ite

i

lognrtho

c

a r t i c l e i n f o

Article history:Received 26 May 2009Accepted 30 July 2009Available online 18 August 2009

Keywords:

metabolism, such as osteonecrosis of the jaws, have been reported forpatients treated with BPs [5,6]. Under these circumstances, thedevelopment of strategies for local administration of BPs becomesmandatory. However, the great afnity of these compounds forcalcium ions hinders the direct synthesis of hybrid calciumphosphate

view of the possible biological applications of these materials.In this study, we investigated the deposition of thin lms of HA

nanocrystals with different alendronate content directly on Tita-nium substrates in order to synthesize suitable coatings combiningthe bioactivity of HA with the local availability of Alendronate. Toreach this goal, we extended Matrix Assisted Pulsed Laser Evapo-ration (MAPLE) to thedepositionofAlendronate-dopedHAcoatings.MAPLE was developed as an alternative to Pulsed Laser Deposition(PLD) [8,9], necessary for delicate (organic or biologic) material

* Corresponding author. Tel.: 39 051 2099551; fax: 39 051 2099456.

Contents lists availab

Biomat

ev

Biomaterials 30 (2009) 61686177E-mail address: adriana.bigi@unibo.it (A. Bigi).1. Introduction

Recently,we have successfully preparedHAnanocrystalsmodiedwithalendronate, apotentbisphosphonate [1].Bisphosphonates (BPs)are synthetic pyrophosphate analogs, in which the POP group isreplaced by the PCP bridge. BPs are widely used for the manage-ment of specic disorders of bone metabolism, such as Paget bonedisease, osteoporosis, brous dysplasia, myeloma and bone metas-tases [24]. Nevertheless, negative effects of over suppression of bone

crystals because of the undesired formation of amorphous calciumalendronate. Our approach, a slightmodication of a classicalmethodof synthesis of HA in aqueous solution, allows to prepare compositehydroxyapatite nanocrystalswith different alendronate content, up to7.1 wt% [1]. In-vitro tests demonstrated that alendronate is able topromote osteoblast activation and extra-cellular matrix mineraliza-tion, and to inhibit osteoclast proliferation evenwhen incorporated inthe composite nanocrystals [7]. This approach for synthesizingalendronate-containing nanocrystals is therefore a relevant tool inBisphosphonateAlendronate-doped hydroxyapatite lmsMAPLEOsteoblastOsteoclast0142-9612/$ see front matter 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.07.066a b s t r a c t

We applied Matrix Assisted Pulsed Laser Evaporation (MAPLE) in order to synthesize alendronate-hydroxyapatite thin lms on titanium substrates. Alendronate-hydroxyapatite composite nanocrystalswith increasing bisphosphonate content (0, 3.9, 7.1% wt) were synthesized in aqueous medium. Then,they were suspended in deionised water, frozen at liquid nitrogen temperature and used as targets forMAPLE experiments. The depositions were conducted with a KrF* excimer laser source (l 248 nm,tFWHM 25 ns) in mild conditions of temperature and pressure. The obtained thin lms had a goodcrystallinity, which slightly decreases with the increase of alendronate content, and exhibited a porous-like structure. Osteoblast-like MG63 cells and human osteoclasts were cultured on the thin lms up to 14days. In the presence of alendronate, MG63 cells displayed a normal morphology, increased proliferationand higher values of differentiation parameters, namely type I collagen, osteocalcin, and osteoprotegerin/TNF-related activation-induced cytokine receptor ratio. In contrast, osteoclasts showed signicantlyreduced proliferation, and increased level of Caspase 3. Moreover, the coatings synthesized fromhydroxyapatite at relatively high bisphosphonate content (7.1% wt) displayed a reduced production ofTumour Necrosis Factor alpha (TNF-a) and Interleukin 6 (IL-6), suggesting a down-regulatory role ofalendronate on the inammatory reaction. The successful deposition of alendronate modiedhydroxyapatite thin lms yields coatings with enhanced bioactivity, able to promote osteoblast differ-entiation and to inhibit osteoclast proliferation.

2009 Elsevier Ltd. All rights reserved.National Institute for Lasers, Plasma and Radiation Physics, P. O. Box MG 36, 77125 Bucharest-Magurele, RomaniaBiofunctional alendronateHydroxyapatAssisted Pulsed Laser Evaporation

