Keywords
viability - medicine - polymethylmethacrylate - hydroxyapatite - stem cell from human
exfoliated deciduous teeth - osteoblast
Introduction
Periodontal disease, tooth loss, trauma, and infection are some of the factors that
might cause alveolar bone defects.[1] In addition, congenital abnormalities in children such as cleft lip and palate (CLP)
are also often accompanied by defects in the alveolar bone. CLP is one of the most
common forms of congenital abnormalities with an incidence occurring in 1: 500 births
in Asians and Native Americans and approximately 1 in 2,400 to 2,500 births in people
of African.[2]
[3] Untreated alveolar bone defects usually lead to resorption of alveolar bone. The
preservation of the alveolar ridge and the prevention of bone resorption are all achieved
by filling these defects with bone graft material.[4] The gold standard bone graft material is autogenous bone because it has all the
characteristics necessary for bone growth.[5] However, autogenous bone has some limitations, specifically bone availability and
complications. Other bone graft materials, including xenograft, also have some disadvantages,
such as the potential of xenogeneic bone blocks may crack during fixation, which could
hinder the operation and the bone's healing process.[6]
Polymethylmethacrylate (PMMA) is one type of polymer that is commonly used in dentistry
and has been used as a fixation component in orthopaedic implants.[7] The flexible nature of PMMA makes it easy to manipulate the manufacture of biomaterials.
The effort to strengthen the function of PMMA is by adding a bioactive ceramic material,
namely hydroxyapatite (HA) that has excellent osteoconductive and osteointegration
properties.[8] HA is a natural mineral form of calcium apatite and HA in bones is approximately
67 to 70% and has bioactive, biocompatible, and nontoxic properties.[9] Because HA is brittle, mixing it might be challenging; adding PMMA will provide
mechanical structural integrity. The use of scaffolds with porous structures from
bioceramic and polymeric components to support the growth of cells and bone tissue
has been an attraction for a long time as an attractive candidate for biomaterials.[5] Tissue engineering techniques to replace missing or damaged functional tissues and
organs with biomaterials that have good biocompatibility have developed rapidly. Research
on stem cells has grown and both fields of medicine and dentistry have done substantial
study of them. This prompted the researcher to apply the use of stem cells from human
exfoliated deciduous teeth (SHED) from the human oral cavity with a scaffold derived
from a mixture of natural biomaterials, namely mixed PMMA-HA.[6]
[10]
Furthermore, whether this scaffold can be compatible with osteoblasts that are naturally
present in the alveolar bone needs to be proven. Apart from being easy to obtain,
SHED obtained from the pulp of primary children's teeth is an ideal source for bone
regeneration because of its good viability and proliferative potential. SHED also
showed positive results on osteogenic differentiation.[11]
[12] Many studies have demonstrated that SHEDs proliferate more quickly and have greater
differentiation potential than bone marrow mesenchymal stem cells (BMSCs) or even
Dental pulp stem cells (DPSCs).[13]
Therefore, the selection of mixed PMMA-HA materials with SHED is considered because
of their respective advantages that can complement each other as candidates for synthetic
bone graft biomaterials. One of the important aspects in the initial screening and
development of a mixed PMMA-HA as a candidate for synthetic bone graft biomaterials
is the toxicity test, which aims to evaluate the toxicity and safety of these materials
before interacting with the active ingredient, that is a SHED and osteoblast naturally
present in the alveolar bone. According to the Telli et al[14] standard, it is stated that a substance is said to be nontoxic if the percentage
of living cells after exposure to the substance is more than 50%.[15] We, therefore, hypothesize that the mixed PMMA-HA may not associated with toxicity
of SHED and osteoblast.
Considering that the mixed PMMA-HA and its interaction with SHED and osteoblast are
a new proposed biomaterial, to date there has been no research regarding it especially
on the toxicity showed by cell's viability. The aim of this study was to analyze the
toxicity test of mixed PMMA-HA scaffold seeded with SHED and osteoblast in vitro as a candidate of synthetic bone graft biomaterial.
Materials and Methods
This was an experimental laboratory design research with a post-test-only control
group design. The materials used in this study were PMMA (PMMA Granules ; HiMedia.
Laboratories Pvt. Ltd. India), HA (Ceramic Center of the Ministry of Industry of the
Republic of Indonesia), SHED obtained from the isolation of pulp tissue of noncarious
primary teeth (Tissue Bank, Dr. Soetomo), 7F2 osteoblast (American Type Culture Collection,
Manassas, Virginia, United States, CRL-12557), and MTT (Sigma Cat.No.M-5655).
