journal of dentistry 42 (2014) 384–394

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Early dentine remineralisation: Morpho-mechanical assessment Manuel Toledano *, Estrella Osorio, Inmaculada Cabello, Raquel Osorio University of Granada, Faculty of Dentistry, Dental Materials Section, Colegio Ma´ximo de Cartuja s/n, 18071 Granada, Spain

article info

abstract

Article history:

Objectives: The purpose of this study was to evaluate some physical–mechanical and

Received 15 November 2013

morphological changes of demineralised dentine at early stages of dentine remineralisa-

Received in revised form

tion.

14 January 2014

Methods: Extracted human third molars were sectioned to obtain dentine discs. After

Accepted 26 January 2014

polishing the dentine surfaces, three groups were established: (1) untreated dentine – UD, (2) 37% phosphoric acid application for 15 s (partially demineralised dentine – PDD) and (3) 10% phosphoric acid for 12 h, at 25 8C (totally demineralised dentine – TDD). Five

Keywords:

different remineralizing fluids were used for 30 min: chlorhexidine (CHX), artificial saliva

Dentine

(AS), phosphated solution (PS), ZnCl2 and ZnO solutions. Atomic force microscopy (AFM)

Remineralisation

imaging/nano-indentation, surface nano-roughness and fibrils diameter were determined.

Microscopy

X-ray diffraction (XRD), energy dispersive elemental analyses (EDX) and high resolution

Mechanical

scanning electron microscopy analysis (HRSEM) were applied.

Roughness

Results: PDD and TDD preserved some mineral contents. After demineralisation and immersion in all solutions, width of nanomechanical properties and fibrils was increased, and total nanoroughness was decreased. Peritubular and intertubular dentine were remineralised. Conclusion: Mineral exists in PA-demineralised dentine matrix and it is important since it may work as a constant site for further nucleation. The dentine surface remineralisation process may be stimulated as early as 30 min in abiotic conditions, with a pH ranging from 7.0 to 7.5. Clinical significance: The existence of enzymes and remineralising factors within the dentine matrix may facilitate early dentine remineralisation under favourable conditions. This process should be stimulated by new reparative materials. # 2014 Elsevier Ltd. All rights reserved.

1.

Introduction

Demineralisation of dentine is the process of removing mineral ions from the apatite latticework leaving the collagen fibres without support except for the water contained within the dentine, decreasing the mechanical properties of the affected tissue.1 Remineralisation of dentine refers to the

proceeding of restoring the inorganic matrix.2 This remineralisation process physiologically occurs onto demineralised dental surfaces, where the mineral is reabsorbed and damaged crystals are rebuilt.3 Thus, nanometer sized hydroxyapatite (HA) develops and grows within nucleation sites1,4 and a primary template of collagen fibrils to re-establish part of the mechanical properties of the tissue and protect them from hydrolytic and enzymatic degradation.5 Nucleation may

* Corresponding author. Tel.: +34 958243788; fax: +34 958240809. E-mail address: [email protected] (M. Toledano). 0300-5712/$ – see front matter # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdent.2014.01.012

journal of dentistry 42 (2014) 384–394

be enhanced by the presence of interfaces which allow aggregation and densification of the liquid-like prenucleation clusters.6 Biomimetic mineralisation imitates the natural process of mineralisation, simulating the natural formation process of mineral crystals on the surface of organic and inorganic matrix without using harsh conditions.2 It has been shown that dentine, even in the absence of cells, is able to actively participate in tissue reparative processes. It contains matrixbonded bioactive molecules, enzymes and growth factors that may be liberated and activated through different mechanisms in order to complete reparative processes.7 Different dentine remineralizing agents have been proposed. Chlorexidine in solution produces digluconate anions which may result in gradual precipitation in the presence of other mono- and divalent cations being in the substrate.8 Similarly, zinc may not only act as a MMPs inhibitor, but also influencing signalling pathways and stimulating a metabolic effect in hard tissue mineralisation9 and remineralisation processes.10 Zinc has also been shown to inhibit dentine demineralisation11 and somehow facilitates enamel remineralisation.10 The sites which need a remineralisation strategy in dentine could be root caries, erosion of cervical area, affected dentine and the exposed collagen incompletely infiltrated.12 The analysis of the limited literature available on dentine remineralisation at 30 min of immersing demineralised collagen in remineralizing solutions indicates that specific studies on the relationship between the dentine micronanostructure and the physicochemical properties of this treated substrate, at this time point, should be performed. The combination of mechanical data with microscopy techniques appears to be a valuable tool to be applied in dentine remineralisation studies.13 The surface properties of dentine and their influence on remineralisation have insufficiently been investigated.2 AFM nano-indentation performed in hydrated dentine, where force and indenter displacement are simultaneously recorded and the elastic modulus and hardness are determined from the load displacement curve, is a suitable method for the determination of the visco-elasticity of the demineralised dentine and its effective remineralisation.14,15 The mechanical properties determination integrates factors such as microstructure, mineral density and location of mineral within the organic matrix.16 The mineral phase in collagenous hard tissues such as bone and dentine is classified as intrafibrillar apatites, which are deposited within or immediately adjacent to gap zones of the collagen molecules and extend along the microfibrillar spaces within the fibril; and extrafibrillar apatites, which are located within the intersticial spaces separating the collagen fibrils. Previous studies have shown that intrafibrillar apatites play a significant role in the mechanical properties of mineralised tissues.13,14 Therefore, the aim of this study was to investigate some physical, chemical, mechanical and morphologic changes of demineralised dentine specimens after 30 min of immersion in different remineralizing solutions. The null hypothesis that was established is that nanomechanical properties, nanoroughness and morphological characteristics did not differ

