Effect of Doping Element and Electrolyte’s pH on the Properties of Hydroxyapatite Coatings Obtained by Pulsed Galvanostatic Technique

: Hydroxyapatite (HAp) is the most widely used calcium phosphate as a coating on metal implants due to its biocompatibility and bioactivity. The aim of this research is to evaluate the effect of the pH’s electrolyte and doping element on the morphology, roughness, chemical, and phasic composition of hydroxyapatite-based coatings obtained by pulsed galvanostatic electrochemical deposition. As doping elements, both Sr and Ag were selected due to their good osseoinductive character and antibacterial effect, respectively. The electrolytes were prepared at pH 4 and 5, in which speciﬁc concentrations of Sr, Ag, and Sr + Ag were added. In terms of morphology, all coatings consist in ribbon-like crystals, which at pH 5 appear to be a little larger. Addition of Sr did not affect the morphology of HAp, while Ag addition has led to the formation of ﬂower-like crystals agglomeration. When both doping elements were added, the ﬂowers like agglomerations caused by the Ag have diminished, indicating the competition between Sr and Ag. X-Ray Diffraction analysis has highlighted that Sr and/or Ag have successfully substituted the Ca in the HAp structure. Moreover, at higher pH, the crystallinity of all HAp coatings was enhanced. Thus, it can be said that the electrolyte’s pH enhances to some extent the properties of HAp-based coatings, while the addition of Sr and/or Ag does not negatively impact the obtained features of HAp, indicating that by using pulsed galvanostatic electrochemical deposition, materials with tunable features dictated by the function of the coated medical device can be designed. Author Contributions: Conceptualization, and E.U.; methodology, C.M.C.; validation, A.V. and M.T.; investigation, E.U., A.C.P. and D.M.V.; data curation, C.M.C. and A.V.; writing—original preparation, E.U. and D.M.V.; writing—review and editing, C.M.C., D.M.V. and A.V.; visualiza-tion, E.U. and


Introduction
In the past years, the use of implants has grown considerably, driven by ageing of populations and the desire of the patients to maintain the same level of activity and quality of life [1].
Among the metallic biomaterials, titanium is the most used due to its excellent properties such as the modulus of elasticity close to that of bone, biocompatibility, and excellent corrosion resistance, being considered the "gold standard" [2,3]. Titanium, due to its bioinert character does not favor the formation of a direct connection with the hard tissue nor the initiation of the bone remodeling mechanism in the early stages after implantation [4].
Imparting multifunctionality of bioinert metals such as Ti and its alloys is generally achieved through surface modification techniques [5], such as coating the metallic biomaterials with bioactive ceramics and/or polymers to improve their osseointegration and bioactivity [6]. or alkaline treatment). To the best of our knowledge, there are only a few research on the Sr and Ag co-doped hydroxyapatite coatings obtained through electrochemical deposition [23,34].
To achieve this goal, different pH values of 4 and 5 were used, since a pH value higher than 4.5 leads to obtaining coatings with high crystallinity [35,36]. Besides this, the concentration of electrolytes used does not allow to modify the electrolyte's pH at values higher than 5. Any attempt to further increase the pH leads to the formation of insoluble ionic solid (precipitate) in the electrolyte. According to the literature [37][38][39], if the pH of the electrolyte is lower than 4.5, other phases are obtained unless a high current density or high deposition temperature is applied.

Coating Preparation
The undoped and doped HAp-based coatings with Sr and/or Ag were obtained on pure titanium substrate (cp-Ti, grade 2). The cp-Ti samples were sliced from a titanium bar with a diameter of 20 mm purchased from BibusMetals, AG (Essen, Germany) under the form of discs with a thickness of 2 mm.
The cp-Ti samples were ground on a SiC metallographic paper of different grits, from 320 to 800. After the grinding process, the samples were rinsed with ultrapure water, degreased using isopropyl alcohol, and cleaned in acetone for 30 min in an ultrasonic bath (Bandelin, Berlin, Germany). Subsequently they were rinsed with ultrapure water (ASTM I) and dried in air.
