Comparative analysis of S100 protein expression in healthy and irreversibly inflamed human dental pulps: A systematic review

1. Introduction

The dental pulp (DP) is a highly intricate connective tissue innervated by trigeminal sensory neurons, primarily types A and C (Henry and Hargreaves, 2007). Pain occurs when the densely innervated pulp-dentine complex becomes inflamed (Bergenholtz, 1981; de arruda et al., 2023; Morse, 1977). Rapid responses to acute pain are stimulated by type A myelinated fibers situated in the farthest layer of the coronal DP (Bergenholtz, 1981). In DP, type C nerve fibers are responsible for the slow transmission of pain sensation in response to noxious stimuli (de arruda et al., 2023; Morse, 1977). This involves a plethora of events, like neuro-immune and neuro-vascular changes, ultimately leading to degenerative changes in DP (de arruda et al., 2023). Currently, DP sensibility tests are the most commonly used tools to determine pulp status based on pulpal neural innervation (de arruda et al., 2023). However, literature has shown that the classification of pain depending upon the clinical tests only is not entirely dependable for arriving at an endodontic diagnosis (Bjørndal et al., 2019; de arruda et al., 2023). Hence, various classifications have been proposed, combining clinico-histological conditions to arrive at an accurate diagnosis, which can result in a diagnostic dilemma (Bjørndal et al., 2019; de arruda et al., 2023). The correlation between the clinical symptoms and histology of the dental pulp is often obscure, leading to diagnostic challenges (Bjørndal et al., 2019; de arruda et al., 2023; Yildirim, 2024). No link has been fully elucidated between pain and irreversible changes in DP, as demonstrated by previous studies (de arruda et al., 2023). Hence, there is a need to devise alternative diagnostic tests to match the clinical diagnosis and symptoms.

The levels of various proteins, such as S100 protein, can be accurately assessed in pulpal blood to aid in the diagnosis of pulp conditions. These S100 proteins are calcium‐binding, neuronal regulatory proteins used to evaluate the DP’s nerve structures (de arruda et al., 2023). A large variety of cellular functions, such as cell growth, proliferation, differentiation, apoptosis, calcium homeostasis, energy metabolism, inflammation, and migration, are carried out by S100 proteins (Jungbluth et al., 2023; Khorasani et al., 2018). Additionally, S100 is an important part of host’s acquired and innate immune system (Sorci et al., 2010). It is linked to varied manifestations of gene expressions leading to a plethora of diseases (Cizkova et al., 2021). Inflammation of DP leads to upregulation of several variants of S100 protein, such as S100-A7, -A8, and -A9 (Sorci et al., 2010) (Jungbluth et al., 2023). The nerve fiber density in healthy, as well as inflamed human pulp, and possibly compromised integrity in pulpal inflammation with the progress of inflammation, are investigated using S100 proteins, which are expressed mainly by Schwann cells (Jungbluth et al., 2023; Khorasani et al., 2018).

McLachlan et al., showed that there is an increased expression rate for S100-A8, -A9, -A12, and -A13 in DP of carious teeth in comparison to non-carious teeth (McLachlan et al., 2004). On the contrary, Khorasani et al., demonstrated reduced levels of S100-A12 in human DP with inflammation. In carious teeth, S100-A7 is secreted by matrix metalloproteinases 20 (MMP-20) (Komichi et al., 2019). Charoenpong et al., corelated the relation between the mechanical stress on S100-A7 and osteoclast differentiation. They found that there is a significant upregulation of S100-A7 levels in DP of orthodontically treated teeth. Additionally, it was shown that S100-A7 stimulated the differentiation of monocytes into odontoclasts (Charoenpong et al., 2019). Gallorini et al., opined that S100-A4 expression is increased 10- to 100-fold in odontoblasts and can therefore be used as a possible marker for these cells. S100-A4 is an integral part of inflammation and mineralization processes (Gallorini et al., 2021a). In irreversible pulpitis, expression of various S100 proteins is altered in comparison to normal DP tissue (Jungbluth et al., 2023). Therefore, S100 protein expression is considered important in different inflammatory changes of the DP. However, the results of previous studies regarding S100 proteins are contradictory. Hence, the current systematic review was conducted to evaluate the tissue-specific localization of S100 proteins in normal and inflamed DP.

