Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Applied Surface Science 255 (2009) 6686–6690 Contents lists available at ScienceDirect Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc Research on micro-structure and hemo-compatibility of the artificial heart valve surface Xia Ye a,b, Yun-liang Shao a, Ming Zhou a,*, Jian Li a, Lan Cai a a b Center of Photonics Fabrication, Jiangsu University, Zhenjiang 212013, PR China School of Mechanical Engineering, Jiangsu Teachers University of Technology, Changzhou 213015, PR China A R T I C L E I N F O A B S T R A C T Article history: Received 29 October 2008 Received in revised form 22 January 2009 Accepted 22 February 2009 Available online 4 March 2009 In order to seek the method to improve the hemo-compatibility of artificial mechanical heart valve, the surface of rabbit’s heart valve was observed using the scanning electron microscopy (SEM). The results showed that the dual-scale structure which consists of cobblestones-like structure of 8 mm in underside diameter and 3 mm in height, and the fine cilia of about 150 nm in diameter, was helpful to the hemocompatibility of the heart valve. Therefore, the polydimethylsiloxane (PDMS) surface with hierarchical micro-structure was fabricated using femtosecond laser fabrication technique and soft lithography. At the same time, the tests of apparent contact angle and platelet adhesion on both smooth and textured PDMS surfaces were carried out to study their wettability and hemo-compatibility. The results demonstrated that the surface with textured structure displayed more excellent wettabililty and anticoagulation property than that of smooth surface. The apparent contact angle of textured surface enhanced from 113.18 to 163.68 and the amount of adsorbed platelet on such surface was fewer, no distortion and no activation were found. ß 2009 Elsevier B.V. All rights reserved. PACS: 81.40.z 87.19.R 87.85.jj Keywords: Heart valve PDMS Micro-structure Superhydrophobicity Hemo-compatibility 1. Introduction The replacement of artificial heart valve is the only way to save the life of the patient whose heart valve is badly pathologically changed by rheumatic heart disease, bacterium caused endocarditis disease and so on. The artificial heart valves used in clinic are classified into bioprosthetic valve and mechanical valve. The biocompatibility and anti-thrombus property of the bioprosthetic valve are quite good, but its durability remains a problem, because it is easy to calcify and to be torn. The average lifetime of bioprosthetic valve is only 5–8 years [1]. So far, there is no effective way to improve its durability. In contrast, the main part of mechanical valve is made of non-biological materials, such as titanium alloy, stainless steel, low temperature isotropic carbon (LTIC) and polyurethane and so on [2], therefore the mechanical valve has a relatively long durability, which can be used as long as 30–50 years. However, the mechanical valve has poor hemocompatibility. In order to decrease the risk of thrombosis complication, the patient who is implanted the mechanical valve should be treated with anticoagulant every day [3]. Even if they are * Corresponding author. Tel.: +86 511 88791458; fax: +86 511 88791288. E-mail address: [email protected] (M. Zhou). 0169-4332/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2009.02.068 treated with sufficient anticoagulant, thromboembolism is still founded in 1–2% patients every year [4]. It has been more than 40 years for the clinical application since Harken et al. [5] first replaced the aortic valve with artificial caged ball valve and Starr and Edwards [6] replaced the mitral valve with ball valve at the same year in 1960. However, up to now, there is no satisfying valve which enjoys good hemo-compatibility and long durability in clinic. Accordingly, it is an urgently key problem to be solved to improve the hemo-compatibility of the mechanical valve. In recent years, all researches about improving the hemo-compatibility of the mechanical valve focus on two aspects: one is to seek new materials with good hemo-compatibility, and another is to modify the artificial surface by using coating or film. The most important studies related to the second aspect are as follows: Li et al. [7] implanted nitrogen ion on the LTIC by ion beam; Dion et al. [8] deposited diamond-like carbon (DLC) film on valve