The biological properties and bioactive potential of hydraulic calcium silicate–based materials have been documented by numerous studies. Still, the rapid introduction of new material compositions with different setting mechanisms into the market calls for an updated biological and chemical profiling of each new composition before its clinical use. This highlights the importance and relevance of
in vitro studies as a preliminary approach for the assessment of new materials. However, results from these assays should be interpreted with caution, since the behavior of the tested materials could be potentially influenced by a number of external factors in the clinical setting [
23].
Experimental setup
The present study investigated the effects of three calcium-silicate based materials on the viability, migration, proliferation, morphology, and adhesion of hPDLSCs. Although recommendations laid down in the ISO specification 10993-5 suggest using established cell lines in cytotoxicity assays, freshly isolated human hPDLSCs were used in this study as they may get in contact with such materials
in vivo [
23]. Although present in a lesser quantity than periodontal ligament fibroblasts and osteoblasts, hPDLSCs are considered a prime cell source for periodontal regeneration [
32] and are supposed to be involved in healing of apical periodontitis and tissue regeneration [
33]. This approach is in line with several previous comparable studies [
20,
21,
34], while one study used human bone marrow-derived mesenchymal stem cells instead of hPDLSCs [
35]. Dental stem cells, as a subpopulation of multipotent mesenchymal stromal cells (MSCs), share a specific mesenchymal-like phenotype, but also exhibit individual characteristics, which could result in a different response to external stimuli [
36]. For example, hPDLSCs exhibit a higher osteogenic potential compared to hDPSCs [
19] and multipotent stem cells derived from pulp tissue of human exfoliated deciduous teeth (SHEDs) [
37]. This justifies the need for a separate assessment and categorization of the biological behavior of the different dental stem cell variants.
The minimum criteria for the identification of MSCs have been developed by the International Cellular Therapy Association. Accordingly, MSCs should adhere to plastic surfaces under standard culture conditions and express CD105, CD73, and CD90, no expression of CD45, CD34, CD14, and CD11b. MSCs should be able to differentiate into different cells such as osteoblasts, adipocytes, and chondroblasts in vitro [
38]. Therefore, primary antibodies raised against CD105, CD90, CD73, CD34, and CD45 were used in the present study for characterization of the isolated PDL cells as mesenchymal stem cells. This approach has been further described in more detail previously [
23].
According to ISO standards, cell viability tests can be evaluated after 24, 48, and 72 h. Although the cell viability test periods are generally stated as the 1st and 3rd days in the literature, the cell viability was evaluated at the end of the 7th day in this study in order to evaluate the long-term toxic effects of the materials [
39]. Other comparable studies also investigated cell viability after 7 days [
34,
35]. The use of three dilutions (1:1, 1:2, and 1:4) was performed to simulate the clinical conditions, in which the tested materials can be placed on the remaining dentin thicknesses of 0.01 to 0.25 mm or directly on pulp exposures. Therefore, the concentration of the material that reaches viable pulp tissue may differ [
17].
It is well known that bioactive materials release substances that could potentially delay or enhance the healing process in the periradicular tissues. For this reason, the wound healing assay was performed to predict how the coordinated migration of hPDLSCs would occur after exposure to the tested materials. There is a direct correlation between cell migration and cell viability [
40].
All tested materials in the present study were incubated as set discs. According to a recent systematic review, this is in accordance with most
in vitro studies on the biological interaction between CSBMs and dental stem cells [
41]. Although the use of freshly mixed materials can better predict the biologic response of dental stem cells, the use of set materials is superior for prediction of their delayed/long-term response [
42]. Future studies should include both preparations, to provide a comprehensive biological profiling of the tested repair materials [
43].
Findings
Regarding cell migration for the 1:1 and 1:2 dilutions at day 1, pH value, and calcium ion release, Biodentine achieved significantly better results than both other materials. With regard to cell viability of the 1:1 and 1:2 dilutions and cell attachment, Biodentine and NeoPutty were significantly superior compared to TheraCal PT. Therefore, the null hypothesis of this study was rejected.
