Microhardness Measurements on Tooth and Alveolar Bone in Rodent Oral Disease Models

In this research, we focused on establishing accurate and consistent microhardness testing. Microhardness testing is the traditional method. But for now, the methodology is not sufficiently described in previous publications.

As we demonstrated in the result, for immediate microhardness reduction depends on the indentation sites of the enamel ike inner, middle, and outer layers. Our results suggest that the indentation site is important to retain accurate and consistent results. The scientific gap is that the previous studies is not sufficiently described the microhardness testing methods, especially the variation sites.

Due to this, microhardness result are not consistent between studies. To fill this gap, we established this protocol for accurate and effective evaluation. Our established protocol will advance mineralized tissue testing in enamel, dentin, and alveolar bone in periodontal disease, bone resorption, and all cancer bone invasion models.

We will focus on how environmental factors affect dental-craniofacial pathology. For example, fluoride and organofluorine PFAS. PFAS is manmade forever chemicals.

Our microhardness protocol will contribute to evaluating how PFAS affect mechanical properties in mineral tissues in tooth development periodontal bone resorption, and oral cancer bone invasion. To begin, coat the inside service of a mounting cup with a thin layer of petrolatum. Pour the resin and hardener into a plastic cup.

Use a wooden spatula to carefully mix the resin and hardener for at least two minutes without any bubble formation. Next, place the dental samples in a horizontal orientation parallel to the bottom of the mounting cup. Pour the mixed resin into the cup to cover the specimen.

Now place the mounting cup containing the specimen on a hot plate at 50 degrees Celsius for at least eight hours. Once the resin is cured, remove the resin with the specimen from the mounting cup. Remove the burrs.

Use an advanced grinder polisher with rough water-resistant abrasive paper to arrange the specimen's plane and the opposite side plane as parallel and flat under water flooding. With a precision sectioning saw, trim the external shape of the resin block to make a rectangle with round corners. Then use an ultrasonic cleaner to remove the debris and particles from the block.

To polish the block, place a rough water-resistant abrasive paper on the grinder. Transfer the block onto the abrasive paper. Now hold the block and pour water over it.

Simultaneously polish the specimen's evaluation surface on the grinder polisher. Change the abrasive paper to the grit 800 P2400 and place the resin block on it. Repeat polishing under water flooding.

Clean the block with an ultrasonic cleaner. Then use finer abrasive papers to perform serial polishing in the given order. Next, place a lapping film on the grinder polisher table without rotation.

Place the resin block on the lapping film. Under water cooling, manually polish the specimen's evaluation surface on the lapping film. Move the sample block vertically, horizontally, and diagonally for the same number of seconds under water injection with strokes of two to three centimeters.

Remove debris and particles with an ultrasound cleaner. Then change the abrasive paper to the next size before polishing the resin block on it. When polishing this properly performed, the resin specimen sticks to the lapping film.

When the final polish has been completed, the specimen should have a surface with a mirror finish. Degrease and dehydrate the surface with 100%ethanol. Set the loading force of a microhardness tester to 25 grams force and the loading duration to 10 seconds.

Place the polished resin block containing an embedded mouse incisor sample on the stage of the instrument. Make six indentation points on each enamel layer and in each dentin region. Measure the length of the two diagonals to calculate the Vickers microhardness value.

Next, replace the incisor resin block with a resin block containing the mouse alveolar bone. Now make three to six indentation points on each mesial and distal side of the bone from the alveolar crest. Indent the alveolar bones between the first and second molar and second and third molar.

The microhardness of all enamel layers of the control mice was lower than the dentin in the cervical region. In the middle and tip regions, the enamel microhardness of each layer was significantly higher than dentin. Among the three enamel layers, microhardness increased from the inner to the outer enamel in each middle and tip region.

The enamel microhardness of the fluoride-treated teeth was less than the dentin in the middle region. The microhardness decreased significantly from the inner to the outer enamel layers. The microhardness values of the alveolar bones affected by periodontal diseases showed a lower tendency relative to the control bones.