EGYPTIAN Vol.

64, 3703:3712, October, 2018

DENTAL JOURNAL I.S.S.N 0070-9484

Fixed Prosthodontics, Dental materials, Conservative Dentistry and Endodontics

www.eda-egypt.org • Codex : 122/1810

REMINERALIZATION OF INITIAL ENAMEL LIKE LESIONS

WITH CHICKEN EGG SHELL POWDER SOLUTION VERSUS
AMORPHOUS CALCIUM PHOSPHATE

Hebatallah M Taher* and Rania E Bayoumi **

ABSTRACT
Aim: To compare the efficacy of chicken egg shell solution powder (CESP) and amorphous
calcium phosphate (ACP) on the remineralization of enamel subsurface lesions.

Materials and Methods: Twenty bovine central incisor teeth were used in this study. The
microhardness and the minerals content of all specimens were initially assessed using Vickers
hardness tester and EDXA respectively (positive control group).The specimens were artificially
demineralized and then reassessed directly after demineralization (negative control group). All
demineralized specimens were randomly classified into 2 equal groups,group I :treated with Chicken
eggshell powder solution (10 specimens), while, groupII: treated with ACP (10 specimens). The
remineralizing agent were applied twice daily for 15 min each for seven successive days. Finally all
specimens were reassessed for minerals content (Ca and PO4 weight %) and surface microhardness.
The data were statistically analyzed using repeated measures analysis of variance (ANOVA), the
significance level was set at P ≤ 0.05.

Results: Microhardness was significantly decreased in all specimens after demineralization

and then it was significantly increased after exposure to therapeutic solutions. There was a
significant increase (P<0.001) in both Calcium and Phosphorus levels after remineralization using
both agents, as detected by EDAX. However, there was no significant difference between the two
remineralized groups.

Conclusions: Within the limitations of this in vitro study, it can be concluded that both
remineralizing agents were similarly able to increase the microhardness and tooth remineralization.
However, being natural products, CESP can be considered as an optimal alternative to the
commercial ones.

KEY WORDS: Demineralization, EDAX, Eggshell, Enamel, remineralization, surface

microhardness

* Associate Professor of Restorative Dentistry, Cairo University and Modern Sciences and Art in Egypt.
** Lecturer, Biomaterials Department, Faculty of Dentistry (girls), El-Azhar University.
(3704) E.D.J. Vol. 64, No. 4 Hebatallah M Taher and Rania E Bayoumi

INTRODUCTION ment and the application of ACP-containing prod-

Enamel demineralization usually begins ucts, especially in the dental fields. It is also used
with dissolution of the subsurface carbonated as filler in ionomer cements to fill carious lesions
hydroxyapatite crystals under the effect of low or as a colloidal suspension in toothpastes, chewing
pH of surrounding acidic media. That’s creating gums or mouthwashes to promote remineralization
fine micro pores and gaps in between the enamel of carious lesions and/or to prevent tooth deminer-
rods (1). At pH 5.5 H+ ions, produced by the bacterial alization(11). As the ACP is not a stabilized structure,
byproducts or any acidic activity, react preferentially it requires a two-phase delivery system to keep the
with the phosphate group of the enamel crystals, calcium and phosphorus components from reacting
altering the (PO4) − 2 ions to (HPO4)− 2 ions which, with each other before use. (12,13)
once formed, no more crystal lattice can be formed
Throughout the ages humans have relied on
and at the same time H+ ions are buffered. This leads
nature to benefit for their basic needs, as medicines
to enamel dissolution which indicates the beginning
of early enamel loss. (2, 3) for the treatment of a wide spectrum of diseases. (14)
Chicken egg shell solution powder (CESP) has been
Management of initial carious lesions should tested in various fields regarding its potential medical
be of a preventive rather than curative approach.
use. It is known as Ca rich source which contains
Enamel repair occurs through the deposition of
about 93% (w/w) elemental Ca with a high degree
depleted minerals in the demineralized defects
of bioavailability due to its high CaCO3content. (15)A
at a molecular level. This process needs neutral
previous study found that the high pH of the chicken
pH at which sufficient calcium and phosphate ions
egg shell solution along with its rich bioavailable
are available in the surrounding environment. So,
remineralizing the exhausted enamel surface with calcium content provided it with great potentiality
the needed calcium and phosphate ions,increased its to favor the remineralization process.(16) Therefore,
resistance to further acid challenge, particularly with further research is necessaryon the efficacy of CESP
the use of enhanced remineralization treatments. (4) for remineralization of early enamel lesions.

