In vitro fibroblast response to polyurethane organosilicate nanocomposites

In vitro fibroblast response to polyurethane organosilicate nanocomposites K. E. Styan,1 D. J. Martin,2 L. A. Poole-Warren1 1 Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052 Australia 2 Division of Chemical Engineering, School of Engineering, University of Queensland, QLD 4072 Australia Received 19 January 2007; revised 15 June 2007; accepted 28 June 2007 Published online 9 November 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31667 Abstract: The term nanocomposite refers to organic:inorganic composites where one phase, typically the inorganic phase, has dimensions on the nanoscale. Several authors have noted the potential benefit of biomedical application of nanocomposite technology, and have suggested using quaternary ammonium compounds (QAC) as an organic modification to enhance dispersion of nanoparticles within polymer matrices. This study aimed to examine fibroblast responses in vitro to a range of nanocomposites using different organic modifiers. Composite materials were prepared from a polyether urethane (PEU) and various unmodified and organically modified montmorillonite (MMT) nanoparticles. QAC and amino undecanoic acid (AUA) modified-MMT were added to PEU at loadings ranging from
1 to 15 wt %. Composites with organically modified QAC and AUA particles displayed partially exfoliated and

intercalated silicate morphology, respectively. Nanocomposites showed increases in ultimate tensile properties for materials with lower QACMMT loadings. However QAC was shown to significantly inhibit cell growth following release from PEU-QACMMT under extraction conditions mimicking those of the physiological environment. Materials containing silicate modified using AUA were cytocompatible. The results of this study suggest that QAC may be unsuitable as organic modifiers for nanoparticles destined for biomedical use. Alternative modifiers based on AUA confer equivalent dispersion and are of low toxicity. Ó 2007 Wiley Periodicals, Inc. J Biomed Mater Res 86A: 571–582, 2008

INTRODUCTION

Achieving optimal dispersion of nanoparticles in composites is of prime importance since materials with poor nanoparticle dispersion are unlikely to have improvements in their physical properties. A typical MMT layer 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).3 Layers naturally stack in groups on the order of 1000 units that need to be delaminated for generation of nanocomposites. Usually, the cationic exchange capacity (CEC) of the layered silicate is exploited to organically modify the inorganic silicate and increase its compatibility with the organic polymer/prepolymer/monomer. Quaternary ammonium compounds (QAC) are the most frequently employed organic modification (OM), however theoretically any positively charged species is capable of cationic exchange onto the MMT. The current study focuses on biomedical application of polyurethane (PU) nanocomposites, where the proposed enhancement of mechanical and barrier properties may have significant advantages over conventional PU. In these material systems, another critical design specification is the appropriate biological performance, or biocompatibility. Previous studies

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. Specifically, Usuki et al.1 synthesized a Nylon-6 composite by polymerizing e-caprolactam within the layers of a natural silicate, montmorillonite (MMT), and showed using X-ray diffraction (XRD) and transmission electron microscopy (TEM) that the silicate was dispersed on the nanoscale. In a subsequent study, Kojima et al.2 reported that for a composite containing 4.7 wt % MMT the tensile strength, elastic modulus, and the heat distortion temperature increased to 140, 168, and 234% (respectively) of Nylon-6 alone. Correspondence to: L. A. Poole-Warren; e-mail: l.poolewarren@ unsw.edu.au Contract grant sponsor: The University of New South Wales Contract grant sponsor: Australian Research Council Discovery Grant ; contract grant number: DP0558561

Ó 2007 Wiley Periodicals, Inc.

Key words: nanocomposite; polyurethane; silicate; biocompatibility; organic modification

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have shown intercalated silicate dispersion, maintenance or improvement of mechanical properties and enhanced barrier properties in solution cast intercalated nanocomposites fabricated using the biomedical PU BiospanTM and Cloisite1 15A (OM: dimethyl ditallow ammonium chloride) or Nanomer 1.30TC (OM: octadecyl ammonium chloride).4,5 The same studies proposed that the reported barrier property enhancement could be advantageous to thin-walled blood-contacting devices such as the blood sacs in ventricular assist devices, where high material strength and low permeability are required, but did not support this proposal with biological evaluation studies. Other research groups have recognized the potential for silicate particles to influence drug release kinetics from hydrogels and have conducted studies using silicates organically modified using QAC to support their hypotheses.6–10 These hydrogel-silicate nanocomposites showed promise in the proposed applications, however nanocomposite biocompatibility was not evaluated. The components of the composite in the current study were a polyether urethane (PEU) with similar chemistry to polymers typically used in medical applications, and a layered silicate MMT, both of which are not expected to possess inherent cytotoxicity. Layered silicates are used in topical and oral pharmaceutical applications as fillers, and in food production processes as filtering and/or clarifying agents,11 and as long as they are not released in vivo should have acceptable biological performance. However, the OM may have a large influence on biocompatibility depending on its chemical properties and whether it is released or exposed in vivo. QACs are the most frequent class of OM used in the nanocomposite literature. They are used commercially in clinical, domestic, and food manufacturing environments due to their antibacterial activity, however, they have been associated with some cytotoxicity and lysis of mammalian cells.12–14 Although implantation of benzalkonium chloride-containing intravenous catheters into human subjects showed no adverse recipient effects,15 the biological performance must be evaluated for any composite material containing OM. Alternative OM that might not introduce biocompatibility issues include a range of alkyl chains with protonated terminal amine groups. Amino undecanoic acid (AUA) was selected in the current study as it possesses amine and carboxyl functionality located at opposite ends of a C10 alkyl chain. When acidified to a pH below the isoelectric point (pI), AUA has a structure similar to that of a QAC in that it contains both a cationic head group and a long alkyl chain. AUA and similar molecules have been used as an OM with some success in the literature,16,17 although the resulting AUA-containing materials have not Journal of Biomedical Materials Research Part A

