Accepted: 21 December 2017

DOI: 10.1111/edt.12382

COMPREHENSIVE REVIEW

Which is the most recommended medium for the storage and

transport of avulsed teeth? A systematic review

Samira Adnan1 | Maham M. Lone1 | Farhan R. Khan2  | Syeda M. Hussain2 | Sana E.

Nagi2

1
Operative Dentistry, Jinnah Sindh Medical
University, Karachi, Pakistan
2
Operative Dentistry, Aga Khan University
Hospital, Karachi, Pakistan
Correspondence
Farhan Raza Khan, Operative Dentistry, Aga
Khan University Hospital, Karachi, Pakistan.
Email: farhan.raza@aku.edu
Abstract
Background/Aims: A wide variety of materials has been researched for their use as
potential storage media for avulsed teeth, but it is essential to recognize the medium
most recommended for improvement of the prognosis of avulsed teeth. The aim of
this systematic review was to identify the most recommended medium to store and
transport avulsed teeth based on the survival of periodontal ligament (PDL) cells as
determined by in vitro studies.
Methods: Only laboratory-­based experimental studies on PDL cells found on adult
permanent teeth were included. Data were collected using PubMed, CINAHL plus
(EBSCO host), and the Cochrane Library, along with Google Scholar and a hand search.
The key terms employed were permutations of [avulsed permanent teeth* OR dental
avulsion* OR knocked out teeth*] AND [storage media* OR transport media* OR bio-
logical transport* OR PDL cell viability* OR PDL cell survival*]. A customized data ex-
traction pro forma was used to extract the data and to evaluate the quality and risk of
bias.
Results: The initial search yielded 978 articles, but only 67 were selected. Milk was the
most recommended individual medium followed by Hank’s balanced salt solution.
Among natural products other than milk, propolis and coconut water were most fre-
quently recommended. Recommendations were based on maintenance of PDL cell
viability followed by ease of availability, low cost, and long shelf life.
Conclusions: Natural products are more effective in maintaining the PDL cell viability
compared to synthetic products. Some storage media recommendations were also
based upon practical aspects. Although natural products other than milk have more
recommendations as a group, milk is the most recommended storage medium indi-
vidually, based not only on PDL cell viability, but also practical considerations.

KEYWORDS
cell viability, periodontal ligaments, storage media, tooth avulsion, transport media

1 |  INTRODUCTION
Tooth avulsion is defined as the complete loss of a tooth from the
alveolar socket as a result of trauma,1 which can be caused by a fall,
road traffic accident, assault, sports, or occupational injury.2,3 This
form of dental trauma comprises 1-­11% of all traumatic injuries to
the permanent dentition.4 Maxillary central incisors are the most fre-
quently involved teeth with children aged 7-­10 years commonly af-
fected. Avulsion can be diagnosed clinically and radiographically by
the complete absence of the tooth from its socket. An avulsed tooth

Dental Traumatology. 2018;34:59–70. wileyonlinelibrary.com/journal/edt   © 2018 John Wiley & Sons A/S. |  59
Published by John Wiley & Sons Ltd
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60       ADNAN et al.

