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Low-power laser therapy for repairing acute and chronic-phase bone lesions
Research in Veterinary Science 94 (2013) 105–110
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Low-power laser therapy for repairing acute and chronic-phase bone lesions F.C.D. Mota a, M.A.A. Belo b,⇑, M.E. Beletti c, R. Okubo d, E.J.R. Prado b, R.V.P. Casale b a
College of Veterinary Medicine, Federal University of Uberlândia (UFU), Uberlândia, MG, Brazil Pharmacology and Clinical Pathology Laboratory, Camilo Castelo Branco University (UNICASTELO), Descalvado, SP, Brazil c Institute of Biomedical Sciences, Federal University of Uberlândia (UFU), Uberlândia, MG, Brazil d Bioengineering Laboratory, University of São Paulo (USP), Ribeirão Preto, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 18 January 2012 Accepted 3 July 2012
Keywords: Low-power laser therapy Fracture consolidation Osteotomy Rat
a b s t r a c t To evaluate the therapeutic activity of low-power laser (InGaAlP: 670 nm/30 mW), at doses of 90 J/cm2, on the process of acute and chronic-phase repair of bone lesions of Wistar rats. Sixty-three adult males were divided into nine groups subjected to bone injury, in order to form the following treatments: T1 (control); T2 (acute-phase); T3 (chronic-phase) which were subdivided into three subgroups (n = 7), analyzed on the 9th, 17th and 28th days post-surgery, after a period of daily treatment with laser. The animals with acute-phase treatment presented a more extensive endochondral ossification process. Laser-treated animals showed significant increases in serum alkaline phosphatase levels and had an effect on biomechanical property, resulting in a gradual increase in bone stiffness. Laser therapy aided the bone consolidation process and favored the physiopathologic mechanisms involved in bone tissue repair, and its effects were more prominent when treatment started during the acute phase of the injury. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Non-consolidation of bone defects is a major challenge for healthcare professionals in different fields (Nascimento et al., 2010). It has been described as one of the conditions leading to the development of pseudoarthrosis and this, in turn, may be responsible for complications that, even after applying modern surgical techniques, do not allow patients to achieve a cure (Nolte et al., 2001). Low-power laser therapy is an alternative treatment that favors regenerative processes in biological tissues, such as in fracture repairs (Guzzardella et al., 2001; Brito, 2004; Javadieh et al., 2009; Fávaro-Pípi et al., 2010), skin lesions (Lucas et al., 2003), nerve tissue (Padua et al., 1999) and muscle tissue (Amaral et al., 2001). It is believed that the irradiation from low-power lasers act on cell components that are sensitive to certain wavelengths, such that photons are absorbed. These trigger chemical reactions, especially in mitochondria, lysosomes and membranes, thereby modifying ion transportation (Karu, 1987) and causing increased levels of ATP and prostaglandins. This contributes towards partial acceleration of the tissue repair during the inflammatory phase (Mester et al., 1985) and stimulates early formation of collagen through improving tissue organization and promoting greater numbers of cells for synthesis of extracellular material (Amaral et al., 2001), as well as, reducing the formation of ROS (reactive
oxygen species) to act beneficially on oxidative processes that involve the activity of enzymes as catalase and superoxide dismutase (Silveira et al., 2009). In osteogenesis, low-power laser also acts to improve the mechanical properties of the bone callus (Lurger et al., 1998), through increasing the number of osteoblasts (Freitas et al., 2000; Stein et al., 2005; Lirani-Galvão, 2006) and fibroblasts in the callus at the fracture (Trelles and Mayayo, 1987) and thus stimulating bone matrix synthesis (Freitas et al., 2000). Despite many studies, the effects of the wavelength, beam type, energy level and intensity and the exposure regimen of the therapeutic laser continue to be unexplained (Coombe et al., 2001; Marino et al., 2003). In addition, there are no specific dosimetry and mechanism of action determinations for different cell types (Coombe et al., 2001). According to Silva et al. (2010), the various application protocols and different materials and wavelengths that have been used make it difficult to compare the results and to choose a therapeutic protocol. Within this context, the present study aimed to evaluate the therapeutic activity of low-power laser (InGaAlP: 670 nm/30 mW), at doses of 90 J/cm2, on the process of acute and chronic-phase repair of bone lesions of Wistar rats. 2. Material and methods 2.1. Site and animals
⇑ Corresponding author. Address: Av. Hilário da Silva Passos 950, Parque Universitário, 13690-000 Descalvado, SP, Brazil. Tel.: +55 1935958500. E-mail address: [email protected] (M.A.A. Belo). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.07.009
This study was conducted in the Pharmacology and Animal Toxicology Laboratory of UNICASTELO, in Descalvado, SP, in
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accordance with experimental protocol No. 2466-2686/09, which had been approved by the institution’s Ethics Committee. Seventy 12-month-old male Wistar rats of mean weight 300 g were used. They were kept in polypropylene boxes that were lined with wood shavings, at room temperature with continual air circulation and under natural light and dark periods, and they received water and animal feed ad libitum. 2.2. Experimental design Sixty-three adult males were randomly divided into three groups (n = 21) and were subjected to a full-depth bone injury of 2 mm in diameter, which was created in the proximal portion of the tibia, in both pelvic limbs. Thus, the following treatment groups were formed: T1 = control animals, which received the application protocol with the equipment switched off; T2 = animals whose treatment started 24 h after the surgical procedure (acute phase); T3 = animals whose treatment started on the fifth day after surgery (chronic phase). A fourth experimental group of seven animals (T4) was formed to serve as a physiological standard for the serum alkaline phosphatase biochemical analysis. These animals did not undergo any injury or treatment. The treatments T1, T2 and T3 were subdivided into three subgroups of seven animals each, which were analyzed on the 9th, 17th and 28th days after surgery. 2.3. Surgical procedures The animals were anesthetized using an association of 10% ketamine hydrochloride (0.2 ml/kg) and 2% xylazine hydrochloride (0.07 ml/kg), applied intramuscularly. Ten minutes after the anesthesia had been applied, the medial region of the animals’ hind legs (both limbs) were shaved, including the knee joint. With the animals placed in dorsal decubitus, antisepsis was performed using 2% polyvinylpyrrolidone followed by 3% iodine/alcohol. The presence of analgesia was verified and then a longitudinal incision was made in the medial face of the leg, in the proximal region of the tibia. The musculature of this region was pushed aside with the aid of anatomical forceps, so as to expose the bone tissue. Following this, with the aid of a drill bit coupled to a slow-rotation mini-drill, under constant irrigation with physiological solution, a full-depth bone failure of 2 mm in diameter was created around 8 mm distally from the knee joint, in both pelvic limbs. After inspection of the osteotomized region and the adjacent soft tissues, the area was cleaned with physiological solution to remove detritus. The skin and musculature were then brought together by means of mattress sutures, using 3-0 nylon thread. The postoperative care was done only in the wound area using a solution of sodium chloride at 0.9%. No single medication has been carried out in this period, in order to avoid interferences in the results, mainly because the laser therapy provides anti-inflammatory activity.
