Biomaterials 30 (2009) 5352–5363

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Time-dependent degradation of titanium osteoconductivity: An implication

of biological aging of implant materials
Wael Att a,1, Norio Hori a,1, Masato Takeuchi b, Jianyong Ouyang c, Yang Yang c, Masakazu Anpo b,
Takahiro Ogawa a, *
a
Laboratory of Bone and Implant Sciences (LBIS), The Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics,
Biomaterials and Hospital Dentistry, UCLA School of Dentistry, Los Angeles, CA, USA
b
Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, Sakai, Japan
c
Department of Materials Science and Engineering, UCLA School of Engineering and Applied Science, Los Angeles, CA, USA

a r t i c l e i n f o a b s t r a c t

Article history: The shelf life of implantable materials has rarely been addressed. We determined whether osteo-
Received 30 April 2009 conductivity of titanium is stable over time. Rat bone marrow-derived osteoblasts were cultured on new
Accepted 19 June 2009 titanium disks (immediately after acid-etching), 3-day-old (stored after acid-etching for 3 days in dark
Available online 11 July 2009
ambient conditions), 2-week-old, and 4-week-old disks. Protein adsorption capacity, and osteoblast
migration, attachment, spread, proliferation and mineralization decreased substantially on old titanium
Keywords:
surfaces in an age-dependent manner. When the 4-week-old implants were placed into rat femurs, the
Bone–titanium integration
biomechanical strength of bone–titanium integration was less than half that for newly processed
Hydrocarbon
Osseointegration implants at the early healing stage. More than 90% of the new implant surface was covered by newly
Total hip replacement generated bone compared to 58% for 4-week-old implants. This time-dependent biological degradation
Dental implant was also found for machined and sandblasted titanium surfaces and was associated with progressive
accumulation of hydrocarbon on titanium surfaces. The new surface could attract osteoblasts even under
a protein-free condition, but its high bioactivity was abrogated by masking the surface with anions. These
results uncover an aging-like time-dependent biological degradation of titanium surfaces from bioactive
to bioinert. We also suggest possible underlying mechanisms for this biological degradation that provide
new insights into how we could inadvertently lose, and conversely, maximize the osteoconductivity of
titanium-based implant materials.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Despite the growing demand for such implant procedures in

Titanium forms a passive and protective surface oxide layer once
exposed to the atmosphere. As a result of chemical stability of this
oxidized surface (primarily TiO2), titanium has superior resistance
to corrosion and is non-cytotoxic. Owing to a bone-forming
phenomenon at the TiO2 interface called ‘‘osseointegration’’ or
‘‘bone–titanium integration’’, titanium implants are widely used as
anchors in reconstructive and restorative surgeries for osteoporotic
fractures, diseased joints, and edentulous jaws.
* Corresponding author. Laboratory for Bone and Implant Sciences (LBIS), The
Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of
Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of
Dentistry, 10833 Le Conte Avenue (B3-081 CHS), Box 951668, Los Angeles, CA
90095-1668, USA. Tel.: þ1 310 825 0727; fax: þ1 310 825 6345.
E-mail address: togawa@dentistry.ucla.edu (T. Ogawa).
1
These authors contributed equally to this work.
a universally aging society, several issues directly related to the
biological capability of titanium remain unresolved. In orthopedic
implants, the treatment outcome includes a high percentage of
revision surgery ranging from 5 to 40% [1–5] (average, 25%) [6]. The
average functional lifetime of the orthopedic titanium implants
used currently is only 10–25 years [7], and the additional surgeries
required after implant failure are a significant burden for patients
and the society. For dental implants, the protracted healing time
required before fabricating prosthetic teeth has been a challenge to
reduce patient morbidity. For dental and orthopedic implants,
various systemic conditions impair the potential of bone healing
around implants [8–12] and may limit this application and
compromise the success rate [13,14]. Rapid and firm establishment
of bone–titanium implant integration has therefore been a persis-
tent challenge in both these fields [15–17]. In addition, titanium is
widely used as a tissue engineering scaffold and coating material
for various medical devices [18–22], and there is a significant

