Hindawi Publishing Corporation

International Journal of Biomaterials

Volume 2013, Article ID 513680, 10 pages
http://dx.doi.org/10.1155/2013/513680

Review Article
Variations to the Nanotube Surface for Bone Regeneration
Christine J. Frandsen, Karla S. Brammer, and Sungho Jin
Materials Science and Engineering Program, University of California, San Diego, La Jolla, CA 92093-0411, USA
Correspondence should be addressed to Sungho Jin; [email protected]
Received 1 June 2012; Accepted 31 March 2013
Academic Editor: Tadashi Kokubo
Copyright 2013 Christine J. Frandsen et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
The complex mechanisms of the bone cell-surface interactions are yet to be completely understood, and researchers continue to
strive to uncover the fully optimized implant material for perfect osseointegration. A particularly fascinating area of research
involves the study of nanostructured surfaces, which are believed to enhance osteogenic behavior, possibly due to the mimicry
of components of the extracellular matrix of bone. There is a growing body of data that emphasizes the promise of the titanium
oxide (TiO2 ) nanotube architecture as an advanced orthopedic implant material. The review herein highlights findings regarding
TiO2 nanotube surfaces for bone regeneration and the osteogenic effects of minute changes to the surface such as tube size and
surface chemistry.

1. Characteristics and Function of

Normal Bone

2. The Evolution of Biomedical

Materials Technology

Bone is a complex tissue that has the ability to heal and

regenerate itself [1], and is continuously in the cycle of
remodeling from before birth until death [2]. The process of
bone modeling and remodeling typically occurs to help the
bone adapt to mechanical forces or to replace microdamaged
bone with new, stronger bone [2]. Occasionally bone defects
will form that are unable to heal on their own, either due to
bone disease or trauma. In these cases, bone reconstruction
is necessary, which requires osteoproduction (colonization of
osteogenic stem cells at defect site), osteoinduction (induced
bone formation), osteoconduction (growth of bone on a
surface), osseointegration (stable anchorage of an implant
achieved by direct bone-to-implant contact), mechanical
stimulation, and vascularization [1, 3, 4]. In many cases an
orthopedic implant is needed in order to stabilize the defect
and provide support for new bone to grow. In order for
orthopedic surgery to be successful, a strong and lasting
connection between the implant and the interfacing bone
tissue must be quickly established. A large part of current
orthopedics research is centered on designing the material
surface to more readily recruit bone forming cells to that
interface.

The technology and design of materials for bone implants

have evolved tremendously over the past 50 years, through
what Hench and Polak defines as three generations of
biomedical materials [5]. First-generation biomedical materials (of the 60s and 70s) were designed solely to achieve a
suitable combination of physical properties to match those
of the replaced tissue with a minimal toxic response in
the host [5]. In the late 70s to early 80s, the focus of
biomaterials design shifted from solely a bioinert tissue
response to include a bioactive tissue response which would
trigger a controlled reaction in vivo [5]. Bioactive materials
reached clinical use by the mid of 1980s for orthopedic
and dental applications, including bioactive glasses, ceramics,
and composites [5]. Resorbable biomaterials (which dissolve into the body) were also introduced to the market,
and resorbable polymer sutures became routinely used. At
this point, biomaterials properties were beginning to be
designed to match the body tissue at the microscale. The
third generation of biomaterials has evolved as the search
continues for an orthopedic implant technology that provides
a stable osseointegration that routinely out-lives the patient.
The aim of third-generation biomaterials is that the material

2
is designed in such a way that it would stimulate certain
cellular responses at the molecular level [5]. By modifying the
surfaces at the molecular and nanoscale levels, researchers
have been able to direct cell proliferation, differentiation, and
extracellular matrix (ECM) production and organization [5].
Great progress has been made in the research behind thirdgeneration biomaterials; however, the complex mechanisms
of the bone cell-surface interactions are yet to be completely
understood, and researchers continue to strive to uncover the
fully optimized implant material for perfect osseointegration.
Third-generation, or nanostructured biomaterials research, has uncovered many interesting aspects of cellsurface interaction, and many believe that these materials will
provide the optimal implant surfaces of the future. Although
this review focused specifically on the nanotube surface
architecture for bone regeneration, the reader is encouraged
to read some interesting and informative literature on the
more generic topic of nanostructured surfaces for osteogenesis, including [614].

