Catalyst-free synthesis of high elongation degradable polyurethanes containing varying ratios of isosorbide and polycaprolactone: physical properties and biocompatibility

Article

Abstract

Biocompatible and biodegradable polyurethanes were prepared with fixed aliphatic diisocyanate level and varying ratios of isosorbide, and PCL diol via a simple one-shot polymerization without a catalyst. The successful synthesis of the polyurethanes was confirmed by gel permeation chromatography, 1H-nuclear magnetic resonance and Fourier transform-infrared spectroscopies and the thermal properties were determined by differential scanning calorimetry and showed glass transition temperatures of around 30–35 °C. The degradation tests were performed at 37 °C in phosphate buffer solution (approx. pH 7.3) and showed a mass loss of around 5 % after 12 weeks, except for the polymer with the highest isosorbide content which showed an initial rapid mass loss. The in vitro cytocompatibility test results following culture of osteoblasts on the polymer surface showed that relative cell number on all of the polyurethane films after 5 days of cultured on polymer films was lower compared to the proliferation rate on the optimized tissue culture plastic. These polymers offer significant promise due to the simplicity of the synthesis and the controlled degradation.

1 Introduction

Synthetic biodegradable polymers have great potential for surgical use as control over their mechanical properties and degradation rates may be achieved for a particular application [1]. These polymers are especially useful in tissue engineering approaches due to their potential ability to enable cell adhesion, migration, proliferation and differentiation [2, 3] and their temporary nature. These materials could also be implanted in the human body by injection, having potential applications in the areas of sustained drug delivery, gene delivery and tissue engineering [4].

Polyurethanes can be prepared from reaction between alcohols and isocyanate forming urethane linkages. There are various methods for fabricating polyurethanes either with organic solvents or solvent-free. The most widely used is the one-shot process, where direct mixing of monomers and concurrent addition of catalysts and other additives are used [5]. Recently, many papers on biodegradable polyurethanes have appeared [6]. Polyurethanes are a class of biodegradable polymers that have been applied as a tissue-engineering scaffold as they show low cytotoxicity in vitro and in vivo [7, 8, 9, 10, 11]. Polyurethanes created by reacting polyesters or polyethers with polyisocyanates provide matrices with good mechanical and physical properties, and with the flexibility and haemocompatibility needed for use in biomedical applications [12, 13, 14]. In fact, a number of non-biodegradable urethanes have been utilized in blood-contact applications such as, heart valves, dialysis membranes, breast implants, aortic grafts, and bone adhesives [12, 13].

For tissue engineering, biodegradable polyurethanes can be obtained following two strategies, using either biodegradable soft segments or biodegradable hard segments. In the first case, polyurethanes such as those containing PCL have been obtained [15, 16] along with PLA- [17], PEO- [18, 19] and poly(3-hydroxybutyrate diol)- [20]. On the other hand, as the hard segment is formed by the diisocyanate and the chain extender, it also provides two possibilities to obtain biodegradable polyurethanes. The diisocyanate can be designed or the chain extender can be chosen from a variety of biologically relevant molecules. For example, biodegradable hard segments have been obtained with aliphatic diisocyanate such as 1,4-butane diisocyanate [15], ethyl lysine diisocyanate [16], hexamethylene diisocyanate(HDI) [21] and methyl lysine diisocyanate [22].

Dianhydrohexitols(DAHs): 1,4:3,6-dianhydro-D-glucitol or isosorbide; 1,4:3,6-dianhydro-d-mannitol or isomannide; and 1,4:3,6-dianhydro-l-iditol or isoidide are good examples of such raw materials which are produced from the glucose of natural feedstock (i.e. starch). The most important differences among the DAHs isomers are the position of the two hydroxyl and proton groups that provide variations in spatial configuration, chemical properties, and physical properties. The bond chirality in the DAHs is a fundamental parameter for modulation of polymers properties based on them [23, 24, 25, 26]. These alcohols derived from renewable agro-resources [27] have attracted considerable attention in the field of materials science [28, 29, 30, 31]. As to their non-toxicity, chirality, and rigidity, isosorbide is a promising material for bio-based monomers [32]. It has been considered in various polymer systems like polyester, polyether, and polyurethane.

