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

Background Regeneration of periodontal tissues is a major goal of periodontal therapy. Dental pulp stem cells (DPSCs) show mesenchymal cell properties with the potential for dental tissue engineering. Enamel matrix derivative (EMD) and platelet-derived growth factor (PDGF) are examples of materials that act as signaling molecules to enhance periodontal regeneration. Mineral trioxide aggregate (MTA) has been proven to be biocompatible and appears to have some osteoconductive properties. The objective of this study was to evaluate the effects of EMD, MTA, and PDGF on DPSC osteogenic differentiation. Methods Human DPSCs were cultured in medium containing EMD, MTA, or PDGF. Control groups were also established. Evaluation of the achieved osteogenesis was carried out by computer analysis of alkaline phosphatase (ALP)-stained chambers, and spectrophotometric analysis of alizarin red S-stained mineralized nodules. Results EMD significantly increased the amounts of ALP expression and mineralization compared with all other groups (P < 0.05). Meanwhile, MTA gave variable results with slight increases in certain differentiation parameters, and PDGF showed no significant increase in the achieved differentiation. Conclusions EMD showed a very strong osteogenic ability compared with PDGF and MTA, and the present results provide support for its use in periodontal regeneration.


Background
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][7][8][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][16][17][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.

Cell isolation and characterization
Dental pulp stem cells in the primary cultures started to appear in 5-14 days and became attached to the plate surfaces (Fig. 1a). Cells from the second passage successfully formed multiple colonies, with around 50 cells per colony (Fig. 1b). Flow cytometry analyses confirmed positive expressions of stromal cell-associated markers, with negative expressions of hematopoietic and endothelial markers (Fig. 1g). Cells that underwent osteogenic induction showed increased ALP staining compared with negative control cells (Fig. 1c, d), while cells cultured in the adipogenic medium exhibited several oil red O-positive lipid granules (Fig. 1e, f).

Material application ALP staining
The samples showed different degrees of ALP staining (Fig. 2). One-way ANOVA revealed significant differences among the compared groups (P < 0.0001) ( Table 1).
In contrast, MTA gave inconsistent findings, although it increased the ALP activity in a similar manner to the reference control when evaluated by the average optical density, the material resulted in reductions of the other parameters compared with the reference control, although those reductions were not always significant (P > 0.05).
With regard to PDGF, ALP expression generally revealed lower results compared with the reference control for the three parameters respectively, and these reductions were consistently significant (P < 0.05; Table 1).

Alizarin red S staining
There were obvious differences in the amounts of mineralization among the groups (Fig. 3). One-way ANOVA revealed these differences to be significant (P < 0.0001) ( Table 2).
The EMD group had a significantly increased amount of mineralized nodule formation compared with all other groups, giving a mean absorbance of 1.2 ± 0.13 (P < 0.05).
Although the mean absorbance of the PDGF group (0.09 ± 0.01) appeared to be slightly different than the other groups, these differences were statistically nonsignificant (P > 0.05; Table 2).

Discussion
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 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  [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.

Conclusions
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.

Isolation of stem cells
Human DPSCs were isolated and characterized by the authors in the Stem Cell Unit, King Saud University, Kingdom of Saudi Arabia (unpublished data). Teeth were collected from patients after they provided signed informed consent, according to a protocol approved by the institutional ethical committee (College of Dentistry Research Center-CDRC). Briefly, the pulp contents of freshly extracted molar teeth were combined and subjected to 20-40 minutes of enzymatic digestion using collagenase type I (1 mg/ml) and dispase (5000 caseinolytic units). Subsequently, the cells were allowed to grow under regular cell culture conditions (37°C , 5 % CO 2 ), using Dulbecco's modified Eagle's medium (DMEM) supplemented with 20 % fetal bovine serum (FBS), 1 % penicillin-streptomycin (Pen-Strept), and 1 % non-essential amino acids (all purchased from Gibco-Invitrogen, USA).

Characterization of stem cells Colony forming unit-fibroblasts (CFU-F)
CFU-F were evaluated by culturing 2.5 × 10 3 cells at the second passage in 6-cm culture dishes. At day 14, the cells were fixed with 1 % paraformaldehyde, stained with 0.5 % crystal violet, and subjected to microscopic evaluation using a phase-contrast inverted light microscope (Zeiss, Leica, Germany).

Flow cytometry
Fourth passage cells (1.5 × 10 6 ) were washed with FACS buffer (1× phosphate-buffered saline, 5 % FBS, 0.1 % sodium azide), and diluted in 1.5 ml of phosphate-buffered saline. Next, PE-conjugated mouse anti-human CD146, CD73, CD29, and HLA-DR, FITC-conjugated mouse anti-human CD34, CD90, CD45, CD13, and CD31, and APC-conjugated mouse anti-human CD105, CD14, and CD44 antibodies were prepared in dark (all from BD Biosciences, USA, except for the monoclonal antibody against human CD105, which was purchased from R&D Systems, USA) and utilized. In each FACS tube, 100 μl of cells was mixed with 10 μl of the corresponding antibody, and incubated for 30 minutes in the dark at 4°C. The expressions of cellular markers were assessed using a Becton Dickinson FACSCalibur Flow Cytometer (BD Biosciences, USA), and the resulting data were analyzed using Cell Quest Pro Software Version 3.3, BD bioscience, USA).

Material application
Initially, a pilot study was carried out to evaluate three different concentrations for each material, and the concentrations yielding the highest amount of differentiation were selected for the comparisons (Fig. 4). Thereafter, cells at the fourth passage were cultured and divided into five groups as shown below. The achieved differentiation was analyzed by evaluation of ALP expression through ALP staining and calcium ion deposition through alizarin red S staining.

ALP activity
Cells were plated on 8-chamber slides at the density of 0.02 × 10 6 cells/chamber and allowed to attach and grow to 50 % confluency. Thereafter, the slides were divided into the above-mentioned five different groups and regular or osteogenic medium was applied accordingly. On day 5, the cells were fixed and stained for ALP with Naphthol-AS-TR-phosphate solution (Sigma, UK). Next, the chambers were evaluated under a high-resolution digital microscope where the whole stained chambers were scanned with a ScanScope slide scanner (Aperio Technologies Inc., USA) at 40× objective magnification. The digital images of  repeated three times independently, giving nine different readings for each trial.

Statistical analysis
Data was analyzed using SPSS statistical software (version 16.0; SPSS, USA). Descriptive statistics (mean and standard deviation) were used to describe the quantitative outcome variables. One-way analysis of variance (ANOVA) was used to compare the mean values of outcome variables across the categorical variables (groups), followed by a post-hoc Tukey test for pairwise comparisons. Values of P < 0.05 were considered to indicate statistical significance.

Competing interest
The authors declare that they have no competing interests.

Authors contributions
SA participated in different aspects of laboratory studies including cell characterization, and material application, in addition to preparing of the primary draft for this paper. NA helped in the development of the main research idea, prepared the basic study design, and provided critical review for whole paper writing. Additionally, she arranged for obtaining the dental test materials. AD provided general technical support especially in cell characterization and differentiation analysis, in addition to his role in getting all basic laboratory materials. MN have helped in cellular osteogenic and adipogenic differentiation studies, and supervised the writing of the technical part of the study (materials and methods). All authors read and approved the final manuscript.
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