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The scaffold based tissue engineering materialized for bone tissue therapy. Gelatin-glutaraldehyde cross linked scaffold was prepared by solvent casting -porogen leaching method. It was characterized by FTIR and SEM microphotograph analysis. Absence of peak at waves no. 1625 cm?1 in ATR-FTIR indicated formation of cross-linking. FE-SEM micrograph showed honeycomb pad like structure with high porosity. Methanolic extract of Withania somnifera (Ashwagandha) root extract induced MC3T3 E1 osteoblast cell adhesion and proliferation on porous gelatin scaffold. GC-MS analysis pointed out presence of 4-amino- 2-ethyl-3-methylquinoline, an active phyto-chemicals having tissue regeneration potential. High anti-oxidant capacity down regulates cell death mechanism by scavenging free radical. The biocompatible gelatin scaffold has RGD moiety that attune the MC3T3 E1 osteoblast cell adhesion. Withania somnifera root extract may boost up cell proliferation on scaffold. Therefore treatment with Withania somnifera root extract may be the new approaches for designing bone tissue scaffold for bone tissue therapy.
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Objective: To investigate the effect of an aqueous extract of Protaetia brevitarsis (AEPB) on osteogenesis using preosteoblast MC3T3-E1 cells and zebrafish larvae. Methods: Flow cytometric analysis was used to measure the cytotoxicy. Alkaline phosphatase activity was detetmined using p-nitrophenyl phosphate as a substrate. Calcium deposition was detected using alizarin red staining along with osteogenic marker expression in preosteoblast MC3T3E1 cells. In addition, vertebral formation in zebrafish larvae was detected using calcein staining and osteogenic gene expression. Results: AEPB highly promoted the expression of osteogenic markers including runt-related transcription factor 2, osterix, and alkaline phosphatase, along with elevated levels of mineralization in MC3T3-E1 cells. Moreover, AEPB accelerated vertebral formation in zebrafish larvae accompanied by upregulated expression of osteogenic genes. FH535, an inhibitor of Wnt/β-catenin, suppressed AEPB-induced osteogenic gene expression and vertebral formation, indicating that AEPB stimulates osteogenesis by activating the Wnt/β-catenin signaling pathway. Conclusions: AEPB stimulates osteoblast differentiation and bone formation by activating β-catenin. Therefore, AEPB is a promising material that induces osteogenesis, and is useful for the treatment of bone resorption diseases.
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Objective@#To compare the efficiency and biocompatibility of four different silanes on immobilizing c(RGDfK) peptide on titanium surface.@*Methods @# After alkali-heat treatment (group OH), the titanium surface was treated with 3-aminopropyltriethoxysilane (APTES) (group OHAP), 3-chloropropyltriethoxysilane (CPTES) (group OHCP) (3-mercaptopropyltrimethoxysilane (MPTS) (group OHMPT) and 3-isobutyryloxy propyltrimethoxysilane(γ- MPS) (group OHMPS) to immobilize the c(RGDfK) cyclic peptide and constructa titanium-silane-c(RGDfK) coating. The NT group was the blank control group. The surface morphology and wettability of the coatings were detected using scanning electron microscopy and contact angle measurement. The elemental composition of the titanium surface was analyzed using X-ray photoelectron spectroscopy. After fluorescent staining with 4’,6-diamino-2-phenylindole (DAPI) and phalloidin, the adhesion of mouse preosteoblast MC3T3-E1 cells on the surface of the materials was observed using laser confocal microscopy. Cell counting kit-8 (CCK-8) and alkaline phosphatase (ALP) activity assays were used to evaluate the proliferation and osteogenic differentiation of MC3T3-E1 cells on the surface of the materials, respectively. @*Results @#Scanning electron microscope observation showed a spongy-like 3-dimensional network formed on the titanium surface after alkali-heat treatment with silane-c(RGDfK) coating adhesion. The wettability of each group was greatly improved compared to the untreated titanium surface. The element ratios of Si/Ti and amide-N/Ti in the OHMPS group were the highest. The OHAP group exhibited the best cell adhesion effect. The cell proliferation and ALP activity of the OHAP, OHMPT, and OHMPS groups were significantly higher than the control group (P <0.05); there was no statistical difference between the OHCP group and the control group.@*Conclusion @#MPTS, CPTES and γ-MPS covalently immobilized cyclic peptide c(RGDfK) on the titanium surface, which promoted adhesion, proliferation and osteogenic differentiation of MC3T3-E1 cells. Theγ-MPS conjugated C (RGDfK)cyclic peptide exhibited the best effect. MPTS, CPTES and γ-MPS coupled with c(RGDfK) cyclic peptides had similar biological properties.
