Kirthika et al, J Dent Res Dent Clin Dent Prospects, 2021, 15(1), 16-21

doi: 10.34172/joddd.2021.004
TUOMS
[Link] PRESS

Original Article

Comparative evaluation of compressive and flexural strength, fluoride

release and bacterial adhesion of GIC modified with CPP-ACP, bioactive
glass, chitosan and MDPB
ID
Natarajan Kirthika1* , Sampath Vidhya2, Venkatappan Sujatha2, Sekar Mahalaxmi2, Renganathan Senthil Kumar3
1
Department of Conservative Dentistry and Endodontics, Karpaga Vinayaga Institute of Dental Sciences, Tamil Nadu, India
2
Department of Conservative Dentistry and Endodontics, SRM Dental College, SRM Institute of Science and Technology,
Chennai, India
3
Department of Conservative Dentistry and Endodontics, Adhiparasakthi Dental College & Hospital, Melmaruvathur, India

ARTICLE INFO Abstract

Article History: Background. This study evaluated the incorporation of casein phosphopeptide-amorphous
Received: 11 Aug. 2020 calcium phosphate (CPP-ACP), calcium sodium phosphosilicate bioactive glass (BAG), chitosan
Accepted: 26 Oct. 2020 (CH), and methacryloyloxydodecylpyridinium bromide (MDPB) on the compressive and flexural
ePublished: 13 Feb. 2021 strength, fluoride (F‒) release, and bacterial adhesion of conventional glass-ionomer cement
(C-GIC).
Keywords: Methods. Modifications were implemented by adding CPP-ACP, BAG, and CH to the glass
Bacterial adhesion powder, while MDPB-GIC was prepared by incorporating MDPB to the liquid of C-GIC.
Bioactive glass Custom-made molds were used for specimen preparation. Compressive and flexural strengths
Chitosan were evaluated using a universal testing machine. F‒ release was calculated with Erichrome
CPP-ACP cyanide reagent, using UV-spectrophotometry, at two time intervals of 24 hours and seven
Fluoride release days. For bacterial adhesion, the test specimens were exposed to the bacterial suspension of
Glass-ionomer cement Streptococcus mutans and Lactobacillus acidophilus for 4 hours, and the adherent bacteria were
MDPB quantified using colorimetry as the optical density (OD).
Results. The incorporation of MDPB increased the flexural strength of C-GIC, with no effect
on its compressive strength. CH significantly improved the compressive and flexural strength;
modifications with CPP-ACP, BAG, and MDPB significantly improved the flexural strength of
C-GIC. While MDPB-GIC released significantly higher F‒ at 24 hours, CPP-ACP- and BAG-
modified GICs were comparable to C-GIC on day 7. C-GIC exhibited the highest bacterial
adhesion, and MDPB-GIC showed the least. The data were analyzed with one-way (ANOVA),
and pairwise comparisons were made with Tukey HSD tests.
Conclusion. Hence, it can be concluded that the incorporation of CPP-ACP, BAG, and CH
improved the mechanical properties of C-GIC, whereas MDPB improved the resistance of
C-GIC to bacterial adhesion.

Introduction of thermal expansion, which is similar to that of tooth

Recurrent caries has been the most frequent cause structure, make it suitable for a wide variety of clinical
of failure of dental restorations. Fluoride-releasing applications. Despite these advantages, GIC has certain
restorative materials were introduced to overcome this drawbacks, such as brittleness and porosity, which result
disadvantage. Among the various commercially available in poor mechanical properties, such as low wear resistance
fluoride-releasing materials, glass-ionomer cement (GIC) and fracture toughness.2
has the highest fluoride release. Resin-modified glass- The composition of GIC has been experimented with
ionomer cement (RMGIC), compomer, and alkasite, the incorporation of a wide variety of biologically active
or ion-releasing composite have evolved over the years materials. Modifications of GIC with casein phosphopeptide-
to harness the advantages of composite resins and GIC. amorphous calcium phosphate nanocomplexes (CPP-ACP),
However, the fluoride release of these newer materials is calcium sodium phosphosilicate bioactive glass (BAG),
still less than GIC.1 Certain inherent properties of GIC, and chitosan (CH) have been reported in the literature.
such as anticariogenicity, biocompatibility, adhesion to The incorporation of CPP-ACP to GIC has shown that the
enamel, dentin, and composite, and its low coefficient localization of CPP to amorphous calcium phosphate of the

