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 materi-
als results in gingival inflammation and secondary caries.

Purpose. The purpose of this in vitro study was to compare 10 commonly used provisional fixed prosthodontic ma-
terials (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 hydrophobic-
ity.

Material and methods. Surface roughness was assessed by perthometer and hydrophobicity by contact angle mea-
surements. 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 Mann-
Whitney 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 fluores-
cence 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 cor-
relation between bacterial adhesion and surface roughness or hydrophobicity was not confirmed. Bisacrylate compos-
ite resins and acrylic polymethyl methacrylates had significantly lower adhesion potentials than improved methacry-
lates. (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.

a
Postdoctoral Research Fellow, Department of Prosthetic Dentistry.
b
Engineer, Department of Prosthetic Dentistry.
c
Professor and Chairman, Department of Prosthetic Dentistry.
Buergers et al
462
The attachment of certain micro-
organisms 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 den-
ture stomatitis, gingival inflamma-
tion, and secondary caries, which
may consequently lead to unhealthy
complications.1,2 Microbiological ad-
hesion testing has primarily focused
on restorative materials such as amal-
gam, glass ionomers, and composite
resins.3-5 In contrast, fewer studies on
prosthodontic and implant materials
have been published, and investiga-
tions 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 lat-
est class of materials is formed by bi-
sacrylate composite resins, which are
comparable to composite resins used
for direct restoration therapy. They
consist of an organic matrix and inor-
ganic fillers. These provisional materi-
als are exposed to bacterial coloniza-
tion to a greater degree than definitive
restorations due to the higher surface
roughness and, generally, to an infe-
rior fitting interface. This is especially
true when they are worn for an ex-
tended period of time.
The quantity and quality of bac-
terial accumulation on specific sub-
strata is determined by variable sur-
face characteristics.8 High surface
roughness values, meaning surfaces
with pits and grooves, significantly
promote adhesion of bacteria by re-
ducing the influence of shear forces
on initially attaching bacteria.9,10 Sub-
strata with high surface free energy
values are known to enhance adhe-
sion of bacteria.11-14 In addition, the
chemical composition of a material,
The Journal of Prosthetic Dentistry
its zeta potential, and the surface hy-
drophobicity strongly influence the
bacterial adhesion process.8,12,15,16 An
increased zeta potential, which refers
to the electrostatic potential generat-
ed by the accumulation of ions on the
surface, results in decreased bacterial
attachment.16 Generally, hydrophobic
microorganisms prefer hydrophobic
substrata, and bacteria with hydro-
philic properties prefer hydrophilic
materials.8,12 Moreover, bacterial ad-
hesion 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 Strepto-
coccus mutans (S. mutans), are known to
play an important role in the patho-
genesis 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 elec-
tron microscopy, radiolabelling, and
direct plate counting, as described by
An et al.8
As a rapid, reproducible, and sim-
ple assay for the precise quantification
of adhering bacteria, fluorometric
techniques have recently gained in-
creasing recognition.18,20,21 In the pres-
ence of viable microorganisms, non-
fluorescent starting substances are
metabolized to fluorescent markers,
which can then be recorded by micros-
copy or an automated fluorescence
reader. In the case of the fluorescence
dye, Alamar Blue, nonfluorescent re-
sazurin is reduced to fluorescent reso-
rufin (highly fluorescent).22 The exact
mechanism of this nontoxic reduction
reaction is assumed to occur intracel-
lularly 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 asso-
ciated amount of living organisms is
used for the quantification of adher-
ing bacteria.23,25,26
The purpose of this in vitro study
was to observe the adhesion poten-
Volume 98 Issue 6
tials of 10 commonly used provisional
fixed prosthodontic materials and the
particular class to which they belong.
The growth and plaque-forming abili-
ties of Streptococcus mutans on these
different materials were investigated
with a spectrofluorometric method
in combination with scanning elec-
tron microscopy. Furthermore, the
influence of specific physico-chemical
surface characteristics (physical con-
figuration, surface roughness, and
hydrophobicity) on the susceptibility
to adhere to S. mutans was investigat-
ed. Finally, the hypothesized correla-
tion between surface roughness and
quantity of bacterial adhesion was
examined. The research hypothesis
was that different provisional fixed
prosthodontic materials would ex-
hibit 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 informa-
tion. All tested materials are commer-
cially available and widely used. They
were chosen without any particular
rationale, but each class of material
was represented. In addition, 2 con-
trol materials were used. Glass (Paul
Marienfeld GmbH, Lauda-Koenigs-
hofen, Germany) is generally consid-
ered to be extremely smooth and often
used in bacterial adhesion studies.27
The second control material, Sinfony
(3M ESPE, St. Paul, Minn) is a popu-
lar 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 cus-
tom metal mold with calibrated circu-
lar holes. Each material was prepared
according to the manufacturer’s in-
structions, inserted into the mold,
and covered immediately with 2 glass
slides (Alfred Becht GmbH, Offen-
burg, Germany) on the top and bot-
tom to prevent formation of an oxy-
Buergers et al
December 2007 463

