Bone Healing

Stein A, Benayahu D, Maltz L, Oron U.

Photomed Laser Surg. 2005 Apr;23(2):161-6

Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel.

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.

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Khadra M, Kasem N, Haanaes HR, Ellingsen JE, Lyngstadaas SP.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004 Jun;97(6):693-700.

Department of Oral Surgery and Oral Medicine, University of Oslo, Norway. maawan@odont.uio.no

OBJECTIVE: To evaluate the effect of low-level laser therapy (LLLT), using a GaAlAs diode laser device, on bone healing and growth in rat calvarial bone defects.

STUDY DESIGN: An animal trial of 4 weeks' duration was conducted using a randomized blind, placebo-controlled design. Standardized round osseous defects of 2.7 mm diameter were made in each parietal bone of 20 rats (n=40 defects). The animals were randomly divided into an experimental and a control group of 10 animals each. In the experimental group, a GaAlAs diode laser was applied immediately after surgery and then daily for 6 consecutive days. The control group received the same handling and treatment, but with the laser turned off. Five rats from each group were killed on day 14 and the remainder on day 28 postoperatively. From each animal, tissue samples from one defect were prepared for histochemistry and samples from the contralateral defect for histology. Levels of calcium, phosphorus, and protein were determined by using atomic absorption spectrometry, colorimetry, and photometry, respectively. Student t-test and Mann-Whitney were used for statistical analyses.

RESULTS: At both time points the tissue samples from the experimental animals contained significantly more calcium, phosphorus, and protein than the controls. Similarly, histological analyses disclosed more pronounced angiogenesis and connective tissue formation, and more advanced bone formation in the experimental group than in the controls.

CONCLUSION: LLLT may enhance bone formation in rat calvarial bone defects.

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Ueda Y, Shimizu N.

J Clin Laser Med Surg. 2003 Oct;21(5):271-7.

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 low-level 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 non-irradiation 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.

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Nicola RA, Jorgetti V, Rigau J, Pacheco MT, dos Reis LM, Zangaro RA.

Lasers Med Sci. 2003;18(2):89-94.

Vale of Paraiba University, Sao Jose dos Campos, SP, Brazil.

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.

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Guzzardella GA, Fini M, Torricelli P, Giavaresi G, Giardino R.

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

Department of Experimental Surgery, Codivilla-Putti Research Institute/Rizzoli Orthopaedic Institute, Italy. gaetanoantonio.guzzardella@ior.it

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.

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Nissan J, Assif D, Gross MD, Yaffe A, Binderman I.

J Oral Rehabil. 2006 Aug;33(8):619-924.

Department of Oral Rehabilitation, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel. nissandr@post.tau.ac.il

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 (Gallium-Arsenide) 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.

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Weber JB, Pinheiro AL, de Oliveira MG, Oliveira FA, Ramalho LM.

Photomed Laser Surg. 2006 Feb;24(1):38-44.

School of Dentistry, Pontificia Universidade Catolica 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.

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Gerbi ME, Pinheiro AL, Marzola C, Limeira Junior Fde A, Ramalho LM, Ponzi EA, Soares AO, Carvalho LC, Lima HV, Goncalves TO.

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

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 (Gen-derm).

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.