Decreased Bone Volume and Bone Mineral Density in the Tibial Trabecular Bone Is Associated with Per2 Gene by 405 nm Laser Stimulation

Low-level laser therapy/treatment (LLLT) using a minimally invasive laser needle system (MILNS) might enhance bone formation and suppress bone resorption. In this study, the use of 405 nm LLLT led to decreases in bone volume and bone mineral density (BMD) of tibial trabecular bone in wild-type (WT) and Per2 knockout (KO) mice. Bone volume and bone mineral density of tibial trabecular bone was decreased by 405 nm LLLT in Per2 KO compared to WT mice at two and four weeks. To determine the reduction in tibial bone, mRNA expressions of alkaline phosphatase (ALP) and Per2 were investigated at four weeks after 405 nm laser stimulation using MILNS. ALP gene expression was significantly reduced in the LLLT-stimulated right tibial bone of WT and Per2 KO mice compared to the non-irradiated left tibia (p < 0.001). Per2 mRNA expression in WT mice was significantly reduced in the LLLT-stimulated right tibial bone compared to the non-irradiated left tibia (p < 0.001). To identify the decrease in tibial bone mediated by the Per2 gene, levels of runt-related transcription factor 2 (Runx2) and ALP mRNAs were determined in non-irradiated WT and Per2 KO mice. These results demonstrated significant downregulation of Runx2 and ALP mRNA levels in Per2 KO mice (p < 0.001). Therefore, the reduction in tibial trabecular bone resulting from 405 nm LLLT using MILNS might be associated with Per2 gene expression.

Low-level laser irradiation (LLLT) can scatter across the skin surface, limiting penetration to the deep bone layers. Thus, LLLT has been directly applied to the bone site of interest through tissue incision. In addition, initial photon density and therapeutic efficacy of LLLT is reduced by light-tissue interaction, such as absorption and scattering [13,14]. LLLT (660 nm wavelength) using a MILNS has been developed to overcome these light limitations in tissue. The effectiveness of this technique for preventing trabecular bone loss has been demonstrated in previous research [13,15,16].
The circadian clock genes Period 2 (Per2) and Cryptochrome 2 (Cry2) regulate distinct pathways in bone volume; Cry2 chiefly influences the osteoclasts, and Per2 acts on osteoblasts, indicating that Per2 and Cry2 differentially balance bone formation [17,18]. Mammalian Cry protein with flavin adenine dinucleotide (FAD) serves as a blue-light photoreceptor of 405 nm wavelength in murine cells [19,20]. The Kushibiki group has shown that LLLT (405 nm) can promote osteogenesis and reduce adipogenesis of mouse mesenchymal stromal cells (MSCs) by inducing translocation of Cry1 and Per2 proteins and decreasing Cry1 mRNA level. LLLT can effectively control the in vitro fate of MSCs as a therapeutic strategy by suppressing Cry transcription [19,20]. Therefore, the present study demonstrated that 405 nm laser stimulation using MILNS applied in vivo tibial trabecular bone in WT and Per2 gene KO mice to investigate bone volume and BMD with an in vivo micro-CT and expressions of Per2, Runx2, and ALP mRNAs through real-time quantitative polymerase chain reaction.

