d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 1080–1089

Available online at www.sciencedirect.com

jo ur nal ho me pag e: www.int l .e lsev ierhea l th .com/ journa ls /dema

Salivary protein adsorption and Streptococccusgordonii adhesion to dental material surfaces

Helmut Schweikla,∗, Karl-Anton Hillera, Ulrich Carla, Rainer Schweigera,Andreas Eidta, Stefan Ruhlb, Rainer Müller c, Gottfried Schmalza

a Department of Operative Dentistry and Periodontology, University of Regensburg Medical Centre, 93042Regensburg, Germanyb State University of New York at Buffalo, School of Dental Medicine, Department of Oral Biology, 213A Foster Hall,Buffalo, NY 14214, United Statesc Institute of Physical and Theoretical Chemistry, University of Regensburg, Universitätsstrasse 31, 93053Regensburg, Germany

a r t i c l e i n f o

Article history:

Received 20 September 2012

Received in revised form

25 July 2013

Accepted 25 July 2013

Keywords:

Dental materials

Protein absorption

Bacterial adhesion

Saliva

a b s t r a c t

Objectives. The initial adhesion of microorganisms to clinically used dental biomateri-

als is influenced by physico-chemical parameters like hydrophobicity and pre-adsorption

of salivary proteins. Here, polymethyl methacrylate (PMMA), polyethylene (PE), polyte-

trafluoroethylene (PTFE), silicone (Mucopren soft), silorane-based (Filtek Silorane) and

methacrylate-based (Tetric EvoCeram) dental composites, a conventional glassionomer

cement as well as cobalt–chromium–molybdenum (Co28Cr6Mo) and titanium (Ti6Al4V) were

tested for adsorption of salivary proteins and adhesion of Streptococcus gordonii DL1.

Methods. Wettability of material surfaces precoated with salivary proteins or left in

phosphate-buffered saline was determined by the measurement of water contact angles.

Amounts of adsorbed proteins were determined directly on material surfaces after biotiny-

lation of amino groups and detection by horseradish peroxidase-conjugated avidin-D. The

same technique was used to analyze for the binding of biotinylated bacteria to material

surfaces.

Results. The highest amount of proteins (0.18 �g/cm2) adsorbed to hydrophobic PTFE sam-

ples, and the lowest amount (0.025 �g/cm2) was detected on silicone. The highest number

of S. gordonii (3.2 × 104 CFU/mm2) adhered to the hydrophilic glassionomer cement surface

coated with salivary proteins, and the lowest number (4 × 103 CFU/mm2) was found on the

hydrophobic silorane-based composite. Hydrophobicity of pure material surfaces and the

number of attached microorganisms were weakly negatively correlated. No such correla-

tion between hydrophobicity and the number of bacteria was detected when surfaces were

coated with salivary proteins.

Significance. Functional groups added by the adsorption of specific salivary proteins to mate-

rial surfaces are more relevant for initial bacterial adhesion than hydrophobicity as a

physical property.

© 2013 Academy

∗ Corresponding author. Tel.: +49 941 944 6142; fax: +49 941 944 6025.E-mail address: helmut.schweikl@klinik.uni-regensburg.de (H. Schw

0109-5641/$ – see front matter © 2013 Academy of Dental Materials. Puhttp://dx.doi.org/10.1016/j.dental.2013.07.021

of Dental Materials. Published by Elsevier Ltd. All rights reserved.

eikl).blished by Elsevier Ltd. All rights reserved.

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. Introduction

he development of biofilms or dental plaque in the oral cav-ty on dental hard and soft tissues, as well as on a multitude oflinically used biomaterials for restorative purposes, is a mainause of common dental diseases including caries, gingivitis,eriodontitis, and peri-implantitis [1,2]. It is a unique fea-ure in the oral cavity that aggregates of microorganisms formn natural and artificial surfaces which are persistently cov-red with salivary proteins [2–4]. Clinically applicable dentalaterials such as alloys, metals, composites, acrylic resin den-

ures, glass ionomer cements or silicones cover a wide rangef distinct surface properties including topography, elementomposition, functionality, electrical charge, or wettability.hese various properties often combine in an individual mate-ial resulting in a highly complex set of parameters whichetermine biofilm formation.

The formation of biofilms begins with the initial reversibledhesion of microorganisms from saliva, which is driven byhe surface energies of material surfaces and organisms in

particular environment. The relevance and effectiveness ofan der Waals forces and Coulomb forces as well as hydropho-ic, electrostatic, or Lewis acid–base interactions dependsn the range of interactions. Among these non-covalentorces, hydrophobic interactions in the medium range of theanometer scale are considered most relevant in water, andlectrostatic interactions are principal forces in the shortange as well [2,5]. These forces also influence the adsorptionf salivary constituents and carbohydrates to form an acquiredellicle on oral surfaces, a proteinaceous layer which containsnzymes, glycoproteins and other macromolecules [2–4]. Itas reported that hydrophobic surfaces may attract higher

