Acellular Synthesis of a Human Enamel‐like Microstructure

Abstract

Dental enamel is the outermost layer of teeth and the hardest mineralized tissue in the human body. It consists of nanorod-like hydroxyapatite (HA) crystals arranged into a highly organized micro-architectural unit called an enamel prism. These special units play an important role in determining the unique physicochemical properties of dental enamel. Cells (ameloblasts) and enamel proteins are thought to be intimately involved in vivo in producing this unique structure. The aim of this research was to create a similar structure directly by crystal growth without using cells and/or enamel proteins. Using a hydrothermal method we have been able to synthesize these prism-like structures, consisting of fluorapatite (FA) crystals, which are similar to the dimensions of those seen in human enamel. Dental pulp stem cells (DPSCs) were cultured on these crystals and showed the excellent biocompatibility of the FA crystals. The growth mechanism of this structure is outlined and its use in tissue repair is discussed. Ninety-five percent (by volume) of human enamel is comprised of nanorod-like calcium hydroxyapatite crystals, which have an approximate cross section of 25–100 nm and an undetermined length of 100 nm to 100 lm or longer along the c-axis. The typical human enamel prism structure is approximately 5 lm in cross section, and can span the entire enamel thickness, that is, approximately 1–2 mm in length. The prevailing theory is that the ameloblasts secrete amelogenin, a major enamel protein constituting approximately 90 % of all organic matrix material in developing enamel, and this protein plays a vital role in enabling crystallites to form a well-organized prism pattern. These prisms project from the dentino–enamel junction to the enamel surface. The prisms are aligned parallel to each other and separated by an interprismatic structure to form a densely compacted enamel layer. Unlike other calcified tissues, such as dentin and bone, there are no living cells in the mature enamel. The ameloblast cells die after the enamel is formed. Thus, when the enamel is damaged, the body has no ability to regenerate it. The approach to create artificial bone and tooth structures has attracted the interest of many researchers. Kniep and co-workers have described a fluorapatite–gelatine system that resembles the biosystem hydroxyapatite–collagen in both bone and dentine. Recently, Yamagishi et al. have reported a paste of fluoridated hydroxyapatite that could be used to repair a small carious lesion. Although the interface between the precipitated layer from the paste and the tooth surface contains elongated crystals some of which are orientated towards the tooth surface, the unique enamel prism structure is not visible from the transmission electron microscopy (TEM) images. In our previous paper, we reported a way to mimic the natural biomineralization process to create these special structures by modifying synthetic hydroxyapatite nanorods with surfactants that allow the nanorods to self-assemble into an enamel prismlike structure at a water/air interface. However, those self-assembled enamel prism-like structures are small, only about 400 nm in length and 100 nm in cross section, and dispersed randomly. Very recently, Fowler et al. reported that they were able to synthesize an HA bundle structure directly from a solution containing the surfactant bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT), water, and oil. These bundles were only 750 nm to 1 lm in length, 250–350 nm wide and they were dispersed randomly. These unique structures, therefore, have limited applications, and the introduction of other chemicals, for example AOT may cause unnecessary biological effects. The development of nanotechnology has created many ways to grow 1D nanostructures. Among them, the hydrothermal method is a widely adopted technique to create nanorods, nanowires, and whiskers and has already been shown to be an effective way to create long hydroxyapatite nanorods and whiskers. In this study, we demonstrate a direct growth method to produce fluorapatite dental enamel prismlike structures using a hydrothermal technique. A film of compacted well-aligned FA crystals, which was grown on metal plates, had a structure C O M M U N IC A TI O N S