Mixed PMMA and HA Manufacturing
In this study, a preliminary test was performed to determine the ratio between groups.
This comparison is based on the HA content in the bones. The procedure for making
mixed PMMA-HA scaffold was performed by weighing PMMA and HA scaffold was done by
weighing 1 g of PMMA, 2 mL of acetone, and 4 g of HA powder for a 20:80 ratio; 1.5 g
of PMMA, 3 mL of acetone, and 3.5 g of HA powder for a 30:70 ratio; 2 g of PMMA, 4 mL
of acetone, and 3 g of HA powder for a 40:60 ratio, respectively.
PMMA that has been weighed was put into a bottle and mixed with acetone, then stirred
briefly until the PMMA grains were submerged in acetone, then left in the refrigerator
at a temperature of −30° for 24 hours. After 24 hours, the HA powder was added to
the PMMA solution and then stirred using a spatula over a magnetic stirrer until it
became homogeneous. After the PMMA:HA mixture became homogeneous, it was poured into
a mold with 5 mm of diameter and height as shown in [Fig. 1].[16]
Fig. 1 Mixed polymethylmethacrylate and hydroxyapatite material. (A) Polymethylmethacrylate granules. (B) Hydroxyapatite from Balai Besar Keramik Indonesia. (C) Three groups of mixed polymethyl methacrylate and hydroxyapatite.
After that, a freeze-drying process was performed. The mixed PMMA and HA that had
been freeze-dried was subjected to gamma radiation sterilization at the Indonesian
Nuclear Energy Agency (BATAN).
Isolation and Culture of Stem Cells from Human Exfoliated Deciduous Teeth
The SHED was collected from deciduous teeth using the following criteria: #73 persistence
of deciduous tooth, free of cavities, no root resorption, and a vital and undamaged
pulp was retrieved after tooth extraction from a healthy, 8-year-old pediatric patient.
Patient anonymity was maintained and written informed consent was obtained from the
patient's parents. The ethical clearance was approved by the Ethical Research Committee
Faculty of Medicine Universitas Airlangga no. 239/HRECC. FODM/V/2021 covered for human
sampling.
The pulp tissue was placed in the medium within a 15 mL conical tube and placed in
a cool box to be immediately sent to the GDC Tissue Bank Dr. Soetomo Surabaya ([Fig. 2]). Then the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies,
Gibco BRL, United States) with the addition of 20% fetal bovine serum (FBS, Biochrom
AG, Germany), 5 mL L-glutamine (Gibco Invitrogen, United States), 100 U/mL penicillin-G,
100 g/mL streptomycin, and 100 g/mL kanamycin (Gibco Invitrogen, 25, United States).[17]
Fig. 2 Stem cell from human exfoliated deciduous teeth isolation protocol. (A) #73 persistence of deciduous tooth, free of cavities, and no root resorption. (B–C) The dental pulp cavity was opened using drills with sterilized round bur. (D) Pulp tissue was taken in the medium within a 15 ml conical tube.
After 3 days, the medium was discarded to take off the portion of the cell that was
not connected to the plate and place it in a new medium. At this stage, fibroblast
growth factor-2 was added. After the cells were confluent, they were passaged using
0.05% trypsin-ethylenediamine tetraacetic acid (EDTA) and after that the cells were
washed and cultured again in 60- or 100-mm tissue culture dishes (Corning). After
the confluent cells are repassed, and the cells can be used for research ([Fig. 3A]).[17]
Fig. 3 Characterization of stem cell from human exfoliated deciduous teeth. (A) Morphology of isolated and unstained MSCs demonstrating a typical mesenchymal stem
cell shape characterization of adherent spindle-shaped MSCs cells in culture. (B) Flow cytometry analysis of passage 3 mesenchymal stem cells culture for CD105, CD90,
CD45, and CD73cells. (C) Calcium deposition can be seen after staining with Alizarin Red in the osteogenic
test culture.
Characterization of Stem Cells from Human Exfoliated Deciduous Teeth
SHEDs were washed twice with phosphate buffered saline (PBS) containing 2% fetal bovine
serum (Gibco; Thermo Fisher Scientific, Inc.) and were incubated for 30 minutes at
room temperature with antibodies against CD45-FITC, CD73-FITC, CD90-PE, and CD105-PE.
All antibodies were used at a dilution of 1:100 and purchased from BD Biosciences.
Samples were diluted up to 1 μL and read with flow cytometry (FACS Callibur, BD).
