Low-level laser irradiation enhances the proliferation and osteogenic differentiation of PDLSCs via BMP signaling

Abstract

The aim of this in vitro study was to evaluate the effects of low-level laser therapy (LLLT) at different energy intensities on proliferation and osteogenesis of periodontal ligament stem cells (PDLSCs). We designed one control group, without irradiation and four testing groups, treated with LLLT (Nd:YAG;1064 nm) at 2, 4, 6, and 8 J/cm² for human PDLSCs. Cell proliferation was measured using colony-forming unit fibroblast assay and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Osteogenic capacity of cells was determined by alkaline phosphatase (ALP) staining, ALP activity assay, Alizarin Red S staining, and the gene levels of runt-related transcription factor 2 (Runx2), ALP, osteocalcin, and bone morphogenetic protein 2 (BMP2). The effects of LLLT on secretion of TNF-α and IL-1β in PDLSCs were measured by enzyme-linked immunosorbent assay. BMP/Smad pathway was measured through the expression of Smad1/5/8 phosphorylation (P-Smad1/5/8). LDN-193189, an inhibitor of the BMP/Smad pathway, was used to explore the underlying effects of BMP/Smad signaling on the process of LLLT regulating the proliferation and osteogenesis of PDLSCs. Our results demonstrated LLLT could promote the proliferation and osteogenesis of PDLSCs at 2-6 J/cm² and LLLT at 8 J/cm² significantly suppress osteogenic differentiation of PDLSCs. Moreover, LLLT stimulated the secretion of TNFα and IL-β1. Finally, we found the irradiation positively modulates the P-Smad1/5/8 level. When the cells were treated with LDN-193189, the proliferation and osteogenic effects of LLLT on PDLSCs were attenuated. In conclusion, LLLT may upregulate the proliferation and bone formation ability of PDLSCs via the BMP/Smad signaling.

Keywords: BMP/Smad pathway; Low-level laser therapy (LLLT); Osteogenesis; Periodontal ligament stem cells (BMSCs); Proliferation.