Abstract
Background/Aim: Bone marrow cells contain nonhematopoietic cells with the ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Mechanical stress influences osteoblast differentiation of bone marrow cells into osteogenic, chondrogenic, and adipogenic lineages, measurable as the abundance of alkaline phosphatase-positive (ALP+) colony-forming unit-fibroblasts (CFU-F); however, the effect of diode laser irradiation on osteoblast differentiation is unknown. The aim of this study was to analyze the effects of photobiomodulation on the osteogenic differentiation of mesenchymal stem cells in the bone marrow, using the CFU-F assay. Materials and Methods: Bone marrow cells isolated from rat tibiae were cultured and irradiated with a diode laser (wavelength 808 nm) at a total energy of 0 J (control), 50 J, and 150 J. Results: On day 7 after irradiation, ALP+ CFU-F were most abundant in the 50 J group and the least abundant in the 150 J group. Mineralized nodule formation was observed after long-term culture (21 days). Compared with the control group, there were significantly more nodules in the 50 J group and significantly fewer nodules in the 150 J group. Osteocalcin mRNA expression was highest in the 50 J group, and there was no difference between the control and 150 J groups. Conclusion: Irradiation with 50 J was effective in stimulating osteogenesis in bone marrow stem cells. These findings suggest that diode laser irradiation can induce osteogenesis in rat bone marrow cells in an energy-dependent manner, and appears suitable for application in bone regeneration therapy.
It is well known that bone tissue is sensitive to mechanical loading. Wolff’s law (1) and Frost’s mechanostat theory (2) state that bone metabolism is influenced by and adapts to mechanical loading. Clinically, in oral lesions such as torus mandibularis and palatinus, characteristic bone formation can be seen in the mandible and palate, respectively. It is considered that bone formation in lesions can be induced by bite force (3). Several studies (3-5) have reported that osteocytes act as mechanosensors in directing bone formation in bone tissue and/or resorption and that their abundance reflects mechanical loading. Robling and Bonewald demonstrated that osteocytes produce sclerostin, an antagonist of the Wnt signal pathway that inhibits bone formation in osteoblasts under unloading; and that osteocytes decrease the expression of sclerostin to accelerate bone formation in response to loading (6). Therefore, mechanical loading is an important factor in inducing bone formation that is applicable to regenerative bone therapy.
Laser irradiation induces the dual effects of tissue vaporization and activation, the former in high reactive-level laser therapy (7), and the latter in low reactive-level laser therapy (LLLT) by photobiomodulation (7-9). Photobiomodulation can be applied for regeneration therapy in various tissues (8). We have previously reported that photobiomodulation by carbon dioxide laser (10) and Er:YAG laser (11) could inhibit the expression of Sclerostin in osteocytes and that laser irradiation has the same effects as mechanical loading in inducing bone formation. These results indicate that in addition to mechanical loading, photobiomodulation could also be employed for bone regenerative therapy. In addition to the use of carbon dioxide and Er:YAG lasers, it has been demonstrated in animal experiments that diode laser (12) and Nd:YAG laser (13) can induce new bone formation in rat tibiae, with confirmation that photobiomodulation can be applied to bone regeneration therapy regardless of laser wavelength.
Bone marrow contains undifferentiated mesenchymal cells and hematopoietic cells that differentiate into osteoblasts (14-16) and osteoclasts (17, 18), respectively. It is considered that bone metabolism exerted by osteoblasts and osteoclasts originating in bone marrow cells is influenced by mechanical loading. Sasaki and Nakamura (19) reported changes in bone marrow cell development in a tail-suspended mouse model that simulated the conditions associated with a lack of mechanical loading. They showed that the number of alkaline phosphatase (ALP)-positive colony-forming units-fibroblastic (ALP+ CFU-F) and mineralized nodules were significantly reduced in bone marrow cell cultures from tail-suspended mice compared with control bone marrow cells. In addition, Menuki et al. (20). reported that bone marrow cell cultures from mice that performed a climbing exercise as a physiological mechanical loading model exhibited an increased number of ALP+ CFU-F and Oil Red O-positive cells compared with control bone marrow cells. These results indicate that both osteoblast differentiation and bone formation by bone marrow cells are influenced by mechanical loading.
It is considered that photobiomodulation can increase the number of ALP+ CFU-F and induce bone formation in bone marrow cells. We have previously reported that the volume of new bone formation in the bone marrow space was much higher in a diode laser irradiation group than in the control group (12). However, there are few reports regarding the effects of photobiomodulation on bone marrow cells.
The aim of this study was to confirm the effects of photobiomodulation of diode laser irradiation on bone marrow cell differentiation and to evaluate whether photobiomodulation can be applied to bone regenerative therapy as effectively as mechanical loading.
Materials and Methods
The study protocol was approved by the institutional review board of Meikai University, Saitama, Japan (B 2104). The experimental procedure met the standard for animal care under the authority of Meikai University, Saitama, Japan.
