Synthetic Hydroxyapatite Inhibits Bisphosphonate Toxicity to the Oral Mucosa In Vitro

Abstract

Medication-related osteonecrosis of the jaw (MRONJ) is a side effect of bisphosphonate therapy, characterised by exposed necrotic bone. The soft tissues of the oral mucosa no longer provide a protective barrier and MRONJ patients experience pain, infections and difficulties eating. We hypothesised that hydroxyapatite (Ca₅(PO₄)₃(OH)) could reduce bisphosphonate concentrations and protect the oral mucosa by exploiting bisphosphonate's calcium binding affinity. The effect of zoledronic acid (ZA) and pamidronic acid (PA) on the metabolism of oral fibroblasts, oral keratinocytes and three-dimensional oral mucosa models was investigated and then repeated in the presence of hydroxyapatite granules. Without hydroxyapatite, ZA and PA significantly reduced the metabolic activity of oral cells in a dose-dependent manner. Both drugs reduced epithelial thickness and 30 µM ZA resulted in loss of the epithelium. Hydroxyapatite granules had a protective effect on oral cells, with metabolic activity retained. Oral mucosa models retained their multi-layered epithelium when treated with ZA in the presence of hydroxyapatite granules and metabolic activity was comparable to controls. These results demonstrate hydroxyapatite granules protected oral soft tissues from damage caused by bisphosphonate exposure. Porous hydroxyapatite granules are currently used for socket preservation and this data suggests their potential to prevent MRONJ in at-risk patients.

Keywords: BRONJ; MRONJ; fibroblasts; keratinocytes; osteonecrosis; osteonecrosis of the jaw; pamidronate; pamidronic acid; zoledronate; zoledronic acid.

Figure 1

(A) A stereomicroscopy image of a PermaBone^® hydroxyapatite granule. (B) A scanning electron microscopy image of pre-granulated PermaBone^® hydroxyapatite.

Figure 2

Cell metabolic activity over 72 h in the presence of bisphosphonates measured by MTT assay. (A) Human oral fibroblasts with pamidronic acid, (B) human oral fibroblasts with zoledronic acid, (C) immortalised human oral keratinocytes with pamidronic acid and (D) 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 * p ≤ 0.05.

Figure 3

The metabolic activity of oral mucosa models when treated with (A) pamidronic acid and (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 * p ≤ 0.05. (C–I) 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 (C) control medium; (D) 50 µM, (E) 75 µM and (F) 50 µM pamidronic acid; (G) 1 µM, (H) 10 µM and (I) 30 µM zoledronic acid, respectively, for 7 days. Representative images used.

Figure 4

(A) Human oral fibroblast and (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 * p ≤ 0.05.

Figure 5

The metabolic activity of oral mucosa models when treated with control medium or 30 µM zoledronic acid in the presence of hydroxyapatite granules at (A) 10 days and (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 * p ≤ 0.05. (C–F) 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 (C) control medium, (E) 30 µM zoledronic acid, (D) control medium and HA granules and (F) 30 µM zoledronic acid and HA granules, respectively, for 7 days. Representative images used.