Engineering alginate for intervertebral disc repair

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

4 (2011) 1196–1205

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journal homepage: www.elsevier.com/locate/jmbbm

Research paper

Engineering alginate for intervertebral disc repair Johannes L. Bron a,b,∗ , Lucienne A. Vonk b,c , Theodoor H. Smit a,b , Gijsje H. Koenderink d a Department of Orthopaedic Surgery, VU University Medical Center, Amsterdam, The Netherlands b Skeletal Tissue Engineering Group Amsterdam (STEGA) and Research Institute MOVE, The Netherlands c Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University

Amsterdam, Amsterdam, The Netherlands d Biological Soft Matter group, FOM Institute for Atomic and Molecular Physics (AMOLF), Amsterdam, The Netherlands

A R T I C L E

I N F O

A B S T R A C T

Article history:

Alginate is frequently studied as a scaffold for intervertebral disc (IVD) repair, since it

Received 14 January 2011

closely mimics mechanical and cell-adhesive properties of the nucleus pulposus (NP) of the

Received in revised form

IVD. The aim of this study was to assess the relation between alginate concentration and

2 April 2011

scaffold stiffness and find preparation conditions where the viscoelastic behaviour mimics

Accepted 4 April 2011

that of the NP. In addition, we measured the effect of variations in scaffold stiffness on the

Published online 24 April 2011

expression of extracellular matrix molecules specific to the NP (proteoglycans and collagen) by native NP cells. We prepared sample discs of different concentrations of alginate (1%–6%)

Keywords:

by two different methods, diffusion and in situ gelation. The stiffness increased with

Alginate

increasing alginate concentration, while the loss tangent (dissipative behaviour) remained

Intervertebral disc

constant. The diffusion samples were ten-fold stiffer than samples prepared by in situ

Tissue engineering

gelation. Sample discs prepared from 2% alginate by diffusion closely matched the stiffness

Gene expression

and loss tangent of the NP. The stiffness of all samples declined upon prolonged incubation

Rheology

in medium, especially for samples prepared by diffusion. The biosynthetic phenotype of native cells isolated from NPs was preserved in alginate matrices up to 4 weeks of culturing. Gene expression levels of extracellular matrix components were insensitive to alginate concentration and corresponding matrix stiffness, likely due to the poor adhesiveness of the cells to alginate. In conclusion, alginate can mimic the viscoelastic properties of the NP and preserve the biosynthetic phenotype of NP cells but certain limitations like long-term stability still have to be addressed. c 2011 Elsevier Ltd. All rights reserved. ⃝

1.

Introduction

Transplantation systems based on scaffolds seeded with stem cells or native cells offer a promising means to repair aged, damaged, or diseased tissues (Hubbell, 2003). Accordingly, there has been much recent effort to design scaffolds that mimic the bioadhesive and physical characteristics

of natural extracellular matrices found in tissues and can thus promote tissue-specific cell phenotype (Huebsch and Mooney, 2009; Lutolf et al., 2009). A variety of tissues can already be engineered by this approach, including artery, skin, cartilage, bone, ligament, and tendon. Scaffold stiffness has been recognized as an especially important cue to

∗ Corresponding address: Department of Orthopaedic Surgery, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands. Tel.: +31 20 44 45242. E-mail addresses: [email protected], [email protected] (J.L. Bron). c 2011 Elsevier Ltd. All rights reserved. 1751-6161/$ - see front matter ⃝ doi:10.1016/j.jmbbm.2011.04.002

