Author’s Accepted Manuscript

Dentin pretreatment with 45S5 and niobophosphate

bioactive glass: Effects on pH, antibacterial,
mechanical properties of the interface and
microtensile bond strength

José Bauer, Allana Silva e Silva, Edilausson

Moreno Carvalho, Paulo Vitor Campos Ferreira,
Ceci Nunes Carvalho, Adriana Pigozzo Manso, www.elsevier.com/locate/jmbbm

Ricardo Marins de Carvalho

PII: S1751-6161(18)31025-7
DOI: https://doi.org/10.1016/j.jmbbm.2018.10.029
Reference: JMBBM3043
To appear in: Journal of the Mechanical Behavior of Biomedical Materials
Received date: 17 July 2018
Revised date: 22 October 2018
Accepted date: 23 October 2018
Cite this article as: José Bauer, Allana Silva e Silva, Edilausson Moreno
Carvalho, Paulo Vitor Campos Ferreira, Ceci Nunes Carvalho, Adriana Pigozzo
Manso and Ricardo Marins de Carvalho, Dentin pretreatment with 45S5 and
niobophosphate bioactive glass: Effects on pH, antibacterial, mechanical
properties of the interface and microtensile bond strength, Journal of the
Mechanical Behavior of Biomedical Materials,
https://doi.org/10.1016/j.jmbbm.2018.10.029
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Dentin pretreatment with 45S5 and niobophosphate bioactive glass: Effects on pH,
antibacterial, mechanical properties of the interface and microtensile bond strength
José Bauera,*, Allana Silva e Silva a, Edilausson Moreno Carvalho a, Paulo Vitor Campos
Ferreira b, Ceci Nunes Carvalho c, Adriana Pigozzo Manso d, Ricardo Marins de Carvalho d
a
University Federal of Maranhão (UFMA), Discipline of Dental Materials, School of
Dentistry, Av. dos Portugueses, 1966, Zip Code 65080-805, São Luis, Maranhão, Brazil.
b
University of Campinas (UNICAMP), Department of Restorative Dentistry, Dental
Materials Division, School of Dentistry, Av. Limeira, 901, Piracicaba 13414-903, SP, Brazil.
c
University Ceuma (UNICEUMA), Department of Restorative Dentistry, School of Dentistry,
R. Josué Montello, 1, Renascença II, Zip Code 65075-120 São Luis, Maranhão, Brazil.
d
University of British Columbia (UBC), Department of Oral Biological and Medical Sciences,
Division of Biomaterials, Faculty of Dentistry, 368-2199 Wesbrook Mall, Vancouver, British
Columbia V6T 1Z3, Canada.

*Corresponding author: José Bauer, Universidade Federal do Maranhão (UFMA),

Departamento de Odontologia I, Curso de Odontologia, Rua dos Portugueses, 1966, Campus
Universitário do Bacanga, Phone: +55 (98) 3272-9541, CEP: 65080-805, São Luís – MA.
bauer@ufma.br

Abstract:

Objectives:

The aim of the study was to evaluate the effect of bioactive glass (45S5 and NbG)

suspensions on bond strength (µTBS), hardness, modulus of elasticity, pH and antibacterial

activity of the resin-dentin interfaces after 3 months.

Methods:

Groups with different concentrations (5% and 20%) of two types of glass (45S5 and NbG),

and a control group (distilled water) were studied. Twenty-five extracted human third molars

were etched with phosphoric acid. The means of the two-way ANOVA and Holm-Sidak tests

(α=5%). The antimicrobial activity data were analyzed by the Kruskal-Wallis test (α=5%).

Results:

The interactions were significant among groups for µTBS (p=0.033). Significant reductions in

µTBS were observed after 3 months storage in PBS for the Control and 5% NbG Groups.
Suspensions with 5% and 20% 45S5 glass and 20% NbG resulted in stable µTBS values and

increased hardness after 3 months. Both 20% suspensions (45S5 and NbG) increased the

elastic modulus. A significant greater reduction in bacterial growth was observed with the use

of 20% 45S5.

Conclusion:

Rewetting dentin with the suspension of 20% 45S5 glass prevented the reduction in bond

strength; increased hardness; modulus of elasticity of the resin-dentin interface, and

demonstrated antibacterial activity against Streptococcus mutans.

