In vivo biostability of polyurethane–organosilicate nanocomposites

Acta Biomaterialia 8 (2012) 2243–2253

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In vivo biostability of polyurethane–organosilicate nanocomposites Katie E. Styan a,⇑,1, Darren J. Martin b, Anne Simmons a, Laura A. Poole-Warren a a b

Graduate School of Biomedical Engineering, University of New South Wales, NSW 2052, Australia Division of Chemical Engineering, School of Engineering, University of Queensland, QLD 4072, Australia

a r t i c l e

i n f o

Article history: Received 4 October 2011 Received in revised form 1 February 2012 Accepted 1 March 2012 Available online 8 March 2012 Keywords: Nanocomposite Polyurethane Silicate Organosilicate Biostability

a b s t r a c t Organically modified layered silicates were incorporated into a polyether soft-segment polyurethane to form composites of at least delaminated morphology. The primary organic modifier was a quaternary ammonium compound; however, one composite included an alternative amino undecanoic acid-modified silicate. The composites’ biostability was assessed in an in vivo ovine model over a period of 6 weeks. Attenuated total reflectance–Fourier transform infrared analysis and semi-quantitative scanning electron microscopy image rating indicate a significant enhancement of the base polyurethane biostability with the inclusion of silicate at 3 wt.%. The potential effect at 15 wt.% was confounded by probable leaching of the quaternary ammonium compound affecting the tissue response. The amino undecanoic acid composite compared favourably with the quaternary ammonium compound composite of equivalent silicate loading, and offers the promise of a more favourable tissue response. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Polyurethane (PU) has, historically, been disadvantaged by having poor biostability in vivo. Such degradation is significant as it introduces a risk of loss of device function and/or compromised biocompatibility. Poly(ester)urethanes were among the earliest biomedical PUs. The problem of the ester’s hydrolytic instability was initially addressed by poly(ether)urethanes (PEUs), but these have since been shown to be susceptible to degradation via oxidation of the ether linkages [1,2], and are thus currently limited to short-term applications in vivo. A PU originating from past work in our laboratory includes a polydimethylsiloxane (PDMS; – (CH3)3–Si–O–[Si–O–(CH3)2]n–Si–(CH3)3–) soft segment and has significantly increased biostability suitable for long-term in vivo applications [3–5]. A current area of research in our laboratories is organosilicate nanocomposites. Nanocomposites are well represented in the literature and have been the focus of much interest since researchers discovered mechanical property trends that were unique in the field of composites [6,7]. Nanocomposites have been explored predominantly for industrial application, with few studies aiming to exploit the enhancement of mechanical and barrier properties for application in biomaterials. However, increasingly of late, studies have explored the potential of silicates to modulate drug release from hydrogels (e.g. [8,9]). ⇑ Corresponding author. Tel.: +61 293853905; fax: +61 296632108. E-mail address: [email protected] (K.E. Styan). Present address: Commonwealth Scientific and Industrial Research Organisation, Materials Science and Engineering, VIC 3069, Australia. 1

In the present study, we explore the hypothesized potential of layered silicates in a PEU matrix to mitigate in vivo degradation. Organosilicate nanocomposites have not been assessed in the published literature for in vivo biostability as far as we are aware. Our hypothesis was based on the fact that one layer of montmorillonite (MMT), a commonly used silicate, is 250 nm in two dimensions and 1 nm in the other, giving an aspect ratio of 250 and total surface area of over 700 m2 g–1 [10]. Layers naturally stack in groups on the order of 1000 units, but these can be at least partially delaminated by exploiting the cationic exchange capacity (CEC) of the layered silicate to organically modify the inorganic silicate and increase its compatibility with the organic polymer matrix. It is now well accepted that the dispersed high surface area particles confer barrier effect properties to the polymer. Accordingly, we hypothesized that the included partially delaminated silicate layers would act as a barrier to attack and ingress of degradation species in vivo, thus protecting ether linkages and inhibiting degradation.

2. Materials and methods 2.1. Poly(ether)urethane PEU with chemical structure illustrated in Fig. 1 was used in this study. The polymer was supplied by Urethane Compounds (Melbourne, Australia) and contained 65 wt.% 1000 g mol–1 poly(tetramethylene oxide) polyol ether soft segment, 4,40 -diphenylmethane diisocyanate and 1,4-butanediol as the chain extender. The components were combined in the molar ratio 100:7.5:46.3, respectively,

1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.03.004

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Fig. 1. Molecular structure of PEU.

