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Magnesium and strontium doped octacalcium phosphate thin films by matrix assisted pulsed laser evaporation
Journal of Inorganic Biochemistry 107 (2012) 65–72
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Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio
Magnesium and strontium doped octacalcium phosphate thin films by matrix assisted pulsed laser evaporation Elisa Boanini a, Paola Torricelli b, Milena Fini b, Felix Sima c, Natalia Serban c, Ion N. Mihailescu c, Adriana Bigi a,⁎ a b c
Department of Chemistry “G. Ciamician”, University of Bologna, 40126 Bologna, Italy Laboratory of Preclinical Surgical Studies, Research Institute Codivilla Putti, Rizzoli Orthopaedic Institute, Bologna, Italy National Institute for Lasers, Plasma and Radiation Physics, P. O. Box MG 36, 77125 Bucharest-Magurele, Romania
a r t i c l e
i n f o
Article history: Received 28 July 2011 Received in revised form 28 October 2011 Accepted 8 November 2011 Available online 19 November 2011 Keywords: Octacalcium phosphate coating Strontium Magnesium MAPLE Osteoblast
a b s t r a c t Octacalcium phosphate (OCP) is a promising alternative to hydroxyapatite as biomaterial for hard tissue repair. In this study we successfully applied Matrix Assisted Pulsed Laser Evaporation (MAPLE) to deposit Mg and Sr doped OCP (MgOCP and SrOCP), as well as OCP, thin films on titanium substrates. OCP, Mg-substituted and Sr-substituted OCP were synthesized in aqueous medium, then were suspended in deionised water, frozen at liquid nitrogen temperature and used as targets for MAPLE experiments. The depositions were carried out using a KrF* excimer laser source (λ = 248 nm, τFWHM = 25 ns) in mild conditions of temperature and pressure. The results of X-ray diffraction, infrared spectroscopy, scanning electron microscopy and energy dispersive spectroscopy investigations revealed that the OCP thin films are deposited in the form of cauliflower-like aggregates and droplets, as well as crystal fragments, with a homogeneous distribution of magnesium and strontium on the surface of the coatings. Human osteoblast-like MG-63 cells were cultured on the different biomaterials up to 14 days. MgOCP and SrOCP coatings promote osteoblast proliferation and differentiation with respect to OCP. Under these experimental conditions, the production of procollagen-type I, transforming growth factor-β1, alkaline phosphatase and osteocalcin indicated that the level of differentiation of the cells grown on the different coatings increased in the order OCP b MgOCP b SrOCP. © 2011 Elsevier Inc. All rights reserved.
1. Introduction An implant material must provide suitable functionality and biological response. In the orthopedic field, metallic materials are widely employed for load bearing implants and inner fixation devices thanks to their relevant mechanical properties: high tensile and yield strength, good resistance to fatigue, deformation, and corrosion [1]. However, their integration with bone tissue depends on the chemistry and physics of the implant surface. Calcium phosphate coatings have been extensively applied in order to enhance the ability of the biomaterials to create a bond with the living host tissue. To this end, a number of physical and chemical methods, including plasma spraying, magnetron sputtering, ion beam coating, electrophoretic deposition, pulsed laser deposition, sol–gel and biomimetic processes, are currently employed [1–7]. Hydroxyapatite, Ca10(PO4)6(OH)2, (HA), is considered to be one of the best biocompatible coatings, providing a highly bioactive and
⁎ Corresponding author. Tel.: + 39 051 2099551; fax: + 39 051 2099456. E-mail address: [email protected] (A. Bigi). 0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2011.11.003
resorbable interface in contact with bone; it is utilized in most commercially available coated metallic prostheses. However, octacalcium phosphate, Ca8H2(PO4)6·5H2O (OCP), which exhibits high osteoconductive characteristics and a speed of resorption that far exceeds that of HA [8–10], has been shown to be the precursor phase of biological apatites, having a very similar lattice surface structure to hexagonal HA. The triclinic structure of OCP can be described as having “apatitic layers”, where Ca 2 + and PO43 − ions occupy the same positions as in HA, alternated to “hydrated layers”, where structural water molecules are interdispersed between cations and anions [11,12]. Its solubility (higher than that of HA) and easy hydrolysis into nanocrystalline apatite constitute potential advantages of OCP with respect to crystalline HA. The stimulating effect of OCP toward new bone formation has been related to its rapid conversion into apatite in a biological setting [13–15]. OCP displays a complex stoichiometry and structure. OCP thin films are therefore quite difficult to deposit by means of physical methods, and most OCP coatings were deposited from supersaturated solutions and by electrochemical methods [16–18]. We previously optimized the experimental conditions to deposit nanocrystalline OCP thin films on Ti substrate by Pulsed Laser Deposition, PLD, technique [4,19]. According to the results of the X-ray diffraction analysis, the coatings displayed an amorphous-poorly crystalline structure, as
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confirmed by High Resolution Transmission Electron Microscopy, HRTEM, observations that showed the presence of crystalline nanodomains interdispersed in an amorphous phase [4,20]. The concept that the biological performance of calcium phosphate based materials might be improved by the addition of bioactive ions, has stimulated a number of studies on ion-substituted or ion-doped HA coatings [21,22]. In particular, the beneficial effect of the presence of carbonate, magnesium, strontium, manganese, and silicon in HAbased coatings deposited on metallic substrates has been demonstrated [23–32]. However, there are few reports on the deposition of ion-substituted or ion-doped OCP on metallic substrates. This is because even in the absence of ionic substitution, the complex stoichiometry and structure of OCP make it difficult to deposit this calcium phosphate [4,16–18]. In this paper we explored the possibility of depositing thin films of ion-substituted OCP, namely magnesium substituted OCP (MgOCP) and strontium substituted OCP (SrOCP), on Ti substrates. To accomplish this goal we applied Matrix Assisted Pulsed Laser Evaporation (MAPLE), which provides a more gentle mechanism than PLD for transferring different compounds, including large molecular weight molecules [33,34]. It is generally recognized that PLD is limited in case of doped compounds because of different ablation thresholds of composing materials. This can cause fractionated vaporization of the components, loss of dopant and component segregation in the deposited films. In case of MAPLE, this is avoided by protection of the frozen solvent. This is the first attempt to deposit OCP and ion-substituted OCP thin films by MAPLE technique. Moreover, to the best of our knowledge, ion-substituted OCP coatings have not been prepared up to now. The thin films synthesized in this study were submitted to structural and morphological characterization, and to in vitro tests of their compatibility to support growth and differentiation of osteoblast-like cells MG63. MG63 activity and differentiation were investigated using the most common markers of osteoblast metabolism: alkaline phosphatase (ALP) that is a specific indicator of osteoblast activity; type I collagen (CICP), that is the major component of the extra-cellular bone matrix; osteocalcin (OC), that is the major non-collagenous protein of the bone matrix. ALP activity increases in the first phase of cell proliferation, while the production of CICP and the synthesis of OC are related to the following phases of differentiation and of extra-cellular matrix production and mineralization [35]. A growth factor and a cytokine were also chosen to investigate osteoblast response to the innovative coatings: Transforming growth factor β1 (TGF-β1) is a growth factor that modulates cell proliferation and enhances the deposition of extra-cellular matrix through different pathways, both promoting synthesis and inhibiting degradation [36]; Interleukin 6 (IL-6) is an indicative pro-inflammatory cytokine that has a major role in the mediation of inflammation and is also implicated in bone remodeling, since it stimulates the first stages of osteoblast differentiation [37]. 2. Materials and methods 2.1. Synthesis and characterization of OCP, MgOCP and SrOCP nanocrystals OCP nanocrystals were synthesized by dropwise addition of 250 ml of 0.04 M Ca(CH3COO)2·H2O over a period of 60 min into 750 ml of a phosphate solution containing 5 mmol of Na2HPO4·12H2O and 5 mmol of NaH2PO4·H2O (Carlo Erba) starting pH 5 [38]. The reaction was carried out at 70 °C with smooth mechanical stirring. The precipitate was stored in contact with the mother solution for 10 min, filtered, repeatedly washed with bidistilled water and dried at 37 °C. The same procedure was applied for the syntheses performed in the presence of Mg2 + or Sr2 + in solution [39]: Mg(CH3COO)2·4H2O and Sr (CH3COO)2·1/2H2O were respectively used together with Ca(CH3COO)2·H2O to prepare solutions with a [M2 + / (Ca 2 + + M2 +)]× 100 ratio of 10, and a total cation concentration of 0.04 M.
