Biomaterials 23 (2002) 3833–3841

Osteointegration of bioactive glass-coated zirconia in healthy bone:

an in vivo evaluation
V. Stanica,b, N. Nicoli Aldinic,*, M. Finic, G. Giavaresic, R. Giardinoc,d, A. Krajewskie,
A. Ravagliolie, M. Mazzocchie, B. Dubinia,b, M.G. Ponzi Bossia,b, F. Rustichellia,b
a
Institute of Physical Sciences, University of Ancona, Ancona, Italy
b
INFM—National Institute for the Physics of The Matter—Unit of Ancona, Italy
c
Experimental Surgery Department, Research Institute Codivilla Putti-Rizzoli Orthopaedic Institute via di Barbiano 1/10, 40136 Bologna, Italy
d
Chair of Surgical Pathophysiology, University of Bologna Medical School, Bologna, Italy
e
Institute for Technological Research on Ceramics of Irtec-CNR, Faenza, Italy
Received 2 July 2001; received in revised form 12 February 2002; accepted 21 March 2002

Abstract

Osteointegration of yttria stabilised tetragonal zirconia (YSTZ), either coated with bioactive glass named RKKP bioglazes
(RKKPs) or uncoated, was evaluated in an animal model. RKKPs-coated and uncoated (controls) YSTZ cylinders were implanted
in the distal femoral epiphyses of 14 Sprague Dawley rats under general anaesthesia. At the experimental times of 30 and 60 days
after sacrifice, histomorphometry and SEM microanalysis were performed on methylmethacrylate-embedded undecalcified sections
to determine the osteointegration rate. At 30 days, a significantly higher affinity index was demonstrated in vivo by
histomorphometric evaluation in RKKPs-coated versus uncoated YSTZ implants (po0:05); at 60 days, the coated implants
behaved better than controls (affinity index of +32% ), but the difference observed lay within the statistical uncertainty.
SEM analysis demonstrated better bone adhesion to the material in RKKPs-coated YSTZ at both 30 and 60 days. These findings
suggest that YSTZ coated with the bioactive glass named RKKPs enhances osteointegration of ceramics. r 2002 Elsevier Science
Ltd. All rights reserved.

Keywords: Bioactive glass; Zirconia; Ceramics; Osteointegration

1. Introduction properties. Yttria stabilised tetragonal zirconia (YSTZ)1

Coating is a common procedure when manufactur-
ing prosthetic devices [1–2]. This treatment is directed
at improving the surface properties of the device
when the biomaterial used acts mainly as a good
mechanical support but requires improvement of its
surface properties to promote the process of osteointe-
gration [3,4].
To make the surface of a device bioactive, various
types of coatings were considered [5–7]. Among these,
bioactive glasses showed promising features in terms of
osteogenic and osteoconductive qualities.
Zirconia bioceramics are widely used in dentistry [8,9]
and orthopaedics [10] due to their attractive mechanical
*Corresponding author. Tel.:+39-051-6366786; fax: +39-051-
6366580.
E-mail address: nicolo.nicolialdini@ior.it (N. Nicoli Aldini).
is stiff, wear resistant and chemically inert [11]. The
current authors tested various materials in healthy and
osteopenic bone, and YSTZ proved to perform better
when implanted in the healthy bone of rats [12]. On the
contrary, a significant decrease in the affinity index was
observed when the material was implanted in rats with
ovariectomy-induced osteopenia [13]. Promising results
were obtained using the bioactive glass RKKP bioglazes
(RKKPs)2 in both healthy and pathological bone,
where it showed good biocompatibility and osteogenic
properties [14].
1
YPSTZ corresponds in this case to the following composition:
94.5% ZrO2+5.5% Y2O3.
2
RKKP code and bioactive glass composition are ISTEC-CNR
trademark and patent, respectively; the term ‘‘bioglaze’’ is reserved
trademark for ISTEC-CNR.