mounts of salivary proteins than hydrophilic materials, sug-esting a high affinity of salivary proteins to polymer materials6–9]. Yet, the parameters influencing the adsorption of proteino surfaces appear to be more complex since contradictoryesults have also been previously reported [10]. The high-st amounts of proteins were found to adsorb to hydrophilicodel surfaces and the lowest amounts were detected on

ydrophilic PEG- and hydrophobic CF3-modified surfaces. Itas also suggested that surface-dependent adsorption of

alivary proteins might vary widely with the adsorption ofarticular proteins [2,8–10]. Most relevant, depending on theifferent physicochemical properties of various surfaces, theomposition of a thin layer of proteins differentially adsorbedrom complex fluids like saliva is most likely more impor-ant than the absolute amount of proteins adsorbed to apecific biomaterial surface. There is experimental evidencehich suggests that specific ligands of salivary proteins sup-ort the initial adhesion of bacteria to surfaces. For instance,wo dominant and unique groups of salivary proteins, proline-ich proteins (PRPs) and salivary mucins, are compounds ofhe acquired pellicle and provide adhesion sites for receptorroteins of oral bacteria [3]. Nevertheless, it has been previ-usly suggested that the adsorption of proteins from saliva

nto surfaces leveled out differences in surface propertiesf different materials depending on the thickness, compo-ition, and conformation of proteins in the adsorbed layer2–4]. Most noteworthy, it was observed that, depending on

0 1 3 ) 1080–1089 1081

the protein species, wettability of hydrophilic surfaces withwater decreased after adsorption of proteins, whereas watercontact angles decreased on hydrophobic surfaces [2,10].

It was the aim of the present investigation to gain moreinsight into the parameters responsible for the initial adhesionof an oral microorganism to a variety of clinically relevant den-tal restorative materials. The formation of an artificial modelbiofilm was beyond the scope of this investigation. We hypoth-esized that hydrophilicity/hydrophobicity of a dental materialsurface influences the initial adhesion of an early colonizerlike Streptococcus gordonii. We first analyzed the water contactangle of pure and protein-coated surfaces of various dentalrestorative materials, amounts of human salivary proteinsadsorbed to material surfaces, and the initial adhesion of S.gordonii. Finally, these data were used to calculate the cor-relation between hydrophilicity/hydrophobicity of a materialsurface, protein adsorption, and bacterial adhesion.

2. Materials and methods

2.1. Materials and chemicals

Polymethylmethacrylate (PMMA, Palapress) was obtainedfrom Heraeus Kulzer GmbH (Hanau, Germany), poly-ethylene (PE; ultra high molecular weight polyethylene),cobalt–chromium–molybdenum (Co28Cr6Mo), and titanium(Ti6Al4V) were purchased from Zimmer GmbH (Winterthur,Switzerland). The silicone Mucopren soft came from Ket-tenbach GmbH (Eschenburg, Germany), a silorane-baseddental composite (Filtek Silorane) and a glassionomer cement(Ketac Molar) was obtained from 3M ESPE AG (Seefeld,Germany), the methacrylate-based composite material TetricEvoCeram came from Ivoclar Vivadent (Schaan, Lichten-stein), and polytetrafluoroethylene (PTFE) specimens wereprovided by the Institute of Chemistry (University of Regens-burg). Caso-bouillon was obtained from VWR International(Darmstadt, Germany). Biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester (sulfo-NHS-LC-biotin) was obtainedfrom Pierce (Rockford, IL USA) and fluorescein isothiocyanate(FITC)-labeled avidin-D was bought from Vector LaboratoriesInc. (Burlingame, CA, USA). Tween 20 was purchased fromSigma–Aldrich (Taufkirchen, Germany). All other chemicalsand organic solvents were purchased in the highest availablepurity from VWR.

2.2. Sample preparation

Polytetrafluoroethylene (PTFE), polyethylene (PE), titanium(Ti6Al4V) and cobalt–chromium–molybdenum (Co28Cr6Mo)samples were cut from rods with a diameter of 11.3 mmand a thickness set to 2 mm. Titanium and CoCrMo samplesurfaces were cleaned by sonication in acetone, toluol, ace-tone, ethanol, and water for 3 min each; PTFE and PE weresonicated in ethanol and water only. Teflon rings with aninner diameter of 11.3 mm and 2 mm in height were used to

prepare specimens between two glass plates to obtain flatsurfaces of PMMA, silicone (Mucopren soft), silorane-basedand methacrylate-based dental composite as well as a glassionomer cement (Ketac Molar). Surface roughness expressed

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as median peak-to-valley values (Ra) were found between0.04 �m (PMMA) and 0.53 �m (PE) using a perthometer (S6P,Mahr GmbH, Göttingen, Germany). PMMA, silicone (Muco-pren soft), silorane- and methacrylate-based dental compositematerials were prepared according to the manufacturer’sinstructions. These specimens were stored in demineralizedwater at 25 ◦C for seven days. Glass ionomer cement (KetacMolar) specimens were first allowed to set for seven min-utes when prepared between glass plates, and then stored fortwenty-four hours in a humidified chamber.

