In vitro assessment of zinc apatite coatings on titanium surfaces

Author’s Accepted Manuscript IN VITRO ASSESSMENT OF ZINC APATITE COATINGS ON TITANIUM SURFACES Iveth Yessenia Ortiz, Aline Raybolt dos Santos, Andrea Machado Costa, Elena Mavropoulos, Marcelo Neves Tanaka, Marcelo Henrique Prado da Silva, Sergio de Souza Camargo www.elsevier.com/locate/ceri

PII: DOI: Reference:

S0272-8842(16)31048-3 http://dx.doi.org/10.1016/j.ceramint.2016.06.203 CERI13219

To appear in: Ceramics International Received date: 25 March 2015 Revised date: 22 June 2016 Accepted date: 30 June 2016 Cite this article as: Iveth Yessenia Ortiz, Aline Raybolt dos Santos, Andrea Machado Costa, Elena Mavropoulos, Marcelo Neves Tanaka, Marcelo Henrique Prado da Silva and Sergio de Souza Camargo, IN VITRO ASSESSMENT OF ZINC APATITE COATINGS ON TITANIUM SURFACES, Ceramics International, http://dx.doi.org/10.1016/j.ceramint.2016.06.203 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

IN VITRO ASSESSMENT OF ZINC APATITE COATINGS ON TITANIUM SURFACES

Iveth Yessenia Ortiz1a, Aline Raybolt dos Santos2b, Andrea Machado Costa3c, Elena Mavropoulos4d, Marcelo Neves Tanaka4e, Marcelo Henrique Prado da Silva3f, Sergio de Souza Camargo Jr.1,5g

1

Metallurgical and Materials Engineering Program - COPPE - Universidade Federal do Rio de Janeiro – UFRJ, Rio de Janeiro, RJ. 2

Department of Dental Protheses and Material Science - Universidade Federal do Rio de Janeiro – UFRJ, Rio de Janeiro, RJ.

3

Materials Science Program - Instituto Militar de Engenharia - IME, Rio de Janeiro, RJ. 4

5

Brazilian Center for Physics Research - CBPF, Rio de Janeiro, RJ.

Nanotechnology Engineering Program - COPPE - Universidade Federal do Rio de Janeiro – UFRJ, Rio de Janeiro, RJ.

a. [email protected], b. [email protected], c. [email protected], d. [email protected], e. [email protected], f. [email protected], g. [email protected]

ABSTRACT In this paper, coatings of hydroxyapatite partially substituted with zinc (ZnHA) were produced on titanium substrates by a two-step hydrothermal process using a precursor solution rich in calcium, phosphate and zinc. Activation of titanium surfaces was performed by oxidation with an acidic HF / HNO3 solution. The coated substrates were then converted into HA by immersion in an alkali 0.1M NaOH solution. The ZnHA samples were characterized by several techniques and their in vitro behavior was studied in comparison to hydroxyapatite (HA) and titanium (Ti - control) samples. A uniform and homogeneous calcium-deficient carbonate apatite coating was obtained for all samples, both doped and undoped with zinc. The percentage of zinc incorporated in the coatings is 7 at. %, and the Ca/P ratio is 1.61 (± 0.01) for both types of samples, suggesting that Zn is incorporated substitutionally, replacing Ca atoms into the HA structure. The incorporation of Zn in the HA structure changed the crystals morphology, reduced crystals sizes and decreased the deposition rate showing that zinc is an inhibitor of the growth of HA crystal. X-ray diffraction showed that HA is the single crystalline phase present after alkali treatment. The coating adhesion strength was evaluated in terms of the critical load (Lc) obtained from scratch tests and no significant difference was found between the two tested groups, indicating the good adhesion of ZnHA to Ti substrates. The in vitro response of human osteoblasts (HOB) exposed to the surfaces of HA and ZnHA coatings was evaluated. The results of Live/Dead tests showed cell viability for all samples surfaces, but the adhesion and proliferation tests showed that ZnHA samples presented better adhered and spread cells compared with HA. ZnHA coatings presented cells with elongated or polygonal shapes and clearly more spread than HA. Quantitative analysis showed that there was a significantly higher number of cells adhered to ZnHA coatings compared to HA, indicating the zinc incorporation stimulates osteoblast proliferation.

