Biomaterials 22 (2001) 987}994

In vitro characterisation of zirconia coated by bioactive glass

M.Bosetti , E.Verne`

, M.Ferraris, A.Ravaglioli, M.Cannas *

Department of Medical Sciences, Human Anatomy, University Eastern Piemont **A. Avogadro++, Via Solaroli 17, 28100 Novara, Italy

Materials Science and Chemical Engineering Department, Polytechnic of Torino, Torino, Italy

Institute for Technological Research on Ceramics, (IRTEC), CNR Faenza, Italy

Received 29 February 2000; accepted 2 August 2000

Abstract

An in vitro evaluation of a biomedical device, which combines the mechanical properties of zirconia substrates with the bioactivity

of two di!erent glass layers (AP40 and RKKP), was performed.In this work, data on di!erent kinds of analysis were reported both on
as-sintered zirconia samples and on RKKP- and AP40-coated zirconia substrates.Structure, composition and morphology of the
apatite layer growth on the coated samples after 30 days of soaking in an acellular simulated body #uid, serum protein adsorption,

"broblasts and human osteoblast-like cells adhesion, growth, morphology and biochemical aspects were studied.Results of soaking

test in SBF, revealed the growth of an apatite layer on the surface of the glass-coated samples.Proteins adsorbed to the materials were
analysed by sodium dodecyl sulphate}polyacrylamide gel electrophoresis and results evidenced that the two glass-coated materials
bound a higher amount of total protein than did the zirconia substrate.Fibroblasts and osteoblast-like cells cultured on RKKP- and
AP40-coated zirconia showed a higher proliferation rate, leading to con#uent cultures with higher cell density and a generally better
expression of osteoblast alkaline phosphatase activity in comparison with zirconia substrate.In conclusion, our results indicate that
the surface chemical characteristics of the two glass coatings AP40 and RKKP, with no great di!erences between them, substantially
enhance zirconia integration with bone cells at least in vitro.This e!ect may be of signi"cance in the stability of glass-coated zirconia
orthopaedic and dental implants.

2001 Elsevier Science Ltd.All rights reserved.

Keywords: Zirconia; In vitro biocompatibility; Osteoblast-like cells; Glass coating

1. Introduction

During the last decade, a large number of biomaterials

have been proposed as arti"cial bone "llers for repairing
bone defects.Among these materials, a distinction can be
made according to their relative surface reactivity
with surrounding tissue: (1) toxic when tissue dies,
(2) non-toxic, biologically inactive when tissue forms
a non-adherent "brous capsule around the implant, (3)
non-toxic and bioactive when the tissue forms an inter-
facial bond with the implant, and (4) non-toxic dissolu-
tion of the implant when tissue replaces implant.

To combine the mechanical properties of a high-

strength inert ceramic with the speci"c properties of bioac-
tive glasses, composite materials based on high-density
zirconia substrates [1] coated by bioactive glasses were

* Corresponding author.Tel.

: #390-321-660-632; fax: #390-321-

660-632.

E-mail address: [e-mail] (M. Cannas).

suggested.In order to characterise in vitro the interac-
tions between the bioactive materials and the adjacent
tissues, studies of serum protein adsorption allowed to
give supposition about the possibility that a conditioning
layer on the material surfaces may in#uence cell attach-
ment and orientation.

As a support to the usually performed test to evaluate the

bioactivity of materials by observing the growth of a hy-
droxyapatite layer on their surface after soaking in acellular
solutions which simulate the ion composition of human
plasma [2], our in vitro cell model was chosen to investigate
the behaviour of osteoblast-like cells cultured on the two
glass coatings, di!ering from each other due to the presence
of small amounts of tantalum and lanthanum oxides.

In this work, the growth of apatite after soaking in

simulated body #uid (SBF), protein adsorption and
cytocompatibility of zirconia substrates covered by
bioactive glass layers were evaluated in vitro, by using
uncoated zirconia as reference material.