Adriana Bigi a,*, Elisa Boanini a, Chiara Capuccini a, MFelix Sima c, Paola Torricelli b

aDepartment of Chemistry G. Ciamician, University of Bologna, via Selmi 2, 40126 Bob Laboratory of Preclinical Surgical Studies, Research Institute Codivilla Putti Rizzoli O

journal homepage: www.elsAll rights reserved.thin lms deposited by Matrix

lena Fini b, Ion N. Mihailescu c, Carmen Ristoscu c,

a, Italypaedic Institute, Bologna, Italy

le at ScienceDirect

erials

ier .com/locate/biomater ia ls

2.3. 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 mg/mlstreptomycin). Cells were detached from culture asks by trypsinization, andcentrifuged; cell number and viability were checked with trypan blue dye exclu-sion test. MG-63 osteoblast-like cells were plated at a density of 2104 cells/ml in24-well plates containing eight sterile samples for each material: uncoated Tita-nium (Ti) as reference, HA coated Ti (HA), HA-AL7 coated Ti (HA-AL7), HA-AL28coated Ti (HA-AL28). The same concentration of cells was seeded in empty wellsfor control experiment (CTR). The medium was changed with DMEM supple-mented with b-Glycerophosphate (108 M) and Ascorbic acid (50 mg/ml) to activateosteoblasts. Plates were cultured up to 14 days in standard conditions, at 37

C

with 95% humidity and 5% CO2. For the production of osteocalcin the culturemediumwas enriched with 1,25(OH)2D3 48 h before the end of each experimentaltime (7 and 14 days).

2.4. Osteoblast adhesion, spreading, proliferation and toxicity

rials 30 (2009) 61686177 6169transfer. MAPLE essentially differs from PLD by target preparation,laser-material interaction and transfer mechanisms. It providesa more gentle mechanism for transferring different compounds,including large molecular weight species, and it is expected toensure an improved stoichiometric transfer, a more accurate thick-ness control and a higher uniformity of obtained coatings.

MAPLE has been successfully applied for transferring of organicpolymer [10,11] and biomolecules, bovine serum albumin [12], silkprotein [13,14] brinogen blood proteins [15], urease [16], andrecently a novel biopolymer-HA composite [17].

This is the rst attempt to synthesize hydroxyapatite andalendronate-doped hydroxyapatite thin coatings by MAPLE tech-nique. Previous studies on BPs-enriched HA coatings were based onbisphosponate absorption from solution or grafting onto hydroxy-apatite coatings [18,19]. At variance, we have directly depositedalendronate-modied HA thin lms on titanium substrates. Thebiological functionality of the obtained layers was tested bymonitoring osteoblast-like cells MG63 and human osteoclasts.

2. Materials and methods

2.1. Synthesis and characterization of HA and HA-AL nanocrystals

HA nanocrystals were grown as previously reported [1,7], in N2 atmosphere bydropwise addition of (NH4)2HPO4 to Ca(NO3)2 4H2O solution of pH adjusted to 10with NH4OH. For the syntheses of alendronatehydroxyapatite nanocrystals, thealendronate solution was dropped under stirring in the reaction vessel immediatelyafter completion of the phosphate addition. The precipitate was maintained incontact with the reaction solution for 5 h under stirring at 90

C, then centrifuged at

10,000 rpm for 10 min and repeatedly washed with CO2-free distilled water. Theproduct was dried at 37

C overnight. Three series of samples (HA, HA-AL7, and HA-

AL28) were synthesized using alendronate concentrations of 0, 7 and 28 mM.Powder X-ray diffraction patterns were recorded using a PANalytical XPert PRO

powder diffractometer. CuKa radiation was used (l 0.154 nm, 40 mA, 40 kV). Datawere obtained in the range of 2q from 10 to 60

(0.02

/step, 10 s/step).

The powder X-ray diffraction pattern of the solid products conrmed that thecompounds grown in the presence of different alendronate concentrations wereconsisting of hydroxyapatite as the sole crystalline phase [1,7]. Alendronate contentwas determined spectrophotometrically via complex formation with Fe(III) ionsusing a Varian Cary50Bio instrument (l 290 nm) [20]. Alendronate content of HA-AL7 and HA-AL28 was 3.9 and 7.1 wt%, respectively.

2.2. Synthesis and characterization of HA and HAAL coatings

Disk-shaped (12 mm diameter, 0.5 mm thick) grade 2 Ti substrates weremechanically polished and subsequently submitted to chemical etching to get anextended active surface [21].

For the preparation of the target, 0.25 g of powder sample suspended in 5 mldistilled water were carefully stirred, homogenized and frozen at 77 K in liquidnitrogen. After freezing, the target was mounted inside the reaction chamber androtated during experiments to avoid overheating and drilling by the multipulse laserirradiation. The depositions were performed in a dynamic pressure of 101 Torr. Thesubstrate was placed parallel to the target at a separation distance of 4 cm andmaintained at 30

C during deposition. A pulsed KrF* laser source (l 248 nm,

sFWHMz25 ns) operating at 10 Hz was used for the irradiation of the targets. 20,000subsequent pulses were applied at an incident laser uence of 0.75 J cm2 for thesynthesis of each structure. During the application of the multipulse laser irradia-tion, the target was continuously cooled down with liquid nitrogen.

