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Effect of water vapor on the surface characteristics and cell compatibility of zein films
Colloids and Surfaces B: Biointerfaces 69 (2009) 109–115
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effect of water vapor on the surface characteristics and cell compatibility of zein films Hua-Jie Wang a , Jian-Xi Fu b , Jin-Ye Wang b,∗ a b
College of Life Science and Biotechnology, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China
a r t i c l e
i n f o
Article history: Received 29 August 2008 Received in revised form 3 October 2008 Accepted 17 November 2008 Available online 25 November 2008 Keywords: Zein film Higher relative humidity Contact angle Compatibility
a b s t r a c t In this study, zein microsphere films were fabricated via a solution casting method, and their surfaces were subsequently treated with a higher relative humidity. Changes in surface properties were characterized by contact-angle measurement and scanning electron microscopy (SEM). Human hepatoma cells (BEL-7402) were used as model cells to evaluate cell adhesion, spreading and proliferation on zein films before and after treatment. Additionally, the adhesion and morphologies of rat platelets on zein films were also investigated to evaluate the blood compatibility of the films. The results showed that the hydrophilicity of zein films was changed after treatment, and the surface morphology varied, gradually becoming more smooth and the microspheres in the films disappeared. As the result, the adhesion and proliferation of BEL-7402 cells were significantly improved, while HUVECs were more sensitive than BEL7402 cells to cell–substrate interactions. Adhesion of rat platelets could be inhibited on the treated zein films. The results suggest that the synergistic actions of hydrophilic properties and micro morphologies are responsible for the cell behaviors. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The compatibility of tissue engineering materials is one important factor that limits their application in the tissue engineering field because the main interactions between a tissue and an implant occur at the atomic layers of an implant. Therefore, improvement of the material–cell interaction interface that forms the basis of cell adhesion and proliferation allows for maintenance of the normal cell phenotype and promotion of differentiation through the activation of specific gene expression. The material–cell interaction is affected by many factors, such as wettability (hydrophilicity/hydrophobicity), surface free energy, chemistry, charge, roughness, and rigidity [1]. Many advances have been made in understanding events at the interface between tissues and implants and in developing methods for controlling these events [2]. For example, proteins can be adsorbed on the surface after implantation into a living system in less than a second and a monolayer of adsorbed protein forms in seconds to minutes; the specific adsorbed protein can further alter the function of a cell. Arima and Iwata prepared self-assembled monolayers of alkanethiols with a wide range of wettabilities, and showed that the adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) and Hela cells were affected by surface wettability
∗ Corresponding author. Tel.: +86 21 54925330; fax: +86 21 64166128. E-mail address: [email protected] (J.-Y. Wang). 0927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2008.11.015
and functional groups, which controlled the adsorption of serum proteins [3]. Zein, soluble in aqueous alcohol solutions, is the major storage protein of corn. Zein has been used widely, especially in the food industrial field [4]. Our previous work has indicated that zein has good biocompatibility with HUVECs, liver cells and mouse fibroblast cells [5,6]. After preparing zein microspheres by the phase isolation technique, we prepared a zein film using the microspheres by the casting method. Interestingly, we found that the surface morphology of the zein film could change during the storage process. In this study, the effect of water vapor on the surface characteristics of zein films was investigated by scanning electron microscopy (SEM) and static water contact-angle analysis, and their cell compatibility and blood compatibility were analyzed by cell culture in vitro and by the platelet adhesion test. 2. Materials and methods 2.1. Preparation of zein films The zein microsphere thin film was prepared on a glass matrix at 37 ◦ C according to our previous work [5,6]. In brief, a zein microsphere suspended solution was formed in a 40% aqueous mixture of ethanol based on the phase isolation technique. Then, the above solution was poured onto glass surfaces, forming a thin layer, and dried through volatilization at 37 ◦ C or by spinning at 500 rpm for 10 min. The films were then stored for the desired time periods (0,
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12, 24, 72, and 144 h) under conditions of moist steam at 37 ◦ C. The films were dried at 37 ◦ C and stored in a desiccator for future use. 2.2. Morphology observation Zein films were mounted on stubs and coated in a vacuum with gold, and the surface morphologies then were investigated by scanning electron microscopy (JSM-6390LV, JEOL, Japan).
