Synthesis and characterization of biodegradable polyurethane films based on HDI with hydrolyzable crosslinked bonds and a homogeneous structure for biomedical applications

Materials Science and Engineering C 52 (2015) 22–30

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Synthesis and characterization of biodegradable polyurethane films based on HDI with hydrolyzable crosslinked bonds and a homogeneous structure for biomedical applications Breno Rocha Barrioni ⁎, Sandhra Maria de Carvalho, Rodrigo Lambert Oréfice, Agda Aline Rocha de Oliveira, Marivalda de Magalhães Pereira Department of Metallurgical Engineering and Materials, Federal University of Minas Gerais, Av. Presidente Antônio Carlos, 6627, School of Engineering, Belo Horizonte, MG 31270-901, Brazil

a r t i c l e

i n f o

Article history: Received 11 November 2014 Received in revised form 31 January 2015 Accepted 22 March 2015 Available online 24 March 2015 Keywords: Biodegradable polyurethane Films Crosslinked polyurethane Biomaterial Biodegradable polymer Mechanical tests

a b s t r a c t Synthetic biodegradable polymers are considered strategic in the biomaterials field and are used in various applications. Among the polymers used as biomaterials, polyurethanes (PUs) feature prominently due to their versatility and the ability to obtain products with a wide range of physical and mechanical properties. In this work, new biodegradable polyurethane films were developed based on hexamethylene diisocyanate (HDI) and glycerol as the hard segment (HS), and poly(caprolactone) triol (PCL triol) and low-molecular-weight poly(ethylene glycol) PEG as the soft segment (SS) without the use of a catalyst. The films obtained were characterized by structural, mechanical and biological testing. A highly connected network with a homogeneous PU structure was obtained due to crosslinked bonds. The films showed amorphous structures, high water uptake, hydrogel behavior, and susceptibility to hydrolytic degradation. Mechanical tests indicated that the films reached a high deformation at break of up to 425.4%, an elastic modulus of 1.6 MPa and a tensile strength of 3.6 MPa. The materials presented a moderate toxic effect on MTT assay and can be considered potential materials for biomedical applications. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Polymers are suitable materials for different biomedical applications [1–6]. Several biodegradable polymers, both synthetic and natural, have been used in this field, such as collagen, chitosan, polyester, and polyamide, among others. Many of these polymers, however, do not have mechanical and physical properties comparable to natural tissues [7,8]. Polyurethanes (PUs) are a very important group of polymers because of their good mechanical properties and versatility [9]. PUs are composed of alternating hard and soft blocks that can lead to separate microphases under appropriate conditions to form hard and soft domains [10]. The soft segments are generally composed of polyester, polyether, or polycarbonate polyols, whereas the hard segments are produced by reactions between a diisocyanate and low-molecular weight diol or diamine chain extender [1,10]. The soft segment provides the elastomeric character to the polymer backbone, while the hard segment usually provides extra strength due to the hydrogen bonding

⁎ Corresponding author. E-mail addresses: [email protected] (B.R. Barrioni), [email protected] (S.M. de Carvalho), rorefi[email protected] (R.L. Oréfice), [email protected] (A.A.R. de Oliveira), [email protected] (M.M. Pereira).

http://dx.doi.org/10.1016/j.msec.2015.03.027 0928-4931/© 2015 Elsevier B.V. All rights reserved.

involving urethane linkages [11]. Changing the chemical composition, the molecular weight and the ratio of hard and soft segments can lead to polymers with different physical and physicochemical properties and, subsequently, different biodegradability properties, to suit the intended application [11,12]. Biodegradable PUs applied as biomaterials are reported by several researchers [1,2,9–13], and their properties can be tailored by the proper choice of raw materials. The rate of polyurethane degradation is extremely dependent on the soft segment structure [14] composed of the polyols. The common polyols employed in biodegradable PUs include poly(propylene glycol) (PPG), poly(ethylene glycol) (PEG), poly(caprolactone) (PCL), and glycolic acid, among others. PEG exhibits attractive properties, including non-toxic degradation products, the absence of antigenicity and immunogenicity, solubility in water and organic solvents, and hydrophilicity [15]. PCL is frequently used as the soft segment in degradable polyurethanes because it can be hydrolyzed and its degradation products are non-toxic and can be metabolized [12]; however, it is rather hydrophobic and has a low degradation rate [15]. The use of polyols with higher functionality can lead to polyurethanes with crosslinked bonds. In general, a crosslinked structure prevents water from easily reaching the ester/ether groups, reducing the hydrolytic degradation capacity. Nevertheless, in some cases the presence of a triol crosslinker can prevent the aggregation of segments through hydrogen bonding of the hard segment, and increase the aggregation

