2.1

Specimen selection and preparation

The experimental group consisted of eight specimens of freshly extracted human premolars with root canal treatment performed at least two years before extraction. The exclusion criteria associated with root canal infection were: history of a previous root canal infection, current radiological signs of acute or chronic periapical infection and macroscopically visible deterioration of periapical tooth cement and dental socket which were examined after extraction. All teeth were filled with gutta-percha. The control group consisted of eight specimens of intact (vital) teeth. The specimens of both vital and devitalized groups were divided according to the age of individuals at the moment of extraction into two categories: young (18–22 years of age) and old (55–60 years of age). All teeth were extracted for orthodontic or periodontal reasons at Department of Oral Surgery, School of Dentistry, University of Belgrade according to the protocol approved by The Ethical Committee of The School of Dentistry, University of Belgrade and for every tooth the Patient’s Informed Consent was obtained. The teeth were cleaned, stored frozen and analyzed shortly after the extraction. Each specimen was taken from different tooth.

The whole teeth were mounted in epoxy resins (Mecaprex KM-U, Grenoble, France) and were cut longitudinally (parallel with the tooth canal) to provide 1 mm thick sections using a diamond-saw microtome (Leica SP1600, Nussloch (Germany) under constant water irrigation ( Fig. 1 A ). A low-speed diamond wheel saw 650 with water soluble coolant (South Bay Technology Inc., San Clemente, CA, USA) was used to obtain dentin specimens ( Fig. 1 B). To avoid influence of endodontic irrigants on dentin the specimens were cut at least 2 mm away from the root wall ( Fig. 1 B). At this point influence of endodontic irrigants cannot be expected . The specimens were approximately 3 mm × 3 mm × 1 mm in size.

Schematic sequence illustrating the procedure of obtainment of the specimen. Each tooth was cut longitudinally (parallel with the tooth canal) to provide 1 mm thick sections (A). Red area on picture A refers the longitudinal section from which the specimen was obtained. Blue section on picture B shows the exact specimen location. (For interpretation of the references to color in this legend, the reader is referred to the web version of the article.)

2.2

Atomic force microscopy (AFM) imaging

Each dentin specimen was ultrasonicated for 5 min to remove any possible debris and organic dirt as previously suggested and then placed horizontally onto the sample disk, and imaged by Multimode quadrex Scanning Probe Microscopy (SPM) with NanoscopeIIIe controller (Veeco Instruments Inc., New York, NY, USA) under ambient conditions. Height and phase images were simultaneously acquired under standard AFM tapping mode using a commercial SNC (solid nitride cone) AFM probe (NanoScience Instruments, Inc. Phoenix, Arizona, USA). As specified by the manufacturer, the cantilever was 125 μm in length, with force constant 40 N/m, the tip radius less than 10 nm and resonant frequency of 275 kHz. At least 10 images were taken per specimen to account for possible spatial variability. Images of various sizes were made: 3 μm × 3 μm, 2 μm × 2 μm, 1 μm × 1 μm, 500 nm × 500 nm and 300 nm × 300 nm. The scans were taken with 256 lines per scan (256 × 256 pixels). The images were acquired from various locations of intertubular dentin to assure representativeness of the observed properties. Particular care was taken to prepare and analyze all specimens in the same manner to assure validity of inter-specimen comparisons. Hydroxyapatite crystal size was determined by measuring maximum dimension of each crystal using Veeco Nanoscope III software (version 5.31r1) like in previous studies . Following estimation of sample size using MedCALC software ver. 9.1.0.1, about 100 crystals per study group were considered for the quantitative analysis. Statistical analysis was performed using analysis of variance in SPSS ver.15 and the values p < 0.05 were considered statistically significant. Tapping mode AFM is able to map simultaneously the topography of the specimen (height image) and the compositional variations (phase image) of the specimen’s surface . Variations in material properties lead to a phase lag of the cantilever oscillation. This phase lag is simultaneously monitored by the AFM control electronics and transformed into phase image. Specifically, the relationship of phase-shifts and energy dissipation enables linking experimental data to materials properties such as stiffness, viscoelasticity, etc. Therefore, phase image gives non-quantitative information about specimen material properties .