experiences severed vascular and nerve supply resulting in pulp death,
4–6
especially in a mature permanent tooth with a closed apex. Avulsion
also causes tearing of the periodontal ligament (PDL) as the tooth is
completely displaced from the socket, resulting in damage to the PDL
cells. These cells form part of the attachment apparatus of the tooth,
and hence, subsequent successful replantation of the avulsed tooth
into its socket is greatly dependent on the presence of viable PDL cells
on the root surface.7
If an avulsed tooth is retrieved from the site of the accident, there
are two possibilities in terms of management. The ideal situation in-
volves meticulous handling of the avulsed tooth by the patient or an
attendant (in case of a minor patient) and immediate replantation into
the socket. This approach prevents any further damage to the PDL
cells, and tissue repair is initiated immediately. The second scenario,
much more frequently encountered, is the tooth not being immedi-
ately replanted, but rather brought to the dentist for replantation.8 In
the latter course of management, the avulsed tooth must not be al-
lowed to dry and should be placed in a storage or transport medium as
soon as possible, until it can be replanted.9 This step is crucial in deter-
mining the subsequent outcome of replantation. An appropriate stor-
age medium ensures the viability of the PDL cells present on the root
surface of the avulsed teeth. Over the years, the composition, formu-
lation, temperature, pH, and other aspects of many potential storage
media have been the focus of research.10 A wide variety of materials
has been researched for their role as potential transport media11 in the
quest for finding the ideal medium which could maintain the viability
of the PDL cells attached to the root surface of the avulsed tooth.
Without a suitable storage medium, the PDL cells are likely to expe-
rience further damage, resulting in poor prognosis for the replanted
5
tooth. Therefore, it is essential that the choice of storage medium for
avulsed teeth is based on scientific observations.
The aim of the review was to identify the most recommended
storage medium that can be utilized for storage and transport of
avulsed teeth, based on PDL cell viability as determined by in vitro
studies.
2 | MATERIALS AND METHODS
The PICO question was as follows: What is the most recommended
medium to store and transport avulsed teeth?
The following PICO model for systematic reviews was applied:
Population: Human PDL cells (isolated from avulsed permanent teeth).
Intervention: Placement of PDL cells of an adult avulsed tooth in the
storage medium.
Comparison: Placement in no storage medium, a different storage me-
dium, or the same storage medium with variability in composition,
concentration or temperature, serving as its own comparator.
Outcome: Survival of PDL cells in terms of viability, increase in num-
bers, no change in morphology, or any other positive outcome as
determined by the laboratory parameters.
The protocol for the present review was registered with Prospero
(an international prospective register for systematic reviews with reg-
istration ID: CRD42013003558). The literature search was conducted
using three major health sciences databases, PubMed (NLM), CINAHL
Plus (EBSCO host), and the Cochrane Library including publications from
1970 till March 2017. The following key terms and their different permu-
tations were used as follows: [avulsed permanent teeth* OR dental avul-
sion*OR knocked out teeth*] AND storage media* OR transport media*
OR biological transport* OR PDL cell viability* OR PDL cell survival*. An

PubMed 905
Cochrane 22
CINAHL Plus 51
TOTAL 978

Duplications 56
Animal-based studies 164
Reviews/guidelines/surveys 174
Techniques/prevention 248
Other forms of dental trauma
Case reports 54
Studies assessing clinical
Survival of teeth 14

Final selection for review 67

F I G U R E   1   Study flow diagram
ADNAN et al. |
      61

T A B L E   1   Criteria for inclusion,

Laboratory-­based experimental
exclusion, intervention recorded, and
Inclusion criteria studies on human adult PDL cells
features of PDL cells assessed
Exclusion criteria Studies on primary teeth or fractured
teeth
Animal-­based studies
Publications in any other language
besides English
Case reports, literature reviews
Studies assessing clinical survival of
teeth
Details of intervention Number of storage media included in
selected study
Type of storage media
Time for which PDL cells placed in
storage medium
Temperature at which PDL cells
stored in storage medium
Method of assessment
Recommended medium and reason
for recommendation
Features of PDL cells assessed Viability (maintained, enhanced)
Mitotic ability
Expression of growth factors

additional search using Google Scholar was performed along with a hand
search. Gray literature was not explored. The flowchart depicting the
study selection methodology is shown in Figure 1. The selection of the
studies was completed according to the inclusion and exclusion criteria
as stated in Table 1.
The studies that fulfilled the basic inclusion criteria were assessed
independently by four reviewers for their suitability. A customized
data extraction form was developed to collect data from the included
studies. Any titles that were duplicated or not pertaining to the re-
search questions were excluded at this point, with reasons for exclu-
sion identified on the form. The study was included if it met the initial
criteria based on three key points; (i) Use of PDL cells, (ii) Use of some
form of transport/storage medium, and (iii) The description of the out-
come in terms of PDL cell survival. If the study failed to meet any one
of the criteria, it was excluded from the review.
To assess the quality of the included studies, each item in the
data extraction form was assigned a specific value, according to the
amount of information that could be obtained from the individual
study. These scores were then added, and the quality of the paper
was graded as fair, good, or excellent according to the maximum score
obtained. Extraction of data from the selected studies and their quality
assessment was performed by four independent reviewers (SA, MML,
SMH, and SEN). Each reviewer was also required to assess the data
extraction form completed by other reviewers. Any discrepancy that
arose during the data extraction or when assessing the quality of the
research paper was resolved through discussion with a senior col-
league (FRK) and mutual consensus developed. In the same manner,
any risk of bias in the included study was also determined. The details
of the studies included in the present systematic review are presented
in Table 2.
Data analysis was performed to report the storage medium recom-
mended by the individual studies in terms of survival of the PDL cells
(as determined under laboratory conditions using various methods of
assessment). The reviewers were not blinded regarding the authors,
journals, date of publication, or results.
3 | RESULTS
A total of 978 references were initially identified from the computer-
ized search, but 843 were excluded after screening the titles. A fur-
ther 68 studies which included case reports and assessment of clinical
survival of teeth were excluded after further appraisal. Sixty-­seven
studies that assessed the PDL cells survival were finally included and
analyzed. The full text was retrieved for 63 studies, while only ab-
stracts were available for the remaining four studies.
For the purpose of data extraction and ease of analysis, the nu-
merous storage media identified in the studies were divided into broad
groups according to their formulation and composition. These groups
included the following: Saliva and its variants, Milk and derivatives,
Hank’s Balanced Salt solution (HBSS) and its variants, Saline and its
variants, other specialized media and Natural products other than milk.
The list of these media along with the number of studies recommend-
ing each is shown in Table 3.
The results suggest that from 1976 till 2000, most studies recom-
mend “milk or its derivatives” as the most suitable storage medium for
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62       ADNAN et al.