3. Metal Oxide Nanotubes

Owing to attractive properties such as the high surface-tovolume ratios and size-dependent properties, nanostructured
materials have been at the center of a large body of innovative
research in science and technology. In particular, nanostructured surfaces are at the focal point of tissue engineering
research due to findings which have demonstrated that
cells will respond to and be directed by dimensions in the
nanometer regime (<100 nm), even as small as 10 nm height
[15, 16]. Although there are many methods for the fabrication
of precisely defined nanostructured surfaces, one of the
most simple and inexpensive processes for nanostructure
formation is electrochemical anodization. Other common
procedures for nanostructure fabrication involve a complicated series of steps which often can only be applied to a
perfectly flat substrate (i.e., nanolithography). In contrast,
electrochemical anodization can be applied to substrates of
various 2D and 3D geometries and shapes, as well as sizes
ranging from very small to potentially unlimited proportions.
This method is also attractive because it is most commonly
applied to titanium, which is one of the most widely used
materials in bone implant technology. Consequently, the
review herein is specifically concentrated on variations to
the nanotube surface formed by electrochemical anodization
in a fluorine containing electrolyte for orthopedic device
applications.
The formation of titanium oxide (TiO2 ) nanotube arrays
via electrochemical anodization was first reported by Gong
et al. in 2001 [17]. Since their discovery, researchers have
achieved better control of nanotube formation through such
methods as varying electrolyte concentration and pH, inorganic and aqueous solvents, and so forth. Extensive control
is now possible for nanotube morphology and dimensions
including diameter, length, length-to-diameter ratio, wallthickness, tube shape (conical versus cylindrical), transparency, and even doping [18]. Such ability to precisely
control nanotube formation via electrochemical anodization

International Journal of Biomaterials

has great promise for their utilization in the orthopedics
industry.
In addition, researchers have been able to apply the same
technique of electrochemical anodization to other metals,
including zirconium (Zr) [19] and tantalum (Ta) [20] thus
forming nanotube arrays of these metal oxides. The work
discussed in this review will focus solely on titanium oxide
(TiO2 ) and zirconium oxide (ZrO2 ) nanotubes, as well as
variations to the TiO2 surface and corresponding effects on
osteoblast and mesenchymal stem cell growth and function.
Electrochemical anodization of titanium (Ti) or other
metal foils involves a two-electrode electrochemical cell with
a platinum (Pt) foil cathode and metal foil (titanium or other
metal of choice) anode which are held at a constant potential (see Figure 1). The traditional method of anodization
utilizes a hydrofluoric-acid-(HF-) based electrolyte; however,
researchers have also used ammonium fluoride and other
chemicals as the fluorine ion source. Additionally, the original
electrolyte solutions were aqueous based; since then, it has
been found that increased control of nanotube morphology
can be achieved using inorganic solvents [21], and increased
mechanical robustness can be achieved using the addition
of acetic acid to the HF electrolyte in a 1 : 7 ratio [18]. A
trend in nanotube growth, that is, consistent, no matter the
electrolyte concentration, is increasing nanotube diameter
with increasing applied voltage. The reader is directed to a
thorough review by Mor et al. for a detailed mechanistic
model of nanotube formation by electrochemical anodization
[18].

4. Osteoblast and Mesenchymal

Stem Cell Growth and Functionality on
TiO2 Nanotubes
In 2006, TiO2 nanotubes were demonstrated by Oh et al.
to significantly accelerate osteoblast adhesion and proliferation and enhance bone mineral formation when compared to nonmodified titanium surfaces [22]. These initial
experiments were performed on TiO2 nanotubes of 100 nm
diameter and 300 nm height, with a wall thickness of
10 nm. Popat et al. reported similar observations in 2007 on
the higher adhesion, proliferation rates, and bone forming
ability of marrow stromal cells on TiO2 nanotoubular surfaces (80 nm diameter, 400 nm height) when compared to
those grown on flat titanium surfaces [23]. The intriguing
phenomena in these studies of differing cell types triggered
further investigation into the effects of nanotube geometry
on osteogenic behavior. As was mentioned previously, precise
control of the nanotube diameter is possible by varying the
applied voltage during anodization. In order to further understand the influence of the nanotube architecture on bone
cell behavior, a series of studies were performed in which
the lateral spacing of the nanotube system was varied by
altering the nanotube diameter from 30, 50, 70, and 100 nm,
as depicted in Figure 2. The average surface roughness (Ra)
and contact angle measurements for the corresponding flat
Ti and various nanotube surfaces are reported in Figure 2(b).

