“Bone response to anodized zirconium implants. A preliminary in vivo approach”
Maria R. Katunar, Andrea Gomez Sanchez, Josefina Ballarre, Silvia Ceré
Division Corrosion-INTEMA, Universidad Nacional de Mar del Plata-CONICET, Juan B. Justo 4302,
((7600) Mar del Plata, Argentina
ABSTRACT
Most metals used as cementless implants undergo some kind of surface modification before clinical
insertion. These modifications are performed to promote biological reactions at the interface mainly
influencing the biological events that lead to bone formation. It is known that the texturing and /or
chemical alterations of material surfaces may lead to long-term integration in bone, so implant topography
is critical to the success of bone-anchored implants.
Zirconium (Zr) is promising materialfor intra-osseous implants for its favorable resistance to corrosion,
osseointegration capability and lower metal ions migration to the biological surroundings when it is
compared with stainless steel and titanium alloys.
The purpose of our preliminary study is to investigate the effect of anodization treatment on Zr as
permanent implant on cellular proliferation and bone deposition in the surrounding of the implant,
Inmunohistochemical staining using the antibody anti-PCANA was successfully done on undecalcified
sections of rats in polymethyl metacrylate embedded sections where cellular proliferation around metallic
implants was evaluated. The results showed that anodization process would increase the number of
proliferating cell. In vivo bone formation was analyzed by polychrome fluorescent labeling of bone, using
calcium-binding fluorochromes that are deposited at the site of active mineralization. In our study the new
bone around implant labeled with calcein and alizarin complexone fluorochromes was quantified by
morphology using fluorescence microscopy and revealed that bone formation in the surrounding of the
implants occurs continuously when evaluating at 45 and 60 days after implantation. These preliminary
results demonstrated that anodization process would benefit not only cellular proliferation around implant
but also it encouraged the mineralization process.
Key Words: osseointegration, zirconium, anodization process, cell proliferation, fluorescent labelling
1-INTRODUCTION
In the last years biomaterials research has been focused on developing novel surface modification to
achieve a complete integration between a biomedical device and cells and tissue minimizing scar tissue
formation (1).It is known that a rapid established, strong and long lasting connection between an implant
and bone is essential for the clinical success of orthopaedic and dental implants (2,3).
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Bone is a vital dynamic connective tissue that has evolved to maintain a balance between its two major
functions: provision of mechanical integrity for locomotion and modulation and control of mineral
homeostasis (4). Mineralized bone is continuously resorbed by osteoclast and new bone remodelling is
highly regulated with maintenance of normal integrity and structure (5).
Many experiments have demonstrated that bone cells react differently with surfaces with different
topographies (6-12) and the understanding of the process of cell/materials interaction is a very important
topic in the biomedical devices area. The technology of surface modifications has been extensively studied
in order to promote osseointegration around the implant and many studies have demonstrated that
chemically treated surfaces can enhanced the adhesion and proliferation of osteogenic cells (13,14),
precipitation of apatite (15), and the expression of bone-related genes and proteins (16,17). The term
Osseointegration has been defined as a direct bone-to-metal interface contact without interposition of nonbone tissue as a direct structural and functional connection between ordered, living bone and the modified
surface of the implant (18). This concept has been defined at multiple levels such as clinically (19),
anatomically (20), histologically and ultrastructurally (21).Currently, an implant is considered as
osseointegrated when there is no progessive relative movement between the implant and the bone where it
has direct contact and there is not fibrous tissue around the implant
Zr is a promising material for permanent implants.The good performance of zirconium has been mainly
attributed to its surface oxide film. The presence on a native ZrO2 oxide (zirconia) on zirconium surface
determines the low corrosion rate of the material, and therefore the low metal ion release to the biological
media.
For dental and orthopedic implants, many materials and surface modification have been examined
experimentally in vivo and in vitro and the histological and inmunohistological characterization of boneimplant interface is of great interest and allowed to evaluate the biology response of the healing response
to the implant surface.
Several works has established that key biological processes, including protein adsorption, cell
proliferation, and gene expression can be controlled by using chemical methods to modify the surface
properties of biocompatibility materials (22).
Surface modification induced by anodization in the
conditions presented in this work corresponds to a surface design criteria based on the modification of
chemical and topological features in the nanometric range with the aim of promoting osseointegration of
zirconium permanent implants.
