|Year : 2020 | Volume
| Issue : 2 | Page : 38-44
Resistance of chicken egg shell powder treated demineralized enamel to further acidic challenges
Abdelrahman E Elshik BSC , Wedad M Etman, Thuraia M Genaid
Department of Restorative Dentistry, Faculty of Dentistry, Tanta University, Tanta, Egypt
|Date of Submission||18-Feb-2019|
|Date of Acceptance||12-Jul-2019|
|Date of Web Publication||26-Sep-2020|
Abdelrahman E Elshik
Faculty of Dentistry, Tanta University
Source of Support: None, Conflict of Interest: None
The purpose of this in-vitro study was to quantitatively evaluate the remineralization potential of chicken egg shell powder (CESP) solution on early demineralized enamel lesions and the resistance of the treated enamel surfaces to further acidic challenges.
Patients and methods
A 3 × 3 mm enamel windows were done on the buccal surface of 36 sound, freshly extracted human first premolars for orthodontic purposes. Specimens were randomly divided according to the period of treatment with CESP solution into two equal groups (18 each). Ten specimens from each group were examined throughout the steps of the study with energy dispersive radiograph analysis for their calcium (Ca) and phosphorus (P) content. Enamel surface topography of the remaining eight specimens was examined under scanning electron microscope (two specimens for each step). Initial enamel lesions were created by immersion of the specimens separately in 10 ml of a demineralizing solution at 37°C for 96 h. For treatment specimens of group I were individually immersed in CESP solution once daily for 12 min and kept in artificial saliva at 37°C for the rest of the day, for 7 consecutive days. Specimens of group II were treated twice daily with a 12 h interval between both immersions following the cycle mentioned before. The treated specimens were then subjected to a 5 days' pH cycling regimen consisting of demineralization for 3 h, and remineralization for 21 h. The collected data were tabulated and statistically analyzed.
Group II recorded a statistically significant higher Ca value and Ca/P ratio after treatment and pH cycling compared to group I (P = 0.000) as detected by independent t test. Regarding the P content value recorded after treatment it was significantly lower for group II versus group I (P = 0.000). While no significant difference was found between both groups after pH cycling (P = 0.755). Scanning electron microscope findings were found supporting the previous results.
CESP solution provided a significant remineralization potential of the human initially demineralized enamel lesions.
Keywords: scanning electron microscope-energy dispersive radiograph, egg shell powder, enamel remineralization
|How to cite this article:|
Elshik AE, Etman WM, Genaid TM. Resistance of chicken egg shell powder treated demineralized enamel to further acidic challenges. Tanta Dent J 2020;17:38-44
|How to cite this URL:|
Elshik AE, Etman WM, Genaid TM. Resistance of chicken egg shell powder treated demineralized enamel to further acidic challenges. Tanta Dent J [serial online] 2020 [cited 2020 Oct 31];17:38-44. Available from: http://www.tmj.eg.net/text.asp?2020/17/2/38/296173
| Introduction|| |
Numerous studies have been carried out, which have helped to increase the knowledge about dental caries and reduce its prevalence. However, according to the world oral health report, dental caries remains a major dental disease .
In the past two decades, caries research has focused on the development of methodologies to gain minerals other than those available in saliva . Since saliva has low concentration of minerals thus a limited ability to remineralize early enamel lesions .
One of the attractive natural sources that can be used to provide minerals is the chicken egg shell. Its powder was studied in various fields , and was found to contain not only calcium but also other elements such as fluoride (F) and strontium which have a positive effect on bone and dental metabolism ,. Researchers also utilized it to synthesize hydroxyapatite with excellent properties ,.
Although many studies have been done to understand the effects of different remineralizing agents on early enamel carious lesions, there is still inadequate information regarding the benefits of using chicken egg shell powder (CESP) for demineralized enamel surface . And only few studies ,, were performed to evaluate its potential to prevent dental caries progression. While, its protective effect against further acidic challenge has never been addressed to date in literature.
Many techniques have been used to evaluate the mineral content in a lesion including microradiography, contact microradiography, dynamic secondary ion mass spectrometry, microdensitometric scans, and energy dispersive radiograph analysis scans.
One of the currently used microanalytical techniques, is the EDX analysis, which is employed to quantitatively estimate the amount of minerals present in a given tooth sample , combined with scanning electron microscope (SEM) which gives the topographical pictures and is used to assess the surface changes seen on enamel . In addition, it gives quantification of various elements such as Ca, P, F, etc., in both atomic and weight percentage .