Keywords: zinc, hydroxyapatite, coatings, titanium surfaces, implants, human osteoblasts.

1.

INTRODUCTION

The search for optimization of coating processes and surface treatments of biocompatible metals like titanium aiming bone-bonding has been subject of many researches [1- 4]. Hydroxyapatite (HA) is used as bone graft and bioactive coating on titanium implants because its chemical composition is similar to that of bone, and its bioactivity allows chemical connection to the bone tissue. HA stoichiometric formula is Ca10(PO4)6(OH)2, with a molar Ca/P ratio of 1.67. Different molar ratios are associated with the presence of other calcium phosphate phases or ionic substitutions in the HA structure [5]. Due to its complex structure, HA enables anionic and cationic isomorphic substitutions. Pure HA does not occur on a macroscopic scale in biological systems. Biological apatite contains impurities, is not stoichiometric and is mainly found in enamel, dentin and bone. The main impurity found is the carbonate group (CO3)2-. Other impurities include ions such as Na+, Mg2+, K+, Cl-, F- and others [6]. Because of its deficiency in calcium of about 10 at. %, natural HA is called calcium deficient hydroxyapatite, and when carbonate is present in the structure, the it is called carbonated hydroxyapatite [7]. In an attempt to mimic natural HA, recent studies have shown that the incorporation of other elements in the HA structure, like Zn and Sr, can improve some of its properties [6, 8 - 11]. Zinc is a structural component of some important enzymes, proteins and bone. When incorporated into HA, zinc may have positive effects; at low concentrations it can stimulate osteoblasts proliferation, allowing the formation of bone matrix and increasing the alkaline phosphatase activity, besides inhibiting osteoclast action [10 12]. For this reason, coating titanium surfaces with hydroxyapatite partially substituted

with zinc (ZnHA) may contribute positively to the successful bone-bonding of the implants. Among the various methods for HA deposition, the biomimetic method, proposed by Kokubo et al. in 1990, tries to mimic the natural process of biological apatite formation and consists of the immersion of bioactive chemically treated titanium in a simulated body fluid (SBF) solution for a long period of time [13]. In order to reduce the deposition time, calcium and phosphate ion concentrations in the solution have been increased and different treatments to activate the substrate surface have been proposed [14 - 16]. Adhesion of the coatings can be enhanced by etching the substrate surface, thus increasing surface roughness. Recent studies show that when the titanium surface is chemically activated by acidic treatment, an intermediate oxide layer between the bioactive precipitated apatite and the metal substrate is created, improving the nucleation and growth of calcium phosphates. This procedure simulates the process of bone biomineralization [17]. The biological response of osteoblastic cells includes cell attachment, cell growth, proliferation and functional activity. Previous studies have demonstrated that ZnHA exhibits good biocompatibility [9, 12] and good performance under in vitro and in vivo testing [11, 18]. However, others authors reported that no significant differences resulted from the addition of Zn to calcium phosphate compounds. This may be due to the low Zn concentration incorporated into the material. Sogo et al. found no significant difference on osteoblastic cell proliferation on rat skulls between pure β-TCP and βTCP with a zinc content of 0.11 wt. % [9]. In the present study, ZnHA coatings were produced on titanium by a hydrothermal process and their compositional, structural, morphological and adhesion properties were

characterized in comparison to pure HA coatings. Then, in vitro tests were performed seeding human osteoblastic cells (HOB) on HA, ZnHA and Ti (control) surfaces in order to evaluate the cytocompatibility, cell attachment and cell proliferation.

2.

