Journal of Taibah University Medical Sciences (2022) 17(6), 1014e1020

Taibah University

Journal of Taibah University Medical Sciences

www.sciencedirect.com

Original Article

The use of mucoadhesive oral patches containing epigallocatechin-3-

gallate to treat periodontitis: an in vivo study
Dur Muhammad Lashari, MPil a, b, Mohammed Aljunaid, MDS a, c, f,
Yasmeen Lashari, MPil d, e, Huda Rashad Qaid, MDS a, f, Rini Devijanti Ridwan, PhD g, *,
Indeswati Diyatri, PhD g, Nejva Kaid, MPharm h and Baleegh Abdulraoof Alkadasi, PhD i
a
Doctoral Program of Dental Medicine, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia
b
Faculty of Oral Biology Dental Department Bolan Medical College Quetta, Pakistan
c
Department of Oral and Dental Medicine, Faculty of Medicine, Taiz University, Taiz, Yemen
d
Doctoral Program, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia
e
Bolan Medical College & Hospital Quetta, Department of Microbiology (Pathology), Quetta, Pakistan
f
Faculty of Oral and Dental Medicine, AL-saeed University, Taiz, Yemen
g
Department of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia
h
Master Program of Drug and Cosmetic Manufacturing Technology, Department of Pharmaceutical Technology, Faculty of
Pharmacy, Yeditepe University, Istanbul, Turkey
i
Head of Oral Medicine and Periodontology Department, Faculty of Dentistry, Ibb University, Ibb, Yemen

Received 17 March 2022; revised 15 May 2022; accepted 27 June 2022; Available online 7 July 2022

