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Total Panax notoginseng saponin (PNS) has been extensively used to treat a variety of diseases, such as bone fractures, soft tissue injuries, etc. In this study, mouse calvaria-original osteoblastic MC3T3-E1 cells were cultured in various concentrations of PNS (0.005-5 mg/mL) during the period (1, 5, 14, and 23 d). At the endpoint, the osteogenic capacity of MC3T3-E1 cells was investigated by measuring the alkaline phosphatase (ALP) activity, the deposited calcium, and the expression of osteogenic-related markers, including bone collagen type 1 (Col1) and osteocalcin (OCN). Compared with all groups in each period, the most pronounced effect was observed at the concentration range between 0.05 and 0.5 mg/mL (P < 0.05) and the cell proliferation with PNS treatment was found during the whole osteogenic period. Moreover, cellular ALP activity with PNS was increased during 7, 14, and 21 d and cell mineralization with PNS was enhanced in 14 and 21 d. Furthermore, the differentiation markers Col1 and OCN increased in the PNS-treated cells. Our work suggests that PNS may stimulate the osteogenesis process which contains osteoblastic proliferation, differentiation, and mineralization by increasing cellular ALP activity, extracellular matrix mineralization, and osteoblast-associated molecules in the osteoblasts.

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Recently, interest in fish byproduct-derived bioactive components as potential ingredients for treating and preventing osteoporosis is increasing. In this study, we investigated the antiosteoporotic effects of mackerel Scomber japonicus extracts in MC3T3-E1 preosteoblasts in vitro and ovariectomized rats in vivo. Scomber japonicus neutrase-assisted protein hydrolysate (SJNH) treatment enhanced alkaline phosphatase (ALP) activity and osteoprotegerin (OPG) level, and attenuated receptor activator of nuclear factor κB ligand (RANKL) level in MC3T3-E1 cells. In an osteoporotic animal model, ovariectomized (OVX) rats were administered SJNH (200 mg/kg/day) for 8 weeks. SJNH prevented OVX-induced bone mineral density (BMD) loss and restored bone structural compartment loss based on micro-computed tomography (CT). In addition, SJNH administration normalized bone remodeling by regulating bone regeneration and resorption-related proteins such as ALP, osteocalcin (OC), and tartrate-resistant acid phosphatase 5b (TRACP-5b) as well as calcium contents in serum. Taken together, our findings suggest that SJNH can promote antiosteoporotic effects both in vitro and in vivo. Thus, SJNH might be a potent therapeutic agent or functional food ingredient for preventing osteoporosis. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of microRNA (miR) - 196a on osteogenic differentiation of MC3T3-El cells by target regulation of his tone deacetylase 9 (HDAC9). Methods MC3T3-El cells were divided into control group (Cont) group, induction group, miR-196a-mimics-NC group, miR-196a-mimics group, miR-196a-inhibitor-NC group, miR-196a-inhibitor group, miR-196a-mimics+pCMV-HDAC9-NC group, and miR-196a-mimics+pCMV-HDAC9 group. Osteogenic induction was performed after transfection according to the group. Quantitative fluorescent PCR was performed to measure the expression of miR-196a and HDAC9 in MC3T3-El cells. Alkaline phosphatase (ALP) activity was measured with a commercial kit. Alizarin red staining was performed to observe the degree of mineralization. Western blotting was performed to measure the expressions of HDAC9, ALP, Runt-related transcription factor 2 (RUNX2), collagen I (COLl), osteopontin (OPN), Histone H3, and Histone H3 (acetyl K9, K 14 and K23). Results Compared to those in the Cont group, the expression of miR-196a, the expressions of ALP, Runx2, COLl, and OPN proteins, ALP activity, mineralization degree, and the acetylation levels of Histone H3 K9, K14, and K23 sites in MC3T3-E I cells in the induction group increased (P < 0. 05), but the expression of HDAC9 mRNA and protein decreased (P < 0. 05). Transfection of miR-196a-mimics was able to significantly increase miR-196a expression, to decrease HDAC9 expression, and to increase ALP, Runx2, COL 1, and OPN protein expression, ALP activity, mineralization degree, and Histone H3 acetylation. Transfection of miR-196a-inhibitor had the opposite effect. MiR-196a was able to target down-regulation of HDAC9 expression. Overexpression of HDAC9 was able to partially reverse the promoting effect of miR-196a mimics on osteogenic differentiation of MC3T3-E I cells. Conclusion MiR-196a target down-regulates HDAC9 expression, increases hi stone acetylation level, and promotes osteogenic differentiation of MC3T3-El cells. [ABSTRACT FROM AUTHOR]

Osteoporosis is an age-related disorder with enormous public health burden with an urgent need for better diagnosis and treatment. Herein, we have investigated the potential role of jatrorrhizine, a plant bioactive, in osteoporosis since it is known to exhibit a wide array of biologic and pharmacologic activities. Using MC3T3-E1 cells, a clonal murine cell line of immature osteoblasts derived from mice, we examined the effect of jatrorrhizine on various parameters of cell function. While jatrorrhizine exhibited no effect on MC3T3-E1 cell viability, it caused reversal of dexamethasone-induced suppression of calcium deposition and alkaline phosphatase activity, elevation of osteogenic differentiation related proteins, and decrease in the level of histone deacetylase 4. These observations led us to conclude that jatrorrhizine ameliorates osteogenic differentiation in dexamethasone treated MC3T3-E1 cells via histone deacetylase 4 suggesting its potential application in the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

