On-Chip AC Electrothermal Pump for Pulsatile Perfusion.

In vacuum devices such as vacuum interrupters (VIs), surface flashover events are one of the obstacles for developing their higher performance. It is believed that such a surface flashover in vacuum has a close relationship with a charging phenomenon on the insulator surface. Therefore, it is important to grasp the charging characteristics on the inner surface of the VI. In this study, we have measured the charge distribution characteristics inside a cylindrical type insulator employed as a model insulator of VI after the AC voltage (50 Hz) application. This cylindrical insulator has shield rings to relax the electric field at triple junction at both ends of the insulator. We investigate the charging characteristics for samples having different gaps between the shield and insulator. As a result, in the case of first AC voltage application to each sample, the vacuum side of the cylindrical insulators were charged to a certain value. As repeating the AC voltage application several times, the amount of electric charge on the insulator surface was gradually reduced in each sample. However the reduction rate of the sample with the narrow clearance is larger than wide one. In addition, the sample with the wide clearance was charged on a larger surface area than that of the narrow clearance.

Effect of AC electric fields on the stabilization of premixed bunsen flames

Survivin Is a Master Regulator of Mesenchymal Stem Cell Functions and Potential Therapeutic Target for Mesenchymal Stem Cell Expansion

We designed photo-crosslinkable polymer brushes with dimethylmaleimide moieties, in order to demonstrate dynamic stimulation of cell differentiation in mesenchymal stem cells (MSCs). The polymer brushes were synthesized by surface-initiated reversible addition fragmentation chain transfer polymerization using dimethylmaleimide ethyl methacrylate and methyl methacrylate on a chain transfer agent-immobilized glass surface. The polymer brushes were crosslinked by photodimerization of the dimethylmaleimide moieties within polymer chains with stem cells present on the surface. In order to evaluate the effects of in situ photo-induced crosslinking of the polymer brushes on gene expression of stem cells, human bone marrow MSCs were cultured under static and dynamic culture conditions for 7 days. Expression of the osteocalcin (Ocn) gene in MSCs was used as an indicator of osteoblast differentiation under dynamic culture conditions. Structural conversion from non-crosslinked polymer brushes to crosslinked polymer brushes increased the expression of Ocn by 1.4-fold in the presence of adhered cells, compared with non-crosslinked polymer brushes under static culture conditions. These results suggest that MSCs recognized surface conversion from non-crosslinked to crosslinked structures, which resulted in altered differentiation lineages. Therefore, photo-crosslinkable surfaces with dimethyl maleimide moieties are potential novel materials for dynamically stimulating MSC differentiation.

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP(1)) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.

Advances in microfabrication technology have enabled precise control of surface geometry, which strongly influences cellular behavior, including adhesion, alignment, and differentiation. However, previous studies have employed diverse substrate materials and fabrication conditions, making it difficult to rigorously evaluate the pure geometric effects of the microstructures. Consequently, variations in physicochemical and mechanical properties, such as surface chemistry and stiffness, have confounded the interpretation of geometry-specific effects. To clarify the influence of microgeometry on cell behavior, particularly cell differentiation, stripe- and mesh-patterned polystyrene substrates were used to systematically investigate the relationship between surface geometry and cell behavior. Human mesenchymal stem cells (hMSCs) and C2C12 myoblasts were seeded on the substrates, and their adhesion morphology and alignment were observed using calcein-AM staining. Osteogenic and myogenic differentiation were subsequently induced, and the expression of differentiation markers was analyzed by immunostaining and RT-qPCR. In hMSCs, osteogenic differentiation was promoted in geometries that facilitated intercellular contact, whereas it was suppressed in highly confined geometries, such as stripes and meshes with greater ridge heights. In C2C12 myoblasts, a clear enhancement of myogenic differentiation was observed on striped substrates, where cells exhibited elongated morphologies aligned with the grooves, accompanied by an elevated expression of myogenin and dystrophin. These findings indicate that the differentiation-promoting or differentiation-suppressive effects of microgeometry are cell type-dependent and are governed by cellular alignment, intercellular interactions, and adhesion morphology. The insights gained from this study may contribute to the rational design of next-generation regenerative scaffolds and highlight the potential applications of microgeometric substrates in drug-screening platforms.

