Effect of various sterilization methods on the bioactivity of laser
ablation pseudowollastonite coating
F. A. Zuleta, P. Velasquez, P. N. De Aza
Instituto de Bioingenieria, Universidad Miguel Hernández, Elche, Alicante, Spain
Received 2 November 2009; revised 8 March 2010; accepted 14 April 2010
Published online 24 June 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31667
Abstract: Sterilization is required for using any material or
device in contact with the human body. The aim of this work
was to investigate the effect of four sterilization methods
(steam autoclave, hydrogen peroxide plasma, ethylene oxide,
and gamma sterilization) on the surface chemistry and in
vitro bioactivity of pseudowollastonite (psW) coatings in tita-
nium alloys substrates. psW coatings in Ti-6Al-4V substrates
obtained by laser ablation technique were sterilized and
immersed in Kokubo’s simulated body fluid (SBF) up to 30
INTRODUCTION
A biomaterial is a substance that has been engineered to take
a form which, alone or as part of a complex system, is used to
direct, by control of interactions with components of living
systems, the course of any therapeutic or diagnostic proce-
dure, in human or veterinary medicine.1 Because biomaterials
are selected based on the combination of mechanical, physical,
and biological properties, investigators have found the need to
enhance biocompatibility. The essential requirement for an ar-
tificial material to bond to living bone is believed to be the
formation of a biologically active apatite-like layer on their
surfaces in the body.2–5 Limited number of ceramic materials
called bioactive materials such as bioglass,2 hydroxyapatite
(HA),5 glass-ceramic A-W,6 diopside,7,8 and pseudowollastonite
(psW)9,10 are generally known to possess the ability to form
bone like apatite in the body environment.
Unfortunately, in most of the cases, mechanical strength
of these materials were not enough to use them as bulk
implants, and hence, their applications have been limited to
less loaded portions. Nevertheless, their field of application
can be expanded if used as coatings on metallic implants. In
this way, the benefits of the bioactive material are combined
together with the mechanical performance of metallic
implants. The most common example of this type of combi-
nation is titanium coated with HA.11–13 Our past and cur-
rent studies demonstrated the formation of a HA-like layer
on the surface of psW ceramic ‘‘in vitro’’14,15 and
‘‘in vivo.’’9,10,16,17 This finding is very significant, as it indi-
cates that the psW can be physically and chemically inte-
grated into the structure of living bone tissue, and therefore,
could be suitable for repair or replacement of living bone.
Correspondence to: P. N. De Aza; e-mail: piedad@umh.es
Contract grant sponsor: CICYT; contract grant number: MAT2006-12749-C02-02
Contract grant sponsor: Generalitat Valenciana; contract grant number: ACOMP/2009/173
V
C 2010 WILEY PERIODICALS, INC.
days. No changes in the chemical composition were noted af-
ter sterilization. However, a Ca/P-layer of different thickness,
identified as hydroxyapatite (HA) like was developed on all
the samples after soaking, although, the ethylene oxide steri-
lized samples present a nonhomogeneous and
55.9% thin-
ner HA-like layer. V C 2010 Wiley Periodicals, Inc. J Biomed Mater
Res Part B: Appl Biomater 94B: 399–405, 2010.
Key Words: bioceramics, sterilization, coating, bioactivity, in vitro
Because sterilization is the final step in manufacturing
any implant device, the effect of sterilization processes on
the modification of the first surface layers of these biomate-
rial surfaces must be clearly understood and characterized.
Furthermore, the relation between the physico–chemical fac-
tors and the tissue response must also be investigated.
Moreover, the requirement for a well-controlled surface
before implantation emphasizes these needs. Shabalovskaya
et al. reported on the correlation between the surface chem-
ical composition and some sterilization treatments.18,19 A
similar study on commercially pure titanium also showed
the potential damaging effect of some sterilization treat-
ments such as ethylene oxide and steam autoclaving on the
in vitro biological response.20 In spite of a wide application
of bioceramics in dentistry and medicine, surprisingly, there
are only a few publications devoted to the influence of the
sterilization conditions on the chemical properties of biocer-
amics.21–23
So, it appears to be both interesting and important to
study the influence of different sterilization methods on the
chemical properties of the bioceramics. The aim of this
work was to study the effects of the most accessible sterili-
zation techniques on the surface chemistry and in vitro bio-
activity of psW coatings in titanium.
