In vitro research in Dentistry ideally uses human teeth as the first option, due to the excellent substrate for testing mechanical properties and tissue response to dental materials (Arango-Santander et al., 2020). However, the limited number and, sometimes, poor quality of extracted human teeth (e.g., large cavitated carious lesions and extensive wear), along with ethical concerns involved in their use, are known limitations (Teruel et al., 2015, Yassen et al., 2011).

Alternative natural substrates have been widely used to address the disadvantages of using human teeth, especially bovine substrates (Comar et al., 2012, Isidor et al., 1999), mainly due to their ease of acquisition in large quantities, their significantly larger flat surface, and the absence of caries lesions that could affect the results (Yassen et al., 2011). Consequently, they have been used in various studies, such as tests of dental materials (Ahiropoulos et al., 2008); de Abreu et al., (2023)), dental erosion (Hertel et al., 2023, Schestakow et al., 2024), and dental caries (Braga et al., 2024, Kharbot et al., 2024) investigations, among others. There are mixed reports in the literature regarding the use of bovine substrates, with some authors stating that human and bovine enamel and dentin are very similar (Möhring et al., 2023, Teruel et al., 2015), while other authors advise extreme caution when analyzing results with bovine teeth in general (Fonseca et al., 2008, Olek et al., 2020, Ortiz-Ruiz et al., 2018), as there are certain differences between human and bovine teeth in both dentin and enamel.

Given that features such as the microstructure, chemical and mechanical composition of bovine dental enamel have been reported to be similar to those of human dental enamel, some authors claim that bovine enamel could be used as an alternative in dental research (Arango-Santander et al., 2020, Turssi et al., 2010, Wang et al., 2021, Möhring et al., 2023). Bovine tooth enamel has been reported to present a similar calcium content to human enamel (Turssi et al., 2010). The gradual decrease in calcium content from the enamel surface to the enamel-dentin interface in humans is also found in bovine enamel (Davidson et al., 1973). However, the actual mineral volume varies significantly, with a mean of ∼ 90 % mineral volume for permanent human enamel (Sousa et al., (2006), (Sousa et al., 2009), compared to a mean of ∼ 80 % for deciduous bovine enamel (Robinson et al., 1988). Major components (mineral, organic, and water) have been measured in bulk bovine enamel only (Robinson et al., 1988, Teruel et al., 2015). Regarding the composition of bulk enamel from permanent bovine incisors, data from the control group of permanent human enamel used to validate the methodology indicated an organic volume of 11.0 % (Teruel et al., 2015), which is much higher than the 0.7–4.8 % organic volume range reported for permanent human enamel using more sensitive chemical techniques (Robinson et al., 1971). This discrepancy raises serious concerns about the accuracy of the reported absolute organic volume in permanent bovine enamel. Measurements at various locations within the enamel layer are crucial for studying variations in optical, mechanical, and compositional properties across histological zones under different conditions (developing, sound, carious, and fluorotic enamel), however, but such data are lacking for bovine enamel. These are important gaps in the literature.

Investigating water content would be particularly important as evidence indicates that bovine enamel is three times more permeable than human enamel (Featherstone & Mellberg, 1981). However, such higher permeability was measured after submitting bovine enamel to cariogenic challenge. No permeability data exists on sound bovine enamel. The amount of water removed at room temperature, an important characteristic of dental enamel, has been reported to be approximately 25 % of the total free water in sound human enamel. However, this trait has never been tested in other species. The remaining water content, known as firmly bound water, is directly bound to the mineral surface. It has been suggested that the fracture toughness of enamel would decrease with an increase in exposed mineral surface (Baldassarri et al., 2008), which, in turn, would present a higher firmly bound water content. The relationship between firmly bound water and enamel toughness has not yet been investigated.

Considering the gaps in the literature cited above, this in vitro study aimed (i) to provide spatially resolved data on bovine enamel composition (mineral, organic, total water, firmly and loosely bound water volumes, and permeability), and (ii) investigate the association between enamel composition and optical behavior (birefringence) and fracture toughness of bovine enamel.