JJOD-2131; No. of Pages 13

Dicalcium phosphate (CaHPO4�2H2O) precipitationthrough ortho- or meta-phosphoric acid-etching:Effects on the durability and nanoleakage/ultra-morphology of resin–dentine interfaces

Victor Pinheiro Feitosa a, Maria Giulia Bazzocchi b, Angelo Putignano c,Giovanna Orsini c, Arlinda Luzi Luzi d, Mario Alexandre Coelho Sinhoreti a,Timothy F. Watson e, Salvatore Sauro e,f,*aDivision of Dental Materials, Piracicaba Dental School, Piracicaba, BrazilbDepartment of Endodontic, Section of Pediatric Dentistry, Dental School, University of Bologna, Bologna, ItalycDepartment of Clinical Sciences and Stomatology, Polytechnic University of Marche, Ancona, ItalydDepartment of Restorative Dentistry and Endodontics, Universidad CEU Cardenal Herrera Valencia, SpaineBiomaterials, Biomimetics and Biophotonics, King’s College London Dental Institute, London, UKfDental Biomaterials Science and Minimally Invasive Dentistry, Universidad CEU Cardenal Herrera Valencia, Spain

j o u r n a l o f d e n t i s t r y x x x ( 2 0 1 3 ) x x x – x x x

a r t i c l e i n f o

Article history:

Received 2 February 2013

Received in revised form

13 August 2013

Accepted 19 August 2013

Available online xxx

Keywords:

Bond durability

Etch-and-rinse adhesives

Meta-phosphoric acid

Resin–dentine interface ultra-

morphology

Confocal microscopy

Calcium-staining technique

FTIR vibration analysis

a b s t r a c t

Objectives: To compare the effects of two etching procedures using meta-phosphoric (MPA)

or ortho-phosphoric acid (OPA) on dentine demineralisation, resin–dentine bonds durability

and interface nanoleakage/ultra-morphology.

Methods: Middle-dentine specimens were etched using 37% OPA (15 s) or 40% MPA (60 s) and

submitted to infrared spectroscopy (FTIR) or ultra-morphology dye-assisted (calcium-stain-

ing) confocal microscopy (Ca-CLSM). A three-step etch-and-rinse adhesive was formulated,

applied onto dentine and light-cured for 30 s before composite build-up. After 24 h, the

dentine-bonded specimens were cut into 1 mm2 beams; half were immediately submitted

to microtensile bond strength (mTBS) and half stored in DW for six months. The mTBS results

were analysed with repeated-measures ANOVA and Tukey’s test ( p < 0.05). Further teeth were

bonded and prepared for interface nanoleakage/ultra-morphology confocal evaluation.

Results: FTIR and Ca-CLSM analyses showed dicalcium phosphate dihydrate (Brushite)

precipitation in MPA-etched dentine and on the bottom (front of demineralisation) of the

OPA-etched dentine. Statistical analysis showed similar mTBS for both etching procedures

after 24 h. The mTBS of specimens in OPA-group dropped significantly ( p < 0.05) after six

month; the specimens in the MPA group showed no statistically difference ( p > 0.05). CLSM

depicted no evident sign of nanoleakage within the resin–dentine interface of the MPA-

treated specimens, while the specimens in OPA-group presented intense nanoleakage and

interface degradation.

Conclusion: The use of MPA (60 s) as an alternative dentine conditioning agent in etch-and-rinse

bonding procedures may be a suitable strategy to create more durable resin–dentine bonds.

# 2013 Elsevier Ltd. All rights reserved.

* Corresponding author at: Dental Biomaterials Science and Minimally Invasive Dentistry, Universidad CEU Cardenal Herrera en Valencia,C/Luis Vives, 1. Alfara del Patriarca, 46115 Valencia, Spain. Tel.: +36 961 36 90 00; fax: +44 020 71881823.

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: www.intl.elsevierhealth.com/journals/jden

E-mail addresses: salvatore.sauro@uch.ceu.us, salvatore.sauro@kcl.ac.uk, salvatore.sauro@me.com (S. Sauro).

Please cite this article in press as: Feitosa VP, et al. Dicalcium phosphate (CaHPO4�2H2O) precipitation through ortho- or meta-phosphoric acid-etching: Effects on the durability and nanoleakage/ultra-morphology of resin–dentine interfaces. Journal of Dentistry (2013), http://dx.doi.org/10.1016/j.jdent.2013.08.014

0300-5712/$ – see front matter # 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jdent.2013.08.014

JJOD-2131; No. of Pages 13

j o u r n a l o f d e n t i s t r y x x x ( 2 0 1 3 ) x x x – x x x2

Fig. 1 – Chemical structures of ortho- and meta-phosphoric

acids showing the protonable sites and acid dissociation

constants.

