Bioactive effects of a calcium/sodiumphosphosilicate on the resin–dentineinterface: a microtensile bondstrength, scanning electronmicroscopy, and confocal microscopystudy

Profeta AC, Mannocci F, Foxton RM, Thompson I, Watson TF, Sauro S. Bioactiveeffects of a calcium/sodium phosphosilicate on the resin–dentine interface: a microten-sile bond strength, scanning electron and confocal microscopy study.Eur J Oral Sci 2012; 120: 353–362. © 2012 Eur J Oral Sci

This study evaluated, through microtensile bond strength (lTBS) testing, the bioac-tive effects of a calcium/sodium phosphosilicate (BAG) at the resin–dentine interfaceafter 6 months of storage in phosphate buffer solution (PBS). Confocal laser scan-ning microscopy (CLSM) and scanning electron microscopy (SEM) were also per-formed. Three bonding protocols were evaluated: (i) RES-Ctr (no use of BAG),(ii) BAG containing adhesive (BAG-AD), and (iii) BAG/H3PO4 before adhesive(BAG-PR). The dentin-bonded specimens were prepared for lTBS testing, whichwas carried out after 24 h or 6 months of storage in PBS. Scanning electron micros-copy ultramorphology analysis was performed after debonding. Confocal laser scan-ning microscopy was used to evaluate the morphological and nanoleakage changesinduced by PBS storage. High lTBS values were achieved in all groups after 24 hof storage in PBS. Subsequent to 6 months of storage in PBS the specimens createdusing the BAG-AD bonding approach still showed no significant reduction inlTBS. Moreover, specimens created using the BAG-AD or the BAG-PR approachshowed an evident reduction of nanoleakage after prolonged storage in PBS. The useof BAG-containing adhesive may enhance the durability of the resin–dentine bondsthrough therapeutic/protective effects associated with mineral deposition within thebonding interface and a possible interference with collagenolytic enzyme activity(matrix metalloproteinases) responsible for the degradation of the hybrid layer.

Andrea C. Profeta, FrancescoMannocci, Richard M. FoxtonIan Thompson, Timothy F. Watson,Salvatore Sauro*

Biomaterials, Biomimetics and BiophotonicsResearch Group (B3), King’s College LondonDental Institute, Guy’s Hospital, London, UK

Salvatore Sauro, Dental Biomaterials Science,King’s College London Dental Institute, Floor17 Guy’s Tower, London SE1 9RT, UK

Telefax: +44-207-1881823E-mail: salvatore.sauro@kcl.ac.uk

Key words: adhesion durability; bioactiveglass; bonded-dentine interface

Accepted for publication May 2012

Bioactive materials are often used in operative den-tistry due to their ability to interact actively withdental hard tissues, inducing calcium-phosphates (Ca/P) deposition in the presence of body fluids or saliva(1–4).

Whereas remineralisation of enamel lesions can beachieved predictably (5, 6), there is little information onwhether it is possible to remineralise specific mineral-deficient areas within the resin–dentine interface (i.e.hybrid layers) (2). Some polyalkanoate cements mayinduce crystal growth within gaps in the bonded interfaceafter long-term storage in water (7). Furthermore, bioac-tive, ion-releasing materials, such as calcium-phosphate(Ca/P) cements, have the potential to encourage dentineremineralisation by mineral precipitations (8–11).

PETERS et al. (12) showed the presence of a highermineral content [determined by electron probe elemen-tal micro-analysis (EPMA) techniques] and an increasein microhardness along the interface of resin-bondedcaries-affected dentine, following the application ofmaterials containing Ca/P cements. Bioactive calcium/sodium (Ca/Na) phosphosilicates, such as Bioglass45S5 (BAG), are able to induce deposition of hydroxy-carbonate apatite (4, 13–15). Although bioactive glasseshave previously been used for dentine remineralisationby direct application onto demineralised dentinal tissuewhen dispersed in water solutions (4, 14), there is littleinformation about the potential therapeutic effects ofBAG on the resin–dentine interface when used duringetch-and-rinse bonding procedures.

Eur J Oral Sci 2012; 120: 353–362DOI: 10.1111/j.1600-0722.2012.00974.xPrinted in Singapore. All rights reserved

� 2012 Eur J Oral Sci

European Journal ofOral Sciences

Therefore, this study was devised to assess the bioac-tive effects of BAG during etch-and-rinse dentine-bond-ing procedures on the resin–dentine interface. This aimwas accomplished by evaluating the microtensile bondstrength (lTBS) of specimens after 24 h and 6 monthsof storage in PBS. Fractographic analysis was also per-formed through scanning electron microscopy (SEM).The ultramorphology and nanoleakage analysis of theresin-bonded dentine was executed using confocal laserscanning microscopy (CLSM).