0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 1 1 9 - 9
3834 V. Stanic et al. / Biomaterials 23 (2002) 3833–3841
On the basis of these data, RKKPs-coated YSTZ
samples were manufactured to evaluate whether this
combination—ceramics and bioactive glass—is effective.
After a preliminary in vitro evaluation [15], an in vivo
study was conducted in the healthy bone of rats, where
RKKPs-coated and uncoated YSTZ samples had been
implanted to study the behaviour of the material. The
results obtained are investigated in the present report.
2. Materials and methods
2.1. Sample production
The YSTZ samples were obtained from highly pure,
medical grade powders (yttria stabilised ZrO2, Y-PSZ
stabilised with 3 mol% Y2O3, or 5.5% wt; Tosoh
TZ3YB, Japan). Cylindrical rods (4 mm in length,
2 mm in diameter) were prepared by extrusion of a
paste containing organic additives and 90% wt YSTZ
powders. After drying the extruded samples were fired in
a laboratory kiln with the following thermal cycle:
increase at the rate of 1001C/h up to a final temperature
of 15501C, steady temperature for 1 h and cooling at the
rate of 2001C/h. All sintered samples were accurately
sized and rectified. Rods were first cut with a diamond
saw and their ends were then rectified. The consistency
of the YSTZ ceramic bodies obtained, proved to be
homogeneously compact and without apparent poros-
ity.
The bioactive glass RKKPs was applied as an enamel
to the surface of the YSTZ samples obtained by
brushing their powders (grains Fo40 mm) with a
suitable slurry (binding polymers, water and finely
powdered bioactive glass). After drying the coated
samples were fired in a laboratory kiln at 12801C to
obtain a vitreous coating (bioglazes) tightly adhering to
the YSTZ ceramic substrate. Before implantation, rods
were sterilised in autoclave at 1201C for 20 min.
Further details of YSTZ sintering and bioactive glass
coating of samples are described elsewhere [16,17].
Ultimate tensile adhesion strength (UTAS) stretch
tests3 and profilometry analyses were performed on all
samples, either RKKPs-coated or uncoated, in order to
acquire adequate information on some physical and
mechanical properties likely to be involved in bone
adhesion. The UTAS tests showed a bond strength of
3273 MPa.
The following data were gathered by means of
profilometry tests on YSTZ samples coated with
bioactive glass: uncoated YSTZ Ra=1.2670.17,
Rt=10.2871.20; RKKPs coated YSTZ Ra=0.377
0.20, Rt=3.2771.57.
3
Bond strength test: B SEN 582: 1984 standard.
Table 1
Affinity Index (A.I.) of uncoated YSTZ and RKKPs-coated YSTZ
samples (Mean7SD, no.=7)
A.I. (%)
YSTZ RKKPs-YSTZ
30 days 45717 72724a
60 days 56732 74717
Student’s t-test.
a
A.I. (po0:05), RKKPs-YSTZ vs. uncoated YSTZ.
Fig. 1. Surface of non-implanted YSTZ (888  ).
2.2. In vivo studies
To investigate the implant behaviour in the bone, an
in vivo study was performed following the European
and International rules.4 Fourteen Sprague Dawley
adult female rats (Charles River, Calco, Lecco, Italy),
350725 g b.w., were used. They were housed in
