In the context of regenerative endodontics, this study investigated the effect of OT on the viability and osteo/odontogenic differentiation of human stem cells from the apical papilla (hSCAPs). Regenerative medicine, including regenerative endodontic therapies, relies on three key elements of scaffold, growth and differentiation factors, and stem cells (Breuil, Trojani et al. 2021).
Recently, mesenchymal stem cell populations have been isolated and characterized from dental tissues. Human stem cells from apical papilla (hSCAPs) residing in the apical papilla of immature permanent teeth represent a novel population of dental mesenchymal stem cells that demonstrate a high proliferative capacity, self-renewal ability, and low immunogenicity(Sonoyama, Liu et al. 2008, Pereira, Longo et al. 2012, Kang, Fan et al. 2019, Karkehabadi, Rahmati et al. 2023). Moreover, substantial evidence indicates that hSCAPs are capable of differentiating into various cell lineages including osteogenic, odontogenic, neurogenic, adipogenic, chondrogenic, and hepatogenic lineages, which may serve as a promising cell source for stem cell-based therapy (Sonoyama, Liu et al. 2008, Dong, Yao et al. 2013, Patil, Kumar et al. 2014).Comparative analysis showed that hSCAPs exhibit a higher proliferation rate compared to dental pulp stem cells and periodontal ligament stem cells (Sonoyama, Liu et al. 2006, Sonoyama, Liu et al. 2008, Lei, Yan et al. 2011, Chen, Xiong et al. 2013). Also these cells express higher levels of survivin (an anti-apoptotic protein) and human telomerase reverse transcriptase (hTeRT) compared to dental pulp stem cells. Both survivin and hTeRT are crucial factors for promoting cell proliferation.
In order to maximize the potential of mesenchymal stem cells, various priming techniques such as drug administration, cytokines, growth factors, or unique culture conditions have been investigated. Reports indicate that OT exerts an anabolic effect on bone metabolism through actions on various mechanisms involved. It has extensive central and peripheral functions and its receptor is found in diverse tissues. Oxytocin is not merely a primary neurohypophyseal hormone but also an anabolic bone hormone. It regulates the calcification of hard tissues and controls the differentiation of human mesenchymal stem cells (Tamma, Colaianni et al. 2009, Altay, Dede et al. 2020). The oxytocin receptor exists in the pulp-dentin complex and periodontal ligament, and it has been shown to reduce the immune response in human dental pulp stem cells during inflammation by inhibiting the production of CXCL10(Kumagai, Shindo et al. 2022) ,as well as stimulate odontoblast differentiation and increase dentin formation (Kato and Yokose 2021). However, its effects on dental mesenchymal stem cells like hSCAPs regarding proliferation, differentiation and gene expression remain unclear.
Various methods are used to assess cell viability. MTT (methyl thiazolyl tetrazolium) assay, as a standard method for assessing cytotoxicity, is being widely used for different stem cells(Karkehabadi, Abbasi et al. 2023). According to the results of this experiment in the present study, at all three concentrations (25, 50 and 100 nM), OT had no cytotoxic effects on hSCAPs at 24, 48 or 72 hours. The 100 nM concentration of OT had significant impact on hSCAPs proliferation at24, 48 and 72 hours. 25 and 50 nM of OT had significant effect only after 24 hours. Because the concentration of 100 nM OT had a constant trend in all three time points, we used this concentration to investigate gene expression and ALP activity tests in order to obtain more reliable results.
Similar to the results of our study, in a study by Noiseux et al.(Noiseux, Borie et al. 2012) mesenchymal stem cells treated with OT showed lower apoptosis rate in compared to the control group and stimulates the protection of mesenchymal stem cells through intrinsic PI3K/Akt and ERK pathways and the secretion of cytoprotective factors. In another study, Nazli Jafarzadeh et al.(Jafarzadeh, Javeri et al. 2014) examined the effect of OT on the viability of adipose-derived stem cells. Their MTT test demonstrated the proliferative effect of OT on adipose-derived stem cells. They attributed this effect to OT-induced increase in the number of cells in S and G2/M mitotic phases. Falah Nejad et al.(Fallahnezhad, Piryaei et al. 2018) in a study investigated the effect of OT and low-level laser on bone marrow-derived mesenchymal stem cells at 24, 48 and 72 hours. They found that OT had a significant effect on the survival and proliferation of bone marrow-derived mesenchymal stem cells at 48 and 72 hours, and also, OT reduced the rate of cell apoptosis. The effect of OT on periodontal ligament (PDL) cells was examined by Bin Ge et al.(Ge, Liu et al. 2019) They found that 50 nM OT concentration significantly increased the growth and proliferation rate of PDL cells. No statistical difference was found between 50 and 100 NM of OT, in terms of cell proliferation. They attributed this effect to saturation theory (Singh, Swami et al. 2016), whereby when all receptors become saturated with ligands, even at the higher ligand concentration, there is no receptor available for binding. In our study, the highest proliferative effect was seen with 100 nM OT, which may be due to differences in the cell type studied. Given the different OT receptor expression in different mesenchymal stem cells, the OT receptor on hSCAPs may not reach saturation at 100 nM OT.