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after immersing partially demineralised dentine samples in some remineralizing solutions.

2.

Materials and methods

2.1.

Specimens preparation

Twenty-four extracted non-carious human third molars were obtained from young patients (20–26 years old) with informed consent from donors, under a protocol approved by the Institution review board. The teeth were stored in 0.1% (w/v) thymol solution at 4 8C and used within one week after extraction. Teeth were sectioned at the cementum–enamel junction to remove the roots, after organic debris/calculus extraction. Mineralised dentine discs (0.75 mm  0.08 mm thick and 6.0  0.01 mm diameter) were obtained from the mid-coronal portion of each tooth using a diamond saw under water cooling. Dentine discs were polished through SiC abrasive papers from 800 up to 4000 grits (Struers LaboPol-4) followed by final polishing steps performed using diamond pastes (Buehler-MetaDi, Buehler Ltd., Lake Bluff, IL, USA) through 1 mm down to 0.25 mm. Eighteen specimens were partially demineralised with 37% phosphoric acid for 15 s, at 25 8C (partially demineralised dentine-PDD) following the method that described in Carrilho et al.17 PDD discs were rinsed in deionised water under constant stirring at 4 8C for 72 h. Dentine discs were dried over anhydrous calcium sulphate (8 h). Specimens were rehydrated in 0.9% NaCl containing 10 U/ml 1 of penicillin G and 300 mg/ ml of streptomycin for 24 h (pH 7.0). Fifteen discs were assigned to the following five immersion solutions and placed in Eppendorf tubes: (i) artificial saliva (AS) containing 50 mM Hepes (Applichem, Darmstadt, Germany), 5 mM CaCl2, 0.001 mM ZnCl2, 150 mM NaCl, 100 U/ml 1 of penicillin, and 1000 mg/ml 1 of streptomycin (pH 7.2); (ii) 40 mM chlorhexidine digluconate in AS (pH 7.4) (CHX); (iii) phosphate solution (PS) containing 50 mM Hepes (Applichem, Darmstadt,Germany), 1.5 mM CaCl2, 0.90 mM KH2PO4, 2 ppm sodium fluoride, 100 U/ml 1 of penicillin, and 1000 mg/ml 1 of streptomycin; (iv) 3.33 mg/ml of zinc chloride was added to the AS (pH 7.0) (ZnCl2); (v) 10 wt% zinc oxide was added to AS (pH 7.0) (ZnO). Specimens were incubated at 37 8C for 30 min. Chemicals used in this study are described in Table 1. Nine further dentine discs of the following groups were also analyzed to serve as controls: (1) Untreated dentine (UD), (2) PDD, and (3) totally demineralised dentine with 10% phosphoric acid for 12 h at 25 8C (TDD). Discs were rinsed in deionised water for 72 h.17 All specimens were polished with 4000-grit SiC abrasive papers and cleaned in an ultrasonic bath (Model QS3, Ultrawave Ltd, Cardiff, UK) containing deionised water [pH 7.4] for 5 min.

2.2.

