article WS

JJOD-1986; No. of Pages 7

Review

Salivary proteins as a biomarker for dental caries— A systematic review

Carla Martins a,b, Ana Karla Buczynski a, Lucianne Cople Maia a, Walter Luiz Siqueira b, Gloria Fernanda Barbosa de Araujo Castro a,* a Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil b Schulich Dentistry and Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada

j o u r n a l o f d e n t i s t r y x x x ( 2 0 1 2 ) x x x – x x x

a r t i c l e i n f o

Article history:

Received 6 September 2011

Received in revised form

26 October 2012

Accepted 28 October 2012

Keywords:

Dental caries

Salivary proteins

Saliva

Systematic review

a b s t r a c t

Aim: To develop a systematic review based on the relation between salivary proteins and

dental caries by comparing subjects with and without caries experience and to evaluate

whether salivary proteins can be considered biomarkers for dental caries or not.

Methods: An electronic search was performed in the PubMed Medline, Ovid Medline, ISI Web

of Science, Medline, Cochrane Library, Lilacs, Scielo, BBO, Paho and Wholis databases

applying the following MeSH terms: ‘‘dental caries’’ OR ‘‘tooth demineralization’’ OR ‘‘dental

caries susceptibility’’ OR ‘‘dental enamel solubility’’ AND ‘‘salivary proteins and peptides’’

OR ‘‘saliva’’ AND ‘‘proteins’’. To be eligible for the systematic review, the observational

controlled studies had to have groups with and without caries experience. Studies with high

risk of bias were excluded.

Results: From a total of 188 identified studies, only seven were included in this systematic

review. Four studies were classified as ‘‘low risk of bias’’ and three as ‘‘moderate risk of

bias’’. Three studies reported a relation between salivary proteins and dental caries.

Conclusion: There was not sufficient evidence to establish salivary proteins as a biomarker

for this disease although three of the seven studies showed a relation between salivary

proteins and dental caries in terms of protein phenotypes, total protein concentration and

protein molecular weight.

# 2012 Elsevier Ltd. All rights reserved.

Available online at www.sciencedirect.com

journal homepage: www.intl.elsevierhealth.com/journals/jden

1. Introduction

Dental caries is the most common infectious oral disease in

human beings1 and is an oral health problem in many

countries. It is a multifactorial disease and bacteria are the

cause of caries lesions. However, additional factors have

demonstrated an influence on the acceleration or slowing

down of the development of new caries lesions. Consequently,

* Corresponding author at: Caixa Postal: 68.066, CCS – Cidade Universita E-mail address: [email protected] (Gloria Fernanda Barbos

Please cite this article in press as: Martins C, et al. Salivary proteins as a b (2012), http://dx.doi.org/10.1016/j.jdent.2012.10.015

0300-5712/$ – see front matter # 2012 Elsevier Ltd. All rights reserved http://dx.doi.org/10.1016/j.jdent.2012.10.015

many researchers have been encouraged to study caries-

inducing factors, as well as defense mechanisms against

dental caries.2

As saliva surrounds soft and hard oral tissues and contains

the necessary elements required for host protection, it could

be expected to be a useful biomarker for oral diagnostics,3 as

has been observed in some studies with emphasis on

proteomic analysis. Based on proteomic analysis, potential

salivary biomarkers for some diseases such as, oral cancer,4

´ ria, Rio de Janeiro, RJ, CEP: 21941-971, Brazil. Tel.: +55 21 78486390. a de Araujo Castro)

iomarker for dental caries—A systematic review. Journal of Dentistry

.

j o u r n a l o f d e n t i s t r y x x x ( 2 0 1 2 ) x x x – x x x2

JJOD-1986; No. of Pages 7

Fibromyalgia syndrome5 and Sjögren’s syndrome6 have

already been identified.

The identification of salivary proteins as biomarkers for

dental caries would allow a subject to be classified as a caries-

susceptible individual when a salivary biomarker was present.

