712 2024 The Combined Effect of Two Natural Extracts on Enamel Remineraliza;on (in vitro Study) Vol 13 No 2 (2024) DOI 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu The Combined Effect of Two Natural Extracts on Enamel Remineralization (in vitro Study) Shatha A. Abbas1, Alhan A. Qasim1, Rusul J. Hadi1, Sarah M. Khammas3, Asmaa M. Khammas1 1College of Den*stry, University of Baghdad, Baghdad, Iraq 2Department of Den*stry, Al Hikma University College, Baghdad, Iraq Abstract Objec&ve: Dental caries is a chronic infec/ous disease that is prevalent worldwide in all age groups. Numerous a9empts have been made to develop conserva/ve approaches to halt caries progression and restore enamel defects. This study aimed to inves/gate the effect of applying grape seed extract and chicken eggshell extract on the microhardness of demineralized enamel in permanent teeth. Methods: Forty-eight sound upper first premolars were used. Following demineraliza/on with the demineralizing solu/on for 96 hours, they were distributed into four groups consistent with the treatment agent used: group A was treated with casein phosphopep/de amorphous calcium phosphate (as a control group), group B was treated with grape seed extract, group C was treated with chicken eggshell extract solu/on, and group D was treated with grape seed extract followed by chicken eggshell extract solu/on. Vickers microhardness measurements were performed on sound enamel, aNer demineraliza/on and remineraliza/on. Results: Paired T-test, one-way ANOVA, and Tukey’s HSD test were used for sta/s/cal analysis. Enamel microhardness was significantly reduced following demineraliza/on (p=0.000) and significantly increased aNer remineraliza/on, with group D showing the highest values (mean microhardness=218.99). Conclusion: Grape seed extract and chicken eggshell extract solu/ons have a synergis/c effect on enamel remineraliza/on which was interpreted from the increase in surface microhardness values. Keywords: Caseins, Eggshell, Enamel, Grape Seed Extract, Tooth Remineraliza/on. Cita:on: Abbas S, et al. (2024) The combined effect of two natural extracts on enamel remineraliza:on (in vitro study). Den:stry 3000. doi:10.5195/d3000.2024.712 Received: August 21, 2024 Accepted: September 1, 2024 Published: October 3, 2024 Copyright: ©2024 Abbas S, et al. This is an open access ar:cle licensed under a Crea:ve Commons AUribu:on Work 4.0 United States License. Email: Shatha.a@codental.uobaghdad.edu.iq Introduction Dental caries is a dynamic process that is influenced by the balance of pathological and defensive mechanisms [1]. The remineralization of dental tissues is characterized by the re-deposition of minerals, particularly calcium, and phosphate, which are supplied by therapeutic agents into the gaps between hydroxyapatite crystals of demineralized enamel, resulting in mineral gain [2]. Casein phosphopeptide amorphous calcium phosphate (CPP-ACP) is a supplement for calcium and phosphate ions derived from milk protein [3]. The anti-cariogenic mechanisms have been suggested to keep the enamel minerals calcium and phosphate supersaturated in the dental plaque, thereby interrupting biofilm formation, hindering bacterial adhesion on the enamel surface, and buffering pH reduction in the oral environment [4]. Thousands of years ago, therapeutic agents were prepared from natural products that have been considered promising sources for new medicines, particularly in oral diseases, such as dental caries [5]. Chicken eggshell powder (CESP) has various medical applications owing to its elevated calcium concentration (approximately 93%) and increased bioavailability [6]. CESP can be used as a coating material in dental implants [7] and as a remineralizing agent against The Combined Effect of Two Natural Extracts on Enamel Remineraliza;on (in vitro Study) Vol 13 No 2 (2024) DOI 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu dental caries and enamel erosion with promising results [8-10]. Grape seed extract (GSE) showed a noticeable effect on the prevention and restoration of demineralized teeth by stabilizing the collagen matrix and inhibiting its breakdown. It has several beneficial properties, but high levels of proanthocyanidins are thought to be responsible for its role in caries prevention [11]. Previous studies used the two extracts separately for enamel remineralization. This study aimed to evaluate the combined effect of CESP and GSE on the microhardness of demineralized enamel. The null hypothesis states that there is no synergistic effect between CESP and GSE in remineralization of enamel. Material and Methods Tooth Preparation Upper first premolars extracted from humans were used, any teeth that had cracks or dental caries, as shown by a magnifying lens, were excluded. Rubber cup and non-fluoridated pumice were used to polish the samples, which were then stored in deionized water containing 0.1% thymol [12]. Each tooth was coated with nail varnish (acid-resistant), except for a window of 2 × 2 mm (in dimension) left in the middle of the buccal surface. These