The role of salivary proteins in early childhood caries: a scoping review

2.1 Study design

This scoping review was conducted using a structured methodological approach based on the Joanna Briggs Institute (JBI) framework and guided by the updated Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines [7, 8]. These guidelines ensured a transparent, systematic, and replicable approach to identifying and mapping existing literature related to salivary proteins and ECC, while addressing heterogeneity in study designs, methodologies, and outcome measures.

2.2 Research questions and study tools

This review aimed to explore the association between salivary proteins and ECC. The focused research question was:

“What salivary proteins have been investigated in relation to ECC, and how do their concentrations differ between CA and CF children?”.

To address this question, a scoping review was conducted using the Population, Concept and Context (PCC) framework. The Population (P) of interest included children in early childhood, typically aged six years and below, without systemic diseases. The Concept (C) focused on salivary proteins, including but not limited to lactoferrin, immunoglobulins, mucins, and proline-rich proteins. The Context (C) involved the presence or absence of ECC, with studies examining differences or associations between CA and CF groups.

2.3 Eligibility criteria

Children aged 0–6 years were included, consistent with the accepted definition of early childhood caries and to capture the full developmental period during which ECC may occur. For consistency throughout this review, children with one or more CA lesions Decayed, Missing, Filled Teeth (DMFT/Decayed, Missing, Filled Surfaces (DMFS) ≥1 or equivalent diagnostic criteria) are referred to as CA, while children with no clinical evidence of caries (DMFT/DMFS = 0) are referred to as CF.

2.4 Information sources and search strategy

A comprehensive literature search was conducted using four major electronic databases: PubMed, EBSCOhost (Dentistry & Oral Sciences Source), ScienceDirect, and Google Scholar. These databases were selected to ensure broad coverage of relevant biomedical and dental research. The search strategy combined controlled vocabulary (such as Medical Subject Headings (MeSH) terms in PubMed) and free-text keywords, including “early childhood caries”, “saliva”, and “salivary proteins”, linked using Boolean operators (AND, OR) to optimize sensitivity and specificity. The search was conducted in accordance with PRISMA-ScR guidelines, limited to English-language, peer-reviewed publications published between January 2020 and August 2025, and excluded grey literature to ensure data quality and comparability. Additional studies were identified by manually screening the reference lists of key articles and relevant reviews. The search strings and details are provided in Table 1.

2.5 Selection of evidence sources (study selection process)

The screening process was conducted independently by two researchers, who reviewed all retrieved records based on the following criteria: (ⅰ) studies that involved children in early childhood (typically under six years old) who were free from systemic diseases, and (ⅱ) studies that examined salivary proteins in the context of ECC, either through clinical or in vitro analysis. Eligibility assessment was guided by the predefined inclusion and exclusion criteria. Discrepancies during study selection were resolved through discussion, and, when necessary, a third reviewer was consulted to reach consensus. The rationale for each inclusion and exclusion criterion is outlined in Table 2.

2.6 Data charting process and data items

Extraction and synthesis of information from the included articles were summarized and presented in tables (Supplementary Table 1, Ref. [9, 10, 11, 12, 13, 14, 15, 16, 17, 18]; Supplementary Table 2, Ref. [9, 10, 11, 12, 13, 14, 15, 16, 17, 18]), organized under descriptive, methodological, and thematic categories corresponding to the objectives and research questions of this scoping review. To ensure systematic reporting and consistency throughout the review process, a charting table was developed during the protocol stage to record relevant information, including author, year, country, study population, salivary proteins investigated, methods of analysis, and main findings.

The data extraction process was conducted using a structured charting form, which was piloted independently by two researchers on a sample of three articles. Following this pilot, the form was refined to ensure alignment with the review objectives and the PCC (Population, Concept, Context) framework. The finalized charting table was then applied consistently across all included studies and updated iteratively throughout the review process as new insights emerged.

A critical evaluation of the evidence regarding the association between salivary proteins and ECC was performed. The charted data were mapped to address the following core review questions:

1. What types of salivary proteins are associated with ECC?

2. What are the differences in salivary protein profiles between CA and CF children?

3. What laboratory or clinical methods are commonly used to identify salivary proteins in studies related to ECC?

4. What is the magnitude of the association between salivary proteins and caries status, and what are the implications for their use as predictive biomarkers?

The extracted data were analyzed based on the type of protein, saliva collection and processing methods, analytical approach (e.g., Western blot, ELISA, proteomics), and population characteristics. Most studies quantified targeted proteins using enzyme-linked immunosorbent assays (ELISA), reflecting a hypothesis-driven approach. In contrast, a smaller number of studies employed proteomic techniques, such as liquid chromatography–mass spectrometry (LC–MS/MS), enabling broader protein profiling. Variations were also observed in sample preparation procedures, including centrifugation conditions, protein normalization strategies, and the use of commercial versus in-house assay kits. Attention was given to studies comparing CA and CF children, either clinically or via in vitro experiments. Differences in study design, diagnostic criteria for ECC, and sample handling methods were extracted and summarized to facilitate comparison across studies and highlight sources of analytical heterogeneity.

