Abstract
Bioglass 45S5 is a silica-based bioactive glass capable of depositing a layer of hydroxyl carbonate apatite on the surface of the glass when immersed in body fluids. The present paper studies a new technique for treating early human dental enamel caries lesions by using a paste composed of 45S5 bioglass and phosphoric acid. Artificial caries lesions were induced in enamel flat surfaces by means of a decalcification solution. All specimens were exposed to a brushing-abrasion challenge to test the durability of any newly formed layer resulting from the application of 45S5 bioglass paste. The specimens treated with bioglass paste showed complete coverage with a layer of brushite crystals. The brushing-abrasion challenge did not statistically affect the percentage of enamel coverage with the crystalline layer formed by the application of bioglass ( p < 0.05). These crystals were converted to hydroxyapatite crystals when stored in artificial saliva for 14 days. The current technique suggests the possibility of restoring incipient enamel erosive lesion with an abrasion durable layer of hydroxyapatite crystals.
1
Introduction
Dental caries affects large sector of the population worldwide. It results from the production of acid by plaque bacteria during fermentation of dietary sugars. This causes under-saturation of saliva and plaque fluids with regard to hydroxyapatite constituents, causing demineralization of enamel . Such enamel lesions in most of the cases have a lesion body with a normally mineralized surface and thus can remineralize naturally at a slow rate or by using current remineralizing agents, i.e. NaF, phosphorus calcium phosphate, etc. provided that there is no further sugar challenge occurring . However, these remineralizing strategies encounter many limitations if the enamel surface is irreversibly lost due to an erosive challenge, making it more difficult to replace the lost enamel.
Techniques for enamel reconstruction have been a challenging topic of research in material sciences and dentistry. Enamel consists of a highly organized hierarchical microstructure made up of carbonated hydroxyapatite nanocrystals, 50–70 nm in width, 20–25 nm in thickness, with a length to width aspect ratio over 1000. These features provide dental enamel its high strength and extreme hardness . Moreover, mature enamel is acellular, and has a mineral content of 95 mass percent and does not remodel. All of these factors contribute to the difficulty of repairing dental enamel tissue .
Many previous studies aimed at the repair of early enamel lesions by the in vitro synthesis of enamel-like hydroxyapatite crystals including: producing enamel-like crystals by mixing (EDTA-Ca-Na 2 ) salt and NaH 2 PO 4 ·H 2 O on a metallic plate under the pressure and temperature of an autoclave ; hydrothermal transformation of octacalcium phosphate (OCP) rods to HAP nanorods in the presence of urea and heating to 100 °C , a paste composed of calcium phosphate mixed with high concentration of hydrogen peroxide ; fluorapatite cement inserted in large enamel defects . However, the majority of these synthetic methods was developed under conditions that were extremely difficult to replicate clinically or were unsuitable for restoring incipient microscopic enamel lesions. Moreover, most of these agents were not evaluated for abrasion durability, although it is one of the most important factors governing the clinical success of any restorative procedure in the oral cavity.
Bioglass ® 45S5 (of nominal weight composition: 45% SiO 2 , 24.5% Na 2 O, 24.5% CaO, 6% P 2 O 5 ) is a bioactive implant material that stimulates bone repair . In an aqueous environment, this material undergoes a series of reactions, resulting in the formation of a surface layer made of hydroxyapatite and/or hydroxycarbonate apatite .
Bioglass ® 45S5 exhibits a high bioactivity index and bonds to both bone and soft connective tissues . It is currently used in prosthetic devices for both bone and dental implants because it has many similarities to hard tissues found in oral and body environment .
We have recently reported the possibility of using a paste containing Bioglass ® 45S5 and 50% phosphoric acid to form an “interaction layer” capable of occluding the dentinal tubule orifices and thus can be used as an aid for the treatment of dentin hypersensitivity lesions . This “interaction layer” represents insoluble reaction products formed by applying bioglass gel to tooth mineral.
The current study aimed at examining the possibility of forming a Bioglass ® 45S5-phosphoric acid paste “interaction layer” on the artificially induced enamel caries lesions using the field emission scanning electron microscopy (FE-SEM) coupled with energy dispersive X-ray spectroscope (EDS), and X-ray diffraction (XRD). Moreover, the brushing-abrasion durability of the newly formed layer was examined.