In order to characterise the "broblasts and osteoblast-

like cells and their behaviour, cell morphology and

0142-9612/01/$ - see front matter

2001 Elsevier Science Ltd.All rights reserved.

PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 2 6 4 - 7

Table 1
Simulated body #uid composition used to study in vitro the apatite
self-grown layer

Ion

Na

> K> Mg> Ca> Cl\

HCO

\

HPO

\

SO

\

pH

(m

M

) 142.0 5.0

1.5

2.5

147.8 4.2

1.0

0.5

7.25

proliferation, cell protein content and alkaline phos-
phatase activity have been studied.

2. Materials

Zirconia substrates (d"6.00$0.05 g cm

\, corre-

sponding to a relative density of 98.5$0.05% with
respect to the theoretical one) sintered by IRTEC-CNR,
Faenza, Italy, and obtained by 3% YO-stabilised zir-

conium oxide powders, with an average grain size of
about 1

m diameter, were used.Samples of 230 mm

surface area were used (10

;11.5;2 mm).

As coating materials two bioactive glasses were used

[3,4], which have the following chemical composition
(wt%):

E AP40: 44.30 SiO, 24.50 -Ca(PO), 18.60 CaO,

4.60 NaO, 0.19 KO, 2.82 MgO, 4.99 CaF.

E RKKP: 43.82 SiO, 24.23 -Ca(PO), 18.40 CaO,

4.55 NaO, 0.19 KO, 2.79 MgO, 4.94 CaF, 0.99

TaO, 0.09 LaO.

Small amounts of TaO and LaO were added to

furnish a possible nucleus of deposition for ions involved
in bone formation.It was demonstrated, by in vitro
experiments, that the presence of these oxides could mod-
ify the surface properties of the glass and in#uence the
protein absorption kinetics [5].The bioactive glasses
were prepared by melting of the starting products in
a platinum crucible at 14503C in a laboratory furnace for
2 h.The melted glasses were simply quenched in cold
water.

The coarse-granulated glasses were powdered by an

alumina vibratory ball mill and sieved selecting the
granulometry in the range 40}100

m.An optimised

thermal treatment was carefully developed to coat zirco-
nia substrates by amorphous AP40 and RKKP.As
described elsewhere [6}8], the preparation of the coat-
ings consisted in covering the zirconia substrate by a slip
or by dry glass powders, then heating them at temper-
atures slightly above the liquidus temperature, obtaining
layers with a thickness of about 150 ($40)

m.After the

"ring process, the coated samples were simply annealed

in order to obtain tension-free glass coatings.

Each coating was characterised by optical and scann-

ing electron microscopy (SEM * Philips 525 M) and
compositional analysis (EDS) (Model EDAX 9100, Phi-
lips); X-ray di!ractometric analyses (PW 1710, Philips
Electronic Instruments, Mahwah, NJ) were carried out in
order to study the possible micro crystalline phases for-
med in the applied layer.The adhesion of the coatings on
zirconia was tested by the Vickers indentation method at
the coating/zirconia interface [6,7].

In order to perform the protein adsorption and

cytocompatibility analyses, all samples were sterilised by

dry heat for 3 h at 1503C.The soaking test in SBF did not
need any preliminary treatment of the samples.

3. Methods

3.1. Soaking in simulated body yuid

Four coated samples were soaked in an acellular SBF,

having the same ion concentration as the human plasma.
This solution, whose composition is reported in Table 1,
was prepared by dissolution of high-purity reagents in
distilled water, and was bu!ered at pH 7.25 with 50 m

M

tris-hydroxymethyl amino ethane and 45 m

M

hydrochlo-

ric acid.

The temperature was maintained at 373C.Each sample

was soaked in 15 ml SBF in a polyethylene bottle, with-
out stirring.After 30 days they were removed from the
solution, gently washed in distilled water, and dried at
room temperature.The soaked samples were then char-
acterised by scanning microscopy and compositional
analyses, in order to observe the modi"cation of their
surface.The samples for SEM observations were simply
dried and covered by a thin gold layer to guarantee the
conductivity.