The FDA has approved isosorbide is for GRAS (generally recognized as safe) materials [33]. It has been generated by potential industrial applications that the preparation of isosorbide nitrates used in cardiac or vascular disease and alkyl derivatives used as solvents in pharmaceutical or cosmetic compositions [23].

To target our polymer design, we considered the following factors : (a) as a degradation mechanism, we chose hydrolysis to minimize individual differences in degradation characteristics caused by enzymes [34]; (b) to provide a hydrolyzable chemical bond, we chose ester and urethane; and (c) we required that specific monomers be nontoxic; (d) we could control hardness and degradation rate. To design biocompatible and biodegradable polyurethane, generally, aliphatic diisocyanates are chosen, because their degradation product is non-toxic. Polyesters such as PCL are also chosen as they can be hydrolytically degraded to give caproic acid. We chose isosorbide, as the di-alcohol monomer because it satisfies these requirements. The polyurethane presented here has been synthesized before but they were synthesized two-step polymerization and used catalyst [35].

In this study, we present the synthesis of a family of biocompatible and biodegradable polyurethanes synthesized by a simple catalyst-free, one-shot polymerization [36] of hexamethylene diisocyanate (HDI), PCL diol (Mw 2000), and isosorbide. In order to avoid the potential toxicity, we did not use any chemical catalysts.

The chemical structure was confirmed by 1H nuclear magnetic resonance (NMR) and Fourier transform-infrared (FT-IR) spectroscopy, and the physical properties were determined by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). We also measured the mechanical properties, degradation rate and cytocompatibility which in general correlated with the ratio of isosorbide to PCL diol.

2 Experimental section

2.1 Materials and instruments

Isosorbide (98 %) and hexamethylene diisocyanate (HDI, 99 %) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polycaprolactonediol2000(CAPA 2201A = Mw 2000) was purchased from Kangnam Chemical Company and phosphate buffered saline(approx. pH 7.3) was obtained from OXOIDLTD. (Basingstoke, Hampshire, England) All chemicals were used without further purification.

2.2 Preparation of polyurethane

The polyurethane series were prepared by the one-shot method shown in Scheme 1. The ingredients were mixed together at once and allowed to cure [36]. Briefly, synthesis was carried out in a 100 mL round-bottomed four neck flask with reactant stoichiometry of 1:1 of diisocyanate:di-alcohol(PCL diol and isosorbide) under a dry nitrogen atmosphere. A nitrogen flushed four neck flask equipped with a mechanical stirrer, thermometer, and condenser was charged with 27.37 mmol of PCL diol(54.74 g) and 27.37 mmol of isosorbide (4.0 g) [reactant stoichiometry and precursor weights are given as an example for PU1 (see Table 1)]. The flask was maintained at 80 °C and stirred under a nitrogen atmosphere. After the solid contents were melted completely, 54.74 mmol of HDI (9.21 g) was added to the reaction system and stirred for 2 min. The sample was allowed to stand in a Teflon dish to cure for 12 h at 150 °C. The synthesized polyurethanes were dissolved in N,N-dimethylformamide (DMF) and the solution was precipitated into a large amount of isopropyl alcohol and washed with isopropyl alcohol. The product was dried at 40 °C for 72 h under vacuum and stored in desiccators. PU 2, PU 3, and PU 4 were prepared by similar procedures as described above. Polymer yields of 89, 77, 60 and 53 % were obtained, respectively. The formulation used is reported in Table 1.
Scheme 1

Polyurethane series synthesis

Table 1

Polyurethane series with different isosorbide and PCL diol contents

Polyurethane

Mole ratio/weight (g)

Yield (%)

HDI (g)

Isosorbide (g)

PCL diol

(Mw 2000) (g)

