Effect of calcium phosphate nanocomposite on in vitroremineralization of human dentin lesions

Abstract

Objective

Secondary caries is a primary reason for dental restoration failures. The objective of this study was to investigate the remineralization of human dentin lesions in vitro via restorations using nanocomposites containing nanoparticles of amorphous calcium phosphate (NACP) or NACP and tetracalcium phosphate (TTCP) for the first time.

Methods

NACP was synthesized by a spray-drying technique and incorporated into a resin consisting of ethoxylated bisphenol A dimethacrylate (EBPADMA) and pyromellitic glycerol dimethacrylate (PMGDM). After restoring the dentin lesions with nanocomposites as well as a non-releasing commercial composite control, the specimens were treated with cyclic demineralization (pH 4, 1 h per day) and remineralization (pH 7, 23 h per day) for 4 or 8 weeks. Calcium (Ca) and phosphate (P) ion releases from composites were measured. Dentin lesion remineralization was measured at 4 and 8 weeks by transverse microradiography (TMR).

Results

Lowering the pH increased ion release of NACP and NACP-TTCP composites. At 56 days, the released Ca concentration in mmol/L (mean ± SD; n = 3) was (13.39 ± 0.72) at pH 4, much higher than (1.19 ± 0.06) at pH 7 (p < 0.05). At 56 days, P ion concentration was (5.59 ± 0.28) at pH 4, much higher than (0.26 ± 0.01) at pH 7 (p < 0.05). Quantitative microradiography showed typical subsurface dentin lesions prior to the cyclic demineralization/remineralization treatment, and dentin remineralization via NACP and NACP-TTCP composites after 4 and 8 weeks of treatment. At 8 weeks, NACP nanocomposite achieved dentin lesion remineralization (mean ± SD; n = 15) of (48.2 ± 11.0)%, much higher than (5.0 ± 7.2)% for dentin in commercial composite group after the same cyclic demineralization/remineralization regimen (p < 0.05).

Significance

Novel NACP-based nanocomposites were demonstrated to achieve dentin lesion remineralization for the first time. These results, coupled with acid-neutralization and good mechanical properties shown previously, indicate that the NACP-based nanocomposites are promising for restorations to inhibit caries and protect tooth structures.

Introduction

Approximately 166 million tooth cavity restorations were placed in 2005 in the Unites States , with at least half of the posterior direct restorations using composites . Composites are increasingly used because of their excellent esthetics and direct-filling capability . Advances in resin compositions, filler particles and the resin–filler interface have improved the composite properties . However, the lifetime of composite restorations is limited by inferior properties such as polymerization shrinkage/stress formation, fracture, abrasion and wear resistance, and marginal leakage . Marginal leakage can result in the formation of secondary caries, the main reason for composite restoration failures .

A promising approach to combat caries is to use composites containing calcium phosphate (CaP) particles. These composites have been shown to release calcium (Ca) and phosphate (P) ions and remineralize tooth lesions in vitro , in situ in the oral environment , and in vivo in human volunteers . Mineral growth in tooth lesions can be stimulated by increasing the calcium and phosphate concentrations within the lesion to levels greater than those existing in oral fluids. Indeed, enamel subsurface lesions were remineralized by a CPP-ACP solution . In this approach, CPP-ACP was included in a sugar-free chewing gum to control dental caries via active remineralization and salivary stimulation. Additionally, ACP was added to sealants to release supersaturating levels of calcium and phosphate ions, driving the solution thermodynamics toward formation of apatite . A drawback with previous CaP composites for dental restorations was that these composites used traditional CaP particles and had low mechanical properties, which were inadequate for bulk restoratives .

Recent studies reported novel nanocomposites containing CaP and CaF 2 nanoparticles with sizes of about 50–100 nm . Nanoparticles of amorphous calcium phosphate (NACP) with a size of 116 nm were synthesized via a spray-drying technique . Nanocomposites containing NACP are advantageous because of the small size and high surface area of the nanoparticles . A previous study showed that the NACP nanocomposite had mechanical properties 2-fold those of traditional CaP composites . The NACP nanocomposite neutralized acid attacks, while commercial controls failed to neutralize the acid . In addition, composites containing CaP nanoparticles released substantially more ions than that with micrometer-sized particles at the same filler level , and CaP nanocomposites possessed much higher strength, fracture toughness, and wear resistance than traditional CaP composites . Recently, NACP nanocomposites were shown to remineralize lesions in human enamel in an in vitro model . Additionally, NACP nanocomposite was shown to reduce caries in enamel in a human in-situ model . Enamel remineralization was partially enabled by the presence of residual seed mineral crystals, which resulted in apatite mineral formation from the diffusion of calcium and phosphate ions into the carious lesion . However, the previous studies focused on enamel without testing the effect of NACP nanocomposite on dentin .