AFM imaging and nano-indentation

Three dentine specimens from the untreated dentine (UD) and the totally demineralised dentine (TDD) groups were selected for characterization. Three additional specimens from the partially demineralised dentine (PDD), stored in each of the

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Table 1 – Chemicals used in the study. Chemicals (batch number) 50% Ortho-phosphoric acid (0000124351) Sodium chloride (0000341223) Chlorhexidine digluconate 20% (w/v) aqueous solution (9418600021) Anhydrous calcium sulphate, Drierite, 6 mesh (14796TJ) Sodium fluoride, dibasic, anhydrous (0000315723) Calcium chloride anhydrous (0000208286) Potassium dihydrogen phosphate (0000216657) HEPES buffer, Sterile, pH 8.0 –1 M/l – (8D007674) Penicillin G potassium salt (1386789) Streptomycin sulphate salt (026K8901) Zinc chloride (0000144046) Zinc oxide(0000325265)

2.4. Manufacturer

Panreac Quı´mica, Barcelona, Spain Guinama, Valencia, Spain Sigma–Aldrich, St. Louis, MO, USA Panreac Quı´mica, Barcelona, Spain

Applichem, Darmstadt, Germany Sigma–Aldrich, St. Louis, MO, USA

five different media (chlorexidine digluconate, artificial saliva, phosphated solution, zinc chloride and zinc oxide) for 30 min, were studied. 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, USA) was employed in this study. The indentation processes were undertaken using a Berkovich diamond indenter with a tip radius of approximately 20 nm. Nanohardness (Hi) and Young Modulus (Ei) were measured in wet at randomly distributed points of the intertubular dentine. Ten indentations were performed with a load of 4000 nN and time functions of 10 s (10 s loading and 10 s unloading) were performed on each dentine disc. The distance between each indentation was kept constant by adjusting the distance intervals in 5 (1) mm steps and the load function. Three dentine surfaces (4 mm  4 mm) per group were evaluated using an AFM (Multimode Nanoscope V, Veeco Metrology group, Santa Barbara, CA) in the tapping mode, using a calibrated vertical-engage ‘‘E’’ piezo-scanner (Digital Instrument, Santa Barbara, CA). Digital images were taken in a liquid environment (water-covered or ethanol-covered). A 10 nm-radius silicon nitride tip (Veeco, Santa Barbara, CA) was attached to the end of an oscillating cantilever that intermittently contacted the surface at the lowest point of the oscillation. Changes in vertical position of the AFM tip at resonance frequencies near 330 kHz provided the height of the images registered as bright and dark regions.

2.3.

Nanoroughness measurements.

Three 15 mm  15 mm and three 500 nm  500 nm digital images were recorded from each surface with a slow scan rate (0.1 Hz). The 15 mm  15 mm images were analyzed quantitatively. For each image, five randomized boxes (3 mm  3 mm) were created to examine the intertubular, peritubular and total roughness of dentine (N = 45). Nanoroughness (Ra, in nanometer) was measured using a proprietary software (Nanoscope Software version V7).

Fibril diameter assessment

Collagen fibril diameter was determined from 500 nm  500 nm images by section analysis using data that had been modified only by plane fitting. The collagen fibril diameter was preferentially determined from fibrils that were exposed along their complete widths. Five fibrils were analyzed from each image. Measurements were corrected for tip broadening18 by the equation e = 2r, where e is the error in the horizontal dimension and r is the tip’s radius.19 As the normality and homoscedasticity assumptions of the data were valid, numerical data were analyzed with ANOVA and Student–Newman–Keuls multiple comparison tests, with statistical significance preset at a = 0.05.

2.5. High resolution scanning electron microscopy (HRSEM), energy dispersive (SEM/EDX) and X-ray diffraction (XRD) analyses Specimens were fixed in a solution of 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer for 24 h, rinsed three times in 0.1 mol/L sodium cacodylate buffer, and postfixed in 1% osmium tetraoxide solution for 2 h. They were then rinsed in distilled water and dehydrated in an ascending ethanol series (30%, 50%, 70%, 80%, 95%, and 100%) for 15 min each. Critical point drying was performed automatically. They were then sputter-coated with carbon by means of a sputter-coating Nanotech Polaron-SEMPREP2 (Polaron Equipment Ltd., Watford, UK) and observed with a high resolution scanning electron microscope (HRSEM Gemini, Carl Zeiss, Oberkochen, Germany) at an accelerating voltage between 10 and 20 kV. Energy-dispersive analysis was performed in selected points using an X-ray detector system (EDX Inca 300, Oxford Instruments, Oxford, UK) attached to the HRSEM. XRD analysis was performed on non-carbon coated dentine surfaces of UD, PDD and TDD. The crystal phases in the specimens were analyzed using X-Ray diffraction analysis under a diffractometer Bruker D8 Advance (XRD Bruker Corporation, Wien, Austria) conditions were Cu Ka radiation in 308 u u scan.

