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Article

Electrophoretic Deposition of Green-Synthesized Hydroxyapatite on Thermally Oxidized Titanium: Enhanced Bioactivity and Antibacterial Performance

by
Mariana Relva
1,
Daniela Santo
2,
Ricardo Alexandre
3,
Pedro Faia
4,*,
Sandra Carvalho
2,5,
Zohra Benzarti
2,6 and
Susana Devesa
2,*
1
University of Coimbra, Physics Department, Rua Larga, 3004-516 Coimbra, Portugal
2
University of Coimbra, Centre for Mechanical Engineering, Materials and Processes, Advanced Production and Intelligent Systems, Department of Mechanical Engineering, Rua Luís Reis Santos, 3030-788 Coimbra, Portugal
3
Inovatools Portugal LDA, Rua da Indústria Metalúrgica, 593 Cumeiras Embra, 2430-528 Marinha Grande, Portugal
4
University of Coimbra, Centre for Mechanical Engineering, Materials and Processes, Electrical and Computer Engineering Department, FCTUC, Polo 2, Pinhal de Marrocos, 3030-290 Coimbra, Portugal
5
IPN—LED&MAT—Instituto Pedro Nunes, Laboratório de Ensaios, Desgaste e Materiais, Rua Pedro Nunes, 3030-199 Coimbra, Portugal
6
University of Sfax, Laboratory of Multifunctional Materials and Applications (LaMMA), Department of Physics, Faculty of Sciences of Sfax, Soukra Road km 3.5, B.P. 1171, Sfax 3000, Tunisia
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2025, 15(15), 8598; https://doi.org/10.3390/app15158598
Submission received: 20 June 2025 / Revised: 29 July 2025 / Accepted: 30 July 2025 / Published: 2 August 2025
(This article belongs to the Special Issue Sustainable Nanomaterials for Biotechnological and Medical Applications: Current Developments)
Browse Figures
Versions Notes

Abstract

Titanium alloys such as Ti-6Al-4V are widely used in biomedical implants due to their excellent mechanical properties and biocompatibility, but their bioinert nature limits osseointegration and antibacterial performance. This study proposes a multifunctional surface coating system integrating a thermally oxidized TiO2 interlayer with a hydroxyapatite (HAp) top layer synthesized via a green route using Hylocereus undatus extract. The HAp was deposited by electrophoretic deposition (EPD), enabling continuous coverage and strong adhesion to the pre-treated Ti-6Al-4V substrate. Structural, morphological, chemical, and electrical characterizations were performed using XRD, SEM, EDS, Raman spectroscopy, and impedance spectroscopy. Bioactivity was assessed through apatite formation in simulated body fluid (SBF), while antibacterial properties were evaluated against Staphylococcus aureus. The results demonstrated successful formation of crystalline TiO2 (rutile phase) and calcium-rich HAp with good surface coverage. The HAp-coated surfaces exhibited significantly enhanced bioactivity and strong antibacterial performance, likely due to the combined effects of surface roughness and the bioactive compounds present in the plant extract. This study highlights the potential of eco-friendly, bio-inspired surface engineering to improve the biological performance of titanium-based implants.

1. Introduction

Titanium alloys, particularly Ti-6Al-4V, have been extensively used in biomedical applications such as orthopedic implants, dental prosthetics, and cardiovascular devices due to their high mechanical strength, low density, excellent corrosion resistance, and superior biocompatibility [1,2,3,4]. Due to these attributes, Ti-6Al-4V is widely chosen for long-term in vivo applications that demand mechanical reliability and biological safety [2].
Ti-6Al-4V is classified as an α + β titanium alloy, offering a balance of strength, ductility, and thermal stability [2,3,4]. Its biocompatibility is primarily attributed to the spontaneous formation of a passive titanium dioxide (TiO2) layer, which acts as a protective barrier against corrosion and reduces metal ion release. This oxide layer regenerates rapidly when damaged and promotes osseointegration, thereby improving implant stability over time [2].
Despite its advantages, Ti-6Al-4V also presents several limitations. It is expensive to produce and difficult to machine due to its high hardness. Additionally, it is prone to wear under certain mechanical conditions and exhibits poor thermal conductivity. Another concern is its tendency toward biofilm development, which can undermine the long-term performance of biomedical implants [2,4]. These drawbacks have led to the development of surface modification strategies to improve the alloy’s biological and mechanical performance.
A frequently used strategy consists of generating a TiO2 layer through anodization or thermal oxidation. Ehlert et al. [5] demonstrated that modifying the Ti-6Al-4V surface with a nanoporous TiO2 layer significantly improved the physicochemical properties and osseointegration potential of the substrate. This layer improves wear resistance and energy dissipation under mechanical loading—critical factors for load-bearing implants. Furthermore, TiO2 acts as a suitable interlayer for HAp coatings by enhancing mechanical bonding and adhesion strength [5]. Due to its high hardness, biocompatibility, and antibacterial properties, TiO2 has proven to be a multifunctional interface for implant applications [6].
To further enhance bioactivity, HAp is frequently employed as a surface coating due to its chemical and structural similarity to bone mineral. HAp is non-toxic, biocompatible, and osteoconductive and promotes long-term osseointegration [7,8,9,10,11]. Moreover, HAp presents valuable properties such as sinterability, resorbability, and controlled solubility, all of which contribute to its suitability for biomedical applications [10,12,13]. In recent years, green synthesis methods have gained attention for the production of HAp, using plant extracts as bio-templates. These approaches reduce the need for toxic reagents or high-energy processes, offering a more sustainable alternative [14,15]. In this work, HAp was synthesized using extracts from Hylocereus undatus cladodes, a cactus species rich in flavonoids, terpenoids, and phenolic compounds, offering an eco-friendly and functional enhanced route for biomedical surface coatings [15].
Among the available deposition techniques, electrophoretic deposition (EPD) has been extensively explored for HAp applications, particularly when using pre-synthesized powders or doped HAp derived from green methods [16,17,18,19]. EPD is a process where charged particles in a suspension migrate under the influence of an electric field and are then deposited as a uniform layer onto the substrate [16,17,18,19]. The process is simple, low-cost, and adaptable to a wide range of materials and geometries [17,19]. Farrokhi-Rad et al. [18] prepared HAp coatings with controlled porosity using EPD and carbon black as a porogen agent, showing the method’s adaptability. Zhitomirsky et al. [20] demonstrated the feasibility of depositing chemically precipitated HAp on Ti-6Al-4V as early as 1997.
By incorporating a TiO2 interlayer, EPD enables the development of coatings with enhanced adhesion, biological performance, and potential antibacterial activity [19]. The bioactivity of these coatings is typically assessed by their ability to form an apatite layer when immersed in simulated body fluid (SBF), which reflects their bone-bonding capacity [21,22]. Simultaneously, growing concerns over implant-associated infections have led to the development of coatings with antibacterial properties. Both TiO2 and calcium phosphate-based coatings have demonstrated antibacterial effects through mechanisms including ion release, surface charge interaction, and photocatalytic activity [23]. Coatings that combine these materials are therefore promising for multifunctional applications, providing both enhanced osseointegration and antibacterial activity.
In this study, we aimed to develop a multifunctional coating system for Ti-6Al-4V implants by integrating a thermally oxidized TiO2 interlayer with a green-synthesized HAp top layer deposited via EPD. Our approach differs from conventional strategies by combining bio-inspired, plant-mediated HAp, formation of the TiO2 interlayer through a brief thermal treatment at moderate temperature, and non-toxic EPD processing. This integrated method results in coatings that demonstrate enhanced biological performance and pronounced antibacterial properties while maintaining a low environmental impact, which collectively defines the novelty and significance of this work.
The structural, chemical, morphological, and electrical properties of the coatings were thoroughly characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), Raman spectroscopy, and impedance spectroscopy. Furthermore, their bioactivity and antibacterial performance were also evaluated in vitro to assess their potential in enhancing the biological performance of titanium-based biomedical implants.

