Debrier Et Al 2020

Received: 20 January 2020 | Revised: 21 April 2020 | Accepted: 21 April 2020
DOI: 10.1111/edt.12564
COMPREHENSIVE REVIEW
Storage of an avulsed tooth prior to replantation: A systematic

review and meta-analysis
Niels De Brier1 | Dorien O1 | Vere Borra1,2 | Eunice M. Singletary3 |

David A. Zideman4 | Emmy De Buck1,2,5 | On behalf of the International Liaison Committee
on Resuscitation First Aid Task Force
1
Centre for Evidence-Based Practice
(CEBaP), Belgian Red Cross, Mechelen, Abstract
Belgium Background/Aim: It is crucial to store an avulsed tooth appropriately to preserve
2
Cochrane First Aid, Belgian Red Cross,
the viability of the periodontal ligament cells prior to replantation. The aim of this
Mechelen, Belgium
3
Department of Emergency Medicine,
systematic review was to identify the best available evidence for the effectiveness
University of Virginia School of Medicine, of any technique available to laypeople for storing an avulsed tooth compared with
Charlottesville, VA, USA
4
storage in milk or saliva.
Thames Valley Air Ambulance,
Stokenchurch, UK Methods: The following databases were searched (September 2019): Cochrane
5
Department of Public Health and Primary Library, MEDLINE, and Embase. Two reviewers independently considered trial eligi-
Care, KU Leuven, Leuven, Belgium
bility, then extracted and analyzed data, and assessed the risk of bias. The certainty
Correspondence of the body of evidence was appraised according to the GRADE methodology.
Niels De Brier, Centre for Evidence-Based
Results: Out of 4118 references, 33 studies were included and reported 23 compari-
Practice (CEBaP), Belgian Red Cross,
Motstraat 42 Mechelen, Belgium. sons of which 10 were synthesized in a meta-analysis. The limited evidence available
Email: [email protected]
favors storing an avulsed tooth in Hank's balanced salt solution (pooled SMD 2.47, 95%
Funding information CI [1.59;3.34], P < .00001), propolis solution (pooled SMD 1.73, 95% CI [1.12;2.33],
Foundation for Scientific Research of the
P < .00001), oral rehydration salts (pooled SMD 4.16, 95% CI [2.10;6.23], P < .0001),
Belgian Red Cross
rice water, and cling film compared with storage in milk. The cell viability rate was
significantly lower in teeth stored in saline solutions (pooled SMD −4.35, 95% CI
[−7.55;−1.14], P = .008), tap water, buttermilk, castor oil, GC Tooth Mousse, and tur-
meric extract than those stored in milk. There is insufficient evidence to recommend
for or against temporary storage of an avulsed tooth in saliva compared with alternative
solutions. The certainty of evidence was considered low to very low due to limitations
in study design, indirect study populations and outcome measures, and imprecision.
Conclusion: Although milk was shown to extend the periodontal ligament cell vi-
ability before replantation compared with saline or tap water, the following media
have also demonstrated efficacy at preserving the cell viability: Hank's balanced salt
solution, propolis, oral rehydration salts, rice water, and cling film.
KEYWORDS
avulsed teeth, evidence-based practice, first aid, storage, transport
See Acknowledgements for the authors present in International Liaison Committee on Resuscitation First Aid Task Force.
© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Dental Traumatology. 2020;00:1–24. wileyonlinelibrary.com/journal/edt | 1

2 | De BRIER et al.
1 | I NTRO D U C TI O N containers have thus far not been described in treatment guidelines
for avulsed teeth. There is a great need to identify effective inter-
Tooth avulsion is commonly caused by a fall, sports injury, traffic ventions that can preserve PDL cell viability of avulsed teeth prior
accident, or assault1–4 and accounts for 0.6 to as many as 20.8% to receiving professional assistance and, hence, improve long-term
of all traumatic dental injuries.1,5 The majority of dental trauma in- tooth survival.
volves the anterior teeth, including the maxillary central and lateral The aim of this systematic review was to identify the best avail-
incisors.1 Treating trauma to permanent teeth is costly,6 and missing able scientific evidence on the effectiveness of storage methods
an anterior tooth may lead to functional, psychological, and esthetic for avulsed teeth which are available to laypeople. In this context,
problems.7,8 Tooth avulsion is defined as the complete displacement the following research question was formulated in “PICO” (Patient,
9
of a tooth from the alveolar socket as a result of trauma, and it is Intervention, Comparison, Outcome) format: Among adults and chil-
characterized by compromised neurovascular supply, pulp necrosis, dren with an avulsed permanent tooth (P), does using any storage
and loss of periodontal ligament (PDL) cells.10–13 The PDL is a layer media, container, or technique prior to replantation (I), compared
of dense fibrous connective tissue which surrounds the root and at- with storage in whole milk or the patient's saliva (C), change success
14
taches the tooth to its alveolar socket. Its healing represents the of replantation, tooth survival or viability, infection rate, pain, mal-
primary outcome measure when assessing interventions for tooth function (eating, speech), and color of the tooth (O)? This systematic
avulsion.15 review was performed as part of the development of evidence-based
Immediate replantation of the avulsed tooth at the site of in- treatment recommendations by the First Aid Task Force of the
jury has been suggested to result in the greatest chance of tooth International Liaison Committee on Resuscitation (ILCOR).
survival, but a first aid provider may lack the required skills and the
willingness to attempt this procedure.3,16,17 In such situations, it is
recommended to temporarily store the avulsed tooth in a medium 2 | M ATE R I A L S A N D M E TH O DS
3,16
capable of preserving PDL cell viability. In the first instance, it
is of utmost importance to minimize the extra-alveolar dry time in The systematic literature review was performed according to a
order to avoid desiccation of the PDL cells that are left on the root predefined protocol submitted to PROSPERO (registration num-
surface.3,15,18 Prolonging the duration of dry storage induces necro- ber CRD42020152903) and was reported in accordance with
sis of the PDL cells after 30 to 60 minutes and directly diminishes the the Preferred Reporting Items for Systematic Reviews and Meta-
chances of healing after replantation.13,18,19 Hence, the tooth should Analyses (PRISMA) checklist (available with authors upon request). 24
be placed in an appropriate storage medium as soon as possible prior Studies were eligible if they addressed the PICO question and
to seeking assistance with replantation. The ideal storage medium met the following inclusion and exclusion criteria:
exhibits no or minimal microbial contamination, is readily available
or accessible, and has a physiologically compatible pH and osmolal- • Population: Included: adults and children with an avulsed or ex-
ity for maintaining PDL cell viability. 20 The pH of the environment tracted permanent tooth. There were no restrictions on causes
should be around 6.6-7.8 in order to sustain cell growth, while the of tooth avulsion or tooth extraction, treatments (mouthwash,
osmolality affects water absorption of the cells and is optimally medication use, or pulp extirpation), and types of replantation
230-400 mOsmol/kg. 21 procedures. Excluded: studies using cultured cells of the PDL or
Physiological storage media such as Hank's balanced salt solution extracted animal teeth.
(HBSS), milk, saline, or the victim's own saliva have been proposed by • Intervention: Included: all solutions, containers, and techniques
several guideline providers to temporarily store the avulsed tooth in which can be used to store an avulsed or extracted tooth (follow-
an out-of-hospital setting.3,16,18 The use of alternative storage solu- ing dry storage) and which are available to laypeople. Excluded:
tions including propolis, egg white, coconut water, or Ricetral has solely dry storage of the avulsed or extracted tooth and all solu-
been recommended in comparison with whole milk, but only based tions or techniques unavailable to laypeople such as cell culture
16
on very low certainty evidence. Previously published systematic media (eg, Dulbecco's modified Eagle's medium and Ham's F-10).
reviews regarding storage of avulsed teeth have mainly focused on • Comparison: Included: patient's saliva and cow's milk with vary-
in vitro cultured PDL cells and they have agreed that milk could be ing fat content. Excluded: other milk types (eg, goat milk, probi-
considered as the most suitable storage medium for avulsed teeth, otic milk, and buttermilk). Of note, these other milk types were
based on descriptive analysis of cell viability and assessment of avail- included as intervention solutions for storing an avulsed or ex-
ability and convenience. 21,22 However, the evidence provided by the tracted tooth.
use of cultured cell lines is too indirect to guide first aid care. Other • Outcome: Included: infection rate, tooth survival or viability, pain,
systematic reviews were limited to the use of storage media derived malfunction (eating and speech), color of the tooth, and success of
10,23
from plant material or HBSS solely as potential interventions. replantation. Excluded: financial costs.
Furthermore, past systematic reviews solely focused on the effec- • Study design: Included: (a) the studies of a systematic review if
tiveness of different solutions for storing an avulsed tooth but alter- the search strategy and selection criteria were clearly described
native storage techniques such as plastic bags, cling film, wraps, and and if at least three electronic databases were searched; (b)
De BRIER et al. | 3
experimental studies: (quasi- or non-) randomized controlled TA B L E 1 Search strategies