1. Introduction

Bonding procedures performed using etch-and-rinse adhe-

sives require necessarily an acid pre-treatment of the dental

hard substrates to consent the establishment of a micro-

mechanical interlocking; Michael Buonocore in 1955 firstly

demonstrated the importance to acid-etch the enamel surface

using ortho-phosphoric acid (OPA) prior resin composite

restorations.1

Twenty five years later Fusayama2 introduced a new acid-

and-rinse concept, demonstrating that dentine and enamel

could be simultaneously etched using OPA before bonding

procedures and resin composite restorations. Nowadays OPA

remains the main acid-conditioner in adhesive dentistry as it

allows clinicians to remove the smear layer and expose a layer

(6–9 mm) of demineralised collagen network.3,4 These demi-

neralised collagen fibrils remain occupied by water after

rinsing procedures5 preventing their collapse during resin

monomers diffusion (i.e. adhesive application).6 However, the

presence of water in the demineralised dentine may represent

a problem if not completely replaced during adhesive

application; residual water may prevent the creation of an

ideal encapsulation of the collagen fibrils and achievement of

an optimal and durable bonding interface.3,7

Direct evidence of hydrolytic8,9 and enzymatic degrada-

tion5,6 of hybrid layers demonstrated the inability of etch-and-

rinse bonding approaches to accomplish these aims; it is

particularly true when ‘‘simplified’’ adhesive systems are

employed in dentine bonding. Indeed, these latter bonding

systems have higher concentration of hydrophilic monomers

which lower the vapour pressure of solvents (i.e. ethanol and/

or acetone) and water and their ability to evaporate from the

etched-dentine. The presence of residual solvent/water within

the hybrid layer jeopardises the mechanical properties10 and

the degree of polymerisation of resin monomers compromis-

ing the longevity of the resin composite restorations.11,12

However, acid-etched dentine represents a functional

substrate for potential therapeutic treatments during bonding

procedures,13 such as application of collagen cross-linkers14,15

and matrix metalloproteinases (MMPs)/cysteine cathepsins

inhibitors.16,17

Furthermore, it has been shown that a suitable strategy to

increases the quality and the longevity of the resin–dentine

interface created with etch-and-rinse adhesives requires the

use of cheleting agents such as ethylenediaminetetraacetic-

acid (EDTA) with a more neutral pH6,7 rather than traditional

etchants based on OPA17,18;

Breschi et al.19 showed through high resolution SEM

evaluation how the use of maleic or citric acid may be able

to remove the smear layer and part of the smear plugs from

the dentinal tubules. However, it is important to consider that

the depth of dentine demineralisation accomplished by these

alternative etching procedures depend on the pH of the

solution (pH 0.7 < 1.4); furthermore, the use of maleic acid in

enamel etching may give controversial bond strength

results.20

Meta-phosphoric acid (MPA) is an interesting chemical

substance which produces metaphosphates in aqueous

solution through the ionisation of one hydrogen ion [H+].

Please cite this article in press as: Feitosa VP, et al. Dicalcium phosphate (etching: Effects on the durability and nanoleakage/ultra-morphology of r10.1016/j.jdent.2013.08.014

Metaphosphate anions present an effective cariostatic activity

in enamel, in particular when used in form of sodium

trimetaphosphate since the excellent chelating affinity for

calcium ions [Ca+2].20,21 Furthermore, metaphosphates

obtained by hydrolysis of trimetaphosphate in aqueous

environment may be used to cross-link the dentine collagen

fibrils and evoke biomimetic remineralisation processes.22,23

However, no investigation has highlighted the effects of the

MPA, when used as dentine conditioner in etch-and-rinse

bonding procedures, on the resin–dentine interfaces after

prolonged water storage.

The aim of this in vitro study was to compare the effects of

two etching procedures performed using a 40% meta-

phosphoric acid (MPA) or traditional 37% ortho-phosphoric

acid solution (OPA) on the mTBS (24 h and 6-month water

storage) and ultra-morphology/nanoleakage of resin–dentine

interfaces created with a standard etch-and-rinse adhesive.

The chemical and ultra-morphology changes induced by the

use of OPA or MPA when applied onto the dentine surface were

also characterised through vibrational spectroscopy (ATR-

FTIR) and dye-assisted Ca-staining confocal microscopy (Ca-

CLSM). The first null hypothesis to be tested is that there

would be no chemical and/or morphological difference

between the OPA-treated and MPA-etched dentine. The

second null hypothesis is that no difference would be found

in mTBS of bonded-dentine specimens created both on OPA-

etched or MPA-etched dentine after 6-month of water storage.

2. Materials and methods

2.1. Specimen preparation

Caries-free human molars (age 20–40) extracted for surgical

reasons under a protocol approved by the institutional review

board were used in this study; these teeth were stored in

distilled water for no more than one month (4 8C). The selected

teeth were sectioned below the cemento-enamel junction

(Accutom-50; Struers, Copenhagem, Denmark) using a water-

cooled diamond saw (330-CA RS-70300; Struers) to remove the

roots. The occlusal surface was made flat (Meta-Serv 3000

CaHPO4�2H2O) precipitation through ortho- or meta-phosphoric acid-esin–dentine interfaces. Journal of Dentistry (2013), http://dx.doi.org/

Table 1 – Composition of the etch-and-rinse adhesiveused in this study.

Standard resin bonding system

Bond resin Primer resin

(pH �6.5) (pH �6.8)

40 wt% E-BisADM 20 wt% E-BisADM

30 wt% TEGDMA 15 wt% TEGDMA

30 wt% HEMA 15 wt% HEMA

– 50 wt% ethanol

E-BisADM: ethoxylated-Bisphenol A dimethacrylate; TEGDMA:

triethyleneglycoldimethacrylates; HEMA: 2-hydroxyethyl metacry-

late.

CQ, camphoroquinone (0.50 wt%); EDAB, 2-ethyl-dimethyl-4-ami-

nobenzoate (0.50 wt%); DPIHP, diphenyliodonium hexafluoropho-

sphate (0.50 wt%) were added as wt% at the end of the final resin

monomers blend (100 wt%) formulation.

j o u r n a l o f d e n t i s t r y x x x ( 2 0 1 3 ) x x x – x x x 3

JJOD-2131; No. of Pages 13

Grinder-Polisher, Buehler Lake Bluff, IL, USA) using ascending

SiC abrasive papers 180#-to-320#-grit (Versocit; Struers,

Copenhagen, Denmark) under constant water irrigation to

provide a standardised smear-layer in middle dentine surface.

The dentine specimens were divided in two principal

groups and acid-etched for 15 s using 37% ortho-phosphoric

acid (OPA; pH < 1) or 40% meta-phosphoric acid (MPA; pH 2.6)

for 60 s (Fig. 1); these were finally rinsed with deionised water

(DW) for 15 s. The two acids (powder purity > 98%) were

purchased from Sigma–Aldrich (Sigma–Aldrich Chemical,

Gillingham, UK).

