Volume 85 • Number 4

The Effect of a-Tocopherol and Selenium

on Human Gingival Fibroblasts and
Periodontal Ligament Fibroblasts
In Vitro
Nejat Nizam,* Feridun Discioglu,† Isil Saygun,† Vehbi Bal,† Ferit Avcu,‡ Cansel Kose Ozkan,§
and Muhittin Abdulkadir Serdari

Background: The aim of the present study is to evalu-

ate the effect of a-tocopherol and selenium on gingival
fibroblasts (GFs) and periodontal ligament fibroblasts
(PDLFs) in terms of proliferation, basic fibroblast growth
factor (bFGF) release, collagen type I synthesis, and wound
healing.
Methods: Primary cultures of human GFs and PDLFs were

I
t is well known that all mammalian
isolated. Four test groups and a control group free of medi- cells use oxygen to produce energy,
cation was formed. In group E, 60 mM a-tocopherol was and, as metabolic byproducts, re-
used, and in groups ES1, ES2, and ES3, the combination active oxygen species (ROS) are formed
of 60 mM a-tocopherol with 5 · 10-9 M, 10 · 10-9 M, and continuously. ROS are essential for
50 · 10-9 M selenium was used, respectively. Viability, pro- the normal function and metabolism
liferation, bFGF, and collagen type I synthesis from both of the cells,1 but they are also capable
cell types were evaluated at 24, 48, and 72 hours, and heal- of initiating lipid peroxidation and
ing was compared on a new wound-healing model at 12, 24, damaging cell membrane and DNA.2,3
36, 48, and 72 hours. In normal physiology, there is a con-
Results: a-Tocopherol alone significantly increased the tinuous balance between ROS activity
healing rate of PDLFs at 12 hours and increased bFGF and and antioxidant defense capacity;
collagen type I release from GFs and PDLFs at 24, 48, and however, when there is a reduction in
72 hours. The a-tocopherol/selenium combination signifi- the antioxidant defense or increase in
cantly enhanced the proliferation rate of both cells at 48 ROS production or activity, oxidative
hours, decreased the proliferation of PDLFs at 72 hours, stress results, leading to potential
and increased the healing rate of GFs at 12 hours and PDLFs damage.1 Excessive production of ROS
at 12 and 48 hours. bFGF and collagen type I synthesis was has been associated with pathogenesis
also increased in both cell types at 24, 48, and 72 hours by of many inflammatory diseases, in-
a-tocopherol/selenium combination. cluding periodontitis.1,2,4
Conclusion: a-Tocopherol and a-tocopherol/selenium Antioxidants can be classified as en-
combination is able to accelerate the proliferation rate and dogenous antioxidants, which are syn-
wound-healing process and increase the synthesis of bFGF thesized by the body, including catalase,
and collagen type I from both GFs and PDLFs. J Periodontol superoxide dismutase, glutathione per-
2014;85:636-644. oxidase, and exogenous antioxidants,
which are obtained through the diet,
KEY WORDS
such as carotenoids, ascorbic acid, and
Antioxidants; collagen; fibroblast growth factors; tocopherols. a-Tocopherol is one of the
fibroblasts; guided tissue regeneration, periodontal; wound subspecies of vitamin E and regarded as
healing. the most important and effective lipid-
soluble antioxidant and vital for main-
* Department of Periodontology, Faculty of Dentistry, Ege University, _Izmir, Turkey. taining cell membrane integrity against
† Department of Periodontology, Center of Dental Sciences, Gulhane Military Medical
Academy, Ankara, Turkey. lipid peroxidation.5 Besides the antioxi-
‡ Department of Hematology, Gulhane Military Medical Academy. dant capacity, vitamin E was shown to
§ Department of Pharmaceutical Technology, Gulhane Military Medical Academy.
i Department of Biochemistry, Gulhane Military Medical Academy.
doi: 10.1902/jop.2013.130184