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been tested for cytotoxicity. The similarity of AUA with biological macromolecules such as the alkyl chain amino acids (for example amino oleic acid) suggests lowered toxicity relative to QAC. The objective of this study was to compare QAC and AUA OM methods for their impact on both the dispersion of the nanoparticles and the mammalian cell response to the material. Specifically, the study aimed to determine the importance of OM for MMT dispersion in a PU matrix and the comparative ability of QAC and AUA-modified MMT to disperse within this polymer matrix. Also studied were the in vitro cytotoxicity of leachables from the QAC and AUAcontaining materials in order to determine the potential for both inhibition of cell growth and cytotoxicity.

MATERIALS AND METHODS Poly(ether)urethane The base PEU used contained 1000 g/mol poly(tetramethylene oxide) polyol (PTMO), 4,40 diphenylmethane diisocyanate (MDI), and 1,4-butanediol (BDO) as the chain extender and was supplied by Urethane Compounds (Melbourne, Australia). The components were combined in the ratio 100:7.5:46.3, respectively, with 0.003 dibutyltin dilaurate added as catalyst,18 thus the PEU contained
65 wt % PTMO (soft segment).

Nanoparticles MMT of chemical formula Na0.33[(Al1.67Mg0.33)Si4O10(OH)2]H2O sourced from Southern Clay Products (Texas, USA) and CEC of 92.6 mEq/100 g19was used as the base silicate. An organically modified MMT, Cloisite 30B (referred to as QACMMT) with QAC methyl tallow bis-2-hydroxyethyl ammonium chloride as an OM was also sourced from Southern Clay Products [see Fig. 1(a)]. The alkyl chain in this OM had a range of lengths including 5% C14, 30% C16, and 65% C18, designated QAC-C14, QAC-C16, and QAC-C18, respectively. A second organosilicate was prepared by modifying the MMT with AUA as an OM as shown in Figure 1(b) (referred to as AUAMMT: Aldrich). Figure 1(c) shows the AUA as it would occur at physiological pH. AUAMMT was prepared by stirring MMT in distilled, deionised Milli-Q water at 1 wt % for 24 h followed by addition of AUA at 0.2 g/100 g MMT (110% CEC). The solution was then acidified using 10M HCl to a pH just below pH 4 (the pI of the similar NH2CH2COOH is 4.89), and left to stir vigorously for a further 24 h at
608C. Product was isolated by 10 min of centrifugation at 30,100g and 158C, and then dried at 608C overnight. Dried organosilicate was crushed in a glass mortar and pestle, sieved with a 325-mesh (45 lm) sieve, and then redried at 608C for several h before use. Nanocomposites were then prepared from the PEU and either MMT alone (no OM), QACMMT, or AUAMMT.

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OM OM MMT  ¼ OM nanoparticle þ1 MMT

ð1Þ

MMT nanoparticle OM MMT ¼ 3 3 PEU PEU nanoparticle OM

ð2Þ

OM MMT OM ¼ 3 PEU PEU MMT

ð3Þ

Nanocomposite morphological characterization 1

Figure 1. Molecular structure of (a) QAC on Cloisite 30B, (b) AUA at pH < 4, and (c) AUA at pH  4 (e.g. in vivo pH 7.4).

Nanocomposite preparation A 5 wt % solution of PEU in dimethylacetamide (DMAc, Sigma Aldrich) was prepared at 608C for 7 days. A 5 wt % suspension of QACMMT in toluene (Sigma Aldrich) was also prepared. As a polar activator, methanol (Univar) and distilled deionised water were then added at 30 wt % and 6 wt % of the QACMMT mass, respectively. The suspension was vigorously stirred for 30 min, and placed in an ultrasonic water bath for 10 min. MMT and AUAMMT were added as dry nanoparticle powder directly to the PEU/DMAc solution. The PEU/DMAc solution was combined with the nanoparticle in appropriate proportions then stirred on a magnetic stirrer for at least 5 h at
508C to result in composites with loadings ranging from
1 to 15 g nanoparticle/100 g PEU. Following stirring, mixtures were poured into glass pour plates 220 3 70 mm2. DMAc was removed at 608C under a dry air atmosphere at partial vacuum of 400–500 mbar in a laboratory vacuum oven (Binder VD). After 24 h, the material films were peeled from the glass and subjected to a further 24 h at the same conditions while resting on a Teflon1 sheet. Materials were designated as polymer-nanoparticle-nanoparticle loading, for example PEU-QACMMT-3 was a material containing 3 g QACMMT/100 g PEU. Materials were prepared with nanoparticle loadings from 1 to 15 wt % as detailed in Table I.