standard conditions (room temperature 2070.51C;
relative humidity 5575%; 12 h light and 12 h darkness)
and were supplied with food (4RF18, Mucedola SRL,
Settimo Milanese MI, Italy) and water ad libitum.
Animals were randomly divided into two groups of
seven each, according to the experimental times: Group
A—30 days and Group B—60 days. RKKPs-coated
YSTZ samples were implanted in the left side of each
animal while uncoated YSTZ was used for the right side.
General anaesthesia was induced by 87 mg/kg keta-
mine (Ketavet Farmaceutici Gellini, Aprilia, Latina,
Italy) and 13 mg/kg xylazine (Rompun, Bayer Italy SpA,
Milan, Italy). The animals were placed supine, and the
lateral aspects of the thigh and knee were shaved. The
lateral surface of the knee joint was then exposed
through a vertical incision to reach the femoral condyle,
4
Italian D.L. January 27, 1992, no. 116; ISO 10993-2; NIH no.
#A5424-01
 V. Stanic et al. / Biomaterials 23 (2002) 3833–3841
where a 4 mm-deep hole was created with a 2 mm-
diameter drill. A sample was then placed into the hole
and pressed until complete insertion. Afterwards, the
skin was closed with interrupted stitches. The same
procedure was performed both on the right and on
the left sides. Postoperatively, the animals were
administered analgesics (0.01 ml/100 g ketoprofen) and
antibiotics (0.06 ml/100 g flumequine). At the selected
experimental times, the animals were pharmacologically
euthanized under general anaesthesia, and both femurs
were explanted and cleaned of soft tissues for morpho-
logical investigations.
2.3. Histology and histomorphometry
Both femurs were fixed in 4% buffered paraformal-
dehyde, then dehydrated in graded series of alcohols
Fig. 2. (a) Surface of the RKKPs glass coating on the YSTZ substrate
(914  ). (b) A backscatter micrograph of the cross-section of the glass
coating (RKKPs) on the YSTZ substrate shows the interface without
any crack. The thickness of the bioactive glass coating in this area is
about 100 mm. 1—YSTZ substrate, 2—grey region, 3—RKKPs
coating.
3835
from 50% to 100% and embedded in methylmethacry-
late (Merck, Schuchardt, Hohenbrunn, Germany).
Cross-sections of 70 and 140 mm were obtained with a
Leica SP 1600 diamond saw microtome (Leica SpA,
Milan, Italy); they were stained with Fast Green and
observed with a Zeiss Axioscop microscope (Carl Zeiss,
SpA, Arese, Italy). Histomorphometry was performed
with Kontron KS 300 software (Kontron Electronics,
Eching bei Munchen, Germany). Three consecutive
sections were measured for each sample. The affinity
index was determined: it was defined as the length of
bone contact/the total length of the bone–implant
interface  100%:
2.4. Analysis by scanning electron microscope
The 140 mm-thick sections obtained with the above-
mentioned procedure, were used for this purpose.
Fig. 3. A backscatter micrograph of the uncoated YSTZ used as
control 30 days after surgery (18  ).
Fig. 4. A backscatter micrograph of the uncoated YSTZ 60 days after
surgery. 1—YSTZ, 2—bone (22  ).
3836 V. Stanic et al. / Biomaterials 23 (2002) 3833–3841