2.3. Salivary protein preadsorption to materialsurfaces

Unstimulated human whole saliva collected from two healthymales (24 and 29 years) was pooled and sterile filtered usingmembrane filters of 0.2 �m pore size (Sartorius, Germany). Theprotein content was determined by the bicinchoninic acid pro-tein assay (BCA, Pierce, Rockford, IL USA) and samples werethen stored at −20 ◦C until use. Material specimens were sep-arately placed into each well of a 24-well plate filled with1.2 ml saliva (0.6 mg/ml protein) and incubated for 30 min atroom temperature. For measuring wettability of the materi-als surfaces as well as bacterial adhesion (see Sections 2.4and 2.6) after protein adsorption, the specimens were washedthree times for 10 min with phosphate-buffered saline (PBS) toremove excess protein and with water to remove salt. Speci-mens covered with PBS were used as controls. Detection of theamounts of adsorbed salivary proteins was performed directlyon material surfaces by a chemiluminescence-based detectionmethod as described below.

2.4. Wettability of material surfaces

Material specimens precoated with salivary proteins or left inphosphate-buffered saline were first dried in a desiccator overnight. Water contact angles on material surfaces were thenmeasured using the sessile drop method with a P1 goniometerof Erna (Tokyo, Japan). Two microliter-droplets were advancedtoward the samples by a syringe tip until the droplets madecontact with the sample surfaces. Left and right contact angleswere read for each droplet exactly 30 s after deposition atthe surface and these values were then averaged. Dropletswere analyzed on six randomly selected pure surfaces andtwelve surfaces coated with salivary proteins for each mate-rial. Material surfaces with contact angles higher than 63◦

were considered hydrophobic [11].

2.5. Detection of salivary protein adsorption onmaterial surfaces

Amounts of adsorbed proteins were determined directly onmaterial surfaces by chemiluminescence as described by usin detail elsewhere [10]. Amino groups were first biotiny-lated with sulfo-NHS-LC-biotin (Pierce, Rockford, IL, USA),and then detected by binding of horseradish peroxidase-

conjugated avidin-D. For detection of chemiluminescence,each material surface was overlaid with a luminol-containingsolution and immediately exposed to a light-sensitive film (X-ray film). For establishment of standard curves, serial dilutions

( 2 0 1 3 ) 1080–1089

of salivary proteins were dotted in duplicate onto nitrocel-lulose membranes (Minifold I dot-blot system (Schleicher &Schuell, Dassel, Germany)) to immobilize the proteins ontonitrocellulose membranes (Schleicher & Schuell). Signals onX-ray films were densitometrically analyzed using Optimassoftware (version 6.2; Optimas Corporation, Bothell, WA, USA)after scanning the films utilizing an image scanner. A standardcurve between amounts of salivary proteins and inverse greylevels was then fitted to the data (TableCurve 2D V5.01; Sys-tat Software Inc., Chicago, IL, USA). The amounts of salivaryproteins attached to material surfaces were calculated afterdensitometric analyses of signals on X-ray films obtained fromstandard curves and specimens as described.

2.6. Bacterial culture and detection of adherentbacteria

Cultures of S. gordonii strain DL1 (Challis) were grown as sta-tionary suspension cultures in brain heart infusion (VWR)in a microaerophilic atmosphere (5% CO2) for 24 h at 37 ◦C.Then, the cells were washed in PBS, and suspensions of 108

cells per ml in PBS containing 1 mM CaCl2 and 1 mM MgCl2were prepared after measuring the optical density at 600 nm.Biotinylation and in situ detection of surface-attached bacte-ria was performed as described [12]. The microorganismswere labeled with sulfo-NHS-LC-biotin, and the number ofbacteria attached to each material surface was calculated.Material specimens precoated with salivary proteins or leftin PBS were incubated for one hour at 4 ◦C with biotiny-lated bacteria to allow for binding and the analysis of initialbacterial adhesion. The specimens were then washed withPBS to remove unbound bacteria, and subsequently incu-bated with a solution of horseradish peroxidase-conjugatedavidin-D in TBS/0.1% Tween20 for 30 min. After that, the spec-imens were washed again with TBS-Tween20, for detection ofchemiluminescence, each material surface was overlaid witha luminol-containing solution and immediately exposed toa light-sensitive film (X-ray film). Standard curves of labeledbacteria were prepared from serial dilutions of bacterial sus-pensions and immobilized on nitrocellulose membranes usinga Minifold I dot-blot system (Schleicher & Schuell). Thenumbers of microorganisms identified on material surfaceswere calculated after densitometric analyses of signals onX-ray films obtained from standard curves and specimensas described using the Optimas software (version 6.2, Opti-mas Corporation, Bothell, WA, USA). Standard curves betweennumbers of microorganisms and inverse grey levels were thenfitted to the data (TableCurve 2D V5.01; Systat Software Inc.,Chicago, IL, USA).

