J Periodontol October 2005

Human Gingival Fibroblast Integrin

Subunit Expression on Titanium
Implant Surfaces
Thomas W. Oates,* Steven C. Maller, Jason West,* and Bjorn Steffensen*

Background: Implant surface characteristics have been shown to

modify cell behavior and regulate integrin expression. Integrin expression and resultant integrin-mediated cellular activity are essential components of tissue healing and homeostasis. Although both
osseous and soft tissue healing around dental implants are critical
to clinical success, there is limited information available on the
effect of implant surfaces on integrin expression in soft tissues. Therefore, the aim of this study was to examine integrin expression for gingival fibroblasts on titanium surfaces and the influence of titanium
surface roughness on integrin expression and cell morphology.
Methods: Human gingival fibroblasts were cultured on smooth
(polished) and rough (sand-blasted acid-etched) titanium surfaces
and a cell culture plastic (control) surface. To analyze integrin expression, total RNA was isolated from experimental and control cells,
and levels of integrin subunit mRNA were assessed by reverse transcription-polymerase chain reaction (RT-PCR) using primers specific
for the a2, a4, a5, av, and b1 integrin subunits and aldolase (internal
control). PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE), confirmed via DNA sequencing, and quantified using computer-assisted densitometry. The expression of the integrin
subunits was analyzed at the protein level using flow cytometry, as
well as fluorescence and confocal laser microscopy. Cell morphology
was evaluated using scanning electron microscopy (SEM).
Results: Our experiments demonstrated cellular expression of
the a2, a4, a5, av, and b1 integrin subunits at both mRNA and protein levels on all surfaces. In addition, the a4 and b1 mRNA levels
were significantly increased on smooth titanium relative to plastic
surfaces (P <0.05) with intermediate mRNA levels found on the
rough titanium surfaces. The smooth titanium surfaces exhibited
a flat monolayer of cells, while rough titanium surfaces showed cells
orienting themselves along surface irregularities.
Conclusions: These results demonstrate the presence of multiple integrin subunits in human gingival fibroblasts grown in contact
with titanium implant surfaces and that titanium surface roughness
alters cellular morphology but appears to have limited effects on
integrin expression. This study provides insight into the complicated cellular and molecular events occurring at the implant surface
that may be critical to optimizing the soft tissue interactions with the
soft tissue-implant interface. J Periodontol 2005;76:1743-1750.
KEY WORDS
Fibroblasts; gingival; implants; integrin; titanium.
* Department of Periodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX.
United States Air Force (USAF), Washington, DC, Department of Periodontics, Royal Air Force,
Lakenheath, U.K.

mplant surface roughness has

a significant effect on cellular
behavior.1 For example, the titanium (Ti) surface topography
modifies cell attachment, orientation, spreading, proliferation, differentiation, and protein expression. In
particular, the roughness of the
titanium surface induces altered
expression of integrin subunits and
production of bone-related extracellular matrix proteins by human
osteoblasts.2-7 It has been proposed
that the cellular interactions with
different implant surfaces translate
into enhanced osseointegration on
a clinical level.8,9 While the effects
of surface roughness have been well
characterized for osteoblastic cells
and bone, the increasing emphasis
on soft tissue management and
esthetics associated with dental implants requires similar consideration for gingival cell interactions
with implant surfaces.
Cellular interactions with the extracellular environment primarily
rely on integrin receptors. The integrins are a family of transmembrane glycoproteins and the major
type of receptors by which cells attach to extracellular molecules such
as collagen and fibronection.10-14
These receptors also contribute to
the regulation of gene expression
via inside-out and outside-in signal
transduction.3 The extracellular domains of integrins interact directly
1743

