Replication Data for: Improvement of osteoblast adhesion, viability, and mineralization via restoring cell cytoskeleton after bisphosphonate discontinuation in vitro

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Replication Data for: Improvement of osteoblast adhesion, viability, and mineralization via restoring cell cytoskeleton after bisphosphonate discontinuation in vitro
Patntirapong, Somying; Champakerdsap, Chunya; Mathaveechotikul, Pichaya; Vatanasilp, Apichaya, 2024, "Replication Data for: Improvement of osteoblast adhesion, viability, and mineralization via restoring cell cytoskeleton after bisphosphonate discontinuation in vitro", https://doi.org/10.48331/scielodata.G7NCSX, SciELO Data, V1, UNF:6:TzhLckb/KHUlQ3pudtqQjw== [fileUNF]
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Description
Introduction: Bisphosphonate is prescribed to treat excessive bone resorption in osteoporotic patients. However, its use is associated with potential adverse effects such as medication-related osteonecrosis of the jaw, prompting the introduction of the drug holiday concept in patients before dentoalveolar surgery. Furthermore, discontinuation of bisphosphonate has been studied in vivo, human and animal models. However, it is not known whether this approach could affect bone cells in vitro. Therefore, the objective of this study was to investigate the potential effects of bisphosphonate discontinuation on pre-osteoblast and osteoblast activities in vitro. Methodology: Pre-osteoblasts (MC3T3) and osteoblasts were treated with bisphosphonate (alendronate) at concentrations 1, 5, and 10 µM. Then, alendronate was withdrawn at different time points. Negative control was untreated cells (0 µM), while positive control was cells incubated with alendronate throughout the experiment. Cell viability, cell adhesion, cell cytoskeleton, mineralization, and gene expressions were investigated. Results: Pre-osteoblasts and osteoblasts showed a decrease in cell viability after treatment with 5-10 μM alendronate for 4 days or longer. Two days of alendronate discontinuation significantly increased cell viability compared with positive control. However, these levels did not reach the levels of the negative control. Bone nodule formation was reduced by alendronate. Discontinuation of alendronate regained bone nodule formation. Longer discontinuation periods were more effective in recovering nodule formation compared to shorter periods. The addition of alendronate resulted in an increase in the percentage of dead cells; this decreased when alendronate was withdrawn. Alendronate affected cell cytoskeleton by disassembling actin stress fiber. Also, the number of cell adhesion and cell morphological parameters were impacted by alendronate. Discontinuation of alendronate restored cell adhesion and these parameters. Overall, the highest improvement after alendronate discontinuation was seen at 10 µM. However, alendronate treatment and discontinuation did not affect osteoblast gene expressions. Conclusion: Discontinuation of alendronate help recover the negative effects of the drug on cell viability, cell adhesion, and mineralization via restoring cell cytoskeleton. Our data suggested the benefits of the drug holiday and/or intermittent strategies for alendronate administration at the cellular level. (2024-06-25)
Subject Medicine, Health and Life Sciences
Keyword Diphosphonates, Osteoblasts, Cytoskeleton
Related Publication Patntirapong S, Champakerdsap C, Mathaveechotikul P, Vatanasilp A. Improvement of osteoblast adhesion, viability, and mineralization via restoring cell cytoskeleton after bisphosphonate discontinuation in vitro. J Appl Oral Sci. 2024;32:e20240034. doi: 1678-7757-2024-0034
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Cell viability of pre-osteoblasts and osteoblasts after ALN treatment and discontinuation at various time points. MC3T3 cells were incubated with 1, 5, and 10 µM ALN (A1, A5, and A10). Negative control was abbreviated as A0. The “S” was abbreviated for stoppage of ALN. N = 4
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Examination of osteoblast nodule formation and viability. Osteoblasts were incubated with 1, 5, and 10 µM ALN (A1, A5, and A10). (A) Micrographs demonstrate nodule formation after treatment and cessation of ALN. (B) Quantification of alizarin red-positive area (mm2). (C) Quantification of alizarin red staining. (D) Osteoblast viability at 20 days. N = 8
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Evaluation of viable cells. (A) Micrographs of pre-osteoblast depicting cell morphology. (B) Number of live cells after 7-day cell culture. (C) Percent of dead cells after 7-day cell culture. Arrowhead indicated round cells. Scale bar = 100 µm. N = 4
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Investigation of cell adhesion after treatment and cessation of ALN. (A) Confocal images of nuclei and cell cytoskeleton. (B) Number of DAPI/field. (C) Stress fiber intensity. (D) Stress fiber density. (E) Cell area (µm2). (F) Cell perimeter. (G) Area/perimeter (A/P). (H) Aspect ratio. (I) Circularity. (J) Solidity. Scale bar = 100 µm.
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Gene expressions. Two conditions of treatment and cessation were investigated. Gr 1: Tx1d S1d, Gr 9: Tx4d S3d. Dashed lines are mRNA expressions of A0. N = 3
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A schematic diagram demonstrates experimental plan. (A) Time periods of ALN treatment and discontinuation for cell viability. (B) Time periods of ALN treatment and discontinuation for mineralization. (C) Cell adhesion and cell cytoskeleton processing
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MTT assay demonstrates pre-osteoblast cell viability. The cells without treatment grew gradually from 2 days to 7 days. ALN treatments (1, 5, and 10 µM) affected cell viability
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This study demonstrated the effects of ALN and ALN discontinuation in a pre-osteoblast/an osteoblast. ALN and ALN discontinuation affected cell cytoskeleton (red color), which led to changes in cell morphology, cell adhesion, cell viability, and bone nodule formation. ALN, alendronate; discon., discontinuation; A/P, area/perimeter; AR, aspect ratio
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3 Variables, 38 Observations UNF:6:jOgApQSchn44yxTgNWRXiw==
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