Effects of Low-Level Er:YAG Laser Irradiation on Proliferation and Calcification of Primary Osteoblast-Like Cells Isolated From Rat Calvaria.

Hiromi Niimi,Shogo Maekawa,Tsuyoshi Shimohira,Hiroshi Kadokura,Sayaka Katagiri,Masahiro Hatasa,Akira Aoki,Tomomitsu Hirota,Yosuke Tsuchiya,Kazuki Watanabe,Satoshi Yokose,Takanori Iwata,Yujin Ohsugi

doi:10.3389/fcell.2020.00459

Abstract

Several reports have shown that the photo-bio-modulation of cells by various lasers has favorable biological effects. However, the effects of low-level Er:YAG laser irradiation on osteoblasts remain unclear. The purpose of this study was to evaluate the effects of low-level Er:YAG laser irradiation on proliferation and osteogenic differentiation of primary osteoblast-like cells isolated from the calvariae of 3–5-day-old Wistar rats. Cells were irradiated by Er:YAG laser at energy fluences of 2.2, 3.3, and 4.3 J/cm2, respectively. After irradiation, cell surface temperatures were measured and cell proliferation was evaluated by flow cytometry and CCK-8. Calcification was evaluated by measuring areas of Alizarin red S staining after 7, 14, and 21 days culture in osteoinductive medium. Gene expression in non-irradiated and laser-irradiated cells was evaluated by qPCR at 3, 6, and 12 h, as well as 1, 3, 7, and 14 days after irradiation. Microarray analysis was performed to comprehensively evaluate the gene expression of non-irradiated and irradiated cells at 3.3 J/cm2 at 6 h after irradiation. No pronounced increase of cell surface temperature was induced by irradiation. Irradiation did not affect osteoblast-like cell proliferation. Osteoblast-like cell calcification was significantly increased 7 days after Er:YAG laser irradiation at 3.3 J/cm2. Bglap expression was significantly increased in cells irradiated at 3.3 J/cm2 6 h post-irradiation. Microarray analysis showed that irradiation at 3.3 J/cm2 caused an upregulation of inflammation-related genes and downregulation of Wisp2. Gene set enrichment analysis also clarified enrichment of inflammation-related and Notch signaling gene sets. In conclusion, low-level Er:YAG laser irradiation at 3.3 J/cm2 enhanced calcification of primary osteoblast-like cells via enhanced Bglap expression and enriched Notch signaling.

Highlights

IntroductionSome types of lasers have been reported to have biostimulation effects on osteoblasts; for example, we previously observed enhanced differentiation and calcification of MC3T3-E1 by blue laser irradiation (Mikami et al, 2018) and others have reported that human osteoblast proliferation and differentiation were promoted in vitro following irradiation by He-Ne (Stein et al, 2005) or Nd:YAG lasers (Arisu et al, 2006)
Regarding Er:YAG laser, which is most effectively used in periodontal regenerative therapy (Aoki et al, 2015), we previously reported that low-level irradiation increased proliferation of MC3T3-E1 (Aleksic et al, 2010)
Since calcification of osteoblast-like cells was significantly increased after 3.3 J/cm2 Er:YAG laser irradiation, we evaluated Runx2, Sp7, Alpl, Bglap, Msx1, Msx2, and Dlx5 mRNA expression (Figures 4A1–G3)

Summary

Introduction

Some types of lasers have been reported to have biostimulation effects on osteoblasts; for example, we previously observed enhanced differentiation and calcification of MC3T3-E1 by blue laser irradiation (Mikami et al, 2018) and others have reported that human osteoblast proliferation and differentiation were promoted in vitro following irradiation by He-Ne (Stein et al, 2005) or Nd:YAG lasers (Arisu et al, 2006). Irradiation by Ga-Al-As diode laser was reported to promote proliferation, differentiation, and bone-nodule formation of primary osteoblast-like cells isolated from rat calvariae (Ozawa et al, 1998; Ueda and Shimizu, 2003; Shimizu et al, 2007). Grassi et al (2011) showed that low-level laser treatment enhanced cell calcification, but not proliferation in osteoblast-like cells

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