Injectable Doubly Cross-Linked Microgels for Improving the

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Injectable Doubly Cross-Linked Microgels for Improving the Mechanical Properties of Degenerated Intervertebral Discs Amir H. Milani,† Anthony J. Freemont,‡ Judith A. Hoyland,‡ Daman J. Adlam,‡ and Brian R. Saunders*,† †

Biomaterials Research Group, Manchester Materials Science Centre, School of Materials, University of Manchester, Grosvenor Street, Manchester, M13 9PL United Kingdom ‡ Regenerative Medicine, Developmental Biomedicine Research Group, School of Medicine, Stopford Building, University of Manchester, Oxford Road, Manchester, M13 9PT United Kingdom S Supporting Information *

ABSTRACT: The use of injectable pH-responsive doubly cross-linked microgels (DX microgels) to improve the mechanical properties of degenerated intervertebral discs is demonstrated for the first time. The microgel comprised methyl methacrylate (MMA), methacrylic acid (MAA), ethyleneglycol dimethacrylate (EGD) and glycidyl methacrylate (GM) and was poly(MMA/MAA/EGD)-GM. The GM facilitated covalent interparticle cross-linking. The DX microgels are shown to have tunable mechanical properties. Degeneration of model bovine intervertebral discs (IVDs) was induced using collagenase. When injected into degenerated IVDs the DX microgels were shown to improve the strain, modulus, toughness and resilience. The extent of mechanical property improvement was an increasing function of DX microgel concentration, suggesting tunability. Cytotoxicity studies showed that the DX microgel was biocompatible under the conditions investigated. The results of this study imply that injectable DX microgels have good potential as a future regenerative medicine strategy for restoring the mechanical properties of degenerated load-bearing soft tissue, such as IVDs. work13,14 required a temperature of 50 °C which is too high for use in the body. DX microgels do not redisperse in water or flow under shear unless this is excessive.13 The ability of DX microgels to support biomechanically meaningful loads within IVDs is important for them to be considered as potential injectable gels for restoring the mechanical properties of degenerated IVDs. The rationale for the present study was that, based on our previous work,11,13,14 it should be possible to establish an injectable DX microgel that formed at 37 °C and would have the ability to restore the mechanical properties of degenerated IVDs. The motivation for the present study arose from the major healthcare problem of degeneration of the intervertebral disk (DIVD). DIVD is a key source of pain that is becoming more prevalent in our aging population. Lower back pain, which is often due to DIVD, affects 1 million workers in the US annually and is responsible for more absences from work than any other musculoskeletal injury.15 The biological and biomechanical changes associated with DIVD are well-known.16−18 Injectable biomaterials have received considerable attention because of their potential to provide a minimally invasive tissue engineer-

1. INTRODUCTION Hydrogels with increasing complexity offer excellent promise for regenerative medicine applications.1−4 Microgels can be considered as colloidal hydrogels and are attracting considerable research interest.5−9 They have been investigated for potential use in regenerative medicine10,11 and cell culture.12 pH-responsive microgels are cross-linked polymer colloid particles (submicrometer size) that exhibit swelling when the pH equals the pKa. We are investigating injectable microgel dispersions as a means for restoring the mechanical properties of degenerated intervertebral discs (IVDs). Proof-of-principle studies have involved injection of IVDs with physically gelled singly cross-linked (SX) poly(ethylacrylate)-based microgel dispersions.11 (SX is used here to highlight cross-linking that occurs within the microgel particles.) Physically gelled microgel dispersions flow under shear and redisperse in water.13 To overcome these major limitations to potential biomaterial application we recently established a method for covalently interlinking physically gelled microgel dispersions.13 Our approach provided pH-responsive hydrogels comprised of microgels that were doubly cross-linked (DX). We use the term DX microgel to emphasize the second level of covalent cross-linking between the microgel particles which interlinks them to form a covalently linked hydrogel. DX microgels are space-filling and potentially injectable. However, all previous © 2012 American Chemical Society

Received: May 17, 2012 Revised: July 21, 2012 Published: August 10, 2012 2793

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Scheme 1. Injectable Doubly Crosslinked Microgels for Reinforcing Degenerated Soft Tissuea

a

(a) Shows general method for preparing doubly crosslinked (DX) microgels using singly crosslinked (SX) GM-M-EGD microgel particles. The physical gel formed immediately and subsequently cured. The double red lines depict vinyl groups and the single red lines depict elastically effective chains inter-linking microgel particles. (b) Shows the contents of the twin syringe used to inject the dispersion into a tissue-free space within degenerated soft tissue. APS is ammonium persulfate.

