A preliminary study of healing of diode laser versus scalpel incisions in rat oral tissue: a comparison of clinical, histological, and immunohistochemical results Camillo D’Arcangelo, DDS,a Franca Di Nardo Di Maio, DDS, PhD,b Gianni Domenico Prosperi, DDS, PhD,c Eugenio Conte, MD, DDS,d Monica Baldi, MD, DDS,e and Sergio Caputi, MD, DDS,f Chieti and Padova, Italy UNIVERSITY “G. D’ANNUNZIO”, FACULTY OF MEDICINE, DEPARTMENT OF ORAL SCIENCE, UNIT OF RESTORATIVE DENTISTRY, AND THE MULTIDISCIPLINARY UNIT OF LASER, ISOMED Objective. The aim of this preliminary study was to compare wound healing of rat oral tissues after surgical procedure with diode laser or scalpel. Healing was evaluated histologically, immunohistochemically, and by measurement of 2 nitric oxide synthase isoforms (eNOS and iNOS) as intracellular messenger molecules with important immune functions. The instruments were also evaluated for performance and ease of use. Study design. Twenty-four standardized incisions were performed in the hard palate of 12 male Wistar rats. Each rat received 2 incisions on the opposite sides of the palate by using a steel scalpel (control group) and a diode laser (808 nm) at a power output of 4 W and 6 W (test group). Histological and immunohistochemical analyses were performed on tissue samples after 7 and 14 days. The expression of eNOS and iNOS was confirmed by RT-PCR (reverse transcriptase-polymerase chain reaction) and Western blot analysis. Results. Scalpel repair was found to be equivalent to or better than laser repair at the intervals measured. Histological analysis showed that incision wound repair after laser surgical procedure was related to parameters and beam characteristics. Diode laser at a power output of 6 W showed the worst results of tissue repair, especially after 7 days. On the contrary, the extent of epithelial damage lateral to the wound edge and the extent of collagen denaturation were near equal with scalpel incision and laser irradiation at 4 W after 14 days. Biochemical analysis of RT-PCR and Western blots also confirmed histological results with a greater concentration of eNOS and iNOS after 7 days of laser surgical procedure. Conclusions. Clinical and histological findings change over time for different treatments. Diode laser tends to produce more pronounced changes than conventional scalpel surgical procedure (due to tissue thermal damage), with corresponding greater inflammatory reaction and delay in tissue organization only at the initial stage. Thus, long-term histology is critical for predicting treatment results. The clinical use of low-level diode laser for tissue welding of oral mucosa should be investigated further, since it appears to be a good alternative to scalpel incision and suture repair. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:764-73) Steel scalpel and laser systems are widely utilized as effective tools in soft-tissue surgical procedure. A scalpel is commonly employed because of its ease of use, accuracy, and minimal damage to tissues. However, scalpels do not provide a good hemostasis, which is important on highly perfused tissues such as in the oral cavity. The use of low-level laser as a therapeutic agent a Professor of Restorative Dentistry. Dentistry. c Dentistry. d Multidisciplinary Unit of Laser, ISOMED. e Multidisciplinary Unit of Laser, ISOMED. f Professor of Prosthetic Dentistry; Director of Department of Oral Science. Received for publication Oct 09, 2005; returned for revision Jul 13, 2006; accepted for publication Aug 01, 2006. 1079-2104/$ - see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2006.08.002 b 764 started with Mester1 in 1971, who studied its effect on the acceleration of wound healing in rats. With laser irradiation, the major advantages are the production of local hemostasis—thereby creating a virtually bloodless surgical field— bacterial elimination, and contactfree incision.2,3 Postoperative pain is also notably minimized with laser incisions. On the contrary, the disadvantage of laser is the induction of thermocoagulation and vacuolization of tissue artefacts. To minimize unwanted thermal damage, the use of different kinds of laser has been investigated in recent years. However literature regarding laser therapy is controversial, either