Bacterial Adhesion and Biofilm Formation on
Direct, Tooth-Colored Restorative Materials:
An in vitro Study
Nathaniel Denson1, Martha Wells1, David A Tipton2, Franklin Garcia Godoy2 and Jegdish P Babu2*
1Department of Pediatric Dentistry, University of TN Health Science Center, USA
2Department of Bioscience Research, University of TN Health Science Center, USA
Submission: March 30, 2018 ; Published: April 11, 2018
*Corresponding author: Jegdish P Babu, Professor, Department of Bioscience Research, College of Dentistry, University of TN Health Science Center, Memphis, TN, USA, Tel: (901)4484342; Email: email@example.com
How to cite this article: Nathaniel D, Martha W, David A T, Franklin G G, Jegdish P B. Bacterial Adhesion and Biofilm Formation on Direct, Tooth-Colored
Restorative Materials: An in vitro Study. Adv Dent & Oral Health. 2018; 8(3): 555736. DOI: 10.19080/ADOH.2018.08.555736
Dental restorative materials are routinely used to restore carious lesions, but over time, they may fail, leading to secondary dental caries. The longevity of restorations appears to depend upon their resistance to bacterial adhesion and biofilm formation.
Objective:To investigate susceptibility of four restorative composite materials to bacterial colonization and biofilm formation.
Methods:20 circular discs (8x2mm) of four restorative materials, Esthet-X ® HD (Dentslpy), Filtek™ Bulk Fill (3M ESPE), Fuji II® LC (GC America), and Activa™ BioActive-Restorative™ (PulpDent) were prepared and de-contaminated. Streptococcus mutans ATCC 700610, and mixed bacterial oral plaque, were cultured for 24 h, and bacteria were suspended to 1x107 cells/ml. For adhesion assays, quadruplicate composite discs were incubated with one ml S. mutans for 24 h. Biofilms of S. mutans and mixed bacterial plaque were grown on quadruplicate discs by inoculating them with one ml of bacterial suspension and incubated for 3 weeks. In both assays the bacterial number on each disc was determined by MTT assay.
Results:Fuji and Activa had greater percentages of adherent bacteria (22.8 ± 3.9 and 18.94 ± 4.7, respectively) than Esthet-X (8.12 ± 1.22) and Filtek (5.6 ± 0.94) (p<0.03). Both Fuji and Activa also supported significantly greater (p<0.002) biofilm growth than Esthet-X and Filtek.
Conclusions:Composite materials appear to differ in their ability to facilitate bacterial adhesion and biofilm formation. The differences in bacterial biofilm formation and retention on the surfaces of the restorative materials demonstrated in this study may be helpful to dentists in selecting restorative composite materials for dental restorations.
A permanent, esthetic restorative material that could be placed directly in the mouth to restore cavities in teeth has long been an important contribution in restorative dentistry and oral health. In fact, the uses of direct esthetic restorations have overtaken amalgam, becoming the most common treatment for minimally invasive dental procedures . These esthetic composite materials can be placed as temporary, intermediate, or permanent restorations . However, the term “permanent restoration” can be deceiving, as no dental restoration is truly permanent. Restorations have a limited lifespan which is primarily based on the material used, although other factors contribute to failure such as individual’s age, oral hygiene and risk of caries as well as the skill of the dentist placing technique-sensitive materials [2,3].
One of the primary causes of failure of a composite restoration is secondary or recurrent caries. Recurrent caries occurs when
a restoration leaks and allows the formation of a cavity beneath
the existing restoration. This process requires a susceptible restoration along with bacterial adhesion and accumulation. Different restorative materials have properties such as surface roughness and antibacterial components that modulate this process. However, previous in vitro research has shown that demineralization depth and degradation of the restorative material is bacteria-dependent. Restoration longevity, therefore, is linked to susceptibility towards bacterial colonization [4-6].
Bacterial colonization in the mouth leads to the creation of a biofilm. The formation of superficial biofilm on a dental surface is a complex phenomenon and different key factors are involved . First, formation of salivary pellicle on the biomaterial by adsorption of host saliva proteins . The next step involves the adhesion of the microbial cells, when bacteria begin to anchor. At this stage, the colonization of the surface takes place, as described
by Hannig . This adhesion of the bacteria to the salivary pellicle is
critical for plaque formation . In the plaque, minimal numbers
of specific bacteria must be present for the cariogenic process
to occur . It is not certain how much of a role the restorative
material plays, but different studies suggest that several materials
may have antibacterial activity or may even induce the growth
of several bacteria . Evidence show that there is a need for
development of alternative resins that do not enhance bacterial
growth leading to secondary caries and restorative failure, and the
need to evaluate biofilm formation on the new resins .
Despite numerous studies on the morphology of oral biofilms
only limited information is available on bacterial adhesion,
especially on the surface of new restorative materials. Still less
is known about bacterial adherence on new bioactive materials
which have recently been proposed as restorative materials
with enhanced biological, physical and mechanical properties
compared to traditional composites [13,14]. There is also little
information on bacterial adhesion to restorative materials after
application of a salivary pellicle. This is essential to mimic the
oral environment, as previous research shows that presence of a
human salivary pellicle significantly affects biofilm formation on
many types of restorative materials [4,15-17].