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guide cell differentiation and extracellular matrix (ECM) production (Breuls et al., 2008; Discher et al., 2005; Engler et al., 2007) and this knowledge is now increasingly being implemented in tissue engineering strategies (Discher et al., 2009). The mechanical characteristics of many tissues have been documented over the recent years, facilitating the development of new generations of 3D scaffolds mimicking these features (Breuls et al., 2008; Iatridis et al., 1996). Our own research over the past years has focused on tissue engineering strategies to repair damaged intervertebral discs (IVDs) (Bron et al., 2009b,a, 2010). The IVD is a cartilaginous structure that lies between adjacent vertebrae, where it acts as a shock absorber and allows motion of the otherwise rigid vertebral column (Roberts et al., 2006). The IVD consists of a collagenous outer annulus fibrosus (AF) which surrounds the gelatinous inner nucleus pulposus (NP). Ageing is accompanied by loss of water and proteoglycans from the gelatinous NP, which becomes more fibrous, resulting in a more rigid IVD. Although these changes are to some extent physiological, they may result in symptomatic degenerative disc disease (Roberts et al., 2006). In some patients, early degeneration of the AF may result in a posterior tear through which the NP can extrude (disc herniation), compromising the neurological structures (spine and nerve roots) that the vertebral column usually protects. The current clinical solution is to evacuate the herniated NP material (discectomy), thereby relieving the compressed nerves (Hegewald et al., 2008). There are, however, serious adverse effects of disc herniation and subsequent discectomy on spinal biomechanics resulting in discogenic back pain that seriously affects the quality of life in many patients. Much research is therefore directed towards the restoration of the herniated disc either by replacement or regenerative approaches. Ideally, current discectomy procedures should be combined with the replacement of the lost NP material by a scaffold with (native or stem-) cells initiating disc regeneration instead of degeneration (Hegewald et al., 2008). In a recent study, we showed that the mechanical properties of the NP can be mimicked using dense scaffolds of collagen I, which is a natural extracellular matrix protein (Bron et al., 2009b). The scaffold stiffness approached that of the NP, but the viscous modulus was lower. Aside from the difference in viscous behaviour, type I collagen is not an optimal replacement of the NP, which is predominantly composed of type II collagen and proteoglycans. Other 3D scaffold materials, such as alginate, agarose and chitosan, have also been studied for NP regeneration, and these might allow a closer match both from a mechanical and a biochemical point of view (Gruber et al., 1997; Leone et al., 2008; Saad and Spector, 2004). Alginate is most often studied since it is inexpensive and does not evoke adverse tissue reactions (Leone et al., 2008; Li et al., 2008; Nunamaker et al., 2007). Alginate is a naturally occurring, water soluble polysaccharide block copolymer composed of β-L-mannuronic acid (M) and α-L-guluronic acid (G) that can be ionically crosslinked by divalent ions, such as calcium (Larsen and Haug, 1971). The resulting matrix has a stiffness which is determined by the alginate concentration and by the ratio between G and M blocks (Nunamaker et al., 2007). Other conditions such as gelation temperature and type of

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crosslinker also influence the final network structure and ensuing mechanical properties (Augst et al., 2006). The aim of this study was to design alginate scaffolds with viscoelastic properties that mimic those of the NP and to assess the biosynthetic response of native NP cells. We therefore investigated the effects of variations in alginate concentration on the viscoelastic (rheological) characteristics of scaffolds. In addition, we compared two different methods of inducing alginate gelation, by diffusion and by ‘in situ’ gelation. In diffusion-induced gelation, calcium ions are allowed to diffuse into the alginate gel via a porous membrane, leading to crosslinking (Nunamaker et al., 2007). “In situ” gelation is performed by mixing insoluble calcium with the alginate solution and then releasing calcium ions within the solution by enzymatically decreasing the pH level (Kuo and Ma, 2001). Since it has been documented that alginate scaffolds rapidly loose their stiffness in vivo (Nunamaker et al., 2007), we monitored the time-dependent stiffness during prolonged incubation in cell culture medium. Finally, to determine whether variations in alginate concentration affect cell behaviour, we cultured native cells isolated from goat NP and annulus fibrosus (AF) in alginate beads of different alginate concentrations (2%, 4% and 6%). We monitored the gene expression levels of the main natural components of the ECM of the NP (types I and II collagen and aggrecan) up to 4 weeks. The gene expression levels were compared to gene expression levels found in chondrocytes from articular cartilage (AC), for which extensive studies have been performed of the preservation of phenotype in alginate (De et al., 2004; Hauselmann et al., 1994; Lin et al., 2009; Masuda et al., 2003).

2.

Materials and methods

2.1. Preparation of alginate sample discs by calcium diffusion Freeze dried alginate (LVCR sodium alginate, Monsanto, San Diego, CA) was dissolved in water containing 0.9 wt% sodium chloride. Alginate solutions at four different concentrations (1, 2, 4, and 6 wt%) were sterilized by autoclaving (121 ◦ C, 15 min). Sample discs were prepared by pouring 2 ml of alginate solution into tissue culture inserts (25 mm, pore size 0.4 µm; Nunc, Roskilde, Denmark). The inserts were placed in Petri dishes containing an aqueous solution of 500 mM calcium chloride, and a polycarbonate filter membrane (thickness 8 mm) was placed on top, which was irrigated with 2 ml of the calcium solution. After two hours at room temperature, alginate gelation was finished and the sample discs were removed from the culture inserts. The samples intended for analysis after prolonged storage in medium were transferred to 6-well plates containing 5 ml Dulbecco’s Modified Eagles Medium (DMEM, Gibco, Paisley, UK) supplemented with 1% streptomycin, penicillin and amphotericin B (all from Gibco). The medium was refreshed every three days. Samples were assayed at three time points (0, 1 and 10 days), using five separate samples for each time point and each alginate concentration.

