TISSUE ENGINEERING: Part C

Volume 22, Number 8, 2016

ª Mary Ann Liebert, Inc.
DOI: 10.1089/ten.tec.2016.0025

METHODS ARTICLE

Antioxidant N-Acetylcysteine and Glutathione Increase

the Viability and Proliferation of MG63 Cells
Encapsulated in the Gelatin Methacrylate/VA-086/Blue
Light Hydrogel System
Chih-Hsin Lin, MS,1 Kai-Fung Lin, MS,1 Kwei Mar, DDS,2
Shyh-Yuan Lee, DDS, DScD,1,3 and Yuan-Min Lin, DDS, PhD1,3

Photoencapsulation of cells inside a hydrogel system can provide a suitable path to establish a gel in situ for
soft tissue regeneration applications. However, the presence of photoinitiators and blue or UV light irradia-
tion can result in cell damage and an increase of reactive oxygen species. We here evaluate the benefits of an
antioxidant pretreatment on the photoencapsulated cells. We study this by evaluating proliferation and viability
of MG63 cells, which we combined with a gelatin methacrylate (GelMA) hydrogel system, using the photo-
initiator, VA-086, cured with 440 nm blue light. We found that blue light irradiation as well as the presence of
1% VA-086 reduced MG63 cell proliferation rates. Adding a short pretreatment step to the MG63 cells,
consisting of the antioxidant molecules N-acetylcysteine (NAC) and reduced glutathione (GSH), and optimizing
the GelMA encapsulation steps, we found that both NAC and GSH pretreatments of MG63 cells significantly
increased both proliferation and viability of the cells, when using a 15% GelMA hydrogel, 1% VA-086, and 1-
min blue light exposure. These findings suggest that the use of antioxidant pretreatment can counteract the
negative presence of the photoinitiators and blue light exposure and result in a suitable environment for
photoencapsulating cells in situ for tissue engineering and soft tissue applications.

Introduction applications, primarily due to its biocompatibility and ease of

gelation.4–6 Gelatin gels have also been utilized for delivery of

H ydrogels have been of great interest to biomaterial

scientists for many years1,2 because they offer three-
dimensional (3D) networks comprising hydrophilic polymers
and can absorb up to 99% of water. A common approach in
tissue engineering is to use hydrogels for encapsulating cells
into 3D cultures. In addition, hydrogels are commonly used
for cell delivery, where cells are suspended in a liquid pre-
cursor solution and then delivered in vivo to the position of
interest. By curing the hydrogel precursor solution directly at
the site of interest, the hydrogel can be created in situ from
aqueous precursors and can form complex shapes that adhere
and conform to tissue structures.
There are many different hydrogel materials that can be
used for cell encapsulation, offering a range of properties to
select from for a specific application. In this study, we chose
gelatin, a derivative of collagen that is formed by breaking the
natural triple-helix structure of collagen into single-strand
molecules.3 Gelatin has found many uses in tissue engineering
growth factors to promote vascularization of engineered new
tissue.7 However, a downside of gelatin is possible variability
from source to source, limited antigenicity, and instability at
body temperature.8,9 Causing gelation with thermal methods is
challenging because the temperature changes required make
manipulation difficult. Photopolymerization offers a simple
alternative that can control the gelation process by modifying
the gelatin with methacrylic anhydride, adding several meth-
acrylate groups at its side chains. This modification can be
carried out by reacting methacrylic anhydride with the amine-
containing side groups of gelatin, turning gelatin into a light-
curable gelatin methacrylate (GelMA).10–12 GelMA has been
gaining popularity as a biomaterial since it is inexpensive and
has tunable physical and biochemical properties. However, the
degradation rate of GelMA can only be modified by changing
the concentration, making it difficult to use.13 Some studies of
gelatin preparations for regenerative medicine applications
have been obtained with endotoxin.14

1
Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan.
2
Department of Dentistry, Taipei City Hospital, Taipei, Taiwan.
3
Department of Stomatology, Taipei Veterans General Hospital, Taipei, Taiwan.

