|  |  |  | | | | | TE-RegenMed-StemCell feed | | | | | | | | | | | | | | | | | | | Synthetic sulfonyl-hydrazone-1 positively regulates cardiomyogenic microRNA expression and cardiomyocyte differentiation of induced pluripotent stem cells. J Cell Biochem. 2011 Mar 28; Authors: Quattrocelli M, Palazzolo G, Agnolin I, Martino S, Bouché M, Anastasia L, Sampaolesi M Induced pluripotent stem cells (iPSCs) are obtained from adult cells through overexpression of pluripotency factors. iPSCs share many features with embryonic stem cells (ESCs), circumventing ethical issues, and, noteworthy, match donor's genotype. iPSCs represent therefore a valuable tool for regenerative medicine. Cardiac differentiation of ESCs can be enhanced via microRNAs (miRNAs) and small chemical compounds, which probably act as chromatin remodelers. Cardiomyogenic potential of iPSCs is currently intensely investigated for cell therapy or in vitro drug screening and disease modeling. However, influences of small compounds on iPSC-related cardiomyogenesis have not yet been investigated in details. Here, we compared the effects of two small molecules, bis-peroxo-vanadium (bpV) and sulfonyl-hydrazone-1 (SHZ) at varying concentrations, during cardiac differentiation of murine iPSCs. SHZ (5μM) enhanced specific marker expression and cardiomyocyte yield, without loss of cell viability. In contrast, bpV showed negligible effects on cardiac differentiation rate and appeared to induce Casp3-dependent apoptosis in differentiating iPSCs. Furthermore, SHZ-treated iPSCs were able to increase beating foci rate and upregulate early and late cardiomyogenic miRNA expression (miR-1, miR-133a and miR-208a). Thus, our results demonstrate that small chemical compounds, such as SHZ, can constitute a novel and clinically-feasible strategy to improve iPSC-derived cardiac differentiation. © 2011 Wiley-Liss, Inc. PMID: 21445862 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Allogeneic mesenchymal stem cells-agents of immune deviation. J Cell Biochem. 2011 Mar 28; Authors: English K, Mahon BP Adult mesenchymal stem cells possess a remarkably diverse array of immunosuppressive characteristics. The capacity to suppress the regular processes of allogeneic rejection, have allowed the use of tissue mismatched cells as therapeutic approaches in regenerative medicine and as agents of immune deviation. This review describes recent advances in understanding the mechanistic basis of MSC interaction with innate immunity. Particular emphasis is placed on the effect of Toll like receptor signalling on MSC and a hypothesis that innate immune signals induce a "licensing switch" in MSC is put forward. The mechanisms underlying MSC suppression of T cell responses and induction of regulatory populations are surveyed. Conflicting data regarding the influence of MSC on B cell function are outlined and discussed. Finally the limits to MSC mediated immune modulation are discussed with reference to the future clinical application of novel cell therapies. © 2011 Wiley-Liss, Inc. PMID: 21445861 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Rational optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells with ultra-high efficiency and fast kinetics. Cell Res. 2011 Mar 29; Authors: Chen J, Liu J, Chen Y, Yang J, Chen J, Liu H, Zhao X, Mo K, Song H, Guo L, Chu S, Wang D, Ding K, Pei D The ectopic expression of several transcription factors can restore embryonic cell fate to cultured somatic cells and generate induced pluripotent stem cells (iPSCs), revealing a previously unknown pathway to pluripotency. However, this technology is currently limited by low efficiency, slow kinetics and multi-factorial requirement. Here we show that reprogramming can be improved and dramatically accelerated by optimizing culture conditions. First, we developed an optimized defined medium, iCD1, which allows Oct4/Sox2/Klf4 (OSK)-mediated reprogramming to achieve ultra-high efficiency (∼10% at day 8). We also found that this optimized condition renders both Sox2 and Klf4 dispensable, although the elimination of these two factors leads to lower efficiency and slower kinetics. Our studies define a shortened route, both in timing and factor requirement, toward pluripotency. This new paradigm not only provides a rationale to further improve iPSC generation but also simplifies the conceptual understanding of reprogramming by defined factors.Cell Research advance online publication 29 March 2011; doi:10.1038/cr.2011.51. PMID: 21445094 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Evaluation of 3D nano-macro porous bioactive glass scaffold for hard tissue engineering. J Mater Sci Mater Med. 2011 Mar 29; Authors: Wang S, Falk MM, Rashad A, Saad MM, Marques AC, Almeida RM, Marei MK, Jain H