jbmr increased chondrocyte adhesion on anodized titanium
TRANSCRIPT
Increased Chondrocyte Adhesion on Nanotubular Anodized Titanium
Kevin Burns, and Chang Yao, and Thomas J. Webster*
Divisions of Engineering and Orthopaedics
Brown University, Providence, RI 02912 USA
*Contact Author:
Thomas J. Webster
Associate Professor
Divisions of Engineering and Orthopaedics
Brown University
Providence, RI 02912 USA
Tel: 401-863-2318
Fax: 401-863-2319
E-mail: [email protected]
Abstract
Previous studies have demonstrated increased osteoblast (bone-forming cells)
functions (including adhesion, synthesis of intracellular collagen, alkaline phosphatase
activity, and deposition of calcium-containing minerals) on titanium anodized to possess
nanometer features compared to their unanodized counterparts. Such titanium materials
were anodized to possess novel nanotubes also capable of drug delivery. Since titanium
has not only experienced wide spread commercial use in orthopedic but also in cartilage
applications, the objective of the present in vitro study was for the first time to investigate
chondrocyte (cartilage synthesizing cells) functions on titanium anodized to possess
nanotubes. For this purpose, titanium was anodized in dilute hydrofluoric acid at 20 V for
10 minutes. Results showed increased chondrocyte adhesion on anodized titanium with
nanotube structures compared to unanodized titanium. Importantly, the present study also
provided evidence why. Since material characterization studies revealed significantly
greater nanometer roughness and similar chemistry as well as crystallinity between
nanotubular anodized and unanodized titanium, the results of the present study highlight
the importance of the nanometer roughness provided by anodized nanotubes on titanium
for enhancing chondrocyte adhesion. In this manner, the results of the present in vitro
study indicated that anodization might be a promising quick and inexpensive method to
modify the surface of titanium-based implants to induce better chondrocyte adhesion for
cartilage applications.
Keywords: titanium; anodization; chondrocyte; nanotopography; surface
roughness; cartilage
Introduction
Titanium is known as a “valve metal”, i.e. when it is exposed to air, water and
other oxygen containing atmospheres, an oxide layer spontaneously forms on its surface
to protect the underlying metal [1]. For this reason, titanium-based alloys have excellent
corrosion resistance and good biocompatibility. Also, due to its light weight and
appropriate mechanical properties, titanium and its alloys are widely used in orthopedic
and cartilage applications. In fact, several companies now have cartilage implant products
based on titanium used in orthopedic applications [2]. However, the inability for
chondrocytes (cartilage synthesizing cells) to adhere and subsequently form new cartilage
tissue on titanium has remained problematic [3]. Clearly, for such patients who
simultaneously have bone and cartilage tissue damage, a titanium-based implant that can
serve to regenerate both tissues would be most beneficial.
Interactions between implants and cells mainly depend on surface properties like
topography, roughness, chemistry, and wettability [4-11]. To improve implant integration
into surrounding bone and cartilage, various surface treatments have been attempted to
modify the topography and chemistry of titanium [12]. Among these methods,
anodization is receiving much attention because it can conveniently and inexpensively
create biologically-inspired micro-rough surfaces with different chemical compositions
[1, 13-16]. For example, for bone, Yang et al. found that the anodic titanium oxide films
facilitated the formation of apatite; thus, calling these films bioactive materials [15]. In
addition, Sul found significantly higher in vivo torque removal values due to increased
bone to metal contacts when S, P, Ca were incorporated into anodized titanium implants
compared to unanodized controls [16].
Studies have also focused on the geometry of the anodized structures formed on
titanium. Specifically, Gong et al. [17] used hydrofluoric acid (HF) as an electrolyte and
successfully produced controllable and ordered nanotube arrays on anodized titanium
surfaces which could further mimic the nanofiber like geometry of natural entities in
bone and cartilage. Yao et al. furthered the study of nanotubular anodized titanium and
reported greater osteoblast (bone forming cell) adhesion, synthesis of alkaline
phosphatase, collagen, and deposition of calcium on nanotubular anodized titanium
compared to nanoparticulate anodized titanium and unanodized titanium [18]. Some
studies further described a possible mechanism by measuring greater fibronectin and
vitronectin (both proteins important for mediating osteoblast adhesion [19,20]) adsorption
on nanotubular anodized compared to unanodized titanium [18]. Lastly, recent studies
have incorporated various pharmaceutical agents into the nanotubes anodized into
titanium to promote their versatile use in a wide range of tissue regeneration applications
[21].