International Journal of Biomaterials

Pt

Ti

Electrolyte
solution

Power supply

Figure 1: Schematic drawing of the electrochemical anodization

setup. Fabrication variables include voltage, electrolyte concentration, temperature, and pH.

5. Effect of Nanotube Size on

Osteogenic Behavior
Most bone implant materials are placed in direct contact with
both adult bone and bone marrow tissue and thus are exposed
to two main cell types: osteoblasts (bone cells) and mesenchymal stem cells (bone marrow cells). In order to develop
an understanding of the role of the TiO2 nanotube surface
in vitro, the behavior of osteoblast cells and mesenchymal
stem cells (MSCs) was studied on a series of nanotube sizes
shown in Figure 2. Experimental conditions and sample
preparation techniques were held constant in both studies,
while only the cell type was varied. The cell morphology
of both osteoblast and MSCs were analyzed using scanning
electron microscopy (SEM). In both studies, an increase
in cell elongation was observed as a function of nanotube
diameter, as shown in Figure 3. On the flat Ti substrate, both
cell types are flat, spread out, and round-shaped; they are
somewhat flat and rounded on 30 nm nanotubes, and they
become progressively elongated as the nanotube diameter
is increased to 50 nm diameter and beyond. It is evident
that the nanotubes with diameters of 70 and 100 nm induce
extraordinary cell elongation (see red arrows and brackets)
after 24 h of culture. In addition, the elongated leading edges
of lamellipodia (yellow arrows) of both cell types indicate that
the cell morphologies are more mobile on the 70 and 100 nm
nanotubular surfaces.
The number of cells that adhered to each surface was
measured as a function of incubation time. The results of the
MSC study are shown in Figure 4(a), and the results of the
osteoblast study are shown in Figure 4(c). The highest number of adhered cells in both studies was found on the 30 nm
diameter nanotube surface. In addition, the cell elongation
of both experiments was quantified by calculating the ratio of
cell length to width; the data of the MSC experiment is shown

in Figure 4(b), and the data from the osteoblast experiment is

shown in Figure 4(d). As was observed in the SEM images in
Figure 3, both cell types become increasingly elongated as the
nanotube pore size increases. However, comparing the cell
adhesion versus the cell elongation in Figure 4, it is apparent
that these phenomena follow opposite trends as a function of
nanotube diameter.
The results of the osteoblast and MSC cell studies indicate
that the nanotube dimensions play an important role in the
initial cell response to the surface, as indicated by the cell
adhesion and elongation behaviors. The mechanism through
which a cell senses and attaches to a surface is through surface
receptors called integrins, which in fact do not sense the
surface, but proteins adhered to the surface. Thus, in order to
understand the behavior of cells on the nanotube topography,
it is important to investigate the manner with which proteins
are adsorbed onto the substrates. Figure 5 shows scanning
electron microscope (SEM) images of proteins adsorbed onto
the flat Ti and 30, 50, 70, and 100 nm diameter nanotube
surfaces after 2 hours of incubation in cell culture medium.
While the presence of protein aggregates is infrequent on Ti,
there is an abundance of aggregates on the 30 nm nanotubes.
However, the proteins on the larger diameter 70 and 100 nm
nanotubes are few and are spaced farther apart. It is evident
from these micrographs that the nanotube diameter causes
distinct differences in the number and placement of proteins
on the surface.
The phenomena of cell adhesion versus elongation on the
nanotube surfaces can be explained by the pattern of protein
adsorption on each of the substrates. The small pore size of
the 30 nm surface allows for proteins to adsorb in a more
tightly knit fashion, which enables the cells to adhere easily.
Additionally, the 30 nm substrate does not direct the cells
to move/stretch in any specific direction since proteins are
everywhere. In contrast, the placement of proteins on the
larger (70 and 100 nm) nanotube topographies encourages
cell spreading due to the adsorption of proteins only on the
nanotube wall rims, thus inducing a fixed distance between
proteins and encouraging the cell to spread in order to find
adhesion proteins. It is probable that the cells are required to
expand their filopodia across larger distances, thus inducing
the elongated cell shape. This would also affect the ability of
the cell to adhere to the surface, which explains the lower
number of adhered cells on the larger diameter substrates.
In addition to the observations of cell adhesion and
elongation, the MSC and osteoblast cell behavior were also
analyzed in terms of bone-forming functionality. In the
MSC study, quantitative polymerase chain reaction (PCR)
analysis was performed in order to estimate the relative
transcript levels of alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN) gene expressions. The
presence of these three genes is important because ALP is
an enzyme produced by cells which indicates their boneforming ability, while OCN and OPN are proteins found in
bone. The data from the PCR analysis, shown in Figure 6(a),
demonstrates significantly higher gene expression of all three
genes on the larger diameter (70 and 100 nm) substrates
when compared to the flat Ti and smaller diameter (30
and 50 nm) substrates, indicating osteogenic differentiation.

