Teeth are ectodermal organs formed through a reciprocal relationship between the oral epithelium and the mesenchyme (Puthiyaveetil et al. 2016). Even though, in the grand scheme of things, deciduous and permanent teeth are organs that function in the same manner, many differences have been observed between them, including in the dental pulp (Arnold et al. 2019; Bardellini et al. 2016; Nukaeow et al. 2022).

Despite several studies having been conducted on the pulp proteome of permanent teeth, the corresponding proteome of deciduous teeth has not been investigated prior to this study. Analysis of biological process term enrichment showed proteins expressed in the deciduous dental pulp to have general functional classifications of cellular process, biological regulation, metabolic process, response to stimulus, localisation, and developmental process. These are similar to the terms identified for the dental pulp proteome of permanent teeth, as reported in previous studies (Eckhard et al. 2015; Eckhardt et al. 2014; Feridouni Khamaneh et al. 2021). Regarding molecular function terms, the top five functions in the dental pulp of deciduous teeth were binding, catalytic activity, transcription regulator activity, molecular function regulator activity and ATP-dependent activity. These likewise showed similarity with permanent dental pulp (Eckhard et al. 2015; Eckhardt et al. 2014; Feridouni Khamaneh et al. 2021), except for transcription regulator activity and ATP-dependent activity, which were more highly represented in deciduous dental pulp. Analysis of cellular component terms found the pulp proteome of deciduous teeth to be largely associated with cellular anatomical entity and protein-containing complex components also reported for the pulp proteome of permanent teeth (Eckhard et al. 2015; Eckhardt et al. 2014; Feridouni Khamaneh et al. 2021).

Despite these categorical similarities, OPLS-DA of pulp proteomes revealed the two dentitions to have completely separate clustering and discriminating characteristics, indicative of significant differences between them. Moreover, the statistical analysis identified 736 DEPs between deciduous and permanent teeth, with the majority being up-regulated in the dental pulp of deciduous teeth. KEGG pathway analysis and cluster enrichment analysis were performed to identify and map DEPs to signalling pathways and revealed the DEPs to be involved in TNF signalling, nuclear factor kappa B (NF-κB) signalling, and odontoclast/osteoclast differentiation. As with the total DEP set, most pathway-associated DEPs were highly expressed in deciduous dental pulp.

TNF signalling activation can promote cell differentiation, proliferation and apoptosis, and thereby ultimately lead to cell death or survival (Aggarwal 2003; Holbrook 2019). The TNF signalling pathway is additionally crucial in the immune response and closely related to the process of inflammation (Van Loo and Bertrand 2023). TNF-superfamily members primarily activate NF-κB, JUN N-terminal kinase, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/ERK2) (Aggarwal 2003). According to the pathway analysis of DEPs, the main avenue of TNF signal transduction in pulp tissue may involve the NF-κB and MAPK pathways. In particular, dual-specificity mitogen-activated protein kinase kinase 1 (MAP2K1), an essential component for transduction and activation of MAPK signalling cascades (Avruch 2007), was up-regulated in the dental pulp of deciduous teeth. Likewise, matrix metalloproteinase-3 (MMP-3), an enzyme that degrades extracellular matrix components and is a well-known inflammatory mediator (Wan et al. 2021), was discovered to be up-regulated in the deciduous dental pulp. Increased MMP-3 production has been reported to be associated with TNF-α signalling activation of the NF-κB and p38-MAPK pathways in cementoblasts, potentially leading to the destruction and remodelling of periodontal tissues (Sanchavanakit et al. 2015).

The NF-κB signalling pathway is a well-established pathway known for regulating immune and inflammatory responses through the activation of a variety of genes involved in the inflammatory process (Lawrence 2009). Regulation of NF-κB signalling begins with the binding of pro-inflammatory cytokines, such as TNF-α or interleukin-1β (IL-1β), to their associated receptors, which initiates the downstream activation of the pathway (Chen and Chen 2013; Lawrence 2009). Such downstream signalling requires activation of the inhibitor of NF-κB kinase (IKK) complex, which consists of the IKKα subunit, IKKβ subunit and NF-κB essential modulator (NEMO). Activation of the IKK complex then leads to degradation of the inhibitor of NF-κB (IκB) and release of NF-κB molecules (Lawrence 2009; Solt et al. 2009). Downstream activation of the NF-κB pathway by IL-1β stimulation could also involve activation of ubiquitin E3-protein ligase, which eventually results in phosphorylation of IKKβ, leading to NF-κB activation (Chen and Chen 2013). In this study, proteins associated with NF-κB signalling, namely IL-1β, the E3 ubiquitin-protein ligase TRIM38 (TRI38) and NEMO, were found to be highly expressed in the dental pulp of deciduous teeth. Meanwhile, ubiquitin carboxyl-terminal hydrolase (CYLD), a tumour suppressor and deubiquitinating enzyme that inhibits NF-κB activity (Leeman and Gilmore 2008), was concurrently downregulated in the pulp of deciduous teeth.