3.2. Protein adsorption method

The materials were incubated with 1 ml foetal bovine

serum (FBS, Sigma, Milan) for 1 h at room temperature.
After removal of serum, samples were washed four times
in phosphate-bu!er saline and incubated at 903C for
10 min in 200

l of a sodium dodecyl sulphate

}polyac-

rylamide gel electrophoresis (SDS}PAGE) sample bu!er
(0.015

M

Tris-HCl, pH 6.8, containing 2.5% (w/v)

glycerol, 1.25% (v/v)

-mercaptoethanol, 0.5% (w/v)

SDS, and 0.01% (w/v) bromophenol blue). Samples were
denatured by boiling for 3 min, centrifuged at 12,000g,
and then a 10

l volume was loaded onto a 10% separat-

ing polyacrylamide gel with a 4% stacking gel.Elec-
trophoresis was conducted at 100 V by a Biorad
Mini-Protean II electrophoresis system (Milan, Italy)
according to Laemli [9].Gels were visualised by a silver-
staining method [10].

3.3. Cell culture method

Human "broblast MRC5 cell line derived from

normal lung tissue (ATCC CCL 171) and cultured at

988

M. Bosetti et al. / Biomaterials 22 (2001) 987}994

Fig.1. Light microscopy appearance of con#uent human trabecular
osteoblast-like cells morphology, evidencing alkaline phosphatase ac-
tivity in the con#uent cultures.Micrograph at 400

; magni

"cation.

1

;10 cell/cm material in Dulbecco

's modi"ed Eagle's

medium (DMEM, Sigma) supplemented with 10% (v/v)
foetal

bovine

serum

(FBS,

Sigma),

penicillin

(100 U ml

\), streptomycin (100 g ml\) and 0.03%

(v/v)

L

-glutamine was used for preliminary cytocompati-

bility tests.

Osteoblast-like cells were obtained by enzymatic isola-

tion from adult human bone, trabecular fragments ob-
tained at surgery are treated as described in the literature
[11].Brie#y, particles 3}5 mm in diameter are plated in
90 mm tissue culture dishes with 0.2}0.6 g of bone/dish.
Treatment with bacterial collagenase removes any

"broblastic cells and undi!erentiated osteoblastic cells.

The bone explants in 10 ml ISCOVE'S supplemented
with 20% FBS and containing 50 U/ml penicillin,
15

g/ml streptomycin and 2 m

M

glutamine are cultured

at 373C in 95% air/5% CO.Outgrowths of cells from

the bone fragments appear within one week and form
a con#uent monolayer at 3}4 weeks.The isolated bone
cells had been characterised including osteoblastic mor-
phology, alkaline phosphatase expression and hormone
responsiveness (PTH, 1.25(OH)D) (Fig.1).

With the described isolation procedure, we reproduc-

ibly obtained an osteoblast-like cell population with
90}95% viability as established from trypan blue exclu-
sion tests.

For histological observations we chose a cell density of

2

;10 cells/cm to obtain intact cultures for longer cul-

ture periods, because after con#uence it is often observed
that the formed cell layer detaches spontaneously from
the

underlying

substrate.A

higher

cell

density,

1

;10 cells/cm was chosen for the cultures used for

biochemical determinations to obtain a well-de"ned sig-
nal with the spectroscopic techniques used.The choice

for these cell densities was based on pilot studies.To
promote attachment, the cells were allowed to adhere to
the disks at 373C for 1 h prior to the addition of the total
culture medium.To evidence the only e!ect of the mate-
rials on cell morphology and biochemistry, osteoblasts
were cultured in ISCOVE's medium supplemented with
only 5% hot inactivated foetal bovine serum and 2 m

M

glutamine.

3.4. Cell behaviour

Specimens were evaluated using #uorescence micro-

scopy.Because the chosen materials were not suited for
normal transmitted light microscopy, we observed cell
morphology and distribution with #uorescence micro-
scopy.After 6 h (adhesion test) and after 5 days (prolifer-
ation test) the materials were rinsed in PBS, "xed for
20 min at 603C and stained for 5 min in a 0.025% Acri-
dine Orange solution, a nucleic acid stain [12].Cell
morphology and cell number on each material were
evaluated using a #uorescent microscope connected to
a GIPS Image processing software with an I.T.I. PCV
board image analyser [13].Fibroblast and osteoblast cell
numbers was referred to a surface area of 0.167 mm

and

was evaluated at 250

; microscope magni

"cation.

