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Biological evaluation of the effect of magnetron sputtered
Ca/P coatings on osteoblast-like cells in vitro
J. E. G. H u lsh o ff K. van D ijk J. P. C. M. van der W aerden J. G. C. W o lk e
L. A. G in s e l
J. A. Jansen1'*
JDepartment of Oral Function , Laboratory of Biomaterials, Dental School , University of Nijmegen, P .O . Box 9101,
6500 HB Nijmegen , The Netherlands; 2Department of Biomaterials, School of Medicine, University of Nijmegen,
Rijnsburgerweg 10, 2333 A A Leiden, The Netherlands; 3Department of Cell Biology and Histology, Faculty of Medical
Sciences, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
,
1
, 1
,
1
/
'
2
,
3
A rat bone marrow cell culture was used to evaluate the
osteogenic potential of amorphous and crystalline thin cal­
cium phosphate (Ca/P) coatings. The coatings were depos­
ited on titanium discs using a radiofrequency magnetron
sputter procedure. Amorphous and crystalline plasma
spray Ca/P coated and noncoated titanium discs served as
reference material. The cellular behavior was analyzed with
quantitative (attachment and proliferation rates) and qual­
itative (scanning electron microscopy) techniques. No sig­
nificant differences were found in cell attachment and pro­
liferation rates between the various materials. Scanning
electron microscopy showed extracellular matrix formation
after 18 days of culture on amorphous plasma-sprayed and
the two types of magnetron sputtered coatings. Further­
more, no severe degradation of the magnetron sputtered
coatings was observed. They even appeared to induce ap­
atite formation, On basis of the results, we conclude that
magnetron sputtering appears to be a promising method to
manufacture bioactive ceramic coatings. © 1995 John Wiley
INTRODU CTION
iments have b egu n on the application of m agnetron
sputtering for the production of Ca/P coatings on
metal and plastic substrates.
Radiofrequent (RF) m agnetron sputter-coating is
p erfo rm ed u s in g com m ercially availab le sp u tter
equipm ent, In a preliminary study, w e sh o w ed that
this technique produces highly a d h esiv e, uniform
coatings.9 Energy-dispersive X-ray analysis, X-ray
diffraction, and atomic absorption spectrom etry co n ­
fir m e d th a t th e s p u t t e r e d la y e r s w e r e w e l l crystallized Ca/P ceramic w ith a Ca/P ratio varying
betw een 1.9 and 2.5. In vitro and in vivo experim ents
dem onstrated the biocompatibility of the coatings.
Based on these results, w e concluded that m agnetron
sputtering is a prom ising m ethod for forming a Ca/P
coating onto an implant material. N everth eless, s e v ­
eral problems, e .g ., the endurance and the Ca/P ratio
of the coating, have to be solved before clinical use of
m agnetron sputtered implant system s can be consid­
ered. To elucidate these problems and to gain more
insight into the physicochem ical and biological p rop ­
erties of the sputtered coatings extensive in vitro and
in vivo experim ents have to be done. In this article w e
present the first stage of the biological evaluation by
using rat bone marrow cell cultures to investigate the
Since about 10 years, the application of calcium
phosphate (Ca/P) coatings has becom e an accepted
technique to improve the biologic behavior of metallic
dental and orthopaedic im plants.1 Currently, plasmaspraying is the most frequently used m ethod for the
deposition of Ca/P coatings on implant materials.
However, despite the described beneficial effect of
these coatings on the bone response,
there is also
so m e concern regarding their lon g term perfor­
m ance.7 For example, it is reported that the Ca/P coat­
ing resorbs over time and loses its mechanical integ­
rity by lack of adherence.8 Since the clinical conse­
q u e n c e s of th e se fin d in g s still h a v e n o t b e e n
understood completely, more know ledge and expe­
rience has to be obtained about the characteristics of
Ca/P coatings. To achieve this goal, besides the fur­
ther improvement of the plasma-spray technique, the
efficacy of more appropriate deposition m ethods has
to be investigated. Therefore, in our laboratory exper­
«
i-
*To whom correspondence should be addressed.
& Sons, Inc,
Journal of Biomedical Materials Research, Vol. 29, 967-975 (1995)
© 1995 John Wiley & Sons, Inc.