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2.2. Preparation of alginate sample discs by in situ gelation Insoluble calcium carbonate powder was mixed at a concentration of 100 mM with alginate solutions in 0.9% NaCl (2%, 4% or 6% alginate) and stirred. The mixture was acidified by adding the enzyme Glucono Delta-Lactone (GDL, Sigma Chemical Co. (St. Louis, MO)) to a final concentration of 80 mM. A volume of 2 ml of the acidified mixture was injected into the wells of 12-well (well diameter 22 mm) plates using a syringe. After 2 h at room temperature, the gelled sample discs were removed from the wells and transferred to the rheometer for analysis. The samples intended for analysis after prolonged storage in medium were transferred to 6-well plates containing 5 ml DMEM supplemented with 1% antibiotics. The medium was refreshed every three days. Samples were assayed at two time points (0 and 10 days), using five separate samples for each time point and each alginate concentration. The samples after 10 days of incubation showed irregular edges and were therefore reduced to a size of 20.0 mm with a cork borer.

2.3.

Rheometry

The viscoelastic properties of the alginate discs were measured using a stress-controlled rheometer (Paar Physica MCR501, Anton Paar, Graz, Austria) equipped with a temperature-controlled steel bottom plate and 20 or 40 mmdiameter steel top plates. The alginate discs showed some variability in diameter after incubation in culture medium, due to variable degrees of shrinkage. Since variations in sample size complicate the interpretation of rheological data, we equalized the sample diameters using cork borers. Samples prepared by diffusion were reduced to a diameter of 20 mm at t = 0 and 15.4 mm for t = 1 and 10 days. In situ gelled samples were perfectly circular directly after gelation, with a diameter of 22 mm; they were measured using a 40 mm top plate. After 10 days incubation, the samples showed some edge irregularities. To exclude any edge effects, the incubated samples were reduced to a diameter of 20 mm, matching the diameter of the 20 mm top plate. For samples with a diameter smaller than the diameter of the rheometer top plate, the absolute values of the shear moduli were corrected as described earlier (Bron et al., 2009b). To prevent sample slippage, self-adhesive sandpaper (CP918C P180, VSM Abrasives, O’Fallon, Missouri, USA) was attached to both plates. The discs were loaded between the plates, and the top plate was lowered until the sample was in good contact with both plates. The tests were performed at a temperature of 37 ◦ C in a humidified chamber. To exclude any time-dependent relaxation during the tests, the samples were first equilibrated for 10 min. During this time, the normal force decreased to values below 0.25 N in all samples. Subsequently, we probed the frequency-dependent shear moduli by performing frequency sweep measurements over an angular frequency range of 0.2–200 rad s−1 at a strain amplitude of 1%, well within the linear regime. Finally, to test the strength of the alginate discs, we subjected them to sinusoidally oscillating shear at a fixed frequency of 0.5 Hz and gradually increasing strain amplitude, until a maximum

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of 1000% strain or until sample failure occurred. The shear modulus G∗ (ω) follows from the ratio between stress (σ) and strain amplitude (γ). G∗ is a complex quantity with an elastic (or storage) modulus (G′ ) and viscous (or loss) modulus (G′′ ). The absolute magnitude of the shear modulus, |G∗ |, was calculated using |G∗ | = ((G′ )2 + (G′′ )2 )0.5 . The ratio G′′ /G′ is referred to as the loss tangent, since it equals the tangent of the phase angle difference between stress and strain (tan δ). Data reported represent the mean ± S.E. from 5 samples per condition.

2.4.