Hai-Honghua medicinal liquor (HHML), an external Chinese herbal formula preparation, is often applied to treat freshly closed tibia/fibular fractures, ankle fractures, and other bone-related disorders, but the related molecular mechanism is unclear. To evaluate the therapeutic effect of HHML in patients with tibial/fibular and ankle fractures, and to explore its related possible mechanism. A total of 182 patients with tibia/fibular fractures and 183 patients with ankle fractures were enrolled in this study. A randomized, controlled, unblinded clinical trial was designed to evaluate the therapeutic effect of HHML on tibial/fibular and ankle fractures. The chemical compositions of HHML were analyzed by the HPLC-Q-Extractive MS/MS. Furthermore, a rat tibial fracture model was established to evaluate the therapeutic effects of HHML in promoting fracture healing, and the mouse embryonic osteoblasts cell line of MC3T3-E1 was further carried out to explore the mechanisms of HHML on osteoblast differentiation. In the clinical evaluation, HHML treatment significantly shortened the time for pain and swelling in patients with tibial/fibular fractures (P < 0.01) and ankle fractures (P < 0.01), and the incidence of complications was significantly reduced as well. Subsequently, 116 constituents were identified from HHML via HPLC-Q-TOF-MS/MS analysis. In vivo , no obvious changes in weight were observed in HHML-treated rats. Moreover, the levels of bone formation markers (including osteocalcin (OCN), N-terminal propeptide of type I procollagen (PINP), alkaline phosphatase (ALP), calcium (Ca) and substance P) in rat serum were significantly increased in HHML-treated rats compared with model rats (P < 0.05). Micro-CT analysis showed bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th) of the HHML-treated rats were significantly increased (P < 0.05, vs. Model) while trabecular separation (Tb.Sp) and structure model index (SMI) values were significantly reduced (P < 0.05, vs. Model). Histological analysis showed that HHML treatment promoted the healing of fractures and cartilage repair, and increased the osteoblasts and collagen fibers. Furthermore, our results also revealed HHML could promote MC3T3-E1 cells proliferation and osteoblast differentiation via regulation of the runt-related transcription factor 2 (RUNX2), bone alkaline phosphatase (BALP), and OCN by activating phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway, which confirmed by adding PI3K chemical inhibitor of LY294002. HHML treatment is a reliable remedy for fractures in tibial and ankle by promotion of osteogenic differentiation via activation of PI3K/Akt pathway. [Display omitted] • HHML was first evaluated by clinical trial and analyzed by HPLC-MS. • HHML promoted fracture healing in patients. • HHML facilitated the formation of callus and the increase of bone trabecular and bone mineral density. • HHML promoted osteoblast differentiation and mineralization in MC3T3-E1 cells via PI3K/Akt. [ABSTRACT FROM AUTHOR]

BACKGROUND: MicroRNA-98-5p (miR-98-5p) can inhibit the high mobility group AT-HOOK 2 (HMGA2). The phosphatidylinositol 3-kinase/alkaline phosphatase/glycogen synthase kinase-3β (PI3K/AKT/GSK-3β) signaling pathway is involved in cell proliferation. During the fracture healing process, it is unclear whether miR-98-5p can act on the PI3K/Akt/GSK-3β pathway by activating HMGA2 to promote the proliferation of osteoblasts. OBJECTIVE: To investigate the mechanisms of microRNA-98-5p (miR-98-5p) in promoting osteoblast proliferation and differentiation by regulating HMGA2 and PI3K/Akt/GSK-3β pathway. METHODS: MC3T3-E1 cells transfected with miR-98-5p mimics and HMGA2 plasmid by Lipofectamine 2000 were divided into a miR-98-5p mimics group and a HMGA2 overexpression group, respectively. MC3T3-E1 cells transfected with dimethyl sulfoxide and scrambled plasmids were divided into a mimic control group and a scramble group. Cells transiently transfected with miR-98-5p inhibitor using liposomes were as a miR-98-5p inhibitor group. Normal MC3T3-E1 cells without treatment were used as a blank control group. RESULTS AND CONCLUSION: There were no significant differences in HMGA2, PI3K/Akt/GSK-3β, alkaline phosphatase activity and mRNA level between the blank control group and the mimic control group. Compared with the mimic control group, the HMGA2 and PI3K/Akt/GSK-3β protein expression, cell differentiation and proliferation, alkaline phosphatase activity and mRNA level were significantly reduced in the miR-98-5p mimics group. The interaction between miR-98-5p and HMGA2 predicted by Targetscan7.1 showed that compared with the mimic control group, the HMGA2 protein and mRNA were reduced in the miR-98-5p mimic group, while compared with the miR-98-5p mimic group, the HMGA2 protein and mRNA had an increase in the miR-98-5p inhibitor group. There were no significant differences in osteoblast proliferation, alkaline phosphatase activity and mRNA between the blank control group and the scramble group. Compared with the scramble group, the PI3K/Akt/GSK-3β expression, cell differentiation and proliferation, alkaline phosphatase activity and mRNA in the HMGA2 overexpression group were significantly increased. To conclude, miR-98-5p can inhibit the HMGA2 and PI3K/Akt/GSK-3β expressions in MC3T3-E1 cells, inhibit cell proliferation and differentiation. HMGA2 is possible the direct target of miR-98-5p. [ABSTRACT FROM AUTHOR]

