Shape-Memory Bionanocomposites Based on Chitin Nanocrystals

Article pubs.acs.org/Biomac

Shape-Memory Bionanocomposites Based on Chitin Nanocrystals and Thermoplastic Polyurethane with a Highly Crystalline Soft Segment Ainara Saralegi,† Susana C. M. Fernandes,† Ana Alonso-Varona,‡ Teodoro Palomares,‡ E. Johan Foster,§ Christoph Weder,§ Arantxa Eceiza,† and Maria Angeles Corcuera*,† †

‘Materials + Technologies’ Group, Department of Chemical and Environmental Engineering, Polytechnic School, University of the Basque Country UPV/EHU, Plaza de Europa 1, 20018, Donostia-San Sebastián, Spain ‡ Department of Cellular Biology and Histology, Faculty of Medicine and Odontology, University of the Basque Country UPV/EHU, B Sarriena, s/n, 48940, Leioa-Bizkaia, Spain § Adolphe Merkle Institute and Fribourg Center for Nanomaterials, University of Fribourg, Route de l′Ancienne Papeterie, CH-1723 Marly, Switzerland

ABSTRACT: Shape-memory bionanocomposites based on a naturally sourced segmented thermoplastic polyurethane and chitin nanocrystals were synthesized, and their mechanical properties and thermally activated shape-memory behavior were studied. The chitin nanocrystals were incorporated during the synthesis of the prepolymer made from a castor oil-based difunctional polyol and hexamethylene diisocyanate. The polymerization was completed by addition of propanediol, as a cornsugar based chain extender, bringing the weight content of components from renewable resources to >60%. Thermal analysis of the bionanocomposites revealed a phase-separated morphology, which is composed of soft and hard domains, which bestow the material with two melting transitions at 60 and 125 °C, that are exploitable for a shape memory effect. The soft segment is responsible for temporary shape fixing, while the hard segment crystallites are responsible for the permanent shape. The introduction of small amounts (0.25−2 wt %) of chitin nanocrystals was found to increase the crystallinity of the hard segment by way of nucleation, which in turn improves the shape recovery considerably. The thermally activated shape-memory behavior of the synthesized bionancomposites is exploitable with a programming and release temperature of 60 °C. The materials display good in vitro cell response, as shown by short-term cytotoxicity assays, and therefore, the bionanocomposites appear to be potentially useful for biomedical applications.

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industrial applications, such as adaptive medical devices,9−11 implants for minimally invasive surgery,12,13 sensors and actuators,14,15 and heat-shrink tubing and films.16 Segmented thermoplastic polyurethanes (STPUs)17−19 represent an important family of shape-memory polymers. These physically cross-linked semicrystalline block copolymers are composed of soft segments (SS), usually formed by a polyether or polyester macroglycol, and hard segments (HS)

INTRODUCTION

In the last decades, the interest in stimuli-responsive or ‘smart’ materials, which have the ability to change their properties in response to an external stimulus or to changes in the surrounding environment, has grown considerably, due to their potential application in a multitude of active structures and devices.1 Shape-memory polymers represent a subset of smart materials that can memorize temporary shapes and revert to their permanent shape upon exposure to an external stimulus such as heat,2 light,3−5 moisture,6,7 or a magnetic field.8 Their ability to change their shape under a predetermined stimulus makes them attractive for advanced biomedical devices and © 2013 American Chemical Society

Received: September 16, 2013 Revised: October 31, 2013 Published: November 4, 2013 4475