International Journal of Biomaterials

30 nm

50 nm
Sample

70 nm

100 nm

100 nm

Roughness
Ra (nm)

Contact
angle ( )

Ti

9.7

54
[ref. 30]

30 nm

13.0

11

50 nm

12.7

70 nm

13.5

100 nm

13.2

(a)

(b)

Figure 2: Physical characterization of different-sized nanotube surfaces. (a) SEM micrographs of self-aligned TiO2 nanotubes with different
diameters. The images show highly ordered nanotubes with four different pore sizes between 30100 nm. (b) Table with average roughness
( ) and surface contact angle measurements for Ti and 30100 nm TiO2 nanotube surfaces.
Ti

= 30 nm

50 nm

70 nm

100 nm

(a) Mesenchymal stem cells, 24 h

Ti

30 nm

50 nm

70 nm

100 nm

100 m
(b) Osteoblast cells, 24 h

Figure 3: SEM micrographs of (a) human mesenchymal stem cells (hMSCs) and (b) osteoblast cells cultured on flat Ti and 30, 50, 70, and
100 nm diameter TiO2 nanotube surfaces after 24 h of culture (scale bar, 100 m). Red arrows (a) and brackets (b) emphasize extraordinary
cell elongation; yellow arrows indicate elongated leading edges of lamellipodia.

Similarly, alkaline phosphatase activity of the osteoblast cells

was measured on each of the experimental culture substrates.
The results from the osteoblast study portray the same trend
of increasing ALP activity with increasing nanotube diameter
(Figure 6(b)). These results are evidence that the nanotube
diameter causes an upregulation in the markers of bone
formation.
Since the cell elongation and cell functionality followed
the same increasing trends as a function of nanotube diameter, it can be speculated that there is a correlation between
the two phenomena. Interestingly, in addition to the highly
elongated cell shape on the large diameter nanotube surfaces,
the cell nuclei on these surfaces were also elongated (by 20
25%, data not shown). It is likely that the elongation of the
cell nuclei is a result of the stretching of the cytoskeletal
morphology of the cell. Researchers have indicated that
cytoskeletal reorganization can cause nuclei distortion, which

may promote differences in DNA behavior due to mechanical restraints within the nuclei [24, 25]. Therefore, it is
evident that the large diameter nanotube substrate induces
cell elongation and thus nuclei distortion, which may cause
osteoblast and MSCs to produce markers of bone formation
and osteogenic differentiation more readily than on a flat
substrate.
The overall trends of the nanocue effects on osteoblast
and stem cell morphology and fate can be summarized by
the schematic illustration in Figure 7. It was observed that
with increasing nanotube diameter cell adhesion growth
decreased (solid red line), in a similar manner as protein
particle density (broken red line). In contrast, both osteoblast
and MSCs demonstrated a higher degree of osteogenic
differentiation (solid blue line) with increasing nanotube
size, analogous to the trend of cell elongation (broken blue
line).

International Journal of Biomaterials

20.000

20

=4

Cell elongation (length/width ratio)

Cell number (cells/cm2 )

15.000
12.500

10.000
7.500
5.000
2.500

= 20

18

17.500

16

14
12
10
8
6

0
2 hrs

12 hrs

7 days

24 hrs

Ti

Incubation time
Ti
50 nm
100 nm

30 nm

30 nm
70 nm

100 nm

(b) Mesenchymal stem cells

30000

25000

12
#

20000
=4

10000

5000

Elongation (length/width)

Cell number (cells/cm 2 )

70 nm

2 hours
24 hours

(a) Mesenchymal stem cells

15000

50 nm

TiO2 nanotube size (dia.)

