1 Introduction

Thrombogenic behavior of dental implant materials has gained much attention during recent years [1,2,3]. Titanium and titanium alloys have proven to be among the most thrombogenic materials, it has been suggested that, the thrombogenic properties of titanium surfaces might be contributed to their good osseointegrating properties and the early blood cell -titanium interactions might play a key role in peri-implant bone healing response [2, 4]. During implantation the first tissue that comes into contact with the implant surface is blood [5]. This initial interaction may influence clot formation, which starts immediately around the implant by platelet aggregation and activation of clotting factors. Peri-implant tissue healing begins immediately after implant insertion by initial blood clotting and formation of a fibrin scaffold at the peri-implant wound site [6]. The contact between the blood and implant surface results in protein adsorption and cell activation, which stimulate healing by platelet activation, matrix formation and initial wound healing [2, 3].

Titanium (Ti) and its alloys have been widely used in dentistry and orthopedics, because of their superior biocompatibility and good mechanical properties. The Ti–6Al–4 V alloy has been used widely as a high-strength biomedical alloy [7]. Moreover, titanium surfaces react with oxygen when exposed to air or water and form a thin film (4–6 nm) of protective oxide. This native nanocrystalline titanium dioxide (TiO2) film results in superior corrosion resistance and good biocompatibility. However, this film is usually non-uniform, mechanically weak and does not enhance the wound healing process [8], in addition there is no direct interaction of titanium surfaces with biologic molecules such as proteins and cells as result of their bioinert nature [9,10,11]. Therefore, numerous surface modification techniques have been investigated to obtain a stable and uniform TiO2 surface to improve osseointegration, bioactivity and bactericidal properties. Nanoscale modification of titanium surfaces has been shown to influence molecular and cellular activity and promote tissue healing [12, 13]. Several methods have been developed to produce optimal nanotopography. These techniques include anodic oxidation [14], a sol-gel coating method [15], and chemical or hydrothermal methods [16]. Among these, hydrothermal treatment has recently attracted attention due to its relative simplicity and flexibility to produce anatase crystalline TiO2 coating for improved bioactivity and enhanced osteoconductivity [17].

Although most dental implant studies have focused on osseointegration, a successful treatment depends on both hard and soft-tissue integration. The surface modification of the transmucosal area improves the soft tissue attachment [18, 19], preserves the crestal bone [20], hinders the bacterial biofilm adhesion [21] and facilitates firm attachment between the implant abutment and the surrounding soft tissue [22, 23]. Therefore, optimal soft-tissue healing would prevent bacterial penetration, reduce inflammation and induce gingival regeneration.

The chemical and physical surface properties, such as surface chemistry, surface topography, roughness and energy, have been shown to affect the initial cell response at the cell-material interface, ultimately affecting the rate and quality of new tissue formation [24]. The wettability of a material has been considered as a predictive indicator of cytocompatibility [25], it has been shown to enhance the protein and platelet adhesion on titanium surfaces [26, 27]. Many studies have shown that hydrophilic surfaces promote the early stage of cell adhesion, proliferation, differentiation and bone mineralization compared to hydrophobic surfaces [28, 29]. Hydrophilic surfaces can influence the bonding strength, promote protein adsorption and enhance cell adhesion compared to hydrophobic surfaces [30]. Implant surface topographies may affect protein adsorption, cell adhesion, and enhance cell proliferation, differentiation, and extracellular matrix formation [31,32,33].