Recently, nano-macro dual-porous, three-dimensional (3D) glass structures were developed for use as bioscaffolds for hard tissue regeneration, but there have been concerns regarding the interconnectivity and homogeneity of nanopores in the scaffolds, as well as the cytotoxicity of the environment deep inside due to limited fluid access. Therefore, mercury porosimetry, nitrogen absorption, and TEM have been used to characterize nanopore network of the scaffolds. In parallel, viability of MG 63 human osteosarcoma cells seeded on scaffold surface was investigated by fluorescence, confocal and electron microscopy methods. The results show that cells attach, migrate and penetrate inside the glass scaffold with high proliferation and viability rate. Additionally, scaffolds were implanted under the skin of a male New Zealand rabbit for in vivo animal test. Initial observations show the formation of new tissue with blood vessels and collagen fibers deep inside the implanted scaffolds with no obvious inflammatory reaction. Thus, the new nano-macro dual-porous glass structure could be a promising bioscaffold for use in regenerative medicine and tissue engineering for bone regeneration. PMID: 21445655 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | The therapeutic potential of human multipotent mesenchymal stromal cells combined with pharmacologically active microcarriers transplanted in hemi-parkinsonian rats. Biomaterials. 2011 Feb;32(6):1560-73 Authors: Delcroix GJ, Garbayo E, Sindji L, Thomas O, Vanpouille-Box C, Schiller PC, Montero-Menei CN Multipotent mesenchymal stromal cells (MSCs) raise great interest for brain cell therapy due to their ease of isolation from bone marrow, their immunomodulatory and tissue repair capacities, their ability to differentiate into neuronal-like cells and to secrete a variety of growth factors and chemokines. In this study, we assessed the effects of a subpopulation of human MSCs, the marrow-isolated adult multilineage inducible (MIAMI) cells, combined with pharmacologically active microcarriers (PAMs) in a rat model of Parkinson's disease (PD). PAMs are biodegradable and non-cytotoxic poly(lactic-co-glycolic acid) microspheres, coated by a biomimetic surface and releasing a therapeutic protein, which acts on the cells conveyed on their surface and on their microenvironment. In this study, PAMs were coated with laminin and designed to release neurotrophin 3 (NT3), which stimulate the neuronal-like differentiation of MIAMI cells and promote neuronal survival. After adhesion of dopaminergic-induced (DI)-MIAMI cells to PAMs in vitro, the complexes were grafted in the partially dopaminergic-deafferented striatum of rats which led to a strong reduction of the amphetamine-induced rotational behavior together with the protection/repair of the nigrostriatal pathway. These effects were correlated with the increased survival of DI-MIAMI cells that secreted a wide range of growth factors and chemokines. Moreover, the observed increased expression of tyrosine hydroxylase by cells transplanted with PAMs may contribute to this functional recovery. PMID: 21074844 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Filter-well technology for advanced three-dimensional cell culture: perspectives for respiratory research. Altern Lab Anim. 2010 Dec;38 Suppl 1:49-65 Authors: Bérubé K, Pitt A, Hayden P, Prytherch Z, Job C Cell culture has long been a valuable tool for studying cell behaviour. Classical plastic substrates are two-dimensional, and usually promote cellular proliferation and inhibit differentiation. Understanding cell behaviour within complex multicellular tissues requires the systematic study of cells within the context of specific model microenvironments. A model system must mimic, to a certain degree, the in vivo situation, but, at the same time, can significantly reduce its complexity. There is increasing agreement that moving up to the third dimension provides a more physiologically-relevant and predictive model system. Moreover, many cellular processes (morphogenesis, organogenesis and pathogenesis) have been confirmed to occur exclusively when cells are ordered in a three-dimensional (3-D) manner. In order to achieve the desired in vivo phenotype, researchers can use microporous membranes for improved in vitro cell culture experiments. In the present review, we discuss the applications of filter-well technology for the advanced 3-D cell culture of human pulmonary cells. PMID: 21275484 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Manipulating co-continuous polymer blends to create PCL scaffolds with fully interconnected and anisotropic pore architecture. J Appl Biomater Biomech. 