Bacterial adhesion of Streptococcus mutans to provisional fixed prosthodontic material

Bacterial adhesion of Streptococcus mutans to provisional fixed prosthodontic material Ralf Buergers, DDS, MS,a Martin Rosentritt, MS,b and Gerhard Handel, DDS, MS, PhDc Regensburg University Medical Center, Regensburg, Germany

Statement of problem. Bacterial adhesion and formation of dental plaque on provisional fixed prosthodontic materials results in gingival inflammation and secondary caries. Purpose. The purpose of this in vitro study was to compare 10 commonly used provisional fixed prosthodontic materials (2 acrylic polymethyl methacrylates, 2 improved methacrylates, and 6 bisacrylate composite resins), based on their susceptibility to adhere to Streptococcus mutans, and examine the influence of surface roughness and hydrophobicity. Material and methods. Surface roughness was assessed by perthometer and hydrophobicity by contact angle measurements. Streptococcus mutans suspension was incubated with 15 disk-shaped specimens for each material (10 x 2 mm) and examined with the fluorescence dye, Alamar Blue/resazurin, and an automated multidetection reader. Glass and the veneering composite resin, Sinfony, served as controls. Statistical analysis was performed using the MannWhitney U-test in combination with the Bonferroni adjustment. Additionally, scanning electron micrographs were made. Results. Median surface roughness values ranged between 0.04 μm and 0.08 μm, and median contact angles between 46.5 and 71 degrees. High relative fluorescence intensities (>10,000) were found for Snap, UniFast LC, and CronMix K plus, moderate values (5000-10,000) for Trim, Temphase, Structur Premium, and PreVISION CB, and lowest fluorescence intensities (<5000) were found for Cronsin, Protemp 3 Garant, and Luxatemp. Scanning electron micrographs displayed streptococcal monolayers on all investigated surfaces, indicating initial bacterial adhesion. Conclusions. The quantity of bacterial adhesion differed significantly among the assessed provisional materials. A correlation between bacterial adhesion and surface roughness or hydrophobicity was not confirmed. Bisacrylate composite resins and acrylic polymethyl methacrylates had significantly lower adhesion potentials than improved methacrylates. (J Prosthet Dent 2007;98:461-469)

Clinical Implications

Provisional fixed prosthodontic materials are commonly used in prosthodontics and are often worn for long periods of time. When used for patients who are prone to gingival inflammation or secondary caries, materials with low bacterial adhesion potentials, such as bisacrylate composite resins and acrylic PMMAs, are preferred.

Postdoctoral Research Fellow, Department of Prosthetic Dentistry. Engineer, Department of Prosthetic Dentistry. c Professor and Chairman, Department of Prosthetic Dentistry. a

b

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Volume 98 Issue 6 The attachment of certain microorganisms to specific surfaces in the human oral cavity and the resulting formation of dental plaque on teeth and dental materials are primary causes for oral diseases such as denture stomatitis, gingival inflammation, and secondary caries, which may consequently lead to unhealthy complications.1,2 Microbiological adhesion testing has primarily focused on restorative materials such as amalgam, glass ionomers, and composite resins.3-5 In contrast, fewer studies on prosthodontic and implant materials have been published, and investigations regarding the bacterial adhesion to provisional fixed prosthodontic materials are even more limited.6,7 These materials may be classified by the type of resin. Acrylic polymethyl methacrylates (PMMA) belong to the oldest group of provisional materials. PMMA fine particles are mixed with monomer liquid and combined with polymerized methyl methacrylate. The improved methacrylates are based on monofunctional acrylate monomers with a high molecular weight. The latest class of materials is formed by bisacrylate composite resins, which are comparable to composite resins used for direct restoration therapy. They consist of an organic matrix and inorganic fillers. These provisional materials are exposed to bacterial colonization to a greater degree than definitive restorations due to the higher surface roughness and, generally, to an inferior fitting interface. This is especially true when they are worn for an extended period of time. The quantity and quality of bacterial accumulation on specific substrata is determined by variable surface characteristics.8 High surface roughness values, meaning surfaces with pits and grooves, significantly promote adhesion of bacteria by reducing the influence of shear forces on initially attaching bacteria.9,10 Substrata with high surface free energy values are known to enhance adhesion of bacteria.11-14 In addition, the chemical composition of a material,