The measuring conditions for all images and all the specimens were constant to allow comparison of the results obtained during AFM characterization. Drive frequency of 87.68 kHz was maintained constant during the imaging, while the drive amplitude was 851 mV. Origin Lab 8.0 program was used for curve fitting and determination of peak positions and areas under the curve.

2.2.1

AFM imaging: power spectral density and fractal dimension

Power spectral density (PSD) analysis was performed on AFM topography images using the software WSxM (WSxM v5.0, developed by Horcas et al. ). Power spectral density is an advanced approach for roughness analysis that arises from Fourier decomposition of an image into the waves of particular wavelengths . PSD analysis reveals characteristics of the surface structure and describes the contribution of various morphological elements to the surface roughness . The power spectral density data were extracted from the images of various sizes and the data points collected from all images belonging to one specimen were then represented on a single graph showing log PSD vs. log spatial frequency of the specimen. The data points were fitted linearly as previously suggested , obtaining the slope of the trendline in each specimen ( p < 0.05). In addition, fractal dimension (FD) – a descriptor of the complexity of object’s structure corresponding to visual perception of roughness – was calculated from log PSD vs. log spatial frequency graphs according to the following equation: FD = 0.5*(7 − β ) ( β = absolute value of the slope of PSD trendline) .

2.3

Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX)

To provide adequately flat surface for microanalyses, the dentin specimens were polished using carbide papers of increasing smoothness (from 600 up to 4000 grit) under constant water irrigation on semiautomatic Unipol 810 polishing machine (MTI Corporation, Richmond, CA, USA). The specimens were ultrasonicated to remove any polishing-related debris and were left to dry naturally at room temperature.

Following sputter coating with gold (Au) in Ion Sputter Coater (Bal-Tec SCD 005, Leica Microsystems, Nussloch, Germany; WD 50 mm, 30 mA, 90 s), the specimens were mounted on a scanning electron microscope (SEM, JEOL JSM 6460 LV, Peabody, MA, USA) equipped with electron dispersive X-ray spectroscopy (EDX, OXFORD-Inca X sight). For EDX analysis, the system was operated at 20 kV, with constant working distance in line with previous studies . Spectra of atomic composition of the specimens’ surface were obtained by spot EDX analysis in five spots located in intertubular dentin in each specimen, under visual control of SEM. The EDX software automatically evaluates relative contribution of each of the detected elements within the region of interest to a total of 100%.

2.4

Micro-Raman spectroscopy

Micro-Raman spectroscopy measurements were performed using DXR Raman Microscope (Thermo Scientific, Waltham, MA, USA). Nd:YAG doubled frequency line of 532 nm was used as an excitation source, whereas the applied power was 10 mW. Objective with 10× magnification was used with the resulting average spot diameter of 2.1 μm on the specimen. All the spectra were collected at room temperature in high resolution mode, in the range (300–1800) cm ⁻¹ , from the dentin tooth area of the teeth specimens ( Fig. 1 ). Spectra acquisition and background fluorescence correction were performed in software package OMNIC (Thermo Scientific, Waltham, MA, USA). Raman spectra were analyzed using OriginPro 8 Software. Multiple-point baseline correction and smoothing were performed as previously suggested . Raman band positions were compared with those of dentin and apatite known from the literature . A selected subregion of each spectrum was fitted with Lorentzian profile .