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through covalent bonds, with the formation of a homogeneous structure with no phase separation. The disordering of the polymer matrix and the reduced movement freedom of the molecular chain caused by the chemical crosslinking allow the ester/ether groups to be exposed to water, improving hydrolytic degradation [16]. In the hard segment, the polyfunctional isocyanate can be aromatic, aliphatic, cycloaliphatic or polycyclic. Aliphatic diisocyanates yield PUs that are less rigid but have better oxidative and ultraviolet stabilities [17]. Some commonly used reagents to prepare biodegradable PU are 1,4-butanediisocyanate (BDI) and isophorone diisocyanate (IPDI), among others. 1,6-Hexamethylene diisocyanate (HDI) is an aliphatic diisocyanate often used in the preparation of biomedical PU [11,18–20], with a relatively non-toxic degradation by-product, diamine 1,6-hexanediamine, and that also provides good mechanical properties to the final product. Degradation products of polyurethanes based on aromatic diisocyanates like 4,4′-methylenediphenyl diisocyanate (MDI) and toluene diisocyanates (TDI) are toxic. Accordingly, aliphatic diisocyanates such as HDI, IPDI and BDI, are replacing aromatic ones in designing biodegradable polyurethanes as they have been reported to degrade into nontoxic decomposition products [18,21–23]. By introducing hydrolysable chain extenders into the hard segment, it is possible to increase the degradation rate of polyurethanes [12]. Conventional chain extenders used in PU formulations are 1,4-butanediol (BDO), 1,2-ethanediol and 1,2ethanediamine. Diamines are usually more reactive than diols or triols. Glycerol is a non-toxic polyol that is soluble in water and contains hydroxyl groups. When used as a chain extender, it provides a crosslinked structure in the polyurethane and can also increase the thermal stability [24]. Modified crosslinked PUs can behave as hydrogels, absorbing large amounts of water without dissolving, which is an important quality for several biomedical applications [25]. The aim of this study is to develop new compositions of polyurethane based on HDI diisocyanates and biodegradable polyols with high functionality, forming a crosslinked network that can behave as a hydrogel polymer capable of hydrolytic degradation, for possible application in the biomedical field. This work obtains polyurethane films based on HDI and glycerol as the hard segment and PCL triol and PEG as the soft segment. PEG hydrophilicity can be an important mechanism for improving the PU′s affinity for water. The glycerol and PCL triol can crosslink, creating an interconnected network structure with a mixture of soft and hard segments, thus providing hydrogel characteristics and good mechanical properties to the material. Additionally, glycerol is a hydrolysable chain extender, increasing the hydrolytic degradation rate. The promising results demonstrated by this new material indicate that it is a candidate for applications in the biomaterial field. 2. Materials and methods 2.1. Synthesis of biodegradable polyurethanes films HDI, PCL triol 900 g·mol− 1 and PEG 600 g·mol− 1 were obtained from Sigma-Aldrich. Glycerol and ethyl acetate were obtained from Synth Brazil. Polyurethane films were prepared by the pre-polymer process. First, PCL triol 900 and PEG 600 were added to the reactor, without any catalyst. The temperature was kept at 55 °C. After the melting of PCL and homogenization, HDI was slowly added to the reactor. The temperature and mixing were maintained for 2 h. 15 mL of the ethyl acetate (solvent) was then added. After mixing, glycerol was added to the reactor. The temperature was reduced to 40 °C, and the system was mixed for another 30 min. All processes were performed under a nitrogen atmosphere. Films were produced by casting the product into polypropylene molds and allowing them to dry at room temperature for 24 h and then for 72 h at 60 °C. In the hard segment, HDI was kept at 34% and glycerol at 5% (mass composition). The mass composition of the polyols was varied in the