Restorative materials are susceptible to bacterial biofilm
formation, and this affects the integrity of the materials and
ultimately oral health. Biofilm formation appears to be influenced
by hydrophobic and hydrophilic interactions between the bacteria
and the restorative material surfaces. Additionally, restorative
material’s chemical composition and surface smoothness also
play roles in biofilm formation . The aim of this study
was to evaluate Streptococcus mutans and mixed bacterial
plaque bacterial biofilm formation on four different unpolished
restorative materials following the application of a salivary
pellicle. The major bacteria species in areas of carious lesions are
different from those in areas without caries . In this study,
cariogenic bacteria, S. mutans and the mixed bacterial plaque
which represent the average human oral flora, were employed to
test for adhesion and biofilm formation by these microorganisms
on the restorative materials, Fuji II LC, Esthet-x HD, Filtek Bulk Fill,
and Activa BioActive.
The hypothesis tested in the present study was that differences
in the chemical nature and morphological properties of dental
restorative materials affect the formation of microbial biofilms
on them, and that some materials may actually promote bacterial
The four restorative materials, Fuji II LC (GC Corporation,
Tokyo, Japan), Esthet-x HD (Dentsply Caulk, Milford, DE), Filtek
Bulk Fill (3M ESPE, St. Paul, MN), and Activa BioActive (PulpDent
Corporation, Watertown, MA) were made into multiple discs. The
restorative materials were placed into round metal molds (8 mm
by 2mm) and light cured for 20 seconds to form discs. The discs
were decontaminated by soaking in 70% ethanol for 10 minutes
and placing them under a UV light for 24 hours. The sterility of
discs was confirmed by incubating the discs in Todd-Hewitt Broth
(THB; Difco, MI, USA) for 24 hours at 37°C. The discs were rinsed
with sterile PBS and then each disc was incubated with 0.5 ml of
human clarified, pooled saliva (see below) for 24 hours. The salivacoated
discs were rinsed with PBS and then used in the study.
Paraffin stimulated whole human saliva (Protocol approved
by the University IRB) was collected from a single laboratory
individual in a cup placed in ice. The saliva was then centrifuged
(8,000 x g) for 10 min, and then the supernatant was collected and
kept frozen until needed for the study
Streptococcus mutans 700610 obtained from ATCC (Rockville,
MD) were grown in THB for 24 hours at 37°C. A standard
suspension (1x107 cells/ml) of bacteria was prepared. Five
discs prepared from each restorative material were incubated
with 1.0 ml of bacterial suspension in a 48-well culture dish for
24 hours at 37°C, and then rinsed with PBS to remove the nonadherent
bacteria. The discs were then incubated with 0.3 ml
of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) labeling reagent (Sigma Aldrich Chemical Co. St. Louis,
MO) for 4 hours and then the solubilizing agent supplied by the
manufacturer was added and incubated overnight. An aliquot (0.1
ml) of supernatant from each disc was placed in a 96-well flat
bottom microtiter plate and optical density reading was read at
570 nm using a BMG Spectrostar spectrophotometer (BMG labtech
Inc. Cary, NC). The background absorbance measured at 690 nm
was subtracted. Discs incubated with THB media alone served as
negative control. The method is based on the conversion of water
soluble MTT compound to an insoluble formazan product. Viable
cells with active metabolism convert MTT into formazan, however,
dead cells lose this ability. The number of adherent bacteria on
each disc was determined by comparing the absorbance value to
the values obtained from the standard curve prepared with known
Saliva coated restorative discs were incubated with 1.0 ml of
standard suspension of S. mutans or with a mixed bacterial plaque
sample and incubated for 21 days at 37°C. During this period,
every 48 hours 0.1 ml of supernatant media was discarded and
replaced with fresh THB, in order to provide nourishment for
the growing biofilm. The dental plaque was obtained from three
laboratory individuals (Approved by the University IRB) and
pooled. The pooled plaque sample was grown in Todd-Hewitt
broth (THB) for 24 hours at 37°C and used in the study. Gram stain of the culture revealed both gram positive and gram negative
cocci and bacillus. No effort was made to grow the anaerobes
from the pooled plaque sample. At the end of 21 days, discs were
rinsed with Phosphate Buffered Saline (PBS) to remove un-bound
bacteria and the bacterial load on each disc was assessed using the
MTT reagent as described above.
All experiments were performed a minimum of three times,
with five samples at each time. The data were expressed as the
means ± SD and were analyzed using a one-way ANOVA and
Scheffe’s F procedure for post hoc comparisons, using STATVIEW®-
software (SAS Institute, Cary, NC, USA). The significance level
adopted was 5% (P < 0.05).
The saliva-coated composite discs (5 from each group) were
tested for adhesion of S. mutans 700610 by incubating the discs
with 1x107 bacteria/ml for 24 hours at 37°C in a 48-well culture
plate. The results (Figure 1) demonstrate the differences in S.
mutans adherence to the various restorative composite discs.
