3.1 Surface characterization
Figure 1 shows the representative SEM images of the titanium dioxide nanotubes modified by the different bioactive factors. It is obvious that after different surface modification processes, the surface nanotube structure remains intact. Compared with TNT-Dopa, the surface immobilization of PAA and the loading of GS and Zn2+ gradually reduced the diameter of the nanotube and increased the thickness of the tube wall. Furthermore, the chemical group changes on the surface were examined by ATR-FTIR. It can be seen from Fig. 2a that there was almost no infrared absorption on the unmodified titanium surface. Our previous work showed that the anodized TiO2 nanotubes have a small amount of hydroxyl on the surface [18], but it cannot be directly used to immobilize the bioactive molecules. To solve this problem, a polydopamine coating was prepared on the surface of TiO2 nanotubes, followed by the grafting of polyethyleneimine (PEI) to create a positively charged surface, PAA with negative charges was then immobilized on the surface by electrostatic interaction to construct a negatively charged surface. Finally, in order to improve the biocompatibility and antibacterial properties, gentamicin and zinc ions were introduced on the PAA-modified surface by layer-by-layer technique and ion chelation, respectively. The results of Fig. 2a show that the stretching vibration and in-plane bending vibration of-NH bond and -OH bond appeared on TNT-Dopa surface at 1590 cm-1 and 3300 cm-1, and the stretching vibration of -OH bond occurred around 3700 cm-1, indicating that the polydopamine coating had been successfully prepared on the surface. After grafting of PEI, the in-plane bending vibration and stretching vibration of -CH2 bond can be observed at 1462 cm-1 and 2832 cm-1, and the bending vibration of -NH bond of primary amine and secondary amine and the stretching vibration of C-N bond can be detected at 1200 cm-1 and 1656 cm-1, indicating that PEI had successfully covalently linked with polydopamine coating. For TNT-PAA/GS, the stretching vibrations of C = O bond and -COOH bond appeared at 1519 cm-1 and 1694cm-1, respectively, suggesting that PAA and GS were successfully self-assembled onto the PEI-modified surface. In order to further clarify the surface element composition, the surface element composition of the modified sample was further analyzed by XPS. Fig. 2b is the XPS diagram of the different samples, and the element compositions are shown in Table 1, it can be seen that the characteristic peaks of C1s (285.2eV) and N1s (400.3eV) appeared on the TNT-Dopa. After the immobilization of PAA, the characteristic peak of O1s (531.8eV) increased obviously, concurrently the characteristic peak of C1s (285.2eV) and carbon content on the surface was reduced, indicating that PAA was successfully grafted onto the surface. For the TNT-PAA/GS, the increased nitrogen content indicated that GS was successfully loaded on the surface. The occurrence of Zn2p peak (1020.9eV) on TNT-PAA/GS-Zn proved that the Zn ions were successfully chelated to the surface.
Table 1 The surface element concentration of the different samples measured by XPS.
Sample
|
Atomic concentration(at.%)
|
Ti
|
O
|
C
|
N
|
Zn
|
Ti
|
61.12
|
29.86
|
9.02
|
0
|
0
|
TNT-Dopa
|
9.28
|
14.30
|
71.83
|
4.59
|
0
|
TNT-PAA
|
9.97
|
30.42
|
56.41
|
3.20
|
--
|
TNT-PAA/GS
|
3.27
|
31.77
|
56.20
|
8.76
|
--
|
TNT-PAA/GS-Zn
|
1.86
|
28.78
|
59.73
|
6.26
|
3.37
|
3.2 GS and Zn2+ release profile, surface hydrophilicity and protein adsorption
In order to characterize the release profile of gentamicin and Zn2+, TNT-PAA/GS-Zn was immersed in PBS solution for different times, and gentamicin and Zn ions were collected to measure the release kinetics curves. As can be seen from the release curve of Fig. 3a, both gentamicin and zinc ions released for more than 14 days. After immersed in PBS solution for 4 hours, the release concentration of gentamicin reached 3.9 μg/ml, and the total release concentration was 13.54 μg/ml at 14th days. Previous studies have shown that the working concentration of gentamicin is 4-20 μg/ml [27], therefore the continuous antibacterial activities can last at least 14 days. At the same time, it can also be seen from Fig. 3a that there was an obvious burst release period of one day for gentamicin and Zn2+. The release rate was relatively large before 1 day, and gentamicin reached 5.5 μg/ml, more than 40% of the total release in 14 days. After one day, the release rates of gentamicin and zinc ions became stable. After 14 days, the total concentration of zinc ion was 0.63 mg/l. It was reported that zinc ion concentration of 0.49-5.2mg/l can promote cell viability, proliferation, adhesion and migration, therefore the released Zn2+ content was within the range of cell physiological concentration.