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samples PU3 (3% PCL Triol 900 and 58% PEG 600) and PU12 (12% PCL Triol 900 and 49% PEG 600). 2.2. Material characterization The structure of the films was analyzed using a Thermo Scientific Nicolet 6700-OMNI Smart Accessory Spectrum Fourier transform infrared (FTIR) spectrometer. Spectra were collected in the mid-infrared range from 600 to 4000 cm−1 in the ATR mode, with 64 scans per spectrum at 4 cm−1 resolution. X-ray Diffraction (XRD) spectra of the films were collected on a Philips PW1700 series automated powder diffractometer using Cu Kα radiation at 40 kV/40 mA. Data were collected between 4.05 and 89.95° with a step of 0.06° and a dwell time of 1.5 s. The measurements of synchrotron small angle X-ray scattering (SAXS) were performed using the beam line of the National Synchrotron Light Laboratory (LNLS, Campinas, Brazil). The photon beam used in the LNLS SAXS beam line comes from one of the 12 bending magnets of the electron storage ring. The white photo beam is extracted from the ring through a high-vacuum path. After passing through a thin beryllium window, the beam is monochromatized (λ = 1.608 Ǻ) and horizontally focused by a cylindrically bent monochromater at the detection plane. The X-ray scattering intensity, l(q), was experimentally determined as a function of the scattering vector “q” whose modulus is given by q = (4π/λ)sin( ), where λ is the X-ray wavelength and is half the scattering angle. Each SAXS pattern corresponds to a data collection time of 900 s. The parasitic scattering intensity produced by the collimating slits was subtracted from the experimental scattering intensity of each sample. All SAXS patterns were corrected for the non-constant sensitivity of the PSD, for the time varying intensity of the direct synchrotron beam and for differences in the sample thicknesses. Because of the normalization procedure, the SAXS intensities for all samples are expressed in the same arbitrary units so that they can be directly compared. An Exstar 7200 from Seiko — SII Nanotechnology Inc. was utilized for the thermal analyses. Thermogravimetric analysis (TG) was carried out in the range of 25–700 °C in Nitrogen (flow = 20 mL min−1) at a heating rate of 10 °C min− 1. Samples with masses of approximately 8 mg were used in a platinum can. Differential scanning calorimetry (DSC) was performed in the range of − 50 to 150 °C. Samples were first cooled to − 50 °C. Then, the samples were heated from − 50 to 100 °C. After they had been cooled from 100 °C to − 50 °C, a second heating was carried out from −50 to 150 °C. The scans were performed at the heating/cooling rate of 10 °C min−1 in a nitrogen atmosphere (flow = 40 mL min−1). Mechanical tests of the materials were performed on an EMICDL300 universal testing machine at ambient temperature. A 50 N load cell and a minimum of 5 test pieces were used in each test. The tensile tests of the films were based on ASTM D 882-12, using a crosshead speed of 20 mm/min. The contact angle analysis was performed on a Goniometer Pixelink model DGD Int DI with the application of 6 μL of deionized water to the PU film surface at room temperature. Ten scans were performed on each sample. For the water absorption test, PU films were dried at 60 °C for 4 h and then weighed and immersed in deionized water at room temperature. Samples were removed after predetermined periods of immersion, placed on a filter paper to remove surface water and weighed again. Assays were performed in triplicate. The content of water absorption from each sample was calculated according to Eq. (1), where Mt is the mass after immersion in deionized water, and Ms is the dry mass of the samples. The results presented are the average of three samples for each experiment, with standard deviation lower than 5%. Water Absorption ð%Þ ¼

ðMt − Ms Þ  100 Ms

1

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2.3. Degradation study

3. Results and discussion

SBF (Simulated Body Fluid) solution was prepared by dissolving NaCl (7.996 g), NaHCO 3 (0.350 g), KCl (0.224 g), K 2 HPO 4·3H2 O (0.228 g), MgCl2 ·6H2 O (0.305 g), CaCl2 (0.228 g) and Na2 SO 4 (0.071 g), all supplied by Synth Brazil, in 1 L of distilled water. The solution was buffered at pH 7.4 by adjusting the volume of trihydroxymethylaminomethane and HCl (Merck) at 36.5 °C. Samples of the films (0.1–0.2 g) were placed in vials containing approximately 20 mL of the solution and kept in a water bath at a constant temperature of 37 °C. The degradation was observed for 90 days. Samples were removed at different intervals of time, washed with distilled water and dried in an oven at 60 °C for 24 h. The remaining weight of the films was evaluated by mass loss according to Eq. (2), where Mi is the initial sample mass and Mf is the mass after degradation. The results presented are the average of three samples for each experiment, with standard deviation lower than 5%.