Fig. 5. A prosthesis of YSTZ coated with RKKPs bioactive glass 30 Fig. 8. A backscatter micrograph of YSTZ coated with RKKPs
days after surgery (8  ). bioactive glass 30 days after surgery. 1—YSTZ, 2 (grey region)—
region of glass diffusion through the YSTZ substrate, 3—RKKPs
bioactive glass, 4—bone (35  ).

Fig. 6. A prosthesis of YSTZ coated with RKKPs 60 days after

surgery. 1—YSTZ, 2—bioactive glass RKKPs, 3—bone (21  ).

Fig. 9. A backscatter micrograph of the prosthesis of YSTZ 60 days

Fig. 7. A backscatter micrograph of YSTZ coated with the RKKPs after surgery (above); the enlarged area of the ‘‘B’’ interface showing
bioactive glass 30 days after surgery. 1—YSTZ, 2 (grey region)— the areas where EDS analysis was performed. 1—YSTZ, 2—bone
region of glass diffusion through the YSTZ substrate, 3—bone (88  ). (below) (273  ).

Observations were performed using a Philips SEM XL
20 scanning electron microscope (SEM). The samples
were coated with graphite to avoid the increase of the
static charge through SEM analysis.
Backscattered electrons respond to composition
(atomic number or compositional contrast), local speci-
men surface inclination (topographic or shape contrast),
crystallography (electron channelling) and internal
magnetic fields (magnetic contrast).
This method allows to look into the interior of the
biological specimens and obtain a backscattered-elec-
tron signal from 1 to 2 mm—inclusions below the surface
of the specimen. Becker and Geoffrey [18] have provided
an excellent review of the application of backscattered
imaging to histochemical studies.
The energy-dispersive spectrometer (EDS) system is
now the commonest X-ray measurement system to be
found in the SEM laboratory. It offers a means of
rapidly evaluating the constituents of a sample; major
constituents (10 wt% or more) can be identified in only
10 s, and a 100 s accumulation is often sufficient for
identification of minor elements (of the order of 1 wt%).
In addition to a rapid qualitative analysis, the EDS
system allows for an accurate quantitative analysis, too
[19].
3. Results
All the animals survived until the scheduled time of
sacrifice. No general or local complications were
encountered.
120
100
80
Ca-Zr weights (%)
60
40
20
-20
-20
Fig. 10. A graph of the areas analysed by EDS in Fig. 11. Ca and Zr weights (%) along the bone–YSTZ interface ‘‘B’’. 1—region of YSTZ; 2—newly
formed bone.
V. Stanic et al. / Biomaterials 23 (2002) 3833–3841 3837
3.1. Histology and histomorphometry
Histology demonstrated a regular architectural pat-
tern of the bone surrounding the implants on 30 and 60
days after implantation. Trabecular bone was regularly
arranged around the RKKPs-coated YSTZ in the
absence of connective capsule.
Table 1 reports the average histomorphometric
results.
30 days (group A): At 30 days, the affinity index
revealed significant differences between uncoated and
RKKPs-coated YSTZ, with a better behaviour of the
coated material.
60 days (group B): The RKKPs-coated YSTZ showed
a higher affinity index than the uncoated sample.
However, the difference observed lay within the
statistical uncertainty.
3.2. SEM analysis
3.2.1. Before implantation
Fig. 1 shows the surface of the YSTZ substrate alone:
it is not homogenous and presents roughs, cavities and
pores. A rough and non-homogeneous surface of the
YSTZ substrate facilitates the adhesion of RKKPs.
Fig. 2a shows the RKKPs bioglass surface after
deposition on the YSTZ substrate, and Fig. 2b a cross-
section of the sample. The latter was obtained by
backscattered electron imaging and shows how the
bioactive glass interacted with the YSTZ substrate. The
active glass deposited on the surface underwent partial
absorption with intergranular penetration into the
ca
zr
1
zirconia
2
bone
0 20 40 60 80 100 120 140
Depth (
m)
3838 V. Stanic et al. / Biomaterials 23 (2002) 3833–3841

YSTZ substrate during firing. This can explain the
darker (grey) zone on the right, the thickness of which
turned out to be about 105 mm under SEM observation.
The remaining external layer of glass alone was found to
be about 75715 mm. Consequently, the overall thickness
of the bioactive glass layer was 180 mm. A microphoto-
graph of this description is shown in Fig. 2b.
3.3. After implantation
3.3.1. Uncoated YSTZ
The uncoated samples showed implants surrounded
by cancellous bone with regular trabecular architecture
on postoperative day 30 (Fig. 3) and 60 (Fig. 4).
Nevertheless, Figs. 3 and 4 clearly depict a low degree
of bone–prosthesis contact. Low affinity was observed
in all of the samples examined by SEM. In Fig. 4 the
prosthesis is surrounded by a black border, which means
low affinity between uncoated YSTZ and bone and
corresponds to the areas of connective tissue observed
by optical microscopy.
As already observed in Figs. 9 and 4, the prosthesis is
surrounded by a black border indicating low affinity
between uncoated YSTZ and bone [20].
3.4.2. RKKP-coated YSTZ
Fig. 11 shows an enlarged area at the interface where
the points subject to analysis are represented by white
dots: the first one in YSTZ lies at about 145 mm from the
bioactive glass–bone interface, the last one in the bone
lies at about 40 mm from the same interface.
Fig. 12 reports Ca, Zr and Si weights (%) along this
bone–YSTZ interface. Bioactive glass components (Si
and Ca, in particular) confirm previous findings about
diffusion through the YSTZ substrate from the outside
(grey region). As expected, the external layer of
bioactive glass alone turned out to be slightly reduced
by about 40 mm as compared to Fig. 12, since its
thickness was about 35 mm. Such finding could be