2. Materials and Methods

2.1. Materials

For the synthesis of HAp, Hylocereus undatus cladodes were collected from an adult plant in a garden in Coimbra, Portugal, in the month of October, and di-phosphorus pentoxide (P2O5, 97%, Scharlau, Barcelona, Spain), calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, 98%, Scharlau, Barcelona, Spain), and sodium hydroxide (NaOH, 98%, Panreac, Barcelona, Spain) were used.
For the EPD of HAp, a Ti-6Al-4V substrate with dimensions of (2.5 × 2.5) cm2 and thickness of 1.10 mm was used. The substrates were previously washed with ethanol, C2H6O (99% v/v, Valente & Ribeiro, Alcanena, Portugal), and hydrogen peroxide, H2O2, (3% w/v, 10 vol, AGA, Prior Velho, Portugal) was used as the exclusive medium for the EPD process.
The deionized water used was self-produced using ultra-pure water equipment (Model HLO 5UV, Hydrolab, Genas, France).

2.2. Synthesis of Hydroxyapatite

To synthesize HAp, an extract of Hylocereus undatus was first prepared by heating 15 g of cladode in 250 g of deionized water at 80 °C for 2.5 h on a heating plate. After this time, the mixture was filtered and stored.
Subsequently, 8.52 g of P2O5 and 47.2 g of Ca(NO3)2·4H2O were mixed, and the prepared extract was slowly added to the precursors. This mixture was kept under continuous stirring at 45 °C for 2 h. The pH was adjusted to 12 using an aqueous NaOH solution, maintaining the stirring and temperature conditions for an additional hour. The obtained material was aged for 2 days at room temperature, after which it was dried in a furnace at 150 °C for 3 h. The resulting powder was then heat-treated at 500 °C for 2 h.
This procedure is schematically represented in Figure 1.

2.3. Pre-Treatment of the Titanium Plate

Prior to deposition, the plates were cleaned with ethanol to remove surface contaminants. Following this, the substrates were thermally treated in the furnace at 400 °C for 1 h, with a heating rate of 10 °C/min. This heat treatment promoted the formation of a thin interlayer of titanium dioxide (TiO2) on the surface, which served to improve the adhesion of the coating.

2.4. Electrophoretic Deposition Procedure

For the electrophoretic deposition, a suspension was prepared using 80 mL of H2O2 and 1 g of the synthesized powder, which, as will be detailed later, comprised 0.34 g of HAp and 0.66 g of NaNO3. To ensure a homogeneous dispersion, the mixture was stirred for 45 min prior to deposition.
A programmable power supply (EA-PSI 9360-15, Elektro-Automatik, Viersen, Germany) was used to regulate the current density, in combination with a digital multimeter (2831E, B&K Precision, Yorba Linda, CA, USA) configured as an ammeter.
The Ti-6Al-4V plate (Jacquet, São João, Portugal), on which Kapton® tape was applied to restrict the deposition area to one side, served as the cathode, while a platinum wire with a diameter of 0.25 mm (99.9%, Merck, Rahway, NJ, USA) was used as the anode.
The deposition parameters are outlined in Table 1.

