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Effects of diameters and crystals of titanium dioxide nanotube arrays on blood compatibility and endothelial cell behaviors
Colloids and Surfaces B: Biointerfaces 184 (2019) 110521
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effects of diameters and crystals of titanium dioxide nanotube arrays on blood compatibility and endothelial cell behaviors Zhihao Gonga,b,1, Youdong Huc,1, Fan Gaob, Li Quanb, Tao Liub, Tao Gongb, Changjiang Pana,b,
T ⁎
a
Faculty of Material Science and Engineering, Nanjing Technology University, Nan’jing, 211816, China Faculty of Mechanical and Material Engineering, Huaiyin Institute of Technology, Huai’an, 223003, China c Department of Geriatrics, The Affiliated Huai'an Hospital of Xuzhou Medical College, Huai'an, 223003, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Titanium dioxide nanotube arrays (TNTAs) Protein adsorption Endothelial cells Blood compatibility
Titanium dioxide nanotube arrays (TNTAs) have attracted extensive attention in the fields of biomaterials and biomedicine due to their unique tubular structure and good biocompatibility. In this paper, TNTAs with different nanotube diameters and lengths were in situ prepared on the titanium surface by the anodic oxidation, and their crystal structures were further changed by annealing treatment. The effects of TNTAs with different diameters and crystals on the blood compatibility and endothelial cell behaviors were investigated. The results showed that TNTAs with the diameter of 30∼90 nm can be obtained by controlling the anodization voltage, and annealing treatment did not obviously change the diameters and lengths of the nanotube arrays. However, annealing treatment can transform the amorphous TNTAs into the anatase structure. The diameter and crystal structure of the nanotube arrays played a key role in the surface wettability and protein adsorption. The nanotube array with larger diameter displayed better surface hydrophilicity as compared to the pristine titanium, and annealing treatment further enhanced the hydrophilicity. As compared to the pristine titanium, the nanotube array structure had the characteristic of selective protein adsorption, and the nanotube array can promote the bovine serum albumin (BSA) adsorption and prevent the fibrinogen (FIB) adsorption, however, the increase of nanotube diameter could reduce BSA adsorption and increase FIB adsorption. Besides, the nanotube array with anatase structure can promote BSA adsorption while reduce FIB adsorption. Therefore, the TNTAs with smaller diameter and anatase crystal had good blood compatibility and cell compatibility, they can not only reduce platelet adhesion and hemolysis rate but also increase endothelial cell adhesion and proliferation. In conclusion, the nanotube arrays of the present study can be used to improve the cell compatibility and blood compatibility of the titanium implants.
1. Introduction Cardiovascular disease has become the first killer threatening the human health. The medical devices with excellent properties and performances play an important role in the treatment of the cardiovascular diseases. Titanium-based biomaterials are widely used in the vascular stents, thrombus filters and other intravascular medical devices, however, surface thrombosis and endothelial dysfunction caused by the implants often lead to implant failure [1]. It is of great scientific and practical significance for the application of titanium-based biomaterials to regulate the behaviors of blood and endothelial cells by changing the surface properties, and further to enhance the performances and functions of materials or devices. Based on the understanding of the interface biological responses
between human blood and endothelial cells and implant biomaterials, depositing inorganic coatings [2], preparing the polymer layer [3] or immobilizing the bioactive molecules [4–7] on the materials surface can not only improve the blood compatibility but also promote endothelium repair to some extent. However, these surface modification strategies cannot completely inhibit thrombus formation or promote complete endothelium healing [8]. In recent years, nanomaterials with unique nanotube array structures have gained extensive attention in the fields of bone implants, drug delivery, tumor therapy, and blood contact biomaterials. The preparation of nanotube arrays on the titanium surface can not only maintain the good mechanical properties and corrosion resistance of the titanium itself, but also build a unique nanotubular structure on the surface to improve the biocompatibility. In addition, the nanotube arrays on the titanium surface have high specific
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Corresponding author at: Faculty of Mechanical and Materials Engineering, Huaiyin Institute of Technology, Huai'an, 223003, China. E-mail address: [email protected] (C. Pan). 1 These authors contributed equally to this work and should be considered co-first authors. https://doi.org/10.1016/j.colsurfb.2019.110521 Received 24 June 2019; Received in revised form 20 August 2019; Accepted 21 September 2019 Available online 23 September 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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structure of the nanotube arrays were observed by scanning electron microscope (SEM, FEI Quanta250) after sprayed with a gold layer. The surface element concentration of the sample was analyzed by EDS (IMA X-Max 20, Britain). X-ray Diffraction (XRD, Switzerland, SCINTXTRA) was used to examine the surface crystal structure before and after annealing treatment, and the scan scope ranged from 15° to 85°. In order to further clarify the surface chemical structure of the original titanium and titanium dioxide nanotubes, the surface of different samples was characterized by X-ray photoelectron spectroscopy (XPS, VG Science, East Grinstead, UK). Water contact angle measurement (DSA25, KrüssGmbH, Germany) was carried out to characterize the surface hydrophilicity. The water contact angle test was done by the sessile drop method at room temperature using the distilled water. Five parallel samples were measured and the values were averaged.
surface area, controllable nanotube diameter and length [9,10], which can provide an excellent platform for the functionalization and then allow more cells to adhere and proliferate on the surface [11–13], leading to better biocompatibility. TNTAs are the excellent candidate for the bone-substituted materials because they can promote the preferential growth of bone tissue and thus make the connections between implant and bone more stable [14]. Moreover, the TNTAs can promote differentiation and proliferation of bone marrow mesenchymal stem cells, osteoblasts and chondrocytes, and greatly improve the integration ability between the implants and the surrounding bone tissues [15]. On the other hand, the unique hollow nanotube structure can be loaded with a variety of drugs, bioactive substances or nanoparticles to improve the biocompatibility and antibacterial properties of the vascular implants [16–19]. Although there are many reports on TNTAs, most of them focus on the bone biomaterials and there are few reports concerning the effects of the nanotube diameter and its crystal structure on the blood compatibility and endothelial cell behaviors. Moreover, there is still much controversy about the blood compatibility of TNTAs. Some studies have found that the surface of TNTAs hardly causes blood reaction, while others have found that TNTAS can cause thrombosis [20]. Therefore, it is necessary to further clarify and explore how the diameter and crystal structure of the TNTAs affect the hemocompatibility and endothelial cell behaviors. At the same time, protein adsorption on the surface is also an important factor affecting the cell compatibility and blood compatibility of TNTAs. The adsorbed extracellular matrix proteins can enhance the adhesion and proliferation of endothelial cells [21]. However, a large number of coagulation factors can be released after the fibrinogen adsorption, leading to fibrin coagulation and platelet aggregation [22]. Therefore, it is also urgent to explore and clarify the protein adsorption behaviors of TNTAs with the different structures and crystals. In order to elucidate the effects of the structural characteristics of TNTAs, such as nanotube diameter, nanotube length and crystal structure, on the blood compatibility and endothelial cell behaviors, an anodic oxidation method was used to in situ prepare a dense array of TiO2 nanotubes on the titanium surface. The diameters and lengths of the nanotube arrays were controlled by changing the oxidation voltages, and the crystal structure was changed by annealing treatment. The effects of the diameters and crystal structures of the nanotube array on the physical and chemical properties and biocompatibility were investigated.
2.3. Protein adsorption The adsorption behaviors of two major plasma proteins (fibrinogen and albumin) were measured by the BCA method. The samples were firstly immersed in ethanol for 30 min and then in PBS (phosphate buffer) for 10 h. Thereafter, the samples were separately placed in 1 mg/ml of BSA (albumin) and 1 mg/ml FIB (fibrinogen) solutions for adsorbing 2 h at 37℃. The samples were washed twice with PBS and then desorbed ultrasonically for 30 min in 2 ml SDS (sodium dodecyl sulfate, 1% wt). Taking 150 μl eluent and 150 μl BCA working solution to mix for reacting one hour at 60℃, and then 200 μl of the reaction liquid was put into a 96-well plate. The absorbance at 562 nm was measured by a Microplate Reader (Bio-Tek, Eons), and the adsorption capacity of the protein was calculated by the standard curve. 2.4. Blood compatibility 2.4.1. Platelet adhesion Platelet-rich plasma (PRP) was obtained by centrifuging fresh human whole blood of a healthy volunteer at 1500 rpm for 15 min. 200 μL PRP was dropped on each sample surface to cover the whole surface. After incubating at 37℃ for 2 h, the samples were washed with PBS. The adhered platelets were fixed with 2.5% glutaraldehyde (in PBS) for 24 h and then washed with PBS. Finally, the samples were dehydrated by 30%, 50%, 75%, 90%, 100% ethanol solutions for 10 min each in sequence, followed by thoroughly dried in an air atmosphere. The samples were coated gold in vacuum and then observed by a scanning electron microscopy (SEM, FEI Quanta 250). The images with large magnification were used to analysis platelet morphologies. Five images with small magnifications for the each sample were randomly selected to calculate the number of the platelets adhered to the surface, and the values were averaged.
2. Materials and methods 2.1. Preparation of titanium dioxide nanotube arrays The pure titanium (TA2, purity 99.6%) bar with a diameter of 15 mm was cut into 2 mm thick plates. The plates were successively polished by silicon carbide papers from 400 to 2000 mesh and then ultrasonically cleaned by acetone, ethanol and distilled water for 10 min, respectively. After dried by the compressed air flow, the samples were immersed in 50 ml electrolyte (ethylene glycol solution containing 0.25% wt NH4F and 6 ml of deionized water) for anodization for 3 h, and the anodic voltage was controlled at 30 V, 40 V, 50 V, and 60 V, respectively. After anodization, the sample was ultrasonically washed with ethylene glycol for 30 min, and then washed with alcohol for 5 min. The original titanium plates and the samples obtained by the anodization were named as Ti, TNTAs-30, TNTAs-40, TNTAs-50, and TNTAs-60. The obtained samples were further treated in an air atmosphere at 500℃ for 3 h, and then air-cooled. They were named as Ti-A, TNTAs-30A, TNTAs-40A, TNTAs-50A, and TNTAs-60A.