PU 1

8/(9.21)

4/(4.00)

4/(54.74)

89

PU 2

8/(9.21)

5/(5.00)

3/(41.06)

77

PU 3

8/(9.21)

6/(6.00)

2/(27.37)

60

PU 4

8/(9.21)

7/(7.00)

1/(13.69)

53

2.3 Polymer characterization

1H-NMR spectra for the synthesized polyurethanes were recorded with a BrukerAvance 600 spectrometer at 600 MHz and performed at ambient temperature with 5 % (w/v) polymer solution in CDCl3. Tetramethylsilane was used as the internal reference.

FT-IR spectra were obtained at room temperature using a Bio-Rad Excaliber TS-3000MX (Bio-Rad, Tokyo, Japan) in the range 4,000–800 cm−1. A 2.5 % solution of polymer in chloroform was placed directly onto a KBr pellet (Sigma-Aldrich, USA). Subsequent evaporation of chloroform at 50 °C under vacuum was performed for 2 h. The spectra were also checked for evidence of residual solvent.

2.4 Molecular weight

The weight average (Mw) and number average (Mn) molecular weights of polyurethanes were measured by GPC, using a Futecs NP-4000 instrument (Futecs, South Korea) equipped with a model P-4000 pump, a model AT-4000 column oven, GPC KF-804 column and a Shodex (Shodex, Japan) R1-101 refractive index detector. Tetrahydrofuran (THF) was used as the eluent at a flow rate of 1.0 mL/min, and a sample concentration of 2.5 mg/mL was used. Polystyrene was used as a standard.

2.5 Thermal analysis

DSC data were recorded with a PerkinElmer PYRIS Diamond DSC instrument. Specimens (approximately 10 mg) were sealed in a DSC aluminum pan before being placed in the calorimeter. The samples were cooled to −70 °C, and then heated to 250 °C at a rate of 10 °C min−1 using a nitrogen atmosphere. Thermogravimetric analysis (TGA) tests were conducted on the samples using a ShimadzuTGA 50 (Shimadzu, Japan) equipment operating from 30 to 600 °C at a heating rate of 10 °C min−1 and under a nitrogen atmosphere. The thermal decomposition temperature was defined as the temperature corresponding to the maximum rate of weight loss.

2.6 Preparation of polymer films

Following synthesis of the polymer, films of the polymer for mechanical testing and degradation testing measurement were prepared by solvent casting in DMF at a 10 % polymer concentration followed by air drying to give films of thickness 0.2 mm.

2.7 Mechanical properties

Tensile strength and elongation at break of the polyurethanes were measured on an Instron universal testing machine (Model 3344, Instron Engineering Corp., Canton, MA, USA) at a crosshead speed of 10 mm min−1 at room temperature. The sample was prepared with a dumbbell-shaped cutter. The thickness and width of the specimens were 0.2 mm and 5 mm, respectively. The length of the sample between the grips of the testing machine was 15 mm. Five sample measurements were conducted for each polyurethane, and the results were averaged to obtain a mean and standard deviation.

2.8 Contact angle and surface energy

The wetting ability of polymer surface was evaluated on the basis of the contact angle measurements using a PHX 300 contact angle equipment (S.E.O, South Korea). Tests were carried using the sessile drop mode with ultra-pure water that was used to represent polar characteristics. Droplets of approximately 5 μL of the test liquid were placed on polymer specimens using a manual syringe. The drop profile was then recorded at 1 s intervals for 1 min, and the measurements were carried out on triplicate samples. Evaluation of the contact angle values was carried out using PHX 300 software. The polyurethane series were dried for 24 h prior to contact angle measurement. The calculation of the surface energy was carried out using Girifalco-Good-Fowkes-Young method via PHX software.

