| Abstract|| |
Aim: This study evaluated the effect of three collagen cross-linking agents – proanthocyanidins (grape seed extract [GSE] and green tea extract [GTE]) and glutaraldehyde [GA] on microshear bond strength (μSBS) of caries-affected dentin (CAD)-resin complex.
Materials and Methods: Freshly extracted 96 teeth with caries up to the middle third of dentin were sectioned through the deepest part of the occlusal fissure, perpendicular to the long axis of the crown. Caries was excavated with large round bur until firm dentin was obtained, confirmed by visual inspection and tactile examination. Flat occlusal dentin surfaces were treated as follows: Group-1 – 6.5% GSE (n = 30), Group-2 – 2% GTE (n = 30), Group-3 – 5% Glutaraldehyde (n = 30), Group-4 – control group (no agents) (n = 6). Each group was further divided into Subgroup A - Etch-N-Rinse 15s, Subgroup B - Etch-N-Rinse 45s, and Subgroup C - Self-etch. Two increments of 1.5-mm thick composite (Tetric-N-Ceram – Ivoclar Vivadent) with a 1-mm diameter were built-up. Each sample was subjected to μSBS test in Universal Testing Machine. Student's t-test was done for intragroup comparison and one-way ANOVA for intergroup comparison.
Results: Statistically significant difference was present in mean μSBS, with Group 1B showing the best results and Group 4C, the least.
Conclusions: Thus, the application of these collagen cross-linkers, to CAD, increases μSBS and promises a new approach to improve dentin bond strength.
Keywords: Caries-affected dentin; cross-linking agents; etch and rinse; glutaraldehyde; grape seed extract; green tea extract; self-etch; shear bond strength
|How to cite this article:|
Govil SA, Asthana G, Sail VA. Bonding strategies to deal with caries-affected dentin using cross-linking agents: Grape seed extract, green tea extract, and glutaraldehyde – An in vitro study. J Conserv Dent 2023;26:108-12
|How to cite this URL:|
Govil SA, Asthana G, Sail VA. Bonding strategies to deal with caries-affected dentin using cross-linking agents: Grape seed extract, green tea extract, and glutaraldehyde – An in vitro study. J Conserv Dent [serial online] 2023 [cited 2023 Feb 6];26:108-12. Available from: https://www.jcd.org.in/text.asp?2023/26/1/108/367918
| Introduction|| |
In the present era, adhesive technology has gained popularity due to minimally invasive techniques and optimal esthetics of composites. However, the major problem is low durability, caused mainly by interfacial failure.
Long-term studies revealed that bonding to dentin deteriorated more readily than that to the enamel. Most studies have used sound dentin as the bonding substrate. However, in clinical settings, the substrate being bonded may often involve caries-affected dentin (CAD), in which the bond strength is lower than that of sound dentin. This may be due to differences in morphological, chemical, and physical characteristics of CAD. Consequently, measures to improve the longevity of dentin bonding need to be taken.
Dentin biomodification, i.e., dentin treatment with collagen cross-linking agents, is one such approach for providing longer durability of adhesive restorations. These agents are classified by the type of biomodification they cause: physical or chemical. Chemical agents can be natural or synthetic. Natural agents include proanthocyanidins (PA), cardol and cardonal, Epigallocatechin 3-gallate (EGCG), etc., whereas synthetic agents include aldehydes such as glutaraldehyde (GA) and carbodiimide.
PAs are a class of bioflavonoids found in natural sources such as grape seed extract (GSE), green tea extract (GTE), pine bark extract, and cranberries. They have the ability to bind to proline-rich proteins, like collagen, and facilitate enzyme proline hydroxylase activity essential for collagen biosynthesis. This combined cross-linking potential and anti-collagenolytic effects might be beneficial in preventing the degradation of dentin collagen within the hybrid layer. GSE and GTE can be added either to the adhesives or used as a primer.