tration of zein, the concentration of the organic phase, the mixing speed, and so on. Therefore, a film with various particle sizes could be obtained from the microspheres using film-preparation techniques. In the present study, we mainly discuss the effect of water vapor on zein films, including surface properties and biological properties. 3.1. Effect of higher relative humidity conditions on surface wettabilities and morphologies
2.3. Contact-angle measurements Wettabilities of the target films were evaluated as static water contact angles using a contact-angle goniometer (JC2000A, Shanghai, China) [7]. Briefly, a droplet of water (0.5 l, ultrapure grade) was put on the surfaces of different materials and the images were photographed, with temperature and moisture being kept constant during the course of the experiments (23 ◦ C and 68%, respectively). The angle between the baseline of the drop and the tangent at the drop boundary was measured. Measurements were performed at least in triplicate on three different batches of material. 2.4. Platelet adhesion assays in vitro Platelet adhesion was performed according to our previous work [5]. The platelets were obtained by centrifuging anticoagulant blood from an SD rat at 1500 rpm for 10 min, and the supernatant plateletrich plasma at 3000 rpm for another 10 min at 4 ◦ C. The platelets were resuspended in PBS, and the number of platelets was diluted to 5 × 106 cells. Zein microsphere films were placed into contact with 50 l of the platelets and left at 37 ◦ C for 60–180 min. The platelets were removed and the films were rinsed three times with PBS [8]. The adhered platelets were fixed by immersing the film into a 2.5% solution of glutaraldehyde in PBS for at least 2 h at 4 ◦ C. Samples were freeze-dried, and then sputter-coated with gold for SEM observation. The number of platelets adhered was determined via a colorimetric method [9]. Briefly, 150 l of 4-nitropheny-phosphate disodium salt was added to each well and the samples were placed at 37 ◦ C for 60 min. The absorption was determined at a wavelength of 405 nm after stopping the reaction with 100 l of 2N NaOH. 2.5. Cell culture The human hepatoma cell line BEL-7402 was used for the in vitro test. The cells were incubated at 37 ◦ C in a 95% humidified air/5% CO2 atmosphere and seeded onto target films in a 96-well Corning culture microplate at 2.4 × 104 cells/ml after digestion with 0.25% trypsin/0.002% EDTA. 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was used to assess the adhesion and proliferation of BEL-7402 cells after 4 h, 24 h, 48 h and 72 h of culture. The morphologies and distribution of cells were observed through a fluorescence microscope (IX71, Olympus, Japan) after being stained with acridine orange fluorescent dye in PBS (pH 7.2) for 5 min.
In order to present the data clearly, the original and treated zein films for different periods are abbreviated as Z0h , Z12h , Z24h , Z72h , and Z144h , respectively. The contact angles of zein films were measured to compare the wettabilities of their surfaces by the sessile drop technique, as shown in Fig. 1. It has been reported that the water contact angle of a hydrophobic surface is over 90◦ [10]. During the treatment, the water contact angles of zein films gradually increased (from 52.0 ± 2.9◦ to 71.8 ± 3.0◦ ) over 24 h, and then gradually decreased to 62.2 ± 5.2◦ when the time was prolonged to 144 h, which indicates relative hydrophobic properties compared to the original zein film. Fig. 2 shows SEM images of zein films during water vapor treatments for different periods. Obvious differences in the surface morphologies of the original and treated zein films were observed. The original zein films were composed of round particles with a size in the nanometer range and the particles conglutinated with each other to form an integrated film. After water vapor treatment, the surface increasingly smooth, but the outline of the particles of the film still remained after 12 h; however, a smooth film was formed after a 24 h treatment, and a few small pores could be observed. This change from Z0h to Z144h was mainly due to the conglutination of zein according to our previous report [5]. To date, the structure of zein in solution has been investigated by various physico-chemical studies, and the amino acid sequence of zein is well known [11]. Zein is a hydrophobic protein due to a high amount of the hydrophobic amino acids such as leucine, proline, alanine, and phenylalanine, which was the main reason for the zein film displaying a water contact angle over 50◦ (Fig. 1). Lakshmannan and Dhathathreyan [12] reported that some peptides could vary from non-polar to polar when they changed from an air/water interface to a solid/air interface, and the zein protein surface could also restructure or reorient in response to different environments. That is to say, zein will orient its polar chemical components toward the interface when the zein film surface is exposed to an aqueous or highly polar environment if given sufficient mobility. Therefore, zein films show hydrophilic properties. The zein molecule is amphiphilic, just as Kim and Xu reported [13]. This property causes the formation of macromolecular micelle-globular aggregates with the hydrophilic moiety exposed to the surface and the hydrophobic moieties clumped together in
2.6. Statistical analysis The number of independent replications is listed individually for each experiment. All data are expressed as mean ± standard deviation and analyzed by Fisher’s method for multiple comparison, and the statistical significance was accepted at p < 0.05. 3. Results and discussion It was shown that the particle size of the zein microspheres could be affected by many factors during the process, such as the concen-
Fig. 1. Changes in the water contact angle of the original zein film and zein films treated with water vapor for 12, 24, 72, and 144 h. (*) Represents a significant difference compared with 0 h samples.
