| A Novel Signaling by Vitamin A/Retinol Promotes Self Renewal of Mouse ES Cells by Activating PI3K/Akt Signaling Pathway Via IGF-1 Receptor.
November 6, 2009 at 9:55 am
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| | A Novel Signaling by Vitamin A/Retinol Promotes Self Renewal of Mouse ES Cells by Activating PI3K/Akt Signaling Pathway Via IGF-1 Receptor. Stem Cells. 2009 Nov 4; Authors: Chen L, Khillan JS Pluripotent embryonic stem (ES) are potential source of all types of cells for regenerative medicine. ES cells maintain pluripotency through a complex interplay of different signaling pathways and transcription factors including leukemia inhibitory factor (LIF), Nanog, Sox2 and Oct3/4. Nanog however, plays a key role in maintaining the pluripotency of mouse and human ESCs. Phosphoinositde 3-kinase (PI3K) signaling pathway that is activated in response to growth factors and cytokines also plays a critical role in promoting the survival and proliferation of ES cells. Our earlier studies have revealed that retinol the alcohol form of vitamin A enhances expression of Nanog and prevents differentiation of ES cells in long term cultures. Normally vitamin A/retinol is associated with cell differentiation via its potent metabolite retinoic acid. Thus far, no direct function has been ascribed to retinol itself. In these studies we demonstrate for the first time that retinol directly activates phosphoinositide 3 (PI3) kinase signaling pathway through IGF-1 receptor/insulin receptor substrate 1 (IRS-1) by engaging Akt/PKB-mTORC1 and mTORC2 complexes indicating a growth factor like function of vitamin A. Further, ES cells do not express enzymes to metabolize retinol into retinoic acid and lack receptors for retinol transport into the cytoplasm indicating that retinol signaling is independent of retinoic acid. The studies present a novel system to investigate how extracellular signals control the self renewal of ES cells which will be important for high quality ES cells for regenerative medicine. PMID: 19890980 [PubMed - as supplied by publisher] |
| Gelatin Hydrogel Prepared by Photo-initiated Polymerization and Loaded with TGF-beta1 for Cartilage Tissue Engineering.
November 6, 2009 at 9:55 am
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| | Gelatin Hydrogel Prepared by Photo-initiated Polymerization and Loaded with TGF-beta1 for Cartilage Tissue Engineering. Macromol Biosci. 2009 Nov 3; Authors: Hu X, Ma L, Wang C, Gao C Gelatin is a nature-derived protein having good cytocompatibility, and widely used in tissue engineering particularly in a form of a hydrogel. To obtain the hydrogel with good enough mechanical properties, however, measures are still need to be taken. In this work, the gelatin molecule was modified with methacrylic acid (MA) to obtain crosslinkable gelatin (GM), which formed a chemically crosslinked hydrogel by photoinitiating polymerization. The gelation time could be easily tuned and showed an inverse relationship with the GM concentration. After photo-irradiation for 20 min there was no detectable double carbon bond in the hydrogen spectrum of high resolution magic angle spinning nuclear magnetic resonance spectroscopy ((1)H HR-MAS NMR). With the increase of the GM concentration, storage modulus and loss modulus of the hydrogels increased, but their swelling ratio and mesh size decreased. Weight loss of the hydrogels was also affected by the polymer concentration. Transform growth factor-beta1 (TGF-beta1) was incorporated into the GM hydrogel to improve its bioactivity. In vitro chondrocyte culture showed that the GM hydrogel had indeed good performance to support chondrocyte growth and maintain chondrocytic phenotype. Incorporation of TGF-beta1 could further improve the biological activity in terms of cell proliferation and extracellular matrix secretion. PMID: 19890886 [PubMed - as supplied by publisher] |
| Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells Under Xeno-Free Conditions.
November 6, 2009 at 9:55 am
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| | Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells Under Xeno-Free Conditions. Stem Cells. 2009 Nov 3; Authors: Rodríguez-Pizà I, Richaud-Patin Y, Vassena R, González F, Barrero MJ, Veiga A, Raya A, Izpisúa Belmonte JC The availability of induced pluripotent stem cells (iPSC) has created extraordinary opportunities for modeling and perhaps treating human disease. However, all reprogramming protocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirely devoid of xenobiotics. We first developed a xeno-free cell culture media that supported the long-term propagation of human embryonic stem cells (hESC) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derived primary cultures of human dermal fibroblasts under strict xeno-free conditions (XF-HFF), and show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin (trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G (VSV-G)-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free human iPSC lines were generated, which could be continuously passaged in xeno-free conditions and maintained characteristics indistinguishable from hESC, including colony morphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiation ability in vitro and in teratoma assays. Overall, the results presented here demonstrate that human iPSC can be generated and maintained under strict xeno-free conditions and provide a path to GMP-applicability that should facilitate the clinical translation of iPSC-based therapies. PMID: 19890879 [PubMed - as supplied by publisher] |
| Dynamic mechanical properties of the tissue-engineered matrix associated with individual chondrocytes.