792
NAC AND GSH INCREASE MG63 VIABILITY IN THE GelMA SYSTEM
The GelMA solution can be photopolymerized into a
hydrogel at body temperature (37C) in the presence of
photoinitiators and light sources.15 A photoinitiator is
a compound that absorbs UV or visible light and converts
the absorbed light energy into chemical energy. These
changes occur in the form of initiating species, that is, free
radicals or cations. The photoinitiation systems used in
cell encapsulation include 2-hydroxy-1-[4-(hydroxyethoxy)
phenyl]-2-methyl-1-propanone (Irgacure 2959), 2,2¢-azobis
[2-methyl-n-(2-hydroxyethyl)propionamide] (VA-086), and
lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).16,17
We chose VA-086 as the photoinitiator because it was re-
ported to be less toxic than Irgacure 2959.16 VA-086 is a
type I photoinitiator, which undergoes a unimolecular bond
cleavage upon irradiation to yield free radicals that can
cause cell damage. For example, free oxygen radicals con-
tribute to oxidative damage to DNA and play an important
role in the etiology of cancer and atherosclerosis and are
also implicated in studies on aging.18 VA-086 is typi-
cally cured with UV light between 365 and 385 nm wave-
length. We decided to use 440 nm, which while off-peak
for VA-086 photoinitiation, allows for more biocompatible
irradiation.16
Oxygen can be toxic to cells at higher concentrations. In
its ground state, molecular O2 is relatively unreactive. Its
partial reduction gives rise to reactive oxygen species (ROS)
such as singlet oxygen, superoxide radical anions, and hy-
drogen peroxide.19 The presence of intracellular oxygen can
initiate a chain of reactions at the cellular level that causes
damage to critical cell biomolecules. These radicals are at-
oms, molecules, or ions, which are highly toxic and have a
single unpaired electron. Environmental stress, such as UV
and free radicals generated during photopolymerization,
can induce a higher intracellular ROS level through multiple
mechanisms. Therefore, a trade-off exists between the need
to supply light energy to the photoinitiator to generate the
free radicals necessary to assist in the gelation and the need
to maintain a safe environment for the cells embedded in the
hydrogel. This trade-off motivated our choice for 440 nm
blue light over the perhaps more commonly used higher
energy UV radiation.
To further reduce the effects of free radicals generated
during photopolymerization, antioxidants can be added to
the culture medium. The antioxidant molecules can stabilize
or deactivate free radicals and reduce cell damage. Anti-
oxidants can be endogenous to the cell or they can be ob-
tained exogenously as a part of the diet or growth medium.
Under conditions of heavy oxidative stress, endogenous
antioxidants may not offer sufficient protection, and die-
tary antioxidants may be added to maintain optimal cellular
functions.
The antioxidant glutathione (GSH) is one of the most im-
portant nonenzymatic removal systems for exogenous and
endogenous free radicals.20 In cells, GSH plays an important
role in maintaining the redox state. It is an abundant tri-
peptidyl molecule and plays important roles in protecting
cells against oxidative stress-induced cellular damage, de-
toxifying xenobiotics, and in drug metabolism. GSH can
maintain the exogenous antioxidants (such as vitamins C
and E) in their reduced forms and help reduce the damage
caused by UV light to skin. There are three amino acids in
the GSH structure: glutamate, glycine, and cysteine.21 The
793
thiol group in the cysteine residue of the GSH acts as a
reducing agent, donating the reducing equivalent (H+ plus
e-) to other unstable molecules (e.g., ROS). Cysteine can
limit the rate of GSH synthesis during times of oxidative
stress. N-acetylcysteine (NAC) is the acetylated precursor
of both the amino acid l-cysteine and reduced GSH. In
addition, NAC is an effective scavenger of free radicals as
well as a major contributor to maintenance of the cellular
GSH status. For example, NAC was shown to be involved in
treatments of different diseases, including cancer, cardio-
vascular diseases, and human immunodeficiency virus (HIV)
infections.22 The mechanism of action for NAC is the scav-
enging of H2O2 and peroxide, and it thus may play a role in
preventing cancer.
In this study, we investigate how the presence of GSH
and NAC can be of benefit when using a blue light-activated
hydrogel system consisting of gelatin methacrylate and VA-
086. We chose cells from the MG63 osteosarcoma cell line
to be encapsulated because this cell line has previously been
used as an osteoblast mode in vitro.23,24
Materials and Methods
Synthesis of methacrylated gelatin
GelMA was synthesized according to Benton et al.10; 1 g
of gelatin was added to 10 mL of phosphate-buffered saline
(PBS) and heated at 50C until the gelatin was dissolved
completely. Methacrylic anhydride was added by stirring into
the mixture until the target volume (1 mL) was reached at a
constant rate of 0.1 mL/min. The reaction was then allowed to
proceed for 3 h at 50C, after which the solution was diluted
fivefold with warm (55C) PBS. This was followed by dial-
ysis with tubular dialysis membranes (Orange Scientific) of
6000–8000 molecular weight for 1 week against 40C dis-
tilled water to remove the methacrylic acid and salts. The
solution was lyophilized for 1 week to obtain a white sponge-
like foam, which was stored at -80C for further reaction.
Culturing of the MG63 osteosarcoma cells
MG63 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine
serum and 1% Penicillin–Streptomycin–Amphotericin B
antibiotics. Cells were kept in a humidified incubator at
37C in the presence of 5% CO2.
Evaluation of the curing time using
the upside-down method and viscometer
Hydrogels were prepared in either 12.5% or 15% (w/v)
of gelatin in DMEM, with 1% VA-086 by weight. GelMA
and VA-086 were weighed and then added into the DMEM
and mixed. Dried glass vials were filled with 1 mL hy-
drogel solution and irradiated under a custom-made 10 W,
440 nm blue LED light source (60 mW/cm2). The light was
placed one centimeter below the bottom of the vial at room
temperature (25C) for different periods of time. The ge-
lation start time was defined as the time at which the
mixture no longer floated when the vial was turned upside
down. The viscosity change of the GelMA during blue
light curing was also recorded using a Brookfield rheom-
eter (DV3TRV Rheometer; Brookfield, Middleboro, MA).
One milliliter of GelMA was loaded into a transparent glass
794 LIN ET AL.