2.1

Specimen selection and preparation

The experimental group consisted of eight specimens of freshly extracted human premolars with root canal treatment performed at least two years before extraction. The exclusion criteria associated with root canal infection were: history of a previous root canal infection, current radiological signs of acute or chronic periapical infection and macroscopically visible deterioration of periapical tooth cement and dental socket which were examined after extraction. All teeth were filled with gutta-percha. The control group consisted of eight specimens of intact (vital) teeth. The specimens of both vital and devitalized groups were divided according to the age of individuals at the moment of extraction into two categories: young (18–22 years of age) and old (55–60 years of age). All teeth were extracted for orthodontic or periodontal reasons at Department of Oral Surgery, School of Dentistry, University of Belgrade according to the protocol approved by The Ethical Committee of The School of Dentistry, University of Belgrade and for every tooth the Patient’s Informed Consent was obtained. The teeth were cleaned, stored frozen and analyzed shortly after the extraction. Each specimen was taken from different tooth.

The whole teeth were mounted in epoxy resins (Mecaprex KM-U, Grenoble, France) and were cut longitudinally (parallel with the tooth canal) to provide 1 mm thick sections using a diamond-saw microtome (Leica SP1600, Nussloch (Germany) under constant water irrigation ( Fig. 1 A ). A low-speed diamond wheel saw 650 with water soluble coolant (South Bay Technology Inc., San Clemente, CA, USA) was used to obtain dentin specimens ( Fig. 1 B). To avoid influence of endodontic irrigants on dentin the specimens were cut at least 2 mm away from the root wall ( Fig. 1 B). At this point influence of endodontic irrigants cannot be expected . The specimens were approximately 3 mm × 3 mm × 1 mm in size.

Schematic sequence illustrating the procedure of obtainment of the specimen. Each tooth was cut longitudinally (parallel with the tooth canal) to provide 1 mm thick sections (A). Red area on picture A refers the longitudinal section from which the specimen was obtained. Blue section on picture B shows the exact specimen location. (For interpretation of the references to color in this legend, the reader is referred to the web version of the article.)

2.2

Atomic force microscopy (AFM) imaging

Each dentin specimen was ultrasonicated for 5 min to remove any possible debris and organic dirt as previously suggested and then placed horizontally onto the sample disk, and imaged by Multimode quadrex Scanning Probe Microscopy (SPM) with NanoscopeIIIe controller (Veeco Instruments Inc., New York, NY, USA) under ambient conditions. Height and phase images were simultaneously acquired under standard AFM tapping mode using a commercial SNC (solid nitride cone) AFM probe (NanoScience Instruments, Inc. Phoenix, Arizona, USA). As specified by the manufacturer, the cantilever was 125 μm in length, with force constant 40 N/m, the tip radius less than 10 nm and resonant frequency of 275 kHz. At least 10 images were taken per specimen to account for possible spatial variability. Images of various sizes were made: 3 μm × 3 μm, 2 μm × 2 μm, 1 μm × 1 μm, 500 nm × 500 nm and 300 nm × 300 nm. The scans were taken with 256 lines per scan (256 × 256 pixels). The images were acquired from various locations of intertubular dentin to assure representativeness of the observed properties. Particular care was taken to prepare and analyze all specimens in the same manner to assure validity of inter-specimen comparisons. Hydroxyapatite crystal size was determined by measuring maximum dimension of each crystal using Veeco Nanoscope III software (version 5.31r1) like in previous studies . Following estimation of sample size using MedCALC software ver. 9.1.0.1, about 100 crystals per study group were considered for the quantitative analysis. Statistical analysis was performed using analysis of variance in SPSS ver.15 and the values p < 0.05 were considered statistically significant. Tapping mode AFM is able to map simultaneously the topography of the specimen (height image) and the compositional variations (phase image) of the specimen’s surface . Variations in material properties lead to a phase lag of the cantilever oscillation. This phase lag is simultaneously monitored by the AFM control electronics and transformed into phase image. Specifically, the relationship of phase-shifts and energy dissipation enables linking experimental data to materials properties such as stiffness, viscoelasticity, etc. Therefore, phase image gives non-quantitative information about specimen material properties .

