Abstract
Objectives
The objective of this study was to investigate the effects of poly (amido amine) (PAMAM), composite with nanoparticles of amorphous calcium phosphate (NACP), and the combined PAMAM + NACP nanocomposite treatment, on remineralization of demineralized dentin in a cyclic artificial saliva/lactic acid environment for the first time.
Methods
Dentin specimens were prepared and demineralized with 37% phosphoric acid for 15 s. Four groups were prepared: (1) dentin control, (2) dentin coated with PAMAM, (3) dentin with NACP composite, (4) dentin with PAMAM + NACP. Specimens were treated with a cyclic artificial saliva/lactic acid regimen for 21 days. Acid neutralization and calcium (Ca) and phosphate (P) ion concentrations were measured. The remineralized dentin specimens were examined by scanning electron microscopy (SEM) and hardness testing.
Results
NACP nanocomposite had mechanical properties similar to commercial control composites (p > 0.1). NACP composite had acid-neutralization and Ca and P ion release capability. PAMAM or NACP composite each alone achieved remineralization and increased the hardness of demineralized dentin (p < 0.05). PAMAM + NACP nanocomposite achieved the greatest mineral regeneration in demineralized dentin and the greatest hardness increase in demineralized dentin, which approached the hardness of healthy dentin (p > 0.1).
Significance
The superior remineralization efficacy of PAMAM + NACP was demonstrated for the first time. PAMAM + NACP induced remineralization in demineralized dentin in an acid challenge environment, when conventional remineralization methods such as PAMAM did not work well. The novel PAMAM + NACP composite approach is promising for a wide range of dental applications to inhibit caries and protect tooth structures.
1
Introduction
In the USA, approximately 166 million tooth cavity restorations were placed annually . Resin composites, consisting of fillers in a polymer matrix, are increasingly popular due to their esthetics, direct-filling ability and enhanced performance . However, reports showed that “the two main challenges are secondary caries and bulk fracture” . Caries at restoration margins is a frequent reason for failure , and replacement of failed restorations accounts for 50–70% of all restorations placed . Replacement dentistry costs $5 billion annually in the USA . In addition, the prevalence of tooth root caries increases with patient aging, which is a growing public health issue due to the rapid increases in the elderly population, coupled with substantial increases in tooth retention in seniors . Gingival recession due to aging, periodontal disease or traumatic tooth-brushing habits can increase the susceptibility to root caries . Furthermore, low salivary flow in seniors and patients with dry mouths further contribute to biofilm and plaque buildup and the occurrence of root caries. Indeed, root caries occurrence in the USA increased rapidly with aging, from 7% among young people, to 56% in seniors of ≥75 years of age . Therefore, there is an increasing need for the development of effective caries-inhibition strategies.
The process of dental caries is now well-understood . Acid-producing bacteria feed on fermentable carbohydrates and produce organic acids as byproducts . These acids dissolve hydroxyapatite minerals, forming a caries lesion . If this process continues, cavitation occurs. Prior to cavitation, a subsurface lesion with partial demineralization is present, and this lesion can be reversed and remineralized under appropriate conditions . Remineralization is the natural repair process for caries lesions . However, natural remineralization can only overcome a certain level of caries (acid) challenges. When bacterial acid challenge is severe, natural remineralization is insufficient to halt or reverse the caries process . Therefore, the development of remineralization approaches is needed to help combat caries and protect the teeth.
Efforts were made to coat remineralization biomaterials onto tooth lesion surfaces . One class of such remineralization biomaterials involved the use of high poly (amido amine) (PAMAM) dendrimer . PAMAM are highly-branched polymers and characterized by the presence of internal cavities, a large number of reactive end groups with well-defined sizes and shapes . In recent years, several types of PAMAM dendrimers were used as nucleation templates to induce tooth remineralization . Amine-terminated PAMAM (PAMAM-NH 2 ) was effective at regenerating minerals on the surfaces of dentin and collagen fibrils . In addition, polyhydroxy terminated PAMAM (PAMAM-OH) had a moderate remineralization ability and could induce dentinal tubule occlusion . Carboxylic-terminated PAMAM (PAMAM-COOH) could absorb calcium (Ca) and phosphate (P) ions within collagen fibrils to form intrafibrillar minerals . Furthermore, phosphate-terminated PAMAM (PAMAM-PO 3 H 2 ) successfully remineralized human dentin in an animal model .