FIG. 1. (A) The spectrum

of the blue LED light. (B)
Viscosity change of the
GelMA solution during light
curing. (C) Gelation time
of the GelMA hydrogel.
(D) Compressive modulus
of the GelMA hydrogel
(***p < 0.001, n = 3). GelMA,
gelatin methacrylate. Color
images available online at
www.liebertpub.com/tec

container and light-cured from the bottom. A V-74 spindle
with a rotation speed of 15 rpm was used for the viscosity
test. Data were collected every 5 s, and the readings be-
tween 10% and 90% torque were recorded and expressed in
centipoise (cP).
Compression test
Uniaxial compression measurements were performed on a
universal testing machine at room temperature in compres-
sion mode. All measurements were tested at a crosshead
speed of 1 mm/min until the samples were compressed by
70% in height.
MTT assay
An MTT assay was used to measure the proliferation
rate of MG63 cells 24 h after they were exposed to blue
light for 1, 2, and 4 min. The cells were pretreated with
different concentrations of the antioxidants, GSH and NAC
(0, 10, 20, and 30 mM), which were dissolved in DMEM
and then incubated at 37C for 20 min. The cells were then
washed in PBS, 1% VA-086 was added, and exposed to
blue light for 1, 2, and 4 min. The cell proliferation rate
was then measured by MTT assay. For photoencapsulated
MG63 cells, after the above steps, the cells were washed
in PBS and isolated by trypsin, and then mixed with
the GelMA solution in DMEM, with the photoinitiator,
VA-086, included (see previous methods for details).
Evaluation of cell growth was done by MTT assay at 1, 3,
and 7 days after MG63 photoencapsulation, both for cells
with and without the antioxidant pretreatment. Ten mi-
croliter MTT solution (5 mg/mL in sterile PBS) was added
into each well and incubated for 3 h at 37C. After incu-
bation, the medium was removed and 200 mL DMSO
(Sigma-Aldrich) was added to each well and incubated for
30 min; 100 mL of test sample taken from each well was