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Fig. 2. Scanning electron micrographs of the original zein film (A) and zein films treated with water vapor for 12 h (B), 72 h (C), and 144 h (D).
the aqueous ethanol solution with ethanol content of lower than 90%. The zein aggregates precipitate as microspheres by the end, when the ethanol content is decreased to less than 40%, in which zein is insoluble (Fig. 2). In addition, zein is able to form a viscoelastic protein network during mixing through a structural rearrangement if the protein is held and mixed at 35 ◦ C, above its glass transition temperature of 28 ◦ C, and at >20% moisture content. This structural change is due to changes in zein secondary structure from ␣-helix to -sheet structures [14,15]. According to the result shown in Fig. 2, after water vapor treatment for over 24 h, zein microspheres disappeared and a smooth zein film formed instead, which further supported the conclusions made upon observation. Morphological changes of the zein film induced by water vapor treatment is similar to that immersed in solution directly, as shown in our previous study, in which sizes of microspheres increased with immersing time of the film in PBS, adjacent microspheres fused with each other [5]. 3.2. Effect of surface treatment on platelet adhesion onto zein films Platelet adhesion onto films from plasma is an important test for evaluating the blood compatibility of materials [16]. We performed a blood platelet adhesion assay on zein films through exposure to fresh rat platelets using a Corning plate as the control, and did a quantified analysis using a colorimetric method based on the activity of the platelet enzyme acid phosphatase [9]. Fig. 3 shows the relative number of platelets adhered on zein films. Compared with the control, zein films significantly inhibited platelet adhesion (p < 0.05). Moreover, the numbers of adhering platelets on the treated films were lower than in the untreated group, except for the Z144h film group. SEM examination clearly showed the adhesion and distribution of platelets on the films (Fig. 4), which was the same result seen using the colorimetric assay. Platelets adhered as single cells and not as aggregates, with only small and sporadic aggregates being detectable. The platelets did not spread over the surface, and maintained a round morphology, which indicates that the platelets
were unactivated. Both results indicated a better hemocompatibility of zein films compared to the control plate. Platelet adhesion mainly depends on the type and conformation of adsorbed plasma proteins [8,17], while plasma protein adsorption strongly depends on the surface characteristics of blood contact materials [9], e.g., surface hydrophobicity, charge and morphology. The higher the particle surface charge density, the more proteins are adsorbed, which may be mainly driven by Coulomb forces [18]. A hydrophobic surface could be more suitable for protein adsorption than a hydrophilic surface [19]. According to results of the contact-angle test (Fig. 1), the treated films were more hydrophobic than the untreated films. The hydrophobicity of Z24h and Z72h were the strongest, and there was no significant difference between Z0h and Z12h . Such properties resulted in more protein adsorption on the treated films. It has been reported that serum albumin is one of the most important transport proteins and also the most abundant plasma protein, so adsorption of albumin will limit the adhesion of platelets [20]. Furthermore, the existence and the maintenance of conformation or orientation of serum albumin on
Fig. 3. Platelet adhesion for 60 min on different zein films treated with water vapor for 0, 12, 24, 72 and 144 h, respectively. (*) Represents a statistically significant difference compared with the Z0h group; (**) represents a statistically significant difference compared with other groups (p < 0.05, n = 3).
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Fig. 4. SEM observation of blood platelets adhered for 60 min onto different zein films treated with water vapor for 0 h (A), 12 h (B), 24 h (C), 72 h (D) and 144 h (E). Bars in the left and right columns represent 100 m and 10 m, respectively.
different matrixes might result in reduced platelet adhesion compared with the control [21]. According to these results, it can be concluded that the surface platelet compatibility of zein films could be adjusted by a
film surface treatment. In previous work, we have demonstrated that zein microsphere films had good properties with regard to resistance to platelet adhesion compared with glass. It was speculated that protein types and the conformation of adsorbed
H.-J. Wang et al. / Colloids and Surfaces B: Biointerfaces 69 (2009) 109–115
Fig. 5. The adhesion behaviors of BEL-7402 on the original zein film and water vaportreated zein films after 4 h of cell seeding, (*) represents a significant difference compared to plate control (p < 0.01, n = 3).