3.1. Structural characterization

Remaining Mass ð%Þ ¼ 100−

ðMi − M f Þ  100 Mi

2

Accelerated hydrolytic degradation tests under basic conditions were performed by placing the samples of the films (0.3–0.4 g) into small containers with 15 mL of NaOH 1 mol L−1 and leaving them in an oven at 60 °C for time periods of 2, 6, 18 and 24 h. After this period, the samples were taken from the container, cleaned with distilled water and dried at 60 °C for 24 h. The remaining mass was calculated according to Eq. (2). 2.4. Toxicity assay by MTT The human osteoblasts (SAOS) were kindly provided by Prof. Goes of the Department of Immunology and Biochemistry, UFMG. The cells were cultured in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) penicillin G sodium (10 units·mL− 1), streptomycin sulfate (10 mg·mL− 1 ) and 0.25 amphotericin-b all from Gibco BRL (NY, USA) in a humidified atmosphere of 5% CO2 at 37 °C. The cells were used for experiments on passage nine. All biological tests were conducted according to ISO standards 109935:1999 (Biological evaluation of medical devices; Part 5: tests for in vitro cytotoxicity) (3-(4,5-dimethylthiazol-2yl) 2,5-diphenyl tetrazolium bromide) (MTT). SAOS cells from passage nine were plated (1 × 104 cells well− 1) in a 24-well plate. Cell populations were synchronized in a serum free medium for 24 h. Next, the medium was aspirated and replaced with a medium containing FBS. Samples (1 mg·mL− 1) of each biomaterial were used. Samples were sterilized using ultraviolet radiation for 30 min on each side of the samples. Controls consisted of cells in 10% DMEM medium with phosphate buffered saline (PBS) (10 ×) for the positive control and sterile chips derived from Eppendorf polypropylene (1 mg mL− 1) from Eppendorf (Hamburg, Germany) for the negative control. After 24 h, all media were aspirated and replaced with 210 μL culture medium with serum in each well. Next, 170 μL of MTT (5 mg·mL− 1) from Sigma-Aldrich (St. Louis, MO, USA) was added to each well and incubated for 4 h followed by incubation for 5 min with isopropanol/4% HCl. Then, 100 μL was removed from each well and transferred to a 96-well plane and the absorbance was measured using a Varioskan Reader (Thermo Scientific) with a 595 nm filter. The values obtained were expressed as a percentage of viable cells according to Eq. (3): Cell viability ð%Þ Absorbance of samples and SAOS cells  100 ¼ : Absorbance ðcontrolÞ

ð3Þ

Transparent and flexible films of polyurethane were obtained by the method described (photo shown in Fig. 1). No catalyst was used during the synthesis. The absence of a catalyst simplifies the preparation process by eliminating the catalyst removal step, and may help to improve the biocompatibility of the materials [26]. The analysis of PU structures by infrared spectroscopy (FTIR) has been reported by several groups [1,10,27–29], and the results were used for reference. Some typical functional groups of polyurethanes were observed through the FTIR analysis, as shown by the spectra in Fig. 2(a). The absence of absorbance at 2250–2270 cm−1, which is related to NCO groups, indicates that the reaction proceeded until complete conversion of the isocyanate. A small shoulder was observed near 3500 cm−1, which is related to free N–H. The absorbance in the region near 3330 cm−1 indicates that most of the N–H groups are hydrogen bonded. Polyurethanes are capable of forming hydrogen bonds in which the N–H group of the urethane linkage is the proton donor. The bands at 1750–1730 cm−1 are assigned to the stretching vibration of free C_O groups in urethane. The shift to lower values of this band indicates the presence of C_;O hydrogen bonding stretching in urethanes. The band at 1715 cm−1 was observed in PU films, confirming the completion of hydrogen bonds in the urethane carbonyl, and the band at 1625 cm−1 is consistent with the hydrogen bonding in amides. An increase in PCL triol content causes an increase in the relative intensity of the symmetric and asymmetric stretching of C–H groups (3000–2840 cm− 1 ) and symmetric deformation of CH2 groups (1475–1450 cm−1) of the polymeric materials. The bands at 1535 and 1520 cm−1 are assigned to, respectively, C–N stretching and N–H deformation. The bands at 1150–940 cm−1 are associated with symmetric stretching of N–CO–O and stretching of C–O–C bonds in polyurethanes. The XRD patterns of PU films are shown in Fig. 2(b). The diffractograms show that both PU3 and PU12 were basically amorphous, with only a broad diffraction band at 2 = 21.6° and no defined diffraction peaks. It is known that PCL with molecular weight less than 2000 g·mol−1 is usually amorphous, as is PEG with low molecular weight, which can reduce the crystallinity of the final product [19,30]. Furthermore, PCL triol and glycerol allow the formation of crosslinked bonds in the hard and soft segments, which can reduce both the mobility of the segments and their abilities to separate phases and pack into crystals, creating an interconnected polymeric network [19,31–33]. Polyurethanes with amorphous structures can degrade more rapidly than those with semicrystalline segments [3], which contributes to the aim of this work. When polymers are placed in an

Fig. 1. Digital photography of a polyurethane film sample.

