Do whey protein beverages affect the microhardness of composites? A laboratory study.

Background: Whey protein supplements might be a potential risk factor for the restoration surface due to its pH and lactose content. Aim: The study aimed to evaluate the effect of whey protein beverages on the microhardness of four restorative composites with different microstructures (Filtek Z 350XT, Filtek Z 250XT, Filtek Bulk Fill (FBF), and Filtek P 60). Materials and Methodology: Forty disc-shaped samples from each material (in total 160) were prepared and wet polished. The samples of each group were randomly divided into four subgroups and subjected to four immersion media namely, whey protein Concentrate (WPC), whey protein Isolate (WPI), whey protein water (WPW), and distilled water (DW). The immersion protocol was performed twice for 10 min daily for 30 days followed by immersion in artificial saliva for another 10 min and stored in DW till the next cycle. The pre immersion and post immersion microhardness of all the samples were recorded with a Microhardness Tester FM-800. Data obtained were statistically analyzed using SPSS 25 software. Results and Conclusions: All composites exhibited decline in microhardness, except for FBF. P 60 composites which showed the highest decline in microhardness. WPC and WPI caused the greatest reduction in microhardness compared to WPW. Copyright:

INTRODUCTION

The concept of healthy eating to maintain physical fitness has promoted the consumption of various types of health supplements amongst all age groups of people. In the early 20th century, whey proteins were added to this list of available supplements owing to their nutritional significance. Following this trend, a huge spike was seen in whey protein market size which expanded at a compound annual growth rate of 9.8% from 2019 to 2020.[1]

Whey proteins, obtained from cow's milk are an excellent source of branch-chained amino acids.[2] They also induce the effects of bioactive compounds such as lactoferrin, immunoglobulins, glutamine, and lactalbumin.[3] Historically, these supplements were consumed by athletes only, but the inadequacies of modern diet and sedentary lifestyle have made us explore faster and easier sources of nutrition. Further, advances in processing technologies have enabled the purification and separation of whey proteins into concentrate and isolate.[4]

These supplements are available in various products like infant formulas, sports bars, beverages, powders, etc., but powders derive maximum benefits. Mostly whey proteins are available as powders which can be mixed in water and milk or they are also available as acidic beverages.

Despite being a nutritional boon they may contain acidulants, carbohydrates (lactose) which can create an acidic environment in the oral cavity and may change the structure of composite resins, thus, hampering the longevity of the restorations.[5]

As commercially available whey protein beverages are acidic in nature, they pose potential risk to teeth and restorative materials.[67] Furthermore, their viscous nature, results in a longer contact time with the tooth and the restorative material compared to other aerated drinks. Moreover, whey protein shakes are often taken post exercise leading to a decreased salivary flow which ultimately results in inadequate rinsing and buffering of demineralizing acids on tooth surfaces and thus, increasing their potential for erosion.[8]

Therefore, keeping in mind the erosive potential of whey protein beverages, a conscious effort was made to evaluate the effect of whey protein beverages on the microhardness of different restorative materials in the present study.

In the present study, the null hypothesis tested was that Whey protein beverages have no effect on different restorative materials.

MATERIALS AND METHODOLOGY

Sample preparation

The study was approved by the institutional ethics committee (SDC/E. C/2019/700). Four commercially available dental resin composites with varied matrix and filler compositions were used in this study [Table 1].

Table 1

Composition of P-60, Z 350XT, Z 250XT and bulk fill composites (as per manufacturer data)

Group	Material	Manufacturer	Monomers	fillers
Group 1	Bulk fill posterior	Filtek bulk fill posterior 3M ESPE, St.Paul, MN, USA	Bis-GMA, AUDMA UDMA, DDDMA	4-11 nm, ytterbium trifluoride (100 nm), zirconia/silica 76.5 wt% 58.4 vol%
Group 2	Microhybrid composite	Filtek P60 posterior restorative 3M ESPE, St.Paul, MN, USA	TEGDMA has been replaced with a blend of UDMA and Bis-EMA (6)	Zirconia/silica 0.01 µm-3.5 µm 61 vol%
Group 3	Nanofilled composite	Filtek Z350 XT universal restorative 3M ESPE, St.Paul, MN, USA	Bis-GMA, UDMA, TEGDMA, PEGDMA and Bis-EMA resins	Zirconia-silica (5-20 nm) 72.5 wt% 55.6 vol%
Group 4	Microhybrid composite	Filtek Z 250 XT universal restorative 3M ESPE, St.Paul, MN, USA	Bis-GMA, UDMA, Bis-EMA 1 mm glass, ceramic microfillers, 20-60 nm SiO2	1 mm glass, ceramic microfillers, 20-60 nm SiO2 78 wt% 66 vol%

