Silk fibroin nanoparticle reinforcement in glass ionomers: a shear bond strength study

Abstract

Glass ionomer cement (GIC) is a widely used material in Dentistry that has some limitations. Silk fibroin (SF) is a natural polymer with potential benefits. Thus, this study has incorporated SF into two GICs.

Aims  Evaluate the influence of this modification on shear bond strength (SBS) and characterize the materials with scanning electron microscopy (SEM).

Methods  Two types of GICs, Maxxion R (M) and Ketac Molar (KM), were used. For each GIC, four formulations were prepared: a control (0% SF) and three experimental concentrations (1%, 3%, and 5% SF nanoparticles). A total of 160 bovine tooth specimens were prepared for SBS analysis, with 20 specimens allocated to each of the 8 experimental groups (2 GIC types x 4 SF concentrations). Subsequently, each group was subdivided into two subgroups of 10 specimens each: one subjected to thermocycling and the other serving as a non-thermocycled control. Additionally, 8 specimens (one from each GIC/SF concentration group) were prepared for SEM analysis. Results were analyzed using the Shapiro-Wilk test, followed by two-way ANOVA and Duncan’s complementary test, at a significance level of 5%.

Results  Concentrations showed statistically significant differences after thermocycling (p = 0.01) for M, revealing an increase in SBS (G1M = 0.69±0.18; G2M = 1.95±0.17; G3M = 1.96±0.17; G4M = 1.54±0.15). KM showed SBS values significantly higher with the addition of SF, with no differences between concentrations (p = 0.001) (G1KM = 1.19±0.21; G2KM = 4.47±0.75; G3KM = 4.31±0.68; G4KM = 3.62±0.54). SEM analysis revealed an increase in cracks with higher concentrations of SF.

Conclusions  Finally, the incorporation of 1% SF nanoparticles into GICs resulted in a significant increase in SBS, particularly following thermocycling.

Keywords
Glass ionomer cements; Bombyx; Fibroins; Nanoparticles; Shear strength

Introduction

Glass ionomer cement (GIC) is a commonly used material in dentistry¹, especially in procedures such as restorations, cementations, and sealants for pits and fissures². Its widespread adoption stems from its unique combination of properties, including fluoride release, biocompatibility, and chemical adhesion to dental hard tissues¹. Structurally, GICs consist of a powder phase (calcium fluoride, aluminum oxide, silicate) and a liquid phase (polymeric water-soluble acid), often supplemented with components like tartaric acid and itaconic acid to control setting kinetics¹,³.

Despite these advantages, GICs exhibit inherent limitations that significantly impact their long-term clinical performance². These include low mechanical resistance to abrasion and fracture toughness, reduced translucency, friability, low wear resistance, and high dissolution in water sorption⁴-⁶. Such deficiencies can compromise the durability and integrity of restorations, particularly in areas subjected to occlusal forces, leading to premature failures and the need for frequent replacements. Consequently, extensive research efforts have focused on modifying GICs to overcome these shortcomings, often through the incorporation of various nanoparticles. Examples include silver diamine fluoride (aimed at enhancing antibacterial properties and bond strength)⁷, nanohydroxyapatite (for improved physical-biological properties and remineralization potential)⁴,⁸, nanosilver (for superior mechanical and antimicrobial characteristics)⁹, and silica nanoparticles (with varying effects on bond strength)¹⁰. These studies highlight a clear trend toward leveraging nanotechnology to optimize GIC performance, yet underscore the importance of material selection and concentration.

Silk fibroin (SF), a natural protein polymer derived from the silkworm Bombyx mori, has emerged as a highly promising biomaterial with a long history of use beyond the traditional textile industry¹¹. Its unique molecular structure, comprising disulfide bond-linked light and heavy chains¹², confers a remarkable set of properties, including exceptional mechanical strength and toughness, excellent biocompatibility, processing flexibility, and resistance to chemical and microbial degradation¹³-¹⁵. These attributes make SF an attractive candidate for a wide array of biomedical and biotechnological applications, from tissue engineering scaffolds to drug delivery systems. In dentistry, SF has demonstrated versatile applications, showing notable performance in the prevention of dental caries¹⁶, the fight against periodontal disease¹⁷, and even in pulp tissue engineering¹⁸.

