Biomineralization potential and biological properties of a new tantalum oxide (Ta2O5)-containing calcium silicate cement

Abstract

Objective: The present study evaluated the biological effects and biomineralization potential of a new tantalum oxide (Ta₂O₅)-containing material designed for vital pulp therapy or perforation repair (NeoMTA 2), compared to NeoMTA Plus and Bio-C Repair.

Material and methods: Human dental pulp stem cells (hDPSCs) were exposed to different eluates from NeoMTA Plus, NeoMTA 2, and Bio-C Repair. Ion release from each material was determined using inductively coupled plasma-optical emission spectrometry (ICP-MS). The biological experiments performed were MTT assays, apoptosis/necrosis assays, adhesion assays, migration assays, morphology evaluation, and reactive oxygen species (ROS) production analysis. Biomineralization was assessed by Alizarin red S staining. Finally, osteo/odontogenic gene expression was determined by real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). Data were analyzed using one-way ANOVA followed by Tukey's multiple comparison test.

Results: NeoMTA 2 displayed a significantly higher calcium release compared to the other materials (p < 0.05). When hDPSCs were cultured in presence of the different material eluates, all groups exhibited similar hDPSC viability and migration rates when compared to untreated cells. Substantial cell attachment and spreading were observed in all materials' surfaces, without significant differences. hDPSCs treated with NeoMTA 2 displayed an upregulation of ALP, Col1A1, RUNX2 (p < 0.001), ON, and DSPP genes (p < 0.05), and showed the highest mineralization potential compared to other groups (p < 0.001). Finally, the more concentrated eluates from these materials, specially NeoMTA Plus and NeoMTA 2, promoted higher ROS production in hDPSCs compared to Bio-C Repair and control cells (p < 0.001), although these ROS levels did not result in increased cell death.

Conclusions: The new tantalum oxide (Ta₂O₅)-containing material shows an adequate cytocompatibility and the ability to promote biomineralization without using chemical osteogenic inducers, showing great potential as a new material for vital pulp therapy.

Clinical relevance: NeoMTA 2 seems to be a promising material for vital pulp therapy. Further studies considering its biocompatibility and biomineralization potential are necessary.

Keywords: Bioactivity; Ion-releasing materials; NeoMTA; Vital pulp therapy biomineralization.

Fig. 1

SEM–EDS analysis results for NeoMTA 2 (A), NeoMTA Plus (B), and Bio-C Repair (C) disks (n = 9). The first column presents SEM micrographs of each material (scale bar: 100 μm). The second column illustrates the EDS plots with the correspondent peaks detected. The third column classifies the list of elements present per material by weight and atomic weight

Fig. 2

Mitochondrial viability assay. In vitro cytotoxicity of hDPSCs after exposure to extracted medium prepared from Neo MTA Plus, Neo MTA 2, and Bio-C Repair. Data are presented as absorbance values (570 nm) at 24, 48, and 72 h of exposure of the material eluates to hDPSCs, compared to the control. *p < 0.05; **p < 0.01; ***p < 0.001. Each experimental condition was performed in triplicate for each VPT material and analyzed in three independent experiments

Fig. 3

Migration was evaluated using wound healing assays. Cells were exposed to undiluted (1/1) and diluted (1/2 and 1/4) eluates from materials. The control condition consisted of cells maintained in normal growth medium. Graphical results are presented as mean relative wound closure (RWC) percentages at each of the time points, relative to the total wound area at 0 h. Asterisks designate significant differences compared to the control. *p < 0.05; **p < 0.01; ***p < 0.001. Each experimental condition was performed in triplicate for each VPT material and analyzed in three independent experiments

Fig. 4

Cell attachment. Sample disks with the aforementioned standardized dimensions were obtained (n = 15) for each of the materials and allocated into three groups (n = 5). Representative SEM micrographs illustrate the adhesion of hDPSCs directly seeded on Neo MTA Plus, Neo MTA 2, and Bio-C Repair. Magnifications: × 100, × 300, and × 1500. Scale bars: 500 μm, 100 μm, and 30 μm

Fig. 5

Analysis of changes in cell morphology, actin cytoskeleton structure, and organization on hDPSCs after treatment with NeoMTA Plus, NeoMTA 2, and Bio-C Repair by confocal fluorescence microscopy. F-actin fibers were stained with AlexaFluor™ 594–conjugated phalloidin (red), whereas cell nuclei were counterstained with DAPI (blue). Confocal fluorescence microscopy images shown are representative from n = 3 separate experiments. Scale bar: 100 μm. Each experimental condition was performed in triplicate for each VPT material and analyzed in three independent experiments

Fig. 6

Flow cytometry analysis of cell apoptosis and necrosis induced by the different vital pulp material extracts on hDPSCs by annexin V-PE/7-AAD staining. Numbers inside representative dot plots represent percentages of live (Q4 quadrants), early apoptotic (Q3 quadrants), and late apoptotic and necrotic cells (Q1 and Q2 quadrants). Bar graphs show mean ± SD from n = 3 separate experiments. Percentages of live cells were significantly decreased compared to the control, *p < 0.05; **p < 0.01; ***p < 0.001, respectively

Fig. 7

Reactive oxygen species (ROS) production. CM-H₂DCFDA staining was used to evaluate intracellular ROS production in hDPSCs exposed to ion-releasing materials. Bar graph showing percentages of positive cells for ROS production in each experimental condition is shown and represented as mean ± SD from n = 3 separate experiments. Percentages of CM-H₂DCFDA-positive cells were significantly increased compared to the control, *p < 0.05; **p < 0.01; ***p < 0.001, respectively

Fig. 8

The expression of osteo/odontogenic genes detected by RT-qPCR. Data are expressed as mean ± SD and relative to GAPDH gene expression. *p < 0.05; **p < 0.01; ***p < 0.001. Each experimental condition was performed in triplicate for each VPT material and analyzed in three independent experiments

Fig. 9

Calcium deposition, as a final product of the odonto/osteogenic differentiation process, was assessed by using Alizarin Red S staining on day 21 (*p < 0.05; **p < 0.01; ***p < 0.001). Each experimental condition was performed in triplicate for each VPT material and analyzed in three independent experiments