Fibroblast Integrin Expression on Implant Surfaces

with ligand molecules and indirectly with other cells,

while the intracellular domains of the receptors interact with cytoskeletal complexes, which include such
molecules as vinculin, talin, actin, and tropomysin.13,15 The integrin a and b subunits link together
non-covalently to form ab heterodimers. The heterodimeric associations between subunits provide the
potential for a large number of integrin subunit
interactions and great versatility in integrin-mediated
activity.12,14
A number of integrin subunits have been identified
on cells of the periodontium. These include the integrin subunits a2, a4, a5, av, b1, and b3 that have been
identified in gingival tissue.16,17 With the critical role
of integrin binding in regulating cellular activity and
extracellular interactions, the specific patterns of expression of integrins on gingival fibroblasts may have
a direct impact on the soft tissue-implant interface.
Furthermore, integrin expression in the periodontium
may be dependent upon the surface topography of the
implant surface. Therefore, the examination of integrin expression may provide both a tool for assessing
the biological effects of surface characteristics and
the basis for optimizing dental implant surfaces. The
present series of experiments provide, to our knowledge, the first demonstration of integrin expression
for human gingival fibroblasts on both smooth and
rough titanium implant surfaces.
MATERIALS AND METHODS
Titanium Disks
Two types of titanium disks with well-characterized
surface roughness were used in this investigation:
a smooth polished titanium (PT) surface and a rough,
sand-blasted acid-etched (SLA) surface. The smooth
PT disks had surfaces with an average R(a) value of
0.54 mm, whereas the rough (SLA) titanium disks had
an average R(a) value of 4.14 mm.18,19 Surface characteristics of representative specimens were visualized at 300 to 2,500 with a cold field emission
scanning electron microscope (SEM) with secondary and backscattered electron capability to assure
consistency in surface preparation.
Cell Culture
Gingival fibroblast populations were cultured from human gingival tissue explants derived from healthy interproximal premolar and molar papillae according to
methods previously described.20 Briefly, the tissue explants were incubated in Dulbeccos modified Eagles
medium (DMEM) containing 10% heat-inactivated
fetal bovine serum (FBS), 5% L-glutamine, 100 U/ml
penicillin, and 100 mg/ml streptomycin and 50 mg/ml
fungizone to establish primary cultures. For experiments, cells were utilized between passages two
through six, with cells of the same passage used within
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Volume 76 Number 10

each experiment. A total of six cell populations were

utilized for all experiments.
For all experiments, cells were cultured on 15-mm
titanium disks placed in 24-well culture platesi; control cells were cultured directly on the cell culture treated plastic surfaces of the 24-well plates. To determine
the approximate time to reach subconfluence and
confluence, cells were plated on plastic and titanium
disks in preliminary experiments and examined at
various time points with SEM. These preliminary experiments determined the specific time points for cell
cultures at subconfluence (5 days) and at confluence
(10 days). For mRNA experiments, both subconfluent
and confluent cultures were assessed. For assessments of protein expression, cells were grown to subconfluent levels prior to analysis.
Scanning Electron Microscopy
Initially, an experiment was designed to evaluate both
cell morphology and times of cell subconfluence and
confluence on both the smooth and rough titanium
disks. The cells were allowed to grow on either smooth
or rough titanium disks and evaluated over time. At
the appropriate times (48 hours, 96 hours, 6 days,
and 9 days), the medium was removed and the cells
were washed twice with phosphate buffered saline
(PBS) slowly. The cells were subsequently fixed with
2% gluteraldehyde, processed for SEM, and examined. Surfaces were visualized at 800 with a cold field
emission scanning electron microscope at 15 kV.
mRNA Levels
Total RNA was extracted using guanidiumphenol-chloroform# according to the manufacturers
instructions. The purified RNA was solubilized in 25
ml diethylpyrocarbonate (DEPC)-treated water and
quantified using spectrophotometry (absorbance at
260 nm). For the experiments, human gingival fibroblasts were grown to confluence in 100-mm tissue
culture plates, trypsinized, and seeded at 2 103
cells/well onto 7-mm2 smooth or rough titanium disks
or plastic (control) in 24-well tissue culture plates,
with one plate serving as the source for total RNA
for each surface condition (plastic, smooth, or rough)
and each cell density (subconfluent or confluent).
The reverse transcription-polymerase chain reaction (RT-PCR) used 1 mg total RNA per reaction, and
the number of cycles used during all assays was within
the exponential phase of DNA amplification for each
target mRNA, as detailed previously.21,22 Primer sequences, as previously reported, were used to assay
each total RNA sample for aldolase and a2, a4, a5,

Institut Straumann AG, Waldenburg, Switzerland.

JEOL 6400 FEC, JEOL USA, Peabody, MA.
Costar, Corning, NY.
JEOL 6400 FEC, JEOL USA.
RNAzol B, Biotech, Houston, TX.

