Abstract
Objective
The aim was to characterize the variations in the structure and surface dehydration of acid demineralized intertubular dentin collagen network with the variations in dentin depth and time of air-exposure (3, 6, 9 and 12 min). In addition, to study the effect of these variations on the tensile bond strength (TBS) to dentin.
Methods
Phosphoric acid demineralized superficial and deep dentin specimens were prepared. The structure of the dentin collagen network was characterized by AFM. The surface dehydration was characterized by probing the nano-scale adhesion force ( F ad ) between AFM tip and intertubular dentin surface as a new experimental approach. The TBS to dentin was evaluated using an alcohol-based dentin self-priming adhesive.
Results
AFM images revealed a demineralized open collagen network structure in both of superficial and deep dentin at 3 and 6 min of air-exposure. However, at 9 min, superficial dentin showed more collapsed network structure compared to deep dentin that partially preserved the open network structure. Total collapsed structure was found at 12 min for both of superficial and deep dentin. The value of the F ad is decreased with increasing the time of air-exposure and is increased with dentin depth at the same time of air-exposure. The TBS was higher for superficial dentin at 3 and 6 min, however, no difference was found at 9 and 12 min.
Significance
The ability of the demineralized dentin collagen network to resist air-dehydration and to preserve the integrity of open network structure with the increase in air-exposure time is increased with dentin depth. Although superficial dentin achieves higher bond strength values, the difference in the bond strength is decreased by increasing the time of air-exposure. The AFM probed F ad showed to be sensitive approach to characterize surface dehydration, however, further researches are recommended regarding the validity of such approach.
1
Introduction
Dentin has been characterized by Marshall et al. as a biologic composite of collagen matrix filled with nanometer-sized calcium-deficient, carbonate-rich apatite crystallites dispersed between parallel micron-sized hypermineralized, collagen poor, hollow cylinders (dentinal tubules containing peritubular dentin). Superficial dentin has few dentinal tubules and composed mainly of intertubular dentin, whereas deep dentin is composed mainly of large dentinal tubules with much less intertubular dentin matrix . The dentin structure and properties are changed with depth as reported by many researchers. Kinney et al. reported that the intertubular dentin becomes softer as deep dentin is tested. In addition, the water content of dentin is lowest in superficial dentin compared to deep dentin . However, the average bulk mineral composition may be relatively constant, as function of dentinal depth, even though the amount of collagen rich intertubular dentin gradually decreases with depth . The amount of collagen fibrils per unit volume of dentin decreases from superficial dentin to deep dentin . The structural integrity and mechanical properties of the collagen fibrils of acid demineralized deep and superficial dentin play an important role in the determination of bond strength and its durability .
Atomic force microscope (AFM) was used extensively, in recent years, as a tool to study the surface morphology and roughness, collagen fibrils structure, and mechanical properties of human dentin . Atomic force microscope is not only a tool to image and measure the topography of solid surfaces at high resolution. It can also be used to measure force-versus-distance curves. Such force curves provide valuable information on local material properties such as adhesion force, elasticity, hardness and surface charge densities . Detailed description of the techniques, interpretation and applications of force curve measurement by AFM is clarified in more specialized references . However, in brief, a force vs. distance curve is a graph of the vertical force on the cantilever tip as a function of the extension of the piezoelectric scanner tube. A force curve is generated at a single location on a specimen surface by measuring how much the cantilever bends during one or more “sweeps” (up and down movements) of the scanner. Variations in the shape of force curves taken at different locations indicate variations in the local nano-scale properties of the specimen surface. The shape of the curve is also affected by contaminants and surface lubricants, as well as the water content of the surface layer of the specimen when operating an AFM in air.
Pashley et al. clarified the difference in the structure of the hybrid layer between superficial and deep dentin. In the first, most of hybrid layer is composed of demineralized and resin-hybridized intertubular dentin with only an occasional resin tags penetrating from the overlying adhesive layer into, relatively narrow, funnel-shaped dentinal tubules. In deep dentin the tubules are so numerous and large that little intertubular dentinal matrix is available. Consequently, resin tags represent a major fraction of bonded surfaces near the pulp. Theoretically, resin tags could contribute to resin retention if they are firmly bonded to the walls of the tubules . In addition to resin tags, hybrid layer formation in demineralized intertubular dentin should also contribute to resin retention in proportion to the amount of etched intertubular dentin that is available for bonding in deep dentin. Therefore, the relative contribution of resin tags and hybrid layer to overall bond strength should be varied with dentin depth because the sum of the cross-sectional area of resin tags and that of the hybrid layer is constant .