UDMA: Urethane dimethacrylate, TEGDMA: Triethylene glycol dimethacrylate, Bis-GMA: Bisphenol A-glycidyl methacrylate, AUDMA: Aromatic urethane dimethacrylate, DDDMA: 1,10-decandiol dimethacrylate, Bis-EMA: Bisphenol A diglycidyl methacrylate ethoxylated; PEGDMA: Polyethylene glycol dimethacrylate

A total of 40 disc-shaped samples were prepared from each type of resin composite according to ISO specification ISO4049/2000.[9] A mold (diameter of 6 mm and depth of 2 mm) was prepared from a plastic straw. Then, each material was squeezed in a single increment into the prepared mold with matrix strip (Mylar strip, SS White Co., Philadelphia, USA) placed on both the open ends of the disc to obtain a flat polymerized surface. The discs were cured from both sides for 40 s with a LED curing unit (Guilin Woodpecker Medical Instrument Co. Ltd, China; Light output: 1000 mW/cm2) operated in standard mode, with the guide tip placed perpendicular to the sample surface emitting an irradiance of 460 Mw/cm2and the irradiance value was repeatedly measured by the radiometer.

The samples were kept in a humid environment at 37°C for 24 h to allow postirradiation hardening of composite restorations before subjecting them to finishing and polishing. The hardened samples were then wet-polished in planar motion using coarse, medium, fine and superfine abrasive discs Super-Snap (Shofu Inc, Kyoto, Japan) for 10 s each, following which they were ultrasonically cleaned in distilled water (DW) for 10 min.

The samples were then subjected to microhardness evaluation taken with a computer-aided Microhardness Tester FM-800 series (Future-Tech Corp, Kanagawa, Japan).

Solution preparation

The samples were exposed to three different immersion media namely; Whey protein Isolate (WPI), Whey protein concentrate (WPC), and Whey Protein Water (WPW).

For preparing the respective solutions, one scoop (25 grams) of WPI (Isopure, Strawberry flavored, WPI, USA) and WPC (Muscleblaze, Whey concentrate, unflavoured, Gurugram, Haryana, India) were individually mixed in 250 mL of lukewarm water while the solution was constantly stirred with a rod to avoid formation of lumps.

The WPW (Muscleblaze, WPW, Cola flavor, Gurugram, Haryana, India) solution was poured from a prepackaged container into a beaker while DW acted as a control. The pH of all three solutions was recorded with a digital pH meter (Vishal, Haryana, India) and noted.

Immersion protocol

Samples were further divided into four subgroups (n = 5), according to the beverages in which they were immersed, namely Subgroup A (DW), Subgroup B (WPI), Subgroup C (WPC) and Subgroup D (WPW). The immersion protocol comprised 2 immersion cycles of 10 min each in their respective solutions followed by immersion in artificial saliva (Wet Mouth, ICPA Health products, India) for another 10 min for 30 days daily. After immersion in artificial saliva, all the samples were blotted dry and stored in DW (pH-6.8) till the next cycle in accordance with Kanchanavasita's study who stated that DW had no effect on microhardness values till 1 year.[10]

Postimmersion, the samples were again tested for microhardness. The Vickers Hardness Number for each sample was determined by calculating an arithmetic mean of three indentations made under 50 g load for 15 s.

Statistical analysis

The pre immersion and post immersion data was utilized to calculate percentage change, mean percentage change in micro hardness and standard deviation. Differences in micro hardness post exposure to different beverages were determined by application of unpaired “t”-test. The level of significance was set at P < 0.5 and Statistical Package for the Social Sciences, version 25 software (IBM, CA, USA) was used for statistical analysis.

RESULTS

The mean percentages change in microhardness values for four different experimental groups post immersion in four different media are represented in the given Tables 2 and 3. The Bulk Fill composite resins exhibited a significant rise in microhardness, whereas the other three composite resins namely P 60, Z 250XT, and Z 350 XT showed a decline in microhardness values post immersion. The Graph 1 also depicts that samples prepared with P 60 composite resins immersed in WPC and WPI showed maximum reduction in microhardness values in comparison to other experimental groups.

