Abstract
Objectives
Calcium phosphate (CaP) composites with Ca and P ion release can remineralize tooth lesions and inhibit caries. But the ion release lasts only a few months. The objectives of this study were to develop rechargeable CaP dental composite for the first time, and investigate the Ca and P recharge and re-release of composites with nanoparticles of amorphous calcium phosphate (NACP) to achieve long-term inhibition of caries.
Methods
Three NACP nanocomposites were fabricated with resin matrix of: (1) bisphenol A glycidyl dimethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) at 1:1 mass ratio (referred to as BT group); (2) pyromellitic glycerol dimethacrylate (PMGDM) and ethoxylated bisphenol A dimethacrylate (EBPADMA) at 1:1 ratio (PE group); (3) BisGMA, TEGDMA, and Bis[2-(methacryloyloxy)ethyl] phosphate (BisMEP) at 2:1:1 ratio (BTM group). Each resin was filled with 20% NACP and 50% glass particles, and the composite was photo-cured. Specimens were tested for flexural strength and elastic modulus, Ca and P ion release, and Ca and P ion recharge and re-release.
Results
NACP nanocomposites had strengths 3-fold of, and elastic moduli similar to, commercial resin-modified glass ionomer controls. CaP ion recharge capability was the greatest for PE group, followed by BTM group, with BT group being the lowest ( p < 0.05). For each recharge cycle, CaP re-release reached similarly high levels, showing that CaP re-release did not decrease with more recharge cycles. After six recharge/re-release cycles, NACP nanocomposites without further recharge had continuous CaP ion release for 42 d.
Significance
Novel rechargeable CaP composites achieved long-term and sustained Ca and P ion release. Rechargeable NACP nanocomposite is promising for caries-inhibiting restorations, and the Ca and P ion recharge and re-release method has wide applicability to dental composites, adhesives, cements and sealants to achieve long-term caries-inhibition.
1
Introduction
Resin composites and adhesives are increasingly used in dental restorations . Tooth cavity restorations cost the United States approximately $46 billion annually . Secondary (recurrent) caries is a frequent reason for the failure of dental restorations , and replacing the failed restorations accounts for 50–70% of all restorations performed . According to the minimally-invasive treatment concept, more dentin tissues are recommended to be preserved, which are accompanied by a certain amount of caries-infected and caries-affected dentin with residual bacteria in the prepared cavity . In addition, microleakage along the restoration margins can provide pathways for new invading bacteria from the oral environment . The bacteria growth and biofilm formation can produce acids to demineralize the tooth structure and cause caries.
A promising approach to combat caries is the development of calcium phosphate (CaP) composites that can release calcium (Ca) and phosphate (P) ions . Traditional CaP composites contained CaP particles with sizes of 1–55 μm and achieved successful remineralization of tooth lesions . Re-incorporation of minerals into the demineralized dentin matrix is important since the precipitated mineral may serve as sites for further nucleation, and the remineralized tissues may be more resistant to degradation . Recently, nanocomposites containing nanoparticles of amorphous calcium phosphate (NACP) with a mean particle size of 116 nm were developed . The 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 can rapidly neutralize lactic acid solutions at a cariogenic pH of 4 and increase the pH to a safe level of above 6 . The NACP nanocomposite successfully 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 secondary caries at the enamel-restoration margins in vivo, reducing the enamel mineral loss at the margins to 1/3 of the mineral loss associated with a control composite without NACP .
However, a major drawback of CaP composites is that the Ca and P ion release lasts for only weeks to months, and then the ion release is diminished over time. Previous studies measured Ca and P ion release from composites to at most a couple of months . However, clinicians and patients would expect the composite restoration to be effective in vivo for much longer than a few months (e.g., for 10 or 20 years). Therefore, it would be highly desirable for the CaP composite to be able to recharge and re-release Ca and P ions, thereby to release Ca and P ions indefinitely and provide long-term caries-inhibition capability. However, literature and patent searches revealed no report on rechargeable calcium phosphate dental composites and resins.