Thus the current hypothesis was to proof that CESP solution was able to remineralize enamel simulated carious lesions and the resistance of these treated enamel surfaces to further acidic challenges.
| Patients and Methods|| |
This experimental study was approved by the Research Ethics Committee of the Faculty of Dentistry, Tanta University. All Patients are informed about the research and agreed to use their extracted teeth in this research. A total of 36 sound, freshly extracted human first premolars were collected, cleaned from all soft debris and/or calculus and polished using prophy polishing paste (Proxy; Ivoclar Vivadent, Schaan, Liechtenstein). Modeling wax pieces (Cavex, Holland, the Netherlands) measured 3 × 3 mm were placed in the middle third of the buccal surfaces of all teeth. All around the crown surfaces of the teeth was coated with two layers of a transparent acid resistant nail varnish; the wax was then removed exposing 3 × 3 mm enamel windows . The prepared specimens were randomly divided into two equal groups (18 specimens each) according to the period of treatment with CESP solution and were color coded to be differentiated. Ten specimens in each group were examined with EDX analysis for their Ca andPcontent through the sequential steps of the study. The enamel surface topography of the remaining eight specimens in each group was evaluated under SEM (JEOL Ltd., Japan) (two specimens for each step). Calcined organic CESP was obtained by the process of calcination. Fifteen organic chicken eggs obtained from a local chicken farm were collected. Egg contents were removed and the egg shells cleaned with distilled water and kept in boiling water at 100°C in a clean stainless-steel pot for 10 min to facilitate removal of the membrane . Cleaned egg shells were allowed to dry in the room temperature and crushed using a sterile mortar and pestle (ZOGEAR, Shanghai, China), sift to have a homogenous powder and the large particles were crushed once again. The calcination process was done by heating the homogenously crushed particles at 900°C in a dental laboratory muffle furnace (Kerr Corporation, USA) for 1 h under atmospheric pressure then powdered once again to be very fine . The prepared powder was analyzed for its individual elemental composition percentage using EDX (JEOL Ltd.), which revealed roughly (93% CaO, 1.6% P) of its composition, the particle size of the powder was analyzed by SEM under a magnification ×20 000 [Figure 1].
|Figure 1: Scanning electron micrograph of the calcined CESP illustrating the particle size of the powder. CESP, chicken egg shell powder.|
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To prepare 3% concentration of the remineralizing CESP solution, 1 g of the calcined powder was dissolved in 33.3 ml of sterile deionized water the clear fluid at the top was collected to be used for treatment of specimens . Its chemical composition Ca(OH)2 was detected at the Faculty of Science, Tanta University.
To create initially demineralized enamel lesions, specimens were immersed separately in test tubes containing 10 ml of demineralizing solution at 37°C for 96 h . For treatment; specimens of group I were individually immersed in CESP solution in test tubes once daily for 12 min at 37°C , and kept in artificial saliva at 37°C for the rest of the day, for 7 consecutive days. While specimens of group II were immersed in the solution twice daily with a 12 h interval between both immersions following the cycle mentioned before. The treated specimens in both groups were then subjected to a pH cycling regimen consisting of demineralization for 3 h using the previously mentioned demineralizing solution, followed by immersion in a specially prepared remineralizing solution for 21 h, this cycle was repeated for 5 consecutive days . The Ca andPwt. % values as well as the Ca/P ratio throughout the steps of the study were collected, tabulated, and statistically analyzed using SPSS, version 20 (SPSS Inc., Chicago, Illinois, USA).
| Results|| |
Analysis of variance test revealed a highly statistically significant difference (P = 0.000) among the mean wt. % values of Ca, Pcontent and Ca/P ratio recorded in the different steps of the study in both groups [Table 1] and [Table 2]. Statistical comparison between both groups using independent t test revealed that; after treatment a significant difference was found between the mean Ca and P values of both groups (P = 0.001 and 0.000), respectively. In addition, the Ca/P ratio was significantly different comparing both groups (P = 0.001) [Table 3].
|Table 1: Statistical analysis of the mean values of the calcium, phosphorus, and calcium/phosphorus ratio in group I throughout the steps of the study|
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|Table 2: Statistical analysis of the mean values of the calcium, phosphorus, calcium/phosphorus ratio in group II throughout the steps of the study|
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|Table 3: Independent t test of the mean (wt. %) values of calcium, phosphorus content and calcium/phosphorus ratio between both groups|
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When the treated specimens were subjected to further acidic challenge through pH cycling, similar findings were found regarding Ca content and the Ca/P ratio with a high statistical significant difference (P = 0.000). While no significant difference was found (P = 0.755) between thePcontent mean values in both groups, respectively.