The clinical success and longevity of any restorative material are critically dependent on its ability to establish and maintain a strong and durable bond with the dental surface, effectively resisting the complex forces acting within the oral cavity¹⁹. Shear bond strength (SBS), specifically, quantifies the material’s resistance to forces that attempt to slide the restorative material past the tooth structure²⁰. Therefore, achieving higher and more reliable SBS values directly translates to improved bonding and enhanced clinical longevity²¹.

Considering the known mechanical limitations of GICs and the exceptional mechanical properties of SF, the incorporation of SF nanoparticles presents a compelling strategy to reinforce GICs and potentially improve their adhesive performance. However, despite the significant potential of SF and the ongoing need to enhance GIC properties, there is a notable paucity of research specifically investigating the effect of SF nanoparticle incorporation on the SBS of GIC, particularly under simulated oral aging conditions like thermocycling. This gap in the literature underscores the necessity for comprehensive studies to determine the efficacy and optimal incorporation parameters of SF in GICs.

Thus, despite their recognized advantages, glass ionomer cements (GICs) present inherent limitations, particularly concerning their long-term mechanical properties and adhesive stability in the dynamic oral environment. Addressing these challenges is critical for improving the durability and clinical longevity of GIC restorations. While various nanomaterials have been explored to overcome these drawbacks, the potential of silk fibroin (SF), a biocompatible natural biopolymer renowned for its exceptional mechanical strength and structural integrity, remains largely unexplored as a reinforcing agent for GICs, specifically concerning its impact on their bond strength to dental substrates. This pioneering study aimed to comprehensively investigate the effects of SF nanoparticle incorporation on a crucial physical-chemical property: the shear bond strength of two widely used restorative GICs (Maxxion R and Ketac Molar). We systematically evaluated three distinct SF concentrations (1%, 3%, and 5%) and assessed the crucial influence of thermocycling, which simulates the long-term thermal and mechanical stresses within the oral cavity.

By enhancing the shear bond strength, especially under aging conditions, this research directly addresses a key challenge in GIC performance, paving the way for more durable and clinically reliable dental restorations and advancing the field of restorative dentistry by offering a novel approach to reinforce these important materials. Furthermore, the surface morphology of the modified materials was critically examined using scanning electron microscopy (SEM) to elucidate the microstructural effects of SF incorporation and correlate them with observed mechanical changes. Based on these aims, the null hypothesis of this study was that the incorporation of SF nanoparticles would not affect the GIC shear bond strength, regardless of the presence or absence of thermocycling.

Material and Methods

This study employed a controlled experimental design to investigate the effect of SF nanoparticle incorporation on the SBS of two distinct GICs, Maxxion R and Ketac Molar. Specimens were prepared from bovine teeth and assigned to various groups based on GIC type and SF concentration (0%, 1%, 3%, and 5%). The influence of thermocycling, simulating oral aging, was also assessed. The primary outcome measured was SBS, while SEM was utilized to characterize the material surfaces following SF incorporation.

GICs used were Maxxion R (FGM, Joinville - SC, Brazil) and Ketac Molar (3M ESPE, Seefeld, Germany). Their compositions and manufacturers are detailed in Table 1. For clarity and ease of reference, Maxxion R will be referred to as M and Ketac Molar as KM throughout the text. Both GICs were modified by incorporating SF nanoparticles at three distinct concentrations: 1%, 3%, and 5% by weight.

Table 1
Detailed composition and manufacturers of the GICs used in the study.

	Composition	Manufacturer
Maxxion	After mixing the phases: aluminofluorosilicate glass, polycarboxylic acid, tartaric acid, calcium fluoride, radiopacifiers, and water.	FGM, Joinville - SC, Brazil
Ketac Molar	Liquid: polialquenoic acid, tartaric acid, and water. Powder: fluorosilicate glass, Al-Ca-La copolymer (acrylic acid and maleic acid).	3M ESPE, Seefeld, Germany

All GIC manipulations, including powder-to-liquid ratios and mixing times for both commercial and SF-modified formulations, strictly adhered to the respective manufacturers’ instructions.