Oates, Maller, West, Steffensen

J Periodontol October 2005

av, and b1 integrin subunits.21-23 DNA sequencing of

amplicons performed at the University of Texas
Health Science Center at San Antonio (UTHSCSA)
DNA Core Facility confirmed that the integrin subunits and aldolase corresponded with published gene
sequences in GenBank. Following amplification, PCR
products were separated on 2% agarose gels, which
were stained with ethidium bromide and visualized using ultraviolet illumination. Bands were analyzed from
captured digital images** to measure area and for
density using a 256-point gray scale. Measurements
of area and density were standardized within each
gel relative to the control (plastic at subconfluence),
which was assigned a value of 1. In addition, the data
were standardized between reactions relative to the
area and densities of bands obtained from the internal
reaction control (aldolase). This quantitative approach enabled comparisons between bands, i.e.,
mRNA levels, within each gel. The experiment was
performed one time for each of the six cell populations
cultured with all molecules assessed within each RNA
sample. For analysis, results were expressed as the
mean values of area density.
Protein Expression
Immunofluorescence analysis was used to assess the
expression of each of the integrin subunits (a2, a4, a5,
av, and b1) at the protein level. For all fluorescence
analyses, specific mouse anti-human integrin monoclonal antibodies and mouse anti-human immunoglobulin (Ig) G control antibodies were used as
primary staining agents. The cells were then stained
for 1 hour at 37
C with a 1:100 antibody concentration
(determined by preliminary experiments; data not
shown). After washing the cell layer twice with DMEM,
FITC-conjugated goat anti-mouse secondary antibody was added at a 1:400 concentration (also determined by preliminary experiments; data not
shown) for 1 hour at room temperature, followed by
rinsing three times with DMEM. After staining, the
cells were analyzed using flow cytometry. In addition, confocal laser and fluorescence microscopy were
utilized to confirm integrin proteins.
Statistical Analysis
The amplified products on gels following RT-PCR were
analyzed for band area and density using digital image
analysis as described above. We analyzed each protein (aldolase, a2, a4, a5, av, and b1) separately. For
each replication (cell population), the measurement
(band area/density) was expressed as a ratio to the
measurement for subconfluent control. These ratios
were analyzed in an analysis of variance (ANOVA)
for randomized complete blocks24 with cell population considered as block. Data for subconfluent control was not included in the ANOVA because all

values were 1.00 (that is, no variability). Residual

analysis confirmed that the assumptions underlying
the ANOVA were reasonably well satisfied. We compared among the three conditions for subconfluent
and confluent. Within subconfluent, the means for
smooth and rough were compared to the control by
comparing to 1.00. Comparisons among means were
Bonferroni adjusted.
RESULTS
Integrin Subunit mRNA Levels
Integrin subunit mRNA was amplified as distinct
bands with an expected molecular sequence for each
of the integrin subunits under study (a2, a4, a5, av, and
b1). All subunits were expressed by gingival fibroblasts grown on smooth and rough titanium surfaces
as well as tissue culture plastic (control) surfaces at
both subconfluence and confluence (Fig. 1). As an
indication of overall cell numbers, aldolase mRNA
levels were significantly (P <0.01) decreased on both
smooth and rough titanium surfaces for cells grown at
subconfluent levels compared to tissue culture plastic
(control) surfaces (Table 1). At confluence, the aldolase levels on both smooth and rough surfaces were
not significantly reduced compared to the plastic surface. The comparison of integrin mRNA levels to the
control surface showed that the a4 levels were significantly (P <0.05) increased by 54% on smooth titanium surfaces at subconfluence, and b1 were found
to be increased by 38% and 52% (P <0.05) on the
rough and smooth titanium surfaces, respectively,
at subconfluence. In most conditions, the relative levels of integrin mRNA were found to be elevated compared to control levels, though not reaching statistical
significance. Also, the smooth surfaces had a consistent trend toward higher mRNA levels for each of the
integrin subunits compared to rough surfaces. The
greatest increases were found at subconfluence for
the a2 subunit. Though not statistically significant,
smooth surfaces had a 77% increase and rough surfaces had a 65% increase relative to the plastic.
Integrin Subunit Protein Expression
Integrin subunit-specific antibody labeling with flow
cytometric analysis was used to identify integrin subunit proteins on cells grown on the titanium surfaces.
Consistent with the mRNA expression, integrin subunit proteins were detected for all subunits under
investigation on each surface (Fig. 2). No obvious
differences were identified for any of the integrins between surface conditions. However, subtle rightward
curve shifts were apparent for a2 and a5 consistent
**

ImagePro Plus, Media Cybernetics, Silver Spring, MD.

