Effect of fluoride, chlorhexidine or Nd:YAG on the progression of root dentin demineralization after removal of the demineralized organic matrix

Abstract Quantification of collagen degradation is an important parameter to evaluate dentin caries for preventive aid. Objectives: Evaluate preventive methods against root collagen degradation by the hydroxyproline assay (HYP) and microradiography technique (MRT). Methodology: Five bovine root dentin blocks were obtained and subjected to an artificial demineralization process by acetate buffer (pH 5) to induce carious lesion formation. Samples were subjected to the following therapeutic treatments: 1) 0.12% chlorhexidine for 1 min, 2) 2% fluoride for 1 min, 3) Nd:YAG Laser (400 μm diameter optical fiber, 10 Hz frequency, 60 mJ/pulse energy, 48 J/cm2 energy density, in noncontact mode for 10 s), 4) deionized water (control) for 1 min, 5) MRT control group (without treatment and removal of collagen). Samples were exposed to degradation by a collagenase enzyme for five days. The enzyme solution was collected, by colorimetry in a spectrophotometer, from the collagen matrix for the hydroxyproline release analysis. The same samples were subjected to an additional two days of demineralization to induce the progression of mineral loss. Samples were analyzed by MRT for the visualization of their degraded areas (estimation of lesion depth and mineral loss). ANOVA was applied to compare hydroxyproline release rates. MRT data were subjected to the Kruskal-Wallis test, followed by the Dunn’s test. Comparisons between the initial five-day and the subsequent two-day demineralization processes were performed by repeated t-test or Wilcoxon (p<0.05) measurements. Results: The amount of HYP released from the dentin samples failed to show significant differences among the groups (p=0.09). Fluoride and chlorhexidine were able to interact with the samples, reducing the progression of dentin caries after removal of the demineralized organic matrix. CHX was the only treatment able to show significant lower lesion depth than the negative control. Conclusion: Chlorhexidine and fluoride were effective in reducing root caries progression.


Introduction
As advances in dentistry have led to significant improvements to the population's oral health, people not only live but also retain their own teeth longer. 1 This has been inevitably associated with the frequent detection of root exposure due to gingival recession and, consequently, the development of root carious lesions affecting older adults. 2 Preventing these situations saves time and money and benefits individuals' quality of life.
Root carious lesions develop as a consequence of mineral loss associated with collagenolytic degradation. 3 Once demineralized, the superficial dentin organic matrix is exposed, becoming susceptible to enzymatic degradation. 4 The degradation of the demineralized organic matrix (DOM) can increase the progression of dentin carious lesions. 5 This process occurs by the activity of host enzymes, such as matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs), present in saliva and dentin. Such enzymes are activated in acidic pH and degrade the exposed collagen, as with CCs and MMPs in acidic and neutralized pH, respectively. 3 Theoretically, if the collagen fibril scaffold of the DOM is preserved and appropriate mineral supplementation is provided, dentin remineralization may occur. Therefore, inhibiting its degradation may be of interest to avoid the progression of dentin caries. 6 Different agents (i.e., fluoride, chlorhexidine, and laser) have served as prevention by reducing mineral loss and/or DOM degradation by physical and chemical changes on its substrate. 5,7 Fluoride (F) plays an important role in the control of root carious lesions by reducing caries progression rates and inducing the arrest of active lesions. 8 Professional fluoride products, applied on cleaned surfaces, allow the precipitation of globule-like CaF 2 on the tooth surface, which acts as a mechanical barrier and F reservoir to interact with teeth during demineralization-remineralization processes. 9 Chlorhexidine (CHX) is known to have antimicrobial effect and to protect the DOM against degradation 5 with the potential to control root dentin caries. 10 The literature has reported that CHX reduces the self-degradation of collagen fibrils by inhibiting host-derived protease activity (MMPs and CCs) in demineralized dentin. 7,[11][12][13] High power laser irradiation can also inhibit demineralization, if applied under specific parameters such as wavelength, density, pulse width, and repetition rate. Studies have found that Nd:YAG laser (1064 nm) enhances the acid-resistance of dental hard tissues by modifying their chemical and physical structure. 14 This modification includes melting, carbonate reduction, and α-or β-tricalcium phosphate and tetracalcium phosphate formation, which are less soluble than hydroxyapatite. 14 It also changes the bands attributed to the collagen matrix. 15 However, even though Nd:YAG has been tested for caries control, controversial results have been reported on its effects on dentin. 16 Furthermore, concerns about heating, photoabsorber presence, and correct laser parameters are still under investigation. 14,17,18 Considering the three possible mechanisms of action (reducing mineral loss, preventing DOM degradation, and/or promoting physical and chemical changes on the substrate), this study aimed to investigate the effect of 2% NaF (professional application), 0.12% CHX, and Nd:YAG laser irradiation (60 mJ) on the progression of dentin demineralization when the DOM is subjected to enzymatic degradation. We performed a hydroxyproline assay (HYP) to analyze DOM degradation (HYP), and transverse microradiography (MRT), to measure lesion progression. Our null hypotheses were: 1) our treatments fail to reduce or avoid DOM degradation (measured by HYP assay) when compared to a negative control, and 2) our treatments fail to reduce dentin mineral loss progression (measured by MRT) when compared to a negative control.