In the Petrocelli et al.(Petrocelli, Abruzzo et al. 2023) study examining the effect of OT on the proliferation of adipose-derived stem cells, although OT had no cytotoxic effects on the cells, no proliferative effect was found by this agent. They showed that the expression of genes involved in cell proliferation and cell cycle including MKI67, CCND1, CDKN2A, CDKN1A, and TP53, was not affected in OT-treated cells. The differences compared to our study might be due to differences in the cell types and the genes examined.
In summary, the present study results are consistent with other conducted studies showing that OT has no cytotoxic effects on mesenchymal stem cells and enhances mesenchymal stem cell proliferation while protecting them from apoptosis, although it should be kept in mind that the effects of OT on mesenchymal stem cell proliferation may differ depending on cell type and OT concentration used.
During osteoblastic differentiation, alkaline phosphatase (ALP) activity is an early marker that is easily identified by standard ALP staining methods (Fukuyasu, Kayashima et al. 2022). The mineralization of the extracellular matrix (ECM), which is crucial for the formation of hard tissues, involves two key sequential steps: 1) the initial synthesis of the collagen network, and 2) the deposition of hydroxyapatite crystals catalyzed by ALP. ALP is thought to facilitate mineral deposition in the early stages of odontoblastic differentiation (Ruparel, de Almeida et al. 2013, Karkehabadi, Abbasi et al. 2023).
In this study, ALP assay was performed with 100 nM OT at 7, 14 and 21 days of osteogenic differentiation, and at all three time points, OT increased the ALP activity. ALP activity trends for two groups of OT treated and control, indicates of early differentiation stage activity of this enzyme. Alizarin red staining is a reliable tool for staining mature mineralized skeletal structures (Bensimon-Brito, Cardeira et al. 2016). After 21 days, all three concentrations of OT (25, 50, and 100 nM) led to increased formation of mineralization nodules compared to the control group. Quantitative analysis of this experiment indicates of higher mineralization achieved by OT treatment.
Consistent with our findings, Bin Ge et al.(Ge, Liu et al. 2019) showed that 10 and 50 nM OT significantly increased mineralization and extracellular calcium deposition after 21 days. Yuka Kato et al. (Kato and Yokose 2021) investigated the impact of OT on mouse dental pulp stem cells. Their ALP assay results after 21 days demonstrated that the number of mineralized nodules was higher in cells treated with OT compared to the control group. Furthermore, in mice lacking the OT receptor, the number of mineralized nodules decreased. Falahnezhad et al.(Fallahnezhad, Piryaei et al. 2018) showed that OT significantly increased ALP activity in bone marrow stem cells at 7, 14, and 21 days. Liu Feixiang et al.(Feixiang, Yanchen et al. 2023) proposed that OT induces osteoblasts differentiation and mineralization by promoting OTR translocation into the nucleus. OT may also modulate the OPG/RANKL ratio in osteoblasts by eliciting intracellular Ca2+ release and nitric oxide production, thereby exerting regulatory effects on osteoclasts. Furthermore, OT can stimulate osteocyte and chondrocyte activity to increase bone mass and improve bone microstructure.
