71 Journal of Conservative Dentistry | Jan-Feb 2013 | Vol 16 | Issue 1

Address for correspondence:

Dr. Tulsi Sanghavi, K.M. Shah Dental College and Hospital,
Sumandeep Vidhyapeeth, Waghodiya, Piparia, Gujarat, India.
E-mail: tulsi.h.sanghavi@gmail.com
Date of submission : 11.06.2012
Review completed : 19.06.2012
Date of acceptance : 21.09.2012
Original Article
Evaluation and comparison of efficacy of three
different storage media, coconut water, propolis, and
oral rehydration solution, in maintaining the viability
of periodontal ligament cells
Tulsi Sanghavi, Nimisha Shah, Vaishali Parekh, Kiran Singbal
Department of Conservative Dentistry, K.M. Shah Dental College and Hospital, Sumandeep Vidhyapeeth, Waghodiya, Piparia,
Gujarat, India
A b s t r a c t
Background: Two of the most critical factors affecting the prognosis of an avulsed tooth after replantation are extra oral dry time
and the storage medium in which the tooth is placed before treatment is rendered. However, the ability of a storage/transport
medium to support cell viability can be more important than the extra oral time to prevent ankylosis and replacement resorption.
Aim: Purpose of this study was evaluation and comparison of efficacy of a new storage medium, oral rehydration solution (ORS)
with coconut water, and propolis in maintaining the viability of periodontal ligament (PDL) cells by using a collagenase-dispase
assay.
Materials and Methods: 40 teeth were selected with intact crown which were advised for Orthodontic extraction having healthy
PDL. Teeth were then randomly divided into three experimental storage solution groups. Other 10 were divided into positive
and negative control groups (5 each).
Statistical Analysis and Result: The results were statistically analyzed with analysis of variance and multiple range by using
post hoc tests. The results of the prevailing study indicated that coconut water group demonstrated a significantly higher
number of viable PDL cells than propolis 50%, and ORS. There was no significant difference between coconut water and
propolis 50% groups.
Keywords: Coconut water, oral rehydration solution, propolis
INTRODUCTION
Dento-alveolar trauma is a very common occurrence out of
all injuries and frequently associated with avulsion injury.
Avulsion injury is associated with compromised esthetic.
[1]

The reported incidence of tooth avulsions ranges from 1%
to 16% of all traumatic injuries occurring in permanent
dentition.
[2]
Avulsion injury, one of the most severe forms of dental
trauma, is characterized by complete displacement of the
tooth from its alveolar socket. Because of the complexity
of avulsion, neurovascular supply is severely compromised
and usually results in loss of pulp vitality. A vital periodontal
membrane (PDM) has been found to be of ultimate
importance for the successful healing of replanted teeth.
Two of the most critical factors affecting the prognosis
of an avulsed tooth after replantation are extra oral
dry time and the storage medium in which the tooth
is placed. However, the ability of a storage/transport
medium to support cell viability is more important than
the extra oral time to prevent ankylosis and replacement
resorption.
[3]
Different storage mediums recommended are saliva, saline,
milk, viaspan, HBSS, gatorade, propolis, coconut water.
[4,5]
Propolis, a substance obtained from the honeybee extract,
is a potent antimicrobial, antioxidant, anti-inflammatory,
antibacterial, antifungal, antiviral, and tissue regenerative
agent.
[6]
In general, propolis is composed of 50% resin and
vegetable balsam, 30% wax, 10% essential and aromatic
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DOI:
10.4103/0972-0707.105303
Sanghavi, et al.: Evaluation and comparison of effcacy of three different storage media
Journal of Conservative Dentistry | Jan-Feb 2013 | Vol 16 | Issue 1 72
oils, 5% pollen, and 5% various other substances including
organic debris. Martin, Pileggi and zan et al.
[7]
found
propolis to be a superior transport medium to HBSS or
milk in terms of maintaining periodontal ligament (PDL)
cell viability after avulsion.
The biologically pure, tender coconut water is readily
accepted by the body because it is sterile and thus, used as
a blood plasma substitute. Gopikrishna et al.
[8]
found that
coconut water was superior to HBSS or milk in terms of
maintaining PDL cell viability after avulsion and storage.
Another storage media oral rehydration solution (ORS)
which contains sodium 75 mmol [1725 mg], glucose
75 mmol [13.5 gm, 1.35%], potassium 20 mmol [782 mg],
chloride 65 mmol [2801 mg], and base (citrate) 10 mmol has
also been tested as its content having similarity with HBSS
and Gatorade. It is a hypotonic solution with osmolarity
between 270 and 300 mOsm/L.
