Catalyst-free synthesis of high elongation degradable polyurethanes containing varying ratios of isosorbide and polycaprolactone: physical properties and biocompatibility
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.
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 . 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 .
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 . Recently, many papers on biodegradable polyurethanes have appeared . 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- , PEO- [18, 19] and poly(3-hydroxybutyrate diol)- . 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 , ethyl lysine diisocyanate , hexamethylene diisocyanate(HDI)  and methyl lysine diisocyanate .
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  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 . 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 . 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 .
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 ; (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 .
In this study, we present the synthesis of a family of biocompatible and biodegradable polyurethanes synthesized by a simple catalyst-free, one-shot polymerization  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
Polyurethane series with different isosorbide and PCL diol contents
Mole ratio/weight (g)
(Mw 2000) (g)
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
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
Molecular weights and thermal properties of polyurethane series
31.14 ± 0.37
61.4 ± 4.4
49.1 ± 4.48
31.85 ± 0.45
58.3 ± 0.6
52.3 ± 0.45
32.12 ± 0.22
56.5 ± 5.7
54.0 ± 5.8
42.50 ± 0.32
54.6 ± 1.3
55.9 ± 1.3
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.
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.
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 .
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  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 . In general, the Tg value of soft segments decreases with increasing soft-segment molecular weight and decreasing diisocyanate content  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 . For the polyurethanes synthesized in this study, Tg increased with decreasing content and molecular weight of the soft segment as expected .
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)  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 . 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 .
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.
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.
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).
- 1.Hollinger JO, Battistone GC. Biodegradable bone repair materials: synthetic polymers and ceramics. Clin Orthop Relat Res. 1986;207:290–305.Google Scholar
- 2.Kohn J, Abramson S, Langer R. In: Ratner BD, Hoffman AS, Schoen FJ, Lemons JL, editors. Biomaterials sciences an introduction to materials in medicine [chapter 2]. London: Elsevier; 2006.Google Scholar
- 12.Lamba MK, Woodhouse KA, Cooper SL. Polyurethanes in biomedical applications. Washington, DC: CRC Press; 1998.Google Scholar
- 13.Gogolewski S. Biomedical polyurethanes. In: Arshady R, editor. Desk reference of functional polymers. Syntheses and application. Am Chem Soc: Washington, DC; 1996. p. 657–698.Google Scholar
- 15.Heijkants RGJC, van Calck RV, van Tienen TG, de Groot JH, Uma PB, Pennings AJ, Veth RPH, Schouten AJ. Uncatalyzed synthesis, thermal and mechanical properties of polyurethanes based on poly(-caprolactone) and 1,4-butanediisocyanate with uniform hard segment. Biomaterials. 2005;26:4219–28.CrossRefGoogle Scholar
- 21.Gorna K, Gogolewski S. Novel biodegradable polyurethanes for medical applications. In: Agrawal CM, Parr JE, Lin ST, editors. Synthetic bioresorbable polymers for implants. ASTM STP 1396. West Conshohocken: American Society for Testing and Material and Materials; 2000. p. 39–57.CrossRefGoogle Scholar
- 24.Kricheldorf HR. ‘‘Sugar diols’’ as building blocks of polycondensates. Polym Rev. 1997;37:599–631.Google Scholar
- 27.Bourbonnais G. Les molecules de la vie: Les glucides. London: Chapman and Hill; 2003. p. 67–83.Google Scholar
- 33.Malhotra SV, Kumar V, East A, Jaffe M. Applications of corn-based chemistry. Bridge-Wash-Nat Acad Eng. 2007;37:17–24.Google Scholar
- 35.Gorna K, Gogolewski S. Biodegradable porous polyurethane scaffolds for tissue repair and regeneration J Biomed Mater Res A. doi:10.1002/jbm.a.
- 50.Lelah MD, Cooper SL. Polyurethanes in medicine. Boca Raton: CRC; 1986.Google Scholar
- 54.Stokes K, Anderson JM, McVenes JM. Polyurethane elastomer biostability. J Biomater Appl. 1995;9:321–54.Google Scholar