2.7. Scanning electron microscopy (SEM)

Incubation of materials with S. gordonii was performed asdescribed. The specimens were then washed with PBS, fixedfor 30 min by the addition of glutaraldehyde to a final con-

centration of 2.5%, washed again with PBS and water, andair dried. Specimens were mounted on stubs and tilted by30◦ in the SEM chamber. Attached bacteria were visualizeddirectly by a Quanta 400 FEG scanning electron microscope

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FEI, Frankfurt, Germany) in low vacuum mode (0.08 Torr) using large-field detector.

.8. Statistical analysis

ater contact angles on material surfaces, amounts ofdsorbed salivary proteins, and numbers of attached S. gor-onii on material surfaces are shown as medians includinghe 25–75 % quartiles summarized from measurements inepeated independent experiments. The number of individ-al values is indicated in the figure legends. Differencesetween median values were statistically analyzed using theann–Whitney U-test (SPSS/PC+, Version 18.0 SPSS, Chicago,

L, USA) for pairwise comparisons among groups at the 0.05evel of significance. Correlation analyses were performedsing best fits and corresponding equations (TableCurve 2D5.01; Systat Software Inc., Chicago, IL, USA). Correlations areresented in graphs created using SigmaPlot 8.0 (Systat Soft-are Inc.)

. Results

.1. Salivary protein adsorption to biomaterials

he amounts of salivary proteins adsorbed on dental bioma-erial surfaces were quantitated using signals obtained aftermino group labeling of salivary proteins in known amountsmmobilized on nitrocellulose to create sigmoid transitionurves (Fig. 1A). The signal intensities on X-ray films, whichriginated from defined amounts of salivary proteins follow-

ng amino group detection by a chemiluminescence-basedssay, correlated with the protein amounts immobilized onhe membranes. Signals from salivary protein standards ana-yzed by densitometry and inverse grey levels were thenlotted against amounts of protein (Fig. 1A). Standard curvesere prepared in each single experiment and used to trans-

orm signals obtained from biomaterial surfaces coated withuman saliva into amounts of adsorbed proteins. Consid-rable variations in the amounts of salivary proteins wereetected on the various surfaces. The highest amount of pro-eins (0.18 �g/cm2) was detected on polytetrafluoroethylenePTFE) samples, a surface analyzed as most hydropho-ic (Fig. 2A). Differences between the amounts of proteinsetected on PTFE and the hydrophobic surface of polyethylene

PE) were weakly significant (p = 0.041). However, it appeareds if hydrophobicity of the pure surfaces was not a parameterhich primarily influenced protein absorption since the low-

st amounts of salivary proteins (0.025 �g/cm2) were detectedn silicone surfaces which did not test significantly different inydrophobicity than PTFE (Fig. 2A). Likewise, almost identicalmounts of salivary proteins were detected on surfaces thatre quite different in hydrophobicity such as the glassionomerement (Ketac Molar) and a silorane-based composite mate-ial (Filtek Silorane), and similar amounts of proteins wereven found on titanium and CoCrMo surfaces (Figs. 1B and 2A).

lthough pure surfaces of the dental composite material (Tet-

ic EvoCeram) were as hydrophobic as PMMA, a significantlyigher amount of proteins adhered to the composite (Figs. 1Bnd 2A).

0 1 3 ) 1080–1089 1083

3.2. Salivary protein adsorption related tohydrophobicity of biomaterial surfaces

Advancing water contact angles were determined on purebiomaterial surfaces and surfaces coated with salivary pro-teins as well. A wide range of contact angles were observedindicating super-hydrophilic as well as hydrophobic surfaces(Fig. 2A). On PTFE samples water contact angles were as highas 115.5◦, but conversely, the surface of a glassionomer cement(Ketac Molar) was completely wettable by water after coat-ing with salivary proteins. Pure material surfaces with watercontact angles between 97.5◦ und 115.5◦ were consideredhydrophobic (PE, silicone, PTFE), other materials with con-tact angles between 64.0◦ (PMMA) und 75.0◦ (Filtek Silorane)were less hydrophobic (PMMA, Filtek Silorane, Tetric EvoCe-ram, titanium, CoCrMo), and the surface of a glassionomercement (Ketac Molar; 46.5◦) was hydrophilic (Fig. 2A). The coat-ing of the surfaces with salivary proteins decreased contactangles of water on most surfaces significantly. In particu-lar, the decrease in the contact angles of water to titanium(38.5◦), CoCrMo (35.7◦), Filtek Silorane (37.5◦) and PMMA (43.7◦)created hydrophilic surfaces. On the other hand, the watercontact angle to the hydrophobic PE (92.2◦) surface did notchange significantly after protein coating. Noteworthy is thatthe protein coating of the surface of the glass ionomer cement(Ketac Molar) produced a surface completely wettable by water(Fig. 2B).

Correlation analysis between contact angles by water(median values) and the amounts of salivary proteinsabsorbed to the different surfaces did not indicate a clearand useful relationship due to a low correlation coefficient(r2 = 0.34) (Fig. 2B). While relatively little differences were foundin the amounts of proteins on pure surfaces with water con-tact angles below 75◦, hydrophobic biomaterials, as indicatedby high contact angles (97.5–115.5◦), varied greatly in theamounts of absorbed proteins (Fig. 2B). Thus, the amounts ofproteins which attach to highly hydrophobic surfaces are notpredictable.