MATERIALS AND METHODS
The starting material for this study was psW ceramic syn-
thesized by a solid stated reaction from a stoichiometric
mixture of calcium carbonate and high purity washed Bel-
gian sand (99.9 wt %). Details of psW preparation and
399
characterization can be found in previous publications.14–16
Pellets of synthesized psW were axially pressed in a press-
ing die of 30 mm of diameter at 150 MPa. A 1 wt % of a so-
lution of 0.11 wt % of a plasticizer Zusoplast 91/11 was
added and further sintered at 1350 C for 2 h. The sintered
disk had a diameter of about 25 mm and a relative density
of about 96%. This disk was used as target for pulsed laser
deposition.
The coatings substrates were 11 cm2 of Ti-6Al-4V with a
thickness of 1.5 mm. All the samples were obtained by follow-
ing the procedure already described in a previous work.13
This procedure combines the pulsed laser deposition and the
laser surface treatment. First, Ti-6Al-4V substrates were
coated with psW through pulsed laser deposition from a bulk
psW target. A pulsed Nd:YAG laser, delivering 10 ns pulses of
70 mJ at a wavelength of 355 nm and a repetition rate of 10
Hz, was used for laser ablation. The laser fluence on the psW
target was 2.5 J/cm2. The substrates were placed at 4 cm in
front of the target, and during deposition, the substrates were
at 550 C in a 10 Pa oxygen atmosphere. Each coating resulted
from 36,000 laser pulses.
After deposition, the samples were treated in air with a
continuous wave Nd:YAG laser to improve the density of the
coatings and their adhesion to the substrate. The laser
beam, with a wavelength of 1064 nm and a power of 35 W,
was focused on the coatings surface at 36 kW/cm2. The
laser beam scanned their entire area at a speed of 250
mm/s line by line with steps of 100 lm.
Twelve pellets of samples were processed under clinical
conditions by common sterilization process used in hospi-
tals including steam autoclave, hydrogen peroxide plasma,
ethylene oxide, and gamma sterilization irradiation steriliza-
tions. Nonsterilized psW-titanium samples were used as
controls
Steam autoclave (EVS 425.2.M)
Sealed bags with the samples were sterilized in autoclave
according to the standard procedure DIN 58946 used in
medicine. A set of samples were sterilized at 121 C. Briefly,
a single run of the steam sterilization procedure in auto-
clave includes three stages: temperature increasing up to
121 C during 15 min with simultaneous pressure increasing
from 0 to 1.4 mbar, sterilization at 121 C during 15 min at
pressure of 1.4 mbar, followed by fast (1–2 min) tempera-
ture reduction to 25 C. Thus, the total time of a single run
of the steam sterilization procedure is about 45 min.
Hydrogen peroxide plasma (Sterrad)
Plasma sterilization treatment was carried out in a large
volume microwave plasma (LMPTM) reactor. A set of sam-
ples were sterilized exposing to a 58% hydrogen peroxide
plasma for 15 min. The gas was introduced into the cham-
ber at an operating pressure of 500 MTorr, and plasma was
excited with 400 W of microwave power.
Ethylene oxide (Steri Vac EOE-M.)
The units were sterilized by exposing to a 100% ethylene
oxide atmosphere for 156 min at 55 C and a gas concentra-
400 ZULETA, VELASQUEZ, AND DE AZA
tion of 900 mg/L. After sterilization, the samples are aer-
ated with warm air flow (37 C) at atmospheric pressure for
330 min to remove residual ethylene oxide, and stored in
indicator bags and sealed.
Gamma sterilization (Rhodotron TT200)
Gamma irradiation sterilization with a 60Co irradiator was
performed by Ionmed S.A. (Cuenca, Spain). Randomized
samples were packed and sealed in indicator bags and
exposed to gamma irradiation in three cycles at doses of
18.71 kGy, 19.17 kGy, and 18.86 kGy, resulting in a dose of
56.34 kGy.