Powder X-ray diffraction patterns were recorded using a PANalytical X'Pert PRO powder diffractometer. CuKα radiation was used (λ = 0.154 nm, 40 mA, 40 kV). Data were obtained in the range of 2θ from 3 to 60° (0.05°/step, 10 s/step). The powder X-ray diffraction pattern of the solid products confirmed that the compounds synthesized in the presence of Mg 2 + and Sr 2 + were constituted of octacalcium phosphate as the sole crystalline phase. Calcium, magnesium and strontium contents in the solid products were determined by means of a Dionex DX100 chromatography system equipped with a Dionex CD20 conductivity detector. Powders were previously dissolved in 0.1 M HCl. The data show that Mg and Sr contents were 0.6 and 5.5 at.% respectively. These analytical contents correspond well with the observed small, but appreciable, variations in the unit cell dimensions of OCP indicating Mg and Sr substitution for Ca in MgOCP and SrOCP respectively [39]. 2.2. Synthesis and characterization of OCP, MgOCP and SrOCP coatings Disk-shaped (12 mm diameter, 0.5 mm thick) grade 2 Ti substrates were mechanically polished and submitted to chemical etching to get an extended active surface [40]. They were ultrasonically cleaned in acetone, ethanol and deionized water and blown dried with high purity N2 gas before use for thin film deposition. The suspension consisting of 0.12 g of OCP powder in 5 ml distilled water was ground with agate balls for 60 min at 360 rot/min in a Retsch centrifugal ball mill suitable for wet grinding. Next, the mixture was collected in a Berzelius glass, mechanically stirred on a Velp Scientifica Vortex agitator, ultrasonicated for 5 min and stirred again. Finally, it was poured into a copper cup which was immediately immersed in liquid nitrogen to serve as a solid target (Scheme 1 — left side). The target was then mounted on a cooling device inside the reaction chamber which maintained it frozen during multipulse laser irradiation. It was rotated during irradiation to avoid local overheating and laser beam drilling and consequently to circumvent the transfer of non-uniform films [34]. The depositions were performed using a dynamic pressure of 10 − 1 Torr. The collector was placed parallel to the target at 4 cm separation distance of and maintained at a temperature of 150 °C during deposition using a conventional 2 inch diameter heater (Scheme 1 — right side). A pulsed KrF* laser source (λ = 248 nm, τFWHM ≈ 25 ns) operating at 7 Hz was used for the multipulse irradiation of the targets. 20,000 subsequent pulses were applied at an incident laser fluence of 0.75 J.cm − 2 for the synthesis of each coating. The laser beam was incident at 45° to the target surface. During the application of the multipulse laser irradiation, the target was constantly cooled down with liquid nitrogen. XRD analyses of deposited samples were performed using a PANalytical X'Celerator powder diffractometer. CuKα (λ = 0.154 nm) radiation was used (40 mA, 40 kV). Two significant angular ranges for OCP were explored: from 3° to 10° and from 25° to 34° (step size of 0.05° and time/step of 1000 s). Morphological investigations of thin films were performed using a Philips XL-20 Scanning Electron Microscope operating at 15 kV. The samples were sputter coated with gold before examination. Energy dispersive X-ray spectrometry (EDS) analyses were performed on all specimens. For Fourier Transform Infrared Spectroscopy (FTIR) investigation, thin films were deposited onto double face polished silicon substrates under identical conditions as those utilized for Ti coatings. FTIR studies were carried out using an IR microscope (Shimadzu Automated FT-IR Microscope (AIM-FTIR) 8800 apparatus) in transmission mode. The microscope provides a control stage movement, aperture setting and focusing directly from the PC screen. 40 scans were performed on square apertures of 120 μm 2 over a distance of 600 μm. The investigated domain was set from 5000 to 650 cm − 1 with a resolution of 4 cm − 1 wavenumber. Cell experiments were carried out on coatings sterilized by gamma-rays (Cobalt-60) at a dose of 25 kGy.
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Scheme 1. MAPLE experimental setup.