2
Materials and methods
2.1
Enamel specimens preparation
Sixty non-carious third molars were used in this study following guidelines approved by Tokyo Medical and Dental University Ethical Committee. The teeth were sectioned to remove their buccal surfaces using water-cooled diamond saw microtome (1600 Microtome, Leitzwetzlar, Germany) and then ground flat with water-cooled silicon carbide discs (600- and 1200-grade papers; Buehler), and wet-polished using diamond paste (1 μm; Buehler), to obtain flat enamel surfaces. Two layers of protective nail varnish to protect half of the exposed enamel area leaving a treatment enamel window of 2 mm 2 . All the enamel surfaces were challenged by a buffered demineralization solution composed of (CaCl 2 2.2 mM/L, NaH 2 PO 4 2.2 mM/L, acetic acid 50 mM/L, pH 4.5) for 4 days . All of the demineralized enamel specimens were randomly assigned into 4 groups having 15 specimens in each group, Table 1 : Group I, no bioglass was applied and no brushing-abrasion was done; Group II, bioglass was applied, but no brushing-abrasion was done, Group III, no bioglass was applied, however, the specimens were exposed to brushing-abrasion challenge, Group IV, bioglass was applied and the specimens were exposed to brushing-abrasion challenge.
| Group I | Group II | Group III | Group IV | |
|---|---|---|---|---|
| Demineralization | + | + | + | + |
| Bioglass | − | + | − | + |
| Brushing-abrasion | − | − | + | + |
2.1.1
45S5 bioglass application
One-tenth of a gram of 45S5 bioglass powder (NovaMin ® , 5 μm average particle, NovaMin Technology, Alachua, FL, USA), composed of 24.5 wt% Na 2 O, 24.4 wt% CaO, 6 wt% P 2 O 5 , and 45 wt% SiO 2 , was mixed on a glass slab for 1 min by spatula with 0.2 mL of 50 wt% phosphoric acid that was prepared by the dilution of 85 wt% phosphoric acid (Wako Chemicals, Osaka, Japan) in distilled water to form a gel (pH 2) . The acidic gel was immediately applied to specimens of Groups II and IV by microbrush (Microbrush International, Grafton, WI, USA). A layer of bonding agent (Clearfil SE Bond, Kuraray Medical, Tokyo, Japan) was immediately applied over the 45S5 bioglass-phosphoric-acid gel and then light-cured ( Table 2 ).
| Materials | Composition | Procedures | |
|---|---|---|---|
| 45S5 bioglass (NovaMin ® Technology, USA) | Weight percent | 45% SiO 2 , 24.5% Na 2 O, 24.4% CaO, 6% P 2 O 5 | Mix 0.1 g of 45S5 bioglass to 0.2 mL of phosphoric acid |
| Clearfil SE Bond (Kuraray, Osaka, Japan) | Primer: | MDP, HEMA, Water, PI, accelerators, CA | Apply self-etching primer (20 s) |
| Adhesive: | MDP, HEMA, MFM, PI, accelerators, CA. Microfiller | Apply adhesive, gently air dry, light cure (10 s) | |
| Clearfil AP-X (Kuraray, Osaka, Japan) | Bis-GMA, TEG-DMA barium glass filler (85 wt%), PI, accelerators | Apply and light cure for (40 s) | |
2.2
Storage of specimens
After the acidic challenge for Groups I and III and after application of bioglass in Group II (demineralized, bioglassapplied/not abraded) and Group IV (demineralized/bioglassapplied/abraded); all specimens were placed for 24 h in a remineralizing medium (1.0 mM CaCl 2 , 3.0 mM KH 2 PO 4 , 100 mM acetate, 100 mM NaCl, 0.02%, NaN 3 ; pH 6.3) .
After the storage period for the specimens of Groups II and IV, the thin layer of the bonding agent covering the bioglass mixture was gently removed by means of an excavator, and then rinsed with water spray for 30 s.