The results obtained for Biodentine are corroborated by several previous studies, which confirmed the excellent level of cytocompatibility of this CSBM [
20,
35,
44‐
46]. Biodentine possesses the potential to induce proliferation and osteogenic differentiation of dental stem cells [
35,
46], as this CSBM has been shown to upregulate the odontogenic marker dentin sialophosphoprotein [
20,
45]. A consistent finding is that Biodentine promoted mineralized nodule deposition in about 21 days [
20,
44] and that exposure of Biodentine to phosphate-buffered solutions resulted in precipitation of apatite crystalline structures on its surface [
47,
48]. Also, the good attachment of stem cells on the surface of Biodentine, as found in the present study, is in agreement with previous reports [
35].
To a certain extent, these properties of Biodentine are related to the distinct calcium ion release and the relatively high pH of this CSBM, as Ca
2+ is necessary for the differentiation, proliferation, and mineralization of cells [
34]. Ca
2+ has an impact on the activity of pyrophosphatase, which induces dentin mineralization [
49]. An alkaline pH has an impact on transforming growth factor-β1 (TGF-β1) release, and TGF-β1 may induce osteoblastic proliferation and differentiation of odontoblasts [
50]. The present results reveal that both pH values as well as calcium ion release were significantly higher for Biodentine than for the two other CSBMs tested. These findings are corroborated by previous studies, confirming the capacity of Biodentine to release calcium ions [
31,
46,
47,
51,
52] and to create an alkaline pH [
31,
44,
47,
50,
52,
53].
NeoPutty obtained similar results compared to Biodentine regarding cell viability, cell migration for dilutions of 1:1 and 1:2 after day 1 and for the 1:4 dilution at all observation times, and cell attachment. To date, limited scientific evidence regarding the biological properties of NeoPutty is available, Sun et al. [
18] reported that NeoPutty exhibited a higher biocompatibility than another CSBMs (EndoSequence BC RRM putty (Brasseler, Savannah, GA, USA), while Lozano-Guillén et al. [
17] reported similar biocompatibility of NeoPutty to its predecessor (NeoMTA Plus) and MTA, both studies used human dental pulp stem cells [
54]. In line with these findings, another study investigated the effects of NeoPutty on human dental pulp stem cells and human periodontal ligament fibroblasts and found that these repair materials showed acceptable cytocompatibility profiles on both cell types [
18]. It is worthy to mention that NeoPutty contains calcium aluminate that has been shown to have adequate biocompatibility after subcutaneous implantation in rats [
55] and also supported the acquisition of osteogenic cell phenotypes in vitro [
56].
According to the present results, the resin-modified material TheraCal PT showed significantly inferior results than Biodentine in all tests and displayed significantly worser results than NeoPutty regarding cell migration for the 1:4 dilution and the 1:1 and 1:2 dilutions after day 1, also for the cell attachment and pH value results. These observations are in full congruence with previous studies [
31,
34] but partially disagrees the results of two other investigations [
20,
21] that demonstrated favorable cytocompatibility on human DPSCs with mineralized nodule formation and bioactive properties [
20,
21], as well as upregulation of the osteogenic markers osteonectin and runt-related transcription factor 2 [
20]. Contrarily, Küden et al. [
34] showed that TheraCal PT decreased the number of viable hDPSCs significantly from day 1 to day 3, and at day 7 viable cells were no longer detected. The authors correlated this adverse effect to the time-dependent monomer release of TheraCal PT and showed that this material released marked amounts of TEGDMA (triethylene glycol dimethacrylate) at days 3 and 7 [
34]. Moreover, in accordance with the present results, Elbanna et al. [
31] reported that TheraCal PT merely slightly increased the pH value up to about 8.7 after 14 days and that the calcium ion release was very limited, as also reported by Küden et al. [
34]. On the whole, the authors concluded that TheraCal PT exhibited only limited bioactivity [
31].
In general, the present findings support the results of a systematic review [
7] in as far as the two resin-free CSBMs tested consistently provided significantly better results than the resin-based material.