2.5. Characterization Techniques

HAp was structurally characterized with XRD using a Rigaku Smartlab diffractometer (Tokyo, Japan) with Cu-Kα (λ = 1.54060 Å) radiation, operating at 40 kV and 50 mA. The measurements were performed in a theta/2theta geometry, with a step size of 0.02°. Raman spectroscopy was conducted using a Renishaw in ViaTM (Wotton-under-Edge, UK) confocal Raman spectrometer, equipped with a 532 nm green laser.
The surface morphology of the Ti-6Al-4V substrate, TiO2 pre-coating, and HAp coating was analyzed by scanning electron microscopy (SEM) using a Hitachi SU3800 microscope (Tokyo, Japan) in secondary electron mode. The elemental composition and Ca/P ratio were assessed by energy-dispersive X-ray spectroscopy (EDS) using a Bruker Nano System (Berlin, Germany), set at an accelerating voltage of 10 keV.
To estimate the thickness of the TiO2 pre-coating, the reflectivity and color of the coatings were measured using a Gretagmacbeth ColorEye® XTH spectrophotometer (Grand Rapids, MI, USA). This equipment measures the specular and diffuse reflection components, in a wavelength range between 360 and 750 nm, and the color coordinates in the CIE-L*a*b* color space, which is the most commonly used approach for human color perception [24].
Electrical impedance spectroscopy measurements were accomplished using an Agilent 4294A, in air, at room temperature and 37 °C, across a frequency range from 0.15 MHz to 40 MHz. In this study, three distinct samples were analyzed, with two acquisitions performed for each sample: the untreated substrate, the alloy after thermal oxidation, referred to as the TiO2 pre-coating, and the thermally oxidized sample further modified by the deposition of a HAp layer. The integrity of the measured complex impedance data was ensured by the Agilent equipment, which includes inbuilt Kramers–Kronig transformations [25].

2.6. Bioactivity Experiments

The bioactivity of the samples was assessed by immersing them in 20 mL of SBF at 37 °C, which closely replicates the ionic composition of human blood plasma, in accordance with the international standard ISO/FDIS 23317 [26,27,28].
Following a 15-day incubation period, the samples were removed, thoroughly rinsed with Milli-Q water, and dried in a desiccator. To analyze the formation of calcium phosphate compounds and monitor variations in the surface Ca/P ratio, a field emission scanning electron microscope (Hitachi SU3800, Tokyo, Japan) coupled with an energy-dispersive X-ray was employed (Bruker Nano System). The accelerating voltage was 10 keV, and the analyses were performed in secondary electron mode. To ensure statistical validation, three measurements were conducted across different zones of the sample.

2.7. Antibacterial Activity

2.7.1. Halo Inhibition Assay and SEM Analysis

The antibacterial efficacy of the coatings was evaluated against the Gram-positive bacterium Staphylococcus aureus (ATCC 6538). The Halo test was performed to evaluate the antibacterial activity. Prior to testing, the samples were sterilized and photoactivated by exposing them to ultraviolet (UV) light for one hour.
The Halo test was performed to evaluate the antibacterial activity. Prior to testing, the samples were sterilized under UV light exposure for one hour. Bacterial inocula were prepared by culturing a single colony in 30 mL of Tryptic Soy Broth (TSB, Frilabo, Milheirós, Portugal), incubated overnight at 37 °C with agitation at 120 rpm. The optical density (OD) of the inoculum was measured at 620 nm and properly diluted in culture media to 1.5 × 108 CFU/mL. A 100 µL aliquot of the bacterial suspension was uniformly spread onto Tryptic Soy Agar (TSA, Frilabo, Milheirós, Portugal) Petri dishes. Once the agar solidified, individual samples were gently placed onto the surface, ensuring direct contact between the coated side and the medium. To evaluate the antibacterial activity of the Hylocereus undatus extract, a sterile filter paper disk was soaked in the extract solution (at the same concentration used for HAp synthesis) and placed on the agar surface. All plates were incubated at 37 °C for 24 h. Following incubation, the presence or absence of inhibition zones (clear halos indicating bacterial growth suppression) was observed. All the experiments were repeated with at least three independent assays. Subsequent to the halo assay, the samples were rinsed with distilled water and subjected to a graded ethanol dehydration series (50%, 70%, 80%, 95%, and 100% v/v) for 10, 10, 10, 10, and 20 min, respectively. The dehydrated samples were then stored in a desiccator, sputter-coated with a thin layer of gold using a Cressington 108 Sputter Coater, and subsequently analyzed by SEM (Hitachi SU3800) in both secondary electron (SE) and backscattered electron (BSE) modes.

2.7.2. Quantitative CFU Counting

For quantitative assessment, the bacterial suspension was adjusted to a final concentration of 5 × 106 CFU/mL in TSB. A 2 mL aliquot was added to each well of a 24-well plate containing the respective coated or uncoated samples. The plates were incubated at 37 °C for 24 h under static conditions. Post-incubation, each sample was gently washed three times with sterile phosphate-buffered saline (PBS) to remove non-adherent bacteria.
To detach adherent bacteria, the samples were placed in a bath sonicator at 50 Hz for 10 min. The recovered bacterial suspensions were serially diluted (10-fold), plated in triplicate onto TSA plates, and incubated at 37 °C for 24 h. After incubation, colony-forming units (CFUs) were counted, and the number of viable bacteria per sample was calculated by multiplying the colony count by the corresponding dilution factor.
Additionally, the antibacterial rate was further calculated using the following equation:
Antibacterial   rate   ( % )   =   [ 1   −   ( C F U s a m p l e C F U c o n t r o l   ) ] × 100
where CFUcontrol is the number of viable bacteria recovered from the uncoated substrate, and CFUsample refers to each test condition. All measurements were performed in triplicate from at least three independent experiments.

2.8. Statistical Analysis

CFU data were analyzed using one-way analysis of variance (ANOVA), followed by Sidak’s multiple comparisons test to assess statistical differences between coated surfaces and the uncoated control. Statistical analyses were performed using GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA). Differences were considered statistically significant at p < 0.05.

3. Results

3.1. Characterization of Hydroxyapatite

HAp powder was obtained via the sol–gel method using Hylocereus undatus extract as a natural precursor. After drying, the resulting powder was characterized to confirm its phase composition and structural properties.
Figure 2 shows the XRD patterns of the sample after heat treatment at 500 °C, alongside the standard reference for HAp (ICDD-00-064-0738).
The sample’s diffraction pattern displays peaks corresponding to hydroxyapatite, which align with the reference, confirming the formation of crystalline HAp. In addition, peaks corresponding to a secondary phase, sodium nitrate (NaNO3), are evident and marked in the figure. Quantitative phase analysis of the XRD data was carried out by Rietveld refinement using Profex software (version 5.0.1). The quantitative analysis revealed that the sample consisted of 34.11% HAp and 65.89% NaNO3. The presence of NaNO3 in the powder is considered advantageous, as it promotes an increase in the ionic strength of the EPD medium, thereby enhancing conductivity [29,30].
Figure 3 presents the Raman of the same sample. Although the characteristic bands of HAp are present, the spectrum is clearly dominated by the peaks attributed to NaNO3, observed at 189 cm−1, 721 cm−1, 1068 cm−1, 1387 cm−1, and 1677 cm−1, consistent with values reported in the literature [31]. These findings corroborate the results obtained from XRD analysis.

