Antiresorption implant coatings based on calcium alendronate and octacalcium phosphate deposited by matrix assisted pulsed laser evaporation

Colloids and Surfaces B: Biointerfaces 136 (2015) 449–456

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Antiresorption implant coatings based on calcium alendronate and octacalcium phosphate deposited by matrix assisted pulsed laser evaporation Elisa Boanini a,∗ , Paola Torricelli b , Lucia Forte a , Stefania Pagani b , Natalia Mihailescu c , Carmen Ristoscu c , Ion N. Mihailescu c , Adriana Bigi a a

Department of Chemistry “G. Ciamician”, University of Bologna, 40126 Bologna, Italy Laboratory of Preclinical Surgical Studies, Research Institute Codivilla Putti—Rizzoli Orthopaedic Institute, via di Barbiano, 40136 Bologna, Italy c National Institute for Lasers, Plasma and Radiation Physics, P. O. Box MG 36, 77125 Magurele, Ilfov, Romania b

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Article history: Received 24 July 2015 Received in revised form 8 September 2015 Accepted 24 September 2015 Available online 28 September 2015 Keywords: Alendronate Octacalcium phosphate MAPLE Osteoblast Osteoclast In-vitro co-cultures.

a b s t r a c t The integration of an implant material with bone tissue depends on the chemistry and physics of the implant surface. In this study we applied matrix assisted pulsed laser evaporation (MAPLE) in order to synthesize calcium alendronate monohydrate (a bisphosphonate obtained by calcium sequestration from octacalcium phosphate by alendronate) and calcium alendronate monohydrate/octacalcium phosphate composite thin films on titanium substrates. Octacalcium phosphate coatings were prepared as reference material. The powders, which were synthesized in aqueous medium, were suspended in deionised water, frozen at liquid nitrogen temperature and used as targets for MAPLE experiments. The transfer was conducted with a KrF* excimer laser source ( = 248 nm,  FWHM ≤ 25 ns) in mild conditions of temperature and pressure. XRD, FTIR and SEM analyses confirmed that the coatings contain the same crystalline phases as the as-prepared powder samples. Osteoblast derived from stem cells and osteoclast derived from monocytes of osteoporotic subjects were co-cultured on the coatings up to 14 days. Osteoclast displayed significantly reduced proliferation and differentiation in the presence of calcium alendronate monohydrate, pointing to a clear role of the coatings containing this bisphosphonate on inhibiting excessive bone resorption. At variance, osteoblast production of alkaline phosphatase and type I pro-collagen were promoted by the presence of bisphosphonate, which also decreased the production of interleukin 6. The positive influence towards osteoblast differentiation was even more enhanced in the composite coatings, thanks to the presence of octacalcium phosphate. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Bisphosphonates (BPs) are widely used for the treatment of specific disorders of bone metabolism associated with conditions of bone loss, such as osteoporosis, Paget’s disease and bone metastases [1–3]. The central carbon atom of BPs binds two phosphonate groups and two covalently bound sidechains, R1 and R2 (Figure S1). It has been shown that the affinity for biological apatite is increased when R1 is a hydroxyl group [4,5]. Moreover, the presence of a nitrogen atom in R2 sidechain enhances the anti-osteoporotic potency and influences bone affinity [6–8]. The relevant antiresorption action of amino-BPs (N-BPs) is related to their inhibition of farnesyl diphosphate synthase, a major enzyme in the mevalonate pathway. The inhibition prevents the prenylation of small

∗ Corresponding author. E-mail address: [email protected] (E. Boanini). http://dx.doi.org/10.1016/j.colsurfb.2015.09.044 0927-7765/© 2015 Elsevier B.V. All rights reserved.