windows were flattened with Sof-Lex Disks (3M ESPE, USA) in an advanced manner [13]. Sample The sample size was calculated using G power 3.0.10 program [14]. Alpha error of probability=0.05 two-sided was used and 95% was the power of the study [15]. The sample size was 48 teeth (each group consisted of 12 samples). The samples were randomly distributed into four groups after demineralization. Treatment with different agents was conducted for 14 days. Group A: Treatment with CPP-ACP cream (GC Tooth Mousse, GC Co, USA) for 3 min daily, then the cream was wiped off with a cotton piece [16] (as control). Group B: Treatment with grape seed extract solution for 2 min daily [17]. Group C: Treatment with CESP solution for 5 min daily. Group D: Treatment with grape seed extract solution for 2 min, then with CESP solution for 5 min. Following the treatment procedure, the samples were washed with deionized water (2 min for each separately) daily and stored until the next day. Preparation of Grape Seed Extract Solution GSE powder (Bulk Supplements, USA) was dissolved in a phosphate buffer solution (0.025 M KH2PO4, pH=7.4) to obtain a concentration of 6.5% [18]. Preparation of CESP Solution The calcination protocol of chicken eggshells was performed according to the World Property Intellectual Organization [19]. A gram of the resultant powder was dissolved with 20 ml of acetic acid (concentration =4%), and only the clear solution was used as a remineralizing agent [20]. Demineralizing Protocol The following concentrations were used to prepare demineralizing solution: acetic acid (0.05 M), sodium dihydrogen orthophosphate dehydrate (2.2 mM), and calcium chloride (2.2 mM). The solution pH was 4.4 (modified by the addition of 1M potassium hydroxide). Each tooth was immersed separately for four consecutive days (96 h) to stimulate enamel demineralization The Combined Effect of Two Natural Extracts on Enamel Remineraliza;on (in vitro Study) Vol 13 No 2 (2024) DOI 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu [21]. The solution was changed every day. Finally, the samples were washed thoroughly and kept in deionized water. Measurement of Enamel Microhardness The microhardness was measured using a digital Vickers microhardness tester (Fischer Technology, Inc., USA) for sound enamel, following demineralization, and after treatment with the selected agent. The load was 500g for 30 sec. For each reading, the tooth was subjected to three indentations, and the average was calculated to denote its hardness value. Statistical analysis SPSS-26 program was used for analysis. One-way ANOVA, Paired T-test, and Tukey’s HSD test were used for statistical analysis. Results Baseline surface microhardness (SMH) was not significantly different among the groups (p=0.11, F-value=2.14), as shown in Figure (1). Following demineralization, SMH significantly decreased in all samples (p=0.000). Table 1 describes the difference among the groups at the sound and demineralization stages. Following treatment with the remineralizing agents, enamel SMH significantly increased (p=0.000, F=203.87). SMH increased compared with that after demineralization (p=0.000) (Table 2). Tukey's test revealed that group D had the highest SMH value followed by group C (with a non-significant difference between them p=0.94) then groups A and B (Table 3). Discussion Dental tissue remineralization is the preferred preventive measure for dental caries [22]. Many studies have focused on the use of natural products for disease management [5,23]. The present study selected GSE and CESP because they are available and economical and have advantageous properties. Baseline SMH was not significantly different among the groups, indicating a relative approximation in the mechanical appropriates of the samples. Following demineralization, enamel microhardness significantly reduced. Initial enamel lesions have fewer minerals, resulting in a reduced microhardness value [8]. Following remineralization, enamel microhardness significantly increased in all samples. Group D showed the highest SMH indicating that the combination GSE and CESP had a synergistic effect on remineralizing the enamel surface. A possible explanation is that gallic acid, a major constituent of GSE [24], promotes the deposition of CESP minerals, primarily on the enamel surface. This is consistent with a preceding study which concluded that GSE could enhance enamel remineralization by increasing SMH [25]. Mirkarimi et al. (2013) reported the remineralizing property of GSE by forming insoluble deposits on enamel by using a scanning electron microscope and reported elevated microhardness value following demineralization [26]. Yassen and Safy (2018) compared GSE with sodium fluoride (NaF) by VMH and found a non-significant difference between them in dentin remineralization [27]. Amin et al. (2019) found that GSE could increase SMH but the value is lower than that achieved by NaF [28]. The current study demonstrated the GSE remineralizing efficacy as evident by a significant increase in SMH of group B