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Fig. 6. Proliferation assay of BEL-7402 cells on the original zein film and water vaportreated zein films for 3 days; a Corning plate was chosen as the control. (*) Represents significant differences compared to the control; (**) represents significant differences compared to the original zein film; (***) represents significant differences compared to both of the control and the original zein film (p < 0.05, n = 3).
3.3. Effect of surface treatment on cell behavior on zein films plasma protein determine the resistance ability of film surfaces to platelets, and that the hydrophobic nature of zein microsphere films is the key factor for the protein adsorption [5]. During water vapor treatment, the morphology and topology characteristics of the film surfaces were the main factors altered, which resulted in wettability variation. Therefore, we deduced that the improved anti-platelet adhesion could be attributed to the microcosmic structure, such as the micro- or nanostructure (Figs. 1 and 2).
For an ideal artificial material, biocompatibility is one of the most important properties, and much effort is being devoted to methods of modifying surfaces of existing biomaterials to achieve desired biological responses. These mainly include physicochemical methods (surface energy, surface charge, and surface composition have been altered with the aim of improving implant interfaces) and morphological methods (alterations in surface
Fig. 7. Morphologies and distributions of BEL-7402 cells cultured on the plate control (A), the original zein film (B), and the zein films treated with water vapor for 12 h (C), 24 h (D), 72 h (E), and 144 h (F) for 1 day (A1 –F1 ), and 3 days (A3 –F3 ). All scale bars represent 62.4 m.
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Fig. 7. (Continued ).
morphology and roughness have been used to influence cell and tissue responses to implants). For example, Zhang et al. [22] reported improving the platelet-adhesive resistance ability of cellulose membranes by grafting with sulfo ammonium zwitterionic vinyl monomers, and this method is mainly based on altering the functional groups of the surface. In addition, surface wettability also affects cell behaviors on the artificial material, as previous work has shown that a hydrophilic surface is suitable for cell growth, especially with a water contact angle of 40–60◦ [1,3]. Of course, some other surface characteristics, such as surface free energy, chemistry, charge, roughness, and rigidity, have an effect on the film surface compatibility. However, in this study, the films had the same chemical composition; therefore, change in the contact angles (or hydrophilic properties) was the only factor that might result in a different cell-zein film interaction. BEL-7402 cells were cultured on the original and treated zein films for 4 h, and cell adhesion was determined by the MTT method. As shown in Fig. 5, there were higher cell numbers on the zein films than on the Corning plate control. Cell adhesion on the surfaces increased and then decreased with a prolonged time period of water vapor treatment of the zein films. Also, the cells adhered more to surfaces with moderate hydrophilicity than to relatively more hydrophobic surfaces, but no significant difference could be observed. Work has been done to show that the cell–substrate adhesion is controlled by the wettability of the surface [2], and it is notable that a surface with about a 55◦ contact angle was considered as the most suitable to HUVEC adhesion [1]. According to the above
result of the contact-angle test, although the treated zein films had different contact angles, there were no differences among them. This result suggested that the BEL-7402 adhesion properties of zein films might mainly depend on the material itself. Fig. 6 shows the proliferation of BEL-7402 on different surfaces. It can be seen that there was significant enhancement of cell proliferation and viability on zein films compared with that on the plate control (p < 0.05, n = 3). Moreover, there was also a significant enhancement of cell viability on Z72h and Z144h films, but no statistical difference in cell viability for Z12h and Z24h films compared with the original zein film on the first day of cell culture. In addition, a significant difference in cell viability appeared only in the Z24h film group after 2 days of cell culture and in Z72h and Z144h films groups after 3 days of cell culture. In order to visualize cell morphologies on different matrix surfaces, cells were stained using acridine orange and observed with a fluorescence microscope (IX71, Olympus, Japan). Fig. 7 shows a series of sequential images of BEL-7402 cells adhered onto the original and treated zein films. BEL-7402 cells were distributed uniformly on the surfaces of the plate control and the treated zein films, and an almost confluent monolayer was formed for these groups on the third day. 4. Conclusions In this work, surface treatment by water vapor on zein films is proposed to functionalize the surface, and we concluded that the platelet adhesion on zein films could be inhibited by this treatment, especially after 12 h of treatment. This was mainly because
