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Human Gingival Fibroblast IntegrinSubunit Expression on Titanium

Implant SurfacesThomas W. Oates,* Steven C. Maller, Jason West,* and Bjorn Steffensen*

Background: Implant surface characteristics have been shown tomodify cell behavior and regulate integrin expression. Integrin ex-pression and resultant integrin-mediated cellular activity are essen-tial components of tissue healing and homeostasis. Although bothosseous and soft tissue healing around dental implants are criticalto clinical success, there is limited information available on theeffect of implant surfaces on integrinexpression in softtissues. There-

fore, the aim of this study was to examine integrin expression for gin-gival fibroblasts on titanium surfaces and the influence of titaniumsurface roughness on integrin expression and cell morphology.

Methods: Human gingival fibroblasts were cultured on smooth(polished) and rough (sand-blasted acid-etched) titanium surfacesand a cell culture plastic (control) surface. To analyze integrin ex-pression, total RNA was isolated from experimental and control cells,and levels of integrin subunit mRNA were assessed by reverse tran-scription-polymerase chainreaction (RT-PCR) using primers specificfor thea2,a4,a5,av, andb1integrin subunits and aldolase (internalcontrol). PCR products were analyzed by polyacrylamide gel electro-phoresis (PAGE), confirmed via DNA sequencing, and quantified us-ing computer-assisted densitometry. The expression of the integrinsubunits was analyzed at the protein level using flow cytometry, aswellas fluorescence and confocal laser microscopy. Cell morphologywas evaluated using scanning electron microscopy (SEM).

Results: Our experiments demonstrated cellular expression ofthea2, a4, a5,av, andb1integrin subunits at both mRNA and pro-tein levels on all surfaces. In addition, the a4and b1mRNA levelswere significantly increased on smooth titanium relative to plasticsurfaces (P

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with ligand molecules and indirectly with other cells,while the intracellular domains of the receptors inter-act with cytoskeletal complexes, which include suchmolecules as vinculin, talin, actin, and tropomy-sin.13,15 The integrin a and b subunits link togethernon-covalently to formab heterodimers. The hetero-

dimeric associations between subunits provide thepotential for a large number of integrin subunitinteractions and great versatility in integrin-mediatedactivity.12,14

A number of integrin subunits have been identifiedon cells of the periodontium. These include the integ-rin subunits a2, a4, a5, av, b1, and b3that have beenidentified in gingival tissue.16,17 With the critical roleof integrin binding in regulating cellular activity andextracellular interactions, the specific patterns of ex-pression of integrins on gingival fibroblasts may havea direct impact on the soft tissue-implant interface.

Furthermore, integrin expression in the periodontiummaybe dependent upon the surface topography of theimplant surface. Therefore, the examination of integ-rin expression may provide both a tool for assessingthe biological effects of surface characteristics andthe basis for optimizing dental implant surfaces. Thepresent series of experiments provide, to our knowl-edge, the first demonstration of integrin expressionfor human gingival fibroblasts on both smooth andrough titanium implant surfaces.

MATERIALS AND METHODS

Titanium DisksTwo types of titanium disks with well-characterizedsurface roughness were used in this investigation:a smooth polished titanium (PT) surface and a rough,sand-blasted acid-etched (SLA) surface. The smoothPT disks had surfaces with an average R(a) value of0.54mm, whereas the rough (SLA) titanium disks hadan average R(a)value of 4.14 mm.

18,19 Surface char-acteristics of representative specimens were visual-ized at 300 to 2,500 with a cold field emissionscanning electron microscope (SEM) with second-ary and backscattered electron capability to assure

consistency in surface preparation.

Cell CultureGingival fibroblast populations were cultured from hu-man gingival tissue explants derived from healthy in-terproximal premolar and molar papillae according tomethods previouslydescribed.20Briefly, thetissue ex-plants were incubated in Dulbeccos modified Eaglesmedium (DMEM) containing 10% heat-inactivatedfetal bovine serum (FBS), 5% L-glutamine, 100 U/mlpenicillin, and 100mg/ml streptomycin and 50mg/mlfungizone to establish primary cultures. For experi-ments, cells were utilized between passages two

through six, with cells of the same passage used within

each experiment. A total of six cell populations wereutilized for all experiments.

For all experiments, cells were cultured on 15-mmtitanium disks placed in 24-well culture platesi; con-trol cells were cultured directly on the cell culture trea-tedplastic surfaces of the 24-well plates. To determine

the approximate time to reach subconfluence andconfluence, cells were plated on plastic and titaniumdisks in preliminary experiments and examined atvarious time points with SEM. These preliminary ex-periments determined the specific time points for cellcultures at subconfluence (5 days) and at confluence(10 days). For mRNA experiments, both subconfluentand confluent cultures were assessed. For assess-ments of protein expression, cells were grown to sub-confluent levels prior to analysis.