2.5. Histopathology All the animals were sacrificed at the end of the respective number of days of observation (9, 17 or 28 days after surgery), by means of an overdose of sodium thiopental intraperitoneally, as recommended by the American Veterinary Medical Association (2001) for scientific research animals. After sacrifice, tibias of the animals were disconnected at knee level and tarsal level. The right tibias were sent for histological analysis and the left knees for biomechanical analysis. The specimens destined for histological examination underwent decalcification by immersing them in 8% trichloroacetic acid and passing an electric current through them, as described by Gonçalves and Oliverio (1965), and were then processed using a routine technique. The histological thin sections were stained using hematoxylin-eosin and Mallory’s trichrome. A blinded experienced pathologist performed histopathologic analyses. 2.6. Biochemical analysis Immediately after the animals had been sacrificed, 2 ml of blood was collected from the abdominal aortic artery, without anti-coagulant. After centrifuging the blood samples (5000 rpm/10 min), the animals’ serum was separated to determine the serum alkaline phosphatase levels. This was done in a semi-automated biochemical analyzer (LabQuest). 2.7. Biomechanical assay The tibias destined for mechanical testing were cleaned and soaked in 0.9% physiological solution, and were then frozen until the time of the mechanical tests. These tests were performed on a universal testing machine (EMICÒ, model DL10000). The tibias were subjected to mechanical flexion tests at three points. The load was applied to the tibia transversally, on its posterior face, by means of an upper pin above a brass accessory, with spacing of 10 mm between the supports. This distance was determined such that the best coupling between the tibia and the accessory would be achieved, and so that the application force would fall on the fracture site. The tests were carried out using a load cell of 50 kgf. The speed used for applying the load was 1 mm/min, with a preload of 5 N and accommodation time of 30 s, for all the tests. The values recorded by the universal testing machine were transcribed for the Tesq software version 1.0, which was used to calculate the mechanical properties of maximum strain and stiffness. 2.8. Statistical analysis The data were subjected to analysis of variance (ANOVA), after ascertaining that the data distribution was normal, using the GraphPad PrismÒ software, version 5.0. Multiple comparisons were measured using Tukey’s test with a 95% confidence interval, in accordance with Snedecor and Cochran (1980).
2.4. Laser equipment and treatment protocols 3. Results BIOSETÒ laser equipment (Phisiolux Dual P. 4050) was set up as a low-power indium-gallium-aluminum-phosphorus (InGaAlP) laser source, with a wavelength of 670 nm and power of 30 mW (continuous). This was applied at doses of 90 J/cm2 for a period of 4 min and 33 s on each limb that had undergone the operation. The animals in both laser-treated groups (T2 and T3) were each subjected to 4, 9 or 19 sessions, depending on the length of the scheduled observation period. The animals with acute-phase treatment (T2) and chronic-phase (T3) initiated the lasertherapy 24 h and 5 days after surgery, respectively, always in accordance with the order of the surgical procedure.
3.1. Histological analysis The control group animals (T1) that were examined on the 9th day after the operation showed the presence of bone fragments and blood coagulum that were undergoing reabsorption, at the center of the lesion. Peripherally, it was noted that the connective tissue was highly cellular, with the presence of fibroblasts, macrophages, osteoclasts and osteoblasts. All the animals in this group presented the beginnings of an intramembranous ossification process and small areas of endochondral ossification, i.e. hypertrophy
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of chondrocytes and ossification of the cartilaginous matrix (Fig. 1A). On the 17th day after surgery, the general picture presented by the control rats (T1) consisted of intense invasion of dense connective tissue inside the lesion, with little formation of bone tissue. This came from the periphery, in intramembranous form (Fig. 2A). On the 28th day after surgery, the lesion was seen to be completely filled with bone tissue, with the characteristics of endochondral ossification. In some of the animals, small areas of dense connective tissue could be noted (Fig. 3A). The animals with acute-phase treatment (T2) examined on the 9th day after surgery presented bone fragments and coagulum at the center of the lesion, undergoing reabsorption, i.e. resembling
the control group at the same observation time. However, later on, both the center of the lesion and the periphery presented intense areas of ossification, basically of endochondral type (Fig. 1B). On the 17th day after surgery, the T2 rats presented complete filling of the lesion with endochondral bone (Fig. 2B). While non-treated animals showed that the lesion had been completely filled with bone on the 28th day after surgery (Fig. 3B). The rats with chronic-phase treatment (T3) examined on the 9th day after surgery presented results that were very similar to those of the control group at this observation time, with the beginnings of an intramembranous ossification process and small areas of endochondral ossification (Fig. 1C).