0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2009.06.040
 W. Att et al. / Biomaterials 30 (2009) 5352–5363
demand to improve titanium bioactivity for therapeutic and
experimental purposes.
Biologically, a crucial question remains unaddressed: Why does
bone tissue not form entirely around titanium implants? The
implant area eventually covered by bone (bone–titanium contact
percentage) remains at 45  16% [23], or 50–65% [24], which is far
below the ideal 100%. Most implants fail because of an incomplete
establishment, or early/late destructive changes at the bone–
implant interface [25–27]. A limited supply of stem cells has been
the only hypothetical explanation for this incomplete bone gener-
ation [28,29].
We hypothesized that the bioactivity of titanium changes over
time. The objective of this study was to examine the effects of the
age of titanium on various in vitro behaviors and functions of
osteoblasts cultured on titanium substrates and an in vivo potential
of bone–titanium integration. Additionally, potential factors on
titanium surfaces responsible for the age-related alteration of
osteoconductivity were explored. Orthopedic and dental implant
products are sold as storable medical devices. These products age in
an unavoidable manner during their inventory and distribution, as
well as during storage before use. The age of titanium-based
experimental materials and therapeutic devices has never been
standardized or even described in the literature. Addressing this
nature of titanium, therefore, will have clinical and scientific
significance.
2. Materials and methods
2.1. Titanium samples and surface characterization
Disks (diameter, 20 mm; thickness, 1.5 mm) and cylindrical rods (diameter,
1 mm; length, 2 mm) were prepared from commercially pure titanium (grade 2).
Surfaces of the titanium samples were prepared by acid-etching with 67% (w/w)
sulfuric acid (H2SO4) at 120  C for 75 s, machine turning, or sandblasting with 50 mm
Al2O3 particles for 1 min at a pressure of 3 bar. They were placed in a sealed container
and stored in a dark room (temperature, 23  C; humidity, 60%) for 0 h (new), 3 days, 2
weeks and 4 weeks. Hydrophilicity of titanium surfaces was measured using an
automated contact angle measuring device (DCA-VZ, Kyowa Interface Science, Sai-
tama, Japan) as the contact angle of 1 ml of H2O. Additional image analyses were
performed to evaluate the area of spread of 10 ml H2O on titanium disks. The chemical
composition of titanium surfaces was evaluated by electron spectroscopy for
chemical analysis (ESCA). ESCA was performed by X-ray photoelectron spectroscopy
(XPS) (Multiprobe Surface Science System, Omicron Nanotechnology, Eden Prairie,
MN, USA) under high vacuum conditions (6  107 Pa).
2.2. Protein adsorption assay
Bovine serum albumin, fraction V (Pierce Biotechnology, Inc., Rockford, IL) was
used as a model protein. Three hundred microliters of protein solution (1 mg/ml
protein/saline) was pipetted onto either the new acid-etched titanium surface,
3-day-old surface, 2-week-old surface, and 4-week-old surface. After incubation for 1
and 24 h at 37  C, nonadherent proteins were removed and mixed with micro-
bicinchoninic acid (Pierce Biotechnology) at 37  C for 60 min. The amount of the
removed albumin, as well as the total amount of albumin inoculated, was quantified
using a microplate reader at 562 nm. The rate of albumin adsorption was calculated as
the percentage of albumin adsorbed to titanium surfaces relative to the total amount.
2.3. Osteoblastic cell culture
As established previously [30], bone marrow cells isolated from the femurs of
8-week-old male Sprague–Dawley rats were placed into alpha-modified Eagle’s
medium supplemented with 15% fetal bovine serum, 50 mg/ml ascorbic acid, 10 mM
Na-b-glycerophosphate, 108 M dexamethasone, and antibiotic–antimycotic solu-
tion. Cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37  C.
At 80% confluency, the cells were detached using 0.25% trypsin–1 mM EDTA–4Na and
seeded onto either the new and differently aged titanium disks (3 days old, 2 weeks
old, and 4 weeks old) at a density of 4  104 cells/cm2. The culture medium was
renewed every three days.
2.4. Cell attachment, density, and proliferation assays
Initial attachment of cells was evaluated by measuring the amount of cells
attached to titanium substrates after 2 h and 24 h of incubation. The propagated cells
5353
were quantified in terms of cell density at 2 and 5 culture days using WST-1 based
colorimetry (WST-1, Roche Applied Science, Mannheim, Germany). The culture well
was incubated at 37  C for 4 h with 100 ml tetrazolium salt (WST-1) reagent. The
amount of formazan product was measured using an ELISA reader at 420 nm. The
proliferative activity of cells was measured by BrdU incorporation during DNA
synthesis. At day 2 of culture, 100 ml of 100 mM BrdU solution (Roche Applied
Science) was added to the culture wells and incubated for 10 h. After trypsinizing
cells and denaturing DNA, cultures were incubated with anti-BrdU conjugated with
peroxidase for 90 min and reacted with tetramethylbenzidine for color develop-
ment. Absorbance at 370 nm was measured using an ELISA reader (Synergy HT,
BioTek Instruments, Winooski, VT).
2.5. Migration assay
Migration of cells to titanium surfaces was examined using a cell invasion assay
(345-024K, Trevigen, Gaithersburg, MD). The assay was established to investigate
chemotaxis, migration and/or invasion of various cells [31–33]. The assay uses
a transwell containing 8-mm pore-size inserts made of polyethylene terephthalate
(PET) and measures the ability of cells to migrate from the upper compartment to
the lower compartment through these pores. Cells were seeded into the upper
compartment and a titanium disk was placed at the bottom of the lower compart-
ment. The cells that had penetrated into the lower compartment after 2 h of incu-
bation were quantified by a plate reader after staining with calcein AM. The
percentage of the migrated cells was then calculated relative to the total number of
cells seeded.
2.6. Morphology and morphometry of cells
Confocal laser scanning microscopy was used to examine cell morphology and
cytoskeletal arrangement of the osteoblasts seeded onto titanium surfaces. After 3 h
of culture, cells were fixed in 10% formalin and stained using the fluorescent dye
rhodamine phalloidin (actin filament, red color; Molecular Probes, OR). Cultures
were also immunochemically stained with mouse anti-vinculin monoclonal anti-
body (Abcam, Cambridge, MA), followed by a FITC-conjugated anti-mouse
secondary antibody (Abcam). The area, perimeter and Feret’s diameter of cells were
quantified using an image analyzer (ImageJ, NIH, Bethesda, ML).
2.7. Alkaline phosphatase (ALP) activity
ALP activity of osteoblasts was examined at day 7 using culture area- and
colorimetry-based assays. Cells were washed twice with Hanks’ solution, and
incubated with 120 mM Tris buffer (pH 8.4) containing 0.9 mM naphthol AS-MX
phosphate and 1.8 mM fast red TR for 30 min at 37  C. The ALP-positive area on the
stained images was calculated as [(stained area/total dish area)  100] (%) using the
image analyzer. For colorimetry, the culture was rinsed with double-distilled water
(ddH2O), followed by the addition of 250 ml p-Nitrophenylphosphate (LabAssay ATP,
Wako Pure Chemicals, Richmond, VA), and then incubated at 37  C for 15 min. ALP
activity was evaluated as the amount of nitrophenol released through the enzymatic
reaction and measured at 405 nm using an ELISA reader.
2.8. Mineralization assay
The mineralization capability of cultured osteoblasts was examined by colori-
metry-based quantification of calcium deposition at day 14. Cultures were washed
with PBS and incubated overnight in 1 ml of 0.5 M HCl solution with gentle shaking.
The solution was mixed with o-cresolphthalein complexone in an alkaline medium
(calcium binding and buffer reagent, Sigma, St. Louis, MO) to produce a red calcium-
cresolphthalein complexone complex. Color intensity was measured by an ELISA
reader at 575 nm absorbance.
2.9. Gene expression analysis
Gene expression was analyzed by reverse transcription-polymerase chain
reaction (RT-PCR). Total RNA in human MSCs cultured in the osteogenic induction
medium was extracted using TRIzol (Invitrogen, Carlsbad, CA) on a purification
column (RNeasy, Qiagen, Valencia, CA). Following DNAse I treatment, reverse tran-
scription of 0.5 mg of total RNA was performed using MMLV reverse transcriptase
(Clontech, Carlsbad, CA) in the presence of oligo(dT) primers (Clontech). PCR reac-