dx.doi.org/10.1021/bm401385c | Biomacromolecules 2013, 14, 4475−4482

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the soft segment was poly(butylene sebacate)diol (CO2) with a number-average molecular weight of 1900 g mol−1, derived from castor oil and consisting of sebacic acid copolymerized with 1,4-butanediol, already used in previous studies.21,22 Isolation of α-Chitin Nanocrystals. α-Chitin nanocrystals were isolated by acid hydrolysis of chitin powder using 3 M HCl at 100 °C for 90 min under vigorous stirring and refluxing, in order to hydrolyze the amorphous regions of the chitin. The ratio of HCl to chitin powder was kept at a concentration of 30 mL g−1.37 After acid hydrolysis, the suspension was diluted 10fold with distilled water and washed by centrifugation, removing the supernatant and replacing by the same amount of distilled water in each centrifugation, until the supernatant was colorless. Afterward, the suspension was transferred to dialysis membranes (Spectra Por 6 MWCO 8000) and dialyzed for 5 days, changing the water every 12 h. To breakup any remaining chitin nanocrystal aggregates, the suspension was subjected to ultrasonic treatment in a horn sonicator operated at 20% amplitude for 10 min (Vibracell 75043 from Bioblock Scientific). Finally, a loose powder was obtained by freezedrying the CHNC suspension at a concentration of 0.05 mg mL−1. The degree of acetylation of the α-chitin sample was estimated by elemental analysis,40 obtaining a value of 92%, and this data was corroborated by 13C NMR, according to the method used by Duarte et al.41 Synthesis of Polyurethane Bionanocomposites. The synthesis of polyurethane bionanocomposites with CHNC was carried out by an in situ polymerization process according to a previously established procedure.31 In the first step, a prepolymer was synthesized from a reaction between HDI and CO2 at 100 °C for 6 h using THF as solvent (the concentration of the resulting prepolymer was 75 mg mL−1), in the presence of CHNCs, dispersed before the reaction by sonication. In the second step, the chain extender, 1,3propanediol, was added in stoichiometric ratio (determined by titration of remaining isocyanate groups) to the prepolymer (the total NCO/OH ratio in the bionanocomposite was equal to 1), and the mixture was stirred for 2 h at 100 °C. Finally, the mixture was poured into a Teflon dish, and was cured at 60 °C for 48 h under vacuum, to remove the solvent. The hard segment (HS) content of as-synthesized bionanocomposites was 17 wt %, when using a molar ratio of 1:2:1 of the starting materials (polyol:diisocyanate:chain extender). As a comparison, neat polyurethane was synthesized using a similar procedure without adding CHNCs. Therefore, neat polyurethane, denoted as STPU17, and bionanocomposites with different CHNC contents ranging from 0.25 to 2 wt % were synthesized and denoted as STPU17/CHNC-X, where X is the amount of CHNCs added (with respect to the total weight of polyurethane bionanocomposite (polyol + diisocyanate + chain extender + CHNCs) and expressed as weight percentage (wt %)). The obtained films were used as-synthesized for thermal and mechanical characterization. Characterization Techniques. Atomic force microscopy (AFM) images of the isolated CHNCs were performed in tapping mode on a Nanoscope IIIa scanning probe microscope (Multimode Digital Instruments) equipped with an integrated silicon tip/cantilever (125 μm in length and with ca. 300 kHz resonant frequency). The measurements were performed at a scan rate of 1.0 Hz, using a scan head with a maximum range of 16 μm × 16 μm. Several regions were scanned and images with similar features were obtained. For sample preparation, a droplet of dilute CHNC suspension obtained by acid hydrolysis