trial (RCT), controlled before and after studies, or controlled in-
Cochrane library
terrupted time series; and (c) observational studies: cohort and
1. [mh “Tooth Injuries”] OR [mh “Tooth Replantation”] OR
case-control studies, controlled before and after studies, and con-
((tooth:ti,ab,kw OR teeth:ti,ab,kw OR denta*:ti,ab,kw OR
trolled interrupted time series. Excluded: cross-sectional studies, dento*:ti,ab,kw OR [mh “Periodontal Ligament”] OR “periodontal
case series, qualitative studies, conference abstracts, and PhD ligament”:ti,ab,kw) AND (avuls*:ti,ab,kw OR replant*:ti,ab,kw))
theses. 2. [mh “Tissue Preservation”] OR stor*:ti,ab,kw OR preserv*:ti,ab,kw
OR transport*:ti,ab,kw OR [mh “Organ Preservation Solutions”]
• Other: No language criteria were used as long as an English ab-
OR [mh “Saliva”] OR saliva:ti,ab,kw OR [mh “Sodium Chloride”]
stract was provided. The review did not report on data from stud- OR saline:ti,ab,kw OR [mh “Milk”] OR milk:ti,ab,kw OR
ies reporting only means, but no SDs, effect sizes, and P-values. [mh “Water”] or water:ti,ab,kw OR solution*:ti,ab,kw OR
propolis:ti,ab,kw OR [mh “Propolis”] OR tea:ti,ab,kw OR [mh
“tea”] OR (egg NEAR/3 (white OR raw OR albumen OR glair OR
The search was conducted in three electronic databases on
glaire)):ti,ab,kw OR [mh “Egg White”] OR ice:ti,ab,kw OR [mh
September 2, 2019: MEDLINE (via the PubMed interface), Embase “Ice”] OR [mh “Sodium Fluoride”] OR “sodium fluoride”:ti,ab,kw
(via the Embase.com interface), and the Cochrane Library. Search OR ((cling OR plastic OR stretch) NEXT/1 (wrap OR film OR
strategies were developed by two reviewers (NDB and VB) for each foil)):ti,ab,kw OR bag:ti,ab,kw OR container:ti,ab,kw OR
box:ti,ab,kw
database by using both index and free-text terms (Table 1). After
3. 1 AND 2
removing duplicates, two reviewers (NDB and DO) independently
MEDLINE (via the PubMed interface)
screened the title and abstract of the references identified by the
1. “Tooth Injuries”[Mesh] OR “Tooth Replantation”[Mesh]
search and, in a next step, assessed the full text of potentially rel-
OR ((tooth[TIAB] OR teeth[TIAB] OR denta*[TIAB] OR
evant manuscripts based on the inclusion and exclusion criteria. dento*[TIAB] OR “Periodontal Ligament”[Mesh] OR “periodontal
Disagreements were resolved by consensus or by a third reviewer ligament”[TIAB]) AND (avuls*[TIAB] OR replant*[TIAB]))
(VB). The reference lists of all selected studies and relevant system- 2. “Tissue Preservation”[Mesh] OR stor*[TIAB] OR preserv*[TIAB]
OR transport*[TIAB] OR “Organ Preservation Solutions”[Mesh]
atic reviews were screened to identify additional studies addressing
OR “Saliva”[Mesh] OR saliva[TIAB] OR “Sodium Chloride”[Mesh]
the PICO question. OR saline[TIAB] OR “Milk”[Mesh] OR milk[TIAB] OR
Data concerning study characteristics and effect sizes were ex- “Water”[Mesh] OR water[TIAB] OR solution*[TIAB] OR
tracted independently by two reviewers (NDB and DO). Risk ratios propolis[TIAB] OR “Propolis”[Mesh] OR tea [TIAB] OR
(RR) for dichotomous outcomes or mean differences (MD) or stan- “Tea”[Mesh] OR (egg[TIAB] AND (white[TIAB] OR raw[TIAB]
or albumen[TIAB] OR glair[TIAB] OR glaire[TIAB])) OR “Egg
dardized mean differences (SMD) for continuous outcomes were cal-
White”[Mesh] OR ice[TIAB] OR “Ice”[Mesh] OR “Sodium
culated along with their confidence intervals (CI) and P-values by the Fluoride”[Mesh] OR “sodium fluoride”[TIAB] OR ((cling[TIAB] OR
reviewers using Review Manager 5.3. P-values less than .05 were con- plastic[TIAB] OR stretch[TIAB]) AND (wrap[TIAB] OR film[TIAB]
sidered significant. When appropriate, random-effects meta-analy- OR foil[TIAB])) OR bag[TIAB] OR container[TIAB] OR box[TIAB]
3. “Letter”[Publication Type] OR “Comment”[Publication Type] OR
ses were carried out using the generic inverse variance method for
“Editorial”[Publication Type] OR “Case Reports”[Publication Type]
continuous data or the Mantel-Haenszel method for dichotomous OR News[Publication Type]
outcomes. The results were visualized in a forest plot including all 4. 1 AND 2 NOT 3
studies with their (pooled) estimate and 95% CI. Heterogeneity was Embase (via the Embase.com interface)
evaluated by combining visual inspection of the forest plot with the 1. ‘tooth injury’/exp OR ‘endodontics’/exp OR ((tooth:ab,ti OR
I2 statistic and the chi-squared test. Heterogeneity was considered teeth:ab,ti OR denta*:ab,ti OR dento*:ab,ti OR ‘periodontal
to be substantial if CIs were clearly not overlapping, the I2 statistic ligament’/exp OR ‘periodontal ligament’:ab,ti) AND (avuls*:ab,ti
OR replant*:ab,ti))
values reached 75% or higher, and the chi-squared test had a P-value
2. ‘tissue preservation’/exp OR stor*:ab,ti OR preserv*:ab,ti OR
smaller than .10. When a meta-analysis was not feasible, that is, due transport*:ab,ti OR ‘preservation solution’/exp OR ‘saliva’/exp OR
to substantial differences between studies, or when only one study saliva:ab,ti OR ‘sodium chloride’/exp OR saline:ab,ti OR ‘milk’/exp
was identified, the results were tabulated and a narrative description OR milk:ab,ti OR ‘water’/exp OR water:ab,ti OR solution*:ab,ti
OR propolis:ab,ti OR ‘propolis’/exp OR tea:ab,ti OR ‘tea’/exp OR
of the results was provided.
(egg NEAR/3 (white OR raw OR albumen OR glair OR glaire)):ab,ti
The certainty of evidence was rated according to the GRADE OR ‘egg white’/exp OR ice:ab,ti OR ‘ice’/exp OR ‘sodium
approach (Grading of Recommendations, Assessment, Development fluoride’/exp OR ‘sodium fluoride’:ab,ti OR ((cling OR plastic OR
and Evaluation). 25 The online available GRADEpro Guideline stretch) NEXT/1 (wrap OR film OR foil)):ab,ti OR bag:ab,ti OR
container:ab,ti OR box:ab,ti
Development Tool (GDT), developed by the GRADE Working
3. [editorial]/lim OR [letter]/lim OR ‘case report’/de
Group, was used to construct the GRADE evidence profile tables 4. 1 AND 2 NOT 3
to present effect sizes and the assessment of the certainty of evi-
dence (http://www.gradepro.org/). The certainty of evidence can be
downgraded due to shortcomings in each of the following criteria: by two reviewers (NDB and DO) using the “Risk of Bias 2” for ran-
study design, inconsistency, indirectness, imprecision, or publication domized studies26 and “ROBINS-I” tool for non-randomized and
bias. The limitations in study design were independently evaluated observational studies. 27 Study design is not considered as a risk of
4 | De BRIER et al.
bias feature in “ROBINS-I,” and, therefore, the initial certainty for studies were included. Two additional studies were finally identified
27
the body of evidence from all included studies was high. The final after screening reference lists of included studies and systematic re-
level of evidence can be graded as high, moderate, low, or very low. views retrieved with the database searches, leading to a total of 33
included studies.
Table 2 summarizes the characteristics of the included studies
3 | R E S U LT S concerning study design, population, comparison, and outcome
measures. More than half of the included studies were RCTs (n = 19;
The systematic literature search yielded 5115 records, and, once the 58%), seven were non-RCTs (21%), and seven were prospective co-
duplicates were removed, 4118 references were screened based on hort studies (21%). About 66% (n = 22) of the included studies were
title and abstract. In the next step, the full text of 125 potentially published in the last ten years (2009-2019) with 45% (n = 15) in the
relevant studies was assessed for eligibility (Figure 1). After reaching last 5 years.
consensus, 30 studies were excluded based on the comparison (no In all controlled experimental studies (both RCT and non-RCT),
data on milk or saliva provided), 23 on publication type (mostly nar- extracted teeth were used to simulate the storage of avulsed teeth
rative reviews and conference abstracts), 16 on intervention (eg, no prior to replantation. The teeth were mostly atraumatically ex-
data on storage media provided and intervention not feasible to lay- tracted for orthodontic reasons, and the age of the patients was
people), 15 on population (cultured cell lines), four on outcome (no relatively low (mostly adolescents and young adults). Information
link between intervention and outcome provided), two on language, on patient characteristics was absent in 15 of the 26 experimental
and one on study design (cross-sectional nature). Furthermore, the studies. Teeth with caries, restorations, or periodontal diseases were
full text of three other studies was unavailable. At this stage, 31 generally excluded prior to randomization. The prospective cohort
Records idenfied through database searching:

n = 5115
Idenficaon
Cochrane Library: n = 67
MEDLINE: n = 1043
Embase: n = 4005
Duplicates removed:
n = 997
Records a er duplicates removed:

n = 4118
Screening
Records excluded based on tle

and abstract:
n = 3993
Full-text arcles assessed for eligibility:

n = 125
Full -textarcles excluded: n = 94

• Comparison: n = 30
Eligibility
• Publicaon type: n = 23
• Intervenon: n = 16
• Populaon: n = 15
• Outcome: n = 4
• Other: n = 3
• Language: n = 2
• Study design: n = 1
Full -textarcles included a er