2.4.2. Hemolysis rate The hemolysis assay was done in accordance with ISO 109934:2009. The fresh human blood was centrifuged for 10 min at 1500 rev/ min, and the supernatant was removed. The precipitated red blood cells were washed three times with PBS until the supernatant did not show red. The resulting red blood cells were mixed with PBS to obtain a 2% suspension (2 mL red blood cells plus physiological saline to 100 mL). The test samples were immersed into the above diluted suspension solution, and incubated in 37℃ water bath for 1 h. The solution was centrifuged at 3000 rpm for 5 min, and the supernatant was added into a 96-well plate. The absorbance value (A) was measured at 450 nm by a microplate reader. For controls, the mixture of 2 mL erythrocyte and 98 mL physiological saline was used as the negative control, and the mixture of 2 mL erythrocyte and 98 mL ultrapure water was used as the positive control. The hemolysis ratio was calculated according to the following formula:
2.2. Surface characterization
HR(%)=(A－A1)/(A2－A1)×100%
The surface morphologies, cross-sectional morphologies and tubular 2
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structure of the nanotube arrays and new crystalline phases appeared. The formation of new phase has important influences on the properties and functions of titanium [24]. According to the results calculated by jade software, the contents of anatase on the surfaces of TNTAs were about 100% (30 V), 86% (40 V), 73% (50 V), 65% (60 V), indicating that not all TNTAs can be fully changed into anatase phase by annealing treatment. As shown in Fig. 2, the TNTAs layer became thicker with the increase of the anodization voltage, when the same annealing time was applied, the relative thin TNTAs layer, such as TNTAs-30, can be completely converted into anatase while the thicker TNTAs cannot be fully changed into anatase crystal. The chemical composition of the modified surface was further studied by X-ray photoelectron spectroscopy (XPS). Fig. 1b shows the typical XPS survey spectra of the original and modified titanium surfaces. The original titanium substrate showed Ti2p, O1s and other weaker elements, indicating that the titanium sample itself had been oxidized naturally and received some pollution. After anodic oxidation, the peak value of O1s increased significantly, and concurrently F1s also appeared, indicating that an oxide layer containing F element appeared on the surface. After annealing, F element disappeared on the TNTAs surface because F exists only in fluoride on the surface and it can be completely removed by heat treatment [25]. Fig. 1c and d show the high resolution spectra of Ti2p peak for the pristine titanium and TNTAs-60A. It can be seen that the pure titanium Ti2p (Fig.1c) can be divided into four peaks: Ti (454.88 eV), Ti-N (455.81 eV), O-Ti-O (458.78 eV), O-Ti-Ca (457.48 eV) [26]. They accounted for 62.35%, 18.04%, 16.56% and 3.05% respectively. Because titanium plates were exposed to air for a long time, which resulted in an oxide layer naturally. Ti2p (Fig.1d) of TNTAs-60A can be divided into two main peaks: O-Ti-O (459.87 eV) and O-Ti-Ca (457.48 eV) [26]. The binding energy value of O-Ti-O peak was slightly higher than that of pure titanium, and the proportion of OTi-O increased from 18.04% to 91.32%, suggesting that the anatase titanium structure was indeed prepared on the titanium surface.
Where HR is the hemolysis rate (%), A is the absorbance of the test sample (%), A1 is the negative control absorbance (%), A2 is the positive control absorbance (%). 2.5. Endothelial cell adhesion and proliferation The samples were first sterilized by UV light and placed in a 24-well cell culture plate. Subsequently, 1.5 mL of 5 × 104 cells/mL endothelial cell (HUVEC, ECV304, Cobioer, Nanjing, China) suspension was seeded on the substrate and incubated 1 and 3 d at 37℃, respectively. The incubation was carried out in a humidified atmosphere containing 5% CO2. The cell culture medium was DMEM/F-12 (Hyclone) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. After the predetermined incubation time, the samples were washed 3 times with PBS. The cells were stained successively by rhodamine and 4′,6-diamidino-2-phenylindole (DAPI). 200 μl rhodamine (1:1000 in PBS) was added to each sample surface for incubating 20 min, and then the sample was washed 3 times with PBS. Finally, 200 μl of DAPI (1:400 in PBS) was added to sample surface for 3 min, and the sample was washed 3 times with PBS. The stained sample was observed by fluorescence microscopy (Zeiss, inverted A2). At the same time, another group samples were sequentially dehydrated 10 min by 30%, 50%, 70%, 90%, 100% ethanol solutions for each sample. The adherent cells were observed by scanning electron microscopy (SEM, FEI Quata 250) after sputter-coated a gold layer. For cell proliferation, endothelial cells were cultured in the same way. After cultured with sample for 1, 3 days, respectively, 0.5 ml of cck-8 solution (cck-8: cell culture solution = 9:1) were added on each sample, incubated 3.5 h at 37 °C. The absorbance of the medium was measured by a microplate reader (Bio-Tek Eons) at 450 nm. Triplicate parallel samples were measured and the values were averaged for each sample. 3. Results and discussion
3.2. Surface morphologies and elemental analysis 3.1. XRD and XPS Fig. 2 shows the typical SEM images of the different samples which were anodized at different voltages. It can be clearly seen that a compact and regular hollow nanotube array structure was formed on the surface after anodic oxidation. The diameters of the nanotube arrays were about 30 ± 2 nm (30 V), 50 ± 5 nm (40 V), 70 ± 8 nm (50 V), 90 ± 7 nm (60 V). According to the mechanism of anodic oxidation, the tubular structure was produced by the corrosion from the surface to the interior. More F− ions could accumulate on the surface during anodic oxidation when larger anodization voltage was applied, leading to relative serious corrosion and larger diameter nanotube. At the same time, the corrosion rate increased with the increase of voltage, therefore the length of nanotubes increased with the increase of voltage when the same oxidation time was applied. As can be seen from the cross-sectional view of the nanotube arrays (Fig. 2 a3～d3), the length of the nanotube array can reach about 5 μm, 7 μm, 15 μm, and 22 μm respectively. Therefore, the higher the voltage, the thicker the anodized film formed on the surface. The anodized nanotube array was further subjected to a high-temperature at 500℃, the nanotube was expanded outward by about 5～10 nm, and the tube wall was thickened by 1～ 2 nm. However, annealing treatment had no influence on the length of the nanotubes. It was presumably considered that the larger volume of anatase TiO2 after annealing treatment cause the nanotubes to expand outward, and the anatase TiO2 gradually merged into the nanotube wall by forming larger crystallites, resulting in the enlargement of the nanotubular structure and rougher surface [27]. Although a small portion of the surface nanotubes were damaged, the length of the nanotubes did not change significantly. Table 1 shows the elemental content of the different samples measured by EDS. It can be seen that the surfaces of anodized TNTAs samples mainly consisted of three elements of Ti, O and F. The surface
Titanium and its alloys are widely used in the blood contact materials and devices, such as thrombosis filter and artificial heart valves. It has been reported that the biocompatibility of titanium, especially blood compatibility, is related to the thickness of the oxide layer and the crystal structure. Anodization can produce an oxide layer with nanotube array structure on the titanium surface, and annealing treatment can further change the crystal structure of the nanotube arrays. In the present study, XRD was firstly used to characterize the surface crystal structure changes of the anodized titanium before and after annealing treatment. Fig. 1a shows the XRD patterns of the different samples. It can be clearly seen that no new diffraction peaks besides the titanium peaks can be detected on the pristine titanium surface after annealing treatment at 500℃, indicating that the crystal structure of the titanium surface had hardly changed. Compared with pure titanium, there was almost no change in the diffraction pattern after anodization and only peaks associated with the Ti substrate can be found, indicating that no new crystalline phase was formed during the anodization process. It has been reported that the oxide barrier layer on the titanium surface is semi-crystalline, while the synthesized TNTAs are amorphous [23]. As can be seen from Fig. 1a, with the increase of anodic oxidation voltage, the intensity of Ti diffraction peaks increased. It was considered that the diameter and length of the amorphous TNTAs increased with the increase of the voltage (as shown in Fig. 2), and the size of the amorphous TNTAs became larger, resulting in an increase in the diffraction peak. After annealing treatment at 500℃, besides the diffraction peaks of Ti, the (101), (103), (004), (112), (211), (204) diffraction peaks of anatase-type titanium dioxide appeared at 2θ = 25.37, 37.03, 37.88, 38.61, 55.10 and 62.74, respectively, demonstrating that annealing treatment changed the surface crystal 3
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Fig. 1. The XRD spectra of the different samples (a), (b) the XPS survey spectra of Ti and TNTAs samples (b), the high-resolution XPS spectra of Ti2p in Ti (c) and TNTAs-60A (d).
of the TNTAs sample mainly formed TiO2 oxide film layer with a small amount of F element attached to the surface. The reason for the existence of F element is that in the process of anodic oxidation, F− in the electrolyte forms (NH4)2TiF6 on the surface by etching the TiO2 oxide layer [11]. As the voltage increased, the content of F element basically showed an upward trend. The higher the voltage, the more F− formed by the anode, and thus the more fluorides introduced. In the process of annealing treatment, NH4F can be generated from (NH4)2TiF6 on the surface of TNTAs, which resulted in the decrease of F element, and consequently changed the contents of Ti and O elements on the TNTAs surface. The ratio of Ti and O elements in all TNTAs samples was close to 3:2, suggesting that the oxide film formed on the surface was mainly TiO2.