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Hypertension is a prevalent systemic disease in the elderly, who can suffer from several pathological skeletal conditions simultaneously, including osteoporosis. Benidipine (BD), which is widely used to treat hypertension, has been proved to have a beneficial effect on bone metabolism. In order to confirm the osteogenic effects of BD, we investigated its osteogenic function using mouse MC3T3-E1 preosteoblast cells in vitro. The proliferative ability of MC3T3-E1 cells was significantly associated with the concentration of BD, as measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cell cycle assay. With BD treatment, the osteogenic differentiation and maturation of MC3T3-E1 cells were increased, as established by the alkaline phosphatase (ALP) activity test, matrix mineralized nodules formation, osteogenic genetic test, and protein expression analyses. Moreover, our data showed that the BMP2/Smad pathway could be the partial mechanism for the promotion of osteogenesis by BD, while BD might suppress the possible function of osteoclasts through the OPG/RANKL/RANK (receptor activator of nuclear factor-κB (NF-κB)) pathway. The hypothesis that BD bears a considerable potential in further research on its dual therapeutic effect on hypertensive patients with poor skeletal conditions was proved within the limitations of the present study.
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BACKGROUND: Studies have shown that the loss of sex combing protein 1 (Asxl1) can lead to the occurrence of bone dysplasia and bone defects, but the relationship between this factor and bone destruction in the microenvironment of apical periodontitis has not been reported. OBJECTIVE: To study the effect of Asxl1 on proliferation and differentiation of osteoblasts in an inflammatory microenvironment. METHODS: MC3T3-E1 cells were excited by lipopolysaccharide to establish an in vitro inflammatory microenvironment. The best concentration and optimal action time of lipopolysaccharide were screened by cell counting kit-8 test. MC3T3-E1 cells were then stimulated with 20 mg/L lipopolysaccharide for 24 hours. The expression levels of Asxl1 protein and mRNA were detected by immunofluorescence and real-time PCR, respectively. After lipopolysaccharide stimulated the formation of inflammatory microenvironment, Asxl1-Si RNA was transfected for 24 hours, cell counting kit-8 was applied to detect the activity of cell proliferation, and real-time PCR was used to detect the expression levels of Asxl1 and osteogenic related genes ALP and RUNX2 mRNA. RESULTS AND CONCLUSION: After lipopolysaccharides stimulation of MC3T3-E1 cells, the expression levels of Asxl1 protein and mRNA were decreased. Under the inflammatory microenvironment, the proliferation activity of MC3T3-E1 cells transfected with Asxl1-Si RNA for 24 hours was significantly decreased, and the expression levels of Asxl1, ALP and RUNX2 mRNA were markedly decreased. These findings indicate that Asxl1 may influence the proliferation and differentiation of osteoblasts by involvement in the process of inflammatory reaction, thereby participating in bone destruction.
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BACKGROUND: Mechanical stress can influence the proliferation and differentiation of MC3T3-E1 cells and trigger differential expression of miR-132-3p. However, further research is warranted concerning whether tensile stress can influence the proliferation and differentiation of osteoblasts by regulating miR-132-3p. OBJECTIVE: To determine the expression of osteogenic differentiation markers and miR-132-3p in MC3T3-E1 cells under 12% cyclic stretch and to explore the effect of miR-132-3p on cell proliferation and differentiation. METHODS: MC3T3-E1 cells were loaded with 0% and 12% tensile stress, and alkaline phosphatase activity, osteocalcin mRNA and miR-132-3p expression levels were detected. MC3T3-E1 cells were transiently transfected with miR-132-3p mimics and a negative control transfection group was set up. The expression of alkaline phosphatase, osteocalcin and Runx2 mRNA in transfected cells were detected by qRT-PCR, and the effect of miR-132-3p on cell proliferation were detected by cell counting kit-8 assay. RESULTS AND CONCLUSION: The alkaline phosphatase activity and osteocalcin mRNA expression were down-regulated in MC3T3-E1 cells under 12% stretch stress (P < 0.01), and the expression of miR-132-3p was significantly increased (P < 0.05). QRT-PCR results showed the expression levels of osteogenic differentiation markers alkaline phosphatase activity, osteocalcin, and Runx2 mRNA in miR-132-3p mimics group were significantly decreased after intracellular transfection of miR-132-3p (P < 0.05). Compared with the negative control transfection group, the cell proliferation in the miR-132-3p mimic group was decreased at 24, 48, and 72 hours after transfection (P < 0.001), and the most obvious reduction was observed after 48-hour transfection. These findings indicate that 12% cyclic tensile stress can negatively regulate the proliferation and differentiation ability of MC3T3-E1 cells by overexpressing miR-132-3p.