636
J Periodontol • April 2014
have anti-inflammatory properties,6 the ability to re-
duce tumor development,7,8 and to increase growth
factor release from fibroblasts.9 It was also demon-
strated that vitamin E accelerated gingival wound
healing10 and had a significant protective effect
against alveolar bone loss in vivo.11 Selenium is the
essential cofactor of glutathione peroxidase, which
detoxifies hydrogen peroxide and other organic per-
oxides. It is effective against oxidative stress, but,
when combined with vitamin E, it has a synergistic
effect.8,12 It was also demonstrated on a rat model
that the combination of vitamin E and selenium was
protective against ROS-induced collagen break-
down.13
Basic fibroblast growth factor (bFGF), an endog-
enous polypeptide growth factor, has numerous bi-
ologic activities, such as cell growth, angiogenesis,
migration, differentiation, morphogenesis, wound
healing, and tissue repair.14,15 Moreover, bFGF
stimulates the proliferation of cells, including fibro-
blasts,16 osteoblasts,17 cementoblasts,18 and endo-
thelial cells,19 which are essential cells for periodontal
wound healing and regeneration. Collagen type I is
one of the major extracellular protein components of
periodontal tissues and is produced by many cells,
including periodontal ligament fibroblasts (PDLFs)
and gingival fibroblasts (GFs). Remodeling of colla-
gen type I in periodontal ligament (PDL) and other
connective tissues is regulated at one level by
bFGF,20 and synthesis of the collagen after injury
seems to be essential for the wound-healing pro-
cess.21
Beyond the antioxidant properties of a-tocoph-
erol and selenium, other effects attributable to
specific interactions of vitamin E with proteins such
as enzymes or transcription factors are possible.22
Vitamin E and the combination of selenium may
affect the function of PDLF and GF cells and may
increase growth factor release, proliferation rate,
and extracellular matrix (ECM) component pro-
duction; hence, periodontal wound healing and
regeneration may be accelerated. Still, little is
known about the mechanism and the biologic ef-
fects of vitamin E and selenium on oral tissues
other than their antioxidative characteristics.
Therefore, the aim of the present study is to
evaluate whether a-tocopherol and the combination
with selenium affect bFGF release and collagen
type I synthesis from PDLFs and GFs and whether
these antioxidants increase the proliferation rate of
the cells and accelerate wound healing in vitro.
MATERIALS AND METHODS
The study was performed from May 9, 2011
through May 3, 2012, and the protocol was ap-
proved by the Internal Review and Ethics Board at
Nizam, Discioglu, Saygun, et al.
the Gulhane Military Medical Academy, Ankara,
Turkey (Protocol no. 175, July 6, 2011). Tissue
samples were taken from systemically healthy male
volunteers who were aged 20 to 31 years (mean
age: 24 years). Each participant signed an informed
consent statement.
Isolation of Human GF and PDLF Cells
Gingival tissue samples were taken from the healthy
gingiva of the patients undergoing second-stage
dental implant surgery, and PDL specimens were
obtained from the middle third of permanent pre-
molar teeth extracted because of orthodontic rea-
sons. After the gingival tissues were excised and the
teeth were extracted, they were placed in Dulbecco
modified Eagle medium (DMEM)¶ supplemented
with 100 U/mL penicillin G# and 100 mg/mL
streptomycin** and were taken to the laboratory
within 10 minutes. The gingival tissues were minced
into 1- to 2-mm pieces, the PDL specimens were
harvested using periodontal curets, and all samples
were then placed into 25-cm2 tissue culture dishes
containing DMEM supplemented with 100 U/mL
penicillin G, 100 mg/mL streptomycin, 1 mmol/L
†† The
L-glutamine, and 10% fetal calf serum (FCS).
cultures were incubated in a humidified atmosphere
at 37C and 5% CO2. After they reached 80% to
90% confluence, the cells were detached using
trypsin–EDTA solution B (0.05% EDTA and 0.25%
trypsin with phenol red)‡‡ at 37C for 10 minutes,
and subcultures were formed in culture plates.
Preparation of Vitamin E and Selenium Solutions
Before the initiation of the study, a pilot study was
conducted, and cytotoxicity/proliferation was tested
using different doses of a-tocopherol (ranging from
20 to 180 mM) and selenium (ranging from 0.01
to 50 · 10-9 M) to find out the optimum dose
range for GFs and PDLFs. The results of the pilot
study indicated that the proliferation rate was at
a higher rate for both cell types when the dose of
a-tocopherol was 60 mM and the dose of selenium
was 5 to 50 · 10-9 M (data not shown). On the
basis of the results of the pilot study, a single dose
of a-tocopherol and three different doses of sele-
nium were used in the present study. Five groups
were formed, including four test groups and one
control group: 1) group E: 60 mM a-tocopherol; 2)
group ES1: 60 mM a-tocopherol plus 5 · 10-9
M selenium; 3) group ES2: 60 mM a-tocopherol
plus 10 · 10-9 M selenium; 4) group ES3: 60 mM
a-tocopherol plus 50 · 10-9 M selenium; and 5)
group C: a control group free of medication.
¶ Invitrogen, Grand Island, NY.
# Invitrogen.
** Invitrogen.
†† Invitrogen.
‡‡ Biological Industries Israel Beit Haemek, Kibbutz Beit Haemek, Israel.
637
Vitamin E and Periodontal Cells Volume 85 • Number 4

