1 Introduction

Titanium and its alloys are widely used as implant materials in orthopaedic surgery because of their good biocompatibility with bone. However, a way to make them bioactive is to coat them with calcium phosphate ceramics which are able to form a real bond with the surrounding bone tissue in vivo [1–4]. Many methods are developed and used to prepare calcium phosphate coatings onto implant surfaces, for example, plasma spray [5–7], sol–gel [8–10], pulsed laser-deposition [11, 12], electrophoretic method [13–15] and electrochemical deposition [16–20]. This last method has a variety of advantages compared to other methods and especially to plasma spray which is the actual industrial standard method: the coating process occurs at low temperature, the thickness and the chemical composition are controlled.

However, when using high current density, a large amount of H2 bubbles is produced at the vicinity of the cathode leading to non uniform and weakly adherent coatings [21]. In order to solve these problems, pulsed electrodeposition may be used [22]. Indeed, the use of a relaxation time between two deposition times (pulse cycle) strongly reduces H2 bubbles emission allowing the calcium phosphate coating to be adequately deposited.

When calcium phosphates are immersed into biological medium, they are subject to a dissolution process immediately followed by the precipitation of a bone-like apatite phase that point out the bioactivity of the prosthetic material [23]. This process modifies the surface morphology and the thickness of the coating. The present investigation follows, as a function of immersion time, the dissolution/precipitation and corrosion processes into Dulbecco’s Modified Eagle Medium (DMEM) of a calcium phosphate coating elaborated by pulsed electrodeposition. The results are systematically compared to those obtained for stoichiometric hydroxyapatite used as a reference since its behaviour in biological solution is well established. Moreover, the surface modification of the coating as a function of immersion time is also studied.

2 Experimental

2.1 Electrodeposition

Figure 1a represents schematically the experimental setup of the electrolytic cell used for calcium phosphate coatings electrodeposition as described previously [24]. The electrolyte was prepared by dissolving 0.042 M Ca(NO3) ·2 4H2O and 0.025 M NH4(H2PO4) in ultra-pure water. The measured pH value was 4.4 and the temperature fixed at 60°C.

Fig. 1
figure 1

Principle of pulsed electrodeposition current: a schematic view of the experimental set-up and b pulsed current evolution as a function of time

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The Ti6Al4V substrates were disks of 12 mm diameter and 4 mm thickness. Prior to coating, they were etched in a mixture of acids consisting of nitric acid (HNO3, 6% in volume) and hydrofluoric acid (HF, 3% in volume). Then, they were ultrasonically cleaned in acetone and in ultra-pure water. Electrodeposition was performed by pulsing the current as indicated in Fig. 1b. A cycle is composed of a time deposition t d = 1 min with a current density j d = −15 mA cm−2 followed by a break time t b = 2 min (j b = 0 mA cm−2). The number of deposition cycles was fixed to five which correspond to a total deposition time of 15 min.

After electrodeposition the specimens were dried and annealed at 550°C during 2 h in order to stabilize the coatings and to improve their adhesion to the substrates [24].

The stoichiometric hydroxyapatite (HAP) coatings used as reference for dissolution studies were obtained by using the same electrodeposition procedure associated with 9% of hydrogen peroxide into electrolyte as we described in a recent work [25].

2.2 Scanning electron microscopy and X-ray analysis

The coatings morphologies and the coating/substrate cross-sections were observed using a LaB6 electron microscope (JEOL JSM-5400LV) operating at 0–30 kV. This microscope is associated to an ultra-thin window Si(Li) detector for X-ray measurements (GENESIS, Eloïse SARL, France). The specimens are coated with a conductive layer (Au–Pd for SEM micrographs and carbon for X-ray microanalysis). The X-ray spectra were acquired at primary beam energy of 15 kV with an acquisition time of 100 s. For the quantitative analysis (concentration and thickness), commercial software (GENESIS, Eloïse SARL, France) associated with an original procedure for coating analysis developed in our laboratory is used [26–28]. Several measurements were carried out to calculate a mean value of Ca/P atomic ratio and coating thickness t with standard deviation.