with 0.003 dibutyltin dilaurate added as a catalyst. This is modelled on PellethaneÒ 2363-80A. 2.2. Layered silicates A commercially available organically modified MMT, CloisiteÒ 30B (QACMMT; Southern Clay Products), was used. QACMMT had the quaternary ammonium compounds (QAC) methyl tallow bis-2-hydroxyethyl ammonium chloride as an organic modifier. This QAC is derived from animal adipose tissue, with the tallow indicating an alkyl chain of varying carbon atoms in length: 5% C14, 30% C16 and 65% C18. As a control, MMT (Na0.33[(Al1.67Mg0.33)Si4O10(OH)2]H2O; Southern Clay Products), of CEC 92.6 m Eq/ 100 g, was organically modified with amino undecanoic acid (AUA; Aldrich) by a method described previously [11]. The organically modified MMT thus prepared was referred to as AUAMMT. 2.3. Composite preparation Composites were prepared from PEU and either (i) QACMMT or (ii) AUAMMT by a solvent casting method. Composites with loadings ranging from 1 to 15 g nanoparticle/100 g PEU (1–15 wt.%) were prepared as detailed in Table 1. For details of the preparation methods and characterization of the resultant material, the reader is referred to our previous publication [11].

2.4.1. In vivo ovine model Methods were based on those published previously [3–5]. Briefly, dumb-bells were punch-cut from composite materials and strained to 150% over polymethylmethacrylate (PMMA) holders. The holders were engraved and tracked by a random code that was not broken until completion of the analysis. Strained samples were washed for 30 min in 2% Decon90 with manual agitation, rinsed at least six times in Milli-Q water, subjected to EtO sterilization and then degassed at room temperature for at least 7 days. All samples were taken from a single material batch, with the number of samples tested detailed in the following sections. Ethics approval was granted by the Animal Care and Ethics Committee at The University of New South Wales (ACE #03/81). Samples were implanted subcutaneously in the dorsal region of sheep (healthy crossbred males, weighing 40–60 kg and 2 years Table 1 Material loadings for PEU, PEU-QACMMT and PEU-AUAMMT materials.

PEU PEU-QACMMT-1 PEU-QACMMT-3 PEU-QACMMT-15 PEU-AUAMMT-1

2.4.2. Scanning electron microscopy Samples were coated with a 20–30 lm thick layer of conductive gold using a Dynavac SC150 sputter coater and analysed using a Cambridge Stereoscan 360 SEM operated at an accelerating voltage (extrahigh tension) of 20 kV, with a typical working distance of 20–27 mm. The system for objectively rating degradation employed was developed by Martin et al. [4]. SEM images from five set locations along the neck region of each sample were collected at 50, 150, and 500 magnification per location, for each of four replicate samples, taken one per sheep. One image at 10 Table 2 Biostability-relevant infrared absorption wave numbers.

2.4. Composite biostability evaluation

Material

of age) for a time period of 6 weeks. Replicate samples were implanted into each of four sheep. One sample was assigned for histological analysis and another randomly selected post-assay for Fourier transform infrared (FTIR) analysis, while four samples were reserved for scanning electron microscopy (SEM) imaging and rating. Unimplanted control samples were also prepared (three per material) for FTIR and SEM analysis. Explanted samples were washed in 0.1 M NaOH for 5 days to remove attached biological tissue and proteins, rinsed in Milli-Q water, washed in 2% Decon-90 for 3 days to remove fatty deposits, rinsed again with Milli-Q water for 4 h and finally dried in a laminar flow hood. Unimplanted control samples were washed by the same process for consistency. Samples remained strained on the PMMA holders during both FTIR and SEM analysis.

Loading (g/100 g PEU) Nanoparticle

Silicate

OM

0 1.0 3.0 15.0 0.9

0 0.8 2.4 12.1 0.8

0 0.2 0.6 2.9 0.1

Wavenumber (cm1) Literature

Experimental

1038 1081

1038b 1075 ± 9.49

1100

1099 ± 1.31

1174

1176 ± 0.17

1208 1219 1251 1365 1446 1529 1597 1703

1208b 1220 ± 0.77 1251 ± 0.24 1367 ± 0.16 1446b 1530 ± 0.69 1597 ± 0.60 1699 ± 0.57

1730

1728 ± 0.43

2797 2852 2922 2938 3305

2795 ± 0.41 2852 ± 0.36 2919b 2938 ± 0.88 3322 ± 0.79

Description of proposed peak assignment a

Stretching of the Si–O–Si bonds in MMT Aliphatic symmetric stretching of the hard segment ether C–O–C Aliphatic asymmetric stretching of the softsegment ether C–O–C Bending of branched ether C–O–C degradation product Twisting of CH2 Stretching of C–N Wagging of CH2 Bending of CH2 Bending of CH2 Stretching of C–N and bending of N–H Stretching of C=C in aromatic ring Stretching of hard segment C=O hydrogen bonded to amine Stretching of non-hydrogen bonded hard segment C=O Symmetric stretching of C–H in –CH2O– Asymmetric stretching of CH2CH Dichroic parallel stretching of CH2 Asymmetric stretching of CH2 Stretching of N–H hydrogen bonded to carbonyl

a Average of all materials (unimplanted and implanted), not correcting for the Si– O–Si peak. b Experimental peak not well defined, so the theoretical peak location was taken.