3.2. Characterization of the Substrate and Pre-Coating Layer

Prior to the deposition of HAp, the Ti-6Al-4V alloy substrate underwent surface pre-treatment to improve coating adhesion, addressing one of the major limitations of electrodeposition or electrophoretic deposition of HAp on metal surfaces—namely, the poor adhesion of the HAp layer to the implant surface. Typically, interfacial delamination of the HAp layer is initiated by vertical cracking throughout the coating thickness, a phenomenon already reported for the HAp–Ti-6Al-4V interface. Such delamination can lead to inflammation in surrounding tissues, resulting in bone loss and even implant loosening [5].
This drawback can be overcome by creating a stable intermediate layer that bridges the titanium alloy surface and the HAp coating. In this regard, a TiO2 layer is a favorable option as an interfacial layer between the HAp coating and the titanium substrate. The mechanical compatibility of TiO2 with HAp, along with its chemical affinity for both HAp and titanium, enhances the bonding strength of the HAp coating to the metallic substrate [5,6].
As mentioned before, to form this interfacial TiO2 layer, the Ti-6Al-4V plates were subjected to a thermal oxidation process.
Figure 4 illustrates the spectral reflectivity curves and the simulated colors derived from these reflectivity values, represented by the coordinates L, a, and b* in the CIELAB colorimetric space, to estimate the TiO2 pre-coating thickness. Additionally, the figure includes photographs of the Ti-6Al-4V surface before and after thermal oxidation, providing a visual comparison of the color changes. These three numerical values uniquely define the color of a surface for a given illuminant and observer. Specifically, L* indicates the lightness of the sample, while a* and b* are the chromatic coordinates: a* varies from green (−a*) to red (+a*), and b* varies from blue (−b*) to yellow (+b*). Together, these parameters determine the perceived color of the material examined [32].
The Ti-6Al-4V substrate, shown in its original unoxidized state, appears silver in color and exhibits reflectivity rates ranging from approximately 30% at 360 nm to about 52% at 750 nm. This smooth increase in reflectivity with wavelength is characteristic of a metallic surface with minimal oxide interference. The relatively low overall reflectivity confirms the alloy’s classification as a high-loss metal, consistent with descriptions found in the literature [33].
After heat treatment at 40 °C for 1 h, the titanium alloy exhibits a distinctive U-shaped spectral reflectance curve—starting at approximately 24%, dropping to a minimum of around 5% at 440 nm, and gradually increasing to about 38% at 750 nm. This pattern is typical of thin-film interference resulting from the formation of an oxide layer, as seen in anodized and thermal-oxidized titanium alloys. The selective absorption near 440 nm corresponds to the visually observed yellow–orange coloration.
This minimum at 440 nm corresponds to destructive interference. The wavelength at which this minimum occurs can be related to the oxide film thickness by the expression [32,34,35]:
2 n d = m + 1 2 λ
where λ is the wavelength of the incident light, d is the thickness of the oxide layer, n is the refractive index of the oxide, and m is the order of interference. Assuming normal incidence and considering first-order interference (m = 0) with a refractive index n = 2.4 [32] for the TiO2 layer, the oxide thickness is estimated to be approximately 46 nm.
The impact of this oxide layer on surface color is further demonstrated by the shift in CIELAB color coordinates. Prior to thermal oxidation, the alloy exhibits values of L = 73.01*, a = 0.96*, and b = 7.21*, indicating a relatively high lightness and near-achromatic character, consistent with its visually perceived silver appearance. These low a* and b* values correspond to a desaturated tone, typical of metallic surfaces with minimal oxide interference [36].
Following heat treatment, the color coordinates shift significantly to L = 49.65*, a = 9.57*, and b = 36.09*, indicating a substantial reduction in lightness and a marked increase in chromaticity. This transition reflects the development of a colored interference film on the surface. The positive a* and b* values confirm a shift toward a warmer hue, dominated by red and yellow components [33].
To further characterize the perceived color, the hue angle (h°)—calculated from the arctangent of the b*/a* ratio—yields a value of 75.15°, placing the color firmly within the yellow–orange region of the CIELAB color space [32,37]. This hue corresponds well with the visually observed coloration and aligns with the interference pattern described, where destructive interference at 440 nm selectively reduces blue reflectance. This analysis supports the conclusion that the observed color change arises from thin-film interference effects associated with the oxide layer formed during thermal oxidation.
From this point forward, the thermally oxidized Ti-6Al-4V surface will be referred to as the “TiO2 pre-coating” condition, denoting the substrate state prior to the electrophoretic deposition of HAp.
Figure 5a presents the SEM image of the untreated Ti-6Al-4V substrate, which exhibits a relatively smooth and homogeneous surface. The corresponding EDS spectrum (Figure 5b) confirms the elemental composition of the substrate, establishing a baseline for comparison with the thermally oxidized surface.
After thermal treatment at 400 °C for 1 h, a thin TiO2 layer is formed on the surface of the substrate. Although the surface morphology, shown in Figure 5c, appears similar to that of the untreated substrate in Figure 5a, the EDS analysis in Figure 5d, including the corresponding quantitative data table, indicates an increase in oxygen content, confirming the formation of the oxide layer.
Further confirmation is provided by Raman spectroscopy, as shown in Figure 6, which displays characteristic peaks of the rutile phase of TiO2 at approximately 142, 440, and 611 cm−1 corresponding to the symmetries B1g, Eg, and A1g, respectively [38,39,40,41]. Additionally, a peak at 250 cm−1 is observed, which is attributed to second-order or disorder-induced scattering, associated with multi-phonon processes [39,40,41]. The presence of these well-defined peaks confirms the formation of a crystalline rutile phase.
The experimental Raman band assignments and corresponding literature values are summarized in Table 2.
The combined SEM, EDS, Raman, and reflectivity analyses confirm the successful formation of a TiO2 interlayer on the Ti-6Al-4V substrate, enhancing its surface chemistry and morphology in preparation for HAp EPD.

