In vitro histological evaluation of the surgical margins made by different laser wavelengths in tongue tissues

Background Lasers have become standard tools for the surgical treatment of oral lesions. The purpose of this study is to determine the surgical margins and histologically evaluate the tissue thermal effects induced by different types of surgical instruments. Material and Methods Cuts were made in pork tongues’ mucosa with different lasers (Er:YAG at 2W with and without air / water spray and at 4W with and without air / water spray; CO2 at 3.5W and 7W in pulsed mode and at 7W in continuous mode; the diode laser at 3.5W and boost 3.5W in pulsed mode; Nd:YAG at 6W, 40Hz and electroscalpel at 5W and conventional scalpel as control. Macroscopic and microscopic morphological changes were evaluated. Results The results of this study showed that the surgical instruments that caused greater tissue damage extension were: the Nd:YAG laser (670.68μm), the diode 3.5W and boost PW (626.82μm), the CO2 7W CW (571.18μm), the CO2 at 7W PW (485.45μm), the diode 3.5W PW (456.15μm), the electroscalpel (409.57μm) and lastly the CO2 laser 3.5W PW (306.19μm) and Er:YAG (74.66μm) laser, regardless of power, mode or air / water spray used. An association between the Tissue Damage Extension and the Degree of Carbonization (r = 0.789; P = 0.01), and an association between the Tissue Damage Extension and Regularity of the Incision were found (r = -, 299; P = 0.01). Conclusions The results of this study suggest that lasers can be used in soft tissues biopsies of the oral cavity, enabling a correct histopathological analysis, as long as the biological effects of each laser type are considered. The Er:YAG laser revealed its potential for biopsies of the oral mucosa ensuring a successful histological evaluation and the CO2 laser at 3,5W in pulsed mode presented itself as the best choice for surgeries with hemostasis. Key words:CO2 laser, diode laser, Er:YAG laser, laser surgery, Nd:YAG laser, oral mucosa, thermal effect.


Introduction
Lasers have become standard tools for the surgical treatment of oral lesions. The use of laser technology in the surgical treatment of oral lesions aims to provide benefits to both the surgeon and the patient (1). The acquired clinical experience over the past decades ensures a number of advantages in the use of laser versus scalpel during soft tissue surgery, including a high degree of decontamination of the surgical field, minimal postoperative bleeding and a significant decrease in pain and postoperative inflammation (1)(2)(3)(4). During the application of laser in oral soft tissues, the light energy is transformed into thermic energy that turns into heat on the target tissue to produce the wanted effect (5). This photothermal effect can produce changes in the tissues, and if the soft tissues are to be examined by an optical microscope, artifacts can make the histopathological interpretation difficult. Therefore the reduction of peri-incisional damage is crucial in oral pathology (6,7). Different types of laser have shown utility and efficiency in dentistry, including CO 2 , Er:YAG, diode or Nd:YAG lasers (8). The CO 2 laser, due to its affinity with water, has become a highly used instrument in the treatment of oral mucosa lesions by oral surgeons (9). Its penetration is poor, which makes the CO 2 laser particularly suited for being used close to critical anatomical structures (10)(11)(12). The CO 2 laser is an ideal tool for a clean bloodless surgical field because of its hemostatic capacity in vessels with less than 0.5 mm diameter (8). For the treatment of vascular lesions in vessels with more than 7-mm diameter, like oral hemangiomas, some authors (13) advocate the Nd:YAG or diode laser. Nevertheless, the strong coagulation effect can lead to artifacts that may influence the histological diagnosis (1). The Er:YAG laser promotes rapid healing due to the short side thermal effect it generates (10,14). However, its drawback is that the interventions won't be so hemostastic as the ones using the CO 2 , Nd:YAG or diode laser (10). The diode and the Nd:YAG lasers are less absorbed by water and more absorbed by hemoglobin and melanin thus having a deeper effect on tissues (15). Nevertheless, in general dentistry it is now a widely accepted treatment aid, with a broad range of applications in oral soft tissue surgery (16). There are only a few studies that have systematically analysed atypical cytological or structural changes in oral epithelium, or its association with different lasers and power (17). Most of the described cases have used the extent of the hyalinised tissue or coagulated tissue adjacent to the irradiated margins to measure the results, and only occasionally were cytological artifacts in the incision considered. Few authors described the type of laser considered suitable for soft tissue biopsy (5).