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BACKGROUND: Preliminary study has found that osteopractic total flavone can promote osteogenic differentiation of MC3T3-E1 cells on the surface of nano-bone material, but the underlying mechanism needs to be studied in depth. OBJECTIVE: To investigate the actin mechanism of osteopractic total flavone combined with nano-bone material on MC3T3-E1 cells. METHODS: MC3T3-E1 cells were co-cultured with nano-bone material, and 100 mg/L and 250 mg/L osteopractic total flavone were treated as drug intervention, including 10 μg/L transforming growth factor-β as positive control. The samples were divided into eight groups: (1) Normal group; (2) DKK1 group: Wnt pathway inhibitor DKK1 (0.1 mg/L) blocks Wnt/β-catenin signaling pathway; (3) DKK1+transforming growth factor-β group; (4) DKK1+100 mg/L osteopractic total flavone group; (5) DKK1+250 mg/L osteopractic total flavone group; (6) DKK1+ nano-hydroxyapatite/collagen+transforming growth factor-β group; (7) DKK1+nano-hydroxyapatite/collagen+100 mg/L osteopractic total flavone group; (8) DKK1+nano-hydroxyapatite/collagen+250 mg/L osteopractic total flavone group. Cells in each group were harvested after 24 and 48 hours of intervention. Immunofluorescence labeling was used to observe the binding of Wnt and LRP in osteoblasts in the Wnt/β-catenin pathway. The expression of β-catenin, LRP 5, GSK-3β, Cyclin D1, and RUNX2 was detected by real-time polymerase chain reaction and western blot assay. RESULTS AND CONCLUSION: (1) Confocal laser scanning microscope showed that obvious brown and yellow staining was shown in the DKK1+transforming growth factor-β group, DKK1+250 mg/L osteopractic total flavone group, DKK1+nano-hydroxyapatite/ collagen+transforming growth factor-β group, and DKK1+nano-hydroxyapatite/collagen+250 mg/L osteopractic total flavone group, indicating that Wnt and LRP combined better than other groups. (2) Real-time polymerase chain reaction and western blot assay results showed that osteopractic total flavone could promote the expression of β-catenin, LRP5 and RUNX2, and downregulated GSK3β expression. These findings confirm that osteopractic total flavone can promote the differentiation and proliferation of osteoblasts by activating the Wnt/β-catenin signaling pathway. Gene activation induced by osteopractic total flavone was dose-dependent.
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BACKGROUND: β-ecdysterone as a “phytoestrogen” has the ability to stimulate protein synthesis, promote carbohydrate and lipid metabolism, relieve hyperglycemia and hyperlipidemia, protect endothelial cells from apoptosis and induce their proliferation. Some scholars have reported that it also plays an important role in the treatment of osteoporosis, fractures and other bone inflammatory diseases. OBJECTIVE: To observe the effect of β-ecdystrone on the proliferation of mouse pre-osteoblasts(MC3T3-E1 cells) in vitro, and to explore whether β-ecdysterone can induce osteogenic differentiation of MC3T3-E1 at a safe dose. METHODS: The fourth generation MC3T3-E1 cells were cultured in the osteogenic induction medium for 7, 10, 14, 21, and 28 days. The osteogenic differentiation proteins(alkaline phosphatase, type I collagen, osteopontin, and calcified nodules) were detected at different time points, to identify whether the cells have the ability of osteogenic differentiation. MC3T3-E1 cells were then seeded into the induction medium containing different final concentrations of β-ecdysterone(0.01, 0.1, 1, 10, 100 µmol/L). The proliferation activity of the cells was detected by cell counting kit-8 method at days 1, 2, 3, 4, 5, 6, and 7 after induction. The control group(general induction medium group) and the experimental group(general induction medium + β-ecdysterone) were cultured under the same conditions, and the expression levels of osteogenic marker proteins in each group of cells at different time periods were determined. RESULTS AND CONCLUSION: In the MC3T3-E1 cells stimulated by the osteogenic induction medium, alkaline phosphatase staining and type I collagen florescence staining showed higher expression at day 10 of induction, and this was also confirmed by detection of alkaline phosphatase activity(P 0.05). The expression of alkaline phosphatase and type I collagen was higher in the experimental group than in the control group at day 10 of induction. The expression of osteopontin and osteocalcin in the cells was higher at day 14 of induction, and there was no significant difference in the calcified nodule staining between the two groups at day 28 of induction. These findings indicate that β-ecdysterone can promote the proliferation of MC3T3-E1 cells in vitro and induce MC3T3-E1 cells to differentiate into osteoblasts at a safe dose.
