Physico-Chemical Properties and In Vitro Antifungal Evaluation of Samarium Doped Hydroxyapatite Coatings

: Hydroxyapatite (HAp) and samarium doped hydroxyapatite, Ca 10 − x Sm x (PO 4 ) 6 (OH) 2 , x Sm = 0.05, (5SmHAp), coatings were prepared by sol-gel process using the dip coating method. The stability of 5SmHAp The toxicity of the 5SmHap was also investigated in vitro using HeLa cell line. suspensions and 84% in the case of the HAp coatings and also 83% in the case of 5SmHAp suspensions


Preparation of HAp and 5SmHAp Coatings
The HAp and 5SmHAp coatings have been deposited on the Si wafer by the sol-gel process using the dip coating method in agreement with previous studies [25]. The Si substrate was washed with ethanol absolute before coating. The 5SmHAp layer was dried at 100 • C for 4 h and heat treated at 500 • C for 2 h. Cooling was done at a rate of 5 • C/min.

Characterization Methods
Ultrasonic measurements were performed on 100 mL of concentrated suspension of 5SmHAp [22,26]. The digitalized ultrasonic signals were recorded on the digital oscilloscope (General-Electric, Krautkramer, Germany) at a very precise interval of 5.00 s. In order to have an accurate evaluation of the stability of the 5SmHAp suspension, the double distilled water (the most stable suspension) was chosen as the reference fluid, under the same experimental conditions.
The morphology of the 5SmHAp suspensions was analyzed using transmission electron microscopy (TEM) with a CM 20 (Philips-FEI, Hillsboro, OR, USA) transmission electron microscope having a Lab6 filament (Agar Scientific Ltd., Stansted, UK), which operates at 200 kV. The particle size distributions from the TEM micrographs was performed by measuring approximately 700 particles from different regions of the sample.
The morphology of 5SmHAp suspensions and coatings were evaluated by scanning electron microscopy (SEM) using a HITACHI S4500 microscope (Hitachi, Ltd., Tokyo, Japan). The 5SmHAp suspensions were prepared on a conductive carbon tape with double adhesion, dried and introduced in the microscope. The microscope was equipped with energy-dispersive X-ray spectroscopy (EDX) (Ametek EDAX Inc., Mahwah, NJ, USA) attachment operating at 20 kV. The particle size distributions from the SEM micrographs was performed by measuring approximately 200 particles from different regions of the sample. The surface morphology of the coatings was analyzed using atomic force microscopy (AFM), in a non-contact mode. The measurements were performed using a NT-MDT NTEGRA Probe NanoLaboratory system (NT-MDT, Moscow, Russia). The data was recorded with a silicon NT-MDT NSG01 cantilever coated with a 35 nm gold layer having a tetrahedral tip. The AFM micrographs were acquired on surface areas of 3 × 3 µm 2 . The data analysis of the 2D surface topographies as well as the 3D representation of the AFM images were performed with the aid of Gwyddion 2.55 software [27].
The functional groups present in the prepared gel and coating were identified by Fourier-transform infrared spectroscopy (FTIR) analysis using a SpectrumBX spectrometer in the case of the prepared gel and by Fourier-transform infrared spectroscopy -Attenuated total reflection (FTIR-ATR) spectroscopy using a Perkin Elmer SP-100 spectrometer (Waltham, MS, USA), for the coatings. The spectra were taken in the spectral range of 400 to 4000 cm −1 with a resolution of 4 cm −1 . The curve fitting analysis of the spectra was achieved using a nonlinear least-squares data-fitting algorithm [28].
X-ray Photoelectron Spectroscopy (XPS) analysis was performed using a VG ESCA 3 MK II XPS installation (E kα = 1486.7 eV). The vacuum analysis chamber pressure was P~3 × 10 −8 torr. The XPS register spectrum involved an energy window w = 20 eV with the resolution R = 50 eV and 256 recording channels. The XPS spectra were analyzed using Spectral Data Processor v 2.3 (SDP) software.

In Vitro Antifungal Activity
The effects of the 5SmHAp coatings against fungi cells were assessed using the reference fungal strain Candida albicans ATCC 10231 acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). The in vitro antifungal assays were performed with fungal suspensions of approximately 10 5 -10 6 colony forming units (CFU)/mL obtained from 15 to 18 h bacterial cultures as previously reported [29]. The qualitative assay of the fungal biofilm development, after incubation at 24, 48, and 72 h, was assessed by the visualization of the adherent fungal cells on the substrates using atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). For the AFM and SEM observation, the 5SmHAp coatings were removed from the C. albicans ATCC 10231 culture medium after 24, 48, and 72 h of incubation, then washed with sterile saline solution and fixed using cold methanol. For the CLSM visualization, the 5SmHAp coatings after 24, 48, and 72 h of incubations with C. albicans fungal culture, were washed with sterile saline buffer solution (PBS) to remove the unattached fungal cells. After that, the unattached cells were fixed with cold methanol and stained in the dark with propidium iodide (PI) for 10 min at room temperature. After the PI staining, the excess of label marker was removed using filter paper. The samples were visualized directly after staining. The CLSM studies were performed both in reflection and fluorescence modes using a Leica TCS-SP confocal microscope (Leica Camera AG, Wetzlar, Germany), equipped with a PL FLUOTAR (40_ NA 0.7) objective and an Ar ion laser with a laser line at 488 nm.
The quantitative assays of the antifungal activity of both 5SmHAp suspensions and coatings were done using an adapted method (E2149-10; ASTM International) [30,31], as previously described [32]. The experiments were performed in triplicate and the results of the results were expressed as mean ± SD. Moreover, the 3D representation of the SEM and CLSM images were obtained Image J software (ImageJ 1.51j8, National Institutes of Health, Bethesda, MD, USA) was used [33].

In Vitro Cytotoxic Assay
The toxicity of the HAp and 5SmHAp solutions and coatings was assessed using the HeLa cell line (Sigma-Aldrich Corp., St. Louis, MO, USA). The in vitro interaction of the HeLa cells with the samples was studied at three different time intervals (24,48, and 72 h) using the methodology previously described in Predoi et al. [34]. A cell viability using live/dead cell double staining kit (purchased from Merck/Sigma-Aldrich) was also performed. The kit allowed the simultaneously staining of both viable and dead cells. The number of live and dead cells were numbered and represented as function of percentage from the number of total cells. The experiments were performed in triplicate and the data represented as mean ± SD.