its zeta potential, and the surface hydrophobicity strongly influence the bacterial adhesion process.8,12,15,16 An increased zeta potential, which refers to the electrostatic potential generated by the accumulation of ions on the surface, results in decreased bacterial attachment.16 Generally, hydrophobic microorganisms prefer hydrophobic substrata, and bacteria with hydrophilic properties prefer hydrophilic materials.8,12 Moreover, bacterial adhesion differs between the various bacterial species. Most of the previous studies refer to streptococci bacteria, since they belong to the group of the so-called “early colonizing bacteria”17 and, especially in the case of Streptococcus mutans (S. mutans), are known to play an important role in the pathogenesis of caries.18 Previous studies describe various in vitro methods for quantifying the adhesion of specific bacterial species to defined dental substrata.2-7,9,11,13-16,19 Examples of such methods include scanning electron microscopy, radiolabelling, and direct plate counting, as described by An et al.8 As a rapid, reproducible, and simple assay for the precise quantification of adhering bacteria, fluorometric techniques have recently gained increasing recognition.18,20,21 In the presence of viable microorganisms, nonfluorescent starting substances are metabolized to fluorescent markers, which can then be recorded by microscopy or an automated fluorescence reader. In the case of the fluorescence dye, Alamar Blue, nonfluorescent resazurin is reduced to fluorescent resorufin (highly fluorescent).22 The exact mechanism of this nontoxic reduction reaction is assumed to occur intracellularly via enzyme activity, or in the medium as a chemical reaction.23,24 The well-known correlation between the amount of reduction of resazurin to fluorescent resorufin and the associated amount of living organisms is used for the quantification of adhering bacteria.23,25,26 The purpose of this in vitro study was to observe the adhesion poten-

The Journal of Prosthetic Dentistry

tials of 10 commonly used provisional fixed prosthodontic materials and the particular class to which they belong. The growth and plaque-forming abilities of Streptococcus mutans on these different materials were investigated with a spectrofluorometric method in combination with scanning electron microscopy. Furthermore, the influence of specific physico-chemical surface characteristics (physical configuration, surface roughness, and hydrophobicity) on the susceptibility to adhere to S. mutans was investigated. Finally, the hypothesized correlation between surface roughness and quantity of bacterial adhesion was examined. The research hypothesis was that different provisional fixed prosthodontic materials would exhibit different potentials to adhere to streptococci, according to their class of material and their specific surface characteristics.

MATERIAL AND METHODS Table I lists all assessed provisional fixed prosthodontic materials, and includes the manufacturer information. All tested materials are commercially available and widely used. They were chosen without any particular rationale, but each class of material was represented. In addition, 2 control materials were used. Glass (Paul Marienfeld GmbH, Lauda-Koenigshofen, Germany) is generally considered to be extremely smooth and often used in bacterial adhesion studies.27 The second control material, Sinfony (3M ESPE, St. Paul, Minn) is a popular microhybrid veneering composite resin material. Fifteen specimens were prepared for each material. Uniform, disk-shaped specimens (10 x 2.0 mm in height) were prepared using a custom metal mold with calibrated circular holes. Each material was prepared according to the manufacturer’s instructions, inserted into the mold, and covered immediately with 2 glass slides (Alfred Becht GmbH, Offenburg, Germany) on the top and bottom to prevent formation of an oxy-

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Table I. Material class, manufacturer information, surface roughness, and contact angle values (median; 25/75%) of assessed provisional fixed prosthodontic materials

Surface Roughness (µm)

Contact Angle (Degrees)

Brand Name (Lot Numbers)

Manufacturer

Cronsin (01110358/0107104)

Merz Dental GmbH, Lutjenburg, Germany

0.08 (0.04/0.08)

63.0 (60.0/68.0)

Trim (0112652/0011680)