‫ﺍﻟﻤﻠﺨﺺ‬ ‫ ﺗﻢ ﺃﺧﺬ ﻋﻴﻨﺔ ﻣﻦ ﺍﻟﻘﺎﻃﻊ ﺍﻟﻤﺮﻛﺰﻱ ﺍﻟﺴﻔﻠﻲ ﻭﺗﺤﻠﻴﻠﻪ ﺑﺎﺳﺘﺨﺪﺍﻡ‬،‫ ﺑﻌﺪ ﺍﻟﻌﻼﺝ‬21
.‫ ﻓﻲ ﺃﻧﺴﺠﺔ ﺍﻟﻠﺜﺔ‬،"‫ﺍﻟﻜﻴﻤﻴﺎﺀ ﺍﻟﻤﻨﺎﻋﻴﺔ ﻟﻜﻞ ﻣﻦ "ﺃﻭ ﺑﻲ ﺟﻲ" ﻭ"ﺭﺍﻧﻜﻞ" ﻭ"ﺭﺍﻧﻚ‬
‫ ﺗﻄﺒﻴﻖ ﺍﻟﺪﻭﺍﺀ ﺍﻟﻤﻮﺿﻌﻲ ﻣﺜﻞ ﺍﻟﺮﻗﻌﺔ ﺍﻟﻔﻤﻮﻳﺔ ﺍﻟﻤﺨﺎﻃﻴﺔ ﻻ ﻳﻬﻴﺞ‬:‫ﺃﻫﺪﺍﻑ ﺍﻟﺒﺤﺚ‬
‫ ﺍﻟﻤﺮﻛﺐ‬.‫ﺍﻟﻐﺸﺎﺀ ﺍﻟﻤﺨﺎﻃﻲ ﻭﻗﺎﺩﺭ ﻋﻠﻰ ﺯﻳﺎﺩﺓ ﻧﻔﺎﺫﻳﺔ ﺍﻷﺩﻭﻳﺔ ﺇﻟﻰ ﺃﻧﺴﺠﺔ ﺍﻟﻔﻢ‬ ‫ ﺯﺍﺩﺕ ﺗﻌﺒﻴﺮﺍﺕ "ﺭﺍﻧﻚ" ﻭ "ﺃﻭ ﺑﻲ ﺟﻲ" ﻓﻲ ﺟﻤﻴﻊ‬،‫ ﺑﻌﺪ ﺍﻟﻌﻼﺝ‬:‫ﺍﻟﻨﺘﺎﺋﺞ‬
‫ﺍﻟﻤﻌﺮﻭﻑ ﺑﺎﺳﻢ "ﺇﻱ ﺟﻲ ﺳﻲ ﺟﻲ" ﻋﺒﺎﺭﺓ ﻋﻦ ﻣﻜﻮﻧﺎﺕ ﻧﺸﻄﺔ ﻟﻬﺎ ﺗﺄﺛﻴﺮﺍﺕ ﻋﺎﻟﻴﺔ‬ ‫ﺍﻟﻤﺠﻤﻮﻋﺎﺕ ﻣﻊ ﺃﻋﻠﻰ ﺯﻳﺎﺩﺓ ﻓﻲ ﺍﻟﻤﺠﻤﻮﻋﺔ ﺍﻟﻤﻌﺎﻟﺠﺔ ﺑﻤﺴﺘﺨﻠﺺ "ﺇﻱ ﺟﻲ ﺳﻲ‬
‫ ﻛﺎﻥ ﺍﻟﻐﺮﺽ ﻣﻦ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﻫﻮ ﺗﺤﻠﻴﻞ‬.‫ﻣﻀﺎﺩﺓ ﻟﻠﺠﺮﺍﺛﻴﻢ ﻭﻣﻀﺎﺩﺓ ﻟﻼﻟﺘﻬﺎﺑﺎﺕ‬ ‫ ﻛﻤﺎ ﺃﻇﻬﺮﺕ ﺍﻟﺘﺤﻠﻴﻼﺕ ﺍﻧﺨﻔﺎﺿﺎ ﻓﻲ‬.‫ﺟﻲ" ﻣﻘﺎﺭﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺘﻴﻦ ﺍﻷﺧﺮﻳﻴﻦ‬
‫ﺍﻹﻣﻜﺎﻧﺎﺕ ﺍﻟﻌﻼﺟﻴﺔ ﻟﻠﺮﻗﻌﺔ ﺍﻟﻤﺨﺎﻃﻴﺔ ﺍﻟﻔﻤﻮﻳﺔ ﺍﻟﺘﻲ ﺗﺤﺘﻮﻱ ﻋﻠﻰ ﻣﺮﻛﺐ "ﺇﻱ ﺟﻲ‬ ‫ ﻭ‬14 ‫ ﻭ‬7 ‫ ﻭ‬5 ‫ ﻭ‬3 ‫ﺗﻌﺒﻴﺮﺍﺕ ﺭﺍﻧﻜﻞ ﻓﻲ ﺟﻤﻴﻊ ﺍﻟﻤﺠﻤﻮﻋﺎﺕ ﺑﻌﺪ ﺍﻟﻌﻼﺝ ﻓﻲ ﺍﻷﻳﺎﻡ‬
،(‫ﺳﻲ ﺟﻲ" ﻓﻲ ﻧﻤﻮﺫﺝ ﺍﻟﺘﻬﺎﺏ ﺍﻟﻠﺜﺔ ﻭﺗﺄﺛﻴﺮﻩ ﻋﻠﻰ ﺃﻭﺳﺘﻴﻮﺑﺮﻭﺗﻴﺠﻴﺮﻳﻦ )ﺃﻭ ﺑﻲ ﺟﻲ‬ ‫ ﻭﻛﺎﻥ ﻟﻠﻌﻼﺝ ﺑﺎﺳﺘﺨﺪﺍﻡ ﻣﺴﺘﺨﻠﺺ "ﺇﻱ ﺟﻲ ﺳﻲ ﺟﻲ" ﺃﻗﻞ ﺗﻌﺒﻴﺮﺍﺕ ﺭﺍﻧﻜﻞ ﻓﻲ‬.21
