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Therapeutic effects of novel resin bonding systemscontaining bioactive glasses on mineral-depleted areaswithin the bonded-dentine interface
Salvatore Sauro • Raquel Osorio • Timothy F. Watson •
Manuel Toledano
Received: 27 November 2011 / Accepted: 27 February 2012 / Published online: 1 April 2012
� Springer Science+Business Media, LLC 2012
Abstract This study aimed in evaluating the effects of
two experimental resin bonding systems containing con-
ventional Bioglass 45S5 (BAG) or Zinc-polycarboxylated
bioactive glass (BAG-Zn) micro-fillers on the resin-bonded
dentine interface after storage in a simulated body fluid
solution (SBFS). Three resin bonding systems were for-
mulated: Resin-A: (BAG containing); Resin-B; (BAG-Zn
containing); Resin-C (no filler). The ability of the experi-
mental resins to evoke apatite formation was evaluated
using confocal Raman spectroscopy. Acid-etched dentine
specimens were bonded, and prepared for AFM/nano-
indentation analysis in a fully-hydrated status to evaluate
the modulus of elasticity (Ei) and hardness (Hi) across the
interface at different SBFS storage periods. Further resin–
dentine specimens were tested for microtensile bond
strength after 24 h or 3 months of SBFS storage. SEM
examination was performed after de-bonding and confocal
laser microscopy was used to evaluate the ultramorphology
of the interfaces and micropermeability. The resin A and B
showed a consistent presence of apatite (967 cm-1),
reduced micropermeability within the resin–dentine inter-
face and a significant increase of the Ei and Hi along the
bonded-dentine interface after prolonged SBFS storage.
Bond strength values were affected by the resin system
(P \ 0.0001) and by storage time (P \ 0.0001) both after
24 h and 3 months of SBFS storage. In conclusion, resin
bonding systems containing bioactive fillers may a have
therapeutic effect on the nano-mechanical properties and
sealing ability of mineral-depleted resin–dentine interface.
1 Introduction
The development of innovative bioactive ion-releasing
restorative materials with a therapeutic ability to remineralise
mineral-depleted sites within the bonded-dentine interface is
currently one of the main targets of the dental biomaterial
research [1–5]. In vitro [6, 7] and in vivo [3, 4] studies
demonstrated that remineralisation may be achieved by
increasing the mineral content and the mechanical properties
(i.e. hardness and modules of elasticity) of dental hard tissues.
Peters et al. [3] showed by electron probe elemental micro-
analysis (EPMA) techniques, an increase mineral content in
the caries affected dentine using resin-based materials con-
taining calcium phosphate cements. Moreover, specific
restorative materials such as glass ionomer cements (GIC) or
resin-modified glass ionomer cements (RMGIC) may also
promote mineral precipitation in residual caries affected
dentine [5, 8, 9]. Ryou et al. [8] have recently demonstrated
that the remineralisation of such demineralised zones within
the hybrid layers may be a potential means for preserving the
durability of the resin–dentine interface [8, 12]. Indeed,
poorly resin-infiltrated hybrid layers created in acid-etched
dentine may be characterised by collagen degradation within
a resin-sparse demineralised zone which frequently remains
subsequent to bonding procedures [10, 11]. However, the use
of Zinc-containing materials may reduce the collagen deg-
radation mediated by matrix metalloproteinases (MMPs)
within the poorly infiltrated hybrid layers and carious dentine
by protecting sensitive cleavage sites of collagen within the
demineralised dentine [13].
S. Sauro (&) � T. F. Watson
Biomaterials, Biomimetics and Biophotonics (B3 group), King’s
College London Dental Institute, Guy’s Dental Hospital, London
SE1 9RT, England, UK
e-mail: [email protected]
S. Sauro � R. Osorio � M. Toledano
Dental Materials, School of Dentistry, University of Granada,
Colegio Maximo, Campus de Cartuja, Granada, Spain
123
J Mater Sci: Mater Med (2012) 23:1521–1532
DOI 10.1007/s10856-012-4606-6
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Bioglass 45S5, a high biocompatible calcium/sodium
phosphate-phyllosilicate originally developed as a bone
conductive material but chemically similar to natural tooth
mineral, is one of the foremost bioactive materials with an
excellent ability to induce calcium phosphate (Ca/P) pre-
cipitation and subsequent crystallisation into hydroxyapa-
tite [HA: Ca10(PO4)6(OH)2] when immersed in protein-
free simulated body fluid solution (SBFS) or saliva
[14]. Whereas Bioglass 45S5 has been successfully used
for dentine remineralisation when directly applied on
demineralised dentinal tissue (16), the development of resin-
based restorative materials containing bioactive micro-fill-
ers with remineralising effects on the mineral-depleted areas
within the bonded-dentine interface remains an important
target to accomplish.
