K. AVSEC et al.: EFFECT OF STERILIZATION ON THE SURFACE PROPERTIES OF Ti6Al7Nb ALLOY ...
59–64
EFFECT OF STERILIZATION ON THE SURFACE PROPERTIES OF
Ti6Al7Nb ALLOY FEMORAL STEMS
VPLIV STERILIZACIJE NA POVR[INSKE LASTNOSTI
FEMORALNIH KOL^NIH KOMPONENT ENDOPROTEZ IZ
Ti6Al7Nb ZLITINE
Klemen Avsec
1,2
, Marjetka Conradi
3
, Monika Jenko
3,4
, Bo{tjan Kocjan~i~
1,2
,
Mojca Debeljak
5
, Matev` Goren{ek
6
, Drago Dolinar
1,2,3*
1
Department for Orthopaedic Surgery, University Medical Centre Ljubljana, Zalo{ka 9, 1000 Ljubljana, Slovenia
2
Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
3
MD-RI Institute for Materials Research in Medicine, Bohori~eva 5, 1000 Ljubljana, Slovenia
4
Institute of Metals and Technology, Lepi pot 11, 1000 Ljubljana, Slovenia
5
University Rehabilitation Institute, Republic of Slovenia, Linhartova 51, 1000 Ljubljana, Slovenia
6
MD Medicina, Bohori~eva 5, 1000 Ljubljana, Slovenia
Prejem rokopisa – received: 2020-07-28; sprejem za objavo – accepted for publication: 2020-08-19
doi:10.17222/mit.2020.141
We investigated thirty retrieved femoral components (cement-less stems made from Ti6Al7Nb alloy) of prematurely failed hip
endoprostheses, due to aseptic loosening, infection and low-grade infection and two new stems (after their expiry date) for com-
parison. The main aim was to find the impact of cleaning and sterilization procedures of new and retrieved stems on the surface
properties, i.e., surface wetting, roughness and surface chemistry. Our hypothesis was that the effect of cleaning and sterilization
should not affect the meaningful difference of surface properties of retrieved and new stems of hip cement-less endoprostheses.
Clean, new and retrieved stems of hip endoprostheses were sterilized using classical steam autoclave AC, procedure protocol
(121 °C, 1.25 bar, 20 min). Surface wettability was determined by measuring the static contact angle of a water drop, the rough-
ness was measured by optical 3D system. The surface chemistry was analysed by X-ray Photoelectron Spectroscopy (XPS). In
wettability results, the contact angle varied from 70.6° to 91°, and the surface roughness (Sa) varied from 4.2 μm to 4.7 μm for
all thirty measured stems. We found that surface properties measured on three different stem positions (neck, middle and root)
differ significantly, we can conclude that corundum (Al2O3) grit-blasted surface used to obtain the necessary roughness for
better osteointegration is not homogeneous. The XPS results of new and retrieved implants that prematurely failed for different
reasons showed minor differences of TiO2 thin oxide native film thickness: 6.2 nm for the new stem and 5.2 nm for retrieved
(prematurely failed due to aseptic loosening, infection or low grade infection, consisting primarily of TiO2). The results confirm
our hypothesis that the effect of cleaning and sterilization on surface properties of new and retrieved stems is negligible.
