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Spectral Absorption of Varnish

Dentin hypersensitivity (DH) is defined as a short, sharp, well-localized pain in response to thermal, tactile, osmotic, or chemical stimuli which cannot be attributed to any other form of dental pathology 1.It is better to regard as a symptom complex, rather than a disease .It occurs as a result of exposed dentin 2.The prevalence of dentin hypersensitivity is > 40% of adults worldwide3.The cervical area of teeth is the most affected site 4. Premolars are the most common teeth affected5.There are many causes for dentin sensitivity. Enamel loss and cementum denudation are the major contributing factors 1and they occur as a result of:attrition, abrasion, erosion, abfraction, gingival recession, bleaching, periodontal treatment.
Hypersensitive teeth have a larger number and wider diameter dentinal tubules (DTs) than those of nonsensitive teeth 6.According to Brannstrom’s hydrodynamic theory 7, the movement of fluid across the DTs stimulates the mechano-receptors in or near the pulp, the occlusion or decreasing the radius of the open DTs reduces dentin permeability and, proportionally, decreases the degree of DH 8.
Various desensitizing agents have been recommended to treat DH, but studies revealed that they are ineffective or not long lasting 9.An ideal desensitizing agent should be effective, not irritant to the pulp, relatively painless, being easy to apply and spread, rapid action, and permanently effective, and should not cause teeth discoloration 10.Dental varnish containing fluoride is an effective agent in the treatment of DH 11. Hansen found that >50% of hypersensitive teeth can be treated with sodium fluoride (NaF) varnish 12 by occluding the DTs through calcium fluoride crystal precipitation 13..
This held true if there is sufficient calcium in the saliva to react with fluoride, but the formation of calcium fluoride  could be limited by decreasing salivary calcium as in abnormal function of salivary glands14. For this reason, NaF varnish with tri-calcium phosphate (TCP), which provides both fluoride and calcium, may help to increase formation of calcium fluoride on tooth surfaces.
But unfortunately, this NaF varnish could be brushed away by the toothbrush, especially from central orifices of the DTs 15.So it is a relatively short term treatment 16.
Recently, the clinical use of lasers had increased. For the management of DH, the mechanism of action differ between lasers, for (low-level) power lasers, it act directly on the pulp nerve endings causing analgesia by inducing change in neural transmission, while for (high-level) power lasers, it provoke melting  with recrystallization in the dentine surface resulting in occlusion of DTs17.
Several studies reported that Nd: YAG, CO2 (high-level) are useful in the occlusion of DTs 18-21 . Diode laser was suggested to treat DH. Diode laser (low-level) can act directly by depressing neural transmission of stimuli. But long term success in clinical use is questionable 22, and in higher degrees of DH its efficacy seems poor.
Few studies have reported that (high-level) diode laser 810 and 980nm can seal DTs 23, and no study yet investigate the effect of (high-level)diode laser 940nm in the occlusion of DTs, like other types of (high-level) lasers.
Additionally, a combination of desensitizing agent with laser treatment showed some cumulative efficacy 152425, and favored the long-term treatment of the desensitizers than when they are used alone17.
Therefore, the aim of this study was to evaluate, under a scanning electron microscope (SEM), the efficacy of using diode laser (940nm) with or without 5 % NaF varnish with TCP in the occlusion of DTs, and the safe parameters to the dental pulp.
Hundred adult human upper premolar teeth of two roots extracted for orthodontic purpose had been used. Crowns with caries, restorations, or fractures were discarded. They were stored in 4°C distilled water containing 0.1% thymol to inhibit microbial growth until use.
Teeth apices were mounted in acrylic resin,  the cementum was removed by 70 times strokes as stated by Coldiron et al26using a periodontal curette, then teeth were immersed for one minute in 17% ethylenediaminetetraacetic acid (EDTA)  (dental products SA   Co,. Switzerland) solution for smear layer removal, then rinsed with distilled water in an ultrasonic bath for 15 minutes, and dried with gauze. An area of (3X4 mm), in the buccal surface of cervical third of the tooth, was marked to determine the lasing area. Twenty teeth were prepared, two per group, for SEM analysis. To assess temperature and thickness measurement, eighty premolars were used, ten per group. In the lingual surface, opposite to the lasing area, a hole was made by diamond fissure bur until reach the pulp cavity to insert a thermocouple.