2.9 Degradation test

Polyurethane film (Ф = 12 mm, n = 5) degradation was quantified by changes in dry weight. The polyurethane samples were degraded for 2, 4, 8, and 12 weeks. Dry films were weighed (w0) and immersed in a conical tube containing 10 mL phosphate buffered solution (approx. pH = 7.3). The degradation was conducted at 37 ± 1.5 °C in an incubator (Leec Limited, Colwick Nottingham, England). Samples were taken at intervals, rinsed with water, dried in a vacuum oven for 2 days at 50 °C, and weighed (wt), after which they were discarded. The weight remaining was calculated as:
$$ {\text{Weight}}\,{\text{remaining}}\,\left( \% \right) = {\text{W}}_{\text{t}} /{\text{W}}_{0} \times 100. $$

2.10 Cell culture

Polyurethane films (n = 3, ϕ = 14 mm) were sterilized by soaking them in 50, 70 and 100 % ethanol for 30 min prior to use and then they were dried for 2 h. Rat bone marrow mesenchymal stromal cells, were maintained in standard T75 tissue culture flasks in normal growth medium composed of α-MEM (Invitrogen, Paisley, UK) supplemented with 10 % fetal bovine serum (Gibco, Daejeon, South Korea) and 1 % penicillin/streptomycin (Gibco, Daejeon, South Korea). Prior to cell seeding, sections were cut from films of the synthesized polyurethanes, placed into wells of 24 well plates and seeded with 1 × 104 cells suspended in 1 mL normal growth medium and maintained at 37 °C with 5 % CO2 for subsequent time course analysis of cell number.

2.11 Cell proliferation

Cells were cultured on the polyurethane films (n = 3) (the films were held down on the bottom of the culture plates with Polytetrafluoroethylene (PTFE) insert rings) in 24 well plates for 1, 3, and 5 days. The cell proliferation was determined at these time periods using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay according to the manufacturer’s instructions (CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit; CellTiter 96®, Promega, Madison, WI, USA). MTS is transformed by mitochondrial enzyme, which is active in living cells, to yield a purple of formazan products. The intensity of the color produced is directly related to the number of viable cells, and thus to their proliferation in vitro. The cells were then incubated for a 1, 3 and 5 days and cell proliferation measured at these time periods by the MTS reagent. MTS solution was freshly prepared in growth medium. 300 μL of MTS reagent were added to each well, followed by incubation in humidified, 5 % CO2 atmosphere. Absorbance was measured at 490 nm using a spectrophotometer (Bio-Rad, Seoul, South Korea). A blank experiment detecting cell-free background absorbance was also performed in parallel. Results were expressed as relative MTS activity as compared to control conditions (cell-free background absorbance).

3 Results and discussions

FT-IR is useful for rapid characterization of the functional groups present in the polymers. The FT-IR spectrum (Fig. 1 and 2) confirmed that the polyurethane structures had a pronounced free amide group (–NH–) peak at 3,300–3,380 cm−1, a carbonyl group (C=O) stretching transmittance at 1,730–1,680 cm−1 [37] and hydrogen bonded NH stretching bands were seen at 1,526 cm−1 [38]. Asymmetric and symmetric CH2 stretching bands were seen in the region around 2,940 and 2,860 cm−1, respectively. The C–C stretching vibration bands and the C–H bending vibration band were seen in the range from 1,500 to 600 cm−1 [39]. In the Fig. 1, polyurethane samples showed the complete disappearance of the isocyanate peaks at 2,280 cm−1 [40] indicating that there were no unreacted isocyanate groups all polyurethane samples. After polymerization, PCL diol and isosorbide showed disappearance of the –OH peaks in the region around 3,440–3,540 cm−1 and 3,370 cm−1, respectively. There appeared the new peak at 1,526 cm−1 which was caused by amide (N–H) bending vibration of polyurethanes. In the Fig. 2, the IR spectra of PU1, PU2, PU3 and PU4 showed obvious differences in the intensity and shape of ester related [41] absorption bands at 1,730 and 1,690 cm−1; the band shift to 1,690 cm−1 indicated that ratio of isosorbide and PCL diol influenced the chemical structure of the ester groups. In addition, the amide (N–H) stretching peak intensity assigned at 3,330 and 1,526 cm−1 showed obvious signs of increasing from PU1 to PU4, indicating that urethane bonds were formed between the isosorbide and isocyanate (–N=C=O) group of HDI [37, 38]. Overall, we confirmed that the synthesis was successful.
Fig. 1

