THE MOST RECOMMENDED STORAGE AND TRANSPORT MEDIA FOR AULSED TEETH A systematic review

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Ugnė Vyšniauskaitė

5th_{Course, group 12}

THE MOST RECOMMENDED STORAGE AND

TRANSPORT MEDIA FOR AULSED TEETH

A systematic review

Master’s Thesis

Supervisor

Assistant, Živilė Kristina Matulaitienė

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

FACULTY OF ODONTOLOGY

THE CLINIC OF ORAL CARE AND PEDIATRIC DENTISTRY

THE MOST RECOMMENDED STORAGE AND TRANSPORT MEDIA FOR AULSED TEETH

Master’s Thesis

The thesis was done by student

Ugnė Vyšniauskaitė, 5th_{course, group 12 Supervisor Assistant, Živilė Matulaitienė} (name surname, year, group) (degree, name surname)

………. ………

(signature) (signature)

……… 20…. ……… 20….

(day/month) (day/month)

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EVALUATION TABLE OF THE MASTER’S THESIS

OF THE TYPE OF SYSTEMIC REVIEW OF SCIENTIFIC LITERATURE Evaluation: ... Reviewer: ...

(scientific degree. name and surname)

Reviewing date: ...

Compliance with MT

No. MT parts MT evaluation aspects requirements and_evaluation

Yes Partially No

1 _Summary Is summary informative and in compliance with the _{thesis content and requirements?} 0.3 0.1 0

2 (0.5 point) Are _essence?keywords In compliance with the Thesis 0.2 0.1 0

3 Introduc- Are the novelty, relevance and significance of the _{work justified in the introduction of the thesis?} 0.4 0.2 0

tion, aim _{Are the problem, hypothesis, aim and tasks formed}

4 and tasks _{clearly and properly?} 0.4 0.2 0

(1 point)

5 Are the aim and tasks interrelated? 0.2 0.1 0

6 Selection Is the protocol of systemic review present? 0.6 0.3 0

criteria of Were the eligibility criteria of articles for the

7 the studies, selected protocol determined (e.g., year, language, 0.4 0.2 0

search publication condition, etc.)

methods and Are all the information sources (databases with

8 _{(3.4 points)}strategy dates _{identify additional studies) described and is the last}of coverage, contact with study authors To 0.2 0.1 0 search day indicated?

Is the electronic search strategy described in such a way that it could be repeated (year of search, the

9 last search day; keywords and their combinations; 0.4 0.1 0 number of found and selected articles according to

the combinations of keywords)?

Is the selection process of studies (screening,

10 eligibility, included _{applicable, included in the meta-analysis)}in systemic review or, If 0.4 0.2 0

described?

Is the data extraction method from the articles

11 (types of investigations, participants, interventions, 0.4 0.2 0 analysed factors, indexes) described?

Are all the variables (for which data were sought

12 and any assumptions and simplifications made) 0.4 0.2 0

listed and defined?

Are the methods, which were used to evaluate the

13 Risk of bias of individual studies and how this 0.2 0.1 0

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information is to be used in data synthesis,

described?

14 Were the principal summary measures (risk ratio, _{difference in means) stated?} 0.4 0.2 0 Is the number of studies screened: included upon

15 assessment for eligibility and excluded upon giving 0.6 0.3 0 the reasons in each stage of exclusion presented?

Are the characteristics of studies presented in the

16 Systemiza- included articles, according to which the data were _{extracted (e.g., study size, follow-up period, type of} 0.6 0.3 0

tion and

respondents) presented?

analysis of

Are the evaluations Of beneficial or Harmful

data

outcomes for each study presented? (a) Simple

17 (2.2 points) _{summary data for each intervention group; b) effect} 0.4 0.2 0

estimates and confidence intervals)

Are the extracted and systemized data from studies

18 presented in the tables according to individual 0.6 0.3 0

tasks?

19 Are _{relevance indicated?}the main findings summarized and is their 0.4 0.2 0

20 Discussion Are the limitations of the performed systemic 0.4 0.2 0

(1.4 points) review discussed?

21 Does _results?author present the interpretation of The 0.4 0.2 0

22 _Conclusions Do the conclusions reflect the topic, aim and tasks _{of the Master’s thesis?} 0.2 0.1 0

23 (0.5 points) Are the conclusions based on the analysed material? 0.2 0.1 0

24 Are the conclusions clear and laconic? 0.1 0.1 0

25 Is the references list formed according to The 0.4 0.2 0 requirements?

Are the links of the references to the text correct?

26 Are the literature sources cited correctly and 0.2 0.1 0

References precisely?

27 (1 point) Is the scientific level Of references suitable For 0.2 0.1 0

Master’s thesis?

Do the cited sources not older than 10 years old

28 form at least 70% of sources, and the not older than 0.2 0.1 0 5 years – at least 40%?

Additional sections, which may increase the collected number of points

29 Annexes Do the presented annexes help to understand The +0.2 +0.1 0

analysed topic?

Practical _{Are the practical recommendations suggested and}

30 recommen- _{are they related to the received results?} +0.4 +0.2 0

dations

Were additional methods of data analysis and their

31 results used and described (sensitivity analyses, +1 +0.5 0 meta-regression)?

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Was meta-analysis applied? Are the selected 32 statistical methods indicated? Are the results of +2 +1 0

each meta-analysis presented?

General requirements, non-compliance with which reduce thenumberof points

33 Is the thesis volume sufficient (excluding 15-20 pages <15 pages

annexes)? _{(-2 points)} _{(-5 points)}

34 Is the thesis volume increased -2 points -1 point

artificially?

