Glancing Angle Deposition of Zn-Doped Calcium Phosphate Coatings by RF Magnetron Sputtering

Zn-substituted hydroxyapatite with antibacterial effect was used in radiofrequency (RF) magnetron deposition of calcium phosphate coating onto Tiand Si-inclined substrates. The development of surface nanopatterns for direct bacteria killing is a growing area of research. Here, we combined two approaches for possible synergetic antibacterial effect by manufacturing a patterned surface of Zn-doped calcium phosphate using glancing angle deposition (GLAD) technique. A significant change in the coating morphology was revealed with a substrate tilt angle of 80◦. It was shown that an increase in the coating crystallinity for samples deposited at a tilt angle of 80◦ corresponds to the formation of crystallites in the bulk structure of the thin film. The variation in the coating thickness, uniformity, and influence of sputtered species energy on Si substrates was analyzed. Coatings deposited on tilted samples exhibit higher scratch resistance. The coating microand nano-roughness and overall morphology depended on the tilt angle and differently affected the rough Ti and smooth Si surfaces. GLAD of complex calcium phosphate material can lead to the growth of thin films with significantly changed morphological features and can be utilized to create self-organized nanostructures on various types of surfaces.


Introduction
The demand for implants that can efficiently conduct bone defect regeneration increases significantly with the aging population [1].It is known that the bioinert metals that are used in implantology and orthopedics do not provide the desired bioactivity and osteoconductivity; therefore, the surface of such medical devices is usually modified with bioactive coatings [2].Moreover, the demand for antibacterial or bactericidal surfaces and coatings has increased steadily in recent years.It has been noted that the modern approach to surface modification should take into account both coating functions: osteoconduction properties and antibacterial effect [3].From that perspective, substituted hydroxyapatites (HAs) are one of the most promising materials in this area of research.It has been shown that the easiness of atomic doping or substitution in HA reveals its potential to be used in various clinical cases [4][5][6].Most importantly, it has been shown that inorganic antibacterial metallic ions (e.g., Ag, Cu, Zn, Sr) can be introduced into the HA lattice and improve its antibacterial properties [7][8][9][10][11][12][13][14].However, there is a physical approach to antibacterial surface production.It has been shown that the creation of nanopatterns is a powerful tool for directing stem cell fate.Another advantage is that high aspect ratio nanopatterns are capable of killing bacteria and preventing biofilm formation [15].
Modern approaches to healthcare are aiming to produce implants with biomimetic properties [16].These properties are crucial to ensuring a desirable biological response to the newly implanted material, in such a manner that the cells, which are adhered to the surface of such scaffolds, can function in a way that is similar to physiological conditions.From that point of view, the formation of a coating that consists of different types of surface gradient structures and with variations in the level of roughness on the submicro-and nanoscale could be of significant interest for biomimetic purposes [17,18].The possibility of manipulating nanotopography has attracted much attention in recent years [19][20][21][22].This has fostered the development of film deposition techniques aimed at enhancing these morphological characteristics.
In the field of optics and microelectronics, physical vapor deposition (PVD) of thin films, allowing the deposition of porous and/or columnar-like structured coatings, has been available for some years.For this purpose, the use of an oblique angle geometrical configuration-or as it is also known, glancing angle deposition (GLAD) method-is frequently exploited [23][24][25][26][27].
The GLAD configuration has emerged as a promising tool for the deposition of nanostructured thin films.This method has mostly been applied in the field of metallic coatings or dielectric coatings for optical systems, and the use of such technology in the field of biomaterials started recently [28].Such coatings are usually represented by inclined columnar-like structures with variations at the nano-roughness level and a direction of growth allowing for the deposition of patterned structures with different shapes and sizes.GLAD has a wide range of applications in various fields, such as sensors [29], optics [30,31], or solar cells [32].This type of coatings has attracted attention due to the anisotropic nature of the formed films, their peculiar mechanical properties, and the possibility of controlling the level of porosity [33] and roughness [34].Most researchers who have utilized the GLAD method have used line-of-sight methods of deposition, such as, for example, thermal evaporation.However, the effect of self-shadowing is applicable in many PVD techniques, such as magnetron sputtering.
An emerging method for bioactive coating deposition in the field of PVD is radiofrequency (RF) magnetron sputtering.Magnetron sputtering is widely used in the formation of coatings for various applications.The continuous interest of scientists in this method is due to the possibility of modifying the coating structure and its physicochemical properties by varying the deposition parameters [2].There is a significant interest in radiofrequency (RF) magnetron sputtering of bioactive calcium phosphate (CaP) thin films [35,36].This method allows for the deposition of CaP coatings with a high level of adhesion to the substrate.Moreover, RF magnetron sputtering is well known as a highly controllable deposition method.However, to date, a feasible method of calcium phosphate coating deposition in GLAD geometry via RF magnetron sputtering has not yet been reported.
Here, we aimed to develop a method for depositing thin Zn-substituted HA (Zn-HA)-based coatings that are formed by hierarchical gradient surface features with the use of GLAD geometry.This method allows us to manipulate the coating roughness on the submicron and nanoscale level and pursue the possible synergetic antibacterial effect of a nanopattern and the antibacterial activity of Zn.

