Osteogenic differentiation of dental pulp stem cells under the influence of three different materials

The major goal of periodontal therapy is to regenerate tooth-supporting structures destroyed by periodontal disease [1]. Periodontal tissue engineering involves complex interactions between different cells and signaling molecules, as well as biological scaffolds [2].

In an attempt to mimic the original developmental events, the integrated use of precursor cell populations with specific biologic stimulants is under investigation [3, 4]. Stem cells represent primitive non-specialized cells with wide capabilities for differentiation and tissue regeneration. To date, mesenchymal stem cells have been successfully isolated from several body organs [5], including multiple tissues with dental origins [6‐9]. Such dental tissue-derived stem cells were found to retain potent capacity for specific differentiation into dental tissue-forming cells [6, 10, 11]. Gronthos and colleagues successfully isolated human dental pulp stem cells (DPSCs), and proved both their multipotency and self-renewal capability [11, 12]. Further studies confirmed their findings [13, 14]. This multipotency, in addition to their relative accessibility, made DPSCs an appealing source of cells for application in regenerative medicine [15‐18]. In fact, several papers have proved their superiority in different aspects, including osteogenic differentiation [19, 20], which supported their use for regeneration of craniofacial defects [21, 22], as well as alveolar bone defects [23, 24]. Additionally, the similar embryonic origins of dental pulp cells and periodontal cells [25] and their presence within protective layers of tooth structure have encouraged their use for periodontal tissue regeneration [26, 27].

Studies on tissue engineering have used biological mediators to selectively enhance the recruitment of cellular populations into periodontal wounds [28]. Enamel matrix derivative (EMD) is a protein harvested from developing porcine teeth that has been reported to induce cementum formation and periodontal regeneration [29]. At the cellular level, EMD was proven to have regulatory effects on multiple periodontal cell types [28, 30].

Platelet-derived growth factor (PDGF) is a very powerful regulatory factor that initiates nearly all wound healing events. The main function of PDGF is to stimulate cell replication (mitogenesis) of healing-capable stem cells and partially differentiated osteoprogenitor cells, which are part of the connective tissue–bone healing cellular make-up [31]. Significant increases in bone and cementum formation have been reported histologically [32]. At the cellular level, PDGF increased the number of collagen-synthesizing cells [33] and stimulated bone sialoprotein transcription [34].

Another material with the ability to induce regeneration is mineral trioxide aggregate (MTA). MTA is a mixture of dicalcium silicate, tricalcium silicate, tricalcium aluminate, gypsum, and tetracalcium aluminoferrite [35]. Torabinejad et al. [36] reported a favorable biologic performance of MTA when in direct contact with bone, through the deposition and formation of hydroxyl apatite on its surface. The material was also found to enhance cellular production of type I collagen, osteocalcin, alkaline phosphatase (ALP), bone sialoprotein, and osteopontin [37]. A systematic review on the histological responses of the periodontium to the material concluded that MTA promoted healing toward regeneration [38].

The above findings suggest similar clinical performances for the three materials with no previous attempts for direct comparisons. Accordingly, the purpose of the present study was to examine and compare the effects of EMD, PDGF, and MTA on the osteogenic differentiation of DPSCs.

In this study, successful isolation of dental pulp cells was achieved through the application of enzymatic digestion with certain modifications to the protocol of Gronthos et al. [11]. The obtained cells underwent several investigations to evaluate their properties. According to the International Society for Cellular Therapy [39], the minimal criteria for defining multipotent mesenchymal stromal cells include: (1) adherence to plastic dishes; (2) multipotent differentiation potential; and (3) expressions of specific stromal surface markers (CD73, CD90, CD105) with lack of expressions of hematopoietic markers (CD45, CD34, CD14 and/or CD11b, CD19, CD79α) and the HLA-DR marker. The isolated cells in this study presented all of the above features.

Different material concentrations were evaluated, and the concentrations with the best differentiation were selected. These concentrations were 200 μg/ml for EMD, 5 ng/ml for PDGF, and 0.05 mg/ml for MTA. The same concentrations were previously used in other studies [34, 40, 41]. In this study, computer analysis for ALP activity and a semiquantitative evaluation technique for alizarin red S staining were selected, as these two techniques were reported to give results with relative sensitivity, and have been applied in previous studies [42, 43].

For EMD, the results revealed significant increases in ALP expression and abundant mineralization enhancement following its application. These findings are in accordance with several other studies evaluating the effects of this material on multiple cell lines [40, 44‐48]. Duan et al. [44] found that EMD enhanced the osteogenic differentiation of induced pluripotent stem cell, as evidenced by increases in RUNX2 mRNA expression. Kémoun et al. [45, 46] evaluated the effects of EMD on follicular cells [45] and periodontal ligament stem cells [46]. In both studies, EMD was found to enhance ALP release and calcium deposition, in addition to the elevation of several mineralization markers. Another study by Guven et al. [47] found that Emdogain was the most effective material for enhancing both proliferation and odontogenic differentiation of human tooth germ stem cells through the evaluation of ALP activity, Von Kossa staining, and RT-PCR analyses for dentin sialophosphoprotein (DSPP), and immunostaining for collagen type I and DSPP. A study by Wang et al. [48] found that Emdogain enhanced the mineralization of DPSCs as well as their osteogenic/odontogenic marker expression. However, studies with contradictory findings are also available [49, 50]. It was reported that EMD might not have appreciable effects on osteoblastic differentiation in periodontal ligament cells [49] or rat bone marrow cells [50]. Although the exact control mechanism remains unclear, these effects were explained by differences in the degrees of cellular immaturity, i.e. the material was thought to enhance cellular proliferation of more immature cells, but differentiation of cells at later stages of maturity [51].

In the present study, MTA gave inconsistent findings. The material revealed mineralization enhancement in comparison with the reference control, reductions in certain ALP parameters (percent total positive staining area and histological score), and maintenance of other parameters (average optical density). Although Yasuda et al. [52] and Lee et al. [53] reported that MTA increased ALP production and/or mineralized nodule formation compared with control cells, both Koh et al. [54] and Nakayama et al. [55] reported similar ALP expression between MTA-treated cells and negative control cells. These inconsistencies suggest that further evaluation of the different parameters guiding and affecting the performance of this material is warranted.

With regard to PDGF in the present study, it was observed that ALP expression generally revealed lower results in comparison with the negative control group as well as all of the other material groups, and the differences were always significant. Regardless of the material’s action in proliferative enhancement, PDGF-BB appeared to have no additional benefit for osteogenic differentiation, according to the parameters evaluated in this study. Several other authors observed similar results [33, 56]. In fact, PDGF enhanced bone collagen degradation [33], and disrupted or inhibited bone matrix formation [56]. Nakashima et al. [57] found that PDGF increased DNA synthesis, while causing 40–65 % inhibition of ALP activity. Tanaka and Liang [58] reported that the material exerted no effect on cellular ALP activity or collagen synthesis. Yokose et al. [59] reported that PDGF-BB significantly reduced the ALP activity of DPSCs.

Favorable cell-surface interactions with EMD were demonstrated, including ALP expression and abundant mineralization. EMD gave superior results compared with MTA and PDGF regarding osteogenic differentiation of DPSCs. The effects of MTA on osteogenesis of DPSCs were inconclusive and further studies are required. Moreover, our data on PDGF did not support its ability to induce osteogenic differentiation of DPSCs. However, PDGF did facilitate cell attachment and growth, suggesting a different mechanism of action that worth further investigation.