Direct Covalent Grafting of Phytate to Titanium Surfaces through Ti

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Direct Covalent Grafting of Phytate to Titanium Surfaces through Ti-O-P Bonding Shows Bone Stimulating Surface Properties and Decreased Bacterial Adhesion. Alba Córdoba, Margarita Hierro-Oliva, Miguel Angel Pacha, Maria Coronada Fernández, Joan Perelló, Bernat Isern, María Luisa Gonzalez-Martin, Marta Monjo, and Joana Maria Ramis ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02533 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 21, 2016

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ACS Applied Materials & Interfaces

Direct Covalent Grafting of Phytate to Titanium Surfaces through Ti-O-P Bonding Shows Bone Stimulating Surface Properties and Decreased Bacterial Adhesion Alba Córdoba, † Margarita Hierro-Oliva, ‡, # Miguel Ángel Pacha, ‡, # María Coronada Fernández, ‡, # Joan Perelló, §Bernat Isern, § María Luisa González-Martín, ‡, # Marta Monjo, †,* and Joana M. Ramis.†,* †

Group of Cell Therapy and Tissue Engineering, Research Institute on Health Sciences

(IUNICS), University of Balearic Islands. Ctra. Valldemossa km 7.5, 07122 Palma de Mallorca, Spain; Instituto de Investigación Sanitaria de Palma, 07010 Palma, España ‡

Departamento de Física Aplicada, Facultad de Ciencias, Universidad de Extremadura. Badajoz,

Spain #

Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine

(CIBER-BBN), Spain. §

Laboratoris Sanifit, ParcBIT, Palma de Mallorca, Spain.

KEYWORDS: phytic acid, coating, bioactive, T-bag method, S. sanguinis, S.mutans, implant.

ACS Paragon Plus Environment

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ABSTRACT. Myo-inositol hexaphosphate, also called phytic acid or phytate (IP6), is a natural molecule abundant in vegetable seeds and legumes. Among other functions, IP6 inhibits bone resorption. It is adsorbed on the surface of hydroxyapatite, inhibiting its dissolution, and decreasing the progressive loss of bone mass. We present here a method to directly functionalize Ti surfaces covalently with IP6, without using a crosslinker molecule, through the reaction of the phosphate groups of IP6 with the TiO2 layer of Ti substrates. The grafting reaction consisted of an immersion in an IP6 solution to allow the physisorption of the molecules onto the substrate, followed by a heating step to obtain its chemisorption, in an adaptation of the T-Bag method. The reaction was highly dependent on the IP6 solution pH, only achieving a covalent Ti-O-P bond at pH 0. We evaluated two acidic pre-treatments of the Ti surface, to increase its hydroxylic content, HNO3 30% and HF 0.2%. The structure of the coated surfaces was characterized by XPS, ToF-SIMS and ellipsometry. The stability of the IP6 coating after three months of storage and after sterilization with gamma irradiation was also determined. Then, we evaluated the biological effect of Ti-IP6 surfaces in vitro on MC3T3-E1 osteoblastic cells, showing an osteogenic effect. Finally, the effect of the surfaces on the adhesion and biofilm viability of oral microorganisms S. mutans and S. sanguinis was also studied, and found that Ti-IP6 surfaces decreased the adhesion of S. sanguinis. A surface that actively improves osseointegration while decreases bacterial adhesion could be suitable for use in bone implants. INTRODUCTION Myo-inositol hexaphosphate (IP6), also called phytic acid, or phytate in its salt form, is the major phosphate store in plant seeds, and it is also present in all animal organs and tissues.1,2 Different biological functions have been described for IP6.3 It acts as a potent crystallization inhibitor of calcium salts (calcification),4 but it also has been described as an antioxidant


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(inhibitor of hydroxyl radical formation),5 and even as an anti- cancer agent.6,7 IP6 functions as an inhibitor of bone resorption in animal models of osteoporosis, it is adsorbed on the surface of hydroxyapatite, the mineral constituent of bone, and inhibits its dissolution, thereby decreasing the progressive loss of bone mass.8 In addition, a positive action of IP6 at cellular level inhibiting osteoclastogenesis, the bone remodelling process, without impairing the differentiation of cells to osteoblasts, the bone forming cells, has also been described.9–11 Besides its described effects on bone, polyphosphates have shown interesting antimicrobial effects, inhibiting the growth of several gram-positive and gram-negative bacteria, including oral bacteria;12 and their antibacterial effects have been related to the ability of polyphosphates to chelate divalent cations, contributing to cell division inhibition and loss of cell wall integrity. In fact, IP6 has also been reported to have synergetic antibacterial effects in the presence of sodium chloride in E. coli.13 Recently, the synthesis of biomaterials containing phytic acid has been reported. Chen et al. developed a biocompatible IP6-magnesium coating, based on the chelating ability of IP6.14 Li et al. optimized this coating with a composite of IP6-magnesium and bioglass,15 and found that the coating decreased the degradation rate of a magnesium alloy and could be used for temporary biodegradable implants. It is known that phosphates and phosphonates can covalently link to metal oxide surfaces, like TiO2.16,17 In fact, alkylphosphonic acids are commonly used as crosslinker agents to functionalize surfaces with other molecules to tune the surface properties to those of interest.18,19 Phosphonate linkers present the advantage of being more stable than other commonly used coupling agents like silanes, which suffer from hydrolytic instability in aqueous environments at


