Evaluation of remineralization potential of chia seed and flaxseed pastes in comparison with fluoride varnish on enamel white spot lesions

Introduction

Dental caries is an ongoing process marked by repeat­ed phases of demineralization and remineralization [1]. The balance between predisposing factors (i.e., acidoge­nic bacteria, fermentable sugars, and saliva) and protective elements (i.e., antibacterial substances, adequate salivary flow, and remineralizing minerals, such as fluo­ride, phosphate, or calcium) determines whether a lesion is progressing or reversing [2]. The initial clinical indication of enamel demineralization is the presence of white spot lesions (WSLs) on the enamel surface [3]. WSLs indicate diminution of minerals in a subsurface region located beneath an intact enamel layer, and can appear as a visible chalky, and white opaque area. If the enamel is still partially demineralized, it can be easily remineralized, especially with the existence of a flawless enamel surface [4].
Early management of enamel demineralization using preventive procedures is better than curative methods [2]. Several conservative modalities can be employed for WSLs treatment, including oral care measures, application of casein phosphopeptide, and fluorides (varnishes or mouthwashes), ozone, and xylitol [5].
Historically, fluoride has played a venerable role in caries prevention, and it is considered the gold standard for inhibiting demineralization. It enhances enamel remineralization and offers antibacterial activity [6]. Demineralization persists until the pH increases, triggering remineralization. This process involves the deposition of remineralizing ions, forming fluorapatite that exhibits greater resistance to acid dissolution [5].
However, the widespread use of fluoride increases the incidence of dental fluorosis along with the deve­lopment of drug resistance, especially in children [7]. Additionally, there is increasing apprehension among parents about the use of fluoride and other antimicrobial agents, which have many adverse effects on the digestive system [2]. Moreover, scientific and public communities expressed their concerns regarding the safety of using fluorides with high concentrations, especially with the development of holistic dentistry and fluoride categorization as a neurotoxic chemical agent [7].
Nowadays, natural sources gain a great deal of attention in the dental field, especially in developing countries due to their economic positions. These materials offer effective, safe, minimal side effects, and affordable options for dental applications [1]. Previous trials revealed antibacterial activity of some natural herbal plants, including aloe vera [8] and rosemary [1], which can be used for caries prevention. Furthermore, a study conducted in vitro indicated that extracts from plants may serve as viable options to chemical substances used for enamel remineralization, such as flaxseed paste and aloe vera gel [3].
Chia are small seeds obtained from the annual herb Salvia hispanica L., a plant in the mint family, Lamiaceae [9]. Recently, the popularity of chia seeds within the medical community can be attributed to their considerable nutritional and therapeutic advantages [10]. Also, high mineral composition of chia seeds in terms of calcium, phosphorus, potassium, and magnesium [11], indicates that chia may play a potential role in enamel remineralization.
Flaxseeds are a member of genus Linum in Linaceae family, often referred to as linseed. Flaxseeds rank among the oldest and most important agronomic oilseed crops used for industrial and food purposes [11]. Flaxseed’s Latin’s name mean “extremely beneficial”, because all of the seed’s parts are exploited, either raw or processed [10]. Flaxseed contains ions and biologically active components, which increase the interest of research domain [12].
In the dental field, there has been a limited number of studies assessing the remineralization capabilities of flaxseed on enamel [3, 13]. To date, there is no research concerning the remineralizing potential of chia seed extract on tooth tissues. Therefore, the current research aimed to evaluate the remineralization capability of chia seed and flaxseed pastes in comparison with fluoride varnish on enamel WSLs, in terms of calcium and phosphorus ions weight percent and microhardness.

Material and methods

Sample size calculation

A power analysis was conducted to ensure sufficient power for testing the null hypothesis, claiming that no significant differences exist in mineral content among the various groups under investigation. By setting an alpha (α) level of 0.05 and a beta (β) level of 0.2 (resulting in a power of 80%), along with an effect size of 0.487 derived from prior research [14], the total sample size required was 52, with 13 samples per group. Calculation of the sample size was completed using R statistical analysis software, version 4.3.2 for Windows (R Core Team, 2023; R: A language and environment for statistical computing, R Foundation for Statistical Computing, Vienna, Austria; URL https://www.R-project.org).

