To investigate the effect of photo-activation of riboflavin either by ultraviolet (UVA) or visible blue light (BL) on the biodegradation resistance, strength of demineralized dentin matrix, bond strength to dentin and resin/dentin interface morphology.
Dentin beams were demineralized, treated with 0.1% or 1% riboflavin solution for 5 min and photo-activated with UVA or BL for 20 s. The ultimate tensile strength (UTS) and hydroxyproline (HYP) release were assessed after 24 h collagenase challenge. For micro-tensile bond strength (μTBS) testing and resin/dentin interface morphology investigation, dentin was acid-etched, crosslinked with riboflavin and bonded with an etch-and-rinse adhesive system. Riboflavin was photo-activated separately with UVA or BL followed by photo-polymerization of the bonding resin with BL (two-step) or both riboflavin photo-activation and bonding resin photo-polymerization were done in one-step using BL.
Significant improvement in the UTS and biodegradation resistance against collagenase challenge was found when riboflavin was photo-activated either with UVA or BL. However, UVA showed more significant improvement compared to BL. After 4 months of water-storage, both UV and BL two-step photo-activation methods significantly preserved higher values of the μTBS compared to the non-crosslinked control group, where UVA showed significantly higher μTBS than BL.
Although UVA most effectively activated riboflavin, visible blue light showed to be a promising substitute for UVA as it is clinically more applicable and acceptable, and still managed to increase the biodegradation resistance, enhance the mechanical properties of dentin collagen and improve and maintain the bond strength and interface integrity after short-term water storage.
The structural integrity and mechanical properties of collagen fibrils directly affect the quality of the bond strength and its durability . Adhesive resin monomers infiltrate and encapsulate the exposed collagen fibrils to form what is known as the hybrid layer . When there is inadequate infiltration, exposed collagen fibrils are vulnerable for degradation and nano-leakage . Etch-and-rinse dentin adhesives involve the application of an acid conditioner, followed by priming and bonding resin monomers to the demineralized dentin collagen fibrils . Resin infiltration and hybrid layer formation is a sensitive procedure due to the delicate nature of dentin collagen fibrils, which can easily collapse due to dehydration interfering with monomer diffusion and consequently affect the quality of the hybrid layer .
Dentin collagen reinforcement and strengthening through collagen crosslinking might be of importance to improve bond strength and durability and structural integrity of the resin/dentin interface with time against enzymatic and/or hydrolytic degradation through the formation of inter- and intra-molecular crosslinks . Several studies reported the use of grape seed extract, proanthocyanidins, and other crosslinking agents for crosslinking of the dentin collagen . However, many variables related to the clinical applicability, suitability and biocompatibility of such compounds in adhesive dentistry are still questionable and need further investigations.
Riboflavin activated by ultraviolet A (UVA), is a crosslinker, introduced as a new treatment for keratoconus and has proven to promote collagen type I crosslinking and increase the biomechanical strength of the human cornea by 300% . Riboflavin is an appropriate candidate for crosslinking dentin collagen due to its biocompatibility and its ability to produce free radicals when photo-activated with spectral range from UV to visible light . These free radicals, or so-called reactive oxygen species such as O 2 and O 2 − , are released when riboflavin is photo-activated and light is absorbed, forming covalent crosslinks between adjacent collagen molecules . It has been proposed that the observed reduction in histidine and tyrosine during crosslinking and the formation of ditryosine, dimer of tyrosine, is a possible mechanism in collagen aggregation mediated through riboflavin .
Recently, Cova et al. concluded that the effect of UVA-activated riboflavin to increase the immediate bond strength to dentin, stabilized adhesive interface, and inhibited dentin matrix metalloproteinases, thereby increasing durability of resin/dentin bonds. Riboflavin is a strong free-radical producing agent when activated by light with maximum absorption peaks at wavelengths of 270, 366 and 445 nm . Although the use of UVA was proven effective as a photo-activation method of riboflavin for collagen crosslinking , the safety issues regarding the use of UVA and its practicality for dental use should be considered. Conventional blue-light halogen-lamp curing units might be a possible alternative for UVA light sources to activate riboflavin owing to its ready availability and its ease and safe use in dentistry.