the anodization, which can form hydrogen bond with water and thus enhanced the hydrophilicity. With the increase of anodic oxidation voltage, the hydrophilicity was enhanced, and even super hydrophilicity can be achieved, for example, the water contact angle of TNTAs-60A was below 5 degrees. It is well known that the hydrophilicity of the material depends both surface chemical structure and surface morphologies. As can be seen from Fig. 2, with the increase of voltage, the diameter of nanotube arrays became larger and the surface became rougher. The porous/rough titanium dioxide structure film inhales water through capillary force, thus making the substrate exhibit better hydrophilic properties [31,32]. The hydrophilicity of the anodized nanotube arrays increased significantly after the annealing treatment, and the water contact angle was reduced to less than 5 degrees, indicating that the material displayed super hydrophilic properties. It was considered that the increased diameter and larger roughness of TNTAs after annealing treatment contributed to the improved hydrophilicity. In addition, it can be seen from Table 1 that the surfaces of the anodized samples contains hydrophobic fluorine elements. After annealing treatment, the surface fluorine element almost disappeared, therefore its hydrophilicity was further improved. At the same time, the amorphous part of TiO2 was transformed into anatase structure after annealing treatment at 500℃. The anatase type of TNTAs had better hydrophilicity than amorphous TNTAs [33]. The protein adsorption was further investigated by BCA method. Generally speaking, when the implant material is in contact with human blood, what occurs first is the interaction between the plasma proteins and the materials. The types and properties of plasma protein adsorbed on the surface play an important role in the subsequent blood reactions and cell behaviors. There are two main proteins in human blood, albumin and fibrinogen. Generally speaking, when biomaterials come into contact with human blood, albumin and fibrinogen compete to adsorb on the surface. The albumin adsorption can reduce the platelets adhesion and activation, and therefore inhibit the occurrence of blood coagulation and prolong the coagulation time [34], leading to better hemocompatibility. In contrast, the adsorption and denaturation of FIB could deteriorate the blood compatibility of materials. In the
3.3. Surface hydrophilicity and protein adsorption In general, hydrophilic surfaces can adsorb large amounts of water molecules, thereby reducing non-specific protein adsorption and further preventing platelet adhesion. For titanium oxide nanotubes arrays, it has been reported that it can prevent fibrinogen adsorption and denaturation [28], therefore improve blood compatibility. On the other hand, the hydrophilic surfaces have shown to activate plasma coagulation [29] in some cases, which can deposit fibrin onto the surfaces to which platelets have numerous membrane-bound receptors [30]. This kind of inter-relationship between coagulation and platelet adhesion greatly complicates TNTAs structure-property relationships. In order to clarify the relationship between the surface properties of TNTAs and the biocompatibility, the surface wettability was studied by measuring the water contact angle. Fig. 3a shows the water contact angle results of the different samples. It can be clearly seen that the hydrophilicity was significantly enhanced after the preparation of the TNTAs on the Ti surface, and the hydrophilicity was further improved by annealing treatment. A hollow nano-tubular structure was in situ produced on the pristine titanium surface by anodization, which can accommodate water into the inner part of the tube. At the same time, a large amount of oxygen was introduced on the surface and inside the nanotube after 4
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Fig. 2. The typical SEM images of TNTAs on Ti surface. a1～d1 shows the TNTAs anodized at 30 V (a1), 40 V (b1), 50 V (c1), 60 V (d1). a2～d2 were the corresponding SEM images after annealing heating and a3～d3 shows the cross sectional morphologies of a1～d1.
specific surface area and thus have a larger contact area with protein. Therefore, with the increase of the nanotube diameter, the amount of BSA adsorbed on the surface decreased. At the same time, as shown in Fig. 3a, the nanotubes arrays with larger diameter displayed better hydrophilicity, which can also inhibit the non-specific BSA adsorption [35] to some degree. In addition to the specific surface area and hydrophilicity, protein adsorption also depends on the surface charge properties of the materials and protein, which may promote or inhibit protein adsorption by the electrostatic interaction [36]. It was reported that the TNTAs by anodization on the titanium had negative charges [37]. The relative larger specific surface area of TNTAs with smaller diameter had more negative charges, which can promote positive charged BSA adsorption. After annealing treatment, due to the increased roughness and the occurrence of anatase crystal structure, the BSA adsorption on all samples surfaces increased to different degrees. Similarly, the amount of BSA adsorbed on the anatase TNTAs-30A surface was the most due to the electrostatic adsorption. It is well known that the crystallinity of the TNTAs is closely related to the adsorption of BSA. Different crystal structures can lead to changes in the surface properties, which further influences BSA adsorption [38]. After
Table 1 Atomic content of surface elements on Ti and TNTAs samples. Sample
Ti TNTAs-30 TNTAs-40 TNTAs-50 TNTAs-60 TNTAs-30A TNTAs-40A TNTAs-50A TNTAs-60A
Atomic concentration (at.%) Ti
O
F
100 56.12 54.91 57.47 54.39 61.39 60.27 59.92 61.55
0 34.55 35.17 32.16 32.84 38.02 39.12 38.87 36.89
0 9.33 9.92 10.37 10.77 0.58 0.90 1.21 1.56
present study, BSA and FIB adsorption on the different samples were investigated, and the results are shown in Fig. 3b and c. It can be seen that the BSA adsorption on the TNTAs surface increased obviously as compared to the pristine titanium, suggesting that preparation of TNTAs can promote albumin adsorption, which could lead to better blood compatibility. The TNTAs with smaller diameter have the larger 5
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Fig. 3. The water contact angle (a), the amounts of BSA (b) and FIB (c) adsorption of the different samples. Data for each sample were taken from three different substrates, after which they were expressed as mean ± SD.
Taking the above results into consideration, it can be seen that the nanotube arrays with the different diameters and lengths were produced successfully on the titanium surface by anodic oxidation. Compared with bare titanium, the substrates with nanotube arrays can promote BSA adsorption and prevent FIB adsorption. Therefore, the nanotube arrays showed the characteristics of selective albumin adsorption. The tube dimension, array thickness, and crystal structure had important effects on the protein adsorption behaviors. The increase of the nanotube diameter could lead to a slight decrease in the BSA adsorption and an increase of FIB adsorption to some extent. The anatase structure of TNTAs could help improving the protein adsorption selectivity of the nanotube arrays.
annealing treatment, amorphous TNTAs were partially transformed into anatase structure, which changed the charge properties of TNTAs, resulting in an increase in BSA adsorption. The fibrinogen adsorption is closely related to the platelets adhesion and the cell adhesion and proliferation. The adsorption of fibrinogen can easily cause a large number of platelets to adhere to the material surface, release a large amount of blood coagulation factors, and further induce thrombosis [39]. In this study, the FIB adsorption behaviors on the different TNTAs surfaces were further studied. Fig. 3c shows the FIB adsorption concentration on the different sample surfaces. It can be seen that FIB content adsorbed on the bare titanium surface was the highest among all samples. After the formation of nanotube arrays by anodic oxidation, the FIB adsorption concentration on the surface decreased significantly. Studies have shown that FIB will preferentially be adsorbed on the hydrophobic surface [40]. The surface of the amorphous TNTAs was hydrophilic as compared to bare titanium, which resulted in less FIB adsorption on the TNTAs surface. With the increase of the amorphous TNTAs diameter, the FIB adsorption increased due to the increased hydrophilicity (As shown in Fig. 3a). Meanwhile, it has been reported that the FIB adsorption capacity increased with the increase of the oxide layer thickness [41]. The length of nanotube arrays increased with the increase of anodic oxidation voltage, which represented the increased thickness of the TNTAs, and therefore the TNTAs with larger lengths can promote FIB adsorption. It was presumably concluded that the influence of the amorphous TNTAs length was much greater than that of hydrophobicity, leading to the increased FIB adsorption with the increase of nanotube array diameter. After annealing treatment, the surface roughness and hydrophilicity of the substrates was further improved due to the formation of anatase-type TNTAs, therefore the amount of FIB adsorbed on the surface decreased as compared to the anodized samples. The FIB adsorption capacity on the anatase TNTAs surface decreased with the increase of tube diameter and length. It was speculated that the electronic band gap configuration of anatase TNTAs reduced the tension between the surface and FIB, resulting in less FIB adsorption [42]. The smaller diameter nanotubes may exhibit smaller tension between the surface and the FIB, and thus less FIB was adsorbed.
3.4. Blood compatibility Platelet adhesion and activation have been considered as a major cause of thrombosis. Early inflammation and thrombosis of the vascular implants are associated with platelet activation. The adherent platelets can promote aggregation with other platelets, and release platelet and pro-coagulant agonists [43]. Therefore, platelet adhesion represents one of the main methods to explore the blood compatibility of biomaterials. Fig. 4a and b are the typical SEM image of platelets adhered on the different samples surfaces, and Fig. 4 c shows the number of platelets adhered on the different samples surfaces. It can be clearly seen that more platelets were attached on the titanium surface, and the adhered platelets on the bare titanium surface not only extended pseudopodia, but also some platelets had been fully activated (the inset in Fig. 4a-a1), indicating that the pristine titanium had limited anticoagulation. Previous studies and our results (Fig. 3) shows that the hydrophobic surface can adsorb more fibrin [44,45], which can deteriorate the blood compatibility. After the anodization, a regular nanotube array structure was produced on the surface. As mentioned above, the fibrinogen adsorption on the nanotube array surfaces can be significantly reduced (Fig.3c), which would help inhibit the conversion of fibrinogen to fibrin, prevent fibrin from clotting into blocks, thereby reducing the chance of thrombosis [46]. Therefore, the platelets adhered to the TNTAs surface were much less than that of the pristine 6
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Fig. 4. The SEM images of platelets adhered on the surface of Ti and TNTAs samples before (a) and after annealing treatment (b). (a1: Ti; a2: TNTAs-30; a3: TNTAs40; a4: TNTAs-50; a5: TNTAs-60; b1: Ti-A; b2: TNTAs-30A; b3: TNTAs-40A; b4: TNTAs-50A;b5: TNTAs-60A). The inset images shows the typical single platelet morphology on the corresponding substrates. (c) The number of adhered platelets on different samples. Five parallel samples were measured and the results were expressed as mean ± SD. (d) The hemolysis rate of Ti and TNTAs samples.