The measuring conditions for all images and all the specimens were constant to allow comparison of the results obtained during AFM characterization. Drive frequency of 87.68 kHz was maintained constant during the imaging, while the drive amplitude was 851 mV. Origin Lab 8.0 program was used for curve fitting and determination of peak positions and areas under the curve.

2.2.1

AFM imaging: power spectral density and fractal dimension

Power spectral density (PSD) analysis was performed on AFM topography images using the software WSxM (WSxM v5.0, developed by Horcas et al. ). Power spectral density is an advanced approach for roughness analysis that arises from Fourier decomposition of an image into the waves of particular wavelengths . PSD analysis reveals characteristics of the surface structure and describes the contribution of various morphological elements to the surface roughness . The power spectral density data were extracted from the images of various sizes and the data points collected from all images belonging to one specimen were then represented on a single graph showing log PSD vs. log spatial frequency of the specimen. The data points were fitted linearly as previously suggested , obtaining the slope of the trendline in each specimen ( p < 0.05). In addition, fractal dimension (FD) – a descriptor of the complexity of object’s structure corresponding to visual perception of roughness – was calculated from log PSD vs. log spatial frequency graphs according to the following equation: FD = 0.5*(7 − β ) ( β = absolute value of the slope of PSD trendline) .

2.3

Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX)

To provide adequately flat surface for microanalyses, the dentin specimens were polished using carbide papers of increasing smoothness (from 600 up to 4000 grit) under constant water irrigation on semiautomatic Unipol 810 polishing machine (MTI Corporation, Richmond, CA, USA). The specimens were ultrasonicated to remove any polishing-related debris and were left to dry naturally at room temperature.

Following sputter coating with gold (Au) in Ion Sputter Coater (Bal-Tec SCD 005, Leica Microsystems, Nussloch, Germany; WD 50 mm, 30 mA, 90 s), the specimens were mounted on a scanning electron microscope (SEM, JEOL JSM 6460 LV, Peabody, MA, USA) equipped with electron dispersive X-ray spectroscopy (EDX, OXFORD-Inca X sight). For EDX analysis, the system was operated at 20 kV, with constant working distance in line with previous studies . Spectra of atomic composition of the specimens’ surface were obtained by spot EDX analysis in five spots located in intertubular dentin in each specimen, under visual control of SEM. The EDX software automatically evaluates relative contribution of each of the detected elements within the region of interest to a total of 100%.

2.4

Micro-Raman spectroscopy

Micro-Raman spectroscopy measurements were performed using DXR Raman Microscope (Thermo Scientific, Waltham, MA, USA). Nd:YAG doubled frequency line of 532 nm was used as an excitation source, whereas the applied power was 10 mW. Objective with 10× magnification was used with the resulting average spot diameter of 2.1 μm on the specimen. All the spectra were collected at room temperature in high resolution mode, in the range (300–1800) cm ⁻¹ , from the dentin tooth area of the teeth specimens ( Fig. 1 ). Spectra acquisition and background fluorescence correction were performed in software package OMNIC (Thermo Scientific, Waltham, MA, USA). Raman spectra were analyzed using OriginPro 8 Software. Multiple-point baseline correction and smoothing were performed as previously suggested . Raman band positions were compared with those of dentin and apatite known from the literature . A selected subregion of each spectrum was fitted with Lorentzian profile .

3.1

AFM

3.1.1

AFM topography and crystal size analysis

AFM topography images revealed granular organization of dentin apatite structure ( Fig. 2 A and B ). Quantitative analysis of the observed surfaces demonstrated that the mean size of mineral crystals was larger in devitalized when compared with healthy teeth (young: 51.4 ± 19.5 nm vs. 36.2 ± 14.4 nm, old: 35.2 ± 14.5 nm vs. 32.6 ± 13.7 nm; p < 0.001).

