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PVA hydrogel composite for its potential use as artificial cornea biomaterial
Materials Science and Engineering C 30 (2010) 214–218
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Preparation and in vitro characterization of BC/PVA hydrogel composite for its potential use as artificial cornea biomaterial Jiehua Wang a, Chuan Gao b, Yansen Zhang b, Yizao Wan b,c,d,⁎ a
School of Agriculture and Bioengineering, Tianjin University, Tianjin 300072, PR China School of Materials Science and Engineering, Tianjin University, Tianjin 300072, PR China Key Laboratory of Advanced Ceramics and Machining Technology, Ministry of Education, Tianjin University, Tianjin 300072, PR China d Research Institute of Composite Materials, Tianjin University, Tianjin 300072, PR China b c
a r t i c l e
i n f o
Article history: Received 26 March 2009 Received in revised form 2 September 2009 Accepted 14 October 2009 Available online 22 October 2009 Keywords: Bacterial cellulose Poly(vinyl alcohol) Artificial cornea
a b s t r a c t In order to investigate the potential use for bacterial cellulose (BC) as a novel artificial cornea replacement, BC/ poly(vinyl alcohol) (BC/PVA) hydrogel composites were synthesized by freezing-thaw method. The BC/PVA composites were characterized by UV–Vis spectrophotometer (UV–Vis), X-ray diffraction (XRD), thermogravimetric (TG) analysis, mechanical property tests and scanning electron microscope (SEM) analyses. Our results showed that the resultant BC/PVA composites exhibited desirable properties as artificial cornea replacement biomaterial including high water content, high visible light transmittance and suitable UV absorbance, increased mechanical strength and appropriate thermal properties. Results of this work revealed that the BC/PVA composites exhibited some promising characteristics as artificial cornea composite material and may be improved further for its realistic applications. © 2009 Elsevier B.V. All rights reserved.
1 . Introduction Corneal disease is a leading cause of blindness and by estimation 10 million people worldwide have lost their sight due to corneal disease or illness. In hopes of making corneal transplants more widely available, researchers have designed bioengineered corneas from different sources in hope to closely resemble the eye's natural cornea. The optimal corneal replacement would be mechanically strong, optically clear, capable of robust integration with surrounding ocular tissue, permeable to nutrients, and supportive of surface epithelialization [1]. Moreover, it would be resistant to protein adsorption to prevent complications leading to opacification and visual loss [1]. So far, there is currently no widely accepted cornea substitute available in despite of its clearly identified need. One candidate material for a biocompatible artificial cornea is poly (vinylalcohol) (PVA). PVA is a representative water soluble polymer and its aqueous solution can form both chemical and physical gels depending on the preparation condition. Low temperature crystallized PVA has light transparency and nutrition permeability [2]. However, pure PVA has low cell affinity. Earlier clinical trials had frequently failed because corneal epithelial down growth occurred between the host cornea and the materials, and the materials were
⁎ Corresponding author. School of Materials Science and Engineering, Tianjin University, Tianjin 300072, PR China. Tel./fax: +86 22 87898601. E-mail address: [email protected] (Y. Wan). 0928-4931/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2009.10.006
finally rejected from the host cornea. The main reason of this rejection is the weak adhesion between the host cornea and the prosthesis. PVA–collagen and PVA–collagen–hydroxylapatite composites have been prepared by Kobayashi et al. in order to enhance the supporting adhesion and growth of corneal epithelium [3]. On the other hand, PVA could be blended or grafted with starch and polyvinyl pyrrolidone (PVP). However, PVA–starch and PVA–PVP composites had low tensile strength [4,5]. Bacterial cellulose (BC) is composed of nano-sized fibril network and due to its unique specific structure and properties such as high purity, high water holding capacity, high tensile strength and excellent biocompatibility, materials based on BC has become increasingly important and open an innovative pool for biomaterial development in medical applications including wound dressings [6], artificial skin [7], artificial blood vessels [8] and tissue engineering scaffold [9,10]. Meanwhile, the nano level porous structure of BC could easily facilitate the permeance of oxygen and nutrients. BC has favorable mechanical properties and its strength could meet the requirements for surgical sutures and maintain the intraocular pressure. BC has a definite pellucidness and favorable mechanical properties which could meet the requirements for surgical sutures and maintain the intraocular pressure. Although showing a little higher refractive index as 1.4–1.5 [11,12] than natural cornea of which refractive index is 1.367 [13], BC/ resin composites can still be used as a potential artificial cornea biomaterial candidate as their refractive index is very close to polydimethylsiloxane (refractive index = 1.41 [14,15]) and polyvinylpyrrolidone coated PDMS (refractive index = 1.43 [16]) which have
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been utilized in artificial cornea applications already [15–17]. In addition, the collagen which has a lower refractive index (1.35) [18] than natural corneal is generally accepted as artificial cornea as well [19]. In an attempt to increase the potential of BC as an artificial cornea biomaterial, we take advantage of both the stability of artificial polymers (PVA) and the biocompatibility and high mechanical properties of BC and innovatively designed a hybrid polymers composed of PVA and BC by freezing-thaw method. In another work by Millon et al. [20], BC suspension was used to produce a nanocomposite for the replacement of soft tissues such as cardiovascular or other connective tissues. In this study, we tried a complete preparation procedure with the aim to prepare biocompatible, flexible and one-piece artificial corneas. The aim of this work is to evaluate the material aspects of BC/PVA composites as artificial cornea including water content, light transmittance, structure, thermal stability and mechanical properties, which were analyzed by gravimetric procedure, UV–Vis spectrophotometer (UV–Vis), X-ray diffraction (XRD), thermogravimetric analysis (TG), mechanical tests and electron scanning microscopy (SEM). Our results demonstrated that the optical properties, thermal stabilities and mechanical characteristics of the BC/PVA composites could meet the requirements of artificial cornea materials and thus were considered to be a more promising biomaterial candidate for cornea replacement. In vitro cell experiments are underway and the specific implantation experiments in vivo are planned These results concerning biocompatibility will be reported in our further works.