GPTases signaling proteins and, as a consequence, interferes with many osteoclast activities, eventually leading to cell apoptosis [4]. BPs also display anti-angiogenic properties [7] and anti-tumoral activity [8]. As a matter of fact, BPs have been shown to prevent and delay skeletal complications in patients with bone metastases from solid tumors or osteolytic lesions from multiple myeloma [9–10]. The major limits of BPs oral administration are poor bioavailability and development of gastrointestinal disorders [11]. On the other hand, adverse side effects, namely osteonecrosis of the jaw and atypical fractures, have been recently reported in patients receiving high doses by intravenous formulation [12–14]. Alternative modes of administration, such as local release at specific bone sites, could allow to reduce the doses of systemic administration and prevent adverse side effects [15]. We recently showed that alendronate (AL), one of the most potent N-BPs, can sequester calcium from insoluble calcium phosphates and yield precipitation of a crystalline BP, calcium alendronate monohydrate, CaAL• H2 O [16,17]. This compound, both alone and especially when associated to octacalcium

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Fig. 1. X-ray diffraction patterns (a) and FT-IR spectra (b) of crystalline powders obtained at increasing AL concentration in solution.

phosphate (OCP), exhibits a remarkable inhibition of osteoclast proliferation and activity, whereas it enhances osteoblast differentiation [17]. In this work, we explored the possibility to deposit thin films of OCP at different contents of CaAL• H2 O on titanium (Ti) substrates in order to get coatings able to offer a suitable interface for bone tissues thanks to the presence of OCP, and to provide a local availability of BP. In fact, although metallic materials, such as Ti, fulfill the requirements of a successful orthopaedic implants, in terms of strength, toughness and resistance to wear and corrosion [18], incomplete osteointegration and stress shielding are the main causes of bone implant failures [19]. Coating the metallic surface with a thin film of calcium phosphate provides improved implant fixation to hard tissues [20,21]. Among the numerous different methods available to coat metallic substrates [20], MatrixAssisted Pulsed Laser Evaporation (MAPLE) allows transferring a variety of compounds, including organic molecules and proteins, with an accurate control of thickness and stoichiometry [22–24]. Herein, we applied MAPLE to transfer and deposit composite crystals at different OCP and CaAL• H2 O content directly on Ti substrates in order to grow coatings with improved properties. This is the first attempt to synthesize in situ CaAL• H2 O–OCP coatings as an inorganic–organic assembling system by MAPLE technique. The biological performance of the coatings was investigated through in vitro tests carried out using co-cultures of osteoblast derived from stem cells and osteoclast derived from monocytes of osteoporotic subjects. 2. Materials and methods 2.1. Synthesis and characterization of crystalline powders OCP was synthesized by dropwise addition of 0.04 M Ca(CH3 COO)2 · H2 O (250 ml) over a period of 60 min into a phosphate solution (750 ml) containing Na2 HPO4 ·12H2 O (5 mmol) and NaH2 PO4 ·H2 O (5 mmol) at a starting pH of 5. 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. Reaction of OCP and AL was carried out in bidistilled water in the presence of two different concentrations of sodium alendronate trihydrate (Chemos), that is 8 and 20 mM. Resulting solid samples have been labeled AL8 and AL20, respectively. The reaction

was performed on 500 mg OCP/250 ml solution at 30 ◦ C, stirring for 72 h. Then the products were centrifuged at 5000 rpm for 10 min, repeatedly washed with double distilled water and dried at 37 ◦ C. X-ray diffraction (XRD) analysis was carried out by means of a PANalytical X’Pert PRO powder diffractometer equipped with a fast X’Celerator detector. CuK␣ radiation was used ( = 0.15418 nm, 40 mA, 40 kV). For phase identification, the 2 range was investigated from 3 to 40 degrees with a step size of 0.067◦ and time/step of 100 s. BP content was determined spectrophotometrically via complex formation with Fe(III) ions using a Varian Cary50Bio instrument ( = 290 nm) [25]. For infrared absorption analysis, spectra were recorded by a Shimadzu 8400S instrument in the 1500–400 cm−1 range, with a resolution of 4 cm−1 and a total of 50 scans/experiment. Morphological investigation was performed by scanning electron microscopy (SEM) using a Philips XL20 instrument operating at 15 kV. The samples were sputter-coated with Au before.