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Objective::To investigate the role and mechanism of Testudinis Carapax et Plastrum aqueous extract in promoting osteogenic differentiation of mouse preosteoblast cell line(MC3T3-E1) by regulating nuclear transcription factor-κB(NF-κB) inflammation microenvironment. Method::MC3T3-E1 cells were cultured in vitro, and osteogenic induction (OI) was performed. Testudinis Carapax et Plastrum was prepared and treated the cells. Cells were devided into control group, osteogenic induction group and Testudinis Carapax et Plastrum (20 mg·L-1)with osteogenic induction group. The proliferation of MC3T3-E1 was detected by cell counting kit-8 (CCK-8), and the optimum concentration of intervention was determined. MC3T3-E1 differentiation and osteogenic mineralization were assayed using alkaline phosphatase (ALP) and Alizarin red staining (ARS), respectively. The expressions of NF-κB p65, NF-κB p105, interleukin-6(IL-6), ALP and Collagen-Ⅰ(COL-Ⅰ) mRNA were detected by Real-time PCR. Result::The results of CCK-8 showed that the proliferation of MC3T3-E1 did not change statistically with time, but it showed an upward trend, while the proliferation at 20 mg·L-1 was more obvious than other groups. The ALP and ARS showed that the positive staining rate of osteogenic induction group and Testudinis Carapax et Plastrum with osteogenic induction group were higher than control group.Real-time PCR results showed that on the 7th day in culture, the expression of NF-κB p105 and IL-6 mRNA in Testudinis Carapax et Plastrum with osteogenic induction group was significantly lower than that in control group (P<0.01), and the expression of ALP and COL-Ⅰ mRNA was significantly upregulated(P<0.05), on the 14th day, the expression of NF-κB p65, NF-κB p105 and IL-6 mRNA in Testudinis Carapax et Plastrum with osteogenic induction group was significantly lower than that in control group (P<0.01). The expression of ALP and COL-Ⅰ mRNA was significantly increased (P<0.05, P<0.01). Conclusion::Testudinis Carapax et Plastrum aqueous extract can promote osteogenic differentiation of MC3T3-E1 via a mechanism associated with the regulation of inhibition of NF-κB inflammatory microenvironment.
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Objective::To observe the effect of sanggenone C (SanC) on the proliferation and differentiation of mouse MC3T3-E1 osteoblasts induced by dexamethasone (DEX), and to explore its mechanism. Method::Molecular docking was conducted between SanC and Runt-associated transcription factor 2(Runx2) protein structure obtained by homologous modeling. MC3T3-E1 cells were jointly treated by different concentrations of SanC (8, 16, and 32 μmol·L-1) and 1 μmol·L-1 DEX, and then cell counting kit-8(CCK-8) method was used to detect the effect of SanC on the proliferation of MC3T3-E1 osteoblasts. The alkaline phosphatase (ALP) activity of MC3T3-E1 osteoblasts was determined by reagent kit and the formation of mineralized bone nodules were detected by alizarin red staining. Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression of Runx2, ALP and Osterix. The protein expression of Runx2 was detected by Western blot. Result::The docking score of SanC and Runx2 was -9.78.As compared with the normal group, DEX group significantly reduced the cell survival rate (P<0.01), and the greatest difference occurred on the seventh day. As compared with DEX group, SanC could significantly promote the cell proliferation of MC3T3-E1 (P<0.01), in which 32 μmol·L-1 SanC had the largest difference in proliferation rate on seventh day. As compared with the normal group, the expression of Runx2, ALP and Osterix mRNA increased to a certain extent in DEX group(P<0.01). As compared with DEX group, the expression levels of Runx2, ALP and Osterix mRNA were up-regulated in different concentration groups of SanC in a dose-dependent manner (P<0.01). As compared with the normal group, the expression of Runx2 protein in DEX group decreased significantly (P<0.05), and as compared with DEX group, the expression of Runx2 protein in cells under the intervention of SanC increased significantly (P<0.01). Conclusion::SanC can promote the proliferation, differentiation and mineralization of MC3T3-E1 osteoblasts, and the mechanism may be related to the up-regulation of Runx2 expression.