Fig. 1. Histological appearance (longitudinal cut) of the tissue filling in the osteotomy in the animals analyzed on the 9th day after surgery. (A) T1 (control group), (B) T2 (underwent laser therapy 24 h after receiving the injury) and (C) T3 (underwent laser therapy 5 days after receiving the injury). Note the presence of connective tissue (+), bone fragments undergoing reabsorption {}, areas of endochondral ossification (arrow) and blood coagulum undergoing reabsorption (N). (50X – Mallory’s trichrome).
Fig. 2. Histological appearance (longitudinal cut) of tissue filling in the osteotomy in the animals analyzed on the 17th day after surgery. (A) T1 (control group), with intense invasion of dense connective tissue. (B) T2 (underwent laser therapy 24 h after receiving the injury), area completely filled with endochondral bone {}. (C) T3 (underwent laser therapy 5 days after receiving the injury), small areas of endochondral ossification (arrow) and intense invasion of dense connective tissue (+). (50X – Mallory’s trichrome).
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served that there was a significant increase (P < 0.05) in the circulating serum levels of this enzyme in the animals treated with laser (T2 and T3), compared with the control group rats (T1). However, the serum alkaline phosphatase analysis performed on the 17th and 28th days after surgery revealed alkaline phosphatase levels that were not significantly higher (P > 0.05) in the animals treated with laser. The highest mean values measured at each analysis time (305 and 315.57, respectively) were observed in the animals that underwent acute-phase treatment (T2). Fig. 4 clearly demonstrates the effect of the bone repair process on the serum alkaline phosphatase levels. It can be seen that there was significantly greater serum activity of this enzyme (P < 0.05) in all the groups that were subjected to bone injury (T1, T2 and T3), compared with the standard values for animals that were not treated and not subjected to any surgical procedure (T4), at all the times analyzed. In relation to the maximum strain (Table 1), no statistically significant difference between the treatments was observed. However, administration of laser treatment during the acute phase (T2) resulted in mean increases in the maximum strain values in the evaluations carried out on the 9th and 17th days after surgery (Table 1). With regard to the mechanical property of stiffness, no significant differences were observed (P > 0.05) in comparisons between the treatments at any time during the experimental period. However, it was observed that the laser treatment had an effect on this biomechanical property, resulting in a gradual increase (P < 0.05) in bone stiffness between the analysis times. The increase was most significant in the animals that started to receive treatment during the acute phase of the injury (T2) (Table 1).
4. Discussion
Fig. 3. Histological appearance (longitudinal cut) of tissue filling in the osteotomy in the animals analyzed on the 28th day after the surgery. (A) T1 (control group); (B) T2 (underwent laser therapy 24 h after receiving the injury); (C) T3 (underwent laser therapy 5 days after receiving the injury). All presented the lesion completely filled in with endochondral bone {}. (50X – Mallory’s trichrome).
Most of the animals that started to receive treatment in the chronic phase (T3) and which were examined on the 17th day after surgery presented areas of endochondral ossification and dense connective tissue at the lesion site. There was more connective tissue than ossification (Fig. 2C). However, on the 28th day after surgery, these animals presented a bone repair response that was similar to that of the rats with acute-phase treatment (T2), with the lesion completely filled with bone. At this time it was not possible to distinguish between the treatment types (Fig. 3C). 3.2. Biochemical and biomechanical analysis The mean alkaline phosphatase values are presented in Fig. 4. In the analysis carried out on the 9th day after surgery, it was ob-
Although the histopathological analysis performed on the 9th day after induction of the bone injury presented similar results in all three experimental groups, intense areas of endochondral ossification were observed at the center and the periphery of the lesions in the animals that started to receive treatment during the acute phase. These findings were accompanied by higher levels of circulating alkaline phosphatase and higher mean values in the biomechanical test on maximum strain. In the same period, only non-treated animals still showed resorption of dead bone tissue and presence of phagocytic cells such as osteoclasts. Formed by the fusion of cells of the monocyte-macrophage cell line (Väänänen et al., 2000), osteoclasts are multinucleated cells found in the initial phase of bone repair by removing its mineralized matrix and breaking up the organic bone. One hypothesis that can explain these observations is that the low-power laser may have modulated the local inflammatory process, thereby promoting faster reabsorption of exudates and also increasing the level of phagocytic activity (Marino et al., 2003; Nicola et al., 2003). These results became more evident with the evolution of the tissue repair process, since greater bone formation was found on the 17th day after surgery, in both groups of rats with laser treatment compared with the control animals. However, the rats that started to receive treatment during the acute phase presented lesions that had been completely filled with endochondral bone, while the animals that started to receive treatment during the chronic phase presented lesions with areas of both endochondral ossification and dense connective tissue, with greater quantities of the latter. This difference probably occurred because the laser acted by stimulating proliferation or differentiation of osteoprogenitor mesenchymal cells that were still immature, but without any effect in areas where mature cells that had already become differentiated predominated (Marino et al., 2003).
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Fig. 4. Mean values and statistical analysis of variance1 for serum alkaline phosphatase levels observed in Wistar rats subjected to the different treatments. 1Means followed by the same letter did not differ according to Tukey’s test (P > 0.05). Upper-case letters compare the treatments on each day analyzed and lower-case letters compare each treatment on the different days analyzed.