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physiological pHs.20 The use of robust and stable coatings under physiological conditions in biomedical applications is of high interest, and phosphates or phosphonates bound to metal oxides are stable in these conditions. Given the positive effect of IP6 on bone cells

9–11

and its antibacterial properties, we

hypothesized that IP6 once covalently bound onto an implant surface could act as a multifunctional bone implant. Since IP6 possesses six phosphate groups, we tested if it could covalently bind directly to titanium substrates, without the need of a crosslinker molecule. We present here a method to functionalize Ti surfaces with IP6 through Ti-O-P bonds, directly, without using a crosslinker molecule, through the reaction of the phosphate groups of IP6 with the hydroxilated TiO2 layer of Ti substrates, in an adaptation of the T-Bag method used to functionalize surfaces with phosphonates.21,22 We evaluated two acidic pre-treatments of the surface to increase its hydroxylic content, using either HNO3 30% or HF 0.2%. The effect of the IP6 solution pH was studied resulting a key parameter. The structure of surfaces was characterized by XPS, ToF-SIMS and ellipsometry. The stability of the IP6 coating after three months storage and after sterilization with gamma irradiation was determined. Then, we evaluated the biological effect of Ti-IP6 surfaces in vitro on MC3T3-E1 osteoblastic cells, measuring the cytotoxicity and the cells metabolic activity, as well as the expression of genes related to osteogenesis after 14 days of culture and the cell mineralization after 21 days. The primary colonizers in the oral microbiota for both mucosal and tooth surfaces are usually streptococci.23 Oral streptococci account for 20% of all supragingival microorganisms present in the oral biofilm,24 but constitute about 80% of the initial colonizers during early biofilm development.25 Therefore, the effect of the surfaces on the


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adhesion and biofilm viability of oral microoorganims Streptococcus mutans and Streptococcus sanguinis was also studied. EXPERIMENTAL SECTION Materials. Machined Ti coins, c.p. grade IV, 6.2 mm diameter and 2 mm height, were purchased to Implantmedia (Lloseta, Spain) and cleaned as described elsewhere.26 cGMP Hexasodium Phytate (myo-inositol hexakisphosphate sodium salt, IP6Na6), purity 87.3%, was supplied by Almac Sciences (Craigavon, UK). Deionized water was obtained from a Millipore system (Billerica, MA, USA). Nitric acid 69.5% and fluorhydric acid 40%, reagent grade, were purchased to Fisher Scientific (Madrid, Spain). Hellmanex III solution was purchased to Hellma Hispania (Badalona, Spain). Fabrication of titanium surfaces covalently functionalized with IP6. For XPS, ToF-SIMS, ellipsometry and microbiological analysis the Ti disks were mirror polished prior to modification (Phoenix 4000, Buehler GmbH, Düsseldorf, Germany) as previously described.26 For cell culture analysis, the surface of Ti coins was blasted with TiO2 particles prior to modification, to give a mean surface roughness (Sa) of 1.67 ± 0.16 µm. All glassware was cleaned with a 2% Hellmanex solution. In first place, the surface of Ti coins was passivated and hydroxilated either with diluted HNO3 or HF. On one hand, Ti disks were passivated in 3:7 (v:v) HNO3-H2O for 30 min, rinsed thoroughly with water, immersed in water for 24 h at room temperature, and dried under a N2 flow. On the other hand, Ti disks were immersed in 0.2% HF for 90 s at room temperature, rinsed repeatedly with water, and dried under a N2 flow.


