UNIVERSIDADE ESTADUAL PAULISTA
“JÚLIO DE MESQUITA FILHO”
Instituto de Ciência e Tecnologia
Campus de São José dos Campos
SYSTEMATIC REVIEW DOI: https://doi.org/10.4322/bds.2024.e4556
1
Braz Dent Sci 2024 Oct/Dec;27 (4): e4556
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
In vitro
evaluation of plant-based storage media compared to Hanks
Balanced Salt Solution for avulsed teeth: a systematic review
Avaliação
in vitro
de meios de armazenamento fitoterápicos comparados à Solução Salina Balanceada de Hanks para dentes
avulsionados: uma revisão sistemática
Ayushma CHAKRAVORTY1 , Vignesh RAVINDRAN1 , Ganesh JEEVANANDAN1
1 - Saveetha University, Saveetha Institute of Medical and Technical Sciences, Saveetha Dental College and Hospitals, Department of
Pediatric and Preventive Dentistry. Chennai, India.
How to cite: Chakravorty A, Ravindran V, Jeevanandan G.
In vitro
evaluation of plant-based storage media compared to Hanks Balanced
Salt Solution for avulsed teeth: a systematic review. Braz Dent Sci. 2024;27(4):e4556. https://doi.org/10.4322/bds.2024.e4556
ABSTRACT
Background: Tooth avulsion, complete loss of tooth from trauma, affects 1-11% of permanent teeth injuries, often
in children aged 7-10. Diagnosis involves examining severed vascular and nerve supply, leading to pulp death and
periodontal ligament (PDL) damage. Successful replantation depends on viable PDL cells. Immediate replantation
is preferred to minimize PDL compromise. Alternatively, the tooth can be taken to a dental professional, requiring
careful storage to maintain PDL cell viability. Natural products are explored for storage due to potential benets.
This study reviews plant effects on avulsed tooth tissue restoration, assessing if plant-based preservation aids PDL
re-establishment post-replantation. Objective: The aim is to systematically review literature on herbal storage
mediums compared to Hanks Balanced Salt Solution (HBSS) for avulsed permanent teeth using in-vitro studies.
Design: Relevant literature from PubMed, Google Scholar, Web of Science, Cochrane, and Scopus was reviewed.
Only laboratory-based studies on PDL cells from adult permanent teeth were included. From 692 initial articles,
19 were selected. RoB 2 was used for quality assessment. Results: Of 692 articles, 19 were selected. Green tea
extract was most recommended, followed by Morinda citrifolia and HBSS. Propolis and coconut water were also
frequently recommended. Recommendations were based on PDL cell viability, availability, cost, and shelf life.
Conclusion: Plant-based preservation has positive effects on PDL cell viability in avulsed teeth. Further research
is needed to validate clinical impacts and explore regenerative treatments.
KEYWORDS
Dental avulsion; HBSS; PDL cell viability; Plant derivatives; Transport media.
RESUMO
Introdução: A avulsão dentária, caracterizada pela perda completa de um dente devido a trauma, representa
de 1 a 11% das lesões dentárias permanentes, principalmente em crianças entre 7 e 10 anos. O rompimento do
suprimento vascular e nervoso leva à necrose pulpar e danos ao ligamento periodontal (LP), cuja viabilidade
é essencial para o sucesso da reimplantação. Embora a reimplantação imediata seja o ideal para minimizar o
comprometimento do LP, caso não seja possível, o dente deve ser transportado a um prossional em condições
de armazenamento que preservem a viabilidade celular do LP. Este estudo revisa os efeitos desses meios na
viabilidade do LP de dentes avulsionados, comparando-os à Solução Salina Balanceada de Hanks (HBSS).
Objetivo: Revisar sistematicamente estudos in vitro que avaliaram meios de armazenamento toterápicos
em comparação à HBSS para dentes permanentes avulsionados. Métodos: Foram analisados artigos das bases
PubMed, Google Scholar, Web of Science, Cochrane e Scopus. Apenas estudos laboratoriais em células do LP
de dentes permanentes adultos foram incluídos. De 692 artigos encontrados, 19 foram selecionados. O risco de
viés foi avaliado pelo RoB 2. Resultados: O extrato de chá verde foi o meio mais indicado, seguido por Morinda
citrifolia, HBSS. Própolis e água de coco também foram frequentemente recomendados. As recomendações
2
Braz Dent Sci 2024 Oct/Dec;27 (4): e4556
Chakravorty A et al. In vitro evaluation of plant-based storage media compared to Hanks Balanced Salt Solution for avulsed teeth: a systematic review
Chakravorty A et al.
In vitro
evaluation of plant-based storage media compared to
Hanks Balanced Salt Solution for avulsed teeth: a systematic
review
INTRODUCTION
Tooth avulsion is the complete loss of a tooth
from its alveolar socket, typically occurring due to
trauma such as a fall, road trafc accident, assault,
sports injuries, or occupational incidents [1].