Key Words: Adhesive, dentin, bioactive glass, microtensile, mechanical properties,

antibacterial activity.

1. Introduction
Recently, several studies have been published about the use of bioactive materials in
Dentistry (Crovace et al., 2016; Niu et al., 2012; 2014a; 2014b). Therefore, the traditional
inert fillers have been substituted by bioactive glass particles for the development of
restorative materials that favor remineralization, inhibition of enzymes, prevention of
bacterial growth and pH control (Osorio et al., 2012; Waltimo et al., 2007; Xu et al., 2011;
2009).
Several studies have attempted to incorporate bioactive particles into dental materials:
resin composites (Hyun and Ferracane, 2016; Hyun et al., 2015; Khvostenko et al., 2016;
2013), paste used for the treatment of hypersensitivity, glass ionomer cement (De Caluwé et
al., 2017), adhesive system (Bauer et al., 2016; Carneiro et al., 2018; Sauro et al., 2012c),
cements for pulp protection (Gandolfi et al., 2015; Prati and Gandolfi, 2015), endodontic
cements (Osorio et al., 2012) and gutta-percha (C. N. Carvalho et al., 2015). However, the
incorporation of bioactive particles into resin materials is complex, particularly in adhesive
systems in which the presence of acidic monomers may lead to reactivity with the glass
particles (Bauer et al., 2016). Therefore, the addition of bioactive glass to resinous materials
appears to result in controversial outcomes in terms of bond strength values (Bauer et al.,
2016; Carneiro et al., 2018; Profeta et al., 2013; 2012; Sauro et al., 2012a; 2012b), perhaps

2
because of the incompatibility between the highly reactive acidic monomers and the also
reactive glasses.
While the incorporation of bioactive glasses into adhesives can be challenging, the
direct application of bioactive glasses in an aqueous suspension to the dentin surface prior to
adhesive application may present more satisfactory results (Profeta et al., 2012). The particles
dispersed in water are capable of diffusing into the demineralized zone of dentin more
efficiently, providing intimate contact between the glass (Vollenweider et al., 2007) and the
sound dentin at the bottom of the demineralized zone, which is necessary for inducing
mineralization (Wang et al., 2011).
Some bioactive glasses have previously been used for remineralization of dentin,
when dispersed in aqueous solutions (Vollenweider et al., 2007) and for dentin rewetting
before adhesive procedures with promising results (Bauer et al., 2018). However, there is
little evidence about the effect of suspensions with different bioactive glasses (45S5 and
NbG) for rewetting dentin in conventional adhesive procedures and their impact on
mechanical properties and bond strength of the resin-dentin interface. In addition to function
as a remineralization agent, the presence of bioactive particles at the resin-dentin interface
may also control bacterial growth (Martins et al., 2011; Waltimo et al., 2007; 2009; Zehnder
et al., 2006) and prevent the recurrence of caries under the restorative material (T. K.
Vaidyanathan and J. Vaidyanathan, 2009).
Thus, the aim of this study was to evaluate the effect of suspensions of different
bioactive glasses used for rewetting dentin, on the microtensile bond strength, hardness and
modulus of elasticity of the resin-dentin interface after 3 months storage. In addition, the
antimicrobial properties of the suspensions were evaluated against Streptococcus mutans.
2. Materials and Methods
2.1. Bioactive Glasses
Two types of bioactive glasses were used: a commercial type (45S5, Sylc, OSspray
Ltd, London, United Kingdom) and an experimental glass based on niobophosphate (NbG),
prepared in accordance with previous studies (Bauer et al., 2018; C. N. Carvalho et al., 2015).
The NbG and 45S5 glasses were crushed in a vibrating system (8000M, Mixer/Mill, SPEX
SamplePrep, NJ, USA) with tungsten carbide grinding balls-vial set (SPEX SamplePrep, NJ,
USA) for 30 minutes. After grinding, the resultant powder passed through a series of sieves of
150μm - 75μm - 53μm - 38μm - 20μm (Hogentogler & Co., Inc, Columbia, MD, USA).

