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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 ( DOI: 10.1002/jbm.b.31667

Abstract: Sterilization is required for using any material or days. No changes in the chemical composition were noted af-
device in contact with the human body. The aim of this work ter sterilization. However, a Ca/P-layer of different thickness,
was to investigate the effect of four sterilization methods identified as hydroxyapatite (HA) like was developed on all
(steam autoclave, hydrogen peroxide plasma, ethylene oxide, the samples after soaking, although, the ethylene oxide steri-
and gamma sterilization) on the surface chemistry and in lized samples present a nonhomogeneous and 55.9% thin-
vitro bioactivity of pseudowollastonite (psW) coatings in tita- ner HA-like layer. V C 2010 Wiley Periodicals, Inc. J Biomed Mater

nium alloys substrates. psW coatings in Ti-6Al-4V substrates Res Part B: Appl Biomater 94B: 399–405, 2010.
obtained by laser ablation technique were sterilized and
immersed in Kokubo’s simulated body fluid (SBF) up to 30 Key Words: bioceramics, sterilization, coating, bioactivity, in vitro

INTRODUCTION Because sterilization is the final step in manufacturing

A biomaterial is a substance that has been engineered to take any implant device, the effect of sterilization processes on
a form which, alone or as part of a complex system, is used to the modification of the first surface layers of these biomate-
direct, by control of interactions with components of living rial surfaces must be clearly understood and characterized.
systems, the course of any therapeutic or diagnostic proce- Furthermore, the relation between the physico–chemical fac-
dure, in human or veterinary medicine.1 Because biomaterials tors and the tissue response must also be investigated.
are selected based on the combination of mechanical, physical, Moreover, the requirement for a well-controlled surface
and biological properties, investigators have found the need to before implantation emphasizes these needs. Shabalovskaya
enhance biocompatibility. The essential requirement for an ar- et al. reported on the correlation between the surface chem-
tificial material to bond to living bone is believed to be the ical composition and some sterilization treatments.18,19 A
formation of a biologically active apatite-like layer on their similar study on commercially pure titanium also showed
surfaces in the body.2–5 Limited number of ceramic materials the potential damaging effect of some sterilization treat-
called bioactive materials such as bioglass,2 hydroxyapatite ments such as ethylene oxide and steam autoclaving on the
(HA),5 glass-ceramic A-W,6 diopside,7,8 and pseudowollastonite in vitro biological response.20 In spite of a wide application
(psW)9,10 are generally known to possess the ability to form of bioceramics in dentistry and medicine, surprisingly, there
bone like apatite in the body environment. are only a few publications devoted to the influence of the
Unfortunately, in most of the cases, mechanical strength sterilization conditions on the chemical properties of biocer-
of these materials were not enough to use them as bulk amics.21–23
implants, and hence, their applications have been limited to So, it appears to be both interesting and important to
less loaded portions. Nevertheless, their field of application study the influence of different sterilization methods on the
can be expanded if used as coatings on metallic implants. In chemical properties of the bioceramics. The aim of this
this way, the benefits of the bioactive material are combined work was to study the effects of the most accessible sterili-
together with the mechanical performance of metallic zation techniques on the surface chemistry and in vitro bio-
implants. The most common example of this type of combi- activity of psW coatings in titanium.
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 MATERIALS AND METHODS
‘‘in vivo.’’9,10,16,17 This finding is very significant, as it indi- The starting material for this study was psW ceramic syn-
cates that the psW can be physically and chemically inte- thesized by a solid stated reaction from a stoichiometric
grated into the structure of living bone tissue, and therefore, mixture of calcium carbonate and high purity washed Bel-
could be suitable for repair or replacement of living bone. gian sand (99.9 wt %). Details of psW preparation and

Correspondence to: P. N. De Aza; e-mail:

Contract grant sponsor: CICYT; contract grant number: MAT2006-12749-C02-02
Contract grant sponsor: Generalitat Valenciana; contract grant number: ACOMP/2009/173