Based on this, the individual could take part in an oral health

programme in order to control diet and hygienic habits to

prevent dental caries development.

However, studies concerning salivary proteins and dental

caries have presented conflicting results. Thus, considering

Evidence Based Dentistry, the present study aimed to develop

a systematic review based on the relation between salivary

proteins and dental caries by comparing subjects with and

without caries experience. Thus, we can evaluate the

possibility of whether salivary proteins can be considered

biomarkers for dental caries or not.

2. Data

This systematic review included controlled observational

studies which evaluated the relation between salivary proteins

and dental caries by comparing subjects with and without

caries experience. Subjects with caries experience were

considered to be those who presented a DMF > 0 while the

subjects without caries experience were those with a DMF = 0.

3. Sources

An electronic search was performed in the PubMed Medline,

Ovid Medline, ISI Web of Science and Medline databases

1. Definition of inclusion criteria;

2. Definition of exclusion criteria;

3. Subjects not exposed to systemic or

development;

4. Description of the dental caries diagn

5. Radiographic exam for dental caries

6. Experienced examiner for dental car

7. Calibrated examiner;

8. Salivary collection description;

9. Salivary analysis description;

10. Statistical analysis description;

11. Groups paired in terms of gender, a

12. Blinded study.

Chart 1 – Quality assessment (criteria to eval

Please cite this article in press as: Martins C, et al. Salivary proteins as a b (2012), http://dx.doi.org/10.1016/j.jdent.2012.10.015

applying the following combinations of MeSH (Medical Subject

Heading) terms: ‘‘dental caries’’ OR ‘‘tooth demineralization’’

OR ‘‘dental caries susceptibility’’ OR ‘‘dental enamel solubili-

ty’’ AND ‘‘salivary proteins and peptides’’ OR ‘‘saliva’’ AND

‘‘proteins’’. The same search strategy was used in Lilacs,

Scielo, BBO, Paho and Wholis Databases, but this time

included the MeSH terms in Portuguese and Spanish.

Additionally, the Cochrane Library was searched for system-

atic reviews about ‘‘dental caries’’ and ‘‘salivary proteins’’. The

database search which included studies published between

1950 and June 2011 was carried out by two of the authors (CM

and AKB). All searches were limited to ‘‘humans’’. Moreover, a

hand search of the references and a ‘‘related articles’’ link

search from the selected papers were performed.

Both authors checked the titles and abstracts of the

identified studies independently. Non-relevant studies were

discarded. Then, the full text of the remaining studies was

read. The relevant studies were chosen and those that fulfilled

the inclusion criteria were selected for this systematic review.

If the two authors carrying out the selection process did not

agree, the opinion of a third author (LCM) was taken into

consideration to reach a consensus.

4. Study selection

Controlled observational studies composed of two groups:

one without caries experience (DMF = 0) and the other with

caries experience (DMF > 0) were initially selected. Only

studies concerning permanent dentition with healthy indi-

viduals who were not taking any medication that could affect

the salivary composition were included. In vitro or in situ

topical fluoride during tooth

osis criteria;

diagnosis;

ies diagnosis;

ge and fluoride exposition;

uate the risk of bias of selected studies).

iomarker for dental caries—A systematic review. Journal of Dentistry

j o u r n a l o f d e n t i s t r y x x x ( 2 0 1 2 ) x x x – x x x 3

JJOD-1986; No. of Pages 7

studies, studies about root caries, reviews and case reports

were excluded. The studies which were identified through the

electronic search using the cited keywords but did not

evaluate the relation between salivary proteins and dental

caries by comparing subjects with and without caries

experience were excluded from this systematic review as

they were considered to have a different research focus.