The thin lms proved particular adherence to Ti substrates as demonstrated bythe absence of any delamination or other visible morphological defects after transferbetween laboratories, till the completion of the in-vitro tests.

XRD measurements were performed on the coatings using a PANalytical XCel-erator powder diffractometer. CuKa (l 0.154 nm) radiationwas used (40 mA, 40 kV).The 2q range was from 25

to 34

with a step size of 0.05

and time/step of 1000 sec.

Morphological investigations of the synthesized thin lms were performedusing a Philips XL-20 Scanning Electron Microscope operating at 15 kV. The sampleswere sputter-coated with gold before examination.

For AFM imaging a Veeco Nanoscope 3D instrument was used. The samples wereanalyzed in tapping mode using a E scanner (maximum scan size 15 mm) and phos-phorus (n) doped silicon probes (spring constant 2080 N/m; resonance frequency250290 kHz; nominal tip radius

Fig. 2. SEM micrographs of thin lms deposited from: (a) HA, (b) HA-AL7, (c) HA-AL28. Bars 2 mm. (d) AFM image of the surface of a thin lm deposited from HA.

A. Bigi et al. / Biomaterials 30 (2009) 616861776170in 2.5% glutaraldehyde, in pH 7.4 phosphate buffer 0.01 M for 1 h and dehydrated ina graded ethanol series. After a passage in hexamethyldisilazane, the samples wereair dried. The samples were sputter-coated with Pd prior to examination witha Philips XL-20 Scanning Electron Microscope.

2.7. Osteoclast culture

Peripheral human blood obtained from healthy adult volunteers was used for

osteoclast cultures. Density gradient centrifugation was used to separate the

Fig. 3. Phalloidin staining of culture after 24 h from seedingmononuclear cells from the other elements of blood. Briey, a volume of peripheralblood was diluted 1:1 with pre-warmed PBS and carefully layered on an equalvolume of Histopaque1077 in a 50 ml tube. The tube was centrifuged with 400 g atroom temperature for 30 min. After centrifugation, the mononuclear cells accu-mulated at the interface between PBS and Histopaque were collected and trans-ferred to another tube. 10 ml of PBS were then added and the tube was centrifugedwith 250 g for 10 min. The pellet was suspended in 1 ml of culture medium(DMEM 10% FBS). Trypan-blue method was used to assess viability and to countcells in a Neubauer chamber. The cells were plated on thin slides ( 10 mm) of: (a) Ti, (b) HA, (c) HA-AL7, (d) HA-AL28. Bars 20 mm.

cortical bone (CTR) and samples of Ti, HA, HA-AL7, HA-AL28 in 24-wells culture plateand incubated at 37

C in 5% CO2. After 24 h the non-adherent cells were washed off

to dispose the culture of contaminating lymphocytes. Accordingly, only the adherentmonocytes were used for culture and the medium was replaced with osteoclastdifferentiation medium (DMEM 10% FBS 107 M PTH, 25 ng/ml M-CSF, 30 ng/mlRANKL). Cells were cultivated for up to 14 days.

After 14 days TRAP-staining and the measure of resorbed area were performed.The TRAP-staining of cells cultured on CTR bone slides was done strictly respectingthe manufacturers instructions (SIGMA, Buchs, Switzerland). Positive cells stain redwith varying intensity. For the measurement of resorbed area in the pit-assay, boneslides with cultured cells were washed with PBS, incubated in 5% sodium hypo-chlorite for 10 min, washed twice with water and stained with 0.1% toluidine blue.The pits developed blue to purple colour.

On experimental samples WST1 test was performed at 7 and 14 days.Phalloidin staining was performed on samples at 14 days, as described above.For the measure of apoptosis, cells of each groups were collected, lysed, andfrozen at 80 C to be assayed for Caspase 3 (ELISA test, Bender Medsystems,Wien, A). Supernatant was collected for the evaluation of Transforming GrowthFactor b1 (ELISA Quantikine TGF-b1 Immunoassay, R&D Systems, MN, USA). Theresults were corrected for total protein amount.

2.8. 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 repeatedthree times and the results presented are the mean of the triplicate values. Data are

reported asmean standard deviations (SD) at a signicance level of p< 0.05. Afterhaving veried normal distribution and homogeneity of variance, a one-wayANOVA was done for comparison between groups. Finally, the Scheffes post hocmultiple comparison tests were performed to detect signicant differencesbetween groups.

3. Results and discussion

Matrix Assisted Pulsed Laser Evaporation has been successfullyemployed to deposit thin lms of HA powders at increasingalendronate content (0, 3.9, 7.1 wt%) on Ti substrates.

3.1. Structural and morphological characterization of the thin lms

Typical X-ray diffraction patterns of the thin lms are shown inFig. 1. All the patterns are consistent with the presence of

Table 1Measure of the surface covered by adhered cells on biomaterials after 24 h fromseeding by computer image analysis system. The results are given as percentage ofcell area measured in 4 elds of observation. t-test: *HA-AL28 versus HA (p< 0.05).

Group Ti HA HA-AL7 HA-AL28

Percentage of sample surface coveredby cells at 24 h from seeding

21.2 0.8 19.5 0.8 20.5 1.6 22.0 0.2*

Table 2Proliferation and synthetic activity of MG63 control group at 1, 7 and 14 days ofculture. Cells are grown on the polystyrene culture plate for control of experiment.

Experimental time 24 h 7 Days 14 Days

WST1 0.817 0.065 1.085 0.103 3.085 0.121LDH (U/L) 1.40 0.49 / /ALP (mmol pNPP/min) 1.51 0.73 2.59 0.75OC (ng/ml) / 1.2 0.4 1.9 0.3CICP (ng/ml) / 8.7 0.7 12.2 0.9OPG/RANKL ratio / 425 22 624 62IL-6 (pg/ml) / 0.61 0.05 0.63 0.11TNF-a (pg/ml) / 3.5 1.5 1.52 0.04MMP-13 (pg/ml) / 0.49 0.01 0.72 0.13

A. Bigi et al. / Biomaterials 30 (2009) 61686177 6171Fig. 4. (a) Proliferation of MG63 (WST1 tests) after 1, 7 and 14 days of culture on sam*** p< 0.0001); 1 day: *HA-AL7 versus Ti, HA; *HA-AL28 versus HA; 7 days: ***HA versus Tproduction by MG63 osteoblast-like cells on Ti, HA, HA-AL7 and HA-AL28 samples after 2signicant differences were detected.ples of Ti, HA, HA-AL7 and HA-AL28. Mean sd, n 3. (* p< 0.05; ** p< 0.005;i, HA-AL7, HA-AL28; 14 days: *HA versus HA-AL7, HA-AL28. (b) Lactate dehydrogenase4 h from seeding. Mean sd, n 3. (* p< 0.05; ** p< 0.005; *** p< 0.0001). No

n sai. (bA. (d

A. Bigi et al. / Biomaterials 30 (2009) 616861776172Fig. 5. Differentiation and synthetic activity of MG63 after 7 and 14 days of culture o*** p< 0.0001): (a) ALP. 7 days: ns; 14 days: **HA, HA-AL28 versus Ti; *HA-AL7 versus T(c) CICP. 7 days: ***HA versus Ti, HA-AL7, HA-AL28; 14 days: *HA-AL7, HA-AL28 versus Hhydroxyapatite as the sole crystalline phase. The slight increase ofthe broadening of the diffraction peaks when increasing alendro-nate concentration is in agreement with the one observed on the

HA; 14 days: ***Ti versus HA, HA-AL7, HA-AL28.

Fig. 6. SEM images of human osteoblasts MG63 on (a) Ti, (b) HA, (c) Hmples of Ti, HA, HA-AL7 and HA-AL28. Mean sd, n 3. (* p< 0.05; ** p< 0.005;) OC. 7 days: *HA-AL7 versus HA; *HA-AL28 versus Ti; **HA-AL28 versus HA; 14 days: ns.) OPG/RANKL ratio. 7 days: *HA-AL7 versus HA; *HA-AL28 versus Ti; **HA-AL28 versusas-synthesized powders [1,7]. It is indicative for a modest decreaseof the length of the crystalline domains as the alendronate contentin the apatite nanocrystals increases up to 7.1%.

AAL7, and (d) HAAL28 after 14 days of culturing. Bars 10 mm.

SEM images of the thin lms (Fig. 2ac) show a morphologyquite different from the granular surface characteristic of theapatitic coatings deposited by PLD [8]. The lms exhibit a porous-like structure, with pores dimension of 24 mm, while only fewgrains are visible. Wemention that the peculiar morphology of thethin lms could be characteristic to the deposition technique. Atvariance with PLD, MAPLE uses a cryogenic composite target (adilute mixture of the material to be deposited). The incident laserpulse initiates in this case two photothermal processes in thematrix: the evaporation of the frozen composite target, and therelease of the material into the chamber. The solvent moleculesare evaporated and evacuated by the pumping system. Thematerial molecules gather sufcient kinetic energy throughcollective collisions with the evaporating solvent molecules to betransferred in gas phase to the substrate. Water evaporation takesplace during the transfer and it continues on the substrate, whichcould explain the origin of the pores evidenced by SEM. Themorphology of the coatings does not show signicant differences

depending on the alendronate content (Fig. 2ac). In goodagreement, the roughness parameters, Ra, Rq and Rt, evaluated byAFM analysis are quite similar for the different coatings. Averagevalues were: Ra 0.3210.037 mm, Rq 0.608 0.045 mm,Rmax 2.105 0.095 mm. A typical AFM image is presented inFig. 2d.