2.3. In vitro studies of OCP, MgOCP and SrOCP coatings 2.3.1. Osteoblast culture MG-63 human osteoblast-like cells were cultured in DMEM medium (Sigma, UK) supplemented with 10% FCS, and antibiotics (100 U/ ml penicillin, 100 μg/ml streptomycin). Cells were detached from culture flasks by trypsinization, and centrifuged. Cell number and viability were checked with trypan blue dye exclusion test. MG-63 osteoblast-like cells were plated at a density of 2 × 104 cells/ ml in 24-well plates containing six sterile samples for each biomaterial: titanium coated with OCP (reference), MgOCP, and SrOCP. The same concentration of cells was seeded into empty wells as a control (CTR) to determine the effects of the titanium with OCP coatings. The medium was changed with DMEM to which β-Glicerophosphate (10 − 2 M) and Ascorbic acid (50 μg/ml) were added to activate the osteoblasts. Plates were cultured under standard conditions, at 37 °C ± 0.5 with 95% humidity and 5% CO2 ± 0.2 up to 14 days. For the production of osteocalcin the culture medium was enriched with 1,25(OH)2D3 (10− 9 M) 48 h before end of each experimental time. 2.3.2. Osteoblast proliferation Cell proliferation and viability (3, 7, and 14 days) was monitored by WST1 (WST1, Roche Diagnostics GmbH, Manheim, Germany) colorimetric reagent test. This viability test is based on the reduction of a tetrazolium salt to a soluble formazan salt by a reductase of the mitochondrial respiratory chain that is active only in viable cells. 50 μl of WST1 solution and 450 μl of medium (final dilution: 1:10) were added to the cell monolayer, and the multi-well plates were incubated at 37 °C for the next 4 h. Supernatants were quantified spectrophotometrically at 450 nm with a reference wavelength of 625 nm. Results of WST1 are reported as optical density (OD) and can be directly correlated with the cell number. Phalloidin staining (specific for cellular actin filaments) was performed at 14 days to assess cell adhesion and colonization of samples: Cultures were washed in PBS and fixed in a solution of 4% formaldehyde in PBS for 15 min at 37 °C; then the samples were permeabilized in 0.5% Triton X-100 for 15 min, washed in PBS, and a FITC-conjugate phalloidin solution (1:100 in PBS) was added and incubated for 30 min at 37 °C. After washing, samples were examined by
fluorescence microscopy and the images were evaluated by a computerized image analysis system (Kontron KS 300 software, Kontron Electronic GmbH, Eching bei Munchen, Germany). 2.3.3. Osteoblast activity and differentiation At the end of the experimental times (3, 7 and 14 days) the supernatant was collected from all wells and centrifuged to remove particulates, if any. Aliquots of the supernatants were dispensed into Eppendorf tubes for storage at −70 °C and assayed for Alkaline Phosphatase activity (ALP, Ostase BAP immunoenzymatic assay, Immuno Diagnostic Systems, UK), Type I Collagen (CICP, Procollagen I C-terminal propeptide Immunoassay kit, Uscn Life Sciences, UK), Osteocalcin (OC, enzyme Immunoassay kit, Bender MedSystems, Vienna, A), Transforming Growth Factor-β1 (TGF-β1, Quantikine Immunoassay, R&D Systems, MN, USA), and Interleukin 6, (IL-6, Quantikine Immunoassay, R&D Systems, MN, USA). All the measured concentrations and activities were normalized by Total Protein content (TP, Total Protein micro-Lowry kit, SIGMA) to take into account the differences in cell growth. 2.3.4. Cell morphology Samples for each material, at the end of the experiment, were processed for Scanning Electron Microscopy (SEM): osteoblasts grown on the materials were fixed in 2.5% glutaraldehyde, in pH 7.4 phosphate buffer 0.01 M for 1 h and dehydrated in a graded ethanol series. After a passage in hexamethyldisilazane, the samples were air dried. The samples were sputter-coated with Pd prior to examination with a Philips XL-20 Scanning Electron Microscope. 2.3.5. Statistical analysis Statistical evaluation of data was performed using the software package SPSS/PC + Statistics™ 10.1 (SPSS Inc., Chicago, IL USA). The experiment was repeated three times and the results presented are the mean of six values. Data are reported as mean ± standard deviations (SD) at a significance level of p b 0.05. After having verified normal distribution and homogeneity of variance, a one-way ANOVA was done for comparison between groups. Finally, the Scheffé's post hoc multiple comparison tests were performed to detect significant differences between groups.
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3. Results and discussion This work indicates that MAPLE can be extended to deposit substituted OCP as well. 3.1. Structural and morphological characterization Fig. 1 shows typical X-ray diffraction patterns (XRD) of the thin films (Fig. 1b–d) compared with that recorded from pure OCP powder (Fig. 1a). The comparison indicates that all the coatings are constituted of OCP, as shown by the presence of the strong low angle reflection at 4.7° 2θ, which is diagnostic for OCP and corresponds to the 100 reflection, and of the series of reflections in the 30–34° 2θ range. The data indicate that the milder conditions of deposition of MAPLE provided a higher degree of OCP crystallinity with respect to PLD [4]. As a matter of fact, the XRD pattern of OCP coatings deposited on Ti substrates by means of PLD was reported to display just a shoulder at about 4.7° 2θ and a very broad peak around 32–33° 2θ [4]. Fig. 2 presents scanning electron microscopic (SEM) images of OCP-coated titanium substrates. The deposition appears quite homogeneous (Fig. 2a). At higher magnification, the presence of crystal fragments, together with cauliflower-like aggregates and droplets, is clearly visible in the SEM images of the coatings. (Fig. 2b). The crystals appear embedded in the thin film, and covered with the aggregates and
droplets, which constitute the bulk of the coating. The presence of crystal fragments is the main difference with respect to the morphology of OCP coatings deposited by PLD, which was characterized by the presence of cauliflower-like aggregates and droplets [4]. We previously showed that Sr 2 + and Mg 2 + can substitute for Ca 2 + into OCP structure, causing a variation of the unit cell parameters and a reduction of OCP stability as a function of ion content [39]. Although MgOCP and SrOCP were prepared using the same ion concentration in solution of 10 at.%, the ion content of the strontium and magnesium solid powders were found to be 5.5 and 0.6 at.%, respectively. These values are in agreement with the relatively different destabilizing roles these ions had on OCP structure [39]. The presence of Mg 2 + and Sr 2 + did not affect appreciably the morphology of the deposited films, which looked quite similar for the different samples. The Electron Dispersive Spectroscopy (EDS) maps shown in Fig. 3 indicate a homogeneous distribution of magnesium and strontium on the surface of the thin films. FTIR results (Fig. 4) confirmed the composition of the coatings. The strong absorption band (red color) at 1120 cm − 1 corresponding to PO42 + is indicative of OCP presence whereas the weak absorption “shoulder” (green–yellow) between 3250 and 3750 cm − 1 is due to H2O presence. Moreover, FTIR microscopy data confirmed the uniform composition of the investigated surface (600 μm scanning distance on 120 × 120 μm apertures) for both MgOCP (Fig. 4a) and SrOCP (Fig. 4b) films. The small fluctuation in absorbance
Fig. 1. Powder X-ray diffraction patterns of (a) OCP powder and of the thin films deposited on Ti substrates from: (b) MgOCP, (c) SrOCP, (d) OCP. In each plot, the intensities of the 25–34° 2θ range are magnified by 5.
Fig. 2. SEM micrographs of thin films deposited from OCP sample. In the image at higher magnification (b) some of the crystals and droplets on the cauliflower-like aggregates and droplets which constitute the bulk of the coating are labeled with (C) and (D) respectively.