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magnification was also collected at the centre location. Two examiners, blinded to the sample identity, individually rated all images according to a rating scheme, whereby degradation at 10, 50, 150 or 500 resulted in a score of 10, 5, 2 or 1, respectively. A material rating ranging from 0 (no surface feature or degradation evident in any image) to 50 (surface features or degradation evident in all images or sample broken) was determined, and the ratings from the examiners were averaged for plotting. Error bars are 1 standard deviation of all data points. Statistical methods suitable for non-normal samples were applied. Briefly, where a Kruskal– Wallisone-way analysis of variance indicated a statistically significant difference, multiple comparisons of test samples with control samples were performed using Dunnett’s method. For normally distributed data, Student’s t-test may also have been performed.

was baselined at numerous locations across the spectral range using the supplied software (Opus), and the data normalized to the C–C bond of the PEU aromatic ring at 1414 cm1[12] using Microsoft Excel. Also shown in Table 2 are the wave number assignments used in the data processing. Data are presented as traces of absorbance intensity vs. wave number, or as a comparison of the intensities at important peak wave numbers. In the latter, for a particular wave number of interest the deviation of the implanted test material absorbance intensity from the unimplanted PEU control absorbance was calculated and then taken as a ratio to the unimplanted PEU control absorbance, as shown in Eq. (1), to enable simultaneous plotting of data for numerous absorption wave numbers.

deviation ¼ 2.4.3. Attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy ATR-FTIR spectroscopy was conducted on a single sample from 4000 to 500 cm1, at a resolution of 2 cm1 for a total of 64 scans, on a Bruker IFS 55/S spectrometer. A diamond crystal ATR attachment was used. Absorbance wave numbers associated with poly(ether)urethane and silicates were collected from various sources [2,3,12–14] and are listed in Table 2. The ATR-FTIR trace

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Atest;implanted  APEU;unimplanted  100% APEU;unimplanted

ð1Þ

2.5. In vivo inflammatory response 2.5.1. Histology Samples for histology were explanted with fibrous capsules intact and placed directly into 10% neutral buffered formalin. The

Fig. 2. SEM images of (a) PEU, (b) PEU-QACMMT-1, (c) PEU-AUAMMT-1, (d) PEU-QACMMT-3 and(e) PEU-QACMMT-15 materials implanted in sheep for 6 weeks. Magnification is 10 (the scale bar is 2 mm). The sample chosen is the worst case.

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sample side of the holder within the capsule was later determined and marked with Indian ink. An annulus of the capsule was then cut and histological sectioning and staining conducted. Briefly, sections were cut transverse to the direction of sample strain and at about the point halfway longitudinally along the dumb-bell. Sections were treated for 12 h in a Miles Tissue Tek Vacuum Infiltration Processor to exchange aqueous regions with paraffin wax, embedded in further paraffin wax, sectioned at 5 lm, mounted on glass microscope slides, and stained with haematoxylin and eosin Y (H&E). Note that the PMMA holder was removed prior to sectioning, and that the sample may have been lost with the PMMA holder and/or during the wash process. 3. Results 3.1. Biostability SEM images of explanted materials displaying the qualitatively worst degradation observable at 10 magnification are shown in Fig. 2. A higher-magnification image of the far left of each of the Fig. 2 samples is shown in Fig. 3. Severe cracks and degradation

are evident on implanted PEU control materials. Much less severe cracks are seen on 1 wt.% composites. The average rating for each of four sample replicates per material (one per animal) is displayed in Fig. 4a for PEU-QACMMT and Fig. 4b for PEU-AUAMMT. Unimplanted materials received ratings of below 10. That some of these unimplanted silicate-containing materials received a non-zero score was due to surface discoloration rather than degradation, which had been observed previously in other studies in our laboratory. In some cases, this surface discoloration would have affected the implanted scores too, but, if so, it would have artificially elevated the degradation score so that the degradation would have appeared worse than in actuality, and thus would not have compromised the assay. The implanted PEU control material received an average rating approaching 50, with some sample replicates achieving a score of 50, which indicates that degradation was observed in most regions and for most magnifications. Implanted PEU-QACMMT-1 had an average rating not statistically significantly greater than PEU due to more congruent sample replicate ratings and surface discoloration; however, as the silicate loading was increased to 3and 15 wt.%, a statistically significant reduction in rating was seen. The average rating of

Fig. 3. SEM images of (a) PEU, (b) PEU-QACMMT-1, (c) PEU-AUAMMT-1, (d) PEU-QACMMT-3 and (e) PEU-QACMMT-15 materials implanted in sheep for 6 weeks. Magnification is 150 (the scale bar is 200 lm). The sample is the same as in Fig. 3.

