Bio-Medical Materials and Engineering 25 (2015) S47–S55 DOI 10.3233/BME-141227 IOS Press
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Structure, physical properties, hemocompatibility and cytocompatibility of starch/zein composites Xing Liu a,∗ , Yi Xie a,∗ , Wei Li b , Wen Sheng c , Yinping Li a , Zan Tong a , Hong Ni b , Celine Huselstein d , Xiong Wang d and Yun Chen a,∗∗ a
Department of Biomedical Engineering, School of Basic Medical Science, Wuhan University, Wuhan, China b Faculty of Life Science, Hubei University, Wuhan, China c Department of Medical Images, Gongan County People’s Hospital, Gongan, China d UMR 7365 CNRS – Université de Lorraine, Ingénierie Moléculaire et Physiopathologie Articulaire, Biopôle, Vandœuvre-lès-Nancy, France Abstract. A series of composite films were prepared from glycerol-plasticized starch and zein by intensive mixing and hot press. The structure and physical properties of the starch/zein (SZ) composite films were characterized by scanning electron microscope (SEM), optical microscopy and water contract angle testing. The hemocompatibility and cytocompatibility of SZ films were evaluated by plasma recalcification time, hemolysis assay and cell culture experiment. SEM and optical observation showed that starch and zein domains can be differed in the films and in a two phase separation status. Glycerol affects the surface hydrophilicity/hydrophobicity of the films. The hemocompatibility and cytocompatibility evaluation showed that SZ composites are anticoagulant materials with no hemolysis and low cytotoxicity. The SZ composites maybe have potentials for applications as biomaterials. Keywords: Starch, zein, hot press, biomaterials
1. Introduction Starch is an abundant natural polymer. Due to its biodegradability, biocompatibility, renewability, availability and economic feasibility, starch-based composites have been widely used as biodegrable materials and partially taken the place of petroleum derived polymers [1,2]. In the biomedical field, starch-based materials showed potentials for the applications of such as bone replacement implants [3], bone cements [4], 3D scaffolds [5], drug delivery [6,7] and plasma substitutes [8]. Unfortunately, poor mechanical properties and easy solubility in water have limited starch’s wide applications [9]. Many efforts have been taken to overcome the shortcomings by modifying starch using physical or chemical methods. Previous studies showed that blending starch with hydrophobic proteins, for example, zein, is an effective way to improve its water-sensitivity, mechanical properties and maintain *
These authors contributed equally to this work. Address for correspondence: Yun Chen, Department of Biomedical Engineering, School of Basic Medical Science, Wuhan University, Wuhan 430071, China. Tel.: +86 27 68759509; E-mail: [email protected]. **
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its biodegradability at the same time [9–12]. Zein, a kind of prolamine, is the major storage protein of corn [13]. Due to its negative nitrogen balance and weak water solubility, zein is seldom used for food [12]. Based on the thermoplastic and hydrophobic properties, zein has potentials to be used as fiber, coating, adhesive, ceramic, ink, cosmetic, textile, chewing gum and biodegradable plastics [13]. Previous work indicated that starch/zein composites could be prepared by glycerol-plasticized and hot press or extruded [9,10,12]. Most of these work focused on the fabrication of starch/zein composites and evaluation of the mechanical properties, water-resistancy and distributions of the two phases. In fact, starch and zein can be separately used as biomaterials. For example, porous three-dimensional zein scaffolds with good biocompatibility and mechanical properties as biomaterials for tissue engineering have been fabricated and evaluated [14,15]. Furthermore, zein microspheres have been prepared and used in drug delivery system [16,17]. Zein/inorganic composites can be used as scaffolds for bone tissue engineering [18]. So, we hypothesized that the starch/zein composites will have good hemocompatibility and biocompatibility. However, until now, almost no work reported the potentials applications of zein/starch composites as biomaterials. Thus, in this work, we fabricated a series of starch/zein composites by intensive mixing and hot process, and their physical properties and hemocompatibility and cytocompatibility were preliminary evaluated. 2. Experimental section 2.1. Materials Pea starch (AccuGelTM ) from Canadian Yellow Peas was supplied by Nutri-Peas, Canada (Portage la Prairie, Canada). Zein (protein content >90%) was from Ruixing Pharmaceutical Raw Materials Co. Ltd (Gaoyou, China). Both starch and zein were vacuum-dried at 70°C for 12 h before use. Iodine (99.99%) and Potassium iodide (>99.99%) were purchased from Aladdin Chemistry Co. Ltd, Shanghai, China. Glycerol (>99.0%) and other chemicals were purchased from Sinopharm Chemical Reagent Co. Ltd, China. 2.2. Preparation of starch/zein composite films Starch (S) and zein (Z) powders were mixed as the weight percent of zein (Wz ) from 0, 10, 30, 50, 70, 90 to 100 wt%, respectively. Then glycerol (35 wt% based on the weight of S and Z) were added to the mixture and premixed in a mortar. After stored at 4°C for 3 days, the mixtures were processed in a SUYUAN SU-70 intensive mixer (Changzhou SUYAN Science & Technology Co. Ltd, China) at 100°C and 30 rpm for 6 min × 3 times. The mixed material was placed into a mould and compressed to a film using a thermo-compressor (Wuhan Qien, China) at 130°C and 20 MPa for 14 min. The films were coded as SZ-n, where n was the value of Wz . All the films were vacuum-dried at 70°C for 12 h, and then cooled and kept in a desiccator before further testing. 2.3. Characterization and evaluation of structure, physical properties, hemocompatibility and cytocompatibility of SZ-n composite films 2.3.1. Morphology of the films The dried films were frozen by liquid nitrogen and then fractured immediately. The cross-section surface of the films were coated with gold and observed by scanning electron microscope (SEM, VEGA 3 LMU, TESCAN, Czech).