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intensity along z axis over the 600 μm scanning distance could be due to the film thickness variation, in agreement with the observed morphological features characterized by a hilly microrelief (Fig. 2b). 3.2. Osteoblast adhesion, spreading, proliferation and activity Both magnesium and strontium occur in the composition of the hard tissues of vertebrates. In particular, Strontium content is relatively higher at the regions of high metabolic turnover of bone and its beneficial effects in the treatment of diseases associated to the loss of bone mass are well documented [41–43]. Magnesium plays a key role on bone metabolism [44,45]. Osteoblast-like MG-63 cells were cultured on OCP, MgOCP and SrOCP up to 14 days. The results indicate that the presence of Mg 2 + and Sr 2 + in the OCP coatings had a positive influence on osteoblast proliferation and differentiation. Composition and surface characteristics of a biomaterial greatly influence cellular behavior and play an essential role in osteoblastic adhesion and proliferation. In agreement with this hypothesis, after adhesion and a significant rapid proliferation from day 3 to 7 in CTR and experimental groups (pb 0.0001), at day 14 cell proliferation decreased in the OCP group, whereas it continued to increase slightly in the in MgOCP and SrOCP groups. As measured by WST1, proliferation of osteoblasts grown on MgOCP (7 and 14 days) and SrOCP (14 days), as well as on CTR, was significantly higher when compared to OCP group (Fig. 5a). In vitro differentiation of osteoblasts is associated with the increase of alkaline phosphatase (ALP) activity and production of collagen type I (CICP) in the first phases of culture, followed by synthesis
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of osteocalcin (OC) in the mineralization stage. The results presented in Fig. 5b–d show that Mg 2 + and Sr 2 + ions influenced osteoblast cultures; however their effect on cell differentiation and matrix deposition were different. ALP was significantly enhanced in experimental groups, when compared to CTR: significant higher values were found in OCP (7 days, p b 0.05), MgOCP (7 and 14 days, p b 0.05), and SrOCP (7 days, p b 0.05, and 14 days, p b 0.0001). Moreover, SrOCP showed a higher activity than OCP (14 days, p b 0.0001) and MgOCP (3 and 14 days, p b 0.05). CICP production on OCP, MgOCP, and SrOCP was significantly higher (pb 0.0001) than CTR at 7 days. At 14 days MgOCP showed the highest production of CICP, when compared to the other groups (pb 0.0001). OC synthesis showed no differences among CTR, OCP and MgOCP groups, while SrOCP values were significantly higher both at 7 (p b 0.005) and 14 days (b0.05), when compared to the other groups. To resume, the most relevant effect of MgOCP was on collagen type I production, while SrOCP primarily stimulated ALP activity and OC synthesis. Moreover both ions enhanced osteoblast proliferation, when compared to OCP. These data are in very good agreement with those previously reported for Mg 2 +-containing and Sr 2 +-containing HA coatings deposited from a supersaturated CaP solution on metallic substrates [26], and confirm the important role of magnesium and strontium on osteoblast proliferation and differentiation. No differences were found in IL-6 level, in all experimental times, proving that the addition of Mg 2 + and Sr 2 + to OCP did not have cytotoxic or inflammatory effects on osteoblast-like cells, even when compared to CTR (Fig. 5e).
Fig. 3. Energy dispersive X-ray spectrometry (EDS) maps recorded for the coatings: (a) MgOCP; (b) SrOCP; The different colors used for Ca2 + (blue), Mg2 + (red) and Sr2 + (yellow) enlight the homogeneous distribution of the ions on the coating.
Fig. 4. FTIR microscopy plots recorded for the coatings: (a) MgOCP; (b) SrOCP; Blue is for weaker, yellow for intermediate while red is for stronger IR absorption.
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Fig. 5. Proliferation, differentiation and synthetic activity of osteoblasts after 3, 7 and 14 days of culture on samples of CTR, OCP, MgOCP, and SrOCP. Mean ± sd, n = 6. (* = p b 0.05; ** = p b 0.005; *** = p b 0.0001).a. WST1. 7 days: *CTR, MgOCP vs SrOCP; 14 days: ***CTR, MgOCP vs OCP; **SrOCP vs OCP.b. ALP. 3 days: *SrOCP vs MgOCP; 7 days: *OCP, MgOCP, SrOCP vs CTR; 14 days: ***SrOCP vs CTR, OCP; *SrOCP vs MgOCP; *MgOCP vs CTR, OCP.c. CICP. 7 days: ***OCP, MgOCP, SrOCP vs CTR; 14 days: ***MgOCP vs CTR, OCP, SrOCP.d. OC. 7 days: **SrOCP vs CTR, OCP, MgOCP; 14 days: *SrOCP vs CTR, MgOCP. e. IL6. ns. f. TGFb1. 3 days: *CTR vs OCP, MgOCP, SrOCP; 7 days: *MgOCP vs SrOCP; 14 days: **CTR, MgOCP, SrOCP vs OCP.
TGF-β1 activity is known to enhance or inhibit osteoblast proliferation and differentiation, including secretion of bone matrix proteins, depending on cell maturation state [46]. TGF-β1 level significantly increased from 3 to 7 days, and then decreased at 14 days in all groups. Incorporation of Mg 2 + and Sr 2 + into OCP caused an increased production of TGF-β1 at 14 days in comparison to OCP alone (p b 0.005). Phalloidin staining was performed to assess cell proliferation and morphology on different substrates. Cell behavior is influenced at a great extent by surface topography and chemical composition that affect first cell adhesion, and then cell proliferation and colonization on biomaterial surface. The image analysis of Phalloidin staining showed no differences among the different coatings on osteoblast morphology (Fig. 6a–c). The cells on the surfaces of the three groups displayed the characteristic shape of osteoblasts. The percent coverage by cells adhering to the surface of the different coatings was measured at 14 days by an image analysis system. The following are the mean values of ten fields for each sample: OCP = 84 ± 3%, SrOCP = 90 ± 4% and MgOCP = 95 ± 7%. The values measured for SrOCP and MgOCP were significantly higher (p b 0.05) than that found for OCP alone; they are consistent with WST1 proliferation data (Fig. 5a). SEM images of the cells cultured up to 14 days on the different materials showed good attachment and spreading and exhibited
numerous lamellipodia and filopodia extensions (Fig. 7). In particular, osteoblasts grown on MgOCP and SrOCP exhibited a greater development of extra-cellular matrix with respect to those grown on OCP, in agreement with the results obtained for the differentiation markers. It can be inferred that the presence of Mg 2 + or Sr 2 + added to OCP in the coating influenced osteoblast behavior, enhancing cell proliferation and differentiation. In particular, the comparison of the results obtained for the differentiation markers on the different coatings indicates that the degree of cell differentiation on the different coatings follows the order OCP b MgOCP b SrOCP. 4. Conclusions Thin films of OCP, MgOCP and SrOCP were deposited by matrix assisted pulsed laser evaporation on Ti substrates. The coatings, composed of crystal fragments, cauliflower-like aggregates and droplets, exhibited a higher crystallinity than OCP films obtained by pulsed laser deposition and displayed a homogeneous distribution of magnesium and strontium on their surfaces. The increased proliferation, and enhanced activity and differentiation of the cells grown on SrOCP and MgOCP with respect to those cultivated on undoped OCP films or on polystyrene plate controls demonstrated that ion-doping improves
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Fig. 6. Phalloidin staining displaying cell adhesion and colonization after 14 days of culture of osteoblast grown on (a) OCP, (b) MgOCP and (c) SrOCP coatings of Ti substrates.
the beneficial effect of OCP on bone cells, suggesting that MgOCP and SrOCP coatings could be usefully applied on Ti implants for orthopedic use.
Abbreviations
OCP Octacalcium phosphate MgOCP Magnesium doped octacalcium phosphate SrOCP Strontium doped octacalcium phosphate MAPLE Matrix assisted pulsed laser evaporation AIM-FTIR Automated infrared microscopy-fourier transform infrared SEM Scanning electron microscopy XRD X-ray diffraction EDS Energy dispersive X-ray spectrometry ALP Alkaline phosphatase activity OC Osteocalcin CICP Type I collagen IL-6 Interleukin 6
Acknowledgments This research was carried out with the financial support of MIUR. The Romanian authors acknowledge with thanks the financial support of this work by PN09390101.
Fig. 7. SEM images of human osteoblasts MG63 on (a) OCP, (b) MgOCP and (c) SrOCP after 14 days of culturing. The cells exhibit good attachment and spreading. Numerous lamellipodia and filopodia extensions can be appreciated in all the images. Moreover, the surface of the cells in (b) and (c) exhibits a greater development of extra-cellular matrix than in (a).