characterization can be found in previous publications.14–16 tion of 900 mg/L. After sterilization, the samples are aer-
Pellets of synthesized psW were axially pressed in a press- ated with warm air flow (37 C) at atmospheric pressure for
ing die of 30 mm of diameter at 150 MPa. A 1 wt % of a so- 330 min to remove residual ethylene oxide, and stored in
lution of 0.11 wt % of a plasticizer Zusoplast 91/11 was indicator bags and sealed.
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 Gamma sterilization (Rhodotron TT200)
deposition. Gamma irradiation sterilization with a 60Co irradiator was
The coatings substrates were 11 cm2 of Ti-6Al-4V with a performed by Ionmed S.A. (Cuenca, Spain). Randomized
thickness of 1.5 mm. All the samples were obtained by follow- samples were packed and sealed in indicator bags and
ing the procedure already described in a previous work.13 exposed to gamma irradiation in three cycles at doses of
This procedure combines the pulsed laser deposition and the 18.71 kGy, 19.17 kGy, and 18.86 kGy, resulting in a dose of
laser surface treatment. First, Ti-6Al-4V substrates were 56.34 kGy.
coated with psW through pulsed laser deposition from a bulk After being ultrasonically washed in acetone and rinsed
psW target. A pulsed Nd:YAG laser, delivering 10 ns pulses of in deionized water, specimens were soaked in 100 mL of
70 mJ at a wavelength of 355 nm and a repetition rate of 10 simulated body fluid (SBF) in polyethylene bottles at
Hz, was used for laser ablation. The laser fluence on the psW 36.5 C. The immersion period of the specimens in SBF was
target was 2.5 J/cm2. The substrates were placed at 4 cm in 21 days. This period was chosen based on the results from
front of the target, and during deposition, the substrates were previous in vitro experiments performed in the SBF.14,15,24
at 550 C in a 10 Pa oxygen atmosphere. Each coating resulted At periodic intervals, the pellets were removed from the
from 36,000 laser pulses. fluid and were left to dry in air at room temperature.
After deposition, the samples were treated in air with a Surface of the samples were characterized before and af-
continuous wave Nd:YAG laser to improve the density of the ter the sterilization treatments. The crystalline structure of
coatings and their adhesion to the substrate. The laser the coatings was analyzed by X-ray diffractometry (XRD)
beam, with a wavelength of 1064 nm and a power of 35 W, and Raman spectroscopy in the range 100–1200 cm1. The
was focused on the coatings surface at 36 kW/cm2. The surface morphology and composition were investigated in a
laser beam scanned their entire area at a speed of 250 scanning electron microscope (SEM) using a Hitachi S-
mm/s line by line with steps of 100 lm. 3500N, equipped with detectors of secondary and backscat-
Twelve pellets of samples were processed under clinical tered electrons and fitted with X-ray energy-dispersive spec-
conditions by common sterilization process used in hospi- trometer (EDS). The coatings thickness was measured with
tals including steam autoclave, hydrogen peroxide plasma, a surface profilometer.
ethylene oxide, and gamma sterilization irradiation steriliza- The structural changes of the psW coatings after immer-
tions. Nonsterilized psW-titanium samples were used as sion in SBF for various periods were first analyzed by thin-
controls film X-ray diffraction (XRD), which enables analysis of up to
a 1-lm thick layer, depending on the incident X-ray beam
Steam autoclave (EVS 425.2.M) angle. The morphology of the surface and the cross sections
Sealed bags with the samples were sterilized in autoclave of the specimens were examined by SEM and EDS. The cross
according to the standard procedure DIN 58946 used in sections studied in the SEM were polished to 1 lm finish
medicine. A set of samples were sterilized at 121 C. Briefly, using diamond paste, gently cleaned in an ultrasonic bath,
a single run of the steam sterilization procedure in auto- and carbon coated. X-ray elemental maps of Si, Ca, and P of
clave includes three stages: temperature increasing up to the specimens were also obtained. The thickness of the pre-
121 C during 15 min with simultaneous pressure increasing cipitation layer formed on the psW coatings surfaces were
from 0 to 1.4 mbar, sterilization at 121 C during 15 min at evaluated from the SEM photographs of the cross section of
pressure of 1.4 mbar, followed by fast (1–2 min) tempera- the soaked psW coatings samples. Additional changes in
ture reduction to 25 C. Thus, the total time of a single run ionic concentration, using inductively couple plasma atomic
of the steam sterilization procedure is about 45 min. emission spectroscopy (ICP), were determined.
The product of the surface reaction was subsequently
Hydrogen peroxide plasma (Sterrad) characterized using transmission electron microscopy (TEM)
Plasma sterilization treatment was carried out in a large technique. Jeol Jem 2010 microscope was operated at 200
volume microwave plasma (LMPTM) reactor. A set of sam- keV, and 80 cm camera length condition was applied for
ples were sterilized exposing to a 58% hydrogen peroxide selected area diffraction patterns (SAD). These specimens
plasma for 15 min. The gas was introduced into the cham- were prepared by careful removal of the reaction layer from
ber at an operating pressure of 500 MTorr, and plasma was the specimen’s surface using razor blade, and dispersing the
excited with 400 W of microwave power. powder on the surface of alcohol in Petri dish. The powder
specimens were then collected on carbon coated copper
Ethylene oxide (Steri Vac EOE-M.) TEM grids of 200 mesh and carbon coated. Electron beam
The units were sterilized by exposing to a 100% ethylene transparent particles were chosen for TEM examination by
oxide atmosphere for 156 min at 55 C and a gas concentra- SAD, high-magnification imaging, and EDS analysis.