3.5. Biochemical evaluation

Alkaline phosphatase activity and cell protein content

were measured.

Quantitative

alkaline

phosphatase

activity

(AP,

a marker for osteoblast expression) of the culture was
determined by an assay based on the hydrolysis of
p-nitrophenylphosphate to p-nitrophenol.Cultures were
collected after 1, 5 and 10 days of culture, rinsed three
times with PBS placed in 100

l PBS and sonicated.To

this solution, we added 100

l of substrate (1 m

M

p-nitro-

phenyl-phosphate in 1

M

diethanolamine#1 m

M

MgCl

pH 9.8). The mixture was incubated at 373C until the
colour was comparable with a standardised series
(a 20 m

M

p-nitrophenol solution) in about 15}30 min.All

samples, including the standardised series, were mea-
sured in duplicate on a Bio-Rad micro plate photospec-
trometer reader at 410 nm.

Cell protein content was measured using a commercial

protein quanti"cation kit (Bio-Rad): the optical densities
were read at 540 nm against a calibration curve using
bovine serum albumin as a standard.The results were
expressed in

g protein per 10 cells.

3.6. Statistics

Statistical analysis of the data was carried out using

a Dell computer equipped with SPSS for Windows soft-
ware.The Duncan test was performed to compare
adhesion and proliferation results for the four materials

M. Bosetti et al. / Biomaterials 22 (2001) 987}994

989

Fig.2. Top view of (a) AP40- and (b) RKKP-coated zirconia after 30 days soaking in acellular SBF.Note the surface covered by a self-grown layer
with the typical globular morphology of apatite.

tested; p-value was obtained from the ANOVA table.The
conventional 0.05 level was considered to re#ect statis-
tical signi"cance.

4. Results

4.1. Glass-coating characterisation

The morphological and structural characterisation

revealed that neither compositional modi"cations nor
crystallisation of additional phases occurred during the
coating preparation.XRD analyses demonstrated that
the coatings were completely amorphous and EDS ana-
lyses showed the presence of all the most signi"cative
signals of the elements belonging to the glass coatings
without a signi"cative zirconia di!usion through the
glass [7].As reported elsewhere [7], the adhesion be-
tween the substrate and the glass coatings was provided
by the in"ltration of the melted glass among the zirconia
granules in the "rst 50

m of the substrate.In this way,

a

`compositea intermediate layer (zirconia granules in

a glass matrix) was obtained, providing a very good
adhesion between the glass coating and the substrate and
a gradual modi"cation of the mechanical properties from
the substrates to the coatings.Almost the same behav-
iour was observed both in the AP40- and RKKP-coated
samples.A detailed description of the shear test results is
given in Ref.[7].

4.2. Soaking in simulated body yuid results

In Fig.2a the top view of an AP40-coated zirconia

sample after 30 days of soaking in SBF is reported.Its
surface is completely covered by a self-grown layer with
the typical globular morphology of apatites nucleated

from aqueous solutions.Fig.2b reports the top view
of a RKKP-coated zirconia sample after 30 days in
SBF.The higher magni"cation evidences the globular
morphology and the cracks due to the drying process.
Fig.3a and b show, respectively, the cross sections
of an AP40 and an RKKP coating on zirconia after
soaking in SBF, where four zones can be noticed:
(1) zirconia, (2) the intermediate composite layer of
zirconia plus glass, (3) the glass coating and (4) a
self-grown layer.The self-grown layer is about 100

m

thick.

The EDS analysis performed on the layer growth on

the coated samples revealed Si, P and Ca, with a Ca/P
weight ratio of 2.3, close to the theoretical value for
apatites (2.15). Fig. 4 shows the XRD pattern of an
AP40-coated sample after 30 days in SBF compared with
that of hydroxyapatite.Due to incomplete crystallisation
of the self-grown layer the peaks in the former pattern are
very weak, but a clear correspondence of these signals
with those of hydroxyapatite is evident.

The reactivity of AP40 and RKKP-coatings is very

similar.No signi"cant di!erences in terms of thickness
and structure of the self-grown apatite layer were found,
despite the slight di!erence in their chemical composi-
tion.