Figure 1. IVDs containing DX microgels. (a) Twin chamber mixing syringe used to inject mixtures for DX microgel preparation into an IVD. (b) Degenerated IVDs containing DX microgel (arrows) after compression experiments and dissection. NP is the nucleus pulposus (c) Shows the geometry used for the compression experiments. ho and h are the initial NP height and height under a given compressive stress, respectively.

ing solution to DIVD.19 One example is an injectable protein polymer that forms cross-linked polymer chains in vivo.20 Other approaches include the use of hyaluronan,21−23 alginate,24 poly(methyl methacrylate) beads,25 poly(NIPAM)PEG/PEI cross-linked gel17 (NIPAM, PEG and PEI are Nisopropylacrylamide, polyethyleneglycol and poly(ethyleneimine)) and polycaprolactone electrospun fibres combined with agarose gels.26 Our microgel differs from these examples because our gel phase is almost completely constructed prior to injection. The microgel particles simply

require swelling and interlinking. Our preformed microgel particles are swollen by pH adjustment and cross-linked across the particle surfaces using free-radical chemistry. Our microgels also differ from other injectable approaches because they are pH-responsive and have high built-in swelling pressures. This insures that they are highly hydrated. Here, we investigated a microgel-based injectable gel which was mechanically stable (Scheme 1). We used vinyl-functionalized microgel particles consisting of methyl methacrylate (MMA), methacrylic acid (MAA), ethyleneglycol dimethacry2794

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rheology and swelling measurements were prepared by rapidly vortex mixing the two mixtures for at least 2 min. In the case of injection into IVDs a twin-syringe biomaterial delivery system (L-System, Medmix) was used. This consisted of a twin-syringe dispenser and a mixer (Scheme 1b and Figure 1a). This dispenser has been used by other groups27 and ensured good mixing of the individual mixtures. In the case of the 11 wt. % DX microgel (termed DX11) Mixture 1 was made by adding 30 μL APS (0.05 M) to 370 μL of GM-M-EGD (24.5 wt. %). Mixture 2 comprised 100 μL of a solution containing NaOH (2M), TEMED 10 wt. %, and DI water (300 μL). In the case of the 15 wt. % DX microgel (termed DX15) mixture 1 was prepared by adding 60 μL of APS (0.05 M) to 740 μL of GM-M-EGD (20 wt. %). Mixture 2 comprised 160 μL of a solution of NaOH (2 M), TEMED 10 wt. %, and DI water (40 μL). The final pH of the DX microgels was 7.5. 2.5. Physical Measurements. Titration measurements were performed using a Mettler Toledo titration unit in the presence of a supporting electrolyte (0.1 M NaCl). Photon correlation spectroscopy (PCS) measurements were performed using a BI-9000 Brookhaven light scattering apparatus (Brookhaven Instrument Cooperation), fitted with a 20 mW HeNe laser and the detector was set at a scattering angle of 90°. The particle volume swelling ratio (Q) was obtained using the hydrodynamic diameter measured by PCS at a given pH (dh) and that measured at pH 4 (dh(4)) using eq 1.

late (EGD) and glycidyl methacrylate (GM). The microgel consists of poly(MMA/MAA/EGD)-GM. The GM functionalization provided a means for permanently interlocking neighboring microgel particles. The mechanism (Scheme 1a) involves a pH-triggered particle swelling (by alkaline treatment) which causes formation of a shear-thinning physical gel of swollen SX microgel particles. Our physical gel formed because the effective volume fraction of the swollen particles approached values greater than that required for particles to move past each other and a glassy state formed (particles became kinetically trapped). In this state the gel has interpenetrating microgel particles. Accordingly, the vinyl groups were closely located and subsequently reacted to interlink the particles (Scheme 1a). In contrast to our previous DX microgel studies,13 here we use an accelerator (TEMED) to enable cross-linking at 37 °C. The present study aimed to establish the concept of using injectable DX microgels to support biomechanically meaningful loads. We confined our measurements to the early stages after DX microgel formation. We did not consider long-term cycling of loads, which was beyond the scope of the study. We begin this study by characterizing the microgels and the new DX microgels. The DX microgels were then formed, by injection, within degenerated model IVDs (Figure 1b) and compressive stress vs strain data measured. We then examine biocompatibility data of the DX microgels. Compared to the earlier work11,13 the present studies examine DX microgels that are injectable (formed at 37 °C) and are based on MMA. We also show data that demonstrate load support for degenerated IVDs using an injectable DX microgel that is permanently cross-linked and inherently mechanically stable for the first time.