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

Surface Contact
Material Brand Name Roughness Angle
Class (Lot Numbers) Manufacturer (µm) (Degrees)

Acrylic PMMA Cronsin Merz Dental GmbH, 0.08 (0.04/0.08) 63.0 (60.0/68.0)
(01110358/0107104) Lutjenburg, Germany

Trim Harry J. Bosworth Co, 0.08 (0.08/0.08) 71.0 (70.0/72.0)

(0112652/0011680) Chicago, Ill

Improved Snap Coltene/Whaledent AG, 0.08 (0.08/0.08) 62.0 (61.0/64.0)

methacrylates (11406/92911) Altstatten, Switzerland

UniFast LC GC Corp, Tokyo, Japan 0.04 (0.04/0.06) 67.0 (62.0/68.0)

(0005121/0604101)

Bisacrylate Protemp 3 Garant 3M ESPE, St. Paul, Minn 0.04 (0.04/0.06) 55.0 (52.0/58.0)
composite resins (B 279384)

Luxatemp Automix DMG, Hamburg, Germany 0.04 (0.04/0.04) 65.0 (64.0/68.0)

(511431)

Temphase Kerr Corp, Orange, Calif 0.04 (0.04/0.04) 53.0 (50.0/55.0)

(003742)

Structur Premium VOCO, Cuxhaven, Germany 0.04 (0.04/0.08) 50.5 (48.0/53.0)

(711470)

PreVISION CB Heraeus Kulzer, 0.04 (0.04/0.04) 48.0 (47.0/51.0)

(135072) Hanau, Germany

CronMix K plus Merz Dental GmbH 0.04 (0.04/0.06) 46.5 (43.0/48.0)

(01420081)

Control material 1 Glass Marienfeld, < 0.01 44.5 (44.0/46.0)

Lauda-Koenigshofen, Germany

Control material 2 Sinfony 3M ESPE 0.04 (0.04/0.08) 45.0 (42.0/48.0)

(159764)

gen-inhibited layer. UniFast LC was
additionally light polymerized for 1
minute on both sides using a light-po-
lymerization unit (Heliolux DLX1; Ivo-
clar 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-
Buergers et al
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 ma-
terial. Materials with roughness val-
ues below 0.2 μm were regarded as
smooth, since no further direct influ-
ence 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 hydro-
phobicity of all test and control ma-
terial surfaces was evaluated by mea-
suring distilled water contact angles.
Disks were cleaned with acetone (Ar-
cos 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). Precise-
ly 30 seconds after careful deposition
of the drop with a syringe, contact
angles were measured with a goni-
ometer (G1; ERNA, Tokyo, Japan) at
25°C room temperature by using the
horizontal projection technique. The
4 measured contact angles per speci-
men 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 min-
utes 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 fluo-
rescence 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 auto-
mated multidetection reader (FLUO-
star Optima; BMG Labtech, Offen-
burg, Germany) at wavelengths of
530 nm excitation and 590 nm emis-
sion. One ml of PBS was added to
each well and the autofluorescence
was subsequently determined. The
buffer was then removed, 1 ml of bac-
terial 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-
The Journal of Prosthetic Dentistry
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 determi-
nation of fluorescence after bacterial
adhesion was performed as described
above. The fluorescence of pure phos-
phate buffered saline (0-control), of
buffer and resazurin (dye-control),
and of pure bacterial solution (bac-
teria-control) served as control refer-
ences.
Five specimens of each provisional
fixed prosthodontic material were ad-
ditionally used for scanning electron
microscopy (SEM) verification. The
specimens with the adhering bacteria
were rinsed in PBS, fixed with metha-
nol, and air dried. The test specimens
were then mounted on aluminum
stubs and sputter-coated with 99.99%
gold (PROVAC, Balzers Corp, Liech-
tenstein). Specimens were examined
with a scanning electron microscope
(magnification x1700) (Stereoscan
240; Cambridge Instruments, Cam-
bridge, UK). The area covered with
adhering bacteria was marked and
quantified with an image analysis pro-
gram (Optimas 6.2; Media Cybernet-
ics, Bethesda, Md). The Mann-Whit-
ney 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 methac-
rylates, 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 measure-
ment. Presented are the median values
and the 25th and 75th percentiles. All
10 materials assessed showed simi-
lar median roughness values, ranging
between 0.04 and 0.08 μm. There-
Volume 98 Issue 6
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 methacry-
late material UniFast LC had the sig-
nificantly smoothest surfaces, while
acrylic PMMAs and the improved
methacrylate Snap had rougher sur-
faces (Table II).
Table I additionally presents the
median values and 25th and 75th per-
centiles from the contact angle mea-
surements. The median contact angles
of the tested provisional fixed prosth-
odontic materials ranged between
46.5 and 71.0 degrees. In general,
there were significant differences be-
tween contact angles of test materials
and controls (66 pairs; α=.00076). All
tested provisional fixed prosthodontic
materials had significantly higher con-
tact angles than control material glass
(44.5 degrees). Not considering Luxa-
temp 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 ad-
hesion 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 ap-
proximately 3000 and 16,000. The
lowest fluorescence with median val-
ues below 5000, indicating low bacte-
rial adhesion, were found for Cronsin
(median value of relative fluorescence
intensity: 4321), Protemp 3 Garant
(4273), and Luxatemp (3286). Mod-
erate 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 in-
tensities, with median values of over
10,000. No significant differences (6
pairs; α=.00076) could be found be-
tween the relative fluorescence inten-
Buergers et al
December 2007 465