3.3. Characterization of Coatings

To evaluate the effect of an additional surface pre-treatment on the quality of HAp coatings, thermally oxidized Ti-6Al-4V substrates were subjected to two different pre-electrophoretic deposition conditions: one set was previously immersed in SBF, while the other received no SBF treatment. It is hypothesized that the initial HAp formed during SBF immersion may act as a nucleation layer, thereby enhancing the coverage and uniformity of the subsequently deposited HAp coating. Following these pre-treatment steps, both sets of samples underwent HAp electrophoretic deposition, followed by surface characterization.
Figure 7 shows SEM micrographs of HAp coatings deposited via EPD onto thermal-oxidized substrates subjected to two different surface treatments. In Figure 7a, the SBF-treated surface is presented, which exhibits a low HAp content, with a dispersed and uneven deposition. In contrast, Figure 7b reveals a surface with significantly higher HAp content, characterized by a denser and more uniform HAp layer on the oxidized substrate that did not undergo SBF pre-treatment. The surface coverage is more extensive, and the HAp particles are clearly visible.
These results clearly demonstrate that the sample without SBF pre-treatment achieved better EPD performance, due to the formation of a weak interface that reduced the final adhesion of HAp to the thermally oxidized substrate. Therefore, this condition was selected for further development and characterization due to its superior HAp adhesion and more substantial coating formation.
To further analyze the properties of the coating formed, a comprehensive characterization was carried out on the non-SBF-treated sample.
Figure 8 presents the SEM image of the HAp coating electrophoretic deposited on the Ti-6Al-4V thermal-oxidized substrate without prior SBF treatment. The surface displays a dense and heterogeneous layer composed of well-distributed HAp agglomerates. To evaluate the chemical composition of the coating, EDS analyses were conducted in three distinct regions, as marked in the image. The corresponding EDS data, presented in Table 3, indicate a Ca/P ratio ranging from 1.87 to 1.90, which is slightly higher than the stoichiometric ratio of 1.67. This enrichment in calcium is commonly observed in electrodeposited coatings and suggests the formation of a calcium-rich surface layer [42,43]. Additionally, calcium-rich HAp is commonly observed in syntheses performed in a highly alkaline pH medium [44].
Figure 9a shows an SEM micrograph of the HAp coating electrophoretic deposited on the Ti-6Al-4V oxidized substrate, while Figure 9b provides higher magnification of the green highlighted section, where elemental quantification measurements by EDS were performed, as reported in Table 3. Figure 9c,d display the elemental mapping of the elements Ca and P, respectively, confirming their uniform distribution across the analyzed area.
To further confirm the presence of HAp on the TiO2 pre-coated substrate, Raman analysis was performed. Figure 10 shows the Raman spectrum of the HAp coating, in which the characteristic bands at 432 cm−1, 451 cm−1, 580 cm−1, 593 cm−1, 609 cm−1, 963 cm−1, and 1046 cm−1, highlighted in yellow, clearly indicate the deposition of HAp [45,46,47,48].
Studies suggest that electric and electromagnetic energy can be applied exogenously to accelerate bone formation and healing, largely due to surface charges and electrical conductivity that promote bone cell differentiation and proliferation. To gain deeper insight into these effects, one of the most comprehensive approaches is to examine the electrical properties of biomaterials through impedance spectroscopy. In this context, the electrical properties of the modified surfaces were also analyzed using this technique [49,50,51].
The complex impedance Z* is a frequency-dependent parameter that characterizes the electrical response of a material and is defined as Z* = Z′ + jZ″, where Z′ is the real part (resistive component) and Z″ is the imaginary part (reactive or capacitive component). The impedance magnitude, ∣Z∣, commonly used for comparative analysis, is derived from these components as ∣Z∣ = (Z′2 + Z″2)1/2 [52,53]. In this study, the measured values of Z″ and Z″ were used to calculate and plot ∣Z∣ to evaluate the electrical properties of the different surface modifications.
Figure 11 presents the impedance magnitude ∣Z∣ as a function of the frequency for the Ti-6Al-4V substrate, the thermally oxidized substrate (referred to as TiO2 pre-coating), and the hydroxyapatite (HAp)-coated sample. At room temperature, both the bare alloy and the TiO2 pre-coating exhibit perceptibly lower impedance in the low-frequency region when compared to the HAp-coated surface. This behavior is expected, as in ambient air, impedance is primarily governed by the resistive and capacitive characteristics of surface layers. The hydroxyapatite layer, being more resistive and less conductive than the metallic substrate or thin oxide film, acts as an effective electrical barrier, increasing the overall impedance. In contrast, the bare and oxidized surfaces facilitate easier displacement or leakage currents, resulting in reduced impedance. Upon heating to 37 °C, the impedance of the HAp coating decreases, and the corresponding curve converges with those of the substrate and TiO2 pre-coating, indicating enhanced charge transport due to thermally activated conductivity. This temperature-driven convergence is consistent with the behavior expected of thermally stimulated conduction mechanisms in solid-state systems.
At physiological temperature, the observed decrease in impedance of the HAp-coated sample—typically indicative of increased electrical conductivity—may enhance cellular metabolism. Conductive implants facilitate intercellular electrical signaling, thereby promoting essential cellular functions such as adhesion and migration, which are critical for effective tissue integration [54].