Harry J. Bosworth Co, Chicago, Ill

0.08 (0.08/0.08)

71.0 (70.0/72.0)

Snap (11406/92911)

Coltene/Whaledent AG, Altstatten, Switzerland

0.08 (0.08/0.08)

62.0 (61.0/64.0)

UniFast LC (0005121/0604101)

GC Corp, Tokyo, Japan

0.04 (0.04/0.06)

67.0 (62.0/68.0)

Protemp 3 Garant (B 279384)

3M ESPE, St. Paul, Minn

0.04 (0.04/0.06)

55.0 (52.0/58.0)

Luxatemp Automix (511431)

DMG, Hamburg, Germany

0.04 (0.04/0.04)

65.0 (64.0/68.0)

Temphase (003742)

Kerr Corp, Orange, Calif

0.04 (0.04/0.04)

53.0 (50.0/55.0)

Structur Premium (711470)

VOCO, Cuxhaven, Germany

0.04 (0.04/0.08)

50.5 (48.0/53.0)

PreVISION CB (135072)

Heraeus Kulzer, Hanau, Germany

0.04 (0.04/0.04)

48.0 (47.0/51.0)

CronMix K plus (01420081)

Merz Dental GmbH

0.04 (0.04/0.06)

46.5 (43.0/48.0)

Control material 1

Glass

Marienfeld, Lauda-Koenigshofen, Germany

< 0.01

44.5 (44.0/46.0)

Control material 2

Sinfony (159764)

3M ESPE

0.04 (0.04/0.08)

45.0 (42.0/48.0)

Material Class Acrylic PMMA

Improved methacrylates

Bisacrylate composite resins

gen-inhibited layer. UniFast LC was additionally light polymerized for 1 minute on both sides using a light-polymerization unit (Heliolux DLX1; Ivoclar Vivadent, Schaan, Liechtenstein), 100 W, at a distance of 2 cm from the tip. Each specimen was polished using a polishing machine (MotoPol 8; Buehler GmbH, Dusseldorf, Ger-

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many) and wet abrasive paper discs (Buehler Ltd, Lake Bluff, Ill) with a grit of 1000, 2000, and 4000. Specimens were stored in distilled water for 10 days before further processing. A stylus instrument (Perthometer S6P; Perthen, Gottingen, Germany) was used to determine the surface roughness of all specimens. Rough-

ness measurements were performed on 3 sites of 3 specimens of each material. Materials with roughness values below 0.2 μm were regarded as smooth, since no further direct influence on the bacterial adhesion would be expected below this limit.28 All specimens were cleansed with ethanol (70%) and fixed into 48-well plates

464 (Sarstedt, Newton, NC). The hydrophobicity of all test and control material surfaces was evaluated by measuring distilled water contact angles. Disks were cleaned with acetone (Arcos Organics, Geel, Belgium) and air dried. The sessile drop method was performed on 5 specimens of each material. Two calibrated droplets (2.0 μl) were assessed on each specimen with 2 measurements for each droplet (right and left contact angle). Precisely 30 seconds after careful deposition of the drop with a syringe, contact angles were measured with a goniometer (G1; ERNA, Tokyo, Japan) at 25°C room temperature by using the horizontal projection technique. The 4 measured contact angles per specimen were then averaged. The contact angle varied typically within the range of ±5 degrees of the mean. S. mutans (strain NCTC 10449; DSMZ, Braunschweig, Germany) was cultivated in sterile trypticase soy broth (BBL Trypticase Soy Broth; BD Diagnostics, Franklin Lakes, NJ) supplemented with yeast extract (BD Diagnostics). The bacterial solution was centrifuged at 18°C for 5 minutes at 2000 rpm and washed twice with phosphate buffered saline (PBS) (Sigma-Aldrich, St. Louis, Mo). The optical density of the suspension was adjusted to 0.3 at 540 nm with a spectrophotometer (Genesys 10S; Thermo Fisher Scientific, Waltham, Mass). The oxidation-reduction fluorescence dye, Alamar Blue/resazurin (0.007536 g/10 ml) (Sigma-Aldrich) was used to determine the amount of bacterial adhesion. Fluorescence intensities were recorded by an automated multidetection reader (FLUOstar Optima; BMG Labtech, Offenburg, Germany) at wavelengths of 530 nm excitation and 590 nm emission. One ml of PBS was added to each well and the autofluorescence was subsequently determined. The buffer was then removed, 1 ml of bacterial solution was added to each well, and the 48-well plates (Sarstedt) were incubated with resazurin (15 µl) at 37°C for 150 minutes before fluores-