showing beneficial effects1,4-8 or no effect at all.9-14 It is common to find investigations that have failed to demonstrate effects because of the choice of incorrect parameters.15,16 Therefore, the existence of various types of lasers showing different capabilities of interaction with tissues should be taken into account, as OOOOE Volume 103, Number 6 well as failure in dosimetry, the mode of application, and the animal model used. The basic principle of the biomodulation of cells by laser therapy is that irradiation at a specific wavelength is able to alter cellular behavior by acting on the mitochondrial respiratory chain17 or on membrane calcium channels,18 promoting an increase in cell metabolism and proliferation.19 These effects give rise to an excellent coagulation ability and to peculiar healing mechanisms rather different than those occurring after scalpel incision with 2 parallel margins that perfectly coincide with each other. Although tissue studies comparing scalpel with laser have been done,14,20-24 an immunohistochemical and biochemical comparison of the effects of scalpel and diode laser on rat oral mucosa tissues has not been reported. Thus, to increase the application of laser treatment modality in clinical dentistry, a larger number of studies are necessary. This should include histological investigations emphasizing different aspects of the biology of tissue repair, such as cell differentiation and maturation, cellular and vascular proliferation, extracellular matrix production, and inflammatory response.12,14,25,26 Based on the aforementioned considerations, healing wound repair after scalpel and laser surgical procedure could also be investigated by evaluating the effect of various molecules with important immune function. Nitric oxide (NO) has been identified as a major biological signal, exerting both intercellular and intracellular effects. It has been implicated in the modulation of platelet function and in regulation of blood flow, macrophage cytotoxicity, and neurotransmission.27 Moreover, NO is considered an effector of the innate immune system, with important immune functions.28 The innate immune system is a set of rapid host responses to pathogens. Cells of the innate immune system—macrophages, neutrophils and natural killer cells— use pattern recognition receptors to recognize molecular patterns associated with pathogens29; activated macrophages then inhibit pathogen replication by releasing a variety of effector molecules, including NO. Nitric oxide is produced by a group of isoenzymes collectively termed NO synthase (NOS).30 Three distinct isoforms of NOS have been cloned to date: the endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) NOS.28 The eNOS and nNOS are constitutive isoforms that can rapidly synthesize small amounts of NO following receptor stimulation.31 iNOS is mainly involved in the inflammatory processes. Proinflammatory stimuli trigger resident and immigrant inflammatory cell populations, thus inducing iNOS.32 iNOS produces large amounts of NO for sustained periods of time, which has a role in a nonspecific immune response, acting as toxic agent in infections.28,31-33 D’Arcangelo et al. 765 Histochemical identification of nicotinamide-adeninedinucleotide-phosphate-diaphorase, one possible marker of NOS34, and immunohistochemical detection of NOS were used for localization of NOS in different animal and human models.35-40 Based on the aforementioned considerations, the aim of this preliminary study was to investigate the histological and immunohistochemical changes of the rat oral mucosa after conventional steel scalpel and diode laser therapy at 4 W and 6 W output powers. Healing was histologically evaluated after 7 and 14 days with microscope analysis and immunohistochemically by measurement of 2 nitric oxide synthase isoforms (eNOS and iNOS) as intracellular messenger molecules with important immune functions. Clinical aspects were also compared and tools were evaluated for performance and ease of use. MATERIAL AND METHODS Laser devices In the present study, a continuous mode diode laser (LaserSmile; Isomed, Albignaseco, Padova, Italy) emitting at 808 nm was employed. The radiation generated from this surgical tool is produced by the solid components