Photobiomodulation Of 620nm Noncoherent Red Light Irradiation On Rat Bone Marrow Mesenchymal Stem Cells

Object:(1) To obtain purity and stable bone marrow mesenchymal stem cells (MSCs) culutured in vitro;(2) To investigate the effect of red light emitted from light-emitting diode (LED) on bone marrow MSCs with or without osteogenic supplements;(3) To investigate the activation of mTOR in photoinduced proliferation of rat MSCs;(4) To investigate the expression activation of mTOR in photoinduced proliferation of rat MSCs:Method:(1) MSCs were isolated by density gradient centrifugation combined adherence culture, observed under optical microscope and detected by flow cytometry. The cells were induced into osteoblasts, fat cells and chondrogenic cells, and identified by von kossa staining, oil red O staining and alcian blue staining; (2) MSCs with and without osteogenic supplements both were divided into four groups and each group was irradiated at dose of 0.1.2. and 4J/cm2. Cellular proliferation was evaluated by using CCK-8 and 5-ethynyl-2'-deoxyuridine (EdU) fluorescence staining. The alkaline phosphatase (ALP) activity, mineralization formation. and expression of osteoblast master genes (Coll a 1, Alpl, Bglap and Runx2) were monitored as indicators of MSCs' differentiation toward osteoblasts. In groups without osteogenic supplements, red light at all doses significantly stimulated cellular proliferation, whereas the osteogenic phenotype of MSCs was not enhanced;(3) MSCs were irradiated once by 620nm red light with radiation energies 0.5 J/cm2, 1J/cm2.2J/cm2. Cellular proliferation was evaluated by CCK-8 assay. The contents and phosphorylation of mTOR were detected by western blot; (4) MSCs were irradiated once by 620nm red light with radiation energies 1 J/cm2 2J/cm2,4J/cm2. Von kossa staining was used to evaluate oste-differentiation of MSCs. Expressions of circadian clock genes (Cry1,Cry2,Per1,Per2,Clock,BMAL1) were monitored with RT-PCR;Result:(1) MSCs were adhered on the bottom of dishes. CD29 and CD44 were highly expressed, CD34 were at low expression level. Von kossa staining showed that a three-dimensional nodular structure of mineralized tissue was formed. Oil red O stained the fats in cells. and Typeâ…¡collagen was stained by alcian blue:(2) In groups without osteogenic supplements, red light at all doses significantly stimulated cellular proliferation, whereas the osteogenic phenotype of MSCs was not enhanced. In groups with osteogenic supplements, red light increased ALP activity and mineralized nodule formation, and stimulated expression of Bglap and Runx2. whereas the cellular proliferation was decelerated:(3) CCK-8 assay indicated that irradiated cells significantly proliferated. This effect was confirmed by higher phosphorylation of mTOR:(4) More mineralized nodules formed in irradiated groups, and the expressions of Cry2, Per2 and Clock were significantly higher than control;Conclusion:(1) Purity and stable bone marrow MSCs were obtained through density gradient centrifugation combined adherence culture.(2) Nonconherent red light can promote proliferation but not induce osteogenic differentiation of MSCs in normal media, while it enhances osteogenic differentiation and decelerates proliferation of MSCs in media with osteogenic supplements.(3) mTOR signal pathway may involve in red light irradiation triggered cell proliferation.(4) Circadian clock genes may involve in photoinduced osteogenic differentiation of MSCs.