A Standardized In Vitro Procedure For Evaluating Surface Microhardness Changes In Nanohybrid Resin Composites After Whey Protein Beverage Exposure

Summary

This protocol describes a standardized in vitro method for evaluating the effects of whey protein beverages on resin composite microhardness. The approach enables controlled assessment of dietary exposure using cyclic immersion and Vickers testing, providing a reproducible model for investigating the interaction between nutritional beverages and restorative biomaterials.

Abstract

The aim of this in vitro study was to evaluate the effect of different whey protein beverages on the surface microhardness of three nanohybrid resin composites. A total of 90 disc-shaped specimens were prepared from three nanohybrid resin composites (n = 30 per material) and subdivided into three groups (n = 10) according to the immersion medium: whey concentrate, whey isolate, and distilled water. The specimens were immersed for 10 min twice daily for 30 days. Surface microhardness was measured at baseline and after immersion using a Vickers microhardness tester, and hardness change was calculated as ΔVHN. The data were analyzed using two-way repeated measures ANOVA and post-hoc tests. The analysis revealed that composite type, immersion solution, and their interaction had statistically significant effects on ΔVHN values (p < 0.0001). The highest hardness loss was observed in the injectable composite exposed to whey concentrate (ΔVHN: 10.4 ± 2.1), whereas the lowest change was recorded in the highly filled composite stored in distilled water (ΔVHN: 0.9 ± 0.4). Immersion in whey concentrate resulted in significantly greater hardness loss in all composites compared to distilled water (p < 0.05). Whey isolate also produced significantly greater hardness loss in all composites except the highly filled composite, in which no statistically significant difference was observed (p = 0.076). These findings indicate that whey protein beverages may reduce the surface microhardness of resin composites, with the extent of degradation depending on both material composition and beverage formulation.

Introduction

The long-term success of resin composite restorations depends on their resistance to complex oral environmental challenges, including mechanical loading, dietary exposure, and chemical degradation^1,2. Nanohybrid resin composites are widely used due to their favorable esthetic and mechanical properties, particularly surface microhardness, which serves as an important indicator of resistance to wear and structural deterioration^3,4.

Previous investigations have demonstrated that exposure to acidic beverages such as fruit juices, carbonated drinks, and energy beverages can adversely affect composite restorations by reducing surface microhardness and altering surface integrity^5,6,7,8. These degradative effects are influenced by multiple factors, including pH, exposure time, and filler–matrix composition. However, most existing experimental models rely on highly acidic solutions, which may not accurately reflect the chemical challenges encountered in contemporary dietary habits.

In contrast, whey protein beverages have gained widespread popularity due to their nutritional value and increasing consumption among fitness-oriented individuals^9,10. Despite their near-neutral pH, these beverages contain lactose, proteins, and other organic components that may interact with the resin matrix and influence material stability. Therefore, evaluating their effects requires a protocol that considers not only acidity but also organic composition and repeated exposure patterns.

The overall goal of this protocol is to provide a standardized and reproducible in vitro method for assessing the effects of whey protein beverages, specifically concentrate and isolate formulations, on the surface microhardness of nanohybrid resin composites. This method enables controlled simulation of daily dietary exposure through cyclic immersion, thereby improving the experimental relevance of in vitro testing conditions.

Compared with conventional acidic-beverage exposure models, this protocol offers several advantages. First, it allows investigation of material degradation under near-neutral pH conditions, allowing evaluation of material responses beyond acid-driven effects. Second, the cyclic immersion design better reflects real-life consumption patterns. Third, the method facilitates comparative evaluation of different composite formulations based on structural characteristics such as filler content and resin matrix composition.

This protocol is particularly suitable for researchers aiming to evaluate the chemical stability of restorative materials under nutritionally relevant conditions, as well as for studies investigating the interaction between dietary components and dental biomaterials. It may also be adapted to test alternative beverage formulations, surface treatments, or newly developed composite materials.

Protocol

The reagents and the equipment used are listed in the Table of Materials.