Materials and Methods
Commercially pure titanium samples (99.58 Ti, 0.12 O, 0.18 Fe, 0.07 C, 0.04 N, and 0.01 H wt %) of 10 × 10 × 1 mm 3 size provided by "VSPO-AVISMA" (Perm, Russia) were used as substrates.The samples were sequentially polished by silicon carbide paper of 120, 480, 600, and 1200 grit.Prior to deposition, the substrates were cleaned in an ultrasonic bath of distilled water for 10 min.Furthermore, commercially available Si wafers (1 0 0) provided by "ELMA" (Zelenograd, Russia) were used as substrates in all the deposition runs.
In Figure 1, the deposition process is schematically shown.The samples were mounted on the custom-made sample holder at different angles to the flux of the sputtered material.Furthermore, commercially available Si wafers (1 0 0) provided by "ELMA" (Zelenograd, Russia) were used as substrates in all the deposition runs.
In Figure 1, the deposition process is schematically shown.The samples were mounted on the custom-made sample holder at different angles to the flux of the sputtered material.The sample holder allows for the simultaneous placing of substrates at 0°, 60°, or 80° to ensure the same sputtering conditions for all the deposited samples.
The sputtering target was Zn-substituted hydroxyapatite.This material was chosen because hydroxyapatite resembles the mineral fraction of bone and is used for many implant coatings, while the addition of Zn can provide faster bone growth together with an antibacterial effect [37].A Zn-HA powder was prepared by mechanochemical synthesis at the Institute of Chemistry and Mechanochemistry, Russian Academy of Sciences, Novosibirsk, Russia.The mechanochemical synthesis was carried out according to the reaction: The prepared powder was used as a precursor for the preparation of a target for sputtering.Its detailed characterization can be found in our previous report [38].The powder was pressed and then sintered in air at 1100 °C for 1 h.The chemical composition of the powder and the target was confirmed by X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) [39].
To identify the optimal deposition parameters for our experiment, we reviewed the literature dealing with GLAD, considering the substrate material, sputtered material, deposition parameters, and type of formed coatings.In Table 1, the deposition parameters for GLAD of various materials and their properties are summarized.The sample holder allows for the simultaneous placing of substrates at 0 • , 60 • , or 80 • to ensure the same sputtering conditions for all the deposited samples.
The sputtering target was Zn-substituted hydroxyapatite.This material was chosen because hydroxyapatite resembles the mineral fraction of bone and is used for many implant coatings, while the addition of Zn can provide faster bone growth together with an antibacterial effect [37].A Zn-HA powder was prepared by mechanochemical synthesis at the Institute of Chemistry and Mechanochemistry, Russian Academy of Sciences, Novosibirsk, Russia.The mechanochemical synthesis was carried out according to the reaction: The prepared powder was used as a precursor for the preparation of a target for sputtering.Its detailed characterization can be found in our previous report [38].The powder was pressed and then sintered in air at 1100 • C for 1 h.The chemical composition of the powder and the target was confirmed by X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) [39].
To identify the optimal deposition parameters for our experiment, we reviewed the literature dealing with GLAD, considering the substrate material, sputtered material, deposition parameters, and type of formed coatings.In Table 1, the deposition parameters for GLAD of various materials and their properties are summarized.

DC magnetron sputtering
The expansion of the well-known model of the Thornton spatial zone, including the inclination angle of the substrate as an additional degree of freedom Ti Si (100) L = 22 cm For α = 70 • , a columnar morphology was formed consisting of vertically aligned and well-separated columns with diameters ranging from 50 to 100 nm.For angles of α = 80 • and α = 85 • , the structure was rather similar.The manipulation of the substrate led to the formation of a zigzag columnar structure.The films prepared in the range of 40-85 • showed an increase in the RMS value of roughness from 7 to 55 nm.
[41] DC magnetron sputtering Influence on structural and optical properties ZnO Si (100) The typical columnar structure was inclined and compact.SEM images showed that the typical columnar structure was inclined, and the column inclination angle was changed from 0 to 34 • .From XRD analysis, it was shown that the strains in the ZnO films decreased with the substrate tilt angle.t = 180 min P = 0.64 Pa W = 400 W [42] Reactive magnetron sputtering Effect on the refractive index ZnO Si (100) L = 14; 28.5 cm The films were porous and of an inclined columnar structure, with columns tilting in the direction of the incident flux.The deposited AZO films had nanocolumnar structures.In tilted angle sputtering, the nanocolumns were tilted away from the surface normal to the incident AZO flux direction.When the substrate was not rotated, the nanocolumn inclination increased with apparently distinguishable grain boundaries as the tilted angle became larger.
[ The GLAD ZnO films were mixtures of columnar structure and pores.The highly-oriented columnar structure of slanted columns indicated that the GLAD ZnO films were anisotropic with the long axis parallel to the columnar growth direction.This anisotropic structure will introduce an anisotropic dependence into the optical, electrical, thermal, and magnetic properties of the films.
Even though most GLADs were performed at target-to-substrate distances larger than 70 mm, we decided to perform deposition at a distance of 45 mm, as it is consistent with most published research papers dealing with sputtering of CaPs [47][48][49] and ensures that the particle-free mean path is significantly higher than the target-to-substrate distance.Moreover, it is known that ZnO and hydroxyapatite share the same type of lattice structure (hexagonal system).Taking into account the fact that there is a correlation between the lattice type of sputtered material and the type of the formed coating structure, we hypothesized that using similar deposition parameters to those used for ZnO would result in the formation of Zn-doped calcium phosphate nanocolumns [42,46,50].
A vacuum installation with a planar magnetron operating at a frequency of 13.56 MHz (TPU, Tomsk, Russia) was utilized for GLAD.The high-quality Zn-HA target was used as the cathode of the magnetron.The sputtering power density was set to 3.15 W/cm 2 .Prior to the deposition, the system was pumped to a base pressure of 5 × 10 −6 Torr, and then, Ar gas was passed through at a controlled rate to maintain the pressure needed for the experimental deposition.The deposition time was set to 3 h, and the target-to-substrate distance was 45 mm.It is important to note that the target-to-substrate distance varied for tilted samples due to the inclination; therefore, the upper part of the sample was as close to the surface of the magnetron as 35 mm.The samples were deposited at a working gas pressure of 0.8 mTorr at a gas flow of 3.7 sccm.Each set of samples with angles varied from 0 to 80 • was deposited in a single run.
The thicknesses of the deposited films were measured using spectroscopic ellipsometry with an ELLIPS-1891 SAG setup (Rzhanov Institute of Semiconductors Physics of SB RAS, Novosibirks, Russia).The surface morphology and composition of the coatings were examined with scanning electron microscopy using an ESEM Quanta 400 FEG scanning electron microscope (SEM, FEI Company, Hillsboro, OR, USA) operating in a high vacuum.The samples were coated with gold-palladium for 15 s before the SEM, which was equipped with energy-dispersive X-ray spectroscopy (EDX; Genesis 4000, SUTW-Si(Li)detector, EDAX, Inc., Mahwah, NJ, USA) and was operating in a high vacuum study in such a way that no alteration of the surface morphology was induced.The estimated thickness of the deposited Au-Pd layer was 4-5 nm.The acceleration voltage for the SEM was 20 kV.To determine the level of crystallinity of the studied samples and estimate the crystallite size of the deposited thin CaP coatings, a Panalytical Empyrean X-ray diffractometer with a Cu Kα radiation source (λ = 1.54 Å; 40 kV and 40 mA) was used.The coatings were investigated by grazing incidence X-ray diffraction (GIXRD) with an incident beam angle of Ψ = 1.0 • (with respect to the sample surface) and at 2θ range from 5 • to 90 • with a step size of 0.05 • .For qualitative phase analysis with the Bruker software Diffrac.Suite EVA 4.2.2, the patterns of hydroxyapatite (#09-0432) and titanium (#44-1294) from the ICDD database were used as a reference.After the instrumental correction derived from a standard powder sample LaB6 from the NIST ( [51]; a(LaB6) = 4.15689 Å), quantitative Rietveld analysis for the calculation of the lattice parameters and the crystallite sizes was performed with the Bruker software TOPAS 5.0.For the morphology visualization and calculation of the surface roughness level of the samples, a JPK NanoWizard Atomic Force microscope (AFM, JPK, Berlin, Germany) was used.The coating-to-substrate adhesion evaluation was carried out with the scratch-test method on a CSM Macro Scratch Tester Revetest (CSM Instruments, Needham Heights, MA, USA) with an indenter of 20 µm in radius.The maximum indentation load was 10 N. The scratch length was set to 5 mm.In order to obtain statistically meaningful data, each measurement was repeated at least three times per sample.

