Keywords
medicinal signaling cells - wound healing - biomaterial - metabolite - biocompatibility
Introduction
Sharp orthodontic appliances can lead to oral tissue injury. Traumatic oral ulcers
are frequently related to sharp orthodontic brackets or wires that irritate the oral
mucosa. The destruction of the oral epithelium is marked by irregular laceration or
ulcer surrounding the erythema area and nerve ending exposure. Traumatic oral ulcers
caused by sharp orthodontic appliances can cause unpleasant and painful sensation.[1] According to the Khan et al study, oral mucosa lesions—such as 40% patients with
erosion and 45% patients with ulceration—frequently occur during fixed orthodontic
treatment.[2] Traumatic oral ulcer incidences range from 60 to 81%. An estimated 47% traumatic
oral ulcer incidences occurred in adults with the most unpleasant sensation during
orthodontic treatment; 29% of adolescents report ulcers as the second-most discomfort
aspect of orthodontic treatment.[1]
[3]
Painful sensation is part of the inflammatory response during healing that occurs
~24 hours.[4] Oral ulcers normally heal in ~10 to 14 days, but sometimes if there is infection,
it can prolong and delay the healing process.[1]
[4] The infection stimulates pro-inflammatory cytokines such as tumor necrosis factor-α
and interleukin-1β. Those pro-inflammatory cytokines stimulate the synthesis of matrix
metalloprotease-9, which can lead to Extracellular Matrix degradation.[5] There are so many normal commensal flora in the oral cavity. Although saliva has
antimicrobial properties like human beta defensin-2 as antimicrobial peptide (AMPs),
the microenvironment in the oral mucosa is very challenging for oral ulcer healing
due to pathogen microorganisms in the oral cavity that can induce an inflammatory
response.[6]
[7]
So many products and drugs, with different formulations and various active compounds,
were used to treat oral ulcers.[8]
[9] The Karavana Hizarcioğlu et al study mentioned that chlorhexidine or benzydamine
hydrochloride is no better than a placebo mouthwash. Ulcers treated with benzydamine
hydrochloride gel reduced 33% more than ulcers in control groups, but they were still
not healed until the 12th day.[9] Ernawati and Puspa and Puspasari et al studies used herbal compounds such as propolis
gel to treat oral ulcers, while an Apriasari et al study used mauli banana stem extract
gel as an anti-inflammatory agent to treat oral ulcers, but the healing process accelerated
after a few days.[4]
[5]
[10]
Medicinal signaling cells or mesenchymal stem cells (MSC) possessed the ability to
accelerate wound healing.[11] During MSC culture in medium, MSC secreted a lot of beneficial cytokines, AMPs,
molecules, and growth factors in the culture medium that were very useful.[12]
[13] After MSC was harvested, the MSC culture medium was discarded and become medical
waste. The MSC culture medium can be purified and can be used again as biomaterial
to accelerate oral ulcer healing.[14]
We would like to make the promising biomaterial, which has great biocompatibility
and potential ability to accelerate oral ulcer healing by MSCM (oral based). The aim
of this study was to analyze the analyze the biocompatibility and potential of MSCM
(oral based) accelerating oral ulcer healing (in vitro). This is important and useful
because traumatic oral ulcer is a major problem during fixed orthodontic treatment
that has yet to be resolved.
Materials and Methods
This study was conducted in an experimental laboratory with a descriptive analysis
observational study and a randomized control group design. The study received ethical
clearance with number 289/HRECC.FODM/XII/2017 from Faculty of Dental Medicine, Universitas
Airlangga, Surabaya.
Formulation of Medicinal Signaling Cells Metabolite (Oral Based)
The MSCM (oral based) gel was made in accordance with the procedures of the Stem Cell
Research and Development Center by mixing 10 ml of MSC metabolite with 2% of carboxymethyl
cellulose sodium ([Fig. 1]).[14]
Fig. 1 Medicinal signaling cells metabolite (oral-based) product.
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide or MTT assay
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (or MTT assay) was performed
using human gingival somatic cell (GSC) culture in accordance with the procedure prescribed
in the Prahasanti et al study.[15] GSCs were isolated and cultured according to the procedure prescribed in the Nugraha
et al study.[16]
[17]
[18]
Furthermore, the samples were divided into two groups: cells treated with MSCM (oral
based) were the treatment group and cells without MSCM (oral based) were the control
group. The number of living cells was then observed by calculating CD50 with the MTT
assay. MTT absorbency was indicated with a crystal formazan blue purple colored was
examined by means of enzyme-linked immunosorbent assay reader using 550 to 620 nm
wavelength. The MTT assay was done at the Stem Cell Research and Development Center,
Surabaya, Indonesia.
Scratch Test
A scratch test was done at the Stem Cell Research and Development Center, Surabaya,
Indonesia. The scratch test was performed to know the ability of MSCM (oral based)
to stimulate human somatic cell proliferation and migration, assessed with cell migration
into the cell-free field, in accordance with the Rantam study.[14] Human somatic cells were cultured in an m24 culture plate with estimated 5.10[4] cells in each well, then incubated at 37°C in an incubator (Sigma Aldrich, US).
Each well was scratched with blue tip (Sigma Aldrich, US) with plus (+) shape. The
samples were divided into four groups (n = 1): A. Control group; B. 0 hour treated with MSCM (oral based); C. 3 hours treated
with MSCM (oral based); D. 12 hours treated with MSCM (oral based). The culture plate
of each group was examined by means of a light microscope with 100x magnification
(Olympus, Japan). Images were taken with a camera (Olympus, Japan).
