Effect of In-Ga-Al-P Diode Laser Irradiation on Angiogenesis in Partial Ruptures of Achilles Tendon in Rats ABSTRACT

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1 Photomedicine and Laser Surgery Volume 23, Number 5, 2005 Mary Ann Liebert, Inc. Pp Effect of In-Ga-Al-P Diode Laser Irradiation on Angiogenesis in Partial Ruptures of Achilles Tendon in Rats ANA C.B. SALATE, 1 GISELE BARBOSA, 1 PATRÍCIA GASPAR, 1 PAULO U. KOEKE, 1 NIVALDO A. PARIZOTTO, 1 BENEDITO G. BENZE, 2 and DAIANE FOSCHIANI 2 ABSTRACT Objective: This study was conducted to analyze the effect of different irradiances of low-level laser therapy (LLLT) on angiogenesis after partial rupture of Achilles tendon of rats. Background Data:... Methods: Ninety-six animals were divided into three groups subject to treatment during 3, 5, and 7 days post-lesion. Thirty-two animals were used in each group. The groups were further divided into four subgroups with eight animals in each, receiving In-Ga-Al-P laser (660 nm) treatment at (1) mean output of 10 mw, (2) 40 mw during 10 sec, (3) a sham subgroup, and (4) a non-treatment subgroup. Each animal was subjected to a lesion of the Achilles tendon by dropping a 186-g weight from a 20-cm height over the tendon. Treatment was initiated 6 h post-injury for all the groups. Blood vessels were colored with India ink injection and were examined in a video microscope. Results: Laser exposure promoted an increase in blood vessel count when compared to controls. The 40-mW group showed early neovascularization, with the greatest number of microvessels after three laser applications. The 10-mW subgroup showed angiogenesis activity around the same time as the sham laser group did, but the net number of vessels was significantly higher in the former than in the controls. After seven irradiations, the subgroup receiving 40 mw experienced a drop in microvessel number, but it was still higher than in the control groups. Conclusions: LLLT of different intensities seems to promote neovascularization in damaged Achilles tendons of rats after partial rupture compared to controls. INTRODUCTION TENDO are the interface between the contractile and noncontractile structures of the body through which controlled forces are applied in creating movement. Connective tissue injuries, such as tendon rupture and ligamentous strains, are common. Unlike most soft tissues, which require 7 10 days to heal, the primary healing of tendons is believed to take at least 6 weeks, during which time they are normally protected in an immobilization cast, which might predispose to complications that retard post-operative rehabilitation. 1 Angiogenesis (i.e., the development of new blood vessels) is a vital part of wound healing, by re-establishing circulation at the injury site, thus limiting ischemic necrosis and permitting repair. 2 Therefore, any treatment that can initiate and promote angiogenesis may aid in healing. Neovascularization restores the supply of oxygen and nutrients to the new tissue, and accommodates the high metabolic demands and continued growth of the reparative cells, permitting them to migrate, divide, and synthesize. 3 After injury, the basement membrane of the capillaries adjacent to the wound-bed breaks down under the influence of proteinases released from endothelial cells. These cells form pseudopodia that protrude through the gaps in the basement membrane and then migrate away from the parent vessel into the perivascular space. Capillary sprouts formed in this way then vacuolize to create a central lumen and eventually connect to another sprout or capillary to form capillary loops, 1Laboratory of Electro-Thermo-Phototherapy, Department of Physiotherapy, Federal University of São Carlos, São Paulo, Brazil. 2Department of Statistics, Federal University of São Carlos, São Carlos, Brazil. 470

