Low intensity laser therapy in bone healing and osteoporosis

Lasers Med Sci. 2011 Nov 18. [Epub ahead of print]

Effects of the combination of low-level laser irradiation and recombinant human bone morphogenetic protein-2 in bone repair.
Rosa AP, de Sousa LG, Regalo SC, Issa JP, Barbosa AP, Pitol DL, de Oliveira RH, de Vasconcelos PB, Dias FJ, Chimello DT, Sissere S.

Source Department of Morphology, Stomatology and Physiology, School of Dentistry at Ribeiro Preto, University of So Paulo, Avenida do Caf, s/n Bairro Monte Alegre, 14040-904, Ribeiro Preto, So Paulo, Brazil. Abstract Low-level laser irradiation (LLLI) and recombinant human bone morphogenetic protein type 2 (rhBMP-2) have been used to stimulate bone formation. LLLI stimulates proliferation of osteoblast precursor cells and cell differentiation and rhBMP-2 recruits osteoprogenitor cells to the bone healing area. This in vivo study evaluated the effects of LLLI and rhBMP-2 on the bone healing process in rats. Critical bone defects were created in the parietal bone in 42 animals, and the animals were divided into six treatment groups: (1) laser, (2) 7 ?g of rhBMP-2, (3) laser and 7 ?g of rhBMP-2, (4) 7 ?g of rhBMP-2/monoolein gel, (5) laser and 7 ?g rhBMP-2/monoolein gel, and (6) critical bone defect controls. A gallium-aluminum-arsenide diode laser was used (wavelength 780 nm, output power 60 mW, beam area 0.04 cm(2), irradiation time 80 s, energy density 120 J/cm(2), irradiance 1.5 W/cm(2)). After 15 days, the calvarial tissues were removed for histomorphometric analysis. Group 3 defects showed higher amounts of newly formed bone (37.89%) than the defects of all the other groups (P?<?0.05). The amounts of new bone in defects of groups 1 and 4 were not significantly different from each other (24.00% and 24.75%, respectively), but were significantly different from the amounts in the other groups (P?<?0.05). The amounts of new bone in the defects of groups 2 and 5 were not significantly different from each other (31.42% and 31.96%, respectively), but were significantly different from the amounts in the other groups (P?<?0.05). Group 6 defects had 14.10% new bone formation, and this was significantly different from the amounts in the other groups (P?<?0.05). It can be concluded that LLLI administered during surgery effectively accelerated healing of critical bone defects filled with pure rhBMP-2, achieving a better result than LLLI alone or the use of rhBMP-2 alone. Lasers Med Sci. 2011 Nov 9. [Epub ahead of print]

Development of a minimally invasive laser needle system: effects on cortical bone of osteoporotic mice.
Kang H, Ko CY, Ryu Y, Seo DH, Kim HS, Jung B.

Source Department of Biomedical Engineering, Yonsei University, 234 Maeji-ri, Heungup-myun, Wonju-si, Gangwon-do, 220-710, Korea. Abstract Many studies have shown the positive effects of low-level laser therapy in the treatment of bone disease. However, laser radiation is scattered in the skin surface which reduces the initial photon density for tissue penetration and consequently the therapeutic efficacy. We developed a minimally invasive laser needle system (MILNS) to avoid laser scattering in tissue and investigated its stimulatory effects in the cortical bone of osteoporotic mice. The MILNS was designed to stimulate cortical bone directly by employing fine hollow needles to guide 100 ?m optical fibers. The study animals comprised 12 mice which were subjected to sciatic denervation of the right hind limb and were randomly divided into two groups, a sham group and a laser group which were treated using the MILNS for 2 weeks without and with laser irradiation, respectively. In vivo micro-CT images were taken to analyze the structural parameters and bone mineral density. After 2 weeks of treatment with the MILNS, the relative changes in mean polar moment inertia, cross-section thickness, and periosteal perimeter were significantly higher in the laser group than in the sham group. Moreover, the distribution of bone mineral density index was higher in the laser group. The MILNS was developed as a minimally invasive treatment modality for bone disease and resulted in positive therapeutic efficacy in the cortical bone of osteoporotic mice. Lasers Med Sci. 2011 Aug 13. [Epub ahead of print]

Effect of low-level laser therapy after rapid maxillary expansion on proliferation and differentiation of osteoblastic cells.
da Silva AP, Petri AD, Crippa GE, Stuani AS, Stuani AS, Rosa AL, Stuani MB. Source

Department of Pediatric Dentistry and Othodontic, School of Dentistry of Ribeirao Preto, University of Sao Paulo, Av do Cafe, s/n, 14040-904, Ribeirao Preto, SP, Brazil.
Abstract

The aim of this study was to investigate the osteoblastic activity of cells derived from the midpalatal suture upon treatment with low-level laser therapy (LLLT) after rapid maxillary expansion (RME). A total of 30 rats were divided into two groups: experimental I (15 rats with RME without LLLT) and experimental II (15 rats with RME + LLLT). The rats were euthanized at 24 h, 48 h, and 7 days after RME, when the osteoblastic cells derived from the rats' midpalatal suture were explanted. These cells were cultured for periods up to 17 days, and then in vitro osteogenesis parameters and gene expression markers were evaluated. The cellular doubling time in the proliferative stage (3-7 days) was decreased in cultured cells harvested from the midpalatal

suture at 24 and 48 h after RME + LLLT, as indicated by the increased growth of the cells in a culture. Alkaline phosphatase activity at days 7 and 14 of the culture was increased by LLLT in cells explanted from the midpalatal suture at 24 and 48 h and 7 days after RME. The mineralization at day 17 was increased by LLLT after RME in all periods. Results from the realtime PCR demonstrated that cells harvested from the LLLT after RME group showed higher levels of ALP, Runx2, osteocalcin, type I collagen, and bone sialoprotein mRNA than control cells. More pronounced effects on ALP activity, mineralization, and gene expression of bone markers were observed at 48 h after RME and LLLT. These results indicate that the LLLT applied after RME is able to increase the proliferation and the expression of an osteoblastic phenotype in cells derived from the midpalatal suture. Endocr Regul. 2010 Oct;44(4):155-63.

Possible role of low level laser therapy on bone turnover in ovariectomized rats.
Saad A, El Yamany M, Abbas O, Yehia M. Source

Physiology Department, Medical Research Institute, Alexandria University, Egypt.

Abstract OBJECTIVE:

The aim of this study was to assess the effect of low level laser therapy (LLLT) on bone turnover markers in ovariectomized rats.
METHODS:

Thirty adult female albino rats were divided into three groups; Group 1: 10 sham- operated control rats; Group 2: 10 bilaterally ovariectomized rats (OVX); Group 3: 10 OVX rats exposed to LLLT. LLLT was applied on the neck and shaft of femur, five times per week for 8 weeks. The dose applied on each point was 1000 Hertz, 5 Watts for 30 seconds with a total dose of 15 mJoule/cm. At the end of experiment, blood samples were collected and sera were separated for determination of serum calcium (Ca), inorganic phosphorus (Pi), osteocalcin and alkaline phosphatase (ALP). In addition, a 24 hour urine sample was also collected from each rat for the determination of urinary calcium, phosphorous and deoxypyridinoline (U-DPD)/creatinine.
RESULTS:

Significant increase in serum Ca, Pi , ALP, osteocalcin and significant decrease in UDPD/creatinine in LLLT exposed group was found as compared to the other two groups. Bone morphological findings revealed the increase in calcium deposition and alkaline phosphatase of femoral bones in LLLT exposed group as compared to sham-operated and OVX rats. The

software image analysis showed increased osteoblast numbers, decreased osteoclast numbers and increased compact bone thickness in LLLT exposed group. Significant positive correlations was obtained between osteoblast numbers and serum Ca , Pi, ALP and osteocalcin in LLLT exposed group ,while a significant negative correlation was noticed with U-DPD.
CONCLUSION:

The use of LLLT was found effective in enhancing bone formation and decreasing bone resorption in the osteoporotic OVX rats. Further studies are necessary to investigate the effect of different parameters of LLLT as wave length, duration and also numbers of sessions. The potential use of LLLT in postmenopausal women with osteoporosis is needed to be verified. J Biomed Opt. 2011 Jul;16(7):078001.

Low-level laser therapy, at 60 J/cm(2) associated with a Biosilicate() increase in bone deposition and indentation biomechanical properties of callus in osteopenic rats.
Fangel R, Se?rgio Bossini P, Cla?udia Renno A, Araki Ribeiro D, Chenwei Wang C, Luri Toma R, Okino Nonaka K, Driusso P, Antonio Parizotto N, Oishi J. Source

Federal University of Sa?o Carlos, Department of Physiotherapy, Rod. Washington Luiz, km 235, Sa?o Carlos, Sao Paulo 13600-970 BrazilFederal University of Sao Paulo, Department of Bioscience, Av. Ana Costa, 95, Santos, Sao Paulo 11050240, BrazilFederal University of Sa?o Carlos, Department of Biology, Rod. Washington Luiz, km 235, Sa?o Carlos, Sao Paulo 13600970 BrazilFederal University of Sao Paulo, Department of Physiotherapy, Av. Ana Costa, 95, Santos, Sao Paulo 11050240, BrazilFederal University of Sa?o Carlos, Department of Statistics, Rod. Washington Luiz, km 235, Sao Carlos, Sao Paulo 13565-2081, Brazil.
Abstract

We investigate the effects of a novel bioactive material (Biosilicate()) and low-level laser therapy (LLLT), at 60 J/cm(2), on bone-fracture consolidation in osteoporotic rats. Forty female Wistar rats are submitted to the ovariectomy, to induce osteopenia. Eight weeks after the ovariectomy, the animals are randomly divided into four groups, with 10 animals each: bone defect control group; bone defect filled with Biosilicate group; bone defect irradiated with laser at 60 J/cm(2) group; bone defect filled with Biosilicate and irradiated with LLLT, at 60 J/cm(2) group. Laser irradiation is initiated immediately after surgery and performed every 48 h for 14 days. Histopathological analysis points out that bone defects are predominantly filled with the biomaterial in specimens treated with Biosilicate. In the 60-J/cm(2) laser plus Biosilicate group, the biomaterial fills all bone defects, which also contained woven bone and granulation tissue. Also, the biomechanical properties are increased in the animals treated with Biosilicate associated to lasertherapy. Our results indicate that laser therapy improves bone repair process in

contact with Biosilicate as a result of increasing bone formation as well as indentation biomechanical properties. Lasers Med Sci. 2011 May 20. [Epub ahead of print]

The effects of low-level laser irradiation on differentiation and proliferation of human bone marrow mesenchymal stem cells into neurons and osteoblasts-an in vitro study.
Soleimani M, Abbasnia E, Fathi M, Sahraei H, Fathi Y, Kaka G. Source

Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran, soleim_m@modares.ac.ir .
Abstract

Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for use in regenerative medicine. Several studies have shown that low-level laser irradiation (LLLI) could affect the differentiation and proliferation of MSCs. The aim of this study was to examine the influence of LLLI at different energy densities on BMSCs differentiation into neuron and osteoblast. Human BMSCs were cultured and induced to differentiate to either neuron or osteoblast in the absence or presence of LLLI. Gallium aluminum arsenide (GaAlAs) laser irradiation (810 nm) was applied at days 1, 3, and 5 of differentiation process at energy densities of 3 or 6 J/cm(2) for BMSCs being induced to neurons, and 2 or 4 J/cm(2) for BMSCs being induced to osteoblasts. BMSCs proliferation was evaluated by MTT assay on the seventh day of differentiation. BMSCs differentiation to neurons was assessed by immunocytochemical analysis of neuron-specific enolase on the seventh day of differentiation. BMSCs differentiation to osteoblast was tested on the second, fifth, seventh, and tenth day of differentiation via analysis of alkaline phosphatase (ALP) activity. LLLI promoted BMSCs proliferation significantly at all energy densities except for 6 J/cm(2) in comparison to control groups on the seventh day of differentiation. LLLI at energy densities of 3 and 6 J/cm(2) dramatically facilitated the differentiation of BMSCs into neurons (p?<?0.001). Also, ALP activity was significantly enhanced in irradiated BMSCs differentiated to osteoblast on the second, fifth, seventh, and tenth day of differentiation (p?<?0.001 except for the second day). Using LLLI at 810 nm wavelength enhances BMSCs differentiation into neuron and osteoblast in the range of 2-6 J/cm(2), and at the same time increases BMSCs proliferation (except for 6 J/cm(2)). The effect of LLLI on differentiation and proliferation of BMSCs is dose-dependent. Considering these findings, LLLI could improve current in vitro methods of differentiating BMSCs prior to transplantation. Photomed Laser Surg. 2011 Feb 9. [Epub ahead of print]

Low-Level Laser Therapy Induces Differential Expression of Osteogenic Genes During Bone Repair in Rats.
Fvaro-Ppi E, Ribeiro DA, Ribeiro JU, Bossini P, Oliveira P, Parizotto NA, Tim C, de Arajo HS, Renno AC. 1 Department of Physiotherapy, Federal University of So Carlos , So Carlos, SP-Brazil .
Abstract

Abstract Objectives: The aim of this study was to measure the temporal pattern of the expression of osteogenic genes after low-level laser therapy during the process of bone healing. We used quantitative real-time polymerase chain reaction (qPCR) along with histology to assess gene expression following laser irradiation on created bone defects in tibias of rats. Material and Methods: The animals were randomly distributed into two groups: control or laserirradiated group. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation started 24 h post-surgery and was performed for 3, 6, and 12 sessions, with an interval of 48?h. A 830?nm laser, 50?J/cm(2), 30 mW, was used. On days 7, 13, and 25 post-injury, rats were sacrificed individually by carbon dioxide asphyxia. The tibias were removed for analysis. Results: The histological results revealed intense new bone formation surrounded by highly vascularized connective tissue presenting slight osteogenic activity, with primary bone deposition in the group exposed to laser in the intermediary (13 days) and late stages of repair (25 days). The quantitative real-time PCR showed that laser irradiation produced an upregulation of BMP-4 at day 13 post-surgery and an upregulation of BMP4, ALP, and Runx 2 at day 25 after surgery. Conclusion: Our results indicate that laser therapy improves bone repair in rats as depicted by differential histopathological and osteogenic genes expression, mainly at the late stages of recovery. Photomed Laser Surg. 2011 Feb 8. [Epub ahead of print]

Application of Low-Level Laser Irradiation (LLLI) and rhBMP-2 in Critical Bone Defect of Ovariectomized Rats: Histomorphometric Evaluation.
Sissere S, Sousa LG, Issa JP, Iyomasa MM, Pitol DL, Barbosa AP, Semprini M, Sebald W, Bentley MV, Regalo SC. 1 Facultie of Dentistry, University of So Paulo , Ribeiro Preto, So Paulo, Brazil .

Abstract

Abstract Objectives: The aim of this study was to evaluate the osteogenic potential of recombinant human bone morphogenetic protein-2 (rhBMP-2) and low-level laser irradiation (LLLI), isolated or combined in critical bone defects (5?mm) in parietal bone using ovariectomized female rats as an experimental animal model. Materials and Methods: Forty-nine female Wistar rats, bilaterally ovariectomized (OVX), were divided into seven treatment groups of seven animals each: (I) laser in a single application, (II) 7??g of pure rhBMP-2, (III) laser and 7??g of pure rhBMP-2, (IV) 7??g of rhBMP-2/monoolein gel, (V) laser and 7??g of rhBMP-2/monoolein gel, (VI) laser and pure monoolein gel, and (VII) critical bone defect controls. The low-level laser source used was a gallium aluminum arsenide semiconductor diode laser device (??=?780?nm, D?=?120?J/cm(2)). Results: Groups II and III presented higher levels of newly formed bone than all other groups with levels of 40.57% and 40.39%, respectively (p?<?0.05). The levels of newly formed bone of groups I, IV, V, and VI were similar with levels of 29.67%, 25.75%, 27.75%, and 30.64%, respectively (p?>?0.05). The area of new bone formation in group VII was 20.96%, which is significantly lower than groups I, II, III, and VI. Conclusions: It was concluded that pure rhBMP-2 and a single dose of laser application stimulated new bone formation, but the new bone formation area was significantly increased when only rhBMP-2 was used. Additionally, the laser application in combination with other treatments did not influence the bone formation area. Lasers Med Sci. 2011 Jan 19. [Epub ahead of print]

Evaluation of the osteogenic effect of low-level laser therapy (808 nm and 660 nm) on bone defects induced in the femurs of female rats submitted to ovariectomy.
R Poppi R, Da Silva AL, Nacer RS, Vieira RP, de Oliveira LV, Santos de Faria Jnior N, de Tarso Camilo Carvalho P. Postgraduate Program in Health and Development in the West Central Region, Federal University of Mato Grosso do Sul, Campo Grande, Brazil.
Abstract

The present study aimed to evaluate the effects of LLLT (660- and 808-nm wavelengths) on the process of repairing bone defects induced in the femurs of female rats submitted to ovariectomy. Bilateral ovariectomies were performed on 18 female Wistar rats, which were divided into control and irradiated groups after the digital analysis of bone density showed decreased bone mass and after standardized drilling of the femurs. The irradiated groups received 133 J/cm(2) of

AsGaAl (660-nm) and InGaAlP (880-nm) laser radiation. The animals were euthanized on days 14 and 21 after the bone defects were established. Detailed descriptive histological evaluations were performed, followed by semi-quantitative histomorphometry. The results from days 14 and 21 showed that the irradiated groups presented increased density of osteoblasts, fibroblasts, and immature osteocytes on the tissue surface compared with the control (non-irradiated) groups (p?<?0.05). Additionally, inflammatory infiltrate evaluations showed that LLLT decreased the accumulation of leukocytes when compared to the control treatment (p?<?0.05). We concluded that, in our experimental model, both wavelengths (660-nm and 880-nm) inhibited the inflammatory process and induced the proliferation of cells responsible for bone remodeling and repair. Photomed Laser Surg. 2010 Dec;28(6):823-30.

Laser-induced alveolar bone changes during orthodontic movement: a histological study on rodents.
Habib FA, Gama SK, Ramalho LM, Canguss MC, Santos Neto FP, Lacerda JA, Arajo TM, Pinheiro AL . Centro de Ortodontia e Ortopedia Facial Prof. Jos Edimo Soares Martins, School of Dentistry, Federal University of Bahia (UFBA), Salvador, Brazil.
Abstract

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. Photomed Laser Surg. 2010 Oct;28 Suppl 2:S89-97. Epub 2010 Oct 7.

Raman spectroscopy validation of DIAGNOdent-assisted fluorescence readings on tibial fractures treated with laser phototherapy, BMPs, guided bone regeneration, and miniplates.
Pinheiro AL, Lopes CB, Pacheco MT, Brugnera A Jr, Zanin FA, Canguss MC, Silveira L Jr. Center of Biophotonics, School of Dentistry, Federal University of Bahia, Salvador, BA, Brazil. albp@ufba.br
Abstract

OBJECTIVES: We aimed to assess through Raman spectroscopy and fluorescence the levels of calcium hydroxyapatite (CHA) and lipids and proteins in complete fractures treated with internal rigid fixation (IRF) treated or not with laser phototherapy (LPT) and associated or not with bone morphogenetic proteins (BMPs) and guided bone regeneration (GBR). BACKGROUND: Fractures have different etiologies and treatments and may be associated with bone losses. LPT has been shown to improve bone healing. METHODS: Tibial fractures were created on 15 animals and divided into five groups. LPT started immediately after surgery, repeated at 48-h intervals. Animal death occurred after 30 days. RESULTS: Raman spectroscopy and fluorescence were performed at the surface. Fluorescence data of group IRF + LPT + Biomaterial showed similar readings to those of the group IRF-no bone loss. Significant differences were seen between groups IRF + LPT + Biomaterial and IRF + LPT; IRF + LPT + Biomaterial; and IRF + Biomaterial; and between IRF + LPT + Biomaterial and IRF. CH groups of lipids and proteins readings showed decreased levels of organic components in subjects treated with the association of LPT, biomaterial, and GBR. Pearson correlation showed that fluorescence readings of both CHA and CH groups of lipids and proteins correlated negatively with the Raman data. CONCLUSIONS: The use of both methods indicates that the use of the biomaterials associated with infrared LPT resulted in a more-advanced and higher quality of bone repair in fractures treated with miniplates and that the DIAGNOdent may be used to perform optical biopsy on bone. Endocr Regul 2010 Oct;44(4):155-63.

Possible role of low level laser therapy on bone turnover in ovariectomized rats.
Saad A, Yamany ME, Abbas O, Yehia M. Abstract Objective. The aim of this study was to assess the effect of low level laser therapy (LLLT) on bone turnover markers in ovariectomized rats. <br />Methods. Thirty adult female albino rats were divided into three groups; Group 1: 10 sham- operated control rats; Group 2: 10 bilaterally ovariectomized rats (OVX); Group 3: 10 OVX rats exposed to LLLT. LLLT was applied on the neck and shaft of femur, five times per week for 8 weeks. The dose applied on each point was 1000 Hertz, 5 Watts for 30 seconds with a total dose of 15 mJoule/cm2. At the end of experiment, blood samples were collected and sera were separated for determination of serum calcium (Ca), inorganic phosphorus (Pi), osteocalcin and alkaline phosphatase (ALP). In addition, a 24 hour urine sample was also collected from each rat for the determination of urinary calcium, phosphorous and deoxypyridinoline (U-DPD)/creatinine. <br />Results. Significant increase in serum Ca, Pi , ALP, osteocalcin and significant decrease in U-DPD/creatinine in LLLT exposed group was found as compared to the other two groups. Bone morphological findings revealed the increase in calcium deposition and alkaline phosphatase of femoral bones in LLLT exposed group as compared to sham-operated and OVX rats. The software image analysis showed increased osteoblast numbers, decreased osteoclast numbers and increased compact bone thickness in LLLT exposed group. Significant positive correlations was obtained between osteoblast numbers and serum Ca , Pi, ALP and osteocalcin in LLLT exposed group ,while a significant negative correlation was noticed with U-DPD.<br />Conclusion. The use of LLLT was found effective in enhancing bone formation and decreasing bone resorption in the osteoporotic OVX rats. Further studies are necessary to investigate the effect of different parameters of LLLT as wave length, duration and also numbers of sessions. The potential use of LLLT in postmenopausal women with osteoporosis is needed to be verified. Keywords: Ovariectomy Laser Calcium Phosphorus Alkaline phosphatase Osteocalcin Osteoporosis. Photomed Laser Surg. 2010 Oct 12. [Epub ahead of print]

Comparison of the Effects of Electrical Stimulation and Low-Level Laser Therapy on Bone Loss in Spinal Cord&#x2013;Injured Rats.
Medalha CC, Amorim BO, Ferreira JX, Ol iveira P, Pereira RM, Tim C, Lirani-Galy Xooe3 O AP, da Silva Ol, Renno AC, 1 Department of Bioscience, Federal University of S&#x00E3;o Paulo (UNIFESP) , Santos, SP, Brazil . Abstract

Abstract Objective: This study investigated the effects of low-level laser therapy (LLLT) and electrical stimulation (ES) on bone loss in spinal cord&#x2013;injured rats. Materials and Methods: Thirty-seven male Wistar rats were divided into four groups: standard control group (CG); spinal cord&#x2013;injured control (SC); spinal cord&#x2013;injured treated with laser (SCL; GaAlAs, 830&#x2009;nm, CW, 30&#x2009;mW/cm, 250&#x2009;J/cm²); and spinal cord&#x2013;injured treated with electrical field stimulation (SCE; 1.5&#x2009;MHz, 1:4 duty cycles, 30 mW, 20&#x2009;min). Biomechanical, densitometric, and morphometric analyses were performed. Results: SC rats showed a significant decrease in bone mass, biomechanical properties, and morphometric parameters (versus CG). SCE rats showed significantly higher values of inner diameter and internal and external areas of tibia diaphyses; and the SCL group showed a trend toward the same result (versus SC). No increase was found in either mechanical or densitometric parameters. Conclusion: We conclude that the mentioned treatments were able to initiate a positive bone-tissue response, maybe through stimulation of osteoblasts, which was able to determine the observed morphometric modifications. However, the evoked tissue response could not determine either biomechanical or densitometric modifications. Lasers Med Sci. 2010 Sep;25(5):727-32. Epub 2010 Jun 3.

