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Bone Regeneration

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.

Lasers Surg Med. 2009 Apr;41(4):298-304.
PMID: 19347943 [PubMed - indexed for MEDLINE]

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 TGF-beta2, 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.

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.

J Clin Laser Med Surg. 2003 Oct;21(5):271-7.
PMID: 14651794 [PubMed - indexed for MEDLINE]

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.

Bone Stimulation by Low Level Laser - A Theoretical Model for the Effects

Philip Gable, B App Sc P.T. G Dip Sc Res (LLLT) MSc, Australia Jan Tunér, D.D.S., Sweden

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 bi-layer. 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 Dörtbudak (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 i.a. 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, A R et al (2001). The effect of low level laser irradiation on osteoblastic cells. Clin Ort Res. 4: 3-14.

Dörtbudak, 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, C. P. (1995). “Does Laser enhance bruising in acute sporting injuries.” Aust. J. Physio. 41(4): 267-269.

Gabel, C. P. (1995). The effect of LLLT on slow healing wounds and ulcers. Health Sciences. Darwin, Northern Territory.

Guzzardella, G A et al (2002). Laser stimulation on bone defect healing: An in vitro study. Lasers Med Sci. 17(3): 216-220.

Guzzardella, G A 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, T. I. (1988). “Molecular mechanism of the therapeutic effects of low intensity laser radiation.” Lasers in Life Science 2: 53-74.

Karu, T. I. (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 lymphaitic system contribution. Personal Communication to C. P. Gabel. Adelaide Oct 1999.

Pouyssegur, J. (1985). “The growth factor-activatable Na+/H+ exchange system: a genetic approach. In Karu, T.I. 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: 453-455.

Pyczek, 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, K. C. (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.

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