GA is predominately used as a fixative that cross-links collagenous biomaterials. GA contains two aldehyde groups which react with amino groups of lysyl or hydrolysyl polypeptide residues in collagen, forming reducible Schiff-base crosslinks. Its ability to resist biodegradation in collagen molecules and produce irreversible cross-links helps to keep the network in a relatively expanded state; an important prerequisite for chemical dehydration by ethanol and interdiffusion of HEMA monomer. GA can be added as an additive to the adhesives or used as a primer.
Few studies have been done to investigate the effect of collagen cross-linking agents –proanthocyanidins (GSE and GTE) and GA on the microshear bond strength (μSBS) of CAD-resin complex. The present study evaluated the effect of 6.5% GSE, 2% GTE, and 5% GA on μSBSs on CAD.
The null hypothesis was (1) there is no difference in the μSBS of CAD after the application of various cross-linking agents. (2) there is no difference in μSBS of CAD using etch-n-rinse, extended etching, and self-etch bonding strategies.
| Materials and Methods|| |
After ethical approval from the institutional ethics committee, freshly extracted human molars, with occlusal caries, were selected from a bulk of extracted teeth. The teeth were radiographed and 96 teeth with radiographic signs of dental caries extending up to the middle third of dentin were selected. To standardize the depth of preparation, they were sectioned through the deepest part of the occlusal fissure, perpendicular to the long axis of the crown, to expose the carious lesion. Caries was excavated with a large round bur, followed by a double-ended hand excavator (Dentsply Maillefer B190, USA) to reach CAD [Figure 1]a.
|Figure 1: (a) After sectioning through the deepest part of the occlusal fissure (for standardization of samples), perpendicular to the long axis of the crown, a carious lesion was exposed. Caries was excavated to reach CAD. (b) Composite built 1 mm in diameter and 1.5-mm thickness on CAD. (c) Sample mounted on Universal Testing Machine (UNITEST-10) and subjected to a μSBS test using a metal attachment placed as close as possible to the composite/dentin interface. CAD: Caries-affected dentin, μSBS: Microshear bond strength|
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CAD was confirmed by visual inspection and tactile examination. Glassy dentin, dark yellow, or slightly brownish is classified as CAD. Clinically, firm dentin is resistant to hand excavation and can only be removed by exerting pressure.
The dentin surfaces were then treated as follows:
Group 1 (n = 30) – 6.5% GSE (Inlife Pvt Ltd, India) applied on CAD for 60 s with a microapplicator brush, rinsed with water thoroughly for 10 s and blot dried.
Group 2 (n = 30) – 2% GTE (Biotrex Nutraceuticals, Ahmedabad, India) was applied for 60 s, and blot dried.
Group 3 (n = 30) – 5% GA (Sigma Aldrich, USA) was applied for 60 s, and blot dried.
Group 4 – control group (n = 6) – No cross-linking agent was applied.
All four groups were further divided into A, B, and C according to the bonding strategy used, i.e., etch-n-rinse or self-etch, and duration of etching.
Subgroups 1A, 2A, 3A, and 4A-Etch-N-Rinse strategy
Acid etching for 15 s with 37% phosphoric acid gel (N-ETCH – Ivoclar Vivadent), then rinsed thoroughly with water and collagen cross-linking agent applied.
Two coats of bonding agent (Tetric N-Bond Total-Etch Dental Adhesive – Ivoclar Vivadent) [Table 2] were applied on CAD and light cured for 20 s (Bluephase C5 curing light [500 mW/cm2] – Ivoclar Vivadent).
Subgroups 1B, 2B, 3B, and 4B-Etch-N-rinse strategy
Acid etching for 45 s with 37% phosphoric acid gel (N-ETCH – Ivoclar Vivadent), then rinsed thoroughly with water and collagen cross-linking agent applied.
Two coats of bonding agent (Tetric N-Bond Total-Etch Dental Adhesive – Ivoclar Vivadent) [Table 2] were applied on CAD and light cured for 20 s.
Subgroups 1C, 2C, 3C, and 4C – Self-etch strategy
Collagen cross-linking agent was used, followed by the application of two coats of bonding agent (Tetric N-Bond Universal Adhesive – Ivoclar Vivadent) [Table 2] and light cured for 20 s.