The electrochemical deposition was carried out in a standard electrochemical cell configured with three electrodes: working electrode (WE), reference electrode (RE), and auxiliary electrode (AE). The reference electrode was a saturated calomel electrode (SCE), while the auxiliary electrode was a platinum plate. The prepared cp-Ti discs were mounted in a Teflon samples holder, with an exposed area to the electrolyte of~2 cm 2 . Figure 1 shows the electrochemical cell configuration used during the electrochemical experiments of undoped and doped HAp coatings deposited on cp-Ti substrate. The electrochemical deposition was performed with a potentiostat/galvanostat PARSTAT MC equipped with PMC-1000 module (Princeton Applied Research-AMETEK, Berwyn, PA, USA). a higher value of the electrolyte's pH leads to the release of a smaller amount of hydrogen, obtaining more uniform and compact layers [31].
Aim of this research is to develop suitable approaches for Sr and Ag single-doped and co-doped hydroxyapatite-based coatings on Ti substrate, through pulsed galvanostatic deposition technique, without any additional treatments (i.e., high temperature treatments or alkaline treatment). To the best of our knowledge, there are only a few research on the Sr and Ag co-doped hydroxyapatite coatings obtained through electrochemical deposition [23,34].
To achieve this goal, different pH values of 4 and 5 were used, since a pH value higher than 4.5 leads to obtaining coatings with high crystallinity [35,36]. Besides this, the concentration of electrolytes used does not allow to modify the electrolyte's pH at values higher than 5. Any attempt to further increase the pH leads to the formation of insoluble ionic solid (precipitate) in the electrolyte. According to the literature [37][38][39], if the pH of the electrolyte is lower than 4.5, other phases are obtained unless a high current density or high deposition temperature is applied.

Coating Preparation
The undoped and doped HAp-based coatings with Sr and/or Ag were obtained on pure titanium substrate (cp-Ti, grade 2). The cp-Ti samples were sliced from a titanium bar with a diameter of 20 mm purchased from BibusMetals, AG (Essen, Germany) under the form of discs with a thickness of 2 mm.
The cp-Ti samples were ground on a SiC metallographic paper of different grits, from 320 to 800. After the grinding process, the samples were rinsed with ultrapure water, degreased using isopropyl alcohol, and cleaned in acetone for 30 min in an ultrasonic bath (Bandelin, Berlin, Germany). Subsequently they were rinsed with ultrapure water (ASTM I) and dried in air.
The electrochemical deposition was carried out in a standard electrochemical cell configured with three electrodes: working electrode (WE), reference electrode (RE), and auxiliary electrode (AE). The reference electrode was a saturated calomel electrode (SCE), while the auxiliary electrode was a platinum plate. The prepared cp-Ti discs were mounted in a Teflon samples holder, with an exposed area to the electrolyte of ~2 cm 2 . Figure 1 shows the electrochemical cell configuration used during the electrochemical experiments of undoped and doped HAp coatings deposited on cp-Ti substrate. The electrochemical deposition was performed with a potentiostat/galvanostat PARSTAT MC equipped with PMC-1000 module (Princeton Applied Research-AMETEK, Berwyn, PA, USA). The chemical composition of the electrolyte is shown in Table 1. The electrolyte was prepared by successive addition of reagents Ca(NO3)2·4H2O, NH4H2PO4, AgNO3, Sr(NO3)2 in ultra-pure water (ASTM I). The reagents were purchased from Sigma Aldrich (St. Louis, MO, USA), and were of analytical grade. All electrolytes were prepared with respect to the Ca/P ratio of 1.67 [35,36]. The chemical composition of the electrolyte is shown in Table 1. The electrolyte was prepared by successive addition of reagents Ca(NO 3 ) 2 ·4H 2 O, NH 4 H 2 PO 4 , AgNO 3 , Sr(NO 3 ) 2 in ultra-pure water (ASTM I). The reagents were purchased from Sigma Aldrich (St. Louis, MO, USA), and were of analytical grade. All electrolytes were prepared with respect to the Ca/P ratio of 1.67 [35,36].