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2.3.2. Observation of two-phases distribution To analysis the distribution of the two phases (starch and zein), the films were stained by iodine. 0.1 g I2 was dissolved to 30 ml 1% KI solution and diluted 10 times for use. The films were cut into slices for transection staining. A drop of I2 –KI solution was added and dispersed evenly to each sample, staining for 1 min, then washed slightly by dd-H2 O for three times. The samples were observed using an OLYMPUS CX 31 light microscope after drying. 2.3.3. Measurement of water contact angle The POWEREACH JC2000D1 contact angle system (Shanghai Zhongchen Digital Technic Apparatus Co., Ltd, Shanghai, China) was used for water contact angles measurement. A water droplet was deposited on each sample surface, and a coupling camera device and image analysis software were used to measure the contact angle values. To determine the impact of the plasticizer (glycerol) on the surface hydrophility, both original and de-glycerol films were tested. The films were soaked in water for 24 h for glycerol dissolution. Two samples were tested for at least 6 times to calculate the average values. 2.3.4. Blood biocompatibility evaluation 2.3.4.1. Plasma recalcification time (PRT). The fresh blood was drawn from a healthy New Zealand rabbit, anticoagulated and centrifuged at 3000 rpm for 30 min at room temperature to obtain plateletpoor plasma (PPP). Then SZ-n films (0.1 cm × 0.1 cm) were soaked in 200 µl PPP at 37°C for 30 min 100 µl calcium chloride solution (0.025 mol/l) was immediately added to PPP. The mixture was kept until the first silky fibrin appeared. The time interval between the addition of calcium chloride and the first silky fibrin appeared was recorded as plasma recalcification time (PRT). Tissue culture polystyrene plate (TCPS) was used as a control. Each sample was tested four times. 2.3.4.2. Hemolysis assay. SZ-n films (0.1 cm × 0.1 cm) were rinsed and washed with distilled water and then normal saline for three times in a tube. Then 10 ml normal saline was added in each tube and placed in water bath 37°C for 30 min. 0.2 ml diluted whole blood was added into each tube, and kept in water bath 37°C for 60 min. Distilled water and normal saline were used as positive and negative control, respectively. The tubes were centrifuged at 1500 rpm for 10 min and then the absorbency of the supernatant was measured at 545 nm with a spectrophotometer (Lambda 25 PerkinElmer, USA). Each sample was repeated for three times. The hemolysis rate was calculated as following: HR (%) = (AS − AN)/(AP − AN) × 100, where AS, AP, AN were the absorbencies of the experimental samples, positive control and negative control, respectively. 2.3.5. Cytocompatibility evaluation SZ-n films were cut into 2 cm × 2 cm pieces and sterilized by UV-radiation for 30 min for each side. Then the pieces were placed into 24-well tissue culture polystyrene plates. Before seeding cells on the plate, each sample was washed by PBS and RMPI1640 medium three times, respectively. L929 cells were seeded onto the films with a density of 2 × 105 cells, and the plate without films was the control. The culture plates were incubated at 37°C in 5% CO2 humidified atmosphere for 24 h. After that, the samples and the control plates were washed with PBS for three times, and fixed in 2.5% glutaraldehyde overnight. The cells cultured on the films were observed by SEM after normal drying and gold coating.