⎛ d ⎞3 Q = ⎜⎜ h ⎟⎟ ⎝ dh(4) ⎠

(1)

SEM measurements were obtained using a Philips FEGSEM instrument. The number-average diameters (dn(SEM)) were calculated from at least 100 particles per sample using Image J (NIH). The program measured the area of each particle. The area was then converted to diameter using the assumption that the particle crosssection was circular. Samples were dried at room temperature or by freeze-drying. Dynamic rheology measurements were performed using a TA Instruments AR G2 temperature-controlled rheometer with an environmental chamber. A plate geometry (diameter of 20 mm) with a solvent trap was used. The gap was 1000 μm. For the strain amplitude measurements a frequency of 1 Hz was used. A strain of 1% was used for the frequency sweep measurements. 2.6. Load Testing Measurements. Static compression tests were carried out on unconstrained bovine caudal (tail) IVDs using a ZwickRoell servo-hydraulic testing instrument and a purpose-built load cell. Bovine IVDs have been shown to be good biomechanical models for human IVDs.28 Bovine tails were obtained from young animals (18− 30 months) from an abattoir on the day of slaughter and placed in a freezer (193 K). Samples were obtained by slicing a prefrozen cow tail and removing all tissue surrounding the disk and end plate bones. We used collagenase to experimentally degenerate the NPs within bovine IVDs. This involved injection of 0.15 mL of collagenase type II (10 g/L) and followed by reaction for 18 h at 37 °C. Degeneration conditions identical to those used here have established that preinjection of the IVD with collagenase produced a broadly spherical, tissue-free space within the NP interior.11 The degenerated IVDs were subsequently injected with the mixtures required to prepare the DX microgels (see section 2.4, Scheme 1, and Figure 1a). The pH increase produced a (shear thinning) physical gel (Figure 3a(ii)) which was injected into the tissue-free space created by the collagenase treatment. The physical gel rapidly cured at 37 °C and produced a DX microgel within about 5 min. An example digital photograph of a dissected IVD after compression is shown in Figure 1b. The DX microgel is evident, which had been dissected along with the NP. The DX microgels occupied about 15 vol. % of the NP within these experiments. There was no evidence of fragmentation or migration (extrusion) of any of the DX microgels under the conditions used for these experiments. The geometry of the loading experiments is depicted in Figure 1c. The data measured were in the form of compressive stress (σ) vs strain (ε). The latter was calculated using

2. MATERIALS AND METHODS 2.1. Materials. MMA (99%), MAA (99%), EGD (98%), and GM (97%) were supplied by Sigma-Aldrich U.K. and used as received. Ammonium persulfate (APS, 98%), sodium dodecyl sulfate (SDS, 98.5%), chloroform (99%), and N,N,N′,N′-tetramethylethylenediamine (TEMED, 99%) were also supplied by Aldrich and used as received. The water used was doubly distilled and deionized. 2.2. Poly(MMA/MAA/EGD) Microgel Synthesis. The method used to prepare the poly(MMA/MAA/EGD) microgel (abbreviated here as M-EGD) was described fully in a previous publication.13 The microgel was prepared using seed-feed emulsion polymerization. The only difference is that the present M-EGD particles were prepared using a lower cross-linker concentration. The comonomer solution used to prepare the particles (and seed) contained MMA (66.45 wt. %), MAA (33.2 wt. %), and EGD (0.35 wt. %). 12.5 wt. % of the comonomer solution was used to prepare the seed particles and emulsion polymerization was conducted in the presence of APS and SDS at 80 °C. The remaining comonomer solution was fed into reaction mixture over a period of 90 min. The M-EGD particles were extensively dialyzed using water. 2.3. Poly(MMA/MAA/EGD)-GM Microgel Synthesis. GMfunctionalized microgel particles were prepared using the method described earlier.13 They are abbreviated as GM-M-EGD. GM was reacted with M-EGD dispersed in water at a pH of about 3.5 and a temperature of 50 °C for 8 h. The particles were washed extensively with chloroform and separated using water washing. 2.4. Doubly Cross-Linked Microgel Preparation. The DX microgels were prepared by combining two separate mixtures. The first mixture (mixture 1) contained APS and GM-M-EGD. The GM-MEGD dispersions remained fluid when the pH was less than or equal to about 6.2. This is a pH which is congruent with potential biomaterial applications. The second mixture (mixture 2) contained NaOH, TEMED, and water. The DX microgels that were studied using SEM, 2795