Osteogenic Differentiation (Alizarin Red Staining)
SHED was induced for osteogenic differentiation (OsteoMAX-XFTM Differentiation Medium)
according to the manufacturer's instruction. Briefly, 2 × 104 cells per well were added in a 48-well plate and grown until confluence. Confluent
cells of 0.5 mL OsteoMAX-XFTM differentiation medium were added to each well. This
medium change corresponds to differentiation day 1. On day 3, 0.25 mL of the medium
was removed from each well and replaced it with 0.5 mL of fresh OsteoMAX-XFTM differentiation
medium. For all subsequent medium changes, 0.5 mL of the medium was removed from each
well and replaced it with 0.5 mL of fresh OsteoMAX-XFTM differentiation medium. Medium
changes should occur every 3 days for 14 to 17 days. After 14 to 17 days of differentiation,
osteocytes could be fixed and stained for alkaline phosphatase (Cat. No. SCR004) or
with Alizarin Red (Cat. No. ECM815) for mineralization.
Osteoblast Cell Culture
7F2 osteoblast (American Type Culture Collection, Manassas, VA, United States, CRL-12557)
was cultured in DMEM media supplemented with 10% fetal bovine serum and streptomycin
penicillin. Cells were cultured in 75 cm2 flasks and allowed to grow until confluent. The cultures were incubated at 37°C with
5% CO2 and the culture medium was changed every 48 to [18]
The cleaned media was rinsed with PBS and then added 1 to 2 mL of the trypsin–EDTA
solution. Flask was left at 37°C incubators until the cells were released. The cell
suspension was then centrifuged at 2000 rpm for 10 minutes, pelleted the suspension
back into the new medium, aspirated, put into a new flask, and then subcultured before
the cells became confluent. The osteoblast cells were counted using hemocytometer
and seeded in 96 well plates with a concentration of 2 × 105/well.[18]
MTT Assay
Cells viability was evaluated by cytotoxicity test using MTT assay. SHED at passages
4 to 5 and osteoblast cells at passages 4 were prepared 80% confluent. There were
two big groups in this study; each group consisted four groups. There were four groups
in this study, control group (without PMMA/ HA scaffold), group 1 (with 20/80 PMMA-HA
scaffold), group 2 (with 30/70 PMMA-HA scaffold), and group 3 (with 40/60 PMMA-HA
scaffold). Five repetitions were performed in each group so that the total sample
was 40 scaffolds. The cells were harvested until become single cells and homogenized
in the culture medium. The cells were planted in 96 well plates with a concentration
of 2 × 105/well and the empty wells were left blank. CO2 was incubated in an incubator for 24 hours until the cells adhered perfectly.
The test material was prepared in the form of a mixture of PMMA and HA with concentration
groups of 20/80, 30/70, and 40/60, and immersed in the culture medium. Soaking medium
of 100 µL was added for each type of test material into the well. It was incubated
again for another 24 hours. A total of 25 µL MTT was added to each well and it was
incubated again for 4 hours. Then the medium and MTT were discarded. dimethyl sulfoxide
(DMSO) of 200 µL was added to each well, and when the color changed into purple, then
200 µL of PBS was added to an empty well and inserted into the enzyme-linked immunosorbent
assay (ELISA) reader.
The absorbance of each well was read at a wavelength of 595 nm. The cytotoxicity is
expressed as cytotoxic dose, the concentration of the substance inhibiting cell growth
by 50% (CD50).[19]
Statistical Analysis
Statistical analysis was performed using IBM SPSS Statistics Software, version 25.0
(IBM Corp., Armonk, New York, United States). The data were statistically analyzed
by using one-way analysis of variance (ANOVA) followed by least significant difference
(LSD) test, considering the level of significance p-value less than 0.05.
Results
Isolation, Culture, and Characterization of Stem Cells from Human Exfoliated Deciduous
Teeth
SHEDs were cultured in nonosteogenic culture media. SHEDs were subcultured until it
reaches passage 3. All adherent cells showed spindle-shaped morphology under electron
microscope ([Fig. 3A]). SHEDs within three passages were shown to be MSCs by flow cytometry. SHEDs expressed
the mesenchymal stem cell surface markers (CD90, and CD105), but were negative for
CD45 and CD73 markers ([Fig. 3B]). Osteogenic potential differentiation of SHED was confirmed by the presence of
calcium deposits on Alizarin Red S staining on day 14 ([Fig. 3C]).