Photocatalysis of TiO2 has been investigated extensively during the past 20 years. This technology provides self-cleaning, self-sterilizing, and more recently antibacterial TiO2 coatings functions based on the photo-induced hydrophilicity and decomposition reaction [34]. It has been shown that, photocatalytic bactericidal effect of TiO2 can be obtained by several coating methods [35, 36]. Ultraviolet (UV) light treatment has been applied to enhance biological properties of titanium surface, by altering its physicochemical properties without altering the implant surface topographical or morphological features. When anatase TiO2 is irradiated with UV light having a wavelength shorter than 385 nm, an electron-hole pair is generated. The adsorbed molecules such as oxygen and water will rapidly be reduced and oxidized to produce superoxide ions (O2−) and hydroxyl radicals (OH−), respectively. These can react with organic material, such as adherent bacteria and mineralizing them into CO2 and H2O [37]. Besides the previously menitoned antimicrobial activity UV-treated TiO2 surfaces have demonstrated superhydrophilicity and stain-proofing properties [38, 39]. Based on in vivo study by Aita et al. (2009) [39] the UV treatment of titanium enhances bone bonding capacity. They showed that UV treatment increased implant push-out values over three times at week 2 of healing. They also reported about 72% BIC around UV-treated implants which was 2.5 times greater than that seen around untreated implants. BIC enhanced until week 4 reaching almost 100% with less than 1% soft tissue intrusion between the de novo bone and implant surface as opposed to 21% around untreated implants. UV treated titanium implant surfaces enhance osteoconductive capacity and improve protein adsorption [40, 41]. In addition, it is believed to increase the rates of attachment, proliferation and differentiation of osteoblast cells as shown in numerous in vitro studies [39,40,41]. These biological improvements were attributed to the generation of superhydrophilicity and hydrocarbon elimination [42, 43]. Furthermore, UV light treatment on various topographical titanium surfaces has been shown to reduce the attachment and biofilm formation of wound pathogens [44]. These data suggests that UV photofunctionalization can be an effective measure to improve implant success in dental and orthopedic applications.

A previous in vitro study by the authors have demonstrated that nanostructured TiO2 implant surfaces obtained by sol-gel and hydrothermal coating methods enhance surface wettability and improve human gingival fibroblast function in terms of adhesion and rate of proliferation. UV light treatment clearly enhances the wettability of all titanium surfaces.

The purpose of this study was to evaluate the blood and platelet responses to nanostructured TiO2 coatings and to investigate the effect of UV light treatment on blood clotting ability, platelet activation and protein adhesion. To the best of the author’s knowledge, there have not been attempts to evaluate the effect of UV light treatment on blood and platelet response of hydrothermally treated TiO2 coatings.

2 Materials and methods

2.1 Sample preparation

In this study, Ti-6Al-4V (α + β) titanium alloy discs with a diameter of 1 cm and a thickness of 1 mm were used as substrate. The discs were ground using silicon carbide grinding paper of 1200 grit with an Ra value of 0.15 µm, and ultrasonically washed with acetone and ethanol (5 + 5 min) and dried in air before any surface treatments were carried out.

2.2 Surface treatments

Discs were divided into three groups, the following surface treatments were examined; the first group was coated with a sol-gel derived MetAlive™ coating (MA); the second group was treated with the hydrothermal method (HT); and the third group represented a negative control (NC) which was left untreated. All specimens were rinsed with acetone for 5 min and then in ethanol for 5 min, followed by thorough drying before testing. The sol-gel and hydrothermal treatments were performed by ID creation Ltd (Turku, Finland). The oxide thickness were in range of 100 to 200 nm. Both coatings consisted of anatase form of TiO2. However, the sol–gel coating is thicker than hydrothermal coatings. The content of anatase relative to amorphous phase was not calculated. Instead the relative photocatalytic activity to prove the efficacy of the crystals was measured. HT surfaces were at least equally or more reactive than sol–gel coatings at all times.

2.2.1 Sol–gel coating preparation

Sol–gel treatment was used to prepare the nanoporous TiO2 thin film on the titanium substrate. The sol was made as originally described by Peltola et al. (1998) [45]. In short, solution I contained commercially available tetra isopropyl orthotitanate, Ti {OCH (CH3)2}4, this was dissolved in absolute ethanol (solution I). Ethyleneglycol monoethylether (CH3CH2OCH2CH2OH) deionized water, and fuming hydrochloric acid (HCl 37%) were dissolved in ethanol (solution II). Solutions I and II were mixed together rapidly and stirred effectively for 3 min. The sol was kept at 0 °C during aging and the dip-coating process. The coating procedure was initiated after 24 h of sol-aging, and samples were coated with five layers. After layer deposition, the substrates were then sintered at 500 °C for 10 min., afterward the coatings were cleaned ultrasonically in acetone for 5 min, 5 min in ethanol, and finally dried at the ambient temperature.

