In Vitro Effect of Grape Seed Extract as an Intracanal Medication on the Mechanical Properties of DentinABSTRACT
Objective: To evaluate the mechanical properties of dentin under the effect of grape seed extract (GSE) as intracanal medication (ICM).
Material and Methods: Ninety-six single-rooted bovine teeth were divided into groups, as follows: control group (Ctrl), without any dentin treatment; groups treated with Ca(OH)2 (Ca7, Ca15 and Ca30); and GSE (GSE7, GSE15 and GSE30), with direct contact between dentin and ICM for different durations of 7, 15 and 30 days. The samples underwent tests of maximum fracture load (FL), flexural strength (σf), cohesive strength (σc), and elastic modulus (E) of the remaining root. For statistical analysis, one-way ANOVA was used, followed by Dunnett's test to verify whether ICMs interfere with dentin properties. Two-way ANOVA (ICM and application time) and Tukey's test were also used, considering the groups treated with different ICM at different intervals. All analyses were performed at p ≤ 0.050.
Results: GSE had a less pronounced effect on dentin's mechanical properties compared to Ca(OH)2. GSE showed less interference with FL (p=0.000), σf (p=0.002), and σc (p=0.000). It was observed that the duration of exposure to ICM had a negative impact on FL (p=0.002) and σc (p=0.031). ICM (p=0.080) and the application time (p=0.985) did not significantly affect E. Moreover, the application time of ICM did not have a significant impact on σf (p=0.169).
Conclusion: Grape seed extract did not affect the mechanical properties of bovine dentin when used as intracanal medication.
Keywords:
Calcium Hydroxide; Dentistry; Flexural Strength; Grape Seed Extract; Root Canal Therapy.
Introduction
Intracanal medications (ICMs) are recommended in endodontics to reduce the remaining microorganisms inside the root canal system [1-3]. Thus, using substances with good antimicrobial properties and low toxicity is indicated, and calcium hydroxide [Ca(OH)2] is the intracanal substance most used in endodontics [4]. However, when used in high concentrations and for long periods, sodium hypochlorite and calcium hydroxide can cause chemical and structural changes in dentin composition, such as collagen degradation and demineralization [5,6].
These alterations may reduce dentin flexural strength and microhardness, thereby interfering with root fracture resistance [6,7], especially when they persist for a long time [7]. When used as ICM, Ca(OH)2 decreases root fracture resistance and increases the microhardness of root dentin [6], also harming the bond strength of resinous endodontic cement to root dentin [8].
Considering the disadvantages associated with Ca(OH)2, alternative substances such as grape seed extract (GSE) are studied to increase the success rate of endodontic treatment [9]. This element has good antimicrobial activity [10] and promotes high adhesive strength of filling materials to root dentin over time [11]. A previous study [1] evaluated the antimicrobial efficacy of grape seed extract medication against Enterococcus faecalis, Staphylococcus aureus, and Candida albicans biofilms. The authors stated that GSE showed higher antibacterial activity against all species investigated compared to Ca(OH)2, and the lowest microbial count was obtained using a combination of GSE and Nd: YAG laser (p<0.001). So, it was concluded that GSE can be considered as an alternative to Ca(OH)2 dressing.
However, the potential interference of GSE with dentin properties requires elucidation. Therefore, this study evaluated the effect of GSE and Ca(OH)2 as intracanal medication for 7, 15, and 30 days on the mechanical properties of dentin, testing the hypothesis that the long-term use of both GSE and Ca(OH)2 interferes with the mechanical properties of dentin, but GSE results are most favorable.
Material and Methods
Ethical Aspects
Considering that the teeth used in the study were obtained from cattle carcasses previously slaughtered for meat and by-product consumption, the Ethics Committee for the Use of Animals at the University of Passo Fundo/RS (CEUA/UPF/RS) exempted the project from ethical review.