‫ ﻭﻣﻨﺸﻂ ﺍﻟﻤﺴﺘﻘﺒﻼﺕ‬،(‫ﺏ )ﺭﺍﻧﻜﻞ‬-‫ﻭﻣﻨﺸﻂ ﻣﺴﺘﻘﺒﻼﺕ ﻟﺠﻴﻦ ﺍﻟﻌﺎﻣﻞ ﺍﻟﻨﻮﻭﻱ ﻛﺎﺑﺎ‬ ‫ ﺃﻇﻬﺮﺕ ﻧﺘﺎﺋﺞ ﺍﻻﺧﺘﺒﺎﺭ ﺍﻟﻼﺣﻖ ﻋﺪﻡ‬.‫ﺟﻤﻴﻊ ﺍﻷﻳﺎﻡ ﻣﻘﺎﺭﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺎﺕ ﺍﻷﺧﺮﻯ‬
.(‫ﺏ )ﺭﺍﻧﻚ‬-‫ﻟﻠﻌﺎﻣﻞ ﺍﻟﻨﻮﻭﻱ ﻛﺎﺑﺎ‬ ‫ﻭﺟﻮﺩ ﻓﺮﻕ ﺫﻱ ﺩﻻﻟﺔ ﺇﺣﺼﺎﺋﻴﺔ ﺑﻴﻦ ﻣﺠﻤﻮﻋﺔ ﺍﻟﺘﺤﻜﻢ ﻭﻣﺠﻤﻮﻋﺔ ﺍﻟﺪﻭﻛﺴﻴﺴﻴﻜﻠﻴﻦ ﻓﻲ‬
."‫ﺍﻟﺘﻌﺒﻴﺮ ﻋﻦ "ﺃﻭ ﺑﻲ ﺟﻲ" ﺃﻭ"ﺭﺍﻧﻜﻞ" ﺃﻭ"ﺭﺍﻧﻚ‬
‫ ﺍﺳﺘﺨﺪﻡ ﻧﻤﻮﺫﺝ ﺍﻟﺘﻬﺎﺏ ﺍﻟﻠﺜﺔ ﺍﻟﻤﺤﺮﺽ ﻓﻲ ﺍﻟﺠﺮﺫﺍﻥ ﺍﻟﻨﺮﻭﻳﺠﻴﺔ‬:‫ﻃﺮﻕ ﺍﻟﺒﺤﺚ‬
‫ ﻣﻞ ﻣﻦ ﺑﻜﺘﻴﺮﻳﺎ "ﻭﺣﻴﺪﺍﺕ ﺍﻟﺨﻠﻴﺔ ﺍﻟﺒﻮﺭﻓﻴﺮﻳﻨﻴﺔ ﺍﻟﻠﺜﻮﻳﺔ" ﺑﻔﻮﺍﺻﻞ‬0.03 ‫ﺑﺎﺳﺘﺨﺪﺍﻡ‬ ‫ ﻳﻤﻜﻦ ﺃﻥ ﻳﻘﻠﻞ ﻣﺴﺘﺨﻠﺺ"ﺇﻱ ﺟﻲ ﺳﻲ ﺟﻲ" ﻣﻦ ﺗﻌﺒﻴﺮ ﺭﺍﻧﻜﻞ ﻭﻳﺰﻳﺪ‬:‫ﺍﻻﺳﺘﻨﺘﺎﺟﺎﺕ‬
‫ ﺗﻢ ﻋﻼﺝ ﺍﻟﺘﻬﺎﺏ ﺍﻟﻠﺜﺔ ﻟﺪﻯ ﺍﻟﻔﺌﺮﺍﻥ‬.‫ﺯﻣﻨﻴﺔ ﻟﻤﺪﺓ ﻳﻮﻣﻴﻦ ﻓﻲ ﺍﻟﻘﻮﺍﻃﻊ ﺍﻟﻤﺮﻛﺰﻳﺔ ﺍﻟﺴﻔﻠﻴﺔ‬ ‫ ﻣﻦ ﺍﻟﻤﻔﺘﺮﺽ ﺃﻥ ﻣﺴﺘﺨﻠﺺ"ﺇﻱ ﺟﻲ ﺳﻲ ﺟﻲ" ﻗﺎﺩﺭ ﻋﻠﻰ‬."‫"ﺃﻭ ﺑﻲ ﺟﻲ" ﻭ"ﺭﺍﻧﻚ‬
‫ ﺃﻭ ﺍﻟﺮﻗﻌﺔ ﺍﻟﻔﻤﻮﻳﺔ ﺍﻟﻤﺨﺎﻃﻴﺔ‬،(‫ﺑـﺎﻟﺮﻗﻌﺔ ﺍﻟﻔﻤﻮﻳﺔ ﺍﻟﻤﺨﺎﻃﻴﺔ )ﻣﺠﻤﻮﻋﺔ ﺍﻟﺘﺤﻜﻢ‬ .‫ﻣﻨﻊ ﻓﻘﺪﺍﻥ ﺍﻟﻌﻈﺎﻡ ﺍﻟﺴﻨﺨﻴﺔ ﻓﻲ ﺍﻟﺘﻬﺎﺏ ﺍﻟﻠﺜﺔ‬
‫ ﺃﻭ ﺍﻟﺮﻗﻌﺔ ﺍﻟﻔﻤﻮﻳﺔ ﺍﻟﻤﺨﺎﻃﻴﺔ ﺍﻟﻤﺤﺘﻮﻳﺔ ﻋﻠﻰ ﻣﺮﻛﺐ‬،‫ﺍﻟﻤﺤﺘﻮﻳﺔ ﻋﻠﻰ ﺍﻟﺪﻭﻛﺴﻴﺴﻴﻜﻠﻴﻦ‬
‫ ﻭ‬14 ‫ ﻭ‬7 ‫ ﻭ‬5 ‫ ﻭ‬3 ‫ ﻓﻲ ﺍﻷﻳﺎﻡ‬.‫ ﺃﻳﺎﻡ‬5 ‫ ﻳﻮﻡ ﻟﻤﺪﺓ‬/ ‫ ﺳﺎﻋﺔ‬1 ‫"ﺇﻱ ﺟﻲ ﺳﻲ ﺟﻲ" ﻟﻤﺪﺓ‬ ‫ ﻓﻘﺪﺍﻥ ﺍﻟﻌﻈﺎﻡ ﺍﻟﺴﻨﺨﻴﺔ؛ ﺇﻳﺒﻴﻐﺎﻟﻮﻛﺎﺗﺸﻴﻦ؛ ﺭﻗﻌﺔ ﻓﻤﻮﻳﺔ؛ ﺍﻟﺘﻬﺎﺏ‬:‫ﺍﻟﻜﻠﻤﺎﺕ ﺍﻟﻤﻔﺘﺎﺣﻴﺔ‬
‫ﺟﺎﻻﺗﻲ‬-3 ‫ﺍﻟﻠﺜﺔ؛ ﺑﻜﺘﻴﺮﻳﺎﺍﻟﺒﻮﺭﻓﻴﺮﻭﻣﻮﻧﺎﺱ؛ ﻳﺒﻴﻐﺎﻟﻮﻛﺎﺗﺸﻴﻦ‬