2.2. Deposition and characterization of coatings Disk-shaped Ti substrates (12 mm diameter and 0.5 mm thick) were mechanically polished and submitted to chemical etching before use as collectors. They were clean in ultrasonic sequential baths of acetone, alcohol and deionized water. For the preparation of one target used in MAPLE experiments, deionized H2 O based solution containing 0.12 g of OCP powder was suspended in 10 ml and subsequently stirred, homogenized and frozen in a special copper holder at 77 K in liquid nitrogen. The holder containing the solid target was then mounted inside a vacuum chamber. The schematic of the setup is presented in Figure S2. During exposure to multipulse laser irradiation, the target was maintained frozen by continuous liquid nitrogen flow inside a supporting cooler device and continuously rotated with 80 rpm to avoid drilling and improve the overall quality of the deposited films. A pulsed KrF* laser source ( = 248 nm,  FWHM ≤ 25 ns) operating at 5 Hz was employed for the irradiation of the target. 20,000 subsequent pulses were applied at an incident laser fluence of 0.73 J cm−2 for the synthesis of each assembly (the corresponding pulse energy was of 280 mJ). The deposition was carried out in a residual pressure of 13 Pa. The substrate was facing the target at a separation distance of ∼4 cm, while its temperature was kept constant at 30 ◦ C during deposition. Iden-

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Fig. 2. SEM images of crystalline powders showing characteristic morphologies of the two component crystalline phases: OCP and CaAL·H2 O.

Fig. 3. X-ray diffraction patterns (a) and FT-IR spectra (b) of thin coatings obtained after MAPLE transfer of different crystalline powders.

tical MAPLE conditions were applied for the deposition of AL8 and AL20 powders. The deposited coatings were labeled cOCP, cAL8, and cAL20. XRD measurements on the coatings were performed using a PANalytical X’Pert PRO powder diffractometer equipped with a fast X’Celerator detector. CuK␣ radiation was used ( = 0.154 nm, 40 mA, 40 kV). The 2 range was investigated from 3 to 12◦ (2) with a step size of 0.05◦ and time/step of 3000 s. FTIR spectrometry study on coatings was conducted by a Shimadzu 8400S instrument in the 1500–400 cm−1 range, with a resolution of 4 cm−1 and a total of 50 scans/experiment. The coating thickness was evaluated by profilometry using a stylus profiler XP2 from Ambios Technology. For this investigation, half of Ti substrate was shielded with tape during deposition in order to evaluate the level difference between top of the coating and uncoated Ti. A continuous scanning with 10 ␮m s−1 speed was used for depth evaluation from the top to the bottom of the coatings (Ti uncoated surface after tape removal). For statistics, we have performed measurements of three identical samples. Morphological investigations of thin films were carried out by SEM with a Philips XL20 instrument operating at 15 kV. The samples were sputter coated with Au before examination. For AFM imaging a Veeco Nanoscope 3D instrument was used. The samples were analyzed in tapping mode using a E scanner (maximum scan size 15 ␮m) and phosphorus (n) doped

silicon probes (spring constant 20–80 N/m; resonance frequency 250–290 kHz; nominal tip radius <10 nm). Roughness parameters, namely arithmetic mean roughness (Ra), root-square roughness (Rq), and the vertical distance between the highest and lowest points within the evaluation length (Rt), were recorded. Release of Calcium from coatings was performed in physiological solution (NaCl 0.9%) up to 7 days using a Thermo Instrument S series atomic absorption spectrometer. The volume of the release solution utilized for each sample at each experimental time was 2 ml. Results from this analysis represent the mean value of three different determinations. Cell experiments were carried out on coatings deposited on Ti substrates and sterilized by gamma-rays (Cobalt-60) at a dose of 25 kGy.

2.3. In vitro co-cultures Peripheral human blood was obtained from osteoporotic adult volunteers (Rizzoli Orthopedic Institute Ethic Committee approval n. 33154, 12/09), who presented a recent diagnosis by densitometry and who have not yet started osteoporosis therapy. Density gradient centrifugation was used to separate the mononuclear cells from the other elements of blood. Briefly, a volume of peripheral blood was diluted 1:1 with pre-warmed PBS, carefully layered on an equal volume of Histopaque 1077, and was centrifuged

