Biomaterials 30 (2009) 61686177

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Biofunctional alendronateHydroxyapatite thin lms deposited by Matrix

Assisted Pulsed Laser Evaporation
Adriana Bigi a, *, Elisa Boanini a, Chiara Capuccini a, Milena Fini b, Ion N. Mihailescu c, Carmen Ristoscu c,
Felix Sima c, Paola Torricelli b
a
b
c

Department of Chemistry G. Ciamician, University of Bologna, via Selmi 2, 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

a b s t r a c t

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

We applied Matrix Assisted Pulsed Laser Evaporation (MAPLE) in order to synthesize alendronatehydroxyapatite thin lms on titanium substrates. Alendronate-hydroxyapatite composite nanocrystals
with 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 for
MAPLE 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 good
crystallinity, which slightly decreases with the increase of alendronate content, and exhibited a porouslike structure. Osteoblast-like MG63 cells and human osteoclasts were cultured on the thin lms up to 14
days. In the presence of alendronate, MG63 cells displayed a normal morphology, increased proliferation
and higher values of differentiation parameters, namely type I collagen, osteocalcin, and osteoprotegerin/
TNF-related activation-induced cytokine receptor ratio. In contrast, osteoclasts showed signicantly
reduced proliferation, and increased level of Caspase 3. Moreover, the coatings synthesized from
hydroxyapatite at relatively high bisphosphonate content (7.1% wt) displayed a reduced production of
Tumour Necrosis Factor alpha (TNF-a) and Interleukin 6 (IL-6), suggesting a down-regulatory role of
alendronate on the inammatory reaction. The successful deposition of alendronate modied
hydroxyapatite thin lms yields coatings with enhanced bioactivity, able to promote osteoblast differentiation and to inhibit osteoclast proliferation.
2009 Elsevier Ltd. All rights reserved.

Keywords:
Bisphosphonate
Alendronate-doped hydroxyapatite lms
MAPLE
Osteoblast
Osteoclast

1. Introduction
Recently, we have successfully prepared HA nanocrystals modied
with alendronate, a potent bisphosphonate [1]. Bisphosphonates (BPs)
are synthetic pyrophosphate analogs, in which the POP group is
replaced by the PCP bridge. BPs are widely used for the management of specic disorders of bone metabolism, such as Paget bone
disease, osteoporosis, brous dysplasia, myeloma and bone metastases [24]. Nevertheless, negative effects of over suppression of bone
metabolism, such as osteonecrosis of the jaws, have been reported for
patients treated with BPs [5,6]. Under these circumstances, the
development of strategies for local administration of BPs becomes
mandatory. However, the great afnity of these compounds for
calcium ions hinders the direct synthesis of hybrid calcium phosphate

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

E-mail address: adriana.bigi@unibo.it (A. Bigi).
0142-9612/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2009.07.066

crystals because of the undesired formation of amorphous calcium

alendronate. Our approach, a slight modication of a classical method
of synthesis of HA in aqueous solution, allows to prepare composite
hydroxyapatite nanocrystals with different alendronate content, up to
7.1 wt% [1]. In-vitro tests demonstrated that alendronate is able to
promote osteoblast activation and extra-cellular matrix mineralization, and to inhibit osteoclast proliferation even when incorporated in
the composite nanocrystals [7]. This approach for synthesizing
alendronate-containing nanocrystals is therefore a relevant tool in
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 Titanium substrates in order to synthesize suitable coatings combining
the bioactivity of HA with the local availability of Alendronate. To
reach this goal, we extended Matrix Assisted Pulsed Laser Evaporation (MAPLE) to the deposition of Alendronate-doped HA coatings.
MAPLE was developed as an alternative to Pulsed Laser Deposition
(PLD) [8,9], necessary for delicate (organic or biologic) material