Results
The quality of the obtained coatings by sol-gel process is influenced by many factors. The most significant element is the stability of the concentrated suspension from which the coatings are obtained. A more accurate way of evaluating the stability of the suspensions is represented by ultrasound measurements. This analysis allows evaluation of the stability of the concentrated solution as opposed to the other traditional methods in which the suspension needs to be diluted. Due to the dilution, important information regarding the stability of the suspension can be lost. Figure 1 shows information regarding the stability of the concentrated suspension of 5SmHAp obtained by the ultrasound measurements. The digitalised ultrasonic signals are recorded on the digital oscilloscope at a very precise interval of 5.00 s (Figure 1a). The peak amplitudes evolve in time in an unusual way for this suspension, compared to many others studied previously. There is an initial slowly decreasing amplitude, followed by a rising amplitude after 2400 s and a hyperpolic decreasing amplitude up to the end of the experiment (5000 s). Time delays between the first echo that was recorded in the analyzed suspension and the equivalent echo in the reference fluid, allowed a precise determination of the velocity of ultrasounds through the sample, for each signal that was recorded. The result for the velocity in the reference fluid was c 0 = 1478.27 m/s while the velocity in the analyzed sample was c = 1492.07 m/s and the temperature at which the experiment was performed was 23.3 • C. The ultrasound velocity in the sample is very close to the velocity in pure water and has a negligible variation during the experiment. To characterize the evolution in time of the sample's stability the evolution of amplitude in time was also evaluated (Figure 1b). The amplitude of the first echo was the only one measured with sufficient accuracy. The parameter that gives quantitative information on the stability of the suspension is closely related to the slope of the amplitude of the first echo vs. time. The value of the stability parameter computed by the algorithm was s = 1 A m dA dt = 1.3 × 10 −4 ·s −1 , in which A m is the averaged amplitude of the signals. Ignoring the local maximum at t = 2400 s, the overall stability parameter can be estimated as s = 9 × 10 −4 ·s −1 . The value obtained for the stability parameter shows a very good stability of 5SmHAp concentrate suspension.
Coatings 2020, 10, x FOR PEER REVIEW 5 of 24 digital oscilloscope at a very precise interval of 5.00 s (Figure 1a). The peak amplitudes evolve in time in an unusual way for this suspension, compared to many others studied previously. There is an initial slowly decreasing amplitude, followed by a rising amplitude after 2400 s and a hyperpolic decreasing amplitude up to the end of the experiment (5000 s). Time delays between the first echo that was recorded in the analyzed suspension and the equivalent echo in the reference fluid, allowed a precise determination of the velocity of ultrasounds through the sample, for each signal that was recorded. The result for the velocity in the reference fluid was c0 = 1478.27 m/s while the velocity in the analyzed sample was c = 1492.07 m/s and the temperature at which the experiment was performed was 23.3 °C. The ultrasound velocity in the sample is very close to the velocity in pure water and has a negligible variation during the experiment. To characterize the evolution in time of the sample's stability the evolution of amplitude in time was also evaluated ( Figure 1b). The amplitude of the first echo was the only one measured with sufficient accuracy. The parameter that gives quantitative information on the stability of the suspension is closely related to the slope of the amplitude of the first echo vs. time. The value of the stability parameter computed by the algorithm was 1.3 10 • , in which Am is the averaged amplitude of the signals. Ignoring the local maximum at t = 2400 s, the overall stability parameter can be estimated as 9 10 • . The value obtained for the stability parameter shows a very good stability of 5SmHAp concentrate suspension. The relative spectral amplitude vs. time for the first echo and attenuation vs. time for the spectral components of echo 1 revealed the complex information concerning the stability of the analyzed concentrated suspension (Figure 2). Figure 2a shows the initial highest amplitude ratio of 0.55 for the frequency of 2 MHz and the lowest of 0.25 at 8 MHz. For accuracy, all values obtained are relative to the values of the reference fluid (double distilled water) measured under the same conditions. Before the peak at value of time t = 2400 s, the amplitudes at all selected frequencies are slowly decreasing. After the peak, the amplitudes decrease continuously, which is atypical for a dispersion after 5000 s. It can be expected that after a much longer period, the relative amplitudes will tend to 1, when most of the particles will settle at the bottom of the container. As shown in Figure 2b  The relative spectral amplitude vs. time for the first echo and attenuation vs. time for the spectral components of echo 1 revealed the complex information concerning the stability of the analyzed concentrated suspension (Figure 2). Figure 2a shows the initial highest amplitude ratio of 0.55 for the frequency of 2 MHz and the lowest of 0.25 at 8 MHz. For accuracy, all values obtained are relative to the values of the reference fluid (double distilled water) measured under the same conditions. Before the peak at value of time t = 2400 s, the amplitudes at all selected frequencies are slowly decreasing. After the peak, the amplitudes decrease continuously, which is atypical for a dispersion after 5000 s. It can be expected that after a much longer period, the relative amplitudes will tend to 1, when most of the particles will settle at the bottom of the container. As shown in Figure 2b   The morphology of the HAp and 5SmHAp suspension was investigated through TEM and SEM studies. The results of these studies for the HAp suspension are depicted in Figure 3a,b. Also, in Figure 4a,b are presented the SEM and TEM micrographs obtained on the 5SmHAp suspension. In addition, the particle size distribution obtained from TEM and SEM measurements are presented in Figure 3c,d for HAp suspension and in Figure 4c   The morphology of the HAp and 5SmHAp suspension was investigated through TEM and SEM studies. The results of these studies for the HAp suspension are depicted in Figure 3a,b. Also, in Figure 4a,b are presented the SEM and TEM micrographs obtained on the 5SmHAp suspension. In addition, the particle size distribution obtained from TEM and SEM measurements are presented in Figure 3c,d for HAp suspension and in Figure 4c,d for the 5SmHAp suspension.  In the TEM micrographs (Figures 3a and 4a) it could be observed that the HAp and 5SmHAp suspensions consists of particles with nanometric dimensions and ellipsoidal morphology. The mean particle size estimated by TEM studies is around 19 nm in the case of 5SmHAp suspension and about  In the TEM micrographs (Figures 3a and 4a) it could be observed that the HAp and 5SmHAp suspensions consists of particles with nanometric dimensions and ellipsoidal morphology. The mean particle size estimated by TEM studies is around 19 nm in the case of 5SmHAp suspension and about In the TEM micrographs (Figures 3a and 4a) it could be observed that the HAp and 5SmHAp suspensions consists of particles with nanometric dimensions and ellipsoidal morphology. The mean particle size estimated by TEM studies is around 19 nm in the case of 5SmHAp suspension and about 17 nm for the 5SmHAp suspension. Also, could be noticed that the presence of the samarium in the sample induces a slight decrease of particle size.
The results of the SEM investigations conducted on the HAp and 5SmHAp suspensions presented in Figures 3b-d and 4b-d revealed that for the both samples the nanoparticles tend to agglomerate and exhibit an elongated morphology. Furthermore, the mean particle size obtained by SEM studies was around 20 nm for the 5SmHAp suspension and about 18 nm for the 5SmHAp suspension. Therefore, it can be seen that the results of SEM studies are in good agreement with those obtained through TEM studies.