Sample preparation
This study was approved by the local Institutional Review Board (protocol number 06/2017). In total, 50 bovine incisors were selected, cleaned, and immersed in deionized water until use. Tooth crowns were sectioned 2 mm from the cementoenamel junction with a diamond wheel (Dremel, Campinas, SP, Brazil). The roots were placed in a universal cutting machine with a trephine diamond bur to obtain round dentin samples measuring 6 mm in diameter and 1.5 mm in thickness.
Samples were polished in a polishing machine (DP-10 Panambra, SP, Brazil) using 120, 600, and 1200 grit silicon carbide (SiC) papers (Extec. Corp. Erios, São were applied on the samples. Sample surfaces were divided into five parts, two of which were covered with nail varnish to act as a control area ( Figure 1[1]).
Dentin samples were then subjected to a demineralization solution to create an incipient lesion, according to the protocol reported by Islam,et al. 19 (2012). Samples were demineralized by an acetate buffer (0.1 mol/L; pH 5.0, v = 30 ml/sample) for five days at 37°C.

Treatments
After demineralization, one coat of blue nail varnish was applied to another 1/5 of the sample surface to protect the demineralized area ( Figure 1 Effect of fluoride, chlorhexidine or Nd:YAG on the progression of root dentin demineralization after removal of the demineralized organic matrix 1 [4]. An MRT control group, only demineralized for five plus two days, without treatment and DOM removal, was included for comparisons.
All dentin samples were washed for 5 min in deionized water, sectioned transversally by low-speed diamond discs, and polished to obtain slices with 100-120 µm. These dentin slices were fixed into a sample-holder together with an aluminum calibration step wedge with 14 steps.

Hydroxyproline (HYP) data
The amount of HYP released from the dentin samples failed to be significantly different among the groups, as Figure 2 shows (ANOVA, p=0.09).

MRT data
We found that five-day demineralization produced a lesion with a mean 2485 (±699.6) %vol.μm mineral loss and a 114 (±42) μm lesion depth. Removal of the demineralized organic matrix failed to increase demineralization regardless of the treatment, which we expected since the protocol can only remove the unprotected collagen matrix rather than its mineral content.
Even though we found no differences among the treatments with respect to mineral loss after the 2 nd demineralization, further two-day demineralizing challenges induced the progression of mineral loss in dentin samples in the negative control and laser groups, whereas, mineral loss failed to progress in the other groups. This result shows that fluoride and chlorhexidine were able to interact with the teeth, reducing the progression of dentin demineralization after DOM removal (Table 1 and Figure 3). # sound area, * lost area, DOM -demineralized organic matrix. There is a mineralized area (radio space layer) below the DOM, followed by the front of demineralization (radiolucent layer)  The second treatment elected for this study was chlorhexidine (CHX), considered the gold standard treatment to inhibit host proteases. 31 According to Baca, et al. 32 (2012), the effectiveness of CHX is related to concentration and frequency of application; the higher the concentration, the better the effect.