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3. Results and discussion 3.1. Morphology of the original materials and the films SEM images of the original starch, zein, and the composite films are shown in Fig. 1. The original starch and zein present elliptical spherical structure and polyhedral-like tubular structure, respectively. The morphology of the films varied with the increasing of zein. SEM images showed the characteristic starch domains, but the original zein morphology could not be observed, indicating phase separation between the hydrophilic starch and the hydrophobic zein occured which corresponded to the iodine staining results. This maybe results from poor adhesion between starch and zein, even glycerol was used as plasticizer. 3.2. Distribution of two phases Optical micrographs of iodine-staining films are shown in Fig. 2. Two distinct phases are observed. The blue or purple zone is starch and the amorphous gray zone is the zein protein. The occurrence of two phase present immiscible morphology, as shown in SEM. Optical micrographs clearly illustrate the phase distribution of the films as the composite ratio changed. SZ-0 film appears almost totally blue and the SZ-100 film presents totally gray. As the starch content exceeds 50%, starch zone is dyed into blue fibrillar structure; while as the content is lower than 50%, starch zone presents characteristic oval-shaped particles. It is interesting to observe that the colour of starch alters from blue to purple as the increasing
Fig. 1. SEM photography of original starch, zein and fractured SZ-n (n = 0, 10, 30, 50, 70, 90 and 100) films.
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Fig. 2. Optical micrographs of two phase distribution in SZ-n films. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-141227.)
Fig. 3. Water contact angle of SZ-n films with glycerol (a) and without glycerol (b).
of zein content. We hypothesis that starch particles were embed in zein as the zein content is more than 50%, and the preparation methods also influence the two phase distribution, leading the different colour. 3.3. Contact angle measurement Contact angle is a parameter to evaluate the surface hydrophobicity or hydrophilicity of films. The water contact angle with (a) and without (b) glycerol of the films are shown in Fig. 3. In Fig. 3(b), it is very clear that the water contact increased with an increase of zein content without glycerol in the films. It means that the hydrophilicity of the films only depends on the nature of the component ratio
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and decreased when zein content increased. This is due to the lower hydrophilicity of zein than starch. However, the change trend of water contact angle in the SZ films containing glycerol looks strange. The hydrophilicity of the films firstly increased with an increase in zein until zein content is 50%, and then decreased and kept no change when zein content increased to 100%. This can be explained by the hydrophilicity of glycerol itself and the interactions between glycerol and starch and between glycerol and zein. SZ-0 contains starch and glycerol, both components have good miscibility, which resulted in homogeneous distribution of glycerol in starch. The film showed hydrophilicity due to the nature of starch and glycerol. SZ-100 contains zein and glycerol, both components are immiscibility. In this case, glycerol could not well plasticize zein component and a layer of glycerol “adhere” on the surface of the films, which resulted in a higher hydrophilicity than SZ-0. It looks that water firstly contacts with glycerol but not directly contacts with zein, so the hydrophilicity of the films just depended on glycerol. In the films containing starch, zein and glycerol, when zein content is higher than starch, some glycerol looks “adhere” on the surface of the films, which resulted in a higher hydrophilicity. From the result of water contact measurement, the hydrophilicity of the SZ films greatly depended on that glycerol was removed or not. For example, if the films were used in the environment with water, the glycerol may be easily dissolved in water and the hydrophilicity of the films would change obviously. 3.4. Blood compatibility When blood contracts with the surface of foreign body, it may bring about coagulation reaction, leading thrombosis or foreign body reaction. Plasma recalcification time is a common way to evaluate in vitro anticoagulation. Figure 4 shows the plasma recalcification time (PRT) values of control and SZ-n composites. The average PRT values of SZ-n composites are all higher than control, and as the zein content increases, the PRT values increase. There is a significant difference between SZ-n composites and control (p < 0.05). The results indicate that SZ-n composites showed higher anticoagulant ability as zein content increased, which is due to the nature of zein’s hemocompatibility.
Fig. 4. The plasma recalcification time (PRT) values of control and SZ-n films.
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Erythorocyte-induced hemolysis in vitro is considered to be another reliable and important parameter to evaluate the hemocompatibility. Distilled water and saline are used as positive and negative controls, respectively. The hemolysis rate of controls and SZ-n films are summarized in Table 1. According to ISO 10993-4 1999, HR less than 5% is regarded to be non-hemolysis. HR of SZ-n film composites are all less than 5%, especially HR of neat zein is as low as 0.80%, and the highest HR of SZ-50 is 3.68%. The results indicate that the SZ-n films do not induce hemolysis when contact with red blood cells. 3.5. Morphology observation of the cells cultured on films Cell attachment, growth, and differentiation on the surface of the material are very important parameters to evaluate the cytocompatibility of biomaterial. Figure 5 shows the SEM photography of L929 cells cultured for 24 h on the surfaces of SZ-n films. The shuttle or round shaped L929 cells expanded and grew well on the surface when the zein content is more than 50%. As shown in the images, the surface of the SZ-n (n = 0, 10 and 30) films is too rough to support the cells to grow on them, only very few cells Table 1 The hemolysis ratio of SZ-n (n = 0, 10, 30, 50, 70, 90 and 100) films Samples Normal saline (negative control) Distilled water (positive control) SZ-0 SZ-10 SZ-30 SZ-50 SZ-70 SZ-90 SZ-100
Optical density at 545 nm 0.0090 ± 0.0011 0.996 ± 0.025456 0.0223 ± 0.016197 0.012 ± 0.00321455 0.023 ± 0.004583 0.03666 ± 0.007638 0.02766 ± 0.006429 0.026 ± 0.010392 0.008 ± 0.001732
HR (%) 0 100 2.24 1.24 2.31 3.68 2.78 2.61 0.80
Fig. 5. SEM images of L929 cells cultured on SZ-n film for 24 h.
