Luciana Maria Pedreira Ramalho

Computerized Morphometric Assessment of the Effect of Low-Level Laser Therapy on Bone Repair: An Experimental Animal Study

OBJECTIVE The aim of this study was to evaluate morphometrically the amount of newly formed bone after GaAlAs laser irradiation of surgical wounds created in the femur of rats. BACKGROUND DATA Low-level laser therapy (LLLT) has been used in several medical specialties because of its biomodulatory effects on different biological tissues. However, LLLT is still controversial because of contradictory reports. This is a direct result of the different methodologies used in these works. MATERIALS AND METHODS In this study, 40 Wistar rats were divided into four groups of 10 animals each: group A (12 sessions, 4.8 J/cm2 per session, observation time of 28 days); group C (three sessions, 4.8 J/cm2 per session, observation time of 7 days). Groups B and D acted as nonirradiated controls. The specimens were routinely processed to wax and cut at 6-microm thickness and stained with H&E. For computerized morphometry, Imagelab software was used. RESULTS Computerized morphometry showed a significant difference between the areas of mineralized bone in groups C and D (p = 0.017). There was no difference between groups A and B (28 days; p = 0.383). CONCLUSION It is concluded that, under this experimental condition, LLLT increased bone repair at early bone healing.

DOSE AND WAVELENGTH OF LASER LIGHT HAVE INFLUENCE ON THE REPAIR OF CUTANEOUS WOUNDS

OBJECTIVE The objective of the present study was to compare histologically the effect of GaAlAs (lambda 830 nm, phi approximately 2 mm(2), 35 mW) and InGaAlP (lambda 685 nm, phi approximately 2 mm(2), 35 mW) lasers, alone or in association with doses of 20 or 50 J/cm(2) on cutaneous wounds in the dorsum of the Wistar rat. BACKGROUND DATA The healing time of surgical wounds is of extreme importance and it is usually associated with a post-operative period free of infection and with less pain and inflammation. MATERIALS AND METHODS Sixty Wistar rats were divided into seven groups: Group I - control (non-irradiated); Group II - lambda 685 nm, 20 J/cm(2); Group III - lambda 830 nm, 20 J/cm(2); Group IV - lambda 685 nm and lambda 830 nm, 20 J/cm(2); Group V - lambda 685 nm, 50 J/cm(2)); Group VI - lambda 830 nm, 50 J/cm(2); and Group VII - lambda 685 nm and 830 nm, 50 J/cm(2). The animals were sacrificed 3, 5, and 7 days after surgery. RESULTS Light microscopic analysis using H&E and Picrosírius stains showed that, at the end of the experimental period, irradiated subjects showed increased collagen production and organization when compared to non-irradiated controls. Inflammation was still present in all groups at this time. CONCLUSION Group IV (lambda 830 nm and lambda 685 nm, 20 J/cm(2)) presented better results at the end of the experimental period. It is concluded that low-level light therapy (LLLT) can have a positive biomodulatory effect on the repair of cutaneous wounds.

Effect of low level laser therapy on the repair of bone defects grafted with inorganic bovine bone

The aim of this study was to assess histologically the effect of LLLT (lambda 830 nm) on the repair of standardized bone defects on the femur of Wistar albinus rats which were grafted with inorganic bovine bone Gen-ox(R). Three randomized groups were studied: group I (control, n=6); group II (Gen-ox, n=9) and group III (Gen-ox + LLLT, n=9). The animals were irradiated every 48 h during 15 days; the first irradiation was performed immediately after the procedure. The animals were irradiated transcutaneuosly at four points around the defect. At each point a dose of 4 J/cm(2) was given (Ø approximately 0.6 mm, 40 mW) and the total dose per session was 16 J/cm(2). The animals were killed by an overdose of general anesthetic 15, 21 and 30 days after surgery. The specimens were routinely processed by embedding in paraffin, serially cut and stained with H&E and Picrosirius and analyzed under light microscopy. The results showed evidence of a more advanced repair in the irradiated group when compared to the non-irradiated groups. The repair of the irradiated group was characterized by both increased bone formation and amount of collagen fibers around the graft within the cavity from the 15th day after surgery, also considering the osteoconductive capacity of the Gen-ox. We conclude that LLLT had a positive effect on the repair of bone defects implanted with inorganic bovine bone.