C C C 0021-9304/95/080967-09
HULSHOFF ET AL.
968
Na (3-glycerophosphate (Sigma), 10~8M dexamethasone (Sigma) and antibiotics at one-tenth of the con­
centration described above. Finally, cultures were in­
cubated in a humidified atmosphere of 95% air, 5%
C 0 2 at 37°C. The phase contrast photomicrograph of
Figure 1 shows the general appearance of a primary
culture. After 5 to 7 days in culture, cells were har­
vested by trypsin treatment (0.25% w/v trypsin) and
used for the experiments.
MATERIALS A N D M ETHODS
Materials
Commercially pure titanium (cpTi) discs with a di­
ameter of 12 m m were used. They were polished to
for 15 min in
alcohol. After drying, the discs were left untreated or
provided w ith four different Ca/P coatings. Tw o
ty p es of Ca/P coatings w e re d e p o s ite d u s in g a
plasma-spray technique. The first was used as a p ­
plied (HA-PS), the second was subjected to a heat
treatment for 2 h at 6O0°C after coating deposition
(HA-PS/ht). The thickness of these coatings w as
about 50 (xm.
Two other types of Ca/P coating were produced
using the RF m agnetron sputter technique, The s p u t­
ter process was done at a pow er level of 800 W a n d a
process pressure of 5 x 10_ c mbar using argon gas.
The titanium discs were m o u n ted on a water-cooled
substrate holder. O ne coating was produced w ith a
rotating substrate holder (Ca/P-r), while the other
in an
was c
indexed position (Ca/P-i), The thickness of the m a g ­
netron sputtered coatings varied betw een 2.5-4,0
|xm.
All deposited coatings were characterized by scan­
ning electron microscopy
, X-ray
(XRD),
Before use in
were autoclaved for 30 min at 120°C
, all discs
cells were confirmed by several parameters, includof c a J c iu m p h o s p h a te m a te ria l by Von K o ssa's
m ethod, and immunostaining. For the immunocytochemical identification we used a specific monoclonal
antibody ( E l l ) 13 against a cell membrane associated
antigen of rat osteoblasts. A goat-antimouse/FITC
conjugate was used to locate the monoclonal anti­
body. We noted that 12-day-old cultures on micro­
scopic glass slides showed a positive anti-osteoblast
reaction. No attempts were made to detect the pos­
sible influence of different substrates on the osteo­
blast expression of the cell cultures.
C ell attachment assay
The test substrates were positioned on the bottom
of sterile 24-well plates (Greiner, Greiner B.V., Alp h e n a/d Rijn, The Netherlands) and a total of 1.0 mL
of culture medium containing 5 x 104 cells was added
to each substrate. The cultures were incubated for 8 h
at 37°C in 5% C 0 2/ai:r. After incubation, the wells
w ere w ashed twice using phosphate buffered saline
(PBS) to remove nonattached cells. Then the substrates were taken out of the wells and placed into
counting tubes. To detach the attached cells, 1 ml, of
trypsin was added and the tube was placed for 8 min
at 37°C. Iso tone solutio n was added a nd the cells
Cell isolation and culture
Osteoblast-like cells were prepared using the rat
bone marrow (RBM) culture m eth o d as described by
r10 Davies/11 a n d De Bruijn.12
Briefly, both femora of young adult male Wistar
rats (weight 100-120 g, age 40-43 days) were rem oved
and washed four times with a-Minimal Essential M e­
dium (a MEM, Gibco, Life Technologies B.V., Breda,
The Netherlands), containing 0.5 mg/mL gentamycin
(Gibco) and 3.0 |±g/mL fu n g izo n e (Gibco). A fter­
wards, the epiphyses were cut off and the diaphyses
flushed out, using a MEM supplem ented with 15%
fetal calf serum (FCS, heat induced at 56°C for 35 min,
Gibco), 50 jxg/mL of freshly prepared ascorbic acid
(Sigma, Chemical Co., St. Louis, MO., USA), 10 m.M
F ig u re 1.
marrow cell culture.
a
969
IN VITRO E V A L U A T IO N O F M A G N ETR O N SPUTTERED Ca/P COATINGS
were counted u sin g a Coulter Counter. After the
counting procedure the p resen ce of nondetached
cells was checked b y scanning electron microscopy of
the various substrate surfaces*
Two runs of experim ents were carried out. In each
run all materials w ere present in quintuple.
dium, but w ithout cells. After incubation, the discs
were processed for sca n n in g electron m icroscopy.