3.

Results

3.1.

AFM imaging and nano-indentation

Mean nanohardness (Hi) and Young modulus (Ei) values were affected by immersion solution, (F = 9.20/P < 0.001 and F = 44.81/P < 0.001, respectively). Interactions were also significant (P < 0.001). Mean values and standard deviations for each group are displayed in Table 2. PDD + ZnO and PDD showed similar Hi, lower than specimens immersed in the rest of remineralising solutions. Ei was practically similar in all samples, and higher than UD after immersion. Demineralised dentine obtained the lowest nanomechanical performance. AFM images, illustrated with the 3-D AFM Images 15 mm  15 mm and 500 nm  500 nm resolutions, are shown for each group in Figs. 1–6. Differences in the surface topography and morphology of UD (Fig. 1a), PDD (Fig. 1b) and TDD (Fig. 1c) dentine, and PDD immersed in CHX (Fig. 2),

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Table 2 – Mean and standard deviation of nanohardness (GPa) and Young’s Modulus (GPa) measured on dentine surfaces after the different experimental treatments of immersion, for 30 min. Dentine

Nanohardness (Hi)

Young moduli (Ei)

UD PA-PDD PA-TDD PDD + CHX PDD + AS PDD + PS PDD + ZnCl2 PDD + ZnO ANOVA results

2.82 (1.01) A 1.71 (0.95) B 0.003 (0.001) C 2.45 (1.12) A 3.19 (2.00) A 3.44 (1.07) A 3.48 (1.50) A 1.84 (1.06) B F = 9.20 P < 0.001

22.16 (1.95) c 2.80 (0.60) d 0.51 (0.19) e 35.53 (10.41) b 41.56(13.80) ab 48.25(20.02) a 44.78 (7.18) ab 42.73 (16.57) ab F = 44.81 P < 0.001

UD, untreated dentine; PA, phosphoric acid; PDD, partially demineralised dentine; TDD, totally demineralised dentine; CHX, chlorhexidine; AS, artificial saliva; PS, phosphated solution; ZnCl2, zinc-chloride solution; ZnO, zinc-oxide solution. Identical letters indicate no significant difference after Student–Newman–Keuls test (P < 0.05).

AS (Fig. 3), PS (Fig. 4), ZnCl2 (Fig. 5) and ZnO (Fig. 6) were established. Height variations in the sample are represented by differences in grey scale, where white represents the highest features and black the lowest features of the scale. The grey level at each position on the surface represents a different depth on the sample surface. Demineralised dentine surfaces presented the most irregular and eroded profile, corresponding with the most aggressive effect. From a morphological point of view, PDD + CHX showed the most irregular intertubular dentine surface among specimens. The rest of immersed samples showed a smoother and globular-like structure of surfaces, more pronounced in the specimens bathed in zinc-based media.

3.2.

Nanoroughness measurements

Mean nanoroughness (SRa) is shown in Table 3. Mean total roughness values decreased after immersing PDD in the different solutions, without differences among them.

Fig. 2 – AFM image of dentine surface demineralised with PA for 15 s (PDD) and immersed in chlorhexidine digluconate (CHX), for 30 min.

PDD + CHX reflected the highest intertubular, and one of the lowest tested peritubular SRa values, among solutions.

3.3.

Fibril diameter assessment

Fibril diameters decreased after demineralisation, and were influenced by the different remineralizing solution (Table 4). Significant differences run as follows: UD > PDD + ZnO > PDD + ZnCl2 > PDD + PS = PDD + AS = PDD + CHX > PA-PDD = PA-TDD.

3.4. High resolution scanning electron microscopy (HRSEM), energy dispersive (SEM/EDX) and X-ray diffraction (XRD) analyses HRSEM images of partially demineralised dentine (PDD) surfaces immersed or not, for 30 min, in the different

Fig. 1 – AFM images of dentine surfaces of (a) untreated dentine (UD), (b) partially demineralised dentine (PDD) and (c) totally demineralised dentine (TDD).

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Fig. 3 – AFM image of dentine surface demineralised with PA for 15 s (PDD) and immersed in artificial saliva (AS), for 30 min.

