Abstract
Dental treatment has been shifted towards more conservative approaches that focus on caries identification at early stage, remineralization of dental surfaces, and preservation of tooth structure. So that, newly safe alternative methods for teeth remineralization have to be created. There is minimal evidence to support the use of eggshell powder (ESP) for remineralizing enamel and Cuttlefish bone powder (CBP) as an alternative calcium source in bone replacement.The aim of this study is to evaluate the capacity of eggshell powder (ESP) and cuttlefish bone powder (CBP) solutions to remineralize early initiated demineralized lesions of enamel in permanent teeth. 40 extracted premolars were divided into four groups: Group1 (which received no treatment), Group2 (which was subjected todemineralizing solution), Group3 (which was subjected todemineralizing solution and then treated with CBP solution), and Group4 (which was subjected todemineralizing solution and then treated with ESPsolution). All groups were prepared forscanning electron microscope (SEM), X-ray microanalysis (EDAX), and microhardness evaluation. The demineralization process significantly changed the surface structure of the enamel, resulting in erosive lesions. CBP and ESP solutions both had a reparative effect, enhanced surface morphology, and increased Ca and P content and microhardness.
Graphical abstract
1
Introduction
Dental caries is classified as one of the most frequent chronic disorders worldwide [ ]. According to the World Health Organization (WHO), there is an extensive dispersion of caries epidemiology, featuring 60–90% caries prevalence among children in schools globally [ ]. Repairing carious lesions with tooth preparation and restorative material is an irreversible cycle that leads toloss of tooth structure over time [ ]. The major components of contemporary dental caries therapy are preventive treatment and early control of beginning carious lesions [ ]. Remineralizing agents serve a significant role in remineralization and the prevention of dental caries [ ].Fluoride is considered a powerful remineralizing agent for the prevention of dental caries. It plays a significant role in enhancing the resistance of tooth structure to acid demineralization by forming fluoro-apatite crystals in enamel [ ].The continuous use of fluoridated toothpastes, on the other hand, raised the risk of dental fluorosis during the dental development phase. Wright et al. (2014) [ ] conducted a systematic evaluation of fluoride toothpaste effectiveness and safety in children under the age of six. They concluded that“fluoride toothpaste was found to be useful in the prevention of dental cavities.” However, ingestion of pea-sized quantities or more, on the other hand, increases the risk of moderate fluorosis. Many studies have been conducted togaininformation about dental caries and minimize its prevalence, but dental caries remains a serious dental condition, based on the World Oral Health report [ ].
Caries researchershave spent the last two decades focusing on developing methods to get minerals for teeth enamel from sources other than those found in saliva [ ]. Since saliva contains few minerals, it has a limited potential to remineralize early enamel defects [ ]. Specialists are looking for distinctive materials that are unsurpassed and more appealing [ ]. Until now, hydroxyapatite has been produced using primitive materials such as eggshells and animal skeletons [ , ]. Sea-going live organisms, for example: coral, nacre, and cuttlefish, have been used recently to promote bone repair [ ].
Despite several studies on the effects of various remineralizing agents on early enamel carious lesions that have been conducted [ ], there is currently little evidence on the advantages of employing eggshell powder (ESP) for remineralizing tooth enamel [ ], and the remineralizing impact of cuttlebone on human enamel has never been investigated through scientific research.
Cuttlefish bone (CB), also known as cuttlefish spine, is the inside shell of marine species known as cuttlefish (Sepia), belonging to the phylum mollusca, class cephalopod, and arrangement Sepiidae [ ]. CB is mostly composed of calcium carbonate. A few studies focused on the use of cuttlefish bone as a calcium source for bone replacements [ ]. CB is commonly used and considered a marine standard filler materialfor osteoporosis [ ].
ESP is considered a natural calcium ion source, itis composed of 94% calcium phosphate, 4% organic matter, 1% magnesium carbonate, and trace amounts of strontium, fluoride, manganese, zinc, and copper and plays a critical function in bone and tooth metabolism [ ]. Researchers demonstrated that ESP could generate high-quality hydroxyapatite [ , ]. Kattimani et al. [ ] showed in 2014 that eggshell-derived hydroxyapatite is a versatile novel regenerative material that could be used as analternative to bone graft due to its biocompatibility, absence of disease transmission hazards, ease of use, endless supply, cost-effectiveness, efficiency, and capacity to be made in a cost-effective manner.When ESP minerals become in contact with caries-like lesions in the enamel, they permeate into the superficial layer and restrict the surface porosities [ ]. In 2016, Haghgoo et al. [ ] stated that ESP solution may be utilized as a remineralizing agent in preventive dentistry as a supplement or may be an alternative to fluoride application.