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h ho

(2)

where h and ho were the nucleus pulposus (NP) heights during and before strain, respectively (Figure 1c). The value for ho was determined using X-ray images obtained of the samples prior to compression. The stress vs strain data were used to determine an instantaneous effective aggregate modulus value, Keff A , which was obtained from the gradient of the plots. The IVD samples were tested in three different conditions: normal, degenerated, and microgel injected. In normal condition the samples were tested without any treatment and were used immediately after being prepared. From this point onward collegenase-treated samples will be referred to as degenerated. PBS was injected into degenerated samples before conducting the loading tests. For degenerated IVDs containing DX microgel, samples were injected with GM-M-EGD (as described in section 2.4) after degeneration. Loading tests were performed on each sample by applying a strain (about 0.3). The first loading and unloading run was a conditioning cycle. Stress and strain data were recorded during the subsequent loading and unloading cycles. The time for each load and unload cycle was the same (1 min). Each sample went through a total of five cycles (one conditioning cycle) with hold time of 20 min between each cycle as relaxing time. Each loading or unloading sweep involved about 200 individual data points. A summary of the measurement conditions used for the samples is shown in Table 1. The two controls are the normal (nondegenerated) IVD and the degenerated IVD. The degenerated sample was injected with PBS solution (0.15 M).

Figure 2. Microgel particle morphology and pH-dependent swelling. Panels a and b show SEM images for M-EGD and GM-M-EGD, respectively. Panels c and d show the variation of hydrodynamic diameter and swelling ratio, respectively, with pH for M-EGD and GM-M-EGD. The lines are guides for the eye.

Table 1. Details of the IVD Samples Investigated sample ID PBS DX11 DX15 normal

conditions degenerated IVD injected with 0.15 M PBS solution degenerated IVD injected with 11 wt. % GM-M-EGD and doubly cross-linked degenerated IVD injected with 15 wt. % GM-M-EGD and doubly cross-linked as supplied IVD (not degenerated)

samples tested

The particles are spherical and have dn(SEM) values in the range of 102−107 nm (Table 2). Figure 2c shows the variation of the

7 5

Table 2. Characterisation Data for the Microgels Investigated in This Study

8 7

abbreviation GM-MEGD M-EGD

2.7. Cytotoxicity. Human nucleus pulposus (NP) cells, cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and antibiotic/antimycotic (Gibco) at 37 °C in a humidified 5% CO2 atmosphere, were seeded at a density of 3 × 104 cells per well into 24-well culture dishes containing 15 mm sterile glass coverslips. After 72 h, sterile toroidshaped DX microgels were introduced to the wells and cultured for up to a further 7 days. The toroid-shaped DX microgels, which partially rested on top of the cells, enabled regions of cells to be in contact, or not in contact, with the gel surfaces within the same culture experiment. For example the cells located underneath the middle of the toroid-shaped DX microgels were not in contact with the gel. Cells located directly below the toroid part were in contact with the gel. Cell viability was assessed at day 1, 4, and 7 following the introduction of the microgels versus controls by Live/Dead assay (Invitrogen). A cytotoxicity experiment that mimicked conditions used to form a DX microgel within an IVD was conducted using a DX microgel reaction mixture containing 10 wt. % microgel particles. Further details can be found in the Supporting Information. 2.8. Statistical Analysis. Statistical analysis was conducted on the compressive load testing data using a Student’s t-test. We considered a confidence level of P < 0.05 to be significant. The load testing data shown in this work are reported as the mean ± standard deviation. The error bars shown for Figures 6 and 7 correspond to the standard deviations.

mol. % MAA

mol. % GMa

dn(SEM)/nm (CV)b

dh(4)c/ nm

Q7.4d/ nm

pKae

23.3

18.4

102 (16)

114

20.2

6.4

107 (18)

114

11.4

6.5

41.7

a

Calculated from the difference in the mol. % MAA before and after functionalization using potentiometric titration data (Figure S1). b Number-average diameters determined from SEM images. The number in brackets is the coefficient of variation. cHydrodynamic diameter at pH 4. dVolume swelling ratio at pH 7.4 from eq 1. e Apparent pKa value obtained from data shown in Figure S1.

hydrodynamic diameter over a range of pH values. Both MEGD and GM-M-EGD had the same hydrodynamic diameter (dh(4)) at pH 4. This indicates that GM-functionalization occurred without aggregation. These diameters are only slightly higher than the dn(SEM) values (Table 2), which indicates that the particles were fully collapsed at pH 4. This shows that eq 1 can be used to estimate Q values for both microgels. It can be seen from Figure 2c,d that the particles exhibited pH-dependent swelling. The Q values increased, and swelling had begun (Figure 2d), when the pH reached the pKa values (Table 2) for the microgel particles. The GM-M-EGD particles swelled more strongly than the M-EGD particles despite the fact that they contained a lower MAA content (Table 2). A similar effect has been observed earlier for related GMfunctionalized microgels.13 This is attributed to rinsing of the GM-M-EGD particles by chloroform during purification (section 2.3) which disrupted hydrophobic (reversible) intersegment physical cross-links within the particles.