Fluorochrome are fluorescent labels with calcium affinity is a widely spread standard technique in skeletal
research, simply and efficient for the dynamics of bone formation in combination with histology. (23) In
this technique, different types of fluororchromes are injected in the organism at different moments of
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ossification, they bind to the available calcium that is precipitating in the mineralization area giving
information about the calcification profile.
the purposes of our study were to investigate the effect of anodization treatment on Zr as permanent
implants on cell proliferation using PCNA as cellular marker and to perform a preliminary study about the
rate of new bone formation around the metallic implants employing the fluorochrome label approach .
2.
MATERIALS AND METHODS
2.1 Implants
In vivo experiments were conducted in total in six Wistar adult rats (weight 350 ± 50 g), according to rules
of the ethical committee of the Bioethics Committee HIEMI-HIGA, October 2011), taking care of
surgical procedures, pain control, standards of living and appropriated death. Rats were anaesthetized with
fentanyl citrate and droperidol (Janssen-Cilag Lab, Johnson and Johnson, Madrid, Spain) according to
their weight and the region of surgery surface was cleaned with antiseptic soap. The animals were placed
in a supine position and the implantation site was exposed through the superior part of the tibia’s internal
face. A region of around 0.5 cm diameter was scraped in the tibia and femur plateau and a hole was drilled
using a hand drill of 0.15 cm diameter bur at low speed. The implantation site was irrigated with
physiological saline solution during the drilling procedure for cleaning and cooling proposes. The implants
Zr0 (without treatment) and the implants Zr30 (with anodized treatment 30V), were placed by press fit
into tibia extending into the medullar canal. Conventional X-ray radiographs were taken before retrieving
the samples for control purposes.
2.2 Inmunohistochemistry
The animals were sacrificed with an overdose of intraperitoneal fentanyl citrate and droperidol after 60
days and the bone with implants was retrieved. The retrieved samples were cleaned from surrounding soft
tissues and fixed in neutral 10 wt % formaldehyde for 24 h. Then they were dehydrated in a series of
alcohol–water mixtures followed by a methacrylated solution and finally embedded in methyl
methacrylate (PMMA) solution and polymerized. The PMMA embedded blocks were cut with a low speed
diamond blade saw (Buehler GmbH) cooled with water. Sections were made 100 µm thick sections for
inmunohistochemical assays. The mounted sections were deacrylated for 48hs with (2-methoxy-ethyl)
acetate (Merck Biomaterials), the solutions were changed three times for 5 minutes with ethanol in
decreasing concentrations. Sections were then rehydrated with distilled water. For PCNA antibody,
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specimen were irradiated twice for 10 min with microvave (850W) in citrate buffer pH:6. To inhibit
endogenous peroxidase activity, tissue sections were previously dehydrated, treated with 0.5% v/v H 2O2 in
methanol for 30 min at room temperature, and rehydrated. The sections were treated for 1 h with 3% v/v
normal goat serum in phosphate buffer saline (PBS) to block non-specific binding sites. After two rinses in
PBS plus 0.025% v/v Triton X-100 (PBS-X), sections were incubated for 48 h at 4ºC with the primary
antibody PCNA (1/500, rabbit, gift from Dr Alicia Brusco laboratory). After five rinses in PBS-X, sections
were incubated for 1 h at room temperature with biotinylated secondary antibodies diluted 1:200. After
further washing in PBS-X, sections were incubated for 1 h with avidin-biotin –enzyme complex
(Vectastain ABC-HRP Kit, Vector, Burlingame, CA). Sections were then washed 5 times in PBS and
twice in 0.1 M acetate buffer, pH 6 (AcB), and development of peroxidase activity was carried out with
0.035% w/v 3,30-diaminobenzidine hydrochloride (DAB) plus 2.5% w/v nickel ammonium sulphate and
0.1% v/v H2O2 dissolved in AcB. After the enzymatic reaction step, sections were washed 3 times in AcB
and once in distilled water. Finally, sections were mounted on gelatine-coated slices; air dried and covers
slipped using Permount for light microscope observations. The antibody as well as the streptavidin
complex was dissolved in PBS containing 1% v/v normal goat serum and 0.3% v/v Triton X-100, pH 7.4.
2.3 Histomorphometry
After 63 days of implantation, three rats with Zr0 implants and three rats Zr30 implants (all individuals
derived from different litters) were deeply anesthetized with Ketamine/Xylasine (75mg/kg, 10mg/kg).