seeded wells from that of the

blank wells. The rate of pro-
liferation was then calculated
as the percentage of absor-
bance values compared with
control cells. The experiments
were repeated in triplicate.
Preparation of Wound-
Healing Model
A precise and reproducible
wound-healing model was de-
signed to evaluate the peri-
odontal wound-healing process
in vitro. Silicone inserts,## two
rectangular wells separated by
a 500-mm-wide barrier, were
placed in 35-mm-wide cell
culture dishes with 500-mm
relocation grids.*** Each well
Figure 1.
In vitro wound-healing model. The initial wound was 500 mm wide. A) GFs at baseline, 24 hours, and 36 of the silicone inserts was filled
hours. B) PDLFs at baseline, 24 hours, and 48 hours. C, Control. with 70 mm media containing
104 cells and incubated in
a humidified atmosphere at
Commercially available (+)-a-tocopherol acetate§§ 37C and 5% CO2. When the wells (0.22 cm2 each)
and sodium seleniteii were used for the preparation were totally covered, the silicone inserts were gently
of the solutions. (+)-a-Tocopherol acetate at 40 mL removed, forming a cell-free 500-mm-wide area. The
was diluted in 125 mL ethanol, and 8.65 mg sodium cell culture dishes were gently washed using phos-
selenite was dissolved in 100 mL distillated water. phate-buffered saline and filled with 2 mL fresh
The precalculated amounts of each solution were medium prepared for control and test groups
added to DMEM supplemented with 100 U/mL (groups C, E, ES1, ES2, and ES3). Five wound-
penicillin G, 100 mg/mL streptomycin, 1 mmol/L healing models were used for each group and each
L-glutamine, and 10% FCS until desired concen- cell type; therefore, a total of 50 cell culture dishes
trations were reached for each test group. After and silicone inserts were used.
filtering using 20-mm filters, the medium was kept The photographs of the cell-free area were taken
in a cool and dry place away from light until used in every 12 hours using the relocation grids as the
the study. reference points. The media were changed every 24
Cell Viability and Proliferation hours, and the supernatants were collected and
Proliferation and viability were determined using kept at -80C. The test lasted for 72 hours for each
metabolic XTT (2,3-bis-[2-methoxy-4-nitro-5-sul- group and cell type. Photographs were analyzed
fophenyl]-2H-tetrazolium-5-carboxanilide) assay.¶¶ using an image analysis program,††† and the cell-
XTT is converted by viable cells to formazan, which free areas were calculated using the grids of the cell
is a colored product and is proportional to the culture dishes as the reference distances (each
number of vital cells. GFs and PDLFs were in- square = 500 · 500 mm) (Fig. 1).
cubated with a final concentration of 200 mg/mL Assay for bFGF and Collagen Type I
XTT and 5 mM reducing agent phenazine metho- Supernatants harvested during wound-healing ex-
sulfate for 4 hours at 37C. periments were used for the detection of bFGF and
Six vertical lines of 96-well plates were used. The collagen type I levels. Quantitative measurement of
first line was free of cells and contained 100 mL human bFGF was performed using enzyme-linked
media; other lines included 1 · 104 cells in each immunosorbent assay (ELISA) kits‡‡‡ as described
well and contained 100 mL medium, prepared in
advance for groups C, E, ES1, ES2, and ES3. §§ Sigma-Aldrich, Saint Louis, MO.
Proliferation and viability tests were performed at ii Sigma-Aldrich.
¶¶ Biological Industries Israel Beit-Haemek.
24, 48, and 72 hours. The absorbance was read at ## Ibidi, Martinsried, Germany.
570 nm, and net absorbance values were calculated *** Ibidi.
††† NIH ImageJ for Windows, National Institutes of Health, Bethesda, MD.
by subtracting the average absorbance values of ‡‡‡ RayBiotech, Norcross, GA.

