Poly(amido amine) and calcium phosphate nanocomposite remineralization of dentin in acidic solution without calcium phosphate ions

Abstract

Objective

Patients with dry mouth often have an acidic oral environment lacking saliva that provides calcium (Ca) and phosphate (P) ions. However, there has been no study on dentin remineralization by placing samples in an acidic solution without Ca and P ions. Previous studies used saliva-like solutions with neutral pH and Ca and P ions. Therefore, the objective of this study was to investigate a novel method of combining poly(amido amine) (PAMAM) with a composite of nanoparticles of amorphous calcium phosphate (NACP) on dentin remineralization in an acidic solution without Ca and P ions for the first time.

Methods

Demineralized dentin specimens were tested into four groups: (1) dentin control, (2) dentin coated with PAMAM, (3) dentin with NACP nanocomposite, (4) dentin with PAMAM plus NACP composite. Specimens were treated with lactic acid at pH 4 without initial Ca and P ions for 21 days. Acid neutralization and Ca and P ion concentrations were measured. Dentin specimens were examined by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and hardness testing vs. remineralization efficacy.

Results

NACP composite had mechanical properties similar to commercial control composites (p > 0.1). NACP composite neutralized acid and released Ca and P ions. PAMAM alone failed to induce dentin remineralization. NACP alone achieved mild remineralization and slightly increased dentin hardness at 21 days (p > 0.1). In contrast, the PAMAM + NACP nanocomposite method in acid solution without initial Ca and P ions greatly remineralized the pre-demineralized dentin, restoring its hardness to approach that of healthy dentin (p > 0.1).

Significance

Dentin remineralization via PAMAM + NACP in pH 4 acid without initial Ca and P ions was demonstrated for the first time, when conventional methods such as PAMAM did not work. The novel PAMAM + NACP nanocomposite method is promising to protect tooth structures, especially for patients with reduced saliva to inhibit caries.

Introduction

In the United States, 166 million tooth cavity restorations are placed annually, costing $46 billion per year . Composites are popular due to their esthetics, direct-filling ability and enhanced performance . However, secondary caries is a frequent reason for failure , and replacement of failed restorations accounts for 50–70% of all restorations placed . Among dental patients, individuals with a higher caries risk often have a higher occurrence of initial and secondary caries . Saliva plays an important role in the inhibition of dental caries . In healthy individuals, the saliva fluid covers the dental hard tissues at all times. The constant flow of saliva helps remove food debris and bacteria by swallowing . Saliva has a strong buffer capacity due to its components of bicarbonates, phosphates, and urea . With frequent flows, saliva can penetrate into bacteria biofilms, neutralize the acids produced by cariogenic bacteria, and increase the local pH . In addition, saliva contains high concentrations of calcium (Ca) and phosphate (P) ions, which help to decrease the solubility of hydroxyapatite, the major component of teeth. The large amounts of Ca and P ions in saliva also favor and promote tooth remineralization .

However, many people suffer from reduced salivary gland functions; indeed, about 30% of the population reports dry mouth . Many factors can cause xerostomia or hyposalivation, such as excessive intake of alcohol, certain medicines, nutrition deficiency, certain systemic diseases, salivary dysfunction, Sjögren’s syndrome, etc. . Saliva flow can drop to only 15%–33% of its normal level due to hyposalivation . This means minimal saliva penetration into biofilm to neutralize acids and supply Ca and P ions . Low salivary flow occurs often in seniors . Root caries in the United States increased with aging, from 7% among young people, to 56% in seniors of ≥75 years of age . In addition, patients with head and neck cancers taking radiation therapy can have extreme saliva reduction , causing rampant radiation caries. To date, there is no effective method for people with dry mouth to inhibit caries. While saliva substitutes are used to relieve the sensation of dry mouth, they offer little to protect the teeth .

In addition, natural remineralization via saliva can only overcome a relatively low level of caries challenges. When bacterial acid challenge is severe, natural remineralization is insufficient to halt the caries process . Therefore, nucleation templates were applied to tooth lesion surfaces to promote remineralization . This strategy is somewhat effective for healthy individuals. However, traditional remineralization agents are ineffective for patients with severe saliva reduction . For example, casein phosphopeptide amorphous calcium phosphate (CPP-ACP) did not reduce radiation caries progression . There is a need to develop a new remineralization method that is effective in an acidic and Ca and P ion-deficient environment to protect teeth, especially for patients with reduced saliva.

Poly(amido amine) (PAMAM) dendrimers are highly-branched polymers with a central core and a large amount of reactive functional groups . They can serve as nucleation templates to induce remineralization . For example, amine-terminated PAMAM (PAMAM-NH 2 ) regenerated minerals in demineralized dentin and collagen fibrils . Polyhydroxy-terminated PAMAM (PAMAM-OH) induced dentinal tubule occlusion . Carboxylic-terminated PAMAM (PAMAM-COOH) could absorb Ca and P ions in collagen fibrils to form intrafibrillar minerals . Phosphate-terminated PAMAM (PAMAM-PO 3 H 2 ) remineralized the demineralized dentin in an animal model .