2011 Mar 23; Authors: Guarino V, Guaccio A, Ambrosio L Purpose: Polymer blending is an attractive route to obtain new materials with superior properties. Control of their characteristics may be initially achieved by manipulating the phase morphology with different aspects (i.e, lamellar, dispersed or fibrillar) to form tailored architectures for different application fields. In particular, co-continuous microstructures due to the interpenetration of two different polymer phases in three dimensions are particularly interesting because they are able to offer a fully interconnected network with improvement of mechanical properties and fluid permeability for tissue engineering applications. Materials and Methods: Macro/microporous substrates with controlled architecture were obtained by blending hydrophylic and hydrophobic polymers, i.e, polycaprolactone (PCL) and poly(ethylene oxide)(PEO) respectively, in a co-continuous state by a twin-screw extruder. Results: In accordance with the leaching-based approaches traditionally used in scaffold manufacturing, the removal of water soluble phases (i.e., PEO and sodium chloride (NaCl) crystals) enables a fully interconnected porous network to be formed, whereas post-extrusion stretching of the melt blend allowsa desired elongation of polymer phases to be imparted before the polymer leaching, thus providing a controlled pore alignment. Conclusion: the proposed investigation confirms that polymer blending is a promising approach to realize structurally organized platforms able to guide the regeneration processes of hierarchically organized tissues (i.e, tendons, muscles, ligaments and nerves). PMID: 21445831 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Clustered integrin α5β1 ligand displays model fibronectin-mediated adhesion of human endometrial stromal cells. Biochem Biophys Res Commun. 2011 Mar 25; Authors: Li Z, Kreiner M, van der Walle CF, Mardon HJ Progress towards endometrial tissue engineering for modelling endometrial diseases and infertility is frustrated by the inability to mimic the fibronectin (FN) extracellular matrix required by human endometrial stromal cells (EnSCs). Here we show that this is because of the requirement to present integrin α5β1 (the FN receptor) ligands in specifically oriented, polyvalent displays; by engineering controlled self-assembly of the 9(th)-10(th) type III FN domain pair (FIII9-10, the minimal integrin α5β1 ligand) immobilised in a specific orientation to cell culture surfaces. The fraction of adherent EnSCs seen to spread increased significantly for the multimeric ligand surfaces in the order: tetramer > trimer > dimer > monomer. The extent of EnSC spread morphology also increased in the same order, with the tetrameric ligand supporting a morphology most similar to that supported by FN. Our data suggest that only higher-order multimers of FIII9-10 will fully promote cell spreading mediated through integrin α5β1 binding. PMID: 21443861 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Pore size regulates cell and tissue interactions with PLGA-CaP scaffolds used for bone engineering. J Tissue Eng Regen Med. 2011 Mar 28; Authors: Sicchieri LG, Crippa GE, de Oliveira PT, Beloti MM, Rosa AL A common subject in bone tissue engineering is the need for porous scaffolds to support cell and tissue interactions aiming at repairing bone tissue. As poly(lactide-co-glycolide)-calcium phosphate (PLGA-CaP) scaffolds can be manufactured with different pore sizes, the aim of this study was to evaluate the effect of pore diameter on osteoblastic cell responses and bone tissue formation. Scaffolds were prepared with 85% porosity, with pore diameters in the ranges 470-590, 590-850 and 850-1200 µm. Rat bone marrow stem cells differentiated into osteoblasts were cultured on the scaffolds for up to 10 days to evaluate cell growth, alkaline phosphatase (ALP) activity and the gene expression of the osteoblast markers RUNX2, OSX, COL, MSX2, ALP, OC and BSP by real-time PCR. Scaffolds were implanted in critical size rat calvarial defects for 2, 4, and 8 weeks for histomorphometric analysis. Cell growth and ALP activity were not affected by the pore size; however, there was an increase in the gene expression of osteoblastic markers with the increase in the pore sizes. At 2 weeks all scaffolds displayed a similar amount of bone and blood vessels formation. At 4 and 8 weeks much more bone formation and an increased number of blood vessels were observed in scaffolds with pores of 470-590 µm. These results show that PLGA-CaP is a promising biomaterial for bone engineering. However, ideally, combinations of larger (∼1000 µm) and smaller (∼500 µm) pores in a single scaffold would optimize cellular and tissue responses during bone healing. Copyright © 2011 John Wiley & Sons, Ltd. PMID: 21446054 