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5 minutes

The authors declare that no funding was received for this study.

The reagents and the equipment used are listed in the Table of Materials.
1. Specimen preparation
<ol>
	<li>Prepare a total of 90 disc-shaped specimens from three nanohybrid resin composites (n = 30 per material).</li>
	<li>Fabricate specimens using cylindrical clear silicone molds (6 mm diameter &#215; 2 mm thickness) to ensure standardized dimensions.</li>
	<li>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.</li>
	<li>Polymerize the specimen using a blue LED light-curing unit (&#8776;440&#8211;480 nm emission spectrum) positioned at approximately 1 mm from the surface with an output intensity of 1000 mW/cm2 for 20 s.</li>
	<li>Verify curing light intensity using a radiometer before each polymerization cycle to ensure consistent light exposure.</li>
	<li>Store the prepared specimens in distilled water at 37 &#176;C for 24 h to allow post-polymerization and simulate initial oral conditions. 
	NOTE:&#160;Specimens may be stored in distilled water at 37 &#176;C for up to 24 h before further processing.</li>
</ol>
2. Finishing and polishing procedure
<ol>
	<li>Finish and polish the upper surfaces of all specimens using aluminum oxide&#8211;impregnated polishing discs. All polishing procedures should be performed by a single operator to ensure surface standardization.</li>
	<li>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.</li>
	<li>Replace polishing discs after every five specimens to maintain consistent abrasive efficiency and ensure surface standardization.</li>
	<li>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.</li>
	<li>Mark the unpolished surface of each specimen with a unique identification code to ensure consistent orientation during subsequent testing.</li>
	<li>Store specimens in distilled water at 37 &#176;C for 24 h prior to baseline microhardness testing. 
	NOTE:&#160;Specimens may be stored in distilled water at 37 &#176;C for up to 24 h before baseline testing.</li>
</ol>
3. Baseline microhardness measurement
<ol>
	<li>Perform baseline Vickers microhardness testing (VHN) following previously published protocols using a Vickers microhardness tester11.</li>
	<li>Apply a 200 g load for 15 s dwell time.</li>
	<li>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.</li>
	<li>Calculate mean baseline VHN.</li>
	<li>Perform measurements by a single calibrated operator to minimize variability. 
	NOTE: Specimens may be stored in distilled water at 37 &#176;C for up to 24 h before immersion.</li>
</ol>
4. Preparation of immersion media
<ol>
	<li>Prepare two whey protein formulations: whey protein concentrate (WC) and whey protein isolate (WI) using commercially available powders.</li>
	<li>Dissolve 25 g of whey protein powder in 200 mL of distilled water (0.125 g/mL) to prepare the immersion solutions.</li>
	<li>Mix the solutions using a laboratory shaker for 60 s until complete dissolution is achieved.</li>
	<li>Allow the solutions to equilibrate at room temperature for 5 min to minimize foam formation and ensure homogeneity.</li>
	<li>Measure and record the pH of each solution using a calibrated digital pH meter before immersion.</li>
	<li>Prepare fresh whey solutions prior to each immersion cycle to ensure consistent chemical composition and avoid protein degradation.</li>
	<li>Use 10 mL of immersion solution per specimen to ensure complete submersion during each cycle.</li>
</ol>
5. Immersion procedure
<ol>
	<li>Divide each composite group into three subgroups (n = 10).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Store the specimens in distilled water between immersion cycles at 37 &#176;C until the next exposure cycle.</li>
	<li>Apply this procedure only to the whey-exposed groups. Maintain the control group continuously in distilled water (DW) at 37 &#176;C throughout the experimental period. 
	NOTE: The immersion protocol may be paused between cycles and resumed on the following day without affecting experimental consistency.</li>
	<li>Continue the immersion protocol for 30 days.</li>
</ol>
6. Post-immersion microhardness measurement
<ol>
	<li>Rinse specimens with distilled water and gently dry.</li>
	<li>Perform Vickers microhardness testing using the same parameters (200 g, 15 s).</li>
	<li>Record three indentations and calculate the mean post-immersion VHN.</li>
	<li>Determine &#916;VHN for each specimen.</li>
</ol>
7. Statistical analysis
<ol>
	<li>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.</li>
	<li>When appropriate, calculate &#916;VHN values as descriptive outcome measures to express the magnitude of microhardness change for each specimen.</li>
	<li>Perform post-hoc pairwise comparisons using Tukey&#8217;s honestly significant difference (HSD) test.</li>
	<li>Calculate 95% confidence intervals for all group means.</li>
	<li>Report effect sizes using partial eta-squared (&#951;2p) for ANOVA results.</li>
	<li>Compare pH values between whey protein solutions using Welch&#8217;s t-test.</li>
	<li>Set the level of statistical significance at p &#60; 0.05.</li>
	<li>In addition to absolute &#916;VHN values, calculate percentage microhardness change using the following formula: [(baseline VHN &#8722; post-immersion VHN) / baseline VHN] &#215; 100.</li>
</ol>