3.3. Bacterial adhesion and quantification

The number of S. gordonii which adhered to biomaterialsurfaces was determined by coupling avidin–horseradish per-oxidase. First, defined numbers of S. gordonii were immobilizedin serial dilutions on nitrocellulose membranes to createa standard curve. Then, signal intensities from standards(colony forming units; CFUs) were analyzed by densitometry,transformed into inverse grey levels and plotted against theknown numbers of microorganisms (Fig. 3A).

Adhesion of S. gordonii was detected with all materialsurfaces, but differences in the numbers of microorgan-isms found on the various surfaces were relatively small(Fig. 3B). Yet, the number of bacteria on a single surface var-ied depending on the coating. Noteworthy is that the highestnumber of bacteria (3.2 × 104 CFU/mm2) adhered to the com-pletely wettable glass ionomer cement surface coated with

salivary proteins, and the lowest number (4 × 103 CFU/mm2)was detected on the hydrophobic pure silorane composite(Filtek Silorane) (Fig. 3B). Remarkable was a slight increasein the numbers of S. gordonii on PTFE (p = 0.0), PE, PMMA,

1084 d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 1080–1089

Fig. 1 – Salivary proteins as detected by a chemiluminescence-based assay. (A) Signals recorded on X-ray film obtained fromserial dilutions of salivary proteins (whole saliva) immobilized on nitrocellulose membranes and a corresponding standardcurve after densitometric analysis. Inverse grey levels (dots) from duplicates were fitted to corresponding proteinconcentrations to create a standard curve with corresponding 95% confidence limits (B) Amounts of human whole saliva(salivary proteins) adsorbed to various material surfaces. Bars represent median values plus 25% and 75% percentiles

expe

calculated from three replicates in at least five independent

silicone, the dental acrylic composite (Tetric EvoCeram) andthe glass ionomer cement (Ketac Molar) coated with sali-vary proteins. Although differences between the numberof bacteria attached to pure and coated surfaces were notsignificant for all materials, the number of bacteria whichadhered to the silorane composite (Filtek Silorane) signifi-cantly (p < 0.001) increased about four-fold on surfaces coated

with salivary proteins. Important to note is that the samenumber of bacteria attached to the silorane were detectedon titanium and CoCrMo coated with salivary proteins, and

A B

PT

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MA

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WS

Fig. 2 – Water contact angles and adsorption of salivary proteinssalivary proteins (whole saliva) on surfaces. Bars represent mediand right contact angles on six (phosphate-buffered saline = PBSbetween median values of whole human saliva (salivary proteincontact angles (specimens stored in PBS) shown as a linear regre95% confidence limits; correlation coefficient r2 = 0.34.

riments (n = 15).

these surfaces even displayed the same contact angles withwater as the silorane surface. However, in contrast to the silo-rane material, the number of bacteria decreased on titanium(p < 0.005) and CoCrMo (p < 0.015) surfaces coated with sali-vary proteins. Noticeable and broad variations in the numberof bacteria between experiments and individual specimenswere detected on coated polyethylene (PE) and glass ionomer

cement (Ketac Molar) surfaces as well as on pure PMMA,although S. gordonii was homogeneously distributed on thesesurfaces as shown representatively with silicone (Fig. 4A–D).

Contact Angle [°]

1201101009080706050Saliva

ry P

rote

ins [µ

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0.00

0.05

0.10

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. (A) Water contact angles before and after adsorption ofan values plus 25% and 75% calculated from averages of left) and twelve (whole saliva = WS) specimens. (B) Correlationss) attached to material surfaces and corresponding waterssion line (y = −0.0291 + 1.26 × 10−3x) with corresponding

d e n t a l m a t e r i a l s 2 9 ( 2 0 1 3 ) 1080–1089 1085

Fig. 3 – Detection and quantification of attached bacteria. (A) Signals recorded on a X-ray film obtained from serial dilutionsof S. gordonii DL1 immobilized on nitrocellulose membranes and a corresponding standard curve after densitometricanalysis. (B) Amounts of S. gordonii DL1 attached to various material surfaces. PBS = phosphate-buffered saline; WS = wholes entil

(atr

Fm

aliva. Bars represent median values plus 25% and 75% perc

A correlation between the contact angles with watermedian values) on pure surfaces of the various biomateri-

ls and the number of bacteria attached to them was foundo be linear (Fig. 5A). The two variables were negatively cor-elated as indicated by a negative slope. A relatively low

ig. 4 – Adhesion of S. gordonii DL1 to silicone surfaces. Scanningicroorganisms in different areas of silicone specimens.

es calculated from ten independent measurements (n = 10).