For thickness assessment, the teeth were sectioned horizontally through the mid-length of the marked lasing area.
Samples were divided into ten groups, the control group which was received no treatment, NaF white varnish (Clinpro White Varnish, 3M ESPE, USA) alone group, 940 nm diode laser EPIC™ (BIOLASE, San Clement, CA, USA) without and with varnish at 0.8, 1.6, 2 and 3Wgroups (see table 1).
The used laser settings: continuous, noncontact mode 1 mm distance, exposure time 10 seconds , power densities 323.8 , 647, 809.7 ,1214.57 W/cm2 for the following output power: 0.8, 1.6, 2 , 3 W respectively, laser fiber tip diameter: 300 Mm which was held at 45 degree angle to the irradiated surface.
For the varnish groups, 5% NaF varnish with TCP was used, after thoroughly mixing, two thin layer were applied and allowed to set in the presence of saliva. The artificial saliva was prepared following the protocol used by Karlinsey et al27. After samples were lased, they were immersed in artificial saliva for 24 h to ensure complete treatment period according to manufacture instructions. Then surface area treated with varnish was brushed manually using soft brush (Sensodyne, Glaxo Smirth Kline co., UK) for 10 seconds to mimic patient teeth brushing. 
Temperature Measurement
Temperature assessment was done by immersion part of the root in water, which was heated by a waterbath (BS-11, Korea) for teeth temperature stabilization at 37 ± 0.5°C. A K type thermocouple was used, which was connected to a digital multilogger thermometer (Amprobe TMD-56, Everett, WA, USA), with an accuracy of ±0.05%. The temperature was recorded every second. A thermoconductor grease of 1W/MK thermal conductivity (Hutixi co., China) was injected inside the pulp chamber, to ensure maximal thermal conduction between thermocouple probe and the pulp cavity. Then a horizontal tooth sectioning through the lasing area was done to measure the dentin thickness by a Vernier caliper (TOPEX Sp. S.K., Warsaw, Poland).
SEM analysis
Samples fixation and dehydration, in an ascending ethanol series, were done following the protocol used by Marchesan et al 28. After dehydration, the specimens were left to dry for 24h, then fixed on aluminum stubs and metallized with a layer of gold, using vacuum evaporation. The samples were analyzed by SEM (Inspect S50, Czech Republic) and were observed under 2000x, 5000x magnification.
Spectral absorption of varnish
The absorption spectra of varnish (Clinpro White Varnish) was determined with a Spectrophotometer (Biotech co. Ltd, UK), calibrated in the spectral range from 900 to1000 nm. Two thin coating of varnish on a glass slide was analyzed, then we subtracted the resulted absorbance from glass slide absorbance to measure varnish absorbance alone.
1-Temperature and thickness measurement
The data obtained from temperature and thickness measurement from all the tested groups were statistically analyzed using SPSS Statistics 23 (IBM Corp., NY, USA).
For temperature assessment, descriptive statistics were done to find the means, standard deviations, maximum and minimum value. (Table 2)
The pulp temperature rise ranged from 0.4°C to 3°C and from 0.2°C to 2.8°C for the diode laser (940nm) groups without and with varnish respectively. The maximum recorded value was 3°C with group 2V+.To check if the obtained data are normally distributed, Shapiro-Wilk test was implemented, and the test statistics showed that data were normally distributed ( > 0.05)
To test if the groups were statistically different, Independent-Sample T test was used. The results displayed no significant different between laser without and with varnish groups for the same parameters. (See graph 1)
For thickness measurement, descriptive statistics were done (Table 3)
To investigate normal distribution of data, Shapiro-Wilk test revealed that data were normally distributed (P>0.05).
To examine if the groups were statistically different, One-way ANOVA test was used and the obtained descriptive level was (0.980), which revealed that the groups were not significantly different.
