The Effects of Cu-doped TiO 2 Thin Films on Hyperplasia , Inflammation and Bacteria Infection

In the present work, different concentrations of Cu ion (1, 2, 5 and 10 wt %) were doped in the TiO2 film by a sol-gel method and dip coating process. The morphology of the Cu-doped TiO2 films were characterized by scanning electron microscopy (SEM) and the results showed that the doped Cu made no change to the TiO2 films. The nitric oxide (NO) release experiment showed that these Cu-doped surfaces showed the ability of catalytic decomposition of exogenous donor S-nitroso-N-acetyl-penicillamine (SNAP) to generate (NO). Based on fluorescence analysis and CCK-8 quantitative results, such films had the ability to inhibit smooth muscle cells adhesion, proliferation and migration with SNAP in vitro. The macrophage adhesion assay and anti-bacterial test proved that such Cu-doped TiO2 films also possessed anti-inflammatory and anti-bacterial abilities. All the abilities above showed positive correlation with the amounts of the doped Cu. This study suggested that the Cu-doped TiO2 films were capable of generating physiological levels of NO in the presence of endogenous donor S-nitrosothiols (RSNO), endowing the TiO2 films with anti-hyperplasia, anti-inflammatory and anti-bacterial abilities.


Introduction
The principal requirement for cardiovascular implanted materials is biocompatibility, and a genuinely biocompatible cardiovascular implanted material should perform its functions with good anti-coagulation, anti-hyperplasia and anti-inflammation abilities, simultaneously [1,2].A cardiovascular implanted device, such as a stent, with excellent biocompatibility, will contribute better clinical efficacy to cardiovascular disease and its complications [3,4].Restenosis and thrombosis are the major complications for stent implantation [5,6].Thrombosis is mainly due to fibrinogen activation and denaturation, causing a series of subsequent reactions (e.g., activation of platelets and envelope of red blood cell) after implantation [7].Restenosis is caused by the proliferation and migration of vascular smooth muscle cells (VSMCs) in the vessel wall in response to acute vessel wall injury induced by angioplasty [8].The application of drug-eluting stent (DES) systems has revolutionized coronary intervention therapy benefiting from their ability to dramatically reduce the possibility of in-stent restenosis through blunting VSMC proliferation [9,10].However, the results from certain recent meta analyses indicate that drug-eluting coatings result in the subsequent serious complication of late angiographic stent thrombosis (LAST), suggesting insufficient function in inhibiting the excessive proliferation and migration of the VSMCs [11,12].So, enhancing the function of inhibiting VSMCs becomes a problem which urgently needs to be resolved for the cardiovascular stent.Lots of clinical cases have proved that inflammation and bacterial infections also should be considered during vascular stent implantation [13,14].Compelling evidence for the importance of inflammation and atherosclerosis at both the basic and clinical level has evolved in parallel.Inflammation involved in the various stages of atherosclerosis-from initiation, through progression, and ultimately, the thrombotic complications of atherosclerosis [15,16].Moreover, a certain therapeutic has been shown to reduce coronary heart disease and limit the inflammation treatment [17].In addition, bacterial composition on the implant materials may cause of peri-implantitis, which will influence the success and survival rates of implant materials [18,19].Therefore, antibacterial efficacy must be considered during medical devices' implantation.
Titanium dioxide (TiO2) has been widely used in terms of electronics, optics and medicine for its unique optical, electrical and chemical properties [20].In recent years, TiO2 has attracted wide attention for its potential application in cardiovascular implanted biomaterials [21,22].Several studies have demonstrated that the TiO2 film possesses excellent blood compatibility and cell compatibility while its anti-hyperplasia and anti-inflammation functions need further improvement [23,24].In the meantime, the applications of TiO2 films doped by nanostructures in antibacterial purposes, cell growth and differentiation have been recently reported [25][26][27].The physiological vascular endothelial layer inspired surface functionalization provides a good choice to solve this problem.Nitric oxide (NO) produced by healthy endothelial tissue is the most important functional molecules on anti-hyperplasia and anti-inflammation of the cardiovascular system [28,29].Furthermore, lots of researches have proved that NO donating materials surfaces can significantly improve their functions of anti-coagulant, anti-hyperplasia, preventing microbial growth [30] and enhancing wound healing [31][32][33].In order to overcome the storage limitation of NO donating materials surfaces, a potentially NO generating material would be used to enhance the biocompatibility of the implanted devices.It is well known that Cu(II)/Cu(I) ions can catalytically decomposes endogenous S-nitrosothiols (RSNO) to release NO in vivo [34][35][36][37][38]. Hence, it should be possible to utilize endogenous reducing equivalents (e.g., ascorbate, L-glutathione (GSH), and cysteine (CySH)) and the endogenous NO precursors (e.g., S-nitrosoglutathione (GSNO), S-nitrosocysteine (CysNO), and S-nitrosoalbumin (Alb-SNO) in blood to generate locally enhanced NO levels at the biomaterials-blood interface by immobilizing catalytic Cu(II)/Cu(I) sites [39].So, TiO2 films doped with Cu have been suggested to have better blood and cell compatibility, and anti-hyperplasia and anti-inflammation functions, simultaneously.Our recent studies showed that Cu-doped TiO2 thin films could effectively suppress platelet adhesion, activation and aggregation [39], while the Cu-doped TiO2 films' capability of anti-hyperplasia and anti-inflammation was not reported, and the anti-bacterial property of the Cu-doped TiO2 films was not investigated.
In this work, we prepared the Cu-doped TiO2 films by sol-gel method because sol-gel method for preparing film has many advantages unmatched by other methods, such as the use of simple equipment, achieving film formation at normal temperature, quantitative doping, and effective control of the film composition and microstructure [23,39].As described in previous work, different concentrations of Cu were doped into the TiO2 films [39], but the Cu-induced catalytic activity films could not in situ induce NO releasing without donor; therefore, S-nitroso-N-acetyl-penicillamine (SNAP) must participate in the cell compatibility tests and NO release detection since exogenous SNAP would decompose to release NO in the presence of catalytic agents.The influences of Cu addition and concentrations on the surface morphology were characterized by scanning electron microscopy (SEM), and the anti-hyperplasia, anti-inflammation and anti-bacterial functions of the films were also investigated, respectively.