Scanning electron microscope
Enamel surface topography throughout the study; was represented by two specimens for each step confirming the previously obtained results this was represented in [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7].
|Figure 2: Scanning electron micrograph of mature sound enamel surface clarifying the smooth aprismatic layer of surface enamel.|
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|Figure 3: Scanning electron micrograph of initially demineralized enamel showing dissolution of the interprismatic substance (arrows).|
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|Figure 4: Scanning electron micrograph of a specimen of group I after treatment showing clear orientation of enamel prisms with remineralization of interprismatic substance (red arrows) and remineralization of enamel prisms bodies (green arrows). T, tail of enamel rods.|
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|Figure 5: Scanning electron micrograph of a specimen of group II showing remineralization of the enamel bodies, tails and interprismatic spaces (green arrows). Yellow arrow, remaining pores in the subsurface of enamel.|
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|Figure 6: Scanning electron micrograph of cross section of rods of mature enamel after pH cycling of group I specimen showing clear remineralized interprismatic spaces (red arrows) and a sort of mineral elimination from the body of prisms forming a porous surface (green arrows).|
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|Figure 7: Scanning electron micrograph after pH cycling of remineralized enamel for group II specimen showing resistance to further demineralization by acids presented by remineralization of interprismatic substance (red arrows) and remineralization of inorganic crystals of the bodies of enamel rods (green arrows). Yellow arrow, remaining pores in the subsurface of enamel.|
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| Discussion|| |
Over many years the cornerstone of the non-invasive management of incipient carious lesions was fluoride, but its ability to promote net remineralization is limited mainly because its action is confined by the availability of Ca and PO4-3 ions . This has led to the search for new remineralization technologies.
Previous investigations  were performed to find out a way to take an advantage of recycling the egg shells to be used for remineralization. Many researches had been done to evaluate its use as a human dietary oral Ca supplement and for the treatment of osteoporosis [20,,.
CESP was currently produced by the calcination process converting the raw chicken egg shells into a pure powder free of pathogens. A 3% solution of calcined CESP in distilled water was prepared following the method described by Haghgoo reporting that immersion of teeth with initial enamel carious lesions in 10% NHA solution showed the same efficacy of 3% CESP solutions for remineralizing such lesions ,.
The selection of human teeth was based on many studies that utilized human enamel for de/remineralization reporting more reliable clinically applied results ,,,. In the present research the teeth type ( first premolars) and age range (16–25) was selected to standardize the enamel thickness and elimination of variables as possible.
To mimic an optimum temperature for storage, the cleaned examined teeth were incubated at 37°C throughout all the steps of the study .
Initially demineralized enamel lesions were created by keeping the specimens in the demineralizing solution for 96 h ,. The concentration of both Ca and PO4-3 in this solution was at 50% saturation level, causing dissolution of the subsurface enamel only, thus simulating the naturally occurring early enamel lesions which have an intact surface layer .
The selection of the Ca andPcontent values as well as their ratio for statistical analysis was based on the fact that these elements are the main chemical elements affecting the hardness of human tooth enamel . Thus, their values play an important role in enamel condition and health.
Concerning the collected data, EDX measured the Ca andPcontents at the sound state (baseline before any treatment) and after each step of the study. In sound enamel, normal amounts of Ca and P levels have been found with a mean Ca/P ratio (1.9). Which was nearly similar to the results found by Majithia et al.  reporting a ratio of 1.82. SEM examination of a representative sample clarified the normal appearance of enamel rods. The enamel surface layer was generally smooth and homogenous as was shown in [Figure 2], which was the same features of sound enamel described by Neves et al. .
However, after initial demineralization of enamel the Ca and P levels decreased to statistically significant levels. This finding came in agreement with Majithia et al.  recording similar values using the same demineralization protocol, relating this finding to the under saturation of the demineralizing solution with respect to these minerals causing their shift from the tooth surface . Other authors , confirmed these results using SEM examination revealing patterns of crystal dissolution of demineralized enamel prisms similar to those currently recorded in [Figure 3].