Volume 98 Issue 6 cence determination. The mixture of bacterial solution and resazurin was extracted by suction, the wells were washed twice with distilled water, and 1 ml of PBS was added. The determination of fluorescence after bacterial adhesion was performed as described above. The fluorescence of pure phosphate buffered saline (0-control), of buffer and resazurin (dye-control), and of pure bacterial solution (bacteria-control) served as control references. Five specimens of each provisional fixed prosthodontic material were additionally used for scanning electron microscopy (SEM) verification. The specimens with the adhering bacteria were rinsed in PBS, fixed with methanol, and air dried. The test specimens were then mounted on aluminum stubs and sputter-coated with 99.99% gold (PROVAC, Balzers Corp, Liechtenstein). Specimens were examined with a scanning electron microscope (magnification x1700) (Stereoscan 240; Cambridge Instruments, Cambridge, UK). The area covered with adhering bacteria was marked and quantified with an image analysis program (Optimas 6.2; Media Cybernetics, Bethesda, Md). The Mann-Whitney U-test in combination with the Bonferroni adjustment (α=.00076 for all materials, 66 pairs; α=.0083 for acrylic PMMAs versus improved methacrylates, 6 pairs; α=.0018 for bisacrylate composite resins versus acrylic PMMAs or improved methacrylates, 28 pairs each) was used to detect differences in prevalence and calculated using statistical software (SPSS 11.5 for Windows; SPSS Inc, Chicago, Ill).

RESULTS Table I shows the results of the surface roughness determination for all materials by perthometer measurement. Presented are the median values and the 25th and 75th percentiles. All 10 materials assessed showed similar median roughness values, ranging between 0.04 and 0.08 μm. There-

The Journal of Prosthetic Dentistry

fore, all surfaces could be regarded as smooth. Within this high level of smoothness significant differences between the specific materials were observed. The bisacrylate composite resins and the improved methacrylate material UniFast LC had the significantly smoothest surfaces, while acrylic PMMAs and the improved methacrylate Snap had rougher surfaces (Table II). Table I additionally presents the median values and 25th and 75th percentiles from the contact angle measurements. The median contact angles of the tested provisional fixed prosthodontic materials ranged between 46.5 and 71.0 degrees. In general, there were significant differences between contact angles of test materials and controls (66 pairs; α=.00076). All tested provisional fixed prosthodontic materials had significantly higher contact angles than control material glass (44.5 degrees). Not considering Luxatemp Automix (21 pairs; α=.00023), the contact angles of the bisacrylate composite resins were significantly lower than those of acrylic PMMAs and improved methacrylates (Table III). The results of the fluorometric adhesion measurements are presented in Figure 1 as median values and 25th and 75th percentiles of the relative fluorescence intensity (no units). The median values varied between approximately 3000 and 16,000. The lowest fluorescence with median values below 5000, indicating low bacterial adhesion, were found for Cronsin (median value of relative fluorescence intensity: 4321), Protemp 3 Garant (4273), and Luxatemp (3286). Moderate median values between 5000 and 10,000 were found for Trim (8237), Temphase (9488), Structur Premium (8594), and PreVISION CB (7380). Snap (15,366), UniFast LC (11,115), and CronMix K plus (10,843) had the highest relative fluorescence intensities, with median values of over 10,000. No significant differences (6 pairs; α=.00076) could be found between the relative fluorescence inten-

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December 2007

Table II. Statistical analysis (P values) of surface roughness (left and down) and relative fluorescence intensity (right and up); Mann-Whitney U-test in combination with Bonferroni adjustment (a=.00076, 66 pairs)

