IADR Abstract Archives
Nanohydroxyapatite Seeded Crystal Growth Determined in a Novel Steady-State Assay.
Objectives: Nano-crystals of hydroxyapatite (“nanohydroxyapatite”) are of increasing interest in formulating products targeting dentine sensitivity. We aimed to 1) develop, validate and use a novel in vitro nucleation/crystal growth (IVNCG) assay; 2) identify minimum amounts of nanohydroxyapatite seed crystals required to produce statistically significant mineral accumulation under steady state conditions and 3) identify composition and morphology of mineral formed.
Methods: The IVNG assay equipment comprised of two reservoirs containing 6.5 mM Ca(NO3)2.4H2O and 3.9 mM (NH4)2HPO4 in 20 mM HEPES, 150 mM NaCl respectively, separated by a partition containing multiple sample wells, each covered with dialysis membrane. System validation used positive (poly-glutamate in agarose) and negative (agarose only) controls (n=20 and 16 respectively). Mineral deposition in each well was measured by spectrophotometric determination of phosphate after 5 days at 37 oC. Nanohydroxyapatite was then seeded in agarose at concentrations of 10, 20 and 30 µg/mL (n=10) and compared to controls. Crystals recovered from the wells were analysed by SEM and EDS. Calcium and phosphate levels in the buffers were determined.
Results: The IVNCG showed statistically significant differences in mineral accumulation between positive and negative controls (p>0.05). Minimum nanohydroxyapatite seeding density required to produce statistically significant differences compared to the positive control was 30 µg/mL (p>0.05). Analysis of recovered crystals indicated distinct differences in morphological and Ca:P ratios between samples. No depletion in buffer calcium and phosphate concentrations was detected throughout the experiment.
Conclusions: Our data suggest that dissolution of nanohydroxyapatite during the early, undersaturated phase of the experiment may result in loss of seeds for subsequent crystal growth when seeding is <30 µg/mL. We conclude that the IVNCG assay, which is simple to make and cheap to use, can provide a screen for potential therapeutics aimed towards dentine sensitivity and mineral precipitation.
Methods: The IVNG assay equipment comprised of two reservoirs containing 6.5 mM Ca(NO3)2.4H2O and 3.9 mM (NH4)2HPO4 in 20 mM HEPES, 150 mM NaCl respectively, separated by a partition containing multiple sample wells, each covered with dialysis membrane. System validation used positive (poly-glutamate in agarose) and negative (agarose only) controls (n=20 and 16 respectively). Mineral deposition in each well was measured by spectrophotometric determination of phosphate after 5 days at 37 oC. Nanohydroxyapatite was then seeded in agarose at concentrations of 10, 20 and 30 µg/mL (n=10) and compared to controls. Crystals recovered from the wells were analysed by SEM and EDS. Calcium and phosphate levels in the buffers were determined.
Results: The IVNCG showed statistically significant differences in mineral accumulation between positive and negative controls (p>0.05). Minimum nanohydroxyapatite seeding density required to produce statistically significant differences compared to the positive control was 30 µg/mL (p>0.05). Analysis of recovered crystals indicated distinct differences in morphological and Ca:P ratios between samples. No depletion in buffer calcium and phosphate concentrations was detected throughout the experiment.
Conclusions: Our data suggest that dissolution of nanohydroxyapatite during the early, undersaturated phase of the experiment may result in loss of seeds for subsequent crystal growth when seeding is <30 µg/mL. We conclude that the IVNCG assay, which is simple to make and cheap to use, can provide a screen for potential therapeutics aimed towards dentine sensitivity and mineral precipitation.