It has been recently revealed that ESP could improve bone mineral density in vitro as well as stimulate chondrocyte differentiation and cartilage formation [ ]. Unfortunately, little research has been conducted to investigate the effect of ESP on the remineralization of incipient enamel carious lesions [ ].
Based on preventive dentistry, nonsurgical care of early carious lesions is mandatory, and efficient therapeutic options to promote non-operative management of early caries lesions (remineralization) are required [ ]. The aim of this study is to evaluate the capacity of ESP and CBP solutions to remineralize early initiated demineralized enamel in permanent teeth. The null hypothesis was that CBP and ESP solutions had a remineralizing effect on demineralized enamel, enhanced surface morphology, and increased Ca and P content and microhardness.
2
Materials and methods
2.1
Samples
Forty human premolars have been extracted lately for orthodontic purposes. The teeth were cleaned and rinsed under running water after being checked by a light microscope (2X) to ensure that there were no cavities, calculus, or surface abnormalities.
2.2
Study design
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Control –ve group (Group 1) : During the experiment, 10 teeth were placed in ready-made artificial saliva and stored at 37 °C in an incubator.
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Experimental Groups: 30 teeth were subjected to the demineralization phase (which will be described below in detail) and then divided into three equal groups (10 teeth each):
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Control + ve group (Group 2) : teeth were stored in artificial saliva and stored at 37 °C in an incubator.
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CBP (Group 3) : teeth were immersed in a 10% solution of CBP for 3 min daily for 7 days.
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ESP (Group 4) : teeth were immersed in a 10% solution of ESP for 3 min daily for 7 days.
2.3
Teeth preparation
Teeth samples were kept in an incubator at 37 °C and individually placed in artificial saliva that had already been prepared for the study. A square window measuring 3 by 3 mm was drawn in the middle-cervical third of the buccal enamel surface of every tooth specimen. In addition, the remaining specimen was given two coats of waterproof acrylic varnish, and the apical foramina of the teeth were occluded with the same acrylic varnish to prevent fluid infiltration via the foramen and, once dry, covered in tin foil [ , ].
Following that, the specimens were placed in individual test tubes containing freshly prepared artificial saliva and kept in an incubator for 48 h at 37 °C. The artificial saliva (pH 6.57) had been made by combining sterile deionized water 99.6%, potassium chloride 0.12%, sodium chloride 0.08%, magnesium chloride 0.01%, carboxymethyl cellulose 0.10%, dibasic potassium phosphate 0.03%, and calcium chloride 0.01%.
2.4
Technique of demineralization
The tooth specimens from Groups 2, 3, and 4 were removed from artificial saliva and immersed individually in test tubes filled with a freshly prepared demineralizing solution (pH = 4.2; 2.2 mMol calcium chloride, 0.05 M, acetic acid, 2.2 mMol sodium phosphate, and 1 M potassium hydroxide; daily changes made to prevent saturation) [ ] for 96 h at 37 °C. After that, the specimens were removed and cleaned with distilled water for 5 s to counteract the demineralizing solution’s effect. They were then placed back in artificial saliva at 37 °C in an incubator until the next research phase.
2.5
Technique of remineralization
For remineralization in Groups 3 and 4, tooth specimens were individually immersed in freshly prepared remineralizing solutions (CBP and ESP) for 3 min daily, then placed back in artificial saliva at 37 °C in an incubator. These operations were carried out once a day for seven days.
Preparation of CBP: Squares of CB were taken from the NorthCoast Sea beach, gently cleaned with distilled water, and dried in free air to erase their fragrance. After drying, the clean cuttlebone was powdered and properly homogenized to a mesh size of 60–100 [ ]. Preparation of CBP solution: By dissolving 10 g of manufactured CBP in 100 ml of distilled water, a 10% solution of CBP was created [ ].