2 LLLT in Neovascularization 471 which subsequently either disappear or develop into larger vessels. 2 Since capillary growth occurs during inflammation and formation of granulation tissue, and is accompanied by an increased accumulation of leucocytes, it has been suggested that initiation of the process of angiogenesis may be due to the release of some substance or substances from these cells. 4 Macrophages also play a crucial role in producing angiogenic factors. 4,5 Polverini et al. 5 demonstrated that macrophages could induce angiogenesis in vivo and also stimulate endothelial cell proliferation in vitro. Vascular endothelial growth factor (VEGF) is a secreted mitogen specific for endothelial cells and an extremely potent angiogenic stimulator. The increase of these factors could affect endothelial cell proliferation and, consequently, neovascularization. 6 Fibroblast growth factor (FGF) promotes chemotaxis, proliferation, and differentiation of healing cells. FGFs can be considered natural mediators of the tissue repair process since they stimulate the growth fibroblasts and endothelial cells, and enhance chemotactic and mitogenic activities of vascular cells in vitro and angiogenic activities in vivo. 7 Some authors 8 have indicated that the healing of tenotomized Achilles tendon is considerably improved following He-Ne laser treatment in animals. The parameters that can be used to improve angiogenesis in a skin flap model have been described by investigators. 9 The use of LLLT in stimulating growth factors and consequently improving tissue repair has been widely accepted. 6,7,10 However, the mechanism by which laser light facilitates tissue healing remains poorly understood due to the dose-dependent response of this kind of therapy. To date, only a few experiments have been performed investigating the relevance of laser irradiation to tissue repair. The purpose of our study was to find out if LLLT could promote angiogenesis, an essential component of tissue repair, in partial ruptures of rat Achilles tendon and to analyze the effect of different irradiances in neovascularization. MATERIALS AND METHODS A total of 96 male Wistar rats (Rattus norvegicus albinus), weighing g, were used. The animals were housed in cages provided with rat chow and water ad libitum, and were maintained on a 12-h photoperiod. An equal number of rats were randomly assigned to groups submitted to laser treatment on 3, 5, and 7 days or to sham treatment on 3, 5, and 7 days post-lesion, starting on the same day of rupture. The procedure followed the National Guide for Animal Experimentation in Brazil ( and the guide of the National Academy of Sciences, USA. Lesion procedure FIG. 1. Model of partial Achilles tendon lesion with a weight of 186 g fallen from a height of 20 cm. The 96 animals were weighed and anesthetized with Ketamine by intraperitoneal injection of 95 mg/kg. Thereafter, the right hind limb was placed in traction, and the Achilles tendon was positioned in the equipment to make a partial lesion (Fig. 1). A weight of 186 g placed 20 cm above the animal was dropped on the tendon and subsequently removed. The potential energy of the fall was mj. The rat was kept under observation until recovery from anesthesia. Laser treatment The right Achilles tendon of the animals received laser treatment with continuous radiation at a 660 nm in a single point produced by a In-Ga-Al-P laser (MMOptics-Twin Laser model class 3, Brazil) at 10 sec with contact application. One group (24 animals) received 10 mw mean output with a dose of 2.5 J/cm 2 every day in the same period, during 3 (n = 8), 5 (n = 8), and 7 days (n = 8) starting the first irradiation in the same day of lesion at about 6 h later. The second group (24 animals) received 40 mw with a dose of 10 J/cm 2 on the same way as the first group. The sham group (n = 24) was submitted to the laser treatment but the equipment was turned off. Another group (n = 24), after rupture, remained in their cages for 3 days (n = 8), 5 days (n = 8), and 7 days (n = 8) without any treatment. Assessment of vascularity and tendon excision One day after the last treatment, the rats were anesthetized with Ketamine (95 mg/kg), and an injection of 1 ml of India ink was made on the iliac artery under manual pressure. The adequacy of the perfusion of the limb with the solution was judged by the appearance of ink solution at the rat paw. The animals were then euthanized with appropriate dose of ethyl ether, and the Achilles tendon was excised as follows. The tendon was separated from the surrounding tissue and sharp transverse cuts were made below the musculotendinous junction and above the calcaneal insertion. These specimens were immersed in a Bouin s solution for 6 h, and dehydrated by immersion in serial dilutions of alcohol (70%, 90%, 95%, and 100%) and then immersed in xylene for 3 h. Serial sections of 7 µm were stained with hematoxylin-eosin (HE) for analysis under light microscope (Zeiss). In all groups, a total of five areas of equal size were determined, and the number of vessels were counted in these areas by a video measuring system (Sony). In ruptured tendons, the