Therefore, the objectives of this study were to develop rechargeable calcium phosphate dental composite for the first time, and to investigate the effects of different resin matrices on the CaP recharge and re-release efficacy. A previous NACP nanocomposite using bisphenol A diglycidyl methacrylate (BisGMA)-based matrix with high levels of Ca and P ion release and good mechanical properties served as a control . Two new NACP nanocomposites were synthesized. One contained pyromellitic glycerol dimethacrylate (PMGDM) and ethoxylated bisphenol A dimethacrylate (EBPADMA) at a mass ratio of 1:1. The other contained Bis[2-(methacryloyloxy)ethyl] phosphate (BisMEP). PMGDM and BisMEP were selected because both are acidic adhesive monomers , and may chelate with calcium and phosphate ions from a recharge solution to achieve the recharge capability. The following hypotheses were tested: (1) rechargeable calcium phosphate composites can be developed and the recharge efficacy will depend on matrix resin type; (2) the re-release of Ca and P ions from the NACP nanocomposite will be maintained over time and not decrease with increasing the number of recharge/re-release cycles; (3) the rechargeable NACP nanocomposite will possess mechanical properties matching or exceeding commercial control fluoride-rechargeable restorative materials.
2
Materials and methods
2.1
NACP nanocomposite fabrication
NACP [Ca 3 (PO 4 ) 2 ] were synthesized via a spry-drying technique as previously described . Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution. The concentrations of Ca and P ions were 8 mmol/L 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 with a median size of 1.4 μm (Caulk/Dentsply, Milford, DE) were silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n-propylamine as previously described .
Three types of matrix resins were prepared to fabricate the NACP nanocomposite. For type 1, a resin of BisGMA and triethylene glycol dimethacrylate (TEGDMA) (Esstech, Essington, PA) at 1:1 mass ratio was rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4-N,N-dimethylaminobenzoate, following previous studies . BisGMA and TEGDMA are common dental resins used frequently in non-rechargeable commercial dental restoratives and, as such, provide a control to compare with the novel, rechargeable NACP nanocomposites. This resin using BisGMA and TEGDMA is referred to as BT resin.
For type 2, acidic monomer PMGDM and dimethacrylate EBPADMA (Sigma–Aldrich, St., Louis, MO) were mixed at a mass ratio of 1:1 to form the matrix resin . This is referred to as PE resin. The combination of PMGDM and EBPADMA were used experimentally in resin-based calcium phosphate cements . Experiments showed that PMGDM and EBPADMA had a cytotoxicity similar to that reported for other dental dimethacrylates, but significantly less than the cytotoxicity of BisGMA .
For type 3, BisGMA, TEGDMA, and acidic monomer BisMEP (Sigma-Aldrich) were mixed at a mass ratio of 2:1:1 to form the matrix resin . This is referred as BTM resin. BisMEP has been used as a component in commercial self-etch primer systems (Tyrian SPE/One-Step Plus, Bisco, Schaumburg, IL) and in experimental amorphous calcium phosphate (ACP)-based composites . The 0.2% camphorquinone and 0.8% ethyl 4-N,N-dimethylaminobenzoate were the same in all three groups.
Each aforementioned resin was filled with mass fractions of 20% NACP and 50% glass particles to form a readily-mixed and cohesive paste. Each composite paste was placed into a stainless steel mold of 2 mm × 2 mm × 25 mm, and light-cured (Triad 2000, Dentsply, York, PA) for 1 min on each open side. The three types of experimental composites are denoted BT-NACP, PE-NACP, and BTM-NACP nanocomposites, respectively.