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with 600 rpm at room temperature for 30 min. After centrifugation, the mononuclear cells accumulated at the interface between PBS and Histopaque were collected and washed twice in PBS. The pellet was resuspended in 1 ml of culture medium (DMEM, basal medium + 10% FBS, 1% antibiotics); cell number and viability were checked with trypan blue dye exclusion test for experiment. Monocytes at a concentration of 5 × 105 cells/ml were differentiated in 12-well plates and incubated at 37 ◦ C in a humidified 95%air/5%CO2 atmosphere (standard condition). After 24 h the nonadherent monocytes were washed off to dispose the culture of contaminating lymphocytes, so that only the adherent monocytes were used for culture. DMEM additioned with macrophage colonystimulating factor (MCSF, 25 ng/ml) and receptor activator for ␬B factor ligand (RANKL, 30 ng/ml) were used for osteoclast differentiation. Primary human stem cells were cultured in DMEM at standard conditions and at 90% confluence cells were subcultured in osteogenic differentiating medium (DMEM additioned with ␤glicerophosphate 10−2 M, dexamethasone 10−4 M, and ascorbic acid 50 ␮g/ml) and then seeded (3 × 105 cells/cm2 ) onto 6 samples of each material: Ti as reference, Ti coated with OCP, AL8 and AL20 (cOCP, cAL8, cAL20). After 1 week experimental biomaterials with osteoblasts (OB) were transferred in the same wells where osteoclasts (OC) were grown as a crown in the bottom, to be co-cultured up to 14 days. Both cell types were cultured in osteoblast:osteoclast differentiation medium. Single cultures of osteoblasts (CTR OB) and osteoclasts (CTR OC), and co-cultures obtained by seeding OB in transwell inserts 0.4-␮m pore size (Millipore, Ireland) and OC in the bottom of the same well (CTR Co) were also prepared as control without materials. 2.4. Cell proliferation OC and OB proliferation and viability at the end of the experimental time were evaluated by WST1 colorimetric reagent test (Roche Diagnostics GmbH, Manheim, Germany). This assay is based on the reduction of tetrazolium salt to a soluble formazan salt

by a reductase of the mitochondrial respiratory chain, active only in viable cells. 100 ␮l of WST1 solution and 900 ␮l 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 directly correlated with the cell number. Adhesion of OB to the surface of materials was assessed by Phallodin staining. Briefly, samples 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 (Sigma–Aldrich, Steinheim, Germany) 1:100 in PBS was added for 30 min at 37 ◦ C. After washing, samples were examined by fluorescence microscope and images taken from the edge or the centre of all samples were recorded. 2.5. Osteoclastogenesis TRAP-staining was performed to assess osteoclast differentiation according to manufacturer’s instructions (SIGMA, Buchs, Switzerland). The positive cells developed red colour of different intensity. The number of TRAP-positive multinucleated cells (three or more nuclei for each cell) was counted under the microscope using a semiautomatic software (NIS-Elements AR 4.30.01) and results are given as percentage of CTR OC considered as 100%. 2.6. Osteoclast and osteoblast activity At the end of the experimental time the supernatant was collected from all wells and centrifuged to remove particulates, if any. Aliquots were dispensed in Eppendorf tubes for storage at −70 ◦ C and assayed for Cathepsin K (CTSK elisa kit, Cusabio Biotech, Wuhan, China), Alkaline Phosphatase (ALP, immunoassay kit, USCN Life Science, Wuhan, China), Type I Pro-Collagen (COLL1, immunoassay kit, USCN), Osteoprotegerin (OPG, enzyme

Fig. 4. SEM images of coatings displaying the characteristic morphologies of the two component crystalline phases – OCP and CaAL·H2 O – after MAPLE transfer.

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After having verified normal distribution and homogeneity of variance, a one-way ANOVA was done for comparison between groups. Finally, post hoc multiple comparison tests were performed to detect significant differences among experimental groups and controls. Pearson test was performed to detect correlation between data. 3. Results and discussion 3.1. Characterization of the crystalline powders

Fig. 5. Effect of cOCP and cAL on cell Proliferation of OB, and OC, after 14 days of coculture compared to Ti as reference material. Proliferation of single and co-culture for CTR groups. The Scheffé’s post hoc multiple comparison tests were performed to detect significant differences between groups (*p < 0.05; ** p < 0.005; *** p < 0.0001). OC: **Ti vs cAL8, cAL20, ***cOCP vs cAL8, cAL20.