Red blood cells are one of the main components in human blood. When blood contacts with biomaterials, the red cells may be destroyed and the red blood protein is released, resulting in occurrence of hemolysis and further causing blood clotting [50]. Hemolysis rate is one of the important indicators to characterize the interaction between biomaterials and red blood cells. According to the international ISO10993-4 standard, if the hemolysis rate (HR) of the material is less than 5%, the material meets the requirements of hemolysis rate. If the HR of the material is more than 5%, the material has a hemolysis effect and is not suitable for use as a blood contact material. In order to further investigate the relationship between nanotube array structure and blood compatibility, the hemolysis assay was further carried out. Fig. 4d shows the hemolysis rate of the different materials. It can be seen that the hemolysis rate of all samples was less than 5%, indicating that the hemolysis did not occur on all samples. Compared with the pure titanium, the hemolysis rate of the material after the anodization decreased significantly, indicating that the nanotube arrays on the surface can further significantly improve the blood compatibility. However, although the hemolysis rate increased with the increase of tube diameter, it was still significantly less than the hemolysis rate of the control titanium. After annealing treatment at 500℃, the hemolysis rate of each sample decreased slightly. The hemolysis rate of TNTAs30A was the lowest, indicating that it had the best blood compatibility.
titanium. As the diameter of nanotube increased, more and more platelets adhered to the amorphous TNTAs surfaces, and the spreading state became more obvious. It was presumably due to the fact that the amorphous TNTAs with the larger diameter increased platelet adhesion by adsorbing more fibrin. In general, rapid adsorption of plasma proteins on the outer surface of the sample results in platelet adhesion [47]. It was also possible that fluoride ions in the electrolyte doped into TNTAs during anodic oxidation induced platelet adhesion and activation. The larger diameter TNTAs with the longer nanotube length are capable of adsorbing more fibrin, thereby increasing platelet adhesion [48]. It can be seen from the above elemental distribution results (Table 1) that the nanotube arrays with larger diameter had the more fluoride ions on the surface, which may arouse more aggregation of platelets. The number of platelets adhered on the titanium surface decreased slightly after annealing treatment at 500℃, however, the activation status of platelets did not improve (the inset of Fig.4b-b1).The number of platelets adhered on the TNTAs surface was significantly reduced after the annealing treatment, and the activation of platelets was also inhibited. The anatase type TNTAs-30A had the best blood compatibility according to the platelet adhesion. Moreover, the platelets adhered on the anatase-type TNTAs surface were rounded, and they can keep intact morphologies and may return to the blood circulation. The anticoagulant of anatase TNTAs was much better than that of the amorphous TNTAs because the former surface was more hydrophilic than the latter, which could prevent non-specific adsorption (such as fibrinogen) and reduce platelet adhesion and activation by increasing the hydration layer [49]. The anatase-type TNTAs-30A surface adsorbed the least FIB, and the ability to induce platelet aggregation and activation was relatively poor. Moreover, the BSA adsorbed on the surface of TNTAs-30A was also the most, which was helpful for inhibiting the platelet adhesion and reducing the probability of thrombosis.
3.5. Endothelial cell adhesion and proliferation Generally speaking, the cells will change their morphologies to achieve the integration of material and cells after contacting biomaterials. The entire progress of cell adhesion and spreading consists of cell attachment, filopodia growth, cytoplasmic webbing, flattening of the cell mass and the ruffling of peripheral cytoplasm [51]. Cell adhesion is widely used to study cellular behaviors on the biomaterials 7
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Fig. 5. The typical fluorescent images of endothelial cells adhered on the surface of Ti and TNTAs samples.
Fig. 6. The typical SEM images of endothelial cells adhered on the surface of Ti and TNTAs samples.
surfaces. In this study, cell adhesion behavior was firstly studied by cell fluorescence staining, and the morphologies of adherent cells were further observed by SEM. Fig. 5 and Fig. 6 shows the typical fluorescent images and SEM images of endothelial cells attached on the different samples surfaces, respectively. It can be seen that the number of endothelial cells attached on the bare titanium surface was relatively small (Fig. 5), and the attached endothelial cells mainly exhibited
round shape, indicating that almost all cells showed limited spreading (Fig. 6). However, after annealing treatment at 500℃, the number of round cells on the surface decreased significantly (Fig. 5), and more cells displayed spreading state (Fig. 6). After anodic oxidation, the number of endothelial cells increased (Fig. 5), suggesting that the nanotube arrays can enhance cell adhesion. With the increase of the nanotube diameter, the number of the adherent cells on the surface was 8
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more endothelial cells can be attached on the surface for proliferation. The absorbance of the endothelial cells attached on the pure titanium surface and amorphous TNTAs for 1, 3 day increased significantly after annealing treatment. Usually, endothelial cells are easy to attach on the hydrophilic surfaces than on the hydrophobic surfaces. After annealing, the surface of TNTAs samples showed super hydrophilicity, therefore the proliferation of endothelial cells on the TNTAs-A surface was more obvious. Higher concentration of fibrin adsorption can prevent the expansion of protein and reduce cell adhesion [57]. The surface of TNTAs-30A with smaller diameter can adsorb less FIB, so the amount of endothelial cells on the surface was more than that of other samples.
reduced (Fig. 5), and the morphologies of the cells was similar to that of on the pure titanium surface after annealing treatment (Fig. 6). Studies have shown that the fate of cells in the nanotube layer is determined by the microenvironment, and 15–30 nm is the optimal diameter for cell adhesion, migration and differentiation [52]. When the diameter exceeds 100 nm, cell adhesion and proliferation hardly occur, and even induce cell apoptosis [50]. The circular shape of nanotubes can promote cell formation, cell differentiation and proliferation [53]. The larger diameter of nanotubes could force cells to grow outward and find the substrate for the protein deposition, which can greatly affect the cell adhesion performance [54]. Therefore, the number of endothelial cells adhered to the surface of TNTAs-30 was the largest. After annealing treatment, the endothelial cells adhesion on the TNTAs surface increased significantly (Fig. 5). Similarly, anatase TNTAs-30A had the best cell adhesion, and the cell morphologies was fully spread out (Fig. 6). It not only showed polygonal shape, but also has many fine villi formed by cytoplasmic processes on the surface (Fig. 6). Some studies have shown that the hydrophilic sample surface can improve cell adhesion and proliferation by exchanging and adsorbing extracellular matrix proteins [55]. Therefore, TNTAs-A with better hydrophilicity can adhere more cells and the cells had better spreading morphologies. CCK-8 was carried out to further investigate the cell proliferation. Fig. 7 shows the CCK-8 values of the endothelial cells on the different samples. It can be clearly seen that the absorbance of the endothelial cells cultured on the surface of bare titanium and amorphous TNTAs for one day had no obvious difference, however the proliferation of the endothelial cells on the amorphous TNTAs with smaller diameter surface was the best. Studies have shown that the diameter and spacing of the nanotubes are important factors affecting cell acquisition of protein, ions and nutrients [56]. Endothelial cells tend to adhere to the smaller diameter nanotubes, leaving more growth factors for cell proliferation. Therefore, the proliferation of endothelial cells for nanotubes with smaller diameter was better. Our results suggested that TNTAs-30 had the best suitable diameter for endothelial cell adhesion, and it also displayed an excellent proliferation performance. At the same time, cells can adhere to these functional surfaces through non-receptor binding forces (electrostatic interactions), leading to the improved cell proliferation. TNTAs have negative charges on the surface, which can promote cell adhesion and proliferation. Due to the fact that the TNTAs with the smaller diameter had more negative charges on the surface,
4. Conclusion In this study, orderly and regular titanium dioxide nanotube arrays were successfully prepared on the titanium surface by the anodic oxidation, and the anatase nanotube arrays were obtained by the annealing treatment. The diameter, length and crystal structure of the nanotube arrays significantly affected the surface wettability, protein adsorption, blood compatibility as well as endothelial cell adhesion and proliferation. TNTAs prepared by anodic oxidation had good hydrophilicity, and the hydrophilicity was further enhanced by annealing treatment. TNTAs showed the characteristics of selective adsorption of BSA. The increase of nanotube diameter could lead to a slight decrease in BSA adsorption, but it could promote FIB adsorption to some extent. The anatase structure would improve the protein adsorption selectivity of nanotube arrays. Therefore, the surface of TNTAs samples with smaller diameter (e.g., TNTAs-30A) can promote BSA adsorption and reduce FIB adsorption, thereby improve the blood compatibility and endothelial cell adhesion and proliferation. The results demonstrated that the nanotube arrays prepared on the titanium surface could not only improve the blood compatibility of titanium, but also promote the adhesion and proliferation of endothelial cells. Therefore, the method of the present study can be used for the surface modification of blood contacting biomaterials to simultaneously improve the blood compatibility and cell compatibility of vascular endothelial cells. Acknowledgements This work is financially supported by the Natural Science
Fig. 7. The results of CCK-8 detection after 1, 3d growth of endothelial cells on Ti and TNTAs samples. 9
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Foundation of China (31870952), Natural Science Foundation of Jiangsu Province of China (BK20181480), the International S&T Cooperation Projects of Huai’an City of China (HAC201703), The Key Program for Natural Science Foundation of the Jiangsu Higher Education Institutions of China (17KJA530002), Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX18-0348).