During the electrochemically assisted deposition of HAp coatings, the electrolyte was maintained at a constant temperature of 75 ± 0.5 • C using a magnetic stirrer/RCT with heater (IKA, Staufen im Breisgau, Germany). The deposition temperature of 75 • C was selected because it favors the electrochemical deposition of crystalline HAp with enhanced features [40]. Moreover, for homogenization of the electrolyte, the solution was constantly stirred during deposition. The pulsed galvanostatic deposition technique was applied according to the following design: i ON = −0.85 mA/cm 2 was applied for 1 s, followed by a break (i OFF = 0 mA/cm 2 ) for 2 s. This cycle was repeated for 900 times, and thus the entire deposition process took 45 min. After the electrochemical depositions of the undoped and doped HAp coatings, the samples were rinsed with distilled water and dried in a hot air jet.
The current density was selected based on previous reports [28,[41][42][43] and was selected because at low current density HAp crystals exhibit a retardant precipitation rate near the cathode, and thus the crystal grows steadily, showing a greater crystal texture [44]. Nevertheless, all the other electrochemical parameters involved in the deposition process must also be taken into consideration when developing coatings with tunable properties.

Characterization
The morphology and elemental composition for the undoped and doped hydroxyapatite coating were analyzed using a scanning electron microscope (SEM) equipped with an X-ray energy dispersive spectrometer (EDS), Phenom ProX, (Phenom World, Eindhoven, The Netherlands).
The phase composition was investigated by X-ray diffraction, using a SmartLab X-Ray (Rigaku, Tokyo, Japan) diffractometer with CuKα radiation (λ = 1.5406 Å) in the range θ/2θ of 20 • -80 • at an incident angle of 3 • . For this purpose, a medium resolution parallel beam optical system was used. The crystallite size was calculated using the Debye-Scherrer mathematical relation (Equation (1)) for the plane (002), being the most intense diffraction peak of electrochemically obtained HAp coatings [45], while the structural properties of the samples under study were obtained from XRD diffractograms.
where K = 0.9 is a constant shape factor for HAp with hexagonal structure, β is the full width at half maximum (FWHM) measured in radian of (002) diffraction plane, and θ in degrees, indicates the Braggs diffraction angle, λ = 1.5406 Å, is the wavelength of CuKα radiation.

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The roughness of the coatings was investigated using a Dektak 150 (Veeco Instruments, Plainview, NY, USA) contact profilometer over a length of 3 mm for 60 s. From the profiles obtained were determined parameters Ra (the arithmetic average of the absolute deviations of the roughness profile from the mean value), Rq (the square root of the arithmetic mean of the squares of the deviations profile from the mean value), and R Sk (factor asymmetry of the evaluated profile). For each sample a number of 10 measurements were performed and the average along with the obtained standard deviation was considered. Figure 2 shows the macroscopic appearance along with the SEM images at different magnifications of the obtained coatings for both pH values, of 4 and 5, respectively. Based on macroscopic images of the coatings after deposition, it can be observed that all coatings are fully covering the surface of the cp-Ti metallic substrate, and no defects were visible to the naked eye. Thus, from this point of view, it can be stated that the coatings were successfully achieved with respect to the selected electrochemical deposition parameters employed in this study. It is widely recognized that the electrochemically synthesized hydroxyapatite can nucleate and grow under various morphologies which sensitively depend on the specific growth conditions [54,55]. In galvanostatic technique, higher reaction rates for phosphate and hydroxyl ion are enforced, influencing the chemical precipitation reaction and thus, the layer morphology and growth rates [56]. Of course, the electrolytes should not be neglected, since a more acidic pH leads to the formation of a higher quantity of hydrogen gas and thus the reactions are more energetic [31]. Nevertheless, because the coatings are deposited in pulses, the repeatedly interrupted process of nucleation and growth of precipitates at, theoretically, nearly constant ion concentrations lead to structures that are usually more compact and uniform.