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PEU-QACMMT-15 is almost zero. The SEM image ratings shown in Fig. 4b seem slightly better for PEU-AUAMMT-1 than for PEUQACMMT-1, although not statistically significant. The ATR-FTIR traces for unimplanted PEU, PEU-QACMMT and PEU-AUAMMT are shown in Fig. 5a (1800–800 cm1) and Fig. 5b (3400–2600 cm1). Unimplanted PEU-QACMMT-1, PEUAUAMMT-1 and PEU-QACMMT-3 show similar infrared absorbance to unimplanted PEU at all wave numbers, validating the direct comparison of implanted composites with unimplanted PEU as a control. PEU-QACMMT-15 had an obvious divergence at 1100–900 cm1 and more subtle divergences at 2922 and 2938 cm1. The ATR-FTIR traces for unimplanted PEU control, implanted PEU control, and implanted PEU-QACMMT and implanted PEUAUAMMT composites are presented in Fig. 6a (1340–800 cm1), 6b (1640–1340 cm1), 6c (1800–1640 cm1) and 6d (3400– 2600 cm1). Figs. 7 and 8 collate the most significant information contained in Fig. 6 for PEU-QACMMT and PEU-AUAMMT, respectively, by presenting the absorbance of each composite at a particular wave number as a quantitative deviation from the absorbance of unimplanted PEU control at that same wave number. Thus, by the definition used in Eq. (1), a ‘‘negative deviation’’ occurs when the test material absorbance is lower than the unimplanted PEU control absorbance (e.g. for 1081 cm1 in Fig. 7a) and a ‘‘positive deviation’’ occurs when the test material absorbance is higher than the unimplanted PEU control absorbance (e.g. 1174 cm1 in Fig. 7b). A ‘‘deviation’’ equal to zero means that the absorbances of the test material and of the unimplanted PEU control are identical: the desired case of no degradation measurable by ATR-FTIR. Thus, in Fig. 7 each line represents a wave number and following a line from left to right illustrates the deviation from unimplanted PEU control as the QACMMT loading is increased from 0 to 15 wt.%. For almost all wave numbers of interest, the largest absorbance deviation from control was seen for implanted PEU control. Also, for almost all wave numbers, when QACMMT was added at 1 wt.%, the deviation from control was decreased relative to that of implanted PEU control. For all wave numbers, as the loading of QACMMT was increased from 1 to 3 wt.%, the deviation was further decreased, and at a QACMMT loading of 15 wt.% the deviation from unimplanted control approached zero. In Fig. 8, the deviation from PEU for each wave number is presented for both PEUAUAMMT-1 and PEU-QACMMT-1. At most wave numbers the absorbance of implanted PEU-AUAMMT-1 is closer to unimplanted PEU control than to implanted PEU-QACMMT-1, which in turn is closer than implanted PEU control.

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4. Discussion 4.1. Silicate dispersion Styan et al. [11] contains a morphological study of the materials used in the present study. There, PEU-QACMMT and PEUAUAMMT composites were considered to be nanocomposites of partially exfoliated morphology, with some layers completely delaminated, and of intercalated morphology, with layer spacing expanded but the stacked nature retained, respectively. Most studies aim for complete exfoliation, believing it to provide the greatest property enhancement, but many, if not most, of the published studies on solution-cast PU organosilicate nanocomposites achieve at best only partially exfoliated morphology [15–19]. PEU-QACMMT-15 displayed significant silicate alignment, which had increased with silicate loading. Overall, the dispersion of QACMMT appears greater than that of AUAMMT. Favourable thermodynamics resulting from the hydroxyl functionality of both the QAC and AUA were likely the primary driving force for silicate dispersion in PEU, specifically hydrogen bonding between the QAC/AUA and the hard segment urethane group N and the soft-segment C–O–C [20,21].

