Journal of Biomaterials and Nanobiotechnology, 2011, 2, 293-300
doi:10.4236/jbnb.2011.23036 Published Online July 2011 (http://www.SciRP.org/journal/jbnb)
293
Macrophage Inflammatory Response to TiO2
Nanotube Surfaces
Lisa M. Chamberlain1,2, Karla S. Brammer1, Gary W. Johnston1, Shu Chien2, Sungho Jin1*
1Materials Science & Engineering, University of California, San Diego, USA; 2Department of Bioengineering, Institute of Engineer-
ing in Medicine, University of California, San Diego, USA.
Received February 9th, 2011; revised March 2nd 2011; accepted April 18th, 2011.
ABSTRACT
It is well known that the native oxide layer on titanium (Ti) implants is responsible for its superior biocompatibility and
tissue integration. Recent efforts have targeted titanium dioxide (TiO2) as a good candidate for surface modification at
the nanoscale, leading to improved nanotextures for enhancing host integration properties. Here we explore the in vitro
inflammatory response of macrophages to TiO2 nanotube surface structures with different diameters (30, 50, 70, and
100 nm) created by a simple electrochemical anodization process. This work was designed to study the nanosize effect
for controlling and optimizing inflammatory response to a Ti implant surface utilizing nanotechnology. Using intracellu-
lar staining and flow cytometry for detecting macrophage TNF cytokine expression, we have found that 70 nm diameter
nanotube surfaces have the best advantage in terms of diameter size by producing the weakest inflammatory response,
compared to a commercially available Ti surface without oxide modification. We also present cell-freedata on free
radical scavenging using the nanotube surfaces with different diameters to test the removal of nitric oxide from solution;
again, our findings indicate that 70 nm titanium dioxide nanotubes exhibit optimal removal of nitric oxide from solution,
making them excellent candidates for use in medical devices that would benefit from decreased inflammatory response.
Keywords: Macrophage, Inflammation, Nitric Oxide, Nanotube, TiO2
1. Introduction
Medical devices, which are commonly used to improve
the health of patients, serve to repair joints, reopen blood
vessels, and trigger electrical stimuli among numerous
applications. Implantation of medical devices is compli-
cated by the creation of trauma caused by the necessary
surgery which is neededfor the placement of these de-
vices [1]. The natural wound healing sequel to this sur-
gical trauma is in turn challenged by the presence of me-
dical devices, leading to an alternate wound healing re-
sponse around the device generally termed the “foreign
body response.” The foreign body response is an inflam-
matory response to the implanted material, and the inten-
sity of this immediate inflammatory response to im-
planted devices directly impacts healing around and tis-
sue integration of the implant, as well as down-the- road
functioning of the medical device [1].
Because the body “sees” the surface of the implant,
many methods to change the surface properties of bio-
materials have been undertaken in hopes of 1) improving
the desired native cell/tissue adhesion and overall host
integration, 2) reducing the unwanted inflammatory re-
sponse of macrophages and various defense cells, and 3)
eliminating the subsequent foreign body response and
rejection of the medical device. One method of inquiry
towards improving the integration of materials with na-
tive tissue, has been to incorporate nanotopography on
the surface, which presents a surface with features that
are on the same nanoscale as the in vivo biological mate-
rials such as biomolecules or enzymes, proteins and ex-
tracellular matrices (e.g. collagen), cell surface receptors
or integrins, etc. that are nanometer in dimension. When
incorporated into culture surfaces, nanotopographies can
vary greatly, e.g., nanoneedles, nanorods, nanoporesor-
nanospheres, etc.
In terms of immune cell reactions to nanotopography,
previous studies on macrophages grown on zinc oxide
nanorods exhibited low viability on tall, thin nanorod
surfaces of approximately 50 nm diameter [2]. On the
other hand, titanium dioxide surfaces with nanofeatures
slightly larger in diameter (~70 nm) and shorter in height
have demonstrated much better macrophage adhesion
and survival, but exhibited less production of inflamma-

Macrophage Inflammatory Response to TiO2 Nanotube Surfaces 294
tory cytokines by the macrophages cultured on the
nanotopographic surfaces compared to a flat surface of
titanium without a nanostructure [3]. Glass nanowires
have also demonstrated a trend of greater in vitro inflam-
matory response with taller than shorter nanofeatures,
although the same study did not detecta significant trend
for inflammatory response related to the height for poly-
mer nanofeatures [3]. In addition, the culture of macro-
phages on patterned nanofeatures using several different
polymers exhibited no significant differences in the re-
lease of inflammatory cytokines [4]. Overall, it appears
that there is no clear trend regarding macrophage re-
sponse and inflammation due to nanotopography (for
review see reference [5]). Therefore further studies to
elucidate the effect of nanotopography on macrophage
response would be extremely valuable for understanding
and controlling the inflammatory cell behavior for im-
plantable devices.