formed from the reaction of a diisocyanate with a low molecular weight diol, referred to as chain extender.20−22 Due to the thermodynamic incompatibility between the soft and hard segments, these polyurethanes phase separate and are good candidates for shape-memory applications. The SS can act as the switching segment (responsible for shape fixity), which requires that they exhibit a glass transition or melting transition in a suitable temperature range. On the other hand, HSs serve as net points and provide the rubber elasticity required for shape recovery.23−26 Polyurethanes generally exhibit high biocompatibility, which has triggered substantial interest in polyurethane shape-memory materials for biomedical applications.27−29 We recently studied the thermally activated shape-memory effect in a family of biobased thermoplastic polyurethanes made from castor oil-derived polyols, corn-sugar-derived propanediol (PD) as a chain extender, and hexamethylene diisocayanate.30 These segmented polyurethanes were synthesized in bulk with a content of renewable matter over 60 wt %, which is very attractive from the viewpoint of sustainability.21 The soft segment (phase formed by the polyol) is semicrystalline and displays a melting point around 60 °C, which is ideal for biomedical applications where damaging of surrounding tissue by application of 60 °C is not a crucial issue, such as bone tissue engineering and wound dressing.27−29 An in-depth investigation of the shape-memory properties of a series in which the content of hard and soft segments was systematically varied revealed outstanding shape fixity (>97%, imparted by the crystalline soft segments) at low hard segment contents, while shape recovery values were not as good (50%) for these compositions. At low hard segment contents, polyurethanes do not contain a sufficiently large number of physical cross-links formed by the hard segments, so that the driving force for shape recovery is low. We show here that the introduction of small amounts (0.25−2 wt %) of chitin nanocrystals (CHNC) leads to a significant improvement of the shape memory properties, on account of a nucleating effect that is imparted to the hard segment phase. The approach was used before in shape-memory nanocomposites of polyurethanes with cellulose nanocrystals,31−35 but instead of using cellulose nanocrystals as renewable nanofillers for reinforcement like most works in the literature do, chitin nanocrystals were used in this study. CHNCs have excellent properties including biocompatibility, biodegradability, lack of toxicity, and advantageous absorption properties.36−39 Therefore, the final properties of the bionanocomposites can be improved, both in terms of mechanical properties and biocompatibility, over their pure polymer counterparts. To this end, bionanocomposites with different CHNC contents were synthesized by in situ polymerization, and the thermal, mechanical and shapememory properties were evaluated, as well as preliminary studies of cellular response and cytotoxicity.

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MATERIALS AND METHOD Materials. α-Chitin powder from Mahtani Chitosan PVT. Ltd. (India) was used as the CHNC source. Hydrochloric acid (HCl) used for acid hydrolysis was purchased from SigmaAldrich (37% w/w) and further diluted. Tetrahydrofuran (THF) purchased from Sigma-Aldrich (>99.9%) was used as solvent. 1,6-Hexamethylene diisocyanate (HDI) was kindly supplied by Bayer under the trade name Desmodur H. 1,3Propanediol (PD) derived from corn-sugar and used as a chain extender was provided by Quimidroga SA. The polyol used as 4476


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Figure 1. Chemical structure (a) and AFM height (b) and phase (c) images of rod-like chitin nanocrystals; height profile (d) related to the red line in the height AFM image.

did not add any extra information, it was not included in this work. Dynamic mechanical analysis (DMA) and thermally activated shape-memory (TASM) properties of the neat polyurethane and STPU/CHNC bionanocomposites were carried out in tensile mode on an Eplexor 100 N analyzer from Gabo and on a DMA Q-800 from TA Instruments, respectively. Samples were prepared by cutting strips from the synthesized films with a width of 4 mm. For DMA measurements, a constant frequency of 10 Hz and a scanning rate of 2 °C min−1 from −100 to 150 °C was used. The TASM properties were measured as follows: first of all, samples were heated and equilibrated at 60 °C, above the soft segment melting temperature. Then, a force ramp of 0.1 N min−1 was applied until a strain of 50% was reached. Subsequently, maintaining the stress applied, samples were cooled to 15 °C at a rate of 5 °C min−1, and the stress was released while the temperature was maintained at 15 °C. Finally, samples were heated at a rate

was deposited on mica by spin-coating at 2000 rpm for 130 s, and residual water was evaporated at ambient temperature. The dispersion of CHNC in the synthesized STPU17/ CHNC bionanocomposites was characterized by AFM, as described earlier. Samples were cross-sectioned using a Leica EM FC6 cryo-ultramicrotome equipped with a diamond knife and operating at −120 °C. Differential scanning calorimetry (DSC) measurements of the synthesized neat polyurethane and bionanocomposites were performed using a Mettler Toledo 822e instrument, equipped with a robotic arm and with an electric intracooler as refrigeration unit. The thermal behavior of the samples was evaluated (approximately 10 mg of sample sealed in an aluminum pan) under a constant dry nitrogen atmosphere from −60 to 220 °C, at a scanning rate of 20 °C min−1. The crystallization process was also followed by cooling the samples from 220 to −60 °C at a scanning rate of 10 °C min−1. Moreover, a second heating run was also performed, but as it 4477


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of 5 °C min−1 to the recovery temperature of 60 °C. More than one cycle was applied to each sample in order to probe any hysteresis of the shape-memory properties of the synthesized neat polyurethane and bionanocomposites. Measuring the length of the samples at the different stages of each cycle and using eqs 1 and 2, strain fixity (Rf) and strain recovery (Rr) values were determined, which are characteristic values for any shape-memory polymer.17−19 R f (N ) =

ε u (N ) × 100 εm(N )

(1)

R r(N ) =

εm(N ) − εf (N ) × 100 εm(N ) − εf (N − 1)

(2)

assays were conducted in triplicate and mean values and their standard deviations were estimated.