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

transfer. MAPLE essentially differs from PLD by target preparation,

laser-material interaction and transfer mechanisms. It provides
a more gentle mechanism for transferring different compounds,
including large molecular weight species, and it is expected to
ensure an improved stoichiometric transfer, a more accurate thickness control and a higher uniformity of obtained coatings.
MAPLE has been successfully applied for transferring of organic
polymer [10,11] and biomolecules, bovine serum albumin [12], silk
protein [13,14] brinogen blood proteins [15], urease [16], and
recently a novel biopolymer-HA composite [17].
This is the rst attempt to synthesize hydroxyapatite and
alendronate-doped hydroxyapatite thin coatings by MAPLE technique. Previous studies on BPs-enriched HA coatings were based on
bisphosponate absorption from solution or grafting onto hydroxyapatite coatings [18,19]. At variance, we have directly deposited
alendronate-modied HA thin lms on titanium substrates. The
biological functionality of the obtained layers was tested by
monitoring 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 by
dropwise addition of (NH4)2HPO4 to Ca(NO3)2 4H2O solution of pH adjusted to 10
with NH4OH. For the syntheses of alendronatehydroxyapatite nanocrystals, the
alendronate solution was dropped under stirring in the reaction vessel immediately
after completion of the phosphate addition. The precipitate was maintained in

contact 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. The

product was dried at 37 C overnight. Three series of samples (HA, HA-AL7, and HAAL28) 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). Data


were 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 the
compounds grown in the presence of different alendronate concentrations were
consisting of hydroxyapatite as the sole crystalline phase [1,7]. Alendronate content
was determined spectrophotometrically via complex formation with Fe(III) ions
using a Varian Cary50Bio instrument (l 290 nm) [20]. Alendronate content of HAAL7 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 were
mechanically polished and subsequently submitted to chemical etching to get an
extended active surface [21].
For the preparation of the target, 0.25 g of powder sample suspended in 5 ml
distilled water were carefully stirred, homogenized and frozen at 77 K in liquid
nitrogen. After freezing, the target was mounted inside the reaction chamber and
rotated during experiments to avoid overheating and drilling by the multipulse laser
irradiation. The depositions were performed in a dynamic pressure of 101 Torr. The
substrate was placed parallel to the target at a separation distance of 4 cm and

maintained at 30 C during deposition. A pulsed KrF* laser source (l 248 nm,
sFWHM z25 ns) operating at 10 Hz was used for the irradiation of the targets. 20,000
subsequent pulses were applied at an incident laser uence of 0.75 J cm2 for the
synthesis of each structure. During the application of the multipulse laser irradiation, the target was continuously cooled down with liquid nitrogen.
The thin lms proved particular adherence to Ti substrates as demonstrated by
the absence of any delamination or other visible morphological defects after transfer
between laboratories, till the completion of the in-vitro tests.
XRD measurements were performed on the coatings using a PANalytical XCelerator powder diffractometer. CuKa (l 0.154 nm) radiation was 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 performed
using a Philips XL-20 Scanning Electron Microscope operating at 15 kV. The samples
were sputter-coated with gold 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 mm) and phosphorus (n) doped silicon probes (spring constant 2080 N/m; resonance frequency
250290 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.
Cell experiments were carried out on coatings deposited on Ti substrates sterilized by gamma-rays (Cobalt-60) at a dose of 25 kGy.

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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/ml
streptomycin). Cells were detached from culture asks 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 eight sterile samples for each material: uncoated Titanium (Ti) as reference, HA coated Ti (HA), HA-AL7 coated Ti (HA-AL7), HA-AL28
coated Ti (HA-AL28). The same concentration of cells was seeded in empty wells
for control experiment (CTR). The medium was changed with DMEM supplemented with b-Glycerophosphate (108 M) and Ascorbic acid (50 mg/ml) to activate

osteoblasts. 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 culture
medium was enriched with 1,25(OH)2D3 48 h before the end of each experimental
time (7 and 14 days).
2.4. Osteoblast adhesion, spreading, proliferation and toxicity
24 h after seeding, Phalloidin staining was performed to assess cell adhesion and
colonization of samples. The cultures were washed in PBS and xed in a solution of