Elemental Composition
The EDS analysis (Table 2) revealed the presence of Ti from cp-Ti used as substrate, but also the elements characteristic for hydroxyapatite (Ca, P, and O) as well as the doping elements (Ag and Sr), indicating that the coatings were successfully achieved by pulse galvanostatic technique.
Because both elements Ag and Sr ions substitute the Ca ions found in the HAp lattice, the (Ca + M)/P ratio was calculated [43,[57][58][59], where M is the doping element, Sr, or/and Ag, respectively. In Table 2 is presented the chemical composition of all the coatings obtained at different pH values. In terms of Ca/P ratio evolution, the obtained coatings registered a ratio below the stoichiometric one of 1.67, indicating that all coatings are Cadeficient, which according to some studies may be beneficial, since calcium phosphate's SEM images are presented in Figure 2 and highlight that the coatings morphology consists of long and wide ribbon-like crystals with a thin in nanometric level range, regardless of the electrolyte's pH value. A possible explanation of this morphology could be attributed to electrolyte concentration [51]. The preferential direction in the crystallization process is perpendicular to the surface of the substrate along the c-axis, which corresponds to the (002) reflection, this being characteristic for the coatings obtained via electrochemical techniques [43,52].
At higher magnification, it can be noticed that the electrolyte's pH induces slight changes in terms of ribbons size, highlighting that at pH 5 the ribbons are slightly narrower. Since the dimension of the ribbon-like crystals could not be measured due to their threedimensional orientation, only a qualitative observation could be made. Therefore, the difference between the coatings obtained at the two pH's can be represented by the crystal's density reported to the investigated area. Based on this, it can be highlighted that at more acidic pH (pH 4) the HAp crystals appear to be smaller and denser, while at higher pH (pH 5) the crystals present some increment of the crystals, forming a more uniform layer, which seems to have lower density. Thus, it can be said that by using a higher pH value, a more uniform layer consisting of crystals with similar dimensions can be obtained.
From the point of view of the doping elements added in the electrolyte, the following were observed. Even though at smaller magnification, the addition of Sr did not induce significant changes on the HAp morphology, at higher one it was observed that the crystals are narrower compared to undoped HAp coatings.
In the case of HAp doped with Ag, the predominant morphology consists in ribbons, coupled with flower-like agglomerations. These findings are consistent with the ones found in the literature [53], being usually met in electrochemical methods.
In the coatings in which both doping elements were added (H-Sr-Ag), the morphology consists of ribbons and flower-like crystals. Moreover, it was also noted that at the pH 5, the number of flowers-like agglomeration significantly decreases, emphasizing that these modifications are in strong correlation with the electrolyte's pH value.
It is widely recognized that the electrochemically synthesized hydroxyapatite can nucleate and grow under various morphologies which sensitively depend on the specific growth conditions [54,55]. In galvanostatic technique, higher reaction rates for phosphate and hydroxyl ion are enforced, influencing the chemical precipitation reaction and thus, the layer morphology and growth rates [56]. Of course, the electrolytes should not be neglected, since a more acidic pH leads to the formation of a higher quantity of hydrogen gas and thus the reactions are more energetic [31]. Nevertheless, because the coatings are deposited in pulses, the repeatedly interrupted process of nucleation and growth of precipitates at, theoretically, nearly constant ion concentrations lead to structures that are usually more compact and uniform.