For mechanical testing, four commercial materials were included as comparative controls to provide a range of commercial mechanical properties. A resin-modified glass ionomer (RMGI) cement (Vitremer, 3 M, St. Paul, MN) consisted of fluoroaluminosilicate glass, and a light-sensitive, aqueous polyalkenoic acid. According to the manufacturer, indications include Class III, V and root-caries restoration, Class I and II in primary teeth, and core-buildup. A powder/liquid ratio of 2.5/1 was used yielding a filler mass fraction of 71.4%, according to the manufacturer. Another RMGI (Ketac Nano, 3 M) consisted of polycarboxilic acid modified with methacrylate groups and fluoroaluminosilicate glass, with a filler level of 69%. It is a two-part, paste/paste system and dispensed using the Clicker Dispensing System. It is recommended for small Class I restorations, and Class III and V restorations. In addition, two composites also served as controls. A nanocomposite Renamel (Cosmedent, Chicago, IL) consisted of nanofillers of 20–40 nm at 60% filler level in a multifunctional methacrylate ester resin. According to the manufacturer, Renamel is indicated for Class III, IV, and V restorations. Another nanocomposite Heliomolar (Ivoclar, Amherst, NY) contained 66.7% of nanofillers of 40–200 nm of silica and ytterbium-trifluoride with fluoride-release (Heliomolar, Ivoclar, Amherst, NY). According to the manufacturer, Heliomolar is indicated for Class I, II, III, IV and V restorations. All specimens were light-cured as described above and treated in the same manner.
2.2
Mechanical testing
All specimens were stored at 37 °C for 24 h. Flexural strength and elastic modulus of specimens were measured using a three-point flexural test 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 was calculated by: S = 3 P max / L (2 bh 2 ), where P max is the fracture load, L is span, b is specimen width and h is thickness. Elastic modulus was calculated by: E = ( P / d )( L 3 /[4 bh 3 ]), where load P divided by displacement d is the slope in the linear elastic region.
2.3
Ca and P ion release from NACP nanocomposites
Three groups were tested for Ca and P ion release: BT group, PE group, and BTM group. The commercial controls were used for mechanical testing, but not in Ca and P ion release experiments because they did not release Ca and P ions. A sodium chloride (NaCl) solution (133 mmol/L) was buffered to pH 4 with 50 mmol/L lactic acid to measure ion release, simulating a cariogenic condition . As in previous studies , three specimens of approximately 2 mm × 2 mm × 12 mm were immersed in 50 mL of solution to yield a specimen volume/solution of 2.9 mm 3 /mL. This was similar to a specimen volume per solution of about 3.0 mm 3 /mL in a previous study . The concentrations of Ca and P ions released from the specimens were measured at 1, 3, 5, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70 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 ion concentrations via a spectrophotometric method (DMS-80 UV–visible, Varian, Palo Alto, CA) using known standards and calibration curves . Six batches of specimens were tested and averaged for ion release for each group. The released ions were reported in cumulative concentrations . This ion release from NACP nanocomposite was termed “virgin release”, to differentiate from the subsequent recharge and re-release.
2.4
Recharge of CaP composite and re-release of ions
The procedures of recharge and re-release measurement are illustrated in Fig. 1 . First, NACP nanocomposite specimens were immersed in pH 4 solution to measure ion release as described in Section 2.3 . At 70 d, the ion measurement showed that the ion concentration had plateaued and there was no further release. The composite specimens were removed from the 70-d solution and rinsed with water for 5 min. The specimens were then immersed in a flesh 50 mL solution at pH 4. Then Ca and P ion release was further measured for 7 d, which confirmed that the ion release was indeed exhausted and there was no further release, as indicated by the two arrows at the lower left corner in Fig. 1 .
