Journal of International Dental and Medical Research ISSN 1309-100X Encapsulation Beta Tri Calcium Phosphate

http://www.jidmr.com Aprilia and et al

Effect of Encapsulation Beta Tri Calcium Phosphate (β -TCP)

from the Synthesis of Anadara Granosa Shell as Pulp Capping Material
against Inflammatory Cytokines IL-10 and TGF- β
Aprilia1,2, Sri Kunarti1*, Theresia Indah Budhy1, Rima Parwati Sari2
1.Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.
2.Faculty of Dental Medicine, Universitas Hang Tuah, Surabaya, Indonesia.

Abstract
Pulp capping treatment is an endodontic treatment that aims to maintain the vitality of the
pulp. Beta-tricalcium phosphate (β-TCP) has a chemical composition close to the structure of bones
and teeth that can stimulate odontoblast to form dentin bridge. β-TCP can be obtained from
anadara granosa hydrothermal process.
This research intends to determine the effect of β-TCP encapsulation of anadara granosa
clamshell against IL-10 and TGF β expression in dentin reparative formation. Thirty-six male Wistar
rats were divided into six groups: control group (-), no-treatment group, CaOH group, and treatment
with β-TCP. Application of pulp capping material on the M1 upper right tooth of Wistar rats that had
been prepared for class I cavities. After seven days and 14 applications, the rats were euthanized,
the prepared teeth were taken, and then IL-10 and TGFβ were examined.
The data were then analyzed statistically (p<0.05). The analyzed data showed significant
differences in each group.
Experimental article(J Int Dent Med Res 2022; 15(4): 1522-1528)
Keywords: β-TCP, calsium hydroxide, IL-10, TGFβ, pulp capping.
Received date: 30 June 2022 Accept date: 04 September 2022

Introduction seal1. Mechanical trauma to the pulp is most

common during deep and extensive carious
The development of restorative materials cavity cleaning2. According to research by Al-
is ongoing, aiming to produce restorative Hisayat3, among 204 perforated pulpitis teeth,
materials that have improved physical and there were 90 teeth (44.11%) perforated due to
mechanical properties and are biocompatible for mechanical trauma and 114 teeth (55.9%)
clinical application. Several in vitro studies have perforated due to caries.
found that placing restorative materials in dentin Minor trauma to the pulpal ceiling does
can be potentially toxic, thus damaging the pulp. not cause damage to the odontoblast cell layer;
Requirements for pulp capping materials must then, these cells can activate their tertiary dentin
meet biocompatibility requirements that are formation to protect the pulp from further
acceptable to the body and will not harm pulp damage. When the trauma is more profound and
and soft tissues of the oral cavity. Pulp capping affects the odontoblast layer, it will create a
material should have ideal traits, such as dentin bridge formation process that requires the
stimulating reparative dentin formation, help of progenitor cells that can differentiate into
maintaining pulp vitality, being bactericidal or odontoblasts2.
bacteriostatic, adhesive to dentin and restorative Pulp capping treatment is an endodontic
materials, resistant to pressure during restorative treatment that intends to maintain the vitality of
placement and during the restoration period, the pulp4. Pulp capping refers to placing a thin
sterile, radiopaque, and providing bacteria layer of dental material on the surface of the
dentin, which aims to maintain the vitality of the
dental pulp. The material that has been used and
*Corresponding author: is the gold standard is calcium hydroxide. These
Sri Kunarti, materials a lack soluble and cause cell surface
Faculty of Dental Medicine, Universitas Airlangga,
Jl. Mayjen Prof. Dr. Moestopo no. 47, death5,6.
Surabaya, 60132, Indonesia. Several long-term studies have proven
E-mail: [email protected] that Ca(OH)2 is less adaptable to dentin, is less

