Diffusion of Hydroxyl Ions from Calcium Hydroxide and Aloe vera Pastes

different calcium hydroxide (CH) pastes containing Aloe vera. Sixty single-rooted bovine teeth were used. The tooth crowns were removed, the root canals were instrumented and the specimens were assigned to 4 groups (n=15) according to the intracanal medication: Group CH/S – CH powder and saline paste; Group CH/P – CH powder and propylene glycol paste; Group CH/A – calcium hydroxide powder and Aloe vera gel paste; Group CH/A/P – CH powder, Aloe vera powder and propylene glycol paste. After placement of the root canal dressings, the teeth were sealed coronally and apically with a two-step epoxy adhesive. The teeth were placed in identified flasks containing deionized water and stored in an oven with 100% humidity at 37 °C. After 3 h, 24 h, 72 h, 7 days, 15 days and 30 days, the deionized water in the flasks was collected and its pH was measured by a pH meter. The obtained data were subjected to statistical analysis at a significance level of 5%. The results demonstrated that all pastes provided diffusion of hydroxyl ions through the dentinal tubules. The combination of Aloe vera and CH (group CH/A) provided a constant release of calcium ions. Group CH/A/P showed the highest pH at 24 and 72 h. In conclusion, the experimental pastes containing Aloe vera were able to enable the diffusion of hydroxyl ions through the dentinal tubules. Diffusion of Hydroxyl Ions from Calcium Hydroxide and Aloe vera Pastes


Introduction
Calcium hydroxide (CH) is the standard material for root canal dressing, being widely used in Endodontics (1).This drug was introduced in Endodontics in 1920 by Hermann and has been used in different clinical situations since then (2).The effects of CH include antimicrobial activity (2)(3)(4)(5), mechanical blocking to avoid re-infection of the root canal (5,6), capacity to limit root resorption (7,8), induces formation of mineralized tissue (5,6) and promotes repair of periapical and adjacent tissues (2,4).
The antimicrobial activity and capacity to limit root resorption are related to the alkalinizing action of calcium hydroxide, which in turn is a consequence of its ionization into hydroxyl ions (7)(8)(9).Tronstad et al. (8) suggested that CH in the root canals promotes a pH increase on the external root surface, thus promoting alkalinization of resorption areas on the root surface, by diffusion of hydroxyl ions through the dentinal tubules.Other important characteristic of the dissociation of CH into hydroxyl ions is the ability to inactivate bacterial lipopolysaccharide (LPS), a toxin produced by Gram-negative bacteria commonly found in the root canal system (4,6).
When applied inside the root canal, CH must be able to diffuse through the apical foramen, ramifications, secondary and accessory canals, as well as through the dentinal tubules, reaching areas of root resorption, areas contaminated by microorganisms and adjacent tissues, to promote its action (4,8,10).
Aloe vera is a plant from the liliacea family commonly known as "babosa".It is a cactus with thorny lance-shaped green leaves and growing fast in warm and dry weather (18,19).After extraction of gel from its leaves, it has been therapeutically used in ancient and modern cultures throughout the world due to its great reputation as a healing and anti-inflammatory agent.Thus, it presents wide utilization in the health, food and cosmetic fields (18-20).Several studies with Aloe vera have demonstrated its positive qualities in relation to healing (18), antimicrobial activity (21), formation of mineralized barrier in pulp capping (22), antifungal activity and anti-inflammatory properties against lipoxygenase and COX-2 (23).Also, a study conducted on acemannan, a mucopolysaccharide extracted from the Aloe vera leaf gel, demonstrated that Ion diffusion from Aloe vera paste this substance plays a significant role in the healing process of oral wounds, by induction of fibroblast proliferation and stimulation of keratinocyte growth factor-1 (KGF-1), vascular endothelial growth factor (VEGF) and expression of type I collagen (24).
After confirming the antimicrobial capacity and biocompatibility of the Aloe vera gel, its use as vehicle for the CH paste may be considered.Therefore, it is of interest to determine the diffusion of hydroxyl ions of this paste through the dentinal tubules, which is fundamental for the action of a CH paste.
This study evaluated the diffusion of hydroxyl ions through the dentinal tubules from different CH pastes containing Aloe vera.The tested hypothesis is that the pastes containing Aloe vera diffuse through the dentinal tubules.

Ethical Aspects
The study was approved by the Institutional Review Board in Animal Studies -CEUA of Araçatuba School os Dentistry, UNESP (Process #02390-2011).