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Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio
Magnesium and strontium doped octacalcium phosphate thin films by matrix assisted pulsed laser evaporation Elisa Boanini a, Paola Torricelli b, Milena Fini b, Felix Sima c, Natalia Serban c, Ion N. Mihailescu c, Adriana Bigi a,⁎ a b c
Department of Chemistry “G. Ciamician”, University of Bologna, 40126 Bologna, Italy Laboratory of Preclinical Surgical Studies, Research Institute Codivilla Putti, Rizzoli Orthopaedic Institute, Bologna, Italy National Institute for Lasers, Plasma and Radiation Physics, P. O. Box MG 36, 77125 Bucharest-Magurele, Romania
a r t i c l e
i n f o
Article history: Received 28 July 2011 Received in revised form 28 October 2011 Accepted 8 November 2011 Available online 19 November 2011 Keywords: Octacalcium phosphate coating Strontium Magnesium MAPLE Osteoblast
a b s t r a c t Octacalcium phosphate (OCP) is a promising alternative to hydroxyapatite as biomaterial for hard tissue repair. In this study we successfully applied Matrix Assisted Pulsed Laser Evaporation (MAPLE) to deposit Mg and Sr doped OCP (MgOCP and SrOCP), as well as OCP, thin films on titanium substrates. OCP, Mg-substituted and Sr-substituted OCP were synthesized in aqueous medium, then were suspended in deionised water, frozen at liquid nitrogen temperature and used as targets for MAPLE experiments. The depositions were carried out using a KrF* excimer laser source (λ = 248 nm, τFWHM = 25 ns) in mild conditions of temperature and pressure. The results of X-ray diffraction, infrared spectroscopy, scanning electron microscopy and energy dispersive spectroscopy investigations revealed that the OCP thin films are deposited in the form of cauliflower-like aggregates and droplets, as well as crystal fragments, with a homogeneous distribution of magnesium and strontium on the surface of the coatings. Human osteoblast-like MG-63 cells were cultured on the different biomaterials up to 14 days. MgOCP and SrOCP coatings promote osteoblast proliferation and differentiation with respect to OCP. Under these experimental conditions, the production of procollagen-type I, transforming growth factor-β1, alkaline phosphatase and osteocalcin indicated that the level of differentiation of the cells grown on the different coatings increased in the order OCP b MgOCP b SrOCP. © 2011 Elsevier Inc. All rights reserved.
1. Introduction An implant material must provide suitable functionality and biological response. In the orthopedic field, metallic materials are widely employed for load bearing implants and inner fixation devices thanks to their relevant mechanical properties: high tensile and yield strength, good resistance to fatigue, deformation, and corrosion [1]. However, their integration with bone tissue depends on the chemistry and physics of the implant surface. Calcium phosphate coatings have been extensively applied in order to enhance the ability of the biomaterials to create a bond with the living host tissue. To this end, a number of physical and chemical methods, including plasma spraying, magnetron sputtering, ion beam coating, electrophoretic deposition, pulsed laser deposition, sol–gel and biomimetic processes, are currently employed [1–7]. Hydroxyapatite, Ca10(PO4)6(OH)2, (HA), is considered to be one of the best biocompatible coatings, providing a highly bioactive and
⁎ Corresponding author. Tel.: + 39 051 2099551; fax: + 39 051 2099456. E-mail address: [email protected] (A. Bigi). 0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2011.11.003
resorbable interface in contact with bone; it is utilized in most commercially available coated metallic prostheses. However, octacalcium phosphate, Ca8H2(PO4)6·5H2O (OCP), which exhibits high osteoconductive characteristics and a speed of resorption that far exceeds that of HA [8–10], has been shown to be the precursor phase of biological apatites, having a very similar lattice surface structure to hexagonal HA. The triclinic structure of OCP can be described as having “apatitic layers”, where Ca 2 + and PO43 − ions occupy the same positions as in HA, alternated to “hydrated layers”, where structural water molecules are interdispersed between cations and anions [11,12]. Its solubility (higher than that of HA) and easy hydrolysis into nanocrystalline apatite constitute potential advantages of OCP with respect to crystalline HA. The stimulating effect of OCP toward new bone formation has been related to its rapid conversion into apatite in a biological setting [13–15]. OCP displays a complex stoichiometry and structure. OCP thin films are therefore quite difficult to deposit by means of physical methods, and most OCP coatings were deposited from supersaturated solutions and by electrochemical methods [16–18]. We previously optimized the experimental conditions to deposit nanocrystalline OCP thin films on Ti substrate by Pulsed Laser Deposition, PLD, technique [4,19]. According to the results of the X-ray diffraction analysis, the coatings displayed an amorphous-poorly crystalline structure, as
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confirmed by High Resolution Transmission Electron Microscopy, HRTEM, observations that showed the presence of crystalline nanodomains interdispersed in an amorphous phase [4,20]. The concept that the biological performance of calcium phosphate based materials might be improved by the addition of bioactive ions, has stimulated a number of studies on ion-substituted or ion-doped HA coatings [21,22]. In particular, the beneficial effect of the presence of carbonate, magnesium, strontium, manganese, and silicon in HAbased coatings deposited on metallic substrates has been demonstrated [23–32]. However, there are few reports on the deposition of ion-substituted or ion-doped OCP on metallic substrates. This is because even in the absence of ionic substitution, the complex stoichiometry and structure of OCP make it difficult to deposit this calcium phosphate [4,16–18]. In this paper we explored the possibility of depositing thin films of ion-substituted OCP, namely magnesium substituted OCP (MgOCP) and strontium substituted OCP (SrOCP), on Ti substrates. To accomplish this goal we applied Matrix Assisted Pulsed Laser Evaporation (MAPLE), which provides a more gentle mechanism than PLD for transferring different compounds, including large molecular weight molecules [33,34]. It is generally recognized that PLD is limited in case of doped compounds because of different ablation thresholds of composing materials. This can cause fractionated vaporization of the components, loss of dopant and component segregation in the deposited films. In case of MAPLE, this is avoided by protection of the frozen solvent. This is the first attempt to deposit OCP and ion-substituted OCP thin films by MAPLE technique. Moreover, to the best of our knowledge, ion-substituted OCP coatings have not been prepared up to now. The thin films synthesized in this study were submitted to structural and morphological characterization, and to in vitro tests of their compatibility to support growth and differentiation of osteoblast-like cells MG63. MG63 activity and differentiation were investigated using the most common markers of osteoblast metabolism: alkaline phosphatase (ALP) that is a specific indicator of osteoblast activity; type I collagen (CICP), that is the major component of the extra-cellular bone matrix; osteocalcin (OC), that is the major non-collagenous protein of the bone matrix. ALP activity increases in the first phase of cell proliferation, while the production of CICP and the synthesis of OC are related to the following phases of differentiation and of extra-cellular matrix production and mineralization [35]. A growth factor and a cytokine were also chosen to investigate osteoblast response to the innovative coatings: Transforming growth factor β1 (TGF-β1) is a growth factor that modulates cell proliferation and enhances the deposition of extra-cellular matrix through different pathways, both promoting synthesis and inhibiting degradation [36]; Interleukin 6 (IL-6) is an indicative pro-inflammatory cytokine that has a major role in the mediation of inflammation and is also implicated in bone remodeling, since it stimulates the first stages of osteoblast differentiation [37]. 2. Materials and methods 2.1. Synthesis and characterization of OCP, MgOCP and SrOCP nanocrystals OCP nanocrystals were synthesized by dropwise addition of 250 ml of 0.04 M Ca(CH3COO)2·H2O over a period of 60 min into 750 ml of a phosphate solution containing 5 mmol of Na2HPO4·12H2O and 5 mmol of NaH2PO4·H2O (Carlo Erba) starting pH 5 [38]. The reaction was carried out at 70 °C with smooth mechanical stirring. The precipitate was stored in contact with the mother solution for 10 min, filtered, repeatedly washed with bidistilled water and dried at 37 °C. The same procedure was applied for the syntheses performed in the presence of Mg2 + or Sr2 + in solution [39]: Mg(CH3COO)2·4H2O and Sr (CH3COO)2·1/2H2O were respectively used together with Ca(CH3COO)2·H2O to prepare solutions with a [M2 + / (Ca 2 + + M2 +)]× 100 ratio of 10, and a total cation concentration of 0.04 M.
