Guanidine hydrochloride embedded polyurethanes ... - KUNDOC.COM

Materials Science and Engineering C 59 (2016) 1025–1037

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Guanidine hydrochloride embedded polyurethanes as antimicrobial and absorptive wound dressing membranes with promising cytocompatibility Maryam Sahraro, Hamid Yeganeh ⁎, Marziyeh Sorayya Polyurethane Department, Iran Polymer and Petrochemical Institute, P.O. Box: 14965/115, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 17 June 2015 Received in revised form 4 November 2015 Accepted 13 November 2015 Available online 14 November 2015 Keywords: Wound dressing Antimicrobial Polyurethane Poly(hexamethylene guanidine hydrochloride)

a b s t r a c t Preparation and assessments of novel absorptive wound dressing materials with efficient antimicrobial activity as well as very good cytocompatibility were described in this work. An amine terminated poly(hexamethylene guanidine hydrochloride) was prepared and used as curing agent of different epoxy-terminated polyurethane prepolymers. The structures of prepared materials were elucidated by evaluation of their 1H NMR and FTIR spectra. The recorded tensile strength of membranes confirmed the excellent dimensional stability of the film type dressings even at fully hydrated conditions. Therefore, these dressings could protect the wound bed from external forces during the healing period. The structurally optimized dressing membranes could preserve the desired moist environment over the wounded area, as a result of their balanced equilibrium, water absorption and water vapor transmission rate. Therefore, a very good condition for stimulation of self-healing of wound bed was attained. Also, owing to the presence of guanidine hydrochloride moieties embedded into the structure of dressings, efficient antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans were detected. In vitro cytotoxicity assay of the prepared dressings revealed cytocompatibility of these materials against fibroblast cells. Therefore, they could support cell growth and proliferation at the wounded area. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Skin generally needs to be covered with a suitable dressing immediately after it is damaged [1]. Unfortunately, there is no single dressing suitable for all types of wounds [2]; therefore, to design an effective wound dressing the condition of wound and its surrounding skin should be considered [3]. High performance wound dressings should provide warm and moist environment over the wound bed [4,5], and enable effective oxygen circulation, absorbing excess exudates from the wound without leakage to the surface of dressing and allowing water vapor transmission at an effective rate to prevent wound desiccation. The dressing should also be non-adherent to the wounded tissue to prevent further damages during changing the dressing [6]. Ultimately, it should provide good mechanical protection [7,8]. It is now widely accepted that preserving moist environment over wound bed can increase the chance of bacteria growth and infection, since wound exudates are enriched with nutrients. Therefore, for exuding wounds, application of dressings with antimicrobial function is preferred [9]. Different antimicrobial agents were utilized to impart

⁎ Corresponding author. E-mail address: [email protected] (H. Yeganeh).

http://dx.doi.org/10.1016/j.msec.2015.11.038 0928-4931/© 2015 Elsevier B.V. All rights reserved.

antimicrobial activity in the dressing membranes. Oxidants, such as peroxides [10], cationic biocides like quaternary ammonium [11,12] and phosphonium salts [13], guanidinium and biguanidinium chloride salts [14–17], halamines and halamides [18] as well as heavy metals such as silver [19,20] are common antimicrobial agents used for preserving wounds against bacterial or fungal infections. Among cationic biocidals, guanidine hydrochloride containing materials with broad-spectrum effectiveness against Gram-positive/Gram-negative bacteria and fungal strains has attracted interest [21–23]. More importantly, this class of water soluble biocidals showed low toxicity against mammalian cells [21,24–26]. For instance, a biocompatibility index greater than 1 was reported for poly(hexamethylenebiguanide) (PHMBG) [27]; therefore, this material was impregnated into different wound dressing membranes [14,17,28] such as Kendall® A.M.D., Telfa® A.M.D., Excilon® A.M.D. and Kerlix® A.M.D. It is worth to mention that gradual release of impregnated biocidals may cause problems such as possible penetration into the human body and entry into blood circulation [29]. Chemical bonding of biocidal moieties to the backbone of wound dressings is an efficient method for resolving this possible problem [29]. The physical state of wound dressings can also influence their performance considerably. If there are no high amounts of wound exudates, foam type dressings may cause wound bed desiccation. In contrast, maceration of the periwound due to saturation of foam dressings may occur,

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if such foam type dressings were used for highly exuding wounds [2]. Dry cotton gauze type dressings have also their own limitations such as low fluid handling capacity and possible adherence to wound bed [30]. Thin film type nonporous dressings with proper wound exudate absorption and water vapor transmission rate are the best candidate for maintaining moist environment over the wounded area. Surprisingly, preparation of film type polymeric wound dressing membranes containing chemically anchored guanidinium active moieties with proper physico-mechanical and biological properties was rarely considered. Therefore, preparation of a novel category of such wound dressings with permanent antibacterial activity, good fluid handling capacity, fine tensile strength even at hydrated state and high biocompatibility was considered in this work. To fulfill these goals, epoxy-terminated polyurethane prepolymers (EPUs) were prepared. An oligomeric amine terminated poly(hexamethylene guanidine hydrochloride) (PHMG) was also synthesized and used for simultaneous crosslinking and introduction of antimicrobial guanidinium chloride moieties in the backbone of wound dressing membranes. All the prepared materials were fully characterized and potential suitability of these membranes for use as wound dressings was evaluated through assessments of their physical, mechanical and biological properties.

2. Experimental

Table 1 Different formulations of EPUs. Sample

GEPU GDEPU

PEG (g)

50.00 50.00

HDI (g)

16.82 16.82

Glycidol (g)

GDGE (g)

– 21.50

7.86 –

Epoxy content (mol kg−1) T

E

1.35 2.66

0.99 1.99

T: theoretical, and E: experimental values.

2.3. Synthesis of amine-terminated poly(hexamethylene guanidine hydrochloride) (PHMG) PHMG was synthesized by a two-step polymerization method according to the reported procedure with minor modification [21]. GHC (10.10 g, 0.1 mol) and HMDA (18.62 g, 0.15 mol) were placed into the three-necked round-bottomed flask equipped with a mechanical stirrer, a thermometer, a gas outlet and an oil bath. The mixture reacted at 120 °C for 2 h, and then at 160 °C for 5 h. During the reaction, the evolved ammonia by-product gas was neutralized by bubbling through an aqueous HCl solution. At the end of reaction, the slightly yellow, viscous liquid product was solidified upon cooling. It was then placed in a vacuum oven at 80 °C for one day to remove any residual ammonia.