Cell culture. Bone marrow cells were collected from the left and right tibiae of seven 10-week-old female Sprague-Dawley rats, using the methods described by Nishida et al. (21). Nucleated cells were counted with a hemocytometer and inoculated at a density of 4×106 cells/well in 6-cell multiwell plates (Falcon Labware, Lincoln Park, NJ, USA). The cells were cultured with alpha-modified minimum essential medium (MEM; Gibco BRL, Gaithersburg, MD, USA) containing 10% calf serum (Gibco BRL), 300 μg/ml β-glycerophosphate, and 50 μg/ml ascorbic acid (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). Phenol red-free medium was used in the experiment to prevent the diode laser from being absorbed by the color red. The medium was changed every two days, and the cells were incubated for up to 21 days.
Laser irradiation. A diode laser (Filio Yoshida Dental Mfg. Co. Ltd., Tokyo, Japan) was used in the experiment. Three irradiation conditions with total energies of 0 J, 50 J, and 150 J were established, as in a previous study (12). The irradiation parameters are summarized in Table I. Guide laser irradiation without diode laser to the cells was used as the control (0 J group). Laser irradiation of the cultured cells was performed once per day for four days after the start of culture.
Irradiation parameters.
CFU-F assay. To examine the properties of CFU-F isolated from the bone marrow of rat tibiae, nucleated cells were cultured for 10 days. The cells were fixed with 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Corporation) for 20 min at room temperature, and alkaline phosphatase (ALP) activity in CFU-F was then determined by enzyme histochemistry using an ALP stain kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. The cells were counterstained with Kernechtrot solution, and the total number of CFU-F and ALP+ CFU-F was counted. Colonies containing more than 50 cells were considered CFU-F.
ALP and Von Kossa staining. On day 21, the cells were fixed with neutral buffered formalin and stained with ALP (ALP stain kit, Takara Bio Inc.) and Von Kossa stain (4% silver nitrate, Fujifilm Wako Pure Chemical Corporation). The numbers of mineralized nodules in each well of a 6-well plate were counted using a light microscope.
Real-time quantitative PCR. Total RNA was extracted from the cultured cells on day 21 using RNAiso Plus kit (Takara Bio Inc.) according to the manufacturer’s protocol. First-strand cDNA was synthesized from each total RNA using a PrimScript RT Master Mix kit (Takara Bio Inc.). Real-time PCR reactions were performed by Thermal Cycler Dice (Takara Bio Inc.) and SYBER Green Real-Time Master Mix (Takara Bio Inc.). The PCR cycling conditions were as follows: 95°C for 10 s followed by 40 cycles at 95°C for 5 s and 60°C for 20 s. Primers were purchased from Takara Bio Inc. and the sequences were as follows; β-actin forward 5′-TGA CAGGATGCAGAAGGAGA-3′ and reverse 5′-TAGAGCCACC AATCCACACA-3′; Osteocalcin forward 5′-AGACTCGGCG CACCTCAA-3′ and reverse 5′-AAGCGGGTGTAGTGCAGCTC-3′. The target gene expression level was normalized to that of β-actin in the sample. The 2−ΔΔCt method was applied to determine differences in relative expression.
Statistical analysis. Data are expressed as the mean±standard deviation (SD). Data were analyzed using the Kruskal-Wallis H test, and differences in the means were assessed using the Mann-Whitney U-test with Bonferroni correction. Statistical significance was defined as p<0.05.
Results
The formations of CFU-F stained with ALP and Kernechtrot are shown in Figure 1. Total colonies composed of ALP-positive (arrows) and -negative (arrowheads) cells are present in the cells (Figure 1). Among the three groups, CFU-F colonies were most abundant in the 50 J group (Figure 2A), followed by the 150 J group. There was no significant difference between the control and 150 J groups (p>0.05) (Figure 2B). The number of ALP+ CFU-F colonies (Figure 2B) and the ratio of ALP+ CFU-F to total CFU-F colonies (Figure 2C) were significantly highest in the 50 J group (p<0.05). In the evaluation of bone formation in ALP+ CFU-F performed after a long culture period (21 days), nodules stained using von Kossa stain were seen in ALP-positive colonies (Figure 3A). The histological findings are presented in Figure 3B. The number of mineralized nodules was higher in the 50 J laser group and lower in the 150 J group than that in the control group (p<0.05) (Figure 3C). Figure 4 shows the mRNA expression of Osteocalcin in bone marrow cells on day 21 after laser irradiation. The expression of Osteocalcin in each group correlated with the results of the number of mineralized nodules. Osteocalcin mRNA expression was higher in the 50 J group and lower in the 150 J group than that in the control group (p<0.05) (Figure 4).
Colony-forming unit-fibroblasts (CFU-F) of bone marrow cells isolated from rat tibiae on day 7 in culture. The cells were irradiated with diode laser as described in Materials and Methods. CFU-F of all groups were stained with alkaline phosphatase (ALP) and Kernechtrot. Arrows indicate ALP-positive colonies, and arrowheads indicate ALP-negative colonies.
The total number of colony-forming unit-fibroblasts (CFU-F) (A), the number of alkaline phosphatase (ALP)-positive CFU-F (B), and the ratio of ALP-positive CFU-F to total CFU-F (C). Data are presented as means±SD of five samples. *p-value<0.05.