2.2. Characterisation of the OPA and MPA-etched dentine

Three middle-dentine specimens were submitted to ATR-FTIR

vibrational analysis (FTIR) before and after acid-etching

treatment using OPA or MPA.24 FTIR analysis was performed

in wet (excess of water removed using absorbent paper points)

or dry (excess of water totally removed by 10 s air-drying)

using the ATR-FTIR Spectrometer (Perkin-Elmer Spectrum

One; Perkin-Elmer, Beaconsfield, UK) in the region of 650–

4000 cm�1 with a resolution of 4 cm�1 and 64 number of scans

for each spectrum. The ATR area had a 2 mm diameter and the

IR radiation penetration was approximately 3–5 mm. In order

to place the specimens in good contact with the ATR crystal, a

moderate pressure was applied (5 psi) during the measure-

ment to achieve high quality results, but at the same time, to

avoid compression of the demineralised collagen to the

underlying mineralised dentine. A control FTIR analysis was

performed for the commercial high purity (>97%) hydroxyap-

atite and brushite (Sigma–Aldrich). To minimise the homoge-

neity variability of the specimens, three spectra were recorded

in different positions of the surface.25

Further four specimens per group were prepared as

previously described and submitted to the dye-assisted

calcium-staining confocal microscopy analysis (CLSM-Ca) in

order to evaluate the morphology features induced by the two

different etching agents (OPA or MPA).25 In details, two

specimens were immediately immersed in a 0.5 wt% calci-

um-chelating dye solution26 (Xylenol Orange; Sigma–Aldrich)

for 24 h at 37 8C (pH 7.2), while two specimens were immersed

for 24 h at 37 8C in a 0.1 wt% dye water/ethanol (50v%/50v%)

solution (FITC – fluorescein isothiocyanate; Sigma–Aldrich)

able to covalently bind collagen.27 Subsequent to the dye

immersion, the specimens were copiously rinsed with water

and treated in an ultrasonic water-bath for 5 min and then

submitted to confocal microscopy evaluation. The confocal

microscopy analysis was performed using a confocal laser

scanning microscope (DM-IRE2 CLSM; Leica, Heidelberg,

Germany) equipped with a 63�/1.4 NA oil immersion lens

and a 488-nm Argon/Krypton ions laser (Fluorescein excita-

tion) or 568-nm Helium/Neon ions laser. The emission

fluorescence was recorded at 512–538 nm and 585–650 nm,

respectively; control dentine (sound – no treatment) speci-

mens were also employed in the Ca-CLSM analysis. CLSM

images were obtained with a 1 mm Z-step (Z-stack) or X-step

(X-stack) to optically section the specimens to a depth up to

25 mm. The Z-stack and X-stack scans performed along the

interface were arbitrarily pseudo-coloured by the same

operator for better exposure and compiled into single

Please cite this article in press as: Feitosa VP, et al. Dicalcium phosphate (etching: Effects on the durability and nanoleakage/ultra-morphology of r10.1016/j.jdent.2013.08.014

projections or 3D images using the Leica image-processing

software (Leica).9 The configuration of the system was

standardised and used throughout the entire investigation.

Each slab was completely investigated and micrographs

representing the most common features observed along the

etched dentine surfaces were captured and recorded.

2.3. Adhesive formulation and bonding procedures

An experimental etch-and-rinse adhesive (primer and bond)

was formulated and used in this study in order to standardise

the chemical composition by having constant concentration

of monomers, solvents, initiators and co-initiators. A neat

resin blend mixture was formulated using 30 wt% 2-hydro-

xyethyl metacrylate (HEMA: Aldrich Chemical Co., Gillingham,

UK), 30 wt% triethylene-glycol-dimethacrylate (TEGDMA),

40 wt% ethoxylated-Bisphenol-A-dimethacrylate (E-BisADM),

(Esstech Essington, PA, USA). The resin blend was made photo-

polymerisable by adding 0.5 wt% camphoroquinone (CQ:

Aldrich Chemicals), 0.5 wt% ethyl-4-dimethylaminobenzoate

(EDMAB: Aldrich Chemicals) and 0.5 wt% diphenyliodonium-

hexafluorophosphate13 (DPIHP: Aldrich Chemicals).

Five-minute sonication (Model QS3; Ultrawave Ltd., Cardiff,

156 UK) and 2-day shaking (Orbital Shakers PSU-20i; Cole

Fisher 157 Scientific Ltd., Loughborough, UK) were required to

yield well-mixed resin mixtures (i.e. primer and bond).13

Subsequently, a resin primer system was created by mixing

50 wt% absolute ethanol (Aldrich) and 50 wt% of neat co-

monomer resin blend; the neat resin (solvent-free) was also

used as a resin bond system. The detailed composition of the

DBA used in this study is shown in Table 1.

The bonding procedures were performed by applying two

consecutive coats of the experimental primer on the etched-

dentine specimens and gentle air-drying for 5 s. Two conse-

cutive coats of the experimental solvent-free resin bond were

immediately applied, spread thin with moisture-free air-blast

and light-cured for 30 s using a halogen light-curing unit

(Translux EC; KulzerGmBh, Bereich Dental, Werheim,

Germany). The output intensity (>600 mW/cm2) was moni-

tored with a Radiometer (Demetron Model 100; Demetron,

Danbury, CT, USA). Composite build-ups (6 mm) were layered

CaHPO4�2H2O) precipitation through ortho- or meta-phosphoric acid-esin–dentine interfaces. Journal of Dentistry (2013), http://dx.doi.org/

Table 2 – Microtensile bond strength values (MPa) todentine when resin adhesive was applied in OPA or MPAtreated dentine.

Dentine conditioning(groups)

Deionised waterstorage (AGEING)

24 h 6 months

Ortho-phosphoric

acid (OPA)

48.4 (8.3)a * (0/50)

[5/35/60]

17.6 (14.3)b (11/39)

[55/40/5]

Meta-phosphoric

acid (MPA)

49.2 (8.3)a (0/50)

[0/65/35]

51.1 (7.8)a (0/50)

[0/86/13]

Values are mean (SD) in MPa.

In columns, same superscripts letters indicate no differences

( p > 0.05).

(*) Differences between different period of storage.

Premature failures were included in the statistical analysis as zero

values and are indicated in parentheses (for instance 18/22 means

that there were 18 premature failures and 22 testable beams).