Cronsin Trim Cronsin

Snap

UniFast LC

Protemp Luxa3 Garant temp

PreCronTem- Structur VISION Mix CB K plus phase Premium

Glass Sinfony

—

.529

.003

.009

.912

.579

.035

.190

.089

.015

.529

.063

Trim

.258

—

.035

.165

.393

.247

.436

1.000

.684

.165

.796

.190

Snap

.258

1.000

—

.436

<.0001*

<.0001*

.089

.015

.043

.247

.004

.353

UniFast LC

.258

.014

.014

—

.005

.004

.529

.089

.165

.971

.029

.796

Protemp 3 Garant

.258

.014

.014

1.000

—

.631

.004

.143

.075

.002

.393

.009

Luxatemp Automix

.050

.436

.436

—

.015

.105

.023

.004

.190

.009

Temphase

.113

.004

.004

.730

.730

.730

—

.247

.393

.436

.075

.631

Structur Premium

.730

.113

.113

.436

.436

.113

.258

—

.853

.063

.481

.218

PreVISION CB

.113

.004

.004

.730

.730

.730

1.000

.258

—

.165

.218

.481

CronMix K plus

.258

.014

.014

1.000

1.000

.436

.730

.436

.730

—

.015

.796

<.0001*

<.0001*

<.0001*

—

.123

1.000

.258

.436

<.0001*

—

Glass Sinfony

<.0001* <.0001*

<.0001* <.0001* <.0001* <.0001* .730

.113

.113

.436

<.0001* .436

<.0001* <.0001* .113

.258

*Significant difference

Table III. Statistical analysis (P values) of contact angle values; Mann-Whitney U-test in combination with Bonferroni adjustment (a=.00076, 66 pairs)

Cronsin Trim

Snap

UniFast LC

Protemp Luxa3 Garant temp

Cronsin

—

.001

.703

.304

.001

Trim

—

—

<.0001*

.001

<.0001*

Snap

—

—

—

.043

<.0001*

.013

UniFast LC

—

—

—

—

<.0001*

.970

*Significant difference

Buergers et al

.305

PreCronTem- Structur VISION Mix CB K plus phase Premium .001

Glass Sinfony

<.0001*

<.0001*

<.0001* <.0001*

<.0001*

<.0001*

<.0001*

<.0001* <.0001*

<.0001*

<.0001*

<.0001*

<.0001*

<.0001* <.0001*

<.0001*

<.0001*

<.0001*

<.0001*

<.0001* <.0001*

<.0001*

<.0001* <.0001*

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Table III. continued (2 of 2) Statistical analysis (P values) of contact angle values; Mann-Whitney U-test in combination with Bonferroni adjustment (a=.00076, 66 pairs)

Cronsin Trim

Snap

UniFast LC

Protemp Luxa3 Garant temp

PreCronTem- Structur VISION Mix CB K plus phase Premium

Glass Sinfony

Protemp 3 Garant

—

—

—

—

—

<.0001*

.254

.028

.005

<.0001* <.0001*

<.0001*

Luxatemp Automix

—

—

—

—

—

—

<.0001*

<.0001*

<.0001*

<.0001* <.0001*

<.0001*

Temphase

—

—

—

—

—

—

—

.357

.060

.0001*

.0001*

.0001*

Structur Premium

—

—

—

—

—

—

—

—

.300

.017

.021

.015

PreVISION CB

—

—

—

—

—

—

—

—

—

.063

.005

.049

CronMix K plus

—

—

—

—

—

—

—

—

—

—

.535

.849

Glass

—

—

—

—

—

—

—

—

—

—

—

.679

Sinfony

—

—

—

—

—

—

—

—

—

—

—

—

*Significant difference

30

20

10

0 Cr on s

in Tri m Pro Un Snap tem ifa p 3 st LC Ga Lu rant xa tem Str T uc emp p tur ha Pr e Pr e m s e V iu Cr ISIO m on N mi C xK B Plu s Gl as s Sin f on y

Relative Fluorescence Intensity

40

1 Relative fluorescence intensity (no unit) of 10 provisional fixed prosthodontic materials and 2 control materials (median; 25%/75%).