3.2. Osteoblast adhesion, spreading, proliferation and toxicity

The biocompatibility of biomaterials is very closely related tocell behaviour when in contact with them, being particularly con-nected to cell adhesion on their surface. Surface characteristics ofmaterials, as e.g. topography, chemistry or surface energy, play anessential role in osteoblast adhesion on biomaterials. Thus, theattachment, adhesion and spreading belong to the rst phase ofcell-material interaction. The quality of this rst phase will inu-ence the cell capacity to proliferate and to differentiate when incontact with the implant [22,23]. Phalloidin stains actin laments

s of

A. Bigi et al. / Biomaterials 30 (2009) 61686177 6173Fig. 7. Pro-degradation and pro-inammation products of MG63 after 7 and 14 day

** p< 0.005; ***p< 0.0001): IL-6. 7 days: ns; 14 days: *HA-AL28 versus Ti. TNF-a. 7 daysAL28 versus HA, HA-AL7; 14 days: **Ti, HA-AL28 versus HA; * Ti, HA-AL28 versus HA-AL7.culture on samples of Ti, HA, HA-AL7 and HA-AL28. Mean sd, n 3. (* p< 0.05;

: *HA-AL28 versus HA; 14 days: *HA-AL28 versus Ti, HA, HA-AL7. MMP-13. 7 days: *HA-

thereby characterizing cytoskeletal organization and cell spreading.Phalloidin staining was performed to assess cell adhesion, cellspreading and initial proliferation on different substrates. Theimages analysis of Phalloidin staining 24 h after seeding did notshow differences in osteoblast morphology (Fig. 3ad). Indeed cellsadhered on all surfaces and exhibited their characteristic shape.The area percentage covered by cells adhering onto the surface ofdifferent biomaterials, evaluated by an image analysis system, ispresented in Table 1 as the mean of ten elds for each sample. Thevalue obtained for HA-AL28 groupwas signicantly higher than theone for HA (p< 0.05), while no differences were observed amongother groups.

The osteoblast proliferation was assessed at 1, 7 and 14 days bythe WST1 test. The data in Fig. 4a showed that osteoblasts grewregularly on all substrates when compared to control (cells onculture plates without biomaterials, Table 2). Moreover, HA-AL7and HA-AL28 groups were signicantly higher than the HA (1, 7 and14 days) and Ti (1 and 7 days) ones.

The evaluation of cytotoxicity was performed by the LDH assayafter 24 h. LDH is a cytoplasmic enzyme present within allmammalian cells. Plasma membrane is normally impermeable toLDH and the enzyme is abnormally released into the extracellularuid when the membrane is damaged. The release of LDH istherefore a sensitive and accurate marker for measuring the

-ALus Cple

A. Bigi et al. / Biomaterials 30 (2009) 616861776174Fig. 8. (a) Osteoclasts proliferation after 7 and 14 days of culture on samples of Ti, HA, HA***HA-AL7 versus CTR, Ti, HA; *** HA-AL28 versus CTR, Ti, HA, HA.AL7; 14 days: * Ti versAL28 versus CTR, Ti. (b) Caspase 3 values of osteoclasts culture for 14 days on sam

*** p< 0.0001). 7 days: ns; 14 days: * CRT versus Ti, HA; , HA-AL7 versus Ti, HA; **HA-AL7,on samples of Ti, HA, HA-AL7 and HA-AL28. Mean sd, n 3. (* p< 0.05; ** p< 0.005;7 and HA-AL28. Mean sd, n 3. (* p< 0.05; ** p< 0.005; ***p< 0.0001). 7 days:TR; ** Ti versus HA; HA-AL7 versus CTR; HA-AL28 versus HA; ***HA-AL7 versus Ti; HA-s of Ti, HA, HA-AL7 and HA-AL28. Mean sd, n 3. (* p< 0.05; ** p< 0.005;

HA-AL28 versus CTR (c) TGF-b1 production by osteoclasts after 7 and 14 days of culture*** p< 0.0001).7 days: ns; 14 days: * HA-AL7 versus Ti; HA-AL28 versus CTR, Ti, HA.

toxicity of biomaterials in in vitro biocompatibility studies [24]. Theresults, normalized to TP amount, do not show any differencesamong groups (Fig. 4b), indicating that none of the differentsubstrates stimulated a cytotoxic response by osteoblasts after 24 h.

3.3. Osteoblast activity and differentiation

Osteoblast activity and differentiation were evaluated throughmeasurements at 7 and 14 days on culture supernatant of thefollowing parameters: ALP and CICP as early differentiationmarkers, OC as later mineralization marker [25], and OPG andRANKL as index of bone formation/resorption balance during thelast stage of differentiation [26]. In fact, the in vitro differentiation ofosteoblasts is associated with the increase of ALP, the deposition ofcollagen type I and the subsequent production of OC.