3. RESULTS AND DISCUSSION 3.1. Microgel characterization. Figure 2a,b shows SEM images for M-EGD and GM-M-EGD particles, respectively. 2796

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From the difference of the MAA contents for the microgel before and after functionalization (Table 2) the extent of functionalization for GM-M-EGD is estimated as 18.4 mol.% This value is about 2.5 times higher than reported for related microgels elsewhere.13,14 The present microgel was prepared using 0.17 mol.% EGD, which is much lower than the value of 0.5 mol % used for earlier work.13,14 We propose that a lower EGD content (and SX cross-linking) facilitated migration of GM into the particles and enhanced vinyl-functionalization. This new finding implies that tuning EGD concentration may be a method to tune GM incorporation for GM-E-BDD microgels. 3.2. Doubly cross-linked microgel characterization. Mixtures of the GM-M-EGD dispersion (Figure 3a(i)) with NaOH solution were produced using the dual-chamber mixing syringe (shown in Figure 1a) and were injectable shear-thinning physical gels. See Figure 3a(ii). Inclusion of APS and TEMED

Figure 4. Mechanical properties of as-made DX microgels. Panels a and b show frequency sweep dynamic rheology data for the as-made DX microgels.

G′) values had low frequency dependencies; this is expected for covalent networks. G′ and tan δ values for the DX microgels are shown in Table 3. Table 3. Mechanical Properties for DX Microgels

a

DX microgel

state (concentration)

Q

G′/kPaa

tan δa

DX11 DX15 DX11(S) DX15(S)

as made (11%) as made (15%) swollen (11%) swollen (15%)

8.7 6.5 10.0 8.5

72.9 134 54.6 74.4

0.030 0.042 0.045 0.056

Values obtained at a frequency of 1 Hz using a strain of 1%.

The G′ values for DX11 (72.9 kPa) and DX15 (134 kPa) (see Table 3) are the highest values reported for DX microgels at these respective particle concentrations (11 and 15 wt. %). We propose that the superior elasticity is due to a combination of the higher extent of functionalization for the GM-M-EGD particles used here as well as the use of the accelerator. The tan δ values were only about 0.03−0.04. Given that a tan δ value of zero is expected for a perfectly covalent gel this shows that the proportion of energy that was stored elastically in these gels was very high compared to that dissipated. It follows that these DX microgels had few imperfections, e.g., dangling chains and loops that did not support stress. We could not be certain of the extent of swelling that would occur for DX microgels within IVDs, Therefore, we also investigated the unconfined swelling of the DX microgels. This corresponds to the maximum swelling possible. The Q values (Figure 5a) increased within the first 1 day and then reached near constant values. It is important to note that there was no evidence of fragmentation for the DX microgels. This confirms that they were covalent hydrogels. DX15 exhibited significantly less swelling than DX11 (Figure 5a and Table 3). This is because of the higher particle concentration since the particles act as multifunctional cross-linkers. The mechanical properties of the equilibrium swollen DX microgels (after 8 days of swelling) were also studied. Frequency sweep G′ and tanδ data are shown in Figure 5b,c, respectively. The swollen gels retained high G′ values (Table 3). Interestingly, the frequency dependence of the tan δ values was insignificant when swollen (Figure 5c). These systems show ideal elastic behavior.29,30 This is attributed to elongation of the elastically effective chains throughout the swollen DX microgels. 3.3. Degenerated Intervertebral Discs Containing DX Microgels. Figure 6 shows average σ vs ε data for the four IVD sample types considered (Table 1). Each σ vs ε curve (loading or unloading) shown is the average of a number of experiments. Each data point shown in Figure 6 is the average of 5 to 8 experiments. All of the data are shown in Figure S2 − S5. The

Figure 3. DX microgel preparation and morphology. (a) Images of the initial fluid (i), shear-thinning physical gel (ii), and covalently interlinked DX microgel (iii). (b) SEM image of freeze-dried DX microgel. The arrows identify microgel particles located at the surface.

prior to mixing followed by injection enabled rapid formation of DX microgels at 37 °C (Figure 3a(iii)). The morphology of freeze-dried DX microgels was examined using SEM (Figure 3b). The particulate nature of the freeze-dried surface can be seen. The particles evident at the surface are comparable in size to those shown in Figure 2b. The mechanical properties of the DX microgels were investigated using dynamic rheology. Frequency sweep dynamic rheology data for the as-made DX11 and DX15 microgels are shown in Figure 4a,b. G′ and G” are the storage and loss modulus, respectively. They represent the energy stored or dissipated per unit strain. The G′ and tan δ (= G″/ 2797

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Figure 5. Swelling and mechanical properties of unconfined DX microgels. (a) Volume swelling ratio as a function of time for DX microgels in pH 7.5 buffer. Panels b and c show frequency sweep data for the DX microgels after swelling for 8 days. The lines in panel a are a guide to the eye.