Effect of GLAD on Coating Thickness and Its Uniformity along the Substrate Surface
The key for variation in the coating surface topography when the GLAD method is used lies in the self-shadowing effect [23].During the film growth, adatoms migrate to the substrate and encounter the surface in a way that causes peak and valley regions to grow.Due to the inclination of the substrate, the peaks receive more atoms compared with the valleys.As the peaks grow higher, a shadow region, which receives a lower number of atoms, is formed behind them.Consequently, the GLAD coatings have a preferential direction of growth and may even be porous.Also, in most of the reports dealing with GLAD, an increase in root-mean-square (RMS) surface roughness is usually observed when compared with the normal degree deposition [25,26,29].
Even though research on GLAD coatings is evolving extensively, there are few reports on the coating uniformity of the substrate surface and the variation in its thickness.Here, we show the thickness variation on the Si substrates measured by ellipsometry.In Figure 2, the coating thickness of the substrates is placed in parallel to the target of the magnetron (0 • ), and inclination angles of 60 GLAD coatings have a preferential direction of growth and may even be porous.Also, in most of the reports dealing with GLAD, an increase in root-mean-square (RMS) surface roughness is usually observed when compared with the normal degree deposition [25,26,29].
Even though research on GLAD coatings is evolving extensively, there are few reports on the coating uniformity of the substrate surface and the variation in its thickness.Here, we show the thickness variation on the Si substrates measured by ellipsometry.In Figure 2, the coating thickness of the substrates is placed in parallel to the target of the magnetron (0°), and inclination angles of 60° and 80° are presented.With the increase in the inclination angle, the coating thickness decreases gradually.We believe that this effect is associated with substrate re-sputtering.It is known that when the substrate is in close proximity to the plasma source, impinging energetic ions of Ar + and O − induce re-sputtering of the coating from the substrate [28].Moreover, the deposition parameters were chosen in such a way that the mean free path of the sputtered atoms was as large as possible while maintaining a relatively high power density.In this way, the kinetic energy of the sputtered material and the generated ions is close to the original level due to the negligible number of collisions they experience.It not only enables better adatom mobility, but also higher substrate heating and hence higher re-sputtering.In addition, a shorter target-substrate distance will increase the rate of deposition but at the expense of film uniformity [29].This was the case for the CaP coatings deposited in our experiments.For the samples deposited at less than an inclination angle of 80°, the coating thickness was measured for the upper part (closer to the magnetron source) and the lower part of the sample.Ellipsometry showed that the coating thickness was inhomogeneous along the sample, and the thickness difference between the upper and lower part of the coating was up to 110 ± 25 nm for some samples.Interestingly, the closer the placement of the sample to the plasma source, the thinner the film deposited.We believe that this effect is due to substrate re-sputtering and energetic ions bombardment.

Effect of GLAD on Coating Topography and Roughness
In Figure 3, SEM images show the topography of CaP coatings deposited at different angles of 0°, 60°, and 80°.The coatings deposited without tilting (0°) show globular-like surface features.The samples inclined at 60° during the deposition decreased in size, and the surface structure elements changed their shape.The surface grains were orientated along the incidence direction of the sputtered particles independent of working gas pressure.This effect was even more pronounced for the samples deposited at a tilt angle of 80°.An alteration in the working gas pressure led to a reduction in surface elements sizes.When the deposition process was performed at a lower working pressure (here: 0.8 mTorr), surface elements tended to be more homogeneously spread on the surface.With the increase in the inclination angle, the coating thickness decreases gradually.We believe that this effect is associated with substrate re-sputtering.It is known that when the substrate is in close proximity to the plasma source, impinging energetic ions of Ar + and O − induce re-sputtering of the coating from the substrate [28].Moreover, the deposition parameters were chosen in such a way that the mean free path of the sputtered atoms was as large as possible while maintaining a relatively high power density.In this way, the kinetic energy of the sputtered material and the generated ions is close to the original level due to the negligible number of collisions they experience.It not only enables better adatom mobility, but also higher substrate heating and hence higher re-sputtering.In addition, a shorter target-substrate distance will increase the rate of deposition but at the expense of film uniformity [29].This was the case for the CaP coatings deposited in our experiments.For the samples deposited at less than an inclination angle of 80 • , the coating thickness was measured for the upper part (closer to the magnetron source) and the lower part of the sample.Ellipsometry showed that the coating thickness was inhomogeneous along the sample, and the thickness difference between the upper and lower part of the coating was up to 110 ± 25 nm for some samples.Interestingly, the closer the placement of the sample to the plasma source, the thinner the film deposited.We believe that this effect is due to substrate re-sputtering and energetic ions bombardment.