Keywords: hip endoprostheses, stem, Ti6Al7Nb alloy, cleaning, steam autoclave sterilization, surface properties, wettability,
roughness, XPS
Raziskali smo trideset uporabljenih femoralnih komponent brezcementnih endoprotez kolka iz Ti6Al7Nb zlitine, ki so
pred~asno odpovedale zaradi enega od slede~ih najpogostej{ih vzrokov: asepti~nega omajanja, oku`be, nizke stopnje oku`be. Za
primerjavo smo raziskali dve novi femoralni komponenti. Glavni namen raziskave je bil ugotoviti vpliv ~i{~enja in sterilizacije
novih in uporabljenih femoralnih komponent na povr{inske lastnosti kot so: omo~enje, hrapavost in povr{inska kemija. Na{a
hipoteza je bila, da vpliv ~i{~enja in sterilizacije bistveno ne vpliva na razlike povr{inskih lastnosti novih in uporabljenih
femoralnih komponent kol~nih endoprotez. ^iste, nove in uporabljene femoralne komponente kol~nih endoprotez smo
sterilizirali po klasi~nem postopku v pari v avtoklavu (AC), po protokolu (121 °C, 1.25 bar, 20 min). Omo~ljivost povr{ine je
bila dolo~ena z meritvami kontaktnega kota vodne kapljice, hrapavost je bila izmerjena z opti~nim 3D sistemom na treh mestih
in sicer na vratu, v sredini in korenu femoralne komponente. Povr{ino smo analizirali z rentgensko fotoelektronsko
spektroskopijo (XPS). Rezultati omo~ljivosti, merjeni na treh razli~nih mestih (vrat, sredina, koren), na vseh tridesetih
femoralnih komponentah brezcementnih endoprotez kolka, so slede~i: kontaktni kot od 70.6° do 91°; hrapavost (Sa) od 4,2 μm
do 4,7 μm. Rezultati omo~ljivosti in hrapavosti, merjeni na treh razli~nih mestih novih in uporabljenih femoralnih komponent,
ka`ejo opazne razlike, iz ~esar lahko sklepamo, da povr{ina obdelana s peskanjem s korundnimi delci (Al2O3), ki je potrebna za
bolj{o osteointegracijo, ni homogena. Rezultati XPS-analize povr{ine novih in uporabljenih vsadkov, ki so pred~asno
odpovedali zaradi razli~nih vzrokov, ka`ejo minimalne razlike v debelini TiO2: pasivne tanke oksidne plasti 6,2 nm za nove
vsadke in 5,2 nm za uporabljene, ki so pred~asno odpovedali zaradi asepti~nega omajanja, oku`be ali nizke stopnje oku`be.
Dobljeni rezultati potrjujejo na{o hipotezo, da je vpliv ~i{~enja in sterilizacije na povr{inske lastnosti novih in uporabljenih
vsadkov, zanemarljiv.
Klju~ne besede: kol~ne endoproteze, Ti6Al7Nb zlitina, ~i{~enje, sterilizacija v avtoklavu, povr{inske lastnosti, omo~ljivost,
hrapavost
1 INTRODUCTION
Total hip joint arthroplasty (THA) is the most suc-
cessful surgical method for relieving pain, correcting de-
formities and treating degenerative hip-joint disease and
trauma
1–7
The Ti6Al7Nb alloy is the most commonly
used metallic implant material for the femoral stem and
the acetabular cup of hip endoprostheses, and has been in
clinical use since 1986.
1–6
The Ti6Al4V alloy, as we have
already described in our previous paper,
8
has been the
most popular metallic implant material used in THA
Materiali in tehnologije / Materials and technology 55 (2021) 1, 59–64 59
UDK 614.48:620.191:544.722.3:669.017.3 ISSN 1580-2949
Original scientific article/Izvirni znanstveni ~lanek MTAEC9, 55(1)59(2021)
*Corresponding author's e-mail:
dolinardrago@gmail.com (Drago Dolinar)

(and is still widely used in the USA), but in recent de-
cades Ti6Al7Nb has become a very popular material be-
cause of its better biocompatibility due to the replace-
ment of vanadium (toxic when released over certain
critic levels) with niobium.
The surface roughness of the cementless stem of a
hip endoprosthesis is very important for good osseo-
integration. With the aim of improving the osseointeg-
ration of orthopaedic implants, many surface-modifica-
tion strategies have been developed, focusing on the
biomaterial surface properties and health costs.