Chemicon International, Temecula, CA.
Chemicon International.
FACSCaliber flow cytometer, Institute of Biotechnology, San Antonio,
TX.

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Fibroblast Integrin Expression on Implant Surfaces

Volume 76 Number 10

Figure 1.
Representative RT-PCR gels for a2, a4, a5, av, and b1 as indicated. Lanes are indicated for standard markers (M), cells at the time of initial
seeding (0), cells grown on tissue culture plastic (C), smooth titanium (PT), and rough titanium (SLA). Lanes indicate assays performed at cellular
subconfluence (day 6) or confluence (day 9).

with increasing receptor levels on the smooth and

rough surfaces relative to plastic.
The expression of each of the integrin subunit
receptor proteins was confirmed using fluorescence
microscopy and laser confocal microscopy. Visualization of subunits identified the integrins as primarily
associated with the cell periphery (Fig. 3). These findings were consistent for each subunit and surface under consideration.
Cell Morphology
In Figure 4, the smooth titanium surface had
small surface irregularities, whereas marked surface
roughness and porosity were noted for the rough titanium surface. The cell morphology varied with sur1746

face roughness of the titanium disks. Cells grown on

the smooth titanium surface tended to grow in a flat
monolayer with cells oriented in a parallel manner,
while cells grown on the rough titanium surface
tended to orient themselves according to surface irregularities and presented numerous cellular extensions bridging irregularities in the surface.
DISCUSSION
Overall, this investigation supports the concept that integrin subunits contribute importantly to gingival fibroblast cell attachment on titanium surfaces. Evaluation
at the mRNA level showed that each of the integrin subunits under study (a2, a4, a5, av, and b1) was expressed
by gingival fibroblasts grown in contact with smooth

Oates, Maller, West, Steffensen

J Periodontol October 2005

Table 1.

Relative Quantitation of Integrin Subunit mRNA Levels (mean standard deviation)

on Tissue Culture Plastic (control) and Titanium Surfaces
Subconfluent

Confluent

Protein

Control

Smooth

Rough

Control

Smooth

Rough

Aldolase

1*

0.73 0.10*

0.82 0.18*

0.96 0.23

0.91 0.15

0.78 0.09

a2

1.77 0.49

1.65 1.11

1.03 0.21

1.80 0.98

1.24 0.58

a4

1.54 0.39

1.24 0.74

1.24 0.35

1.64 0.32

1.40 0.17

a5

1.07 0.19

0.95 0.30

1.14 0.32

1.32 0.64

1.24 0.34

aV

1.32 0.41

1.18 0.62

1.34 0.47

1.48 0.51

1.19 0.38

b1

1.52 0.45

1.38 0.62

1.35 0.60

1.35 0.41

1.26 0.20

* P <0.01.
P <0.05.

Figure 3.
Representative fluorescence (A) and confocal laser microscopic (B)
images of fluorescently stained gingival fibroblasts are shown for the
b1 integrin subunit. Arrows highlight several areas of positive staining
along cellular membrane and extensions.

Figure 2.
Representative histograms of fluorescence activated cell sorter (FACS)
analysis of integrin subunits for gingival fibroblasts grown on smooth
or rough titanium surfaces. The black curve represents staining under
control conditions with non-specific antibody; the gray curve represents
cells grown on a tissue culture (control) plastic surface; the blue curve
represents cells grown on a smooth surface; and the red curve
represents cells grown on a rough surface.

and rough titanium surfaces as well as plastic (control)

surfaces at subconfluence and at confluence. This
observation confirms and expands our previous identification of these integrin subunits in the periodontal tissues.16 In addition, our results also confirmed the
presence of each integrin subunit investigated at the
protein level on cultured gingival fibroblasts. The confocal laser and fluorescence microscopic results identified the presence of integrins primarily at the
periphery of the fibroblasts and in conjunction with cellular extensions. This corresponds to the localization
of integrin receptors reported in focal adhesions and
cellular extensions reported in the literature.16
Also, these findings are consistent with previous reports that have shown surface roughness to alter cell
orientation.1,25-27 Interestingly, cells grown on the
smooth surfaces tended to break down easily when
processed for scanning electric microscopy, whereas
those grown on the rough surfaces seemed to be more
resistant to breakdown and largely appeared intact.
One may speculate that the greater resilience of cells
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Fibroblast Integrin Expression on Implant Surfaces