Comparative study of the effects of low-intensity pulsed ultrasound and low-level laser therapy on bone defects in tibias of rats.
Fvaro-Ppi E, Feitosa SM, Ribeiro DA, Bossini P, Oliveira P, Parizotto NA, Renno AC. Department of Physiotherapy, Federal University of So Carlos, Rodovia Washington Lus (SP310), Km 235, So Carlos, SP, Brazil.
Abstract

The aim of this study was to investigate and to compare the effects of low intensity ultra-sound (LIPUS) and low-level laser therapy (LLLT) during the process of bone healing by means of histopathological and morphometric analysis. The animals were randomly distributed into three groups of 30 animals each: the control group (bone defect without treatment); the laser-treated group: (bone defect treated with laser), and the LIPUS-treated (bone defect treated with ultrasound). Each group was further divided into three different subgroups (n = 10) and on days 7, 13, and 25 post-injury, rats were killed with an intra-peritoneal injection of general anesthetic. The rats were treated with a 30-mW/cm(2) low-intensity pulsed ultrasound and a 830-nm laser at 50 J/cm(2). The results showed intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition being observed in the group exposed to laser in the intermediary (13 days) and late stages of repair (25 days). This was confirmed by morphometric analysis in which significant statistical differences (p < 0.05) were noticed when compared to the control. No remarkable differences were noticed in the specimens treated with ultrasound with regard to the amount of newly formed bone in comparison to the control group. Taken together, our results indicate that laser therapy improves bone repair in rats as depicted by histopathological and morphometric analysis, mainly at the late

stages of recovery. Moreover, it seems that this therapy was more effective than US to accelerate bone healing. Photomed Laser Surg. 2010 Aug;28 Suppl 1:S157-65.

Red-light light-emitting diode irradiation increases the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells.
Li WT, Leu YC, Wu JL. Department of Biomedical Engineering, Chung Yuan Christian University, Chung-Li, Taiwan, Republic of China. wtli@cycu.edu.tw
Abstract

OBJECTIVE: The objective of this study was to investigate the effects on the proliferation and osteogenic differentiation of rat mesenchymal stem cells (MSCs) by using red-light lightemitting diode (LED) irradiation. BACKGROUND DATA: Low-level light irradiation (LLLI) has been shown to enhance proliferation and cytokine secretion of a number of cells. MSCs are capable of regenerating various mesenchymal tissues and are essential in supporting the growth and differentiation of hematopoietic stem cells within the bone marrow. MATERIALS AND METHODS: Rat bone marrow MSCs were treated with single or multiple doses of LLLI from an LED array (630 nm) at the irradiances of 5 and 15 mW/cm(2), and radiant exposures of 2 and 4 J/cm(2). The proliferation, clonogenic potential, and osteogenic differentiation of MSCs were evaluated after illumination. RESULTS: The growth of MSCs was enhanced by red-light LLLI, and the effect became more obvious at low cell density. A single dose of LLLI led only to a short-term increase in MSCs proliferation. A maximal increase in cell proliferation was observed with multiple exposures of LLLI at 15 mW/cm(2) and 4 J/cm(2). The number of colony-forming unit fibroblasts increased when cells were illuminated under the optimal parameter. During osteogenesis, significant increases (p < 0.01) in both alkaline phosphatase and osteocalcin expressions were found in the MSCs that received light irradiation. CONCLUSION: Our data demonstrated that MSCs proliferation was enhanced by multiple exposures to LLLI from 630-nm LEDs, and cell growth depended on the plating density. Furthermore, multiple dose of LLLI could enhance the osteogenic potential of rat MSCs. Lasers Med Sci. 2010 Jul;25(4):559-69. Epub 2010 Feb 26.

Low-level Er:YAG laser irradiation enhances osteoblast proliferation through activation of MAPK/ERK.
Aleksic V, Aoki A, Iwasaki K, Takasaki AA, Wang CY, Abiko Y, Ishikawa I, Izumi Y. Section of Periodontology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan.
Abstract

Although the use of high-level Er:YAG laser irradiation has been increasing in periodontal and peri-implant therapy, the effects of low-level Er:YAG laser on surrounding tissues and cells remain unclear. In the present study, the effects of low-level Er:YAG laser irradiation on osteoblast proliferation were investigated. Cells of the osteoblastic cell line MC3T3-E1 were treated with low-level Er:YAG laser irradiation with various combinations of laser settings (fluence 0.7-17.2 J/cm(2)) and in the absence or presence of culture medium during irradiation. On day 1 and/or day 3, cell proliferation and death were determined by cell counting and by measurement of lactate dehydrogenase (LDH) levels. Further, the role of mitogen-activated protein kinase (MAPK) pathways in laser-enhanced cell proliferation was investigated by inhibiting the MAPK pathways and then measuring MAPK phosphorylation by Western blotting. Higher proliferation rates were found with various combinations of irradiation parameters on days 1 and 3. Significantly higher proliferation was also observed in laser-irradiated MC3T3-E1 cells at a fluence of approximately 1.0-15.1 J/cm(2), whereas no increase in LDH activity was observed. Further, low-level Er:YAG irradiation induced the phosphorylation of extracellular signal-regulated protein kinase (MAPK/ERK) 5 to 30 min after irradiation. Although MAPK/ERK 1/2 inhibitor U0126 significantly inhibited laser-enhanced cell proliferation, activation of stress-activated protein kinases/Jun N-terminal kinase (SAPK/JNK) and p38 MAPK was not clearly detected. These results suggest that low-level Er:YAG laser irradiation increases osteoblast proliferation mainly by activation of MAPK/ERK, suggesting that the Er:YAG laser may be able to promote bone healing following periodontal and peri-implant therapy. Photomed Laser Surg. 2010 Jul 22. [Epub ahead of print]

Low-Level Laser Therapy Stimulates Mineralization Via Increased Runx2 Expression and ERK Phosphorylation in Osteoblasts.
Kiyosaki T, Mitsui N, Suzuki N, Shimizu N. 1 Department of Orthodontics, Nihon University School of Dentistry , Chiyoda-ku, Tokyo, Japan . Abstract

Abstract Objective: This study examined the effects of low-level laser therapy (LLLT) on osteoblasts via insulin-like growth factor I (IGF-I) signal transduction. Background: Because orthodontic treatment is usually accompanied by bone formation, if bone formation can be promoted, the treatment and retention periods will be shorter. Recently, we reported the stimulatory effects of LLLT on bone formation. It was dependent on increased IGF-I, which plays an essential role in the anabolic regulation of bone metabolism. However, the signal transduction of IGF-I stimulated by LLLT was not elucidated. Materials and Methods: Mouse osteoblastic MC3T3-E1 cells were cultured with or without LLLT (0.96-3.82 J/cm(2)), and the expression of IGF-I and Runt-related transcription factor 2 (Runx2) and the phosphorylation of extracellular-signal-regulated kinase (ERK) were determined by using real-time PCR and Western blot analysis. Results: LLLT at 1.91 J/cm(2) significantly increased the expression of IGF-I and Runx2 and of ERK phosphorylation. Cyclolignan picropodophyllin (PPP; an IGF-I receptor inhibitor) partly inhibited the LLLT-induced expression of these factors. Moreover, when conditioned medium from the LLLT (1.91 J/cm(2)) cells was added to the MC3T3-E1 culture, the calcium content in the mineralized nodules increased significantly. PPP or noggin [a bone morphogenetic protein (BMP) antagonist] partly inhibited the LLLT-induced change in calcium content, and the addition of both PPP and noggin inhibited most of the LLLT-induced change in calcium content. Conclusion: These results suggest that LLLT stimulates in vitro mineralization through increased IGF-I and BMP production, through Runx2 expression and ERK phosphorylation in osteoblasts. Med Oral Patol Oral Cir Bucal. 2010 Jul 1;15(4):e616-8.

Histological evaluation of the effect of low-level laser on distraction osteogenesis in rabbit mandibles.
Kreisner PE, Blaya DS, Gaio L, Maciel-Santos ME, Etges A, Santana-Filho M, de Oliveira MG. School of Dentistry, Pontifcia Universidade Catlica do Rio Grande do Sul, Porto Alegre, RS, Brazil. Abstract OBJECTIVES: This study evaluated the action of low level laser therapy (LLLT) on the percentage of newly formed bone in rabbit mandibles that underwent distraction osteogenesis (DO). STUDY DESIGN: Ten rabbits underwent bone lengthening according to the following protocol: Latency 3 days; Activation 7 days 0.7 mm/d; and Consolidation 10 days. The control group was composed of 4 rabbits. The experimental group, composed of 6 rabbits, received infrared GaAlAs LLLT (wavelength=830 nm, P=40 mW) according to the following protocol: point dose of 10 J/cm(2) applied directly on the bone site that underwent DO during bone consolidation at 48-hour intervals. RESULTS: The percentage of newly formed bone was greater in the LLLT group (57.89%) than in the control group (46.75%) (p=0.006). CONCLUSION: The results suggest that LLLT had a positive effect on the percentage of newly formed bone. Better-quality bone sites may allow early removal of the osteogenic distractors, thus shortening total treatment time.

Photomed Laser Surg. 2010 Jun;28(3):411-6.

Effect of biostimulation on healing of bone defects in diabetic rats.

Akyol UK, Gngrm? M. Ataturk University, Faculty of Dentistry, Department of Oral and Maxillofacial Surgery, Erzurum, Turkey. utkanakyol@yahoo.com
Abstract

BACKGROUND AND OBJECTIVE: The aim of this study was to investigate the effects of biostimulation on healing of bone defects in diabetic rats. STUDY DESIGN/ MATERIAL AND METHODS: Twenty-eight Wistar rats weighting 250 to 300 g were used for this study. Diabetes was chemically induced with streptozotocin, and 14 nondiabetic and 14 diabetic rats were included in the study. The distal epiphysis of the right and left femurs of the diabetic rats were perforated with a surgical bone drill. This surgical procedure was performed on the left femurs of normal rats too. The wound on the right side of each diabetic rat received laser stimulation. The left femur of each nondiabetic (normal) rat served as a control. The rats were assigned to three experimental groups: (1) normal bur (control group); (2) diabetic bur; (3) diabetic bur + biostimulation. RESULTS: There was a significant difference among all groups in substantia spongiosa formation on day 10. According to the Mann-Whitney U test, there was a difference between Groups 1 and 2. A significant difference was noted between Groups 2 and 3 as well as between Groups 1 and 3 and between Groups 2 and 3 in union at 20 d of healing. CONCLUSIONS: Substantia spongiosa formation was slightly more evident in Groups 1 and 3 than in Group 2. Also, there was more union in Group 3 than in the other groups on day 20. As a result, it can be concluded that low-level laser therapy (808 nm laser at 10 J/cm(2)) can have a beneficial effect on spongiosa in diabetic bone repair when five treatments are administered with 2 d intervals between treatments. Lasers Med Sci. 2010 Jun 3. [Epub ahead of print]

Comparative study of the effects of low-intensity pulsed ultrasound and low-level laser therapy on bone defects in tibias of rats.
Fvaro-Ppi E, Feitosa SM, Ribeiro DA, Bossini P, Oliveira P, Parizotto NA, Renno AC. Department of Physiotherapy, Federal University of So Carlos, Rodovia Washington Lus (SP310), Km 235, So Carlos, SP, Brazil.

Abstract The aim of this study was to investigate and to compare the effects of low intensity ultra-sound (LIPUS) and low-level laser therapy (LLLT) during the process of bone healing by means of histopathological and morphometric analysis. The animals were randomly distributed into three groups of 30 animals each: the control group (bone defect without treatment); the laser-treated group: (bone defect treated with laser), and the LIPUS-treated (bone defect treated with ultrasound). Each group was further divided into three different subgroups (n = 10) and on days 7, 13, and 25 post-injury, rats were killed with an intra-peritoneal injection of general anesthetic. The rats were treated with a 30-mW/cm(2) low-intensity pulsed ultrasound and a 830-nm laser at 50 J/cm(2). The results showed intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition being observed in the group exposed to laser in the intermediary (13 days) and late stages of repair (25 days). This was confirmed by morphometric analysis in which significant statistical differences (p < 0.05) were noticed when compared to the control. No remarkable differences were noticed in the specimens treated with ultrasound with regard to the amount of newly formed bone in comparison to the control group. Taken together, our results indicate that laser therapy improves bone repair in rats as depicted by histopathological and morphometric analysis, mainly at the late stages of recovery. Moreover, it seems that this therapy was more effective than US to accelerate bone healing. Biol Trace Elem Res. 2010 Mar;133(3):342-9. Epub 2009 Jul 4.

Therapeutic effect of organic gallium on ovariectomized osteopenic rats by decreased serum minerals and increased bone mineral content.
Ma Z, Fu Q. Department of Orthopaedics, Sheng Jing Hospital, China Medical University, Shenyang, 110004, People's Republic of China.
Abstract

The purpose of this study was to verify the effect of organic gallium on ovariectomized osteopenic rats. Thirty Wistar female rats used were divided into three groups: (1) shamoperation rats (control), (2) ovariectomized (OVX) rats with osteopenia, and (3) OVX rats with osteopenia treated with organic gallium. Treatments were performed over an 8-week period. At sacrifice, the fifth lumbar vertebral body, one tibia, one femur, and the fourth lumbar vertebrae were removed, subjected to micro-CT for determination of trabecular bone structure, and then processed for histomorphometry to assess bone turnover. The femoral neck was used for mechanical compression testing. Treatment with organic gallium increased bone volume in OVX animals. Organic gallium-treated animals had significant increases in trabecular and cortical thickness and bone strength. The plasma total calcium and inorganic phosphate concentrations in OVX rats decreased and bone mineral content in the lumbar vertebrae and femur increased after treatment with organic gallium. These data provide an important proof of concept that organic

gallium may represent a powerful approach to treating or reversing severe osteoporosis in humans. Osteoporos Int. 2010 Mar 4. [Epub ahead of print]

Laser 904 nm action on bone repair in rats with osteoporosis.

Pires-Oliveira DA, Oliveira RF, Amadei SU, Pacheco-Soares C, Rocha RF. Programa de Ps Graduao-Unicastelo, Departamento Curso de Odontologia, Universidade Camilo Castelo Branco-Unicastelo So Paulo, Rua Carolina Fonseca 584, Itaquera, So Paulo, So Paulo, CEP-08.230-030, Brazil, deisepyres@yahoo.com.br. The aim of the present study was to determine the action of AsGA laser irradiation on bone repair in the tibia of osteopenic rats. The animals were randomly divided into eight experimental groups according to the presence of ovarian hormone (sham group) or the absence of the hormone (OVX group), as well as being irradiated or non-irradiated. Low-level 904-nm laser (50 mJ/cm(2)) accelerated the repair process of osteopenic fractures, especially in the initial phase of bone regeneration. INTRODUCTION: The development of new techniques to speed the process of bone repair has provided significant advances in the treatment of fractures. Some attention recently focused on the effects of biostimulation on bone. METHODS: Forty-eight adult rats were randomly divided into eight experimental groups (six animals in each group) according to the presence of ovarian hormone (sham group) or absence of the hormone (ovariectomized (OVX) group) as well as being irradiated or non-irradiated. For the application of low-level laser therapy, the animals were anesthetized with one third of the dose sufficient to immobilize the animal and irradiated with AsGa laser (904 nm, 50 mJ/cm(2) for 2 s, point form and in contact). The control animals received the same type of manipulation as the irradiated animals, but with the laser turned off. Half of the animals were killed 7 days following the confection of the bone defect, and the other half were killed 21 days after the surgery. After complete demineralization, the tibias were cut cross-sectionally in the central region of the bone defect and embedded in paraffin blocks. The blocks were then cut in semi-seriated slices and stained with hematoxylin and eosin. RESULTS: There was new bone formation in the animals in the OVX group with laser treatment killed after 7 days (p < 0.001). The lowest percentage of bone formation was observed in the OVX without laser killed after 7 days (p > 0.05). All animals killed after 21 days exhibited linear closure of the lesion. CONCLUSION: Low-level 904-nm laser (50 mJ/cm(2)) accelerated the repair process of osteopenic fractures, especially in the initial phase of bone regeneration. Photomed Laser Surg. 2010 Feb;28(1):45-9.

Effect of low-level laser therapy and calcitonin on bone repair in castrated rats: a densitometric study.
Nascimento SB, Cardoso CA, Ribeiro TP, Almeida JD, Albertini R, Munin E, Arisawa EA.

Biomedical Vibrational Spectroscopy Laboratory, Universidade do Vale do Paraba, So Jos dos Campos, So Paulo, Brazil.
Abstract

OBJECTIVE: To investigate the healing of bone defects in male rats treated with salmon calcitonin, low-level laser therapy (LLLT), or both. BACKGROUND: Healing of bone defects still represents a challenge to health professionals in several areas. In this article, the effect of calcitonin in combination with LLLT on bone repair was studied. Densitometry was used as a valuable tool for the measurement of bone regeneration. METHODS: Sixty male Wistar rats underwent bilateral castration surgery before the creation of a surgical bone defect. The animals were randomly divided into four groups: control, treated with calcitonin (Ca), treated with LLLT (La), and treated with calcitonin and LLLT (CaLa). Groups Ca and CaLa received 2 IU/kg of synthetic salmon calcitonin intramuscularly three times a week. Groups La and CaLa received laser therapy using a gallium-aluminum-arsenide laser (10 mW, 20 J/cm(2), wavelength 830 nm). Control animals were submitted to sham irradiation. The animals were sacrificed 7, 14, and 21 days after surgery, and bone defects were analyzed using densitometry. RESULTS: The CaLa group had a higher degree of bone regeneration 14 and 21 days after surgery. CONCLUSIONS: The La and CaLa had significantly higher bone mineral density than the control and Ca groups. J Orthop Surg Res. 2010 Jan 4;5(1):1. [Epub ahead of print]

Effects of low power laser irradiation on bone healing in animals: a meta-analysis.

Bashardoust Tajali S, Macdermid JC, Houghton P, Grewal R. ABSTRACT: Purpose: The meta-analysis was performed to identify animal research defining the effects of low power laser irradiation on biomechanical indicators of bone regeneration and the impact of dosage. Methods: We searched five electronic databases (MEDLINE, EMBASE, PubMed, CINAHL, and Cochrane Database of Randomised Clinical Trials) for studies in the area of laser and bone healing published from 1966 to October 2008. Included studies had to investigate fracture healing in any animal model, using any type of low power laser irradiation, and use at least one quantitative biomechanical measures of bone strength. There were 880 abstracts related to the laser irradiation and bone issues (healing, surgery and assessment). Five studies met our inclusion criteria and were critically appraised by two raters independently using a structured tool designed for rating the quality of animal research studies. After full text review, two articles were deemed ineligible for meta-analysis because of the type of injury method and

biomechanical variables used, leaving three studies for meta-analysis. Maximum bone tolerance force before the point of bone fracture during the biomechanical test, 4 weeks after bone deficiency was our main biomechanical bone properties for the Meta analysis. Results: Studies indicate that low power laser irradiation can enhance biomechanical properties of bone during fracture healing in animal models. Maximum bone tolerance was statistically improved following low level laser irradiation (average random effect size 0.726, 95% CI 0.08 1.37, p 0.028). While conclusions are limited by the low number of studies, there is concordance across limited evidence that laser improves the strength of bone tissue during the healing process in animal models. Lasers Med Sci. 2010 Jan;25(1):73-7. Epub 2009 Apr 28.

Effect of low-level laser therapy on the fracture healing process.

Kazem Shakouri S, Soleimanpour J, Salekzamani Y, Oskuie MR. Tabriz University of Medical Sciences, Tabriz, Iran. sk0531ir@yahoo.com
Abstract

Low-level laser therapy (LLLT) is a biophysical form of intervention in the fracture-repair process, which, through several mechanisms, accelerates the healing of fractures and enhances callus formation. The effect of laser on fracture healing is controversial. Some authors affirm that LLLT can accelerate bone formation by increasing osteoblastic activity. The objective of our study was to evaluate the effect of laser therapy on fracture healing. Thirty rabbits were subjected to tibial bone open osteotomies that were stabilized with external fixators. The animals were divided into two study groups: laser group and control group. Callus development and bone mineral density were quantitatively evaluated by CT; the animals were then killed and the fractures were assessed for biomechanical properties. The results demonstrated that the increasing rate of bone mineral density was higher in the laser (L) group than in the control (C) group. CT at 5 weeks revealed a mean callus density of 297 Hounsfield units (HU) for the control group and 691 HU for the L group, which was statistically significant (P = 0.001). In the L group, the mean recorded fracture tension was 190.5 N and 359.3 N for healed and intact bones, respectively, which was statistically significant (P < 0.001). The result of the study showed that the use of laser could enhance callus development in the early stage of the healing process, with doubtful improvement in biomechanical properties of the healing bone; therefore, laser therapy may be recommended as an additional treatment in non-union fractures in humans. Med Oral Patol Oral Cir Bucal. 2009 Dec 29. [Epub ahead of print]

Histological evaluation of the effect of low-level laser on distraction osteogenesis in rabbit mandibles.
Kreisner PE, Blaya D, Gaio L, Maciel-Santos ME, Etges A, Santana-Filho M, de Oliveira MG.

Av. Cel. Lucas de Oliveira, 1841/203, Petrpolis CEP: 90460-001 Porto Alegre, RS, Brazil, mogerhardt@yahoo.com.br. Objectives: This study evaluated the action of low level laser therapy (LLLT) on the percentage of newly formed bone in rabbit mandibles that underwent distraction osteogenesis (DO). Study design: Ten rabbits underwent bone lengthening according to the following protocol: Latency 3 days; Activation 7 days 0.7 mm/d; and Consolidation 10 days. The control group was composed of 4 rabbits. The experimental group, composed of 6 rabbits, received infrared GaAlAs LLLT (wavelength=830 nm, 40 mW) according to the following protocol: point dose of 10 J/cm(2) applied directly on the bone site that underwent DO during bone consolidation at 48hour intervals. Results: The percentage of newly formed bone was greater in the LLLT group (57.89%) than in the control group (46.75%) (p=0.006). Conclusion: The results suggest that LLLT had a positive effect on the percentage of newly formed bone. Better-quality bone sites may allow early removal of the osteogenic distractors, thus shortening total treatment time. J Mater Sci Mater Med. 2009 Nov 27. [Epub ahead of print]

Low level laser therapy does not modulate the outcomes of a highly bioactive glass-ceramic (Biosilicate((R))) on bone consolidation in rats.
Oliveira P, Ribeiro DA, Pipi EF, Driusso P, Parizotto NA, Renno AC. Department of Physiotherapy, Federal University of So Carlos, Rodovia Washington Lus (SP310), Km 235, So Carlos, SP, Brazil. The main purpose of the present work was to evaluate if low level laser therapy (LLLT) can improve the effects of novel fully-crystallized glass-ceramic (Biosilicate((R))) on bone consolidation in tibial defects of rats. Forty male Wistar rats with tibial bone defects were used. Animals were divided into four groups: group bone defect control (CG); group bone defect filled with Biosilicate((R)) (BG); group bone defect filled with Biosilicate((R)), irradiated with LLLT, at 60 J cm(-2) (BG 60) and group bone defect filled with Biosilicate((R)), irradiated with LLLT, at 120 J cm(-2) (BG 120). A low-energy GaAlAs 830 nm, CW, 0.6 mm beam diameter, 100 W cm(-2), 60 and 120 J cm(-)(2) was used in this study. Laser irradiation was initiated immediately after the surgery procedure and it was performed every 48 h for 14 days. Fourteen days postsurgery, the three-point bending test revealed that the structural stiffness of the groups CG and BG was higher than the values of the groups BG60 and BG120. Morphometric analysis revealed no differences between the control group and the Biosilcate((R)) group. Interestingly, the groups treated with Biosilicate((R)) and laser (BG 60 and BG120) showed statistically significant lower values of newly formed bone in the area of the defect when compared to negative control (CG) and bone defect group filled with Biosilicate (CB). Our findings suggest that although Biosilicate((R)) exerts some osteogenic activity during bone repair, laser therapy is not able to modulate this process. Photomed Laser Surg. 2009 Oct 27. [Epub ahead of print]

Effect of Biostimulation on Healing of Bone Defects in Diabetic Rats.