2.2.1
Brushing-abrasion challenge
After the storage period, specimens of Groups III (demineralized/abraded) and Group IV (demineralized/bioglass/abraded) were attached to an acrylic resin block and then exposed to 6000 cycles of brushing-abrasion challenge ( Fig. 1 ) using a tabletop machine (TTC3-1-2020-10B, IAI Co., Shizouka, Japan). The brushing-abrasion challenge was performed using a nylon bristle toothbrush (PROSPEC adult hard, GC Corp., Tokyo, Japan.) attached to a lever arm operating in back and forth motions (load of 250 gf, 150 mm/stroke, 100 stroke/min) under wet conditions. The percentage of newly formed mineral layer coverage resulting from bioglass application in Groups I and IV was detected by FE-SEM top surface examination and the results were compared statistically p < 0.05.
2.2.2
FE-SEM/EDS interface preparation
Five specimens from each group were conditioned for 20 s by the self-etching primer of Clearfil SE-Bond, bonded according to manufacturer’s instructions ( Table 2 ), and then light-cured. Building up of light-curing resin composite (Clearfil AP-X, Kuraray-Medical) was carried out. The specimens were stored in de-ionized water for 24 h, then sectioned perpendicular to the interface to give 1.5-mm-thick slabs. The transverse sectioned surfaces were ground and then polished with diamond pastes down to 0.25 μm. The polished surfaces of the slabs were etched using an ion milling system (EIS-IE, Elionix, Tokyo, Japan), gold coated and examined by FE-SEM/EDS (S-4500, Hitachi High Technology, Hitachinaka, Japan) . Line scans were done across the treated enamel surfaces for the following elements: phosphorus, calcium and silicon.
2.2.3
FE-SEM top surface examination
Five specimens from each group were gradually dehydrated in an ascending ethanol series (50–100%), gold coated and examined by FE-SEM/EDS. Two images were obtained from the center of each specimen with magnifications of 1500×. The newly formed mineral layer was characterized for Groups II and IV using the EDS analysis for the calcium and phosphorus content. The percentage of enamel surface coverage by the newly formed mineral layer on the SEM images was determined for Groups II and IV using Image J (version 1.40g, National Institutes of Health, USA), and statistically compared p < 0.05. If the tested enamel surface was totally covered with brushite crystals (characterized by the EDS), so, the percentage of enamel surface coverage by the brushite crystals was considered 100%.
2.2.4
XRD examination of the enamel surfaces
Five specimens from each group were examined by an X-ray diffractometer (RAD IIA, Rigaku Denki, Tokyo, Japan) with Cu Kα radiation of 40 kV using a Ni filter. The diffraction intensities were measured four times for each specimen by scanning in the range of 2 θ (i.e. 10–60° in 0.1° steps for 2 s per point). The same enamel specimens for Groups II and IV were examined after 7 days and 14 days of storage in the remineralizing solution described previously to detect the nature of the calcium-phosphate crystals formed on enamel.
2.3
Statistical analysis
The effect of brushing-abrasion on the newly formed mineral layer was detected by comparing the percentage of enamel coverage by the newly formed mineral layer for Groups II and IV using Wilcoxon Signed-Rank test ( p < 0.05).
2
Materials and methods
2.1
Enamel specimens preparation
Sixty non-carious third molars were used in this study following guidelines approved by Tokyo Medical and Dental University Ethical Committee. The teeth were sectioned to remove their buccal surfaces using water-cooled diamond saw microtome (1600 Microtome, Leitzwetzlar, Germany) and then ground flat with water-cooled silicon carbide discs (600- and 1200-grade papers; Buehler), and wet-polished using diamond paste (1 μm; Buehler), to obtain flat enamel surfaces. Two layers of protective nail varnish to protect half of the exposed enamel area leaving a treatment enamel window of 2 mm 2 . All the enamel surfaces were challenged by a buffered demineralization solution composed of (CaCl 2 2.2 mM/L, NaH 2 PO 4 2.2 mM/L, acetic acid 50 mM/L, pH 4.5) for 4 days . All of the demineralized enamel specimens were randomly assigned into 4 groups having 15 specimens in each group, Table 1 : Group I, no bioglass was applied and no brushing-abrasion was done; Group II, bioglass was applied, but no brushing-abrasion was done, Group III, no bioglass was applied, however, the specimens were exposed to brushing-abrasion challenge, Group IV, bioglass was applied and the specimens were exposed to brushing-abrasion challenge.
