Effect of diode laser depigmentation on gingival tissue of dogs

Sara H Elbanna 1 *, Ahmed M Halawa 2 , Hanaa M ElShenawy 3 and Dina M Abdel Khalek 4 . 1. Assistant lecturer of Oral Biology, Faculty of Dentistry, MSA University, Egypt. 2. Professor of Oral Biology, head of Oral Biology Department, Faculty of Dentistry, Ain Shams University,Egypt. 3. Assistant Professor in Laser Dental Applications, Oral Surgery and Medicine Department, National Research Center,Egypt. 4. Lecturer of Oral Biology, Faculty of Dentistry, Ain Shams University,Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5(1), 741-750 742 Gingival hyperpigmentation is increased pigmentation beyond the normally expected degree of the oral mucosa. Several physiologic and pathologic factors can cause hyperpigmentation (Dummett and Gupta, 1964). However, the most common cause is physiologic or ethnic hyperpigmentation which is genetically determined and is clinically manifested as variable amounts of diffuse or multifocal melanin pigmentation in different ethnic groups (Hegde et al., 2013).
Numerous intrinsic and extrinsic factors, including body distribution, ethnicity/gender differences, variable hormone-responsiveness, genetic defects, hair cycle-dependent changes, age, UV-R, climate/season, toxin, pollutants, chemical exposure and infestations, are responsible for a whole range of responses in melanosome structure and distribution (Costin and Hearing, 2007).
Depigmentation is the treatment of removal of melanin hyperpigmentation.Various methods have been used for this procedure including gingivectomy, electrosurgery,cryosurgery, chemically using phenol and alcohol and abrasion with diamond bur (Atsawasuwan et al., 2000).
Laser has been used in dentistry since the beginning of the 1980s. Recent research has centered on using diode laser for oral surgery of the tongue and gingiva and to remove infected epithelium in chronic periodontitis. Diode laser has offered some advantages over the others, such as easy gingival reshaping, reduced need of local anesthesia and dry operative field due to excellent hemostasis associated with significant decrease in pain and inflammatory postoperative score. Minimal scarring and satisfactory clinical outcome on the long term have also been achieved.Moreover, there is evidence in the recent literature of successful depigmentation using diode laser (Hedge et al., 2013).

Aim of the study:-
The aim of the present study was to evaluate the effect of diode laser irradiation on gingival depigmentation, histologically, histochemically and ultrastructurally.

Materials and methods:-
Twenty-eight adult male dogs were evenly divided into 4 main groups; groups (I, II, III and IV) correspondent to post-operative follow-up periods of (1, 2, 4 and 8 weeks) respectively. Specimens of each group were divided into 2 groups; control (A) and experimental (B), where specimens of group B (right side) were selected from contralateral area to group A (left side).
The melanin pigmented gingiva at the region of the canines was ablated by diode laser irradiation with a flexible, fiber-optic delivery system (320 microns diameter) in a contact technique, air cooling handpiece with 3 W continuous mode under standard protective measures. The procedure was performed on pigmented areas until blister formation occurred. Laser ablation started from the mucogingival junction towards the free gingival margin including the inter-dental papilla. The laser beam was guided in brush stoke movements until the entire pigmented spot had disappeared without causing any bleeding which was beneficial for clear visualization(Agarwal et al., 2014). This procedure was repeated until the desired depth of tissue removal was achieved. Remnants of the ablated tissue were removed using sterile gauze damped with saline so as to allow clear visibility of the field. Gingival specimens were examined by light microscope using Hematoxyline and Eosin stain and Masson Fontana special stain and by electron microscope.

Results:-
Histologically, before laser irradiation, the gingiva of dogs showed extensive physiologic pigmentation of a brown color. The specimens showed that most of the melanin is located in the basal and suprabasal layers. After 1, 2, 4 and 8 weeks of the laser irradiation, the specimens were free of the the brown coloration. Complete re-epithelialization was seen after 1 week (subgroup IB) of the application. Some cytoplasmic vacuoles were seen in the granular layer after 2 weeks (subgroup IIB) of the irradiation. Also the granular cells were of faintly stained and less dense cytoplasm. A remarkable thickening in the keratin layer of subgroup IIIB (4 weeks after irradiation) was observed. Normal epithelial cells were seen after 8 weeks (subgroup IVB) of laser irradiation. Lamina propria was normal with some inflammatory cell infltration (Fig. 1).