Akyol UK, Gngrm? M. Ataturk University , Faculty of Dentistry, Department of Oral and Maxillofacial Surgery, Erzurum, Turkey . Abstract Background and Objective: The aim of this study was to investigate the effects of biostimulation on healing of bone defects in diabetic rats. Study Design/Material and Methods: Twenty-eight Wistar rats weighting 250 to 300 g were used for this study. Diabetes was chemically induced with streptozotocin, and 14 nondiabetic and 14 diabetic rats were included in the study. The distal epiphysis of the right and left femurs of the diabetic rats were perforated with a surgical bone drill. This surgical procedure was performed on the left femurs of normal rats too. The wound on the right side of each diabetic rat received laser stimulation. The left femur of each nondiabetic (normal) rat served as a control. The rats were assigned to three experimental groups: (1) normal bur (control group); (2) diabetic bur; (3) diabetic bur + biostimulation. Results: There was a significant difference among all groups in substantia spongiosa formation on day 10. According to the Mann-Whitney U test, there was a difference between Groups 1 and 2. A significant difference was noted between Groups 2 and 3 as well as between Groups 1 and 3 and between Groups 2 and 3 in union at 20 d of healing. Conclusions: Substantia spongiosa formation was slightly more evident in Groups 1 and 3 than in Group 2. Also, there was more union in Group 3 than in the other groups on day 20. As a result, it can be concluded that low-level laser therapy (808 nm laser at 10 J/cm(2)) can have a beneficial effect on spongiosa in diabetic bone repair when five treatments are administered with 2 d intervals between treatments. Lasers Med Sci. 2009 Sep;24(5):689-95. Epub 2008 Sep 12.

Morphometric and histological analysis of low-power laser influence on bone morphogenetic protein in bone defects repair.
Denadai AS, de Carvalho Pde T, dos Reis FA, Belchior AC, Pereira DM, Dourado DM, Silva IS, de Oliveira LV. Postgraduate Program on Health and Development in West Central Region, Federal University of Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, Brazil.
Abstract

Bone morphogenetic proteins (BMPs) are secreted signaling molecules belonging to the transforming growth factor-beta (TGF-beta) superfamily. The objective of this study was to determine how gallium-aluminum-arsenium (GaAlAs) 650 nm laser influenced the action of BMPs on bone defects created in rat femurs. The sample consisted of 24 male albino Wistar rats. Group 1 was composed of rats with bone defects filled with bone-inducing substance, with the

application of low-power laser. Group 2 contained rats with bone defects filled with a boneinducing substance, without the application of low-power laser. Group 3 rats had bone defects not filled with a bone-inducing substance, with the application of low-power laser. Group 4 rats had bone defects and no treatment (control group). A bone defect was produced with drills. In groups 1 and 2 the defects were filled with a bone-inducing substance. The animals were treated with GaAlAs (50 mW) laser, energy density 4 J/cm(2), for 80 ss on a 1 cm(2) area. Groups 2 and 4 were used as control. Bone samples were removed for histological procedures and morphometric analysis on the 7th, 14th and 21st days after surgery. Results obtained were subjected to statistical analysis. Rejection level for the null hypothesis was 0.05. Statistical differences were found in the comparison between group 1 (G1), G2, G3 and G4 [analysis of variance (ANOVA); P < 0.0134]. There was a statistically significant correlation between groups 1 and 4 (P < 0.01). The results of other correlations by Tukey's post-hoc test were: group 1 vs group 3 (P = 0.341), group 1 vs group 2 (P = 0.862), group 2 vs group 4 (P = 0.061), group 2 vs group 3 (P = 0.744), and group 3 vs group 4 (P = 0.249). We concluded that the association of low-power laser with a bone-inducing substance produced better results than when low-power laser or BMPs were used alone. Photomed Laser Surg. 2009 Aug;27(4):641-6.

The effects of infrared low-level laser therapy on healing of partial osteotomy of tibia in streptozotocin-induced diabetic rats.
Javadieh F, Bayat M, Abdi S, Mohsenifar Z, Razi S. Anatomy Department, Medical Faculty, Shahid Beheshti University, MC, Tehran, Iran. OBJECTIVE: The effects of low-level laser therapy (LLLT) on a bone defect model in streptozotocin-induced diabetic (STZ-D) rats was examined. BACKGROUND DATA: LLLT accelerates bone fracture repair in healthy animals, but its effect in diabetic animals is unclear. METHODS: Twenty-eight rats were divided into five groups: 1 (diabetes, no LLLT), 2 (diabetes, LLLT high dose), 3 (diabetes, LLLT low dose), 4 (no diabetes, no LLLT), and 5 (no diabetes, LLLT low dose) Diabetes was induced by a single injection of STZ in rats of groups 1, 2, and 3. A bone defect was made in the right tibia of rats in all groups. The defect in groups 2, 3, and 5 was treated with LLLT (890 nm, 70 W, 3000 Hz, circular beam shape, and 1 cm(2) spot size). Doses of 23.3 J/cm(2) (530 s) for group 2 and 11.6 J/cm(2) (265 s) for groups 3 and 5 were applied three times a week. The right tibias were collected 42 days after surgery and subjected to three-point bending test on a material testing machine (MTM) until fracture occurred. Data was automatically recorded on the MTM formed the load-deformation curve. RESULTS: MannWhitney test showed that LLLT with 11.6 J/cm(2) significantly increased bending stiffness and maximum force in diabetic rats compared with group 1 (both p = 0.041). CONCLUSION: LLLT in an experimental diabetic model enhanced bone repair with a higher bending stiffness and maximum force compared to the control group. Lasers Med Sci. 2009 Jul;24(4):527-33. Epub 2008 Jul 15

Effect of soft laser and bioactive glass on bone regeneration in the treatment of bone defects (an experimental study).
AboElsaad NS, Soory M, Gadalla LM, Ragab LI, Dunne S, Zalata KR, Louca C. Department of Oral Medicine and Periodontology, Faculty of Dentistry, Mansoura University, Mansoura, Egypt. This study aimed to investigate the influence of low-power gallium-aluminium-arsenide (GaAlAs) laser [830 nm, continuous wave (CW), 40 mW and fluence 4 J/cm(2)] on the healing of surgically created bone defects in rats treated with bioactive glass graft material. Surgical bone defects were created in the mandibles of 36 Wistar rats divided into two groups, each consisting of 18 rats. Group I was treated with bioactive glass plus laser irradiation. Group II was treated with graft material only. The animals were killed at 4 weeks, 8 weeks and 12 weeks postoperatively for histological examination. Laser irradiation had significantly accelerated bone healing at 4 weeks and 8 weeks in comparison with that at the sites not irradiated. However at 12 weeks, complete healing of the defects had occurred with no difference detected. Our results have confirmed the positive effect of soft laser in accelerating bone regeneration. Lasers Med Sci. 2009 Jun 23. [Epub ahead of print

Effects of low-level laser therapy on bone formation after distraction osteogenesis.

Hbler R, Blando E, Gaio L, Kreisner PE, Post LK, Xavier CB, de Oliveira MG. School of Physics, Pontifcia Universidade Catlica do Rio Grande do Sul (PUCRS), Av. Ipiranga, 6681, Prdio 10, sala 222, Caixa Postal 1429, 90619-900, Porto Alegre, RS, Brazil. This study evaluated the effect of low-level laser therapy (LLLT) on the chemical composition, crystallinity and crystalline structure of bone at the site of distraction osteogenesis. Five rabbits were subjected to distraction osteogenesis (latency = 3 days; rate and frequency = 0.7 mm/day for 7 days; consolidation = 10 days), and three were given LLLT with arsenide-galliumaluminum (AsGaAl; 830 nm, 40 mW): 10 J/cm(2) dose per spot, applied directly to the distraction osteogenesis site during the consolidation stage at 48 h intervals. Samples were harvested at the end of the consolidation stage. X-ray fluorescence and X-ray diffraction were used to analyze chemical composition, crystallinity and crystalline structure of bone at the distraction osteogenesis site. The analysis of chemical composition and calcium (Ca) and phosphorus (P) ratios revealed greater mineralization in the LLLT group. Diffractograms showed that the crystalline structure of the samples was similar to that of hydroxyapatites. Crystallinity percentages were greater in rabbits that were given LLLT. Crystallinity (41.14% to 54.57%) and the chemical composition of the bone at the distraction osteogenesis site were similar to the that of the control group (42.37% to 49.29%). The results showed that LLLT had a positive effect on the biomodulation of newly formed bone.

Lasers Med Sci. 2009 May;24(3):347-52. Epub 2008 Jul 22.

Effect of low-power gallium-aluminum-arsenium laser therapy (830 nm) in combination with bisphosphonate treatment on osteopenic bone structure: an experimental animal study.
Diniz JS, Nicolau RA, de Melo Ocarino N, do Carmo Magalhes F, de Oliveira Pereira RD, Serakides R. Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraba, So Jos dos Campos, SP, Brazil.
Abstract

Laser therapy is able to modulate cell metabolism and accelerate the repair of fracture. Little attention has been given to the effect of laser on bone with osteopenia or osteoporosis. The purpose of our study was to verify the effect of laser therapy in combination with bisphosphonate on osteopenic bone structure. The 35 Wistar female rats used were divided into five groups: (1) sham-operation rats (control), (2) ovariectomized (OVX'd) rats with osteopenia, (3) OVX'd rats with osteopenia treated with laser, (4) OVX'd rats with osteopenia treated with bisphosphonate and (5) OVX'd rats with osteopenia treated with bisphosphonate and laser. Groups 3 and 5 were given daily 6 mg doses of bisphosphonate orally. Groups 4 and 5 underwent low level laser therapy [gallium-aluminum-arsenium (GaAlAs) laser, 830 nm, 50 mW and 4 J/cm(2)] on the femoral neck and vertebral segments (T13-L2). Both treatments were performed over an 8-week period. Rats from the osteopenic control and osteopenic + laser groups presented marked osteopenia. In the osteopenic + bisphosphonate group, the trabecular bone volume in vertebra L2 was significantly greater than in the osteopenic control group. Notably, in the association between laser and bisphosphonate, the trabecular bone volume was significantly greater in vertebrae L2 and T13 and was similar to that in the sham-operation control group. It was concluded that the laser therapy associated with bisphosphonate treatment was the best method for reversing vertebral osteopenia caused by the ovariectomy. Lasers Surg Med. 2009 Apr;41(4):298-304.

Superpulsed laser irradiation increases osteoblast activity via modulation of bone morphogenetic factors.
Saracino S, Mozzati M, Martinasso G, Pol R, Canuto RA, Muzio G. Department of Experimental Medicine and Oncology, University of Turin, Corso Raffaello 30, 10125 Turin, Italy.

Abstract

BACKGROUND AND OBJECTIVE: Laser therapy is a new approach applicable in different medical fields when bone loss occurs, including orthopedics and dentistry. It has also been used to induce soft-tissue healing, for pain relief, bone, and nerve regeneration. With regard to bone synthesis, laser exposure has been shown to increase osteoblast activity and decrease osteoclast number, by inducing alkaline phosphatase (ALP), osteopontin, and bone sialoprotein expression. Studies have investigated the effects of continuous or pulsed laser irradiation, but no data are yet available on the properties of superpulsed laser irradiation. This study thus aimed to investigate the effect of superpulsed laser irradiation on osteogenic activity of human osteoblast-like cells, paying particular attention to investigating the molecular mechanisms underlying the effects of this type of laser radiation. STUDY DESIGN/MATERIALS AND METHODS: Human osteoblast-like MG-63 cells were exposed to 3, 7, or 10 superpulsed laser irradiation (pulse width 200 nanoseconds, minimum peak power 45 W, frequency 30 kHz, total energy 60 J, exposure time 5 minutes). The following parameters were evaluated: cell growth and viability (light microscopy, lactate dehydrogenase release), calcium deposits (Alizarin Red S staining), expression of bone morphogenetic factors (real-time PCR). RESULTS: Superpulsed laser irradiation decreases cell growth, induces expression of TGFbeta2, BMP-4, and BMP-7, type I collagen, ALP, and osteocalcin, and increases the size and the number of calcium deposits. The stimulatory effect is maximum on day 10, that is, after seven applications. CONCLUSIONS: Reported results show that superpulsed laser irradiation, like the continuous and pulsed counterparts, possesses osteogenic properties, inducing the expression of molecules known to be important mediators of bone formation and, as a consequence, increasing calcium deposits in human MG-63 cells. Moreover, the data suggest a new potential role for PPARgamma as a regulator of osteoblast proliferation. Photomed Laser Surg. 2009 Apr;27(2):309-15.

Low-intensity pulsed laser irradiation affects RANKL and OPG mRNA expression in rat calvarial cells.
Xu M, Deng T, Mo F, Deng B, Lam W, Deng P, Zhang X, Liu S. School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China. xumin@hkbu.edu.hk

Abstract

OBJECTIVE: This study aimed to investigate the effect of low-intensity pulsed laser (LIPL; 650 nm, 2 mW) irradiation on mRNA expression of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) in rat calvarial cells. MATERIALS AND METHODS: Cultured cells were treated with LIPL irradiation of 1.14 J/cm(2) (group A) or 2.28 J/cm(2) (group B), and non-irradiated cells (group C) were used as controls. The changes in cell numbers, alkaline phosphatase (ALP) activity, RANKL, and OPG mRNA expression in the three study groups was determined using MTT, UV/VIS spectrophotometry, and RT-PCR analyses. RESULTS: The cell numbers in groups A and B increased significantly (7.52% and 8.80%, respectively), as did ALP activity (71.95% and 88.20%, respectively), compared with group C (p < 0.001). Meanwhile, RANKL and OPG mRNA expression in group A were 51.06% lower and 3.35 times higher, respectively, than those seen in the controls (p < 0.05), and the RANKL:OPG mRNA ratio in group A was 81.82% lower than that in group C (p < 0.005). CONCLUSION: LIPL irradiation may directly promote osteoblast proliferation and differentiation, and indirectly inhibit osteoclast differentiation, by downregulating the RANKL:OPG mRNA ratio in osteoblasts. Thus LIPL irradiation may play an important role in bone remodeling, and should be valuable for the treatment of bone diseases such as osteoporosis. Lasers Med Sci.s 2009 Mar;24(2):234-40. Epub 2008 Apr 17.

Bone repair following bone grafting hydroxyapatite guided bone regeneration and infra-red laser photobiomodulation: a histological study in a rodent model.
Pinheiro AL, Martinez Gerbi ME, de Assis Limeira F Jr, Carneiro Ponzi EA, Marques AM, Carvalho CM, de Carneiro Santos R, Oliveira PC, Nia M, Ramalho LM. Department of Propedeutica and Clnica Integrada, Laser Center, School of Dentistry, Federal University of Bahia, Salvador, BA, Brazil. albp@ufba.br
Abstract

The aim of the investigation was to assess histologically the effect of laser photobiomodulation (LPBM) on a repair of defects surgically created in the femurs of rats. Forty-five Wistar rats were divided into four groups: group I (control); group II (LPBM); group III (hydroxyapatite guided bone regeneration; HA GBR); group IV (HA GBR LPBM). The animals in the irradiated groups were subjected to the first irradiation immediately after surgery, and it was repeated every day for 2 weeks. The animals were killed 15 days, 21 days and 30 days after surgery. When the groups irradiated with implant and membrane were compared, it was observed that the repair of

the defects submitted to LPBM was also processed faster, starting from the 15th day. At the 30th day, the level of repair of the defects was similar in the irradiated groups and those not irradiated. New bone formation was seen inside the cavity, probably by the osteoconduction of the implant, and, in the irradiated groups, this new bone formation was incremental. The present preliminary data seem to suggest that LPMB therapy might have a positive effect upon early wound healing of bone defects treated with a combination of HA and GBR. Lasers Med Sci. 2009 Mar;24(2):195-201. Epub 2008 Feb 29.

Low-level laser therapy modulates cyclo-oxygenase-2 expression during bone repair in rats.
Matsumoto MA, Ferino RV, Monteleone GF, Ribeiro DA. Department of Oral Maxillofacial Surgery, School of Dentistry, University of the Sacred Heart (USC), Bauru, SP, Brazil.
Abstract

The goal of this study was to analyze the role of cyclo-oxygenase-2 following bone repair in rats submitted to low-level laser therapy. A total of 48 rats underwent surgery to inflict bone defects in their tibias having been randomly distributed into two groups: negative control and laser exposed group, ie, the animals were treated with low-level laser therapy by means of gallium arsenide laser at 16 J/cm(2). The animals were killed after 48 h, 7 days, 14 days, or 21 days. The tibias were removed for morphological, morphometric, and immunohistochemistry analysis for cyclo-oxygenase-2. Statistical significant differences (P < 0.05) were observed in the quality of bone repair and quantity of formed bone between groups 14 days after surgery in the laser exposed group. In the same way, cyclo-oxygenase-2 immunoreactivity was more intense in bone cells for intermediate periods evaluated in this group. Taken together, such results suggest that low-level laser therapy is able to improve bone repair in the tibia of rats after 14 days of surgery as a result of an up-regulation for cyclo-oxygenase-2 expression in bone cells. Wien Klin Wochenschr. 2008;120(3-4):112-7.

Initial effects of low-level laser therapy on growth and differentiation of human osteoblast-like cells.
Stein E, Koehn J, Sutter W, Wendtlandt G, Wanschitz F, Thurnher D, Baghestanian M, Turhani D. Department of Cranio-Maxillofacial and Oral Surgery, Medical University of Vienna, Austria.

Abstract

Low-level laser therapy is a clinically well established tool for enhancement of wound healing. In vitro studies have also shown that low level laser therapy has a biostimulatory effect on cells of different origin. The aim of this in vitro study was to investigate the initial effect of low-level laser therapy on growth and differentiation of human osteoblast-like cells. SaOS-2 cells were irradiated with laser doses of 1 J/cm2 and 2 J/cm2 using a diode laser with 670 nm wave length and an output power of 400 mW. Untreated cells were used as controls. At 24 h, 48 h and 72 h post irradiation, cells were collected and assayed for viability of attached cells and alkaline phosphatase specific activity. In addition, mRNA expression levels of osteopontin and collagen type I were assessed using semi-quantitative RT-PCR. Over the observation period, cell viability, alkaline phosphatase activity and the expression of osteopontin and collagen type I mRNA were slightly enhanced in cells irradiated with 1 J/cm2 compared with untreated control cells. Increasing the laser dose to 2 J/cm2 reduced cell viability during the first 48 h and resulted in persistently lower alkaline phosphatase activity compared with the other two groups. The expression of osteopontin and collagen type I mRNA slightly decreased with time in untreated controls and cells irradiated with 1 J/cm2, but their expression was increased by treatment with 2 J/cm2 after 72 h. These results indicate that low-level laser therapy has a biostimulatory effect on human osteoblast-like cells during the first 72 h after irradiation. Further studies are needed to determine the potential of low-level laser therapy as new treatment concept in bone regeneration. J Contemp Dent Pract. 2008 Sep 1;9(6):41-8.

Histologic study of the effect of laser therapy on bone repair.

Blaya DS, Guimares MB, Pozza DH, Weber JB, de Oliveira MG. Centro Universitrio Franciscano, Santa Maria, RS, Brazil.
Abstract

AIM: This study used histologic analysis and HE staining to evaluate laser biomodulation of bone repair in cavities made in the femurs of rats that underwent non-ablative laser irradiation. METHODS AND MATERIALS: Eighteen male Wistar rats weighing 300 to 400 grams were randomly assigned to three groups of six animals each. A surgical defect site was produced with a trephine about 2 mm in diameter under abundant irrigation. In Group I the complete surgical protocol to produce a bone defect was followed but without laser radiation (control). In Group II a continuous wave 830 nm infrared laser was used at 10 J/cm2 and 50 mW at each point of the surgical site. In Group III a continuous wave 685 nm infrared laser at 10J/cm2 and 35 mW was used at each point of surgical site. The animals were irradiated at intervals of 48 hours beginning immediately after the preparation of the defect and were sacrificed on the 15th, 21st, and 30th days. Slides were studied by means of descriptive analysis.

RESULTS: Greater degrees of new bone formation and vertical regeneration were found in the irradiated groups than in the control group. CONCLUSION: Laser therapy in this study protocol was efficient in promoting bone repair. CLINICAL SIGNIFICANCE: The use of laser technology has been used to improve the clinical results of bone surgeries and to promote a more comfortable postoperative period and quicker healing. Photomed Laser Surg. 2008 Aug;26(4):301-6.

Laser-needle therapy for spontaneous osteonecrosis of the knee.

Banzer W, Hbscher M, Schikora D. Department of Sports Medicine, Goethe-University Frankfurt/Main, University of Paderborn, Frankfurt/Main, Germany. banzer@sport.uni-frankfurt.de
Abstract

OBJECTIVE: This case report describes the treatment of a 63-year-old patient with spontaneous osteonecrosis of the knee (SONK). BACKGROUND DATA: SONK usually appears in the elderly patient without the typical risk factors for osteonecrosis. It is characterized by acute and sudden pain, mostly occurring at the medial side of the knee joint. Symptoms usually worsen with physical activity and improve with rest. Besides physical therapy, limited weight-bearing and the use of analgesics and nonsteroidal anti-inflammatory drugs, we propose low-level laser therapy (LLLT) as a conservative treatment option. METHODS: LLLT was carried out using laser needles emitting radiation with wavelengths of 685 and 885 nm, and a power density of 17.8 W/cm(2). Therapy sessions lasted 60 min and were performed daily over a period of 3 mo. The total irradiation dose emitted by 8 laser needles in 60 min of treatment was 1008 J. RESULTS: Magnetic resonance imaging revealed distinct restitution of the spongiosa edema 5 wk after treatment onset, and the final check-up at 35 wk demonstrated complete restoration of integrity. CONCLUSION: The present case report provides the first indication that laser-needle therapy may be a promising tool for complementary and alternative therapeutic intervention for those with SONK. Photomed Laser Surg. 2008 Aug;26(4):401-4.

Evaluation of low-level laser therapy of osteoblastic cells.