10

= 20

8
6
4
2

0
2 hrs

12 hrs

24 hrs

48 hrs

7 days

Incubation time

Flat Ti
30 nm
50 nm

70 nm
100 nm
(c) Osteoblast cells

0
Flat Ti

30 nm

50 nm
70 nm
Nanotube size

100 nm

2 hrs
24 hrs
(d) Osteoblast cells

Figure 4: Comparative graphs showing the influence of nanotube diameter on cell number and elongation at early incubation time points.
MSC cell number versus incubation time (a) and MSC elongation (length to width ratio) as a function of nanotube diameter at 2 and 24 h
(b). Osteoblast cell number versus incubation time on each substrate (c) and osteoblast cell elongation as a function of nanotube diameter at
2 and 24 h (d).

The findings of these two studies give light to increased

understanding of the role of nanostructure dimensions for
enhanced biomaterial surface design. However, the nanotube
size in these studies was restrained to a maximum diameter
of 100 nm due to the limitations of TiO2 anodization in
an aqueous hydrofluoric acid electrolyte as was used for
preparation of these surfaces. Anodization methods that

enable the fabrication of larger diameter nanotube arrays

have been reported, even to as large as 350 nm diameter
using an electrolyte consisting of diethylene glycol with low
concentrations of hydrofluoric acid (HF) [26]. With careful
control of anodization protocols, eventual progress may be
made which enables the growth of mechanically strong
nanotubes with large diameter pore openings. It would be of

International Journal of Biomaterials

Ti

30 nm

50 nm

100 nm

70 nm

200 nm

Figure 5: SEM micrographs showing protein adsorption on the surfaces of flat Ti and 30, 50, 70, and 100 nm diameter TiO2 nanotubes after
2 h of incubation in growth medium.
1.6

=3
=3

0.7

1.2

Alkaline phosphatase activity

(mmol/hour/g protein)

Relative transcript levels

1.4

1.0
0.8
0.6
0.4
0.2

0.6
0.5
0.4
0.3

0.2

0.1

0.0
Positive
control

Ti

30 nm

50 nm

70 nm

100 nm

0.0

24

Culture substrates
ALP
OCN
OPN

Flat Ti
30 nm
50 nm
(a) Mesenchymal stem cells

48
Incubation time (hours)
70 nm
100 nm

(b) Osteoblast cells

Figure 6: Comparative graphs showing the trend of MSC and osteoblast cell functionality with increasing nanotube diameter. (a) Quantitative
PCR analysis for ALP, OCN, and OPN after 3 week mesenchymal stem cell culture. Plastic cell culture plate with osteogenic inducing media
was used as a positive control for osteogenic differentiation. (b) ALP activity after 24 and 48 h of osteoblast incubation.

great interest to further investigate the osteoblast and MSC

behaviors at diameters beyond 100 nm.
The size effect of the nanodimensions of TiO2 nanotube
surfaces raises the question of whether the trend is unique
to the TiO2 nanoarchitecture fabricated via electrochemical
anodization or if osteogenic behavior would be similar on
various sizes of nanotubes of different surface chemistries.
In 2009 an interesting study was published by Bauer et al.
in which the size selective behavior of MSCs was analyzed
on ZrO2 nanotubes as well as TiO2 nanotubes coated with a
conformal layer of AuPd [27]. Brammer et al. observed that
the different surface chemistries did not affect the diameter
dependence of cell adhesion or proliferation. However, a
more recent study by the Jin lab has demonstrated that
various surface chemistries do affect multiple cell types
differently [28], as will be described in detail in the following
section.