following demineralization. Furthermore, CESP has a high calcium concentration and elevated pH [29], causing a thorough blockage of surface pores and a net increase in enamel The Combined Effect of Two Natural Extracts on Enamel Remineraliza;on (in vitro Study) Vol 13 No 2 (2024) DOI 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu microhardness8. Preceding studies stated that CESP could increase SMH [9] and decrease the surface roughness of enamel affected by erosive lesion [20]. CESP also increases SMH and decreases lesion depth of demineralized enamel [10], in addition to increasing the Ca/P ratio and acid resistance of enamel [30]. This finding can clarify the increase in the enamel microhardness of both groups (D & C with a non- significant difference between them) following exposure to CEPS solution, and the value is higher than those of other groups. No previous study used the same combination to compare with this result. CCP-ACP has remineralizing properties, which have been proven previously. The CPP/ACP paste treatment of the enamel surface results in filling the inter- prism voids and, to some extent, covering the enamel prisms with a layer-like structure [16], thereby increasing the SMH of group A samples. The demineralization procedure used in the current study induced the loss of minerals from the subsurface layer along with preserving calcium and phosphate in the superficial layer [31]. VMH measurement is a quick, nondestructive method for the assessment of surface layer changes in demineralization and remineralization [32]. Flat and polished samples were used for standardization and elimination of naturally occurring variances between teeth that result in different reactions to acid dissolution [33]. Finally, consistent with the obtained outcomes, the null hypothesis was rejected, and the presence of a synergistic effect between GSE and CESP in enamel remineralization was confirmed. Conclusion GSE and CESP had a positive role in enamel remineralization. The combination improved the mineral gain and enhanced the mechanical properties of the enamel surface following demineralization, as shown by the increased SMH values. The findings of this study are limited because it was conducted in vitro. Future clinical studies should verify the outcome presented. Additionally, different application techniques, time intervals, and variables are recommended to investigate the combined effect of these extracts. Ethical Approval This study was approved by the Research Ethics Committee of the College of Dentistry, University of Baghdad (Reference number: 721, Date 28-12-2022). Conflicts of interest The authors declare no competing interest. References 1. Obaid SF, Munther S, Abbas MH. 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Ren Y-F, Zhao Q, Malmstrom H, Barnes V, Xu T. Assessing fluoride treatment and resistance of dental enamel to sos drink erosion in vitro: applicakons of focus variakon 3D scanning microscopy and stylus profilometry. Journal of Denkstry. 2009;37(3):167-76. 33. Hemingway C, Parker D, Addy M, Barbour M. Erosion of enamel by non-carbonated sos drinks with and without toothbrushing abrasion. Briksh Dental Journal. 2006;201(7):447-50. PMID: 17031352. The Combined Effect of Two Natural Extracts on Enamel Remineraliza;on (in vitro Study) Vol 13 No 2 (2024) DOI 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu Figure 1. Baseline enamel microhardness. Table 1. Microhardness at baseline and a=er demineraliza@on. Groups Baseline Mean ± SD After Demineralization Mean ± SD Mean Difference ± SE 95% Confidence Interval of the Difference t-Test Correlation P- Value Lower Upper A 342.18 ± 0.54 175.62 ± 4.83 166.56 ± 1.42 163.43 169.68 117.23 -0.104- 0.000 B 343.03 ± 2.85 174.50 ± 3.34 168.53 ± 0.93 166.47 170.58 180.46 0.464 0.000 C 343.75 ± 1.31 176.22 ± 1.75 167.53± 0.52 166.37 168.68 319.89 0.326 0.000 D 342.51 ± 0.52 176.31 ± 3.24 166.20 ±0.98 164.03 168.37 168.41 -0.248- 0.000 The Combined Effect of Two Natural Extracts on Enamel Remineraliza;on (in vitro Study) Vol 13 No 2 (2024) DOI 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu Table 2. Microhardness a=er demineraliza@on and a=er remineraliza@on . Study Group Demineraliza@on Mean ± SD Remineraliza@on Mean ± SD Mean Difference ± SE 95% Confidence Interval of the Difference t-Test Correla@on P-Value lower Upper A 175.62 ± 4.83 198.68 ± 4.09 -23.05±1.92 -27.28 -18.82 -12.003- -.104- 0.000 B 174.50 ± 3.34 180.94 ± 2.95 -6.44±1.25 -9.20 -3.68 -5.138- 0.053 0.000 C 176.22 ± 1.75 217.84 ± 2.19 -41.62±0.90 -43.60 -39.64 -46.239- -0.238- 0.000 D 176.31 ± 3.24 218.99 ± 6.80 -42.68±1.18 -45.28 -40.07 -35.992 0.904 0.000 Table 3. Post hoc test (Tukey HSD) for groups microhardness at the remineraliza@on stage. Groups Mean Difference ± SE P-Value 95% Confidence Interval Lower limit Upper limit A & B 17.73 ± 1.78 0.000 12.9660 22.5073 A& C 19.16 ± 1.78 0.000 23.9373 14.3960 A& D 20.31 ± 1.78 0.000 25.0806 15.5394 B & C 36.90 ± 1.78 0.000 41.7640 32.1327 B & D 38.04 ± 1.78 0.000 42.8173 33.2760 C & D 1.14 ± 1.78 0.918 5.1940 3.6273