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Immediately after hydroxilation, coins were immersed horizontally in a freshly prepared 1.5 mM IP6 aqueous solution at pH 0, 1, 2, 3 or 4, without stirring, in a closed container for 48 h, to allow adsorption of phytate on the Ti surface. Then, under a dry N2 atmosphere, samples were removed from the solution and immediately heated at 140 ºC under vacuum conditions for 30 min, rinsed thoroughly with water and dried under a N2 flow. Ti-IP6 surfaces were stored in vacuumed sealed bags and kept at -20ºC until use. Samples for in vitro cell cultures were prepared in aseptic conditions. Samples for microbiological analyses were sterilized under 25 KGy gamma irradiation, since this is the most commonly validated dose used to sterilize medical products.27 Physic-chemical characterization of Ti-IP6 surfaces Chemical structure of the surfaces by XPS. XPS analyses were performed using a K-Alpha (Thermo Scientific) system, with monochromatic Al Kα (1486.68 eV) X-ray source with a spot size of 300 µm and 50.4 W (12 kV × 4.2 mA) power. For each sample, two survey spectra as well as high resolution spectra were acquired for C, O, P, N and Ti elements on the sample surface. At least two sample replicates were analyzed for each group (n=2). XPS depth profiles were determined using an Ar+ sputtering gun operated at 3 kV. Adhesion Tape Tasting. A strip of pressure sensitive tape was pressed firmly across the surface of the covered samples. After 30 seconds the tape was removed by a rapid pull force applied approximately perpendicular to test area. Afterwards, the surface was analysed by XPS as previously described. Film thickness by ellipsometry. Ellipsometric measurements were made with a phase modulated UVISEL (Horiba Jovin Yvon) ellipsometer in the 0.6-6.5 eV energy range at an


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incidence angle of 70° and at room temperature. Experimental data were fitted to a model of the sample to determine the film thickness. Composition of the surfaces by ToF-SIMS. The ToF-SIMS measurements were carried out with a ToF-SIMS5 (ION TOF) equipped with a Bi+ primary ion source operated at 25 kV. Negative ion spectra were taken on two samples replicates for each group (n=2). The total ion dose used to acquire each spectrum was about 1012 ions/cm2 with an analyzed area of 150 x 150 µm2. Calibration of the mass spectra in the negative mass mode was based on H-, C-, CH-, CH2-, O-, OH-, OH2-, C2-, C3-. Biological effect in vitro of Ti-IP6 on osteoblastic MC3T3-E1 cells Cell Culture. The mouse osteoblastic cell line MC3T3‐E1, (DSMZ, Braunschweig, Germany) was maintained in α‐minimum essential media (Gibco, Grand Island, NY , US) supplemented with 10% fetal bovine serum (PAA Laboratories GmbH) and 1%‐ penicillin–streptomycin (Sigma, St. Louis, MO, USA) at 37 °C in a humidified atmosphere of 5% CO2. Cells were seeded on the surfaces at a plating density of 7000 cells per well in 96‐well plates. Cell culture media was refreshed every 48 h during 14 days. Cytotoxicity of surfaces and cell metabolic activity. The presence of lactate dehydrogenase (LDH) activity in the culture media after 48 h of cell seeding was used as an index of cell death. LDH activity was determined according to the manufacturer's kit instructions (Cytotoxicity Detection kit, Roche Diagnostics, Mannheim, Germany) from supernatants of six sample replicates for each group (n=6). Positive control was cell culture media from cells incubated with 1% Triton X‐100. Negative control was cell culture media from cells cultured on tissue culture plastic (TCP). Background control was the LDH activity contained in the assay medium (cell


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culture media not incubated with cells), and this absorbance value was substracted from all other values to calculate the percentage of cytotoxicity. Cytotoxicity was calculated according to Equation 1: Exp.value-Low Control

Cytotoxicity (%)=100× High Control-Low Control

(1)

Total metabolic activity of cells cultured on the surfaces was quantified 48 h after seeding using Presto Blue reagent (Life Technologies, Carlsbad, CA) from six sample replicates for each group (n=6) following the manufacturer’s protocol and 1 h of incubation time. Gene expression of bone stimulating markers by Real Time RT-PCR. After 14 days of cell culture, RNA was isolated using Tripure® (Roche Diagnostics, Mannheim, Germany), according to the manufacturer's protocol. 950 ng of RNA were reverse transcribed to cDNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA), following the supplier’s protocol. Real-time PCR was performed using LightCycler FastStart DNA Master PLUS SYBR Green I (Roche Diagnostics) following the manufacturer’s instructions using specific primers (Table S1, Supporting Information). All samples were normalized by the geometric mean of the expression levels of reference genes m18s and mGapdh and fold changes were related to the control groups using the method described by Pfaffl.28 Bonelike nodule formation by calcein blue staining. After 21 days of MC3T3‐E1 culture on Ti and Ti-IP6 groups, using machined substrates, bonelike nodule formation was evaluated by calcein blue staining of two sample replicates, following the methodology described in reference 29