Another approach for remineralization is via calcium phosphate (CaP) composites . Resins containing nanoparticles of amorphous calcium phosphate (NACP) released high levels of Ca and P ions . NACP nanocomposite rapidly neutralized acids , and remineralized enamel lesions in vitro . In a human in situ model, NACP nanocomposite inhibited caries at the enamel-restoration margins . It would be interesting to combine PAMAM with NACP. PAMAM can absorb Ca and P ions for remineralization ; however, it relies on the supply of Ca and P ions which may be deficient in cases of dry mouth. To date, there has been no report on combining PAMAM with NACP composite, except our pilot study which used a cyclic artificial saliva/lactic acid regimen, where remineralization relied on the artificial saliva containing Ca and P ions . It did not investigate dentin remineralization in an acidic environment without Ca and P ions.

The objectives of this study were to develop a novel remineralization method that is effective even in an acidic solution without any initial Ca and P ions, and to investigate the effects of combining PAMAM with NACP nanocomposite on dentin remineralization, acid neutralization, and dentin hardness. It was hypothesized that: (1) PAMAM alone could not remineralize dentin in a lactic acid solution; (2) NACP nanocomposite could neutralize the acid, release Ca and P ions and promote dentin remineralization in lactic acid; (3) the novel PAMAM + NACP composite combined method would achieve the greatest remineralization in dentin, and effectively restore the hardness for pre-demineralized dentin.

Materials and methods

PAMAM synthesis

PAMAM dendrimers were synthesized following a previous study . 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, which 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 that were called “full-generations”. The first and second generations of PAMAM dendrimers are linear molecules, while the third generation is a sphere macromolecule with more functional ending groups, which supplies more nucleation locations to absorb Ca and P ions during remineralization. The present study employed the third generation of PAMAM-NH 2 (G3-PAMAM-NH 2 ) (Chenyuan Dendrimer, 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 have a concentration of 1 mg/mL to form the PAMAM solution, following a previous study .

NACP nanocomposite fabrication

NACP [Ca 3 (PO 4 ) 2 ] were synthesized using a spry-drying technique as described previously . Briefly, calcium carbonate and dicalcium phosphate were dissolved into an acetic acid solution to obtain final Ca and P ion concentrations of 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 method 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 . 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 resin is referred to as EBPM. To test the effect of NACP filler level, the following four composites were made where the resin amount was increased when the NACP amount was increased to obtain a cohesive paste:

  • (1)

    10% NACP + 65% glass + 25% EBPM;

  • (2)

    20% NACP + 50% glass + 30% EBPM;

  • (3)

    30% NACP + 35% glass + 35% EBPM;

  • (4)

    40% NACP + 20% glass + 40% EBPM.

Two commercial composites were used as controls in mechanical testing. A nanocomposite (Heliomolar, Ivoclar, Amherst, NY) contained 66.7% of nanofillers of 40–200 nm of silica and ytterbium-trifluoride. According to the manufacturer, 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. Another nanocomposite Renamel (Cosmedent, Chicago, IL) consisted of nanofillers of 20–40 nm at 60% filler level in a multifunctional resin of diurethane dimethacrylate and butanediol dimethacrylate. Renamel is indicated for Class III, IV, and V restorations. Each 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.

Mechanical testing

All composite specimens were stored 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 .

Preparation of dentin specimens

The use of extracted human teeth was approved by the University of Maryland Institutional Review Board. Extracted healthy adult third molars were collected from the dental school clinics. Teeth were disinfected in a 0.005% promodyne solution for 4 h and stored at 4 °C in distilled water. Dentin squares of 4 × 4 mm with a 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 microhardness of the dentin surfaces was tested with a Vickers diamond indenter (Tukon 2100B, Instron, Canton, MA). All the dentin samples used in subsequent experiments had hardness values of 0.53–0.58 GPa. The dentin squares were acid-etched with 37% phosphoric acid for 15 s to create demineralization, following a previous study . Since our aim was to prevent secondary caries, especially focusing on the challenge with patients having dry mouths in which the caries lesion was removed and large amounts of collagen fibrils were present due to the dentin bonding process, we chose this demineralization method for the present study . The demineralized dentin squares 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).

Remineralization in acidic solution without any initial Ca and P ions

The demineralized dentin specimens were randomly divided into four groups and treated as described below.

  • (1)

    Control group . Each demineralized dentin specimen was coated with 100 μL of distilled water and then air dried, to serve as a control .