The aim of this study was to evaluate the ability of two
experimental resin bonding systems containing Bioglass
45S5 (BAG) or Zinc-polycarboxylated bioactive glass
(BAG-Zn) to induce therapeutic effects on the bonded-
dentine interface after SBFS ageing. The null hypothesis to
be tested is that the inclusion of bioactive micro-fillers
within the composition of the resin bonding systems
encourages no remineralisation on the mineral-depleted
areas within the bonded-dentine interface.
2 Materials and methods
2.1 Bioactive fillers and experimental bonding systems
preparation
A BAG micro-filler was produced by melting 46.1 mol%
SiO2, 26.9 mol% CaO, 24.4 mol% Na2O and 2.5 mol%
P2O5 in a platinum crucible at 10�C/min up to 1,100�C and
maintained for 1 h. Subsequently the temperature was raised
to 1450�C (10�C/min) and maintained for further 30 min.
The glass melt was rapidly quenched in water (~20�C),
dehydrated in absolute ethanol for 3 h, dried overnight in a
furnace at 110�C, milled and finally sieved (\20 lm). The
BAG-Zn micro-filler was produced by mixing 80 wt% BAG
with 20 wt% Zinc oxide (Aldrich Chemical Co, Gillingham,
UK) and mixed (ratio 1:2) with 10% polycarboxylic acid
solution (PAA: MW 1800; Aldrich Chemical Co.) for 5 min
(40 rpm). The mixture was dried in a furnace at 80�C for
12 h. The final product was finally milled and sieved as
previously described. A resin co-monomer blend was for-
mulated using three hydrophobic monomers (UDMA,
BisGMA, TEGDMA: Esstech Essington, PA, USA) a
hydrophilic monomer (HEMA) and absolute ethanol
(Aldrich Chemical Co.). The resin solution was made light-
curable by adding 0.5 wt% of camphoroquinone and
1.0 wt% of ethyl 4-dimethylaminobenzoate (Aldrich
Chemical Co.). 5-min sonication (Model QS3, Ultrawave
Ltd, Cardiff, UK) and 2-day shaking (Orbital Shakers PSU-
20i, Cole Fisher Scientific Ltd, Loughborough, UK) were
required to achieve a well-mixed resin blend. Three exper-
imental resin bonding systems were subsequently created:
(1) Resin A: 60 wt% resin blend/40 wt% BAG; (2) Resin B:
60 wt% resin blend/40 wt% BAG-Zn; (3) Resin-C: 100%
resin solution (no filler). The detailed composition of each
resin bonding system is shown in Table 1.
2.2 Confocal Raman spectroscopy evaluation
Three disks for each resin were created using a TEFLON
cylinder mold (8 mm in dm and 1 mm thick) and a light-
curing system (600 mWcm2, Optilux VLC, Demetron,
Research Co., CT, USA) for 1 min. A computer-controlled
confocal laser Raman apparatus equipped with a Leica
DM/LM optical microscope with a 209/0.40 NA objective
and CCD detector attached to a modular research spec-
trograph (Renishaw InVia; Renishaw plc, Gloucheshire,
UK) was used to evaluate the apatite precipitation after
24 h, 1 month (1 m) and 3 months (3 m) of SBFS aging. A
near-infrared diode laser spot size (\1 lm) operating at
785 nm was used to induce the Raman scattering effect
(200–1,100 cm-1). The entire surface of the specimens
was examined with steps of 20.0 lm on the X and Y axes
using a computer-motorised stage and analysed for the
peak of apatite at 967 cm-1 using the software Wire 3.2
(Renishaw), [15]. A SBF solution containing NaCl,
NaHCO3, KCl, K2HPO4, MgCl2, CaCl2, and (CH2OH)3
CNH2/HCl (6 N) to buffer the pH (7.4) was replaced every
72 h.
2.3 Dentine specimen preparation and bonding
procedures
Human caries-free molars extracted for surgical reasons
under a protocol approved by an institutional review board
were stored in deionised water (pH 7.1) at 4�C for no
longer than one 1 month. Dentine crown specimens were
prepared by cutting the roots 1 mm beneath the cemento–
enamel junction (CEJ) using a hard tissue microtome
(Isomet 11/1180, Buehler, Coventry, UK) equipped with a
diamond embedded blade (XL 12205, Benetec Limited,
London, UK). The occlusal enamel was then removed with
a second parallel cut in order to expose a middle coronal
dentine. A standard and more clinically relevant smear
layer was created using 180-grit SiC paper for 30 s under
constant water irrigation. The specimens were divided into
three groups based on the tested bonding systems (A, B, C)
and conditioned using a 35% orthophosphoric acid solution
(H3PO4; Aldrich Chemical, Co.) for 15 s followed by a
copious water rinse (5 s). The acid-etched specimens were
air-dried for 2 s, bonded with the experimental adhesives
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within a period of 20 s and immediately light-cured for
30 s (600 mW cm2, Optilux VLC, Demetron, Research
Co., CT, USA). A flowable resin composite (X-FlowTM,
Dentsply, Caulk, UK) was placed incrementally in two
1 mm layers and light-cured for 40 s. The detailed appli-
cation mode is shown in Table 1.