Elemental Composition
The EDS analysis (Table 2) revealed the presence of Ti from cp-Ti used as substrate, but also the elements characteristic for hydroxyapatite (Ca, P, and O) as well as the doping elements (Ag and Sr), indicating that the coatings were successfully achieved by pulse galvanostatic technique. Because both elements Ag and Sr ions substitute the Ca ions found in the HAp lattice, the (Ca + M)/P ratio was calculated [43,[57][58][59], where M is the doping element, Sr, or/and Ag, respectively. In Table 2 is presented the chemical composition of all the coatings obtained at different pH values. In terms of Ca/P ratio evolution, the obtained coatings registered a ratio below the stoichiometric one of 1.67, indicating that all coatings are Ca-deficient, which according to some studies may be beneficial, since calcium phosphate's constitution in human bones is non-stoichiometric [60][61][62].
By comparing the same coating type at the two pH's selected, it can be observed that a higher pH leads to an enhancement of the Ca/P ratios (Figure 3a), with values closer to the stoichiometric one. In terms of doping element, it can be observed that irrespective of the pH value (Figure 3a), the H-Ag coatings have registered the highest Ca/P ratio, of 1.62 at pH 4 and 1.64 at pH 5, while the lowest were obtained for the H-Sr coatings, of 1.45 at pH 4 and 1.49 at pH 5. By co-doping the HAp with Sr and Ag, the obtained ratios were found to be between the highest and lowest values, with values of 1.51 at pH 4 and 1.54 at pH 5. Overall, it can be said that the addition of Ag increases the Ca/P ratio, Sr decreases it, while when both elements are added, it can be observed that the two elements are competing each other, leading to a ratio that is slightly higher than the one obtained for the H-Sr coatings, indicating that the addition of Ag in the H-Sr coatings has a beneficial effect.

Phase Composition
The XRD diffractograms of the undoped and doped HAp presented in Figure 4, revealed that all obtained coatings consist of hydroxyapatite (HAp, card no. 09-0432) as the main phase in accordance with the International Center for Diffraction Data (ICDD), regardless the pH of the electrolyte. A small diffraction peak located at 30.31° was assigned to monetite (CaHPO4) according to ICDD card No. 01-071-1759, indicating the presence of a secondary phase. Monetite is an important calcium phosphate precursor that promotes cell proliferation, being recognized for its osseoinductive and osteoconductive character [69]. Another important aspect highlighted by the EDS analysis was that by increasing the pH value from 4 to 5, the at.% of Ag was reduced from 1.80 to 0.86 in the co-doped coatings (H-Sr-Ag), and from 1.67 to 1.08 for the H-Ag coatings, indicating that even though the initial silver concentration is the same, the pH value strongly influences the Ag content in the HAp-based coatings (Figure 3b). A similar observation was also made by Narendran et al. [63], which has evidenced that by increasing the pH value, the Ag amount decreases. Oppositely, the Sr quantity remains approximately at the same values (between 3.40 at.% and 3.52 at.%), irrespective of the pH level or the coating type (H-Sr and/or H-Sr-Ag).
The silver quantity and its chemical stability within the material is of crucial importance since a quantity of 6 wt.% of Ag [64], can induce not just the desired antibacterial effect but also cell apoptosis, which in medical applications must be avoided or, if not possible, at least to be reduced at minimum. In accordance with the in vitro assays on human osteoblasts, an Ag content between 2 wt.% and 4 wt.% provides a fine balance between cytotoxicity and antimicrobial activity [64,65]. Within this context, it must be said that the H-Ag and H-Sr-Ag coatings at pH 5 had values lower than 4 wt.%, of 3.02 wt.%, and 2.40 wt.%, respectively, while at pH 4 both coatings had values of~4.8 wt.% (Figure 3b).
In a study performed by Ciuca et al. [17] it was proven that 2 at.% of Ag is sufficient to enhance the resistance to candida albicans, a Gram-positive bacteria, known to affect prosthetic joints [66][67][68], while Badea et al. [20] highlighted that even a small quantity of Ag (0.7 at.%) into hydroxyapatite coatings can improve the antibacterial properties of hydroxyapatite.
Since the amount of Ag is of critical importance, a balance between these two characteristics, antibacterial effect, and biocompatibility [59], must be taken into consideration when designing a material. Thus, based on these findings, it can be stated that by using a pH of 5, the Ag amount can be obtained within its optimum concentration in terms of antibacterial effect without affecting the cell behavior.