After being ultrasonically washed in acetone and rinsed
in deionized water, specimens were soaked in 100 mL of
simulated body fluid (SBF) in polyethylene bottles at
36.5 C. The immersion period of the specimens in SBF was
21 days. This period was chosen based on the results from
previous in vitro experiments performed in the SBF.14,15,24
At periodic intervals, the pellets were removed from the
fluid and were left to dry in air at room temperature.
Surface of the samples were characterized before and af-
ter the sterilization treatments. The crystalline structure of
the coatings was analyzed by X-ray diffractometry (XRD)
and Raman spectroscopy in the range 100–1200 cm1. The
surface morphology and composition were investigated in a
scanning electron microscope (SEM) using a Hitachi S-
3500N, equipped with detectors of secondary and backscat-
tered electrons and fitted with X-ray energy-dispersive spec-
trometer (EDS). The coatings thickness was measured with
a surface profilometer.
The structural changes of the psW coatings after immer-
sion in SBF for various periods were first analyzed by thin-
film X-ray diffraction (XRD), which enables analysis of up to
a 1-lm thick layer, depending on the incident X-ray beam
angle. The morphology of the surface and the cross sections
of the specimens were examined by SEM and EDS. The cross
sections studied in the SEM were polished to 1 lm finish
using diamond paste, gently cleaned in an ultrasonic bath,
and carbon coated. X-ray elemental maps of Si, Ca, and P of
the specimens were also obtained. The thickness of the pre-
cipitation layer formed on the psW coatings surfaces were
evaluated from the SEM photographs of the cross section of
the soaked psW coatings samples. Additional changes in
ionic concentration, using inductively couple plasma atomic
emission spectroscopy (ICP), were determined.
The product of the surface reaction was subsequently
characterized using transmission electron microscopy (TEM)
technique. Jeol Jem 2010 microscope was operated at 200
keV, and 80 cm camera length condition was applied for
selected area diffraction patterns (SAD). These specimens
were prepared by careful removal of the reaction layer from
the specimen’s surface using razor blade, and dispersing the
powder on the surface of alcohol in Petri dish. The powder
specimens were then collected on carbon coated copper
TEM grids of 200 mesh and carbon coated. Electron beam
transparent particles were chosen for TEM examination by
SAD, high-magnification imaging, and EDS analysis.
STERILIZATION METHODS ON BIOACTIVITY PSEUDOWOLLASTONITE COATING

FIGURE 1. SEM micrographs of the (A) surface of the psW-coating
before sterilization (control sample). (B) Steam autoclave sterilized
sample as a representative of all the sterilized samples.
RESULTS
Figure 1(A) shows SEM photograph of the surface micro-
structure of the coating after laser treatment. It can be seen
that the coating is characterized by a compact and rough
surface, with some partially melted particles. The analysis of
the composition of the coatings by EDX gives the following
atomic ratios: Si/Ca ¼ 0.9 and O/Ca ¼ 2.9. These values are
very close to those of the psW: Si/Ca ¼ 1 and O/Ca ¼ 3.
The microstructure of the steam autoclave method sterilized
psW-coating sample as a representative of all the samples
studied is shown in Figure 1(B). The picture is similar to
that of the control sample [Figure 1(A)] presenting a
smooth surface with some partially melted particles.
The results obtained with XRD on the psW-coating sam-
ples before and after sterilization are shown in Figure 2.
The XRD of the psW powder used as a raw material for the
coating is also include for comparative purposes. From Fig-
ure 2(A), it can be seen that the psW powder has high crys-
tallinity and there are only peaks corresponding to psW
(high-temperature form of wollastonite-2M). Some changes
can be noted in Figure 2(B), where in addition to the sharp
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | AUG 2010 VOL 94B, ISSUE 2
ORIGINAL RESEARCH REPORT
FIGURE 2. X-ray diffraction of (A) pseudowollastonite powder (B)
psW-coating before sterilization (control sample). (C) Steam auto-
clave, (D) Sterrad, (E) ethylene oxide, and (F) gamma sterilized sam-
ples. (*) ¼ Pseudowollastonite and () ¼ Ti-6Al-4V substrate.
peaks corresponding to psW, they appear those of the sub-
strate. The results obtained from Figure 2(B,F) demonstrate
that there are no differences before and after sterilization.
The diffracted peaks can be unequivocally identified as psW
plus some sharp peaks of the titanium substrate.