X-ray diffraction Diffractograms were taken on samples from cast sheet using a Siemens D5000 wide angle XRD generating CuKa radiation of wavelength 0.15406 nm from 1.88 to
6–108 2h at a rate of 0.088/min, a 0.018 step size, and with source conditions of 40 kV, 35 mA. Parameters used for the PEUMMT materials were 18/min and 0.028 step size, since peaks were of higher intensity. A 2 mm anti-scatter slit and 0.05 mm receiving slit were used to increase the signal to noise ratio at the lower angles. The raw data was processed using the XRD software’s fitting and deconvolution function, applying the pseudo-Voigt function to manually selected peaks. This data was converted to 2h data using Bragg’s Law [Eq. (4)] where k is the wavelength of the incident X-ray beam (nm), d is the distance (nm) between the silicate layers (from the top of one layer to the top of the next layer), and h is the incident beam angle (radians). Displacement correction (shift) was conducted using Eq. (5) where s is the sample thickness (mm), R is the goniometer radius (mm, 200 mm for the Siemens D5000), and h is the incident X-ray beam angle (radians) and the shift was added using Microsoft Excel. Data was then converted to d (nm) [using a rearrangement of Eq. (4)]. Results are expressed as plots of the shifted pseudo-Voigt fitted curve TABLE I Material Loadings for PEU-MMT, PEU-QACMMT, and PEU-AUAMMT Materials Loading

Thermal gravimetric analysis PEU-QACMMT and PEU-AUAMMT composites were analyzed by thermal gravimetric analysis (TGA) to determine percent organic component present. A Perkin Elmer Pyris 1 TGA operated with an air atmosphere and a 20 mL/min N2 purge was used to determine percent organic component (the loading of OM on the MMT, that is, the OM/MMT) with OM weight loss occurring from
100 to 8008C. Scans were collected from 50–8008C at 308C/min. Nanoparticle loadings were calculated as both g silicate/ 100 g PEU and g OM/100 g PEU using the measured OM/MMT and Eqs. (1), (2), and (3) as shown in Table I.

Material

g nanoparticle/ 100 g PEU

g silicate/ 100 g PEU

g OM/ 100 g PEU

PEU-MMT-1 PEU-MMT-3 PEU-MMT-7 PEU-MMT-15 PEU-QACMMT-1 PEU-QACMMT-2 PEU-QACMMT-3 PEU-QACMMT-7 PEU-QACMMT-15 PEU-AUAMMT-1 PEU-AUAMMT-3 PEU-AUAMMT-7

1 3 7 15 1 2 3 7 15 1.0 3.0 6.4

1 3 7 15 0.8 1.6 2.4 5.7 12.1 0.8 2.4 5.5

0 0 0 0 0.2 0.4 0.6 1.3 2.9 0.2 0.6 0.9

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TABLE II Mechanical Properties of all Materials Material

Number of Samples/Batches

PEU PEU-MMT-1 PEU-MMT-3 PEU-MMT-7 PEU-MMT-15 PEU-QACMMT-1 PEU-QACMMT-2 PEU-QACMMT-3 PEU-QACMMT-7 PEU-QACMMT-15 PEU-AUAMMT-1 PEU-AUAMMT-3 PEU-AUAMMT-7

27/12 4/2 5/1 4/2 4/2 9/5 4/3 5/3 19/5 16/4 7/2 7/2 8/2

Ultimate Strain (% 6 S.D.) 481 469 439 469 431 480 503 476 477 450 503 457 465

6 6 6 6 6 6 6 6 6 6 6 6 6

Ultimate Stress (MPa 6 S.D.)

33 5 21a 24 11b 13 17 10 16 20b 13c 42 16

56 47 40 47 31 60 62 45 41 28 57 54 40

6 6 6 6 6 6 6 6 6 6 6 6 6

Elastic Modulus (MPa 6 S.D)

11 3a 4a 8 3a 7 8 5 7b 5b 6 8 4b

13 13 16 22 35 15 19 23 30 45 13 18 30

62 61 6 1a 6 1a 6 5b 6 3b þ 1b 6 1b 6 7b 6 12b þ1 6 2a 6 3b

a Significantly different to PEU (b) or corresponding (by closest loading) PEU-QACMMT (c) respectively (student’s t-test, 95% confidence).

of XRD intensity versus 2h, and as shifted silicate spacing, d (nm). The MMT silicate spacing is referred to herein as the natural spacing, while for QACMMT and AUAMMT, spacing is referred to as organosilicate basal spacing. When multiple peaks were detected in composite materials, peaks were referred to as first peak and second peak, and are presented from right to left on the graphs. k ¼ 2d sin u D2u ¼

2s cos u R

ð4Þ ð5Þ

Transmission electron microscopy A sample of
1 3
4 mm2 dimension was inserted into a holder and frozen in a sucrose solution using liquid nitrogen. TEM sections were then prepared using a Reichert Ultracut E cryo-ultramicrotome equipped with a diamond knife and operated at 2808C. Sections were placed on 400-mesh copper grids and analyzed in a Hitachi H7000 TEM operated with a beam current of 100 keV. Several sections from the one sample were prepared and imaged, with at least five images at each magnification being collected per material.