Experimental Design
Ninety-six single-rooted bovine teeth were obtained from a licensed slaughterhouse and stored at 4°C in a saline solution until testing. The powder of 99% Ca(OH)2 (Biodinamica, Ibiporã, PR, Brazil) and 97% GSE (Shopping LINE, São Paulo, SP, Brazil) was added to 0.9% saline in a glass plate, aided by a #50 spatula (SSWhite Duflex, Juiz de Fora, MG, Brazil) until obtaining pastes with “putty” consistency. The intracanal medications (ICMs) remained in direct contact with dentin for 7, 15, or 30 days, according to the experimental groups: Ctrl (without ICM application), GSE7, GSE15, GSE30, Ca7, Ca15, and Ca30.
Maximum Fracture Load (FL)
For the maximum fracture load (FL) test, seventy roots with closed apex were sectioned under water cooling using a metallographic cutter (Biopdi - Equipamentos para Ensaio de Materiais, Vila Mariana, SP, Brazil), resulting in 15-mm-long straight roots. Reference marks were made on the root surfaces using an overhead projector pen and a digital caliper (Vonder, Curitiba, PR, Brazil), 2 mm below the upper cut, 2 mm above the root apex, and the third mark was halfway between these two. The teeth had a standard buccolingual diameter of 6.5 mm in the cervical third, 5.5 mm in the middle third, and 4 mm in the apical third, and a standard mesiodistal diameter of 5.0 mm in the cervical third, 4.5 mm in the middle third, and 3.5 mm in the apical third.
The root canals were ground with a #1090 cylindrical diamond bur (KG Sorensen, Cotia, SP, Brazil) under abundant irrigation until obtaining 1-mm-thick dentin walls in all roots, verified by a digital caliper adapted for the inner surface of the canals. Distilled water was used to remove the material from the root canal preparation. The roots were soaked in acrylic resin (Artigos Odontológicos Clássico Ltda., Campo Limpo Paulista, SP, Brazil) using PVC plastic cylindrical molds (Tigre Brasil, Osasco, SP, Brazil) with thicknesses of ½ inch and heights of 20 mm, maintaining a 3-mm root exposure to simulate the biological space.
The samples were divided into experimental groups according to the ICM and application time: GSE was applied for 7, 15, or 30 days, and Ca(OH)2 was used for 7, 15, or 30 days. The ICM of each group was inserted with a 21-mm #4 lentulo drill (Dentsply Sirona, York, PA, USA). After filling, a sterile cotton ball was applied, and the upper opening was sealed with a temporary restorative glass ionomer cement restoration (Maxxion R, FGM Dental Group, Joinville, SC, Brazil). The teeth were incubated at 37 °C and 100% humidity for different observation times. After each period, the ICM was removed with abundant saline irrigation. The specimens were tested in a universal testing machine (EMIC DL 2000, São José dos Pinhais, PR, Brazil) under compressive load with a blunt tip device applied vertically to the coronal surfaces of the roots at a loading speed of 1 mm/minute until fracture. The maximum fracture load was recorded in Newtons (N) (Figure 1a)
a. Sequence of bovine teeth selection, crown removal, root preparation, acrylic resin basis, test substance application times according to the experimental group, and load-bearing capacity test; b. Sequence of crown removal, dentin microbar achievement, test substance application times according to the experimental group, three-point bending test, flexural strength equation, and elastic modulus equation; c. Sequence of root preparation, longitudinal root section, hourglass dentin specimen preparations, test substance application times according to the experimental group, and ultimate tensile strength test.
Flexural Strength (σf)
For the flexural strength (σf) test, the crowns of eight teeth were removed, and the roots were sectioned to produce rectangular dentin microbars (5.0 mm in length, 0.20 mm in thickness, and 1.7 mm in width). The microbars were randomly divided into five experimental groups (n = 10). The samples were placed in Eppendorf tubes and remained submerged in the test substances. At the end of the period, they were abundantly irrigated with 5 mL of saline to remove the medications until no visible trace of them remained, simulating a clinical condition. The three-point bending test (3PBT) was performed in a universal testing machine using a device with a 3-mm extension support. A load was applied to the center of the upper surface of the specimen at a speed of 0.5 mm/minute until fracture. Flexural strength (in MPa) was calculated using the equation 3PL/2bh2, where P is the maximum fracture load (N), L is the distance between support points (mm), b is the sample width (mm), and h is the sample height (mm) (Figure 1b).