As previously reported, immersion of OCP in AL solution provokes precipitation of crystalline calcium alendronate, thanks to AL recruitment of Ca ions from OCP [17]. CaAL• H2 O amount in the composite materials can be tailored by varying AL concentration in solution [17,26]. The reaction is completed in three days and the relative content of CaAL• H2 O in the final solid samples amounts to 48 wt% for AL8 (the remaining 52 wt% is still OCP) and to 100 wt% for AL20. In agreement, the XRD patterns of AL8 show the presence of the characteristic peaks of both OCP and CaAL• H2 O, which is the sole crystalline phase present in AL20 (Fig. 1a) [17]. Similarly, SEM images show that AL20 is constituted just of tiny, long rod-like crystals characteristic of CaAL• H2 O, whereas AL8 exhibits also the presence of big plate-like crystals characteristic of OCP (Fig. 2). The FTIR absorption spectra shown in Fig. 1b confirm the different composition of the samples. The spectrum of pure OCP displays the absorption bands characteristic of PO4 stretching and bending modes, respectively in the regions 1100–1000 cm−1 and 630–450 cm−1 , besides the absorption bands between 1300 and 1100 cm−1 , and 960 and 840 cm−1 , due to HPO4 stretching [27]. We note that the IR spectrum of AL20 is similar to that reported for sodium alendronate and for calcium alendronate monohydrate [28,29]. In particular, the relevant absorption bands at 1135 and 1094 cm−1 have been associated to O P O and C C O bond stretches, whereas those at 940–916 cm−1 are connected only to motion of O H O and H O P, while the 613–580 cm−1 absorption bands are related to modes of the phosphate group [28]. The absorption bands present in the FTIR spectrum of AL8 clearly indicate the contribution of both OCP and AL. 3.2. Structural and morphological characterization of the coatings

Immunoassay kit, Boster Biological Technology, Ca, USA), and Receptor Activator for Nuclear factor kB Ligand (RANKL, enzyme Immunoassay kit, Boster), Interleukin-6 (IL6, enzyme Immunoassay kit, Boster). Co-culture medium additioned with RANKL was evaluated and subtracted from RANKL samples values. 2.7. Osteoblast morphology At the end of the experiment, samples of each material were prepared for 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 before examination with a Philips XL-20 instrument operating at 15 kV. 2.8. Statistical analysis Statistical evaluation of data was performed using the software package SPSS/PC+ StatisticsTM 10.1 (SPSS Inc., Chicago, IL USA). The presented results are the mean of six values. Data are reported as mean ± standard deviations (SD) at a significance level of p < 0.05.

The XRD patterns of the thin films are consistent with those of the powder samples utilized for their deposition (Fig. 3a). At low angle, the X-ray diffraction pattern of cOCP thin film presents just the characteristic peak of OCP at 4.7◦ 2, whereas the XRD pattern of cAL20 shows just a peak at 9.1◦ 2, in agreement with the presence of CaAL• H2 O as the unique crystalline phase. The contribution of both crystalline phases, OCP and CaAL• H2 O, can be discriminated in the XRD pattern of cAL8 (Fig. 3a). Similarly, the crystalline phases present in the different thin films can be identified also by FTIR absorption spectra (Fig. 3b), even if the absorption bands are generally less resolved with respect to those shown in the spectra of the corresponding powder samples (see Fig. 1b). The results of thickness measurements indicate no significant differences between cOCP, cAL8 and cAL20, which display mean values approaching 100 nm. It was previously shown that the topography of layers so thin as 100 nm do not affect cellular behavior [30]. Moreover, the roughness parameters, Ra, Rq and Rt, evaluated by AFM analysis (Figure S3) are quite similar for the different samples. Average values were: Ra = 0.211 ± 0.011 ␮m, Rq = 0.170 ± 0.007 ␮m, Rt = 1.288 ± 0.010 ␮m. SEM images of the cOCP thin films display a quite homogeneous surface, as visible from Fig. 4. In agreement with previous

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Fig. 6. Fluorescence detection of Phallodin staining showing actin filaments of OB adhered to material surface. Images on the bottom were taken on the center of the samples, whereas those on the top are of OB grown on the edge of samples (10×).