Another approach to inhibiting caries was the development of calcium phosphate (CaP) composites, which could release Ca and P ions and remineralize tooth lesions . Traditional CaP composites used CaP particles of 1–55 μm and had low mechanical properties that were inadequate for bulk restoratives . Recently, nanoparticles of amorphous calcium phosphate (NACP) with a mean size of 116 nm were synthesized . NACP nanocomposites released high levels of Ca and P ions while having mechanical properties 2-fold those of traditional CaP composites . The NACP nanocomposite was “smart” and rapidly neutralized acid solutions, raising a cariogenic pH of 4 to a safe pH of nearly 6 . NACP nanocomposite remineralized enamel lesions in vitro, achieving a remineralization that was 4-fold that of a commercial fluoride-releasing composite . In a human in situ model, the NACP nanocomposite inhibited caries at the enamel-restoration margins, reducing enamel mineral loss to 1/3 that of a control composite .
However, literature search revealed no report on dentin remineralization via NACP nanocomposite. Furthermore, there has been no report on the effect of combining PAMAM with NACP nanocomposite on remineralization. In addition, previous remineralizations induced by PAMAM were achieved in a neutral pH environment . There has been no report on the remineralization effect of PAMAM in an acid challenge environment, even though acid challenges are often encountered orally.
Because acids dissolve hydroxyapatite minerals in teeth , remineralization in an acid challenge environment is more difficult to achieve, is more realistic, and needs to be investigated. PAMAM is an excellent nucleation template and can rapidly absorb Ca and P ions to cause remineralization; however, it cannot neutralize acids and raise pH to prevent demineralization. In contrast, NACP can neutralize acids and release Ca and P ions to decrease the damage caused by acids, and the high ion concentrations can facilitate remineralization . However, the lack of nucleation template on the demineralized dentin surface could limit its remineralization effect . Therefore, it would be highly desirable to combine NACP with PAMAM to achieve the double benefits of remineralization-promotion and demineralization-prevention.
Accordingly, the objective of this study was to investigate the effects of PAMAM, NACP nanocomposite, and PAMAM + NACP nanocomposite on dentin remineralization in a cyclic artificial saliva/acid challenge environment for the first time. It was hypothesized that: (1) PAMAM treatment would significantly remineralize the demineralized dentin in the cyclic artificial saliva/acid regimen; (2) NACP nanocomposite would greatly increase the Ca and P ion concentrations, neutralize the acid and promote dentin remineralization in the cyclic artificial saliva/acid treatment; (3) The combined PAMAM + NACP nanocomposite method would achieve much greater remineralization in dentin than PAMAM or NACP alone.
2
Materials and methods
2.1
PAMAM synthesis
PAMAM dendrimers were synthesized as described previously . Briefly, the divergent synthesis of PAMAM dendrimers included a two-step interactive sequence to produce amine-terminated structures. Iterative sequencing involved alkylation with methyl acrylate (MA) followed by amidation with excess 1,2- ethylenediamine (EDA). The alkylation step produced ester-terminated intermediates that were called “half-generations”. The second step involved amidation of the ester-terminated intermediates with a large excess of EDA to produce amine terminated intermediates, which were called “full-generations”. The first and second generations are linear molecules, while the third generation is a sphere molecule with more functional groups, which ensures that it could absorb more Ca and P ions during remineralization. Previous studies showed that PAMAM-NH 2 effectively remineralized the demineralized dentin . The present study used the third generation PAMAM-NH 2 (G3-PAMAM-NH 2 ), which was obtained commercially (Chenyuan Dendrimer Tech., Weihai, China). In this article, the term “PAMAM” refers to G3-PAMAM-NH 2 . PAMAM solution was prepared by dissolving 50 mg of PAMAM powder in 50 mL of distilled water to achieve a concentration of 1 mg/mL . This concentration was chosen because a previous study showed that the 1 mg/mL PAMAM solution could achieve both good biocompatibility and superior remineralization ability .
2.2
NACP nanocomposite fabrication
NACP [Ca 3 (PO 4 ) 2 ] were synthesized via a spray-drying technique . Briefly, calcium carbonate and dicalcium phosphate were dissolved into an acetic acid solution. The Ca and P ion concentrations were 8 and 5.333 mmol/L, respectively. The solution was sprayed into a heated chamber to evaporate the water and volatile acid. The dried particles were collected by an electrostatic precipitator. This yielded NACP with a mean particle size of 116 nm . As a co-filler, barium boroaluminosilicate glass particles (1.4 μm median size, Caulk/Dentsply, Milford, DE) were silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n -propylamine as previously described .