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can still be observed on the films. Since influenced by the films themselves, the cell density cannot be distinguished accurately, but the increasing trend of the cell density can be observed as the zein content increased. 4. Conclusions Starch/zein films were successfully fabricated by intensive mixing and hot press. The SEM images from the films showed the starch domains and zein domains occurred in the films, which also was proved by the results from the two phase distribution observed by optical microscopy. The results of water contract angle showed that glycerol as the plasticizer affected the surface hydrophilicity/hydrophobicity of the films. The results from hemocompatibility evaluation suggested that SZ-n composites are anticoagulant materials and had no hemolysis. The results from cell culture manifest that the composites have low cytotoxicity to L929 cells when zein content was more than 50%, showing potentials for biomedical applications. Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, 2010CB732203), National Natural Science Foundation of China (81171480) and “Program Cai Yuanpei 2013-2015” (CSC No. 201304490192 and 201304490191) from China Scholarship Council. References [1] L. Avérous, Biodegradable multiphase systems based on plasticized starch: a review, J. Macromol. Sci. 44 (2004), 231– 274. [2] J. Loercks, Properties and applications of compostable starch-based plastic material, Polym. Degrad. Stabil. 59 (1998), 245–249. [3] R.L. Reis and A.M. Cunha, Characterization of two biodegradable polymers of potential application within the biomaterials field, J. Mater. Sci. Mater. Med. 6 (1995), 786–792. [4] R.L. Reis, S.C. Mendes, A.M. Cunha and M.J. Bevis, Processing and in vitro degradation of starch/EVOH thermoplastic blends, Polym. Int. 43 (1997), 347–352. [5] M.E. Gomes, V.I. Sikavitsas, E. Behravesh, L.R. Rui and G.M. Antonios, Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three-dimensional scaffolds, J. Biomed. Mater. Res. Part A 67 (2003), 87–95. [6] C. Elvira, J.F. Mano, J.S. Roman and R.L. Reis, Starch-based biodegradable hydrogels with potential biomedical applications as drug delivery systems, Biomaterials 23 (2002), 1955–1966. [7] C.K. Simi and T.E. Abraham, Hydrophobic grafted and cross-linked starch nanoparticles for drug delivery, Bioproc. Biosys. Eng. 30 (2007), 173–180. [8] R.C.G. Gallandat Huet, A.W. Siemons, D. Baus, W.T. van Rooyen-Butijn, J.A.M. Haagenaars, W. van Oeveren and F. Bepperling, A novel hydroxyethyl starch (Voluven® ) for effective perioperative plasma volume substitution in cardiac surgery, Can. J. Anesth. 47 (2000), 1207–1215. [9] E. Corradini, A.J.F. Carvalho, A.A.S. Curvelo, J.M. Agnelli and L.H.C. Mattoso, Preparation and characterization of thermoplastic starch/zein blends, Mater. Res. 10 (2007), 227–231. [10] E. Habeych, A.J. van der Goot and R. Boom, In situ compatibilization of starch–zein blends under shear flow, Chem. Eng. Sci. 64 (2009), 3516–3524. [11] E. Habeych, B. Dekkers, A.J. van der Goot and R. Boom, Starch–zein blends formed by shear flow, Chem. Eng. Sci. 63 (2008), 5229–5238. [12] E. Corradini, E. Souto de Medeiros, A.J.F. Carvalho, A.A.S. Curvelo and L.H.C. Mattoso, Mechanical and morphological characterization of starch/zein blends plasticized with glycerol, J. Appl. Polym. Sci. 101 (2006), 4133–4139.
