Background Osteoprotegerin （OPG） and osteoprotegerin ligand （OPGL） play an important role in human bone metabolism. The aim of this research was to detect the expression of OPG and OPGL during human marrow stromal...Background Osteoprotegerin （OPG） and osteoprotegerin ligand （OPGL） play an important role in human bone metabolism. The aim of this research was to detect the expression of OPG and OPGL during human marrow stromal cells （hMSC） differentiation into osteoblasts （OB）, and to observe their effect on osteoclasts （OC） formation in vitro to investigate bone metabolism mechanisms. Methods hMSCs were obtained from human bone marrow specimens using gradient centrifugation method, before being purified and incubated with differentiation medium to develop along the human osteoblasts （hOB） pathway. Morphology observation, biochemical detection and cell staining were performed during hMSC differentiation. OPG and OPGL mRNA levels were detected by reverse transcription-polymerase chain reaction. OPG and OPGL protein expression were determined by Western blotting. We further obtained OC progenitor cells from mice bone marrow and co-cultured with differentiating MSCs. We assessed the effect of OPG and OPGL on OC formation by identifying tartrate resistant acid phosphatase （TRAP） positive multinuclear cells. Results Optimal hMSC survival and purification were observed, along with stable biochemical indexes. Alkaline phosphatase secretion increased significantly and mineralization nodules appeared in the process of cell differentiation. OPG mRNA and protein level increased significantly, while OPGL mRNA and protein level decreased. Average levels of OPG mRNA and protein were about 2.5-fold higher than the control, while OPGL mRNA and protein levels were reduced by about one-half. In the group co-culturing with undifferentiated MSC or added OPGL, we found TRAP positive and multi- nuclear OC formation. However, OC formation was absent in the group co-culturing with differentiated MSC or added OPG. Conclusions During hMSC differentiation into hOB, OPG secretion increased rapidly and OPGL production decreased significantly. The OPG/OPGL ratio was also increased, while OC formation was inhibited and bone abso展开更多
BACKGROUND Cigarette smoking(CS)is the most common method of consuming tobacco.Deleterious effects on bone integrity,increased incidence of fractures,and delayed fracture healing are all associated with CS.Over 150 of...BACKGROUND Cigarette smoking(CS)is the most common method of consuming tobacco.Deleterious effects on bone integrity,increased incidence of fractures,and delayed fracture healing are all associated with CS.Over 150 of the 6500 molecular species contained in cigarette smoke and identified as toxic compounds are inhaled by CS and,via the bloodstream,reach the skeletal system.New technologies designed to develop a reduced-risk alternative for smokers are based on electronic nicotine delivery systems,such as e-cigarettes and tobacco heating systems(THS).THS are designed to heat tobacco instead of burning it,thereby reducing the levels of harmful toxic compounds released.AIM To examine the effects of THS on osteoprogenitor cell viability and function compared to conventional CS.METHODS Human immortalized mesenchymal stem cells(n=3)and primary human preosteoblasts isolated from cancellous bone samples from BG Unfall Klinik Tübingen(n=5)were osteogenically differentiated in vitro with aqueous extracts generated from either the THS 2.4“IQOS”or conventional“Marlboro”cigarettes for up to 21 d.Cell viability was analyzed using resazurin conversion assay(mitochondrial activity)and calcein-AM staining(esterase activity).Osteogenic differentiation and bone cell function were evaluated using alkaline phosphatase(AP)activity,while matrix formation was analyzed through alizarin red staining.Primary cilia structure was examined by acetylatedα-tubulin immunofluorescent staining.Free radical production was evaluated with 2’,7’-dichlorofluoresceindiacetate assay.RESULTS Our data clearly show that THS is significantly less toxic to bone cells than CS when analyzed by mitochondrial and esterase activity(P<0.001).No significant differences in cytotoxicity between the diverse flavors of THS were observed.Harmful effects from THS on bone cell function were observed only at very high,non-physiological concentrations.In contrast,extracts from conventional cigarettes significantly reduced the AP activity(by two-fold)and matrix 展开更多
文摘Background Osteoprotegerin （OPG） and osteoprotegerin ligand （OPGL） play an important role in human bone metabolism. The aim of this research was to detect the expression of OPG and OPGL during human marrow stromal cells （hMSC） differentiation into osteoblasts （OB）, and to observe their effect on osteoclasts （OC） formation in vitro to investigate bone metabolism mechanisms. Methods hMSCs were obtained from human bone marrow specimens using gradient centrifugation method, before being purified and incubated with differentiation medium to develop along the human osteoblasts （hOB） pathway. Morphology observation, biochemical detection and cell staining were performed during hMSC differentiation. OPG and OPGL mRNA levels were detected by reverse transcription-polymerase chain reaction. OPG and OPGL protein expression were determined by Western blotting. We further obtained OC progenitor cells from mice bone marrow and co-cultured with differentiating MSCs. We assessed the effect of OPG and OPGL on OC formation by identifying tartrate resistant acid phosphatase （TRAP） positive multinuclear cells. Results Optimal hMSC survival and purification were observed, along with stable biochemical indexes. Alkaline phosphatase secretion increased significantly and mineralization nodules appeared in the process of cell differentiation. OPG mRNA and protein level increased significantly, while OPGL mRNA and protein level decreased. Average levels of OPG mRNA and protein were about 2.5-fold higher than the control, while OPGL mRNA and protein levels were reduced by about one-half. In the group co-culturing with undifferentiated MSC or added OPGL, we found TRAP positive and multi- nuclear OC formation. However, OC formation was absent in the group co-culturing with differentiated MSC or added OPG. Conclusions During hMSC differentiation into hOB, OPG secretion increased rapidly and OPGL production decreased significantly. The OPG/OPGL ratio was also increased, while OC formation was inhibited and bone abso
文摘BACKGROUND Cigarette smoking(CS)is the most common method of consuming tobacco.Deleterious effects on bone integrity,increased incidence of fractures,and delayed fracture healing are all associated with CS.Over 150 of the 6500 molecular species contained in cigarette smoke and identified as toxic compounds are inhaled by CS and,via the bloodstream,reach the skeletal system.New technologies designed to develop a reduced-risk alternative for smokers are based on electronic nicotine delivery systems,such as e-cigarettes and tobacco heating systems(THS).THS are designed to heat tobacco instead of burning it,thereby reducing the levels of harmful toxic compounds released.AIM To examine the effects of THS on osteoprogenitor cell viability and function compared to conventional CS.METHODS Human immortalized mesenchymal stem cells(n=3)and primary human preosteoblasts isolated from cancellous bone samples from BG Unfall Klinik Tübingen(n=5)were osteogenically differentiated in vitro with aqueous extracts generated from either the THS 2.4“IQOS”or conventional“Marlboro”cigarettes for up to 21 d.Cell viability was analyzed using resazurin conversion assay(mitochondrial activity)and calcein-AM staining(esterase activity).Osteogenic differentiation and bone cell function were evaluated using alkaline phosphatase(AP)activity,while matrix formation was analyzed through alizarin red staining.Primary cilia structure was examined by acetylatedα-tubulin immunofluorescent staining.Free radical production was evaluated with 2’,7’-dichlorofluoresceindiacetate assay.RESULTS Our data clearly show that THS is significantly less toxic to bone cells than CS when analyzed by mitochondrial and esterase activity(P<0.001).No significant differences in cytotoxicity between the diverse flavors of THS were observed.Harmful effects from THS on bone cell function were observed only at very high,non-physiological concentrations.In contrast,extracts from conventional cigarettes significantly reduced the AP activity(by two-fold)and matrix