November 6, 2009 at 9:55 am
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| | Dynamic mechanical properties of the tissue-engineered matrix associated with individual chondrocytes. J Biomech. 2009 Nov 2; Authors: Lee B, Han L, Frank EH, Chubinskaya S, Ortiz C, Grodzinsky AJ The success of cell-based tissue engineering approaches in restoring biological function will be facilitated by a comprehensive fundamental knowledge of the temporal evolution of the structure and properties of the newly synthesized matrix. Here, we quantify the dynamic oscillatory mechanical behavior of the engineered matrix associated with individual chondrocytes cultured in vitro for up to 28 days in alginate scaffolds. The magnitude of the complex modulus (|E*|) and phase shift (delta) were measured in culture medium using Atomic Force Microscopy (AFM)-based nanoindentation in response to an imposed oscillatory deformation (amplitude approximately 5nm) as a function of frequency (f=1-316Hz), probe tip geometry (2.5mum radius sphere and 50nm radius square pyramid), and in the absence and presence of growth factors (GF, insulin growth factor-1, IGF-1, and osteogenic protein-1, OP-1). |E*| for all conditions increased nonlinearly with frequency dependence approximately f(1/2) and ranged between approximately 1 and 25kPa. This result, along with theoretical calculations of the characteristic poroelastic relaxation frequency, f(p), ( approximately 50-90Hz) suggested that this time-dependent behavior was governed primarily by fluid flow-dependent poroelasticity, rather than flow-independent viscoelastic processes associated with the solid matrix. |E*(f)| increased, (f) decreased, and the hydraulic permeability, k, decreased with time in culture and with growth factor treatment. This trend of a more elastic-like response was thought to be associated with increased macromolecular biosynthesis, density, and a more mature matrix structure/organization. PMID: 19889416 [PubMed - as supplied by publisher] |
| Urodynamic evaluation of fesoterodine metabolite, doxazosin and their combination in a rat model of partial urethral obstruction.
November 6, 2009 at 9:55 am
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| | Urodynamic evaluation of fesoterodine metabolite, doxazosin and their combination in a rat model of partial urethral obstruction. BJU Int. 2009 Nov 3; Authors: Füllhase C, Soler R, Gratzke C, Brodsky M, Christ GJ, Andersson KE OBJECTIVE To evaluate the urodynamic effects of fesoterodine, a new antimuscarinic agent, alone and combined with doxazosin, in a rat model of partial urethral obstruction (PUO), as 35-83% of men with bladder outlet obstruction (BOO) secondary to benign prostatic hyperplasia (BPH) have overactive bladder (OAB) syndrome, and as the combination of alpha(1)-adrenoceptor- and muscarinic-receptor antagonists has been proposed to be beneficial for these patients. MATERIALS AND METHODS Thirty-seven male Sprague-Dawley rats (250 g) had surgically induced PUO; 2 weeks later they were evaluated by cystometry with no anaesthesia or any restraint. After a 1-h period either 5-hydroxymethyl tolterodine (5-HMT, the active metabolite of fesoterodine, previously known as SPM 7605), doxazosin or a combination of both, was given intravenously (0.1 mg/kg body weight), and cystometry was continued for another 45 min. Fifteen healthy, age-matched rats served as a control. RESULTS At 2 weeks after surgery the obstructed rats had an greater bladder weight, threshold pressure (TP) and micturition frequency (MF), and lower bladder capacity (BCap) and micturition volume (MV) than the controls. 5-HMT did not cause urinary retention in obstructed rats, but decreased TP, maximum pressure (MP), spontaneous bladder activity (SA) and, paradoxically, increased MF. Doxazosin alone decreased TP, MP, MF and increased BCap and MV. 5-HMT and doxazosin together did not depress the ability to empty the bladder, and showed decreased TP, MP and SA. CONCLUSIONS 5-HMT, alone and in combination, did not impair the voiding ability in obstructed rats. Doxazosin counteracted some of the 'negative' effects of 5-HMT in this model (increase of MF) and did not attenuate the 'positive' effects (decrease of bladder SA). In this model, the combination of 5-HMT and doxazosin appeared to be urodynamically safe and well tolerated. PMID: 19888972 [PubMed - as supplied by publisher] |
| The tumorigenicity of diploid and aneuploid human pluripotent stem cells.
November 6, 2009 at 9:55 am
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| | The tumorigenicity of diploid and aneuploid human pluripotent stem cells. Cell Cycle. 2009 Dec 14;8(23) Authors: Blum B, Benvenisty N Human embryonic stem cells (HESCs) and induced pluripotent stem cells (HiPSCs) offer an immense potential as a source of cells for regenerative medicine. However, the ability of undifferentiated HESCs and HiPSCs to produce tumors in vivo presents a major obstacle for the translation of this potential into clinical reality. Therefore, characterizing the nature of HESC- and HiPSC-derived tumors, especially their malignant potential, is extremely important in order to evaluate the risk involved in their clinical use. Here we review recent observations on the tumorigenicity of human pluripotent stem cells. We argue that diploid, early passage, HESCs produce benign teratomas without undergoing genetic modifications. Conversely, HESCs that acquired genetic or epigenetic changes upon adaptation to in vitro culture can produce malignant teratocarcinomas. We discuss the molecular mechanisms of HESC tumorigenicity and suggest approaches to prevent tumor formation from these cells. We also discuss the differences in the tumorigenicity between mouse embryonic stem cells (MESCs) and HESCs, and suggest methodologies that may help to identify cellular markers for culture adapted HESCs. PMID: 19887907 [PubMed - as supplied by publisher] |
| Pre-clinical Models for Oral and Periodontal Reconstructive Therapies.
November 6, 2009 at 9:55 am
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| | Pre-clinical Models for Oral and Periodontal Reconstructive Therapies. J Dent Res. 2009 Nov 3; Authors: Pellegrini G, Seol YJ, Gruber R, Giannobile WV The development of new medical formulations (NMF) for reconstructive therapies has considerably improved the available treatment options for individuals requiring periodontal repair or oral implant rehabilitation. Progress in tissue engineering and regenerative medicine modalities strongly depends on validated pre-clinical research. Preclinical testing has contributed to the recent approval of NMF such as GEM 21S((R)) and INFUSE((R)) bone grafts for periodontal and oral regenerative therapies. However, the selection of a suitable preclinical model for evaluation of the safety and efficacy of a NMF remains a challenge. This review is designed to serve as a primer to choose the appropriate pre-clinical models for the evaluation of NMF in situations requiring periodontal or oral reconstruction. Here, we summarize commonly used pre-clinical models and provide examples of screening and functional studies of NMF that can be translated into clinical use. PMID: 19887682 [PubMed - as supplied by publisher] |
| Plasticity and recovery of skeletal muscle satellite cells during limb regeneration.