In addition to bone, cartilage tissue also possesses a unique nanostructure rarely
duplicated in synthetic materials. Specifically, chondrocytes are naturally accustomed to
interacting with a well-organized nanostructured collagen matrix. Despite the role that
titanium can (and currently does) play in both orthopedic and cartilage applications, and
the natural nanostructure of cartilage, few (if any) reports exist investigating chondrocyte
functions on titanium anodized to possess biologically-inspired nanotubes.
Materials and Methods
Titanium substrates
Titanium foil (10 x 10 x 0.2 cm; 99.2 % pure; Alfa Aesar) was cut into 1 x 1 cm
squares using a metal abrasive cutter (Buchler 10-1000; Buehler LTS, IL). All the
substrates were then cleaned with liquid soap (VWR) and 70 % ethanol (AAPER) for 10
min in an aqua sonicator (Model 50 T; VWR). Substrates were then dried in an oven
(VWR) at about 65 C for 30 min to prepare them for anodization. After anodization, all
the substrates were ultrasonically washed in an aqua sonicator with acetone
(Mallinckrodt) for 20 min and 70 % ethanol for 20 min.
Borosilicate glass (Fisher Scientific; 1.8 cm diameter) was used as a reference
material in the present study. The glass coverslips were degreased by soaking in acetone
for 10 min, sonicating in acetone for 10 min, soaking in 70 % ethanol for 10 min, and
sonicating in ethanol for 10 min. Lastly, the coverslips were etched in 1 N NaOH (Sigma)
for 1 hour at room temperature.
Anodization process
Prior to anodization, the titanium substrates were immersed in an acid mixture (2
ml 48% HF, 3 ml 70 % HNO3 (both Mallinckrodt Chemicals) and 100 ml DI water) for 5
min to remove the naturally formed oxide layer. Some of the acid-polished substrates
were then immediately treated by anodization.
The titanium substrates served as an anode in the anodization process while an
inert platinum sheet (Alfa Aesar) was used as a cathode (Figure 1). The anode and
cathode were connected by copper wires and were linked to a positive and negative port
of a 30V / 3A power supply (SP-2711; Schlumberger), respectively. During processing,
the anode and cathode were kept parallel with a separation distance of about 1 cm, and
were submerged into an electrolyte solution in a Teflon beaker (VWR). According to
previous studies [17, 21], dilute hydrofluoric acid (1.5 wt %) was used as an electrolyte.
Previous studies have determined the evolution of the resulting anodized titanium
structures in order to determine the exact parameters necessary to form nanotubes [18].
The potential between the anode and cathode was kept constant at 20 V. All anodizations
were completed for 10 min in this study. After anodization, all substrates were rinsed
thoroughly with deionized (DI) H2O, dried in an oven at about 65 C for 30 min, and
sterilized in an autoclave at 120 C for 30 min.
Substrate surface characterization
Surface morphologies of the unanodized and anodized titanium substrates were
mainly characterized using a JEOL JSM-840 Scanning Electron Microscope and a
Hitachi S4800 Field Emission Scanning Electron Microscope for ultra-high
magnifications. All samples were sputter-coated with AuPd before imaging using a
HUMMER I sputter-coater for 3 min.
Surface roughness of the substrates was measured by an Atomic Force Microscope
(AFM, Multimode SPM Digital Instruments Veeco). The typical tip (NSC15;
Mikromasch) curvature radius used in the present study was less than 10 nm. The
measurements were conducted in ambient air under tapping mode with a scan rate of 2
Hz. The scan area was 1 x 1 µm. The root mean square (rms) roughness, relative surface
area, and z direction depth were estimated with the aid of Nanoscope imaging software.
To determine the composition of surface oxide formed on titanium, both unanodized
and anodized nanotubular substrates were also examined by an X-ray Photoelectron
Spectroscope (XPS, Surface Science Instruments X-probe Spectrometer). This instrument
has a monochromatized Al Kα X-ray and a low energy electron flood gun for charge
neutralization. X-ray spot size for these acquisitions was on the order of 800 µm. The
take-off angle was ~55o; a 55 o take-off angle measures about 50 Å sampling depth. The
Service Physics ESCAVB Graphics Viewer program was used to determine peak areas.