After the completion of bonding, composite buildup was done in all groups. For standardization of the samples, for μSBS testing, composite was built 1 mm in diameter and 1.5-mm thickness on CAD (Tetric N-Ceram composite – Ivoclar Vivadent) [Figure 1]b, and light cured for 40 s.
Each sample was fixed in acrylic resin block and mounted on the Universal Testing Machine (UNITEST-10). Samples were subjected to a μSBS test using a metal attachment placed as close as possible to the composite/dentin interface [Figure 1]c. The test was run at a crosshead speed of 1.0 mm/min. The force required for failure (Newton) was divided by the surface area (mm2) to calculate shear bond strength in MPa.
Data were analyzed using SPSS version 23 (SPSS ver 23, IBM Inc, Illinois, NY, USA). Descriptives, Student's t-test was done for intragroup comparison, i.e., etch-n-rinse 15s, etch-n-rinse 45s, and self-etch groups. One-way ANOVA was done for intergroup comparison, i.e., GSE, GTE, and GA.
| Results|| |
Means [Graph 1] and standard deviations from the μSBS test are shown in [Table 1]. One-way ANOVA done for intergroup comparison showed that there is a statistically high significant difference present in mean μSBS between various cross-linking agents used for pretreatment (P < 0.001). Student's t-test done for intragroup comparison revealed that statistically high significant difference is present in mean μSBS in etch-n-rinse 15s, 45s, and self-etch subgroups (P < 0.001). Intragroup comparison between subgroups 4A and 4B and between subgroups 4B and 4C was statistically significant (P < 0.05). However, intragroup comparison between Groups 4A and 4C shows t = 3.603 which was statistically nonsignificant (P > 0.05).
μSBSs achieved in all groups, in ascending order were as follows: 4C <4A <4B ≤3C <3A ≤2C ≤3B <1C ≤2A ≤1A <2B <1B.
| Discussion|| |
Bonding to dentin can be considered a form of tissue engineering. Nevertheless, adhesion to dentin is still challenging due to the following factors: (1) Presence of considerable proportion of water and organic material. (2) Presence of smear layer and smear plugs on dentin surface. (3) Degradation of collagen fibrils over time by matrix metalloproteinases (MMPs). (4) Incomplete infiltration of resin monomers at the bottom of the hybrid layer. (5) Alteration in dentin structure due to aging, caries, and pretreatment with various chemicals such as H2O2 and NaOCl.,
Most knowledge about dentin bonding has been limited to normal dentin, it is important that adhesives should prove their effectiveness when bonded to clinically most commonly available substrate – CAD.
Although mineral depositions occlude dentinal tubules in CAD, it is still softer than normal dentin, with about half the hardness. The mean hardness value of normal dentin (KHN = 52–57) is approximately twice than that of CAD (KHN = 25). CAD is identified clinically according to various criteria, including hardness and color (visual and tactile examination). Caries removal methods have been developed that attempt to define this endpoint (e.g., self-limiting burs and chemomechanical removal). Most methods have been validated in vitro, but lack sufficient clinical validation, while some are even detrimental, for example, staining using caries detector dyes.,
The bond strengths to CAD have been reported to be 20%–50% lower than to sound dentin. Dentin reactions to caries initiate readily after caries affect the enamel, so it is common, those adhesive restorations will be bonded to different degrees of altered dentin in a clinical setting. Hence, reported bond strengths to sound dentin are of no predictive value.,
MMPs are a family of over 20 host-derived proteolytic enzymes with the ability to degrade the majority of extracellular matrix components. The acid-etching procedure may lead to the release and activation of pro-MMPs within the mineralized dentin. This causes collagenolytic and gelatinolytic activities within the hybridized dentin. Activated host MMPs are liable for almost complete disappearance of portions of hybrid layers, from resin–dentin bonds. Thus, bond strength decreases over time. Cysteine cathepsins may be activated in mildly acidic environments, which cause the activation of matrix-bound MMPs. To inactivate these, and to stabilize the hybrid layer, collagen cross-linking agents have been invented that prevent degradation of resin–dentin bond, as shown in a study by Asthana et al.