35 Does the thesis structure satisfy the -1 point -2 points requirements of Master’s thesis?

36 Is the thesis written in correct language, -0.5 point -1 points scientifically, logically and laconically?

37 Are there any grammatical, style or -2 points -1 points

computer literacy-related mistakes?

38 Is text consistent, integral, and are the -0.2 point -0.5 points volumes of its structural parts balanced?

General

39 Amount of plagiarism in the thesis. >20%

require- _{(not evaluated)}

ments Is the content (names of sections and sub-

40 sections and enumeration of pages) in -0.2 point -0.5 points compliance with the thesis structure and

aims?

Are the names of the thesis parts in

41 compliance with the text? Are the titles of -0.2 point -0.5 points sections and sub-sections distinguished

logically and correctly?

42 Are there explanations of the key terms -0.2 point -0.5 points and abbreviations (if needed)?

Is the quality of the thesis typography

43 (quality of printing, visual aids, binding) -0.2 point -0.5 points

good?

*In total (maximum 10 points):

*Remark: the amount of collected points may exceed 10 points.

Reviewer’s comments: _________________________________________________________ ________________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ____________________________________________________________________

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ABBREVIATIONS:

HBSS- Hanks Balanced Salt Solution

DMEM-Dulbecco’s modified Eagle’s medium PJ- Pomegranate juice

NC-100- Nucleocounter

MTT- 3- [4, 5-demethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide

MTS- 4, 5-demethylthiazolyl-2]-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium PDL- Periodontal ligament

NRU- Neutral red update

PDLF-Periodontal Ligament Fibers GT- Green tea

GTE- Green tea extract

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The most recommended storage and transport media for avulsed teeth- a systematic review

SUMMARY

Objective. The purpose of this systemic review was to evaluate and identify the most

recommended natural medium to store and transport avulsed teeth.

Material and methods. According to the PRISMA recommendation, publications of this

systemic review were selected through PubMed and ResearchGate on English studies dated from 2011 to 2021. In the results it was shown 313 publications (abstracts). Later, 64 articles related to the topic were revised. 8 articles matched up to the criteria and were included in this systematic review.

Result. 8 articles were used in the study which met the inclusion criteria. All of articles in vitro.

Few different types of milk were tested, and results showed that goat milk, low fat milk and cooled milk maintain periodontal cells viability most efficiency.

Conclusion. Cooled milk has ability to maintain PDL cells viability up to 120 hours. Green tea

extract suggested as suitable storage media for avulsed teeth despite that pH and osmolality values unsuitable for cell growth.

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Labiausiai rekomenduojamos laikymo ir transportavimo terpės avulsiniems dantims-sisteminė apžvalga

SANTRAUKA

Tikslas. Odontologijoje viena svarbiausių užduočių yra išsaugoti natūralius dantis, kad būtų

išlaikyta okliuzija, pilnavertis krantymas, kaulo tankis ir tūris. Avulsinis dantis apibūdinamas kaip visiškai išmuštas dantis ir teikiaga, kad tai yra sudėtingiausia danties trauma. Reimplantacija yra vienas iš gydimo metodikų, tačiau sėkminga reimplantacija priklauso nuo periodonto audinių

gyvibindumo. Dėl šios priežasties iškėlėme tikslą nustatyti veiksmingiausia naturalia terpę

periodonto lastelės gyvibingumui palaikyti.

Medžiaga ir metodas. Laikantis PRISMA rekomendacijų, publikacijos buvo ieškomos dviejuose

duomenų bazėse – PubMed ir ResearchGate, anglų kalbos bibliotekoje, atrinktoje nuo 2011 iki 2021 metų. Iš viso idetifikuoti 313 straipsnių. 64 publikacijos peržiūrėtos ir išanalizuotos,

perskaičius pilnus straipsnių tekstus. Pritaikius atrankos kriterijus galutinei analizei, buvo atrinkti 8 mokslinės publikacijos.

Rezultatai. Į sisteminę atžvalga įtraukti 8 straipsniai, kurie atitiko kriterijus. Visi straipsniai in vitro. Išanalizavus skirtingas pieno rūšis, tyrimai prarodė ožkos pieno, liesaus pieno ir atvėsinto

pieno efektivumas įvertintas geriausia atsižvelgiant į periodonto ląstelės išlaikymo gyvibingumą.

Išvados. Atvėsintas pienas ilgiausiai išlaiko periodonto raiščius gyvibingus iki 120 valandu, nei

kambario temperatūros pienas. Žalios arbatos ekstraktas tinkama terpė laikyti avulsinius dantis,

nepaisant nepalankių pH ir osmoliariško periodonto ląstelės gyvibingumui palaikyti.

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INTRODUCTION

Avulsion of teeth is one of the most acute forms of dental trauma and it is quite common in

the young, where root formation is still incomplete [1]. Tooth avulsion is a complex traumatic

injury characterised by the rupture of the neurovascular bundle and periodontal ligament (PDL) exposing the tooth to the outer environment [2].The main etiological factors are trauma after fighting and sports, as well as falls and bumps against hard objects and/or the floor [3]. The age

group of 7-10 years appears to be most affected [4], when the alveolar bone is resilient and offers

minimal resistance to extrusive forces [2]. The incidence of tooth avulsion accounts approximately 1-16% of all traumatic injuries in the permanent dentition and 7-21% in the

primary dentition [3]. Despite the fact that tooth avulsion is a complex trauma, tooth can be saved

and apply appropriate treatment. The appropriate treatment for an avulsed tooth is its immediate reimplantation [1]. Therefore, the ideal treatment of choice at the time of avulsion should be immediate replantation, in order to reestablish the natural nutrient supply to the periodontal ligament cells [5].