Tensile testing Tensile tests were conducted on strip-shaped samples as recommended by ASTM D 882.20 Strips of from 7 to 12 mm width were cut from solution cast materials and 33 mm or 45 mm gauge lengths were marked on samples using a soft-tipped marker. Thickness was measured using a digital micrometer at the extreme and mid positions of the marked gauge length region and the three measures were averaged. ASTM D 88220 suggests a variation in thickness of no more than 10% over the gauge length be used however this was difficult to achieve in this system. Actual thickness variations were 12% for PEU, 13% for Journal of Biomedical Materials Research Part A

PEU-MMT, 19% for PEU-QACMMT and 13% for PEUAUAMMT. An INSTRON 4302 was then used to obtain force versus elongation data, from which ultimate strain (e), ultimate stress (s), and elastic modulus (E), were calculated using Eqs. (6), (7), and (8). Strips were gripped using the combination of line contact and smooth rubber faces at a grip pressure of 500 kPa, and were tested at a strain rate of 100 mm/min until failure. A two-tailed student’s t-test with the assumption of equal variances was conducted to compare among materials. Results are expressed as mean 61 standard deviation. The number of samples and batches from which the mechanical property data was obtained are shown in Table II. elongation breakage length initial e¼ 3 100ð%Þ length

ð6Þ

initial

s¼

forcejbreakage ðNÞ cross.sectional.areajinitial ðm2 Þ

ðPaÞ

stressðPaÞ E¼ ðPaÞ strainð%Þ 100 linear.toe.region

ð7Þ

ð8Þ

Nanocomposite biological characterization In vitro mammalian cell response In accordance with AS ISO 10993,21 samples were punch cut from cast materials to give an area of 6 cm2, or weighed to give 0.1–0.2 g, per mL of extraction vehicle. Samples were washed with 2% Decon-90 for 24 h, rinsed with MilliQ water for 2 days, sterilized with 100% ethylene oxide gas and allowed at least 7 days to degas. The samples were extracted at 378C for 24 h in complete media (Eagle’s minimum essential media (EMEM, Sigma) þ 10 vol % fetal calf

IN VITRO FIBROBLAST RESPONSE TO PU ORGANOSILICATE NANOCOMPOSITES

serum (FCS, Gibco)) supplemented with 2% penicillin/ streptomycin (P/S, CSL Biosciences). Controls of PEU, silastic (negative, Dow Corning), latex (positive, gloves), and duplicate extraction controls (glass vials) were included. A fibroblast monolayer (L929 mouse fibroblasts, ATCC CCL-1) was prepared by seeding at 105 cells/mL in complete media and incubating for 24 h at 378C and 5% CO2. After removal of media and washing of the cell layer, extraction fluid was applied to the cell monolayers and incubated for 48 h. Null samples were not exposed to extraction fluid, but were replenished with fresh complete media and incubated under identical conditions. After 48 h, monolayers were washed with Dulbecco’s phosphate buffered saline without calcium and magnesium (DPBS, Sigma), trypsinised (0.12% trypsin, 0.02% ethylene diamine tetra-acetic acid (EDTA), 0.04% glucose, JRH Biosciences), and assessed by flow cytometry. Fluorescent beads (Bangs Laboratories) were added at a ratio of
1:10 with cells to allow quantitation. Fluorescent propidium iodide (PI, Sigma-Aldrich) was added to assess cellular membrane integrity at a working concentration of 1 lg/ mL. The cell/bead mixture was analyzed on a Becton Dickinson FACSort flow cytometer, with final data manipulation conducted using WinMDI 2.8 and Microsoft Excel software. Measured events were plotted as PI fluorescence versus forward scatter. Null samples were used to determine the baseline PI fluorescence, with events falling below this baseline considered PI negative (PI2) membrane intact cells (likely viable), and those above considered PI positive (PIþ) membrane compromised cells (likely dead). The total cell numbers used for the majority of calculations are a sum of the PIþ and PI2 cell populations. Where cell growth was inhibited after contact with extractables, materials were sequentially extracted over ten consecutive days to determine whether cytotoxic leachables could be completely extracted. Briefly, extraction fluid was removed from n 5 3 material samples of each type after 24 h of extraction, and fresh extraction media added. This procedure was repeated ten times and the extracts applied cells as described. Where toxicity was observed, modified nanoparticles alone were tested and the results compared with unmodified MMT silicates. Briefly, nanoparticles were extracted in complete media for 24 h at 0.007 g/mL, which is equivalent to extraction of PEU-QACMMT-7 at 0.1 g/mL. After removal of the silicate by filtration with a 0.45 lm syringe filter, a standard in vitro cell growth assay as described above was conducted on the extraction fluid and dilutions of 1021 and 1022. All assays were conducted in triplicate and were repeated at least three times on different days for both PEU-QACMMT and PEU-AUAMMT materials. Total cell counts obtained for PEU from all assays conducted were pooled and averaged. Total cell counts for test materials were then presented as a ratio with a control, which was either the extraction controls described above or the average PEU value. Plots show the test : control ratio on the yaxis, and the nanoparticle loading or material type on the x-axis. A test sample was considered bio-inhibitory or cytotoxic if the test : control ratio was below 0.7. Membrane integrity data is presented as the percent of total cell population that is PI2. A two-tailed student’s t-test with the assumption of equal variances was conducted to compare

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the cell response to materials with equivalent nanoparticle loading.