2.2.2 Hydrothermal treatment

Following titanium substrate ultrasonic washing with ethanol and distilled water for 5 min, a hydrothermal suspension was prepared using reagent grade chemicals. This was prepared by dissolving Titanium dioxide (TiO2), purified water, 1:10 diluted tetra methyl-ammonium hydroxide (TMAH) (N(CH3)4+OH)−, and mixed for 5 min. The hydrothermal suspension was added to the titanium discs which were laid at the bottom of Teflon containers consists of a Teflon inner vessel and a stainless steel jacket. Next, the vessel was kept at 150 ± 10 °C in a constant-temperature oven for 48 h. Subsequent to the hydrothermal treatment period, the titanium discs were removed from the vessel and cooled in air. All the discs were washed with distilled water in an ultrasonic bath for 10 min.

2.3 UV light treatment

A total of (69) substrates in three different groups (MA, HT and NC) were treated with UV light for 1 h under ambient conditions using a 36 W puritec HNS germicidal UV lamp (Osram GmbH; Germany), with dominant wavelength of 254 nm. Analysis of blood clotting, platelet activation and protein adsorption were conducted immediately following the UV light treatment (fresh surface). This was compared with non-UV treated titanium discs.

2.4 Surface characterization

The chemical composition of the hydrothermal surfaces (HT) was examined by X-ray photoelectron spectroscopy (XPS). To compare the material surface, the samples were allowed to stay in contact with air for 2 weeks after the production. HT coated samples were treated with UV directly as received from storage for various times according to the description in materials and methods, whereas the native surface (before UV) was analyzed directly as received from storage. The XPS measurements were carried out by using a Physical Electronics Quantum 2000 instrument using a monochromatic Al Kα X-ray excitation, operated at 24.4 W. The diameter of the analysis spot was 100 μm.

The surface topography of the substrates was characterized using field-emission scanning electron microscopy (SEM). An approximately 20 nm thick gold layer was applied on samples with a sputter coater, and secondary electron images were recorded with (SEM).

2.4.1 Contact angle measurements

The equilibrium contact angles of each sample were measured using the sessile drop method described by Jong et al. (1982) [46], with a contact angle meter (KSVCAM100 KSV, Instrument LTD, Finland). The contact angles were determined on UV and non UV treated substrates by using the Young- Laplace equation. A drop was deposited on the surface of the specimen and imaged for 20 s by collecting at least 120 images. The contact angles on both side of the droplet and their mean values were calculated in room temperature. Three liquids were used as a probe for SFE calculations: ultrapure water, diiodomethane and formamide pro analysis. The result was the mean value of at least 6 drops on each specimen for each liquid.

2.4.2 Surface free energy calculations

The SFE of the substrates were calculated using the Owens-Wendt (OW) approach. The total (γTOT), dispersive (γD) and the polar (γP) SFE components were calculated. Three liquids were used as a probe for SFE calculations (Distilled water ultrapure water, Diiodomethane >99% purity and Formamide pro analysis), every group has at least six measurements and the result was the mean of SFE calculated.