FTIR measurements were conducted in order to investigate the presence of the vibrational bands characteristic to the HAp structure in the samples. In Figures 5 and 6 are presented the general FTIR absorbance spectra of HAp and 5SmHAp coatings and suspension along with their FTIR deconvoluted spectra in the 400-700 cm −1 and 900-1200 cm −1 spectral regions.
SEM studies was around 20 nm for the 5SmHAp suspension and about 18 nm for the 5SmHAp suspension. Therefore, it can be seen that the results of SEM studies are in good agreement with those obtained through TEM studies.
FTIR measurements were conducted in order to investigate the presence of the vibrational bands characteristic to the HAp structure in the samples. In Figures 5 and 6 are presented the general FTIR absorbance spectra of HAp and 5SmHAp coatings and suspension along with their FTIR deconvoluted spectra in the 400-700 cm −1 and 900-1200 cm −1 spectral regions.
In the general FTIR absorbance spectra ( Figure 5) of the studied samples have been identified the peaks belonging to the main vibrational modes of the adsorbed water (H-O-H), phosphate (PO4 3− ) and hydroxyl (OH − ) groups characteristic of the hydroxyapatite structure. The presence of the peak at about 635 cm −1 corresponds to the stretching librational mode (νL) of (OH − ) group [35,36]. At the same time, the presence of the peak at around 3570 cm −1 is due to stretching mode of (OH − ) groups. The presence of wide peaks in the 1600-1700 cm −1 and 3200-3600 cm −1 spectral regions are assigned to H-O-H bands of lattice water [35][36][37]. The presence of the peaks at around 1045 and 1093 cm −1 denote the presence of ν3 of (PO4 3− ) group [35,36]. Furthermore, the peaks at around 604 and 568 cm −1 belong to ν4 of (PO4 3− ) group. In agreement with the studies conducted by Iconaru et al. [35] a well crystalized hydroxyapatite structure is highlighted by the presence of the vibrational bands at around 635 cm −1 and at about 3570 cm −1 (which correspond to the OH − groups from the structure of hydroxyapatite). On the other hand, it can be seen that the vibration bands are more intense in the case of 5SmHAp coatings compared to HAp and 5SmHAp suspensions. This behaviour is atributed to the the fact that the 5SmHAp coatings are more crystaline comparative to the other two studied samples. Also, in the case of 5SmHAp coatings a slight displacement of the position of the peaks was observed.  main components. In addition, our studies revealed that to have a good fit for the 5SmHAp suspension are needed six main components for the 400-700 cm −1 spectral region and four components for the 900-1200 cm −1 spectral region. Furthermore, an increase in the intensity of the subbands from the both deconvoluted regions specific to the 5SmHAp coatings could be noticed.This behavior is due to the superior crystallinity of the deposited coatings compared to that of the nanoparticles in suspensions. In order to analyze the surface chemical elemental composition in the 5SmHAp coatings XPS measurements were conducted. The XPS general spectrum of the coating with xSm = 0 (HAp) and xSm = 0.05 (5SmHAp) was presented in Figure 7. The results indicated that elements such as C, O, Ca, and P were observed in the scan of HAp and 5SmHAp general spectrum (Figure 7a,b). In the scan of 5SmHAp XPS general spectra Sm was observed, too. and hydroxyl (OH − ) groups characteristic of the hydroxyapatite structure. The presence of the peak at about 635 cm −1 corresponds to the stretching librational mode (ν L ) of (OH − ) group [35,36]. At the same time, the presence of the peak at around 3570 cm −1 is due to stretching mode of (OH − ) groups. The presence of wide peaks in the 1600-1700 cm −1 and 3200-3600 cm −1 spectral regions are assigned to H-O-H bands of lattice water [35][36][37]. The presence of the peaks at around 1045 and 1093 cm −1 denote the presence of ν 3 of (PO 4 3− ) group [35,36]. Furthermore, the peaks at around 604 and 568 cm −1 belong to ν 4 of (PO 4 3− ) group. In agreement with the studies conducted by Iconaru et al. [35] a well crystalized hydroxyapatite structure is highlighted by the presence of the vibrational bands at around 635 cm −1 and at about 3570 cm −1 (which correspond to the OH − groups from the structure of hydroxyapatite). On the other hand, it can be seen that the vibration bands are more intense in the case of 5SmHAp coatings compared to HAp and 5SmHAp suspensions. This behaviour is atributed to the the fact that the 5SmHAp coatings are more crystaline comparative to the other two studied samples. Also, in the case of 5SmHAp coatings a slight displacement of the position of the peaks was observed. Figure 6 depicts the deconvoluted FTIR spectra of the HAp suspension 5SmHAp coatings and suspension (green lines represents the individual subbands) in the 400-700 cm −1 and 900-1200 cm −1 spectral regions. In Figure 6a,b it can be observed that in order to obtain a good fit of the experimental data obtained oh HAp suspension, five subbands are needed in the spectral region 450-700 cm −1 , while for the spectral domain between 900 and 1200 cm −1 , eight subbands are needed. Also, it can be seen that in the case of 5SmHAp coatings in order to obtain a good fitting in the 400-700 cm −1 spectral region are needed six main components while for the 900-1200 cm −1 spectral region are needed four main components. In addition, our studies revealed that to have a good fit for the 5SmHAp suspension are needed six main components for the 400-700 cm −1 spectral region and four components for the 900-1200 cm −1 spectral region. Furthermore, an increase in the intensity of the subbands from the both deconvoluted regions specific to the 5SmHAp coatings could be noticed.This behavior is due to the superior crystallinity of the deposited coatings compared to that of the nanoparticles in suspensions.