A total of 160 specimens were prepared using bovine teeth. These specimens were initially divided into two main categories based on the GIC type (M or KM), with 80 specimens allocated to each GIC. Within each GIC type, specimens were further subdivided into four experimental groups (n=20 specimens per group), designated as follows:

Maxxion R (M) Groups:

• G1M (Control): Maxxion R without SF incorporation (n=20)

• G2M (1% SF): Maxxion R with 1% SF incorporation (n=20)

• G3M (3% SF): Maxxion R with 3% SF incorporation (n=20)

• G4M (5% SF): Maxxion R with 5% SF incorporation (n=20)

• Ketac Molar (KM) Groups:

• G1KM (Control): Ketac Molar without SF incorporation (n=20)

• G2KM (1% SF): Ketac Molar with 1% SF incorporation (n=20)

• G3KM (3% SF): Ketac Molar with 3% SF incorporation (n=20)

• G4KM (5% SF): Ketac Molar with 5% SF incorporation (n=20)

Subsequently, each of these 8 specific groups (G1M, G2M, G3M, G4M, G1KM, G2KM, G3KM, and G4KM) was further subdivided into two subgroups (n=10 specimens per subgroup): one subjected to thermocycling simulation and the other serving as a non-thermocycled control. This resulted in a total of 80 thermocycled specimens (40 for M and 40 for KM) and 80 non-thermocycled specimens (40 for M and 40 for KM), summing to the 160 specimens used for SBS analysis.

Additionally, one representative specimen from each of the 8 specific groups (G1M, G2M, G3M, G4M, G1KM, G2KM, G3KM, and G4KM) was prepared explicitly for surface examination using scanning electron microscopy (SEM). These 8 specimens were prepared separately and were not included in the SBS testing pool.

Synthesis of nanoparticles and incorporation into GIC

The synthesis began with the acquisition of Bombyx mori cocoons, with no prior treatment of the fibers to preserve their properties. The processes of degumming, dissolution, dialysis, and lyophilization were performed using 5 g of shredded silkworm cocoons. All steps followed the protocol described by Torres et al.¹¹ (2023). The particles employed in this research exhibited nanometric dimensions, with their size predominantly ranging from 250 to 400 nm, as comprehensively characterized by Torres et al.¹¹ (2023).

The SF nanoparticles were incorporated into powder exclusively in a proportion of 1%, 3% and 5% by weight. These values were measured using an analytical precision balance (Urano - UA 220/0.0001) for powder dosing. After weighing, the mixture was placed in a plastic Eppendorf tube and stirred using a stirrer (Digital Amalgamator YG-100 – Kondentech) for 30 seconds to obtain a homogeneous dispersion of the particles. The materials were handled under controlled temperature conditions (23 ± 1 °C) and relative humidity (50 ± 5%).

Sample preparation

The selected bovine teeth were thoroughly cleaned using curettes, water, pumice, and a brush coupled to a micromotor. Subsequently, teeth were stored in distilled water at 4°C. The detailed sequence for preparing the specimens is visually presented in Figure 1.

Figure 1
Adaptation from Borsatto et al.²² (2013) and Torres et al.²³ (2005). Specimens preparation. A: Sections of the root portions and crowns in mesio-distal direction. B: Inclusion in acrylic resin. C: Wear of specimens. D: Circular area delimitation. E, F, G, H: metallic locking device. I: Specimens.