4% formaldehyde in PBS for 15 min at 37 C. Then, the samples were permeabilized
with 0.5% Triton X-100 for 15 min, washed in PBS, and a FITC-conjugate phalloidin

solution (1:100 in PBS) was added for 30 min at 37 C. After washing, samples were
examined by uorescence microscope and the images evaluated by a computerized
image analysis system (Kontron KS 300 software, Kontron Electronic GmbH, Eching
bei Munchen, Germany).
Cell proliferation and viability (1, 7, and 14 days) was monitored by WST1 (WST1,
Roche Diagnostics GmbH, Manheim, Germany) colorimetric reagent test. The assay
is based on a tetrazolium salt that is reduced to a soluble formazan salt by
a reductase of the mitochondrial respiratory chain, active only in viable cells. 100 ml
of WST1 solution and 900 ml of medium (nal 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 quantied spectrophotometrically at 450 nm with a reference
wavelength of 640 nm. Results of WST1 are reported as optical density (OD) and
directly correlated with the cell number. 24 h after seeding, the supernatant was
assayed for Lactate Dehydrogenase (LDH, kinetic assay, Media Diagnostics, Forl`, I).
2.5. Osteoblast activity and differentiation
At the end of experimental time (7 or 14 days), 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 Alkaline Phosphatase activity (ALP, kinetic assay, Media Diagnostics, Forl`, I), Type I Collagen (CICP,
Prolagen-C enzyme Immunoassay kit, Metra Biosystem, CA, USA), Osteocalcin (OC,
enzyme Immunoassay kit, Bender MedSystems, Vienna, A), Osteoprotegerin (OPG,
enzyme Immunoassay kit, Bender MedSystems, Vienna, A), Receptor Activator for
Nuclear factor K B Ligand (RANKL, enzyme Immunoassay kit, Bender MedSystems,
Vienna, A), Interleukin 6, (IL-6, Quantikine IL-6 Immunoassay, R&D Systems, MN,
USA), Tumour Necrosis Factor-a (TNF-a, Immunoassay, R&D Systems, MN, USA), proMatrix Metalloproteinase-13 (MMP-13 Immunoassay, R&D Systems, MN, USA), at 7
and 14 days, Total Protein content (TP, Total Protein micro-Lowry kit, SIGMA) at 1, 7,
and 14 days. All measured concentrations and activities were normalized by TP to
take into account the differences in cell growth.
2.6. 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 xed

Fig. 1. Powder X-ray diffraction patterns of the thin lms deposited from: (a) HA, (b)
HA-AL7, (c) HA-AL28.

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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.
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.
2.7. Osteoclast culture
Peripheral human blood obtained from healthy adult volunteers was used for
osteoclast cultures. Density gradient centrifugation was used to separate the

mononuclear cells from the other elements of blood. Briey, a volume of peripheral
blood was diluted 1:1 with pre-warmed PBS and carefully layered on an equal
volume of Histopaque1077 in a 50 ml tube. The tube was centrifuged with 400 g at
room temperature for 30 min. After centrifugation, the mononuclear cells accumulated at the interface between PBS and Histopaque were collected and transferred to another tube. 10 ml of PBS were then added and the tube was centrifuged
with 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 count
cells in a Neubauer chamber. The cells were plated on thin slides ( 10 mm) of

Fig. 3. Phalloidin staining of culture after 24 h from seeding: (a) Ti, (b) HA, (c) HA-AL7, (d) HA-AL28. Bars 20 mm.

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

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

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

24 h

7 Days

14 Days

Group

Ti

Percentage of sample surface covered

by cells at 24 h from seeding

21.2  0.8 19.5  0.8 20.5  1.6 22.0  0.2*

WST1
LDH (U/L)
ALP (mmol pNPP/min)
OC (ng/ml)
CICP (ng/ml)
OPG/RANKL ratio
IL-6 (pg/ml)
TNF-a (pg/ml)
MMP-13 (pg/ml)

0.817  0.065
1.40  0.49

1.085  0.103
/
1.51  0.73
1.2  0.4
8.7  0.7
425  22
0.61  0.05
3.5  1.5
0.49  0.01