This type of dental trauma constitutes 1-11%
of all traumatic injuries to permanent teeth,
with maxillary central incisors being the most
commonly affected, especially among children
aged 7-10 years [2]. Children most commonly
suffered trauma between the ages of 8 and 11
years, with boys having a greater incidence [3-5].
Clinical and radiographic examination, revealing
the absence of the tooth from its socket, is used as
diagnostic criteria [6]. When a tooth is avulsed, it
presents with severed vascular and nerve supply,
leading to pulp death, particularly in mature
permanent teeth with a closed apex. Avulsion also
results in the tearing of the periodontal ligament
(PDL), causing damage to PDL cells that play a
crucial role in tooth attachment [7]. It causes
functional, psychological, and aesthetic challenges
[6]. The success of replanting an avulsed tooth
into its socket largely depends on the presence of
viable PDL cells on the root surface [7].
After retrieving an avulsed tooth from the
incident site, two management approaches
become prominent. The preferred method
involves careful handling of the avulsed tooth,
either by the patient or an attendant (in the
case of a minor patient), leading to immediate
replantation into the alveolar socket. This
careful approach aims to minimize further
compromise to the integrity of periodontal
ligament (PDL) cells, facilitating the prompt
initiation of tissue repair. In a more commonly
encountered situation, the avulsed tooth is not
immediately replanted but is instead brought to
a dental professional or medical professional for
subsequent replantation [8,9]. In such cases, it is
crucial to prevent desiccation of the avulsed tooth
and promptly immerse it in an appropriate storage
or transport medium until the optimal moment
for replantation arises [6,8]. The effectiveness of
this interim storage step signicantly inuences
the course of the subsequent replantation
outcome, as a carefully chosen storage medium
ensures the viability of PDL cells on the root
surface of avulsed teeth.
An optimal storage medium should be
able to maintain the viability of periodontal
ligament (PDL) cells, a crucial factor for
broblast repopulation on the root surface to
prevent the adherence of osteoclasts in that
region [10]. The exploration of natural products
as alternative sources of medications has gained
attention in the field of complementary and
alternative medicine, leading to numerous
studies on their use in tissue repair [11]. The
bioactive compounds within medicinal plants
contribute to their pharmacological effects, and
various phytochemicals have been scrutinized
to understand the therapeutic impact of natural
products. In comparison to synthetic products,
natural products may offer greater efcacy in
preserving the viability of PDL cells [12].
Several phytotherapies have been explored
as potential plant-based storage mediums for
avulsed teeth due to their bioactive properties that
promote tissue repair and maintain cell viability.
Natural products like green tea, aloe vera,
propolis, coconut water, honey, neem, turmeric,
Morinda citrifolia (noni), pomegranate, and
ginger exhibit anti-inammatory, antioxidant,
and antimicrobial effects. Compounds such as
catechins, curcumin, avonoids, and phenolic
acids in these plants enhance periodontal
ligament (PDL) cell viability, support broblast
repopulation, and prevent osteoclast activity.
These phytotherapies provide a promising
alternative to synthetic storage mediums,
warranting further investigation for their clinical
applicability in preserving and restoring avulsed
tooth tissues. Given this existing research, there is
a requirement for a systematic review to ascertain
which plants yield the most favorable effects.
The objective of this study was to delineate the
effects of plants on the restoration of tissues
in avulsed teeth and to ascertain whether a
consideraram viabilidade celular, disponibilidade, custo e vida útil. Conclusão: Meios de armazenamento à base
de toterápicos mostraram benefícios na preservação do LP de dentes avulsionados. Mais estudos são necessários
para conrmar os impactos clínicos e explorar tratamentos regenerativos.
PALAVRAS-CHAVE
Avulsão dentária; HBSS; Viabilidade celular; Medicamento toterápico; Meio de transporte.
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Braz Dent Sci 2024 Oct/Dec;27 (4): e4556
Chakravorty A et al. In vitro evaluation of plant-based storage media compared to Hanks Balanced Salt Solution for avulsed teeth: a systematic review
Chakravorty A et al.
In vitro
evaluation of plant-based storage media compared to
Hanks Balanced Salt Solution for avulsed teeth: a systematic
review
plant-based preservation medium enables the
re-establishment of the periodontal ligament
(PDL) following tooth avulsion and replantation.
MATERIALS AND METHODS
Protocol
The Preferred Reporting Items for Systematic
Reviews and Meta-Analysis 2020 Statement
and the Cochrane Handbook were followed
in the preparation of the review’s protocol -
CRD42024520907 (PROSPERO Registration
number). This review’s main question was: What
are the effects of various plants or plant-based
materials on the tissue repair of avulsed teeth
after replantation when compared to using Hanks
Balanced Salt Solution (HBSS)?
