Hydroxyapatite delivery to dentine tubules using carboxymethyl cellulose dental hydrogel for treatment of dentine hypersensitivity ()
1. INTRODUCTION
One of the common clinical problems concerning human teeth is the occurrence of dentine hypersensitivity [1-3] . It is considered to be an oral health concern among adults worldwide [4]. The incidence of hypersensitivity ranges from 10% - 30% of the general population [5]. Dentine hypersensitivity is defined as a transient pain arising in response to chemical, thermal, tactile or osmotic stimuli [6]. This problem can be caused by the exposure of dentine tubules as a result of enamel wear or gingival recession, which allows intradentinal fluid movement and leads to dentinal hypersensitivity [7-9] . This causes an excitation in the nerves inside the tooth exposed through the openings of the dentine tubules [10]. Although dentinal hypersensitivity is not considered life-threatening or a serious dental injury, the resulting uncomfortable and unpleasant sensation, variously described as dull or sharp, vague or specific and intermittent or constant, may dictate the diet adopted by those affected, limiting their choices of food [5].
Procedures aimed at reducing or eliminating dental hypersensitivity range from simple home desensitizing therapies to more complicated clinical processes such as surgery, pulpectomy or laser treatments [11,12] . An ideal desensitizing agent should not irritate or endanger the integrity of the pulp, should be painless immediately upon application and thereafter, be easy to apply, display rapid effect, be long-lasting or permanent and should not damage the teeth or gums, such as by discoloration. Many treatments have been devised according to these criteria, but no gold standard has been established [13].
One of the materials being utilized to treat hypersensitivity is hydroxyapatite (HAp). HAp is considered to be chemically similar to enamel. The ultra-structure of dental enamel consists of inorganic components distributed as rod and prisms of hexagonal HAp crystals [14]. HAp is also used as a component of bone substitutes and has become a mainstay material for clinical use due to its bioactivity and biocompatibility [15]. The similarities between HAp and human enamel have spurred interest in use of HAp in the remediation of damaged enamel [16- 18]. The use of HAp as a filler in dental restoration presents advantages that include intrinsic radio-opaque response, enhanced polishing ability, improved wear performance and reduced expense, compared to materials commonly used in dental health care [19].
The aim of the present study was the delivery of HAp to dentine tubules using carboxymethyl cellulose (CMC) hydrogel. CMC is a natural biodegradable and biocompatible anionic polymer obtained from natural cellulose by chemical modification. CMC polymers are often used as thickeners and additives in the food and chemical industries [20,21]. CMC is enriched in hydroxyl groups that can be used to prepare hydrogels easily with fascinating and adjustable structures and properties to suit the required applications [22]. In one study, CMC hydrogel was used to deliver stannous fluoride to control dentinal hypersensitivity [23]. Presently, HAp-containing CMC hydrogels were characterized physically and chemically, and were tested for in vitro biocompatibility. As well, the HAp-amended hydrogels were applied to dentine samples as a means of occluding dentinal tubules.
2. EXPERIMENTAL PROCEDURES
2.1. Hydrogel Preparation
The ingredients utilized in the study are summarized in Table 1. Hydrogels were prepared using CMC mixed with glycerol in a beaker using a magnetic stirrer until homogeneous. As the mixing of glycerol/CMC was being conducted, a solution of distilled water and sorbitol was also stirred until homogeneous. The two solutions were combined and continuously mixed for 1 h.