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RESULTS AND DISCUSSION Shape-memory bionanocomposites based on chitin nanocrystals (CHNCs) and segmented thermoplastic polyurethane (STPU) were synthesized by in situ polymerization, adding different amounts of CHNCs (0.25−2 wt %). CHNCs were isolated by acid hydrolysis, using HCl. A semicrystalline polyol derived from castor oil (CO2) and a corn-sugar based chain extender (propanediol, named as PD) were used as starting materials in order to maximize the content of carbon from renewable resources. The bionanocomposite films obtained after polymerization of the reaction mixture under pressure at 60 °C for 48 h were used as-synthesized. Morphology of CHNCs and Polyurethane Bionanocomposites. The structure and size distribution of isolated CHNCs deposited from a dilute solution were analyzed by AFM (Figure 1). The micrographs show indeed nanocrystals on the nanometer scale with rod-like morphology. In order to eliminate the effect of tip radius on width measurements,43 the diameter of the CHNCs was determined from the height profiles (Figure 1d), according to the Nanoscope V analysis software, resulting in an average diameter of 11.1 ± 1.4 nm and a length of 176 ± 34 nm, corresponding to an average aspect ratio of 15.8 ± 4.1. AFM was also used to characterize the dispersion of CHNCs in the synthesized bionanocomposites. Figure 2 shows height

where εm is the maximum strain reached in the cyclic tensile test, εu is the fixed strain of the film unloaded and stabilized at 15 °C, εf is the final strain of the sample after shape recovery, and N is the number of cycles. Stress−strain measurements of STPU17/CHNC bionanocomposites and neat polyurethane were performed using a MTS Insight 10 instrument with a load cell of 250 N. Dogbone-shaped specimens were used for tensile tests, according to ASTM D 1708-93 standard. Five replicates of each material were used to measure Young’s modulus (E), yield strength (σy), tensile strength at maximum elongation (σmax), and strain at break (εb), calculated from the load−displacement data, where the deformation was measured by the crosshead displacement. Moreover, toughness in the elastic region (8% strain) and total toughness were also measured integrating the area under stress−strain curves obtained by tensile tests. Finally, to assess in vitro cell response, short-term cytotoxicity evaluation of the polyurethane bionanocomposites was carried out using L-929 murine fibroblast cells, following ISO 10993 recommendations.42 To prepare extracts of test materials, samples with an area of 6 cm2 were rinsed with MilliQ water, sterilized with 100% ethylene oxide gas, and allowed at least 7 days to degas. Sterilized film samples were incubated separately in standard cell culture medium (Dulbecco’s modified Eagle’s medium [Sigma Chemicals Co, USA] plus 10% fetal calf serum [Gibco] and supplemented with antibioticantimycotic solution [Sigma]) at 37 °C for 24 h to obtain the extracted culture media. In addition, L-929 murine fibroblasts were seeded and allowed to grow in 96-well microplates at a density of 4 × 103 cells/well in the presence of standard culture medium for 24 h before the experiments. In the cytotoxicity test, cultures were treated for 24, 48, and 72 h with the extracted media. As controls, standard culture medium (control) were used, high-density polyethylene (HDPE, negative control, USP Rockville, USA), and polyvinyl chloride (PVC, positive control, Portex, UK). To evaluate cell viability and proliferation, metabolic activity of viable cells was determined using the colorimetric assay MTT (Cell Proliferation Kit I MTT, Roche). This test is based on reduction of 3-(4,5-dimethyltriazol-2-yl)-2,5 diphenyltetrazolium bromide on formazan in the mitochondria of living cells. The cell number per well was proportional to the amount of formazan crystals and was determined by measuring the absorbance at 540 nm using a microplate reader (ELISA). Viability (%) was calculated as following: [A]test/[A]control × 100, where [A]test is the absorbance of the sample cells and [A]control is the absorbance of the negative control cells.42 All