Liquid chromatography mass spectroscopy Where toxicity of extracts was observed, they were analyzed using a reverse-phase liquid chromatography separation system (Thermofinnigan Surveyor) and an electrospray ionization mass spectrometer (LCQ Deca XP plus) (LCMS). The eluting solvent used had initial composition 97.9% water, 2% acetonitrile, and 0.1% formic acid, and was gradually changed to 100% acetonitrile over the 40 min run. The solvent flow rate was set to 200 lL/min, with 10 lL of sample being analyzed. The chromatography column had a C8 stationary phase, internal dimensions 100 3 2.1 mm2, and pore size 7 lm (Aquapore, Applied Biosystems). In these studies, the QAC containing materials were associated with toxicity and therefore chromatography traces were scanned for molecules of molecular weight (MW) corresponding to the QAC: that is 316.5 (QAC-C14), 344.6 (QAC-C16), 372.6 (QAC-C18) 6 0.5 to allow for instrumental error. MMT composites were used as controls and AUAMMT was not tested.

RESULTS Silicate dispersion Silicate dispersion was assessed using XRD and TEM. Raw XRD peaks were diffuse and tended to increase in intensity and area with silicate loading as the number of constructively interfering diffractions increased. It is likely that a range of silicate spacings and/or orientations was present in all materials. The shifted silicate spacings are shown in Table III and TABLE III XRD Peaks of MMT, QACMMT, and AUAMMT Nanoparticles, and PEU-MMT, PEU-QACMMT, and PEU-AUAMMT Materials Silicate Spacing (nm)a Material

First peak

Second peak

MMT PEU-MMT-1 PEU-MMT-7 PEU-MMT-15 QACMMT PEU-QACMMT-1 PEU-QACMMT-2 PEU-QACMMT-3 PEU-QACMMT-7 PEU-QACMMT-15 AUAMMT PEU-AUAMMT-1 PEU-AUAMMT-3 PEU-AUAMMT-7

1.24 1.29 1.30 1.25 1.82 1.74 1.78 1.84 1.73 1.77 1.39 1.70 1.67 1.68

3.35 3.66 3.23 3.32 n/a 2.91 2.79 2.84

a One silicate layer thickness plus space between layers (
0.97 nm).

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2.9 nm. Data compares well with that reported by the silicate manufacturer of 1.17 nm and 1.85 nm for MMT and QACMMT, respectively.19,22 Representative TEM images of PEU-MMT, PEUQACMMT, and PEU-AUAMMT materials are presented in Figure 3(a–f). TEM images of PEU control materials contained no observable inclusions at any magnification, and are thus not presented. MMT is evident as dark inclusions existing only as large clumps on the order of
1000 to
5000 nm dispersed throughout the sample. QACMMT is evident as fine groupings of few silicate layers with thickness of approximately several nm, as well as small clumps on the order of
100 to 500 nm. The thickness of the silicate groups and the number of clumps tends to increase with the QACMMT loading. AUAMMT is evident as both large and small organosilicate clumps of
2000 nm and
200 to
600 nm in dimension, respectively. Higher magnification images of PEU-QACMMT and PEU-AUAMMT materials are presented in Figure 4(a–d), where the overall dispersion of QACMMT appears greater. This supports the XRD results which also suggest that the layered silicate spacing was slightly greater in QACMMT compared with the AUAMMT.

Mechanical properties

Figure 2. XRD traces for (a) MMT and QACMMT nanoparticles, and PEU-QACMMT materials, and (b) MMT, QACMMT, and AUAMMT nanoparticles, and PEUAUAMMT materials. The XRD intensity has been adjusted to increase presentation clarity. Pseudo-voigt deconvolution and displacement correction have been applied.