of the laser, and energy is rapidly absorbed by tissue and water, with minimal collateral thermal damage and tissue charring. Laser radiation was employed on the oral mucosa of rats at 4 W and 6 W output powers to evaluate the healing process after 7 and 14 days. The delivery system consisted of a fiberoptic tube terminating in a handpiece with a tip of 400 ␮m in diameter and 8 mm in length. Laser tip was at a distance of approximately 0.5 cm from the epithelial surface during laser exposure (for about 6 seconds). Animal model and experimental groups classification The present preliminary study was carried out after being approved by the Committee of Animal Experimentation of G. D’Annunzio University, Chieti, Italy. Twelve Wistar male white rats, all 1 to 2 years old, were kept in individual metal cages at room temperature, with 12 hours of light per day and 50% relative humidity. They received a standard pelleted laboratory diet and water ad libitum. Before the experimental procedures, rats were randomly divided into 4 groups of 3 rats each. Two parallel incisions (approximately 15-20 mm in length) were performed in the hard palate of each rat. One incision was performed on the left side of the palate by using a steel scalpel (Bard-Parker number 15; control side), while the other incision was performed on the right side by using a diode laser at different output powers (4 W and 6 W; test side). The tissues were incised without any elevation of a full 766 D’Arcangelo et al. OOOOE June 2007 mucoperiosteal flap. The scalpel incisions were then sutured, whereas no suturing was needed for the laser wounds. Rats were killed at intervals of 7 and 14 days after surgical procedure, and the tissues were analyzed to compare histological and immunohistochemical alterations in each group at different times: (1) group 1: scalpel (control side), laser 4 W (test side), with histological and immunohistochemical analysis after 7 days; (2) group 2: scalpel (control side), laser 6 W (test side), with histological and immunohistochemical analysis after 7 days; (3) group 3: scalpel (control side), laser 4 W (test side), with histological and immunohistochemical analysis after 14 days; and (4) group 4: scalpel (control side), laser 6 W (test side), with histological and immunohistochemical analysis after 14 days. means of an overdose of carbon dioxide at intervals of 7 and 14 days after surgical procedure, for groups 1 and 2 and groups 3 and 4, respectively. Specimens measuring approximately 10 ⫻ 20 mm were then removed from the control (left) and test (right) sides of each animal. The samples were immediately frozen at ⫺80°C for 48 hours in liquid nitrogen, and alternate 7 ␮m sections were cut using a cryostat (Frigocut2800, Reichert-Jung, Germany) and put on treated chromealum gel slides, at room temperature. The serial histological sections were stained with hematoxylin-eosin by using standard procedures and then were examined by light microscopy or processed for immunohistochemical analyses. Finally, the remaining tissue was centrifuged and managed for the eNOS and iNOS molecular analysis. Presurgical treatment and surgical procedure The mucosa of the hard palate was selected for the oral mucous membrane wounds because of its accessibility. The rats were anaesthetized with an intraperitoneal injection of Zoletil 20 solution (chloral hydrate tiletamine ⫹ chloral hydrate zolazepam; Verbac Laboratories, Carros, France) given slowly as a dose of 80 mg/kg of body weight. After anesthesia, the intraoral surgical field together with the handpiece and fiber of the laser device were sterilized with Betadine sol solution (Viatris GmbH & Co. KG, Frankfurt, Germany). Two types of wounds were introduced with both stainless steel scalpel and a continuous wave mode diode laser. On the left side of the palate of each rat (control side), the tissues were incised using a steel scalpel (Bard-Parker number 15), accomplished by suturing with 4-0 Ethibond suture (Ethicon, Pomezia, Italy). Tissue incisions on the right side of the palate (test side) were performed using a continuous wave mode diode laser, and no suturing was needed. During irradiation, the laser tip beam was kept perpendicular to the irradiated tissue surface, and the laser beam was constantly