Fig. 5 – AFM image of dentine surface demineralised with PA for 15 s (PDD) and immersed in a ZnCl2 (ZnCl2) solution, for 30 min.

remineralizing solutions are provided in Figs. 7–12. PDD exposed a reticular structure (Fig. 7). Some clustered granular structure of spherical grains was observed to form around the collagen fibrils. After immersion, peritubular and intertubular dentine surfaces were covered by a mineral layer that impeded to observe dentine collagen fibres, except in the case of dentine surfaces immersed in ZnCl2 solutions where collagen fibres could clearly be seen, and partially in ZnO solution. EDX analyses were performed at randomized intertubular dentine sites and the most representative spectra are also shown in

Fig. 6 – AFM image of dentine surface demineralised with PA for 15 s and immersed in a ZnO (ZnO) solution, for 30 min.

Fig. 4 – AFM image of dentine surface demineralised with PA for 15 s (PDD) and immersed in phosphated solution (PS) for 30 min.

the ensuing figures (Figs. 7–12). Ca and P presence was proved in all samples, except for PDD. Additionally, Zn appeared in PDD immersed in both ZnCl2 and ZnO solutions. Higher Ca/P ratio was attained in dentine surfaces immersed in CHX, ZnCl2 and ZnO solutions if compared to surfaces immersed in AS or PS. XRD spectra are presented in Fig. 13. XRD analysis of untreated dentine presents characteristic pattern of HAP. After partial demineralisation (PDD) or total demineralisation (TDD), HAP crystals remained at the tested surfaces.

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Table 3 – Mean and standard deviation of nanoroughness Sra (nm) measured on dentine surfaces after the different experimental treatments of immersion, for 30 min. Dentine

Peritubular

Intertubular

Total

UD PA-PDD PA-TDD PDD+ CHX PDD + AS PDD + PS PDD + ZnCl2 PDD + ZnO ANOVA results

XX 17.58 (7.72) B 24.76 (8.42) A 6.24 (2.12) CD XX 9.65 (3.54) C 4.64 (0.74) D 4.47 (2.01) CD F = 26.86 P < 0.001

7.34 (2.54) d 36.25 (9.01) a 26.39 (12.35) b 20.19 (3.36) c 12.51 (2.13) d 12.72 (1.44) d 10.96 (2.91) d 8.88 (2.54) d F = 31.33 P < 0.001

10.09 (2.21) B 164 (19.52) A 133.75 (29.09) A 62.70 (12.78) B 25.05 (9.59) B 41.72 (28.69) B 14.64 (6.27) B 34.83 (32.04) B F = 22.58 P < 0.001

UD, untreated dentine; PA, phosphoric acid; PDD, partially demineralised dentine; TDD, totally demineralised dentine; CHX, chlorhexidine; AS, artificial saliva; PS, phosphated solution; ZnCl2, zinc-chloride solution; ZnO, zinc-oxide solution. Identical letters indicate no significant difference after Student–Newman–Keuls test ( p < 0.05). XX, due to the absence of morphological differences it could not be measured.

Table 4 – Mean and standard deviation of fibrils width (nm) after the different experimental treatments of immersion, for 30 min. Dentine treatment

Fibrils width

UD PA-PDD PA-TDD PDD + CHX PDD + AS PDD + PS PDD+ ZnCl2 PDD + ZnO

441.87 (75.2) A 60.85 (5.91) E 68.38 (4.86) E 137.58 (31.58) D 162.43 (24.8) D 178.89 (28.65) D 247.28 (22.17) C 349.33 (69.63) B

Fig. 7 – HRSEM images and EDX spectra of PAdemineralised dentine surface.

lesion, while an outer zone would correspond with the most severe demineralised substrate, e.g., the TDD. In general, demineralisation promoted a decrease of both Hi and Ei (Table 2), an increase in nanoroughness in

UD, untreated dentine; PA, phosphoric acid; PDD, partially demineralised dentine; TDD, totally demineralised dentine; CHX, chlorhexidine; AS, artificial saliva; PS, phosphated solution; ZnCl2, zinc-chloride solution; ZnO, zinc-oxide solution. Values with identical numbers indicate no significant difference between dentine treatments, using Student–Newman–Keuls test ( p < 0.05).

4.

Discussion

This study sought to gain insights into the role of different solutions leading to remineralisation and mechanical recovery of phosphoric acid demineralised dentine. Information on the surface properties of the demineralised dentine and their influence on remineralisation are lacking.2 Nanoindentation is the most commonly applied means of testing the mechanical properties of materials or substrates.20 The lowest Hi and Ei values were obtained by totally demineralised dentine (TDD). When dentine was partially demineralised (PDD), nanomechanical properties were somehow higher (Table 2). Different dentine demineralisation conditions are expected to influence the mechanical properties of dentine surfaces. From a clinical standpoint, PDD might correspond with an inner zone at the bottom of the carious

Fig. 8 – HRSEM images and EDX spectra of dentine surface demineralised with PA for 15 s (PDD) and immersed in chlorhexidine digluconate (CHX), for 30 min.

