**3. Interaction of Cells with Nanostructured Surfaces**

Apart from exploiting the NP–membrane interactions in biomedical applications, the applications of micro- and nano-structured surfaces (Figure 2) in biomedicine have also attracted significant attention. Among them, of particular interest is the design of nanotopographic features such as biomimetic interfaces for implantable devices [47–49]. In vitro results have shown that the surface features on the nanometer scale stimulate and control several molecular and cellular events on tissue/implant interfaces, which can be observed by differences in the cell morphology, orientation, cytoskeleton organization, proliferation, and gene expression [47–49]. In the last decade, TiO<sup>2</sup> nanotubes fabricated via electrochemical anodization (Figures 3 and 4) have attracted significant attention toward medical applications [1,50].

**Figure 2.** Encapsulation and endocytosis of TiO<sup>2</sup> particles (microbeads) in the cells of urinary bladder (transmission and scanning electron microscopy). Reprinted from [32,33] with the permission of AIP Publishing.

**Figure 3.** TiO<sup>2</sup> nanostructured surfaces: (**a**) microbeads, (**b**) nanotubes, (**c**) nanowires and (**d**) octahedral nanoparticles. Partially reprinted (**a**,**c**) by permission from Springer Nature: Springer Protoplasma [51] (2016).

Among others, nanotopography on TiO<sup>2</sup> surfaces exhibits reduced activation and the aggregation of platelets [52], and the controlled adhesion and proliferation of endothelial and smooth muscle cells [3]. The excellent potential of TiO<sup>2</sup> nanotubes in medicine and biotechnology is mainly due to their high effective surface area, increased surface charged density [47,48], and the possibility to vary their geometry (diameter and length), which could be specially designed/adapted for a desired biological response (cell selectivity). TiO<sup>2</sup> nanotubes have also been shown to increase selective protein adsorption [53] and, thus, they enhance the biological response. For example, studies have shown that TiO<sup>2</sup> nanotubes increase bone growth/regeneration, are antibacterial, and reduce inflammation [54,55]. Moreover, the endothelium formed on the surfaces with nanoscale topography exhibits an enhanced expression of anti-thrombogenic genes, providing a more extended coagulation cascade, probably due to a thicker oxide layer and specific topography [49].

Several reports have shown that nanotopography significantly influences cell behaviors, i.e., adhesion, proliferation, and differentiation [47,56,57]. It was recently shown that the specific nanotopography of TiO<sup>2</sup> used for cardiovascular stents can improve their bio-/hemo-compatibility via the increased adhesion and growth of human coronary artery endothelial cells and reduced adhesion and activation of platelets [3]. The surface nanostructurization of titanium (specifically the formation of TiO<sup>2</sup> nanotubes with different diameter) was shown to alter physicochemical properties, such as wettability [2] and surface chemistry, which consequently affect the interactions of TiO<sup>2</sup> nanostructured surfaces with cells [3,47,58]. In vivo studies have shown improved endothelization and reduced neointimal thickening on nanostructured stents compared to bare-metal stents [58]. Moreover, the study performed by Peng et al. [59] showed that the TiO<sup>2</sup> nanotubular surface significantly enhances endothelial cell proliferation, while, at the same time, the growth of vascular smooth muscle cells is reduced.

**Figure 4.** Scanning electron microscope (SEM) images of TiO<sup>2</sup> nanotubular surface (**A**) and the TiO<sup>2</sup> nanotubular surface that was exposed to the isolate of extracellular vesicles (white arrow) from blood (**B**). Material deposited also on the top surface of the TiO<sup>2</sup> nanotube walls is denoted by a black arrow. Adapted with permission from [60,61].

Surface nanotopography may thus strongly influence the adhesion and proliferation of cells [47]. Experimental studies of the interaction between lipid bilayers and solid surfaces have indicated that the specific structure of the surface can induce even the phase transitions in the membrane upon its adhesion to the surface [62].