3.4. Bioactivity Response

The ability of the coatings to promote apatite formation in SBF is a key indicator of their bioactivity and potential for osseointegration in biomedical applications [55]. To assess this, SBF immersion tests were conducted on the uncoated Ti-6Al-4V substrate, the TiO2 pre-coated substrate, and the HAp-coated substrate. Figure 12 presents SEM images of each sample before and after a 15-day incubation period in SBF at 37 °C.
As observed in Figure 12a,b, the uncoated substrate maintained uniform and smooth individual grain surfaces, with no evident signs of apatite formation after SBF exposure, indicating negligible bioactivity. The TiO2 pre-coated sample (Figure 12c,d) exhibited a slight increase in surface porosity after immersion, possibly due to minor surface reactions; however, the absence of distinguishable apatite crystals suggests limited bioactivity. In contrast, the HAp-coated sample (Figure 12e,f) showed a notable transformation. Prior to immersion, the surface presented the typical rough and porous morphology associated with HAp coatings. After 15 days in SBF, the formation of a dense layer of spherical, globular deposits was evident—an indication of extensive apatite (Ca+P) precipitation. This morphological change is a strong indicator of the coating’s bioactivity tendency. The EDS spectrum of the post-immersion surface, present in Figure 13, confirmed the composition of the deposited layer as apatite, with dominant peaks of calcium (Ca), phosphorus (P), and oxygen (O). Trace amounts of sodium (Na), magnesium (Mg), and chlorine (Cl), originating from the SBF solution, were also detected, while titanium (Ti) signals are attributed to the underlying substrate.
Before immersion in SBF, the HAp coating exhibited a Ca/P atomic ratio of 1.90, which is slightly higher than the stoichiometric value of 1.67 for pure hydroxyapatite (Table 3). This deviation suggests a calcium-enriched surface, likely resulting from the synthesis conditions, particularly the highly alkaline pH [44]. Following 15 days of immersion in SBF, the Ca/P ratio increased further to 2.10. This rise can be attributed to the selective adsorption and incorporation of calcium ions (Ca2+) from the SBF solution onto the HAp surface during the nucleation and growth of new apatite phases. Although SBF contains both calcium and phosphate ions, its ionic exchange dynamics tend to favor the accumulation of calcium—especially on already calcium-rich surfaces—resulting in the formation of a calcium-rich apatite layer.
These results confirm the superior bioactivity tendency of the HAp coating, in stark contrast to the inert behavior observed for the uncoated and TiO2-coated substrates. This clearly demonstrates the effectiveness of the electrodeposited HAp layer in promoting in vitro bone-like mineralization and its potential for enhancing osseointegration in biomedical implants.

3.5. Antibacterial Activity

The antibacterial performance of the different surfaces was assessed against the Gram-positive bacterium Staphylococcus aureus, using a combination of qualitative and quantitative methods. Initially, antibacterial effects were assessed qualitatively through a halo inhibition assay and SEM in SE and BSE modes after 24 h of incubation (Figure 14). S. aureus was selected for this proof-of-concept study due to its clinical relevance as one of the primary pathogens associated with implant-related infections [56]. Its strong ability to adhere to biomaterial surfaces and form biofilms makes it a representative and challenging model organism for evaluating antibacterial performance in titanium-based implant applications.
Although no visible inhibition zones were detected in the halo assay, SEM analysis revealed distinct differences in bacterial colonization across the tested surfaces. The uncoated titanium substrate exhibited extensive bacterial adhesion, indicating no inherent antibacterial activity. The TiO2 pre-coated surface exhibited a moderate reduction in bacterial density, possibly explained by its photocatalytic activity [57] and the higher surface energy of titanium oxide [58]. According to Liu L. et al., TiO2 surfaces exhibit higher surface energy than uncoated titanium controls, which may promote the adsorption of proteins. These adsorbed proteins could subsequently interact with bacterial cell membranes, preventing bacterial attachment to the surface, thereby reducing adhesion and inhibiting biofilm formation [58].
Notably, the HAp-coated surface exhibited a dramatic reduction in bacterial presence, with no S. aureus cells detected, suggesting that the surface possesses significant antibacterial activity. BSE imaging was instrumental in distinguishing bacterial cells from HAp particles. Due to the higher atomic numbers of calcium and phosphorus, HAp regions appeared brighter in BSE mode, whereas bacterial cells—mainly composed of lighter elements such as carbon and nitrogen—appeared darker. This contrast facilitated the accurate interpretation of surface bacterial adhesion and/or colonization.
To quantitatively validate these observations, colony-forming unit (CFU) assays were conducted after 24 h of incubation (Figure 15).
The uncoated substrate exhibited the highest bacterial load, confirming its lack of antibacterial activity. In contrast, the TiO2 pre-coated surface demonstrated substantial antibacterial efficacy. Although SEM images revealed the presence of bacterial cells on the TiO2-coated surface, this observation does not necessarily contradict the high antibacterial rate, as SEM cannot differentiate between metabolically active and inactive bacteria. Remarkably, the HAp-coated surface not only achieved the highest antibacterial efficacy (~98.8% CFU reduction) but also showed no detectable bacterial cells in SEM micrographs, indicating a strong correlation between the structural evidence and the viability-based quantitative data. As discussed above, the antibacterial effect of the TiO2 coating is primarily attributed to its photocatalytic activity and increased surface energy. However, the superior and potentially synergistic antibacterial performance of the HAp-coated surface suggests the involvement of additional mechanisms. In this study, HAp was synthesized using a green, bio-inspired method with Hylocereus undatus (dragon fruit) extract as the natural reaction medium. This extract contains a variety of bioactive compounds—including flavonoids, terpenoids, chlorophyll, quercetin, and phenolic acids—that may contribute both to HAp crystallization and its antibacterial activity [59,60,61]. To support this hypothesis, the antibacterial activity of Hylocereus undatus extract was independently assessed via an agar diffusion assay (Figure 16).
To further support this hypothesis, the antibacterial activity of the Hylocereus undatus extract was independently evaluated through an agar diffusion test (Figure 16).
A clear inhibition halo was observed around the paper disk soaked in the extract solution, accompanied by a visible reduction in bacterial cells on the treated surface. These results support the hypothesis that the antibacterial efficacy of the HAp-coated surface is not solely attributable to its composition or surface topography but is also amplified by the presence of phytochemicals derived from the H. undatus extract. This combination of physical and biochemical antimicrobial mechanisms positions the developed coating as a promising multifunctional material for bioactive, infection-resistant biomedical applications. Importantly, these quantitative CFU findings align with the qualitative SEM observations, which showed a marked reduction or absence of S. aureus cells on the HAp-coated surfaces. Together, these results confirm the strong antibacterial potential of the HAp-based coating and support its applicability in infection-resistant biomaterials.