Ethoxylated bisphenol A dimethacrylate (EBPADMA, Sigma-Aldrich, St. Louis, MO) and pyromellitic dianhydride glycerol dimethacrylate (PMDGDM, Esstech, Essington, PA) were mixed at 1:1 mass ratio . This resin was render light-curable with 0.2% camphorquinone and 0.8% ethyl 4- N , N -dimethylaminobenzoate, following previous studies . This EBPADMA-PMDGDM resin was referred to as EBPM. EBPM resin was filled with mass fractions of 30% NACP and 40% glass particles to form a cohesive paste. The composite paste was placed into a mold of 2 × 2 × 25 mm, and light-cured (Triad 2000, Dentsply, York, PA) for 1 min on each open side.
Two commercial composites served as controls in mechanical testing. Heliomolar (Ivoclar, Amherst, NY) served as control composite I. The fillers consisted of silica and ytterbi-trifluoride nanoparticles at a filler mass fraction of 66.7%. Heliomolar is indicated for Class I and Class II restorations in the posterior region, Class III and Class IV anterior restorations and Class V restorations, according to the manufacturer. Another composite (Renamel Microfill, Cosmedent, Chicago, IL) served as control II. It consisted of fillers of 40 nm–0.2 μm at 60% filler level by volume in a multifunctional resin of diurethane dimethacrylate and butanediol dimethacrylate. Renamel is indicated for Class III–V restorations. All specimens were light-cured in the same manner as described above.
2.3
Mechanical testing
Composite specimens were incubated at 37 °C for 24 h, and then fractured in three-point flexure with a 10-mm span at a crosshead-speed of 1 mm/min on a computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) . Flexural strength (S) was calculated as: S = 3P max L/(2bh 2 ), where P is the fracture load, L is span, b is specimen width and h is thickness. Elastic modulus (E) was calculated as: E = (P/d)(L 3 /[4bh 3 ]), where load P divided by displacement d is the slope in the linear elastic region .
2.4
Preparation of dentin specimens
Caries-free human molars were collected from the dental school clinics following a protocol approved by the University of Maryland Institutional Review Board. Teeth were disinfected in a 0.005% promodyne solution for 4 h and stored at 4 °C in distilled water. Dentin squares of 4 mm × 4 mm with an approximately thickness of 1.0 mm were prepared by cutting perpendicular to the long axis of the tooth 4 mm above the cemento-enamel junction using a diamond blade (Buehler, Lake Bluff, IL, USA). The dentin specimens were acid-etched with 37% phosphoric acid for 15 s to create demineralization, following a previous study . The demineralized dentin specimens were sonicated in distilled water for 10 min to remove any debris and then stored at 4 °C in phosphate-buffered saline (PBS, pH 7.4) before use.
2.5
Remineralization in an acid challenge environment
The demineralized dentin squares were randomly divided into four groups:
- (1)
Each demineralized dentin square was coated with 100 μL of distilled water, and then air dried to serve as a negative control .
- (2)
Each demineralized dentin square was coated with 100 μL of the PAMAM solution which was kept on dentin for 1 h to ensure that PAMAM macromolecules were immobilized on dentin, and then the specimen was rinsed with water to remove any loose PAMAM . PAMAM macromolecules could be immobilized on demineralized dentin by both electrostatic interactions and size-exclusion features of collagen fibrils . The 100 μL of PAMAM solution was used because it could completely immerse and cover the dentin surface to ensure that the PAMAM macromolecules could bind to the demineralized dentin, following a previous study .
- (3)
Each demineralized dentin square was placed in contact with three NACP composite bars of 2 × 2 × 12 mm . Three bars were used following previous studies where three bars of the same dimensions were used for Ca and P ion release measurements . This could enable the results to be compared with previous studies, and three bars could indicate the situation of multiple restorations in the oral cavity.
- (4)
Each demineralized dentin square was first coated with 100 μL of PAMAM solution, and then three NACP composite of 2 × 2 × 12 mm were placed on dentin specimen.
These four groups are denoted Control group (no PAMAM, no NACP), PAMAM group, NACP group, and PAMAM + NACP group, respectively. NACP here refers to NACP nanocomposite. Six specimens were tested for each group (n = 6). A 1.5 mL conical vial was used to store each sample which was completely immersed in 1 mL of a solution below.