FIG. 2. Images of LIVE/DEAD assays of MG63 cells (6 · 104/cm2) cultured for 24 h in different concentrations of
GelMA solution medium. (A) Control. (B) 12.5% GelMA solution. (C) 15% GelMA solution. White scale bar equals
200 mm. Color images available online at www.liebertpub.com/tec
NAC AND GSH INCREASE MG63 VIABILITY IN THE GelMA SYSTEM
added into a 96-well plate for absorbance measurements
using an ELISA reader at 570 nm with a reference wave-
length of 620 nm.
Antioxidant pretreatment and photoencapsulation
of MG63 cells in hydrogel
Before cell encapsulation, a 1% solution of the photo-
initiator, VA-086, in PBS was prepared and stored at -80C.
Fifteen percent of w/v GelMA was dissolved into the so-
lution. To test the effect of NAC and GSH, MG63 cells
cultured on tissue culture plastics were pretreated with
10 mM NAC, 20 mM NAC, 10 mM GSH, and 20 mM GSH
for 20 min each. As experimental control, no antioxidant
pretreatment was performed. Cells were then trypsinized,
counted, and spinned down. The cell pellets were re-
suspended in the polymer solution with VA-086, until the
cells were evenly distributed (at 106 cells/mL). Twenty
microliters of cell-containing hydrogel solution in each 24-
well cell culture dish was irradiated under blue LED light at
room temperature (25C) to initiate the photopolymeriza-
tion. This step should be performed rapidly to minimize cell
settling before light exposure. After the gels cross-linked,
they were washed with PBS and cultured in DMEM. On
days 1, 3, and 7 after seeding, samples were collected to
conduct MTT assay and Live/Dead assay test.
FIG. 4. Cell proliferation rate of MG63 cells treated with different concentrations of NAC (A) and GSH (B) after
exposure to blue light for 0, 1, 2, and 4 min. MTT was performed 24 h after light exposure (**p < 0.01, ***p < 0.001).
GSH, glutathione; NAC, N-acetylcysteine.
795
FIG. 3. (A) MTT absor-
bance and (B) ROS level
of MG63 cells (105/well)
irradiated under blue light
for 0, 1, 2, and 4 min each
(**p < 0.01; ***p < 0.001).
ROS assay
ROS tests (Cell Biolabs, Inc.) were performed according
to the manufacturer’s protocol, using the cell-permeable
fluorogenic probe, 2¢,7¢-dichlorodihydrofluorescein diace-
tate (DCFH-DA), which is first diffused into the cells and
then deactelyated into nonfluorescent 2¢,7¢-dichlorodihydro-
fluorescein (DCFH). ROS rapidly oxidized DCFH into 2¢,
7¢-dichlorodihydrofluorescein (DCF). The fluorescence in-
tensity was measured using a fluorometric plate reader at
480/530 nm.
Live/Dead cell viability assay
To test for cytotoxicity after GelMA photoencapsulation,
we used a Live/Dead assay. Live cells were detected
through the presence of intracellular esterase activity. An
enzymatic conversion of the nonfluorescent cell-permeant
calcein AM into the intensely fluorescent calcein was con-
ducted. Ethidium homodimer-1 (EthD-1) enters through
damaged cell membranes and binds to nucleic acids. This
produces a bright red fluorescence in dead cells. The solu-
tion consists of 2 mM calcein AM and 4 mM EthD-1 as a
working solution, which was added directly to the GelMA
hydrogel. The hydrogel was incubated at room temperature
for 30 min, and cells were imaged under a fluorescence
microscope and labeled.
796
FIG. 5. ROS levels of MG63 cells treated with different concentrations of NAC (A) and GSH (B) after exposure to blue
light for 0, 1, 2, and 4 min (*p < 0.05, **p < 0.01, ***p < 0.001). ROS, reactive oxygen species.
Statistical analysis
All experimental results are expressed as mean – standard