*Corresponding Author: Natarajan Kirthika, Email: [Link]@[Link]

© 2021 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (http://
[Link]/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited.
 Kirthika et al

tooth surface increases the anticariogenicity by maintaining from 1-bromododecane and 3-hydroxypyridine by the
a state of supersaturation concerning the tooth mineral. Its reflux method. Finally, MDPB was prepared by mixing
anticariogenic effect is further potentiated by its interaction the prepared hydroxy dodecylpyridinium bromide and
with fluoride (F‒) ions present in GIC, which produces a methacryloyl chloride at a ratio of 1:1. The obtained
stabilized amorphous calcium fluoride phosphate phase.2,3 product, MDPB, was further confirmed by an infrared (IR)
Petri et al showed that the incorporation of CH to spectrometer. The obtained IR spectrum of the prepared
conventional GIC (C-GIC) considerably improved its compound was compared with the standard compound,
flexural strength and F‒ release.4,5 The incorporation of and the frequencies were assigned (Figure 1).
BAG into GICs resulted in better dentin mineralization, as
it induced calcium phosphate precipitation on its surface Preparation of experimental materials
in contact with saliva.6 C-GIC (group I) was prepared by dispensing two scoops
Methacryloyloxydodecylpyridinium bromide (MDPB) of powder and two drops of liquid (Type II GIC, GC India
is a quaternary ammonium compound obtained by Dental Pvt. Ltd., India) on a moisture-impervious paper
substituting the hydroxyl group at the terminal end pad and mixing with an agate spatula in a folding motion
of hydroxy dodecylpyridinium bromide with the for 25‒30 seconds to obtain a paste-like consistency.
methacryloyl group. This increases the antibacterial Modifications of GIC with CPP-ACP (group II), BAG
activity of the parent compound. MDPB has been a (group III), and CH (group IV) were implemented by
breakthrough in the development of non-agent-releasing dispensing 180 mg of CPP-ACP paste (RecaldentTM,
antibacterial restoratives.7 Imazato et al confirmed the GC Corporation, Japan), BAG powder (45S5, IFGL Bio
bactericidal activity of MDPB-modified monomer against Ceramics Limited, Kolkata, India), and 36 mg of CH
seven species of oral streptococci.8 powder (Sigma-Aldrich Co. LLC, USA) to 180 mg of type
Despite improvements in the mechanical properties of II GIC powder on separate paper pads and mixing the
various modified formulations of GIC, bacterial adhesion ingredients thoroughly. To these powders, 120 mg of type
on its surface still remains a concern as it predisposes the II GIC liquid was added and mixed using an agate spatula
microenvironment at the tooth‒restoration interface to in a folding motion for 25‒30 seconds to obtain a paste-
secondary caries. F‒ release from GIC further deteriorates like consistency. MDPB-GIC (group V) was prepared
its surface integrity, favoring the adhesion of oral flora.9 by first mixing 60 mg of MDPB liquid and type II GIC
Several studies have shown that incorporating MDPB into liquid thoroughly. To this, 360 mg of type II GIC powder
resinous materials like bonding agents and composite was then incorporated and mixed to obtain a paste-like
resins, resulted in a significant decrease in recurrent consistency.
caries and bacterial adhesion to the biomaterial surface.10
No study has evaluated the effect of incorporating MDPB In vitro testing
on the mechanical properties, F‒ release, and bacterial Compressive and flexural strengths
adhesion of C-GIC. Hence, this in vitro study aimed to Compressive and flexural strength testing was carried
evaluate the effect of incorporating CPP-ACP, BAG, CH, out following ISO specifications 9917-1:2007 and
and MDPB on the compressive and flexural strength, 9917-2:2007, respectively. Stainless steel molds of two
F‒ release, and bacterial (Streptococcus mutans and dimensions (6 × 4 mm and 10 × 2 × 2 mm) were prepared
Lactobacillus acidophilus) adhesion of C-GIC. for compressive strength and flexural strength testing,
respectively. Fifteen specimens per group were prepared
Methods from the experimental materials for each strength test.
Preparation of MDPB During setting, the top and bottom portions of the molds
The chemicals used to prepare MDPB were of analytical were covered with Mylar strips to obtain a smooth surface.
grade. 1-bromododecane and 3-hydroxypyridine were
procured from Spectrochem Pvt. Ltd., Mumbai, India.
Hydroquinone, acrylic acid, and benzoyl chloride were
procured from Merck, Mumbai, India. MDPB was
synthesized by the reaction of hydroxy dodecylpyridinium
bromide and methacryloyl chloride.
0.72 g of acrylic acid was dissolved in 10 mL of distilled
ethanol. This solution was added to 1.4 g of benzoyl
chloride under 5 mL of 0.1 g of hydroquinone at a
temperature of 72‒76°C. The obtained product, acryloyl
chloride, was again dissolved in methanol and further
subjected to re-distillation at a temperature of 72‒76°C to
obtain methacryloyl chloride. Hydroquinone was added
as a catalyst to prevent the polymerization of acrylic Figure 1. The final product was confirmed as MDPB from the frequencies of
acid. Hydroxy dodecylpyridinium bromide was prepared infrared spectrophotometry.