In order to analyze the surface chemical elemental composition in the 5SmHAp coatings XPS measurements were conducted. The XPS general spectrum of the coating with x Sm = 0 (HAp) and x Sm = 0.05 (5SmHAp) was presented in Figure 7. The results indicated that elements such as C, O, Ca, and P were observed in the scan of HAp and 5SmHAp general spectrum (Figure 7a,b). In the scan of 5SmHAp XPS general spectra Sm was observed, too. The high resolution of C 1s, Ca 2p, O 1s, P 2p and Sm 3d are exhibited in Figure 8. The highresolution C 1s spectra for the coating with xSm = 0 (HAp) and xSm = 0.05 (5SmHAp) are presented in Figure 8a,b. The peak in the C 1s region for HAp coating presented one component of the binding energy (BE) of 284.8 eV attributed to C-C bonds was used as a reference (Figure 8a). Three peaks in the binding energy (BE) region of C 1s were observed (Figure 8b). The peak of C 1s at 284.8 eV attributed to C-C bonds was used as a reference. Peak of C 1s at 286.74 eV can be assigned to C-O bonds while peak at 289.63 eV can be assigned to C=O bonds. In Figure 8c,d the high-resolution Ca 2p spectra for HAp and 5SmHap coatings is presented. The peaks in the BE region of Ca 2p exhibits a well-defined doublet with two components (Ca 2p3/2 and Ca 2p1/2). For the 5SmHAp coatings the BE shift slightly from 347.2 eV (for the HAp coatings) to 347.8 eV for the 5SmHAp coatings. The peak located at about 347.2 eV highlights that the calcium atoms are bound to a phosphate group (PO4 3− ). Kaciulis et al. showed [38] that the shift of the maximum to a higher value of the binding energy in the case of hydroxyapatite doped with different ions shows that the obtained sample is well crystallized. The high resolution XPS spectra of oxygen O 1s for pure HAp (xSm = 0) coating was shown in the Figure 8e. The peak at BE of 531.4 eV was assigned to hydroxyl groups that are the result of water or oxygen chemisorption [39]. The O 1s photoelectron peak was deconvoluted into two components (Figure 6f). The component located at 531.4 eV is characteristic to O 1s peak in HAp structure while the peak at 534.1 eV was attributed to the O 1s in adsorbed water [40,41]. The second component of O 1s was observed in the coatings obtained by sol-gel method [38,41]. In previous studies, Gaggiotti et al. [42] reported that the EB = 531.8 eV correspond to a position of the hydroxyl ion (OH − ). Kawabe et al. [43] appreciate that the two oxygen species (O-and OH), may be attributed to the peak at binding energy of 531.2 eV. Moreover, according to previous studies [44][45][46] the peak position of chemisorbed oxygen species O-could be attributed to a binding energy between EB = 531.0-531.5 eV. The P 2p photoelectron peak of pure HAp coating is shown in Figure 8g. The peak associated to P 2p revealed one component after the deconvolution data processing that was located at around EB = 133.1eV. The peak pf P 2p of 5SmHAp coating (Figure 8h) was deconvoluted into two components at 133.01 and 134.04 eV BE in agreement with previous studies [47]. The high resolution of C 1s, Ca 2p, O 1s, P 2p and Sm 3d are exhibited in Figure 8. The high-resolution C 1s spectra for the coating with x Sm = 0 (HAp) and x Sm = 0.05 (5SmHAp) are presented in Figure 8a,b. The peak in the C 1s region for HAp coating presented one component of the binding energy (BE) of 284.8 eV attributed to C-C bonds was used as a reference (Figure 8a). Three peaks in the binding energy (BE) region of C 1s were observed (Figure 8b). The peak of C 1s at 284.8 eV attributed to C-C bonds was used as a reference. Peak of C 1s at 286.74 eV can be assigned to C-O bonds while peak at 289.63 eV can be assigned to C=O bonds. In Figure 8c,d the high-resolution Ca 2p spectra for HAp and 5SmHap coatings is presented. The peaks in the BE region of Ca 2p exhibits a well-defined doublet with two components (Ca 2p3/2 and Ca 2p1/2). For the 5SmHAp coatings the BE shift slightly from 347.2 eV (for the HAp coatings) to 347.8 eV for the 5SmHAp coatings. The peak located at about 347.2 eV highlights that the calcium atoms are bound to a phosphate group (PO 4 3− ). Kaciulis et al. showed [38] that the shift of the maximum to a higher value of the binding energy in the case of hydroxyapatite doped with different ions shows that the obtained sample is well crystallized. The high resolution XPS spectra of oxygen O 1s for pure HAp (x Sm = 0) coating was shown in the Figure 8e. The peak at BE of 531.4 eV was assigned to hydroxyl groups that are the result of water or oxygen chemisorption [39].
The O 1s photoelectron peak was deconvoluted into two components (Figure 6f). The component located at 531.4 eV is characteristic to O 1s peak in HAp structure while the peak at 534.1 eV was attributed to the O 1s in adsorbed water [40,41]. The second component of O 1s was observed in the coatings obtained by sol-gel method [38,41]. In previous studies, Gaggiotti et al. [42] reported that the EB = 531.8 eV correspond to a position of the hydroxyl ion (OH − ). Kawabe et al. [43] appreciate that the two oxygen species (O-and OH), may be attributed to the peak at binding energy of 531.2 eV. Moreover, according to previous studies [44][45][46] the peak position of chemisorbed oxygen species Ocould be attributed to a binding energy between EB = 531.0-531.5 eV. The P 2p photoelectron peak of pure HAp coating is shown in Figure 8g. The peak associated to P 2p revealed one component after the deconvolution data processing that was located at around EB = 133.1eV. The peak pf P 2p of 5SmHAp coating (Figure 8h) was deconvoluted into two components at 133.01 and 134.04 eV BE in agreement with previous studies [47]. Previous studies on hydroxyapatite layers [48] revealed that the P 2p photoelectron line consists of a single component assigned to a peak at binding energy of 133.4 eV. On the other hand, the precedent XPS analysis [49] indicated that the binding energy of the photoelectron peaks for P and Ca are characteristic to their full oxidation states (P 5+ and Ca 2+) for hydroxyapatite. The high resolution spectra of the Sm 2p3 is shown in Figure 8i. The peak corresponding to the binding energy of 1082.93 eV can be assigned to Sm 3d5/2 [50]. In agreement with previous studies [51], the binding energy of Sm 3d5/2 as revealed in Figure 8i suggested that the chemical state of Sm is +3. The XPS results provided obvious information for the successful doping of HAp with samarium. Figure 9 shows the XRD diffractogram and SEM micrograph of HAp (a,c) and 5SmHAp coatings (b,d). The elemental mapping analysis of the chemical constituents of 5SmHAp coatings (Ca (a), O (b), P (c), Sm (d)) is also presented in Figure 10. In the SEM micrograph (Figure 9d) obtained on the 5SmHAp coatings could be noticed that the surface of the coatings is uniform and continuous with a granular morphology (granules formed by agglomeration of nanoparticles). Previous studies on hydroxyapatite layers [48] revealed that the P 2p photoelectron line consists of a single component assigned to a peak at binding energy of 133.4 eV. On the other hand, the precedent XPS analysis [49] indicated that the binding energy of the photoelectron peaks for P and Ca are characteristic to their full oxidation states (P 5+ and Ca 2+ ) for hydroxyapatite. The high resolution spectra of the Sm 2p3 is shown in Figure 8i. The peak corresponding to the binding energy of 1082.93 eV can be assigned to Sm 3d5/2 [50]. In agreement with previous studies [51], the binding energy of Sm 3d5/2 as revealed in Figure 8i suggested that the chemical state of Sm is +3. The XPS results provided obvious information for the successful doping of HAp with samarium. Figure 9 shows the XRD diffractogram and SEM micrograph of HAp (a,c) and 5SmHAp coatings (b,d). The elemental mapping analysis of the chemical constituents of 5SmHAp coatings (Ca (a), O (b), P (c), Sm (d)) is also presented in Figure 10. In the SEM micrograph (Figure 9d) obtained on the 5SmHAp coatings could be noticed that the surface of the coatings is uniform and continuous with a granular morphology (granules formed by agglomeration of nanoparticles). In order to determine the crystal structure phase of