Modeling experiments on the packing density of molecules within tendon collagen fibrils indicate that there may be significant space between the collagen molecules for tissue fluid as water. As long as the interfibrillar spaces, which from interconnected channels having capillary dimensions, are in hydrated state and maintaining an open-structure, pathways are provided for monomers diffusion through collagen fibrils network . This water may be lost with dehydration, as a result of air-exposure, leading to shrinkage and collapse of the collagen fibrils network . As stated by Nakabayashi and others for hybrid layer formation, intertubular dentin must be demineralized to expose the open collagen fibrils network of the dentinal matrix to create diffusion pathway for monomer infiltration. Therefore, the presence of water is crucial to the maintenance of the structure and the strength of the demineralized dentin collagen matrix and consequently higher bond strength values are obtained with wet surfaces .
The current study investigated the hypothesis that the variations in dentin depth and air-exposure time affect the performance, collagen fibrils network structure and the bonding efficacy of acid demineralized intertubular dentin. Therefore, the first aim of this study was to determine whether the structural changes of the intertubular dentin collagen fibrils network as a result of air-exposure following phosphoric acid etching differ with dentin depth and air-exposure time. The second aim was to characterize the dehydration from the surface of acid etched intertubular dentin collagen network as a function of dentin depth and air-exposure time by probing the nano-scale adhesion force between atomic force microscope (AFM) tip and intertubular dentin surface as a new experimental approach. Finally, to study the effect of the variations in the structure of the acid demineralized collagen fibrils network and surface dehydration, with the variations in dentin depth and air-exposure time, on the bond strength to dentin using an alcohol-based dentin self-priming adhesive.
2
Materials and methods
Dentin specimens used in this study for both of AFM surface characterization and bond strength evaluation were prepared from randomly selected non-carious and non-restored mandibular third molars. All teeth were recently extracted, less than 3 months from the time of the study. All patients were of age range of 25–30 years old. All extracted teeth were stored in 0.5% chloramines T solution for 2 weeks then in distilled water at 4 °C until use .
2.1
Preparation of dentin specimens for AFM study
Eight teeth were used for AFM study from which eight superficial (Sd) and eight deep (Dd) dentin disks, of approximately 2 mm thickness, were prepared. From each tooth one superficial and one deep dentin disks were prepared to decrease the variation between specimens. For the preparation of the superficial dentin disks, occlusal enamel was removed perpendicular to the teeth long-axis with a diamond disk mounted to a milling machine (Nouvage AG, Fräsgerät AF 30, Switzerland) using slow-speed under water cooling until the superficial dentin surface approximately 1 mm below the DEJ was exposed. Then the superficial dentin disks, having an approximate thickness of 2 mm, were cut parallel to the exposed superficial dentin surfaces. After cutting of the superficial dentin disks, the exposed dentin surfaces, which approximately 3 mm below the DEJ, were considered as deep dentin. Then the deep dentin disks were prepared in a similar manner as in superficial dentin disks. The dentin disks were cut into two equal halves to produce 16 superficial and 16 deep dentin specimens for the AFM study. The dentin specimens were sequentially wet-grinded with 600, 800, 1000 and 1500 grit SiC polishing papers to produce relatively smooth surfaces for AFM characterization and to creates standardized smear layer. All specimens were then ultrasonically cleaned in deionized water for 15 min and then stored in distilled water at 37 °C for 24 h before AFM study.
2.2
Atomic force microscope (AFM) study
An atomic force microscope (Autoprobe CP-II, Veeco, CA, USA) was used to characterize the etched intertubular dentin collagen fibrils network structure and to probe the nano-scale adhesion force between AFM silicon nitride tip and etched intertubular dentin surface to characterize surface dehydration as a function of both of dentin depth and air-exposure time.