materials; Ali et al. [9] prepared the Cr-modified diamond-like carbon (Cr-DLC) films using the magnetron sputtering technique and Grill et al. [10] prepared Si-DLC films using the same technique; Huang et al. [11] synthesized the Ti-coating by ion beam; Leng et al. [12] deposited the duplex films, consisting of layers of Ti–O and Ti–N, using plasma immersion ion implantation and deposition method; Chen et al. [13] deposited Ti–O coatings doped with Ta and synthesized Ti(Ta5+)O2 thin films using magnetron sputtering and thermal X. Ye et al. / Applied Surface Science 255 (2009) 6686–6690 oxidation procedures; Bolz and Schaldach [14] deposited a coating of amorphous hydrogenated silicon carbide by plasma-enhanced chemical vapor deposition (PE-CVD). All the above studies show that the blood compatibility of the artificial heart valve can be increased by the surface modification technologies such as surface coating and deposition of thin film. However, due to the curved surface of the artificial heart valve, it is difficult to ensure the uniformity of the coating or film. Furthermore, the adhesive force between the coating layer and matrix also affect the working life of the artificial heart valve. Polydimethylsiloxane (PDMS) is a curable inorganic polymer with desirable properties. It is transparent, non-fluorescent, biocompatible and nontoxic, and has been used extensively as a biomaterial in catheters, drainage tubing, insulation for pacemakers, membrane oxygenators, and ear and nose implants [15], since its initial use for topographic cell substrates [16]. In addition, PDMS is chemically inert, thermally stable, permeable to gases, simple to handle and manipulate, exhibits isotropic and homogeneous properties, and can conform to submicron features to develop micro-structures [17–19]. In this paper, the micro-structure on the surface of the rabbit’s heart valve was studied using scanning electron microscopy (SEM). And the hierarchical structure on PDMS surface was prepared by femtosecond laser fabrication technique and soft lithography technology. The hemo-compatibility of such surface was also examined. The purpose of the present study is to explore the effect of surface micro-structure on the blood compatibility and provide a basis for the research of the artificial heart valve. 2. Experimental 2.1. Observation on the micro-structure of biological prototype The heart of the rabbit was took out and irrigated with normal saline. Then the left atrium and ventricle were cut open with scissors, the mitral valve was removed and fixed in solution of glutaraldehyde (Res Group Co., Ltd. chemical reagents). After low temperature freeze-drying (ES-2030 vacuum freeze-drying device, Hitachi Japan) in the 10 8C for 2–3 h, the valve was treated by spray-gold (E-1010 ion sputtering device, Hitachi Japan) and observed using the scanning electron microscope (S-3000N scanning electron microscope SEM, Hitachi Japan). 2.2. Preparation of the bionic valve surface The sample with a micro-structure on the surface, which is similar to the surface of biological prototype, was fabricated using soft lithography. In order to obtain the patterned mold, pit structure on the K9 glass surface was fabricated using femtosecond laser. After pattering, the mold was exposed to Trichloro(1H,1H,2H,2Hperfluorooctyl)silane(CF3(CF2)5CH2CH2SiCl3, Aldrich) for 2 h in a low-pressure chamber, allowing an easy removal of the PDMS membrane from it. PDMS gel was generally prepared from a 10:1 mixture (by weight exactly) of silicone elastomer and catalyst (Sylgard 184, Dow Corning, Co.), poured onto the patterned K9 glass, and left to settle for 10 min, so that the trapped air bubbles could emerge to the surface. After the removal of all the air bubbles, the patterned K9 glass with PDMS gel was put into a vacuum chamber (100 8C) for 30 min. After curing, the PDMS sample was gently peeled from the mold. The sample was observed using scanning electron microscope (SEM, JSM-840A, JEOL LTD., Japan) 2.3. Contact angle measurements Contact angle was used to determine the wettability of regular arrays micro-structure surface. The static contact angle (CA) of 6687 water on the sample surface was measured in laboratory atmosphere at room temperature using the sessile drop method of a contact angle goniometer (OCAH200, Dataphysics, Germany). A deionized water drop of 1 mL was deposited on the surface, and each reported angle was calculated as the average of five measurements in different points. In order to compare the wettability of textured surface with that of smooth surface, the CA of the water on the flat PDMS surface was also measured. 