Figure 2. Height (left) and three-dimensional (right) AFM images of the neat polyurethane (STPU17) (a) and the bionanocomposite STPU17/CHNC-2 (b), obtained from cryo-ultramicrotomed sample cross section.

and three-dimensional AFM images of STPU17 and STPU17/ CHNC-2, obtained from cryo-ultramicrotomed sample cross section, where an effective CHNC dispersion (white dots) can be observed (Figure 2b). The reaction between hydroxyl groups from CHNC surface and isocyanate groups from polyurethane precursors, either HDI or prepolymer, resulted in polyurethane chains anchored to CHNCs, improving the dispersion of nanocrystals in the polyurethane.31 Furthermore, the addition of CHNCs caused an increase in bionanocomposites surface roughness. Thermal Analysis. The thermal properties of the neat polyurethane and the bionanocomposites, were studied by differential scanning calorimetry (DSC). The DSC traces show endothermic transitions around 60 and 125 °C, related to soft 4478


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pronounced sharp exothermic peak associated with soft phase crystallization around 30 °C, a weak exothermic signal associated with the crystallization of the hard phase around 75−80 °C, which increased in temperature and enthalpy with increasing CHNC content, confirming that CHNCs indeed act as nucleation agents for the hard phase. Dynamic mechanical analysis experiments (Figure 4) were conducted to probe the storage modulus (E′) and tan δ of the

phase (TmSS) and hard phase melting temperatures (TmHS), respectively. Table 1 summarizes the melting temperatures and Table 1. Soft Segment Melting Temperature (TmSS) and Enthalpy (ΔHmSS), and Hard Segment Melting Temperature (TmHS) and Enthalpy (ΔHmHS), As Well As the Relative Crystallinity Values of the Soft (χc(SS)) and Hard (χc(HS)) Segments Obtained from DSC Measurements for the Neat Polyurethane (STPU17) and the Bionanocomposites Synthesized by in Situ Polymerization sample STPU17 STPU17/ CHNC-0.25 STPU17/ CHNC-0.5 STPU17/ CHNC-0.75 STPU17/ CHNC-1 STPU17/ CHNC-2

TmSS (°C)

ΔHmSS (J g−1)

χc(SS)

TmHS (°C)

ΔHmHS (J g−1)

χc(HS)

62.5 62.1

51.2 49.6

1.0 1.0

120.3 121.8

8.0 8.6

1.0 1.1

60.8

49.7

1.0

123.4

9.0

1.2

59.4

48.3

0.9

122.6

9.7

1.3

61.0

46.9

0.9

124.3

12.0

1.5

60.8

47.4

0.9

126.4

12.4

1.6

enthalpies for these transitions, as well as the relative crystallinity values (relative crystallinities to the neat polyurethane, without CHNCs) calculated by eq 3:44

χc =

ΔHm ω·ΔH100

Figure 4. Storage modulus (E′) and tan δ as a function of temperature for films of neat polyurethane (STPU17) and the bionanocomposites.

(3)