Mineralized nodules of colony-forming unit-fibroblasts (CFU-F) stained with alkaline phosphatase (ALP) and von Kossa (A and B) and the number of mineralized nodules (C) in each group on day 21 in culture. Data are presented as means±SD of five samples. *p-value<0.05. Bar: 200 mm.
Osteocalcin expression in bone marrow cells on day 21 of culture. The cells were irradiated with diode laser as described in Materials and Methods. Data are presented as means±SD of five samples. *p-value<0.05.
Discussion
Bone marrow cells exert important roles in bone metabolism as stem and progenitor cells, which differentiate into mesenchymal cells comprising osteoblasts, chondroblasts, adipocytes, and myoblasts (21). Aging and metabolic bone diseases affect the number of ALP+ CFU-F (22-24), and changes in this number can indicate alterations in the nature of osteoprogenitor cells in the bone marrow. Thus, it can be useful for exploring the mechanism of osteogenesis in bone marrow. Nishida et al. (21) reported that intermittent parathyroid hormone administration induced osteogenesis that was apparent as an increase in ALP+ CFU-F. Accordingly, we used this assay system to examine the effects of laser irradiation on bone formation in the bone marrow.
We have previously reported that CO2 and Er:YAG laser have the same photobiomodulation effect as mechanical stress on bone formation and that photobiomodulation and mechanical stress both suppress sclerostin in osteocytes (10, 11).
Sclerostin is a well-known antagonist of the Wnt signaling pathway (25). Its suppression by mechanical stress and laser photobiomodulation is considered the mechanism by which bone formation is stimulated in osteoblasts. In addition, it has been reported that mechanical stress controls osteoblast differentiation in bone marrow cells (19, 20, 26, 27). Mechanical loading has been shown to increase ALP+ CFU-F numbers in bone marrow cells of the tibia (20), whereas ALP+ CFU-F numbers decreased in bone marrow cells in the tibiae of tail-suspended animals (19). These results indicate that mechanical stress stimulated undifferentiated mesenchymal cells and induced osteoblast differentiation in bone marrow cells. We hypothesized that in addition to mechanical stress, laser irradiation can also induce osteoblast differentiation in bone marrow cells by photobiomodulation. To the best of our knowledge, the present study is the first to report the effect of photobiomodulation using laser irradiation on osteoblast differentiation in bone marrow cells.
In the present study, laser irradiation at 50 J energy most strongly stimulated osteoblast differentiation in bone marrow cells, whereas 150 J energy had no photobiomodulation effects on osteoblast differentiation in bone marrow cells and the high energy inhibited mineralized nodule formation. These results demonstrate that laser irradiation at 150 J inhibits the osteogenic differentiation of bone marrow cells. Frost’s mechanostat theory (2) proposes strain magnitude as the stimulus for bone functional adaptation, and states that the effect of mechanical stress on bone metabolism is to increase bone formation; however, excessive mechanical stress induces immature bone tissue. Given that laser irradiation stimulus and mechanical stress have the same effect on bone marrow cells, irradiation energy of 50 J could be suitable for stimulating osteoblast differentiation and increasing bone formation; however, that of 150 J might provide excessive stimulus. A recent study has reported the interesting result that the effects of laser irradiation on the bone healing process in vivo varied depending on the irradiation energy (12). In that study, rat tibiae that received bone defects were irradiated daily with 40 J of diode laser for 14 days and bone formation was analyzed on days 7 and 14 of the healing process. Compared with controls that were not irradiated, bone volume in tibial bone marrow spaces irradiated with the laser was higher on day 7 but lower on day 14. These results support our findings. We consider that 7 days might be a suitable duration for laser energy irradiation, but that a duration of 14 days would be too long to stimulate bone formation in the bone marrow according to Frost’s mechanostat theory (2). These results suggest that laser irradiation has similar effects as mechanical stress regarding bone formation. However, at 150 J, there was a slight increasing trend in total CFU-F numbers, whereas there was a decreasing trend in the ratio of ALP+ CFU-F to total CFU-F compared with that of the control group. This finding indicates that energy at 150 J selectively inhibited osteogenic differentiation of bone marrow cells. Further investigation is required to elucidate the mechanism.
Conclusion
Our results indicate that 50 J irradiation was effective in stimulating osteogenesis in bone marrow stem cells. In other words, these findings suggest that diode laser irradiation can induce osteogenesis in rat bone marrow cells in an energy-dependent manner, and appears suitable for application to bone regeneration therapy.
Acknowledgements
This study was partially supported by JSPS KAKENHI Grant No. 22K10020.
Footnotes
Authors’ Contributions
EI, NK, TY, and SY designed the research; EI, NK, HK, and YK conducted the research; EI and TT performed statistical analysis; SY had primary responsibility for the final content. All Authors have read and approved the final manuscript.
Conflicts of Interest
All Authors have no conflicts of interest to declare in relation to this study.
- Received April 16, 2024.
- Revision received May 20, 2024.
- Accepted May 21, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