Teeth selection and samples preparation

Twenty-six human permanent premolars extracted for orthodontic reasons were used in the present study. The research approval was granted by the Scientific Research Ethical Committee, Faculty of Medicine, Fayoum University, with a research code of R 608 – session 122 – 17/9/2024. Inform consents were obtained from all patients, who donated their teeth for this study. Sample’s collection and storage were conducted in compliance with appropriate rules and regulations. Teeth were rinsed under running water to remove any attached soft tissue and plaque, and subsequently preserved in distilled water at 4ºC for maximum duration of one month. The roots of the teeth were severed 2 mm beneath the cemento-enamel junction, while the crown portion was divided longitudinally into buccal and lingual enamel blocks. Enamel surfaces underwent abrasion using sandpaper discs with grits of 800, 1,000, and 1,200, followed by polishing with rubber cups and paste to ensure a plane finish. Following polishing, the specimens were subjected to ultrasonic cleaning (Wisd, WUC-D06H, Daihan Scientific Co., Ltd., Korea) with deionized water for 15 minutes to eliminate any residual debris. After that, they were preserved in distilled water until further testing.
Baseline assessment of the surface topography was applied to determine calcium (Ca) and phosphate (P) ions weight percent on the enamel surface using environmental scanning electron microscope (ESEM; Prisma E, Thermo Fisher Scientific) attached to an EDX unit. The specimens were adhered to aluminum stubs utilizing regular diameter carbon sticky tape. ESEM exa­mination of each specimen was executed at accelerating voltage of 30 kilovolts, and the analysis of all groups was done at 1000× magnification.
Surface microhardness was measured at baseline using digital display Vickers microhardness tester (model HVS-50, Laizhou Huayin Testing Instrument Co., Ltd., China), with Vickers diamond indenter and a 20× objective lens. For 15 seconds, a 100 g load was placed on the surface of every specimen, where three indentations were made, each evenly spaced, and a minimum distance of 0.5 mm from one another maintained. Using a built-in scaled microscope, diagonal lengths of the indentations were assessed, and Vickers values were subsequently converted into microhardness values. Calculation of microhardness was performed using the following formula:
HV = 1.854 · P/d2,
where HV is Vickers hardness in kgf/mm2, P is the load in kgf, and d is the length of diagonals in mm.

Specimens grouping

Based on the type of remineralizing agent, the specimens were allocated into four groups randomly (n = 13): 1) DW (distilled water) group: negative control group, no remineralizing agent applied; 2) WV (white varnish) group: positive control group, fluoride ClinproTM white varnish (5% sodium fluoride), TCP (tri-calcium phosphate), alcohol, water, resin, mint flavors; 3M ESPE, St. Paul USA); 3) CS (chia seeds) group: chia seeds paste; and 4) FS (flaxseeds) group: flaxseeds paste. Both chia seed and flaxseed extracts and pastes were specifically prepared at Extraction Lab, Faculty of Pharmacy, October 6 University.

Fabrication of enamel white spot lesions

Artificial enamel WSLs were created by immersing the enamel specimens in a demineralizing solution (10 mM NaH2PO4–2H2O, 50 mM acetic acid, 2.2 mM CaCl2–2H2O, 100 mM NaCl, 1 ppm NaF, 5 mM NaN3), with pH 4.5 [15]. Demineralization was done at 37°C for four days, and subsequently, they were washed in deionized water using an ultrasonic cleaning device for 15 minutes to stop demineralization [16]. The surface was then air-dried to obtain chalky white appearance of WSLs [16]. This procedure was done for all groups, except for the DW group, and the specimens were assessed for Ca and P ions weight percent and microhardness.

Preparation of chia seed and flaxseed extracts and pastes

A total of 750 grams of each type of seeds (chia and flaxseeds) were weighed using a four-digit scale (Libror AEG 220, Denver Instrument Company, USA), and the seeds were crushed using an electric blender (Mouli­nex Genuine, France). The resulting powder (100 grams) from each type of seeds was macerated in 200 milliliters of 100% ethanol for 72 hours. The filtrates were assembled and evaporated to dryness using a vacuum apparatus (Büchi Labortechnik AG 9230, Flawil, Switzerland). Subsequently, they were subjected to freezing-drying process to produce a dark brown amorphous powder extract. Finally, 25 grams of chia seed extract and 25 grams of flaxseed extract were added to a toothpaste formulation, created as follows: 1 gram of sodium lauryl sulfate and 0.5 grams of sorbitol were dissolved in 25 grams of deionized water; then, 3 grams of carboxymethyl cellulose and 7 grams of glycerin were added, the mixture was well-stirred, resulting in a smooth gel. The final paste was stored in a sealed tube until further use [17].

