Enamel microhardness test

A sticker was placed on the buccal surface of the teeth measuring 2 × 2 mm, and its surrounding area was sealed with two layers of nail varnish. The sticker was then removed, and excess material was rinsed with distilled water. This was done to standardize the tooth surface subjected to materials. The microhardness of the tooth surface was measured by applying a 50-g load for 10 s to five points of each sample using a Vickers diamond indenter (Wolpert, Darmstadt, Germany). The Vickers hardness number (VHN) was determined by calculating the ratio of load (kilogram force) to the surface area (mm²) using the formula below [9]:

Where F is the applied load in kilogram force and A is the surface area in square millimetres.

After obtaining the VHN, the mean microhardness of the five points was calculated and reported as the microhardness of enamel. All teeth were then demineralized to create incipient caries.