Initially, root portions were carefully removed using a cutting machine (Minitom, Struers A/S, Copenhagen, DK-2610, Denmark), and the crowns were sectioned in a mesio-distal direction to obtain standardized tooth fragments (Figure 1A). These fragments were then embedded in self-curing acrylic resin within PVC cylinders (Figure 1B) to facilitate handling and standardization. Following this, the embedded specimens were polished using a polishing machine under refrigeration (Struers A/S, Copenhagen, Denmark) with 180-600 grit sandpaper disks to achieve flat, standardized surfaces of superficial dentin (Figure 1C). After polishing, specimens were washed and stored in distilled water at room temperature for 24 hours. To define the bonding area, a circular area with a diameter of 3mm was delimited on the prepared dentin surface of each specimen using perforated adhesive tape and a 3mm-diameter circular punch (Figure 1D). To ensure impartiality and minimize bias, specimens were then randomly assigned to their respective experimental groups using a computer-generated random number sequence. No dentin conditioner (polyacrylic acid) was applied before GIC insertion, to isolate and evaluate the effect of SF incorporation on SBS, without the influence of additional surface treatments.

For the restoration phase, a custom-designed Teflon locking device was employed. This device, composed of two half-circle parts, fits together to form a precise circular hole when assembled (illustrated in Figure 1E). After manipulation of the GICs according to the manufacturer’s instructions, using a plastic spatula, the material was carefully inserted into this device. Matrices were adapted over the device to ensure that the GIC cylinder was precisely positioned over the previously isolated circular area on the dentin surface (Figure 1F-H). The GICs were allowed to undergo their initial setting within the device, strictly adhering to each manufacturer’s recommended setting times (approximately 4-5 minutes for M and 2.5-4 minutes for KM). Following this initial setting period, specimens were carefully removed from the device, and the matrices were taken off. The resulting GIC cylinder, with dimensions of Ø 4 x 3mm, was thus firmly adhered to the dentin surface (Figure 1I). All prepared specimens were then stored in distilled water at 37ºC for 24 hours before further analysis.

Degradation of the adhesive interface

To simulate the degradation of the adhesive interface, the restored specimens were removed from distilled water and subjected to simulated oral cavity conditions. Thermal cycling was performed in a thermocycling machine (Ética Equipamentos Científicos S/A, São Paulo, SP, Brazil). Each cycle consisted of immersing the specimens alternately in water baths at two different temperatures: 5°C and 55°C. The specimens were kept in each bath for 30 seconds, with a transfer time of 7 seconds between baths. The experiment was conducted for a total of 12,000 thermal cycles, equivalent to a six-month simulation period of exposure to oral conditions. This regimen was adopted based on established protocols in previous literature, recognized for simulating clinically relevant aging conditions²²,²⁴. After thermal cycling, the specimens were once again stored in water at 37°C.

Shear bond strength (SBS) analysis

Once identified within their respective groups, specimens were tested for SBS using a Universal Testing Machine (Mod. MEM 2000; EMIC Ltda, São José dos Pinhais, PR, Brazil). The tests were conducted at a speed of 0.5mm/min with a 50Kgf load cell. Adhesive strength was recorded in Kgf/cm and converted into MPa.

Statistical analysis

The sample size (n=20 per GIC type, divided into subgroups of n=10 for each condition) was determined based on similar previous studies in the literature evaluating SBS of GICs with nanoparticle incorporation, ensuring sufficient statistical power to detect relevant differences.

Adhesive strength values measured in MPa were used for statistical analysis. The data were subjected to the Shapiro-Wilk test followed by two-way ANOVA and Duncan’s multiple range test, with a significance level set at 5%. The statistical analysis was performed using the SPSS statistical program version 20.0 (Chicago, IL, USA).

Results

Table 2 presents the mean values and standard deviations of SBS for the M groups before and after thermocycling. A statistically significant difference in SBS values (p=0.01) was observed when evaluating the different concentrations of SF incorporated into GIC after the 6-month thermocycling simulation (12,000 cycles). While a reduction in SBS values was noted for G1M (control) after thermocycling, G2M, G3M, and G4M (with SF) subjected to thermocycling presented a significant increase in SBS compared to G1M.

Table 2
Mean values and standard deviation (in MPa) of Maxxion R before and after thermocycling.

Maxxion (M)	Concentration				p-value
	G1 Control	G2 (1% SF)	G3 (3% SF)	G4 (5% SF)
No thermocycling	2,33±0,24aA	2,14±0,40abA	1,33±0,31bcA	1,33±0,23bcA	0,01
With thermocycling	0,69±0,18cB	1,95±0,17baA	1,96±0,17baA	1,54±0,15baA

• Different lowercase letters indicate statistically significant differences among columns (p < 0.05).
• Different capital letters indicate statistically significant differences between rows (p < 0.05).

