Comparative evaluation of the resin–dentin interface using different matrix metalloproteinase inhibitors: A confocal laser scanning microscopic study

Abstract

Context:

The hybrid layer integrity is critical for durable resin–dentin bonding and is compromised by activation of matrix metalloproteinases (MMPs).

Aims:

To evaluate and compare hybrid layer thickness and resin tag penetration depth following pretreatment with maritime pine bark extract, green apple extract, and chlorhexidine in simulated erosive dentin using confocal laser scanning microscopy.

Settings and Design:

In vitro experimental study.

Materials and Methods:

Thirty-three extracted intact human teeth were used. After exposing the dentin, samples were treated with 10% citric acid to simulate erosive dentin. After acid etching, the samples were divided into three groups (n = 11): 6.5% maritime pine bark extract, 6.5% green apple extract, and 2% chlorhexidine. A fluorescent dye was incorporated into the adhesive; specimens were restored with composite resin and analyzed under confocal laser scanning microscopy.

Statistical Analysis:

One-way analysis of variance with post-hoc comparisons was performed. Significance was set at P < 0.05.

Results:

Hybrid layer thickness (F = 4.477, P = 0.020, standard deviation [SD] =81.621) and depth of resin tag penetration (F = 6.027, P = 0.006, SD = 186.558) in Group 3 (chlorhexidine) as MMP inhibitor showed a statistically significant difference between the groups, with the mean scores being highest in Group 3, followed by Group 1 (maritime pine bark extract) and Group 2 (green apple extract).

Conclusion:

Hybrid layer thickness and resin tag penetration depth following pretreatment with Group 3 were the highest and statistically significantly compared with Group 1 and Group 2

INTRODUCTION

In modern dentistry, bonding a resin composite securely to dentin is crucial for an intact and long-lasting. When resin infiltrates acid-etched, moisture-laden dentin, it forms the hybrid layer, a fine mesh that anchors the restoration effectively.[1] The hybrid layer is the area that exists between the tooth substrate and the adhesive materials. This concept was given by Nakabayashi et al.[2] The permeability of the dentin substrate on which an adhesive is adhered has a significant effect on this hybrid layer.[3,4]

The smear layer in the etch and rinse method poses a significant barrier to the dentin substrate’s permeability and hybrid layer development. Neither resin tag production nor hybridization can take place unless the smear layer is eliminated. The dentin needs to be acid-etched in order to remove the layer of debris and smear plug. Acid etching decalcifies the inter-tubular and peritubular dentin, helps in the opening of the dentinal tubules, promotes adhesive permeability, and eliminates the smear layer.[5] Too much collagen can hinder uniform resin penetration.[5,6] Incomplete infiltration results in vulnerable zones within the hybrid layer, which increases microleakage, which further leads to secondary caries and postoperative sensitivity.[7,8]

This hybrid layer, a mesh of resin-reinforced collagen, is fundamental to adhesion quality. Dentin contains latent matrix metalloproteinases (MMPs) and cysteine cathepsins, which gets into an active form by the carious process and acid-etching.[9] When these proteases are activated, they break down the exposed collagen in the hybrid layer, rendering the restoration less probable to survive.

One effective approach to counteract degradation is the use of MMP inhibitors (MMPIs). These agents preserve hybrid layer integrity and extend bond longevity.[10]

Chlorhexidine (CHX) is the most widely studied MMPI in dentistry. In vitro studies confirm that applying 2% CHX post-etch significantly preserves bond strength over 6 months, with fewer failures occurring within the hybrid layer. These findings consistently support CHX’s role in maintaining resin–dentin bond over time.[11]

Proanthocyanidins (PAs) - polyphenols found in plants-offer a two-fold advantage: they both inhibit MMPs and cross-link collagen, reinforcing the hybrid layer structurally and chemically.

Pycnogenol, an extract from maritime pine bark, is rich in proanthocyanidins. Apples are also a significant dietary source of PAs.[8] Yet, surprisingly, no studies have explored the use of these extracts in dental bonding applications, especially as natural MMP inhibitors and collagen cross-linkers. This represents an appealing research frontier.