Eligibility criteria
A PICOS criterion was developed to help with
the search strategy and ascertain which studies
should be included in the present systematic
review [13]. The following is the PICOS formula:
• Population (P) - Human PDL cells (isolated
from avulsed permanent teeth)
• Intervention (I) - Plant-based/ derived,
herbs or medicinal plant compounds used
as storage mediums
• Comparison (C) - HBSS
• Outcome (O) - PDL cell viability
• Study Design (S): Original research
investigating the effects of different
storage mediums on Human PDL cells in
In-vitro
studies was included; overviews,
narrative reviews, letters to the editor, brief
communications, case reports, and case
series were not.
For this review, only articles that were
published were reviewed. Only articles published
in the English language were included in the
review process.
INFORMATION SOURCES AND SEARCH
STRATEGY
To nd all peer-reviewed studies relevant to
the review’s question, a comprehensive search of
PubMed, Cochrane CENTRAL, Scopus and Web
of Science, was conducted for articles published
between January 2014 and January 2024 (10
years). When developing thorough search
strategies, each database’s unique vocabulary and
syntactical restrictions were considered.
The following MeSH terms were used in search
strategy (((avulsed permanent tooth) OR (dental
avulsion)) OR (dental trauma)) OR (tooth injury)
AND ((((((((((((((((((((herbal derivatives) OR
(coconut water)) OR (pomegranate juice)) OR
(green tea extract)) OR (plants)) OR (herb)) OR
(Plant derivatives)) OR (plants)) OR (Green tea
extract)) OR (Aloe Vera)) OR (transport media))
OR (biological transport)) OR (Nigella sativa))
OR (Cocos nucifera)) OR (castor oil)) OR (Red
mulberry)) OR (Marmosa Rubra)) OR (Salvia
ofcinalis)) OR (Punica granatum)) OR (Dragon’s
blood sap)) OR (soy milk) AND ((Hanks balanced
salt solution) OR (HBSS)) OR (storage medium)
AND (pdl cell viability) OR (periodontal ligament
viability).
When more information on a particular
article was required, attempts were made to
contact the respective authors.
Study screening and selection
One author carried out the search strategy
from the individual databases. The overall
titles obtained were scanned and evaluated
independently by two authors, to select the most
relevant articles. The research work that were
duplicated in multiple databases were eliminated.
In case of any disagreement between the authors
the selection was made after a debate between
the authors. When the information provided in
the title was insufcient, assessment of abstracts
were done. Based on the inclusion and exclusion
criteria the research articles were chosen. When
the information provided by the abstract proved
to be unsatisfactory, the full text was evaluated.
The references of all the selected articles were
checked as they might have been missed out in
the databases due to various reasons. A manual
search was conducted, and the reference lists of all
papers were reviewed to identify any additional
studies that were not found in the computerized
search. A screening of the electronic databases
identied 692 records, of which 24 were excluded
after the removal of duplicates and 633 were
excluded after title screening. 35 articles were
screened, out of which 14 were excluded as
they did not match the inclusion criteria. 19
full-text articles satised the eligibility criteria
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Braz Dent Sci 2024 Oct/Dec;27 (4): e4556
Chakravorty A et al. In vitro evaluation of plant-based storage media compared to Hanks Balanced Salt Solution for avulsed teeth: a systematic review
Chakravorty A et al.
In vitro
evaluation of plant-based storage media compared to
Hanks Balanced Salt Solution for avulsed teeth: a systematic
review
of the targeted research and were covered
in this systematic review. Figure 1 gives the
PRISMA ow diagram. Desired information such
as the design of the study, bibliographic data,
characteristics of the participants, intervention
and outcome were recorded.
Data extraction
Two reviewers (AC) and (VR) carried out
the data extraction. Information like year of
publication, study design, tooth origins, storage
media, viability evaluation, period of storage, and
results were taken from the studies that satised
the requirements for inclusion and eligibility and
entered into a Microsoft Excel (Microsoft Corp.,
Redmond, WA, USA) spreadsheet.
Assessment of risk of bias
The Risk of Bias tool (RoB 2) for randomized
trials provided by the Cochrane Handbook
for Systematic Reviews was used to assess the
quality of the included studies. The included
studies were evaluated using the RevMan 5.4.1
software for the following domains: random
sequence generation and allocation concealment
(selection bias), blinding of participants and
personnel (performance bias), blinding of
outcome assessment (detection bias), absence of
incomplete outcome data assessment (attrition
bias), bereft from baseline imbalance (reporting
bias) and adequate reliability. The risk of bias
evaluation was carried out independently by both
authors, who resolved any disagreement through
discussions.
Figure 1 - Flowchart of the retrieved studies through the selection process.