3.085  0.121
/
2.59  0.75
1.9  0.3
12.2  0.9
624  62
0.63  0.11
1.52  0.04
0.72  0.13

HA

HA-AL7

HA-AL28

cortical bone (CTR) and samples of Ti, HA, HA-AL7, HA-AL28 in 24-wells culture plate

and 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 adherent
monocytes were used for culture and the medium was replaced with osteoclast
differentiation medium (DMEM 10% FBS 107 M PTH, 25 ng/ml M-CSF, 30 ng/ml
RANKL). 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 respecting
the manufacturers instructions (SIGMA, Buchs, Switzerland). Positive cells stain red
with varying intensity. For the measurement of resorbed area in the pit-assay, bone
slides with cultured cells were washed with PBS, incubated in 5% sodium hypochlorite 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, and

frozen at 80 C to be assayed for Caspase 3 (ELISA test, Bender Medsystems,
Wien, A). Supernatant was collected for the evaluation of Transforming Growth
Factor b1 (ELISA Quantikine TGF-b1 Immunoassay, R&D Systems, MN, USA). The
results were corrected for total protein amount.

/
/
/
/
/
/

reported as mean  standard deviations (SD) at a signicance level of p < 0.05. After
having veried normal distribution and homogeneity of variance, a one-way
ANOVA was done for comparison between groups. Finally, the Scheffes post hoc
multiple comparison tests were performed to detect signicant differences
between groups.

3. Results and discussion

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

2.8. Statistical analysis

3.1. Structural and morphological characterization of the thin lms

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 the triplicate values. Data are

Typical X-ray diffraction patterns of the thin lms are shown in

Fig. 1. All the patterns are consistent with the presence of

Fig. 4. (a) Proliferation of MG63 (WST1 tests) after 1, 7 and 14 days of culture on samples of Ti, HA, HA-AL7 and HA-AL28. Mean  sd, n 3. (* p < 0.05; ** p < 0.005;
*** p < 0.0001); 1 day: *HA-AL7 versus Ti, HA; *HA-AL28 versus HA; 7 days: ***HA versus Ti, HA-AL7, HA-AL28; 14 days: *HA versus HA-AL7, HA-AL28. (b) Lactate dehydrogenase
production by MG63 osteoblast-like cells on Ti, HA, HA-AL7 and HA-AL28 samples after 24 h from seeding. Mean  sd, n 3. (* p < 0.05; ** p < 0.005; *** p < 0.0001). No
signicant differences were detected.

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Fig. 5. Differentiation and synthetic activity of MG63 after 7 and 14 days of culture on samples of Ti, HA, HA-AL7 and HA-AL28. Mean  sd, n 3. (* p < 0.05; ** p < 0.005;
*** p < 0.0001): (a) ALP. 7 days: ns; 14 days: **HA, HA-AL28 versus Ti; *HA-AL7 versus Ti. (b) OC. 7 days: *HA-AL7 versus HA; *HA-AL28 versus Ti; **HA-AL28 versus HA; 14 days: ns.
(c) CICP. 7 days: ***HA versus Ti, HA-AL7, HA-AL28; 14 days: *HA-AL7, HA-AL28 versus HA. (d) OPG/RANKL ratio. 7 days: *HA-AL7 versus HA; *HA-AL28 versus Ti; **HA-AL28 versus
HA; 14 days: ***Ti versus HA, HA-AL7, HA-AL28.

hydroxyapatite as the sole crystalline phase. The slight increase of

the broadening of the diffraction peaks when increasing alendronate concentration is in agreement with the one observed on the

as-synthesized powders [1,7]. It is indicative for a modest decrease

of the length of the crystalline domains as the alendronate content
in the apatite nanocrystals increases up to 7.1%.

Fig. 6. SEM images of human osteoblasts MG63 on (a) Ti, (b) HA, (c) HAAL7, and (d) HAAL28 after 14 days of culturing. Bars 10 mm.