638
J Periodontol • April 2014 Nizam, Discioglu, Saygun, et al.

previously.23 Detection of collagen type I was

achieved using ELISA kits§§§ as described by the
manufacturer.

Statistical Analyses
A statistical software package was used for the
statistical analysis.iii One-sample Kolmogorov-
Smirnov test was used to evaluate the distribution
of the variables. For non-normal distribution
(bFGF, collagen type I levels, and in vitro wound-
healing percentages), Kruskal–Wallis test was used
to compare the difference among groups, and
Mann–Whitney U test was performed to test the
significance of pairwise differences using Bonfer-
roni correction to adjust for multiple comparison.
For normally distributed variables (viability/pro-
liferation), one-way analysis of variance test was
used, and Tukey honestly significant difference test
served as the post hoc test. The differences were
considered to be significant when P <0.05.

RESULTS
The proliferation rate of GF cells was significantly
higher in group ES2 compared with control and test
groups at 48 hours (P <0.05). However, the pro-
liferation rates were similar for all groups at the 24-
and 72-hour evaluations (Fig. 2).
There was no statistical significant difference
among groups in the proliferation rates of PDLFs at
24 hours. The rate was significantly higher in group
ES3 at 48 hours (P <0.05) and significantly lower in
group ES1 at 72 hours compared with the other test
and control groups (P <0.05) (Fig. 2).
The cell-free surface area covered with GF cells
in the wound-healing model was significantly higher
in the test groups compared with the control group
at 12 hours (P <0.05). Similar coverage areas were
calculated for all groups at other observation in-
tervals (Table 1).
The in vitro wound area covered with PDLFs was
significantly higher for all test groups compared
with the control group at 12 hours (P <0.05). It was
also significantly higher in group ES2 compared
with the control, E, ES1, and ES3 groups at 48
hours (P <0.05). Coverage amounts were similar for
all groups at the 24-, 36-, and 72-hour observations
(Table 2).
bFGF released from GF and PDLF cells was
significantly higher in groups E and ES1, re-
spectively, compared with the control group and
other test groups at 24, 48, and 72 hours (P <0.05).
There was no statistically significant difference
between other groups for both GFs and PDLFs
during the entire evaluation period (Fig. 3).
At 24 hours, collagen type I synthesized from
GFs was significantly higher in groups E, ES1, and
Figure 2.
Effect of a-tocopherol and a-tocopherol/selenium combination on
proliferation/viability of GFs (A) and PDLFs (B). *Significant difference
compared with groups C, E, ES1, and ES3. †Significant difference
compared with groups C, E, ES1, and ES2. ‡Significant difference
compared with groups C, E, ES2, and ES3. Bars represent positive
standard deviations.
ES3 compared with the control group, but the
collagen type I level was significantly lower in group
ES2 compared with group E at the same time point
(P <0.05). Collagen type I levels were significantly
higher in all test groups compared with the control
group at the 48- and 72-hour evaluations (P <0.05)
(Fig. 4).
Collagen type I levels of PDLFs were significantly
higher in all the test groups compared with the
control group at all time points (P <0.05). At 48
hours, it was statistically higher in group E com-
pared with groups ES1, ES2, and ES3 (P <0.05).
The collagen type I levels of PDLFs were found to
be significantly higher in groups E and ES1 com-
pared with groups ES2 and ES3 at the 72-hour
evaluation (P <0.05) (Fig. 4).
DISCUSSION
It was reported previously that vitamin E has bi-
ologic effects on human cells in addition to the
§§§ Cusabio Biotech, Wuhan, China.
iii SPSS v.15.0, IBM, Chicago, IL.

