The endoplasmic reticulum (ER) functions as a site for protein folding, lipid biosynthesis, and calcium homeostasis (Sarvani et al., 2017). Numerous proteins, either destined for cell-surface or for secretion, undergo process of synthesis and modification such as folding and assembly (Schröder and Kaufman, 2005). Trafficking and translocation of proteins in the ER would cause loads of stress to the ER regulation due to the large number of proteins that need to be executed (Hartl and Hayer-Hartl, 2009). However, several molecular chaperones, such as multi-domain families of proteins, are located at the lumen of the ER, which help in achieving correct protein folding and assembly (Saibil, 2013). The process of balancing between the load of protein synthesis and its protein folding ability is referred to as ER homeostasis. When there is a disruption in ER homeostasis, ER stress can be triggered which is caused by the presence of mis-folded and unfolded proteins (Ron and Harding, 2012). The unfolded protein response (UPR) activates in response to ER stress when ER capacity for protein folding is deficient (Kozutsumi et al., 1988; Zimmer et al., 1999). UPR activation alleviates ER stress by increasing ER abundance, decreasing synthesis of proteins, and augmenting the ER chaperone protein synthesis to sustain homeostatic balance; however, when ER stress could not be restored by UPR, apoptosis takes place (Merksamer and Papa, 2010; Hetz, 2012).

Dentin is typically classified into formation stages, namely: primary, secondary, and tertiary (Goldberg et al., 2011). During odontogenesis, primary dentin is formed by odontoblasts until the tooth becomes functional. Secondary dentin is formed after root completion and continues lifelong. Particularly, in the pathological condition, odontoblasts form a specific dentin—the tertiary dentin. Tertiary dentin, whether reactionary or reparative, is formed in response to an external stimulus, caries, or abrasion (Angelova Volponi et al., 2018). Reactionary dentin is formed from pre-existing odontoblast, and reparative dentin, which is elaborated by odontoblast-like cells form after the death of the original odontoblasts (Cajazeira Aguiar and Arana-Chavez, 2007; Goldberg et al., 2011). Proper regeneration of dentin still remains as an unmet solved problems in dental fields. Tissue regeneration requires high fidelity of restoration of structure including vesicle secretion for tissue function (Simon and Smith, 2014; Angelova Volponi et al., 2018). Odontoblasts and pulp cells react against the damage through reinitiating differentiation of odontoblasts from stem cells and progenitor cells in dental pulp resulting secretion of the reparative dentin matrix for normal physiologic function of tooth (Simon and Smith, 2014; Angelova Volponi et al., 2018). During dentin formation, ER stress regulation plays a significant role especially in the secretory stage (Cameron et al., 2015). Previous report suggested that ER stress would affect formation of dentin. For instance: when ER stress regulator gene, Tmbim6 was knocked out, the dentin and enamel structures were disturbed by altering odontoblast and enamel differentiation (Aryal et al., 2019). Mutated dentin forming genes of which could cause Dentinogenesis Imperfecta and Dentin Dysplasia may cause a high level of ER stress (Brookes et al., 2014; Brookes et al., 2017; Liang et al., 2019). Odontoblasts and pulp cells react against the damage by reinitiating differentiation of the odontoblasts from stem cells and progenitor cells in the dental pulp resulting in the secretion of the reparative dentin matrix for normal physiologic function of the tooth (Brookes et al., 2014).

4-Phenylbutyric acid (4PBA) is an FDA-approved drug intended to treat congenital diseases in the urea cycle (Iannitti and Palmieri, 2011). However, 4PBA is a known chemical chaperone that functions as an ER stress antagonist (Zeng et al., 2017); a low-molecular weight compound which stabilizes protein conformations, enhancing the protein folding ability of ER and protein trafficking to alleviate ER stress (Yam et al., 2007). A number of previous studies have also proved the therapeutic effects of 4PBA in proteostasis. Mainly, 4PBA facilitates metabolic syndrome and metabolism such as obesity and diabetes (Ozcan et al., 2009; Luo et al., 2010; Wu et al., 2020). The chemical chaperone 4PBA is also known to alleviate ER stress-mediated cell death and genetic disorders caused by protein mis-folding (Sawada et al., 2008; Singh et al., 2008) and protein mis-folding genetic disorders (Vij et al., 2006; Singh et al., 2008). Furthermore, various effects of 4PBA in inflammatory disorders, neurological diseases, and cancers are also well known (Luo et al., 2010; Kim et al., 2013).

Pulp cavity preparation is the most well-known method of dentin-pulp restoration. In this procedure, the tooth itself receives stress even in protein modulation and secretion of ER. This study hypothesizes that 4PBA, not applied in dental treatment yet, is involved in the modification and secretion of protein matrix for dentin formation by attenuating ER stress when added to the process of pulp cavity preparation.