2.4 AFM imaging and nano-indentation
Three bonded-dentine specimens for each group were lon-
gitudinally sectioned in slabs (Thickness: 1.5 mm) and
polished through SiC abrasive papers from 800 up to
4000-grit. A final polishing procedure was performed using
diamond pastes (Buheler-MetaDi, Buheler Ltd. Lake Bluff,
IL, USA) through 1 lm down to 0.25 lm. The specimens
were treated in ultrasonic bath (Model QS3, Ultrawave Ltd,
Cardiff, UK) containing deionised water (pH 7.4) for 5 min
at each polishing step. An atomic force microscope (AFM -
Nanoscope V, Digital Instruments, Veeco Metrology group,
Santa Barbara, CA, USA) equipped with a Triboscope
indentor system (Hysitron Inc., Minneapolis, MN) and a
water immersion Berkovich-tip indenter (radius *20 nm)
was employed for the imaging and indentation processes
in a fully-hydrated status. Six indentations with a load of
4000 nN and a time function of 10s were performed in a
straight line starting from the adhesive layer down to the
intertubular dentine at different SBFS storage periods (24 h,
1 m, 3 m) in order to evaluate the modulus of Ei and Hi.
Three indentation lines were executed in five different
mesio-distal positions along the interface. The distance
between each indentation was kept constant by adjusting the
distance intervals in 5 (±1) lm steps and the load function
[16, 17]. ANOVA was performed including Ei or Hi as a
dependent variable. Resin adhesive type (A/B/C), period of
SBFS storage (24 h/1 m/3 m), and position along the
interface (1st–6th) were considered as independent vari-
ables. Analysis of interaction was also executed. Student–
Newman–Keuls test was used for multiple compari-
sons. Statistical analysis was set at a significance level of
a = 0.05.
2.5 Confocal laser scanning microscopy evaluation
(CLSM)
Three acid-etched dentine specimens for each group were
bonded, as previously described, with the experimental
resins doped with 0.2 wt% Sulfo-Rhodamine 101 (SR-101:
Sigma Chemicals, St. Louis, MO, USA). The pulp chamber
of the specimens was exposed and half of the specimens
were immediately filled with 0.1 wt% water solution of
Lucifer Yellow CH (LY: Sigma Chemicals) for 3 h
[17, 18] while, the remnant half part was immersed in
SBFS for 3 m and subsequently filled with LY. The
specimens were washed with deionised water in an ultra-
sonic bath for 2 min, sectioned into slabs (1.5 mm) and
finally polished using 4000-grit SiC. A confocal laser
Table 1 Composition of the experimental resin bonding system used in this study
Resin Chemical composition Application mode and bonding procedures
Resin A 35% UDMA
3.6% Bis-GMA
3.4% TEGDMA
15% HEMA
10% Absolute ethanol
33% Bioglass 45S5
• Etching with 35%-H3PO4 for 15s
• Abundant rinse with deionised water
• Air-dried for 2 s,
• Application of a first layer of each experimental adhesives for 10 s
• Air-dried for 5s at maximum stram power
• Application of a second layer of each experimental adhesives for 10 s
• Gently air-dried for 2 s,
• Light-cure for 30 s
• Apply resin composite and light-cure
Resin B 35% UDMA
3.6% Bis-GMA
3.4% TEGDMA
15% HEMA
10% Absolute ethanol
33% Zn-polycarboxilated bioactive glass
Resin C 55% UDMA
4.5% Bis-GMA
10.5% TEGDMA
18% HEMA
12% Absolute ethanol
0.5 wt% of camphoroquinone (CQ) and 1.0 wt% of ethyl 4-dimethylaminobenzoate (EDMB) were also included within the composition of the resins
Abbreviations: BisGMA 2,2-bis[4-(2-hydroxy-3-methacryloylpropoxy)]-phenyl propane, TEGDMA triethyleneglycol dimethacrylate, HEMA2-hydroxyethylmethacrylate, UDMA urethane dimethacrylates, Absolute ethanol: 100% ethylic alcohol
J Mater Sci: Mater Med (2012) 23:1521–1532 1523
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scanning microscope (CLSM: Leica SP2 CLSM, Heidel-
berg, Germany) equipped with a 63x/1.4 NA oil immersion
lens, a 488 nm argon/helium and a 633 nm krypton ion
laser illumination was used to analysed the ultra-mor-
phology and the micropermeability along the resin–dentine
interfaces. Reflection and fluorescence optical images were
captured 5 lm below the outer surface up to 25 lm depth
(1 lm z-step) and converted in topographic single projec-
tions using the Leica SP2 CLSM image-processing soft-
ware (Leica, Heidelberg, Germany). The configuration of
the system was standardised and used at the same level for
the entire investigation.