Cu-doped TiO2 Thin Films Preparation
Cu-doped TiO2 thin films with different Cu concentrations were prepared as described in the previous work [39].At first, silicon wafers were cut into small plates (7 mm × 7 mm) and sequentially cleaned in acetone, alcohol, and distilled water (dH2O) ultrasonically for 10 min three times, respectively.Subsequently, Ti(OC4H9)4 was added to the mixture of ethanol and acetic acid under vigorous stirring to form a solution.The ratio of each reagent was Ti(OC4H9)4:C2H5OH:CH3COOH:C5H8O2 = 9:36:5:1.After this, a certain proportion of Cu(NO3)2•3H2O was added to the solution, the molar ratio of Cu and Ti was 1% (labeled as Cu1), 2% (labeled as Cu2), 5% (labeled as Cu3) and 10% (labeled as Cu4), respectively.Then, Cu-doped TiO2 thin films were deposited on silicon substrates by a dip-coating process at room temperature with the withdrawing speed of 20 mm/min.Dip-coating and preannealing at 100 °C for 10 min were repeated and multilayers of Cu-doped TiO2 thin films can be obtained.This procedure was operated three times.The total film thickness was estimated to be 130 nm.Finally, each sample was thermally treated at 450 °C for 1 h.The detailed process of preparation of the Cu-doped TiO2 thin films was shown in Figure 1.

Surface Morphology Characterization
The surface morphology and roughness of the Cu-doped TiO2 thin films were characterized by atomic force microscopy (AFM, JPK Instruments, Berlin, Germany) in tapping mode [40].The crystalline structure of the Cu doped TiO2 films were studied by X-ray diffraction (XRD, X' Pert ProMPD, PANalytical B.V., Almelo, The Netherlands) [39].The amounts of the doped Cu on each TiO2 samples were measured by a typical micro BCA method [41].