Phase Composition
The XRD diffractograms of the undoped and doped HAp presented in Figure 4, revealed that all obtained coatings consist of hydroxyapatite (HAp, card no. 09-0432) as the main phase in accordance with the International Center for Diffraction Data (ICDD), regardless the pH of the electrolyte.

Phase Composition
The XRD diffractograms of the undoped and doped HAp presented in Figure 4, revealed that all obtained coatings consist of hydroxyapatite (HAp, card no. 09-0432) as the main phase in accordance with the International Center for Diffraction Data (ICDD), regardless the pH of the electrolyte. A small diffraction peak located at 30.31° was assigned to monetite (CaHPO4) according to ICDD card No. 01-071-1759, indicating the presence of a secondary phase. Monetite is an important calcium phosphate precursor that promotes cell proliferation, being recognized for its osseoinductive and osteoconductive character [69].
Even though, the XRD analysis was performed in grazing mode, the XRD diffractograms also showed the diffraction peaks specific to the titanium substrate according to ICDD card No. 044-1298, and indirectly suggesting that the coatings are not very thick.
With respect to the main phase of the coatings, attributed to HAp, the maximum diffraction intensity found at 2θ of ~26° indicates that the preferential orientation is in the direction of the c-axis, according to (002) plan, which is in good agreement with the SEM images, in which was shown that the HAp crystals grow perpendicular on the substrate surface. These findings are similar to the ones already reported in the literature [70][71][72] and which have proven that crystalline HAp with a c-axis orientation increases the A small diffraction peak located at 30.31 • was assigned to monetite (CaHPO 4 ) according to ICDD card No. 01-071-1759, indicating the presence of a secondary phase. Monetite is an important calcium phosphate precursor that promotes cell proliferation, being recognized for its osseoinductive and osteoconductive character [69].
Even though, the XRD analysis was performed in grazing mode, the XRD diffractograms also showed the diffraction peaks specific to the titanium substrate according to ICDD card No. 044-1298, and indirectly suggesting that the coatings are not very thick.
With respect to the main phase of the coatings, attributed to HAp, the maximum diffraction intensity found at 2θ of~26 • indicates that the preferential orientation is in the direction of the c-axis, according to (002) plan, which is in good agreement with the SEM images, in which was shown that the HAp crystals grow perpendicular on the substrate surface. These findings are similar to the ones already reported in the literature [70][71][72] and which have proven that crystalline HAp with a c-axis orientation increases the capacity of osseointegration, by promoting cell proliferation, and presenting a higher chemical stability.
No specific peaks of Ag and/or Sr in metallic form were identified by XRD analysis. Due to the differences among the ionic radius of Ca 2+ (0.99 Å), Sr 2+ (1.2 Å), and Ag + (1.28 Å), the diffraction peaks shifted toward smaller angles (Figure 4) with the addition of Sr 2+ and Ag + denoting an expansion in the crystal lattice caused by the partial incorporation of Ag and/or Sr into HAp, rather than just a surface modification [23,73].
In Table 3 is presented the degree of crystallinity, lattice parameters, and crystallite sizes.
A narrower peak suggests that both lattice parameters and the crystallinity increase [74]. Thus, compared to HAp, addition of the doping elements with higher ionic radius than Ca has led to an increment of the lattice parameters a and c, which is in good agreement with other studies [75][76][77]. However, by increasing the electrolyte's pH to 5, the lattice parameters presented slightly higher values for the same coating type. The same trend was also noted for the crystallinity degree which had values between 46% and 50%, but also for the crystallite dimension, indicating that a higher pH value favors the development of more crystalline HAp-based coatings. It can be observed that the H-Sr coatings, irrespective of pH value, present very close crystallite dimension and crystallinity. By correlating these results ( Figure 5) with EDS analysis, which highlighted that Sr is found in approximately equal amounts (3.52 at.% at pH 4 and 3.45 at.% at pH 5), it can be stated that the electrolyte's pH does not have such a great effect on the quantities of Sr as dopant in HAp structure, nor the crystallite dimension or crystallinity. Thus, as a general observation, it can be said that XRD analysis has confirmed that the HAp-based coatings are doped with Ag and/or Sr and that by increasing the pH value from 4 to 5, the crystallinity degree increases.