Included
reference list search: n = 2
F I G U R E 1 PRISMA flowchart for the

Studies included: identification and selection of eligible
n = 33
studies
De BRIER et al. | 5
TA B L E 2 Characteristics of the included studies
Reference,
Country Study design Population Comparison Outcome
Abraham Experimental: randomized 40 extracted premolars Intervention: HBSS (n = 10) or 100% aloe vera Number of viable
et al33 India controlled trial gel (n = 10) vs Control: low-fat cow's milk PDL cells
(n = 10)
Dry: 30 min; Storage: 45 min
Ahangari Experimental: randomized 60 extracted anterior Intervention: propolis 10%, 0.04 mg/mL Percentage of viable
et al28 Iran controlled trial single root teeth (n = 10), propolis 50%, 0.2 mg/mL (n = 10), PDL cells
HBSS (Save-A-Tooth) (n = 10), or egg white
(n = 10) vs Control: milk (n = 10)
Dry: 30 min; Storage: 60 or 180 min
Andreasen Observational: 94 replanted teeth Intervention: saline (n = 12) or another Pulp healing or pulp
et al12 prospective cohort with incomplete root person's saliva (n = 5) vs Control: patient's necrosis
Denmark study formation at the time of saliva (n = 17) Follow-up period of
injury at least 1 year
Andreasen Observational: 322 patients with 400 Intervention: saline (n = 33) or another Periodontal healing
et al19 prospective cohort avulsed and replanted person's mouth (n = 9) vs Control: patient's Follow-up period of
Denmark study permanent incisors mouth (n = 9) or saliva (n = 33) at least 1 year
Bağ and Experimental: non- 18 extracted third molar Intervention: HBSS (n = 6) vs Control: whole Number of viable
Yildirim29 randomized controlled teeth milk (n = 6) PDL cells after
Turkey trial Storage: 30 to 60 min or 12 h proliferation (cell
growth)
Caglar et al38 Experimental: non- 36 extracted single-rooted Intervention: Lactobacillus reuteri solution Number of viable
Turkey randomized controlled second premolars (n = 6), HBSS (n = 6), or saline (n = 6) vs PDL cells
trial Control: milk (n = 6)
Caglar et al39 Experimental: non- 36 extracted single-rooted Intervention: Bifidibacterium animalis DN Number of viable
Turkey randomized controlled second premolars 173010 containing probiotic yogurt (n = 6), PDL cells
trial HBSS (n = 6), or saline (n = 6) vs Control: milk
(n = 6)
Chappuis and Observational: 34 patients with 45 Intervention: Dentosafe® box (n = 16) vs Periodontal healing
Von Arx13 prospective cohort avulsed permanent teeth. Control: milk (n = 17) or saliva (n = 5) Follow up at 3, 6,
Switzerland study Patient's age ranged from and 12 months
6 to 48 y (mean age: 21 y)
Chen and Experimental: randomized 30 extracted single-rooted Intervention: epigallocatechin-3-gallate Percentage of viable
Huang54 controlled trial teeth (EGCG) (n = 10) or HBSS (n = 10) vs Control: PDL cells
China whole pasteurized milk (n = 10)
Storage: 120 min
D'Costa et al45 Experimental: randomized 40 extracted premolars Intervention: coconut water (n = 10) or saline Percentage of viable
India controlled trial (n = 10) vs Control: milk (n = 10) PDL cells
Mud/sand: 30 min; Storage: 45 min
Dhimole et al51 Experimental: randomized 90 extracted single-rooted Intervention: neem (A indica) (n = 30) or Percentage of viable
India controlled trial premolar teeth. Patient's turmeric (C longa) extract (n = 30) vs Control: PDL cells
age ranged from 13 to milk (n = 30)
28 y. Dry: 15 min; Storage: 30 min
Doyle et al67 Experimental: randomized 49 extracted molars, Intervention: HBSS (n = 15) vs Control: vitamin Percentage of viable
USA controlled trial premolars, and anterior D homogenized milk (n = 15) PDL cells
teeth. Patient's age Dry: 30, 60, or 90 min; Storage: 15 min
ranged from 18 to 65 y.
Gopikrishna Experimental: randomized 55 extracted teeth (mean Intervention: coconut water (n = 15) or HBSS Number of viable
et al53 India controlled trial age: 24 y) (n = 15) vs Control: milk (n = 15) PDL cells
Hegde et al46 Experimental: non- 30 extracted premolars Intervention: 0.9% saline (n = 10) or GC Tooth Percentage of viable
India randomized controlled Mousse Plus (n = 10) vs Control: chilled PDL cells
trial homogenous milk (n = 10)
Storage: 30 or 60 min
(Continues)
6 | De BRIER et al.
TA B L E 2 (Continued)
Reference,
Karayilmaz Observational: 66 children with a total Intervention: saliva (n = 3) vs Control: milk Healing with a
et al4 Turkey prospective cohort of 93 avulsed anterior (n = 3) normal PDL or
study permanent teeth. with surface
Patient's age ranged resorption
from 6 to 16 y (mean age: The patients were
10.2 ± 2.5 y). followed for 1 to
8 y.
Khademi Experimental: randomized 100 extracted permanent Intervention: egg white (n = 25), HBSS Percentage of viable
et al30 Iran controlled trial premolars (n = 25), or tap water (n = 25) vs Control: milk PDL cells
(n = 25)
Storage: 1, 2, 4, 8, or 12 h
Kokkali et al50 Experimental: randomized 55 extracted premolars. Intervention: coconut water (n = 15) or Number of viable
India controlled trial Patient's age ranged from buttermilk (n = 15) vs Control: cow's milk PDL cells
17 to 20 y. (n = 15)
Lekic et al35 Experimental: non- 20 premolars extracted Intervention: saliva and thereafter HBSS Clonogenic capacity
Canada randomized controlled from 11 patients. (n = 5) vs Control: saliva and thereafter milk of PDL cells (%)
trial Patient's age ranged from (n = 5)
11 to 14 y. Storage: 15 min saliva and (15-30 min HBSS
or milk or 30-60 min HBSS or milk)
Martin and Experimental: randomized 70 extracted single-rooted Intervention: saline (n = 12), HBSS (n = 12), Number of viable
Pileggi43 USA controlled trial teeth (mean age: 45 y) propolis 50%, 0.2 mg/mL (n = 12), or PDL cells (cells/mL)
propolis 100%, 0.4 mg/mL (n = 12) vs
Control: milk (n = 12)
Nabavizadeh Experimental: randomized 40 extracted single-rooted Intervention: castor oil (n = 10) or HBSS Percentage of viable
et al40 Iran controlled trial teeth (mean age: 22.5 y) (n = 10) vs Control: milk (2.5% fat) (n = 10) PDL cells
Patil et al48 Experimental: non- 32 extracted single-rooted Intervention: saline (0.9%, sterile solution Number of viable
USA randomized controlled premolars. Patient's age for lenses) (n = 12) vs Control: vitamin D PDL cells
trial ranged from 13 to 28 y. pasteurized whole milk (n = 12)
Pileggi et al41 Experimental: non- 33 extracted teeth Intervention: HBSS, saline, or water vs Control: Percentage of viable
USA randomized controlled milk (numbers unspecified) PDL cells
trial Dry: 30 min; Storage: 120 min
Pohl et al49 Observational: 28 avulsed and replanted Intervention: Dentosafe® box (n = 8) or saline Functional healing
Germany prospective cohort teeth (upper incisors) (n = 7) vs Control: milk (n = 6) Mean observation
study from 24 patients. period was
Patient's age ranged from 31.2 months
7.1 to 17.3 y (mean age:
10.3 ± 2.6 y).
Prueksakorn Experimental: randomized 96 premolars extracted Intervention: propolis, 2.5 mg/mL (n = 10) or Percentage of viable
et al31 controlled trial from 96 patients. HBSS (n = 10) vs Control: UHT milk (n = 10) PDL cells
Thailand Patient's age ranged from Dry: 30 min; Storage: 180 min
8 to 24 y.
Rajendran Experimental: randomized 30 extracted single-rooted Intervention: Ricetral (n = 10) or HBSS (n = 10) Number of viable
et al42 India controlled trial teeth vs Control: milk (n = 10) PDL cells
Sharma et al52 Experimental: randomized 45 extracted premolars Intervention: aloe vera gel (n = 15) or egg Number and
India controlled trial white (n = 15) vs Control: pasteurized, percentage of
homogenized milk with fat content of viable PDL cells (4
3 g/100 mL (n = 15) x 1 mm2 areas)
Mud: 15 min; Storage: 30 min
(Continues)
De BRIER et al. | 7
Reference,
44
Sharma India Experimental: randomized 45 extracted premolars Intervention: rice water (n = 15) or egg Number and
controlled trial white (n = 15) vs Control: pasteurized, percentage of
homogenized milk with fat content of viable PDL cells (4
3 m/100 mL (n = 15) x 1 mm2 areas)
Mud: 15 min; Storage: 30 min
Subramaniam Experimental: randomized 130 premolars extracted Intervention: HBSS (Save-A-Tooth system) Number of viable
et al34 India controlled trial from healthy children. (n = 40) or oral rehydration salt-liquid (ORS- PDL cells
Patient's age ranged from L) (n = 40) vs Control: cold whole bovine milk
13 to 18 y. (3.0% fat) (n = 40)
Dry: 30 or 60 min; Storage: 45 or 90 min
Sunil et al47 Experimental: randomized 48 extracted mandibular Intervention: HBSS (n = 8), saline solution Number of viable
India controlled trial premolars (n = 8), or tender coconut water (n = 8) vs PDL cells
Control: milk (n = 8)
Talebi et al32 Experimental: randomized 60 extracted premolars Intervention: HBSS (n = 15) or tap water Percentage of viable
Iran controlled trial (n = 15) vs Control: pasteurized skim milk PDL cells
(n = 15)
Dry: 30 min; Storage: 1, 3, 6, or 24 h
The extracted teeth were analyzed at the
four different timepoints (60 analyses per
study group).
Wang et al37 Observational: 196 replanted teeth from Intervention: saline (n = 38) vs Control: milk Functional healing
China prospective cohort 157 patients (94 boys (n = 26) or saliva (n = 18) The mean follow-up
study and 63 girls). Patient's period was 4.0 y
age ranged from 6 to 16 y
(mean age: 9.9 y).
The maxillary central
incisor was the most
frequently avulsed tooth
(150/196).
Werder et al11 Observational: 42 avulsed permanent Intervention: Dentosafe® box (n = 8) or saline Periodontal healing,
Switzerland prospective cohort teeth from 37 patients. (n = 9) vs Control: milk (n = 16) Median follow-up
study Patient's age ranged period: 2.8 y
from 6 to 62 y (mean age:
16.3 y).
Zeissler- Experimental: randomized 35 third molars extracted Intervention: SOS Zahnbox® (n = 7), sterile The probability and
Lajtman controlled trial (21 females and 14 males, isotonic saline solution (n = 7), tap water rate of cell growth
et al36 mean age: 26.5 ± 8.52 y). (n = 7), or cling film (n = 7) vs Control: UHT
Switserland milk (n = 7)
Storage: 120 min
studies involved patients presenting at hospitals or medical centers extracted tooth in HBSS compared with storing it in milk. Twelve
with avulsed teeth which were surgically replanted and followed up studies reported on the storage of the avulsed or extracted teeth
for at least one year. In line with the experimental studies, the mean in saline; four on storage in coconut water, egg white, or tap water;
age of the patients with replanted avulsed teeth was 21 years or three on storage in propolis solutions or the Dentosafe® box; and
lower. Injured teeth with pre-existing root canal treatment, accom- two on storage in aloe vera gel, probiotic media, or oral rehydra-
panying fractures or previous restorations, were mostly excluded. tion salts (ORS) compared with milk. Finally, single studies assessed
Cases in which avulsed teeth could not be replanted were also not each of the following comparisons: storage in buttermilk, castor oil,
taken into account. cling film, epigallocatechin-3-gallate (EGCG), GC Tooth Mousse, rice
While about 1400 avulsed or extracted teeth were used in the water, saliva, and thereafter HBSS, neem extract, SOS Zahnbox®, or
included studies to address the PICO question, the number of teeth turmeric extract. Five studies evaluated the storage of avulsed or
used in the intervention or control groups was rather low (n = 3 to extracted teeth in saliva compared with storage in a Dentosafe® box,
n = 38). In total, 23 different comparisons which are relevant in a milk, another person's saliva/mouth, or saline solutions. In the ex-
first aid setting were reported in the included studies. A majority perimental studies, the teeth were typically allowed to dry for about
of the studies (n = 17) evaluated the effectiveness of storing an 30 minutes (in mud or sand) to simulate the first aid situation prior
8 | De BRIER et al.
TA B L E 3 Synthesis of findings and certainty assessment according to the GRADE methodology
Certainty assessment № of patients
Other
No of studies Study design Risk of bias Inconsistency Indirectness Imprecision considerations Intervention
Interventions showing benefit on the preservation of tooth or cell viability