error of the mean. Comparative studies of means were an-
alyzed using one-way ANOVA with a statistical significance
of p < 0.05. In all figures, the significance at p < 0.05, 0.01,
and 0.001 was labeled as *, **, and ***, respectively.
Results
Viscosity change, gelation time, and compressive
modulus of GelMA hydrogels cured by blue
LED irradiation
The viscosity of 12.5% and 15% GelMA solution in-
creases rapidly at 55 and 40 s, respectively (Fig. 1C). These
numbers are longer than the gelation time of 33.3 – 1.5 and
29.3 – 1.1 s when measured by the upside-down method
(Fig. 1B). The compressive modulus of 12.5% and 15% of
GelMA hydrogels is 9.28 – 0.47 and 16.46 – 1.01 kPa, re-
spectively (Fig. 1D). The compressive modulus of the GelMA
hydrogel increases as the GelMA concentration increases, as
expected. The 2.5% difference in the GelMA concentration
results in a statistically significant increase in the compressive
modulus of GelMA ( p < 0.001).
The viscosity increase of the GelMA solution was non-
linear. Before light curing, the GelMA solution has a low
FIG. 6. Cell proliferation of MG63 cells photo-
encapsulated in GelMA, evaluated by MTT assay. In com-
parison with the control, all antioxidant pretreatments
significantly reduced the ROS during photoencapsulation
(***p < 0.001).
LIN ET AL.
viscosity, comparable with water. At the start of gelation,
when the GelMA begins to photopolymerize, the viscos-
ity increases. As the GelMA hardens, eventually the shear
loading exceeds the maximum shear stress of GelMA gel,
and the rotating spindle resulted in gel rupture. At this point,
a zigzag curve was observed (Fig. 1C). Although the gela-
tion starting time was determined using a viscometer,
the change in viscosity does not fully represent the regular
static photopolymerization process because the viscosity
measurement was done in a dynamic environment. How-
ever, the viscosity measurement still can provide a simple
way to evaluate the gelation behavior.
Cytotoxicity test of GelMA prepolymers
Cytotoxicity is one of the critical factors determining
whether the prepared GelMA is pure enough for further cell
encapsulation experiments. Figure 2 shows the live and dead
images of MG63 cells cultured with 12.5% and 15% (w/v)
for 24 h. No dead cells (red fluorescence) were found in the
control group and the two experimental groups, suggesting
that the prepared GelMA is not cytotoxic to MG63 cells.
The effects of blue LED light irradiation on MG63
proliferation and ROS levels
Some studies showed that blue light can induce mito-
chondrial DNA damage and produce free radicals in cer-
tain cells.25 To test the effect of cells exposed to blue
light, MG63 cells (2 · 104/well) were irradiated for 0, 1, 2,
and 4 min, rinsed with 1· PBS, and then cytotoxicity was
evaluated by MTT assay. Figure 3A shows that 1, 2, and
4 min of blue light exposure causes a reduction of MG63
cell proliferation rates, which is most significant at a 4-min
exposure time ( p < 0.01). On the other hand, ROS levels
rapidly increased after blue light exposure of 1, 2, and 4 min
(Fig. 3B). This suggests that blue light exposure has a
negative effect on cell proliferation rates and on ROS levels.
The effects of NAC and GSH pretreatment on MG63
exposed to VA-086 with blue LED light exposure
MG63 cells were seeded in a 24-well dish and pretreated
with 0–30 mM of NAC (Fig. 4A) and 0–30 mM of GSH
(Fig. 4B) using a volume of 300 mL for 20 min. Three
NAC AND GSH INCREASE MG63 VIABILITY IN THE GelMA SYSTEM 797