129 PAPERS EX

Subjects medica

No comparisons susceptible sub

Not in humans:

Review: 21

Different aim: 82

TITLE AND ABSTRACT

EVALUATION

INITIAL SEARCH

(n=188 studies)

FULL-TEXT

EVALUATION

49 PAPERS EXC

No comparisons

susceptible sub

Different aim: 15

PAPERS SELECTED FOR THE SYSTEMATIC

REVIEW

(n=7 studies)

2 PAPERS EXC

Classified as “h

QUALITY

ASSESSMENT

Fig. 1 – Stages of the stud

Please cite this article in press as: Martins C, et al. Salivary proteins as a b (2012), http://dx.doi.org/10.1016/j.jdent.2012.10.015

Furthermore, based on the checklist of items that should be

included in reports of observational studies, according to

STROBE Statement,7 twelve criteria were selected to assess the

methodological quality of the identified studies regarding the

risk of bias (Chart 1). The studies which presented at least 8 of

the 12 evaluated criteria were considered as ‘‘low risk of bias’’;

those which presented from 4 to 7 of the criteria were

CLUDED - REASONS:

lly compromised: 20

between caries-resistant and caries-

jects: 4

2

LUDED - REASONS:

between caries-resistant and caries-

jects: 33

LUDED - REASON:

igh risk of bias”

ies selection process.

iomarker for dental caries—A systematic review. Journal of Dentistry

j o u r n a l o f d e n t i s t r y x x x ( 2 0 1 2 ) x x x – x x x4

JJOD-1986; No. of Pages 7

considered ‘‘moderate risk of bias’’; and those studies which

presented 3 criteria or less were considered ‘‘high risk of bias’’.

Moreover, considering the risk of bias (low, moderate and

high), the studies were also classified as studies with high,

moderate and low evidence, respectively. When classified as

‘‘high risk of bias’’, which also means with low evidence, the

studies were excluded from this systematic review.

5. Results

The electronic searches retrieved 186 non-duplicated

records; one additional paper was identified through the

‘‘related articles’’ link and another one from the hand search.

After checking the titles, abstracts and full texts of the

identified studies nine studies were selected for this system-

atic review. However, two of them (Anderson and Mandel8

and Cowman et al.9) were excluded because they were

classified as ‘‘high risk of bias’’, leaving a total of seven

studies for this systematic review, as shown in Fig. 1. From the

selected studies, four were classified as ‘‘low risk of bias’’10–13

and the other three studies as ‘‘moderate risk of bias’’14–16.

Table 1 shows the quality assessment and Table 2 sum-

marizes the description of the selected studies.

One of the selected studies16 divided the sample in more

than two groups (healthy: 49 individuals completely free of

caries; history of caries: 49 subjects with amalgam or resin

fillings and currently free of caries; and active caries: 47

patients with multiple cavities, including enamel and

dentine). However, for our analysis, the subjects with

history of caries and active caries were considered as

belonging to the same group (group of individuals with

caries experience). The study developed by Tulunoglu

et al.13 had two groups (with and without caries experience)

subdivided in four other groups according to gender and age

(7–10 yrs and 11–15 yrs). However for our systematic review

Table 1 – Quality assessment of the selected studies.

Criteria

Anderson et al., 1982

Stuchell and Mandel, 1983

Dod et al.,

Inclusion criteria X X X

Exclusion criteria X X

No exposition to fluoride during

tooth development

X X

Dental caries diagnosis criteria X X X

Radiographic exam X

Experienced examiner X

Calibrated examiner X

Salivary collection description X X X

Salivary analysis description X X X

Statistical analysis description X X X

Paired groups

Blinded study

Risk of bias Moderate Low Low

Level of evidence Moderate High High

Note: X means that the criterion was present.

Please cite this article in press as: Martins C, et al. Salivary proteins as a b (2012), http://dx.doi.org/10.1016/j.jdent.2012.10.015

we only considered the two groups (with and without caries

experience).

