Abstract
Background/Aim
The aim of this study was to examine the effects of diode laser irradiation (910 nm) on bone formation in tibiae with bone defects in estrogen-deficient rats. Micro-computed tomography (micro-CT) was performed for 3-dimensional (3D) morphological evaluation of newly formed bone tissues.
Materials and Methods
Rats underwent sham operation (Sham) or ovariectomy (OVX), and bone defects were created in the tibiae, which were then subjected to diode laser irradiation for seven days in the laser groups (Sham-laser or OVX-laser groups). Tibiae with bone defects from Sham or OVX groups were irradiated with a guide light instead of a laser, serving as control groups (Sham-Cont or OVX-Cont groups). The tibiae were exposed to laser irradiation every day over a period of seven days. After irradiation, the tibiae underwent micro-CT, and then 3D reconstruction was performed for analysis of new bone formation.
Results
Estrogen deficiency induced osteoporosis in the tibiae of OVX rats. Laser irradiation induced greater new bone formation in the region of bone defects in both Sham- and OVX-laser groups compared to the Sham- and OVX-Cont groups, respectively. No significant difference in the volume of new bone formation was seen between Sham- and OVX-laser groups.
Conclusion
Laser irradiation could induce new bone formation in the region of bone defects in both Sham and OVX rats. This suggests that laser irradiation has potential for bone regeneration therapy in cases of postmenopausal osteoporosis.
Keywords: Diode laser, OVX, bone defect, bone formation, tibia, rat
Introduction
With the advent of super-aged societies, the number of patients with osteoporosis is expected to increase (1). Postmenopausal osteoporosis is caused by estrogen deficiency and results in a drastic decrease in bone volume (2). This greatly increases the risk of bone fracture in patients with postmenopausal osteoporosis. For these reasons, new treatments to regenerate bone in osteoporosis patients would be very attractive. Recently, laser irradiation of bone tissue has been demonstrated to cause osteocytes to inhibit the expression of sclerostin, an antagonist of the Wnt canonical pathway. Suppression of sclerostin expression leads to the induction of bone formation (3-5). In addition, diode laser irradiation of bone marrow cell cultures has been shown to stimulate osteoblast differentiation, as revealed by an increase in the number of alkaline phosphatase (ALP)-positive colony-forming unit-fibroblasts (CFU-F) (6). These results indicated that photobiomodulation using laser irradiation can be applied for bone regeneration therapy in specific osteoporosis patients.
In this study, to confirm photobiomodulation of osteoporotic bone tissues by laser irradiation, we created bone defects in the tibiae of ovariectomized (OVX) rats and performed diode laser irradiation in the area of the bone defects. The aim of this study was to confirm whether diode laser irradiation could be applied for bone regeneration therapy in estrogen-deficient rats and to obtain basic data contributing to the development of bone regeneration therapy.
Materials and Methods
Experimental design. All experiments were approved by the Animal Ethics Committee of Meikai University (approval nos. A2204 and A2310). Twenty female Sprague-Dawley rats were used at 10 weeks old in this experiment. Ten rats underwent ovariectomy as the OVX group and the others underwent Sham operation (Sham group) according to the methods described previously by Yokose et al. (7). Two weeks postoperatively, bone defects were created in both the left and right tibiae using a dental steel bur (diameter, 1.0 mm) at high speed (15,000 rpm) under saline irrigation in all rats. Defects were located approximately 25 mm above the calcaneus base (Figure 1). Twenty-four hours after defect creation, all rats entered the irradiation phase of the experiment.
Figure 1.
A bone defect is created in the tibia using a dental bur. The defect is located above the calcaneus as described in the Materials and Methods section.
Irradiation conditions. Five rats in each group received laser irradiation in the region of the tibial defects (Sham-Laser or OVX-Laser groups). A diode laser (Lumix2; USA Laser Biotech Inc., Richmond, VA, USA) with a wavelength of 910 nm was used in the experiment. The experimental parameters were: modulation, 100%; frequency, 30 kHz; energy, 120 J; exposure time, 7.5 min (8). The tibiae in the laser groups were exposed to laser irradiation every day for a period of seven days. After seven days of laser exposure, the tibiae were obtained for morphological analysis. As controls, the remaining tibiae with bone defects were exposed to irradiation from a guide light instead of a laser for the same duration (Sham-Cont and OVX-Cont groups).