The aim of this study is to investigate the effect of photo-activation of riboflavin either by UVA or visible blue light (BL) on: (1) the biodegradation resistance and tensile strength of demineralized dentin matrix and (2) the variation in bond strength and resin/dentin interface morphology with short-term water storage using an etch-and-rinse dentin adhesive system. The null hypotheses tested were: first; pretreatment with photo-activated (UVA or BL) riboflavin would not significantly affect the ultimate tensile strength (UTS), collagenase-mediated biodegradation resistance of demineralized dentin–collagen matrix and the bond strength to dentin. Second; photo-activation of riboflavin either with UVA or BL has no significant difference on UTS, biodegradation resistance and bond strength.
Materials and methods
In this study, the effect of riboflavin (RF) photo-activated by UVA or visible blue light (BL) on the strength and biodegradation rate of the demineralized dentin matrix was evaluated by ultimate tensile strength test (UTS) and hydroxyproline (HYP) release rate, respectively. In addition, the variations in the bond strength and resin/dentin interface morphology with short-term water storage were investigated by micro-tensile bond strength (μTBS) and SEM, respectively. Non-carious and non-restored human molars, which were extracted for clinical purposes, were collected and used in this study and approved by the Institutional Review Board of the National University of Singapore. Teeth were stored in 0.2% thymol at 4 °C and used within 3 months following extraction.
Ultimate tensile strength (UTS) testing
The occlusal enamel was removed 1 mm below DEJ using a low speed diamond saw (Buehler, Lake Bluff, IL, USA) under water cooling, and dentin discs of 1.7 ± 0.1 mm in diameter were obtained from mid-coronal dentin by sectioning the cervical and occlusal portions of each crown. The discs were sectioned into 0.5 ± 0.1 mm thick beams in the mesio-distal direction and further trimmed by cylindrical bur (#557D, Brasseler, Savannah, GA, USA) to a rectangular dimension of 0.5 mm thickness × 1.7 mm width × 6.5 mm length. The beams were made into hour-glass shaped specimens with a neck area of 0.5 × 0.5 ± 0.1 mm at middle, with the use of a cylindrical diamond bur. The beams were then fully demineralized in 10% phosphoric acid for 5 h , which was verified by X-ray, and then thoroughly rinsed in distilled water for 5 min at room temperature.
The demineralized beams were immediately divided into three control groups and four experimental groups ( n = 13/group) according to the photo-activation/crosslinking method. In the first control group (DW), beams were placed in distilled water for 5 min and no crosslinking or photo-activation was done. The beams of the second control group (DW/BL) were placed in distilled water for 5 min, gently air dried followed by 20 s exposure to a blue-light (BL) tungsten/halogen-lamp curing unit (Curing Light 2500; 3M ESPE, MN, USA) with maximum spectral output range of 470–480 nm and 600 mW/cm 2 output at a distance of 10 mm from the light source and 7 mm illuminated diameter. One specimen was irradiated separately at each time in air and room temperature in the hydrated moist condition. The third control group (DW/UVA) was exactly treated as the previous group, except UVA ( λ = 368 nm) light source (Dymax, BlueWave, 75 UV curing light spot) was used for 20 s at ∼10 mW/cm 2 placed at 10 mm distance from the specimen surface. The UVA was delivered by 70° wave-guide with an illuminating diameter of 7 mm. As for BL, one specimen was irradiated with UVA separately at each time in air and room temperature in the hydrated moist condition. The power outputs of the UVA and BL light sources were frequently monitored and checked with an optical power-meter (Newport, USA). The following two experimental groups (0.1% RF/UVA and 0.1% RF/BL) were treated for 5 min with 0.1% solution of riboflavin, gently air dried and photo-activated either by UVA or BL, as previously described. The final two experimental groups (1% RF/UVA and 1% RF/BL) were treated in the same way as the two previous groups respectively, except 1% riboflavin solution was applied. The 0.1% and 1% riboflavin solutions were prepared from riboflavin-5-phosphate (Sigma–Aldrich) dissolved in distilled water and the pH was adjusted at approximately 7. The prepared riboflavin solutions were kept in light-proof test tubes to avoid any light activation of riboflavin before use and applied to dentin specimens (15 μL) at room temperature (21 °C).