Effect of GLAD on Coating Topography and Roughness
In Figure 3, SEM images show the topography of CaP coatings deposited at different angles of 0 • , 60 • , and 80 • .The coatings deposited without tilting (0 • ) show globular-like surface features.The samples inclined at 60 • during the deposition decreased in size, and the surface structure elements changed their shape.The surface grains were orientated along the incidence direction of the sputtered particles independent of working gas pressure.This effect was even more pronounced for the samples deposited at a tilt angle of 80 • .An alteration in the working gas pressure led to a reduction in surface elements sizes.When the deposition process was performed at a lower working pressure (here: 0.8 mTorr), surface elements tended to be more homogeneously spread on the surface.
Coatings 2018, 8, x FOR PEER REVIEW 3 of 17 In Figure 4, the dispersion of the size of the surface features for the cases of GLAD at 0° and 80° is presented.The mean grain size of the coating decreased from 210 to 190 nm with the increasing tilt angle.It is well known that the coating deposited by magnetron sputtering insignificantly changes the surface topography [52].In most cases, the deposited coating repeats the topography of the initial substrate surface.Even after polishing, the Ti surface still had some marks after machining that could potentially disturb or alter the growth of the surface coating's elements.Therefore, polished Si samples were used alongside the Ti substrates in all the deposition runs, as they do not provide additional ambiguity in the understanding of coating growth on a non-smooth surface topography.Figure 5 shows SEM images of CaP on the Si substrate deposited at 0°, 60°, and 80° tilt angles and at a pressure of 0.8 mTorr.This was the pressure that promoted the dispersion of the most homogeneous surface features and minimized the particle collision during deposition most effectively.In Figure 4, the dispersion of the size of the surface features for the cases of GLAD at 0 • and 80 • is presented.The mean grain size of the coating decreased from 210 to 190 nm with the increasing tilt angle.It is well known that the coating deposited by magnetron sputtering insignificantly changes the surface topography [52].In most cases, the deposited coating repeats the topography of the initial substrate surface.Even after polishing, the Ti surface still had some marks after machining that could potentially disturb or alter the growth of the surface coating's elements.Therefore, polished Si samples were used alongside the Ti substrates in all the deposition runs, as they do not provide additional ambiguity in the understanding of coating growth on a non-smooth surface topography.Figure 5 shows SEM images of CaP on the Si substrate deposited at 0°, 60°, and 80° tilt angles and at a pressure of 0.8 mTorr.This was the pressure that promoted the dispersion of the most homogeneous surface features and minimized the particle collision during deposition most effectively.It is well known that the coating deposited by magnetron sputtering insignificantly changes the surface topography [52].In most cases, the deposited coating repeats the topography of the initial substrate surface.Even after polishing, the Ti surface still had some marks after machining that could potentially disturb or alter the growth of the surface coating's elements.Therefore, polished Si samples were used alongside the Ti substrates in all the deposition runs, as they do not provide additional ambiguity in the understanding of coating growth on a non-smooth surface topography.
Figure 5 shows SEM images of CaP on the Si substrate deposited at 0 • , 60 • , and 80 • tilt angles and at a pressure of 0.8 mTorr.This was the pressure that promoted the dispersion of the most homogeneous surface features and minimized the particle collision during deposition most effectively.Interestingly, the deposition of the CaP coating on polished Si resulted in the appearance of surface grains in smaller quantities.Apparently, the Ti surface with defects, cavities, and an overall rougher topography induced more nucleation sites for subsequent grain formation compared with Si.Even though the initial surface of the Si samples was smooth and without pronounced defects or impurities, grain nucleation still took place in regions that were energetically favorable.Even though the surface of coated Si deposited at the normal incidence was rather smooth, it was possible to find rare globular-like shaped grains on its surface.In Figure 5, some of the grains that were found on the smooth surface of the sample deposited without tilting are shown.For samples deposited at an angle of 60°, the concentration of surface elements increased together with their size.Moreover, in some cases, grains were comprised of smaller structural elements and had complex topography.We assume that this could be a transition state before the grains coalescence into bigger surface structural elements.With an increase in tilt angle to 80°, the effect of self-shadowing became more pronounced and resulted in the appearance of surface grains of different sizes.In this case, the surface grains covered the whole surface of the sample and left no place for a region without complex topography.From this, we can conclude that an RF magnetron GLAD method for CaP can have a topographical effect on smooth and rough surfaces of different materials.Álvarez et al. [23] performed mathematical modeling with experimental validation for Ti thin films deposited by RF magnetron sputtering.The proposed model correlated well with the experimental results.It was solely based on ballistic deposition phenomena, indicating that the simulations realistically reproduced the competitive growth mechanism involved in GLAD experiments.However, a modeling of a far more complex structure (hydroxyapatite) still needs to be performed.
Figure 6 shows a cross-section SEM of thin films deposited under tilt angles of 0° (a) and 80° (b) at 100000× magnification and films under tilt angles of 0° (c) and 80° (d) at 200000× magnification on Si substrates.For the untilted samples, the cross section is presented by an amorphous calcium phosphate layer without any visible structural features.In contrast, coatings deposited at 80° inclination show an anisotropic, elongated type of inner structure orientated towards the particle source represented by small crystallites.EDX data obtained from both types of samples showed that the Ca/P ratio equals 1.8-1.9,while the content of Zn does not exceed 1 at.%.It is worth noting that the inner structure corresponds to the surface topography of the samples.In that way, the surface of the coatings deposited on the untilted samples is smooth with a few globular-like features (Figure Even though the initial surface of the Si samples was smooth and without pronounced defects or impurities, grain nucleation still took place in regions that were energetically favorable.Even though the surface of coated Si deposited at the normal incidence was rather smooth, it was possible to find rare globular-like shaped grains on its surface.In Figure 5, some of the grains that were found on the smooth surface of the sample deposited without tilting are shown.For samples deposited at an angle of 60 • , the concentration of surface elements increased together with their size.Moreover, in some cases, grains were comprised of smaller structural elements and had complex topography.We assume that this could be a transition state before the grains coalescence into bigger surface structural elements.With an increase in tilt angle to 80 • , the effect of self-shadowing became more pronounced and resulted in the appearance of surface grains of different sizes.In this case, the surface grains covered the whole surface of the sample and left no place for a region without complex topography.From this, we can conclude that an RF magnetron GLAD method for CaP can have a topographical effect on smooth and rough surfaces of different materials.Álvarez et al. [23] performed mathematical modeling with experimental validation for Ti thin films deposited by RF magnetron sputtering.The proposed model correlated well with the experimental results.It was solely based on ballistic deposition phenomena, indicating that the simulations realistically reproduced the competitive growth mechanism involved in GLAD experiments.However, a modeling of a far more complex structure (hydroxyapatite) still needs to be performed.
Figure 6 shows a cross-section SEM of thin films deposited under tilt angles of 0 • (a) and 80 • (b) at 100,000× magnification and films under tilt angles of 0 • (c) and 80 • (d) at 200,000× magnification on Si substrates.For the untilted samples, the cross section is presented by an amorphous calcium phosphate layer without any visible structural features.In contrast, coatings deposited at 80 • inclination show an anisotropic, elongated type of inner structure orientated towards the particle source represented by small crystallites.EDX data obtained from both types of samples showed that the Ca/P ratio equals 1.8-1.9,while the content of Zn does not exceed 1 at.%.It is worth noting that the inner structure corresponds to the surface topography of the samples.In that way, the surface of the coatings deposited on the untilted samples is smooth with a few globular-like features (Figure 5a), and a cross-section of this sample (Figure 6a,c) is represented by smooth featureless layer.In the case of 80 • , tilting small crystallites do not only belong to the surface (Figure 5b) but are also visible throughout the coatings' inner structure (Figure 6b,c).Moreover, predominant growth towards the particle flux is detectable, and the angle of structural inclination β is calculated to be in the range of 60 • -65 • .As it is mentioned in the literature [53], the tilt angle is always smaller than the vapor incidence angle (β < θ).In the paper by Grüner et al. [53], it is stated that, as a first order approximation, a linear relationship between the tilt angle and the angle of incidence β ∼ = 0.71θ can be obtained for GLAD of silicon.A similar, linear behavior can be found for obliquely deposited germanium and molybdenum thin films covering a wide range of melting points.However, we could speculate that, in our case, this coefficient is going to be in a range of 0.75-0.8according to the SEM measurements.Taking into account the fact that the melting point of silicon (1414 • C) is relatively close to the melting point of hydroxyapatite (1570 • C) [54], this coefficient could indicate the differences in the lattices of the sputtered materials.Most published papers dealing with GLAD focused on materials with relatively simple oxide or monocrystal types of structures with a higher level of symmetry when compared with calcium phosphates.In turn, during the sputtering of CaP, various sputtered species travel to the substrate and are later are involved in the condensation step.Therefore, we assume that this fact is responsible for the difference in the calculated coefficient and overall difference in the thin film structure.
Coatings 2018, 8, x FOR PEER REVIEW 5 of 17 5a), and a cross-section of this sample (Figure 6a,c) is represented by smooth featureless layer.In the case of 80°, tilting small crystallites do not only belong to the surface (Figure 5b) but are also visible throughout the coatings' inner structure (Figure 6b,c).Moreover, predominant growth towards the particle flux is detectable, and the angle of structural inclination β is calculated to be in the range of 60°-65°.As it is mentioned in the literature [53], the tilt angle is always smaller than the vapor incidence angle (β < θ).In the paper by Gruner et al.
[53], it is stated that, as a first order approximation, a linear relationship between the tilt angle and the angle of incidence β ≅ 0.71θ can be obtained for GLAD of silicon.A similar, linear behavior can be found for obliquely deposited germanium and molybdenum thin films covering a wide range of melting points.However, we could speculate that, in our case, this coefficient is going to be in a range of 0.75-0.8according to the SEM measurements.Taking into account the fact that the melting point of silicon (1414 °C) is relatively close to the melting point of hydroxyapatite (1570 °C) [54], this coefficient could indicate the differences in the lattices of the sputtered materials.Most published papers dealing with GLAD focused on materials with relatively simple oxide or monocrystal types of structures with a higher level of symmetry when compared with calcium phosphates.In turn, during the sputtering of CaP, various sputtered species travel to the substrate and are later are involved in the condensation step.Therefore, we assume that this fact is responsible for the difference in the calculated coefficient and overall difference in the thin film structure.In Figure 7, an AFM visualization of CaP coatings deposited on Ti with and without tilting is shown.According to Sarkar and Pradhan [55], the RMS values of film roughness increase with the increasing oblique angle of deposition.However, in our case, the RMS for tilted samples were smaller than for samples without inclination: 17 nm for the 80° sample and 28 nm for the sample without inclination (0°), respectively.We believe that this is because small surface grains, which homogeneously cover the initial rough Ti surface, are reducing the RMS compared with the sample without any In Figure 7, an AFM visualization of CaP coatings deposited on Ti with and without tilting is shown.According to Sarkar and Pradhan [55], the RMS values of film roughness increase with the increasing oblique angle of deposition.However, in our case, the RMS for tilted samples were smaller than for samples without inclination: 17 nm for the 80 • sample and 28 nm for the sample without inclination (0 • ), respectively.We believe that this is because small surface grains, which homogeneously cover the initial rough Ti surface, are reducing the RMS compared with the sample without any inclination.In the case of the normal incidence of sputtered particles, round-shaped grains are formed on top of the Ti surface, artificially increasing the RMS.AFM measurements allow us to have a 3D image of the surface topography and reveal an out-of-plane growth of surface grains (Figure 7c).inclination.In the case of the normal incidence of sputtered particles, round-shaped grains are formed on top of the Ti surface, artificially increasing the RMS.AFM measurements allow us to have a 3D image of the surface topography and reveal an out-of-plane growth of surface grains (Figure 7c).In order to exclude the impact of the substrates' surface topography on the coating's roughness, AFM measurements were performed for polished Si samples as well (Figure 8).For the samples deposited at the normal incidence, it was almost impossible to detect distinct surface features.In an untilted sample, some structural elements were found.Surface elements are represented in the form of pyramids, which possibly represent artifacts rather than the actual shape.Nevertheless, the RMS value for the untilted sample was very low (down to 200 pm).In order to exclude the impact of the substrates' surface topography on the coating's roughness, AFM measurements were performed for polished Si samples as well (Figure 8).For the samples deposited at the normal incidence, it was almost impossible to detect distinct surface features.In an untilted sample, some structural elements were found.Surface elements are represented in the form of pyramids, which possibly represent artifacts rather than the actual shape.Nevertheless, the RMS value for the untilted sample was very low (down to 200 pm).
Coatings 2018, 8, x FOR PEER REVIEW 6 of 17 inclination.In the case of the normal incidence of sputtered particles, round-shaped grains are formed on top of the Ti surface, artificially increasing the RMS.AFM measurements allow us to have a 3D image of the surface topography and reveal an out-of-plane growth of surface grains (Figure 7c).In order to exclude the impact of the substrates' surface topography on the coating's roughness, AFM measurements were performed for polished Si samples as well (Figure 8).For the samples deposited at the normal incidence, it was almost impossible to detect distinct surface features.In an untilted sample, some structural elements were found.Surface elements are represented in the form of pyramids, which possibly represent artifacts rather than the actual shape.Nevertheless, the RMS value for the untilted sample was very low (down to 200 pm).However, the RMS of the GLAD sample was 5 nm, i.e., was significantly higher than that of the untilted sample.Moreover, these results are in good correlation with GLAD-related research, as RMS values are usually found to increase with the increasing oblique angle of deposition [24].Such changes in morphology may lead to a significant decrease in the values of the contact angle of the coatings.