639
Vitamin E and Periodontal Cells Volume 85 • Number 4

Table 1.
The Percentage of Cell-Free Area Occupied by GFs in the In Vitro Wound-Healing Model

Group 12 Hours 24 Hours 36 Hours 48 Hours 72 Hours

C 3.4 (2.0 to 4.6)* 58.9 (58.9 to 59.1) 88.1 (87.4 to 91.7) 100 (100 to 100) 100 (100 to 100)
E 12.7 (12.5 to 14.9) 79.0 (50.1 to 92.5) 90.1 (70.1 to 100) 100 (100 to 100) 100 (100 to 100)
ES1 11.0 (7.8 to 19.0) 65.5 (53.1 to 71.0) 94.3 (84.9 to 96.5) 100 (100 to 100) 100 (100 to 100)

ES2 17.2 (16.4 to 19.2) 64.2 (57.2 to 75.4) 85.2 (83.9 to 100) 100 (100 to 100) 100 (100 to 100)
ES3 11.3 (10.1 to 12.5) 49.4 (45.2 to 55.8) 78.4 (70.3 to 83.9) 100 (100 to 100) 100 (100 to 100)
Values are shown as median (minimum to maximum).
* Significant difference compared with groups E, ES1, ES2, and ES3.

Table 2.
The Percentage of Cell-Free Area Occupied by PDLFs in the In Vitro Wound-Healing Model

Group 12 Hours 24 Hours 36 Hours 48 Hours 72 Hours

C 2.0 (1.8 to 2.9)* 15.5 (7 to 15.5) 43.1 (21.4 to 49.8) 67.5 (49.0 to 72.0) 100 (100 to 100)
E 5.3 (5.2 to 7.4) 21.9 (21.8 to 38.8) 42.5 (39.1 to 63.4) 74.7 (71 to 94.2) 100 (100 to 100)
ES1 6.8 (5.2 to 8.4) 20.2 (17.4 to 23.1) 43.5 (41.0 to 45.9) 64.8 (50.4 to 79.1) 91.3 (82.7 to 100)

ES2 10.3 (10.2 to 14.0) 18.1 (18.0 to 18.1) 50.5 (45.8 to 57) 96.9 (77.0 to 100)† 100 (100 to 100)
ES3 4.1 (4.0 to 8.8) 17.6 (12.4 to 22.0) 44.6 (25.2 to 51.1) 71 (59.9 to 77.2) 99.3 (97.0 to 100)
Values are shown as median (minimum to maximum).
* Significant difference compared with groups E, ES1, ES2, and ES3.
† Significant difference compared with groups C, E, ES1, and ES3.

antioxidative characteristics.9 Although it was dem-
onstrated that vitamin E accelerated gingival wound
healing10 and was protective against alveolar bone
loss in vivo,11 little is known about its effect on
periodontal tissues. To the best of the authors’
knowledge, this is the first study to evaluate the
biologic effects of vitamin E on GFs and PDLFs in
terms of proliferation, wound healing, bFGF, and
collagen type I synthesis. Because the combination
of vitamin E with selenium has a synergistic ef-
fect,8,12 the effect of the combination is also tested
in the present study.
It was demonstrated previously that a-tocopherol
enhances the proliferation of cultured endothelial
cells,24 inhibits the proliferation of human aorta
smooth muscle and mouse fibroblasts, but does not
affect the proliferation of human osteosarcoma and
mouse monocyte macrophages.25,26 The results of
the present study show that a-tocopherol does not
affect the proliferation rate of GFs and PDLFs.
These findings together with previously published
data24-26 may suggest that the effect of a-tocoph-
erol on proliferation could be related to the type of
the cell.
Although a-tocopherol alone did not make any
difference in the proliferation of GFs and PDLFs, the
combination with selenium at the dose of 10 · 10-9
M (group ES2) and 50 · 10-9 M (group ES3),
respectively, significantly increased the proliferation
of the cells at 48 hours compared with the other
groups. These findings may indicate that addition of
selenium to a-tocopherol could increase the pro-
liferation of both cell types in a time-dependent
manner. The proliferative effect of the selenium/
vitamin E combination could also be attributed to
their synergistic effect because no proliferative ef-
fect of selenium was observed in the pilot study
(data not shown). An interesting finding of the
present study is that there is no difference in the
proliferation rates among the groups at 72 hours
for GFs, and the rate was lowest in the ES1 group
for PDLFs. A limitation of the present study could
be the intervals between the evaluation time points
(24 hours) because the proliferation rates in the
present study may not represent the data between
these time points. PDLFs in group ES1 might have
covered the wells before the evaluation time points,
and contact inhibition could have occurred that can