The modes of failure are expressed in percentage in the brackets

[adhesive/mix/cohesive].

j o u r n a l o f d e n t i s t r y x x x ( 2 0 1 3 ) x x x – x x x4

JJOD-2131; No. of Pages 13

using a light-cured flowable resin composite (TetricEvoFlow;

Ivoclar-Vivadent, Schaan, Liechtenstein) in six 1-mm-thick

increments. The resin–dentine specimens were stored in DW

for 24 h at 37 8C before further processing.

2.4. Microtensile bond strength test (mTBS)

Five Resin–dentine specimens were created in middle-dentine

as previously described for each experimental group (OPA and

MPA). They were sectioned with a diamond wafering blade in

both X and Y directions across the resin–dentine interface to

obtain beams with cross-sectional area of 1 mm2. By excluding

peripheral beams showing the presence of residual enamel,

only the central 10 beams were used from each tooth. Thus,

there were 10 beams � 5 teeth = 50 beams in each sub-group

(Table 2).

Half of the beams for each group were tested after 24 h and

half tested after 6 months of DW storage DW (37 8C) in a

testing apparatus (Bencor Multi-T; Danville Engineering Co.,

Danville, CA, USA) mounted on a universal testing machine

(Instron 4411; Instron Corporation, Canton, MA, USA) at a

crosshead speed of 0.5 mm/min. Bond strength results were

calculated in MPa and the values of the beams from the same

restored tooth were averaged and the mean bond strength

was used as statistical unit for the statistical analysis.8

However, prior the execution of the micro-mechanical test

(mTBS), a pilot study was performed to establish the most

appropriate etching time for the 40% MPA. It was observed

that the minimal application time required in order to achieve

a reliable micro-tensile bond strength was 60 s; this latter did

not clearly changed when exciding the 60 s of MPA applica-

tion (Unpublished results).

The mTBS (MPa) data for each group were subjected to a

repeated measures ANOVA (24 h and 6 months) and Tukey’s

post hoc test for pair-wise comparisons (a = 0.05). Bond

strengths of sticks from the same bonded-tooth were averaged

and the mean was used as a statistical unit. The pre-test failed

beams were set as 0 MPa and were included in the statistical

analysis.

Please cite this article in press as: Feitosa VP, et al. Dicalcium phosphate (etching: Effects on the durability and nanoleakage/ultra-morphology of r10.1016/j.jdent.2013.08.014

Modes of failure were classified as adhesive (A), mixed (M),

or cohesive (C) when the failed bonds were examined at 30� by

stereoscopic light microscopy. Ten fractured specimens

exhibiting the most frequently observed failure pattern (i.e.

mixed or cohesive) and the mTBS close to the mean of each

respective group were selected and processed for SEM ultra-

morphology analysis.8 They were first dehydrated through

ascending alcohols (40–90%) for 3 min each step and finally

immersed for 10 min in absolute ethanol. The specimens were

subsequently exsiccated in a dry incubator (40 8C; 24 h) and

then mounted on aluminium stubs with carbon cement. They

were sputter-coated with gold (SCD 004 Sputter Coater; Bal-

Tec, Vaduz, Liechtenstein) and imaged using a scanning

electron microscope (SEM S-3500; Hitachi, Wokingham, UK)

with an accelerating voltage of 15 kV at increasing magnifica-

tion from 60� to 3000�.

2.5. Confocal interfacial ultra-morphology andnanoleakage evaluation

Three further resin–dentine specimens for each group were

created using the resin primer and bond doped with 0.1 wt%

rhodamine-B (Rh-B; Sigma Chemicals, St. Louis, USA) and

employed for the confocal microscopy analysis (CLSM).14

The specimens were serially sectioned across the adhesive

interface to obtain resin–dentine slabs with a thickness of

1.0 mm. These slabs (n = 10/group) were then divided into 2

subgroups based on the period of storage in DW (24 h/6 m).

Subsequently, these specimens were coated with 2 layers of

fast-setting nail varnish applied 1 mm from the resin–

dentine interfaces and immersed in 1 wt% aqueous fluores-

cein dye solution for 24 h.28 The specimens were then

treated in an ultrasonic water bath for 2 min and polished

using ascending (#1200-to-#4000) SiC abrasive papers (Ver-

socit; Struers) on a water-cooled rotating polishing machine

(Meta-Serv 3000; Buehler). A final ultrasonic water bath

treatment (5 min) completed the specimen preparation for

the CLSM evaluation.

The microscopy examination was performed using a 63�/

1.4 NA oil immersion lens using a 488 nm (fluorescein

excitation) or a 568 nm ions laser illumination. The reflec-

tion imaging was performed using the 488 nm laser. The

emission fluorescence was recorded at 488–510 nm and 570–

590 nm, respectively. CLSM reflection and fluorescence

images were obtained with 1 mm z-step to section optically

the specimens to a depth up to 20 mm below the surface. The

Z-stack scans were compiled into single projections and

arbitrarily pseudo-coloured as previously described. The

configuration of the system was standardised and used

throughout the entire investigation. Each resin–dentine

interface was firstly completely analysed and then only 5

optical images were randomly captured; those representing

the most common features of nanoleakage observed along

the bonded interfaces.14

3. Results

The FTIR spectra recorded in sound, etched-dentine speci-

mens (MPA and OPA), commercial hydroxyapatite and

CaHPO4�2H2O) precipitation through ortho- or meta-phosphoric acid-esin–dentine interfaces. Journal of Dentistry (2013), http://dx.doi.org/

j o u r n a l o f d e n t i s t r y x x x ( 2 0 1 3 ) x x x – x x x 5

JJOD-2131; No. of Pages 13

brushite (control powders) are shown in Fig. 2.The spectra

generated from the dentine surface before OPA or MPA acid-

etching presented phosphate bands at 885–1180 cm�1 (phos-

phate stretching mode) representative of mineral components

and amide bands from organic components (1200–1725 cm�1).