They were perfused through the cardiac left ventricle, initially with 15 ml of a cold saline solution
containing 0.05% w/v NaNO2 plus 50 IU of heparin and subsequently with 150 ml of a cold fixative
solution containing 4% paraformaldehyde in 0.1 mol/l phosphate buffer, pH 7.4. The retrieved samples
were cleaned from surrounding soft tissues and fixed in neutral 10 wt% formaldehyde for 24 h. Then they
were dehydrated in a series of alcohol – water mixtures followed by a methacrylated solution and finally
embedded in methyl methacrylate (PMMA) solution and polymerized. The PMMA embedded blocks were
cut with a low speed diamond blade saw (Buehler GmbH) cooled with water. Sections were made 100 µm
thick sections for dynamic histomorphometry assays.
2.3.1 Bone labeling with fluorochromes:
Time course of bone formation was analyzed by polyfluorochromic markers using fluorescence
microscopy. The polychrome sequential labeling of mineralising tissue according to Rhan (24) was
performed. The polyfluorochrome tracers, Calcein (C) 30mg/kg and Alizarine Complexone (AC) 30mg/kg
were administrated by an intraperitoneal inoculation at 45 and 60 days after the implantation surgery
respectively. The animals were sacrificed three days after the last injection (Figure 1).The sections were
4
viewed at a magnification of 40x and 200X under an epifluorescence microscopy Nikon Eclipse Ti
(Nikon, Japan) for evidence of fluorochrome double-labeled bone ingrown in the proximity of the
anodized surface implant.
Figure 1: Time line showing the protocol of fluorochrome injections. T0: implantation day; T 45: C, Calcein injection, T 60:
AC, Alizarin complexone; S: Sacrified
2.3.4 Morphometry:
The morphometry of the new bone formed around the implant was analyzed in the area indicated by a box
(Figure 4).The extent of newly formed bone around the implant was measured in 200x fluorescence
microscopy images for each type of implant. At 45 and 60 days after implantation, the distances from the
bone surface facing the implant to the calcein labeled line and alizaline complexone-labeled line, were
measured to evaluate the amount of newly formed bone. The nomenclature and and symbols used in
conventional bone histomorphometry are those describe by Parfitt et al (25). The parameter evaluated was:
The mineral apposition rate (MAR, µm/day) is the rate at which mineral accretion occurs at a remolding
site during the period of bone formation. MAR is a fundamental histomorphometric variable, and it is a
reliable measure of osteoblast function (26)
2.3 Superficial treatment
Specimens of 1mm diameter and 4-5 cm length of commercially pure zirconium (99,5%) were used. A
copper wire conveniently isolated on one extreme of the sample was used as electrical contact.
Anodizing treatment was carried out in a two electrode cell. The auxiliary electrode was a stainless steel
mesh that acts simultaneously as a reference electrode. The specimens were anodized in 1mol/L
H3PO4 solution at a constant potential between 3 y 30 V with respect to the reference electrode for 60
minutes. Phosphoric acid was selected as the anodizing electrolyte with the aim of promoting the
incorporation of P to the oxide film. The anodizing solution was prepared by diluting concentrated
5
H3PO4 (Aldrich) in deionized water (18.2 MΩ.cm, Millipore).Before and after each test the samples were
cleaned with acetone, dried in air and stored in a dryer.
2.4 Statistics
In this study, the data were showed in the form of mean value±SD. Differences between the groups were
assessed by one-way ANOVA with Tukey post-hoc test was performed using GraphPad In Stat version
3.00 (Graph Pad Software). A p value < 0.05 was considered significant for all statistical analyses.
3. RESULTS
3.1 Inmunohistochemistry
The inmunohistochemistry expresión of PCNA positive cells was analyzed in Zr permanent implants
subjected to anodized treatment. Figure 1 showed the inmunohistochemical expression of PCNA+ cells
around Zr0 and Zr30 volts anodized implant. It is possible to note that PCNA inmunoreactivity was
mainly associated to the cell body in both implants sixty days after the implantation. (Fig 2A and Fig 2B)
B
A
Implant
PCNA+ cells
PCNA+ cells
Implant
Figure 2: Photographs showing PCNA+ cells /u.area in the proximity of the zirconium metallic implants Zr0 (A) and Zr30 ( B).
Scale barr: 50μm
When we quantified the number of PCNA positive cells per unit area around the implants, it is possible to
note that there is no significant difference between both surface treatments (Figure 3)
6
+/
n°cells PNCA u.area
0.00075
0.00050
0.00025
0.00000
Zr0
Zr30
Figure 3: Quantitative analysis of the number of PCNA positive cells per unit area both in zirconium anodized (30 volts) and
anodized (30votls) metallic implants. Values are reported as mean ± SEM.* P<0.05.