Table 1 Mean values (and their respective standard deviations) and statistical analysis of variancea for the biomechanical test. Biomechanical parameter
Treatmentb
9
17
28
Maximum strain (N/m2)
T1 T2 T3
3419331 ± 129517Aa 3817048 ± 165110Aa 3496061 ± 235463Aa
3889131 ± 177548Aa 4133334 ± 190551Aa 3610022 ± 566372Aa
4893996 ± 604780Aa 4995820 ± 557305Aa 5105094 ± 161281Aa
Stiffness (N/mm)
T1 T2 T3
284.80 ± 63.78Aa 286.05 ± 9.42Aa 282.09 ± 24.59Aa
300.29 ± 52.47Aa 324.85 ± 12.40Ab 295.75 ± 9.56Aa
365.19 ± 33.14Aa 372.87 ± 14.51Ac 379.67 ± 57.63Ab
Analysis time (days after surgery)
a Means followed by the same letter did not differ according to Tukey’s test (P > 0.05). Upper-case letters compare the treatments on each day analyzed and lower-case letters compare each treatment on the different days analyzed. b T1 = control; T2 = acute fase; T3 = chronic fase.
On the 17th day, the biochemical analysis on serum alkaline phosphatase and the biomechanical test on maximum strain presented the same trend as observed on the 9th day of evolution of the regenerative process, thus corroborating the histopathological findings. In the literature, other studies have demonstrated the therapeutic activity of low-power laser in bone reabsorption and formation in fractures (Nicola et al., 2003), through direct stimulation of osteoid formation (Lirani-Galvão, 2006) and increased numbers of osteoblasts, as well as activation of bone matric synthesis (Freitas et al., 2000; Lirani-Galvão, 2006). Through this, the incorporation of intracellular calcium is increased (Coombe et al., 2001). The activity of alkaline phosphatase in bones is not fully known, but it has been demonstrated that this enzyme is essential for normal bone formation (Fedde et al., 1999; Knoch et al., 2005). Its presence is indicative of the activity of osteoblasts and osteoclasts (Garnero and Delmas, 1988), and it is considered to be a good marker for bone formation and a guide for the degree of osteoblast activity (Brito, 2004). In this study, the alkaline phosphatase levels increased during the bone healing process, as a result of the stimulus from the injury, in comparison with the physiological standard. However, these values were greater in the groups that underwent laser therapy. These findings suggest the hypothesis that the observed increase in the serum levels of this enzyme are associated with a more advanced stage of fracture repair in the
treated groups, which presented greater areas of ossification than seen in the control rats. In this phase, the laser modulates the inflammatory process, thus altering the activity level and the numbers of osteoblasts and osteoclasts, which is reflected in the alkaline phosphatase activity (Barushka et al., 1995). Throughout the study, higher serum activity values for alkaline phosphatase were observed in the animals treated with laser during the acute phase of the lesion (T2). This reveals that there was greater activity of osteoblasts promoted by laser therapy action, and this was confirmed through the histopathological findings and the biomechanical test. Treatment with laser during the acute phase resulted in higher maximum strain values in the analyses on the 9th and 17th days and greater stiffness throughout the trial, compared with the other treatments. Although these findings were not statistically significant, these results seem to indicate that laser treatment at the initial stage of bone healing favors the consolidation process, associated with endochondral ossification in the lesions of the animals that were analyzed on the 9th day after surgery, and complete filling of the lesion with endochondral bone on the 17th day. According to Lirani-Galvão (2006), bones with lower quantities of fibrocartilaginous tissue and greater ossification tend to be stronger. Low-power laser therapy (InGaAlP: 670 nm/30 mW) at doses of 90 J/cm2 was shown to aid in the bone consolidation
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process in lesions induced at the proximal extremity of the tibia in Wistar rats, and these effects were more significant when the treatment was started during the acute phase of the tissue repair process. Conflict of interest statement We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this research that could have influenced its outcome. The manuscript has been read and approved by all named authors and there are no other persons who satisfied the criteria for authorship but are not listed. The order of authors listed in the manuscript has been approved by all of us. Acknowledgments The authors are grateful for BIOSET company for providing the equipment Phisiolux Dual Laser P.4050 and the technical help of Luana Fatoretto during this experimental trial. This research was sponsored by Camilo Castelo Branco University. References Amaral, A.C., Parizotto, N.A., Salvani, T.F., 2001. Dose-dependency of low-energy He–Ne Laser effect in regeneration of skeletal muscle in mice. Lasers in Medical Science 16, 44–51. American Veterinary Medical Association, 2001. Report of the AVMA panel on euthanasia. Journal of American Veterinary Medical Association 218, 669. Barushka, O., Yaakobi, T., Oron, U., 1995. Effect of low energy (He–Ne) irradiation on the process of bone repair in the rat tibia. Bone 16, 47–55. Brito, M.A.P., 2004. Analysis with the use of spectrophotometry in bone repair by laser biomodulation. Dissertation (Masters in Biomedical Engineering) – Research and Development Institute, University of Vale do Paraíba, 65p. Coombe, A.R., Ho, C.T., Darendeliler, M.A., Hunter, N., Philips, J.R., Chapple, C.C., Yum, L.W., 2001. The effects of low level laser irradiation on osteoblastic cells. Clinical Orthopaedics and Related Research 4, 3–14. Fávaro-Pípi, E., Feitosa, S.M., Ribeiro, A., Bossini, P., Oliveira, P., Parizotto, N.A., Renno, A.C.M., 2010. Comparative study of the effects of low-intensity pulsed ultrasound and low-level laser therapy on bone defects in tibias of rats. Lasers in Medical Science 25, 727–732. Fedde, K.N.N., Blair, L., Silverstein, J., Coburn, S.P., Ryan, L.M., Weinstein, R.S., Waymire, K., Narisawa, S., Millan, J.L., Macgregor, G.R., Whyte, M.P., 1999. Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. Journal of Bone and Mineral Research 14, 2015–2026. Freitas, I.G.F., Baranauskas, V., Cruz-Höfling, M.A., 2000. Laser effects on osteogenesis. Applied Surface Science 154–155, 548–554. Garnero, P., Delmas, P.D., 1988. Biochemecal markes of bone turnover: applications for osteoporosis. Endocrinology and Metabolism Clinics of North America 27, 303–323.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Low-power laser therapy for repairing acute and chronic-phase bone lesions F.C.D. Mota a, M.A.A. Belo b,⇑, M.E. Beletti c, R. Okubo d, E.J.R. Prado b, R.V.P. Casale b a
College of Veterinary Medicine, Federal University of Uberlândia (UFU), Uberlândia, MG, Brazil Pharmacology and Clinical Pathology Laboratory, Camilo Castelo Branco University (UNICASTELO), Descalvado, SP, Brazil c Institute of Biomedical Sciences, Federal University of Uberlândia (UFU), Uberlândia, MG, Brazil d Bioengineering Laboratory, University of São Paulo (USP), Ribeirão Preto, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 18 January 2012 Accepted 3 July 2012