Fig. 7. SEM micrographs of osteoblasts after 14 days of culture.

data on OCP thin films deposited by MAPLE [31], the presence of crystal fragments, together with cauliflower-like aggregates and droplets, is evidently visible at higher magnification. Much smaller rod-like crystals, clearly discernible from the big plate-like crystals of OCP, can be appreciated in SEM images of cAL8 and cAL20 films. These small crystals, which display the characteristic morphology of CaAL• H2 O [17], are the only ones which can be distinguished in SEM images of cAL20 thin films. Degradation of the coatings has been investigated through determination of calcium release in physiological solution. The results are in agreement with the different solubility of CaAL• H2 O and OCP [32,33], and indicate that Ca release from cAL20 is about 14 mg/L after 6 h and it increases up to about 18 mg/l at 2 days, after which it remains constant. Ca release from cOCP is much lower, whereas the amounts released from cAL8 assume intermediate values (Fig. S4). 3.3. In vitro study OB derived from stem cells and OC derived from monocytes of osteoporotic subjects were used in the present study in attempt to mimic in vitro the pathophysiological microenvironment and to evaluate the effects of calcium alendronate local administration. Results of proliferation tests of OB and OC co-cultured in differentiating medium onto Ti, cOCP, cAL8, cAL20 and CTRs are shown in Fig. 5. OB cultured on experimental materials, reference and CTR groups regularly proliferated as demonstrated by WST1 absorbance values and no viability differences were found among groups. The good level of viability on experimental samples was also demonstrated by actin fluorescent staining (Fig. 6), both on the center and

on the edge of samples: the center of sample surface was fully covered by a cell monolayer. The images taken on the edges, in which the cells were less confluent, allowed to verify cell morphology that did not differ among groups. Moreover, at 14 days OB appeared attached and well spread, and rich of filopodia on all the samples, as shown in the SEM images reported in Fig. 7. These results confirm that OB viability is supported on cOCP coating and that OB proliferation is not significantly inhibited by the presence of the BP, both when present together with OCP (cAL8) and when it is the only component of the coating (cAL20). On the contrary, osteoclastogenesis from OC precursors was widely affected by the presence of CaAL• H2 O in the coatings. OC on cAL8 and cAL20 groups showed significantly lower viability as compared to OC on CTRs, Ti, and cOCP groups (Fig. 5). The analysis of TRAP staining images allows to assess that OC regularly differentiate after 1 week of culture in CTR groups and onto both Ti and cOCP samples (Fig. S5). It is known that OC resorbing activity in osteoporosis is more active than normal, with consequent impaired balance between bone resorption and bone formation. Microenvironmental and systemic factors in osteoporosis strongly activate OC. The presence of the BP in the coatings dramatically reduced the number of differentiated OC. In fact, monocytes cultured onto cAL8 and cAL20 showed a significantly reduced percentage of multinucleated cells (25% and 11% respectively), in comparison to Ti (97%) and cOCP (102%). We previously demonstrated that the differentiation and activity of OC derived from monocytes precursor was greatly inhibited by CaAL• H2 O both alone and in combination with OCP [17]. The present study indicates that the presence of CaAL• H2 O into Ti coatings exhibits a similar effect on osteoporotic OC, as also stated by

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Fig. 8. Metabolic activity of OC and OB measured in cell culture supernatant after 14 days of co-culture with cOCP, cAL8, cAL20, and Ti as reference materials. Evaluation of single and co-culture for CTR groups are also presented. The T3 Dunnett post hoc multiple comparison tests were performed to detect significant differences between groups (*p < 0.05; **p < 0.005; ***p < 0.0001) CTSK: ***Ti vs cAL8, cAL20; **cOCP vs cAL8, cAL20; *CTR OC Co vs CTR OC ALP: *Ti vs cOCP, cAL8, cAL20; *cAL8 vs cAL20; *CTR OB Co vs CTR OB COLL1: ***cAL8 vs Ti; **cAL8 vs cOCP, cAL20; **cAL20 vs Ti OPG/RANKL ratio: *cOCP vs Ti; **cAL8 vs Ti; *cAL8 vs cOCP; ***cAL20 vs Ti; **cAL20 vs cOCP; *CTR OB Co vs CTR OB IL6: *cOCP vs Ti, cAL20; **cOCP vs cAL8; **CRT OB Co vs CTR OB.