3
2.2. Preparation of the Suspensions
Four suspensions were prepared with different concentrations of bioactive glasses, and
one Control:
1. Control Group: Distilled water without glass particles
2. 5% NbG Suspension: 0.2 g NbG in 5 ml distilled water
3. 5% 45S5 Suspension: 0.2 g 45S5 in 5 ml distilled water
4. 20% NbG Suspension: 1 g NbG in 5 ml distilled water
5. 20% 45S5 Suspension: 1 g 45S5 in 5 ml distilled water
The suspensions were stirred for 48 hours (Model 210T, Thermix Fisher, Waltham,
MA, USA) and then allowed to sediment in sealed glass vials. Just before use, the vial
containing the suspension was stirred again for 10 seconds (Bauer et al., 2018).
2.3. pH measurement
Distilled water had its pH adjusted either to 4 with HCl or to 7 with NaOH. NbG and
45S5 glass particles were added at concentrations of 5 and 20% (as above). After mixing for
60 seconds, the pH change was monitored over different time intervals (1 minute, 1 hour, 24
hours, 48 hours and 7 days) using a pH meter (Quimis, Model Q838-F Diadema, SP, Brazil).
2.4. Antibacterial activity
The NbG and 45S5 powders were sterilized by autoclaving at 120°C for 15 min.
Aliquots of frozen stocks of Streptococcus mutans (UA159) were placed on Brain Heart
Infusion (BHI; Sigma-Aldrich, St Louis, MO, USA) agar plates and incubated at 37°C for 48
h. Colony-Forming Units (CFU) were collected and transferred to tubes containing BHI broth
supplemented with 1% sucrose and grown until the late exponential phase. In order to form a
microbial inoculum, the density of the suspension was adjusted to that of a standard solution
0.5 of the McFarland scale, resulting in a suspension with an approximate concentration of
108 CFU/mL. Aliquots of 100 µl of BHI supplemented with 1% sucrose were added to each
well of a sterile 96-well plate followed by 100 µl of the experimental suspensions and
controls (NbG 5%, NbG 20%, 45S5 5%, 45S5 20%, chlorexidine 0.12%, NaCl 0.9%) and 10
µl inoculum (n=6). The plate was incubated at 37°C for 24h. After 24h had elapsed, an
aliquot of 100 µl from each well was transferred to tubes containing 1ml of sterile 0.9% NaCl
and vortexed vigorously. Aliquots of these suspensions were serially diluted up to 10-8 and 2
drops of 10 µl of each dilution were inoculated on BHI agar (BD, Sparks, USA) to determine
the number of CFUs. The plates were incubated at 37°C in 10% CO2. for 48h. After 48h had
elapsed, CFU were counted under a stereomicroscope and the results were expressed as log10
CFU/mL.

4
2.5. Microtensile Bond Strength Test
Twenty-five sound human extracted third molars were collected and stored in a 0.5%
chloramine solution until they were used. The study protocol was submitted to the Research
Ethics Committee of the University for evaluation (UBC CREB: H13-01451).
Flat dentin surfaces were obtained after removing the occlusal enamel with a diamond
disc at low speed (Isomet 5000, Buehler, Illinois, USA) under water cooling. The roots were
removed by transverse section below the cement-enamel junction. The exposed dentin was
abraded (Unipol 1210, MTI Corporation, CA, USA) under water cooling with silicon carbide
abrasive papers of different granulations (#180 e #320) using each grain for 20s.
The teeth were randomly assigned into 5 different groups as previously described
(n=5). The dentin surface was etched with 35% phosphoric acid without BAC (Bisco,
Schaumburg, IL, USA) for 15s, washed with a jet of water for the same time and dried with
jets of air for 30s. The dentin was then rewetted with one of the four suspensions (or control)
for 10 s (Microbrush, Applicator regular – Ref X-80250P, Bisco, USA). A light jet of air was
applied to remove excess suspension, leaving the surface wet for bonding. Two consecutive
coats of the adhesive system (One Step, Bisco, Inc., Schaumburg, IL, USA) were applied on
the dentin with a microbrush for 10s, followed by a jet of air applied for 10s at a distance of
20cm, and light curing for 10s at 1500 mW/cm² (Bluephase 20i, Ivoclar Vivadent AG, Schaan,
Liechtenstein). After this, the surface was covered with composite resin (ÆLITE, Bisco, Inc.,
Schaumburg, IL, USA) in 2 mm increments, and polymerized for 40s. The bonded specimens
were placed in vials containing Phosphate Buffered Saline (PBS, Dulbecco’s Phosphate
Buffered Saline, Sigma Adrich, St Louis, MO, USA) and kept in an oven at 37°C for 24h.
The bonded teeth were sectioned along two perpendicular axes, perpendicular to the
resin-dentin interface, using a diamond disc under water cooling to obtain rectangular-shaped
test specimens with a cross-sectional area of approximately 1 mm2. The total number of test
specimens obtained from cutting the 5 teeth were equally divided for evaluation in two
different time intervals (24h) and after 3 months of storage in PBS.
Before performing the test, the cross-section of each test specimen was obtained by
measuring with a digital caliper (Absolute Digimatic, Mitutoyo, Tokyo, Japan), and then
fixed to the testing devices with cyanoacrylate glue in gel form (Pegamil Bond Gel, Buenos
Aires, Argentina). In the previously designated time interval, the test specimens were
submitted to the microtensile bond strength test in a universal test machine (Instron 3342,
Canton, MA, USA). The data noted were expressed in MPa (maximum load, in N/area of tests
specimens, in mm2).