Histochemical staining. Tibiae were fixed in 10% neutral formalin (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) for 48 h and decalcified with K-CX (Falma Inc., Tokyo, Japan) containing hydrochloric acid at 4˚C according to the manufacturer’s instructions. Specimens were embedded in paraffin and serial sections were stained using hematoxylin and eosin (HE).
Micro-computed tomography (micro-CT) and 3-dimensional (3D) analysis. A ScanXmate-RX (Comscan Teeno, Kanagawa, Japan) was used. The scanner was set at a voltage of 29 kV and a current of 250 μA. Bone defects in the tibiae were evaluated in three dimensions (XY, YZ, and XZ sections) using Amira software (version 2020.2.; Thermo Fisher Scientific, Waltham, MA, USA). A region of interest (ROI) that included newly formed trabecular bone was placed at 6 mm region covering both sides of the defect in each tibia. The ROI was traced in each dimension semi-automatically and 3D models were reconstructed. The microstructural index of bone volume (BV) was calculated from 3D models. To confirm the presentation of osteoporosis, trabecular BV in the metaphysis of each tibia from Sham or OVX rats was also analyzed using the same methods described above.
Statistical analysis. Data are presented as mean±standard deviation (SD). Data were analyzed using the Kruskal-Wallis H-test, and differences in means were assessed using the Mann-Whitney U-test with Bonferroni correction. Statistical significance was defined as p<0.05.
Results
For Sham-Cont (Figure 2A) and OVX-Cont (Figure 2B) rats, 3D models showed trabecular bone (blue color, Figure 2A and B) and BV (Figure 2C) in the tibial metaphysis. Trabecular BV was significantly decreased in OVX-Cont as compared with that in Sham-Cont, confirming that estrogen deficiency caused osteoporosis in OVX rats. In each group, 3D models showed new bone formation in the defect region and bone marrow space (blue color, Figure 3). The BV of new bone formation was lower in OVX-Cont than in Sham-Cont, but no significant differences were seen between Sham-Cont and OVX-Cont (Figure 3A, C, and E). Laser irradiation induced formation of a larger amount of new bone in Sham‑Laser and OVX‑Laser (Figure 3B and D) than in Sham‑Cont or OVX‑Cont (Figure 3A and C). Measured values for the BV of newly formed bone tissue in the region of tibial defects were significantly larger in Sham- and OVX-Laser than in Sham- or OVX-Cont (Figure 3E). Interestingly, no significant differences were apparent between Sham-Laser and OVX-Laser groups. HE staining of the tibiae demonstrated histological features of newly formed bone tissues (Figure 4). The spaces of defects in the cortical bones in all groups were filled with newly formed woven bone that had spread into the marrow spaces. Laser irradiation induced a larger amount of woven bone in the Sham- and OVX-Laser groups compared to the Sham- or OVX-Cont groups.
Figure 2.
Three-dimensional images of trabecular bone volume (BV) as shown by blue color in the primary spongiosa of the tibiae in Sham (A) and OVX (B) rats. Trabecular BV is significantly lower in OVX rats than in Sham rats (C). Results represent mean±SD (n=5). *p<0.05 compared with the Sham.
Figure 3.
Three-dimensional models of newly formed bone tissue as shown by blue color in the region of tibial defects in Sham-Cont (A), Sham-Laser (B), OVX-Cont (C), and OVX-Laser (D) groups. E) Measured bone volume (BV) in each group. Results represent mean±SD (n=5). p<0.05 compared with the control. NS: Not significant.
Figure 4.
Histological features of newly formed bone tissues (asterisk) in the region of tibial defects in Sham-Cont (A), Sham-Laser (B), OVX-Cont (C), and OVX-Laser (D) groups. Bar: 200 μm.