A. HBSS vs milk
Cell viability after 15-min to 24-h immersion
13 RCT and Serious Not serious Serious Not serious None 206
non-RCTd
Cell viability after 45-min immersion

2 Non-RCT Very serious Not serious Serious Serious None 6
1 Non-RCT Very serious Not serious Serious Serious None
1 RCT Serious Not serious Serious Serious None 10
B. Saliva and thereafter HBSS vs saliva and thereafter milk
Cell viability after 30- to 60-min immersion
1 Non-RCT Serious Not serious Serious Serious None 5
C. Oral rehydration salts vs milk
Cell viability after 45- to 90-min immersion (immersion after 30- or 60-min drying)
D. Propolis solution vs milk

E. Cling film vs milk

Rate of cell growth (7 to 14 d) after 120-min storage
F. Rice water vs milk
Interventions showing less benefit on the preservation of tooth or cell viability

G. Saline solution vs milk
5 RCT and Serious Serious Serious Serious None 52
non-RCTd
De BRIER et al. | 9
Effect
Relative
Control (95% CI) Absolute (95% CI) Certainty Importance References
206 - SMD 2.47 SD higher (1.59 ⨁⨁◯◯LOW CRITICAL Abraham et al33 Ahangari et al28
higher to 3.34 higher) Bag and Yildirim29
Chen and Huang54
Doyle et al67 Gopikrishna et al53
Khademi et al30
Martin and Pileggi43
Prueksakorn et al31
Rajendran et al42
Subramaniam et al34
Sunil et al47
Talebi et al32
6 - Median 507 500 highera ⨁◯◯◯ VERY LOW CRITICAL Caglar et al38,39
c
- MD 3.55 lower ⨁◯◯◯ VERY LOW CRITICAL Pileggi et al41
10 - Median 4.08% lowera ⨁◯◯◯ VERY LOW CRITICAL Nabavizadeh et al40
5 - MD 1 lowerb ⨁◯◯◯ VERY LOW CRITICAL Lekic et al35

5 - MD 2.4 higherb ⨁◯◯◯ VERY LOW CRITICAL
50 - SMD 4.16 SD higher (2.1 ⨁◯◯◯ VERY LOW CRITICAL Rajendran et al42 Subramaniam et al34
higher to 6.23 higher)
32 - SMD 1.73 SD higher (1.12 ⨁◯◯◯ VERY LOW CRITICAL Ahangari et al28
higher to 2.33 higher) Martin and Pileggi43
Prueksakorn et al31
7 - MD 0.45 higherb ⨁◯◯◯ VERY LOW CRITICAL Zeissler-Lajtman et al36

7 - MD 0.41 higherb ⨁◯◯◯ VERY LOW CRITICAL
15 - MD 11 higher (5.29 higher ⨁◯◯◯ VERY LOW CRITICAL Sharma44

to 16.71 higher)
52 - SMD 4.35 SD lower (7.55 ⨁◯◯◯ VERY LOW CRITICAL D'Costa et al45
lower to 1.14 lower) Hegde et al46
Martin and Pileggi43
Patil et al48
Sunil et al47
(Continues)
10 | De BRIER et al.
Other

1 Non-RCT Serious Not serious Serious Serious None
Functional and periodontal healing
3 Prospective Very serious Not serious Not serious Serious None 23/54 (42.6%)
cohort study
H. Tap water vs milk

1 Non-RCT Very serious Not serious Serious Serious None
I. GC Tooth Mousse vs milk

J. Buttermilk vs milk
K. Turmeric extract vs milk

L. Castor oil vs milk

Interventions showing conflicting effects on the preservation of tooth or cell viability
M. Aloe vera gel vs milk
1 RCT Serious Serious Serious Serious None 10
N. Coconut water vs milk

De BRIER et al. | 11
Effect
Relative
6 - Median 376 000 highera ⨁◯◯◯ VERY LOW CRITICAL Caglar et al38,39
c
- MD 12.79 lower ⨁◯◯◯ VERY LOW CRITICAL Pileggi et al41
18/48 RR 1.20 75 more per 1000 (from ⨁◯◯◯ VERY LOW CRITICAL Pohl et al49
(37.5%) (0.74 to 98 fewer to 356 more) Werder et al11
1.95) Wang et al37
- MD 45.42 lowerc ⨁◯◯◯ VERY LOW CRITICAL Pileggi et al41
15 - MD 18.53% lower (23.53 ⨁◯◯◯ VERY LOW CRITICAL Talebi et al32

lower to 13.53 lower)
lower to 5.4 lower)
5 - MD 2% lower (3.39 lower ⨁◯◯◯ VERY LOW CRITICAL Hegde et al46

to 0.61 lower)
5 - MD 2.3% lower (3.91 ⨁◯◯◯ VERY LOW CRITICAL
15 - MD 12 646 lower ⨁◯◯◯ VERY LOW CRITICAL Kokkali et al50

(14 084.66 lower to
11 208.48 lower)
30 - MD 8.35% lower (11.29 ⨁◯◯◯ VERY LOW CRITICAL Dhimole et al51

10 - Median 8.17% lowera ⨁◯◯◯ VERY LOW CRITICAL Nabavizadeh et al40
10 - MD 571.7 lower (875.1 ⨁◯◯◯ VERY LOW CRITICAL Abraham et al33

15 - MD 14.2 higher (8.72 ⨁◯◯◯ VERY LOW CRITICAL Sharma et al52
(Continues)
Other
Note: Significant (P < .05) results are indicated in gray.

