Mouse Pre-osteoblast MC3T3-E1 Research Articles

An axial distribution of seeding, proliferation, and osteogenic differentiation of MC3T3-E1 cells and rat bone marrow-derived mesenchymal stem cells across a 3D Thai silk fibroin/gelatin/hydroxyapatite scaffold in a perfusion bioreactor

In cell culture, a perfusion bioreactor provides effective transportation of nutrients, oxygen, and waste removal to and from the core of the scaffold. In addition, it provides mechanical stimuli for enhancing osteogenic differentiation. In this study, we used an axial distribution of cell numbers, alkaline phosphatase (ALP) enzyme activity, and calcium content across 4 cross-sections of 10mm thick scaffold, made of Thai silk fibroin (SF)/gelatin (G)/hydroxyapatite (HA), as a tool to evaluate the suitable perfusion flow rate. These evaluations cover all cellular developmental phases starting from seeding, to proliferation, and later osteogenic differentiation. Mouse pre-osteoblastic MC3T3-E1 cell lines were used as a cell model during seeding and proliferation. The bioreactor seeded scaffold provided more uniform cell distribution across the scaffold compared to centrifugal and agitation seeding, while the overall number of adhered cells from bioreactor seeding was slightly lower than agitation seeding. The dynamic culture using 1ml/min perfusion flow rate (initial shear stress of 0.1dyn/cm2) enabled statistically higher MC3T3-E1 proliferation, ALP activity, and calcium deposition than those observed in the static-culturing condition. However, the perfusion flow rate of 1ml/min seemed not to be enough for enhancing ALP expression across all sections of the scaffold. Rat bone marrow derived stromal cells (rMSC) were used in the detachment test and osteogenic differentiation. It was found that perfusion flow rate of 5ml/min caused statistically higher cell detachment than that of 1 and 3ml/min. The perfusion flow rate of 3ml/min gave the highest rMSC osteogenic differentiation on a SF/G/HA scaffold than other flow rates, as observed from the significantly highest number of ALP enzyme activity and the calcium content without any significant cell growth. In addition, all of these parameters were evenly distributed across all scaffold sections.

Fabrication of a highly ordered hierarchically designed porous nanocomposite via indirect 3D printing: Mechanical properties and in vitro cell responses

Abstract Design and development of biodegradable scaffolds with highly uniform and controlled internal structure that stimulate tissue regeneration are the focus of many studies. The aim of this work is to apply a modified three-dimensional (3D) printing process to fabricate polymer-matrix composites with controlled internal architecture. Computationally-designed plaster molds with various pore sizes in the range of 300–800 μm were prepared by employing 3D printing of a water-based binder. The molds were converted to e-polycaprolactone (PCL) and PCL/bioactive glass (BG) composite scaffolds by solvent casting and freeze drying methods. Optical and electron microscopy studies revealed that the pore structure was retained without shape distortion of the scaffolds. The wall structure of the pores was composed of a homogeneous fibril interconnected porous surface. The mechanical properties of the scaffolds were evaluated by uniaxial compression and nano-indentation tests. In vitro cell studies by MC3T3-E1 mouse preosteoblast cells showed that the prepared 3D scaffolds supported cell adhesion, tissue growth and cell differentiation. The effect of pore geometry and bioactive glass particles on the mechanical properties and tissue growth were evaluated. The results of mechanical examinations and in vitro cell responses determined the potential of the process for the fabrication of non-load bearing 3D scaffolds.

Combining bone morphogenetic proteins-2 and -6 has additive effects on osteoblastic differentiation in vitro and accelerates bone formation in vivo.

While only two members of the bone morphogenetic protein subfamily (BMP-2 and -7) are approved to be used in combination with collagen type I in orthopaedic surgery, other BMPs are known to also be highly osteoinductive. Although all the osteogenic BMPs signal through Smad-1/-5/-8 phosphorylation, they show different preferences for the available BMP receptors. In this work we studied the effect of combining two osteogenic BMPs (-2 and -6), which belong to different groups within the subfamily and have different affinities to the existing BMP receptors. Both the growth and in vitro differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells (MSCs) were studied, as well as in vivo ectopic bone formation when the BMPs were intramuscularly implanted in rats with collagen type I sponges as carriers. The results show that these two growth factors have additive effects on the osteoblastic differentiation of cells in vitro and that their combination might be helpful to accelerate in vivo osteogenesis while reducing the amount of each individual BMP used.

MEK5 suppresses osteoblastic differentiation

Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and is activated by its upstream kinase, MAPK kinase 5 (MEK5), which is a member of the MEK family. Although the role of MEK5 has been investigated in several fields, little is known about its role in osteoblastic differentiation. In this study, we have demonstrated the role of MEK5 in osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells.We found that treatment with BIX02189, an inhibitor of MEK5, increased alkaline phosphatase (ALP) activity and the gene expression of ALP, osteocalcin (OCN) and osterix, as well as it enhanced the calcification of the extracellular matrix. Moreover, osteoblastic cell proliferation decreased at a concentration of greater than 0.5 μM. In addition, knockdown of MEK5 using siRNA induced an increase in ALP activity and in the gene expression of ALP, OCN, and osterix. In contrast, overexpression of wild-type MEK5 decreased ALP activity and attenuated osteoblastic differentiation markers including ALP, OCN and osterix, but promoted cell proliferation. In summary, our results indicated that MEK5 suppressed the osteoblastic differentiation, but promoted osteoblastic cell proliferation. These results implied that MEK5 may play a pivotal role in cell signaling to modulate the differentiation and proliferation of osteoblasts.Thus, inhibition of MEK5 signaling in osteoblasts may be of potential use in the treatment of osteoporosis.

Integrin-β1, not integrin-β5, mediates osteoblastic differentiation and ECM formation promoted by mechanical tensile strain.

BackgroundMechanical strain plays a great role in growth and differentiation of osteoblast. A previous study indicated that integrin-β (β1, β5) mediated osteoblast proliferation promoted by mechanical tensile strain. However, the involvement of integrin-β in osteoblastic differentiation and extracellular matrix (ECM) formation induced by mechanical tensile strain, remains unclear.ResultsAfter transfection with integrin-β1 siRNA or integrin-β5 siRNA, mouse MC3T3-E1 preosteoblasts were cultured in cell culture dishes and stimulated with mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz applied once a day for 1 h over 3 or 5 consecutive days. The cyclic tensile strain promoted osteoblastic differentiation of MC3T3-E1 cells. Transfection with integrin-β1 siRNA attenuated the osteoblastic diffenentiation induced by the tensile strain. By contrast, transfection with integrin-β5 siRNA had little effect on the osteoblastic differentiation induced by the strain. At the same time, the result of ECM formation promoted by the strain, was similar to the osteoblastic differentiation.ConclusionIntegrin-β1 mediates osteoblast differentiation and osteoblastic ECM formation promoted by cyclic tensile strain, and integrin-β5 is not involved in the osteoblasts response to the tensile strain.Electronic supplementary materialThe online version of this article (doi:10.1186/s40659-015-0014-y) contains supplementary material, which is available to authorized users.

Layer-by-layer assembly of peptide based bioorganic-inorganic hybrid scaffolds and their interactions with osteoblastic MC3T3-E1 cells.

In this work we have developed a new family of biocomposite scaffolds for bone tissue regeneration by utilizing self-assembled fluorenylmethyloxycarbonyl protected Valyl-cetylamide (FVC) nanoassemblies as templates. To tailor the assemblies for enhanced osteoblast attachment and proliferation, we incorporated (a) Type I collagen, (b) a hydroxyapatite binding peptide sequence (EDPHNEVDGDK) derived from dentin sialophosphoprotein and (c) the osteoinductive bone morphogenetic protein-4 (BMP-4) to the templates by layer-by-layer assembly. The assemblies were then incubated with hydroxyapatite nanocrystals blended with varying mass percentages of TiO2 nanoparticles and coated with alginate to form three dimensional scaffolds for potential applications in bone tissue regeneration. The morphology was examined by TEM and SEM and the binding interactions were probed by FITR spectroscopy. The scaffolds were found to be non-cytotoxic, adhered to mouse preosteoblast MC3T3-E1 cells and promoted osteogenic differentiation as indicated by the results obtained by alkaline phosphatase assay. Furthermore, they were found to be biodegradable and possessed inherent antibacterial capability. Thus, we have developed a new family of tissue-engineered biocomposite scaffolds with potential applications in bone regeneration.

Roles of hydroxyapatite allocation and microgroove dimension in promoting preosteoblastic cell functions on photocured polymer nanocomposites through nuclear distribution and alignment.

This study clarifies how hydroxyapatite (HA) allocation and microgroove dimension affect mouse preosteoblastic MC3T3-E1 cell functions on microgrooved substrates of polymer nanocomposites. Using replica molding from micromachined silicon wafer templates, we fabricated photocured poly(ε-caprolactone) triacrylate (PCLTA)/HA nanocomposite substrates with parallel microgrooves (two groove widths of 5 and 15 μm and one groove depth of 5 μm). Four types of microgrooved substrates were made: "homogeneous" ones of PCLTA and PCLTA/HA with uniform distribution and two "heterogeneous" laminated microgrooved substrates with HA only in the PCLTA matrix in the ridges or bottom. These substrates were used to regulate MC3T3-E1 cell attachment, proliferation, alignment, nuclear circularity and distribution, and mineralization. MC3T3-E1 cell attachment and proliferation were much higher on the microgrooved substrates of PCLTA/HA than on those of PCLTA, in particular, on the 5 μm wide microgrooved substrate with PCLTA/HA ridges and PCLTA bottom. The shape and distribution of MC3T3-E1 cytoskeleton and nuclei were altered by the substrate topography and HA allocation. For 5 μm wide heterogeneous microgrooved substrates with HA only in the ridges, MC3T3-E1 cells exhibited better spreading perpendicular to the microgrooves but tended to extend along the microgrooves containing HA in the bottom. The widest cells and the roundest/largest cell nuclei were observed on the heterogeneous substrate with PCLTA/HA ridges, while the narrowest cells with the best elongation were found on the homogeneous PCLTA/HA substrate. The trend in MC3T3-E1 cell mineralization on the substrates was consistent with that in cell/nuclear elongation. Osteocalcin mRNA expression was significantly higher on the PCLTA/HA substrates than on the PCLTA ones and also on the microgrooved substrates of PCLTA/HA than on the flat ones, regardless of the groove width of 5 or 15 μm.

Influence of MC3T3-E1 preosteoblast culture on the corrosion of a T6-treated AZ91 alloy.

This study investigated the corrosion of artificially aged T6 heat-treated Mg-9%Al-1%Zn (AZ91) for biomedical applications. Corrosion tests and surface analysis were completed both with and without a monolayer of mouse preosteoblast MC3T3-E1 cells cultured on the sample. Electrochemical impedance spectroscopy (EIS) and inductively coupled plasma mass spectroscopy (ICPMS) were used to explore the corrosion processes after either 3 or 21 days of AZ91 incubation in cell culture medium (CCM). The EIS showed both the inner layer resistance (Rin ) and outer layer resistance (Rout ) were lower for samples without cells cultured on the surface at 3 days (Rin = 2.64 e4 Ω/cm(2) , Rout = 140 Ω/cm(2) ) compared to 21 days (Rin = 3.60 e4 Ω/cm(2) , Rout = 287 Ω/cm(2) ) due to precipitation of magnesium and calcium phosphates over time. Samples with preosteoblasts cultured on the surface had a slower initial corrosion (3 day, Rin = 1.88 e5 Ω/cm(2) , Rout = 1060 Ω/cm(2) ) which was observed to increase over time (21 day, Rin = 2.99 e4 Ω/cm(2) , Rout = 287 Ω/cm(2) ). Changes in the corrosion processes were thought to be related to changes in the coverage provided by the cell layer. Our results reveal that the presence of cells and biological processes are able to significantly influence the corrosion rate of AZ91.

Ophiopogonin D: A new herbal agent against osteoporosis

Excessive reactive oxygen species (ROS) play an important role in the development of osteoporosis. Ophiopogonin D (OP-D), isolated from the traditional Chinese herbal agent Radix Ophiopogon japonicus, is a potent anti-oxidative agent. We hypothesized that OP-D demonstrates anti-osteoporosis effects via decreasing ROS generation in mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells and a macrophage cell line RAW264.7 cells. We investigated OP-D on osteogenic and osteoclastic differentiation under oxidative status. Hydrogen peroxide (H2O2) was used to establish an oxidative damage model. In vivo, we established a murine ovariectomized (OVX) osteoporosis model. Then, we searched the molecular mechanism of OP-D against osteoporosis. Our results revealed that OP-D significantly promoted the proliferation of MC3T3-E1 cells and improved some osteogenic markers. Moreover, OP-D reduced TRAP activity and the mRNA expressions of osteoclastic genes in RAW264.7 cells. OP-D suppressed ROS generation in both MC3T3-E1 and RAW264.7 cells. OP-D treatment reduced the activity of serum bone degradation markers, including CTX-1 and TRAP. Further research showed that OP-D displayed anti-osteoporosis effects via reducing ROS through the FoxO3a-β-catenin signaling pathway. In summary, our results indicated that the protective effects of OP-D against osteoporosis are linked to a reduction in oxidative stress via the FoxO3a-β-catenin signaling pathway, suggesting that OP-D may be a beneficial herbal agent in bone-related disorders, such as osteoporosis.

Mechanical Strain Affects Some Microrna Profiles in Pre-Oeteoblasts.

MicroRNAs (miRNAs) are important regulators of cell proliferation, differentiation and function. Mechanical strain is an essential factor for osteoblast proliferation and differentiation. A previous study revealed that a physiological mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz applied once a day for 1 h over 3 consecutive days promoted osteoblast differentiation. However, the mechanoresponsive miRNAs of these osteoblasts were not identified. In this study, we applied the same mechanical tensile strain to in vitro cultivated mouse MC3T3-E1 pre-osteoblasts and identified the mechanoresponsive miRNAs. Using miRNA microarray and qRT-PCR assays, the expression patterns of miRNAs were evaluated and 5 of them were found to be significantly different between the mechanical loading group and the control group: miR-3077-5p, 3090-5p and 3103-5p were significantly upregulated and miR-466i-3p and 466h-3p were downregulated. Bioinformatics analysis revealed possible target genes for these differentially expressed miRNAs. Some target genes correlated with osteoblast differentiation. These findings indicated that the mechanical strain changed the expression levels of these miRNAs. This might be a potential regulator of osteoblast differentiation and responses to mechanical strain.

Effect of surface topography and bioactive properties on early adhesion and growth behavior of mouse preosteoblast MC3T3-E1 cells.

The effects of bioactive properties and surface topography of biomaterials on the adhesion and spreading properties of mouse preosteoblast MC3T3-E1 cells was investigated by preparation of different surfaces. Poly lactic-co-glycolic acid (PLGA) electrospun fibers (ES) were produced as a porous rough surface. In our study, coverslips were used as a substrate for the immobilization of 3,4-dihydroxyphenylalanine (DOPA) and collagen type I (COL I) in the preparation of bioactive surfaces. In addition, COL I was immobilized onto porous electrospun fibers surfaces (E-COL) to investigate the combined effects of bioactive molecules and topography. Untreated coverslips were used as controls. Early adhesion and growth behavior of MC3T3-E1 cells cultured on the different surfaces were studied at 6, 12, and 24 h. Evaluation of cell adhesion and morphological changes showed that the all the surfaces were favorable for promoting the adhesion and spreading of cells. CCK-8 assays and flow cytometry revealed that both topography and bioactive properties were favorable for cell growth. Analysis of β1, α1, α2, α5, α10 and α11 integrin expression levels by immunofluorescence, real-time RT-PCR, and Western blot and indicated that surface topography plays an important role in the early stage of cell adhesion. However, the influence of topography and bioactive properties of surfaces on integrins is variable. Compared with any of the topographic or bioactive properties in isolation, the combined effect of both types of properties provided an advantage for the growth and spreading of MC3T3-E1 cells. This study provides a new insight into the functions and effects of topographic and bioactive modifications of surfaces at the interface between cells and biomaterials for tissue engineering.

Bruton tyrosine kinase (Btk) suppresses osteoblastic differentiation.

The Tec family of nonreceptor tyrosine kinases has been shown to play a key role in inflammation and bone destruction. Bruton tyrosine kinase (Btk) has been the most widely studied because of its critical role in B cells. Furthermore, recent evidence has demonstrated that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. The role of Btk in osteoblastic differentiation has not been well elucidated. In this study, we demonstrated the role of Btk in osteoblastic differentiation and investigated the effects of a Btk inhibitor on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells, primary calvarial osteoblasts, and bone marrow stromal ST2 cells. Btk expression was detected in all three cell lines. Btk inhibition stimulated mRNA expression of osteoblastic markers (alkaline phosphatase, osteocalcin, and osterix) and promoted mineralization of the extracellular matrix. In addition, Btk knockdown caused increased mRNA expression of osteoblastic markers. Furthermore, Btk inhibition suppressed the phosphorylation of mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NFκB), and protein kinase Cα (PKCα). Our results indicate that Btk may regulate osteoblastic differentiation through the MAPK, NFκB, and PKCα signaling pathways.

Electrospinning of aniline pentamer-graft-gelatin/PLLA nanofibers for bone tissue engineering

Blends of aniline pentamer-graft-gelatin (AP-g-GA) and poly(l-lactide) (PLLA) were electrospun to prepare uniform nanofibers as biomimetic scaffolds. The nanofibers exhibited good electroactivity, thermal stability and biodegradability. The biocompatibility of the nanofibers in vitro was evaluated by the adhesion and proliferation of mouse preosteoblastic MC3T3-E1 cells. The cellular elongation was significantly greater on electroactive AP-g-GA/PLLA nanofibers than on PLLA nanofibers. Moreover, the AP-g-GA/PLLA nanofibers stimulated by an electrical pulsed signal could promote the differentiation of MC3T3-E1 cells compared with pure PLLA nanofibers. Our results demonstrated that the biodegradable and electroactive AP-g-GA/PLLA nanofibers had potential application in vivo as bone repair scaffold materials in tissue engineering.

Ginsenoside Rh1 induces mouse osteoblast growth and differentiation through the bone morphogenetic protein 2/runt-related gene 2 signalling pathway.

This study aimed to investigate the stimulative and pharmacological effects of ginsenoside Rh1 (hereinafter referred to as: Rh1) on differentiation and mineralization of osteoblast and its possible mechanism of action on the expression of bone morphogenetic protein 2 (BMP-2)/Runt-related gene 2 (Runx2) signalling pathways using mouse preosteoblastic MC3T3-E1 cell line as in-vitro model. An in-vitro stimulative activity of Rh1 was assessed by analyzing alkaline phosphatase activity (ALP), type-I collagen (Coll-I) synthesis, mineralization and glutathione content. Its antioxidant activity was measured by evaluating the reactive oxygen species (ROS) production in the presence of antimycin A (AMA), one of the mitochondrial dysfunction factors. The level of BMP-2/Runx2 signal-regulated osteoblast-specific proteins such as osteocalcin (OCN), Coll-I and ALP were detected using Western blot analysis. Rh1 was capable to stimulate cell growth, ALP activity, Coll-I synthesis, mineralization and glutathione content in the MC3T3-E1 cells. BMP-2 and Runx2 expression were also increased by Rh1 concentration dependently. Additionally, Rh1 also showed inhibitory action on the level of ROS production enhanced by AMA in MC3T3-E1 cells. Rh1 could increase the expression level of BMP-2/Runx2 signal-regulated osteogenic markers such as ALP, Coll-I and OCN. Rh1, a protopanaxatriol type's active ingredients of Panax ginseng Meyer, possesses osteoblast differentiation, osteogenic stimulatory and anti-oxidative activity.

Melatonin promotes osteoblastic differentiation of MC3T3-E1 cells under hypoxic condition

Melatonin has been shown to influence cell differentiation, where it can either stimulate or suppress cell division depending on its concentration or the cell type examined. A number of in vitro and in vivo studies have suggested beneficial effect of melatonin on promotion of osteoblastic differentiation. However, its precise mechanisms and the signaling pathway involved in the process, especially under hypoxic conditions have remained unknown. This presented study was performed to determine whether melatonin could promote osteoblastic differentiation of mouse preosteoblastic MC3T3-E1 cells and their mineralization activities under hypoxic conditions.

Enhancement of cell ingrowth, proliferation, and early differentiation in a three-dimensional silicon carbide scaffold using low-intensity pulsed ultrasound.

Concerns over the use of autografts or allografts have necessitated the development of biomaterials for bone regeneration. Various studies have been performed to optimize the cultivation of osteogenic cells using osteoconductive porous scaffolds. The aim of this study was to evaluate the osteogenic efficiency of bone cell ingrowth, proliferation, and early differentiation in a silicon carbide (SiC) porous ceramic scaffold promoted with low-intensity pulsed ultrasound. MC3T3-E1 mouse preosteoblasts were seeded onto scaffolds and cultured for 4 and 7 days with daily of 20-min ultrasound treatment. The cells were evaluated for cell attachment, morphology, viability, ingrowth depth, volumetric proliferation, and early differentiation. After 4 and 7 days of culture and ultrasound exposure, the cell density was higher in the ultrasound-treated group compared with the sham-treated group on SiC scaffolds. The cell ingrowth depths inside the SiC scaffolds were 149.2±27.3 μm at 1 day, 310.1±12.6 μm for the ultrasound-treated group and 248.0±19.7 μm for the sham control at 4 days, and 359.6±18.5 μm for the ultrasound-treated group and 280.0±17.7 μm for the sham control at 7 days. They were significantly increased, that is, 25% (p=0.0029) and 28% (p=0.0008) increase, respectively, with ultrasound radiation force as compared with those in sham control at 4 and 7 days postseeding. The dsDNA contents were 583.5±19.1 ng/scaffold at 1 day, 2749.9±99.9 ng/scaffold for the ultrasound-treated group and 2514.9±114.7 ng/scaffold for the sham control at 4 days, and 3582.3±325.3 ng/scaffold for the ultrasound-treated group and 2825.7±134.3 ng/scaffold for the sham control at 7 days. There was a significant difference in the dsDNA content between the ultrasound- and sham-treated groups at 4 and 7 days. The ultrasound-treated group with the SiC construct showed a 9% (p=0.00029) and 27% (p=0.00017) increase in the average dsDNA content at 4 and 7 days over the sham control group, respectively. Alkaline phosphatase activity was significantly increased by the treatment of ultrasound at 4 (p=0.012) and 7 days (p=0.035). These results suggested that ultrasound treatment with low-intensity acoustic energy facilitated the cellular ingrowth and enhanced the proliferation and early differentiation of osteoblasts in SiC scaffolds.

Enhanced bone cell functions on poly(ε-caprolactone) triacrylate networks grafted with polyhedral oligomeric silsesquioxane nanocages

Poly(ε-caprolactone) triacrylate (PCLTA) developed in our laboratory is a photo-crosslinkable, injectable, and biodegradable polymeric biomaterial for diverse tissue engineering applications. To engineer its physical properties for bone regeneration, we incorporated PCLTA networks with a photo-reactive methacryl isobutyl polyhedral oligomeric silsesquioxane (POSS), which is a silicon-based monomer with a nano-sized cage. Homogeneous nanohybrid networks were prepared by photo-crosslinking POSS with two PCLTAs having molecular weights of ∼7000 and ∼20,000 g/mol at the POSS weight compositions (ϕPOSS) of 0–20%. The lower-molecular-weight PCLTA resulted in amorphous networks while the higher one resulted in semi-crystalline networks. POSS nanocages tethered in the PCLTA networks greatly enhanced the mechanical and rheological properties, but did not significantly alter the surface wettability and the capability of adsorbing serum proteins from cell culture media. Better mouse pre-osteoblastic MC3T3-E1 cell attachment, spreading, and proliferation were found on the stiffer PCLTA20k networks than on the PCLTA7k ones, and on the networks with ϕPOSS of 10–20% than the networks containing no POSS. Mineralization of MC3T3-E1 cell cultured for two weeks showed a significantly higher alkaline phosphatase activity and more mineralized nodules on the PCLTA20k networks with ϕPOSS of 10–20%, in correlation with their enhanced mechanical properties. The present results indicated that this series of nanohybrid PCLTA/POSS networks with improved mechanical properties and osteoconductivity has great potential as scaffolding materials for bone repair and regeneration.

Gene delivery from supercharged coiled-coil protein and cationic lipid hybrid complex

A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP·DNA(8:1)·FG lipoproteoplex complex demonstrated enhanced transfection of β-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat·FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP·DNA(8:1)·FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery.

Plasma Modification of DLC Films and the Resulting Surface Biocompatibility

Diamond-like carbon (DLC) films were synthesized on a p-type silicon wafer using radio-frequency plasma composed of a mixture of Ar and C2H2 (ratio of 7 to 28). NH3 plasma treatment of as-grown DLC substrate was carried out to generate surface-terminal amino groups while oxidation of as-grown DLC was performed in O2 plasma. X-ray photoelectron spectroscopy (XPS) was used to characterize the different surface functions formed on DLC surfaces. Water contact angle measurements indicate different wetbility of modified surfaces. The cell (Mouse MC3T3-E1 pre-osteoblasts) morphology and proliferation were monitored to evaluate the biocompatibility of the modified DLC surfaces. A cell count kit-8 (CCK-8 Beyotime) was employed to determine quantitatively the viable pre-osteoblasts. The cell viability assay shows that osteoblast proliferation are improved on NH3 and O2 plasma-treated DLC surface after culturing for 1day, 2days and 3 days. The cell-surface interactions are studied by fluorescence microscopy. There are more osteoblasts as well as better spreading on the aminated and oxidized surfaces after culturing for 3 days. In summary, compared to the as-grown sample, the modified DLC shows better biocompatibility.

Dioscin promotes osteoblastic proliferation and differentiation via Lrp5 and ER pathway in mouse and human osteoblast-like cell lines.

BackgroundDioscin, a typical steroid saponin, is isolated from Dioscorea nipponica Makino and Dioscorea zingiberensis Wright. It has estrogenic activity and many studies have also reported that dioscorea plants have an effect in preventing and treating osteoporosis. However, the molecular mechanisms underlying their effect on osteoporosis treatment are poorly understood. Therefore, the present study aims to investigate the mechanism (s) by which dioscin promotes osteoblastic proliferation and differentiation in mouse pre-osteoblast like MC3T3-E1 cells and human osteoblast-like MG-63 cells.ResultsWe found that dioscin (0.25 μg/ml, 0.5 μg/ml, and 1.0 μg/ml) promoted MC3T3-E1 cells and MG-63 cells proliferation and differentiation dose dependently. Western blot analysis results showed that estrogen receptor α (ER-α), estrogen receptor β (ER-β), β-catenin and Bcl-2 protein expression increased after MC3T3-E1 cells were treated with dioscin. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that dioscin could increase the ratio of osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) and up-regulate the level of Lrp5 and β-catenin. And by RNA interference analysis, we proved that the effect of dioscin increasing the ratio of OPG/RANKL was dependent on Lrp5 pathway. In addition, we also found that these effects of dioscin were abolished by ICI 182, 780 (100 nM), an antagonist of ER, indicating that an ER signaling pathway was also involved. We also found that dioscin (0.25 μg/ml, 0.5 μg/ml, and 1.0 μg/ml) induced MG-63 cells proliferation and differentiation in a dose-dependent manner. Western blot analysis results indicated that ER-α, ER-β and β-catenin protein expression increased after MG-63 cells were treated with dioscin.ConclusionsThe current study is the first to reveal that dioscin can promote osteoblasts proliferation and differentiation via Lrp5 and ER pathway.