European Polymer Journal 45 (2009) 32493256

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European Polymer Journal

journal homepage: www.elsevier.com/locate/europolj

Biodegradable elastomer for soft tissue engineering

L.Y. Lee, S.C. Wu, S.S. Fu, S.Y. Zeng, W.S. Leong, L.P. Tan *
School of Materials Science and Engineering, Nanyang Technological University, N4.1-1-30 Nanyang Avenue, Singapore 639798, Singapore

a r t i c l e

i n f o

a b s t r a c t
In this work we present the synthesis of a biodegradable, elastomeric material with a wide range of mechanical properties. The synthesis of the material was done by condensation polymerization of malic acid and 1,12dodecandiol. The synthesized materials have low Youngs modulus ranging from about 1 to 4 MPa and a high elongation at break of 25737% depending on the crosslinking density of the system. The cell growth observed under microscope showed good proliferation at 3 days of culture indicating good biocompatibility and support of L929 cells growth. The fabrication of 3D scaffold from these materials using the super critical CO2 foaming method was also attempted. This method of scaffold fabrication is appropriate for materials that are easily hydrolysable and it also has the advantage of being a solvent free process. These materials are generally soft, biocompatible and biodegradable making them suitable for tissue engineering of soft tissues that are elastic in nature like muscles and blood vessels. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 20 May 2009 Received in revised form 9 July 2009 Accepted 22 July 2009 Available online 29 July 2009 Keywords: Biodegradable elastomers Soft tissue engineering Supercritical CO2 foaming Scaffold

1. Introduction Design of new materials has been one of the many challenges in the eld of biomedical engineering. Most of the common biodegradable synthetic biopolymers are linear thermoplastics that are relatively stiff in nature which results in a very poor mechanical compliance with the soft and elastic native tissues [13]. Therefore biodegradable elastomers development has been receiving much attention lately. There have been a number of developments in biodegradable elastomers in the recent years. There are two main classes of synthetic biodegradable elastomers; thermoplastic [47] and thermoset [813]. While thermoplastic elastomer materials offer synthetic control and ease of processing, the majority of these materials contain crystalline domains and the elasticity is not as superior as the thermosetting elastomers. Thermoset elastomers are rubbery at 37 C giving them a highly elastic property. However, many of these elastomers require challenging and costly synthesis methods and some also require use

of initiators and catalysts which may be detrimental for cells survival [1417]. Some elastomers may be less synthetically challenging to prepare but the mechanical properties are still quite high relative to some soft tissues and hence may be more suitable for the stiffer soft tissues like cartilage and tendon [18]. In this work, we report the synthesis, characterization and scaffold fabrication method for an elastomeric biodegradable polymer that has a wide range of mechanical properties. The tensile strength can range from 0.21 to 4.16 MPa and Youngs modulus from 0.98 to 4.04 depending on the curing conditions which can be easily modied. 2. Materials and methods 2.1. Synthesis of poly (diol malates) Malic acid and 1,12 dodecandiol used in this study were purchased from SigmaAldrich, USA at 99% purity for both materials. Different curing conditions and molar ratios of acid to diol have been attempted in this study as summarized in Table 1. It is to be noted that crosslinking could take place due to the fact that the diacid contains a secondary alcohol.

* Corresponding author. Fax: +65 6790 9081. E-mail address: lptan@ntu.edu.sg (L.P. Tan). 0014-3057/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2009.07.016