Fluoride
For this reason, we sought a balance between concentration and frequency in this study to avoid side effects such as tooth staining or taste impairment. 33 It has been shown that generalized tooth staining can occur if CHX is used in high concentrations and for a prolonged period. 34  The last treatment used in this study was the Nd:YAG laser, due to its ability to fuse and resolidify the substrate, acting on its organic and inorganic portions. 14,15,17 In this treatment, hydroxyapatite is transformed into tricalcium B-phosphate, increasing the acid resistance of the tissue. This fact is relevant because, as reported in the literature, exposed dentine is more susceptible to cavities than enamel, and laser can increase the relative mineral content of the dentin. 25 Thus, to obtain the desired effects via laser, the correct selection of parameters is fundamental. 36 Energy density, power, frequency, and contact or not of the optical fiber with the substrate are certainly responsible for the great variability of results found in the literature. Therefore, the parameters selected for this study were 60 mJ of energy, 48 J/cm 2 of energy density, and 10 Hz of frequency, 100 μs in non-contact mode due to thermal melting and resolidification processes shown by laser irradiation in dentin. 37 Nd:YAG wavelengths show reduced absorption by dental hard tissues and promote local, controlled temperature rises (less than 5.5ºC when 60 mJ is applied for 1 min in non-contact mode), leading to morphological and compositional changes in this substrate. 15,20 Specimen storage in a collagenase solution provided collagenolytic activity after demineralization of the root dentin, also previously applied by Kato, et al. 7 (2012) and Islam, et al. 6 (2016). This methodology simulates the degradation of the carious process, in which, after demineralization in acid pH, the organic matrix is exposed to hydrolysis, accelerated by the enzymatic activity of host MMPs, cysteines, and cathepsins. 5 Collagenase can promote "in vitro" degradation, releasing essential amino acids, which constitute collagen, in the storage solution. This release can be detected by colorimetric methods.
Among these amino acids is hydroxyproline, which was analyzed in this study according to the literature. 6,7 It is known that 90% of the dry mass of the DOM consists of type I collagen, which contains around 10% of HYP in its mass, whereas other proteins contain little or none of this amino acid. 38 In our results, the assessed treatments were unable to reduce collagen degradation since they failed to decrease the amount of HYP released from the dentin samples, as Figure 2 shows. Thus, we accepted the 1 st null hypothesis since our treatments failed to reduce DOM degradation, as measured by the HYP assay. Reddy and Enwemeka 39 (1996)  Presumably, the amount of exposed DOM was higher in the case of erosion than those induced by cariogenic challenges, such as in this study, which may justify the differences. Based on these observations, we suggest that future studies should verify the influence of the demineralization degree and the thickness of the remaining DOM on the results of HYP release.
Our results are in agreement with Walter, et al. 41 (2008), who also used the HYP method to evaluate collagen degradation in root dentin after treatments to promote the stability of the collagen matrix and prevent root cavities. Thus, it is important to stress that other forms of evaluation should be used to complement the analysis of how caries lesion progresses in dentin. Effect of fluoride, chlorhexidine or Nd:YAG on the progression of root dentin demineralization after removal of the demineralized organic matrix To calculate mineral loss and lesion progression between both demineralization challenges, we applied MRT since it is considered the gold-standard method to quantify the degree of demineralization and show the profile of the lesion. 25 Even though we found no differences among the treatments, the groups that received the one-minute topical application of CHX (0.12%) or F (9000 ppm) showed a lower demineralization progress. Furthermore, CHX also reduced lesion depth progression after two additional days of demineralization. Therefore, we reject our 2 nd null hypothesis. Based on our results, CHX was unable to decrease protease activity by HYP assay, as shown in previous works. 5 However, it was able to reduce demineralization progression, which may be due to its precipitation on the surface, as shown previously. 12 As it occurs for F (by CaF 2 precipitates), CHX might also have some physical effect by occluding tubules of the dentin surface and protecting the tissue against further demineralization.
Even though this study was unable to detect low HYP release by chlorhexidine, the inhibitory effect of chlorhexidine on MMPs is attributed to a chelating mechanism since the inhibition of MMP-2 and MMP-9 could be prevented by the addition of calcium chloride binding chlorhexidine. It was also discussed how chlorhexidine might affect essential sulfhydryl groups and/or cysteine present in the active site of MMPs. At salivary concentrations above 0.2%, the inhibitory action of chlorhexidine might also relate to protein denaturation. 25 Fluoride, on the other hand, proved to be effective in reducing mineral loss, but failed to affect lesion depth (Tables 1 and 2). These results may relate to the fact that fluoride has a higher reaction on lesion surfaces, improving mineral content, even though it failed to penetrate sufficiently deep to impair or reduce acid penetration and lesion progression at its deeper portions. 29 We observed that, regardless of the treatment, DOM removal failed to increase demineralization since the proposed protocol can remove unprotected collagen, but not its mineral content. 25 However, when we compared results within the same group, we found no significant progression in lesion depth, except for the negative control without DOM compared to the control with DOM, showing that the presence of the DOM may decrease the depth of the acid penetration.
On the other hand, CHX was the only treatment able to decrease lesion depth progression, which may be due to surface precipitation, as discussed above.
Despite the benefits of applying laser to promote morphological and/or chemical changes to the dentin surface, 14 the induction of cracks and macroscopic voids may impair its action against demineralization, which justifies its lack of a protective effect in this study. Laser parameters are the key to obtaining good results with this technique. The literature shows huge differences between the protocols of laser application, making direct comparisons impossible. 14,20 Thereby, studies should evaluate different parameters and/or the association of laser and fluoride or a photoabsorber to better understand the real contribution of this technology to the field. Future research should also conduct SEM images and chemical analyses to provide more information about the process. Therefore, the field needs further studies to find the most adequate treatment, concentrations, treatment associations, and application times under models which simulate caries development under biofilm growth. Research should also explore the decreased lesion depth progression we observed in the CHX group and confirm our results under models closer to in vivo conditions.

Conclusions
No treatment was able to reduce DOM degradation by HYP release. However, F and CHX reduced mineral loss progression, which is very promising when considering their clinical indication.