X-ray maps (256 × 200 pixels) of the coatings surface were obtained by scanning a 68 μm × 54 μm area with 0.05 s per pixel time (which corresponded to a total acquisition time of about 45 min).

3D reconstruction of the coating surfaces was carried out using commercial software (MeX, Alicona, France). This method consists in a combination of stereoscopic images obtained by inclination of the sample in SEM. Therefore, digital elevation model (DEM) and roughness profiles of the surface specimen are obtained.

2.3 Scanning transmission electron microscopy (STEM)

A Philips CM30 operating from 100 to 300 kV was used to study the morphology of the samples at a nanometre level. The cross-sections were prepared using a specific method developed in our laboratory and previously described [25].

2.4 X-ray diffraction analysis

For the phase composition investigation, powder was scratched from the coating and analysed with an X-ray diffractometer Bruker D8 Advance. The X-ray pattern data were collected from 2θ = 10° to 45° using a monochromatic CuKα radiation at the step of 0.04 degrees with a count time of 12 s at each step. The calcium phosphate structures were identified basing on JCPDS files and the percentage of crystallinity was determined following standard ISO 13779-3 [29].

2.5 Dissolution experiments

The electrodeposited coatings were immersed in triplicate in 7 ml of Dulbecco’s Modified Eagle Medium: DMEM (Table 1) at 37°C in humidified atmosphere containing 5% CO2. The different incubation periods tested were 1, 4 and 12 h and 1, 2, 4, 7, 14, 21 and 28 days. After these immersion times, the specimens were retrieved, rinsed by dipping in distilled water and then dried and kept at 37°C for SEM analysis and the media were collected to determine the Ca and P amounts using ICP-AES (Induced Coupled Plasma–Atomic Emission Spectroscopy, VARIAN Liberty Série II). Measurements were performed in triplicate. The measured values were systematically compared to those of HAP coating (reference) measured in the same experimental conditions.

Table 1 Chemical composition of DMEM
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2.6 Statistical analysis

Significance of data obtained from ICP-AES experiments was assessed using a non parametric Mann & Whitney Test (StatXact 7.0, Cytel Inc, Cambridge, MA, USA). Difference between two conditions was considered significant when P < 0.05.

2.7 In vitro electrochemical corrosion studies

Potentiodynamic polarization experiments were used to evaluate the corrosion behaviour of electrodeposited coatings on Ti6Al4V before and after immersion into DMEM. The temperature was maintained at 37°C. The polarization curves (potential variation as a function of current density) were determined by increasing the potential at a scan rate of 1.0 mV s−1. Measurements were performed in triplicate.

3 Results

The SEM micrograph of the electrodeposited coating shown in Fig. 2a indicates that the coating is composed of small needles and crystallites. The Scanning transmission electron microscopy (STEM) micrograph of Fig. 2b confirms these observations and shows that the needles size is less than 0.2 μm. Quantitative analysis from X-ray spectrum of Fig. 2c give a Ca/P atomic ratio of 1.60 ± 0.02 and a thickness t = 7.9 ± 0.6 μm. XRD pattern of Fig. 2d shows that the coating is composed of an apatitic phase of low crystallinity with typical diffraction peaks at 2θ = 25.9°–31.8°–32.2°–32.9°–34°–39.8° corresponding to hydroxyapatite (JCPDS 09-0432). The calculated percentage of crystallinity was 65%. The combination of Scanning electron microscopy and X-ray analysis (SEM-EDXS) and XRD results indicates that the obtained coating corresponds to a calcium deficient hydroxyapatite (Ca-def HAP) [30].

Fig. 2
figure 2

Characterization of the electrodeposited coating: a SEM and b STEM micrographs, c EDXS spectrum and d XRD pattern

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Next, these coatings were immersed in DMEM until 28 days. In order to show the evolution of calcium and phosphorus concentration in DMEM collected after each time period, we determined by ICP-AES the ratios Caspec/Castand and Pspec/Pstand.

The Caspec and Pspec (specimen) are respectively, the measured calcium and phosphorus concentrations in DMEM which were in contact with the coatings.