T A B L E   2   Included studies, transport media assessed, method of PDL viability assessment, and the recommended medium

Author(s), ref no. Media assessed Method of assessment Recommended media

67
Kristerson et al Tissue culture media, liquid Not mentioned Tissue culture media, liquid nitrogen
nitrogen
Blomlof et al44 Milk, saliva, EMEM Trypan blue staining/exclusion Whole milk
39
Blomlof et al Saliva, milk, tap water, 3-­H urine leakage test Milk, saline
saline
Patil et al40 Milk, saline Number counted by hemocytometer Milk, saline
Alacam et al58 HBSS, custodial solution, LDH measurements HBSS, custodial solution
sterile saline
Olson et al41 Milk, Gatorade, Save-­a-­ MTS assay Milk, Save-­A-­Tooth with PGDF
Tooth, Save-­a-­Tooth +
PDGF, EMEM
Harkacz et al42 Tap water, saliva, skimmed Bio-­Rad model 2550 EIA plate reader Skim milk (Oaks farm’s brand)
milk (Oak farm’s brand),
2% milk, whole milk, 0.5%
milk, 1% milk, gatorade
Lekic et al78 Saliva, milk, HBSS, α-­MEM Clonogenic capacity Saliva
Ashkenazi et al43 Culture media, α-­MEM, Trypan blue staining/exclusion HBSS, milk
milk, HBSS, Viaspan,
conditioned medium
Marino et al45 Regular pasteurized milk, Cell proliferation assay Long shelf life milk
long shelf life milk,
Save-­A-­Tooth, MEM, Tap
water
Ashkenazi et al59 HBSS, culture medium, Trypan blue staining/exclusion HBSS, culture media
α-­MEM, Viaspan
Ashkenazi et al60 Viaspan, HBSS, α-­MEM, Trypan blue staining/exclusion HBSS, alpha MEM
α-­MEM with 15% fetal
calf serum, antibiotics
(penicillin 100 unit/mL,
Gentamicin 50 μg/mL,
Fungizone 0.3 μg/mL)
Pearson et al46 Whole milk, reconstituted Cell proliferation assay Enfamil (baby formula milk)
powdered milk, evapo-
rated milk, Similac, Enfamil
Vajrabhaya et al68 HBSS, conditioned medium Staining with neutral red dye, observed Culture media
with spectrophotometer
Sigalas et al61 Culture medium, Milk, Trypan blue staining/exclusion HBSS, culture media
HBSS, Soft Wear contact
lens solution, Opti Free
contact lens solution, Solo
Care contact lens solution,
Gatorade, Tap water
Martin et al12 HBSS, milk, saline, propolis Number counted by hemocytometer Propolis 20%
50%, propolis 100%
Al-­Nazhan et al69 Bausch and Lomb (Renu) Morphology using SEM Renu, Optifree
contact lens solution, Ciba
Vision (Titmus) contact
lens solution, Alcon
(Opti-­free) contact lens
solution, EMEM
Ozan et al13 Propolis 10%, Propolis Trypan blue staining/exclusion Propolis 10%
20%, Long-­shelf life light
milk with lower fat
content, HBSS, DMEM

(Continues)
ADNAN et al. |
      63

T A B L E   2   (Continued)