Pires Oliveira DA, de Oliveira RF, Zangaro RA, Soares CP. Laboratrio de Dinmica de Compartimentos Celulares, Instituto de Pesquisa e Desenvolvimento, UNIVAP, So Jos dos Campos, So Paulo, Brazil.
Abstract

OBJECTIVE: The purpose of the present study was to evaluate the effect of biomodulation on osteoblastic cells using a gallium-aluminium-arsenide diode laser. BACKGROUND DATA: Low-level laser therapy (LLLT) is a non-pharmacological therapeutic resource to which biological tissues respond well, producing such effects as the acceleration of bone formation and bone repair. MATERIALS AND METHODS: Osteoblastic cell cultures (OFCOL II) were irradiated with a gallium-aluminium-arsenide diode laser (GaAlAs lambda = 830 nm; 50 mW; 3 J/cm(2); 600microm-diameter optical fiber) and divided into two groups: group 1irradiated cells, and group 2non-irradiated cells. Irradiation occurred at 24-h intervals for a total of 3 d. After each interval, the cells were marked with Mito Tracker Orange dye to assess the biostimulatory effect on mitochondrial activity and cell proliferation using an MTT assay. RESULTS: Intense grouping of mitochondria in the perinuclear region was observed at 24 h and 48 h following irradiation. Changes from a filamentous to a granular appearance in mitochondrial morphology and mitochondria distributed throughout the cytoplasm were observed 72 h following proliferation. Such changes led to an in vitro proliferation process, as confirmed by the MTT assay. CONCLUSION: LLLT has shown itself capable of altering mitochondrial activity and the population of OFCOL II cells. Photomed Laser Surg. 2008 Feb;26(1):55-60.

Infrared laser light further improves bone healing when associated with bone morphogenic proteins: an in vivo study in a rodent model.
Gerbi ME, Marques AM, Ramalho LM, Ponzi EA, Carvalho CM, Santos Rde C, Oliveira PC, Nia M, Pinheiro AL. School of Dentistry, Federal University of Pernambuco, Recife, PE, Salvador. OBJECTIVE: This study assessed histologically the effect of laser photobiomodulation (LPBM) on the repair of surgical defects created in the femurs of Wistar rats treated or not treated with

bone morphogenic proteins (BMPs) and organic bovine bone graft. BACKGROUND DATA: This paper is part of an ongoing series of works in which biomaterials are used in association with LPBM. Several previous reports by our group have shown that the use of laser photobiomodulation improves the treatment of bone defects. MATERIALS AND METHODS: Forty-eight adult male Wistar rats were divided into four randomized groups: group I (control, n = 12); group II (LPBM, n = 12); group III (BMPs + organic bovine bone graft, n = 12); and group IV (BMPs + organic bovine bone graft + LPBM, n = 12). The irradiated groups received seven irradiations every 48 h, beginning immediately after the surgical procedure. The laser therapy (lambda = 830 nm, 40 mW CW, varphi = 0.6 mm) consisted of 16 J/cm(2) per session divided equally over four points (4 J/cm(2) each) around the defect. The subjects were sacrificed after 15, 21, and 30 d, and the specimens were routinely embedded in wax, stained with hematoxylin and eosin and sirius red, and analyzed under light microscopy. RESULTS: The results showed histological evidence of increased deposition of collagen fibers (at 15 and 21 d), as well as an increased amount of well-organized bone trabeculae at the end of the experimental period (30 d) in the irradiated animals versus the non-irradiated controls. CONCLUSION: The use of LPBM with BMPs and organic bovine bone grafts increases the positive biomodulating effects of laser light. J Oral Rehabil. 2008 Dec;35(12):925-33.

Low-level laser therapy improves bone repair in rats treated with antiinflammatory drugs.
Ribeiro DA, Matsumoto MA. Department of Biosciences, Federal University of Sao Paulo, UNIFESP, Santos, SP, Brazil. daribeiro@unifesp.br
Abstract

Nowadays, selective cyclooxygenase-2 non-steroidal anti-inflammatory drugs have been largely used in surgical practice for reducing oedema and pain. However, the association between these drugs and laser therapy is not known up to now. Herein, the aim of this study was to evaluate the action of anti-COX-2 selective drug (celecoxib) on bone repair associated with laser therapy. A total of 64 rats underwent surgical bone defects in their tibias, being randomly distributed into four groups: Group 1) negative control; Group 2) animals treated with celecoxib; Group 3) animals treated with low-level power laser and Group 4) animals treated with celecoxib and lowlevel power laser. The animals were killed after 48 h, 7, 14 and 21 days. The tibias were removed for morphological, morphometric and immunohistochemistry analysis for COX-2. Statistical significant differences (P < 0.05) were observed in the quality of bone repair and quantity of formed bone between groups at 14 days after surgery for Groups 3 and 4. COX-2 immunoreactivity was more intense in bone cells for intermediate periods evaluated in the laserexposed groups. Taken together, such results suggest that low-level laser therapy is able to improve bone repair in the tibia of rats as a result of an up-regulation for cyclooxygenase-2 expression in bone cells.

Photomed Laser Surg. 2008 Aug;26(4):371-7.

Does the use of laser photobiomodulation, bone morphogenetic proteins, and guided bone regeneration improve the outcome of autologous bone grafts? An in vivo study in a rodent model.
Torres CS, dos Santos JN, Monteiro JS, Amorim PG, Pinheiro AL. Centro Baiano de Estudos Odontolgicos, Especializao em Cirurgia e Traumatologia Bucomaxilofaciais, Universidade Federal da Bahia, Salvador, Brazil.
Abstract

OBJECTIVE: The aim of the present investigation was to histologically assess the effect of laser photobiomodulation (LBPM) on the repair of autologous bone grafts in a rodent model. BACKGROUND DATA: A major problem in modern dentistry is the recovery of bone defects caused by trauma, surgical procedures, or pathologies. Several types of biomaterials have been used to improve the repair of these defects. These materials are often associated with procedures of guided bone regeneration (GBR). MATERIALS AND METHODS: Twenty four animals were divided into four groups: group I (control); group II (LPBM of the bone graft); group III (bone morphogenetic proteins [BMPs] + bone graft); and group IV (LPBM of the bed and the bone graft + BMPs). When appropriate the bed was filled with lyophilized bovine bone and BMPs used with or without GBR. The animals in the irradiated groups received 10 J/cm(2) per session divided over four points around the defect (4 J/cm(2)), with the first irradiation immediately after surgery, and then repeated seven times every other day. The animals were humanely killed after 40 d. Results: The results showed that in all treatment groups, new bone formation was greater and qualitatively better than the untreated subjects. Control specimens showed a less advanced repair after 40 d, and this was characterized by the presence of medullary tissue, a small amount of bone trabeculi, and some cortical repair. CONCLUSION: We conclude that LPBM has a positive biomodulatory effect on the healing of bone defects, and that this effect was more evident when LPBM was performed on the surgical bed intraoperatively, prior to the placement of the autologous bone graft. Lasers Med Sci. 2008 Jul;23(3):313-7. Epub 2007 Sep 20.

Effect of IR laser photobiomodulation on the repair of bone defects grafted with organic bovine bone.
Mrquez Martnez ME, Pinheiro AL, Ramalho LM.

School of Dentistry, Pernambuco University, Camaragibe, Pernambuco, Brazil.

Abstract

A major problem on modern dentistry is the recovery of bone defects of different etiologies. Biomaterials are used to improve the repair of these defects. Previous studies have shown positive effects of Laser Photobiomodulation (LPBM) on the repair of both soft and bone tissues. This study assessed histologically the effect of LPBM on the repair of surgical defects on the femur of rats filled with lyophilized bovine bone. The animals were divided into three groups: group I (control); group II (graft); group III (graft + LPBM). The animals on the irradiated groups received 16 J/cm(2) per session divided into four points around the defect being the first irradiation immediately after surgery and repeated at every 48 h during 2 weeks. Animal death occurred 15, 21, and 30 days after surgery. The specimens were routinely processed and stained with H/E and Sirius red and analyzed by light microscopy. There was histological evidence of improved collagen fiber deposition at early stages of the healing; increased amount of wellorganized bone trabeculae at the end of the experimental period on irradiated animals. It is concluded that LPBM has positive biomodulative effect on the healing process bone defects. Lasers Med Sci. 2008 Jul;23(3):313-7. Epub 2007 Sep 20.

Effect of IR photobiomodulation on the repair of bone defects grafted with organic bovine bone.
Mrquez Martnez ME, Pinheiro AL, Ramalho LM. School of Dentistry, Pernambuco University, Camaragibe, Pernambuco, Brazil. A major problem on modern dentistry is the recovery of bone defects of different etiologies. Biomaterials are used to improve the repair of these defects. Previous studies have shown positive effects of Laser Photobiomodulation (LPBM) on the repair of both soft and bone tissues. This study assessed histologically the effect of LPBM on the repair of surgical defects on the femur of rats filled with lyophilized bovine bone. The animals were divided into three groups: group I (control); group II (graft); group III (graft + LPBM). The animals on the irradiated groups received 16 J/cm(2) per session divided into four points around the defect being the first irradiation immediately after surgery and repeated at every 48 h during 2 weeks. Animal death occurred 15, 21, and 30 days after surgery. The specimens were routinely processed and stained with H/E and Sirius red and analyzed by light microscopy. There was histological evidence of improved collagen fiber deposition at early stages of the healing; increased amount of wellorganized bone trabeculae at the end of the experimental period on irradiated animals. It is concluded that LPBM has positive biomodulative effect on the healing process bone defects. Photomed Laser Surg. 2008 Feb;26(1):37-46

Nd:YAG laser biostimulation in the treatment of biphosphonateassociated osteonecrosis of the jaw: clinical experience in 28 cases.
Vescovi P, Merigo E, Manfredi M, Meleti M, Fornaini C, Bonanini M, Rocca JP, Nammour S. Unit of Oral Pathology and Medicine, Section of Dentistry, Department of ENT/Dental/Ophthalmological and Cervico-Facial Sciences, University of Parma, Parma, Italy. paolo.vescovi@unipr.it OBJECTIVE: To research an efficient treatment for the management of bisphosphonateassociated osteonecrosis. BACKGROUND DATA: Necrosis of the jawbone has recently been described in association with systemic bisphosphonate therapy with drugs including zoledronic acid, pamidronate, and alendronate. The extent and clinical characteristics of bisphosphonate-associated osteonecrosis (BON) of the jaw are extremely variable, and range from the presence of fistulae in the oral mucosa or orofacial tissues, to large exposed areas of necrotic bone within the oral cavity. Clinical signs and symptoms commonly reported include pain, swelling, the presence of pus, loose teeth, ill-fitting dentures, and paresthesias of the inferior alveolar nerve when the necrosis affects the mandible. Fractures have also been reported. The treatment of BON of the jaw is still controversial since no therapy has proven to be efficacious as shown by the literature on the subject. MATERIALS AND METHODS: In this study we report results achieved with 28 patients affected by BON of the jaw, who received treatment with the Nd:YAG laser alone or in combination with conventional medical or surgical treatment. Clinical variables such as severity of symptoms, presence of pus, and closure of mucosal flaps before and after therapy were evaluated to establish the effectiveness of laser irradiation. The 28 patients with BON were subdivided into four groups: eight patients were treated with medical therapy only (antibiotics with or without antimycotics and/or antiseptic rinses), six patients were treated with medical and surgical therapy (necrotic bone removal and bone curettage), six patients were treated with medical therapy associated with laser biostimulation, and eight patients were treated with medical therapy associated with both surgical therapy and laser biostimulation. RESULTS: Of the 14 patients who underwent laser biostimulation, nine reported complete clinical success (no pain, symptoms of infection, or exposed bone or draining fistulas), and three improved their symptomatology only, with a follow-up of between 4 and 7 mo. CONCLUSIONS: While the results reported in this study are not conclusive, they indicate that laser therapy has potential to improve management of BON. Lasers Surg Med. 2007 Dec;39(10):788-96.

Effect of low-level laser therapy on bone repair: histological study in rats.

Pretel H, Lizarelli RF, Ramalho LT. Department of Orthodontics and Pediatric Dentistry, School of Dentistry of Araraquara, So Paulo State University, Rua Humait 1680, Araraquara, SP, Brazil. hpretel@hotmail.com
Abstract

BACKGROUND AND OBJECTIVES: Bone remodeling is characterized as a cyclic and lengthy process. It is currently accepted that not only this dynamics is triggered by a biological process, but also biochemical, electrical, and mechanical stimuli are key factors for the maintenance of bone tissue. The hypothesis that low-level laser therapy (LLLT) may favor bone repair has been suggested. The purpose of this study was to evaluate the bone repair in defects created in rat lower jaws after stimulation with infrared LLLT directly on the injured tissue. STUDY DESIGN/MATERIALS AND METHODS: Bone defects were prepared on the mandibles of 30 Holtzman rats allocated in two groups (n = 15), which were divided in three evaluation period (15, 45, and 60 days), with five animals each. control group-no treatment of the defect; laser group-single laser irradiation with a GaAlAs semiconductor diode laser device (lambda = 780 nm; P = 35 mW; t = 40 s; Theta = 1.0 mm; D = 178 J/cm(2); E = 1.4 J) directly on the defect area. The rats were sacrificed at the pre-established periods and the mandibles were removed and processed for staining with hematoxylin and eosin, Masson's Trichrome and picrosirius techniques. RESULTS: The histological results showed bone formation in both groups. However, the laser group exhibited an advanced tissue response compared to the control group, abbreviating the initial inflammatory reaction and promoting rapid new bone matrix formation at 15 and 45 days (P<0.05). On the other hand, there were no significant differences between the groups at 60 days. CONCLUSION: The use of infrared LLLT directly to the injured tissue showed a biostimulating effect on bone remodeling by stimulating the modulation of the initial inflammatory response and anticipating the resolution to normal conditions at the earlier periods. However, there were no differences between the groups at 60 days. Photomed Laser Surg. 2007 Dec;25(6):487-94.

Effect of lower-level laser therapy on rabbit tibial fracture.

Liu X, Lyon R, Meier HT, Thometz J, Haworth ST. Musculoskeletal Functional Assessment Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53201, USA. xcliu@mcw.edu

Abstract

OBJECTIVE: The purpose of the study was to demonstrate the biological effects of low-level laser therapy (LLLT) on tibial fractures using radiographic, histological, and bone density examinations. METHODS: Fourteen New Zealand white rabbits with surgically induced mid-tibial osteotomies were included in the study. Seven were assigned to a group receiving LLLT (LLLT-A) and the remaining seven served as a sham-treated control group (LLLT-C). A low-energy laser apparatus with a wavelength of 830 nm, and a sham laser (a similar design without laser diodes) were used for the study. Continuous outflow irradiation with a total energy density of 40 J/cm(2) and a power level of 200 mW/cm(2) was directly delivered to the skin for 50 seconds at four points along the tibial fracture site. Treatment commenced immediately postsurgery and continued once daily for 4 weeks. RESULTS: Radiographic findings revealed no statistically significant fracture callus thickness difference between the LLLT-A and LLLT-C groups (p > 0.05). However, the fractures in the LLLT-A group showed less callus thickness than those in LLLT-C group 3 weeks after treatment. The average tibial volume was 14.5 mL in the LLLT-A group, and 11.25 mL in the LLLT-C group. The average contralateral normal tibial volume was 7.1 mL. Microscopic changes at 4 weeks revealed an average grade of 5.5 and 5.0 for the LLLT-A group and the LLLT-C group, respectively. The bone mineral density (BMD) as ascertained using a grey scale (graded from 0 to 256) showed darker coloration in the LLLT-A group (138) than in the LLLT-C group (125). CONCLUSION: The study suggests that LLLT may accelerate the process of fracture repair or cause increases in callus volume and BMD, especially in the early stages of absorbing the hematoma and bone remodeling. Further study is necessary to quantify these findings. Lasers Med Sci. 2007 Oct 31; [Epub ahead of print]

Relevance of laser irradiance threshold in the induction of alkaline phosphatase in human osteoblast cultures.
Haxsen V, Schikora D, Sommer U, Remppis A, Greten J, Kasperk C. Faculty of Science, Biophotonics Group, University of Paderborn, Paderborn, Germany, volker.haxsen@urz.uni-heidelberg.de . Induction of matrix synthesis by low-level laser has been demonstrated extensively. However, the question of dose- or power intensity-dependency is under-investigated. To address this issue we chose human osteoblast cell cultures and measured their alkaline phosphatase (ALP) activity after laser irradiation. The cell cultures were irradiated periodically by 690 nm radiation via optical transmission fiber-based laser needles, reaching into the culture dishes. The osteoblasts showed no induction of ALP activity when we used a single laser needle stimulation with a laser

irradiance of 51 mW/cm(2), an increase of approximately 43% at 102 mW/cm(2) irradiance (two needles per well) and a ninefold increase at 204 mW/cm(2) irradiance (four needles per well), leaving the temperature of the culture medium unaffected. We concluded that the osteoblastic response in ALP activity to a laser stimulus shows a logarithmic relationship, with a distinct threshold, rather than a linear dose-dependency. Secondly, the laser irradiance, rather than the dose, is relevant for the impact of the laser. Photomed Laser Surg. 2007 Aug;25(4):275-80.

The effects of laser irradiation on osteoblast and osteosarcoma cell proliferation and differentiation in vitro.
Renno AC, McDonnell PA, Parizotto NA, Laakso EL. Physical Therapy Department, Federal University of Sao Carlos, Sao Carlos, Brazil. a.renno@unifesp.br
Abstract

OBJECTIVE: The aim of this study was to investigate the effects of 670-nm, 780-nm, and 830nm laser irradiation on cell proliferation of normal primary osteoblast (MC3T3) and malignant osteosarcoma (MG63) cell lines in vitro. BACKGROUND: Some studies have shown that laser phototherapy is able to stimulate the osteogenesis of bone tissue, increasing osteoblast proliferation and accelerating fracture consolidation. It has been suggested that laser light may have a biostimulatory effect on tumor cells. However, the mechanism by which the laser acts on cells is not fully understood. MATERIALS AND METHODS: Neonatal, murine, calvarial, osteoblastic, and human osteosarcoma cell lines were studied. A single laser irradiation was performed at three different wavelengths, at the energies of 0.5, 1, 5, and 10 J/cm(2). Twenty-four hours after laser irradiation, cell proliferation and alkaline phosphatase assays were assessed. RESULTS: Osteoblast proliferation increased significantly after 830-nm laser irradiation (at 10 J/cm(2)) but decreased after 780-nm laser irradiation (at 1, 5, and 10 J/cm(2)). Osteosarcoma cell proliferation increased significantly after 670-nm (at 5 J/cm(2)) and 780-nm laser irradiation (at 1, 5, and 10 J/cm(2)), but not after 830-nm laser irradiation. Alkaline phosphatase (ALP) activity in the osteoblast line was increased after 830-nm laser irradiation at 10 J/cm(2), whereas ALP activity in the osteosarcoma line was not altered, regardless of laser wavelength or intensity. CONCLUSION: Based on the conditions of this study, we conclude that each cell line responds differently to specific wavelength and dose combinations. Further investigations are required to investigate the physiological mechanisms responsible for the contrasting outcomes obtained when using laser irradiation on cultured normal and malignant bone cells.

J Photochem Photobiol B. 2007 Jul 27;88(1):11-5. Epub 2007 May 1.

The therapeutic effect of low-level laser on repair of osteochondral defects in rabbit knee.
Kamali F, Bayat M, Torkaman G, Ebrahimi E, Salavati M. Department of Physical Therapy, University of Social Welfare and Rehabilitation, Tehran, Iran. fahimehkamali@hotmail.com
Abstract

INTRODUCTION: Low level laser therapy (LLLT) has been shown to enhance collagen production and wound healing but its effect on cartilage repair from biomechanical point of view is not known yet. The aim of present study was to evaluate the biomechanical behaviour of repairing osteochondral defect in rabbits which received a pulsed low-level gallium-arsenide (Ga-As) laser irradiation. MATERIALS AND METHODS: Osteochondral defects with 5mm diameter and 4mm in depth induced by drilling in right femoral patellar grooves of 41 adolescent male rabbits. They were divided into experimental and control groups. Experimental group received pulsed Ga-As (890nm) laser irradiation with energy density of 4.8J/cm(2). The rabbits in control group received placebo LLLT with shut-down equipment. The control defects were allowed to heal spontaneously. Each group were divided into three subgroups: A, B and C. Subgroups A, B and C were sacrificed on 4, 8, and 16 weeks after surgery. The knee joint were removed, and the defects were examined biomechanically by in situ-indentation method. The thickness, instantaneous and equilibrium indentation stiffness was measured during the test. Data were analysed using ANOVA and independent sample t-test. RESULT: While no difference was observed in the repaired cartilage biomechanical properties among 4th, 8th, 16th weeks in study groups. The equilibrium indentation stiffness of experimental group was significantly higher in 8th week in comparison with control group. CONCLUSION: LLLT significantly enhances the stiffness of repairing tissue in the 8th week post injury in osteochondral defects in rabbits. Implant Dent. 2007 Jun;16(2):204-11.

Histologic comparison of light emitting diode phototherapy-treated hydroxyapatite-grafted extraction sockets: a same-mouth case study.
Brawn PR, Kwong-Hing A.

343 Railway Street, Vancouver, British Columbia, Canada. brawn_peter@yahoo.ca

Abstract

BACKGROUND: The stimulating effect of red and near-infrared (NIR) laser phototherapy on bone regeneration and growth has been shown in a number of in vitro and animal studies. However, the effect of NIR phototherapy on the bone regeneration of hydroxyapatite (HA) treated extraction sockets has not been previously demonstrated. MATERIALS AND METHODS: An investigational Biolux extraoral light emitting diode phototherapy device was used daily for 21 days postextraction and socket grafting with HA (Osteograf LD300) unilaterally. Bone regeneration of the phototherapy-treated and nontreated side was compared in same-mouth extraction sockets. RESULTS: Histologic evaluations showed enhanced bone formation and faster particle resorption associated with the phototherapy-treated socket graft compared with the nonphototherapy-treated socket. CONCLUSIONS: The accelerated bone healing in the phototherapy-treated HA socket graft may provide faster implant placement compared to non-phototherapy-treated socket grafts. J Oral Maxillofac Surg. 2007 Feb;65(2):168-76.

Low-level laser effect on mandibular osteogenesis.

Miloro M, Miller JJ, Stoner JA. Section of Oral and Maxillofacial Surgery, University of Nebraska Medical Center, Omaha, NE 68198-5180, USA. mmiloro@unmc.edu Abstract PURPOSE: The purpose of this study was to determine whether low-level laser (LLL) application during distraction osteogenesis could accelerate bone regeneration and decrease the length of the consolidation phase and thereby reduce potential patient morbidity. MATERIALS AND METHODS: Nine adult female New Zealand white rabbits underwent bilateral mandibular corticotomies and placement of unidirectional distraction devices (KLSMartin LP, Jacksonville, FL). Each rabbit served as its own internal control. After a latency of 1 day, distraction progressed bilaterally at 1 mm per day for 10 days. Immediately after each device activation, the experimental side, chosen randomly, was treated with real LLL (Laser Medical Systems, Hedehusene, Denmark) of 6.0 J x 6 transmucosal sites in the area of the distraction gap. Radiographs were taken presurgically, immediately postsurgically, and weekly until sacrifice, and the bone was analyzed using a semiquantitative 4-point scale (Bone Healing

Score [BHS]). Three animals each were sacrificed at 2, 4, and 6 weeks postdistraction, and each hemimandible was prepared for histologic examination in a blinded fashion. RESULTS: Ten millimeters of distraction was achieved in each rabbit bilaterally. Radiographically, the BHS was higher for the LLL-treated group at all time periods. Histologically, the area of new bone trabeculation and ossification was more advanced for the LLL-treated group, with less intervening fibrovascular intermediate zone in the bony regenerate, at all time periods. The formation of a complete inferior border occurred sooner in the treatment group than in the controls. CONCLUSIONS: LLL accelerates the process of bone regeneration during the consolidation phase after distraction osteogenesis. The adjunctive use of LLL may allow a shortened period of consolidation and therefore permit earlier device removal, with the avoidance of morbidity associated with prolonged device retention. Minerva Stomatol. 2007 Jan-Feb;56(1-2):27-30.