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Fig. 9 – HRSEM images and EDX spectra of dentine surface demineralised with PA for 15 s (PDD) and immersed in artificial saliva (AS), for 30 min.

Fig. 11 – HRSEM images and EDX spectra of dentine surface demineralised with PA for 15 s (PDD) and immersed in a ZnCl2 (ZnCl2) solution, for 30 min. (a) peritubular and intertubular dentine and (b) peritubular dentine.

Fig. 10 – HRSEM images and EDX spectra of dentine surface demineralised with PA for 15 s (PDD) and immersed in phosphated solution (PS), for 30 min.

comparison to the remineralised substrate (Table 3), and a decrease in the bandwidth of collagen fibrils (Table 4). After demineralisation, the dentine surfaced appeared roughened (Figs. 1b, 1c and 7) (Table 3), as compared with the normal

tissue (Fig. 1a), and the peritubular mineral vanished, thus widening the tubule lumens (Fig. 7). The typical staggered pattern of collagen fibrils due to the characteristic 67 nm periodicity (Fig. 1a) was not visible after demineralisation (Fig. 1b and c), which is consistent with fibrils containing remnant intrafibrillar or extrafibrillar mineral,4,21 as confirmed our X-ray diffraction (XRD) studies which demonstrated the presence of hydroxyapatite in both demineralised substrates (Fig. 13). The existence of these crystals is a crucial finding, as they can induce and facilitate further dentine remineralisation.22 Replacing mineral within type I collagen is critical to establish the normal mechanical properties of dentine that forms the bulk of tooth.23 In the present study, nanohardness (Hi) and modulus of elasticity (Ei) of PDD immersed in all remineralizing solutions increased (in Ei, exceeding UD),

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Fig. 12 – HRSEM images and EDX spectra of dentine surface demineralised with PA for 15 s (PDD) and immersed in a ZnO (ZnO) solution, for 30 min.

except when specimens were kept in ZnO, which showed similar Hi values than the partially demineralised substrate (Table 2). Similar performance was obtained by Bertassoni et al.,4 who attained 9.7 GPa in Ei, following the continuous approach of mineral growth, at 24 h. Our results showed 2.80 GPa after 30 min of immersion, but experimental setting, viscoelastic responses and experimental artefacts24 make comparisons difficult. Thereby, the null hypothesis should only be accepted, at this point. Complementarily, PDD substrate immersed in CHX experimented a reduction in nanoroughness at peritubular, intertubular and total scale (Table 3), suggesting an indirect sight of

Fig. 13 – X-ray diffraction patterns of (a) untreated dentine (UD), (b) partially demineralised dentine (PDD) and (c) totally demineralised dentine (TDD) surfaces. Vertical bars indicate hydroxylapatite peaks position.

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intrafibrillar remineralisation. Comparable roughness parameters to our peritubular SRa values (Table 3) for dentine remineralisation strategies, based on proteins and time points combinations, have reported21 a range of values from 6.5 to 10 nm, at 5 h of immersion. Different methodology makes comparisons difficult, but authors agree that a roughness decrease may be associated to a role of mineral maturation.21 Images of specimens remineralised with CHX revealed fibrillar structures with significant topographical changes that suggest a preferential organization of mineral within the surface of collagen fibrils, in an intrafibrillar manner.12 The remineralisation procedure made the fibrils to exhibit a growing width of 138 nm (Table 4) (Fig. 2), and under higher magnification the prototypical 67-nm-D-periodicity banding of collagen fibrils21 was observed. New mineral, attached to the collagen network, tried to occlude the entrance of tubules (Fig. 2), though HRSEM image (Fig. 8) showed that these apertures remained empty and permeable. Dentine surface appeared covered by mineral deposits, as it became visible completely mineralised at both peritubular and intertubular, though differentiated. Spectrum from EDX showed elemental composition of Ca and P (Fig. 8). The presence of this nucleating surface induces structural and compositional changes that enable the denser packing of the clusters and their subsequent fusion to form amorphous calcium phosphate and ultimately apatite crystals.6,25,26 Kim et al.12 have established that to promote a growth centre, collagen structure should be sound in addition to the presence of residual mineral crystals. The first condition was accomplished, as the collagen Raman spectra collected from the partially demineralised dentine13 was almost identical to the mineralised dentine substrate, indicating that the demineralised collagen was not denatured after acid etching.13,27 The mineral-inductive capacity of partially demineralised dentine (PDD) may be explained, in addition, by the small but definitive fraction of remaining polyanionic proteins, strongly attached to the collagenous matrix.28 The peptides responsible for mineral induction are located between the two collagen binding sites.29 Secondly, the presence of residual mineral was similarly, identified in the demineralised layer, in the region across the demineralised/mineralised dentine,27 and shown in the Fig. 13, where the presence of hydroxyapatite peaks are clearly observed at the partially demineralised substrate. Final remineralisation will be then facilitated around the remaining mineral crystals by obtaining mineral sources,12 from artificial saliva (AS) (CaCl2, ZnCl2 and NaCl), in solution with CHX, in the present study. The organic phosphate of dentine collagen (more abundant in PDD than in TDD) is required for mineral induction. As a result, phosphate from dentine and additional calcium from dentine and AS could precipitate in the sound collagen scaffold and initiate the remineralisation process, as deposition of Ca-P minerals were detected at the remineralised dentine surface (Fig. 8). Immersion of PDD in AS promoted the highest Hi and Ei values. Clinically, saliva is readily available to potentially remineralize. Remineralisation of dentine substrate may depend upon the quality and quantity of the mineral and/or organic component remaining in the structure30; therefore, calcium and phosphate of simulated saliva formulations can