Several labs have been investigating the use of TiO2
nanotubes as a titanium surface modification to the na-
tive metal-oxide for use as an improved biomaterial sur-
face, and many interesting cellular responses have been
observed with these materials [6-10]. This type of TiO2
surface nano-configuration is advantageous in regulating
many positive cell and tissue responses for various ap-
plications for tissue engineering and regenerative medi-
cine; therefore it was chosen as the primary substrate of
investigation in this research on the macrophage inflam-
matory cell response. Previously, when comparing the
effect of different diameters or inner pore sizes of TiO2
nanotubes, it was found that there were distinct size re-
gimes for controlling the behaviors of osteoblast [7],
chondrocyte [11], and mesenchymal stem cells [8,10,12].
As well, a study comparing 20 nm vs. 200 nm pores of
aluminum oxide revealed unique differences in the in-
flammatory response of macrophages [13]. Because pore
size seems to play an important role in controlled cell
behavior, here we investigate the potential influence of
differently sized TiO2 nanotubes on the in vitro inflam-
matory responses of macrophages, and we hypothesize
that the geometry of the nanotube may have significant
impacts on the inflammatorycell behavior.
2. Materials and Methods
2.1. Substrate Preparation
Annealed titanium foil (0.25 mm thick) was purchased
from Alpha Aesar (Ward Hill, MA) and cut into 2 cm × 5
cm samples. Samples were cleaned with an ultrasonic
cleaner for an hour in acetone, liberally rinsed with DI
water, and dried overnight in a 60˚C oven. Nanotube
surfaces were prepared in a 1:7 volumetric ratio of acetic
acid (99.99% purity, Sigma–Aldrich) to 0.5% w/v hydro-
fluoric acid in water (48% w/v, EM Science, USA) by
anodizing 2 cm × 5 cm samples for 30 minutes at differ-
ent voltages (5 V, 10 V, 15 V, and 20 V). Surfaces were
then rinsed liberally with DI water for at least 30 seconds
and dried in a 60˚C oven overnight. Following the crea-
tion of the TiO2 nanotubes, samples of these and un-
treated titanium were placed in a tube furnace and baked
at 500˚C for 2 hours to anneal the material and crystallize
the fabricated amorphousTiO2 to anatase phase for opti-
mal cell culture conditions, as the effect of crystallinity
has already been determined and previously reported [6,
14]. Samples were then cut into 1 cm × 1 cm pieces for
cell culture, and baked again in a tube furnace at 260˚C
for 2 hours to ensure the degradation of any endotoxin
that may have been introduced to the samples through
handling. Samples were further sterilized in an autoclave
prior to use in cell culture and oxygen radical elimination
experiments. The presence of nanotubes was visualized
by scanning electron microscopy (SEM, XL30, FEI Co.,
USA).
2.2. Primary Cell Sourcing
Pathogen-free female C57BL/6 mice, 6 to 8 weeks old,
were purchased from Charles River Laboratories. Mice
were maintained in the University of California San
Diego animal facilities, and were given sterile water, and
mouse chow for the duration of the experiments. Animal
guidelines for the care and use of laboratory animals
have been observed; all experimental protocols used in
this study were approved by the Institutional Animal
Care and Use Committee of the University of California
San Diego.
Bone marrow cells were harvested from murine tibias
and femurs and differentiated into macrophage cells us-
ing previously described methods [15-17]. Bone marrow
cells were flushed from long bones, and then differenti-
ated into bone-marrow derived macrophages (BMMΦ)
by incubating in complete DMEM (cDMEM: DMEM
supplemented with 10% heat inactivated FBS, 10% of
supernatant from L-929 fibroblast cells (ATCC, Manas-
sas, VA), 1% penicillin-streptomycin (Gibco®, Carlsbad,
CA), 0.01M Hepes buffer (Gibco®, Carlsbad, CA), 1mM
sodium pyruvate (Gibco®, Carlsbad, CA), and 1% of a
100X MEM non-essential amino acids solution (Gibco®,
Carlsbad, CA)). The cells were cultured for 7 days on ti-
ssue culture treated dishes, with media changes every 2
days. Adherent day 7 cultured cells were selected as ma-
ture macrophages for further studies. This protocol has
been shown to produce a mature macrophage phenotype
[16]. Replicates are defined as cells from different mice.