* Corresponding address: Department of Oral Biology, Faculty of Abstract

Dental Medicine, Universitas Airlangga, Jalan. Prof. Dr. Moestopo
No 47, Surabaya, 60132, Indonesia. Objectives: The application of topical drugs such as
E-mail: rini-d-r@fkg.unair.ac.id (R.D. Ridwan)
mucoadhesive oral patches (MOPs) do not irritate the
Peer review under responsibility of Taibah University.
mucosa and are able to increase the permeability of drugs
to oral tissue. Epigallocatechin-3-gallate (EGCG) is an
active ingredient that exhibits significant antibacterial
Production and hosting by Elsevier and anti-inflammatory effects. The purpose of this study

1658-3612 Ó 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.jtumed.2022.06.006
 D.M. Lashari et al. 1015

was to analyze the therapeutic potential of a mucoadhe- osteoprotegerin (OPG). Consequently, these proteins are crit-
sive oral patch containing EGCG (MOP-EGCG) in a ical for hemostatic control.4
model of periodontitis and investigate its effects on the As compared to other drug administration options, the
expression of osteoprotegerin (OPG), receptor activator mucoadhesive drug delivery system represents a much safer
of nuclear factor-kappa B ligand (RANKL) and receptor option. This system has many benefits compared to other
activator of nuclear factor-kB (RANK). administrative techniques. This method provides us with a
controlled method for the release of drugs at a specific
Methods: A model of periodontitis was induced in Rattus target site with extended retention times at the target site.
novergicus used Porphyromonas gingivalis by applying One important benefit of this type of system is that it
0.03 ml of bacteria locally with 1
1010 colony-forming avoids the first phase of metabolism.5 The process by
units (CFU) seven times at 2-day intervals in the central which polymers adhere to biological substances with
lower incisors. Periodontitis was then treated with MOP mucus or the epithelium surface is called mucoadhesion.
(control), a mucoadhesive oral patch containing doxycy- A substrate containing a bio-adhesive polymer can sup-
cline (MOP-doxy) or MOP-EGCG for 1 h/day for 21 days. port the transfer of drugs to target sites and control the
On days 3, 5, 7, 14 and 21 after treatment, the central lower release of drug dosage over an extended period. Studies of
incisor was biopsied and analyzed by immunohistochem- mucoadhesive polymers have indicated that this is a good
istry for RANK/RANKL and OPG expression in the method for mucoadhesion and identified some factors that
gingiva tissue. can affect the mucoadhesive properties of a polymer. Both
natural and synthetic polymers are used in the preparation
Results: MOP-EGCG extract significantly reduced the of mucoadhesive oral patches.6
expression of RANKL and increased the expression of A mucoadhesive drug delivery system is a safer delivery
OPG and RANK (p < 0.05) when compared to the system when compared to other drug administration
MOP-doxy and MOP groups. routes and has several advantages over conventional
administration methods. Hence, in this study, we analyzed
Conclusion: MOP-EGCG extract reduced the expression the therapeutic potential of a mucoadhesive oral patch
of RANKL and increased the expression of OPG and (MOP) containing EGCG (MOP-EGCG) in a periodon-
RANK, thus suggesting that MOP-EGCG can inhibit the titis model induced by Porphyromonas gingivalis and
loss of alveolar bone in periodontitis. investigated its effect on the expression of RANK/
RANKL and OPG.
Keywords: Bone loss; Epigallocatechin-3-gallate; Oral patch;
Periodontitis; Porphyromonas gingivalis Materials and Methods