1. Specimen preparation

1. Prepare a total of 90 disc-shaped specimens from three nanohybrid resin composites (n = 30 per material).
2. Fabricate specimens using cylindrical clear silicone molds (6 mm diameter × 2 mm thickness) to ensure standardized dimensions.
3. Place the composite material into the molds in a single increment (2 mm thickness) and cover the surface with a clear microscope slide to obtain a flat surface.
4. Polymerize the specimen using a blue LED light-curing unit (≈440–480 nm emission spectrum) positioned at approximately 1 mm from the surface with an output intensity of 1000 mW/cm² for 20 s.
5. Verify curing light intensity using a radiometer before each polymerization cycle to ensure consistent light exposure.
6. Store the prepared specimens in distilled water at 37 °C for 24 h to allow post-polymerization and simulate initial oral conditions.
   NOTE: Specimens may be stored in distilled water at 37 °C for up to 24 h before further processing.

2. Finishing and polishing procedure

1. Finish and polish the upper surfaces of all specimens using aluminum oxide–impregnated polishing discs. All polishing procedures should be performed by a single operator to ensure surface standardization.
2. Apply polishing sequentially using medium, fine, and superfine discs for 10 s each under water cooling at approximately 10,000 rpm with light pressure and a unidirectional rotational movement.
3. Replace polishing discs after every five specimens to maintain consistent abrasive efficiency and ensure surface standardization.
4. After each polishing step, rinse specimens with distilled water for 10 s to remove debris and gently air dry for 5 s before proceeding to the next disc.
5. Mark the unpolished surface of each specimen with a unique identification code to ensure consistent orientation during subsequent testing.
6. Store specimens in distilled water at 37 °C for 24 h prior to baseline microhardness testing.
   NOTE: Specimens may be stored in distilled water at 37 °C for up to 24 h before baseline testing.

3. Baseline microhardness measurement

1. Perform baseline Vickers microhardness testing (VHN) following previously published protocols using a Vickers microhardness tester¹¹.
2. Apply a 200 g load for 15 s dwell time.
3. Make three indentations per specimen with at least 1 mm spacing. Perform indentations on the central surface area of each specimen to avoid edge effects.
4. Calculate mean baseline VHN.
5. Perform measurements by a single calibrated operator to minimize variability.
   NOTE: Specimens may be stored in distilled water at 37 °C for up to 24 h before immersion.

4. Preparation of immersion media

1. Prepare two whey protein formulations: whey protein concentrate (WC) and whey protein isolate (WI) using commercially available powders.
2. Dissolve 25 g of whey protein powder in 200 mL of distilled water (0.125 g/mL) to prepare the immersion solutions.
3. Mix the solutions using a laboratory shaker for 60 s until complete dissolution is achieved.
4. Allow the solutions to equilibrate at room temperature for 5 min to minimize foam formation and ensure homogeneity.
5. Measure and record the pH of each solution using a calibrated digital pH meter before immersion.
6. Prepare fresh whey solutions prior to each immersion cycle to ensure consistent chemical composition and avoid protein degradation.
7. Use 10 mL of immersion solution per specimen to ensure complete submersion during each cycle.

5. Immersion procedure

1. Divide each composite group into three subgroups (n = 10).
2. Immerse specimens in the assigned solutions for 10 min, twice daily. Select immersion duration and frequency based on previously established in vitro beverage exposure models designed to simulate repeated daily consumption patterns⁷.
3. After each immersion cycle, rinse the specimens with distilled water and place them in artificial saliva for 10 min to simulate short-term salivary exposure.
4. Store the specimens in distilled water between immersion cycles at 37 °C until the next exposure cycle.
5. Apply this procedure only to the whey-exposed groups. Maintain the control group continuously in distilled water (DW) at 37 °C throughout the experimental period.
   NOTE: The immersion protocol may be paused between cycles and resumed on the following day without affecting experimental consistency.
6. Continue the immersion protocol for 30 days.

6. Post-immersion microhardness measurement

1. Rinse specimens with distilled water and gently dry.
2. Perform Vickers microhardness testing using the same parameters (200 g, 15 s).
3. Record three indentations and calculate the mean post-immersion VHN.
4. Determine ΔVHN for each specimen.

7. Statistical analysis

1. Analyze Vickers microhardness values using a repeated measures ANOVA model, with time point (baseline and post-immersion) as the within-subject factor and composite type and immersion solution as between-subject factors.
2. When appropriate, calculate ΔVHN values as descriptive outcome measures to express the magnitude of microhardness change for each specimen.
3. Perform post-hoc pairwise comparisons using Tukey’s honestly significant difference (HSD) test.
4. Calculate 95% confidence intervals for all group means.
5. Report effect sizes using partial eta-squared (η²p) for ANOVA results.
6. Compare pH values between whey protein solutions using Welch’s t-test.
7. Set the level of statistical significance at p < 0.05.
8. In addition to absolute ΔVHN values, calculate percentage microhardness change using the following formula: [(baseline VHN − post-immersion VHN) / baseline VHN] × 100.

Results

Descriptive statistics

The mean ΔVHN (±SD) values of the composite-solution combinations are presented in Table 1.

...

Resin composite	Solution	ΔVHN (Mean ± SD)	95% CI	n
EA

Discussion

The present protocol provides a standardized approach to evaluating the chemical impact of whey protein beverages on resin composite microhardness. Critical steps include uniform specimen fabrication, controlled light-curing conditions, and standardized Vickers testing parameters. Maintaining consistent specimen dimensions and curing intensity is essential, as variations may influence the degree of conversion and mechanical stability. Additionally, strict control of immersion duration and temperature ensures reproducibil...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aluminum oxide polishing discs	RubyPlaton, Inci Dental Tibbi Malz. San. ve Tic. Ltd. Sti., Istanbul, Turkey	5714	Medium, fine, superfine, used for finishing and polishing
Analytical balance			Used for weighing materials
Artificial saliva solution	DIN 53160-1, Testonic Laboratory, Istanbul, Turkey		Used for storage between immersion cycles
Digital pH meter	Hanna Instruments, Woonsocket, RI, USA	HI2211	Measurement of beverage pH
Distilled water	Not specified	Not applicable	Used as control solution and for specimen storage
Glass microscope slide	Ningbo Trueline Imp. & Exp. Co., Ltd, Zhejiang, China	7102	Used to obtain a flat specimen surface during composite placement
Graduated cylinder	Not specified	Not applicable	Used for measuring liquid volumes
Highly filled nano hybrid resin composite (EA)	Estelite Asteria (Tokuyama Dental, Tokyo, Japan)	10942	Used as restorative material; differs in filler content and resin matrix composition
Incubator	Memmert Universal, Memmert GmbH + Co. KG, Schwabach, Germany	UN55	Used for storage at 37 °C
Injectable nano hybrid resin composite (GUI)	G-ænial Universal Injectable (GC Corporation, Tokyo, Japan)	12365	Used as restorative material; differs in filler content and resin matrix composition
Laboratory beaker	Not specified	Not applicable	Used for preparation of whey protein solutions
Laboratory shaker	Not specified	Not applicable	Used for mixing and homogenization of solutions
LED light-curing unit and radiometer	Woodpecker LED curing unit (Guilin Woodpecker Medical Instrument Co., Ltd., Guangxi, China)	FD-300	Polymerization and  verification of light intensity
Micromotor handpiece	FX205, NSK Ltd., Kanuma, Japan	C1056002	Used in polishing procedure
Moderately filled nano hybrid resin composite (FU)	Filtek Universal (3M ESPE, St. Paul, MN, USA)	6570A2	Used as restorative material; differs in filler content and resin matrix composition
Silicone mold	Not specified	Not applicable	Disc-shaped, 6 mm diameter × 2 mm thickness, used for specimen fabrication
Vickers microhardness tester	HMV, Shimadzu Corporation, Kyoto, Japan	HMV-G31	Vickers hardness number (VHN) testing
Whey protein concentrate powder	Whey Protein Strawberry Flavor, Proteinocean Gida AS., Ankara, Turkey		Lactose content: ~1–2%, protein content: ~85–90%
Whey protein isolate powder	Isolate Whey Chocolate Flavor, Proteinocean Gida AS., Ankara, Turkey		Lactose content: ~4–6%, protein content: ~70–80%