3.3.2. RKKPs-coated YSTZ

All of the samples examined by SEM revealed very
good contact at the material–bone interface in the 30
day (Fig. 5) and 60 day (Fig. 6) groups. In fact,
micrographs did not show any black border around
the prosthesis. The bone surrounding the implant
showed regular trabecular architecture. These latter
findings can be interpreted as evidence of good
osteointegration.
Images in Figs. 7 and 8 were acquired with the BSE
technique. Fig. 7 shows three regions: the substrate of
YSTZ, the depth of bioactive glass RKKPs penetration
into YSTZ and bone. Fig. 8 reveals four regions: YSTZ,
the penetration area by RKKPs into YSTZ, RKKPs
and bone [20].

3.4. EDS analysis

3.4.1. Uncoated YSTZ

Backscatter imaging of uncoated YSTZ at 60 days
after surgery is shown in Fig. 9. The interface ‘‘B’’ is
enlarged, which is the area where EDS analysis was
performed. A graph of the areas, which were the subject
of the EDS analysis, is shown in Fig. 10. There is no
evident Ca diffusion to YSTZ and no Zr diffusion to
bone.
The graph in Fig. 10 is based on the microanalysis of
each white dot depicted in Fig. 9. It shows a transitional
area sized 83–100 mm which corresponds to the dark Fig. 11. A backscatter micrograph of the prosthesis of YSTZ coated
with RKKPs bioactive glass 60 days after surgery (above); the
band shown in Fig. 9 and representing the connective
enlarged area of the ‘‘A’’ interface showing the areas where EDS
tissue. Such finding confirms the lack of direct contact analysis was performed (below). 1—YSTZ, 2—grey region, region of
between the surface of the YSTZ prosthesis and bone in bioactive glass diffusion through YSTZ, 3—bioactive glass, 4—bone
this area. (500  ).