correlation coefficient (r2 = 0.55), however, indicated that therelationship was not certain (Fig. 5A). Furthermore, a correla-

tion between the contact angles with water (median values)on surfaces coated with salivary proteins and the number ofattached bacteria revealed a correlation coefficient close to

electron microscopy shows an equal distribution of

1086 d e n t a l m a t e r i a l s 2 9

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Fig. 5 – Correlation analyses. (A) Correlations betweenmedian values of water contact angles (specimens stored inPBS) and median values of S. gordonii DL1 attached tomaterial surfaces shown as a linear regression line(y = 34,121.6 − 230.09x) with corresponding 95% confidencelimits; correlation coefficient r2 = 0.44. (B) Correlationsbetween water contact angles (specimens covered withsalivary proteins) and median values of S. gordonii DL1attached to material surfaces shown as a linear regressionline (y = 15,134.9 − 5.5x) with corresponding 95% confidencelimits; correlation coefficient r2 = 0.00. (C) Correlationsbetween amounts of salivary proteins (whole saliva) andmedian values of S. gordonii DL1 attached to materialsurfaces shown as a linear regression line(y = 17,039.5 − 3886.2x) with corresponding 95% confidencelimits; correlation coefficient r2 = 0.00.

hydrophobic domains adsorb onto PE and silicone. Since dry-ing the material surface is necessary for the measurementof static contact angles to water, attached salivary proteinswill most likely change their three-dimensional conformation.

zero (r2 = 0.0), which indicated no relationship between the twovariables (Fig. 5B). Similarly, a correlation between the abso-lute amounts of salivary proteins and the number of attachedbacteria was found to be uncertain due to a low correlationcoefficient (r2 = 0.00) (Fig. 5C).

( 2 0 1 3 ) 1080–1089

4. Discussion

The accumulation of proteins, bacteria, or cells from body flu-ids or neighboring tissues in surfaces of biomaterials in theoral cavity or elsewhere in the human body is a complexprocess regulated by parameters including surface rough-ness, hydrophobicity, or chemical charge and functionality[1,2]. Here, we characterized the influence of the wettability(hydrophilicity/hydrophobicity) on the absorption of salivaryproteins and the adhesion of the oral microorganism S. gor-donii to chemically heterogeneous smooth biomaterials usedin dentistry and medicine.

It has been suggested that biomaterials with hydrophilicsurfaces tend to be more resistant to protein adsorption andbacterial adhesion than hydrophobic ones [13,14]. Since thebiomaterials analyzed face an aqueous solvent in a phys-iological environment, wettability of theses surfaces wasdetermined by the measurement of water contact angles usingthe sessile drop method. Measurement of static water contactangles is an established method following an accepted andstandardized protocol. Although the information obtainedmay have limitations compared to dynamic measurementsit provides sound information on wettability of a material’ssurface [15,16]. Surfaces that allow for contact angles bywater greater than 63◦ were considered hydrophobic in arecent study [11]. Following these criteria, the surfaces ofmost of the materials analyzed in the present investiga-tion were hydrophobic with the exception of the hydrophilicsurface of the glass ionomer cement. While PTFE, PE andsilicone surfaces were highly hydrophobic, the surfaces ofPMMA, the composites Tetric EvoCeram and Filtek Siloraneas well as titanium and CoCrMo were considered moderatelyhydrophobic. The hydrophobic properties of these materialsurfaces corresponds very well with findings reported by oth-ers previously [11,17,18]. Notably, the contact angles detectedwith PTFE, PE, or even silicone correspond very well withcontact angles detected on silicon model surfaces after immo-bilization of fluoralkyl chains (–CF3). Likewise, lower contactangles as detected with PMMA, a silorane composite mate-rial, and acrylic composite or titanium and CoCrMo weremore related to a hydrophobic surface obtained after immo-bilization of hydrocarbon chains (–CH3). The contact angledetected with the surface of the glass ionomer cement wasvery similar to a model surface modified by the introductionof polyethylene glycol [10]. Contact angles of water on mosthydrophobic surfaces decreased after coating with salivaryproteins. Yet, water contact angles to the hydrophobic PE andsilicone surfaces remained unchanged after protein coating,although salivary proteins were detected on these surfacesand the amount of proteins on each material was significantlydifferent. Currently, it remains unclear how the water struc-ture near these two hydrophobic surfaces is still disturbedafter protein adsorption, and thus this area requires furtherinvestigation to identify the specific proteins which adherefrom saliva. One may speculate that primarily proteins with

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et, polar chemical groups including keto and amino groupsf the protein backbone as well as functional side chains ofhe various amino acids will still interact with water allow-ng for the estimation of water contact angles. Similar tour observations, protein adsorption to chemically modifiedelf-assembled monolayer surfaces (SAM) indicated that wett-bility of the surfaces was changed by the adsorption ofrotein layers of various origins. In general, it appeared thatydrophobic surfaces became more hydrophilic after proteinoating and vice versa [12]. These observations on the coatingf dental materials with salivary proteins were interpreted as

leveling out of distinct surface-free energies found on pureurfaces [19].