The Castand and Pstand (standard) are respectively, the calcium and phosphorus concentrations in DMEM alone measured in the same experimental conditions.

The obtained results are presented in Fig. 3. For the HAP coating (reference), the data demonstrated that Caspec/Castand (Fig. 3a) and Pspec/Pstand (Fig. 3b) ratios do not exhibit any variations (P > 0.05) before 2 days of immersion. Then, significant 21 and 27% decreases (P < 0.05) are observed from 2 to 14 days of immersion (for Caspec/Castand and Pspec/Pstand ratios respectively) which stabilized thereafter (P > 0.05). Concerning the Ca-def HAP coating, the results highlight a significant decrease from 4 h to 14 days of immersion for both elements (48 and 50% respectively for Caspec/Castand and Pspec/Pstand ratios). Finally, the values reached a plateau until 28 days of immersion in DMEM. Based on ICP-AES results, we focused the SEM characterizations of the immersed specimens on the main delays 1, 7 and 14 days. The results are presented on Fig. 4. One may observe a progressive change of the coating morphology especially after 14 days of immersion where the precipitated coating is homogenous and less porous than 1 day sample. The SEM cross-sections of Fig. 5 show that the precipitated coating thickness is enhanced reaching about 35 μm with an improved link to the substrate. These observations are confirmed by elemental X-ray maps of Fig. 6 showing that calcium and phosphorus distributions are more homogenous from 1 to 14 days compared to those of non-immersed coating. Moreover, the X-ray intensity of titanium substrate decreases significantly, mainly at 7 and 14 days which confirms the important thickness increase of the precipitated coating.

Fig. 3
figure 3

\( \left( {{\frac{{{\text{Ca}}_{\text{spec}} }}{{{\rm Ca}_{\text{stand}} }}}} \right) \)a and \( \left( {{\frac{{{\text{P}}_{\text{spec}} }}{{{\rm P}_{\text{stand}} }}}} \right) \)b ratios measured by ICP-AES as a function of immersion time in DMEM. Shown are the mean values of nine measures ± SD. Gray solid line represents DMEM alone as reference, black dashed line and black solid line represent HAP standard coating and Ca-def HAP coating respectively. $ and * mean P < 0.05 compared to the previous time value for HAP standard coating and Ca-def HAP coating respectively

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Fig. 4
figure 4

SEM micrographs of the coating showing the modification of the morphology after 1, 7 and 14 days of immersion time in DMEM

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Fig. 5
figure 5

Cross-section SEM micrographs showing the thickness and the coating/substrate interface evolutions before and after 1, 7 and 14 days of immersion time in DMEM

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Fig. 6
figure 6

EDXS elemental maps of the coating as a function of immersion time in DMEM

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On the other hand, to illustrate the decrease of the porosity, 3D-SEM reconstructions were carried out. Figure 7 shows topographic images of the samples from 0 to 14 days of immersion. The corresponding profiles (Fig. 8) clearly indicate an important reduction of depth/height differences. The corresponding roughness values presented in Table 2 underline the decrease observed as a function of immersion time. For example, the arithmetic roughness (Ra) of the coating decreases from 4.9 μm before immersion to 2.9 μm after 14 days of immersion.

Fig. 7
figure 7

3D-SEM reconstruction of the coating surface before and after 1, 7 and 14 days of immersion time in DMEM

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Fig. 8
figure 8

Roughness profile of the coating before and after 1, 7 and 14 days of immersion time in DMEM

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Table 2 Roughness parameters of the calcium phosphate coating surface as a function immersion time in DMEM
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Furthermore we hypothesized that Ca-def HAP coatings modifications can alter corrosion properties of the samples. Figure 9 shows polarization curves of the uncoated Ti6Al4V (a) compared to Ca-def HAP/Ti6Al4V at 0 day (b) and 7 days (c) of immersion. A shift to nobler values can clearly be observed for coated Ti6Al4V samples. The corrosion potential value increased from −0.514 V for the uncoated Ti6Al4V to −0.394 V for the Ca-def HAP/Ti6Al4V sample. After 7 days of immersion, the corrosion potential reaches 0.104 V indicating that the pulsed electrodeposited coating acts as a protective barrier for the titanium substrate and improves its corrosion protection in biological liquid.