Figure 2(a,b). The natural spacing of MMT of 1.24 nm increased marginally to
1.30 nm following inclusion in PEU at any loading. Contrarily, the MMT natural spacing increased to 1.82 nm and 1.39 nm with the intercalation of the QAC and AUA, respectively, and most PEU composites of QACMMT and AUAMMT contained two peaks. PEU-QACMMT materials contained a first peak at the QACMMT organosilicate basal spacing of (1.8 nm, and a second peak at an expanded silicate spacing of
3.2 to
3.7 nm. This second peak was not observed for the material with lowest organosilicate loading, PEU-QACMMT-1. PEU-AUAMMT materials contained a first peak that was significantly greater at
1.7 nm than the AUAMMT organosilicate basal spacing of 1.39 nm, and a further expanded second peak at
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The ultimate mechanical properties and the elastic moduli for all materials are presented in Table II. In comparison to PEU control, ultimate strain was reduced upon incorporation of MMT with some loadings resulting in significantly lowered values and ultimate stress was significantly lowered across all MMT loadings except PEU-MMT-7. Elastic modulus significantly increased for all MMT loadings except 1 wt %. The inclusion of nanoparticles of QACMMT resulted in maintenance of PEU ultimate strain until the highest loading of 15 wt %. The lower QACMMT loadings of 1 and 2 wt % caused an increase in the ultimate stress (not significant), however as the loading increased the ultimate stress was significantly lowered compared to PEU. It should be noted that the host PEU employed in this work had an inherently high tensile strength with respect to other published values for similar materials.16,23–25 Elastic modulus increased with QACMMT loading as for MMT, however the rate of increase was significantly greater with QACMMT inclusion. Inclusion of AUAMMT caused similar changes in PEU mechanical properties as QACMMT, however the increased stress at low loadings was not observed and the elastic modulus of PEUAUAMMT-3 was significantly lower than the PEUQACMMT-3 modulus.

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Figure 3. TEM images of (a) PEU-MMT-1 and (b) PEU-MMT-7 at 35003 showing the large clumps of silicate and a scale bar of 5000 nm. Higher magnification images were not shown as most were featureless; The remaining TEM images are at 10,0003 magnification showing (c and d) PEU-QACMMT-1 and PEU-QACMMT-7 and (e and f) PEU-AUAMMT-1 and PEU-AUAMMT-7, with the scale bar representing 2000 nm, respectively.

In vitro mammalian cell response The cell response to silicone negative controls and latex positive control materials was as expected in all assays confirming the validity of the assay. The cell response to PEU-MMT, PEU-QACMMT, and PEU-AUAMMT materials is presented in Figure 5(a). PEU-MMT materials did not significantly inhibit cell growth at any MMT loading. PEU-AUAMMT materials also caused minimal cell growth inhibition at all AUAMMT loadings tested, and materials with higher AUAMMT loadings tended to cause greater cell growth reduction. PEU-QACMMT materials caused a dramatic reduction in the number of cells present following ex-

posure to the sample extraction fluid. The membrane integrity of cells following exposure to PEU and PEU-QACMMT and PEU-AUAMMT material extracts is presented in Figure 5(b). The majority of the cell population was not affected by exposure to PEU or PEU-AUAMMT extracts, however membrane permeability was affected by PEU-QACMMT extracts. Specifically, there appeared to be increased membrane damage with increased QACMMT loading, although this was not significant. In vitro cell response assays were also used to assess extracts of the QACMMT nanoparticles compared with the unmodified MMT particles. MMT extraction fluid did not affect cell growth while QACMMT extraction fluid caused significant reducJournal of Biomedical Materials Research Part A

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Figure 4. TEM images at 60,0003 magnification of (a) PEU-QACMMT-1 and (b) PEU-QACMMT-7; (c) PEU-AUAMMT-1 and (d) PEU-AUAMMT-7, with the scale bar representing 200 nm. These high magnification images show the silicate tactoids separating into individual layers.

tion of cell numbers. The entire cell population experienced disruption to the membrane permeability to PI following exposure to QACMMT extraction fluid, except after a hundred-fold dilution of the extract was conducted (data not shown). The results of the repeated extraction assay are presented in Figure 6. PEU-QACMMT materials showed reduced cell growth inhibition with successive extractions over 10 days, however unacceptable levels of inhibition were still present after the tenth successive extraction.


21 min residence time period [see Fig. 7(d)], however these PEU leachate species did not have MW equivalent to that of QAC. PEU-QACMMT-1 and PEU-QACMMT-15 material extraction fluids contained species whose MW matched that of the QAC, with the amount appearing to increase with the QACMMT loading as shown in Figure 7(e,f).

Liquid chromatography mass spectroscopy

Silicate dispersion

Figure 7(a–f) show the partial Liquid Chromatography Mass Spectroscopy (LCMS) data for complete media alone and complete media extracts of MMT and QACMMT nanoparticles, PEU, and PEUQACMMT materials. There was no observable difference between the data for complete media, and MMT extracts [see Fig. 7(a,b)]. The QACMMT extract however was markedly different, with a group of species eluting between
19 and
21 min [see Fig. 7(c)]. Further, peaks were detected when these eluting species’ mass spectrometric data was scanned for presence of species with MW equivalent to that of the QAC. The extraction of PEU controls also resulted in leachables that elute within the
19 to

On the basis of XRD and TEM analysis PEUQACMMT and PEU-AUAMMT materials were concluded to be nanocomposites of partially exfoliated and intercalated morphology, respectively. Contrarily, nanocomposites were not formed by incorporation of MMT without OM into PEU. PEU-MMT materials can thus be described as unintercalated microcomposites. Approximately half of the studies presented in the literature on solution cast PU organosilicate nanocomposites concluded partially exfoliated morphology.16,17,25–27 In TEM images of PEUQACMMT both finely-dispersed groups (exfoliated) and small clumps or tactoids (intercalated) of silicate layers were observed, while in images of PEU-

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DISCUSSION

IN VITRO FIBROBLAST RESPONSE TO PU ORGANOSILICATE NANOCOMPOSITES

Figure 5. The effect of 24 h, 378C complete media extracts of PEU-MMT, PEU-QACMMT, and PEU-AUAMMT materials on (a) the growth of mouse fibroblasts, and (b) mouse fibroblast membrane permeability to PI. Mean 6 1 standard deviation. Number of assay replicates shown below data.