moved to prevent tissues from overheating, necrosis, and carbonization. In groups 1 and 3, the power output was set at 4 W, whereas in groups 2 and 4 it was set at 6 W. The laser was calibrated by the manufacturer before the study. All of the surgical procedures were performed by the same operator under aseptic conditions. After the surgical procedures, the rats were then returned to their cages, without activity limitations. eNOS and iNOS immunohistochemical procedures Ten sections of each group were treated with a blocking agent containing 5% goat normal serum and 0.1% serum albumin in a phosphate buffered saline (PBS) solution, at room temperature for 30 minutes in a moist chamber. Afterward, sections were rinsed in TRIS-HCl for 5 minutes and incubated with the primary rabbit antibodies against eNOS and iNOS for 15 minutes (both antibodies were diluted 1:100 in PBS, Santa Cruz Biotech Inc., Santa Cruz, CA). After incubation with the primary antibodies, sections were rinsed with TRISHCl and incubated with the secondary goat anti-rabbit antibody for 30 minutes. Then, sections were rinsed as aforementioned and incubated with peroxidase antiperoxidase complex for 10 minutes. Antibody complexes were visualized as red precipitates, and sections were counterstained with hematoxylin.31,40 Postsurgical treatment and histological procedures To prevent postsurgical infections, antibiotics (Terramycin 0.5 mL, IM) were administered until 4 days following surgical procedure. The rats were killed by eNOS and iNOS biochemical procedures RT-PCR. Total RNA was extracted using 1 mL RNazol (Cinna/Biotecx, Houston, TX) with 20 ␮g Escherichia coli rRNA (Boehringer Ingelheim GmbH, Germany) employed as a carrier. A mixture (20 ␮g) of M-MLV transcriptase (Perkin-Elmer, Boston, MA), 1 mM dNTP, 2.5 ␮M random primers, and 1 U/␮L RNase inhibitor (GE Healthcare Europe GmbH, Milan, Italy), for 30 minutes at 42°C, was used for transcription. The PCR was amplified through an Eppendorf Mastercycler 5330 (Eppendorf, Milan, Italy),, for 60 seconds at 72°C. For eNOS and iNOS cDNA, a 2.0 mM MgCl2 solution was employed. The sequences of the primers used were 5=-TGTCTGTCTGCTGCTAG-3= (sense) e 5=-CTCTCCAGGCACTTC AGGC-3= (antisense) for eNOS, and 5=-AGTGATGGCAAG CACGACTTC-3= (sense) and 5=-TCTGTCACTCGCTCACCACGG-3= (antisense) for iNOS. An internal OOOOE Volume 103, Number 6 D’Arcangelo et al. 767 Fig. 3. Western blot analysis of eNOS proteins obtained after 7 days from scalpel incision; laser incision at 4 W, and laser incision at 6 W. Fig. 1. Occurrence of mRNA of eNOS in 300 base pair (bp) RT-PCR eNOS by RT-PCR after 14 days. The standard band is on the right lane (S 488 bp). Fig. 4. Western blot analysis of iNOS proteins obtained after 7 days from scalpel incision; laser incision at 4 W, and laser incision at 6 W. tase conjugate substrate kit, Bio-Rad; Figs. 3 and 4).12-14 Fig. 2. Occurrence of mRNA of iNOS in 340 bp by RT-PCR after 14 days. The standard band is on the right lane (S 488 bp). standard of 18S rRNA was used for this reaction (Figs. 1 and 2).12,13 Western blot analysis Fifty ␮g of proteins from the specimen of each group was obtained by homogenizing tissues with a solution (PBS 10%, N-P40 10%, sodium-deoxycholate 10%, SDS 10%) and with protease inhibitors containing aprotinin, leupeptin, and Na3VO4 (Sigma Aldrich Co., St. Louis, MO). Proteins were divided by electrophoresis on a 7.5% polyacrylamide-SDS gel (Bio-Rad, Milan, Italy) and were put on a nitrocellulose membrane at 4°C (Bio-Rad) in a glycine-methanol solution. Primary antibodies against eNOS and iNOS were incubated for 12 hours. The nitrocellulose membrane was rinsed in tris buffered saline, incubated with the secondary antibody, and conjugated with alkaline phosphatase for 2 hours; it was then rinsed and developed in an alkaline solution with nitro blue tetrazolium (alkaline phospha- Histomorphometry and statistics Following immunohistochemical procedures, sections were analyzed by Leitz Dialux 22 optical microscope (Leica, Heidelberg, Germany). The quantitative evaluations of the immune reaction were performed by determination of the integrated optical density (IOD) changes with a digital image analysis. Five slides for each group were analyzed. For data processing, each