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spectrophotometer (TU1810PC, Beijing) and the percentage of light transmittance of hydrogel samples at 543 nm was measured following the method described by Saito et al. [22]. The composition and structure of BC/PVA composites were determined after vacuum drying by X-ray diffraction (XRD) (Rigaku D/Max 2500 V/PC X-ray diffractometer, Japan) using CuKα radiation generated at 40 kV and 200 mA; the range of diffraction angle (2Theta) was 10–30° at a scanning speed of 2°/min. Thermogravimetric analysis (TG) was carried out with a STA-449PC/ 4/H Luxx simultaneous TG-DTA/DSC apparatus (Netzsch, Germany) with a heating rate of 10 °C/min in the range of 25–800 °C under a flowing N2 atmosphere. The tensile strength and elongation at break of the composite samples were tested by a tensile machine (Reger-3050, Beijing) in accordance with ASTM D882-09 standard. Tests were done in Krebs solution at a temperature of 37 °C with a crosshead speed of 10 mm/ min. Young's modulus, maximum load and displacement at maximum load were recorded as indications of the mechanical properties of the various samples. Tensile fracture surfaces were examined by scanning electron microscopy (JSM-6700F FE-SEM, JEOL). Prior to SEM observation, all samples were sputter coated with a thin layer of gold to avoid electrical charging. 3. Results and discussion 3.1. Water content
2. Materials and methods 2.1. Preparation of the BC/PVA composites PVA purchased from Tianjin Tian Da Tian Long Sci. & Tech. Co., Ltd. was analytical reagent and used as received without further purification. The preparation and purification procedure of BC was conducted as described previously [21]. The thickness of BC membranes was controlled by adjusting the length of bacteria growing time in the incubator. BC membrane with 0.3 mm thickness was immersed in an optimized PVA water solution in 80 °C water bath for 24 h and then frozen at −20 °C for 24 h followed by thawing at room temperature for 12 h. After dehydration by evaporating water at room temperature, the membrane was hydrolyzed in deionized water until swollen to equilibrium. The mass fraction of BC in the dry composite prepared by vacuum drying at 80 °C was calculated supposing the aqueous solution in BC membrane has been displaced totally by the PVA solution. The designation of BC/PVA composites with different BC content was illustrated in Table 1. 2.2. Characterization of BC/PVA composites Water absorption of the BC/PVA composite was determined by gravimetric procedure. The water content of the composite was calculated by using the formula: Mt(wt.%) = (Wh−Wd)/Wh × 100%, where Wh and Wd are the weight of hydrated composite and dried composite respectively. The hydrogel samples were examined for transparency by scanning within the visible range of wavelengths (390–780 nm) with a UV–Vis Table 1 The composition of BC/PVA composite membrane with different BC content. PVA solution used for immersing BC membrane BC content of the resultant BC/PVA (w/v %)
(wt.%)
5 9 13
27 17 12
Water content in the BC/PVA composites was affected by the concentration of BC. Pure PVA had water content of 83.4 ± 0.41% (Table 2). The water content in the BC/PVA composites was significantly lower than the pure PVA hydrogel and this might be interpreted by the lower water content in the BC part of the composite and the change in network structure caused by BC integration and interactions formed between BC and PVA chain [23]. For example, the interactions between different components in a composite could make the complex inter- and intra-chain hydrogen bond stronger and thus reduce the amount of bound water. It is worth to note that the water content in the BC/PVA hydrogel composites (67–73%) is close to that of human natural cornea (78%) [24], so that it could meet the requirement for the water content as artificial cornea. 3.2. Light transmittance Fig. 1 showed the light transmittance curves of BC/PVA composites with different BC loadings. In curve a, pure PVA hydrogel not only had a 95% light transmittance to visible lights, but also had a high transmittance to UV. The BC/PVA composites had a visible light transmittance of 92%–97%, 90%–94% and 81%–90% when the BC content was 12% (curve b), 17% (curve c) and 27% (curve d) respectively. More importantly, the composites had a good blocking ability to UV lights, which made them good candidate materials as artificial cornea. Yano et al. have made composite using dry BC with transparent resin and achieved a light transmittance of 80% [25]. They attributed such a high light transmitting ability to the nano-effect, e.g. when the size of the fiber in the fiber/resin composite is less than one tenth of the wavelength of visible light, the scattering of lights can be ignored
Table 2 Water content of BC/PVA composites. BC content (wt.%)
Water content (wt.%)
0 12 17 27
83.4 ± 0.41 72.6 ± 1.07 73.2 ± 1.08 67.4 ± 0.91
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Fig. 1. Transmittance curves of BC/PVA composites with different BC mass fractions of (a) 0%, (b) 12%, (c) 17%, (d) 27% and (e) pure BC.