Fluoride containing products have long been rec- Thus the aim of this in vitro study was to com-
ognized as they promote caries lesion remineraliza- pare the efficacy of amorphous calcium phosphate
tion and inhibits demineralization of tooth surfaces (ACP) and Chicken eggshell powder (CESP) in
subjected to acids related to the caries process (5). the remineralization of initial enamel subsurface
However, utilizing fluoride alone seems question- lesions using quantitative energy dispersive X-ray
able as the formation of each fluorapatite molecule analysis (EDXA) and microhardness analysis. The
requires calcium and phosphate in addition to fluo- null hypothesis was that CESP has the same potent
ride ions.(6) Thus, it is necessary to find an efficient, remineralizing effect as ACP material.
safe alternative to fluoride to completely prevent
caries and remineralize the incipient enamel lesions. MATERIALS AND METHODS
Amorphous calcium phosphate(ACP) has been
Materials:
widely applied in biomedical field due to its excel-
lent bioactivity, high cell adhesion, adjustable bio- Two different remineralizing agents were used in
degradation rate and good osteoconduction(7-9) .The the current study; commercial relief ACP and labo-
first quantitative studies on synthetic ACP were ratory prepared egg shell solution. Materials, com-
done in the mid-1960s(10). Afterward, more and positions, manufacturers and batch number of the
more attention has been attracted in the develop- materials used in this study are shown in table I
REMINERALIZATION OF INITIAL ENAMEL LIKE LESIONS WITH CHICKEN EGG (3705)

TABLE (I) Materials, compositions, manufacturers and batch number of the used materials.

Commercial Product
Materials and Manufacturer/ Compositions Batch Number
Laboratory Prepared
5%potassium nitrate, 0.22% sodium fluoride,
0.375% amorphous calcium phosphates,water, po-
Relief ACP Dash Philips USA 16314001
loxamer 338, natural peppermint, calcium nitrate,
sodium phosphate, sodium saccharin
94% calcium carbonate,1% calcium phosphate, 1%
Egg shell powder Laboratory prepared ...................
magnesium carbonate, 4% organic matrix (17)
(Na-3PO4 (3.90mM), NaCl (4.29mM), KCI
Artificial saliva Laboratory prepared (17.98mM), CaCl2 (1.10mM), MgCl2 (0.08mM), ..................
H2SO4 (0.50mM), NaHCO3 (3.27 mM)(18)
50 mM acetic acid derivation, 2.25 mM CaCl2
Demineralizing solution Laboratory prepared ...............
2H2O, 1.35 mMKH2PO4; 130 mm KCl(19,20)

Methodology: Tester (Wilsonminiaturized scale hardness analyzer,

display Tukon1102 Germany) with a Vickers dia-
Specimen preparation:
mond indenter and a 20X objective lens. A load of
Twenty sound bovine incisor teeth were selected 100g was applied to the surface of the specimens for
for this study. The teeth were examined using 20 seconds. Three indentations, which were equally
magnifying loupes at 3X (Univet custom made placed over a circle and not closer than 0.5 mm to
lopes, Italy) and head light to ensure the absence of the adjacent indentations, were made on the surface
any deformity or pathological defects. The selected of each specimen. The diagonal lengths of inden-
teeth were thoroughly cleaned to remove debris or tations were measured by built-in scaled microm-
any attached periodontal tissue using a hand scaler eter and values were changed over into Vicker’s
Scaler 10A, NOVA instruments Ltd, Berkshire, UK) numbers. The values were averaged to produce one
then they were stored in deionized water containing hardness value for each specimen Micro-hardness
0.2% thymol solution. Radicular portions of all values were obtained utilizing the following equa-
teeth were removed with a slow speed diamond saw tion: HV=1.854 P/d2 (21) Where, HV was Vickers
(Hard tissue microtome,Bronwill,E.McGranthinc, hardness in Kg/mm2, P was the load in Kg and d
MA,USA) under water irrigation. The coronal was the average length of the diagonals in mm.
portion of each tooth was imbedded in self-cured
acrylic resin with labial surface facing upward.The Quantitative elemental analysis (weight %) by
EDXA:
labial enamel surfaces of the specimens were ground
using silicon carbide paper (grades 600-1200) The mineral content of all positive control speci-
under water irrigation to produce more consistent mens was assessed quantitatively using Energy Dis-
reproducible flat enamel surfaces. persive X-ray analysis (EDXA) (Ametek, Materials
Analysis Division, Netherlands), computer con-
Baseline micro-hardness test trolled software Genesis using an accelerating volt-
The baseline microhardness was evaluated for age of 20–25 kV. Elemental level including Calcium
all specimens (positive control group/ n=20 speci- (Ca) and Phosphorus (P) were evaluated in weight
mens) using Digital Display Vickers Microhardness percentage.
(3706) E.D.J. Vol. 64, No. 4 Hebatallah M Taher and Rania E Bayoumi