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effects of diameters and crystals of titanium dioxide nanotube arrays on blood compatibility and endothelial cell behaviors Zhihao Gonga,b,1, Youdong Huc,1, Fan Gaob, Li Quanb, Tao Liub, Tao Gongb, Changjiang Pana,b,
T ⁎
a
Faculty of Material Science and Engineering, Nanjing Technology University, Nan’jing, 211816, China Faculty of Mechanical and Material Engineering, Huaiyin Institute of Technology, Huai’an, 223003, China c Department of Geriatrics, The Affiliated Huai'an Hospital of Xuzhou Medical College, Huai'an, 223003, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Titanium dioxide nanotube arrays (TNTAs) Protein adsorption Endothelial cells Blood compatibility
Titanium dioxide nanotube arrays (TNTAs) have attracted extensive attention in the fields of biomaterials and biomedicine due to their unique tubular structure and good biocompatibility. In this paper, TNTAs with different nanotube diameters and lengths were in situ prepared on the titanium surface by the anodic oxidation, and their crystal structures were further changed by annealing treatment. The effects of TNTAs with different diameters and crystals on the blood compatibility and endothelial cell behaviors were investigated. The results showed that TNTAs with the diameter of 30∼90 nm can be obtained by controlling the anodization voltage, and annealing treatment did not obviously change the diameters and lengths of the nanotube arrays. However, annealing treatment can transform the amorphous TNTAs into the anatase structure. The diameter and crystal structure of the nanotube arrays played a key role in the surface wettability and protein adsorption. The nanotube array with larger diameter displayed better surface hydrophilicity as compared to the pristine titanium, and annealing treatment further enhanced the hydrophilicity. As compared to the pristine titanium, the nanotube array structure had the characteristic of selective protein adsorption, and the nanotube array can promote the bovine serum albumin (BSA) adsorption and prevent the fibrinogen (FIB) adsorption, however, the increase of nanotube diameter could reduce BSA adsorption and increase FIB adsorption. Besides, the nanotube array with anatase structure can promote BSA adsorption while reduce FIB adsorption. Therefore, the TNTAs with smaller diameter and anatase crystal had good blood compatibility and cell compatibility, they can not only reduce platelet adhesion and hemolysis rate but also increase endothelial cell adhesion and proliferation. In conclusion, the nanotube arrays of the present study can be used to improve the cell compatibility and blood compatibility of the titanium implants.
1. Introduction Cardiovascular disease has become the first killer threatening the human health. The medical devices with excellent properties and performances play an important role in the treatment of the cardiovascular diseases. Titanium-based biomaterials are widely used in the vascular stents, thrombus filters and other intravascular medical devices, however, surface thrombosis and endothelial dysfunction caused by the implants often lead to implant failure [1]. It is of great scientific and practical significance for the application of titanium-based biomaterials to regulate the behaviors of blood and endothelial cells by changing the surface properties, and further to enhance the performances and functions of materials or devices. Based on the understanding of the interface biological responses
between human blood and endothelial cells and implant biomaterials, depositing inorganic coatings [2], preparing the polymer layer [3] or immobilizing the bioactive molecules [4–7] on the materials surface can not only improve the blood compatibility but also promote endothelium repair to some extent. However, these surface modification strategies cannot completely inhibit thrombus formation or promote complete endothelium healing [8]. In recent years, nanomaterials with unique nanotube array structures have gained extensive attention in the fields of bone implants, drug delivery, tumor therapy, and blood contact biomaterials. The preparation of nanotube arrays on the titanium surface can not only maintain the good mechanical properties and corrosion resistance of the titanium itself, but also build a unique nanotubular structure on the surface to improve the biocompatibility. In addition, the nanotube arrays on the titanium surface have high specific
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Corresponding author at: Faculty of Mechanical and Materials Engineering, Huaiyin Institute of Technology, Huai'an, 223003, China. E-mail address: [email protected] (C. Pan). 1 These authors contributed equally to this work and should be considered co-first authors. https://doi.org/10.1016/j.colsurfb.2019.110521 Received 24 June 2019; Received in revised form 20 August 2019; Accepted 21 September 2019 Available online 23 September 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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structure of the nanotube arrays were observed by scanning electron microscope (SEM, FEI Quanta250) after sprayed with a gold layer. The surface element concentration of the sample was analyzed by EDS (IMA X-Max 20, Britain). X-ray Diffraction (XRD, Switzerland, SCINTXTRA) was used to examine the surface crystal structure before and after annealing treatment, and the scan scope ranged from 15° to 85°. In order to further clarify the surface chemical structure of the original titanium and titanium dioxide nanotubes, the surface of different samples was characterized by X-ray photoelectron spectroscopy (XPS, VG Science, East Grinstead, UK). Water contact angle measurement (DSA25, KrüssGmbH, Germany) was carried out to characterize the surface hydrophilicity. The water contact angle test was done by the sessile drop method at room temperature using the distilled water. Five parallel samples were measured and the values were averaged.
surface area, controllable nanotube diameter and length [9,10], which can provide an excellent platform for the functionalization and then allow more cells to adhere and proliferate on the surface [11–13], leading to better biocompatibility. TNTAs are the excellent candidate for the bone-substituted materials because they can promote the preferential growth of bone tissue and thus make the connections between implant and bone more stable [14]. Moreover, the TNTAs can promote differentiation and proliferation of bone marrow mesenchymal stem cells, osteoblasts and chondrocytes, and greatly improve the integration ability between the implants and the surrounding bone tissues [15]. On the other hand, the unique hollow nanotube structure can be loaded with a variety of drugs, bioactive substances or nanoparticles to improve the biocompatibility and antibacterial properties of the vascular implants [16–19]. Although there are many reports on TNTAs, most of them focus on the bone biomaterials and there are few reports concerning the effects of the nanotube diameter and its crystal structure on the blood compatibility and endothelial cell behaviors. Moreover, there is still much controversy about the blood compatibility of TNTAs. Some studies have found that the surface of TNTAs hardly causes blood reaction, while others have found that TNTAS can cause thrombosis [20]. Therefore, it is necessary to further clarify and explore how the diameter and crystal structure of the TNTAs affect the hemocompatibility and endothelial cell behaviors. At the same time, protein adsorption on the surface is also an important factor affecting the cell compatibility and blood compatibility of TNTAs. The adsorbed extracellular matrix proteins can enhance the adhesion and proliferation of endothelial cells [21]. However, a large number of coagulation factors can be released after the fibrinogen adsorption, leading to fibrin coagulation and platelet aggregation [22]. Therefore, it is also urgent to explore and clarify the protein adsorption behaviors of TNTAs with the different structures and crystals. In order to elucidate the effects of the structural characteristics of TNTAs, such as nanotube diameter, nanotube length and crystal structure, on the blood compatibility and endothelial cell behaviors, an anodic oxidation method was used to in situ prepare a dense array of TiO2 nanotubes on the titanium surface. The diameters and lengths of the nanotube arrays were controlled by changing the oxidation voltages, and the crystal structure was changed by annealing treatment. The effects of the diameters and crystal structures of the nanotube array on the physical and chemical properties and biocompatibility were investigated.
2.3. Protein adsorption The adsorption behaviors of two major plasma proteins (fibrinogen and albumin) were measured by the BCA method. The samples were firstly immersed in ethanol for 30 min and then in PBS (phosphate buffer) for 10 h. Thereafter, the samples were separately placed in 1 mg/ml of BSA (albumin) and 1 mg/ml FIB (fibrinogen) solutions for adsorbing 2 h at 37℃. The samples were washed twice with PBS and then desorbed ultrasonically for 30 min in 2 ml SDS (sodium dodecyl sulfate, 1% wt). Taking 150 μl eluent and 150 μl BCA working solution to mix for reacting one hour at 60℃, and then 200 μl of the reaction liquid was put into a 96-well plate. The absorbance at 562 nm was measured by a Microplate Reader (Bio-Tek, Eons), and the adsorption capacity of the protein was calculated by the standard curve. 2.4. Blood compatibility 2.4.1. Platelet adhesion Platelet-rich plasma (PRP) was obtained by centrifuging fresh human whole blood of a healthy volunteer at 1500 rpm for 15 min. 200 μL PRP was dropped on each sample surface to cover the whole surface. After incubating at 37℃ for 2 h, the samples were washed with PBS. The adhered platelets were fixed with 2.5% glutaraldehyde (in PBS) for 24 h and then washed with PBS. Finally, the samples were dehydrated by 30%, 50%, 75%, 90%, 100% ethanol solutions for 10 min each in sequence, followed by thoroughly dried in an air atmosphere. The samples were coated gold in vacuum and then observed by a scanning electron microscopy (SEM, FEI Quanta 250). The images with large magnification were used to analysis platelet morphologies. Five images with small magnifications for the each sample were randomly selected to calculate the number of the platelets adhered to the surface, and the values were averaged.
2. Materials and methods 2.1. Preparation of titanium dioxide nanotube arrays The pure titanium (TA2, purity 99.6%) bar with a diameter of 15 mm was cut into 2 mm thick plates. The plates were successively polished by silicon carbide papers from 400 to 2000 mesh and then ultrasonically cleaned by acetone, ethanol and distilled water for 10 min, respectively. After dried by the compressed air flow, the samples were immersed in 50 ml electrolyte (ethylene glycol solution containing 0.25% wt NH4F and 6 ml of deionized water) for anodization for 3 h, and the anodic voltage was controlled at 30 V, 40 V, 50 V, and 60 V, respectively. After anodization, the sample was ultrasonically washed with ethylene glycol for 30 min, and then washed with alcohol for 5 min. The original titanium plates and the samples obtained by the anodization were named as Ti, TNTAs-30, TNTAs-40, TNTAs-50, and TNTAs-60. The obtained samples were further treated in an air atmosphere at 500℃ for 3 h, and then air-cooled. They were named as Ti-A, TNTAs-30A, TNTAs-40A, TNTAs-50A, and TNTAs-60A.