FIG. 7. Fluorescent images

of LIVE/DEAD assays of
MG63 cells photo-
encapsulated in GelMA: (A)
nontreated control and MG63
cells pretreated with (B)
10 mM NAC, (C) 10 mM
GSH, (D) 20 mM NAC, and
(E) 20 mM GSH for 20 min.
The columns (from left to
right) are GelMA hydrogels
cultured for 1, 3, and 7 days,
respectively. White scale bar
equals 200 mm. Color images
available online at www
.liebertpub.com/tec

hundred microliters of 1% VA-086 was added to each well,
followed by 1–4 min of blue light exposure. The results
showed no significant difference in the absorbance value of
the MTT assay for the NAC pretreatment (Fig. 4A). In
contrast, cells pretreated with 10–30 mM GSH proliferated
significantly better than the controls after 4 min of blue
light irradiation (Fig. 4B). The 10 and 20 mM GSH pre-
treatment showed the highest MG63 proliferation rates
( p < 0.001). We also confirmed the ROS levels in MG63
cells under the same experimental conditions. The data
showed that 10 mM of NAC pretreatment significantly
decreased ROS levels after 2 and 4 min of blue light ex-
posure (Fig. 5A). In addition, the level of ROS generation
decreased for 10–30 mM GSH pretreatment and 2 and
4 min of blue light irradiation (Fig. 5B). The above results
indicate that both NAC and GSH can reduce ROS levels on
MG63 cells and that GSH is more effective than NAC for
ROS level reduction.
Evaluation of untreated and antioxidant-pretreated
MG63 cells photoencapsulated in hydrogel
Untreated MG63 cells and MG63 cells pretreated with
NAC and GSH were encapsulated, and after 1, 3, and
7 days, tested with MTT (Fig. 6) and Live/Dead assays
(Fig. 7). Cells were found to survive after photoencapsula-
tion, and cells pretreated with NAC and GSH exhibited
higher proliferation rates than controls (Fig. 6). During days
1 and 3, GSH and NAC showed very similar results in the
MTT measurements for both 10 and 20 mM concentrations.
On day 7, however, 20 mM of GSH induced slightly higher
cell proliferation rates than 20 mM of NAC (Fig. 6).
798
The images obtained by the Live/Dead assay show that
most cells encapsulated in GelMA hydrogel survive, as seen
by the green fluorescence throughout the 7-day period under
all conditions (Fig. 7A–E). For the controls, we observed a
higher occurrence of red fluorescent cells, that is, dead cells,
than for any antioxidant-treated MG63 cells on day 1, 3, or 7
(Fig. 7A–E).
Discussion
The compressive strength of our light-cured GelMA is
comparable with that reported in the literature.26 The
compressive strength of GelMA is suitable for applications
such as regeneration of soft tissues, where the material
does not bear higher physiologic loads. One such example
is the regeneration of periodontal tissues in the periodontal
pocket, where osteoblasts containing GelMA solution can
be injected into periodontal pocket, followed by blue light
curing. The GelMA solution can thus be turned into gel
in situ and hold the osteoblasts in the periodontal pocket,
and also act as a barrier that prevents gingiva cells from
ingrowth into the periodontal pocket. For this example, a
low compressive strength of GelMA does not affect its
clinical potential.
Based on the results from the live and dead assay, which
we used to test for the biocompatibility of the GelMA so-
lution, it was found that GelMA did not kill cells (Fig. 2).
However, the morphology of MG63 cells in the control
group was slightly different from the cells incubated with
GelMA, suggesting that our GelMA solution may have re-
sulted in a slight increase of cytotoxicity.
There are many studies on the effects of visible and UV
radiation and the photochemical damage in neural retina and
retinal pigment epithelium (RPE).25,27–29 Some in vivo studies
have shown that the death of blue light-irradiated RPE cells
occurs through apoptotic mechanisms.30,31 Blue light damage
may induce free radical production in some cells, resulting in
phototoxicity. Our MTT assay tested for phototoxicity, and
longer blue light exposure caused damage to MG63 cells
(Fig. 3), with increases in light intensity or exposure duration