Fourier transform-infrared (FT-IR) spectra of (a) polyurethane, (b) HDI, (c) isosorbide, (d) PCL diol 2000

Fig. 2

Fourier transform-infrared (FT-IR) spectra of (a) PU 1, (b) PU 2, (c) PU 3, and (d) PU 4

The chemical structure of polyurethane was characterized by 13C NMR spectra (Fig. 3) and 1H NMR spectra (Fig. 4). In the 13C NMR spectra (Fig. 3b) of hexamethylene diisocyanate shows the isocyanate (–N=C=O) carbon peak 122 ppm. After the polymerization reaction, hexamethylene diisocyanate showed disappearance of the isocyanate carbon peaks in the region around 122 ppm (Fig. 3a). The 13C peak of isocyanate carbon of the new urethane linkage was observed around 156 ppm (Fig. 3a). This shift was attributed to the attachment of the hydroxyl group to the isocyanate functional groups in the formation of the urethane bond. The Fig. 4a shows the 1H NMR spectrum of polyurethane in CDCl3, in which all proton signals belonging to isosorbide (Fig. 4c), PCL diol 2000 (Fig. 4d), and HDI (Fig. 4b) segments are confirmed. The signals (Fig. 4c) occurring from 3.0 to 4.8 and at 2.3 ppm could be reasonably assigned to methylene protons and hydroxy proton of isosorbide, respectively. The peaks of isosorbide shifted to the urethane bond from 3.5 to 5.2 ppm. Then the hydroxyl proton of isosorbide was disappeared after polymerization. In addition, 3.1 ppm (Fig. 4a) could be reasonably assigned to amine proton of urethane linkage.
Fig. 3

13C Nuclear magnetic resonance (NMR) spectra of (a) PU 1, (b) HDI, (c) isosorbide, (d) PCL diol 2000

Fig. 4

1H Nuclear magnetic resonance (NMR) spectra of (a) PU 1, (b) HDI, (c) isosorbide, (d) PCL diol 2000

The molecular weight and thermal properties of the polyurethanes are summarized in Table 2. The weight average molecular weights (Mw) ranged from 103,336 to 19,139. Polyurethanes with higher levels of isosorbide may have yielded lower Mw compared to polyurethanes with low levels of isosorbide due to presence of a secondary hydroxyl group which is lower in reactivity compared to the primary hydroxyl group [42] of PCL diol. For the PU series presented in this paper, the effect of the lower reactive secondary hydroxyl of isosorbide appears tobe quite evident with PU 1 which has the lowest isosorbide content and showing a higher Mw. This is also supported by the polymer yield data.
Table 2

Molecular weights and thermal properties of polyurethane series

 

Mwa

Mnb

PDIc

Tg (°C)d

Tm (°C)e

Water (°)f

Surface energyg

PU 1

103,336

50,986

2.0267

31.14 ± 0.37

61.4 ± 4.4

49.1 ± 4.48

PU 2

57,937

28,075

2.0637

31.85 ± 0.45

58.3 ± 0.6

52.3 ± 0.45

PU 3

41,485

20,321

2.0415

32.12 ± 0.22

56.5 ± 5.7

54.0 ± 5.8

PU 4

19,139

16,789

1.1399

42.50 ± 0.32

54.6 ± 1.3

55.9 ± 1.3

aThe weight average molecular weights

bThe number average molecular weights

cPolydispersity index. Mw/Mn

dGlass transition temperature

eMelting temperature

fWater contact angle

gSurface energy (mN/m)