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Development of conductive polymer with carbon nanotubes for regenerative medicine applications. Conf Proc IEEE Eng Med Biol Soc. 2010;2010:815-8 Authors: Antoniadou EV, Cousins BG, Seifalian AM Multi-wall carbon nanotube (MWCNT)/polymer composites are hybrid materials that combine numerous mechanical, electrical and chemical properties and thus, constitute ideal biomaterials for a wide range of regenerative medicine applications. Although, complete dispersion of MWCNT in a polymer matrix has rarely been achieved, in this study we have studied the dispersibility of MWCNT in POSS-PCU, a novel polymer based on polyprolactone and polycarbonate polyurethane (PCU) with an incorporated polyhedral oligomeric silsesquioxane (POSS). Furthermore, we developed a computational model that can visualise MWCNTs in order to predict the range of dispersibility and provide a 3-D mathematical model that can predict the chemical concentration for ideal nanocomposites. PMID: 21096308 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | The effect of elastin on chondrocyte adhesion and proliferation on poly (ɛ-caprolactone)/elastin composites. Biomaterials. 2011 Feb;32(6):1517-25 Authors: Annabi N, Fathi A, Mithieux SM, Martens P, Weiss AS, Dehghani F The aim of this study was to demonstrate the effect of elastin on chondrocyte adhesion and proliferation within the structure of poly (ɛ-caprolactone) (PCL)/elastin composites. The homogenous 3D structure composites were constructed by using high pressure CO(2) in two stages. Porous PCL structures with average pore sizes of 540 ± 21 μm and a high degree of interconnectivity were produced using gas foaming/salt leaching. The PCL scaffolds were then impregnated with elastin and cross-linked with glutaraldehyde (GA) under high pressure CO(2). The effects of elastin and cross-linker concentrations on the characteristics of composites were investigated. Increasing the elastin concentration from 25mg/ml to 100mg/ml elevated the amount of cross-linked elastin inside the macropores of PCL. Fourier transform infrared (FTIR) analysis showed that elastin was homogeneously distributed throughout the 3D structure of all composites. The weight gain of composites increased 2-fold from 15.8 ± 0.3 to 38.3 ± 0.7 (w/w) % by increasing the elastin concentration from 25mg/ml to 50mg/ml and approached a plateau above this concentration. The presence of elastin within the pores of PCL improved the water uptake properties of PCL scaffolds; the water uptake ratio of PCL was enhanced 100-fold from 0.030 ± 0.005g liquid/g polymer to 11.80 ± 0.01g liquid/g polymer, when the elastin solution concentration was 50mg/ml. These composites exhibited lower compressive modulus and energy loss compared to pure PCL scaffolds due to their higher water content and elasticity. In vitro studies show that these composites can support primary articular cartilage chondrocyte adhesion and proliferation within the 3D structures. These results demonstrate the potential of using PCL/elastin composites for cartilage repair. PMID: 21115195 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | High-density collagen gel tubes as a matrix for primary human bladder smooth muscle cells. Biomaterials. 2011 Feb;32(6):1543-8 Authors: Micol LA, Ananta M, Engelhardt EM, Mudera VC, Brown RA, Hubbell JA, Frey P Tissue-engineered grafts for the urinary tract are being investigated for the potential treatment of several urologic diseases. These grafts, predominantly tubular-shaped, usually require in vitro culture prior to implantation to allow cell engraftment on initially cell-free scaffolds. We have developed a method to produce tubular-shaped collagen scaffolds based on plastic compression. Our approach produces a ready cell-seeded graft that does not need further in vitro culture prior to implantation. The tubular collagen scaffolds were in particular investigated for their structural, mechanical and biological properties. The resulting construct showed an especially high collagen density, and was characterized by favorable mechanical properties assessed by axial extension and radial dilation. Young modulus in particular was greater than non-compressed collagen tubes. Seeding densities affected proliferation rate of primary human bladder smooth muscle cells. An optimal seeding density of 10(6) cells per construct resulted in a 25-fold increase in Alamar blue-based fluorescence after 2 wk in culture. These high-density collagen gel tubes, ready seeded with smooth muscle cells could be further seeded with urothelial cells, drastically shortening the production time of graft for urinary tract regeneration. PMID: 21074843 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | The influence of biological motifs and dynamic mechanical stimulation in hydrogel scaffold systems on the phenotype of chondrocytes. Biomaterials. 