2.1. Materials Poly(ethylene glycol) (PEG, Mn 1000 g mol−1, Merck) was dried at 80 °C under vacuum for 24 h. Trace of moisture was removed from this polyol through azeotropic distillation with toluene. 1, 6-Hexamethylene diisocyanate (HDI) and dibutyltin dilaurate (DBTDL) were purchased from Merck. 2,3-Epoxy-1-propanol (GD), 1,6-hexamethylene diamine (HMDA, 99%) and guanidine hydrochloride (GHC, 99%) were purchased from Aldrich and used as received. Glycerol diglycidyl ether (GDGE, technical grade) was purchased from Aldrich and dried at 60 °C in a vacuum oven for 4 h prior to use. The measured epoxy content of GDGE was 7.30 mol kg−1. Tetrahydrofuran (THF) and toluene were purchased from Merck and dried by distillation over sodium wire. Phosphatebuffered saline (PBS) was prepared by dissolving NaCl (5.85 g), KH2PO4 (0.6 g), and Na2HPO4 (6.4 g) all from Merck in distilled water and the final volume was adjusted to 1 l. The pH valve was then correlated to 7.4 by HCl or NaOH solutions (0.2 M). Mouse L-929 fibroblast cells were supplied from Pasteur Institute of Iran. Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), and Candida albicans (ATCC 10231) were provided by the Iranian Research Organization for Science and Technology (Tehran, Iran).

2.2. Synthesis of epoxy-terminated polyurethanes (GEPU and DGEPU) The procedure reported in our previous publications [11,31] was followed for the preparation of these materials. PEG was placed in a three-necked, round-bottomed flask along with pre-dried THF. HDI was dropped into the flask and the reaction was continued at 70 °C until the NCO content reached the predetermined theoretical value as determined by back titration method described in ASTM D2572-97 [32]. The reaction temperature was reduced to room temperature and then GD or GDGE diluted in THF was dropped into the reaction mixture slowly. To compensate the lower reactivity of the secondary hydroxyl groups of GDGE, one drop of DBTDL catalyst was added to the reaction flask. The reaction was continued until no NCO group peak at 2270 cm−1 was detected in the FTIR spectra of samples collected from the reactor. The reaction content was poured into a Teflon coated pan and freed from solvent via heating at 60 °C under vacuum for 12 h. The epoxy content of EPUs was determined by the method reported in Ref [33]. Different formulations of epoxy-terminated polyurethane prepolymers are collected in Table 1.

2.4. Preparation of wound dressing membranes (CEPU1–5) The required amounts of epoxy-functional materials and PHMG crosslinker were dissolved in the proper solvent as shown in Table 2. The molar ratio of the terminal NH groups of curing agent and epoxy groups of epoxy-functional materials (GEPU and DGEPU) were set at 1.3 to 1 for all formulations except for CEPU5 in which the ratio of 2.5 to 1 was selected. The resulting homogenous mixtures were poured slowly to a Teflon mold (10 × 10 × 0.1 cm3) and placed in an oven at the proper temperature (Table 2). The resulting membranes were subjected to gel content measurement to find an insight about the level of curing reaction and network formation. The prepared membranes were extracted with ethanol (70%) before subjecting to further characterization and property assessments. 2.5. Measurement of PHMG molecular weight To find an insight regarding molecular weight of the prepared PHMG, the data generated by Zhang et al. [21] was utilized. At first, the viscosity of the dilute solutions of PHMG (0.75, 1.5, 2.25 and 3.0 g dl−1) was determined using an Ubbelohde viscometer (at 25 °C ± 0.05). Since PHMG was a polycation in water, the measurements were performed in aqueous NaCl solutions (3 g dl−1) to diminish the polyelectrolyte effect [34]. Based on the Huggins and Kraemer equations [35] and a double extrapolation of reduced viscosity (ηred) and inherent viscosity (ηinh) curves to infinite dilution, the intrinsic viscosity ([η]) of the synthesized PHMG was determined as 0.0145. With changing the reaction condition, PHMG at different molecular weights was prepared by Zhang et al. [21]. They

Table 2 Different formulations of wound dressing membranes. Samples

GEPU/GDEPU/GDGE/PHMG (g)

Solvent

Curing

Gel %

CEPU1 CEPU2 CEPU3 CEPU4 CEPU5

4/–/–/0.72 3/–/0.14/0.72 –/4/–/1.37 −/3/0.25/1.37 4/–/–/1.38

H2O H2O/DMFa H2O H2O/DMFa H2O

12 h/60 °C 12 h/60 °C 12 h/100 °C 12 h/100 °C 12 h/60 °C

95 ± 4 99 ± 2 76 ± 7 73 ± 6 85 ± 4

a

70 to 30 v/v ratio of H2O to DMF.


calculated the [η] of these polymers and measured their Mn values using vapor-phase osmometry technique. These data were utilized for plotting of a calibration curve, and then the following equation was fitted to this curve: Mn ¼ 20805½η þ 270:4:

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and tan δ versus temperature were recorded for each sample. Cross-link density (νc) of the dressings was estimated from their storage modulus in the rubbery region by applying an equation from the statistical theory of rubber elasticity. E0 ¼ Φνc RT:

By replacing in this equation the measured value of [η], the Mn of PHMG was determined as 572 g mol−1. In this equation, E′ is the storage modulus at the rubbery region obtained from DMA curves, R is gas constant, T is the absolute temperature and Φ is the front factor, which is equal to unity for ideal rubbers.

2.6. Spectroscopic methods 1

H NMR spectra were recorded on a 400 MHz Bruker instrument (model Avance 400, Germany) using CDCl3 and D2O as solvents. FTIR spectra were obtained using Bruker IFS 48 instruments. All spectra were obtained under air as a function of time with 16 scans at a resolution of 4 cm−1 and a spectral range of 500–4000 cm−1. 2.7. Dynamic mechanical thermal analysis (DMA) DMA was performed on a Tritec 2000 DMA instrument in tensile mode, temperature range of −100 to 200 °C, heating rate of 3 °C min−1 and a frequency of 1 Hz. The values of storage modulus, loss modulus

2.8. Determination of tensile properties Measurement of tensile strength, elongation-at-break, and modulus of samples under dry and hydrated states were carried out using an universal tensile tester (Instron 6025) with a crosshead speed of 50 mm min−1. Samples were cut into bars of 50 mm length and 5 mm width. The tests were performed at room temperature using five specimens of each sample. The tensile properties of hydrated samples were measured using bars equilibrated in PBS for 48 h.