Discussion
OVX rats have been demonstrated to be a good model for osteoporosis, and experiments using OVX animals have facilitated the development of many pharmacotherapies for osteoporosis (9-11). In addition, mechanical stress can provide suitable treatment for various bone diseases, including osteoporosis (12-14). In fact, Oxlund et al. reported that mechanical stress had the effect of inhibiting bone resorption as well as stimulating bone formation in OVX rats (14). We recently reported that photobiomodulation using laser irradiation could promote bone formation under the same mechanism involved in mechanical stress (4,5). Iso et al. (6) reported that diode laser irradiation could stimulate osteoblast differentiation in bone marrow cells containing undifferentiated mesenchymal stem cells. We also confirmed that both CO2 and Er:YAG laser irradiation inhibited the expression of sclerostin, an antagonist of the Wnt canonical and bone morphogenetic protein (BMP) pathways, and these mechanisms indicated why laser irradiation can induce bone formation in a same manner similar to mechanical stress (4,5). We therefore hypothesized that laser irradiation can be applied as a regeneration therapy for osteoporotic bone tissues.
Comparison of trabecular BV in the tibial metaphysis of OVX rats with that of Sham rats confirmed the onset of osteoporosis induced by estrogen deficiency. The 3D models showed that new bone formation was induced in the region of bone defects in the tibiae of both Sham and OVX rats. Although no significant differences in BV were evident between Sham-Cont and OVX-Cont rats, BV in OVX-Cont tended to be impaired compared with that in Sham-Cont rats. This has been supported by several experiments (15-17) and reinforces the notion that the bone repair process is delayed under conditions of estrogen deficiency.
Laser irradiation induced new bone formation in the region of tibial defects in both Sham and OVX rats. The most noteworthy finding was that the BV of newly formed bone tissues in the region of tibial defects was comparable between OVX-Laser rats and Sham-Laser rats, and the effects of laser irradiation on bone formation were also confirmed through histological analysis of the tibiae in Sham and OVX rats. These results indicated that laser irradiation directly affected osteoblast differentiation and function regardless of whether estrogen was present. Wronsky et al. (18) reported that the bones in OVX rats showed a high turnover and both osteoblasts and osteoclasts were simultaneously activated under estrogen deficiency. They concluded that bone loss was associated with elevated histo-morphometric indices of bone resorption and formation. Such findings support our results, and laser irradiation has been postulated to selectively affect osteoblasts to stimulate bone formation. We recently demonstrated that sclerostin expression in osteocytes inhibited laser irradiation (3,4). Sclerostin is a well-known antagonist of both Wnt and BMP signaling pathways, inhibiting bone formation by osteoblasts (3,19-21). This effect of laser irradiation on osteocytes is thought to be one mechanism by which bone formation increases. In addition, undifferentiated mesenchymal stem cells in the bone marrow have been shown to be stimulated toward differentiation into osteoblasts by laser irradiation (6). These mechanisms might be involved in the bone formation induced by laser irradiation in the defect regions of both Sham-Laser and OVX-Laser rats.
Furthermore, our results suggest that the mechanism of bone formation by osteoblasts stimulated with laser irradiation during the repair of injured bone tissues under estrogen deficiency may be the same as in normal conditions, indicating that laser irradiation can induce sufficient bone formation by osteoblasts, even in the deficiency of estrogen. However, the present results are insufficient to fully clarify the mechanisms involved, and further investigation is necessary.
Conclusion
Laser irradiation induced the formation of new bone tissue in the region of tibial defects in estrogen-deficient rats. This suggests that diode laser irradiation has potential applications in bone regeneration therapy for postmenopausal osteoporosis.
Conflicts of Interest
The Authors have no conflicts of interest to report in relation to this study.
Author’s Contributions
Y.F and S.Y performed all experiments and wrote the article. M.K, N.U., and R.I. supported the micro-CT analysis. N.U. and S.Y. edited and revised the article.
Acknowledgements
This study was supported in part by a Grant-in-Aid for Scientific Research (22K10020) from the Ministry of Education, Science, and Culture of Japan.
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