The purpose of this study is to determine the macroscopic and microscopic morphological changes in the surgical margins in tongue tissue (ex vivo) induced by different surgical instruments, including various types of laser.
-Evaluation Tools The emission for each laser parameters used were those recommended by the manufacturer for soft tissue surgery, and some other variants were selected by the researchers for the purpose of the study. Each surgical instrument used, and its respective parameters, correspond to each tongue incision: CO 2 laser by DEKA® Smart US-20D with a wavelength of 10,6μm was used with a no-contact handpiece for three different types of application: 3.5W in pulsed mode (PW) at 50Hz, 7W PW at 50Hz and 7W in continuous mode (CW). Another laser used was the Nd:YAG by DEKA® Smart A10 with a wavelength of 1.06μm using fiber of 300 μm, 6W power with contact mode and frequency of 40Hz. The Er:YAG laser by DEKA® Smart 2940 D plus with a wavelength of 2,940μm was used with a no-contact piece for four different types of applications: 2W 10Hz and 0.2J short pulse with air / water spray, 2W at 10 Hz and 0.2J short pulse without air / water spray, 4W 10Hz and short pulse 0.4J with air / water spray and 4W 10Hz and 0.4J short pulse without air / water spray. The diode laser of LITE-MEDICS® with a wavelength of 980nm was used in contact mode for two different applications: 3.5W and 3.5W Boost PW. It was also used a Servotome electroscalpel by SATELEC® at 5W of power, and for the specimens control a scalpel blade number 15 by KIATO® was used.
-Data Collection Procedures • Surgical procedure The samples were stored at 2-4ºC during transportation and 100% humidity to prevent tissue degradation as reported in the literature (5,18). The surgical technique was achieved by directing the laser beam perpendicularly to the dorse of the tongue. Samples were collected by the same dentist to prevent errors from interindividual differences. A second operator then placed the samples in sterile containers with formalin buffered at 10%. The samples were sectioned with a minimum margin of 10mm from the study cut.
• Macroscopic evaluation Based on the criteria of Cercadillo-Ibarguren et al. (5) with respect to the tissue carbonisation, we proceeded to the macroscopic evaluation of the incision based on a scale of 0 to 4, in which 0 corresponds to no color detected in the incision, 1 corresponds to a brownish color on the surface of the incision, 2 when brown is detected deep into the edges of the incision, 3 to classify a black color on the surface of the incision and 4 to black in depth.
• Histologic evaluation The specimens were fixed, dehydrated and embedded in paraffin. Serial sections were performed with 3µm thickness. They were conventionally stained with haematoxylin-eosin (HE) and were also dyed with Masson Tricrome (TM) to control false positives. Overall, we obtained 240 histological preparations (120 HE and 120 with TM), and they were evaluated on a ZEISS Axio®optical microscope with Axiovision® software (release 4.6.3). The histological variables assessed at specimens' level are based on the criteria established by Vescovi et al. (2). Epithelial changes in the core include core, cytoplasmic and membrane modifications, and possible loss of intraepithelial and subepithelial adhesion; modification of connective tissue including charring and desiccation; morphology and regularity of the incision on a scale of 0 to 4 in which they were classified as "regular" (≥2) when it presents a smooth, linear border mostly of incisional margin, and as "irregular" (<2) in the presence of a rough and uneven edge in most of the incision, where level 4 represents the highest quality and 0 the worst incisional quality; Extent of Thermal Tissue Damage (ETTD) expressed in microns by measuring the greatest distance from the edge of the incision to the end of the laser thermal damage in the tissue. At the same time, a photographic file was compiled. The samples were coded and a double blind analysis for each type of laser setup used was made by two pathologists to reach a consensus for each case.