Abbreviations: CI, confidence interval; MD, mean difference; RR, risk ratio; RCT, randomized controlled trial; SMD, standardized mean difference.
a
Absolute effect (ie, MD and CI) could not be calculated because the data are expressed as median values;
b
CI could not be calculated because the standard errors are not described;
c
Absolute effect could not be calculated because the number of teeth in each group is not given;
d
RCT and non-RCT studies were pooled since they were very similar regarding study population (extracted teeth), experimental design (controlled
immersion of teeth in storage media), and outcome analysis (microscopic evaluation of viable PDL cells). The only difference is that the non-RCT
studies did not mention the randomization process in the article.
to storing them in the solutions or films for 15 minutes to 24 hours. with storage in milk. In addition, six storage interventions had a less
It is of note that only five experimental studies stored the extracted beneficial impact on the preservation of cell viability than milk and
teeth for longer than 120 minutes. 28–32 two interventions suffered from conflicting evidence. Finally, for the
In most of the experimental studies (n = 23), the researchers as- other nine comparisons, there was evidence neither in favor of the
sessed the viability of the PDL cells as a measure for tooth viability. intervention nor in favor of the control.
The analysis of cell viability was similar across the different studies: Several storage techniques were associated with improved pres-
After storage of the teeth, the PDL cells were harvested, stained with ervation of tooth or cell viability. It was reported that storing an
0.4% (w/v) trypan blue, and counted under a light microscope with a avulsed tooth in (saliva and thereafter) HBSS, ORS, propolis solu-
hemocytometer. Viable cells have intact cell membranes and do not tions, cling film, and rice water resulted in a significantly higher PDL
absorb the blue stain, while the dye readily penetrates into the cyto- cell viability rate compared with storage in milk (Table 3).
plasm of the non-viable cells with damaged cell membranes.33,34 This Firstly, a meta-analysis of 22 comparisons from 12 experimental
method is considered one of the most accurate analyses to detect studies involving 412 extracted teeth revealed that the cell viabil-
the remaining viable cells. 22 Other outcome measures were related ity rates of the teeth stored in HBSS for 15 minutes up to 24 hours
to clonogenic capacity or cell growth. 29,35,36 The observational stud- were significantly higher than those stored in milk (pooled SMD 2.47,
ies evaluated the success of replantation by clinical and radiographic 95% CI [1.59;3.34], P < .00001), with substantial heterogeneity be-
examinations of functional and periodontal healing of the avulsed tween trials (non-overlapping CIs, I2 = 89%, and Chi2 test P < .0001)
and replanted teeth. These outcomes involved healing in the ab- (Figure 2, comparison 1.1.1). Based on visual inspection of the forest
sence of surface resorption, replacement resorption, inflammatory plot, the study of Khademi et al30 was excluded from the meta-anal-
resorption, and/or ankylosis.11,19,37 ysis since the very large effect estimates in favor of storing teeth
The synthesis of findings of all included studies is summarized in in HBSS were considered as outliers in the analysis. However, this
Tables 3 and 4. The experimental studies for each of the following exclusion did not reduce the reported I2. Moreover, excluding the
comparisons were pooled: HBSS, ORS (including Ricetral), propolis, experimental study of Bag and Yildirim29 from the meta-analysis
saline solutions, and egg white vs milk (Figure 2). Further, prospective based on its methodological shortcomings (extracted teeth were not
cohort studies evaluating the following five comparisons were also randomly assigned to the study groups, see below) did not influence
pooled: saline solutions compared with milk or saliva, Dentosafe® the statistical heterogeneity (data not shown). Four other studies
box compared with milk, saliva compared with milk, and another per- could not be included in the meta-analysis because the effect size
son's saliva/mouth compared with patient's saliva/mouth (Figure 3). (ie, mean difference and/or CI) could not be calculated from median
A narrative overview of the results of the studies not included in the cell viability values,38–40 or the number of teeth in the experimental
meta-analysis is given below. Among the 23 comparisons evaluating or control group was not provided.41 It is of note that the latter stud-
the effect of storage on the viability of avulsed or extracted teeth, ies were not able to demonstrate a statistically significant difference
six showed positive effects on the viability of the PDL cells compared in cell viability when storing an extracted tooth in HBSS or milk for
Effect
Relative
10 - MD 23% higher (19.57 ⨁◯◯◯ VERY LOW CRITICAL D'Costa et al45

15 - MD 339.4 higher (331.65 ⨁◯◯◯ VERY LOW CRITICAL Gopikrishna et al53
15 - MD 9293.33 lower ⨁◯◯◯ VERY LOW CRITICAL Kokkali et al50
(10 930.14 lower to
7656.52 lower)
8 - MD 260.12 higher (236.43 ⨁◯◯◯ VERY LOW CRITICAL Sunil et al47
45 minutes. Furthermore, Lekic et al35 showed that the clonogenic Storage techniques showing less benefit compared with milk on
capacity of extracted teeth stored in saliva (15 minutes) and there- the preservation of tooth or cell viability are summarized in Table 3.
after HBSS (45 minutes) was significantly higher than those stored Firstly, a meta-analysis of six comparisons from three RCTs and
in saliva and thereafter milk. However, a statistically significant two non-RCTs involving 104 extracted teeth showed that the cell
difference in clonogenic capacity could not be demonstrated after viability rate was significantly lower in extracted teeth stored in sa-
15-minute storage in HBSS or milk following 15 minutes pre-immer- line solutions for 30 to 120 minutes compared with those stored in
sion in saliva. milk (pooled SMD −4.35, 95% CI [−7.55;−1.14], P = .008) (Figure 2,
Secondly, the effectiveness of ORS for storing an avulsed tooth comparison 1.1.4).43,45–48 The heterogeneity across the trials was
was assessed. When pooling five effect estimates from two RCTs in substantial (non-overlapping CIs, I2 = 94%, and Chi2 test P < .0001),
34,42
a meta-analysis involving 100 extracted teeth, the cell viability and one study, Sunil et al,47 showed that the teeth stored in saline
rate of the teeth stored in solutions made of ORS (including Ricetral) solution had significantly higher cell viability rates compared with
was found to be higher after 45 to 90 minutes of storage than those teeth stored in milk (SMD 3.81, 95% CI [2.00;5.62], P < .0001), in
stored in milk (pooled SMD 4.16, 95% CI [2.10;6.23], P < .0001) and contrast to all other studies. Excluding the studies of Hegde et al46
there was substantial heterogeneity between the trials (non-over- and Patil et al48 from the meta-analyses based on their method-
lapping CIs, I2 = 87%, and Chi2 test P < .0001) (Figure 2, comparison ological limitations in the randomization process (see below) did
1.1.2). not lower the statistical heterogeneity. In the meta-analysis, the
Thirdly, in order to study the effectiveness of propolis solutions standard deviations of the mean values extracted from the study of
(from 0.04 mg propolis/mL to 2.5 mg/mL) for storing extracted teeth, Hegde et al46 were imputed based on experimental errors from the
four comparisons from three RCTs (86 extracted teeth in total) were other included studies. Three other non-RCTs could not be included
included in a meta-analysis providing evidence that propolis solu- in the meta-analysis because the absolute effect size could not be
tions were more effective than milk to preserve PDL cell viability calculated from median cell viability values,38,39 or the number of
(pooled SMD 1.73, 95% CI [1.12;2.33], P < .00001) (Figure 2, com- teeth in each study group was not provided.41 These studies failed to
parison 1.1.3). 28,31,43 The heterogeneity was low (overlapping CIs, show a difference in PDL cell viability rate with immersion in either
2 2
I = 17%, and Chi test P = .31). saline solution or milk. Similarly, three prospective cohort studies
Finally, the evidence on cling film and rice water relies on sin- of 102 avulsed teeth failed to show a difference in healing after re-
gle experimental studies. Compared with storage in milk, Zeissler- plantation following storage in either saline solution or milk (pooled
Lajtman et al36 showed that the extracted teeth wrapped in cling RR 1.20, 95% CI [0.74;1.95], P = .47). There was no heterogeneity
film for 2 hours was associated with the highest rate of cell growth (I2 = 0) (Figure 3, comparison 1.1.1).11,37,49 No absolute effects (ie,
44
(P = .033), and Sharma identified an increased rate of cell viability MD and CI) and P-values could be calculated from data of the studies
of extracted teeth immersed in rice water (P < .00001). of Zeissler-Lajtman et al.36
TA B L E 4 Synthesis of findings which could not demonstrate an effect on the preservation of tooth or cell viability and certainty
assessment according to the GRADE methodology
Certainty assessment No of patients
Other
®
A. Dentosafe box vs milk
Periodontal and functional healing
cohort study
B. Egg white vs milk