Reimplantation of a tooth within 5 minutes usually ensure prompt return of the PDL cells to normal function [6]. In case of delayed in replantation as short as 8 min may decrease the

probability' of periodontal healing to less than 50% [7]. After 30 minutes of dry storage, virtually all of the PDL cells remaining in the tooth root are likely to have become necrotic [6]. The immediate transportation and reimplantation of the avulsed teeth are rarely achieved, principally because of stress and lack of dental knowledge from parents/tutors at the moment of the accident [3]. So, the PDLF should be incubated in a physiological storage medium to maintain its viability during transportation to the dental office because dry storage of avulsed teeth leads to the death of PDL cells of the root [8].

Storage media is one of the critical factors affecting an avulsed tooth replantation success. The ideal storage medium should have the capability of preserving the cellular PDL so that the cells could go through the process of mitosis, and form clones of the damaged fibroblasts of the PDL, and its generating cells [9]. Compatible physiological pH and osmolality, antioxidant property, no or minimal microbial contamination, maintained at an appropriate temperature, high availability, and low cost and also should be readily accessible, especially to families with

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coconut water, tap water, aloe vera. An avulsed tooth can be placed not only in HBSS, but in natural and synthetic solutions, like milk, coconut water, contact lens solution, which can be

found at home or at site of accident. Storage media is desired to help preserve the viability of

PDL cells following avulsion until replantation [11].

A wide variety of solutions suitable to store teeth exists, but it is not clear which storage medium is the most suitable to achieve efficiency to maintain viability of PDL cells as long as possible, seeing as immediate replantation impossible. For these reasons this title is relevant and significant. Thus, the aim of our work is to systematize the available scientific articles and to find out which natural storage media is most effectiveness to maintain viability of PDL cells in extra-oral environment.

Aim:

The aim is to evaluate, compare and to find out the most suitable natural and synthetic storage media for avulsed teeth for transportation to dental office.

Tasks:

1. To find out general storage and transport medias suitable for avulsed teeth.

2. To answer questions which natural storage media is the most effective in cell viability maintenance.

3. To comprehend what effect has pH and osmolality in different storage medias. 4. To find out the most favorable pH and osmolality for PDL cell viability.

Hypothesis:

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1. SELECTION CRITERIA OF THE STUDIES, SEARCH METHODS

AND STARTEGY

1.1. The protocol for systematic review

This systematic review was approved by LUHS Bioethics centre, approval CODE (BEC-OF-114, ANNEX Nr.1). Following the PRISMA (Preferred Reporting Item for Systematic Review and Meta-Analyses) statement [12]. Publications were selected through PubMed and ResearchGate. The following key words and their combinations “storage media”, “tooth

avulsion”, “propolis”, “periodontal ligament”, “milk” was used to search and proved with a help of Medical subject Headings (MeSH) dictionary. The comprehensive search was restricted to English language articles, published from 2011 to 2021. In the result it was shown 313 articles. The duplicated articles were excluded, and 64 articles related to the topic were revised, of which 8 consistent with the subject of this review were qualified regarding to PICOS criteria (table 1).

1.2 Inclusion and exclusion criteria

The selection of articles was based on PICOS model and selected keywords. The systematic analysis of the scientific literature included studies, which correspond to criteria presented in Table No.1.

Table No.1 Articles search criteria according to PICOS

Population (P)

Publications are included if: Publications are excluded if:

• studies with different storage media performed only with human teeth;

• in vitro;

• sudies with different storage media performed on animals;

• in vivo;

Intervention (I) (participants)

Publications are included, which; Publications are excluded, which;

• solutions suitable for avulsed tooth transportation and storage;

• other types of medicaments for dental hygiene ex.: mouthrinse, tooth pastes;

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Publications are included, if contain;

Publications are excluded, if contain; • comparing different storage

media, characteristics, quality to keep PDL cell viability as long as possible;

• different concentrations, storage time and temperature;

• comparing other rinsing fluids with storage media for avulsed teeth;

• liquids do not have effect on PDL cell viability;

Outcome (O) (results)

Publications are included,

describing;

Publications are excluded, describing; • present positive results after used

appropriate storage media;

• negative influence on tooth

reimplantation;

• unsuitable storage media which can cause ankylosis and rooth resorption;

Study characteristics (S)

Publications are included, which: Publications are excluded, which :

• articles published in english language;

• articles published between 2011 and 2021;

• research type: cohort studies, prospective and retrospective clinical trial, prospective and

retrospective review and

observational trials;

• articles published not in english language;

• articles published before 2011;

• research type: metal-analyses,

systematic literature review, one case study, case reports;

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14 1.3. Literature search strategy

Following the PRISMA guidelines, publications of this systemic review were selected through PubMed and ResearchGate databases. The different keyword combinations were used, and Table 2 presents the numbers of results we found.

Table No.2 Keyword combinations and results

Keyword combinations Results

in PubMed Results in Research Gate Search date

“Storage media” AND“tooth avulsion” 34 37 From

2021.03 .19 to 2021.03 .21 “Storage media”AND“periodontal ligament” 35 94

“Storage media”AND “propolis” 9 27

“Storage media”AND“tooth

avulsion”AND “milk”

22 7

“Periodontal ligament”AND“propolis” 12 35

The search and selection of articles was carried out in two stages. At first stage, the title and abstracts of the articles were reviewed, articles that correspond the selection criteria were selected. The duplicated articles were rejected. During the second stage, full-text articles were reviewed, and 9 publications consistent to the PICOS criteria were included in the literature analysis. The whole process of article selection with the use of PRISMA flow chart is representing in image 1 (see in 2.1 section).