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Braz Dent Sci 2024 Oct/Dec;27 (4): e4556
Chakravorty A et al. In vitro evaluation of plant-based storage media compared to Hanks Balanced Salt Solution for avulsed teeth: a systematic review
Chakravorty A et al.
In vitro
evaluation of plant-based storage media compared to
Hanks Balanced Salt Solution for avulsed teeth: a systematic
review
RESULTS
A screening of the electronic databases
identied 692 records, of which 24 were excluded
after the removal of duplicates and 633 were
excluded after title screening. 35 articles were
screened, out of which 14 were excluded as
they did not match the inclusion criteria. 19
full-text articles satised the eligibility criteria of
the targeted research and were covered in this
systematic review.
The characteristics of the included studies
were determined. The articles included in
vitro studies into the effects of plant use on
avulsed periodontal ligament tissues in humans.
However, each study presented a different type
of evaluation on the effects in the tissues, with
the methods being described in Table I.
The systematic review encompasses
studies employing various storage mediums
for preserving periodontal ligament (PDL) cells
post-tooth avulsion. Pomegranate juice (PJ) was
investigated in three studies [14,22,30], in which
the study by Sara Hojjati et al. [14] revealed
promising results, with a 7.5% concentration
exhibiting the highest viability of PDL cells. Green
Tea Extract (GTE), was explored in ve studies
[15,17,27-29], demonstrated varied outcomes,
emphasizing the signicant effects of GTE on
cell viability, Adeli et al. [17] showed that GTE
was better than HBSS, Alavi et al. [21] showed
that higher concentrations of GTE and the
combination of GTE with aloe vera (AV) showed
increased cell viability, both Ghamsempour et al.
[15] and Abdelfattah et al. [27] studies showed
no significant difference between HBSS and
GTE, P. Mahendra et al. [28] was the only study
where HBSS showed more cell viability. AV
was studied in six articles [18,21,22,26,28,29]
Fulzele et al. [18] showed that both HBSS Figure 3 - Risk of bias summary represented in traffic light plot.
Figure 2 - Risk of bias reported as percentage across all included studies.
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Chakravorty A et al. In vitro evaluation of plant-based storage media compared to Hanks Balanced Salt Solution for avulsed teeth: a systematic review
Chakravorty A et al.
In vitro
evaluation of plant-based storage media compared to
Hanks Balanced Salt Solution for avulsed teeth: a systematic
review
and AV were effective in maintaining PDL cell
viability, S. Sepolia et al. [26] showed most
viability with AV. Navit et al. [21], Babaji et al.
[22] and P. Mahendra et al. [28] were the studies
where HBSS showed more cell viability. Milk,
including Skimmed Milk (SMilk), Whole Milk
(WMilk), Coconut Milk (CMilk), Probiotic Milk
(PMilk) and Almond Milk (AMilk), featured
prominently in 5 studies [17,20,23,25,32]. Two
studies were plant-based milk extract studies.
The study by D. Saini et al. [20] showed HBBS
was better than CM in maintaining cell viability,
its efficacy in maintaining PDL cell viability
across different temperatures. Combination of
AV and propolis emerged as potential mediums
in various studies [18,20,26,31]. Additionally,
HBSS featured prominently in several studies
[14-21,23,24,26,30], highlighting its prevalence
as a standard medium. Other notable studies
explored DMEM [16,17,27], Save-A-Tooth®
system’s HBSS (SAVE) [19,25] and AMilk [32],
contributing valuable insights into alternative
storage solutions. This categorization provides a
comprehensive overview of the diverse storage
mediums investigated in the included studies.
Among the 19 studies evaluating the effect
of plants on avulsed teeth, 15 showed positive
effects of plant treatment on the cells of the PDL.
In addition, all studies evaluated the primary
cultures of cells. No studies were found that used
cells of other dental tissues, such as cementum,
gingiva or alveolar bone.
About 14 studies evaluated cell viability
using different means of verication. Trypan blue
was used 11 times. The MTT assay was used in
eight studies, the neutral red assay was used in
three studies.
About 4 studies evaluated the temperature
of the storage medium. The temperatures
recorded in these studies were 4°C, 5°C, 20°C,
Table I - Study characteristics of included studies
Sl.
No. Studies Teeth type/
number Storage media Methodology
Cell viability
evaluation
method
Storage time Main results
1. Sara Hojjati et al.
2014 [14]
PDL cells
from freshly
extracted
orthodontic
premolars.
HBSS,
pomegranate
juice (PJ),
tap water.
Isolation of
PDL cells after
placement
in storage
solution.
Neutral Red
assay.
1,3,6 and 24
hours storage
time in all
mediums.
7.5% concentration
of PJ showed most
viability, 1% of PJ was
as effective as HBBS.
2. M. Ghasempour et al.
2015 [15]
44 freshly
extracted teeth.