Scheme 1. Synthetic routes followed for the preparation of EPUs.

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2.9. Gel content measurement To evaluate the network formation, gel content of dressing membranes was determined. The vacuum dried samples were weighed accurately and then extracted by THF in a Soxhlet extractor for 24 h. The insoluble part was dried at 50 °C and weighed. The gel content was defined as follows: Gel content% ¼ Wd =Wi 100; where Wi and Wd designate the initial weight and weight of dried membrane after extraction. The values reported were the average of three measurements. 2.10. Measurements of surface and bulk hydrophilicity Surface hydrophilicity of membranes was determined by measuring the water droplet contact angle at six different positions of membrane surface for each sample. The contact angle was determined via running Image J 1.44p software on the digital pictures taken from the interfaces of membranes and droplets. The values reported were the average of six measurements. The equilibrium water absorption (EWA%) and the equilibrium water content (EWC%) were calculated for all the prepared samples. For this purpose, the completely dried and accurately weighed films were soaked in PBS at room temperature until the swelling equilibrium was attained (about 48 h). The weight of swelled membrane was determined after being gently wiped with filter paper to remove the surface liquid. EWA% and EWC% were determined using the following equations: EWA% ¼ ½ðWs −Wd Þ=Wd 100 EWC% ¼ ½ðWs −Wd Þ=Ws 100; where Wd and Ws are the weights of dry and swelled membranes. The values reported were the average of three measurements. The moisture permeability of the membranes was determined by the measurement of water vapor transmission rate (WVTR) across the material as expressed by the protocol reported in ASTM E96/E96M [36]. The WVTR was calculated using the following equation: WVTR ¼ ½ðWi −Wt Þ=A T; where WVTR is expressed in g m−2 day−1, A is the area of cup mouth (m2), and Wi and Wt are the weight of water containing cup before and after placing in the oven at 37 °C and 35% humidity. The values reported are the average of three measurements. 2.11. Mapping and estimation of the guanidinium groups' content The content and distribution of guanidinium groups embedded into the backbone of membranes were determined indirectly by measuring the amount of Cl− counter ion by elemental analysis using energy dispersive X-ray (EDX) analyzer system. For this purpose, a scanning electron microscope (SEM, Tescan, Vega II, and Czech) equipped with EDX system (Oxford Instrument, INCA, England) was used. 2.12. Evaluation of cytocompatibility Cytocompatibility of the prepared dressings was evaluated by a microscopic study of L929 fibroblast cell morphology after direct contact with samples, as well as tetrazolium dye-based colorimetric assay (MTT assay) according to our previously reported procedures [11]. The samples were sterilized by incubation at 120 °C for 15 min before each test. In the “direct contact test”, the evaluation of cell morphology present on the interface of samples and culture plate was performed after an incubation time of 48 h. To check whether the cytotoxic agents may possibly leach out from the

Fig. 1. FTIR spectra of a) GDEPU, b) PHMG, c) CEPU1, and d) CEPU3.

wound dressing membranes, the freshly synthesized dressings were immersed in the culture medium for 3 days at 37 °C and then MTT assay was performed on extracted leachates using L-929


fibroblast cells. The percentage of relative cell viability was calculated according to following equation: cell vailability% ¼

ODsample −ODpositive control ; ODnegative control

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different samples was performed with one-way analysis of variance with the Tukey post-hoc tests. The significance level was set at p ≤ 0.05.

3. Results and discussion

where OD designates the optical density. A growth medium, containing cells but no membrane was tested as negative control and a growth medium, containing membrane but no cell was tested as positive control. Each measurement was repeated at least five times. 2.13. Evaluation of antimicrobial activity Antimicrobial activity of the prepared membranes was evaluated using “colony forming count” method. Bacteria at an inoculated concentration of 2 × 108 CFU ml−1, and dressing membranes with the dimensions of 1 cm × 1 cm were used throughout the tests. The procedure reported in ASTM E 2180-07 [37] was followed and the details of experiments were also described in our previous article [20]. The percentage of microorganism reduction was calculated according to the following equation: log10 X1 þ log10 X2 þ log10 X3 3 ða−bÞ 100; Reduction% ¼ a

Geometric mean ¼

where X designates the number of organisms recovered from the incubation period control or incubation period treated samples, a designates the antilog of the geometric mean of organisms recovered from the incubation period control samples and b designates the antilog of the geometric mean of organisms recovered from the incubation period treated samples. 2.14. Statistical analysis Statistical analyses were performed via the PASW Statistics program package, version 18 (SPSS, Chicago, IL). Comparison of obtained data for

3.1. Synthesis and spectroscopic characterization of prepared materials Polyurethane based framework was selected in the present study for the preparation of wound dressing membranes with desirable biocompatibility (Scheme 1). To increase hydrophilicity of the final materials, PEG segment with high potency for establishing hydrogen-bonding type interactions with water molecules was embedded into the EPU structures. As well, for maintaining dimensional stability of dressings at highly swelled condition the crosslinked version of polyurethane was selected. To this end, epoxy-terminated polyurethane intermediates were selected, since high reactivity of epoxy groups towards amine based curing agents led to efficient formation of network without facing problems of toxic free isocyanate residues [11,38]. Also, in contrast to isocyanate-terminated prepolymers, EPUs were not moisture sensitive and they were stable materials under conventional conditions [11]. PEG containing EPUs were completely water soluble; therefore, network formation through subsequent reaction with the appropriate curing agent could be performed in aqueous media. Two different EPUs (GEPU and GDEPU) with an average of two and four reactive epoxy groups were prepared in this study (Scheme 1). Synthesis and characterization of GEPU were fully described in our previous publications [39]. The structure of GDEPU was also elucidated by FTIR and 1H NMR spectroscopic methods. In the FTIR spectrum of GDEPU (Fig. 1a), the characteristic bands of urethane groups were detected at 3326–3334 cm−1 (N–H stretching), 1714–1716 cm−1 (NHCOO stretching), and 1529–1531 cm−1 (C–N stretching, combined with N–H out of plan bending). The peaks of epoxy groups also appeared at 848 and 951 cm−1. There was no sign of peak at 2270 cm−1, which confirmed the complete reaction of the NCO groups during the preparation of this intermediate compound. 1H NMR spectrum of GDEPU was also in accordance to its proposed structure. Protons of epoxy groups (N1, N2 and M) appeared at about 2.61–3.17 ppm [40], which confirmed the preservation of

Fig. 2. 1H NMR spectrum of GDEPU.