3.2. Inflammatory response A single representative histological image of the sample-side of the capsule is contained in Fig. 9a–e. Images of the PMMA side are not shown. Qualitatively, the capsules of all test materials excepting PEU-QACMMT-15 appeared to be composed of fibrous tissue. The body’s response seems to have increased in severity with silicate loading, since, with the exceptions discussed below, the observed qualitative capsular thickness was greater for samples of higher QACMMT loading. That said, the capsular thickness of the 1 wt.% materials was not obviously thicker than that of PEU control. Comparing the PEU-AUAMMT1 and PEU-QACMMT-1 materials, the cellularity and fibrous morphology visually appeared similar. PEU-QACMMT-3 had a much thicker capsule wall on both the PMMA side and the sample side of the capsule; however, the cellularity and fibrous morphology appeared normal. The sample side capsule of PEU-QACMMT-15 was virtually non-existent and was severely lacking in fibrous structure, while the PMMA side was present but smaller than for implanted PEU control.

Fig. 4. Degradation ratings of SEM images of unimplanted and implanted PEU control and (a) PEU-QACMMT and(b) PEU-QACMMT-1 compared to PEU-AUAMMT1. A rating of 50 or 0 indicates visual evidence of degradation at all or no magnifications, respectively. ⁄⁄Statistically significant difference (p < 0.05). Data points are the average of one rating from each of two raters, for four replicate samples of each material. Values are mean ± one standard deviation.

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Fig. 5. ATR-FTIR scans of unimplanted PEU control, PEU-QACMMT and PEU-AUAMMT at wave numbers from (a) 1800–800 cm1 and (b) 3400–2600 cm1.

4.2. Validation of assay for assessment of in vivo biostability Two factors needed validation before the implanted silicatecontaining samples could be analysed. The first was to confirm that degradation in PEU control is typical of environmental stress cracking (ESC), as seen in previous studies in this laboratory [3–5]. The SEM images for implanted PEU control (Figs. 2 and 3) shows the degradation to be manifested as microscopic surface cracks when in the presence of mechanical stress such as that applied by the PMMA holders, which is typical. Then, comparing ATR-FTIR traces for unimplanted PEU control to those for implanted PEU control in Fig. 6, it is further evident that implanted PEU control underwent classic ESC. Most characteristic is the oxidative degradation of both hard-and soft-segment ether, as evidenced by reduced absorbance at 1081 and 1100 cm1, respectively, and by introduction of branched ether degradation products at 1174 cm1. Hydrolytic degradation of the ‘‘inter-phase region’’, the urethane junction between the hard and soft segments, is also indicated by loss of non-hydrogen-bonded carbonyl at 1730 cm1, carbon–nitrogen stretching at 1219 cm1 and the ether mentioned previously at 1081 cm1 [2,3]. That such severity of ESC was observed on the PEU material suggests that an implantation period of 6 weeks is of a sufficient time length to allow evaluation of any biostability conferred by the silicate.

The second factor requiring validation is that inclusion of the silicate does not result in a change in the ATR-FTIR traces of the unimplanted samples, as such a change would invalidate the comparison of implanted silicate-containing samples with implanted PEU control. Unimplanted composites of 1and 3 wt.% had ATR-FTIR traces comparable to that of unimplanted PEU control, as shown in Fig. 5a and b. Unfortunately, for PEU-QACMMT-15 the unimplanted ATR-FTIR trace varied significantly from that of unimplanted PEU control, particularly at 2922 and 2938 cm1 and in the region of 1100–900 cm1. It is suggested that the difference in the unimplanted PEU-QACMMT-15 trace arises as a consequence of the silicate’s presence, and that the effect is significant at 15 wt.% and not the lower loadings due to ATR-FTIR sensitivity. The absorbance increases at 2922 and 2938 cm1 are related to the vibration of the C–H bond in methylene groups (CH2) in the alkyl chain in the QAC (data not shown). The absorbance differences at 1100–900 cm1 are more interesting, and are suggested to be a result of (i) absorbance at 1038 cm1 due to stretching of the Si–O–Si bonds in the layered silicate and (b) silicate–ether interaction lowering the ether 1081 cm1 vibration. The outcome is that the affected 1100– 900 cm1 region of the PEU-QACMMT-15 trace was not considered in the analysis of the degree of biostability of PEU-QACMMT-15. This excluded the important ether degradation at 1081 cm1, but was inconsequential as other peaks, particularly the generation of

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Fig. 6. ATR-FTIR scans of unimplanted PEU control and implanted PEU control, PEU-QACMMT and PEU-AUAMMT at wave numbers from (a) 1340–880 cm1, (b) 1640– 1340 cm1, (c) 1800–1640 cm1 and (d) 3400–2600 cm1.