2.4. Platelet adhesion in vitro In order to test the potential hemo-compatibility of the materials, platelet adhesion studies were conducted since platelet adhesion was one of the most important steps during blood coagulation on artificial surface [20–22]. Textured PDMS surface and smooth PDMS surface which was used to show a contrast were washed in the ultrasonic wave cleaning machine (Kunshan Ultrasound Machines Ltd.) with deionized water for three times and then equilibrated in phosphate buffer solution (PBS, pH 7.4) for 120 min. In the meantime, fresh human blood added with anticoagulant citrate dextrose (9:1) (provided by Jiangbin Hospital in Zhenjiang) was centrifuged at 1000 rpm for 10 min to obtain platelet-rich plasma (PRP). Then two kinds of PDMS surface were incubated in it at 37 8C for 120 min. After being washed three times with PBS to remove non-adherent platelets, the samples were soaked in 2.5 vol% glutaraldehyde for 60 min to fix the adhered platelets. Then they were washed with PBS (PH 7.4) again, and dehydrated in a series of gradual ethanol/distilled water mixtures from 10% to 100% (20% ethanol increment) for 30 min each. After drying at room temperature, the samples surface were treated by spary-gold using magnetron sputtering technique and tested by SEM. The results of SEM experiments were shown with representative micrographs. Eight to 10 fields of view were chosen randomly to obtain good statistical density of the platelet adhesion. The flat PDMS sample was taken as a contrast. The final number of platelets adhering to the surface was expressed as a percentage of the control. 3. Results and discussion 3.1. Characterization of biological prototype surface The SEM image of the surface of the rabbit’s mitral valve was shown in Fig. 1. It could be evidently seen from images that there were some regular cobblestones-like on the heart valve surface which seemed very smooth by unaided eye. The underside diameter of each cobblestone-like was about 8 mm, and the height was about 3 mm. Fig1c showed a high-resolution SEM image of a single cobblestone-like, where hierarchical structure was in the forms of tenuous villus in average diameters of 150 nm and height of 500 nm on each cobblestone-like. According to the Wenzel theory [23] and Cassie and Baxter theory [24], this hierarchical structure on the surface of the heart valve, which is similar to that of lotus leaf surface [25], may be very useful to modify the wettability of heart valve surface and reduce the amount of adsorbed platelet on the surface, thus decreases the form of thrombus on the heart valve surface. 3.2. Characterization of artificial surface According to the structure of the biological prototype, the regular array mastoid structure was designed on the sample surface. And the geometry size of mastoid was as follows: basal diameter a, spacing b and height h, as in sketched in Fig. 2. In this experiment, the size a was 60 mm, b was 40 mm and h was 30 mm. Fig. 3a showed the SEM image of the mastoid of the textured PDMS X. Ye et al. / Applied Surface Science 255 (2009) 6686–6690 6688 Fig. 1. The heart valve of rabbit (a) mitral valve; (b) large-scale SEM image of the micro-structure of valve surface and (c) magnified image on a single cobblestone of b, and some nano tenuous villus on it. Table 1 Contact angle of surface of the sample (8). Contact angle Mastoid structure surface Smooth PDMS surface Fig. 2. Sketch of mastoid micro-structure: a is the basal diameter of a single mastoid, b is the space between two mastoids and h is the height of mastoid. surface. It was seen from the images that the surface quality of PDMS surface with mastoid array structure was very good. There were almost no surface distortion and damnification on the surface. Moreover, in the enlarged SEM image of a single mastoid (Fig. 3b), the fine structure could be observed on the mastoid. In fact, the protrusion with sizes from 1 to 4 mm, which provided the hierarchical structure on the artificial PDMS surface, enhanced the hydrophobicity of the surface. 