The aim of this study was to isolate human mesenchymal stem cells (MSCs) from the gingiva (GMSCs) and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs) and dermal stem cells (DSCs) cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F) and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP) activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN) and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation). Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM) was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM β-glycerophosphate (βGP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM βGP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number or APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes. J. Cell. Biochem. 64:295–312. © 1997 Wiley-Liss, Inc.

Polycaprolactone nanofibers are used as scaffolds in the field of tissue engineering for tissue regeneration or drug delivery. Polycaprolactone (PCL) is a biodegradable hydrophobic polyester used to obtain implantable nanostructures, which are clinically applicable due to their biological safety. Polydatin (PD), a glycosidic precursor of resveratrol, is known for its antioxidant, antitumor, antiosteoporotic, and bone regeneration activities. We aimed to use the osteogenic capacity of polydatin to create a biomimetic innovative and patented scaffold consisting of PCL-PD for bone tissue engineering. Both osteosarcoma cells (Saos-2) and mesenchymal stem cells (MSCs) were used to test the in vitro cytocompatibility of the PD-PCL scaffold. Reverse-phase (RP) HPLC was used to evaluate the timing release of PD from the PCL-PD nanofibers and the MTT assay, scanning electron microscopy, and alkaline phosphatase (ALP) activity were used to evaluate the proliferation, adhesion, and cellular differentiation in both osteosarcoma and human mesenchymal stem cells (MSCs) seeded on PD-PCL nanofibers. The proliferation of osteosarcoma cells (Saos-2) on the PD-PCL scaffold decreased when compared to cells grown on PLC nanofibers, whereas the proliferation of MSCs was comparable in both PCL and PD-PCL nanofibers. Noteworthy, after 14 days, the ALP activity was higher in both Saos-2 cells and MSCs cultivated on PD-PCL than on empty scaffolds. Moreover, the same cells showed a spindle-shaped morphology after 14 days when grown on PD-PCL as shown by SEM. In conclusion, we provide evidence that nanofibers appropriately coated with PD support the adhesion and promote the osteogenic differentiation of both human osteosarcoma cells and MSCs.

Voltage-gated potassium (Kv) channels are known regulators of cellular electrical properties in excitable cells such as neurons and muscle cells. The Kv channels also play roles in cell proliferation, differentiation and cell survival in non-excitable cells. In the present study, we focused on the role of Kv channels in osteogenic differentiation using human mesenchymal stem cells (hMSCs) and the osteoblast-like MG-63 and SaOS-2 cell lines. Use of a non-specific Kv channel blocker, tetraethylammonium (TEA), confirmed that blockade of Kv channels in hMSCs increased their mineral contents density, an indicator of osteogenic differentiation. We also used two osteoblast-like cell lines, MG-63 and SaOS-2 cells, to focus on the regulation of osteogenic properties by Kv channel blockers. We screened the mRNA and protein expression of Kv channels in MG-63 and SaOS-2 cells and compared the relative expression of Kv channels during osteogenic differentiation. We confirmed that the expression level of Kv7.3 was significantly changed, while blockade of Kv7.3 using the selective Kv7.3 blocker, linopirdine and XE991, increased osteogenic differentiation in MG-63 and SaOS-2 cells. In contrast, the use of Kv7 opener, flupirtine, caused no significant change in osteogenic differentiation in either cell line. In conclusion, Kv channels, especially the Kv7 families, are involved in osteogenic differentiation and have potential functions in hMSCs, MG-63 and SaOS-2 cells during osteogenic differentiation. Blockade of Kv7.3 may be important in regulation of osteogenic differentiation.