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

SEM images of the thin lms (Fig. 2ac) show a morphology

quite different from the granular surface characteristic of the
apatitic coatings deposited by PLD [8]. The lms exhibit a porouslike structure, with pores dimension of 24 mm, while only few
grains are visible. We mention that the peculiar morphology of the
thin lms could be characteristic to the deposition technique. At
variance with PLD, MAPLE uses a cryogenic composite target (a
dilute mixture of the material to be deposited). The incident laser
pulse initiates in this case two photothermal processes in the
matrix: the evaporation of the frozen composite target, and the
release of the material into the chamber. The solvent molecules
are evaporated and evacuated by the pumping system. The
material molecules gather sufcient kinetic energy through
collective collisions with the evaporating solvent molecules to be
transferred in gas phase to the substrate. Water evaporation takes
place during the transfer and it continues on the substrate, which
could explain the origin of the pores evidenced by SEM. The
morphology of the coatings does not show signicant differences

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depending on the alendronate content (Fig. 2ac). In good

agreement, the roughness parameters, Ra, Rq and Rt, evaluated by
AFM analysis are quite similar for the different coatings. Average
values were: Ra 0.321  0.037 mm, Rq 0.608  0.045 mm,
Rmax 2.105  0.095 mm. A typical AFM image is presented in
Fig. 2d.
3.2. Osteoblast adhesion, spreading, proliferation and toxicity
The biocompatibility of biomaterials is very closely related to
cell behaviour when in contact with them, being particularly connected to cell adhesion on their surface. Surface characteristics of
materials, as e.g. topography, chemistry or surface energy, play an
essential role in osteoblast adhesion on biomaterials. Thus, the
attachment, adhesion and spreading belong to the rst phase of
cell-material interaction. The quality of this rst phase will inuence the cell capacity to proliferate and to differentiate when in
contact with the implant [22,23]. Phalloidin stains actin laments

Fig. 7. Pro-degradation and pro-inammation products of MG63 after 7 and 14 days of culture on samples of Ti, HA, HA-AL7 and HA-AL28. Mean  sd, n 3. (* p < 0.05;
** p < 0.005; *** p < 0.0001): IL-6. 7 days: ns; 14 days: *HA-AL28 versus Ti. TNF-a. 7 days: *HA-AL28 versus HA; 14 days: *HA-AL28 versus Ti, HA, HA-AL7. MMP-13. 7 days: *HAAL28 versus HA, HA-AL7; 14 days: **Ti, HA-AL28 versus HA; * Ti, HA-AL28 versus HA-AL7.

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thereby characterizing cytoskeletal organization and cell spreading.

Phalloidin staining was performed to assess cell adhesion, cell
spreading and initial proliferation on different substrates. The
images analysis of Phalloidin staining 24 h after seeding did not
show differences in osteoblast morphology (Fig. 3ad). Indeed cells
adhered on all surfaces and exhibited their characteristic shape.
The area percentage covered by cells adhering onto the surface of
different biomaterials, evaluated by an image analysis system, is
presented in Table 1 as the mean of ten elds for each sample. The
value obtained for HA-AL28 group was signicantly higher than the
one for HA (p < 0.05), while no differences were observed among
other groups.

The osteoblast proliferation was assessed at 1, 7 and 14 days by

the WST1 test. The data in Fig. 4a showed that osteoblasts grew
regularly on all substrates when compared to control (cells on
culture plates without biomaterials, Table 2). Moreover, HA-AL7
and HA-AL28 groups were signicantly higher than the HA (1, 7 and
14 days) and Ti (1 and 7 days) ones.
The evaluation of cytotoxicity was performed by the LDH assay
after 24 h. LDH is a cytoplasmic enzyme present within all
mammalian cells. Plasma membrane is normally impermeable to
LDH and the enzyme is abnormally released into the extracellular
uid when the membrane is damaged. The release of LDH is
therefore a sensitive and accurate marker for measuring the