Powder X-ray diffraction patterns were recorded using a PANalytical X'Pert PRO powder diffractometer. CuKα radiation was used (λ = 0.154 nm, 40 mA, 40 kV). Data were obtained in the range of 2θ from 3 to 60° (0.05°/step, 10 s/step). The powder X-ray diffraction pattern of the solid products confirmed that the compounds synthesized in the presence of Mg 2 + and Sr 2 + were constituted of octacalcium phosphate as the sole crystalline phase. Calcium, magnesium and strontium contents in the solid products were determined by means of a Dionex DX100 chromatography system equipped with a Dionex CD20 conductivity detector. Powders were previously dissolved in 0.1 M HCl. The data show that Mg and Sr contents were 0.6 and 5.5 at.% respectively. These analytical contents correspond well with the observed small, but appreciable, variations in the unit cell dimensions of OCP indicating Mg and Sr substitution for Ca in MgOCP and SrOCP respectively [39]. 2.2. Synthesis and characterization of OCP, MgOCP and SrOCP coatings Disk-shaped (12 mm diameter, 0.5 mm thick) grade 2 Ti substrates were mechanically polished and submitted to chemical etching to get an extended active surface [40]. They were ultrasonically cleaned in acetone, ethanol and deionized water and blown dried with high purity N2 gas before use for thin film deposition. The suspension consisting of 0.12 g of OCP powder in 5 ml distilled water was ground with agate balls for 60 min at 360 rot/min in a Retsch centrifugal ball mill suitable for wet grinding. Next, the mixture was collected in a Berzelius glass, mechanically stirred on a Velp Scientifica Vortex agitator, ultrasonicated for 5 min and stirred again. Finally, it was poured into a copper cup which was immediately immersed in liquid nitrogen to serve as a solid target (Scheme 1 — left side). The target was then mounted on a cooling device inside the reaction chamber which maintained it frozen during multipulse laser irradiation. It was rotated during irradiation to avoid local overheating and laser beam drilling and consequently to circumvent the transfer of non-uniform films [34]. The depositions were performed using a dynamic pressure of 10 − 1 Torr. The collector was placed parallel to the target at 4 cm separation distance of and maintained at a temperature of 150 °C during deposition using a conventional 2 inch diameter heater (Scheme 1 — right side). A pulsed KrF* laser source (λ = 248 nm, τFWHM ≈ 25 ns) operating at 7 Hz was used for the multipulse irradiation of the targets. 20,000 subsequent pulses were applied at an incident laser fluence of 0.75 J.cm − 2 for the synthesis of each coating. The laser beam was incident at 45° to the target surface. During the application of the multipulse laser irradiation, the target was constantly cooled down with liquid nitrogen. XRD analyses of deposited samples were performed using a PANalytical X'Celerator powder diffractometer. CuKα (λ = 0.154 nm) radiation was used (40 mA, 40 kV). Two significant angular ranges for OCP were explored: from 3° to 10° and from 25° to 34° (step size of 0.05° and time/step of 1000 s). Morphological investigations of thin films were performed using a Philips XL-20 Scanning Electron Microscope operating at 15 kV. The samples were sputter coated with gold before examination. Energy dispersive X-ray spectrometry (EDS) analyses were performed on all specimens. For Fourier Transform Infrared Spectroscopy (FTIR) investigation, thin films were deposited onto double face polished silicon substrates under identical conditions as those utilized for Ti coatings. FTIR studies were carried out using an IR microscope (Shimadzu Automated FT-IR Microscope (AIM-FTIR) 8800 apparatus) in transmission mode. The microscope provides a control stage movement, aperture setting and focusing directly from the PC screen. 40 scans were performed on square apertures of 120 μm 2 over a distance of 600 μm. The investigated domain was set from 5000 to 650 cm − 1 with a resolution of 4 cm − 1 wavenumber. Cell experiments were carried out on coatings sterilized by gamma-rays (Cobalt-60) at a dose of 25 kGy.
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Scheme 1. MAPLE experimental setup.