Effect of GLAD on Coating Crystallinity and Scratch Resistance
It is not rare that CaP coatings deposited by RF magnetron sputtering have a low level of crystallinity [56].Frequently, CaP-deposited coatings are annealed in order to reveal peaks from crystalline Zn-HA [7].However, in our case, we decided to perform a GIXRD of the untreated sample, as the temperature treatment will significantly change the internal structure of the CaP. Figure 9 shows representative X-ray powder diffraction patterns of the coatings deposited on Ti substrates at oblique angles of 0° and 80°.The GIXRD measurements indicate a formation of mostly amorphous CaP coating with a broad halo at 2θ = 30°, which can be attributed to hydroxyapatite with a crystallite size 2(1) nm as determined by the Rietveld refinement.The slightly increased intensity of the amorphous halo of the hydroxyapatite coating deposited under 80° (Figure 9b) compared with the hydroxyapatite coating under 0° might be caused by the increased crystallinity of the HA coating.The calculated lattice parameters of Ti (a = 2.951 and c = 4.685 Å) agree well with the ICDD database, confirming that the RF magnetron sputtering does not change the structure of the metallic substrate (as expected).Only a small difference between the Ti substrates was observed in the intensity of diffraction peak (002) at 2θ = 38.4°,which was caused by the typical rolling texture in hexagonal metals, including Ti [57][58][59].It is worth noting, that the increased crystallinity of the samples inclined to 80° correlates with the formation of small-sized crystallites on the surface and in the cross-section structure of the GLAD samples (Figure 6b,d).
(a) However, the RMS of the GLAD sample was 5 nm, i.e., was significantly higher than that of the untilted sample.Moreover, these results are in good correlation with GLAD-related research, as RMS values are usually found to increase with the increasing oblique angle of deposition [24].Such changes in morphology may lead to a significant decrease in the values of the contact angle of the coatings.