5
 The fractured specimens were analyzed by stereomicroscope (Koso Optical and
Electronical Instrumental, Nanjing, Jiangsu, China) at 40X magnification. Fracture patterns
were classified as follows: 1) adhesive; 2) mixed at the resin-dentin interface; 3) cohesive in
dentin; 4) cohesive in resin composite. Two specimens from each group were randomly
selected to further examination by Scanning Electron Microscopy (TM3030, Hitachi, Japan).
2.6. Hardness and Modulus of Elasticity
For the hardness and modulus of elasticity tests, five teeth were used. Flat dentin
surfaces were obtained after removing the occlusal enamel with a diamond disc at low speed
under water cooling and the roots were removed by transverse section below the cement-
enamel junction. They were sectioned in the mesio-distal and vestibular-lingual directions to
obtain 20 fragments. The 20 fragments were distributed into 5 experimental groups (n=4).
The fragments were bonded in the same manner as for the µTBS test. The bonded
fragments were embedded in transparent acrylic resin (Orthodontic Resin, Dentsply Caulk,
Milford, DE, USA). After the resin had set, the resin-dentin interfaces were polished with
1200/2400-grit SiC paper and 1 μm diamond paste by using a polishing cloth (Unipol 1210,
MTI Corporation, CA, USA). The modulus of elasticity and hardness along the resin-dentin
interface were evaluated after 24 h and after 3 months storage in PBS in an oven at 37°C.
The measurements were obtained by using an ultra-microdurometer (DUH-211S,
Shimadzu Corporation, Kyoto, Japan). The indenter used was of the pyramidal-triangular type
(Berkovich indenter - 115°), with a maximum force of 19.6 mN. Each indentation was
performed with a load-unload cycle at a speed of 4.44 mN/s. The hold period at the maximum
load enabled creep around the contact site to ensure minimal creep during unloading and
thereby generate a reliable elastic modulus value from the slope of the unloading curve. The
software associated with the ultra-microdurometer analyzes the data and calculates the
hardness and the elastic modulus from the load – displacement curve recorded (Angker et al.,
2003). Four indentations were made in the hybrid layer (as close as possible to the adhesive
layer), in a straight line parallel to resin-dentin interface, with a constant distance of 5μm (±1)
between them. The modulus of elasticity and hardness across the resin-dentin interface were
evaluated with the specimens in a fully hydrated status.
2.7. Statistical Analysis
Statistical analysis was performed using the SigmaPlot 13 software (SigmaPlot v. 13.0,
Systat Software Inc., San Jose, USA). The normality and equality of variance assumptions
were statistically analyzed by Shapiro-Wilk test and Brown-Forsythe test. For antibacterial
activity the normality was violated, thus the data were analyzed by using the Kruskal-Wallis

6
 (post hoc Student-Newman-Keuls) and µTBS, hardness and modulus of elasticity data were
submitted to two-way ANOVA (suspension vs. time) and Holm-Sidak tests (=0.05).