Artificial saliva solution was prepared by dissolving, in distilled water, 1.5 mmol/L CaCl 2 , 0.9 mmol/L KH 2 PO 4 , 130 mmol/L KCl, 1.0 mmol/L NaN 3 and 20 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and adjusting to pH 7.0 with KOH (1 mmol/L) . Artificial saliva is a solution to mimic the human saliva, which can supply Ca and P ions for the remineralization . In addition, a sodium chloride (NaCl) solution (133 mmol/L) was buffered to pH 4 with 50 mmol/L lactic acid to simulate a cariogenic condition (referred to as “lactic acid solution”) . Each day, each specimen of the aforementioned four groups was immersed in 1 mL of fresh artificial saliva for 23 hours (h), and then in 1 mL of lactic acid solution for 1 h at 37 °C, following previous studies . This was repeated for 21 days. The 1-h duration in the lactic acid solution approximated the accumulated acid challenge times in a 24-h period orally .
2.6
Acid neutralization
At 1, 3, 5, 7, 10, 14 and 21 days, the pH values of lactic acid solutions of the four groups were measured. Every day, each sample was immersed in the artificial saliva solution for 23 h, and in the lactic acid solution for 1 h. During that 1 h in lactic acid solution, the pH was monitored with a combination pH electrode (Orion, Cambridge, MA). The pH in artificial saliva was also measured.
2.7
Ca and P ion concentrations measurement
At 1, 3, 5, 7, 10, 14 and 21 days, the Ca and P ion concentrations in lactic acid and artificial saliva solution were measured. At each time, the 1 mL solution was removed and replaced by fresh solution. The collected solution was analyzed for Ca and P ion concentrations via a spectrophotometric method (DMS-80 UV–vis, Varian, Palo Alto, CA) using known standards and calibration curves, following previous studies .
2.8
Scanning electron microscopic (SEM) examination
To examine whether there were minerals regenerated in the demineralized dentin surface and in the dentinal tubules, after 21 days, all dentin squares were removed from the solutions. The dentin square was cut with a diamond saw (Buehler, Lake Bluff, IL) into two halves along the midline. One half was used for observing the occlusal section (the observed surface was perpendicular to the tubule axis). The other half was used for observing the longitudinal section (the observed surface was parallel to the tubule axis). The dentin samples were sonicated in water for 10 min to remove the debris caused by the cutting. They were then immersed in 1% glutaraldehyde in PBS for 4 h at 4 °C. They were rinsed with PBS and subjected to graded ethanol dehydrations, and then rinsed with 100% hexamethyldisilazane . Then the dentin samples were sputter-coated with gold and examined via scanning electron microscopy (SEM, JEOL 5300, Peabody, MA).
2.9
Hardness measurement
The hardness of dentin was measured for the four groups after 21 days. A hardness tester (Tukon 2100B, Instron, Canton, MA) was used with a Vickers diamond indenter at a load of 20 g and a dwell time of 10 s . Six indentations were made in each dentin, and six dentin specimens were tested for each group. In addition, hardness values of sound dentin without treatments, and dentin after acid-etching but without the 21 days treatment, were also measured.
2.10
Statistical analysis
All data were checked for normal distribution with the Kolmogorov–Smirnov test. One-way and two-way analyses of variance (ANOVA) were performed to detect the significant effects of the variables. Tukey’s multiple comparison tests were used at a p value of 0.05.
2
Materials and methods
2.1
PAMAM synthesis
PAMAM dendrimers were synthesized as described previously . Briefly, the divergent synthesis of PAMAM dendrimers included a two-step interactive sequence to produce amine-terminated structures. Iterative sequencing involved alkylation with methyl acrylate (MA) followed by amidation with excess 1,2- ethylenediamine (EDA). The alkylation step produced ester-terminated intermediates that were called “half-generations”. The second step involved amidation of the ester-terminated intermediates with a large excess of EDA to produce amine terminated intermediates, which were called “full-generations”. The first and second generations are linear molecules, while the third generation is a sphere molecule with more functional groups, which ensures that it could absorb more Ca and P ions during remineralization. Previous studies showed that PAMAM-NH 2 effectively remineralized the demineralized dentin . The present study used the third generation PAMAM-NH 2 (G3-PAMAM-NH 2 ), which was obtained commercially (Chenyuan Dendrimer Tech., Weihai, China). In this article, the term “PAMAM” refers to G3-PAMAM-NH 2 . PAMAM solution was prepared by dissolving 50 mg of PAMAM powder in 50 mL of distilled water to achieve a concentration of 1 mg/mL . This concentration was chosen because a previous study showed that the 1 mg/mL PAMAM solution could achieve both good biocompatibility and superior remineralization ability .