despite of the difference in the index of refraction of these two materials [25]. BC is composed of nano-sized fibrils connecting each other by hydrogen bonds to from a fiber ribbon of 30–100 nm wide and 3–8 nm thick, so that their nano-size could effectively dismiss the light scattering and achieve a good transparency. Work by Nogi et al. revealed that variation of the BC content in the composites only slightly affected its transmittance [26]. In this work, despite of the obvious difference in the refractive exponent of PVA (1.54) and BC (1.57), the BC/ PVA hydrogel composite still achieved a light transmittance higher than 90% probably due to the nano-effect of BC nano-fibrils. Wet BC membrane (curve e) had a light transmittance of 60–88% in the range of 400–900 nm. This could be due to that the high water content (>90%) [9] in the wet BC membrane can effectively attenuate the light scattering and lead to a high light transmittance. The absorbance for UV light is crucial for ideal artificial cornea in terms of preventing the damage of UV to the internal eye tissue. Fig. 1 also showed that in the range of 200–400 nm UV lights, the transmittance of both wet and dry BC membranes declined obviously with the decrease of the wavelength, which demonstrated their good absorbing ability for UV lights. 3.3. Structure analysis
Fig. 2. The XRD patterns of BC/PVA composites with different BC mass fractions of (a) 27%, (b) 17% and (c) 12%.
temperature (40–130 °C) for BC (curve a) was attributed to the evaporation of absorbed moisture [28,29]. Physically adsorbed and hydrogen bond linked water molecules were lost at this first stage. It could be observed that until 130 °C all samples were considered as thermally stable with minor mass losses apart from that attributed to humidity. The residue of BC was 17.5% (Table 3) which coincided with that detected by Barud et al. [30]. TG curves of different BC/PVA composites and pure PVA revealed three main weight loss regions. The first region, at a temperature of 100–130 °C, was due to the evaporation of physically weak and chemically strong bound water. The weight loss was about 4%–5%. The second transitional region, at around 250–360 °C appeared due to the degradation of the side-chain of the polymer [31]. The total weight loss corresponding to this stage was about 70%–80%. The weight loss occurred at 420–450 °C was assigned to the third stage due to the cleavage C-C backbone of polymer, the so called carbonation, with a total weight loss at about 95% at 800 °C, as listed in Table 3 [31]. In literature, the PVA/starch [32] and PVA/caprolactam [33] composites exhibited similar thermo gravimetric behaviors. The TG test was considered as much more sensitive method for determining thermal stability [34]. Suñol et al. declared that a higher
The XRD results of BC/PVA composites were shown in Fig. 2 and basically, BC/PVA XRD profiles exhibited the characteristic peaks of both BC (14.7°, 16.8° and 22.6° in the crystal region) and PVA (19.5°, 23.7° and 27.8° in the crystal region). Note that with the increase of BC content, the intensity of BC-related peaks increased and that of PVA-related peaks decreased along with the enlarged peak width at half-height (FWHM). Deduced from the Scherre formula, the degree of crystallinity of PVA gradually decreased with increasing BC content. This indicated that BC content had effects on both the cross-linking density of the network and the degree of crystallinity of PVA, which was consistent with the report of Nishio and Manley for their plant cellulose–BC composite system [27]. In current work, this phenomenon could be explained by that in the BC/ PVA composites, BC strongly interacted with the hydroxide radical of PVA and the tangling between them caused steric effect and destroyed the highly-organized arrangement of PVA, and in turn caused the decrease of its degree of crystallinity. 3.4. Thermal properties Fig. 3 was the TG curves of BC, PVA and BC/PVA with different BC contents. The early minor weight loss observed at initial low
Fig. 3. TGA curves of BC/PVA composites with different BC mass fractions of (a) pure BC, (b) 27%, (c) 17%, (d) 12% and (e) 0%.
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Table 3 Thermal characteristics of TG curves in Fig. 3. Curve
a
b
c
d
e
Tonset (°C) Residuals (wt.%)
310 17.5
268 5.5
257 4.5
252 4.2
249 5.4
onset temperature (Tonset) was associated with higher thermal stability [35]. Table 3 showed the Tonset of the samples calculated for the TG curves. The Tonset of pure PVA was 249 °C, which was comparable to the results reported by Chen et al. [36]. BC exhibited the highest thermal stability with a Tonset of 310 °C [30] when compared to PVA and BC/PVA composites. With increasing BC content in BC/PVA composites, the thermal stability of the composites was enhanced, which was demonstrated by the increasing Tonset values. The increase of Tonset could be attributed to the formation of the hydrogen bonding between BC and PVA, which also indicated the good compatibility of BC and PVA. Fig. 4. Stress–strain curves of BC/PVA composites with different BC mass fractions of (a) 0%, (b) 12%, (c)17% and (d)27%.