Table 2

Comparison of mean percentage change in micro-hardness values between different pairs of sub groups in bulk-fill, P 60, Z 250 and Z 350 composites by using unpaired t-test

Pair of sub-groups	Group-1 (bulk fill), P	Group-2 (P-60), P	Group-4 (Z-250), P	Group-3 (Z-350), P
DW and WPI	0.9006**, >0.05	0.0004*, <0.05	0.7570**, >0.05	0.0003*, <0.05
DW and WPC	0.8448**, >0.05	0.0000*, <0.05	0.4366**, >0.05	0.0009*, <0.05
DW and WPW	0.0290*, <0.05	0.1302**, >0.05	0.6250**, >0.05	0.0176*, <0.05
WPI and WPC	0.7511**, >0.05	0.4888**, >0.05	0.5237**, >0.05	0.6540**, >0.05
WPI and WPW	0.0260*, <0.05	0.0029*, <0.05	0.8390**, >0.05	0.4231**, >0.05
WPC and WPW	0.0393*, <0.05	0.0000*, <0.05	0.5898**, >0.05	0.5861**, >0.05

*A significant difference between groups at 0.05 level of significance. (P<0.05), **No significant difference between groups at 0.05 level of significance. (P>0.05). WPC: Whey protein concentrate, WPI: Whey protein isolate, WPW: Whey protein water, DW: Distilled water

Table 3

Comparison of mean percentage change in microhardness values between different pair of groups post immersion in distilled water, whey protein isolate, whey protein concentrate and whey protein water using unpaired t-test

Pair of sub-groups	DW, P	WPI, P	WPC, P	WPW, P
Bulk fill and Z 250XT	0.0000*, <0.05	0.1664**, >0.05	0.4821**, >0.05	0.0222*, <0.05
Bulk fill and P 60	0.0000*, <0.05	0.0000*, <0.05	0.0000*, <0.05	0.0002*, <0.05
Bulk fill and Z 350XT	0.0000*, <0.05	0.0002*, <0.05	0002*, <0.05	0.0414*, <0.05
P 60 and Z 250XT	0.0296*, <0.05	0.0000*, <0.05	0.0000*, <0.05	0.0007*, <0.05
P 60 and Z 350XT	0.0665**, >0.05	0.0006*, <0.05	0.0000*, <0.05	0.7961**, >0.05
Z 250XT and Z 350XT	0.3067**, >0.05	0.0000*, <0.05	0.0007*, <0.05	0.0116*, <0.05

*A significant difference between groups at 0.05 level of significance. (P<0.05), **No significant difference between groups at 0.05 level of significance. (P>0.05). WPC: Whey protein concentrate, WPI: Whey protein isolate, WPW: Whey protein water, DW: Distilled water

Graph 1

Comparison of mean percentage change in microhardness for four different groups post immersion in distilled water (DW), whey protein isolate (WPI), Whey Protein Concentrate (WPC) and whey protein water (WPW)

DISCUSSION

Ever since the discovery of Bis-GMA by Dr. R Bowen in 1962, Resin-based composites continue to be the supreme restorative material owing to their universal usage, easy manipulation, reliable mechanical properties, and excellent esthetics.

Most of the composite resins that were developed were specific to the restoration type, i.e., materials designed for the high aesthetic requirement in anterior teeth or high strength in posterior teeth. Although, continuous research and development have narrowed down the gaps between strength and esthetics, yet some materials possess exclusive monomers to compensate for polymerization shrinkage and ease their placement in posterior restorations.

Filtek Posterior 60 composite resin (P 60) is one such special blend of high molecular weight monomers exclusively designed for direct and indirect posterior restorations while Filtek Bulk Fill (FBF) with its unique class of Addition-Fragmentation monomers was designed to create simpler and faster posterior restorations.

In addition, there are multipurpose restorative materials like nano-hybrids and the newly introduced nanotechnology-derived nano-filled resin composites. The Filtek Z 250XT (nano-hybrid) is an excellent combination of aesthetics, outstanding wear resistance, and flexural strength while Filtek Z 350XT, formulated with precise manipulation of filler architecture have properties like fluorescence and opalescence which impart these materials life-like characteristics.

Since these materials have varied compositions and filler loading, they were purposely chosen to observe their interactions with whey protein beverages.

Composite resin restorations are subjected to a vivid range of physical and chemical conditions in the mouth, which greatly influence the in vivo degradation of resin composites.[11] Degradation over time is inevitable and can be predicted by observing the decrease in wear resistance and loss of substance under varied intra-oral conditions. Voltarelli et al. stated that a material's microhardness correlates with it resistance to intra-oral softening, compressive strength, and degree of conversion.[12] Thus, in the present study, changes in the microhardness were measured to evaluate the degradation of dental materials exposed to different whey protein beverages. The period for immersion was chosen for 10 min based on the study done by Santin et al.[7]

In the dynamic oral cavity, degradation of composite resins can be attributed to two types of interactions taking place between the resin composites and the dietary agents consumed. One, the acidic pH of dietary products may erode the resin matrix, second, the hygroscopic agents like proteins, carbohydrates (lactose), etc., when deposited on restorative surfaces might increase the water sorption of composite resins owing to the high water binding affinity of these substances.[131415] Moreover, the surface degradation of restorative materials is also dependent on the filler and matrix composition of a specific material.[16]