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Table 1 Different curing conditions and molar ratios of acid to diol attempted in this study. Polymer Molar ratio of acid to diol Equivalent ratio of COOH to OH functional group 2:3 2:3 2:3 2:3 2:5 2:5 2:5 No. of post curing days 140 C A B C D E F G 1:1 1:1 1:1 1:1 1:2 1:2 1:2 21 36 5 12 36 16 22 160 C 0 0 16 17 0 13 31

with the control. A cell solution with cell density 5 105/ ml of L929 mouse broblast is prepared for cell seeding. 100 ll of cell solution is added onto each well plate and are incubated at 37 C and 5% CO2 for 1 h before adding 500 ll of complete medium to each scaffold. The cells were observed under the Nikon ecLirse TS 100 Optical Microscope at the 3rd day of culture. 2.4. Scaffold fabrication using supercritical CO2 (SCCO2) For scaffold fabrication, a partially cured polymer was placed inside the supercritical CO2 chamber and then heated up to the saturation temperature (Tsaturation) at 120 C. The pressure in the chamber was increased to saturation pressure (Psaturation) of 3500 psi once the desired saturation temperature is achieved. The whole process of scaffold fabrication is as shown in the steps below:

The required amount of malic acid and diol were mixed in a three neck ask and purged with high purity nitrogen gas. The set up was heated to 140 C by silicon oil bath under constant stirring. The solution at this stage is denoted as the pre-polymer. Once the chemicals were homogeneously mixed, the material was then poured into a mold and sent for post curing. After post curing, the materials were rinsed in water to remove residue monomers and are then ready for characterization. 2.2. Material characterization Indication of crosslinking was monitored by following the changes in the functional groups using Attenuated Total Reectance (ATR); Fourier Transform InfraRed (FTIR) Perkin Elmer GX2000. Before the runs, the FTIR system was purged with nitrogen overnight to remove any moisture inside the system. The background spectrum was taken in air at 4 cm1 resolution for 16 times accumulations. Mechanical properties of the elastomers were tested using Instron Microtester 5848. Samples were cut into rectangular shape of 6 0.5 18 mm (W T L). The crosshead speed was set at 5 mm/min. The Youngs modulus that was obtained was used to calculate the crosslinking density based on the theory of rubber elasticity using the following equation below.

3500 psi2 h ! 500 psi10 min ! 3500 psi1 h ! 100 psi post cure in SCCO2 Machine overnight:
3. Results 3.1. Polymer characterization Fig. 1a and b show the FTIR analysis of the elastomers and pre-polymer. The elastomers showed very similar prominent peaks at 16901750 cm1 which could be assigned to carbonyl (C@O) groups. The peaks at about 2931 cm1 is assigned to methylene groups which were found in all the spectra of the elastomers. The broad peaks centered at 3475 cm1 are assigned to the hydrogen bonded hydroxyl group stretching vibration. It is very obvious that this peak showed up much more strongly in the pre-polymers compared to the elastomers. For example, in Fig. 1a, the ratio of the OH peak relative to the methylene peak is about 0.68 and 0.063 for the pre-polymer and elastomer D, respectively. In Fig. 1b, the ratio was 0.900.11 for the pre-polymer and elastomer G, respectively. This reected the uncured OH groups from the diol in the pre-polymer. The crosslinking density of the elastomers presented in Fig. 2 showed an increase of crosslinking density with the rst step (140 C) curing time as expected. However, the second step curing (at 160 C) seems to be more effective in increasing the crosslinking density. The crosslinking density for the rst group of elastomers (A to D) seemed to have reached saturation at curing condition of C. 3.2. Mechanical properties Typical stress strain curves of the elastomers are shown in Fig. 3; exhibiting elastomeric characteristics. After sample failure, no permanent deformation was found in all the samples which also indicates elastomeric behavior of the materials. The tensile strength and Youngs modulus ranged from 0.21 0.04 to 4.16 0.90 MPa and 0.98 0.17 to 4.04 0.78 MPa, respectively, depending on the crosslinking density and acid to diol molarity. As seen in Table 2,

N E=3RT
3

where n is the crosslinking density (mol/m ), E is the Youngs modulus (Pa); R is the universal gas constant (8.3144 J/mol K); T is the absolute temperature (K), which is 298 K in this case. The degradation studies were obtained by monitoring the weight loss over immersion time. Each sample was immersed in 15 mL PBS solution and incubated at 37 C. The PBS solution would be changed frequently to maintain the pH at around 7.4. Weight loss was obtained by comparing the initial weight (W0) and the weight measured at xed intervals (Wt).