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)

Uni- Protemp Pre- Cron-

Fast 3 Luxa- Tem- Structur VISION Mix
Cronsin Trim Snap LC Garant temp phase Premium CB K plus Glass Sinfony

Cronsin — .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 .258 .014 .014 1.000 — .631 .004 .143 .075 .002 .393 .009
Garant

Luxatemp .050 <.0001* <.0001* .436 .436 — .015 .105 .023 .004 .190 .009
Automix

Temphase .113 .004 .004 .730 .730 .730 — .247 .393 .436 .075 .631

Structur .730 .113 .113 .436 .436 .113 .258 — .853 .063 .481 .218
Premium

PreVISION .113 .004 .004 .730 .730 .730 1.000 .258 — .165 .218 .481
CB

CronMix .258 .014 .014 1.000 1.000 .436 .730 .436 .730 — .015 .796
K plus

Glass <.0001* <.0001* <.0001* <.0001* <.0001* <.0001* <.0001* <.0001* <.0001* <.0001* — .123

Sinfony .730 .113 .113 .436 .436 .113 .258 1.000 .258 .436 <.0001* —

*Significant difference

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

Uni- Protemp Pre- Cron-

Fast 3 Luxa- Tem- Structur VISION Mix
Cronsin Trim Snap LC Garant temp phase Premium CB K plus Glass Sinfony

Cronsin — .001 .703 .304 .001 .305 .001 <.0001* <.0001* <.0001* <.0001* <.0001*

Trim — — <.0001* .001 <.0001* <.0001* <.0001* <.0001* <.0001* <.0001* <.0001* <.0001*

Snap — — — .043 <.0001* .013 <.0001* <.0001* <.0001* <.0001* <.0001* <.0001*

UniFast LC — — — — <.0001* .970 <.0001* <.0001* <.0001* <.0001* <.0001* <.0001*

*Significant difference
Buergers et al
466 Volume 98 Issue 6

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

Uni- Protemp Pre- Cron-

Fast 3 Luxa- Tem- Structur VISION Mix
Cronsin Trim Snap LC Garant temp phase Premium CB K plus Glass Sinfony

Protemp 3 — — — — — <.0001* .254 .028 .005 <.0001* <.0001* <.0001*

Garant

Luxatemp — — — — — — <.0001* <.0001* <.0001* <.0001* <.0001* <.0001*

Automix

Temphase — — — — — — — .357 .060 .0001* .0001* .0001*

Structur — — — — — — — — .300 .017 .021 .015

Premium

PreVISION — — — — — — — — — .063 .005 .049

CB

CronMix — — — — — — — — — — .535 .849

K plus

Glass — — — — — — — — — — — .679

Sinfony — — — — — — — — — — — —

*Significant difference

40
Relative Fluorescence Intensity

30

20

10

0
in
m
Un Snap
p 3 st LC
Lu rant

uc emp p
Pr e Pr e m s e
Cr ISIO m
xK B
s
s
y
Plu
as
f on
tem

C
s
Tri

iu
ha
on

Gl
N
Ga

Sin
tem ifa

xa
Cr

mi
T
tur

V
on
Pro

Str

1 Relative fluorescence intensity (no unit) of 10 provisional fixed

prosthodontic materials and 2 control materials (median; 25%/75%).