MATERIALS AND METHODS

Treatment of titanium substrates Commercially pure titanium (cp-Ti) substrates were cut into small squares of 10 x 10 x 1 mm3, grounded with 400 to 600 grit SiC sandpapers and then cleaned with deionized water. Substrates were subsequently immersed in a hydrofluoric and nitric acid solution [HF / HNO3] for 30 seconds at a concentration of 70 ml H2O, 28 ml HNO3, 2 ml HF, followed by an ultrasonic cleaning with acetone and deionized water and then dried in an oven at 80 °C for 10 minutes.

Preparation of precursor solution A saturated solution of Ca2+ and PO43- ions (SSCP) was prepared for the deposition of the HA coatings on titanium substrates as described elsewhere [16]. During the preparation, 0.5 M calcium hydroxide (Ca(OH)2), 0.3 M phosphoric acid (H3PO4) and 1 M lactic acid (C3H6O3) were successively dissolved in 750 ml of deionized water at constant stirring. For the coatings containing zinc, a saturated solution of Ca2+ and PO43ions and Zn2+ (SSCP-Zn) was prepared replacing 10 mol. % of calcium hydroxide for zinc nitrate as described by Prado et al. [19]. The solution was stirred for 24 hours at room temperature, controlling the pH to be around 3.7.

Coating deposition process

For the deposition of HA coatings, pretreated titanium samples were immersed in 250 ml of SSCP and heated in a water bath at 80 °C for 1 h. In case of ZnHA coatings, the substrates were immersed in 250 ml of SSCP-Zn, heated in a water bath at 80 °C for 12 hours. Crystals precipitated on the metal surface producing a continuous adhered coating. After deposition of the coatings, both types of samples were placed in a solution of 0.1 M NaOH at 60 °C for 24 hours.

Characterization Test materials were divided in three groups according to the different surface treatments: Ti coated with hidroxyapatite (HA), Ti coated with hydroxyapatite partially substituted with zinc (ZnHA) and polished cp-Ti (Ti). The phases present in the coating materials were identified by X-ray diffraction in a PANalytical X'Pert X-ray diffraction system operating with CuKα ( = 0.1542 Å) X-ray source with a grazing incident angle of 2.5° and a step rate of 0.05° per second, from 10° to 80°. The coatings morphology was observed by scanning electron microscopy (SEM - JEOL JSM 5800LV) operated at an accelerating voltage of 20 kV in the low vacuum mode. Semi-quantitative element analysis was obtained by energy-dispersive X-ray analysis (EDX). To identify the chemical groups present in the coatings, HA and ZnHA were scraped off the substrates for Fourier transform infrared spectroscopy (FTIR) analysis using the KBr pellet technique. Elemental quantitative analysis for the presence of Ca, P and Zn were made using X-ray fluorescence (XRF) spectroscopy (Vulcan X-ray Fluorescence Spectrometer). Scratch tests were carried out in order to determine the adhesion of the HA and ZnHA coatings to the Ti substrates using a Universal Mechanical Tester (Bruker UMT-2). This

test involves dragging a Rockwell diamond indenter across the surface of the sample while the load applied to the indenter is linearly increased. The lateral (friction) force Ff and the acoustic emission (AE) signal are continuously monitored during the experiment. A maximum normal load of 20 N was employed, and the loading rate and sliding speed were 5.3 N/s and 1.66 mm/s, respectively. The resulting scratch scars of approximately 6 mm in length were examined with an optical microscope and the coating adhesion strength was evaluated by the critical load (Lc) [20]. For each sample nine scratch tests were performed and the average values and standard deviations of Lc were obtained. Coating thickness was measured by stylus profilometry (Bruker Dektak XT). The film thickness was determined as the average value of five different scans.