<ol>
	<li>Ferracane JL. Resin composite&#8212;state of the art. Dent Mater. 2011;27(1):29&#8211;38.</li>
	<li>Ilie N, Hickel R. Resin composite restorative materials. Aust Dent J. 2011;56(Suppl 1):59&#8211;66.</li>
	<li>Sideridou ID, Karabela MM, Vouvoudi EC. Dynamic mechanical properties and degree of conversion of light-cured dimethacrylate-based dental resins. Dent Mater. 2008;24(6):737&#8211;743.</li>
	<li>Erdemir U, Yildiz E, Eren MM, Ozel S. Surface hardness evaluation of different composite resin materials. J Dent. 2012;40(1):17&#8211;22.</li>
	<li>Aliping-McKenzie M, Linden RWA, Nicholson JW. The effect of Coca-Cola and fruit juices on the surface hardness of glass-ionomers and composite resins. J Oral Rehabil. 2004;31(11):1046&#8211;1052.</li>
	<li>Tahmassebi JF, Duggal MS, Malik-Kotru G, Curzon MEJ. Soft drinks and dental health: A review of the current literature. J Dent. 2006;34(1):2&#8211;11.</li>
	<li>Borges AFS, Caneppele TMF, Luz MAAC, Pucci CR. Effect of acidic drinks on surface roughness and microhardness of composite resins. J Appl Oral Sci. 2014;22(6):547&#8211;553.</li>
	<li>Ehlen LA, Marshall TA, Qian F, Wefel JS, Warren JJ. Acidic beverages increase the risk of in vitro. tooth erosion. Nutr Res. 2008;28(5):299&#8211;303.</li>
	<li>Vasconcelos QDJS, Bachur TPR, Arag&#227;o GF. Whey protein supplementation and its potentially adverse effects on health: a systematic review. Appl Physiol Nutr Metab. 2021;46(1):27&#8211;33.</li>
	<li>Kaur N, Nikhil V. Do whey protein beverages affect the microhardness of composites? A laboratory study. J Conserv Dent. 2022;25(1):72&#8211;77.</li>
	<li>Garc&#237;a-Contreras R, Scougall-Vilchis RJ, Acosta-Torres LS, Arenas-Arrocena MC, Garc&#237;a-Gardu&#241;o R, de la Fuente-Hern&#225;ndez J (2015) Vickers microhardness comparison of 4 composite resins with different types of filler.&#160;J Oral Res. 4(5):313&#8211;320.&#160;</li>
	<li>Ferracane JL. Hygroscopic and hydrolytic effects in dental polymer networks. Dent Mater. 2006;22(3):211&#8211;222.</li>
	<li>Ilie N, Hickel R. Investigations on mechanical behaviour of dental composites. Clin Oral Investig. 2009;13(4):427&#8211;438.</li>
	<li>Lussi A, Jaeggi T. Erosion&#8212;diagnosis and risk factors. Clin Oral Investig. 2008;12(Suppl 1):S5&#8211;S13.</li>
	<li>Hannig M, Joiner A. The structure, function and properties of the acquired pellicle. Monogr Oral Sci. 2006;19:29&#8211;64.</li>
</ol>

All authors declare that they have no conflicts of interest.

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...

The long-term success of resin composite restorations depends on their resistance to complex oral environmental challenges, including mechanical loading, dietary exposure, and chemical degradation1,2. 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 deterioration3,4.
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 integrity5,6,7,8. These degradative effects are influenced by multiple factors, including pH, exposure time, and filler&#8211;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 individuals9,10. 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.

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

a standardized in vitro procedure for evaluating surface microhardness changes in nanohybrid resin composites after whey protein beverage exposure

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

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 &#916;VHN. 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 &#916;VHN values (p &#60; 0.0001). The highest hardness loss was observed in the injectable composite exposed to whey concentrate (&#916;VHN: 10.4 &#177; 2.1), whereas the lowest change was recorded in the highly filled composite stored in distilled water (&#916;VHN: 0.9 &#177; 0.4). Immersion in whey concentrate resulted in significantly greater hardness loss in all composites compared to distilled water (p &#60; 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.