tion was performed using Taq DNA polymerase (EX Taq, Takara Bio, Madison, WN) to
detect type I collagen and osteocalcin mRNA using the primer designs and PCR
conditions optimized previously [30]. PCR products were visualized on 1.5% agarose
gel with ethidium bromide staining. Band intensity was detected and quantified
under UV light and normalized to GAPDH mRNA.
2.10. Pretreatment of titanium disks
Protein adsorption and cell attachment on pretreated titanium disks were also
evaluated to identify the role of surface electric charge of titanium surfaces in
determining their bioactivity. Acid-etched titanium disks were incubated for 24 h at
5354 W. Att et al. / Biomaterials 30 (2009) 5352–5363
room temperature in 1 ml of 0.1 M NaCl or CaCl2. Titanium disks incubated in ddH2O
served as controls. Disks were then washed three times with ddH2O and left to
completely dry at room temperature for 3 h.
2.11. Animal surgery
Eight-week-old male Sprague–Dawley rats were anesthetized with 1–2% iso-
flurane inhalation. Their left legs were shaved and scrubbed with 10% providone–
iodine solution. The distal aspect of the left femurs was carefully exposed by skin
incision and muscle dissection. The flat surface of the distal femur was selected for
implantation. The implant site was prepared 9 mm from the distal edge of the femur
by drilling with a 0.8 mm round burr and then enlarged using reamers (#ISO 090
and 100). Profuse irrigation with sterile isotonic saline solution was used for cooling
and cleaning. One cylindrical titanium rod was inserted into each prepared hole per
femur. Surgical sites were then closed in layers. Muscle and skin were sutured
separately with resorbable suture thread. The University of California at Los Angeles
Chancellor’s Animal Research Committee approved this protocol and all experi-
mentation was performed in accordance with the United States Department of
Agriculture guidelines of animal research.
2.12. Implant biomechanical push-in test
The implant biomechanical push-in test was used to assess the biomechan-
ical strength of bone–implant integration, and is described elsewhere [8,34].
Femurs containing a cylindrical implant were harvested at week 2 and 4 of
healing and embedded into auto-polymerizing resin with the top surface of the
implant being horizontal. MicroCT was used to confirm that implants were free
from cortical bone support to the lateral and bottom sides of the implant. The
testing machine (Instron 5544 electromechanical testing system, Instron, Canton,
MA) equipped with a 2000 N load cell and a pushing rod (diameter, 0.8 mm) was
used to load the implant vertically downward at a crosshead speed of 1 mm/min.
The push-in value was determined by measuring the peak of the load–
displacement curve.
2.13. Histological preparation
Femurs containing an acid-etched implants were harvested at weeks 2 and 4
post-implantation and fixed in 10% buffered formalin for 2 weeks at 4  C. Specimens
were dehydrated in an ascending series of alcohol rinses and embedded in light-
curing epoxy resin (Technovit 7200VLC, Hereaus Kulzer, Weinheim, Germany)
without decalcification. Embedded specimens were sawed perpendicular to the
longitudinal axis of the cylindrical implants at a site 0.5 mm from the apical end of
the implant. Specimens were ground to a thickness of 30 mm with a grinding system
(Exakt Apparatebau, Norderstedt, Germany). Sections were stained with Goldner’s
trichrome stain and observed via light microscopy.
2.14. Histomorphometry
A 40 magnification lens and a 4 zoom on a computer display were used for
computer-based histomorphometric measurements (Image Pro-plus, Media Cyber-
netics, Silver Spring, MD). To identify the tissue structure in detail, microscopic
magnification up to 400 was used. We previously established implant histo-
morphometry that discriminates between implant-associated bone and nonim-
plant-associated bone [24]. Based on this method, the circumferential zone within
50 mm of the implant surface was defined as an area of interest.
Bone–implant contact (%) ¼ (sum of the length of bone–implant contact)/
(circumference of the implant)  100, where the bone–implant contact was defined
as the interface at which bone tissue was located within 20 mm of the implant
surface without intervention of soft tissue.
Bone volumeð%Þ [ ðbone area in the area of interestÞ=ðarea of interestÞ 3 100:
2.15. Statistical analyses
The number of samples was 3 for all studies, except for cell morphometry
(n ¼ 10), the implant push-in test (n ¼ 5) and bone histomorphometry (n ¼ 4). Two-
way ANOVA was used to examine the effects of culture/healing time and aging of
titanium. If necessary, the post-hoc Bonferroni test was used as a multiple
comparison test; p < 0.05 was considered significant. If data were available at only
one time point, one-way ANOVA was used to determine the differences among
different aging groups. Correlations between biological parameters and physico-
chemical parameters of titanium surfaces were determined by least-mean-square
approximation.
3. Results
3.1. Protein adsorption and initial osteoblast behaviors on
differently aged titanium
We first focused on acid-etched titanium surfaces to examine
the effect of titanium aging (new, 3-day-old, 2-week-old, and
4-week-old surfaces) on their bioactivity. The amount of albumin
adsorbed to titanium surfaces during 1 h and 24 h incubation
significantly decreased on the aged surfaces in an age-dependent
manner (Fig. 1A). The 4-week-old disks exhibited only one-quarter
the albumin adsorption of the new disks after 1 h, and this age-
dependent attenuation was substantial even after 24 h. The
number of rat bone marrow-derived osteoblasts attached to tita-
nium surfaces during 2 h and 24 h incubation decreased signifi-
cantly with an increase in titanium age (Fig. 1B). The number of
cells that migrated to titanium surfaces through 8-mm holes during
2 h incubation also decreased depending on the age of titanium
surfaces (Fig. 1C). Confocal microscopic images of osteoblasts 3 h
after seeding onto the new and 4-week-old titanium surfaces
revealed a distinctive contrast in cell morphology and the estab-
lishment of focal adhesion (Fig. 1D). The cells were noticeably larger
with an extensive expression of vinculin on the new surface.
Cytomorphometric evaluations demonstrated that the area,
perimeter, and Feret’s diameter of the cells were significantly
greater for the new surfaces than for the 4-week-old surfaces,
indicating a considerable delay in cell spread and cytoskeletal
development on the 4-week-old surface.
3.2. Osteoblast proliferation on differently aged titanium
The cell density was significantly reduced on days 2 and 5 on the
old titanium surfaces; the number of cells was reduced to one-half
on the 4-week-old surface as compared to the new surface at both
time points (Fig. 2A). To determine whether the lower cell density
was ascribed to decreased cell attachment upon seeding or
a possible impairment of subsequent cellular propagation, BrdU
incorporation into DNA was measured relative to cell number. The
rate of cell proliferation on old surfaces decreased in an age-
dependent manner (Fig. 2B).
3.3. Osteoblast differentiation on differently aged titanium
The alkaline phosphatase (ALP)-positive area of the culture
decreased on aged titanium surfaces (Fig. 2C); in the 4-week-old
titanium culture on day 7, it was less than one-half that on the new
surface. Calcium deposition in the mineralizing cultures was simi-
larly reduced on day 14 (Fig. 2D). To determine whether these
attenuating changes in functional phenotypes occurred because of
reduced cell density, possibly impaired differentiation, or both, ALP
activity, which was optically quantified and standardized relative to
cell number, was examined. There were no significant differences in
cell-based ALP activity among the new and differently aged tita-
nium disks (Fig. 2E). The expression level of collagen I and osteo-
calcin evaluated by RT-PCR was also not significantly different
among the differently aged groups (Fig. 2F).
3.4. Biomechanical strength of bone–implant integration
The strength of bone–implant integration evaluated by the
push-in test was substantially impaired depending upon the age of
titanium implants (Fig. 3). The push-in value for new implants at
the early healing stage of week 2 was 2.2 times that for 4-week-old
implants, i.e., mechanical retention obtained with the 4-week-old
implants was less than 50% of that obtained with the new implants.
 W. Att et al. / Biomaterials 30 (2009) 5352–5363 5355