The Journal of Prosthetic Dentistry Buergers et al

December 2007 467
sities of the materials with low fluo-
rescence values (Cronsin, Protemp 3
Garant, Luxatemp) and the control
glass (4644). Only Snap showed sig-
nificantly higher fluorescence inten-
sity 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 methacry-
lates had significantly higher relative
fluorescence values than the other
material classes; there was no signifi-
cant difference between acrylic PM-
MAs and bisacrylate composite resins 2 Scanning electron micrograph of S. mutans adhered to Cronsin
(Table II). There was neither a correla- (x1700 magnification).
tion between the amount of bacterial
adhesion and the surface roughness,
nor between quantity of bacterial ad-
hesion 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 coloniza-
tion pattern on all assessed material
surfaces, with a varying number of
adhering microorganisms. A bacte-
rial monolayer was observed on all
examined surfaces, indicating bacte-
rial adhesion rather than bacterial ac-
cumulation. Single streptococci and
small aggregates of organisms were 3 Scanning electron micrograph of S. mutans adhered to Temphase
found on the materials with low ad- (x1700 magnification).
hesion values, Cronsin (Fig. 2), Luxa-
temp, 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 Premi-
um, Trim, and PreVISION CB. Larger
and structured bacterial aggregates
with a high number of bacteria dem-
onstrated heavy bacterial adhesion to
UniFast LC (Fig. 4), Snap, and Cron-
Mix K plus.