R ESU LTS
Characterization o f th e Ca/P coatin gs
Cell proliferation assay
RBM cell su sp en sion s, containing 5 X 104 cells,
were seeded on the experimental substrates as de­
scribed before and incubated at 37°C in 5% C 0 2/air.
After 8 h of incubation, the nonattached cells were
removed by PBS rinses. To each well 1 mL of fully
supplem ented m ed iu m w a s added and the specimens were incubated for 5 days. The m edium was
changed every other day. At the end of the incubation period, the m edium w a s discarded and the substrates were rinsed twice w ith PBS. Then, the sub­
strates were taken out of their w ells and placed into
counting tubes. After detachm ent by trypsinization,
isotone solution w a s added to count the number of
adherent cells using a Coulter Counter. Similar to the
attachment assay, noncoated and coated surfaces
were checked on the presence of nondetached cells.
The presented results are based on the average of
two separate experim ents. In each experiment all ma­
terials were present in quintuple,
Cell m orphology assay
RBM cell su sp en sio n (200 jxL per well, containing 1
x 104 cells) w as added to the test substrates as pre­
viously described for the cell attachment and prolif­
eration experiments. The cultures were incubated for
6 and 18 days at 37°C in 5% C 0 2/air. The culture
medium was changed every other day. After the var­
ious incubation periods, the nonattached cells were
removed by PBS rinses. The attached cells were fixed
in situ with 2% v/v glutaraldehyde in 0.1 M sodium
cacodylate buffered solution for 30 min at 4°C, rinsed
twice in cacodylate buffered solution, followed by de­
hydration through a graded series of ethanol. Subse­
quently, the specim ens w ere dried by tetramethylsilane. Finally, after sputter-coating with gold, they
were examined u sin g a Philips SEM-500 scanning
electron m icroscope at an accelerating voltage of
12 kV.
The possible influence of culture medium on the
noncoated and Ca/P coated titanium discs was also
examined. Therefore, som e discs were incubated for
6 and 18 days w ith fully supplem ented culture me-
A complete description of the characteristics of the
coatings has already b e e n g iv en e ls e w h e r e .14'15
In summary, SEM exam ination revealed that the
specimens were co v ered w ith an uniform coating.
Further, it w as o b served that the Ca/P~r coatings had
a sm oother surface m icrostructure, w h ile the Ca/P-i
surfaces show ed a polycrystalline appearance.
XRD patterns d em o n stra ted that the H A -PS and
HA-PS/ht coatings s h o w e d an am orphous/crystalline
structure. The crystallinity of the H A-PS coatings appeared to be 60%. After heat-treatm ent (HA-PS-ht
specimen) the crystallinity of the coatings increased
slightly to 65%.
In Figure 2 XRD patterns of the Ca/P-i and Ca/P-r
are sh o w n . T he a n a ly s is o f th e d iffr a c to g r a m s
show ed that the in d e x e d procedure resulted in a crystaIline Ca/p coatin g w ith a preferred (001) crystallographic orientation w ith the C-axis perpendicular on
the substrate surface (reflections 002, '102, 112, 202,
respectively; 25.9, 28.1, 32.4, and 34.0° 2-T heta).
Coatings prepared w ith the rotating substrate holder
show ed an am orp h ou s structure, w ith o u t an y sp e ­
cific reflection lines.
Infrared m e a s u r e m e n ts of the p lasm a-sp rayed ,
samples sh ow ed ab sorp tion bands characteristic of
P— O and O— H b o n d s. In the spectra of both sp u t­
tered coatings no O H b o n d s w ere detectable, T h ese
coatings sh ow ed a w id e peak over the region from
2800 to 4000 c m “ 1 indicative for w ater absorption at
the surface.16
Cell attachment
Table I sh ow s th e results of the osteoblast attach­
m ent experiments. Statistical testing of the findings,
using an one-w ay a n a ly sis of variance (A N O V A ) and
a multiple com parison p roced u re (N ew m a n - Keuls)
revealed that no sign ifican t differences existed be­
tween the attachm ent p ercen ta g es of RBM cells to the
various materials. D ata are unavailable for Ca/P-i
coatings due to d e ta c h m e n t of the coating during cell
trypsinization, w h ic h o b stru cted the orifice of the
counting tube. SEM in sp ectio n dem on strated that no
cells were left on th e various substrates after trypsin­
ization.