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branched ether degradation products at 1174 cm1, could be relied upon. 4.3. Biostability and inflammatory response of composites in vivo The data strongly supports the hypothesis of enhanced biostability for PEU organosilicate composites when compared to neat PEU control. SEM images clearly show a reduction in visible surface degradation with increased QACMMT loading, as in Figs. 2–4. The SEM image ratings show that the 3 and 15 wt.% samples are significantly less degraded, and it is thought that the stringency of the rating scale was not sensitive enough to display small differences such as are thought to exist for the 1 wt.% materials. This qualitative assessment was strengthened by the ATR-FTIR data, as evident in the traces in Fig. 6, and most readily understood by consideration of the processed data in Fig. 7. Of most importance is that absorbance, irrespective of wave number and direction of deviation from control, tends to approach that of unimplanted PEU control as the silicate loading is increased. PEU-QACMMT-3 offers 50% improved biostability over PEU control, and PEU- QACMMT-1 results in some minor enhancement of biostability. The implanted PEU-QACMMT-15 absorption trace is nearly identical to that of unimplanted PEU control at wave numbers 3400–2600 and 1800–1640 cm1, suggesting a near complete avoidance of in vivo degradation at the urethane linkages and much reduced degradation in other regions of the PEU chain. However, there is some concern as to the origin of this effect, as discussed in the following paragraphs. Previous in vitro studies have observed leaching of, and subsequent cellular response to, organic modifiers from within PU–silicate composites [11,22]. Further, independent studies have observed Sepiolite inhalation to produce macrophage agglomerates and/or multinucleate giant cells that are persistent for up to 3 months [23]. Sharma et al. [24] also recently observed oxidative stress and genotoxicity in liver cells when exposed to zinc oxide nanoparticles. Thus, it is plausible that the QAC and/or silicate could be leaching from PEU-QACMMT and inciting surrounding cells to (i) produce a more corrosive environment than typical and/or (ii) die, resulting in a less corrosive environment than typical. This is supported by preliminary histology conducted on explanted samples, as in Fig. 9. That is, the QAC is interfering in an assessment of the affect provided by the silicate layers per se, since we no longer have control of all variables or consistent treatment among samples. However, an argument for protection by the silicate can still be made. For PEU-QACMMT-1, the capsular thickness appeared similar to that around PEU and cells appeared healthy. There was likely a slight protective benefit to the QACMMT inclusion, as seen in some SEM ratings and in the ATR-FTIR data. For PEU-QACMMT-3, the capsular thickness was much thicker than that around PEU, but the cells appeared healthy, suggesting a much stronger inflammatory response. Despite this, there was a significant protective benefit to the QACMMT inclusion, as seen in significantly different SEM ratings and in the ATR-FTIR data. In this case, it seems fair to conclude that the biostability enhancement observed was due to the silicate inclusion and may even be an understatement of that possible from an otherwise equivalent composite including a less irritating organic modifier. For PEU-QACMMT-15, the material clearly caused severe tissue degradation as the capsule was almost nonexistent. Thus, it is not possible to attribute the extreme biostability enhancement seen in SEM ratings and ATR-FTIR data entirely to a silicate protective effect. However, it is likely that at least some of the 15 wt.% effect is due to the silicate presence, based on the positive 1 and 3 wt.% data. Since this issue was pre-empted, PEU-AUAMMT-1 was included in this study as a partial control. Unfortunately, animal constraints meant that 3 and 15 wt.% controls could not be included in this

Fig. 7. The effect of increasing QACMMT loading on the maintenance, to the level of unimplanted PEU control, of biostability relevant PEU infrared absorptions. Shown are wave numbers whose test material absorbance was (a) lower than that of unimplanted PEU control and (b) higher than that of unimplanted PEU control.

Fig. 8. Comparison of the deviation of biostability-relevant PEU infraredabsorptions from control levels between PEU-QACMMT-1 and PEU-AUAMMT-1 composites. Also shown, as a reference, is implanted PEU data.

study. A loading of 1 wt.% was chosen over the higher loadings as it was thought that if enhanced biostability was seen at the lowest AUAMMT loading then this could be extrapolated to higher loadings

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Fig. 9. Light microscopy images of histological sections of the sample side of fibrous capsules of (a) PEU, (b) PEU-QACMMT-1, (c) PEU-QACMMT-3, (d) PEU-QACMMT-15 and (e) PEU-AUAMMT-1. Magnification in  10 (the scale bar (red) is 200 lm). Staining is with H&E.