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To study the osteoprotective effect of 1,2,3,4,6-pentyl-O-galloyl-beta-D-glucose (PGG) its anti-osteoblast apoptosis related mechanism was investigated. A model of zebrafish osteoporosis induced by prednisolone (Pred, 25 μmol·L-1) was established in vivo, and calcein staining was used to detect the effect of PGG on the bone area of zebrafish. Bone marrow mesenchymal stem cells were cultured in vitro, and the number of calcified nodules was observed by alizarin red staining, and the relevant indexes of osteoblast differentiation runt-related transcription factor 2 (Runx 2), osteocalcin (OCN) mRNA level were detected by qRT-PCR. The osteoblast cell line MC3T3-E1 cells was cultured in vitro, and 400 μmol·L-1 hydrogen peroxide (H2O2) was used to intervene the injury to detect the effect of PGG on osteoblasts under oxidative stress. The effect of PGG on osteoblast activity was detected by MTT assay. The effect of PGG on apoptosis was observed by Hoechst 33342 staining. Western blot was used to detect the expression of Bcl-2, Bax, nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). DCFH-DA fluorescence staining for detection of reactive oxygen species (ROS) levels. JC-1 staining was used to detect mitochondrial membrane potential levels. The results showed that PGG could significantly increase the vertebral area of the zebrafish model when compared with the model group. On the 14 th day of osteoblast differentiation, the number of calcified nodules in the PGG group was significantly increased when compared with the control group and the mRNA levels of Runx 2 and OCN were also significantly increased. In addition, under oxidative stress, PGG could increase osteoblast viability, significantly reduce the number of apoptotic cells, and increase the ratio of Bcl-2/Bax. Fluorescence staining results show that PGG decreased intracellular ROS fluorescence density and increased mitochondrial membrane potential. Western blot data showed that PGG could promote the expression of Nrf2 in the nuclear and enhance the expression of downstream protein HO-1. In conclusion, PGG could improve osteoporosis in zebrafish, and this effect may be related to the regulation of Nrf2/HO-1 signaling pathway to improve mitochondrial dysfunction, anti-oxidative stress in osteoblast apoptosis and promote bone formation. This study provides new ideas and clues for the discovery of anti-osteoporosis drugs.
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OBJECTIVE@#To investigate the relationship between necroptosis and apoptosis in MCET3-E1 cell death induced by glucocorticoids.@*METHODS@#MC3T3-E1 cells were incubated with 10-6 mol/L dexamethasone followed by treatment with the apoptosis inhibitor z-VAD-fmk (40 μmol/L) or the necroptosis inhibitor necrostatin-1 (40 μmol/L) for 2 h. At 72 h after incubation with dexamethasone, the cells were harvested to determine the cell viability using WST-1 assay and the rate of necrotic cells using annexin V/PI double staining; the percentage of apoptotic cells was determined using Hoechst staining. The mitochondrial membrane potential and the level of ATP in the cells were also evaluated. Transmission electron microscopy was used to observe the microstructural changes of the cells. The expressions of RIP-1 and RIP-3 in the cells were detected by Western blotting.@*RESULTS@#At a concentration of 10-6 mol/L, dexamethasone induced both apoptosis and necroptosis in MC3T3- E1 cells. Annexin V/PI double staining showed that inhibition of cell apoptosis caused an increase in cell necrosis manifested by such changes as mitochondrial swelling and plasma membrane disruption, as shown by electron microscopy; Hoechst staining showed that the percentage of apoptotic cells was significantly reduced. When necroptosis was inhibited by necrostatin-1, MC3T3-E1 cells showed significantly increased apoptosis as shown by both AV/PI and Hoechst staining, and such changes were accompanied by changes in mitochondrial membrane potential and ATP level in the cells.@*CONCLUSIONS@#In the process of dexamethasone-induced cell death, necroptosis and apoptosis can transform reciprocally accompanied by functional changes of the mitochondria.