2011 Feb;32(6):1508-16 Authors: Appelman TP, Mizrahi J, Elisseeff JH, Seliktar D Primary bovine chondrocytes and PEG-based hydrogels were used to investigate the effects of scaffold composition and architecture on the cellular response to large dynamic compressive strain stimulation. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic scaffolds. Second passage articular chondrocytes were encapsulated into four different scaffold compositions: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only and subjected to 15% dynamic compressive strain at 1-Hz frequency. Cellular response was evaluated in terms of cell number, glycosaminoglycans (GAGs), collagen type II and collagen type I accumulation in the constructs following 24h and 28 days of stimulated and static culture. Stimulation of the constructs resulted in an increase in the cell number in all scaffolds, with no statistical difference measured among them. Dynamic stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the GAGs by 33%, 53.4%, 240.5%, and 284.5%, compared to their static controls. The permissive PEG and PA scaffolds showed a significantly larger relative increase in the GAGs in comparison to the other scaffolds tested. Collagen type II content in the PF, PA and PEG constructs increased by 78%, 1266% and 896% respectively, compared to their static controls. Permissive constructs showed a significantly larger relative increase and final absolute values of GAGs and type II collagen, compared to the PF constructs. Immunostaining for collagen type I, an indicator for chondrocyte de-differentiation, indicated that stimulation inhibited its production. Correlation maps between scaffold properties highlighted the major differences between permissive and instructive scaffolds. These results support the hypothesis that both compressive strain and scaffold bioactivity have an important effect on the chondrocyte metabolic response to mechanical stimulation, and that the 3-D environment surrounding chondrocytes can actively participate in translating mechanical stimulation to the resident cells. PMID: 21093907 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Labeling of mesenchymal stem cells with bioconjugated quantum dots. Methods Mol Biol. 2011;680:61-75 Authors: Shah BS, Mao JJ Quantum dots (QDs) are semiconductor nanocrystals, and recently they have been shown as effective probes for cell labeling. Due to their unique spectral, physical, and chemical properties, QDs can concurrently tag multiple intercellular and intracellular components of live cells for time ranging from seconds to months. Different color QDs can label different cell components that can be visualized with fluorescent microscopy or in vivo. Here, we provide a detailed protocol for labeling postnatal and differentiated stem/progenitor cells with bioconjugated quantum dots. For example, peptide CGGGRGD is immobilized on CdSe-ZnS QDs with free carboxyl groups. These bioconjugates label selected integrins on cell membrane of human mesenchymal stem cells (hMSCs). QD concentration and incubation time to effectively label hMSCs is optimized. We discovered that bioconjugated QDs effectively label hMSCs not only during population doubling, but also during multi-lineage differentiation into osteoblasts, chondrocytes, and adipocytes. Undifferentiated and differentiated stem cells labeled with bioconjugated QDs can be readily imaged by fluorescent microscopy. Thus, quantum dots represent an effective cell labeling probe and an alternative to organic dyes and fluorescent proteins for cell labeling and cell tracking. PMID: 21153373 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Cell infiltration and growth in a low density, uncompressed three-dimensional electrospun nanofibrous scaffold. Biomaterials. 2011 Feb;32(6):1583-90 Authors: Blakeney BA, Tambralli A, Anderson JM, Andukuri A, Lim DJ, Dean DR, Jun HW A limiting factor of traditional electrospinning is that the electrospun scaffolds consist entirely of tightly packed nanofiber layers that only provide a superficial porous structure due to the sheet-like assembly process. This unavoidable characteristic hinders cell infiltration and growth throughout the nanofibrous scaffolds. Numerous strategies have been tried to overcome this challenge, including the incorporation of nanoparticles, using larger microfibers, or removing embedded salt or water-soluble fibers to increase porosity. However, these methods still produce sheet-like nanofibrous scaffolds, failing to create a porous three-dimensional scaffold with good structural integrity. Thus, we have developed a three-dimensional cotton ball-like electrospun