4% sodium alginate in water into a bath of 102 mM CaCl2 or BaCl2
[2,3] through a 25G needle with rapid random motion of the needle tip
through the bath. After 5 minutes in the bath, the supernatant was
decanted, and the felt was washed twice in phosphate-buffered saline
(PBS) containing 1.3 mM CaCl2 or BaCl2. The supernatant was again
decanted, and the felt was freeze-dried, then sterilized by soaking
overnight in 70% ethanol and freeze-dried again. This procedure
produced mechanically stable, randomly oriented felt with
approximately 240 µm fiber diameter (Fig. 1). Alginate felts were
chemically modified with polypeptide residues using a previously
described protocol [4]. Briefly, alginate was placed into a solution of
sulfo-NHS (N-hydroxy-sulfosuccinimide) and EDC (1-ethyl(dimethylaminopropyl) carbodiimide) at a molar ratio of 1:2 (sulfoNHS: EDC) and 1:20 (EDC: uronic acid monomers) and incubated for 5
minutes. Peptide RGD (GGGGRGDY), RGE (GGGGRGEY) or a water
control were added at 500 mg per gram of alginate and incubated for 20
hours. The resulting modified alginate was dialyzed for 3 days against
PBS to remove unbound peptide and chemical residues, then rinsed
thoroughly in PBS.
AF cells culture on the scaffold and gene expression: Canine AF
cells were achieved from collagenase and pronase digestion of minced
annulus fibrosis tissue. The cells were loaded into the alginate scaffolds
with different cross-linker (BaCl2 or CaCl2) and polypeptide coating
(Blank control, RGD or RGE) by incubating overnight on a shaker table.
The cell/scaffold constructs were then culture in 6-well plates for 7 days,
with media changed every two days.
Culture medium was
DMEM/Ham’s-12 containing 10% fetal bovine serum, penicillin
(100U/ml) and streptomycin (100U/ml). At the end of the experiment,
constructs were snap-frozen and collected for RNA extraction. Gene
expression was analyzed using real-time RT-PCR for collagen I,
collagen IIB, aggrecan and decorin.
Data were analyzed using ANOVA with Turkey’s post-hoc test, with
significance set at p<0.05.
the BaCl2 constructs, and they appeared more gelatinous than the fibrous
BaCl2 condition.
Real time RT-PCR results indicated that the AF cells can express
collagen I, collagen II, aggrecan and decorin genes on all the alginate
scaffolds. Collagen I and aggrecan genes showed higher relative
expression than Collagen II and decorin (approximately 105-fold higher).
In comparing the crosslinking ions, CaCl2 showed a significantly higher
expression of collagen I, collagen II, decorin and aggrecan than BaCl2
(p<0.05). In comparing the peptide treatments, there were no significant
differences in expression level for all genes between the RGD, RGE and
blank scaffold group (Fig. 2). There was also no significant interaction
between the cation and peptide treatments in all genes tested.
Figure 2: Relative gene expression on different scaffolds. Data are
displayed as percentage of CaCl2-blank control. N=4.
DISCUSSION
Alginate has found wide biotechnology applications as a material for
the encapsulation of a variety of cells or for wound dressing because its
biocompatibility, hydrophilicity, and relative low cost. In tissue
engineering, crosslinked alginate has been used extensively as a delivery
matrix for cartilage reconstruction [5, 6]. However, most of these
investigations used cells embedded within the polymerized gel. From an
anatomical viewpoint, this kind of gel scaffold may not be as suitable for
the annulus fibrosus. Therefore our present studies produced a nonwoven alginate felt. It is highly likely that reconstruction of a threedimensional shape can be made using this alginate sponge, to fit to a
degenerated annulus fibrosus. Our results show that canine annulus
fibrosus cells can grow on this kind of alginate scaffold and express
several functional genes. While the CaCl2 crosslinker demonstrated
better support for gene expression, the BaCl2 condition may be more
suitable, due to its better mechanical integrity. Further studies will
attempt to stabilize the CaCl2 condition and/or stimulate gene expression
in the BaCl2 condition. Peptide modification of the gels did not produce
any obvious improvement for all of the genes explored; however, this
may be more a function of batch-to-batch variability than a difference in
responsiveness. These preliminary results demonstrate that a non-woven
alginate sponge can act as a matrix for annulus fibrosus cells growth,
that the gel has good potential as a three-dimensional scaffold for
intervertebral disc tissue engineering. Future work will focus on the
scaffold shape modification and further characterization of the system.
ACKNOWLEDGEMENTS
CJH is supported by the McCaig Centre for Joint Injury and Arthritis
Research. This work was funded by NSERC under grant number
288259.
Figure 1. Left: alginate felt (left: hydrated, right: freeze-dried). Right:
Individual fibers have a mean diameter of 240 µm.
RESULTS
The CaCl2 condition was not as stable as the BaCl2, as the felts began
to degrade after approximately 4 days in culture. At the end of the
experiment, the CaCl2 constructs were approximately ¼ of the mass of
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2004 [4] Rowley et al, Biomaterials 1999, 20,45-53 [5] Fragonas E et al,
Biomaterials 2000,21(8):795-801 [6] Mierisch CM et al, J Bone Joint
Surg Am. 2003 Sep;85-A(9):1757-67.
51st Annual Meeting of the Orthopaedic Research Society
Poster No: 1724