In vitro evaluation All experiments were performed using human osteoblasts cells (HOB). The cell cultures were maintained using Dulbecco’s modified Eagle’s medium (DMEM) low glucose (GIBCO) and supplemented with 10% fetal bovine serum (FBS, Soromed) maintained at 37 °C in a humidified atmosphere of 5% CO2/air. The cells were lysed with 0.05 % trypsin / 0.02 % EDTA in PBS solution and cultured on sample surfaces using 1x104, 5x104 or 1x105 cells/sample. All samples were identified and sterilized by gamma radiation (cobalt 60 source, 15 KGy, 19.72 Gy/min, total 12 h) before each analysis.

Viability assay Viability of the cells seeded on samples surface was assessed after 24 h using a live/dead kit detection assay (Invitrogen). The cells were incubated with live/dead

reagents simultaneously staining with green-fluorescent calcein-AM (AM-Ca) to indicate intracellular esterase activity present in viable cells and red-fluorescent ethidium homodimer-1 (EthD-1) to indicate loss of plasma membrane integrity of nonviable cells for 30 min at 37ºC in the dark. Cells were washed with PBS for 5 min and images acquired by an AxioVision 4.8.1 (Zeiss) fluorescent microscope. The corresponding green and red fluorescence were detected at 645 and 530 nm, respectively, using specific band-pass fluorescence filters. Image Pro Plus 6.0 software were used to image processing and overlay. Two samples for each group were analyzed in 4 fields at the same magnification for each sample.

Osteoblast cell adhesion and morphology The adhesion and morphology of cell-surfaces interaction were performed by scanning electron microscopy. HOB cells were seeded at a density of 5x104 cells/sample and examined after 30, 120 and 240 minutes (n = 3 per material for each time point). Cells were washed with 0.01 M PBS to remove non-adherent cells and fixed using Karnovsky's solution (2.5 % glutaraldehyde, 8 % PFA, 0.1 M sodium cacodylate) for 1 hour at room temperature, washed 3 times with 0.2 M sodium cacodylate buffer and post-fixed with osmium tetroxide (1 % osmium in cacodylate 1 %). Fixed cells were washed with 0.2 M cacodylate and dehydrated by consecutively adding ethanol/distilled water mixtures containing 30, 50, 70, 90, and 100 % volumes of ethanol and critical point drying (BAL-TEC DPC 030) using CO2 as ethanol substitute. The samples were metallized with a thin gold film (Emitec) and observed by SEM.

Cell proliferation and cytoskeleton distribution

HOB cells were seeded on the samples surfaces at a cell density of 1 x 105 cells. The area of the cell-material interaction on each sample was restricted with Pyrex cloning cylinders and cultured under standard cell culture conditions during 1, 2 and 3 days. The samples were then washed three times with phosphate-buffered saline (PBS) to remove non-adherent cells. The remaining cells were fixed with 4 % paraformaldehyde solution for 10 minutes, washed with PBS, and permeabilized with PBS-Triton (0.1 %) for 10 minutes. Samples were washed with PBS containing 3 % BSA (bovine serum albumin Sigma-Aldrich A4503, w/v) for 30 minutes, labeled with Alexa Fluor 546 phalloidin, diluted in BSA (1 %) and Triton (0.1 %) for 30 min, washed with PBS and stained with 1:1000 DAPI (40.6 to 20-diamidino-phenylindole dihydrochloride) for 15 minutes at room temperature to obtain nucleus imaging. The staining samples were examined through a fluorescence microscope (Axio Observer A1, Zeiss, Germany) and five different fields were registered for subsequent quantification using Image Pro Plus 6.0 software (Media Cybernetics, Inc., USA). All experiments were performed in duplicate.

Statistical analysis Statistical analysis were performed using GraphPad Prism software (version 5.0) (GraphPad Software Inc, CA, USA). Statistical significance of differences between experimental groups obtained on cell trials were assessed by one-way analysis of variance (ANOVA) and T-test (p <0.05).

3.

