Key words:
Acidic drinks - esthetic restorative materials - glass ionomer cement -
Streptococcus mutans
INTRODUCTION
The use of resins based materials for direct and indirect restorations has now entered
the daily clinical practice; parallel to restorative composite resins, only for direct
restorations, glass ionomer cements (GIC) are used for their self adhesive capacity
and antibacterial ability.[1]
[2]
[3]
[4]
One of the problems concerning adhesive restorative procedures is represented by the
secondary infiltration of the restoration. This is due to the presence of a marginal
gap between the filling and the tooth surface. This occurrence is more frequent among
GIC. Moreover, it is one of the most likely causes of resurfacing of previous fillings.[5]
[6] One of the biggest problems for these materials is the marked susceptibility to
the accumulation of bacterial plaque.[7]
[8] In the execution of direct restorations, due to the shrinkage stress of polymerization,
it is possible the genesis of marginal discrepancies in which the cariogenic bacteria
contained in plaque can give rise to a secondary infiltration.[9]
[10] An increase in plaque retention places patients at risk for secondary caries adjacent
to the composite resin margins, and additionally, the formation of biofilm may result
in gingival inflammation.[11] For indirect restorations, this issue, even if present, is limited to the cement
adhesive layer.
The strong tendency to accumulate plaque for composite materials thus plays a central
role in the formation of secondary caries.[12]
[13] The formation of oral plaque on the surface of restorative materials, and in general
on the surface of the teeth, is a complex process that is determined both by the salivary
and bacterial conditions of the oral cavity, but also by the eating habits and the
surface on which it is formed.[14]
[15] Moreover, common acidic beverages (cola, energy drinks) can produce erosion of restorative
materials.[16] The distribution and form of the fillers, the composition of the resin matrix, and
the silane surface treatment of the filler particles significantly affect the surface
degradation of the composite materials.[17] Composites are complex heterogeneous materials formed from a resin base in which
are embedded the filler particles, coated with a bonding agent, of different origin
with variable size and shape. This implies that the surface of such materials can
be a heterogeneous interface of particles distributed on different physical chemical
phases: commercially, available restorative composite resins will present different
surfaces in terms of roughness and polishing depending on the filler used.[18]
[19]
[20]
[21] At the same way, GIC are heterogeneous in terms of formulation, and in addition,
the release of fluoride ions causes a further deterioration of the surface that over
time will facilitate the adhesion of bacterial plaque.[1] However, GIC are generally used where additional protection against tooth decay
is required, especially in children, since potentially reduce microleakage adhering
to tooth structure, inhibit the growth of cariogenic bacteria, and neutralize the
acids produced by these bacteria by releasing ions.[22]
[23] The surface morphology of the restoration therefore plays a crucial role in adherence
of bacterial plaque, both for composite resins and GIC.[24]
[25]
[26]
The purpose of this research was to evaluate and compare bacterial adhesion on five
esthetic restorative materials and three GIC after acidic drink exposure. The null
hypothesis of the study was that there is no significant difference in bacterial adhesion
values among the different restorative materials after exposure to acidic drink.
MATERIALS AND METHODS
Specimens’ preparation
Five esthetic restorative materials and three GIC were evaluated in this study [Table 1].
Table 1:
Esthetic restorative materials specified for direct restorations used in this study
|
Material
|
Manufacturer
|
Type
|
LOT
|
|
Filtek Supreme XTE
|
3M ESPE, St Paul, MN, USA
|
Nanofilled composite
|
N801824
|
|
Ceram.X Universal
|
Dentsply De Trey, Konstanz, Germany
|
Nanohybrid composite with prepolymerized fillers
|
1608000937
|
|
Essentia
|
GC Corporation, Tokyo, Japan
|
Microfilled hybrid composite
|
1601121
|
|
Admira Fusion
|
Voco, Cuxhaven, Germany
|
Nanohybrid ormocer-based composite
|
1630296
|
|
Estelite Asteria
|
Tokuyama Dental Corporation, Tokyo, Japan
|
Supranano spherical hybrid composite
|
066E16
|
|
ChemFil Rock
|
Dentsply De Trey, Konstanz, Germany
|
Glass ionomer
|
1607000503
|
|
Equia Forte Fil
|
GC Corporation, Tokyo, Japan
|
Glass ionomer
|
150810A
|
|
IonoStar Plus
|
Voco, Cuxhaven, Germany
|
Glass ionomer
|
1631408
|
|
Equia Forte Fil + Equia Forte Coat
|
GC Corporation, Tokyo, Japan
|
Glass ionomer + multifunctional monomer coating
|
150810A
|
|
IonoStar Plus + Easy Glaze
|
Voco, Cuxhaven, Germany
|
Glass ionomer + nano-filled coating
|
1631408
|
Materials were polymerized into silicon rings (external diameter 9 mm, internal diameter
6 mm, thick 2 mm) to obtain identical specimens.[27] Cavities of these rings were slightly overfilled with material, covered with Mylar
Matrix Strip (Henry Schein, Melville, NY, USA), pressed between two glass plates and
polymerized for 40 s on each side using a curing unit (Celalux II, Voco, Cuxhaven,
Germany). One light polymerization mode was used for each material standard: 1000
mW/cm2 for 40 s. The light was placed perpendicular to the specimen surface, at distance
of 1.5 mm or less.[27] The upper surface of each specimen was then polished with fine and superfine polishing
disks (Sof Lex Pop On; 3M ESPE, St. Paul, MN, USA) to simulate clinical conditions.