Formation of dentin is mainly contributed by cellular physiology of odontoblasts. These specific secreting cells are precisely regulated and modulated by ER regulation and vesicle trafficking to form the specifically featured dentin matrices through protein synthesis, folding, trafficking, secretion, and patterned apposition (Brookes et al., 2014; Aryal et al., 2019; Liang et al., 2019). Previous studies reported that modulation of ER stress would be an important cellular pathway for differentiation and maintenance of dentin (Kim et al., 2014; Brookes et al., 2017). In this study, we hypothesized that in the exposed dentin and pulp complex, ER stress would be occurred unavoidably to repair the dentin structure and maintain the vitality of the pulp as examined in other wound sites (Brookes et al., 2014; Aryal et al., 2019; Liang et al., 2019). In inflammation condition, a number of studies revealed the vicious circle of escalation of pathological conditions between ER stress and inflammation responses (Luo et al., 2010; Qi et al., 2011). Inflammation disturbs protein folding, leading to the accumulation of unfolded or mis-folded proteins inside the ER which in turn results in UPR (Kim et al., 2013). Furthermore, sustained ER stress induces chronic activation of UPR, which would eventually lead to severe inflammation and even cell death (Rao et al., 2001). Recently, studies on ER regulation and vesicle trafficking made use of chemical chaperone treatments to subdue a range of incurable diseases (Singh et al., 2008; Luo et al., 2010; Wu et al., 2020). Similarly, in this study, in order to modulate the excessive inflammatory environment and promote dentin regeneration after pulp exposure, we examined the function of the chemical chaperone 4PBA, one of the candidate drugs for relieving ER stress, an important cellular step for protein formation and secretion in regeneration, and as a drug repositioning approach, in exposed pulp cavity for 3, 5, and 42 days, using a well-established animal model system (Jung et al., 2017; Aryal et al., 2021).

Recent reports elaborated 4PBA has chaperone properties and stabilizes protein conformation in ER which facilitates ER stress and UPR activation (Ozcan et al., 2006; Ozcan et al., 2009). We performed local delivery of 4PBA with concentration of 100 μM which was determined in vitro cell culture condition (Ranga Rao et al., 2018) and it is comparably lower than oral medication for systematic applications (Singh et al., 2008; Basseri et al., 2009; Luo et al., 2010; Brookes et al., 2014). This low concentration of drug local delivery was available in pulp cavity preparation model system and would confirm the merits including harmless, economic and efficient method for treatment. Interestingly, our results showed that local delivery of 4PBA presented the excellent morphological changes with dentin bridge and altered cellular changes in late and early time period of exposed pulp and dentin. Based on results, we proposed that two key factors for successive pulp therapy would be management of ER stress and control of initial inflammatory status of the pulp which influences the quality of newly mineralized hard tissue after treatment. As we expected, 4PBA showed the excellent function for resolving ER stress from the exposed pulp (Figure 1). After precise examinations of localization patterns of ER stress related molecules, GRP78 and HRD1, we suggest that the regulation of inflammation of dental pulp and formation of reparative dentin would be resulted from the proper modulation of ER stress by treatment of 4PBA (Kaufman, 2002; Lee, 2005). HRD1 plays a critical role in ER-associated degradation (ERAD) of mis-folded/unfolded proteins as it protects cells from ER stress-induced cell death (Sun et al., 2015; Wei et al., 2018; Wu et al., 2020). We have observed interesting result after precise examination of localization patterns of HRD1. The differential localization patterns of HRD1 between controls and the 4PBA group is worth noting. The localization pattern of HRD1 in control group showed cytoplasmic positive localization generally at the entire pulp area. In the 4PBA-treated group after 3 days, there was a decrease in the localization compared to controls, and localization pattern was observed only at a limited area of the dentin wall, which was a nuclear localization (Figure 1). These results coincide with a previous report that found that cytoplasmic localization of HRD1 would take place when secretory cells are impaired (Wu et al., 2020). The results support that 4PBA treatment in the exposed dentin alleviates ER stress and ERAD by providing the proper condition of tissue regeneration. In addition, histological observation and the localization pattern of MPO would suggest that 4PBA initiates blood vessel formation for rapid control of inflammation through neutrophil-mediated modulation of inflammation after the pulp exposure.

Moreover, the increased localization patterns of NESTIN and TGF-β1 after 3 days of pulp exposure in our study confirmed the previous report that elevated levels of NESTIN would stimulate cell proliferation and invasion by stimulating the TGF-β1 signaling pathway (Toyono et al., 1997; Bokhari et al., 2016; Niwa et al., 2018). To understand the 4PBA molecular reactions, we examined the micro-CT and histomorphology after 6 weeks of cavity preparation (Figure 3). As we observed at early-onset cellular changes, 4PBA-treated group showed long-term regeneration of reparative dentin in the exposed pulp with dentin bridge formation (Figure 3). Our results showed that 4PBA treatment in exposed pulp and dentin complex would modulate blood vessel formation and also promotes the active odontoblasts to produce reparative dentin through TGF-β1 signaling pathways. We suggest and recommend that 4PBA would be a feasible treatment after pulpal cavity preparation to facilitate dentin regeneration and alleviating ER stress.

Overall, we examined the function of the FDA-approved drug, 4PBA in dentin regeneration as a drug repositioning approach. This attempt would be a plausible answer to extend and develop new drugs and techniques in the dental field. For example, as a drug repositioning strategy, many ER stress-related drugs such as melatonin, simvastatin, and TUDCA should be tested in the dental field (Ozcan et al., 2009; Ranga Rao et al., 2018; Karakida et al., 2019). In addition, we suggest examining the synergistic effects of ER stress-relieving molecules in combination with previously established treatment including triple antibiotic paste and Wnt signaling-related small molecules (Vijayaraghavan et al., 2012; Jung et al., 2017; Faria et al., 2018).