November 6, 2009 at 9:55 am
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| | Plasticity and recovery of skeletal muscle satellite cells during limb regeneration. FASEB J. 2009 Nov 3; Authors: Morrison JI, Borg P, Simon A Salamander limb regeneration depends on local progenitors whose progeny are recruited to the new limb. We previously identified a Pax7(+) cell population in skeletal muscle whose progeny have the potential to contribute to the regenerating limb. However, the plasticity of individual Pax7(+) cells, as well as their recovery within the new limb, was unclear. Here, we show that Pax7(+) cells remain present after multiple rounds of limb amputation/regeneration. Pax7(+) cells are found exclusively within skeletal muscle in the regenerating limb and proliferate where the myofibers are growing. Pax7 is rapidly down-regulated in the blastema, and analyses of clonal derivatives show that Pax7(+) cell progeny are not restricted to skeletal muscle during limb regeneration. Our data suggest that the newt regeneration blastema is not entirely a composite of lineage-restricted progenitors. The results demonstrate that except for a transient and subsequently blunted increase, skeletal muscle satellite cells constitute a stable pool of reserve cells for multiple limb regeneration events.-Morrison, J. I., Borg, P., Simon, A. Plasticity and recovery of skeletal muscle satellite cells during limb regeneration. PMID: 19887652 [PubMed - as supplied by publisher] |
| Lin-Sca-1+CD49fhigh Stem/Progenitors Are Tumor-Initiating Cells in the Pten-Null Prostate Cancer Model.
November 6, 2009 at 9:55 am
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| | Lin-Sca-1+CD49fhigh Stem/Progenitors Are Tumor-Initiating Cells in the Pten-Null Prostate Cancer Model. Cancer Res. 2009 Nov 3; Authors: Mulholland DJ, Xin L, Morim A, Lawson D, Witte O, Wu H We have shown previously that Pten deletion leads to the expansion of subset of prostate cancer cells positive for CK5 and p63. Although this subpopulation may be involved in tumor initiation or progression, studies to date have not functionally validated this hypothesis. Using in vitro sphere-forming assay and in vivo prostate reconstitution assay, we show here the presence of a tumor-initiating subpopulation in the Pten prostate cancer mouse model. Specifically, we show that the Lin(-)Sca-1(+)CD49f(high) (LSC) subpopulation overlaps with CK5(+);p63(+) cells and is significantly increased during prostate cancer initiation and progression and after castration. Mutant spheres mimic the structural organization of the epithelial compartment in the Pten-null primary tumor. Sorted LSC cells from either Pten-null spheres or primary tumors are able to regenerate prostate epithelial structure with cancerous morphology, closely mimicking that of primary cancers. Therefore, the LSC subpopulation is capable of initiating a cancerous phenotype that recapitulates the pathology seen in the primary lesions of the Pten mutant prostate model. [Cancer Res 2009;69(22):8555-62]. PMID: 19887604 [PubMed - as supplied by publisher] |
| Niche recycling through division-independent egress of hematopoietic stem cells.
November 6, 2009 at 9:55 am
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| | Niche recycling through division-independent egress of hematopoietic stem cells. J Exp Med. 2009 Nov 2; Authors: Bhattacharya D, Czechowicz A, Ooi AG, Rossi DJ, Bryder D, Weissman IL Hematopoietic stem cells (HSCs) are thought to reside in discrete niches through stable adhesion, yet previous studies have suggested that host HSCs can be replaced by transplanted donor HSCs, even in the absence of cytoreductive conditioning. To explain this apparent paradox, we calculated, through cell surface phenotyping and transplantation of unfractionated blood, that approximately 1-5% of the total pool of HSCs enters into the circulation each day. Bromodeoxyuridine (BrdU) feeding experiments demonstrated that HSCs in the peripheral blood incorporate BrdU at the same rate as do HSCs in the bone marrow, suggesting that egress from the bone marrow to the blood can occur without cell division and can leave behind vacant HSC niches. Consistent with this, repetitive daily transplantations of small numbers of HSCs administered as new niches became available over the course of 7 d led to significantly higher levels of engraftment than did large, single-bolus transplantations of the same total number of HSCs. These data provide insight as to how HSC replacement can occur despite the residence of endogenous HSCs in niches, and suggest therapeutic interventions that capitalize upon physiological HSC egress. PMID: 19887396 [PubMed - as supplied by publisher] |
| Multiscale 3d scaffolds for soft tissue engineering via multimodal electrospinning.