3.5. Mechanical properties In the work by Favier et al., they indicated that with the increase of fiber content, the latex/cellulose whiskers composite exhibited better tensile strength and Young's modulus [37]. In Table 4, we showed the tensile strength and Young's modulus of BC/PVA composites with various BC content. At BC fiber loadings of 12%, 17% and 27%, the Young's modulus of BC/PVA composites were 24.0 MPa, 38.7 MPa, and 63.0 MPa respectively and tensile strength were 3.9 MPa, 5.8 MPa and 7.2 MPa respectively. We can see that after BC's incorporation, the mechanical properties of the composites enhanced significantly, which could be attributed to that the high strength and modulus of BC nano-fibrils. Another reason could be that BC has numerous hydroxyl group and large specific surface area, so it can form plenty of hydrogen bonds with PVA, which lead to the high mechanical strength for the composites [38]. The human cornea has a tensile strength of about 3.8 MPa [39–41]. The pure PVA hydrogel has a tensile strength of 3.2 MPa in our test and 3.5 MPa reported by Liu et al. [42], which are both lower than that of human cornea. The addition of BC can enhance the tensile strength as BC has excellent mechanical properties. As shown in Table 4, when BC content was 12%, BC/PVA composite hydrogel exhibited a tensile strength of 3.9 MPa which was very similar to that of human cornea. Fig. 4 showed the stress–strain curves of BC/PVA composites. The elongation at break of BC/PVA composites was significantly lower compared to pure PVA. Since BC is the main load-bearing component in the composites, the determinant of the elongation at break for the composite have been shown to be the BC content. The reason for this could be that the hydroxyl groups on the BC surface have chemically formed hydrogen bonds with PVA and changed the special arrangement of BC nano-fibrils which in turn affected the orientation of fibrils under load-bearing conditions and caused the decline in its elongation at break. Typical tensile fracture surfaces of two selected BC/PVA composites, one with 12% (a) and the other with 27% (b) BC fiber content, were displayed in Fig. 5. Note that BC fibers were uniformly
distributed throughout the composites in both cases and fiber ends were clearly visible in two photos. However, the fiber pull-out length is fairly little, which indicated a good fiber–matrix bonding.
Table 4 Mechanical properties of BC/PVA composites with different BC fraction. BC content (wt.%)
Tensile strength (MPa)
Young's modulus (MPa)
0 12 17 27
3.2 ± 0.3 3.9 ± 0.9 5.8 ± 0.6 7.2 ± 0.2
0.5 ± 0.1 24.0 ± 4.8 38.7 ± 5.9 63.0 ± 3.1
Fig. 5. Tensile fracture surfaces of two BC/PVA composites with different BC mass fractions of (a) 12% and (b) 27% revealed by scanning electron microscopy.
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4. Conclusions In this study, by the use of the freezing-thaw method, we have made a hydrogel composite by impregnating various amount of PVA into a transparent bacterial cellulose polymer matrix, and then characterized its water holding capacities, light transmittance, mechanical and thermal properties. The large amount of hydroxyl groups on these two components assures satisfying interfacial interactions through hydrogen bonding, leading to desirable adhesion at the BC/PVA interfaces. Consequently, the as-obtained composite showed high percent light transmittance, improved mechanical properties and excellent thermal properties. Thus this type of hydrogel composite is a very promising optically functional material. Acknowledgements This work is supported by the National Natural Science Foundation of China (grants 50872088 and 50673076), Tianjin Municipal Science and Technology Committee (grant 07JCZDJC07200), the Foundation of Tianjin Key Laboratory of Industrial Microbiology (Tianjin University of Science and Technology), China (no. Wsw-01), the State Key Basic Research (973) Program (grant 2007CB936100) and the National HiTech Research Development (863) Program (2009AA03Z311). References [1] T.V. Chirila, Biomaterials 22 (2001) 3311. [2] H. Kobayashi, Y. Ikada, T. Moritera, Y. Ogura, Y. Honda, J. Biomed. Mater. Res. 26 (1992) 1583. [3] H. Kobayashi, M. Kato, T. Taguchi, T. Ikoma, H. Miyashita, S. Shimmura, K. Tsubota, J. Tanaka, Mater. Sci. Eng. C 24 (2004) 729. [4] C.-Y.H. Sung-Yeng Yang, J. Appl. Polym. Sci. 109 (2008) 2452. [5] D.-J. Kim, I.-S. Park, M.-H. Lee, Ceram. Int. 31 (2005) 577. [6] V.I. Legeza, V.P. Galenko-Yaroshevskii, E.V. Zinov'ev, B.A. Paramonov, G.S. Kreichman, I.I. Turkovskii, E.S. Gumenyuk, A.G. Karnovich, A.K. Khripunov, Bull. Exp. Biol. Med. 138 (2004) 311. [7] D. Klemm, D. Schumann, F. Kramer, N. Hessler, M. Hornung, H.P. Schmauder, S. Marsch, Polysaccharides II 205 (2006) 49. [8] D. Klemm, D. Schumann, U. Udhardt, S. Marsch, Prog. Polym. Sci 26 (2001) 1561. [9] A. Svensson, E. Nicklasson, T. Harrah, B. Panilaitis, D.L. Kaplan, M. Brittberg, P. Gatenholm, Biomaterials 26 (2005) 419. [10] A. Bodin, H. Backdahl, G. Helenius, L. Gustafsson, B. Risberg, P. Gatenholm, Abstr. Pap. — Am. Chem. Soc. 229 (2005) U297-U297.