3. Results
3.1. pH measurement
The pH results obtained demonstrated that 45S5 glass had a higher and faster capacity of
raising and maintaining the pH into highly basic conditions (pH around 11.0-12.0),
irrespective of the initial pH (4 or 7). Conversely, NbG glass was only able to raise the pH to
around 7-8 when the initial pH was 4.0 and no marked changes occurred when the initial pH
was 7.0 (Figure 1).

Figure 1 - pH changes over time for both glasses in different concentrations: (A) initial pH 4.0 and
(B) initial pH 7.0.

3.2. Antibacterial activity

The 45S5 glass presented superior antibacterial activity against Streptococcus
mutans. At the concentration of 20%, it was capable of inhibiting antibacterial activity to an
extent similar to that of the positive control, 0.12% Chlorhexidine. Additionally, at the
concentration of 5%, 45S5 glass resulted in higher antibacterial activity than both 5% and
20% NbG and the negative control, 0.9% NaCl (Figure 2).

7
Figure 2 - Box plot of the antibacterial activity results (Log10 CFU/mL) of the bioactive glass
suspensions in different concentrations tested and of the negative (NaCl) and positive
(chlorhexidine)* controls.

*Different letters indicate statistical difference between the groups (p<0.05)

3.3. Microtensile bond strength, hardness and modulus of elasticity of the resin-dentin
interface
The results of evaluations of the mechanical properties are presented in Table 1.
Table 1 - Mean (Standard Deviation) of microtensile bond strength values (μTBS), hardness
(HN) and modulus of elasticity (GPa) of the resin-dentin interface.
Mechanical Properties
Groups Modulus of Elasticity
μTBS (MPa) Hardness (HN)
(GPa)

8
 3 months
24 h 3 months 24 h 3 months 24 h b a

54.6 (7.6) 37.6 (8.3) 39.0 (2.5) 34.6 (2.0) 1.4 (0.1)
Control 1.3 (0.3) B
Aa Bb Aa Ba
54.4 (12.9) 46.0 (10.7) 34.6 (2.6) 43.1 (2.6) 2.3 (0.4)
45S5 5% 1.7 (0.1) A
Aa ABa Ab Aa
53.3 (7.1) 60.1 (8.7) 31.3 (2.6) 44.3 (7.9) 2.4 (0.2)
45S5 20% 1.5 (0.5) A
Aa Aa Ab Aa
53.5 (12.0) 36.7 (7.0) 37.7 (4.1) 44.1 (6.0) 1.8 (0.1)
NbG 5% 1.7 (0.4) B
Aa Bb Aa Aa
45.8 (12.9) 47.3 (10.1) 36.2 (6.6) 46.5 (4.0) 2.3 (0.4)
NbG 20% 1.8 (0.2) A
Aa ABa Ab Aa
* μTBS and hardness - Different capital letters indicate statistically significant differences in
the same column (p<0.05)
** μTBS and hardness - Different lower case letters indicate statistically significant
differences between storage time (p<0.05)
*** Modulus of Elasticity – Different superscripted letters indicate statistically significant
differences between main factors (p<0.05)
For the microtensile bond strength values (µTBS), analysis demonstrated a significant
interaction between the factors suspension and time (p=0.033). There were no significant
differences among the groups for the 24 h testing (p>0.05). There was a reduction in the bond
strength values after 3 months of storage in the groups that were rewetted with distilled water
(control) and in Group 5% NbG (p>0.05). The 45S5 (5% and 20%) and 20% NbG rewetting
suspensions were capable of maintaining the µTBS values after 3 months of storage.

Figure 3 - Fracture pattern distribution in percentage (number of specimens) analyzed under

stereomicroscope (40X) for all conditions evaluated.

9
The distribution of the fracture patterns is represented in Figure 3. For all groups and in both
intervals, adhesive fractures were predominant.

Figure 4 - Images of the fractured specimens. For Control (A-B), it was possible to observe
demineralization of the dentin substrate, with open tubules and absence of smear layer
(removed by phosphoric acid). In the group 45S5 it is not possible to visualize glass particles
in the resin-dentin interface (C-F). On the other hand, where NbG suspensions were used, the
presence of glass particles could be observed incorporated into the adhesive and within the
dentinal tubules (G-J).