1.4. Study selection

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15 1.5. Variables, assumptions and simplifications

In the articles we were searching information about the PDL cell viability after storage of avulsed teeth or periodontal cells in different medias at various time points. The publications were selected and further analyzed in which different naturals and synthetics medias were compared on extracted human teeth or PDL cells scraped from human teeth in vitro. In studies was included: different time ranges, solutions concentration and storage media characteristics ex.: pH, osmolarity.

Assumption: avulsed tooth stored in synthetic media has better postoperative prognosis after the replantation than tooth stored in natural media.

1.6. Assessment of bias risk

(JBI) risk of bias tool was used to assess included studies. This checklist contains 11 questions that assess specific domains of studies to determine the potential risk of bias and could be answered with ‘yes’, ‘no’ and ‘unclear. The risk of bias for individual studies were detected following by low risk of bias if 70% answer is yes, moderate risk of bias up to 50% yes answer and high risk of bias below 50% yes answer. The tool presented in ANNEX Nr. 2.

Table No. 3 JBI risk of bias quality assessment for cohort studies

Checklist

Study Year Study design Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Q10 Q11 yes % Risk Souza et al [20] 201 1 Retrospectiv e x + + - - + + + - - + 54.5 Moderat e risk Badakha h et al

[15] 2013 Retrospective x + + - - + + + - - + 54.5 Moderate risk Adeli et

al [18] 2016 Retrospective x - + - - + + + - - + 45.5 Low risk Ulusoy

et al [17] 2015 Retrospective x + + - ? + + + - - + 54.5 Moderate risk Anegund

i et al

[21] 2016 Retrospective x + + - - + + + - - + 54.5 Moderate risk Tavassol

i-hojjati

et al [19] 2014 Retrospective x - + - - + + + - - + 45.5 Low risk Saxena

et al

[16] 2011 Retrospective x + + - - + + + - - + 54.5 Moderate risk Hwang

et al [14] 2011 Retrospective x + + - - + ? + - - + 45.5 Low risk

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16 1.7 The principal summary measures

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2. SYSTEMIZATION AND ANALYSIS OF DATA

2.1. The number of studies screened

The publications research was done in PubMed and ResearchGate database, in total of 313 were initially identified. After eliminating duplicate publications, 257 articles remained. After review of the title and abstracts, 193 articles were removed due to non-relevant content. The full text was analyzed for 64 studies. According to exclusion and inclusion criteria, 8 scientific

publications were selected in this systematic review [14-21]. The full and detailed selection of the literature is presented in image 1.

Image 1. PRISMA flow diagram

Id

en

ti

fi

cat

ion •PubMed and ResearchGate database

advanced search (n=312)

Additional records identified

through other sources (n=1)

Records after duplicates removed (n =257) Removal of duplication (n=56) Filtered Records screened (n=257) Full-text articles assessed for eligibility

(n=64)

Exclusion criteria -Not relevant title and abstract records excluded (n=193)

Filtered

Filtered Full-text articles _{reviewed and excluded:}

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18 2.2. The characteristics of studies

Overall, 8 articles were included in systematic review study, while all of the were retrospective studies. All studies were performed in vitro, published from 2011 to 2021year.

In studies were assessed the viability of PDL cells in different storage media at various time intervals. The two methods were chosen for evaluating the viability of dental fibroblasts in different storage media. The first method, and most commonly used, is to scrape off fibroblasts from the root surfaces first and then add then to a storage medium for culturing. The PDL cell viability is evaluated at different stimulated time intervals and the cell line is used for the test in this method. In Anegundi et al study [21] was used another method, the extracted tooth is directly placed into the storage medium. After the pre-determined stipulated time, the cells are isolated with the help of enzymes and the tooth is removed from medium to evaluate the viability of the cells [21]. The systematization of data is present in Annex 3.

2.3. The evaluations of outcomes for study

2.3.1. Evaluation of the differences between various cell viability determination methods

There are different methods to assay cell viability with own advantages or limitation [17]. In Sarena et al study [16] was used three methods for cell viability determination and was found that trypan blue dry exclusion assay is the most sensitive assay compared to neutral red and 3- [4, 5-demethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide (MTT) assay. MTT assay was most common used method in studies [15, 17, 18, 20] despite the fact that MTT and Neutral red update (NRU) are known to show some higher values that the exact [16]. It is a colorimetric assay [17] measuring the viable cells which are metabolically active and is based on using a yellow

tetrazolium salt which changes to insoluble formazan crystal in purple hue [15]. In study [14] was used Nucleocounter (NC-100), but the disadvantage of this method is the inability to measure the

number of cells if it is below 5 x103_{. Therefore, 4,}

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19 Table No. 3 The main advantages and disadvantages of PDL cell viability methods evaluation

References PDL cell viability evaluation Advantages Disadvantages Badakhah et al [15], Ulusoy et al [17], Adeli et al [18], Souza et al [20] MTT assay The accuracy, timesaving, and no need use of radioisotopes

MTT show some higher values that the exact Hwang et al [14] MTS and NC-100 NC-100 provide faster, more efficient and more reliable values. Able to handle a large number of samples with a high degree of precision.

The inability to measure the number of cells if it is below 5 x103 Saxena et al [16] MTT, NRU and Trypan blue dye exclusion assay Trypan blue dye exclusion assay ids the most

sensitive assay among all the assays

MTT and NRU are known to show some higher values that the exact. They are indirect assay.