Biomaterials should have good surface properties to avoid adverse host reactions after contact with organisms [28], in which wettability is an important factor affecting interface biological reactions [29]. The surface hydrophilicity/hydrophobicity is closely related to the protein adsorption and biocompatibility. Generally speaking, the good wettability is helpful to prevent the non-specific protein adhesion and promote the cell adhesion and proliferation [30]. As can be seen from Fig. 3b, the water contact angle of the blank titanium decreased obviously after anodizing, it was considered that the introduction of a large amount of oxygen elements and the formation of the special nano-porous structure can contribute to the excellent hydrophilicity. Due to the introduction of hydrophilic amine groups after the immobilization of dopamine, TNT-Dopa still had excellent hydrophilicity. However, after further the immobilization of PAA, the water contact angle increased significantly, this was mainly because the porous structure on the surface of the material was filled to some extent after the fixation of PEI and PAA (as shown in Fig. 1), which partially changed the surface morphology and made it difficult for water molecules to enter the interior of the nanotubes, so the contact angle increased. After further GS loading, the porous structure of the surface was further filled, and thus the water contact angle also increased although the hydrophilic carboxyl groups were introduced. Finally, because the positively charged zinc ions can chelate with the hydrophilic -COOH groups on the surface, combining with the further porous filling, the water contact angle increased further.
It is well known that protein adsorption is the first event when biomaterials contact blood, and it plays a decisive role in the blood compatibility [31]. Albumin and fibrinogen are the two main proteins in the blood. In general, albumin adsorption can reduce platelet adhesion. On the contrary, the adsorption of fibrinogen could increase the platelet adhesion and activation. Fig. 3c and d shows the adsorption concentrations of bovine serum albumin (BSA) and fibrinogen (Fib) on the different surfaces. As compared to the pristine titanium, the anodized titanium surface can enhance albumin adsorption, while the fibrinogen adsorption decreased to a certain extent, indicating that the anodized TiO2 nanotube array can selectively adsorb albumin. It was considered that the behaviors of protein adsorption were related to the surface wettability, surface morphologies and surface charges. The study showed that when the water contact angle is less than 110 °, fibrinogen would preferentially be adsorbed on the hydrophobic surface [32]. The increase of surface hydrophilicity after anodizing and a small amount of negatively charged hydroxyl groups on the surface were beneficial to the adsorption of positively charged albumin on the surface, but not conducive to the adsorption of negatively charged fibrinogen. The polydopamine have very strong stickiness to lysine-rich proteins [33], so the contents of BSA and fibrinogen adsorbed on TNT-Dopa surface increased significantly. It has been shown that the immobilization of polyacrylic acid on the surface can repel the non-specific protein adsorption because of the hydration layer formed by PAA and the negatively charged character of PAA [34]. Therefore, after the immobilization of PAA on TNT-Dopa, both BSA and fibrinogen adsorption decreased significantly. However, it was worth noting that BSA adsorption returned to the level of titanium oxide nanotubes, while fibrinogen adsorption was slightly higher than that of TNT. Furthermore, after loading GS by layer-by-layer technique, due to the further increase of hydrophobicity, the adsorption content of BSA protein did not change significantly, but the adsorption amount of fibrinogen decreased, indicating that with the increase of self-assembly layers, the introduction of a large number of PAA increased the content of negative charges on the surface, so that the negatively charged fibrinogen was not easily adsorbed on the PAA surface, thus reducing the fibrinogen adsorption. The zinc ions loaded on the surface can chelate with the carboxyl group of PAA to reduce the surface hydrophilicity, at the same time, zinc ion loading reduced the content of negative charges on the surface, so BSA adsorption increased slightly, while the adsorption capacity of fibrinogen further decreased.
3.5 Blood compatibility
Blood compatibility refers to the required response of blood to exogenous substances or materials, which generally refers to the compatibility between materials and various components of blood [35]. Generally speaking, blood compatibility includes three aspects: the interaction between materials and plasma proteins (such as albumin and fibrinogen), the interaction between materials and blood cells (red blood cells, white blood cells, platelets, etc.), and the interaction between materials and coagulation factors.