Roughness
Beside phase composition, and crystallinity of the coatings, surface free energy, porosity, as well as their surface roughness play important parts in the pursuit for enhancing the biomedical performance of implantable materials [6]. Surface roughness is among the characteristics that can affect the cell behavior [78]. Thus, osteoblasts prefer a rougher surface, while fibroblasts prefer a smoother surface [79]. Usually, it is classified into macro and nano-roughness, and each of them influences the properties of the surface differently [80]. Regarding the effect of the roughness on the bacteria attachment, it should be said that this is a very complex process which is controlled by interplay between physicochemical, topographical surface properties, bacterial characteristics, and environmental conditions [81]. Thus, a correlation between bacterial adhesion and just the surface roughness is challenging or even impossible to make, since more aspects need to be taken into Oppositely, Ag appears to be dependent on the pH value, revealing a different behavior. Thus, at higher pH, the crystallite dimension and crystallinity decrease, which was to be expected since a smaller quantity of Ag (1.02 at.%) was observed in H-Ag coatings at pH 5.
By co-doping the HAp coatings with Ag and Sr (H-Sr-Ag), the crystallite dimension and crystallinity have registered an increment at pH 5, compared to pH 4. These results can also be correlated with the elemental composition but also with the competition between Sr and Ag.
Thus, as a general observation, it can be said that XRD analysis has confirmed that the HAp-based coatings are doped with Ag and/or Sr and that by increasing the pH value from 4 to 5, the crystallinity degree increases.

Roughness
Beside phase composition, and crystallinity of the coatings, surface free energy, porosity, as well as their surface roughness play important parts in the pursuit for enhancing the biomedical performance of implantable materials [6]. Surface roughness is among the characteristics that can affect the cell behavior [78]. Thus, osteoblasts prefer a rougher surface, while fibroblasts prefer a smoother surface [79]. Usually, it is classified into macro and nano-roughness, and each of them influences the properties of the surface differently [80]. Regarding the effect of the roughness on the bacteria attachment, it should be said that this is a very complex process which is controlled by interplay between physico-chemical, topographical surface properties, bacterial characteristics, and environmental conditions [81]. Thus, a correlation between bacterial adhesion and just the surface roughness is challenging or even impossible to make, since more aspects need to be taken into consideration.
In Figures 6 and 7 are presented the profile lines specific for each coating and the main roughness parameters.  The asymmetry of the surface distribution also known as skewness (RSk) provides information on the distribution of hills (slope < 0) for an area with deeper valleys, and the distribution of the valleys (slope > 0) for an area with higher hills. Thus, based on this criterion, all the coatings showed a positive asymmetry, suggesting that all surfaces are predominant in hills. Nevertheless, just as observed for Ra parameter, the addition of Ag at lower pH values, generated higher hills compared to the ones obtained at higher pH, and thus a higher RSk, most likely due to the presence of the flower-like crystals.  The asymmetry of the surface distribution also known as skewness (RSk) provides information on the distribution of hills (slope < 0) for an area with deeper valleys, and the distribution of the valleys (slope > 0) for an area with higher hills. Thus, based on this criterion, all the coatings showed a positive asymmetry, suggesting that all surfaces are predominant in hills. Nevertheless, just as observed for Ra parameter, the addition of Ag at lower pH values, generated higher hills compared to the ones obtained at higher pH, and thus a higher RSk, most likely due to the presence of the flower-like crystals. According to the obtained results, it can be stated that by increasing the pH value from 4 to 5, the average roughness (Ra) and root mean square roughness (Rq) parameters present some modifications with respect to the doping elements and pH value as following.