November 6, 2009 at 9:55 am
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| | Multiscale 3d scaffolds for soft tissue engineering via multimodal electrospinning. Acta Biomater. 2009 Oct 31; Authors: Soliman S, Pagliari S, Rinaldi A, Forte G, Fiaccavento R, Pagliari F, Franzese O, Minieri M, Nardo PD, Licoccia S, Traversa E A novel (scalable) electrospinning process was developed to fabricate bio-inspired multiscale 3D scaffolds endowed with a controlled multimodal distribution of fiber diameters and geared towards soft tissue engineering. The resulting materials finely mingle nano- and microscale fibers together. rather than simply juxtapose as commonly found in literature. A detailed proof-of-concept study was conducted on a simpler bimodal poly(epsilon-caprolactone) (PCL) scaffold with modes of the fiber distribution at 600 nm and 3.3 mum. Three conventional unimodal scaffolds with mean diameters of 300 nm, 2.6 mum, and 5.2 mum respectively were used as controls to evaluate the new materials. The characterization of microstructure (i.e. porosity, fiber distribution, and pore structure) and mechanical properties (i.e. stiffness, strength, and failure mode) indicated that the multimodal scaffold had superior mechanical properties (Young's modulus approximately 40 MPa and strength approximately 1 MPa) in comparison to the controls, despite the large porosity ( approximately 90% on average). A biological assessment was conducted with bone marrow stromal cell type (mesenchymal stem cells, mTERT-MSCs). While the new material compared favourably against the controls as far as cell viability (on the outer surface), it outperformed them in terms of cell colonization within the wall. The latter result, which could neither be practically achieved in the controls nor be expected based on current models of pore size distribution, demonstrated the greater openness of the pore structure of the bimodal material that remarkably did not come at the expense of mechanical properties. Furthermore nanofibers were seen to form a nanoweb bridging across neighbouring microfibers, which contributed to boost cell motility and survival. At last standard adipogenic and osteogenic differentiation tests served to demonstrate that the new scaffold does not hinder the multilineage potential of stem cells. PMID: 19887125 [PubMed - as supplied by publisher] |
| Small Intestinal Submucosa Gel as a Potential Scaffolding Material for Cardiac Tissue Engineering.
November 6, 2009 at 9:55 am
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| | Small Intestinal Submucosa Gel as a Potential Scaffolding Material for Cardiac Tissue Engineering. Acta Biomater. 2009 Oct 31; Authors: Crapo PM, Wang Y Cardiac tissue engineering typically utilizes protein-rich scaffolding materials and growth factors to improve cardiac tissue function in vitro and in vivo. The objectives of this preliminary study were (I) to investigate the potential of porcine small intestinal submucosa gel (SIS gel) in cardiac tissue engineering and (II) to compare the function of tissue based on either SIS gel or Matrigel, a tumor-derived benchmark material. Neonatal rat cardiac cells were combined with either SIS gel or Matrigel and cultured on porous elastomeric scaffolds composed of poly(glycerol sebacate) for 13 days. Tissue function was assessed by measuring contraction rates twice daily. Tissue morphology was compared qualitatively by H&E staining. Normalized troponin T expression (troponin T:DNA) was compared using image analysis. SIS gel constructs contracted at significantly higher rates than Matrigel constructs on days 8-11. Normalized troponin T expression was significantly higher in SIS gel constructs compared to Matrigel constructs. In summary, this research demonstrates that: (I) SIS gel can be used to create contractile engineered cardiac tissue; and (II) SIS gel produced engineered cardiac tissues with a more physiologic contraction rate and higher phenotypic protein expression based on basic in vitro examinations performed in this study. PMID: 19887120 [PubMed - as supplied by publisher] |
| Anti-TNF-alpha-Loaded Microspheres as a Prospective Novel Treatment for Crohn's Disease Fistulae.
November 6, 2009 at 9:55 am
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| | Anti-TNF-alpha-Loaded Microspheres as a Prospective Novel Treatment for Crohn's Disease Fistulae. Tissue Eng Part C Methods. 2009 Nov 3; Authors: Foong KS, Patel R, Forbes A, Day RM Background and Aims: Antibodies to tumour necrosis factor alpha (TNF-alpha) have been successful in treating perianal fistulae in Crohn's disease, but current modes of delivery are limited. Microspheres are currently being assessed as scaffolds for tissue engineering and drug delivery devices. The aim of this study was to produce anti-TNF-alpha antibody encapsulated microspheres using thermally induced phase separation (TIPS) and to characterise their behaviour. Methods: Anti-TNF-alpha antibody was encapsulated into the microspheres (100mg infliximab/g poly(lactide-co-glycolide [PLGA]) w/w) using a novel technique combining a vibration encapsulator unit with a TIPS process, using either lyophilized particulate antibody or an aqueous solution of antibody. Microspheres were incubated in phosphate buffered saline for collection of supernatant and assessment of degradation. The amount and biological activity of the encapsulated antibody released from the microspheres was assessed by enzyme-linked immunosorbent assay (ELISA) and its ability to neutralize recombinant human (rh)TNF-alpha in vitro with a cytotoxicity assay. An in vitro wound scratch assay was used to assess the effect of released antibody on fibroblast migration. Ultrastructural characteristics of the different microspheres were characterised by scanning electron microscopy. Results: Highly porous microspheres released anti-TNF-alpha antibody under zero order kinetics and inhibited the cytotoxic activity of rhTNF-alpha, producing a significant increase in cell viability compared with cells treated with rhTNF-alpha alone. This effect was most pronounced with microspheres fabricated by blending lyophilized particulate anti-TNF-alpha antibody into the polymer solution, which also significantly reduced the release of lactate dehydrogenase. Summary and Conclusions: Anti-TNF-alpha antibody encapsulated into highly porous microspheres was released in a controlled manner and exhibited biological activity against TNF-alpha. The technique used to produce TIPS microspheres is rapid and provides high encapsulation efficiency. This technique could also be applied to other therapeutic peptides where rapid fabrication and high yields are required. PMID: 19886803 [PubMed - as supplied by publisher] |
| Mesenchymal stromal cells for cardiovascular repair: current status and future challenges.