Author(s), ref no. Media assessed Method of assessment Recommended media

14
Ozan et al Saliva officinalis (4%, 2.5%, Trypan blue staining/exclusion Saliva officinalis
1.5%, 0.5%) solution,
HBSS, PBS, Tap water
Gopikrishna et al15 Coconut water, HBSS, Milk Trypan blue staining/exclusion Coconut water (fresh)
Gopikrishna et al16 Coconut milk, propolis, Number counted by hemocytometer Coconut milk (fresh)
HBSS, Milk
Ozan et al57 PBS containing 4.0% Trypan blue staining/exclusion HBSS
M. rubra juice, PBS
containing 2.5% M. rubra
juice, PBS containing 1.5%
M. rubra juice, PBS
containing 0.5% M. rubra
juice, HBSS, PBS, tap
water
Cehreli et al70 Casein phosphopeptide- Iodide orange staining Casein phosphopeptide-­amorphous
­amorphous calcium calcium 10−12 dilution
phosphate paste at 10−3,
10−4, 10−6, 10−8, 10−12
dilutions
Moreira-­Neto et al47 Still mineral water, coconut Neutral red assay Whole milk
water, saline, coconut
water with sodium
bicarbonate, milk
Caglar et al48 HBSS, Saline, probiotic Trypan blue staining/exclusion HBSS, probiotic solution, milk
solution (Lactobacillus
reuteri solution), Milk
Souza et al49 Sterile HBSS, non-­sterile Trypan blue staining/exclusion Skim milk (Oaks farm brands)
HBSS, Save-­a-­Tooth,
skimmed milk, MEM,
water
de Souza et al65 Recently prepared HBSS, MTT assay HBSS (recently prepared)
HBSS stored for
6 months, HBSS stored
for 12 months, Save-­a-­
Tooth’s system HBSS,
MEM, Water
Souza et al50 Skimmed milk, whole milk, MTT assay Skimmed milk, whole milk
HBSS, natural coconut
water, industrialized
coconut water Save-­a-­
Tooth’s system HBSS, tap
water, MEM
Mousavi et al74 Standard ORS, HBSS, tap Trypan blue staining/exclusion Oral rehydration solution (ORS)
water
Subramaniam et al75 Oral rehydration salt-­liquid Number counted by hemocytometer Oral rehydration salt liquid (100%)
(ORS-­L)
Rajendran et al62 HBSS, milk, Ricetral Trypan blue staining/exclusion Ricetral, HBSS
17
Hwang et al HBSS, tap water, milk, Number counted by hemocytometer Green tea extract
green tea extract,
commercial green tea
Gjertsen et al33 Propolis 25%, Propolis Apoptosis assay Propolis 50%
50%, Propolis 100%, 0.4%
ethanol/HBSS, cell culture
medium (DMEM/10%
PBS)

(Continues)
 |
64       ADNAN et al.

T A B L E   2   (Continued)

Author(s), ref no. Media assessed Method of assessment Recommended media

36
Silva et al Long-­shelf life coconut MTS assay Soya milk, HBSS, milk
water, long-­shelf life
whole milk, long-­shelf life
soymilk, Gatorade, egg
white, HBSS, DMEM,
distilled water
Moazami et al37 Soymilk, HBSS, powdered Trypan blue staining/exclusion HBSS, soya milk, powdered baby
baby formula milk, formula milk
DMEM, tap water
Chen et al71 Epigallocatechin-­3-­gallate, Trypan blue staining/exclusion Epigallocatechin-­3-­gallate
HBSS, milk
Mahal et al34 HBSS, egg albumen, Number counted by hemocytometer HBSS, egg albumin, Propolis
Propolis
Silva et al38 Soy milk, coconut water, Multiparametric assay Soya milk, HBSS, milk
HBSS, whole milk
Sanghavi et al18 ORS, coconut water, Trypan blue staining/exclusion Coconut water (fresh)
Propolis 50%
Eskandarian et al76 ORS 25%, ORS 50%, ORS MTT assay ORS 50%
100%, DMEM
Badakhsh et al19 Aloe vera (10%, 30%, 50%, MTT assay Egg white
100%-­diluted with
DMEM), egg white,
DMEM
Tavassoli-­Hojjati et al20 Pomegranate juice (1%, Neutral red assay Pomegranate juice 7.5%
2.5%, 5%, 7.5%), 1%
HBSS, tap water
Hiremath et al72 Platelet-­rich fibrin(PRF) Number counted by hemocytometer PRF with PRP
with platelet poor plasma
(PRP), platelet poor
plasma
Ebenezar et al63 De-­ionized water, Number counted by hemocytometer HBSS with l-­DOPA
de-­ionized water with
l-­DOPA, HBSS, HBSS