The dentin specimens were etched for 15 s with 37% phosphoric acid gel (Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) and rinsed ultrasonically in distilled water for 5 min. Then the etched dentin specimens were treated by 6.5 vol.% sodium hypochlorite (NaOClaq) for 120 s as a deproteinizing agent to remove non-collagenous proteins from the extracellular organic matrix and to reveal the collagen fibril network structure. Then all specimens were thoroughly rinsed in distilled water for 5 min and the excess water was removed by gentle blotting with absorbent paper leaving the dentin surface visibly moist. Immediately after surface preparation, dentin specimens were characterized by AFM.
2.2.1
Characterization of collagen fibrils network structure by tapping-mode AFM
Eight specimens of each of superficial and deep dentin were used for the tapping-mode AFM study. For the tapping mode, gold-coated all-silicon cantilevers (Ultralevers™, Thermo-Microscopes, CA, USA) with integrated high aspect ratio conical tips were used. The typical radius of curvature of the scanning tip was 10 nm. The force constant and the resonance frequency of the used cantilevers were 17 N/m and 320 kHz, respectively. The Images were recorded with scan rate of 2 Hz at a scanning area of 3 μm × 3 μm and a resolution of 512 × 512 pixels per image.
All dentin specimens were characterized in air under well-controlled laboratory environments in term of humidity (45 ± 5%) and temperature (21 ± 0.5 °C). Immediately after surface preparation, each specimen was mounted to the AFM and characterized immediately at relatively hydrated state. The average time for specimen mounting and operating the AFM was approximately 3 min. Therefore, the first scan taken for each specimen was preformed approximately at 3 min ( t 1) of air-exposure. After the first scan at time ( t 1), a series of three consecutive scans were preformed at 3 min time intervals from the end of the first scan. Therefore, each specimen, prepared either from superficial or deep dentin, was characterized at 3 ( t 1), 6 ( t 2), 9 ( t 3), and 12 ( t 4) min of air-exposure from the end dentin surface preparation steps. Accordingly, the variations in the structure of the acid demineralized intertubular dentin collagen fibrils network with variations in dentin depth and air-exposure time were characterized.
2.2.2
Probing nano-scale adhesion force ( F ad ) by contact-mode AFM
The remaining superficial ( n = 8) and deep ( n = 8) dentin specimens were used to probe the nano-scale adhesion force between AFM silicon nitride tip and etched intertubular dentin surface as a function of time of air-exposure and dentin depth. Immediately after surface preparation, superficial and deep dentin specimens were characterized by contact-mode AFM at the same environmental conditions and air-exposure time intervals ( t 1– t 4) as previously described.
AFM contact-mode was operated to characterize the force-versus-distance curve (force curve) between the AFM tip and specimen surface. Silicon nitride tips, of 50 nm nominal radii, mounted to contact-mode cantilevers (Type D Microlevers™, Thermo-Microscopes, CA, USA) were used to scan specimens surfaces. The force constant and the resonance frequency of the used cantilevers were 2.1 N/m and 160 kHz, respectively. For each specimen, surface scan of 3 μm × 3 μm area was done at a scan rate of 2 Hz and then a set of force curves were measured for selected 14 points at the intertubular dentin. The same process was repeated for each specimen at each previously described time intervals such that a set of force curves as a function of time of air-exposure ( t 1– t 4) and dentin depth (Sd and Dd) were recorded.
The force curves typically show the deflection of the free end of the AFM cantilever as the fixed end of the cantilever is brought vertically towards and then away from the specimen surface. The deflection of the free end of the cantilever is measured and plotted at many points as the z -axis scanner extends the cantilever towards the surface and then retract it again. Then the nano-scale surface adhesion force ( F ad ) between the AFM tip and intertubular surface was calculated in nano-Newton (nN) from the mean force curve of each specimen at each air-exposure time interval. The adhesion force ( F ad ) was calculated from the difference between the snap-in point (the point of contact between the cantilever tip and specimen surface) and the snap-out point (the point of separation or detachment between the cantilever tip and specimen surface) .