use of relatively large data sets and averaging was essential due to the natural variability of the IVDs. The inset of Figure 6a shows the strain sweep profile used. It can be seen from Figure 6a that the normal (nondegenerated) IVD showed hysteresis of the σ vs ε data. This behavior has been reported by others for bovine IVDs28 and is the result of the inability of the fluid outflow and inflow to match the rate of strain variation. With each successive cycle there is an increase in the final stress reached. This is due to an increased tissue volume fraction in the NP caused by partial dehydration from the applied strain. This general phenomenon is the reason why humans loose height during the day and regain it after long periods in a horizontal position (i.e., sleeping). The degenerated IVD injected with PBS (Figure 6b) shows a much decreased maximum σ and more pronounced hysteresis. Concerning the latter it can be seen that for a given σ (e.g., 0.5 MPa) there is a much larger difference in ε values for the loading and unloading cycle for the degenerated sample containing PBS sample than for the normal IVD (Figure 6a). The experimentally induced degeneration decreased the ability of the NP to reswell upon strain removal over the experimental period used. Figure 6c,d show average σ vs ε data for degenerated IVDs containing DX11 and DX15. Importantly, formation of DX microgel in the degenerated IVD caused an increase in maximum stress and a decrease in hysteresis (cf. Figure 6b). These changes are consistent with our earlier report that used an injected physically gelled SX poly(ethyl acrylate)-based microgel dispersion.11 This is the first time reinforcement has been reported for a DX microgel. Moreover, the present microgel is based on MMA and has greater potential as a biomaterial because it is much closer to the composition of bone cement (which is also based on MMA). In order to quantify the ability to withstand stress we measured the ε value during the load cycle at a stress of 1 MPa (i.e., ε1(L)). We selected σ = 1 MPa because this is a

Figure 6. Stress vs strain data for various IVD samples. Panels a−d show average stress vs strain data for IVDs containing DX microgels. Average data for IVDs containing PBS and also normal IVDs are shown for comparison. The directions of loading and unloading are indicated. The inset for panel a shows the strain profile as a function of time. Panel c also shows the approximate position of the strain for a stress of 1 MPa during the load cycle (ε1(L)). Panels e and f show the ε1(L) and % resilience values for each cycle, respectively. The full data set are shown in Figures S2−S5.

representative compressive stress for a person standing.31 Figure 6e shows average εI(L) values measured as a function of cycle number for each of the systems. (Data for the fourth load sweep are shown in Table 4.) Statistical analysis of the data Table 4. Selected Compression Data for Various IVDsa system PBS DX11 DX15 Normal

ε1(L)b 0.219 0.220 0.189 0.192

± ± ± ±

0.027 0.017 0.017 0.011

toughness/ (kJ/m3) 166 178 230 240

± ± ± ±

41 43 53 44

Rc/% 43.4 54.7 54.6 60.1

± ± ± ±

3.6 4.6 4.7 3.0

d Keff A1(L)/MPa

10.24 13.07 14.29 13.74

± ± ± ±

1.00 1.07 1.59 1.56

These average values are for cycle 4. The numbers after ± are the standard deviations. bStrain at a stress of 1 MPa during the loading cycle. cCompressive resilience (see text). dEffective aggregate modulus at a stress of 1 MPa during the loading cycle. a

showed that the ε1(L) values for the DX15 samples were significantly smaller than those for the PBS samples. Also, the ε1(L) values for the DX15 samples were not distinguishable from the values for the normal IVDs. Accordingly, DX15 provided a statistically significant decrease in strain (less compression) during loading. There was no difference in the ε1(L) values for the DX11 gel and PBS samples. This 2798