the dosage of CTSK, a marker of OC differentiation (Fig. 8): OC cultured in the presence of cAL8 and cAL20 showed significantly lower values of CTSK when compared to Ti and cOCP. These data strictly correlate with WST1 values (Pearson test: direct correlation 0.985, p < 0.0005) demonstrating that CaAL• H2 O influences both cell proliferation and differentiation. Significant difference was also found between CTR OC and CTR OC Co, as shown in Fig. 8. The activity of co-cultured OB is summarized in Fig. 8. ALP and COLL1 were chosen as representative markers of OB differentiation. ALP activity (Fig. 8) was significantly enhanced in cAL8 group in comparison with cAL20 and Ti. COLL1 production (Fig. 8) was higher onto both AL8 and AL20 groups, showing a statistical difference when compared to Ti (vs cAL8, cAL20) and cOCP (vs cAL8). As shown in Fig. 8, co-culture influenced significantly ALP activity, but did not affect COLL1 synthesis. OPG and RANKL are deeply involved in the regulation of osteoclastogenesis, with an outcome related to their ratio. In particular, high levels of OPG/RANKL ratio contribute to down-regulate OC differentiation. Fig. 8 reports the results of OPG/RANKL ratio. Significant differences among groups are the results of significantly lower RANKL concentration in both BP containing groups, while no differences were found in OPG level. On the contrary, in CTR groups the differences were due to OPG level. These results indicate that OPG synthesis was stimulated by the presence of OC, but was not altered by materials. OPG/RANKL ratio was also inversely correlated with WST1 and CTSK OC values (Pearson test: inverse correlation −0.701, p = 0.002 and −0.843, p = 0.0005 respectively). IL6, a cytokine playing an important role in inflammation, is a bone resorption promoting factor, favoring OC differentiation. It is known to be up-regulated in estrogen deficiency, as in postmenopausal osteoporosis. Other authors showed contradictory

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results on the effects of treatment with AL on IL6 production in normal human OB culture [34,35]. Our results obtained in co-culture model demonstrate that CaAL• H2 O is able to decrease IL6 synthesis: IL6 high level found in CTR OB Co was extremely elevated with respect to to CTR OB, but it was significantly reduced in cAL8 (p < 0.005) and cAL20 (p < 0.05) groups when compared to cOCP (Fig. 8). It is important to underline that the results obtained on CTR groups (Fig. 8) confirm significant differences between single culture and co-culture models, in accordance with reports in literature [36,37]. CSTK production, ALP activity, OPG/RANKL ratio, and IL6 stimulation showed very low values in single OC and OB culture when compared to co-cultures. Significant differences detected between CTR OB/CTR OC and CTR Co show that co-culture deeply influences OB and OC metabolism, which is very differently activated when cells were cultured together. Cells recruited from hematopoietic precursors interact with direct and indirect communication through cytokines, growth factors and other molecules secreted in cell microenvironment, and differentiate to complete the phases of bone remodeling. The interplay of OB and OC activity in the balance of remodeling cycle depends on many correlated factors, not last an inflammatory environment that is particularly relevant in osteoporosis [38]. Our study confirms that the co-culture model of OB and OC of osteoporotic origin is suitable for the study of functionalized biomaterials and coatings, and that the effects of in vivo therapeutical local activity of the bisphosphonate may be partially reproduced by in vitro models. Data suggest that the presence of CaAL• H2 O in the coatings is relevant to stimulate OB activity and to inhibit OC differentiation through different pathway. Moreover, the co-presence of OCP further improves OB differentiation. 4. Conclusions MAPLE technique has been successfully applied to synthesize thin films of calcium alendronate monohydrate, octacalcium phosphate, as well as CaAL• H2 O/OCP composite on Titanium substrates. The presence of calcium alendronate in the coatings dramatically inhibits proliferation, differentiation and activity of osteoporotic osteoclast. On the contrary, the bisphosphonate does not affect osteoblast viability, and it promotes their activity. Osteoblast differentiation is further enhanced when the coating is a composite of CaAL• H2 O and OCP, most likely thanks to the good bioactivity of OCP. The data demonstrate that the peculiar characteristics of MAPLE allow to develop implant coatings, which can provide not only a suitable interface for bone growth but also offer the availability of drugs able to prevent undesirable excessive bone resorption. Acknowledgements The authors are grateful for the financial support from the University of Bologna, Rizzoli Orthopaedic Institute and funds 5 × 1000 year 2012 (In vitro and in vivo biocompatibility and bioactivity of biomaterials and scaffolds for bone tissue). Romanian authors acknowledge with thanks the financial support of UEFISCDI under the contracts 19 RO-FR/2014 and PCCA 244/2014. Ms. Iuliana Urzica is gratefully acknowledged for help with profilometry measurements. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.09. 044.

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