The exhausted specimens were then used for recharge. The recharging solutions for Ca and P ions were prepared respectively. The calcium ion recharging solution consisted of 20 mmol/L CaCl 2 and 50 mmol/L HEPES buffer . The phosphate ion recharging solution consisted of 12 mmol/L KHPO 4 and 50 mmol/L HEPES buffer. Each solution was adjusted to a pH of 7.0 using 1 mol/L KOH . Three specimens of approximately 2 mm × 2 mm × 12 mm were immersed in 5 mL of the calcium or phosphate solution and gently shaken on a mixing machine (Analog Vortex Mixer, Fisher Scientific, Waltham, MA) at a power level of 3 for 1 min. This immersion and shaking treatment simulated the movement in the mouth rinsing process when a calcium or phosphate mouth-rinse could be used. Then the specimens were rinsed with running distilled water for 1 min to remove any loosely attached deposits on specimen surfaces (hence only the ions recharged into the interior of the composite will be measured in the subsequent re-release test). This recharge process was repeated three times daily at 9:00 am, 12:00 noon and 5:00 pm for 3 d. All specimens for the BT group, PE group and BTM group were treated in the same manner for comparison of the recharge and re-release efficacy.
The recharged specimens were then immersed in 50 mL of the pH 4 solution as described in Section 2.3 to measure Ca and P ion re-release, as indicated by the third arrow in the bottom of Fig. 1 . In order to test the recharge/re-release cycle repeatedly for many time to investigate the durability, each cycle of re-release measurement lasted for 7 d (the short arrow in Fig. 1 indicates the measurement from 1 d to 7 d in the first cycle). After 7 d of re-release, the specimens were recharged again and tested for re-release, as cycle 2. This was repeated for 6 cycles in the present study as illustrated in Fig. 1 .
After 6 cycles, in order to investigate how long the specimens could further release Ca and P ions, the specimens after the 6th cycle (without further recharge) were immersed in 50 mL of the pH 4 solution as described in Section 2.3 . The measurements of Ca and P ion release from these specimens were continued for an additional 42 d. At 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35 and 42 d, the Ca and P measurements were performed as described in Section 2.3 . For each of the three NACP nanocomposite groups, three batches of specimens were tested and averaged for ion release following previous studies .
2.5
Statistical analysis
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 to compare the data at a p value of 0.05.
2
Materials and methods
2.1
NACP nanocomposite fabrication
NACP [Ca 3 (PO 4 ) 2 ] were synthesized via a spry-drying technique as previously described . Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution. The concentrations of Ca and P ions were 8 mmol/L 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 with a median size of 1.4 μm (Caulk/Dentsply, Milford, DE) were silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n-propylamine as previously described .
Three types of matrix resins were prepared to fabricate the NACP nanocomposite. For type 1, a resin of BisGMA and triethylene glycol dimethacrylate (TEGDMA) (Esstech, Essington, PA) at 1:1 mass ratio was rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4-N,N-dimethylaminobenzoate, following previous studies . BisGMA and TEGDMA are common dental resins used frequently in non-rechargeable commercial dental restoratives and, as such, provide a control to compare with the novel, rechargeable NACP nanocomposites. This resin using BisGMA and TEGDMA is referred to as BT resin.
For type 2, acidic monomer PMGDM and dimethacrylate EBPADMA (Sigma–Aldrich, St., Louis, MO) were mixed at a mass ratio of 1:1 to form the matrix resin . This is referred to as PE resin. The combination of PMGDM and EBPADMA were used experimentally in resin-based calcium phosphate cements . Experiments showed that PMGDM and EBPADMA had a cytotoxicity similar to that reported for other dental dimethacrylates, but significantly less than the cytotoxicity of BisGMA .
For type 3, BisGMA, TEGDMA, and acidic monomer BisMEP (Sigma-Aldrich) were mixed at a mass ratio of 2:1:1 to form the matrix resin . This is referred as BTM resin. BisMEP has been used as a component in commercial self-etch primer systems (Tyrian SPE/One-Step Plus, Bisco, Schaumburg, IL) and in experimental amorphous calcium phosphate (ACP)-based composites . The 0.2% camphorquinone and 0.8% ethyl 4-N,N-dimethylaminobenzoate were the same in all three groups.