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Animals , Mice , 3T3 Cells , Adenosine Triphosphate , Apoptosis , Cell Death , Dexamethasone , Membrane Potential, Mitochondrial , Microscopy, Electron , Mitochondria , NecrosisABSTRACT
Objective: To discuss the effect of Piezo1 mechanically sensitive protein in migration process of mouse MC3T3-E1 osteoblast cells. Methods: The 5th-10th generation mouse MC3T3-E1 osteoblasts were divided into Piezo1-small interfering RNA (siRNA) transfection group (group A), negative control group (group B), and blank control group (group C). Piezo1-siRNA or negative control siRNA was transfected into mouse MC3T3-E1 osteoblasts by siRNA transfection reagent, respectively; group C was only added with siRNA transfection reagent; and the cell morphology was observed under inverted phase contrast microscope and fluorescence microscope, and the transfection efficiency was calculated. The expression of Piezo1 protein was detected by immunofluorescence staining and Western blot. Transwell cell migration assay and cell scratch assay were used to detect the migration of MC3T3-E1 osteoblasts after Piezo1-siRNA transfection. Results: After 48 hours of transfection, group A showed a slight increase in cell volume and mutant growth, but cell colonies decreased, suspension cells increased and cell fragments increased when compared with untransfected cells. Under fluorescence microscope, green fluorescence was observed in MC3T3-E1 osteoblasts of group B, and the transfection efficiency was 68.56%±4.12%. Immunofluorescence staining and Western blot results showed that the expression level of Piezo1 protein in group A was significantly lower than that in groups B and C ( P0.05). Transwell cell migration assay and cell scratch assay showed that the number of cells per hole and the scratch healing rate of cells cultured for 1-4 days in group A were significantly lower than those in groups B and C ( P0.05). Conclusion: Piezo1 knocked down by siRNA can inhibit the migration ability of MC3T3-E1 osteoblast cells.
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This aim of this study was to assess the molecular mechanism of osteoporosis in schizophrenia patients with risperidone use. Here, we investigated the effects of risperidone on cellular proliferation and apoptosis of a preosteoblast cell line, MC3T3-E1. Cell viability and apoptotic rate of MC3T3-E1 were detected by cell counting kit-8 and flow cytometry at a serial dose of risperidone and at different time points, respectively. Bone transformation relevant gene serum osteocalcin (BGP), collagen 1, tumor necrosis factor-α (TNF-α), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels were determined by real-time PCR (qPCR). Their protein expression patterns were evaluated using western blot. The results revealed that risperidone dramatically inhibited MC3T3-E1 cell proliferation in a dose-dependent manner. It also significantly induced MC3T3-E1 cell apoptosis. TNF-α gene and protein levels were greatly enhanced after risperidone treatment. In contrast, BGP, collagen 1, OPG, and RANKL gene and protein levels were markedly downregulated. Our study indicated that risperidone suppressed MC3T3-E1 cell proliferation and induced apoptosis. It also regulated BGP gene and protein expression.
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Animals , Osteoblasts/drug effects , Apoptosis/drug effects , Risperidone/pharmacology , Cell Proliferation/drug effects , Osteocalcin/drug effects , Cell Line , Collagen/drug effects , Tumor Necrosis Factor-alpha/drug effects , Receptor Activator of Nuclear Factor-kappa B/drug effects , Osteoprotegerin/drug effects , Flow CytometryABSTRACT
Objective: To observe the effect of mistletoe polysaccharose on proliferation and apoptosis of osteoblast and to explore the possible mechanism. Methods: The mistletoe polysaccharose was extracted and made different concentrations (0.625 g/L, 1.25 g/L, 2.5 g/L, 5 g/L) to treat mouse skull bone cell line MC3T3-E1. Cell cycle and apoptosis were detected by flow cytometry. Cell proliferation and extracellular alkaline phosphatase secretion were detected by BrdU and alkaline phosphatase ELISA kits. The mRNA expression level of osteoblast related gene was detected by Realtime PCR. Results: Compared with the control group, the result of treatment groups with increasing mistletoe polysaccharose concentrations were as follows: the proportions of S phase and G2/M phase in cell cycle increased and the cell count Annexin V +/PI-significantly decreased, the number of cells increased, extracellular alkaline phosphatase activity obviously decreased. The increasing transcription levels of Runt-related transcription factor 2(Runtx2) and collagen type I alpha 1 (COL1A) were stabled over 2. 5 g/L and the up-regulation of mRNA expression of osteocalcin (OC) was steady at 1.25 g/L. Conclusion: Mistletoe polysaccharose can promote the proliferation and inhibit the apoptosis of MC3T3-E1 cells, and its possible mechanism might be related to down-regulating of extracellular alkaline phosphatase activity and the grow exhanced expression of OC, Runtx2 and COL1A.
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Objective To evaluate the effects of the human recombinant parathyroid hormone [rhPTH (1 -34)] on the initial adhesion of MC3T3-E1 osteoblasts cultured on titanium surfaces of sand-blasted large grift acid-etched (SLA). Methods The optimal concentration of the rhPTH(1-34) action was determined by the CCK8 method. Then the MC3T3-E1 cells were divided into experimental groups treated with the optimal concentration of the rhPTH(1-34) medium and control groups treated with blank medium, and inoculated on SLA-treated titanium plates. DAPI immunocytochemistxy, scanning electron microscopy, and RT-PCR were used to count the cell adhesion of MC3T3-E1 cells on the surface of titanium plates. Morphological changes were observed and the expression of the integrin α1, α5, β1 mRNA in MC3T3-E1 cells was determined. Results Compared with the control group, the adherence of osteoblasts was more than that of the control group. The remodeling of actin filaments, the change of cell morphology, and the speed of adhesion and extension on the titanium plate were faster than that of the control group. The expression of the osteoblast integrin subunit α1, α5 and (31 was higher than that of the control group. Conclusion rhPTH (1-34) can promote the adhesion of MC3T3-E1 cells on pure titanium surface.