2.2
NACP nanocomposite fabrication
NACP [Ca 3 (PO 4 ) 2 ] were synthesized via a spray-drying technique . Briefly, calcium carbonate and dicalcium phosphate were dissolved into an acetic acid solution. The Ca and P ion concentrations were 8 and 5.333 mmol/L, respectively. The solution was sprayed into a heated chamber to evaporate the water and volatile acid. The dried particles were collected by an electrostatic precipitator. This yielded NACP with a mean particle size of 116 nm . As a co-filler, barium boroaluminosilicate glass particles (1.4 μm median size, Caulk/Dentsply, Milford, DE) were silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n -propylamine as previously described .
Ethoxylated bisphenol A dimethacrylate (EBPADMA, Sigma-Aldrich, St. Louis, MO) and pyromellitic dianhydride glycerol dimethacrylate (PMDGDM, Esstech, Essington, PA) were mixed at 1:1 mass ratio . This resin was render light-curable with 0.2% camphorquinone and 0.8% ethyl 4- N , N -dimethylaminobenzoate, following previous studies . This EBPADMA-PMDGDM resin was referred to as EBPM. EBPM resin was filled with mass fractions of 30% NACP and 40% glass particles to form a cohesive paste. The composite paste was placed into a mold of 2 × 2 × 25 mm, and light-cured (Triad 2000, Dentsply, York, PA) for 1 min on each open side.
Two commercial composites served as controls in mechanical testing. Heliomolar (Ivoclar, Amherst, NY) served as control composite I. The fillers consisted of silica and ytterbi-trifluoride nanoparticles at a filler mass fraction of 66.7%. Heliomolar is indicated for Class I and Class II restorations in the posterior region, Class III and Class IV anterior restorations and Class V restorations, according to the manufacturer. Another composite (Renamel Microfill, Cosmedent, Chicago, IL) served as control II. It consisted of fillers of 40 nm–0.2 μm at 60% filler level by volume in a multifunctional resin of diurethane dimethacrylate and butanediol dimethacrylate. Renamel is indicated for Class III–V restorations. All specimens were light-cured in the same manner as described above.
2.3
Mechanical testing
Composite specimens were incubated at 37 °C for 24 h, and then fractured in three-point flexure with a 10-mm span at a crosshead-speed of 1 mm/min on a computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) . Flexural strength (S) was calculated as: S = 3P max L/(2bh 2 ), where P is the fracture load, L is span, b is specimen width and h is thickness. Elastic modulus (E) was calculated as: E = (P/d)(L 3 /[4bh 3 ]), where load P divided by displacement d is the slope in the linear elastic region .
2.4
Preparation of dentin specimens
Caries-free human molars were collected from the dental school clinics following a protocol approved by the University of Maryland Institutional Review Board. Teeth were disinfected in a 0.005% promodyne solution for 4 h and stored at 4 °C in distilled water. Dentin squares of 4 mm × 4 mm with an approximately thickness of 1.0 mm were prepared by cutting perpendicular to the long axis of the tooth 4 mm above the cemento-enamel junction using a diamond blade (Buehler, Lake Bluff, IL, USA). The dentin specimens were acid-etched with 37% phosphoric acid for 15 s to create demineralization, following a previous study . The demineralized dentin specimens were sonicated in distilled water for 10 min to remove any debris and then stored at 4 °C in phosphate-buffered saline (PBS, pH 7.4) before use.
2.5
Remineralization in an acid challenge environment
The demineralized dentin squares were randomly divided into four groups:
- (1)
Each demineralized dentin square was coated with 100 μL of distilled water, and then air dried to serve as a negative control .
- (2)
Each demineralized dentin square was coated with 100 μL of the PAMAM solution which was kept on dentin for 1 h to ensure that PAMAM macromolecules were immobilized on dentin, and then the specimen was rinsed with water to remove any loose PAMAM . PAMAM macromolecules could be immobilized on demineralized dentin by both electrostatic interactions and size-exclusion features of collagen fibrils . The 100 μL of PAMAM solution was used because it could completely immerse and cover the dentin surface to ensure that the PAMAM macromolecules could bind to the demineralized dentin, following a previous study .