2.3
Tensile bond strength (TBS) testing
Dentin/resin-based restorative composite specimens were prepared from freshly extracted mandibular third molars for the tensile bond strength (TBS) testing. The roots of the extracted molars were embedded in self-polymerized resin 3 mm below the cervical line. The occlusal enamel was removed using diamond disk mounted to a milling machine as previously described in the preparation of the specimens for the AFM study under copious water spray to expose flat dentin surface. Half of the teeth were prepared to expose the superficial dentin surfaces, while in the other half the deep dentin surfaces were exposed. The smear layer was standardized as described previously. The exposed dentin surfaces were etched for 15 s with 37% phosphoric acid gel, thoroughly rinsed in distilled water for 5 min and the excess water was removed by gentle blotting with absorbent paper leaving the dentin surface visibly moist.
A single bottle alcohol-based self-priming adhesive (Excite, Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) was applied to the conditioned dentin surfaces. One coat of Excite adhesive was applied over the etched dentin surface and gently agitated for 10 s; excess solvent was removed using a gentle stream of air for 3 s. The Excite was then light-cured for 20 s using a light-curing unit (Cromalux-E, Meca-Physik Dental Division, D-76437 Rastatt, Germany) with a light output of 600 mW/cm 2 . According to the dentin depth and the time of application of the self-priming adhesive (air-exposure time), teeth were divided into the following test groups to simulate what was done in AFM study. Superficial dentin (Sd): Sd( t 1), Sd( t 2), Sd( t 3), and Sd( t 4). Deep dentin (Dd): Dd( t 1), Dd( t 2), Dd( t 3), and Dd( t 4). After the application of the self-priming adhesive at the desired air-exposure times ( t 1– t 4), a light cured resin-based restorative composite material (Tetric Ceram, Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) was applied and cured in four increments for each tooth. The restored teeth were then stored in distilled water at 37 °C for 48 h to complete the curing process.
After storage, each restored tooth was sectioned with diamond disk fixed to a milling machine under copious water spray to prepare dentin/composite slabs of 1 mm thickness from the central part of the tooth. A fine high-speed diamond stone with air/water spray was used to trim the slabs into an hour-glass shape with cross-sectional area of approximately 1 mm 2 at the bonded interface which checked and confirmed by a digital caliper. Twelve ( n = 12) hour-glass shape slabs were tested for each group. All slabs were stored in distilled water at 37 °C for 24 h before mechanical testing. Each slab was fixed to two disposable acrylic resin jigs using cyanoacrylate adhesive. The acrylic jigs were mounted to a universal testing machine (Lloyd Instruments, LR5 series, UK). The test was run at a cross-head speed of 0.5 mm/min until failure. The cross-sectional area of each fractured slab was confirmed using a digital caliper and the respective load was divided over the cross-sectional area to calculate the TBS in MPa.
2.4
Statistical analysis
All data of nano-scale adhesion force ( F ad ) and tensile bond strength (TBS) were expressed as means and standard deviations (SD). Statistical analysis was carried out using SPSS program (Release 15, 2006). Two-way ANOVA was preformed to test the effect of dentin depth, air-exposure time and their interaction on either of F ad and TBS. Tukey–Kramer multiple-comparison post hoc test was used to compare each of F ad and μTBS between the tested groups. The correlation between F ad and TBS through the different air-exposure times ( t 1– t 4) was tested using Pearson’s correlation coefficient. P -values less than 0.05 were considered statistically significant.
2
Materials and methods
Dentin specimens used in this study for both of AFM surface characterization and bond strength evaluation were prepared from randomly selected non-carious and non-restored mandibular third molars. All teeth were recently extracted, less than 3 months from the time of the study. All patients were of age range of 25–30 years old. All extracted teeth were stored in 0.5% chloramines T solution for 2 weeks then in distilled water at 4 °C until use .
2.1
Preparation of dentin specimens for AFM study
Eight teeth were used for AFM study from which eight superficial (Sd) and eight deep (Dd) dentin disks, of approximately 2 mm thickness, were prepared. From each tooth one superficial and one deep dentin disks were prepared to decrease the variation between specimens. For the preparation of the superficial dentin disks, occlusal enamel was removed perpendicular to the teeth long-axis with a diamond disk mounted to a milling machine (Nouvage AG, Fräsgerät AF 30, Switzerland) using slow-speed under water cooling until the superficial dentin surface approximately 1 mm below the DEJ was exposed. Then the superficial dentin disks, having an approximate thickness of 2 mm, were cut parallel to the exposed superficial dentin surfaces. After cutting of the superficial dentin disks, the exposed dentin surfaces, which approximately 3 mm below the DEJ, were considered as deep dentin. Then the deep dentin disks were prepared in a similar manner as in superficial dentin disks. The dentin disks were cut into two equal halves to produce 16 superficial and 16 deep dentin specimens for the AFM study. The dentin specimens were sequentially wet-grinded with 600, 800, 1000 and 1500 grit SiC polishing papers to produce relatively smooth surfaces for AFM characterization and to creates standardized smear layer. All specimens were then ultrasonically cleaned in deionized water for 15 min and then stored in distilled water at 37 °C for 24 h before AFM study.