The Journal of Prosthetic Dentistry

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December 2007 sities of the materials with low fluorescence values (Cronsin, Protemp 3 Garant, Luxatemp) and the control glass (4644). Only Snap showed significantly higher fluorescence intensity than the control material, Sinfony composite resin (11,516). All other materials ranged below Sinfony in terms of relative fluorescence values. In comparing the different classes to each other (6 pairs, α=.0083; 28 pairs, α=.0018), improved methacrylates had significantly higher relative fluorescence values than the other material classes; there was no significant difference between acrylic PMMAs and bisacrylate composite resins (Table II). There was neither a correlation between the amount of bacterial adhesion and the surface roughness, nor between quantity of bacterial adhesion and the hydrophobicity of the tested materials. Examples of scanning electron micrographs are presented in Figures 2 through 4. The adhered S. mutans bacteria showed an analog colonization pattern on all assessed material surfaces, with a varying number of adhering microorganisms. A bacterial monolayer was observed on all examined surfaces, indicating bacterial adhesion rather than bacterial accumulation. Single streptococci and small aggregates of organisms were found on the materials with low adhesion values, Cronsin (Fig. 2), Luxatemp, and Protemp 3 Garant. Short bacterial chains and larger adherent aggregates, a more developed stage in biofilm formation, were observed on Temphase (Fig. 3), Structur Premium, Trim, and PreVISION CB. Larger and structured bacterial aggregates with a high number of bacteria demonstrated heavy bacterial adhesion to UniFast LC (Fig. 4), Snap, and CronMix K plus.

2 Scanning electron micrograph of S. mutans adhered to Cronsin (x1700 magnification).

3 Scanning electron micrograph of S. mutans adhered to Temphase (x1700 magnification).

DISCUSSION The results of the present study support the hypothesis that provisional fixed prosthodontic materials may differ in their susceptibility to

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4 Scanning electron micrograph of S. mutans adhered to UniFast LC (x1700 magnification).

468 adhere to streptococcal bacteria. The assumed influence of certain surface characteristics could not be confirmed. Several in vitro models to quantify adhering microorganisms on dental substrata have been established and applied in the past.19 Recently, fluorescence techniques have resulted in more rapid and reproducible quantification procedures with a minimized number of potential measurement errors.18,19,21 Gaines et al18 developed a microtitre plate-based assay to quantify the adherence of fluorescent-labelled S. mutans with 2´, 7´-bis-(carboxyethyl)-5(6´)-carboxyfluorescein-acetomethyl ester to hydroxylapatite using a spectrofluorometer, which accelerates and simplifies enumeration measurements. In contrast to this method, the resazurin fluorescence assay (Alamar Blue) can indicate the amount of viable bacteria, since there is a direct correlation between the number of living microorganisms and the amount of reduction of resazurin to fluorescent resorufin.23,25,26 SEM observation is especially suited for the microscopic characterization of bacterial morphology and material surfaces or the interactions between them.19 In this study, scanning electron micrographs were used for supplementary verification of the results from fluorescence adhesion tests. Bacterial adhesion, visualized by relative fluorescence intensity, varied significantly between the assessed materials (Table III). Only 3 materials (Cronsin, Protemp 3 Garant, and Luxatemp) were ranked below control material glass, indicating a low susceptibility to adherence by S. mutans. Slightly higher fluorescence intensities were measured on 4 materials (Trim, Temphase, Structur Premium, and PreVISION CB) and significantly higher intensities were found on 3 materials (Snap, UniFast LC, and CronMix K plus). These 3 materials had higher adhesion values than the second control material, Sinfony. In comparison to the control materials, most provi-