Dentin contains 70% carbonated apatite, 20% organic matrix (mostly collagen) and 10% water . When dentin lesions form, the mineral phase is damaged and may be destroyed. As the carious attack progresses, the collagen fibers are exposed and degraded, leading to a decrease in the mechanical properties of dentin . In demineralized dentin, unlike enamel, there are fewer residual mineral seed crystals present, which may make it more difficult to remineralize dentin compared to enamel. Clinically, treatment of carious dentin lesions is dependent on the depth of the lesion. In shallow to moderate lesions, the carious material can be completely removed and restored with composite, amalgam or glass ionomer. In asymptomatic deep lesions, where there is a risk of pulp exposure but restoration of tooth function is possible, partial removal of the carious dentin may be considered the clinically conservative approach. The treatment can involve an attempt to remineralize the demineralized dentin by either indirect pulp treatment or stepwise caries removal. In indirect pulp treatment, most of the carious lesion is removed and the finished cavity preparation is lined with a remineralizing material (calcium hydroxide, resin-modified glass ionomer, etc .) and the final restoration is placed to provide a good seal. Stepwise caries removal is a 2-step process, requiring removal of less carious dentin, followed by an interim placement of glass ionomer cement to aid in remineralization. After several months, remineralization is assessed and, if successful, a permanent restoration is placed . While CaP nanocomposites were shown to release more Ca and P ions and possess much better mechanical properties than traditional CaP composites , the remineralization of dentin caries via nanocomposite containing NACP has yet to be reported.

Accordingly, the objective of this study was to investigate the remineralization of dentin lesions in human teeth in vitro via nanocomposites containing NACP and NACP plus micron-sized tetracalcium phosphate (TTCP) particles. It was hypothesized that: (1) the cyclic demineralization/remineralization regimen would fail to remineralize dentin lesions when restored with the commercial composite; (2) the new NACP and NACP-TTCP nanocomposites would successfully regenerate the mineral lost in dentin; (3) dentin remineralization via NACP and NACP-TTCP nanocomposites would increase with increasing time from 4 to 8 weeks.

Materials and methods

Synthesis of ACP nanoparticles

NACP (Ca 3 [PO 4 ] 2 ) were formed via a spray-drying technique . Briefly, a solution was prepared by adding 1.5125 g of glacial acetic acid (J.T. Baker, Phillipsburg, NJ) into 500 mL of distilled water. Then, 0.8 g of calcium carbonate (CaCO 3 , Fisher, Fair Lawn, NJ) and 5.094 g of dicalcium phosphate anhydrous (DCPA, J.T. Baker) were dissolved into the acetic acid solution. The final Ca and P ionic concentrations were 8 mmol/L and 5.333 mmol/L, respectively. This yielded a Ca/P molar ratio of 1.5, the same as that for ACP. This solution was sprayed through a nozzle (PNR, Poughkeepsie, NY) that was situated on a spray chamber with heated air flow. The water/volatile acid were evaporated into the dry, heated column. The dried particles were collected by an electrostatic precipitator (AirQuality, Minneapolis, MN). The collected powder was examined with X-ray diffractometry (XRD, DMAX2200, Rigaku, Woodlands, TX). This method produced NACP with a mean size of 116 nm .

TTCP and glass fillers

TTCP (Ca 4 [PO 4 ] 2 O) was synthesized from a solid-state reaction between CaHPO 4 and CaCO 3 (Baker Chemical, Phillipsburg, NJ), which were mixed and heated at 1500 °C for 6 h in a furnace (Thermolyne, Dubuque, IA) . The mixture was quenched to room temperature and ground in a blender (Dynamics Corp., New Hartford, CT). The powder was then sieved to obtain TTCP particles with sizes of 1.5–60 μm, with a median of 16 μm. This TTCP powder was then ground in 95% ethanol with a ball-mill (Retsch, Newtown, PA) for 24 h. The particle size distribution was measured via a sedimentation method with the use of a centrifugal particle analyzer (SA-CP3, Shimazu, Kyoto, Japan) as described in a previous study . This yielded a particle size range of 0.2–3.0 μm, with a median of 0.8 μm. In addition, for mechanical reinforcement, barium boroaluminosilicate glass particles with a median diameter of 1.4 μm (Caulk/Dentsply, Milford, DE) were used because it is a typical dental glass filler similar to those in a hybrid composite (TPH, Caulk/Dentsply). The glass particles were silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n -propylamine (mass %) .