3.2 Morphometry analysis
Fluorescent microscopy
A classical image of the area around the implants was shown in Figure 4. The image showed the green and
red lines labeled with Calcein and Alizarin Complexone, in the new bone ingrowth around the metallic
implant.
Bone deposition with fluorescent labels was most notable in photomicrographic images show in Fig.4.At
45 and 60 days after implantation, the double fluorescent labels (green lines and red lines) in the bone
around Zr0 and Zr30 implants were very noticeable where calcein-labeled lines adjoining the marrow
cavity were observed. At 63 days after implantation, the brightness of calcein-line was little diminished;
alizarin complexone-lines were more obvious than the green lines. That was probably as consequences of
the natural resorption process.
To quantify the new bone formed around the metallic implants, he distance from the bone surface facing
the implant to the calcein-labeled line and the alizarin-labeled lines was measured as described in Material
and Methods section.
Figure 4: A cross-sectional image of a tibia with
an anodized zirconium implant (30V), 63 days
after implantation. A longitudinal undecalcified
section is prepared for fluorescence microscopy.
Green (calcein) and red (alizarin complexone)
lines are seen in the new bone laid down around
the implant (Imp) at 45 or 60 days after
implantation, respectively. Bone formation is
analyzed in the area indicated by the box. BM:
bone marrow, CB: cortical bone. Original
magnification: _35. Bar=1mm.
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Figure 5: Fluorescent microscopy images
(200X) were used to measure the mineral
apposition rate. The fluorochrome labels,
Calcein (green) and Alizarin complexone (red)
can be observed near the metallic implant
(black) at 63 days after implantation in a
titanium implant 30 volt anodized. Both
labeled lines were very marked
The mineral apposition rate (MAR, µm/day) was quantitative measured in the bone ingrowth in Zr0 and
Zr30 samples after 63 days of implantation. (Figure 6). It was found that there was a significant increase in
the MAR in the implants that were anodized at 30 volts.
3.5

MAR m/day
3.0
2.5
Figura 6: Quantitative analysis of mineral
apposition rate (MAR) in Zr0 and Zr30 volt
implants. Values are reported as mean ±
SEM. * P<0.05.
2.0
1.5
1.0
0.5
0.0
Zr 0 Volt
Zr 30 Volt
4-DISCUSSION
The direct observation of bone-implant interface is of great interest for the basic material science as well
as for clinical application particularly in orthopaedic and trauma surgery The inmunodetection for markers
of cell proliferation, bone resorption, bone formation and angiogenesis at the undecalcified bone sections
are interesting because they allow the understanding of the complex osseointegration process, including
the study of the cellular activity and the cell-matrix interations at the bone implant interface. (27).After
implantation, the formation of mineralized bone near implants surface requires the colonization of implant
surface by osteoblastic cells, these cells mainly originate from mesenchymal stem cells (MSC) recruited
8
by implant surface. MSC differentiate into osteogenitor cells, and then into osteoblast. Osteoblasts
synthesize osteoid that is mineralized to form new bone (28, 29).
The preliminary results obtained in this study demonstrated that anodized Zr used as permanent implants
showed a slight tendency to increase the number of cells proliferating around the metallic implant when
they are compared with non anodized implant, suggesting that anodization would stimulate the
colonization and differentiation of cells in the bone-implant interface. Nevertheless, complementary assays
should be done to evaluate the expression of important proteins associated to the osseointegration process
as: osterix, osteocalcine, alkaline phosphatase (ALP) and collagen type I as osteoblast markers and the
tatrate-resistant acid phosphatase (TRAP) as a molecular marker for osteoclast (30)
Fluorescent microscopy of the sequential fluorochrome labels revealed the dynamics of bone formation in
different periods of implantation (31, 32). An apposition rate represent in some sense the activity of a team
of osteoblast, but it is important to take in mind that formation rate is influenced by the rate of remodeling
activation and consequently it depends on the number of osteoblast team as well as on their activity. In our
experiments the sequential fluorochrome labeling with the fluorochromes Calcein and Alizarin
complexone demonstrated that bone formation and bone remodelling surroundings implants can be
encouraged if the metallic implants surface are anodized. The results demonstrated that the anodization
treatment can significantly increase the mineralization process and the creation of new bone around the
metallic implant.
On the whole, the findings of this study are a preliminary combination of inmunohistochemistry and
fluorochrome labels assays to evaluate the osseointegration process suggesting that alow voltage
anodization is a promising superficial treatment to improve the structural connection between the bone and
the implant surface.
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