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 Journal of International Dental and Medical Research ISSN 1309-100X Encapsulation Beta Tri Calcium Phosphate
http://www.jidmr.com Aprilia and et al

able to stimulate odontoblast differentiation used. The structure of the oyster shell is
consistently, is cytotoxic to cells, and a high pH generally similar to that of cancellous bone, and
causes Ca(OH)2 to dissolve quickly, resulting in its mechanical properties are similar to that of
tunnel defects 7,1. Some Research concluded that bone. Based on the research of Kamba and
the failure of pulp capping with calcium hydroxide Zakaria14, it appears that the presence of calcium
was about 5-21% within a year, and 20% of teeth carbonate crystals (CaCO3) originating from the
show failure in the first year and 30% after two shell has the potential to mimic the composition,
years of calcium hydroxide used. On research structure, and properties of the shell. The oyster
concluded that the failure of pulp capping with shell has a relatively high CaCO3 mineral
calcium hydroxide was about 15-30% within a composition (98.7%)15. In this study, the anadara
year8. granola shell was treated with a hydrothermal
The research of Maden9 and Nowicka10 method for 18 hours at a temperature of 2000C,
said the application of CaOH caused higher cell and sintering for 3 hours at a temperature of
death/failure of pulp capping treatment compared 9000C resulted in HA (15%), β-TCP (79%), and
to other materials. Within a week, the CaOH2 (6%)16. β-TCP has a chemical
inflammatory layer will be replaced by granulation composition that is close to the structure of
tissue with fibroblasts and blood vessels. bones and teeth17-18. Tricalcium phosphate (TCP)
Progenitor cells are then induced to proliferate is a material that is biodegradable, bioactive, and
and migrate to the wound area, where they has a high solubility level compared to HA, with
differentiate into odontoblast-like cells capable of one of the polymorphs β-TCP which is generally
synthesizing proteins involved in reparative used because of its osteoconductive trait19. β-
dentin formation. The origins of stem/progenitor TCP can promote bone reconstruction and
cells are still under research. If inflammation provide a better barrier than calcium hydroxide in
remains in the pulp, reparative dentin obtaining an open apex, thus providing the same
development is obstructed and will be followed repair19,20. In theory, the biocompatibility of TCP
by pulp necrosis. with a combination of calcium release can make
Research by Tran11 showed the formation TCP stimulate odontoblasts so that dentin
of reparative dentin in mechanical trauma applied bridges can form20.
by Ca(OH)2, showing a tunnel defect. This This research inspired an investigation on
porosity can eventually lead to the entry of odontogenesis. In the process of odontogenesis,
bacteria into the pulp. The tunnel defect is reparative dentin formation occurs because of
formed because the Ca(OH)2 material in the the bruised stimulation to the pulp ceiling.
process of forming the dentin bridge is less than Perforated pulp ceiling due to mechanical trauma
perfect because the sealing ability in the dentin will experience an inflammatory response. The
wall is not optimal and become the initial cause inflammatory response plays an essential role in
of this failure material12. In addition, the dentin both standard and pathological healing. Soon
bridge formed is porous and does not correctly after trauma, the innate immune system is
adhere to the dentin ceiling. This causes failure activated in a response known as Damage
because it does not show a long-term biological Associated Molecular Pattern molecules
seal against bacteria13. The nature of this (DAMPs). The hypoxic environment in the wound
Ca(OH)2 material also has an alkaline pH of 11- stimulates many cell types, including
12, so it can cause local necrosis of the pulp inflammatory cells.
tissue around the injured site11. Along with the influx of neutrophils,
Based on the above, it is necessary to circulating monocytes enter the wound and
look for other alternatives to direct pulp capping differentiate into macrophages in the injured
materials that can be used in dentistry which tissue. The macrophage is the crucial player in
must be able to optimize dentin regeneration so the regeneration process that involves
that the inflammatory process and the impact of phagocytosis, antigen presentation, and
necrosis can be minimized. Utilization of secretion of various cytokines, chemokines, and
essential ingredients from materials can be growth factors (GF) that protect the body from
gathered from the natural environment, such as inflammation. Macrophages have several
anadara granosa shells. phenotypes, such as M1 and M2 (M2a, M2b,
Oyster shell is waste that is not commonly M2c). At the beginning of inflammation, M1 is