Elastic Modulus (E)
The elastic modulus (E, in GPa) was based on flexural strength results, using the equation PL3/4bh3d, where P is the maximum fracture load (N), L is the distance between lower supports (mm), b is the sample width (mm), h is the sample height (mm), and d is deflection (mm) (Figure 1b).
Ultimate Tensile Strength (σt)
For the ultimate tensile strength (σt), the crowns of 18 incisors were removed, and a cut toward the long axis of the tooth was made for each root, obtaining two halves. For each half, three cuts were made perpendicular to the long axis, resulting in four slices from each root (1 mm thickness). The samples were manually ground under abundant irrigation with a #1090 cylindrical diamond bur (KG Sorensen, Cotia, SP, Brazil) until reaching an hourglass shape and a central portion area of approximately 0.8 mm2. The ends of the hourglasses were sealed with varnish to prevent touching the ICMs, and only the central region was in contact with the test substances. The samples were randomly divided (n = 10) and submerged in the test substances as described. The σt test was conducted at a speed of 0.5 mm/min using a universal testing machine. Maximum fracture resistance was recorded in N, and σt was calculated (in MPa) using the equation: F/A, where F is the maximum fracture resistance and A is the cross-sectional area of the narrowest part of the hourglass (mm2) (Figure 1c).
Statistical Analysis
The statistical power of the sample size was calculated before comparing the groups. The Control and GSE30 groups were considered, with α = 0.05, resulting in a power of 0.989 for maximum fracture load (FL), 0.999 for flexural strength (σf), 0.597 for elastic modulus (E), and 0.936 for ultimate tensile strength (σt).
The data passed the normality and equality of variance tests. A one-way ANOVA was used, followed by Dunnett's test to verify whether ICMs interfere with dentin properties, comparing the control group with the experimental groups. Two-way ANOVA (ICM and application time) and Tukey's test were also used, removing the control group from the comparison and considering only the groups treated with different ICMs at different intervals. All analyses were performed at p ≤ 0.05, using the software Minitab 18 and GraphPad Prism 6.
Results
Table 1 presents the results of the mechanical experimental groups. The control group exhibited a higher fracture load (FL) than the other experimental groups that received intracanal medications (ICMs) (p<0.010). Two-way ANOVA showed that the substance used as ICM (p=0.000) and time (p=0.002) interfered with FL results, but the interaction between factors was insignificant (p=0.234). Two-way ANOVA exhibited significance in σf results for the substance factor (p=0.002) but not for time (p=0.169) or interaction (p=0.975). ICMs did not affect dentin elastic modulus (E), showing similar values between the experimental and control groups. Substance (p=0.080), time (p=0.985), and the interaction between both (p=0.979) were insignificant for E. The control group presented statistically superior results to the other experimental groups for σt. Two-way ANOVA showed significance for ICM (p=0.000) and time (p=0.031), and none for their interaction (p=0.399). Figure 2 shows the comparison between the Control Group (dentin) and all other groups exposed to ICMs in relation to the mechanical tests performed, in different time periods.
Graphics showing the comparison between the Control Group (dentin) and all other groups exposed to ICMs in relation to the mechanical tests performed, in different time periods.
Discussion
In this study, the FL and σt results verified that the substance affected dentin properties, with Ca(OH)2 showing less favorable outcomes. The findings of the present study align with those of Hoffmann et al. [12] and Al-Hiyasat et al. [13], who reported that using Ca(OH)2 as an ICM for 14 and 90 days reduced the fracture resistance of root dentin. Yassen and Platt [14] presented similar results, suggesting that prolonged exposure to Ca(OH)2 may weaken dentin, making roots more susceptible to fracture. Likewise, Turk and Fidler [15] reported that applying Ca(OH)2 for more than four weeks harms the bond strength to dentin, potentially increasing the risk of tooth fractures.