Anegundi et al [21] Trypan blue staining Quick easily performed

Only assess the viability of the cell and not actual physiologic health or

metabolic capabilities of the cell

Tavassoli-hojjati et al [19]

Neutral

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20 2.3.2. The milk and milk substitute’s ability to maintain periodontal cell viability

In the studies conducted by Ulusoy et al in 2015 [17] - the only one of the selected articles performed analysis in different types and fats of milk. Extracted 60 caries free human premolars were randomly divided in 3 groups and transferred in each storage medium namely: low fat milk, medium fat milk and high fat milk at room temperature. In 2011 Souza et al study [20], was maintained that the viability of PDL cells affects the incubation temperature. Experiment showed that a lower temperature has a positive effect on cellular viability maintenance [20], so this study tested human PDL cell viability at 5 temperature. Ulusoy et al [17] used human PDL cell scraped off from the root surface of healthy extracted premolars. The cells were exposed to the storage solutions: tap water, Eagle’s maintenance medium (EMM), Hanks Balanced Salt Solution (HBSS), lactose-free milk, whole milk, skimmed milk, soy milk, goat milk, milk with probiotic, propolis, egg white. The goat milk showed the highest capacity to maintain PDL cell viability at all test interval (P<0.001). At first hour was not significant different between the tested all types milks, propolis, and egg white (P>0.05).

The results confirmed that lower milk temperature can maintain periodontal cell viability significantly longer up to 120 hours [20].

2.3.3. The natural and artificial storage media for avulsed teeth

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Hwang et al [14] studied in vitro the possibility of green tea (GT) and green tea extract (GTE) to store avulsed teeth at various time duration. The pH level of GT was 6.294 and its osmolarity value was reported 12 mosmol/kg. The GTE had higher osmolarity value 139 mosmol/kg and pH 5.913. In this study, cell viability was tested by two methods, first using a nucleocounter and second MTS assay. Cell viability exanimated by nucleocounter showed that in GTE solution 97.3% of periodontal cells stay alive for all 24 hours. Comparing GTE with HBSS was not significant differences (P<0.05). GT able to keep alive periodontal cells only until 3 hours compared to GTE, and after 3 hours the viability of cells only less than 40%. The MTS assay showed a similar result as the nucleocounter. There were no significant different between HBSS and GTE. After 3 hours, the GT group had a significantly lower (P<0.05) than those of GTE and HBSS [2].

Badakhsh et al [15] in his study tested Aloe Vera extract as a new storage and transport media for avulsed teeth. Four different concentrations 10%, 30%, 50% and 100% had been used in study and compared with egg white. Aloe Vera extract in different concentrations (10%, 30% and 50%) had the greatest ability to preserve the cell viability at least up to 9 hours and there was no

significant difference among them (P<0.001). Egg white had a significantly lower capacity compared to Aloe Vera extract (10%, 30% and 50%), but was significantly better that Aloe Vera 100% [15].

In vitro experiment Saxene et al [16] evaluate the potential of the propolis as storage media for avulsed teeth in various time (30min, 1, 3, 6, 12 and 24 hours) periods. Also, in this study was examinated as storage media artificial saliva and propolis mixed with Dulbecco’s modified Eagle’s medium

(DMEM). The cell viability was assessed by MTT, neutral red uptake and trypan blue dye exclusion assay. No significant changes were observed in the viability of cells until 3h between the propolis solutions except for artificial saliva (P<0.001) [16]. From 3 hour in artificial saliva was not detected any alive cells 0.0%. Propolis 10%+DMEM has showed the best performance and was able to retain the cell viability up to 79.8±3.4%, 82.7±3.4% and 78.5±5.6% at 24h in MTT, NRU, and trypan blue assays [5]. At 12 and 24 hours propolis 10%+DMEM maintain cell viability more significantly than propolis at 2.5%, 5.0%, 20% and HBSS. Propolis 10% was found to be the most efficacious to preserve the viability of PDL cells among all the

concentrations of propolis used in the study [16].

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water maintains significant higher number of available cells than stored in industrialized coconut water at every time period (P<0.05). At 3 and 6 hours, there were no significant differences between HBSS and natural coconut water (P<0.05), whereas from 24 to 120h, industrialized coconut water and tap water were the worst storage media (P<0.05) [20].

Anegundi et al [21] in 2016 assessed varies different natural (coconut water, egg white, milk, tap water) and artificial (saline, Gatorade, contact lens solution) storage medias at different time intervals from 1 hour to 24 hours. The number of viable cells were counted under a light

microscope and according to the results, tap water maintained the least number of viable cells followed by Gatorade, saline, coconut water and egg albumen, whole milk and lens solution demonstrated higher values. Egg white significantly maintain viability of periodontal cells up to 2 hours, later number of alive cells dramatically decline up 0.0% at 24 hours. In this study, for 1, 2 and 4 hours intervals, coconut water showed insignificant values as compared to milk and egg white. Gatorade solution showed unsatisfactory results compared to others artificial storage medias, while the contact lens solution showed the highest viability up to 24 hours. In the study was founded that milk especially low-fat milk can be used as a storage media up to 6 hours [21]. The all-research dates are listed in Table No. 4.