Phase analysis of the titanium substrates was carried out by X-ray diffraction
(XRD) analysis using a Siemens D500 Diffractometer (Bruker AXS Inc., WI). Copper
Kα radiation (λ=1.5418 Å) scanned the nanotubular anodized samples from 2θ angles of
20 to 60 at a scan speed of 0.5/min with a 0.05 increment. Resulting XRD spectra were
compared to titanium (JCPS # 050682) and titania (rutile and anatase; JCPS # 211276
and JCPS # 211272, respectively) standards.
Cell experiments
Human articular chondrocytes (cartilage-synthesizing cells; Cell Applications
Inc.) were cultured in Chondrocyte Growth Medium (Cell Applications Inc.). Cells were
incubated under standard cell culture conditions, specifically, a sterile, humidified, 5%
CO2, 95% air, 37 °C environment. Chondrocytes used for the following experiments were
at passage numbers below 10. The phenotype of these chondrocytes has previously been
characterized by the synthesis of Chondrocyte Expressed Protein-68 (CEP-68) for up to
21 days in culture under the same conditions [22]. Chondrocytes were seeded at 3,500
cells/cm2 pre samples and were allowed to attach for 4 hours. After the prescribed time
point, non-adherent cells were removed by rinsing with phosphate buffered saline (PBS)
solution. Cells were then fixed, stained with rhodamine phalloidin, and counted according
to standard procedures [18]. Five random fields were counted per substrate and all
experiments were run in triplicate, repeated at least three times.
Statistical analysis
All experiments were run in triplicate and were repeated three different times.
Numerical data were analyzed using standard analysis of variance (ANOVA) techniques;
statistical significance was considered at p < 0.05.
Results
Creation of anodized titanium surfaces possessing nanotubular structures
The unanodized titanium as purchased from the vendor possessed micron rough
surface features as displayed under SEM (Figure 2). After anodization in 0.5 % HF at 20
V for 20 min, the titanium surface was oxidized and possessed nanotubular structures
uniformly distributed over the whole surface (Figure 2). As estimated from these SEM
images, the inner diameter of the nanotubular structures was from 70 to 80 nm (Figure 2).
Surface characterization of anodized titanium substrates
Representative AFM images of unanodized and nanotubular anodized titanium
were characterized by root mean square (rms) and relative surface area (Figure 3; Table
1). Results showed that the unanodized titanium surface was relatively smooth (4.74 nm)
compared to the nanotubular anodized titanium surfaces. Moreover, the rms value was
larger for the nanotubular anodized titanium surface structures (25.54 nm). Further
information on the depth and diameter of the nanometer surface features was obtained
from the AFM images and profiles. It was estimated that the nanotubes were between 100
and 200 nm deep and had an inner diameter approximately 70 to 80 nm, as also
confirmed by SEM (Figure 3).
High resolution X-ray Photoelectron Spectroscopy spots were taken on each
sample to examine Ti 2p binding energy (Table 2). Importantly, other than TiO2, no other
titanium species (for example, TiO and Ti2O3) were present. X-ray Photoelectron
Spectroscopy results also demonstrated that the outermost layers of oxide mainly
contained C, O, Ti, F, and N (Table 3) and were similar between the unanodized and
nanotubular anodized titanium. XRD spectra confirmed the presence of amorphous
titania (no anatase or rutile phase was observed) on both unanodized and nanotubular
anodized titanium (data not shown). In summary, the results of the present study showed
that while the degree of nanometer roughness was much greater for nanotubular anodized
titanium compared to unanodized, chemistry and crystallinity were similar.
Chondrocyte adhesion
For the first time, the results of this study demonstrated greater chondrocyte
adhesion on the nanotubular anodized titanium compared to unanodized titanium (Figures
4 and 5). Since chondrocytes are anchorage dependent cells, increased adhesion on
nanotubular anodized titanium may lead to greater proliferation and synthesis of a
cartilage extracellular matrix. Lastly, the results presented in Figure 4 were normalized to
the surface area provided by AFM characterization studies; thus, they incorporate the
greater surface area of the nanotubular anodized titanium and still showed greater
chondrocyte adhesion.
