DR REM BA (Orcid ID : 0000-0001-8411-6470)

Accepted Article
Article type : Original Article

Full Title: EFFECT OF AVULSION STORAGE MEDIA ON PERIODONTAL LIGAMENT

FIBROBLAST DIFFERENTIATION

rem BA 1, Sibel YILDIRIM 2

1
Dumlupnar University, Faculty of Dentistry, Department of Pediatric Dentistry, Ktahya, Turkey

2
Seluk University, Faculty of Dentistry, Department of Pediatric Dentistry, Konya, Turkey

Correspondence to: rem BA, Dumlupnar University, Faculty of Dentistry, Department of Pediatric
Dentistry, Ktahya, Turkey

Tel: 0090 506 437 9883

e-mail: irem.bag@dpu.edu.tr

Key Words: cell differentiation, fibroblast, periodontal ligament, storage media, tooth avulsion

Running Title: STORAGE MEDIAS EFFECT ON CELL DIFFERENTIATION

Conflict of interest: The authors declare that no conflict of interest exists.

Acknowledgements: We are thankful to Hasan Acar, MD PhD for providing us his labs for our
forever-lasting experiments.

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/edt.12356
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 ABSTRACT

BACKGROUND/AIM: An avulsed tooth must be stored in a solution which

maintains periodontal ligament (PDL) cell viability. The aim of this study was to
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compare the effects of Hanks Balanced Salt Solution (HBSS) and milk on the
differentiation of PDL fibroblasts.

MATERIAL AND METHODS: Eighteen extracted third molars, (n = 3 for

each group), were immersed in HBSS, milk, and Dulbeccos Modified Eagle
Medium-Hams F12 (DMEM-F12) at 4 C for 30-60 min or 12 h. The growth
dynamics of PDL fibroblasts were evaluated with cell proliferation graphics and
population doubling time (PDT) values. Runt-related transcription factor 2 (RUNX2),
receptor activator of nuclear factor kappa-B ligand (RANKL) and collagen type XII
(COL12) expression were used to evaluate the differentiation of PDL fibroblasts.

RESULTS: The percentage of cell numbers and PDT values of groups were
statistically insignificant. In the HBSS groups, RUNX2 expression increased showing
a direction to osteogenic differentiation of PDL fibroblasts. In the DMEM-F12 groups
RANKL expression increased, indicating there was a tendency for osteoclastogenic
differentiation. In the milk groups, RUNX2 expression decreased while other markers
were stable showing PDL fibroblasts could protect fibroblast identity.

CONCLUSIONS: In terms of protecting fibroblast identity and resistance to

differentiation, milk was more effective than HBSS.

INTRODUCTION

Tooth avulsion is defined as the complete displacement of a tooth from its alveolar
1
socket and such injuries demand urgent intervention. During avulsion, the neurovascular
supply surrounding the tooth ruptures and separates. Then fibroblasts and other cells attached
to the root surface degenerate.2 Treatment should include methods to maintain the viability of
the periodontal ligament (PDL) cells preferably by immediate replantation.3,4 The most
serious complication after replantation of a tooth is replacement resorption of the root.

PDL cells have heterogeneous cell populations including fibroblasts, cementoblasts,

osteoblasts and undifferentiated mesenchymal cells. PDL fibroblasts represent the main cell
group in the PDL and they are responsible for the synthesis of the extracellular matrix and
PDL regeneration.5 As seen in all regenerative tissues of the body, the PDL has a
heterogeneous cell population that is capable of differentiation into different relevant cell
types, such as osteoblasts, osteoclasts or cementoblasts.6

In order to maintain the PDL integrity and viability, avulsed teeth should be stored
immediately in an appropriate solution until replantation.3,4 There are many studies about
storage media, such as tap water, saliva, and saline. Milk, Hanks balanced salt solution