Isolation of native cells and cell culture in alginate

Cartilaginous tissues were obtained from skeletally mature female Dutch milk goats (n = 8) that were sacrificed for other studies. All thoracic and lumbar intervertebral discs (IVDs, T1-L2/L6/S1) and articular cartilage (AC) from the glenohumeral joint were collected. The IVDs were dissected to separate the nucleus pulposus (NP) from the annulus fibrosus (AF). To assure an adequate cell number, tissues from two goats were mixed for every measurement. Experiments were performed in quadruplicate. The tissues were dissected and minced, and the cells were released by subjected the tissues to sequential treatments first with DMEM supplemented with 1% foetal bovine serum (FBS, HyClone, Logan, UT, USA), 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B and 2.5% (w/v) Pronase E (Sigma, St. Louis, MO) for 1 h, then with DMEM supplemented with 25% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B and 0.125% (w/v) collagenase (CLS-2, Worthington, Lakewood, NJ) for 16 h at 37 ◦ C. After filtering the cell suspension through a 70 µm pore size cell strainer (BD Biosciences, San Diego, CA), isolated cells were resuspended in an alginate solution (2, 4 and 6 (w/v) in 0.9% NaCl (0.2 µm sterile filtered), creating a suspension of 4 × 106 cells/ml. The suspension was homogenized by slow pipetting and transferred to a sterile syringe. Alginate beads were formed by the diffusion method, dripping ∼10 µL drops of the solution from the syringe needle (26 gauge) into a calcium chloride solution (102 mM). The beads were allowed to gel by inward diffusion of Ca2+ for 10 min at ambient temperature. After washing twice in 0.9% NaCl and twice in DMEM, the alginate beads were transferred to 24-well tissue culture dishes with 10 beads per well (Greiner Bio-one, Kremsmuenster, Austria). The cells were cultured in 500 µl of DMEM per well, supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 50 µg/ml ascorbate-2-phosphate (Sigma). We note that our purpose is to develop a clinical procedure where freshly harvested cells are immediately transplanted back into the patient in an alginate scaffold. For this reason, we did not first do expansion in 2D culture, but characterized gene expression for freshly isolated cells cultured in 3D.

2.5.

Real-time PCR

Alginate beads were dissolved in alginate dissolving buffer (55 mM Na-citrate, 0.15 M NaCl, 30 mM Na2 EDTA, pH 6.8), total RNA was isolated from the cells with the RNeasy mini kit (Qiagen, Gaithersburg, MD), and DNase I treatment was

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performed as described by the manufacturer to remove any contaminating genomic DNA. Total RNA (750 ng) was reverse transcribed using 250 U/ml Transcriptor Reverse Transcriptase (Roche Diagnostics, Mannheim, Germany), 0.08 U random primers (Roche diagnostics), and 1 mM of each dNTP (Invitrogen, Carlsbad, CA) in Transcriptor RT reaction buffer at 42 ◦ C for 45 min followed by inactivation of the enzyme at 80 ◦ C for 5 min. Real-time PCR reactions were performed using the SYBRGreen reaction kit according to the manufacturer’s instructions (Roche Diagnostics) in a LightCycler 480 (Roche Diagnostics). The LightCycler reactions were prepared in 20 µl total volume with 7 µl PCR-H2 O, 0.5 µl forward primer (0.2 µM), 0.5 µl reverse primer (0.2 µM), 10 µl LightCycler Mastermix (LightCycler 480 SYBR Green I Master; Roche Diagnostics), to which 2 µl of 5 times diluted cDNA was added as PCR template. Primers (Invitrogen) used for real-time PCR are listed in Table 1. Specific primers were designed from sequences available in data banks, based on homology in conserved domains between human, mouse, rat, dog and cow. The amplified PCR fragment extended over at least one exon-border (except for 18S). Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (Ywhaz) and hypoxanthine 18S (ribosomal RNA) were used as housekeeping genes and the gene expression levels were normalized using a normal√ ization factor calculated with the equation (Ywhaz x 18S). With the LightCycler software (version 4), the crossing points were assessed and plotted versus the serial dilution of known concentrations of the standards derived from each gene using the Fit Points method. PCR efficiency was calculated by LightCycler software and the data were used only if the calculated PCR efficiency was between 1.85 and 2.0.

2.6.

Statistical analysis

For the rheological measurements, unpaired Students’ T-test was used for statistical analysis. P < 0.05 was considered as significant. For the real-time PCR experiments, Friedman’s non-parametric rank test was used to determine statistically significant differences within an experiment. When statistically significant differences were detected, assessment of differences between individual groups was performed using Wilcoxon’s signed-rank test.

3.

Results

3.1.

Alginate sample discs prepared by different methods

We prepared alginate discs of concentrations between 1 and 6 wt% by two different methods, namely by diffusion of Ca2+ ions from outside or by in situ release of Ca2+ from calcium carbonate inside the alginate. To characterize the viscoelastic properties, we performed small amplitude oscillatory shear tests on the alginate discs. Both series of samples became significantly stiffer with increasing alginate concentration (square symbols, upper panel Fig. 1). However, the samples prepared by diffusion (black squares) were at least ten-fold stiffer than the in situ gelated samples (grey squares) at all alginate concentrations (significant with P <

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Fig. 1 – Linear viscoelastic behaviour of alginate matrices. Upper panel: dependence of scaffold stiffness (G′ , squares), and viscous modulus (G′′ , triangles), on alginate concentration, for samples prepared by diffusion (black symbols) and in situ gelation (grey symbols), measured at 10 rad/s. Bottom panel: dependence of the loss tangent of alginate scaffolds on alginate concentration, for samples prepared by diffusion (black circles) and in situ gelation (grey circles).