A minimum of three replicates were completed for all
experiments.

Macrophage Inflammatory Response to TiO2 Nanotube Surfaces 295
2.3. Macrophage Adhesion and Growth on
Substrates
Mature macrophages were removed from culture sur-
faces by rinsing with non-cationic PBS (Gibco®, Carls-
bad, CA), soaking for 5 minutes in non-cationic PBS,
and finally scraping with a cell scraper. Cell suspensions
were counted, and 500,000 cells were seeded into each
well of a 24-well plate containing one 1cm X 1cm model
material in each well in the complete DMEM. Cells were
allowed to adhere and proliferate for 24 hours. Samples
were then rinsed 3 times with PBS, and fixed by soaking
in a solution of 4% paraformaldahyde in PBS for 20
minutes. Nuclei were stained using DAPI (1:1000, Che-
micon) in PBS overnight and rinsed 3 times with PBS.
Substrates were mounted onto glass slides using Fluor-
mount-G (Southern Biotech), visualized and photo-
graphed using a LEICA DM IRB microscope. Five ran-
dom fields were imaged from each sample, and nuclei
were counted using Image J software (NIH).
2.4. Scanning Electron Microscopy (SEM) for
Cell Morphological Examination
After 24 hours of incubation, the cells on the substrates
were washed with PBS and fixed with 2.5 w/v% glu-
taraldehyde (Sigma, USA) in PBS for 1 hour. After fixa-
tion, they were washed three times with PBS for 15 min-
utes perwash. Then the cells were dehydrated in a graded
series of ethanol (50%, 70%, 90%, and 100% v/v) for 30
minutes each and left in 100% ethanol until they were
dried with acritical point dryer (EMS 850, Electron Mi-
croscopy Science Co., USA). Next, the dried samples
were sputter-coated with metal for SEM (scanning elec-
tron microscopy) examination. The morphology of the
adhered cells were observed using SEM (XL30, FEI Co.,
USA).
2.5. Macrophage Cytokine Expression Following
Exposure to Experimental Substrates
Measurement of intracellular TNF in all cell types from
all substrate cultures were conducted by plating cells at
sub-confluent levels in tissue culture polystyrene (TCPS)
dishes (used as a control), and in 24-well plates with 1
cm × 1 cm substrates with 1 μL/mL of monensin (ebio-
sciences, San Diego, CA). In a separate TCPS dish, 5
μg/mL LPS (E. coli LPS, Sigma–Aldrich, St. Louis, MO)
was added for a positive control. The cells were incu-
bated for 8 hours under normal culture conditions. The
treatment in this protocol stops the export of cellular pro-
ducts, thus allowing for the buildup of cytokines within
the cell. The cells were removed from the culture surface,
fixed and permeabilized in suspension using a fixation
and permeabilization kit (ebiosciences, San Diego, CA)
and finally stained for intracellular tumor necrosis factor,
TNF-
 (clone MP6-XT22, rat IgG1). Antibodies were
purchased from eBioscience (San Diego, CA) as direct
conjugates of FITC, and PE, respectively. Data acquisi-
tion and analysis for this study were done using a FAC-
Scan (BD Biosciences, Mountain View, CA), CellQuest
TM software (BD Biosciences, San Jose, CA), and
WinMDI software (Joseph Trotter, The Scripps Research
Institute, San Diego, CA). Data presented represent at
least three replicates, with each replicate of cells coming
from a separate mouse.
2.6. Surface Interaction with Nitric Oxide
To investigate the nitric oxide quenching properties of
difference surfaces, an NO donor DPTA-NO was used to
make a solution of known NO concentration, and a 500
µL sample of this solution was then subjected to each of
the surfaces for 15minutes. Samples in duplicate were
taking from each well and a Griess reagent was used to
stabilize the NO concentration by converting it into ni-
trite (NO2). The measured concentration of NO2 in each
of the extracted samples correlates to the remaining con-
centration of NO after the surfaces have scavenged some
of free radicals.The concentration of the total nitrate was
determined from the absorbance at wavelength λ = 550
nm by using a UV-Vis spectrophotometer (BiomateTM 3,
Thermo Electron Co., USA) and calculated with the aid
ofa dilution standard curve.