The results obtained with Raman spectroscopy on the
psW-coating samples before and after sterilization are
shown in Figure 3. These results demonstrate that no signif-
icant chemical changes happen when the samples are steri-
lized. Like the results of XRD, no differences before and af-
ter sterilization were observed for all the samples
investigated.
Figure 4 shows the SEM micrographs of the psW-coating
control, the steam autoclave, and ethylene oxide sterilized
samples after different soaking times into SBF. Steam, Ster-
rad, and gamma samples present similar behavior than the
psW-coating control with a similar type of the precipitates.
After 7 days of immersion, the material surface is covered
by a layer of small globular particles smaller than 5 lm in
diameter, some of which reaches the size of 6 lm after 30
days of soaking. After 1 week, these globular particles cover
homogeneously the whole surface of the material. The par-
ticles morphology does no further change with soaking
time, although the density of the layer increases.
FIGURE 3. Raman spectra of the (A) control sample (nonsterilized)
and (B) steam autoclave, (C) Sterrad, (D) EtO, and (E) gamma steri-
lized samples (All the peaks correspond to pseudowollastonite.).
401
FIGURE 4. SEM images of the samples surfaces. Control sample (nonsterilized), steam autoclave, and EtO sterilized samples after immersion
into SBF during (A) 7 days and (B) 30 days.

SEM micrographs of ethylene oxide sterilized samples,
namely, EtO in short, after different soaking times into SBF
are also shown in Figure 4. The behavior detected was simi-
lar to that previously mentioned, although a difference
should be pointed out. After soaking time of 7 days, isolated
aggregates of globular particles are detected on the surface,
partially covering the surface. EDX analysis shows that these
spherical particles are constituted by calcium and phospho-
rous. After 14 days of immersion, these aggregates of par-
ticles grow in size. This feature does not further change and
the surface is not fully covered by the spherical particles af-
ter 30 days of immersion [Figure 4(B), EtO)]. After 30 days,

402 ZULETA, VELASQUEZ, AND DE AZA STERILIZATION METHODS ON BIOACTIVITY PSEUDOWOLLASTONITE COATING

FIGURE 5. Ca/P-layer thickness change as a function of soaking time
into SBF: Control sample (nonsterilized) and steam autoclave, Sterrad,
EtO, and gamma sterilized samples.
the globular particles reach a size between 3 and 3.9 lm in
diameter.
Figure 5 shows the relationship between the soaking
time and the thickness of the Ca/P-layer developed on the
surface of the psW-coating. In the control sample (nonsteri-
lized), a significant thickness of Ca/P-layer has been formed,
reaching a total thickness of
24.3 lm (61 lm) after 30
days. The formation rate gradually slows from
2.3 lm per
day, after 7 days of soaking, to 0.5 lm per day, after pro-
longed soaking (4th week of soaking). That is to say, the
measurements reveal that the Ca/P-layer formation kinetic
depends directly on the square root of soaking time. In the
sterrrad, stem, and gamma sterilized samples, the morphol-
ogy of the surface product was the same, although the reac-
tion rate and the thickness of the layer after 30 days were
slight smaller. In Steam sterilized samples, the total final
thickness of the Ca/P-layer after 30 days of soaking is 23
lm (61 lm), in Sterrad sterilized samples is 21 lm (61
lm) and for Gamma method its 18.4 lm (61 lm).
The main difference was in the EtO sterilized samples.
Here it is worth to point out that the layer detected in the
EtO sterilized samples was not of uniform structure and
density throughout the sample. In the areas which the layer
was present a very low-layer formation rate of about 1 lm
per day, up to 9.4 lm (61 lm), after 7 days of soaking, was
detected. The layer reached a total thickness of 12.1 lm
(61 lm) within these zones.