Keywords: Low-power laser therapy Fracture consolidation Osteotomy Rat
a b s t r a c t To evaluate the therapeutic activity of low-power laser (InGaAlP: 670 nm/30 mW), at doses of 90 J/cm2, on the process of acute and chronic-phase repair of bone lesions of Wistar rats. Sixty-three adult males were divided into nine groups subjected to bone injury, in order to form the following treatments: T1 (control); T2 (acute-phase); T3 (chronic-phase) which were subdivided into three subgroups (n = 7), analyzed on the 9th, 17th and 28th days post-surgery, after a period of daily treatment with laser. The animals with acute-phase treatment presented a more extensive endochondral ossification process. Laser-treated animals showed significant increases in serum alkaline phosphatase levels and had an effect on biomechanical property, resulting in a gradual increase in bone stiffness. Laser therapy aided the bone consolidation process and favored the physiopathologic mechanisms involved in bone tissue repair, and its effects were more prominent when treatment started during the acute phase of the injury. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Non-consolidation of bone defects is a major challenge for healthcare professionals in different fields (Nascimento et al., 2010). It has been described as one of the conditions leading to the development of pseudoarthrosis and this, in turn, may be responsible for complications that, even after applying modern surgical techniques, do not allow patients to achieve a cure (Nolte et al., 2001). Low-power laser therapy is an alternative treatment that favors regenerative processes in biological tissues, such as in fracture repairs (Guzzardella et al., 2001; Brito, 2004; Javadieh et al., 2009; Fávaro-Pípi et al., 2010), skin lesions (Lucas et al., 2003), nerve tissue (Padua et al., 1999) and muscle tissue (Amaral et al., 2001). It is believed that the irradiation from low-power lasers act on cell components that are sensitive to certain wavelengths, such that photons are absorbed. These trigger chemical reactions, especially in mitochondria, lysosomes and membranes, thereby modifying ion transportation (Karu, 1987) and causing increased levels of ATP and prostaglandins. This contributes towards partial acceleration of the tissue repair during the inflammatory phase (Mester et al., 1985) and stimulates early formation of collagen through improving tissue organization and promoting greater numbers of cells for synthesis of extracellular material (Amaral et al., 2001), as well as, reducing the formation of ROS (reactive
oxygen species) to act beneficially on oxidative processes that involve the activity of enzymes as catalase and superoxide dismutase (Silveira et al., 2009). In osteogenesis, low-power laser also acts to improve the mechanical properties of the bone callus (Lurger et al., 1998), through increasing the number of osteoblasts (Freitas et al., 2000; Stein et al., 2005; Lirani-Galvão, 2006) and fibroblasts in the callus at the fracture (Trelles and Mayayo, 1987) and thus stimulating bone matrix synthesis (Freitas et al., 2000). Despite many studies, the effects of the wavelength, beam type, energy level and intensity and the exposure regimen of the therapeutic laser continue to be unexplained (Coombe et al., 2001; Marino et al., 2003). In addition, there are no specific dosimetry and mechanism of action determinations for different cell types (Coombe et al., 2001). According to Silva et al. (2010), the various application protocols and different materials and wavelengths that have been used make it difficult to compare the results and to choose a therapeutic protocol. Within this context, the present study aimed to evaluate the therapeutic activity of low-power laser (InGaAlP: 670 nm/30 mW), at doses of 90 J/cm2, on the process of acute and chronic-phase repair of bone lesions of Wistar rats. 2. Material and methods 2.1. Site and animals
⇑ Corresponding author. Address: Av. Hilário da Silva Passos 950, Parque Universitário, 13690-000 Descalvado, SP, Brazil. Tel.: +55 1935958500. E-mail address: [email protected] (M.A.A. Belo). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.07.009
This study was conducted in the Pharmacology and Animal Toxicology Laboratory of UNICASTELO, in Descalvado, SP, in
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accordance with experimental protocol No. 2466-2686/09, which had been approved by the institution’s Ethics Committee. Seventy 12-month-old male Wistar rats of mean weight 300 g were used. They were kept in polypropylene boxes that were lined with wood shavings, at room temperature with continual air circulation and under natural light and dark periods, and they received water and animal feed ad libitum. 2.2. Experimental design Sixty-three adult males were randomly divided into three groups (n = 21) and were subjected to a full-depth bone injury of 2 mm in diameter, which was created in the proximal portion of the tibia, in both pelvic limbs. Thus, the following treatment groups were formed: T1 = control animals, which received the application protocol with the equipment switched off; T2 = animals whose treatment started 24 h after the surgical procedure (acute phase); T3 = animals whose treatment started on the fifth day after surgery (chronic phase). A fourth experimental group of seven animals (T4) was formed to serve as a physiological standard for the serum alkaline phosphatase biochemical analysis. These animals did not undergo any injury or treatment. The treatments T1, T2 and T3 were subdivided into three subgroups of seven animals each, which were analyzed on the 9th, 17th and 28th days after surgery. 2.3. Surgical procedures The animals were anesthetized using an association of 10% ketamine hydrochloride (0.2 ml/kg) and 2% xylazine hydrochloride (0.07 ml/kg), applied intramuscularly. Ten minutes after the anesthesia had been applied, the medial region of the animals’ hind legs (both limbs) were shaved, including the knee joint. With the animals placed in dorsal decubitus, antisepsis was performed using 2% polyvinylpyrrolidone followed by 3% iodine/alcohol. The presence of analgesia was verified and then a longitudinal incision was made in the medial face of the leg, in the proximal region of the tibia. The musculature of this region was pushed aside with the aid of anatomical forceps, so as to expose the bone tissue. Following this, with the aid of a drill bit coupled to a slow-rotation mini-drill, under constant irrigation with physiological solution, a full-depth bone failure of 2 mm in diameter was created around 8 mm distally from the knee joint, in both pelvic limbs. After inspection of the osteotomized region and the adjacent soft tissues, the area was cleaned with physiological solution to remove detritus. The skin and musculature were then brought together by means of mattress sutures, using 3-0 nylon thread. The postoperative care was done only in the wound area using a solution of sodium chloride at 0.9%. No single medication has been carried out in this period, in order to avoid interferences in the results, mainly because the laser therapy provides anti-inflammatory activity.