Thirty cylindrical specimens of each material were prepared in this manner. After
polymerization and during the experimentation, the specimens were stored in distilled
water at 37°C and 100% humidity. Each material was tested 4 weeks after polymerization.
Immersion in acidic drink
Each material is represented by 30 specimens. The 30 specimens of each esthetic restorative
materials were randomly attributed to three groups (n = 10): specimens of Group 1 were used as control, specimens of Group 2 were immersed
in 50 ml of acidic drink (Coca Cola/Coca Cola Company, Milano, Italy) for 1 day, and
specimens of Group 3 were immersed in 50 ml of acidic drink (Coca Cola/Coca Cola Company,
Milano, Italy) for 7 days. The specimen of the given material pertaining to a specific
group was removed from the beverage using tweezers, sterilized in autoclave at 121°C
and packed in dry plastic sterile bags before being tested with bacteria.[27]
[28]
Bacterial growth condition
A strain of Streptococcus mutans (CCUG35176) obtained from the culture collection of the University of Göteborg was
used for the in vitro adhesion tests. S. mutans was cultured in Brain Heart Infusion (BHI, Difco, CA, USA) supplemented with 10%
(v/v) heat inactivated horse blood serum (Oxoid, Milan, Italy) to improve its growth.
The culture of S. mutans was statically incubated under aerobic conditions for 16 h at 37°C. This culture,
used as source for the experiments, was reduced at a final density of 1 × 1010 cells/mL as determined by comparing the OD600 of the sample with a standard curve
relating OD600 to cell number.[29]
Assessment of bacterial adhesion
After extensive washing of each materials, 100 μL of an overnight growth culture (107 bacteria/mL) was seeded onto each sample test placed at the bottom of a 24 well plate
(Celbio, Milan, Italy) and incubated at 37°C for 4 h in static conditions. The choice
of this time of incubation is due to the fact that biofilm formation in the oral cavity
normally occurs in 2–4 h. After incubation, loosely adhering bacteria were removed
by gently washing the samples tests with PBS. Three sample tests of each experimental
condition were used for total viable count (TVC). Briefly, the samples with bacterial
cells were dispersed into 1 mL sterile Ringer solution (Oxoid, Milan, Italy) by vortex
for 3 min.[10] Serial dilutions of the bacterial cells suspensions were prepared, and 0.1 mL of
each dilution was deposited onto BHI agar (Bacto agar, Difco, CA, USA) plates. The
plates were incubated for 24–48 h at 37°C and the number of colonies counted. Mean
TVC values were calculated for each sample, and the results are expressed as colony
forming units (CFU) per mL.[30]
Statistical analysis
First, data were assessed to be normal by means of Shapiro–Wilk normality test. The
analysis of variance (differences among substrates at each condition and differences
about treatment, per substrate) was carried out using two way ANOVA followed by Bonferroni’s
post hoc tests. Analyses were performed using Prism 4.0 (GraphPad Software, La Jolla, CA,
USA). Two tailed P = 0.05 were considered statistically significant.
RESULTS
To evaluate the S. mutans ability to adhere to different restorative materials with or without soft drink treatment,
a TVC assay was performed. The results are shown in [Table 2] and collectively represented in [Figure 1].
Figure 1: Colony forming units values of Streptococcus mutans cells adherent to restorative materials. Results were expressed as colony forming
units/mL
Table 2:
Colony-forming unit values of Streptococcus mutans cells adherent to restorative materials
|
Material
|
Mean of bacterial adhesion±SD
|
|
Control
|
After 1 day in soft drink
|
After 1 week in soft drink
|
|
Results were expressed as mean of bacterial adhesion±SD. §No significant difference
among data. SD: Standard deviation
|
|
Filtek Supreme XTE
|
1.50±0.70×102
|
3.450±0.21×103
|
2.0325±0.75×104
|
|
Ceram.X Universal
|
1.075±0.74×103
|
2.625±0.41×103
|
1.7425±0.26×104
|
|
Essentia
|
4.050±0.14×103§
|
5.175±0.32×103§
|
1.3800±0.64×104
|
|
Admira Fusion
|
1.50±0.42×102
|
4.075±0.46×103
|
1.1200±0.58×104
|
|
Estelite Asteria
|
5.50±0.31×102
|
8.300±0.60×103§
|
1.2600±0.78×104§
|
|
ChemFil Rock
|
2.550±0.35×103
|
2.5375±0.45×104§
|
1.9925±0.24×104§
|
|
Equia Forte Fil
|
4.825±0.51×103
|
1.3325±0.67×104
|
4.1750±0.87×104
|
|
Iono Star Plus
|
3.225±0.84×103
|
1.8400±0.41×104§
|
1.3725±0.53×104§
|
|
Equia Forte Fil + Equia Forte Coat
|
1.00±0.23×102
|
2.2075±0.75×104§
|
2.3125±0.75×104§
|
|
IonoStar Plus + Easy Glaze
|
1.100±0.49×103
|
4.275±0.36×103
|
1.4800±0.34×104
|
The bacterial adherence values to the untreated materials (the control) are ranging
from 102 to 5 × 103 CFU. Filtek Supreme XTE, Admira Fusion, and Equia Forte Fil + Equia Forte Coat exhibited
the lowest bacterial adhesion values, whereas the CFU values for Ceramχ, Estelite
Asteriam and Iono Star + coat were statistically significant (P < 0.05) and higher than the materials above mentioned. The highest bacterial adhesion
values were showed for Essentia, ChemFil Rock, Equia Forte Fil, and IonoStar Plus
(P < 0.05).
