Research Article: Can nano-hydroxyapatite permeate the oral mucosa? A histological study using three-dimensional tissue models

Date Published: April 23, 2019

Publisher: Public Library of Science

Author(s): Shogo Komiyama, Ryosuke Miyasaka, Keiichiro Kikukawa, Roslyn Hayman, Salik Hussain.


Nano-hydroxyapatite is used in oral care products worldwide. But there is little evidence yet whether nano-hydroxyapatite can enter systemic tissues via the oral epithelium. We investigated histologically the ability of two types of nano-hydroxyapatite, SKM-1 and Mi-HAP, to permeate oral epithelium both with and without a stratum corneum, using two types of three-dimensional reconstituted human oral epithelium, SkinEthic HGE and SkinEthic HOE respectively with and without a stratum corneum. Both types of nano-hydroxyapatite formed aggregates in solution, but both aggregates and primary particles were much larger for SKM-1 than for Mi-HAP. Samples of each tissue model were exposed to SKM-1 and Mi-HAP for 24 h at concentrations ranging from 1,000 to 50,000 ppm. After treatment, paraffin sections from the samples were stained with Dahl or Von Kossa stains. We also used OsteoSense 680EX, a fluorescent imaging agent, to test for the presence of HAP in paraffin tissue sections for the first time. Our results for both types of nano-hydroxyapatite showed that the nanoparticles did not penetrate the stratum corneum in SkinEthic HGE samples and penetrated only the outermost layer of cells in SkinEthic HOE samples without stratum corneum, and no permeation into the deeper layers of the epithelium in either tissue model was observed. In the non-cornified model, OsteoSense 680EX staining confirmed the presence of nano-hydroxyapatite particles in both the cytoplasm and extracellular matrix of outermost cells, but not in the deeper layers. Our results suggest that the stratum corneum may act as a barrier to penetration of nano-hydroxyapatite into the oral epithelium. Moreover, since oral epithelial cell turnover is around 5–7 days, superficial cells of the non-keratinized mucosa in which nanoparticles are taken up are likely to be deciduated within that time frame. Our findings suggest that nano-hydroxyapatite is unlikely to enter systemic tissues via intact oral epithelium.

Partial Text

Nanomaterials are generally defined as entities with at least one dimension in the range of 1–100 nm [1]. In the European Union, nanomaterial has been officially defined as meaning ‘a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm–100 nm’ [2].

Recently nanomaterials have come to be used in various fields and the use of nanotechnology in oral care products is increasing worldwide. However, there is still insufficient data on the behavior of nanomaterials in the oral cavity, including whether nanoparticles could enter the bloodstream and systemic tissues via the oral mucosa. We focused on toothpaste which accounts for a large share of oral care products. Toothpastes containing HAP, which is the main component of tooth enamel, are now widely available some of them containing n-HAP. We carried out what we believe to be the first study of its kind using 3-D reconstituted oral mucosal tissue to examine histologically whether n-HAP particles can permeate the oral mucosa, and our results suggest that n-HAP is unlikely to enter the systemic tissues via this route.

This study was a first-step experiment to investigate whether n-HAP used in oral care products is likely to enter the systemic tissues via the oral mucosa. Histological investigation showed that neither of the two different types of n-HAP particles used in our study penetrated the stratum corneum of the 3-D oral epithelial model with stratum corneum that we used, though nanoparticles of both types were observed in the cytoplasm and around the membrane of cells in the outermost layers of the 3-D oral epithelial model without stratum corneum, regardless of their size or concentration. Moreover in no case was the presence of n-HAP detected in the deeper layers of the epithelium in either model. In the actual oral mucosa, there are defense mechanisms at work, such as salivary mucin, the mucus membrane and certain barrier functions of mucosal epithelial cells, which are not present in the 3-D reconstituted tissue models. Furthermore, since the exposure dosage of n-HAP used in this study was much larger than the likely exposure during actual toothbrushing, it was concluded that n-HAP particles are very unlikely to enter the blood stream or systemic tissue via intact oral mucosa.