BACKGROUND: Growth factors are one of the key elements for bone formation, remodeling and repair. The combination of multiple growth factors to induce osteogenic differentiation of bone stem cells has certain advantages compared with single ones. However, what combination of concentrations can play the best inducing effect has not been fully documented. OBJECTIVE: To explore the effects of transforming growth factor-β combined with bone morphogenetic protein-2 on the proliferation and differentiation of mouse MC3T3-E1 cells. METHODS: Different concentrations of transforming growth factor-β and bone morphogenetic protein-2 were combined to cultivate MC3T3-E1 cells. The cells were then divided into six groups, including one control group and five experimental groups, and were tested at different time points. The proliferation of the cells w as determined by MTT method. Real-time quantitative RT-PCR was used to detect Runx2 and osteocalcin gene expression. Alkaline phosphatase staining was used to observe the viability and function of osteoblasts. Alizarin red staining was used to observe the amount of calcium nodules. RESULTS AND CONCLUSION: Whether transforming growth factor-β and bone morphogenetic protein-2 were used alone or in combination could significantly promote the proliferation of MC3T3-E1cells, promote the expression of Runx2 and osteocalcin, and increase the alkaline phosphatase activity and mineralization ability (P < 0.05). The osteogenic effect of two growth factors in combination was positively correlated with the concentration. Even the combination at a low concentration can achieve or surpass the effect of single use. The optimal combined concentration was 1 μg/L for transforming growth factor-β and 100 μg/L for bone morphogenetic protein-2. Therefore, transforming growth factor-β and bone morphogenetic protein-2 used alone or in combination can significantly promote the proliferation and osteogenesis of osteoblasts, which focus on different aspects and interact with each other at different stages of proliferation and differentiation. Within the concentration range of this study, 1 μg/L transforming growth factor-β plus 100 μg/L bone morphogenetic protein-2 show the best promotion effect. [ABSTRACT FROM AUTHOR]

BACKGROUND: Previous studies have found that cinnamaldehyde can promote the biological properties of osteoblasts in high glucose environment. OBJECTIVE: To observe the effect of cinnamaldehyde on proliferation and differentiation of osteoblasts under high glucose with electrospinning membrane as carrier. METHODS: The polycaprolactone membranes were prepared by electrospinning. MC3T3-E1 cells in logarithmic growth phase were divided into four groups: blank control group was added with high glucose medium. Polycaprolactone group was added with polycaprolactone membrane scaffold and high glucose medium. Drug group was added with high glucose medium containing cinnamaldehyde. Combined group was added with polycaprolactone membrane and high glucose medium containing cinnamaldehyde. CCK-8 method was used to detect cell proliferation and alkaline phosphatase kit was used to detect alkaline phosphatase activity at 1, 4 and 7 days of culture. RT-qPCR was used to detect the expression of osteocalcin mRNA at 7 days. RESULTS AND CONCLUSION: (1) CCK-8 assay showed that the cell proliferation of polycaprolactone group, drug group and combined group was faster than that of blank control group at each time point (P < 0.05). Cell proliferation of combined group was faster than that of polycaprolactone group and drug group at 4 and 7 days (P < 0.05), and cell proliferation of drug group was faster than that of polycaprolactone group at 4 and 7 days (P < 0.05). (2) The alkaline phosphatase activity of polycaprolactone group, drug group, and combined group was higher than that of blank control group at 4 and 7 days (P < 0.05). The alkaline phosphatase activity of combined group was higher than that of polycaprolactone group and drug group at 4 and 7 days (P < 0.05). The alkaline phosphatase activity of drug group was higher than that of polycaprolactone group at 7 days (P < 0.05). (3) The expression of osteocalcin mRNA in the four groups from high to low was the combined group, drug group, polycaprolactone group, and blank control group; there were significant differences between the two groups (P < 0.05). (4) Results suggest that polycaprolactone electrospinning membrane and cinnamaldehyde can promote the proliferation and differentiation of osteoblasts in high glucose environment. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stem cells isolated from the dental pulp of primary and permanent teeth can be differentiated into different cell types including osteoblasts. This study was conducted to compare the morphology and osteogenic potential of stem cells from exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSC) in granular hydroxyapatite scaffold (gHA). Preosteoblast cells (MC3T3-E1) were used as a control group. Methodology: The expression of stemness markers for DPSC and SHED was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR). Alkaline phosphatase assay was used to compare the osteoblastic differentiation of these cells (2D culture). Then, cells were seeded on the scaffold and incubated for 21 days. Morphology assessment using field emission scanning electron microscopy (FESEM) was done while osteogenic differentiation was detected using ALP assay (3D culture). Results: The morphology of cells was mononucleated, fibroblast-like shaped cells with extended cytoplasmic projection. In RT-PCR study, DPSC and SHED expressed GAPDH, CD73, CD105, and CD146 while negatively expressed CD11b, CD34 and CD45. FESEM results showed that by day 21, dental stem cells have a round like morphology which is the morphology of osteoblast as compared to day 7. The osteogenic potential using ALP assay was significantly increased (p < 0.01) in SHED as compared to DPSC and MC3T3-E1 in 2D and 3D cultures. Conclusion: gHA scaffold is an optimal scaffold as it induced osteogenesis in vitro. Besides, SHED had the highest osteogenic potential making them a preferred candidate for tissue engineering in comparison with DPSC. [ABSTRACT FROM AUTHOR]

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). At day 14 of induction, osteopontin immunocytochemical staining also indicated significant expression. Alizarin red staining results demonstrated that the number of calcified nodules increased significantly after osteogenic induction, and there were more calcified nodules with larger diameter and darker color at day 28 than at day 21. After treatment with β-ecdysterone, the proliferative activity of MC3T3-E1 cells reached the peak at day 5 after induction. The optimal concentrations of β-ecdysterone were 0.01 and 0.1 μmol/L. There was no significant difference between the two concentrations (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. [ABSTRACT FROM AUTHOR]