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of different wettabilities due to varying surface morphologies. However, BEL-7402 cell functions (such as cell adhesion and cell proliferation) could be enhanced by coating with a zein film, while they did not response significantly to changes in the surface properties of zein films. Acknowledgements This study was supported by the National Program on Key Basic Research Projects of China (973 Program, 2005CB724306), the National Natural Science Foundation of China (30870635), and the National Hi-Tech Research and Development Plan (863 Project) of China (2002AA327100). References
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effect of water vapor on the surface characteristics and cell compatibility of zein films Hua-Jie Wang a , Jian-Xi Fu b , Jin-Ye Wang b,∗ a b
College of Life Science and Biotechnology, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China
a r t i c l e
i n f o
Article history: Received 29 August 2008 Received in revised form 3 October 2008 Accepted 17 November 2008 Available online 25 November 2008 Keywords: Zein film Higher relative humidity Contact angle Compatibility
a b s t r a c t In this study, zein microsphere films were fabricated via a solution casting method, and their surfaces were subsequently treated with a higher relative humidity. Changes in surface properties were characterized by contact-angle measurement and scanning electron microscopy (SEM). Human hepatoma cells (BEL-7402) were used as model cells to evaluate cell adhesion, spreading and proliferation on zein films before and after treatment. Additionally, the adhesion and morphologies of rat platelets on zein films were also investigated to evaluate the blood compatibility of the films. The results showed that the hydrophilicity of zein films was changed after treatment, and the surface morphology varied, gradually becoming more smooth and the microspheres in the films disappeared. As the result, the adhesion and proliferation of BEL-7402 cells were significantly improved, while HUVECs were more sensitive than BEL7402 cells to cell–substrate interactions. Adhesion of rat platelets could be inhibited on the treated zein films. The results suggest that the synergistic actions of hydrophilic properties and micro morphologies are responsible for the cell behaviors. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The compatibility of tissue engineering materials is one important factor that limits their application in the tissue engineering field because the main interactions between a tissue and an implant occur at the atomic layers of an implant. Therefore, improvement of the material–cell interaction interface that forms the basis of cell adhesion and proliferation allows for maintenance of the normal cell phenotype and promotion of differentiation through the activation of specific gene expression. The material–cell interaction is affected by many factors, such as wettability (hydrophilicity/hydrophobicity), surface free energy, chemistry, charge, roughness, and rigidity [1]. Many advances have been made in understanding events at the interface between tissues and implants and in developing methods for controlling these events [2]. For example, proteins can be adsorbed on the surface after implantation into a living system in less than a second and a monolayer of adsorbed protein forms in seconds to minutes; the specific adsorbed protein can further alter the function of a cell. Arima and Iwata prepared self-assembled monolayers of alkanethiols with a wide range of wettabilities, and showed that the adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) and Hela cells were affected by surface wettability
∗ Corresponding author. Tel.: +86 21 54925330; fax: +86 21 64166128. E-mail address: [email protected] (J.-Y. Wang). 0927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2008.11.015
and functional groups, which controlled the adsorption of serum proteins [3]. Zein, soluble in aqueous alcohol solutions, is the major storage protein of corn. Zein has been used widely, especially in the food industrial field [4]. Our previous work has indicated that zein has good biocompatibility with HUVECs, liver cells and mouse fibroblast cells [5,6]. After preparing zein microspheres by the phase isolation technique, we prepared a zein film using the microspheres by the casting method. Interestingly, we found that the surface morphology of the zein film could change during the storage process. In this study, the effect of water vapor on the surface characteristics of zein films was investigated by scanning electron microscopy (SEM) and static water contact-angle analysis, and their cell compatibility and blood compatibility were analyzed by cell culture in vitro and by the platelet adhesion test. 2. Materials and methods 2.1. Preparation of zein films The zein microsphere thin film was prepared on a glass matrix at 37 ◦ C according to our previous work [5,6]. In brief, a zein microsphere suspended solution was formed in a 40% aqueous mixture of ethanol based on the phase isolation technique. Then, the above solution was poured onto glass surfaces, forming a thin layer, and dried through volatilization at 37 ◦ C or by spinning at 500 rpm for 10 min. The films were then stored for the desired time periods (0,
110
H.-J. Wang et al. / Colloids and Surfaces B: Biointerfaces 69 (2009) 109–115
12, 24, 72, and 144 h) under conditions of moist steam at 37 ◦ C. The films were dried at 37 ◦ C and stored in a desiccator for future use. 2.2. Morphology observation Zein films were mounted on stubs and coated in a vacuum with gold, and the surface morphologies then were investigated by scanning electron microscopy (JSM-6390LV, JEOL, Japan).