2.2
Atomic force microscope (AFM) study
An atomic force microscope (Autoprobe CP-II, Veeco, CA, USA) was used to characterize the etched intertubular dentin collagen fibrils network structure and to probe the nano-scale adhesion force between AFM silicon nitride tip and etched intertubular dentin surface to characterize surface dehydration as a function of both of dentin depth and air-exposure time.
The dentin specimens were etched for 15 s with 37% phosphoric acid gel (Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) and rinsed ultrasonically in distilled water for 5 min. Then the etched dentin specimens were treated by 6.5 vol.% sodium hypochlorite (NaOClaq) for 120 s as a deproteinizing agent to remove non-collagenous proteins from the extracellular organic matrix and to reveal the collagen fibril network structure. Then all specimens were thoroughly rinsed in distilled water for 5 min and the excess water was removed by gentle blotting with absorbent paper leaving the dentin surface visibly moist. Immediately after surface preparation, dentin specimens were characterized by AFM.
2.2.1
Characterization of collagen fibrils network structure by tapping-mode AFM
Eight specimens of each of superficial and deep dentin were used for the tapping-mode AFM study. For the tapping mode, gold-coated all-silicon cantilevers (Ultralevers™, Thermo-Microscopes, CA, USA) with integrated high aspect ratio conical tips were used. The typical radius of curvature of the scanning tip was 10 nm. The force constant and the resonance frequency of the used cantilevers were 17 N/m and 320 kHz, respectively. The Images were recorded with scan rate of 2 Hz at a scanning area of 3 μm × 3 μm and a resolution of 512 × 512 pixels per image.
All dentin specimens were characterized in air under well-controlled laboratory environments in term of humidity (45 ± 5%) and temperature (21 ± 0.5 °C). Immediately after surface preparation, each specimen was mounted to the AFM and characterized immediately at relatively hydrated state. The average time for specimen mounting and operating the AFM was approximately 3 min. Therefore, the first scan taken for each specimen was preformed approximately at 3 min ( t 1) of air-exposure. After the first scan at time ( t 1), a series of three consecutive scans were preformed at 3 min time intervals from the end of the first scan. Therefore, each specimen, prepared either from superficial or deep dentin, was characterized at 3 ( t 1), 6 ( t 2), 9 ( t 3), and 12 ( t 4) min of air-exposure from the end dentin surface preparation steps. Accordingly, the variations in the structure of the acid demineralized intertubular dentin collagen fibrils network with variations in dentin depth and air-exposure time were characterized.
2.2.2
Probing nano-scale adhesion force ( F ad ) by contact-mode AFM
The remaining superficial ( n = 8) and deep ( n = 8) dentin specimens were used to probe the nano-scale adhesion force between AFM silicon nitride tip and etched intertubular dentin surface as a function of time of air-exposure and dentin depth. Immediately after surface preparation, superficial and deep dentin specimens were characterized by contact-mode AFM at the same environmental conditions and air-exposure time intervals ( t 1– t 4) as previously described.
AFM contact-mode was operated to characterize the force-versus-distance curve (force curve) between the AFM tip and specimen surface. Silicon nitride tips, of 50 nm nominal radii, mounted to contact-mode cantilevers (Type D Microlevers™, Thermo-Microscopes, CA, USA) were used to scan specimens surfaces. The force constant and the resonance frequency of the used cantilevers were 2.1 N/m and 160 kHz, respectively. For each specimen, surface scan of 3 μm × 3 μm area was done at a scan rate of 2 Hz and then a set of force curves were measured for selected 14 points at the intertubular dentin. The same process was repeated for each specimen at each previously described time intervals such that a set of force curves as a function of time of air-exposure ( t 1– t 4) and dentin depth (Sd and Dd) were recorded.