The evaluation of ALP activity showed no differences amonggroups at 7 days, while at 14 days the Ti group presented signi-cantly lower values than the others (Fig. 5a). The production of CICPwas signicantly higher for both HA-AL7 and HA-AL28 groups ascompared to HA (7 and 14 days) and Ti (7 days) (Fig. 5c). Also, thelevel of OC at 7 days was signicantly higher for both HA-AL7 andHA-AL28 than for HA and Ti groups, even if at 14 days no differ-ences where found among groups (Fig. 5b). According to theseresults, osteoblasts show a higher rate of proliferation and earlierdifferentiation in the presence of alendronate.

OPG and RANKL are also involved in bone metabolism.Specically, the ratio of these factors is believed to play a key roleon the rate of osteoclastogenesis and the net outcome of boneformation/resorption. Alendronate not only signicantly

and 14 days, demonstrating that alendronate inuences osteo-blast metabolism. It increased the release of soluble OPG relativeto RANKL and favoured the bone-forming event (inhibiting thebone resorption). An increased OPG/RANKL ratio should thereforefavour the bone formation and contribute to successfulosteointegration.

SEM images of osteoblasts grown on the different materials for14 days showed good cells attachment and spreading (Fig. 6ad). Inthe presence of AL (Fig. 6c and d) the cells appear even more at-tened and display more lopodia than those grown on control HA(Fig. 6b) and on Ti (Fig. 6a).

Il-6 and TNF-a were chosen as indicative for pro-inammatorycytokine and growth factor. In fact, Il-6 has a major role in themediation of the inammatory and immune responses initiated byinfections or injuries. Moreover, an increase of its level is related toan osteopenic state of bone tissue [27]. TNF-a is a pleiotropiccytokine that plays a key role in both inammation and apoptosis[28]. The results presented in Fig. 7a and b suggest a down-regu-latory effect of alendronate upon osteoblasts production of both IL-6 and TNF-a, in good agreement with the signicant reduction oftheir levels observed at the highest AL concentration.

Finally, a matrix-metalloproteinase was tested to assess thestimulation of degradative enzymes. Matrix Metallo-Proteinases(MMPs) constitute a family of endopeptidases that function inthe breakdown of the extracellular matrix (ECM). They play animportant role in many normal physiological processes, such asembryonic development, morphogenesis, reproduction andtissue remodelling [29]. MMP-13 participates in cleavage of type Icollagen and bronectin. Therefore, MMP-13 is likely to play

A. Bigi et al. / Biomaterials 30 (2009) 61686177 6175improved the OPG production (compare in Fig. 5d HA-AL28 at 14days with the other groups, p< 0.05), but also provoked a reduc-tion of RANKL expression (compare HA-AL7 and HA-AL28 at 14days with the other groups, p< 0.05). The OPG/RANKL ratio(Fig. 5d) was signicantly higher in alendronate groups both at 7Fig. 9. Phalloidin staining of osteoclast culture at the end of experimea crucial role in the modulation of extracellular matrix degra-dation and cell-matrix interactions [30]. Our results (Fig. 7c)showed that at 7 days HA-AL28 group reached the highestsignicant level, while at 14 days an important decrease wasdetected. The initial high activity, followed by a rest, seems tontal time: (a) Ti, (b) HA, (c) HA-AL7, (d) HA-AL28. Bars 50 mm.

expression of osteoprotegerin and rank ligand and the support of osteoclastformation by stromal-osteoblast lineage cells is developmentally regulated.

rialsindicate cell stimulation for remodelling, rather than degradationof extracellular matrix.

3.4. Osteoclast culture

Fig. 8ac show the proliferation, Caspase 3 and TGF-b1 produc-tion of osteoclasts when cultured on the different materials. Thepresence of alendronate signicantly affected cell viability, apoptosisand growth factor level. Both at 7 and 14 days, theWST1 valuesweresignicantly reduced inHA-AL7 andHA-AL28groups, in respectwiththe control or the other groups (Fig. 8a). The Caspase 3 plays a crucialrole in the cascade of apoptotic pathways, activating cleavage ofproteins critical for cell survival [31]. The data presented in Fig. 8bshow that at 14 days the HA-Al7 and HA-Al28 groups displaya signicantly higher level of Caspase 3 as compared to the othergroups. It can be inferred that the presence of bisphosphonate notonly negatively inuenced osteoclast proliferation and differentia-tion (in agreementwith osteoclastWST1 and osteoblast OPG/RANKLratio results) but it even induced osteoclast apoptosis, as revealed byCaspase 3 results [32].