C. Epigallocatechin-3-gallate vs milk
D. Neem extract vs milk

E. Probiotic media vs milk

F. Saliva vs milk
Periodontal and functional healing
cohort study
G. Another person's saliva/mouth vs patient's saliva

Periodontal and pulp healing
cohort study
H. Dentosafe® box vs saliva
Periodontal healing
cohort study
I. Saline solutions vs saliva
Functional, periodontal and pulp healing
cohort study
Abbreviations: CI, confidence interval; MD, mean difference; RCT, randomized controlled trial; RR, risk ratio; SMD, standardized mean difference.
a
Absolute effect (ie, MD and CI) could not be calculated because the data are expressed as median values.
Two studies reported on the effectiveness of tap water for stor- groups but also showed that the extracted teeth stored in tap water
ing an extracted tooth. One RCT including 30 extracted teeth for for 45 minutes had a lower cell viability rate than those stored in
each comparison showed less benefit for cell viability after 60-min- milk.41 No absolute effect sizes (ie, MD and CI) and P-values could be
32
ute to 24-hour immersion in tap water compared with milk. One calculated from mean values from the studies of Khademi et al30 and
non-RCT did not report the number of extracted teeth in the study Zeissler-Lajtman et al.36
Effect
Relative (95%
Control CI) Absolute (95% CI) Certainty Importance References
21/39 (53.8%) RR 1.20 (0.61 108 more per 1000 (from 210 ⨁◯◯◯ VERY LOW CRITICAL Chappuis and Von Arx13
to 2.36) fewer to 732 more) Pohl et al49
Werder et al11
25 - SMD 0.14 SD higher (0.52 ⨁◯◯◯ VERY LOW CRITICAL Ahangari et al28 Khademi et al30
lower to 0.8 higher) Sharma et al44,52
10 - MD 9.8% higher (8.66 lower ⨁◯◯◯ VERY LOW CRITICAL Chen and Huang54
to 28.26 higher)
30 - MD 1.98% lower (4.54 lower ⨁◯◯◯ VERY LOW CRITICAL Dhimole et al51
to 0.58 higher)
6 - median 116 000 highera ⨁◯◯◯ VERY LOW CRITICAL Caglar et al38
6 - median 995 000 highera ⨁◯◯◯ VERY LOW CRITICAL Caglar et al39
22/46 (47.8%) RR 0.96 (0.65 19 fewer per 1000 (from 167 ⨁◯◯◯ VERY LOW CRITICAL Chappuis and Von Arx13
to 1.43) fewer to 206 more) Wang et al37
Karayilmaz et al4
4/14 (28.6%) RR 1.00 (0.31 0 fewer per 1000 (from 197 ⨁◯◯◯ VERY LOW CRITICAL Andreasen et al12
to 3.18) fewer to 623 more) Andreasen et al19
3/5 (60.0%) RR 0.83 (0.35 102 fewer per 1000 (from ⨁◯◯◯ VERY LOW CRITICAL Chappuis and Von Arx13
to 1.98) 390 fewer to 588 more)
16/63 (25.4%) RR 0.97 (0.53 8 fewer per 1000 (from 119 ⨁◯◯◯ VERY LOW CRITICAL Andreasen et al12
to 1.79) fewer to 201 more) Andreasen et al19
Wang et al37
One non-RCT (n = 20) found that teeth stored in GC Tooth Mousse Conflicting evidence for the preservation of tooth or cell viability
had significantly lower cell viability rates than those stored in milk after was found in two storage techniques. Two RCTs evaluating storage
30- and 60-minute immersion.46 Compared with milk, three RCTs includ- of 50 extracted teeth in aloe vera gel for 45 minutes found conflict-
ing 20 to 60 extracted teeth showed lower cell viability rates after 30- to ing results. Sharma et al52 demonstrated significantly greater cell via-
45-minute immersion in buttermilk,50 turmeric extract,51 and castor oil.40 bility rates from storage of extracted teeth in aloe vera gel compared
F I G U R E 2 Study-specific standardized mean differences (intervention vs. milk) in cell viability. Standardized mean difference ( ) of the
respective study together with a 95% confidence interval (CI). The size of the square represents the weight of the study in the meta-analysis.
Weights are from random-effects analysis. Mean overall effect (♦) together with a 95% CI. The immersion time is given between brackets.
Codes a, b, and c: 30-, 60-, and 90-min drying of avulsed teeth prior to immersion in medium, respectively
F I G U R E 3 Study-specific risk ratios (intervention vs. control) in healing of the replanted teeth. Risk ratio ( ) of the respective study
together with a 95% confidence interval (CI). The size of the square represents the weight of the study in the meta-analysis. Weights are
from random-effects analysis. Mean overall effect (♦) together with a 95% CI. Andreasen 1995a corresponds to Andreasen et al12 and
Andreasen 1995b to Andreasen et al.19
with storage in milk, while Abraham et al33 found lower cell viability Lastly, storage techniques which could not demonstrate an ef-
rates following storage of extracted teeth in aloe vera gel compared fect on the preservation of tooth or cell viability were also identi-
with milk (Table 3). fied. As outlined in Table 4, there was evidence neither in favor of
The effect of coconut water for storing extracted teeth also storing an avulsed tooth in egg white, Dentosafe® box, probiotic
suffers from conflicting evidence originating from four RCTs in- media, neem extract, or EGCG nor in favor of storing it in milk prior
cluding 96 extracted teeth. Three RCTs showed improved cell via- to replantation. A statistically significantly higher cell viability rate,
bility rates after storing extracted teeth for 45 minutes in coconut using egg white as a storage medium compared with milk, could not
water compared with milk, 45,47,53 while the fourth RCT showed be demonstrated in a meta-analysis (Figure 2, comparison 1.1.5)
lower cell viability rates following storage in coconut water for including 50 extracted teeth from three RCTs (SMD 0.14, 95% CI
45 minutes. 50 [−0.52;0.80], P = .68). 28,30,44,52 The data from Khademi et al30 were
excluded from the meta-analysis since the heterogeneity (I2) was people performing the interventions were probably aware of the as-
2
lowered from 79% to 18% (overlapping CIs and Chi test P = .30) with signed intervention, no deviations from the intended interventions
little, if any, impact on the pooled effect sizes. It is of note that both were expected that were likely to have affected the outcome at this
studies of Sharma et al44,52 reported identical data on the effective- stage. Moreover, in all RCTs, the outcome data were available for all
ness of egg white for storage of an extracted tooth compared with randomized extracted teeth. Since the assessment of cell viability
storage in milk and the data were hence analyzed as a single result under a microscope involves some judgment of the researcher, there
in the meta-analysis. were serious concerns regarding the risk of bias in the measure-
The use of the Dentosafe® box for storing 71 avulsed teeth was ment of the outcome when the outcome assessor was not blinded
evaluated in three prospective cohort studies (Figure 3, comparison to the intervention. Only three studies reported that the cells were
1.1.2). A difference in success of replantation of an avulsed tooth counted by researchers who were blinded to the identity of the study
could not be demonstrated with storage in the Dentosafe® box groups. 28,31,50 Martin and Pileggi43 reported that the cell viability was
compared with milk (pooled RR 1.20, 95% CI [0.61;2.36], P = .59, independently assessed by two researchers to minimize the risk of
I2 = 52%).11,13,49 Zeissler-Lajtman et al36 studied the use of the SOS bias due to human errors. Finally, it was judged that studies reporting
®
Zahnbox (commercial rescue box) for preserving extracted teeth, on cell growth rate or on number of viable PDL cells likely selected
but no absolute effects and P-values could be calculated from the the outcome results from multiple measurements or data analyses,
provided mean values. Regarding storage in probiotic media, two respectively.33,34,36,42,43,47,50,53 In the outcome domain, percentage
observational studies each with 12 extracted teeth38,39 reported of viable PDL cells is clearly the most reported measurement and it is
only median cell viability values, and thus, absolute effects could not not appropriate to only show the number of viable PDL cells since it
be calculated. These studies were not able to demonstrate benefit is not corrected for the total amount of cells in the sample. As a con-
or harm of immersion in probiotic media when compared with milk sequence, percentage of viable PDL cells was preferred over number
(P > .05). The benefit of storage in neem extract51 and EGCG for cell of viable PDL cells or outcomes dealing with cell growth. In general,
viability of extracted teeth, respectively, could also not be demon- the included studies had serious limitations in their study design due
strated when compared with milk.54 to risk of bias regarding the randomization process, measurement of
Last but not least, five prospective cohort studies evaluating the outcome, and/or selection of the reported results. The studies
the impact of storage of an avulsed tooth in saliva on the success of of Ahangari et al28 and Prueksakorn et al31 had only some concerns
replantation were identified. Three of these studied the impact of arising from the randomization process.
storage of 72 avulsed teeth on the success of replantation (Figure 3, For the included non-RCT and observational studies, the lim-
comparison 1.1.3) and provided evidence neither in favor of storing itations in study design are shown in Figure 5. For the non-RCTs,
an avulsed tooth in saliva nor in favor of storing it in milk (pooled there is limited, if any, bias due to confounding except for the study
2 4,13,37
RR 0.96, 95% CI [0.65;1.43], P = .86, I = 0%). No differences of Lekic et al35 since the extracted teeth were not standardized
could be demonstrated for the success of replantation when com- prior to inclusion. The prospective cohort studies from Andreasen
paring storage of avulsed teeth in saline solutions (pooled RR 0.97, et al12,19 controlled for confounding factors by using a paired sam-
95% CI [0.53;1.79], P = .92, I2 = 6%),12,19,37 or another person's sa- ple approach. Thus, in each case with a certain storage period in
2 12,19
liva (pooled RR 1.00, 95% CI [0.31;3.18], P = 1.00, I = 0%), with saliva, they selected a case with a similar storage period in another
storage in the patient's saliva (Figure 3, comparisons 1.1.4 and 1.1.5, medium. Similarly, Pohl et al49 and Wang et al37 grouped their re-
13
respectively). For success of replantation, Chappuis and Von Arx sults for the impact of storage media on successful replantation of
also found no statistically significant differences between storage of avulsed teeth based on different dry and wet storage times. While
®
avulsed teeth in the Dentosafe box or saliva. Karayilmaz et al4 only controlled for the storage time, Chappuis and
In a next step, the certainty of the included studies was as- Von Arx13 and Werder et al11 only showed unadjusted outcome
sessed. For the RCTs, an overview of the limitations in study design results. It is of note that the studies of Andreasen et al,12,19 Pohl
for the included studies individually is shown in Figure 4 (“Risk of et al,49 and Wang et al37 performed a multivariate logistic regression
Bias 2” tool). All but one study noted that the study was random- model to control for confounding factors, but no adjusted data on
ized without further details of the randomization process. Sharma the storage media of interest were provided. No inappropriate se-
et al52 performed the allocation of the extracted teeth based on the lection of extracted teeth or patients was detected in the non-RCT
chit-pull system. The allocation concealment was not described in and prospective cohort studies, respectively. In the latter studies,
the included RCTs, but there were no apparent baseline differences avulsed teeth which could not be replanted were logically excluded.
between the study groups due to problems with the randomization Moreover, in the same studies, there were some concerns regard-
process. The extracted teeth in the studies were standardized since ing the classification of the interventions since results on storage
teeth with caries, periodontal diseases, and restorations were mostly media and time were commonly grouped based on the judgment of
33
excluded prior to randomization. It is of note that Abraham et al, the researchers. The information used to define the intervention
Martin and Pileggi,43 and Rajendran et al42 highlighted that the tooth groups was based on patient's records upon arrival at the hospi-
extraction procedure may have induced variable trauma poten- tal. There were no concerns about missing outcome data except
tially leading to variability of PDL cell viability counts. Although the for the results of Andreasen et al12 and Wang et al37 since 16 and
F I G U R E 4 Methodological quality
summary: Review authors' judgments
about each methodological quality item
using the “Risk of Bias 2” tool for each
included study with low risk of bias
(green), some concerns (yellow), or high
risk of bias (red)
122 teeth, respectively, were lost to follow up and excluded from unquestionably downgraded for inconsistency (−1) as a result of con-
analysis. They were excluded because early complications led to the tradictory data for the following comparisons: aloe vera gel, coconut
extraction of the replanted tooth, but it was not possible to assess water, or saline solution vs milk since there is, in each case, one study
whether the proportion of missing patients was similar across inter- suggesting a significant opposite effect on cell viability than in other
ventions. Since the evaluation of cell viability (microscopic analysis) studies. It is of note that inconsistency is important when it reduces
and success of replantation (radiographic and clinical assessment) the confidence in the results. As a consequence, although the het-
clearly involve the judgment of the unblinded researchers, there erogeneity for the comparisons between HBSS or ORS and milk was
were serious concerns regarding the risk of bias in measurement of substantial (non-overlapping CIs, I2 > 75 and Chi2 test P < .10), rating
35
outcomes. Regarding selection of the reported results, Lekic et al down for inconsistency was not relevant since the differences were
and Bag and Yildirim29 showed outcome data on clonogenic capac- between small and large effects of the intervention.55 For the exper-
ity and growth of PDL cells of extracted teeth while, based on the imental studies, the reviewers also downgraded for indirectness (−1)
body of evidence, it is common to measure the viability. Finally, the since extracted instead of avulsed teeth were included and because