The null hypotheses to be tested in this study were:(i) the use of BAG employed during bonding proce-dures has no effect on the bond strength, and (ii) thepresence of BAG does not reduce nanoleakage withinthe demineralised ‘poorly-infiltrated’ areas within theresin-dentine interface.

Material and methods

Specimen preparation

Caries-free human third molars, extracted for surgical rea-sons from 20- to 40-yr-old patients, were used in thisstudy. The treatment plan of any of the involved patients,who had given informed consent for use of their extractedteeth for research purposes, was not altered by this study.The study was conducted in accordance with the ethicalguidelines of the Research Ethics Committee (REC) formedical investigations.

The teeth were stored in deionised water (pH 7.1) at 4°C and used within 1 month after extraction. The coronaldentine specimens were prepared by sectioning the roots1 mm beneath the cemento–enamel junction (CEJ) with ahard tissue microtome (Accutom-50; Struers, Copenhagen,Denmark) using a slow-speed, water-cooled diamondwafering saw (330-CA RS-70300; Struers) (Fig. 1). A 180-grit silicon carbide (SiC) abrasive paper mounted on awater-cooled rotating polishing machine (Buehler Meta-Serv 3000 Grinder-Polisher; Buehler, Dusseldorf,Germany) was used (30 s) to remove the diamond sawsmear layer and to replace it with a standard and moreclinically relevant smear layer (16).

Experimental bonding procedures and formulation ofresin adhesives

A resin co-monomer blend was formulated by using ahydrophobic, cross-linking dimethacrylate monomer –bisphenyl-A-glycidyl methacrylate (Bis-GMA; Esstech, Es-sington, PA, USA) – and a hydrophilic monomer –2-hydroxyethyl methacrylate (HEMA; Sigma-Aldrich,Gillingham, UK). In order to obtain a dental bondingsystem with chemical affinity to calcium (present indentine and BAG), an acidic functional monomer – 2,5-dimethacryloyloxyethyloxycarbonyl-1,4-benzenedicarboxylicacid (PMDM; Esstech Essington) – was also includedwithin the composition of the resin blend. Subsequently,the resin blend was made light-curable by a binaryphotoinitiator system based on camphoroquinone (CQ;Sigma-Aldrich) and 1,2-ethyl-dimethyl-4-aminobenzoate(EDAB; Sigma-Aldrich). This resin co-monomer blendwas used to formulate the experimental primer and thebonds used in this study (Table 1).

A BAG (Sylc; OSspray, London, UK) with particlesize < 10 lm was employed in the etch-and-rinse bondingprocedures using two different experimental approaches:(i) BAG-AD (30wt% BAG included within the composi-tion of a resin adhesive as a bioactive microfiller), and (ii)BAG-PR (BAG applied directly onto H3PO4-etched/wetted dentine before bonding procedures). The neat adhe-sive, with no BAG, served as the control (RES-Ctr)(Fig. 1).

In detail, a water wet-bonding dentine substrate wasachieved by water-rinsing, for 15 s, the dentine surfacesacid-etched with 37% phosphoric acid solution (H3PO4)(Sigma-Aldrich) and gently blowing off (for 2 s) excesswater to leave a wet reflective-surface.

The control bonding procedure (RES-Ctr) was accom-plished by applying two consecutive coats of an ethanol-solvated resin primer [50 wt% absolute ethanol (Sigma-Aldrich) and 50 wt% of neat co-monomer resin blend] anda layer of the neat co-monomer resin blend (Table 1) withina period of 20 s. Light-curing was immediately performedfor 30 s (>600 mW/cm�2, Optilux VLC; Demetron,Danbury, CT, USA).

The first experimental bonding procedure (BAG-AD)was performed by applying the same ethanol-solvatedresin primer onto H3PO4-etched dentine, as previouslydescribed, followed by a layer of bonding resin containingBAG (Table 1; Fig. 1). The bonding and the light-curingprocedures were executed as previously described for theRES-Ctr group.

The second experimental bonding procedure (BAG-PR)was performed as follows. The 37% H3PO4 solution(Sigma-Aldrich). was applied onto the dentine surface for15 s. Then, 0.05 g of BAG powder was placed onto theH3PO4-etched wet dentine surface, spread immediately for10 s using a cotton pellet, and finally rinsed with copiousamounts of deionised water for 15 s (Fig. 1). The primer/bond application and the light-curing procedures were per-formed as previously described for the RES-Ctr group.