Nitric Oxide (NO) Release in Vitro
The release of NO catalyzed by Cu-doped TiO2 thin films was examined using Savillee-Griess reagent [42].As a stable degradation product of NO, the content of nitrite could represent the release of NO.In short, a 200 μL solution containing 65 μM SNAP and 30 μM GSH were added onto the surface of all the samples (Cu0, Cu1, Cu2, Cu3 and Cu4).Then, SNAP was detected before and after the catalytic decomposition for 1 h, 2 h and 3 h, respectively, by reacting with equal-volume Savillee-Griess reagent, and the absorption was read at 540 nm.The amount of releasing NO was obtained by calculating the decrease of SNAP after catalytic decomposition.

Isolation and Culture of HUASMC
Human umbilical arterial smooth muscle cells (HUASMCs, Maternal and Child Health Hospital of Chengdu, China) were obtained from neonatal umbilical cord by the traditional method [43].In brief, the human umbilical cord was thoroughly rinsed with PBS buffer to remove the residual blood.Then, tearing the outer connective tissue and peeling the outer fibroblast layer so the artery was resected from the umbilical cord.Subsequently, the umbilical artery longitudinally broke away, and the ECs inside were gently scraped by a sharp tweezer.After that, the muscle tissue was sufficiently washed with PBS buffer, and then cut into small pieces.The tissue pieces were seeded into a culture flask, cultured with 5 mL medium F12 containing 0.5 mL FBS, and incubated at 37 °C in a humidified atmosphere containing 95% air and 5% CO2.The tissue blocks were removed after the HUASMCs migrated to the flask wall.Replicated cultures were performed by trypsinization as cells were approaching confluence.Cells were fed with freshly prepared growth medium every 24 h.The third passage of SMCs with the concentration of 5 × 10 4 cells/mL was used.HUASMCs derived from new born umbilical vein were approved by the Maternal and Child Health Hospital Institutional Review Board, and none of the research performed violated the Helsinki Declaration [44].

HUASMC Morphology and Proliferation
A rhodamine staining assay was used for observing the morphology and number of the HUASMC on each samples after cultured for 1 day and 3 days, respectively.The samples cultured with HUASMCs were rinsed with phosphate buffer solution (PBS, pH = 7.4) at 37 °C three times, then fixed with 4% paraformaldehyde for 12 h at room temperature.After a rinsing step, the samples were immunofluorescence stained with rhodamine (70 μM each sample) under dark conditions for 15 min.Washed three times again, the samples were air dried under dark conditions.At last, all samples were observed under a Leica DMRX fluorescence microscope.HUASMC proliferation was investigated by Cell Counting Kit-8 (CCK-8, Boshide, Wuhan, China) after incubation for 1 and 3 days, respectively [45].

HUASMC Migration
Firstly, the medical titanium foil was cut into a rectangular size of 1 cm × 1 cm.Cleaned by a conventional method, titanium foils were half cover with different concentrations of Cu-doped TiO2 thin films (Cu0, Cu1, Cu2, Cu3 and Cu4) as the method described above.All the samples were bent at right angles from the middle with tweezers, so that one side of the titanium foils is titanium, the other side is film.Titanium is placed at the bottom to culture cells to obtain a confluent monolayer.Then, HUASMCs were seeded onto samples with a density of 5 × 10 5 cells/cm 2 .After culture for 6 h (the HUASMCs formed a confluent monolayer on this half untreated titanium), the rectangular of specimen was vertically turned over and transferred to a new well plate, and was covered by the new culture medium.All the specimens were cultured for 1 day in an incubator at 37 °C.All the culture experiments above were conducted in sterile conditions.After fluorescence staining as above, the samples were observed by a Leica DMRX fluorescence microscope (Leica, Heidelberg, Baden-Württemberg, Germany).