RESULTS

Figure 1 (a) shows the X-ray diffractogram of HA coating deposited on titanium surface from SSCP solution. The only phase present is monetite (JCPDS -01-070-1425), which is the most stable phase of calcium phosphate at the deposition conditions. A micrograph obtained by SEM of the monetite coating shows microcrystals with a platelike morphology (Figure 1 b). When zinc is added to the saturated solution (SSCP-Zn), the X-ray diffractogram shown in Figure 1 (c) reveals the formation of a second phase identified as parascholzite (JCPDS-00-035-0495) in addition to monetite. The incorporation of Zn to the solution also resulted in a great reduction of crystals sizes and changed the crystals morphology, as can be observed from the comparison of Figures 1 b and d. After the immersion of the coatings in NaOH solution for 24 hours at 60 °C, a dissolution and reprecipitation process occurred converting the coatings to hydroxyapatite. The X-ray diffractogram shows hydroxyapatite (JCPDS-00-009-0432) as the single phase in both cases (Figure 2). The SEM micrographs obtained for the HA coatings show a continuous layer composed of plate-like crystals and the presence of nanocrystals on the surface of these plates (Figure 3 a and b). The addition of zinc to the solution resulted in changes of the crystals morphology, as shown in Figure 3 (c) and (d). The reprecipitation process of ZnHA crystals is also observed. As before, a great reduction of crystals sizes was observed as a result of zinc incorporation. An average thickness of 100 µm was measured for HA coatings while for ZnHA coatings the average thickness is about 18 µm. The significantly smaller thickness of ZnHA when compared to the HA coatings can be attributed to the fact that zinc acts as a growth inhibitor of HA crystals.

The FTIR spectra obtained for HA and ZnHA coatings are shown in Figure 4. In case of HA, the main bands related to the (PO4)3- groups are observed at 1092, 1045, 960, 602 and 570 cm-1 and to the (OH)- groups at 3570 (not shown in the figure) and 635 cm-1. These peak positions agree well with those observed by Peña et al. for HA [21]. The FTIR spectrum of the ZnHA coatings, on the other hand, shows the presence of the main bands associated to the (PO4)3- groups at 1121, 1082, 1037, 972, 942, 605 and 547 cm-1, which also agree well with the peaks observed for a biphasic material composed of HA and -tricalcium phosphate (-TCP) with Ca/P ratio of about 1.54 [21]. Semiquantitative analysis of chemical composition by XRF showed that the molar ratio Ca/P of the HA coating is 1.60 and the molar ratio (Ca + Zn) / P is 1.61 for the ZnHA coating, both values corresponding to calcium deficient hydroxyapatite. This ratio is approximately the same for both coatings, suggesting that Zn is incorporated substitutionally, replacing Ca atoms into the HA structure. The ratio Zn / (Ca + Zn) is 7 at. %, which is slightly lower than the predicted value of 10 at. %, demonstrating the efficiency of the hydrothermal method. The adhesion of the coatings to the Ti substrates was determined by scratch tests. Figure 5 (a) shows the applied load (FZ), friction force (Ff) and acoustic emission (AE) as a function of time during a scratch test performed on a HA coating. Since a gradual increase of the friction force is observed during the tests, SEM micrographs (Figure 5b) have been used to identify the points where coating delamination initiates (Lc1) and where there is complete delamination of the HA coating (Lc2), exposing the Ti substrate completely. In Figure 5 (b), the dark areas on the scratch are associated to the coating material and the light areas to the Ti substrate. These observations were confirmed by