Results
The structural parameters to quantify tibial trabecular bone changes using micro-CT systems were shown in the 405 nm laser-irradiated WT and Per2 KO mice, respectively ( Figure 1). The bone microarchitecture values of BV/TV, Tb.Sp, Tb.N, Conn.Dn, and BMD were significantly changed in the 405 nm laser-irradiated WT and Per2 KO mice at four weeks compared to two weeks, respectively (BV/TV, Tb.Sp, and Tb.N, p < 0.01; Conn.Dn and BMD, p < 0.001) ( Figure 1). Three-dimensional (3D) images of decreased trabecular bone were seen in Per2 KO mice compared to WT mice that received 405 nm laser irradiation at 0, 2 and 4 weeks (Figure 2A). Changes in structural parameters were evaluated with total bone volume and BMD in WT mice and Per2 KO mice at 0, 2 and 4 weeks ( Figure 2B,C). Total bone volume and BMD were significantly reduced in Per2 KO mice compared to WT mice (p < 0.001).
To measure the reduction of tibial bone affected by LLLT using MILNS, levels of ALP and Per2 mRNA were investigated in the 405 nm laser-irradiated WT and Per2 KO mice at four weeks, respectively ( Figure 3). ALP mRNA level was significantly reduced in right tibial bone of WT and Per2 KO mice compared to left tibia, respectively (p < 0.001) ( Figure 3A). In addition, Per2 mRNA level in WT mice was significantly lower in right tibial bone compared to left tibia (p < 0.001) ( Figure 3B).
To identify the decrease in tibial trabecular bone via Per2 gene, levels of Runx2 and ALP mRNA were determined in non-irradiated WT and Per2 KO mice, demonstrating significant down-regulation of Runx2 and ALP mRNA levels in Per2 KO mice compared to WT mice (p < 0.001) ( Figure 4A,B). Per2 mRNA was expressed in WT mice and was not detected in Per2 KO mice ( Figure 4C). Therefore, the reduction in tibial trabecular bone by 405 nm laser irradiation according to MILNS might be associated with Per2 genes. Dotted line indicates relative variation at the start of LLLT using MILNS. The tibia was directly irradiated with LLLT using the MILNS (405 nm, 5 mW, 3 J/cm 2 , 600 s). Mice were irradiated five days/week for four weeks. At two and four weeks after laser irradiation, BV/TV (%), Tb.Th (mm), Tb.Sp (mm), Tb.N (mm −1 ), Tb.Pf (mm −1 ), SMI, Conn.Dn (mm −3 ), and BMD (g/cm 3 ) were measured from two-dimensional images obtained using CT-AN 1.8. Data are expressed as mean ± SEM (n = 9) at each point and were subjected to statistical analysis, ** p < 0.01; ***p < 0.001. LLLT: Low-level laser therapy/treatment; MILNS: minimally invasive laser needle system; WT: wild-type; BV/TV: structural parameters including bone volume fraction; Tb.Th: trabecular thickness; Tb.Sp: trabecular separation; Tb.N: trabeculae number; Tb.Pf: trabecular bone pattern factor; SMI: structure model index; Conn.Dn: connective density; BMD: bone mineral density.    and c and f, four weeks. The tibia was directly irradiated with LLLT using the MILNS (405 nm, 5 mW, 3 J/cm 2 , 600 s). Mice were irradiated five days/week for four weeks. Scale bar, 0.5 mm; At 0, two, and four weeks after laser irradiation, total bone volume (mm 3 ) (B) and bone mineral density (g/cm 3 ) (C) were measured on two-dimensional images obtained using CT-AN 1.8. Data are expressed as mean ± SEM (n = 9) and were subjected to statistical analysis, * p < 0.05; *** p < 0.001.  and c and f, four weeks. The tibia was directly irradiated with LLLT using the MILNS (405 nm, 5 mW, 3 J/cm 2 , 600 s). Mice were irradiated five days/week for four weeks. Scale bar, 0.5 mm; At 0, two, and four weeks after laser irradiation, total bone volume (mm 3 ) (B) and bone mineral density (g/cm 3 ) (C) were measured on two-dimensional images obtained using CT-AN 1.8. Data are expressed as mean ± SEM (n = 9) and were subjected to statistical analysis, * p < 0.05; *** p < 0.001.