2.3. In vitro studies of OCP, MgOCP and SrOCP coatings 2.3.1. Osteoblast culture MG-63 human osteoblast-like cells were cultured in DMEM medium (Sigma, UK) supplemented with 10% FCS, and antibiotics (100 U/ ml penicillin, 100 μg/ml streptomycin). Cells were detached from culture flasks by trypsinization, and centrifuged. Cell number and viability were checked with trypan blue dye exclusion test. MG-63 osteoblast-like cells were plated at a density of 2 × 104 cells/ ml in 24-well plates containing six sterile samples for each biomaterial: titanium coated with OCP (reference), MgOCP, and SrOCP. The same concentration of cells was seeded into empty wells as a control (CTR) to determine the effects of the titanium with OCP coatings. The medium was changed with DMEM to which β-Glicerophosphate (10 − 2 M) and Ascorbic acid (50 μg/ml) were added to activate the osteoblasts. Plates were cultured under standard conditions, at 37 °C ± 0.5 with 95% humidity and 5% CO2 ± 0.2 up to 14 days. For the production of osteocalcin the culture medium was enriched with 1,25(OH)2D3 (10− 9 M) 48 h before end of each experimental time. 2.3.2. Osteoblast proliferation Cell proliferation and viability (3, 7, and 14 days) was monitored by WST1 (WST1, Roche Diagnostics GmbH, Manheim, Germany) colorimetric reagent test. This viability test is based on the reduction of a tetrazolium salt to a soluble formazan salt by a reductase of the mitochondrial respiratory chain that is active only in viable cells. 50 μl of WST1 solution and 450 μl of medium (final dilution: 1:10) were added to the cell monolayer, and the multi-well plates were incubated at 37 °C for the next 4 h. Supernatants were quantified spectrophotometrically at 450 nm with a reference wavelength of 625 nm. Results of WST1 are reported as optical density (OD) and can be directly correlated with the cell number. Phalloidin staining (specific for cellular actin filaments) was performed at 14 days to assess cell adhesion and colonization of samples: Cultures were washed in PBS and fixed in a solution of 4% formaldehyde in PBS for 15 min at 37 °C; then the samples were permeabilized in 0.5% Triton X-100 for 15 min, washed in PBS, and a FITC-conjugate phalloidin solution (1:100 in PBS) was added and incubated for 30 min at 37 °C. After washing, samples were examined by
fluorescence microscopy and the images were evaluated by a computerized image analysis system (Kontron KS 300 software, Kontron Electronic GmbH, Eching bei Munchen, Germany). 2.3.3. Osteoblast activity and differentiation At the end of the experimental times (3, 7 and 14 days) the supernatant was collected from all wells and centrifuged to remove particulates, if any. Aliquots of the supernatants were dispensed into Eppendorf tubes for storage at −70 °C and assayed for Alkaline Phosphatase activity (ALP, Ostase BAP immunoenzymatic assay, Immuno Diagnostic Systems, UK), Type I Collagen (CICP, Procollagen I C-terminal propeptide Immunoassay kit, Uscn Life Sciences, UK), Osteocalcin (OC, enzyme Immunoassay kit, Bender MedSystems, Vienna, A), Transforming Growth Factor-β1 (TGF-β1, Quantikine Immunoassay, R&D Systems, MN, USA), and Interleukin 6, (IL-6, Quantikine Immunoassay, R&D Systems, MN, USA). All the measured concentrations and activities were normalized by Total Protein content (TP, Total Protein micro-Lowry kit, SIGMA) to take into account the differences in cell growth. 2.3.4. Cell morphology Samples for each material, at the end of the experiment, were processed for Scanning Electron Microscopy (SEM): osteoblasts grown on the materials were fixed in 2.5% glutaraldehyde, in pH 7.4 phosphate buffer 0.01 M for 1 h and dehydrated in a graded ethanol series. After a passage in hexamethyldisilazane, the samples were air dried. The samples were sputter-coated with Pd prior to examination with a Philips XL-20 Scanning Electron Microscope. 2.3.5. Statistical analysis Statistical evaluation of data was performed using the software package SPSS/PC + Statistics™ 10.1 (SPSS Inc., Chicago, IL USA). The experiment was repeated three times and the results presented are the mean of six values. Data are reported as mean ± standard deviations (SD) at a significance level of p b 0.05. After having verified normal distribution and homogeneity of variance, a one-way ANOVA was done for comparison between groups. Finally, the Scheffé's post hoc multiple comparison tests were performed to detect significant differences between groups.
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3. Results and discussion This work indicates that MAPLE can be extended to deposit substituted OCP as well. 3.1. Structural and morphological characterization Fig. 1 shows typical X-ray diffraction patterns (XRD) of the thin films (Fig. 1b–d) compared with that recorded from pure OCP powder (Fig. 1a). The comparison indicates that all the coatings are constituted of OCP, as shown by the presence of the strong low angle reflection at 4.7° 2θ, which is diagnostic for OCP and corresponds to the 100 reflection, and of the series of reflections in the 30–34° 2θ range. The data indicate that the milder conditions of deposition of MAPLE provided a higher degree of OCP crystallinity with respect to PLD [4]. As a matter of fact, the XRD pattern of OCP coatings deposited on Ti substrates by means of PLD was reported to display just a shoulder at about 4.7° 2θ and a very broad peak around 32–33° 2θ [4]. Fig. 2 presents scanning electron microscopic (SEM) images of OCP-coated titanium substrates. The deposition appears quite homogeneous (Fig. 2a). At higher magnification, the presence of crystal fragments, together with cauliflower-like aggregates and droplets, is clearly visible in the SEM images of the coatings. (Fig. 2b). The crystals appear embedded in the thin film, and covered with the aggregates and
droplets, which constitute the bulk of the coating. The presence of crystal fragments is the main difference with respect to the morphology of OCP coatings deposited by PLD, which was characterized by the presence of cauliflower-like aggregates and droplets [4]. We previously showed that Sr 2 + and Mg 2 + can substitute for Ca 2 + into OCP structure, causing a variation of the unit cell parameters and a reduction of OCP stability as a function of ion content [39]. Although MgOCP and SrOCP were prepared using the same ion concentration in solution of 10 at.%, the ion content of the strontium and magnesium solid powders were found to be 5.5 and 0.6 at.%, respectively. These values are in agreement with the relatively different destabilizing roles these ions had on OCP structure [39]. The presence of Mg 2 + and Sr 2 + did not affect appreciably the morphology of the deposited films, which looked quite similar for the different samples. The Electron Dispersive Spectroscopy (EDS) maps shown in Fig. 3 indicate a homogeneous distribution of magnesium and strontium on the surface of the thin films. FTIR results (Fig. 4) confirmed the composition of the coatings. The strong absorption band (red color) at 1120 cm − 1 corresponding to PO42 + is indicative of OCP presence whereas the weak absorption “shoulder” (green–yellow) between 3250 and 3750 cm − 1 is due to H2O presence. Moreover, FTIR microscopy data confirmed the uniform composition of the investigated surface (600 μm scanning distance on 120 × 120 μm apertures) for both MgOCP (Fig. 4a) and SrOCP (Fig. 4b) films. The small fluctuation in absorbance
Fig. 1. Powder X-ray diffraction patterns of (a) OCP powder and of the thin films deposited on Ti substrates from: (b) MgOCP, (c) SrOCP, (d) OCP. In each plot, the intensities of the 25–34° 2θ range are magnified by 5.
Fig. 2. SEM micrographs of thin films deposited from OCP sample. In the image at higher magnification (b) some of the crystals and droplets on the cauliflower-like aggregates and droplets which constitute the bulk of the coating are labeled with (C) and (D) respectively.