Ó 2022 The Authors. Published by Elsevier B.V. This is an

open access article under the CC BY license Materials
(http://creativecommons.org/licenses/by/4.0/).
Epigallocatechin-3-gallate (EGCG) concentrate from
green tea extract (Camellia sinensis) was obtained from
Xi’An Rongsheng Biotechnology Co., Ltd. (Shaanxi,
China). Polyethylene glycol (PEG) was obtained from
Introduction Schuchardt OHG (Germany). PEG 4000 was obtained from
SigmaAldrich (St. Louis, MO). Sodium carboxymethyl cel-
Periodontitis refers to inflammation of the periodontal tis- lulose (CMC-Na) and propylene glycol (PEG) was obtained
sues and is characterized by alveolar bone resorption.1 from Teknis (Indonesia) and doxycycline was obtained from
Periodontitis is one of the most common infectious diseases Kimia Pharma.
and is derived from largely uncharacterized communities of
oral bacteria that grow as biofilms on teeth and gingival Preparation of mucoadhesive oral patches
surfaces in periodontal pockets. The current Global Burden
of Disease Study reports that periodontitis is the sixth most Mucoadhesive oral patch (MOP) preparation was per-
prevalent global disease with an overall prevalence of up to formed using the solvent casting technique. Patches were pre-
20% of the world’s population.2 The inflammatory response pared using CMC-Na and PG was used as a plasticizer to
of immune cells is activated by bacterial invasion. In later improve the performance and release characteristics of the
stages of disease, this causes the loss of both soft and hard patch.
supporting structures of the teeth. Thus far, only a few types CMC-Na (0.6 g) was dissolved in 30 ml of warm
of bacteria have been categorized as infectious agents of distilled water; then PG (2.5 g) was added under contin-
periodontitis.3 The successful treatment of periodontitis uous stirring to obtain a suitable viscosity dispersion. The
inhibits tissue destruction, stimulates osteointegration and mixture has been poured into Petri dishes, stored at 4  C
inhibits osteoclastogenesis. Osteointegration and osteoclasto for 48 h to remove entrapped air bubbles, and finally oven-
genesis include the activation and interaction of receptor dried at 40  C. All patches were uniform, homogeneous,
activator of nuclear factor-kB (RANK), receptor activator of and free from bubbles; the thickness of the patch was
nuclear factor-kappa B ligand (RANKL), and 0.3 mm.
1016 The use of mucoadhesive oral patches

Preparation of mucoadhesive oral patches containing EGCG anesthesia agent. The lower anterior incisive region of the
and mucoadhesive oral patches containing doxycycline mandible was biopsied.

The process used to create MOP-EGCG and MOPs RANK/RANKL expression

containing doxycycline (MOP-doxy) were similar to those
used for the preparation of blank patches. We dissolved RANK is a protein expressed in the osteoclasts of alveolar
CMC-Na (0.6 g) in 30 ml of warm distilled water with bone tissue. We used an anti-RANK (Biogear Indonesia)
manual stirring. Then, EGCG (100 mg) and doxycycline monoclonal antibody to semi-qualitatively investigate the
(100 mg) were added to the CMC-Na solution separately and expression of RANK/RANKL in experimental animals by
stirring was continued until the mixture became homoge- immunohistochemical (IHC) methods with a light micro-
neous. Then, PEG (1 g) was added with continuous stirring scope (400
magnification); positive staining was seen as a
until suitable viscosity dispersion was obtained. The mixtures brown color.
were then poured into Petri dishes and oven-dried at 40  C.
The final thickness of the patch was 0.3 mm and contained OPG expression
15 mg EGCG and 3 mg doxycycline.
OPG is a protein expressed in the osteoblasts of alveolar
Experimental model bone tissue. We used an anti-OPG (Biogear Indonesia)
monoclonal antibody to semi-qualitatively investigate the
The study protocol was approved by The Faculty of expression of OPG by IHC methods with a light microscope
Veterinary Medicine, Airlangga University Laboratory. This (400
magnification); positive staining was seen as a brown
was a laboratory experimental study with a post-test-only color.
control group design. In total, we used 45 healthy male
Wistar rats (Rattus norvegicus), aged between 5 and 6 Statistical analysis
months with a body weight of 250e300 g; all rats were
characterized by energetic movement and were acclimatized Data analyses were conducted with SPSS (version 25).
for 1 week prior to experimentation. The experimental ani- Data is shown in the form of mean  standard deviation
mals were received by the Faculty of Veterinary Medicine, (SD). Normality tests were conducted with the Kolmogorov
Airlangga University Animal Laboratory. According to Smirnov test and differences between groups were tested by
Lemeshow,7 a sample of 45 models was required, with each one-way analysis of variance and Tukey’s post hoc test. A
group containing three animals for a total of 15 groups. statistically significant difference was defined as p < 0.05.