For the hardness values (HN), analysis demonstrated a significant interaction between
the factors suspension and time (p=0.006). There were no significant differences among the
groups for the 24 h testing (p>0.05). There was an increase in the hardness values after 3
months of storage in the groups that were rewetted with 45S5 (5% and 20%) and 20% NbG
and (p>0.05).
For the modulus of elasticity the interaction of the factors (suspension and time) was
not significant, only the difference between the main factors suspension and time (p=0.086).
There was an increase in the values of modules after three months of storage (p<0.001). The

10
groups rewetted with 45S5 (5% and 20%) and 20% NbG presented the highest values of
modulus of elasticity when compared to the groups rewetted with distilled water and 5% NbG
(p>0.05).
4. Discussion
This study demonstrated that the use of bioactive glass suspensions prior to bonding
with an etch-and-rinse adhesive had an influence on the bond strength, hardness and modulus
of elasticity of dentin adjacent to the resin-dentin interface. Additionally, the bioactive glasses
presented different ability to increase the pH of the medium and antibacterial activity against
S. mutans, depending on the concentration and type of glass. The use of 45S5 and NbG
suspensions in higher concentrations (20%) was capable of maintaining the µTBS after 3
months of storage; promote the increase in hardness and modulus of elasticity of the hybrid
layer in the resin-dentin interface.
The presence of bioactive glass at the resin-dentin interface, obtained by rewetting the
dentinal substrate with the suspensions did not influence the bond strength in the 24h
evaluation. These results are in agreement with those of other studies that sought to
incorporate these particles into the resin-dentin interface in different ways, such as application
by means of airborne glass particle abrasion on the surface (E. M. Carvalho et al., 2015; Leal
et al., 2017) or their inclusion in adhesive systems (Bauer et al., 2016; Carneiro et al., 2018;
Profeta et al., 2013).
Studies have demonstrated that bioactive glass particles are capable of releasing ions,
such as Ca+2 and PO4-3, promoting mineral deposition on the demineralized dentin substrate
(C. N. Carvalho et al., 2016a; 2016b; Vollenweider et al., 2007), and the capacity to form
apatite in conditions similar to those of the oral medium (Sauro et al., 2013; 2015; Tauböck et
al., 2014). Moreover, reports have indicated that bioactive materials may reduce collagen
degradation (Tezvergil-Mutluay et al., 2017) caused by the action of MMPs (Osorio et al.,
2012). With the mineral deposition, the ions released in the bioactive glasses can aggregate
with MMP-2 and MMP-9, forming complexes (CaP-MMP) with high molecular weight and
restricted mobility, which might reduce the proteases activity (Makowski and Ramsby, 2004).
The creation of a moderately alkaline environment at the resin-dentin interface, due to
ionic exchanges between the glass and the physiological medium may have contributed to a
lower activity of pH dependent MMPs. These factors may explain the findings of this study,
in which bioactive 45S5 and NbG suspensions - especially in high concentrations - were used,
thus making it possible to observe the maintenance of the bond strength after storage for 3
months.