Fig. 9
figure 9

Polarization curves of a uncoated Ti6Al4V substrate, b as-deposited Ca-def HAP coating and c Ca-def HAP coating after 7 days of immersion in DMEM

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4 Discussion

In this work we have elaborated and characterized a suitable biomaterial for titanium alloy prosthesis coating. Its bioactivity is greater than one of HAP, the gold standard material in the field [31].

We have performed biomaterial synthesis using pulsed electrodeposition of calcium phosphate to avoid the problems linked to H2 bubbles generation at the Ti6Al4V cathode [24]. When the current density is applied, reduction of water occurs at the cathode as:

$$ 2{\text{H}}_{2} {\text{O}} + 2{\text{e}}^{-} \to {\text{H}}_{2} + 2{\text{OH}}^{-} $$
(1)

Then, the pH at the vicinity of the cathode increases, therefore reaction (1) is followed by acid–base reactions of phosphate ions such as:

$$ {\text{H}}_{2} {{\text{PO}}_{4}^{}}^{-} + {\text{OH}}^{-} \to {{\text{HPO}}_{4}^{}}^{2-} + {\text{H}}_{2} {\text{O}} $$
(2)
$$ {{\text{HPO}}_{4}^{}}^{2-} + {\text{OH}}^{-} \to {{\text{PO}}_{4}^{}}^{3-} + {\text{H}}_{2} {\text{O}} $$
(3)

At these pH values (more than 13), the minority specie is HPO4 2− and the predominant specie is PO4 3− as shown by Eliaz et al. [32]. Therefore, calcium deficient hydroxyapatite (Ca-def HAP) phase precipitates as:

$$ 9.5\,{\text{Ca}}^{2 + } + 5.5\,{{\text{PO}}_{4}^{}}^{3-} + 0.5\,{{\text{HPO}}_{4}^{}}^{2-} + 0.5\,{\text{OH}}^{ - } \to {\text{Ca}}_{9.5} \left( {{\text{HPO}}_{4} } \right)_{0.5} \left( {{\text{PO}}_{4} } \right)_{5.5} \left( {\text{OH}} \right)_{0.5} $$
(4)

Indeed, the SEM-EDXS and XRD characterizations of the biomaterial have highlighted a calcium-deficient hydroxyapatite (Ca-def HAP). To investigate on this coating’s bioactivity in biological fluid, we performed immersion experiments followed by morphological examinations of the samples to show their surface modifications. In fact, as it is well described [33], the calcium phosphate coating is dissolved initially releasing Ca2+ and PO4 3− ions into the solution. With increasing the ions concentrations, the solution becomes super-saturated with those ions resulting in a re-precipitation of apatite. The precipitation rate in the case of Ca-def HAP is more pronounced than that of HAP (reference). For example, the Caspec/Castand ratio passed from 1 at 0 day to 0.48 at 28 days in the case of Ca-def HAP and only to 0.7 in the case of HAP. The precipitation process is more rapid in the case of Ca-def HAP than in HAP. The Caspec/Castand ratio of Ca-def HAP reached the 0.7 value through 2 days compared to 28 days for HAP.

The dissolution/precipitation process of the coating was also illustrated across the morphological changes observed by SEM, especially the improvement of the bond between the titanium alloy substrate and the Ca-def HAP coating as it is a critical parameter in coated-implant lifespan [34]. In addition, the increase of the coating’s thickness and the decrease of its roughness observed by SEM cross-sections and X-ray elemental maps play an important role on its corrosion behaviour as confirmed by polarization measurements.

5 Conclusion

In this work, the bioactivity of a Ca-def HAP coating on titanium alloy elaborated by pulsed electrodeposition was investigated. It is demonstrated that the dissolution/precipitation process into biological liquid (DMEM) is more pronounced in comparison with stoichiometric HAP coating used as reference. The characterization of the precipitated coating exhibited a compact and thick coating leading to an improvement of its corrosion protection in biological liquid. We believe that this biomaterial will be considered as an alternative to the stoichiometric HAP usually employed in implant industry.