2.5. Histopathology All the animals were sacrificed at the end of the respective number of days of observation (9, 17 or 28 days after surgery), by means of an overdose of sodium thiopental intraperitoneally, as recommended by the American Veterinary Medical Association (2001) for scientific research animals. After sacrifice, tibias of the animals were disconnected at knee level and tarsal level. The right tibias were sent for histological analysis and the left knees for biomechanical analysis. The specimens destined for histological examination underwent decalcification by immersing them in 8% trichloroacetic acid and passing an electric current through them, as described by Gonçalves and Oliverio (1965), and were then processed using a routine technique. The histological thin sections were stained using hematoxylin-eosin and Mallory’s trichrome. A blinded experienced pathologist performed histopathologic analyses. 2.6. Biochemical analysis Immediately after the animals had been sacrificed, 2 ml of blood was collected from the abdominal aortic artery, without anti-coagulant. After centrifuging the blood samples (5000 rpm/10 min), the animals’ serum was separated to determine the serum alkaline phosphatase levels. This was done in a semi-automated biochemical analyzer (LabQuest). 2.7. Biomechanical assay The tibias destined for mechanical testing were cleaned and soaked in 0.9% physiological solution, and were then frozen until the time of the mechanical tests. These tests were performed on a universal testing machine (EMICÒ, model DL10000). The tibias were subjected to mechanical flexion tests at three points. The load was applied to the tibia transversally, on its posterior face, by means of an upper pin above a brass accessory, with spacing of 10 mm between the supports. This distance was determined such that the best coupling between the tibia and the accessory would be achieved, and so that the application force would fall on the fracture site. The tests were carried out using a load cell of 50 kgf. The speed used for applying the load was 1 mm/min, with a preload of 5 N and accommodation time of 30 s, for all the tests. The values recorded by the universal testing machine were transcribed for the Tesq software version 1.0, which was used to calculate the mechanical properties of maximum strain and stiffness. 2.8. Statistical analysis The data were subjected to analysis of variance (ANOVA), after ascertaining that the data distribution was normal, using the GraphPad PrismÒ software, version 5.0. Multiple comparisons were measured using Tukey’s test with a 95% confidence interval, in accordance with Snedecor and Cochran (1980).
2.4. Laser equipment and treatment protocols 3. Results BIOSETÒ laser equipment (Phisiolux Dual P. 4050) was set up as a low-power indium-gallium-aluminum-phosphorus (InGaAlP) laser source, with a wavelength of 670 nm and power of 30 mW (continuous). This was applied at doses of 90 J/cm2 for a period of 4 min and 33 s on each limb that had undergone the operation. The animals in both laser-treated groups (T2 and T3) were each subjected to 4, 9 or 19 sessions, depending on the length of the scheduled observation period. The animals with acute-phase treatment (T2) and chronic-phase (T3) initiated the lasertherapy 24 h and 5 days after surgery, respectively, always in accordance with the order of the surgical procedure.
3.1. Histological analysis The control group animals (T1) that were examined on the 9th day after the operation showed the presence of bone fragments and blood coagulum that were undergoing reabsorption, at the center of the lesion. Peripherally, it was noted that the connective tissue was highly cellular, with the presence of fibroblasts, macrophages, osteoclasts and osteoblasts. All the animals in this group presented the beginnings of an intramembranous ossification process and small areas of endochondral ossification, i.e. hypertrophy
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of chondrocytes and ossification of the cartilaginous matrix (Fig. 1A). On the 17th day after surgery, the general picture presented by the control rats (T1) consisted of intense invasion of dense connective tissue inside the lesion, with little formation of bone tissue. This came from the periphery, in intramembranous form (Fig. 2A). On the 28th day after surgery, the lesion was seen to be completely filled with bone tissue, with the characteristics of endochondral ossification. In some of the animals, small areas of dense connective tissue could be noted (Fig. 3A). The animals with acute-phase treatment (T2) examined on the 9th day after surgery presented bone fragments and coagulum at the center of the lesion, undergoing reabsorption, i.e. resembling
the control group at the same observation time. However, later on, both the center of the lesion and the periphery presented intense areas of ossification, basically of endochondral type (Fig. 1B). On the 17th day after surgery, the T2 rats presented complete filling of the lesion with endochondral bone (Fig. 2B). While non-treated animals showed that the lesion had been completely filled with bone on the 28th day after surgery (Fig. 3B). The rats with chronic-phase treatment (T3) examined on the 9th day after surgery presented results that were very similar to those of the control group at this observation time, with the beginnings of an intramembranous ossification process and small areas of endochondral ossification (Fig. 1C).