Periodontitis model Results

P. gingivalis (ATCC 33 277) was produced in media RANK expression

containing tryptic soy broth (TSB) and then raised in
anaerobic conditions for 18e24 h. Bacterial colonies were IHC analyses revealed increased RANK expression in all
taken and transferred in 3 ml of BHI broth media then groups post-treatment on days 3, 5, 7, 14 and 21; the MOP
incubated at 37  C for 18 h. The bacterial suspension was group had the lowest levels of RANK expression (Figure 2a).
synchronized with a 0.5 McFarland standard (1010 colony- When compared with other groups, treatment with MOP-
forming units (CFU)/ml) and taken with a micropipette EGCG extract resulted in the highest expression levels of
and dropped onto the surface of Mueller-Hinton agar media RANK on days 3, 5, 7, 14 and 21 (Figures 1 and 2a). Tukey’s
and incubated for 24 h at 37  C in a 5% CO2 anaerobic post hoc test results showed that there were significant
atmosphere.8,9 differences in RANK expression between the MOP and the
The periodontitis model was achieved by injecting MOP-EGCG group after 3, 5, 7, 14 and 21 days (p < 0.002,
P. gingivalis (0.03 ml containing 1
1010 CFU) into the p < 0.001, p < 0.001, p < 0.001, and p < 0.001, respectively)
mandibular gingival incisive sulcus of the anterior teeth of and between the MOP and the MOP-doxy group after day 21
animals seven times at 2-day intervals using a disposable (p ¼ 0.013). However, there was no significant difference in
syringe (0.5 cc). RANK expression between the MOP and MOP-doxy groups
after 3, 5, 7 and 14 days (p ¼ 0.165, p ¼ 0.300, p ¼ 0.645, and
Periodontitis treatment application p ¼ 0.054 respectively).

Once periodontitis had developed, each group of animals RANKL expression

received treatment after been anesthetized with 10% keta-
mine (0.1 ml/100 g body weight) delivered by intramuscular IHC analyses revealed a reduction in the expression levels
injection. Each group received MOP-EGCG, MOP-doxy or of RANKL in all groups post-treatment on days 3, 5, 7, 14
a blank patch for 1 h/day for 5 days. The patch was cut and and 21 (Figures 1 and 2b). Treatment with MOP-EGCG
sized into small pieces to fit the incisive gingival mucosa of resulted in the lowest RANKL expressions on days 3, 5, 7,
the rat’s jaw. Animals were killed on days 3, 5, 7, 14 and 21 14 and 21 when compared to the other groups (Figures 1 and
after the commencement of treatment. Ketamine (0.4 ml/ 2b). Tukey’s post hoc test results showed that there were
100 g body weight) by intramuscular injection was used as an significant differences in the expression of RANKL
 D.M. Lashari et al.
Figure 1: Immunohistochemical staining of RANK/RANKL and OPG expression in periodontitis.

1017
 1018
The use of mucoadhesive oral patches
Figure 2: Expression levels of RANK, RANKL and OPG. The same letter on each a day of observation indicates significant differences, as determined by the Tukey-HSD test (p < 0.05).
 D.M. Lashari et al. 1019