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Fig. 4. tan δ curves recorded at different frequencies.

Fig. 3. DMA curves of CEPU1–5.

these groups during the course of synthesis. Formation of urethane bonds was elucidated by detection of methylene groups attached to urethane oxygen and nitrogen at about 4.3 (H) and 3.2 (G) ppm, respectively. Urethane NH groups appeared at about 7.2 ppm [41] and methylene groups of PEG moieties were also detected at about 3.8 ppm. Other protons of GDEPU were marked in Fig. 2. PHMG was synthesized and characterized according to a known method with minor difference regarding the molar ratio of HMDA to GHC. The excess amount of HMDA to GHC was applied to prepare amine-terminated low molecular weight oligomer. In the FTIR spectrum of PHMG (Fig. 1b), the characteristic peaks of the guanidine group were detected at 3300 (N–H stretching), 1650 (C_N stretching), 1630 (N–H bending) and 1348 cm−1 (C–N stretching). Peaks at 1620, 1560 cm−1 were also attributed to bending vibration of NH+ 2 moieties. The desired antimicrobial wound dressing membranes (CEPU1– CEPU5) were easily prepared through heating of the aqueous mixtures consisting of PHMG with either GEPU or GDEPU (Scheme 1). The epoxy functionality of EPUs was utilized as a structural factor for the control of the tensile strength of dressing membranes. In addition, a certain amount of GDGE as a low molecular weight reactive epoxy containing material was added to some of the formulations to increase the crosslink

density and consequently improve the mechanical strength of the resulting dressings. To know about the possible effect of guanidinium chloride groups' content on biological activity of dressings, the preparation of a sample (CEPU5) with excess amount of PHMG was also examined (Table 2). The prepared materials were subjected to gel content measurement using THF/H2O 80/20 w/w as solvent. High gel content values of dressing membranes obtained from GEPU prepolymer confirmed the suitability of the curing reaction condition applied to the preparation of the CEPU1 and CEPU2 samples (Table 2). However, under the same curing condition (60 °C, 12 h), lower gel content was recorded for the networks derived from GDEPU prepolymer. Increasing the reaction temperature to 100 °C improved the gel content slightly. As well, increasing the concentration of PHMG resulted in an increase of gel content of the network. The cured materials were studied by FTIR spectroscopy. Representative examples are provided in Fig. 1c and d. Investigation of these spectra showed that, the characteristic peaks of the urethane groups at 3326–3334, 1714–1716, and 1529–1531 cm−1 were preserved. Extra peaks due to guanidinium moieties were also detected at 1650, 1630, 1350, 1620, and 1560 cm−1. Meanwhile, a considerable reduction in height of peaks at 951 and 848 cm−1 (epoxy rings) was observed for the dressings derived from GEPU prepolymer. It was implied that under the employed reaction conditions, significant

Table 3 DMTA results of prepared membranes. Code

Cross-link density (mol/cm3)

Tg1

Tc

Tg2


1433 1866 438 655 828

−30 −28 −47 −42 −19

– – 43 43 –

– – 88 90 –


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Table 5 Hydrophilicity results of CEPU1–5.x Code

EWC (%)

EWA (%)

WVTR (g m−2 day−1)

Contact angle


64.8 ± 0.4a,b 63.1 ± 0.1b 66.6 ± 0.8a 67.5 ± 2a 74.2 ± 0.6c

184.5 ± 3.1a 170.8 ± 0.6b 199.7 ± 8.1a 208.3 ± 19.3a 287.4 ± 9.1c

1114 ± 17a,b 1175 ± 38a 1171 ± 58b 1218 ± 44b 1261 ± 39c

64.2 ± 2.4a 70.4 ± 1.5b 65.5 ± 3.2a 67.1 ± 1.9a 56.2 ± 5.1c

x According to analysis of variances the difference between quantities with similar superscripts (a, b, c and d) is not significant (p N 0.05) for data in each column.

Fig. 5. Stress–strain curves for CEPU1–5 at a) dry and b) hydrated states.

amounts of epoxy groups reacted with amine groups of PHMG. However, under the same condition, a higher concentration of unreacted epoxy rings (higher height of peaks at 951 and 848 cm−1) remained for the dressings made from the DGEPU prepolymer. 3.2. Viscoelastic and tensile properties of CEPU1–5 DMA was utilized for studying viscoelastic behavior of CEPUs. Variation of storage modulus (E′), loss modulus (E″) and loss tangent (tan δ) vs. temperature is shown in Fig. 3. Investigation of DMA curves showed diverse behavior for samples derived from different EPUs (GEPU and GDEPU). GEPU based samples (CEPU1, CEPU2, CEPU5) exhibited one

peak in their tan δ curves at about − 30 °C; however, for CEPU3 and CEPU4 samples derived from GDEPU, three different transitions at about −45, 45 and 88 °C were detected. For better characterization of these peaks, DMA study was conducted at three different frequencies (0.1, 1 and 10 Hz) and the possible shift to higher temperature with the increase of the frequency was monitored [42]. In Fig. 4, the tan δ curves of the CEPU1 and CEPU3 samples as representative materials made from GEPU and GDEPU prepolymers are collected. The position of the peak observed for the GEPU based sample shifted to the higher temperature upon increasing the frequency; therefore, this peak was attributed to a second order transition (Tg) related to the soft segment of the network composed of PEG moiety. For the GDEPU based sample, the positions of the first and the third transitions were frequency dependent; however, no change in the position of second transition was detected upon frequency variation. The first and the third peaks were related to Tg of the soft and hard domains of networks composed of PEG and PHMG moieties, respectively. The second transition at about 45 °C was attributed to crystallization of PEG soft segment (Tc). In fact, during the course of the analysis when tensile mode of DMA was selected, alignment and crystallization of soft segment occurred. This phenomenon is common for elastomers with a low degree of crosslink density [43–46]. Therefore, it was concluded that the crosslink density of network made from GDEPU was much lower than the corresponding material made from GEPU. Crosslink density (υc) of all samples was calculated (Table 3) and interestingly, lower crosslink density was obtained for GDEPU based samples despite the higher functionality of the epoxy groups. This occurrence could be attributed to the incomplete reaction of the epoxy groups. This finding was in accordance to the lower gel content of GDEPU based films and higher degree of the remaining epoxy groups' peaks in FTIR spectra of cured samples derived from the GDEPU prepolymer. The main reason suggested for this observation was lower reactivity of the glycidyl groups present in the structure of GDEPU in comparison to glycidylcarbamate functions available in the structure of GEPU. A close inspection of literature data validated this suggestion [47].The lower crosslink density of GDEPU prepolymer provides enough degree of freedom for PEG segments to form an independent crystalline domain. Investigation of storage modulus variation vs. temperature showed higher modulus at glassy state for samples derived from GDEPU (Fig. 3); however, the modulus of the rubbery plateau region was lower for these samples in comparison to those materials derived from GEPU prepolymer. This phenomenon could be related to higher physical