Effect of superpulsed laser irradiation on bone formation in a human osteoblast-like cell line.
[Article in English, Italian] Martinasso G, Mozzati M, Pol R, Canuto RA, Muzio G. Department of Medicine and Experimental Oncology, Turin University, Turin, Italy.
Abstract

AIM: The effect superpulsed of low-level laser therapy (SLLLT) on bone regeneration has been the focus of recent research. This preliminary study investigated the effect of superpulsed laser irradiation on proliferation and bone formation in human osteoblast-like cells MG-63. METHODS: Human osteoblast-like cells MG-63 were exposed every 24 h to superpulsed lowlevel laser produced by the device Lumix 2 HFPL Dental (Fisioline snc, Verduno, Cuneo, Italy); the experimental protocol comprised 4 days of treatment. At each experimental time, cell proliferation and some markers of osteoblast activity were evaluated. RESULTS: Numbers of laser-treated cells increased starting from day 2 of treatment. The ability of SLLLT irradiation to stimulate bone production was evaluated by determining the expression of osteocalcin and alkaline phosphatase, proteins involved in calcium nodule formation. These proteins increased markedly after 3 days of laser treatment. CONCLUSIONS: These preliminary results show that repeated SLLLT irradiation stimulates cell proliferation in human osteoblast-like cells and, importantly, increases the expression of proteins essential for bone formation.

Photomed Laser Surg. 2006 Oct;24(5):642-5.

Effects of 830-nm laser light on preventing bone loss after ovariectomy.

Renno AC, de Moura FM, dos Santos NS, Tirico RP, Bossini PS, Parizotto NA. Department of Physiotherapy, Federal University of So Carlos, So Carlos, Brazil.
Abstract

OBJECTIVE: The aim of this study was to investigate the effects of low-level laser therapy (LLLT; infrared, 830 nm) on the bone properties and bone strength of rat femora after ovariectomy (OVX). BACKGROUND DATA: Osteoporosis affects 30% of postmenopausal women, and it has been recognized as a major public health problem. Based on the stimulatory effects of LLLT on proliferation of bone cells, we hypothesized that LLLT would be efficient in preventing bone mass loss in OVX rats. Methods: Forty female rats were divided into four groups: sham-operated control (SC), OVX control (OC), sham-operated irradiated at a dose of 120 J/cm(2) (I120), and OVX irradiated at a dose of 120 J/cm(2) (O120). Animals were operated at the age of 90 days. Laser irradiation was initiated 1 day after the operation and was performed three times a week, for 2 months. Femora were submitted to a biomechanical test and a physical properties evaluation. RESULTS: Maximal load of O120 was higher than in control groups. Wet weight, dry weight, and bone volume of O120 did not show any difference when compared with SC. CONCLUSION: The results of the present study indicate that LLLT was able to prevent bone loss after OVX in rats. However, further studies are needed to investigate the effects of different parameters, wavelengths, and sessions of applications on OVX rats J Oral Rehabil. 2006 Aug;33(8):619-924.

Effect of low intensity laser irradiation on surgically created bony defects in rats.
Nissan J, Assif D, Gross MD, Yaffe A, Binderman I. Department of Oral Rehabilitation, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel. nissandr@post.tau.ac.il

Abstract

Low intensity lasers have been used by clinicians to improve healing and reduce pain in humans. Lasing also results in new bone formation around hydroxyapatite implants and a significant increase in the total bone area. However, the exact mechanism of cell biostimulation by laser is still unclear. This study biochemically assessed the effects of low intensity laser (GalliumArsenide) using 4 and 22.4 mW cm(-2) power density on the bone healing process after surgically creating bony cavities in rat mandibles. Rats (n = 24) were divided into two groups each treated with specific energy, 4 or 22.4 mW cm(-2), for 3 min each day post-surgery. Surgical cavities were created on both sides of the mandible: the left served as an untreated control, the right was treated with laser. All rats were sacrificed after 1, 2 and 4 weeks of treatment. In the newly formed callus, accumulation of radiocalcium and alkaline phosphatase activity was measured to indicate osteogenic activity. One-way anova with repeated measures showed that the low intensity laser using 4 mW cm(-2) power density significantly increased radiocalcium accumulation from 2 weeks post-surgery, whereas 22.4 mW cm(-2) had no effect. No changes were noted in the activity of alkaline phosphatase with the laser treatment. These results suggest that laser therapy of low power density is effective on the bone healing process in artificially created osseous cavities by affecting calcium transport during new bone formation. Acta Biomed. 2006 Aug;77(2):109-17.

Bone necrosis of the jaws associated with biphosphonate treatment: a report of twenty-nine cases.
Merigo E, Manfredi M, Meleti M, Guidotti R, Ripasarti A, Zanzucchi E, D'Aleo P, Corradi D, Corcione L, Sesenna E, Ferrari S, Poli T, Bonaninil M, Vescovi P. Unit of Oral Pathology and Medicine, Section of Odontostomatology, Department of ENT/Dental/Ophthalmological and Cervico-Facial Sciences, University of Parma, Parma, Italy. elisabetta.merigo@unipr.it Bone necrosis of the jaws is often related to head and neck radiotherapy, to surgical procedures at maxillary or mandibular level but also to various local and systemic factors such as haematological diseases, haemoglobinopathies and systemic lupus eritematosus; its pathogenesis maybe associated with defects of vascularization. Bisphosphonate are synthetic analogues of pyrophosphate used for the treatment of hypercalcemia in patients with malignancies and bone metastasis and for the treatment of many other disorders such as metabolic bone diseases, Paget's disease, and osteoporosis; their pharmacological activity is related to the inhibition of the osteoclastic function which leads to resorption and reduction of bone vascularization. Since the end of 2003 Bisphosphonate-associated Osteonecrosis (BON) has become an increasing problem and the test of that is the increase of the relative published case report and case series. Here we report 29 cases of bone necrosis of the jaws in patients treated with pamidronate (Aredia), zoledronate (Zometa) and alendronate: 15 underwent surgical procedures and 14 occurred spontaneously. Among these patients (21 females, 8 males; mean age between 45 and 83 years); 14 were treated for bone metastasis, 12 for multiple myeloma and 3 for osteoporosis. Bone

necrosis involved only maxilla in 7 patients, only mandible in 20 patients and both in 2 patients. Six patients had multiple osteonecrotic lesions, 3 contemporary lesions and 3 non contemporary. In these patients we performed 3 kinds of therapy, associated or not: medical therapy (with antibiotic drugs, antimycotics and antiseptic mouthwashes), surgical therapy with curettage or sequestrectomy and Nd:YAG laser biostimulation. Acta Cir Bras. 2006;21 Suppl 4:63-8.

Effect of 650 nm low-power laser on bone morphogenetic protein in bone defects induced in rat femors.
Carvalho Pde T, Silva IS, Reis FA, Belchior AC, Facco GG, Guimares RN, Fernandes GH, Denadai AS. Department of Physiotherapy, UNIDERP, Brazil. ptpaulo@terra.com.br
Abstract

PURPOSE: To investigate the influence of 650 nm GaAlAs laser on the action of bone morphogenetic protein (BMP) in bone defects produced in rat femurs. METHODS: The sample consisted of 12 male albino Wistar rats (Rattus norvegicus). The animals were randomly divided into four experimental groups. After undergoing anesthesia, the fur was removed from the lateral face of the right thigh and surgical dissection was performed to view the femur region. A bone defect was created using a spherical diamond-tipped drill bit. In groups 1 and 2, the defect was filled with a paste of Gen-Tech bone-inducing substance. The animals were treated with GaAlAs laser, at a predetermined dose of joules/cm(2) for 80 seconds, over an area of 1 cm(2). Groups 2 and 4 were used as controls. Bone samples were removed to perform histological procedures and morphometric analyses on the 7th, 14th and 21st days after the operation. The results obtained were subjected to statistical analysis using ANOVA variance according to two criteria, with four repetitions, followed by the post hoc t test. The rejection level for the nullity hypothesis was 0.05 or 5% (alpha < or = 0.05). RESULTS: In comparisons between G1, G2, G3 and G4, p = 0.024 was observed. In statistical comparisons using the t test for paired samples, only G1 vs. G4 presented a statistically significant result (p = 0.021). CONCLUSION: The association of low-power laser application and Gen-Tech bone-inducing substance achieved a better result than laser application alone or BMP use alone. Photomed Laser Surg. 2005 Aug;23(4):382-8

Assessment of bone repair associated with the use of organic bovine bone and membrane irradiated at 830 nm.
Gerbi ME, Pinheiro AL, Marzola C, Limeira Jnior Fde A, Ramalho LM, Ponzi EA, Soares AO, Carvalho LC, Lima HV, Gonalves TO. School of Dentistry, Federal University of Bahia, Salvador, Brazil. OBJECTIVE: The aim of the present investigation was to assess histologically the effect of LLLT (GaAIAs, 830 nm, 40 mW, CW, (Phi) approximately 0.6 mm, 16 J/cm(2) per session) on the repair of surgical defects created in the femur of the Wistar Albinus rat. The defects were filled to lyophilized bovine bone (Gen-ox), organic matrix) associated or not to GTR (Genderm). BACKGROUND DATA: A major problem on modern Dentistry is the recovery of bone defects caused by trauma, surgical procedures or pathologies. Several types of biomaterials have been used in order to improve the repair of these defects. These materials are often associated to procedures of GTR. Previous studies have shown positive effects of LLLT on the repair of soft tissue wounds, but there are a few on its effects on bone healing. METHODS: Surgical bone defects were created in 42 animals divided into five groups: Group I (control, 6 animals); Group II (Gen-ox, 9 animals); Group III (Gen-ox + Laser, 9 animals); Group IV (Gen-ox + Gen-derm, 9 animals); Group V (Gen-ox + Gen-derm + Laser, 9 animals). The animals on the irradiated group received 16 J/cm(2) per session divided into four points around the defect (4 J/cm(2)) being the first irradiation immediately after surgery and repeated seven times at every 48 h. The animals were humanly killed after 15, 21, and 30 days. RESULTS: The results of the present investigation showed histological evidence of improved amount of collagen fibers at early stages of the bone healing (15 days) and increased amount of well organized bone trabeculae at the end of the experimental period (30 days) on irradiated animals compared to non irradiated ones. CONCLUSIONS: It is concluded that a positive biomodulative effect on the healing process of one defect associated or not to the use of organic lyophilized bone and biological bovine lyophilized membrane on the femur of the rat. Photomed Laser Surg. 2008 Aug;26(4):401-4

Evaluation of low-level laser therapy of osteoblastic cells.

Pires Oliveira DA, de Oliveira RF, Zangaro RA, Soares CP. Laboratrio de Dinmica de Compartimentos Celulares, Instituto de Pesquisa e Desenvolvimento (UNIVAP), So Jos dos Campos, So Paulo, Brazil.

Abstract Objective: The purpose of the present study was to evaluate the effect of biomodulation on osteoblastic cells using a gallium-aluminium-arsenide diode laser. Background Data: Lowlevel laser therapy (LLLT) is a non-pharmacological therapeutic resource to which biological tissues respond well, producing such effects as the acceleration of bone formation and bone repair. Materials and Methods: Osteoblastic cell cultures (OFCOL II) were irradiated with a galliumaluminium-arsenide diode laser (GaAlAs lambda = 830 nm; 50 mW; 3 J/cm(2); 600-mumdiameter optical fiber) and divided into two groups: group 1-irradiated cells, and group 2-nonirradiated cells. Irradiation occurred at 24-h intervals for a total of 3 d. After each interval, the cells were marked with Mito Tracker Orange dye to assess the biostimulatory effect on mitochondrial activity and cell proliferation using an MTT assay. Results: Intense grouping of mitochondria in the perinuclear region was observed at 24 h and 48 h following irradiation. Changes from a filamentous to a granular appearance in mitochondrial morphology and mitochondria distributed throughout the cytoplasm were observed 72 h following proliferation. Such changes led to an in vitro proliferation process, as confirmed by the MTT assay. Conclusion: LLLT has shown itself capable of altering mitochondrial activity and the population of OFCOL II cells. Wien Klin Wochenschr. 2008 Feb;120(3-4):112-117

Initial effects of low-level laser therapy on growth and differentiation of human osteoblast-like cells.
Stein E, Koehn J, Sutter W, Wendtlandt G, Wanschitz F, Thurnher D, Baghestanian M, Turhani D. Department of Cranio-Maxillofacial and Oral Surgery, Medical University of Vienna, Austria. Low-level laser therapy is a clinically well established tool for enhancement of wound healing. In vitro studies have also shown that low level laser therapy has a biostimulatory effect on cells of different origin. The aim of this in vitro study was to investigate the initial effect of low-level laser therapy on growth and differentiation of human osteoblast-like cells. SaOS-2 cells were irradiated with laser doses of 1 J/cm(2) and 2 J/cm(2) using a diode laser with 670 nm wave length and an output power of 400 mW. Untreated cells were used as controls. At 24 h, 48 h and 72 h post irradiation, cells were collected and assayed for viability of attached cells and alkaline phosphatase specific activity. In addition, mRNA expression levels of osteopontin and collagen type I were assessed using semi-quantitative RT-PCR. Over the observation period, cell viability, alkaline phosphatase activity and the expression of osteopontin and collagen type I mRNA were slightly enhanced in cells irradiated with 1 J/cm(2) compared with untreated control cells. Increasing the laser dose to 2 J/cm(2) reduced cell viability during the first 48 h and resulted in persistently lower alkaline phosphatase activity compared with the other two groups. The

expression of osteopontin and collagen type I mRNA slightly decreased with time in untreated controls and cells irradiated with 1 J/cm(2), but their expression was increased by treatment with 2 J/cm(2) after 72 h. These results indicate that low-level laser therapy has a biostimulatory effect on human osteoblast-like cells during the first 72 h after irradiation. Further studies are needed to determine the potential of low-level laser therapy as new treatment concept in bone regeneration. Lasers Med Sci. 2008 Feb 29 [Epub ahead of print]

Low-level laser therapy modulates cyclo-oxygenase-2 expression during bone repair in rats.
Matsumoto MA, Ferino RV, Monteleone GF, Ribeiro DA. Department of Oral Maxillofacial Surgery, School of Dentistry, University of the Sacred Heart (USC), Bauru, SP, Brazil. The goal of this study was to analyze the role of cyclo-oxygenase-2 following bone repair in rats submitted to low-level laser therapy. A total of 48 rats underwent surgery to inflict bone defects in their tibias having been randomly distributed into two groups: negative control and laser exposed group, ie, the animals were treated with low-level laser therapy by means of gallium arsenide laser at 16 J/cm(2). The animals were killed after 48 h, 7 days, 14 days, or 21 days. The tibias were removed for morphological, morphometric, and immunohistochemistry analysis for cyclo-oxygenase-2. Statistical significant differences (P < 0.05) were observed in the quality of bone repair and quantity of formed bone between groups 14 days after surgery in the laser exposed group. In the same way, cyclo-oxygenase-2 immunoreactivity was more intense in bone cells for intermediate periods evaluated in this group. Taken together, such results suggest that low-level laser therapy is able to improve bone repair in the tibia of rats after 14 days of surgery as a result of an up-regulation for cyclo-oxygenase-2 expression in bone cells. Photomed Laser Surg. 2007 Dec;25(6):487-94

Effect of lower-level laser therapy on rabbit tibial fracture.

Liu X, Lyon R, Meier HT, Thometz J, Haworth ST. Musculoskeletal Functional Assessment Center, Medical College of Wisconsin, Milwaukee, Wisconsin. Objective: The purpose of the study was to demonstrate the biological effects of low-level laser therapy (LLLT) on tibial fractures using radiographic, histological, and bone density examinations.

Methods: Fourteen New Zealand white rabbits with surgically induced mid-tibial osteotomies were included in the study. Seven were assigned to a group receiving LLLT (LLLT-A) and the remaining seven served as a sham-treated control group (LLLT-C). A low-energy laser apparatus with a wavelength of 830 nm, and a sham laser (a similar design without laser diodes) were used for the study. Continuous outflow irradiation with a total energy density of 40 J/cm(2) and a power level of 200 mW/cm(2) was directly delivered to the skin for 50 seconds at four points along the tibial fracture site. Treatment commenced immediately postsurgery and continued once daily for 4 weeks. Results: Radiographic findings revealed no statistically significant fracture callus thickness difference between the LLLT-A and LLLT-C groups (p > 0.05). However, the fractures in the LLLT-A group showed less callus thickness than those in LLLT-C group 3 weeks after treatment. The average tibial volume was 14.5 mL in the LLLT-A group, and 11.25 mL in the LLLT-C group. The average contralateral normal tibial volume was 7.1 mL. Microscopic changes at 4 weeks revealed an average grade of 5.5 and 5.0 for the LLLT-A group and the LLLT-C group, respectively. The bone mineral density (BMD) as ascertained using a grey scale (graded from 0 to 256) showed darker coloration in the LLLT-A group (138) than in the LLLT-C group (125). Conclusion: The study suggests that LLLT may accelerate the process of fracture repair or cause increases in callus volume and BMD, especially in the early stages of absorbing the hematoma and bone remodeling. Further study is necessary to quantify these findings. Photomed Laser Surg. 2007 Aug;25(4):275-80.

The effects of laser irradiation on osteoblast and osteosarcoma cell proliferation and differentiation in vitro.
Renno AC, McDonnell PA, Parizotto NA, Laakso EL. Physical Therapy Department, Federal University of Sao Carlos, Sao Carlos, Brazil. a.renno@unifesp.br OBJECTIVE: The aim of this study was to investigate the effects of 670-nm, 780-nm, and 830nm laser irradiation on cell proliferation of normal primary osteoblast (MC3T3) and malignant osteosarcoma (MG63) cell lines in vitro. BACKGROUND: Some studies have shown that laser phototherapy is able to stimulate the osteogenesis of bone tissue, increasing osteoblast proliferation and accelerating fracture consolidation. It has been suggested that laser light may have a biostimulatory effect on tumor cells. However, the mechanism by which the laser acts on cells is not fully understood. MATERIALS AND METHODS: Neonatal, murine, calvarial, osteoblastic, and human osteosarcoma cell lines were studied. A single laser irradiation was performed at three different

wavelengths, at the energies of 0.5, 1, 5, and 10 J/cm(2). Twenty-four hours after laser irradiation, cell proliferation and alkaline phosphatase assays were assessed. RESULTS: Osteoblast proliferation increased significantly after 830-nm laser irradiation (at 10 J/cm(2)) but decreased after 780-nm laser irradiation (at 1, 5, and 10 J/cm(2)). Osteosarcoma cell proliferation increased significantly after 670-nm (at 5 J/cm(2)) and 780-nm laser irradiation (at 1, 5, and 10 J/cm(2)), but not after 830-nm laser irradiation. Alkaline phosphatase (ALP) activity in the osteoblast line was increased after 830-nm laser irradiation at 10 J/cm(2), whereas ALP activity in the osteosarcoma line was not altered, regardless of laser wavelength or intensity. CONCLUSION: Based on the conditions of this study, we conclude that each cell line responds differently to specific wavelength and dose combinations. Further investigations are required to investigate the physiological mechanisms responsible for the contrasting outcomes obtained when using laser irradiation on cultured normal and malignant bone cells. Lasers Surg Med. 2007 Jul;39(6):551-9.

Low-intensity laser irradiation stimulates bone nodule formation via insulin-like growth factor-I expression in rat calvarial cells.
Shimizu N, Mayahara K, Kiyosaki T, Yamaguchi A, Ozawa Y, Abiko Y. Department of Orthodontics, Nihon University School of Dentistry, 1-8-13 Kanda, Surugadai, Chiyoda-Ku, Tokyo101-8310, Japan. shimizu-n@dent.nihon-u.ac.jp
Abstract

BACKGROUND AND OBJECTIVE: We previously reported that low-intensity laser irradiation stimulated bone nodule formation through enhanced cellular proliferation and differentiation. However, the mechanisms of irradiation are unclear. Thus, we attempted to determine the responsibility of insulin-like growth factor (IGF)-I for the action observed. STUDY DESIGN/MATERIALS AND METHODS: Osteoblast-like cells were isolated from fetal rat calvariae and cultured with rat recombinant (r) IGF-I, IGF-I-antibody (Ab), and/or the cells were irradiated once (3.75 J/cm(2)) with a low-intensity Ga-Al-As laser (830 nm). The number and area of bone nodules formed in the culture were analyzed, and IGF-I expression was also examined. RESULTS: Treatment with rIGF-I significantly stimulated the number and area of bone nodules. This stimulatory effect was quite similar to those by laser irradiation, and this stimulation was abrogated dose-dependently by treatment with IGF-I-Ab. Moreover, laser irradiation significantly increased IGF-I protein and gene expression. CONCLUSION: The stimulatory effect of bone nodule formation by low-intensity laser irradiation will be at least partly mediated by IGF-I expression.

Photomed Laser Surg. 2006 Dec;24(6):735-40.

Comparative study of how low-level laser therapy and low-intensity pulsed ultrasound affect bone repair in rats.
Lirani-Galvao AP, Jorgetti V, Da Silva OL. Bioengineering Department, University of Sao Paulo, Sao Paulo, Brazil. Objective: This study aimed to compare the consequences of low-level laser therapy (LLLT) and low-intensity pulsed ultrasound (LIPUS) on bone repair. Background Data: Many studies have assessed the effects of LLLT and LIPUS on bone repair, but a comparison of them is rare. Methods: Male Wistar rats (n = 48) with tibial bone osteotomy were used. One group had the osteotomized limb treated with LLLT (GaAlAs laser, 780 nm, 30 mW, 112.5 J/cm(2)) and the second group with LIPUS (1.5 MHz, 30 mW/cm(2)), both for 12 sessions (five times per week); a third group was the control. After 20 days, rats were sacrificed and had their tibias submitted to a bending test or histomorphometric analysis. Results: In the bending test, maximum load at failure of LLLT group was significantly higher (p < 0.05). Bone histomorphometry revealed a significant increase in osteoblast number and surface, and osteoid volume in the LLLT group, and a significant increase in eroded and osteoclast surfaces in the LIPUS group. Conclusion: LIPUS enhanced bone repair by promoting bone resorption in the osteotomy area, while LLLT accelerated this process through bone formation. Photomed Laser Surg. 2006 Oct;24(5):642-5

Effects of 830-nm Laser Light on Preventing Bone Loss after Ovariectomy.

Renno AC, Moura FM, Santos NS, Tirico RP, Bossini PS, Parizotto NA. Department of Physiotherapy, Federal University of Sao Carlos, Sao Carlos, Brazil. Objective: The aim of this study was to investigate the effects of low-level laser therapy (LLLT; infrared, 830 nm) on the bone properties and bone strength of rat femora after ovariectomy (OVX).

