Molecular and morphological surface analysis: effect of filling pastes and cleaning agents on root dentin

Abstract The quality of the dentin root is the most important factor for restoration resin sealing and drives the outcome of endodontic treatment. Objective This study evaluated the effect of different filling pastes and cleaning agents on the root dentin of primary teeth using Fourier-transformed Raman spectroscopy (FT-Raman), micro energy-dispersive X-ray fluorescence (µ-EDXRF) and scanning electron microscopic (SEM) analysis. Material and Methods Eighty roots of primary teeth were endodontically prepared and distributed into 4 groups and filled according to the following filling pastes: Control-no filling (CP), Calen®+zinc oxide (CZ), Calcipex II® (CII), Vitapex® (V). After seven days, filling paste groups were distributed to 4 subgroups according to cleaning agents (n=5): Control-no cleaning (C), Ethanol (E), Tergenform® (T), 35% Phosphoric acid (PA). Then, the roots were sectioned and the dentin root sections were internally evaluated by FT-Raman, µ-EDXRF and SEM. Data was submitted to two-way ANOVA and Tukey tests (α=0.05). Results Regarding filling pastes, there was no significant difference in organic content. CP provided the lowest calcium values and, calcium/phosphoric ratio (Ca/P), and the highest phosphoric values. For cleaning agents there was no difference in organic content when compared to the C; however, T showed significantly higher calcium and Ca/P than PA. All groups showed similar results for phosphorus. The dentin smear layer was present after use of the cleaning agents, except PA. Conclusion The filling pastes changed the inorganic content, however they did not change the organic content. Cleaning agents did not alter the inorganic and organic content. PA cleaned and opened dentin tubules.


Introduction
After root canal treatment of primary teeth, the tooth can be restored with an adhesive material, which 22 . However, the The composition of the materials, such as oily excipient and calcium hydroxide, would act as contaminants and decrease bonding strength in dentin 9,12 . In addition, polymerized in clean dentin surface, for a successful retention of adhesive restoration 15 . Thus, in order to obtain satisfactory restoration longevity, the dentin surface has to be free of debris and with ideal features to allow for an adequate hybridization of the resin monomers and dentin 5 and the restoration sealing are the most important factors for the endodontic treatment's outcome 6 .
on severely damaged primary anterior teeth need to Calcium hydroxide's effect on the physical and sealing properties of canal sealers was investigated 10 .
It was observed that Calcipex ® paste, composed of calcium hydroxide, barium sulfate and distilled water was easy to handle, and also easily removed 10 . In addition, Vitapex ® , a silicon oil-based, composed of calcium hydroxide, iodoform and silicone paste after cleaning the root surface. Therefore, it is supposed that Vitapex ® and Calcipex ® when used as removed depending on the excipient contained.
In order to clean and prepare the root canal surface for dentin bonding, cleaning agents can be used to anionic detergent solution (sodium diethylene glycol lauryl ether sulfate) can be used to clean the dentin liquids and suspension of the debris that results from root preparation. In addition, it could also 14 . Ethanol is another cleaning agent that can be used to remove some oil is an organic solvent 26 . The use of ethanol on dentin promotes drying, inducing a hydrophobic dentin of resin monomers to wet dentin, increasing resin retention 23 .
Phosphoric acid used as an etching agent is used in the substrate surface during adhesive procedures, it can also Benetello considered as a surface cleaning agent that can clean dentin, since it removes the peritubular dentin and exposes the collagen matrix without changing the intrinsic characteristics of the inorganic dentin content 2,3 .
of the canal preparation and cleaning procedures. It modify the dentin surface 18 and can jeopardize the bonding strength of adhesive restorative materials, compromising the endodontic treatment's success.
agents, the evaluation of organic and inorganic content of dentin is an important factor for observing alterations in the dentin and could be helpful for selecting the best cleaning agent to be used in filled roots, without reducing adhesion. The calcium hydroxide-based pastes can alter root dentin properties such as hardness and modulus of elasticity 29 . In addition, depending on size and shape, the calcium hydroxide particles can penetrate into the dentin tubules 11 , occluding them and be detrimental to adhesion. On the other hand, cleaning agents, such of dentin 20 . Therefore, anionic detergent solution removed the smear layer and opened the dentin tubule 17 ; its use combined with phosphoric acid demineralizes the dentin, which could modify both the organic and the inorganic content 16 . Therefore, the dentin structure and composition can be evaluated through sensitive analysis of the dentin's chemical aspects to determine inorganic transformed Raman spectroscopy (FT-Raman) and micro energy-dispersive X-ray fluorescence  [1][2][3]13 . Also, the scanning electron microscopic (SEM) can be used to observe details, post-treatment, of the root canal dentin surface of primary teeth.
Thus, the aim of this in vitro study was to verify agents on primary teeth root dentin. The hypothesis with different cleaning agents, affect the molecular and surface features of the root canal dentin surface of primary teeth.