The commercial brushite and the dentine surface etched

with MPA showed bands at 3544, 3491, 3290, 3163 and 2955 due

to the O–H stretching of water (H2O), while the OPA-treated

dentine showed an higher H2O band at 3200–3400 cm�1; the

H2O band was clearly less intense in the dried specimens. The

commercial brushite also showed the presence of H2O at

1653 cm�1 (bending mode), O–H in-plane bending at 1219 cm�1

and H2O oscillating motion at 791 cm�1. The dentine surface

treated using MPA showed the presence of collagen in the

same region (C O: 1650 cm�1 – amide I) and (CNH: 1540 cm�1 –

amide II). The PO stretching peaks of the commercial brushite

as well as that of MPA-treated the specimens were observed at

1134, 1057, and 987 cm�1. Sound dentine presented the PO

peaks at 961 (n1), 1019 (n3 – asymmetric stretching mode of

hydroxyapatite) and carbonate bands at 1400–1500 cm�1.

The OPA-treated dentine showed low intensity peaks at

1080 and 1030 cm�1 related to the P–OR esters. Furthermore, P–

O(H) stretching was found at 876 cm�1 in all the specimens,

except for the OPA-treated dentine. In summary, the FTIR

survey showed the presence of brushite after etching the

dentine surface using MPA, whereas no signs of superficial

minerals (3–5) mm were detected in OPA-treated dentine.

Fig. 2 – FTIR-ATR characterisation of the dentine surface before a

Bands at 3200–3400 cmS1 represent the O–H stretching of water

hydroxyapatite and acid-etched dentine surfaces; brushite also

in-plane bending at 1219 cmS1 and in oscillating motion (librati

found at 1134, 1057, and 987 cmS1, while hydroxyapatite presen

mode of hydroxyapatite) and carbonate bands at 1400–1500 cm

collagen is visible in at 1650 cmS1 (C O: amide I) and 1540 cmS

Please cite this article in press as: Feitosa VP, et al. Dicalcium phosphate (etching: Effects on the durability and nanoleakage/ultra-morphology of r10.1016/j.jdent.2013.08.014

However, when these specimens were over-dried and the

demineralised collagen collapsed, it was possible to detect the

representative peaks of brushite from the underlying front of

demineralisation (Fig. 2). The CLSM-Ca analysis confirmed the

precipitation of calcium-minerals (Fig. 3A) along 8–10 mm of

the MPA-etched dentine (Fig. 3A1); however, only 5–7 mm of

fluorescein-stained mineral-depleted collagen were detected

(Fig. 3B1 and B2). Conversely, the dentine specimens treated

using OPA (Fig. 3C) showed a thicker layer (12–14 mm) of

fluorescein-stained demineralised collagen and only 3–4 mm

of calcium-minerals deposition (Fig. 3C1) underneath

this layer.

The microtensile bond strength (mTBS) was affected by the

different acid-etching procedure employed in this study

( p < 0.05); means, standard deviations, number of pre-test

failures and the spreading of failure patterns are depicted

in Table 2.

In details, it was observed that subsequent to 24 h of DW

storage, the mTBS values of the specimens in the groups OPA

and MPA were similar ( p > 0.05). After 6 months of ageing in

DW, the mTBS of OPA group statistically dropped (�64%)

compared to the results obtained after 24 h. Conversely, the

mTBS of MPA group remained stable after ageing with no

statistical difference ( p > 0.05) compared to the control group

(24 h in DW).

Different fractures were observed after etching treatments

in both periods of storage. In particular after 24 h, the

nd after acid-etching using ortho- or meta-phosphoric acid.

(H2O) both in commercial (control powders) brushite/

showed the H2O bending mode at 1653 cmS1 and the O–H

on mode) at 791 cmS1. The PO stretching of the brushite is

ts the PO peaks at 961 (n1), 1019 (n3 – asymmetric stretchingS1. P–O(H) stretching is found at 876 cmS1. The dentine1 (CNH: amide II).

CaHPO4�2H2O) precipitation through ortho- or meta-phosphoric acid-esin–dentine interfaces. Journal of Dentistry (2013), http://dx.doi.org/

Fig. 3 – Dye-assisted calcium-staining confocal microscopy characterisation of the OPA and MPA-etched dentine. Confocal

single Z-stack projection (calcium-staining fluorescence) of the dentine surface treated using MPA for 60 s (A) showing a

clear presence of Ca-minerals (8–10 mm) along the demineralised substrate (A1–X-stack projection). However, it is also

possible to observe both in the Z-stack image (B1) and in the 3D reconstructed image (B2) that the thickness of collagen

within this demineralised layer (collagen-staining fluorescence) is in a range of 6–8 mm.

The confocal single Z-stack projection (calcium-staining fluorescence) of the dentine surface treated using OPA (C) shows

only a slight presence of Ca-minerals deposited (3–4 mm) within the demineralised substrate (C1–X-stack projection). In

this case, the OPA-treated dentine surface submitted to fluorescence collagen-staining technique (D) presented both in

the Z-stack image (D1) and in the 3D image (B2) that the thickness of the demineralised collagen was in a range of 12–

14 mm.

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Please cite this article in press as: Feitosa VP, et al. Dicalcium phosphate (CaHPO4�2H2O) precipitation through ortho- or meta-phosphoric acid-etching: Effects on the durability and nanoleakage/ultra-morphology of resin–dentine interfaces. Journal of Dentistry (2013), http://dx.doi.org/10.1016/j.jdent.2013.08.014

Fig. 4 – SEM micrographs showing fractures at low (300T) and higher (2500T) magnification captured in dentine side of de-

bonded beams subsequent to mTBS testing. (A) The beams of the OPA group tested after 24 h of storage in DW, which de-

bonded prevalently in mixed mode (arrow), present remnant collagen fibrils (open arrows) and totally or partially

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specimens etched using OPA mainly de-bonded in cohesive

(60%) and mixed (35%) mode, while after 6 months of DW

ageing a remarkable number of prematurely failed sticks was

attained and the main failures occurred during the mTBS test

were adhesive (55%) and mixed (40%) mode. Conversely, the

specimens etched with MPA showed no adhesive failure both

after 24 h and 6 months of DW storage. The main failure mode

observed after 24 h during the mTBS test was mixed (65%) and

cohesive (35%). Subsequent to prolonged DW ageing (6

months), the specimens debonded mainly in mixed mode

(86%).