Green tea
extract (GTE),
HBSS (positive
control), water
(negative
control).
Isolation of
PDL cells after
placement
in storage
solution.
Tryptan blue
exclusion
technique.
Specimens
immersed for
1,3 and 15 hours
at 4°C.
No significant difference
was found between
HBSS and GTE at all
time intervals.
3F. Ozan et al.
2015 [16]
PDL cells
obtained from
extracted
permanent
teeth.
HBSS,
Dulbecco’s
Modified Eagles
Medium (DMEM)
(control),
C.Spinosa,
light milk.
Isolation of PDL
cells prior to
incubation.
Tryptan blue
exclusion
technique.
Monitored every
5 minutes for
26 hours.
DMEM and C.Spinosa
had higher cell value
index than HBSS and
light milk. C.Spinosa
had better results than
DMEM but was not
statistically signifcant
4. Adeli et al.
2016 [17]
PDL cells
from freshly
extracted third
molars.
Dulbecco’s
Modified
Eagles Medium
(DMEM), HBSS,
tap water, whole
milk, hypotonic
sucrose solution,
Green Tea
Extract (GTE),
GTE + sucrose.
Isolation of PDL
cells prior to
incubation.
MTT assay. 1, 2, 4 and 24
hours at 37°C.
GTE was better than
HBSS at 2, 4 and 24
hours, DME at 2 hours,
and milk at 4 hours.
5. Fuzele et al.
2016 [18]
PDL cells
from freshly
extracted third
molars.
Hanks Balanced
Salt Solution
(HBSS), Aleo
Vera gel (AV),
packaged
drinking water.
Isolation of PDL
cells prior to
incubation.
Tryptan blue
dye exclusion
test with
haemocytometer.
15,30,60,90 and
120 minutes.
Both HBSS and AV are
effective in maintaining
PDL cells.
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Braz Dent Sci 2024 Oct/Dec;27 (4): e4556
Chakravorty A et al. In vitro evaluation of plant-based storage media compared to Hanks Balanced Salt Solution for avulsed teeth: a systematic review
Chakravorty A et al.
In vitro
evaluation of plant-based storage media compared to
Hanks Balanced Salt Solution for avulsed teeth: a systematic
review
Sl.
No. Studies Teeth type/
number Storage media Methodology
Cell viability
evaluation
method
Storage time Main results
6. M de Souza et al.
2017 [19]
PDL cells
from freshly
extracted third
molars.
5°C and 20°C,
in skimmed
Milk (SMilk),
whole milk
(WMilk), HBSS,
Save-A-Tooth®
system’s HBSS
(Save), natural
coconut water,
propolis,and egg
white (Egg).
Isolation of PDL
cells prior to
incubation.
MTT assay.
3, 6, 24, 48, 72, 96,
and 120 hours,
14 plates at 5°C
and 7 at 20°C.
At 5°C, SMilk and
WMilk were better than
HBSS in maintaining
cell viability, from 24
hours onward. At 20°C,
HBSS was the best
storage medium at 96
and 120 hours. At both
temperatures, from 6
hours onward, Coconut,
Propolis and Egg were
less effective than
SMilk, WMilk, and HBSS,
the lowest temperature
undermined the
effectiveness of HBSS
from 24 hours and
favored SMilk and
WMilk, from 96 and
48 hours onward,
respectively.
7. D. Saini et al.
2017 [20]
69 freshly
extracted
premolars.
HBSS (control),
coconut milk,
probiotic milk
(PM).
Isolation of
PDL cells after
placement
in storage
solution.
0.5% tryptan
blue staining.
Stored dry-
20 minutes and
then 30 minutes
in storage
media.
PM and HBSS are better
than coconut milk. No
significant difference
between PM and HBSS.
8. Navit et al.
2017 [21]
58 freshly
extracted
premolars.
HBSS, coconut
water, aloe vera
(AV), saline.
Isolation of
PDL cells after
placement
in storage
solution.
Haemocytometer.
Air dried for
30 minutes,
stored in different
mediums for 45
minutes followed
by 30 minutes
with collagenase
and dispase II.
HBSS most effective
then coconut water and
then AV.
9. Babaji et al.
2017 [22]
50
orthodontically
extracted teeth.
HBSS, propolis,
aloe vera (AV),
pomegranate
juice (PJ).
Isolation of
PDL cells after
placement
in storage
solution.
Tryptan blue
test.
Immediately
placed in various
storage medium
for 45 minutes,
controls were
bench dries for
8 hours then
placed in dipase
and collagenase.
Propolis showed more
viable cells than HBSS,
then AV and last PJ.
10. Nabavizadeh et al.
2018 [23]
40 freshly
extracted teeth.
Two control
groups, HBSS,
milk, castor oil.