To the best of our knowledge, no studies using maritime pine bark extract and apple extract as MMPI have been carried out till date. To bridge this gap, the present study aimed to test maritime pine bark extract and apple extract as natural agents for preserving the resin–dentin bond. As these extracts are natural plant-based rich in PA.

MATERIALS AND METHODS

Ethical approval

This in vitro study was approved by the Institutional Review Board of Dr. D. Y. Patil Dental College and Hospital, Pimpri (Approval No. DYPDCH/IEC/08/2024).

Thirty-three noncarious intact, single-rooted permanent human teeth were collected. After extraction, all teeth were cleaned using an ultrasonic scaler, sterilized by autoclaving, and stored in 0.1% thymol solution until used. Each tooth was embedded in cold-cure acrylic resin to allow secure handling. The occlusal enamel was carefully removed using a slow-speed diamond disc under continuous water irrigation to expose the dentin surface. The remaining dentin thickness was standardized at approximately 1.5–2 mm, and the surface was polished with 600-grit silicon carbide paper.

To simulate erosive dentin, exposed dentin samples were immersed in 10% citric acid for 4 h, washed with normal saline to remove all remnants. The dentin surfaces were then etched with 35%–37% phosphoric acid for 15 s, rinsed thoroughly with water for 10 s and dried with blotting paper, keeping the dentin surface moist.

The specimens were randomly divided into three groups (n = 11 each). Pretreatments applied to the acid-etched dentin included (Group 1) - 6.5% w/v maritime pine bark extract (Greendorse) in distilled water, (Group 2) - 6.5% w/v green apple extract (vital herbs) in distilled water and (Group 3) - 2% chlorhexidine (Dentochlor) as a positive control, each left on the surface for 15–20 s and air-dried with a blotting paper.

A Rhodamine B fluorescent dye (0.1% w/v) was mixed into the 5^th generation dentin bonding agent (Adper Single Bond 2; 3M ESPE) to enable visualization under confocal microscopy. The bonding agent was applied and light-cured for 20 s, followed by incremental placement of composite resin (Filtek Z350; 3M ESPE) using plastic cylindrical tubes (2 mm × 5 mm). Light curing was done for each 2-mm layer for 20 s. After finishing and polishing, all specimens were stored in normal saline for 24 h.

Each tooth was sectioned longitudinally into 1-mm thick slices.[12] Samples were air-dried and examined under a Confocal Laser Scanning Microscope (Carl Zeiss LSM 710) using a 10 × objective at 561–570 nm excitation. Confocal microscopy allows detailed optical sectioning of the resin–dentin interface without physical destruction, providing high-resolution images of hybrid layer formation (thickness) and resin tag penetration (depth was measured).[13]

Statistical analysis

Data were subjected to one-way ANOVA to compare mean hybrid layer thickness and depth of penetration across groups using software IBM SPSS Statistics version 21, (IBM Corporation (International Business Machines Corporation), Armonk, New York, U.S.A.). A post-hoc test was used for intergroup comparisons. Significance was set at P < 0.05.

RESULTS

Hybrid layer thickness

Comparison of the hybrid layer thickness mean score in all the groups was done. It showed a statistically significant difference between the groups, with the mean score of Group 3 (chlorhexidine, mean = 162 μm) to be highest, followed by Group 1 (Maritime pine, mean = 133 μm) and 2 (green apple, mean = 77.82 μm) [Figure 1]. Intergroup comparison in all the groups showed a statistically significant difference between Groups 2 and 3 (P = 0.020) [Table 1]. However, no significant difference was seen between Groups 1 and 2 and Groups 1 and 3. The mean thickness of hybrid layer formation in Group 3 was maximum, followed by Groups 1 and 2, respectively.

Figure 1.

Confocal laser scanning microscopy image with measurements of depth of penetration and resin tag penetration at ×10: (a) Maritime pine bark; (b) Green apple; (c) Chlorhexidine

Table 1.