-Analysis procedure data The data analysis was obtained by descriptive and inferential statistics, using the SPSS-22.0 software (Statistical Package for Social Sciences). Given that the null hypothesis (H0) to the Kolmogorov-Smirnov normality test is that data is normally distributed, and as the result of P-value was (P < 0.05) for the variables under study, we reject the null hypothesis (H0) and we assume that the sample does not follow a normal distribution in the variables under study. Thus, nonparametric tests like Spearman correlation test, Mann-Whitney test, Kruskal-Wallis test and the Chi-Square test were used.

-Macroscopic evaluation
With the exception of the Er:YAG laser with mean values of 0 and 1, all the other surgical instruments showed a significant charring average value as can be seen in instruments with the highest number of tissue changes were the electroscalpel and Nd:YAG laser, and the one with fewer changes, particularly at epithelial level, was the Er:YAG laser, regardless of power, mode or spray used (Fig. 1). Significant differences were found in ETTD between the number of tissue changes in Score Nuclei (P < 0.001); Score Cytoplasm (P < 0.001) and Connective Score (P < 0.001). It was observed a higher ETTD in the presence of a greater number of changes within each score. Although not statistically significant, an association between the type of surgical instrument and its power and the tissue changes (instrument and power Vs picnotic Core / core spindle / core hyperchromatic / cytoplasmic hyperchromatism / cell fusion (P < 0.001); instruments and power vs adherence loss (P = 0.02); instruments and power vs carbonization (P = 0.035), except for change "dissection" (P = 0.214)) was found. It was also possible to behold statistically significant differences (P <0.001) in ETTD related to the types of surgical instruments used. The values of ETTD for surgical instrument, power, mode and air / water spray are shown in table 3 and (Fig. 2) regardless of their power, mode or air / water spray (Fig.  1). It was found that there is an average lower ETTD in the presence of air / water spray and a higher average in the absence of air / water spray of the Er:YAG laser, although the difference was not statistically significant (P = 0.123). As expected, the scalpel control specimens demonstrated no thermal damage at the margins of the incision (Fig. 1l). Table 4 shows the values of the regularity of the incision for surgical instrument, power, mode and air / water spray. The most regular incision was obtained with the CO 2 laser at 3,5W in pulsed mode and the less regular incision with the Nd:YAG laser. An association between ETTD and the regularity of the incision was found (r = -, 299; P = 0.01).

Discussion
There are several studies on the use of laser in oral biopsy of soft tissue, but only some of them focus on the damage caused by this device in peri-incisional margins of tissue samples, and only a few include associated variables, as the power, wavelength or emission mode.    Recent studies (15,(18)(19)(20) have shown that the CO 2 , Er:YAG, Nd:YAG and diode lasers proved to be ideal devices for oral soft tissue with little thermal damage, allowing a correct histological diagnosis. We have showed in this paper that the artifacts caused by different types of laser are limited to a small area of tissue and don't affect the entire fragment. The instruments with the highest degree of carbonization of the tissue were the Nd:YAG and CO 2 7W CW lasers, and the one with the lowest charring was the Er:YAG laser. The paper by Cercadillo-Ibarguren et al. (5) regarding macroscopic classification, states that the CO 2 and diode lasers always produced higher values of carbonization, probably due to higher power level, continuous mode and because of the wavelength used in their research (830nm). The present study found a significant association between ETTD and carbonization degree, showing that the higher the degree of charring caused by the surgical instrument, the higher the ETTD induced on the specimen. In this case the Nd:YAG and CO 2 7W CW lasers with grade 4 carbonization also had the highest ETTD values. While the Er:YAG laser with the lowest values of thermal damage caused a non-relevant carbonization without air / water spray and no carbonization at all with air / water spray regardless of the power used. These results seem to indicate that in soft tissue surgery of the oral mucosa, the carbonization degree may be a clinical indicator of the damage that is being induced in the tissue.
Concerning the regularity of the incision, the best result was obtained with the CO 2 laser, regardless of mode or power, and the worst result with the Nd:YAG laser. These results confirm the ones in the study of Merigo et al. (18), who reported good and regular results with the CO 2 laser but weaker ones with the Nd:YAG laser. The intermediate quality of the incisions with the Er:YAG laser are concurrent with the results of these authors and do not seem to be related to the presence of air / water spray. When we compared the incision regularity with ETTD, we observed that more regular cuts of the incision corresponded to cases with lower ETTD. The results of Vescovi et al.