Effect of GLAD on Coating Crystallinity and Scratch Resistance
It is not rare that CaP coatings deposited by RF magnetron sputtering have a low level of crystallinity [56].Frequently, CaP-deposited coatings are annealed in order to reveal peaks from crystalline Zn-HA [7].However, in our case, we decided to perform a GIXRD of the untreated sample, as the temperature treatment will significantly change the internal structure of the CaP. Figure 9 shows representative X-ray powder diffraction patterns of the coatings deposited on Ti substrates at oblique angles of 0 • and 80 • .The GIXRD measurements indicate a formation of mostly amorphous CaP coating with a broad halo at 2θ = 30 • , which can be attributed to hydroxyapatite with a crystallite size 2(1) nm as determined by the Rietveld refinement.The slightly increased intensity of the amorphous halo of the hydroxyapatite coating deposited under 80 • (Figure 9b) compared with the hydroxyapatite coating under 0 • might be caused by the increased crystallinity of the HA coating.The calculated lattice parameters of Ti (a = 2.951 and c = 4.685 Å) agree well with the ICDD database, confirming that the RF magnetron sputtering does not change the structure of the metallic substrate (as expected).Only a small difference between the Ti substrates was observed in the intensity of diffraction peak (002) at 2θ = 38.4• , which was caused by the typical rolling texture in hexagonal metals, including Ti [57][58][59].It is worth noting, that the increased crystallinity of the samples inclined to 80 • correlates with the formation of small-sized crystallites on the surface and in the cross-section structure of the GLAD samples (Figure 6b,d).However, the RMS of the GLAD sample was 5 nm, i.e., was significantly higher than that of the untilted sample.Moreover, these results are in good correlation with GLAD-related research, as RMS values are usually found to increase with the increasing oblique angle of deposition [24].Such changes in morphology may lead to a significant decrease in the values of the contact angle of the coatings.