It is known that TGF-b1 is a multifunctional regulator of differ-entiation and activity of both osteoblast and osteoclasts. Stimulatedosteoclasts release active TGF-b1 [33]. Active, resorbing osteoclastsare capable of activating TGF-b1, which in turn attenuates furtherbone resorption by impairing osteoclastogenesis and promotesbone formation through chemotactic attraction and stimulation ofproliferation and differentiation of osteoblast [34]. Statisticalanalysis of TGF-b1 results (Fig. 8c) showed that at 7 days there wereno differences among all group. Osteoclasts grown on HA-AL7 andHA-AL28 showed the same activity of other groups withoutAlendronate. These results suggests that in the rst days of contactwith biomaterials, osteoclasts were still active, while at 14 dayssignicant lower values were found in both HA-AL7 and HA-AL28groups, demonstrating that osteoclasts number and activity wasthen reduced, as conrmed caspase 3 and proliferation results at 14days. The analysis of the phalloidin staining performed after 14days of osteoclast culture conrmed the signicant reduction of thecell number on the alendronate containing coatings (Fig. 9).

4. Conclusions

Crystalline alendronate-doped hydroxyapatites with differentbisphosphonate content have been successfully deposited on Tita-nium substrate by MAPLE technique. The presence of alendronatein the hydroxyapatite thin lms has an opposite effect on osteoclastand on osteoblast cells. It inhibits osteoclast proliferation anddifferentiation, and promotes their apoptosis. At variance, alendr-onate has a benecial inuence on osteoblast growth, viability andearlier differentiation. The data demonstrate that it is possible touse MAPLE to synthesize coatings coupling the bioactivity of HAwith the local availability of alendronate, and accordingly suitableto promote bone formation and prevent bone resorption.

Acknowledgements

The authors acknowledge with thanks the partial support of thisresearch under the project New biomimetic calcium phosphatecoatings for metallic implants (mobility exchange in the 15th

Italian-Romanian Executive Programme of S&T Co-operation).

Appendix

Figures with essential colour discrimination. Certain gures in

A. Bigi et al. / Biomate6176this article, in particular Figs. 2 and 9, are difcult to interpret inEndocrinology 2000;141:476876.[27] Kishimoto T, Akira S, Taga T. Interleukin-6 and its receptor: a paradigm for

cytokines. Science 1992;258(5082):5937.black and white. The full colour images can be found in the on-lineversion, at doi:10.1016/j.biomaterials.2009.07.066.

References

[1] Boanini E, Gazzano M, Rubini K, Bigi A. Composite nanocrystals provide newinsight on alendronate interaction with hydroxyapatite structure. Adv Mater2007;19:2499502.

[2] Fleisch H. Bisphosphonates in bone disease, from the laboratory to the patient.San Diego: Academic Press; 2000.

[3] Adami S. Bisphosphonate antifracture efcacy. Bone 2007;41:S815.[4] Chesnut III C, Skag A, Christiansen C, Recker R, Stakkestad JA, Hoiseth A, et al.

Effects of oral ibandronate administered daily or intermittently on fracturerisk in postmenopausal osteoporosis. J Bone Miner Res 2004;19:12419.

[5] Ott SM. Long-term safety of bisphosphonates. J Clin Endocrinol Metab2005;90:18979.

[6] Woo SB, Hellstein JW, Kalmar JR. Systematic review: bisphosphonates andosteonecrosis of the jaws. Ann Intern Med 2006;144:75361.

[7] Boanini E, Torricelli P, Gazzano M, Giardino R, Bigi A. Alendronatehydroxy-apatite nanocomposites and their interaction with osteoclasts and osteoblast-like cells. Biomaterials 2008;29:7906.

[8] Bigi A, Bracci B, Cuisinier F, Elkaim R, Fini M, Mayer I, et al. Human osteoblastresponse to pulsed laser deposited calcium phosphate coatings. Biomaterials2005;26:23819.

[9] Capuccini C, Torricelli P, Sima F, Boanini E, Ristoscu C, Bracci B, et al. Stron-tium-substituted hydroxyapatite coatings synthesized by pulsed-laser depo-sition: in vitro osteoblast and osteoclast response. Acta Biomater 2008;4:188593.

[10] Bubb DM, Wu PK, Horwitz JS, Callahan JH, Galicia M, Vertes A, et al. The effectof the matrix on lm properties in matrix-assisted pulsed laser evaporation.J Appl Phys 2002;91:20558.

[11] Toftmann B, Papantonakis MR, Auyeung RCY, Kim W, OMalley SM, Bubb DM,et al. UV and RIR matrix assisted pulsed laser deposition of organic MEH-PPVlms. Thin Solid Films 2004;453454:17781.

[12] Sagawa J, Nagare S, Senna M. Preparation and properties of bovine serumalbumin thin lms by pulsed laser deposition. Appl Surf Sci 2005;244:6114.

[13] Tsuboi Y, Goto M, Haya A. Pulsed laser deposition of silk protein: effect ofphotosensitized-ablation on the secondary structure in thin deposited lms.J Appl Phys 2001;89:791721.