2.5 Blood-clotting time measurement

The thrombogenicity of the titanium substrates was assessed using fresh human blood with a whole blood kinetic clotting time method [47, 48]. Static and air contacting condition were performed. Blood was taken from a healthy unmedicated adult female volunteer by venipuncture into vacutainer tubes. In order to prevent contamination with tissue thromboplastin caused by needle puncture, the first 3 ml of the taken blood was disposed. Then, 100 µl volume of blood was immediately added to the surface of the UV and non-UV treated substrates, 96 titanium substrates were used. Each of the experimental groups (HT; MA; NC) had 32 substrates. Half of the substrates in each group (n = 16) were UV treated while the remaining 16 were left without UV treatment. All substrates were settled in 12-well plates and incubated at room temperature for 10, 20, 40, and 60 min. For each time point four replicates (n = 4) per each group were used, and the experiment was repeated twice. At the end of each time point, the substrates were incubated with 3 ml of ultrapure water for 5 min. The addition of ultrapure water lysed the red blood cells that had not been trapped in a thrombus and then free hemoglobin was dispersed in water. For clotting time measurement, each well was sampled in triplicate (200 µl each) and transferred to a 96-well plate. Free hemoglobin concentration in the water was assessed by measuring the absorbance at 570 nm using an ELISA plate reader. The size of the clot is inversely proportional to the absorbance value.

2.6 Platelets adhesion and morphology

A platelet adhesion test was conducted to evaluate the quantity, morphology and the adhesion behavior of the platelets on the UV and non-UV treated titanium substrates. Fresh human blood was drawn from a healthy adult volunteer as described above. The whole blood was treated with an anticoagulant (0.109 M solution of sodium citrate) at a dilution ratio of 9:1 (blood/sodium citrate solution). Then platelet-rich plasma (PRP) was obtained by centrifuging the anticoagulated blood at 1500 rpm for 15 min at room temperature. Each of the experimental groups (HT; MA; NC) had 6 substrates (n = 18). In each group 3 substrate were UV treated and 3 were not UV treated. 100 µl of PRP was carefully added to the substrates surface and then incubated at 37 °C for 1 h. The substrates were then rinsed thoroughly three times with phosphate-buffered saline (PBS) to remove weakly adherent platelets. The adhered platelets were fixed with 2.5% glutaraldehyde solution for 2 h. Eventually, all substrates were rinsed in PBS and dehydrated at increasing alcohol concentrations. Carbon sputtering coating of 10–20 nm thick was applied to the specimens and then analyzed by scanning electron microscopy (SEM). FE-SEM images at different magnifications were collected for each substrate to examine the platelet morphologies.

2.7 Protein adsorption and analysis

UV and non-UV treated titanium substrates were rolled for 1 h at room temperature in tubes containing human plasma that was diluted with PBS at a ratio of 1:4 (n = 12). Then the specimens were washed twice with PBS for 2 min. Collection of absorbed proteins was made essentially according to Tanner et al. (2002) [49], with some modifications. Proteins bound to the substrates were desorbed by rubbing the top and bottom surfaces of each substrate with three applicator sticks (Quick-Stick® Dentsolv AB, Saltsjö-Boo, Sweden) wetted with 4 µl of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) buffer (1 mM Na phosphate buffer, 2% SDS, 0.003% bromophenol blue) and then, with one dry applicator stick. The tips of the sticks were collected in an Eppendorf-tube to which 20 µl of buffer was added. The tube was heated in boiling water for 7 min. The tubes were then perforated with a needle and placed in larger tubes that collected the sample solutions after centrifugation for 2 min (13.000 × g, 2 min). Samples of duplicate specimens (from each surface) were collected in the same tube. The protein solutions were analyzed by SDS-PAGE and silver staining with the use of gradient Mini-Protean TGX gels (4–12%; Bio-Rad laboratories, Berkeley, CA, USA). The resultant gels were observed and images were taken using an imaging system (ChemiDoc MP, Bio-Rad laboratories, Hercules, CA, USA). To evaluate the adsorption of fibronectin on the surfaces, the same procedure was repeated by rolling the specimens in a solution of 0.125 mg/ml bovine fibronectin (F4759 Sigma, Sigma-Aldrich, St. Louis, MO, USA).

2.8 Statistical analysis

Statistical analysis was performed using the SPSS v.23.0 software package (IBM SPSS Inc.). To analyze the differences among several means, the data were analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Differences were considered significant at 95% confidence level, with p-values below 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001).