Discussion
While the results of this study showed promise for nanotubular anodized titanium
for cartilage applications, additional reports have indicated promise for other anodized
materials for bone regeneration [25]. Specifically, extensive studies have not only been
conducted for titanium as previously mentioned, but also, aluminum. Increased osteoblast
proliferation and matrix production (higher Ca/Al, P/Al ratio) were observed on anodized
ordered nano-porous alumina (pore diameter about 100 nm) compared to amorphous
alumina and non-anodized aluminum after 4 weeks of culture [25]. These aluminum
feature sizes and structures were similar to the presently studied nanotubular anodized
titanium. Although no reports exist determining the functions of chondrocytes on
anodized aluminum, similar promoted functions of osteoblasts on anodized titanium and
aluminum imply that numerous traditional orthopedic implant materials could possibly be
anodized to promote their efficacy.
The presence of the unique nanotubular anodized structures in the present study
needs to be emphasized. Specifically, other anodized structures have been formulated on
titanium, most noteably, nanoparticulate anodized structures [18]. Although not
addressed in the current study, a previous study demonstrated greater osteoblast function
on nanotubular compared to nanoparticulate anodized titanium [18]. Anodized titanium
with heterogeneous nanoparticles is present as an intermediate structure between
unanodized and nanotubular anodized titanium [18]. In addition, the ability to form
nanotubes capable of drug delivery further suggests the continued promise of these novel
structures for numerous tissue engineering applications; this study adds cartilage to the
list.
Importantly, by design, this study also provided evidence as to why chondrocyte
adhesion was promoted on nanotubular anodized titanium; understanding changes in
materials properties that alter cell responses is often missing in today’s research. Clearly,
changes in both topography and chemistry after anodization of titanium may influence
chondrocyte adhesion. To better understand the role that topography played in this study
to promote chondrocyte adhesion, it was necessary to eliminate the influence of
chemistry and crystallinity. This study did provide evidence that unanodized and
nanotubular anodized titanium had similar chemistry and crystallinity. Although further
investigation is required, results of this study suggested that the nanotubular surface
topography resulting from titania anodization was a major factor that influenced greater
chondrocyte adhesion.
Although the mechanism(s) of enhanced osteoblast functions on nanotubular
anodized titanium has been elucidated [18], those relating to chondrocytes have not.
Some thoughts can be made, however, in this respect. First, the unique nanotube
structures provided more surface area and more reactive sites for initial protein
interactions that may mediate chondrocyte adhesion. Although the chondrocyte adhesion
results were normalized to the increased surface area of nanotubular anodized titanium,
changes in protein interactions may have promoted greater chondrocyte adhesion. For
example, it was reported that the initial adsorption of vitronectin increased on
nanotubular anodized titanium [22, 23]. It is also possible that the unique nanotube
structures (inner diameter 70 to 80 nm, a few hundred nm deep) might be sites for
preferential adsorption of proteins (vitronectin is 15 nm in length [23] and fibronectin is
about 130 nm long [24]) to mediate chondrocyte adhesion.
Changes in topography could also affect surface wettability and surface
potential, which are all known to influence chondrocyte responses. For example, it has
been reported that cells prefer to attach on surfaces with higher surface energy [25, 26].
Generally, surface wettability is influenced by both the chemistry and topography and it
increases with roughness if the ideal smooth surface is hydrophilic [27]. Zhu et al.
reported that titanium anodized in 0.2 M H3PO4 and 0.03 M calcium
glycerophosphate/0.15 M calcium acetate had contact angles between 60 and 90, which
corresponds to hydrophilic surfaces, and some of the anodized substrates had better
wettability than much smoother unanodized titanium substrates [9]. Although more
experiments would be needed to verify this, similar events may be happening on titanium
substrates anodized in HF in the present study to promote chondrocyte adhesion.