A final composite build-up (5 mm) was constructed oneach specimen using a light-cured resin composite (Filtek

Fig. 1. Schematic illustrating the experimental study design.Human third molars were used to prepare standardised den-tine surfaces. The three different bonding approaches wereperformed using specific components and application proce-dures. Bis-GMA, bisphenyl-A-glycidyl methacrylate; HEMA,2-hydroxyethyl methacrylate; PMDM, 2,5-dimethacryloyloxy-ethyloxycarbonyl-1,4-benzenedicarboxylic acid.

354 Profeta et al.

Z250; 3M-ESPE, St Paul, MN, USA) in five incrementallayers (of 1 mm thickness). Each layer of composite wasindividually light cured for 20 s. The resin-bonded dentinespecimens were stored in PBS for 24 h or 6 months at 37°C. The PBS was composed of (in g/l) CaCl2 (0.103),MgCl2.6H2O (0.019), KH2PO4 (0.544), KCl (17), andHEPES (acid) buffer (4.77), and the pH was 7.4.

lTBS and SEM fractography and failure analysis

Twenty dentine-bonded specimens from each group weresectioned using a slow-speed water-cooled diamond wafer-ing blade (Struers) mounted on a hard-tissue microtome(Isomet 11/1180; Buehler) in both x and y directionsacross the adhesive interface to obtain matchsticks withcross-sectional areas of 0.9 mm2. By excluding peripheralbeams showing the presence of residual enamel, only theremaining matchsticks (n = 10–15) were selected to createthree groups with the same total number of resin–dentinespecimens in each group (n = 280). The exact width ofeach matchstick was checked using a calliper (MitutoyoCD15; Mitutoyo, Kawasaki, Japan) and half of them(n = 140) were tested after 24 h of storage in PBS and theremaining half (n = 140) were tested after 6 months ofstorage in PBS at 37°C. The lTBS test was performedusing a microtensile jig in a LAL300 linear actuator(SMAC Europe; Horsham, UK) with a LAC-1 high-speedcontroller single axis with a built-in amplifier and at thefollowing settings: stroke length = 50 mm, peakforce = 250 N, displacement resolution = 0.5 mm, andcrosshead speed = 1 mm min�1. Bond-strength data werecalculated and expressed in MPa, the lTBS values ofsticks from the same restored teeth were averaged, and themean bond strength was used as one statistical unit forthe statistical analysis. The lTBS (mean-MPa) data for

each group were analysed using a repeated-measures ANO-

VA and Tukey’s post-hoc test for pairwise comparisons(a = 0.05).

The mode of failure was classified as percentage ofadhesive, mixed, or cohesive. The failed bonds were exam-ined at 930 magnification using a stereoscopic microscope(Leica M205A; Leica Microsystems, Wetzlar, Germany).

Five representative debonded specimens for each groupthat failed in mixed or adhesive modes were selected forultramorphology analysis of the fractured surface (SEMFractography). They were dried overnight and mounted onaluminium stubs with carbon cement, then sputter-coatedwith gold (SCD 004 Sputter Coater; Bal-Tec, Vaduz, Liech-tenstein) and examined using SEM (S-3500; Hitachi, Wok-ingham, UK) with an accelerating voltage of 15 kV and aworking distance of 25 mm at increasing magnifications.

Confocal microscopy ultramorphology andnanoleakage evaluation

A further three dentine specimens from each group werebonded, as previously described, with the primer/bondresins doped with 0.1 wt% rhodamine-B (Rh-B: Sigma-Aldrich, St Louis, MO, USA) and employed for theconfocal microscopy analysis (18, 19). The specimens wereserially sectioned across the adhesive interface to obtainresin–dentine slabs (of 1 mm thickness). The resin–dentineslabs (n = 10 per group) were then divided into two sub-groups based on the period of storage in PBS (24 h or6 months) (Fig. 2). Subsequent to the storage period, thespecimens were coated with two layers of fast-setting nailvarnish applied 1 mm from the resin–dentine interfacesand immersed in 1 wt% aqueous fluorescein (Sigma-Aldrich) solution for 24 h. The specimens were thentreated in an ultrasonic water bath for 2 min and polished

Table 1

Composition of the experimental bonding procedures/adhesive systems used in this study

Experimental bondingprocedures Dentine conditioning

Chemical composition (wt%) of the adhesivesystems

Primer Bond

BAG-AD 37% H3PO4 solution, 15 s immediately followedby the application of a 0.05 g of BAG onH3PO4 wet dentine

20.21 wt% BisGMA15.54 wt% PMDM14.25 wt% HEMA50.00 wt% Absoluteethanol

37.50 wt% BisGMA16.80 wt% PMDM15.70 wt% HEMA30.00 wt% BAG

BAG-PR 37% phosphoric acid solution, 15 s – H3PO4: 20.21 wt% BisGMA15.54 wt% PMDM14.25 wt% HEMA50.00 wt% Absoluteethanol