An increase in S. mutans cells adhesion to Coca Cola treated samples was observed. Moreover, the treatment
of Filtek Supreme XTE, Ceram • χ Universal, Essentia, Admira Fusion, Equia Forte Fil, and IonoStar Plus + coat with
acidic drink for long time (7 days) increases their susceptibility to be colonized
compared to the same samples treated for a shorter time (24 h) (P < 0.05). After 24 h acidic drink treatment, Filtek Supreme XTE, Ceram • χ Universal,
Essentia, Admira Fusion, and IonoStar Plus + IonoStar Plus Coat showed the lowest
values of bacterial adhesiveness (ranging from 2625 to 5175 × 103 CFU) (P < 0.05), without differences among themselves (P > 0.05). Instead, Estelite Asteria, ChemFil Rock, Equia Forte Fil, IonoStar Plus,
and Equia Forte Fil + Equia Forte Coat showed the highest values (from 083 to 253
× 104 CFU) (P < 0.05), without differences among themselves (P > 0.05). No significant difference between samples treated for 7 days with acidic
drink was observed (P > 0.05).
DISCUSSION AND CONCLUSIONS
DISCUSSION AND CONCLUSIONS
Bacterial plaque is an aggregate of microorganism that adheres tenacely to each other
and to the dental surfaces; it is a biofilm wrapped in an extracellular polymer matrix
secreted by the bacterial flora of the oral cavity. After only few minutes of brushing,
the teeth are covered with a film obtained from a layer of salivary mucoproteins that
is rapidly colonized by the microorganisms present in the oral cavity: complex processes
such as salivary pellicle formation, pellicle adsorption to the surface, passive transport
of bacteria to the pellicle surface, coadhesion, and multiplication are the protagonists
of oral plaque formation.[31] Modern restorative materials appear less affine to bacterial biofilm, consequently
reducing and delaying oral plaque formation.[10]
During the initial stages of colonization, quantitatively, the bacterial adhesion
to the restorative materials is related to the intrinsic physicochemical properties
of restorative materials; however, also, the types of bacteria present in the biofilm
and the active and passive adhesion mechanisms affect the development of oral plaque.[32]
[33] The growth and development of dental biofilm can be stimulated both in vivo and in vitro: to conduct the study analyzing simultaneously all the material in similar condition,
it was decided to proceed by an in vitro research.[31] An in vitro biofilm model allows to the use of aseptic and removable samples, which should be
discrete, representative, and reproducible. Extended exposure to soft drink was used
to deteriorate and alters the surface of the materials tested in this study and creates
a more suitable substrate for the formation and adhesion of bacterial plaque; the
use of acidic soft drink is served to mimic the aging process of restorative materials
that inevitably occur in the oral environment: the result is a degradation of the
surface layer of the restorative material with consequent creation of a rougher surface.
Regarding the glass ionomer group, no significant reduction in bacterial adhesion
was recorded, even though they are known for their significant release of fluoride.
On the contrary, all the GIC of the study appeared as the most adhesive surfaces.
This behavior can be explained by the reduced contact time between bacteria and GIC.
As investigated in other studies, the fluoride released by these materials requires
a variable time span (from 48 h to 7 days) to correctly express its antibacterial
activity.[33]
[34] The objective of this in vitro study was to evaluate and compare bacterial adhesion in relation to the different
types of materials and the timing of exposure of such materials to acidic drink. The
results of this investigation suggest that surface morphology and roughness of restorative
materials is critical for bacterial adhesion and a correlation can be established.
In Group 1, differences among materials were recorded. the highest values were recorded
for GIC that appear to be rougher than composites.[35] The use of coat reduces adhesion values in the control group, but the action of
acids alters its surface very quickly, raising the values of adhesion to the amount
of the other materials in Groups 2 and 3. All materials have demonstrated a statistically
significant increase in terms of bacterial adhesion after exposure to acidic drink,
which in most cases increases with exposure to harmful factor. Within the limits of
the present in vitro study, bacterial adhesion seems to be related to erosion of restorative materials
caused by acids.
Financial support and sponsorship
Nil.