Volume 76 Number 10

Figure 4.
Representative scanning electron micrographs of gingival fibroblast morphology after 2, 6, and 9 days in culture on smooth or rough titanium
surfaces (original magnification 800).

on the rough titanium surfaces reflects a stronger

mode of attachment. However, the mechanism remains to be better understood.
The upregulation of a4 and b1 subunit RNA levels
highlights the potential for these integrin subunits to
play an important role for cells interacting with titanium surfaces. The a4 integrin contributes importantly
to binding of fibronectin.14,28,29 Fibronectin is a major
component of serum and the periodontal tissues
critical to wound healing.30 It is thought to play a
critical role in early wound healing events such as
cell migration and adhesion consistent with tissue
development.31 Interestingly, the upregulation found
on the mRNA level was not consistent with findings on
the protein level, but the significance of this difference
is not fully explained by the current investigation.
It has been suggested previously that cells grown
on rough titanium surfaces have decreased proliferation and more differentiation, and these more differentiated cells may downregulate integrin expression.32
Our findings do not provide strong support for this,
but it is possible that there are elevated levels of integrin receptors on smooth titanium relative to rough
titanium surfaces as suggested by our findings. This
would be consistent with surface topography influencing cellular differentiation. However, this would be in
contrast to several findings. A recent report has shown
that increases in surface roughness were associated
with increases in integrin expression for osteoblastic
cells.33 This is also supported by a second study of osteoblasts in which a2, a5, and b1 all showed increases
in expression with increasing roughness.34
1748

It is also noteworthy that the b1 integrin subunit

mRNA was expressed at higher levels on the titanium
surfaces compared to the plastic surfaces, but, as was
found with the a4 subunit, this is not consistent with
findings on the protein level. It may be that the b1 subunit is expressed differentially on the protein level as
suggested by the extra PCR bands and the slight leftward shift with flow cytometry. This would be consistent
with the alternative splicing of the b1 integrin subunit,
which has been reported.35,36 However, sequencing
done in the present study failed to yield sequences
consistent with alternatively spliced variants of the
b1 integrin gene, and further investigation is needed.
The differential expression of integrins on cells in
contact with implants could provide the basis for novel
therapeutic modalities and rational modifications of
implant surfaces. For example, the attachment of specific integrins to the implant could be enhanced by
coating the surfaces with short amino acid sequences
corresponding to the integrin binding sites on the
extracellular matrix molecules. The a2b1 binds the
Asp-Gly-Glu-Ala peptide from collagen with high affinity.37 Likewise, other integrins preferentially bind
the Arg-Gly-Asp sequence.38 This principle is indeed
already in use with the P15 peptide, which corresponds to the cell-binding site on collagen and has
shown promise as an adjunct to periodontal healing
when coated on appropriate bone replacement graft
materials.
In summary, this research demonstrates that gingival fibroblast morphology, consistent with osteoblastic behavior, is influenced by titanium surface

J Periodontol October 2005

roughness characteristics. Furthermore, it has shown,

for the first time to our knowledge, the presence of
multiple integrin subunits expressed by human gingival fibroblasts when grown in contact with both
smooth and rough titanium implant surfaces. The
presence of the each of the investigated integrin subunits (a2, a4, a5, av, and b1) was confirmed at the protein level, showing that the mRNA is indeed being
translated to its respective protein. Collectively, our
findings demonstrate the complexity of the soft tissue-implant interface and the potential for implant
surface characteristics to influence the biology of
the soft tissue-implant interface at both the cellular
and molecular levels. Only through a complete characterization of this interface can we move toward the
development of the optimal implant surface to manage the dental soft tissue-implant complex.
ACKNOWLEDGMENT
The views expressed in this article are those of the
authors and are not to be construed as official or as
reflecting the views of the United States Air Force or
the Department of Defense.
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Correspondence: Dr. Thomas W. Oates, Department of
Periodontics, University of Texas Health Science Center at
San Antonio, 7703 Floyd Curl Drive, San Antonio, TX
78229-3900. Fax: 210/567-6858; e-mail: oates@uthscsa.
edu.
Accepted for publication March 10, 2005.