40.00 wt% Bis-GMA31.50 wt% PMDM28.50 wt% HEMA

RES-Ctr 37% phosphoric acid solution, 15 s – H3PO4: 20.21 wt% BisGMA15.54 wt% PMDM14.25 wt% HEMA50.00 wt% Absoluteethanol

40.00 wt% Bis-GMA31.50 wt% PMDM28.50 wt% HEMA

Bis-GMA, bisphenyl A glycidyl methacrylate; HEMA, hydrophilic 2-hydroxyethyl methacrylate; PMDM, 2,5-dimethacryloyloxy-ethyloxycarbonyl-1,4-benzenedicarboxylic acid.At the end of the formulation of the resins, 0.25 wt% camphoroquinone (CQ) and 1.0 wt% 2-ethyl-dimethyl-4-aminobenzoate(EDAB) were added to the resin mixture.

Effects of a bioactive glass on the resin–dentine interface 355

using SiC abrasive papers of ascending grit (#1200 to#4000) (Versocit; Struers) on a water-cooled rotating pol-ishing machine (Buehler Meta-Serv 3000 Grinder-Polisher;Buehler). A final treatment in an ultrasonic water bath(5 min) completed the specimen preparation for the confo-cal microscopy evaluation (Fig. 2).

The microscopy examination was performed using aconfocal laser scanning microscope (Leica SP2 CLSM; Le-ica, Heidelberg, Germany) equipped with a 63 9 /1.4 NAoil-immersion lens and using 488-nm argon/helium (fluo-rescein excitation) or 568-nm krypton (rhodamine excita-tion) laser illumination. The reflection imaging wasperformed using the argon/helium laser. Confocal laserscanning microscopy reflection and fluorescence imageswere obtained with a 1-lm z-step to section optically thespecimens to a depth up to 20 lm below the surface (18).The z-axis scans of the interface surface were arbitrarilypseudo-coloured by two selected operators and compiledinto single projections using the Leica image-processingsoftware (Leica). The configuration of the system wasstandardised and used at the same settings for the entireinvestigation. Each resin–dentine interface was completelyinvestigated and then five optical images were randomlycaptured. Micrographs representing the most common fea-tures of nanoleakage observed along the bonded interfaceswere captured and recorded (19).

Results

lTBS and SEM fractography and failure analysis

The BAG-bonding technique vs. storage time was statisti-cally significant only for the BAG-AD group (P = 0.001);no significant reduction of the lTBS values was observedafter 6 months of storage in PBS (P > 0.05). On the otherhand, significant lTBS reductions were observed in boththe BAG-PR and RES-Ctr groups (P < 0.05) after pro-longed storage in PBS (6 months). The lTBS results(expressed asMean and SD) are presented in Table 2.

High lTBS values were achieved in all groups after24 h of storage in PBS, with failures occurring mainlyin cohesive mode in all groups; in contrast, importantchanges in the lTBS were observed after 6 months ofstorage in PBS. For instance, the lTBS of specimens inthe RES-Ctr group (no BAG) showed a significant(P < 0.05) decrease after 6 months of storage in PBSand failed mostly in adhesive mode (66%). The speci-mens stored for 24 h in PBS that fractured in mixedmode were characterised by the presence of exposeddentinal tubules with spare extruded resin tags(Fig. 3A2). Conversely, the surface of the specimensthat failed in adhesive mode after 6 months of storagein PBS presented several ‘funnelled’ dentinal tubuleswith no exposed collagen fibrils (Fig. 3A3). The resin–dentine specimens of the BAG-AD group maintained ahigh lTBS (P > 0.05) after 6 months of storage in PBS(23.89 ± 7.74 MPa). In these specimens the failure wasprevalent in cohesive (43%) and mixed (40%) modes(Fig. 3B1) and the SEM analysis of the fractured sur-face revealed a dentine surface predominantly coveredby residual resin and mineral crystals embedded withina resin/collagen network (Fig. 3B3).

The specimens of the BAG-PR group, where theBAG powder was applied onto acid-etched/wetted

Fig. 2. Schematic illustrating the composite-tooth matchsticks(1 mm) prepared using a water-cooled diamond saw, stored inPBS for 24 h or 6 months, and then subjected to microtensilebond strength (lTBS) testing and scanning electronmicroscopy failure analysis. This schematic also illustrateshow composite-tooth slabs were prepared, stored in PBS for24 h or 6 months, and evaluated by confocal laser scanningmicroscopy.