HU LSHOFF ET AL
970
counts
A
600
500
400
300
200
Ti
100
o
24
22
20
26
28
34
32
30
36
38
40
36
38
40
[2 0 ]
counts
B
1,800
CM
O
O
1,600
1,400
1 ,2 0 0
1,000
I
I
I
b
CM
800
600
400
I
«
i
200
0
20
22
24
26
28
30
32
34
[2 6 ]
Figure 2. X-ray diffraction pattern of the magnetron sputtered coatings: (A) amorphous Ca/P-r coating; (B) crystalline
Ca/P-i coating,
Cell proliferation
The results of the proliferation assay of RBM cells
on the various substrate materials are given in Table
L SEM examination of the trypsinized substrates, af­
ter the 5-day culturing period of the proliferation as­
say, demonstrated incom plete removal of the cells
from HA-PS-ht surfaces (Fig. 3). Therefore, these
data were excluded from the statistical analysis. One
w ay analysis of variance (ANOVA) and m ultiple
comparison procedure (N ew m an-K euls) revealed no
significant difference betw een titanium and the other
materials.
Cell morphology
Scanning electron microscopy show ed a compara­
ble cell morphology on all test surfaces. Cells cultured
on cpTi surfaces formed multilayers. N o systematic
signs of bonelike tissue formation were observed
(Fig. 4).
The morphology of RBM cells cultured on the dif­
ferent plasma-sprayed coatings was similar as de-
TABLE I
Numbers of Attached and Proliferated Rat Bone Marrow
Cells Cultured on the Various Substrate Materials
A ttachm ent
Proliferation
Results:
Substrate
M ean
SEMa
M ean
SEM
cpTi
HA-PS
HA-PS/ht
Ca/P-r
Ca/P-i
22624
22400
23928
28736
---
272
1408
1000
2736
---
218322
226395
145743
255854
249080
38948
38545
13117
53650
53846
SEM represents standard error of the mean.
F ig u re 3. S canning m icrograph sho w in g cells left on a
p lasm a-sp ray coated substrate left after trypsinization; bar
= 12.8 |xm.
scribed by De Bruijn. In short: after culture periods
of 6 days the cells were spread and exhibited filopodia s p a n n in g the m acropores produced by the
plasma-spraying. Extracellular matrix formation was
observed after 18 days of culture. No difference in
coverage with extracellular matrix existed between
the heat treated and nonheated plasma-sprayed coat­
ings.
The cellular response to the sputter-coated ceramic
substrates was identical to the plasma-sprayed sub­
strates, although the occurring processes were easier
to follow due to the smooth surface texture of the
sputtered coatings. Following incubation, the RBM
cells adhered and spread over the sputtered Ca/P sur­
face, resulting in a confluent cell layer at the sixth
day. After 18 days formation of a granular mineral­
ized layer was seen. No differences in appearance
and coverage of this ECM layer was observed be­
tween both types of sputtered Ca/P ceramic (Figs. 5
Figure 5. Scanning m icrographs of RBM cells c u ltu re d foi­
l s days on a Ca/P-r coated specim en. Four different layers
can be observed: (A) RBM cell m ultilayer, (B) g ra n u la r m in ­
eralized layer, (C) Ca/P-r coating, a n d (D) original tita n iu m
surface. The coating is covered w ith small spherulites. The
specimens show n u m ero u s cracks, As sh o w n by the c o n ­
tinuation of cell structures at both sides of th ese cracks,
they are caused by the drying m e th o d u sed for SEM p r e p ­
aration. (a) bar = 6.7 |xm; (b) bar = 3.3 fxm.