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Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Preparation and in vitro characterization of BC/PVA hydrogel composite for its potential use as artificial cornea biomaterial Jiehua Wang a, Chuan Gao b, Yansen Zhang b, Yizao Wan b,c,d,⁎ a
School of Agriculture and Bioengineering, Tianjin University, Tianjin 300072, PR China School of Materials Science and Engineering, Tianjin University, Tianjin 300072, PR China Key Laboratory of Advanced Ceramics and Machining Technology, Ministry of Education, Tianjin University, Tianjin 300072, PR China d Research Institute of Composite Materials, Tianjin University, Tianjin 300072, PR China b c
a r t i c l e
i n f o
Article history: Received 26 March 2009 Received in revised form 2 September 2009 Accepted 14 October 2009 Available online 22 October 2009 Keywords: Bacterial cellulose Poly(vinyl alcohol) Artificial cornea
a b s t r a c t In order to investigate the potential use for bacterial cellulose (BC) as a novel artificial cornea replacement, BC/ poly(vinyl alcohol) (BC/PVA) hydrogel composites were synthesized by freezing-thaw method. The BC/PVA composites were characterized by UV–Vis spectrophotometer (UV–Vis), X-ray diffraction (XRD), thermogravimetric (TG) analysis, mechanical property tests and scanning electron microscope (SEM) analyses. Our results showed that the resultant BC/PVA composites exhibited desirable properties as artificial cornea replacement biomaterial including high water content, high visible light transmittance and suitable UV absorbance, increased mechanical strength and appropriate thermal properties. Results of this work revealed that the BC/PVA composites exhibited some promising characteristics as artificial cornea composite material and may be improved further for its realistic applications. © 2009 Elsevier B.V. All rights reserved.
1 . Introduction Corneal disease is a leading cause of blindness and by estimation 10 million people worldwide have lost their sight due to corneal disease or illness. In hopes of making corneal transplants more widely available, researchers have designed bioengineered corneas from different sources in hope to closely resemble the eye's natural cornea. The optimal corneal replacement would be mechanically strong, optically clear, capable of robust integration with surrounding ocular tissue, permeable to nutrients, and supportive of surface epithelialization [1]. Moreover, it would be resistant to protein adsorption to prevent complications leading to opacification and visual loss [1]. So far, there is currently no widely accepted cornea substitute available in despite of its clearly identified need. One candidate material for a biocompatible artificial cornea is poly (vinylalcohol) (PVA). PVA is a representative water soluble polymer and its aqueous solution can form both chemical and physical gels depending on the preparation condition. Low temperature crystallized PVA has light transparency and nutrition permeability [2]. However, pure PVA has low cell affinity. Earlier clinical trials had frequently failed because corneal epithelial down growth occurred between the host cornea and the materials, and the materials were
⁎ Corresponding author. School of Materials Science and Engineering, Tianjin University, Tianjin 300072, PR China. Tel./fax: +86 22 87898601. E-mail address: [email protected] (Y. Wan). 0928-4931/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2009.10.006
finally rejected from the host cornea. The main reason of this rejection is the weak adhesion between the host cornea and the prosthesis. PVA–collagen and PVA–collagen–hydroxylapatite composites have been prepared by Kobayashi et al. in order to enhance the supporting adhesion and growth of corneal epithelium [3]. On the other hand, PVA could be blended or grafted with starch and polyvinyl pyrrolidone (PVP). However, PVA–starch and PVA–PVP composites had low tensile strength [4,5]. Bacterial cellulose (BC) is composed of nano-sized fibril network and due to its unique specific structure and properties such as high purity, high water holding capacity, high tensile strength and excellent biocompatibility, materials based on BC has become increasingly important and open an innovative pool for biomaterial development in medical applications including wound dressings [6], artificial skin [7], artificial blood vessels [8] and tissue engineering scaffold [9,10]. Meanwhile, the nano level porous structure of BC could easily facilitate the permeance of oxygen and nutrients. BC has favorable mechanical properties and its strength could meet the requirements for surgical sutures and maintain the intraocular pressure. BC has a definite pellucidness and favorable mechanical properties which could meet the requirements for surgical sutures and maintain the intraocular pressure. Although showing a little higher refractive index as 1.4–1.5 [11,12] than natural cornea of which refractive index is 1.367 [13], BC/ resin composites can still be used as a potential artificial cornea biomaterial candidate as their refractive index is very close to polydimethylsiloxane (refractive index = 1.41 [14,15]) and polyvinylpyrrolidone coated PDMS (refractive index = 1.43 [16]) which have
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been utilized in artificial cornea applications already [15–17]. In addition, the collagen which has a lower refractive index (1.35) [18] than natural corneal is generally accepted as artificial cornea as well [19]. In an attempt to increase the potential of BC as an artificial cornea biomaterial, we take advantage of both the stability of artificial polymers (PVA) and the biocompatibility and high mechanical properties of BC and innovatively designed a hybrid polymers composed of PVA and BC by freezing-thaw method. In another work by Millon et al. [20], BC suspension was used to produce a nanocomposite for the replacement of soft tissues such as cardiovascular or other connective tissues. In this study, we tried a complete preparation procedure with the aim to prepare biocompatible, flexible and one-piece artificial corneas. The aim of this work is to evaluate the material aspects of BC/PVA composites as artificial cornea including water content, light transmittance, structure, thermal stability and mechanical properties, which were analyzed by gravimetric procedure, UV–Vis spectrophotometer (UV–Vis), X-ray diffraction (XRD), thermogravimetric analysis (TG), mechanical tests and electron scanning microscopy (SEM). Our results demonstrated that the optical properties, thermal stabilities and mechanical characteristics of the BC/PVA composites could meet the requirements of artificial cornea materials and thus were considered to be a more promising biomaterial candidate for cornea replacement. In vitro cell experiments are underway and the specific implantation experiments in vivo are planned These results concerning biocompatibility will be reported in our further works.