with l-­DOPA
Sharma et al21 Milk, aloe vera, egg white Trypan blue staining/exclusion Aloe vera
Bharath et al35 HBSS, ringer lactate Use of optical microscope and optical Ringer lactate, tender coconut water,
solution, tender coconut calorimeter green tea extract
water, green tea extract
Sricholpech et al22 HBSS, Royal jelly, UHT low MTT assay Royal jelly
fat milk
Subramaniam et al64 Oral hydration salt-­liquid Trypan blue staining/exclusion ORS-­liquid, HBSS
(ORS-­L), HBSS, milk
Choi et al51 Milk Microassay data analysis, reactive oxygen Whole milk
species, Western blot analysis
Ghasempour et al23 Green tea extract, HBSS, Trypan blue staining/exclusion Green tea extract
water
Caglar et al24 Probiotic yogurt, HBSS, Trypan blue staining/exclusion Probiotic solution
saline, milk
Jabarifar et al77 HBSS, tap water, ORS 25%, MTT assay, apoptosis assay ORS (100%)
ORS 50%, ORS 100%

(Continues)
ADNAN et al. |
      65

T A B L E   2   (Continued)

Author(s), ref no. Media assessed Method of assessment Recommended media

25
Martins et al 10% Dragon’s blood sap, Trypan blue exclusion test and MTT assay Dragon’s blood sap
phosphate buffered saline,
DMEM, whole milk,
Water
Souza et al26 Skim milk, natural coconut MTT assay Natural coconut water
water, Whole milk, freshly
prepared HBSS,
Save-­a-­Tooth, industrial-
ized coconut water, tap
water, MEM
Ulusoy et al52 UHT long-­shelf life MTT assay Goat milk
lactose-­free cow milk,
UHT long-­shelf life whole
cow milk, UHT long-­shelf
life skimmed cow milk,
UHT long-­shelf life soy
milk, UHT long-­shelf life
goat milk, UHT long-­shelf
life follow on milk with
probiotic, 20% Propolis,
egg white, tap water,
EMM
Prueksakorn et al27 HBSS, UHT processed milk, Trypan blue staining/exclusion 2.5 mg/mL Thai propolis extract
Thai propolis extract
(0.25 mg/mL, 0.5 mg/mL,
1 mg/mL, 2.5 mg/mL,
5 mg/mL, 10 mg/mL), PBS
Saini et al53 Coconut milk, probiotic Trypan blue staining/exclusion Probiotic milk
milk, HBSS
Saluja et al54 Low-­fat (3%) milk, medium Trypan blue staining/exclusion Light milk with low-­fat content
fat (3.5%) milk, high-­fat
(4%) milk
Sharma et al28 Regular pasteurized milk, Trypan blue staining/exclusion Rice water
rice water, egg white
Anegundi et al29 Cranberry juice, pomegran- Neutral red assay Cranberry juice
ate juice, plum, guava,
green tea extract
Fulzele et al66 HBSS, aloe vera gel, Trypan blue staining/exclusion HBSS
packaged drinking water
Hedge et al55 0.9% Saline, chilled Trypan blue staining/exclusion Chilled milk
homogenous milk, GC
tooth mousse plus
Lee et al73 Save-­a-­Tooth, EMT Quantifying ATP with luminescent dye EMT toothsaver
toothsaver (Dentosafe),
HBSS, water
Adeli et al30 HBSS, pasteurized long-­life MTT assay Green tea extract
whole milk, hypotonic
sucrose solution, green
tea extract, green tea
extract with sucrose,
DMEM, distilled water
Reis et al31 Soya milk, whole milk, XTT assay, neutral red uptake test, Soya milk
HBSS, DMEM Sulforhodamine test (SRB)
Babaji et al32 HBSS, Propolis, aloe vera, Trypan blue staining/exclusion Propolis (50% in 0.4% ethanol)
pomegranate juice

(Continues)
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66       ADNAN et al.