Table 2

Mean and standard deviation (SD) of microtensile bondstrength values (MPa) obtained for the different experimentalgroups and percentage distribution of failure mode after micro-tensile bond strength testing; total number of beams (tested

stick/pre-load failure)

lTBS – mean ± SD(N of tested/pre-failed beams)

% Failure [A/M/C]24 h test 6 month test

BAG-AD A126.91 ± 3.43(140/0)[0/10/90]

B123.89 ± 7.75(135/5)[17/40/43]

BAG-PR A127.20 ± 3.92(140/0)[0/9/91]

A213.35 ± 5.32(134/6)[56/11/33]

RES-Ctr A129.12 ± 4.75(140/0)[0/4/96]

A218.18 ± 5.66(133/7)[66/10/24]

For each horizontal row: values with identical numbers indi-cate no significant difference.For each vertical column: values with identical letters indicateno significant difference using Student-Newman–Keuls test(P > 0.05).

356 Profeta et al.

dentine before application of the adhesive system,showed a significant decrease in lTBS values (P < 0.05)after prolonged storage in PBS (table 2). These speci-mens failed mainly in adhesive mode (56%) after6 months of storage in PBS, and the SEM fractograph-ic analysis showed that the fracture during lTBS test-ing occurred along the intertubular dentine, leaving anintact peritubular dentine and a consistent precipitationof mineral inside the dentinal tubules (Fig. 3C3).

Confocal microscopy ultramorphology andnanoleakage evaluation

The CLSM investigation showed that all the bondingprocedures used in this study were able to create a

resin diffusion within the demineralised dentine(hybrid layer 7–9 lm) and several resin tags into thedentinal tubules (Fig. 4A). Nevertheless, the resin–den-tine interfaces of the specimens created in the threegroups showed evident fluorescein penetration (nano-leakage) within the hybrid layer and along the den-tinal tubules after 24 h of storage in PBS (Fig. 4B,C).The experimental bonding approach used to bond thespecimens of the BAG-PR group created resin–dentineinterfaces characterised by the presence of mineraldeposits inside the dentinal tubules and within thehybrid layer (Fig. 4D).

The prolonged storage in PBS induced importantchanges in terms of ultramorphology and nanoleakage.For instance, the resin–dentine interface of the RES-

A1 A2 A3

B1 B2 B3

C1 C2 C3

Fig. 3. Scanning electron microscopy images of failure modes of the resin-bonded specimens created using the three differentbonding approaches tested. (A) Micrograph of the failure mode (cohesive) of the resin control bonded to etched dentine (37%H3PO4) after 24 h of storage in PBS (A1). At higher magnification (A2) it was possible to observe the presence of some exposeddentinal tubules, but most remained obliterated by resin tags. No exposed collagen fibrils were visible on the dentine surface, anda well resin-hybridised hybrid layer was present (pointer). At 6 months (A3), the resin–dentine interfaces created with the controlresin (RES-Ctr; containing no bioactive filler) showed only a few resin tags inside the dentinal tubules and no collagen fibrils werevisible on a dentine surface characterised by funnelled dentinal tubules (pointer). (B) Micrograph of the failure mode (mixed) ofthe calcium/sodium phosphosilicate-containing adhesive (BAG-AD) bonded to dentine, after 6 months of storage in PBS (B1). Athigher magnification (B2) no exposed dentinal tubules or exposed collagen fibrils were observed; the dentine surface was wellresin-hybridised (pointer). After 6 months of storage in PBS (B3), the debonded resin–dentine interface showed the presence ofresin tags remaining inside the dentinal tubules and mineral crystals embedded within a preserved collagen network (pointer).(C) Micrograph of the failure mode (adhesive) of BAG applied directly onto H3PO4-etched/wetted dentine before bonding (BAG-PR) after 24 h of storage in PBS (C1). At higher magnification (C2) it was possible to observe the presence of some exposeddentinal tubules, while most remained obliterated by resin tags containing few BAG particles. No exposed collagen fibrils werepresent on the dentine surface (pointer). At 6 months testing (C3), the resin–dentine interface created with the BAG-PR showed adentine surface characterised by the presence of remineralised dentinal tubules obliterated by mineral crystals. It is interesting tonote how the fracture occurred along the intertubular dentine leaving an intact peritubular dentine around the mineral-obliterateddentinal tubule (pointer). rt, resin tags; t, dentinal tubules.