2.6 Micro-tensile bond strength (lTBS)
Further five bonded-dentine specimens for each group were
sectioned in both X and Y directions to obtain match-sticks
of 0.9 mm2. They were tested after 24 h or 3 m of SBFS
storage using a customised microtensile jigs on a linear
actuator (SMAC Europe Ltd., Horsham, West Sussex, UK).
Mean bond strength values were analysed by two-way
ANOVA and Student–Newman–Keuls multiple compari-
sons. Statistical analysis was performed at a significance
level of a = 0.05. Modes of failure were classified as per-
centage of adhesive (A) or mixed (M) or cohesive (C) using
a 30x USB digital-microscope (CY-800B). Five represen-
tative fractured specimens for each group were sputter-
coated with gold (SCD 004 Sputter Coater; Bal-Tec, Vaduz,
Liechtenstein) and examined with SEM (S-3500; Hitachi,
Wokingham, UK) with an accelerating voltage of 15 kV
and a working distance of 15 mm.
3 Results
3.1 Confocal Raman spectroscopy evaluation
No presence of apatite was detected after 24 h of SBFS
storage but Resin A showed a consistent presence of a
prominent peak at 967 cm-1 both after 1 m and 3 m of
SBFS storage. Conversely, Resin B presented an irregular
detection of apatite after 1 m; a prominent and reliable
peak at 967 cm-1 was detected after 3 m of SBFS storage.
No apatite formation was identified on the specimens cre-
ated using the Resin C after 3 m of SBFS storage (Fig. 1).
3.2 Nano-mechanical properties and AFM/CLSM
microscopy
The nano-mechanical properties (Ei and Hi) were influenced
by resin adhesive type, position along the interface (1st–6th)
and storage time (P \ 0.01), (Table 2 and Fig. 2). Interac-
tions between factors were also significant (P \ 0.001). In
detail, the first indentation performed on the adhesive layer of
the Resin A showed a significant reduction in Ei and Hi after
prolonged SBFS storage (1 m and 3 m) (Fig. 2A, B). Con-
versely, both the second indentation values corresponding to
the hybrid layer (HL) and the third value performed in the
bottom of the HL showed a statistical increase of the Ei and
Hi (P \ 0.01). No significant changes were observed along
the intertubular dentine in the 4th, 5th and 6th position sub-
sequent to prolonged SBFS storage. The AFM imaging
showed a scarcely pronounced indentation mark on the HL
after 24 h of SBFS storage (Fig. 3A). On the contrary, a well
shaped indentation mark was observed both after 1 m
(Fig. 3B) and 3 m (Fig. 3C) of SBFS storage. The confocal
microscopy showed after 24 h an intense micropermeability
within the HL after 24 h (Fig. 3D, E) while, a strong
reflection signal was detected subsequent to 3 m of SBFS
storage (Fig. 3F). The experimental Resin B showed higher
Ei and Hi (P \ 0.01) (Fig. 2C, D) and a pronounced inden-
tation mark both on the HL and bottom of HL after prolonged
SBFS storage (Fig. 4A). The bonded-dentine interface cre-
ated with Resin B (Fig 4B) was affected by important
micropermeability at 24 h (Fig. 4C) which appeared limited
and localised at the bottom of a very reflective HL after 3 m
of SBFS storage (Fig 4D). Resin C showed a statistical
reduction in Ei and Hi (P \ 0.01) of the adhesive layer, HL
and bottom of HL after prolonged SBFS storage (Fig. 2E, F);
a severe micropermeability (Fig. 5A) and an evident gap
were observed within the interface (Fig. 5B).
Fig. 1 Confocal Raman spectroscopy of the resin bonding systems
tested in this study. The resins A, B and C show no peak at 967 cm-1
(apatite) after 24 h of SBFS storage. The outer surfaces of resin A and
B were characterised by a constant presence of apatite after prolonged
SBFS storage (3 m)
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3.3 Micro-tensile bond strength (lTBS) and failure
mode analysis
Bond strength values were affected by the resin type
(F = 8.50; P \ 0.0001) and by storage time (F = 182.58;
P \ 0.0001). Interactions were also significant (P \0.001), (Table 3). Reliability of the model was 77%. In
detail, resin A showed lTBS values up to 44.4 ± 7.4 MPa
and generally failed in a mixed mode after 24 h. The
prolonged SBFS storage (3 m) induced in these dentine-
bonded specimens a significant lTBS drop (P \ 0.05) and
a failure mode prevalently mixed and adhesive. However,
when the 3 m SBFS-aged match-sticks failed in an adhe-
sive mode (Fig. 6A1) were analysed using SEM it was
possible to observe a surface still covered by residual resin
(Fig. 6B1) and mineral crystals embedded in a resin/col-
lagen scaffold-like structure (Fig. 6B2). In contrast, the
match-sticks tested after 24 h showed an exposed dentine
surface with a few crystals only inside the dentinal tubules
(Fig. 6B3). The match-sticks created with the resin B
achieved lTBS values up to 36.6 ± 11.3 MPa after 24 h of
SBFS storage and failed generally in the mixed mode.