experimental frame was digitized into 512 ⫻ 512 pixels by a Sony video camera connected to a Leica Quantimet 500 plus (Leica Cambridge Ltd., Cambridge, England), and the change in IOD was determined using ISO transmission density (Kodak CAT 152-3406, Eastman Kodak Company, Rochester, NY) as a standard.31,40,41 The inflammatory response on the various group tissues were classified as grade 1 (low inflammatory grade), grade 2 (medium inflammatory grade), and grade 3 (high inflammatory grade). Statistical analysis All results were expressed as mean ⫾ SD and processed for statistical analysis. ANOVA test was performed between all groups. Probability of null hypoth- 768 D’Arcangelo et al. OOOOE June 2007 Fig. 5. Immunohistochemical localization of eNOS in the sections. A, Scalpel, 7 days; B, scalpel, 14 days; C, laser 4 W, 7 days; D, laser 4 W, 14 days; E, laser 6 W, 7 days; F, laser 6 W, 14 days (hematoxylin-eosin, original magnification ⫻10). eNOS was visualized in the endothelial cells (fine arrows), fibroblasts (thick arrows), and in some gigant cells (arrowheads; E and F). esis of ⬍5% (P ⬍ .05) was considered as statistically significant. RESULTS When compared with traditional surgical procedure, several valuable characteristics of diode laser were found. Laser produced extremely precise surgical incisions that did not require suture. The intrasurgical bleeding appeared enormously reduced, simplifying the surgical phase. The optic microscope observation evidenced histological differences among the various groups based on the inflammatory cells and on the organization of the connective tissue after 7 and 14 days from treatment (Figs. 5 and 6). The inflammatory responses on the various group tissues are reported in Table I. Healing after traditional surgical procedure (control side) Group 1 and Group 2. Histological analysis evidenced an inflammatory infiltration constituted by neu- trophils, polymorphous-nucleation, and some giant cells; the blood vessels appeared almost normal (Figs. 5 and 6). After 7 days, the eNOS found at the level of endothelial cells appeared all along the borders of the incision (Fig. 5); the iNOS appeared especially in the zone of the inflammatory infiltration (Fig. 6). Group 3 and Group 4. The observable histological results were characterized by scarce inflammatory cells, numerous fibroblasts with intense proliferative activity, and absence of vascular phenomena (Figs. 5 and 6). After 14 days, the eNOS increased its presence along the borders of the incisions, but the enzymatic levels were normal (Fig. 5); the iNOS showed a decrement due to the reduction of the inflammatory cells (Fig. 6). Healing after laser surgical procedure (test side) Group 1. The histological results after laser incision at 4 W output power showed a strong inflammatory infiltration with predominance of macrophages, giant cells and plasma cells, hyperemia, and cellular disorganization. Edema and cellular metaplasia were also OOOOE Volume 103, Number 6 D’Arcangelo et al. 769 Fig. 6. Immunohistochemical localization of iNOS in the sections. A, Scalpel, 7 days; B, scalpel, 14 days; C, laser 4 W, 7 days; D, laser 4 W, 14 days; E, laser 6 W, 7 days; F, laser 6 W, 14 days (hematoxylin-eosin, original magnification ⫻25). iNOS immunopositivity was characteristic mainly in the accumulated leukocytes and in the area adjacent to that of dense leukocytic infiltration (arrows). Table I. Inflammation at different times based on the histological differences of the various groups Surgery technique Group 1 Group 2 Group 3 Group 4 Scalpel Laser 4 Scalpel Laser 6 Scalpel Laser 4 Scalpel Laser 6 W W W W Inflammatory grading Grade Grade Grade Grade Grade Grade Grade Grade 2 3 2 3 1 1 1 2 Histological analysis After 7 days After 7 days After 14 days After 14 days visible. The enzymatic values for the eNOS and iNOS at the incision zone were high (Figs. 5 and 6). Group 2. By analyzing the test sites after laser incision at 6 W, necrosis of the external cellular layer and metaplasia of the connective tissue were documented. The inflammatory cells found were above all macrophages and giant cells. The enzymatic location for eNOS, and particularly for iNOS, appeared higher than those in group 1 (Figs. 5 and 6). Group 3. The histological results with