Demineralizing Protocol was done to increase the mixture alkalinity and to

make sure that it was pathogen free.(22,23)
The early artificial demineralization of enamel
subsurface was achieved by immersing the
Preparation of Eggshell Powder Solution:
specimens into glass container containing 20ml
of demineralizing solution. The solution was One gram of CEP was dissolved in 20 ml of
adjusted with a 1.0 M of NaOH to a pH of 5.0 4% acetic acid (India Chemicals, Pvt ltd. Mumbai,
using pH meter (Deluxe deep vision, model no: India) in a test tube. The clear fluid which was
101, California, USA), at room temperature for 72 collected at the top was then transferred to a beaker
hours. (19,20) Microhardness and elemental analysis .The pH of the solution was measured by using a
were revaluated for the demineralized specimens pH meter(Deluxe deep vision, model no: 101,
(negative control group/ n=20 specimens). California, USA) and it was adjusted to 11.7 (16)

Grouping of the specimens Application of the remineralizing agents

Specimens were divided into two groups(n=10 The demineralized specimens were immersed in
specimens/gp) according to the remineralizing the egg shell powder solution for 15 minutes/twice
material used, as follow : daily for seven consecutive days. During this seven
days, the specimens were stored in artificial saliva
Group (I): Remineralized using prepared egg
with pH = 7.2. A fresh egg shell powder solution was
shell solution.
used daily to enhance the process of remineralization.
Group (II): Remineralized using amorphous cal- While, in the second group, specimens were painted
cium phosphate ACP (Relief, Zoom Philips, USA) using disposable brushes with the ACP using the
same testing period. Finally, Microhardness and
Preparation of Chicken Eggshell Powder: elemental analysis were conducted again after the
Powder was prepared by the calcination remineralization procedure.
protocol given by World Property Intellectual
Statistical analysis
organization (22). Chicken Egg Shell has about
95 % of calcium carbonate which is converted into Numerical data were explored for normality by
basic calcium oxide by calcination process. Twenty checking the distribution of data and using tests
chicken eggs were obtained from a local hatchery, of normality (Kolmogorov-Smirnov and Shapiro-
the contents were removed and the eggshells were Wilk tests). All data showed normal (parametric)
cleaned in distilled water. The eggshells were then distribution. Data were presented as mean and
kept in hot water bath at 100°C for 10 minutes standard deviation (SD) values. One-way ANOVA
followed by removing the inner membrane. These was used to compare between the groups. Tukey’s
eggshells were then crushed using a sterile mortar post-hoc test was used for pair-wise comparisons.
and pestle. The crushed particles then heated at The significance level was set at P ≤ 0.05. Statistical
1200°C in a furnace (Lava Furnace 200,3M,USA)TM analysis was performed with IBM® SPSS® Statistics
to go through the calcination process. This process Version 20 for Windows.

® IBM Corporation, NY, USA.

® SPSS, Inc., an IBM Company.
REMINERALIZATION OF INITIAL ENAMEL LIKE LESIONS WITH CHICKEN EGG (3707)

RESULTS Results of quantitative elemental analysis

The changes in concentrations of the total
Microhardness
mineral components of bovine enamel in the
One-way ANOVA results showed a statistically different groups are summarized in table (2) and
significant difference between the groups at graphically represented in figure(2)
P<0.001. Result revealed that the (+ve) control
One-way ANOVA results showed that the (+ve)
group had the statistically significantly highest
control group had the statistically significantly
mean vicker’s hardness number (295.1 ±25.6 VHN)
highest mean minerals content (35.9 ±6.4 Ca wt %
while, the demineralization group (-ve control)
and 15.2 ±3.1 P wt%). While demineralized group
showed the statistically significantly lowest mean
(-ve control gp) showed the statistically significantly
hardness (204.1 ±41.2.1 VHN). Moreover, there
lowest mean minerals content (24.1 ±4.5 Ca wt %
was no statistically significant difference between
and 9.7 ± 2.2 P wt %). Irrespective of the treatment
Egg Shell groups (gp I) and ACP (gp II) (235.9
group there was no statistically significant difference
±30.5 VHN and 244.8 ± 51.1. VHN respectively);
between GpI and Gp II; both showed statistically
both showed a statistically significantly higher mean
significantly higher mean minerals content than -ve
hardness than demineralization group and lower
control group and lower mean mineral content than
hardness than (+ve) control group as summarized
+ve control group.
in table (1) and graphically represented in figure (1).