Resin B showed a significant lTBS reduction after pro-
longed SBFS storage (3 m) with a failure mode mixed and
adhesive remnant resin and mineral crystals was detected
by SEM on the fractured surfaces (Fig. 6C1, C2). Mineral
crystals were observed only inside the dentinal tubules in
specimens aged for 24 h as shown in Fig. 6B4. The resin C
attained lTBS values up to 37.4 ± 14.7 MPa and failed
prevalently in mixed mode after 24 h. These values sig-
nificantly dropped subsequent to prolonged SBFS storage
(3 m) and the fractured surface was characterised by the
presence of numerous exposed dentinal tubules (Fig. 6D1)
and collagen fibrils (Fig. 6D2–3).
4 Discussion
The nano-mechanical properties and the ultramorphology/
micropermeability of the bonded-dentine interface were
influenced considerably by the experimental resins sub-
sequent to SBFS storage. Therefore, the null hypothesis
that the inclusion of bioactive micro-fillers within the
composition of the resin-based restorative materials has no
Table 2 Mean and standard deviation of AFM nano-mechanical properties (Ei/Hi) along the resin–dentine interface
Ei–24 h Ei–1 month Ei 3–months Hi–24 h Hi–1 month Hi–3 months
Resin A
1st 3.22 ± 1.10 A1 2.30 ± 0.70 A2 1.85 ± 0.40 A2 0.17 ± 0.03 A1 0.11 ± 0.02 A2 0.07 ± 0.03 A2
2nd 1.51 ± 0.48 B1 2.17 ± 0.67 A2 2.35 ± 0.70 B2 0.03 ± 0.01 B1 0.09 ± 0.01 A2 0.09 ± 0.03 A2
3rd 8.40 ± 2.43 C1 10.3 ± 1.58 B2 11.5 ± 2.66 C2 0.37 ± 0.10 C1 0.48 ± 0.11 B2 0.57 ± 0.10 B3
4th 15.9 ± 1.95 D1 16.3 ± 1.38 C1 16.70 ± 0.95 D1 0.73 ± 0.12 D1 0.72 ± 0.06 C1 0.72 ± 0.06 C1
5th 16.9 ± 0.85 DE1 18.0 ± 1.44 D1 17.7 ± 0.82 DE1 0.75 ± 0.06 D1 0.73 ± 0.07 C1 0.73 ± 0.07 C1
6th 17.7 ± 1.30 E1 18.5 ± 0.96 D1 18.8 ± 0.70 E1 0.85 ± 0.08 E1 0.89 ± 0.09 D1 0.89 ± 0.09 D1
Resin B
1st 2.33 ± 0.47 A1 1.71 ± 0.84 A1 1.53 ± 0.53 A2 0.33 ± 0.04 A1 0.21 ± 0.12 A2 0.20 ± 0.14 A2
2nd 1.68 ± 0.50 B1 1.77 ± 0.37 A1 3.17 ± 0.95 B2 0.04 ± 0.01 B1 0.07 ± 0.02 B2 0.09 ± 0.01 B2
3rd 7.43 ± 1.91 C1 9.71 ± 1.79 B2 12.7 ± 1.35 C3 0.47 ± 0.11 C1 0.43 ± 0.11 C1 0.60 ± 0.09 C2
4th 16.0 ± 2.45 D1 16.8 ± 1.39 C1 16.8 ± 1.39 D1 0.72 ± 0.14 D1 0.70 ± 0.04 D1 0.73 ± 0.04 D1
5th 17.2 ± 0.94 E1 17.4 ± 0.73 CD1 17.9 ± 0.61 DE1 0.78 ± 0.10 DE 0.76 ± 0.07 D1 0.75 ± 0.05 D1
6th 18.6 ± 0.80 E1 18.7 ± 0.86 D1 18.6 ± 0.56 E1 0.81 ± 0.08 E1 0.82 ± 0.13 E1 0.80 ± 0.13 E1
Resin C
1st 2.97 ± 0.28 A1 2.12 ± 0.12 A1 2.02 ± 0.15 A1 0.27 ± 0.07 A1 0.24 ± 0.04 A1 0.19 ± 0.02 A3
2nd 1.13 ± 0.16 B1 0.00 ± 0.00 B2 0.00 ± 0.00 B2 0.04 ± 0.01 B1 0.00 ± 0.02 B2 0.00 ± 0.00 B2
3rd 5.77 ± 1.21 C1 5.51 ± 0.96 C1 5.40 ± 1.55 C1 0.44 ± 0.09 A1 0.29 ± 0.06 A2 0.26 ± 0.06A A2
4th 15.0 ± 3.05 D1 11.4 ± 0.72D2 11.8 ± 1.54 D2 0.68 ± 0.05 C1 0.66 ± 0.04 C1 0.68 ± 0.05 C1
5th 16.8 ± 1.17 DE1 16.9 ± 1.52 E1 16.1 ± 1.00 E1 0.76 ± 0.05 D1 0.74 ± 0.07 D1 0.73 ± 0.04 C1
6th 17.9 ± 0.53 E1 17.8 ± 0.72 E1 18.1 ± 1.08 E1 0.81 ± 0.03 E1 0.81 ± 0.06 E1 0.76 ± 0.09 C1
Mean (S.D) of AFM nano-properties (Ei/Hi) of the dentine-bonded interfaces using the three different experimental resins used in this study
Same letter indicates no differences in columns (P [ 0.05) along different indentation position. Same number indicates no differences in rows for
different SBFS storage time (P [ 0.05); [Ei or Hi]
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therapeutic effects of remineralisation on the mineral-
depleted areas with the bonded-dentine interface must be
rejected.
Indeed, the results obtained during the AFM nano-
indentation assessment after prolonged SBFS storage
showed a statistical increase of the nano-properties (Ei and