  • (2)

    PAMAM . Each demineralized dentin was coated with 100 μL of PAMAM solution, which was maintained on dentin for 1 h, and then the specimen was rinsed with distilled water to remove any loose PAMAM . PAMAM could be immobilized on demineralized dentin by size-exclusion features of collagen fibrils and electrostatic interactions . The 100 μL was used because it could cover the dentin surface .

  • (3)

    NACP . The 40% NACP was selected for remineralization testing. This was because the mechanical test showed that 10–40% NACP in EBPM had similar mechanical properties, and previous studies showed that higher NACP filler level had more Ca and P ion releases . Each dentin square was placed in contact with three NACP composite bars of 2 × 2 × 12 mm , used because when immersed in 1 mL solution, this would yield a composite/solution volume ratio of 0.14/1, the same as that in a previous study .

  • (4)

    PAMAP + NACP . Dentin was coated with 100 μL of PAMAM solution, and then three NACP nanocomposite of 2 × 2 × 12 mm were placed on dentin specimen as in (3).

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 the following solution. 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 sample of the four groups was immersed in 1 mL of fresh lactic acid solution for 24 h (h) at 37 °C. This was repeated for 21 days (d). The 24 h every day of lactic acid solution immersion simulated the most challenging oral environment of patients with the most severe saliva reduction or even saliva deficiency . The rationale was that if dentin remineralization could be achieved in this most extremely challenging condition, then remineralization would be more readily achieved under more favorable oral conditions.

Acid neutralization

At 1, 3, 5, 7, 10, 14 and 21 d, the pH of the lactic acid solutions of the four groups was measured. Every day, each sample was immersed in the lactic acid solution for 24 h. During the 24 h, the pH was monitored with a combination pH electrode (Orion, Cambridge, MA).

Ca and P ion concentrations measurement

At 1, 3, 5, 7, 10, 14 and 21 d, the Ca and P ion concentrations were measured for the lactic acid solution where the samples were immersed. 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-visible, Varian, Palo Alto, CA) using known standards and calibration curves, following previous studies .

SEM and EDS examinations

To determine whether there were regenerated minerals precipitated in the demineralized dentin, at 21 d, the dentin specimens were removed from the lactic acid solution. Each dentin was cut with a diamond saw (Buehler, Lake Bluff, IL) into two halves along the midline. One half was used to examine the occlusal section (the observed surface was perpendicular to the tubule axis). The other was used to examine the longitudinal section (the observed surface was parallel to the tubule axis). They were immersed in 1% glutaraldehyde in PBS for 4 h at 4 °C, subjected to graded ethanol dehydrations, and rinsed with 100% hexamethyldisilazane . Then they were sputter-coated with gold and examined via scanning electron microscopy (SEM, JEOL 5300, Peabody, MA). Meanwhile, the chemical components of the remineralized dentin were examined from the SEM images using energy dispersive spectroscopy (EDS, INCA350, Oxford, UK).

Hardness measurement

The hardness of dentin was measured for the four groups at 7, 14, and 21 d. 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, yielding 36 indentations per group at each time period. In addition, hardness of healthy untreated dentin, and dentin after acid-etching but without the lactic acid solution immersion, were also measured as comparative controls.

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.

Materials and methods

PAMAM synthesis

PAMAM dendrimers were synthesized following a previous study . 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, which 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 that were called “full-generations”. The first and second generations of PAMAM dendrimers are linear molecules, while the third generation is a sphere macromolecule with more functional ending groups, which supplies more nucleation locations to absorb Ca and P ions during remineralization. The present study employed the third generation of PAMAM-NH 2 (G3-PAMAM-NH 2 ) (Chenyuan Dendrimer, 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 have a concentration of 1 mg/mL to form the PAMAM solution, following a previous study .

NACP nanocomposite fabrication

NACP [Ca 3 (PO 4 ) 2 ] were synthesized using a spry-drying technique as described previously . Briefly, calcium carbonate and dicalcium phosphate were dissolved into an acetic acid solution to obtain final Ca and P ion concentrations of 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 method 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 . 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 resin is referred to as EBPM. To test the effect of NACP filler level, the following four composites were made where the resin amount was increased when the NACP amount was increased to obtain a cohesive paste:

  • (1)

    10% NACP + 65% glass + 25% EBPM;

  • (2)

    20% NACP + 50% glass + 30% EBPM;

  • (3)

    30% NACP + 35% glass + 35% EBPM;

  • (4)

    40% NACP + 20% glass + 40% EBPM.

Two commercial composites were used as controls in mechanical testing. A nanocomposite (Heliomolar, Ivoclar, Amherst, NY) contained 66.7% of nanofillers of 40–200 nm of silica and ytterbium-trifluoride. According to the manufacturer, 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. Another nanocomposite Renamel (Cosmedent, Chicago, IL) consisted of nanofillers of 20–40 nm at 60% filler level in a multifunctional resin of diurethane dimethacrylate and butanediol dimethacrylate. Renamel is indicated for Class III, IV, and V restorations. Each 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.