HAp and 5SmHAp coating, the X-ray diffraction (XRD) was performed using Cu Kα radiation (λ = 1.5406 Å). The XRD patterns of the HAp coating were presented in Figure 9a. Figure 9b shows the XRD patterns of the 5SmHAp coating with xSm = 0.05. The XRD peaks were indexed by the hexagonal phase of pure hydroxyapatite (JCPDS no. 09-0432). It should be noted that no other secondary phases due to Sm 3+ ions doped hydroxyapatite were observed after the analysis. This result highlights that Sm 3+ ions substituted Ca 2+ ions of 5SmHAp without changing the crystal structure. The diffraction peaks of HAp and 5SmHAp coating correspond to (002) The morphology of the HAp and 5SmHAp coatings surface topography has been investigated using atomic force microscopy. The results of the AFM investigations regarding the surface morphology of the HAp and 5SmHAp coatings are depicted in Figure 11a-d. In order to determine the crystal structure phase of HAp and 5SmHAp coating, the X-ray diffraction (XRD) was performed using Cu Kα radiation (λ = 1.5406 Å). The XRD patterns of the HAp coating were presented in Figure 9a. Figure 9b shows the XRD patterns of the 5SmHAp coating with x Sm = 0.05. The XRD peaks were indexed by the hexagonal phase of pure hydroxyapatite (JCPDS no. 09-0432). It should be noted that no other secondary phases due to Sm 3+ ions doped hydroxyapatite were observed after the analysis. This result highlights that Sm 3+ ions substituted Ca 2+ ions of 5SmHAp without changing the crystal structure. The diffraction peaks of HAp and 5SmHAp coating correspond to (002) (Figure 10a-d).
In the Figure 10a-d can be seen that the main chemical constituents of the 5SmHAp coatings were Ca, O, P, and Sm. Moreover, our studies revealed that all the chemical constituents were evenly distributed in coatings.
The morphology of the HAp and 5SmHAp coatings surface topography has been investigated using atomic force microscopy. The results of the AFM investigations regarding the surface morphology of the HAp and 5SmHAp coatings are depicted in Figure 11a-d.    Figure 11 presents the AFM 2D micrograph and the 3D representation of the HAp and 5SmHAp surface topography of the HAp and 5SmHAp coatings. The results of the AFM studies emphasized that all the investigated coatings have the morphology of a uniformly deposited layer. Furthermore, the 2D representation of the surface highlighted that there is no visible evidence of the existence of cracks or fissures and that the deposited coatings both in the case of HAp and 5SmHAp samples consist of uniformly distributed nanoaggregates. The AFM results showed that the surface topography of the 5SmHAp coatings was also homogenous having a roughness (R RMS ) value of 21.45 nm. In addition, the roughness (R RMS ) value for the HAp coatings resulted from the AFM studies was 12.55 nm. The results obtained by AFM studies are in good agreement with the SEM studies, which also revealed that the coatings are uniform and homogenous.
The adhesion of the C. albicans cells on the surface of 5SmHAp coatings was also investigated by AFM studies. For this purpose, the surface of HAp and 5SmHAp coatings incubated with C. albicans fungal cells at three different time intervals was studied using AFM topography. The results of the AFM surface topography of the HAp and 5SmHAp coatings incubated with C. albicans fungal cells for 24, 48, and 72 h are presented in Figure 12. The 2D AFM images of the 5SmHAp coatings surface highlighted that the C. albicans cell development was inhibited by the coatings and that the inhibition rates were correlated with the incubation time. The AFM images collected on an area of 20 × 20 µm 2 evidenced that the C. albicans development was inhibited starting from 24 h and the inhibition continued and accentuated after 48 and 72 h. In the case of the coatings incubated for 72 h, the C. albicans cells were almost completely eradicated from the surface of the 5SmHAp coatings, demonstrating that the incubation time strongly affected the antifungal properties of the 5SmHAp coatings. Furthermore, the AFM images of the HAp coatings incubated with C. albicans fungal cells emphasized that the HAp coatings promoted the development of the fungal cells and provided a good adhesive surface for the C. albicans cells and allowed the development of fungal biofilm. topography of the 5SmHAp coatings was also homogenous having a roughness (RRMS) value of 21.45 nm. In addition, the roughness (RRMS) value for the HAp coatings resulted from the AFM studies was 12.55 nm. The results obtained by AFM studies are in good agreement with the SEM studies, which also revealed that the coatings are uniform and homogenous.
The adhesion of the C. albicans cells on the surface of 5SmHAp coatings was also investigated by AFM studies. For this purpose, the surface of HAp and 5SmHAp coatings incubated with C. albicans fungal cells at three different time intervals was studied using AFM topography. The results of the AFM surface topography of the HAp and 5SmHAp coatings incubated with C. albicans fungal cells for 24, 48, and 72 h are presented in Figure 12. The 2D AFM images of the 5SmHAp coatings surface highlighted that the C. albicans cell development was inhibited by the coatings and that the inhibition rates were correlated with the incubation time. The AFM images collected on an area of 20 × 20 µm 2 evidenced that the C. albicans development was inhibited starting from 24 h and the inhibition continued and accentuated after 48 and 72 h. In the case of the coatings incubated for 72 h, the C. albicans cells were almost completely eradicated from the surface of the 5SmHAp coatings, demonstrating that the incubation time strongly affected the antifungal properties of the 5SmHAp coatings. Furthermore, the AFM images of the HAp coatings incubated with C. albicans fungal cells emphasized that the HAp coatings promoted the development of the fungal cells and provided a good adhesive surface for the C. albicans cells and allowed the development of fungal biofilm. The effects of the 5SmHAp coatings on the adhesion and proliferation of C. albicans were furthermore studied using SEM and CLSM investigations. The images of C. albicans cell development on the surfaces of 5SmHAp coatings after 24, 48, and 72 h of incubation with the C. albicans microbial cells, resulted from SEM observations were presented in Figure 13. The SEM visualization of the 5SmHAp coatings incubated with the fungal cell at different time intervals highlighted that the morphology of the fungal cells was typical to that of the C. albicans fungal strain, having round and oval shapes. Moreover, the SEM images emphasized that the size of the C. albicans fungal cells were in the range of 2.378-4.419 µm. In addition, the SEM analysis revealed that the fungal cells adhesion and development of biofilms on the 5SmHAp coatings was inhibited only after 24 h of incubation. Moreover, the SEM observations highlighted the inhibitory effects of the 5SmHAp coatings against C. albicans fungal strain at all tested time intervals. Furthermore, the results of the SEM visualization evidenced that the inhibitory effect of the 5SMHAp coatings was correlated with the incubation time. The effects of the 5SmHAp coatings on the adhesion and proliferation of C. albicans were furthermore studied using SEM and CLSM investigations. The images of C. albicans cell development on the surfaces of 5SmHAp coatings after 24, 48, and 72 h of incubation with the C. albicans microbial cells, resulted from SEM observations were presented in Figure 13. The SEM visualization of the 5SmHAp coatings incubated with the fungal cell at different time intervals highlighted that the morphology of the fungal cells was typical to that of the C. albicans fungal strain, having round and oval shapes. Moreover, the SEM images emphasized that the size of the C. albicans fungal cells were in the range of 2.378-4.419 µm. In addition, the SEM analysis revealed that the fungal cells adhesion and development of biofilms on the 5SmHAp coatings was inhibited only after 24 h of incubation. Moreover, the SEM observations highlighted the inhibitory effects of the 5SmHAp coatings against C.