120
100
Si-Zr-Ca weights (%)
80
60
40
20
-20
200
Fig. 12. A graph of the areas analysed by EDS in Fig. 9. Si, Zr and Ca weights (%) along the bone–YSTZ interface ‘‘A’’. 1—region of the YSTZ
substrate without the elements diffused from the RKKPs coating; 2–grey region, region of the YSTZ substrate with the elements diffused from the
RKKPs coating; 3—RKKPs; 4—newly formed bone.
ascribed to the mechanisms of activity typical of
bioactive glasses.
4. Discussion
Ceramics can be used as materials for bone segment
replacement when their mechanical properties are
adequate or, alternatively, as coatings for pins, un-
cemented arthroprostheses or screws [21,22]. Further
investigation is needed to increase the osteogenic
capacity of these materials because of the still high rate
of implant failures [23], the reported side effects due to
the in vivo ceramic degradation [24] and the increasing
number of patients with biological drawbacks requiring
specific strategies to enhance bone bonding to bioma-
terials [25,26]. Bioactive glasses are promising materials
recognised as being highly biocompatible. Moreover,
their surface reactivity makes them osteoconductive
when placed in bone tissue [27]. Nowadays, they can be
used with good results for coatings, since they lack those
biomechanical properties required when mechanical
stabilisation is needed.
RKKPs is a bioactive glass that was extensively
investigated by the current authors [16,17]. It provided
favourable results that prompted research on this type
of coating. In the present study the RKKPs-coated
YSTZ samples were evaluated in vivo in an animal
model with healthy bone. The study was performed
using rats to allow for comparison with previous studies
on YSTZ and RKKPs [13]; the current findings
demonstrated the superiority of RKKPs coating over
YSTZ in terms of osteointegration.
V. Stanic et al. / Biomaterials 23 (2002) 3833–3841 3839
si
Zr
Ca
bioglass
3
4 2
grey zone 1
Zirconia substrate
bone
150 100 50 0
Depth (
m)
Histological and histomorphometric analyses con-
firmed that RKKPs coating significantly enhances the
in vivo behaviour of YSTZ at 30 days after implanta-
tion. A similar behaviour was deduced from the average
values calculated at 60 days and showing a persistent
increase of 32% in the affinity index of the glass-coated
surface as compared to uncoated YSTZ, even though no
statistical significance was reached.
SEM micrographs demonstrated enhanced osteointe-
gration in coated samples at both 30 and 60 days. In
fact, they showed very good bone–prosthesis contact in
the RKKPs-coated samples but poor contact in the
uncoated samples.
The EDS analysis showed how the bioactive glass
components (Si and Ca, in particular) diffuse through
the YSTZ support from the interface to a shallow depth
(about 100 mm, see Fig. 12). The depth extent was
greater than the depth (about 40 mm) previously
observed [16]. In the past ceramics have been moulded
using the die-pressing technique, whereas the current
authors chose the extrusion procedure, which produces
slightly less dense ceramics. The glass impregnating the
underlying layer of the YSTZ substrate ensured good
attachment of coating to YSTZ. On the other hand, the
UTAS tests performed showed a bond strength of
3273 MPa, as already highlighted in a previous study
[16]. Table 1 shows two trends of Affinity Index (A.I.)
calculated using the peak values at 30 and 60 days and
proving that RKKPs biological activity accelerates
bone growth, which is slower on uncoated samples and
faster on bioactive glass-coated samples.
The statistical uncertainty characterising the improve-
ment of the affinity index at 60 days in uncoated YSTZ
3840 V. Stanic et al. / Biomaterials 23 (2002) 3833–3841
demonstrated that bone growth slowly continues on
uncoated ceramics, even if the bioactive glass coating
has already reached final and complete osteointegration
at 30 days. Moreover, the material–bone bonding in rats
may be complete at 60 days, whereas intensive bone
remodelling should not occur by that time [28].
Other studies may be conducted to better understand
the meaning of affinity index data and bone quality may
be investigated around other materials that were not
included in the present stage of the research process.
Overall, bone histomorphometry seems to be consistent
with SEM analysis, thus reinforcing the value of these
two techniques. The mechanism of action of the glass
surface in accelerating bone healing around implants is
reported to be of chemical origin and differs from
ceramics, which seem to bind to bone mainly by
mechanical interlocking [28]. The chemical nature of
bioactive glass–bone bonding could be based on the
release of biological active ions from the silicate matrix
and regulated by the composition and presence of other
ions able to lock the molecular network. However, the
matrix surface is submitted to lixiviation with silica gel
formation, due to the exchange of H+ ions coming from
the physiological body fluids and cations from the glass.
A hydroxyapatite layer is then formed and enhances
osteoconduction. Its formation could be promoted by
the terminal Si–OH groups of the gel which are
responsible for the formation of a sufficiently stable
bridging bond through the interposition of Ca2+ [29,30].
The present study demonstrated the promising
properties of RKKPs bioactive glass as a coating
material for prostheses to be implanted in bone. In
particular, an appreciable enhancement of osteointegra-
tion was observed when the prosthesis was coated with
RKKPs. This fact may be of clinical relevance because
osteointegration in trabecular bone is a slow process
also in healthy human bone [31], and any improvement
of this process secondary to the use of a coated
prosthesis is surely of benefit to the patient.
Future investigations should be directed at the study
of the behaviour of the RKKPs bioactive glass in
osteopenic bone, to create experimental conditions
superimposable on those observed in clinical practice.
Acknowledgements
This work was supported by the C.N.R. project
‘‘Studio di biosmalti per il ricoprimento di materiali
inorganici’’ (Legge 95/95-PF 32). Moreover, the authors
would like to thank Claudio Dal Fiume, Nicola
Corrado, Patrizio Di Denia, Patrizia Nini and Franca
Rambaldi (Experimental Surgery Department, Rizzoli
Orthopaedic Institute) and Mario Pergolini (Institute of
Physical Sciences, University of Ancona) for their
technical assistance. The work reported here forms a
part of thesis by V. Stanic.
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