Table 3 presents mean values and standard deviations of SBS for Ketac Molar groups before and after thermocycling. Similar to the Maxxion R group, SBS values for the groups without thermocycling were lower and statistically significant for G2KM, G3KM and G4KM compared to G1KM. Interestingly, when subjected to thermocycling, SBS values were significantly higher at all SF concentrations.

Table 3
Mean values and standard deviation (Mpa) of Ketac Molar before and after thermocycling

Ketac (KM)	Concentration				p-value
	G1 Control	G2 (1% SF)	G3 (3% SF)	G4 (5% SF)
No thermocycling	5,20±0,66aA	3,57±0,85abA	2,20±0,65bcA	2,13±0,32bcA	0,001
With thermocycling	1,19±0,21cB	4,47±0,75aA	4,31±0,68aB	3,62±0,54baA

• Different lowercase letters indicate statistically significant differences among columns (p < 0.05).
• Different capital letters indicate statistically significant differences between rows (p < 0.05).

There was a decrease in SBS in both materials when not subjected to thermocycling, especially in G3 and G4. However, G2, G3 and G4 after thermocycling achieved shear resistance values comparable to G1 without thermocycling, improving the bond strength with the dental system.

When comparing the two materials, initially G1KM exhibited higher values, which were statistically different from G1M (p=0.001). After thermocycling, both materials reduced SBS, with no statistical difference (p=0.392). With G2 of both materials showed similar SBS values before thermocycling, but G2KM still presented significantly higher values after thermocycling (p=0.010). The same situation was repeated for G3KM and G4KM, respectively (p=0.001 and p≤0.001).

Surface analysis

Figure 2 illustrates SEM images of GICs after SF incorporation in different concentrations. Structure differences can be observed as an increase in cracks with higher concentrations of SF added to GIC.

Figure 2
SEM (x1000) of modified GICs. A: Ketac 1% SF; B: Maxxion 1% SF; C: Ketac 3% SF; D: Maxxion 3% SF; E: Ketac 5% SF; F: Maxxion 5% SF.

Discussion

The primary objective of this study was to evaluate the influence of SF nanoparticle incorporation on the SBS of two different GICs, M and KM, under both non-thermocycled and thermocycled conditions. Based on our findings, the null hypothesis, which stated that SF incorporation would not affect GIC SBS, was unequivocally rejected. The results demonstrate a significant influence of SF nanoparticles on the adhesive performance of both GICs, particularly after simulated oral aging via thermocycling, highlighting the potential for biopolymeric reinforcement in these materials.

To address their inherent limitations, GICs have undergone various modifications, as evidenced by extensive research⁴,⁷-¹⁰. This effort is part of a broader trend in the dental field, where incorporating diverse products into materials consistently yields promising results²²,²⁵,²⁶.

Literature presents different outcomes for nanoparticle-modified GICs. Incorporation of silver diamine fluoride improved bond strength without adversely affecting microleakage, supporting its use as a functional additive⁷. Nanosilver-enriched GICs demonstrated superior mechanical and bond strength properties, reinforcing the concept that metallic nanoparticles can act as strengthening agents⁹. Conversely, incorporation of silica nanoparticles at 0.5 wt% did not alter microshear bond strength, showing that not all nanoparticle modifications produce interfacial improvements¹⁰. Studies with nanohydroxyapatite have mainly reported beneficial effects on physical-biological properties, with bond strength effects varying depending on the formulation and protocol⁴,⁸. Within this context, our findings show that 1% SF nanoparticles significantly improved SBS after thermocycling, highlighting their potential as a novel biopolymeric reinforcement. These results align with the general trend that carefully selected and dosed nanoparticles can enhance GIC performance, while also underscoring that composition and concentration are decisive for achieving favorable outcomes.