Odontogenic differentiation from dental pulp cells into secretory odontoblasts is a vital process in tooth development(Thesleff and Nieminen 1996). The process of differentiation from dental pulp cells to odontoblasts involves many molecules at each stage. ALP is considered an early marker of odontogenic and osteogenic differentiation that can facilitate mineral deposition (Wu, Jia et al. 2013, Halling Linder, Ek-Rylander et al. 2017).DSPP and DMP1, which are members of the integrin-binding small integrin-binding N-linked glycoprotein family, are highly expressed in mature odontoblasts and are essential for extracellular matrix mineralization and dentin formation(Kang, Chen et al. 2021). These genes were examined on days 7, 14 and 21 of differentiation to assess the extent of odontogenicity, and a raise in expression was found for the genes at all-time points. As expected, the ALP expression level sharply increased in the early days (7-day time period) of the differentiation process and gradually decreased in expression in subsequent days (days 14 and 21), given that this gene is an early factor in differentiation. This data confirms the data obtained from ALP activity experiment.
In the present study, the RUNX2 and COL1A1 genes were examined to assess the extent of osteogenicity. COL1A1 is one of the early markers in the osteogenic differentiation process, and defects in this gene lead to fragile bones (Marom, Rabenhorst et al. 2020, Dirani, Cuenca et al. 2022).In vitro and in vivo studies show that RUNX2 controls the expression of the major bone matrix protein that resides in the promoter of several osteoblast-specific genes (Liu and Lee 2013, Zhang and Li 2018). The RUNX2 expression level is important for normal bone growth, with decreased RUNX2 expression leading to abnormal bone growth (Komori, Yagi et al. 1997, Otto, Thornell et al. 1997). As seen in this study, the COL1A1 gene expression level was higher in the first week of differentiation compared to the second and third weeks, and reached the lowest level towards the end of the period. Also, its expression level at all-time points was higher in the OT-treated group compared to the control group. The results of the present study showed that the RUNX2 gene expression level gradually increased over the differentiation period. OT also increased the expression of this gene on days 14 and 21 in hSCAPs.
Yuka Kato et al. (Kato and Yokose 2021) demonstrated that in addition to odontoblasts and periodontal ligament cells, the OT receptor is also expressed in dental pulp stem cells. Consistent with our study, they showed that OT increases the gene expression of DSP, Wnt10a, and BGP in dental pulp stem cells.
The results of the study by FalahNejad et al. (Fallahnezhad, Jajarmi et al. 2020) also show that OT significantly upregulates RUNX2 gene expression. They found that on day 21, OT leads to increased RUNX2 expression. Their main finding was that OT increases bone mineralization.
Upregulation of the expression of the ALP, Col 1 and RUNX2 genes by OT on days 7 and 14 was reported by Bin Ge et al.(Ge, Liu et al. 2019) They also reported that OT increased mineralization on day 21, consistent with the findings of the present study.
In their review study, Liu Feixiang et al. (Feixiang, Yanchen et al. 2023) showed that OT can stimulate osteoblast mineralization by promoting translocation of the OT receptor to the osteoblast nucleus. Additionally, by inducing intracellular Ca2+ release and nitric oxide synthesis, OT can regulate the OPG/RANKL ratio in osteoblasts and exert a two-way regulatory effect on osteoclasts. It can also increase osteocyte and chondrocyte activity, which increase bone mass and improve bone microstructure.
The present study's findings align with previous research, indicating that OT promotes the differentiation of stem cells into osteoblasts (Elabd, Basillais et al. 2008, Petrocelli, Abruzzo et al. 2023).
Petrocelli et al. (Petrocelli, Abruzzo et al. 2023) conducted a study to investigate the impact of OT on the expression of the RUNX2 gene in both osteogenic and non-differentiation media. The findings revealed a significant increase in RUNX2 expression when OT was introduced in the osteogenic medium, but there were no notable changes compared to the control group in the non-differentiation medium. Based on these results, the researchers concluded that OT alone is insufficient to induce osteogenesis. They also showed that oxytocin stimulates osteogenesis by increasing autophagy gene markers at the onset of treatment.
In another study by Christian Roux et al.(Roux, Pisani et al. 2020) the impact of OT on the expression of the COL1A1 gene in adipose-derived stem cells was investigated. The results revealed that there were no significant differences in COL1A1 expression between cells treated with OT and the control group. While mesenchymal stem cells derived from various sources possess distinct phenotypic features, it is important to note that not all types of mesenchymal stem cells exhibit similar responses to identical stimuli. This differential response could potentially be attributed to variations in the expression of OT receptors among different types of mesenchymal stem cells.
To our knowledge, the present study is the first to examine the effect of OT on hSCAPs. Our study results indicate that treatment of hSCAPs with OT enhances osteogenic/odontogenic differentiation of these cells. Additionally, OT increased hSCAPs proliferation rate at 100 nM.