Journal of International College of Dentists

2. Materials and Methods

2.1. Study design

The present in vitro study was designed to comparatively evaluate the effect of different dentin pretreatment agents on the push-out bond strength of fiber posts luted with resin cement.

A total of forty freshly extracted human mandibular premolars were selected for the study. The teeth were extracted for orthodontic purposes and collected after obtaining informed consent. The study protocol was approved by the Institutional Research and Development Committee (IRDC) prior to commencement of the study.

Following extraction, the teeth were cleaned of soft tissue remnants and calculus using hand instruments and thoroughly rinsed with distilled water. The specimens were stored in distilled water at room temperature until further use to maintain hydration and preserve dentinal properties.

2.2. Sample selection and preparation

The following inclusion and exclusion criteria were applied:

2.2.1. Inclusion criteria

Freshly extracted human single-rooted, single-canal mandibular premolars. Fully formed apices. Intact roots without cracks, fractures, or structural defects. Teeth without previous endodontic treatment.

2.2.2. Exclusion criteria

Teeth with root resorption. Teeth with immature apices. Teeth with calcified or severely curved canals. Teeth with visible cracks or fractures.

2.3. Root canal treatment

The selected teeth were decoronated at the cementoenamel junction using a circular disc under continuous water irrigation to obtain straight line access and standardize the root length to 16 mm.

The working length was determined using a size 10 K-file, and biomechanical preparation was performed using a rotary nickel-titanium instrumentation system according to the manufacturer's instructions.

During instrumentation, the canals were irrigated with 3% sodium hypochlorite solution (Vishal Dentocare Pvt. Ltd., Ahmedabad, India). Following completion of canal preparation, the canals were rinsed with distilled water and dried using sterile paper points.

The prepared canals were obturated using gutta-percha cones and an epoxy resin-based root canal sealer, AH Plus (Dentsply Sirona, Konstanz, Germany). The specimens were stored at 37°C and 100% humidity for 7 days to allow complete setting of the sealer.

2.4. Post space preparation

After obturation, post spaces were prepared using the corresponding drill system for RelyX Fiber Post (3M ESPE, Seefeld, Germany).

Approximately 5 mm of apical gutta-percha was retained to maintain the apical seal, and the remaining obturation material was removed to create a standardized post space.

The post spaces were irrigated with distilled water and dried using sterile paper points before application of dentin pretreatment agents.

2.5. Preparation of dentin pretreatment solutions

2.5.1. Preparation of 0.5 M 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution

A 0.5 M solution of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl) (Sigma-Aldrich, St. Louis, USA) was prepared by dissolving the required amount of EDC powder in distilled water. The solution was freshly prepared before use to maintain cross-linking activity.

2.5.2. Preparation of 6.5% proanthocyanidin solution

A 6.5% proanthocyanidin (PAC) solution was prepared by dissolving grape seed powder (Yucca Enterprises, Mumbai, India) in distilled water to obtain the desired concentration. The solution was thoroughly mixed to achieve a homogeneous preparation before application.

2.5.3. Preparation of 0.2% chitosan solution

A 0.2% chitosan solution was prepared by dissolving 0.2 g of chitosan powder (HiMedia Laboratories Pvt. Ltd., Mumbai, India) in 100 mL of 1% acetic acid solution (Merck Life Science Pvt. Ltd., Mumbai, India). The mixture was continuously stirred using a magnetic stirrer for 2 hours at room temperature until a uniform solution was obtained. The solution was freshly prepared before dentin pretreatment.

2.6. Group allocation

Forty specimens were randomly divided into four groups (n = 10) according to the dentin pretreatment agent used:

Group I – EDC Group II – Proanthocyanidin Group III – Chitosan Group IV – Control (Saline)

2.7. Dentin pretreatment protocol

Following post space preparation, the respective dentin pretreatment solutions were introduced into the canal space and allowed to interact with the root dentin for a standardized duration.

After treatment, the canals were rinsed with distilled water to remove residual solution and dried gently using sterile paper points before fiber post cementation.

2.8. Fiber post cementation

RelyX Fiber Posts were selected according to the prepared post space dimensions and trial fitted within the canal.

The posts were cleaned as per the manufacturer's recommendations. RelyX U200 self-adhesive resin cement (3M ESPE, St. Paul, USA) was mixed and introduced into the post space using an intracanal delivery tip.

The fiber post was inserted with gentle finger pressure, and excess cement was removed. Light polymerization was performed according to the manufacturer's instructions.