scaffold that consists of an accumulation of nanofibers in a low density and uncompressed manner. Instead of a traditional flat-plate collector, a grounded spherical dish and an array of needle-like probes were used to create a Focused, Low density, Uncompressed nanoFiber (FLUF) mesh scaffold. Scanning electron microscopy showed that the cotton ball-like scaffold consisted of electrospun nanofibers with a similar diameter but larger pores and less-dense structure compared to the traditional electrospun scaffolds. In addition, laser confocal microscopy demonstrated an open porosity and loosely packed structure throughout the depth of the cotton ball-like scaffold, contrasting the superficially porous and tightly packed structure of the traditional electrospun scaffold. Cells seeded on the cotton ball-like scaffold infiltrated into the scaffold after 7 days of growth, compared to no penetrating growth for the traditional electrospun scaffold. Quantitative analysis showed approximately a 40% higher growth rate for cells on the cotton ball-like scaffold over a 7 day period, possibly due to the increased space for in-growth within the three-dimensional scaffolds. Overall, this method assembles a nanofibrous scaffold that is more advantageous for highly porous interconnectivity and demonstrates great potential for tackling current challenges of electrospun scaffolds. PMID: 21112625 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Development and application of human virtual excitable tissues and organs: from premature birth to sudden cardiac death. Altern Lab Anim. 2010 Dec;38 Suppl 1:87-99 Authors: Holden AV The electrical activity of cardiac and uterine tissues has been reconstructed by detailed computer models in the form of virtual tissues. Virtual tissues are biophysically and anatomically detailed, and represent quantitatively predictive models of the physiological and pathophysiological behaviours of tissue within an isolated organ. The cell excitation properties are quantitatively reproduced by equations that describe the kinetics of a few dozen proteins. These equations are derived from experimental measurements of membrane potentials, ionic currents, fluxes, and concentrations. Some of the measurements were taken from human cells and human ion channel proteins expressed in non-human cells, but they were mostly taken from cells of other animal species. Data on tissue geometry and architecture are obtained from the diffusion tensor magnetic resonance imaging of ex vivo or post mortem tissue, and are used to compute the spread of current in the tissue. Cardiac virtual tissues are well established and reproduce normal and pathological patterns of cardiac excitation within the atria or ventricles of the human heart. They have been applied to increase the understanding of normal cardiac electrophysiology, to evaluate the candidate mechanisms for re-entrant arrhythmias that lead to sudden cardiac death, and to predict the tissue level effects of mutant or pharmacologically-modified ion channels. The human full-term virtual uterus is still in development. This virtual tissue reproduces the in vitro behaviour of uterine tissue biopsies, and provides possible mechanisms for premature labour. PMID: 21275487 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Chondrogenesis of human mesenchymal stem cells in fibrin constructs evaluated in vitro and in nude mouse and rabbit defects models. Biomaterials. 2011 Feb;32(6):1495-507 Authors: Park JS, Yang HN, Woo DG, Jeon SY, Park KH In this study, hMSCs encapsulated in a fibrin hydrogel containing heparinized NPs loaded with TGF-β3 (100 ng/ml), or TGF-β3 (100 ng/ml) alone, were subjected to growth factor release and denaturation tests at one, two and four weeks in in vitro culture systems. Additionally, stem cell differentiation was assessed via RT-PCR, real-time quantitative PCR (qPCR), histology, and immunohistochemical assays. In the in vivo studies with nude mouse, when transplanted into nude mice, hMSCs embedded in fibrin hydrogels survived and proliferated more readily in those samples containing TGF-β3-loaded NPs, or TGF-β3 alone, compared to those containing only NPs or the fibrin hydrogel alone. Additionally, RT-PCR, real-time qPCR, histology, Western blotting, and immunohistochemistry analyses revealed that chondrocyte-specific extracellular matrix (ECM) genes and their proteins were expressed at high levels by hMSCs embedded in hydrogels containing TGF-β3-loaded NPs. Finally, the results observed in the rabbit animal model treated with hMSCs embedded in a fibrin hydrogel containing TGF-β3-loaded NPs were also evaluated by the RT-PCR, real-time qPCR, histology, Western blotting, and immunohistochemistry analyses. The in vitro and in vivo results