4.3. Protein adsorption results

Studies

of

serum

protein

adsorption

allowed

giving clues about the formation of a conditioning
layer on the material surfaces.The electrophoretic
patterns of the serum protein eluted from the surfaces
clearly showed an increased protein binding after
coating of the zirconia with AP40 and with RKKP
(Fig.5: lanes 4 and 5) compared to uncoated zirconia
(Fig.5: lane 3).

990

M. Bosetti et al. / Biomaterials 22 (2001) 987}994

Fig.3. Cross section of (a) AP40- and (b) RKKP-coated zirconia after 30 days soaking in acellular SBF.Note four zones: (1) zirconia; (2) zirconia plus
glass; (3) glass; (4) self-grown layer with the typical globular morphology of apatite.

Fig.4. XRD patterns of the self-grown apatite layer formed on AP40-
coated zirconia and of pure hydroxyapatite.

Fig.5.Silver-stained SDS}PAGE electrophoresis of proteins eluted
from the surfaces of zirconia (lane 3) AP40 glass-coated zirconia (lane 4)
RKKP glass-coated zirconia (lane 5).Lane 1: standard molecular
weights.Lane 2: FBS (1:20 dilution) used for the protein adsorption
experiments.

4.4. Cell-material results

The morphology of MRC5 "broblasts stained with

Acridine Orange, after 6 h of adhesion on bioactive
glass-coated zirconia, evidenced a higher cell spread
(Fig.6a) respect to cells observed at the same incubation
time on to zirconia-uncoated substrata (Fig.6b).At
5 days growth AP40- and RKKP-coated zirconia evid-
enced no di!erence between their cell isle organisation
(Fig.6c), characteristic of new tissue formation, whereas,
cells grown on zirconia substrata evidenced no organisa-
tion with lower cell number (Fig.6d).Such small "eld
widths were selected for presentation and comparison
because we have chosen high magni"cation to better
compare cell morphology on the di!erent materials.The

"elds selected are representative of all the specimens and

the quantitative results reported in Fig.7 de"ne better
cell number.

No signi"cant di!erence was observed concerning the

extent of "broblast attachment on to the three substrates

studied (zirconia, AP40- and RKKP-coated zirconia)
while "broblast proliferation showed a signi"cantly
greater ( p(0.05) cell growth on AP40- and RKKP-
coated zirconia than on uncoated control; no statistical
di!erences were seen between the two coated materials
(Fig.7a).Also, osteoblasts evidenced no signi"cant di!er-
ence in cell adhesion onto the three tested materials while
AP40- and RKKP-coating, induced a signi"cant increase
in osteoblast proliferation with p(0.05 (Fig. 7b).

4.5. Biochemical results

Osteoblast-like cells growth on the glass-coated mate-

rials, evidenced an alkaline phosphatase activity similar
to zirconia substrate at 1 day culture (3.91$1.15

M

for

AP40 and 4.69$2.10

M

for RKKP with respect to

5.24$0.18

M

for zirconia).An increased alkaline phos-

phatase activity was observed at 5 and 10 days growth

M. Bosetti et al. / Biomaterials 22 (2001) 987}994

991

Fig.6. Acridine Orange staining of human "broblasts (MRC5).Fluorescence microscopy at 400

; magni

"cation.The four photomicrographs showed

a representative surface area of 0.0682 mm

.High cell spread at 6 h of incubation (adhesion test) was seen in cells cultured on RKKP- and AP40-coated

zirconia (a) and in cells cultured on zirconia substrata used as control (b).Cell isle organisation was evidenced at 5 days growth (proliferation test) when
cells were cultured on RKKP- and AP40-coated zirconia (c).Lower cell growth and organisation was seen when cells were cultured on zirconia
substrata (d).

Fig.7. Fibroblasts and osteoblast-like cells quanti"cation on the three substrates studied.Cell number was the mean of 40 measurements in three
experiments (n"120) and was referred to a surface area of 0.167 mm

. (*) p(0.05. Space represent$followed by standard deviation.

992

M. Bosetti et al. / Biomaterials 22 (2001) 987}994

for the three materials studied with higher levels in AP40-
and RKKP-coated zirconia (9.98$0.80 and 9.94$
2.90

M

, respectively) with respect to zirconia substrate

(6.67$1.98

M

).