synthesized materials as a function of temperature and composition. All DMA curves follow the same general trend and exhibit high E′ values of 2000−3000 MPa below the glass transition of the soft phase (which appears around −30 °C). The introduction of CHNCs led to bionanocomposites with higher storage moduli (the E′ for STPU17 at −75 °C is 1685 MPa, and for STPU17/CHNC-2 it is 2615 MPa), due to the nucleation of the hard phase, as observed by DSC (Figure 3). Furthermore, due to the nucleation effect, the maximum in tan δ, associated with the glass transition temperature of the soft phase, increased in temperature and decreased in intensity with increasing CHNC content. Moreover, a broadening of the peak was observed, because both ordered hard domains and CHNCs restrict amorphous SS chain mobility.46 The sharp decrease in E′ values observed around 50 °C is associated with soft phase melting temperature. Before soft phase melting temperature (at 45 °C), all materials display E′ values of 150−390 MPa, and after TmSS (at 75 °C), they display E′ values of 30−65 MPa. For the neat polyurethane and the bionanocomposites, E′ values drop 20% upon melting of the soft phase, corroborating the results obtained by DSC (constant χc(SS) values in Table 1). Above TmSS, a rubbery plateau in storage modulus is observed, with higher E′ values for the bionanocomposites with high CHNC contents (E′ values of 30 MPa for STPU17 and of 65 MPa for STPU17/CHNC-2), due to the nucleation effect of the hard phase. Finally, at high temperatures, a marked decrease of E′ is observed, owing to the disruption of hard crystalline domains. The addition of CHNCs led to higher thermomechanical stability (the dramatic decrease for STPU17 starts around 120 °C and for STPU17/CHNC-2 around 130 °C), due to the nucleation effect of the hard phase, as observed by DSC. It was observed in the literature that the addition of cellulose nanocrystals lead to thermoset polymers,47 because nanocrystals tend to act as chemical cross-linkers. Nevertheless, in this

where ΔH100 is the heat of fusion of the corresponding phase, soft or hard, without CHNCs and determined by DSC, ω is the weight fraction of polymeric material in the bionanocomposites, and ΔHm is the experimental melting enthalpy value obtained by DSC (Table 1). It can be observed that the melting temperature (TmSS) and relative crystallinity (χc(SS)) of the soft phase, remained nearly constant, independent of the amount of CHNCs incorporated. On the other hand, hard phase melting temperature (TmHS) and relative crystallinity (χc(HS)) values increased upon addition of CHNCs, suggesting a nucleation effect of the nanocrystals, as was also observed by other authors for polyurethane and poly(L-lactide)-based nanocomposites.31,32,45,46 This effect was corroborated by performing DSC cooling scans (Figure 3). The traces show, besides the

Figure 3. DSC cooling scans (cooling rate= 10 °C min−1) for the neat polyurethane (STPU17) and the bionanocomposites. 4479


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Table 2. Mechanical Properties of the Neat Polyurethane (STPU17) and the Bionanocomposites sample STPU17 STPU17/CHNC-0.25 STPU17/CHNC-0.5 STPU17/CHNC-0.75 STPU17/CHNC-1 STPU17/CHNC-2

E (MPa) 238.8 253.7 269.5 279.8 288.8 321.5

± ± ± ± ± ±

6.7 6.8 7.7 7.5 7.6 8.2

σy (MPa) 10.3 11.1 11.8 12.7 13.2 14.9

± ± ± ± ± ±

0.4 0.5 0.4 0.4 0.6 0.5

σmax (MPa) 26.5 16.9 15.4 15.6 14.2 11.5

± ± ± ± ± ±

2.3 1.4 1.1 1.4 1.3 1.2

Ε (%) 762 470 430 406 420 65

± ± ± ± ± ±

42 24 25 22 24 7

resilience (MJ m−3) 0.94 1.36 1.37 1.40 1.43 1.54

± ± ± ± ± ±

0.05 0.06 0.05 0.05 0.04 0.07

total toughness (MJ m−3) 103.6 56.9 47.1 40.5 41.7 8.60

± ± ± ± ± ±

10.3 5.3 4.8 4.1 3.8 0.5

imposed by HS crystallites. Therefore, the SS is responsible for shape fixity and HS for shape recovery.19,23 Figure 5