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 Journal of International Dental and Medical Research ISSN 1309-100X Encapsulation Beta Tri Calcium Phosphate
http://www.jidmr.com Aprilia and et al

characterized by the production of many microparticles is the ability to penetrate the

cytokines of tumor necrosis factor-alpha (TNF-α), intercellular spaces that can only be penetrated
interleukin-1 (IL-1), and interleukin-6 (IL-6), while by colloidal particle size 22.
at a later stage, M2 is found, namely with the Based on the background above,
emergence of anti-inflammatory cytokines such the researchers wanted to find out if there could
as interleukin-10 (IL-10), interleukin-4 (IL-4) and be an increase in IL10 and TGF- β expressions
interleukin-13 (IL-13)21. after the administration of β-TCP.
Calcium/calmodulin signaling regulates
gene expression in osteoblasts. Applying β- Materials and methods
TCP will produce Ca2+, directly hitting
macrophages and increasing Calcium-Sensing Preparation of Anadara granosa
Receptors (CaSR). Extracellular Ca2+ that Oyster shells (Anadara granosa) are
increases Intracellular Ca2+ is activated by the boiled for 30 minutes with boiling water. Anadara
mediator CaSR - to start activating granosa shell was brushed on the outside and
Phospholipase C (PLC), leading to an increase in inside using water and soap without bleach, then
inositol 1,4,5-trisphosphate (IP3). Calcium dried at room temperature. After that, it was
activates calmodulin which activates CaM Kinase ground using a mortar and pestle until the results
II (CaMKII) and calcineurin leading to modulation were in the form of powder and sifted with 200
of expression of member transcription factor mesh to get smaller particles. Next, the
Activator protein 1 (AP-1). The presence of AP-1 hydrothermal process was applied: mixing 10
activation enhances the regeneration process grams of Anadara granosa shell powder
stimulated by various GFs. This process will dissolved in 100 ml of distilled water and 6.9
increase the number of blood vessels due to grams of 0.6 M NH4H2PO4 dissolved in 100 ml of
angiogenesis, an increase in BMP2, which aquadest with a magnetic stirrer for 30 minutes.
increases the differentiation of Mesenchymal The mixed solution was then transferred to the
Stem Cells (MSCs) into odontoblast-like cells, reactor. The reactor was put in an electric oven
and transforming growth factor (TGF-β), which to be heated at a temperature of 200oC for 18
induces Alkaline Phosphatase (ALP) to increase hours. The results were then cooled at room
the secretion of Collagen type I (COL-1)22,23. temperature. After that, the heated powder is first
Odontoblast-like cells will stimulate Runx2 dissolved with 100 ml of distilled water on a
through Smad 1/5/8 through Smad 422. magnetic stirrer and then washed with distilled
Furthermore, Runx2 induces the formation of water using filter paper. Washing was conducted
mature osteoblasts by secreting osteopontin and repeatedly until the reaction product separated
osteocalcin. ALP is also formed through Smad, from distilled water, indicated by the pH returning
which prepares an alkaline atmosphere in the to 7. This was done to remove acidic by-
formed osteoid tissue, thus triggering the products. After the pH returned to 7, the last
formation of COL-1. Osteopontin, osteocalcin, washing was conducted with methanol to limit the
and COL-1 will soon undergo calcification, which agglomeration of TCP particles during drying.
plays a role in the dentin matrix formation, thus Samples were then dried in an electric oven at
accelerating the reparative dentin formation22. 50oC for 4 hours and then sintered at 900°C for 3
Furthermore, Runx2 induces the hours to remove impurities and increase the
formation of mature osteoblasts by secreting crystallinity of the sample.
osteopontin and osteocalcin. ALP is also formed Procedure for making encapsulation
through Smad, which functions to prepare an (β-TCP)
alkaline atmosphere in the formed osteoid tissue, Manufacturing Method β-TCP-Alginate
thus triggering the formation of COL-1. with Gelation Ionotropic Method Aerosolization
Osteopontin and osteocalcin, as well as COL-1, Technique. 0.5 grams of sodium alginate was
will soon undergo calcification which plays a role then dissolved in 50 ml of aqua demineralized
in the formation of the dentin matrix, thus and stirred with a magnetic stirrer to get an
accelerating the process of reparative dentin alginate solution. β-TCP of 0.5 grams is put into
formation22. The use of microparticle technology 50 ml of the alginate solution that has been
as a drug delivery system in dentistry is starting formed and mixed until homogeneous. Solution
to be used. One of the advantages of β-TCP-alginate then dripped with CaCl2 solution

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 Journal of International Dental and Medical Research ISSN 1309-100X Encapsulation Beta Tri Calcium Phosphate
http://www.jidmr.com Aprilia and et al