9–13
Corundum-grit blasting is the most widely used me-
chanical surface treatment for roughening the surfaces of
titanium cementless implants due to its good sur-
face-roughening properties. Corundum-grit blasting in-
troduces contamination of the surface of the Ti6Al7Nb
alloy implant due to retained corundum, which can ad-
versely affect the osseointegration process of the im-
plant. This is in addition to the roughening effects and
the source of the wear particles that could cause de-
bris-related aseptic loosening.
14–16
The retrieved biomaterial devices, i.e., femoral metal-
lic components of hip endoprostheses, that are stored for
the investigation of premature failures, etc., must be
cleaned and sterilized.
Park et al.
17
reported on the effects of cleaning and
sterilization and presented a graphic illustration of pro-
cessing of biomaterials from cleaning to implantation
(Figure 1).
The cleaning procedure removes or reduces the visi-
ble contamination (blood, protein and debris) on the sur-
face, while the sterilization stops the reproduction of mi-
cro-organisms, bacteria, spores and fungi.
17–23
The
following sterilization methods are in use nowadays for
different materials such as Gama irradiation GR
(25KGy), Steam autoclave AC 121 °C, 1.24 bar, 20 min,
Oxygen plasma (OP) and Ultarviolet (UV) 254 nm for
90 minutes.
17
The cleaning and sterilization influence the proper-
ties of new and retrieved Ti6Al7Nb alloy surfaces, which
were studied to find possible changes in terms of
wettability, roughness, morphology, microstructure, cor-
rosion and surface chemistry.
17–20
A major responsibility of healthcare providers is to
minimize patient risks. Therefore, they require additional
attention to the sterilization and quality control process.
According to the US Food and Drug Administration
(FDA), an implant/implantable device is a "device that is
placed into a surgically or naturally formed cavity of the
human body" Ideally, implants and implantable devices
should come individually wrapped and sterile from the
manufacturer.
The surface roughness of a cementless stem is very
important for the good osseointegration of the implant.
In order to enhance the osseointegration of orthopaedic
implants, many surface-modification strategies have been
tried, focusing on the important role of the biomaterial’s
surface properties.
9–13
Grit blasting using corundum or alumina is a com-
monly used surface treatment for roughening the sur-
faces of titanium cementless implants.
In the present study the surface properties, in terms of
wettability, roughness and morphology of retrieved and
new femoral components, i.e., the stems, of cementless
hip endoprostheses, were studied in detail with the aim
to find differences in the surface properties of new and
K. AVSEC et al.: EFFECT OF STERILIZATION ON THE SURFACE PROPERTIES OF Ti6Al7Nb ALLOY ...
60 Materiali in tehnologije / Materials and technology 55 (2021) 1, 59–64
Figure 1: Outline of procedures at the Ti alloy surface during cleaning, sterilization, and implantation, adapted from J. H. Park et al.
17
Figure 2: Sterilization methods: gamma irradiation (25 kGy overnight), steam autoclave (121°C at 1.25 bar, 20 min), oxygen plasma and ultra vi-
olet (254 nm, 90 min)

retrieved femoral components after cleaning and steril-
ization.
The main aim is that our results from retrieved im-
plants can be used as the base knowledge for the im-
provement of existing and the development of new pro-
cedures and manufacturing of better implants with better
longevity.
Our hypothesis is that the effect of cleaning and ster-
ilization on surface properties of new and retrieved stems
is negligible.
2 MATERIALS AND METHODS
2.1. Implants/stems
The retrieved Ti6Al7Nb stems of the cementless hip
endoprostheses were selected from revision surgeries
performed at the Department for Orthopaedic Surgery of
the University Medical Centre (UMC) Ljubljana
(Ljubljana, Slovenia). We also investigated two new
stems (after their expiry date) for comparison.
For this investigation, 30 stems of cementless hip
endoprostheses that prematurely failed due to:
1) aseptic loosening (10 implants),
2) infection (10 implants) and
3) latent infection (10 implants)
were selected.
The interval between the primary hip replacement
and the revision surgery was 36 months to 239 months
for aseptic loosening, 3 months to 36months for infec-
tion and 36 months to 168 months for latent infection.