As a general remark, all coatings have registered higher Ra than the Ti substrate (Ra = 86 nm). By comparing the undoped HAp at the two selected pHs, it can be observed that by increasing the pH level, the Ra parameter also increased with~100 nm. A similar trend is also visible for the coatings doped with Sr, which at pH 5 had an Ra of 403 nm, compared to 248 nm obtained at pH 4. Addition of Ag in the H-Ag or H-Sr-Ag coatings has led to a decrement of the roughness at higher pH value, from 510 and 904 to values of 390 and 684 nm, respectively. These findings can be correlated with the SEM images, which have highlighted that compared to pH 5, at lower pH value, multiple flower-like formations were found. Thus, the presence of these morphologies, can explain the higher roughness obtained at pH 4 for the H-Ag and H-Sr-Ag coatings.
The asymmetry of the surface distribution also known as skewness (R Sk ) provides information on the distribution of hills (slope < 0) for an area with deeper valleys, and the distribution of the valleys (slope > 0) for an area with higher hills. Thus, based on this criterion, all the coatings showed a positive asymmetry, suggesting that all surfaces are predominant in hills. Nevertheless, just as observed for Ra parameter, the addition of Ag at lower pH values, generated higher hills compared to the ones obtained at higher pH, and thus a higher R Sk , most likely due to the presence of the flower-like crystals.
Moreover, by comparing the R Sk parameter of the Ti substrate, with the ones obtained for the coatings, a decrement can be observed indicating that the coatings tend to smoothen the surface. It was also noted that the coatings obtained at pH 5 present a lower R Sk than the ones obtained at pH 4.
In a study performed by Evgeny et al. [82] it was found that increasing positive skewness is an advantage for having good corrosion resistance, while surfaces with negative skewness mainly exhibit pitting corrosion. The surface with negative skewness consists in many grooves which could develop the micro-reaction sites and trap the corrosion products formed at the base of the grooves, which can accelerate the growth of pits. Skewness parameter can also be correlated with other types of investigations such as biodegradation because changes of this parameter could be used to monitor time-dependent biodegradation of materials [83].
Thus, by increasing the pH level, a more uniform surface with an average roughness between 380 and 680 nm, and a tendency toward a symmetrical topography, can be obtained, irrespective of the doping element used.

Conclusions
According to this study, the galvanostatic pulse technique is a versatile technique that allows to obtain uniform coatings and the pH of the electrolyte is among the most important parameters involved in the deposition process. Thus, at a higher pH, more uniform layers were obtained, regardless of the doping element, with a Ca/P ratio closer to the stoichiometric hydroxyapatite and higher crystallinity. The doping elements were successfully substituted in the HAp structure regardless the electrolyte's pH, this aspect being highlighted by XRD analysis, which have also shown that addition of elements with a higher ionic radius than Ca, lead to an increment of the lattice parameters. The crystallite dimension has increased with respect to the doping elements added. Moreover, irrespective of the doping element used, it was shown that the HAp coatings obtained at a pH of 5 present a more uniform surface with an average roughness between 380 and 680 nm, and a tendency toward a symmetrical topography.
Nevertheless, the present study has also highlighted one important aspect, namely that the pH value of the electrolytes has a weak influence on the quantities of Sr as dopant, while Ag is in strong dependency with the pH value at which the electrochemical deposition is carried out, as demonstrated by the SEM, EDS, and XRD analysis.
Within the limitations of the current study, it can be said that the coatings obtained at pH 5 revealed superior properties compared to those obtained at pH 4, and addition of Ag and Sr also play important parts in developing such type of coatings. Nevertheless, the mechanism governing such an enhancement is not clearly understood and further studies must be made. Thus, as future direction, the coatings obtained at higher pH will be further evaluated in terms of electrochemical behavior in synthetic media, along with their biomineralization ability and in vitro cells behavior.