Isolation of
PDL cells after
placement
in storage
solution.
Tryptan blue
assay.
2 hours dry
time for
control groups,
experimental
groups had
30 minutes of
dry time and
then immersed
in mediums for
30 minutes.
Castor oil showed
significantly lower
viability.
11. Özgür Ïlke et al.
2019 [24]
40 freshly
extracted teeth.
HBSS, virgin
olive oil (VOO),
soyabean (SO),
one positive
control, one
negative control.
Isolation of
PDL cells after
placement
in storage
solution.
0.4% Tryptan
blue assay.
Air dried for
30 minutes and
then soaked in
mediums.
SO and VOO had more
viable cells than HBBS.
No significant difference
between SO and VOO.
12. B.D.M Souza et al.
2019 [25]
Human PDL
cells extracted
from permanent
teeth.
Skimmed and
whole milk,
HBSS, Save-A-
Tooth system’s,
coconut water,
propylene
glycol with 20%
propolis, egg
white, tap water.
Isolation of PDL
cells prior to
incubation.
MTT 24, 48, 72,
96, and
120 hours at
20°C and MEM
24, 48, 72, 96,
and 120 hours
at 37 °C.
Incubated till
120 hours at
different temps
(5 5 °C, milk
(5°C, 20°C
and 37°C) tap
water (negative
control) at 5 °C
and 20 °C, for
24 hours.
5 °C, milk maintained
more viable cells
immediately after
exposure, 20 °C, milk
and HBSS were similar
till 24 and 48h, HBSS
superior at 72 hours.
Table I - Continued...
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Chakravorty A et al. In vitro evaluation of plant-based storage media compared to Hanks Balanced Salt Solution for avulsed teeth: a systematic review
Chakravorty A et al.
In vitro
evaluation of plant-based storage media compared to
Hanks Balanced Salt Solution for avulsed teeth: a systematic
review
Sl.
No. Studies Teeth type/
number Storage media Methodology
Cell viability
evaluation
method
Storage time Main results
13. S. Sepolia et al
2020 [26]
90 freshly
extracted teeth.
HBSS, propolis,
aloe vera (AV),
one control.
Isolation of
PDL cells after
placement
in storage
solution.
0.4% Tryptan
blue assay.
Soaked in
mediums for
30 minutes.
AV showed (82%) most
viability followed by
propolis (68%) and
HBSS (66%).
14. Abelfattah et al.
2022 [27]
30 freshly
extracted
premolars.
HBSS,
Dulbecco’s
Modified Eagles
Medium (DMEM)
(control group),
Green tea
extract (GTE).
Isolation of
PDL cells after
placement
in storage
solution.
MTT assay.
1,3,6,12,24,48,
hours viability
seen.
No significant difference
at 1 and 3 hour between
HBBS and GTE. GTE was
superior in 6, 12, 24 and
48 hour.
15. P. Mahendra et al.
2022 [28]
55 freshly
extracted
permanent
teeth.
HBSS, green tea
extract (GTE),
aloe vera (AV), 2
control groups
(one negative,
one positive).
Isolation of
PDL cells after
placement
in storage
solution.
Tryptan blue dye
test.
Air dried for 30
minutes, then
45 minutes in
storage mediums.
Negative control
bench dried
for 8 hours,
positive group
immediately
treated with
collagenase).
HBSS had most
viable cells out of all
experimental groups.
No significant difference
between AV and
GT. all groups were
significantly higher than
negative control and
significantly lower than
positive control.
16. SH Alavi et al.
2022 [29]
Human PDL
cells extracted
from permanent
teeth.
Aloe vera (AV),
green tea
extract (GTE),
combination
of GTE and
AV,HBSS
(positive
control),
culture medium
(negative
control).
Isolation of PDL
cells prior to
incubation.
MTT Assay. 4 hours of
incubation.
Higher concentrations
of green tea and the
combination of the two
extracts significantly
increased cell viability.
Higher concentrations
of Aloe vera had the
least positive effect on
maintaining the viability
of these cells.
17. Thoyalil et al.
2023 [30]
65 freshly
extracted
premolars.
Placentrex,
propolis 10%,
pomegranate
juice (PJ) 5%,
HBSS.
Isolation of
PDL cells after
placement
in storage
solution.
Tryptan blue test
with haemocy-
tometer.
Placed in storage
medium for
45 minutes each,
then placed in
incubator for
30 minutes.
HBSS has most viability,
Placentrex is a better
alternative to PJ and
propolis.
18. Sagare et al.
2023 [31]
65 freshly
extracted
premolars.
HBSS, Morinda
citrifolia
juice, ocimum
sanctum.
Isolation of
PDL cells after
placement
in storage
solution.
MTT assay,
Trypsin dye
exclusion
technique.