tration of zein, the concentration of the organic phase, the mixing speed, and so on. Therefore, a film with various particle sizes could be obtained from the microspheres using film-preparation techniques. In the present study, we mainly discuss the effect of water vapor on zein films, including surface properties and biological properties. 3.1. Effect of higher relative humidity conditions on surface wettabilities and morphologies
2.3. Contact-angle measurements Wettabilities of the target films were evaluated as static water contact angles using a contact-angle goniometer (JC2000A, Shanghai, China) [7]. Briefly, a droplet of water (0.5 l, ultrapure grade) was put on the surfaces of different materials and the images were photographed, with temperature and moisture being kept constant during the course of the experiments (23 ◦ C and 68%, respectively). The angle between the baseline of the drop and the tangent at the drop boundary was measured. Measurements were performed at least in triplicate on three different batches of material. 2.4. Platelet adhesion assays in vitro Platelet adhesion was performed according to our previous work [5]. The platelets were obtained by centrifuging anticoagulant blood from an SD rat at 1500 rpm for 10 min, and the supernatant plateletrich plasma at 3000 rpm for another 10 min at 4 ◦ C. The platelets were resuspended in PBS, and the number of platelets was diluted to 5 × 106 cells. Zein microsphere films were placed into contact with 50 l of the platelets and left at 37 ◦ C for 60–180 min. The platelets were removed and the films were rinsed three times with PBS [8]. The adhered platelets were fixed by immersing the film into a 2.5% solution of glutaraldehyde in PBS for at least 2 h at 4 ◦ C. Samples were freeze-dried, and then sputter-coated with gold for SEM observation. The number of platelets adhered was determined via a colorimetric method [9]. Briefly, 150 l of 4-nitropheny-phosphate disodium salt was added to each well and the samples were placed at 37 ◦ C for 60 min. The absorption was determined at a wavelength of 405 nm after stopping the reaction with 100 l of 2N NaOH. 2.5. Cell culture The human hepatoma cell line BEL-7402 was used for the in vitro test. The cells were incubated at 37 ◦ C in a 95% humidified air/5% CO2 atmosphere and seeded onto target films in a 96-well Corning culture microplate at 2.4 × 104 cells/ml after digestion with 0.25% trypsin/0.002% EDTA. 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was used to assess the adhesion and proliferation of BEL-7402 cells after 4 h, 24 h, 48 h and 72 h of culture. The morphologies and distribution of cells were observed through a fluorescence microscope (IX71, Olympus, Japan) after being stained with acridine orange fluorescent dye in PBS (pH 7.2) for 5 min.
In order to present the data clearly, the original and treated zein films for different periods are abbreviated as Z0h , Z12h , Z24h , Z72h , and Z144h , respectively. The contact angles of zein films were measured to compare the wettabilities of their surfaces by the sessile drop technique, as shown in Fig. 1. It has been reported that the water contact angle of a hydrophobic surface is over 90◦ [10]. During the treatment, the water contact angles of zein films gradually increased (from 52.0 ± 2.9◦ to 71.8 ± 3.0◦ ) over 24 h, and then gradually decreased to 62.2 ± 5.2◦ when the time was prolonged to 144 h, which indicates relative hydrophobic properties compared to the original zein film. Fig. 2 shows SEM images of zein films during water vapor treatments for different periods. Obvious differences in the surface morphologies of the original and treated zein films were observed. The original zein films were composed of round particles with a size in the nanometer range and the particles conglutinated with each other to form an integrated film. After water vapor treatment, the surface increasingly smooth, but the outline of the particles of the film still remained after 12 h; however, a smooth film was formed after a 24 h treatment, and a few small pores could be observed. This change from Z0h to Z144h was mainly due to the conglutination of zein according to our previous report [5]. To date, the structure of zein in solution has been investigated by various physico-chemical studies, and the amino acid sequence of zein is well known [11]. Zein is a hydrophobic protein due to a high amount of the hydrophobic amino acids such as leucine, proline, alanine, and phenylalanine, which was the main reason for the zein film displaying a water contact angle over 50◦ (Fig. 1). Lakshmannan and Dhathathreyan [12] reported that some peptides could vary from non-polar to polar when they changed from an air/water interface to a solid/air interface, and the zein protein surface could also restructure or reorient in response to different environments. That is to say, zein will orient its polar chemical components toward the interface when the zein film surface is exposed to an aqueous or highly polar environment if given sufficient mobility. Therefore, zein films show hydrophilic properties. The zein molecule is amphiphilic, just as Kim and Xu reported [13]. This property causes the formation of macromolecular micelle-globular aggregates with the hydrophilic moiety exposed to the surface and the hydrophobic moieties clumped together in
2.6. Statistical analysis The number of independent replications is listed individually for each experiment. All data are expressed as mean ± standard deviation and analyzed by Fisher’s method for multiple comparison, and the statistical significance was accepted at p < 0.05. 3. Results and discussion It was shown that the particle size of the zein microspheres could be affected by many factors during the process, such as the concen-
Fig. 1. Changes in the water contact angle of the original zein film and zein films treated with water vapor for 12, 24, 72, and 144 h. (*) Represents a significant difference compared with 0 h samples.