Fig. 8. (a) Osteoclasts proliferation after 7 and 14 days of culture on samples of Ti, HA, HA-AL7 and HA-AL28. Mean  sd, n 3. (* p < 0.05; ** p < 0.005; *** p < 0.0001). 7 days:
***HA-AL7 versus CTR, Ti, HA; *** HA-AL28 versus CTR, Ti, HA, HA.AL7; 14 days: * Ti versus CTR; ** Ti versus HA; HA-AL7 versus CTR; HA-AL28 versus HA; ***HA-AL7 versus Ti; HAAL28 versus CTR, Ti. (b) Caspase 3 values of osteoclasts culture for 14 days on samples of Ti, HA, HA-AL7 and HA-AL28. Mean  sd, n 3. (* p < 0.05; ** p < 0.005;
*** p < 0.0001). 7 days: ns; 14 days: * CRT versus Ti, HA; , HA-AL7 versus Ti, HA; **HA-AL7, HA-AL28 versus CTR (c) TGF-b1 production by osteoclasts after 7 and 14 days of culture
on samples of Ti, HA, HA-AL7 and HA-AL28. Mean  sd, n 3. (* p < 0.05; ** p < 0.005; *** p < 0.0001).7 days: ns; 14 days: * HA-AL7 versus Ti; HA-AL28 versus CTR, Ti, HA.

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

toxicity of biomaterials in in vitro biocompatibility studies [24]. The

results, normalized to TP amount, do not show any differences
among groups (Fig. 4b), indicating that none of the different
substrates stimulated a cytotoxic response by osteoblasts after 24 h.
3.3. Osteoblast activity and differentiation
Osteoblast activity and differentiation were evaluated through
measurements at 7 and 14 days on culture supernatant of the
following parameters: ALP and CICP as early differentiation
markers, OC as later mineralization marker [25], and OPG and
RANKL as index of bone formation/resorption balance during the
last stage of differentiation [26]. In fact, the in vitro differentiation of
osteoblasts is associated with the increase of ALP, the deposition of
collagen type I and the subsequent production of OC.
The evaluation of ALP activity showed no differences among
groups at 7 days, while at 14 days the Ti group presented signicantly lower values than the others (Fig. 5a). The production of CICP
was signicantly higher for both HA-AL7 and HA-AL28 groups as
compared to HA (7 and 14 days) and Ti (7 days) (Fig. 5c). Also, the
level of OC at 7 days was signicantly higher for both HA-AL7 and
HA-AL28 than for HA and Ti groups, even if at 14 days no differences where found among groups (Fig. 5b). According to these
results, osteoblasts show a higher rate of proliferation and earlier
differentiation 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 role
on the rate of osteoclastogenesis and the net outcome of bone
formation/resorption. Alendronate not only signicantly
improved the OPG production (compare in Fig. 5d HA-AL28 at 14
days with the other groups, p < 0.05), but also provoked a reduction of RANKL expression (compare HA-AL7 and HA-AL28 at 14
days with the other groups, p < 0.05). The OPG/RANKL ratio
(Fig. 5d) was signicantly higher in alendronate groups both at 7

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and 14 days, demonstrating that alendronate inuences osteoblast metabolism. It increased the release of soluble OPG relative
to RANKL and favoured the bone-forming event (inhibiting the
bone resorption). An increased OPG/RANKL ratio should therefore
favour the bone formation and contribute to successful
osteointegration.
SEM images of osteoblasts grown on the different materials for
14 days showed good cells attachment and spreading (Fig. 6ad). In
the presence of AL (Fig. 6c and d) the cells appear even more attened 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-inammatory
cytokine and growth factor. In fact, Il-6 has a major role in the
mediation of the inammatory and immune responses initiated by
infections or injuries. Moreover, an increase of its level is related to
an osteopenic state of bone tissue [27]. TNF-a is a pleiotropic
cytokine that plays a key role in both inammation and apoptosis
[28]. The results presented in Fig. 7a and b suggest a down-regulatory effect of alendronate upon osteoblasts production of both IL6 and TNF-a, in good agreement with the signicant reduction of
their levels observed at the highest AL concentration.
Finally, a matrix-metalloproteinase was tested to assess the
stimulation of degradative enzymes. Matrix Metallo-Proteinases
(MMPs) constitute a family of endopeptidases that function in
the breakdown of the extracellular matrix (ECM). They play an
important role in many normal physiological processes, such as
embryonic development, morphogenesis, reproduction and
tissue remodelling [29]. MMP-13 participates in cleavage of type I
collagen and bronectin. Therefore, MMP-13 is likely to play
a crucial role in the modulation of extracellular matrix degradation and cell-matrix interactions [30]. Our results (Fig. 7c)
showed that at 7 days HA-AL28 group reached the highest
signicant level, while at 14 days an important decrease was
detected. The initial high activity, followed by a rest, seems to