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influence dentine remineralisation.31 In the present study, no phosphate-based component was included into AS formulation, but Kawasaki et al.32 reported that the immobilized phosphoproteins are thought to supply phosphate for mineralisation, being capable of modulating crystal nucleation and growth, as well as binding to the collagenous network.33 The organic content of dentine is approximately 18% (w/w) of which 90% is collagen and 10% noncollagenous proteins. The majority of the noncollagenous proteins are phosphoproteins; their anionic character may have been involved on biomineralisation.28,30 Removing, by different decalcifying agents,34 the soluble portion of the organic matrix, which contains phosphoproteins, it is essential to remineralisation of demineralised dentine.32 Similarly, it has been speculated that the insoluble phosphoproteins, in decalcified collagen, may serve as a locus for nucleation for mineral and so enable the remineralisation to occur, in vitro,30,35 starting at intertubular dentine,30 just where the nanoroughness parameters attained the lowest values after 30 min of immersion (Table 3). When analyzed by AFM, fibrils reconstituted in AS solution appeared to be well formed with clear D periodic banding patterns (Fig. 3). Using the software analysis all of the reconstituted fibrils had a bandwidth of 162 nm (Table 4), similar to the remineralised fibrils in CHX and PS. Once a crystal was induced on the surface of the fibril, growth of that crystal would be favoured over the nucleation of new crystals within the fibril. Crystal growth would be favoured both from energetic considerations13 and the relative rate of supply of lattice ions to the sites for apatite deposition.36 This mineral deposition was confirmed (by the Ca and P reflected by the EDX spectra and by HRSEM images), and observed on the surface of both peritubular and intertubular dentine, without mineral inside the tubules and a well defined collar of peritubular dentine (Fig. 9). When PS was used as remineralizing media, the highest values of both Hi (in junction with CHX, AS, and ZnCl2) and Ei (in junction with AS, ZnCl2 and ZnO) were obtained. Remineralizing agents seek to promote remineralisation through increases in bioavailable Ca and P species that become incorporated in dentine. It has been reported that supersaturated Ca and P solutions have little effectiveness, per se, to induce dentine remineralisation.37 The PS used in the current study provided high phosphate ion levels, supplied by KH2PO4, that in junction with CaCl2 has been well reported in the literature for remineralisation of demineralised dentine substrates.38 Collagen network was covered by a calcium and phosphate precipitate (Fig. 10), but allowing visual observation of fibrils (Fig. 4). When analyzed by AFM, fibrils reconstituted in the presence of PS appeared also to be rightly formed with a clear periodic banding and an increased average bandwidth of 178.89 nm (Table 4). HRSEM images exhibited a random, but layered, precipitation of mineral on the surface of the dentine matrix, entirely covering the intertubular region and some part of tubules, allowing the observation of a partial collar around the entrance of canaliculi (Fig. 10). Amorphous clumps of mineral precipitates scattered throughout the dense network of plate-like multilayered crystals at the entrances of tubules might have originated the highest values of peritubular roughness (9.65 nm) (Table 3). Immersion of PDD samples in Zn solutions promoted an increase in nanomechanical properties, except in ZnO, whose