BackgroundThe proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMScs) are modulated by a variety of microRNAs (miRNAs). SATB homeobox 2 (SATB2) is a critical transcription factor that contributes to maintain the balance of bone metabolism. However, it remains unclear how the regulatory relationship between miR-103 and SATB2 on HBMScs proliferation and osteogenic differentiation.MethodsHBMScs were obtained from Cyagen Biosciences and successful induced osteogenic differentiation. The proliferation abilities of HBMScs after treatment with agomiR-103 and antagomiR-103 were assessed using a cell counting Kit-8 (CCK-8) assay, and osteogenic differentiation was determined using alizarin red S staining and alkaline phosphatase (ALP) activity assay. The expression levels of miR-103, SATB2, and associated osteogenic differentiation biomarkers, including RUNX family transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), and secreted phosphoprotein 1 (SPP1), were evaluated using real-time qPCR and Western blot. The regulatory sites of miR-103 on SATB2 were predicted using bioinformatics software and validated using a dual luciferase reporter assay. The underlying mechanism of miR-103 on SATB2-medicated HBMScs proliferation and osteogenic differentiation were confirmed by co-transfection of antagomiR-103 and SATB2 siRNA.ResultsThe expression of miR-103 in HBMScs after induction of osteogenic differentiation was reduced in a time-dependent way. Overexpression of miR-103 by transfection of agomiR-103 suppressed HBMScs proliferation and osteogenic differentiation, while silencing of miR-103 by antagomiR-103 abolished these inhibitory effects. Consistently, RUNX2, BGLAP and SPP1 mRNA and protein expression were decreased in agomiR-103 treated HBMScs compared with those in agomiR-NC group. Meanwhile, antagomiR-103 upregulated the mRNA and protein expression levels of RUNX2, BGLAP and SPP1 in HBMScs. Further studies revealed that SATB2 was a direct target gene of miR-103. BMSCs transfected with agomiR-103 exhibited significantly downregulated protein expression level of SATB2, whereas knockdown of miR-103 promoted it. Additionally, rescue assays confirmed that silencing of SATB2 partially reversed the effects of antagomiR-103 induced HBMScs proliferation and osteogenic differentiation.ConclusionsThe present results suggested that miR-103 negatively regulates SATB2 to serve an inhibitory role in the proliferation and osteogenic differentiation of HBMScs, which sheds light upon a potential therapeutic target for treating bone-related diseases.

HAMSCs/HBMSCs coculture system ameliorates osteogenesis and angiogenesis against glucolipotoxicity

Human adipose tissue-derived mesenchymal stem cells (hADSCs) due to easy extraction, relative abundance, in vitro expansion and differentiation potential, frozen storage capability, and ability to secrete cytokines, compared to other stem cells, are appropriate candidate in regenerative medicine. Extremely low-frequency electromagnetic fields (ELF-EMF) and betaine are two safe factors in bone lesions repair. This study was designed to assess the osteogenic differentiation potential of these factors on hADSCs. The samples were collected from women undergoing liposuction after obtaining written consent. The hADSCs were extracted and treated with osteogenesis differentiation medium (OD) as the positive control, with OD and betaine (BET group), with OD and EMF (EMF group), and with OD and betaine and EMF (BET+EMF group) for 21 d; the negative control consisted of cells without treatment. Betaine 10 mM and EMF with 50-Hz frequency, 1-mT intensity (8 h daily), and in the form of sinus wave were used. Osteogenic differentiation was evaluated by Alizarin Red staining, alkaline phosphatase activity, calcium deposition, and real-time PCR. A significant increase in calcium deposition in the BET+EMF group was observed compared to the other groups. The activity of alkaline phosphatase in the positive control and BET groups was increased significantly compared to EMF and BET + EMF groups and a significant increase of this enzyme activity in the BET + EMF compared to EMF group was observed. The expression of RUNX2 and OCN genes in the EMF-treated groups were significantly reduced compared to the non-EMF-treated groups, and BET+EMF showed a significant increase of RUNX2 gene expression as compared the EMF group. The ELF-EMF leads to a decrease in the osteogenic differentiation and the expression RUNX2 and OCN genes in hADSCs. But osteogenic differentiation and RUNX2 gene expression were increased post-induction by betaine. The synergic effect of betaine and EMF on the osteogenic differentiation and related genes expression of hADSCs was higher than EMF.