Table 4 Tensile properties of CEPU1–5 recorded at dry and hydrated states.x Code

Tensile strength (MPa) Dried

CEPU1 CEPU2 CEPU3 CEPU4 CEPU5 x

Elongation at break (%) Wet

a

1.44 ± 0.03 2.46 ± 0.30a,c 12.87 ± 1.30b 14.47 ± 1.32b 4.42 ± 0.29c

Dried a

0.40 ± 0.08 0.62 ± 0.09b,a 0.29 ± 0.04c,a 0.22 ± 0.06c 0.15 ± 0.02d,c

Initial modulus (MPa) Wet

a

63.7 ± 13.9 92.4 ± 11.2a,b 137 ± 20.7b 138 ± 3.8b 341.3 ± 28.3c

Dried a,c

30.7 ± 7.0 38.1 ± 3.9a,d 23.2 ± 0.3b 17.5 ± 1.7c,b 46.8 ± 5.1d

Wet a

3.57 ± 0.80 4.95 ± 0.68a 43.9 ± 13b 138.8 ± 10.3b 2.6 ± 0.8a

According to analyses of variance the difference between quantities with similar superscripts (a, b, c and d) is not significant (p N 0.05) for data in each column.

1.49 ± 0.07a,b,c 1.96 ± 0.22a,c 1.33 ± 0.30b,c 1.51 ± 0.17c 0.38 ± 0.02d

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interaction (hydrogen bonding and ion–ion interaction) between PHMG moieties present at higher concentration in the structure of CEPU3 and CEPU4 samples. As well, the presence of crystalline domains originate from PEG soft segment could intensify these interactions. At higher temperature when these physical interactions were diminished, the behavior of samples was determined solely by the amount of chemically crosslinked bonds. Due to higher susceptibility of glycidylcarbamate functions for the reaction with curing agent, more established networks were formed for CEPU1 and CEPU2 samples. The behavior of CEPU5 sample made through the reaction of excess amount of PHMG was slightly different from other networks obtained from GEPU. Higher weight percent of PHMG led to more physical interactions (hydrogen bonding and ion– ion interactions) at glassy state, but significant reduction in rubbery plateau modulus and lower chemical crosslink density were also detected. It seems that a portion of PHMG chains was not fully reacted with epoxy groups, therefore, these PHMG chains possessed more degree of freedom and acted as dangling short chains. A dressing membrane should preserve its dimensional stability during the wound healing process to protect the wounded area from external impacts [48]. It is well known that intermolecular attraction present in polar dressing materials can be deteriorated by plasticization effect of absorbed exudates. This phenomenon can negatively influence on the

tensile strength of a wound dressing. To study this important characteristic of designed dressings, tensile properties were evaluated at both dry and fully hydrated states (Fig. 5, Table 4). It was found that the final tensile properties of these materials were influenced by two main factors including crosslink density and crystallinity of the polymer backbone. These two factors were dictated by the nature and the number of epoxy groups present in the structure of prepolymers and molar concentration of PHMG curing agent. At dry state, GEPU based membranes showed behavior of lightly crosslinked rubbers with smooth transition in their stress–strain curves. There was no significant difference between strengths of the CEPU1 and CEPU2 samples; however, significant enhancement in tensile properties was detected for CEPU5 made from excess amount of PHMG in comparison to CEPU1. Since the measurement of tensile property was performed at ambient temperature, the behavior of these materials was mainly influenced by the physical interactions of chains. CEPU5 with more proportion of PHMG showed enhanced mechanical properties. In the mean time, despite the lower chemical crosslink density of the GDEPU based samples, higher tensile property was detected for them in comparison to GEPU based membranes. As it was evidenced from stress–strain curves, a pronounced yield point followed by necking and drawing indicative of semi-crystalline polyurethanes was observed.

Fig. 6. Cl atoms maps and elemental analysis results of a) CEPU2, b) CEPU3, and c) CEPU5 derived from EDX.


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Fig. 7. Optical microscopy images of fibroblast cells in direct contact with a) negative control, b) CEPU2, c) CEPU4, and d) CEPU5 after 24 h and 48 h incubation periods.

This result was in strong accordance with DMA findings. Indeed, the tensile property was mainly determined by the presence of crystalline domain of PEG segment and attraction of PHMG segment through hydrogen bonding and coulombic interactions. In the wet state, absorbed water molecules within polymeric networks caused deterioration of physical interactions of polar and guanidine hydrochloride ionic centers. Therefore, tensile property was mainly determined by chemical crosslinking level. Interestingly, GEPU based membranes showed better tensile strength than GDEPU made samples due to their higher crosslink density. Among prepared samples, CEPU2 showed the highest tensile strength due to its high crosslink density.

Table 6 Results of MTT assay for cells contacted with samples or leachates extracted from them.x Code

Cell viability (%) Direct contact

Cell viability (%) Indirect contact

CEPU2 CEPU4 CEPU5

93.9 ± 3. 6a 94.4 ± 4.3a 26.8 ± 2.3b

94.6 ± 3.3 95.5 ± 2.7 84.3 ± 3.4b

x According to analysis of variances the difference between quantities with similar superscripts (a, b, c and d) is not significant (p N 0.05) for data in each column.