Knowledge of the behaviour of individual prosthesis
in certain clinical conditions is very important. We have
selected for detailed investigation the cementless
Zweymuller-ZM type hip endoprostheses, new and re-
trieved, that prematurely failed due to aseptic loosening,
infection and latent infection. Figure 3 shows the stems
of different hip endoprostheses manufacturers.
The retrieved stems were ZM type from an unknown
manufacturer, while the new ones were Smith and
Nephew (London, UK) Ti6Al7Nb stems of cement-less
hip endoprostheses. After the revision surgery, the
retrieved implants were sent for sonication in Ringer’s
solution and afterwards for cleaning and sterilization. All
the retrieved stems were cleaned according to standard
procedures at UMC Ljubljana, which consist of immer-
sion in 2 % microsoap solution, followed by acetone,
isopropanol (xN), 95 % ethanol (xN), and deionized wa-
ter (xN); (xN) is the number of repeated processes. Ster-
ilization was performed by autoclaving according to a
standard protocol at 120 °C and a pressure of 1.25 bar
for 20 min. Afterwards, sterilized stems were kept in
sterile bags in a dry place for further investigations. New
femoral components were cleaned and sterilized at the
manufacturer’s site and the special bags were opened on
site just before the implantation. The cleaning and steril-
ization processes, according to the literature data and our
experiences, do not affect the removal of the particle de-
bris.
10–15
The chemical compositions of the new and re-
trieved implants were determined by X-ray fluorescence
(XRF, Niton XL3t GOLDD+, Thermo Scientific,
Waltham, MA, USA) and inductively coupled plasma
atomic emission spectroscopy (ICP-AES, Agilent 720,
Agilent Technologies, Santa Clara, CA, USA) chemical
analyses. The results of the measurements (Ti 87.8, Al
5.6, Nb 6.4, Fe 0.21, Cr 0.007, Ta 0.02 in mass %) were
in good agreement with the requirements of the ASTM
F1295-05 Standard Specification for Wrought Tita-
nium-6 Aluminum-7 Niobium Alloy for Surgical Im-
plant Applications.
24
Samples for the investigation of the surface proper-
ties (wettability, roughness) and morphology using scan-
ning electron microscopy (SEM – ZEISS crossbeam 550
FIB-SEM, Carl Zeiss AG, Oberkochen,Germany) were
prepared from the new and retrieved stems, cleaned in an
ultra-sonic bath of isopropyl alcohol and dried in dry ni-
trogen gas. The samples for the surface-chemistry analy-
ses of the passive film had polished surfaces.
2.2. Wettability
The static-water contact-angle measurements on the
as-received and sterilized stems were performed at three
characteristic sites (at the root, in the middle and at the
neck) using a surface-energy evaluation system (Advex
Instruments r.o.). Water droplets of 5 μL were deposited
on the surface and the average contact angle was deter-
mined using Young–Laplace fitting from at least five
measurements on each characteristic site. The measure-
ments were carried out at 20 °C and an ambient humidity
of 55 %. An optical 3D metrology system, Alicona Infi-
nite Focus (Alicona Imaging GmbH, Raaba, Austria),
was employed for the surface-roughness measurements
on the as-received and sterilized stems at three character-
istic sites (at the root, in the middle and at the neck).
Three measurements were performed at each site using a
magnification of 20× with a lateral resolution of 0.9 μm
and a vertical resolution of about 50 nm. Subsequently,
K. AVSEC et al.: EFFECT OF STERILIZATION ON THE SURFACE PROPERTIES OF Ti6Al7Nb ALLOY ...
Materiali in tehnologije / Materials and technology 55 (2021) 1, 59–64 61
Figure 3: ZM type of cementless femoral component hip endo-
prostheses of Ti6Al7Nb alloy new, and retrieved femoral stems used
in our research

the IF-Measure Suite (Version 5.1) software was used for
an evaluation of the average surface roughness, S
a
:
S
LL
zxy xy
xy
L L y x
a
dd =
∫ ∫
11
0 0
(,)
where L
x
and L
y
are the x and y acquisition lengths of
the surface and z(x,y) is the height. The size of the ana-
lysed area was 714 μm × 542 μm.