3 472 Salate et al. region of lesion was considered as 1 and the others (region 2, 3, 4, 5) were above (2, 3) and below this (4, 5). Statistical analysis ** ** The nonparametric Friedman test with replicated factors was used to compare the numbers of vessel of the groups treated after tendon lesion across the different groups. The software used was Statistica for Windows (version 5.5, StatSoft, Inc., USA) ** * RESULTS The results of this study indicated that the number of vessels markedly increased in laser-treated rats compared with control groups. Table 1 shows the descriptive results, including mean, median, standard deviation, and maximum and minimum values, observed during the analysis. After 4 days of lesion and a total of three irradiations, the group treated with mean output laser of 40 mw showed an increase in vessel number when compared with the other groups (p < 0.01). The group treated with laser with 10 mw showed an increase compared with the control group that had not received any treatment (p < 0.05; Fig. 2). Histologic examination of the affected tendon in this period made evident that the rats receiving laser treatment showed an organized tissue with a greater amount of microvessels compared to control groups. After five irradiations, all laser-treated groups showed an increased number of microvessels when compared with the sham treatment group (p < 0.01) and the non-treated group (p < 0.01; Fig. 3). When the 10-mW and 40-mW groups were compared, no differences were observed. By the seventh day of treatment, the results were the same as those observed after 5 days, with groups treated with laser showing more vessels when compared with control tendons (p < 0.01; Fig. 4). In this period, collagen fibers were organized, especially in 40-mW group. However, the tissue showed fewer vessels when compared with the periods of 3 and 5 days of treatment. Nevertheless, the number of blood vessels was greater than in the control groups. 0 10mW x 40 mw 10mW x sham group 10mW x non-treated FIG. 2. Graphic of subgroups after 3 days indicating comparison of 10 mw 40 mw; 10 mw sham group; 10 mw non-treated group; 40 mw sham group; 40 mw nontreated group; sham group non-treated group, respectively (*p < 0.05; **p < 0.01; = no statistical significance). Figure 5 shows the differences between the group treated with laser of 40 mw during 3 days and the non-treated group. We noted that the structure of the tissue was better in treated group than the non-treated, with more vessels on the paratenon. Figure 6 displays data on the group treated with 10 mw mean power laser which showed an organized tissue when compared with sham treated group after 5 days of treatment. We could note that the sham group shows more inflammatory cells and the fibroblasts were not aligned in the tissue as in the laser group. DISCUSSION The clinical efficacy of LLLT has been received increasing acceptance as a valid intervention promoting injury repair. 11 Using light microscopy analysis in conjunction with a video system, we found a significant increase on vessel number after 40mW x sham group 40mW x non-treated sham group X non-treated TABLE 1. DATA OF BLOOD VESSEL COUNT INCLUDING MEAN, MEDIAN, STANDARD DEVIATION AND MAXIMUM AND MINIMUM VALUES IN ALL GROUPS IN MORPHOMETRIC ANALYSIS Sham Noln- Sham Non- Sham Non- 10 mw, 40 mw, group, treated, 10 mw, 40 mw, group, treated, 10 mw, 40 mw, group, treated, 3 days 3 days 3 days 3 days 5 days 5 days 5 days 5 days 7 days 7 days 7 days 7 days Mean 8,250 11,325 7,100 6,675 9,200 9,225 5,850 5,975 11,150 9,425 6,425 5,475 Median 8,000 11,000 7,000 6,000 8,500 8,000 5,000 6,000 11,000 9,000 7,000 6,000 Maximum 15,000 19,000 12,000 12,000 16,000 16,000 13,000 11,000 25,000 16,000 11,000 8,000 Minimum 4,000 2,000 3,000 3,000 5,000 5,000 3,000 3,000 1,000 4,000 2,000 3,000 Standard 2,295 3,452 0,312 0,331 0,459 0,463 0,371 0,274 0,704 0,437 0,356 0,199 deviation