Resin composite fabrication

The resin consisted of ethoxylated bisphenol A dimethacrylate (EBPADMA, Sartomer Co., West Chester, PA) and the acidic pyromellitic glycerol dimethacrylate (PMGDM, Esstech, Essington, PA), each at a mass fraction of 49.5%. This was photoactivated with 0.2% camphorquinone and 0.8% ethyl 4- N,N -dimethylaminobenzoate. Two experimental composites were formulated: (1) 40% NACP + 20% silanized glass + 40% resin (referred to as NACP nanocomposite); (2) 40% NACP + 20% TTCP + 40% resin (referred to as NACP-TTCP nanocomposite). Both had a total filler mass fraction of 60%. Additionally, a non-releasing commercial hybrid composite (TPH, Caulk/Dentsply) was included as a control. For mechanical properties, a glass ionomer Fuji II LC (GC America, Alsip, IL) served as a control. Fuji II LC is indicated for Class III and Class V restorations, restoration of primary teeth, and core buildup applications. Fuji II LC is a two part, powder/liquid system. Specimens were made using the manufacturer’s suggested powder/liquid ratio of 3.2/1. In addition, a resin-modified glass ionomer Vitremer (3M, St. Paul, MN) also served as a control. Vitremer consisted of fluoroaluminosilicate glass and a light-sensitive, aqueous polyalkenoic acid. Its indications include Class III, V and root-caries restorations, Classes I and II restorations in primary teeth, core-buildup, and orthodontic cement. A powder/liquid mass ratio of 2.5/1 was used according to the manufacturer. Each material was placed into 2 × 2 × 25 mm molds, photo-cured (Triad 2000, Dentsply, York, PA; light intensity at specimen location was approximately 110 mW/cm 2 ) for 1 min on each open side, and incubated for 24 h at 37 °C before immersion, as described below.

Mechanical testing

For immersion and mechanical testing, a sodium chloride solution (133 mmol/L) was buffered to two pH values: pH 4 with 50 mmol/L lactic acid, and pH 7 with 50 mmol/L HEPES. For each material, the cured specimens were randomly divided into two groups and immersed in the pH 7 and pH 4 solutions, respectively, at 37 °C for 1 day or 4 weeks. Each group was immersed in 200 mL of solution in a sealed polyethylene container. For the 4 weeks groups, the solution was changed every week. For the pH 4 groups, the pH was adjusted with lactic acid to be at pH 4.

A computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) was used to fracture the specimens in three-point flexure with a 10 mm span at a crosshead speed of 1 mm/min. The specimens were wet and not dried, and were fractured within a few minutes after being taken out of the solution. 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.

Calcium and phosphate ion release

A sodium chloride (NaCl) solution (133 mmol/L) was buffered to two different pHs: pH 4 with 50 mmol/L lactic acid, and pH 7 with 50 mmol/L HEPES. Following previous studies , three specimens of approximately 2 × 2 × 12 mm were immersed in 50 mL of solution at each pH, yielding a specimen volume/solution of 2.9 mm 3 /mL. This compared to a specimen volume per solution of approximately 3.0 mm 3 /mL in a previous study . For each solution, the concentrations of Ca and P ions released from the specimens were measured at 1, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days (d). At each time, aliquots of 0.5 mL were removed and replaced by fresh solution. The aliquots were analyzed for Ca and P ions via a spectrophotometric method (SpectraMax M5, Molecular Devices, Sunnyvale, CA) using known standards and calibration curves . The released ions were reported in cumulative concentrations.