Significantly higher bacterial adhesion was found on the Fuji II LC
(P < 0.026), and Activa BioActive discs (P < 0.031) compared to
Esthet-X and Filtek Bulk Fill discs. On an average, 18,253 ± 985
bacteria 15,152 ± 750 bacteria adhered to Fuji II LC and Activa
BioActive composite discs, respectively, while the numbers of
bacteria on Esthet-X and Filtek Bulk Fill were 6,495 ± 495 and
4,793 ± 440, respectively (Figure 1).
Over all higher numbers of tested bacteria of the three week
old biofilms were found on all composite discs than the numbers
we found in adhesion assay. Three- week biofilm study with S.
mutans and mixed bacterial plaque also showed results similar
to adhesion assay (Figure 2). Significantly higher numbers of S.
mutans biofilm bacteria were found on Fuji II LC (24,972 ± 1,880;
P<0.018) and Activa BioActive discs (21,055 ± 1,650; P<0.026)
compared to the numbers on Esthet-X (9,192 ± 580) and Filtek
Bulk Fill (8,173 ± 520). The mixed plaque biofilm bacterial
numbers were higher on all composite discs than the single S.
mutans. The results (Figure 2) show that on an average 41,065
± 3,750 bacteria on Activa BioActive, and on Fuji II LC 43,470 ±
4,955 composite discs, respectively. In comparison, only 12,040 ±
1,250 and 11, 735 ± 1,250 bacteria were found on Esthet-X and
Filtek Bulk Fill, respectively (Figure 2). The result of this study
delineates the differences between composite materials with
respect to microbial colonization on their surfaces.
Several factors have been shown to be important in oral
bacterial adhesion and biofilm formation on hard dental
surfaces and restorations. Certain restorative composites may
have properties that make them more susceptible to dental
biofilm formation which can lead to secondary caries and even
periodontitis. Various in vitro studies have demonstrated that
oral bacteria attachment and biofilm formation occur at a higher
frequency on composites than on the natural teeth in the oral
Biological interactions of microorganism with dental
restorative materials play a crucial role in determining the
successful function of these materials. The aim of the present
study was to compare the in vitro bacterial adhesion and biofilm
formation on four widely used dental restorative materials.
Circular un-polished discs were light cured under similar
conditions, so that differences in adhesion and biofilm formation
would result from the properties and composition of the material
itself. The discs were coated with whole human saliva to simulate
conditions in the oral cavity. Such experiments were conducted
successfully in our laboratory previously .
Our results showed lower bacterial adhesion and biofilm
formation on Filtek discs followed by Esthet-x discs. Bulk fill
composites are newer materials that have limited studies
investigating their properties , but no studies to the knowledge
of the investigators have described biofilm formation on these
materials, especially with mixed bacterial plaque organisms.
Filtek bulkfil has been shown to have spherical particles ranging
from 0.1 to 4.0 micro meters while Esthet-x has particles sizes
of approximately 2.5 micro meters. Filler particle and surface
roughness has been shown to influence bacterial adhesion with
materials with small filler size having lower bacterial adherence.
The Fuji and Activa discs harbored the highest bulk of biofilm, and
facilitated adhesion of S. mutans (Figures 1 & 2). Less bacterial
adhesion to Esthet-x and Filtek discs may be due to the filler
size, which is different from that of Fuji and Activa. Fuji II has
particle sizes of approximately 5.9 micro meters while Activa is
uncharacterized in this regard.
While filler size may be important, these results clearly
demonstrate the nature of composite materials plays a role in the
microbial bioactive interaction. Other studies have shown that
the surface properties of a material, such as hydrophobicity and
ion release, influence bacterial adhesion and biofilm formation.
Higher numbers of bacteria associated with ionomer-containing
restorations were also reported by other investigators . A
higher degree of bacterial biofilm formation on the Fuji discs may
be due to the positive surface charge of this material, which may
facilitate bacterial colonization. Other investigators have shown
that more bacteria adhere to positively charged composites .
Brambilla et al.  showed the same results, that surface charge
of the dental material plays a vital role in microbial adhesion. It
may also be possible that Fuji and Activa adsorb more salivary
receptor molecules than the other two materials tested, however
we did not characterize these potential differences in this study.
Within the limitations of this study, our findings demonstrated
differences in cariogenic bacteria adhesion and biofilm formation
by mixed bacteria obtained from human plaque between four
different restoration materials. However, other factors such
as surface roughness, and preferential adsorption of salivary molecules, which may also affect these processes, were not
investigated in this study. If the integrity of the composite is not
compromised, then it would be beneficial to choose a restoration
which does not favor microbial colonization.
The study demonstrated the influence of nature and type
of dental restorative materials on bacterial colonization by
cariogenic and plaque microorganisms. The result of the study
highlights the differences between restorative materials and may
aid the Dentists in choosing the restorative material which does
not favor the microbial colonization, thus preventing the failure of