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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:
[email protected] (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
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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