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[13] R. Shukla and M. Cheryan, Zein: the industrial protein from corn, Ind. Crop. Prod. 13 (2001), 171–192. [14] J. Dong, Q. Sun and J.Y. Wang, Basic study of corn protein, zein, as a biomaterial in tissue engineering, surface morphology and biocompatibility, Biomaterials 5 (2004), 4691–4697. [15] S.J. Gong, H.J. Wang, Q.S. Sun, S.T. Xue and J.Y. Wang, Mechanical properties and in vitro biocompatibility of porous zein scaffolds, Biomaterials 27 (2006), 3793–3799. [16] E. Mathiowitz, H. Bernstein, E. Morrel and K. Schwaller, Method for producing protein microspheres, U.S. Patent No. 91/06286, 1991. [17] X.M. Liu, Q.S. Sun, H.J. Wang, L. Zhang and J.Y. Wang, Microspheres of corn protein, zein, for an ivermectin drug delivery system, Biomaterials 26 (2004), 109–115. [18] Z.H. Qu, H.J. Wang, T.T. Tang, J.Y. Wang and K.R. Dai, Evaluation of the zein/inorganics composite on biocompatibility and osteoblastic differentiation, Acta Biomater. 4 (2008), 1360–1368.
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Structure, physical properties, hemocompatibility and cytocompatibility of starch/zein composites Xing Liu a,∗ , Yi Xie a,∗ , Wei Li b , Wen Sheng c , Yinping Li a , Zan Tong a , Hong Ni b , Celine Huselstein d , Xiong Wang d and Yun Chen a,∗∗ a
Department of Biomedical Engineering, School of Basic Medical Science, Wuhan University, Wuhan, China b Faculty of Life Science, Hubei University, Wuhan, China c Department of Medical Images, Gongan County People’s Hospital, Gongan, China d UMR 7365 CNRS – Université de Lorraine, Ingénierie Moléculaire et Physiopathologie Articulaire, Biopôle, Vandœuvre-lès-Nancy, France Abstract. A series of composite films were prepared from glycerol-plasticized starch and zein by intensive mixing and hot press. The structure and physical properties of the starch/zein (SZ) composite films were characterized by scanning electron microscope (SEM), optical microscopy and water contract angle testing. The hemocompatibility and cytocompatibility of SZ films were evaluated by plasma recalcification time, hemolysis assay and cell culture experiment. SEM and optical observation showed that starch and zein domains can be differed in the films and in a two phase separation status. Glycerol affects the surface hydrophilicity/hydrophobicity of the films. The hemocompatibility and cytocompatibility evaluation showed that SZ composites are anticoagulant materials with no hemolysis and low cytotoxicity. The SZ composites maybe have potentials for applications as biomaterials. Keywords: Starch, zein, hot press, biomaterials
1. Introduction Starch is an abundant natural polymer. Due to its biodegradability, biocompatibility, renewability, availability and economic feasibility, starch-based composites have been widely used as biodegrable materials and partially taken the place of petroleum derived polymers [1,2]. In the biomedical field, starch-based materials showed potentials for the applications of such as bone replacement implants [3], bone cements [4], 3D scaffolds [5], drug delivery [6,7] and plasma substitutes [8]. Unfortunately, poor mechanical properties and easy solubility in water have limited starch’s wide applications [9]. Many efforts have been taken to overcome the shortcomings by modifying starch using physical or chemical methods. Previous studies showed that blending starch with hydrophobic proteins, for example, zein, is an effective way to improve its water-sensitivity, mechanical properties and maintain *
These authors contributed equally to this work. Address for correspondence: Yun Chen, Department of Biomedical Engineering, School of Basic Medical Science, Wuhan University, Wuhan 430071, China. Tel.: +86 27 68759509; E-mail: [email protected]. **
0959-2989/15/$27.50 © 2015 – IOS Press and the authors. All rights reserved
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its biodegradability at the same time [9–12]. Zein, a kind of prolamine, is the major storage protein of corn [13]. Due to its negative nitrogen balance and weak water solubility, zein is seldom used for food [12]. Based on the thermoplastic and hydrophobic properties, zein has potentials to be used as fiber, coating, adhesive, ceramic, ink, cosmetic, textile, chewing gum and biodegradable plastics [13]. Previous work indicated that starch/zein composites could be prepared by glycerol-plasticized and hot press or extruded [9,10,12]. Most of these work focused on the fabrication of starch/zein composites and evaluation of the mechanical properties, water-resistancy and distributions of the two phases. In fact, starch and zein can be separately used as biomaterials. For example, porous three-dimensional zein scaffolds with good biocompatibility and mechanical properties as biomaterials for tissue engineering have been fabricated and evaluated [14,15]. Furthermore, zein microspheres have been prepared and used in drug delivery system [16,17]. Zein/inorganic composites can be used as scaffolds for bone tissue engineering [18]. So, we hypothesized that the starch/zein composites will have good hemocompatibility and biocompatibility. However, until now, almost no work reported the potentials applications of zein/starch composites as biomaterials. Thus, in this work, we fabricated a series of starch/zein composites by intensive mixing and hot process, and their physical properties and hemocompatibility and cytocompatibility were preliminary evaluated. 