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 (HBSS), ViaSpanTM (DuPont Pharmaceuticals, Wilmington, DE, USA) and modified
Eagles medium (MEM) that have been suggested as ideal storage media for avulsed teeth.7,8
Although Viaspan and HBSS can maintain cell viability for a long time, they are expensive
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products and not readily available.7,9 On the other hand, milk is usually readily available.
Milk has physiological osmolality (230270 mOsm/kg), neutral pH (6.57.2), low bacterial
content, essential nutrients and growth factors.10-15 In previous tooth replantation studies
using HBSS and milk as storage media, there were different results concerning healing with
normal PDL, surface resorption, replacement and inflammatory resorption.7,16-21 Thus, it is
important to understand how milk and HBSS affect healing differently after an avulsed tooth
is replanted.

In addition to the advantages described above, milk is a rich liquid with 120 mg/100
mL calcium content. If an avulsed tooth is placed in milk, PDL cells come into contact with
calcium ions. Previous studies have shown that an increased calcium level raises the
osteogenic marker RUNX2s expression.22 Koori et al.23 have revealed that increased
extracellular calcium levels trigger the osteogenic differentiation of PDL stem cells.23
Additionally, in bone resorption studies, it has been shown that extracellular calcium ions
were an inhibitory factor for osteoclast activity.24,25 The proposed hypothesis suggests that
calcium-rich milk causes PDL fibroblast differentiation.

The differentiation outcome can be displayed by markers of the investigated new cell
type. Collagen type XII (COL12) is one of the fibroblast markers used for the identification
of PDL fibroblasts-specific cell lines.26,27 Runt-related transcription factor 2 (RUNX2) is a
transcription factor and its expression indicates osteoblastic cell differentiation from
pluripotent mesenchymal cells. PDL fibroblasts are positive for RUNX2.28 On the other hand,
PDL fibroblasts also have osteoclastic differentiation potential. Osteoblast cells originating
from PDL fibroblasts synthesize receptor activator of nuclear factor kappa-B ligand
(RANKL) and it is a quite realiable indicator for osteoclastic differentiation.29,30

The aim of this study was to investigate whether HBSS and milk help to maintain the
fibroblastic nature of PDL cells.

MATERIAL AND METHODS

All procedures were performed with informed consent and were approved by the
Ethics Committee of the Faculty of Dentistry, Selcuk University, Konya, Turkey (2013/06).

Human PDL fibroblasts were harvested from the PDL tissue of 18 freshly extracted
third molar teeth, which had no caries, infection, restoration, periodontal disease or
hypoplasia. Figure 1 summarises the main procedures of the study with.

Third molar teeth were extracted with appropriate indications, cleaned with sterile
saline and stored in either HBSS (LONZA, Walkersville, MD, USA) or whole milk (PINAR
MILK, zmir, Turkey) (3 teeth for each group) for 30- 60 min or for 12 h at 4C. DMEM-
F12 (LONZA, Walkersville, MD, USA) was used as a positive control under the same
conditions.

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 At the end of the two storage periods, the teeth were washed gently with phosphate
buffered saline (PBS) (LONZA, Walkersville, MD, USA). Under a laminar flow cabinet, the
PDL tissue was scraped with a sharp blade from the middle-third of the root surface of each
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tooth. This tissue was placed in sterile eppendorf tubes containing culture medium, which had
1 ml DMEM-F12 with 10% fetal bovine serum (FBS) (LONZA, Walkersville, MD, USA)
and 1% penicillin- streptomycin- amphotericin B solution (LONZA, Walkersville, MD,
USA). After the tissue samples were cut into small pieces with sharp scissors, they were
centrifuged at 500 rpm for 1 min, and the supernatant was drained. For enzymatic digestion,
the pellet of packed cells was dissolved in a 1 ml solution of 2 mg/mL collagenase type I and
2 mg/mL dispase (Roche, Mannheim, Germany) and then they were put in a 37 C water bath
for 3045 min. The cells were centrifuged at 1,000 rpm for 2 min, and the supernatant was
drained. The pellet of packed cells was then re-suspended in a T25 tissue culture flask with
culture medium. The culture flasks were incubated at 37 C in 5% CO2 and 95% air. The
culture medium was replaced every three days until cell growth attained confluence. The
primary culture was designated as passage 1 (P1). The cells were detached with a solution
containing 0.25% trypsin and 1M ethylenediamine tetraacetic acid (EDTA) (Gibco Life
Technologies, Bleiswijk, Netherlands). After detachment, the cells were stained with trypan
blue to assess cell viability and to count the viable cells. The cells were counted under an
inverted microscope using a hemocytometer, and 5103 cells per 1 cm2 were seeded into the
T25 tissue culture flask (P2). Cells of the third and fourth passages were used for the
evaluations.31