640
J Periodontol • April 2014
Figure 3.
Effect of a-tocopherol and a-tocopherol/selenium combination on the
release of bFGF from GFs (A) and PDLFs (B). *Significant difference
compared with groups C, ES1, ES2, and ES3. †Significant difference
compared with groups C, E, ES2, and ES3.
explain the lower proliferation rate of the cells at 72
hours.
In vitro periodontal wound-healing models are
often used to evaluate the effect of an agent on
a target cell.27-29 For this purpose, a scratch is
generally made on a cell monolayer, creating a cell-
free area that then will be covered with surrounding
cells. The form of the wound in scratch assays is
mainly dependent on the operator, and scratching
may cause damage to surrounding cells, which is
why it is not precise and reproducible. The in vitro
wound model of the present study also contains
a grid to mark specific areas of the wound, which
can serve as the reference point for digital evalu-
ation. This new periodontal healing model seems to
be precise and reproducible, which could eliminate
the disadvantages of the previous models27-29 and
could be used for in vitro periodontal healing
evaluation.
In the present study, GF and PDLF cells occupied
a significantly higher amount of the wound area at
12 hours compared with control group, but the only
Nizam, Discioglu, Saygun, et al.
Figure 4.
Effect of a-tocopherol and a-tocopherol/selenium combination on the
release of colI (collagen type I) from GFs (A) and PDLFs (B). *Significant
difference compared with groups E, ES1, ES2, and ES3. †Significant
difference compared with groups E, ES1, and ES3. ‡Significant difference
compared with group ES2. § Significant difference compared with group
ES1, ES2, and ES3. kSignificant difference compared with groups ES2 and
ES3.
significant difference among the test groups was
evident for PDLF cells in the ES2 group at 48 hours.
These findings may suggest that a-tocopherol could
affect periodontal healing at an early stage by
stimulating both GFs and PDLFs, and the selenium/
a-tocopherol combination is able to show a time-
dependent effect on PDLFs but at a specific dose for
selenium. These findings support the results of Kim
and Shklar,10 who demonstrated enhanced gingival
wound healing after vitamin E supplementation
in vivo. It is hard to clarify whether the result is
attributable to the synergistic effect between a-to-
copherol and selenium or the effect of selenium
alone, because there was no selenium-alone group
in the present study.
In cell culture studies, ECM components and
growth factors are measured in both the superna-
tants and the extracts of the cells.23,30,31 The main
641
Vitamin E and Periodontal Cells
advantage of using supernatants is the chance to
keep the cells vital and continue the testing process
with the same cells as in the present study. How-
ever, it was demonstrated previously that the col-
lagen type I amount is found at a lower level in
supernatants compared with the extracts and does
not necessarily represent the amount of total col-
lagen type I in the culture.30 Therefore, the amount
of growth factors and ECM components may differ
when the cells are included in the analyses. Be-
cause in the present study the aim is to evaluate the
collagen type I and bFGF synthesis simultaneously
during the in vitro wound-healing process, collagen
type I and bFGF levels were measured using the
supernatants only.
Significantly higher levels of bFGF in groups E
and ES1 for GFs and PDLFs, respectively, may
indicate that a-tocopherol is able to stimulate GF
cells and enhance the release of the growth factor,
whereas PDLFs need the addition of selenium to
show similar effects. It is known that bFGF stimu-
lates the proliferation of fibroblasts,16 osteoblasts,17
and cementoblasts18 and enhances wound healing
and tissue repair.14,15 Therefore, increased levels of
bFGF attributable to the stimulation of GFs could be
a mechanism beyond enhanced gingival wound
healing10 and prevention of alveolar bone loss11 as
shown previously.
Collagen type I is the major extracellular protein
component of periodontal tissues, and production
of the collagen after injury is important for the
periodontal wound-healing process.21 Collagen type
I levels of the present study show that a-tocopherol
alone or the combination with selenium may en-
hance the production of the collagen from both GFs
and PDLFs. These findings together with those of