albicans fungal strain at all tested time intervals. Furthermore, the results of the SEM visualization evidenced that the inhibitory effect of the 5SMHAp coatings was correlated with the incubation time.
formation of a fungal biofilm on their surface. These studies are in good agreement with previous conducted studies regarding the antifungal properties of HAp. The SEM images have revealed that the number of C. albicans fungal cells was considerably diminished after 48 and 72 h of incubation with the 5SmHAp coatings. Furthermore, the 3D representation of the SEM images with the C. albicans attached fungal cells was also performed using Image J software (Image J 1.51j8) [33]. The 3D representation of the SEM images of the 5SmHAp coatings incubated at three different time intervals with C. albicans fungal cells are presented in Figure 14.   In addition, the C. albicans development on the HAp coatings was also assessed by SEM visualization at three different time intervals. The results emphasized that the HAp coatings promoted the development of the fungal cells and aided their proliferation and also allowed the formation of a fungal biofilm on their surface. These studies are in good agreement with previous conducted studies regarding the antifungal properties of HAp. The SEM images have revealed that the number of C. albicans fungal cells was considerably diminished after 48 and 72 h of incubation with the 5SmHAp coatings. Furthermore, the 3D representation of the SEM images with the C. albicans attached fungal cells was also performed using Image J software (Image J 1.51j8) [33]. The 3D representation of the SEM images of the 5SmHAp coatings incubated at three different time intervals with C. albicans fungal cells are presented in Figure 14.
The development and adhesion of C. albicans cells on the surface of 5SmHAp coatings after different time incubation intervals were also examined by CLSM. The images acquired using CLSM proved much more clearly, that the C. albicans cells development was inhibited by the 5SmHAp coatings and that the coatings did not allowed the fungal cells to proliferate and develop biofilms. These results are in good agreement with previous reported studies regarding the fungal properties of other biocomposite layers deposited on Si and Ti substrates [12,29,52,53]. CLSM is an optical imaging technique and a useful tool for imaging, qualitative analysis and quantification of cells [54,55]. Figure 15 shows the CLSM images of C. albicans cell growth on 5SmHAp coatings incubated with the fungal cells at different time intervals. The CLSM visualization was done using propidium iodide to label the fungal cells. The CLSM visualization evidenced that the surviving C. albicans cells were intact with round morphology and a smooth surface for all tested time intervals of incubation on the surface of HAp and 5SmHAp coatings. The results are depicted in Figure 15a-f. Furthermore, the CLSM examination confirmed the results obtained by the qualitative antimicrobial assay and was in good agreement with the results obtained from the SEM analysis. CLSM images highlighted that 5SmHAp coatings after 24 h of incubation exhibited a strong antifungal activity against Candida albicans ATCC 10231 and inhibited the biofilm formation. In addition, the results obtained by CLSM studies have emphasized that the time of incubation influenced the antifungal activity of the 5SmHAp coatings. The antifungal assays performed revealed that the 5SmHAp coatings could have a great potential to be used in medical application as antifungal coatings for medical devices.   The development and adhesion of C. albicans cells on the surface of 5SmHAp coatings after different time incubation intervals were also examined by CLSM. The images acquired using CLSM proved much more clearly, that the C. albicans cells development was inhibited by the 5SmHAp coatings and that the coatings did not allowed the fungal cells to proliferate and develop biofilms. These results are in good agreement with previous reported studies regarding the fungal properties of other biocomposite layers deposited on Si and Ti substrates [12,29,52,53]. CLSM is an optical imaging technique and a useful tool for imaging, qualitative analysis and quantification of cells [54,55]. Figure  15 shows the CLSM images of C. albicans cell growth on 5SmHAp coatings incubated with the fungal cells at different time intervals. The CLSM visualization was done using propidium iodide to label the fungal cells. The CLSM visualization evidenced that the surviving C. albicans cells were intact with round morphology and a smooth surface for all tested time intervals of incubation on the surface of HAp and 5SmHAp coatings. The results are depicted in Figure 15a-f. Furthermore, the CLSM examination confirmed the results obtained by the qualitative antimicrobial assay and was in good agreement with the results obtained from the SEM analysis. CLSM images highlighted that 5SmHAp coatings after 24 h of incubation exhibited a strong antifungal activity against Candida albicans ATCC 10231 and inhibited the biofilm formation. In addition, the results obtained by CLSM studies have emphasized that the time of incubation influenced the antifungal activity of the 5SmHAp coatings. The antifungal assays performed revealed that the 5SmHAp coatings could have a great potential to be used in medical application as antifungal coatings for medical devices. Furthermore, the 3D representation of the CLSM images of C. albicans fungal cells adhered on the surface of the 5SmHAp coatings after being incubated at three different time intervals analyzed using Image J software [33] are presented in Figure 16. The 3D representation display the structure and spatial distribution of C. albicans surviving cells on the 5SmHAp coatings after 24, 48, and 72 h of incubation with the fungal cells. The images exhibited in Figure 15 revealed the spatial distribution of C. albicans surviving cells (red color) along horizontal (coverage) and the vertical (thickness) distributions on the 5SmHAp surfaces. The CLSM images presented in Figure 16 demonstrated that the survival of C. albicans cells have been significantly reduced in the presence of 5SmHAp coatings and that their inhibition was strongly correlated with the incubation time. Furthermore, the 3D representation of the CLSM images of C. albicans fungal cells adhered on the surface of the 5SmHAp coatings after being incubated at three different time intervals analyzed using Image J software [33] are presented in Figure 16. The 3D representation display the structure and spatial distribution of C. albicans surviving cells on the 5SmHAp coatings after 24, 48, and 72 h of incubation with the fungal cells. The images exhibited in Figure 15 revealed the spatial distribution of C. albicans surviving cells (red color) along horizontal (coverage) and the vertical (thickness) distributions on the 5SmHAp surfaces. The CLSM images presented in Figure 16 demonstrated that the survival of C. albicans cells have been significantly reduced in the presence of 5SmHAp coatings and that their inhibition was strongly correlated with the incubation time. The quantitative antifungal properties of the HAp and 5SmHAp coatings deposited on silicium substrate were also assessed using C. albicans ATCC 10231 fungal strain. The graphical representation of C. albicans ATCC 10231 colony-forming units on 5SmHAp suspensions and coatings at three different time intervals (24,48, and 72 h) are depicted in Figure 17.