- (3)
Each demineralized dentin square was placed in contact with three NACP composite bars of 2 × 2 × 12 mm . Three bars were used following previous studies where three bars of the same dimensions were used for Ca and P ion release measurements . This could enable the results to be compared with previous studies, and three bars could indicate the situation of multiple restorations in the oral cavity.
- (4)
Each demineralized dentin square was first coated with 100 μL of PAMAM solution, and then three NACP composite of 2 × 2 × 12 mm were placed on dentin specimen.
These four groups are denoted Control group (no PAMAM, no NACP), PAMAM group, NACP group, and PAMAM + NACP group, respectively. NACP here refers to NACP nanocomposite. Six specimens were tested for each group (n = 6). A 1.5 mL conical vial was used to store each sample which was completely immersed in 1 mL of a solution below.
Artificial saliva solution was prepared by dissolving, in distilled water, 1.5 mmol/L CaCl 2 , 0.9 mmol/L KH 2 PO 4 , 130 mmol/L KCl, 1.0 mmol/L NaN 3 and 20 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and adjusting to pH 7.0 with KOH (1 mmol/L) . Artificial saliva is a solution to mimic the human saliva, which can supply Ca and P ions for the remineralization . In addition, a sodium chloride (NaCl) solution (133 mmol/L) was buffered to pH 4 with 50 mmol/L lactic acid to simulate a cariogenic condition (referred to as “lactic acid solution”) . Each day, each specimen of the aforementioned four groups was immersed in 1 mL of fresh artificial saliva for 23 hours (h), and then in 1 mL of lactic acid solution for 1 h at 37 °C, following previous studies . This was repeated for 21 days. The 1-h duration in the lactic acid solution approximated the accumulated acid challenge times in a 24-h period orally .
2.6
Acid neutralization
At 1, 3, 5, 7, 10, 14 and 21 days, the pH values of lactic acid solutions of the four groups were measured. Every day, each sample was immersed in the artificial saliva solution for 23 h, and in the lactic acid solution for 1 h. During that 1 h in lactic acid solution, the pH was monitored with a combination pH electrode (Orion, Cambridge, MA). The pH in artificial saliva was also measured.
2.7
Ca and P ion concentrations measurement
At 1, 3, 5, 7, 10, 14 and 21 days, the Ca and P ion concentrations in lactic acid and artificial saliva solution were measured. At each time, the 1 mL solution was removed and replaced by fresh solution. The collected solution was analyzed for Ca and P ion concentrations via a spectrophotometric method (DMS-80 UV–vis, Varian, Palo Alto, CA) using known standards and calibration curves, following previous studies .
2.8
Scanning electron microscopic (SEM) examination
To examine whether there were minerals regenerated in the demineralized dentin surface and in the dentinal tubules, after 21 days, all dentin squares were removed from the solutions. The dentin square was cut with a diamond saw (Buehler, Lake Bluff, IL) into two halves along the midline. One half was used for observing the occlusal section (the observed surface was perpendicular to the tubule axis). The other half was used for observing the longitudinal section (the observed surface was parallel to the tubule axis). The dentin samples were sonicated in water for 10 min to remove the debris caused by the cutting. They were then immersed in 1% glutaraldehyde in PBS for 4 h at 4 °C. They were rinsed with PBS and subjected to graded ethanol dehydrations, and then rinsed with 100% hexamethyldisilazane . Then the dentin samples were sputter-coated with gold and examined via scanning electron microscopy (SEM, JEOL 5300, Peabody, MA).
2.9
Hardness measurement
The hardness of dentin was measured for the four groups after 21 days. A hardness tester (Tukon 2100B, Instron, Canton, MA) was used with a Vickers diamond indenter at a load of 20 g and a dwell time of 10 s . Six indentations were made in each dentin, and six dentin specimens were tested for each group. In addition, hardness values of sound dentin without treatments, and dentin after acid-etching but without the 21 days treatment, were also measured.
2.10
Statistical analysis
All data were checked for normal distribution with the Kolmogorov–Smirnov test. One-way and two-way analyses of variance (ANOVA) were performed to detect the significant effects of the variables. Tukey’s multiple comparison tests were used at a p value of 0.05.
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