Background Data: Osteoporosis affects 30% of postmenopausal women, and it has been recognized as a major public health problem. Based on the stimulatory effects of LLLT on proliferation of bone cells, we hypothesized that LLLT would be efficient in preventing bone mass loss in OVX rats. Methods: Forty female rats were divided into four groups: sham-operated control (SC), OVX control (OC), sham-operated irradiated at a dose of 120 J/cm(2) (I120), and OVX irradiated at a dose of 120 J/cm(2) (O120). Animals were operated at the age of 90 days. Laser irradiation was initiated 1 day after the operation and was performed three times a week, for 2 months. Femora were submitted to a biomechanical test and a physical properties evaluation. Results: Maximal load of O120 was higher than in control groups. Wet weight, dry weight, and bone volume of O120 did not show any difference when compared with SC. Conclusion: The results of the present study indicate that LLLT was able to prevent bone loss after OVX in rats. However, further studies are needed to investigate the effects of different parameters, wavelengths, and sessions of applications on OVX rats. J Craniofac Surg. 2006 Mar;17(2):297-301.

Repair of bone defects treated with autogenous bone graft and lowpower laser.
da Silva RV, Camilli JA. Department of Anatomy, Institute of Biology, State University of Campinas, UNICAMP, So Paulo, Brazil.
Abstract

Because bone healing at the graft site is similar to a fracture repair, the purpose of the present study was to evaluate the effects of low-power laser irradiation on the repair of rat skull defects treated with autogenous bone graft. A defect measuring 3 mm in diameter was produced in the left parietal bone and filled with an autogenous bone graft obtained from the right parietal bone. The animals were divided into 3 groups of 20 rats each: nonirradiated control, irradiated with 5.1 J/cm, and irradiated with 10.2 J/cm. The laser (2.4 mW, 735 nm, 3.4 x 10 W/cm, 3-mm spot size) was applied three times per week for 4 weeks. Greater volume of newly formed bone was observed in the irradiated group with 10.2 J/cm. In both irradiated groups, a greater volume of newly formed bone occurred only in the first 2 weeks. The results demonstrated that laser irradiation at the grafted site stimulated osteogenesis during the initial stages of the healing process in a skull defect of the rat and that this effect was dose dependent. Photomed Laser Surg. 2006 Feb;24(1):38-44.

Laser therapy improves healing of bone defects submitted to autologous bone graft.
Weber JB, Pinheiro AL, de Oliveira MG, Oliveira FA, Ramalho LM. School of Dentistry, Pontifcia Universidade Catlica do Rio Grande do Sul, Porto Alegre, Brazil. OBJECTIVE: The aim of the present study was to assess histologically the effect of low-level laser thrapy (LLLT) (lambda 830 nm) on the healing of bone defects associated with autologous bone graft. BACKGROUND DATA: LLLT has been used on the modulation of bone healing because of the photo-physical and photochemical properties of some wavelengths. The use of correct and appropriate parameters has been shown to be effective in the promotion of a positive biomodulative effect on the healing bone. METHODS: Sixty male Wistar rats were divided into four groups: G1 (control), G2 (LLLT on the surgical bed), G3 (LLLT on the graft), and G4 (LLLT on both the graft and the surgical bed). The dose per session was 10 J/cm(2), and it was applied to the surgical bed (G2/G4) and on the bone graft (G3/G4). LLLT was carried out every other day for 15 days (lambda 830 nm, phi = 0.5 cm(2), 50 Mw, 10 J/cm(2)). The dose was fractioned in four points. The animals were sacrificed 15, 21, and 30 days after surgery; specimens were taken and routinely processed (wax, cut, and stain with H&E and Sirius red stains). Light microscopic analysis was performed by a pathologist. RESULTS: In the groups in which the LLLT was used trans-operatively on the surgical bed (G2/G4), bone remodeling was both quantitatively and qualitatively more evident when compared to subjects of groups G1 and G3. CONCLUSION: The present study indicates that the use of LLLT trans-operatively resulted in a positive biomodulative effect on the healing of bone defects associated with autologous bone grafts. Photomed Laser Surg. 2006 Apr;24(2):169-78

Photoengineering of bone repair processes.

Pinheiro AL, Gerbi ME. Laser Center, School of Dentistry, Department of Propedutica and Clnica Integrada, Universidade Federal da Bahia, Canela Salvador, BA, Salvador, Brazil. albp@ufba.br

OBJECTIVE: This paper aims to report the state of the art with respect to photoengineering of bone repair using laser therapy. BACKGROUND DATA: Laser therapy has been reported as an important tool to positively stimulate bone both in vivo and in vitro. These results indicate that photophysical and photochemical properties of some wavelengths are primarily responsible for the tissue responses. The use of correct and appropriate parameters has been shown to be effective in the promotion of a positive biomodulative effect in healing bone. METHODS: A series of papers reporting the effects of laser therapy on bone cells and tissue are presented, and new and promising protocols developed by our group are presented. RESULTS: The results of our studies and others indicate that bone irradiated mostly with infrared (IR) wavelengths shows increased osteoblastic proliferation, collagen deposition, and bone neorformation when compared to nonirradiated bone. Further, the effect of laser therapy is more effective if the treatment is carried out at early stages when high cellular proliferation occurs. Vascular responses to laser therapy were also suggested as one of the possible mechanisms responsible for the positive clinical results observed following laser therapy. It still remains uncertain if bone stimulation by laser light is a general effect or if the isolate stimulation of osteoblasts is possible. CONCLUSION: It is possible that the laser therapy effect on bone regeneration depends not only on the total dose of irradiation, but also on the irradiation time and the irradiation mode. The threshold parameter energy density and intensity are biologically independent of one another. This independence accounts for the success and the failure of laser therapy achieved at lowenergy density levels. Lasers Med Sci. 2005 Dec;20(3-4):138-46. Epub 2005 Nov 16.

Low level laser irradiation stimulates osteogenic phenotype of mesenchymal stem cells seeded on a three-dimensional biomatrix.
Abramovitch-Gottlib L, Gross T, Naveh D, Geresh S, Rosenwaks S, Bar I, Vago R. Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. liata@bgu.ac.il
Abstract

Mesenchymal stem cells (MSCs) seeded on three-dimensional (3D) coralline (Porites lutea) biomatrices were irradiated with low-level laser irradiation (LLLI). The consequent phenotype modulation and development of MSCs towards ossified tissue was studied in this combined 3D biomatrix/LLLI system and in a control group, which was similarly grown, but was not treated by LLLI. The irradiated and non irradiated MSC were tested at 1-7, 10, 14, 21, 28 days of culturing via analysis of cellular distribution on matrices (trypan blue), calcium incorporation to

newly formed tissue (alizarin red), bone nodule formation (von Kossa), fat aggregates formation (oil red O), alkaline phosphatase (ALP) activity, scanning electron microscopy (SEM) and electron dispersive spectrometry (EDS). The results obtained from the irradiated samples showed enhanced tissue formation, appearance of phosphorous peaks and calcium and phosphate incorporation to newly formed tissue. Moreover, in irradiated samples ALP activity was significantly enhanced in early stages and notably reduced in late stages of culturing. These findings of cell and tissue parameters up to 28 days of culture revealed higher ossification levels in irradiated samples compared with the control group. We suggest that both the surface properties of the 3D crystalline biomatrices and the LLLI have biostimulatory effects on the conversion of MSCs into bone-forming cells and on the induction of ex-vivo ossification.
Arq Bras Endocrinol Metabol. 2005 Dec;49(6):891-6. Epub 2006 Mar 16.

Evidences of physical agents action on bone metabolism and their potential clinical use.
[Article in Portuguese] Lirani AP, Lazaretti-Castro M. Departamento de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, SP. analirani@fcr.epm.br The action of physical agents such as low level laser therapy, low-intensity pulsed ultrasound and electrical and electromagnetic fields on bone have been often studied, showing that they are able to promote osteogenesis, accelerate fracture consolidation and augment bone mass. The use of these therapeutic modalities was first based on the finding that bone is a piezoelectric material, that means it can generate polarization when deformed, transforming mechanical energy into electric energy, and this has widen therapeutic possibilities to bony tissue. The present work aims to present evidences of physiologic effects and mechanisms of action of these physical agents on bone metabolism, based on articles published in international scientific literature. Photomed Laser Surg. 2005 Aug;23(4):382-8.

Assessment of bone repair associated with the use of organic bovine bone and membrane irradiated at 830 nm.
Gerbi ME, Pinheiro AL, Marzola C, Limeira Jnior Fde A, Ramalho LM, Ponzi EA, Soares AO, Carvalho LC, Lima HV, Gonalves TO. School of Dentistry, Federal University of Bahia, Salvador, Brazil.

Abstract

OBJECTIVE: The aim of the present investigation was to assess histologically the effect of LLLT (GaAIAs, 830 nm, 40 mW, CW, (Phi) approximately 0.6 mm, 16 J/cm(2) per session) on the repair of surgical defects created in the femur of the Wistar Albinus rat. The defects were filled to lyophilized bovine bone (Gen-ox), organic matrix) associated or not to GTR (Genderm). BACKGROUND DATA: A major problem on modern Dentistry is the recovery of bone defects caused by trauma, surgical procedures or pathologies. Several types of biomaterials have been used in order to improve the repair of these defects. These materials are often associated to procedures of GTR. Previous studies have shown positive effects of LLLT on the repair of soft tissue wounds, but there are a few on its effects on bone healing. METHODS: Surgical bone defects were created in 42 animals divided into five groups: Group I (control, 6 animals); Group II (Gen-ox, 9 animals); Group III (Gen-ox + Laser, 9 animals); Group IV (Gen-ox + Gen-derm, 9 animals); Group V (Gen-ox + Gen-derm + Laser, 9 animals). The animals on the irradiated group received 16 J/cm(2) per session divided into four points around the defect (4 J/cm(2)) being the first irradiation immediately after surgery and repeated seven times at every 48 h. The animals were humanly killed after 15, 21, and 30 days. RESULTS: The results of the present investigation showed histological evidence of improved amount of collagen fibers at early stages of the bone healing (15 days) and increased amount of well organized bone trabeculae at the end of the experimental period (30 days) on irradiated animals compared to non irradiated ones. CONCLUSIONS: It is concluded that a positive biomodulative effect on the healing process of one defect associated or not to the use of organic lyophilized bone and biological bovine lyophilized membrane on the femur of the rat. Photomed Laser Surg. 2005 Apr;23(2):212-5.

Effect of low-intensity laser irradiation on the process of bone repair.

Merli LA, Santos MT, Genovese WJ, Faloppa F. School of Dentistry, Unversidade Cruzeiro Do Sul, So Paulo, Brazil. luizmerli@uol.com.br
Abstract

The effect of low-intensity laser (GaAsAl) irradiation on bone repair in the femurs of mice was investigated. An experimental model of hole injury with surgery drills was used in 20 mouse femurs followed by a study of the effect of low-energy laser irradiation on bone repair. The experimental model was divided into two groups. The first (10 left femurs) received laser irradiation immediately after injury and was followed for different time intervals (24, 48, and 72

h). The right femurs (control group) underwent hole injury but no laser irradiation. The rats were sacrificed after 14 days and the results were analyzed using a quantitative histometrical method. The Mann-Whitney test was used to perform the statistical analysis. Histometrical analysis revealed a more rapid accumulation of reparative new bone in the hole injury of the laserirradiated legs. We conclude that GaAsAl laser irradiation after injury was effective on bone repair when compared to results in the control group. Photomed Laser Surg. 2005 Apr;23(2):161-6.

Low-level laser irradiation promotes proliferation and differentiation of human osteoblasts in vitro.
Stein A, Benayahu D, Maltz L, Oron U. Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, TelAviv, Israel.
Abstract

OBJECTIVES: The aim of the present study was to investigate the effect of low-level laser irradiation on proliferation and differentiation of a human osteoblast cell line. BACKGROUND DATA: It was previously found that low-level laser therapy (LLLT) enhances bone repair in experimental models. MATERIALS AND METHODS: Cultured osteoblast cells were irradiated using He-Ne laser irradiation (632 nm; 10 mW power output). On the second and third day after seeding the osteoblasts were exposed to laser irradiation. The effect of irradiation on osteoblast proliferation was quantified by cell count and colorimetric MTT (dimethylthiazol tetrazolium bromide) assay 24 and 48 h after second irradiation. RESULTS: A significant 31-58% increase in cell survival (MTT assay) and higher cell count in the once-irradiated as compared to nonirradiated cells was monitored. Differentiation and maturation of the cells was followed by osteogenic markers: alkaline phosphatase (ALP), osteopontin (OP), and bone sialoprotein (BSP). A two-fold enhancement of ALP activity and expression of OP and BSP was much higher in the irradiated cells as compared to non-irradiated osteoblasts. CONCLUSION: We conclude that LLLT promotes proliferation and maturation of human osteoblasts in vitro. These results may have clinical implications. Photomed Laser Surg. 2005 Feb;23(1):27-31.

Infrared laser light reduces loading time of dental implants: a Raman spectroscopic study.
Lopes CB, Pinheiro AL, Sathaiah S, Duarte J, Cristinamartins M. IP&D and Department of Dentistry, FCS, UNIVAP, SJ Campos, So Paulo, Brazil.
Abstract

OBJECTIVE: The aim of this study was to assess, through near-infrared Raman spectroscopy (NIRS), the incorporation of hydroxyapatite of calcium (CHA; approximately 960 cm(1))on the healing bone around dental implants submitted or not to low-level laser therapy (LLLT) (lambda830 nm). BACKGROUND DATA: The process of maturation of the bone is important for the success of dental implants, as it improves the fixation of the implant to the bone, allowing the wearing of a prosthesis. LLLT has been suggested as a mean of improving bone healing because of its biomodulatory capabilities. METHODS: Fourteen rabbits received a titanium implant on the tibia; eight of them were irradiated with lambda830-nm laser (seven sessions at 48-h intervals, 21.5 J/cm(2) per session, 10 mW, phi approximately 0.0028 cm(2), 85 J/cm(2) treatment dose), and six acted as control. The animals were sacrificed at 15, 30, and 45 days after surgery. Specimens were routinely prepared for Raman spectroscopy. Twelve readings were taken on the bone around the implant. RESULTS: The results showed significant differences in the concentration of CHA on irradiated and control specimens at both 30 and 45 days after surgery (p < 0.001). Conclusion: It is concluded that LLLT does improve bone healing, and this can be safely assessed by Raman spectroscopy. Swed Dent J Suppl. 2005;(172):1-63.

The effect of low level laser irradiation on implant-tissue interaction. In vivo and in vitro studies.
Khadra M. Department of Oral Surgery and Oral Medicine, Faculty of Dentistry, University of Oslo, Norway. Low-level laser therapy (LLLT) is increasingly used in medicine and dentistry. It has been suggested that LLLT may be beneficial in the management of many different medical conditions, including pain, wound healing and nerve injury. The present thesis is based on a series of in vivo and in vitro experimental studies investigating whether LLLT has the potential to enhance

titanium-implant interaction. Information about LLLT effect on bone healing is fundamental to understand whether LLLT may improve implant-tissue interaction. Thus in the initial study (I), the effect of LLLT on bone healing and growth in rat calvarial bone defects was investigated. It was found that LLLT may accelerate metabolism and/or mineralization during early bone healing. Based on these findings, study II explored the hypothesis that LLLT can enhance implant integration in the rabbit tibial bone. It was shown that LLLT stimulated the mechanical strength of the interface between the implant and bone after a healing period of 8 weeks. Histomorphometrical and mineral analyses showed that the irradiated implants had greater boneto-implant contact than the controls. In the in vitro experiments, cellular responses to LLLT were studied in two cell types: primary cultures of human gingival fibroblasts and human osteoblastlike cells, with special reference to attachment, proliferation, differentiation and production of transforming growth factor beta1 (TGF-beta1). The objectives of studies III & IV were to develop a standardized, reproducible in vitro model for testing a GaAlAs diode laser device and to document the influence of single or multiple doses of LLLT, as a guide to defining the optimal laser dose for enhancing cell activity. A further objective was to investigate the effect of LLLT on initial attachment and subsequent behaviour of human gingival fibroblasts cultured on titanium. While both multiple doses (1.5 and 3 J/cm2) and a single dose (3 J/cm2) enhanced cellular attachment, proliferation increased only after multiple doses (1.5 and 3 J/cm2). Study V concerned the response to LLLT of osteoblast-like cells, derived from human alveolar bone cultured on titanium implant material. In this study LLLT significantly enhanced cellular attachment. Greater cell proliferation in the irradiated groups was observed first after 96 h indicating that the cellular response is dose dependent. Osteocalcin synthesis and TGF-beta1 production were significantly stimulated on the samples exposed to 3 J/cm2. The following conclusions are drawn from the results of these five studies: LLLT can promote bone healing and bone mineralization and thus may be clinically beneficial in promoting bone formation in skeletal defects. It may be also used as additional treatment for accelerating implant healing in bone. LLLT can modulate the primary steps in cellular attachment and growth on titanium surfaces. Multiple doses of LLLT can improve LLLT efficacy, accelerate the initial attachment and alter the behaviour of human gingival fibroblasts cultured on titanium surfaces. The use of LLLT at the range of doses between 1.5 and 3 J/cm2 may modulate the activity of cells interacting with an implant, thereby enhancing tissue healing and ultimate implant success. Photomed Laser Surg. 2004 Jun;22(3):249-53

Molecular structure of the bony tissue after experimental trauma to the mandibular region followed by laser therapy.
Rochkind S, Kogan G, Luger EG, Salame K, Karp E, Graif M, Weiss J. Department of Neurosurgery, Division of Peripheral Nerve Reconstruction, Tel Aviv Sourasky Medical Center, Tel Aviv University, Israel. rochkind@zahav.net.il OBJECTIVE: We investigated the therapeutic efficiency of laser irradiation and Bio-Oss, both and separately, on the post-traumatic regeneration of bone tissue in rats using infrared spectroscopy as an informative and accurate measuring method.

BACKGROUND DATA: The therapeutic effect of low-power laser irradiation on bone tissue regeneration processes in animal models has been studied using morphogenic, biochemical, roentgenographic and electron microscopic measurements. Natural bone minerals, such as BioOss collagen, were recommended for the reconstruction of bone defects in the alveolar process. MATERIALS AND METHODS: 29 male Wistar rats, divided into four random groups in a blinded manner were operated on the right alveolar process. A bone defect was made by penetrating the right alveolar process of the mandible bone using a 3-mm drill. The rats were divided into four groups as follows: Group I, left side served as intact bone and right injured side as the control; Group II, right injured side was treated by organic bovine bone (Bio-Oss); Group III, right side bone defect was treated by HeNe laser (632.8 nm, 35 mW) applied transcutaneously for 20 min to the injured area daily for the following 14 consecutive days; and Group IV, Bio-Oss was placed loosely in the right side defect followed by laser treatment. After 2 weeks, the intact bone and bone replicas of the trauma area were removed and analyzed by infra-red spectroscopy technique. The composition and the structure of the bone tissue mineral substances were determined and compared among the four groups. For quantitative analysis of the regenerative bone process, the Mineralization index was used. An increase in this index indicates regenerative bone processes. RESULTS: The normal state analysis of the IR spectra of the normal alveolar bone tissue within the intervals of 400 to 4000 cm(-1) revealed characteristic absorption bands for the inorganic bone component in spectrum regions 450-1480 cm(-1), and the organic component at 1540-3340 cm(-1). In the case of trauma, the intensity of absorption of the inorganic component was decreased by 54%, and the absorption band became narrow, which can be interpreted as quantitative changes of the bone tissue mineral content. The wavelength characteristics of the inorganic component remained unchanged; that is, the induced trauma under these experimental conditions did not provoke alterations in the structure of the phosphate framework. The organic component showed decreased absorption by 10-15%, compared to the normal bone, and slight displacement of the wavelength, which can be interpreted as changes occurring in the quality of the organic content of the bone tissue. In the Bio-Oss-treated group, the intensity of absorption of the inorganic component increased by 43%, compared to the control injured area; however, there was a decrease of 22.6% in the normal bone. The wavelength characteristics of the inorganic component remained unchanged. The organic component showed similar absorption results in the injured non-treated group and absorption was 10-15% less than in the normal bone. Mineralization Index in the Bio-Oss-treated group was 0.93, compared to 0.63 in the control group and 2.04 in the normal bone. In the laser-treated group, the intensity of absorption of the inorganic component increased by 62, compared to the control injured area, and decreased only 11.4% in the normal bone. The wavelength characteristics of the organic component remained unchanged; that is, the organic component was similar to that of normal bone. Mineralization Index in the laser-treated group increased significantly to 1.86, compared to 0.63 in the control group and 2.04 in the normal bone. In the combined laser and Bio-Oss-treated groups, the intensity of absorption of the inorganic component and organic component was similar to that of normal bone. Mineralization Index in this group increased significantly to 1.98, compared to 0.63 in the control group and 2.04 in the normal bone.

