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....

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 (≈440̵....

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%