prospective cohort studies generated multiple results for different PDL cell viability was assessed as a measure for tooth viability. It is
subgroups, which involves a risk of selective reporting. also of note that the experimental studies commonly used extracted
Lastly, a GRADE assessment of the certainty of the body of ev- (pre)molars for assessing the effectiveness of storage media for pre-
idence is detailed in Tables 3 and 4. The certainty of evidence was serving cell viability, while the (central) incisors are most prone to
downgraded for limitations in study design (see above). The risk of dental injuries. The overall certainty of evidence was further down-
bias was classified as “serious” in the RCTs (−1) and as “very seri- graded (−1) for all but one comparison (HBSS vs milk) because of im-
ous” in the non-RCTs and observational studies (−2). The reviewers precision due to limited sample sizes, large variability of the results,
F I G U R E 5 Methodological quality
summary: Review authors' judgments
about each methodological quality
item using the “ROBINS-I” tool for each
included study with low risk of bias
(green), moderate risk of bias (yellow),
high risk of bias (red), or critical risk of bias
(dark red)
and/or lack of data. As a result, the certainty of evidence was graded systematic reviews attempted to identify the most recommended
as low for the comparison between HBSS and milk and as very low media for storing an avulsed tooth based on descriptive analysis of
for all other comparisons. (cultured) PDL cell viability,10,21,22 this systematic review is the first
to quantitatively report on the comparisons between various stor-
age media and techniques available to laypeople and the currently
4 | D I S CU S S I O N recommended transport solutions: milk and saliva.
Out of 4118 references, the reviewers selected 33 studies (15
The aim of this systematic review was to identify the best available published in the last five years) comparing the efficacy of the use of
evidence on the effectiveness of any solution or technique poten- any solution, medium, or technique for storing an avulsed tooth com-
tially available to laypeople for storing an avulsed tooth prior to pared with storage in milk or (the patient's) saliva. The main finding
replantation. Although some first aid guidelines on storage of an of this systematic review is that the limited evidence available favors
avulsed tooth already exist,3,16,18 the evidence they are based on HBSS, propolis (from 0.04 mg to 2.5 mg per mL 0.4% ethanol), ORS
is limited and they are becoming outdated since multiple individual solutions including Ricetral [ORS solutions containing sodium chlo-
studies fulfilling the eligibility criteria were published in the last five ride, glucose, potassium chloride, and citrate (or extruded rice)],34,42
years. It is generally recommended to transport the avulsed tooth to or cling film compared with cow's milk for storing an avulsed tooth
the emergency services in a glass of milk or in the patient's mouth until replantation can be performed. HBSS is composed of inter alia
(saliva).3 In performing this systematic review, the authors acknowl- glucose and sodium, potassium, calcium, and magnesium ions34
edge that the immediate replantation of an avulsed tooth substan- and is recommended by the International Association of Dental
3,16–18
tially increases its survival and, hence, healing. However, this Traumatology as a storage medium for avulsed teeth.3 Interestingly,
procedure may not be possible in the first aid setting due to the the pH and osmolality values of HBSS (7.2-7.3 and 270-290, respec-
patient‘s concomitant injuries (eg, unconsciousness, bleeding, frac- tively) and ORS (7.0-7.2 and 285-325, respectively) are suitable for
tures, etc.) at the time of accident and lack of knowledge in, and will- cell growth and, hence, for preserving PDL cell viability. 21,30 The ef-
3,12
ingness to undertake, the management of such injuries. The use ficacy of cling film for storing an avulsed tooth can be ascribed to its
of a suitable temporary storage solution or technique for an avulsed ability to retain the thin fluid film on the root surface. The cling film
tooth should not delay efforts at replantation, but it may aid in the keeps the tooth moist and, at the same time, creates a physiological
survival of the tooth before receiving professional help. While recent environment which is necessary for cell survival.36 Lastly, propolis
is a yellowish to brownish resinous beehive product containing a A statistically significant increase in cell viability rates could not
wide range of biologically active compounds such as polyphenols, be demonstrated with use of probiotic media, EGCG (ie, a catechin
to which anti-bacterial and anti-inflammatory properties can be as- found in high concentrations in green tea),54 the Dentosafe® box (ie,
cribed. These properties are held responsible for the good results of a tooth rescue system marketed in Europe for tooth preservation in
propolis solutions in maximizing PDL cell survival.31,43 the first aid setting and containing a tissue culture medium),11,13,49
The choice of a suitable temporary solution or medium for first neem extract, or egg white compared with cow's milk for storing an
aid storage of an avulsed tooth will depend on the setting and avail- avulsed tooth. Surprisingly, there is also limited evidence available
ability of products in each country. HBSS is the main ingredient in the favoring temporary storage of an avulsed tooth in either saliva or
most common commercially marketed tooth preservation product in alternative solutions.
the United States (Save-A-Tooth™; Phoenix Lazarus Inc, Pottstown, Recent systematic reviews have focused mainly on the viability
PA, USA) and can be found in some larger first aid kits designed for of cultured PDL cells and found that of the media which are avail-
dental emergencies in the wilderness or at sporting events. It may, able to laypeople, HBSS, 21–23 milk, 21,22 propolis,10,21,22 coconut
however, not be commonly available at places where traumatic den- water,10,21,22 and aloe vera gel,10,21 were most effective in preserving
3
tal injuries occur such as schools, homes, camps, and sport fields. If the cell viability. In this systematic review, the limited evidence avail-
there is access to special storage media such as HBSS or diluted prop- able favors storing an avulsed tooth in HBSS or propolis compared
olis solutions, the evidence supports their use compared with other with milk prior to replantation but there was conflicting evidence
interventions evaluated in this review. While propolis solutions might regarding the potential of coconut water and aloe vera gel. The re-
be available in African households, most (rural areas) of low- and mid- view by Adnan et al22 also included four studies recommending ORS
dle-income countries will have no or limited access to commercial for storing an avulsed tooth.34,58–60 Similar to the current review,
products such as rescue boxes or Tooth Mousse. Cling film may be Osmanovic et al21 also provided limited evidence that tap water was
a simple and readily available choice in many households and has a not able to effectively preserve the PDL cells. Further, Fagundes
very limited cost. In Europe and Africa, ORS is available in first aid kits et al23 highlighted, based on meta-analyses, that storage in HBSS
and therefore easily applicable in all settings. Also, evidence-based was associated with a higher level of viable PDL cells in comparison
African first aid recommendations have already taken into account with tap water and saline solutions. Finally, one systematic review
that ORS can be prepared based on local ingredients and, hence, its solely dealing with the replantation of avulsed teeth in animals was
use might be recommended for storing an avulsed tooth in rural and unable to draw any conclusion regarding the effectiveness of the
remote regions.56 A potential disadvantage to ORS is the need to re- included storage media due to substantial heterogeneity and limita-
constitute the salts with water. Although evidence from one study tions in study design.61
shows the benefit of immersion in rice water when compared with The major limitation of the current review is that it provides
44
milk, it may not be feasible to apply. The preparation of rice water evidence of only low to very low certainty because of serious con-
(ie, boiling rice in water and allowing to cool) is time-consuming and cerns regarding the design of the included studies, indirectness in
could create a delay in storage of the avulsed tooth. population (extracted teeth instead of avulsed teeth) and outcome
If none of the above choices are available, cow's milk, in any (cell viability instead of tooth viability), and imprecise results (ie, lim-
percentage fat or form, could be considered for temporary stor- ited sample sizes, large variation in the results, and/or lack of data).
age of an avulsed tooth. Several evaluated storage solutions such Moreover, the relatively high heterogeneity observed in the me-
as water and saline failed to show improved PDL cell viability rates ta-analysis comparing HBSS, ORS, and saline solutions with milk as
when compared with milk or saliva. Milk is currently the most rec- potential storage media for an avulsed tooth indicated that there is
ommended medium for storing an avulsed tooth in a pre-hospital substantial variability among the included studies, which can com-
setting, since its pH (6.7) and osmolality values (about 280 mOsm/ promise the reliability of the pooled effect sizes. To tackle the above
kg) are compatible with those of the PDL cells. Further, it contains limitations and to formulate stronger evidence-based conclusions,
the required sugars and minerals and is free of bacteria.3,21,22 On large well-designed cohort studies with traumatic avulsed teeth (in-
the contrary, tap water typically has osmolality values that are too stead of extracted teeth) measuring tooth viability (not cell viability)
low. Although saline solution has an osmolality of 280 mOsm/kg, it and success of replantation are warranted. Designing an RCT with
is deficient in essential nutrients such as magnesium, calcium, and avulsed teeth instead of extracted teeth would be impossible in a
glucose which are required for normal metabolism of PDL cells.57 first aid setting. Furthermore, in the current review, all but five ex-
Due to their hypotonic properties, saline and especially tap water perimental studies dealt with storage times of 15 to 120 minutes.
may induce rapid cellular lysis,30 and they are not suggested for In a West European context, it is reasonable to expect that patients
storing an avulsed tooth compared with storage in milk. In line with with an avulsed tooth can find professional help within 2 hours after
these findings, buttermilk has a relatively low pH (4.2) and osmolality the accident. However, access to emergency care is often delayed in
50
values (142 mOSm/kg) which are not conducive for cell viability. (rural areas of) low- and middle-income countries which may lead to
Finally, despite the antioxidant properties of castor oil and turmeric prolonged storage of the avulsed tooth.62,63 Osmanovic et al21 pro-
40,51
extract, they were also not found to be capable of preserving vided limited evidence that there is a potential interaction effect be-
the PDL cell viability as well as milk. tween storage time and medium on PDL cell viability by reviewing cell
culture experiments. However, when performing subgroup analyses tooth. However, the identified evidence is of low to very low cer-
according to the storage time in the meta-analyses, no significant tainty and results are imprecise especially due to limited sample
subgroup differences were detected (data not shown). Whatever the sizes. Large well-designed cohort studies are warranted to confirm
case, more studies are currently warranted to assess the impact of these findings.
storage time on tooth cell viability in relation to the storage tech-
nique. Lastly, the original wording of this PICO question specified AC K N OW L E D G E M E N T S
the use of whole milk as a comparison. After the initial search re- NDB, DO, VB, and EDB are employees at Belgian Red Cross and re-
sults were reviewed, it was recognized that the studies identified use ceive no other funding. One of the activities of Belgian Red Cross is
cow's milk as the comparator, but with varying percentages of fat providing first aid training to laypeople. This work was made possible
content, and some milk was pasteurized or homogenized. One can through funding from the Foundation for Scientific Research of the
therefore only recommend that cow's milk be used, without a specific Belgian Red Cross (Mechelen, Belgium).
fat percentage. The fat content of milk, pasteurization, homogeniza- Besides VB, ES, and DZ, members of the International Liaison
tion, and the effect of temperature were not evaluated, and there- Committee on Resuscitation First Aid Task Force include Jason
fore, they are a limitation of this review. Although the authors did C. Bendall, David C. Berry, Jestin N. Carlson, Pascal Cassan, Wei-
not systematically search for the effect of the fat content on tooth Tien Chang, Nathan P. Charlton, Therese Djärv, Matthew Douma,
viability, there is one experimental study using cultured cells, indi- Jonathan L. Epstein, Natalie A. Hood, David S. Markenson, Daniel
cating that low-fat milk may be the most appropriate for maintaining Meyran, Aaron Orkin, Tetsuya Sakamoto, Janel M. Swain, and
64
the viability of PDL cells. Temperature could also play a key role Jeffrey A. Woodin
since cooled milk demonstrated greater survival of cultured PDL cells
compared with milk at room temperature.65,66 These studies were C O N FL I C T O F I N T E R E S T
excluded from this systematic review since they dealt with cultured The authors confirm that they have no conflict of interest.
cells instead of extracted or avulsed teeth. In future PICO questions,
(a) storing an avulsed tooth for short times (less than 120 minutes) ORCID
vs long-term storage (more than 120 minutes) (b) storing an avulsed Niels De Brier https://orcid.org/0000-0002-0605-2376
tooth in cooled conditions vs storage at room temperature, (c) stor-
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Received: 20 January 2020 | Revised: 21 April 2020 | Accepted: 21 April 2020