CMC hydrogels were also prepared containing varying ratios of Hap as shown in Table 2. HAp powder was synthesized using microwave-hydrothermal method. Calcium nitrate tetrahydrate (Ca(NO3)2∙4H2O, 98.5%; SAMCHUN Chemicals, Seoul, South Korea) and ammonium phosphate ((NH4)2HPO4, 98.5%; SAMCHUN Chemicals) were used as the starting materials in this preparation. The materials were mixed with water separately and stirred until completely dissolved. The solutions were prepared with the reactants at a weight molar ratio of 1.17 - 1.77. The solutions were combined and stirred for 10 min. The resulting single solution was transferred to a closed-vessel microwave device constructed of perfluoralkoxy (PFA) Teflon and exposed to 1200 W of output power generated by a dual magnetron device using 2.45 GHz frequency microwave radiation. Three milliliters of deionized water was added to the resulting mixture, followed by a three-step procedure. The first step was a warming-up process generated at 250 W for 2 min. The second step was a main reaction process generated at 250 - 650
Table 1. Composition of CMC Hydrogel.
Table 2. Composition of the HAp-modified CMC hydrogel.
W for 4 min. The final step was a 20-min cooling process. After completion of HAp synthesis, both the elimination of residuals in products and the crystallization of powders were achieved by microwave heating at 250 W for 3 min. Samples containing 0%, 10%, 20% and 30% HAp were prepared and used in this experiment.
2.2. Physical and Chemical Analyses
The fabricated hydrogels were analyzed by attenuated reflectance Fourier transform infrared spectroscopy (FTIR) using a Spectrum GX apparatus (PerkinElmer, New Jersey, USA). The IR spectra of the samples were measured over a wavelength range of 4000 - 500 cm−1, and the spectra were collected from 64 scans with a resolution of 4 cm−1. The viscosity of the samples was measured using a Viscotester VT-04F viscometer (Rion, Tokyo, Japan). Three hundred milliliters of each sample was prepared and measured for a certain time range. The pH of the samples was measured using a PHS-3BW pH meter (Bante, Shanghai, China). Measurements were conducted five times for each sample. Dilutions (10%) of each sample were prepared using phosphate buffered saline (PBS) before measurement.
2.3. Cell Culture
M3CT3-E1 cells obtained from Korean Cell Line Bank were maintained following prescribed protocols. Upon confluence, cells were washed with PBS to remove metabolic wastes, media and non-adherent cells prior to detachment with Trypsin-EDTA for 5 minutes. Detached cells were then collected and centrifuged at 1500 rpm for 150 s and re-suspended with fresh minimum essential medium (MEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin antibiotics. Incubation conditions were 37˚C and 5% CO2. Medium was replaced every 2 days.
2.4. Cytotoxicity and Cell Growth Behavior
Determination of the cytotoxicity of the samples was done according to previously published protocols done in our laboratory with modifications in consideration of the consistency of the samples [24]. Samples were immersed in MEM in 1:3 ratio of sample weight to media volume and agitated at 50 rpm under humidified conditions at 37˚C for 24 h. Consequently, cells were seeded in wells of a 24-well plate, 103 cells/ml, and incubated under the aforementioned conditions for 24 h. After incubation, cells were treated with medium supplemented with extract solution in a 50:50 ratio for 1, 2 and 3 days.
Quantization of cell viability was done by a 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay. MTT substrate (5 mg/ml) were supplied to cells and incubated for 4 h. The converted formazan salts were dissolved with dimethysulfoxide and the absorbance was read at 595 nm using a microplate reader (Tecan, Mannedorf, Switzerland). The obtained optical densities were equated with the relative amount of viable cells that were able to convert the substrate.
Live/Dead® cell staining was done during the third day of incubation to visualize the cell growth under prolonged treatment with extract solutions of samples. Stained cells were viewed using a Fluoview 100 confocal laser scanning microscope (Olympus) using 473 nm and 559 nm fluorophore filters for Calcein AM and Ethidium homodimer-1, respectively. Viable cells were stained with Calcein AM and fluoresced green, while dead cells were stained with Ethidium homodimer-1 and fluoresced red.