J Dent Res Dent Clin Dent Prospects, 2021, Volume 15, Issue 1 17
Kirthika et al

After the initial setting, the test specimens were removed GIC, and CH-GIC were significantly higher than C-GIC
from the molds and stored in artificial saliva for 24 hours. and MDPB-GIC (P < 0.05). Among these three, CH-
They were then subjected to compressive and flexural GIC exhibited significantly higher values (P < 0.05). No
loading in a universal testing machine (Instron, Canton, significant difference was observed between the mean
USA) at a 1 mm/min crosshead speed. compressive strength values of C-GIC and MDPB-GIC
(P > 0.05) (Figure 2). The mean flexural strength of all
Fluoride release the modifications was significantly higher than C-GIC
Under ISO specification 19448:2018, five specimens (P < 0.05). Among the four modifications, MDPB-GIC
measuring 6 × 4 mm were prepared using stainless exhibited the least flexural strength (P < 0.05) (Figure 3).
steel molds for each group. After the initial setting,
the specimens were stored in 100 mL of deionized Fluoride release
distilled water at 37°C. F‒ release was evaluated without UV spectrophotometry used in this study to evaluate
replenishing the water, at two time intervals of 24 h F‒ release offers an advantage over other types as it
and seven days with UV spectrophotometry (K. Roy & determines only free fluoride without interference from
Co., India) using Erichrome cyanide (Medilab Exports any type of covalent bond with other ions. At 24 hours, all
Consortium, Haryana, India) as a reagent. the modifications showed significantly higher F‒ release
than C-GIC (P < 0.05). Furthermore, BAG-GIC exhibited
Bacterial adhesion tests significantly higher F‒ release than the other groups
For the bacterial adhesion tests, apart from the (P < 0.05) (Figure 4). On day 7, the means of F‒ release from
experimental groups, the polystyrene strip served as C-GIC, CPP-ACP-GIC, and BAG-GIC were significantly
a positive control (group VI), and plastic mesh served higher than CH-GIC and MDPB-GIC (Figure 4).
as a negative control (group VII). Twenty specimens
measuring 6 × 4 mm were prepared in each group and Bacterial adhesion
randomly divided into two subgroups (n = 10) each, based According to the results of this study, despite its fluoride-
on the two bacterial strains tested. After the initial setting, releasing properties, C-GIC showed significantly
the cement specimens were stored in artificial saliva for 24 higher bacterial adhesion (S. mutans, 2.05 ± 0.1 OD
hours. They were then sterilized in an autoclave at 121°C
for 15 minutes at 15-lbs pressure. Under sterile conditions,
each test material was placed in the well of a 12-well plate
and exposed to a standardized bacterial suspension (2 mL
of fresh broth and 20 µL of cell suspension) in brain heart
infusion (BHI) broth followed by incubation at 37°C for 4
hours. Reference strains of S. mutans and L. acidophilus,
the common cariogenic oral bacteria, were used. After 4
hours, the test materials were retrieved from the culture
broth and washed three times with 5 mL of sterile saline
solution to remove non-adhering cells. The test materials
were then suspended in glass tubes containing 1 mL
of saline solution, and the tubes were transferred to an
Figure 2. Comparison of mean compressive strengths (MPa) of all the groups.
ultrasonic bath cleaner fitted with a test tube holder,
Different alphabets indicate significant difference between the groups
operating at 47 kHz (234 W) and sonicated for 6 minutes to (P < 0.05). SD, standard deviation.
detach the adherent bacteria from the biomaterial surfaces,
bringing them into suspension. The test specimens were
then removed, and 10 mL of fresh broth was added to each
tube. The tubes were again incubated at 37°C for 24 hours.
After incubation, the concentration of the bacteria in the
broth was finally measured using colorimetry (K. Roy &
Co, India). The results were tabulated as optical density
(OD) values.