Atomic force microscopic characterization of dentin in vital and devitalized teeth. AFM 3D topographic images (305 nm × 305 nm) of devitalized (A) and vital dentin (B); corresponding AFM phase images of devitalized (C) and vital dentin (D); differences in surface phase composition of dentin in devitalized and vital teeth (E). For all phase images z -range is 100°.

3.1.2

Power spectral density and fractal analyses

Power spectral density analysis was obtained based on AFM topography images, providing quantitative characterization of the surface structure ( Fig. 3 ). Linear fitting of PSD data ( p < 0.05) revealed differences in the pattern of surface roughness between vital and devitalized teeth, where the PSD slopes showed a trend to steeper values in the vital teeth (young: PSD _slope = −3.985, old: PSD _slope = −3.885) when compared to devitalized cases (young: PSD _slope = −3.646, old: PSD _slope = −3.719).

An illustrative power spectral density graph showing contribution (power) of surface morphological features of various sizes (spatial frequencies) to overall surface roughness. Linear fitting of the PSD data allowed calculation of the PSD slope which is considered as a true material characteristic.

In both young and old cases, the fractal dimension was calculated from the PSD data showing higher values in devitalized specimens when compared to vital specimens (young: 1.677 vs. 1.508; old: 1.640 vs. 1.558), indicating their higher degree of structural complexity and surface roughness. This is in line with larger mineral crystals observed in devitalized dentin. The differences in surface morphology were more pronounced between devitalized and vital teeth in young cases than in the elderly, where healthy elderly teeth already presented a changed surface complexity in comparison with the young.

3.1.3

AFM phase analysis

AFM phase imaging of dentin surfaces mapped spatial distribution of material properties as reflected in different phase shifts of the cantilever during the tip-sample interaction across the specimens’ surface ( Fig. 2 C and D). Further evaluation of the phase images revealed that dentin surfaces reflected two distinct phase peaks on histograms derived from the AFM phase images: the peak corresponding to a lower phase shift (left peak), and the peak corresponding to a higher phase shift during the tip-specimen interaction (right peak) ( Fig. 2 E). Since all the specimens were imaged under the same scanning conditions, it was possible to compare the peak positions between the specimens. The AFM phase images and related histograms showed notable differences in material properties between different groups of specimens ( Fig. 2 C–E). Fig. 2 E shows that the exact position of the peaks, as well as their area under the curve, differed among the groups. In particular, not only that the histograms of phase images of the devitalized dentin showed a slight shift of the left peak to the right when compared to vital dentin, but also the relative area under the right peak increased remarkably in devitalized specimens (in old: 28% vs. 2.5%; in young: 32% vs. 5.3%), revealing that a higher proportion of the devitalized dentin surface was composed of a material producing a higher phase shift during the tip-specimen interaction.

3.2

EDX analysis

EDX analysis showed similar Ca/P ratio between vital and devitalized specimens. The averaged weight Ca/P ratios were: 1.87 ± 0.01 in young vital dentin, 1.85 ± 0.08 in young devitalized dentin, 1.91 ± 0.02 in old vital dentin and 1.90 ± 0.10 in old devitalized dentin ( p > 0.05). However, the average content of calcium was slightly lower in devitalized teeth (32.45 ± 0.03 wt% – young vital dentin, 29.17 ± 0.50 wt% – young devitalized dentin, 30.9 ± 0.30 wt% – old vital dentin and 28.17 ± 0.90 wt% – old devitalized dentin).

3.3

Micro-Raman spectroscopy

In Table 1 Raman shifts observed in Raman spectra are presented (see Fig. 4 A), together with the reference data. Regions of the spectra, characterized by the appearance of the vibrational modes of hydroxyapatite (HAP), other calcium orthophosphates (octacalcium phosphate – OCP, dicalciumphosphatedihydrate – DCPD and tricalcium phosphate – TCP) and collagen, were analyzed.

Table 1

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