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Fig. 2. (a) FTIR spectra and typical absorption bands and (b) X-ray diffraction curves of PU3 and PU12 polyurethane films.

aqueous medium, water may permeate into the amorphous region, promoting water uptake and, consequently, the degradation [1,34]. SAXS data of PU3 and PU12 are shown in Fig. 3. SAXS is an important tool to find evidence of SS and HS phase separation. When scattering peaks are observed within SAXS data, it can indicate the presence of distinct microphases with different and more defined electron densities [35]. Local heterogeneities in the electron density of the material can lead to scattering peaks [36]. Therefore, the data in Fig. 3 imply that PU3 and PU12 both have a homogeneous structure without distinct microphases, as no scattering peaks are observed for either sample. It is known that PUs with structural segments derived from aliphatic diisocyanates have a lower degree of separation than their equivalent PUs obtained from aromatic diisocyanates [37]. The interconnected network formed through the introduction of a triol crosslinker into the PU hard segment, in addition to the introduction of a triol in the soft segment, can cause the reduction in the degree of microphase separation [36]. The presence of a crosslinker increases the aggregation of segments through covalent bonds but decreases the aggregation through hydrogen bonding of the hard segments [16,23]. The reduction in the hydrogen bonding rate and the disordering of the polymer matrix caused by the chemical crosslinking can increase the capacity of water to penetrate and reach ether/ester groups and can consequently increase hydrolytic degradation [16]. Furthermore, the increased crosslinked density and interconnectivity can be important for improving the mechanical properties, such as the maximum strain and tensile strength [38].

Fig. 3. SAXS scattering data obtained for films PU3 and PU12.

3.2. Thermal analysis The thermal stability of a polyurethane elastomer is affected by the raw materials used, the proportions of the hard and soft segments, the density and type of crosslinking bonds, and the chain extender and synthesis method [39]. Fig. 4(a) presents the TG and DTG thermograms of PU3. The initial sample mass reduction of 1–3% observed through TG and DTG, which takes place at approximately 70 °C, is probably the result of physical desorption of volatile organic components from the sample [39]. It's noted through the TG that the material degradation starts at 209 °C, approximately 91% of mass is lost at 475 °C, and the degradation ends at 640 °C. The degradation behavior is better visualized by two peaks in the DTG curve. The first has a maximum rate of weight loss at 271 °C, probably associated with the degradation of the hard segment. The second occurs with a maximum rate at 356 °C, representing the degradation of a higher content of PU3, most likely associated with the soft segment with a contribution from the hard segment. A peak is observed at 391 °C, which can be related to the decomposition of ester bonds from the PCL triol, which are more stable than ether bonds from PEG. The final degradation, which occurs above 450 °C, can be related to advanced chain fragmentation formed by the first and second stages of decomposition, as well as to secondary gasification and dehydrogenation reactions and decomposition of ashes formed in the previous steps [23]. Fig. 4(b) presents the TG and DTG thermograms from PU12. The bimodal character of degradation typical of polyurethanes [40] is better visualized in this analysis. The degradation starts at 210 °C, followed by a first stage of maximum rate of weight loss at 356 °C and 50% of weight loss up to 374 °C. The second stage has a maximum weight loss at 392 °C, with 42% of mass loss up to 482 °C. The first stage of degradation can be assigned to the decomposition of urethane bonds, and the second stage to the decomposition of polyols [31,41]. The thermal decomposition is a complex multi-stage process that is highly dependent on the degradation of the hard segment, which is the first degradation step [42]. This stage is fast, but its rate decreases with a higher content of soft segments [37]. A series of polyurethanes based on PEG, HDI and 1,4-butandiol, with different molecular weight and concentrations, were synthesized and reported elsewhere [42], and the DTG showed two main peaks: the first (~ 285–314 °C) attributed to the higher mass loss rate of the HS, while the later (~ 340–370 °C) accounts for the mass loss rate of SS. Another work [18] reports a PU based on HDI, PCL diol and cysteine with the onset of degradation from 186–198 °C. These results are in accordance with the thermal behavior observed for PU3 and PU12, and the temperature for the onset of degradation increased when compared to HDI based PU without crosslinking.