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spectrophotometer (TU1810PC, Beijing) and the percentage of light transmittance of hydrogel samples at 543 nm was measured following the method described by Saito et al. [22]. The composition and structure of BC/PVA composites were determined after vacuum drying by X-ray diffraction (XRD) (Rigaku D/Max 2500 V/PC X-ray diffractometer, Japan) using CuKα radiation generated at 40 kV and 200 mA; the range of diffraction angle (2Theta) was 10–30° at a scanning speed of 2°/min. Thermogravimetric analysis (TG) was carried out with a STA-449PC/ 4/H Luxx simultaneous TG-DTA/DSC apparatus (Netzsch, Germany) with a heating rate of 10 °C/min in the range of 25–800 °C under a flowing N2 atmosphere. The tensile strength and elongation at break of the composite samples were tested by a tensile machine (Reger-3050, Beijing) in accordance with ASTM D882-09 standard. Tests were done in Krebs solution at a temperature of 37 °C with a crosshead speed of 10 mm/ min. Young's modulus, maximum load and displacement at maximum load were recorded as indications of the mechanical properties of the various samples. Tensile fracture surfaces were examined by scanning electron microscopy (JSM-6700F FE-SEM, JEOL). Prior to SEM observation, all samples were sputter coated with a thin layer of gold to avoid electrical charging. 3. Results and discussion 3.1. Water content
2. Materials and methods 2.1. Preparation of the BC/PVA composites PVA purchased from Tianjin Tian Da Tian Long Sci. & Tech. Co., Ltd. was analytical reagent and used as received without further purification. The preparation and purification procedure of BC was conducted as described previously [21]. The thickness of BC membranes was controlled by adjusting the length of bacteria growing time in the incubator. BC membrane with 0.3 mm thickness was immersed in an optimized PVA water solution in 80 °C water bath for 24 h and then frozen at −20 °C for 24 h followed by thawing at room temperature for 12 h. After dehydration by evaporating water at room temperature, the membrane was hydrolyzed in deionized water until swollen to equilibrium. The mass fraction of BC in the dry composite prepared by vacuum drying at 80 °C was calculated supposing the aqueous solution in BC membrane has been displaced totally by the PVA solution. The designation of BC/PVA composites with different BC content was illustrated in Table 1. 2.2. Characterization of BC/PVA composites Water absorption of the BC/PVA composite was determined by gravimetric procedure. The water content of the composite was calculated by using the formula: Mt(wt.%) = (Wh−Wd)/Wh × 100%, where Wh and Wd are the weight of hydrated composite and dried composite respectively. The hydrogel samples were examined for transparency by scanning within the visible range of wavelengths (390–780 nm) with a UV–Vis Table 1 The composition of BC/PVA composite membrane with different BC content. PVA solution used for immersing BC membrane BC content of the resultant BC/PVA (w/v %)
(wt.%)
5 9 13
27 17 12
Water content in the BC/PVA composites was affected by the concentration of BC. Pure PVA had water content of 83.4 ± 0.41% (Table 2). The water content in the BC/PVA composites was significantly lower than the pure PVA hydrogel and this might be interpreted by the lower water content in the BC part of the composite and the change in network structure caused by BC integration and interactions formed between BC and PVA chain [23]. For example, the interactions between different components in a composite could make the complex inter- and intra-chain hydrogen bond stronger and thus reduce the amount of bound water. It is worth to note that the water content in the BC/PVA hydrogel composites (67–73%) is close to that of human natural cornea (78%) [24], so that it could meet the requirement for the water content as artificial cornea. 3.2. Light transmittance Fig. 1 showed the light transmittance curves of BC/PVA composites with different BC loadings. In curve a, pure PVA hydrogel not only had a 95% light transmittance to visible lights, but also had a high transmittance to UV. The BC/PVA composites had a visible light transmittance of 92%–97%, 90%–94% and 81%–90% when the BC content was 12% (curve b), 17% (curve c) and 27% (curve d) respectively. More importantly, the composites had a good blocking ability to UV lights, which made them good candidate materials as artificial cornea. Yano et al. have made composite using dry BC with transparent resin and achieved a light transmittance of 80% [25]. They attributed such a high light transmitting ability to the nano-effect, e.g. when the size of the fiber in the fiber/resin composite is less than one tenth of the wavelength of visible light, the scattering of lights can be ignored
Table 2 Water content of BC/PVA composites. BC content (wt.%)
Water content (wt.%)
0 12 17 27
83.4 ± 0.41 72.6 ± 1.07 73.2 ± 1.08 67.4 ± 0.91
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Fig. 1. Transmittance curves of BC/PVA composites with different BC mass fractions of (a) 0%, (b) 12%, (c) 17%, (d) 27% and (e) pure BC.