0.05). The sample-to-sample variability was higher for the diffusion series than for the in situ series, as shown by the larger error bars. This indicates that the diffusion samples were less homogeneous than the in situ gelled samples, consistent with prior observations (Nunamaker et al., 2007). The viscous modulus of the samples prepared by diffusion (black triangles) was also significantly larger than that of the in situ polymerized samples (grey triangles). The loss tangent (G′′ /G′ ) was independent of alginate concentration for the diffusion gelated samples (P > 0.05), as shown in the bottom panel of Fig. 1 (black circles). For the in situ gelled samples (grey circles), the 4% and 6% alginate samples had a significantly lower loss tangent than the 2% alginate samples (P < 0.05). The loss tangent of the samples prepared by diffusion (black circles) was significantly higher than that of samples that were gelled in situ (grey circles). These findings implicate that the diffusion samples are stiffer but also have a higher viscosity. To characterize the nonlinear viscoelastic behaviour, we subjected the alginate discs to large amplitude oscillatory shear. The alginate samples prepared by diffusion showed no appreciable linear elastic regime: their shear modulus immediately started to decrease as the strain amplitude was raised, and they failed already at strains of about 100% (black symbols in Fig. 2). In contrast, the samples prepared by in situ gelation were linearly elastic up to strains of about 10%, and thereafter gradually strain-weakened (grey symbols in Fig. 2).

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Table 1 – Primer sequences used for real-time PCR to determine gene expression levels of extracellular matrix components of the NP (collagen types I and II and aggrecan). Target gene Ywhaz 18S Agc Col1a1 Col2a1

Oligonucleotide sequence Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

5′ GATGAAGCCATTGCTGAACTTG3′ 5′ CTATTTGTGGGACAGCATGGA3′ 5′ GTAACCCGTTGAACCCCATT3′ 5′ CCATCCAATCGGTAGTAGCG3′ 5′ CAACTACCCGGCCATCC3′ 5′ GATGGCTCTGTAATGGAACAC3′ 5′ TCCAACGAGATCGAGATCC3′ 5′ AAGCCGAATTCCTGGTCT3′ 5′ AGGGCCAGGATGTCCGGCA3′ 5′ GGGTCCCAGGTTCTCCATCT3′

Annealing temperature (◦ C)

Product size (bp)

56

229

56

151

57

160

57

191

56

195

Ywhaz, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide; 18S, 18S ribosomal RNA; Agc, aggrecan; Col1a1, α1 (I)procollagen; Col2a1, α1 (II)procollagen.

Fig. 2 – Nonlinear viscoelastic response of alginate matrices to large amplitude oscillatory shear. The complex shear modulus of the alginate scaffolds is plotted as a function of strain amplitude. Samples prepared by diffusion gelation (black symbols) gradually strain-weaken and fail at approximately 100% strain, whereas in situ gelled samples (grey symbols) are linearly elastic up to 10% strain, and then gradually weaken. Symbols correspond to alginate concentrations of 2% (squares), 4% (circles), and 6% (triangles).

3.2.

Stability of samples discs in cell culture medium

After prolonged incubation, all samples were visibly weaker than freshly prepared samples. The samples of the lowest concentrations (1% for diffusion and 2% for in situ gelation) had even become too fragile for testing by rheology. After 1 day of incubation in cell culture medium, the diffusion samples already showed a 10% reduction in elastic and viscous modulus (black symbols, upper panel Fig. 3). The decreases of G′ and G′′ were statistically significant at alginate concentrations of 2% (black squares) and 6% samples (black triangles), but not at 4% (black circles; G′ : P = 0.3, G′′ : P = 0.08). After 10 days, the moduli were ten-fold lower than the original value at t = 0(P < 0.05). The samples gelled in situ also showed a significant decline in stiffness, but less (∼40% compared to the initial value), than samples prepared by diffusion (∼90% compared to the initial value), both at an alginate concentration of 4% (grey circles) and 6% (grey triangles), as shown in the upper panel of Fig. 3. After 10 days, there was no longer a significant difference in stiffness between alginate samples of different concentrations or

Fig. 3 – Time-dependence of the linear viscoelastic behaviour of alginate matrices stored in cell culture medium. Upper panel: the complex shear modulus of alginate discs prepared by diffusion gelation (black symbols) or in situ gelling (grey symbols) upon incubation in cell culture medium for 1 and 10 days (at 10 rad/s). Symbols correspond to alginate concentrations of 2% (squares), 4% (circles), and 6% (triangles). After 10 days, no significant differences in stiffness remain. Bottom panel: loss tangent of alginate discs upon incubation in cell culture medium for 1 and 10 days (measured at a frequency of 10 rad/s).