3.3. Contact angle measurements To compare the wettability of smooth PDMS surface with that of PDMS surface with mastoid micro-structure, the static contact angle of water on both surfaces was measured using the sessile droplet method and the results were shown in Table 1. The results showed that the smooth surface was hydrophobic (CA = 113.18) and the micro-structure surface was superhydrophobic u1 u2 u3 u4 u5 uAV 163.6 112.3 166 112.1 163.4 113.5 160.8 113.5 164.4 114.0 163.6 113.1 (CA = 163.68). The shape of water droplet on the both surfaces showed in Fig. 4. It was clearly seen that the droplet sit on the peak of the roughness (Fig. 4b) and the light could pass through the space in the depressions of the rough surface, which indicated that a composite surface (Cassie state) was formed on the textured PDM surface. It is indicated that the hierarchical mastoid microstructure on the surface enhanced the wettability of the surface, although the sizes of the mastoid structure on the PDM surface (50 mm in diameter and 30 mm in height) were greater than that of the cobblestone-like structure (8 mm in diameter and 3 mm in height) on the rabbit’s mitral valve. According to the Cassie–Baxter equation [24], cos uCB ¼ 1 þ f ð1 þ cos u Y Þ where f is an area fraction of solid in contact with the liquid and uY is Young’s contact angle, the geometric parameters of the mastoid structure greatly affect the value of apparent contact angle uCB, namely affect the wettability of the rough surface. The detail of the effect of the different sizes of mastoids on the wettability and hemo-compatibility of the artificial heart valve will be further studied theoretically and experimentally in the future researches. 3.4. Platelets adhesion Adhesion of platelets to foreign materials is one of their most important functions. When blood contacts with the surface for a period of time, there are a certain number of platelets adhesion to Fig. 3. SEM images of the textured PDMS surface (a) SEM of mastoid micro-structure and (b) SEM of a single mastoid, some fine structure on it. X. Ye et al. / Applied Surface Science 255 (2009) 6686–6690 6689 Fig. 4. Photographs of water droplets on the sample surface (a) droplet on the smooth PDMS surface and (b) droplet on the mastoid micro-structure PDMS surface. Fig. 5. Platelets adhesion on the smooth surface: (a) low magnification and (b) high magnification. the surface. Under the surface tension force, platelets are activated. Then platelets are interacted with the material surface, lead to a series of complex reaction, join together, release the coagulation factor, and activate coagulation reaction finally. Platelet–surface interaction is a very complex and dynamic series of events. Platelet adhesion on the surface is invariably followed by the appearance of platelet aggregates and platelets spreading and subsequent thrombus formation that causes potential danger of using artificial heart valve in vivo [25]. Platelet adhesion and activation are the indicators of thrombosis on a biomaterial surface. Utilizing the platelet adhesion test in vitro, the preliminary evaluation on hemo-compatibility of the microstructure surface can be obtained through measuring the number of platelet adhesion. Fig. 5 showed the SEM photograph of the smooth PDMS surface on which platelet adhesion experiment had been tested. There were a large number of blood platelets attached to the smooth surface (Fig. 5a). It showed the morphology of platelets on flat PDMS surface flattened and platelets aggregated. Part of adhered platelets had produced distortion and extended long pseudopod (Fig. 5b). Compared with that of the smooth surface, the number of the attached platelets on the mastoid micro-structure surface was fewer (Fig. 6a). It was seen from the enlarged SEM image (Fig. 6b) of the rough surface, the shape of attached platelets was whole round, without deformation and aggregation. It showed that there was no platelet activation on the mastoid micro-structure surface. Why do different platelet adhesion phenomena respond to the two different surfaces in this way? Possibly, the different hydrophobility is affected in some way. Although the chemical compositions of both surfaces are the same, the wettability of surface is different. That is, the smooth PDMS surface is hydrophobic and the mastoid micro-structure surface is super- Fig. 6. Platelets adhesion on the mastoid micro-structure PDMS surface: (a) low magnification and (b) high magnification. 