The control and experimental groups were then further divided to be stored either for 24 h in distilled water at 37 °C (baseline measurement) or for 24 h in collagenase type I solution at 37 °C. The collagenase solution was prepared by dissolving 100 mg of collagenase type I (Clostridiopeptidase A from Clostridium histolyticum, 125 U/mg, Sigma–Aldrich) in 6 ml of tricine buffer and 3 ml of distilled water. Tricine buffer solution was prepared with 50 mmol/L tricine, 12 mmol/L CaCl 2 and 400 mmol/L NaCl and adjusted to a pH of 7.5. For UTS testing, beams were fixed to a custom-made metallic jig designed for tensile strength measurement by cyanoacrylate adhesive (Zapit, Dental Ventures of America, Corona, CA, USA) and mounted on to a universal testing machine (Instron, 5848 Microtester, USA). Tensile load was applied at the center and parallel to the longitudinal axis of the beam with the direction of the dentinal tubules at a crosshead speed of 1 mm/min through a 50 N load cell until failure. Test was run until failure and the maximum load was divided by the specimen cross-sectional area to calculate the UTS in MPa . All measurements were done in air while the specimens were in hydrated state.
Collagenase-mediated collagen resistance to degradation
The hydroxyproline (HYP) release in the supernatant was measured using an assay kit (BioVision Inc., CA, USA) according to its manufacturer’s instructions. Dentin slabs, with dimensions of 4.5 mm length × 3.5 mm width × 0.5 mm thickness, were prepared from the coronal dentin and demineralized in 10% phosphoric acid for 5 h. The demineralized slabs were rinsed and treated as described previously for UTS testing according to their respective groups ( n = 5 slabs/group). All specimens were then exposed for 24 h to 100 μg ml −1 bacterial collagenase type I in tricine buffer. Then, 100 μl aliquot of the supernatant was collected and hydrolyzed in 12 N HCl at 120 °C for 3 h. Next, 10 μl of hydrolyzed specimen aliquots from each group were transferred to a 96-wellplate and evaporated to dryness under vacuum. After that, 100 μl of chloramine-T buffer reagent (6 μl of chloramine-T added to 94 μl of oxidation buffer) was added to each well containing either the experimental specimen or the standard solution, and incubated for 5 min at room temperature. Then, 100 μl of DMAB reagent (DMAB and perchloric acid, 50 μl each) was added to each well and incubated for 90 min at 60 °C. Spectrophotometer 96-well plate reader (Infinite 200 Tecan, Switzerland) was used to measure the absorbance at 560 nm. Standard curves for the quantity of HYP [0–1 μg ml −1 /well] were generated. HYP content for each specimen was averaged from quadruplicate measurements of each specimen.
Dentin bonding and μTBS testing
The occlusal enamel 1 mm below the DEJ was removed using a low-speed diamond saw under water-cooling and grinded with 600 grit-size silicon-carbide papers (Carbimet; Buehler, Lake Bluff, IL, USA) to create a standardized smear layer using a micro-grinder/polisher machine (Pheonix Beta Polisher/Grinder). In this study, one type of etch-and-rinse dentin adhesive system was used according to manufacturer’s instruction (Adper™ Singlebond 2; 3M ESPE). Dentin surfaces were etched with 35% phosphoric acid gel (3M ESPE) for 10 s and rinsed thoroughly with distilled water. After acid etching, dentin specimens were crosslinked and bonded as described in Table 1 . After dentin bonding, a crown of 4 mm height was built up for each tooth by equal increments, cured with a blue-light halogen-lamp (Curing Light 2500; 3M ESPE, MN, USA) for 20 s using a resin-based restorative composite (Filtek Z350 XT A3; 3M ESPE). The restored teeth were then placed in distilled for 24 h at 37 °C to complete the polymerization reaction. Then, the teeth were sectioned serially in both x – and y -directions across the adhesive interface to obtain resin/dentin beams of approximately 1 mm × 0.9 mm. The prepared beams were then divided to be stored either for 24 h or 4 months in distilled water at 37 °C. Fifteen beams for each group at each storage time were tested. The distilled water was changed weekly for 4 months. After the storage period, beams were fixed to custom-made metallic-jig with cyanoacrylate adhesive mounted to the universal testing machine and stressed to failure using 50 N load cell at cross-head speed of 1 mm/min. The μTBS was calculated by dividing the maximum load to the respective surface area which was reconfirmed by digital caliber.