Mechanical testing

All composite specimens were stored 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 .

Preparation of dentin specimens

The use of extracted human teeth was approved by the University of Maryland Institutional Review Board. Extracted healthy adult third molars were collected from the dental school clinics. Teeth were disinfected in a 0.005% promodyne solution for 4 h and stored at 4 °C in distilled water. Dentin squares of 4 × 4 mm with a 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 microhardness of the dentin surfaces was tested with a Vickers diamond indenter (Tukon 2100B, Instron, Canton, MA). All the dentin samples used in subsequent experiments had hardness values of 0.53–0.58 GPa. The dentin squares were acid-etched with 37% phosphoric acid for 15 s to create demineralization, following a previous study . Since our aim was to prevent secondary caries, especially focusing on the challenge with patients having dry mouths in which the caries lesion was removed and large amounts of collagen fibrils were present due to the dentin bonding process, we chose this demineralization method for the present study . The demineralized dentin squares 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).

Remineralization in acidic solution without any initial Ca and P ions

The demineralized dentin specimens were randomly divided into four groups and treated as described below.

  • (1)

    Control group . Each demineralized dentin specimen was coated with 100 μL of distilled water and then air dried, to serve as a control .

  • (2)

    PAMAM . Each demineralized dentin was coated with 100 μL of PAMAM solution, which was maintained on dentin for 1 h, and then the specimen was rinsed with distilled water to remove any loose PAMAM . PAMAM could be immobilized on demineralized dentin by size-exclusion features of collagen fibrils and electrostatic interactions . The 100 μL was used because it could cover the dentin surface .

  • (3)

    NACP . The 40% NACP was selected for remineralization testing. This was because the mechanical test showed that 10–40% NACP in EBPM had similar mechanical properties, and previous studies showed that higher NACP filler level had more Ca and P ion releases . Each dentin square was placed in contact with three NACP composite bars of 2 × 2 × 12 mm , used because when immersed in 1 mL solution, this would yield a composite/solution volume ratio of 0.14/1, the same as that in a previous study .

  • (4)

    PAMAP + NACP . Dentin was coated with 100 μL of PAMAM solution, and then three NACP nanocomposite of 2 × 2 × 12 mm were placed on dentin specimen as in (3).

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 the following solution. 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 sample of the four groups was immersed in 1 mL of fresh lactic acid solution for 24 h (h) at 37 °C. This was repeated for 21 days (d). The 24 h every day of lactic acid solution immersion simulated the most challenging oral environment of patients with the most severe saliva reduction or even saliva deficiency . The rationale was that if dentin remineralization could be achieved in this most extremely challenging condition, then remineralization would be more readily achieved under more favorable oral conditions.

Acid neutralization

At 1, 3, 5, 7, 10, 14 and 21 d, the pH of the lactic acid solutions of the four groups was measured. Every day, each sample was immersed in the lactic acid solution for 24 h. During the 24 h, the pH was monitored with a combination pH electrode (Orion, Cambridge, MA).

Ca and P ion concentrations measurement

At 1, 3, 5, 7, 10, 14 and 21 d, the Ca and P ion concentrations were measured for the lactic acid solution where the samples were immersed. 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-visible, Varian, Palo Alto, CA) using known standards and calibration curves, following previous studies .

SEM and EDS examinations

To determine whether there were regenerated minerals precipitated in the demineralized dentin, at 21 d, the dentin specimens were removed from the lactic acid solution. Each dentin was cut with a diamond saw (Buehler, Lake Bluff, IL) into two halves along the midline. One half was used to examine the occlusal section (the observed surface was perpendicular to the tubule axis). The other was used to examine the longitudinal section (the observed surface was parallel to the tubule axis). They were immersed in 1% glutaraldehyde in PBS for 4 h at 4 °C, subjected to graded ethanol dehydrations, and rinsed with 100% hexamethyldisilazane . Then they were sputter-coated with gold and examined via scanning electron microscopy (SEM, JEOL 5300, Peabody, MA). Meanwhile, the chemical components of the remineralized dentin were examined from the SEM images using energy dispersive spectroscopy (EDS, INCA350, Oxford, UK).

Hardness measurement

The hardness of dentin was measured for the four groups at 7, 14, and 21 d. 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, yielding 36 indentations per group at each time period. In addition, hardness of healthy untreated dentin, and dentin after acid-etching but without the lactic acid solution immersion, were also measured as comparative controls.

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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Nov 22, 2017 | Posted by in Dental Materials | Comments Off on Poly(amido amine) and calcium phosphate nanocomposite remineralization of dentin in acidic solution without calcium phosphate ions
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