Hydroxyapatite is an extensively used calcium phosphate for bone regenerative applications in orthopaedics and dentistry [1
]. Hydroxyapatite nanoparticles (HAP-NP) are very similar to natural hydroxyapatite in terms of size and crystal structure, which contributes to their use in oral care treatments [2
]. In recent years, HAP-NP have been incorporated in oral care products such as toothpastes and mouthwashes with the aim of treating dental sensitivity by occluding the dentinal tubules that are opened to the dentin surface and connected to the pulp; or with the purpose of promoting enamel remineralisation by replacing calcium and phosphate ions to areas from which minerals have dissolved, restoring its integrity and gloss [2
Safety of cosmetic raw materials has to be demonstrated in order to be approved, yet the Cosmetics Directive establishes the prohibition to test finished cosmetic products and cosmetic ingredients on animals [8
]. Having this in mind and in order to assess the safety of a toothpaste ingredient, it should be taken into account that the toothpaste will be in contact with gingival tissue only for a short period of time (~2 min per 3 times a day); However, this period could be potentially enough for a mouth cellular uptake. In addition, the ingredient could be swallowed and absorbed through the gastrointestinal track and potentially become systemically available [9
To study the biocompatibility of the HAP-NP in human oral gingiva, in vitro 3D histotypic cultures that closely resemble the histology of the native tissue can be used as an animal alternative for cosmetic ingredient testing [10
]. In fact, the use of in vitro replacement alternatives for skin corrosion and skin irritation testing using commercialised human skin models has been validated by the Scientific Committee on Consumer Safety (SCCS) [12
In the last years, SCCS has drawn the attention to possible emerging public health problems like the incorporation of nanomaterials in cosmetic products. In this situation, the SCCS group of scientific experts investigate and issue official opinions that are determinant to define the EU legislation [13
]. Particularly for hydroxyapatite nanoparticles, a safety assessment has been done by the SCCS in 2016, containing an exhaustive bibliographic analysis regarding the toxicological effects of this nanomaterial as a cosmetic ingredient. However, the SCCS opinion about hydroxyapatite nanoparticles was not completely conclusive, since the toxicological effect of nanoparticles may depend on physical aspects such as size and morphology. For that reason, SCCS demands that the toxicological assessment must be performed for each type of nanomaterial individually in order to prove its safety for consumers [14
]. Therefore, the aim of the present study was to assess biocompatibility of a specific HAP-NP to clarify the safety of this ingredient for oral care applications. For that purpose, a commercially available human gingival epithelium reconstructed from primary cells [15
] was used, and parameters such as MTT cell viability, LDH activity, and IL-1alpha production were evaluated. Moreover, in order to study the possibility that HAP-NP could become systemically available through absorption in the gingival tissue, transmission electron microscopy (TEM) images of the tissue after different exposure times were evaluated. Finally, the dissolution behaviour in simulated gastric fluid was investigated, so as to assess the possibility of absorption through the intestinal cells after accidental ingestion.
The present study evidences the safety of the tested HAP-NP aqueous suspension nanoparticles for oral care products, showing no deleterious effect on HGE tissues and a complete dissolution in simulated gastric fluid. All the tests performed were designed to comply with the European policy of non-animal testing for cosmetics safety assessment. A three-dimensional in vitro replacement alternative that resembles human gingival epithelium was used to demonstrate the safety of these HAP-NP for use in oral care products. Cell viability (MTT), cell toxicity (LDH activity), and the pro-inflammatory response (IL-1alpha) induced by a 3.1% HAP-NP at different time points were assessed.
Hydroxyapatite nanoparticles have been incorporated in different commercial oral care products [4
] and according to the manufacturer, the maximum incorporation of this 15.5% HAP-NP aqueous suspension oral care ingredient in a toothpaste is 20% and in a mouthwash is 10%. Considering this, it was decided to test a final concentration of 20% dilution of the HAP-NP aqueous suspension, which corresponds to 3.1% HAP-NP. As regards to the exposure time, taking into account that the recommended as well as applied average time for tooth brushing is 2 min [20
], considerable longer exposure times were evaluated, namely 10 min, 1 h, and 3 h to ensure that the HAP-NP would be safe on this application even with much longer contact times than the expected.