MC3T3‐E1 cells were divided into Blank, miR‐135b mimics, miR‐135b inhibitors, AG490, and miR‐135b inhibitors + AG490 groups. Cell viability was determined by MTT, alkaline phosphatase (ALP) activity by the corresponding kit, and mineralization by alizarin red staining. Furthermore, miR‐135b, osteoblast‐specific genes, and JAK2/STAT3 were detected through quantitative real‐time polymerase chain reaction and Western blotting. MiR‐135b downregulation was identified with increased JAK2 during osteoblast differentiation. JAK2 was confirmed as a target gene of miR‐135b by dual‐luciferase reporter assay. MC3T3‐E1 cells in both miR‐135b mimics and AG490 groups manifested decrease in cell viability, ALP activity, and mineralized nodes, as well as reductions in osteoblast‐specific genes and proteins of JAK2, p‐JAK2, and p‐STAT3, but increase in cell apoptosis. However, opposite changes of the above factors were shown in cells from miR‐135b inhibitors group. Notably, AG490 could reverse promotion effects of miR‐135b inhibitors on osteoblast differentiation. Inhibiting miR‐135b could activate the JAK2/STAT3 signaling pathway, thereby improving the cell viability and promoting the osteoblast differentiation. [ABSTRACT FROM AUTHOR]

Objective: Evidence suggests that microRNAs (miRNAs) regulate the expression of genes involved in bone metabolism. This study aimed to investigate the role of miR-505 in the osteogenic differentiation of MC3T3-E1 cells. Methods: We performed miRNA sequencing to identify differentially expressed miRNAs between MC3T3-E1 cells treated with osteogenic induction medium (OIM) and control cells. Bioinformatics analysis was performed by using the TargetScan and miRDB databases. The expression of miR-505 in MC3T3-E1 cells was detected during osteogenic differentiation. After transfection with miR-505 mimic or miR-505 inhibitor, MC3T3-E1 cells were induced to differentiate into osteoblasts, and the expression of osteogenic differentiation markers (Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), and osterix (OSX)) was detected. Results: miR-505 was the most downregulated miRNA among the differentially expressed miRNAs. The RUNX2 gene was identified as a potential target of miR-505 using the target prediction program. miR-505 expression was downregulated during osteogenic differentiation of MC3T3-E1 cells. The expression of osteogenic marker genes was inhibited in MC3T3-E1 cells after transfection with miR-505. However, the expression of osteogenic marker genes was upregulated after transfection with miR-505 inhibitor. Conclusion: This study is the first to report miR-505 could bind to the RUNX2 gene and thus regulate partly the dysfunction of osteoblasts differentiation, which is expected to be targets for the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of cyclophilin B on MC3T3-E1 cells and the underlying mechanism. CCK-8 assay was used to evaluate the cell activity, polymerase chain reaction to verify the overexpression of cyclophilin B, western blot to detect the protein levels, and clone formation assay to evaluate the proliferation ability. Alkaline phosphatase (ALP) activity was assessed by ALP assessment kit. The results showed that compared with the control group/negative control (NC) group, the cells in the cyclophilin B overexpression group possessed enhanced proliferation ability, higher ALP activity and enhanced mineralization. Compared with the control group, the levels of Oct4, Sox2, Runx2, collagen I, BMP2 and BMPR-II, as differentiation-related proteins, were increased by cyclophilin B. Compared to the NC group, the expression levels of p-JAK2 and p-STAT3 in the cyclophilin B overexpression group were higher, suggesting that cyclophilin B can activate the JAK2/STAT3 signaling pathway. The expression levels of p-JAK2 and p-STAT3 in the cyclophilin B overexpression group were higher than that in the cyclophilin B overexpression + JAK2 inhibitor group, further confirming the hypothesis that the JAK2/STAT3 signaling pathway was activated by cyclophilin B. Taken together, the results suggest that cyclophilin B promotes the proliferation and differentiation of MC3T3-E1 cells via the JAK2/STAT3 signaling pathway and may be a promising therapeutic target for bone injury. [ABSTRACT FROM AUTHOR]

Astragalin (AG) is a biologically active flavonoid compound that can be extracted from a number of medicinal plants. However, the effects of AG on osteoblastic differentiation in mouse MC3T3-E1 cells and on bone formation in vivo have not been studied fully. In this study, we found that the activities of alkaline phosphatase (ALP) and mineralized nodules in MC3T3-E1 cells were both significantly increased after treatment with AG (5, 10, and 20 μM). Meanwhile, the mRNA and protein levels of osteoblastic marker genes in MC3T3-E1 cells after AG treatment were markedly increased compared with a control group. In addition, the levels of BMP-2, p-Smad1/5/9, and Runx2 were significantly elevated in AG-treated MC3T3-E1 cells. Moreover, we found that the protein levels of Erk1/2, p-Erk1/2, p38, p-p38, and p-JNK were also significantly increased in AG-treated MC3T3-E1 cells compared to those in the control group. Finally, in vivo experiments demonstrated that AG significantly promoted bone formation in an ovariectomized (OVX)-induced osteoporotic mouse model. This was evidenced by significant increases in the values of osteoblast-related parameters (BFR/BS, MAR, Ob.S/BS, and Ob.N/B.Pm) and bone histomorphometric parameters (BMD, BV/TV, Tb.Th, and Tb.N.) in OVX mice after AG treatment (5, 10, and 20 mg/kg). Collectively, these results demonstrated that AG may promote osteoblastic differentiation in MC3T3-E1 cells via the activation of the BMP and MAPK pathways and promote bone formation in vivo. These novel findings indicated that AG may be a useful bone anabolic agent for the prevention and treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