Fig. 9. Phalloidin staining of osteoclast culture at the end of experimental time: (a) Ti, (b) HA, (c) HA-AL7, (d) HA-AL28. Bars 50 mm.

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

indicate cell stimulation for remodelling, rather than degradation

of extracellular matrix.

black and white. The full colour images can be found in the on-line
version, at doi:10.1016/j.biomaterials.2009.07.066.

3.4. Osteoclast culture

References

Fig. 8ac show the proliferation, Caspase 3 and TGF-b1 production of osteoclasts when cultured on the different materials. The
presence of alendronate signicantly affected cell viability, apoptosis
and growth factor level. Both at 7 and 14 days, the WST1 values were
signicantly reduced in HA-AL7 and HA-AL28 groups, in respect with
the control or the other groups (Fig. 8a). The Caspase 3 plays a crucial
role in the cascade of apoptotic pathways, activating cleavage of
proteins critical for cell survival [31]. The data presented in Fig. 8b
show that at 14 days the HA-Al7 and HA-Al28 groups display
a signicantly higher level of Caspase 3 as compared to the other
groups. It can be inferred that the presence of bisphosphonate not
only negatively inuenced osteoclast proliferation and differentiation (in agreement with osteoclast WST1 and osteoblast OPG/RANKL
ratio results) but it even induced osteoclast apoptosis, as revealed by
Caspase 3 results [32].
It is known that TGF-b1 is a multifunctional regulator of differentiation and activity of both osteoblast and osteoclasts. Stimulated
osteoclasts release active TGF-b1 [33]. Active, resorbing osteoclasts
are capable of activating TGF-b1, which in turn attenuates further
bone resorption by impairing osteoclastogenesis and promotes
bone formation through chemotactic attraction and stimulation of
proliferation and differentiation of osteoblast [34]. Statistical
analysis of TGF-b1 results (Fig. 8c) showed that at 7 days there were
no differences among all group. Osteoclasts grown on HA-AL7 and
HA-AL28 showed the same activity of other groups without
Alendronate. These results suggests that in the rst days of contact
with biomaterials, osteoclasts were still active, while at 14 days
signicant lower values were found in both HA-AL7 and HA-AL28
groups, demonstrating that osteoclasts number and activity was
then reduced, as conrmed caspase 3 and proliferation results at 14
days. The analysis of the phalloidin staining performed after 14
days of osteoclast culture conrmed the signicant reduction of the
cell number on the alendronate containing coatings (Fig. 9).
4. Conclusions
Crystalline alendronate-doped hydroxyapatites with different
bisphosphonate content have been successfully deposited on Titanium substrate by MAPLE technique. The presence of alendronate
in the hydroxyapatite thin lms has an opposite effect on osteoclast
and on osteoblast cells. It inhibits osteoclast proliferation and
differentiation, and promotes their apoptosis. At variance, alendronate has a benecial inuence on osteoblast growth, viability and
earlier differentiation. The data demonstrate that it is possible to
use MAPLE to synthesize coatings coupling the bioactivity of HA
with the local availability of alendronate, and accordingly suitable
to promote bone formation and prevent bone resorption.
Acknowledgements
The authors acknowledge with thanks the partial support of this
research under the project New biomimetic calcium phosphate
coatings for metallic implants (mobility exchange in the 15th
Italian-Romanian Executive Programme of S&T Co-operation).
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