DISCUSSION

The results of the present study

support the hypothesis that provi-
4 Scanning electron micrograph of S. mutans adhered to UniFast
sional fixed prosthodontic materials
LC (x1700 magnification).
may differ in their susceptibility to
Buergers et al
468
adhere to streptococcal bacteria. The
assumed influence of certain surface
characteristics could not be con-
firmed.
Several in vitro models to quan-
tify adhering microorganisms on den-
tal substrata have been established
and applied in the past.19 Recently,
fluorescence techniques have re-
sulted in more rapid and reproduc-
ible quantification procedures with a
minimized number of potential mea-
surement 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´)-car-
boxyfluorescein-acetomethyl ester to
hydroxylapatite using a spectrofluor-
ometer, which accelerates and simpli-
fies 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 cor-
relation 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 mor-
phology and material surfaces or the
interactions between them.19 In this
study, scanning electron micrographs
were used for supplementary verifica-
tion of the results from fluorescence
adhesion tests.
Bacterial adhesion, visualized by
relative fluorescence intensity, varied
significantly between the assessed
materials (Table III). Only 3 materi-
als (Cronsin, Protemp 3 Garant, and
Luxatemp) were ranked below control
material glass, indicating a low sus-
ceptibility to adherence by S. mutans.
Slightly higher fluorescence intensities
were measured on 4 materials (Trim,
Temphase, Structur Premium, and
PreVISION CB) and significantly high-
er intensities were found on 3 materi-
als (Snap, UniFast LC, and CronMix
K plus). These 3 materials had higher
adhesion values than the second con-
trol material, Sinfony. In comparison
to the control materials, most provi-
The Journal of Prosthetic Dentistry
sional fixed prosthodontic materials
had a low or moderate adhesion ten-
dency for S. mutans. In general, when
the different classes of provisional
materials were compared, bisacrylate
composite resins and acrylic PMMAs
had significantly lower adhesion po-
tentials than improved methacrylates.
In this context, the adhesion of S. mu-
tans may be influenced by the compo-
sition of the investigated provisional
materials. The organic matrix is based
on methacrylate systems with varia-
tions in molecular weight and chemi-
cal backbone structure. Primary and
side chains, as well as resulting po-
lymerization rates, may influence wet-
tability and water uptake. The content
and type of inorganic fillers may also
have direct influence on the surface of
the restoration. Unfortunately, the ex-
act 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 bacte-
rial 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 sus-
ceptibilities to S. mutans adherence are
primarily caused by variable surface
roughness values. Microscopic exami-
nations indicate cracks, grooves, and
pits in substrata are responsible for
initial bacterial attachment.10 In or-
der to minimize the effect of surface
roughness on the quantity of adhe-
sion in this study, all surfaces were
polished equally. Quirynen et al28 in-
dicate 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 me-
dian roughness values varied between
0.04 and 0.0756 μm, whereas no cor-
Volume 98 Issue 6
relation between surface roughness
and quantity of bacterial adhesion
could be found. Additionally, there
seems to be an interaction between
surface roughness and hydrophobic-
ity, 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 mea-
suring hydrophobicity values, which
strongly influence affinity for bacte-
rial adhesion.4,7,12,16 The sessile drop
method, which involves measur-
ing 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 de-
grees. In this method, high contact
angles indicate hydrophobic surfaces.
Remarkably, the hydrophobicity of
the PMMAs and improved methac-
rylates (median values of contact an-
gles from 62.0 to 71.0 degrees) were
higher than the hydrophobicity of bis-
acrylate 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 micro-
graphs (Figs. 2 through 4) confirmed
the results of the fluorescence adhe-
sion tests. Bacterial colonization and
more complex aggregates were found
on materials with high relative fluores-
cence 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 re-
produces specific sections of a whole
specimen. A bacterial monolayer was
found on all assessed specimens, indi-
cating bacterial adhesion rather than
bacterial accumulation.
In contrast to the in vitro situa-
tion, the process of bacterial adhesion
to any surface within the human oral
cavity is influenced by various collat-
eral parameters, such as coadhesion
to other bacterial or fungal species,
Buergers et al
December 2007
the acquired pellicle formed by sali-
vary proteins, and the shear force of
the floating saliva.17 Therefore, flow
chambers and specimens precoated
with saliva are used to mimic intra-
oral 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 susceptibil-
ity to adherence to oral pathogens.
CONCLUSIONS
Within the limitations of this study,
bacterial adhesion on the 3 classes of
material assessed differed significant-
ly. 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 bac-
terial adhesion and surface roughness
and the surface hydrophobicity was
not found, due to the low roughness
values and the comparable hydropho-
bicity values (contact angles) of all