: 10 mg of calcein blue were dissolved in 0.25 ml of KOH 1M and 9.75 ml of distilled water

was added to get a 3.1·10-3 M calcein blue solution. Then, the solution was filtered through a 0.22 µm membrane, added to the culture medium (15 µl per well, to achieve a final concentration


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in each well of 3.1·10-5 M) and incubated for 1h at 37ºC. After, the surfaces were thoroughly washed with PBS, fixed with p-formaldehyde 4% in PBS for 10 min, washed again with PBS and air dried. The stained surfaces were glued to microscope slides and representative images were acquired with a confocal microscope (Leica DMI 4000B equipped with a Leica TCS SPE laser system) for each sample under UV light excitation. Adhesion and biofilm viability of microorganisms on Ti-IP6 surfaces The oral strains used for adhesion and biofilm assays were Streptococcus mutans ATCC 25175 and S. sanguinis ATCC 10556, obtained from the Spanish Type Culture Collection (CECT). All strains were routinely maintained on Brain-Heart Infusion (BHI) agar or broth at 37 ºC in 5% CO2 (Galaxy ® 170S, Eppendorf AG, Hamburg, Deutschland) to obtain cultures. These cultures were incubated in BHI broth with CO2 atmosphere (5%) for 18-24 h at 37 °C. Adhesion experiments on the Ti-IP6 surface. For the adhesion assay, the bacteria were then harvested by centrifugation for 5 min at 1000 g (Sorvall TC6, Dulon, USA) and washed three times with artificial saliva (pH 6.8) pre-conditioned at 37 ºC and prepared according to Gal, J.Y. et al.30 Then, the bacteria were re-suspended in artificial saliva at a concentration of 3 x 108 bacteria·ml-1. A modified Robbins device (MRD) was used for the adhesion assay. Disks were fixed and adjusted to each port of the MRD to ensure laminar conditions. Prior to adhesion experiments, the device was conditioned with artificial saliva at 37 ºC for 15 min. Then, the flow of bacterial suspension was gently kept at a constant rate of 2 ml·min-1 using a peristaltic pump. Ports were removed sequentially at different times: 30, 60, 90, and 120 min, to study the kinetics of the process. Each removed port was replaced by dummy ports. Quantification and viability of retained bacteria from the extracted ports were carried out by epifluorescence microscopy


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(Nikon, Eclipse Ci, Japan) on staining the adhered microorganisms with the kit Live/Dead Baclight L-7012 (Invitrogen SA, Spain) and with automated counting procedures using the software NIS-Elements BR 4.10 (Nikon Instruments INC., USA). Biofilm formation on the Ti-IP6 surface. For biofilm formation assay, the bacterial concentration was spectrophotometrically adjusted to 82% of transmittance (Helios epsilon model, ThermoSpectronic, Thermo Fisher Scientific Inc., Cambridge, UK) at 492 nm wavelength (λ) and diluted 1/100 in order to obtain 1x106 ufc·ml-1 approximately. Ti and Ti-IP6 discs were placed on sterile 96-well flat bottom polystyrene culture plates (Greiner bio-one. Germany). Then, 200 µl of bacterial suspension were cultured on the surfaces at 37 ºC and gentle shaked (20 rpm) for 90 min, to facilitate the adherence. Subsequently, bacterial suspension was carefully removed, fresh sterile BHI was added and cultured at 37 ºC for 24 h. The viability of the bacterial biofilm was quantified using BacTiter-Glo™ Microbial Cell Viability Assay (Promega Corporation, Madison, WI, USA) according to manufacturer's instructions. Culture medium was removed and 200 µl of the BacTiter-Glo ™ Reagent per well were added, gentle shaked (20 rpm) and incubated in the dark for 10 min. This suspension was transferred to 96-well white polystyrene flat-bottom microtiter plates (Greiner bio-one). The light emission reaction (luciferin-luciferase) was measured by a luminometer (Microplate Fluorescent Reader FLX 800, BIO-TEK INSTRUMENTS, INC). Each assay was performed in duplicate and repeated three or more times to confirm reproducibility among different cultures. We used a Scanning Electron Microscope (Quanta 3D FEG, FEI Company, Hillsboro, US) to visualise biofilm formation on the surfaces. The samples were fixed for 12-15 h in 3 wt.% glutaraldehyde at room temperature, then washed with phosphate buffer solution (pH 7.4) and