Immunohistochemical assessment of myofibroblasts and lymphoid cells during wound healing in rats subjected to laser photobiomodulation at 660 nm

OBJECTIVE The goal of this study was to assess the biomodulatory effect of low-level laser therapy (LLLT) on myofibroblasts and T and B cells during wound healing. BACKGROUND DATA Phtotobiomodulation using LLLT has been extensively applied to improve wound healing. MATERIALS AND METHODS Standardized artificial surgical wounds were made on the backs of 24 male rats. Half of them underwent LLLT (20 J/cm(2)) at 660 nm delivered for 7 d. At 8 and 14 d post-surgery the healing wounds were removed and immunohistochemical analysis of myofibroblasts, T cells, and B cells was carried out. The mean of each cell subset was calculated and compared to one another using two-way analysis of variance (ANOVA) and Tukeys test. RESULTS The average number of myofibroblasts was statistically significantly higher in the irradiated group than in the non-irradiated group on the eighth (p = 0.001) but not the 14th (p = 0.555) day. B and T cells were significantly more conspicuous in the irradiated group on both the eighth (p = 0.004 and 0.02, respectively) and 14th days (p = 0.04 and 0.03, respectively). CONCLUSIONS Our results suggest that LLLT facilitates myofibroblastic differentiation during the early stages of the cicatricial repair process. Furthermore, LLLT also appears to modulate the inflammatory response by downregulating lymphocytic proliferation during the wound healing process.

Laser-Induced Alveolar Bone Changes During Orthodontic Movement: A Histological Study on Rodents

OBJECTIVE The aim of this study was to assess by light microscopy changes in alveolar bone during orthodontic movement in rats. BACKGROUND Orthodontic movement causes both removal and deposition of bone tissue. The use of laser phototherapy (LPT) is considered an enhancement factor for bone repair. METHODS Thirty Wistar rats were divided into two groups (n = 15) and subdivided according to animal death (7,13, and 19 days). Half of the animals in each group were treated with LPT during orthodontic movement. After animal death, specimens were processed and underwent histological and semi-quantitative analyses (HE and Sirius red). RESULTS LPT-irradiated specimens showed significantly higher numbers of osteoclasts when compared with controls at both 7 (p = 0.015) and 19 (p = 0.007) days, as well as significant increases in the number of osteoblasts (p = 0.015) between days 7 and 13. The amount of collagen matrix was significantly reduced between days 7 and 13 at both pressure and tension sites in controls (p = 0.015) but not in LPT-treated animals. LPT-treated subjects showed significantly greater deposition of collagen matrix at the pressure site at both the thirteenth (p = 0.007) and nineteenth days (p = 0.001). At the tension site, a significant increase in the amount of collagen matrix was observed in non-irradiated specimens (p = 0.048) between days 7 and 19. CONCLUSIONS LPT caused significant histological changes in the alveolar bone during induced tooth movement, including alterations in the number of both osteoclasts and osteoblasts and in collagen deposition in both pressure and tension areas.

Light microscopic description of the effects of laser phototherapy on bone defects grafted with mineral trioxide aggregate, bone morphogenetic proteins, and guided bone regeneration in a rodent model

We carried out a histological analysis on bone defects grafted with mineral trioxide aggregate (MTA) treated or not with laser, bone morphogenetic protein (BMP), and guided bone regeneration (GBR). Benefits of the use of MTA, laser, BMPs, and GBR on bone repair are well known, but there is no report on their association with laser light. Ninety rats were divided into 10 groups each subdivided into 3. Defects on G II and I were filled with the blood clot. G II was further irradiated with LED. G III and IV were filled with MTA; G IV was further irradiated with laser. G V and VI, the defects filled with MTA and covered with a membrane (GBR). G VI was further irradiated with laser. G VII and VIII, BMPs were added to the MTA and group VIII further irradiated with laser. G IX and X, the MTA + BMP graft was covered with a membrane (GBR). G X was further irradiated with laser. Laser light (λ = 850 nm, 150 mW, 4 J/cm(2) ) was applied over the defect at 48-h intervals and repeated for 15 days. Specimens were processed, cut and stained with H&E and Sirius red and underwent histological analysis. Subjects on group X were irradiated. The results showed different tissue response on all groups during the experimental time. Major changes were seen on irradiated subjects and included marked deposition of new bone in advanced maturation. It is concluded that near infrared laser phototherapy improved the results of the use of the MTA on bone defects.