A New B New C New

3-day-old 3-day-old 3-day-old
60 2-week-old 2-week-old 2-week-old
0.5 20
4-week-old 4-week-old 4-week-old
Protein adsorption (%)

Cell attachment (WST-1)

50 **
**

Migrated cells (%)

0.4
15 **
40
0.3
30
**
10
**
0.2
20
5
10 0.1

0 0 0
1 hour incubation 24 hour incubation 2 hour incubation 24 hour incubation 2 hour incubation

D New surface 4-week-old surface

New 4-week-old

300 80
2000
** ** **

Feret’s diameter (µm)

Perimeter (µm)
60
µm2)

100 µm 100 µm 200

Area (µ

40
1000
100
20

0 0 0

50 µm 50 µm

Fig. 1. Age-dependent degradation of osteoblast affinity of titanium surfaces. Acid-etched titanium disks of different agesdnew (immediately after processing), 3-day-old, 2-week-
old, and 4-week-old surfacesdwere tested. (A) Albumin adsorption to titanium surfaces during 1 h and 24 h of incubation. (B) The number of rat bone marrow-derived osteoblasts
attached to titanium surfaces after incubation for 2 h and 24 h, as evaluated by WST-1 colorimetry. (C) Osteoblasts migrated to the titanium surface through 8 mm pores during 2 h
of incubation quantified by invasion assay. Data are mean  SD (n ¼ 3) for panels A–C. **p < 0.01, indicating a statistically significant effect of titanium aging. (D) Initial spread and
cytoskeletal arrangement of osteoblasts 3 h after seeding onto new and 4-week-old titanium surfaces. Representative confocal microscopic images of cells stained with rhodamine
phalloidin for actin filaments (red) and anti-vinculin (green), along with cytomorphometric evaluations, are presented. Data are mean  SD (n ¼ 10). **p < 0.01, indicating
a statistically significant difference between the new and 4-week-old surfaces.

Even at a late stage of healing (week 4), age-dependent reduction in
the push-in value remained substantial. The push-in value for the
new implants at week 2 of healing was even higher than that of the
4-week-old implants at week 4 of healing (p < 0.05).
3.5. Bone formation around differently aged implants
Histological and histomorphometric analyses of bone were
performed around the new and 4-week-old implants. At week 2 of
healing, newly formed bone was observed around both implants
(Fig. 4A and B). The area adjacent to the implant surface showed
noticeable differences in bone growth between the two implants.
The bone formation was contiguous and extensive around the new
implant (Fig. 4A and E), but localized and fragmented around the
4-week-old implant (Fig. 4B and F), leaving a large area covered by
soft tissue (arrowhead in panels B and F). At week 4 of healing, bone
formed almost entirely around the new implants (Fig. 4C and G),
showing a clear distinction from the 4-week-old implants where
a significant portion of the implant was covered by fibrous tissue
(arrowheads in panels D and H). Bone histomorphometry demon-
strated that the percentage of bone–implant contact for the new
implants was consistently greater than that for the 4-week-old
implants (2.3 times at week 2 and 1.6 times at week 4) (Fig. 4I).
Bone volume associated with the implants was also consistently
greater for the new implants (Fig. 4J).
3.6. Effects of titanium aging on other surface types
We further investigated if the time-dependent degradation in
bioactivity of titanium would occur in surface types other than the
5356 W. Att et al. / Biomaterials 30 (2009) 5352–5363

A New B New
3-day-old 3-day-old
2-week-old 2-week-old
2.5 4-week-old 4-week-old
4
2.0 ** *
Cell density (WST-1)

BrdU incorporation
3

(Od370 nm)/cell
1.5

2
1.0
**
0.5 1

0 0
Day 2 Day 5 Day 2

C New 3-day-old 2-week-old 4-week-old D

New
3-day-old
2-week-old
4-week-old

15
Calcium deposition (mg/dl)
60
New
ALP positive area (%)

50 ** 3-day-old **
2-week-old
40 4-week-old 10

30

20 5

10

0 0
Day 7 Day 14

E F Day 7 Day 14
New New 3-D 2-W 4-W New 3-D 2-W 4-W
3-day-old Collagen I
2-week-old
Osteocalcin
4-week-old
GAPDH
4
Relative ALP activity/cell

New 3-day-old 2-week-old 4-week-old

3
0.6 0.6
Gene expression
(arbitrary unit)