Based on the results of the selected studies, three of them

found a statistically significant difference between subjects

with and without caries experience12,13,16 in terms of salivary

proteins. The study developed by Tulunoglu et al.13 found high

evidence that the total protein concentration increases with

dental caries experience, however, this result was not

corroborated by the other studies15,16. Also, there was

moderate evidence in the study that a protein with 17 kDa

molecular weight was related to dental caries experience.16

However this protein was not identified by the authors.

6. Discussion

The studies selected for this systematic review were those that

best satisfied the minimum criteria to be able to evaluate the

role of salivary proteins related to dental caries. Although

innumerous studies related to dental caries can be found on

the scientific databases, only seven studies were considered

suitable for this analysis.

Nevertheless, from the seven studies selected for the

present systematic review, the majority10,11,14,15 did not

present any evidence of a relation between salivary proteins

and dental caries. For example, the concentration of lysozyme

in parotid and submandibular–sublingual saliva is not by itself

a critical determinant for resistance or susceptibility to

caries.10 Another study14 did not show any evidence of

differences in the overall protein pattern or in the distribution

of proline-rich, parotid acidic, double band, parotid size

variant, parotid middle band, or parotid basic phenotypes

between subjects with and without caries experience. The

authors suggested that due to the wide range of phenotypes,

more studies are necessary to evaluate this relation. Yet, no

significant differences in overall protein composition were

Authors, year

ds 1997

Ayad et al., 2000

Tulunoglu et al., 2006

Shimotoyodome et al., 2007

Roa et al., 2008

X X X X

X X X

X X

X X X X

X

X

X

X X X X

X X X X

X X X X

X X X

Low Low Moderate Moderate

High High Moderate Moderate

iomarker for dental caries—A systematic review. Journal of Dentistry

Table 2 – Description of the selected studies.

Authors, year Country

Sample size (n)

Caries index

Type of saliva Salivary parameters Data analysis Results Other considerations

Anderson

et al., 1982

USA

DMF = 0:46

DMF > 0:47

DMF-T SPS Salivary protein polymorphisms

(Pr, Pa, Db)

x 2 No statistically significant

difference

Criteria for the group of subjects

with caries experience: �5 caries lesions with 3=4 extending

through dentine

Stuchell and

Mandel, 1983

USA

DMF = 0:46

DMF > 0:17

DMF-S SPS and stimulated

and unstimulated

submandibular

and sublingual

saliva

Lysozyme T test No statistically significant

difference

Age: �25 yrs Criteria for the group of subjects

with caries experience: DMF-S

> 15 (�1 new carious lesion within the past yr)

Dodds et al., 1997

USA

DMF = 0:38

DMF > 0:49

DMF-S SPS Histatin, basic and acidic PRPs,

statherin, amylase

and uric acid

ANOVA and linear

regression

No statistically significant

difference

Age: �18–30 yrs Criteria for the group of subjects

with caries experience: �5 decayed tooth surfaces

requiring restoration

Ayad et al., 2000

USA

DMF = 0:9

DMF > 0:9

DMF-S SPS Basic proline-rich peptide Mann–Whitney

and Fisher’s

exact tests

Differences were found in

the phenotypes of proline-rich

proteins expressed by

subjects with and without

caries experience

Age: �50 yrs

Tulunoglu

et al., 2006

Turkey

DMF = 0:40

DMF > 0:40

DMF-S UWS Total proteins Mann–Whitney U-test

and T-test

Total protein concentration

increased in subjects with

caries experience

Age: 7–15 yrs

Criteria for the group of subjects

with caries experience: �5 decayed tooth surface

requiring restoration

Shimotoyodome

et al., 2007

Japan

DMF = 0:9

DMF > 0:12

DMF-S SPS

SWS

Total proteins, amylase,

IgA, glycoprotein

T test, U Mann–

Whitney

No statistically significant

difference

Age: 26–40 yrs (subjects with

caries experience) and 24–45 yrs

(subjects without caries

experience)