2. Experimental section 2.1. Materials Pea starch (AccuGelTM ) from Canadian Yellow Peas was supplied by Nutri-Peas, Canada (Portage la Prairie, Canada). Zein (protein content >90%) was from Ruixing Pharmaceutical Raw Materials Co. Ltd (Gaoyou, China). Both starch and zein were vacuum-dried at 70°C for 12 h before use. Iodine (99.99%) and Potassium iodide (>99.99%) were purchased from Aladdin Chemistry Co. Ltd, Shanghai, China. Glycerol (>99.0%) and other chemicals were purchased from Sinopharm Chemical Reagent Co. Ltd, China. 2.2. Preparation of starch/zein composite films Starch (S) and zein (Z) powders were mixed as the weight percent of zein (Wz ) from 0, 10, 30, 50, 70, 90 to 100 wt%, respectively. Then glycerol (35 wt% based on the weight of S and Z) were added to the mixture and premixed in a mortar. After stored at 4°C for 3 days, the mixtures were processed in a SUYUAN SU-70 intensive mixer (Changzhou SUYAN Science & Technology Co. Ltd, China) at 100°C and 30 rpm for 6 min × 3 times. The mixed material was placed into a mould and compressed to a film using a thermo-compressor (Wuhan Qien, China) at 130°C and 20 MPa for 14 min. The films were coded as SZ-n, where n was the value of Wz . All the films were vacuum-dried at 70°C for 12 h, and then cooled and kept in a desiccator before further testing. 2.3. Characterization and evaluation of structure, physical properties, hemocompatibility and cytocompatibility of SZ-n composite films 2.3.1. Morphology of the films The dried films were frozen by liquid nitrogen and then fractured immediately. The cross-section surface of the films were coated with gold and observed by scanning electron microscope (SEM, VEGA 3 LMU, TESCAN, Czech).
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2.3.2. Observation of two-phases distribution To analysis the distribution of the two phases (starch and zein), the films were stained by iodine. 0.1 g I2 was dissolved to 30 ml 1% KI solution and diluted 10 times for use. The films were cut into slices for transection staining. A drop of I2 –KI solution was added and dispersed evenly to each sample, staining for 1 min, then washed slightly by dd-H2 O for three times. The samples were observed using an OLYMPUS CX 31 light microscope after drying. 2.3.3. Measurement of water contact angle The POWEREACH JC2000D1 contact angle system (Shanghai Zhongchen Digital Technic Apparatus Co., Ltd, Shanghai, China) was used for water contact angles measurement. A water droplet was deposited on each sample surface, and a coupling camera device and image analysis software were used to measure the contact angle values. To determine the impact of the plasticizer (glycerol) on the surface hydrophility, both original and de-glycerol films were tested. The films were soaked in water for 24 h for glycerol dissolution. Two samples were tested for at least 6 times to calculate the average values. 2.3.4. Blood biocompatibility evaluation 2.3.4.1. Plasma recalcification time (PRT). The fresh blood was drawn from a healthy New Zealand rabbit, anticoagulated and centrifuged at 3000 rpm for 30 min at room temperature to obtain plateletpoor plasma (PPP). Then SZ-n films (0.1 cm × 0.1 cm) were soaked in 200 µl PPP at 37°C for 30 min 100 µl calcium chloride solution (0.025 mol/l) was immediately added to PPP. The mixture was kept until the first silky fibrin appeared. The time interval between the addition of calcium chloride and the first silky fibrin appeared was recorded as plasma recalcification time (PRT). Tissue culture polystyrene plate (TCPS) was used as a control. Each sample was tested four times. 2.3.4.2. Hemolysis assay. SZ-n films (0.1 cm × 0.1 cm) were rinsed and washed with distilled water and then normal saline for three times in a tube. Then 10 ml normal saline was added in each tube and placed in water bath 37°C for 30 min. 0.2 ml diluted whole blood was added into each tube, and kept in water bath 37°C for 60 min. Distilled water and normal saline were used as positive and negative control, respectively. The tubes were centrifuged at 1500 rpm for 10 min and then the absorbency of the supernatant was measured at 545 nm with a spectrophotometer (Lambda 25 PerkinElmer, USA). Each sample was repeated for three times. The hemolysis rate was calculated as following: HR (%) = (AS − AN)/(AP − AN) × 100, where AS, AP, AN were the absorbencies of the experimental samples, positive control and negative control, respectively. 2.3.5. Cytocompatibility evaluation SZ-n films were cut into 2 cm × 2 cm pieces and sterilized by UV-radiation for 30 min for each side. Then the pieces were placed into 24-well tissue culture polystyrene plates. Before seeding cells on the plate, each sample was washed by PBS and RMPI1640 medium three times, respectively. L929 cells were seeded onto the films with a density of 2 × 105 cells, and the plate without films was the control. The culture plates were incubated at 37°C in 5% CO2 humidified atmosphere for 24 h. After that, the samples and the control plates were washed with PBS for three times, and fixed in 2.5% glutaraldehyde overnight. The cells cultured on the films were observed by SEM after normal drying and gold coating.