4. Conclusions

This study demonstrated the successful development of a sustainable and multifunctional coating system for Ti-6Al-4V substrates, based on a thermally oxidized TiO2 interlayer and a hydroxyapatite (HAp) top layer synthesized via a green route using Hylocereus undatus extract. Structural and compositional analyses confirmed the effective formation of both layers, with the TiO2 film enabling uniform and adherent electrophoretic deposition of the HAp coating. The bioactivity of the coatings was evidenced by the formation of apatite-like structures after immersion in simulated body fluid (SBF), confirming their osteoconductive potential. The antibacterial properties of the coated surfaces were assessed against Staphylococcus aureus using SEM imaging and CFU quantification. The HAp-coated surface exhibited the most pronounced antibacterial effect, suggesting a combination of multiple mechanisms. These results support the hypothesis that the antibacterial efficacy of the HAp-coated surface is not solely attributable to its composition or surface topography but is also amplified by the presence of phytochemicals derived from the H. undatus extract. Together, these findings highlight the potential of this environmentally friendly approach to produce implant coatings that effectively promote bioactivity and reduce bacterial colonization, representing a promising solution for next-generation orthopedic and dental implants.

Author Contributions

Conceptualization, M.R., D.S. and S.D.; methodology, M.R., D.S. and S.D.; validation, M.R., D.S. and S.D.; formal analysis, M.R., D.S. and S.D.; investigation, M.R., D.S., Z.B., P.F. and S.D.; resources, P.F. and S.C.; data curation, M.R. and D.S.; writing—original draft preparation, M.R. and D.S.; writing—review and editing, M.R., D.S., Z.B., R.A., P.F., S.C. and S.D.; visualization, M.R., D.S. and S.D.; supervision, Z.B., S.C. and S.D.; project administration, R.A. and S.C.; funding acquisition, S.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research is sponsored by national funds through FCT—Fundação para a Ciência e Tecnologia, under projects UIDB/00285—Centre for Mechanical Engineering, Materials and Processes and LA/P/0112/2020. This research was also supported by project DRIVOLUTION—Transition to the Factory of the Future (7141-02/C05-i01.02/2022.PC644913740–00000022–23), which was financed by the PRR—Recovery and Resilience Plan—and by the Next Generation EU European Funds, following NOTICE No. 02/C05-i01/2022.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Schematic representation of HAp synthesis.
Figure 1. Schematic representation of HAp synthesis.
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Figure 2. XRD pattern of the sample obtained and the percentage of each compound.
Figure 2. XRD pattern of the sample obtained and the percentage of each compound.
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Figure 3. Raman spectra of the sample obtained.
Figure 3. Raman spectra of the sample obtained.
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Figure 4. Spectral reflectivity curves of the Ti-6Al-4V surface before and after thermal oxidation, along with the corresponding simulated colors and optical images of the Ti-6Al-4V surface pre- and post-thermal oxidation.
Figure 4. Spectral reflectivity curves of the Ti-6Al-4V surface before and after thermal oxidation, along with the corresponding simulated colors and optical images of the Ti-6Al-4V surface pre- and post-thermal oxidation.
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Figure 5. (a) SEM image and (b) EDS spectrum of the substrate; (c) SEM image and (d) EDS spectrum of the thermal-oxidized substrate.
Figure 5. (a) SEM image and (b) EDS spectrum of the substrate; (c) SEM image and (d) EDS spectrum of the thermal-oxidized substrate.
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Figure 6. Raman spectra of the thermal-oxidized substrate.
Figure 6. Raman spectra of the thermal-oxidized substrate.
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Figure 7. SEM images of HAp coatings on thermal-oxidized substrates: (a) SBF-treated and (b) untreated.
Figure 7. SEM images of HAp coatings on thermal-oxidized substrates: (a) SBF-treated and (b) untreated.
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Figure 8. SEM image of HAp coating on thermal-oxidized substrate without SBF treatment, showing the regions analyzed by EDS.
Figure 8. SEM image of HAp coating on thermal-oxidized substrate without SBF treatment, showing the regions analyzed by EDS.
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Figure 9. SEM and EDS elemental mapping of the HAp coating: (a) SEM image (scale bar: 8 µm); (b) SEM image (scale bar: 6 µm); (c) EDS map showing the distribution of Ca; (d) EDS map showing the distribution of P (scale bar for c and d: 6 µm).
Figure 9. SEM and EDS elemental mapping of the HAp coating: (a) SEM image (scale bar: 8 µm); (b) SEM image (scale bar: 6 µm); (c) EDS map showing the distribution of Ca; (d) EDS map showing the distribution of P (scale bar for c and d: 6 µm).
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Figure 10. Raman spectrum of the HAp coating with the bands assigned to HAp.
Figure 10. Raman spectrum of the HAp coating with the bands assigned to HAp.
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Figure 11. Frequency-dependent impedance magnitude ∣Z∣ for Ti-6Al-4V substrate, TiO2 pre-coating, and hydroxyapatite (HAp)-coated sample, measured in air. The main plot shows the data at room temperature and the inset presents the same measurements at 37 °C.