laser at 4 W showed, after 14 days, an inflammatory infiltration constituted by neutrophils and polymorphonuclear leukocyte; little vasodilatation was found, as well as numerous cells in proliferative activity. Few connective alterations of the tissue and of gingival epithelium were recognized. The enzymatic levels for the eNOS and the iNOS were lower than those of group 1 (Figs. 5 and 6). Group 4. Histological observation 14 days after 6 W laser surgical procedure showed healing characterized by an intense fibroblastic activity, presence of inflammatory infiltration, and marked hyperemia (vasodilatation). Most of the inflammatory cells found were neutrophils, lymphocytes, and numerous fibroblasts in proliferative activity. The phenomena of cellular metaplasia were still partially identifiable. The enzymatic levels of eNOS and iNOS at the 770 OOOOE June 2007 D’Arcangelo et al. Table II. Immunohistochemical analysis* Immunohistochemical analysis Laser 4 W 7 days Laser 4 W 14 days Laser 6 W 7 days Laser 6 W 14 days Scalpel 7 days Scalpel 14 days iNOS eNOS 32 ⫾ 0.03 36 ⫾ 0.03 18 ⫾ 0.035 21 ⫾ 0.04 92 ⫾ 0.06 85 ⫾ 0.04 46 ⫾ 0.05 32 ⫾ 0.035 25 ⫾ 0.04 20 ⫾ 0.02 5 ⫾ 0.002 10 ⫾ 0.03 *Integrated optical density changes. Data are represented as mean ⫾ SD. incision zone appeared lower than those of group 2 but higher than those of group 3 (Figs. 5 and 6). Biochemical results The results of biochemical evaluation of reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analysis for the enzymes eNOS and iNOS confirmed the location and the expression of the enzymes obtained by immunohistochemical techniques (Tables II-IV and Figs. 1-4). With regard to the test sites, the highest values of eNOS were found after 7 days in groups 2 and 1. After 14 days, a decrease of the eNOS was found in group 3; in group 4 a lower decrease was found. The expression of iNOS was higher in group 2 when compared with group 1; after 14 days it decreased in group 3, but still was present in group 4. Figs. 1 and 2 (PT-PCR analysis) clearly show the presence of eNOS 300 base pair (bp) in all groups, with the highest level in the 6 W group. The presence of iNOS 340 bp in the scalpel group is not detectable; however, a marked increase of iNOS 340 bp was found in 4 W and 6 W groups. The internal standard is 18S (488 bp). Figs. 3 and 4 (Western blot analysis) show that all groups contain the eNOS enzyme, with the highest amount in the 6 W group. The iNOS protein was undetectable in the scalpel group. A significant increase of iNOS enzyme was observed in the laser 4 W and the laser 6 W groups. The changes of the IOD graphically showed that the expression of the enzymes varied in the different groups (Figs. 1-4 and Tables II-IV). Significant differences were shown in all 6 groups of eNOS and iNOS IOD (P ⬍ .05). DISCUSSION The differences in healing between surgical wounds produced by scalpel and continuous wave mode of diode laser at different power output were compared. The disadvantage of laser systems is represented by histologically evident thermal destruction around the laser beam incision and thermal damage that may range from a transient heating to protein denaturation, water evaporation, carbonization, or burning.2,3 Wavelength of the laser, power setting (watts), continuous/pulsed mode, pulse duration, pulse frequency, and exposure time are important laser parameters governing the extent of thermal injury to the tissues. Scalpel wounds, in contrast, do not cause any thermal damage but allow extravasation of blood and lymph, causing a more marked inflammatory response with resultant swelling and formation of a scab.2,3,42 This preliminary study was conducted on rats to evaluate, with histological and immunohistochemical analysis, laser surgical procedure performance at specific parameters, thus suggesting further diode laser application in human oral surgical procedures. The differences in healing between surgical wounds produced by scalpel and diode laser at different output powers (4 W and 6 W) were compared at 7 and 14 days after surgical procedure. The laser irradiation parameters followed instructions from the manufacturer and were based on literature. The energy density employed was in accordance with previous studies on humans38,39 and on animals.40 