2.4.2. Hemolysis rate The hemolysis assay was done in accordance with ISO 109934:2009. The fresh human blood was centrifuged for 10 min at 1500 rev/ min, and the supernatant was removed. The precipitated red blood cells were washed three times with PBS until the supernatant did not show red. The resulting red blood cells were mixed with PBS to obtain a 2% suspension (2 mL red blood cells plus physiological saline to 100 mL). The test samples were immersed into the above diluted suspension solution, and incubated in 37℃ water bath for 1 h. The solution was centrifuged at 3000 rpm for 5 min, and the supernatant was added into a 96-well plate. The absorbance value (A) was measured at 450 nm by a microplate reader. For controls, the mixture of 2 mL erythrocyte and 98 mL physiological saline was used as the negative control, and the mixture of 2 mL erythrocyte and 98 mL ultrapure water was used as the positive control. The hemolysis ratio was calculated according to the following formula:
2.2. Surface characterization
HR(%)=(A－A1)/(A2－A1)×100%
The surface morphologies, cross-sectional morphologies and tubular 2
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structure of the nanotube arrays and new crystalline phases appeared. The formation of new phase has important influences on the properties and functions of titanium [24]. According to the results calculated by jade software, the contents of anatase on the surfaces of TNTAs were about 100% (30 V), 86% (40 V), 73% (50 V), 65% (60 V), indicating that not all TNTAs can be fully changed into anatase phase by annealing treatment. As shown in Fig. 2, the TNTAs layer became thicker with the increase of the anodization voltage, when the same annealing time was applied, the relative thin TNTAs layer, such as TNTAs-30, can be completely converted into anatase while the thicker TNTAs cannot be fully changed into anatase crystal. The chemical composition of the modified surface was further studied by X-ray photoelectron spectroscopy (XPS). Fig. 1b shows the typical XPS survey spectra of the original and modified titanium surfaces. The original titanium substrate showed Ti2p, O1s and other weaker elements, indicating that the titanium sample itself had been oxidized naturally and received some pollution. After anodic oxidation, the peak value of O1s increased significantly, and concurrently F1s also appeared, indicating that an oxide layer containing F element appeared on the surface. After annealing, F element disappeared on the TNTAs surface because F exists only in fluoride on the surface and it can be completely removed by heat treatment [25]. Fig. 1c and d show the high resolution spectra of Ti2p peak for the pristine titanium and TNTAs-60A. It can be seen that the pure titanium Ti2p (Fig.1c) can be divided into four peaks: Ti (454.88 eV), Ti-N (455.81 eV), O-Ti-O (458.78 eV), O-Ti-Ca (457.48 eV) [26]. They accounted for 62.35%, 18.04%, 16.56% and 3.05% respectively. Because titanium plates were exposed to air for a long time, which resulted in an oxide layer naturally. Ti2p (Fig.1d) of TNTAs-60A can be divided into two main peaks: O-Ti-O (459.87 eV) and O-Ti-Ca (457.48 eV) [26]. The binding energy value of O-Ti-O peak was slightly higher than that of pure titanium, and the proportion of OTi-O increased from 18.04% to 91.32%, suggesting that the anatase titanium structure was indeed prepared on the titanium surface.
Where HR is the hemolysis rate (%), A is the absorbance of the test sample (%), A1 is the negative control absorbance (%), A2 is the positive control absorbance (%). 2.5. Endothelial cell adhesion and proliferation The samples were first sterilized by UV light and placed in a 24-well cell culture plate. Subsequently, 1.5 mL of 5 × 104 cells/mL endothelial cell (HUVEC, ECV304, Cobioer, Nanjing, China) suspension was seeded on the substrate and incubated 1 and 3 d at 37℃, respectively. The incubation was carried out in a humidified atmosphere containing 5% CO2. The cell culture medium was DMEM/F-12 (Hyclone) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. After the predetermined incubation time, the samples were washed 3 times with PBS. The cells were stained successively by rhodamine and 4′,6-diamidino-2-phenylindole (DAPI). 200 μl rhodamine (1:1000 in PBS) was added to each sample surface for incubating 20 min, and then the sample was washed 3 times with PBS. Finally, 200 μl of DAPI (1:400 in PBS) was added to sample surface for 3 min, and the sample was washed 3 times with PBS. The stained sample was observed by fluorescence microscopy (Zeiss, inverted A2). At the same time, another group samples were sequentially dehydrated 10 min by 30%, 50%, 70%, 90%, 100% ethanol solutions for each sample. The adherent cells were observed by scanning electron microscopy (SEM, FEI Quata 250) after sputter-coated a gold layer. For cell proliferation, endothelial cells were cultured in the same way. After cultured with sample for 1, 3 days, respectively, 0.5 ml of cck-8 solution (cck-8: cell culture solution = 9:1) were added on each sample, incubated 3.5 h at 37 °C. The absorbance of the medium was measured by a microplate reader (Bio-Tek Eons) at 450 nm. Triplicate parallel samples were measured and the values were averaged for each sample. 3. Results and discussion
3.2. Surface morphologies and elemental analysis 3.1. XRD and XPS Fig. 2 shows the typical SEM images of the different samples which were anodized at different voltages. It can be clearly seen that a compact and regular hollow nanotube array structure was formed on the surface after anodic oxidation. The diameters of the nanotube arrays were about 30 ± 2 nm (30 V), 50 ± 5 nm (40 V), 70 ± 8 nm (50 V), 90 ± 7 nm (60 V). According to the mechanism of anodic oxidation, the tubular structure was produced by the corrosion from the surface to the interior. More F− ions could accumulate on the surface during anodic oxidation when larger anodization voltage was applied, leading to relative serious corrosion and larger diameter nanotube. At the same time, the corrosion rate increased with the increase of voltage, therefore the length of nanotubes increased with the increase of voltage when the same oxidation time was applied. As can be seen from the cross-sectional view of the nanotube arrays (Fig. 2 a3～d3), the length of the nanotube array can reach about 5 μm, 7 μm, 15 μm, and 22 μm respectively. Therefore, the higher the voltage, the thicker the anodized film formed on the surface. The anodized nanotube array was further subjected to a high-temperature at 500℃, the nanotube was expanded outward by about 5～10 nm, and the tube wall was thickened by 1～ 2 nm. However, annealing treatment had no influence on the length of the nanotubes. It was presumably considered that the larger volume of anatase TiO2 after annealing treatment cause the nanotubes to expand outward, and the anatase TiO2 gradually merged into the nanotube wall by forming larger crystallites, resulting in the enlargement of the nanotubular structure and rougher surface [27]. Although a small portion of the surface nanotubes were damaged, the length of the nanotubes did not change significantly. Table 1 shows the elemental content of the different samples measured by EDS. It can be seen that the surfaces of anodized TNTAs samples mainly consisted of three elements of Ti, O and F. The surface
Titanium and its alloys are widely used in the blood contact materials and devices, such as thrombosis filter and artificial heart valves. It has been reported that the biocompatibility of titanium, especially blood compatibility, is related to the thickness of the oxide layer and the crystal structure. Anodization can produce an oxide layer with nanotube array structure on the titanium surface, and annealing treatment can further change the crystal structure of the nanotube arrays. In the present study, XRD was firstly used to characterize the surface crystal structure changes of the anodized titanium before and after annealing treatment. Fig. 1a shows the XRD patterns of the different samples. It can be clearly seen that no new diffraction peaks besides the titanium peaks can be detected on the pristine titanium surface after annealing treatment at 500℃, indicating that the crystal structure of the titanium surface had hardly changed. Compared with pure titanium, there was almost no change in the diffraction pattern after anodization and only peaks associated with the Ti substrate can be found, indicating that no new crystalline phase was formed during the anodization process. It has been reported that the oxide barrier layer on the titanium surface is semi-crystalline, while the synthesized TNTAs are amorphous [23]. As can be seen from Fig. 1a, with the increase of anodic oxidation voltage, the intensity of Ti diffraction peaks increased. It was considered that the diameter and length of the amorphous TNTAs increased with the increase of the voltage (as shown in Fig. 2), and the size of the amorphous TNTAs became larger, resulting in an increase in the diffraction peak. After annealing treatment at 500℃, besides the diffraction peaks of Ti, the (101), (103), (004), (112), (211), (204) diffraction peaks of anatase-type titanium dioxide appeared at 2θ = 25.37, 37.03, 37.88, 38.61, 55.10 and 62.74, respectively, demonstrating that annealing treatment changed the surface crystal 3
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Fig. 1. The XRD spectra of the different samples (a), (b) the XPS survey spectra of Ti and TNTAs samples (b), the high-resolution XPS spectra of Ti2p in Ti (c) and TNTAs-60A (d).
of the TNTAs sample mainly formed TiO2 oxide film layer with a small amount of F element attached to the surface. The reason for the existence of F element is that in the process of anodic oxidation, F− in the electrolyte forms (NH4)2TiF6 on the surface by etching the TiO2 oxide layer [11]. As the voltage increased, the content of F element basically showed an upward trend. The higher the voltage, the more F− formed by the anode, and thus the more fluorides introduced. In the process of annealing treatment, NH4F can be generated from (NH4)2TiF6 on the surface of TNTAs, which resulted in the decrease of F element, and consequently changed the contents of Ti and O elements on the TNTAs surface. The ratio of Ti and O elements in all TNTAs samples was close to 3:2, suggesting that the oxide film formed on the surface was mainly TiO2.