2.3 Surface chemistry – XPS analysis
The samples for XPS studies were cut from new and
retrieved stems, grinded and polished and exposed for
24 h at room temperature to the air.
The X-ray photoelectron spectroscopy (XPS) analy-
ses were carried out on a PHI-TFA XPS spectrometer
from Physical Electronics Inc. USA. Samples were pol-
ished before the analyses and exposed to the air for one
day. The native oxide layer was analyzed for thickness
and composition on two Ti6Al7Nb samples. The ana-
lysed area was 0.4 mm in diameter and the analyzed
depth was about 3–5 nm. This high surface sensitivity is
a general characteristic of the XPS method. Sample sur-
faces were excited by X-ray radiation from a monochro-
matic Al source at a photon energy of 1486.6 eV. Quanti-
fication of the surface composition was performed from
the XPS peak intensities, taking into account the relative
sensitivity factors provided by the instrument manufac-
turer.
24–26
We estimate that the relative error of the calcu-
lated concentrations is about 20 % of the reported values.
In order to analyze the in-depth distribution of elements
in the sub-surface region up to 25 nm, XPS depth
profilling was performed in combination with argon-ion
sputtering. Ar ions of energy 3 keV were used. The ve-
locity of the ion sputtering was estimated to be
1.0 nm/min, calibrated on a Ni/Cr multilayer structure of
known thickness.
3 RESULTS AND DISCUSSION
3.1 Wettability and roughness
To analyse the surface wettability, we performed five
static contact-angle measurements with water at three
characteristic spots on each stem: neck, middle and root.
We determined the average contact-angle values at each
site with an estimated error in the reading of ±2.0°. The
average surface-roughness parameter was used to evalu-
ate the morphological characteristics of the stems and the
three sites of the measurements.
Contact-angle and average-surface-roughness mea-
surements at the three characteristic sites on a new stem
before and after sterilization are shown in Table 1.W e
can see that the new stem is moderately hydrophobic and
the wettability is practically the same at all three sites.
Sterilization, however, turns the surface of the stem hy-
drophilic. Interestingly, there is a large difference in the
wettability between the three sites, the root being
strongly hydrophilic compared to the middle and the
neck (Figure 4 and Table 1).
Table 1: Static-water contact angles and average surface roughness,
S
a
, of a new Ti6Al7Nb stem (Smith & Nephew) N1 before and after
sterilization.
before steril-
ization
after steriliza-
tion
Implant N 1 Sa
(μm)  (deg)  (deg)
Site 1 5.209 116.3 66.5 (49–85)
Site 2 5.884 114.8 73.7
Site 3 5.570 115.9 36.8
The average surface roughness is of the same order
for all three sites and is not affected by the sterilization.
We performed additional surface analyses on the
neck, middle and root of the used stems. Contact-angle
K. AVSEC et al.: EFFECT OF STERILIZATION ON THE SURFACE PROPERTIES OF Ti6Al7Nb ALLOY ...
62 Materiali in tehnologije / Materials and technology 55 (2021) 1, 59–64
Figure 5: Static contact-angle measurements of prematurely failed hip
endoprostheses due to: a) aseptic loosening, b) infection and c) latent
infection: there is no large difference regarding the cause of premature
failure (Table 2)
Table 2: Static-water contact angles and average surface roughness,
S
a
, of prematurely failed hip endoprostheses due to: a) aseptic loosen-
ing, b) infection and c) latent infection
a) Sa
(μm)  (°)
Neck 4.3±0.1 82.5±1.5
middle 4.2±0.3 80.4±2.1
Root 4.3±0.1 81.3±1.4
b)
Neck 4.6±0.2 82.1±2.1
middle 4.6±0.2 86.9 ± 1.3
Root 4.7±0.1 91.6 ± 1.9
c)
Neck 4.5±0.1 80.4±1.9
middle 4.5±0.2 70.6±1.8 Figure 4: Static water contact-angle measuring site on new stem

and average-surface-roughness measurements of the pre-
maturely failed hip endoprostheses due to aseptic loosen-
ing, infection and latent infection are shown in Table 2.