Volume 98 Issue 6 sional fixed prosthodontic materials had a low or moderate adhesion tendency for S. mutans. In general, when the different classes of provisional materials were compared, bisacrylate composite resins and acrylic PMMAs had significantly lower adhesion potentials than improved methacrylates. In this context, the adhesion of S. mutans may be influenced by the composition of the investigated provisional materials. The organic matrix is based on methacrylate systems with variations in molecular weight and chemical backbone structure. Primary and side chains, as well as resulting polymerization rates, may influence wettability and water uptake. The content and type of inorganic fillers may also have direct influence on the surface of the restoration. Unfortunately, the exact compositions of the materials are generally proprietary and therefore inaccessible; thus, the conclusions concerning the interaction between bacterial adhesion and composition of the materials remain somewhat speculative. Differences in physico-chemical characteristics are the reason some materials are more prone to bacterial adhesion and plaque formation than others. Surface roughness and surface free energy are the 2 most important determinants of bacterial adhesion.6,12 Furthermore, it is well known from various in vivo and in vitro studies3,9,11,12 that different susceptibilities to S. mutans adherence are primarily caused by variable surface roughness values. Microscopic examinations indicate cracks, grooves, and pits in substrata are responsible for initial bacterial attachment.10 In order to minimize the effect of surface roughness on the quantity of adhesion in this study, all surfaces were polished equally. Quirynen et al28 indicate no correlation between surface roughness and quantity of bacterial adhesion for roughness values below 0.2 μm. The results from the present study confirm the hypothesis that median roughness values varied between 0.04 and 0.0756 μm, whereas no cor-

The Journal of Prosthetic Dentistry

relation between surface roughness and quantity of bacterial adhesion could be found. Additionally, there seems to be an interaction between surface roughness and hydrophobicity, or rather, surface free energy.13,14 Therefore, dominant and variable roughness values would complicate the interpretation of the influence of hydrophobicity values on the quantity of bacterial adhesion. The surface free energy of certain substrata can be evaluated by measuring hydrophobicity values, which strongly influence affinity for bacterial adhesion.4,7,12,16 The sessile drop method, which involves measuring contact angles, is an established method for obtaining hydrophobicity values. Contact angles of the assessed materials were significantly different, ranging between 46.5 and 71.0 degrees. In this method, high contact angles indicate hydrophobic surfaces. Remarkably, the hydrophobicity of the PMMAs and improved methacrylates (median values of contact angles from 62.0 to 71.0 degrees) were higher than the hydrophobicity of bisacrylate composite resins (median values of contact angles from 46.5 to 55.0 degrees), excluding Luxatemp Automix (median contact angle 65.0 degree). The scanning electron micrographs (Figs. 2 through 4) confirmed the results of the fluorescence adhesion tests. Bacterial colonization and more complex aggregates were found on materials with high relative fluorescence intensity, for example, UniFast LC (Fig. 4). Nevertheless, SEM data should be interpreted carefully since it allows no differentiation between viable and dead bacteria and only reproduces specific sections of a whole specimen. A bacterial monolayer was found on all assessed specimens, indicating bacterial adhesion rather than bacterial accumulation. In contrast to the in vitro situation, the process of bacterial adhesion to any surface within the human oral cavity is influenced by various collateral parameters, such as coadhesion to other bacterial or fungal species,

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December 2007 the acquired pellicle formed by salivary proteins, and the shear force of the floating saliva.17 Therefore, flow chambers and specimens precoated with saliva are used to mimic intraoral conditions in adhesion testing.19 These techniques were not used in the present study, because their use makes the interpretation of results more complicated. However, future studies should include such approaches and might therefore lead to reliable and interpretable in vivo investigations and possibly to the development of dental materials with low susceptibility to adherence to oral pathogens.

CONCLUSIONS Within the limitations of this study, bacterial adhesion on the 3 classes of material assessed differed significantly. Bisacrylate composite resins and acrylic PMMAs showed significantly lower bacterial adhesion potentials than improved methacrylates (6 pairs, α=.0083; 28 pairs, α=.0018). A correlation between quantity of bacterial adhesion and surface roughness and the surface hydrophobicity was not found, due to the low roughness values and the comparable hydrophobicity values (contact angles) of all materials.

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