6690 X. Ye et al. / Applied Surface Science 255 (2009) 6686–6690 hydrophobic. It means that the surface energy of the patterned surface is lower than that of the flat surface. So the amount of the adhered platelets on the patterned surface is less than that of the smooth surface. The agglomerate phenomenon and the level of activation apparent of the smooth surface are more obvious than that of the patterned surface. So the clotting reaction of the microstructure surface is slower than that of the smooth surface, which makes better hemo-compatibility of the surface with mastoid micro-structure. Moreover, the nano-structure on the mastoid surface may extremely contribute to improve the hydrophobility of the patterned surface and reduces the number of adhered platelets. 4. Conclusion In this paper, we investigate the micro-structure on the rabbit’s mitral valve surface. It is shown that the dual-scaled structure which consists of both micro cobblestones-like structure and nano tenuous villus, is helpful in reducing the formation of thrombus. Therefore, the PDMS surface with hierarchical regular microstructure is prepared using femtosecond laser fabrication technique and soft lithography. And the mastoid micro-structure surface displays more excellent superhydrophobicity and blood compatibility than that of the smooth surface. Therefore, both surface wettability and surface morphology play important roles in hemocompatibility. An excellent anticoagulation material should have a superhydrophobic merit in order to adsorb platelets as fewer as possible. Meanwhile, it should also have a relatively fine structure. In this way, the coagulation reaction can be delayed. In addition, the present study provides a basic investigation for the design and fabrication of the artificial heart valve. Acknowledgements This research is supported by the National Natural Science foundation of China (50435030, 50775104), the Program for New Century Excellent Talents in Chinese University and Excellent Young Scholars foundation of Jiangsu (BK 200607). References [1] M.M. Black, P.J. Drury, W.B. Tindale, Journal of the Royal Society of Medicine 76 (1983) 667–680. [2] Y.Y. Lu, D.Z. Cui, X.J. Yang, et al. Heat Treatment of Metals 29 (9) (2004) 23–26. [3] K. Barton, A. Campbell, J.A. Chinn, et al. Biomedical Engineering Society (BMES) Bulletin 25 (1) (2001) 3–7. [4] Z.Y. Xu, Continuing Medical Education 20 (10) (2006) 63–65. [5] D.E. Harken, H.S. Soroff, W.J. Taylor, et al. Journal of Thoracic and Cardiovascular Surgery 40 (1960) 744–762. [6] A. Starr, M.L. Edwards, Annals of Surgery 154 (1961) 726–740. [7] C.R. Li, X.H. Wang, Z.H. Zheng, et al. Journal of Functional Materials and Devices 8 (1) (2002) 63–68. [8] I. Dion, X. Roques, C. Baquey, et al. Bio-medical Materials Engineering 3 (1) (1993) 51–55. [9] N. Ali, Y. Kousar, J. Gracio, et al. Thin Solid Films 51 (2006) 59–65. [10] A. Grill, B.S. Meyerson, V.V. Patel, et al. Journal of Applied Physics 61 (8) (1987) 2874–2877. [11] N. Huang, P. Yang, X.T. Zeng, Chinese Journal of biomedical Engineering 16 (3) (1997) 199–205. [12] Y.X. Leng, J.Y. Chen, P. Yang, et al. Surface and Coatings Technology 201 (2006) 1012–1016. [13] J.Y. Chen, Y.X. Leng, X.B. Tian, et al. Biomaterials 23 (2002) 2545–2552. [14] A. Bolz, M. Schaldach, Artificial Organs 144 (4) (1990) 260–269. [15] S.A. Visser, R.W. Hergenrother, S.L. Cooper, Polymers. Biomaterials Science (1996) 50–60. [16] J.A. Schmidt, A.F. von Recum, Biomaterials 12 (4) (1991) 385–389. [17] J.C. McDonald, G.M. Whitesides, Accounts of Chemical Research 35 (2002) 491– 499. [18] T. Murakami, S. Kuroda, Z. Osawa, Journal Colloid and Interface Science 202 (1998) 37–44. [19] J.M.K. Ng, I. Gitlin, A.D. Stroock, et al. Electrophoresis 23 (2002) 3461–3473. [20] A.R. Thompson, L.A. Harker, Manual of Hemostasis and Thrombosis, third ed., Davis FA, Philadelphia, 1983. [21] M.D. Lelah, S.L. Cooper, Polyurethans in Medicine, CRC Press, Boca Raton, Florida, 1986. [22] C. Fougnot, D. Labarre, M. Jozefonvicz, Josefowicz, in: G.W. Hastings, P. Ducheyne (Eds.), Macromolecular Biomaterials, CRC Press, Boca Raton, 1984. [23] R.N. Wenzel, Industrial & Engineering Chemistry Research 28 (1936) 988–994. [24] A.B.D. Cassie, S. Baxter, Transaction of the Faraday Society 40 (1944) 546–551. [25] T. Okada, Y. Ikada, Makromolekulare Chemie – Macromolecular Chemistry and Physics 192 (1991) 1705–1713.