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Fig. 4. Thermogravimetric analysis and derived curves for (a) PU3 and (b) PU12.

The initial degradation pattern is identical in both samples, and a difference is presented only above 368 °C (49% of mass loss), where PU12 exhibits a better thermal stability. It's known that polyurethanes with high crosslink density have good thermal stability [26,43]. Crosslinking increases the number of intermolecular bonds, and consequently, more energy is needed to decompose the polyurethane. Furthermore, crosslinking can also restrict the diffusion of degradation products of the matrix [24]. Additionally, an increase of PCL triol content gives more thermal stability through the ester bonds of polyurethane [39], which resulted in a better thermal stability of PU12. The DSC first and second heating scans of PU3 and PU12 are shown in Fig. 5. The first heating run showed an endothermic transition for both samples at 43 °C that may be related to the melting of remaining crystalline regions rich in PEG. The cooling rate used was not slow enough to allow the recrystallization of these regions, and therefore the same transitions were not observed in the second heating run. The second heating scan obtained from the DSC analysis showed no abrupt transition within a narrow temperature range, but instead showed indistinct transitions in a wide temperature range. If the hard phase and soft phase become completely immiscible in a segmented PU structure, two separate phase transition points are frequently observed as two clearly different glass transition temperatures for the soft and hard segments [37]. The possibility of crosslinked bonds in the soft and in the hard segment by means of PCL triol and glycerol, respectively, results in a very large mixture of phases in the final polymer structure, and it has become more difficult to observe a distinct and well-defined transition for each segment in these samples than in other polyurethane systems [39,43,44]. This result was already noted in the SAXS analysis. 3.3. Mechanical tests Fig. 6 shows the tensile stress–strain curves for the samples. The films showed flexibility and a nonlinear behavior typical of polyurethanes. The average values of maximum strain (εr), tensile strength

(σmax) and elastic modulus (E) are shown in Table 1. The mechanical behavior of polyurethanes is related to several factors, such as crystallinity, concentration and interconnectivity of hard segments, the ability of soft segments to crystallize with the applied stress and also to the presence of hard segments in the soft segment, which provides a mixture of phases [45]. Both samples present an amorphous structure, as already indicated, and contain hard segments with the same composition. The difference in the measured mechanical properties can be related to the difference of the soft segments of the polyurethane samples in this work. PU12 presented lower E and greater εr than did PU3. The increment in PCL triol content, with a consequent increase in the crosslinked density, can be an important factor related to the increased values of σmax and εr [38]. Interconnectivity provided by the increased crosslinking density tends to limit the mobility of the chains and causes an increase in the values of mechanical properties, among other properties [45,46]. 3.4. Degradation study The hydrolytic degradation plays a key role in the development of materials applied to the biomedical field. The hydrolysis of ester-based polymers involves three stages. The first is the incubation stage, in which absorption of water occurs. The second is the induction stage, during which the polymer chain is broken via ester bonds. Finally, there is the erosion stage, wherein the water-soluble entities, such as PEG blocks and oligomers, are dissolved in the buffer solution, with corresponding polymer mass loss [47,48]. Whereas the primary mechanism of hydrolytic degradation of polyurethanes is the hydrolysis of the ester and urethane groups, it is important to check the degree of water absorption of such polymers [23], which can also characterize their hydrophilicity. Fig. 7 presents the absorption rate measured for the samples under study after immersion in deionized water. The water uptake during the first hour was over 60% for both samples and continued to increase for 6 h, at which time the absorption was nearly complete, with only a slight increase after

Fig. 5. Differential scanning calorimetry analysis of (a) first heating run and (b) second heating run of polyurethane films.

B.R. Barrioni et al. / Materials Science and Engineering C 52 (2015) 22–30

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Fig. 6. Tensile stress–strain curves for polyurethanes PU3 and PU12.