between the MOP and MOP-EGCG groups after 3, 5, 7, 14 are becoming increasingly popular. It has been verified that
and 21 days (p < 0.003, p < 0.003, p < 0.001, p < 0.001, and green tea extract exerts antibacterial activity against
p < 0.001, respectively). However, there were no significant P. gingivalis bacteria, the most common pathogenic bacteria
differences in expression between the MOP and the MOP- that cause periodontitis. Green tea is one of the most
doxy groups after 3, 5, 7, 14 and 21 days (p ¼ 0.209, commonly used beverages worldwide and is obtained from the
p ¼ 0.234, p ¼ 0.072, p ¼ 0.072, and p ¼ 0.075, respectively). dried leaves of the plant Camellia sinensis.16 The polyphenol
epigallocatechin-3-gallate is the most important of the cate-
OPG expression chin components of green tea.18 Green tea has been
demonstrated to have scientifically proven benefits and many
IHC analyses revealed an increased in the expression of functional properties; at present, the consumption of green
OPG in all groups post-treatment on days 3, 5, 7, 14 and 21. tea is widely recommended.17 Previous studies have
The MOP group had the lowest levels of OPG expression investigated how the topical application of green tea extract
(Figures 1 and 2c). Treatment with MOP-EGCG led to the gel might reduce infection in the periodontium, thus
highest expression levels of OPG on days 3, 5, 7, 14 and 21 suggesting that the formulation could be used as an adjuvant
when compared to the other groups (Figures 1 and 2c). treatment for regeneration after periodontitis.19
Tukey’s Post hoc testing showed that there were significant Analysis of RANKL showed that there were significant
differences in the expression of OPG between the MOP differences between the negative control group and the
and MOP-EGCG groups after 3, 5, 7, 14 and 21 days positive control group; the treatment group and the negative
(p < 0.001, p < 0.001, p < 0.001, p < 0.002, and p < 0.014, control group; and the positive control group and the
respectively). However, there were no significant differences treatment group. Analysis of OPG revealed that there were
in expression between the MOP and the MOP-doxy groups significant differences between the control negative group
after 3, 5, 7, 14 and 21 days (p ¼ 0.075, p ¼ 0.067, p ¼ 0.075, and the positive control group and the treatment group; and
p ¼ 0.075, and p ¼ 0.129, respectively). between the control negative group, positive control group
and the treatment group.
Discussion In a previous in silico study, we use EGCG compounds as
a ligand for formulas obtained from a Pub Chem database.
This study aimed to determine the levels of RANK/ Targeted compounds of three-dimensional (3D) structures
RANKL and OPG in healed tissue models of periodontitis. underwent energy minimization at PyRx to stabilize the
The results revealed a significant increase in RANK and molecular structure and acquire a bank of data.20 A number
OPG expression, as well as a significant decrease in the of proteins were identified in this previous study, including
expression of RANKL in a rat model of periodontitis. Our sclerostin, TRAP, Rank-rank, osteocalcin, NFATC1,
findings indicate the treatment of periodontitis led to a osterix, and RUNX2; these proteins were identified by the
decrease in number of RANKL receptors but a significant RCSB PDB database. The 3D structures were then down-
increase in both OPG and RANK. Moreover, the number of loaded in the pdb protein format; then, we sterilized the
RANK receptors increased; however, due to the reduction in water molecules and native ligand through the PyMol soft-
the number of RANKL receptors, it was not possible for ware. EGCG binding occurred with NFACT, sclerostin
RANK to fully bind and activate the process of osteogenesis; TRAP, RANK-RANKL, thus demonstrating inhibitory
instead, RANKL bound with OPG in order to bind with effects.21,22
RANK. Overall, this process would lead to a reduction in the There were some limitations to this study that need to be
process of bone resorption. considered. For example, because we needed a long contact
An imbalance in the RANKL/OPG signaling pathway time of 1 h between the mucoadhesive gingival patch and the
during osseointegration, defined by high expression levels of gingiva, we needed to apply an anesthetic with a long dura-
RANKL/OPG, might cause a disruption in tissue wound tion of action. Furthermore, because the patch slides easily
healing, thus increasing the risk of periodontitis. The osteo- from the gingival incisive sulcus, it needs to be held or tied
clastogenic profile of periodontitis has been documented in with a ligature. However, despite of these limitations, the
many previous studies.10,11 Prostaglandin E2 (PGE2), results of this study confirm that the use of EGCG in MOP
interleukin-1-beta (IL-1b), interleukin-6 (IL-6), and tumor can play a role in the treatment of periodontitis, in which
necrosis factor-alpha (TNF-a), in combination with some RANK, RANKL and OPG expression levels can be used as
other mediators of bone metabolism (transforming growth the main indicators of periodontitis. To strengthen our
factor-beta [TGF-b], parathyroid hormone, 1,25- findings, further research is required. For example, we need
dihydroxyvitamin D3, glucocorticoids, and estrogen) have to identify the precise effects of MOP-EGCG on other
been shown to cause effects on osteoclastogenesis by regulating markers of periodontitis such as TNF-a and IL-10. We also
the osteoblastic/stromal cell production of OPG and RANKL need to investigate other subspecies of MOP to evaluate the
and were detected immediately at infected periodontal sites.12 antibacterial activity against periodontopathic bacteria and
Bone loss has also been reported in periodontitis; a previous apply other products such as gels, mouthwashes and tooth-
study showed that changes in the levels of RANKL play a paste that be formulated with EGCG.
vital role in bone loss.13,14 A rise in the expression levels of
Conclusion
RANKL, along with a reduction of OPG expression, have
been observed in gingival crevicular fluid.15
Herbal medicine is an alternative option for treatment and Mucoadhesive oral patches loaded with epigallocatechin-
is not associated with side effects; consequently, herbal options 3-gallate extract prevented alveolar bone damage in a model
1020 The use of mucoadhesive oral patches