Figure 4. S canning electron m icrograph show ing a m ulti­
layer of RBM cells including the underlying titanium su r­
face. A lth o u g h som e globular interfacial matrix formation
can be o b serv ed , it has to be noted that this matrix was not
system atically present; bar = 3.2 \xm.
and 6). Examination of cracks in this layer, which
were due to the drying process, made apparent that
this layer was rather thick (about '1 ¿im). In areas
where parts of the ECM w ere elevated, a fibrillar
mineralization foci were observed. In addition, expo­
sure of the underlying ceramic surface proved that 18
days after incubation a substantial thickness of the
coating was still present. Further inspection revealed
the precipitation of tiny spherulites (about 0.35 fxm)
covering this original coating surface.
This formation of spherulites on the surface of the
sputtered coatings was also seen after 6 days of incu­
bation in fully supplemented culture medium w ith ­
out cells. Figure 7(a) and (b) shows the SEM p h o to ­
graph of these specimens. The small cracks present in
the coatings are a result of the SEM preparation pro­
cess. This kind of spherulites was also formed on the
control HA-PS specimens, while on the heat treated
HULSHOFF ET AL.
972
(b)
(a)
Figure 6.
for 18 d a y s on a Ca/P-i coated specim en. A po rtion of the cell
S c a n n in g micrugic
i in the ECM. F urth er, p arts of
m u ltilay er an d ECM are raised, resu ltin g in a good v iew on
to the
the co atin g b e c am e d e ta c h e d from the titan iu m disc
. (a) b ar = 13 jxm; (b) b a r = 3 fim.
ECM. It can be se e n th a t th e co atin g has follow ed perfectly the
plasma-sprayed surfaces deposits did not develop.
After 18 days of incubation, these spherulites in ­
creased in num ber, as well as in size, on the C’a/P-r,
Ca/P-i, and HA-PS coatings, In contrast, the HA-PS/
ht coating still did n o t sh o w a sign of this kind of
structures (Fie. 8).
D IS C U S S IO N A N D C O N C L U SIO N S
The observations presented here again confirm the
suitability of in vitro experim entation for studying
, but e
uation
on the electron microscopical level. Moreover, cell
culture techniques have the advantage of offering a
other hand, the alkaline phosphatase expression and
parathyroid hormone response, characteristic of osteoblastic phenotype expression,
were
cultures grown on hydroxyapatite. Massas suggested
that, since bone cell populations are heterogeneous,
this increase is caused by the higher capacity of hy­
droxyapatite to support the proliferation and differ­
entiation of osteogenic cells. A similar phenom enon
can have occurred in our RBM cell cultures, resulting
in more osteoblast-like cells together with ECM for­
mation on the Ca/P coated specimens. This supposed
influence of the substrate material upon osteoblast
phenotype is further supported by Lian.19 She dem ­
onstrated that osteoblast proliferation is related to the
.
-I_______ ...........................
................ ................
■
'1 8
__________ _
........
*
for the in vivo behavior of titanium and Ca/P coated
in i
20-22
The rat bone marrow cells attached and grew on all
substrate surfaces. The question was raised w h eth er
this was correlated to the induction and formation of
atrix. For example, no s
of bone like tissue formation could be found on titanium substrates. Although, a definite reason for this
effect of titanium is difficult to express, a possible
is given by M a ssa s.17 He cultured rat parietal bone cells on titanium and hydroxyapatite substrates. After an
were equally proliferating on both materials. O n the
: ... u
in various
h
In contrast to these findings Davies
ECM formation on titanium surfaces. A reason for
this difference may be that in these studies the cells
were enzymatically released from the tissue culture
flasks w ith 0.01% trypsin solution, while we used
0.25% solution. Since various proteolytic enzymes
and concentrations can have a
feet on morphology, growth rate,
activity, : ~ ~ this might result in a
no type an d ECM secretion.
Considering the attachment and
as-
IN VITRO EVALUATION OF MAGNETRON SPUTTERED Ca/P COATINGS
Figure 7. S cann in g m icro g rap h s of the m ag n etro n sp u t­
tered coatings so ak ed in fully su p p le m e n te d culture m e­
d iu m for 6 d ay s. All coatings s h o w cracks, w hich are
caused by th e SEM p re p a ra tio n process. Spherulitic accre­
tions are visible at the surface of both coatings (arrows), (a)
Ca/P-r, bar = 3.2 firn; (b) Ca/P-i, bar = 3.2 \xm.