Adhesion to plastic surfaces of the cells seeded into culture flasks was evaluated
under an inverted microscope. The clonogenic capacity of cells, which is defined as one
progenitor cells ability to form a cell colony, was determined by observing cell colonies
under an inverted microscope.32

In order to determine the cell proliferation dynamics, viable cells were counted and
proliferation graphs were created. The average time it took for a cell population to double the
number of cells, population doubling time (PDT), was calculated with the formula:

PDT= (T-T0) lg2/ (lgNt-lgN0)

T0 was the starting time, T was the end time of the cell culture, while N0 represented
the number of seeded cells and Nt represented the number of harvested cells.33

Statistical analysis of the data was accomplished using the Kolmogorov-Smirnov test
to determine whether the data showed normal distribution or not. As the data showed normal
distribution, parametric tests were used and independent-samples t-tests were done.

On passage 3, cells (5103) were seeded onto coverslips (1818 mm) on a six-well
plate. When cell growth attained confluence, the culture medium was drained and the cells
were gently rinsed three times with PBS. Then, cells were fixed in 3.5% paraformaldehyde
(Merck, Darmstadt, Germany) for 3035 min at room temperature and rinsed with PBS three
times.32

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 For each of the three antibodies (RUNX2, RANKL, COL 12), three positive and one
negative control coverslips were selected from the coverslips belonging to each tooth. After
paraformaldehyde fixation, the coverslips were incubated with a blocking solution containing
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0.1% Triton-X (Life Technologies, Paisley, Scotland) at 37 C for 30 min.

Immunofluorescence staining for RUNX2 (Cell Signaling Technology, Inc. Beverly,

MA, United States), RANKL (Abcam, Cambridge, United Kingdom) and COL12 (Acris
Antibodies, Herford, Germany) (1: 100 dilution) were applied to each cell group at 37C for
2 h. Samples were rinsed three times with PBS. Primary antibody application was followed
by FITC-conjugated secondary antibodies (1:100 dilution) (Cell Signaling Technology, Inc.
Beverly, MA, United States) for 2 h at 37C. Samples were then rinsed with PBS for 10 min.
In order to stain nuclei, cells were incubated with 7-aminoactinomycin D (7AAD)
(BIOLEGEND San Diego, CA, United States) for 20 min at 37C and rinsed three times with
PBS. Non-specific control staining was evaluated on negative control slides by incubating
cells without primary antibodies.
Samples stained with immunofluorescence antibodies were examined with a laser
confocal scanning microscope (Zeiss LSM510, Jena, Germany). Cell photographs were saved
and used to determine the percentage of cells which expressed the mentioned antibodies. The
scores of three samples obtained from each tooth were averaged to give the percentage
values. Then, the values were recorded graphically.

RESULTS
In P1, starting from the first hours of each culture, the cells obtained from the digested
tissue adhered easily to plastic surfaces. In the passage made on the 1014th days of each
culture (P2), the homogenic growth pattern and adhesion to plastic surfaces were observed.
The ability of the cells to adhere to plastic did not show any differences dependent on storage
media and storage time (30- 60 min or 12 h).