Due to the specific titanium surface morphology after anodization, the charge
distribution and arrangement on the surface in the culture medium may also be different
compared to unanodized substrates. For example, the more sharp bottoms and edges of
the nanotubes on titanium may lead to higher charge densities. Different surface charge
densities will lead to different surface electric potential. The Zeta (ξ) potential is the
electric potential at an interface between a solid surface and a liquid. Roessler et al.
reported that a natural titanium oxide layer (about 5 nm) and a thicker, anodized
amorphous titanium oxide layer (about 150 nm) had a Zeta potential around -40 mV and -
55 mV (PH = 7, KCl solution), respectively [32]. Similarly, in the present study, the
anodized titanium surface with nanotube structures may have a different Zeta potential
compared to the unanodized titanium with a thinner natural oxide layer. This would also
influence initial protein adsorption events responsible for increased chondrocyte
adhesion. For instance, earlier studies revealed the highest fibronectin adsorption on
anodized titanium possessing nanotube structures among the unanodized and anodized
titanium as well as higher fibronectin adsorption on anodized titanium possessing nano-
particulate structures compared to unanodized titanium [8].
Due to the integral role that conventional titanium has played in cartilage
implants, whatever the reason for the currently observed enhanced chondrocyte adhesion,
this study clearly suggests that anodized titanium possessing nanotube structures should
be further studied for improved chondrocyte functions.
Conclusions
By selecting proper anodization conditions, nanotubes can be formed on titanium
surfaces with similar chemical composition and crystallinity to the starting unanodized
titanium. The present in vitro study provided the first evidence of enhanced chondrocyte
adhesion on nanotubular anodized titanium compared to unanodized titanium. Due to the
already wide spread use of titanium in cartilage applications, results of the present study
suggests that nanotubular anodized titanium should be further considered for regions of
an implant in which formation is cartilage is desirable.
Acknowledgements
The authors would like to thank the VA Hospital in Providence, RI for funding.
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Table 1: Surface roughness of unanodized and nanotubular anodized
titanium surfaces.
Substrates Relative surface area
Root mean square roughness (nm)
Unanodized titanium 1.018+0.008 4.74+1.87Anodized titanium with
nano-tube structures1.811+0.133* 25.54+3.02*
* p < 0.01 compared to unanodized titanium.
Table 2: Binding energy of the high resolution Ti 2p peaks for unanodized and
nanotubular anodized titanium substrates as examined by
X-ray Photoelectron Spectroscopy.
Substrates PeakBinding Energy
(ev)Area
%
Unanodized titaniumTi 2p3/2 458.8 67.8Ti 2p1/2 464.5 32.1
Anodized titanium with nano-tube structures
Ti 2p3/2 458.7 67.6Ti 2p1/2 464.5 32.4
Table 3: Atomic percentage of selective elements in the outermost layers of
unanodized and anodized titanium substrates as examined by
X-ray Photoelectron Spectroscopy.
Substrates C O Ti N FUnanodized titanium 43.2 41.7 8.3 2.5 2.2
Anodized titanium with nano-tube structures
40.8 42.9 9.0 1.5 2.8
Figure Legends
Figure 1. Schematics of Ti anodization. The two electrode configurations were linked
to a DC power supply. A platinum mesh and Ti disk served as the cathode and anode,
respectively. 1.5% HF was used as an electrolyte contained in a Teflon beaker.
Figure 2. Scanning electron microscopy images of unanodized and nanotubular
anodized Ti. Bars = 1 m for unanodized Ti, and 200 nm (low magnification) and 500
nm (high magnification) for nanotubular anodized Ti.
Figure 3. AFM images of unanodized and nanotubular anodized titanium:
(a) unanodized titanium and (b) anodized titanium with nano-tube-like structures.
The scan area is 1 x 1 µm.
Figure 4. Increased chondrocyte adhesion on nanotubular anodized Ti. Values are
mean + SEM; n = 3; * p < 0.01 compared to glass (reference); ** p < 0.01 compared to
unanodized Ti.
Figure 5. Fluorescent images of increased chondrocyte adhesion on nanotubular
anodized Ti. Stain = Rhodamine phalloidin. Bars = 50 m.
Figures
Figure 1
Unanodized Ti
Nanotubular Anodized Ti; Low Magnification
Nanotubular Anodized Ti; High Magnification
Figure 2
Unanodized Ti
Nanotubular Anodized Ti
Figure 3
Figure 4
Nanotubular Anodized Unanodized Glass
Titanium
***
*
Unanodized Ti
Nanotubular Anodized Ti
Figure 5