The current significant reduction of Ca content after demineralization than that ofPcontent might be attributed to the presence of sodium phosphate in the composition of the utilized demineralizing solution. This was confirmed by the explanation of Hornby et al.  reporting that the presence of sodium phosphate in the demineralizing solution has a protective effect against further loss ofP ions from enamel surfaces.
For remineralization to occur, bioavailable Ca and PO4-3 are essential . Therefore, it was expected that the rich bioavailability of Ca present in CESP solution coupled with its increased pH may enhance the remineralization . In other words, more ions are available for enamel surface remineralization. This explains the current findings in both tested groups showing a statistically significant increase of the values recorded after remineralization versus those of demineralized specimens.
In addition, the Ca and P levels were elevated in both groups to high levels nearly comparable to sound enamel (group I) or even beyond it (group II). Since the formation of hydroxyapatite crystals in the oral environment depends on supersaturated (Ca and PO4-3) ions medium , thus remineralization might be due to deposition of minerals into the porous zone of enamel rather than growth of crystals. SEM examination confirmed the previous results [Figure 4] and [Figure 5].
The currently recorded Ca/P ratio after treatment (2.03 and 2.29 in both groups, respectively) was nearly comparable to that (2.40) reported by Mony et al.  in their study evaluating the remineralization potential of CESP on demineralized enamel lesions by exposing the samples for CESP solution for 21 h for 7 consecutive days. This slightly higher value may be attributed to the difference in the treatment exposure periods between both studies.
To measure the efficiency of the remineralizing material or regimen used in different studies, the treated specimens were re-exposed to many different acidic challenges ranging from pure acids like acetic acid or citric acid, or pH cycling regimens, to even cola based beverages .
In the current study the treated specimens of both groups were exposed to a pH cycling regimen that consisted of alternating demineralization and remineralization according to previous studies ,. The period of the demineralization phase was adjusted to be for 3 h, to simulate the duration of demineralization (low cariogenic challenge) that occurs in the oral cavity .
Regarding the effect of pH cycling on the Ca content values, a significant decrease was recorded in both groups compared to their respective values after remineralization. This finding is confirmed by Majithia et al.  utilizing the same pH cycling protocol.
The SEM representative image of a specimen of group I after pH cycling [Figure 6] revealed elimination of mineral content from core of enamel rods  leaving a porous enamel surface denoting that specimens treated with CESP solution once daily had a lower resistance to further acidic attacks. This may be explained by the lower Ca regain value than group II which revealed more resistant enamel rods to further acidic attacks as represented in [Figure 7]. The increased Ca content by treatment was reported to act as a reservoir to increase the capacity of enamel to resist further ionic loss .
On the other hand, after pH cycling, P level in group I decreased significantly versus that recorded after treatment. While in group II although the treatment has not elevated thePlevels significantly, there was more resistance to further loss ofPions which may be attributed to the higher reservoir of Ca ions in this group that was able to saturate the demineralizing solution. This could be supported by Cummins  reporting that during demineralization, Ca release precedesPrelease.
Therefore, this released Ca saturated the demineralizing solution causing lesser effect on thePcontent. Therefore, using calcium rather than phosphate to suppress the demineralization process would be effective.
So compared to other remineralization studies, CESP could be considered to have a promising future in treating initial enamel lesions due to its natural source and easy bioavailability .
| Conclusion|| |
Under the limitations of this in-vitro study, the following might be concluded:
- CESP could be considered among the natural sources of minerals to be used for remineralization of initially demineralized enamel surface.
- The regimen utilizing CESP solution twice daily to treat initially demineralized enamel was found effective in increasing the Ca, P, and Ca/P ratio in the surface enamel with better results to utilizing the solution once daily.
- CESP solution treated enamel surface resisted further acidic challenges.
| Recommendations|| |
Based on the findings of this in-vitro study, the following recommendations could be drawn:
- Incorporation of the tested CECP solution in a commercial product for further in-vitro and in-vivo investigations.
- Comparative evaluation of the tested solution with the currently available remineralizing agents.
- Assessing the particle size after calcination using laser diffraction analysis to visualize the homogeneity of the powder after calcination.
- Assess the usage of different higher concentration of CESP solution with its potentiality for remineralization.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3]