AUAMMT the finely-dispersed groups are less prevalent and silicate-rich tactoid regions appear denser. Favorable thermodynamics resulting from the hydroxyl functionality of both the QAC and AUA were likely the primary driving force for silicate dispersion in both materials, specifically hydrogen bonding between the nanoparticle OM and the hard segment urethane group N and the soft segment C OC.28,29 However, the CH2CH2OH functionality of the QAC likely provides a more energetically favorable, hydrophilic molecular environment near to the QACMMT surface, thus likely encouraging strong intercalation by PEU and molecular interaction of PEU and QAC organic sequences. The position of the AUA polar OH functionality on the molecule end that is farthest from the silicate surface might actually encourage a ‘stratified’ arrangement with overlaying of AUA by PEU chains as opposed to molecular intermingling, since PEU chains are not expected to associate as readily with this region closer to the silicate surface. The observa-

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tion that the organosilicate basal spacing of QACMMT at 1.82 nm was substantially greater than that of AUAMMT at 1.39 nm partly supports this postulation. The significant difference in organosilicate basal spacing is most likely due to the relative amounts of OM exchanged (see Table I), the shorter length of AUA compared to QAC, and possibly a variation in the interlayer conformation adopted by the OM. That is, the two CH2CH2OH arms on the QAC head group likely cause the OM to extend more perpendicularly away from the silicate surface, while the AUA is likely to lie more parallel to the silicate surface. For PEU-AUAMMT materials, the organosilicate basal spacing of AUAMMT at 1.39 nm was not observed by XRD and instead all AUAMMT was expanded to at least
1.7 nm. This was not the same for PEU-QACMMT materials where all materials had some proportion of QACMMT nanoparticles existing in an undispersable state at
1.8 nm layer spacing. It is suggested that this could be due to inconsistent silicate size, shape, charge density, or OM surface coverage. All PEU-AUAMMT and PEUQACMMT materials also displayed a second expanded layer spacing at
2.85 nm and
3.5 nm, respectively. The exception was PEU-QACMMT-1 which displayed only the organosilicate basal spacing of
1.8 nm, and did not display a second expanded spacing. Supported by supplementary TEM evidence, the absence of an expanded peak in PEU-QACMMT-1 indicates a degree of QACMMT dispersion beyond the limit of detection of the XRD. This proposition is also supported by the analysis conducted during this research being extremely rig-

Figure 6. The effect of 24 h, 378C complete media extracts of QACMMT on the growth of mouse fibroblasts. The complete media was collected and then replaced with fresh complete media each day for 10 consecutive days. Also shown is data for control materials Silastic (S) and Latex (L). Mean 6 one standard deviation. Assay was not repeated (n 5 1); standard deviation generated from intraassay replicates. Journal of Biomedical Materials Research Part A

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Figure 7. LCMS data for (a) complete media, (b) MMT extract, (c) the extract from QACMMT alone, (d) PEU extract, and (e and f) PEU-QACMMT-1 and PEU-QACMMT-15 extracts. For each sample, two sets of data are shown; the upper data set corresponds to all eluting species, while the lower data set is specific to the MW of the QAC. Only the region of interest is shown. X-axis is time (min) to elute through chromatography column. Y-axis is amount of substance eluting.

orous in terms of scatter minimization and data collection time (0.088/min, 0.018 step size) when compared with others presented in the literature.16,30,31 That only PEU-QACMMT-1 has all silicate (except the hypothesized undispersable silicate at
1.8 nm) dispersed beyond the XRD detection limit is supported by the theoretical argument of Vaia et al.32 In this study, it was reported that in order for organosilicate to have a chance of achieving complete exfoliation in a given host polymer, spatial restrictions impose that the volume fraction of silicates must be less than 1/aspect ratio. The natural MMT employed here has an average aspect ratio in the order of 100,33 meaning that true exfoliation will only occur at volume fractions of 1% or less where ‘‘overcrowding’’ of the silicate layers will not limit dispersion. In the case of PEU-AUAMMT, the presence of the second expanded silicate spacing for all loadings suggests that no material achieved complete exfoliaJournal of Biomedical Materials Research Part A

tion, which was in agreement with silicate dispersion observed in TEM images. The majority of literature studies on PU nanocomposites utilize a QAC organic modifier, however Han et al.16 recently reported on an AUA-containing nanocomposite comprising a 5 wt % composite of an AUAMMT with a soft grade MDI/polypropylene oxide/BDO PEU. On the basis of the absence of an XRD peak above 1.58 2h and TEM imaging, an exfoliated nanocomposite was concluded. The XRD operating parameters were not detailed in the study and only a high magnification TEM was included. As a result it is difficult to compare between the current study and the study by Han et al. However, the likely cause of the seemingly better silicate dispersion achieved by Han et al. compared with that of this study is the softer PEU grade and the use of ultrasonic energy during nanocomposite processing. Softer polymers often display improved dispersion