TABLE (1) The mean, standard deviation (SD) values of the microhardness(VHN) of the different groups

+ve control (n = 20) -ve control (n = 20) Gp I (n = 10) Gp II (n = 10)

P-value
Mean SD Mean SD Mean SD Mean SD

295.1 A 25.6 204.1 C 41.2 235.9 B 30.5 244.8 B

51.1 <0.001*

*: Significant at P ≤ 0.05, different superscripts are statistically significantly different.

TABLE (2) The mean and standard deviation (SD) values of minerals content (Ca and P weight %) of the
different groups

+ve control (n = 20) -ve control (n = 20) GpI (n = 10) GpII (n = 10)
P-value
Mean SD Mean SD Mean SD Mean SD

Ca 35.9 A 6.4 24.1 C 4.5 31.2 B 7.4 30.1 B 6.5 <0.001*

P 15.2 A 3.1 9.7 C 2.2 11.5 B 3.0 10.9 B 2.5 <0.001*

*: Significant at P ≤ 0.05, Different superscripts in the same row are statistically significantly different
(3708) E.D.J. Vol. 64, No. 4 Hebatallah M Taher and Rania E Bayoumi

Fig. (1): Column chart representing mean and standard deviation Fig. (2). Column chart representing mean and standard
values for microhardness (VHN) in the different groups deviation values for Calcium and Phosphorus weight %
in the different group

Element Wt % At %

CK 16.82 29.36

OK 31.28 41.00

PK 16.20 10.97

CaK 35.70 18.67

Fig. (3) Representive figure for the elemental analysis by EDX of enamel surface of specimen befor any treatment (+ve control
group)

Element Wt % At %

CK 28.60 42.31

OK 37.00 41.11

PK 10.17 05.83

CaK 24.23 10.75

Fig. (4): Representive figure for the elemental analysis by EDX of enamel surface of specimen after demineralization (-ve control

group)
REMINERALIZATION OF INITIAL ENAMEL LIKE LESIONS WITH CHICKEN EGG (3709)

Element Wt % At %

CK 17.57 29.80

OK 33.95 43.22

PK 15.75 10.36

CaK 32.72 16.63

Fig. (5) Representive figure for the elemental analysis by EDX of enamel surface of specimen after remineralization using egg shell
powder solution (Gp I).

Element Wt % At %

CK 20.55 34.00

OK 32.81 40.76

PK 14.26 09.15

CaK 31.75 15.74

Fig. (6) Representive figure for the elemental analysis by EDX of enamel surface of specimen after remineralization using ACP
varnish (Gp II)

DISCUSSION bioactive glass, casein phosphopeptide, fluoride and

some natural agents.(4,28,29) In the current study, two
The modern dental practice is now continuously
different remineralizing agents were used; the first
shifting towards the concept of minimal invasion
is derived from natural which was egg shell powder
dentistry (MID) which is a conservative concept
solution (CESP) and the second one is a commercial
that mainly emphasizes upon early detection of
available ACP
carious lesions, remineralization of tooth surfaces
and preservation of surrounding tooth structure(24 This study sought to assess and compare the
,25)
. Studies based on this concept revealed that the efficacy of the CESP solution and ACP gel for
incipient enamel caries are reversible provided remineralization of enamel initial like lesions in
that a super saturated state with calcium and bovine teeth. Concerning the CESP, previous X-
phosphorous ions are kept in the adjacent enamel. Ray fluorescence spectroscopic analysis of the
(26,27)
Super saturation can be achieved by using egg shell have shown that it contains around 98%
different remineralizing agents that are able to Calcium, 0.46% of phosphate, 0.53% of Magnesium,
release sufficient amount of Ca and P ions. These 0.18% of Strontium, 0.03% of Potassium and 0.03%
agents include amorphous calcium phosphate, Potassium. This high concentration of bio available
(3710) E.D.J. Vol. 64, No. 4 Hebatallah M Taher and Rania E Bayoumi