Among TiO<sup>2</sup> nanostructured surfaces presented in Figure 3, TiO<sup>2</sup> nanotubular surfaces were synthesized by electrochemical anodization [3,48,53,63], while hydrothermal treatment was employed for synthesizing other structures. To illustrate the interaction of nanostructured surfaces with biological systems, two nanostructuring procedures electrochemical anodization and hydrothermal treatment—were used for the synthesis of TiO<sup>2</sup> nanotubes (Figure 3b) and octahedral nanostructures (Figure 3d), respectively. In the case of electrochemical anodization, nanotubes with a diameter of 100 nm were synthesized by using HF and ethylene gylcol as electrolytes (described in detail in Refs. [60,61]), whereas for the hydrothermal method, Ti foils were exposed to a basic medium of Ti isopropoxide suspension. Briefly, in the hydrothermal method, Ti foil (0.1 mm, 99.6+%, Advent) was washed in acetone, ethanol, and water (in each for 5 min). Afterward, the samples were dried at 70 ◦C in the furnace. Ti isopropoxide (1 mL, 99.999% trace metals basis, Sigma Aldrich) was used as an ion precursor, and KOH (flake, 85%, Alfa Aesar) was added to the solution until the pH reached 10. The cleaned and dried Ti foil was placed at the bottom of a Teflon vessel in a hydrothermal reactor and poured with Ti isopropoxide suspension. The reaction was carried out at 200 ◦C for 24 h. Samples were then vigorously washed with deionized H2O, dried under the stream of N2, and used for further experiments.

As-prepared nanostructured surfaces were used to study interactions with blood platelets to evaluate the materials' potential thrombogenic nature as in [3]. It is of tremendous importance that metal implants, used as vascular stents, inhibit or decrease potentially fatal thrombosis and restenosis conditions. The stent surface should prevent excessive adhesion and aggregation of platelets as it can lead to blood clot formation/thrombosis. The adhesion and activation of platelets on the surface presented in Figure 5 is an indicator of how hemocompatible a material is; the lower platelet adhesion and activation, the higher is material compatibility with blood (hemocompatibility) [64,65]. It has already been recognized that protein adsorption and its conformation play an important role in platelet adhesion. Fibrinogen adsorption has been so far recognized as an important factor determining platelet adhesion and activation [66]. Studies have already shown that surface nanotopography may significantly influence platelet adhesion and activation, mainly due to nanotopography-induced changes in fibrinogen adsorption [66]. The study showed that the confirmation of adsorbed fibrinogen is highly linked to nanotopographic features of the surface. In the present study, other surface features such as wettability, roughness, and chemistry remained unchanged. However, when designing nanomaterials, it is sometimes hard to eliminate other surface features, which may even change over time. It was shown that TiO<sup>2</sup> nanotubular surfaces (Figure 3) tend to age with time and lose their hydrophilic characteristic [2]. The altered surface wettability and slight changes in chemistry significantly influence platelet adhesion. In this case, a decrease in the wettability of nanotubes 15, 50, and 100 nm in diameter resulted in a higher platelet adhesion and activation compared to more hydrophilic nanotubes [3]. However, the optimal surface nanostructure may significantly influence the biological response when other surface features are optimized.

In Figure 6, the scanning electron microscopy (SEM) images of plain Ti foil, TiO<sup>2</sup> nanotubes, and hydrothermally treated Ti foil are presented. TiO<sup>2</sup> nanotubes prepared by electrochemical anodization are 100 nm in diameter, while the nanostructured surface of hydrothermally treated Ti foil consists of octahedral nanoparticles with sizes of about 150–300 nm. More detailed information about surface roughness can be obtained from images taken via atomic force microscopy (AFM). The 3D image of surface topography obtained from AFM reveals that plain Ti foil has no special surface morphology, and the estimated surface roughness (Ra) evaluated from the images is about 11.7 nm. However, both nanostructured surfaces have a higher surface roughness (Ra), about 40 and 49 nm for the TiO<sup>2</sup> nanotubular and hydrothermal surface, respectively. It should be mentioned that in the case of nanotubes, the ability of the AFM tip to enter the hollow interior of the nanotube is limited; however, it gives us some additional information about surface

topography, especially the variation in the height of nanotubes. The evaluated difference in height between nanotubes is about 190 nm. In the case of hydrothermal treatment, the difference in the height of octahedral particles is very similar, about 185 nm. Thus, the main difference between both surfaces is in the width and shape of the nano-features.