Scanning Electron MicroscopyInitially, an experiment was designed to evaluate bothcell morphology and times of cell subconfluence andconfluence on both the smooth and rough titaniumdisks. The cells were allowed to grow on either smoothor rough titanium disks and evaluated over time. Atthe appropriate times (48 hours, 96 hours, 6 days,and 9 days), the medium was removed and the cellswere washed twice with phosphate buffered saline(PBS) slowly. The cells were subsequently fixed with2% gluteraldehyde, processed for SEM, and exam-ined. Surfaces were visualized at800 with a cold fieldemission scanning electron microscope at 15 kV.

mRNA LevelsTotal RNA was extracted using guanidium-phenol-chloroform# according to the manufacturersinstructions. The purified RNA was solubilized in 25ml diethylpyrocarbonate (DEPC)-treated water andquantified using spectrophotometry (absorbance at260 nm). For the experiments, human gingival fibro-blasts were grown to confluence in 100-mm tissueculture plates, trypsinized, and seeded at 2 103

cells/well onto 7-mm2 smooth or rough titanium disksor plastic (control) in 24-well tissue culture plates,with one plate serving as the source for total RNAfor each surface condition (plastic, smooth, or rough)and each cell density (subconfluent or confluent).

The reverse transcription-polymerase chain reac-tion (RT-PCR) used 1mg total RNA per reaction, andthe number of cycles used during all assays was withinthe exponential phase of DNA amplification for eachtarget mRNA, as detailed previously.21,22 Primer se-quences, as previously reported, were used to assayeach total RNA sample for aldolase and a2, a4, a5,

Institut Straumann AG, Waldenburg, Switzerland. JEOL 6400 FEC, JEOL USA, Peabody, MA.i Costar, Corning, NY. JEOL 6400 FEC, JEOL USA.# RNAzol B, Biotech, Houston, TX.

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av,and b1integrin subunits.21-23 DNA sequencing of

amplicons performed at the University of TexasHealth Science Center at San Antonio (UTHSCSA)DNA Core Facility confirmed that the integrin sub-units and aldolase corresponded with published genesequences in GenBank. Following amplification, PCR

products were separated on 2% agarose gels, whichwere stained with ethidium bromide and visualized us-ing ultraviolet illumination. Bands were analyzed fromcaptured digital images** to measure area and fordensity using a 256-point gray scale. Measurementsof area and density were standardized within eachgel relative to the control (plastic at subconfluence),which was assigned a value of 1. In addition, the datawere standardized between reactions relative to thearea and densities of bands obtained from the internalreaction control (aldolase). This quantitative ap-proach enabled comparisons between bands, i.e.,

mRNA levels, within each gel. The experiment wasperformed one time for each of the six cell populationscultured with all molecules assessed within each RNAsample. For analysis, results were expressed as themean values of area density.

Protein ExpressionImmunofluorescence analysis was used to assess theexpression of each of the integrin subunits (a2, a4, a5,av, and b1) at the protein level. For all fluorescenceanalyses, specific mouse anti-human integrin mono-clonal antibodies and mouse anti-human immuno-

globulin (Ig) G control antibodies

were used asprimary staining agents. The cells were then stainedfor1hourat37C with a 1:100 antibody concentration(determined by preliminary experiments; data notshown). After washing the cell layer twice with DMEM,FITC-conjugated goat anti-mouse secondary anti-body was added at a 1:400 concentration (also de-termined by preliminary experiments; data notshown) for 1 hour at room temperature, followed byrinsing three times with DMEM. After staining, thecells were analyzed using flow cytometry. In addi-tion, confocal laser and fluorescence microscopy wereutilized to confirm integrin proteins.

Statistical AnalysisThe amplified products on gels following RT-PCRwereanalyzed for band area and density using digital imageanalysis as described above. We analyzed each pro-tein (aldolase, a2, a4,a5, av, and b1) separately. Foreach replication (cell population), the measurement(band area/density) was expressed as a ratio to themeasurement for subconfluent control. These ratioswere analyzed in an analysis of variance (ANOVA)for randomized complete blocks24 with cell popula-tion considered as block. Data for subconfluent con-

trol was not included in the ANOVA because all

values were 1.00 (that is, no variability). Residualanalysis confirmed that the assumptions underlyingthe ANOVA were reasonably well satisfied. We com-pared among the three conditions for subconfluentand confluent. Within subconfluent, the means forsmooth and rough were compared to the control by

comparing to 1.00. Comparisons among means wereBonferroni adjusted.