2.5.1
Preparation of ESP
Fourteen organic chicken eggs were acquired from a supermarket. The egg contents were removed, and the eggshells were washed with distilled water before being placed in boiling water at 100 °C in a clean stainless-steel kettle for 10 min to assist membrane removal [ ]. Cleaned eggshells were allowed to dry at room temperature before being crushed using a sterile mortar and pestle, sifted to make a homogeneous powder, and the big particles crushed again. The homogeneously crushed particles were calcined by heating them at 900 °C in a muffle furnace (Kerr Company, USA) for 1 h under air pressure before being powdered again [ ].
Preparation of ESP solution : A 10% solution of ESP was prepared by dissolving 10 g of ESP in 100 ml of distilled water [ ].
Daily pH readings of the freshly prepared remineralizing solutions were taken, and the average values for CBP and ESP solutions were found to be 9.2 and 9.44, respectively.
2.6
Experimental procedures
2.6.1
Scanning electron microscope (SEM) and energy dispersive X-ray microanalysis (EDXA) examination
A removable adhesive was used to fix dry teeth to the SEM holder. The buccal surface (one-third of the cervix) was investigated, and the surfaces (Ca), (P), and (C) were weighted using an FEI/Inspect (SEM) – EDXA Unit, Chemical Laboratory Defense, with the S-UTW detector (EDXA Inc., Mahwah, NJ, USA). They were scanned at 30 keV using the LFD secondary electron detector at (X1000) and (X2000) magnifications (spot size 5.5 nm) at each magnification. The EDXA detector has a count rate of 1800–2000 counts per second and a resolution of 132.14 V.
2.6.2
Microhardness (MH) evaluation
A Vickers MH tester (LaizhouHuayin Testing Instrument Co., model HVS-50, Ltd., China) with a Vickers diamond pointer and 20× objective lens was used for MH measurements. For each sample, measurements were obtained at three separate locations. Indents were created roughly 0.5 mm apart. Each measurement was taken for 15 s with a 200 g force directed perpendicular to the cervical buccal surface. The indentations’ diagonal lengths were measured with a built-in micrometer, and all measurements were transformed to Vicker numbers.
The following equation was used to calculate MH:
HV is the Vickers hardness in kgf/mm 2 , P is the load in kgf, and d is the average diagonal length in mm.
2.6.3
Statistical analysis
Sample size calculation was done using power and sample size calculations software (version 3.1.6) for MS Windows by selecting F-test family for one-way ANOVA with effect size f = 1.251558, power (1- β) = 80% and α = 5%, based on the previous study [ ]. The total sample size was 27 samples (7 in each group). The total size was increased to 10 samples per each group (Total: 40) to compensate for the loss of samples before the end of study.
The measurements are reported as mean ± standard deviation (SD). The significance level was chosen at P < 0.05. To compare Ca, P, and C contents, Ca/P ratio, and MH values between research groups, one-way ANOVA and Tukey’s HSD post hoc tests were used. The statistical analysis was carried out with the help of the software Statistical Program for Social Science (SPSS®, IBM 25, USA).
3
Results
3.1
Histological results
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Group 1 (Control –ve): SEM examination of the surface of Group 1 revealed well-defined perikymata grooves, ridges, and ends of the enamel rods with smooth surfaces ( Fig. 1 A and B).
Fig. 1 Scanning electron micrographs for cervical buccal enamel showing: A&B: Group1(C-ve) showing perikymata grooves and ridges (Red arrows), few enamel rod ends (Black arrows) and areas of rodless enamel (yellow dots) (Ax1000 &Bx2000). C&D: Group2 (C + ve), showing grooves of multiple length and directions (Red arrows), some erosive areas (Black arrows) (Cx1000 &Dx2000). E&F: Group3 (CBP) showing diffused mineralized deposits (Blue arrows), small areas of defect (Red arrows), clearly observed enamel rod ends (Black arrows) (Ex1000 &Fx2000). G&H: Group 4 (ESP) showing smooth surface with perikymata grooves (Red arrows), apparent light mineralized sporadic areas (Blue arrows), well-defined erosive areas (Black arrows) (Gx1000 &Hx2000). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) -
Group2 (Control + ve): displayed variable lengths and orientations of grooves, as well as well-marked erosive patches, and an uneven enamel surface ( Fig. 1 C and D).