Fig. 1. Histological appearance (longitudinal cut) of the tissue filling in the osteotomy in the animals analyzed on the 9th day after surgery. (A) T1 (control group), (B) T2 (underwent laser therapy 24 h after receiving the injury) and (C) T3 (underwent laser therapy 5 days after receiving the injury). Note the presence of connective tissue (+), bone fragments undergoing reabsorption {}, areas of endochondral ossification (arrow) and blood coagulum undergoing reabsorption (N). (50X – Mallory’s trichrome).
Fig. 2. Histological appearance (longitudinal cut) of tissue filling in the osteotomy in the animals analyzed on the 17th day after surgery. (A) T1 (control group), with intense invasion of dense connective tissue. (B) T2 (underwent laser therapy 24 h after receiving the injury), area completely filled with endochondral bone {}. (C) T3 (underwent laser therapy 5 days after receiving the injury), small areas of endochondral ossification (arrow) and intense invasion of dense connective tissue (+). (50X – Mallory’s trichrome).
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served that there was a significant increase (P < 0.05) in the circulating serum levels of this enzyme in the animals treated with laser (T2 and T3), compared with the control group rats (T1). However, the serum alkaline phosphatase analysis performed on the 17th and 28th days after surgery revealed alkaline phosphatase levels that were not significantly higher (P > 0.05) in the animals treated with laser. The highest mean values measured at each analysis time (305 and 315.57, respectively) were observed in the animals that underwent acute-phase treatment (T2). Fig. 4 clearly demonstrates the effect of the bone repair process on the serum alkaline phosphatase levels. It can be seen that there was significantly greater serum activity of this enzyme (P < 0.05) in all the groups that were subjected to bone injury (T1, T2 and T3), compared with the standard values for animals that were not treated and not subjected to any surgical procedure (T4), at all the times analyzed. In relation to the maximum strain (Table 1), no statistically significant difference between the treatments was observed. However, administration of laser treatment during the acute phase (T2) resulted in mean increases in the maximum strain values in the evaluations carried out on the 9th and 17th days after surgery (Table 1). With regard to the mechanical property of stiffness, no significant differences were observed (P > 0.05) in comparisons between the treatments at any time during the experimental period. However, it was observed that the laser treatment had an effect on this biomechanical property, resulting in a gradual increase (P < 0.05) in bone stiffness between the analysis times. The increase was most significant in the animals that started to receive treatment during the acute phase of the injury (T2) (Table 1).
4. Discussion
Fig. 3. Histological appearance (longitudinal cut) of tissue filling in the osteotomy in the animals analyzed on the 28th day after the surgery. (A) T1 (control group); (B) T2 (underwent laser therapy 24 h after receiving the injury); (C) T3 (underwent laser therapy 5 days after receiving the injury). All presented the lesion completely filled in with endochondral bone {}. (50X – Mallory’s trichrome).
Most of the animals that started to receive treatment in the chronic phase (T3) and which were examined on the 17th day after surgery presented areas of endochondral ossification and dense connective tissue at the lesion site. There was more connective tissue than ossification (Fig. 2C). However, on the 28th day after surgery, these animals presented a bone repair response that was similar to that of the rats with acute-phase treatment (T2), with the lesion completely filled with bone. At this time it was not possible to distinguish between the treatment types (Fig. 3C). 3.2. Biochemical and biomechanical analysis The mean alkaline phosphatase values are presented in Fig. 4. In the analysis carried out on the 9th day after surgery, it was ob-
Although the histopathological analysis performed on the 9th day after induction of the bone injury presented similar results in all three experimental groups, intense areas of endochondral ossification were observed at the center and the periphery of the lesions in the animals that started to receive treatment during the acute phase. These findings were accompanied by higher levels of circulating alkaline phosphatase and higher mean values in the biomechanical test on maximum strain. In the same period, only non-treated animals still showed resorption of dead bone tissue and presence of phagocytic cells such as osteoclasts. Formed by the fusion of cells of the monocyte-macrophage cell line (Väänänen et al., 2000), osteoclasts are multinucleated cells found in the initial phase of bone repair by removing its mineralized matrix and breaking up the organic bone. One hypothesis that can explain these observations is that the low-power laser may have modulated the local inflammatory process, thereby promoting faster reabsorption of exudates and also increasing the level of phagocytic activity (Marino et al., 2003; Nicola et al., 2003). These results became more evident with the evolution of the tissue repair process, since greater bone formation was found on the 17th day after surgery, in both groups of rats with laser treatment compared with the control animals. However, the rats that started to receive treatment during the acute phase presented lesions that had been completely filled with endochondral bone, while the animals that started to receive treatment during the chronic phase presented lesions with areas of both endochondral ossification and dense connective tissue, with greater quantities of the latter. This difference probably occurred because the laser acted by stimulating proliferation or differentiation of osteoprogenitor mesenchymal cells that were still immature, but without any effect in areas where mature cells that had already become differentiated predominated (Marino et al., 2003).
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Fig. 4. Mean values and statistical analysis of variance1 for serum alkaline phosphatase levels observed in Wistar rats subjected to the different treatments. 1Means followed by the same letter did not differ according to Tukey’s test (P > 0.05). Upper-case letters compare the treatments on each day analyzed and lower-case letters compare each treatment on the different days analyzed.