It is important to understand the thermal behavior of polymers developed for biomedical applications, because it determines the physical properties of the materials and the processability. For example, if the glass transition temperature (Tg) value of the polymer is above that of body temperature, the polymer is more rigid structure [43]. In contrast, if the Tg value is below body temperature, this indicates that the material is in the rubbery state. Figure 5 and Table 2 shows the data obtained from DSC measurements of the four synthesized polyurethanes. DSC analysis was conducted to characterize the thermal behaviors of polyurethanes. Only one glass transition was detected for all samples. The Tg’s of PU 1, PU 2, PU 3, and PU 4 were 31.14, 31.85, 32.12, and 42.5 °C, respectively.
Fig. 5

DSC curves of polyurethanes: PU 1, PU 2, PU 3, and PU 4

Thermogravimetric analysis (TGA) was used to confirm the composition of the copolymer as well as to evaluate the thermal stability of the polyesters. Figure 6 shows the TGA scan results for PU series. PU 1, PU 2, PU 3, and PU 4 showed their 10 % weight loss at about 343, 334, 312, and 303 °C and 99 % weight loss at about 566, 589, 590, and 599 °C, respectively.TGA curves showed an increase in the initial thermal stability of polyurethane by increasing amount of PCL diol. It can be said that primary alcohol of PCL diol participation into the polymer chain length will be increased over molecular weight.
Fig. 6

TGA curves of polyurethanes: PU 1, PU 2, PU 3, and PU 4

Figure 7 shows the ultimate tensile strength and Young’s modulus data for the polyurethanes. The higher molecular weight polyurethane was obtained with PU 1, and this gave a mean stiffness of 8.2 ± 2.1 MPa. The ultimate tensile strength was 9.8 ± 0.95 MPa. Low molecular weight polyurethane was obtained with PU 4, with a mean stiffness of 42 ± 4.1 MPa. The ultimate tensile strength was 5.4 ± 4.1 MPa. There was a relationship between Mw and UTS with increasing UTS with increasing Mw. In addition, there was a relationship between hard segment and stiffness [56, 57]. The stiffness increased with increasing isosorbide (hard segment) content.
Fig. 7

a Ultimate tensile stress (UTS), b Young’s modulus and c example tensile stress-strain curves for polyurethane series, d strain at break

Significant attention has been paid to the surface structures and properties of polymeric materials, because the functional groups on the surface play an important role in the interactions among materials with cell and biological molecules [41, 44]. A contact angle study is a recognized technique to estimate surface properties. As show in Table 2, all of the polyurethane water contact angle were <62°. It is generally agreed that hydrophilic surface have contact angle with water in the range 1°–30°, while hydrophobic surface are greater than 90°. Thus the polyurethane presented here show values somewhere in between these range. As the content of isosorbide which hydrophilic unit monomer increased, the water contact angle of polyurethanes was decreased. The results could suggest that we can control hydrophilicity of polyurethanes.

The degradation characteristics of the polyurethane series was examined in vitro. Polyurethane samples were immersed in a phosphate buffer solution at 37 °C. Figure 8 shows the percentage weight loss of the polymers with time. It is evident that the polyurethane series are undergoing slow degradation. SEM (Model JSM-5410LV, JEOL, Japan) photographs showed the changes in film surface after degradation. Initially all the surfaces appear relatively smooth, with few defects. Between time zero and 2 weeks, PU 1 2 and 3 show a weight loss of around 2 %. However PU 4 shows a loss of around 7 %. For PU 2, 3 and 4, between 2 and 12 weeks there is a continual slow weight loss and at the end of the test, the weight lost is around 3–4 %. For the PU 4 however, the rate of weight loss from 2 to 12 weeks is much slower compared to 0–2 weeks and is very similar to PU 1 to 3. The overall weight loss for PU 4 at 12 weeks is around 10 %. The polyurethane series were undergoing slow degradation then PU 4 had the fastest degradation of the polyurethane series, because the high molecular weight of the polyurethane is slower than the rate of degradation of low molecular weight. For all polymers, the surface became rougher with time (Fig. 9), although the extent of changes differed depending on the type of material tested and is clearly significantly higher for the PU 4.
Fig. 8