T A B L E   2   (Continued)

Author(s), ref no. Media assessed Method of assessment Recommended media

56
de Souza et al Skimmed milk, whole milk, MTT assay Skimmed milk
recently prepared HBSS,
Save-­a-­Tooth system’s
HBSS, natural coconut
water, Propolis, egg white

EMEM, Eagle’s minimum essential medium; HBSS, Hank’s Balanced Salt Solution; LDH, lactate dehydrogenase; PDGF, platelet derived growth factor; α-­
MEM, alpha minimum essential medium; DMEM, Dulbecco’s modified Eagle’s medium; PBS, physiologically buffered saline; ORS, oral rehydration solution;
UHT, ultra heat treatment; EMM, Eagle’s maintenance medium; MTT, 3-­[4,5-­dimethylthiazol-­2-­yl]-­2,5 diphenyltetrazolium bromide assay.

avulsed teeth, whereas from 2001 onwards, more recommendations Specialized media were also recommended in some studies,67–73
are for “natural products other than milk.” There is a plethora of mate- while saline and its variants were specifically recommended in four
rials included in these two broad varieties of media. The different milk studies.74–77 There was only one recommendation for saliva.78 The
products that have been mentioned in the included studies are whole results are summarized in Table 3. Overall, around one-­third of the
milk, skimmed milk, milk with varying fat content, baby formula, long studies recommended multiple storage media rather than a single one.
shelf life milk, goat milk, probiotic milk, and chilled milk. The natural The most common reason for recommendation was based on
products other than milk mentioned are saliva, soya milk, saliva offici- the ability of the medium to maintain PDL cell viability, while a small
nalis, coconut water, probiotic (lactobacillus reuteri) solution, propolis, number of recommendations were based on ease of availability, low
egg white, green tea extract, aloe vera, royal jelly, pomegranate juice, cost, and long shelf life of the storage medium. Only a few media
dragon’s blood sap, rice water, and cranberry juice. were recommended on the basis of actually enhancing the viability of
Overall, 22 studies exclusively recommended natural products as PDL cells.23,25,26,33,70 The methods of assessing PDL cell viability were
12–33
storage media, while two studies recommended them in addition to also evaluated, and Trypan blue staining was found to be the most
HBSS34 and Ringer’s lactate.35 A further three studies also recommend frequently utilized laboratory technique to determine viable PDL cells
36–38
milk and HBSS along with different natural products. Twenty-­ (Figure 2).
one studies recommended milk in some form in addition to the other In terms of the quality of included studies, 65 of the 67 studies
media tested,36–56 while 13 of these studies exclusively recommended were found to be in the “excellent” category, as these studies encom-
milk.39,42,45–47,49–56 HBSS was found to be the second most recom- passed all the relevant information pertaining to the study question.
34,36–38,43,48,57–66
mended individual medium in 16 studies. Of these, The risk of bias in studies was also assessed and is depicted in Figure 3.
four studies57,63,65,66 exclusively recommended HBSS, while 12 stud- In regard to randomization, there was no mention of random sequence
ies recommended HBSS along with other media.34,36–38,43,48,58–62,64 generation in any study; while 33% of the studies mentioned random
allocation of samples.12,15,16,18,19,21,23,24,27,28,32,34,53,54,58,62–64,71,72,74,75
Where only abstracts could be retrieved, the risk of selective report-
T A B L E   3   Recommendations for storage media groups as reported ing was unclear.39,40,43,44,67 However, no such bias could be detected
in the included studies in the full-­text articles. As the included studies were laboratory experi-
Storage medium and their derivatives Number of studies ments, all of the parameters for risk assessments of clinical trials could
not be applied to them.
Saliva and derivates 1
Milk and derivates 13
HBBS and variants 4
4 | DISCUSSION
Saline and variants 4
Specialized culture media 7
Although there are numerous studies that have reviewed and com-
Natural products other than milk 22 pared media for the storage and transfer of avulsed teeth,10,11,79 no
HBBS and milk derivatives 3 systematic review determining the most recommended storage and
Saline and milk derivative 2 transport media for avulsed teeth has been reported in the literature
HBSS and specialized culture media 5 previously. The present systematic review compared the various stor-
Milk and HBSS and natural products other 3 age media in terms of their ability to maintain PDL cell viability, and
than milk in some cases, to even enhance their growth. To determine the actual
HBSS and natural products other than milk 1 physical state of the PDL cells after they had been placed in different
Ringer’s lactate and natural products other 1 storage media, only laboratory-­based studies were selected. These
than milk studies had a relatively more stringent protocol, and their outcome
HBSS and saline variants 1 measurements were relatively straightforward to determine, in com-