Asman et al.,13 who demonstrated that vitamin E
and selenium were protective against ROS-induced
collagen breakdown, suggest that vitamin E and
selenium may be effective on collagen metabolism.
Increased synthesis of collagen type I from GFs and
PDLFs may be related to the positive effect of vi-
tamin E on gingival wound healing as demonstrated
previously by Kim and Shklar.10
In the present study, it is shown that a-tocopherol
alone and/or the combination with selenium may
affect the proliferation, growth factor release, col-
lagen synthesis, and in vitro wound healing of GFs
and PDLFs. It is important to note that the increase
in cell proliferation was observed only at 48 hours
for GFs and PDLFs in groups ES2 and ES3, re-
spectively. However, an increase in collagen type I
synthesis was evident in all evaluation periods in
groups E and ES1 for GFs and PDLFs, respectively.
Although the proliferation rate was lowest for PDLFs
in group ES1 at 72 hours, the collagen type I level
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Volume 85 • Number 4
was highest. Conversely, the amount of growth
factor released was not parallel to the proliferation
rates of the GF and PDLF cells. These findings can
demonstrate that the increased amount of collagen
type I and bFGF in the present study cannot be
solely related to the increased number of the cells.
Because the mechanism beyond these effects still
remains obscure, detection of signaling response or
oxidative stress markers, e.g., nitrotyrosine and
nitric oxide synthase, could be of value for future
studies.
It was demonstrated in the present study that GF
cells expresses higher levels of bFGF without se-
lenium and PDLF cells with selenium (lower con-
centration). Also, the proliferation rate was higher in
the moderate-dose selenium group at 48 hours for
GFs, but it was higher in the high-dose selenium
group for PDLFs. This difference between the fi-
broblast types was not surprising. Although both
are fibroblastic in nature, there are some charac-
teristic differences between the two cell types. In
vitro characteristics of GFs and PDLFs were com-
pared previously by Somerman et al.32 and dem-
onstrated that protein and collagen production was
significantly greater in PDL cells when compared
with that of GFs. In addition, PDL cells had higher
alkaline phosphatase (ALP) levels when compared
with those of GFs. Although both cell types are vital
for periodontal healing and regeneration, they differ
in terms of protein pattern, ALP levels, and collagen
production. It was also shown that GFs and PDLFs
have different responses to high-mobility group box
1 (HMGB1) in terms of proliferation. GFs showed
a proliferative response to HMGB1, whereas PDLFs
did not.33 Distinct responses of GFs and PDLFs to
a-tocopherol and a-tocopherol/selenium combina-
tion in terms of bFGF, collagen type I levels, pro-
liferation/viability, and wound healing are also
observed in the present study probably because of
their characteristic differences as shown pre-
viously.32,33
CONCLUSIONS
Within the limits of the present study, it may be
concluded that a-tocopherol is able to enhance
collagen type I production from GFs and accelerate
in vitro wound healing of PDLFs. a-Tocopherol is
also capable of enhancing bFGF release from both
GFs and PDLFs. Addition of selenium to a-to-
copherol may increase the proliferation rates and
in vitro wound healing of GFs and PDLFs. a-To-
copherol/selenium combination may also enhance
bFGF release from both GFs and PDLFs and in-
crease collagen type I synthesis from PDLFs. Be-
cause a certain dose of selenium seems to be
effective for both cell types, selenium alone needs
J Periodontol • April 2014
to be tested to clarify whether the increased effect
of a-tocopherol/selenium combination is related to
their synergistic effect or whether it is the effect of
selenium alone.
ACKNOWLEDGMENTS
The authors are grateful to M. Pınar Elcxi and Meral
Sarper, Department of Cancer Research, Stem Cell
Laboratory, Gulhane Military Medical Academy, An-
kara, Turkey, for their kind assistance during the
laboratory process. The authors report no conflicts
of interest related to this study.
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Correspondence: Dr. Nejat Nizam, Ege University, Faculty
of Dentistry, Department of Periodontology, 35100 _Izmir,
Turkey. E-mail: nejat.nizam@ege.edu.tr.
Submitted March 17, 2013; accepted for publication May
21, 2013.