To find a correlation between temperature and thickness at each group, Pearson test was done. It showed high negative correlation (P<0.01), which revealed that pulp temperature elevation inversely proportional to remnant dentin thickness.
2- SEM Evaluation.
SEM analysis showed that control group presented open tubules and absence of the smear layer (figure 2 (a) (b)).
For the varnish alone group, only few tubules where occluded (figure 3(a) (b))
For laser alone groups, a narrowing of DTs was observed at 0.8W group (figure 3 (a) (b), and some of occluded tubule observed at 1.6W groups, (figure 5 (a) (b)).While for 2W group, a large number DTs were sealed without signs of cracking or char. (figure 6(a) (b)).At 3W group, laser irradiation provoked some darkened areas indicating dentin carbonization and destruction (figure 7(a) (b)).
Where as in combination groups, more number of DTs were occluded at (0.8, 1.6) WV+ group compared to laser alone groups (figure 8(a) (b), 9(a) (b). At 2WV+ almost optimum sealing of tubules were observed (figure 10(a) (b). At higher output power 3WV+, excessive melting with carbonization track were noticed (Figure 11 (a) (b)).
Absorption spectrum analysis
The absorption spectra of varnish showed that the absorbance at 940 nm was 0.808 (see fig.12).
DH is correlated to the number of exposed tubules on the root surface. The main aim of a successful treatment is the partial or complete occlusion of DTs 29. In this study we tried to assess the ability of diode laser 940nm laser alone or in combination with NaF varnish with TCP to reduce or close opened DTs, regarding effects on dentin morphology and pulp temperature elevation.
A laser wavelength of between 800 and 980nm has a poor absorption in water and hydroxyapatite30This low absorption prevails scattering, or diffused transmission of the laser radiation through the dentin reaching the pulp, and important thermal effects31.
The low absorbed energy by the dentin surface (via its mineral such as phosphate and carbonate) leads to heat accumulation, which gradually increases the surface temperature32.This results in melting and modification of organic matrix layer with an amorphous form and hence DTs sealing28
We utilized continuous mode to ensure scanning the whole dentin surface, noncontact mode to protect the optical fiber from contamination with varnish.
SEM analysis revealed that laser effect on melting grade of the dentin surface was intensified with high power densities, due to increased absorbed energy32. This reflect increased number of sealed DTs. Approximately optimum sealing of DTs occurred in 2WV+ laser with varnish group .But at 3W groups carbonization was noticed indicating irreversible destruction of dentin surface.
In the combination groups the number of occluded DTs more than the laser alone group, appeared to be due to melting and solidification of varnish after absorption of laser beam via its mineral contents, as investigated with absorption spectrum analysis. But it may provoke cracks and destruction at highest energy densities.
This melting and solidification of varnish by laser, reflect its resistance to be brushed away by manual tooth brush after 10s brushing that was done after the lasing, while it can be brushed away when was used alone 15.
We measured pulp temperature elevation to confirm that our parameters are within a 5.5°C pulp safety threshold as was established by Zach and Cohen33. The maximum recorded value was 3°C with an average dentin thickness of 1.897 mm, which was accepted with Krmek et al 34 study, that 3°C is the maximum threshold to not produce irreversible pulpal damage.
The higher absorption in the laser with varnish than laser alone groups may explain their lower pulp temperature rise. However the difference in temperature rise was not significant statistically.
At high power 3W a drop in pulp temperature was noticed, which could be due to carbonization and hence increased dentin surface absorption.
According to our findings, we suggested that NaF varnish with TCP combined with diode laser 940nm irradiation at (1.6, 2) W can be effectively and safely used in the treatment of DTs.
Our results confirmed that 940nm diode laser irradiation (1.6, 2) WV+, continuous mode, irradiation speed: about 1.2 mm/sec for 10 sec, laser fiber tip diameter: 300 Mm can lead to successfully and safely narrowing or sealing of DTs.
The application of NaF varnish with TCP improves the thermal effects of the diodes on the dentin surface, inversely at high power it increases surface destruction.
Further clinical studies need to be accessed in order to confirm these in vitro results before final conclusions can be drawn.

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