The specimens were stored under controlled conditions to allow complete polymerization of the resin cement before mechanical testing.

2.9. Push-out bond strength evaluation

Each root was sectioned perpendicular to its long axis using a precision cutting machine to obtain standardized 2-mm thick slices from the middle third of the root.

Push-out bond strength testing was performed using a universal testing machine. Each specimen was positioned with the apical surface facing upward, and a compressive load was applied using a cylindrical plunger at a crosshead speed of 0.5 mm/min until dislodgement of the fiber post occurred.

The maximum load required for post extrusion was recorded in Newtons (N), and push-out bond strength was calculated in megapascals (MPa) using the formula:

Push-out bond strength (MPa) = Maximum load (N) / Bonded surface area (mm²)

A schematic overview of the specimen preparation and push-out bond strength testing procedure is presented in Figure 1.

2.10. Statistical analysis

Statistical analysis was performed using SPSS software version 21.0 (IBM Corp., NY, USA).

The mean and standard deviation values were calculated for each group.

Intergroup comparison of push-out bond strength values among the four groups was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test.

A p-value < 0.05 was considered statistically significant.

3. Results

Statistical analysis was performed using SPSS software version 21.0 (IBM Corp., NY, USA). Descriptive statistics including mean and standard deviation were calculated for all groups. Intergroup comparison of push-out bond strength values among the four experimental groups was performed using one-way ANOVA, followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant.

The mean push-out bond strength values demonstrated statistically significant differences among the groups (p < 0.001). The highest push-out bond strength was observed in Group I (EDC) with a mean value of 12.8 ± 1.9 MPa, followed by Group II (Proanthocyanidin) (11.8 ± 1.6 MPa), Group III (0.2% Chitosan) (9.8 ± 1.5 MPa), and Group IV (Saline control) (6.0 ± 1.2 MPa) as presented in Table 1.

Tukey’s post hoc analysis revealed that Group I (EDC) and Group II (Proanthocyanidin) exhibited comparable push-out bond strength values with no statistically significant difference between them (p > 0.05). Both groups demonstrated significantly higher bond strength compared with Group III (Chitosan) and Group IV (Saline control) (p < 0.05).

Group III (0.2% Chitosan) demonstrated significantly higher push-out bond strength compared with the saline control group (p < 0.05), indicating improved interaction between the modified dentin substrate and resin cement. However, chitosan showed lower bond strength values compared with EDC and proanthocyanidin groups.

Overall, dentin pretreatment with collagen-modifying agents enhanced fiber post retention, with EDC and proanthocyanidin showing the greatest improvement in push-out bond strength, followed by chitosan, whereas saline-treated specimens demonstrated the lowest values.

4. Discussion

The long-term success of fiber post restorations depends largely on the integrity and durability of the bond between the resin cement and radicular dentin. Although fiber posts exhibit favorable mechanical properties and stress distribution similar to dentin, debonding at the resin cement–dentin interface remains one of the major causes of clinical failure.^[3] The present in vitro study evaluated the effect of different dentin pretreatment agents, including EDC, proanthocyanidin, and chitosan, on the push-out bond strength of fiber posts luted with resin cement.

The push-out test was selected for evaluating bond strength because it provides a reliable assessment of the adhesion between the post and surrounding dentin by generating stress distribution similar to clinical loading conditions.^[11] In the present study, a single type of fiber post system and resin luting cement were used for all specimens to minimize variations related to restorative materials and ensure standardization of the bonding protocol. This allowed the differences observed in push-out bond strength values to be primarily associated with the effect of the dentin pretreatment agents. The middle third of the root was selected for evaluation as it provides a relatively uniform post space geometry and minimizes anatomical variations associated with the coronal and apical regions.^[12]

In the present study, dentin pretreatment with all biomodifying agents resulted in higher push-out bond strength values compared with saline-treated specimens. The increase in bond strength observed with EDC, proanthocyanidin, and chitosan may be attributed to their ability to modify the dentinal collagen matrix, improve substrate stability, and enhance interaction between resin cement and root dentin.