say, there are two other findings that need further
explanation. First, the n u m b er of attached ceils
seeded on Ca/P-i coatings could not be measured by
Coulter Counter. Examination revealed that detached
coating particles obstructed the orifice of the counting
tube. SEM inspection of the Ca/P-i coatings before
their use in the cell studies, showed that these sur­
faces had a polycrystalline appearance. This grain
structure is induced by internal stresses in the coating
due to the combination of heating of the titanium
substrates during the magnetron sputter process and
differences in thermal expansion coefficient to sub­
strate and deposited ceramic coating. Consequently,
after short incubation periods, when still no addi­
tional surface layers are deposited, this stressed Ca/
P-i coating can easily be removed by rinsing proce­
dures as used in the counting process.
A second issue is the incomplete removal of disso­
ciated cells from the heat-treated plasma-spray coated
substrates in the growth experiments. This observa­
973
tion justifies the conclusion that cellular proliferation
has to be considered similar on all investigated s u r­
faces. Besides, it emphasizes the importance of visual
surface inspection in these kinds of experiments.
Finally, the study to the influence of culture m e ­
dium on the Ca/P coated discs revealed that small
spherulites were deposited on Ca/P-i, Ca/P-r, ancl
HA-PS substrates, but not on the HA-PS/ht speci­
mens. This surface behavior corresponds well with
one of our physicochemical studies,26 in which the
dissolution and precipitation properties of am o r­
phous, amorphous/crystalline, and crystalline s p u t­
tered and plasma-sprayed coatings were determined
after incubation in simulated body fluid (SBF). The
Ca, P, and Mg concentration of SBF is almost equal to
human blood plasma.27 The coated specimens were
soaked for 7 days in SBF and the fluid was changed
daily. Spectrometric analysis showed, that for am or­
phous and amorphous/crystalline substrates the Ca
and P concentration of SBF, after an initial increase,
decreased until a constant concentration was o b ­
tained. HA-PS/ht did not change the ion concentra­
tion of SBF. SEM examination of the samples d em o n ­
strated the formation and growth of apatite nuclei on
all surfaces, except for heat treated substrates. O n
basis of the spectrometric data and similar as d e ­
scribed by Li28,29 for silica gel, the start of this precip­
itation process can be attributed to a local supersatu­
ration of both Ca and P caused by dissolution of these
ions from the ceramic coating. T he s u b s e q u e n t
growth of the nuclei is controlled by the Ca and P
ions already present in SBF. Tissue culture media
contain also physiological concentrations of Ca, P,
and Mg ions. In addition, they are buffered at the
same pH as SBF. Therefore, it can be assum ed that,
comparable to SBF, apatite induction processes will
take place after immersion of Ca/P coated specimens
in fully supplemented culture medium without cells.
It will be evident that this apatite formation will in­
fluence the bonding properties and the final b o n e substrate interface. Nevertheless, the full implication
and clinical consequences of this phenom enon for
sputter deposited Ca/P coatings have to be investi­
gated further in transmission electron microscopical
studies. Also, in these experiments, the required
coating thickness to induce the most optimal apatite
deposition has to be determined.
In summary, the experiments dem onstrated that
RBM cells cultured on magnetron sp u ttered Ca/P
coatings stimulated the formation of ECM. Although
the sputtered coatings detached in the short term at­
tachment studies, no severe degradation of the coat­
ings was observed in the prolonged culturing assays.
In contrast even, sputtered coatings appeared to in ­
duce apatite formation. Based on these results, it can
be concluded that magnetron sputtering is a promis-
H U Í,SI i O i I ET A L.
974
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IN VITRO EVALUATION OF M AGNETRON SPUTTERED Ca/P COATINGS
ing m ethod to manufacture bioactive ceramic coat­
ings. If this can be confirmed in vivo , this coating
process may be of advantage over the currently used
techniques.
This study was supported by the Netherlands Technol­
ogy Foundation (STW). In addition, the authors thank Dr.
A. Wetterwald from the Pathophysiological Institute of the
University of Bern, Switzerland, who provided them with
the antibody E ll.
References
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2.
3.
4.
5.
6.
7.
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R. G. T. Geesink and M. T. Manley (eds.), Hydroxylapatite Coatings in Orthopaedic Surgery, Raven Press,
New York, 1993.
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Received June 3, 1994
Accepted February 21, 1995