Histological analysis of the gingival epithelium and connective tissue was performed, and inflammatory cells and vasodilatation were also evaluated. Furthermore, immunohistochemical and biochemical investigations have been performed using iNOS and eNOS as immunohistochemical markers, since many studies have shown the importance of these enzymes that have a role in a nonspecific immune response, acting as a toxic agent in infections.23,24-26 Nitric oxide is an intracellular messenger molecule with important immune functions.23 It is produced by a group of isoenzymes collectively termed NO synthase (NOS),24 and to date 3 distinct isoforms of NOS have been cloned: the endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) NOS.23 In this study, iNOS and eNOS were evaluated in the different groups to compare the inflammatory response after scalpel and laser surgical procedure. Both scalpel and diode laser at 4-W output exhibited wounds with complete healing at 14 days with virtually no difference between the 2 techniques. However, the healing process after laser technique is shown to be worse when compared with scalpel wounds at 4 W and 6 W at 7 days and 6 W at 14 days. The data presented for both surgical techniques clearly showed a progressive im- OOOOE Volume 103, Number 6 D’Arcangelo et al. 771 Table III. PT-PCR analysis* RT-PCR analysis Laser 4 W 7 days Laser 4 W 14 days Laser 6 W 7 days Laser 6 W 14 days Scalpel 7 days Scalpel 14 days iNOS eNOS 34 ⫾ 0.04 38 ⫾ 0.02 17 ⫾ 0.03 23 ⫾ 0.035 87 ⫾ 0.04 79 ⫾ 0.035 48 ⫾ 0.04 36 ⫾ 0.03 24 ⫾ 0.035 22 ⫾ 0.04 8 ⫾ 0.003 14 ⫾ 0.02 *Integrated optical density changes. Data are represented as mean ⫾ SD. Table IV. Western blot analysis* Western blot analysis iNOS eNOS Laser 4 W 7 days Laser 4 W 14 days Laser 6 W 7 days Laser 6 W 7 days Scalpel 7 days Scalpel 14 days 30 ⫾ 0.02 32 ⫾ 0.035 15 ⫾ 0.03 18 ⫾ 0.04 85 ⫾ 0.04 75 ⫾ 0.035 53 ⫾ 0.04 28 ⫾ 0.03 28 ⫾ 0.04 21 ⫾ 0.03 7 ⫾ 0.002 12 ⫾ 0.035 *Integrated optical density changes. Data are represented as mean ⫾ SD. provement of the tissue architecture, a reorganization of the vascular component, and progressive disappearance of the inflammatory infiltration. The analysis of the test sites showed an excellent response to laser incision, but healing parameters were rather low when the tissues were radiated with an output power of 6 W. An excessive absorption of energy can induce thermal damage with necrosis and tissue carbonization. The action of laser at lower power output (4 W) instead reduces the effectiveness of the incision, but also minimizes thermal damage of the tissue. In fact, since different thermal effects are produced in biological tissue exposed to radiation, the right type of laser settings should be carefully chosen depending on the clinical demands and the different tissue characteristics. In such a way, it would be possible to guarantee the maximum clinical effectiveness, avoiding damage to the radiated tissue.41,43 Since diode lasers are absorbed by dark substances such as hemoglobin, their in-depth propagation into tissue is related to the wavelength employed and the coefficient of absorption of the same tissue.42 In the present study, different laser output powers were tested through histological and immunohistochemical analysis to identify which guaranteed a correct compromise in terms of healing and therapeutic effectiveness. On the test sites, the best healing results were observed at an output of 4 W, although the incision was less effective if compared with that of a 6 W output. These findings concur well with previous studies showing that the healing mechanism depends on laser parameters.22 However, our histological analyses showed that healing is not compromised but rather slower, but satisfactory when higher output power (6 W) is used. Bryant et al.22 evaluated the wound healing of soft oral tissues after diode laser irradiation and concluded that the clinical application in oral surgical procedures seems to have a beneficial effect. However, no immunohistochemical analysis was performed after laser therapy. Other studies found that irradiation with diode laser facilitates considerable bacterial elimination, and this could positively influence the healing repair mechanism.6 These results are in contrast with