Increase of bone volume by a nanosecond pulsed laser irradiation is caused by a decreased osteoclast number and an activated osteoblasts

Abstract

The biostimulatory effects of laser irradiation focus not only in the field of soft tissue but also bone formation. Studies have shown that the light of a nanosecond pulsed laser which has a high peak power can produce stress waves in tissue. We have hypothesized that nanosecond pulsed laser irradiation stimulates bone formation. Our aim was to clarify the mechanism of increased bone volume by nanosecond pulsed laser irradiation. Rat femur was irradiated with a Q-switched Nd:YAG laser, which has a wavelength of 1064 nm. The quantification of trabecular architecture using three-dimensional morphometric analysis and measurement of bone mineral density (BMD) using pQCT was performed on day 1, day 3, day 5, and day 7 after laser irradiation. The laser effects on bone cells were also investigated using histological and immunohistochemical analysis. On day 1 after laser irradiation, bone volume (BV/TV), trabecular thickness (Tb.Th), and other parameters of the irradiated group did not significantly differ from the non-irradiation group (control). However, the mean BV/TV, Tb.Th, mineral apposition rate, and BMD of the laser group on day 7 after laser irradiation were significantly greater than those of the control. On histological analysis, the number of TRAP-positive osteoclasts was lower on day 3 after laser irradiation. Osteoblasts with activated clearance were seen in the laser irradiated group on day 1 and day 3. These data reveal that the increased bone volume by nanosecond pulsed laser irradiation causes an increase in osteoblast activity and a decrease in osteoclast number.

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.