Table No. 4 Osmolality and pH comparison at different storage media

Cell viability (%) at different time periods (hours) Recommended media

Author Storage media pH Osmolarity (mosmol/kg) 1 2 3 4 6 9 12 24 GTE Hwang et al [14]

HBSS _7.382 284 _95.1 _{No data} _94.3 _{No data} _96.0 _dataNo _92.9 _93.3

Tap water 7.134 3 48.6 No data 7.4 No data 10.0 data No 0.0 0.0

Milk 6.709 288 60.4 No data 55.8 No data 43.2 data No 45.0 35.1

GT 6.294 12 79.1 No data 32.6 No data 19.8 data No 19.6 22.2

GTE 5.913 139 99.0 No data 100.0 No data 100.0

No

data 98.8 97.2

Adeli et al [18]

DMEM 7.3 305 76.4 55.4 No data 95.3 No data data No data No 75.2

GTE

Tap water 4.8 15 33.0 28.1 No data 9.5 No data data No data No 5.4

HBSS 7.2 280 75.3 68.3 No data 90.7 No data data No data No 60.5

Whole milk 4.6 286 92.1 92.5 No data 93.6 No data data No data No 97.6

GTE 5.4 87 94.2 100.5 No data 105.0 No data data No data No 115.9

GTE +

sucrose 5.2 300 96.1 80.3 No data 98.5 No data data No data No 92.9

Sucrose 5.7 213 30.0 31.0 No data 20.3 No data data No data No 4.8

Badakhsh et al [15]

DMEM 6.87 310 105 No data 100 No data 100 100 data No data No

Aloe vera in 10%, 30% and

50% Aloe vera

10% 7.15 324 96.98 No data 94.47 No data 95.49 96.27 data No data No

Aloe vera

Aloe vera 100% 5.21 296 32.88 No data 29.23 No data 29.92 26.67 No data No data

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23 2.3.4. The most recommended natural storage media for avulsed teeth based on storage time

In all studies, cultures cells were subjected along different natural and artificial solutions at various time intervals. One-way ANOVA was used to detect differences between the groups at each time point [14-19, 21] and P<0.05 was taken to indicate significant differences.

Anegundi et al [21] identified low fat milk statistically significant from medium and high fat milk but can be used as a storage medium up to 6 hours, later low-fat efficacy decreases. Ulusoy et al [17] found that capacity of goat milk to maintain cell viability is the highest between tested milk substitutes until 6hours. Sauza et al [20] compare skimmed milk and whole milk at 5 °C found that cooled milk effectively maintains periodontal cells viability up to 120 hours. Both milk ability to maintain periodontal cells viability similar, however at 120 hours skimmed milk showed higher percentage (35.8%) than whole milk (27,4%).

Image 2. Cell viability (%) in various fat milk based on time

In two studies [14, 18], the green tea and green tea extract was tested in vitro. Hwang et al [14],

results indicated that GTE efficiently to maintain periodontal cells viability is significant at all time periods. However, GT was stated as a short period storage media of 1hour to maintain PDL cell viability, after 1hour efficiency decline. 2016 was stated that GT and GT + sucrose were equally effective to protect the cells [18].

0 10 20 30 40 50 60 70 80 90 100 1h 2h 4h 6h 24h

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24

Anegundi et all [21] in 2016 study, was stat that coconut water showed insignificant values as

compared to milk and egg white. A study done by Souza et al [20], also state that coconut water at 96 and 120 hours performed better than HBSS, while milk showed more significant values than coconut water on various time. Also, Anegundi et al [21] stated that egg white can be perfect medium for avulsed teeth for 2 hours. Conducted by Badakhah et al [15] have shown that egg white can maintain cell viability up to 6 hours, white Ulusoy et al [17] found that egg white maintains periodontal cells save only up to 1 hours.

Tavassoli-Hojjati et al [19] evaluated PJ capacity to save periodontal ligament cells, based on this study 7.5% PJ had the most significant effect on PDL cell viability until 24 hours (P<0.05).

Image 3. Cell viability (%) in tested media based on time

Saxena et al [16] compared four different propolis concentrations, was determined not

significant different among the propolis group. By 24h, propolis 10% was found to be the most efficacious to preserve the viability of PDL cells among all the concentrations of propolis used in the study [16]. In agreement, Ulusoy et al [17] present that propolis is effective solution to

maintain periodontal cells up to 24 hours. Badakhsh et al [15] in 2013 during the study was found aloe vera in 10, 30 and 50% concentrations had the greatest capacity to maintain periodontal cells up to 9 hours. The results of natural storage media presented in tables 5, 6 and 7.

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25

Table No. 5 Cell viability in various natural medium based by short storage time

References Storage media Cell viability %

30min 1h 2hrs

Hwang et

al[14] GT No data 79,1 No data

Hwang et

al [14] GTE No data 99 No data

Adeli et al

[18] GTE + sucrose No data 98 87,5

Anegundi

et al [21] Egg white No data 52,34 52,43

Ulusoy et

al [17] Egg white No data 91,55 No data

Anegundi

et al [21] Coconut water No data 49,99 45

Tavassoli-hojjati et al [19] PJ 7.5% No data 144,43 No data Saxena et al [16] Propolis 10% 96,5 95,2 No data Badakhah

et al [15] Aloe vera 10% No data 96,98 No data

Badakhah

et al [15] Aloe vera 30% No data 98,1 No data

Table No. 6 Cell viability in various natural medium based by medium storage time

References Storage media Cell viability %

3h 4h 6h 9h

Ulusoy et

al [17] Goat milk 109,95 No data 108,47 No data

Hwang et

al [14] GTE 100 No data 100 No data

Adeli et al

[18] GTE + sucrose No data 99,5 No data No data

Tavassoli-hojjati et al [19]

PJ 7.5% 182 No data 219.27 No data

Saxena et

al [16] Propolis 10% 90,4 No data 83,2 No data

Badakhah

et al [15] Aloe vera 10% 94,47 No data 95,49 96,27

Badakhah

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26 Table No. 7 Cell viability in various natural medium based by the longest storage time

References Storage

media

Cell viability %

12h 24h 48h 72h 96h 120h

Hwang et

al [14] GTE 95,6 94 No data No data No data No data

Adeli et al [18]

GTE +

sucrose No data 92 No data No data No data No data

Tavassoli-hojjati et al

[19]

PJ 7.5% No data 206,7 No data No data No data No data

Saxena et al [16]

Propolis

10% 76,8 71,5 No data No data No data No data

Souza et al [20]

Skimmed

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27

3.DISCUSSION

A systematic review of the scientific literature was performed to collect and analyse studies, which examining various storage media capacity to maintain viability of PDL cells at different time points. The purpose of this review was to find the best suitable natural storage media for avulsed teeth.