These findings suggested that both the TNF and NF-κB signaling pathways are activated in the pulp of deciduous teeth. This could imply that the pulp of deciduous teeth is more prone to inflammatory and immune responses than that of permanent teeth, which might help to explain why some pulp therapy outcomes differ between deciduous and permanent teeth (Akhlaghi and Khademi 2015; Sanusi and Al-Bataynehb 2023; Silva et al. 2019).

Physiologic root resorption is a phenomenon unique to the deciduous dentition. The roots of deciduous teeth are only complete for a short period of time; resorption begins within three years after the roots are fully formed and continues throughout the tooth's life (Sheid and Weiss 2012). Even in the absence of a permanent successor, physiologic resorption of deciduous teeth proceeds, but at a relatively slow rate (Harokopakis-Hajishengallis 2007). Animal model research has revealed physiologic root resorption to be a complex early process involving a series of biological events, not just a single event that occurs prior to exfoliation (Lin et al. 2012; Murthy and Bhojraj 2023; Sasaki et al. 1990). In the interest of investigating the protein profiles and gaining a better understanding of pulp functions in deciduous dentition, the teeth used in this study were in the early stages of root resorption (Murthy et al. 2020), with resorption visible only in the apical third of the root.

Odontoclasts and osteoclasts are the cells responsible for root resorption (Harokopakis-Hajishengallis 2007). As one might expect, the enrichment analysis in this study identified odontoclast/osteoclast differentiation as an enriched pathway, with the majority of associated DEPs being up-regulated in the dental pulp of deciduous teeth. It has been confirmed that the functional characteristics, cellular mechanism, enzymatic properties, and differentiation process of odontoclasts are similar to those of osteoclasts, and the two cell types are believed to arise from the same origin (Harokopakis-Hajishengallis 2007; Kamat et al. 2013; Oshiro et al. 2001). TNF and NF-κB signalling are important in the activation of odontoblast/osteoclast differentiation (Harokopakis-Hajishengallis 2007; Kamat et al. 2013; Xiao et al. 2022); in addition, IL-1β, which was found to be highly expressed in the pulp of deciduous teeth, can also directly activate odontoblast/osteoclast differentiation (Harokopakis-Hajishengallis 2007; Kamat et al. 2013).

The activation of TNF and NF-κB inflammatory pathways, along with the differentiation of odontoblasts/osteoclasts in the pulp tissue of deciduous teeth, may shed light on why vital pulp therapy in deciduous teeth, particularly direct pulp capping and pulpotomy with calcium hydroxide, tend to result in internal resorption of deciduous dental pulp, leading to pulp treatment failure (Canoğlu et al. 2022; Jha et al. 2021; Moretti et al. 2008). In contrast, the same approaches have proven to be more effective in permanent teeth, resulting in the formation of a mineralised tissue barrier, and are, therefore, regarded as appropriate for vital pulp therapy in permanent teeth. (Akhlaghi and Khademi 2015; Ricucci et al. 2023).

The findings of this study indicated that there were considerable differences between the dental pulp of the two dentitions, with the pathway of inflammation and hard tissue resorption being particularly active in the pulp of deciduous teeth. Because of these differences, dental practitioners should be aware that even with identical dental procedures, the pulp of deciduous teeth may not respond in the same manner as permanent teeth, potentially leading to differing treatment outcomes. Particularly when performing dental procedures or utilising materials that are prone to triggering inflammatory reactions or odontoblast/osteoclast differentiation, it may result in a failure outcome in deciduous teeth. Understanding these characteristics could aid in determining an appropriate pulp therapy approach for deciduous teeth.

This study has some limitations with regard to donor group age difference, as deciduous teeth are usually extracted during middle or late childhood for reasons of prolonged retention or orthodontic purposes, whereas third molars are typically removed during late adolescence or adulthood. Furthermore, due to the timing of tooth removal, obtaining deciduous and permanent dental pulp from the same person is impractical. The sample size in this study is quite small because most deciduous teeth can naturally be exfoliated; thus, it is difficult to obtain deciduous teeth for which the majority of the root structure is still intact, in this case defined as having more than two-thirds of the root. Nonetheless, the data is sufficient to provide an initial screening of deciduous teeth pulp protein profiles, as well as statistical analysis and comparison of proteins from deciduous and permanent dental pulp. To investigate and expand on these findings, further research should be conducted with a larger sample size and should validate the proteins found to be differentially expressed between dental pulp of different dentitions.