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3. Results and discussion 3.1. Morphology of the original materials and the films SEM images of the original starch, zein, and the composite films are shown in Fig. 1. The original starch and zein present elliptical spherical structure and polyhedral-like tubular structure, respectively. The morphology of the films varied with the increasing of zein. SEM images showed the characteristic starch domains, but the original zein morphology could not be observed, indicating phase separation between the hydrophilic starch and the hydrophobic zein occured which corresponded to the iodine staining results. This maybe results from poor adhesion between starch and zein, even glycerol was used as plasticizer. 3.2. Distribution of two phases Optical micrographs of iodine-staining films are shown in Fig. 2. Two distinct phases are observed. The blue or purple zone is starch and the amorphous gray zone is the zein protein. The occurrence of two phase present immiscible morphology, as shown in SEM. Optical micrographs clearly illustrate the phase distribution of the films as the composite ratio changed. SZ-0 film appears almost totally blue and the SZ-100 film presents totally gray. As the starch content exceeds 50%, starch zone is dyed into blue fibrillar structure; while as the content is lower than 50%, starch zone presents characteristic oval-shaped particles. It is interesting to observe that the colour of starch alters from blue to purple as the increasing
Fig. 1. SEM photography of original starch, zein and fractured SZ-n (n = 0, 10, 30, 50, 70, 90 and 100) films.
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Fig. 2. Optical micrographs of two phase distribution in SZ-n films. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-141227.)
Fig. 3. Water contact angle of SZ-n films with glycerol (a) and without glycerol (b).
of zein content. We hypothesis that starch particles were embed in zein as the zein content is more than 50%, and the preparation methods also influence the two phase distribution, leading the different colour. 3.3. Contact angle measurement Contact angle is a parameter to evaluate the surface hydrophobicity or hydrophilicity of films. The water contact angle with (a) and without (b) glycerol of the films are shown in Fig. 3. In Fig. 3(b), it is very clear that the water contact increased with an increase of zein content without glycerol in the films. It means that the hydrophilicity of the films only depends on the nature of the component ratio
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and decreased when zein content increased. This is due to the lower hydrophilicity of zein than starch. However, the change trend of water contact angle in the SZ films containing glycerol looks strange. The hydrophilicity of the films firstly increased with an increase in zein until zein content is 50%, and then decreased and kept no change when zein content increased to 100%. This can be explained by the hydrophilicity of glycerol itself and the interactions between glycerol and starch and between glycerol and zein. SZ-0 contains starch and glycerol, both components have good miscibility, which resulted in homogeneous distribution of glycerol in starch. The film showed hydrophilicity due to the nature of starch and glycerol. SZ-100 contains zein and glycerol, both components are immiscibility. In this case, glycerol could not well plasticize zein component and a layer of glycerol “adhere” on the surface of the films, which resulted in a higher hydrophilicity than SZ-0. It looks that water firstly contacts with glycerol but not directly contacts with zein, so the hydrophilicity of the films just depended on glycerol. In the films containing starch, zein and glycerol, when zein content is higher than starch, some glycerol looks “adhere” on the surface of the films, which resulted in a higher hydrophilicity. From the result of water contact measurement, the hydrophilicity of the SZ films greatly depended on that glycerol was removed or not. For example, if the films were used in the environment with water, the glycerol may be easily dissolved in water and the hydrophilicity of the films would change obviously. 3.4. Blood compatibility When blood contracts with the surface of foreign body, it may bring about coagulation reaction, leading thrombosis or foreign body reaction. Plasma recalcification time is a common way to evaluate in vitro anticoagulation. Figure 4 shows the plasma recalcification time (PRT) values of control and SZ-n composites. The average PRT values of SZ-n composites are all higher than control, and as the zein content increases, the PRT values increase. There is a significant difference between SZ-n composites and control (p < 0.05). The results indicate that SZ-n composites showed higher anticoagulant ability as zein content increased, which is due to the nature of zein’s hemocompatibility.
Fig. 4. The plasma recalcification time (PRT) values of control and SZ-n films.