HBSS, Hank’s balanced salt solution. parison with clinical studies, in which evaluation of the survival of PDL
ADNAN et al. |
      67

Method used to assess PDL survival fibroblast, with replenishment of the lost metabolites. This ability has
been documented to be sustained for up to 48 hours. It has been rec-
Trypan blue staining ommended that the avulsed tooth be placed in HBSS for 30 minutes
20 before replantation into the socket, regardless of which storage me-
MTT assay
27
dium the tooth was placed in prior to this procedure.83,84
Among the natural products other than milk, six studies recom-
Number counted by
Heamcytometer mended propolis,12,13,27,32–34 and five studies proposed coconut

Neutral red assay
3
Other methods
8
9 than milk had the largest number of studies recommending them
F I G U R E   2   Laboratory methods used to assess the periodontal
ligament cell survival (n = 67)
cells is based on a long follow-­up period and variable interpretation of
clinical and radiographic findings in patients.
The present systematic review demonstrated that the most rec-
ommended transport medium for avulsed teeth is milk. Milk has a
unique combination of nutrients, capable of maintaining PDL cell vi-
ability, and with its physiological pH of 6.5-­7.2, it can be considered
as the best storage medium in most situations.80 The PDL cells have
81
been shown to survive for 2-­6 hours when immersed in milk. Chilled
milk or milk with a lower fat content is considered a better choice
for storing avulsed teeth, but there are some impediments in using
milk as a storage medium. The antigens present in milk could inter-
fere with the process of reattachment of PDL cells when the tooth is
7 20
replanted. Moreover, the milk needs to be fresh and refrigerated.
Sour milk should not be used as it harmful to the PDL cells.82 Recent
studies26,28,30,32 have favored other natural media (other than milk) for
storage of avulsed teeth, but the low cost, presence of nutrients, and
ease of availability make milk a more practical choice than any other
medium.
HBSS is a specially designed storage medium containing essen-
tial nutrients.80 Although it is not easily available in most parts of
the world, it is marketed in some countries as “Save-­a-­tooth” (Save-­
A-­Tooth, PA, USA). It has shown to maintain PDL cell viability and
has demonstrated clonogenic and mitogenic capacity of the PDL
water as storage media for transporting avulsed teeth.15,16,18,26,35
There were four recommendations for green tea extract.17,23,30,35
The rest of the media from this category were recommended in
either only one or two studies. Although natural products other
as a group, when the single most recommended medium was ex-
plored, the number of recommendations for any specific medium
was greatly diluted. No consensus was found for on a storage me-
dium as being the best or ideal. Contrary to the fact that saliva is
biological and easy available, saliva as a storage or transport me-
dium for avulsed teeth was only recommended in one study, which
recommended placing the avulsed tooth in saliva for not more than
30 minutes.78 This is because the presence of enzymes and bacteria
present in saliva exert harmful effects on the PDL cells.39
Propolis is a natural product with anti-­inflammatory properties,
and it is also known to maintain or enhance the PDL cell viability.
Various formulations of propolis have been studied.12,13,32,33 However,
no consideration during its recommendation has been given to the
ease of its availability or the cost.
The recommendation for coconut water is based on the mainte-
nance of PDL cell viability, cost-­effectiveness, and ease of availability.
Coconut water may be easily accessible in some parts of the world,56
and this would make it a viable storage medium in case of an avulsion
for some geographic locations. However, it cannot be recommended
universally due to the practical limitations. Even if ease of availability
is considered, the primary factor in recommending a storage medium,
this may not hold true universally for every part of the world. This
holds true for milk also, where some countries may not have pasteur-
ized milk readily available. Therefore, a practical approach would be to
identify a list of recommended media that can be used in a variety of
locations and situations.
There was a wide variety of methods in the studies included in
the present systematic review that was utilized for the assessment of