Laser phototherapy as a treatment for radiotherapy-induced oral mucositis

Oral mucositis is a harmful side effect of radiotherapy (RT) on the head and neck region. There are encouraging reports on the beneficial aspects of the use of laser light on the treatment of oral mucositis. This paper reports the efficacy of laser phototherapy (LPT) on the treatment of oral mucositis in a patient undergoing RT after surgical removal of a squamous cell carcinoma with osseous invasion of the maxilla. Palatal and commissural lesions were treated with λ660 nm, 40 mW, ∅=4 mm(2), in contact mode, 5 x 2.4 J/cm(2) per point, 14.4 J/cm(2) per session. For treating the lesion on the patients nasal mucosa, LPT (∅=4 mm(2), λ780 nm, 70 mW, 3 x 2.1 J/cm(2) per point, 6.3 J/cm(2) per session, contact mode) was used on the external area of the nose. A single dose (2.4 J/cm(2)) with the λ660 nm laser, as described before, was applied on the entrance of each nostril. LPT was used 3 times/week during 4 weeks. Treatment results indicate that the use of LPT on oral mucositis was effective and allowed the patient to carry on the RT without interruption. However, long-term and controlled clinical trials are necessary to establish both preventive and curative protocols using LPT.

p53 and MDM2 protein expression in actinic cheilitis

Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

Assessment of the Behavior of Myofibroblasts on Scalpel and CO2 Laser Wounds: An Immunohistochemical Study in Rats

OBJECTIVE The aims of this study were to quantitative and statistically assess the presence of myofibroblasts on both conventional and CO(2) laser wounds. BACKGROUND DATA Wound contraction of both traumatic and surgical origin may reduce or limit the function of the tissue. Myofibroblasts are cells involved in the process of wound contraction, which is smaller in CO(2) laser wounds. MATERIALS AND METHODS Thirty-two animals (rattus norvegicus) were divided into four groups and treated using either the CO(2 )laser (groups 1 and 2) or conventional scalpel (groups 3 and 4). The animals were sacrificed eight days post-operatively (groups: 1 and 3) and 14 days after surgery (groups: 2 and 4). The specimens were routinely processed to wax and stained with alpha-smooth muscle actin (alpha SMA) and analyzed under light microscopy (40x) using a calibrated eyepiece and a graticule. Two standard areas around the wound of each slide were selected and used to count the number of myofibroblasts present. RESULTS The results of this study show that it is possible to determine the number of myofibroblasts present in wounds produced by the laser or the scalpel at both eight and 14 days after surgery. However the number of myofibroblasts at day eight was significantly higher than at day 14 (laser, p = 0.007 and scalpel, p = 0.001). The number of cells present in group 3 was significantly higher than in group 1 (p = 0.001). However on the 14th day there was no such difference (p = 0.072). CONCLUSION It is concluded that the small number of myofibroblasts at day eight after wounding with the CO(2) laser may be the reason that contraction on this wound is smaller than the one observed in conventional surgery.

Clear cell odontogenic carcinoma: case report with immunohistochemical findings adding support to the challenging diagnosis

Clear cell odontogenic carcinoma (CCOC) is a rare odontogenic tumor associated with aggressive clinical behavior, metastasis, and low survival. We report a case of CCOC affecting the mandible of a 39-year-old man. The tumor presented a biphasic pattern composed of clear cell nests intermingled with eosinophilic cells and separated by collagenous stroma. Immunoreactivity to cytokeratin (CK), specifically AE1/AE3 and CK 8, 14, 18, and 19 was found, as well as to epithelial membrane antigen (EMA). The tumor cells were negative for S100 protein, CK 13, vimentin, smooth muscle actin, laminin and type IV collagen. Low labeling indices for the proliferation markers Ki-67 and proliferating cell nuclear antigen and to p53 protein might predict a favorable prognosis for the lesion. A surgical resection was performed, followed by adjuvant radiotherapy. A 2-year follow-up has shown no signs of recurrence. The significance of histochemical and immunohistochemical resources in the correct diagnosis of CCOC is analyzed.