indicated that transplanted hMSCs together with TGF-β3 may constitute a clinically efficient method for the regeneration of hyaline articular cartilage. PMID: 21122912 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | A preliminary study comparing the use of allogenic chondrogenic pre-differentiated and undifferentiated mesenchymal stem cells for the repair of full thickness articular cartilage defects in rabbits. J Orthop Res. 2011 Mar 28; Authors: Dashtdar H, Rothan HA, Tay T, Ahmad RE, Ali R, Tay LX, Chong PP, Kamarul T Chondrogenic differentiated mesenchymal stem cells (CMSCs) have been shown to produce superior chondrogenic expression markers in vitro. However, the use of these cells in vivo has not been fully explored. In this study, in vivo assessment of cartilage repair potential between allogenic-derived chondrogenic pre-differentiated mesenchymal stem cells and undifferentiated MSCs (MSCs) were compared. Bilateral full thickness cartilage defects were created on the medial femoral condyles of 12 rabbits (n = 12). Rabbits were divided into two groups. In one group, the defects in the right knees were repaired using alginate encapsulated MSCs while in the second group, CMSCs were used. The animals were sacrificed and the repaired and control knees were assessed at 3 and 6 months after implantation. Quantitative analysis was performed by measuring the Glycosaminoglycans (GAGs)/total protein content. The mean Brittberg score was higher in the transplanted knees as compared to the untreated knee at 6 months (p < 0.05). Quantitative analysis of GAGs was consistent with these results. Histological and immunohistochemical analysis demonstrated hyaline-like cartilage regeneration in the transplanted sites. Significant differences between the histological scores based on O'Driscoll histological grading were observed between contralateral knees at both 3 and 6 months (p < 0.05). No significant differences were observed between the Britberg, O'Driscoll scores, and GAGs/total protein content when comparing defect sites treated with MSC and CMSC (p > 0.05). This study demonstrates that the use of either MSC or CMSC produced superior healing when compared to cartilage defects that were untreated. However, both cells produced comparable treatment outcomes. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res. PMID: 21445989 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | In vivo imaging of the dynamics of different variants of EGFR in glioblastomas. Methods Mol Biol. 2011;680:153-64 Authors: Shah K A number of altered pathways in cancer cells depend on growth factor receptors. The amplification/alteration of the epidermal growth factor receptor (EGFR) has been shown to play a significant role in enhancing tumor burden in a number of tumors, including malignant glioblastomas (GBM). To dissect the role of EGFR expression in tumor progression in mouse models of cancer and ultimately evaluate targeted therapies, it is necessary to visualize the dynamics of EGFR in real time in vivo. Non-invasive imaging based on quantitative and qualitative changes in light emission by fluorescent and bioluminescent markers offers a huge potential to facilitate drug development. Multiple approaches could be used to follow a molecular target or pathway with the fusion of a bioluminescent-fluorescent marker. This unit describes a protocol for simultaneously imaging EGFR activity and progression of GBM in a mouse model. Human glioma cells transduced with lentiviral vectors bearing different combinations of fluorescent and bioluminescent proteins either fused to EGFR or expressed alone can be grown as monolayers and maintained over several passages. The unit begins with a method for transducing glioma cells with lentiviral vectors for stable expression of these fluorescent and bioluminescent markers in vitro, followed by transplantation of engineered glioma cells in mice, and, finally, sequential bioluminescent imaging of EGFR expression and GBM progression in mice. The protocol details characterization of engineered glioma cells in culture, surgical preparation, craniotomy, cell implantation, animal recovery, and imaging procedures to study kinetics of EGFR expression and GBM progression. PMID: 21153380 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Aligned, isotropic and patterned carbon nanotube substrates that control the growth and alignment of Chinese hamster ovary cells. Nanotechnology. 