Discussion
In vivo, Micro-CT system is an effective and noninvasive method for the evaluation of microstructural characteristics of bone tissues. Using micro-CT allows for clear and accurate imaging of internal and external bone structures in the smallest bone fraction [21][22][23][24]. The internal and external architecture of bone has a major impact on its mechanical properties. The mechanical properties of cancellous bone are relevant to its stiffness and strength [21][22][23][24]. The biomechanical stiffness and strength of bone depend on both bone internal architecture and BMD [25]. This study was evaluated by several structural parameters including tibial trabecular bone volume fraction (BV/TV), trabecular separation (Tb.Sp), trabecular number (Tb.N), and connective density (Conn.Dn) (Figure 1) as well as total bone volume ( Figure 2B) and BMD ( Figure 2C). Bone densitometry using quantitative micro-CT systems provides clinical information about BMD implicated in the BV/TV. Both BMD and BV/TV can estimate the stiffness and strength of normal and pathologic trabecular bone induced by osteoporosis or metastatic cancer [26]. BMD has also been used clinically to evaluate osteoporosis and fracture risk [27]. Our findings demonstrated that total changes in total bone volume and BMD from trabecular bone of right tibia were significantly reduced in Per2 KO mice compared to in WT mice due to 405 nm LLLT according to MILNS at 0 to four weeks ( Figure 2B,C). The 3D images obtained from micro-CT clearly identified decreased trabecular bone of right tibia in Per2 KO mice compared to in WT mice (Figure 2A). This result suggests that the reduction in tibial trabecular bone caused by 405 nm LLLT using MILNS is mediated by the Per2 gene. Unfortunately, this study is lack of micro-CT analysis in the trabecular bone of left tibia. However, we showed that the expressions of ALP mRNA was significantly different between the right and left tibial bones ( Figure 3A) This study used micro-CT data to demonstrate that LLLT using MILNS led to significant reductions in tibial trabecular bone. Our results are contrary to previous studies that reported the capability of LLLT using MILNS to stimulate the cortical bone growth of osteoporotic mice, prevent trabecular bone loss in ovariectomized mice, and suppress trabecular bone loss induced by skeletal unloading [13,15,16]. These previous studies reported that LLLT using MILNS has potential to inhibit bone loss at a wavelength of 660 nm, energy of 3 J/cm 2 , output power of 10 mW, and irradiation time 300 s for five days per week for two weeks, indicating that it enhances bone formation and suppresses bone resorption. However, the present study applied a wavelength of 405 nm, energy of 3 J/cm 2 , output power of 5 mW, and irradiation time of 600 s for five days per week for two and four weeks ( Figure 2B,C). The other experiment also demonstrated decreased trabecular bone loss at a wavelength of 405 nm, energy of 3 J/cm 2 , output power of 10 mW, and irradiation time 300 s (data not shown). However, the Kushibiki group used a wavelength of 405 nm, 100 mW/cm 2 , 180 s, and two weeks and demonstrated that LLLT enhanced mesenchymal stromal cell differentiation to osteoblasts in vitro [19,20]. Therefore, an excessively high laser dosage might lead to inhibitory effects on cell growth and proliferation [28][29][30]. Two reviews have reported that LLLT at wavelengths ranging from 600 to 904 nm and output powers of 1-500 mW is very

