Influence of Dental Exposure to Oral Environment on Smear Layer Removal and Collagen Exhibition after Using Different Conditioning Agents

Root surfaces affected by periodontal disease become hypermineralized (1) and contaminated by bacteria (2) and their toxins (3). Scaling and root planning (SRP) is unable to completely decontaminate root surface and results in production of a smear layer (4), which is formed by remnants of calculus, plaque and contaminated dental hard tissues (cement and dentin) (5,6). It might act as a barrier, preventing blood clot adhesion to the root surface (4) and periodontal regeneration (5). Root surface conditioning has been studied to overcome this problem. It is aimed at removing the smear layer and exposing the organic dental matrix rendering root surface more biocompatible, which may increase success in regenerative procedures (7). Citric acid (8,9), ethylenediaminetetraacetic acid (EDTA) (10-12) and tetracycline hydrochloride Influence of Dental Exposure to Oral Environment on Smear Layer Removal and Collagen Exhibition after Using Different Conditioning Agents


INTRODUCTION
Root surfaces affected by periodontal disease become hypermineralized (1) and contaminated by bacteria (2) and their toxins (3).Scaling and root planning (SRP) is unable to completely decontaminate root surface and results in production of a smear layer (4), which is formed by remnants of calculus, plaque and contaminated dental hard tissues (cement and dentin) (5,6).It might act as a barrier, preventing blood clot adhesion to the root surface (4) and periodontal regeneration (5).Root surface conditioning has been studied to overcome this problem.It is aimed at removing the smear layer and exposing the organic dental matrix rendering root surface more biocompatible, which may increase success in regenerative procedures (7).

Influence of Dental Exposure to Oral Environment on Smear Layer Removal and Collagen Exhibition
after Using Different Conditioning Agents (13) presented good results on root conditioning in vitro.Nevertheless, this has not been observed in vivo (14,15).This unexpected result may be explained by the fact that root conditioning is influenced by the type of conditioner, the method of SRP (manual or ultrasonic) (16), the duration of the conditioning procedure (17), the mode of application of the conditioner (18), the concentration and pH of the conditioner (11), and probably the degree of root surface mineralization.This last factor is influenced by the length of time that the root surface is exposed to the oral environment.The degree of dental mineralization influences the demineralization and decontamination produced by chemicals used for root surface modification.
The aim of the present study was to assess the capacity of 3 conditioners (24% EDTA, 1% citric acid, 25% citric acid and 25 mg/mL tetracycline hydrochloride) to remove smear layer and expose organic dental matrix

MATERIAL AND METHODS
This study was approved by the Research Ethics Committee of the Araraquara Dental School, UNESP -Univ.Estadual Paulista (CEP-FOAr #39/07).Seventy teeth were obtained from the undergraduate oral surgery clinic after patients signed an informed consent to use their extracted teeth.Thirty-five teeth had their root surface exposed to the oral environment.Inclusion criteria were used in order to standardize the conditions of these teeth as follows: 1. age of the patients from 35 to 45 years old; 2. root surface exposed due to periodontal breakdown and presenting calculus; 3. probing depth to the selected tooth ≥ 4 mm, with probing bleeding.The other 35 teeth were impacted teeth.The integrity of the cementoenamel junction (CEJ) was an inclusion factor for those teeth.After extraction, teeth were kept in saline (Merck, Darmstadt, Germany) to prevent dehydration and exposed and non-exposed teeth were maintained apart in different receptacles.

Specimen Preparation
Tooth segments were obtained from the mesial/ distal or buccal/lingual sides of the cervical third of the roots by making two parallel grooves with a #2135 cylindrical bur (KG Sorensen, Barueri, SP, Brazil) under copious irrigation.The first groove was positioned horizontally at the CEJ and the second groove was made parallel and 4 mm apical in relation to the first.Only tooth segments that were able to provide the flattest specimens were selected.The area between the two grooves was then scaled with 50 apico-cervical strokes using a sharp #5-6 Gracey curette (Hu-Friedy, Chicago, IL, USA).
Specimens were retrieved using a diamond disc (KG Sorensen), cutting off the crown at the first groove.Then a vertical section was performed to the second groove, producing two specimens of approximately 3x4 mm for each tooth segment.Specimens were stored in saline to prevent dehydration and those obtained from exposed and non-exposed teeth were maintained apart in different receptacles.