Platelets are one of the main components of human blood. Its main functions are clotting and hemostasis as well as repairing damaged blood vessels. The platelet adhesion to the biomaterials surface is a key event of coagulation. Platelet adhesion, aggregation and activation will promote blood coagulation. Therefore, the biomaterials with good blood compatibility should have the role of maintaining normal platelet physiological function, and can effectively prevent platelet adhesion, aggregation and activation [36]. At the same time, the increase of cGMP released from platelets can inhibit platelet activation [37]. In this paper, the adhesion and aggregation of platelets were observed by SEM, and the platelet activation was evaluated by measuring the cGMP. The results are shown in Fig. 4a, b and c. There were a large number of platelets adhered to the blank titanium surface, and the adhered platelets displayed spread state and extended pseudopodia, indicating that the platelets on the titanium surface may have been activated, the results of cGMP further proved this point. After anodization, the number of platelets adhered to the surface was significantly reduced. On the one hand, the anodized TiO2 nanotube arrays had excellent hydrophilic properties, which can reduce platelet adhesion and aggregation; on the other hand, the selective adsorption of albumin by TiO2 nanotube not only contributed to reduce platelet adhesion and aggregation, but also promote the expression of cGMP (Fig. 4c), leading to the improved blood compatibility. For TNT-Dopa, although its hydrophilicity was still excellent, compared with TNT, the number of platelets adhered to the surface was still significantly increased, while the expression of cGMP was also decreased, which could be due to the fact that the polydopamine coating had the ability of non-specific protein adsorption which can significantly increase the fibrinogen adhesion to the surface so as to enhance platelet adhesion and activation. Compared with the blank titanium and TNT, when the composite film of PAA and GS was prepared on the surface, the number of platelets on the surface decreased sharply, and the expression of cGMP also increased, indicating that the anticoagulation was enhanced. It was considered that PAA itself is a substance with good blood compatibility and can effectively reduce fibrinogen adsorption [38]. When zinc ions were loaded on the surface, the adsorption amount of fibrinogen further decreased, while the albumin adsorption increased, which not only reduced the adhesion and aggregation of platelets, but also increased the cGMP release and inhibited platelet activation. On the other hand, zinc ions can make platelets produce more NO signals which can inhibit platelet adhesion and aggregation by inhibiting thromboxane A2 (TXA-2) receptor [39]. Therefore, the loading of zinc ions on the surface further improved the blood compatibility.
The effects of the different materials on red blood cells were further studied. Hemolysis rate is one of the important methods to characterize the interaction between materials and red blood cells. According to the international ISO10993-4 standard, the hemolysis rate (HR) below 5% means that the material meets the requirements, on the contrary, the material with HR more than 5% is not suitable to be used as a blood contacting material. Fig. 4d shows the hemolysis rates of the different samples. The hemolysis rates of all samples were less than 5%, indicating that none of them could cause severe hemolysis. The hemolysis rate of TiO2 nanotubes was lower than that of pure titanium, but the hemolysis rate increased slightly after the preparation of polydopamine coating. When the composite film of PAA and GS was prepared and the zinc ion was loaded, the hemolysis rate further decreased significantly, indicating that PAA and zinc ions can improve the blood compatibility.
Generally speaking, when the biomaterial interacts with human blood, blood coagulation may happen. Blood coagulation is a complex chain process involving a series of stimulus responses in conjunction with coagulation factors and enzymes, whose intent is to stop blood fluxes when a vascular tissue injury occurs [40]. According to the difference of the initial pathway and participating factors, blood coagulation can be divided into two pathways: endogenous coagulation and exogenous coagulation. Among them, the endogenous coagulation pathway is initiated by the activation of factor XII [41], and the activated partial thromboplastin time (APTT) is an important method reflecting the endogenous coagulation pathway, especially the activity of coagulation factor XII [42]. Figure 4e shows the APTTs of the different samples. It can be seen that the clotting time of blank titanium was shorter than that of normal plasma, indicating that it may promote blood coagulation to a certain extent. However, the clotting time of the anodized titanium was longer than that of normal plasma, suggesting that the surface of TiO2 nanotube had good anticoagulation performance. Although the clotting time decreased after dopamine surface modification, the clotting time was significantly prolonged after the immobilization of PAA and the loading of GS and Zn2+, indicating that the subsequent surface modification improved the anticoagulant properties of the materials.