Discussion
In vivo, Micro-CT system is an effective and noninvasive method for the evaluation of microstructural characteristics of bone tissues. Using micro-CT allows for clear and accurate imaging of internal and external bone structures in the smallest bone fraction [21][22][23][24]. The internal and external architecture of bone has a major impact on its mechanical properties. The mechanical properties of cancellous bone are relevant to its stiffness and strength [21][22][23][24]. The biomechanical stiffness and strength of bone depend on both bone internal architecture and BMD [25]. This study was evaluated by several structural parameters including tibial trabecular bone volume fraction (BV/TV), trabecular separation (Tb.Sp), trabecular number (Tb.N), and connective density (Conn.Dn) (Figure 1) as well as total bone volume ( Figure 2B) and BMD ( Figure 2C). Bone densitometry using quantitative micro-CT systems provides clinical information about BMD implicated in the BV/TV. Both BMD and BV/TV can estimate the stiffness and strength of normal and pathologic trabecular bone induced by osteoporosis or metastatic cancer [26]. BMD has also been used clinically to evaluate osteoporosis and fracture risk [27]. Our findings demonstrated that total changes in total bone volume and BMD from trabecular bone of right tibia were significantly reduced in Per2 KO mice compared to in WT mice due to 405 nm LLLT according to MILNS at 0 to four weeks ( Figure 2B,C). The 3D images obtained from micro-CT clearly identified decreased trabecular bone of right tibia in Per2 KO mice compared to in WT mice (Figure 2A). This result suggests that the reduction in tibial trabecular bone caused by 405 nm LLLT using MILNS is mediated by the Per2 gene. Unfortunately, this study is lack of micro-CT analysis in the trabecular bone of left tibia. However, we showed that the expressions of ALP mRNA was significantly different between the right and left tibial bones ( Figure 3A) This study used micro-CT data to demonstrate that LLLT using MILNS led to significant reductions in tibial trabecular bone. Our results are contrary to previous studies that reported the capability of LLLT using MILNS to stimulate the cortical bone growth of osteoporotic mice, prevent trabecular bone loss in ovariectomized mice, and suppress trabecular bone loss induced by skeletal unloading [13,15,16]. These previous studies reported that LLLT using MILNS has potential to inhibit bone loss at a wavelength of 660 nm, energy of 3 J/cm 2 , output power of 10 mW, and irradiation time 300 s for five days per week for two weeks, indicating that it enhances bone formation and suppresses bone resorption. However, the present study applied a wavelength of 405 nm, energy of 3 J/cm 2 , output power of 5 mW, and irradiation time of 600 s for five days per week for two and four weeks ( Figure 2B,C). The other experiment also demonstrated decreased trabecular bone loss at a wavelength of 405 nm, energy of 3 J/cm 2 , output power of 10 mW, and irradiation time 300 s (data not shown). However, the Kushibiki group used a wavelength of 405 nm, 100 mW/cm 2 , 180 s, and two weeks and demonstrated that LLLT enhanced mesenchymal stromal cell differentiation to osteoblasts in vitro [19,20]. Therefore, an excessively high laser dosage might lead to inhibitory effects on cell growth and proliferation [28][29][30]. Two reviews have reported that LLLT at wavelengths ranging from 600 to 904 nm and output powers of 1-500 mW is very helpful in enhancing the proliferation of various cell lines and improving bone healing [1,2]. Tajali et al. [3] performed a meta-analysis through MEDLINE, EMBASE, PubMed, CINAHL, and Cochrane Database of Randomized Clinical Trials published from 1966 to October 2008. Specifically, they looked for studies that highlighted the effects of LLLT on biomechanical properties of bone regeneration and the dose impact in animals. These reports might provide sufficient evidence to support the role of LLLT in animal and human bone healing.
The role of the Per2 gene in bone is not clear, but previous studies have shown that Per2 plays a significant role in regulating bone growth [17,18]. Per2 KO mice demonstrate increases in bone mass and bone volume [17,18]. In children with Smith-Magenis syndrome, a genetic disorder associated with skeletal malformations, the Per2 gene is also expressed with high variability and no Per2 rhythm [31]. Per2 might also influence bone growth by altering p21 cell cycle progression, through its effects on ER-mediated gene expression and its action on parathyroid hormone administration [32][33][34]. However, in the present study, Per2 KO mice showed significantly reduced bone volume and BMD in tibial trabecular bone due to LLLT using MILNS (Figures 1 and 2B,C).
Runt-related transcription factor 2 (Runx2) is a master transcription factor of osteoblast differentiation or osteogenesis. Runx2 expression is upregulated in immature osteoblasts, decreases during bone development, and demonstrates a mature phenotype in osteoblasts, which are required for mature bone formation [35,36]. ALP plays an essential role in the bone formation process and reflects osteoblastic activity [37,38]. ALP is used clinically as a marker of bone formation. LLLT has a biostimulatory effect on bone formation and increases ALP expression or activity. Previous studies have shown that LLLT induces a significant increase in expression of Runx2 and ALP mRNAs [4,[39][40][41][42][43]. LLLT effects on osteoblast proliferation and bone formation involving the increase of Runx2 mRNA [6,8]. The present study, however, demonstrated that Per2 KO mice rather than WT mice significantly downregulated Runx2 and ALP mRNA levels in tibial trabecular bone after LLLT using MILNS (Figure 4). This finding indicates that the reduction of Runx2 and ALP mRNA levels in the tibial trabecular bone caused by 405 nm laser irradiation using MILNS is associated with the decrease of Per2 gene expression.

Experimental Animals
All procedures were performed according to a protocol approved by the Yonsei University of Animal Care Committee (YMC-141112-1). Male inbred 129/Sv WT and mPer2 KO mice at 6 weeks of age (average weight 24.2˘0.8 g) (n = 9) were maintained in the animal facility of Yonsei University, Wonju, Korea. Environment of cage was controlled within standard conditions, temperature (23.5˘1˝C) and humidity (50%˘5%). And also the mice were in controlled light (12:12 h light/dark (LD) schedule; light on at 7:00 a.m.), and were fed ad libitum. Mice were stabilized and synchronized with LD cycle in time-scheduled animal facility for at least 2 weeks.