Treatment Groups
One hundred and forty specimens were obtained, being 70 specimens from teeth with radicular surfaces exposed to the oral cavity and 70 specimens from roots of impacted teeth.Specimens were subjected to different chemical treatments and each chemical treatment had its control group (treated with saline).Parameters used for each chemical treatment (concentration, duration and mode of application) were obtained from previous studies (10,13).Each group was formed by 10 specimens (n=10).Table 1 summarizes conditioning procedures in the experimental groups.

Specimen Preparation for SEM Analysis
After conditioning, specimens were subjected to a method that allows visualization of the collagen matrix in SEM micrographs (19): the 10 specimens of each group were allocated in codified cassettes.Then specimens were dehydrated by immersion in ethanol concentrations of 30, 50, 70, 80, 95 or 100% (1 h in each solution), followed by immersion in a solution of 0.75 mL of 100% ethanol and 0.75 mL of hexamethyldisilazane (HMDS; Sigma-Aldrich Inc., St. Louis, MO, USA) for 30 min and a final immersion in 1 mL of pure HMDS for 10 min.HMDS allows visualization of the collagen matrix in SEM micrographs.Finally, specimens were dried for 48 h in a dehydration jar (Corning, São Paulo, SP, Brazil), mounted on metallic stubs (Senai, São Paulo, SP, Brazil) for sputter coating with 99.99% pure gold.

SEM Analysis
Two SEM micrographs (×1500 and ×3500) from the center of each specimen were obtained with a scanning electron microscope (JEOL JSM-T330A; Jeol Ltd., Peabody, MA, USA).A previously trained blind examiner (12,13) analyzed the micrographs at three moments according to a root surface modification scoring system, as follows: Score 1: absence of smear layer, showing denuded cement, and/or dentin and/or collagen fibers from dental matrix -"clean" specimens (Fig. 1A); Score 2: partially open dentin tubules and no collagen fibers exposure (Fig. 1B); Score 3: presence of smear layer (Fig. 1C); Score 4: smear layer formed by chemical dissolution of dentin surface (over demineralization) (Fig. 1D).The examiner was trained during 3 weeks before performing the analysis.For calibration purposes, SEM micrographs representative of the different scores were selected and 3 evaluations were performed per week with a 48-h interval between each analysis. .The conditioning procedures assigned to groups IIA and IVA were the same, so there was an only experimental group (n=10) to provide results for these two groups.The same is valid for groups IIB and IVB.observed in the 50 mg/mL tetracycline hydrochloride and 1% citric acid groups, while lower frequencies of this score was observed for the same conditioners when applied to teeth exposed to oral environment.The totality of specimens from impacted teeth treated with 24% EDTA and 25% citric acid presented with score 4. Similarly, a high frequency of score 4 was observed when these 2 conditioners were applied to specimens from teeth exposed to oral environment.

DISCUSSION
Root surface conditioning studies present great variability in their results due to many factors, including the degree of root surface mineralization.The duration of root surface exposure to the oral environment (DES-RE process, dental plaque) implies the degree of mineralization (1), which might influence root surface modification.A hypermineralized root surface is harder to demineralize and decontaminate than a less mineralized one.
Figure 2, which presents results of specimens obtained from teeth exposed to oral environment, shows a poor capacity of 50 mg/mL tetracycline hydrochloride and 1% citric acid to remove smear layer (low frequency of score 2).This was expected since these agents present a lower demineralizing capacity (6,9).This finding disagree with the results of a previous study (13), which

Statistical Analysis
Statistical analyses were performed using GraphPad Prism software version 5.05 (Graph Pad Instat, San Diego, CA, USA).A non-parametric analysis of variance (Mann-Whitney test) was applied to compare groups using a p≤0.05.