4 LLLT in Neovascularization mW x 40 mw 10mW x sham group 10mW x non-treated group 40mW x sham group 40mW x non-treated group sham group X non-treated group mW x 40 mw 10mW x sham group 10mW x non-treated group 40mW x sham group 40mW x non-treated group sham group X non-treated group FIG. 3. Graphic of subgroups after 5 days indicating comparison of 10 mw 40 mw; 10 mw sham group; 10 mw non-treated group; 40 mw sham group; 40 mw nontreated group; sham group non-treated group respectively. (**p < 0.01; = no statistical significance). FIG. 4. Graphic of subgroups after 7 days indicating comparison of 10 mw 40 mw; 10 mw sham group; 10 mw non-treated group; 40 mw sham group; 40 mw nontreated group; sham group non-treated group respectively. (**p < 0.01; = no statistical significance). laser therapy in tendon repair when compared with control groups. To our knowledge, this is the first report on the quantitative determination of the tendon number vessels after LLLT. Previous studies evaluating the effects of laser radiation on neoangiogenesis have been limited mainly to in vitro experiments analyzing increases in growth factors elicited by neovascularization events during the wound healing process. There is evidence that LLLT is able to induce neoangiogenesis in vitro and in vivo. In addition, it has been demonstrated that this type of therapy could stimulate certain cellular functions such as protein synthesis and mitotic rate, induce release of growth factors involved in neovascularization such as cytokines, basic fibroblast growth factor, and vascular endothelial growth factor. 12 The literature indicates that light photobioactivation accelerates wound healing and promotes angiogenesis. In vitro studies have been found to modulate the activity of a variety of cells involved in the process of wound healing including macrophages, fibroblasts, mast cells, lymphocytes and endothelial cells. 4,6,7,10,11,13 Immuno-inflammatory cells such as macrophages and T-lymphocytes are capable of producing cytokines and growth factors that control the functions of other cells and regulate the processes of angiogenesis and fibroplasia. 13 A B FIG. 5. Photomicrography of tendons (longitudinal view) submitted to 40-mW laser treatment (A) and non-treated group (B) during 3 days after rupture (arrows, vessels colored with India ink; bar = µm).

5 474 Salate et al. A B FIG. 6. Photomicrography of tendons (longitudinal view) submitted to 10-mW laser treatment (A) and sham-treated group (B) during 5 days after rupture (arrow = vessels colored with India ink; bar = µm). It has been shown that a 660-nm laser-irradiated macrophages produce conditioned media that contain growth factors capable of modulating the proliferation of fibroblasts. 10 By increasing fibroblast production, the proliferative phase of repair could be accelerated, and thus, LLLT could be of considerable benefit in the treatment of tissue injuries. Webb et al. 7 suggest that photoenergy at 660 nm wavelength at given parameters, possibly induced fibroblasts to secrete growth factors like bfgf (basic FGF) that could promote chemotaxis, proliferation and differentiation of vascular endothelial cells in vitro and angiogenesis in vivo. Ghali and Dyson 14 showed that, of the endothelial cell exposed to 660-nm In-Ga-Al-P or 820-nm Ga-Al-As wavelengths from 15-mW laser diodes, only the 660-nm irradiated cells were significantly higher than their controls, suggesting 660-nm LLLT induced the release of mitosis-promoting cytokines and growth factors. Our study showed that the 660-nm LLLT was efficient in the increase of angiogenesis during tendon repair. However, the response of LLLT on cells may be dependent on the parameters used. The mean output of 40 mw during 10 sec promoted an increase of neovascularization starting after 3 days of application and, with mean output of 10 mw, showed more vessels after 5 days of irradiation. On the seventh day, the group irradiated with 40 mw had fewer vessel number than observed on 5-day. In accordance with Enwemeka, 15 we saw a period of fibroplasia and fibrillogenesis begin from about the seventh day, when the number of vessels decreases and returns to normal. The periods of the effect of laser in each treated group seemed to differ between groups. We suppose that the increase in neoangiogenesis in laser treated groups occur due the stimulatory effect of laser in inflammatory cells (macrophages and fibroblasts) to release growth factors specially FGF and VEGF, which are related to be potent angiogenic stimulators. The exact mechanism explaining how low-level laser irradiation stimulates neovascularization on biological tissues has been elucidated yet. The most suggestive evidence for it has been proposed by researchers conducting experimental laboratory studies on animal models and tissue cultures. 