3.1 Study selection

The database search identified 612 records. After removing duplicates and ineligible records, 119 articles remained for title and abstract screening. Following this screening, 119 full-text articles were assessed for eligibility. Of these, 20 articles could not be retrieved, and 89 were excluded for not meeting the inclusion criteria. Ultimately, 10 studies fulfilled the eligibility criteria and were included in this scoping review. Fig. 1 presents the PRISMA-ScR flow diagram illustrating the study selection process and the reasons for exclusion.

Fig. 1.Flowchart of literature search and selection criteria adapted from the PRISMA.

3.2 Primary characteristics of included studies

Supplementary Table 1 summarizes the primary characteristics of the 10 included studies [9, 10, 11, 12, 13, 14, 15, 16, 17, 18]. All studies were conducted in Asia and collectively recruited 646 participants, comprising 313 CA children (48.5%) and 333 CF children (51.5%), with ages ranging from 9 months to 6 years. Most studies utilized unstimulated saliva samples, with only one study employing stimulated saliva [11]. Saliva collection times varied widely (08:00 AM to 12:00 PM), and reported sample volumes ranged from approximately 1 mL to 5 mL.

Caries assessment methods varied across studies. While the Decayed, Missing and Filled (DMF) index (DMFT/DMFS) was most frequently applied, four studies employed alternative approaches, including visual inspection, intraoral examination, designated codes, and caries detector liquid [10, 11, 13, 16]. Criteria for defining caries status also differed. Three studies classified DMFT ≥1 as indicative of CA children [9, 12, 17], while two studies defined CA children as DMFT/DMFS >4 [14, 16]. Conversely, five studies identified children with DMFT/DMFS = 0 as CF [9, 12, 14, 16, 17]. One study further differentiated low-risk children with DMFT scores between 1 and 4 [18]. This variation in caries assessment and classification criteria, including distinctions between CF and low-risk children, represents a key methodological source of heterogeneity across the included studies and should be considered when interpreting the findings.

3.3 Association between salivary proteins and caries

A range of salivary proteins was investigated across the included studies, namely: (1) lysozyme, (2) IgA, (3) histatin-5, (4) submandibular gland androgen regulatory protein 3B (SMR-3B), (5) Mucin-7, (6) cystatin S, (7) α-amylase, (8) MMP-8, (9) MMP-20, (10) statherin, (11) β-defensin, (12) α-defensin-3, (13) cathepsin G, (14) β-defensin-2, (15) MUC1, (16) MUC2, and (17) lactoferrin (Supplementary Table 2). Significant increases in salivary levels were reported in CA children for lysozyme [9], histatin-5 [10], MMP-8 [14], β-defensin [15], α-defensin-3 and cathepsin G [16], and β-defensin-2 [17]. Conversely, elevated levels in CF children were observed for IgA [10, 18], cystatin S [12], α-amylase [13], statherin [15], and MUC1 [18]. No statistically significant associations were noted for MMP-20 [14], statherin [17], lactoferrin, or lysozyme [18]. Notably, conflicting findings emerged for lysozyme and statherin: lysozyme levels were significantly elevated in CA children in one study [9] but showed no significant difference in another [18]. Similarly, both studies on statherin reported decreased levels in CA children, though statistical significance was observed in one study [14] and not in the other [17].

4.1 Upregulated host-defense proteins in CA children

A key finding was the consistent elevation of innate immune proteins including lysozyme, histatin-5, MMP-8, β-defensin, α-defensin-3, cathepsin G, and β-defensin-2 in CA children. These proteins, typically released during microbial challenges or inflammation, suggest that ECC is associated with a heightened inflammatory response.

The increased Lysozyme level, as reported by Maulitasari et al. [9], likely represents a compensatory response to elevated Streptococcus mutans burden. This aligns with prior studies showing lysozyme disrupts bacterial cell walls and rises during oral inflammation. However, Zhang et al. [18] report contradictory findings, indicating that lysozyme alone cannot reliably distinguish ECC status, as its expression may be influenced by gingival health or microbial load rather than caries alone.

Similarly, histatin-5, an antimicrobial peptide with antifungal and antiplaque properties, was markedly elevated in CA children. This supports the hypothesis that its upregulation is an attempt to control cariogenic biofilms. The observed co-variation between histatin-5 and low sIgA further suggests a compensatory mechanism, where deficiencies in mucosal immunity trigger greater reliance on innate peptides [10].