Representative SEM images showing the main failure

features (e.g. Fractography) are displayed in Fig. 4. Briefly,

the specimens of OPA group (24 h) showed the presence of

fractured resin tags inside the dentinal tubules and collagen

fibrils that were possibly detached from tat the bottom of the

hybrid layer during the microtensile test (Fig. 4A). Conversely,

after 6 months of DW storage, most of the fractures occurred

at the bottom of hybrid layers showing a very rough collagen-

free dentine surface due to severe interface degradation and

several patent tubules with residual resin tags (Fig. 4B).

The specimens from the MPA-treated dentine group

showed no clear presence of collagen on the fractured surface

(Fig. 4C). Likewise, the same specimens stored for 6 months in

DW debonded prevalently at the bottom of hybrid layer

leaving a well mineralised surface as well as intratubular

dentine portion; particular remnants of adhesive and resin

tags were also detected (Fig. 4D).

The features of CLSM nanoleakage are presented in

Fig. 5.The hybrid layer of OPA-treated specimens showed

intense fluorescein uptake (Fig. 5A) within the thick resin–

dentine interface (�10–12 mm). Subsequent to the DW ageing

period, it was possible to observe only a partial presence of

nanoleakage within the resin–dentine interface due to the

degeneration of the hybrid layer structure (Fig. 5B). The OPA-

etched specimens created a thicker hybrid layer compared

to that observed in the MPA-treated specimens. The resin–

dentine interface of the MPA group showed high resistance

to fluorescein infiltration; it was only rarely present at the

bottom of the adhesive layer (Fig. 5C and C1). Nevertheless,

an evident fluorescein penetration was detected within the

adhesive layers of the specimens submitted to prolonged

ageing in DW (Fig. 5D and D1).

4. Discussion

The two essential processes involved in dentine etch-and-

rinse bonding procedures are the dissolution of the mineral

obliterated (resin tags) dentinal tubules. (B) In OPA group, the bea

in adhesive mode at the bottom of hybrid layer (open arrow) le

dentinal tubules, probably due to peritubular dentine degradati

The beams of the MPA group tested after 24 h of DW storage d

dentine with no clear presence of exposed collagen fibrils (arrow

fracture is represented by de-bonding along the hybrid layer (op

months of DW storage showed the absence of exposed collage

some fractured resin tags inside the dentinal tubules (arrows) a

(open arrows).

Please cite this article in press as: Feitosa VP, et al. Dicalcium phosphate (etching: Effects on the durability and nanoleakage/ultra-morphology of r10.1016/j.jdent.2013.08.014

phase (i.e. 35–40% phosphoric acid) and the infiltration of the

demineralised collagen matrix with adhesive resin monomers

which polymerise in situ to form the hybrid layer (HL).29,30

However, hybrid layers with a high quality and longevity

characteristics can be only achieved if the acid-etched dentine

is fully resin-infiltrated.31 Unfortunately, complete resin

monomers diffusion within OPA-etched dentine is such a

complex task to achieve during in situ bonding procedures in

vital teeth. Indeed, the resulting poorly-infiltrated phases (i.e.

nano- and micro-porosities) within the resin–dentine inter-

face represent a clear evidence of such a critical condition.32

Such porosities are relatively rich in water content and they

symbolise the main centres for the enzymatic (MMPs 2, 8, 9, 20)

collagen degradation which, in part, contributes to the

reduction of the resin–dentine durability.33–36 Moreover,

hydrolysis of specific resin-monomers (i.e. methacrylates

ester bonds) caused by salivary or bacterial esterases contrib-

utes to the degradation of the hybrid layer jeopardising the

longevity of resin-composite restorations.37–39 Nowadays, it is

of primary importance to find a suitable strategy which may

increase the longevity of adhesive restorations.40–42

The first null hypotheses tested in this study that no

difference would be observed between the OPA-treated and

MPA-etched dentine must be rejected due to the chemical

differences encountered during the FTIR and morphological

CLSM-Ca assessment. The second null hypothesis that no

difference would be found in microtensile bond strength

(mTBS) after 6-month of water storage for the resin–dentine

interfaces created in OPA-etched or MPA-etched dentine

needs to be also rejected as the specimens in OPA-group

showed important differences when compared to those

specimens in MPA-group.

In details, the resin–dentine specimens created in MPA-

etched dentine showed no statistical mTBS drop after 6 months

of DW storage ( p > 0.05), while OPA-etched bonded specimens

showed significant lower mTBS results ( p < 0.05). The speci-

mens of the MPA group showed no adhesive failures after DW

storage; however they mainly de-bonded in mixed (65%) and

cohesive (35%) after 24 h and in mixed mode (86%) after 6

months. Contrariwise, 60% of the specimens in OPA group de-

bonded in cohesive mode, 35% in mixed mode and only 5% of

the specimens failed in cohesive mode after 24 h. The

prolonged DW storage (6 months) caused an evident change

in the failure mode of the specimens in OPA group which de-

bonded prevalently in adhesive mode (55%) and mixed mode

(40%), (Table 2).

