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

Shogo Komiyama,Keiichiro Kikukawa,Ryosuke Miyasaka,Roslyn Hayman,Salik Hussain

doi:10.1371/journal.pone.0215681

Abstract

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.

Highlights

Nanomaterials are generally defined as entities with at least one dimension in the range of 1–100 nm [1]
As a preliminary step to investigate whether n-HAP particles can enter systemic tissues through the oral epithelium, we studied histologically to what extent n-HAP could penetrate the stratified layers in two types of three-dimensional (3-D) reconstituted human oral epithelial models, one with and one without a stratum corneum, in what we believe to be the first study of its kind using n-HAP particles
Two types of n-HAP were used in the present study, both prepared by Sangi Co., Ltd., Japan, one having rod-like nano-scale primary particles and produced by wet chemical synthesis (SKM-1) and the other having smaller, irregularly shapes nanoparticles and produced by similar chemical synthesis followed by grinding in a wet-mill (Mi-HAP)

Summary

Introduction

Nanomaterials are generally defined as entities with at least one dimension in the range of 1–100 nm [1]. Sangi Co., Ltd. Roslyn Hayman is the president of Sangi Co., Ltd. Roslyn Hayman is the president of Sangi Co., Ltd This does not alter the author’s adherence to all the PLOS ONE policies on sharing data and materials, in this study. 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]. The use of biocompatible nanoparticles in drug delivery systems, for example for selective cell-targeting and gene therapy is being widely studied [8]

Methods

Results

Discussion

Conclusion