Effects of a bioactive glass on the resin–dentine interface 357

Ctr group specimens was affected by severe nanoleak-age within the hybrid layer and the presence of a con-tinuous gap between dentine and composite (Fig. 5A).Conversely, the specimens of the BAG-AD groupshowed the presence of a strong reflective mineralmaterial and partial dye penetration within the hybridlayer (Fig. 5B). The resin–dentine interface of speci-mens in the BAG-PR group was affected by partial dyepenetration within a crystallised hybrid layer. However,gaps were also observed between the hybrid and adhe-sive layers (Fig. 5C), probably caused by the samplepreparation procedure before the CLSM analysis.

Discussion

Hybrid layers created using etch-and-rinse adhesivesinclude water-rich, resin-sparse regions that account for2–3% of their entire volume, which increase subsequent

to prolonged aging in fluids (20). The water-rich, resin-sparse regions represent essentially the nanoporositieswithin the demineralised collagen fibrils, created duringadhesive application as a result of incomplete replace-ment of water by resin infiltration (21). This undis-placed water may act as a functional medium for thehydrolysis of suboptimally polymerised resin matricesby esterases and denaturation of collagen via the acti-vation of host-derived matrix metalloproteinases(MMPs), jeopardizing the durability of the resin–dentine interfaces (21–23).

Several methods have been advocated to increase thelongevity of these resin–dentine interfaces, including theinhibition of the MMPs within the hybrid layer (22, 23)and enhancement of the resin infiltration within thedemineralised collagen fibril using more hydrophobicresin monomers and ethanol wet-bonding (21).

Based on the results obtained in this study, the firstnull hypothesis must be partially rejected because the

A B

C D

Fig. 4. Confocal laser scanning microscopy (CLSM) images showing the interfacial characterisation and nanoleakage, after 24 hof storage in PBS, of the resin–dentine interfaces created using the three different bonding approaches tested. (A) Confocal laserscanning microscopy three-dimensional (3D) single-projection (fluorescence mode) image exemplifying the interfacial characteris-tics of the resin–dentine interface created using the control adhesive system (RES-Ctr) applied onto H3PO4-etched dentine. It ispossible to observe a clear hybrid layer (approximate thickness 9 lm) located underneath a thick adhesive layer and long resintags. (B) This CLSM 3D single-projection (fluorescence/reflection mode) image of the resin–dentine interface created using thebioactive calcium/sodium phosphosilicate-containing adhesive (BAG-AD) shows an intense nanoleakage signal from the hybridlayer (pointer) located underneath a thick adhesive layer characterised by the presence of BAG microfiller. The presence of longresin tags is also evident. (C) The resin–dentine interface created using the bonding procedure where the BAG is applied directlyonto H3PO4-etched/wetted dentine (BAG-PR) shows evident dye penetration within the hybrid layer (pointer). Short resin tagsare visible underneath a thick adhesive layer. The reason why only short resin tags could be created during this type of bondingprocedure is shown in (D) where it is possible to observe a strong reflective signal from the demineralised dentine layer (pointer)and inside the dentinal tubules, indicating the presence of mineral particles. a, adhesive layer; c, composite; fl, BAG microfiller; rt,resin tags; t, dentinal tubules.

358 Profeta et al.

use of BAG produced bioactive/protective effects onthe bond strength only when used as resin microfillerwithin the adhesive composition. The second nullhypothesis must be totally rejected as both the experi-mental bonding approaches based on the use of BAGwere able to reduce the nanoleakage within the demin-eralised ‘poorly infiltrated’ areas within the resin–den-tine interface.

In detail, the control bonding procedure (RES-Ctr)and the two experimental bonding approaches used(BAG-AD and BAG-PR) to bond the acid-etched den-tine produced comparably high lTBS values after 24 hof storage in PBS (Table 2). Conversely, a significantdecrease in lTBS (P > 0.05) occurred in all groupsafter storage in PBS for 6 months, except for the speci-mens bonded using the resin adhesive containing BAGmicrofiller (BAG-AD).

The SEM analysis of the fractured specimens of theRES-Ctr group showed, after 24 h of storage in PBS, adentine surface characterised by a hybrid layer that washighly hybridised with resin and no presence of demi-neralised collagen fibrils exposed (Fig. 3A2). Con-versely, these resin–dentine specimens stored for6 months in PBS had a fractured surface characterisedby ‘funnelled’ dentinal tubules, indicating degradationof the demineralised peritubular dentine (Fig. 3A3). Incontrast, the bonded-dentine specimens of the BAG-ADgroup immersed in PBS for 6 months had a frac-tured (adhesive mode) dentine surface, with mineralcrystals embedded within a preserved collagen networkand no evidence of ‘funnelled’ dentinal tubules(Fig. 3B3).