2 0.4 0.4

1 0.2 0.2

0 0 0
Day 7 Day 7 Day 14 Day 7 Day 14
Collagen I Osteocalcin
Fig. 2. Osteoblast proliferation and function on differently aged titanium surfaces. Rat bone marrow-derived osteoblasts were cultured on acid-etched titanium disks with different
agesdnew, 3-day-old, 2-week-old, and 4-week-old surfaces. (A) Cell density at days 2 and 5 of cultures. (B) Cell proliferative activity evaluated by BrdU incorporation per cell at day
2 of culture. (C) Alkaline phosphatase (ALP) activity measured by the ALP-positive (red) area at day 7. Representative images of the stained culture are presented on the top. (D) Total
calcium deposition at day 14, as measured using a colorimetry-based method. (E) ALP activity at day 7 colorimetrically quantified and standardized relative to cell number. (F)
Expression of bone-related genes in osteoblast cultures at days 7 and 14 examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Expression levels were quantified
relative to the level of GAPDH mRNA expression. Data are mean  SD (n ¼ 3) for all panels. **p < 0.01, indicating a statistically significant effect of titanium aging.
 W. Att et al. / Biomaterials 30 (2009) 5352–5363 5357

New acid-etched one. Machined and sandblasted titanium disks were

3-day-old prepared and stored for different periods of time before use in the
60 2-week-old same manner as the acid-etched disks. Cell density decreased on
both types of titanium surfaces in an age-dependent manner
4-week-old
(Fig. 5A and C). ALP staining of the cultures on these surface types
50 was also attenuated, and the range of reduction was as substantial
*
as that observed for the acid-etched surface (Fig. 5B and D).
40
Push-in value (N)

* 3.7. Physicochemical characteristics of differently aged titanium

30 surfaces

We conducted surface characterization of acid-etched titanium to

20
identify factors responsible for its time-dependent biological degra-
dation. The new surface, i.e., the surface obtained immediately after
10 acid-etching, showed superhydrophilic property with a 10-ml water
drop spread over on a 20-mm diameter disk and a contact angle of
0 0.0 (Fig. 6A). As the titanium disks aged, the surface property
Week 2 Week 4 changed from being hydrophilic to hydrophobic. Least-mean-square
Healing time approximation indicated that the contact angle did not significantly
correlate with the albumin adsorption or cell attachment capacities of
Fig. 3. In vivo bone integration ability of differently aged titanium implants. Biome- the titanium surface (R2 ¼ 0.49 and 0.56, respectively; Fig. 6B and C).
chanical strength of bone–implant integration evaluated by the biomechanical push-in
test. Implants with new, 3-day-old, 2-week-old, and 4-week-old acid-etched surfaces
XPS spectra showed peaks of Ti2p, O1s and C1s on the acid-
were compared. Data are mean  SD (n ¼ 5). *p < 0.05, indicating a statistically etched titanium surfaces, but not other peaks (Fig. 6D). The C1s
significant effect of implant aging. peak was considerably higher on the 4-week-old surface than on

Week 2 of healing Week 4 of healing

New implants 4-week-old implants New implants 4-week-old implants
A B C D

µm
200µ

E F G H

100µm

I New J New
4-week-old
Bone - implant contact (%)

10 0 100 4-week-old
*
*
Bone volume (%)

80 80
**
60 60 **

40 40

20 20

0 0
Week 2 Week 4 Week 2 Week 4
Fig. 4. In vivo bone morphogenesis around differently aged titanium implants. Implants with new and 4-week-old acid-etched surfaces were compared. (A–H) Representative
histological images of peri-implant tissue with Goldner’s trichrome stain at an original magnification of 200 for panels (A)–(D), and 400 for panels (E)–(H) are presented. Mean
histomorphometric values with SD (error bars) of bone–implant contact (I) and bone volume (J) are shown (n ¼ 4). **p < 0.01, *p < 0.05, indicating a statistically significant
difference between the new and 4-week-old surfaces.
5358 W. Att et al. / Biomaterials 30 (2009) 5352–5363

A New B 2-week 4-week

New -old -old
2-week-old
4-week-old

4
Cell density (WST-1)

80 New

ALP positive area (%)

2-week-old
3 ** **
60 4-week-old

2 * 40

1 20

0 0
Day 2 Day 5 Day 7

C New D 2-week 4-week

New -old -old
2-week-old
4-week-old

2.5
**
60 New
Cell density (WST-1)

2.0
ALP positive area (%)

2-week-old
50
1.5 4-week-old
40 **
1.0 ** 30
20
0.5
10

0 0
Day 2 Day 5 Day 7

Fig. 5. Osteoblast function on differently aged titanium surfaces with other surface textures. Rat bone marrow-derived osteoblasts were cultured on machined (A, B) and sand-
blasted (C, D) titanium disks of different ages: new (immediately after processing), 2-week-old, and 4-week-old surfaces. Cell density at days 2 and 5 (A, C) and ALP-positive area at
day 7 (B, D) shown as mean  SD (n ¼ 3). *p < 0.05, **p < 0.01, indicating a statistically significant effect of titanium aging.

the new surface. Close examination of the peaks revealed that
a shoulder peak at about 288 eV ascribed to the presence of
oxygen-containing hydrocarbons appeared on old surfaces, as
shown on the 4-week-old surface (Fig. 6E). This was not observed
on the new surface. The atomic percentage of carbon continued to
increase from 16 to 62% as the titanium surfaces aged (Fig. 6F).
Least-mean-square approximation revealed that there was
a significant inverse linear correlation between the atomic
percentage of carbon and the (1) amount of albumin adsorbed to
titanium surfaces and (2) number of attached cells (R2 ¼ 0.91 and
0.98, respectively; Fig. 6G and H). In other words, the lower the
percentage of carbon on the titanium surface, the more the amount
of albumin and cells attached to the surface.
3.8. Protein adsorption and cell attachment to differently aged
titanium with various surface treatments
Albumin adsorption was evaluated on the new and 4-week-old
titanium surfaces at different pH conditions (Fig. 7A). Remarkably
enhanced albumin adsorption to the new surface at pH 7 was
completely abrogated at pH 3 (untreated group in Fig. 7A). We next
treated the new surface with various solutions containing anions
(e.g., Cl) for 24 h and dried it before albumin incubation. The
anion-treated new surface was no longer protein attractive
(Fig. 7A), indicating that its protein-favorable electrostatic property
was neutralized. All new surfaces maintained superhydrophilicity
after the 24-h anion treatment (images of H2O drop on titanium
disks in Fig. 7A). The albumin adsorption of the 4-week-old tita-
nium surface increased only when it was treated with divalent
cations (e.g., Ca2þ) before albumin incubation. The treatment with
divalent cations did not increase albumin adsorption on the new
surface.
The effects of anion and cation treatment of the new and
4-week-old surfaces on cell attachment were examined next
(Fig. 7B). The enhanced osteoblast attachment to the new titanium
surface was eliminated by masking the surface with monovalent
anions. The treatment with divalent cations (Ca2þ) improved
osteoblast attachment to the 4-week-old surface. Similar to protein
 W. Att et al. / Biomaterials 30 (2009) 5352–5363 5359

A New 3-day-old 2-week-old 4-week-old

B C
40 0.5

Albumin adsorption (%)

N.S.

