Production of Osteopontin and Osteocalcin by MG63 Cells In Response to Bone Morophogenetic Proteins and IGF‐1
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Abstract:
Bone morphogenetic proteins (BMPs) are a group of structurally related proteins in the transforming growth factor‐beta (TGF‐beta) family which have been shown to stimulate bone formation in vivo. The hypothesis of this study was that osteoblast‐like cells (OBs) treated with BMP would have similar levels of secretion of osteopontin (OPN) and osteocalcin (OCN) to that of OBs treated with IGF‐1. Specifically, the aims of this study were to evaluate osteoblast‐like cells (MG‐63 cell line) for secretion of both OPN and OCN after treatment with DBM, OP‐1, or insulin‐like growth factor‐1 (IGF‐1) compared to control, at 24, 48, and 72 hours. After each incubation period, ELISA kits were used to determine the levels of osteopontin and osteocalcin production. The results clearly demonstrated a rise of OCN at 48 hours and a fall at 72 hours for all samples, including control groups. However, there was no significant difference between groups (p > 0.05). With the OPN assay, results showed no significant difference between groups until 72 hours (p = 0.022), where the IGF‐1 group was significantly higher than the Control and (p = 0.021, respectively). This information is important for understanding the signaling pathways that may be innervated in the osteoblast following stimulation with growth factors.Keywords:
Osteopontin
Bone remodeling
Objective: Ectomesenchymal stem cells (EMSCs) are considered to be able to differentiate toward a cementoblast/osteoblast phenotype. This study is to investigate Osteopontin and Osteocalcin mRNA expression of Rat ectomesenchymal stem cells under inducing condition of EMPs EMD in vitro. Methods: Cultured EMSCs were exposed to the EMPs- or EMD- or (EMPs+EMD)- conditioned culture media. Osteoblast MC3T3-E1 served as positive control (P) and non-induced EMPs as negative control (N). Then Osteopontin and Osteocalcin mRNA expression were measured by RT-PCR technique. Result: 1) There was expression of Osteopontin mRNA in group EMPs, group EMD and group (EMPs+EMD) after induction for 3 weeks ; 2) There was expression of Osteocalcin mRNA only observed in group (EMPs+EMD). Conclusion: The cultured Rat EMSCs kept non-osteogenic differentiated state by itself; but may have osteogenic or cementogenic potential testified by expression of Osteopontin and Osteocalcin mRNA when induced by EMPs.
Osteopontin
Cementoblast
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To observe the effect of leptin on osteoblast.Human osteoblast primary culture was carried out, and the morphology and function of osteoblast were observed. The effects of different levels of leptin on osteoblast in different days were assessed by MTT colorimetry. Osteocalcin production was measured also.Human osteoblasts were fusiform in shape and were positive for alkaline phosphatase by histochemical staining, positive for osteocalcin by immunofluorescence staining, and positive by Alizarin Reds staining after mineralized upon supplementation with ascorbate and beta-glycerophosphate. On the first, second and third days, the proliferation of osteoblast, cultured with different concentrations of leptin, had no changes. The leptin-stimulated synthesis of osteocalcin of cells was found to be dose-dependent (P < 0.05), but not time-dependent (P > 0.05).The above data indicated that there were no evidences for the effects of leptin on the proliferation of human osteoblast, but leptin could enhance the function of human osteoblast.
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Bone sialoprotein
Osteopontin
Calvaria
Type I collagen
Osteoid
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Osteopontin
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Purpose: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-El osteoblastic cell line, particularly on the expression of osteocalcin and osteopontin. Materials and Methods: Cells were irradiated with a single dose of 0.5, 1,4, and 8 Gy at a dose rate of 5.38 Gy/min using a cesium 137 irradiator. After the specimens were harvested, RNA was extracted on the 3rd, 7th, 14th, and 21st day after irradiation. The RNA strands were reverse-transcribed and the resulting cDNAs were subjected to amplification by PCR. Results: The irradiated cells demonstrated a dose-dependent increase in osteocalcin and a dose-dependent decrease in osteopontin mRNA expression compared with the non-irradiated control group, The amount of osteocalcin mRNA expression decreased significantly at the 3rd day after irradiation of 0,5, 1,4, and 8 Gy, and also decreased significantly at the 3rd, 14th, and 21 st day after irradiation in the 8 Gy exposed group compared with the control group, The degree of osteopontin mRNA expression increased significantly at the 7th day after irradiation of 0,5, 1,4, and 8Gy, Conclusion: These results showed that each single dose of 0,5, 1, 4, and 8 Gy influenced the mRNA expression of osteocalcin and osteopontin associated with the calcification stage of osteoblastic cells, suggesting that each single dose affected bone formation at the cell level.