Weight loss % W 0 W t =W 0 100

2.3. Cell attachment and proliferation

After sterilization of the materials, they were cut into circular shapes and placed in the 24-well plate together

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7
Pre-polymer

6 5 Absorbance 4 3 2 1 0
4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750

B C D

Wavenumber (cm -1)

6
Pre-polymer

Absorbance

4
E

3
F

0 4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 Wavenumber (cm - 1 )
Fig. 1. FTIR spectra of elastomers synthesized from acid to diol ratio of (a) 1:1 and (b) 1:2.

generally, at the molar ratio acid and diol of 1:1 produced elastomers of higher tensile strength and Youngs modulus. Longer curing time and higher curing temperature also led to higher crosslinking density and consequently higher strength and modulus. 3.3. Degradation Degradation was monitored by following the mass loss of the elastomers over immersion time in PBS solution (Fig. 4). For the molar ratio of acid and diol of 1:1, the longer curing time and 2 step curing led to more stable elastomers where

after about a month the mass loss of elastomer A was about 2.8% and elastomers B, and D were less than 1%. For elastomers synthesized from the molar ratio of acid to diol of: 1:2; the trend is similar in that the longer curing time and 2 step curing produced more stable elastomers. Elastomer G has minimal mass loss of <1% after about 30 days as compared to <5% and almost 9% for F and E, respectively. 3.4. Biocompatibility Fig. 5 shows the photomicrographs of theL929 mouse broblast cells seeded onto the elastomers over 3 days of

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700 Crosslinking density (mol/m3) 600 500 400 300 200 100 0 A B C D E F G

Acid to diol ratio = 1:1

Acid to diol ratio = 1:2

Fig. 2. Crosslinking density of the elastomers with different material and curing conditions.

2
Tensile stress (MPa)

1.8 1.6 Tensile stress (MPa) 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0

0.4 0.3 0.2 0.1 0 0 20 40 Tensile strain (%) 60

100

200

300

400 500 Tensile strain (%)

600

700

800

Fig. 3. Typical stress strain curve of the elastomers A; and E in the inset.

Table 2 Mechanical properties and crosslinking density of the elastomers. Polymer A B C D E F G Molar ratio of acid to diol 1:1 1:1 1:1 1:1 1:2 1:2 1:2 Youngs modulus (MPa) 0.98 0.17 3.09 0.42 3.44 0.12 3.50 0.13 1.15 0.34 2.16 0.51 4.04 0.78 Tensile strength (MPa) 0.21 0.04 3.22 0.93 3.88 0.55 4.04 0.66 0.71 0.17 1.86 0.43 4.16 0.90 Elongation (%) 737.48 243.59 25.86 5.48 28.0 4.59 25.27 5.34 48.86 10.87 35.72 6.99 37.07 6.68 Crosslinking density (mol/m3) 132.26 22 415.34 56 462.34 17 470.4 18 155.12 45 290.00 69 543.83 104

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12
A B D E F G

10 Mass loss (%)

0 0 5 10 15 20 Time (days)
Fig. 4. Mass loss of the elastomers with immersion time indicating degradation.

25

30

35

40

culture. The cell attachment and proliferation were evident on all the elastomers tested. 3.5. Scaffold fabrication Fig. 6 shows the SEM picture of the cross section of the scaffold displaying pores of about 100250 lm. The porosity density is 65.6% as measured using Mercury Intrusion Porosimetry.