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intensity along z axis over the 600 μm scanning distance could be due to the film thickness variation, in agreement with the observed morphological features characterized by a hilly microrelief (Fig. 2b). 3.2. Osteoblast adhesion, spreading, proliferation and activity Both magnesium and strontium occur in the composition of the hard tissues of vertebrates. In particular, Strontium content is relatively higher at the regions of high metabolic turnover of bone and its beneficial effects in the treatment of diseases associated to the loss of bone mass are well documented [41–43]. Magnesium plays a key role on bone metabolism [44,45]. Osteoblast-like MG-63 cells were cultured on OCP, MgOCP and SrOCP up to 14 days. The results indicate that the presence of Mg 2 + and Sr 2 + in the OCP coatings had a positive influence on osteoblast proliferation and differentiation. Composition and surface characteristics of a biomaterial greatly influence cellular behavior and play an essential role in osteoblastic adhesion and proliferation. In agreement with this hypothesis, after adhesion and a significant rapid proliferation from day 3 to 7 in CTR and experimental groups (pb 0.0001), at day 14 cell proliferation decreased in the OCP group, whereas it continued to increase slightly in the in MgOCP and SrOCP groups. As measured by WST1, proliferation of osteoblasts grown on MgOCP (7 and 14 days) and SrOCP (14 days), as well as on CTR, was significantly higher when compared to OCP group (Fig. 5a). In vitro differentiation of osteoblasts is associated with the increase of alkaline phosphatase (ALP) activity and production of collagen type I (CICP) in the first phases of culture, followed by synthesis
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of osteocalcin (OC) in the mineralization stage. The results presented in Fig. 5b–d show that Mg 2 + and Sr 2 + ions influenced osteoblast cultures; however their effect on cell differentiation and matrix deposition were different. ALP was significantly enhanced in experimental groups, when compared to CTR: significant higher values were found in OCP (7 days, p b 0.05), MgOCP (7 and 14 days, p b 0.05), and SrOCP (7 days, p b 0.05, and 14 days, p b 0.0001). Moreover, SrOCP showed a higher activity than OCP (14 days, p b 0.0001) and MgOCP (3 and 14 days, p b 0.05). CICP production on OCP, MgOCP, and SrOCP was significantly higher (pb 0.0001) than CTR at 7 days. At 14 days MgOCP showed the highest production of CICP, when compared to the other groups (pb 0.0001). OC synthesis showed no differences among CTR, OCP and MgOCP groups, while SrOCP values were significantly higher both at 7 (p b 0.005) and 14 days (b0.05), when compared to the other groups. To resume, the most relevant effect of MgOCP was on collagen type I production, while SrOCP primarily stimulated ALP activity and OC synthesis. Moreover both ions enhanced osteoblast proliferation, when compared to OCP. These data are in very good agreement with those previously reported for Mg 2 +-containing and Sr 2 +-containing HA coatings deposited from a supersaturated CaP solution on metallic substrates [26], and confirm the important role of magnesium and strontium on osteoblast proliferation and differentiation. No differences were found in IL-6 level, in all experimental times, proving that the addition of Mg 2 + and Sr 2 + to OCP did not have cytotoxic or inflammatory effects on osteoblast-like cells, even when compared to CTR (Fig. 5e).
Fig. 3. Energy dispersive X-ray spectrometry (EDS) maps recorded for the coatings: (a) MgOCP; (b) SrOCP; The different colors used for Ca2 + (blue), Mg2 + (red) and Sr2 + (yellow) enlight the homogeneous distribution of the ions on the coating.
Fig. 4. FTIR microscopy plots recorded for the coatings: (a) MgOCP; (b) SrOCP; Blue is for weaker, yellow for intermediate while red is for stronger IR absorption.
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Fig. 5. Proliferation, differentiation and synthetic activity of osteoblasts after 3, 7 and 14 days of culture on samples of CTR, OCP, MgOCP, and SrOCP. Mean ± sd, n = 6. (* = p b 0.05; ** = p b 0.005; *** = p b 0.0001).a. WST1. 7 days: *CTR, MgOCP vs SrOCP; 14 days: ***CTR, MgOCP vs OCP; **SrOCP vs OCP.b. ALP. 3 days: *SrOCP vs MgOCP; 7 days: *OCP, MgOCP, SrOCP vs CTR; 14 days: ***SrOCP vs CTR, OCP; *SrOCP vs MgOCP; *MgOCP vs CTR, OCP.c. CICP. 7 days: ***OCP, MgOCP, SrOCP vs CTR; 14 days: ***MgOCP vs CTR, OCP, SrOCP.d. OC. 7 days: **SrOCP vs CTR, OCP, MgOCP; 14 days: *SrOCP vs CTR, MgOCP. e. IL6. ns. f. TGFb1. 3 days: *CTR vs OCP, MgOCP, SrOCP; 7 days: *MgOCP vs SrOCP; 14 days: **CTR, MgOCP, SrOCP vs OCP.
TGF-β1 activity is known to enhance or inhibit osteoblast proliferation and differentiation, including secretion of bone matrix proteins, depending on cell maturation state [46]. TGF-β1 level significantly increased from 3 to 7 days, and then decreased at 14 days in all groups. Incorporation of Mg 2 + and Sr 2 + into OCP caused an increased production of TGF-β1 at 14 days in comparison to OCP alone (p b 0.005). Phalloidin staining was performed to assess cell proliferation and morphology on different substrates. Cell behavior is influenced at a great extent by surface topography and chemical composition that affect first cell adhesion, and then cell proliferation and colonization on biomaterial surface. The image analysis of Phalloidin staining showed no differences among the different coatings on osteoblast morphology (Fig. 6a–c). The cells on the surfaces of the three groups displayed the characteristic shape of osteoblasts. The percent coverage by cells adhering to the surface of the different coatings was measured at 14 days by an image analysis system. The following are the mean values of ten fields for each sample: OCP = 84 ± 3%, SrOCP = 90 ± 4% and MgOCP = 95 ± 7%. The values measured for SrOCP and MgOCP were significantly higher (p b 0.05) than that found for OCP alone; they are consistent with WST1 proliferation data (Fig. 5a). SEM images of the cells cultured up to 14 days on the different materials showed good attachment and spreading and exhibited
numerous lamellipodia and filopodia extensions (Fig. 7). In particular, osteoblasts grown on MgOCP and SrOCP exhibited a greater development of extra-cellular matrix with respect to those grown on OCP, in agreement with the results obtained for the differentiation markers. It can be inferred that the presence of Mg 2 + or Sr 2 + added to OCP in the coating influenced osteoblast behavior, enhancing cell proliferation and differentiation. In particular, the comparison of the results obtained for the differentiation markers on the different coatings indicates that the degree of cell differentiation on the different coatings follows the order OCP b MgOCP b SrOCP. 4. Conclusions Thin films of OCP, MgOCP and SrOCP were deposited by matrix assisted pulsed laser evaporation on Ti substrates. The coatings, composed of crystal fragments, cauliflower-like aggregates and droplets, exhibited a higher crystallinity than OCP films obtained by pulsed laser deposition and displayed a homogeneous distribution of magnesium and strontium on their surfaces. The increased proliferation, and enhanced activity and differentiation of the cells grown on SrOCP and MgOCP with respect to those cultivated on undoped OCP films or on polystyrene plate controls demonstrated that ion-doping improves
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Fig. 6. Phalloidin staining displaying cell adhesion and colonization after 14 days of culture of osteoblast grown on (a) OCP, (b) MgOCP and (c) SrOCP coatings of Ti substrates.
the beneficial effect of OCP on bone cells, suggesting that MgOCP and SrOCP coatings could be usefully applied on Ti implants for orthopedic use.
Abbreviations
OCP Octacalcium phosphate MgOCP Magnesium doped octacalcium phosphate SrOCP Strontium doped octacalcium phosphate MAPLE Matrix assisted pulsed laser evaporation AIM-FTIR Automated infrared microscopy-fourier transform infrared SEM Scanning electron microscopy XRD X-ray diffraction EDS Energy dispersive X-ray spectrometry ALP Alkaline phosphatase activity OC Osteocalcin CICP Type I collagen IL-6 Interleukin 6
Acknowledgments This research was carried out with the financial support of MIUR. The Romanian authors acknowledge with thanks the financial support of this work by PN09390101.
Fig. 7. SEM images of human osteoblasts MG63 on (a) OCP, (b) MgOCP and (c) SrOCP after 14 days of culturing. The cells exhibit good attachment and spreading. Numerous lamellipodia and filopodia extensions can be appreciated in all the images. Moreover, the surface of the cells in (b) and (c) exhibits a greater development of extra-cellular matrix than in (a).
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