Application of remineralizing agents

Regarding CS and FS groups, the specimens underwent twice a day brushing for two minutes per session, lasting for 14 days, using the corresponding extract paste and electric toothbrush (Electric toothbrush, Oral-B Vitality, cross action, Braun GmbH, Germany). The samples were brushed by the same operator to ensure standardization. In-between brushing cycles, the samples were stored in distilled water at room temperature, with daily water replacing. For the WV group, ClinproTM white varnish was carefully mixed using a soft brush applicator (3M ESPE, St. Paul, IL, USA), and evenly applied on demineralized enamel according to the manufacturer’s recommendations. The teeth were subsequently evaluated for Ca and P ions weight percent and microhardness.

Statistical analysis

Numerical data were presented as mean and standard deviation (SD) values. They were tested for normality and variance homogeneity by viewing distribution and using Shapiro-Wilk’s and Levene’s tests, respectively. Non-parametric data were evaluated using mixed aligned rank transform (ART) analysis. Comparisons of simple effects were done with error term of the main model and p-value adjusted with false discovery rate (FDR) method. Significance level was set at p < 0.05 for all tests. Statistical analysis was performed with R statistical analysis software version 4.4.1 for Windows (R Core Team, 2024; R: A language and environment for statistical computing, R Foundation for Statistical Computing, Vienna, Austria; URL https://www.R-project.org/).

Results

The comparisons of simple effects presented in Table 1 showed that for all tested remineralizing agents, a notable disparity was observed in Ca and P weights in diffe­rent treatments applied (p < 0.001). For Ca and P weights in both the CS and FS groups, all comparisons were statistically significant, with the highest value measured after remineralization, followed by baseline and demine­ralization values. Regarding Ca weight in the WV group, all pairwise comparisons were statistically significant, with the highest value measured at baseline, followed by remineralization and after demineralization values. While for the P weight, baseline and remineralization values were significantly higher than the demineralization value.
At baseline, the analysis revealed no notable diffe­rences in Ca and P weights among the different remi­neralizing agents measured. After demineralization, Ca and P weights were significantly decreased in all tested groups, except for the DW samples showing significantly higher values. Regarding Ca weights after reminera­lization, the CS group demonstrated significantly higher values than other remineralizing agents, followed by the FS and DW groups, while WV showed the lowest values (Figure 1). As for P weights results after remineralization, the comparison revealed no significant difference among the CS, FS, and DW groups, all of which exhibited higher values than the WV group having significantly lower values (Figure 2). ESEM-EDX analysis of the specimens at different treatment phases (baseline, demineralization, and remineralization) for all tested groups showed a deposition of minerals (Figure 3).
The microhardness results presented in Table 2 for all groups demonstrated a decrease in microhardness values after demineralization. Following remineralization, the analysis revealed no notable differences between the CS, FS, and DW groups, which showed significantly greater values than the WV group having significantly lower values.