(1) support this hypothesis in human oral mucosa studies with Nd:YAG laser at 3.5W and 5W, in which they didn't find a statistically significant difference, although the incision was better and ETTD lower in the specimens obtained with lower power laser. All types of surgical instruments used in this study induced the same Tissue Artefact Changes, mainly located in the basal and suprabasal layers of the lingual epithelium, according to many authors (17,21), and its connective tissue. The surgical instruments that induced the higher number of artifacts were the Nd:YAG laser and electroscalpel mainly at a nuclear level; the Er:YAG laser generated fewer changes at the epithelial level (nucleus and cytoplasm) as described by Merigo et al. (18). However, loss of adherence was higher with the electroscalpel, the diode laser at 3.5W boost and the Nd:YAG laser, while Merigo et al. (18) only found this in the last laser referred.
The electroscalpel produced similar values of tissue changes to the ones obtained with the Nd:YAG laser, which is in line with the comparative study of laser surgery and electrosurgery by Vitale et al. (22) that found the greater damage in electrosurgery biopsies particularly at the epithelial level.
In the ETTD analysis by histological artefact induced by the different surgical instruments, it can be stated that the measure of thermal damage was normally higher in the presence of these tissue changes, suggesting that in surgical margins with higher ETTD, more histological artefacts will be found. This difference in ETTD between the presence and absence of these tissue changes was statistically significant. And while this is an expected result, it strengthens the quality of the sample under study.  (17,18,21,25), which can justify the need to include an additional amount of adjacent healthy tissue that exceeds the expected extent of epithelial thermal damage. The thermal effect of the diode laser in this study was wide, and induced a lower ETTD average at 3.5W than that generated at 3.5W boost. Other authors reported smaller thermal effects, from 321,4μm (26) to 623 μm (15), but a lower power was used in both cases.
Values that are close to the ones in our study, up to 750μm with 3W and 5W power, were reported (18), but using a wavelength of 808nm. Romeo et al. (24) found differences in the thermal effects of the laser diode of 980nm and 808nm in a pig's tongue, with the longer wavelength achieving an extensive general thermal effect; the chorion was corrupted by more than 1.5mm and the epithelium by more than 1 mm with a wide dermoepithelial detachment. With the diode laser at 808nm in pulsed mode, the peri-incisional cell damage was evidently reduced, showing the best results, with a peripheral damage of less than 1mm. Histological evaluation of the specimens revealed a markedly longer ETTD in the group of the incisions with CO 2 at 7W, Nd:YAG and diode lasers compared to the electroscalpel group. This observation has been recognised in some studies (27,28) but has also been contradicted by others (29,30). ETTD induced by surgical instruments was observed, from the highest to the lowest result in: the Er:YAG laser, the CO 2 laser at 3.5W PW, the electroscalpel, the diode laser at 3.5W PW, the CO 2 laser at 7W PW, the CO 2 laser at 7W CW, the diode laser at 3.5W Boost PW and finally the Nd:YAG laser. The small number of tissue changes and lower ETTD induced by Er:YAG laser appears to be an indicator of its potential for soft tissue surgery of the oral mucosa ensuring a successful histological evaluation. However, because it doesn't provide effective hemostatic properties, the laser which indices less tissue damages and has a superior hemostasis capacity is the CO 2 laser at 3.5W PW. Moreover, this laser was the instrument that offered the most regular incisions. The Nd:YAG lasers obtained the worst results in the preservation of peri-incisional tissue, reinforcing the need of an adequate knowledge of its characteristics and appropriate choice of the parameters associated to a training period.
As conclusion, our results show that lasers may be used in soft tissue surgery of the oral cavity, as long as the biological effects related to the use of each type of laser are understood and respected. The Er:YAG laser may be the laser of choice for biopsies of the oral mucosa because of the minimum histological artefacts observed in this paper, ensuring a valid histological evaluation, followed by the CO 2 laser at 3.5W in pulsed mode, especially when the surgeon needs more hemostasis on the surgical field.