Figure 11. Frequency-dependent impedance magnitude ∣Z∣ for Ti-6Al-4V substrate, TiO2 pre-coating, and hydroxyapatite (HAp)-coated sample, measured in air. The main plot shows the data at room temperature and the inset presents the same measurements at 37 °C.
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Figure 12. SEM micrographs of uncoated substrate (a,b), TiO2 pre-coating (c,d) and HAp coating (e,f) before and after 15 days of incubation in SBF at 37 °C.
Figure 12. SEM micrographs of uncoated substrate (a,b), TiO2 pre-coating (c,d) and HAp coating (e,f) before and after 15 days of incubation in SBF at 37 °C.
Applsci 15 08598 g012
Applsci 15 08598 g013
Figure 13. Representative EDS spectra of the HAp coatings after soaking for 15 days in SBF.
Figure 13. Representative EDS spectra of the HAp coatings after soaking for 15 days in SBF.
Applsci 15 08598 g013
Applsci 15 08598 g014
Figure 14. Top-view SEM micrographs acquired in secondary electron (SE) and backscattered electron (BSE) modes, showing Staphylococcus aureus populations after 24 h of incubation on different surfaces: (a,b) uncoated substrate, (c,d) TiO2 pre-coated surface, and (e,f) HAp-coated surface.
Figure 14. Top-view SEM micrographs acquired in secondary electron (SE) and backscattered electron (BSE) modes, showing Staphylococcus aureus populations after 24 h of incubation on different surfaces: (a,b) uncoated substrate, (c,d) TiO2 pre-coated surface, and (e,f) HAp-coated surface.
Applsci 15 08598 g014
Applsci 15 08598 g015
Figure 15. Antibacterial activity of HAp-coated surfaces against Staphylococcus aureus. Viable bacterial counts were quantified after 24 h of incubation on uncoated substrate, TiO2 pre-coated, and HAp-coated surfaces. Data are expressed as mean ± standard deviation (SD) of log10-transformed colony-forming units (CFUs). The right Y-axis indicates the antibacterial rate (%) calculated as the reduction in CFUs relative to the uncoated surface. Statistically significant differences compared to the uncoated substrate are indicated by asterisks (p < 0.01).
Figure 15. Antibacterial activity of HAp-coated surfaces against Staphylococcus aureus. Viable bacterial counts were quantified after 24 h of incubation on uncoated substrate, TiO2 pre-coated, and HAp-coated surfaces. Data are expressed as mean ± standard deviation (SD) of log10-transformed colony-forming units (CFUs). The right Y-axis indicates the antibacterial rate (%) calculated as the reduction in CFUs relative to the uncoated surface. Statistically significant differences compared to the uncoated substrate are indicated by asterisks (p < 0.01).
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Applsci 15 08598 g016
Figure 16. Top-view SEM micrographs of Staphylococcus aureus populations after 24 h of incubation (a) with Hylocereus undatus (dragon fruit) cactus extract and (b) with TSB medium (control). Insets display the antibacterial activity as determined by the inhibition halo test. Yellow circles indicate the sample area, while yellow dashed circles delineate the zones of inhibition.
Figure 16. Top-view SEM micrographs of Staphylococcus aureus populations after 24 h of incubation (a) with Hylocereus undatus (dragon fruit) cactus extract and (b) with TSB medium (control). Insets display the antibacterial activity as determined by the inhibition halo test. Yellow circles indicate the sample area, while yellow dashed circles delineate the zones of inhibition.
Applsci 15 08598 g016
Table 1. Electrophoretic deposition parameters.
Table 1. Electrophoretic deposition parameters.
I (mA)u (V)T (°C)w (rpm)t (hours)
503.7552002
Table 2. Assignment of Raman bands along with the literature values.
Table 2. Assignment of Raman bands along with the literature values.
Experimental
(cm−1)		Challagulla et al.
[40]
(cm−1)		Kernazhitsky et al.
[39]
(cm−1)		Mazza et al.
[38]
(cm−1)		Sacco et al.
[41]
(cm−1)		Band
Assignment
142140144-143B1g
250230235-235-
440430445447447Eg
611590610612612A1g
Table 3. EDS analysis of HAp coating at selected regions.
Table 3. EDS analysis of HAp coating at selected regions.
RegionAtomic % CaAtomic % PCa/P Ratio
165.1634.841.87
265.3034.701.88
365.6234.381.90
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Relva, M.; Santo, D.; Alexandre, R.; Faia, P.; Carvalho, S.; Benzarti, Z.; Devesa, S. Electrophoretic Deposition of Green-Synthesized Hydroxyapatite on Thermally Oxidized Titanium: Enhanced Bioactivity and Antibacterial Performance. Appl. Sci. 2025, 15, 8598. https://doi.org/10.3390/app15158598

AMA Style

Relva M, Santo D, Alexandre R, Faia P, Carvalho S, Benzarti Z, Devesa S. Electrophoretic Deposition of Green-Synthesized Hydroxyapatite on Thermally Oxidized Titanium: Enhanced Bioactivity and Antibacterial Performance. Applied Sciences. 2025; 15(15):8598. https://doi.org/10.3390/app15158598

Chicago/Turabian Style

Relva, Mariana, Daniela Santo, Ricardo Alexandre, Pedro Faia, Sandra Carvalho, Zohra Benzarti, and Susana Devesa. 2025. "Electrophoretic Deposition of Green-Synthesized Hydroxyapatite on Thermally Oxidized Titanium: Enhanced Bioactivity and Antibacterial Performance" Applied Sciences 15, no. 15: 8598. https://doi.org/10.3390/app15158598

APA Style

Relva, M., Santo, D., Alexandre, R., Faia, P., Carvalho, S., Benzarti, Z., & Devesa, S. (2025). Electrophoretic Deposition of Green-Synthesized Hydroxyapatite on Thermally Oxidized Titanium: Enhanced Bioactivity and Antibacterial Performance. Applied Sciences, 15(15), 8598. https://doi.org/10.3390/app15158598

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