Cell attachment (WST-1)

N.S. 0.4
300 60 30
Area of water spread

Contact angle of water

50 0.3
20
200 40
(mm2)

0.2

(degree)
30 10
0.1
100 20
10 0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
0 0 Contact angle of H2O (degree) Contact angle of H2O (degree)
New 3-d 2-w 4-w New 3-d 2-w 4-w
-old - old -old -old - old -old

D E F G H
O1s C1s 80
40 0.5
O Y=-0.49x + 43.0 Y=-0.006x + 0.55

Cell attachment (WST-1)

Ti2p C1s

Albumin adsorption (%)

60 C (R2= 0.91; p<0.05) 0.4 (R2= 0.98; p<0.01)
Atomic percentage

30
Intensity (cps)

Intensity (cps)

4-week 4-week 0.3

-old -old 40
20
0.2

20 10 0.1
New New Ti
0 0 0
600 400 200 292 288 284 0 3d 2 week 4 week 10 20 30 40 50 60 10 20 30 40 50 60
Binding Energy Age of titanium Atomic percentage of Atomic percentage of carbon
Binding Energy (eV)
(eV) carbon

Fig. 6. Age-dependent changes in surface physicochemistry of titanium with respect to their biological effects. (A) Hydrophilicity of new (immediately after acid-etching) and aged
(3-day-old, 2-week-old, and 4-week-old) acid-etched titanium surfaces. Upper panels show the top and side views of titanium disks immediately after a 10 ml droplet of distilled
H2O was placed. Lower panels show the calculated the area of spread on the disks and the contact angle of H2O measured by an automatic contact angle measuring device. Data are
mean  SD (n ¼ 3). Albumin adsorption after 1 h incubation (B) and osteoblast attachment during 2 h incubation (C) plotted against the H2O contact angle on titanium surfaces,
showing no significant correlation between them. (D) X-ray photoelectron spectroscopy (XPS) spectrum for the new and 4-week-old titanium surfaces. (E) Close-up view of the XPS
C1s peak. (F) Change in atomic percentage on the titanium surface with age, i.e., new to 4 weeks old. (G) Plot of albumin adsorption after 1 h incubation against the atomic
percentage of carbon on the titanium surface, showing a significant inverse linear correlation. (H) Osteoblast attachment after 2-h incubation plotted against the atomic percentage
of carbon on the titanium surface, showing their significant inverse linear correlation.

adsorption, even with the divalent cations, cell attachment to the The lower in vivo parameters of the aged implants were in
4-week-old surface was far below that of the new surface. agreement with the impaired migration, attachment, spread, and
The attachment of osteoblasts to titanium surfaces was also proliferation of osteoblasts observed on the aged titanium surfaces.
examined under a serum-free condition. The amount of cells However, the cell-based ALP activity and osteoblastic gene
attached to the new surface was twice that observed on the 4- expressions were not significantly affected by the age of titanium,
week-old surface under this challenging condition (Fig. 7C). Diva- suggesting that the age of titanium was unlikely to modulate the
lent cations did not increase the cell attachment to the 4-week-old rate of osteoblast differentiation and that the age-dependent
surface unlike the result in the serum (þ) cultures (Fig. 7B and C). attenuation in the ALP-positive area and calcium deposition was
probably a result of the compromised initial behavior and response
4. Discussion of osteoblasts, such as the migration, attachment and proliferation
of the cells. More importantly, the reduction of these in vitro
In vivo establishment of implant fixation is the most important parameters was found on aged surfaces of other surface topogra-
factor in determining the clinical capacity of implants as load- phies: machined and sandblasted surfaces, suggesting that this
bearing devices. The biomechanical strength of bone–implant age-dependent biological degradation may be applicable to any
integration evaluated by the push-in test was reduced titanium surface texture of titanium-based materials.
age-dependently and was eventually less than half after 4 weeks of Commercially available orthopedic and dental implants are sold
storage of the implants. The lower strength of bone–implant inte- as storable medical devices. These products invariably age during
gration around the aged implants suggested that the old implant their inventory and distribution, as well as storage before use. This
surfaces involve a smaller volume of newly generated bone, may explain the relatively low bone–titanium contact results in the
a smaller percentage of bone-to-implant contact, or both. Histo- literature [23,24,35,36]. Conversely, from a therapeutic perspective,
logical and histomorphometric analyses demonstrated that both of we may be able to harness the discovered phenomenon of the
these parameters were substantially lower around the 4-week-old biological aging to maximize titanium bioactivity. There is a strong
implants. It should be noted that the biomechanical and histolog- possibility that the innate bioactivity of titanium implant products
ical indices of the aged implants showed lower values not only at is much greater than that obtained from commercially available
the early healing stage of week 2 but also at the late stage of week 4, products after aging during an unidentified time period. This study
indicating that the aging of titanium implants resulted in not only demonstrated that implant fixation was enhanced 2.2 times, and
the delay but also a compromised level of bone–implant bone–implant contact increased to >90% simply by the use of new
integration. titanium surfaces as compared to 4-week-old surfaces. This could
5360 W. Att et al. / Biomaterials 30 (2009) 5352–5363

A 40
pH7 pH3
**
Protein adsorption (%)

30
**

20 **

10

0
Untreated NaCl- CaCl2- H2O- Untreated NaCl- CaCl2- H2O-
treated treated treated treated treated treated

New surfaces 4-week-old surfaces

B 0.2 C 0.2
**

Cell attachment (WST-1)

**
Cell attachment (WST-1)

0.1 ** 0.1
**

0 0
Untreated NaCl- CaCl2- H2O- Untreated NaCl- CaCl2- H2O- Untreated CaCl2- Untreated CaCl2-
treated treated treated treated treated treated treated treated