Each aforementioned resin was filled with mass fractions of 20% NACP and 50% glass particles to form a readily-mixed and cohesive paste. Each composite paste was placed into a stainless steel mold of 2 mm × 2 mm × 25 mm, and light-cured (Triad 2000, Dentsply, York, PA) for 1 min on each open side. The three types of experimental composites are denoted BT-NACP, PE-NACP, and BTM-NACP nanocomposites, respectively.
For mechanical testing, four commercial materials were included as comparative controls to provide a range of commercial mechanical properties. A resin-modified glass ionomer (RMGI) cement (Vitremer, 3 M, St. Paul, MN) consisted of fluoroaluminosilicate glass, and a light-sensitive, aqueous polyalkenoic acid. According to the manufacturer, indications include Class III, V and root-caries restoration, Class I and II in primary teeth, and core-buildup. A powder/liquid ratio of 2.5/1 was used yielding a filler mass fraction of 71.4%, according to the manufacturer. Another RMGI (Ketac Nano, 3 M) consisted of polycarboxilic acid modified with methacrylate groups and fluoroaluminosilicate glass, with a filler level of 69%. It is a two-part, paste/paste system and dispensed using the Clicker Dispensing System. It is recommended for small Class I restorations, and Class III and V restorations. In addition, two composites also served as controls. A nanocomposite Renamel (Cosmedent, Chicago, IL) consisted of nanofillers of 20–40 nm at 60% filler level in a multifunctional methacrylate ester resin. According to the manufacturer, Renamel is indicated for Class III, IV, and V restorations. Another nanocomposite Heliomolar (Ivoclar, Amherst, NY) contained 66.7% of nanofillers of 40–200 nm of silica and ytterbium-trifluoride with fluoride-release (Heliomolar, Ivoclar, Amherst, NY). According to the manufacturer, Heliomolar is indicated for Class I, II, III, IV and V restorations. All specimens were light-cured as described above and treated in the same manner.
2.2
Mechanical testing
All specimens were stored at 37 °C for 24 h. Flexural strength and elastic modulus of specimens were measured using a three-point flexural test 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 was calculated by: S = 3 P max / L (2 bh 2 ), where P max is the fracture load, L is span, b is specimen width and h is thickness. Elastic modulus was calculated by: E = ( P / d )( L 3 /[4 bh 3 ]), where load P divided by displacement d is the slope in the linear elastic region.
2.3
Ca and P ion release from NACP nanocomposites
Three groups were tested for Ca and P ion release: BT group, PE group, and BTM group. The commercial controls were used for mechanical testing, but not in Ca and P ion release experiments because they did not release Ca and P ions. A sodium chloride (NaCl) solution (133 mmol/L) was buffered to pH 4 with 50 mmol/L lactic acid to measure ion release, simulating a cariogenic condition . As in previous studies , three specimens of approximately 2 mm × 2 mm × 12 mm were immersed in 50 mL of solution to yield a specimen volume/solution of 2.9 mm 3 /mL. This was similar to a specimen volume per solution of about 3.0 mm 3 /mL in a previous study . The concentrations of Ca and P ions released from the specimens were measured at 1, 3, 5, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70 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 ion concentrations via a spectrophotometric method (DMS-80 UV–visible, Varian, Palo Alto, CA) using known standards and calibration curves . Six batches of specimens were tested and averaged for ion release for each group. The released ions were reported in cumulative concentrations . This ion release from NACP nanocomposite was termed “virgin release”, to differentiate from the subsequent recharge and re-release.
2.4
Recharge of CaP composite and re-release of ions
The procedures of recharge and re-release measurement are illustrated in Fig. 1 . First, NACP nanocomposite specimens were immersed in pH 4 solution to measure ion release as described in Section 2.3 . At 70 d, the ion measurement showed that the ion concentration had plateaued and there was no further release. The composite specimens were removed from the 70-d solution and rinsed with water for 5 min. The specimens were then immersed in a flesh 50 mL solution at pH 4. Then Ca and P ion release was further measured for 7 d, which confirmed that the ion release was indeed exhausted and there was no further release, as indicated by the two arrows at the lower left corner in Fig. 1 .