The null hypothesis was rejected for the present study as there were statistically significant differences between various experimental groups. In this study, all materials showed a decrease in microhardness post immersion in the three beverages except for FBF (Group 1) which showed a rise in microhardness post immersion. Such contrasting results could be attributed to the difference in the post-cure polymerization phenomena of different composite resins Tarumi et al. stated that polymerization of light-cured resin-based composites may continue for several days and the data suggests that microhardness correlates with the degree of conversion of composite materials.[17] Par et al. revealed that the degree of conversion significantly increased up to 7 days postcuring for two bulk-fill composites. In vitro studies suggest that an aging period of 24 h or more results in changes in the polymeric network structure of dental composites due to postcure polymerization.[18] The complex chemistry of Bulk Fill composites presumably allows slower crosslinking of the polymeric network which promotes better mobility of reactive species thus supporting the prolonged postcure polymerization.[19]

Jacobsen and Darr concluded that composite resins with low initial hardness present a possibility of movement of nonsaturated chains and further participation in the polymerization reaction.[20]

The results obtained in this study are consistent with Alshali et al. who revealed that FBF exhibit the lowest initial microhardness and that microhardness increases within a few days.[21]

The other three composite resins had high initial microhardness suggesting restricted movement of unpolymerized species to involve in further reaction and hence did not show rise in microhardness.[20]

DW had no effect on microhardness values of bulk-fill composites but other whey protein beverages restricted the rise in microhardness due to the greater water sorption effect owing to their high lactose content and acidic pH. Hence, this explains the least rise in microhardness of samples dipped in WPW.

In the present study, the highest decline for P 60 composite resins (Group 2) was in agreement with the statement that composite resins with a lower filler loading tend to show greater decline in mechanical properties in comparison to composite resins with a higher filler loading.[22] As P 60 composite resins have the lowest filler loading (61% by weight) out of all the materials used, the highest drop in microhardness could be explained.

The Z 350XT (Group 3) and Z 250XT (Group 4) also exhibited a drop in microhardness when exposed to different protein beverages but the drop in Z 350XT samples was significantly greater than the samples made with Z 250XT which could be understood by the lower filler loading (72.5% by weight) of Z 350XT composite resins in comparison to Z 250XT (82% by weight).[22]

Moreover, Gonclaves suggested that Z 350XT samples presented with higher water sorption and solubility than hybrid composite resins.[23] The higher water sorption of Z 350XT samples might have contributed to increased leaching of ions and even contributed in greater hydrolysis of ester bonds of the silane coupling agent, eventually resulting in a greater decline in microhardness.

The study suggested that the microhardness declined in all the four subgroups namely DW, WPI, WPC, and WPW irrespective of the material utilized except for FBF (Group 1). On exposure to the aqueous media, there is an increase in the concentration of hydroxyl ions which tend to break the siloxane network of the matrix resulting in auto-catalytic cycle of surface degradation and softening of composite resins.[24]

One unique finding of this study was that for all the groups, the highest decline was seen in samples dipped in WPC and WPI while the lowest was seen in samples dipped in DW.

The explanation for the same lies in the viscous nature of the WPI and WPC, which leave gritty residues on composite discs. Lactose being an integral component of virtually all whey protein powders tends to increase their stickiness and moisture absorption ability.[25] Hence, when samples were dipped in these immersion media, the whey proteins adhered to the surfaces of the discs and result in more water sorption in comparison to samples dipped in DW.

The decline in WPC samples was higher than WPI owing to the greater lactose content in WPC.[24] In addition, proteins present in whey powders have an innate water-binding affinity which would result in more softening of the discs in whey protein beverages than in DW.[26]

Another interesting finding of this study was that the samples immersed in WPW showed lesser drop in microhardness than WPI and WPC, despite having a lower pH (3.9) than WPI. (5.9) and WPC (5.7). One possible explanation for the same could be the greater washing and buffering effect by the saliva in WPW samples due to their nonsticky nature.[6] Moreover, Ireland et al. suggested that a drink which does not adhere to the hard tissues/restorative materials will be flushed faster by the action of saliva.[27]

One of the major limitations of this study was its inability to replicate the complex oral environment. Clinically, the effect of compounding variables such as temperature and masticatory forces was not taken into consideration.

CONCLUSIONS

Modern dental restorative materials behave differently when exposed to different types of whey protein beverages. According to the present study, the P 60 composite resins were significantly affected by exposure to WPC and WPI while FBF showed greater resistance to alterations in microhardness. In addition, other factors like viscosity, lactose concentration, and the pH of the beverages were found to play an important role in determining the resin surface stability.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.