[9]
Hence, the purpose of this study was evaluation and
comparison of efficacy of three different storage
mediumORS, coconut water and propolis in maintaining
the viability of PDL cells by using a collagenase-dispase assay.
MATERIALS AND METHODS
40 teeth were selected with intact crown and close apices
which were advised for orthodontic extraction having
healthy PDL.
After a traumatic extractions, the teeth were held with
forceps from the coronal region, and the coronal 3 mm
of PDL was scraped with a curette to remove cells that
might have been damaged.
The teeth were then randomly divided into three
experimental storage solution groups,
Group 1 - Tender coconut water
}
10 samples per group Group 2 - Propolis 50%
Group 3 - Flavored ORS.
Positive and negative control groups consisted of five
samples each.
Tender coconut water (Group 1):
Freshly open tender coconut water was used for each
sample.
Preparation of propolis 50% (Group 2):
Propolis was made into 50% concentration using 0.4%
ethanol solution.
Propolis 50% was prepared by adding 50 mg ground
propolis per 250 ml of the 0.4% ethanol solution.
Before submersion of teeth in propolis, solutions were
shaken for 15 minutes.
Preparation of ORS (Group 3):
1 teaspoon of ORS powder was taken in 200 ml (one
glass) of distal water and stirred.
New experimental solution was made every time.
Teeth in experimental groups were dried for 30 minutes
(including time taken for curetting coronal PDL cells),
followed by a 45-minute immersion in one of the three
storage solution groups.
The teeth in positive control group after extraction
was immediately treated with dispase and
collagenase.
The teeth in negative control group were bench-dried
for 8 hours, with no follow- up storage solution time,
and then placed in the dispase and collagenase.
After drying and soaking of each experimental teeth,
2.5 ml of stock solution containing grade II dispase
and collegenase were added to teeth and incubated
for 30 min at 37C. After incubation, 50 L of fetal
bovine serum was added to each tube with help of
micropipette.
All tubes were then centrifuged for 4 minutes at
1000 rpm and supernatant was removed with sterile
micropipettes.
The cells were labeled with 0.4% trypan blue for
determination of viability. The number of viable
PDL cells was counted under light microscope with
hemocytometer at 40 magnification.
Statistical analysis
Results were statistically analyzed using analysis of
variance and post hoc tests [Table 1].
The level of significance was 5% (P < 0.05).
Statistical analysis showed a significant difference
among the groups. Tukey test showed significantly
higher number of viable PDL cells in coconut water
group than propolis 50% and ORS.
There was no significant difference between coconut
water and propolis 50% groups. However, both
coconut water and propolis 50% groups demonstrated
a significantly higher number of viable PDL cells than
ORS [Table 2].
All experimental solution groups were significantly
lower than positive control and higher than negative
control group.
DISCUSSION
Avulsion injuries are the worst of dentoalveolar injuries.
Usually, avulsion involves single tooth, and the most
frequently avulsed tooth is maxillary central incisor.
Table 1: Standard deviation of test groups
Number Mean/cumm SD SE
Coconut water 10 28.7000 4.11096 1.30000
Propolis 10 26.3000 3.12872 0.98939
ORS 10 13.7000 3.46570 1.09595
Total 30 22.9000 7.53543 1.37578
SD: Standard deviation, SE: Standard Error
Sanghavi, et al.: Evaluation and comparison of effcacy of three different storage media
73 Journal of Conservative Dentistry | Jan-Feb 2013 | Vol 16 | Issue 1
The healing pattern of an avulsed tooth after
replantation will depend upon the healing potential
of each cellular component of the tissues involved.
Prognosis of a replanted tooth will depend on minimal
damage to the PDL, which is critical for regeneration of
the attachment apparatus and also to protect root from
resorption.
[10,11]
Extra oral time and storage conditions are the most
crucial factors in determining the viability of the
remaining PDL cells, and thus the survivability of the avulsed
tooth. Studies have shown that an avulsed tooth can
be replanted without complications after 1-3 hours of
being placed in suitable storage conditions.
[5]
An ideal storage medium would be one that is capable of
preserving the viability, mitogenicity, and clonogenic capacity
of the damaged PDL in order to facilitate repopulation
of the denuded root surface thereby preventing further
root resorption.
[12]
The storage medium should have a
physiological osmolarity and pH and should be maintained
at an appropriate temperature to allow optimal cell growth
or survival. Finally, the ideal storage media should be readily
available for use in emergency situations.