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Fig. 2. Scanning electron micrographs of the original zein film (A) and zein films treated with water vapor for 12 h (B), 72 h (C), and 144 h (D).
the aqueous ethanol solution with ethanol content of lower than 90%. The zein aggregates precipitate as microspheres by the end, when the ethanol content is decreased to less than 40%, in which zein is insoluble (Fig. 2). In addition, zein is able to form a viscoelastic protein network during mixing through a structural rearrangement if the protein is held and mixed at 35 ◦ C, above its glass transition temperature of 28 ◦ C, and at >20% moisture content. This structural change is due to changes in zein secondary structure from ␣-helix to -sheet structures [14,15]. According to the result shown in Fig. 2, after water vapor treatment for over 24 h, zein microspheres disappeared and a smooth zein film formed instead, which further supported the conclusions made upon observation. Morphological changes of the zein film induced by water vapor treatment is similar to that immersed in solution directly, as shown in our previous study, in which sizes of microspheres increased with immersing time of the film in PBS, adjacent microspheres fused with each other [5]. 3.2. Effect of surface treatment on platelet adhesion onto zein films Platelet adhesion onto films from plasma is an important test for evaluating the blood compatibility of materials [16]. We performed a blood platelet adhesion assay on zein films through exposure to fresh rat platelets using a Corning plate as the control, and did a quantified analysis using a colorimetric method based on the activity of the platelet enzyme acid phosphatase [9]. Fig. 3 shows the relative number of platelets adhered on zein films. Compared with the control, zein films significantly inhibited platelet adhesion (p < 0.05). Moreover, the numbers of adhering platelets on the treated films were lower than in the untreated group, except for the Z144h film group. SEM examination clearly showed the adhesion and distribution of platelets on the films (Fig. 4), which was the same result seen using the colorimetric assay. Platelets adhered as single cells and not as aggregates, with only small and sporadic aggregates being detectable. The platelets did not spread over the surface, and maintained a round morphology, which indicates that the platelets
were unactivated. Both results indicated a better hemocompatibility of zein films compared to the control plate. Platelet adhesion mainly depends on the type and conformation of adsorbed plasma proteins [8,17], while plasma protein adsorption strongly depends on the surface characteristics of blood contact materials [9], e.g., surface hydrophobicity, charge and morphology. The higher the particle surface charge density, the more proteins are adsorbed, which may be mainly driven by Coulomb forces [18]. A hydrophobic surface could be more suitable for protein adsorption than a hydrophilic surface [19]. According to results of the contact-angle test (Fig. 1), the treated films were more hydrophobic than the untreated films. The hydrophobicity of Z24h and Z72h were the strongest, and there was no significant difference between Z0h and Z12h . Such properties resulted in more protein adsorption on the treated films. It has been reported that serum albumin is one of the most important transport proteins and also the most abundant plasma protein, so adsorption of albumin will limit the adhesion of platelets [20]. Furthermore, the existence and the maintenance of conformation or orientation of serum albumin on
Fig. 3. Platelet adhesion for 60 min on different zein films treated with water vapor for 0, 12, 24, 72 and 144 h, respectively. (*) Represents a statistically significant difference compared with the Z0h group; (**) represents a statistically significant difference compared with other groups (p < 0.05, n = 3).
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Fig. 4. SEM observation of blood platelets adhered for 60 min onto different zein films treated with water vapor for 0 h (A), 12 h (B), 24 h (C), 72 h (D) and 144 h (E). Bars in the left and right columns represent 100 m and 10 m, respectively.
different matrixes might result in reduced platelet adhesion compared with the control [21]. According to these results, it can be concluded that the surface platelet compatibility of zein films could be adjusted by a
film surface treatment. In previous work, we have demonstrated that zein microsphere films had good properties with regard to resistance to platelet adhesion compared with glass. It was speculated that protein types and the conformation of adsorbed
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Fig. 5. The adhesion behaviors of BEL-7402 on the original zein film and water vaportreated zein films after 4 h of cell seeding, (*) represents a significant difference compared to plate control (p < 0.01, n = 3).