SHED and Osteoblast Viability
The cytotoxicity effect of mixed PMMA-HA on SHED and osteoblast that represent cells
viability were assessed using the MTT assay method based on the absorbance value detected
by ELISA reader to see the number of cells in optical density units and converted
into the cell viability formula. The percentage of viability of SHED and osteoblast
cells against the mixed PMMA-HA can be seen in [Figs. 4] and [5]. [Fig. 4] showed the 20/80 groups has the highest mean percentage of SHED's viability of 87.03%
and the percentage of osteoblast's viability of 123.6%. The data obtained were homogeneous
and normally distributed, tested with the one-sample Kolmogorov–Smirnov test. To determine
the difference in viability of SHED and osteoblast against the mixed PMMA-HA, statistical
calculations were performed using one-way ANOVA, obtained p-value less than 0.05. The results of this analysis showed that there was a significant
difference in the viability of SHED and osteoblast against the mixed PMMA-HA. LSD
analysis showed significant difference between each group of mixed PMMA-HA.
Fig. 4 Effects of different mixed polymethylmethacrylate and hydroxyapatite (PMMA-HA) on
viability of stem cell from human exfoliated deciduous teeth (SHED). Statistically significant (p < 0.05; least significant difference test differences in values compared with the
control value (untreated) are indicated by an SPSS. Graphics represent the means and ± standard
deviation from three independent determinations performed in five replicates.
Fig. 5 Effects of different mixed polymethylmethacrylate and hydroxyapatite (PMMA-HA) on
viability of osteoblast. Statistically significant (p < 0.05; least significant difference) test differences in values compared with the
control value (untreated) are indicated by an SPSS. Graphics represent the means and ± standard
deviation from three independent determinations performed in five replicates.
Discussion
The purpose of tissue engineering is to develop tissue reconstruction that is useful
for restoring, maintaining, repairing, or enhancing the function of tissues that are
damaged or lost due to physiological, pathological, and mechanical conditions or trauma.[20] Three important components in tissue engineering are stem cells/progenitor cells,
signaling, and scaffold.[15]
[21] These three important components are known as the tissue engineering triad because
they are arranged in such a way that they resemble the natural regeneration that occurs
in cells, tissues, and organs.[22] Scaffold is a porous solid biomaterial with a three-dimensional shape that was designed
to deliver sufficient nutrients, gases, and regulatory factors to allow interactions
between cells and biomaterials, cell adhesion, and extracellular matrix deposits to
decay at a controlled rate according to the rate at which the material was deposited,
tissue regeneration and minimize inflammatory reactions.[23] Scaffold can be made from synthetic bone graft material that has the ability to
induce bone formation. One of the ideal requirements of synthetic bone graft is that
it is biocompatible or not toxic.[19]
[24]
Cytotoxicity refers to cell damage, where cells can die due to necrosis or apoptosis
(programmed cell death).[20]
[25] Cytotoxicity test is a method to determine whether a substance is toxic to certain
cells. The parameter of toxicity test is cell viability. One of the methods to assess
the cytotoxicity of a substance is by an enzymatic test with MTT assay reagent. This
method was chosen because it has good sensitivity in evaluating the cytotoxicity of
the test material.[26] It also has a relatively fast procedure step and is easy to retest when needed.
This method measures the metabolic activity of cell growth after exposure to the material
test. The basic principle is to measure cellular activity based on the activity of
the enzyme succinate dehydrogenase in the mitochondria of cells to reduce the tetrazolium
salt MTT. This enzyme will react with MTT and form purple formazan crystals whose
amount is proportional to the activity of living cells because these crystals are
impermeable to dead cell membranes.[26] The parameter used for the cytotoxic test is the IC50 value (50% inhibition concentration). The IC50 value is a concentration value that indicates the inhibition of cell proliferation
by 50% and the potential toxicity of material to cells. IC50 value can indicate the potential of the material as cytotoxic. This value is a benchmark
for conducting a cell kinetics observation test. The greater the IC50 value, the lower toxicity of the material to cells.[27]
PMMA-HA cytotoxicity check was performed on SHED and osteoblast cultures as a candidate
of synthetic bone graft materials that is expected to regenerate bone in alveolar
defects. This is based on the role of SHED and osteoblast as active cells responsible
for osteogenic differentiation and bone matrix formation.[11] Based on Su Min Lee's research,[28] which considered SHED's potent osteogenic potential as well as its successful use
in the regeneration of teeth and the bone regeneration of the craniofacial region.