EDS analysis. In case of the test shown in Figure 5 (a), Lc1 and Lc2 were determined to be 5.6 N and 10 N, respectively. At the positions corresponding to Lc1 and Lc2 both the friction force and the acoustic emission show significant variations (marked by arrows). The results of a scratch test on a ZnHA of coating are shown in Figures 5 (c) and (d). In this case, critical loads Lc1 = 4.9 N and Lc2 = 9.2 N have been determined. Also in this case, the friction force and the acoustic emission signals presented significant variations at the corresponding positions. Table 1 shows the average values and standard deviations of the critical loads Lc1 and Lc2 obtained for HA and ZnHA coatings. The results of the Live/Dead tests for both HA and ZnHA groups showed high proportions of viable cells (Figure 6) without significant differences between the two groups, with a value of (82.18 ± 5.5) % (n = 9) for the samples coated with HA and a value of (79.32 ± 3.4) % (n = 9) for the samples coated with ZnHA. None of the analyzed coatings showed dead cells. The osteoblasts cell adhesion and morphology could be analyzed from the electron micrographs of the coated (HA and ZnHA) and uncoated surfaces shown in Figure 7. The presence of spherical osteoblasts was observed after 30 minutes for the three groups, lateral spread to one or more sides, with cytoplasmic protrusions and lamellipodia and the presence of microvilli-like structures only in case of the ZnHA group. After 120 minutes, differences between the three groups started to become evident. In case of the uncoated titanium sample (Figure 7 d), cells start to spread laterally to several sides and some cytoplasmic protrusions and lamellipodia can be seen. The cells on HA coatings (Figure 7 e) are still spreading and show the presence of the filopodia structures. Samples coated with ZnHA present well-adhered and wellspread cells, as shown by the presence of microvilli-like structures (Figure 7 f). The

majority of cells exhibited a flat shape after 240 minutes, suggesting that cells were well-fixed onto the surfaces. In the case of HA samples (Figure 7 h), well-spread cells are observed. For the ZnHA samples (Figure 7 i), the cells establish a connection through the interaction receptor ligand and start to spread onto the surface. The results of cell proliferation tests, e.g., the number of cells adhered to HA and ZnHA surfaces after 1, 2 and 3 days compared to the control group (cp-Ti), are shown in Figure 8. The control group (cells seeded in highly adherent cover slips) presented larger number of cells than both HA and ZnHA. Otherwise, the number of cells adhered to ZnHA is significantly higher than on HA coatings along the timeline of culture (1, 2 and 3 days). Direct fluorescence staining of actin cytoskeleton (with phalloidin) and nucleus (DAPI) of HOB cells cultured for 3 days on HA and ZnHA coatings and on control group (glass slides) are shown in Figure 9. Scattered cells with large nuclei were observed for the control group (Figure 9 a), while HA coatings showed fusiform cells with surrounding filopodes (Figure 9 b). ZnHA coatings (Figure 9 c) presented elongated- or polygonalshaped cells, with numerous filopode expansions and clearly more scattered than the cells of the HA group.

4.

DISCUSSION

In this study, a modification was introduced to the hydrothermal coating method previously reported by Prado da Silva [16]. In this method, the use of a chemical activation treatment of the titanium surface allows the oxidation of the surface, significantly enhancing the nucleation and growth of the hydroxyapatite crystals and resulting in an accelerated growth and higher crystalline density [17]. The presence of

OH– charges on the surface of the pretreated substrate has been shown to play an important role in the nucleation process of monetite [22]. During the process of hydrothermal deposition of the precursor phase on the previously activated substrate surface, the presence of two different phases was observed, monetite and parascholzite. Monetite is considered to be a precursor of hydroxyapatite and is the most stable calcium phosphate phase at pH 3.7 and 80 oC [16]. Parascholzite appears as a consequence of the incorporation of zinc in the structure. Other authors reported the presence of parascholzite for Zn concentrations up to 18 wt. % in hydroxyapatite [23]. It was also observed that the nucleation and growth rates were lower in SSCP-Zn compared to that of SSCP. This may be due to the lower oxygen affinity of zinc as compared to calcium, which can be related to the lower electronegativity difference between oxygen and the metal atom, increasing the energy barrier for nucleation and growth [19]. In the second stage of the deposition process, the coating composed of monetite and parascholzite is converted into hydroxyapatite by a hydrothermal conversion in an alkaline medium. The most probable mechanism of this conversion is a continuous process of dissolution and re-precipitation as described by Prado da Silva [16]. It is suggested that there is a continuous dissolution of the precursor with a consequent enrichment of Ca2+ and PO43- ions in the solution. When thermodynamic equilibrium is reached, HA is precipitated on the surface of the precursor. HA and ZnHA samples presented very similar XRD patterns, suggesting the formation of a single crystalline hydroxyapatite phase. This result suggests that Ca2+ ions were replaced by Zn2+ ions in the crystal structure of HA.