November 6, 2009 at 9:55 am
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| | Mesenchymal stromal cells for cardiovascular repair: current status and future challenges. Future Cardiol. 2009 Nov;5(6):605-17 Authors: Mathiasen AB, Haack-Sørensen M, Kastrup J Ischemic heart disease is the most common cause of death in most industrialized countries. Early treatment with stabilizing drugs and mechanical revascularization by percutaneous coronary intervention or coronary bypass surgery has reduced the mortality significantly. In spite of improved offers of treatments in patients with heart failure, the 1-year mortality is still approximately 20% after the diagnosis has been established. Treatment with stem cells with the potential to regenerate the damaged myocardium is a relatively new approach. Mesenchymal stromal cells are a promising source of stem cells for regenerative therapy. Clinical studies on stem cell therapy for cardiac regeneration have shown significant improvements in ventricular pump function, ventricular remodeling, myocardial perfusion, exercise potential and clinical symptoms compared with conventionally treated control groups. The results of most studies are promising, but there are still many unanswered questions. In this review, we explore present preclinical and clinical knowledge regarding the use of stem cells in cardiovascular regenerative medicine, with special focus on mesenchymal stromal cells. We take a closer look at sources of stem cells, delivery method and methods for tracking injected cells. PMID: 19886787 [PubMed - in process] |
| Sonic Hedgehog promotes angiogenesis and osteogenesis in a co-culture system consisting of primary osteoblasts and outgrowth endothelial cells.
November 6, 2009 at 9:55 am
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| | Sonic Hedgehog promotes angiogenesis and osteogenesis in a co-culture system consisting of primary osteoblasts and outgrowth endothelial cells. Tissue Eng Part A. 2009 Nov 3; Authors: Dohle E, Fuchs S, Kolbe M, Hofmann A, Schmidt H, Kirkpatrick J A number of previous studies documented the angiogenic potential of outgrowth endothelial cells (OEC) in vitro and in vivo and provided evidence that therapeutic success could depend on co-culture or co-implantation strategies. Thus, deeper insight into the molecular mechanisms underlying this pro-angiogenic effect of co-cultures might provide new translational options for tissue engineering and regenerative medicine. One promising signaling pathway in bone repair involved in neoangiogenesis and bone formation, is the sonic hedgehog (Shh) pathway. In this paper we focus on the effect of Shh on the formation of microvessel-like structures and osteoblastic differentiation in co-cultures of primary osteoblasts (pOB) and OEC. Already after 24 hours of treatment, Shh leads to a massive increase in microvessel-like structures compared to untreated co-cultures. Increased formation of angiogenic structures seems to correlate with the upregulation of vascular endothelial growth factor (VEGF) or angiopoietins (Ang-1, Ang-2) studied at both the mRNA and protein-level. In addition, treatment with cyclopamine, an inhibitor of hedgehog signaling, blocked the formation of microvessel-like structures in the co-cultures. However, exogenous Shh also resulted in the upregulation of several osteogenic differentiation markers in real time PCR, as well as in an increased mineralization and alkaline phosphatase (ALP) activity. The present data highlight the central role of the Shh pathway in bone regeneration and vascularization. Furthermore sonic hedgehog might have the potential to improve both angiogenesis and osteogenesis in clinical applications in the future. PMID: 19886747 [PubMed - as supplied by publisher] |
| Nylon-3 Copolymers that Generate Cell-Adhesive Surfaces Identified by Library Screening.
November 6, 2009 at 9:55 am
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| | Nylon-3 Copolymers that Generate Cell-Adhesive Surfaces Identified by Library Screening. J Am Chem Soc. 2009 Nov 3; Authors: Lee MR, Stahl SS, Gellman SH, Masters KS Polymers in the nylon-3 family contain subunits derived from beta-amino acids, which are linked to one another via amide bonds. Thus, the nylon-3 backbone is homologous to the alpha-amino acid-based backbone of proteins. This molecular-level homology suggests that nylon-3 materials might be intrinsically protein-mimetic. The experiments described here explore this prospect in the context of cell adhesion, with tissue engineering as a long-range goal. We have evaluated a small library of sequence-random nylon-3 copolymers for the ability to render surfaces attractive to NIH 3T3 fibroblast adhesion and spreading. Library screening was accomplished in a high-throughput, parallel mode via attachment of the copolymers in a two-dimensional array to a modified glass surface. Significant variations in fibroblast adhesion and spreading were observed as a function of nylon-3 subunit identity and proportion. Several of the nylon-3 copolymers supported cell adhesion and morphology that was comparable, or even superior, to that achieved on positive control substrates such as tissue culture polystyrene and collagen-coated glass. Moreover, studies conducted under serum-free conditions demonstrated that specific nylon-3 derivatives supported cell adhesion independently of serum protein adsorption. Although cell adhesion was diminished in the absence of serum, particular copolymers demonstrated an ability to support substantially greater cell adhesion than any of the other conditions, including the positive controls. The nylon-3 copolymers that were most effective at promoting adhesion to a modified glass surface proved also to be effective at promoting adhesion when attached to a PEG-based hydrogel, demonstrating the potential for these copolymers to be used in tissue engineering applications. PMID: 19886604 [PubMed - as supplied by publisher] |
| Stem cells in stroke--appraisal of literature.
November 6, 2009 at 9:55 am
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| | Stem cells in stroke--appraisal of literature. J Indian Med Assoc. 2009 Jun;107(6):400, 402 Authors: Bhattacharya AK, Banik KK The repair of human brain after stroke appears unrealistic as there is loss of many different neurons and glial cells. The functional improvement after stroke or neurorestorative process includes neurogenesis, angiogenesis and synaptic plasticity. Stem cell therapy has the potential in inducing all of the above neurorestorative processes--thus facilitating functional recovery. The replacement therapy includes stem cells derived from various tissues which can generate functional neurons and glia. Stem cell research is an exciting new avenue in the field of stroke management. PMID: 19886378 [PubMed - in process] |
| Engineering osteogenesis and chondrogenesis with gene-enhanced therapeutic cells.