Discussion

The current study was designated to evaluate the remineralization potential of chia seed and flaxseed pastes in comparison with fluoride varnish on enamel WSLs, in terms of calcium and phosphorus ions weight percent and microhardness.
Dental caries principally results from the disturbance in demineralization and remineralization cycles. The earliest clinical evidences of enamel deminerali­zation are WSLs, which are limited to the superficial enamel layer [16]. WSLs indicate diminution of mine­rals in a subsurface region located beneath an intact enamel layer. However, if these lesions remain untreated and mineral loss continues, they can eventually result in cavitation [18]. Currently, the research work focuses on remineralization of the demineralized enamel, especially if it has no localized breakdown [6].
Enamel remineralization is influenced by several factors, including salivary pH that can buffer the plaque pH and provide a reservoir of minerals for remineralization [5]. However, the remineralization process facilitated by saliva is relatively slow and insufficient to achieve a net gain in minerals, resulting in a minimal enhancement of structural integrity of lesions [18].
Fluoride’s efficacy is fundamentally constrained by the presence of phosphate and calcium ions; despite the advantages it provides [4]. Even though various formulations are used in remineralization, such as calcium-phosphate-based agents and tricalcium phosphate fluo­ride [19], frequent use of chemicals and synthetic pro­ducts is unhealthy; therefore, natural extracts have become popular in the dental field as they are safe compared with chemical products [20].
Recently, researchers have highlighted natural plant compounds as promising agents enhancing the oral health, offering a viable substitute to synthetic chemicals for plaque control, antimicrobials [8], and remineralization [3]. Chia and flax seeds have gained scientific attention because of their excellent nutrient profile, non-conventional sources of protein, omega fatty acid contents, minerals, and several other bioactive compounds [13]. Therefore, chia seed and flaxseed pastes were investigated in the present study.
Different methods can evaluate enamel reminerali­zation, including surface microhardness, mineral analy­sis of Ca/P ions, polarized light microscopy, and scanning electron microscopy. The current study employed microhardness evaluation and energy dispersive X-ray (EDX) spectroscopy to investigate the remineralization potential of pastes derived from chia and flax seeds extracts. Both techniques are effective for examining the enamel surface and analyzing its mineral composition. Furthermore, EDX offers quantitative evaluation of subtle variations in mineral composition, and provides important information on the elemental makeup of treated enamel surfaces [6, 21].
Damage associated with WSLs arises from demineralization that is triggered by acidic conditions [16]. Therefore, the enamel demineralization in this study was performed following the methodology used by Hua et al. [15], resulting in WSLs production. The findings indicate that after demineralization, microhardness and Ca and P weights were markedly reduced in all experimental groups, exception for the control group. The observed outcome can be explained by the surface disintegration of hydroxyapatite and demineralization of the enamel outer layer due to acids [22]. This process leads to the formation of microporosities and a chalky white appearance of the enamel, known as WSLs [23]. This observation aligns with previous study [24] that demonstrated the solubility of minerals under acidic conditions.
An increase in calcium and phosphorus weights was observed for the CS, FS, and WV groups following remi­neralization when compared with values recorded after demineralization. This may be attributed to the movement of mineral ions into the external layer, which decreases the surface porosity [6], thus, emphasizing the notable variations in Ca and P weights for remineralized and demineralized enamel. Additionally, this can be correlated with the microhardness results of the current study.
Chia is an herbaceous plant known for its seeds possessing a glossy and smooth surface, with colors ranging from white to brown. Traditionally, these seeds have been utilized for a variety of therapeutic purposes [13]. In modern times, chia seeds are frequently added to numerous food products, such as baked beverages and dairy items, due to their remarkable nutritional advantages and health-promoting properties [11]. Chemical analyses of chia seeds revealed a variety of constituents, including vitamins A, B1, B2, and B3 as well as high percentage of fatty acids, carbohydrates, proteins, and mine­rals [11]. Additionally, these seeds are packed with flavonoids, i.e., caffeic acid, myricetin, and quercetin [25]. This diverse composition supports their potential use in multiple applications, such as food industry, personal care, and pharmaceutical applications [11]. Chia seeds possess various therapeutic properties, e.g., cardioprotective effects, body weight regulation, antioxidant activity as well as benefits related to cancer prevention, hypertension reduction, and neuroprotection [26]. Furthermore, they are acknowledged for their anti-inflammatory, antiplatelet, antidepressant, and anxiolytic effects [13]. Chia seeds have been employed as a laxative, an analgesic, in the treatment of anemia and dermatitis, and as an immune system enhancer [9].