both leading to increased cytotoxicity, as expected.
Supplementation of NAC (a precursor of GSH, the body’s
main antioxidant) increases GSH levels and supports the
antioxidant and nitric oxide systems during infection, in-
flammation, stress, and exposure to toxins.32,33 High NAC
concentrations (5 and 10 mM) can reduce cell death and
restore mitochondrial activity after a 24-h cotreatment.34
The duration of the antioxidant pretreatment is critical.
MG63 cells are human osteosarcoma cells, and ROS may be
necessary for tumor cells.35 The antioxidant may therefore
inhibit the generation and accumulation of intracellular ROS
if the treatment is too long. In our experiments, 20 min of
NAC and GSH pretreatment time was a suitable treatment
duration, above which the NAC started to show certain
levels of cytotoxicity (data not shown).
The choice of the photoinitiator concentration is impor-
tant. In all our experiments, we chose 1% as the concen-
tration for VA-086 because other studies with up to 1%
(w/v) of VA-086 reported low cytotoxicity.36 At these
concentrations, sufficient VA-086 is present to enable
photocross-linking, yet without significant cell death.37 To
generate radicals, the photoinitiator requires light irradia-
LIN ET AL.
tion. Therefore, we combined the VA-086 photoinitiator
with the blue light source to evaluate the effects of NAC and
GSH on reducing oxidative stress during the initiation step
of the polymerization. Concentrations of 10 or 20 mM NAC
did not protect MG63 cells against the radicals generated
from VA-086 under blue light irradiation (Fig. 4A). How-
ever, concentrations of 10–30 mM GSH effectively pro-
tected MG63 cells from VA-086 and blue light exposures
up to 4 min. The reduction of ROS is reflected in the in-
creased absorbance value of the MTT assay (Fig. 4B). Be-
cause the VA-086/blue light exposure assay was performed
immediately after the antioxidant treatment, we think that
NAC may not have been converted to replenish the GSH
in time, explaining why NAC did not help to reduce ROS.
This hypothesis was supported by the results from the ROS
tests (Fig. 5).
At the initiation of polymerization, radicals are created
from initiators excited by UV or visible light. If there is no
monomer present in the reaction solution, the radicals re-
combine. The recombination occurs on the microsecond
timescale. However, the radicals generated from VA-086
during light exposure can also damage cells, especially for
exposure durations of 4 min. Therefore, for our cell encap-
sulation experiments, we selected durations of 1 min. With a
monomer present, free radical generation will continue be-
yond the blue light exposure time. This results in an in-
creased free ROS damage to cells. However, with 20 min of
antioxidant pretreatment, the encapsulated MG63 cells can
survive and proliferate significantly better than experimental
controls (Figs. 6 and 7). Among all samples, 20 mM of GSH
showed the highest cell survival rates after 7 days, effec-
tively reducing the free radicals generated during photo-
encapsulation. Additional experiments may be performed to
further optimize the dosage, treatment time, and frequency
of the antioxidant pretreatment for photoencapsulation.
Conclusion
In this study, we encapsulated MG63 cells using a Gel-
MA/VA-086/blue light hydrogel system. It was found that
blue light can replace UV light to initiate photoencapsula-
tion, opening the possibility of enabling cell therapy in
clinical settings using standard, dental, handheld blue light
devices. We further discovered that NAC and GSH pretreat-
ments can effectively reduce the oxidative stress damage
created during photoencapsulation. This novel photoencap-
sulation protocol can be applied to additional cell types to
increase cell survival and proliferation rates.
Acknowledgment
This project was funded by the Ministry of Science and
Technology, Taiwan (Project no. NSC101-2314-B-010-037).
Disclosure Statement
No competing financial interests exist.
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