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demonstrates a particle concentration dependence and hence the potential for tuning the stress−strain behavior. The area under a stress vs strain curve can be equated to the toughness.32 Here, we calculated the compression toughness.33 Toughness values from the fourth load cycle appear in Table 4. The toughness of the DX15 system is significantly higher than that of the degenerated IVDs injected with PBS. Furthermore, the toughness of the DX15 system is not significantly different to the normal IVD. We can conclude that the DX15 injection effectively restored the toughness of degenerated IVDs to normal values. The resilience is a measure of the ability of a material to deform reversibly without energy loss.34 The compressive resilience (R) is the energy recovered after removal of the compressive stress divided by the total deformation energy.35 Average %R values were calculated from the σ vs ε data shown in Figure 6a−d. The %R values appear in Figure 6f. It can be seen that the degenerated samples (PBS injection) were much less resilient than the normal IVDs. There was no statistically significant difference between the %R values for the degenerated IVDs containing DX11 or DX15 over the cycles. Importantly, injection DX11 or DX15 significantly improved the resilience of the degenerated IVDs. At Cycle 4 the %R values for the degenerated IVDs containing DX11 or DX15 were 91% of the value for the normal IVD. For comparison, the %R value for the PBS-containing IVD was only 72% of the value for the normal IVD at cycle 4. The Keff A value is often used as a measure of elasticity for nonuniform natural materials such as cartilage36 and NPs.37 Here, we determined average Keff A values from the instantaneous gradients of each of the σ vs ε curves (Figure S2−S5) that were used to construct the average σ vs ε curves shown in Figure 6a−d. The full Keff A vs ε data set are shown in Figure S6−S9. The average Keff A values are shown in Figure 7a−d. For clarity, only the loading cycles have been considered. The variation of Keff A with ε for the normal IVD (Figure 7a) shows a gradual increase at first, followed by a linear increase with strain. It can be seen from Figure 7b that degeneration (PBS sample) reduces both the Keff A values as well as its rate of increase with ε. Figure 7c,d show that injection of the microgels increased both the Keff A values as well as the gradients. It can be seen that the degenerated IVDs injected with DX15 (Figure 7d) showed the closest Keff A vs ε profiles to those for the normal IVDs (Figure 7a). The injected DX microgels had a major influence on the effective aggregate modulus values at 1 MPa (Keff A1(L)). This can be seen from Figure 7e. Even the use of the lowest DX microgel concentration (DX11) caused a significant increase in Keff A1(L) and this approached the normal IVD value after the fourth load ramp. The DX microgel provided a major increase in the stiffness of the degenerated IVDs, as determined by Keff A1(L) values. There was no significant difference between the Keff A1(L) values for the IVDs containing DX15 gel and the normal IVD. Under biomechanically meaningful loads the DX microgel (DX11 or DX15) restored Keff A for the degenerated IVD to normal values. 3.4. Proposed Mechanism for Reinforcement of Degenerated IVDs by DX Microgels. The mechanical properties of IVDs under cyclic compression have been successfully described using a biphasic model.37 The biphasic model 38 considered the NP to be a mixture of an incompressible solid phase (collagen/proteoglycan/NP cells) and incompressible fluid phase (interstitial water). Tissue

Figure 7. Modulus vs strain behaviors for various IVDs. Panels a−d show average effective aggregate modulus values as a function of strain during the load cycles for the IVDs. The same legend applies to each of these panels. Panel e shows the modulus values for each system at 1 MPa. The data for this panel were obtained using those shown in Figures S2−S5 (see text). All data are shown in Figures S6−S9.

compressibility arises from fluid transport into or out of the solid matrix which is porous and permeable. Stress dissipation occurs due to fluid flow resulting in time-dependent stress vs 39 strain behavior. The equation for Keff A is

K Aeff = KA + Π

(3)

For eq 3, KA is the aggregate modulus and Π is the rate of change of osmotic pressure with dilation. The contributions for each of the two terms are frequency dependent. When the frequency (f) is smaller than the characteristic frequency ( fc) fluid can flow out of the pores in response to stress and the magnitude of Π approaches zero. The stress is then supported by the solid phase, which deforms. When f is greater than fc there is a finite resistance to fluid flow and a pressure gradient builds up within the NP. This results in a dominant Π value and is the common state within human IVDs for daily activity. The value for fc is given by the following equation:39

fc =

KAko h2

(4)

where ko and h are the average permeability and thickness, respectively. To consider the mechanism for reinforcement we first estimate the value for fc (eq 4). The value for ko was reported as 9 × 10−16 m4/Ns by Johannessen et al.37 for normal human NP tissue. The value for KA for NP tissue can be calculated as 15.5 kPa using the values of shear modulus37 (G, 5.8 kPa), Poisson ratio (ν, 0.2) and the following equation: 2799

dx.doi.org/10.1021/bm3007727 | Biomacromolecules 2012, 13, 2793−2801

Biomacromolecules KA =

2Gν + 2G 1 − 2ν

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period of these experiments as judged by the lack of dead cells for the control sample. A cytotoxicity experiment was also conducted using an injectable DX microgel formulation. For this experiment a DX microgel containing 10 wt. % of particles was used (DX10). When compared to DX11, the DX10 microgel contained an APS concentration that was three times that used for DX11. It also contained a TEMED concentration that was about half that used for DX11. DX10 formed a robust gel rapidly and was placed in an insert after 15 min of reaction. The media present was in contact with NP cells and was changed once after 3 h. (See the Supporting Information for details.) The % cell viability (as analyzed by MTT assay) decreased to 81.0% after 2 days and then stabilized at 79.2% after 5 days (Figure S10). Although DX10 was not prepared in exactly the same way as DX11 or DX15, these data are encouraging in the context of a future injectable DX microgel therapy for DIVD.