Group I (EDC) demonstrated the highest push-out bond strength (12.8 ± 1.9 MPa) among all groups. The improved bonding performance of EDC may be attributed to its collagen cross-linking ability. EDC acts as a zero-length cross-linking agent by activating carboxyl groups present within collagen molecules, resulting in the formation of stable amide bonds between adjacent collagen chains. This cross-linking mechanism increases collagen stiffness, reduces susceptibility to enzymatic degradation, and improves the mechanical properties of dentin.^[5] However, despite its superior bonding performance, the synthetic nature of EDC has stimulated the search for naturally derived dentin biomodifiers. Moreover, the efficacy of EDC is concentration- and application time-dependent, with variations in these parameters influencing the extent of collagen stabilization and its interaction with dentinal tissues.^[4] Therefore, naturally derived agents such as proanthocyanidins and chitosan have gained attention due to their collagen-stabilizing properties, antioxidant potential, and favorable biocompatibility, offering promising alternatives for enhancing dentin bonding durability.

Group II (Proanthocyanidin) demonstrated comparable push-out bond strength values (11.8 ± 1.6 MPa) to EDC, with no statistically significant difference observed between the two groups. The enhanced bonding ability of grape seed-derived proanthocyanidin (GSE-PAC) may be attributed to its strong interaction with dentinal collagen fibrils. PACs are oligomeric flavonoids containing multiple hydroxyl groups that facilitate hydrogen bonding and hydrophobic interactions with collagen, resulting in increased intermolecular cross-linking within the dentin matrix.^[13] This biomodification improves collagen rigidity, enhances resistance against enzymatic degradation, and provides a more stable substrate for resin cement interaction. However, studies have shown that the clinical application of GSE-PAC may be influenced by its concentration-dependent activity and variability in composition, as the quantity and structure of proanthocyanidins can differ according to extraction methods and natural sources.^[14] These limitations have encouraged the exploration of other naturally derived biomodifiers with additional functional properties.

Group III (0.2% Chitosan) demonstrated improved push-out bond strength (9.8 ± 1.5 MPa) compared with the saline control group, although the values were lower than those observed in the EDC and proanthocyanidin groups. The favorable effect of chitosan may be attributed to its unique physicochemical properties, including biocompatibility, antimicrobial activity, and interaction with dentinal components. The protonated amino groups present in chitosan molecules can interact electrostatically with negatively charged dentinal structures and may contribute to modification of the collagen matrix. Additionally, hydrogen bonding and intermolecular interactions between chitosan and dentinal collagen may enhance collagen stability and improve the interaction between resin cement and radicular dentin.^[15] However, the lower bond strength compared with EDC and proanthocyanidin may be related to its comparatively weaker collagen cross-linking potential. Further investigations evaluating chitosan in nanoparticulate form may be warranted, as chitosan nanoparticles offer increased surface area and may enhance interaction with dentinal collagen networks and substrate penetration, potentially improving dentin biomodification efficacy and long-term bond durability.

The saline control group exhibited the lowest push-out bond strength (6.0 ± 1.2 MPa), representing the baseline adhesion achieved without dentin biomodification. In untreated dentin, exposed collagen fibrils remain more susceptible to enzymatic degradation by endogenous proteolytic enzymes such as matrix metalloproteinases (MMPs) and cysteine cathepsins, which may compromise the stability of the resin–dentin interface.^[2][4] The significantly higher bond strength observed in biomodifier-treated groups highlights the importance of dentin substrate modification before fiber post cementation.

The present study has certain limitations. Being an in vitro investigation, it does not completely replicate intraoral conditions such as thermal cycling, mechanical fatigue, and long-term enzymatic degradation. Further studies incorporating aging protocols, cyclic loading, and evaluation of long-term bond durability are required to validate the clinical applicability of these dentin biomodifying agents.

Overall, the findings indicate that pretreatment of radicular dentin with collagen-modifying agents significantly enhances fiber post retention. Among the tested materials, EDC and proanthocyanidin demonstrated the greatest improvement in push-out bond strength, whereas chitosan showed promising potential as a natural biomaterial-based pretreatment agent.

5. Conclusion

Dentin pretreatment with collagen-modifying agents significantly improved the push-out bond strength of fiber posts compared with untreated group. EDC and proanthocyanidin demonstrated comparable and higher bond strength values, while 0.2% chitosan showed improved adhesion compared with untreated dentin. These findings suggest that dentin biomodification may enhance fiber post retention and improve the resin cement–dentin interface.

6. Authors Contributions

Tanvi Pimpale: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Writing – original draft, Writing – review editing, Gaurav Jain: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review editing, Sonali Verma: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Writing – original draft, Writing – review editing, Pradyumna Misra: Conceptualization, Project administration, Supervision.

7. Source of Funding

None

8. Conflicts of Interest

None