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Abstract Flavones have the potential of being used as a dietary supplement for bone health promotion beyond calcium and vitamin D. Recent studies have showed that flavones enhanced bone formation and inhibited bone resorption by affecting osteoblast and osteoclast differentiation through various cell signaling pathways. In this study, we investigated the effects of a new flavone (2R,3S)-pinobanksin-3-cinnamate, isolated from the metabolites of the endophytic fungus Penicillium sp. FJ-1 of Acanthus ilicifolius L., Acanthaceae, on osteoblast differentiation by using MC3T3-E1 cells. It was observed that (2R,3S)-pinobanksin-3-cinnamate promoted osteoblast differentiation, as evidenced by increased mineralization process and alkaline phosphatase activity, as well as expression of genes encoding the bone differentiation. Moreover (2R,3S)-pinobanksin-3-cinnamate treatment upregulated the gene expression of wingless-type MMTV integration site family, bone morphogenetic protein and runt-related transcription factor 2, and protein expression of phosphor-Smad1/5/8, β-catenin and runt-related transcription factor 2 in MC3T3-E1 cells. The osteoblast differentiation effects induced by (2R,3S)-pinobanksin-3-cinnamate were attenuated by the bone morphogenetic protein antagonist Noggin, and wingless-type MMTV integration site family signaling pathway inhibitors Dickkopf-1. Co-treatment with adenosine 30,50-cyclic monophosphate and guanosine 30,50-cyclic monophosphate pathway inhibitors, H89 and KT5823, respectively, reversed the (2R,3S)-pinobanksin-3-cinnamate-induced activations of p-Smad1/5/8, β-catenin, and runt-related transcription factor 2. Our data demonstrated that (2R,3S)-pinobanksin-3-cinnamate promoted the osteoblast differentiation of MC3T3-E1 cells, at least partially through the adenosine 30,50-cyclic monophosphate and guanosine 30,50-cyclic monophosphate signaling pathways, providing the scientific rational to develop (2R,3S)-pinobanksin-3-cinnamate against bone loss-associated diseases.
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BACKGROUND: Sand blasted titanium (Ti) is commonly used in designing endosseous dental implants due to its biocompatibility and ability to form bonds with bone tissues. However, titanium implants do not induce strong interactions with teeth bones. To increase strong interactions between Ti disk implants and teeth bones, the L-glutamic acid grafted hydroxyapatite nanorods (nHA) were immobilized on albumin modified Ti disk implants (Ti-Alb). METHODS: For modification of Ti disk implants by nHA, the L-glutamic acid grafted nHA was synthesized and then immobilized on albumin modified Ti disk implants. Fourier transformed infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscope; energy dispersive spectroscopy and confocal laser scanning microscopy were used to confirm the modification of Ti disk implants. The bioactivity of nHA-modified Ti disk implants was evaluated by seeding MC3T3-E1 cells on Ti-nHA implants. RESULTS: Characterization techniques have confirmed the successful modification of Ti disk implants by L-glutamic acid grafted nHA. The nHA-modified Ti disk implants have shown enhanced adhesion, proliferation and cytotoxicity of MC3T3-E1 cells in comparison to pristine Ti implants. CONCLUSION: The modification of Ti implants by L-glutamic acid grafted nHA has produced highly osteogenic Ti disk plants in comparison to pristine Ti disk implants due to the formation of bioactive surfaces by hydroxyapatite nano rods on Ti disk implants. Ti-nHA disk implants showed enhanced adhesion, proliferation, and MC3T3-E1 cells viability in comparison to pristine Ti disk implants. Thus nHA might be to be useful to enhance the osseointegration of Ti implants with teeth bones.