The results show that there is no noticeable difference re-
garding the cause of premature failure due to these rea-
sons. All the surfaces are poorly hydrophilic close to the
hydrophobic regime and there is no significant difference
in the average surface roughness at different sites and on
different stems.
Surface-roughness measurements of a new hip
cementless Zweymüller stems of three different produc-
ers: a) Smith &Nephew S
a
= 6.7 μm, b) Alloclasic varial
S
a
= 5.0 μm and c) Alloclasic Zweymüller S
a
= 4.7 μm,
measured by Alicona instrument described in our previ-
ous paper.
14
3.2 XPS surface analysis
When exposed to air or water at room temperature, ti-
tanium alloys spontaneously form thin TiO
2
oxide films
(a passive film) of 4–6 nm thickness immediately. A sta-
ble, thin oxide film protects the Ti alloys from pitting,
intergranular and crevice corrosion and are responsible
for the excellent biocompatibility.
XPS depth profiles of native oxide film that forms on
the fresh surface, after grinding and polishing and expo-
sure to the air for 24 h of new and retrieved stems are
shown in Figures 6 and 7. The oxide layer on the new
and retrieved samples consisted of Ti-oxide, Al-oxide
and Nb-oxide. This was evidenced by the Ti 2p
3/2
peak at
458.6 eV, characteristic for Ti(4+) in a TiO
2
-like environ-
ment, the Nb 3d
5/2
peak at 207.0 eV, characteristic for the
Ni(5+) oxidation state, and the Al 2p peak at 74.0 eV,
characteristic for the Al(3+) oxidation state.
In our recent preliminary investigation of retrieved
implants that prematurely failed for different reasons
(aseptic loosening, infection, latent infection) using the
surface-analysis method XPS, we noticed minor differ-
ences and proceeded with a detailed investigation of the
implant surfaces prematurely failed due to aseptic loos-
ening, infection and low-grade infection.
5 CONCLUSIONS
The present study establishes the effect of cleaning
and sterilization on the surface properties of the new and
retrieved femoral components of a Ti6Al7Nb alloy of
cementless hip endoprostheses that were provided by the
Orthopaedic Clinic of the University Medical Centre
Ljubljana.
We found that sterilization, steam autoclaving at
121°C, 1.25 bar, for 20 minutes, affects the wetting prop-
erties of the new and retrieved Ti6Al7Nb alloy, turning
the initially moderately hydrophobic stem to hydrophilic.
The analyses of retrieved stems showed the poorly hy-
drophilic nature of all the samples, regardless of the fail-
ure.
The thicknesses of the thin oxide films on the
Ti6Al7Nb (primarily of TiO
2
) were estimated using XPS
depth profiling. The Ti, O, Al, C and Nb peaks were de-
tected in the XPS analysis. The estimated oxide thick-
ness of new implant was 6.2 nm and 5.2 nm for retrieved
one, consisting primarily of TiO
2
.
The results confirm our hypothesis that the effect of
cleaning and sterilization on surface properties of new
and retrieved stems is negligible.
Acknowledgement
This research was funded by the Slovenian Research
Agency Grant ARRS (P2-0132 Institute of Metals and
Technology) and Tertiary Projects of the Department of
Orthopaedic Surgery of University Medical Centre
Ljubljana UKCLJ20180128 and UKC20190145.
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64 Materiali in tehnologije / Materials and technology 55 (2021) 1, 59–64