In vitro degradation profile of polyurethane series. Residual weight is shown as a function of degradation time

Fig. 9

Scanning electron micrographs of hydrolytic degraded polyurethanes in phosphate buffer solution during 3 months. a PU 1, b PU 2, c PU 3, and d PU 4

MTS assay provides a good estimate of cell survival based on bioreduction of MTS to aqueous soluble colored formazan crystals accomplished by dehydrogenase enzymes found in metabolically active cells [45]. Figure 10 shows the results of MTS absorbance values for rat bone marrow stromal cells (MSC) adhesion to and proliferation on the polyurethane films compared to the control (tissue culture plastic). The MTS assay was implemented to measure MSC cells growth and since the MTS assay washes off the non-adherent cells and it is specific to viable cells, it reflects not only the ability of the polyurethane film to promote cell adhesion and proliferation, but also its cytotoxicity. In comparison with the control, cells showed lower viability from 1 to 5 days seeded on polyurethane surfaces. It should be borne in mind that the tissue culture plastic is optimized for cell culture and is designed to promote high cell growth rates. However, cells continued to proliferate and increase. Despite the cells showing lower rate of proliferation than the control, it can be suggested that the polyurethane series synthesized here are cytocompatible and may useful for biomedical applications including wound healing, drug delivery, and tissue engineering.
Fig. 10

MTS assay of rat bone marrow stromal cells (MSC) cultured on polyurethanes

Figure 10 MTS assay of rat bone marrow stromal cells (MSC) cultured on polyurethanes wells showed that there were more metabolically active cells on polyurethanes during the first 5 days. Data are mean ± SD. Significantly different from the control (*P < 0.05, **P < 0.01, ANOVA, n = 3). Normalized values shown.

4 Discussion

Medical elastomers are the materials of choice for the design of a number of implantable devices. Cardiovascular implants, artificial skin and tissue adhesion barriers are typical examples. For some applications it would be advantageous if in addition to the elastic properties, the implants had a bioresorbable and/or biodegradable Functionality. Progressive degradation of the implant material may then be accompanied by the formation of the new tissue and/or organ [46, 47, 48, 49]. Biodegradable polyurethane elastomers are candidate materials for such implants [31].

In this study, a family of aliphatic biodegradable/biocompatible polyurethane elastomers were synthesized and the physical, mechanical, and thermal properties were compared. The family of polyurethanes was synthesized by simple one-shot polymerization without any catalyst. There is already data in the literature showing two-step polymerization methods with the addition of a catalyst [35] but these methods needed additional post-polymerization procedures to remove the catalyst and organic solvents via various purification methods. Our one-shot polymerization process is very simple and the absence of a catalyst reduces toxicity effects as well as reducing the need for surfactants. In addition, the chemistry presented here allows can control of the physical properties and degradation rate. The polyurethanes were synthesized using a soft segment polycaprolactonediol and a hard segment isosorbide and the segmented polyurethanes were synthesized with various ratios of soft segments to hard segments and evaluated their physical properties and invitro degradation. The variation of the glass-transition temperature (Tg) of the soft segment as a function of composition or segmental chemical structure has been suggested as an indicator of the degree of microphase separation in thermoplastic polyurethane elastomers [50]. In general, the Tg value of soft segments decreases with increasing soft-segment molecular weight and decreasing diisocyanate content [51] albeit the changes can be quite small. In polyurethanes based on aliphatic diisocyanate, the factors affecting Tg include the crystallization of the soft-segment and hard-segment components, the steric hindrance of the hard-segment unit in a hydrogen bonding process, and the inherent solubility of the hard and soft components [52]. For the polyurethanes synthesized in this study, Tg increased with decreasing content and molecular weight of the soft segment as expected [51].