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dehydrated in a serie of solutions of increasing ethanol concentration (30, 50, 70, 96, and 100 vol. %) for 1 h each. Dried samples were sputter coated with gold and scanned using secondary electrons, low vacuum conditions at 15 kV acceleration voltage and 5 Å spot size. Statistics. For the in vitro MC3T-E1 assays, the data were expressed as mean ± standard error of the mean (SEM) of six sample replicates (n=6). Differences between groups were assessed by Mann-Whitney-test or by Student t-test depending on their normal distribution. The Kolmogorov-Smirnov test was used to assume parametric or non-parametric distributions for the normality tests. Tests were performed with software SPSS® v.17.0 (SPSS, Chicago, IL, USA). For the microbiology studies the data were expressed as mean ± SEM of at least three independent experiments (n=3). Differences between groups were determined by t-test. Levene’s test was performed for equal or not variances by statistical software SPSS v20 (SPSS, Chicago, IL, USA). Results were considered statistically significant at p values≤ 0.05. RESULTS AND DISCUSSION Covalent grafting of phytate to titanium through phosphate groups The Ti surfaces were covalently functionalized with IP6, through the reaction of the phosphate groups of IP6 with the TiO2 layer of Ti substrates (Figure 1), without using a crosslinker molecule. Since the IP6 molecule has six phosphate groups that could bind Ti, there are many possibilities for the coating’s structure, depending on the number of phosphates bound to the substrate, the conformation of the cyclohexane ring, and the orientation of the non-bound phosphates.


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Figure 1. Chemical structure of the Ti-IP6 coating. The IP6 grafting reaction consisted of an immersion of the pre-treated Ti surfaces in an aqueous IP6 solution for 48 h at room temperature, to allow the physisorption of the molecules onto the substrate, followed by a heating step at 140 ºC for 30 min under vacuum conditions, to obtain the chemisorption of the molecules, in an adaptation of the T-Bag method developed by Hanson et al.21 The heat treatment increases the deprotonation rate of the P−OH groups, resulting in the formation of Ti−O−P bonds, enhancing the stability of the monolayer.18,19 The heating step was performed under vacuum conditions to avoid the presence of water during the condensation, since it is reported to negatively affect the bonding.22 pH, together with temperature, are also known to affect the bonding of phosphates and phosphonates to metal oxide surfaces.19 Therefore, we prepared Ti-IP6 surfaces varying the pH of the IP6 solution at pH 0, 1, 2, 3 and 4 (the non-adjusted pH of the 1.5 mM IP6 aqueous solution was 4), and characterized the surfaces obtained by XPS, ToF-SIMS and ellipsometry. Table 1 shows the elemental composition of the surfaces after grafting of IP6 at different solution pHs, as determined by XPS. We evaluated two acidic pre-treatments of the substrate: passivation with HNO3 30% or with HF 0.2%. In all cases, the elements detected were O1s, from the IP6 and the TiO2 layers; C1s, from the IP6 layer and from atmospheric contamination; Ti2p, from the TiO2 and Ti substrate; Na1s and P2s, from the IP6 layer, and N1s, a small contaminant detected also on pure IP6 and passivated Ti (data not shown). In some cases, low amounts of


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Si2p were also detected, due to the silica solution used for polishing the substrates prior to modification. The detection of P2p (from 4.30 to 8.47 at%) indicated the presence of a phytate layer on all the Ti-IP6 surfaces. On control passivated Ti surfaces, P2p was not detected (data not shown). Table 1. XPS elemental composition (at%) of Ti surfaces after grafting of IP6 at different pH of the solution of IP6, and heated at 140 ºC under vacuum conditions. Two pre-treatments of the Ti surface, passivation with HNO3 30% or with HF 0.2% were evaluated.

O1s C1s Ti2p Na1s P2p N1s Si2p

pH0 47.2 23.4 11.0 9.10 5.25 2.17 0.89

Ti-IP6 (HNO3 30%) a pH1 pH2 pH3 52.2 50.7 50.6 21.8 24.2 24.3 15.0 13.2 13.8 4.36 5.67 5.16 5.21 4.54 4.30 0.83 0.88 0.75 0.65 0.76 1.09

pH4 50.5 24.5 14.4 4.01 4.47 1.16 0.97

pH0 51.4 19.8 9.61 7.09 8.47 1.12 1.14

Ti-IP6 (HF 0.2%)a, b pH1 pH2 pH3 51.7 52.9 52.9 22.9 22.0 21.4 11.7 12.7 13.5 4.72 4.01 3.85 7.30 7.49 6.81 0.53 0.59 0.68 0.96 0.53

pH4 51.0 24.2 12.3 3.87 6.91 1.19 0.28

a

Small amounts of Ca2p and Mg1s were also detected (< 1 at%). b On samples pre-treated with HF, F1s was also detected (

Direct Covalent Grafting of Phytate to Titanium Surfaces through Ti

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