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Erythorocyte-induced hemolysis in vitro is considered to be another reliable and important parameter to evaluate the hemocompatibility. Distilled water and saline are used as positive and negative controls, respectively. The hemolysis rate of controls and SZ-n films are summarized in Table 1. According to ISO 10993-4 1999, HR less than 5% is regarded to be non-hemolysis. HR of SZ-n film composites are all less than 5%, especially HR of neat zein is as low as 0.80%, and the highest HR of SZ-50 is 3.68%. The results indicate that the SZ-n films do not induce hemolysis when contact with red blood cells. 3.5. Morphology observation of the cells cultured on films Cell attachment, growth, and differentiation on the surface of the material are very important parameters to evaluate the cytocompatibility of biomaterial. Figure 5 shows the SEM photography of L929 cells cultured for 24 h on the surfaces of SZ-n films. The shuttle or round shaped L929 cells expanded and grew well on the surface when the zein content is more than 50%. As shown in the images, the surface of the SZ-n (n = 0, 10 and 30) films is too rough to support the cells to grow on them, only very few cells Table 1 The hemolysis ratio of SZ-n (n = 0, 10, 30, 50, 70, 90 and 100) films Samples Normal saline (negative control) Distilled water (positive control) SZ-0 SZ-10 SZ-30 SZ-50 SZ-70 SZ-90 SZ-100
Optical density at 545 nm 0.0090 ± 0.0011 0.996 ± 0.025456 0.0223 ± 0.016197 0.012 ± 0.00321455 0.023 ± 0.004583 0.03666 ± 0.007638 0.02766 ± 0.006429 0.026 ± 0.010392 0.008 ± 0.001732
HR (%) 0 100 2.24 1.24 2.31 3.68 2.78 2.61 0.80
Fig. 5. SEM images of L929 cells cultured on SZ-n film for 24 h.
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can still be observed on the films. Since influenced by the films themselves, the cell density cannot be distinguished accurately, but the increasing trend of the cell density can be observed as the zein content increased. 4. Conclusions Starch/zein films were successfully fabricated by intensive mixing and hot press. The SEM images from the films showed the starch domains and zein domains occurred in the films, which also was proved by the results from the two phase distribution observed by optical microscopy. The results of water contract angle showed that glycerol as the plasticizer affected the surface hydrophilicity/hydrophobicity of the films. The results from hemocompatibility evaluation suggested that SZ-n composites are anticoagulant materials and had no hemolysis. The results from cell culture manifest that the composites have low cytotoxicity to L929 cells when zein content was more than 50%, showing potentials for biomedical applications. Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, 2010CB732203), National Natural Science Foundation of China (81171480) and “Program Cai Yuanpei 2013-2015” (CSC No. 201304490192 and 201304490191) from China Scholarship Council. References [1] L. Avérous, Biodegradable multiphase systems based on plasticized starch: a review, J. Macromol. Sci. 44 (2004), 231– 274. [2] J. Loercks, Properties and applications of compostable starch-based plastic material, Polym. Degrad. Stabil. 59 (1998), 245–249. [3] R.L. Reis and A.M. Cunha, Characterization of two biodegradable polymers of potential application within the biomaterials field, J. Mater. Sci. Mater. Med. 6 (1995), 786–792. [4] R.L. Reis, S.C. Mendes, A.M. Cunha and M.J. Bevis, Processing and in vitro degradation of starch/EVOH thermoplastic blends, Polym. Int. 43 (1997), 347–352. [5] M.E. Gomes, V.I. Sikavitsas, E. Behravesh, L.R. Rui and G.M. Antonios, Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three-dimensional scaffolds, J. Biomed. Mater. Res. Part A 67 (2003), 87–95. [6] C. Elvira, J.F. Mano, J.S. Roman and R.L. Reis, Starch-based biodegradable hydrogels with potential biomedical applications as drug delivery systems, Biomaterials 23 (2002), 1955–1966. [7] C.K. Simi and T.E. Abraham, Hydrophobic grafted and cross-linked starch nanoparticles for drug delivery, Bioproc. Biosys. Eng. 30 (2007), 173–180. [8] R.C.G. Gallandat Huet, A.W. Siemons, D. Baus, W.T. van Rooyen-Butijn, J.A.M. Haagenaars, W. van Oeveren and F. Bepperling, A novel hydroxyethyl starch (Voluven® ) for effective perioperative plasma volume substitution in cardiac surgery, Can. J. Anesth. 47 (2000), 1207–1215. [9] E. Corradini, A.J.F. Carvalho, A.A.S. Curvelo, J.M. Agnelli and L.H.C. Mattoso, Preparation and characterization of thermoplastic starch/zein blends, Mater. Res. 10 (2007), 227–231. [10] E. Habeych, A.J. van der Goot and R. Boom, In situ compatibilization of starch–zein blends under shear flow, Chem. Eng. Sci. 64 (2009), 3516–3524. [11] E. Habeych, B. Dekkers, A.J. van der Goot and R. Boom, Starch–zein blends formed by shear flow, Chem. Eng. Sci. 63 (2008), 5229–5238. [12] E. Corradini, E. Souto de Medeiros, A.J.F. Carvalho, A.A.S. Curvelo and L.H.C. Mattoso, Mechanical and morphological characterization of starch/zein blends plasticized with glycerol, J. Appl. Polym. Sci. 101 (2006), 4133–4139.
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