Air dried for
30 minutes,
stored in different
mediums for 45
minutes followed
by 30 minutes
with collagenase
and dispase II.
PDL cells viability
was most in Morinda
citrifolia juice followed
by HBSS followed
by Ocimum sanctum
extract.
19. S.Hussein Adam et al.
2024 [32]
66 wells with
extracted
human PDL cells.
HBSS, almond
milk, control.
Isolation of PDL
cells prior to
incubation.
Annexin V-FITC
fluorescent
cytometry test,
IC50 cytotoxic
assay multiple
range test.
Incubation for
1 hour.
In necrotic, early
apoptosis phase and
late apoptosis phase
HBBS was highly
significant to control
and almond milk,
while in normal intact
cells phase, almond
milk showed highest
significant Q3 apoptotic
cells amongst the other
groups.
Table I - Continued...
37°C and room temperature. The detailed data
are described in Table I.
Another drawback identified in the
assessment of the employed tests is the inadequacy
of data, posing a notable risk of bias. Unlike
the majority of studies that focused solely on
evaluating cell viability, only three studies
conducted multiple tests. Overall, the ndings
lack consistency in suggesting that the studied
9
Braz Dent Sci 2024 Oct/Dec;27 (4): e4556
Chakravorty A et al. In vitro evaluation of plant-based storage media compared to Hanks Balanced Salt Solution for avulsed teeth: a systematic review
Chakravorty A et al.
In vitro
evaluation of plant-based storage media compared to
Hanks Balanced Salt Solution for avulsed teeth: a systematic
review
plants could be recommended for the clinical
management of avulsed teeth.
DISCUSSION
Natural products have proven to be more
effective than synthetic storage mediums for
avulsed teeth, although alternative options exist.
Studies indicate that plants may contribute to the
treatment of periodontal diseases by facilitating
tissue regeneration, osteogenic differentiation,
and mineralisation. However, identifying suitable
plant storage media for tooth preservation
is presently unviable due to the variability
of existing data and several methodological
deciencies. Consequently, the level of evidence
identied in contemporary research is conveyed
through a qualitative analysis, which is the sole
methodology employed.
This analysis revealed multiple plant species
that have been studied for their ability to
maintain PDL cell viability. None of the trials
included, however, offered compelling evidence
that the effect approached the expected degree of
condence. The heterogeneity of data rendered
another systematic review’s assessment of in
vivo experiments insufcient for determining the
optimal storage medium.
It was determined that propolis was the
most extensively studied botanical extract. The
anti-inammatory, anti-cancer, antioxidant, and
regenerative properties of propolis have been
evidenced in numerous studies [29]. The majority
of the studies referenced in this review focused
on Brazilian propolis. Nonetheless, the study
exclusively employed propolis sourced from the
Apis mellifera L. species [30]. This review suggests
that propolis may be an effective component for
maintaining the pulp-density liposome (PDL) of
avulsed teeth due to its impact on cell viability,
anti-inflammatory properties, and osteogenic
differentiation. A recent study examined the
effect of polyphenols on the reduction of bacterial
proliferation utilising Chilean propolis. Because
avulsed teeth are susceptible to contamination
once they are out of the alveolus and exposed
to the environment, it is crucial to evaluate their
antibacterial potential when treating them.
The pharmacological effects of propolis in a
0.4% ethanolic solution may have affected the
outcomes of that investigation. The antimicrobial
properties of the plant media were not, however,
tested in any of the planned research.
The antibacterial actions, inclusion of vital
amino acids, vitamins, and electrolytes of coconut
water prompted its investigation as another plant
medium. Though it is recognised that coconut
water comes from the species Cocos nucifera
L., as cited in some papers of this study, four
articles [22,25,31,32] reported that the coconut
studied was from Thailand. Based on the data
we have, coconut water may have a protective
effect on the survival and health of PDL cells from
avulsed teeth.
The inexpensive and widely-available aloe
vera plant has more than 75 nutrients and a
transparent gel within its leaves. Aloe vera’s
phytotherapeutic characteristics, including its
antibacterial, antioxidant, and anti-inammatory
effects, have been detailed in scientic studies [33].
According to all research, this plant keeps PDL
cells alive, making it an attractive option for tooth
storage. Seven of the research [16,20,22,24] made
use of gel-form Aloe vera. Information regarding
the plant shape was lacking in the other research.
As a potential antibacterial agent, green tea
has been investigated in the eld of dentistry.
In periodontal diseases, the catechins in green
tea extracts have shown that they prevent the
production of osteoclasts and macrophages
by reducing the expression of matrix
metalloproteinase-9 (MMP-9). Avulsed teeth
have also been tested in green tea, which is a
physiological medium and widely available across
the world. When it comes to preserving PDL cells,
green tea outperformed the more typical options
of water and milk in cases of tooth trauma.