New surfaces 4-week-old surfaces New surfaces 4-week-old surfaces

Fig. 7. Protein adsorption and cell attachment to differently aged titanium under various electrostatic conditions. (A) Protein adsorption to new and 4-week-old acid-etched
titanium surfaces with and without pretreatment with various cations/anions under different pH. Data are mean  SD (n ¼ 3). **p < 0.01, indicating a significant difference from the
untreated 4-week-old surface. The hydrophilicity test indicated that all new surfaces are superhydrophilic after ion pretreatment, and all 4-week-old surfaces are hydrophobic, as
illustrated in the images at the bottom. (B) Osteoblast attachment to the new and 4-week-old titanium surfaces with and without various cations/anions pretreatment. Data are
mean  SD (n ¼ 3). **p < 0.01, indicating a significant difference from the untreated 4-week-old surface. (C) Osteoblast attachment to the new and 4-week-old titanium surfaces
with and without CaCl2 pretreatment in a serum-free medium. Data are mean  SD (n ¼ 3). **p < 0.01, indicating a significant difference from the untreated 4-week-old surface.

be a simple but effective therapeutic approach. Given that time-
related biological degradation was demonstrated on multiple
surface types of titanium, this strategy is expected to be applicable
to many currently available titanium-based materials.
The age dependency of titanium bioactivity, which has been
unearthed in this study, yielded 2–7 times differences in the
various measured parameters. This finding may provide a new
important insight into further advancing the research on implant
and other titanium-containing biomaterials. To our knowledge, the
age of titanium implants and other titanium-containing experi-
mental substrates has not been standardized in the literature.
Given the substantial age-dependent abatement in bioactivity,
future studies comparing different titanium surface types, e.g.,
chemical and topographical modifications, may require standardi-
zation of their age or a careful interpretation of the research
outcome. The knowledge gained in the present study may help
establish new guidelines for research design and data interpreta-
tion in this field.
The marked increase in protein adsorption, and cell migration
and attachment on the new surface, as well as the upregulated
expression of focal adhesion proteins, prompted us to explore the
possible presence of chemoattractants specific to the new surface.
As shown in Fig. 1A, albumin adsorption was considerably reduced
on the 4-week-old surface as compared to the new surface. TiO2
surface is known to be electronegatively charged at the physiologic
pH values [37,38] and serum albumin molecules are known to be
negatively charged. It was therefore reasonable to anticipate that
there is not much albumin–titanium surface interaction [37,38]. In
fact, the amount of albumin adsorbed to the old titanium surface
was limited in the present result; however, here is a fundamental
but important question: At what age of titanium surfaces has the
electrostatic status been evaluated. From a practical view point,
regardless of commercially available or in-house manufactured
bulk titanium, the age of titanium surfaces subjected to charac-
terization is unidentified. It is unlikely that freshly exposed tita-
nium surfaces are examined immediately after the processing. The
present results showing increased albumin adsorption on the new
titanium surface led us to hypothesize that new surfaces may have
an electrostatic status different from the old surfaces. To address
this hypothesis, albumin adsorption was evaluated on the new and
4-week-old titanium surfaces at different pH conditions. It turned
out that the remarkably enhanced albumin adsorption to the new
 W. Att et al. / Biomaterials 30 (2009) 5352–5363 5361

Protein Cell

O RGD O Na Cell-inert
Cell-
Ti Ti
attractive
O O Cell-repellent

RGD Cell-
O Ca attractive
O
Ti Cell- Ti
O attractive O
K Cell-inert
New Ti Aged Ti
Cell-repellent
O RGD O
Cell- Ti
Ti
attractive O
O Na Cell-inert

Cell-repellent
O Cell- O
Ti attractive Ti
O O Cell-inert
K

Fig. 8. Schematic description of the proposed mechanism of electrostatic interactions underlying the aged-dependent biological degradation of TiO2 surfaces: the age-dependent
conversion of titanium surfaces from bioactive to bioinert. The new TiO2 surface (left) is abundant in cell-attracting terminals consisting of the RGD sequence or positively charged
TiO2 surface, which serve as chemoattractants without divalent cations such as Ca2þ. The old TiO2 surface (right) involves cell-inert and cell-attractive terminals consisting of
competitive binding of monovalent and divalent cations to negatively charged TiO2 surface, respectively. The surface attracts proteins and cells only with divalent cations. When
there are no sufficient cations available, a part of the old TiO2 surface remains to be negatively charged, leaving these terminals cell-repellent.