Both cell viability and cytotoxicity tests showed that exposure to 3.1% HAP-NP for up to 3 h is safe. To the best of our knowledge, this is the first study evaluating the safety of HAP-NP in HGE. A safety assessment using mucosa-like human corneal epithelial (HCE) tissue model―using human corneal keratinocytes―has also demonstrated similar results, with a lack of cytotoxicity and no changes in IL-1 alpha release [22
]. Usually, safety studies are performed using 2D cell culture models, such as human buccal epithelial cells [23
], 3T3 fibroblasts [22
] on osteoblast-like cells [24
] using different crystallographic nature of the HAP-NP, which can induce different cell responses, as reviewed by Epple [25
The lack of effect in cell viability induced by the positive control can be explained by the relatively low SDS concentration used in this assay, evidencing the low sensitivity of HGE tissue particularly for MTT test, as reported by others [10
]. This probably happens due to the keratinized external layer of this tissue and/or to the relatively low SDS concentration (0.5%) used in this assay. This concentration was selected as is routinely used in this kind of tests in 3D in vitro models and works well for oral epithelium [10
], which lacks the protection to the detergent given by the keratinized external layer. However, although no deleterious effect induced by the positive control could be observed by the MTT test, higher cytotoxicity determined by LDH activity and higher IL-1alpha levels were found. IL-1 cytokines are one of the primary initiators of the dermal pro-inflammatory response and are well-known indicators of acute irritation in reconstructed human epidermis [17
]. In fact, IL-1alpha has been proposed by the SCCS [12
] as a second endpoint in order to obtain better sensitivity. Thus, after 3 h of exposure, 0.5% SDS treatment induced a significantly higher release of IL-1alpha compared to the negative control (10-fold increase) and to the HAP-NP test solution. These results are in agreement with previous observations [26
], showing that elevated levels of IL-1alpha could be detected without any detectable decrease in cell viability in response to endoplasmic reticulum stress.
To test the possibility of cellular uptake of the HAP-NP, two different tests were performed. The first test evaluated the presence or absence of HAP-NP in the gingival mucosa by observation with TEM. Our results confirmed that at the concentrations evaluated and during the exposure times tested there are no cellular uptake by HGE. The second test was performed in case of accidental swallowing and consequent uptake in other zones of the digestive tract. For that purpose, 3.1% HAP-NP suspension was added to SGF to evaluate the gastric dissolution following the recommendations of the FDA [16
] and the exposure doses referred in the SCCS Notes [12
According to FDA guidelines, a conservative conclusion is that a product undergoing 85% dissolution in 15 min under mild dissolution conditions in 0.1 N HCl behaves like a solution and generally should not have any bioavailability problems [16
]. In this work, it was proved that the 3.1% HAP-NP suspension was fully dissolved in half of this time (Table 3
and Figure 5
). This test was done considering the double of the maximum daily concentration of HAP-NP used in a mouthwash in a single ingestion, which is the worst scenario of exposure and ingestion if compared to a toothpaste [12
]. Additionally, it should be highlighted that once the HAP-NP 3.1% suspension (an opaque suspension) is added to the SGF, the dissolution is almost instantaneous leading to a clear solution without any observation of the opacity caused by the HAP-NP particles suspension. This reinforces the conclusion about the safety of the HAP-NP in the case of ingestion, since it will dissolve instantaneously in its Ca2+
ions, in agreement with the Epple’s statement that rules out any adverse health effect by an oral exposition to calcium phosphate [25
]. Therefore, the possibility of HAP-NP absorption by intestinal cells after accidental ingestion was also discarded.