Fourier-Transform Infrared Spectroscopy and Energy Dispersive X-Ray Analysis
The MSCM (oral based) product was examined by means of Fourier transform infrared
spectroscopy (FTIR) to know its functional structure and composition, and to know
the elemental composition of MSCM (oral based), an energy dispersive X-ray (EDX) analysis
was performed. The EDX and FTIR analyses were done accordance with the procedure of
the Prahasanti et al and Nugraha et al studies.[15]
[16] Both analyses was done at the Laboratory of Technical Industrial Faculty, Institute
of Technology Sepuluh Nopember, Surabaya, Indonesia.
Results
The optical density of MSCM (oral based) can be seen in [Fig. 2]. The MTT assay examination result showed that MSCM (oral based) has a high viability
to human somatic cell culture ([Fig. 3]). The scratch test result showed that MSCM (oral based) administration accelerated
the proliferation of somatic cell culture after 12 hours in vitro ([Fig. 4]). The FTIR examination showed a functional structure and composition of MSCM (oral
based) that can be seen in [Fig. 5]. It has Carboxylic Acids & Derivatives bond with peak ~3270.11. The EDX examination
showed that MSCM (oral based) mostly contained Carbon Potassium and it did not contain
heavy metal substances ([Fig. 6]). The composition of MSCM (oral based) can be seen in [Table 1].
Fig. 2 Optical density of medicinal signaling cells metabolite (MSCM) (oral based).
Fig. 3 Cell viability of medicinal signaling cells metabolite (MSCM) (oral based).
Fig. 4 Scratch test to examine somatic cell proliferation ability (in vitro) examined using
electron microscope with 100x magnification (Olympus, Japan). (A) Control group, (B) 0 hour treated with medicinal signaling cells metabolite (MSCM) (oral based); (C) 3 hours treated with MSCM oral-based product; (D) 12 hours treated with MSCM (oral based).
Fig. 5 Fourier-transform infrared spectroscopy result of medicinal signaling cells metabolite
oral-based functional structure and composition.
Fig. 6 Energy-dispersive X-ray result of medicinal signaling cells metabolite oral-based
product.
Table 1
Element composition analysis using EDX of MSCM oral based
|
Element
|
Wt%
|
At%
|
|
Abbreviations: EDX, energy-dispersive X-ray; MSCM, medicinal signaling cells metabolite.
|
|
C K
|
28.06
|
40.81
|
|
N K
|
04.40
|
05.49
|
|
O K
|
26.59
|
29.04
|
|
Ni L
|
01.19
|
00.35
|
|
Na K
|
17.44
|
13.25
|
|
P K
|
00.86
|
00.48
|
|
S K
|
00.87
|
00.47
|
|
Cl K
|
19.67
|
09.69
|
|
K K
|
00.92
|
00.41
|
Discussion
The successfulness oral ulcer treatment depends on several factors such as the elimination
of etiology factor, minimalize the pathogen or commensal microorganism in the oral
niche that can delay healing.[4]
[5]
[10] A biocompatible biomaterial that supports cell, and the potential growth factor
in biomaterial to support endogenous cell metabolic activities is necessary needed.[17]
[18]
[19] MSCM (oral based) has good cell viability in GSCs examined by the MTT assay. GSCs
can be easily isolated with noninvasive methods from free-margin gingiva in the oral
cavity.[20] GSCs are suitable cells for cytotoxicity test purpose.[21] It can be said that MSCM (oral base) is a biocompatible material for application
in the oral cavity. Surprisingly, MSCM (oral based) did not decrease the cell number
during the MTT assay but increased it. MSCs during culture secreted abundant advantageous
growth factors through paracrine and autocrine pathways such as vascular endothelial
growth factor, fibroblast growth factor, and insulin growth factor, which support
cell metabolism, proliferation, and differentiation.[22]
[23]
[24] The scratch test support the fact that growth factor contained in MSCM (oral based)
can increase cell proliferation after 12 hours. MSCM (oral based) stimulates migration
factors such as CXC motif chemokine ligand 12 and stromal-derived factor-1 that made
communication of the cells happen. In addition, cells secreted various growth factors
to support cell proliferation.[24]
[25]
[26]
[27] The Somoza et al study also mentioned that MSCs secreted antimicrobial peptides
such as Cathelicidin against microorganism and Immunomodulation molecules like Indoleamine
2,3-dioxygenase. [12] Those beneficial molecules are very useful for tissue regeneration caused by injury.
At the injury sites, MSCs focus their local immunomodulatory and trophic activities.[24]
[25]
[26]
[27]
The FITR results of this study revealed the function and structure of MSCM (oral based).
FTIR spectroscopy will result in a powerful tool in the study and diagnosis of biological
systems. FTIR spectroscopy is a rapid, noninvasive, accurate, and efficient technique
to examine biomaterial. [15] The structural organization of MSCM (oral based) is an important matrix for retaining
the cells at a specific site and initiating appropriate cell-to-cell interactions
and migration.[22]
EDX is very useful in the biomedical fields because of its high sensitivity to examine
the different elements in tissues. EDX is generally used to improve the biomaterial
or chemotherapeutic agents. EDX is also an important tool to detect nanoparticles.[28] In this study, EDX was performed to examine the composition of MSCM (oral based).
MSCM (oral based) was a safe biomaterial because it did not contain heavy metal substances
such as lead (Pb), nickel (Ni), zinc (Zn), cadmium (Cd), chromium (Cr), copper (Cu),
or mercury (Hg). EDX can be used to detect heavy metal substances due to pollution
in the environmental. Heavy metals are nonbiodegradable substances. These are the
biggest problem for the environment, and they can accumulate and negatively affect
the human body.[29]
Conclusion
MSCM (oral based) has a biocompatible and potential ability to accelerate healing
in oral ulcer in vitro. It would be useful in daily clinical practice in treating
traumatic oral ulcer.