Hi) in definite areas of the bonded-dentine interface created
using the two experimental resins containing BAG or BAG-
Zn micro-fillers. These results are in agreement with those
obtained by Bertassoni et al. [19] who showed that a con-
tinuous delivery of calcium (Ca2?) and phosphates (PO4-3)
ions may induce remineralisation and mechanical recovery
of mineral-deficient dentine due to a fine association of the
minerals with the organic matrix. Balooch et al. [20] dem-
onstrated that the AFM nano-indentation assessment, where
force and indenter displacement are recorded simulta-
neously during the indentation and the elastic modulus and
hardness are determined from the load displacement curve,
is a suitable method to evaluate the visco-elasticity of the
demineralised dentine and the effective remineralisation of
the collagen matrix [21, 22]. The results obtained in this
study confirmed these latter observations showing an HL
Fig. 2 Mean (S.D) of AFM nano-properties (Ei and Hi) of the main
zones along the resin–dentine interface created using the three
different experimental resins. The resin–dentine interface created
using resin A shows reduction of the adhesive layer’s nano-
mechanical properties and increase in the HL and bottom of HL
after SBFS storage (A and B). Resin B presents an increase in Hi
(C) and Ei (D) at the HL and bottom of HL after SBFS storage (1 m
and 3 m). While, an important nano-properties reduction can be
observed in the adhesive layer after prolonged SBFS storage. The
resin–dentine interface created with resin C is characterised by nano-
property reduction both at the HL and bottom of HL after SBFS
storage (E and F)
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with high visco-elasticity characteristics after 24 h of SBFS
storage and a well pronounced indentation mark only after
prolonged SBFS storage (Fig. 3C, 4A).
It is well known that BAG is able to bond hard and soft
tissues and evoke apatite precipitation due to a simultaneous
process where silicic acid Si(OH)4 is firstly released and
then chemisorbed within the demineralised collagen via
electrostatic bonding to form a poly-condensation silica-
gel-layer for the precipitation of Ca2? and PO4-3 [22–27].
Conversely, Zinc-doped bioactive glasses such as Bioglass
46S6 are characterised both by slower poly-condensation
reaction and Ca2? and PO4-3 release when immersed in
SBFS [28, 29]. Furthermore, the use of a polyacrylic acid
may act as a sequestrating agent and favour the aggregation
of Ca/P nano-precursors within the dentine collagen fibrils
regulating the entire process of remineralisation [33, 34].
The confocal micro-Raman spectroscopy results attained in
this study confirmed the slower apatite deposition activity
of the BAG-Zn-containing resin B which showed a con-
sistent presence of apatite only after prolonged (3 m) SBFS
storage. Conversely, the presence of the BAG micro-filler
induced a strong and consistent apatite precipitation
immediately after 1 m of SBFS storage (Fig. 1). Moreover,
the high zinc-content of the BAG-Zn (20 wt%) may have
protected the collagen fibrils from MMPs [13] which are
principally responsible for the degradation process both in
carious dentine [30–32] and within the HL [35, 36]. The
creation of a moderate alkaline environment within the
bonded-dentine interface due to the exchange between Na?
and H?/H3O? ions and the rapid release of Ca2? and PO4-3
species from the bioactive glasses may also have contrib-
uted in the inhibition of pH-dependent MMPs [13, 35, 36].