The SBS values observed in our study, particularly for the non-thermocycled groups, were in some instances lower or comparable to those reported in other studies for KM (e.g., 3.08 MPa cited in Marti et al.²⁷ (2014)). This variation can be attributed to several factors inherent in GIC research. GICs are known for their sensitivity to moisture during their setting phase, which can profoundly affect their final mechanical and adhesive properties²⁸. Furthermore, the bond between GIC and dental tissue is a complex interplay involving the reaction between phosphate ions in the dentin/enamel and carboxylate groups in the GIC’s polyacrylic acid, influenced by variables such as the type of dental substrate, the presence of a smear layer, and the specific application protocol³. While our study aimed for standardized preparation of the dentin surface, these factors could explain some of the observed variabilities and lower absolute bond strength values when compared to studies employing different methodologies or conditioning protocols.

An important practical consideration from our study is that the optimal 1% SF proportion did not adversely affect the handling characteristics of the GICs. The modified materials exhibited satisfactory agglutination and maintained working times and final setting times comparable to their commercial counterparts. This is clinically significant, as it suggests that the integration of SF nanoparticles at this concentration can enhance mechanical properties without compromising the established clinical manipulation protocols, thus preserving the material’s user-friendliness. The SEM analysis provided visual evidence corroborating these findings: at 1% SF incorporation, the GIC structure appeared largely undisturbed. However, the SEM analysis also revealed a crucial insight: higher concentrations of SF (above 3%) were associated with an increase in visible cracks within the GIC matrix. This morphological alteration directly correlates with the observed detrimental effect on bond strength at these higher concentrations. This suggests a critical threshold for SF incorporation. At the same time, low concentrations contribute to reinforcement, excessive loading may lead to nanoparticle agglomeration or structural inhomogeneities that weaken the material rather than strengthen it, serving as stress concentration points that propagate cracks.

It is important to acknowledge the challenges in directly comparing our findings with existing literature, primarily due to the novel nature of incorporating SF nanoparticles into GICs for SBS enhancement. The existing literature on nanoparticle-modified GICs often presents conflicting results, which can be attributed to a multitude of factors, including variations in the type and concentration of nanoparticles, material composition, application methods, tooth preparation protocols, storage conditions, and aging regimens²⁸-³⁰. This inherent variability makes direct, quantitative comparisons difficult and underscores the need for standardized testing protocols in this field. Our study, therefore, serves as a foundational step in exploring SF’s potential in this specific dental application.

Therefore, GIC containing 1% SF nanoparticles represents a promising restorative dental material, particularly for applications where it will be subjected to temperature variations or require enhanced durability in the oral environment. The significant improvement in SBS observed after thermocycling suggests that this modification could lead to more resilient and long-lasting restorations, especially in areas experiencing higher functional stresses. While our findings are encouraging, further research is undoubtedly warranted before clinical application. Future studies should ideally explore a broader spectrum of mechanical properties (e.g., flexural strength, wear resistance), assess long-term degradation in more complex oral environments, and investigate the precise surface interactions between SF nanoparticles and the GIC matrix at a molecular level to fully elucidate the reinforcement mechanisms. Additionally, in vivo studies would be crucial to validate these promising in vitro results and assess the clinical performance and biocompatibility in a living system.

Based on the results obtained, the addition of 1% SF nanoparticles to GICs is the ideal concentration since it does not compromise SBS without thermocycling. Furthermore, under thermal degradation, this modification leads to a significant increase in SBS, indicating potential improvement in GIC performance with the incorporation of SF nanoparticles.

Acknowledgments

Jeferson Aparecido Moreto is CNPq fellow.

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• Data availability:
  Datasets related to this article will be available upon request to the corresponding author.
• Funding:
  This research was supported by the São Paulo Research Foundation (FAPESP) [grant number 2021/14202-0].

Edited by

• Editor:
  Dr. Altair A. Del Bel Cury

Data availability

Datasets related to this article will be available upon request to the corresponding author.

Publication Dates

• Publication in this collection
  20 Apr 2026
• Date of issue
  2026

History

• Received
  5 Aug 2024
• Accepted
  13 Sept 2025