Statistical analysis
The data were analyzed using one-way ANOVA and Tukey
post hoc tests. Significance was set at P < 0.05.

Results
Figure 3. Comparison of mean flexural strengths (MPa) of all the groups.
Compressive and flexural strengths Different alphabets indicate significant difference between the groups
The mean compressive strength of CPP-ACP-GIC, BAG- (P < 0.05). SD, standard deviation.

18 J Dent Res Dent Clin Dent Prospects, 2021, Volume 15, Issue 1
 Kirthika et al

Figure 4. Comparison of mean fluoride release at 24 hours and 7 days.

Figure 5. Comparison of mean bacterial adhesion of Streptococcus mutans.

and L. acidophilus, 2.2 ± 0.1 OD) compared to all the

experimental groups, but less than the plastic mesh
(negative control). The adhesion of S. mutans (0.04 ± 0.01
OD) and L. acidophilus (0.04 ± 0.008 OD) on MDPB-
GIC was significantly less than all the other experimental
cements and even less than the polystyrene strip that
served as a positive control (0.09 ± 0.01 OD). CPP-ACP-
modified GIC exhibited significantly less adhesion of S.
mutans (1.5 ± 0.01 OD) and L. acidophilus (1.3 ± 0.09 OD)
than BAG-GIC, CH-GIC, and C-GIC. Adhesion of S.
mutans (1.5 ± 0.1 OD) and L. acidophilus (1.3 ± 0.03 OD)
on BAG-GIC was significantly less than C-GIC. CH-GIC
showed significantly less adhesion of S. mutans (1.7 ± 0.06 Figure 6. Comparison of mean bacterial adhesion of Lactobacillus acidophilus.

OD) and L. acidophilus (1.4 ± 0.01 OD) than C-GIC

(Figures 5 and 6).
movement of carboxylic acid groups to react with the ions
Discussion leached from the glass particles of GIC. Presumably, this
Compressive strength is indirectly related to flexural and freedom of the primary, secondary, and tertiary pendent
diametral tensile strengths in a complicated way. Although carboxylic acid groups improves the reactivity with Ca2+
direct measurements of tensile strength are inherently and Al3+ ions from the glass, resulting in greater filler‒
valid, problems arise during testing of brittle materials, polymer chemical reaction and increased homogeneity in
like GIC. For these reasons, it has been suggested that the the cement.7
measurement of flexural and compressive strengths offers This could be the reason why the carboxylic acid
the best practical and reliable estimate of tensile strength.7 groups present in long polymer chains of MDPB could
The increased compressive and flexural strengths of CH- have enhanced the formation of salt bridges, improving
GIC, compared to the other groups, could be attributed the flexural strength of the modified GIC in the present
to the presence of several hydroxyl and acetamide groups study, despite a reduction in the volume of PAA by half.
in CH chains, which might bind to the hydroxyl and However, the mechanism beneath this inference needs to
carboxylic groups of polyacrylic acid (PAA) in C-GIC. be studied in future experiments.
This could have reduced the interfacial tension between F‒ neither takes part in the acid‒base setting reaction
the glass-ionomer components, improving mechanical of GIC, nor is it incorporated into the matrix structure.11
performance.4,11 The glass particles of BAG adhere to The highest F‒ release in BAG-GIC at 24 hours could be
the GIC matrix, reinforcing the matrix by serving as attributed to the higher solubility and dissolution rate
filler components.12 C-GIC has a porous structure, with of hydroxycarbonate apatite [HCA, Ca10(PO4)6(OH)2],
the pores acting as stress concentration points. The which is formed on the surface of the BAG in an aqueous
incorporation of CPP-ACP nanoparticles leads to the medium.12,13 Similar to BAG, the incorporation of CH might
obliteration of these pores, resulting in a cross-linked have had a catalytic effect, facilitating the diffusion of F‒
matrix structure. This could lead to an improvement in through the cement matrix towards the external medium.
the mechanical properties of these modified cements.2 Additionally, the release of F‒ ions from the inorganic
The incorporation of an amino acid-derived monomer of matrix seems to be favored when reinforced complexes
MDPB into the PAA of GIC might improve the flexural have been formed.4 Reynolds reported that the release
strength of C-GIC, as shown in a study by Kao et al. It has of F‒ ions from CPP-ACP-modified GIC was promoted
been reported that the longer the polymer chains of amino by the formation of CPP-ACP nanocomplexes.12,14 The
acid-derived monomers, the greater is the freedom of same mechanism could have resulted in an increased F‒