RESULTS
Results from specimens retrieved from teeth exposed to oral environment are presented in Figure 2. Control groups always received score 3. Score 1 was assigned only to 24% EDTA and 25% citric acid groups.Score 2 was the highest score attributed to the 50 mg/mL tetracycline hydrochloride and 1% citric acid groups.Higher frequencies of score 4 were observed in the 24% EDTA and 25% citric acid groups.
Results from specimens retrieved from impacted teeth are presented in Figure 3.Control groups always received score 3, except in the 25% citric acid control group.Score 2 was the highest score attributed to the 50 mg/mL tetracycline hydrochloride and 1% citric acid groups.Score 4 were assigned to 100% of the specimens belonging to the 24% EDTA and 25% citric acid groups.
Figure 4 shows frequency of scores of test groups in impacted teeth and in teeth exposed to oral environment.The highest frequency of score 2 in impacted teeth was  showed good demineralizing capacity to 50 mg/mL tetracycline hydrochloride, which could be attributed to the fact that this study did not control the root surface exposure to the oral environment factor.
The demineralizing action of citric acid has been shown to depend on its concentration (8) and high concentrations of citric acid (9) or EDTA (12) should be avoided since they produce smear layer as a result of over-demineralization, i.e., complete dissolution of the tooth surface.
The high frequency of score 4 in the 24% EDTA gel group has not been shown before (12).This can be attributed to the fact that in the previous study (12) specimens were washed with 10 mL of saline between each 1-min gel application, which may wipe high surface tension gel (10) off the dental surface, reducing hypermineralization occurrence.The results of the present study suggest that while 24% EDTA and 25% citric acid may hyperdemineralize dental surface (producing a "chemical dissolution" smear layer), 50 mg/ mL tetracycline hydrochloride and 1% citric acid have no efficacy in demineralizing supposedly hypermineralized dental surfaces.
Figure 3 shows results obtained in impacted teeth.As expected, 24% EDTA and 25% citric acid caused hyper-demineralization of dental surface.The 50 mg/mL tetracycline hydrochloride and 1% citric acid produced root demineralization and difference between these 2 groups and its controls was statically significant.The higher frequency of scores 1 and 2 in the 50 mg/mL tetracycline hydrochloride group corroborated previous results (13).The high frequency of score 2 in the 1% citric acid group may be explained by the lower degree of dental surface mineralization found in impacted teeth.It can be inferred that less mineralized dental surfaces from impacted teeth are more easily demineralized than hypermineralized dental surfaces from oral environment exposed root surfaces.
Finally, Figure 4 compares the test specimens.It can be seen that the degree of root surface mineralization does not affect results obtained with 24% EDTA and 25% citric acid, probably due to the high demineralizing potential of these agents which produced high frequency of specimens with hypermineralization.Comparing results obtained with 50 mg/mL tetracycline hydrochloride and 1% citric acid, it can be observed a tendency of specimens from impacted teeth to present higher degree of smear layer removal in relation to those from oral environment exposed root surfaces.The degree of mineralization may be responsible for this occurrence, suggesting that less mineralized dental surfaces from impacted teeth can be more easily demineralized than hypermineralized ones retrieved from root surfaces exposed to oral environment.
Under the tested conditions and considering the results obtained, it may be concluded that: 1. 24% EDTA and 25% citric acid produced root surface hyper-demineralization; 2. 50 mg/mL tetracycline hydrochloride and 1% citric acid showed greater smear layer removal capacity than the other two conditioners; 3.Although specimens from impacted teeth showed a tendency to have smear layer more easily removed, no significant difference was found in comparison with specimens obtained from teeth exposed to the oral environment.

Figure 1 .
Figure 1.Root surface modification scores.A (Score 1) = no smear layer, denuded cement and/or dentin and/or collagen fibers from dental matrix -"clean" specimens; B (Score 2) = partially open dentin tubules and no exposed collagen fibers; C (Score 3)= presence of smear layer; D (Score 4) = smear layer formed by chemical dissolution of dentin surface (over demineralization).

Figure 2 .
Figure 2. Frequency distribution of scores among specimens from teeth exposed to oral environment according to the treatment received.

Figure 3 .
Figure 3. Frequency distribution of scores among specimens from impacted teeth according to the treatment received.* Statistically significant different results according to Wilcoxon test (p<0.05).

Figure 4 .
Figure 4. Frequency distribution of scores among specimens from impacted teeth and from teeth exposed to oral environment according to the treatment received.

Table 1 .
Conditioning procedures in the experimental groups.