Morphology of Macrophages
Peritoneal macrophages derived from the abdominal cavity of Sprague Dawley (SD) rats (Dashuo Co., Ltd., Chengdu, China) were isolated using the following method: SD rats were treated by cervical dislocation, immersed in 75% alcohol for 15 min.Next, the rats were set in supine, fixed extremities and extracted the abdominal wall with tweezers.Cutting a small hole in the skin and injection of 10 mL cell culture medium into its intraperitoneal, and then the abdominal cavity was gently massaged about 5-10 min.The cell culture medium was sucked out from abdominal cavity with a dropper and centrifuged at 1200 rpm for 5 min.The supernatant of the centrifuge tube was discarded, hereafter fresh culture medium containing 10% fetal calf serum was added in the centrifuge tube to dilute the concentration of the cell suspension to 1 × 10 5 cells/mL.Then, 1 mL of the cells suspension was added onto each sample (Cu0, Cu1, Cu2, Cu3 and Cu4) in 24-well plates and incubated (37 °C, 5% CO2) for 3 days.All the macrophage culture experiments above were operated in the sterile condition.Thereafter, samples were rinsed for three times with PBS (pH = 7.4), 5 min each time, and fixed in 2.5% glutaraldehyde for 12 h.A rhodamine staining was performed to statistic the number of attached macrophages on each samples directly (15 images each sample), and SEM characterization was performed to observe the morphology of the macrophages on the samples.Before the SEM characterization, samples were dehydrated, dealcoholized, followed with drying by critical point dryer (BAL-TEL CPD 030, Leica, Heidelberg, Baden-Württemberg, Germany), and gold deposited.

Anti-Bacterial Performance Assay
The anti-bacterial experimental procedure was performed under sterile conditions as follows.Firstly, 0.1 g of different concentrations of copper-doped titanium dioxide sol powder by heat treatment was pressed into tablets with a diameter of 10 mm and a thickness of 2 mm, which contained activated bacteria.Secondly, cryopreserved E. coli with inoculation loop was inoculated on agar medium and placed in an incubator at 37 °C, and the third generation activation E. coli loop which was incubated at 37 °C for 48 h was added to an erlenmeyer flasks containing PBS buffer (pH = 7.4), and after a mixing operation, bacterial suspension with a concentration of 5 × l0 6 cfu/mL was obtained.Then, 0.1 mL of bacterial suspension was added drop wise the surface of solidified beef extract peptone agar until the petri dish was covered completely, and was dried in the room temperature for 5 min.Finally, the prepared samples were placed in the bacterial culture medium surface, and incubated in the incubator 37 °C, bacteriostatic ring was observed after 24 h.

Statistical Analysis
In this work, all the experiments were replicated thrice (n = 3), and all the quantitative results were expressed as mean ± standard deviation (SD).The data were analyzed by the software SPSS 11.5 (Chicago, IL, USA).Statistical significance was accepted at a p-value of less than 0.05 (p < 0.05).

Surface Characterization
The morphology and roughness of the Cu0, Cu1, Cu2, Cu3 and Cu4 surfaces observed by AFM are shown in Figure 2. It was obvious that Cu0, Cu1 and Cu2 displayed smooth surfaces, with their roughness 0.40 ± 0.03 nm, 0.42 ± 0.02 nm and 0.43 ± 0.03 nm, respectively.Nevertheless, Cu3 and Cu4 samples showed significantly rougher surfaces compared to Cu0, Cu1 and Cu2 (Ra: Cu0, Cu1 and Cu2 < Cu3 < Cu4).The AFM results suggested that a little amount of Cu doped in the TiO2 could not remarkably change its morphology and roughness, while a large quantity of Cu doped in the TiO2 could significantly increase its roughness and change its morphology.