[5,13]
This study focused on evaluation and comparison of efficacy
of three different storage media-ORS, coconut water and
propolis in maintaining the viability of PDL cells by using a
collagenase-dispase assay.
The biologically pure, tender coconut water is readily
accepted by the body because it is sterile and thus used as
a blood plasma substitute. It also helps to replace fluids,
electrolytes (potassium, calcium, and magnesium), and
sugar lost from the body during heavy physical exercise.
Gopikrishna et al.
[8]
found that coconut water was superior
to HBSS or milk in terms of maintaining PDL cell viability
after avulsion and storage. It has high potassium content
and contains antioxidants linked to a variety of health
benefits. Cytokinins in coconut water may be among its
most beneficial components.
[8,14]
Propolis is a multifunctional material used by bees in the
construction and maintenance of their hives. Recently, it
has attracted much attention as a useful substance applied
in medicine and cosmetics because of its antibacterial and
antifungal activities. In general, propolis is composed of
50% resin and vegetable balsam, 30% wax, 10% essential
and aromatic oils, 5% pollen, and 5% various other
substances.
[7]
Also Ozan et al.
[5]
evaluated the effect of propolis on survival
of PDL and found propolis to be a superior transport
medium to HBSS or milk in terms of maintaining PDL cell
viability after avulsion and storage.
ORS is an easily available rehydration solution. It
contains sodium 75 mmol [1725 mg], glucose 75 mmol
[13.5 gm, 1.35%], potassium 20 mmol [782 mg], chloride
65 mmol [2801 mg], and base (citrate) 10 mmol. It is
a hypotonic solution with osmolarity between 270 and
300 mOsm/L.
[9]
Various techniques have been used to quantitate the
number of viable PDL cells like a stepwise by Reinholdt
et al., Chromogenic stain by Soder et al.
[14]
In the current study to minimize the exposure of cells to
active trypsin and to preserve maximum cell viability, the
root surface was treated with collagenase and dispase
grade II as was performed in the work by Pileggi et al.
[15]
This procedure allowed rapid cell retrieval and maintained
maximum cellular integrity, as was demonstrated by the
positive control samples. This method is more representative
of the actual clinical situation because the cells are not
subjected to long processing times to determine their
viability status. Collagenase and dispase assay provides
a combination of collagenolytic and proteolytic enzymes
required for tissue disaggregation.
[16,17]
Also, we used fetal bovine serum as growth supplement for
cell culture media because of its high content of embryonic
growth promoting factors. When used at appropriate
concentrations, it supplies many defined and undefined
components that have been shown to satisfy specific
metabolic requirements for the culture of cells in vitro.
[18]
The trypan blue exclusion staining technique was used
because it is quick, easily performed, and distinctively
differentiates nonviable cells from viable cells. It is based on
the principle that live cells possess intact cell membranes
that exclude certain dyes.
[19]
In this test, a cell suspension is simply mixed with dye and
then visually examined to determine whether cells take up
or exclude dye. In the protocol presented here, a viable cell
will have a clear cytoplasm whereas a nonviable cell will
have a blue cytoplasm.
[20,21]
Table 2: Post hoc test
Multiple comparisons
Tukey HSD Signi.
Coconut water
Propolis 0.309
ORS 0.000
Propolis
Coconut water 0.309
ORS 0.000
ORS
Coconut water 0.000
Propolis 0.000
*The mean difference is significant at the 0.05 level (P<0.05)
Sanghavi, et al.: Evaluation and comparison of effcacy of three different storage media
Journal of Conservative Dentistry | Jan-Feb 2013 | Vol 16 | Issue 1 74
The results of the prevailing study indicated that coconut
water group demonstrated a significantly higher number of
viable PDL cells than propolis 50%, and ORS. There was no
significant difference between coconut water and propolis
50% groups.
Ideal requirements for a storage media as suggested by
Blomof
[5]
are 290-330 mOsm/L and a pH of 6.6 to 7.8. Even
though the osmolarity of ORS is comparable the pH is
significantly less than the ideal which may be the reason
for the comparatively lesser number of viable PDL cells in
this study.
It remains to be explored, whether the modification of ORS
by addition of common day to day product could increase
its pH to the required level to optimize its efficacy as
storage media.
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How to cite this article: Sanghavi T, Shah N, Parekh V, Singbal
K. Evaluation and comparison of effcacy of three different storage
media, coconut water, propolis, and oral rehydration solution, in
maintaining the viability of periodontal ligament cells. J Conserv
Dent 2013;16:71-4.
Source of Support: Nil, Confict of Interest: None declared.
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