The force curves typically show the deflection of the free end of the AFM cantilever as the fixed end of the cantilever is brought vertically towards and then away from the specimen surface. The deflection of the free end of the cantilever is measured and plotted at many points as the z -axis scanner extends the cantilever towards the surface and then retract it again. Then the nano-scale surface adhesion force ( F ad ) between the AFM tip and intertubular surface was calculated in nano-Newton (nN) from the mean force curve of each specimen at each air-exposure time interval. The adhesion force ( F ad ) was calculated from the difference between the snap-in point (the point of contact between the cantilever tip and specimen surface) and the snap-out point (the point of separation or detachment between the cantilever tip and specimen surface) .
2.3
Tensile bond strength (TBS) testing
Dentin/resin-based restorative composite specimens were prepared from freshly extracted mandibular third molars for the tensile bond strength (TBS) testing. The roots of the extracted molars were embedded in self-polymerized resin 3 mm below the cervical line. The occlusal enamel was removed using diamond disk mounted to a milling machine as previously described in the preparation of the specimens for the AFM study under copious water spray to expose flat dentin surface. Half of the teeth were prepared to expose the superficial dentin surfaces, while in the other half the deep dentin surfaces were exposed. The smear layer was standardized as described previously. The exposed dentin surfaces were etched for 15 s with 37% phosphoric acid gel, thoroughly rinsed in distilled water for 5 min and the excess water was removed by gentle blotting with absorbent paper leaving the dentin surface visibly moist.
A single bottle alcohol-based self-priming adhesive (Excite, Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) was applied to the conditioned dentin surfaces. One coat of Excite adhesive was applied over the etched dentin surface and gently agitated for 10 s; excess solvent was removed using a gentle stream of air for 3 s. The Excite was then light-cured for 20 s using a light-curing unit (Cromalux-E, Meca-Physik Dental Division, D-76437 Rastatt, Germany) with a light output of 600 mW/cm 2 . According to the dentin depth and the time of application of the self-priming adhesive (air-exposure time), teeth were divided into the following test groups to simulate what was done in AFM study. Superficial dentin (Sd): Sd( t 1), Sd( t 2), Sd( t 3), and Sd( t 4). Deep dentin (Dd): Dd( t 1), Dd( t 2), Dd( t 3), and Dd( t 4). After the application of the self-priming adhesive at the desired air-exposure times ( t 1– t 4), a light cured resin-based restorative composite material (Tetric Ceram, Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) was applied and cured in four increments for each tooth. The restored teeth were then stored in distilled water at 37 °C for 48 h to complete the curing process.
After storage, each restored tooth was sectioned with diamond disk fixed to a milling machine under copious water spray to prepare dentin/composite slabs of 1 mm thickness from the central part of the tooth. A fine high-speed diamond stone with air/water spray was used to trim the slabs into an hour-glass shape with cross-sectional area of approximately 1 mm 2 at the bonded interface which checked and confirmed by a digital caliper. Twelve ( n = 12) hour-glass shape slabs were tested for each group. All slabs were stored in distilled water at 37 °C for 24 h before mechanical testing. Each slab was fixed to two disposable acrylic resin jigs using cyanoacrylate adhesive. The acrylic jigs were mounted to a universal testing machine (Lloyd Instruments, LR5 series, UK). The test was run at a cross-head speed of 0.5 mm/min until failure. The cross-sectional area of each fractured slab was confirmed using a digital caliper and the respective load was divided over the cross-sectional area to calculate the TBS in MPa.
2.4
Statistical analysis
All data of nano-scale adhesion force ( F ad ) and tensile bond strength (TBS) were expressed as means and standard deviations (SD). Statistical analysis was carried out using SPSS program (Release 15, 2006). Two-way ANOVA was preformed to test the effect of dentin depth, air-exposure time and their interaction on either of F ad and TBS. Tukey–Kramer multiple-comparison post hoc test was used to compare each of F ad and μTBS between the tested groups. The correlation between F ad and TBS through the different air-exposure times ( t 1– t 4) was tested using Pearson’s correlation coefficient. P -values less than 0.05 were considered statistically significant.
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