The crude polysaccharide CO70 isolated from Curculigo orchioides could alleviate ovariectomy-induced osteoporosis in rats. To clarify the bioactive components, a new heteropolysaccharide (COP70-1) was purified from CO70 in this study, which was consisted of β -D-Man p -(1→, →4)- α -D-Glc p -(1→, →4)- β -D-Man p -(1→, →3,4)- β -D-Man p -(1→, →4,6)- β -D-Man p -(1→, and →4,6)- α -D-Gal p -(1→. COP70-1 significantly promoted the osteoblastic differentiation of MC3T3-E1 cells through improving alkaline phosphatase activity, the deposition of calcium as well as up-regulating the expression of osteogenic markers (RUNX2, OSX, BSP, OCN, and OPN). Furthermore, COP70-1 stimulated the expression of critical transcription factors of the BMP and Wnt pathways, including BMP2, p-SMAD1, active- β -catenin, p-GSK-3 β , and LEF-1. In addition, LDN (BMP pathway inhibitor) and DKK-1 (Wnt pathway inhibitor) suppressed the COP70-1-induced osteogenic differentiation of MC3T3-E1 cells. Therefore, COP70-1 was one of the bioactive constituents of C. orchioides for targeting osteoblasts to treat osteoporosis by triggering BMP/Smad and Wnt/ β -catenin pathways. [Display omitted] • A novel polysaccharide COP70-1 purified from the rhizomes of Curculigo orchioides. • The structure of COP70-1 was characterized for the first time. • COP70-1 promoted osteogenic differentiation and mineralization of MC3T3-E1 cells. • COP70-1 upregulated the levels of osteogenic markers and the ratio of OPG/RANKL. • COP70-1 stimulated osteoblast differentiation by activating BMP and Wnt pathways. [ABSTRACT FROM AUTHOR]

Graphical abstract Two distinct hydroxyapatite (HAP) with ordered rod-like and flower plate-like HAP crystals were deposited directly on titanium surface and MC3T3-E1 cells responds on them were investigated. Highlights • Rod-like and flake-like hydroxyapatite coatings were fabricated. • With different crystal orientation, surface roughness and wettability. • Rod-like one prefers cell spreading, proliferation and osteogenic differentiation. • Might cause topography-dependent coordination with biomolecules. Abstract How the surface topography of hydroxyapatite (HAP) coatings remodels hard tissue is of utmost importance and a contributing factor to ambiguity regarding this subject. Here, HAP coatings with different topographies of a rod-like nanoarray with c-axis orientation and a flake-like micro-flower array with a(b)-axis orientation on a Ti substrate were synthesized via hydrothermal-electrodeposition by controlling the concentration of electrolytes. XRD, TEM and SAED analyses indicated that the rod-like HAP nanoarray was predominant with an orientation of (001), while the HAP micro-flower samples were based on an orientation of (100). Compared to the flake-like HAP, the rod-like nanoarray HAP possessed better hydrophilic properties and lower roughness, which not only enhanced adsorption of specific fibronectin proteins but also promoted the spreading and growth of MC3T3-E1 cells. Runx2, alkaline phosphatase, collagen and osteocalcin were also analyzed by RT-PCR on the two distinctive HAP-coated samples. MC3T3-E1 cells on the rod-like nanoarray coating had higher osteo-related gene expression. This finding suggested that the ordered assembly structure of the HAP might cause topography-dependent coordination with biomolecules for enhancing osteoblast-like cell proliferation and osteogenic differentiation. This study provided an understanding of the surface's features for biomaterials to ensure better bioactivity. [ABSTRACT FROM AUTHOR]

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Shikonin, the predominant naphthoquinone pigment isolated from the Chinese plant Lithospermum erythrorhizon, is anti-inflammatory, antiviral and exerts anticancer effects, amongst other biological activities. However, it is unknown whether shikonin affects bone formation. In the present study, the role of shikonin on cell proliferation was assessed via MTT assay, and shikonin was identified to markedly promote cell growth in a time- and dose-dependent manner in the MC3T3-E1 cell line. In addition, flow cytometric analysis was performed to evaluate the effect of shikonin on the cell cycle, and it was observed that shikonin markedly increased the percentage of S-phase MC3T3-E1 cells to accelerate the G1/S transition. To investigate the potential molecular mechanism by which shikonin enhances bone formation, the changes in bone morphogenic protein-2 (BMP-2), SMAD family member 5 (Smad5), runt related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OC) expression levels induced by shikonin were investigated using western blot analysis and quantitative polymerase chain reaction. The results indicated that shikonin increased the BMP-2 and Smad5 mRNA levels, and upregulated Smad5 and Runx2 protein expression levels to promote osteoblast differentiation. Furthermore, ALP staining was performed, and revealed that shikonin enhanced ALP activity. These results indicate that shikonin promotes cell proliferation and differentiation of MC3T3-E1 cells via the BMP-2/Smad5 signaling pathway. [ABSTRACT FROM AUTHOR]