other findings that revealed that diode laser therapy did not accelerate the healing of oral mucosa after gingivoplasty.7 Other reports based on CO2 laser irradiation found that laser wounds of the oral mucosa tend to show less collagen formation, little wound contraction, and slower epithelial regeneration compared with conventional surgical wounds.44 Possible explanations for the delayed re-epithelialization of laser wounds include inhibitory substances produced by necrotic tissues, physical hindrance caused by the presence of eschar, or heat fixation of adjacent epithelial cells.45,46 Based on the aforementioned considerations, the objective of this preliminary study was to compare the efficacy of diode laser at 2 output powers (4 W and 6 W) with scalpel surgical procedure. In addition, immunohistochemical analysis of iNOS and eNOS expression, as intracellular messenger molecules with important immune functions, was evaluated at the different postsurgical time points of the diode laser wounds and the scalpel wounds. Evaluation of the inflammatory response in this study demonstrated that diode laser wounds (especially 7 days after 6 W output power irradiation) tend to be associated with more inflammatory cells and a higher level of iNOS and eNOS when compared with scalpel wounds. During the present study, we also evaluated the clinical aspects of the use of laser for surgical purpose. The 772 D’Arcangelo et al. extreme effectiveness and the clinical advantages of this technology in the treatment of soft tissue in comparison to the traditional surgical procedure must be underlined. In minor oral surgical procedure, diode laser therapy certainly is less invasive and presents some indisputable advantages such as the elimination of bleeding and suturing, as well as minimal postsurgical pain and edema. In this preliminary study, to eliminate the unwanted thermal damage and consequent loss of substance, the surgical incision was performed by slow but continuous movements of the optic fiber along the area to be treated. This limited the loss of substance and favored the mechanism of healing. In conclusion, although traditional surgical procedure allows an incision without loss of tissue, laser surgical procedure is able to guarantee a good healing and has shown important clinical advantages, especially the excellent ability of incision and good bleeding control. Our result showed that continuous mode diode laser tends to produce more pronounced changes (due to tissue thermal damage) than conventional surgical procedure with corresponding greater inflammatory reaction (highest levels of iNOS and eNOS) and delay in tissue organization only initially (7 days), and especially for the highest parameter of laser radiation (6 W). The best results appeared after laser surgical procedure at 4 W at 14 days (group 3), whereas the least satisfactory results were observed after 7 days in the sites radiated with a power of 6 W (group 2). From this preliminary study, it was found that attention to choosing suitable parameters and opportune clinical use of laser equipment allowed optimization of incisions and a control of bleeding; good clinical follow-up procedure resulted in a drastic reduction of the loss of tissue. All the advantages offered by laser surgical procedure are certainly magnified in patients with hemorrhagic diathesis, which requires strict control of bleeding.47 Thus, the optimal results of our study suggest the use of laser as a first choice in specific clinical situations with an elevated hemorrhagic risk. REFERENCES 1. Mester E. Effect of laser rays on wound healing. Amer J Surg 1971;122:532-5. 2. Pogrel MA, Yen C-K, Hansen A. A comparison of carbon dioxide laser, liquid nitrogen cryosurgery, and scalpel wounds in healing. Oral Surg Oral Med Oral Pathol 1990;69:269-73. 3. Wilder-Smith P, Arrastia A-MA, Liaw L-H, Berns M. Incision properties and thermal effects of three CO2 lasers in soft tissue. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;79:685-91. 4. Kana JS, Hutschenreiter G, Haina D, Waidelich W. Effect of low power density laser radiation on healing of open skin wound in rats. Arch Surg 1981;116:293-6. OOOOE June 2007 5. Mester E, Mester AF, Mester A. 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