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
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-
FIGURE 1. SEM micrographs of the (A) surface of the psW-coating rad, and gamma samples present similar behavior than the
before sterilization (control sample). (B) Steam autoclave sterilized psW-coating control with a similar type of the precipitates.
sample as a representative of all the sterilized samples. 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
RESULTS homogeneously the whole surface of the material. The par-
Figure 1(A) shows SEM photograph of the surface micro- ticles morphology does no further change with soaking
structure of the coating after laser treatment. It can be seen time, although the density of the layer increases.
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
FIGURE 3. Raman spectra of the (A) control sample (nonsterilized)
(high-temperature form of wollastonite-2M). Some changes and (B) steam autoclave, (C) Sterrad, (D) EtO, and (E) gamma steri-
can be noted in Figure 2(B), where in addition to the sharp lized samples (All the peaks correspond to pseudowollastonite.).

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


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,
FIGURE 5. Ca/P-layer thickness change as a function of soaking time
with no discontinuity or bending present. EDS analysis per-
into SBF: Control sample (nonsterilized) and steam autoclave, Sterrad, formed on the thin crystals confirmed the presence of cal-
EtO, and gamma sterilized samples. 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
the globular particles reach a size between 3 and 3.9 lm in literature.26–31 The HA-like crystals which orientation varied
diameter. from (100) did no fulfill the lattice imaging conditions.
Figure 5 shows the relationship between the soaking When the specimen was appropriately oriented, the SAD of-
time and the thickness of the Ca/P-layer developed on the ten displayed (002) arcs corresponding to the 0.344 nm lat-
surface of the psW-coating. In the control sample (nonsteri- tice spacing, indicating on the preferential orientation of the
lized), a significant thickness of Ca/P-layer has been formed, HA-like crystals in the layer. Figure 6(C) displays a good
reaching a total thickness of 24.3 lm (61 lm) after 30 match between the measured values of the lattice spacings
days. The formation rate gradually slows from 2.3 lm per obtained from the SAD pattern [Figure 6(C)] and ASTM data
day, after 7 days of soaking, to 0.5 lm per day, after pro- from HA [Figure 6(D)].
longed soaking (4th week of soaking). That is to say, the
measurements reveal that the Ca/P-layer formation kinetic DISCUSSION
depends directly on the square root of soaking time. In the The in vitro results carried out in SBF for the psW coatings
sterrrad, stem, and gamma sterilized samples, the morphol- in titanium alloys substrates sterilized by steam autoclave,
ogy of the surface product was the same, although the reac- hydrogen peroxide plasma, ethylene oxide, and gamma steri-
tion rate and the thickness of the layer after 30 days were lization irradiation, suggest that all the sterilized materials
slight smaller. In Steam sterilized samples, the total final have in vitro bioactivity as shown by the formation of HA-
thickness of the Ca/P-layer after 30 days of soaking is 23 like phase in SBF solutions and the mechanism of this bio-
lm (61 lm), in Sterrad sterilized samples is 21 lm (61 activity is in agreement with the mechanisms reported by
lm) and for Gamma method its 18.4 lm (61 lm). other researcher.32–34
The main difference was in the EtO sterilized samples. When immersed psW-coating in SBF solution, both dis-
Here it is worth to point out that the layer detected in the solution and HA-like precipitation occur on the surface on
EtO sterilized samples was not of uniform structure and the materials. At early stage, the dissolution of psW-coating
density throughout the sample. In the areas which the layer generally proceeds faster than precipitation. The psW-coat-
was present a very low-layer formation rate of about 1 lm ing dissolved little before the HA-like precipitation process
per day, up to 9.4 lm (61 lm), after 7 days of soaking, was became dominant. When the psW-coating gets in contact
detected. The layer reached a total thickness of 12.1 lm with the SBF, a partial dissolution occurs, producing an ionic
(61 lm) within these zones. exchange of Ca2þ for 2Hþ within the material network, lead-
Changes in the elemental ionic concentrations of SBF af- ing to the formation of silanol groups on the surface of the
ter 30 days of immersion are shown in Table I. The compo-
sition of the original SBF solution is also enclosed for com- TABLE I. Concentration of Caþþ, Si4þ, and HPO2
4 , in SBF
parison. The chemical analysis of the SBF used in the study before and after Immersion During 30 Days of the Control
agreed with the composition of the one reported in the lit- Sample (Nonsterilized) and Steam Autoclave, Sterrad, EtO,
erature.25 It was found that calcium and silicon ion concen- and Gamma Sterilized Samples
trations in SBF slightly increased over the exposure time Samples (mg/L) Caþþ Si4þ HPO2
indicating partial dissolution of the psW-coating. On the SBF control solution 100.36 – 93
other hand, phosphorous ion concentration decreased more Control pellet 104.23 8.64 63.98
significantly because of the precipitation of Ca-P phase on Steam autoclave 102.58 7.582 65.27
the surfaces of the psW-coating. Although the formation of Sterrad 106.39 7.24 74.37
Gamma 101.73 8.45 77.97
Ca-P phase consumed some calcium ions, the calcium ions
EtO 102.17 7.87 83.01
dissolution from the psW-coating were more than those