Storage of an avulsed tooth prior to replantation: A systematic

Niels De Brier1 | Dorien O1 | Vere Borra1,2 | Eunice M. Singletary3 |

avulsed teeth, evidence-based practice, first aid, storage, transport

Dental Traumatology. 2020;00:1–24. ﻿ wileyonlinelibrary.com/journal/edt | 1

experimental studies: (quasi- or non-) randomized controlled TA B L E 1 Search strategies

Records idenfied through database searching:

Records a er duplicates removed:

Records excluded based on tle

Full-text arcles assessed for eligibility:

Full -textarcles excluded: n = 94

Full -textarcles included a er

reference list search: n = 2

F I G U R E 1 PRISMA flowchart for the

TA B L E 2 Characteristics of the included studies

TA B L E 3 Synthesis of findings and certainty assessment according to the GRADE methodology

Certainty assessment № of patients

Interventions showing benefit on the preservation of tooth or cell viability

Cell viability after 45-min immersion

D. Propolis solution vs milk

E. Cling film vs milk

Interventions showing less benefit on the preservation of tooth or cell viability

5 - MD 1 lowerb ⨁◯◯◯ VERY LOW CRITICAL Lekic et al35

7 - MD 0.45 higherb ⨁◯◯◯ VERY LOW CRITICAL Zeissler-Lajtman et al36

15 - MD 11 higher (5.29 higher ⨁◯◯◯ VERY LOW CRITICAL Sharma44

Certainty assessment № of patients

Cell viability after 45-min immersion

H. Tap water vs milk

1 RCT Serious Not serious Serious Serious None 15

1 RCT Serious Not serious Serious Serious None 15

1 RCT Serious Not serious Serious Serious None 15

I. GC Tooth Mousse vs milk

1 Non-RCT Very serious Not serious Serious Serious None 5

K. Turmeric extract vs milk

L. Castor oil vs milk

1 RCT Serious Serious Serious Serious None 15

N. Coconut water vs milk

- MD 45.42 lowerc ⨁◯◯◯ VERY LOW CRITICAL Pileggi et al41

15 - MD 18.53% lower (23.53 ⨁◯◯◯ VERY LOW CRITICAL Talebi et al32

5 - MD 2% lower (3.39 lower ⨁◯◯◯ VERY LOW CRITICAL Hegde et al46

15 - MD 12 646 lower ⨁◯◯◯ VERY LOW CRITICAL Kokkali et al50

30 - MD 8.35% lower (11.29 ⨁◯◯◯ VERY LOW CRITICAL Dhimole et al51

10 - Median 8.17% lowera ⨁◯◯◯ VERY LOW CRITICAL Nabavizadeh et al40

10 - MD 571.7 lower (875.1 ⨁◯◯◯ VERY LOW CRITICAL Abraham et al33

Certainty assessment № of patients

1 RCT Serious Serious Serious Serious None 10

1 RCT Serious Serious Serious Serious None 15

1 RCT Serious Serious Serious Serious None 15

1 RCT Serious Serious Serious Serious None 8

Note: Significant (P < .05) results are indicated in gray.

10 - MD 23% higher (19.57 ⨁◯◯◯ VERY LOW CRITICAL D'Costa et al45

Certainty assessment No of patients

B. Egg white vs milk

D. Neem extract vs milk

E. Probiotic media vs milk

G. Another person's saliva/mouth vs patient's saliva

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Dental Traumatology. 2020;00:1–24. wileyonlinelibrary.com/journal/edt | 1

Records idenfied through database searching:

Records excluded based on tle

Full-text arcles assessed for eligibility:

Full -textarcles excluded: n = 94

Full -textarcles included a er