LLLT Using MILNS
In this study, laser stimulation was performed using the MILNS technique previously developed by the Department of Biomedical Engineering and Yonsei-Fraunhofer Medical Device Lab [13,15,16]. A 130-µm-inner diameter fine needle was used to guide a 100-µm-diameter optical fiber. A diode laser (120 mW, 405 nm; No. ML320G2-11; ThorLabs, Newton, NJ, USA) was used as a light source. The optical power output from the diode laser at the end of the fine needle was set to 10 mW just before irradiation of the bone. The tibia was directly irradiated with the laser (405 nm, 5 mW) for 600 s (energy 3 J/cm 2 ). The bone surface of the right tibia was directly irradiated percutaneously using MILNS at the proximal end of the tibia. The left tibia was not irradiated, but treated with needle. Mice immobilized by a customized restrainer were treated 5 days per week for 2 or 4 weeks without anesthesia.

Measurements of Structural Parameters
We scanned the tibiae before laser stimulation and after 2 or 4 weeks of laser stimulation with an in vivo micro-CT (Skyscan 1076; Bruker, Belgium, Germany) at a resolution of 18 µm 3 under anesthesia. Anesthesia, using a combination of xylazine (0.5 mL/kg; Bayer Korea, Seoul, Korea) and zoletil (0.5 mL/kg; Virbac, Seoul, Korea), was performed during micro-CT scanning. To estimate the effects of LLLT, structural parameters of tibia were quantitatively measured. The structural parameters such as bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm), trabecular number per unit length (Tb.N, mm´1), trabecular bone pattern factor (Tb.Pf, mm´1), structure model index (SMI), connective density (Conn.Dn, mm´3), and bone mineral density (BMD, g/cm 3 ) were analyzed on two-dimensional images obtained by CT-AN 1.8 (Bruker, Germany). We selected the region of interest (ROI) for microCT analyses, 1.8 mm in length at the distal metaphyseal secondary spongiosa (100 slices) from a point that is under 0.54 mm (30 slices) from the end of the proximal growth plate on the tibia.

Preparation of cDNA and Real-Time Quantitative Polymerase Chain Reaction (qPCR)
Total RNAs from tibial bone marrow were isolated from mice sacrificed at 4 weeks after LLLT. Total RNA from each sample was extracted using TRI reagent (MRC, Cincinnati, OH, USA), following the manufacturer's instructions. Samples in 100 µL of TRI reagent were homogenized, and any contaminating genomic DNA was eliminated using the RNase-Free DNase kit (Promega, Madison, WI, USA). GoScript™ Reverse Transcription System (Promega) was used to synthesize cDNA from 2 µg of total RNA, according to the manufacturer's protocol. The gapdh housekeeping gene was used as a constitutive control for normalization. The specific primer pairs used for real-time PCR are listed in Table 1. qPCR were carried out using SYBR Green reagent (Applied Biosystems, Foster City, CA, USA) and the StepOnePlus Systems (Applied Biosystems). PCR conditions included one cycle of 10 min at 95˝C followed by 40 cycles of 15 s at 95˝C, 30 s at 58˝C, and 30 s at 72˝C. The relative gene expression level was calculated using the 2´ Ct method [44]. All samples were analyzed in triplicate and in three independent measures. Table 1. Nucleotide sequences of the primer pairs used for real-time PCR.

Gene
Strand Sequence Accession No.

Statistical Analysis
All the experiments were carried out at least three times. Data are presented as mean˘standard error of the mean (SEM). The significance of differences between groups was determined using Student's t-test with SPSS 17.0 (SPSS Inc., Chicago, IL, USA). p-values less than 0.05 were considered statistically significant.

Conclusions
The results of our study illustrate that LLLT using MILNS plays a critical role in the decrease of bone volume and BMD in tibial trabecular bone in WT and Per2 KO mice. This reduction of bone depended on significant downregulation of Runx2 and ALP mRNA levels in Per2 KO mice compared to WT mice. Future research should investigate whether specific pharmaceutical targeting of the Per2 gene can serve as a new therapeutic avenue to treat bone loss conditions such as osteoporosis.