[14] Taketani I, Nakayama S, Nagare S, Senna M. The secondary structure control ofsilk broin thin lms by post treatment. Appl Surf Sci 2005;24:6236.

[15] Patz T, Cristescu R, Narayan R, Menegazzo N, Mizaikoff B, Messersmith PB,et al. Laser deposition of brinogen blood proteins thin lms by matrixassisted pulsed laser evaporation. Appl Surf Sci 2005;248:4227.

[16] Gyorgy E, Sima F, Mihailescu IN, Smausz T, Megyeri G, Kekesi R, et al.Immobilization of urease by laser techniques: synthesis and application tourea biosensors. J Biomed Mater Res A 2008;89A:18691.

[17] Negroiu G, Piticescu RM, Chitanu GC, Mihailescu IN, Zdrentu L, Miroiu M.Biocompatibility evaluation of a novel hydroxyapatite-polymer coating formedical implants (in vitro tests). J Mater Sci: Mater Med 2008;19:153744.

[18] McLeod K, Anderson GI, Dutta NK, Smart RSC, Voelcker NH, Sekel R, et al.Adsorption of bisphosphonate onto hydroxyapatite using a novel co-precipi-tation technique for bone growth enhancement. J Biomed Mater Res A2006;79A:27181.

[19] Peter B, Pioletti DP, Laib S, Bujoli B, Pilet P, Janvier P, et al. Calcium phosphatedrug delivery system: inuence of local zoledronate release on bone implantosteointegration. Bone 2005;36:5260.

[20] Kuljanin J, Jankovic I, Nedeljkovic J, Prstojevic D, Marinkovic V. Spectropho-tometric determination of alendronate in pharmaceutical formulations viacomplex formation with Fe(III) ions. J Pharm Biomed Anal 2002;28:121520.

[21] Giordano C, Sandrini E, Del Curto B, Signorelli E, Rondelli G, Di Silvio L.Titanium for osteointegration: comparison between a novel biomimetictreatment and commercially exploited surfaces. J Appl Biomater Biomech2004;2:3544.

[22] Anselme K. Osteoblast adhesion on biomateials. Biomaterials 2000;21:66781.[23] Reyes CD, Petrie TA, Burns KL, Schwartz Z, Garca AJ. Biomolecular surface

coating to enhance orthopaedic tissue healing and integration. Biomaterials2007;28:322835.

[24] Rae T. Tissue culture techniques in biocompatibility testing. In: Williams DF,editor. Techniques of biocompatibility testing. Boca Raton: CRC Press; 1986.p. 8193.

[25] Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentia-tion interrelationship during progressive development of the osteoblastphenotype. Endocr Rev 1993;14:42442.

[26] Gori F, Hofbauer LC, Dunstan CR, Spelberg TC, Khosla S, Riggs BL. The

30 (2009) 61686177[28] MacEwan DJ. TNF ligands and receptors a matter of life and death. Br JPharmacol 2002;135:85575.

[29] NagaseH,Woessner Jr JF.Matrixmetalloproteinases. JBiolChem1999;274:214914.[30] Nakashima A, Tamura M. Regulation of matrix metalloproteinase-13 and

tissue inhibitor of matrix metalloproteinase-1 gene expression by WNT3A andbone morphogenetic protein-2 in osteoblastic differentiation. Front Biosci2006;11:166778.

[31] Wolf BB, Green DR. Suicidal tendencies: apoptotic cell death by caspase familyproteinase. J Biol Chem 1999;274:2004952.

[32] Hughes DE, Wright KR, Uy HL, Sasaki A, Yoneda T, Roodman GD, et al.Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo.J Bone Miner Res 1995;10:147887.

[33] Bonewald LF, Oreffo R0C, Seyedin S, Mundy GR. Isolated osteoclasts activatelatent TGF. J Bone Miner Res 1988;3:S98.

[34] Janssens K, ten Dijke P, Janssens S, Van Hul W. Transforming growth factor-b1to the bone. Endocr Rev 2005;26:74374.

A. Bigi et al. / Biomaterials 30 (2009) 61686177 6177

Biofunctional alendronate-Hydroxyapatite thin films deposited by Matrix Assisted Pulsed Laser EvaporationIntroductionMaterials and methodsSynthesis and characterization of HA and HA-AL nanocrystalsSynthesis and characterization of HA and HA-AL coatingsOsteoblast cultureOsteoblast adhesion, spreading, proliferation and toxicityOsteoblast activity and differentiationCell morphologyOsteoclast cultureStatistical analysis

Results and discussionStructural and morphological characterization of the thin filmsOsteoblast adhesion, spreading, proliferation and toxicityOsteoblast activity and differentiationOsteoclast culture

ConclusionsAcknowledgementsReferences