work, due to the low amount of nanocrystals added and due to the lower amount of hydroxyl functional groups that chitin nanocrystals present, comparing with cellulose nanocrystals, CHNCs act as nucleation agents rather than chemical crosslinkers, obtaining thermoplastic bionanocomposites even with 2 wt % of CHNCs. Therefore, all the synthesized bionanocomposites presented the before mentioned dramatic decrease in E′ values after the melting temperature of the hard phase, typical behavior presented by thermoplastic materials. Mechanical Properties. Table 2 summarizes the mechanical data obtained by tensile tests, including Young’s modulus (E), yield strength (σy), tensile strength at maximum elongation (σmax), strain at break (εb), and toughness. It can be observed from the values in the table that the addition of CHNCs leads to bionanocomposites with higher Young’s modulus and yield strength values, and therefore toughness in the elastic region increases, as it is a direct measurement of the area under stress−strain curves in the elastic region. CHNCs act as nucleation agents for the hard phase, increasing hard phase crystallinity and therefore material stiffness, thus increasing E, σy, and toughness in the elastic region (resilience), and decreasing strain at break values. Moreover, the use of highly crystalline SS leads to stiffer materials (E values of 240−320 MPa), comparing with the data obtained in previous works of the group (E values of 20−35 MPa) for polyurethanes based on crystalline HS (1,6-hexamethylene diisocyanate-1,4-butanediol) but poorly crystalline SS poly(caprolactone-b-polytetradydrofuran-b-polycaprolactone)diol).31 On the other hand, σmax and total toughness decrease with the addition of CHNCs. The addition of CHNCs and the nucleation effect of hard phase restrict amorphous SS orientation and crystallitzation under strain, decreasing σmax values and therefore the total area under the stress−strain curve, which is a direct measurement of the toughness. Finally, regarding strain at break values, as can be observed in Table 2, bionanocomposites with 2 wt % of chitin nanocrystal content present a huge drop in εb values, due to the nucleation effect of the HS phase and the formation of a physically crosslinked network of hard domains, which restricted the mobility of the amorphous segments, decreasing the elongation at break values. A similar behavior was observed for polyurethane nanocomposites synthesized with similar cellulose nanocrystal contents,31 as well as for polyurethanes synthesized with high hard segment contents.21 Thermally Activated Shape-Memory Properties. In order to assess the thermally activated shape-memory properties of the neat polyurethane and the synthesized bionanocomposites, thermo-mechanical cyclic tensile tests were performed, as detailed in the experimental section. The temperature selected for shape memory testing (i.e., programming and release) was 60 °C, a temperature at which the materials show a rubbery plateau, due to the mobility of the amorphous phase of the SS and the restricted molecular motion

Figure 5. Shape fixity (a) and shape recovery (b) values obtained for the first two shape-memory cycles for the neat polyurethane (chitin nanocrystal content= 0%) and the bionanocomposites as a function of chitin nanocrystal content.

summarizes the values determined for shape fixity (Rf) and shape recovery (Rr) in the first two thermo-mechanical cycles. On one hand, as can be observed in Figure 5a, the shape fixity remained nearly constant at a very high level of 97−98%, irrespective of the amount of CHNCs added. This is consistent with the observation made by DSC and DMA that the addition of CHNCs did not affect SS crystallinity, the segment responsible for shape fixity. The Rf values determined in the first and second thermo-mechanical cycles were almost nearly constant, because the soft segment crystallizes efficiently and does not show a dependence of thermo-mechanical history, which is important to retain its function as switching segment for several cycles.25,35 It can be observed from Figure 5b, that the most important benefit of the addition of CHNCs is the substantial increase of 4480


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the Rr values in both first and second thermo-mechanical cycles. Owing to the low HS content of STPU17, the shape recovery of the neat polyurethane in the first thermo-mechanical cycle was quite low (52%), because the HS content was not sufficiently high to provide enough network points in the form of physical cross-links and thereby the elasticity required to restore the original shape.48 Nevertheless, with the addition of CHNCs, significant improvement in Rr was observed, due to the nucleation effect of hard phase, increasing physical crosslinks and thereby Rr values. For the first thermo-mechanical cycle, Rr values increased from 52% (neat polyurethane) to 67% (0.25 wt % of CHNCs) and 74% (2 wt % of CHNCs). On the other hand, for the second thermo-mechanical cycle, Rr values increased from 53% (neat polyurethane) to 87% (0.25 wt % of CHNCs) and 98% (2 wt % of CHNCs). As was observed in previous studies,30 plastic and nonreversible deformation occurs during the first thermo-mechanical cycle. Nevertheless, in the second thermo-mechanical cycle, less plastic and nonreversible deformation occurs (a phenomenon known as hysteresis), resulting in higher Rr values. In both cases (first and second thermo-mechanical cycles), the biggest jump in Rr values was observed for low CHNC contents, and therefore bionanocomposites with outstanding shape-memory properties were obtained adding small amounts of CHNCs. Finally, Figure 6 presents the stress−strain-temperature diagram for several consecutive shape-memory cycles, starting