Changes in the elemental ionic concentrations of SBF af-
ter 30 days of immersion are shown in Table I. The compo-
sition of the original SBF solution is also enclosed for com-
parison. The chemical analysis of the SBF used in the study
agreed with the composition of the one reported in the lit-
erature.25 It was found that calcium and silicon ion concen-
trations in SBF slightly increased over the exposure time
indicating partial dissolution of the psW-coating. On the
other hand, phosphorous ion concentration decreased more
significantly because of the precipitation of Ca-P phase on
the surfaces of the psW-coating. Although the formation of
Ca-P phase consumed some calcium ions, the calcium ions
dissolution from the psW-coating were more than those
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | AUG 2010 VOL 94B, ISSUE 2
ORIGINAL RESEARCH REPORT
consumed. This finding is in agreement with the fact that
the EtO samples do not present the surface fully covered by
the globular particles after 30 days of immersion, as can be
observed in Figure 4((B), EtO).
TEM was used to examine the ultra structure of the sur-
face product formed after the exposure of Steam autoclave
psW-coating sample to SBF for 30 days. High-resolution lat-
tice image was performed on the Ca/P crystals formed at
the surface of steam autoclave psW-coating sample. The
individual’s crystals were found to grow in close contact
forming continuous phase. (002) Lattice plane image of
0.344 nm spacing were well resolved in many areas [Figure
6(A)]. The resolved lattices were found to be defect free,
with no discontinuity or bending present. EDS analysis per-
formed on the thin crystals confirmed the presence of cal-
cium and phosphorous in the region [Figure 6(B)]. The
measured lattice spacing (obtained from the image negative)
matches well the values of 0.82 nm for HA reported in the
literature.26–31 The HA-like crystals which orientation varied
from (100) did no fulfill the lattice imaging conditions.
When the specimen was appropriately oriented, the SAD of-
ten displayed (002) arcs corresponding to the 0.344 nm lat-
tice spacing, indicating on the preferential orientation of the
HA-like crystals in the layer. Figure 6(C) displays a good
match between the measured values of the lattice spacings
obtained from the SAD pattern [Figure 6(C)] and ASTM data
from HA [Figure 6(D)].
DISCUSSION
The in vitro results carried out in SBF for the psW coatings
in titanium alloys substrates sterilized by steam autoclave,
hydrogen peroxide plasma, ethylene oxide, and gamma steri-
lization irradiation, suggest that all the sterilized materials
have in vitro bioactivity as shown by the formation of HA-
like phase in SBF solutions and the mechanism of this bio-
activity is in agreement with the mechanisms reported by
other researcher.32–34
When immersed psW-coating in SBF solution, both dis-
solution and HA-like precipitation occur on the surface on
the materials. At early stage, the dissolution of psW-coating
generally proceeds faster than precipitation. The psW-coat-
ing dissolved little before the HA-like precipitation process
became dominant. When the psW-coating gets in contact
with the SBF, a partial dissolution occurs, producing an ionic
exchange of Ca2þ for 2Hþ within the material network, lead-
ing to the formation of silanol groups on the surface of the
TABLE I. Concentration of Caþþ, Si4þ, and HPO2
4 , in SBF
before and after Immersion During 30 Days of the Control
Sample (Nonsterilized) and Steam Autoclave, Sterrad, EtO,
and Gamma Sterilized Samples
Samples (mg/L) Caþþ Si4þ HPO2
4
SBF control solution 100.36 – 93
Control pellet 104.23 8.64 63.98
Steam autoclave 102.58 7.582 65.27
Sterrad 106.39 7.24 74.37
Gamma 101.73 8.45 77.97
EtO 102.17 7.87 83.01
403

FIGURE 6. (A) HRTEM micrograph of the HA-like phase formed during
exposure to SBF after 30 days (steam autoclave sterilized sample), (B)
EDX analysis, (C) SAD pattern of the phase, and (D) lattice spacing of
the HA-like phase, taken from SAD of (C) and ASTM data.
psW-coating. Later, there is a partial dissolution of amor-
phous silica as SiO23 . This fact enhances the formation of
crystallization nuclei for the HA-like phase, which can be
formed from the high concentration of Ca2þ and HPO2 4
present in the media. Before immersion in SBF, the psW-
coating had an average thickness of 18 lm (61 lm). After
immersion in SBF for 30 days, its thickness decreased, for
all the sterilization methods, in average up to 12 lm (61
lm), and the precipitated HA-like grew uniformly in average
to a thickness of 21.62 lm (61 lm) for the control and all
sterilized samples except EtO, where the precipitation is not
homogeneous.