surface at pH 7 was completely abrogated at pH 3. This was because
the isoelectric point of albumin lies between 4.7 and 4.9, and at pH
values <4.7, it undergoes a neutral to basic transition and is posi-
tively charged. The isoelectric point of TiO2 between 5.6 and 6.1 also
contributed to this abrogation. We next treated the new surface
with anions for 24 h and dried it before albumin incubation. The
anion-treated new surface was no longer protein attractive as
shown in Fig. 7A, suggesting that its protein-favorable electrostatic
property was neutralized. It should be noted that all new surfaces
tested maintained superhydrophilicity after the 24-h anion treat-
ment (Fig. 7A). Meanwhile, the albumin adsorption of the 4-week-
old titanium surface increased only when it was treated with
divalent cations (e.g., Ca2þ) before albumin incubation. This agreed
with the established mechanism of titanium-protein interaction in
which divalent calcium cations deposited onto monovalent nega-
tive titanium surfaces play a bridging role between negative protein
molecules and the TiO2 surface [37,38]. However, treatment with
divalent cations did not increase albumin adsorption on the new
surface. These results indicated that the new titanium surface is
electropositive, as opposed to the long-established understanding
of negatively charged titanium surfaces, and uncovered an unex-
pected role of this new surface property in achieving a new level of
bioactivity. The new level of bioactivity was well represented by the
fact that the amount of albumin adsorption to the 4-week-old
surface was substantially lower than that on the bare new titanium
surface, even with the divalent cation treatment. In addition,
isoelectric control or anion treatment of the new surfaces domi-
nantly regulated protein adsorption, superseding the effect of
superhydrophilicity. These results suggest that the unique elec-
trostatic status of the new titanium surface serves as a chemo-
attractant for proteins, and is the critical factor in determining
titanium bioactivity.
If protein adsorption is enhanced, subsequent cell attachment
should be enhanced in correlation with an increased cell-protein
interaction via ligand-specific binding (e.g., integrin–RGD interac-
tion). This hypothesis was supported by enhanced cell attachment
to the new surfaces compared to the old surfaces, as described
earlier. Anion and cation treatment of the new and 4-week-old
surfaces further indicated the following (Fig. 7B): (1) the enhanced
osteoblast attachment to the new titanium surface was eliminated
by masking the surface with monovalent anions, (2) divalent
cations (Ca2þ) are necessary to improve osteoblast attachment to
the 4-week-old surface; and (3) similar to protein adsorption, even
with the divalent cations, cell attachment to the 4-week-old surface
was far below that of the new surface.
We also examined whether the new titanium surface attracts
osteoblasts via electrostatic forces without cell-protein interaction.
It was likely that there may be a direct interaction between the
positively charged new titanium surface and negatively charged
biological cells. This hypothesis was supported by the result
showing that the amount of cells attached to the new surface was
twice that observed on the 4-week-old surface in serum-free
cultures. Divalent cations were not as effective at increasing cell
attachment on the 4-week-old surface as in the serum (þ) culture,
indicating that proteins are crucial to enable cell-titanium inter-
action on this aged surface.
We have schematically represented the proposed mechanism of
protein and cell attachment to titanium surfaces of different age
(Fig. 8). The titanium bulk on the right (aged Ti) indicates the cell-
titanium interaction around titanium surfaces, which has been
proposed as an underlying mechanism of the ‘‘bioinert’’ surface
[37,38]. The surface has a net negative charge as reported in the
literature and must therefore first be bridged by divalent cations
such as Ca2þ to attract anionic proteins [37–39]. Cells will then get
5362 W. Att et al. / Biomaterials 30 (2009) 5352–5363
attached to the titanium surface via the RGD sequence recognition
of the protein. Direct cell attachment to the bridging cation sites,
skipping the protein binding phase, is unlikely to occur (Fig. 7C).
Only divalent cations function as bridging agents: therefore
competitive binding of monovalent cations, such as Naþ and Kþ,
may block the anion sites of the titanium surface for Ca2þ binding,
making a large part of the titanium surface protein- and cell inert
[38]. If divalent cations are insufficient, old titanium surfaces may
remain protein- and cell repellent. The scheme on the left (new Ti)
represents a newly proposed mechanism that renders the new
surface ‘‘bioactive.’’ The positively charged surface of the newly
processed titanium allows both proteins and cells (most of which
are anionic) to directly attach to the titanium surface without
divalent cations, largely creating a protein- and cell-attractive
interface. A probable direct cell–titanium interaction has also been
proved in the absence of proteins in the present results (Fig. 7C).
Unlike TiO2 surfaces, one of the advantages of hydroxyapatite as an
implantable and coating material stems from its positive surface
charge that allows interaction with proteins and cells without
bridging ions [37,39–41]. Hydroxyapatite has therefore been cate-
gorized as a bioactive material, whereas TiO2 has been categorized
as a bioinert material. The present results and proposed mecha-
nisms provide a scientific cue of re-evaluating and re-defining
biological properties of titanium-based biomaterials.
Mechanisms linking the progressive deposition of hydrocarbons
on titanium surfaces and the electrostatic status of such surfaces
require further investigation. Titanium constantly absorbs organic
impurities such as carbon and hydrocarbons from the atmosphere,
water and cleaning solutions [42–44]. The currently used titanium
implants are routinely found to be contaminated with hydrocar-
bons [45–48], which suggests that such contamination, particularly
with contaminants having a carbonyl moiety, is unavoidable during
storage under ambient conditions. Although the cause–effect
relationship between hydrocarbons and time-related degradation
of osteoconductivity needs to be determined mechanistically, there
was indeed a strong inverse correlation. In light of this finding, the
amount of hydrocarbons adsorbed on the TiO2 surface at implan-
tation seems to be crucial in determining the initial affinity level for
osteoblasts and consequently, the level of new bone formation and
degree of bone–titanium integration. A recent study demonstrated
that UV treatment of titanium effectively removes hydrocarbons
from the surface, resulting in the enhancement of its osteo-
conductivity [49]. In line with the present results, this approach
may open a new avenue in the therapeutics and science of titanium
implants.
5. Conclusions
This study tested a hypothesis that the bioactivity of titanium
surfaces changes during their aging. Rat bone marrow-derived
osteoblasts were cultured on new titanium disks (immediately after
either acid-etching, machining, or sandblasting), 3-day-old (stored
after processing for 3 days in dark ambient conditions), 2-week-old,
and 4-week-old disks. Osteoblast migration and attachment to
titanium, proliferation, alkaline phosphatase positive area, and
mineralized nodule area in osteoblastic cultures were substantially
lower on the old titanium disks in an age-dependent manner. The
biomechanical strength of bone–titanium integration for the
4-week-old titanium implants measured in a rat femur model was
less than half that for new implants. At week 4 of healing, more than
90% of the new titanium surface was covered by newly generated
bone compared to 58% for 4-week-old implants. The levels of
protein adsorption and osteoblast attachment to titanium surfaces
were inversely correlated with the amount of hydrocarbon on
titanium surfaces which increased age-dependently, but not with
the level of hydrophilicity. The increased bioactivity of new surfaces
was completely abrogated under the acidic condition or by treating
the surface with anions, while maintaining its superhydrophilicity.
The cell attachment was significantly higher on the new surface
than on the old surfaces even in the serum-free culture condition.
These results uncover an aging-like time-dependently degrading
osteoconductivity of titanium. We suggest that newly processed
titanium surfaces are bioactive and that the surfaces degrade
substantially over time to the level of so-called ‘‘bioinert’’. We also
suggest possible underlying mechanisms for this nature of titanium
that provide new insights into how we could inadvertently and
unavoidably lose, and conversely, maximize the osteoconductivity
of titanium-based implant materials.
Acknowledgements
This work was supported by JAMSEA. This study was conducted
in a facility constructed with support from the Research Facilities
Improvement Program, grant no. C06RR014529, of the National
Center for Research Resources, National Institute of Health.
Appendix
Figures with essential color discrimination. The majority of the
figures in this article have parts that are difficult to interpret in
black and white. The full color images can be found in the on-line
version, at doi:10.1016/j.biomaterials.2009.06.040.
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