Hydrophilicity of a glass ionomer cement surfaces asetected here is plausible. Although the glass ionomer surfaceainly contained carbon and oxygen, inorganic componentsere detected in the surface layer as well. It was reported

hat the glass ionomer surface was strongly basic with a rela-ively large polar energy component [20]. In contrast to what

ight be expected, the surface of the glass ionomer cementKetac Molar) was completely wettable by water after salivarotein coating. The reason why the polar solvent water may

nteract so intensively with proteins adsorbed to this surfaceemains unclear. It is possible that the glass ionomer sur-ace attracted small ions from the solvent, or exposed moreharged components which electrostatically bind acidic, basic,nd polar amino acid side chains of salivary proteins, leadingo an increase in surface charges which are then shielded byydrating water [21].

The amounts of salivary proteins found on the various sur-aces varied to a great extent. The highest amount of salivaryroteins adsorbed to the most hydrophobic PTFE surfaces andigh amounts of proteins were found on the hydrophobic sur-

ace of PE as well. It was repeatedly reported that salivaryroteins adsorbed preferably on hydrophobic surfaces, androteins, in general, had a higher affinity to hydrophobic poly-ers than to hydrophilic surfaces. Even the formation of a

ental pellicle as a thin film of proteins adsorbed from salivaay be supported by hydrophobic forces [2]. Hydrophobic

urfaces may adsorb more protein than hydrophilic surfacesue to a greater number of possible adsorption-promoting

nteractions, but it has also been discussed that some pro-eins preferentially adsorb to hydrophilic surfaces because of aigh charge-dependent surface affinity [21]. The present find-

ngs do not support the hypothesis that hydrophobicity ofhe pure surfaces was the parameter primarily responsibleor protein absorption. The lowest amount of salivary pro-eins was detected on silicone, a surface as hydrophobic asTFE, whereas almost the same amounts of salivary proteinsdsorbed to the hydrophilic surface of a glass ionomer cementKetac Molar) or a silorane-based composite. Taken together,hese findings and correlation analyses in the present investi-ation suggest that a relationship between the hydrophobicityf the various pure surfaces and the amounts of salivary pro-eins absorbed is uncertain and not diagnostically useful as aesult of a low correlation coefficient. It is possible that pro-

eins in the different salivary fractions adsorb differentiallyo surfaces of varying hydrophobicity as suggested earlier [2].

recent study on the adhesion of salivary proteins utilizingwo-dimensional gel electrophoresis indicated that protein

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patterns on human enamel, as well as on the dental mate-rials titanium and poly(methyl methacrylate), were different[22]. Amounts of salivary proteins formed on the denture basematerial PMMA in the human oral cavity in vivo were in thesame range as detected in our present in vitro investigation.Moreover, predominantly salivary proteins were detected inthe pellicle, but large individual variations were reported inthe adsorbed amounts of proteins [23].

Furthermore, the amount of proteins adsorbed onto a par-ticular surface after a short time period does not correspondto the tendency and affinity of a surface to attract a protein.Salivary proteins might bind only weakly to hydrophobic sur-faces and could be desorbed easily [5]. Likewise, it was reportedthat ceramic crowns with a hydrophobic surface accumulatedalmost no plaque, indicative of low shear stress resistance[24]. Thus, protein adsorption to biomaterial surfaces is arather complex process driven by a variety of parameterswhich are difficult to control. Hydrophobicity is only one factoramong many including functionality of chemical groups, sol-vent or fluid, pH, ions and ionic strength, which influence thestructure of water at a material surface, as well as the func-tionality of proteins [6,14,21]. Moreover, it has been observedthat the adsorption of salivary proteins to hydrophobic andhydrophilic surfaces is a very rapid process. Proteins areobviously adsorbed with varying binding strengths dependingon the nature of the surface, and an increase in ionic strengthcauses larger amounts to be adsorbed on both hydrophobicand hydrophilic surfaces [5]. Consequently, information onsurface energy alone does not allow for the prediction of theamount and type of specific protein adsorbed from a partic-ular environment. Nevertheless, it appears that, in general, aprotein layer on the surface of biomaterials adds to the sumof the functional groups available and specific proteins mayfunction as ligands for cell and bacterial adhesion.

The initial adhesion of the oral microorganism S. gordoniiDL1 to the various biomaterials tested in the present studywas directly detected by in situ quantification utilizing a bacte-rial overlay technique. This method was originally developedand successfully used in the quantification of various bacte-rial strains on self-assembled monolayers functionalized bychemical modification. Bacterial adhesion was differentiallyquantitated after variation of such experimental parame-ters as bacterial strain, surface wettability, and prior proteinadsorption [12]. With this method, S. gordonii DL1 was foundto be attached to all biomaterial surfaces. Although bioadhe-sion is a complex process, microorganisms adhere to smoothdental material surfaces per se, not only to those coated withsalivary proteins [25].

Hydrophobicity was considered a physico-chemical factorprimarily influencing bacterial adhesion to materials surfaces[12,18]. In the present investigation, the highest amounts ofS. gordonii DL1 were detected on the hydrophilic surface ofa glass ionomer cement, whereas a relatively small numberof cells were found on the hydrophobic surfaces of PTFE or asilorane-based composite (Filtek Silorane). Notably, the sameratio of the number of S. gordonii DL1 found attached to the

hydrophobic silorane-based composite and a methacrylate-based composite was reported by others after a similarexposure period [17]. In contrast, a higher amount of bacterialcells were detected on the highly hydrophobic PTFE surface

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than on the silorane-based composite. These findings indicatethat parameters other than hydrophobicity, like functionalityof the surface including charged groups, may be more relevantfor the adhesion of microorganisms.

