Background

Stem cell-based technologies are an ideal source for regenerative medicine, immunological studies, and cell therapy because they induce tissue repair and regeneration [1, 2]. Mesenchymal stem cells (MSCs) play a key role in tissue regeneration treatment. They are rapidly adherent, clonogenic, and capable of extended proliferation in vitro [3]. In addition, they maintain stem cell properties such as self-renewal, long-term viability, and differentiation potential into mesodermal origin osteocytes, chondrocytes, and adipocytes [4, 5]. As a result of their capacity to differentiate into various cell types, MSCs play a key role in tissue and organ regeneration and have recently attracted great interest in tissue engineering [6, 7].

Even though MSCs can be isolated from many sources, such as cord blood, bone marrow, or adipose tissue [8], a very promising source is the relatively easily obtainable dental tissue. There are five types of human dental stem cells: dental pulp stem cells (DPSCs) [9], stem cells from exfoliated deciduous teeth (SHED) [10], periodontal ligament stem cells (PDLSCs) [11], dental follicle stem cells [12], and stem cells from apical papilla [13]. These MSCs express specific MSC markers, such as CD29, CD73, CD90, CD105, and CD166, and can differentiate into odontoblasts, chondrocytes, and adipocytes under appropriate circumstances [9, 10]. DPSCs can easily be isolated from the dental pulp tissue of newly extracted teeth, making the procedure relatively more straightforward and avoiding ethical dilemmas [14]. DPSCs are commonly used in the regeneration and reconstruction of dental structures in addition to bone tissue engineering after undergoing osteogenic differentiation [15, 16].

Tissue engineering techniques, including the use of MSCs, often require scaffolds and cytokines serving as inductive factors [17]. Some inflammatory cytokines alter stem cell functions as well as immune or inflammatory cells [18]. In vitro studies have revealed that cytokines can affect the differentiation process of mesenchymal progenitor cells during tissue formation. Most of these in vitro studies have used MSCs by isolating them because of their ability to adhere to plastic [19, 20].

Cytokines are commonly used superior markers of inflammation, modulating immune and inflammatory responses [21]. Tumour necrosis factor-α (TNF-α) is defined as a proinflammatory cytokine expressed in injured tissues as well as in ischaemic situations [22]. TNF-α also plays a major role in the repair process of injured tissues and promotes MSC recruitment [23,24,50, 51]. The two most commonly used techniques for isolating DPSCs from dental pulp tissue are the explant method and enzymatic digestion. Hilkens et al. [52] reported no difference in the tissue differentiation potential of DPSCs regarding the isolation method. In our study, the enzymatic digestion method used to isolate DPSCs was based on previous studies [9, 10]. After isolation, the cells displayed fibroblast-shaped morphology and were adherent to plastic Petri plates. Flow cytometry analysis declared that the cells showed positive expression for CD29, CD105, CD146, and CD73 markers and negative for CD3, CD4, and CD20 markers, which agree with the criteria of the International Society of Cellular Therapy [48]. Osteogenic, chondrogenic, and adipogenic differentiation procedures were performed on characterised DPSCs, and Alizarin red, Alcian blue, and oil red stains were used, respectively, to determine their differentiation into cell lines in accordance with previous studies [2, 53, 54]. Tarte et al. [55] also used staining procedures to compare the proliferation and differentiation of SHEDs and PDLSCs, but used von Kossa staining instead of Alcian blue to determine chondrogenic differentiation. MSCs can differentiate in vitro spontaneously or by the induction of biologically active molecules [56]. DPSCs can also proliferate and differentiate, as can other stem cells of dental origin [2, 9, 10, 51].

Effect of cytokine on differentiation

Cytokines modulate immune and inflammatory responses and are markers of inflammation [21, 57]. In many situations, certain tissues need to be regenerated due to injury. Whether the tissue injury is caused by microorganisms (e.g. pulpitis) or trauma (e.g. bone fractures), proinflammatory cytokines are the superior markers of inflammatory responses [32, 58]. Both positive and negative impacts of cytokines on MSC differentiation and tissue healing have been reported [32, 47]. The present study determined how DPSCs might behave in an inflammatory environment set up with some key proinflammatory cytokines TNF-α, IL-1β, and IL-6. Many previous in vitro and in vivo studies have evaluated their roles in osteogenic and chondrogenic differentiation of MSCs. Kondo et al. [59]. indicated that in the early stages, TNF-α, IL-1β, and IL-6 contribute to fracture healing and bone remodelling. **e et al. also [54] demonstrated that IL-6 promotes osteogenic differentiation in BM-MSCs in vitro. In vitro studies demonstrating the effects of TNF-α, IL-1β and IL-6 have mostly involved osteogenesis with MSCs other than dental origin and have not directly compared their effects on differentiation [32, 47]. Contrary to our results, Liu et al. [51] demonstrated that TNF-α promoted the osteogenic differentiation of DPSCs in vitro. Similarly, Feng et al. [25] demonstrated that TNF-α activates the NF-κB pathway and promotes osteogenic differentiation of DPSCs in vitro. Another in vitro study showed increased calcium deposits following IL-1β pretreatment when culturing BM-MSCs in osteogenic medium.

On contrary, Kondo et al. [59] also reported bone resorption can be induced under IL-6 stimulation. Lacey et al. [32] compared the effects of TNF-α and IL-1β on the osteogenic capacity of murine MSCs and found that these cytokines inhibited MSC differentiation to osteoblasts, which agrees with our findings. Liu et al. [51] investigated osteogenic differentiation of DPSCs promoted by TNF-α; this was similar to our study with the difference of evaluating transcriptome changes. Additionally, relatively long-term exposure to inflammatory mediators were reported to suppresses DPSC differentiation ability [60].

Considering the chondrogenic differentiation of BM-MSCs, Mumme et al. demonstrated the most intense staining for cartilage with low-dose IL-1β (10 and 50 pg/mL) [41]. The discrepancies in the outcomes among these studies and our study can be explained by the differences in the concentrations of proinflammatory cytokines and in the origin of the stem cells.

A limitation of the present study was that it was an in vitro analysis and not in vivo. The cytokine concentrations used in the study were used as the highest possible concentrations to assess the differentiation potential of DPSCs into the inflammatory niche. These concentrations may not be similar in vivo. In addition, the differentiation potential of DPSCs may vary in the presence of anti-inflammatory drugs such as anti-TNF-α, anti-IL6, which are used in some autoimmune or inflammatory diseases. Nonetheless, our findings might be useful for further studies for understanding the mechanisms and outcomes of DPSC differentiation with specific cytokine modulation both in vitro and in vivo because the functions and expressions of proinflammatory cytokines during certain tissue differentiations remain unclear in vivo [54]. In addition, stem cells of dental origin are expected to be preferred more frequently in future research because they are easy to obtain. Future studies should be designed to include different concentrations of inflammatory cytokines, evaluation of gene expression, and use of dental stem cells with different origins.

Conclusion

Our results indicated that DPSCs are highly proliferative MSCs in terms of osteogenic, chondrogenic, and adipogenic differentiation. In the present in vitro study, TNF-α, IL-1β, and IL-6 were demonstrated to inhibit DPSC differentiation and tissue formation. Further studies, including in vivo applications with different dental MSCs origins and diverse amount, type and appliance durations are required to more comprehensively understand the underlying molecular mechanisms for application in stem cell therapies.