4. Discussion The spectra are very similar across the elastomers which are not unexpected as they have the same chemical structures. Nevertheless, the spectrum of the pre-polymer has a signicantly stronger peak at 3475 cm1 clearly showing higher concentration of OH groups from the uncured diol group in the pre-polymer. As crosslinking proceeded with longer curing time and temperature, the peak clearly diminished. The crosslinking occurrence was also afrmed by monitoring the change in the contact angle of the elastomers with curing time. The contact angle of pre-polymer was about 32 and as curing time increases, the contact angle reached a plateau at about 87 (results not shown). The pre-polymer having hydrophilic carboxyl and hydroxyl groups would show a low contact angle but as polymerization takes place, there is a reduction of these free groups and concurrent increase in ester group leading to the increase in the contact angle. The mechanical properties of the elastomers are highly dependant on the crosslinking density which in turn is controlled mainly by the curing condition. The curing was done in either 1 or 2 steps. 1 step curing was not as effective as 2 steps in increasing the crosslinking density of the elastomers. Equimolar of acid and diol produced elastomers that are more highly crosslinked and stronger

in general. By changing the molar ratio of acid to diol to 1:2 while maintaining the same curing conditions, the crosslinking density reduced by about three times and the tensile strength and Youngs modulus dropped by about 3 and 4.5 times, respectively. This is accompanied by a signicant 2 times increase in the elongation at break. The decrease in the stiffness when diol concentration is increased is not unexpected as the relative concentration of acid which serves as the crosslinker in this case is decreased. Hence with the decrease of crosslinker concentration the crosslinking density decreases. However, this reduction could be favorable for application in scaffolds for soft tissues. These results showed that the mechanical properties of the elastomers are wide ranging and more importantly easily adjustable. Cell proliferation was observed to be comparable to control for all the elastomers indicating good biocompatibility. Although there might be some unreacted monomer residues in the system, they did not seem to cause any adverse reactions to the cells. This is favorable because the degradation products may probably be similar to the starting materials. The degradation as observed by following the mass loss over about a months time is as wide ranging as the mechanical properties. Samples with shorter curing time and 1 step curing generally showed a higher mass loss within the stipulated time. The degradation is controlled by the crosslinking density of the same system. As crosslinking density increases it stabilizes the elastomers by two ways: increase in density (compactness) causing a decrease in water diffusivity and increase in hydrophobicity causing less water penetration. Hence the degradation of the system is closely tied to the crosslinking density of the same system. As shown in Table 2, the mass loss trend is in agreement with the crosslinking density of the elastomers. Super critical CO2 foaming was used to fabricate the 3D scaffold of the equimolar elastomer. This method has not been widely used and is in fact very suitable for crosslink-

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Fig. 5. Photomicrographs of L929 cell proliferation on the elastomers A, B, C, D, E, F, G and control. The scale bar on control is applicable to all other micrographs.

ing systems. Elastomers being elastic and not dissolvable means pore creation has to be done before or during the crosslinking process. The normal methods of scaffold fabri-

cation like phase separation, rapid prototyping, ber bonding and electrospinning may be difcult or even impossible to be used for elastomeric materials. Salt leaching method

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Fig. 6. SEM picture showing the cross section of the scaffold fabricated using super critical CO2 foaming.

may be another option but however, for materials of this nature they are prone to hydrolysis and hence may not be the best method. Super critical CO2 foaming approach is ideal as it is free from solvent and water and crosslinking process can take place concurrently with pore creation. From Fig. 6, the pores created using this method are shown to be interconnected with pore size ranging from about 100250 lm and porosity of 65.5%. This result shows that supercritical CO2 foaming is a promising method for scaffold fabrication of elastomers and may be especially useful if solvent is a concern and as well as for moisture sensitive material. 5. Conclusion The elastomers synthesized using condensation polymerization of malic acid and diol exhibit a wide range of mechanical properties, biodegradable and are all biocompatible. The mechanical properties can be easily tweaked by changing the curing conditions like curing time, temperature or curing steps. Molarity of the acid and diol could also be used as a means to produce elastomers of different properties. Being the crosslinker, the acid component could be increased or decreased to produce stronger and weaker elastomers, respectively. Lastly, the diol chain length could also be a simple means to alter mechanical properties. The elastomers may nd applications in tissue engineering such as blood vessels, heart muscle and soft tissues in general. Acknowledgements The authors Lee L.Y. and S.C. Wu contributed equally to this work. The authors would like to thank A*Star, Singapore and NTU for the nancial support.