The addition of zinc to the precursor solution changed the crystals morphology as observed by SEM. Also, a great decrease of the crystals sizes was observed. This is in agreement with other works where zinc was shown to decrease nucleation and growth of HA crystals when used at high concentrations [12, 19]. The average thickness of the HA coatings is much larger than that obtained by other methods [16], while the average thickness of the ZnHA coatings is significantly lower than that of HA coatings, which can also be attributed to the fact that zinc acts as a growth inhibitor of HA. Some authors, however, observed a broadening of XRD peaks as zinc content increases, indicating that zinc incorporation may result in a decreased crystallinity [24]. However, in our case the crystal size reduction was not enough to affect the obtained XRD spectra. The observed molar ratio Ca/P for the HA coating and the molar ratio (Ca + Zn) / P for ZnHA coating correspond to calcium deficient hydroxyapatite [25]. Matsunaga [26] found that hydroxyapatite doped with Zn2+ tends to be poor in calcium, since Zn2+ ions occupy the Ca2+ positions. The Zn2+ ions incorporated in the HA is not limited to ion exchange, but also to produce Ca2+ complex defects which may act as suitable sites for Zn and other substitutions such as carbonate. In the case of a carbonated HA, the neutralization of the charges occurs due to calcium deficiency. In the present study, the presence of (CO3)2- groups were observed by FTIR both for HA and ZnHA samples, indicating that the incorporation of carbonate groups into the coatings occurs regardless they are doped with zinc or not. The results of the compositional analysis showed that the incorporation of a zinc content of about 7 at. % was obtained without changes in the carbonated HA crystalline structure. The amount of zinc that HA can incorporate without changing its structure is

still quite controversial. The ease of substitution of Ca2+ by Zn2+ apatite changes depending on the synthesis process. Since Ca2+ vacancy concentration in calciumdeficient HA obtained by a solution precipitation method is generally below 10 at. % (1.50 < Ca/P <1.67), it is likely that the substitution of Zn2+ in this type of synthesis is limited to several at. % [26]. Sogo et al. [27], conducted a study on the most suitable molar (Ca + Zn) / P ratio for ceramic composites ZnTCP/HA, obtaining a zinc content in the range of 1.60 to 0.316 at. % was effective for promoting bone formation. Kawamura et al., [28] reported that the optimal content of zinc found for β-ZnTCP and ZnHA is at least 0.316 wt. % and a maximum of 0.63 wt. % for promoting in-vivo bone formation. Miao et al. [29] coated a Ti alloy with a layer of fluorinated hydroxyapatite containing 9 at. % of zinc and showed that Zn plays an important role in enhancing bone growth when used as bioactive coating on metal implants. The results of the scratch tests showed that the critical loads obtained for HA and ZnHA are very similar (see Table 1). This suggests that the incorporation of zinc does not affect the adhesion of the coatings when compared with conventional HA. However, the different thickness of the two types of coatings may be a factor that influences the critical load. For instance, Blind et al. [30] reported in case of HA coatings that the critical load increases as the film thickness decreases. Also, the different crystals morphologies and sizes of the two coatings may influence the mechanisms that dissipate the stress from the applied load [31]. However, this effect cannot be easily evaluated. Nevertheless, the fact that the much thinner ZnHA films present similar critical loads to the thicker HA films is an indication of the good adhesion of ZnHA to Ti substrates.