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3.3. Fluid handling capacity of dressings Dehydration of the wound surface can lead to eschar formation and adherence of the dressing to the wounded tissue. Presence of excessive exudate is also associated with maceration of surrounding tissue, malodor, and high risk of infection that eventually can lead to increased wound management costs and patient morbidity [48,49]. Manipulation of these important tasks can be performed by using proper wound dressings. When dressing is in contact with exudates of wound, it absorbs exudates and swelling of the dressing occurs. This helps to reduce any space that may exist between the dressing and the wound, which in theory should insulate and maintain an optimum wound temperature. Therefore, the ability of dressing materials to retain the absorbed fluid directly above the wounded area is an important characteristic of functional dressing materials. To evaluate the fluid-handling capacity of the prepared dressings, physical parameters including EWC, EWA and WVTR were determined (Table 5). The desirable values of EWA and EWC for wound dressing certainly depend on the type and position of the wounded area. For example, full thickness wound with large amount of exudates needs to cover with dressings having high absorptive capacity [49] and low exudates wounds like bedsore needs dressing with low to moderate ability for exudate absorption. Investigation of recorded results (Table 5) obviously showed that all of the prepared dressings had very good ability for absorption and consequently evaporation of excessive moisture from the

wounded area. In fact the fluid absorbed by the dressing was no longer available to wet the surrounding skin through sideways spread or ‘lateral wicking’, and lost to the atmosphere by evaporation. For better judgment regarding this important issue, the WVTR values for some wellknown commercially available dressings were considered. For example, the WVTR values of Dermiflex® (J&J), Tegaderm®, Bioclusive® (J&J), Op Site® (J&J), Metoderm (Conva tec Ltd.), Duoderm (Conva tec Ltd.), Biobrain®, Lyofoam, Geliperms (Geistlich Ltd.) and Vigilons (Bard Ltd.) are 90 ± 3, 491 ± 41, 394 ± 12, 792 ± 32, 823 ± 45, 886 ± 60, 1565 ± 51, 3052 ± 684, 9009 ± 319 and 9360 ± 34 g m−2 day−1, respectively [50]. The dressings prepared in the preset study had WVTR values in the range of 1114–1261 g m−2 day−1. Hence, the prepared membranes were found to be proper candidates for dressing of the wounds with moderate exudates. 3.4. Evaluation of guanidine group distribution within dressing membranes Uniform distribution of guanidinium groups in the membrane backbone is a vital factor for ensuring homogenous performance of prepared membranes against bacteria. To estimate this issue, the membranes were washed with plenty of double distillated water, and then the amount of guanidine moieties were calculated via measurement of the chloride counter ion wt.% by EDX analysis. The related maps for the representative samples are shown in Fig. 6. Fortunately, due to covalent bonding of antibacterial guanidinium moieties to dressing membranes,

Fig. 8. Agar disk diffusion test for prepared membranes: a) control, b) CEPU1, and c) CEPU3.


all samples showed homogenous distribution of chlorine counter ions. As well, these functions were preserved after several washing and sterilization procedures. CEPU3 with higher percentage of curing agent exhibited the densest distribution of chlorine ions; therefore, better antimicrobial activity was expected for this sample. 3.5. Evaluation of cytocompatibility and antimicrobial activity of dressing membranes An ideal wound dressing should be non-cytotoxic and biocompatible. For evaluating the cytocompatibility of the prepared dressing membranes, the behavior of cultured L-929 fibroblast cells on these materials was monitored for 24 and 48 h. Results for selected samples are shown in Fig. 7. Fortunately, no pronounced cell debris and changes in morphology, such as cell lysis, loss of spindle shape and detachment from bottom was observed for the cells cultured on the prepared dressing membranes except for the CEPU5 sample. The appropriate cytocompatibility of prepared

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samples is a consequence of using suitable starting materials i.e. EPU with confirmed biocompatibility [11,39] and guanidinum based disinfectant with high biocompatibility index [27] for the preparation of the final networks. However, the behavior of the CEPU5 sample was not appropriate. According to data recorded for viscoelastic and tensile properties of this sample, it was suggested that this compound contains PHMG dangling moieties. The toxicity of this sample might be attributed to the free terminal amine groups of these dangling PHMG chains. Since excess amount of HMDA was used for the preparation of PHMG, therefore, this compound contains terminal free amines. Consequently, as excess amount of PHMG (in comparison to epoxy content of GEPU) was used for the preparation of CEPU5, therefore, the presence of free amine groups was expected for this sample. Higashi has detected toxic behavior for 1,6-hexanediamine moiety (the chain segment exists in the structure of CEPU5) [51]. To examine this theory and find better insight regarding the cytocompatibility of the prepared membranes, MTT assay

Fig. 9. Bactericidal and fungicidal activities of a) CEPU1, b) CEPU2, and c) CEPU4 samples against S. aureus, P. aeruginosa, and C. albicans strains.

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was performed on the membranes and leachates extracted from samples after 3 days. Results for the selected samples are collected in Table 6. Excellent cell viability was recorded for CEPU2 and CEPU4 samples, when MTT assays were performed directly on these samples or on their extracted leachates (indirect assays). It is interesting to note that very good cell viability was recorded for cells cultured in a medium containing leachates extracted from CEPU5. This observation is a good support for our hypothesis regarding the origin of cytotoxicity of CEPU5 sample. The antimicrobial activity of dressing membranes was qualitatively detected by disk diffusion method and quantified by colony count technique. The results of disk diffusion method for two stains of Gram positive and Gram negative bacteria are depicted in Fig. 8. Since guanidine groups were covalently attached to the membranes' backbone and there was no possibility for the release of these active groups from the polymers, no considerable zone of inhibition around these specimens was detected. Meanwhile, no bacterial colonies were grown on both specimens' surface and area of agar plate under each sample. As a result, the prepared polymers had contact killing activity. The quantified results regarding antimicrobial activity of the dressing materials obtained from colony count method are collected in Fig. 9. Excellent bactericidal and fungicidal activities against all of the studied microorganisms with 100% reduction were recorded. The antimicrobial activity of the dressings with embedded cationic guanidinium groups was attributed to destructive interaction of these quaternary ammonium functions with the cell wall and/or cytoplasmic membranes of microorganisms. 4. Conclusion Wound dressing membranes were prepared by thermal curing reaction of different epoxy-terminated polyurethane prepolymers with amine-terminated poly(hexamethylene guanidine hydrochloride). The resulting dressings showed promising bactericidal and fungicidal activities due to the presence of cationic guanidinum groups chemically embedded to the backbone of the prepared membranes. Simultaneously, these formulations of prepared dressings with proper concentration of active guanidinum groups showed excellent cytocompatibility against fibroblast cells. The physical and mechanical properties of dressings were influenced by their chemical structures and concentration of their constituents. Chemical and physical interactions including hydrogen bonding and ion–ion interactions of polymeric chains were responsible for the recorded macroscopic properties. The hydrophilicity and WVTR for most of the prepared membranes guaranteed the presence of moist environment over the wounded bed. The optimum characteristics of the CEPU1 and CEPU2 samples showed very good potential of these materials for use as antimicrobial, absorptive wound dressing suitable for moderately exuding wounds. References [1] S. Lu, W. Gao, H.Y. Gu, Construction, application and biosafety of silver nanocrystalline chitosan wound dressing, Burns 34 (2008) 623–628, http://dx.doi.org/10.1016/ j.burns.2007.08.020. [2] J. Boateng, K. Matthews, Wound healing dressings and drug delivery systems: a review, J. Pharm. Sci. 97 (2008) 2892–2923, http://dx.doi.org/10.1002/jps. [3] P. Zahedi, I. Rezaeian, S.-O. Ranaei-Siadat, S.-H. Jafari, P. Supaphol, A review on wound dressings with an emphasis on electrospun nanofibrous polymeric bandages, Polym. Adv. Technol. 77–95 (2009), http://dx.doi.org/10.1002/pat.1625. [4] L.G. Ovington, Advances in wound dressings, Clin. Dermatol. 25 (2007) 33–38, http://dx.doi.org/10.1016/j.clindermatol.2006.09.003. [5] L.L. Bolton, K. Monte, L.A. Pirone, Moisture and healing: beyond the jargon, Ostomy Wound. Manage. 46 (2000) 51S–62S. [6] Z. Abdali, H. Yeganeh, A. Solouk, R. Gharibi, M. Sorayya, Thermoresponsive Antimicrobial Wound Dressings via Simultaneous Thiol-ene Polymerization and In Situ Generation of Silver Nanoparticles, RSC Adv. (2015), http://dx.doi.org/10.1039/C5RA11618J. [7] C. Weller, G. Sussman, Wound dressings update, J. Pharm. Pract. Res. 36 (2006) 318–324, http://dx.doi.org/10.1002/j.2055-2335.2006.tb00640.x. [8] S. Rajendran, Advanced Textiles for Wound Care, CRC Press, 2009.