2. Methods

2.1 Protocol and registration

Preferred Reporting Items for Systematic Reviews and Meta-Analyses, 2020 (PRISMA 2020) guidelines and the Cochrane Handbook for Systematic Reviews were followed while planning for this meta-analysis. It was listed at PROSPERO under registration code CRD42024557251 (Fig. 1).

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Fig. 1.

PRISMA 2020 flow diagram

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3. Results

4. Discussion

Trauma-induced inflammation of DP tissues may lead to root resorption due to the release of various cytokines (DE ARRUDA et al., 2023). In such inflammation, dental and periodontal stem cells play a primary role in osteoclast differentiation. Moreover, locally present immune cells (such as macrophages) release various pro‐inflammatory cytokines to attract osteoclast precursors during the inflammation. The occurrence of odontoclasts in the resorption zone has been shown in several studies (Galler et al., 2021; Sasaki, 2003). Resorption of dentin occurs through odontoclasts, which are osteoclast-like cells located in the DP. Expression of cathepsin K, tartrate‐resistant acid phosphatase, H⁺‐ATPase, and MMP‐9 is characteristic of odontoclasts (Babaji et al., 2017). During pulpitis, there is an increase in extravascular fluid movement that may lead to a manyfold increase in intrapulpal pressure, as the DP tissue is encased by a mineralized tissue (Heyeraas and Berggreen, 1999; Yildirim, 2024). When such mechanical stress is experienced by the DP cells, the cells secrete inflammatory mediators, among which is S100-A7. The differentiation of monocytes into osteoclasts/odontoclasts and enhancement of osteoclast resorption activity is promoted by S100-A7, contributing to root resorption (DE ARRUDA et al., 2023; Galler et al., 2021).

A comparative analysis was done between irreversibly inflamed (symptomatic and asymptomatic) and healthy DPs for the expression of thirteen different S100 genes. Both groups had significantly altered gene expression patterns, which established pulpitis as a clinical diagnosis. The expression of the majority of the S100 genes in SIP was decreased compared with healthy and AIP. Under the conditions of SIP, S100-A1, -A2, -A3, -A4, -A6, -A10, and -A13 were shown to be down-regulated, whereas these genes seem to be less affected in AIP. An endogenic pro-inflammatory factor, S100-A12, is considered a Damage-Associated Molecular Pattern (DAMP) molecule. Its expression is observed in several inflammatory diseases and can lead to the exacerbation of inflammation. One of the investigations in this review showed that, in comparison to healthy, non-inflamed pulp, mRNA levels of S100-A12 were considerably lower in inflamed pulp (Jungbluth et al., 2022). Given the inflammatory state of the inflamed pulp, it was anticipated that S100-A12 expression would rise; nevertheless, the findings run counter to the hypothesis. Toll-like receptors (TLRs) are important for identifying DAMPs and causing inflammation in injured tissues (Piccinini and Midwood, 2010). Previous investigations have shown that irritated pulp increases TLR expression levels, particularly TLR2 (Lee et al., 2021; Mutoh et al., 2007). According to the results of this study, the S100-A12 molecule, which is recognized by the Receptor for Advanced Glycation End Products (RAGE), does not play an important role in inflamed pulp. Detection and localization of S100 proteins in normal, symptomatic, and AIP were found using immunohistologic microscopy (Sorci et al., 2010). However, in inflamed DP, a significant alteration in S100 protein was seen at various locations, particularly in the stroma, odontoblast region, the pulp, and around the calcification bodies.