The SEM analysis of the de-bonded specimens in the OPA

group (24 h) showed that the fractured surface were char-

acterised by the presence of some collagen fibrils which were

ms tested after 6 months of DW storage de-bonded mainly

aving a rough dentine surface characterised by wide open

on, and very few residual fractured resin tags (arrow). (C)

e-bonded prevalently in mixed mode showing a fractured

) but a uniform presence of remaining resin adhesive; the

en arrows). (D) The specimens in MPA group tested after 6

n fibrils but a fractured dentine surface characterised by

nd small remnants of hybrid and adhesive layers

CaHPO4�2H2O) precipitation through ortho- or meta-phosphoric acid-esin–dentine interfaces. Journal of Dentistry (2013), http://dx.doi.org/

Fig. 5 – Single projections (reflection/fluorescence) obtained during the CLSM ultramorphology/nanoleakage analysis of

the resin–dentine interfaces created in MPA or OPA etched-dentine. (A) Resin–dentine interface created in OPA-etched

dentine showing a thick hybrid layer (HL) completely infiltrated by fluorescein (nanoleakage) due to imperfect resin

infiltration within the demineralised collagen matrix (porous hybrid layer). It is also possible to observe long resin-tags

penetrating several microns into the dentinal tubules (t). (B) Resin–dentine interface created in OPA-etched dentine and

stored in DW for 6 months. This image evidently show degradation of the HL characterised by micro-imperfections

(pointers) located between the dentine and the adhesive-(a)/composite-(c) layers. However, although part of the hybrid

layer (HL) is preserved within the resin–dentine, the entire interface is affected by severe nanoleakage. (C) Resin–dentine

interface created in MPA-etched dentine showing a thick adhesive layer (a) and short resin-tags (rt). Note the limited

presence of fluorescein (nanoleakage) within the resin–dentine interface (C1). (D) Resin–dentine interface created in MPA-

etched dentine aged in DW for 6 months shows limited presence of nanoleakage (fluorescein infiltration) within the resin–

dentine interface. Nevertheless, it is clear to observe the presence of dye penetration throughout the entire thickness of

the adhesive layer (D1).

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probably poorly resin-infiltrated (Fig. 4A). The specimens from

the same group de-bonded prevalently at the bottom of hybrid

layer after 6 months of DW storage leaving a dentine surface

with several patent tubules presenting a large diameter, likely

due to degradation of the intratubular dentine (Fig. 4B).

Conversely, the specimens created on the MPA-etched dentine

showed no presence of collagen fibrils both after 24 h and 6

months of DW storage (Fig. 4C and D) indicating that the

demineralised dentine collagen network may have been better

infiltrated by resin monomers; residual adhesive resin and

Please cite this article in press as: Feitosa VP, et al. Dicalcium phosphate (etching: Effects on the durability and nanoleakage/ultra-morphology of r10.1016/j.jdent.2013.08.014

fractured resin tags were often observed on the de-bonded

specimens (Fig. 4B).

The confocal microscopy analysis performed in wet

condition (oil immersion specimens) supported the features

attained in the mTBS/failure mode analysis by showing intense

fluorescein nanoleakage (Fig. 5A) within the resin–dentine

interfaces of the OPA group submitted to 24 h of DW storage; a

severe degradation of the hybrid layer was observed subse-

quent to prolonged DW immersion (Fig. 5B). On the other

hand, the resin–dentine interface of the MPA-etched

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specimens showed high resistance to fluorescein infiltration

both after 24 h (Fig. 5C and C1) and 6 months of DW storage

(Fig. 5D and D1) with no sign of degradation at the resin–

dentine interface.

The results obtained in this study may be possibly

attributed to the milder acidity and the particular acid-

ionisation constant (Ka) of MPA (Fig. 1) which contributed to

the creation of a thinner partially demineralised collagen layer

(Fig. 3D) characterised by an essential presence of mineral

precipitation before and after rinsing procedures (Figs. 2 and

3A). The etching time is dependent on the pH, buffer capacity

and number of hydrogen ions available for deprotonisation in

the chemical nature of the etching agent.51 MPA differs in

many aspects from OPA; in particular, it has only one

hydrogen ion available for deprotonisation, a mild pH (2.5

with �40% concentration) and a great buffering ability.

Meanwhile, OPA has three protonable hydrogen ions and a

very low pH (<1.0 with 35–37% concentration).

FTIR analysis performed in wet and dry conditions showed

the precipitation of dicalcium phosphate (Brushite: CaH-

PO4�2H2O) in the MPA-etched dentine specimens. Conversely,

the specimens etched with OPA showed the presence of

brushite only when over-dried and the demineralised collagen

collapsed; it may be hypothesised that the rinsing procedure

removed most of the calcium phosphates from the surface of

the OPA-etched dentine.

It has been reported that the extra-fibrillar and the intra-

fibrillar mineral of dentine surfaces etched with OPA are

totally dissolved; this may increase the risk for demineralised

fibrils to dehydrate and collapse during the bonding proce-

dures.43 Indeed, in case of collagen matrix shrinkage the

individual collagen fibrils touch each other and create

chemical interactions (mainly H-bonds) which then require

a great effort from the solvated co-monomers to diffuse

around the fibrils and break these chemical bonds to allow

optimal resin encapsulation.6,44,45 Moreover, it has been

demonstrated through micro-Raman spectroscopy the impact

of water present within the demineralised dentine on the

development of an ideal resin–dentine interface; the hybrid

layer is mainly characterised by collagen and HEMA, with poor

contribution from the hydrophobic components (i.e. Bis-

GMA).7,46

In this regard, we hypothesise that the thinner demi-

neralised collagen layer created by MPA-etching, along

with the presence of residual brushite crystallites and a

lower presence of water within the collagen fibrils (Fig. 2),

may have prevented the shrinkage of the collagen matrix

during infiltration of co-monomers, thus facilitating the

diffusion of the adhesive within the demineralised den-

tine.18,47 A remarkable feature which may support this

hypothesis can be found in the SEM results of the de-

bonded specimens created in MPA-etched dentine, which

showed the absence of recession in the mineralised

peritubular dentine (Fig. 4C) with the maintenance of the

diameter of the tubules lumen of the tubules (intact

intratubular dentine); these features were not observed

in the de-bonded specimens created in OPA-etched

(Fig. 4D). This correlation with the characteristics of the

intratubular dentine is in accordance with previous

observations reported by Marshall et al.6

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A further possible explanation for the results attained with

the specimens of the MPA group may be attributed to the

presence of brushite precipitation (Figs. 2 and 3A) within the

partially demineralised dentine which might have protected

sensitive cleavage sites of collagen from the MMP degradation.