the anodization, which can form hydrogen bond with water and thus enhanced the hydrophilicity. With the increase of anodic oxidation voltage, the hydrophilicity was enhanced, and even super hydrophilicity can be achieved, for example, the water contact angle of TNTAs-60A was below 5 degrees. It is well known that the hydrophilicity of the material depends both surface chemical structure and surface morphologies. As can be seen from Fig. 2, with the increase of voltage, the diameter of nanotube arrays became larger and the surface became rougher. The porous/rough titanium dioxide structure film inhales water through capillary force, thus making the substrate exhibit better hydrophilic properties [31,32]. The hydrophilicity of the anodized nanotube arrays increased significantly after the annealing treatment, and the water contact angle was reduced to less than 5 degrees, indicating that the material displayed super hydrophilic properties. It was considered that the increased diameter and larger roughness of TNTAs after annealing treatment contributed to the improved hydrophilicity. In addition, it can be seen from Table 1 that the surfaces of the anodized samples contains hydrophobic fluorine elements. After annealing treatment, the surface fluorine element almost disappeared, therefore its hydrophilicity was further improved. At the same time, the amorphous part of TiO2 was transformed into anatase structure after annealing treatment at 500℃. The anatase type of TNTAs had better hydrophilicity than amorphous TNTAs [33]. The protein adsorption was further investigated by BCA method. Generally speaking, when the implant material is in contact with human blood, what occurs first is the interaction between the plasma proteins and the materials. The types and properties of plasma protein adsorbed on the surface play an important role in the subsequent blood reactions and cell behaviors. There are two main proteins in human blood, albumin and fibrinogen. Generally speaking, when biomaterials come into contact with human blood, albumin and fibrinogen compete to adsorb on the surface. The albumin adsorption can reduce the platelets adhesion and activation, and therefore inhibit the occurrence of blood coagulation and prolong the coagulation time [34], leading to better hemocompatibility. In contrast, the adsorption and denaturation of FIB could deteriorate the blood compatibility of materials. In the
3.3. Surface hydrophilicity and protein adsorption In general, hydrophilic surfaces can adsorb large amounts of water molecules, thereby reducing non-specific protein adsorption and further preventing platelet adhesion. For titanium oxide nanotubes arrays, it has been reported that it can prevent fibrinogen adsorption and denaturation [28], therefore improve blood compatibility. On the other hand, the hydrophilic surfaces have shown to activate plasma coagulation [29] in some cases, which can deposit fibrin onto the surfaces to which platelets have numerous membrane-bound receptors [30]. This kind of inter-relationship between coagulation and platelet adhesion greatly complicates TNTAs structure-property relationships. In order to clarify the relationship between the surface properties of TNTAs and the biocompatibility, the surface wettability was studied by measuring the water contact angle. Fig. 3a shows the water contact angle results of the different samples. It can be clearly seen that the hydrophilicity was significantly enhanced after the preparation of the TNTAs on the Ti surface, and the hydrophilicity was further improved by annealing treatment. A hollow nano-tubular structure was in situ produced on the pristine titanium surface by anodization, which can accommodate water into the inner part of the tube. At the same time, a large amount of oxygen was introduced on the surface and inside the nanotube after 4
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Fig. 2. The typical SEM images of TNTAs on Ti surface. a1～d1 shows the TNTAs anodized at 30 V (a1), 40 V (b1), 50 V (c1), 60 V (d1). a2～d2 were the corresponding SEM images after annealing heating and a3～d3 shows the cross sectional morphologies of a1～d1.
specific surface area and thus have a larger contact area with protein. Therefore, with the increase of the nanotube diameter, the amount of BSA adsorbed on the surface decreased. At the same time, as shown in Fig. 3a, the nanotubes arrays with larger diameter displayed better hydrophilicity, which can also inhibit the non-specific BSA adsorption [35] to some degree. In addition to the specific surface area and hydrophilicity, protein adsorption also depends on the surface charge properties of the materials and protein, which may promote or inhibit protein adsorption by the electrostatic interaction [36]. It was reported that the TNTAs by anodization on the titanium had negative charges [37]. The relative larger specific surface area of TNTAs with smaller diameter had more negative charges, which can promote positive charged BSA adsorption. After annealing treatment, due to the increased roughness and the occurrence of anatase crystal structure, the BSA adsorption on all samples surfaces increased to different degrees. Similarly, the amount of BSA adsorbed on the anatase TNTAs-30A surface was the most due to the electrostatic adsorption. It is well known that the crystallinity of the TNTAs is closely related to the adsorption of BSA. Different crystal structures can lead to changes in the surface properties, which further influences BSA adsorption [38]. After
Table 1 Atomic content of surface elements on Ti and TNTAs samples. Sample
Ti TNTAs-30 TNTAs-40 TNTAs-50 TNTAs-60 TNTAs-30A TNTAs-40A TNTAs-50A TNTAs-60A
Atomic concentration (at.%) Ti
O
F
100 56.12 54.91 57.47 54.39 61.39 60.27 59.92 61.55
0 34.55 35.17 32.16 32.84 38.02 39.12 38.87 36.89
0 9.33 9.92 10.37 10.77 0.58 0.90 1.21 1.56
present study, BSA and FIB adsorption on the different samples were investigated, and the results are shown in Fig. 3b and c. It can be seen that the BSA adsorption on the TNTAs surface increased obviously as compared to the pristine titanium, suggesting that preparation of TNTAs can promote albumin adsorption, which could lead to better blood compatibility. The TNTAs with smaller diameter have the larger 5
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Fig. 3. The water contact angle (a), the amounts of BSA (b) and FIB (c) adsorption of the different samples. Data for each sample were taken from three different substrates, after which they were expressed as mean ± SD.
Taking the above results into consideration, it can be seen that the nanotube arrays with the different diameters and lengths were produced successfully on the titanium surface by anodic oxidation. Compared with bare titanium, the substrates with nanotube arrays can promote BSA adsorption and prevent FIB adsorption. Therefore, the nanotube arrays showed the characteristics of selective albumin adsorption. The tube dimension, array thickness, and crystal structure had important effects on the protein adsorption behaviors. The increase of the nanotube diameter could lead to a slight decrease in the BSA adsorption and an increase of FIB adsorption to some extent. The anatase structure of TNTAs could help improving the protein adsorption selectivity of the nanotube arrays.
annealing treatment, amorphous TNTAs were partially transformed into anatase structure, which changed the charge properties of TNTAs, resulting in an increase in BSA adsorption. The fibrinogen adsorption is closely related to the platelets adhesion and the cell adhesion and proliferation. The adsorption of fibrinogen can easily cause a large number of platelets to adhere to the material surface, release a large amount of blood coagulation factors, and further induce thrombosis [39]. In this study, the FIB adsorption behaviors on the different TNTAs surfaces were further studied. Fig. 3c shows the FIB adsorption concentration on the different sample surfaces. It can be seen that FIB content adsorbed on the bare titanium surface was the highest among all samples. After the formation of nanotube arrays by anodic oxidation, the FIB adsorption concentration on the surface decreased significantly. Studies have shown that FIB will preferentially be adsorbed on the hydrophobic surface [40]. The surface of the amorphous TNTAs was hydrophilic as compared to bare titanium, which resulted in less FIB adsorption on the TNTAs surface. With the increase of the amorphous TNTAs diameter, the FIB adsorption increased due to the increased hydrophilicity (As shown in Fig. 3a). Meanwhile, it has been reported that the FIB adsorption capacity increased with the increase of the oxide layer thickness [41]. The length of nanotube arrays increased with the increase of anodic oxidation voltage, which represented the increased thickness of the TNTAs, and therefore the TNTAs with larger lengths can promote FIB adsorption. It was presumably concluded that the influence of the amorphous TNTAs length was much greater than that of hydrophobicity, leading to the increased FIB adsorption with the increase of nanotube array diameter. After annealing treatment, the surface roughness and hydrophilicity of the substrates was further improved due to the formation of anatase-type TNTAs, therefore the amount of FIB adsorbed on the surface decreased as compared to the anodized samples. The FIB adsorption capacity on the anatase TNTAs surface decreased with the increase of tube diameter and length. It was speculated that the electronic band gap configuration of anatase TNTAs reduced the tension between the surface and FIB, resulting in less FIB adsorption [42]. The smaller diameter nanotubes may exhibit smaller tension between the surface and the FIB, and thus less FIB was adsorbed.
3.4. Blood compatibility Platelet adhesion and activation have been considered as a major cause of thrombosis. Early inflammation and thrombosis of the vascular implants are associated with platelet activation. The adherent platelets can promote aggregation with other platelets, and release platelet and pro-coagulant agonists [43]. Therefore, platelet adhesion represents one of the main methods to explore the blood compatibility of biomaterials. Fig. 4a and b are the typical SEM image of platelets adhered on the different samples surfaces, and Fig. 4 c shows the number of platelets adhered on the different samples surfaces. It can be clearly seen that more platelets were attached on the titanium surface, and the adhered platelets on the bare titanium surface not only extended pseudopodia, but also some platelets had been fully activated (the inset in Fig. 4a-a1), indicating that the pristine titanium had limited anticoagulation. Previous studies and our results (Fig. 3) shows that the hydrophobic surface can adsorb more fibrin [44,45], which can deteriorate the blood compatibility. After the anodization, a regular nanotube array structure was produced on the surface. As mentioned above, the fibrinogen adsorption on the nanotube array surfaces can be significantly reduced (Fig.3c), which would help inhibit the conversion of fibrinogen to fibrin, prevent fibrin from clotting into blocks, thereby reducing the chance of thrombosis [46]. Therefore, the platelets adhered to the TNTAs surface were much less than that of the pristine 6
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Fig. 4. The SEM images of platelets adhered on the surface of Ti and TNTAs samples before (a) and after annealing treatment (b). (a1: Ti; a2: TNTAs-30; a3: TNTAs40; a4: TNTAs-50; a5: TNTAs-60; b1: Ti-A; b2: TNTAs-30A; b3: TNTAs-40A; b4: TNTAs-50A;b5: TNTAs-60A). The inset images shows the typical single platelet morphology on the corresponding substrates. (c) The number of adhered platelets on different samples. Five parallel samples were measured and the results were expressed as mean ± SD. (d) The hemolysis rate of Ti and TNTAs samples.
