Development, Establishment, and Validation of a Model for the Mineralization of Periodontium Remodelling Cells: Cementoblasts
Abstract
:1. Introduction
2. Results
2.1. Cementoblast Calcification Increases with Increasing Phosphate Concentrations
2.2. Cementoblast Calcification Increases with Increasing Calcium Concentrations
2.3. Increasing Phosphate Concentrations in Combination with Increased Calcium Concentrations Augment Cementoblast Calcification
2.4. Increasing Calcium Concentrations in Combination with Increased Phosphate Concentration Augment Cementoblast Calcification
2.5. Cementoblast Calcification Decreases with Increasing Fetal Calf Serum Concentrations
2.6. Cementoblast Calcification Is Dependent on Ascorbic Acid Concentrations, Independent of Dexamethasone Supplementation
2.7. Cementoblast Calcification Is Dependent on β-Glycerolphosphate Concentrations
2.8. Visualization and Quantification of the Calcification of Cementoblasts by Von Kossa Staining and Alizarin Red Staining
2.9. Different Calcification Pathways Are Triggered by Different Calcification-Inducing Supplements
2.10. Kinases Influencing Osteogenic Pathways Are Phosphorylated by the Calcification-Inducing Supplements
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Basic Incubation Medium of Cementoblasts
4.3. Inducers of Calcification Processes in Cementoblasts for Experiments
4.4. Quantification of the Calcium Concentration of Cementoblasts
4.5. Quantification of Protein Concentration of Cementoblasts
4.6. Visualization and Quantification of the Calcification of Cementoblasts by Von Kossa Staining
4.7. Visualization and Quantification of the Calcification of Cementoblasts by Alizarin Red Staining
4.8. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) Analyses of mRNA Expression
4.9. Kinase Assay
4.10. Statistics
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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CM 1 | CM 2 | CM 3 | CM 4 | CM 5 | CM 6 | |
---|---|---|---|---|---|---|
FCS [Vol%] | 2.5 | 2.5 | 5 | 5 | 5 | |
NaH2PO4 [mmol L−1] | 0.95 | 0.65 | ||||
Na2HPO4 [mmol L−1] | 0.95 | 0.65 | ||||
CaCl2 [mmol L−1] | 2.5 | 0.2 | ||||
L-ascorbic acid [µmol L−1] | 75 | 75 | 75 | |||
β-glycerolphosphate [mmol L−1] | 10 | 12.5 | 7.5 | |||
Dexamethasone [nmol L−1] | 10 | 5 | 5 |
Experiment | DMEM | P/S [%] | Na2HPO4: NaH2PO4 (1:1) [mmol L−1] Final Conc. | CaCl2 [mmol L−1] Final Conc. | FCS [%] | β-Glycerol-Phosphate [mmol L−1] | Ascorbic Acid [µmol L−1] | Dexame-Thasone [nmol L−1] |
---|---|---|---|---|---|---|---|---|
Phosphate or calcium concentration dependency | ||||||||
Control | + | 1 | 0.9 | 1.8 | 2.5 | – | – | – |
CM (Phosphate) | + | 1 | 1.3, 1.8, 2.3, 2.8, 3.3, 3.8, 4.3 | 1.8 | 2.5 | – | – | – |
CM (CaCl2) | + | 1 | 0.9 | 2.3, 2.8, 3.3, 3.8, 4.3, 4.8, 5.3 | 2.5 | – | – | – |
Phosphate and calcium concentration dependency | ||||||||
Control | + | 1 | 0.9 | 1.8 | 2.5 | – | – | – |
CM (Phosphate & fixed CaCl2) | + | 1 | 1.3, 1.6, 1.9, 2.2, 2.5, 2.8, 3.1 | 2 | 2.5 | – | – | – |
CM (Fixed phosphate & CaCl2) | + | 1 | 1.1 | 2.1, 2.4, 2.7, 3.0, 3.3, 3.6, 3.9 | 2.5 | – | – | – |
FCS concentration dependency | ||||||||
Control | + | 1 | 0.9 | 1.8 | 1, 2.5, 5, 7.5, 10, 12.5, 15 | – | – | – |
CM | + | 1 | 0.9 | 1.8 | 1, 2.5, 5, 7.5, 10, 12.5, 15 | 10 | 75 | – |
Ascorbic acid concentration dependency | ||||||||
Control | + | 1 | 0.9 | 1.8 | 5 | – | – | – |
CM | + | 1 | 0.9 | 1.8 | 5 | 10 | 25, 50, 75, 100, 125, 250 | – |
CM with dexamethasone | + | 1 | 0.9 | 1.8 | 5 | 10 | 25, 50, 75, 100, 125, 250 | 10 |
β-glycerolphosphate concentration dependency | ||||||||
Control | + | 1 | 0.9 | 1.8 | 5 | – | – | – |
CM | + | 1 | 0.9 | 1.8 | 5 | 1.5, 2.5, 5, 7.5, 10, 12.5, 15 | 75 | – |
CM with dexamethasone | + | 1 | 0.9 | 1.8 | 5 | 1.5, 2.5, 5, 7.5, 10, 12.5, 15 | 75 | 10 |
Gene | Nucleotide Sequence | |
---|---|---|
β-actin | Forward | 5′-CAACGAGCGGTTCCGATG-3′ |
Reverse | 5′-GCCACAGGATTCCATACCCAA-3′ | |
Bone sialoprotein (Lbsp) | Forward | 5′-TGAGTGACAGCCGGGAGAAC-3′ |
Reverse | 5′-AAGAAAGTAGCGTGGCCGGT-3′ | |
Osteopontin (Opn) | Forward | 5′-CTTTCACTCCAATCGTCCCTAC-3′ |
Reverse | 5′-GCTCTCTTTGGAATGCTCAAGT-3′ |
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Bhargava, S.; Jankowski, J.; Merckelbach, E.; Roth, C.E.; Craveiro, R.B.; Wolf, M. Development, Establishment, and Validation of a Model for the Mineralization of Periodontium Remodelling Cells: Cementoblasts. Int. J. Mol. Sci. 2023, 24, 13829. https://doi.org/10.3390/ijms241813829
Bhargava S, Jankowski J, Merckelbach E, Roth CE, Craveiro RB, Wolf M. Development, Establishment, and Validation of a Model for the Mineralization of Periodontium Remodelling Cells: Cementoblasts. International Journal of Molecular Sciences. 2023; 24(18):13829. https://doi.org/10.3390/ijms241813829
Chicago/Turabian StyleBhargava, Shruti, Joachim Jankowski, Erik Merckelbach, Charlotte Elisa Roth, Rogerio Bastos Craveiro, and Michael Wolf. 2023. "Development, Establishment, and Validation of a Model for the Mineralization of Periodontium Remodelling Cells: Cementoblasts" International Journal of Molecular Sciences 24, no. 18: 13829. https://doi.org/10.3390/ijms241813829