In the areas which the layer was detected, a low-layer
formation rate of about 1 lm per day was measured. After
7 days of soaking, the layer reached 9.4 6 1 lm attaining,
in these areas, a total thickness of 12.1 lm (61 lm). This
mean that, in the EtO sterilized samples, the total Ca/P-layer
formed, after 30 days of immersion was about 55.9% thin-
ner than in the other sterilized samples. This is in agree-
ment with the previously mentioned results obtained by
SEM during the surface studies [Figure 4(B), EtO] where the
EtO sterilized samples do not present, after 30 days of
immersion, the surface fully covered by the Ca/P globular
particles. On the other hand, the ICP-AES results of phos-
phorous ion concentration in SBF (Table I) shows that the
minor depletion in P corresponds to the EtO sterilized sam-
ples. One of the reasons for this difference, in the EtO steri-
lized samples, is due to some residual ethylene gas that pos-
sibly is present on the surface of the material after
404 ZULETA, VELASQUEZ, AND DE AZA
sterilization.35–39 As it was reported elsewhere,35–39 there
are problems with the toxicity and carcinogenicity of the re-
sidual ethylene gas in materials. The residual ethylene gas
should also affect the nucleation and crystal growth of HA-
like. Our next step will be to implant the sterilized materials
to study the tissue responds.
On the other hand, the aspect of all sterilized ceramic
surfaces after 30 days of soaking is comparable with that
shown by other silica-based materials. Generally, the HA-like
formation occurs in two stages: a previous formation of
globular particles followed by the apparition of aggregates,
which after 10–15 days of soaking are being to be indistin-
guishable.24,32,33,39 The comparison of the results obtained
for all sterilized materials points to a disperse nucleation of
an HA-like phase on EtO sample. Globular aggregates can be
detected from the 1st week of soaking and are heterogene-
ously distributed over EtO sample surface. Instead, the other
sterilized methods samples seems to nucleate homogene-
ously so that the observed layer is formed from a larger
number of crystallization nuclei homogeneously distributed
since the 1st week of immersion. The individual growth of
these leads to the HA-like layer formation. The comparison
of the results obtained for all sterilized materials points also
to a disperse nucleation of an HA-like phase on EtO sample.
Globular aggregates can be detected from the 1st week of
soaking and are heterogeneously distributed over EtO sam-
ple surface. Instead, the other sterilized methods samples
seems to nucleate homogeneously so that the observed
layer is formed from a larger number of crystallization
nuclei homogeneously distributed since the 1st week of
immersion. The individual growth of these leads to the HA-
like layer formation.
CONCLUSIONS
The influence of four sterilization methods (steam autoclave,
hydrogen peroxide plasma, ethylene oxide, and gamma steri-
lization) on the surface chemistry and the in vitro bioactiv-
ity of psW coatings in Ti-6Al-4V substrates were
investigated.
The XRD and Raman spectroscopy results appeared basi-
cally similar; it has been shown that there was no signifi-
cant modification of the psW-coating structure in the sur-
face of the samples by the four sterilization methods used.
SEM observation, after the SBF soaking, showed essen-
tially identical surface morphology regardless of the sterili-
zation by ethylene oxide, where the HA-like layer does not
fully cover the surface of the samples after 30 days of
immersion. One of the reasons for this difference is due to
some residual ethylene gas that possibly is present on the
surface of the EtO sterilized samples after sterilization. This
residual ethylene gas should affect the nucleation and crys-
tal growth of HA-like.
The sterilized materials have in vitro bioactivity with the
formation of an HA-like layer on the surface of all the sam-
ples, although the ethylene oxide sterilized samples present
a
55.9% thinner and nonhomogeneous HA-like deposition
throughout the samples. Therefore, we recommended not
using the EtO sterilized method for this type of materials.
STERILIZATION METHODS ON BIOACTIVITY PSEUDOWOLLASTONITE COATING

ACKNOWLEDGMENTS
The authors thank Dr. Luna from Ionmed SA (Cuenca, Spain)
for the gamma sterilization and Dr. F. A. Lopez Prats for the
steam autoclave, hydrogen peroxide plasma, and ethylene ox-
ide sterilizations.
REFERENCES
1. Williams DF. On the nature of biomaterials. Biomaterials 2009;30:
5897–5909.
2. Hench LL, Spinter RJ, Allen WC, Greenlee TK, Jr. Bonding mech-
anisms at the interface of ceramic prosthetic materials. J Biomed
Mater Res 1971;2:117–141.
3. De Aza PN, Luklinska Z, Santos C, Guitian F, De Aza S. Mecha-
nism of bone-like apatite formation on a bioactive implant in
vivo. Biomaterials 2003;24:1437–1445.
4. De Aza PN, De Aza AH, De Aza S. Crystalline bioceramic materi-
als. Bol Soc Esp Ceram Vidrio 2005;44:135–145.
5. Clèries L, Fernández-Pradas JM, Morenza JL. Bone growth on and
resorption of calcium phosphate coatings obtained by pulsed
laser deposition. J Biomed Mater Res 2000;49:43–52.
6. Ohtsuki C, Aoki Y, Kokubo T, Bando Y, Neo M, Yamamuro T,
Yacamura T. Characterization of apatite layer formed on bioactive
glass-ceramic A-W. Bioceramics 1992;5:87–94.
7. Nonami T, Tsutsumi S. Study of diopside ceramics for biomateri-
als. J Mater Sci Mater Med 1999;10:475–479.
8. De Aza PN, Luklinska ZB, Anseau MR. Bioactivity of diopside ce-
ramic in human parotid saliva. J Biomed Mater Res B Appl Bio-
mater 2005;73:54–60.
9. De Aza PN, Luklinska ZB, Martı́nez A, Anseau MR, Guitian F, De
Aza S. Morphological and structural study of pseudowollastonite
implants in bone. J Microsc-Oxford 2000;197:60–67.
10. De Aza PN, Luklinska ZB, Anseau MR, Guitian F, De Aza S. Trans-
mission electron microscopy of the interface between bone and
pseudowollastonite implant. J Microsc-Oxford 2001;201:33–43.
11. Pawlowski L. Thick laser coatings: A review. J Therm Spray Tech-
nol 1999;8:279–294.
12. Ding ST, Ju CP, Chern Lin JH. Morphology and immersion behav-
ior of plasma-sprayed hydroxyapatite/bioactive glass coating. J
Mater Sci Mater Med 2000;11:183–190.
13. Fernández-Pradas JM, Serra P, Morenza JL, De Aza PN. Pulser
laser deposition of pseudowollastonite coatings. Biomaterials
2002;23:2057–2061.
14. De Aza PN, Guitian F, De Aza S. Bioactivity of wollastonite
ceramics: In vitro evaluation. Scr Metall Mater 1994;31:1001–1005.
15. Carrodeguas RG, De Aza AH, De Aza PN, Baudin C, De Aza S,
Arribas JJ, Lopez-Bravo A. Assessment of natu ral and synthetic
wollastonite as source for bioceramics preparation. J Biomed
Mater Res A 2007;83:484–495.
16. De Aza PN, Luklinska ZB, Anseau MR, Guitian F, De Aza S. Mor-
phological studies of pseudowollastonite for biomedical applica-
tion. J Microsc-Oxford 1996;182:24–31.
17. Minarelli-Gaspar AM, Saska S, da Cunha LR, Bolini PDA, Carrode-
guas RG, De Aza AH, Pena P, De Aza PN, De Aza S. Comparison
of the biological behaviour of wollastonite bioceramics prepared
from synthetic and natural precursors. Key Eng Mater 2008;
361–363:1083–1086.
18. Shabalovskaya S, Andereeg J. Surface spectroscopic characteriza-
tion of NiTi equiatomic shape memory alloys for implants. J Vac
Sci Technol A 1995;13:2624–2632.
19. Shabalovskaya S, Cunnick J, Andereeg J, Harmon B, Sachdeva R.
Preliminary data on in vitro study of proliferative rat spleen cell
response to NiTi surfaces characterized using ESCA analysis. In:
Proceedings of the International Conference on Shape Memory
and Superelasticity; Pacific Grove, CA, 1997.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | AUG 2010 VOL 94B, ISSUE 2
ORIGINAL RESEARCH REPORT
20. Vezeau PJ, Koorbusch GF, Draughn RA, Keller JC. Effects of
multiple sterilization on surface characteristics and in vitro bio-
logic responses to titanium. J Oral Maxillofac Surg 1996;54:
738–746.