Osteoblast-like cell protein content, expressed in

g

protein per 10

cells, evidenced no statistical di

!erences

between zirconia- and glass-coated zirconia (11.11$
4.51

g for RKKP and 15.96$4.92 g for AP40)

with lower levels in

cells on

zirconia substrate

(7.23$1.42

g).

5. Discussion and conclusions

The goal of this investigation was to test the hypothesis

that the surface chemical characteristics of two di!erent
glass coatings, AP40 and RKKP, increase zirconia integ-
ration into bony tissues.The results obtained here clearly
show that these coatings permit the combination of the
mechanical performances of zirconia with the properties
of new bioactive glasses which demonstrates, with respect
to zirconia a better compatibility related to the aspects
considered.As discussed in a previous work [7], the com-
posite layer of zirconia plus glass formed during the
coating preparation (Fig.3a and b) guarantees a gradual
modi"cation of the mechanical properties from the sub-
strate toward the glass coating, assuring a good
adherence of the coating to the substrate.

As reported in the literature [2,14,15], a glass can be

considered bioactive if the formation of a hydroxyl}car-
bonate apatite layer on its surface after soaking in
a simulated body #uid is observed.The glasses used in
this work are based on SiO as initiator of the amorph-

ous network and they contain several alkaline ions
(mainly Ca

> and Na>).They are considered bioactive

because they can develop the apatite layer by a mecha-
nism widely discussed in the literature [15] based on
alkaline ions leaching, surface dissolution and formation
of a silica-gel layer, which provides good sites for the
nucleation and growth of apatite.The result obtained in
this study by EDS analysis of the layer growth on the
AP40- and RKKP-coated samples is correlated to the
apatite formation, according to its growth mechanism on
bioactive glasses [7].In this case, the bioactivity mecha-
nism was maintained even after the coating process.In
fact, previous studies [6}8] revealed that the time and
temperature conditions developed in order to prepare the
glass coatings did not change their original chemical
composition (for example, because of deep reactions with
the zirconia substrate).This is a necessary condition in
order to assure the bioactivity of the glass, which could
be a!ected by the presence of small amounts of some
multivalent cations [6,16].

Apatite-forming ability seems to be a very important

surface reaction in forming a bond between tissue and
bioactive materials [17] followed by adsorption of biolo-

gical structural proteins that play an important role in
the process of attachment, spreading and proliferation of
cells on the material surface [18].Our observations evid-
enced that the increased apatite formation on the two
glass coatings with respect to zirconia substrata is ac-
companied by an increased deposition of serum proteins.
Although cells have been demonstrated to adhere to
materials aspeci"cally [19], the adsorption on the surface
of speci"c receptors favours the rearrangement of the
cytoskeleton toward a pattern similar to that adopted by
the cell within the physiological extracellular matrix,
thus, promoting its appropriate metabolic functions [20].
For these reasons, biomaterials able to support the ad-
sorption of extra cellular matrix proteins with receptor
sites for tissue cells represent materials of choice for those
applications in which the integration of the prosthesis
within the tissue is required.Results from this study show
that both glass coatings studied increase serum protein
adsorption, enhance "broblasts and osteoblast-like cells
adhesion, spreading and growth, favouring micro-
scopic tissue/cell in growth and clinical implant "xation
improvement.Furthermore, the enzyme alkaline phos-
phatase, prominently associated with osteoblast matura-
tion and calci"cation [21], was used here as a marker
and con"rmed the better osteoblast expression in the two
glass-coated zirconia.In conclusion, our results indicate
that the surface chemical characteristics of the two glass
coatings AP40 and RKKP, with no great di!erences
between them, substantially enhance zirconia integration
with bone cells.

Acknowledgements

This work was realised under MURST Program

`Innovative Materialsa (L.95/1995) with contract: `Stu-
dio di biosmalti per la ricopertura di materiali inor-
ganici

a (Pos.118.2 * Roma, 29.05.1997).

We are grateful to Ing M.Mazzocchi and Ing A.

Krajewski.

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