2.7. Statistical Significance
All bar graphs are displayed as the mean ± standard error.
Sigma Plot software (2001) which specializes in scien-
tific data analysis and presentation, was utilized for
demonstrating statistical significance for the assays. One-
way ANOVAs were performed using the pairwisemulti-
ple comparison procedure.
3. Results
3.1. Substrate Properties Are Consistent with
Previously Published Results
The experimental TiO2 nanotube surfaces appear similar
to previously fabricated surfaces [6-8,11,12,18], and have
similar size characteristics and discrete uniform geome-
tries (Figure 1). Figure 1 shows highly ordered, verti-
cally aligned nanotube structures with different diameters
fabricated by varying the anodization potential. Applied
voltage for creating nanotube surfaces with different di-
ameters were from the same as in previous studies: 5 V =
30 nm, 10 V = 50 nm, 15 V = 70 nm, 20 V = 100 nm.
3.2. Macrophage Adhesion to Substrates
Following 24 Hours of Culture
As seen in Figure 2, the number of adhered macrophages

Macrophage Inflammatory Response to TiO2 Nanotube Surfaces 296
Figure 1. Physical characteristics of titanium dioxide nano-
tube surfaces. SEM images of nanotube surfaces and meas-
urements of nanotube phy sical dimensions are repr oducible
and consistent with previous reports.
to unmodified commercial Ti (with thin native oxide
layer) and anodized TiO2 nanotube substrates is inde-
pendent of oxide structure. The average number of cells
per 10Xmicroscopic field ranged from 150 on titanium to
230 on 30 nm TiO2 nanotube surfaces, with the anodized
TiO2 surfaces generally supporting more cell adhesion.
Although there was a general trend of a reduction in the
number of adherent macrophages as the nanotube size
increased, this was not statistically significant. No sig-
nificant differences in cell adhesion among the substrates
were observed.
3.3. Macrophage Morphology on the
Experimental Substrates
Figure 3 illustrates SEM micrographs of macrophages
on the Ti and TiO2 nanotube surfaces after 24 hours of
incubation, which is an adequate time to reveal the acute
inflammatory response onthe different surfaces. The re-
action of the cells to the surfaces can be seen from cell-
spreading, ruffled membranes, and extended filipodia.
On the smaller 30 nm diameter and flat Ti, there seemed
to be more filipodia extensions, with a spiky appearance
of the cell, as shown by the arrows in Figure 3. Larger
spreading areas with less filipodia are seen on the 50 -
100 nm TiO2 surfaces. Both the cell spreading and filipo-
dia extension are typical signs of macrophage activation.
3.4. Production of the Inflammatory Cytokine
TNF
Tumor necrosis factor (TNF) is a cytokine involved in
inflammation and is a member of a group of cytokines
Figure 2. Macrophage adhesion to substrates is independent
of nanotexture. No significant differences were observed in
the number of adherent macrophages on substrates follow-
ing 24 hours of culture.
Figure 3. Macrophage morphology. The SEM images show
macrophages on the experimental surfaces after 24 hours of
incubation. Arrow indicate abundant filipodia extensions on
the Ti and 30 nm TiO2 nanotubes. Dotted lines show cell
outlines and large spreading area on the 50 - 100 nm TiO2
nanotubes surfaces.
that stimulate the acute phase reaction. This cytokine was
used to test the inflammatory response of the macro-
phages to the different surfaces. As presented in Figure 4,
the production of TNF per cell varied on the different
substrates. Mean channel fluorescence (MCF) values
exhibited several significant differences for cells grown
on the various substrates. MCF for cells grown on tissue
culture polystyrene was 8.85, a value significantly lower
than that for cells cultured on flat titanium surfaces
(9.81). Cells grown on 30, 50 and 100 nm surfaces had
no significant differences in MCF values compared to
other surfaces (9.43, 9.45 and 9.40, respectively). How-
ever, cells cultured on 70 nm surfaces had significantly
lower MCF values than flat Ti (9.13). Percent positive
data show no significant differences among the culture
substrates.