Red blood cells are one of the main components in human blood. When blood contacts with biomaterials, the red cells may be destroyed and the red blood protein is released, resulting in occurrence of hemolysis and further causing blood clotting [50]. Hemolysis rate is one of the important indicators to characterize the interaction between biomaterials and red blood cells. According to the international ISO10993-4 standard, if the hemolysis rate (HR) of the material is less than 5%, the material meets the requirements of hemolysis rate. If the HR of the material is more than 5%, the material has a hemolysis effect and is not suitable for use as a blood contact material. In order to further investigate the relationship between nanotube array structure and blood compatibility, the hemolysis assay was further carried out. Fig. 4d shows the hemolysis rate of the different materials. It can be seen that the hemolysis rate of all samples was less than 5%, indicating that the hemolysis did not occur on all samples. Compared with the pure titanium, the hemolysis rate of the material after the anodization decreased significantly, indicating that the nanotube arrays on the surface can further significantly improve the blood compatibility. However, although the hemolysis rate increased with the increase of tube diameter, it was still significantly less than the hemolysis rate of the control titanium. After annealing treatment at 500℃, the hemolysis rate of each sample decreased slightly. The hemolysis rate of TNTAs30A was the lowest, indicating that it had the best blood compatibility.
titanium. As the diameter of nanotube increased, more and more platelets adhered to the amorphous TNTAs surfaces, and the spreading state became more obvious. It was presumably due to the fact that the amorphous TNTAs with the larger diameter increased platelet adhesion by adsorbing more fibrin. In general, rapid adsorption of plasma proteins on the outer surface of the sample results in platelet adhesion [47]. It was also possible that fluoride ions in the electrolyte doped into TNTAs during anodic oxidation induced platelet adhesion and activation. The larger diameter TNTAs with the longer nanotube length are capable of adsorbing more fibrin, thereby increasing platelet adhesion [48]. It can be seen from the above elemental distribution results (Table 1) that the nanotube arrays with larger diameter had the more fluoride ions on the surface, which may arouse more aggregation of platelets. The number of platelets adhered on the titanium surface decreased slightly after annealing treatment at 500℃, however, the activation status of platelets did not improve (the inset of Fig.4b-b1).The number of platelets adhered on the TNTAs surface was significantly reduced after the annealing treatment, and the activation of platelets was also inhibited. The anatase type TNTAs-30A had the best blood compatibility according to the platelet adhesion. Moreover, the platelets adhered on the anatase-type TNTAs surface were rounded, and they can keep intact morphologies and may return to the blood circulation. The anticoagulant of anatase TNTAs was much better than that of the amorphous TNTAs because the former surface was more hydrophilic than the latter, which could prevent non-specific adsorption (such as fibrinogen) and reduce platelet adhesion and activation by increasing the hydration layer [49]. The anatase-type TNTAs-30A surface adsorbed the least FIB, and the ability to induce platelet aggregation and activation was relatively poor. Moreover, the BSA adsorbed on the surface of TNTAs-30A was also the most, which was helpful for inhibiting the platelet adhesion and reducing the probability of thrombosis.
3.5. Endothelial cell adhesion and proliferation Generally speaking, the cells will change their morphologies to achieve the integration of material and cells after contacting biomaterials. The entire progress of cell adhesion and spreading consists of cell attachment, filopodia growth, cytoplasmic webbing, flattening of the cell mass and the ruffling of peripheral cytoplasm [51]. Cell adhesion is widely used to study cellular behaviors on the biomaterials 7
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Fig. 5. The typical fluorescent images of endothelial cells adhered on the surface of Ti and TNTAs samples.
Fig. 6. The typical SEM images of endothelial cells adhered on the surface of Ti and TNTAs samples.
surfaces. In this study, cell adhesion behavior was firstly studied by cell fluorescence staining, and the morphologies of adherent cells were further observed by SEM. Fig. 5 and Fig. 6 shows the typical fluorescent images and SEM images of endothelial cells attached on the different samples surfaces, respectively. It can be seen that the number of endothelial cells attached on the bare titanium surface was relatively small (Fig. 5), and the attached endothelial cells mainly exhibited
round shape, indicating that almost all cells showed limited spreading (Fig. 6). However, after annealing treatment at 500℃, the number of round cells on the surface decreased significantly (Fig. 5), and more cells displayed spreading state (Fig. 6). After anodic oxidation, the number of endothelial cells increased (Fig. 5), suggesting that the nanotube arrays can enhance cell adhesion. With the increase of the nanotube diameter, the number of the adherent cells on the surface was 8
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more endothelial cells can be attached on the surface for proliferation. The absorbance of the endothelial cells attached on the pure titanium surface and amorphous TNTAs for 1, 3 day increased significantly after annealing treatment. Usually, endothelial cells are easy to attach on the hydrophilic surfaces than on the hydrophobic surfaces. After annealing, the surface of TNTAs samples showed super hydrophilicity, therefore the proliferation of endothelial cells on the TNTAs-A surface was more obvious. Higher concentration of fibrin adsorption can prevent the expansion of protein and reduce cell adhesion [57]. The surface of TNTAs-30A with smaller diameter can adsorb less FIB, so the amount of endothelial cells on the surface was more than that of other samples.
reduced (Fig. 5), and the morphologies of the cells was similar to that of on the pure titanium surface after annealing treatment (Fig. 6). Studies have shown that the fate of cells in the nanotube layer is determined by the microenvironment, and 15–30 nm is the optimal diameter for cell adhesion, migration and differentiation [52]. When the diameter exceeds 100 nm, cell adhesion and proliferation hardly occur, and even induce cell apoptosis [50]. The circular shape of nanotubes can promote cell formation, cell differentiation and proliferation [53]. The larger diameter of nanotubes could force cells to grow outward and find the substrate for the protein deposition, which can greatly affect the cell adhesion performance [54]. Therefore, the number of endothelial cells adhered to the surface of TNTAs-30 was the largest. After annealing treatment, the endothelial cells adhesion on the TNTAs surface increased significantly (Fig. 5). Similarly, anatase TNTAs-30A had the best cell adhesion, and the cell morphologies was fully spread out (Fig. 6). It not only showed polygonal shape, but also has many fine villi formed by cytoplasmic processes on the surface (Fig. 6). Some studies have shown that the hydrophilic sample surface can improve cell adhesion and proliferation by exchanging and adsorbing extracellular matrix proteins [55]. Therefore, TNTAs-A with better hydrophilicity can adhere more cells and the cells had better spreading morphologies. CCK-8 was carried out to further investigate the cell proliferation. Fig. 7 shows the CCK-8 values of the endothelial cells on the different samples. It can be clearly seen that the absorbance of the endothelial cells cultured on the surface of bare titanium and amorphous TNTAs for one day had no obvious difference, however the proliferation of the endothelial cells on the amorphous TNTAs with smaller diameter surface was the best. Studies have shown that the diameter and spacing of the nanotubes are important factors affecting cell acquisition of protein, ions and nutrients [56]. Endothelial cells tend to adhere to the smaller diameter nanotubes, leaving more growth factors for cell proliferation. Therefore, the proliferation of endothelial cells for nanotubes with smaller diameter was better. Our results suggested that TNTAs-30 had the best suitable diameter for endothelial cell adhesion, and it also displayed an excellent proliferation performance. At the same time, cells can adhere to these functional surfaces through non-receptor binding forces (electrostatic interactions), leading to the improved cell proliferation. TNTAs have negative charges on the surface, which can promote cell adhesion and proliferation. Due to the fact that the TNTAs with the smaller diameter had more negative charges on the surface,
4. Conclusion In this study, orderly and regular titanium dioxide nanotube arrays were successfully prepared on the titanium surface by the anodic oxidation, and the anatase nanotube arrays were obtained by the annealing treatment. The diameter, length and crystal structure of the nanotube arrays significantly affected the surface wettability, protein adsorption, blood compatibility as well as endothelial cell adhesion and proliferation. TNTAs prepared by anodic oxidation had good hydrophilicity, and the hydrophilicity was further enhanced by annealing treatment. TNTAs showed the characteristics of selective adsorption of BSA. The increase of nanotube diameter could lead to a slight decrease in BSA adsorption, but it could promote FIB adsorption to some extent. The anatase structure would improve the protein adsorption selectivity of nanotube arrays. Therefore, the surface of TNTAs samples with smaller diameter (e.g., TNTAs-30A) can promote BSA adsorption and reduce FIB adsorption, thereby improve the blood compatibility and endothelial cell adhesion and proliferation. The results demonstrated that the nanotube arrays prepared on the titanium surface could not only improve the blood compatibility of titanium, but also promote the adhesion and proliferation of endothelial cells. Therefore, the method of the present study can be used for the surface modification of blood contacting biomaterials to simultaneously improve the blood compatibility and cell compatibility of vascular endothelial cells. Acknowledgements This work is financially supported by the Natural Science
Fig. 7. The results of CCK-8 detection after 1, 3d growth of endothelial cells on Ti and TNTAs samples. 9
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Foundation of China (31870952), Natural Science Foundation of Jiangsu Province of China (BK20181480), the International S&T Cooperation Projects of Huai’an City of China (HAC201703), The Key Program for Natural Science Foundation of the Jiangsu Higher Education Institutions of China (17KJA530002), Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX18-0348).
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