Tooth section preparation

This study (Protocol #: 29749) received an exemption from the University of Maryland Institutional Review Board, as it was deemed not human subjects research. Teeth were collected from clinics and had no personal information of the donors. Twenty caries-free teeth were disinfected in a 0.005% promodyne solution for 4 h. The roots of each tooth were removed at a location approximately 5 mm below the cemento-enamel junction and embedded with composite resin ( Fig. 1 A). The upper third of the crown was removed to expose the dentin surface and a single cavity with dimensions 6 × 3 × 1 mm was machined into the dentin surface under constant water irrigation. The enamel surface was coated with varnish and the dentin surface was exposed to 25 mL of demineralizing solution (8.7 mmol/L CaCl 2 , 8.7 mmol/L KH 2 PO 4 , 0.05 ppm NaF, 75 mmol/L acetic acid, pH 4.0—adjusted with KOH) for 48 h. Each cavity was filled with either NACP composite, NACP-TTCP composite, or TPH control without the use of a bonding agent and light cured for 2 min (Triad 2000, Dentsply International, York, PA, USA). Five teeth were restored for each of the above conditions. Each restored tooth was wet cut into sections approximately 120 μm thick using a diamond blade (Buehler, Lake Bluff, IL) on a Buehler Isomet low-speed saw at the highest speed setting. Additionally, unrestored teeth were also sectioned and acted as a negative control. 3–6 tooth sections were collected from each restored tooth. To assist in the alignment of the “before” and “after” microradiographic images, 200-mesh Maxtaform Copper/Rhodium transmission electron microscopy (TEM) grids (Electron Microscopy Sciences, Hatfield, PA) were cut under a stereomicroscope into rows encompassing at least one full grid and adhered to each tooth section.

Fig. 1
Schematic of experimental setup for the dentin demineralization study. (A) Cavity preparation and tooth sectioning. (B) Assembly of tooth section between glass slides to be used in the cyclic demineralization/remineralization treatment.

Transverse microradiography

Contact microradiographs of the tooth sections before treatment were produced on holographic film (Integraf LLC, Kirkland, WA, USA) exposed for 30 min to Cu Kα radiation (80 kV, 3 mA; Faxitron Model #43855A, Hewlett Packard, McMinnville, OR, USA) according to a previous study . An aluminum step-wedge was used to estimate the mineral density .

Assembly of specimens and pH cycling experiment

After the initial contact microradiographs were taken, the tooth sections were assembled for the cyclic demineralization/remineralization protocol by sandwiching between layers of parafilm and a plastic coverslip and then wrapped again with parafilm and sandwiched between glass microscope slides as depicted in Fig. 1 B, with the restoration edge exposed.

The demineralizing solution consisted of: 3.0 mmol/L CaCl 2 , 1.8 mmol/L K 2 HPO 4 , 0.1 mol/L lactic acid, mass fraction 1% carboxymethylcellulose, and a pH of 4.0 (adjusted with KOH) . The remineralizing solution consisted of 1.2 mmol/L CaCl 2 , 0.72 mmol/L K 2 HPO 4 , 2.6 μmol/L F, 50 mmol/L HEPES buffer (pH 7.0 adjusted with KOH) . 20 mL of fresh demineralizing or remineralizing solution was used per specimen for each immersion with continuous magnetic stirring. The specimens were immersed in demineralizing solution for 1 h and remineralizing solution for 23 h at 37 °C. This was repeated for 8 weeks. Contact microradiographs were retaken and the remineralization after treatment was determined.

Microradiography analysis

The developed film was fixed to a glass slide and observed under an optical microscope (Olympus BX50F, Olympus, Japan). Digital images were captured with a digital microscope camera (RGB/YC/NTSC, Microimage Video Systems, Boyerstown, PA, USA) with an intensity resolution of 256 gray levels and a horizontal spatial resolution of 1.25 μm/pixel. Digitized images were analyzed with the ImageJ software (NIH, Bethesda, MD). This is illustrated in Fig. 2 . A rectangular selection the width of one square of the copper grid was made between gridlines and perpendicular to the tooth surface; starting in sound dentin, passing through the demineralized lesion, and extending outside the tooth surface. The “Plot Profile” option was then used to average pixels in a line and to generate and plot the grayscale profile from the exterior to the interior of the tooth which has a direct correlation to mineral density. The profiles collected before and after pH cycling were plotted and aligned via the TEM grids and normalized by using the aluminum step-wedge as a standard . Changes in lesion depth (L d , defined as the distance in μm from the point of peak mineral density in the surface layer to the point where mineral content reaches 95% of sound dentin), mineral loss (ΔZ; volume fraction % × 1.25 μm) were compared for each imaged area before and after the treatment. The percent change in mineral content given by the integrated ΔZ values across the depth of each lesion before and after treatment was calculated according to equation to yield remineralization : Δ(ΔZ)% = [(ΔZ before − ΔZ after )/(ΔZ before )] × 100. The remineralization Δ(ΔZ) obtained for all image areas of NACP composite, NACP-TTCP composite, and TPH was used to indicate remineralization or further demineralization of the lesions as a result of pH-cycling treatment.