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Fig. 6. Proliferation assay of BEL-7402 cells on the original zein film and water vaportreated zein films for 3 days; a Corning plate was chosen as the control. (*) Represents significant differences compared to the control; (**) represents significant differences compared to the original zein film; (***) represents significant differences compared to both of the control and the original zein film (p < 0.05, n = 3).
3.3. Effect of surface treatment on cell behavior on zein films plasma protein determine the resistance ability of film surfaces to platelets, and that the hydrophobic nature of zein microsphere films is the key factor for the protein adsorption [5]. During water vapor treatment, the morphology and topology characteristics of the film surfaces were the main factors altered, which resulted in wettability variation. Therefore, we deduced that the improved anti-platelet adhesion could be attributed to the microcosmic structure, such as the micro- or nanostructure (Figs. 1 and 2).
For an ideal artificial material, biocompatibility is one of the most important properties, and much effort is being devoted to methods of modifying surfaces of existing biomaterials to achieve desired biological responses. These mainly include physicochemical methods (surface energy, surface charge, and surface composition have been altered with the aim of improving implant interfaces) and morphological methods (alterations in surface
Fig. 7. Morphologies and distributions of BEL-7402 cells cultured on the plate control (A), the original zein film (B), and the zein films treated with water vapor for 12 h (C), 24 h (D), 72 h (E), and 144 h (F) for 1 day (A1 –F1 ), and 3 days (A3 –F3 ). All scale bars represent 62.4 m.
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Fig. 7. (Continued ).
morphology and roughness have been used to influence cell and tissue responses to implants). For example, Zhang et al. [22] reported improving the platelet-adhesive resistance ability of cellulose membranes by grafting with sulfo ammonium zwitterionic vinyl monomers, and this method is mainly based on altering the functional groups of the surface. In addition, surface wettability also affects cell behaviors on the artificial material, as previous work has shown that a hydrophilic surface is suitable for cell growth, especially with a water contact angle of 40–60◦ [1,3]. Of course, some other surface characteristics, such as surface free energy, chemistry, charge, roughness, and rigidity, have an effect on the film surface compatibility. However, in this study, the films had the same chemical composition; therefore, change in the contact angles (or hydrophilic properties) was the only factor that might result in a different cell-zein film interaction. BEL-7402 cells were cultured on the original and treated zein films for 4 h, and cell adhesion was determined by the MTT method. As shown in Fig. 5, there were higher cell numbers on the zein films than on the Corning plate control. Cell adhesion on the surfaces increased and then decreased with a prolonged time period of water vapor treatment of the zein films. Also, the cells adhered more to surfaces with moderate hydrophilicity than to relatively more hydrophobic surfaces, but no significant difference could be observed. Work has been done to show that the cell–substrate adhesion is controlled by the wettability of the surface [2], and it is notable that a surface with about a 55◦ contact angle was considered as the most suitable to HUVEC adhesion [1]. According to the above
result of the contact-angle test, although the treated zein films had different contact angles, there were no differences among them. This result suggested that the BEL-7402 adhesion properties of zein films might mainly depend on the material itself. Fig. 6 shows the proliferation of BEL-7402 on different surfaces. It can be seen that there was significant enhancement of cell proliferation and viability on zein films compared with that on the plate control (p < 0.05, n = 3). Moreover, there was also a significant enhancement of cell viability on Z72h and Z144h films, but no statistical difference in cell viability for Z12h and Z24h films compared with the original zein film on the first day of cell culture. In addition, a significant difference in cell viability appeared only in the Z24h film group after 2 days of cell culture and in Z72h and Z144h films groups after 3 days of cell culture. In order to visualize cell morphologies on different matrix surfaces, cells were stained using acridine orange and observed with a fluorescence microscope (IX71, Olympus, Japan). Fig. 7 shows a series of sequential images of BEL-7402 cells adhered onto the original and treated zein films. BEL-7402 cells were distributed uniformly on the surfaces of the plate control and the treated zein films, and an almost confluent monolayer was formed for these groups on the third day. 4. Conclusions In this work, surface treatment by water vapor on zein films is proposed to functionalize the surface, and we concluded that the platelet adhesion on zein films could be inhibited by this treatment, especially after 12 h of treatment. This was mainly because
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of different wettabilities due to varying surface morphologies. However, BEL-7402 cell functions (such as cell adhesion and cell proliferation) could be enhanced by coating with a zein film, while they did not response significantly to changes in the surface properties of zein films. Acknowledgements This study was supported by the National Program on Key Basic Research Projects of China (973 Program, 2005CB724306), the National Natural Science Foundation of China (30870635), and the National Hi-Tech Research and Development Plan (863 Project) of China (2002AA327100). References
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