*2.2. Exclusion Criteria*

Histopathological presence of dysplasia,

Systemic diseases such as HIV, HBV and HCV infections, celiac disease or physiological conditions such as pregnancy,

Having performed treatment for hyperkeratosis and leucoplakia in the 2 months prior to the clinical trial.

During the first visit clinical data of each patient was collected and the informed consent to the treatment was signed. The patients were also asked about their habits, in order to identify those, which could be detrimental. The study was approved by the Clinical Research Ethics Committee (CEIC) (code 09/093), following the principles of Helsinki for human experimentation.

After collecting this data and having examined the patient both from a systemic and dental point of view, an examination of the lesions was carried out, thus evaluating:


As previously stated, the biopsy and its subsequent histological examination were fundamental: the presence of cellular atypia, dysplasia or malignant lesions could be excluded, thus making the patient suitable for receiving laser treatment.

The biopsy was performed at the most significant point of the lesion, in order to also remove a part of the healthy tissue, useful for identification in the subsequent histological examination. First of all a 1.8 mL vial of mepivacaine and adrenaline 1:100,000 (OPTOCAIN, Molteni Dental, Milan, Italy) at the perilesional level were inoculated. After having disinfected the oral mucosa with 0.2% chlorhexidine, the part of tissue to be extracted was delimited with a 4 or 5 mm biopsy punch and subsequently extracted with a scalpel, taking care to remove all the layers of the mucosa. A suture was carried out with simple detached stitches in 4/0 silk, which was removed 7 days later.

The sample was fixed in 10% formalin (BIO-OPTICA, Milan, Italy) and sent to the pathological anatomy laboratory of the Dr. Peset University Hospital in Valencia. Here it was included in paraffin then 5-micron thick sections were made, stained with haematoxylin and eosin and analysed under an optical microscope. Approximately two weeks later the histological examination report was received.

The treatment consisted of four sessions a week and a follow-up visit one month after the last treatment session. *Surgical technique*: the surgical technique provided no anaesthesia and a superficial and fast touch of the lesion, in this way we could observe the removal of the pathological area from the underlying mucosa, which was then removed manually with the aid of anatomical tweezers. Once the white area was removed the underlying mucosa appeared to be clinically normal and healed for second intention.

The protocol stated that at each session before treatment and in the final follow-up session:


During each session some aspects that were presented during the seven days since the previous session were assessed in particular:


To perform the treatment a diode laser was used which had a wavelength of 940 nm, the fibre had a length of 9 mm and diameter of 300 microns; a pulsed mode was selected with a Ton corresponding to Toff and corresponding to 10−<sup>3</sup> s, the period corresponds to the summary of Ton and Toff that is 2 × 10−<sup>3</sup> s. It is also known that the frequency and the period of every electromagnetic wave are inversely proportional to each other for the equation ν = 1 *T* ; reporting the equation in our case shows that

$$v = \frac{1}{2 \cdot 10^{-3}} = \frac{10^3}{2} = 5000\text{ Hz}.\tag{1}$$

When using the pulsed mode, there is both a peak power and average power where the latter is defined as the percentage of Ton in the period. In this case it stands at 50%. Having set the peak power at 1.8 W, the average constant power during the emission corresponds to 0.9 W; this allows intervention without using anaesthetic devices, unless specifically requested by the patient.

Before laser-assisted treatment the fibre was activated, using the special supplied activator. Both the operator and the patient wore protective glasses. The laser was used with a contact on the mucosa for about 1 min, the tissue was then left to cool for 30 s and the whole sequence was repeated in order to perform a total of four sessions in one visit. After each visit the patient was instructed to avoid too hot and too spicy foods for a few days.