The confocal microscopy analysis offered further evi-
dence on the therapeutic ability of the two experimental
resins containing bioactive micro-fillers showing a strong
reflection signal and a reduced micropermeability along the
entire bonded-dentine interface after prolonged SBFS
storage (Figs. 3F and 4D). Conversely, a clear microper-
meability within a gap-affected interface was observed
within the dentine bonded with resin C (Fig. 5B). Similar
results were also observed in previous studies [37, 38]
which showed a progressive dehydration and water
replacement by mineral crystals within the denuded col-
lagen matrices of bonded-dentine interfaces.
Ion-releasing resin-based restorative materials, e.g.
resin-modified glass ionomer cements (RMGIC), are char-
acterised by an important water sorption phenomenon
which represents a key factor for their therapeutic activity
Fig. 3 (A) AFM topographic image showing the indentations per-
formed in a straight line from the adhesive layer (a) along the bonded-
dentine interface created with the resin A. The second indentation was
obtained from the HL (pointer) and the third value from the bottom of
the HL. The remnant indentations were performed along the intertu-
bular dentine (d). It is possible to see that the indentation mark
corresponding to the HL is scarcely visible (pointer). The appearance
of the first resin tags (t) after the bonding interface indicates the bottom
of the HL. The indentation mark performed on the HL of resin A stored
in SBFS for 1 m (B) and for 3 m (C) results well pronounced. (D) A
CLSM single projection image showing a bonded-dentine interface
(resin A) characterised by a clear HL, long resin tags penetrating the
dentinal tubules (t) and several filler lacunas (FL) within the adhesive
layer. In fluorescence mode it is possible to observe a porous HL
completely infiltrated by the fluorescent dye LY (pointer), (E) while,
in reflection/fluorescence mode it is possible to note a strong reflection
signal both from the dentinal tubules and from an HL only slightly
affected by LY micropermeability (arrow), (F)
J Mater Sci: Mater Med (2012) 23:1521–1532 1527
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on demineralised dental tissues [39]. The resin blend solu-
tion used to formulate the resin bonding systems tested in
this study was specifically created (Table 1) to achieve an
essential resin elution/degradation during the SBFS storage
in order to favour ion-exchange and mineral precipitation
within the dentine-bonded interface [10, 11]. This is the
reason why the dentine-bonded specimens presented high
lTBS values after 24 h and an important lTBS reduction
(P \ 0.05) subsequent to 3 months of SBFS storage
(Table 2). Nevertheless, the SEM ultra-morphology analy-
sis performed in the 3 months SBFS-aged specimens cre-
ated with resin A and B showed a de-bonded surface
characterised by a remineralised resin-dentine matrix
(Fig. 6B2, C2). These results are in accordance with those
shown by Liu et al. [40] who identified large mineral plates
within a water-entrapped polymer hydrogel matrix corre-
sponding to the basal region of the filled water channels and
agglomerates of apatite nanocrystals within the gap region
of the water channel. However, it is also possible that the
crystallised SBFS-aged bonded-dentine interface created
using resin A and B may have had similar mechanical
characteristics as well as those created by GIC applied on
PAA-etched dentine when submitted to tensile tests. Indeed,
previous studies indicated that tensile or shear bond
strengths were rarely above 5 MPa [41, 42] while, the lTBS
of the GICs bonded to dentine was 12 ± 15 MPa with a
failure mode prevalently adhesive and mix [42]. Yip et al.
[43] stated that the results obtained from GICs bonded to
dentine under tensile stress does not represent their true
adhesive strength but only ultrastructural observation of the
bonding interface or of the fractured interface may indicate
the real bonding ability of conventional GIC to dentine.
However, further studies are ongoing on the formulation of
resin bonding systems containing bioactive micro-fillers
and biomimetic agents which may induce higher intra and
extra fibrils remineralisation and higher bond strength
results after prolonged SBFS aging.
In conclusion, a specific formulation of resin bond-
ing systems containing bioactive fillers such as BAG or
BAG-Zn may offer the possibility of increasing the
nano-mechanical properties of Hi and Ei and reduce the
micropermeability along the dentine-bonded interface by
therapeutic remineralisation of imperfect mineral-depleted
areas. This particular approach may be suitable for the
current concept of minimally invasive cavity preparation
followed by therapeutic restorative materials which may
stabilise the carious lesion and/or create an optimal envi-
ronment to repair the demineralised dental tissues.
Fig. 4 (A): AFM topographic
image showing the resin–
dentine interface created with
resin B after SBFS (3 m)
storage showing a clear
indentation mark on the HL.