The SEM ultramorphology analysis of the fracturedspecimens (adhesive mode) of the RES-PR groupstored for 24 h in PBS demonstrated the presence ofdentinal tubules obliterated by resin tags and noexposed collagen fibrils (Fig. 3C2). Interestingly, when

this type of dentine-bonded specimen was immersed inPBS for 6 months it was possible to detect a fractureddentine surface characterised by dentinal tubules oblit-erated by mineral crystals and a distinctive fracturealong the intertubular dentine, which left an intact peri-tubular dentine (Fig. 3C3).

Possible explanations for such longevity attained indentine-bonded specimens created using BAG-AD afterprolonged storage in PBS may be as follows:

(i) The presence of BAG within the resin–dentineinterface may have induced the release of a silicicacid, such as Si(OH)4, and a subsequent polycon-densation reaction between the silanols com-pounds and the demineralised collagen viaelectrostatic, ionic, and/or hydrogen bonding (13,24, 25), which interfered with the ability of MMPs– although BAG is not a direct MMP inhibitor –to execute their collagenolytic and gelatinolyticactivities. A study by OSORIO et al. (26) showedthat it is possible to reduce the collagen-degrada-tion process by using specific chemical com-pounds, such as zinc oxide, which interfere withthe zinc-binding and calcium-binding catalyticdomains of MMPs.

(ii) The precipitation of an amorphous calcium phos-phate (ACP) on the polycondensate SiO2-rich tem-plate of nucleation (3, 5, 13, 18) induced by thedissolution and immediate reaction between Ca2+

and PO43� species from BAG may have also

favoured the formation of a high-molecular-weightcomplex (Ca/P–MMPs), which restricted theactivities of MMP-2 and MMP-9 within thehybrid layer (27). However, the ability of specificbioactive glass, such as Bioglass 45S5, to modulateand/or reduce the presence of collagens I, II, andIII, osteocalcin, osteonectin, and osteopontin, has

A B C

Fig. 5. Confocal laser scanning microscopy (CLSM) images showing the interfacial characterisation and nanoleakage, after6 months of storage in PBS, of the resin–dentine interfaces. (A) Confocal laser scanning microscopy three-dimensional single-pro-jection (fluorescence/reflection mode) image of the resin–dentine interface created using the control adhesive system (RES-Ctr)applied onto H3PO4-etched dentine. It is possible to note the presence of evident dye diffusion (nanoleakage) within the hybridlayer and inside the dentinal tubules (pointer). A gap is present between the dentine and the composite. (B) The resin–dentineinterface created using the bonding approach where the bioactive calcium/sodium phosphosilicate-containing adhesive (BAG-AD)is applied onto H3PO4-etched dentine shows partial dye diffusion within a hybrid layer characterised by a strong reflective signal(pointer). (C) The resin–dentine interface created using the bonding procedure where the BAG is applied directly onto H3PO4-etched/wet dentine (BAG-PR) shows a crystallised reflective layer (pointer) characterised by low dye penetration (nanoleakage). Apronounced gap can be seen between the adhesive layer and the composite. It is also possible to observe the remaining reflectivemineral materials on the fractured edge of the adhesive layer (arrows). a, adhesive layer; c, composite; g, gap.

Effects of a bioactive glass on the resin–dentine interface 359

also been demonstrated in bone-regenerationstudies (24).

(iii) The release of Na+ and Ca2+ ions from BAG,and the incorporation of H3O

+ protons into theglass particles, may have created an optimal alka-line environment (5, 18) within the resin–dentineinterface that interfered with the activity ofMMPs, which are very acidic-pH dependent (22,23).

(iv) The bioactive remineralisation induced by BAGmay have decreased the distribution of the water-rich, resin-sparse regions within the hybrid layer(2, 18) via silanols polycondensation and subse-quent ACP/AH remineralisation, which probablyinterfered with the water-dependent hygroscopicand hydrolytic degradation of the polymer net-work (28).

The confocal microscopy evaluation performed after6 months of storage in PBS indicated that both theexperimental bonding approaches used in this study(BAG-AD and BAG-PR) created a resin–dentine inter-face affected only by partial dye penetration (nanoleak-age) within a hybrid layer characterised by thedeposition of a strong reflective mineral (Fig. 5B,C).

Whereas it is reasonable to believe that the hybridlayer of the specimens created using the BAG-ADapproach remineralised as a result of the bioactive/bi-omimetic activity of Bioglass 45S5 after prolonged(6 months) storage in PBS (17, 18, 25), a completelydifferent bioactive phenomenon may have occurredwithin the resin–dentine interface created by directlyapplying the BAG on the demineralised H3PO4-wetteddentine, as a significant decrease of lTBS was attainedafter prolonged storage in PBS (Table 2).