2011 May 20;22(20):205102 Authors: Abdullah CA, Asanithi P, Brunner EW, Jurewicz I, Bo C, Azad CL, Ovalle-Robles R, Fang S, Lima MD, Lepro X, Collins S, Baughman RH, Sear RP, Dalton AB Here we culture Chinese hamster ovary cells on isotropic, aligned and patterned substrates based on multiwall carbon nanotubes. The nanotubes provide the substrate with nanoscale topography. The cells adhere to and grow on all substrates, and on the aligned substrate, the cells align strongly with the axis of the bundles of the multiwall nanotubes. This control over cell alignment is required for tissue engineering; almost all tissues consist of oriented cells. The aligned substrates are made using straightforward physical chemistry techniques from forests of multiwall nanotubes; no lithography is required to make inexpensive large-scale substrates with highly aligned nanoscale grooves. Interestingly, although the cells strongly align with the nanoscale grooves, only a few also elongate along this axis: alignment of the cells does not require a pronounced change in morphology of the cell. We also pattern the nanotube bundles over length scales comparable to the cell size and show that the cells follow this pattern. PMID: 21444962 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Evaluation of 3D nano-macro porous bioactive glass scaffold for hard tissue engineering. J Mater Sci Mater Med. 2011 Mar 29; Authors: Wang S, Falk MM, Rashad A, Saad MM, Marques AC, Almeida RM, Marei MK, Jain H Recently, nano-macro dual-porous, three-dimensional (3D) glass structures were developed for use as bioscaffolds for hard tissue regeneration, but there have been concerns regarding the interconnectivity and homogeneity of nanopores in the scaffolds, as well as the cytotoxicity of the environment deep inside due to limited fluid access. Therefore, mercury porosimetry, nitrogen absorption, and TEM have been used to characterize nanopore network of the scaffolds. In parallel, viability of MG 63 human osteosarcoma cells seeded on scaffold surface was investigated by fluorescence, confocal and electron microscopy methods. The results show that cells attach, migrate and penetrate inside the glass scaffold with high proliferation and viability rate. Additionally, scaffolds were implanted under the skin of a male New Zealand rabbit for in vivo animal test. Initial observations show the formation of new tissue with blood vessels and collagen fibers deep inside the implanted scaffolds with no obvious inflammatory reaction. Thus, the new nano-macro dual-porous glass structure could be a promising bioscaffold for use in regenerative medicine and tissue engineering for bone regeneration. PMID: 21445655 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | A time to replace animal models?- A clinical perspective: reflections on the keynote address. Altern Lab Anim. 2010 Dec;38 Suppl 1:iii-v Authors: Bhogal N PMID: 21404723 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Luciferase expression and bioluminescence does not affect tumor cell growth in vitro or in vivo. Mol Cancer. 2010;9:299 Authors: Tiffen JC, Bailey CG, Ng C, Rasko JE, Holst J Live animal imaging is becoming an increasingly common technique for accurate and quantitative assessment of tumor burden over time. Bioluminescence imaging systems rely on a bioluminescent signal from tumor cells, typically generated from expression of the firefly luciferase gene. However, previous reports have suggested that either a high level of luciferase or the resultant light reaction produced upon addition of D-luciferin substrate can have a negative influence on tumor cell growth. To address this issue, we designed an expression vector that allows simultaneous fluorescence and luminescence imaging. Using fluorescence activated cell sorting (FACS), we generated clonal cell populations from a human breast cancer (MCF-7) and a mouse melanoma (B16-F10) cell line that stably expressed different levels of luciferase. We then compared the growth capabilities of these clones in vitro by MTT proliferation assay and in vivo by bioluminescence imaging of tumor growth in live mice. Surprisingly, we found that neither the amount of luciferase nor biophotonic activity was sufficient to inhibit tumor cell growth, in vitro or in vivo. These results suggest that luciferase toxicity is not a necessary consideration when designing bioluminescence experiments, and therefore our approach can be used to rapidly generate high levels of luciferase expression for sensitive imaging experiments. PMID: 21092230 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Effects of intracoronary stem cell transplantation in patients with dilated cardiomyopathy. J Card Fail. 2011 Apr;17(4):272-81 Authors: Vrtovec B, Poglajen G, Sever M, Lezaic L, Domanovic D, Cernelc P, Haddad F, Torre-Amione G We investigated clinical effects of intracoronary transplantation of CD34+ cells in patients with dilated cardiomyopathy (DCM). PMID: 21440864 [PubMed - in process] | | | | | | | | | |  | |  | |  |
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