The quantitative results emphasized that the fungal colony forming unity development was inhibited from the early phase of adherence in the case of 5SmHAp both suspensions and coatings. The colony forming unit count (CFUc) assay showed a significant decrease of the number of colonies in the case of both 5SmHAp suspensions and coatings compared to the number of colonies formed in the case of the control culture. Furthermore, the results emphasized that the antifungal activity of 5SmHAp suspensions was higher than that of 5SmHAp coatings for all tested intervals. The results obtained by the qualitative and quantitative studies conducted, evidenced that the 5SmHAp suspensions and coatings inhibited the C. albicans fungal strain development. The results are in good agreement with previous reported data [10,53,[56][57][58] and emphasized that samarium ions exhibit antifungal properties.  The quantitative antifungal properties of the HAp and 5SmHAp coatings deposited on silicium substrate were also assessed using C. albicans ATCC 10231 fungal strain. The graphical representation of C. albicans ATCC 10231 colony-forming units on 5SmHAp suspensions and coatings at three different time intervals (24,48, and 72 h) are depicted in Figure 17. The quantitative antifungal properties of the HAp and 5SmHAp coatings deposited on silicium substrate were also assessed using C. albicans ATCC 10231 fungal strain. The graphical representation of C. albicans ATCC 10231 colony-forming units on 5SmHAp suspensions and coatings at three different time intervals (24,48, and 72 h) are depicted in Figure 17.
The quantitative results emphasized that the fungal colony forming unity development was inhibited from the early phase of adherence in the case of 5SmHAp both suspensions and coatings. The colony forming unit count (CFUc) assay showed a significant decrease of the number of colonies in the case of both 5SmHAp suspensions and coatings compared to the number of colonies formed in the case of the control culture. Furthermore, the results emphasized that the antifungal activity of 5SmHAp suspensions was higher than that of 5SmHAp coatings for all tested intervals. The results obtained by the qualitative and quantitative studies conducted, evidenced that the 5SmHAp suspensions and coatings inhibited the C. albicans fungal strain development. The results are in good agreement with previous reported data [10,53,[56][57][58] and emphasized that samarium ions exhibit antifungal properties.  The quantitative results emphasized that the fungal colony forming unity development was inhibited from the early phase of adherence in the case of 5SmHAp both suspensions and coatings. The colony forming unit count (CFUc) assay showed a significant decrease of the number of colonies in the case of both 5SmHAp suspensions and coatings compared to the number of colonies formed in the case of the control culture. Furthermore, the results emphasized that the antifungal activity of 5SmHAp suspensions was higher than that of 5SmHAp coatings for all tested intervals. The results obtained by the qualitative and quantitative studies conducted, evidenced that the 5SmHAp suspensions and coatings inhibited the C. albicans fungal strain development. The results are in good agreement with previous reported data [10,53,[56][57][58] and emphasized that samarium ions exhibit antifungal properties.
The antifungal properties of HAp suspensions and HAp coatings were also investigated and a free C. albicans culture was used as positive control C+. The results demonstrated that both HAp suspensions and HAp coatings encouraged the proliferation of the fungal cells and influenced positive their growth for all tested time intervals. These results are in good agreement with previously reported studies regarding the lack of antimicrobial properties of hydroxyapatite [20,59,60]. Moreover, previous studies [15,[56][57][58][59][60][61][62][63][64] reported that the antifungal effect of samarium could be explained by the fact that samarium ions have the ability to attach to the cell membrane leading to changes of its permeability. Furthermore, samarium ions that are released from the composite matrix could disrupt the bacterial membrane integrity thus affecting a multitude of cellular processes such like adhesion, ion conductivity and cell signaling [61].
The results of the qualitative and quantitative antifungal assays are in agreement with the previous data presented in the literature and highlighted that the Sm 3+ ions present in the hydroxyapatite matrix of the 5SmHAp coatings is responsible for the antifungal activity of the coatings. Moreover, the data also suggested that the antifungal activity of both the 5SmHAp suspensions and coatings are strongly correlated with the incubation time. The qualitative studies conducted by AFM, SEM and CLSM suggested that the adhesion of C. albicans fungal cells was greatly reduced by the samarium doped composites. These results are similar with those reported in the literature, regarding the adherence of S. aureus, S. epidermidis and P. aeruginosa strains on composites doped with samarium [15]. The results reported by the authors showed that compared to glass-reinforced hydroxyapatite composites (GR-HA) composite the S. aureus, S. epidermidis and P. aeruginosa microbial cell development was considerably inhibited in the Sm doped composites and that the microbial cell reduction was correlated with the Sm content. The antibacterial effect of the composites were attributed also to the release of samarium ions from the composites. Thus, the development of 5SmHAp coatings with enhanced osteoblastic cell response given by the hydroxyapatite matrix and possessing antibacterial activity due to the presence of samarium ions could lead to a better outcome of bone graft implantation. The choice of Si as a substrate started from the intention to extend the applicability of these biocompatible materials with antimicrobial properties from dental field to devices that could be used in tissue engineering [62] or human prosthetic [63] in order to prevent postoperative infections. Ruffino and Torrisi [64] in their studies regarding the influence of interaction between film and substrate on the nanoscale film morphology showed that between Ag films deposited on SiO 2 /Si and TiO 2 /Ti substrates exist various differences. Also Jouanny et al. [65] reported that between the TiO 2 thin films deposited on the Si and Ti-6Al-4V substrate respectively, are differences from mechanical point of view. According to the studies reported by Barry et al. [66] the nature of the substrate could influence the surface roughness and coating thickness but there are no significant differences between the interface properties and the coating composition depending on the type of alloy. Moreover, Ferraris et al. [67] reported that the substrate also influences the biological properties of the samples.