(B): This CLSM single
projection captured from the
same resin-dentine specimens
(resin B) shows a clear HL with
long resin tags penetrating the
dentinal tubules (t) and a
substantial presence of BAG-Zn
filler (FL) within the adhesive
layer. (C): In reflection/
fluorescence lacune mode it is
possible to see a resin–dentine
interface characterised by a
porous HL completely
infiltrated by the LY dye
(pointer) after 24 h of SBFS
storage. (D): The CLSM single
projection image captured from
resin–dentine interfaces (resin
B) after prolonged SBFS
storage (3 m) showing a strong
reflection signal both in HL and
dentinal; a limited
micropermeability at the bottom
of the HL is also evident
(pointer)
1528 J Mater Sci: Mater Med (2012) 23:1521–1532
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Fig. 5 (A): This CLSM reflection/fluorescence single projection
shows the resin–dentine interface created with resin C characterised
by a HL affected by severe micropermeability and a filler-free
adhesive layer (a). (B): This type of bonded-dentine interface showed
signs of degradation (pointer) between a remnant porous HL
infiltrated by LY dye after prolonged SBFS storage. No indentation
mark is visible both in the AFM image obtained in the specimens
stored for 24 h (C) and 3 m in SBFS (D). It is also possible to note the
presence of a gap between the adhesive layer (a) and the dentine
(d) indicating a severe degradation of the HL
Table 3 Mean and standard deviation of microtensile bond strength values in MPa obtained for the different experimental groups and per-
centage distribution of failure mode after microtensile bond strength testing; total number of beams (tested stick/pre-load failure)
lTBS-Mean ± SD (N of beams) % Failure [A/M/C] t P
24 h test 3 Month test
Resin A
(BAG)
B144.42 ± 7.43
(28/0) [15/70/15]
B215.02 ± 8.15
(25/3) [41/59/0]
11.66 \0.0001
Resin B
(BAG-Zn)
A136.60 ± 11.32
(30/0) [12/68/20]
A28.15 ± 5.12
(29/1) [72/26/2]
11.44 \0.0001
Resin C (No Filler) A137.44 ± 14.75
(31/0) [12/70/18]
C223.78 ± 10.11
(29/2) [32/68/0]
3.64 \0.001
For each horizontal row: values with identical numbers indicate no significant difference. P and t values are displayed at the right column
For each vertical column: values with identical letters indicate no significant difference using Student–Newman–Keuls test (P [ 0.05)
Failure mode [A adhesive, M mixed, C cohesive]
J Mater Sci: Mater Med (2012) 23:1521–1532 1529
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Fig. 6 Scanning electron microscopy images of failure modes of
resin A (A1), resin B (A2) and resin C (A3) bonded to H3PO4-etched
dentine. Resin A (B1) shows a fracture surface after 3 m of SBFS
storage characterised by the presence of remnant resin (pointer) and
mineralised tissue (pointer). At higher magnification (B2) it was
possible to observe the presence of mineral crystals embedded in a
remnant resin/collagen scaffold-like structure (pointer). Conversely, a
limited presence of mineral crystals inside the tubules can only be
observed (pointer) in the specimens stored in SBFS for 24 h (B3).
Also the specimens created of the resin B group immersed in SBFS
for 3 m show remnant resin (pointer) on the fractured surface (C1).
At higher magnification it was possible to observe the presence of
mineral crystals (pointer) and no exposed collagen fibrils (C2). Also
in this case, the specimens aged in SBFS for 24 h show very few
mineral crystals embedded in the resin tags inside the tubules
(pointer) (C3). The SEM specimens of resin C group show a fractured
surface characterised by the presence of many exposed dentinal
tubules and remnant resin tags (D1). At higher magnification, it was
possible to note the presence of collapsed collagen fibrils (pointer),
exposed dentinal tubules (t) and remnant resin tags (rt) (C2). The 24 h
SBFS-aged bonded-dentine specimens showed a few exposed
dentinal tubules (pointer) and no exposed collagen fibrils (D3)
1530 J Mater Sci: Mater Med (2012) 23:1521–1532
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Acknowledgments This work was supported by grants CICYT/
FEDER MAT2008-02347, CICYT/FEDER MAT2011-24551,
JA-P07-CTS2568 and JA-P08-CTS-3944. This article presents inde-
pendent research commissioned by the National Institute for Health
Research under the Comprehensive Biomedical Research Centre at
Guy’s & St. Thomas’ Trust. The views expressed in this publication
are those of the author(s) and not necessarily those of the NHS, the
NIHR or the Department of Health. The authors also acknowledge
support from the Centre of Excellence in Medical Engineering funded
by the Wellcome Trust. The authors have no financial affiliation or
involvement with any commercial organization with direct financial
interest in the materials discussed in this manuscript. Any other
potential conflict of interest is disclosed.
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