November 6, 2009 at 9:55 am
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| | Engineering osteogenesis and chondrogenesis with gene-enhanced therapeutic cells. Curr Opin Mol Ther. 2009 Aug;11(4):404-10 Authors: Hao J, Varshney RR, Wang DA For bone and cartilage regeneration, a direct dose of growth factors or the viral or non-viral vector-mediated delivery of growth factor genes to the site of osteal or chondral wounds has disadvantages. These limitations include the short half-life and instability of the proteins, resulting in low efficacy with the repeated administration of the therapy, and the nonspecific targeting of the therapy that elevates general toxicity and systemic immunogenicity. To address these challenges, the focus of gene therapy for bone and cartilage repair has shifted in recent years to the use of autologous cells, typically osteocytes or chondrocytes, or their progenitors, transfected with therapeutic genes; the cells are cultivated in vitro before in vivo transplantation. These gene-enhanced therapeutic cells provide sustained autocrine/paracrine stimulation and localized gene expression. An important advantage of the cell-based approach is that factors contributing to off-target toxicity and immunogenicity are metabolically cleared during the in vitro incubation of the transfected cells prior to being administered to the transplant recipients. This review focuses on gene therapy approaches for treating bone and joint disorders, and specifically discusses the development of cell-based delivery approaches. PMID: 19649985 [PubMed - indexed for MEDLINE] |
| Mesenchymal stem cells targeting the GVHD.
November 6, 2009 at 9:55 am
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| | Mesenchymal stem cells targeting the GVHD. Sci China C Life Sci. 2009 Jul;52(7):603-9 Authors: Wang L, Zhao RC Acute graft-versus-host disease (GVHD) occurs after allogeneic hematopoietic stem cell transplant and is a reaction of donor immune cells against host tissues. About 35%-50% of hematopoietic stem cell transplant (HSCT) recipients will develop acute GVHD. It is associated with considerable morbidity and mortality, particularly in patients who do not respond to primary therapy, which usually consists of glucocorticoids(steroids). Most of the available second-line and third-line treatments for steroid-refractory acute GVHD induce severe immunodeficiency, which is commonly accompanied by lethal infectious complications. Mesenchymal stem cells (MSCs) have been shown to mediate immunomodulatory effects. The recently elucidated immunosuppressive potential of mesenchymal stem cells has set the stage for their clinical testing as cellular immunosuppressants, MSCs have been used in patients with steroid-refractory acute GVHD, and encouraging responses have been obtained in many studies. The utility of MSCs for the treatment of GVHD is becoming clear. PMID: 19641864 [PubMed - indexed for MEDLINE] |
| Characterization of engineered tissue construct mechanical function by magnetic resonance imaging.
November 6, 2009 at 9:55 am
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| | Characterization of engineered tissue construct mechanical function by magnetic resonance imaging. J Tissue Eng Regen Med. 2009 Aug;3(6):477-85 Authors: Neu CP, Arastu HF, Curtiss S, Reddi AH Non-invasive magnetic resonance imaging (MRI) is a technology that enables the characterization of multiple physical phenomena in living and engineered tissues. The mechanical function of engineered tissues is a primary endpoint for the successful regeneration of many biological tissues, such as articular cartilage, spine and heart. Here we demonstrate the application of MRI to characterize the mechanical function of engineered tissue. Phase contrast-based methods were demonstrated to characterize detailed deformation fields throughout the interior of native and engineered tissue, using an articular cartilage defect model as a study system. MRI techniques revealed that strain fields varied non-uniformly, depending on spatial position. Strains were highest in the tissue constructs compared to surrounding native cartilage. Tissue surface geometry corresponded to strain fields observed within the tissue interior near the surface. Strain fields were further evaluated with respect to the spatial variation in the concentration of glycosaminoglycans ([GAG]), critical proteoglycans in the extracellular matrix of cartilage, as determined by gadolinium-enhanced imaging. [GAG] also varied non-uniformly, depending on spatial position and was lowest in the tissue constructs compared to the surrounding cartilage. The use of multiple MRI techniques to assess tissue mechanical function provides complementary data and suggests that deformation is related to tissue geometry, underlying extracellular matrix constituents and the lack of tissue integration in the model system studied. Specialized and advanced MRI phase contrast-based methods are valuable for the detailed characterization and evaluation of mechanical function of tissue-engineered constructs. PMID: 19530259 [PubMed - indexed for MEDLINE] |
| Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts.
November 6, 2009 at 9:55 am
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| | Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts. FASEB J. 2009 Oct;23(10):3325-34 Authors: Vandenburgh H, Shansky J, Benesch-Lee F, Skelly K, Spinazzola JM, Saponjian Y, Tseng BS Identification of factors that improve muscle function in boys with Duchenne muscular dystrophy (DMD) could lead to an improved quality of life. To establish a functional in vitro assay for muscle strength, mdx murine myoblasts, the genetic homologue of DMD, were tissue engineered in 96-microwell plates into 3-dimensional muscle constructs with parallel arrays of striated muscle fibers. When electrically stimulated, they generated tetanic forces measured with an automated motion tracking system. Thirty-one compounds of interest as potential treatments for patients with DMD were tested at 3 to 6 concentrations. Eleven of the compounds (insulin-like growth factor-1, creatine, beta-hydroxy-beta-methylbutyrate, trichostatin A, lisinopril, and 6 from the glucocorticoid family) significantly increased tetanic force relative to placebo-treated controls. The glucocorticoids methylprednisolone, deflazacort, and prednisone increased tetanic forces at low doses (EC(50) of 6, 19, and 56 nM, respectively), indicating a direct muscle mechanism by which they may be benefitting DMD patients. The tetanic force assay also identified beneficial compound interactions (arginine plus deflazacort and prednisone plus creatine) as well as deleterious interactions (prednisone plus creatine inhibited by pentoxifylline) of combinatorial therapies taken by some DMD patients. Since mdx muscle in vivo and DMD patients respond in a similar manner to many of these compounds, the in vitro assay will be a useful tool for the rapid identification of new potential treatments for muscle weakness in DMD and other muscle disorders. PMID: 19487307 [PubMed - indexed for MEDLINE] |
| Different sensitivity of human endothelial cells, smooth muscle cells and fibroblasts to topography in the nano-micro range.