Comparison of hybrid layer thickness mean scores of all the groups

Hybrid layer thickness	n	Mean±SD	F	P
Group 1	11	133.00±79.712	4.477	0.020
Group 2	11	77.82±21.507
Group 3	11	162.00±81.621
Total	33	124.27±73.955

One-way ANOVA, P<0.05. Group 1: Maritime Pine Bark, Group 2: Green Apple, Group 3: Chlorhexidine, n: Number of samples per group, SD: Standard deviation, ANOVA: Analysis of variance

Resin tag penetration depth

Comparison of the depth of penetration mean score in all the groups showed a statistically significant difference between the groups, with the mean score of Group 3 (mean = 431.27 μm) being the highest, followed by Group 1 (mean = 382.09 μm) and Group 2 (mean = 230 μm) [Figure 1]. Intergroup comparison in all the groups showed a statistically significant difference between Groups 2 and 3 (P = 0.006) [Table 2]. However, no significant difference was seen between Groups 1 and 2 and Groups 1 and 3. Mean length of resin tag formation in Group 3 (chlorhexidine) was maximum, followed by Group 1 and Group 2, respectively.

Table 2.

Comparison of depth of penetration mean scores of all the groups

Depth of penetration	n	Mean±SD	F	P
Group 1	11	382.09±121.665	6.027	0.006
Group 2	11	230.00±103.332
Group 3	11	431.27±186.558
Total	33	347.79±162.506

One-way ANOVA, P<0.05. Group 1: Maritime Pine Bark, Group 2: Green Apple, Group 3: Chlorhexidine, n: Number of samples per group, SD: Standard deviation, ANOVA: Analysis of variance

DISCUSSION

Dentin bonding is not only a technical step but also its a cornerstone of lasting and effective dental restorations. It determines how long a restoration retains and how well it performs over time.

Because dentin is rich in organic matter, especially Type I and III collagen fibers. This organic content makes bonding more challenging. When the bond fails, it can lead to-microleakage, secondary caries, and posttreatment sensitivity.[11]

These issues can often be avoided if we properly “hybridize” the dentin surface, which is essential for a durable bond. The smear layer is a thin film of debris left behind after preparing the tooth. It must be removed to allow quality and quantity of hybrid layer and resin tag penetration are some of the criteria for the success of adhesive restoration. Thats why the etch-and-rinse method is so popular to remove the smear layer, opens up the tubules, and let adhesive penetrate deeply into dentinal tubules.

Bonding works differently depending on the state of the dentin. In caries-affected dentin, the hybrid layer is more uneven and exposes collagen more, which makes it more prone to weak bonds, and it is more vulnerable to water damage and enzymatic degradation.[14]

Inside dentin, there are enzymes called MMPs and cysteine cathepsins.[15] Under normal conditions, they are dormant. However when dentin is etched with acid before bonding, these enzymes are activated and start breaking down collagen fibers. That weakens the bond over time.

Preventing this degradation, one promising solution is the use of MMP inhibitors (MMPIs).[16] When used correctly, these inhibitors help stabilize the hybrid layer and make the adhesive bond last longer. As an inhibitor of MMP, commonly used chlorhexidine is a synthetic cationic bis-guanide. CHX’s ability to inhibit proteases seems to be concentration-dependent. The suppression of MMP-2 and MMP-9 at low concentrations may be due to a cation chelating mechanism.[17] CHX changes the configurations of the MMP active site and binds to dentine electrostatically. When compared to the other groups in the current investigation, Group 3 - CHX had the highest values for resin tags and hybrid layer thickness.

Komori M, et al. concluded that over a 6-month period of storage in artificial saliva, chlorhexidine pretreatment significantly mitigated the decline in bond strength observed in sound dentin specimens; however, in caries-affected dentin specimens, the treatment did not yield a statistically significant preservation of bond strength compared to the untreated control group.[18]

De Munck et al. investigated the effects of including CHX into adhesive primers and the ability of adhesives to extract and stimulate endogenous MMPs from dentin. Unlike etch and rinse adhesives, mild SE adhesives are unable to eliminate and activate endogenous MMPs.[19]

According to Ekambaram M. et al., applying chlorhexidine (CHX) directly improves both sound and caries-affected dentin, demonstrating enhanced bond strength even after a full year.[20] CHX only maintains the stability of the resin-dentin bond for a year, as Montagner et al. showed.[21]