(0.5-gram CaCl2 powder in 25 ml of distilled M1 pulp chamber. Before preparation, rats were
water) until fiber shaped by continuously stirring anesthetized using ketamine HCL and xylazine at
using a magnetic stirrer for 120 minutes at a a dose of 0.11 mL/100g body weight
speed of 1000 rpm. After that, the suspension intramuscularly.
formed was centrifuged at a speed of 2500 rpm On the 7th and 14th days, experimental
for 6 minutes, then the liquid was discarded to animals from each group (6 groups) were
separate the microspheres from the CaCl2 sacrificed under anesthesia using ketamine-
solution. The microspheres were filtered, and the acepromazine with a dose ratio of 0.1 ml: 0.1 ml,
wet microspheres were then weighed. After that, then decapitated by taking the maxillary bone
microspheres were dried by freeze drying at a along with the left upper three molars jaw
temperature of -80oC (freezing process for 30 approximately ±3 cm, then put in a 10%
minutes and drying process for 12 hours). formaldehyde buffer solution in order to prevent
Pre-clinical trials on experimental the tissue from breaking down, harden the tissue
animals and increase the tissue affinity against the paint.
This research used white rat experimental After the tissue fixation process, the
animals (Rattus norvegicus strain Wistar), which decalcification process was conducted using
is a true experimental with a random selection of EDTA for two months.
animals. The samples used were 36 tails which After the tooth tissue is soft enough, it is
were divided into six groups, with criteria processed to become paraffin blocks through the
selected were adult males aged 4-5 months, dehydration stage, clearing, impregnation, and
body weight 250-300 grams, the characteristics embedding. After that, the tooth tissue in paraffin
of clear eyes characterize physical health, shiny was cut using a microtome, with a tissue slice
fur, agile movements, and good or not soft faces thickness of 0.5 cm, followed by staining with
and there was no caries in the maxillary first hematoxylin-eosin to get macrophages, and then
molar. This research was conducted upon prepared to examine the expression of cytokines
approval from the veterinary ethics committee of using the kit: IL-10 and TGF-β. Expression was
the Dentistry Faculty of Airlangga University, viewed under a light binocular microscope
Surabaya. (Olympus brand) at 400x magnification. For
The acclimatization procedure in this macrophages that express cytokines, cells brown
research was conducted for seven days before ones were counted as positive.
rats were randomly chosen and divided into six The data gathered were analyzed to get an
groups: negative control group (K(-)-1) with overview of the distribution and a summary to
seven days of treatment, negative control group clarify the results. Then, the hypothesis was
(K(-)-2) with 14 days treatment, positive control tested using the One-way ANOVA parametric
group (K(+)-1) with seven days treatment, statistical test, followed by the LSD test.
positive control group (K (+)-2) with 14 days
treatment, treatment group (P-1) with seven days Results
treatment, treatment group (P-2) with 14 days
treatment. Results of the average description
In the negative control group, the upper showed that the group with the highest IL-10
first molars were prepared and filled with Glass expression was in the treatment group that
Ionomer Cement. In contrast, the positive control applied -TCP of Anadara granosa shell on day
group conducted direct pulp capping using the 14, while the lowest was in the negative control
application of CaOH + aquadest in a ratio of 1:1 group on day 7. The increase in IL-10 expression
and filled with Glass Ionomer Cement, while the was seen on day 14, higher than on day 7 in
treatment group conducted direct pulp capping each treatment group. On the seventh day, the
with encapsulation applications. β-TCP from highest -TCP treatment group compared with the
anadara granosa shell + aquadest in a ratio of positive control group (CaOH) and the negative
1:1 and filled with Glass Ionomer Cement. control group on day 14.
On the first day of the experiment, all
groups of experimental animals were prepared
for class I preparation with a low-speed round bur
(0.84 mm diameter) to reach the right maxillary

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 Journal of International Dental and Medical Research ISSN 1309-100X Encapsulation Beta Tri Calcium Phosphate
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dentin in the b-TCP group on day 7 (E) and day

14 (F). IHC staining with monoclonal IL-10.
Observations using a light microscope at
magnification of 400 times.

Data presentation from observations of IL-

10 expression in macrophage cells can be seen
Table . Effect of administration of materials on in Table 1 and Figure 1. Pathological conditions
direct pulp capping treatment against IL-10 where IL-10 expression was seen in
expression. macrophages (Figure 2)
The results of average description
showed that the group with the highest TGF-ß
expression was found in the treatment group that
was applied to β-TCP from Anadara granosa
shell on day 14, while the lowest was in the
negative control group on day 7. An increase in
TGF-ß expression seen on the 14th day was
higher than the 7th day in each treatment group.
Data presentation from observations of TGF-ß
expression in macrophage cells can be seen in
table 2 and Figure 3. Pathological conditions
where TGF- expression was seen in
Figure 1. Tabel of IL-10 expression in pulp macrophages (Figure 4).
capping.