In our study, we used 6.5% GSE, 2% GTE, and 5% GA as primer for an application time of 60 s, which is clinically acceptable.
While making intragroup comparisons in Group 1, the highest mean μSBS was of subgroup B (30.80 MPa), i.e., etch-n-rinse for 45 s, followed by subgroup A (27.93 MPa) and least in subgroup C and self-etch group (25.28 MPa).
In CAD, the intertubular dentin is hypomineralized, but dentinal tubules are occluded with mineral deposits. Owing to repeated cycles of demineralization and remineralizations, larger calcium phosphate crystals are formed, which are less soluble in acidic conditions than normal apatite. The partial removal of intratubular deposits by additional acid etching could increase dentinal tubule permeability. The formation of funnel-shaped resin tags and resin infiltration into the lateral branches through dentinal tubule anastomosis might have contributed to the increased bond strengths. Arrais et al. concluded that extended etching for 45 s improves bonding to CAD.
On the other hand, in self-etch strategy, due to their higher pH, they cannot dissolve and remove acid-resistant mineral deposits in dentinal tubules of CAD. Therefore, an etch-n-rinse adhesive system might be superior to self-etch systems, although acid etching could not completely dissolve the mineral deposits in dentinal tubules of CAD., This is in accordance with the study, where lower bond strengths were obtained with single bond universal on CAD.
For the same plausible explanations, intragroup comparisons in Ggroups 2, 3, and 4, also yielded the highest mean μSBS of subgroup B, followed by subgroup A and least in subgroup C. Thus, the second null hypothesis that there is no difference in μSBS values of various bonding strategies was rejected.
In intergroup comparisons, among all subgroups A, B, and C, there is statistically significant difference present in the mean μSBSs, with values in order as Group 1 (GSE) >Group 2 (GTE) >Group 3 (GA) >Group 4 (No cross-linking agent). Thus, the first null hypothesis that there are no significant differences between μSBSs of various cross-linking agents stands rejected.
GSE significantly improved the bond strength of deep dentin. Similar results were found by Srinivasulu et al. GTE increases the bond strength of affected dentin, where the occurrence of MMPs and cysteine cathepsins is more. This could be attributed to MMP inhibitory mechanisms. The studies evaluating GTE and EGCG solutions on dentin are scarce, and most studies are on sound dentin, so the comparison of results is difficult.
A study, showed that 5% GA primer application for 1 min significantly improved bond strengths; however, it was done on normal dentin.. Macedo et al. used GSE and GA on sound as well as CAD and concluded that the application of these agents to dentin significantly improved the microtensile bond strength. However, proanthocyanidins interacted with proteins to induce cross-links by four different mechanisms: Covalent, ionic, hydrogen bonding, and hydrophobic; thus, seemed to have more ability to interact with collagen and increase the mechanical properties of dentin, when compared to GA.
The results of the present study show that pretreatment of CAD with cross-linking agents decreases collagenase degradation, reduces water absorption, and increases mechanical properties.
Limitations of the study
As this is an in vitro study, actual clinical conditions could not be simulated. Further in vivo studies over longer periods are needed to substantiate the same.
| Conclusions|| |
Within the limitations of the present study, the following conclusions were drawn:
- CAD treated by 6.5% GSE, 2% GTE, and 5% GA, resulted in increased μSBS (in the order of GSE >GTE >GA) due to the inactivation of MMPs and cystine cathepsins, enhancing the durability of resin–dentin bond
- μSBSs were higher in etch-n-rinse adhesives than self-etch adhesives
- Increasing the etching time to 45s, increased μSBS to CAD, due to the solubilization of acid-resistant mineral deposits in tubule lumens.
Thus, the application of these collagen cross-linkers to CAD promises a new approach to improve dentin bond strength.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Shrusti Ajay Govil
G-404, Shantiniketan-3, Opposite RAF Camp, S. P. Ring Road, Vastral, Ahmedabad - 382 418, Gujarat
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]