November 6, 2009 at 9:55 am
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| | Different sensitivity of human endothelial cells, smooth muscle cells and fibroblasts to topography in the nano-micro range. Acta Biomater. 2009 Sep;5(7):2460-6 Authors: Biela SA, Su Y, Spatz JP, Kemkemer R Cell adhesion, orientation and migration are influenced by surface topographies in the micrometer and nanometer range. In this work, we demonstrate the stimulation by topographical signals of human fibroblast cells (FCs), endothelial cells (ECs) and smooth muscle cells (SMCs). We systematically quantified the contact guidance alignment and directed migration of FCs, ECs and SMCs adhering to grooved substrates with lateral dimensions of 2-10microm and depths of 50-200nm. We found a common quantitative response characteristic of all three cell types: contact guidance significantly increased when the cells were cultured on substrates with smaller lateral dimensions or deeper grooves. Despite their general behavior, the three cell types exhibited a cell-type specific sensitivity to the groove patterns. The minimum groove depth to induce an orientation response and change cell shape was 50nm for FCs and about two times deeper for ECs and SMCs. The degree of alignment and directed migration of the FCs along the grooves was significantly stronger than for the ECs and SMCs. We demonstrate that ECs and SMCs can be stimulated by topographical signals but are less sensitive than FCs. PMID: 19410529 [PubMed - indexed for MEDLINE] |
| Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion.
November 6, 2009 at 9:55 am
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| | Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion. Acta Biomater. 2009 Sep;5(7):2441-50 Authors: Damodaran G, Collighan R, Griffin M, Navsaria H, Pandit A Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides, which facilitates cell adhesion, migration and proliferation. In this study, we evaluated the cell adhesion properties of a tailored laminin-332 alpha3 chain tethered to a type I collagen scaffold using microbial transglutaminase (mTGase) by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide A: PPFLMLLKGSTREAQQIVM) or lysine (peptide B: PPFLMLLKGSTRKKKKG). The degree of cross-linking was studied by amino acid analysis following proteolytic digestion and the structural changes in the modified scaffold further investigated using Fourier transform infrared spectroscopy and atomic force microscopy. Fibroblasts were used to evaluate the cellular behaviour of the functionalized collagen scaffold. mTGase supports cell growth but tethering of peptide A and peptide B to the mTGase cross-linked collagen scaffold caused a significant increase in cell proliferation when compared with native and mTGase cross-linked collagen scaffolds. Both peptides enabled cell-spreading, attachment and normal actin cytoskeleton organization with slight increase in the cell proliferation was observed in peptide A when compared with the peptide B and mTGase cross-linked scaffold. An increase in the amount of epsilon(gamma-glutamyl) lysine isopeptide was observed in peptide A conjugated scaffolds when compared with peptide B conjugated scaffolds, mTGase cross-linked scaffold without peptide. Changes in D-spacing were observed in the cross-linked scaffolds with tethered peptides. These results demonstrate that mTGase can play a bifunctional role in both conjugation of the glutamine and lysine containing peptide sequences and also in the cross-linking of the collagen scaffold, thus providing a suitable substrate for cell growth. PMID: 19364681 [PubMed - indexed for MEDLINE] |
| A poly(L-lactic acid) nanofibre mesh scaffold for endothelial cells on vascular prostheses.
November 6, 2009 at 9:55 am
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| | A poly(L-lactic acid) nanofibre mesh scaffold for endothelial cells on vascular prostheses. Acta Biomater. 2009 Sep;5(7):2418-28 Authors: François S, Chakfé N, Durand B, Laroche G The absence of neoendothelium covering the intimal surface of small-diameter PET vascular prostheses is known to be one cause of failure following implantation in humans. Protein coatings currently used to seal porous textile structures have not shown evidence of in vivo neoendothelium formation. In this study, we covered the inner wall of textile prostheses with a biodegradable synthetic scaffold made of poly(l-lactic) acid (PLLA) nanofibres obtained by an air-spinning process we developed that produces nanofibres by stretching a solution of polymer with a high-speed compressed air jet. The air spinning was designed to process a scaffold that would support good endothelial cell proliferation. Our innovative process enabled us to very rapidly cover textile samples with PLLA nanofibres to determine the influence of air pressure, polymer solution flow rate and polymer concentration on fibre quality. High air pressure was shown to induce a significant number of ruptures. High polymer flow rate stimulated the formation of polymer droplets, and the fibre diameter mean increased for the 4% and 7% polymer concentrations. The adherence and proliferation of bovine aortic endothelial cells was assessed to compare prosthesis samples with or without the PLLA nanofibre scaffold and PET film. The PLLA nanofibres displayed a significantly better proliferation rate, and enabled endothelial cells to proliferate in the monolayer. Our novel approach therefore opens the door to the development of partially degradable textile prostheses with a blood/textile interface that supports endothelial cell proliferation. PMID: 19345622 [PubMed - indexed for MEDLINE] |
| Biofunctionalized electrospun silk mats as a topical bioactive dressing for accelerated wound healing.