According to Koshy et al. [16], the weakening caused by Ca(OH)2 in the root dentin has not been completely elucidated. One hypothesis is that it occurs due to the action of such a substance on the organic matrix of dentin, which is composed of collagen fibers. Calcium hydroxide would break the bond between these fibers and hydroxyapatite (inorganic dentin portion) due to its high alkalinity, triggering the denaturation of the proteins and proteoglycans that make this connection. These factors would make the dentin structure more rigid, less flexible, and consequently more friable [16]. Furthermore, the present study demonstrated that Ca(OH)2 treatment for 30 days presented the lowest flexural strength values. Cvek [17] reported similar results, showing a decrease in the fracture resistance of root dentin when Ca(OH)2 was used as a dressing for an extended period.
Although no statistically significant difference was noted in relation to the elastic modulus in this study, the mean numerical value of the Ca30 Group was the highest, which emphasizes the ability of Ca(OH)2 used as intracanal medication for an extended period to increase dentin stiffness. Castellan et al. [18] characterized the properties of the dentin matrix treated with proanthocyanidins, finding an increase in the elastic modulus of the demineralized dentin as exposure time increased, mainly attributed to the higher rigidity of the collagen matrix. This numerical trend (higher mean value in groups with more prolonged exposure to GSE) was also noted in the present study.
The control group had the highest σt values, followed by GSE for 30 days. Cohesive strength is essential in maintaining the structural properties of root dentin. A significant reduction in this mechanical property can increase root dentin fragility and the likelihood of an unfavorable tooth root fracture [19]. Therefore, assessing the cohesive strength of root dentin after treatment with different ICM protocols is imperative. The present study evaluated the cohesive strength of root dentin according to a previous analysis by de Andrade Marafiga et al. [20], who produced root dentin hourglasses. Our findings seem to demonstrate that a longer GSE contact time provides better outcomes in the cohesive strength of dentin. Cecchin et al. [10] presented similar results using GSE with the test substance. Furthermore, GSE is reported to be a proanthocyanidin-rich extract that has been shown to have antioxidant properties [10] and antimicrobial activity when used as a disinfection solution, although without the ability to remove the contaminated smear layer. Furthermore, they do not show detrimental effects on the dentin collagen network [9].
This experiment aimed to reproduce, as much as possible, the procedures performed in the clinical environment, using bovine teeth, whose composition and morphology are similar to human teeth, despite presenting a more standardized collagen pattern [21]. Thus, the use of bovine teeth as an experimental model is already scientifically recognized for carrying out in vitro studies testing dentin properties [22] or the bond strength of posts to root dentin [23] after exposure to various intracanal medications or irrigating solutions.
The present study did not consider the variability of dentin in the root thirds (cervical, middle, and apical) because the tooth roots were sectioned to prepare the specimens, which greatly facilitated the removal of the ICMs, except for the Maximum Fracture Load (FL) test. Under clinical conditions, however, it should be taken into account that the ICMs will always be more easily removed in the cervical third, when compared to the middle and apical root thirds [24]. This fact may generate variability in the results obtained for the Flexural Strength (σf), Elastic Modulus (E), and Ultimate Tensile Strength (σt) tests, for GSE or Ca(OH)2.
It cannot be stated from this experiment to what extent the values found have an impact on the integrity of the tooth, nor what would be the acceptable values for the tests carried out in the experimental groups. However, the clinical significance of this study lies in the fact that, overall, GSE demonstrated a significantly milder impact on dentin properties compared to Ca(OH)2. Although the samples in the present study were produced from incisors, it was decided to use vertical loads in the fracture tests, since the intention was not to analyze the behavior of ICMs only in anterior teeth, but also in posterior ones. Akman et al. [25] report the possibility of variations in the teeth, which may significantly contribute to the effect of ICMs. Cecchin et al. [10] suggest a possible tooth darkening after using GSE as ICM. Furthermore, it would be essential to carry out further studies on the antimicrobial properties of GSE over time when used as ICM (for example, evaluating a period of up to 30 days after its application).
Conclusion
Our study reveals that both Ca(OH)2 and GSE exhibit adverse effects on the mechanical properties of dentin. However, it is noteworthy that GSE demonstrated a significantly milder impact on these properties in comparison to Ca(OH)2. These findings underscore the potential utility of GSE as an alternative intracanal medication for preserving the mechanical integrity of dentin. However, further research is warranted to understand its clinical implications fully.
Financial Support
Data Availability
The data used to support the findings of this study can be made available upon request to the corresponding author.
References
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