Random sequence allocation

(selection bias) 67% 33%

Allocation concealment
(selection bias) 100%

Blinding of outcome
assessor 98.5%
(detection bias)
Selective reporting
(reporting bias) 7% 93%

n = 67 studies 0%___________25%_____________50%___________75%_________100%

F I G U R E   3   Overall risk of bias for the High risk of bias Unclear risk of bias Low risk of bias
included studies
 |
68       ADNAN et al.
these PDL cells. Therefore, it was difficult to directly compare studies
with one another. The most common method reported for assessing
the viability of the PDL cells was the Trypan blue exclusion or staining
test. Trypan blue solution in the strength of 0.4% is routinely used as a
cell stain to assess cell viability using the dye exclusion test. This test
is based on the concept that viable cells do not take dye, and it is only
taken up by dead cells which become permeable.18 It is considered one
of the most accurate methods to detect the remaining viable cells. The
cells can also be counted using hemocytometer in the same instance.
A disadvantage of using Trypan blue is that the dye is cytotoxic to
some degree, and also stains the background which can result in in-
accuracy when counting the cells.53 Another method that was used to
determine PDL cell viability was MTT assay (3-­[4,5-­dimethylthiazol-­2
-­yl]-­2,5 diphenyltetrazolium bromide) which is basically a colorimetric
test determining the metabolic activity of cells. This tetrazolium-­based
assay is useful because of its rapid results, objectivity, and ease of ma-
nipulation as well as immediate identification of viable PDL cells.85 The
results of MTT assay are comparable to the histological examination of
the PDL cells in vitro.86
The present systematic review attempted to answer the ques-
tion of the most recommended storage medium using in vitro stud-
ies. Parameters indicating a good prognosis for the replanted avulsed
tooth such as the lack of replacement resorption, external inflamma-
tory resorption, external invasive resorption, pain, and swelling can
only be assessed clinically and over long follow-­up periods. Therefore,
the clinical application of the investigated storage media can only be
established after their use in clinical trials and/or field situations. It is
difficult to conduct clinical trials evaluating the storage media most
suited to storing avulsed teeth as avulsion occurs without warning,
and the traumatized patient may or may not be able to recover the
avulsed tooth from the site of the accident, to bring to the clinic to
be replanted.87 Also, such time may be extremely variable. Hence to
standardize the inclusion criteria dictated by the dry time of the PDL
cells would be very difficult. Thus, identification of a the most rec-
ommended storage medium for avulsed teeth in the context of clin-
ical success/survival warrants a separate systematic review, focusing
exclusively on the clinical trials, and/or case reports where clinical
parameters for the survival of a replanted avulsed tooth (after being
kept in storage/transport media) would be analyzed. This could be
considered the primary shortcoming of the present review, in that no
studies assessing the clinical survival of teeth were included. This was
mainly because the parameters of assessment of clinical success were
different to those of laboratory-­based studies, and this would make
the interpretation of data meaningless. Moreover, the majority of the
studies in the present review did not mention the methods adopted by
their authors to eliminate biases such as random allocation or blinding
(Figure 3). The inferences of the investigation would be more expres-
sive had the methods to control such biases been applied.
Ideally, the most recommended storage medium should be able to
not only maintain PDL cell viability but also enhance it. However, this
enhancement of PDL cell viability was only observed in a few stud-
ies.23,25,26,33,41,70,72 The actual number of cells that remained viable
after being placed in the storage medium could not be compared in the
selected studies because of the great variability in the methodology
of studies. This technical heterogeneity made a generalized assump-
tion next to impossible. The “best” medium encompassing all the ideal
characteristics may yet to be identified, but this systematic review has
managed to identify the most recommended storage media that can
be used to store or transport avulsed teeth.
5 | CONCLUSIONS
Compared to synthetic products, natural products may be more ef-
fective in maintaining the PDL cell viability. Due to the wide variety
of storage media assessed using variable methodologies, the in-
cluded studies could not be compared in all aspects for the qualities
of the storage media. However, the literature suggests milk as the
most viable option in terms of PDL cell viability, ease of availability,
and cost-­effectiveness followed by HBSS. Among the natural prod-
ucts other than milk, propolis and coconut water were frequently
recommended.
CO NFL I C T O F I NT ER ES TS
The authors (SA, MML, FRK, SMH, and SEN) confirm that there are no
competing interests regarding this publication.
AU T HO R S ’ CO NT R I B U T I O N
SA did the literature search and drafted the manuscript. MML did data
collection. FRK contributed to the discussion, assessment of biases in
the primary studies, and critically reviewed the manuscript. SMH and
SE helped with initial data compilation and review of studies. All au-
thors have contributed substantially to the content of the manuscript.
They have read and approved the final manuscript.
O RC I D
Farhan R. Khan  http://orcid.org/0000-0002-5650-6268
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https://doi.org/10.1111/edt.12382