48 h of analysis. The amount of water absorption of the polymer during the study period reached 109% for PU3 and 92% for PU12. When placed in an aqueous medium, water may permeate into the amorphous region of PU samples, which are mainly amorphous, as evidenced by XRD, and are thus favorable for water absorption [34]. Additionally, PUs with higher PCL content absorb less water, while an increase in PEG content results in PUs that are more hydrophilic. The increased PEG content in PU3 relative to PU12 was a crucial factor in water absorption, making PU3 more hydrophilic, as was also indicated by the Contact Angle test with water, where for PU3 the result was 75 ± 2°, whereas for PU12 the result was 86 ± 1°. The smaller the angle, the greater is the interaction with water, and the greater is the material hydrophilicity. Contact angles above 90° indicate hydrophobicity of the surface [49]. Hydrophilicity is important in the biomedical field because the wettability affects the interaction of cells with the materials. PU3 and PU12 showed hydrogel properties, with a crosslinked structure that can absorb large amounts of water. For applications as biomaterials, hydrogels offer the advantage of compatibility with the internal environment of the body, which has a high water content [50]. To verify the induction period, PU films were subjected to hydrolysis in SBF buffer pH 7.4 for 90 days, at 37 °C. The remaining weight of the films after immersion in SBF for different time periods is shown in Fig. 8(a), with values of 90.0 ± 2.7% for PU3 and 87.8 ± 2.6% for PU12 after 90 days of immersion. In general, polyurethanes having soft segments consisting of polyesters are highly susceptible to hydrolysis due to the ester bonds [14], a factor that may have resulted in the greater degradation in SBF for the PU12 samples. The high crosslinking density of the PU chains can lead to the lower degradation rate in SBF, as the degradation of these connections is required prior to the erosion process of the polymer [12,47]. To assess the degradation by mechanisms of hydrolysis, techniques for speeding up the process are used because the verification in vivo is more time consuming and laborious. The degradation process can be accelerated in two ways; first by increasing the temperature of the degradation medium, and second by the addition of hydroxyl ions [47]. The remaining mass of the degraded PUs samples after different periods of time in NaOH 1 mol·L−1 at 60 °C is shown in Fig. 8(b) to illustrate the PU erosion process. Table 1 Mechanical properties of polyurethane samples obtained by tensile testing. Sample

E (MPa)

σmáx (MPa)

εr (%)

PU3 PU12

2.2 ± 0.2 2.0 ± 0.5

2.7 ± 0.6 3.6 ± 0.7

177 ± 25 425 ± 70

Fig. 7. Kinetics of water absorption of polyurethane samples after immersion in deionized water for different periods of time. The results represent the average of three measurements with p b 0.05.

The remaining mass after 48 h of degradation in NaOH solution was 51.0 ± 0.6% and 53.3 ± 0.8% for PU12 and PU3, respectively. The PU sample with a higher PCL triol content (PU12) showed a higher degradation in NaOH, confirming the results obtained in the SBF degradation analysis. It was possible to observe the increased fragility of the material after degradation. The increased pH degrades the soft segment mainly due to the hydrolysis of ester groups but can also degrade the hard segment [51]. Glycerol is a hydrolysable chain extender that can increase the rate of degradation. Fig. 9 presents the results of the FTIR analysis of PU3 before degradation and after 2, 24 and 48 h of degradation in NaOH. Increases in absorption intensity near 1618 cm−1 and 1448 cm−1 after degradation of the sample may be related to the stretching of COO− groups from carboxylic acid that have been formed [28,47]. The hydrolytic degradation of ester groups may form carboxyl and hydroxyl groups [23]. The gradual disappearance of the FTIR absorption band at approximately 1710 cm−1 was also observed and assigned to the C_O stretching of the urethane group with the presence of hydrogen bonds [52]. The absorption in the region between 1250 and 1520 cm−1, related to the amide groups [28,29], showed an intensity reduction. There has been a slight increase in absorption at 3326 cm−1, related to N–H groups. A proposed mechanism for hydrolytic degradation of polyurethanes involves breaking urethane groups, with the formation of amines and hydroxyl groups and the release of CO2 [23,47]. Two hydrolytic degradation pathways of PU samples of this work are schematically shown in Fig. 10. The high concentration of crosslinking and the extensive mixture of phases contribute to the chemical stability of the polyurethanes produced in this work, which slows the degradation process. The PCL hydrolysis at high pH (although not biologically relevant) has major advantages, as it introduces OH and COOH groups on its surface, yielding a hydrophilic substrate that will increase the degradation [51]. For in vivo applications, it is known that bio-molecules significantly increase polymer degradation [23]. The accelerated degradation test shows that the analyzed PU is susceptible to hydrolytic degradation, confirming their biodegradability and indicating their potential for use as biomaterials, because the main mechanism of in vivo degradation is through hydrolysis. PU12 showed a better degradation performance than PU3, with faster rates of degradation in SBF and in basic medium, probably due to the increased content of ester groups, which are highly susceptible to hydrolysis. The PUs used in this study showed relatively slow rates of degradation. Their use would be more suitable for applications as