Macrophage Inflammatory Response to TiO2 Nanotube Surfaces 297
Figure 4. Inflammatory cytokine production per cell follow-
ing 8 hours of culture on substrates. The production of the
inflammatory cytokine TNF was dependent on the surface
of the experimental substrate expressed by mean channel
fluorescence and percent positive cells. *indicates statistical
significance p < 0.0 5 compared to flat titanium surfaces.
3.5. Oxygen Radical Removal from Solution by
Substrates
NO is generated and secreted as free radicals by macro-
phages as part of the natural immune response which
causes a cascade of signaling for inflammation. In order
to predict how the surfaces would scavenge the free ra-
dicals and lower the inflammatory response, a cell-free
experiment was conducted to assess how the surfaces
could clean away or remove free radicals from solution.
In Figure 5, when compared to glass and TCPS (used as
control surfaces), all substrates removed significantly
more NO– from solution, with solutions exposed to glass
and polystyrene having NO– concentrations of 82.9 and
79.6 µM, respectively. Flat Tiand 30nm nanotube sur-
faces had similar NO– concentrations, 75.7 and 75.5 µM,
respectively. Nanotube surfaces with diameters greater
Figure 5. Nitric oxide quenching by surface. The removal of
nitric oxide from solutions following 20 minutes of incuba-
tion with substrates was very dependent on the surface of
the experimental substrate. *indicates statistical signifi-
cance with p < 0.05.
than 30 nm performed even better, with 71.6, 70.7, and
73.4 µM in solutions exposed to 50, 70, and 100 nm
nanotube surfaces, respectively. The 70 nm surface re-
duced the amount of available NO– most effectively
(Figure 5).
4. Discussion
We have chosen to evaluate the response of macrophages
to TiO2 nanotubes with different diameters, with the in-
tention of getting a more complete understanding of the
effect of nanotube size on the inflammatory system for
biomedical implant purposes. Our results have shown
that the nanotube size of the material studied affects the-
macrophage activation. The different activation states
were reflected by morphological changes and secretion
levels of TNF, a proinflammatory cytokine.
The results indicate that macrophage adhesion is higher
on the nanostructured modified surfaces over unmodified
Ti (Figure 2); this agrees with the general notion that
nanoscale surface structuring increases cell adhesion
over microstructures and commercially flat surfaces
[6,7,19,20]. It could be speculated that the increased sur-
face contact area due to the introduction of the nanos-
tructure may be responsible for the increase in macro-
phage adhesion, however further research beyond the
scope of this report is needed to clarify this speculation.
Although there was no significant difference in adhesion
relative to the size of the nanotube, it appears that the
smallest diameter tends to have the greatest number of
adhered cells, which is in agreement with other cell ad-
hesion studies on the nanotube surfaces with varied di-
ameters [8,10,12]. A recent study on macrophages cul-
tured on nanoporous aluminum also showed an increase

Macrophage Inflammatory Response to TiO2 Nanotube Surfaces 298
in adhesion on the small 20 nm pore size incomparison to
the larger 200 nm pore size [13].
Activated macrophagespresent a large number of mor-
phologic, functional, andmetabolic differences from nor-
mal resting cells. They are largerin size, anddisplay pro-
nounced ruffling of the plasma membrane, increased ca-
pacity for adherence and spreading on surfaces, increased
formation of pseudopods, as well as functional differ-
ences [21]. Macrophage spreading is the preliminary
stepin of macrophage activation andconsidered to be an
important marker of this event [22]. The active process
of spreading represents alterations including filipodia and
rough plasma membranes. It appears that cells present on
the experimental surfaces had activated morphologies
withruffled membranes and filipodia extensions. The Ti
and 30 nm TiO2 nanotubes had extensive filipodia,
whereas less filipodia but greater spreading area with
some membrane ruffling was observed on the 50 - 100
nm TiO2 surfaces (Figure 3). Because cells on the Ti and
30 nm TiO2 show more abundant and well-established
filipodia extensions, it can be assumed that there was a
higher degree of activation on these surfaces.Although
less filipodia were observed on the 50 - 100 nm nano-
tubes, cells exhibited a larger spreading area. While the
mechanism for filipodia activation (Ti and 30 nm TiO2)
vs. cell spreading (50 - 100 nm TiO2) on the different
nanotube diameters was not determined in the scope of
this report it would be valuable in the future to shed light
on this morphological phenomenon in determining the
immune response.