despite of the difference in the index of refraction of these two materials [25]. BC is composed of nano-sized fibrils connecting each other by hydrogen bonds to from a fiber ribbon of 30–100 nm wide and 3–8 nm thick, so that their nano-size could effectively dismiss the light scattering and achieve a good transparency. Work by Nogi et al. revealed that variation of the BC content in the composites only slightly affected its transmittance [26]. In this work, despite of the obvious difference in the refractive exponent of PVA (1.54) and BC (1.57), the BC/ PVA hydrogel composite still achieved a light transmittance higher than 90% probably due to the nano-effect of BC nano-fibrils. Wet BC membrane (curve e) had a light transmittance of 60–88% in the range of 400–900 nm. This could be due to that the high water content (>90%) [9] in the wet BC membrane can effectively attenuate the light scattering and lead to a high light transmittance. The absorbance for UV light is crucial for ideal artificial cornea in terms of preventing the damage of UV to the internal eye tissue. Fig. 1 also showed that in the range of 200–400 nm UV lights, the transmittance of both wet and dry BC membranes declined obviously with the decrease of the wavelength, which demonstrated their good absorbing ability for UV lights. 3.3. Structure analysis
Fig. 2. The XRD patterns of BC/PVA composites with different BC mass fractions of (a) 27%, (b) 17% and (c) 12%.
temperature (40–130 °C) for BC (curve a) was attributed to the evaporation of absorbed moisture [28,29]. Physically adsorbed and hydrogen bond linked water molecules were lost at this first stage. It could be observed that until 130 °C all samples were considered as thermally stable with minor mass losses apart from that attributed to humidity. The residue of BC was 17.5% (Table 3) which coincided with that detected by Barud et al. [30]. TG curves of different BC/PVA composites and pure PVA revealed three main weight loss regions. The first region, at a temperature of 100–130 °C, was due to the evaporation of physically weak and chemically strong bound water. The weight loss was about 4%–5%. The second transitional region, at around 250–360 °C appeared due to the degradation of the side-chain of the polymer [31]. The total weight loss corresponding to this stage was about 70%–80%. The weight loss occurred at 420–450 °C was assigned to the third stage due to the cleavage C-C backbone of polymer, the so called carbonation, with a total weight loss at about 95% at 800 °C, as listed in Table 3 [31]. In literature, the PVA/starch [32] and PVA/caprolactam [33] composites exhibited similar thermo gravimetric behaviors. The TG test was considered as much more sensitive method for determining thermal stability [34]. Suñol et al. declared that a higher
The XRD results of BC/PVA composites were shown in Fig. 2 and basically, BC/PVA XRD profiles exhibited the characteristic peaks of both BC (14.7°, 16.8° and 22.6° in the crystal region) and PVA (19.5°, 23.7° and 27.8° in the crystal region). Note that with the increase of BC content, the intensity of BC-related peaks increased and that of PVA-related peaks decreased along with the enlarged peak width at half-height (FWHM). Deduced from the Scherre formula, the degree of crystallinity of PVA gradually decreased with increasing BC content. This indicated that BC content had effects on both the cross-linking density of the network and the degree of crystallinity of PVA, which was consistent with the report of Nishio and Manley for their plant cellulose–BC composite system [27]. In current work, this phenomenon could be explained by that in the BC/ PVA composites, BC strongly interacted with the hydroxide radical of PVA and the tangling between them caused steric effect and destroyed the highly-organized arrangement of PVA, and in turn caused the decrease of its degree of crystallinity. 3.4. Thermal properties Fig. 3 was the TG curves of BC, PVA and BC/PVA with different BC contents. The early minor weight loss observed at initial low
Fig. 3. TGA curves of BC/PVA composites with different BC mass fractions of (a) pure BC, (b) 27%, (c) 17%, (d) 12% and (e) 0%.
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Table 3 Thermal characteristics of TG curves in Fig. 3. Curve
a
b
c
d
e
Tonset (°C) Residuals (wt.%)
310 17.5
268 5.5
257 4.5
252 4.2
249 5.4
onset temperature (Tonset) was associated with higher thermal stability [35]. Table 3 showed the Tonset of the samples calculated for the TG curves. The Tonset of pure PVA was 249 °C, which was comparable to the results reported by Chen et al. [36]. BC exhibited the highest thermal stability with a Tonset of 310 °C [30] when compared to PVA and BC/PVA composites. With increasing BC content in BC/PVA composites, the thermal stability of the composites was enhanced, which was demonstrated by the increasing Tonset values. The increase of Tonset could be attributed to the formation of the hydrogen bonding between BC and PVA, which also indicated the good compatibility of BC and PVA. Fig. 4. Stress–strain curves of BC/PVA composites with different BC mass fractions of (a) 0%, (b) 12%, (c)17% and (d)27%.