3 Results

3.1 Surface characterization

X-ray photoelectron spectroscopy (XPS) indicated signals of Ti, C, and O on HT surfaces before and after UV treatment. HT surface with 60 min UV treatment had higher O and Ti surface concentration (68.3 and 17.0 respectively) in comparison with HT surface before UV treatment (57.0 and 13.3 respectively), and less C (8.5) than before UV treatment (24.5), which indicates less adsorption of CO2 and other organic impurities from the atmosphere. UV treatment for 60 min removed almost 66% of the surface carbon.The SEM experiment was carried out to investigate the surface topography of the substrates. Figure 1 shows SEM images of the substrate surfaces at low and high magnification. The NC substrates showed a smooth surface with some grinding lines spreading over the surfaces. The MA surface showed a uniform smooth surface with extensive cracking, whereas the HT surfaces were fully covered with the coating crystals consisting of nearly spherical nanoparticles of 20–50 nm. The surface doesn’t change in appearance as a result of the UV treatment, and all the UV and NUV have the same surface morphology.

Fig. 1
figure 1

SEM images of the substrate investigated show surfaces topography at low and high magnification

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3.1.1 Contact angle result

The HT group had the lowest water contact angle (40.1°) followed by MA (42.0°), whereas the NC group had the highest contact angle value (56.0°). The UV light treatment significantly enhanced substrates hydrophilicity, after UV treatment the water contact angles dropped for all substrates, being 15.7°, 11.8° and 33.8° for HT, MA and NC respectively (p < 0.001) (Fig. 2).

Fig. 2
figure 2

Mean values and standard deviations (SD) of contact angle measurements on different substrates before ultraviolet (NUV) and after (UV) light treatment. Statistically significant differences were found between the hydrothermal (HT) and MetAlive™ coating (MA) groups and the non-coated (NC) group, and within the same groups before and after UV treatment (***p < 0.001)

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Although there was no significant difference between the HT and MA UV treated groups their contact angles were significantly lower than that of the NC UV group (p < 0.001). The contact angles values for the other two liquids were presented in Fig. 2, the contact angles dropped for all substrates after UV treatment.

3.1.2 Surface free energy calculations

The SFE result is shown in (Fig. 3). Before UV treatment the HT and MA groups showed higher polar (γP), dispersive (γD) and total (γTOT) SFE components compared with NC group. After UV treatment the SFE components were increased for all substrates and there was significant differences between the UV and non UV treated substrates (p < 0.001). Although there was no significant difference between the HT and MA UV treated groups, their SFE components were significantly higher than that of the NC UV group (p < 0.001).

Fig. 3
figure 3

Dispersive (γD), polar (γP), and total (γTOT) components of surface free energy (SFE) calculated using the Owens-Wendt approach

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3.2 Blood clotting

The blood clotting profiles for UV and non-UV titanium substrates at all time points is shown in Fig. 4. The absorbance of the hemolyzed hemoglobin solution varies with time, and the lower the absorbance value the better the thrombogenic behavior. The absorbance values of UV treated groups were lower than non UV treated groups at all time points. The total clotting time for the UV treated HT, MA and NC titanium substrates was almost 40 min compared to 60 min for non-UV substrates, and the UV MA substrates had the lowest absorbance values compared to the other groups at all time points.

Fig. 4
figure 4

Blood clotting profiles for UV and non-UV titanium substrates at all time points, showing the optical density vs. time. Blood is considered totally clotted at an absorbance value of 0.1. Statistically significant differences (*p < 0.05, **p < 0.01, and ***p < 0.001) between the substrate types at same time points

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Although there were no significant differences among the UV treated groups, the total clotting time for the HT, MA and NC UV treated groups were significantly lower than that of the non UV NC group (p = 0.002, 0.004, 0.004) respectively. The UV light treatment significantly enhances coagulation rates.

3.3 Platelet adhesion and morphology

Platelets adhered on all UV and non-UV treated surfaces, which was clearly seen in scanning electron micrographs after 1 h adhesion period (Fig. 5). However, the HT and MA substrates presented more morphological platelet changes, including aggregation, spreading and pseudopod formation, in comparison with the NC substrates. UV treatment did not affect platelet adhesion and activation.