Fig. 2
Transverse microradiographic (TMR) imaging technique for dentin. (A) A typically prepared tooth section, and (B) a corresponding plot profile determined from the grayscale intensities of the TMR image.

One-way and two-way ANOVA were performed to detect the significant effects of the experimental variables. Tukey’s multiple comparison test was used to compare the measured data at a p value of 0.05.

Materials and methods

Synthesis of ACP nanoparticles

NACP (Ca 3 [PO 4 ] 2 ) were formed via a spray-drying technique . Briefly, a solution was prepared by adding 1.5125 g of glacial acetic acid (J.T. Baker, Phillipsburg, NJ) into 500 mL of distilled water. Then, 0.8 g of calcium carbonate (CaCO 3 , Fisher, Fair Lawn, NJ) and 5.094 g of dicalcium phosphate anhydrous (DCPA, J.T. Baker) were dissolved into the acetic acid solution. The final Ca and P ionic concentrations were 8 mmol/L and 5.333 mmol/L, respectively. This yielded a Ca/P molar ratio of 1.5, the same as that for ACP. This solution was sprayed through a nozzle (PNR, Poughkeepsie, NY) that was situated on a spray chamber with heated air flow. The water/volatile acid were evaporated into the dry, heated column. The dried particles were collected by an electrostatic precipitator (AirQuality, Minneapolis, MN). The collected powder was examined with X-ray diffractometry (XRD, DMAX2200, Rigaku, Woodlands, TX). This method produced NACP with a mean size of 116 nm .

TTCP and glass fillers

TTCP (Ca 4 [PO 4 ] 2 O) was synthesized from a solid-state reaction between CaHPO 4 and CaCO 3 (Baker Chemical, Phillipsburg, NJ), which were mixed and heated at 1500 °C for 6 h in a furnace (Thermolyne, Dubuque, IA) . The mixture was quenched to room temperature and ground in a blender (Dynamics Corp., New Hartford, CT). The powder was then sieved to obtain TTCP particles with sizes of 1.5–60 μm, with a median of 16 μm. This TTCP powder was then ground in 95% ethanol with a ball-mill (Retsch, Newtown, PA) for 24 h. The particle size distribution was measured via a sedimentation method with the use of a centrifugal particle analyzer (SA-CP3, Shimazu, Kyoto, Japan) as described in a previous study . This yielded a particle size range of 0.2–3.0 μm, with a median of 0.8 μm. In addition, for mechanical reinforcement, barium boroaluminosilicate glass particles with a median diameter of 1.4 μm (Caulk/Dentsply, Milford, DE) were used because it is a typical dental glass filler similar to those in a hybrid composite (TPH, Caulk/Dentsply). The glass particles were silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n -propylamine (mass %) .

Resin composite fabrication

The resin consisted of ethoxylated bisphenol A dimethacrylate (EBPADMA, Sartomer Co., West Chester, PA) and the acidic pyromellitic glycerol dimethacrylate (PMGDM, Esstech, Essington, PA), each at a mass fraction of 49.5%. This was photoactivated with 0.2% camphorquinone and 0.8% ethyl 4- N,N -dimethylaminobenzoate. Two experimental composites were formulated: (1) 40% NACP + 20% silanized glass + 40% resin (referred to as NACP nanocomposite); (2) 40% NACP + 20% TTCP + 40% resin (referred to as NACP-TTCP nanocomposite). Both had a total filler mass fraction of 60%. Additionally, a non-releasing commercial hybrid composite (TPH, Caulk/Dentsply) was included as a control. For mechanical properties, a glass ionomer Fuji II LC (GC America, Alsip, IL) served as a control. Fuji II LC is indicated for Class III and Class V restorations, restoration of primary teeth, and core buildup applications. Fuji II LC is a two part, powder/liquid system. Specimens were made using the manufacturer’s suggested powder/liquid ratio of 3.2/1. In addition, a resin-modified glass ionomer Vitremer (3M, St. Paul, MN) also served as a control. Vitremer consisted of fluoroaluminosilicate glass and a light-sensitive, aqueous polyalkenoic acid. Its indications include Class III, V and root-caries restorations, Classes I and II restorations in primary teeth, core-buildup, and orthodontic cement. A powder/liquid mass ratio of 2.5/1 was used according to the manufacturer. Each material was placed into 2 × 2 × 25 mm molds, photo-cured (Triad 2000, Dentsply, York, PA; light intensity at specimen location was approximately 110 mW/cm 2 ) for 1 min on each open side, and incubated for 24 h at 37 °C before immersion, as described below.