Experimental Design
The study was conducted on 60 extracted singlerooted bovine teeth, with single and straight root canals.The soft tissues, root cementum and calculi adhered to the root surface were removed with periodontal curettes, avoiding damage to the external surface.The crowns were transversally sectioned by a carborundum disc (Dentorium International, New York, NY, USA) in a low-speed handpiece, at approximately 12,000 rpm (KaVo Dental, Charlotte, NC, USA), irrigated with distilled water, standardizing the root length at 20 mm.Teeth that showed enlarged root canal (K file >#20) were discarded.
The total root canal length was measured by introduction of a size 30 K-file (Maillefer Instruments, Ballaigues, Switzerland) with a rubber stop.When the file tip crossed the apical foramen, the stop was placed on the cervical edge of the root and the length was recorded.The working length was established by subtracting 1 mm from the total root canal length.Roots with working length lower than 19 mm were discarded and replaced.
Biomechanical preparation was performed up to the predetermined limit and the apical end was prepared up to a size 80 K-file with stepback up to size 120 K-file.During instrumentation, the root canals were irrigated with 2.5% NaOCl at each change of file.
After biomechanical preparation, a size 30 K-file was introduced up to the total root canal length to clean the apical foramen.The root canals were dried with absorbent paper points (Tanariman Industrial Ltda, Manacapuru, AM, Brazil), filled with 17% EDTA for 3 min and then rinsed with saline and dried again with absorbent paper points.
The teeth were randomly divided in 4 groups (n=15) according to the root canal dressing pastes prepared by mixing CH powder with different vehicles: Group CH/S (control paste): 1 g CH plus 1.5 mL saline; Group CH/P (control paste): 1 g CH plus 2 mL propylene glycol; Group CH/A (experimental paste 1): 1 g CH plus 1 mL Aloe vera gel; Group CH/A/P (experimental paste 2): 1 g CH, 1 g Aloe vera powder and 2 mL propylene glycol.
The placement of the intracanal medicaments was performed by a manual lentulo spiral (Dentsply).After filling the root canals completely with the pastes, the root canal openings were sealed with temporary sealer (Coltosol; Vigodent SA Indústria e Comércio, Rio de Janeiro, RJ, Brazil).Thereafter, the apical foramen and openings (over the interim sealer) were sealed with a two-step epoxy adhesive.The teeth were placed in flasks containing 30 mL deionized water and kept in an oven at 37 ºC with 100% humidity.
After 3 h, 24 h, 72 h, 7 days, 15 days and 30 days, the pH values of solutions in the flasks were measured using a pH meter (Hanna Instruments Brasil, São Paulo, SP, Brazil), previously calibrated for each group, using standardized buffer solutions with pH 4.0 (acetic acid) and 7.0 (sodium acetate).For each measurement, the pH meter electrode was carefully rinsed with deionized water and dried with absorbent paper to eliminate residues that might interfere with the measurements.

Statistical Analysis
Analysis of variance was performed to test differences between experimental groups and the analyzed time periods.Tukey's post-hoc test was used to analyze significant results.Values of p<0.05 were considered statistically significant.

Results
Figure 1 presents the pH variation in relation to the time periods for the different pastes.The values indicate the means obtained for each period, considering the 15 measurements obtained for each group.The details of the means and standard deviation obtained may be seen in Table 1.
In Group CH/S, the highest pH value was observed at periods of 3 and 24 h, being lower at other periods (p<0.05).There was a significant decrease in pH between 24 and 72 h, yet after this period the pH values remained stable in relation to the value at 24 h, up to 30 days.
In Group CH/P, the highest pH value was observed in periods of 3 h, being lower in other periods (p<0.05).There was significant reduction in pH between 24 h and 15 days, yet at 30 days the pH value increased significantly in relation to the other periods, except for 3 h (p<0.05).
In Group CH/A, the highest pH values were observed in periods of 3 h, 15 days and 30 days (p<0.05).Significant ion release was observed at 3 h, which reduced up to 7 days and increased again at 15 days.
In Group CH/A/P, highest pH values were observed at 24 and 72 h (p<0.05).There was lower ion release at 3 h, which increased in periods of 24 and 72 h, reducing at 7 days and remaining stable thereafter up to 30 days.
Comparison of the study groups evidenced greater ion release at 3 h in Groups CH/S and CH/P (p<0.05).At 24 h, Groups CH/S and CH/A/P presented higher pH values, with significantly higher pH in Group CH/A/P compared to the other groups (p<0.05).At 72 h, Group CH/A/P still presented higher pH than the other groups.At 7 days, the highest pH was observed for Group CH/S, followed by Group CH/A/P.At 15 days, there was no difference in pH between Groups CH/S, CH/A and CH/A/P, with lowest pH values observed in Group CH/P.At 30 days, Group CH/S had a more expressive pH, without difference only with Group CH/A.