CONCLUSION: The results suggest that low-power laser irradiatults suggest that low-power laser irradiation alone and in combination with Bio-Oss enhances bone healing and increases bone repair. Used by the kind permission of the Czech Society for the Use of Laser in Medicine, www.laserpartner.org

Bone Stimulation by Low Level Laser A Theoretical Model for the Effects
Philip Gable, B App Sc PT G Dip Sc Res (LLLT) MSc, Australia Jan Tunr, DDS, Sweden

Abstract
The anecdotal and researched evidence for the effects of Low Level Laser Therapy (LLLT) on the stimulation of bone have been reported for over 20 years. This has been in the form of local as well as systematic effects including contra-lateral effects. Reports of stimulation of rabbit radii fractures and mice femurs were made as early as 1986 and 1987 with irradiated bones healing faster than controls and contra-lateral non-treated fractures similarly demonstrating faster healing times. Over the following decade and a half, further studies have also investigated and demonstrated that LLLT is effective for the stimulation of bone tissue. *** The reasons for this have been attributed to the general effects of LLLT and its ability to increase the rates of healing through mitochondrial ATP production and alteration in the cellular lipid bilayer. Additional hypothesis include the subsequent capacity of irradiated cells to alter their ion exchange rate and thus influence the catalytic effects of the specific enzymes and substrates. These in turn initiate and promote the healing process completing the cascading cycle of events. In the area of bone specific research, Dr. Tony Pohl of the Royal Adelaide Hospital in South Australia, has provided a new theory that postulates that the majority of fluid transfer and exchange within living bone is predominantly influenced by the lymphatic circulation. LLLT is well documented and known as having effects that influence the lymphatic circulation and wound healing process. A coupling of these two areas of theory can demonstrate a positive description and explanation of the predominant effects of LLLT in bone stimulation. In reality, LLLT's effects on bone may well be a further consequence of its actions on the lymphatic circulation. Reports of stimulation of Rabbit radii fractures were made by Tang in 1986 and similar reports by Trelles in 1987 on mice femurs. In both situations the irradiated bones healed faster than the

controls. In another study by Hernandez-Ros, in 1987, LLLT demonstrated stimulation of fresh fractures on Sprague-Dawley rats that were fractured bilaterally. The unexpected results of this study were that the contra-lateral fractured non-treated limb also healed faster than the control group. Over the following decade and a half further studies (Yamada 1991; Pyczek, Sopala et al. 1994; Ozawa 1995; Horowitz 1996; Yaakobi 1996; Saito and Shimizu 1997) have also investigated and demonstrated that LLLT is effective for promoting the stimulation of bone healing. Recently Nicolau and colleagues (2002) from Brazil demonstrated the positive effect of LLLT on the stimulation of bone in mice with latent promotion of bone remodulation at injury sites without changes in bone architecture, increased bone volume and increased osteoblast surface through increased resorption and formation of bone with higher apposition rates. A positive effect on bony implants has been demonstrated by Drtbudak (2002) and Guzzardella (2003). The effect of laser irradiation on osteoblastic cells has been reported by Yamamoto (2001) and Guzzardella (2002). The reasoning for this amelioration in all experimental circumstances, based on electron microscopy as well as macroscopic histological evidence, was concluded to be due to ia improved vascularisation as a consequence of blood vessel formation, absorption of the haematoma, macrophage action, fibroblast proliferation, chondrocyte activity, bone remodeling from increased osteoblastic activity and deposition of calcium salts. These changes and evidence based studies attribute the macro- and microscopic effects to the known and accepted general actions of LLLT and its ability to increase rates of healing through stimulation of ATP production, (Karu 1989; Smith 1990) promoting repair and polarization of the cellular lipid bilayer (Fenyo 1990) as well as LLLT's capacity to affect cells through alterations in their exchange rate of ions (Robinson and Walters 1991) and influences the catalytic effects of the specific enzymes and substrates (Pouyssegur 1985; Karu 1988) which in turn initiate and promote the healing process. More recent work by Dr. Tony Pohl, an internationally renowned Orthopaedic Surgeon from the Royal Adelaide Hospital in South Australia and lecturer at the Adelaide and South Australian Universities, has given rise to a new theory on bone circulation through reconsideration of fluid and protein transfer within bone (Pohl 1999). This theory suggests that the general understanding of the circulatory action within bone has been incorrect. Pohl postulates that the majority of fluid transfer and exchange within the living bone is predominantly influenced by the lymphatic rather than the vascular circulation. This is justified through studies on bone fluid input and output levels that have demonstrated that the venous and arterial aspect of circulation alone cannot account for the demonstrated levels of output nor the presence of free radical molecules which exceed those of the vascular input. Furthermore, the diameter of large protein cells within the bone exceed the diameter of the vessels that form the terminal aspects of the circulatory system making it impossible for them to have been delivered via this system. Consequently, an additional circulatory system must be present that will account for both the increased output and the presence of the large diameter protein cells as well as the free radicals. If LLLT is then considered within the context of this new theory on bone circulation and the contribution of the lymphatic circulation then a further logical reasoned deduction for the action of LLLT on bone stimulation can be made. LLLT has a well documented and known effect

influencing the lymphatic circulation. This has been demonstrated from the early works of Lievens, (1985) that demonstrated the influence of Laser Irradiation on the motricity of the lymphatic system and on the wound healing process. This is supported by several wound studies that demonstrate that the levels of protein rich exudates in non-healing wounds increase markedly from exposure to LLLT. This demonstrated action is determined to be as a result of the increase in lymphatic circulation (Robinson and Walters 1991; Gabel 1995). More recent work at the Flinders Medical Center in Adelaide South Australia has been completed and presented at the World Association of Laser Therapy conference in Tokyo Japan (Anderson, Carati et al. 2002). This study has demonstrated the positive effects of LLLT on the lymphatic circulation and its consequential benefits to the post mastectomy patient. An understanding of the existing knowledge of the effects of LLLT on the lymphatic system combined with the hypothesis of bone fluid transport provides a coupled theory that would demonstrate a positive description and explain of the predominant effects of LLLT in bone stimulation. In the trauma situation of direct or indirect damage to the bone, including fractures and periosteal induced damage such as stress fractures, the tissue damage leads to compromises that include but are not limited to, physical blockage from the trauma and waste / debris, increased fluid and circulatory viscosity from added cellular content within the lymphatics, lower speed motility and energy deficit in the tissue and cells from the loss of ATP production as a general effect from the trauma, cell changes and inability of mitochondria to function at the normal higher level to promote self repair and regeneration. LLLT with its known general effects and specific direct effects on the lymphatic system would act to stimulate mitochondria ATP that increases cellular and circulatory motility as well as directly influencing lymphatic flow. LLLT also promotes increased permeability in interstitial tissue and facial layers (Gabel 1995) reducing stagnation and blockage. These actions would assist the increase in lymphatic flow and consequently the circulation within the affected bone. There is also a hypothetical potential that the presence of LLLT by increasing lymphatic circulation does so by virtue of an increase in the diameter of the lymphatic vessels, not just by increased flow rates within the vessel at an unchanged diameter. This diameter increase, if definitively present, would also explain the presence of large diameter protein cells within the normal bone circulation that cannot be attributed to the vascular circulation and would additionally explain a facilitated process for removal of debris and larger protein cells passing out of traumatized areas that is additionally stimulated by the use of LLLT. Stimulation of bone healing by LLLT has till now has been generally classified as a consequence of the general healing effects of LLLT. In reality LLLT's effect on bone may well be a further consequence of its actions on the lymphatic circulation. References

Anderson, S, Carati, C et al. (2002). Low Level Laser Therapy (LLLT) as a Treatment for Postmatestectomy Lymhoedema. WALT 2002, Tokyo Japan. Coombe, AR et al (2001). The effect of low level laser irradiation on osteoblastic cells. Clin Ort Res. 4: 3-14.

Drtbudak, O et al (2002). Effect of low-power laser irradiation on bony implant sites. Clin Oral Implants Res 13(3):288-292. Fenyo, M. (1990). Theoretical and Experimental Basis of Biostimulation by Bioptron, Bioptron AG, Monchaltorf, Switzerland. Gabel, CP (1995). Does Laser enhance bruising in acute sporting injuries. Aust. J. Physio. 41(4): 267-269. Gabel, CP (1995). The effect of LLLT on slow healing wounds and ulcers. Health Sciences. Darwin, Northern Territory. Guzzardella, GA et al (2002). Laser stimulation on bone defect healing: An in vitro study. Lasers Med Sci. 17(3): 216-220. Guzzardella, GA et al (2003). Osseointegration of endosseous ceramic implants after postoperative low-power laser stimulation: an in vivo comparative study. Clin Oral Implants Res. 14: 226-232. Horowitz, I. et al. (1996). Infrared spectroscopy analysis of the effect of low power laser irradiation on calvarial bone defect healing in the rat (abstract). Laser Therapy 8: 29. Karu, TI (1988). Molecular mechanism of the therapeutic effects of low intensity laser radiation. Lasers in Life Science 2: 53-74. Karu, TI (1989). Photobiology of low-power laser therapy. London, Harwood Academic Publishers. Lievens, P. (1985). The influence of Laser Irradiation on the motricity of the lymphatical system and on the wound healing process. International Congress on Laser in Medicine and Surgery., Bologna. Nicolau, R A., Jorgetti, V, Rigau, J et al. Effect of low power laser Ga-Al-As (660nm) in the bone tissue remodulation in mice Ozawa, Y. et al (1995). Stimulatory effects of low-power laser irradiation on bone formation in vitro. SPIE Proc. 1995 Vol. 1984: 281-288. Pohl, T. (1999). Bone circulation, the lymphatic system contribution. Personal Communication to CP Gabel. Adelaide Oct 1999. Pouyssegur, J. (1985). The growth factor-activatable Na+/H+ exchange system: a genetic approach. In Karu, TI 1988, 'Molecular mechanism of the therapeutic effects of low intensity laser radiation', Lasers in Life Science , vol.2, p.53-74. Trends in Biochemical Science 10: 453455. Pycek, M., Sopala, M et al. (1994). Effect of low-energy laser power on the bone marrow of the rat.. Folia Biol (Krakow) 42(3-4): 151-156. Robinson, B. and Walters, J (1991). The use of low level laser therapy in diabetic and other ulcerations. Journal of British Podiatric Medicine 46(10): 186-189. Saito, S. and. Shimizu, N. (1997). Stimulatory effects of low-power laser irradiation on bone regeneration in midpalatal suture during expansion in the rat. Am J Ortod Dentofac Orthop 11(5): 525-. Smith, KC (1990). Light and life: The photobiological basis of the therapeutic use of radiation from lasers. International Laser Therapy Association Conference, Osaka. Yaakobi, T. et. al. (1996). Promotion of bone repair in the cortical bone of the tibia in rats by low energy laser (He-Ne) irradiation. Calcif Tissue Int. 59(4): 297-300. Yamada, K. (1991). Biological effects of low power laser irradiation on clonal osteoblastic cells (MC3T3-E1). Nippon Seikeigeka Gakkai Zasshi 65(9): 787-799. Yamamoto, M et al (2001). Stimulation of MCM3 gene expression in osteoblast by low level laser irradiation. Laser in Med Sci. Abstract issue. 16(3): 213-217.

Photomed Laser Surg. 2005 Aug;23(4):382-8

Assessment of bone repair associated with the use of organic bovine bone and membrane irradiated at 830 nm.
Gerbi ME, Pinheiro AL, Marzola C, Limeira Jnior Fde A, Ramalho LM, Ponzi EA, Soares AO, Carvalho LC, Lima HV, Gonalves TO. School of Dentistry, Federal University of Bahia, Salvador, Brazil. OBJECTIVE: The aim of the present investigation was to assess histologically the effect of LLLT (GaAIAs, 830 nm, 40 mW, CW, (Phi) approximately 0.6 mm, 16 J/cm(2) per session) on the repair of surgical defects created in the femur of the Wistar Albinus rat. The defects were filled to lyophilized bovine bone (Gen-ox), organic matrix) associated or not to GTR (Genderm). BACKGROUND DATA: A major problem on modern Dentistry is the recovery of bone defects caused by trauma, surgical procedures or pathologies. Several types of biomaterials have been used in order to improve the repair of these defects. These materials are often associated to procedures of GTR. Previous studies have shown positive effects of LLLT on the repair of soft tissue wounds, but there are a few on its effects on bone healing. METHODS: Surgical bone defects were created in 42 animals divided into five groups: Group I (control, 6 animals); Group II (Gen-ox, 9 animals); Group III (Gen-ox + Laser, 9 animals); Group IV (Gen-ox + Gen-derm, 9 animals); Group V (Gen-ox + Gen-derm + Laser, 9 animals). The animals on the irradiated group received 16 J/cm(2) per session divided into four points around the defect (4 J/cm(2)) being the first irradiation immediately after surgery and repeated seven times at every 48 h. The animals were humanly killed after 15, 21, and 30 days. RESULTS: The results of the present investigation showed histological evidence of improved amount of collagen fibers at early stages of the bone healing (15 days) and increased amount of well organized bone trabeculae at the end of the experimental period (30 days) on irradiated animals compared to non irradiated ones. CONCLUSIONS: It is concluded that a positive biomodulative effect on the healing process of one defect associated or not to the use of organic lyophilized bone and biological bovine lyophilized membrane on the femur of the rat.
J Clin Laser Med Surg. 2003 Dec;21(6):383-8.

Effect of 830-nm laser light on the repair of bone defects grafted with inorganic bovine bone and decalcified cortical osseous membrane.
Barbos Pinheiro AL, Limeira Junior Fde A, Marquez Gerbi ME, Pedreira Ramalho LM, Marzola C, Carneiro Ponzi EA, Oliveira Soares A, Bandeira De Carvalho LC, Vieira Lima HC, Oliveira Goncalves T. Laser Center, School of Dentistry, Federal University of Bahia, Salvador, Brazil. albp@ufba.br OBJECTIVE: The aim of this study was to assess histologically the effect of LLLT (lambda830 nm) on the repair of standardized bone defects on the femur of Wistar albinus rats grafted with inorganic bovine bone and associated or not to decalcified bovine cortical bone membrane. BACKGROUND DATA: Bone loss may be a result of several pathologies, trauma or a consequence of surgical procedures. This led to extensive studies on the process of bone repair and development of techniques for the correction of bone defects, including the use of several types of grafts, membranes and the association of both techniques. There is evidence in the literature of the positive effect of LLLT on the healing of soft tissue wounds. However, its effect on bone is not completely understood. MATERIALS AND METHODS: Five randomized groups were studied: Group I (Control); Group IIA (Gen-ox); Group IIB (Gen-ox + LLLT); Group IIIA (Gen-ox + Gen-derm) and Group IIIB (Gen-ox + Gen-derm + LLLT). Bone defects were created at the femur of the animals and were treated according to the group. The animals of the irradiated groups were irradiated every 48 h during 15 days; the first irradiation was performed immediately after the surgical procedure. The animals were irradiated transcutaneously in four points around the defect. At each point a dose of 4 J/cm2 was given (phi approximately 0.6 mm, 40 mW) and the total dose per session was 16 J/cm2. The animals were humanely killed 15, 21, and 30 days after surgery. The specimens were routinely processed to wax, serially cut, and stained with H&E and Picrosirius stains and analyzed under light microscopy. RESULTS: The results showed evidence of a more advanced repair on the irradiated groups when compared to non-irradiated ones. The repair of irradiated groups was characterized by both increased bone formation and amount of collagen fibers around the graft within the cavity since the 15th day after surgery, through analysis of the osteoconductive capacity of the Gen-ox and the increment of the cortical repair in specimens with Gen-derm membrane. CONCLUSION: It is concluded that LLLT had a positive effect on the repair of bone defect submitted the implantation of graft.
Lasers Med Sci. 2003;18(2):89-94.

Effect of low-power GaAlAs laser (660 nm) on bone structure and cell activity: an experimental animal study.
Nicola RA, Jorgetti V, Rigau J, Pacheco MT, dos Reis LM, Zangaro RA. Vale of Paraiba University, Sao Jose dos Campos, SP, Brazil. renatanicolau@hotmail.com Low-level laser therapy (LLLT) is increasingly being used in the regeneration of soft tissue. In the regeneration of hard tissue, it has already been shown that the biomodulation effect of lasers repairs bones more quickly. We studied the activity in bone cells after LLLT close to the site of the bone injury. The femurs of 48 rats were perforated (24 in the irradiated group and 24 in the control group) and the irradiated group was treated with a GaAlAs laser of 660 nm, 10 J/cm2 of radiant exposure on the 2nd, 4th, 6th and 8th days after surgery (DAS). We carried out histomorphometry analysis of the bone. We found that activity was higher in the irradiated group than in the control group: (a) bone volume at 5 DAS (p=0.035); (b) osteoblast surface at 15 DAS (p=0.0002); (c) mineral apposition rate at 15 and 25 DAS (p=0.0008 and 0.006); (d) osteoclast surface at 5 DAS and 25 DAS (p=0.049 and p=0.0028); and (e) eroded surface ( p=0.0032). We concluded that LLLT increases the activity in bone cells (resorption and formation) around the site of the repair without changing the bone structure. Lasers Med Sci. 2003;18(2):78-82.

Effect of low-level laser irradiation on osteoglycin gene expression in osteoblasts.

Hamajima S, Hiratsuka K, Kiyama-Kishikawa M, Tagawa T, Kawahara M, Ohta M, Sasahara H, Abiko Y. Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Abstract

Many studies have attempted to elucidate the mechanism of the biostimulatory effects of lowlevel laser irradiation (LLLI), but the molecular basis of these effects remains obscure. We investigated the stimulatory effect of LLLI on bone formation during the early proliferation stage of cultured osteoblastic cells. A mouse calvaria-derived osteoblastic cell line, MC3T3-E1, was utilised to perform a cDNA microarray hybridisation to identify genes that induced expression by LLLI at the early stage. Among those genes that showed at least a twofold increased expression, the osteoglycin/mimecan gene was upregulated 2.3-fold at 2 h after LLLI. Osteoglycin is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix which was previously called the osteoinductive factor. SLRP are abundantly contained in the bone matrix, cartilage cells and connective tissues, and are thought to regulate cell proliferation,

differentiation and adhesion in close association with collagen and many other growth factors. We investigated the time-related expression of this gene by LLLI using a reverse transcription polymerase chain reaction (RT-PCR) method, and more precisely with a real-time PCR method, and found increases of 1.5-2-fold at 2-4 h after LLLI compared with the non-irradiated controls. These results suggest that the increased expression of the osteoglycin gene by LLLI in the early proliferation stage of cultured osteoblastic cells may play an important role in the stimulation of bone formation in concert with matrix proteins and growth factors. J Clin Laser Med Surg. 2003 Oct;21(5):271-7.Links

Effects of pulse frequency of low-level laser therapy (LLLT) on bone nodule formation in rat calvarial cells.
Ueda Y, Shimizu N. Department of Orthodontics, Nihon University School of Dentistry at Matsudo Chiba, Japan. OBJECTIVE: The purpose of this study was to determine the effect of pulse frequencies of lowlevel laser therapy (LLLT) on bone nodule formation in rat calvarial cells in vitro. BACKGROUND DATA: Various photo-biostimulatory effects of LLLT, including bone formation, were affected by some irradiation factors such as total energy dose, irradiation phase, laser spectrum, and power density. However, the effects of pulse frequencies used during laser irradiation on bone formation have not been elucidated. MATERIALS AND METHODS: Osteoblast-like cells isolated from fetal rat calvariae were irradiated once with a low-energy Ga-Al-As laser (830 nm, 500 mW, 0.48-3.84 J/cm2) in four different irradiation modes: continuous irradiation (CI), and 1-, 2-, and 8-Hz pulsed irradiation (PI-1, PI-2, PI-8). We then investigated the effects on cellular proliferation, bone nodule formation, alkaline phosphatase (ALP) activity, and ALP gene expression. RESULTS: Laser irradiation in all four groups significantly stimulated cellular proliferation, bone nodule formation, ALP activity, and ALP gene expression, as compared with the nonirradiation group. Notably, PI-1 and -2 irradiation markedly stimulated these factors, when compared with the CI and PI-8 groups, and PI-2 irradiation was the best approach for bone nodule formation in the present experimental conditions. CONCLUSION: Since low-frequency pulsed laser irradiation significantly stimulates bone formation in vitro, it is most likely that the pulse frequency of LLLT an important factor affecting biological responses in bone formation.
J Photochem Photobiol B. 2003 May-Jun;70(2):81-9.

Low power laser irradiation improves histomorphometrical parameters and bone matrix organization during tibia wound healing in rats.
Garavello-Freitas I, Baranauskas V, Joazeiro PP, Padovani CR, Dal Pai-Silva M, da CruzHofling MA. Faculdade de Engenharia Eletrica e Computacao, Departamento de Semicondutores Instrumentos e Fotonica, Universidade Estadual de Campinas, Av. Albert Einstein N.400, 13 083-970 Campinas, SP, Brazil. The influence of daily energy doses of 0.03, 0.3 and 0.9 J of He-Ne laser irradiation on the repair of surgically produced tibia damage was investigated in Wistar rats. Laser treatment was initiated 24 h after the trauma and continued daily for 7 or 14 days in two groups of nine rats (n=3 per laser dose and period). Two control groups (n=9 each) with injured tibiae were used. The course of healing was monitored using morphometrical analysis of the trabecular area. The organization of collagen fibers in the bone matrix and the histology of the tissue were evaluated using Picrosirius-polarization method and Masson's trichrome. After 7 days, there was a significant increase in the area of neoformed trabeculae in tibiae irradiated with 0.3 and 0.9 J compared to the controls. At a daily dose of 0.9 J (15 min of irradiation per day) the 7-day group showed a significant increase in trabecular bone growth compared to the 14-day group. However, the laser irradiation at the daily dose of 0.3 J produced no significant decrease in the trabecular area of the 14-day group compared to the 7-day group, but there was significant increase in the trabecular area of the 15-day controls compared to the 8-day controls. Irradiation increased the number of hypertrophic osteoclasts compared to non-irradiated injured tibiae (controls) on days 8 and 15. The Picrosirius-polarization method revealed bands of parallel collagen fibers (parallel-fibered bone) at the repair site of 14-day-irradiated tibiae, regardless of the dose. This organization improved when compared to 7-day-irradiated tibiae and control tibiae. These results show that low-level laser therapy stimulated the growth of the trabecular area and the concomitant invasion of osteoclasts during the first week, and hastened the organization of matrix collagen (parallel alignment of the fibers) in a second phase not seen in control, non-irradiated tibiae at the same period. The active osteoclasts that invaded the regenerating site were probably responsible for the decrease in trabecular area by the fourteenth day of irradiation. J Clin Laser Med Surg. 2002 Apr;20(2):83-7.

Computerized morphometric assessment of the effect of low-level laser therapy on bone repair: an experimental animal study.
Silva Jnior AN, Pinheiro AL, Oliveira MG, Weismann R, Ramalho LM, Nicolau RA. School of Dentistry, Postgraduate Programe on Oral and Maxillofacial Surgery, Pontifcia Universidade Catlica do Rico Grande do Sul, Porto Alegre, Brazil.

Abstract

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 6microm 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. Lasers Med Sci. 2002;17(3):216-20.

Laser stimulation on bone defect healing: an in vitro study.

Guzzardella GA, Fini M, Torricelli P, Giavaresi G, Giardino R. Department of Experimental Surgery, Codivilla-Putti Research Institute/Rizzoli Orthopaedic Institute, Italy. gaetanoantonio. guzzardella@ior.it
Abstract

The aim of this in vitro study was to evaluate whether low-power laser (LPL) stimulation can accelerate bone healing. Bone defects of a standard area were created in the distal epiphysis of 12 femora explanted from six rats, and they were cultured in BGJb medium for 21 days. Six defects were treated daily with Ga-Al-As, 780 nm LPL for 10 consecutive days (lased group, LG), while the remainder were sham-treated (control group, CG). Alkaline phosphatase/total protein (ALP/TP), calcium (Ca), and nitric oxide (NO) were tested on days 7, 14 and 21 to monitor the metabolism of cultured bone. The percentage of healing of the defect area was determined by histomorphometric analysis. After 21 days significant increases were observed in ALP/TP in LG versus CG (p<0.001), in NO in the LG versus CG ( p<0.0005) and in Ca in CG versus LG ( p<0.001). The healing rate of the defect area in the LG was higher than in the CG (

p=0.007). These in vitro results suggest that Ga-Al-As LPL treatment may play a positive role in bone defect healing. Int J Artif Organs. 2001 Dec;24(12):898-902.

Laser technology in orthopedics: preliminary study on low power laser therapy to improve the bone-biomaterial interface.
Guzzardella GA, Torricelli P, Nicoli Aldini N, Giardino R. Experimental Surgery Department, Research Institute Codivilla Putti, Bologna, Italy. gaetanoantonio.guzzardella@ior.it
Abstract

Low Power Laser (LPL) seems to enhance the healing of bone defects and fractures. The effect of LPL in other orthopedic areas such as osteointegration of implanted prosthetic bone devices is still unclear. In the present study, 12 rabbits were used to evaluate whether Ga-Al-As (780 nm) LPL stimulation has positive effects on osteointegration. Hydroxyapatite (HA) cylindrical nails were drilled into both distal femurs of rabbits. From postoperative day 1 and for 5 consecutive days, the left femura of all rabbits were given LPL treatment (Laser Group-LG) with the following parameters: 300 Joule/cm2, 1 Watt, 300 Hertz, pulsating emission, 10 minutes. The right femura were sham-treated (Control Group-CG). At 4 and 8 weeks after implantation, histologic and histomorphometric investigations evaluated bone-biomaterial-contact. Histomorphometry showed a higher degree of osteointegration at the HA-bone interface in the LG Group at 4 (p < 0.0005) and 8 weeks (p < 0.001). These preliminary positive results seem to support the hypothesis that LPL treatment can be considered a good tool to enhance the boneimplant interface in orthopedic surgery. Bull Exp Biol Med. 2001 Apr;131(4):399-402.