3.6 Endothelial cell growth behaviors
Fig. 5a and b shows the fluorescent images and the CCK-8 values of endothelial cells adhered to the different surfaces. It can be clearly seen that the number of cells adhered to the surface of the blank titanium was less than that of other modified samples. After anodizing, the number of adherent cells on the surface increased, and the morphologies of adherent cells displayed spread state, its proliferation was also improved (Fig. 5b). It can be concluded that the surface of pure titanium became more hydrophilic after anodizing, which can improve cell adhesion and proliferation on the surface through the exchange and adsorption of extracellular matrix proteins [43], moreover, the special nanostructure also contributed to cell adhesion and spreading. Dopamine is a chemical substance produced by the central nervous system of the human body. The polydopamine coating on the surface can promote the adhesion and proliferation of endothelial cells [44]. Therefore, the number of endothelial cells adhered to TNT-Dopa increased significantly, and the proliferation performance was further improved. After the preparation of the first PAA layer on PEI-modified TNT surface, due to the cytotoxicity of PEI and the hydrophobicity of PAA, the adhesion of cells to the material surface was reduced, and the morphologies of cells did not spread well, so the proliferation of endothelial cells decreased slightly. When PAA and GS were deposited alternately for 10 times, the thickness of the coating increased, which eliminated the effect of PEI on cells. Therefore, the number of endothelial cells attached on the surface increased again, and the spreading and proliferation of endothelial cells were better than TNT-Dopa. Zinc is an essential micronutrient for human health, and Zn2+ homeostasis in cells is essential for cell function and survival [45]. Zn2+ acts as the first or second messenger and is the signal pathway that triggers physiological functions. In our previous work, zinc ions were doped into TiO2 nanotubes by hydrothermal method. The results showed that the release of zinc ions not only increased the anticoagulant properties of the materials, but also promoted the adhesion and proliferation of endothelial cells [46]. In this study, the zinc ions were loaded on the TNT surface by chelation with PAA. The results of Fig. 5 showed that the release of zinc ions from the surface can significantly promote the adhesion and proliferation of the endothelial cells.
Furthermore, the function expression of endothelial cells was studied. Figure 5c shows the results of VEGF secretion of endothelial cells on the different samples. It can be seen that, compared with the blank titanium, anodization can promote the expression of VEGF in endothelial cells. When the polydopamine coating was prepared on the surface, due to the enhancement of the coating on cell adhesion and proliferation, endothelial cells could express more VEGF. For TNT-PAA and TNT-PAA/GS, the amount of VEGF decreased slightly as compared to TNT-Dopa, but it was not significant. After zinc ion loading, the content of VEGF increased significantly, indicating that the release of zinc ions can promote the up-regulated expression of VEGF in endothelial cells, which was conducive to maintaining the growth of endothelial cells and promoting the surface endothelialization of the implanted materials. Fig. 5d shows the NO secretion of endothelial cells grown on different samples. The NO content released by endothelial cells on the surface increased after anodizing. The NO content for TNT-Dopa was larger because the polydopamine coating can promote the growth of endothelial cells. After further preparation of PAA film and alternating deposition of PAA and GS, the growth state of cells was slightly worse (Fig. 5a). At the same time, the content of VEGF release could also indirectly affect the production of eNOS in endothelial cells, thus affecting the release of NO [47], therefore the NO content decreased slightly. However, it was worth noting that the loading zinc ions can significantly promote the activity of intracellular NOs enzyme and promote the release of NO, and thus enhance the NO expression of endothelial cells.
3.7 Antibacterial activities
Bacterial adhesion is widespread in nature, and there are usually two strategies of antibacterial strategies: killing bacteria and reducing bacterial adhesion [48]. In this study, gentamicin and zinc ions were loaded into titanium oxide nanotubes on the titanium surface to prevent infection. Gram-negative bacteria-Escherichia coli was used as a test strain to evaluate the antibacterial properties of the modified material surface. Fig. 6a shows the SEM images of bacterial adhesion on the different sample surfaces. It can be seen that although titanium had good biocompatibility to endothelial cells, its anti-bacterial adhesion property was poor, and there were a large number of Escherichia coli bacteria adhering to the surface. In contrast, the property of non-specific protein adsorption caused by the excellent hydrophilicity of the nanotubes and a small amount of negative charges on the surface could contributed to inhibit the adhesion of negatively charged Escherichia coli. As the same with cell results, polydopamine coating could make Escherichia coli easier to adhere to the surface, therefore, compared with TNT sample, the adsorption of bacteria increased on TNT-Dopa. When PAA was prepared on the surface, the amount of negative charges increased, which reduced the adhesion of Escherichia coli to the surface. Moreover, after the multilayer film of PAA and GS was prepared, the increased negative charges on the surface and the continuous GS release could further prevent the negatively charged Escherichia coli to stay on the surface. Due to the antibacterial and bactericidal activity of releasing zinc ions [49], there was almost no bacterial adhesion on TNT-PAA/GS-Zn. Fig. 6b shows the antibacterial properties of the different samples, from which it can be seen that pure titanium, TiO2 nanotubes and dopamine coatings had poor bactericidal properties. After further immobilization of PAA, the number of Escherichia coli colonies decreased significantly, indicating that the PAA coating had certain antibacterial properties. When the PAA and GS composite coating was prepared, the release of GS could effectively kill bacteria, so the number of observed colonies decreased significantly. Finally, zinc ion can also inactivate the protein needed by bacteria and cause the condensation of DNA [50], so the antibacterial activity was further improved after Zn2+ loading.