The polyurethanes were found to be flexible with tensile strengths from 5.4 ± 4.1 to 9.8 ± 0.95 MPa, stiffness from 8.2 ± 2.1 to 42 ± 4.1 MPa and breaking strains from 97 to 1,245 % for the polyurethane films. The tensile strengths of the films were comparable with human bladder tissue (2.6 MPa) [55] while the breaking strains were generally greater than that of human bladder. The mechanical properties of the films were shown to be related to the molecular weight. In general, as the molecular weight of the polymer increased, the tensile strength increased. In addition, there was a relationship between hard segment content and stiffness [56, 57]. The polyurethane stiffness increased with increasing isosorbide (hard segment) levels.

The degradation of polyurethanes in the aqueous media proceeded via hydrolysis. The rate of hydrolysis was controlled mainly by the presence of ester bonds and urethane bonds in the main chain and varies considerably with the chemical structure of the polymer. Water reacts with a carboxylic ester bond in polyurethane, which breaks the polymer chains into two shorter ones. One of these ends is then a hydroxyl group, whereas the other end is a carboxyl group. The acidic carboxyl group can accelerate the further hydrolysis of the polyester segments [50, 53, 54].In addition, Biodegradability of polyurethanes depends on their chemical structure, molecular weight, degree of crystallinity, susceptibility to microbial attack, and hydrophilicity [58]. If the polymer is hydrophilic, water molecules can more easily penetrate into the material and give increased rates of hydrolysis of the ester and urethane bonds. The number of carbon chains of diol seems to play an important role in the hydrolytic degradation. The presence of isosorbide imparts a stronger hydrophilicity to the polyurethane segment than does PCL diol, increasing the rate of hydrolytic degradation. In this experiment, the degradation rate was influenced molecular weight and hydrophilicity. As the molecular of the polyurethanes decreased, the degradation rate increased. Furthermore, as the water contact angles decreased, the degradation rate increased. The polyurethanes presented here showed that the hydrophilicity and hard segment contents were found to exert the dominant effect on the degradation rate [58].

There are significant numbers of studies utilizing the MTS or MTT assay for biocompatibility testing. Generally, when cell proliferation on the polymer being tested can be maintained for a 1–2 week time period [59, 60], it can be suggested that the material being tested is cytocompatible and biocompatible. However, if the cell proliferation was progressed further in time, cell proliferation reduces and cell death can occur due to confluency being reached. The cell proliferation on the polyurethanes was evaluated by incubating the rat bone marrow stromal cells (MSC) with the polymer disks over a period of 1, 3 and 5 days at 37 °C. The rat derived MSCs are known to be highly sensitive to culture conditions. Cell viability and activity were assessed via an MTS assay, which is frequently used to assess the cytotoxicity of polymers due to its reliability and sensitivity [61, 62]. The results are shown in Fig. 10. In comparison with the control, cells showed lower viability and activity from 1 to 5 days seeded on polyurethane surfaces. However, the cells did proliferate and increase. Although the cells showed lower rates of proliferation than the control, it can suggested that the polyurethane series presented here are cytocompatible and may be useful for various biomedical applications.

5 Conclusion

The series of polyurethane based on isosorbide polymers were synthesized from hexamethylene diisocyanate by a simple one-shot polymerization with the significant advantage of not requiring a catalyst during the reaction which offers significant clinical advantages related to toxicity. The in vitro cell culture with mouse osteoblasts showed that the cells while suppressed slightly compared to the control (P < 0.05) in general they grew well on these types of polyurethane films up to 5 days. MTS cytotoxicity assay indicated that polyurethane had good biocompatibility. According to the properties that is mechanical properties, surface properties, biodegradability and biocompatibility, these biodegradable polyurethane can be suggested promising materials for cardiovascular, trachea, and bladder applications.

Notes

Acknowledgments

This work was supported by WCU Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. R31-10069).

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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Department of Nanobiomedical Science and WCU Research CenterDankook University Graduate SchoolChungnamSouth Korea
  2. 2.Division of Biomaterials and Tissue EngineeringUCL Eastman Dental InstituteLondonUK

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