Thus, the study’s null hypothesis was disproved. This is the first comprehensive and systematic assessment of S100 protein immunohistochemical investigation in the meticulous diagnosis of inflamed pulpal tissues (Komichi et al., 2019). To determine the potential involvement of the corresponding S100 proteins in calcification processes, pathological defense, and physiological defense, analyses were performed at four distinct anatomic localizations. The odontoblast layer and calcification boundary regions showed accumulation of the proteins that are thought to interact during dentin and pulp stone formation (Goldberg, 2011). Potential involvement of the S100 protein in the pulpal functioning and inflammation was estimated and examined in the vascular walls and pulpal stroma (Pohl et al., 2024).

Excessive accumulation of S100-A1 protein at the odontoblast layer and calcification border areas in normal pulp (NP) and AIP indicates its active role in the calcification process (Gallorini et al., 2021b). Moreover, no reduction of S100-A1 protein in SIP compared with NP was found. In contrast, there was intense staining of pulpal stroma, suggesting that the accretion of S100-A1 protein would be more in SIP than in NP. In the pulpal stroma, S100-A2 was shown to be more prevalent in AIP and SIP than in NP. According to reports, S100-A3 enhanced the incidence of pulpal stroma inflammation, caused significant staining at the calcification boundary areas, and was more prominent in the odontoblast layers of NP, AIP, and SIP. These findings suggest that this protein plays a role in calcification processes.

Unlike the majority of the other S100 proteins at this site, which were distributed over a larger area, the S100-A4 protein accumulated in the inflamed pulpal stroma in a cluster-like pattern (Zhan et al., 2023). In the odontoblast layer, S100-A4 primarily displayed moderate to intense staining, although in two-thirds of the SIP specimens, this position could not be determined (Cai et al., 2022). In the pulpal stroma and odontoblast layer, there were more dispersed round monocytes with strong S100-A4 nuclear staining (Jungbluth et al., 2023).

Even under normal circumstances, S100-A6 appeared to be at a reasonably high level in the pulp tissue, at the calcification sites, and in the vessels; there were no discernible modifications when inflammation occurred (Yamamoto et al., 2015). In the odontoblast layer, it was significantly accumulated in NP and AIP, while SIP was decreased. S100-A7 was only weakly stained, and there were no discernible differences between the odontoblast layer and pulpal stroma under normal and inflammatory settings (Donato et al., 2013). Strong staining was seen in AIP single stroma specimens. On the other hand, all diagnoses had moderate to severe staining at the calcification border locations, while AIP 3030 showed the most prominent staining (Zhou et al., 2020). The odontoblast layer in NP showed moderate to high staining, whereas SIP with S100-A8 and -A9, and AIP with S100-A9, showed significantly less staining. In AIP, the decrease was less noticeable with S100-A8. In the pulp, both proteins were substantially more prevalent in AIP and SIP than in NP. For both proteins, moderate to intense staining was seen at the calcification border areas in all three diagnoses, but S100-A9 showed a notable intensification. Alongside the vessels, extreme stains were observed, but there was no discernible pattern between the groups (Jungbluth et al., 2023; Komichi et al., 2019).

5. Limitations and Future Implications

The included studies have only mentioned the occurrence of various S100 proteins at different anatomic locations in inflamed pulp and have compared them with normal pulp, but future studies can be carried out to describe the importance of their occurrence and their clinical correlation.

6. Conclusions

Proteins S100-A1, -A2, -A3, -A4, -A6, -A8, and -A9 were substantially less prevalent in permanently inflamed DP than in normal DP at several anatomic locations. S100-A7 detected certain trends, although they weren’t very noticeable. In the pulpal stroma, S100-A1, -A2, -A3, -A4, -A8, and -A9 proteins were shown to be substantially more prevalent in AIP and SIP specimens than in NP. S100-A1, -A6, -A8, and -A9 were shown to be substantially less common in SIP than in NP, whereas S100-A9 was found to be much less common in AIP than in NP in the odontoblastic layer. Under specific circumstances, S100-A1, -A2, -A3, -A4, -A6, -A8, and -A9 appear to be involved in the development of pulp stones and localized calcification phenomena of the DP.