(5)

Using an average value for h of 5.5 mm and eq 4 an fc value of 5 × 10−7 s−1 can be calculated. The average value of f used for our experiments was about 8 × 10−3 s−1. Assuming the calculated fc value is comparable to that of bovine NP tissue, then f ≫ fc. It follows that interstitial fluid pressurization made a major contribution to Keff A (eq 3) for these systems. This conclusion is consistent with the G′ range for the DX microgels 73−134 kPa, which is much lower than the Keff A1(L) values of 13.1−14.3 MPa (Table 4). The load data for the PBS system (Figures 6 and 7) show much reduced load support compared to the normal IVDs. We propose that this is due to a decrease of Π because of a lower total proteoglycan content. Injection of DX microgel is proposed to increase the overall charge density of the NP and, presumably, Π. For this mechanism load support, and an increase in Keff A , due to the DX microgel originates from a high swelling pressure which results in a higher trapped water content. This provides an improvement in fluid pressurization. 3.5. Cell Challenge Experiments. We investigated the effect of the DX microgel on cells by placing preprepared, washed, DX15 microgel in direct contact with human NP cells cultured on 13 mm glass coverslips. Figure 8a,b show Live/

4. CONCLUSIONS In this study we investigated the ability of DX microgels to improve the mechanical properties of degenerated IVDs. Unlike previous work,11 the microgels used here can be considered as injectable and form permanent gels that do not redisperse. They were based on MMA. Two DX microgels were prepared at different particle concentrations. The DX15 system was the most successful in that it resulted in a statistically significant improvement in the mechanical properties, i.e., strain, modulus and toughness. Under the conditions used here there is strong evidence that the DX microgels restored the mechanical properties of degenerated IVDs to normal values. The resilience of the degenerated IVDs was also much improved by the DX microgels and reached values of up to 91% of the normal values. This implies that the new injectable DX microgels introduced here have potential to restore the mechanical properties of degenerated IVDs under biomechanically meaningful loads. There is also evidence that the mechanical benefit afforded by the DX microgels is tunable through particle composition with the DX15 microgel system being close to optimal in terms of the key parameters used for characterization (ε1(L) and Keff A1(L)). The DX microgels were shown to not be cytotoxic for the conditions employed here. The data presented here provide a new concept for potential injectable mechanical property restoration for degenerated IVDs. The results should also have relevance to articular cartilage repair. Longer term biomechanical studies and more extensive biocompatible data are required in order for the potential of this new injectable biomaterial to be realized.

Figure 8. Cells in contact with DX microgel. Optical micrographs (top) and Live/Dead assay (bottom) images for adherent human NP cells (on a coverslip) that were in direct contact with DX15 (a and b) and cell regions that were not in contact (controls) with DX15 (c and d). All images were taken after 7 days in culture. The scale bars represent 100 μm.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details concerning the injectable gel cytotoxicity experiments for DX10 provide as well as optical micrographs of NP cells and % alive cells versus time. Potentiometric titration data for microgels, stress vs strain data for all IVDs examined, and modulus vs strain data for all IVDs examined. This material is available free of charge via the Internet at http://pubs.acs.org.

Dead assay staining and phase-contrast images of the cells after 7 days in contact with the DX microgel compared to controls. The cells were not in contact with the DX microgel for the control systems. However, they were in contact with the same media. This was achieved using toroid DX microgel samples. See section 2.7 for details. In each case the cells adopted elongated morphologies and there was no evidence of significant cell death as denoted by the green (live) and red (dead) fluorescent staining. There was no observable change in the cell morphology. This shows that washed DX microgel is inherently biocompatible. Furthermore, there is no evidence of cytotoxic species leaching from the DX microgels over the time

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AUTHOR INFORMATION

Notes

The authors declare the following competing financial interest(s): B.R.S. and A.J.F. are founders of a university spin off company that translates developments in microgel technology into the clinic. 2800

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(35) Debnath, S.; Madhusoothanan, M. J. Eng. Fibers Fabr. 2009, 4, 14−19. (36) Ateshian, G. A.; Chahine, N. O.; Basalo, I. M.; Hung, C. T. J. Biomech. 2004, 37, 391−400. (37) Johannessen, W.; Elliott, D. M. Spine 2005, 30, E724−E729. (38) Mow, V. C.; Kuei, S. C.; Lai, W. M.; Armstrong, C. G. J. Biomed. Eng. 1980, 102, 73−84. (39) Soltz, M. A.; Ateshian, G. A. Ann. Biomed. Eng. 2000, 28, 150− 159.

ACKNOWLEDGMENTS B.R.S. thanks UMIP for funding this work and Prof. Peter Lovell for access to the PCS instrument.

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