Microscopic images of PDL fibroblasts were obtained from different storage media
(HBSS, milk, DMEM-F12) for two time periods (30- 60 min or 12 h). When the microscopic
images were evaluated with morphological terms, typical fibroblastic, spindle-like cell
morphology were observed during the passages (Fig. 2). For all groups, it was observed that
PDL fibroblasts had the ability to form cell colonies from only one cell indicating these cells
had clonogenic capacities.

Figure 3 shows the average cell number ( SD) of groups for P3- P4 passages. In the
milk and DMEM-F12 groups, cell proliferation was decreased by the increased storage time.
Although, in the HBSS group, cell proliferation was increased, no statistically significant
difference was observed among the groups (p>0,05).

Average PDT values ( SD) of the experimental groups were calculated and there was
no statistically significant differences among the groups (p>0,05) (Fig. 4).

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 Markers RUNX2, RANKL, and COL12 were tested on the groups. For every
micrograph (X40, n=3) chosen from all samples (n=3), certain excitation and emission
wavelength values were used. The proportion of strongly expressing and not expressing cells
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was determined as the percentage value. Then, the average of the percentage value was
calculated.

In the HBSS groups, RUNX2 expression was observed by bright spots in the nucleus
in all groups. At the 12 h periods for all media, RUNX2 expression was higher than in the
3060 min periods. While increased RUNX2 and decreased COL12 expressions were
detected in the HBSS groups, RANKL expression was stable at all time periods. Results from
the milk groups showed stable RANKL and COL12 expression, however decreased
expression of RUNX2 was observed. In the DMEM-F12 groups, RANKL expression
significantly increased as storage time changed from 3060 min to 12 h. COL12 expression
was stable, while RUNX2 expressions decreased in this group (Fig. 5).

When the averages of the percentage values of the proportion of strongly expressing
and not expressing cells from the 3060 min groups were compared to the results from the 12
h groups, RUNX2 in the HBSS groups, RANKL in the DMEM-F12 groups, and COL12 in
the milk groups were found to have increased dramatically (Fig. 6).

DISCUSSION

Replacement root resorption can develop after tooth avulsion and the root surface can
be repaired with cementum after surface and inflammatory resorption. During bone
remodeling, the resorbed area on a root surface will be replaced by bone.34,35 Osteoblasts are
the main cells responsible for this situation, which is defined as replacement resorption. It has
been proven that PDL cells can maintain their viability in HBSS and milk.7,9 However, very
few studies have been done to examine the effects of HBSS and milk on the PDL fibroblast
differentiation mechanism. In order to observe the possible effects of the storage solutions on
the differentiation of PDL fibroblasts, the present study investigated PDL fibroblasts identity
by using the markers of three different lineages (osteoblastic, osteoclastic and fibroblastic).

Adhesion to plastic surfaces and clonogenic capacity give information about the
survival of the cells at the injury site. Clonogenic activity of precursor cells attached to the
root surface could lead to desirable regeneration activity by preventing this area from
attaching osteoclasts.36 In the present study, all the PDL cells obtained from the groups
showed high clonogenic capacity and quick adhesion to plastic surfaces. These results
showed that a high proportion of excess precursor cells from PDL cells of representative
avulsed teeth could be cultured effectively. The PDT values were used to evaluate the
proliferation dynamics of PDL fibroblasts and statistically insignificant differences were
found between the PDT values of the cells from all the groups.