21. Dorozhkin V, Schmitt M, Bouler JM, Daculsi G. Chemical transfor-
mation of some biologically relevant calcium phosphates in aque-
ous media during a steam sterilization. J Mater Sci Mater Med
2000;11:779–786.
22. Suwanprateeb J, Tanner KE, Turner S, Bonfield W. Influence of
sterilization by gamma irradiation and of thermal annealing on
creep of hydroxyapatite-reinforced polyethylene composites.
J Biomed Mater Res 1998;39:16–22.
23. De Aza PN, De Aza AH, Herrera A, Lopez-Prats FA, Pena P. The
influence of sterilization technique on the morphology and the
in vitro bioactivity of pseudowollastonite ceramic. J Am Ceram
Soc 2006;89:2619–2624.
24. Alemany MI, Velasquez P, de la Casa-Lillo MA, De Aza PN. Effect
of materialś processing methods on the ‘‘in vitro’’ bioactivity of
wollastonite glass-ceramic materials. J Non-Cryst Solids 2005;351:
1716–1726.
25. Kokubo T, Kushitani H, Sakka S, Kitsugi T, Yamamuro T.
Solutions able to reproduce in vivo surface-structure changes in
bioactive glass-ceramics A-W. J Biomed Mater Res 1990;24:
721–734.
26. Robinson RA. An electron microscopy study of the crystalline
inorganic component of bone and its relationship to organic ma-
trix. J Bone Joint Surg Am 1952;34:389–435.
27. Spechman TW, Norris WP. Bone crystallites as observed by use
of the electron microscope. Science 1957;126:753.
28. Spector M. High resolution electron microscopy study of lattice
image in biological apatites. J Microsc-Oxford 1975;103:55–62.
29. Kerebel B, Daculsi G, Verbaere A. High resolution electron micros-
copy and crystallographic study of some biological apatites.
J Ultrastruct Res 1976;57:266–275.
30. Daculsi G, Legeros RZ, Deudon C. Scanning and transmission
electron microscopy and electron probe analysis of the interface
between implants and host bone. Osseo-coalescence versus
osseo-integration. Scanning 1990;4:309–314.
31. Daculsi G, Legeros RZ, Legeros JP, Mitre D. Lattice defects in cal-
cium phosphate ceramics: High resolution TEM ultrastructural
study. J Biomed Mater Res B Appl Biomater 1991;2:147–152.
32. Ohtsuki C, Kokubo T, Yamamuro T. Mechanism of apatite forma-
tion on CaO-SiO2- P2O5 glasses in a simulated body fluid. J Non--
Cryst Solids 1992;143:84–92.
33. Kokubo T, Hayashi T, Sakka S, Kitsugi T, Yamamuro T. Bonding
between bioactive glasses, glass-ceramics or ceramics in simu-
lated body fluid. Yogyo-Kyokai-Hi 1987;95:785–791.
34. Kokubo T. Surface chemistry of bioactive glass-ceramics. J Non--
Cryst Solids 1990;120:138–151.
35. Harper EJ, Braden M, Bonfield W, Dingeldein E, Wahlig H. Influ-
ence of sterilization upon a range of properties of experimental
bone cements. J Mater Sci Mater Med 1997;8:849–853.
36. Lewis G, Mladsi S. Effect of sterilization method on properties of
Palacos R acrylic bone cement. Biomaterials 1998;19:117–124.
37. Zahraoui C, Sharrock P. Influence of sterilization on injectable
bone biomaterials. Bone 1999;25:63S–65S.
38. Sainz VD, Rodriquez N, Fuentes EG, Guerra M, Arcis SW, Peon
AE, Diaz AJ, Zaldivar SD. Radiation sterilization of abifunctional
cement formulation of hydroxilapatite-plasterpolymers. Ann NY
Acad Sci 1999;18:64–70.
39. Ohura K, Nakamura T, Yamamuro T, Kokubo T, Ebisawa T,
Kotoura Y, Oka M. Bone-bonding ability of P2O5- free CaOSiO2
glasses. J Biomed Mater Res 1991;25:357–365.
405