Roa et al., 2008

Colombia

DMF = 0:49

DMF > 0:96

DMF-T UWS Total proteins and

proteins with different

molecular weight

Kruskal–Wallis,

U-Mann–

Whitney and x2

A 17 kDa molecular weight

protein was related to

caries experience

Age: >18 yrs

Criteria for the group of subjects

with caries experience: 49 with

history of caries and 47 with

active caries

DMF-T, decayed, missing and filled teeth; DMF-S, decayed, missing and filled surfaces; SPS, stimulated parotid saliva; UWS, unstimulated whole saliva; SWS, stimulated whole saliva; Pr, proline-rich;

Pa, parotid acidic; Db, double band; glu, glutamine; his, histatin; arg, arginine; lys, lysine.

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JJOD-1986; No. of Pages 7

found in parotid saliva11 and whole saliva15 between subjects

with and without caries experience.

The one study13 that found statistically significant results

relating to total protein concentration and dental caries

experience was not corroborated by the other studies15,16

selected for this systematic review. Thus, it must be consid-

ered that, probably, there is no relation between total protein

concentration and dental caries. So, we are in agreement with

Banderas-Tarabay et al.17 that further evidence concerning the

role of salivary proteins in modifying the risk for dental caries

is necessary.

It must be pointed out that many variables can influence

the comparison between the selected studies. For example,

the sample size ranged from n = 9 to n = 96 between the

studies. The age and type of saliva (whole saliva, parotid

saliva, submandibular saliva or sublingual saliva) also varied.

Another example that shows heterogeneity between the

selected studies is that two10,16 of them considered the

increment of caries lesion for the group of subjects with

caries experience, but only one observed a statistically

significant difference between the groups.16

The reason for the difficulty to establish a single

variable as predictive for dental caries severity is probably

due to the fact that it has a multifactorial etiology.18 The

exposure of sample subjects to systemic fluoride during

tooth development, for example, can be considered as a

potential confounder. So, systemic fluoride was adopted as

an exclusion criterion for some selected studies.8,10–13 On

the other hand, other variables, such as the use of xylitol

and participation in an oral health programme (with dental

biofilm control) can also be considered as potential

confounders. However, even if such potential confounders

are present, they will not influ have received the same

treatment (fluoride intake, xylitol or biofilm control).

Nevertheless, regarding the salivary composition, it must

be emphasized that the final structure of most proteins and

peptides present in whole saliva is defined by a complex

series of molecular processes. Thus, knowledge about the

composition of saliva in terms of proteins and peptides is

important not just for the information about the function of

this component but also for the growing interest in saliva-

based diagnostics.3

Sensitive proteomic methodologies, which have opened

up new avenues for the characterization of very small

amounts of organic material, including proteins and pep-

tides,19 are recommended for future studies. Besides,

defined criteria are essential to avoid risk of bias due the

multifactorial aetiology of dental caries. Furthermore, we

highlight that salivary proteins present in acquired enamel

pellicle must also be considered as an important tool in

protection against dental caries, since acquired enamel

pellicle is formed on tooth surfaces and has a role in the de/

remineralization processes.

7. Conclusion

The results of the selected studies showed that only three

of the seven studies found any statistically significant

difference between individuals with and without caries

Please cite this article in press as: Martins C, et al. Salivary proteins as a b (2012), http://dx.doi.org/10.1016/j.jdent.2012.10.015

experience in terms of salivary proteins and dental caries.

Although these studies found a relation regarding protein

phenotypes, total protein concentration and protein molec-

ular weight, it is not possible to conclude that there is

sufficient evidence to establish salivary proteins as a

biomarker for dental caries.

Acknowledgments

This study was supported by the Canadian Institutes of Health

Research (CIHR grants No. 97577 and 106657), the Natural

Sciences and Engineering Research Council of Canada (NSERC

grant No. 371813) and Fundação Carlos Chagas Filho de

Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). W.L.S.

is recipient of a CIHR New Investigator Award (grant No.

113166).

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iomarker for dental caries—A systematic review. Journal of Dentistry