Human mesenchymal stem cells (hMSCs) represent a promising treatment approach for tissue repair and regeneration. However, little is known about the underlying mechanisms and the fate of the transplanted cells. The objective of the presented work was to determine the feasibility of PET imaging and in vivo monitoring after transplantation of dopamine type 2 receptor-expressing cells. An hMSC line constitutively expressing a mutant of the dopamine type 2 receptor (D2R80A) was generated by lentiviral gene transfer. D2R80A messenger RNA expression was confirmed by reverse transcriptase-polymerase chain reaction. Localization of the transmembrane protein was analyzed by confocal fluorescence microscopy. The stem cell character of transduced hMSCs was investigated by adipogenic and osteogenic differentiation. Migration capacity was assessed by scratch assays in time-lapse imaging. In vitro specific binding of ligands was tested by fluorescence-activated cell sorting analysis and by radioligand assay using (18)F-fallypride. Imaging of D2R80A overexpressing hMSC transplanted into athymic rats was performed by PET using (18)F-fallypride. hMSCs showed long-term overexpression of D2R80A. As expected, the fluorescence signal suggested the primary localization of the protein in the membrane of the transduced cells. hMSC and D2R80A retained their stem cell character demonstrated by their osteogenic and adipogenic differentiation capacity and their proliferation and migration behavior. For in vitro hMSCs, at least 90% expressed the D2R80A transgene and hMSC-D2R80A showed specific binding of (18)F-fallypride. In vivo, a specific signal was detected at the transplantation site up to 7 d by PET. The mutant of the dopamine type 2 receptor (D2R80A) is a potent reporter to detect hMSCs by PET in vivo.

The involvement of long non-coding RNA (lncRNA) in bone marrow mesenchymal stem cell (MSC) osteogenic differentiation during osteoporosis (OP) development has attracted much attention. In this study, we aimed to disclose how LINC01089 functions in human mesenchymal stem cell (hMSC) osteogenic differentiation, and to study the mechanism by which LINC01089 regulates MSC osteogenesis. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting were performed to analyze LINC01089, miR-1287-5p, and heat shock protein family A (HSP70) member 4 (HSPA4) expression. The osteogenic differentiation of MSCs was assessed through alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and by measuring the levels of osteogenic gene marker expressions using commercial kits and RT-qPCR analysis. Cell proliferative capacity was evaluated via the Cell Counting Kit-8 (CCK-8) assay. The binding of miR-1287-5p with LINC01089 and HSPA4 was verified by performing dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments. LINC01089 expression was reinforced in serum samples of OP patients, but it gradually diminished while hMSCs underwent osteogenic differentiation. LINC01089 knockdown facilitated hMSC osteogenic differentiation. This was substantiated by: the increase in ALP activity; ALP, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) messenger RNA (mRNA) levels; and level of ARS staining. Meanwhile, LINC01089 upregulation resulted in the opposite effects. LINC01089 targeted miR-1287-5p, and the LINC01089 knockdown-induced hMSC osteogenic differentiation was repressed by miR-1287-5p depletion. HSPA4 is a downstream function molecule of the LINC01089/miR-1287-5p pathway; miR-1287-5p negatively modulated HSPA4 levels and attenuated its functional effects. LINC01089 negatively regulated hMSC osteogenic differentiation, at least in part, via governing miR-1287-5p/HSPA4 signalling. These findings provide new insights into hMSC osteogenesis and bone metabolism.