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Using Masson Fontana, the specimens before laser irradiation showed positive reaction throughout the entire epithelial layers. After 1 and 2 weeks (subgroups IB and IIB), there was a remarkable decrease in the melanin concentration. But at 4 and 8 weeks (subgroups IIIB and IVB), there was a little increase in the melanin concentration compared to subgroups IB and IIB but yet didn't reach the baseline (Fig. 2). Our histological and histochemical results were confirmed with the histomorphometry where subgroups IB and IIB were highly statistically significant with our histological results as the melanin concentration in the basal layer was significantly decreased while subgroups IIIB and IVB statistically significant where melanin concentration in basal layer was decreased but yet more than subgroups IB and IIB (Fig. 3).
Electron microscopic examination of the gingiva of the control subgroup showed the basal cells of their two types; the serrated basal cells which are a single layer of cuboidal or high cuboidal cells with protoplasmic processes projecting from their basal surface towards the connective tissue. They contain some melanin granules in their cytoplasm around the nucleus. The other population of the basal cells which are the non-serrated could also be seen. They give rise to a population of cells amplified for cell division. Considerable amount of melanosomes of variable sizes and electron density were seen inside their cytoplasm (Fig. 4a). Melanocytes with many melanin granules were scattered between lower prickle layer. Some melanocytes had a large rounded nucleus and others with elongated or ellipsoid nucleus containing a nucleolus. The nuclear membrane was smooth with regular outline which may however exhibit a single small cleft. The cytoplasm was relatively clear or electron lucent surrounding the nucleus Numerous electron dense melanin granules (melanosomes) of different size and shape correspondent to their maturative stage were seen throughout the cytoplasm (Fig. 4b). Relatively less melanin granules were seen between upper prickle cell layer. The granular cell layer consisted of flatter, wider and larger cells than those of the prickle layer. Their nuclei showed signs of degeneration and pyknosis. A cytoplasmic vacuole surrounded the nucleus and some melanosomes were detected in it (Fig. 4c). The keratin layerpresented some melanosomes and pyknotic nuclei.Gingival specimens of subgroup IB showed that almost all basal cells were devoid of melanin granules meanwhile few of them presented some. The nuclear outline tended to be more irregular (Fig. 4d). most of the prickle cells were devoid of melanin granules and normal desmosomal attachments were seen between them (Fig.  4e). The specimens of subgroup IIB showed the basal cells with variable sized electron dense melanin granules (Fig.  4f). Cells of the prickle layer were almost devoid of melanin granules similar to those of subgroup IB. However, some of them presented cytoplasmic vacuoles (Fig. 4g). Subgroup IIIB showed prominent melanin granules in some of their prickle cells (Fig. 4h).A remarkable thickening of the keratin layer was noticed in this subgroup (Fig. 4i). Abundant melanin granules were seen between adjacent keratinocytes of subgroup IVB (Fig. 4j).

Discussion:-
Laser was used in the current study because its use is currently the most common technique in gingival depigmentation. Laser depigmentation is a superior treatment modality for a number of conditions when compared to traditional techniques in terms of precision cutting, hemostasis and lowering the risk of other complications commonly seen such as pain, edema and infection (Romeo et al., 2014).
Gingival depigmentation performed in this study was carried out by a 970 nm wave length diode laser and a 3 W irradiation power settings as it has near optimal absorption for melanin and hemoglobin (Lagdive et al., 2010). In the present study there was minimal side effects such as the very slight coagulation on the treated surface as laser has been recognized as one of the most effective, comfortable and reliable techniques for the gingival depigmentation (Shenawy et al., 2015).
In the present study, dogs have been chosen for the gingival depigmentation procedures rather than any other experimental animal due to the marked presence of melanin pigments in their gingiva.  declared the presence of melanin pigments in the stratum spinosum and the basal layers of the dog's attached gingiva through a heavy patchy distribution.
It is known that the nature of the pigmentation and the depth or the degree of its penetration into the tissue are decisive factors in the outcome of the laser treatment (Sharon et al., 2000). In the current study, the utilized laser wavelength was 970nm. Such wavelength is known to induce relatively deep tissue penetration (Atsawasuwan et al., 2000).
In the present study, the light microscopic examination of subgroup IB (1 week after laser irradiation) revealed the completion of re-epithelialization and normal maturation of the epithelium manifested by the presence of all layers 744 till the stratum corneum. It also showed the absence of the brown colored melanin pigmentation in the basal cell layer compared to the control subgroup (IA). The results of subgroup IIB (2 weeks after laser irradiation), showed that the basal and prickle cells presented almost normal histological features. The granular cells were of faintly stained cytoplasm. Also some cells showed a relatively large vacuolar spaces. A remarkable thickening in the keratin layer of subgroup IIIB (4 weeks after irradiation) was observed. Normal epithelial cells were seen in subgroup IVB. These findings were supported by Yousuf et al., 2000 who evaluated the possibility of the removal of canine gingival melanin pigmentation in dogs with the semiconductor diode laser using a 3 W in a continuous mode. They also checked for the brown pigmentation after 3 weeks of the laser irradiation and they did not find any repigmentation when examined by H&E stain.
However, in our study using Masson Fontana stain reflected that there was rare amount of melanin in basal and suprabasal layers of the epithelium of the dog gingiva after 1 week (subgroup IB) of the diode laser application. Also after 2 weeks (subgroup IIB) of laser application, Masson Fontana stain revealed very small spots of melanin. And according to the statistical analysis of the histomorphomertic results, these two groups showed a highly statistically significant decrease in melanin concentration in the basal and suprabasal layers of the gingival epithelium in comparison to the baseline treatment of each subgroup.
Masson Fontana results of subgroups IIIB and IVB (4 and 8 weeks postoperative respectively) revealed a slight to moderate appearance of melanin throughout the whole thickness of the epithelium. The statistical analysis of the histomorphometry revealed a statistically significant decrease in amount of melanin in relation to the baseline of each subgroup.
In the present study, the ultrastructural changes observed in the epithelial cells after laser irradiation were consistent with the histological and histochemical examinations.
Despite the different technique of erbium:YAG laser that was utilized by Attwa et al., 2015in treatment of melasma of skin (acquired hyperpigmentation involving cheeks, nose, upper lip and forehead) in humans. Skin of the face was treated with 2 consecutive passes. This treatment was repeated monthly for a total of 6 months. Evaluation was done at baseline, before each session and at 1, 3 and 6 months after final treatment. Similarly, their electron microscopic results seemed similar to those of the current study. Their results showed that there was a decrease in the aggregation of melanin granules within keratinocytes and melanocytes of the epidermis.
In accordance to the results of this study, it may be concluded that the application of diode laser appears to be a safe and effective alternative method for gingival depigmentation procedure. However, the esthetic outcome may not last for long time. Therefore, further studies are recommended with different laser treatment parameters for a better understanding of the potential benefits of laser depigmentation.

Conclusions:-
 Using diode laser of 970nm wave length and 3 watts power caused significant decrease in gingival pigmentation in all follow-up periods.  Gingival pigmentation began to reappear on the 8th week after laser irradiation yet did not reach the baseline.