6. Variations of Nanotube Surface Chemistry

Although nanostructured surface geometries have provided
exciting findings in the latest biomaterials research, only a few

publications have directly compared nanostructures of various surface chemistries [28]. Usually, the surface chemistry is
held constant, while the nanotopography or nanogeometry is
varied. The history of orthopedic implant materials has made
it obvious that body tissues respond differently to surfaces
depending on the type of foreign material [29]. Surface
chemical factors are in fact one of the most significant factors
on the nanoscale that can affect cell-material interactions
[30]. Though the majority of related studies compare only
nanotextured with nontextured surfaces of the same material,
an important addition to this research would be the direct
comparison of the same nanostructure with different surface
chemistries. It is possible that a unique combination of
surface chemistry and nanostructured geometry may provide
a balance of defined characteristics towards an optimized cell
response.
The advantages of the TiO2 nanotube surface topography
for orthopedic applications have been well outlined in prior
sections. Since titanium is one of the most commonly used
orthopedic materials in use today, it is of great interest to
compare any future materials with a well-recognized industry
standard. Therefore, experiments in the Jin lab were designed

International Journal of Biomaterials

Adhesion

7
Differentiation

Cell
elongation

Protein
particle
density

30

100
(nm)
Increasing nanotube diameter

Figure 7: Schematic illustration of the overall trends of nanocue

effects on cell fate and morphology. The change in cell adhesion
and growth without differentiation (solid red line) has the same
trend as protein particle density (broken red line), whereas that of
differentiation (solid blue line) has the same trend as cell elongation
(broken blue line).

to provide a direct comparison of flat Ti and TiO2 nanotubes

with the other potential surface chemistries to advance orthopedic implant technology. In order to accomplish this, the
concept was implemented of maintaining constant nanotube
geometry (i.e., TiO2 nanotubes with 100 nm diameter as
shown in Figure 2(a)), while varying the surface chemistry.

7. Carbon Chemistry Effects on

Osteogenic Behavior
Carbon films deposited on metal in both its amorphous
and crystalline forms have been investigated as potential
biomedical materials, mainly because of the chemical inertness of carbon and its naturally occurring presence in the
human body [3133]. The application of carbon films to
materials that are sensitive to wear, such as Ti and Si, has been
a convenient method that has shown significant potential
for implant coating applications [3440], specifically for
orthopedic implants. The effect of a carbon thin film coating
on the surface of TiO2 nanotubes is thus of interest and will
be discussed in this section.
The nanotube substrates used in this study were prepared
according to standard anodization protocol for the formation
of TiO2 nanotubes with 100 nm diameter and 1 : 3 aspect
ratio as shown in the bottom right corner of Figure 2(a). For
the carbon-coated comparative surface, the TiO2 nanotubes
were deposited with a thin, conformal layer of carbon by DC
sputter deposition methods in order to obtain a nanotube
architecture with a carbon surface chemistry. The carbon
coating was only deposited in a very thin layer onto the
nanotube wall rims, not altering the nanotube architecture in
any way.
Since cell behavior varies depending on cell type, both
osteoblast and osteoprogenitor (mesenchymal stem) cells
were plated in this study onto the experimental surfaces in
order to assess their behavior in response to the surface
chemistry/nanostructure combination. At early incubation

time points, the cellular behavior on the surface in vitro

includes cell adhesion, growth, and morphological orientation/organization. These three behaviors were assessed via
MTT analysis, immunofluorescent cytoskeletal actin staining, and SEM examination (data not shown, the reader is
directed to ref [28] for full details). The results of each of
these assays for both osteoblast and osteoprogenitor cells
at early time points (24 and 48 hours) were insignificantly
different, which indicates that the carbon versus TiO2 surface
chemistry did not affect the initial cell response to the surface.
Both cell types adhered and proliferated equally well on both
the TiO2 nanotube and carbon-coated nanotube surfaces. In
addition, the cell morphology showed no difference.
The ability of osteoblasts and MSCs to mature properly
and readily is a vital part of measuring cellular response
for bone implant purposes. In this study, the two cell types
were cultured for 3 weeks in order to analyze the cells
function over time and to determine whether the surfaces
were inhibiting or promoting bone function. It should be
clarified that the experiments included in this study included
the corresponding flat TiO2 and carbon substrates as control
surfaces. The nanotube substrates were found to enhance
both osteoblast and MSC cellular response when compared
to the flat controls in all aspects of this study, and the data
was thus not included.
In order to assess the behavior of the osteoblast cells, the
alkaline phosphatase (ALP) activity was measured, which is
an enzyme indicative of bone forming ability. Comparative
levels of ALP activity on each substrate are presented in
Figure 8(a) as a function of incubation time. In order
to visually verify the ALP activity quantitative results, the
osteoblast cells were also stained using an ALP staining kit,
as shown in Figure 8(b). The ALP activity indicates that the
bone-forming ability of the osteoblast cells was enhanced on
the TiO2 surface chemistry when compared to the ALP levels
on the carbon surface.
In contrast, the MSCs appear to favor the carbon surface
chemistry, as indicated by the enhanced ALP activity of the
MSCs on the carbon NTs shown in Figure 8(c). Additionally, the degree of osteogenic differentiation and maturation
of the MSCs was analyzed by quantitative PCR analysis
for osteocalcin (OCN) and osteopontin (OPN). OCN and
OPN are two major noncollagenous protein components of
bone extracellular matrix which are considered markers of
osteogenic differentiation as they are solely synthesized and
secreted by osteoblastic cells. The graphs of relative amounts
for each assay are shown in Figure 8(d); the relative gene
expression of both proteins was enhanced on the carbon
surface chemistry when compared to the TiO2 chemistry, and
the OPN was especially upregulated. It is evident from these
findings that the carbon surface chemistry causes an increase
in osteogenic differentiation and function of the MSCs. These
results indicate that MSCs can distinguish between surface
chemistries and crystallinity, as other research groups have
also observed [41].
The results of this study indicate that mesenchymal stem
cells and osteoblast cells respond differently to remarkably different surface chemistries and seem to have different chemical preferences for optimal cell function. While