In this case, a possible explanation for the reducedconfocal nanoleakage may be due to the chemical nat-ure of mineral precipitation that occurred within theresin–dentine interface created as a result of the experi-mental bonding procedures (BAG-PR). Our hypothesisis that the chemical reaction between BAG and H3PO4

solution (Fig. 4D) may have induced the precipitationof dicalcium-phosphate salts (i.e. brushite and mone-tite). BAKRY et al. (17, 29) showed that the acid–basechemical reaction between BAG and H3PO4 mayinduce the formation of brushite via combination ofthe phosphate (released from the BAG and H3PO4)and calcium ions (released from BAG and etched den-tine). The precipitation reaction of the brushite may beresponsible for the creation of an acidic environment(30), which may have evoked the activation of MMPs(22, 23); this situation is also aggravated by the factthat BAG no longer has the ability to create a localised‘protective’ alkaline pH within the resin–dentine inter-face.

Moreover, it is also possible that the BAG/H3PO4

reaction may have altered the chemical and/or physicalcharacteristics of BAG, in particular those responsiblefor the polycondensation of silanols and ACP/HA pre-cipitation (13, 24, 25), which may be fundamental inaltering the activity of MMPs (27), as previously

described. However, even supposing that the reactionbetween hydroxyl ions and Si(OH)4 formed the silanolscompounds and induced the polycondensation reaction,they may have been washed out by application of theair-water jet before application of the primer and bond(31).

Furthermore, a slightly acidic environment may haveremained in loco within the resin–dentine interface dur-ing the prolonged storage in PBS as a result of therelease of H+ from the acidic monomer (2,5-dimethac-ryloyloxyethyloxycarbonyl-1,4-benzenedicarboxylicacid) contained within the resin adhesives (32–34), caus-ing a long-standing, MMP-mediated degradation ofcollagen in both the RES-Ctr and BAG-PR groups. Inaddition, a durable acidic environment may haveinduced supplementary precipitation of dicalcium oroctocalcium phosphates (30, 35) during buffered condi-tion (replacement of PBS) within the microporositiesgenerated by the degradation of the dentine collagenfibrils (Fig. 5C). Indeed, as a result of this probableadditional precipitation of mineral over time, the inter-face created using the BAG-PR bonding technique mayhave achieved mechanical characteristics similar tothose created using glass ionomer cements (GICs)applied onto polyacrylic acid-etched dentine (18, 36,37). This is probably why bond strength reduction andgap formation were observed in the BAG-PR speci-mens. The GIC-bonded interfaces can reach a tensile orshear bond strength of approximately 5 MPa andfrequently prefail during specimen preparation (37, 38).YIP et al. (39) affirmed that the results obtained fromtensile testing of GICs bonded to dentine do not repre-sent the actual strength of such stiff bonded interfacesand that only an accurate ultramorphology analysisusing electron microscopy may reveal the proper bond-ing ability of such restorative materials.

However, it is also important to consider that thehydrophilic characteristics conferred by specific resinmonomers, such as HEMA and PMDM, within thetested adhesives (Fig. 1) may have compromised themechanical properties (i.e. modulus of elasticity) of thehybrid layers (40, 41) as a result of polymer hydrolysisand swelling tensions generated within the polymerchains. In contrast, the BAG microfiller containedwithin the adhesive used in the BAG-AD group mayhave absorbed and used the water not required by thehydrophilic/acid monomers for the bioactive processesof conversion into apatite (18), thus preventing thepolymer network from considerable hygroscopic/hydro-lytic degradation (28).

In conclusion, this study provided preliminary evi-dence for the use of bioactive Ca/Na phosphosilicate,such as Bioglass 45S5, in dentine-bonding proceduresin order to enhance the durability of the resin–dentineinterfaces. However, further in vitro (i.e. transmissionelectron microscopy and atomic force microscopy-nanoindentation examination) and long-term clinicalstudies are required to confirm the protective/therapeu-tic effects of BAG on the resin-dentine interface. Con-focal Raman analysis will be also necessary to confirmthe chemical nature of the mineral precipitates observed

360 Profeta et al.

within the bonded-dentine interfaces created with thetwo experimental BAG-bonding procedures.

Acknowledgements – This article presents independent researchcommissioned by the National Institute for Health Research(NIHR) under the i4i programme and the Comprehensive Bio-medical Research Centre at Guy’s & St Thomas’ Trust. The viewsexpressed in this publication are those of the author(s) and notnecessarily those of the NHS, the NIHR or the Department ofHealth. The authors also acknowledge support from the Centreof Excellence in Medical Engineering funded by the WellcomeTrust.

Conflicts of interest – None.

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