Increase of bone volume by a nanosecond pulsed laser irradiation is caused by a decreased osteoclast number and an activated osteoblasts
Introduction
Physical stress is essential for maintenance of bone metabolism as bone is a mechanosensitive tissue that adapts its mass, architecture, and mechanical properties in response to physical stress [1]. When this balance is altered under conditions such as long-term bed-rest, immobility, space flight, or a microgravity environment, bone loss occurs, with a decrease in bone formation and/or an increase in resorption. Bone loss may result in bone fractures, which in turn impair quality of life. Thus far, the effects of physical stress on bone volume have been investigated using application of physical stress, such as mechanical loading [2], [3], ultrasound modality [4], electromagnetic field exposure [5], and laser light irradiation [6], [7].
Laser light irradiation has been applied in the medical field and possesses biostimulatory effects on wound healing, collagen synthesis [8], and fibroblast proliferation [9]. In addition, laser light appears to increase mitochondrial respiration and ATP synthesis [10], [11]. The laser light is receiving increasing attention not only regarding soft tissue, but also in bone metabolism. Although laser light irradiation has been reported to promote bone repair [12], bone nodule formation, osteoblast differentiation [13], and ALP activity [7], the mechanism of these effects remains unclear. The light energy at low-irradiation doses is absorbed by intracellular chromophores [6], [14]. Different types of laser systems have been used to obtain different biostimulatory effects: ruby, He–Ne, argon-ion, CO2, semiconductor, eximer, and others, administered as a continuous wave, interrupted wave, and pulsed lights. The biological effects of laser irradiation depend on the specifications of the light source (e.g., wavelength, output power, energy density) [15]. Wavelength is an important factor, which relates to penetration of laser light through the biological tissue. The oscillation mode, which changes the biological effects of the laser light, is also important. Interrupted laser light has been reported to be more effective for nodule formation than continuous laser light [13]. In the case of the same energy density, the peak power of pulsed laser light is higher than that of continuous and interrupted laser light. It is well known that a nanosecond pulsed laser light with high-intensity source can produce stress waves in the tissue by rapid heating [16]. We have been investigating the effects of a nanosecond pulsed laser irradiation on bone formation. In a previous study, we demonstrated that a nanosecond pulsed laser light increases bone volume in the femur of unloading rat using histomorphometric analysis [17]. However, the mechanisms of increased bone volume by laser irradiation have remained to be addressed in detail because the analysis of the effect has not been performed using three-dimensional analysis and histological analysis. In addition, it is necessary that the temporal changes after laser irradiation are estimated to clarify the mechanism.
In the present study, to examine circumstantially an increase in bone volume by nanosecond pulsed laser irradiation, we quantified the trabecular architecture using three-dimensional morphometric analysis and measurement of bone mineral density (BMD) using peripheral quantitative computed tomography (pQCT). Moreover, we investigated the effects of lasers on osteoblasts and osteoclasts using histological and immunochemical analysis in order to clarify the mechanism of the increase in bone volume.
Section snippets
Experimental animals
A total of 96 Sprague–Dawley female rats weighing 241.5 ± 4.9 g (mean ± SD), aged 10 weeks, were purchased from Charles River Japan (Yokohama, Japan) and acclimatized for a week before the beginning of the experiment. Our animal use protocol was reviewed and approved by the committee for the care and use of laboratory animals at Matsumoto Dental University.
Laser irradiation and tissue preparation
Rats were randomly divided into 8 groups. The 1dLA, 3dLA, 5dLA, and 7dLA groups included specimens from rats on day 1, day 3, day 5, and day 7
Body weight and size of femur after laser irradiation
The body weight of the rats on day 7 after laser irradiation did not significantly differ from that at the start of the experiment. The length and width of the rat femurs also did not differ among the groups (data not shown). There was no rise in heat in the irradiated region by laser light. Furthermore, no erythema, burns, or inflammatory responses were seen on the skin of the target area of the femur. The skin of the laser irradiated groups showed no abnormal appearance.
Bone morphometric analysis of trabecular bone
The images of the
Discussion
We described the increases of bone mass by a nanosecond pulsed laser light in the previous paper [17]. In the present paper, it was clear that the means BMD and SSI were increased by a nanosecond laser light. In addition, histochemical analysis revealed that number of osteoclast decreased on day 3 and activity of osteoblast accelerated on early stage after laser irradiation.
Bone morphometric analysis using μCT showed that the bone volume and trabecular thickness on day 7 after laser irradiation
Acknowledgments
The authors would like to thank Dr. Y. Ikehara for kindly providing the antibody against TNAP. This work was supported by a Grant-in-Aid for scientific research (A) (2) 14207075, 2002, 2003, 2004 and the foundation of Matsumoto Dental University in 2003.
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