Healing of bone fractures of rat shin and some immunological indices during magnetic laser therapy and osteosynthesis by the ilizarov method.
Baibekov IM, Khanapiyaev UK. Laboratory of Pathological Anatomy, V. Vakhidov National Surgery Center; Institute of Traumatology and Orthopedics, Ministry of Health of Uzbekistan, Tashkent.

Abstract

The effect of magnetic and laser therapy on healing of bone fractures and blood levels of T and B lymphocytes was studied in rats during osteosynthesis by the Ilizarov method. Laser therapy induced changes in cells attesting to stimulation of reparative processes and normalization of immunological parameters. Lasers Med Sci. 2001;16(3):213-7.

Stimulation of MCM3 gene expression in osteoblast by low level laser irradiation.

Yamamoto M, Tamura K, Hiratsuka K, Abiko Y. Department of Biochemistry, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Abstract

Biostimulatory effect of cell proliferation and bone formation by laser irradiation has been reported, however, very little is known about the molecular basis of mechanisms. We previously constructed the cDNA library of mouse osteoblastic cells (MC3T3-E1) which enhanced gene expression by laser irradiation using a subtracted gene cloning procedure. In the present study, we focused on a gene clone, designated as MCL-140, which exhibited the high homology of DNA sequence with mouse minichromosome maintenance (MCM) 3 gene. MCM3 is involved in the initiation of DNA replication as licensing factor in eukaryotic cells. Nucleotide sequence of MCL-140 insert was determined and assessed in the nucleic acid databases. The transcription level of MCL-140 was examined by Northern blot analysis. The DNA sequences of clone MCL140 insert exhibited 96.2% homology with MCM 3 gene coding P1 protein. Higher MCM3 mRNA levels were observed in laser-irradiated cells compared to the levels in non-irradiated cells: furthermore, radiolabelled thymidine incorporation was increased by laser irradiation. These findings suggest that low-level laser irradiation may enhance DNA replication and play a role in stimulating proliferation of osteoblast through the enhancement of the MCM3 gene expression. Clin Orthod Res. 2001 Feb;4(1):3-14

The effects of low level laser irradiation on osteoblastic cells.

Coombe AR, Ho CT, Darendeliler MA, Hunter N, Philips JR, Chapple CC, Yum LW. Discipline of Orthodontics, Faculty of Dentistry, University of Sydney, New South Wales, Australia; Institute of Dental Research, United Dental Hospital, New South Wales, Australia.

Low level laser therapy has been used in treating many conditions with reports of multiple clinical effects including promotion of healing of both hard and soft tissue lesions. Low level laser therapy as a treatment modality remains controversial, however. The effects of wavelength, beam type, energy output, energy level, energy intensity, and exposure regime of low level laser therapy remain unexplained. Moreover, no specific therapeutic window for dosimetry and mechanism of action has been determined at the level of individual cell types. The aim of this study was to investigate the effects of low level laser irradiation on the human osteosarcoma cell line, SAOS-2. The cells were irradiated as a single or daily dose for up to 10 days with a GaAlAs continuous wave diode laser (830 nm, net output of 90 mW, energy levels of 0.3, 0.5, 1, 2, and 4 Joules). Cell viability was not affected by laser irradiation, with the viability being greater than 90% for all experimental groups. Cellular proliferation or activation was not found to be significantly affected by any of the energy levels and varying exposure regimes investigated. Low level laser irradiation did result in a heat shock response at an energy level of 2 J. No significant early or late effects of laser irradiation on protein expression and alkaline phosphatase activity were found. Investigation of intracellular calcium concentration revealed a tendency of a transient positive change after irradiation. Low level laser irradiation was unable to stimulate the osteosarcoma cells utilised for this research at a gross cell population level. The heat shock response and increased intracellular calcium indicate that the cells do respond to low level laser irradiation. Further research is required, utilising different cell and animal models, to more specifically determine the effects of low level laser irradiation at a cellular level. These effects should be more thoroughly investigated before low level laser therapy can be considered as a potential accelerator stimulus for orthodontic tooth movement.
Clin Oral Implants Res. 2003 Apr;14(2):226-32.

Osseointegration of endosseous ceramic implants after postoperative low-power laser stimulation: an in vivo comparative study.
Guzzardella GA, Torricelli P, Nicoli-Aldini N, Giardino R. Department of Experimental Surgery/Codivilla-Putti Research Institute, Rizzoli Orthopaedic Institute, Bologna, Italy. gaetanoantonio. guzzardella@ior.it Stimulation with low-power laser (LPL) can enhance bone repair as reported in experimental studies on bone defects and fracture healing. Little data exist concerning the use of LPL postoperative stimulation to improve osseointegration of endosseous implants in orthopaedic and dental surgery. An in vivo model was used for the present study to evaluate whether Ga-Al-As (780 nm) LPL stimulation can improve biomaterial osseointegration. After drilling holes, cylindrical implants of hydroxyapatite (HA) were placed into both distal femurs of 12 rabbits. From postoperative day 1 and for 5 consecutive days, the left femurs of all rabbits were submitted to LPL treatment (LPL group) with the following parameters: 300 J/cm2, 1 W, 300 Hz, pulsating emission, 10 min. The right femurs were sham-treated (control group). Three and 6 weeks after implantation, histomorphometric and microhardness measurements were taken. A higher affinity index was observed at the HA-bone interface in the LPL group at 3 (P<0.0005)

and 6 weeks (P<0.001); a significant difference in bone microhardness was seen in the LPL group vs. the control group (P<0.01). These results suggest that LPL postoperative treatment enhances the bone-implant interface. Clin Laser Med Surg. 2002; 20: 83-87

Computerized morphometric assessment of the effect of low-level laser therapy on bone repair: an experimental animal study.
Silva Jnior AN, Pinheiro AL, Oliveira MG, Weismann R, Ramalho LM, Nicolau RA. J 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. 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. 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). Laser Med Surg Abstract issue, 2002: 11.

Effects of visible NIR low intensity laser on implant osseointegration in vivo.

Blay A, Blay CC, Groth EB et al. The effects of 680 and 830 nm lasers on osseointegration was studied by Blay. 30 adult rats were divided into three groups; two laser groups and one control. The rats in the two laser groups had pure titanium Frialit-2 implants implanted into each proximal metaphysis of their respective tibias, inserted with a 40 Ncm torque. The initial stability was monitored by means of a resonance frequency analyser. Ten irradiations were performed, 48 hours apart, 4 J/cm2 on two points, starting immediately after surgery. Resonance frequency analysis indicated a significant difference between frequency values at 3 and 6 weeks, as compared to control. At 6 weeks the removal torque in the laser groups was much higher than in the control group. Laser Surg Med. Abstract Issue 2002. abstract 303.

Bone repair of the periapical lesions treated or not with low intensity laser (wavelength=904 nm).
Sousa GR, Ribeiro MS, Groth E B. The effect of bone repair in periapical lesions has been studied by Sousa []. 15 patients with a total of 18 periapical lesions were divided into two groups. One group received endodontic treatment and/or periapical surgery. The patients in the other group were submitted to the same procedure and in addition the lesions were irradiated by GaAs laser, 11 mW, 9 J/cm2. This therapy was performed during 10 sessions with an interval of 72 hours. Bone regeneration was evaluated through X-ray examination. The results showed a significant difference between the laser and the control group in favour of the laser group.
J Clin Laser Med Surg. 2003 Dec;21(6):383-8.

Effect of 830-nm laser light on the repair of bone defects grafted with inorganic bovine bone and decalcified cortical osseous membrane.
Barbos Pinheiro AL, Limeira Junior Fde A, Marquez Gerbi ME, Pedreira Ramalho LM, Marzola C, Carneiro Ponzi EA, Oliveira Soares A, Bandeira De Carvalho LC, Vieira Lima HC, Oliveira Goncalves T. Laser Center, School of Dentistry, Federal University of Bahia, Salvador, Brazil. albp@ufba.br OBJECTIVE: The aim of this study was to assess histologically the effect of LLLT (lambda830 nm) on the repair of standardized bone defects on the femur of Wistar albinus rats grafted with inorganic bovine bone and associated or not to decalcified bovine cortical bone membrane. BACKGROUND DATA: Bone loss may be a result of several pathologies, trauma or a consequence of surgical procedures. This led to extensive studies on the process of bone repair and development of techniques for the correction of bone defects, including the use of several types of grafts, membranes and the association of both techniques. There is evidence in the literature of the positive effect of LLLT on the healing of soft tissue wounds. However, its effect on bone is not completely understood. MATERIALS AND METHODS: Five randomized groups were studied: Group I (Control); Group IIA (Gen-ox); Group IIB (Gen-ox + LLLT); Group IIIA (Gen-ox + Gen-derm) and Group IIIB (Gen-ox + Gen-derm + LLLT). Bone defects were created at the femur of the animals and were treated according to the group. The animals of the irradiated groups were irradiated every 48 h during 15 days; the first irradiation was performed immediately after the surgical procedure. The animals were irradiated transcutaneously in four points around the defect. At each point a dose of 4 J/cm2 was given (phi approximately 0.6 mm, 40 mW) and the total dose per session was 16 J/cm2. The animals were humanely killed 15, 21, and 30 days after surgery. The

specimens were routinely processed to wax, serially cut, and stained with H&E and Picrosirius stains and analyzed under light microscopy. RESULTS: The results showed evidence of a more advanced repair on the irradiated groups when compared to non-irradiated ones. The repair of irradiated groups was characterized by both increased bone formation and amount of collagen fibers around the graft within the cavity since the 15th day after surgery, through analysis of the osteoconductive capacity of the Gen-ox and the increment of the cortical repair in specimens with Gen-derm membrane. CONCLUSION: It is concluded that LLLT had a positive effect on the repair of bone defect submitted the implantation of graft.
J Clin Laser Med Surg. 2003 Oct;21(5):271-7.

Effect of pulse frequency of low-level laser therapy (LLLT) on bone nodule formation in rat calvarial cells.
Ueda Y, Shimizu N. Department of Orthodontics, Nihon University School of Dentistry at Matsudo Chiba, Japan. OBJECTIVE: The purpose of this study was to determine the effect of pulse frequencies of lowlevel laser therapy (LLLT) on bone nodule formation in rat calvarial cells in vitro. BACKGROUND DATA: Various photo-biostimulatory effects of LLLT, including bone formation, were affected by some irradiation factors such as total energy dose, irradiation phase, laser spectrum, and power density. However, the effects of pulse frequencies used during laser irradiation on bone formation have not been elucidated. MATERIALS AND METHODS: Osteoblast-like cells isolated from fetal rat calvariae were irradiated once with a low-energy Ga-Al-As laser (830 nm, 500 mW, 0.48-3.84 J/cm2) in four different irradiation modes: continuous irradiation (CI), and 1-, 2-, and 8-Hz pulsed irradiation (PI-1, PI-2, PI-8). We then investigated the effects on cellular proliferation, bone nodule formation, alkaline phosphatase (ALP) activity, and ALP gene expression. RESULTS: Laser irradiation in all four groups significantly stimulated cellular proliferation, bone nodule formation, ALP activity, and ALP gene expression, as compared with the nonirradiation group. Notably, PI-1 and -2 irradiation markedly stimulated these factors, when compared with the CI and PI-8 groups, and PI-2 irradiation was the best approach for bone nodule formation in the present experimental conditions. CONCLUSION: Since low-frequency pulsed laser irradiation significantly stimulates bone formation in vitro, it is most likely that the pulse frequency of LLLT an important factor affecting biological responses in bone formation.

Lasers Med Sci. 2003;18(2):78-82.

Effect of low-level laser irradiation on osteoglycin gene expression in osteoblasts.

Hamajima S, Hiratsuka K, Kiyama-Kishikawa M, Tagawa T, Kawahara M, Ohta M, Sasahara H, Abiko Y. Nihon University School of Dentistry at Matsudo, Chiba, Japan. Many studies have attempted to elucidate the mechanism of the biostimulatory effects of lowlevel laser irradiation (LLLI), but the molecular basis of these effects remains obscure. We investigated the stimulatory effect of LLLI on bone formation during the early proliferation stage of cultured osteoblastic cells. A mouse calvaria-derived osteoblastic cell line, MC3T3-E1, was utilised to perform a cDNA microarray hybridisation to identify genes that induced expression by LLLI at the early stage. Among those genes that showed at least a twofold increased expression, the osteoglycin/mimecan gene was upregulated 2.3-fold at 2 h after LLLI. Osteoglycin is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix which was previously called the osteoinductive factor. SLRP are abundantly contained in the bone matrix, cartilage cells and connective tissues, and are thought to regulate cell proliferation, differentiation and adhesion in close association with collagen and many other growth factors. We investigated the time-related expression of this gene by LLLI using a reverse transcription polymerase chain reaction (RT-PCR) method, and more precisely with a real-time PCR method, and found increases of 1.5-2-fold at 2-4 h after LLLI compared with the non-irradiated controls. These results suggest that the increased expression of the osteoglycin gene by LLLI in the early proliferation stage of cultured osteoblastic cells may play an important role in the stimulation of bone formation in concert with matrix proteins and growth factors.
Clin Oral Implants Res. 2002 Jun;13(3):288-92.

Effect of low-power laser irradiation on bony implant sites.

Dortbudak O, Haas R, Mailath-Pokorny G. Department of Oral Surgery, Dental School, University of Vienna, Austria. orhun.doerbudak@univie.ac.at This study was designed to examine the effects of low-energy laser irradiation on osteocytes and bone resorption at bony implant sites. Five male baboons with a mean age of 6.5 years were used in the study. Four holes for accommodating implants were drilled in each iliac crest. Sites on the left side were irradiated with a 100 mW low-energy laser (690 nm) for 1 min (6 Joule) immediately after drilling and insertion of four sandblasted and etched (Frialit-2 Synchro) implants. Five days later, the bone was removed en bloc and was evaluated histomorphometrically. The mean osteocyte count per unit area was 109.8 cells in the irradiated group vs. 94.8 cells in the control group. As intra-individual cell counts varied substantially, osteocyte viability was used for evaluation. In the irradiated group, viable osteocytes were found

in 41.7% of the lacuna vs. 34.4% in the non-irradiated group. This difference was statistically significant at P < 0.027. The total resorption area, eroded surface, was found to be 24.9% in the control group vs. 24.6% in the irradiated group. This difference was not statistically significant. This study showed that osteocyte viability was significantly higher in the samples that were subjected to laser irradiation immediately after implant site drilling and implant insertion, in comparison to control sites. This may have positive effects on the integration of implants. The bone resorption rate, in contrast, was not affected by laser irradiation.
Int J Artif Organs. 2001 Dec;24(12):898-902.

Laser technology in orthopedics: preliminary study on low power laser therapy to improve the bone-biomaterial interface.
Guzzardella GA, Torricelli P, Nicoli Aldini N, Giardino R. Experimental Surgery Department, Research Institute Codivilla Putti, Bologna, Italy. gaetanoantonio.guzzardella@ior.it Low Power Laser (LPL) seems to enhance the healing of bone defects and fractures. The effect of LPL in other orthopedic areas such as osteointegration of implanted prosthetic bone devices is still unclear. In the present study, 12 rabbits were used to evaluate whether Ga-Al-As (780 nm) LPL stimulation has positive effects on osteointegration. Hydroxyapatite (HA) cylindrical nails were drilled into both distal femurs of rabbits. From postoperative day 1 and for 5 consecutive days, the left femura of all rabbits were given LPL treatment (Laser Group-LG) with the following parameters: 300 Joule/cm2, 1 Watt, 300 Hertz, pulsating emission, 10 minutes. The right femura were sham-treated (Control Group-CG). At 4 and 8 weeks after implantation, histologic and histomorphometric investigations evaluated bone-biomaterial-contact. Histomorphometry showed a higher degree of osteointegration at the HA-bone interface in the LG Group at 4 (p < 0.0005) and 8 weeks (p < 0.001). These preliminary positive results seem to support the hypothesis that LPL treatment can be considered a good tool to enhance the boneimplant interface in orthopedic surgery.
Stomatologiia (Mosk). 2001;80(2):33-5.

Prevention of inflammatory complications after mandibular osteosynthesis by a combination of low-frequency ultrasound and laser exposure.
[Article in Russian] Tarasenko SV, Agapov VS, Trukhina GM, Techiev SK, Artsibushev VI.

Clinical and laboratory study of the efficiency of separate and combined use of low-frequency ultrasound and laser exposure of the operative wound for prevention of pyoinflammatory complications during mandibular osteosynthesis was carried out. Clinical parameters of wound reparation in the course of healing and microbiological and cytological findings in various methods of treatment are presented. The results evidence a high efficiency of these physical methods, particularly of their combination.
Bull Exp Biol Med. 2001 Apr;131(4):399-402.

Healing of bone fractures of rat shin and some immunological indices during magnetic laser therapy and osteosynthesis by the ilizarov method.
Baibekov IM, Khanapiyaev UK. Laboratory of Pathological Anatomy, V. Vakhidov National Surgery Center; Institute of Traumatology and Orthopedics, Ministry of Health of Uzbekistan, Tashkent. The effect of magnetic and laser therapy on healing of bone fractures and blood levels of T and B lymphocytes was studied in rats during osteosynthesis by the Ilizarov method. Laser therapy induced changes in cells attesting to stimulation of reparative processes and normalization of immunological parameters. J Oral Sci. 2001 Mar;43(1):55-60.

Pulse irradiation of low-power laser stimulates bone nodule formation.

Ueda Y, Shimizu N. Department of Orthodontics, Nihon University School of Dentistry at Matsudo, Chiba, Japan. ueda@mascat.nihon-u.ac.jp
Abstract

Although low-power laser irradiation provides many anabolic effects such as acceleration of bone formation, the effects of different pulse frequencies used during laser irradiation on bone formation have not been elucidated. Osteoblastic cells isolated from fetal rat calvariae were irradiated once with a low-power Ga-Al-As laser (830 nm, 500 mW) in two different irradiation modes; continuous irradiation (CI), and 1 Hz pulsed irradiation (PI). We then investigated the effects on cellular proliferation, bone nodule formation, alkaline phosphatase (ALP) activity, and ALP gene expression. Laser irradiation in both groups significantly stimulated cellular proliferation, bone nodule formation, ALP activity, and ALP gene expression, as compared with the nonirradiation group. Notably, PI markedly stimulated these factors, when compared with the

CI group. Since 1 Hz pulsed laser irradiation significantly stimulates bone formation in vitro, it is most likely that pulse frequency is an important factor affecting biological responses in bone formation. us for orthodontic tooth movement.
Clin Orthod Res. 2001 Feb;4(1):3-14.

The effects of low level laser irradiation on osteoblastic cells.

Coombe AR, Ho CT, Darendeliler MA, Hunter N, Philips JR, Chapple CC, Yum LW. Discipline of Orthodontics, Faculty of Dentistry, University of Sydney, New South Wales, Australia; Institute of Dental Research, United Dental Hospital, New South Wales, Australia. Low level laser therapy has been used in treating many conditions with reports of multiple clinical effects including promotion of healing of both hard and soft tissue lesions. Low level laser therapy as a treatment modality remains controversial, however. The effects of wavelength, beam type, energy output, energy level, energy intensity, and exposure regime of low level laser therapy remain unexplained. Moreover, no specific therapeutic window for dosimetry and mechanism of action has been determined at the level of individual cell types. The aim of this study was to investigate the effects of low level laser irradiation on the human osteosarcoma cell line, SAOS-2. The cells were irradiated as a single or daily dose for up to 10 days with a GaAlAs continuous wave diode laser (830 nm, net output of 90 mW, energy levels of 0.3, 0.5, 1, 2, and 4 Joules). Cell viability was not affected by laser irradiation, with the viability being greater than 90% for all experimental groups. Cellular proliferation or activation was not found to be significantly affected by any of the energy levels and varying exposure regimes investigated. Low level laser irradiation did result in a heat shock response at an energy level of 2 J. No significant early or late effects of laser irradiation on protein expression and alkaline phosphatase activity were found. Investigation of intracellular calcium concentration revealed a tendency of a transient positive change after irradiation. Low level laser irradiation was unable to stimulate the osteosarcoma cells utilised for this research at a gross cell population level. The heat shock response and increased intracellular calcium indicate that the cells do respond to low level laser irradiation. Further research is required, utilising different cell and animal models, to more specifically determine the effects of low level laser irradiation at a cellular level. These effects should be more thoroughly investigated before low level laser therapy can be considered as a potential accelerator stimul Bone. 1998 Apr;22(4):347-54.

Low-energy laser irradiation stimulates bone nodule formation at early stages of cell culture in rat calvarial cells.
Ozawa Y, Shimizu N, Kariya G, Abiko Y.

Department of Orthodontics, Nihon University School of Dentistry at Matsudo, Chiba, Japan.

Abstract

Although the acceleration of bone regeneration by laser treatment has been reported, the mechanisms of action of laser on bone are unclear. To determine the target cells responsible for the action of laser irradiation and roles of irradiation on these cells during bone formation, we investigated the effects of low-energy laser irradiation at various cell culture stages on cellular proliferation, bone nodule formation, alkaline phosphatase activity, and osteocalcin gene expression, employing rat calvarial cells. Osteoblast-like cells isolated from fetal rat calvariae were irradiated once with a low-energy Ga-Al-As laser (830 nm, 500 mW) at various cell culture stages (days 1-16). Laser irradiation at early stages of culture significantly stimulated cellular proliferation, ALP activity, and osteocalcin gene expression thereafter. Furthermore, laser irradiation at earlier stages of culture significantly stimulated a greater number (1.7-fold) and larger area (3.4-fold) of bone nodules that had developed in the culture dish on day 21. However, these effects could not be found by irradiation at a later date. These results suggest that laser irradiation may play two principal roles in stimulating bone formation. One is stimulation of cellular proliferation, especially proliferation of nodule-forming cells of osteoblast lineage, and the other is stimulation of cellular differentiation, especially to committed precursors, resulting in an increase in the number of more differentiated osteoblastic cells and an increase in bone formation. Both bone-formation-stimulating roles may be exhibited by laser irradiation to immature cells only. Lasers Surg Med. 1998;22(2):97-102.

Effect of low-power laser irradiation on the mechanical properties of bone fracture healing in rats.
Luger EJ, Rochkind S, Wollman Y, Kogan G, Dekel S. Department of Orthopedic Surgery B, Tel Aviv Sourasky Medical Center, Israel.
Abstract

BACKGROUND AND OBJECTIVE: Low-power laser irradiation (LPLI) has been found to have a positive effect on bone fracture healing in animal models, based on morphogenic, biochemical, roentgenographic, and electron microscopic measurements. We investigated the effect of LPLI on bone fracture healing in rats using biomechanical methods. STUDY DESIGN/MATERIALS AND METHODS: Two groups of male Wistar rats, divided in a randomized block design in a blinded fashion, each consisting of 25 animals, were subjected to anesthesia and tibial bone fracture with internal fixation. The first group was treated with LPLI (HeNe laser 632.8 nm, 35 mW), applied transcutaneously over 30 minutes to the area of the fracture daily for 14 days. The second group served as a control. After 4 weeks, the tibia was removed and tested at tension up to failure (by a Lloyd LR 50K testing apparatus, UK) in 16 rats

from group I and 15 from group II. The maximal load at failure, the structural stiffness of the tibia (callus stiffness), and the extension maximal load were measured. RESULTS: The maximal load at failure and the structural stiffness of the tibia were found to be elevated significantly in the irradiated group (P = .014 and P = .0023, respectively), whereas the extension maximal load was reduced (P = .015). In addition, gross non-union was found in four fractures in the control group, compared to none in the irradiated group. CONCLUSION: These results suggest that LPLI treatment may play a role in enhancing bone healing.
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