Low‐level ultrahigh‐frequency and ultrashort‐pulse blue laser irradiation enhances osteoblast extracellular calcification by upregulating proliferation and differentiation via transient receptor potential vanilloid 1

Background and Objective Low‐level laser irradiation (LLLI) exerts various biostimulative effects, including promotion of wound healing and bone formation; however, few studies have examined biostimulation using blue lasers. The purpose of this study was to investigate the effects of low‐level ultrahigh‐frequency (UHF) and ultrashort‐pulse (USP) blue laser irradiation on osteoblasts. Study Design/ Materials and Methods The MC3T3‐E1 osteoblast cell line was used in this study. Following LLLI with a 405 nm newly developed UHF‐USP blue laser (80 MHz, 100 fs), osteoblast proliferation, and alkaline phosphatase (ALP) activity were assessed. In addition, mRNA levels of the osteoblast differentiation markers, runt‐related transcription factor 2 ( Runx2 ), osterix ( Osx ), alkaline phosphatase ( Alp ), and osteopontin ( Opn ) was evaluated, and extracellular calcification was quantified. To clarify the involvement of transient receptor potential (TRP) channels in LLLI‐induced biostimulation, cells were treated prior to LLLI with capsazepine (CPZ), a selective inhibitor of TRP vanilloid 1 (TRPV1), and subsequent proliferation and ALP activity were measured. Results LLLI with the 405 nm UHF‐USP blue laser significantly enhanced cell proliferation and ALP activity, compared with the non‐irradiated control and LLLI using continuous‐wave mode, without significant temperature elevation. LLLI promoted osteoblast proliferation in a dose‐dependent manner up to 9.4 J/cm 2 and significantly accelerated cell proliferation in in vitro wound healing assay. ALP activity was significantly enhanced at doses up to 5.6 J/cm 2 , and expression of Osx and Alp mRNAs was significantly increased compared to that of the control on days 3 and 7 following LLLI at 5.6 J/cm 2 . The extent of extracellular calcification was also significantly higher as a result of LLLI 3 weeks after the treatment. Measurement of TRPV1 protein expression on 0, 3, and 7 days post‐irradiation revealed no differences between the LLLI and control groups; however, promotion of cell proliferation and ALP activity by LLLI was significantly inhibited by CPZ. Conclusion LLLI with a 405 nm UHF‐USP blue laser enhances extracellular calcification of osteoblasts by upregulating proliferation and differentiation via TRPV1. Lasers Surg. Med. 50:340–352, 2018. © 2017 Wiley Periodicals, Inc.