Distal-less genes (DLX) play important roles in regulating organism development. DLX2 is crucial for the differentiation and development of the primordium, which determines the subsequent development and phenotype of the maxillofacial skeletal patterns, and is the primary candidate gene that regulates the development of the first branchial arch. The aim of the present study was to investigate the effects of DLX2 overexpression on the osteogenic differentiation of MC3T3-E1 cells in vitro. A DLX2-expression retrovirus vector was constructed by subcloning with a murine stem cell virus (MSCV) and verified by sequencing. MC3T3-E1 cells were transfected with pMSCV-DLX2 and stable clones were selected with puromycin. The mRNA and protein expression levels of DLX2 were determined using quantitative polymerase chain reaction (PCR) and western blot analysis, respectively. In addition, the expression levels of the osteogenic biomarkers, alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor (RUNX)2 and Msh homeobox (MSX)2, were assessed by quantitative PCR. ALP detection and Alizarin red staining were conducted to evaluate the effect of DLX2 overexpression on osteogenic differentiation. The data were analyzed by analysis of variance using the Student-Newman-Keuls method. Successful pMSCV-DLX2 construction, as verified by direct sequencing, enabled DLX2 overexpression in vitro. Enhanced ALP activity and Alizarin red staining were observed in the MC3T3-E1-DLX2 cells when compared with the control group. During osteogenic induction, DLX2 overexpression was demonstrated to upregulate ALP and MSX2 expression at the early stage and OCN expression at the late stage, while no statistically significant difference was observed in RUNX2 expression when compared with the control group. Therefore, DLX2 overexpression in vitro induced the osteogenic differentiation of MC3T3-E1 cells via upregulating bone formation-associated genes, such as ALP and MSX2. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Velvet antlers (VA) have been claimed for centuries to have numerous medical benefits including strengthen bones. To investigate and compare the anti-osteoporotic activities from different sections of VA. Materials and methods: Fresh VA prepared from farmed sika deers (Cervus nippon) was divided into upper (VAU), middle (VAM), and basal (VAB) sections. The chemical constituents and anti-osteoporotic effect of different sections from VA were evaluated using ovariectomized rats. Results: Levels of water-soluble extracts, diluted alcoholic extract, amino acids, testosterone, insulin-like growth factor (IGF)-1and testosterone plus estradiol significantly differed among the different sections. Levels of these constituents were significantly higher in the upper section than in the basal section. Moreover, levels of testosterone and IGF-1 of the VAM were also significantly higher than those of the VAB. Calcium level increased downward from the tip with statistical significance. The strength of vertebrae increased in all VA-treated groups compared to the control, but only treatment with VAU and VAM increased the strength of the femur and the microarchitecure of the trabecular bone. Alkaline phosphatase levels of VAU- and VAM-treated groups significantly decreased, but osteocalcin did not significantly change. Moreover, VAU and VAM dose-dependently increased proliferation and mineralization of MC3T3-E1 cells. Conclusion: Our study provides strong evidence for the regional differences in the effectiveness of velvet antler in treating osteoporosis. However, further studies are needed to elucidate the bioactive chemical constituents associated with the anti-osteoporotic effects of velvet antler. [ABSTRACT FROM AUTHOR]

This study examined the effect of pinacidil, a nonselective adenosine triphosphate-sensitive potassium channel opener, on the function of osteoblastic MC3T3-E1 cells. Pinacidil caused a significant elevation of collagen synthesis, alkaline phosphatase activity, osteocalcin synthesis and mineralization in the cells ( p < 0.05). Pinacidil significantly decreased the production of osteoclast differentiation inducing factors such as TNF-α, IL-6 and receptor activator of nuclear factor-κB ligand in the presence of antimycin A, which inhibits mitochondrial electron transport. Moreover, pinacidil prevented antimycin A-induced reactive oxygen species and nitrotyrosine production. These results demonstrate that pinacidil may have positive effects on skeletal structure. [ABSTRACT FROM AUTHOR]

Abstract: As part of our search for biologically active anti-osteoporotic agents that enhance the differentiation and mineralization of osteoblastic MC3T3-E1 cells, we identified the ginsenoside Rd as the most active compound among ginsenosides. In this study, we showed that Rd stimulates osteoblastic differentiation and mineralization, manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and von Kossa/Alizarin Red staining, respectively. Rd induces the mRNA expression of bone morphogenetic protein-2 (BMP-2) and the secretion of the corresponding protein into media in a concentration-dependent manner. The mRNA expression and enzyme activity of alkaline phosphatase (ALP) were suppressed when MC3T3-E1 cells were exposed to noggin, a BMP-2 antagonist. The level of phosphorylated AMP-activated protein kinase (pAMPK) protein was also up-regulated by Rd in a time- and concentration-dependent manner. Rd-induced ALP activity, mineralization, and BMP-2 production were all inhibited by either Ara-A (AMPK inhibitor) or siRNA targeting AMPK. In addition, we investigated whether Rd-induced BMP-2 transduces signals through the Smad signaling pathways. Rd induced a significant level of phosphorylation of Smad1/5, and this effect was blocked when the cells were transfected with siRNA targeting Smad4, indicating that Smad1/5 must form complex with Smad4 to translocate into the nucleus and regulate the transcription of osteogenic genes. In summary, these results indicate that Rd induces the differentiation and mineralization of MC3T3-E1 cells through the activation of the AMPK/BMP-2/Smad signaling pathways. These findings provide a molecular basis for the osteogenic effect of Rd in MC3T3-E1 cells. [Copyright &y& Elsevier]

Abstract: Liquiritigenin is one of the flavonoids present in Glycyrrhizae radix. In the present study, the effects of liquiritigenin on the function of osteoblastic MC3T3-E1 cells were studied. Liquiritigenin caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, mineralization, and glutathione content in the cells (P <0.05). Moreover, liquiritigenin significantly decreased the production of reactive oxygen species (ROS) and osteoclast differentiation inducing factors such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and receptor activator of nuclear factor-κB ligand (RANKL) in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as a ROS generator. These results demonstrate that liquiritigenin may have positive effects on skeletal structure. [Copyright &y& Elsevier]