Table 2. Effect of administration of materials on

treatmentpulp capping indirecton TGF-
expression.

Figure 2. Pathological conditions where IL-10

expression was seen in macrophages found in
dentin in the negative control group on day 7 (A)
and 14 (B). IL-10 expression on macrophages in
dentin in the CaOH group (+) on day 7 (C) and Figure 3. Tabel of TGF- expression in pulp
14 (D). IL-10 expression on macrophages in capping.

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 Journal of International Dental and Medical Research ISSN 1309-100X Encapsulation Beta Tri Calcium Phosphate
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such as non-reactivity and good

osteoconductivity1. This research tested the
cytokine expression of IL-10 and TGF-ß, an anti-
inflammatory cytokine.

This research showed that on day 14, the

group with β -TCP from the Anadara granosa
shell application showed a significant increase in
IL-10 expression compared to the other groups,
while the lowest was in the untreated group
(negative control) on day 7.
With the ANOVA test or difference test between
groups, it was found that there were significant
differences in each group, both between the 7th
and 14th days in the control group (-), positive
control as well as treatment. Similarly, a
comparison between the 7th and 14th days of the
same group has a significant difference.
IL-10 expression was the highest on day
14 among all groups. The inflammatory response
plays an essential role in both standard and
Figure 4. Pathological conditions in which the pathological healing. Immediately after trauma,
expression of TGF- on macrophages found in the innate immune system is activated in a
dentin in the negative control group on day 7 (A) response known as Damage Associated
and on day 14 (B). Expression TGF- on Molecular Pattern molecules (DAMPs). The
macrophages in dentin in the CaOH group on hypoxic environment in the wound stimulates
day 7 (C) and on day 14 (D). Expression TGF- on many cell types, including inflammatory cells.
Along with the influx of neutrophils, the circulating
macrophages in dentin in the b-TCP group on monocytes enter the wound and differentiate into
day 7 (E) and on day 14 (F). IHC staining with macrophages in the injured tissue. Macrophages
monoclonal IL-10. Observations using a light are critical players in the regeneration process
microscope at magnification of 400 times. that involves phagocytosis, antigen presentation,
and secretion of various cytokines, chemokines,
Discussion and growth factors (GF) that protect the body
from inflammation. At the early inflammation,
This research was conducted to much M1, which is characterized by the
determine the effect of reparative dentin production of the tumor necrosis factor-alpha
formation. On β-TCP (beta-tricalcium (TNF-α) cytokines, interleukin-1 (IL-1), and
phosphate) encapsulation from the anadara interleukin-6 (IL-6) were found, while at a later
granosa shell synthesis, compared with CaOH, stage the presence of M2 was found with the
which is currently still included in the gold appearance of anti-inflammatory cytokines such
standard. β-TCP was obtained from the shell of as interleukin-10 (IL-10), interleukin-4 (IL-4) and
Anadara granosa that was applied with a interleukin-13 (IL-13)21. Based on these findings,
hydrothermal process for 18 hours at a it is said that β-TCP can provide an excellent
temperature of 9000C, sintering for 3 hours to inflammatory response in the pulp capping
produce HA (21%), β-TCP (79%)16. treatment process. IL-10 is a robust anti-
β-TCP has a close chemical composition inflammatory cytokine that suppresses the
to the structure of bones and teeth17,18. β–TCP synthesis of pro-inflammatory cytokines and
can be used as a potential bone substitute chemokines in macrophages by activating
because it is biocompatible, bioresorbable (easily STAT3 signaling26. In particular, high levels of IL-
absorbed), and has osteoconductivity traits18,25. 10 have been proven to be detectable in the
HA and TCP can be categorized as bioceramic dental pulp of teeth with deep carious lesions27
materials with excellent biological properties, and caries-exposed pulp28.

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Transforming growth factor (TGF-ß) is a 11. Tran XV, Gorin C, Willig C., Baroukh B., Pellat B., Decup F.,
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