Descriptive statistics
The mean ΔVHN (±SD) values of the composite-solution combinations are presented in Table 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Resin composite</td>
			<td>Solution</td>
			<td>ΔVHN (Mean ± SD)</td>
			<td>95% CI</td>
			<td>n</td>
		</tr>
		<tr>
			<td rowspan="3">EA</td>
...

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.

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 ...

We examine the impact of whey protein beverages on the surface microhardness of non-hybrid resin composites. Existing methods like standard design beverage exposure conditions limiting reproducibility. This protocol establishes controlled exposure conditions, enabling reliable comparable microhardness assessments of resin composites.To begin, prepare a total of 90 disc-shaped specimens from three nanohybrid resin composites. Fabricate the specimens using cylindrical clear silicone molds. Place the composite material into the molds in a single 2-millimeter thick increment.Cover the surface with a clear microscope slide to obtain a flat surface. Polymerize the specimen for 20 seconds using a blue LED light curing unit at 1, 000 milliwatts per square centimeter, positioned approximately 1 millimeter from the surface. Before each polymerization cycle, verify the curing light intensity with a radiometer to ensure consistent light exposure.Store the prepared specimens in distilled water to complete post-polymerization and simulate oral conditions. Using medium, fine and super fine aluminum oxide impregnated discs, polish the upper surfaces of all specimens sequentially for 10 seconds each under water cooling at 10, 000 revolutions per minute using light pressure and a unidirectional rotational motion. Replace the polishing discs after every five specimens to maintain consistent abrasive efficiency and ensure surface standardization.After each polishing step, rinse the specimens with distilled water for 10 seconds to remove debris. Gently air dry the specimens for five seconds before proceeding to the next disc. Mark the unpolished surface of each specimen with a unique code for consistent orientation and store them in distilled water before baseline microhardness testing.Perform the baseline Vickers microhardness or VHN testing using a VHN tester with a 200 gram load applied for 15 seconds. Make three indentations per specimen with at least 1 millimeter spacing between each indentation. Perform the indentations on the central surface area of each specimen to avoid edge effects.Calculate the mean baseline VHN for each specimen, with all measurements performed by a single calibrated operator to minimize variability. Dissolve 25 grams of whey protein powder in 200 milliliters of distilled water to prepare immersion solutions. Mix the solutions using a laboratory shaker for 60 seconds until complete dissolution is achieved.Allow the solutions to equilibrate at room temperature for five minutes to minimize foam formation and ensure homogeneity. Measure and record the pH of each solution using a calibrated digital pH meter before immersion. Prepare fresh whey solutions prior to each immersion cycle to ensure consistent chemical composition and avoid protein degradation.Use 10 milliliters of immersion solution per specimen to ensure complete submersion during each cycle. Divide each composite group into three subgroups containing 10 specimens each. Immerse the specimens in the designated solutions for 10 minutes twice daily following in vitro beverage exposure models that reflect daily consumption patterns.For whey exposed groups, after each immersion cycle, rinse the specimens with distilled water. Place the specimens in artificial saliva for 10 minutes to simulate short term salivary exposure. Store the specimens in distilled water at 37 degrees Celsius between immersion cycles and continue the immersion protocol for 30 days.Rinse the specimens with distilled water and gently dry them. Perform VHN as demonstrated earlier and calculate the mean post-immersion VHN. Determine the change in VHN or delta VHN for each specimen.Analyze VHN values using a repeated measures ANOVA model with time point as the within-subject factor and composite type and immersion solution as between-subject factors. When appropriate, calculate the delta VHN values as descriptive outcome measures to express the magnitude of microhardness change for each specimen. Perform post-hoc pairwise comparisons using Tukey's Honestly Significant Difference test.Calculate 95%confidence intervals for all group means and report effect sizes using partial eta squared for ANOVA results. Compare hydrogen potential values between whey protein solutions using Welch's t-test and set the level of statistical significance for the analysis. In addition to absolute change in VHN values, calculate percentage microhardness change using the presented formula.The lowest microhardness change was observed in the highly filled composite stored in distilled water, whereas the greatest change was recorded in the injectable composite exposed to whey concentrate. Exposure to whey protein solutions resulted in greater hardness loss compared to distilled water. However, no statistically significant difference was observed for whey isolate in the highly filled composite.Immersion in whey concentrate caused significantly greater microhardness loss compared to distilled water for all tested composites. Specimens exposed to whey isolate also showed higher delta VHN values than those stored in distilled water. This method allows researchers to quantify microhardness change caused by dietary protein beverages in restorative materials.The most important consideration while performing this protocol is maintaining consistent specimen preparation and controlled immersion conditions to ensure reliable results. Future studies can investigate additional beverages, longer exposures and other material properties.

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University of Health Sciences.  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.

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

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