11 Previous studies have suggested that photobiostimulation occurs through the electron transport chain enzymes in mitochondria, by producing ATP, which increase cellular metabolism and function. 16,17 According to Karu, 16 the process involved is the acceleration of the electron transfer in the redox pairs in some sections of the respiratory chain. We observed that the LLLT response to increased neovascularization occurs, but it is dose-dependent. The results support the hypothesis that LLLT has a dose-related effect in modulating cell functions or in the release of growth factors. Studies using cell cultures to improve secretion and release of growth factors are of importance for determining the best combination of parameters in different cell types. CONCLUSIO In conclusion, we have demonstrated that LLLT has an important effect in promoting neovascularization during tendon repair with a significant increase in microvessel number, and the highest output power showed an earlier response than the lesser irradiances. REFERENCES 1. Enwemeka, C.S., and Reddy, K. (2000). The biological effects of laser therapy and other physical modalities on connective tissue repair processes. Laser Ther. 12, Hudlicka, O., and Tyler, K.R. (1986). Angiogenesis. The Growth of the Vascular System. London: Academic Press. 3. Schaffer, C.J., and Nanney, L.B. (1996). Cell biology of wound healing. Int. Rev. Cell Cytol. 169, Young, S.R., and Dyson, M. (1990). The effect of therapeutic ultrasound on angiogenesis. Ultrasound Med Biol. 16, Polverini, P.J., Cotran, R.S., Gimbrone, M.A. Jr., et al. (1977). Activated macrophages induce vascular proliferation. Nature 269, Kipshidze, N., Nikolaychik, V., Keelan, M.H., et al. (2001). Lowpower helium:neon laser irradiation enhances production of vascular endothelial growth factor and promotes growth of endothelial cells in vitro. Lasers Surg. Med. 28, Webb, C., Dyson, M., and Lewis, W.H.P. (1998). Stimulatory effect of 660-nm low-level laser energy on hypertrofic scar-derived fibroblasts: possible mechanisms for increase in cell counts. Lasers Surg. Med. 22, Reddy, G.K., Gum, S., Stehno-Bittel, L., et al. (1998). Biochemistry and biomechanics of healing tendon. Part II. Effects of com-

6 LLLT in Neovascularization 475 bined laser therapy and electrical stimulation. Med. Sci. Sports Exerc Abergel, R.P., Meeker C.A., Lam, T.S., et al. (1984). Control of connective tissue metabolism by lasers: recent developments and future prospects. J. Am. Acad. Dermatol. 11, Young, S., Bolton, P., Dyson, M., et al. (1989). Macrophage responsiveness to light therapy. Lasers Surg. Med. 9, Stadler, I., Evans, R., Kolb, B., et al. (2000). In vitro effects of low-level laser irradiation at 660 nm on peripheral blood lymphocytes. Lasers Surg. Med. 27, Schindl, A., Schindl, M., Schindl, L. (1999). Increased dermal angiogenesis after low-intensity laser therapy for a chronic radiation ulcer determined by a video measuring system. J. Am. Acad. Dermatol. 40, Agaiby, A.D., Ghali, L.R., Wilson, R., et al. (2000). Laser modulation of angiogenic factor production by T-lymphocytes. Lasers Surg. Med. 26, Ghali, L., and Dyson, M. (1992). The direct effect of light therapy on endothelial cell proliferation, in: R. Steiner, and R. Langer (eds.). Angiogenesis: Key Principles Science-Technology-Medicine. Basel: Birkhauser Verlag, pp Enwemeka, C.S. (1989). Inflammation, cellurarity, and fibrillogenesis in regeneration tendon: implications for tendon rehabilitation. Phys. Ther. 69, Karu, T. (1989). Photobiology of low-power laser effects. Health Phys. 56, Passarella, S., Ostuni, A., Atlante, A., et al. (1988). Increase in the ADP/ATP exchange in the rat liver mitochondria irradiated in vitro by helium-neon laser. Biochem. Biophys. Res. Commun. 156, Address reprint requests: Dr. Nivaldo Antonio Parizotto Universidade Federal de São Carlos Departamento de Fisioterapia Rodovia Washington Luís, km 235 Caixa Postal , São Carlos, SP, Brazil parizoto@power.ufscar.br