International Journal of Biomaterials

2.5

Alkaline phosphatase activity

(ng/g protein)

2
TiO2 NT

1.5

Carbon NT

0.5

0 hours

24 hours

48 hours

14 days

Incubation time
30 m

TiO2 NT
Carbon NT

(a) Osteoblast cells

(b) Osteoblast cells

2
3.5

1.6

1.4

2.5

Relative gene expression

Alkaline phosphatase activity

(ng/g protein)

1.8

1.2
1
0.8
0.6

2
1.5
1

0.4

0.5

0.2

TiO2 NT
TiO2 NT

Carbon NT

(c) Mesenchymal stem cells

Carbon NT

Osteocalcin
Osteopontin
(d) Mesenchymal stem cells

Figure 8: Comparison of the degree of osteoblast functionality and MSC differentiation and maturation in late stage culture (3 weeks) on
the TiO2 versus carbon chemistry nanotube surfaces. ALP activity for osteoblasts (a) and MSCs (c) cultured on the nanotube surfaces shows
different favorable chemistries. (b) Fluorescent images showing ALP staining of osteoblast cells verifying data shown in (a). (d) Quantitative
PCR analysis of the MSCs on each surface for osteocalcin and osteopontin, verifying the trend in (c).

osteoblast cells are mature bone cells specific to bone tissue,

mesenchymal stem cells are highly sensitive, unprogrammed
cells and are readily influenced by extracellular factors such
as chemical and topographical cues. Therefore, it is not
surprising that the two cell types have different preferences
of surface chemistry. It is possible that the inclination of
the MSCs for the C-coated surface may be explained by the
fact that bone marrow contains many organic carbon-rich
components. In contrast, bone tissue is composed of more

ceramic/mineral rich components, similar to TiO2 . Perhaps

the different cell types are partial to distinct chemistries
because of the chemical components of their natural extracellular environments in vivo.

8. Conclusions and Future Directions

It is apparent that TiO2 nanotubes as an advanced biomaterial
are capable of strongly affecting cell behavior and that even

International Journal of Biomaterials

minute changes in the nanotube surface can have substantial
results. The fact that such a small range of dimensions as 30
100 nm diameter pore openings can alter cell functionality
has great promise for researchers in the field of bone regeneration. Additionally, since the process of electrochemical
anodization provides such facile methods for altering nanotube dimensions, and is applicable to substrates of 2- and
3D geometries, it is imaginable that these findings would be
appealing. Furthermore, the differing cellular preferences for
various surface chemistries indicate the potential for further
experiments comparing multiple surface chemistries with the
same underlying nanotopography. Unique combinations of
topography and chemistry could provide the ability to tailor
a medical implant surface to a particular tissue type, which
would revolutionize the field of regenerative medicine.

Conflict of Interests
There is no conflict of interests existing in this work.

Acknowledgments
This work was supported by K. Iwama Endowed Chair fund,
the von Liebig grant at UC San Diego, and UC Discovery
Grant no. ele08-128656/Jin.

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