more confidently than a back-extrapolation from, say, 15 wt.% to 1 wt.%. Pleasingly, PEU-AUAMMT-1 seems to have provided a small protective effect on the order of that seen for PEU-QACMMT-1, as seen in the SEM images/ratings and ATR-FTIR traces in Figs. 2–6. Also, when the ATR-FTIR data is summarized as in Fig. 8, PEUAUAMMT-1 is seen to consistently possess slightly less degraded chemical bonds than PEU-QACMMT-1. Since the silicate dispersion of PEU-AUAMMT-1 is poorer than that of PEU-QACMMT-1, perhaps the biostability enhancement observed for PEU-AUAMMT-1 is an under-estimation of that expected if dispersion could be increased. 4.4. Potential mechanisms of biostability enhancement The mechanisms responsible for the observed reduction in degradation are likely to stem from alterations in the barrier properties and mechanical properties that occur in nanocomposites. However, it is difficult to separate the individual contributions of these factors. The modulation of barrier properties characteristic of nanocomposites has been well established in several polymerbased nanocomposite systems (e.g. [25,26]). Diffusion of gases such as oxygen can be significantly retarded in the presence of dispersed silicates [27], and water vapour permeability has also

been shown to decrease significantly [28]. Given that oxidative mechanisms of degradation combined with applied mechanical stress are primarily responsible for ESC in polyurethanes, it is likely that perturbation of the diffusion of reactive oxygen species (ROS) impacts on the degradation rate in nanocomposites. ROS, especially the hydroxyl radical, react with PEU chains by extracting hydrogen ions from methylene groups, leaving a free electron pair (CH) in a process known as hydrogen abstraction. If oxygen is present, a peroxyl radical (COO) forms on the polymer chain that is also capable of hydrogen abstraction, leading to further degradation and cross-linking. A hydrogen ion (H+) is also produced, which may increase the degradation of the PEU ether present in the soft segment and the ester linking the soft and hard segments. The density of the impermeable silicate layers at or very near the surface likely inhibits the access of the ROS to the polymer surface, thereby preventing degradation. Internally, these layers may also inhibit migration of the ROS through the polymer bulk by the so-called ‘‘tortuous path’’ effect. In preliminary studies done in our laboratories, and as seen throughout the literature, silicate inclusion reduces permeability to both water and oxygen. The barrier effect of the silicate layers appears to be akin to the action of the interconnected hard segments of PellethaneÒ 2363-55DA,

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which has much greater biostability than PellethaneÒ 2363-80A [5]. Another consideration is that the PEU hard segments are physically larger than the interlayer silicate spacing measured by XRD and are thus unlikely to exist between the silicate layers [29]. By deduction, it is probable that it is the soft, rather than the hard, segments which are associated with the silicate and are thus offered greatest protection. As the soft segments are more susceptible to degradation, their association with the silicate layers would provide greater biostability enhancement than if it were the hard segment that was most protected. In support of this, the lowering of the PEU ether peak at 1081 cm1 in the PEU-QACMMT-15 composite (Fig. 5a) is suggestive of silicate–ether interaction. Also, any crack beginning through chain cleavage in the soft segment might be hindered upon reaching a silicate layer. Mechanical stress has been shown to increase the severity and rate of degradation in many cases (e.g. [30]), so the in vivo biostability assay used applied stress by straining samples over PMMA holders. A potential contributor that has not been resolved is the changes to the internal structure caused by the applied strain. In previous studies on PU without included silicate [4], straining to 150% over the PMMA holders was done in order to accelerate degradation. With the included silicate layers, however, the effect of the strain may have been different. The PEU of this study combined with a similar organosilicate has been studied by Finnegan et al. [29,31]. Small-angle X-ray scattering(SAXS) results showed that silicate alignment in the direction of strain is detectable, with orientation beginning immediately upon application of strain and significant rotation occurring by 150% strain. This has also been seen by other groups [32]. Conceptually, the path length to migrating species will be shortest if the silicate is aligned broadside to the sample surface and longest if aligned thin side to the sample surface. The application of strain theoretically causes alignment in the direction of the applied strain; thus, pulling the sample over the PMMA holder may have also pulled silicate particles to show a greater broadside surface area parallel to the sample surface. No studies have looked at the effect of silicate orientation on the magnitude of the barrier effect of nanocomposites, due presumably to the difficulty of preparing a series of nanocomposites with various degrees of silicate alignment. However, there is support by studies such as that by Schubert et al. [33], where reduced degradation of a poly(etherurethane urea) elastomer was observed and attributed to the increased orientation and compaction of polyether segments following stretching, which also resulted in lowered oxygen permeability. In the present case, in addition to silicate alignment, it is possible that the soft segment has been oriented and compacted between silicate layers. If these orientation effects were present, the observed biostability enhancement would be overstated compared to the actual situation for an unstrained sample. Promisingly, though, increased soft-segment crystallinity was not detected by ATR-FTIR, as evidenced by the absence of a peak at 996 cm1 and/or one at 1370 cm1 [33], and no change in the soft-segment morphology of very similar composites was detected by SAXS/small-angle neutron scattering[29,31].