3.5. Mechanical properties In the work by Favier et al., they indicated that with the increase of fiber content, the latex/cellulose whiskers composite exhibited better tensile strength and Young's modulus [37]. In Table 4, we showed the tensile strength and Young's modulus of BC/PVA composites with various BC content. At BC fiber loadings of 12%, 17% and 27%, the Young's modulus of BC/PVA composites were 24.0 MPa, 38.7 MPa, and 63.0 MPa respectively and tensile strength were 3.9 MPa, 5.8 MPa and 7.2 MPa respectively. We can see that after BC's incorporation, the mechanical properties of the composites enhanced significantly, which could be attributed to that the high strength and modulus of BC nano-fibrils. Another reason could be that BC has numerous hydroxyl group and large specific surface area, so it can form plenty of hydrogen bonds with PVA, which lead to the high mechanical strength for the composites [38]. The human cornea has a tensile strength of about 3.8 MPa [39–41]. The pure PVA hydrogel has a tensile strength of 3.2 MPa in our test and 3.5 MPa reported by Liu et al. [42], which are both lower than that of human cornea. The addition of BC can enhance the tensile strength as BC has excellent mechanical properties. As shown in Table 4, when BC content was 12%, BC/PVA composite hydrogel exhibited a tensile strength of 3.9 MPa which was very similar to that of human cornea. Fig. 4 showed the stress–strain curves of BC/PVA composites. The elongation at break of BC/PVA composites was significantly lower compared to pure PVA. Since BC is the main load-bearing component in the composites, the determinant of the elongation at break for the composite have been shown to be the BC content. The reason for this could be that the hydroxyl groups on the BC surface have chemically formed hydrogen bonds with PVA and changed the special arrangement of BC nano-fibrils which in turn affected the orientation of fibrils under load-bearing conditions and caused the decline in its elongation at break. Typical tensile fracture surfaces of two selected BC/PVA composites, one with 12% (a) and the other with 27% (b) BC fiber content, were displayed in Fig. 5. Note that BC fibers were uniformly
distributed throughout the composites in both cases and fiber ends were clearly visible in two photos. However, the fiber pull-out length is fairly little, which indicated a good fiber–matrix bonding.
Table 4 Mechanical properties of BC/PVA composites with different BC fraction. BC content (wt.%)
Tensile strength (MPa)
Young's modulus (MPa)
0 12 17 27
3.2 ± 0.3 3.9 ± 0.9 5.8 ± 0.6 7.2 ± 0.2
0.5 ± 0.1 24.0 ± 4.8 38.7 ± 5.9 63.0 ± 3.1
Fig. 5. Tensile fracture surfaces of two BC/PVA composites with different BC mass fractions of (a) 12% and (b) 27% revealed by scanning electron microscopy.
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4. Conclusions In this study, by the use of the freezing-thaw method, we have made a hydrogel composite by impregnating various amount of PVA into a transparent bacterial cellulose polymer matrix, and then characterized its water holding capacities, light transmittance, mechanical and thermal properties. The large amount of hydroxyl groups on these two components assures satisfying interfacial interactions through hydrogen bonding, leading to desirable adhesion at the BC/PVA interfaces. Consequently, the as-obtained composite showed high percent light transmittance, improved mechanical properties and excellent thermal properties. Thus this type of hydrogel composite is a very promising optically functional material. Acknowledgements This work is supported by the National Natural Science Foundation of China (grants 50872088 and 50673076), Tianjin Municipal Science and Technology Committee (grant 07JCZDJC07200), the Foundation of Tianjin Key Laboratory of Industrial Microbiology (Tianjin University of Science and Technology), China (no. Wsw-01), the State Key Basic Research (973) Program (grant 2007CB936100) and the National HiTech Research Development (863) Program (2009AA03Z311). References [1] T.V. Chirila, Biomaterials 22 (2001) 3311. [2] H. Kobayashi, Y. Ikada, T. Moritera, Y. Ogura, Y. Honda, J. Biomed. Mater. Res. 26 (1992) 1583. [3] H. Kobayashi, M. Kato, T. Taguchi, T. Ikoma, H. Miyashita, S. Shimmura, K. Tsubota, J. Tanaka, Mater. Sci. Eng. C 24 (2004) 729. [4] C.-Y.H. Sung-Yeng Yang, J. Appl. Polym. Sci. 109 (2008) 2452. [5] D.-J. Kim, I.-S. Park, M.-H. Lee, Ceram. Int. 31 (2005) 577. [6] V.I. Legeza, V.P. Galenko-Yaroshevskii, E.V. Zinov'ev, B.A. Paramonov, G.S. Kreichman, I.I. Turkovskii, E.S. Gumenyuk, A.G. Karnovich, A.K. Khripunov, Bull. Exp. Biol. Med. 138 (2004) 311. [7] D. Klemm, D. Schumann, F. Kramer, N. Hessler, M. Hornung, H.P. Schmauder, S. Marsch, Polysaccharides II 205 (2006) 49. [8] D. Klemm, D. Schumann, U. Udhardt, S. Marsch, Prog. Polym. Sci 26 (2001) 1561. [9] A. Svensson, E. Nicklasson, T. Harrah, B. Panilaitis, D.L. Kaplan, M. Brittberg, P. Gatenholm, Biomaterials 26 (2005) 419. [10] A. Bodin, H. Backdahl, G. Helenius, L. Gustafsson, B. Risberg, P. Gatenholm, Abstr. Pap. — Am. Chem. Soc. 229 (2005) U297-U297.
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