prepared by different methods. As shown in the bottom panel of Fig. 3, the loss tangent of samples prepared by diffusion (black symbols) and in situ gelling (grey symbols) significantly decreased with increasing incubation time in medium, while being independent of alginate concentration (compare 2%, squares, and 4%, circles). The decrease of loss tangent of the 2% diffusion gelled samples only showed a non-significant decline in the loss tangent after 10 days compared to day 0

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Table 2 – Summary of reported stiffnesses of cartilaginous tissues (Iatridis et al., 1996). Tissue Nucleus pulposus Meniscus Annulus Fibrosus Articular cartilage

G∗ (kPa) 11 100 540 600

and 1 (P = 0.07 and P = 0.16). After 10 days, the loss tangents of all samples were statistically indistinguishable.

3.3.

ECM gene expression

To assess the influence of the alginate matrices on the biosynthetic phenotype of native tissue cells, we cultured NP, AC, and AF cells isolated from goat IVDs and cartilage inside alginate beads with concentrations of 2%–6% alginate. We measured the expression levels of NP-specific extracellular matrix components (collagen types I and II and aggrecan) by real-time PCR. Cells freshly isolated from the AF showed the highest level of type I collagen gene expression and chondrocytes from AC showed the lowest expression level (Fig. 4, T = 0; differences statistically significant with p < 0.05). Upon culturing in alginate beads, there was an increase in type I collagen gene expression by all the cells after 1 week (p < 0.001) which was sustained after 2 and 4 weeks (Fig. 4). However, this increase was not significantly influenced by the alginate concentration over a range of 2%–6% (p > 0.05). The increase in gene expression of type I collagen was strongest for the NP cells. After 4 weeks of culture, the expression level of type I collagen for NP cells was similar to the levels found in AF cells (p = 0.08). The levels found in AC cells consistently remained the lowest (p < 0.05). The highest type II collagen gene expression levels directly after cell isolation were found in AC cells and the lowest in AF cells (Fig. 5 T = 0; differences statistically significant with p < 0.05). The gene expression level of type II collagen for all cell types was decreased significantly after 1 week of culture in alginate (p < 0.001), but thereafter remained constant (Fig. 5, p > 0.3). Levels of type II collagen expression remained the lowest in AF cells (p < 0.05), while NP and AC cells had similar levels of expression after 1 or more weeks culturing in alginate (p = 0.067). Levels of aggrecan gene expression were highest for NP cells after isolation (Fig. 6 T = 0, p < 0.05). Culture in alginate led to a steady decrease of the aggrecan gene expression levels of all three cell types, which was already noticeable after 1 week (p < 0.001). NP cells had higher aggrecan gene expression levels than AF and AC cells (p < 0.05). No significant differences in the gene expression levels for type I and II collagen and aggrecan could be found in any of the cell populations when cultured in beads with alginate concentrations of 2% (white bars), 4% (grey bars), or 6% (black bars) (Figs. 4–6; p > 0.05).

4.

Discussion

The principal aim of the current study was to design alginate scaffolds with viscoelastic properties that mimic those of