In addition, SHED is one of the mesenchymal stem cells that has a high level of sensitivity
to toxic agents so it is often used in toxicity tests. In this study, SHED expressed
the mesenchymal stem cell surface markers (CD90, and CD105), but was negative for
CD45 and CD73 markers. The nonuniform expression of CD73 on MSC may be associated
with the reparative property, as extracellular adenosine catalyzed by the dephosphorylation
activity of CD73 has been proven as a pivotal regulator of local immune responses.[29]
Quantitative data from the research was obtained by measuring the percentage of the
number of living cells in each group. The results of this study showed that all treatment
groups did not have toxic properties against SHED and osteoblast according to the
Telli et al[11] standard, which states that a substance is said to be nontoxic if the percentage
of living cells after exposure to the substance is more than 50%. The results of cell
viability that have been exposed to a mixed PMMA-HA for 24 hours with several concentrations,
cell viability of SHED ranged from 65.79 to 87.03% and cell viability of osteoblast
ranged from 93.48 to 123,6%; so it can be said to be nontoxic. These results were
consistent with Pridanti et al[30] on umbilical cord mesenchymal stem cells at all concentrations due to high Ca/P
ratio on HA. Similar result was shown by Gayathri et al's[31] with adipose-derived mesenchymal stem cells. Additionally, Mostafa's research, which
evaluated the PMMA-HA-MgP nanocomposite combination's compression strength and cytotoxicity
against fibroblast cells, revealed that the HA ratio group with a weight ratio of
7.5% and a weight ratio of 6.12% had the highest compression strength and cell viability
values.
The combination of PMMA-HA did not have a toxic effect because the HA content in mixed
PMMA-HA was directly proportional to the percentage of SHED viability. HA is an inorganic
material that contained approximately 67 to 70% of the bone. HA has good biocompatibility
and bioactivity properties.[10]
[22] The elements present in HA are dominated by elements of calcium (Ca), oxygen (O),
phosphorus (P), and other with minimal amount elements (<5%), namely aluminum (Al),
silica (Si), sodium (Na), and magnesium (Mg).[16]
[32] Ca contributes to the signaling of osteoblast growth and extracellular matrix. The
high amount of calcium would increase the proliferation, due to the increase of calcium
channel expression. Calcium sensing receptor could detect any external Ca2+ concentration change and increase Ca2+ influx. The higher influx in higher calcium ions concentration would induce cellular
responses such as cell proliferation.[30] The increase in extracellular Ca2+ induced expressions of cell growth factors (fibroblast growth factor 2 and transforming
growth factor beta 1) and the levels of cell cycle regulators. Therefore, we expected
that these factors might mediate the increase extracellular Ca2+-induced cell proliferation.[33] A calcium channel overexpression might be inhibited by an excessive concentration
of extracellular Ca2+ ions. Calcium phosphate concentrations would rise as Ca2+ ions moved into the cytoplasm. This increase would cause the endoplasmic reticulum
to produce intracellular Ca2+ ions, which would disrupt intracellular Ca2+ homeostasis and act as a secondary messenger in preserving cell function, predisposing
to mitochondrial-mediated apoptosis.[34]
Phosphorus (P) will regulate the signal of proliferation, differentiation, mineralization
of osteoblasts, and apoptosis of osteoclasts.[24]
[35] When cells are exposed to high quantities of phosphate, osteoblast apoptosis can
be triggered. This could be due to the mitochondrial membrane being severely damaged.[36] Si stimulates DNA synthesis and cell growth in fibroblast and bone cells from mammals.
Si ions stimulate osteogenesis via raising levels of alkaline phosphatase (ALP) and
osteocalcin, dentin sialoprotein (DSP), and mineralization as well as increased proliferation
states. It has been well documented that silica may modify biological responses thus
supporting stem cells and growth factors in the tissue engineering process.[37] Furthermore, the amount of Al and Mg in HA may induce cells apoptosis, which is
determined by dose and duration. Changes in the core morphology indicate that Al2O3 nanoparticles alter cell cycle progression and gene expression. Corrosion products
of Mg can significantly affect metabolic activity and cell proliferation, which in
turn affects cell fusion/differentiation.[38]
[39] However, because the content of Al and Mg in HA is below 5 wt%, it is not expected
to have a toxic effect on cells.
Unlike HA, PMMA has a different nature. PMMA is composed of the elements carbon (C),
oxygen (O), and hydrogen (H), which contribute to the mechanical stability of the
bone graft by increasing the scaffold's structural integrity.[40] The above elements have a positive impact on the viability of SHED and osteoblast.
However, this study was limited to the cytotoxicity test of the mixed PMMA-HA against
SHED and osteoblasts.
Conclusion
According to the research, it was concluded that mixed PMMA-HA was not toxic for the
SHED and osteoblast. This characteristic is the initial requirement to be proposed
as an alternative material for healing alveolar bone defects. Further research studying
the proliferation rate of SHED and osteoblasts after implanted in mixed PMMA-HA is
needed. In vivo animal research is mandatory to confirm the use of PMMA-HA on the alveolar defect
model.