materials.
REFERENCES
1. Deligeorgi V, Mjor IA, Wilson NH. An
overview of reasons for the placement and
replacement of restorations. Prim Dent
Care 2001;8:5-11.
2. Okte E, Sultan N, Dogan B, Asikainen S.
Bacterial adhesion of Actinobacillus acti-
nomycetemcomitans serotypes to titanium
implants: SEM evaluation. A preliminary
report. J Periodontol 1999;70:1376-82.
3. Eick S, Glockmann E, Brandl B, Pfister W.
Adherence of Streptococcus mutans to vari-
ous restorative materials in a continuous
flow system. J Oral Rehabil 2004;31:278-
85.
4. Satou J, Fukunaga A, Satou N, Shintani
H, Okuda K. Streptococcal adherence on
Buergers et al
various restorative materials. J Dent Res
1988;67:588-91.
5. Svanberg M, Mjor IA, Orstavik D. Mutans
streptococci in plaque from margins of
amalgam, composite, and glass-ionomer
restorations. J Dent Res 1990;69:861-4.
6. Sardin S, Morrier JJ, Benay G, Barsotti O. In
vitro streptococcal adherence on prosthetic
and implant materials. Interactions with
physicochemical surface properties. J Oral
Rehabil 2004;31:140-8.
7. Grivet M, Morrier JJ, Benay G, Barsotti O.
Effect of hydrophobicity on in vitro strep-
tococcal adhesion to dental alloys. J Mater
Sci Mater Med 2000;11:637-42.
8. An YH, Friedman RJ. Concise review of
mechanisms of bacterial adhesion to
biomaterial surfaces. J Biomed Mater Res
1998;43:338-48.
9. Morgan TD, Wilson M. The effects of sur-
face roughness and type of denture acrylic
on biofilm formation by Streptococcus
oralis in a constant depth film fermentor. J
Appl Microbiol 2001;91:47-53.
10.Nyvad B, Fejerskov O. Scanning electron
microscopy of early microbial colonization
of human enamel and root surfaces in vivo.
Scand J Dent Res 1987;95:287-96.
11.Taylor RL, Verran J, Lees GC, Ward AJ. The
influence of substratum topography on
bacterial adhesion to polymethyl methacry-
late. J Mater Sci Mater Med 1998;9:17-22.
12.Quirynen M, Bollen CM. The influence of
surface roughness and surface-free energy
on supra- and subgingival plaque forma-
tion in man. A review of the literature. J Clin
Periodontol 1995;22:1-14.
13.Busscher HJ, van Pelt AWJ, de Boer HP, de
Jong HP. The effect of surface roughening
of polymers on measured contact angles of
liquids. Colloids and Surfaces 1984;9:319-
31.
14.Quirynen M. The clinical meaning of the
surface roughness and the surface free
energy of intra-oral hard substrata on the
microbiology of the supra- and subgingival
plaque: results of in vitro and in vivo experi-
ments. J Dent 1994;22 Suppl 1:S13-6.
15.Carlen A, Nikdel K, Wennerberg A, Hol-
mberg K, Olsson J. Surface characteristics
and in vitro biofilm formation on glass
ionomer and composite resin. Biomaterials
2001;22:481-7.
16.Weerkamp AH, Uyen HM, Busscher HJ.
Effect of zeta potential and surface energy
on bacterial adhesion to uncoated and
saliva-coated human enamel and dentin. J
Dent Res 1988;67:1483-7.
17.Whittaker CJ, Klier CM, Kolenbrander PE.
Mechanisms of adhesion by oral bacteria.
Annu Rev Microbiol 1996;50:513-52.
18.Gaines S, James TC, Folan M, Baird AW,
O‘Farrelly C. A novel spectrofluoromet-
ric microassay for Streptococcus mutans
adherence to hydroxylapatite. J Microbiol
Methods 2003;54:315-23.
469
19.An YH, Friedman RJ. Laboratory methods
for studies of bacterial adhesion. J Micro-
biol Methods 1997;30:141-52.
20.Grivet M, Morrier JJ, Souchier C, Barsotti
O. Automatic enumeration of adherent
streptococci or actinomyces on dental alloy
by fluorescence image analysis. J Microbiol
Methods 1999;38:33-42.
21.Logan RP, Robins A, Turner GA, Cockayne
A, Borriello SP, Hawkey CJ. A novel flow
cytometric assay for quantitating adherence
of Helicobacter pylori to gastric epithelial
cells. J Immunol Methods 1998;213:19-30.
22.Fields RD, Lancaster MV. Dual-attribute
continuous monitoring of cell prolif-
eration/cytotoxicity. Am Biotechnol Lab
1993;11:48-50.
23.O‘Brien J, Wilson I, Orton T, Pognan F.
Investigation of the Alamar Blue (resa-
zurin) fluorescent dye for the assessment of
mammalian cell cytotoxicity. Eur J Biochem
2000;267:5421-6.
24.de Fries R, Mitsuhashi M. Quantification
of mitogen induced human lymphocyte
proliferation: comparison of alamarBlue
assay to 3H-thymidine incorporation assay.
J Clin Lab Anal 1995;9:89-95.
25.Voytik-Harbin SL, Brightman AO, Waisner
B, Lamar CH, Badylak SF. Application and
evaluation of the alamarBlue assay for cell
growth and survival of fibroblasts. In Vitro
Cell Dev Biol Anim 1998;34:239-46.
26.Nakayama GR, Caton MC, Nova MP, Pa-
randoosh Z. Assessment of the Alamar Blue
assay for cellular growth and viability in
vitro. J Immunol Methods 1997;204:205-8.
27.Tanner J, Vallittu PK, Soderling E. Ad-
herence of Streptococcus mutans to an
E-glass fiber-reinforced composite and
conventional restorative materials used in
prosthetic dentistry. J Biomed Mater Res
2000;49:250-6.
28.Quirynen M, Bollen CM, Papaioannou W,
Van Eldere J, van Steenberghe D. The influ-
ence of titanium abutment surface rough-
ness on plaque accumulation and gingivitis:
short-term observations. Int J Oral Maxil-
lofac Implants 1996;11:169-78.
Corresponding author:
Dr Ralf Buergers
Department of Prosthetic Dentistry
Regensburg University Medical Center
Franz-Josef-Strauss-Allee
Regensburg, D-93042
GERMANY
Fax: 49-941-944-6171
E-mail: ralf.buergers@klinik.uni-regensburg.de
Acknowledgements
The authors thank Mrs Gerlinde Held for tech-
nical assistance.
Copyright © 2007 by the Editorial Council for
The Journal of Prosthetic Dentistry.