of periodontitis by inhibiting the expression of RANKL and 9. Krismariono A. The decreasing of NFkB level in gingival
increasing the expression of OPG and RANK. junctional epithelium of rat exposed to Porphyromonas gingi-
valis with application of 1% curcumin on gingival sulcus. Dent J
Source of funding (Majalah Kedokt Gigi) 2015; 48(1): 35.
10. Giannopoulou C, Martinelli-klay CP, Lombardi T. Immuno-
histochemical expression of RANKL , RANK and OPG in
This study was supported by the Ministry of Higher Ed- gingival tissue of patients with periodontitis. Acta Odontol
ucation, the Republic of Indonesia in Scheme Penelitian Scand 2012; 70(6): 629e634.
Disertasi Doktor (PDD) 2021 (Grant No. 275/UN3/2021). 11. Chen B, Wu W, Sun W, Zhang Q, Yan F, Xiao Y. RANKL
expression in periodontal disease : where does RANKL come
Conflict of interest from? Biomed Res Int 2014; 2014:731039.
12. Mieko Saika, Daisuke Inoue, Shinsuke Kido,
Toshio Matsumoto. 17b-Estradiol Stimulates Expression of
The authors have no conflict of interest to declare. Osteoprotegerin by a Mouse Stromal Cell Line, ST-2, via Es-
trogen Receptor-a. 17b-Estradiol Stimulates Expression of
Ethical approval Osteoprotegerin by a Mouse Stromal Cell Line, ST-2, via Es-
trogen Receptor-a. 6th, 142. Endocrinology; 2001. pp. 2205e
This study was performed in strict accordance with the 2212 (6).
13. Crotti T, Hirsch R, Soukoulis S, Weedon H, Capone M, Mj A,
Guide for the Care and Use of Laboratory Animals, Na-
et al. Receptor activator NF kappaB ligand (RANKL) and
tional Health Research and Development Ethics Standard
osteoprotegerin (OPG) protein expression in periodontitis.
and Guidelines Council (2017), Minister of Health, Republic J Periodontal Res 2003; 2: 380e387.
of Indonesia. This research study obtained ethical approval 14. Garlet GP, Martins Jr W, Fonseca BA, Ferreira BR, Silva JS.
(Reference 466/HRECC.FODM/X/2020, Approval Date: 9 Matrix metalloproteinases, their physiological inhibitors and
October 2020) from the Research Ethics Commission of the osteoclast factors are differentially regulated by the cytokine
Faculty of Dentistry, Airlangga University, Surabaya. profile in human periodontal disease. J Clin Periodontol 2004;
31(8): 671e679.
Authors contributions 15. Emingil G, Afacan B, Han B, To H, Berdeli A, Atilla G. Dif-
ferential expression of receptor activator of nuclear factor- j B
ligand and osteoprotegerin mRNA in periodontal diseases.
DML, MA, and HRQ carried out the research and J Periodontal Res 2007; 42(4): 287e293.
collected the data. RDR designed and supervised the study, 16. Graham HN. Green tea composition, consumption, and poly-
visualized, and validated the data, acquired funding, and phenol chemistry. Prev Med (Baltim). 1992; 21(3): 334e350.
reviewed draft material. The data were organized, analyzed, 17. Sharma S, Bhuyan L, Ramachandra S, Sharma S, Dash KC,
and interpreted by ID and BAA along with YL and NQ who Dhull KS. Effect of green tea on periodontal health: a Pro-
also reviewed the article. All authors have critically reviewed spective Clinical Study. J Int Oral Heal 2017; 9(2 Mar-Apr):
and approved the final draft and are responsible for the 39e44.
content and similarity index of the manuscript. 18. Samar S, Prasad S, Lanker A, Meer Z, Imranulla M, Pasha S.
Evaluation of green tea extract on gingival and periodontal
status: a randomized controlled pilot study. J Dent Sci 2018;
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