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OBJETIVO: O objetivo deste estudo foi avaliar o efeito da T3 na expressão da osteocalcina, osteopontina e colágeno I durante a diferenciação osteogênica das células-tronco mesenquimais (CTM). MATERIAIS E MÉTODOS: As células da medula óssea de ratas Wistar jovens foram extraídas, cultivadas e separadas em cinco grupos: controle (indiferenciado), diferenciado (estímulo osteogênico) e diferenciado com T3 (10-3 nM, 10-2 nM e 100 nM). Para cada grupo, foram cultivadas quatro amostras que foram analisadas por RT-PCR tempo real aos 7, 14 e 21 dias, para quantificação dos transcritos gênicos para osteocalcina, osteopontina e colágeno I. RESULTADOS: Todos os grupos diferenciados sem T3 ou com T3 independentemente da concentração apresentaram expressão de colágeno I significativamente menor e expressão de osteocalcina e osteopontina significativamente maior em comparação a das CTM indiferenciadas. Mas o grupo T3 100 nM apresentou concentração de osteocalcina mais elevada e semelhante à da cultura de osteoblastos. CONCLUSÃO: Conclui-se que a triiodotironina não altera a expressão de osteopontina e de colágeno pelas CTM, mas aumenta a expressão da osteocalcina durante a diferenciação osteogênica in vitro, sendo esse efeito dose-dependente.
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Objective: To investigate the effects of IGF-1 on bone formation of rabbit osteoblast in vitro.Methods:Osteoblasts were obtained from rabbit costal bone by explant culture technique.The second subculture was exposed to IGF-1 at variant concentrations of 0.1,1,10 and 20ng/ml for 24 and 36 hours,and the effects of IGF-1 on osteoblast proliferation was observed by MTT assay.The contents of BGP were measured by the radioimmunoassay in the culture supernatant harvested after the second subculture were exposed to IGF-1 for 72 and 108 hours.Results:Osteoblast exposed to IGF-1 for 24 and 36 hours at variant concentrations of 0.1,1,10,20ng/ml proliferated significantly compared with the control (P0.001),and significant differences were found between every two groups when osteoblast were exposed to IGF-1 at range from 0.1 to 10ng/ml(P0.05).Levels of osteocalcin in culture supernatant of osteoblast exposed to IGF-1 at variant concentrations for 72 and 108 hours was not statistically significant compared with control group (P0.05).Conclusions:IGF-1 could promote the proliferation of osteoblast,and there were dose-dependent relationship at a limited range from 0.1 to 10ng/ml.IGF-1 had no effect on osteocalcin synthesis of osteoblast.
Subculture (biology)
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Bone morphogenetic proteins (BMPs) are a group of structurally related proteins in the transforming growth factor‐beta (TGF‐beta) family which have been shown to stimulate bone formation in vivo. The hypothesis of this study was that osteoblast‐like cells (OBs) treated with BMP would have similar levels of secretion of osteopontin (OPN) and osteocalcin (OCN) to that of OBs treated with IGF‐1. Specifically, the aims of this study were to evaluate osteoblast‐like cells (MG‐63 cell line) for secretion of both OPN and OCN after treatment with DBM, OP‐1, or insulin‐like growth factor‐1 (IGF‐1) compared to control, at 24, 48, and 72 hours. After each incubation period, ELISA kits were used to determine the levels of osteopontin and osteocalcin production. The results clearly demonstrated a rise of OCN at 48 hours and a fall at 72 hours for all samples, including control groups. However, there was no significant difference between groups (p > 0.05). With the OPN assay, results showed no significant difference between groups until 72 hours (p = 0.022), where the IGF‐1 group was significantly higher than the Control and (p = 0.021, respectively). This information is important for understanding the signaling pathways that may be innervated in the osteoblast following stimulation with growth factors.
Osteopontin
Bone remodeling
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Osteopontin
Bone matrix
Matrix (chemical analysis)
matrix Gla protein
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