Discussion
The hypothesis was accepted since the results showed that the pastes containing Aloe vera may diffuse through the dentinal tubules.
Due to the wide use of CH as intracanal medication, several studies have addressed its diffusion in the dentinal tubules (3,13,17).The diffusion of CH through the apical foramen, ramifications and dentinal tubules is fundamental to eliminate the contamination, inhibit root resorption and induce apical and periapical repair (4,10,25).
Different vehicles have been added to CH powder for preparation of pastes (13,25).These associations aim to facilitate the use of CH, improve the antimicrobial capacity, enhance the ion diffusion through the dentinal tubules, aid in radiopacity, biocompatibility and texture of the material (2,10).In this context, the beneficial qualities of the Aloe vera (18,22) when associated with CH may increase the apical and periapical repair.However, other studies should be performed to obtain better understanding.
In the search for a better antimicrobial capacity of CH against resistant microorganisms, especially against E. faecalis, new vehicles have been investigated, including chlorhexidine (5,25) and propolis (17).
Similar to the studies of Mori et al. (25) and Montero and Mori (17), this investigation demonstrated that the saline and propylene glycol pastes diffused through the dentinal tubules, in which similar levels were observed in both groups, confirming indication of these substances as vehicles for CH paste.
Considering that the efficacy of these substances (saline and propylene glycol) has been demonstrated in the literature as vehicles for CH, they were used as reference groups for comparisons in this study.Thus, they showed an effective dissociation of hydroxyl ions in the first hours as described in literature (10,16,25).Furthermore, after 30 days, both pastes exhibited a similar diffusion of hydroxyl ions to the external root surface (17).
Because of its antimicrobial capacity (21) and beneficial qualities considering the need of repair (18,22), Aloe vera

Ion diffusion from Aloe vera paste
has been suggested in association with CH.Therefore, this study was conducted to investigate the influence of Aloe vera on the diffusion of hydroxyl ions through the dentinal tubules.Since Aloe vera is available both as powder and gel, it was interesting to analyze both presentations, thus two Aloe vera groups were constituted.In the stage of paste preparation, Group CH/S evidenced need of constant handling to enhance its insertion and paste texture after mixing, complicating its insertion in the root canal.Group CH/P presented easy mixing, viscous texture after mixing, and good insertion in the root canal.Group CH/A exhibited easy mixing, viscous texture after mixing and good insertion in the root canal.Group CH/A/P presented difficult mixing, sticky texture after mixing and good insertion in the root canal.The pastes in Groups CH/S, CH/P, CH/A presented white color and the paste in Group CH/A/P exhibited a dark brown color, which evidenced the need for more careful cleaning of the pulp chamber.All pastes were present in the root canals at 30 days after the study.The teeth in Group CH/A/P presented discoloration.
According to the present results, the experimental paste 1 (Group CH/A) diffused constantly through the dentinal tubules, reaching the external root surface.The experimental paste 2 (Group CH/A/P) presented the highest pH at 24 and 72 h, suggesting its use as a fast action medication.The pH increase of Group CH/A/P may be associated to the propylene glycol which is related to the greatest diffusion of hydroxyl ions in the first hours (17).However, the Group CH/P did not show these values, suggesting that Aloe vera facilitated diffusion through the dentinal tubules.Therefore, further studies addressing the biocompatibility and antimicrobial action of experimental pastes are indicated to determine their utilization.
In conclusion, based on the outcomes of this in vitro study, experimental pastes containing Aloe vera were able to enable the diffusion of hydroxyl ions through the dentinal tubules.The CH, Aloe vera and propylene glycol paste (Group CH/A/P) presented the highest pH at 24 and 72 h.After 30 days, all of the studied pastes presented similar diffusion of hydroxyl ions through the dentin.

Figure 1 .
Figure 1.Variation of mean pH values according to the study periods, for the different study groups.

Table 1 .
Means and standard deviations of pH variation with time in all groups d (0.184) Statistically significant difference (p<0.05):a,b; c,d; e,f.