Ca plays a vital role in enamel remineralization when enamel caries-like lesions. The result shown in
CESP applied topically.(16,25) Calcination process the current study was consistent with the results of
was done to obtain pure powder free of pathogens previous study done by Haghgooetal in 2016(33) who
and to increase the alkalinity of powder. This high found that the egg shell solution greatly increased
alkalinity (pH = 11.7) was approved by previous the microhardness of the enamel by improving the
studies as it is the main reason for increased ion remineralization of enamel like lesions.
activity and availability of anions such as hydroxyl
ions and phosphate ions for remineralization. If In the current study, it was expected that CESP
the pH of remineralizing solution is low (acidic) would induce more remineralization than ACP due
there will be more concentration of H+ ions which to its high bioavailability of Ca content, neverthless,
combines with the available anions and thus less no significant difference was noted between the
ions will be available for remineralization.(23,25) results of the specimens remineralized with the
CESP and those remineralized with ACP as shown
The commercial Relief ÒACP was selected as it
in table (2) and figure (1). This could be explained
has a triple action, rebuilds enamel by depositing Ca
and P on the enamel forming hydroxyapatite which by the NaF contents of ACP which had a synergistic
lead to remineralization as proved by Job et al in effect on the remineralization process, combined
2018. Also it reduces sensitivity by occluding the with deposition of extensive amounts of Ca2+ and
dentin tubules as it contain potassium nitrate, in PO4-3 significantly promoting the remineralization
addition to the restoration of the enamel luster to and making it equivalent to CESP.
create a smoother and glossier appearance.(30) Moreover, the results of the present study showed
In order to simulate the subsurface that the calcium and phosphorus levels have been
demineralization lesions while maintaining the significantly increased after remineralization process
superficial enamel layer intact, weak organic both in Gp I and GpII. This might be explained
acetic acid was included in the composition of the by the diffusion of extensive amounts of Ca+2 and
demineralizing agent(1,31). Furthermore, the presence phosphorus ions, as clarified and detected in the
of calcium and phosphate, in the demineralizing elemental analysis (figures 2), into the superficial
solution helped preserve the superficial enamel layer layer of enamel and their deposition leading to
while stimulating mineral loss from the subsurface complete obstruction of all the surface porosities,this
layer. (32) This study was conducted in vitro as it led to the increase in the microhardness values of all
would be difficult to control all the confounding the demineralized specimens.(33) In addition, Gamal
factors in clinical studies such as diet, differences
et al in 2017 (34) demonstrated that the combination
in the flow and composition of saliva, patient
of calcium and the phosphates acted like a scaffold,
cooperation, and interpretation of results (1)
or they could fill the gaps in between the enamel
Microhardness measurement was done as it is crystals, yielding a uniform crystalline enamel
appropriate for a material having fine microstructure, structure with high mineral content which may
non homogenous and prone to cracking like enamel. enhance the remineralization.
Surface microhardness indentation provides
relatively a simple, non-destructive, rapid method.(16) The null hypothesis was accepted as both ACP
Results of this study showed that the microhardness and CESP produced the same remineralizing effect
was significantly decreased following exposure to on the demineralized enamel. However additional
demineralizing solution while it was significantly studies should be conducted to know what are
increased following exposure to remineralizing the optimal concentration, form and maximum
agents (Table 2). This indicates the efficacy of the time of application of CESP to produce efficient
agents used for the remineralization of incipient remineralization.
REMINERALIZATION OF INITIAL ENAMEL LIKE LESIONS WITH CHICKEN EGG (3711)

CONCLUSIONS 13. Shen P, Cai F, Nowicki A, Vincent J, Reynolds E.

Remineralization of enamel subsurface lesions by sugar free
Within the limitations of this in vitro study, it chewing gum containing casein phosphopeptide- amorphous
can be concluded that both remineralizing agents calcium phosphate. J Dent Res 2001;80:2066–70.
were similarly able to increase the microhardness 14. Gordon M. Cragg and David J. Newman. Natural
and tooth remineralization. However, being natural products: a continuing source of novel drug leads. Biochim
products, CESP can be considered as an optimal BiophysActa. 2013 Jun; 1830;6: 3670–95.
alternative to the commercial ones. 15. Park JW, Bae SR, Suh JY, Lee DH, Kim SH, Kim H, et
al. Evaluation of bone healing with eggshell-derived bone
graft substitutes in rat calvaria: a pilot study. J Biomed
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