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
FIGURE 6. (A) HRTEM micrograph of the HA-like phase formed during to a disperse nucleation of an HA-like phase on EtO sample.
exposure to SBF after 30 days (steam autoclave sterilized sample), (B)
EDX analysis, (C) SAD pattern of the phase, and (D) lattice spacing of
Globular aggregates can be detected from the 1st week of
the HA-like phase, taken from SAD of (C) and ASTM data. 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
psW-coating. Later, there is a partial dissolution of amor- nuclei homogeneously distributed since the 1st week of
phous silica as SiO23 . This fact enhances the formation of immersion. The individual growth of these leads to the HA-
crystallization nuclei for the HA-like phase, which can be like layer formation.
formed from the high concentration of Ca2þ and HPO2 4
present in the media. Before immersion in SBF, the psW- CONCLUSIONS
coating had an average thickness of 18 lm (61 lm). After The influence of four sterilization methods (steam autoclave,
immersion in SBF for 30 days, its thickness decreased, for hydrogen peroxide plasma, ethylene oxide, and gamma steri-
all the sterilization methods, in average up to 12 lm (61 lization) on the surface chemistry and the in vitro bioactiv-
lm), and the precipitated HA-like grew uniformly in average ity of psW coatings in Ti-6Al-4V substrates were
to a thickness of 21.62 lm (61 lm) for the control and all investigated.
sterilized samples except EtO, where the precipitation is not The XRD and Raman spectroscopy results appeared basi-
homogeneous. cally similar; it has been shown that there was no signifi-
In the areas which the layer was detected, a low-layer cant modification of the psW-coating structure in the sur-
formation rate of about 1 lm per day was measured. After face of the samples by the four sterilization methods used.
7 days of soaking, the layer reached 9.4 6 1 lm attaining, SEM observation, after the SBF soaking, showed essen-
in these areas, a total thickness of 12.1 lm (61 lm). This tially identical surface morphology regardless of the sterili-
mean that, in the EtO sterilized samples, the total Ca/P-layer zation by ethylene oxide, where the HA-like layer does not
formed, after 30 days of immersion was about 55.9% thin- fully cover the surface of the samples after 30 days of
ner than in the other sterilized samples. This is in agree- immersion. One of the reasons for this difference is due to
ment with the previously mentioned results obtained by some residual ethylene gas that possibly is present on the
SEM during the surface studies [Figure 4(B), EtO] where the surface of the EtO sterilized samples after sterilization. This
EtO sterilized samples do not present, after 30 days of residual ethylene gas should affect the nucleation and crys-
immersion, the surface fully covered by the Ca/P globular tal growth of HA-like.
particles. On the other hand, the ICP-AES results of phos- The sterilized materials have in vitro bioactivity with the
phorous ion concentration in SBF (Table I) shows that the formation of an HA-like layer on the surface of all the sam-
minor depletion in P corresponds to the EtO sterilized sam- ples, although the ethylene oxide sterilized samples present
ples. One of the reasons for this difference, in the EtO steri- a 55.9% thinner and nonhomogeneous HA-like deposition
lized samples, is due to some residual ethylene gas that pos- throughout the samples. Therefore, we recommended not
sibly is present on the surface of the material after using the EtO sterilized method for this type of materials.


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