Nitric Oxide Release in Vitro
In vitro catalytic degradation of SNAP was performed using Saville-Griessreagent to assess the function of NO release (Figure 5).It could be seen that there was no substantial release of NO from the surface of Cu0, which indicated that TiO2 itself could not induce the exogenous donor SNAP to produce NO.Obviously, there were significant NO released from the Cu doped TiO2 samples, and the more Cu doped in the TiO2 films, the faster NO was released from the samples' surfaces (catalytic generation of NO: Cu4 > Cu3 > Cu2 > Cu1).Notably, the NO generation ratios of Cu2, Cu3 and Cu4 samples were within the range of 0.5 × 10 −1 mol/cm 2 •min to 4 × 10 −1 mol/cm 2 •min, which was in the range of NO generation ratios of healthy endothelial cells [39].The results above indicated that the doped Cu could catalyze SNAP in producing NO, and Cu4 sample which was doped with the most Cu, possessed the fastest NO release ratio.

SMC Attachment and Proliferation
Suppression of vascular smooth muscle cells (SMCs) from excessive proliferation is an effective method for reducing the in-stent restenosis and hyperplasia, which is the most serious clinical problem in the treatment of cardiovascular diseases [46].Thus, SMCs' attachment and proliferation test in vitro can be performed to investigate the anti-hyperplasia property of biomaterials.The fluorescence images of the SMCs on all samples after one day and three days of culturing are presented in Figure 6.It seemed that there were more SMCs on no-donor samples surfaces compared to the donor surface, and the more Cu doped in the TiO2 films, the less SMCs that were adherent on the samples, suggesting Cu ions may have some influence on the attached number of SMCs.It also could be seen that the SMCs on the no-donor surfaces showed a synthetic phenotype (polygon morphology), while the SMCs on the donor surfaces presented a contractile phenotype (spindle morphology).This result indicated that the Cu-doped surface could promote the SMCs' phenotypic contraction and inhibit SMCs' excessive proliferation by catalyzing donor release NO. Figure 7 displays the amounts of SMCs on the Cu0, Cu1, Cu2, Cu3 and Cu4 surfaces by CCK-8 assay after one day and three day cultures, respectively.The results showed that increasing doped amounts of Cu in the TiO2 films (Cu1, Cu2, Cu3 and Cu4) could reduce the attachment of SMCs (attached SMC numbers in one day: Cu4 < Cu3 < Cu2 < Cu1 < Cu0), but could not effectively inhibit the SMCs' proliferation because there were no significant differences between all the no-donor samples and the number of SMCs after three days of culture.Notably, the donor samples showed a significantly lower number of SMC compared with the no-donor samples, and the number of SMCs reduced as the amount of doped Cu increased.This result was consistent with the NO release result and the SMC staining result, indicating the amount of doped Cu was positively correlated with the released RSNO and was negatively correlated with the SMCs' attachment and proliferation.The results above demonstrated that the Cu-doped TiO2 films could significantly induce SNAP to release NO and further inhibit SMCs' attachment and proliferation.

HUASMC Migration
It is well known that the proliferation and migration of SMCs on the biomaterials surface could lead to pathological growth of the endothelial cell and further stent restenosis [47].Therefore, SMC migration tests in vitro can be performed as an evaluation of the anti-hyperplasia of the biomaterials.Figure 8 presents the fluorescence images of SMCs' migration on each sample (Cu0, Cu1, Cu2, Cu3 and Cu4).The statistical results show that there are a large number of cultured SMCs migrated on the surface of the no-donor group Cu0, and which had a long migration distance of 1175 ± 25 μm.The doped Cu had a small inhibitory effect on the SMCs' migration, but this effect was not remarkable in the fluorescence images.Notably, the donor group possessed a significant inhibitory effect on the SMCs' migration, and this effect was positively correlated with the amounts of the doped Cu.Accordingly, the Cu4 had the strongest effect in demonstrating SMCs' migration, in which the SMCs only migrated 545 ± 15 μm.This result was consistent with the NO release result and the SMC proliferation result.Thus, the results proved that the Cu doped TiO2 films with suitable SNAP plays an important role in regulating not only SMCs' proliferation but also their migration.

