Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
, 31 (12), 1510-22

Cytotoxicity and Osteogenic Potential of Silicate Calcium Cements as Potential Protective Materials for Pulpal Revascularization

Affiliations
Comparative Study

Cytotoxicity and Osteogenic Potential of Silicate Calcium Cements as Potential Protective Materials for Pulpal Revascularization

Eduardo A Bortoluzzi et al. Dent Mater.

Abstract

Objectives: In pulpal revascularization, a protective material is placed coronal to the blood clot to prevent recontamination and to facilitate osteogenic differentiation of mesenchymal stem cells to produce new dental tissues. Although mineral trioxide aggregate (MTA) has been the material of choice for clot protection, it is easily displaced into the clot during condensation. The present study evaluated the effects of recently introduced calcium silicate cements (Biodentine and TheraCal LC) on the viability and osteogenic differentiation of human dental pulp stem cells (hDPSCs) by comparing with MTA Angelus.

Methods: Cell viability was assessed using XTT assay and flow cytometry. The osteogenic potential of hDPSCs exposed to calcium silicate cements was examined using qRT-PCR for osteogenic gene expressions, alkaline phosphatase enzyme activity, Alizarin red S staining and transmission electron microscopy of extracellular calcium deposits. Parametric statistical methods were employed for analyses of significant difference among groups, with α=0.05.

Results: The cytotoxic effects of Biodentine and TheraCal LC on hDPSCs were time- and concentration-dependent. Osteogenic differentiation of hDPSCs was enhanced after exposure to Biodentine that was depleted of its cytotoxic components. This effect was less readily observed in hDPSCs exposed to TheraCal LC, although both cements supported extracellular mineralization better than the positive control (zinc oxide-eugenol-based cement).

Significance: A favorable tissue response is anticipated to occur with the use of Biodentine as a blood clot-protecting material for pulpal revascularization. Further investigations with the use of in vivo animal models are required to validate the potential adverse biological effects of TheraCal LC on hDPSCs.

Keywords: Biocompatibility; Calcium silicate cements; Cytotoxicity; Mineral trioxide aggregate; Revascularization.

Figures

Figure 1
Figure 1
XTT assay. A. Direct evaluation of effect of calcium silicate cements (N = 12) on the mitrochondrial enzymatic activitiy of human dental pulp stem cells (hDPSCs) after different aging cycles. Values (in means and standard deviations) are expressed as percentages of the dehydrogenase activities of untreated hDPSCs (negative control). For the factor material within each aging cycle, groups designated by horizontal bars at the same level are not significantly different (P > 0.05). For the factor aging cycle within each material, groups connected by dotted lines at the same level are not significantly different (P > 0.05). B. Indirect evaluation of effect of eluents with different dilution factors (N = 12) on the mitrochondrial enzymatic activitiy of hDPSCs. Values represent means and standard deviations. For the factor material within each dilution factor, groups designated by a horizontal bar are not significantly different (P > 0.05). For the factor dilution factor within each material, groups connected by dotted lines are not significantly different (P > 0.05).
Figure 2
Figure 2
Flow cytometry. Representative two-deminersional dot plots of annexin V FITC and ethdium homodimer III-stained hDPSCs that were sorted into healthy (lower left), early apoptotic (lower right), late apoptotic (upper right) and necrotic cells (upper left). A. Untreated hDPSCs (negative control). B. hDPSCs exposed to MTA Angelus. C. hDPSCs exposed to Biodentine. D. hDPSCs exposed to TheraCal LC. E. hDPSCs exposed to IRM (positive control). F. Bar chart comparing the percentage of healthy cells in the negative control and the three calcium silicate cements (N = 3). Values represent means and standard deviations. Groups designated by a horizontal bar are not significantly different (P > 0.05).
Figure 3
Figure 3
mRNA expressions of target osteogenic markers in hDPSCs after exposure to different tricalcium silicate cements in osteogenic differentiation medium. Values represent means and standard deviations. For each osteogenic marker (N = 3), groups with the same colored asterisks are significantly different from the GADPH endogenous control (P < 0.0085).
Figure 4
Figure 4
A. Intracellular ALP enzyme level in untreated hDPSCs and in hDPSCs exposed to the three calcium silicate cements and the IRM positive control (N = 12). Values represent means and standard deviations. For statistical comparison among the untreated hDPSCs and hDPSCs exposed to the calcium silicate cements, groups designated with horizontal bars are not significantly different (P > 0.05). B. Calcium molar contration of extracellular mineral deposits secreted by hDPSCs that were stained by Alizarin red S in the five groups (N = 6). Values represent means and standard deviations. Pairwise comparisons among untreated hDPSCs and hDPSCs exposed to the three calcium silicate cements indicate that all groups are significantly different from one another (P < 0.05). Representative images of Alizarin red S-stain mineral deposits prior to stain extraction and quantification in C. Untreated hDPSCs; D. MTA Angelus; E. Biodentine; F. TheraCal LC.
Figure 5
Figure 5
TEM images of untreated hDPScs before and after switching to the osteogenic differentiation medium. A. Low magnification of a stained section of virgin undifferentiated hDPSCs showing collagen fibrils deposited within the extracellular matrix. Bar = 500 nm. B. High magnification of stained, immature collagen fibrils secreted by undifferentiated hDPSCs. Bar = 100 nm. C. Low magnification of a stained section of hDPSCs after osteogenic differentiation with intracellular (arrowhead) and extracellular (pointer) mineral nodules. The minerals within the nodules were partially dislodged during sectioning. Bar = 1 μm. D. High magnification of unstained, extracellular mineralized nodules secreted by differentiated hDPSCs showing needle-shaped crystallites. Bar = 200 nm.

Similar articles

See all similar articles

Cited by 16 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback