Preosteoblast Cell Line MC3T3-E1 Research Articles

ETS Proto-Oncogene 1 Suppresses MicroRNA-128 Transcription to Promote Osteogenic Differentiation Through the HOXA13/β-Catenin Axis

ETS proto-oncogene 1 (ETS1) has been implicated in osteoporosis (OP), but the exact molecular mechanisms are complex. This work focuses on the impact of ETS1 on the osteogenic differentiation and the molecules involved. A mouse pre-osteoblast cell line MC3T3-E1 was used for in vitro experiments. ETS1 was upregulated during the process of osteogenic differentiation of MC3T3-E1 cells. Overexpression of ETS1 promoted expression of osteogenic markers, alkaline phosphate concentration, and calcareous accumulation in cells. ETS1 was found to specifically bind to miR-128 promoter to suppress its transcription, while miR-128 could target homeobox A13 (HOXA13). Therefore, ETS1 suppressed miR-128 transcription to upregulate HOXA13 expression. Overexpression of HOXA13 promoted the osteogenic differentiation ability of cells and increased the protein level of β-catenin. Either overexpression of miR-128 or downregulation of β-catenin by CWP232228, a β-catenin-specific antagonist, blocked the promoting roles of ETS1 in cells. To conclude, this study provided evidence that ETS1 suppresses miR-128 transcription to activate the following HOXA13/β-catenin axis, therefore promoting osteogenic differentiation ability of MC3T3-E1 cells. This finding may offer novel ideas for OP treatment.

Homeopathic Rhus toxicodendron Induces Cell Adhesions in the Mouse Pre-osteoblast Cell Line MC3T3-e1.

Rhus toxicodendron (R. tox) has been used as a homeopathic remedy for the treatment of inflammatory conditions. Previously, we reported that R. tox modulated inflammation in the mouse chondrocyte and pre-osteoblastic MC3T3-e1 cell line. During the inflammatory process, cells adhere to the extracellular matrix (ECM) and then migrate to the inflammation site. We examine here the process of cell adhesion in MC3T3-e1 cells after their stimulation with homeopathic R. tox. For the cell-substrate adhesion assay, the cultured MC3T3-e1 cells were trypsinized, starved for 1 h in serum-free media, and plated onto culture plates coated with fibronectin (FN), 30c R. tox or gelatin, respectively. The cells were allowed to adhere for 20 min incubation and unattached cells were washed out. Adherent cells were measured using the water-soluble tetrazolium salt-8 assay. The intracellular signals after stimulation of R. tox were examined by analyzing the tyrosine phosphorylation of focal adhesion kinase (FAK), Src kinase, and Paxillin using immunoblot assay. Formation of focal adhesion (FA, an integrin-containing multi-protein structure that forms between intracellular actin bundles and the ECM) was analyzed by immunocytochemistry using NIH ImageJ software. Cell adhesion increased after stimulation with R. tox (FN, 20.50%; R. tox, 44.80%; and gelatin, 17.11% vs. uncoated cells [control]). Tyrosine phosphorylation of FAK, Paxillin, and Src increased compared with that of gelatin when stimulated with R. tox. Additionally, R. tox-stimulated cells formed many FAs (number of FAs per cell, 35.82 ± 7.68) compared with gelatin-stimulated cells (number of FAs per cell, 19.80 ± 7.18) and exhibited extensive formation of actin stress fibers anchored by FAs formed at the cell periphery. Homeopathic R. tox promotes the formation of cell adhesions in vitro.

Impact of photobiomodulation using four diode laser wavelengths of on cationic liposome gene transfection into pre-osteoblast cells

Gene therapy can be an effective treatment modality for some severe genetic diseases. Despite efforts to improve their performance, non-viral gene delivery methods remain inefficient and costly. As an alternative to viral vectors, cationic liposomes have a good safety profile and low immunogenicity, but relatively low transfection efficiency. They may also be toxic to cells at high concentrations. Given these challenges, the present study explored the impact of photobiomodulation (PBM) on cationic liposome plasmid DNA transfection in terms of its efficiency and toxicity, using Lipofectamine 2000 to carry green fluorescent protein (GFP) encoding plasmid DNA, with the pre-osteoblast MC3T3-E1 cell line as the target. Cultures were irradiated using diode lasers (445, 685, 810, or 970 nm) at 200 mW using pulsed mode (50 Hz), with a power density of 104.64 mW/cm2, and irradiance from 6 to 18 joules. To determine transfection efficiency, expression of GFP was assessed using confocal laser scanning microscopy and flow cytometry. Cell viability was evaluated using the MTT assay. PBM using 810 nm and 970 nm lasers significantly enhanced transfection efficiency for GFP, indicating more efficient uptake of plasmid DNA. Conversely, laser irradiation at 445 nm and 685 nm wavelengths reduced the GFP transfection efficiency. Treatment using 685, 810, and 970 nm lasers at 12 J maintained cell viability and prevented toxicity of cationic liposomes. Overall, these findings support the concept that PBM using near infrared laser wavelengths can enhance transfection efficiency and support cell viability when cationic liposomes are used as the vector in gene therapy.

Liraglutide regulates proliferation, differentiation, and apoptosis of preosteoblasts through a signaling network of Notch/Wnt/Hedgehog signaling pathways.

This study aims to investigate whether liraglutide can affect proliferation, osteogenic differentiation and serum deprivation-induced apoptosis of preosteoblast cell line MC3T3-E1 through the Notch, Wnt/β-catenin, and Hedgehog (Hh) signaling pathways. MC3T3-E1 cells were exposed to different treatments (via Notch inhibitor DAPT, an Hh inhibitor cyclopamine, or serum deprivation) or transfections of different siRNAs (targeting glucagon-like peptide-1 receptor (GLP-1R), β-catenin, or Gli1) in the presence or absence of 100 nM liraglutide. Cell proliferation, mRNA levels of osteogenic differentiation-related genes, mRNA and protein levels of the Notch and Hh signaling pathway proteins, and apoptosis-related proteins were assessed. Liraglutide significantly increased proliferation of MC3T3-E1 cells, expression levels of the Notch and Hh signaling pathway proteins and β-catenin, and mRNA levels of osteogenic differentiation-related genes and TCF7L2. Moreover, liraglutide promoted a translocation of β-catenin, increased a ratio of Bcl-2/Bax proteins, reduced serum deprivation-induced apoptosis of MC3T3-E1 cells, and a ratio of caspase-3/procaspase-3. However, a cotreatment with liraglutide and DAPT reversed the alterations. A cyclopamine treatment and knockdowns of GLP-1R, Gli1, and β-catenin significantly reduced the expression of Notch proteins. Furthermore, the knockdown of GLP-1R, β-catenin, or Gli1 significantly increased apoptosis, which could be inhibited by liraglutide. In summary, liraglutide can promote proliferation and differentiation of MC3T3-E1 cells, and inhibit their serum deprivation-induced apoptosis by activating both the Notch and Hh signaling pathways involving β-catenin and Gli1. These results provide a therapeutic foundation that patients with diabetes and osteoporosis may be cured with treatments of liraglutide.

Titanium Dioxide Thin Films Obtained by Atomic Layer Deposition Promotes Osteoblasts' Viability and Differentiation Potential While Inhibiting Osteoclast Activity-Potential Application for Osteoporotic Bone Regeneration.

Atomic layer deposition (ALD) technology has started to attract attention as an efficient method for obtaining bioactive, ultrathin oxide coatings. In this study, using ALD, we have created titanium dioxide (TiO2) layers. The coatings were characterised in terms of physicochemical and biological properties. The chemical composition of coatings, as well as thickness, roughness, wettability, was determined using XPS, XRD, XRR. Cytocompatibillity of ALD TiO2 coatings was accessed applying model of mouse pre-osteoblast cell line MC3T3-E1. The accumulation of transcripts essential for bone metabolism (both mRNA and miRNA) was determined using RT-qPCR. Obtained ALD TiO2 coatings were characterised as amorphous and homogeneous. Cytocompatibility of the layers was expressed by proper morphology and growth pattern of the osteoblasts, as well as their increased viability, proliferative and metabolic activity. Simultaneously, we observed decreased activity of osteoclasts. Obtained coatings promoted expression of Opn, Coll-1, miR-17 and miR-21 in MC3T3-E1 cells. The results are promising in terms of the potential application of TiO2 coatings obtained by ALD in the field of orthopaedics, especially in terms of metabolic- and age-related bone diseases, including osteoporosis.

Injectable hydrogels based on gellan gum promotes in situ mineralization and potential osteogenesis

Bone defects are usually caused by trauma, infection, tumor resection or congenital diseases, and they attract many researchers’ attention to fabricate biomaterials to promote bone regeneration. In this report, we prepared double-network hydrogel by one-step mixing method, in which anion polysaccharide gellan gum (GG) was used as the first network and polymerizable monomer in the presence of crosslinking agent was acted as the second network. Among then, a synthesized monomer named as 2-methacrylamidoethyl dihydrogen phosphate (MDP) with phosphate group was introduced in the second network to promote in situ mineralization. The addition of the second network can significantly increase the mechanical properties of the hydrogel, including both the compressive modulus and ultimate stress of hydrogels increase compared to uncrosslinked groups. To certify the potential application of double-network hydrogel to act as scaffold in bone regeneration, the double-network hydrogels was incubated in simulated body fluid (SBF) for 2 weeks to explore the in situ mineralization capacity of hydrogels. The hydroxyapatite can be observed on the surface of hydrogels, as confirmed by the scanning electron microscope (SEM), energy dispersive spectroscopy (EDS) test and X-ray diffraction test (XRD). In addition, the results of thermal gravimetric analysis (TGA) and colorimetric test indicate that as the concentration of monomer MDP increased, both the quantity and the rate of the generation of hydroxyapatite on the surface of hydrogels increased. The cytotoxicity of hydrogels was also tested by preosteoblast MC3T3-E1 cell line. Therefore, the double-network hydrogels can act as a potential scaffold material in bone regeneration.

PEGylated gold nanoparticles promote osteogenic differentiation in in vitro and in vivo systems

Gold nanoparticles (GNPs) have attracted considerable attention in promoting osteogenic differentiation. However, little is known about their osteogenic effects on cells from various origins, and further in vivo investigation as a promising osteogenic agent upon hydrogel-conjugation is required. Herein, the effects of PEGylated GNPs with the sizes of 4, 18 and 45 nm on osteogenic differentiation in the pre-osteoblastic MC3T3-E1 cell line, human bone marrow-derived mesenchymal stem cells (hBMSCs) and rat bone marrow-derived mesenchymal stem cells (rBMSCs) were comprehensively evaluated. The results showed that GNPs with all of three different sizes had excellent biocompatibility, and their effects on induction of osteogenic differentiation of various cell types were dependent on size. Specifically, 45-nm GNPs showed more pronounced alkaline phosphatase (ALP) activity, mineralized nodule formation and expression of osteogenic marker genes compared with the 18-nm group, while 4-nm GNPs reduced the above osteogenic effects. Meanwhile, β-catenin and p-GSK-3β were upregulated by GNPs-45 nm and GNPs-18 nm, but suppressed by GNPs-4 nm, indicating that Wnt/β-catenin signaling pathway mighy play an important role in GNPs-induce osteogenic differentiation. In addition, specific blocking of Wnt/β-catenin signals by KYA1797K inhibited 45 nm GNPs-induced osteogenic differentiation of MC3T3-E1 cells. Furthermore, 45-nm GNPs conjugated with PEG-hydrogel were chosen for in vivo bone regeneration testing in rabbits with 4-mm femur defects. The results demonstrated that the GNPs stimulated bone regeneration rapidly and effectively. Such comprehensive evaluation of GNPs' effects on in vitro osteogenic differentiation and in vivo bone regeneration could promote their applications in clinical translation.

Synthesis and potential of skipjack tuna bone hydroxyapatite as bone tissue engineering biomaterial

Hydroxyapatite Caio(P04)e(OH)2 is an alloplast material that is used to increase bone regeneration. It can be synthesis by processing natural materials such as fish bones. The purpose of this study was to synthesize hydroxyapatite from natural resources skipjack tuna (Katsuwonus pelamis) bones with precipitation method. Then, characterize hydroxyapatite morfology with FESEM and its biocompatibility using the preosteoblast MC3T3-E1 cell line. Skipjack tuna bone was synthesized into hydroxyapatite through precipitation method. The morphology of hydroxyapatite sample was revealed with field-emission scanning microscope FESEM. While the constituent elements were analyzed using SEM EDAX. Biocompatibility of hydroxyapatite was tested using preosteoblast cell culture. Cells were treated with different hydroxyapatite concentration 200 μglml, 100 μglml and 5 μg/ml. After incubation with C02 5% at 37°C for 24h,48h and 72h the cultore was tested for viability using MTT Cell Viability Assay Kit. Results were reported as optical density. The study showed that skipjack tuna bone produced grain-shaped particles with almost uniform sizes. The surface material appears to be agglomerates and form pores in between. Weight percentages Ca/P ratio for hydroxyapatite from skipjack tuna bones is 1.94. MTT assay showed cell viability after 3 days. These results suggest that skipjack tuna bone hydroxyapatite is has potential as bone engineering biomaterial

Signal transducer and activator of transcription 3 positively regulates osteoblastic differentiation in MC3T3-E1 cells.

Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in osteoblastic differentiation. However, the exact role of STAT3 in osteogenic differentiation of the pre-osteoblastic cell line MC3T3-E1 is still controversial. In this study, we demonstrated that eradication of STAT3 signaling by the inhibitors cryptotanshinone (CPT, a STAT3-specific inhibitor) or STAT3 siRNA both suppressed osteogenic differentiation of MC3T3-E1 cells, with a decrease in alkaline phosphatase (ALP) activity, protein expressions of the osteogenic differentiation markers Collagen I (ColI), ALP, and osteocalcin (OCN), and reduced matrix mineralization capacity at the terminal stage of osteogenic differentiation. However, the inhibition of STAT3 by CPT did not affect MC3T3-E1 cell proliferation. To further clarify the effect of STAT3 on osteogenic differentiation of MC3T3-E1 cells, we forced STAT3 expression and found that this ameliorated osteogenic differentiation. Thus, our results confirmed that STAT3 is a likely positive regulator of osteogenic differentiation in MC3T3-E1 cells. These findings may provide a basis for the development of more efficient and controllable protocols for osteoblastic differentiation and facilitate their use in regenerative medicine. In addition, our results provide novel insights into the effect of the STAT3 antagonist CPT on modulation of osteogenesis.

MicroRNA-142 regulates osteoblast differentiation and apoptosis of mouse pre-osteoblast cells by targeting bone morphogenetic protein 2.

Osteoporosis is a common disease that can seriously impair the physical and mental health of the elderly. However, current treatment is unsatisfactory due to the lack of effective therapeutic targets. Abnormal expression and involvement of microRNA-142 (miR-142) have been identified in many diseases, including bone-related diseases. Herein, we explored the effect of miR-142 on the viability, differentiation and apoptosis of the mouse preosteoblast cell line MC3T3-E1. We observed that the viability of MC3T3-E1 cells was significantly inhibited or promoted after transfection of miR-142 mimic or inhibitor, respectively. The apoptotic rate was dramatically increased by miR-142 mimic and decreased by inhibitor compared with the negative control group. Bcl-2 expression was down-regulated in the miR-142 mimic group and up-regulated in the miR-142 inhibitor group, whereas levels of cleaved caspase-3 and Bax were increased in the miR-142 mimic group and reduced in the miR-142 inhibitor group. Expression changes of Runx2 and Osteocalcin suggest that miR-142 inhibits the differentiation of osteoblast cells. Moreover, the luciferase reporter assay was used to verify that bone morphogenetic protein 2 (BMP2) is a target of miR-142. Overexpression of BMP2 repressed the proapoptotic effect of miR-142 mimic, whereas knockdown of BMP2 abolished the inhibitory effect of miR-142 inhibitor on the apoptosis of MC3T3-E1 cells. Furthermore, up-regulation or down-regulation of miR-142 dramatically decreased or increased the ratio of p-Smad1/5/Smad1 and p-Smad1/5/Smad5, respectively. Collectively, our results imply that miR-142 might influence the viability and differentiation of osteoblast cells by regulating BMP2 and BMP/Smad signaling.

Bioprinting of Alginate-Encapsulated Pre-osteoblasts in PLGA/β-TCP Scaffolds Enhances Cell Retention but Impairs Osteogenic Differentiation Compared to Cell Seeding after 3D-Printing

Background: In tissue engineering, cellularization of scaffolds has typically been performed by seeding the cells after scaffold fabrication. 3D-printing technology now allows bioprinting of cells encapsulated in a hydrogel simultaneously with the scaffold material. Here, we aimed to investigate whether bioprinting or cell seeding post-printing is more effective in enhancing responses of pre-osteoblastic MC3T3-E1 cell line derived from mouse calvaria. Methods: Two methods were used for incorporation of pre-osteoblasts in poly(lactic-co-glycolic acid)/β-tricalcium phosphate (PLGA/β-TCP) scaffolds: first, seeding the cells after scaffold printing, and second, bioprinting of alginate-encapsulated cells between PLGA/β-TCP struts. AlamarBlue® assay was used to determine cell retention and proliferation. Live/dead staining and picrosirius red staining were used to assess cell viability and collagen deposition, respectively. Alkaline phosphatase activity was measured to assess the osteoblastic phenotype of pre-osteoblasts. Results: Cell retention was 51 ± 5% in cell-seeded and 87 ± 3% in bioprinted scaffolds, whereas cell viabilities were 87 ± 2% and 78 ± 4%, respectively. Cell proliferation was higher on cell-seeded scaffolds (day 7: 2.5-fold, day 14: 4.0-fold, day 21: 4.7-fold) compared with bioprinted scaffolds (1.2-fold, 2.1-fold, and 3.9-fold, respectively). At day 21, the voids between PLGA/β-TCP struts were covered with cell-deposited collagenous matrix in both scaffold types. Alkaline phosphatase activity was higher (3.4-fold) on cell-seeded scaffolds compared with bioprinted scaffolds. Conclusion: Our data demonstrate that encapsulation of pre-osteoblasts in alginate and printing inside PLGA/β-TCP scaffolds enhances cell retention but decreases cell proliferation and osteogenic differentiation compared to seeding cells on scaffolds post-printing. This might have important implications for bone tissue engineering. Lay Summary: Bioprinting, i.e., printing of hydrogel-encapsulated cells alone or in combination with scaffold material, presents an attractive tool for fabrication of customized tissue equivalents as opposed to conventional cell seeding on tissue engineering scaffolds. However, the function of the printed cells might be adversely affected by the hydrogel encapsulation and printing process. Our aim was to investigate whether bioprinting of alginate-encapsulated pre-osteoblasts in poly(lactic-co-glycolic) acid/β-tricalcium phosphate scaffold is more effective in enhancing cell retention, proliferation, and osteogenic differentiation as opposed to cell seeding post-printing. Our studies showed that the bioprinted scaffold had enhanced cell retention capacity, but impaired cell proliferation and osteogenic differentiation compared to the cell-seeded scaffold. These findings might have important implications for the improvement of alginate-based bioinks with, e.g., (natural) bioactive peptides for bioprinting of bone tissue engineering scaffolds. [Figure not available: see fulltext.].

Enhancement of local bone formation on titanium implants in osteoporotic rats by biomimetic multilayered structures containing parathyroid hormone (PTH)-related protein

Osteoporosis is a severe health problem causing bone fragility and consequent fracture. Titanium (Ti) implants, used in patients with osteoporotic fractures, are prone to failure because of the decreased bone mass and strength. Therefore, it is of utmost importance to fabricate implants possessing osteogenic properties to improve implant osseointegration. To improve the long-term survival rate of Ti implants in osteoporotic patients, hyaluronic acid/ϵ-polylysine multilayers containing the parathyroid hormone (PTH)-related protein (PTHrP) were deposited on Ti implants by a layer-by-layer (LBL) electro assembly technique. The murine pre-osteoblast cell line MC3T3-E1, possessing a high potential of osteoblast differentiation, was used to evaluate the osteo-inductive effects of Ti-LBL-PTHrP in vitro. In addition, the performance of the Ti (Ti-LBL-PTHrP) implant was evaluated in vivo in a femoral intramedullary implantation in Sprague Dawley rats. The Ti-LBL-PTHrP implant regulated the release of the loaded PTHrP to increase bone formation in the early stage of implantation. The in vitro results revealed that cells on Ti-LBL-PTHrP did not show any evident proliferation, but a high level of alkaline phosphatase activity and osteoblast-related protein expression was found, compared to the uncoated Ti group (p < 0.05). In addition, in vivo micro-CT and histological analysis demonstrated that the Ti-LBL-PTHrP implants could significantly promote the formation and remodeling of new bone in osteoporotic rats at 14 d after implantation. Overall, this study established a profound and straightforward methodology for the manufacture of biofunctional Ti implants for the treatment of osteoporosis.

Carbohydrate responsive element binding protein (ChREBP) negatively regulates osteoblast differentiation via protein phosphatase 2A Cα dependent manner.

Carbohydrate responsive element binding protein (ChREBP) is a major transcription factor of lipogenesis regulated by glucose status in the liver. However, the function of ChREBP in osteogenic differentiation is unclear. The present study examined the role of ChREBP in osteoblast differentiation in MC3T3-E1 preosteoblast cell line. The mRNA expression of ChREBP, protein phosphatase 2A catalytic subunit-α (PP2A Cα) and the osteogenic genes such as, DNA-binding protein inhibitor (Id1), runt-related transcription factor-2 (Runx2), and alkaline phosphatase (ALP) was measured by qPCR and RT-PCR. Runx2, ChREBP, and PP2A Cα, protein levels were evaluated by Western blotting. ALP staining experiment was carried out to evaluate ALP enzyme activity, and a luciferase reporter assay was performed to analyze Runx2 transcriptional activity. Expression of ChREBP and PP2A Cα did not change during bone morphogenetic protein-2 (BMP2)-induced osteoblast differentiation. Overexpression of ChREBP reduced the osteogenic genes (Runx2 and ALP) expression and ALP activity, while knockdown of ChREBP had the opposite effects. Overexpression of PP2A Cα increased ChREBP expression, while inhibition of PP2A Cα using okadaic acid not only inhibited the expression of ChREBP, but also restored the mRNA and protein expression of Runx2 and activity of ALP enzyme. These results demonstrate that ChREBP inhibits BMP2-induced osteoblast differentiation in a PP2A Cα- dependent manner.

Preventive Effects of Chrysanthemum coronarium L. Extract on Bone Metabolism In Vitro and In Vivo.

Osteoporosis is characterized by decreased bone mass and bone microarchitectural failure, leading to an enhanced risk of bone fractures. Chrysanthemum coronarium L. (CC) is a natural plant with powerful antioxidant activity. This study investigated the antiosteoporotic effects of CC extracts in in vitro cell cultures and in vivo bone loss animal models. CC stimulated osteoblast differentiation and mineralized bone formation by osteoblasts by increasing the expression of bone formation markers (alkaline phosphatase, osteoprotegerin, and osteoprotegerin/receptor activator nuclear factor-κB ligand ratio) in the murine preosteoblastic cell line MC3T3-E1. Additionally, CC was found to inhibit osteoclast differentiation by downregulating bone resorption markers (tartrate-resistant acid phosphatase, cathepsin K, dendritic cell-specific transmembrane protein, and calcitonin receptor) in the murine macrophage-like cell line RAW264.7. CC prevented ovariectomy-induced bone loss, preserved trabecular microarchitecture, and improved serum bone turnover markers in an osteoporotic mouse model. These findings suggest that CC extract may be considered as a natural therapeutic or preventive agent for osteoporotic bone loss.

Identification of a novel melatonin-binding nuclear receptor: Vitamin D receptor.

Previous studies confirmed that melatonin regulates Runx2 expression but the mechanism is unclear. There is a direct interaction between Runx2 and the vitamin D receptor (VDR). Herein, we observed a direct interaction between melatonin and the VDR but not Runx2 using isothermal titration calorimetry. Furthermore, this direct binding was detected only in the C-terminal ligand binding domain (LBD) of the VDR but not in the N-terminal DNA-binding domain (DBD) or the hinge region. Spectrophotometry indicated that melatonin and vitamin D3 (VD3) had similar uptake rates, but melatonin's uptake was significantly inhibited by VD3 until the concentration of melatonin was obviously higher than that of VD3 in a preosteoblastic cell line MC3T3-E1. GST pull-down and yeast two-hybrid assay showed that the interactive smallest fragments were on the 319-379 position of Runx2 and the N-terminus 110-amino acid DBD of the VDR. Electrophoretic mobility shift assay (EMSA) demonstrated that Runx2 facilitated the affinity between the VDR and its specific DNA substrate, which was further documented by a fluorescent EMSA assay where Cy3 labeled Runx2 co-localized with the VDR-DNA complex. Another fluorescent EMSA assay confirmed that the binding of the VDR to Runx2 was significantly enhanced with an increasing concentrations of the VDR, especially in the presence of melatonin; it was further documented using a co-immunoprecipitation assay that this direct interaction was markedly enhanced by melatonin treatment in the MC3T3-E1 cells. Thus, the VDR is a novel melatonin-binding nuclear receptor, and melatonin indirectly regulates Runx2 when it directly binds to the LBD and the DBD of the VDR, respectively.

Phlorofucofuroeckol A from Edible Brown Alga Ecklonia Cava Enhances Osteoblastogenesis in Bone Marrow-Derived Human Mesenchymal Stem Cells.

The deterioration of bone formation is a leading cause of age-related bone disorders. Lack of bone formation is induced by decreased osteoblastogenesis. In this study, osteoblastogenesis promoting effects of algal phlorotannin, phlorofucofuroeckol A (PFF-A), were evaluated. PFF-A was isolated from brown alga Ecklonia cava. The ability of PFF-A to enhance osteoblast differentiation was observed in murine pre-osteoblast cell line MC3T3-E1 and human bone marrow-derived mesenchymal stem cells (huBM-MSCs). Proliferation and alkaline phosphatase (ALP) activity of osteoblasts during differentiation was assayed following PFF-A treatment along extracellular mineralization. In addition, effect of PFF-A on osteoblast maturation pathways such as Runx2 and Smads was analyzed. Treatment of PFF-A was able to enhance the proliferation of differentiating osteoblasts. Also, ALP activity was observed to be increased. Osteoblasts showed increased extracellular mineralization, observed by Alizarin Red staining, following PFF-A treatment. In addition, expression levels of critical proteins in osteoblastogenesis such as ALP, bone morphogenetic protein-2 (BMP-2), osteocalcin and β-catenin were stimulated after the introduction of PFF-A. In conclusion, PFF-A was suggested to be a potential natural product with osteoblastogenesis enhancing effects which can be utilized against bone-remodeling imbalances and osteoporosis-related complications.

Perlecan domain I gradients establish stable biomimetic heparin binding growth factor gradients for cell migration in hydrogels

Growth factor gradients orchestrate many biological processes including organogenesis, wound healing, cancer invasion, and metastasis. Heparin-binding growth factor (HBGF) gradients are established in living systems by proteoglycans including the extracellular matrix heparan sulfate proteoglycan, perlecan/HSPG2. Three potential HBGF-binding glycosaminoglycan attachment sites occur in N-terminal domain I of perlecan’s five domains. Our overarching goal was to form stable, biomimetic non-covalently bound HBGF gradients surrounding cells encapsulated in hyaluronate-based hydrogels by first establishing perlecan domain I (PlnD1) gradients. A versatile multichannel gradient maker device (MGMD) was designed and 3D printed, then used to create desired gradients of microparticles in hydrogels. Next, we used the device to covalently incorporate gradients of PEGylated PlnD1 in hydrogels with high-low-high or high-medium-low concentrations across the hydrogel width. Fluorescently-labeled fibroblast growth factor-2 was delivered to hydrogels in phosphate-buffered saline and allowed to electrostatically bind to the covalently pre-incorporated PlnD1, producing stable non-covalent HBGF gradients. To test cell viability after flow through the MGMD, delicate primary human salivary stem/progenitor cells were encapsulated in gradient hydrogels where they showed high viability and continued to grow. Next, to test migratory behavior in response to HBGF gradients, two cell types, preosteoblastic MC3T3-E1 cell line and breast cancer cell line MDA-MB-231 were encapsulated in or adjacent to PlnD1-modified hydrogels. Both cell lines migrated toward HBGFs bound to PlnD1. We conclude that establishing covalently-bound PlnD1 gradients in hydrogels provides a new means to establish physiologically-relevant gradients of HBGFs that are useful for a variety of applications in tissue engineering and cancer biology. Statement of SignificanceGradients of heparin binding growth factors (HBGFs) direct cell behavior in living systems. HBGFs bind electrostatically to gradients of HS proteoglycans in the extracellular matrix creating HBGF gradients. We recreated HBGF gradients in physiological hyaluronate-based hydrogels using a 3D-printed multichannel gradient maker device (MGMD) that created gradients of HS proteoglycan-derived perlecan/HSPG2 domain I. We demonstrated the ability of a variety of cells, including primary salivary stem/progenitor cells, pre-osteoblastic cells and an invasive breast cancer cell line, to be co-encapsulated in gradient hydrogels by flowing them together through the MGMD. The versatile device and the ability to create HBGF gradients in hydrogels for a variety of applications is innovative and of broad utility in both cancer biology and tissue engineering applications.

Osteogenesis activity of isocoumarin a through the activation of the PI3K-Akt/Erk cascade-activated BMP/RUNX2 signaling pathway

Bone formation refers to a series of complex events related to the activities of osteoblasts. In this study, we evaluated the osteogenesis activity of a natural compound named isocoumarin A that was isolated from the rhizomes of Polygonum amplexicaule on the non-transformed preosteoblastic cell line MC3T3-E1 for an in vitro study, and the results revealed that it increased the proliferation and promoted the mineralization of the extracellular matrix of MC3T3-E1 cells after treatment for 3 d in a dose-dependent manner. The cell metabolic activity peaked at 169% at 10 μM, and the activity of alkaline phosphatase (ALP) tripled to 15.94 U/mg compared with the control group. The protein levels of morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX2), ALP, and the mRNA levels of ALP, type I collagen (COL-1), and osteocalcin (OCN) were also upregulated after isocoumarin A administration. The mechanism investigation revealed that these effects were associated with the activation of the p-Akt/p-Erk1/2-activated BMP/RUNX2 signaling pathway. Subsequently, the in vivo investigation on the zebrafish embryos model demonstrated that isocoumarin A (0.30 mM) increased the number of vertebrae (5.38 ± 2.07 pcs) and the vertebral area (433.25 ± 111.77 μm2) in the development process of zebrafish embryos after a 7-day postfertilization (dpf) culture compared with the control group (2.50 ± 1.16 pcs and 209.75 ± 86.40 μm2). Together, these results indicated that isocoumarin A could be viewed as a promising candidate in early drug discovery and development to promote the healing of fractures and postmenopausal osteoporosis.

Adenosine N1-Oxide Exerts Anti-inflammatory Effects through the PI3K/Akt/GSK-3β Signaling Pathway and Promotes Osteogenic and Adipocyte Differentiation.

Previously, we reported that adenosine N1-oxide (ANO), which is found in royal jelly, inhibited the secretion of inflammatory mediators by activated macrophages and reduced lethality in lipopolysaccharide (LPS)-induced endotoxin shock. Here, we examined the regulatory mechanisms of ANO on the release of pro-inflammatory cytokines, with a focus on the signaling pathways activated by toll-like receptor (TLR)4 in response to LPS. ANO inhibited both tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion from LPS-stimulated RAW264.7 cells without affecting cell proliferation. In this response, phosphorylation of mitogen-activated protein kinase (MAPK) family members (extracellular signal-regulated kinase (ERK)1/2, p38 and SAPK/c-Jun N-terminal kinase (JNK)) and nuclear factor-κB (NF-κB) p65 was not affected by treatment with ANO. In contrast, phosphorylation of Akt (Ser473) and its downstream molecule glycogen synthase kinase-3β (GSK-3β) (Ser9) was up-regulated by ANO, suggesting that ANO stimulated GSK-3β phosphorylation via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The phosphorylation of GSK-3β on Ser9 has been shown to negatively regulate the LPS-induced inflammatory response. Activation of PI3K/Akt signaling pathway has also been implicated in differentiation of mesenchymal stem cells into osteoblasts and adipocytes. As expected, ANO induced alkaline phosphatase activity and promoted calcium deposition in a mouse pre-osteoblastic MC3T3-E1 cell line. The ANO-induced differentiation into osteoblasts was abrogated by coincubation with Wortmannin. Furthermore, ANO promoted insulin/dexamethasone-induced differentiation of mouse 3T3-L1 preadipocytes into adipocytes at much lower concentrations than adenosine. The protective roles of PI3K/Akt/GSK-3β signaling pathway in inflammatory disorders have been well documented. Our data suggest that ANO may serve as a potential candidate for the treatment of inflammatory disorders. Promotion of osteogenic and adipocyte differentiation further suggests its application for regenerative medicine.

Osteogenic potential of hexane and dichloromethane fraction of Cissus quadrangularis on murine preosteoblast cell line MC3T3-E1 (subclone 4).

In continuation of the investigation of osteogenic potential of solvent fractions of ethanolic extract of Cissus quadrangularis (CQ), an ancient medicinal plant, most notably known for its bone-healing properties, to isolate and identify antiosteoporotic compounds. In the current study, we report the effect of hexane fraction (CQ-H) and dichloromethane fraction (CQ-D) of CQ on the differentiation and mineralization of mouse preosteoblast cell line MC3T3-E1 (subclone 4). Growth, viability, and proliferation assays revealed that low concentrations (0.1, 1, and 100 ng/ml) of both solvent fractions were nontoxic, whereas higher concentrations were toxic to the cells. Differentiation and mineralization of MC3T3-E1 with nontoxic concentrations of CQ-D and CQ-H revealed that CQ-D delayed the mineralization of MC3T3-E1 cells. However, early and enhanced mineralization was observed in cultures treated with nontoxic concentrations of CQ-H, as indicated by Von Kossa staining and expression profile of osteoblast marker genes such as osterix, Runx2, alkaline phosphatase (ALP), collagen (Col1a1), integrin-related bone sialoprotein (IBSP), osteopontin (OPN), and osteocalcin (OCN). These findings suggest CQ-H as the most efficacious solvent fraction for further investigation to isolate and identify the active compounds in CQ-H.