Mechanical testing

For immersion and mechanical testing, a sodium chloride solution (133 mmol/L) was buffered to two pH values: pH 4 with 50 mmol/L lactic acid, and pH 7 with 50 mmol/L HEPES. For each material, the cured specimens were randomly divided into two groups and immersed in the pH 7 and pH 4 solutions, respectively, at 37 °C for 1 day or 4 weeks. Each group was immersed in 200 mL of solution in a sealed polyethylene container. For the 4 weeks groups, the solution was changed every week. For the pH 4 groups, the pH was adjusted with lactic acid to be at pH 4.

A computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) was used to fracture the specimens in three-point flexure with a 10 mm span at a crosshead speed of 1 mm/min. The specimens were wet and not dried, and were fractured within a few minutes after being taken out of the solution. 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.

Calcium and phosphate ion release

A sodium chloride (NaCl) solution (133 mmol/L) was buffered to two different pHs: pH 4 with 50 mmol/L lactic acid, and pH 7 with 50 mmol/L HEPES. Following previous studies , three specimens of approximately 2 × 2 × 12 mm were immersed in 50 mL of solution at each pH, yielding a specimen volume/solution of 2.9 mm 3 /mL. This compared to a specimen volume per solution of approximately 3.0 mm 3 /mL in a previous study . For each solution, the concentrations of Ca and P ions released from the specimens were measured at 1, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days (d). At each time, aliquots of 0.5 mL were removed and replaced by fresh solution. The aliquots were analyzed for Ca and P ions via a spectrophotometric method (SpectraMax M5, Molecular Devices, Sunnyvale, CA) using known standards and calibration curves . The released ions were reported in cumulative concentrations.

Tooth section preparation

This study (Protocol #: 29749) received an exemption from the University of Maryland Institutional Review Board, as it was deemed not human subjects research. Teeth were collected from clinics and had no personal information of the donors. Twenty caries-free teeth were disinfected in a 0.005% promodyne solution for 4 h. The roots of each tooth were removed at a location approximately 5 mm below the cemento-enamel junction and embedded with composite resin ( Fig. 1 A). The upper third of the crown was removed to expose the dentin surface and a single cavity with dimensions 6 × 3 × 1 mm was machined into the dentin surface under constant water irrigation. The enamel surface was coated with varnish and the dentin surface was exposed to 25 mL of demineralizing solution (8.7 mmol/L CaCl 2 , 8.7 mmol/L KH 2 PO 4 , 0.05 ppm NaF, 75 mmol/L acetic acid, pH 4.0—adjusted with KOH) for 48 h. Each cavity was filled with either NACP composite, NACP-TTCP composite, or TPH control without the use of a bonding agent and light cured for 2 min (Triad 2000, Dentsply International, York, PA, USA). Five teeth were restored for each of the above conditions. Each restored tooth was wet cut into sections approximately 120 μm thick using a diamond blade (Buehler, Lake Bluff, IL) on a Buehler Isomet low-speed saw at the highest speed setting. Additionally, unrestored teeth were also sectioned and acted as a negative control. 3–6 tooth sections were collected from each restored tooth. To assist in the alignment of the “before” and “after” microradiographic images, 200-mesh Maxtaform Copper/Rhodium transmission electron microscopy (TEM) grids (Electron Microscopy Sciences, Hatfield, PA) were cut under a stereomicroscope into rows encompassing at least one full grid and adhered to each tooth section.

Nov 22, 2017 | Posted by in Dental Materials | Comments Off on Effect of calcium phosphate nanocomposite on in vitroremineralization of human dentin lesions
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