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Fig. 8. Remaining mass of polyurethane samples (a) after incubation in SBF solution at 37 °C and (b) after degradation in NaOH solution 1 mol·L−1 at 60 °C for different periods of time. The results represent the average of three measurements with p b 0.05.

biomaterials where a longer time of regeneration of the tissue is needed. Other biodegradable polymers used as biomaterials that have a slow degradation rate are reported in several works, for example, materials based on PCL [53,54], poly(propylene fumarate) [55] and poly(L-lactide) [56]. Furthermore, the mechanical properties are also an important biomaterial selection. For example, cartilage tissue has an elastic modulus in the range of 0.7–15.3 MPa and a tensile strength of 3.7–10.5 MPa [57], values which are comparable with the results obtained for PU12.

Extra procedures can be performed to reduce cytotoxicity and improve biocompatibility. Surface treatments are performed in several applications with specific molecules or other agents to promote greater biocompatibility and biofunctionality of the material, while maintaining their physical and mechanical properties. One example is the synthesis of composites of PU/Bioglass® leading to an increase in the polymer bioactivity and promoting the formation carbonated hydroxyapatite layers on their surfaces, which bind to bone and are thus promising materials for tissue engineering applications.

3.5. Cytotoxicity assay by MTT 4. Conclusions Fig. 11 shows the results of MTT assays for PU films after 24 hour contact of primary osteoblast cultures with samples. This assay is used specifically for mitochondrial function and evaluated the toxicity of the material from the cell viability. Cytotoxicity can be rated based on the cell viability relative to controls, where activity relative to controls is less than 30% (severe cytotoxicity), between 30 and 60% (moderate cytotoxicity), between 60 and 90% (slight cytotoxicity) or greater than 90% (not cytotoxic) [58,59]. There was a reduction of 47.7% in the cell viability compared to control for PU3 and 54.3% for PU12, which can be considered as a moderate cytotoxicity for both samples, but PU12 presented better results than PU3. One hypothesis for the reduction in cell viability is that the hard segment of PU can degrade and the degradation products can have toxic effects on osteoblast cells [60], reducing the cell viability. It is necessary to determine the PU degradation products and study the release of these products to clarify the toxicity effect.

Fig. 9. FTIR of PU3 (a) before degradation, (b) after 2 hour assay, (c) after 24 hour assay and (d) after 48 hour assay (NaOH 1 mol·L−1, 60 °C).

Biodegradable polyurethane films were obtained through the prepolymer method based on HDI reactions. A relatively simple procedure of synthesis was described that uses an aliphatic diisocyanate with related nontoxic degradation products. Different compositions of polyols PEG and PCL triol were used and the reaction proceeded without the use of a catalyst. The presence of PCL triol and glycerol enabled the formation of a crosslinked structure in the samples, creating an interconnected polymeric network. As a consequence, a homogeneous structure without distinct hard and soft segments was formed in the samples and the structure was confirmed through SAXS and DSC analysis. This phase mixture resulted in polymers with an amorphous character, which allows water to penetrate into the structure and increases hydrolytic degradation. The PU water uptake proved to be high, with greater uptake for PU3, and the samples absorbed water without dissolving, which is a hydrogel property reported for several biomedical applications. PU12 showed faster rates of degradation than PU3 in SBF and in basic medium. The degradation results imply that these PUs are more suitable as biomaterials for which a longer period of application is needed. PU12 also presented better thermal stability than PU3, probably as a result of increased PCL triol content. Mechanical tests showed the flexible behavior of the polyurethane films, with the high values of tensile strain for both PU3 and PU12 being the most striking feature. The greater crosslinked content had an influence on the mechanical properties, and PU12 showed better properties. The elastic modulus and tensile

Fig. 10. Schematic hydrolytic degradation pathways for polyurethane samples.

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Fig. 11. Cell viability of PU films by MTT assay for 24 h at a significance level of 0.05% (*represents significant difference compared to control).

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