References
[1] Agrawal CM, Ray RB. Biodegradable polymeric scaffolds for musculoskeletal tissue engineering. J Biomed Mater Res 2001;55: 14150. [2] Wang Y, Ameer GA, Sheppard BJ, Langer R. A tough biodegradable elastomer. Nat Biotechnol 2002;20:6026. [3] Lendlein A, Langer R. Biodegradable, elastic shape-memory polymers for potential biomedical applications. Science 2002; 296:16736. [4] Cohn D, Hotovely Salomon A. Designing biodegradable multiblock PCL/PLA thermoplastic elastomers. Biomaterials 2005;26:2297305. [5] Venkatraman Subbu S, Tan LP, Joso JFD, Freddy YC, Freddy, Xintong Wang. Biodegradable stents with elastic memory. Biomaterials 2006;27:15738. [6] Loefgren A, Renstad R, Albertsson AC. Synthesis and characterization of a new degradable thermoplastic elastomer based on 1,5-dioxepan-2-one and -caprolactone. J App Polym Sci 1995;55: 1589600. [7] Cohn D, Stern T, Gonzalez MF, Epstein J. Biodegradable poly(ethylene oxide)/poly(epsilon-caprolactone) multiblock copolymers. J Biomed Mater Res 2002;59:27381. [8] Yang J, Webb A, Ameer G. Novel citric acid-based biodegradable elastomers for tissue engineering. Adv Mater 2004;16:5116. [9] Liu Q, Tian M, Ding T, Shi R, Zhang L. Preparation and characterization of a biodegradable polyester elastomer with thermal processing abilities. J Appl Polym Sci 2005;98:203341. [10] Ding T, Liu Q, Shi R, Tian M, Yang J, Zhang L. Synthesis, characterization and in vitro degradation study of a novel and rapidly degradable elastomer. Polym Degrad Stab 2006;91:7339. [11] Amsden BG, Wang S, Wyss U. Synthesis and characterization of thermoset biodegradable elastomers based on star-poly(epsilon-caprolactone-co-D,L-lactide). Biomacromolecules 2004;5:1399404. [12] Nagata M, Sato Y. Biodegradable elastic photocured polyesters based on adipic acid, 4-hydroxycinnamic acid and poly(epsiloncaprolactone) diols. Polymer 2004;45:8793. [13] Turunen MP, Korhonen H, Touminen J, Seppala. Synthesis, characterization and crosslinking of functional star-shaped poly(epsilon-caprolactone). J Polym Int 2001;51:92100. [14] Olson DA, Gratton S, DeSimone J, Sheares V. Amorphous linear aliphatic polyesters for the facile preparation of tunable rapidly degrading elastomeric devices and delivery vectors. Am Chem Soc 2006;128:1362533.

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L.Y. Lee et al. / European Polymer Journal 45 (2009) 32493256 [17] Amsden BG, Misra G, Gu F, Younes HM. Synthesis and characterization of a photo-cross-linked biodegradable elastomer. Biomacromolecules 2004;5:247986. [18] Kang Y, Yang J, Khan S, Anissian L, Ameer GA. A new biodegradable polyester elastomer for cartilage tissue engineering. J Biomed Mater Res 2006;77A:3319.

[15] ABrown A, Sheares V. Amorphous unsaturated aliphatic polyesters derived from dicarboxylic monomers synthesized by DielsAlder chemistry. Macromolecules 2007;40:484853. [16] Gautrot J, Zhu XX. Main-chain bile acid based degradable elastomers synthesized by entropy-driven ring-opening metathesis polymerization. Angew Chem Int Ed 2006;45:68724.