A negative correlation between contact angles to waterand the adhesion of S. gordonii DL1 was unexpected heresince hydrophilic surfaces were discussed as being moreresistant to initial bacterial adhesion, in general, thanhydrophobic surfaces [12]. In addition, the adhesion of S.gordonii DL1 to hydrophobic surfaces appears to be ther-modynamically favorable at first sight due to the surfaceproperties of this microorganism. Specific proteins or polypep-tides which may function as fimbrial adhesins on thecell surface may cause hydrophobicity of the cell sur-face [26]. Under the current experimental conditions, wecan not rule out that using phosphate-buffered saline asa solvent created a hydrophilic and charged surface onS. gordonii DL1 with high affinity to a positively chargedsurface of the glass ionomer cement [18,20,27]. To dis-tinguish between the effects caused by hydrophilicity orcharges, future experiments should analyze the adhesion ofS. gordonii DL1 to surfaces of the same hydrophilicity createdby opposite charged groups. Electrostatic forces contributestrongly to the adhesion strength of bacterial cells tohydrophilic and hydrophobic surfaces. It was speculated thatmicroorganisms rapidly bind to hydrophobic surfaces throughweak hydrophobic interactions, but adhesion strength ofbacteria to hydrophilic surfaces was higher than to hydropho-bic surfaces [28]. These suggestions could, at least in part,explain the high number of S. gordonii DL1 attached to the glassionomer surface.

The formation of dental plaque is initiated by the bindingof early colonizing bacteria, including S. gordonii DL1, to recep-tor structures in the pellicle, a thin film mainly composed ofsalivary proteins [2]. However, before the specific reversiblebinding of bacterial adhesins to ligands in the pellicle, the ini-tial adhesion to a protein-coated biomaterial surface occursthrough physico-chemical forces including hydrophobic inter-actions, van der Waals and electrostatic forces as discussed indetail elsewhere [2]. Thus, the influence of the hydrophobicityof biomaterials coated with salivary proteins on the adhesionof S. gordonii DL1 was analyzed in the present investigation.Yet, a correlation between water contact angles on coatedmaterials and the adhesion of S. gordonii DL1 was not detected.Recently, similar results were reported using different den-tal composite materials including a silorane-based composite[17,19].

Due to the lack of correlation between water contact angelsand amounts of microorganisms attached to surfaces, wespeculate that specific ligands of the salivary proteins pro-mote initial adhesion of cells to the coated glass ionomercement. It is likely that these specific compounds are avail-able in very similar quantities, although the absolute amountsof salivary proteins in the various surfaces are different. Aminimal number of ligands including, for instance, MG2 (lowmolecular weight mucin glycoprotein) or PRPs (proline-rich

proteins) would be sufficient for the saturated binding of S. gor-donii DL1 [3,29,30]. Such ligands may bind to adhesins like theserine-rich protein Hsa on the surface of S. gordonii DL1 [31].Very similar to the observation made with a glass ionomer

( 2 0 1 3 ) 1080–1089

cement in the present investigation, the adhesion of S. gor-donii DL1 to hydrophilic silicon wafer surfaces modified withpolyethylene glycol (PEG) was enhanced after coating withsalivary proteins [12]. In this respect, these data do not sup-port a paradigm stating that the adsorption of salivary proteinsequalizes differences in surface properties of different mate-rials [1]. In contrast, they suggest that salivary proteins maynot only mask, but enrich the properties of pure material sur-faces with new functional groups. However, the amounts aswell as the spectrum of salivary proteins specifically adsorbedto pure material surfaces with distinct physico-chemical prop-erties in a particular environment, like the oral cavity, have yetto be determined.

5. Conclusions

The sensitive methods used here allow for a reliable estima-tion of very small amounts of proteins and microorganisms.Thus, the calculation of correlations between wettability ofpure surfaces of materials and the amounts of adsorbed sali-vary proteins or S. gordonii DL1 as well as the correlationbetween the amounts of salivary proteins and the numbersof S. gordonii DL1 attached to material surfaces is also consis-tent. The analysis of the initial adhesion of S. gordonii DL1 tosmooth surfaces of various dental biomaterials revealed a neg-ative correlation between the hydrophobicity of pure surfacesand the number of attached microorganisms. However, nosuch correlation was detected when surfaces were coated withsalivary proteins. Likewise, the number of microorganismsdid not correlate with the total amounts of salivary proteinsadsorbed to materials. These findings, although obtained witha limited number of chemically different materials, suggestthat functional groups added by the adsorption of specific sali-vary proteins to material surfaces are more relevant for initialbacterial adhesion than hydrophobicity as a physical property.

Acknowledgements

We would like to thank G. Ferstl for excellent technical assis-tance (SEM), and the Medical Faculty of the University ofRegensburg for financial support.

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