In the current study, the results of the chia seed group demonstrated high Ca and P weights percent comparing with other groups. The CS group had the highest value after remineralization, more than values recorded for baseline. This may be attributed to the high levels of Ca and P present in chia seeds. 100 grams of chia seeds contain approximately 456-631 mg of calcium, and 860-919 mg of phosphorus ions [10, 11]. In addition to these minerals, chia seeds are rich in potassium and magnesium. Notably, their calcium content surpasses that of other grains, such as rice, barley, or oats. When compared with other grains, chia seeds exhibit elevated concentrations of magnesium, potassium, and phosphorus, among other minerals [9]. Studies on the remine­ralizing possibility of chia seed extracts on WSLs are not currently available in the literature. Our study represents the first research utilizing the chia seed extracts in remi­neralization of WS lesions; therefore, a direct comparison of this outcome is not possible.
Flaxseed has garnered significant attention in diet and disease investigations due to its potential health and nutritional advantages [11]. The seeds are rich in omega fatty acids, proteins, minerals, and various other biologically active substances [10]. Flaxseed has been utilized in the medical field, demonstrating efficacy for a range of conditions, including cardiovascular disease, cancer, arthritis, hypertension, atherosclerosis, diabetes, osteoporosis, and autoimmune, and neurological disorders [13]. Also, it provides a laxative effect and alleviates menopausal symptoms [10]. It has been noted for its properties, such as antiatherogenic, antidiabetic, anti-inflammatory, and antioxidant, in addition to enhancing vascular function [10, 13]. Flaxseed has been effectively utilized in dentistry. It exhibits antimicrobial properties against cariogenic pathogens [12] and enhances the appearance of demineralized tooth surfaces [13]. Therefore, the current work attempted to estimate the quantitative remineralization potentials of flaxseed extract on demineralized enamel.
The results of the FS group after remine­ralization showed statistically significant higher values of Ca weights, surpassing the control group values. However, P weights did not exhibit significant differences among the FS, CS, and DW groups, all of which demonstrated significantly higher values. This may be associated with the high mineral content present in flaxseed, as 100 grams of flaxseed contain about 255 mg of Ca and 642 mg of P in addition to other minerals, such as potassium and magnesium [13]. This can be correlated with the microhardness findings. Moreover, the availability of Ca and P ions facilitates remineralization effects of deminera­lized enamel [3]. This result aligns with previous studies, which employed flaxseeds in remine­ralization of demi­neralized enamel, and reported their ability to reverse demineralization and repair enamel surface [3, 6].
The positive control group used in the present study was ClinproTM white varnish, consisting of five percent sodium fluoride with tri-calcium phosphate. In terms of Ca and P weights following remineralization, the WV group demonstrated markedly reduced values in-between the tested groups. A pronounced difference was observed in the percentage of Ca weight, with the highest value seen at baseline, followed by the remineralization and after demineralization values. However, the P weight showed no significant difference between the baseline and remineralization. Regarding the microhardness results, this group exhibited lower values.
ClinproTM white varnish action depends on the fact that the crystalline structure of hydroxyapatite is able to exchange Ca and P and their deposition on tooth tissues [27]. Moreover, its activation is enhanced by the presence of moisture and saliva, which promotes fluoride release [28]. Our result is compatible with a research conducted by Varma et al. [27], reporting that ClinproTM white varnish is less effective in enamel remi­neralization. The microhardness results align with findings of a previous research conducted by Salinovic et al. [21], which showed that ClinproTM white varnish has less microhardness values. However, it contradicts previous studies, suggesting that ClinproTM white varnish has a greater remineralizing effect [28, 29]. This inconsistency may result from variations in the study design, comparative groups, and application time.
To summarize, in the current study, chia and flax seed pastes were able to remineralize WSLs, thereby offering reliable, effective, and affordable options for dental applications.
Challenges associated with simulating natural variations in the oral environments of humans were the limi­tations of this study. Further in vivo research is necessary to investigate benefits and/or disadvantages of these products. It is crucial to evaluate how saliva and oral pH influence the remineralization effectiveness of these seed pastes. Also, it is important to investigate the effects of brushing techniques, frequency, and long-term implications of chia seed and flaxseed pastes on enamel remineralization. The ability to incorporate these natural extracts into clinically relevant delivery systems, such as dentifrices and mouthwashes, should be additionally considered.

Conclusions

Chia seed and flaxseed pastes improve the calcium and phosphorus content more effectively than fluoride varnish. Moreover, both extract pastes result in greater microhardness values superior to fluoride varnish. The use of chia seed and flaxseed pastes is beneficial in remineralization of enamel WSLs, providing safe, effective, and cost-efficient options for dental care.

Disclosures

1. Institutional review board statement: Research approval was granted by the Scientific Research Ethical Committee, Faculty of Medicine, Fayoum University, with a research code of R 608 – session 122 – 17/9/2024. Written informed consents were obtained from all patients, who donated their extracted teeth used in the study.
2. Assistance with the article: None.
3. Financial support and sponsorship: None.
4. Conflicts of interest: The authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

References