Material and methods
This study was conducted after receiving approval from The Ethics Committee in Research of the Dental School.

Sample selection
Eighty extracted, human anterior primary teeth were cleaned with saline solution to remove any remaining debris and soft tissue. The samples were frozen and stored for no more than 6 months until analysis. The selection criteria included those with at least two thirds of the root length and with no prior exposure to endodontic therapy.

Specimen preparation
The crowns were sectioned at the cementoenamel junction (CEJ) using a high-speed diamond saw with water-cooling and were subsequently discarded. Then, the roots were sectioned longitudinally, and only one slice from each root was selected, 80 specimens were obtained. The specimens were selected under a stereo structural alterations were excluded. Thereafter, the 320-grit SiC paper, and sonicated for 60 seconds with deionized water to remove the residues from the lumen of the root canal of primary teeth. seconds. All specimens were dried with paper points.
After that, they were stored at a relative humidity of 100% at 37°C until the molecular and morphological analyses were conducted ( Figure 1B). The application times of E and T were established in a pilot study. All materials used (including manufacturer details) are described in Figure 2.

Molecular analysis
The cervical third of each lumen of the root canal was analyzed by Fourier-transformed Raman spectroscopy (FT-Raman) and μ-EDXRF spectroscopy in order to evaluate changes in the dentin's organic and inorganic components, respectively. Both analytical methods were nondestructive and the data collection was based on laser or X-ray interactions with the sample, without contact.  The samples were positioned in the sample holder compartment and an IR352 lens collected radiation scattered through 90° on the dentin surface. For each sample, one spectrum was collected at a central point on the cervical dentin root. In order to obtain a good signal to noise ratio, 100 scans were co-added for each spectra. Five spectra were obtained in each group.

FT-Raman spectroscopic analysis
The changes in the organic dentin components were analyzed by comparing the integrated areas  The equipment calibration and the chemical balance were performed as previously reported 25 .
analyzed by μ-EDXRF using three points.

Scanning electron microscopic (SEM) analysis
The specimens were dried and mounted on a holder using double-sided adhesive carbon tape.   (Table 1) paste (Table 1), μ-EDXRF analysis showed that the Ca content and Ca/P ratio of the control group (no Regarding cleaning agents (Table 2), concerning organic content than the ethanol group (p<0.05). In

μ-EDXRF, T showed higher values for Ca content and
Ca/P ratio than the phosphoric acid group. All groups were similar for P content with regards to the μ-EDXRF analysis.
pastes contained calcium in their composition. The CZ titanium and sulfur, but had a higher percentage of in higher proportions than calcium.
Morphological analysis of the root dentin of primary paste amount and smear layers upon the dentin surface for all cleaning agent groups, except when pastes on the dentin surface ( Figure 4)  Discussion pastes associated with different cleaning agents affect the molecular and surface features of the root canal dentin surface of primary teeth was not accepted.
Regarding the FT-Raman and μ-EDXRF, there was no and cleaning agents).
content regarding Ca, P and Ca/P for the μ-EDXRF 1). For Ca and Ca/P, the values (%w) were higher for organic content of dentin when FT-Raman analysis was conducted ( Table 1). The increased Ca values could calcium-based (Table 3) and they remained on dentin surface associated with the smear layer. However, based on the microanalysis of chemical containing pastes, it was noticed that the amount of Ca in the dentin, since CII presented the highest Ca (Table 3) value and it did not provide a higher Ca %w (Table   1) available on the dentin when compared with the phosphate analysis. Similar results were described by Rythén, et al. 21 (2010), in which exfoliated primary teeth showed higher Ca than P on the dentin, the study was performed using X-ray microanalyses.
the organic content (FT-Raman) when the cleaning agents were compared to the control (no cleaning agent), however, the root dentin of primary teeth treated with T showed higher amounts of collagen than those treated with E ( Table 2)    Representative SEM images (Figure 4)  an adequate surface for bonding, as it is commonly dentin 15 . The results obtained from this study are promising, since it does not use up any cleansing agent, but uses two-step etching and adhesive rinsing, the adhesion of restorative materials is feasible, since it is less time consuming. In addition, the use of a self-etching system can be used with E or T or PA, which should be the best choice to cleaning the root paste, since, they did not change the inorganic or organic content and also allowed for a smear layer on the dentin surface.

Conclusion
Based on our findings, regardless of filling pastes, all cleaning agents, in spite of providing some disturbance on the dentin surface, cannot be considered as harmful for cleaning the root dentin.
alter the organic content of primary teeth root dentin.