[9] P.G. Bowler, Wound pathophysiology, infection and therapeutic options, Ann. Med. 34 (2002) 419–427, http://dx.doi.org/10.1080/078538902321012360. [10] P. Niethammer, C. Grabher, A.T. Look, T.J. Mitchison, A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish, Nature 459 (2009) 996–999, http://dx.doi.org/10.1038/nature08119. [11] A. Yari, H. Yeganeh, H. Bakhshi, Synthesis and evaluation of novel absorptive and antibacterial polyurethane membranes as wound dressing, J. Mater. Sci. Mater. Med. 23 (2012) 2187–2202, http://dx.doi.org/10.1007/s10856-012-4683-6. [12] A. Yari, H. Yeganeh, H. Bakhshi, R. Gharibi, Preparation and characterization of novel antibacterial castor oil-based polyurethane membranes for wound dressing application, J. Biomed. Mater. Res. A 102 (2014) 84–96, http://dx.doi.org/10.1002/jbm.a.34672. [13] Y. Xue, Y. Pan, H. Xiao, Y. Zhao, Novel quaternary phosphonium-type cationic polyacrylamide and elucidation of dual-functional antibacterial/antiviral activity, RSC Adv. 4 (2014) 46887–46895, http://dx.doi.org/10.1039/C4RA08634A. [14] W.R. Lee, K.M. Tobias, D.A. Bemis, B.W. Rohrbach, In vitro efficacy of a polyhexamethylene biguanide-impregnated gauze dressing against bacteria found in veterinary patients, Vet. Surg. 33 (2004) 404–411, http://dx.doi.org/ 10.1111/j.1532-950X.2004.04059.x. [15] L. Martineau, P.N. Shek, Evaluation of a bi-layer wound dressing for burn care. II. In vitro and in vivo bactericidal properties. Burns 32 (2006) 172–179, http://dx.doi. org/10.1016/j.burns.2005.08.012. [16] S. Johnson, M. Thomas, M. Walker, Evaluating a dressing impregnated with polyhexamethylene biguanide, Wounds UK 7 (2011) 20–25. [17] T. Eberlein, O. Assadian, Clinical use of polihexanide on acute and chronic wounds for antisepsis and decontamination, Skin Pharmacol. Physiol. 23 (Suppl.) (2010) 45–51, http://dx.doi.org/10.1159/000318267. [18] R. Li, P. Hu, X. Ren, S.D. Worley, T.S. Huang, Antimicrobial N-halamine modified chitosan films, Carbohydr. Polym. 92 (2013) 534–539, http://dx.doi.org/10.1016/j. carbpol.2012.08.115. [19] Z. Ma, H. Ji, D. Tan, Y. Teng, G. Dong, J. Zhou, et al., Silver nanoparticles decorated, flexible SiO2 nanofibers with long-term antibacterial effect as reusable wound cover, Colloids Surf. A Physicochem. Eng. Asp. 387 (2011) 57–64, http://dx.doi. org/10.1016/j.colsurfa.2011.07.025. [20] R. Gharibi, H. Yeganeh, H. Gholami, Z.M. Hassan, Aniline tetramer embedded polyurethane/siloxane membranes and their corresponding nanosilver composites as intelligent wound dressing materials, RSC Adv. 4 (2014) 62046–62060, http://dx. doi.org/10.1039/C4RA11454J. [21] Y. Zhang, J. Jiang, Y. Chen, Synthesis and antimicrobial activity of polymeric guanidine and biguanidine salts, Polymer (Guildf) 40 (1999) 6189–6198, http://dx.doi. org/10.1016/S0032-3861(98)00828-3. [22] L. Feng, F. Wu, J. Li, Y. Jiang, X. Duan, Antifungal activities of polyhexamethylene biguanide and polyhexamethylene guanide against the citrus sour rot pathogen Geotrichum citri-aurantii in vitro and in vivo, Postharvest Biol. Technol. 61 (2011) 160–164, http://dx.doi.org/10.1016/j.postharvbio.2011.03.002. [23] Z. Zhou, D. Wei, Y. Guan, A. Zheng, J.-J. Zhong, Extensive in vitro activity of guanidine hydrochloride polymer analogs against antibiotics-resistant clinically isolated strains, Mater. Sci. Eng. C 31 (2011) 1836–1843, http://dx.doi.org/10.1016/j.msec.2011.08.015. [24] D. Wei, Q. Ma, Y. Guan, F. Hu, A. Zheng, X. Zhang, et al., Structural characterization and antibacterial activity of oligoguanidine (polyhexamethylene guanidine hydrochloride), Mater. Sci. Eng. C 29 (2009) 1776–1780, http://dx.doi.org/10.1016/j. msec.2009.02.005. [25] L. Qian, Y. Guan, B. He, H. Xiao, Modified guanidine polymers: synthesis and antimicrobial mechanism revealed by AFM, Polymer (Guildf) 49 (2008) 2471–2475, http://dx.doi.org/10.1016/j.polymer.2008.03.042. [26] L. Bromberg, T.A. Hatton, Poly(N-vinylguanidine): characterization, and catalytic and bactericidal properties, Polymer (Guildf) 48 (2007) 7490–7498, http://dx.doi. org/10.1016/j.polymer.2007.10.040. [27] G. Müller, A. Kramer, Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity, J. Antimicrob. Chemother. 61 (2008) 1281–1287, http://dx.doi.org/10.1093/jac/dkn125. [28] B. Jiang, J.C. Larson, P.W. Drapala, V.H. Pérez-Luna, J.J. Kang-Mieler, E.M. Brey, Investigation of lysine acrylate containing poly(N-isopropylacrylamide) hydrogels as wound dressings in normal and infected wounds, J. Biomed. Mater. Res. B Appl. Biomater. 100 (B) (2012) 668–676, http://dx.doi.org/10.1002/jbm.b.31991. [29] C.J. Waschinski, J. Zimmermann, U. Salz, R. Hutzler, G. Sadowski, J.C. Tiller, Design of contact-active antimicrobial acrylate-based materials using biocidal macromers, Adv. Mater. 20 (2008) 104–108, http://dx.doi.org/10.1002/adma.200701095. [30] V.J. Jones, The use of gauze: will it ever change? Int. Wound J. 3 (2006) 79–88, http://dx.doi.org/10.1111/j.1742-4801.2006.00215.x. [31] H. Yeganeh, P. Hojati-Talemi, Preparation and properties of novel biodegradable polyurethane networks based on castor oil and poly(ethylene glycol), Polym. Degrad. Stab. 92 (2007) 480–489, http://dx.doi.org/10.1016/j.polymdegradstab.2006.10.011. [32] Standard Test Method for Isocyanate Groups in Urethane Materials or Prepolymers, 1998, http://dx.doi.org/10.1520/D2572-97R10.2. [33] H. Lee, K. Neville, Handbook of Epoxy Resins, 1967. [34] M.E. Zeynali, A. Rabii, H. Baharvand, Synthesis of partially hydrolyzed polyacrylamide and investigation of solution properties (viscosity behaviour), Iran. Polym. J. 13 (2004) 479–484. [35] M. Chanda, Introduction to Polymer Science and Chemistry: A Problemsolving Approach, CRC Press, 2013 (http://books.google.com/books?id= iu1eZOkV34AC&pgis=1). [36] Standard Test Methods for Water Vapor Transmission of Materials, 2002, http://dx. doi.org/10.1520/E0096. [37] Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s), Polymeric or Hydrophobic Materials, 2011, http://dx.doi.org/10.1520/ E2180-07.2.