RESULTS

Integrin Subunit mRNA LevelsIntegrin subunit mRNA was amplified as distinctbands with an expected molecular sequence for eachof the integrin subunits under study (a2,a4,a5, av,andb1). All subunits were expressed by gingival fibro-blasts grown on smooth and rough titanium surfacesas well as tissue culture plastic (control) surfaces atboth subconfluence and confluence (Fig. 1). As an

indication of overall cell numbers, aldolase mRNAlevels were significantly (P

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with increasing receptor levels on the smooth andrough surfaces relative to plastic.

The expression of each of the integrin subunitreceptor proteins was confirmed using fluorescencemicroscopy and laser confocal microscopy. Visuali-zation of subunits identified the integrins as primarilyassociated with the cell periphery (Fig. 3). These find-ings were consistent for each subunit and surface un-der consideration.

Cell MorphologyIn Figure 4, the smooth titanium surface hadsmall surface irregularities, whereas marked surfaceroughness and porosity were noted for the rough tita-

nium surface. The cell morphology varied with sur-

face roughness of the titanium disks. Cells grown onthe smooth titanium surface tended to grow in a flatmonolayer with cells oriented in a parallel manner,while cells grown on the rough titanium surfacetended to orient themselves according to surface ir-regularities and presented numerous cellular exten-sions bridging irregularities in the surface.

DISCUSSION

Overall, this investigation supports the concept that in-tegrin subunits contribute importantly to gingival fibro-blast cell attachment on titanium surfaces. Evaluationat the mRNA level showed that each of the integrin sub-units under study (a2,a4,a5,av,andb1) was expressed

by gingival fibroblasts grown in contact with smooth

Figure 1.Representative RT-PCR gels fora2,a4,a5,av, andb1as indicated. Lanes are indicated for standard markers (M), cells at the time of initialseeding (0), cells grown on tissue culture plastic (C), smooth titanium (PT), and rough titanium (SLA). Lanes indicate assays performed at cellularsubconfluence (day 6) or confluence (day 9).

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and rough titanium surfaces as well as plastic (control)surfaces at subconfluence and at confluence. Thisobservation confirms and expands our previous identi-fication of these integrin subunits in the periodontal tis-sues.16 In addition, our results also confirmed thepresence of each integrin subunit investigated at theprotein levelon culturedgingivalfibroblasts.Theconfo-cal laser and fluorescence microscopic results iden-tified the presence of integrins primarily at theperiphery of the fibroblasts and in conjunction with cel-lular extensions. This corresponds to the localizationof integrin receptors reported in focal adhesions andcellular extensions reported in the literature.16

Also, these findings are consistent with previous re-ports that have shown surface roughness to alter cellorientation.1,25-27 Interestingly, cells grown on thesmooth surfaces tended to break down easily whenprocessed for scanning electric microscopy, whereasthose grown on the rough surfaces seemed to be moreresistant to breakdown and largely appeared intact.

One may speculate that the greater resilience of cells

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 1.77 0.49 1.65 1.11 1.03 0.21 1.80 0.98 1.24 0.58

a4 1 1.54 0.39 1.24 0.74 1.24 0.35 1.64 0.32 1.40 0.17

a5 1 1.07 0.19 0.95 0.30 1.14 0.32 1.32 0.64 1.24 0.34

aV 1 1.32 0.41 1.18 0.62 1.34 0.47 1.48 0.51 1.19 0.38

b1 1 1.52 0.45 1.38 0.62 1.35 0.60 1.35 0.41 1.26 0.20

*P

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roughness characteristics. Furthermore, it has shown,for the first time to our knowledge, the presence ofmultiple integrin subunits expressed by human gingi-val fibroblasts when grown in contact with bothsmooth and rough titanium implant surfaces. Thepresence of the each of the investigated integrin sub-

units (a2, a4, a5, av, and b1) was confirmed at the pro-tein level, showing that the mRNA is indeed beingtranslated to its respective protein. Collectively, ourfindings demonstrate the complexity of the soft tis-sue-implant interface and the potential for implantsurface characteristics to influence the biology ofthe soft tissue-implant interface at both the cellularand molecular levels. Only through a complete char-acterization of this interface can we move toward thedevelopment of the optimal implant surface to man-age the dental soft tissue-implant complex.

ACKNOWLEDGMENT

The views expressed in this article are those of theauthors and are not to be construed as official or asreflecting the views of the United States Air Force orthe Department of Defense.

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Correspondence: Dr. Thomas W. Oates, Department ofPeriodontics, University of Texas Health Science Center atSan Antonio, 7703 Floyd Curl Drive, San Antonio, TX78229-3900. Fax: 210/567-6858; e-mail: oates@uthscsa.edu.

Accepted for publication March 10, 2005.

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