11
 The greatest difference between the composition of 45S5 and NbG is the presence of
niobium in NbG and silica in 45S5. This result in a difference in the layer formed on the
surface of the glass particles. In the surface of bioactive glass containing niobium is formed
Nb–OH, Nb-O-Nb-O or O–P–O–Nb–O– groups that can act as apatite nucleation centers
(Lopes et al., 2014), occupying a role similar to that of the silanol groups (Si–OH) or –O-Si-
O- bonds in 45S5 or S53P4 bioactive glass (Zhang et al., 2008). This difference in the layer
formed on the surface of the particles glass will guide the behavior of pH and ionic release. A
layer formed by Nb-O bonds is less permeable due to more strong chemical bonds, as
compared to the bonds formed by the silanol (Si-OH) groups. This is evident when the ions
release from the 45S5 and NbG glasses are observed in a short evaluation period (10 min and
24h) (Carvalho et al., 2016).
On the other hand, this chemical stability caused by the inclusion of niobium in
phosphate glasses, allows the incorporation of these bioactive particulates for the
development of adhesive systems, without causing an incompatibility with acidic monomers
(Bauer et al., 2016). Besides that, these composition have the advantage of not compromising
the ability of the bioactive particles to form apatite (da Rocha et al., 2013) or phosphate and
carbonate precipitates (Carneiro et al., 2018).
The NbG glass at a low concentration (5%) was incapable of promoting statistically
significant changes with respect to the mechanical properties of the resin-dentin interface
(µTBS, hardness and modulus of elasticity) evaluated in this study. The presence of NbG
glass only had an effect on the mechanical properties when a higher concentration (20%) was
used. This suggests that a larger quantity of the material is required when compared to 45S5.
On the another hand, irrespective of the concentration (5 or 20%), the effect of NbG
on the antimicrobial activity results was not so effective (Bauer et al., 2018). The chemical
stability of this glass may have had an influence on the different findings. The inferior
capacity to raising the pH may explain this result, since the pH could be determinant for an
antibacterial action. pH elevation is known to affect the integrity of the bacterial cytoplasmic
membrane, leading to cellular destruction. Another way, when NbG bioactive glass was
added to gutta-percha showed a reduction in biofilm volume, and resulted in the lowest
number of viable bacteria in the biofilm (C. N. Carvalho et al., 2016b).
Calcium hydroxide has a well known antimicrobial activity, which is influenced by the
speed of dissociation of hydroxyl ions that generate a high pH environment (Siqueira and De
Uzeda, 1998), and this high pH is essential for microbial control because most
microorganisms do not survive pH values higher than 9.0 (Freire et al., 2010). The

12
antibacterial properties of the glass is ascribed to an elevation of pH and also of osmotic
pressure that are caused by the chemical reactions at the glass surface, which take place as
soon as the glass is implanted into the body (Lindfors et al., 2016). A high pH in environment
is very important for hydroxyapatite nucleation (Lu and Leng, 2005) and this helps to explain
the excellent results obtained by the 45S5 bioactive glass irrespective of the concentration
used.
From a clinical restorative perspective, the rapid action on raising the pH and the
release of ions from the 45S5 glass becomes important to protect the collagen fibers not
infiltrated by the adhesive system and would act against the action of the MMPs enzymes
(Tay and Pashley, 2008; 2009). The rapid increase in pH would eliminate bacteria remaining
in the operative procedure (Leal et al., 2017). Some studies have shown that 45S5 glass acts
rapidly in remineralization and protection of collagen fibers, around 10.2h (Saito et al., 2003),
and in the recovery of the mechanical properties of the dentin through the formation of
precipitates (Vollenweider et al., 2007, Wang et al., 2011).
According to the findings of this study, the use of 45S5 and NbG suspensions in
concentrations of 20% were shown to be a technique capable of increasing the stability of
bond to dentin. On the other hand, only the suspension containing 20% of 45S5 glass was
capable of impeding bacterial growth to an extent similar to that of chlorhexidine.
This result becomes valuable due to the large volume of resin restorations that need to
be changed due to recurrent caries localized exactly at the resin-dentin interface. The high
rates of dental restoration failures, pulp treatment, and tooth extractions indicate that the
techniques in use at present are faulty. Therefore, the use of the technique of rewetting dentin
with a suspension containing 20% 45S5 bioglass is interesting, due to both the capacity for
mineral deposition, controlling pH and its potential antibacterial activity.
5. Conclusion
Rewetting demineralized dentin with a 45S5 (20%) suspension was capable of
preventing a reduction in bond strength values; increasing hardness and the modulus of
elasticity of the dentin adjacent to the resin-dentin interface, and presenting antibacterial
activity against Streptococcus mutans.
Acknowledgements
The authors thank the Research Support for Scientific and Technological
Development Foundation of Maranhão [FAPEMA – BEPP 01087/18] and the National
Council of Scientific and Technological Development [CNPq - 237066/2012-2] for
supporting this research. The authors also thank the University British of Columbia (UBC)

13
Start-Up funds granted to RMC and APM.

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Highlights
• Suspension rich in bioactive glass can maintain the bond strength over time
• The presence of glass in the adhesive interface can inhibit bacterial growth
• Bioactive glass in dentin can prevent pH drop and inhibit the action of MMPs
• Bioactive glasses may increase and mechanical strength of conditioned dentin

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