IN VITRO FIBROBLAST RESPONSE TO PU ORGANOSILICATE NANOCOMPOSITES

and properties,4,16,30 and ultrasound has been seen in this laboratory to result in damage and scission of the PEU chain. Thus, the current study and that of Han et al. have almost certainly been conducted on significantly different polymer systems and thus cannot be readily compared. Mechanical properties PEU-QACMMT and PEU-AUAMMT materials displayed similar mechanical integrity except that PEUQACMMT had greater mechanical strength at the lower loadings. Both materials displayed properties that were greater than that of PEU-MMT. Reduced ultimate properties for PEU-MMT materials, particularly ultimate stress, was likely due to material defects caused by chemical incompatibility at the interface between PEU and MMT. It is understandable given the proposed partially exfoliated silicate morphology of PEU-QACMMT materials that these materials display the most improved mechanical strength. In general, properties began to decline beyond 2–3 wt % nanoparticle. It is possible that as the silicate loading increases above 2 wt %, the ‘‘overcrowding’’ of silicate layers restricts the dynamic motion of polymer chains predicted to allow polymer stress shielding and stress transfer to the silicate.32 Also, at the higher organosilicate loadings, the poorly dispersed silicate tactoids become more frequent and larger, and it is likely that poor interfacial connections between the PEU and the organosilicate simply lead to a higher chance of void formation and thus poorer tensile properties when under tensile stress. The observed trends upon addition of QACMMT are in agreement with studies by Finnigan et al. on the same PEU nanocomposite system, although in this study the lowest QACMMT loading tested was 3 wt %.27 In vitro fibroblast response Experimental results suggested that the QAC OM caused inhibition of cell growth and loss of cellular membrane integrity. QAC release was strongly suggested by LCMS analysis of extraction fluids, where it was shown that species of equivalent MW to that of QAC molecules were observed only in extractions of materials containing QACMMT. QACs have been shown to interact with cellular membranes rendering the cell incapable of maintaining homeostasis and thus slowing cell growth and even causing cell death.12,34 That QAC leached from the nanocomposites in sufficient quantities to cause cell growth disruption is not surprising. QAC is associated with the silicate via a relatively weak electrostatic interaction

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as opposed to being covalently bound, and the release of small MW substances from similar PU has been studied previously.35 Further, Edwards et al.36 recently reported that excess QAC is present in Cloisite organosilicates due to incomplete washing after the alkylammonium exchange process by the manufacturer, and such an excess would render more QAC available for leaching than expected. Since the strength of AUA interaction with silicate is likely on the same order of magnitude as that for QAC with silicate, and since AUA is a smaller molecule than the QAC it is probable that a similar or even more rapid release pattern occurred for AUA as for QAC. This suggests that AUA released is less cytotoxic than QAC released. At pH above the pI the AUA amine group is neutral as opposed to positively charged, and the carboxyl group is negatively charged, as shown in Figure 1(c). Also, the anti-bacterial activity of QAC is generally considered to be due to the cationic ammonium head group facilitating insertion of the hydrophobic alkyl chain into the cellular membrane, disrupting maintenance of cell homeostasis, and leading to cell death.37 Thus, at pH 7.4 as in cell culture media, the absence of cationic groups on AUA may explain its reduced capacity for insertion in the cellular membrane leading to cell death. Further, the AUA molecule is C11 in length while the QACMMT QAC is C14 to C18 in length, and longer alkyl chain lengths have been shown to have greater anti-bacterial efficacy.38 Although the in vitro cytotoxicity of PEUQACMMT materials has been demonstrated by this study, the potential for successful in vivo application is unknown. In the proposed applications of PU nanocomposites, the release of nanoparticles is unlikely. However, it is clear that the OM must be carefully evaluated for cytotoxicity given the high likelihood of its release from the material in vivo.

CONCLUSIONS OM of MMT nanoparticles is essential to achieve nanoparticulate dispersion. Unmodified MMT did not disperse on the nanoscale, while both QAC and AUA organically modified MMT displayed intercalated or partially exfoliated dispersion. Composites based on silicates dispersed using QACs showed significant cell growth inhibition and disruption of cell membranes. However, an intercalated dispersion with no associated cell growth inhibition was observed for an AUA modified MMT. AUA modified MMT dispersed in PU represents the first generation of nanocomposite elastomers with improved mechanical properties and with potential for in vivo biomedical usage. Journal of Biomedical Materials Research Part A

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The assistance of the following University of New South Wales staff members is appreciated: Sigrid Fraser (Electron microscopy Unit, TEM), Mark Raftery (Bioanalytical Mass Spectroscopy Facility, LCMS), and Yu Wang (Materials Science, XRD).

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