Pomegranate, which is abundant in
polyphenolic flavonoids, has been previously
studied for their potential effects on tissues
in the mouth. Besides being an antioxidant,
pomegranate also has anti-inflammatory and
antibacterial properties. Research evaluating
PDL cells following avulsed tooth storage in
pomegranate juice is cited in this review [14-16].
According to two papers that were included,
pomegranate juice could be suggested as a good
medium for avulsed teeth.
Cell proliferation and survival are inuenced
by the physiological osmolality, pH, and
temperature. One recent study recommended
looking at a medium’s pH and osmolality before
doing anything else. Reducing the osmolality of
the medium is crucial for cell survival since it
impacts the water uptake of the cells. Biological
10
Braz Dent Sci 2024 Oct/Dec;27 (4): e4556
Chakravorty A et al. In vitro evaluation of plant-based storage media compared to Hanks Balanced Salt Solution for avulsed teeth: a systematic review
Chakravorty A et al.
In vitro
evaluation of plant-based storage media compared to
Hanks Balanced Salt Solution for avulsed teeth: a systematic
review
processes are affected by changes in pH, which
affect all cellular responses. Three of the
experiments maintained their storage media at
4°C, which is about the same as the temperature
in a standard household fridge. On the other
hand [25], was the only study to compare two
temperatures (5°C and 20°C) in this review, and
it demonstrated that keeping the cells at 20°C
generated better results.
As a last stage, we looked at how different
plants affected the periodontal ligament (PDL)
cell survival in avulsed teeth, and we found
that they helped maintain the cells intact.
There is a shortage of evidence in the existing
literature to determine which plants can be
employed as appropriate storage mediums to
alter the results of replantation. To further
understand how maintaining avulsed teeth
in plant-based medium affects distinct cells
involved in tooth reintegration, larger-scale
clinical trials are urgently needed. Improving
the prognosis for avulsed teeth and finding
practical clinical applications are the goals of
this approach. Additionally, it is highly desirable
to identify plants and their derivatives that are
both economical and conveniently available for
persons who have endured trauma and lost a
tooth. It is crucial to have an easily available
and user-friendly tooth preservation medium
on hand in case of accidents. This will allow the
traumatised person to seek replantation and
tissue repair therapies from a dentist without
delay.
The methodological differences across the
publications were a major shortcoming of this
comprehensive study. One difference was that
different investigations employed different plants
to treat periodontal ligament cells. Different tests
were utilised to examine the same outcome, which
further prohibited direct comparison between the
researches. There was a deciency of information
regarding the age and number of cell culture
passages, even though all the articles focused
on human PDL cells in primary culture. Lack of
validated methodologies for analysing in vitro
study evidence levels and bias issues is another
key constraint. Due to their failure to fully address
the research themes provided in this systematic
review, no publications were assessed to be of high
quality. Additional non-biased research is needed
to test the validity of systematic review instruments
in order to remove these constraints.
CONCLUSION
This systematic review revealed a diversity
of studies exploring the utilization of plants for
preserving avulsed teeth. Despite the varied
approaches among the reviewed studies, the
overall results indicate encouraging impacts of
plants on the viability of periodontal ligament
(PDL) cells. The ndings from this comprehensive
analysis offer insights for prospective research,
proposing the need for additional studies to
validate the clinical effects of these plants on the
PDL. Moreover, the review suggests investigating
regenerative treatments involving cells from other
tissues within the periodontal complex.
Author’s Contributions
AC: Conceptualization, Methodology,
Software, Validation, Formal Analysis,
Investigation, Resources, Data Curation, Writing
– Original Draft Preparation, Writing – Review
& Editing, Visualization, Supervision, Project
Administration and Funding Acquisition. VR:
Conceptualization, Validation, Formal Analysis,
Writing – Review & Editing, Visualization,
Supervision, Project Administration. GJ: Writing
– Review & Editing, Visualization, Supervision.
Conict of Interest
No conicts of interest declared concerning
the publication of this article.
Funding
The authors declare that no nancial support
was received.
Regulatory Statement
PROSPERO number- CRD42024520907.
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In vitro
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Hanks Balanced Salt Solution for avulsed teeth: a systematic
review
Chakravorty A et al. In vitro evaluation of plant-based storage media compared to Hanks Balanced Salt Solution for avulsed teeth: a systematic review
Chakravorty A et al.
In vitro
evaluation of plant-based storage media compared to
Hanks Balanced Salt Solution for avulsed teeth: a systematic
review
Date submitted: 2024 Oct 22
Accept submission: 2024 Dec 19
Vignesh Ravindran
(Corresponding address)
Saveetha University, Saveetha Institute of Medical and Technical Sciences,
Saveetha Dental College and Hospitals, Department of Pediatric and Preventive
Dentistry, Chennai, India.
Email: drvigneshpedo@gmail.com
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