2.5. Preparation of Dentine Samples
Caries-free and cleaned human molar teeth were obtained from healthy adults. The teeth samples were submerged in ethanol before being using to obtain dentine disks. The dentine discs were prepared with a thickness of 1.0 mm by sectioning with a RB 205 MetSaw Loaw High Speed Diamond Cutter (R & B, Daejeon, South Korea). Occlusal enamel was removed to expose the middle dentine. The sectioned discs were polished then etched with 6% citric acid for 2 minutes to completely open the dentine tubules. After etching, the samples were ultrasonicated to further expose the dentine occlusions and remove the residual smear layers.
2.6. Experimental Treatment
The dentine discs samples were randomly grouped into four groups with each group to be treated with each fabricated samples respectively. The samples were applied as dental gel in the same manner toothpaste is applied to teeth. Dentine specimens were brushed manually using an electric toothbrush operating at 20,000 rpm. Hydrogel (0.5 g) were applied every treatment and treatments were done three times a day (8 am, 2 pm and 8 pm). Every treatments lasts for 2 minutes at room temperature. After every treatment, the dentine discs were washed with deionized water to remove excess samples.
2.7. Scanning Electron Microscopy (SEM) Observation
The dentine tubules were observed using a JSM-635 scanning electron microscope (JEOL, Tokyo, Japan). The dentine discs samples were fixed in a sample holder and coated with platinum using a SPI-module Sputter coater operating at 7 mA. The observation was conducted before and after treatment with the hydrogel samples.
2.8. Statistical Analyses
The values reported in this study were expressed as the average of three replicates or repetitions unless stated otherwise. The resulting values were analyzed statisticcally using single factor and two-way ANOVA with posthoc correction (Tukey’s and Bonferroni method) using GraphPad Prism 5 with a confidence level of p ≤ 0.05 to determine statistical significance of each result.
3. RESULTS
3.1. Characteristics of the CMC Hydrogels
The different components of the hydrogel were mixed and formed a paste-like consistency. Figure 1(a) shows the experimental procedure done to prepare the hydrogel and the resulting hydrogels with and without HAp are depicted in Figures 1(b) and (c), respectively. Complete incorporation of different concentrations of HAp within the hydrogel mixture was shown in the FTIR spectra of the different samples (Figure 2).
All FTIR representative spectra were recorded in the region of 500 - 4000 cm−1. A broad band ranging from 3000 to 3600 was observed (Figure 2) and could be attributed to the stretch vibration of O-H group. The band at 2940 to 2960 was due to carbon-hydrogen bonding
Figure 1. Experimental procedure in the preparation of the hydrogel (a) and photograph of CMC hydrogel without HAp (b) and CMC hydrogel with Hap (c).
Figure 2. FT-IR spectra of the fabricated CMC hydrogels modified with varying concentrations of HAp.
(C-H) stretching vibration. The carboxyl, methyl and hydroxyl functional groups displayed a wavelength of 1640, 1410 and 1320 respectively. The band at 1460 is assigned to the
. An intense peak was evident at around 1030, consistent with
. More 
peaking were seen in the region of 1080, 960 and 870. The FTIR spectra confirmed the complete blending of the CMC hydrogels and offered a strong indication that HAp was successfully incorporated in the material [25,26].
The samples had the physical characteristics of toothpaste and could be injected out of a syringe or a tube. With the addition of HAp, the viscosity of the hydrogel changed and varied markedly. The addition of HAp increased the viscosity (Figure 3). After 60 min of testing, final viscosity measurements of 33dPa∙s for CHAp0, 105dPa∙s for CHAp10, 345dPa∙s for CHAp20 and 470 CHAp30 were recorded, showing an increasing rate of viscosity as the HAp concentration increased.
Figure 4 shows the pH measurement of the hydrogels formulated with the various HAp concentrations. Compared to the PBS control, the addition of HAp progresssively decreased the pH of the hydrogel. Results of the pH measurement were 7.19 ± 0.018 for CHAp0, 6.83 ± 0.019 for CHAp10, 6.63 ± 0.007 for CHAp20 and 6.43 ± 0.008 for Chap30 compared to the measured value of 7.25 for PBS.