In Vitro Effects Of Low-Level Laser Irradiation On Bone Marrow Mesenchymal Stem Cells Biological Characteristics

Objectives:Bone marrow derived mesenchymal stem cells(BMSCs) have shown to be an appealing source for cell therapy and tissue engineering.Previous studies have confirmed that the application of low-level laser irradiation(LLLI) could affect the cellular process. However,little is known about the effects of LLLI on BMSCs.The aim of this study was designed to investigate the influence of LLLI at different energy densities on BMSCs proliferation,secretion and myogenic differentiation.Materials and Methods:BMSCs were harvested from rat fresh bone marrow and exposed to a 635nm diode laser(60mW;0,0.5,1.0,2.0,or 5.0 J/cm~2).The lactate dehydrogenase(LDH) release was used to assess the cytotoxicity of LLLI at different energy densities.Cell proliferation was evaluated by using 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) and 5-bromo-20-deoxyuridine(BrdU) assay.Production of vascular endothelial growth factor(VEGF) and nerve growth factor(NGF) were measured by enzyme-linked immunosorbent assay(ELISA).Myogenic differentiation, induced by 5-azacytidine(5-aza),was assessed by using immunocytochemical staining for the expression of sarcomericα-actin and desmin.Results:Cytotoxicity assay showed no significant difference between the non-irradiated group and irradiated groups.LLLI significantly stimulated BMSCs proliferation and 0.5 J/cm~2 was found to be an optimal energy density.VEGF and NGF were identified and LLLI at 5.0 J/cm~2 significantly stimulated the secretion.After 5-aza induction,myogenic differentiation was observed in all groups and LLLI at 5.0 J/cm~2 dramatically facilitated the differentiation.Conclusions:LLLI stimulates proliferation,increases growth factors secretion and facilitates myogenic differentiation of BMSCs.Therefore,LLLI may provide a novel approach for the preconditioning of BMSCs in vitro prior to transplantation. Objectives:Recent years have witnessed a growing interest and enthusiasm in the application of bone-marrow derived stem cell-based therapies to repair or regenerate damaged myocardium.However,progress in stem cell therapy is hampered by the poor survival of implanted cells.We hypothesized that low-level laser irradiation(LLLI) preconditioning prior to bone-marrow mesenchymal stem cells(BMSCs) transplantation might remodel the hostile milieu of infarcted myocardium and subsequently enhance early survival and therapeutic potential of implanted cells.Materials and Methods:BMSCs were isolated from male donor rat.Myocardial infarction was induced by left anterior descending artery ligation in female rats.Three weeks later,female rats were randomly divided for LLLI preconditioning study and the following LLLI preconditioning and cell survival study.After chest-opened,a 635 nm,5 mW diode laser was performed with energy density of 0.96 J/cm~2 for 150 seconds for the purpose of myocardial preconditioning.In LLLI preconditioning study,vascular endothelial growth factor(VEGF),glucose-regulated protein 78(GRP78),superoxide dismutase(SOD) and malondialdehyde(MDA) in the infarcted myocardium were evaluated at 1 hour,1 day and 1 week after laser irradiation.In cell survival study,2 millions male BMSCs or no-serum culture media was injected into infarcted myocardium with or without LLLI preconditioning.Cell survival was assayed with quantitative real-time polymerase chain reaction to identify Y chromosome gene and apoptosis was assayed with TUNEL staining. Capillary density,myogenic differentiation and left ventricular function were tested at 1 week.Results:After LLLI preconditioning,increased VEGF and GRP78 expression,as well as the enhanced SOD activity and inhibited MDA production,was observed.Compared with BMSCs transplantation and culture media injection group,although there was no difference in the improved heart function and myogenic differentiation,LLLI preconditioning significantly enhanced early cell survival rate by 2-fold,decreased the apoptotic percentage of implanted BMSCs in infarcted myocardium and thus increased the number of newly formed capillaries.Conclusions:LLLI preconditioning could be a novel non-invasive approach for intraoperative cell transplantation to enhance cell early survival and therapeutic potential.