M. Sahraro et al. / Materials Science and Engineering C 59 (2016) 1025–1037 [38] C.A. Krone, J.T.A. Ely, T. Klingner, R.J. Rando, Isocyanates in flexible polyurethane foams, Bull. Environ. Contam. Toxicol. 70 (2003) 328–335, http://dx.doi.org/10. 1007/s00128-002-0195-2. [39] H. Yeganeh, M.M. Lakouraj, S. Jamshidi, Synthesis and properties of biodegradable elastomeric epoxy modified polyurethanes based on poly(ε-caprolactone) and poly(ethylene glycol), Eur. Polym. J. 41 (2005) 2370–2379, http://dx.doi.org/10. 1016/j.eurpolymj.2005.05.004. [40] F.G. Garcia, B.G. Soares, Determination of the epoxide equivalent weight of epoxy resins based on diglycidyl ether of bisphenol A (DGEBA) by proton nuclear magnetic resonance, Polym. Test. 22 (2003) 51–56, http://dx.doi.org/10.1016/S01429418(02)00048-X. [41] P.A. Mirau, Practical Guide to Understanding the NMR of Polymers, WileyInterscience, 2005. [42] K.P. Menard, Dynamic Mechanical Analysis: A Practical Introduction, Second ed. CRC Press, Boca Raton, FL, 2008. [43] N. Reynolds, H.W. Spiess, H. Hayen, H. Nefzger, C.D. Eisenbach, Structure and deformation behaviour of model poly(ether–urethane) elastomers, 1. Infrared studies, Macromol. Chem. Phys. 195 (1994) 2855–2873. [44] C.E. Wilkes, C.S. Yusek, Investigation of domain structure in urethan elastomers by X-ray and thermal methods, J. Macromol. Sci. Part B Phys. 7 (1973) 157–175.

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[45] M. Niesten, J.W. Ten Brinke, R.J. Gaymans, Segmented copolyetheresteraramids with extended poly(tetramethyleneoxide) segments, Polymer (Guildf) 42 (2001) 1461–1469. [46] E.M. Christenson, J.M. Anderson, A. Hiltner, E. Baer, Relationship between nanoscale deformation processes and elastic behavior of polyurethane elastomers, Polymer (Guildf) 46 (2005) 11744–11754. [47] P.A. Edwards, G. Striemer, D.C. Webster, Novel polyurethane coating technology through glycidyl carbamate chemistry, JCT Res. 2 (2005) 517–527. [48] Z. Peles, M. Zilberman, Novel soy protein wound dressings with controlled antibiotic release: mechanical and physical properties, Acta Biomater. 8 (2012) 209–217, http://dx.doi.org/10.1016/j.actbio.2011.08.022. [49] M. Kokabi, Z.M. Hassan, POLYMER PVA — clay nanocomposite hydrogels for wound dressing, 43 (2007) 773–781, http://dx.doi.org/10.1016/j.eurpolymj.2006.11.030. [50] H.-J. Yoo, H.-D. Kim, Characteristics of waterborne polyurethane/poly(Nvinylpyrrolidone) composite films for wound-healing dressings, J. Appl. Polym. Sci. 107 (2008) 331–338, http://dx.doi.org/10.1002/app.26970. [51] K. Higashi, K. Yoshida, K. Nishimura, E. Momiyama, K. Kashiwagi, S. Matsufuji, et al., Structural and functional relationship among diamines in terms of inhibition of cell growth, J. Biochem. 136 (2004) 533–539.