In the following articles nine natural solutions have been studied: milk, tap water, coconut water, propolis, pomegranate juice (PJ), egg white, green tea (GT), green tea extract (GTE), aloe vera. The shortest incubation time was chosen 30min, whereas the longest storage time was even 120 hours. The prognosis for success of replantation attachment is greatly dependent on the extra-oral dry time to which the avulsed tooth is exposed and placement in a storage medium that is capable of maintaining the viability of the PDL cells [10]. For this reason, in order to have positive results after reimplantation, one of the most important aspect become the

storage/transport media.

Milk has a similar osmotic pressure as periodontal cells, therefore it has been stated as a suitable medium for avulsed tooth storage. The tooth can be hold for up to 6 hours. Milk, like physiological fluid, does not contain the nutrients needed to maintain the viability of periodontal cells, making it unsuitable for long-tern use. Comparing chilled and room temperature milk efficiency was ascertained that milk at 5 temperature, preserve significantly more viable PDL cells than room temperature milk. Chilled milk increased cell viability time up to 120 hours. Saluja et al [1] and Anegundi et al [10] studies different milk fattiness. Milk containing 3% fat maintain cell viability at a significantly greater then medium and high fat milk at 1, 2, 4, 6 hours. Only one study compared different types of milk ability to maintain periodontal cells viability. Ulusoy et al [6] found that goat milk exhibited the highest number of alive cells up to 24 hours. Tap water is easily available at accident place, however the effects on periodontal cells are not only beneficial but also harmful. Tap water is hypotonic solution, it causes lysis of periodontal cells. In studied articles, water was used as a negative group because when a tooth is kept in tap water for a period of time, the cells die. Several studies have been used coconut water as a storage medium for avulsed teeth, but the results were not so good. Anegundi et al [10] and Souza et al [9] observed that coconut water at 37 temperature has less ability to maintain periodontal cells viability that milk.

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showed after 2 hours different between egg white and tap water was not significant. Another study suggests that the egg white can maintain cell viability for up to 6 hours [4]. By 24 hours, propolis 10% was found to be the most efficacious to preserve the viability of PDL cells among all the concentrations of propolis used in study [5]. The higher percentage of viable cells

determined in 7.5% PJ at all times points. Pomegranate juice in 5% and 7.5% concentrations showed significant better results compared with HBSS. The percentages of viable cells peak absorbed at 6 hours (219.27%) in 7.5% PJ. Commercial GT was less effective solution than GTE because it showed hypotonic property and contained other chemical tastes [2]. Adeli et al [7] approved that GTE is more significant more effective than HBSS at 24 hours.

Badakhsh et al [4] evaluate the viability of periodontal cells of avulsed teeth in four different aloe vera concentrations and results suggest that aloe vera (10%, 30%, 50%) had the highest of viable cells up to 9 hours. Aloe vera 100% had the lowest percentage number in preserving periodontal cells viability as same as tap water.

This systematic review has had limitations. The results may have differed due to different chosen methods for measuring periodontal cell viability, because each method had advantages and disadvantages. It could also have been affected by different preparation method of human PDL cells for experiment. The limitation in scientific articles comparing more different types of natural medias. In this way, it would be possible to compare the efficiency of various media with each other in a better way, avoiding errors due to different evaluating methods of cells viability. In the analysis of articles, we lacked a case reports about replantation tooth success after some time stored in natural storage media. Therefore, we were unable to assess the natural storage media efficiency on tooth healing with a normal periodontal ligament after replantation.

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CONCLUSIONS

1. Avulsed teeth can be storage in various media: milk, propolis, coconut water, aloe vera, contact lens solution, pomegranate juice, GT, GTE, egg white, HBSS, saline, Gatorade, saliva, DMEM. 2. Cooled skimmed milk presenting as most suitable storage media for avulsed teeth.

3. The growth of cells take place at a pH of 6.6 to 7.8 and osmolality of 230 to 400 mosmol/kg. The osmolality of 30 mosmol/kg and pH of 7.4 to 7.79 cause cells lysis.

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PRACTICAL RECOMMENDATIONS

1. Provide more information about natural solutions suitable for avulsed tooth storage, which are easily available in accident place or at home.

2. Develop an algorithm understandable to parents, teachers, children and nursing in educational institutions. The algorithm should specify how to handle in the situation and in which media avulsed tooth should be stored until replantation.

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REFERENCES

1. Jain D, Nagarajappa S, Dasar P. natural products as storage media for avulsed tooth. Saudi Endodontic Journal. 2015; 5(2):107.

2. Malhotra N. Current developments in interim transport (storage) media in dentistry: an update. British Dental Journal. 2011 Jul 8: 211(1):29-33.

3. Silva E, Rollemberg C, Coutinho-Filho T, Krebs R, Zaia A. A multiparametric assay to compare the cytotoxicity of different storage media for avulsed teeth. Brazilian Journal of Oral Sciences. 2013 June; 12(2): 90-94.