Fig. 5
figure 5

Scanning electron micrographs of platelet morphologies after 1 h adhesion period on UV and non-UV treated hydrothermal (HT), MetAlive™ coating (MA) and the non-coated (NC) substrates. Inner images are the high magnification of the same titanium substrate

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3.4 Protein adsorption

The protein adsorption analysis showed similar protein-binding profiles on all substrates. The intensities of albumin and fibronectin bands were similar on the HT, MA, and NC groups (Fig. 6). There were no qualitative differences in protein adsorption between the UV and non-UV treated substrates.

Fig. 6
figure 6

Protein-binding profile of plasma run on different titanium substrates, the intensities of albumin bands were similar on the HT, MA, and NC groups. The fibronectin run show no qualitative differences in fibronectin bands between the UV and non-UV treated substrates

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4 Discussion

This study showed that UV treatment improves blood coagulation rate, both on nano-structured oxidized titanium and non-coated titanium surfaces. Enhanced blood response of the UV treated nano-structured TiO2 surfaces has good potential to improve wound healing and tissue integration of various medical implants.

The blood clotting ability of a material has been considered as an important factor in hemostasis. A blood clot represents the connection site between the implant surfaces and the surrounding tissues [50]. It is achieved by the aggregation of platelets and the activation of the humoral coagulation system (clotting factors) [3,4,5]. The contact of blood with the implant surface begins with the adsorption of plasma or serum proteins, extracellular matrix formation and continues through to the activation of connective tissue cells which initiate wound healing [2,3,4,5,6]. The clotting time is the time at which the optical density (OD) becomes equal to 0.1 [51], a lower OD value relates to lower hemoglobin concentration in blood solution which indicates faster thrombous formation on implant surfaces. In this study the OD values of the nano-structured TiO2 surfaces were lower than that of NC Ti-6Al-4V surfaces, which shows that nano-structured surfaces enhance coagulation rates and provided higher thrombogenicity.

Wettability has been recognized as an important surface property of implants, which enhances the protein and platelet adhesion on implant surfaces [26, 27]. The SFE of a solid surface has a strong influence on wetting, adsorption and adhesion activities, and it has been suggested that high surface energy increased bone formation on modified titanium surfaces [52]. Measurement of the wettability and SFE of the materials can be determined by measuring the contact angle on solid surface in the presence of different liquids, and a low contact angle indicates good wetability [25]. The effects of hydrophilicity on protein adhesion have been reviewed by Wilson et al. [30], who reported that protein adsorption is appearently promoted when blood and other biological fluids comes in contact with hydrophilic surfaces which enhances cell adhesion. In contrast hydrophobic surfaces disturb protein structure leading to less accessible cell binding sites, which eventually diminish cell adhesion. Our findings indicated that the hydrophilicity of the nano-structured TiO2 surfaces were stronger than that of NC Ti-6Al-4V surfaces. The differences among the groups we observed are related to the differences in surface topographies and crystalline structures of their outermost surfaces. After UV light treatment, all surfaces became remarkably more wettable to water; the contact angles decreased dramatically whereas the SFE increased enhancing the hydrophilicity of the substrates. The UV treated nano-structured TiO2 surfaces had a low contact angle, as well as high surface energy which indicated their superior wettability compared to the NC titanium surfaces. These findings were in agreement with Wang et al. [53] who showed that UV irradiation of crystalline TiO2 surfaces increased surface hydrophilicity. The XPS measurement shows the differences in the chemical composition of UV and non-UV HT surfaces. A higher carbon contamination was observed on non-UV surfaces compared to UV treated surfaces and carbon content reduces with UV treatment. The UV treatment was performed in air and samples were moved directly into the XPS vacuum chamber so that the influence of further contamination was minimized. However, it cannot be ruled out that some carbon was immediately adsorbed onto the surfaces. Whilst carbon is reducing the relative amounts of oxygen and titanium is increasing within their stoichiometric proportions of TiO2. Taken together, these data (CA, SFE, SEM AND XPS) showed that UV treatment increased surface energy without changing surface topography. UV light treatment clearly improved the wettability of all examined Ti-6Al-4V surfaces, which may explain the reason for enhanced blood clotting and higher platelet activity. Our finding in the present study showed that nano-structured surfaces were further improved by UV treatment. This is probably related to the fact that UV treatment converts already hydrophilic nanostructured Ti alloy surfaces to superhydrophilic and cleans the contaminated hydrocarbons that accumulate on titanium surfaces [39, 40]. The exposure of TiO2 to UV results in excitation of an electron from valence band to conduction band [53]. The excited electron, along with the created positive hole on a superficial layer of TiO2 catalyze the chemical reaction. Titanium surfaces require ionic bridges (divalent cations) such as Ca2+ to attract protein and cells [10, 11] and it seems that there are no direct interactions between titanium surfaces and biologic molecules and/or cells. However, recent studies found that UV-treated titanium surfaces act as a direct attractant for cells because of their improved electrostatic status and the positively charged TiO2 surface can attach directly to negatively charged protein as well as cells and no longer require the ionic bridges [54, 55]. By altering the surface structure, removing the contaminated hydrocarbons and creating surface oxygen vacancies at bridging sites, UV treatment creates direct cell attractants without the help of ionic and organic bridges [39, 42, 43]. The removal of carbon can alter surface charge from electronegative to electropositive [54, 55]. UV treatment-induced superhydrophilicity as well as electropositive charge on titanium surfaces have a regulatory role in determining their bioactivity, which attract negatively charged proteins, such as fibrinogen and albumin as well as blood cells on titanium surfaces without the aid of bridging ions [43, 54, 55].

The nanoscale modification of the implant surface has been widely investigated. This surface modification can alter the surface chemistry and topography of an implant surface, which enhances molecular and cellular activity and influence the initial cell response at the cell-material interface [56]. Platelet adhesion and activation have been extensively studied due to their role in thrombogensis [3, 5, 50]. Platelet adhesion has different activation patterns ranging from a low activated state (round or dentric), to a high activated state (intermediate and fully spread) [57]. In our study the platelet adhesion and activation were more extensive on HT and MA surfaces compared with the NC surface. This is in agreement with Park et al. [2, 50] who showed that blood cell adhesion increased on a rough surface of a titanium substrate compared to a smooth surface. Nygren et al. [58] demonstrated that platelet adhesion and protein adsorption became enhanced when on treated titanium surfaces compared to polished surfaces. After the UV treatment, all groups showed no differences in platelet adhesion and activation. A prolonged UV treatment time for several hours seems likely to improve the fibrinogen adsorption as well as platelet adhesion [59]. Chen et al. (2015) showed that a long UV treatment time (e.g., 240 min) of TiO2 surfaces, may enhance fibrinogen adsorption and platelet adhesion which might be related to an increase of the positive charge and decomposition of absorbed hydrocarbon, and these may promote electrostatic attraction of negatively charged fibrinogen and platelets [26, 51]. However, in our study no additional effect of UV treatment on protein adsorption and platelet adhesion was found probably due to the relatively short UV exposure time (1 h). The differences in surface chemistry of titanium used, UV treatment time and the wavelength used may produce different biological effects.

In blood-contacting devices, such as dental implants, the interactions of the implant material with blood system are extremely important in defining implant’s fate [2, 4]. The formation of blood clot serves as the connection site between the surrounding living tissues and implant surface [50], which bonds wound together with the implant, protects the implant surfaces from the external environment and acts as a barrier against a possible infection. This may enhance early event of wound healing around implant and abutment surfaces.

5 Conclusions

Based on the finding of this in vitro study it can be concluded that, nanostructured titanium dioxide implant surfaces obtained by sol-gel or hydrothermal coating methods increase coagulation rate (short clotting time) and enhanced platelet response when compared with non-coated surfaces. UV light treatment clearly improved thrombogenicity of all examined Ti-6Al-4V surfaces.