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 In terms of mitogenic and clonogenic capacity, milk is one of the most recommended
solutions to maintain the viability of PDL fibroblasts in in vitro studies.37-39 Milk also appears
to be a reliable viability supportive solution in clinical trials.20,21,40,41 In places where avulsion
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injuries usually occur, such as homes, schools, and in the street, milk is readily available.
Skimmed or low-fat milk has been reported to be more suitable for maintaining PDL
fibroblast viability than whole milk.42 However, Wang et al.10 showed that the effect of
skimmed milk on osteogenic differentiation of PDL stem cells was more significant than that
of HBSS.10 Osteogenic differentiation of PDL stem cells triggered by increased extracellular
calcium level23 and increased calcium levels caused high expression of RUNX2 from PDL
cells.22 Extracellular calcium was found to be an inhibitory factor for osteoclastic activity.
The present study showed that RUNX2 expression from PDL fibroblasts decreased when
whole milk was used as a storage media. Contrary to the proposed hypothesis, the results of
the present study showed that calcium-rich milk did not cause differentiation of PDL
fibroblasts and expression of intense COL12 in the milk group showed maintainance of the
fibroblastic genotype.

Cell culture media such as MEM and DMEM have been used as storage media for
avulsed teeth.17,43-45 DMEM is recommended for the short- and long-term storage of avulsed
teeth because it contains 2-4 times more glucose, 4 times more amino acid and vitamin than
MEM. However, DMEM and Hams F12 (DMEM- F12) can provide cell growth without
stimulating premature aging or differentiation of mesenchymal cells.46 For those reasons,
DMEM-F12 was used as a positive control. According to the results of this study, DMEM-
F12 provided a suitable environment for saving fibroblast identity by consistent COL12
expression. However, it also promoted osteoclastogenic activity observed by increased
RANKL expression.

HBSS has been reported as a suitable storage media for avulsed teeth to maintain PDL
cell viability and the morphology of the cells remains unchanged.47 However, even though
the morphology remains unchanged, some markers expressed by cells reveal the identity of
the cell. It was reported that HBSS is less effective for osteogenic differentiation of PDL stem
cells than skimmed milk.10 However, the current results showed that RUNX2 level in HBSS
groups rose when storage time was increased to 12 hours. Similar to these results, Courts et
al.48 also reported that the differentiation ability of PDL cells incubated in milk was lower
than in HBSS.48 These observations can explain why replacement and inflammatory
resorption were seen after replantation of dogs teeth stored in HBSS.17,49 Meanwhile, it
would be reasonable to conclude that replacement resorption is considered a high risk for
teeth stored in HBSS.

In conclusion, although several studies about HBSS reported successful results in

maintaining cell viability, the present study showed HBSS caused osteogenic differentiation

of PDL fibroblasts. Conversely, milk is advisable as storage media for avulsed teeth because

it protects PDL fibroblasts viability and their identity.

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 FIGURE LEGENDS

Figure 1. An overview of all procedures performed in this study.

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Figure 2. Microscopic images of PDLF saved at P2 and P3 passages for all experimental
groups. Periodontal ligament cells of all groups reflected typical fibroblastic, spindle- like
cell morphology.

Figure 3. Average cell number ( SD) of groups according to the cell counting at P3- P4
passages.

Figure 4. Graph showing average PDT values ( SD) of calculated cells.

Figure 5. Confocal microscope images of all groups.

HBSS: RUNX2 expression with bright spots in the nucleus in the 12 h group was higher than
in the 30- 60 min group (A, B). RANKL expression was not clear (C, D). COL12 expression
was observed intensively in cytoplasms of the PDL fibroblasts of 30- 60 min group (E, F).

MILK: RUNX2 expression was high in the 30-60 min group (A, B). In the 30-60 min and 12
h groups, RANKL expression was positive (C, D). COL 12 was observed clearly in both
groups (E, F).

DMEM- F12: Runx2 expression was higher in the 30- 60 min group than in the 12 h group
(A, B). RANKL expression was observed clearly in the 12 h group (C, D). In the 30-60 min
and 12 h groups, COL12 was clearly positive (E, F).

Figure 6. Changes in expression of IF markers (the average of the percentage value of the
proportion of strongly expressing and not expressing cells) with experimental solutions and
time.

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