J Dent Res Dent Clin Dent Prospects, 2021, Volume 15, Issue 1 19
Kirthika et al

release in CPP-ACP-GIC compared to C-GIC at both time exhibited higher F‒ release, but the adhesion of bacterial
intervals in the current study. MDPB-GIC showed no strains to these experimental restorative materials was
significant difference in its F‒ release when compared to much higher compared to MDPB-GIC, which showed the
C-GIC at both time intervals. Imazato et al added MDPB least F‒ release among the experimental groups.
to composite resin and reported that MDPB had an The various parameters in this study were tested
immobilized alkylpyridinium part, which was entrapped under controlled in vitro conditions. Caution should be
in the composite resin matrix.10 A similar entrapment exercised to extend these results to the clinical settings,
could have happened within the solid glass matrix of GIC, where the interplay of multiple factors and complex
with no interference with its F‒ release. environmental changes occur. Future studies should assess
Despite numerous studies on the morphology of the biocompatibility, mixing time, setting time, setting
oral biofilm and its effect on the tooth surface, only reaction, and adhesive properties of these experimental
limited information is available on bacterial adhesion GIC formulations before their effective clinical use. Their
to restorative materials, especially on the surface of F‒ durability in stress-bearing areas and individuals with
releasing restorative materials.14 Hence in this study, the high caries risk also need to be studied.
adhesion of S. mutans and L. acidophilus on the different
modifications of GIC was evaluated using in vitro Conclusion
adhesion tests. In the present study, the cements were The incorporation of CPP-ACP, BAG, and CH improved
in contact with the bacterial inoculum for 4 hours. This the compressive and flexural strengths of C-GIC. The
period was selected because complete biofilm formation incorporation of MDPB did not adversely affect the
in the oral cavity usually occurs in 2‒4 hours.15 mechanical properties and F‒ release but improved the
The increased surface roughness of GIC, coupled resistance of C-GIC to bacterial adhesion.
with the loss of surface integrity associated with fluoride
release, predisposes C-GIC to higher bacterial adhesion.16 Authors’ Contributions
In MDPB-GIC, the strong bactericidal action of MDPB is Conceptualization: NK and SV. Experimental work: NK. Original
draft preparation: NK, SV, VS, SM, and RS. All the authors have read
due to its cationic binding to the bacterial cell wall, which and agreed to the publication version of the manuscript.
disturbs the membrane integrity, subsequently leading to
leakage of cytoplasmic material and bacterial cell lysis.10 Acknowledgments
The polymer chains of MDPB could also have decreased The preparation part of MDPB was guided by Dr. B. Karthikeyan
the surface free energy of GIC, resulting in significantly PhD., the Department of Chemistry, Chidambaram, Tamil Nadu.
reduced bacterial adhesion.17 Reynolds showed that the
Funding
adsorption of CPP-ACP nanoparticles on the surface This research did not receive any specific grant from funding
of enamel increases the surface net negative charge of agencies in the public, commercial, or not-for-profit sectors.
enamel, influencing the long-term interactions with
microbes through the development of repulsive forces.12,14 Competing Interests
CPP-ACP has been shown to delay biofilm formation The authors deny any actual or potential conflicts of interest related
to the study.
by preventing cell-to-cell adhesion of bacteria. These
nanoparticles can also modify the long-term adhesion of Ethics Approval
streptococci by masking the streptococci-related receptors Not Applicable.
on salivary molecules.18 In BAG-GIC, the surface reaction
of BAG with an aqueous medium produces an alkaline References
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