Furthermore, due to the intended purpose of being used in biomedical applications, the toxicity of the HAp and 5SmHAp suspensions and coatings were also assessed using one of the most studied cell line. The toxicity of HAp and 5SmHAp suspensions and coatings was assessed against HeLa cells at three different time intervals (24,48, and 72 h). The results of the cytotoxicity assay obtained by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) are presented in Figure 18. HeLa cell line is the oldest and the most commonly used human cell line and has been extensively used in scientific studies due to its remarkably durable and prolific properties. Since its discovery, the HeLa cells have been intensively and continually used for research into various fields of research such as cancer, AIDS, the effects of radiation and toxic substances, the toxicity of nanoparticles and other types of materials, gene mapping and countless other scientific pursuits. The MTT studies revealed that after 24 h of incubation time, there were no representative differences between the cells viabilities of the HAp and 5SmHAp suspensions. The results have emphasized that after 24 h of incubation, the cell viability of HeLa cells was 90% in the case of the HAp suspensions and 84% in the case of the HAp coatings and also 83% in the case of 5SmHAp suspensions and 82% for the HeLa cells incubated with the 5SmHAp coatings. Moreover, the MTT assay results show that after 48 and 72 h of incubation a slight increase of the HeLa cell viability was observed for all the investigated samples.
The results highlighted that there is a correlation between the incubation time and the cell viability of the samples. Moreover, the MTT suggested that the HAp and 5SmHAp suspensions had better biocompatible properties than the HAp and 5SmHAp coatings. The results obtained in the present study are in good agreement with previous studies regarding the toxicity of hydroxyapatite suspensions and coatings [68][69][70]. In addition, a live/dead cell viability assay was performed in order to quantify the ratio of the live and dead cells after the incubation of the HeLa cells with 5SmHAp solutions and coatings at different time intervals. The results are depicted in Figure 19. The results are in agreement with the MTT assay and evidenced that both the 5SmHAp solutions and coatings did not present any toxicity against the tested cells for all tested time intervals. Furthermore, the data suggested that the percent of dead cells in the case of both samples and for all tested intervals was under 10% from the total number of counted cells.  The MTT studies revealed that after 24 h of incubation time, there were no representative differences between the cells viabilities of the HAp and 5SmHAp suspensions. The results have emphasized that after 24 h of incubation, the cell viability of HeLa cells was 90% in the case of the HAp suspensions and 84% in the case of the HAp coatings and also 83% in the case of 5SmHAp suspensions and 82% for the HeLa cells incubated with the 5SmHAp coatings. Moreover, the MTT assay results show that after 48 and 72 h of incubation a slight increase of the HeLa cell viability was observed for all the investigated samples.
The results highlighted that there is a correlation between the incubation time and the cell viability of the samples. Moreover, the MTT suggested that the HAp and 5SmHAp suspensions had better biocompatible properties than the HAp and 5SmHAp coatings. The results obtained in the present study are in good agreement with previous studies regarding the toxicity of hydroxyapatite suspensions and coatings [68][69][70]. In addition, a live/dead cell viability assay was performed in order to quantify the ratio of the live and dead cells after the incubation of the HeLa cells with 5SmHAp solutions and coatings at different time intervals. The results are depicted in Figure 19. The results are in agreement with the MTT assay and evidenced that both the 5SmHAp solutions and coatings did not present any toxicity against the tested cells for all tested time intervals. Furthermore, the data suggested that the percent of dead cells in the case of both samples and for all tested intervals was under 10% from the total number of counted cells. The MTT studies revealed that after 24 h of incubation time, there were no representative differences between the cells viabilities of the HAp and 5SmHAp suspensions. The results have emphasized that after 24 h of incubation, the cell viability of HeLa cells was 90% in the case of the HAp suspensions and 84% in the case of the HAp coatings and also 83% in the case of 5SmHAp suspensions and 82% for the HeLa cells incubated with the 5SmHAp coatings. Moreover, the MTT assay results show that after 48 and 72 h of incubation a slight increase of the HeLa cell viability was observed for all the investigated samples.
The results highlighted that there is a correlation between the incubation time and the cell viability of the samples. Moreover, the MTT suggested that the HAp and 5SmHAp suspensions had better biocompatible properties than the HAp and 5SmHAp coatings. The results obtained in the present study are in good agreement with previous studies regarding the toxicity of hydroxyapatite suspensions and coatings [68][69][70]. In addition, a live/dead cell viability assay was performed in order to quantify the ratio of the live and dead cells after the incubation of the HeLa cells with 5SmHAp solutions and coatings at different time intervals. The results are depicted in Figure 19. The results are in agreement with the MTT assay and evidenced that both the 5SmHAp solutions and coatings did not present any toxicity against the tested cells for all tested time intervals. Furthermore, the data suggested that the percent of dead cells in the case of both samples and for all tested intervals was under 10% from the total number of counted cells.

Conclusions
Hydroxyapatite and samarium doped hydroxyapatite (x Sm = 0.05) coatings were synthesized by a simple sol-gel route using the dip coating method. Characterization studies of HAp and 5SmHAp nanoparticles in suspension and coatings using different techniques were performed. The stability of 5SmHAp suspension was highlighted by ultrasound measurements. Ultrasound measurements showed that the 5SmHAp suspension had a behavior similar to that of double-distilled water considered as a reference fluid. XPS and XRD studies on 5SmHAp coating confirmed that the Sm 3+ ions have been incorporated into the 5SmHAp synthesized material by substituting the Ca 2+ ions. The uniform distribution of constituent elements on 5SmHAp coating surface was confirmed by elemental mapping analysis. SEM and AFM investigations revealed the uniform and homogenous surface of 5SmHAp coating. In addition, FTIR studies confirmed the presence of the functional groups characteristic to the HAp structure in both samples. The AFM analysis of the surface coatings revealed the obtaining of homogenous and uniform coatings with no cracks or fissures. The antifungal activity of HAp and 5SmHAp coatings and suspensions was investigated using a Candida albicans ATCC 10231 fungal strain. The results of the quantitative assay of the antifungal activity revealed that both 5SmHAp coatings and suspensions inhibited the development of C. albicans fungal strain. The results obtained by the qualitative assays using AFM, SEM CLSM visualization of the C. albicans cell adherence on the surface coatings confirmed the quantitative results and evidenced that the antifungal properties of the coatings were influenced by the incubation time. Furthermore, the cytotoxic assay using HeLa cell line emphasized that both 5SmHAp suspensions and coatings did not present any toxicity against HeLa cells for all tested incubation intervals. Nowadays, following the occurrence of microbial infections affecting public health, the development of novel materials that could be used as a cheap alternative for the obtaining of coatings with high antimicrobial activity over time is of great interest.