This hypothesis seems to be in accordance with the results

obtained by Osorio et al.48 who have recently demonstrated

that the quality and the longevity of the resin–dentine

interface may be increased by using innovative dental

adhesives able to induce proteolytic resistance48 and mineral

precipitations in demineralised dentine explants.49,50

However, future studies are necessary to evaluate if the

brushite precipitation induced by MPA during etching proce-

dures may inhibit MMPs and/or protect the demineralised

dentine collagen degradation. Furthermore, further studies

are already in progress to evaluate the ability of commercial

and experimental etch-and-rinse adhesive systems formulat-

ed with or without the use of functional monomers to bond

MPA-etched enamel as the use of mild acids in enamel etching

may give controversial bond strength results.20

It is important to define that dicalcium phosphates such as

brushite and monetite are stable at low pH (<4.2).52,53 An

interesting paper of Jiang et al.52 has demonstrated that the

transformation from brushite to stable hydroxyapatite is also

possible when immersed in a calcium-ion saturated solution

and through pH increase. Nowadays, this transformation is

used in bio-engineered scaffolds to biomineralise bone

tissues via hydroxyapatite crystallisation.54 In dentistry, this

type of transformation is not new and it has been explored for

some decades55,56; indeed, dentine acid-etching procedures

induce the release of calcium and phosphate which may re-

precipitate as brushite, monetite or octacalcium phosphate

depending on the environmental pH. At pH below 3, such as in

the case of MPA and OPA solutions, brushite is maily

precipitated,53,55 while in very low pH (<1.0) there is the

formation of more ionic forms of calcium and phosphate

rather than brushite, very easily removed by water rinsing.

The low solubility of brushite (created after MPA-etching)

along with the volume of its crystallites57 contributed to the

entrapment of minerals within the demineralised dentine

collagen also after vigorous water rinsing. However, the

possible formation of hydroxyapatite from the brushite

within the demineralised dentine and resin–dentine inter-

face requires further investigation and cannot be confirmed

by our findings.

Although the effectiveness of MPA to create a potential

substrate suitable to create resin–dentine interface with

improved longevity compared to those attained in traditional

OPA-etched dentine was proven in this study (Table 2), it may

be also speculate that metaphosphate anions (MPAa) derived

from the dissociation of the MPA-etchant once applied on

dentine could remain chemically bound to collagen fibrils as

demonstrated by Gu et al.22 and Liu et al.23 when using

hydrolysed sodium trimetaphosphate as a biomimetic analo-

gues of matrix phosphoproteins. This might also favour

dentine collagen remineralisation in presence of super-

saturated solutions containing specific ions (e.g. Ca+2 and

PO43�). However, further investigations are already on going to

confirm this hypothesis of remineralising resin–dentine

interfaces subsequent etching procedures performed using

CaHPO4�2H2O) precipitation through ortho- or meta-phosphoric acid-esin–dentine interfaces. Journal of Dentistry (2013), http://dx.doi.org/

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MPA with or without rinsing step and subsequent application

of ions releasing restorative/adhesive materials.58

It is also important to consider that the experimental

adhesive used in this study was formulated based on a tertiary

photo-initiator/co-initiator system (Table 1) containing cam-

phoroquinone (CQ), 2-ethyl-dimethyl-4-aminobenzoate

(EDAB), and diphenyliodonium hexafluorophosphate (DPIHP;

hydrophilic activator). This latter component (DPIHP) has been

demonstrated to increase the degree of conversion and the

monomer cross-linking ability of adhesive systems containing

hydrophilic monomers such as HEMA12 and/or phosphate

monomers (acidic functional monomer).59 However, DPIHP

seems to offer a greater contribute on the longevity of the

resin–dentine bonds (mTBS results after 6 mouths of DW

storage) only in presence of partially mild-demineralised

dentine collagen (Figs. 2 and 3) or in case self-etch/two-step

adhesive systems were employed.59

We suppose that the differences observed in the mTBS

results attained in this study and in those recently reported by

Leal at al.59 who showed that a model self-etch/two-step

adhesive formulated using a CQ/EDAB/DPIHP system was able

to maintain high mTBS results after one year of DW storage,

may be probably due to the less water content within the

etched-dentine substrate (low nanoleakage expression;

Fig. 5C1 and D1) which facilitated the diffusion and a greater

polymerisation of more hydrophobic resin monomers within

the bonding interface (Fig. 5C and D).

The clinical relevance extrapolated from the results

attained in this study are that alternative etching procedures

using MPA may provide a suitable dentine substrate to create

resin–dentine interfaces with higher durability, despite the

time required for MPA etching is much longer (60 s) than 35–

37% OPA in a clinical scenario (10–15 s).

In conclusion, it possible to affirm that the use of MPA as an

alternative dentine acid etching agent induces reliable

brushite precipitation which benefits the durability and

nanoleakage/ultra-morphology of resin–dentine interfaces.

On-going in vitro studies will highlight the durability and the

ultra-morphology characteristics of the resin–dentine inter-

faces created using commercial etch-and-rinse and self-

etching adhesives applied on MPA-conditioned dentine.

Conflict of interest

There are no known conflicts of interest.

Contribution

Dr. Feitosa performed part of the experimental processes and

wrote the first draft of the manuscript (confocal microscopy

and optical analysis of the fracture).

Dr, Bazzocchi performed part of the experimental process-

es (micro-tensile testing).

Prof. A. Putignano proofread the manuscript and contrib-

uted substantially for the discussion and conclusion of the

manuscript.

Prof. G. Orsini performed part of the experimental

processes (SEM-analysis and proofread the manuscript).

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Prof. A. Luzi Luzi performed the FTIR analysis and

contributed substantially for the discussion and conclusion

of the manuscript.

Prof. M. Sinhoreti performed the statistical analysis.

Prof. T.F. Watson proofread the manuscript and contribut-

ed substantially for the discussion and conclusion of the

manuscript.

Prof. S. Sauro led and supervised the entire experimental

project, proofread the manuscript and contributed substan-

tially for the discussion and conclusion of the manuscript.

Acknowledgments

This article presents independent research commissioned by

the National Institute for Health Research (NIHR) 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 and the Ministry of

Education of Brazil (Capes Grant 6850-12-0).

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