Subject(s)
Bone and Bones , Dental Implants , Durapatite , Fourier Analysis , Glutamic Acid , Microscopy, Confocal , Nanotubes , Osseointegration , Photoelectron Spectroscopy , Spectrum Analysis , Titanium , Tooth , TransplantsABSTRACT
PURPOSE: The Glycyrrhiza uralensis species (Leguminosae) as a medicinal biocompound, and one of its root components, isoliquritigenin (ISL), which is a flavonoid, has been reported to have anti-tumor activity in vitro and in vivo. However, its function in bone formation has not been studied yet. In this study, we tested the effect of Glycyrrhiza uralensis (ErLR) and baked Glycyrrhiza uralensis (EdLR) extracts on osteoblast proliferation, alkaline phosphatase (ALP) activity, and bone-related gene expression in osteoblastic MC3T3-E1 cells. METHODS: MC3T3-E1 cells were cultured in various levels of ErLR (0, 5, 10, 15, 20 µg/mL), EdLR (0, 5, 10, 15, 20 µg/mL), or ISL (0, 5, 10, 15, 20 µM) in time sequences (1, 5, and 20 days). Also, isoliquritigenin (ISL) was tested for comparison to those two biocompound extracts. RESULTS: MTT assay results showed that all three compounds (ErLR, EdLR, and ISL) increased osteoblastic-cell proliferation in a concentration-dependent manner for one day. In addition, both ErLR and EdLR compounds elevated the osteoblast proliferation for 5 or 20 days. Extracellular ALP activity was also increased as ErLR, EdLR, and ISL concentration increased at 20 days, which implies the positive effect of Glycyrrhiza species on osteoblast mineralization. The bone-related marker mRNAs were upregulated in the ErLR-treated osteoblastic MC3T3-E1 cells for 20 days. Bone-specific transcription factor Runx2 gene expression was also elevated in the ErLR- and EdLR-treated osteoblastic MC3T3-E1 cells for 20 days. CONCLUSION: These results demonstrated that Glycyrrhiza uralensis extracts may be useful for preventing osteoporosis by increasing cell proliferation, ALP activity, and bone-marker gene expression in osteoblastic cells.
Subject(s)
Alkaline Phosphatase , Cell Proliferation , Gene Expression , Glycyrrhiza uralensis , Glycyrrhiza , In Vitro Techniques , Miners , Osteoblasts , Osteogenesis , Osteoporosis , RNA, Messenger , Transcription FactorsABSTRACT
<p><b>Background</b>Estrogen, as an important hormone in human physiological process, is closely related to bone metabolism. The aim of this study was to investigate the mechanism of estrogen on osteoblasts metabolism in MC3T3-E1 cells.</p><p><b>Methods</b>We treated the MC3T3-E1 cells with different concentrations of β-estradiol (0.01, 0.1, 1, and 10 nmol/L), observed the morphological changes of the cells, and detected the cell's proliferation and apoptosis of MC3T3-E1 cells. Two transcriptome libraries were constructed and sequenced. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to confirm the differentially expressed genes (DEGs), and then treated the MC3T3-E1 cells with estrogen receptor (ER) inhibitors α and β, respectively, and then examined the expression of Tgfbr1 and Bmpr1a genes. The promoter of Tgfbr1 and Bmpr1a gene was analyzed, and the ER response elements were identified. Finally, ChIP was used to verify the binding of ER to Tgfbr1 and Bmpr1a promoter.</p><p><b>Results</b>In the high-concentration β-estradiol treatment group (1 nmol/L and 10 nmol/L), there was no significant difference in the morphology of the cells under the microscope, 1 nmol/L and 10 nmol/L treated group appeared statistically significant difference in cell apoptosis and proliferation (P < 0.05 and P < 0.01, respectively). We found 460 DEGs compared with the control group. Among the DEGs, there were 66 upregulated genes and 394 downregulated genes. Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that many bone metabolism-related biological processes and cell signaling pathways were disordered. The qRT-PCR verification showed that the expressions of Tgfbr1- and Bmpr1a-related genes in bone metabolism pathway in the 10 nmol/L treatment group were significantly decreased (P < 0.05). ER β was involved in the inhibitory effect of Tgfbr1 and Bmpr1a genes. The bioinformatics of the promoter found that there were three ER response elements in the promoter of Tgfbr1, and there were two ER response elements in Bmpr1a promoter regions. ChIP experiments showed that estrogen could enhance the binding of ERs to Tgfbr1 and Bmpr1a genes.</p><p><b>Conclusions</b>Estrogen can promote the apoptosis and proliferation of osteoblasts simultaneously, and the mechanism may be the joint action of transforming growth factor-beta, Wnt, mitogen-activated protein kinase, and nuclear factor-kappaB bone metabolism-related signaling pathway. Estrogen inhibits the expression of Tgfbr1 and Bmpr1a genes through ER β and affects the metabolism of MC3T3-E1 osteoblasts.</p>