Synthetic Hydroxyapatite Inhibits Bisphosphonate Toxicity to the Oral Mucosa In Vitro
"> Figure 1
<p>(<b>A</b>) A stereomicroscopy image of a PermaBone<sup>®</sup> hydroxyapatite granule. (<b>B</b>) A scanning electron microscopy image of pre-granulated PermaBone<sup>®</sup> hydroxyapatite.</p> "> Figure 2
<p>Cell metabolic activity over 72 h in the presence of bisphosphonates measured by MTT assay. (<b>A</b>) Human oral fibroblasts with pamidronic acid, (<b>B</b>) human oral fibroblasts with zoledronic acid, (<b>C</b>) immortalised human oral keratinocytes with pamidronic acid and (<b>D</b>) immortalised human oral keratinocytes with zoledronic acid. Values normalised to 0 µM at 24 h. One significant outlying value removed. N = 3, n = 3. Error bars = standard deviation (SD). Significance against 0 µM at each time point indicated by * <span class="html-italic">p</span> ≤ 0.05.</p> "> Figure 3
<p>The metabolic activity of oral mucosa models when treated with (<b>A</b>) pamidronic acid and (<b>B</b>) zoledronic acid for 7 days after prior culture at ALI in control medium for 7 days, measured with a resazurin assay. A blank well reading was subtracted before values were normalised to day 7 value for each model, defining day 7 values as 100 % (not shown). N = 3, n = 3. Error bars = SD. Statistical significance against 0 µM at each time point indicated by * <span class="html-italic">p</span> ≤ 0.05. (<b>C</b>–<b>I</b>) H & E-stained sections of oral mucosa models seeded with human oral fibroblasts and immortalised human oral keratinocytes cultured at ALI for 7 days in control medium, then treated with (<b>C</b>) control medium; (<b>D</b>) 50 µM, (<b>E</b>) 75 µM and (<b>F</b>) 50 µM pamidronic acid; (<b>G</b>) 1 µM, (<b>H</b>) 10 µM and (<b>I</b>) 30 µM zoledronic acid, respectively, for 7 days. Representative images used.</p> "> Figure 4
<p>(<b>A</b>) Human oral fibroblast and (<b>B</b>) immortalised human oral keratinocyte metabolic activity over 72 h in the presence of pamidronic acid and zoledronic acid, measured by MTT assay. Bisphosphonates had previously been incubated for 72 h with hydroxyapatite (HA) granules; bisphosphonates incubated with no hydroxyapatite used as a control. Values normalised to control 0 µM. N = 3, n = 3. Error bars = SD. Significance against control indicated by * <span class="html-italic">p</span> ≤ 0.05.</p> "> Figure 5
<p>The metabolic activity of oral mucosa models when treated with control medium or 30 µM zoledronic acid in the presence of hydroxyapatite granules at (<b>A</b>) 10 days and (<b>B</b>) 14 days. Models with no HA cultured as controls, measured with a resazurin assay. Blank well reading subtracted from values before normalising to day 7 values for each model, defining day 7 values as 100 % (not shown). N = 3, n = 2. Error bars = SD. Statistical significance against control indicated by * <span class="html-italic">p</span> ≤ 0.05. (<b>C</b>–<b>F</b>) H & E-stained sections of oral mucosa models seeded with human oral fibroblasts and primary human oral keratinocytes cultured at ALI for 7 days in control medium, then treated with (<b>C</b>) control medium, (<b>E</b>) 30 µM zoledronic acid, (<b>D</b>) control medium and HA granules and (<b>F</b>) 30 µM zoledronic acid and HA granules, respectively, for 7 days. Representative images used.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Microscopy
2.2. Cell Culture
2.3. De-Cellularised Dermis Preparation
2.4. Bisphosphonates
2.5. Two-Dimensional Cell Viability Assays
2.6. Three-Dimensional Cell Culture
2.7. Hydroxyapatite Binding Assays
2.8. Statistics
3. Results
3.1. Microscopy of Hydroxyapatite Granules Confirmed Highly Porous Structure
3.2. Clinically Relevant Pamidronic Acid and Zoledronic Acid Concentrations are Toxic to Oral Mucosa Cells in 2D
3.3. Pamidronic Acid and Zoledronic Acid Reduce Epithelial Thickness and Cause Toxicity to the Oral Mucosa in 3D
3.4. Hydroxyapatite Granules Prevent Pamidronic Acid and Zoledronic Acid Toxicity in 2D
3.5. Hydroxyapatite Prevents Zoledronic Acid Toxicity in 3D
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Bullock, G.; Miller, C.; McKechnie, A.; Hearnden, V. Synthetic Hydroxyapatite Inhibits Bisphosphonate Toxicity to the Oral Mucosa In Vitro. Materials 2020, 13, 2086. https://doi.org/10.3390/ma13092086
Bullock G, Miller C, McKechnie A, Hearnden V. Synthetic Hydroxyapatite Inhibits Bisphosphonate Toxicity to the Oral Mucosa In Vitro. Materials. 2020; 13(9):2086. https://doi.org/10.3390/ma13092086
Chicago/Turabian StyleBullock, George, Cheryl Miller, Alasdair McKechnie, and Vanessa Hearnden. 2020. "Synthetic Hydroxyapatite Inhibits Bisphosphonate Toxicity to the Oral Mucosa In Vitro" Materials 13, no. 9: 2086. https://doi.org/10.3390/ma13092086