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the NP. We showed that the stiffness of alginate scaffolds, crosslinked by diffusion of calcium into the alginate solution, can be varied over two orders of magnitude (between 1 kPa and almost 100 kPa) by varying the alginate concentration. The loss tangent was not affected by variations in polymer concentration. The closest matching of the stiffness as well as loss tangent of a healthy NP, which has a stiffness of 11 kPa and loss tangent of about 0.24 (Iatridis et al., 1996), was found for 2% alginate scaffolds. Other cartilaginous tissues are stiffer than even the most concentrated alginate discs (6%) (Table 2). It is difficult to prepare more concentrated alginate discs because the high viscosity of the pre-gelled solution renders it difficult to process and mould the gel and to mix in cells (Augst et al., 2006). However, this difficulty may be counteracted by stirring the alginate solutions, which are shear-thinning (Augst et al., 2006). Moreover, the stiffness of alginate scaffolds may be tuned by other factors, such as the alginate source, G/M ratio, cross linker type, and temperature (Augst et al., 2006; Kuo and Ma, 2001; Leone et al., 2008; Nunamaker et al., 2007). However, these factors are not always accessible for manipulation in the context of tissue engineering. The G/M ratio depends on alginate source and processing and is therefore usually fixed upon delivery (Kuo and Ma, 2001; Larsen and Haug, 1971). Changes in Ca2+ concentration and temperature influence gelation time and thereby matrix organization and stiffness (Augst et al., 2006), but these variations are not always well tolerated by seeded cells. We also compared samples derived by diffusion gelation to samples prepared by in situ release of Ca2+ (Nunamaker et al., 2007). The “in situ” method has several practical advantages over the diffusion method for clinical applications in tissue engineering. The liquid solution can be injected via a syringe and gelation occurs inside the tissue of interest. Moreover, in situ gelation results in more homogeneous scaffolds with less spatial and sampleto-sample variation in biomechanical properties. Scaffolds prepared by diffusion are notoriously inhomogeneous due to the diffusion kinetics of calcium ions. Although we prepared the diffusion scaffolds under standardized circumstances, the structural inhomogeneity appeared to affect the rheology and may have affected cell phenotype. The stiffness of alginate samples prepared by in situ gelation was much lower than that of the diffusion samples, consistent with prior findings (Li et al., 2008). To assess the applicability of alginate discs for IVD repair, it is also crucial to characterize the biological response of native tissue cells to prolonged culture in an alginate matrix. We therefore screened the effects of alginate scaffolds (prepared by diffusion) on native cells by measuring gene expression of extracellular matrix components that are naturally present in the NP. The relative gene expression levels for types I and II collagen found in native IVD cells after isolation is in line with the well-known collagenous composition of these tissues (Almarza and Athanasiou, 2004). The AF contains a mixture of type I and II collagen, the NP contains more type II collagen than the AF, and the AC contains predominantly type II collagen (Almarza and Athanasiou, 2004; Kuettner and Cole, 2005). Aside from collagens, proteoglycans (mainly aggrecan) are the main components of cartilage. The NP contains the highest amount of proteoglycans and the AF the

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B

C

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Fig. 4 – Gene expression levels for type I collagen of native cells cultured in alginate matrices. Real-time PCR was performed on reverse-transcribed RNA isolated from cells derived from the NP, AF and AC of goat intervertebral discs after 0, 7, 14, and 28 days of culture in alginate beads with a concentration of 2% (white bars), 4% (grey bars) and 6% (black bars). The gene expression level of type I collagen (Col1a1) is normalized by the expression levels of two housekeeping genes (2hk). Data are shown as mean ± SD. Differences between NP, AF, and AC cells are statistically significant with p < 0.05 at all time points and alginate concentrations (except for NP–AF in Fig. 4(D), p = 0.08). The dependence on alginate concentration for each cell type is not statistically significant (p > 0.05). Changes with time are significant for all cells on going from T = 0 to later time points (p < 0.001), and for NP cells there is a significant increase between T = 1 week to 4 weeks (p = 0.03). Otherwise, there are no statistically significant time changes.

lowest amount (Almarza and Athanasiou, 2004; Kuettner and Cole, 2005). This is reflected by the high gene expression levels of aggrecan seen in native NP cells and the low levels found in native AF cells. The differences found between the cell populations after isolation were mostly maintained during culture in alginate beads, suggesting that alginate preserves the phenotypical characteristics of the cells. Although we observed a decrease in the gene expression levels of type II collagen and aggrecan during culture in alginate beads, it has been reported that AC and IVD cells do produce considerable amounts of both proteins under these culture conditions (Chubinskaya et al., 2001; Hauselmann et al., 1994; Masuda et al., 2003; Vonk et al., 2010). In contrast, cells cultured on polystyrene dishes lose the ability to synthesize aggrecan and type II collagen and start to produce more type I collagen (Darling and Athanasiou, 2005). We did not observe any effect of variations in the alginate concentration on gene expression levels of type I and type II collagen or aggrecan by NP, AF or AC cells cultured within alginate gels (Figs. 4–6). This observation is in contrast with observations on other cell types cultured on top of flat elastic hydrogels,

where a pronounced influence of matrix stiffness on ECM synthesis has been documented (Breuls et al., 2008). However, a requirement for a stiffness-responsive cell phenotype is that the cell adheres to the scaffold material via integrin adhesions so that the cell can exert traction forces to the matrix by actomyosin contractility (Discher et al., 2009). Cells have no integrin receptors for alginate and therefore adhere very weakly to alginate matrices (Augst et al., 2006). They can therefore not actively respond to matrix stiffness via focal adhesion sites. Several methods to promote cell attachment to alginate matrices are currently being studied; for instance, by coupling of extracellular matrix proteins such as laminin, collagen, fibronectin, or RGD peptides (Alsberg et al., 2001; Augst et al., 2006; Huebsch et al., 2010; Degala et al., 2011). We note that another reason for the lack of sensitivity to matrix stiffness observed in our study may be the rapid reduction of matrix stiffness upon prolonged exposure to cell culture medium. Similarly, it has been documented that alginate scaffolds rapidly soften after implantation in vivo (Nunamaker et al., 2007). This softening has been attributed to the loss of divalent crosslinking cations at neutral pH

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Fig. 5 – Gene expression levels for type II collagen of native cells cultured in alginate matrices. Real-time PCR was performed on reverse-transcribed RNA isolated from cells derived from the NP, AF and AC of goat intervertebral discs after 0, 7, 14, and 28 days of culture in alginate beads with a concentration of 2% (white bars), 4% (grey bars) and 6% (black bars). The gene expression level of type II collagen (Col2a1) is normalized by the expression levels of two housekeeping genes (2hk). Data are shown as mean ± SD. Differences between NP, AF, and AC cells are statistically significant with p < 0.05 at all time points and alginate concentrations (except for NP-AC in Fig. 4(B), p = 0.067). The dependence on alginate concentration for each cell type is not statistically significant (p > 0.05). There is only a significant change with time for all cells on going from T = 0 to T = 1 or more weeks (p < 0.001).

(Augst et al., 2006). In our study, we observed a 10% decline in stiffness after 1 day and a ten-fold decline after 10 days in medium in samples prepared by diffusion (Fig. 3). The ‘in situ’ gelled samples softened less in medium, but were much softer than samples prepared by diffusion to begin with, so that their stiffness after 10 days was similar to that of the diffusion samples. The consequence of the loss of stiffness is that the 2% alginate scaffold no longer matched the stiffness of the NP after 10 days. Moreover, even the stiffness of the 6% alginate scaffolds was lower than that of the NP after 10 days. Several methods to prevent the loss of stiffness during storage in medium have been reported. Arguably the most straightforward method is to supplement Ca2+ to the medium. However, this strategy is not attractive in the context of NP regeneration, where chondrogenic differentiation is required, since calcium has osteogenic effects. Moreover, the Ca2+ concentration of the environment is difficult to control after in vivo implantation. An alternative strategy is the addition of cationic polyethyleneimine to alginate to increase the resistance to de-crosslinking (Kuo and Ma, 2001, 2008). Finally, the addition of cells is known to have stabilizing effects on the alginate matrix (Augst et al., 2006). We could not test this stabilizing effect with native IVD cells,

because there were not enough cells available for the large sample sizes required for rheology. In fact, limited availability of cells is also a limiting factor for IVD engineering with native cells (Bron et al., 2009a). In patients, the availability of IVD tissue for digestion is even less than in our study, in which we used pooled IVDs derived from goat thoracic spines. An attractive alternative is the use of mesenchymal stem cells (Lutolf et al., 2009), but differentiation towards NP or AF phenotypes is still not fully directional (Bron et al., 2009a). Furthermore, specific phenotypic markers to distinguish both cell types are currently being elucidated (Lee et al., 2007; Rutges et al., 2010; Sakai et al., 2009; Vonk et al., 2010). In conclusion, we showed that the stiffness of alginate scaffolds can be varied by tuning the alginate polymer concentration and can be matched to the stiffness of the NP. Moreover, the biosynthetic phenotype of native IVD cells is maintained upon prolonged culture in alginate matrices. There are still some practical limitations that need to be solved, specifically the long-term mechanical stability in vivo, the bioadhesive properties, and the availability of tissue cells from the patient. Current study underscores the potential of alginate as a scaffold material for IVD engineering, but more importantly reveals some important limitations, which

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Fig. 6 – Gene expression levels for aggrecan of native cells cultured in alginate matrices. Real-time PCR was performed on reverse-transcribed RNA isolated from cells derived from the NP, AF and AC of goat intervertebral discs after 0, 7, 14, and 28 days of culture in alginate beads with a concentration of 2% (white bars), 4% (grey bars) and 6% (black bars). The gene expression level of aggrecan (Acan) is normalized by the expression levels of two housekeeping genes (2hk). Data are shown as mean ± SD. Differences between NP, AF, and AC cells are statistically significant with p < 0.05 at all time points and alginate concentrations. The dependence on alginate concentration for each cell type is not statistically significant (p > 0.05). There is only a significant change with time for all cells on going from T = 0 to T = 1 or more weeks (p < 0.001) and for the AC cells between week 2 and 4 (p = 0.002).

in spite of many promising research over the past decade, still have to be overcome. REFERENCES

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