5. Conclusions Much effort has been directed towards the generation of more biostable PUs. Success has been achieved through replacement of the soft segment with more chemically resistant polymers, most notably polycarbonate and PDMS. The current study is the first to show that biostability can also be improved by the addition of layered silicates. The addition of dispersed QACMMT at loadings lower than 3 wt.% resulted in significantly enhanced biostability, likely

due primarily to decreased material permeability. The addition of 15 wt.% QACMMT superficially seemed to result in a stronger protective effect; however, the impact of a relatively larger immune response on the reduced PEU degradation observed is unknown. A more biocompatible PEU-AUAMMT-1 material of poorer silicate dispersion and likely reduced host response displayed properties comparable toPEU-QACMMT-1. The significance of these results is that: (i) although the QAC had a detrimental effect on surrounding tissue, a less irritating organosilicate may be found (such as AUAMMT) that provides significant biostability enhancements; and (ii) although this study employed a poly(ether)urethane specifically because of its lack of biostability, it is reasonable to extrapolate that a similar effect could be seen for other PUs. Acknowledgements The assistance of Veronika Tatorinoff (Surgery) and Kate Noble (Surgery, SEM), from the University of New South Wales, and Karen Barnes (histological image preparation), from the University of Sydney, is gratefully acknowledged. The authors would also like to acknowledge funding support in the form of both a Faculty of Engineering Research Grant from The University of New South Wales and an Australian Research Council Discovery Grant (DP0558561). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Fig. 9, is difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org.10.1016/j.actbio.2012. 03.004. References [1] Pinchuk L. A review of the biostability and carcinogenicity of polyurethanes in medicine and the new generation of biostable polyurethanes. J Biomater Sci Polym Ed 1994;6(3):225–67. [2] Gunatillake PA et al. Designing biostable polyurethane elastomers for biomedical implants. Aust J Chem 2003;56:545–57. [3] Simmons A et al. Long-term in vivo biostability of poly(dimethylsiloxane)/ poly(hexamethylene oxide) mixed macrodiol-based polyurethane elastomers. Biomaterials 2004;25(20):4887–900. [4] Martin DJ et al. New methods for the assessment of in vitro and in vivo stress cracking in biomedical polyurethanes. Biomaterials 2001;22:973–8. [5] Martin DJ et al. Polydimethylsiloxane/polyether-mixed macrodiol-based polyurethane elastomers: biostability. Biomaterials 2000;21(10):1021–9. [6] Usuki A et al. Synthesis of nylon 6-clay hybrids. J Mater Res 1993;8(5):1179–84. [7] Kojima Y et al. Mechanical properties of nylon 6–clay hybrids. J Mater Res 1993;8(5):1185–9. [8] Thatiparti TR, Tammishetti S, Nivasu MV. UV Curable polyester polyol acrylate/ bentonite nanocomposites: synthesis, characterization, and drug release. J Biomed Mater Res Part B–Appl Biomat 2010;92B(1):111–9. [9] Cypes SH, Saltzman WM, Giannelis EP. Organosilicate–polymer drug delivery systems: controlled release and enhanced mechanical properties. J Control Release 2003;90(2):163–9. [10] Beall, G.W., New conceptual model for interpreting nanocomposite behaviour.In: G.W. Beall and T.J. Pinnavaia (eds.), Polymer–Clay Nanocomposites. Brisbane: John Wiley & Sons, Ltd., 2000, p. 267–279. [11] Styan KE, Martin DJ, Poole-Warren LA. In vitro fibroblast response to polyurethane organosilicate nanocomposites. J Biomed Mater Res 2008;86A:571–82. [12] Dillon JG. Infrared spectroscopic atlas of polyurethanes. Lancaster, PA: Tachnomic Publishing Company, Inc.; 1989. [13] McCarthy SJ et al. In-vivo degradation of polyurethanes transmission–FTIR microscopic characterization of polyurethanes sectioned by cryomicrotomy. Biomaterials 1997;18(21):1387–409. [14] Loo LS, Gleason KK. Fourier transform infrared investigation of the deformation behaviour of montmorillonite in nylon-6/nanoclay nanocomposite. Macromolecules 2003;36:2587–90. [15] Chen T-K, Tien Y-I, Wei K-H. Synthesis and characterization of novel segmented polyurethane/clay nanocomposites. Polymer 2000;41:1345–53. [16] Tien YI, Wei KH. High-tensile-property layered silicates/polyurethane nanocomposites by using reactive silicates as pseudo chain extenders. Macromolecules 2001;34(26):9045–52.

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