The incorporation of chitosan into electro-deposited calcium phosphate (CaP) coatings increases bone marrow stromal cell attachment. We hypothesized that such electrodeposited CaP/chitosan coatings can also enhance the proliferative ability and differentiation potential of osteoblasts. To verify this hypothesis, we cultured osteoblast-like MC3T3-E1 cells on these CaP coatings. It was found that MC3T3-E1 cells cultured on the electrodeposited CaP/chitosan coatings had cell proliferation rates higher than those on the electrodeposited CaP coatings. At the same time, both alkaline phosphatase activity and collagen expression were increased, and both bone sialoprotein and osteocalcin genes were upregulated when MC3T3-E1 cells were cultured on the electrodeposited CaP/chitosan coatings. Additionally, within the range of selected chitosan concentrations in solution, no significant difference was found between the CaP/chitosan coatings. Our results suggest that the electrodeposited CaP/chitosan coatings are favorable to the proliferation and differentiation of MC3T3-E1 cells, which may endow them with great potential for future applications. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the effect of polyphosphoric acid (PPA) pre-treatment of titanium (Ti) on the initial attachment, proliferation, and differentiation of mouse osteoblast-like cells (MC3T3-E1). Materials and methods: Adsorption of PPA to Ti was achieved by immersing Ti disks (15 mm in diameter) into 0, 1, and 10 wt% PPA and 10 wt% orthophosphoric acid (OPA) for 24 h. On each pre-treated Ti disk, 5.0 × 104 MC3T3-E1 cells were seeded, and 1, 3, and 5 h later cell attachment was evaluated. Cell proliferation was also determined 1, 3, and 5 days after cell seed. Cell differentiation was evaluated 5, 10, and 15 days after cell seed using osteoblast marker gene expression. Total RNA was purified from each culture and Type-I collagen, alkaline phosphatase, and osteocalcin mRNA expression levels were measured by real-time reverse transcription polymerase chain reaction. Results: Adsorption of PPA or OPA to Ti significantly accelerated initial cell attachment at every time point ( P<0.0001). As with cell attachment, cell proliferation was also accelerated on the PPA-treated Ti disks in a dose-dependent manner at every time point ( P<0.0001). However, OPA-treated Ti disks did not enhance the cell proliferation. Regarding osteoblastic differentiation, PPA-treated Ti significantly accelerated the Type-I collagen gene expression at 5 and 10 days after cell seed. Regarding alkaline phosphatase and osteocalcin gene expression, no significant difference was found among the different Ti surface conditions. Conclusion: The accelerated cell behavior following Ti pre-treatment with PPA is a promising new strategy to fabricate bioactive Ti implants. [ABSTRACT FROM AUTHOR]

Some practitioners of the Nishino Breathing Method (NBM) were found to have a higher bone density than the average values of age- and gender-matched non-practitioners. Using bone cell culture models, we investigated a possible mechanism behind this observation. For the study of bone mineralization, we performed the following two experiments using cultured osteoblastic MC3T3-E1 cells: (i) Kozo Nishino, a Japanese Ki expert, sent Ki-energy to the cells once for 5 or 10min after they were seeded in culture dishes in the presence of 10% fetal bovine serum (FBS). They were incubated for 72h and the cells were counted. The number in the dish with 10-min Ki-exposure was significantly greater than that in the control (P<0.01 with n=8). We performed a reverse transcription-polymerase chain reaction (RT PCR) study using these cells, but the mRNA expressions did not change significantly. (ii) After cells were incubated for 72 h without Ki-exposure (in the presence of FBS), they were further cultured for 48 h (in the absence of FBS) to promote differentiation. At the beginning of the second culture stage, Ki was applied once for 10 min. Alter 48 h, RT PCR was performed. The mRNA expressions which are related to bone mineralization, such as Runx2, α1(I) collagen, alkaline phosphatase and osteocalcin, increased significantly (P<0.05 and n=4 for all). For the bone resorption study, we used mouse marrow cultures, which can form osteoclast-like cells in the presence of (1 34) parathyroid hormone (PTH), and stimulate resorption. We exposed these cells to Ki-energy twice for the duration of 5 or 10min on day 0 and day 4. On day 7, the cells were counted. The number of osteoclast-like cells in dishes with Ki exposure was significantly smaller than those in control dishes (P<0.05 with n = 5). The difference between 5-min exposure and 10-min exposure was not statistically significant. All of our data suggest that the Ki-effect on osteoporosis should be further explored. [ABSTRACT FROM AUTHOR]

We investigated the effect of γ-irradiation on the activities of MC3T3-E1 cells cultured on γ-irradiated salmon atelocollagen (SAC) scaffolds. SAC-cultured samples were irradiated at doses of 10, 15, and 25 kGy. γ-Irradiation had a significant impact on the activities of MC3T3-E1 cells. The proliferation rates and alkaline phosphatase activities of MC3T3-E1 cells increased with γ-irradiation dose. [Copyright &y& Elsevier]

Sodium-dependent vitamin C transporter (SVCT) 2 facilitates reduced ascorbic acid (AA) transport in MC3T3-E1 osteoblasts. Our previous studies suggested that Zn-induced osteoblast differentiation and Ca2+-, PO43−-stimulated osteopontin (OPN) expression might result from their up-regulation effect on SVCT2 expression and AA uptake. Here, we investigated the role of SVCT2 on osteoblast differentiation by using SVCT2-overexpressing cells. Two clones of SVCT2-introduced cells overexpressed SVCT2 mRNA by 2.8- and 3.1-fold those of control cells, which resulted in obvious increase of AA uptake by 2.1- and 2.4-fold in Vmax with no change in Km. Alkaline phosphatase activity, hydroxyproline content significantly increased in SVCT2-overexpressing cells, and the induction of OPN mRNA was through up-regulation of OPN promoter activity by SVCT2 overexpression. Moreover, SVCT2-overexpressing cells exhibited more ability to promote mineralization and increase calcium deposition under the stimulation of 5 mM β-glycerophosphate. These findings indicate that SVCT2 stimulates osteoblast differentiation and mineralization. [Copyright &y& Elsevier]