A previous study onmacrophage production of in-
flammatory cytokines showed higher production of many
cytokines on flat Ti compared to nanostructured TiO2 [3].
In the current study on TNF expression on similar nanos-
tructured surfaces with the same chemistry but varying
geometries, we observed the same general trend of lower
TNF production by cells on the nanotubes, suggesting
that the nanotopography itself contributes to lowering the
TNF production. No statistically significant correlation-
was observed as a function of the diameter size, but the
largest decrease in TNF values was significant for
macrophages cultured on 70 nm nanotube surfaces (Fig-
ure 4). A possible reason for the lower inflammatory
response in general of the TiO2 nanotubesurfaces could
be due to radical quenching by the surface, aknown
property of TiO2 [23,24], which was observed in our re-
sults with higher levels of NO– quenching on TiO2 nano-
tube surfaces compared to the other experimental materi-
als (Figure 5). Oxygen radical production is a common
occurrence in inflammation [25], and the reduction of
oxygen radicals at the surface of a biomaterial could re-
duce the local inflammatory response. Although the
mechanism for oxygen radical reduction is not explained
by the extent of this report, it appears that the 70 nm di-
ameter nanotubes are most advantageous in terms of
radical reduction for decreasing the inflammatory re-
sponse to the surface. Interestingly, the surfaces that had
the most radical reduction or oxygen quenching, i.e. 50 -
100 nm TiO2 nanotubes (Figure 5), showed the least
amount of filipodia activation (Figure 3) and possibly a
lower degree of inflammatory response of the macro-
phages. However, to shed further light on the complete
understanding the effect of surface nanotopography on
macrophage behaviors, additional studies are needed to
determine the fate of the cells as well as the secretion of
other growth factors.
In summary, this study demonstrated that the TiO2
nanotubes with 70 nm diameter is the surface with the
lowest level of macrophage morphological activation, the
lowest macrophage production of inflammatory cyto-
kines, and the greatest oxygen radical quenching capabil-
ity. Hence, TiO2 nanotubes in the ~70 nm diameter re-
gime ismost promising for implant surfaces for decreased
inflammatory response. It should be noted that larger
nanotubes in the ~70 - 100 nm range elicited a favorable
response in terms of chondrocytes and osteoblast cells
[7,11], mature cells also derived from mesenchymal cells
in general.
While TiO2 nanotubes surfaces are well knownfor or-
thopedic implant technologies, it is advantageous and
beneficial to consider this type of surface nanostructure
for other biomedical implant technologies including vas-
cular stent applications. In our previous work explor-
ingthe possibility of utilizing TiO2 nanotubes as a possi-
ble Ti or NiTi surface modifications in arterial stents, we
have found that the TiO2 nanotube surface structuring is
excellent for the growth, mobility, and endothelialization
of vascular luminal surface [18]. Collectively, the reduc-
tion of inflammation and the quenching of oxygen radi-
cals shown in the present study bolster the potential use
of TiO2 nanotubes for stents, especially be- cause nitric
oxide synthase has been shown to co-localize with athe-
rosclerotic plaques [26].
5. Conclusions
The present work shows that by changing the diameter of
TiO2 nanotubes, in the range of 30 - 100 nm, it is possi-
ble to modulate the macrophage and inflammatory re-
sponse. In addition, these findings open the possibility of
exploiting some of the beneficial material properties of
TiO2 as oxygen radical scavengers. In general, it was
found that TiO2 nanotubes surfaces had lower macro-
phage activation, decreased levels of TNF cytokine ex-
pression, as well as increased ability to quench free radi-
cals, resulting in lower inflammatory effects compared to
conventional Ti. The nanotube surface with ~70 nm di-

Macrophage Inflammatory Response to TiO2 Nanotube Surfaces 299
ameter has the greatest effect in reducing the inflamma-
tory response. This study emphasizes the role of
nanotopography in dictating inflammatory cell responses
and demonstrates that nanotopography can be utilized to
control the inflammatory likelihood of medical implants.
6. Acknowledgements
The authors acknowledge financial support of this re-
search by UC Discovery Grant No. ele08-128656/Jin.
This work was also partially supported by Postdoctoral
Fellowship (for Lisa Chamberlain) through the NHLBI
institutional training grant 5T32HL007089.
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