Figure 7. Absorbance at 540 nm versus incubation time of a positive control (PVC), negative control (HDPE), the neat polyurethane (STPU17), and the bionanocomposite STPU17/CHNC-1 (a) and viability of L-929 murine fibroblast cells as a function of incubation time (b).

proliferate. By contrast, both neat polyurethane and STPU17/ CHNC-1 bionanocomposite displayed markedly nontoxic cell growth, similar to the negative control. Quantification of the cytotoxic response is shown in Figure 7b, where the cell proliferation with respect to the negative control is represented (viability) as a function of incubation time. It is clearly observed that cell viability was higher than 70% in the first 24 h, and indicative nontoxicity. However, after the first 24 h, all samples presented a less pronounced cell proliferation in comparison with the values obtained for negative control, but they still present a greater similarity to the negative control behavior than for positive control. Moreover, ISO 10993-12 only takes into account the data obtained in the first 24 h. Therefore, cytotoxicity studies revealed that the as-synthesized bionanocomposites with excellent thermally activated shape-memory properties are good candidates to use as smart materials in biomedical applications.

Figure 6. Stress−strain-temperature diagram for several consecutive shape-memory cycles, starting with the third cycle, for STPU17/ CHNC-1.

with the third cycle, for STPU17/CHNC-1 (similar results were obtained for the rest of the bionanocomposites, but they are not included in this work for simplification), corroborating cyclic shape-memory properties, as values close to 100% were obtained for Rf and Rr. In Vitro Cell Response Evaluation. Short-term cytotoxicity assays were performed to determine changes in cellular growth, incubating L-929 murine fibroblast cells over 24, 48, and 72 h at 37 °C in the prepared samples. Figure 7a shows the absorbance measurements determined by colorimetric assay versus incubation time for both positive (PVC) and negative (HDPE) controls, as well as for STPU17 and STPU17/ CHNC-1. PVC shows a remarkably toxic effect, as the positive control, wherein the L-929 murine fibroblast cells are unable to

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CONCLUSIONS Bionanocomposites with thermally activated shape-memory properties were synthesized based on chitin nanocrystals and biobased segmented thermoplastic polyurethane by in situ polymerization, where AFM images confirmed an effective dispersion of CHNCs. Thermal properties were measured by DSC, observing a nucleation effect of CHNCs on the hard phase, increasing the melting temperature and crystallinity of 4481


Biomacromolecules

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the hard phase with the addition of CHNCs. However, soft segment crystallinity remained constant, regardless of CHNC content. Regarding thermally activated shape-memory properties, neat polyurethane (STPU17) presented poor shapememory properties, with shape recovery values around 50%. Nevertheless, with the addition of CHNCs, an increase of physical cross-links among hard domains was observed, due to the nucleation effect of CHNCs on the hard phase, increasing shape recovery values and reaching values close to 100% for the second thermo-mechanical cycle. Additionally, polyurethane bionanocomposites synthesized with CHNCs displayed nontoxic behavior, similar to the neat polyurethane and the negative control. Therefore, owing to the shape-memory properties as well as to the good in vitro cell response that the synthesized polyurethane bionanocomposites presented, they seem to be good candidates for use in biomedical applications as smart materials.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (+34)943017186; fax: (+34)943017130; e-mail address: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Basque Government in the frame of Grupos Consolidados (IT-776-13) is gratefully acknowledged. Additionally, A.S. thanks Eusko Jaurlaritza/Gobierno Vasco for ‘Programas de becas para formación y perfeccionamiento de personal investigador’ (BFI-09-167) and for the doctoral mobility grant for accomplishing a 3 months stay at Adolphe Merkle Institute. E.J.F. and C.W. would like to gratefully acknowledge the support from the Adolphe Merkle Foundation. Moreover, technical support provided by SGIker (UPV/ EHU, MINECO, GV/EJ, ESF) is gratefully acknowledged.

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REFERENCES

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