Attachment and Morphology of Macrophages
The attachment and activation of macrophages on a biomaterial's surface could lead to inflammatory response.Therefore, the attachment and activation of macrophages on Cu-doped TiO2 thin films was performed to evaluate the anti-inflammation function of the film.Figure 9 shows the fluorescence images and counting results of the macrophages on Cu0, Cu1, Cu2, Cu3 and Cu4 surfaces.Obviously, the macrophages on the Cu3 and Cu4 were significantly less than the cells on Cu0, Cu1 and Cu2, and the morphology of macrophages showed typical shrinkage, suggesting a non-activated phenotype.SEM micrographs (Figure 10) displayed further results of the macrophages' morphology.The macrophages on the samples of Cu0, Cu1 and Cu2 showed a multilateral and spreading morphology, with many pseudopods stretching out and aggregating, indicating an activated phenotype, while the macrophages on the Cu3 and Cu4 samples showed a round and shrinking morphology, suggesting a non-activated phenotype.This result demonstrated that the more Cu doped in the TiO2 films, the fewer macrophages adherent to and activated on the surfaces.The result also suggested that the Cu doped TiO2 thin film surfaces may be toxic while the human body contains 0.1 g of Cu per 75 kg of body weight and it is widely distributed in the blood, bone and muscle [39].The change of macrophage morphology often accompanied by their immune functions, such as: phage function and secretion function changes [48].Activated macrophages can release specific chemical factors, which induce other inflammatory cells and cytokines to flock to the trauma location and cause inflammation.Thus, the attachment and activation of macrophages on the biomaterials is often detected as an evaluation of their anti-inflammation properties.Therefore, the above results showed that the samples Cu3 and Cu4 had good anti-inflammatory properties.

Antibacterial Experimental Results
Figure 11 depicts the results of the anti-microbial property on the Cu0, Cu1, Cu2, Cu3 and Cu4 samples.The Cu/TiO2 2 mm heat treated tablets were fabricated by a method consistent with the thin film to perform this antibacterial characterization.The Cu0 and Cu1 almost formed no anti-microbial zone, and this may be attributed to the insufficiency of the doped Cu.With the increase of the Cu doped in the TiO2 films, the anti-microbial zone formed and became larger.(Anti-microbial zone size: Cu4 > Cu3 > Cu2 > Cu1).Copper ions-which are positively charged, are easy to bind with negatively charged bacterial protein-further combine with sulfhydryl groups (-SH) of protein causing inactivation of the active enzyme, which leads to bacterium death [49].The results above proved that the Cu doped TiO2 thin film possessed antibacterial properties, which was positively correlated with the amounts of doped Cu; more doped Cu contributed to better antibacterial properties.Therefore, Cu4 possessed the best antibacterial function in this work.

Conclusions
TiO2 films were widely applied on blood contact biomaterials for their excellent blood compatibility and mechanical behavior.In this work, different concentrations of Cu ion were doped in the TiO2 film by a sol-gel method and dip coating process to enhance their biocompatibility.The SMC culture result demonstrated that the Cu-doped TiO2 films could inhibit SMC attachment, proliferation and migration by inducing SNAP to release NO.The macrophages' culture result showed that the Cu-doped TiO2 films could inhibit macrophages' attachment and activation.The anti-bacterial test proved that the Cu-doped TiO2 films could significantly inhibit the growth of bacteria.All the properties above were positively correlated with the amounts of the doped Cu.This Cu-doped TiO2 film was potentially anticipated and applied for the cardiovascular implanted devices.

Figure 1 .
Figure 1.The detailed process of preparation of the Cu-doped TiO2 thin films.

Figure 6 .
Figure 6.The fluorescence staining of SMC on the Cu0, Cu1, Cu2, Cu3 and Cu4 surfaces after one day and three days of culturing, respectively.

Figure 8 .
Figure 8. Migration of SMCs on the Cu0, Cu1, Cu2, Cu3 and Cu4 surfaces after culture of one day.The migration distance was calculated from at least 12 images.