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. 2020 Jan-Dec:29:963689720948497.
doi: 10.1177/0963689720948497.

Dose-Dependent Effects of Zoledronic Acid on Human Periodontal Ligament Stem Cells: An In Vitro Pilot Study

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Dose-Dependent Effects of Zoledronic Acid on Human Periodontal Ligament Stem Cells: An In Vitro Pilot Study

Anna Di Vito et al. Cell Transplant. 2020 Jan-Dec.

Abstract

Bisphosphonates (BPs) are widely used to treat several metabolic and oncological diseases affecting the skeletal system. Despite BPs' well-known therapeutic potential, they also displayed important side effects, among which is BPs-related osteonecrosis of the jaw, by targeting osteoclast activities, osteoblast, and osteocyte behavior. The aim of this study is to evaluate the biological effects of zoledronic acid (ZOL) in an in vitro model of periodontal ligament stem cells (PDLSCs) by using an experimental setting that resembles the in vivo conditions. PDLSCs were treated with different concentrations of ZOL ranging from 0.1 to 5 μM. The effects of ZOL exposure were evaluated on cell viability via 3-[4,5-Dimethylthiaoly]-2,5-diphenyltetrazolium bromide (MTT), cell cycle analysis, apoptosis detection, and immunofluorescence. Quantitative real-time polymerase chain reaction (PCR), colorimetric detection of alkaline phosphatase activity, and Alizarin Red S staining were performed to investigate the osteogenic potential of PDLSCs exposed to ZOL. MTT analysis showed that the viability of PDLSCs exposed to ZOL concentration ≥1.5 μM for 3 and 6 days was significantly lower (P < 0.001) than that of untreated cells. The percentage of apoptotic cells was significantly higher in PDLSCs exposed for 4 days to ZOL at 2 μM (P < 0.01) and 5 μM (P < 0.001) when compared to the control. Moreover, ZOL treatment (3 days) accounted for alterations in cell cycle distribution, with an increase in the proportion of cells in G0/G1 phase and a reduction in the proportion of cells in S phase. Chronic exposure (longer than 7 days) of PDLSCs to ZOL accounted for the downregulation of ALP, RUNX2, and COL1 genes at all tested concentrations, which fit well with the reduced alkaline phosphatase activity reported after 7 and 14 days of treatment. Reduced Col1 deposition in the extracellular matrix was reported after 14 days of treatment. Increased calcium deposits were observed in treated cells when compared to the control cultures. In conclusion, chronic exposure to 1 μM ZOL induced significant reduction of osteogenic differentiation, while ZOL concentrations ≥1.5 μM are required to impair PDLSCs viability and induce apoptosis.

Keywords: cell cultures; human periodontal ligament stem cells; osteogenic differentiation; zoledronic acid.

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Conflict of interest statement

Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Effects of ZOL on the viability and cell cycle distribution of PDLSCs. MTT analysis of PDLSCs at 3 and 6 days of exposure (A). Flow cytometry analyses of PDLSCs upon 3 days of drug treatment. Representative plots of cells in G1, S, and G2 cell cycle phases are shown. Histogram representative of four independent experiments shows percentage of cells in the different cell cycle phases in the presence or absence of ZOL (B). Representative dot plots of dose-dependent apoptosis increase in PDLSCs after dose escalation of ZOL evaluated with annexin-V/PI assay after 4 days, data analysis are shown in the histogram (C). Representative images of phalloidin staining highlighting F-actin rearrangement after 3 days of treatment; please note the presence of blebs (arrowheads) in cells exposed to 2 μM ZOL as well as the loss of actin stress fibers (asterisks) in cells treated with 5 μM ZOL. Nuclei are indicated in blue. Images were collected using Leica TCS SP8 confocal microscopy system (63×; D). Each assay was performed at least three times on biological replicates. Data are presented as the mean. For statistical analysis, Student’s t test was applied. *, P < 0.05; **/b, P < 0.01; ***/c, P < 0.001; DIV: days in vitro; PDLSCs: periodontal ligament stem cells; PI: propidium iodide; ZOL: zoledronic acid.
Figure 2.
Figure 2.
Immunophenotypic profile of PDLSCs after 5 days exposure to ZOL. Flow cytometry analysis showed mean fluorescence intensity of CD73, CD90, and CD105 (data obtained from three patients are reported, indicated as blue, red, and green lines) after dose escalation of ZOL. Please note that a significant reduction of the percentage of CD105-positive cells was only reported after exposure to 5 μM ZOL (A). Representative images of immunofluorescence analysis of vimentin in PDLSCs untreated and treated with ZOL. Nuclei are indicated in blue. Images were collected using Leica TCS SP8 confocal microscopy system (63×; B). Each assay was performed at least three times on biological replicates. Data are presented as the mean. For statistical analysis, Student’s t test was applied. ***, P < 0.001. PDLSC: periodontal ligament stem cell; ZOL: zoledronic acid.
Figure 3.
Figure 3.
Osteogenic differentiation of PDLSCs after chronic exposure to ZOL. Figure shows the schema of the experimental setup (A). Relative mRNA expression level of ALP at DIV3, DIV7, and DIV10 was normalized to GAPDH and displayed as fold increase or decrease respect to untreated cells at DIV3. Please note the upregulation of ALP mRNA in untreated cells during osteogenic differentiation (B). ALP activity was qualitatively evaluated after 7 and 14 days of osteo-induction (C). Relative mRNA expression level of COL1 at DIV3, DIV7, and DIV10 was normalized to GAPDH and displayed as fold increase or decrease respect to untreated cells at DIV3. Please note the upregulation of COL1 mRNA in untreated cells during osteogenic differentiation (D). Representative images of immunofluorescence analysis of Col1 in PDLSCs untreated and treated with ZOL for 7 and 14 days. Nuclei are indicated in blue. Arrows indicate Col1 deposits in extracellular space. Images were collected using Leica TCS SP8 confocal microscopy system (63×; E). Relative mRNA expression levels of RUNX2 and OCN in PDLSCs after differentiation culture for 10 days (F). Detection of the osteogenic differentiation of PDLSCs by Alizarin Red S (G) staining after 31 days of osteo-induction. Arrows indicate calcium deposits. Each assay was performed at least three times on biological replicates. Data are presented as the mean. For statistical analysis, Student’s t test was applied. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ALP: alkaline phosphatase; COL1: collagen type 1; DIV: days in vitro; OCN: osteocalcin; PDLSC: periodontal ligament stem cell; RUNX2: runt-related transcription factor 2; ZOL: zoledronic acid.

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