Surface chemistry should also be considered when designing the biomaterial surface. Using the XPS technique, it is possible to determine the chemical composition of the elements on the top surface (about 5 nm in depth), which interact with the biological environment. The plain, as well as the nanostructured, surfaces were analyzed by X-ray photoelectron spectroscopy (XPS). The results from XPS analysis are presented in (Table 1). An increase in titanium and oxygen concentration after both hydrothermal and anodization processes is observed compared to the control (Ti foil), partially also due to the removal of surface hydrocarbon contamination (lower carbon content). In the case of the anodization process, fluorine is also detected on the surface due to HF used as an electrolyte. Thus, after altering the surface nanotopography, changes in surface chemistry should also be considered, as they may influence biological interactions. As the wettability of surfaces is also highly correlated with biological response, it should be mentioned that both freshly synthesized nanostructured (nanotubular and octahedral) surfaces are hydrophilic with water contact angle (WCA) below 5◦ , while the WCA measured on plain Ti foil exhibits a more hydrophobic characteristic (WCA of about 75◦ ). A detailed description of wettability studies can be found in Ref. [3].

**Table 1.** Atomic concentration (**%**) of detected elements by XPS on: Ti foil, hydrothermally treated Ti foil (HT), and TiO<sup>2</sup> nanotubular (NT) surface (diameter of the nanotubes = 100 nm).


After analyzing the surface properties, the interaction of surfaces with the biological environment should be studied. For the application of biomaterial for vascular implants, the interaction with whole blood should be evaluated. According to Goodman et al. [64], platelet adhesion and activation can be evaluated from their shape. The activation of platelets can be described in three steps: adhesion, spreading/aggregation of platelets, and activation of platelets/formation of a thrombus clot [67]. The morphology of adherent platelets is commonly described as round (R), dendritic (D), spreading dendritic (SD), spreading (S), and fully spreading (FS). Platelets with F and FS are considered activated platelets (Figure 5) [64,68].

**Figure 5.** Platelet's morphology after the adhesion. Their shape is marked as: round (R), dendritic (D), spreading dendritic (SD), spreading (S), and fully spread (FS). Reprinted with permission from Ref. [68] copyright 2014 American Chemical Society.

The interaction of whole blood and Ti surfaces is presented in Figure 6. On the smooth, non-treated Ti foil surface (control), the platelets are preferentially in dendritic form, and platelet-to-platelet adhesion (aggregation) is observed (Figure 6g). Platelets are aggregated and activated as the specific platelet shape change is observed, i.e., the multiple filopodial extensions from the platelet body and lamellipodia formation can be seen (SD and S shape). On the surface of the TiO<sup>2</sup> nanotubular layer, the platelets are extensively spread (S and FS shape), agglomerated, and express lamellipodia and numerous filopodia (Figure 6h). The area of spreading is significantly higher than on the surface of Ti foil.

On the surface of hydrothermally treated Ti foil, individual discoid/dendritic platelets with no obvious pseudopods are observed. Platelets are mainly in the round (R) and dendritic (D) form, indicating low surface interaction. Moreover, compared to the TiO<sup>2</sup> nanotubular layer (Figure 6h), a lower number of platelets is detected on these surfaces (Figure 6i), and platelets seem to not be in the activated form. The results presented in this section suggest that nanostructured surfaces may reduce or even increase platelet adhesion and activation. Although both nanostructured surfaces (i.e., nanotubular and octahedral) have similar surface chemistry (higher concentration of Ti and O atoms, with the addition of F for the case of the nanotubular layer) and wettability (hydrophilic) compared to plain Ti foil, their interaction with platelets seems to differ significantly in attachment and proliferation. Thus, it is important to note that a specific surface nanotopography may significantly affect the surface interaction with platelets. In Refs. [64,68], it was also shown that platelets, endothelial cells, and smooth muscle cells selectively interact with the TiO<sup>2</sup> nanotubes with various diameters, which, again, implies that surface nanotopography plays a significant role in the adhesion of biological material. Hence, by modifying the surface and with the appropriate surface chemistry, the influence of surface hydrothermally treated Ti can potentially prevent thrombosis, which could, to some extent, be correlated with its specific nanotopographic features, as the surface wettability, as well as chemistry, of both nanostructured materials was shown to be very similar.

**Figure 6.** SEM and AFM images of Ti foil (**a**,**d**), TiO<sup>2</sup> nanotubular surface (**b**,**e**), and TiO<sup>2</sup> octahedral surface (**c**,**f**), and the adhered blood platelets on these three kinds of surfaces (**g**–**i**).