Matrix metalloproteinase-8 (MMP-8) emerged as a particularly promising marker, strongly associated with severe ECC, this is biologically plausible, as MMP-8 mediates collagen breakdown in demineralized dentin, directly contributing to lesion progression. In contrast, MMP-20, primarily active during tooth development, did not differ between CA and CF children, indicating limited relevance to active caries [14]. Defensins (β-defensin, β-defensin-2, α-defensin-3) and cathepsin G were also elevated, reflecting epithelial defence and neutrophil activation typical of ECC’s inflammatory environment. Notably, cathepsin G remained significant after adjusting for confounders, suggesting its potential as a robust marker of caries severity [15, 16, 17].

Collectively, these findings indicate that ECC is characterized not only by microbial dysbiosis but also by upregulated mucosal immune and inflammatory responses, likely representing compensatory mechanisms against cariogenic biofilms.

4.2 Protective salivary proteins in caries-free children

CF children consistently showed higher levels of proteins involved in maintaining enamel integrity, microbial clearance and immune regulation. These include secretory IgA (sIgA), cystatin S, α-amylase, statherin and MUC1 [12, 13, 18]. These biomarkers are recognized as key components of the salivary innate immune defense due to their roles in microbial aggregation, inhibition of adhesion and modulation of host-microbe interactions [19].

Secretory IgA (sIgA) plays a critical role in immune exclusion by preventing bacterial adhesion, as widely described in mucosal immunity literature [20]. Most studies reported higher sIgA in CF children, although earlier literature indicates that sIgA may occasionally increase in response to bacterial load [21]. Age was positively associated with sIgA, emphasizing the need for age-standardized comparisons in future research [22].

Salivary α-amylase (sAA) was also higher in CF children, consistent with its role in agglutinating bacteria and clearing carbohydrates from the oral cavity [13]. While some older studies suggested sAA increases with caries activity, differences in population age and caries detection criteria may explain these inconsistencies [23].

MUC1, a low-molecular-weight mucin involved in lubrication and microbial adhesion inhibitions, was more abundant in CF children, whereas MUC2 tended to be higher in CA children [18]. This aligns with mucin biology literature, which demonstrates distinct functional roles of mucin subtypes in oral defense [24], suggesting that different mucins may differentially influence caries susceptibility and warrant further proteomic investigation.

Considering the evidence collectively, only a subset of salivary proteins showed consistent associations with caries status. MMP-8 emerged as the most reliable biomarker, consistently elevated in CA children across multiple studies, reflecting its role in active tissue degradation and inflammation. In contrast, cystatin S, α-amylase, and sIgA were more frequently associated with CF status, suggesting protective functions. Other proteins, including lysozyme, histatin-5, and statherin, showed variable or conflicting results and should be regarded as preliminary candidates requiring further validation.

4.3 Methodological heterogeneity and sources of variability

Although methodological heterogeneity was observed across the included studies, several key sources of variability warrant systematic consideration, as they likely contribute to inconsistencies in reported associations between salivary proteins and early childhood caries.

4.4 Toward a multimarker diagnostic approach

Based on this review, no single salivary protein is sufficient to serve as biomarker for ECC. This aligns with broader biomarker research, where multimarker panels generally outperform single-protein indicators due to the multifactorial nature of disease pathways [25, 26]. Combining multiple salivary proteins—including inflammatory markers (MMP-8 and defensins), protective proteins (statherin, cystatin S and sIgA), and enzymatic regulators (α-amylase)—is likely to improve diagnostic accuracy.

In addition, high-throughput proteomics procedure combined with machine learning offers a promising method for developing a saliva-based diagnostic panel suitable for chairside screening in children. Proteomics has demonstrated strong potential for identifying complex biomarker signatures [27, 28], and machine learning has shown excellent accuracy in classifying caries risk from multidimensional salivary data. However, further validation in large and diverse paediatric populations is needed before these models can be used in routine practice [29].

The development of saliva-based diagnostic kit would align with the goals of minimally invasive dentistry which enable early detection, personalized prevention and timely intervention. Moreover, the kit would particularly be valuable for young children who often struggle with conventional diagnostic procedures.

4.5 Knowledge gaps and future directions

Despite growing interest in salivary proteins as potential biomarkers for ECC, significant gaps remain. Most studies included in this review were cross-sectional, limiting insight into the temporal relationship between salivary protein expression and caries initiation or progression. Longitudinal validation across independent pediatric populations is scarce, and the influence of age-related immune maturation, oral microbiome dynamics, and behavioral factors on salivary protein profiles remains insufficiently characterized.

In addition, several practical and methodological challenges must be addressed before salivary protein panels can be translated into clinical tools. Evidence on assay feasibility, cost-effectiveness, and standardization across laboratories is limited, and common confounding factors—such as diet, oral hygiene, fluoride exposure, and concurrent oral inflammation—are often inadequately controlled. The lack of longitudinal and real-world testing further restricts understanding of how salivary protein profiles perform in routine clinical settings. Addressing these gaps will be essential to determine whether salivary protein panels can serve as reliable, non-invasive adjuncts for ECC risk assessment.