4. Adnan S, Khan F. Storage Media for Avulsed Teeth: A Review. Journal of The Pakistan Dental Association. 2018 July.

5. Thomas T, Gopikrishna V, Kandaswamy D. Comparative evaluation of maintenance of cell viability of an experimental transport media “coconut water” with Hank’s balanced salt solution and milk, for transportation of an avulsed tooth: An in vitro cell culture study. Journal of Conservative Dentistry. 2008 Jan-Mar; 11(1): 22-29.

6. Khinda V, Kaur G, Brar G, Kallar S, Khurana H. Clinical and Practical Implications of Storage Media used for Tooth Avulsion. International Journal of Clinical Pediatric Dentistry. 2017 Apr-Jun; 10(2):158-165

7. Barrett E, Kenny D. avulsed permanent teeth: a review of the literature and treatment guidelines. Endodontics Dental Traumatology Journal. 1997 Aug; 13(4):153-63. 8. Dharati P, Bafna Y. Original research paper readily available storage media for avulsed

teeth in school and community: a review. International Journal of Scientific Research. 2020 October; 9(5):48-51.

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10. Martin M, Pileggi R. A quantitative analysis of Propolis: a promising new storage media following avulsion. Dental Traumatology. 2004 Apr; 20(2):85-9.

11. Vishwanathan S, Nandan N, Sauni Raj N, Anitha C, Rajappa M. Storage Media-A review. European Journal of Medical and Health Sciences. 2019 January; Vol. 4, No.6.

12. Liberati A, Altman D, Tetzlaff J, Mulrow C, Gotzsche P, Ioannidis J et al. The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluwsate healthcare interventions: explanation and elaboration. BMJ. 2009;339(jul21 1): b2700- b2700.

13. Moola S, Mumm Z, Tufanaru C, Aromataris E, Sears K, Sfetcu R, Currie M, Qureshi R, Mattis P, Lisy K, Mu P-F. Chapter 7: Systematic reviews of etiology and risk. In: Aromataris E, Munn Z (Editors). JBI Manual for Evidence Synthesis. JBI, 2020.

14. Hwang J, Choi S, Park J, Kang S. The use of green tea extract as a storage medium for the avulsed tooth. Journal of Endodontics. 2011 Jul;37(7):962-7.

15. Badakhsh S, Eskandarian T, Esmaeilpour T. The use of aloe vera extract as a novel storage media for the avulsed tooth. Iranian Journal of Medical Sciences. 2014 Jul; 39(4): 327-32. 16. Saxena P, Pant V, Wadhwani K, Kashyap M, Gupta S, Pant A. Potential of the propolis as

storage medium to preserve the viability of cultured human periodontal ligament cells: an in vitro study. Dental Traumatology. 2011 Apr; 27(2): 102-8.

17. Ulusoy A, Kalyoncuoglu E, Kaya S, Cehreli Z. Evaluation of goat milk as storage media to preserve viability of human periodontal ligament cells in vitro. Dental Traumatology. 2016 Aug; 32(4): 264-8.

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19. Tavassoli-Hojjati S, Aliasghar E, Babaki F, Emadi F, Parsa M, Tavajohi S, Ahmadyar M, Ostad S. Pomegranate Juice (Punica Granatum): A New Storage Medium for Avulsed Teeth. Journal of Dentistry. 2014 Mar; 11(2): 225-232.

20. Souza B, Luckemeyer D, Reyes-Carmona J, Felippe W, Simoes C, Felippe M. viability of human periodontal ligament fibroblasts in milk, Hank’s balanced salt solution and coconut water as storage media. International Endodontic Journal. 2011 Feb; 44(2): 111-5.

21. Anegundi R, Daruwalla S. Assessment of Viability of Periodontal Ligament Cells in

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ANNEXES

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35 Annex 2. According the Joanna Briggs Institute critical appraisal checklist for cohort studies

yes no unclear

not applicable

1.Were the two groups similar and recruited from the same population?

2.Were the exposures measured similarly to assign people to both exposed and

unexposed groups?

3.Was the exposure measured in a valid and reliable way?

4.Were confounding factors identified?

5.Were strategies to deal with confounding factors stated?

6. Were the groups/participants free of the outcome at the start of the study (or at the

moment of exposure)?

7.Were the outcomes measured in a valid and reliable way?

8.Was the follow up time reported and sufficient to be long enough for outcomes to

occur?

9.Was follow up complete, and if not, were the reasons to loss to follow up described

and explored?

10. Were strategies to address incomplete follow up utilized?

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36 Annex 3

Table No. 3 Systematization of data and characteristics of studies

Author Study of type Model Types of media PDL cell

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37 Ulusoy et al [6] Retrospective Human PDL cells • Tap water • HBSS • Lactose-free milk • Whole milk • Skimmed milk • Soy milk • Goat milk • Milk with probiotic • Propolis • Egg white • EMM 2% MTT assay

Goat milk showed the highest

capacity to maintain cell viability at all test intervals (P<0.001). Anegundi et al [10] Retrospective Extracted human premolars

• Low fat milk • Medium fat

milk

• High fat milk • Coconut water • Egg white • Tap water • Saline • Gatorade • Contact lens solution 0.4% trypan blue exclusion staining

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38 • Propolis 10% + DMEM • Propolis 20% + DMEM Hwang et al [2] Retrospective Human PDL cells • HBSS • Tap water • Milk • GT • GTE NC-100 and MTS assay GTE and HBSS media do not show significant

THE MOST RECOMMENDED STORAGE AND TRANSPORT MEDIA FOR AULSED TEETH A systematic review

Ugnė Vyšniauskaitė