Ricci et al. and Brackett et al. demonstrated that CHX had a positive effect on slowing down the rate at which the resin-dentin bond degraded. However, after 2 years, clinical investigations revealed that CHX did not improve the clinical durability of adhesive restorations.[22,23] While the addition of CHX will increase the bond’s strength, the release of MMP-2 from etch and rinse adhesives may affect the bond’s durability.[24,25]

Kunhappan et al. demonstrated that the depth of adhesive resin penetration varied significantly amongst the three surface pretreatment techniques used in the sandwich method, according to this in vitro confocal laser scanning microscopy investigation. The findings imply that proper surface pretreatment might improve resin infiltration and possibly increase sandwich restorations’ bonding efficacy.[25]

Rahal et al. investigated the relationship between a self-etch adhesive system’s microtensile bond strength, resin tag length, and hybrid layer thickness. It was observed no significant correlation between bond strength and the length of the resin tag or the thickness of the hybrid layer. According to the findings, adhesive chemistry and infiltration quality have a greater influence on bond strength in self-etch adhesives than layer thickness or tag length.[26]

Proanthocyanidin (PA), a natural compound, it strengthens the collagen network by forming strong, covalent cross-links and inhibits MMPs, protecting the collagen from breakdown. In etch-and-rinse systems, the bond between adhesive and dentin is enhanced by using, PA.

It has been demonstrated that proanthocyanidin binds to the collagen matrix and has a greater affinity for proteins, which causes water molecules to be displaced. PA may be able to stay in the collagen matrix for an extended period of time due to the hydrogen connection that forms between PA and collagen molecules. Collagen fibrillar mobility may be decreased by intermolecular, intramolecular, and intermicrofibrillar crosslinking, which could lead to deeper penetration of PA molecules and the detachment of collagen-bound MMPs. The detachable MMPs may be stuck inside the cross-linked collagen fibers and cause their inactivation as a result of the fibrils’ increasing collagen cross-linking.

In the present research, we assessed how various naturally occurring PAs affected resin tag penetration and hybrid layer thickness, including maritime pine bark extract (Group 1), Green apple extract (Group 2) on erosive dentin, where MMPs were prominent. Maritime pine bark shows comparable hybrid layer and resin tags results to Chlorhexidine (Group 3). Green apple extract was effective, but not as much as the other two groups. PA was a highly hydroxylated structure that can interact with proteins and produce an insoluble complex. Better collagen networks and inter-fibrillar volume were generated by Group 3, Group 2, and Group 1 pretreatments in this study, which improved adhesive resin penetration.

The ability of PAs to crosslink has drawn a lot of attention. This improves the demineralized dentin matrix and the strength of the dentin-resin connection. The dentin matrix’s immediate elastic modulus can be raised by PA pretreatment. In addition, CHX and PA can both bind to MMP metal ions in a competitive manner.

In the study, morphological assessment was done, i.e., Hybrid layer thickness and dentinal tubule penetration depth, which are not only criteria for adequate bond strength more functional parametric assessment micro tensile bond strength, nano leakage, interfacial integrity, and long-term durability of restorations are also required as functional parametric analysis to support the hypothesis, which is a limitation of the study.

Limitations of the study

A small sample size is the drawback of the study. In vitro limitations: laboratory conditions can’t fully mimic the complex in vivo environment. Factors such as fluid dynamics, tissue responses, and host immunity are not represented. In vivo studies could confirm whether the bond strength is improved or not in clinical conditions. The study design only takes into consideration the morphological assessment and does not include assessment or quantification of MMP activity and degree of crosslinking.

Implications and future research

Given the encouraging performance of maritime pine bark extract, further research should explore its use in clinical settings, its long-term stability, and optimization of concentration. Investigations into other proanthocyanidin-rich natural sources, standardized formulations, and delivery protocols could open up more biocompatible and cost-effective options for preserving the resin–dentin interface.

CONCLUSION

According to the study’s limitations, chlorhexidine demonstrated the highest, maritime pine bark showed a comparable result to CHX, and green apple showed the least value. The findings support the hypotheses and other studies that were previously presented.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.