Aluminum (Al) is a ubiquitous environmental metal toxicant that causes osteoblast (OB) damage which leads to Al-related bone diseases. Mitochondrial damage plays a key role in Al-related bone diseases, and while mitophagy can clear damaged mitochondria and improve OB function, the relationship between mitophagy and Al-induced OB dysfunction is unknown. To explore the role of mitophagy in Al-induced OB dysfunction in vitro , we used 2 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 0.4 μM Cyclosporin A (CsA) to activate and inhibit mitophagy, respectively. MC3T3-E1 cells were treated with 0 mM AlCl 3 (control group); 2 mM AlCl 3 (Al group); 2 μM CCCP (CCCP group); 2 μM CCCP and 2 mM AlCl 3 (CCCP + Al group); 0.4 μM CsA (CsA group); 0.4 μM CsA and 2 mM AlCl 3 (CsA + Al group). The results showed that Al induced ultrastructural and functional impairment of MC3T3-E1 cells. Compared to the Al group, mitophagy activation caused mitochondrial membrane potentials to collapse, up-regulated PINK1, Parkin, and LC3 expression, down-regulated p62 expression, and increased mitophagosome numbers. Mitophagy activation also reduced Al-induced oxidative stress and MC3T3-E1 cell functional damage, as seen in improvement in cell viability, cellular calcium and phosphorus contents, and collagen I, osteocalcin, and bone alkaline phosphatase gene expression. Mitophagy inhibition had the opposite effects on activation. Overall, these results show that mitophagy can protect against Al-induced OB dysfunction. [Display omitted] • Al caused dysfunction in MC3T3-E1 cells. • Levels of mitophagy were affected by CCCP and CsA. • Mitophagy could mitigate Al-induced ultrastructural damage and oxidative stress. • Mitophagy slowed Al-induced dysfunction in MC3T3-E1 cells. [ABSTRACT FROM AUTHOR]

Cells of the clonal osteogenic cell line MC3T3-E1 were seeded onto a three-dimensional matrix of denatured collagen type 1 and cultured for a period of up to 8 weeks. Specimens were analyzed by histological, enzyme histochemical, immunocytochemical, and ultrastructural methods and by in situ hybridization between day 7 and day 56 after seeding. In 56-day cultures, the MC3T3-E1 cells were arranged in a three-dimensional network and formation of bone-like tissue was indicated by calcification of a newly synthesized collagen type I matrix resembling osteoid and surrounding osteocyte-like cells. The differentiating culture showed high expression of osteocalcin and alkaline phosphatase activity. NIH3T3 fibroblasts used as control cells passed through the network of the substrate forming a confluent monolayer underneath. This culture system offers a potentially powerful model for bone formation in vitro and for investigating the osteogenic potential of bone-derived cells. [ABSTRACT FROM AUTHOR]

Nanodiamonds (NDs) have been used as drug delivery vehicles due to their low toxicity and biocompatibility. Recently, it has been reported that NDs have also osteogenic differentiation capacity. However, their capacity using NDs alone is not enough. To significantly improve their osteogenic activity, we developed icariin (ICA)-functionalized NDs (ICA-NDs) and evaluated whether ICA-NDs enhance their in vitro osteogenic capacity. Unmodified NDs and ICA-NDs showed nanosized particles that were spherical in shape. The ICA-NDs achieved a prolonged ICA release for up to 4 weeks. The osteogenic capacities of NDs, ICA (10 μg)-NDs, and ICA (50 μg)-NDs were demonstrated by alkaline phosphatase (ALP) activity; calcium content; and mRNA gene levels of osteogenic-related markers, including ALP, runt-related transcript factor 2 (RUNX2), collagen type I alpha 1 (COL1A1), and osteopontin (OPN). In vitro cell studies revealed that ICA (50 μg)-ND-treated MC3T3-E1 cells greatly increased osteogenic markers, including ALP, calcium content, and mRNA gene levels of osteogenic-related markers, including ALP, RUNX2, COL1A1, and OPN compared to ICA (10 μg)-NDs or ND-treated cells. These our data suggest that ICA-NDs can promote osteogenic capacity. [ABSTRACT FROM AUTHOR]

Bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, indicating that stimulation of osteoblast migration could be a promising osteoanabolic strategy. We showed that purified δ-tocotrienol (δ-TT, 10 μg/mL), isolated from commercial palm oil (Elaeis guineensis) fraction, stimulates the migration of both MC3T3-E1 osteoblast-like cells and primary human bone marrow mesenchymal stem cells (BMSC) as detected by wound healing assay or Boyden chamber assay respectively. The ability of δ-TT to promote MC3T3-E1 cells migration is dependent on Akt phosphorylation detected by Western blotting and involves Wnt/β-catenin signalling pathway activation. In fact, δ-TT increased β-catenin transcriptional activity, measured using a Nano luciferase assay and pretreatment with procaine (2 µM), an inhibitor of the Wnt/β-catenin signalling pathway, reducing the wound healing activity of δ-TT on MC3T3-E1 cells. Moreover, δ-TT treatment increased the expression of β-catenin specific target genes, such as Osteocalcin and Bone Morphogenetic Protein-2, involved in osteoblast differentiation and migration, and increased alkaline phosphatase and collagen content, osteoblast differentiation markers. The ability of δ-TT to enhance the recruitment of BMSC, and to promote MC3T3-E1 differentiation and migratory behavior, indicates that δ-TT could be considered a promising natural anabolic compound. [ABSTRACT FROM AUTHOR]