Effect of GLAD on Coating Crystallinity and Scratch Resistance
It is not rare that CaP coatings deposited by RF magnetron sputtering have a low level of crystallinity [56].Frequently, CaP-deposited coatings are annealed in order to reveal peaks from crystalline Zn-HA [7].However, in our case, we decided to perform a GIXRD of the untreated sample, as the temperature treatment will significantly change the internal structure of the CaP. Figure 9 shows representative X-ray powder diffraction patterns of the coatings deposited on Ti substrates at oblique angles of 0° and 80°.The GIXRD measurements indicate a formation of mostly amorphous CaP coating with a broad halo at 2θ = 30°, which can be attributed to hydroxyapatite with a crystallite size 2(1) nm as determined by the Rietveld refinement.The slightly increased intensity of the amorphous halo of the hydroxyapatite coating deposited under 80° (Figure 9b) compared with the hydroxyapatite coating under 0° might be caused by the increased crystallinity of the HA coating.The calculated lattice parameters of Ti (a = 2.951 and c = 4.685 Å) agree well with the ICDD database, confirming that the RF magnetron sputtering does not change the structure of the metallic substrate (as expected).Only a small difference between the Ti substrates was observed in the intensity of diffraction peak (002) at 2θ = 38.4°,which was caused by the typical rolling texture in hexagonal metals, including Ti [57][58][59].It is worth noting, that the increased crystallinity of the samples inclined to 80° correlates with the formation of small-sized crystallites on the surface and in the cross-section structure of the GLAD samples (Figure 6b,d).
(a) In Figure 10, the results of CaP coating scratching are shown.Thin film adherence is one of the most important aspects when dealing with physical vapor deposition methods.Lc (critical load) is a function of coating-substrate adhesion, but also involves other parameters related to the shape of the stylus, the mechanical properties of the substrate and the coating, the thickness, and the internal stress.In Figure 10a, a micro-scratch on the surface of the Zn-doped CaP coating on the untilted sample is represented.The Lc was determined during the scratch test to be a relatively small load of 10 N. The first adhesive cracks were observed after Lc = 5.3 N for the untilted sample.The average value of the friction coefficient during the measurements was calculated to be 0. Local detachment of the coating is seen on the side of the track and in several other regions that are identified by arrows in Figure 10a.We concluded that the coating demonstrates sufficient scratch resistance, which is in accordance with the scientific data available in the literature [60,61].In contrast, samples deposited at inclinations of 60° and 80° (Figure 10b,c) show higher scratch resistance.Thus, coating delamination even at the maximum load of 10 N is not observed.The average value of the friction coefficient for both the tilted substrates during the measurements did not exceed 0.25.This fact also contributed to the observed, improved scratch resistance of the tilted samples.We associate In Figure 10, the results of CaP coating scratching are shown.Thin film adherence is one of the most important aspects when dealing with physical vapor deposition methods.L c (critical load) is a function of coating-substrate adhesion, but also involves other parameters related to the shape of the stylus, the mechanical properties of the substrate and the coating, the thickness, and the internal stress.In Figure 10a, a micro-scratch on the surface of the Zn-doped CaP coating on the untilted sample is represented.The L c was determined during the scratch test to be a relatively small load of 10 N. The first adhesive cracks were observed after L c = 5.3 N for the untilted sample.The average value of the friction coefficient during the measurements was calculated to be 0. In Figure 10, the results of CaP coating scratching are shown.Thin film adherence is one of the most important aspects when dealing with physical vapor deposition methods.Lc (critical load) is a function of coating-substrate adhesion, but also involves other parameters related to the shape of the stylus, the mechanical properties of the substrate and the coating, the thickness, and the internal stress.In Figure 10a, a micro-scratch on the surface of the Zn-doped CaP coating on the untilted sample is represented.The Lc was determined during the scratch test to be a relatively small load of 10 N. The first adhesive cracks were observed after Lc = 5.3 N for the untilted sample.The average value of the friction coefficient during the measurements was calculated to be 0.  Local detachment of the coating is seen on the side of the track and in several other regions that are identified by arrows in Figure 10a.We concluded that the coating demonstrates sufficient scratch resistance, which is in accordance with the scientific data available in the literature [60,61].In contrast, samples deposited at inclinations of 60° and 80° (Figure 10b,c) show higher scratch resistance.Thus, coating delamination even at the maximum load of 10 N is not observed.The average value of the friction coefficient for both the tilted substrates during the measurements did not exceed 0.25.This fact also contributed to the observed, improved scratch resistance of the tilted samples.We associate Local detachment of the coating is seen on the side of the track and in several other regions that are identified by arrows in Figure 10a.We concluded that the coating demonstrates sufficient scratch resistance, which is in accordance with the scientific data available in the literature [60,61].In contrast, samples deposited at inclinations of 60 • and 80 • (Figure 10b,c) show higher scratch resistance.Thus, coating delamination even at the maximum load of 10 N is not observed.The average value of the friction coefficient for both the tilted substrates during the measurements did not exceed 0.25.This fact also contributed to the observed, improved scratch resistance of the tilted samples.We associate these results with the high energy of sputtered species impinging the substrate during the condensation phase because of the placement of the inclined substrate closer to the particle source.The influence of bombardment energy on the level of scratch resistance can also be found in the literature [62].