Table 1 Mean values (and their respective standard deviations) and statistical analysis of variancea for the biomechanical test. Biomechanical parameter
Treatmentb
9
17
28
Maximum strain (N/m2)
T1 T2 T3
3419331 ± 129517Aa 3817048 ± 165110Aa 3496061 ± 235463Aa
3889131 ± 177548Aa 4133334 ± 190551Aa 3610022 ± 566372Aa
4893996 ± 604780Aa 4995820 ± 557305Aa 5105094 ± 161281Aa
Stiffness (N/mm)
T1 T2 T3
284.80 ± 63.78Aa 286.05 ± 9.42Aa 282.09 ± 24.59Aa
300.29 ± 52.47Aa 324.85 ± 12.40Ab 295.75 ± 9.56Aa
365.19 ± 33.14Aa 372.87 ± 14.51Ac 379.67 ± 57.63Ab
Analysis time (days after surgery)
a Means followed by the same letter did not differ according to Tukey’s test (P > 0.05). Upper-case letters compare the treatments on each day analyzed and lower-case letters compare each treatment on the different days analyzed. b T1 = control; T2 = acute fase; T3 = chronic fase.
On the 17th day, the biochemical analysis on serum alkaline phosphatase and the biomechanical test on maximum strain presented the same trend as observed on the 9th day of evolution of the regenerative process, thus corroborating the histopathological findings. In the literature, other studies have demonstrated the therapeutic activity of low-power laser in bone reabsorption and formation in fractures (Nicola et al., 2003), through direct stimulation of osteoid formation (Lirani-Galvão, 2006) and increased numbers of osteoblasts, as well as activation of bone matric synthesis (Freitas et al., 2000; Lirani-Galvão, 2006). Through this, the incorporation of intracellular calcium is increased (Coombe et al., 2001). The activity of alkaline phosphatase in bones is not fully known, but it has been demonstrated that this enzyme is essential for normal bone formation (Fedde et al., 1999; Knoch et al., 2005). Its presence is indicative of the activity of osteoblasts and osteoclasts (Garnero and Delmas, 1988), and it is considered to be a good marker for bone formation and a guide for the degree of osteoblast activity (Brito, 2004). In this study, the alkaline phosphatase levels increased during the bone healing process, as a result of the stimulus from the injury, in comparison with the physiological standard. However, these values were greater in the groups that underwent laser therapy. These findings suggest the hypothesis that the observed increase in the serum levels of this enzyme are associated with a more advanced stage of fracture repair in the
treated groups, which presented greater areas of ossification than seen in the control rats. In this phase, the laser modulates the inflammatory process, thus altering the activity level and the numbers of osteoblasts and osteoclasts, which is reflected in the alkaline phosphatase activity (Barushka et al., 1995). Throughout the study, higher serum activity values for alkaline phosphatase were observed in the animals treated with laser during the acute phase of the lesion (T2). This reveals that there was greater activity of osteoblasts promoted by laser therapy action, and this was confirmed through the histopathological findings and the biomechanical test. Treatment with laser during the acute phase resulted in higher maximum strain values in the analyses on the 9th and 17th days and greater stiffness throughout the trial, compared with the other treatments. Although these findings were not statistically significant, these results seem to indicate that laser treatment at the initial stage of bone healing favors the consolidation process, associated with endochondral ossification in the lesions of the animals that were analyzed on the 9th day after surgery, and complete filling of the lesion with endochondral bone on the 17th day. According to Lirani-Galvão (2006), bones with lower quantities of fibrocartilaginous tissue and greater ossification tend to be stronger. Low-power laser therapy (InGaAlP: 670 nm/30 mW) at doses of 90 J/cm2 was shown to aid in the bone consolidation
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process in lesions induced at the proximal extremity of the tibia in Wistar rats, and these effects were more significant when the treatment was started during the acute phase of the tissue repair process. Conflict of interest statement We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this research that could have influenced its outcome. The manuscript has been read and approved by all named authors and there are no other persons who satisfied the criteria for authorship but are not listed. The order of authors listed in the manuscript has been approved by all of us. Acknowledgments The authors are grateful for BIOSET company for providing the equipment Phisiolux Dual Laser P.4050 and the technical help of Luana Fatoretto during this experimental trial. This research was sponsored by Camilo Castelo Branco University. References Amaral, A.C., Parizotto, N.A., Salvani, T.F., 2001. Dose-dependency of low-energy He–Ne Laser effect in regeneration of skeletal muscle in mice. Lasers in Medical Science 16, 44–51. American Veterinary Medical Association, 2001. Report of the AVMA panel on euthanasia. Journal of American Veterinary Medical Association 218, 669. Barushka, O., Yaakobi, T., Oron, U., 1995. Effect of low energy (He–Ne) irradiation on the process of bone repair in the rat tibia. Bone 16, 47–55. Brito, M.A.P., 2004. Analysis with the use of spectrophotometry in bone repair by laser biomodulation. Dissertation (Masters in Biomedical Engineering) – Research and Development Institute, University of Vale do Paraíba, 65p. Coombe, A.R., Ho, C.T., Darendeliler, M.A., Hunter, N., Philips, J.R., Chapple, C.C., Yum, L.W., 2001. The effects of low level laser irradiation on osteoblastic cells. Clinical Orthopaedics and Related Research 4, 3–14. Fávaro-Pípi, E., Feitosa, S.M., Ribeiro, A., Bossini, P., Oliveira, P., Parizotto, N.A., Renno, A.C.M., 2010. Comparative study of the effects of low-intensity pulsed ultrasound and low-level laser therapy on bone defects in tibias of rats. Lasers in Medical Science 25, 727–732. Fedde, K.N.N., Blair, L., Silverstein, J., Coburn, S.P., Ryan, L.M., Weinstein, R.S., Waymire, K., Narisawa, S., Millan, J.L., Macgregor, G.R., Whyte, M.P., 1999. Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. Journal of Bone and Mineral Research 14, 2015–2026. Freitas, I.G.F., Baranauskas, V., Cruz-Höfling, M.A., 2000. Laser effects on osteogenesis. Applied Surface Science 154–155, 548–554. Garnero, P., Delmas, P.D., 1988. Biochemecal markes of bone turnover: applications for osteoporosis. Endocrinology and Metabolism Clinics of North America 27, 303–323.
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