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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
V
C 2010 WILEY PERIODICALS, INC. 399
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.
400 ZULETA, VELASQUEZ, AND DE AZA STERILIZATION METHODS ON BIOACTIVITY PSEUDOWOLLASTONITE COATING
ORIGINAL RESEARCH REPORT
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | AUG 2010 VOL 94B, ISSUE 2 401
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, 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,
402 ZULETA, VELASQUEZ, AND DE AZA STERILIZATION METHODS ON BIOACTIVITY PSEUDOWOLLASTONITE COATING
ORIGINAL RESEARCH REPORT
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | AUG 2010 VOL 94B, ISSUE 2 403
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.
404 ZULETA, VELASQUEZ, AND DE AZA STERILIZATION METHODS ON BIOACTIVITY PSEUDOWOLLASTONITE COATING
ORIGINAL RESEARCH REPORT
ACKNOWLEDGMENTS 20. Vezeau PJ, Koorbusch GF, Draughn RA, Keller JC. Effects of
The authors thank Dr. Luna from Ionmed SA (Cuenca, Spain) multiple sterilization on surface characteristics and in vitro bio-
logic responses to titanium. J Oral Maxillofac Surg 1996;54:
for the gamma sterilization and Dr. F. A. Lopez Prats for the 738–746.
steam autoclave, hydrogen peroxide plasma, and ethylene ox- 21. Dorozhkin V, Schmitt M, Bouler JM, Daculsi G. Chemical transfor-
ide sterilizations. mation of some biologically relevant calcium phosphates in aque-
ous media during a steam sterilization. J Mater Sci Mater Med
2000;11:779–786.
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