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Group 3: exposed visible dispersed light mineral deposits, little areas of defects, and the ends of the enamel rods were clearly shown. It showed a more accentuated continuous amorphous layer of mineral deposition with an irregular structureless appearance. In certain areas, this deposition exhibited a globular appearance with some globules fused together ( Fig. 1 E and F).
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Group 4: distinct perikymata grooves, visible light-mineralized regions, well-defined erosive areas and an amorphous layer deposited on the surface of the enamel. Obliteration of rod ends was detected in some areas, as a result of minerals deposition along their periphery ( Fig. 1 G and H).
3.2
Statistical analysis
3.2.1
EDXA results
There were significant differences between the groups in terms of mean surface Ca, P, and C% wt change ( Tables 1–3 ). In addition, groups 1, 3, and 4 had higher mean Ca values and lower mean C contentsthan group 2. Regarding the experimental groups, G3 had the greatest mean Ca wt%, followed by G4, and G2 had the lowest value. G4 had the greatest mean P wt%, followed by G3, and G2 had the lowest value. G2 has the greatest mean C wt% followed by G4 and G3. G2 has the highest mean Ca/P ratio, followed by G3 and G4 ( Fig. 2 A). There is no statistically significant difference in Ca/P ratio between G1 and G4 ( Table 4 ).
| Source | SS | Df | MS | F = 2027.01744 a |
|---|---|---|---|---|
| Between-Groups | 859.1769 | 3 | 286.3923 | |
| Within-Groups | 3.3909 | 24 | 0.1413 | |
| Pairwise Comparisons | HSD .05 = 0.5543 | Q .05 = 3.9013 | p- value | |
| HSD .01 = 0.6971 | Q .01 = 4.9068 | |||
| G 1 :G 2 | M 1 = 52.00 ± 0.192 | 13.94 | Q = 98.14 | <0.0001 a |
| M 2 = 38.05 ± 0.17 | ||||
| G 1 :G 3 | M 1 = 52.00 ± 0.192 | 1.67 | Q = 11.77 | <0.0001 a |
| M 3 = 50.32 ± 0.287 | ||||
| G 1 :G 4 | M 1 = 52.00 ± 0.192 | 8.22 | Q = 57.87 | <0.0001 a |
| M 4 = 43.77 ± 0,57 | ||||
| G 2 :G 3 | M 2 = 38.05 ± 0.17 | 12.27 | Q = 86.37 | <0.0001 a |
| M 3 = 50.32 ± 0.287 | ||||
| G 2 :G 4 | M 2 = 38.05 ± 0.17 | 5.72 | Q = 40.27 | <0.0001 a |
| M 4 = 43.77 ± 0,57 | ||||
| G 3 :G 4 | M 3 = 50.32 ± 0.287 | 6.55 | Q = 46.09 | <0.0001 a |
| M 4 = 43.77 ± 0.57 | ||||
| Source | SS | df | MS | F = 521.5252 a |
|---|---|---|---|---|
| Between-Groups | 265.5899 | 3 | 88.53 | |
| Within-Groups | 4.074 | 24 | 0.1698 | |
| Total | 269.664 | 27 | ||
| Pairwise Comparisons | HSD .05 = 0.6075 | Q .05 = 3.9013 | p- value | |
| HSD .01 = 0.7641 | Q .01 = 4.9068 | |||
| G 1 :G 2 | M 1 = 20.58 ± 0.38 | 7.88 | Q = 50.58 | <0.0001 a |
| M 2 = 12.71 ± 0.123 | ||||
| G 1 :G 3 | M 1 = 20.58 ± 0.38 | 5.03 | Q = 32.33 | <0.0001 a |
| M 3 = 15.55 ± 0.636 | ||||
| G 1 :G 4 | M 1 = 20.58 ± 0.38 | 1.45 | Q = 9.29 | <0.0001 a |
| M 4 = 19.14 ± 0.102 | ||||
| G 2 :G 3 | M 2 = 12.71 ± 0.123 | 2.84 | Q = 18.25 | <0.0001 a |
| M 3 = 15.55 ± 0.636 | ||||
| G 2 :G 4 | M 2 = 12.71 ± 0.123 | 6.43 | Q = 41.29 | <0.0001 a |
| M 4 = 19.14 ± 0.102 | ||||
| G 3 :G 4 | M 3 = 15.55 ± 0.636 | 3.59 | Q = 23.04 | <0.0001 a |
| M 4 = 19.14 ± 0.102 | ||||
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