This study evaluated and compared the in vitro response of human osteoblasts exposed to the surfaces of HA and ZnHA coatings. Both materials proved to be non-toxic, confirming their biocompatibility and ability to maintain cellular proliferation. The HOB cells adhered to the surface coatings exhibited an obvious increase in cell spreading over time. Initially, the cells responded better to ZnHA surface and over time the best cell spreading was also observed for ZnHA surfaces (Figure 7). It is well known that the surface topography directly influences cell adhesion and cell motility. So, this finding may be associated to the smaller crystals of the ZnHA surface. In contrast, HOB cells seemed to adhere not so easily to the HA surface probably due to the larger crystals sizes. These results are consistent with earlier studies, where other calcium phosphate ceramics with zinc content of 1.20 wt. % were able to significantly promote the in vitro proliferation of MC3T3-E1 cells [13]. Our data showed that an increase in the proliferation of osteoblasts is observed for the coatings doped with zinc as compared with pure HA coatings. Accordingly, Webster et al. reported that the adhesion of human osteoblasts was higher in HA doped Zn+2 in comparison with undoped HA [10].

5.

CONCLUSIONS

In the present study, uniform and homogeneous coatings of calcium deficient, carbonate substituted hydroxyapatite both undoped and doped with zinc were obtained by the hydrothermal method. The incorporation of 7 at. % of Zn2+ occurs substituting Ca2+ ions without changing the HA crystalline structure and resulted in a reduced the deposition rate, suggesting that zinc is an inhibitor of the growth of apatite crystals. No significant

difference between the adhesion of zinc doped and undoped coatings to Ti substrates was observed. Human osteoblast cells grew and proliferated over the two types of coatings proving both materials to be non-toxic. Zinc incorporation into HA enhanced osteoblast proliferation resulting in a larger number of cells adhered to the coatings surface and also more spread and scattered cells. This finding may be associated to the smaller crystals sizes and crystal morphology of ZnHA coatings. In summary, our experiments demonstrated that ZnHA coating on titanium implants may have a potential benefit to improve osseointegration process.

ACKNOWLEDGEMENTS The authors acknowledge the financial support of CNPq funding agency.

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(a)

(b)

(c)

(d)

Figure 1 – X-ray diffractograms and SEM micrographs of coatings before conversion: HA (a and b) and ZnHA(c and d).

Figure 2 – XRD patterns of the HA and ZnHA samples.

(a)

(b)

(c)

(d)

Figure 3 – SEM micrographs coatings surface after conversion: HA (a and b) and ZnHA (c and d).

Transmitance

Zn-HA

HA

1400

1200

1000

800

600 -1

Wavenumber (cm )

Figure 4 – FTIR spectra of HA and ZnHA coatings.

400

(a)

(b)

(c)

(d)

Figure 5 – Results of scratch tests showing the applied load (Fz), friction force (Ff) and acoustic emission signal (AE), a function of time and SEM micrographs showing the scratch surfaces for HA (a and b) and ZnHA (c and d) coatings. The arrows show the positions of the critical loads and the spots where the coating failures occur.

Figure 6 – Percentage of viable cell after 24 hours of cell culture. * Indicates significant difference of ZnHA and HA groups compared to the control group. There is no significant statistic difference between the samples ZnHA and HA. Vertical bars show the mean ± standard deviation (95% confidence).

Figure 7 – SEM micrographs of Ti, HA and ZnHA surfaces after 30, 120 and 240 minutes of culture of osteoblasts.

Figure 8 – Results of cell proliferation tests presented as mean ± standard deviation for control (highly adherent cover slips), HA and ZnHA coatings surfaces after 1, 2 and 3 days of culture.

Figure 9 – Fluorescence microscopy images of actin cytoskeleton (with phalloidin) and nucleus (with DAPI) of HOB cells cultured for 3 days on glass slides (a), HA (b) and ZnHA (c) coatings.

Table 1 – Average values and standard deviations of the critical loads Lc1 and Lc2 for HA and ZnHA coatings.

Coating

Lc1 (N)

Lc2 (N)

HA

5,9 (±0,1)

8,7 (±0,2)

ZnHA

5,3 (±0,2)

9,1 (±0,1)