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: Thomas_Webster@Brown.edu

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