Low-level laser therapy using the minimally invasive laser needle system on osteoporotic bone in ovariectomized mice

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Abstract

This study tested the effectiveness of low-level laser therapy (LLLT) in preventing and/or treating osteoporotic trabecular bone. Mice were ovariectomized (OVX) to induce osteoporotic bone loss. The tibiae of eight OVX mice were treated for 5 days each week for 2 weeks by LLLT (660 nm, 3 J) using a minimally invasive laser needle system (MILNS) which is designed to minimize loss of laser energy before reaching bone (LASER group). Another eight mice received a sham treatment (SHAM group). Structural parameters of trabecular bone were measured with in vivo micro-computed tomography images before and after laser treatment. After LLLT for 2 weeks, the percentage reduction (%R) was significantly lower in BV/TV (bone volume fraction) and Tb.N (trabecular number, p < 0.05 and p < 0.05) and significant higher in Tb.Sp (trabecular separation) and SMI (structure model index, p < 0.05 and p < 0.05) than in the SHAM group. The %R in BV/TV at sites directly treated by LLLT was significantly lower in the LASER group than the SHAM group (p < 0.05, p < 0.05). These results indicated that LLLT using MILNS may be effective for preventing and/or treating trabecular bone loss and the effect may be site-dependent in the same bone.

Introduction

Osteoporosis is a metabolic bone disorder characterized by deterioration of bone structure, low bone mass, and increased bone fragility, leading to increasing fracture risk [1]. Contributing factors in osteoporosis include estrogen deficiency [1], and insufficient weight-bearing activity [2]. Osteoporotic fractures are associated with increased morbidity and mortality as well as limitations in functional mobility [1], [3]. Patients may also experience chronic anxiety, fear, depression, and disabling pain [4]. In addition to significant reductions in the quality of life [5], osteoporosis generates direct and indirect costs for treatment and support, which accumulate over time [5]. Recently, non-pharmacological therapies have been suggested to treat and/or prevent osteoporosis as alternatives or adjuvant to pharmacological intervention, which might occasionally cause undesirable side effects.

Low-level laser treatment (LLLT) is a promising non-pharmacological therapy for bone regeneration. Although the biological mechanisms of LLLT are not fully revealed, it has been applied both clinically and experimentally to promote wound healing and tissue regeneration [6]. Bashardoust Tajali et al. reported that LLLT can facilitate healing of fractures in vivo [7], and trials have recently been conducted to test this application. Studies in vitro generally support the positive effects of LLLT on bone regeneration, increased bone formation [8] and decreased bone resorption [9]. However, attempts to use LLLT as a therapy for bone loss [10], [11], presented conflicting results. Medalha et al. found no beneficial effects of LLLT on biomechanical properties of the tibia in bone loss following spinal cord injury [10]. Diniz et al. suggested that LLLT may be effective in preventing ovariectomy-induced bone loss in rats, but only if combined with bisphosphonates. LLLT alone may not consistently enhance the bone volume fraction of trabecular bone, and when effective, may not prevent osteoporosis [12]. Renno et al. showed that LLLT enhances maximal loading capacity of the femur in ovariectomized osteopenic rats [13]. Because biomechanical properties of bone tissue differ within and between individuals of same genetic background, gender, and age [2], [14]. Moreover, bone adaptation to extrinsic factors and intrinsic factors may depend on its baseline status [15], [16]. However, there were few longitudinal studies on the bone adaptation to LLLT, by comparison to baseline status.

All such evaluations must consider the attenuation of laser energy through absorption, scattering, or/and reflection on and between tissues [17]. In this regard, studies suggest that laser therapy of higher intensity than used at present may be needed to deliver LLLT effectively [13], [17]. Data from Renno et al. show that maximal bone load in osteoporotic bone may be higher after 8 weeks of LLLT with 120 J/cm2 than with 60 J/cm [13]. Ninomiya et al. found that high-intensity pulsed laser can enhance osteoblast activity and reduce osteoclast number, leading to an increase in trabecular bone volume [17]. To build on these findings, we have developed the minimally invasive laser needle system (MILNS), which can deliver LLLT directly to bone with minimal loss of laser energy.

The effects of biophysical stimuli on bone, including ultrasound and partial mechanical loading, may be strongly site-specific. In a previous study (2011) we showed that low-intensity ultrasound stimulation (LIUS) may increase bone quantity at the site of direct treatment, but not at the far end of the same bone [18]. The effects of direct biophysical stimulation may not enhance bone regeneration at locations away from the stimulated site, and the response may differ between locations on the same bone [14]. The spatial distribution of effect relative to the stimulation site must be determined. To the best of our knowledge no study has addressed the spatial distribution of LLLT effects on osteoporotic bone loss following estrogen deficiency.

Recently, in vivo micro-computed tomography (μCT) can generate a time-lapse evaluation of bone response in a small animal, and assess variability in character within an individual bone. These capabilities are important because bone adaptation may differ according to baseline bone status [16] and because the longitudinal effects of LLLT on bone are biologically and clinically relevant. Recently, in vivo μCT has provided new insight into bone adaptation to external stimuli [14], [18]. In this study we evaluated LLLT, using MILNS, as a preventive and therapeutic intervention for trabecular bone loss following estrogen deficiency. We also evaluated the spatial distribution of the LLLT effect in bone relative to the site of laser therapy. These changes in bone treated with LLLT were followed over time by using in vivo μCT.

Section snippets

Animal preparation

All procedures were performed according to a protocol that the Yonsei University Animal Care Committee approved. Twenty 12-week-old virgin female ICR mice were ovariectomized (OVX) bilaterally to induce osteoporosis. The mice were anesthetized during surgery and micro-CT scanning using a combination of xylazine (0.5 mL/kg, Bayer Korea, Korea) and ketamine (1.5 mL/kg, Huons, Korea). Two weeks after OVX, a 39.6% decrease in bone volume fraction (BV/TV %) confirmed significant bone loss. Because

Results

The structural parameters at 0 weeks are shown in Table 2. After 2 weeks of LLLT, the BV/TV and Tb.N tended to decrease in both LASER and SHAM groups, and the Tb.Sp and SMI tended to increase (Fig. 3). The Tb.Th increased slightly in the LASER group, but decreased slightly in the SHAM group. The %Rs of BV/TV and Tb.N were 58.97% and 52.17%, respectively, in the LASER group less than in SHAM (p < 0.05, p < 0.05, respectively). However, the %Rs of Tb.SP and SMI were 53.47% and 63.80%, respectively, in

Discussion

In this study, we found that LLLT, using MILNS as developed by our group, may be effective in preventing and/or treating trabecular bone loss in osteoporosis. Through the use of a fine hollow guide needle with a laser fiber, LLLT can deliver treatment directly to a site of bone loss with minimal attenuation of laser energy (Fig. 1) [19]. We also found that the quantitative and qualitative effects of LLLT at a site in bone may depend on the distance and direction of that site from the site of

Competing interests

None declared.

Funding

National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology.

Ethical approval

Yonsei University Animal Care Committee approved (YWC-090213-1).

Acknowledgements

This research was supported by the Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (2010-00757).

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