November 6, 2009 at 9:55 am
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| | Biofunctionalized electrospun silk mats as a topical bioactive dressing for accelerated wound healing. Acta Biomater. 2009 Sep;5(7):2570-8 Authors: Schneider A, Wang XY, Kaplan DL, Garlick JA, Egles C Materials able to deliver topically bioactive molecules represent a new generation of biomaterials. In this article, we describe the use of silk mats, made of electrospun nanoscale silk fibers containing epidermal growth factor (EGF), for the promotion of wound healing processes. In our experiments, we demonstrated that EGF is incorporated into the silk mats and slowly released in a time-dependent manner (25% EGF release in 170h). We tested these materials using a new model of wounded human skin-equivalents displaying the same structure as human skin and able to heal using the same molecular and cellular mechanisms found in vivo. This human three-dimensional model allows us to demonstrate that the biofunctionalized silk mats, when placed on the wounds as a dressing, aid the healing by increasing the time of wound closure by the epidermal tongue by 90%. The preservation of the structure of the mats during the healing period as demonstrated by electronic microscopy, the biological action of the dressing, as well as the biocompatibility of the silk demonstrate that this biomaterial is a new and very promising material for medical applications, especially for patients suffering from chronic wounds. PMID: 19162575 [PubMed - indexed for MEDLINE] |
| A continuum of cell States spans pluripotency and lineage commitment in human embryonic stem cells.
November 6, 2009 at 3:55 am
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| | A continuum of cell States spans pluripotency and lineage commitment in human embryonic stem cells. PLoS One. 2009;4(11):e7708 Authors: Hough SR, Laslett AL, Grimmond SB, Kolle G, Pera MF BACKGROUND: Commitment in embryonic stem cells is often depicted as a binary choice between alternate cell states, pluripotency and specification to a particular germ layer or extraembryonic lineage. However, close examination of human ES cell cultures has revealed significant heterogeneity in the stem cell compartment. METHODOLOGY/PRINCIPAL FINDINGS: We isolated subpopulations of embryonic stem cells using surface markers, then examined their expression of pluripotency genes and lineage specific transcription factors at the single cell level, and tested their ability to regenerate colonies of stem cells. Transcript analysis of single embryonic stem cells showed that there is a gradient and a hierarchy of expression of pluripotency genes in the population. Even cells at the top of the hierarchy generally express only a subset of the stem cell genes studied. Many cells co-express pluripotency and lineage specific genes. Cells along the continuum show a progressively decreasing likelihood of self renewal as their expression of stem cell surface markers and pluripotency genes wanes. Most cells that are positive for stem cell surface markers express Oct-4, but only those towards the top of the hierarchy express the nodal receptor TDGF-1 and the growth factor GDF3. SIGNIFICANCE: These findings on gene expression in single embryonic stem cells are in concert with recent studies of early mammalian development, which reveal molecular heterogeneity and a stochasticity of gene expression in blastomeres. Our work indicates that only a small fraction of the population resides at the top of the hierarchy, that lineage priming (co-expression of stem cell and lineage specific genes) characterizes pluripotent stem cell populations, and that extrinsic signaling pathways are upstream of transcription factor networks that control pluripotency. PMID: 19890402 [PubMed - in process] |
| A novel nanocomposite polymer for development of synthetic heart valve leaflets.
November 6, 2009 at 3:55 am
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| | A novel nanocomposite polymer for development of synthetic heart valve leaflets. Acta Biomater. 2009 Sep;5(7):2409-17 Authors: Kidane AG, Burriesci G, Edirisinghe M, Ghanbari H, Bonhoeffer P, Seifalian AM A novel nanocomposite polymer with a polycarbonate soft segment (PCU) and polyhedral oligomeric silsesquioxanes (POSS) nanoparticle (POSS-PCU) has been selected for a synthetic heart valve due to its superior biocompatibility and in vivo biostability. However, the development of synthetic heart valves from polymeric materials requires an understanding of the basic mechanical and surface properties of the polymer. In this study, the mechanical properties of POSS-PCU, including tensile strength, tear strength and hardness, were tested and compared to control (PCU). The surface property was analyzed using contact angle measurement and the resistance to platelet adhesion was also investigated. POSS-PCU (hardness 84+/-0.8 Shore A) demonstrated significantly higher tensile strength 53.6+/-3.4 and 55.9+/-3.9Nmm(-2) at 25 and 37 degrees C, respectively) than PCU (33.8+/-2.1 and 28.8+/-3.4Nmm(-2) at 25 and 37 degrees C, respectively). Tensile strength and elongation at break of POSS-PCU was significantly higher than PCU at both 25 and 37 degrees C (P<0.001). POSS-PCU showed a relatively low Young's modulus (25.9+/-1.9 and 26.2+/-2.0Nmm(-2)) which was significantly greater in comparison with control PCU (9.1+/-0.9 and 8.4+/-0.5Nmm(-2)) at 25 and 37 degrees C, respectively, with 100mum thickness. There was no significant difference (P>0.05) in tear strength between POSS-PCU and PCU at 25 degrees C. However, tear strength increased significantly (P<0.001) (at 37 degrees C) as the thickness increased from 100microm (51.0+/-3.3Nmm(-1)) to 200microm (63+/-1.5Nmm(-1)). The surface of POSS-PCU was significantly less hydrophilic than that of PCU. PMID: 19497802 [PubMed - indexed for MEDLINE] | | | 
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