Hi values did not alter after immersion (Table 2). ZnCl2 is soluble and hydrophilic. It rapidly diffuses throughout the substrate,39 probably producing an increase of local ions available for further mineral growth, as crystallinity increased after 30 min of immersion.13 Higher solubility of ZnO when in contact with acid substrates, as some acidic non collagenous proteins (dentine matrix proteins), could also account for the effective release of zinc ions,13,40 which stimulates protein phosphorylation, enhanced calcium deposition,41 and facilitated partial dentinal tubules occlusion by crystals precipitation (Figs. 5 and 6). Son et al.42 stated that based on the theory of the crystals structure, one can expect that if the radii of doped ions (Zn: 0.074 nm) are smaller than Ca (0.099 nm), it is easy for zinc to fill in the vacancy or interstitial sites of crystal lattice presenting as lose certain electrical neutrality. This causes the loss of an equivalently charged ion, and creates more vacancies for point defects. Additionally, collagen fibres diameter was augmented when dentine was immersed in ZnCl2 (247 nm) solution and much more in ZnO (349 nm) (Table 4). Deeper analysis on the signification of this finding deserves future work. AFM (Fig. 5) and HRSEM (Fig. 11) of PDD substrata immersed in ZnCl2 revealed topographical changes suggestive of mineral association with the organic matrix, as opposed to precipitated minerals in platforms on the surface, as observed in ZnO solutions (Figs. 6 and 12). That means that the mineral precursors were more homogenously distributed through the matrix, in ZnCl2, allowing for a finer interaction with the organic network. In the HRSEM images (Fig. 11), a mixture of amorphous clumps of material scattered throughout a dense network of buttons-like material, can be shown. These knob-like structures corresponded to calcified nucleus, as Ca and P were shown (Fig. 11, EDX spectra). Hence, the mineral is more likely to grow through the depth and width of the demineralised substrate, and less likely to form a surface precipitate. The different and heterogeneous precipitation of extrafibrillar apatites contributed minimally to the mechanical properties of mineralised collagen fibrils, conditioning the lower Hi values of ZnO solution, which did not differentiate with PDD (Table 2). HRSEM also showed outer zone mineral which seemed to cover the region filling most of the tubule lumen. In the gradient zone the mineral formed a lip around each tubule lumen detected (Fig. 12). A favourable surface for nucleation of apatite may cause a faster deposition of Ca-P minerals on the surface of demineralised dentine.2 Once surface crystallization took place, the mineral precursors may have been likely sequestered by existing crystallites and thus precipitate, as suggested by HRSEM. These crystals, in turn, covered the demineralised dentine surface and limited the ionic diffusion into the matrix, causing the lower mechanical properties that observed (Table 2). The clinical significance of this aspect is double: (a) there must exist a tight association of the re-growth mineral with the remineralised matrix, thus enabling the mechanical properties of the tissue,4 as the intrafibrillar mineral has been suggested to be crucial for the normal mechanical properties of the tissue24; (b) this may be an adverse effect on remineralisation of deep caries lesions, but may be applied for treatment of dentine hypersensitivity.2 However, when ZnO was used as immersion media, the

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average diameter of the final fibrils was the highest, which represents the maximum width-band that attained, with a strong tendency towards aggregating and forming wider fibrils. If EDX analysis of dentine surfaces immersed in zinc solutions (Figs. 11 and 12) are compared to other experimental groups, differences in Ca/P ratios and presence of zinc were encountered. It may account for the formation of other compounds different to HAP. The formula for stoichiometric HAP is Ca10(PO4)6(OH)2. However, biological apatite is calcium deficient and contains substantial amounts of carbonate.43 Carbonated apatite is a precursor of HAP, but when it is precipitated in the presence of zinc an exchange between Zn2+ and Ca2+ occurs in vitro forming a substituted apatite compound.44 An isomorphous substitution can be obtained when Ca2+ is replaced by Zn2+ into dentine HAP.45 The phosphate may also participate in the formation of other zinc-phosphate compounds.45 In the present study, it has been evidenced that phosphoric acid demineralised dentine is a bioactive matrix, being able to form HAP onto the surface as early as 30 min. The existence of enzymes and remineralizing factors within the dentine matrix may have facilitated remineralisation under favourable conditions.7 It is important to note that dentine was immersed in abiotic solutions, with a stable pH ranging from 7.0 to 7.5. The present data may be a starting point for new laboratory models to better understand the highly complex process of dentine remineralisation. More studies are, therefore, needed to provide definitive conclusions on this matter.

Acknowledgements This work was supported by grants CICYT/FEDER MAT201124551, JA-P08-CTS-3944 and CEI-Biotic UGR. 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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