Conclusions
Two approaches for achieving a possible synergetic antibacterial effect by manufacturing a patterned surface of Zn-doped calcium phosphate using GLAD technique in RF magnetron sputtering were used.Two types of substrates (Ti and Si) with significantly different roughness were chosen.Variations in coating thickness and uniformity take place starting from a tilt angle of 60 • .It was shown that it is possible to manipulate the surface roughness and morphology of the coatings by varying the substrate tilt angle.The columnar structure becomes more prominent at an oblique angle of 80 • , and it is made of inclined Zn-doped CaP columns.A significant change in the coating morphology becomes obvious starting from the substrate tilt angle of 60 • .An increase in the coating crystallinity for samples deposited at a tilt angle of 80 • corresponds to the formation of crystallites in the bulk structure of the thin film.It was shown that GLAD is a powerful technique that allows for the variation of CaP thin film morphology and can be utilized to create self-organized nanostructures that can be used for synergetic antibacterial effect.

Figure 1 .
Figure 1.Schematic image of the deposition process of the Ti and Si samples with varying particle incidence angles at 0°, 60°, and 80° during the radiofrequency (RF) magneton sputtering of a Znsubstituted hydroxyapatite (Zn-HA) target with the arrows indicating the direction of the flux.

Figure 1 .
Figure 1.Schematic image of the deposition process of the Ti and Si samples with varying particle incidence angles at 0 • , 60 • , and 80 • during the radiofrequency (RF) magneton sputtering of a Zn-substituted hydroxyapatite (Zn-HA) target with the arrows indicating the direction of the flux.

Figure 2 .
Figure 2. Plot of the coating thickness against the substrate tilt angle.

Figure 2 .
Figure 2. Plot of the coating thickness against the substrate tilt angle.

Figure 4 .
Figure 4. Dispersion of coating surface grain sizes for samples deposited under tilt angles of 0° (a) and 80° (b) at a working gas pressure of 0.8 mTorr.

Figure 4 .
Figure 4. Dispersion of coating surface grain sizes for samples deposited under tilt angles of 0° (a) and 80° (b) at a working gas pressure of 0.8 mTorr.

Figure 4 .
Figure 4. Dispersion of coating surface grain sizes for samples deposited under tilt angles of 0 • (a) and 80 • (b) at a working gas pressure of 0.8 mTorr.

Figure 5 .
Figure 5. CaP coatings deposited on Si at a working gas pressure of 0.8 mTorr under tilt angles of 0 • (a), 60 • (b), or 80 • (c) on the Si substrates.

Figure 6 .
Figure 6.SEM cross-section images of obliquely deposited Zn-doped CaP thin films deposited on Si under tilt angles of 0° (a) and 80° (b) at 100000× magnification and films under tilt angles of 0° (c) and 80° (d) at 200000× magnification on Si substrates, demonstrating significant changes in the film morphology.The arrows indicate the angle of incidence θ.The columns are tilted by angle β.

Figure 6 .
Figure 6.SEM cross-section images of obliquely deposited Zn-doped CaP thin films deposited on Si under tilt angles of 0 • (a) and 80 • (b) at 100,000× magnification and films under tilt angles of 0 • (c) and 80 • (d) at 200,000× magnification on Si substrates, demonstrating significant changes in the film morphology.The arrows indicate the angle of incidence θ.The columns are tilted by angle β.

Figure 7 .
Figure 7. CaP coatings deposited on Ti at a working gas pressure of 0.8 mTorr under tilt angles of 0° (a) and 80° (b) and three-dimensional (3D) visualization of coating deposited under 80° showing the out-of-plane growth of grains (c).

Figure 7 .
Figure 7. CaP coatings deposited on Ti at a working gas pressure of 0.8 mTorr under tilt angles of 0 • (a) and 80 • (b) and three-dimensional (3D) visualization of coating deposited under 80 • showing the out-of-plane growth of grains (c).

Figure 7 .
Figure 7. CaP coatings deposited on Ti at a working gas pressure of 0.8 mTorr under tilt angles of 0° (a) and 80° (b) and three-dimensional (3D) visualization of coating deposited under 80° showing the out-of-plane growth of grains (c).

Figure 8 .
Figure 8. CaP coatings deposited on Si at a working gas pressure of 0.8 mTorr under inclination angles of 0° (a) and 80° (b) and 3D visualization of coating deposited under 80° (c).

Figure 8 .
Figure 8. CaP coatings deposited on Si at a working gas pressure of 0.8 mTorr under inclination angles of 0 • (a) and 80 • (b) and 3D visualization of coating deposited under 80 • (c).

Figure 8 .
Figure 8. CaP coatings deposited on Si at a working gas pressure of 0.8 mTorr under inclination angles of 0° (a) and 80° (b) and 3D visualization of coating deposited under 80° (c).

Figure 10 .
Figure 10.Optical microscopy of the tracks after scratch testing of the CaP coatings deposited on Ti at a working gas pressure of 0.8 mTorr under inclination angles of 0° (a), 60° (b), and 80° (c).

Figure 9 .
Figure 9. Representative X-ray powder diffractograms (with Rietveld refinement) of CaP coatings deposited on the Ti substrate perpendicular to the flux at 0 • (a) and inclined at 80 • (b).The diffraction peaks of HA and Ti (with Miller indices) are shown.

3 .Figure 9 .
Figure 9. Representative X-ray powder diffractograms (with Rietveld refinement) of CaP coatings deposited on the Ti substrate perpendicular to the flux at 0° (a) and inclined at 80° (b).The diffraction peaks of HA and Ti (with Miller indices) are shown.

Figure 10 .
Figure 10.Optical microscopy of the tracks after scratch testing of the CaP coatings deposited on Ti at a working gas pressure of 0.8 mTorr under inclination angles of 0° (a), 60° (b), and 80° (c).

Figure 10 .
Figure 10.Optical microscopy of the tracks after scratch testing of the CaP coatings deposited on Ti at a working gas pressure of 0.8 mTorr under inclination angles of 0 • (a), 60 • (b), and 80 • (c).

Table 1 .
Deposition parameters and coating properties for glancing angle deposition (GLAD) with magnetron sputtering from the literature.