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| Geron's Clinical Trial: Risk, Front-loading and Costs
July 31, 2010 at 9:47 PM
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| The Vatican doesn't particularly care for the idea. "Unacceptable" was the word it used.
Over at Bioworld Today, it was "the dawn of a new era."
A reader of the New York Times warned of the danger of "front-loaded" arguments that can be detrimental to stem cell research.
What they were talking about is Geron and its re-vitalized clinical trial of an hESC therapy for spinal injuries. The FDA | | |
| Recreating the perivascular niche ex vivo using a microfluidic approach.
July 31, 2010 at 4:10 PM
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| | Recreating the perivascular niche ex vivo using a microfluidic approach. Biotechnol Bioeng. 2010 Jul 29; Authors: Carrion B, Huang CP, Ghajar CM, Kachgal S, Kniazeva E, Jeon NL, Putnam AJ Stem cell niches are composed of numerous microenvironmental features, including soluble and insoluble factors, cues from other cells, and the extracellular matrix (ECM), which collectively serve to maintain stem cell quiescence and promote their ability to support tissue homeostasis. A hallmark of many adult stem cell niches is their proximity to the vasculature in vivo, a feature common to neural stem cells, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue, hematopoietic stem cells, and many tumor stem cells. In this study, we describe a novel 3D microfluidic device (MFD) as a model system in which to study the molecular regulation of perivascular stem cell niches. Endothelial cells (ECs) suspended within 3D fibrin gels patterned in the device adjacent to stromal cells (either fibroblasts or bone marrow-derived mesenchymal stem cells) executed a morphogenetic process akin to vasculogenesis, forming a primitive vascular plexus and maturing into a robust capillary network with hollow well-defined lumens. Both MSCs and fibroblasts formed pericytic associations with the ECs, but promoted capillary morphogenesis with distinct kinetics. Biochemical assays within the niche revealed that the perivascular association of MSCs required interaction between their alpha6beta1 integrin receptor and EC-deposited laminin. These studies demonstrate the potential of this physiologically relevant ex vivo model system to study how proximity to blood vessels may influence stem cell multipotency. (c) 2010 Wiley Periodicals, Inc. PMID: 20672286 [PubMed - as supplied by publisher] | | |
| Gene therapy and angiogenesis in patients with coronary artery disease.
July 31, 2010 at 10:39 AM
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| | Gene therapy and angiogenesis in patients with coronary artery disease. Expert Rev Cardiovasc Ther. 2010 Aug;8(8):1127-38 Authors: Kastrup J Not all patients with severe coronary artery disease can be treated satisfactorily with current recommended medications and revascularization techniques. Various vascular growth factors have the potential to induce angiogenesis in ischemic tissue. Clinical trials have only evaluated the effect of VEGF and FGF in patients with coronary artery disease. The initial small and unblinded studies with either recombinant growth factor proteins or genes encoding growth factors were encouraging, demonstrating both clinical improvement and evidence of angiogenesis. However, subsequent larger double-blind placebo-controlled trials could not confirm the initial high efficacy of either the growth factor protein or the gene therapy approaches observed in earlier small trials. The clinical studies so far have all been without any gene-related serious adverse events. Future trials will focus on whether an improvement in clinical results can be obtained with a cocktail of growth factors or by a combination of gene and stem cell therapy in patients with severe coronary artery disease, which cannot be treated effectively with current treatment strategies. PMID: 20670190 [PubMed - in process] | | |
| Neuregulin/ErbB Signaling Regulates Cardiac Subtype Specification in Differentiating Human Embryonic Stem Cells.
July 31, 2010 at 9:22 AM
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| | Neuregulin/ErbB Signaling Regulates Cardiac Subtype Specification in Differentiating Human Embryonic Stem Cells. Circ Res. 2010 Jul 29; Authors: Zhu WZ, Xie Y, Moyes KW, Gold JD, Askari B, Laflamme MA Rationale: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) exhibit either a "working" chamber or a nodal-like phenotype. To generate optimal hESC-CM preparations for eventual clinical application in cell-based therapies, we will need to control their differentiation into these specialized cardiac subtypes. Objective: To demonstrate intact neuregulin (NRG)-1beta/ErbB signaling in hESC-CMs and test the hypothesis that this signaling pathway regulates cardiac subtype abundance in hESC-CM cultures. Methods and Results: All experiments used hESC-CM cultures generated using our recently reported directed differentiation protocol. To support subsequent action potential phenotyping approaches and provide a higher-throughput method of determining cardiac subtype, we first developed and validated a novel genetic label that identifies nodal-type hESC-CMs. Next, control hESC-CM preparations were compared to those differentiated in the presence of exogenous NRG-1beta, an anti-NRG-1beta neutralizing antibody, or the ErbB antagonist AG1478. We used 3 independent approaches to determine the ratio of cardiac subtypes in the resultant populations: direct action potential phenotyping under current-clamp, activation of the aforementioned genetic label, and subtype-specific marker expression by RT-PCR. Using all 3 end points, we found that inhibition of NRG-1beta/ErbB signaling greatly enhanced the proportion of cells showing the nodal phenotype. Conclusions: NRG-1beta/ErbB signaling regulates the ratio of nodal- to working-type cells in differentiating hESC-CM cultures and presumably functions similarly during early human heart development. We speculate that, by manipulating NRG-1beta/ErbB signaling, it will be possible to generate preparations of enriched working-type myocytes for infarct repair, or, conversely, nodal cells for potential use in a biological pacemaker. PMID: 20671236 [PubMed - as supplied by publisher] | | |
| Reconstructing the lung.
July 31, 2010 at 9:22 AM
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| | Reconstructing the lung. Science. 2010 Jul 30;329(5991):520-2 Authors: Wagner WR, Griffith BP PMID: 20671176 [PubMed - in process] | | |
| Generation of induced pluripotent stem cells in rabbits: potential experimental models for human regenerative medicine.
July 31, 2010 at 9:22 AM
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| | Generation of induced pluripotent stem cells in rabbits: potential experimental models for human regenerative medicine. J Biol Chem. 2010 Jul 29; Authors: Honda A, Hirose M, Hatori M, Matoba S, Miyoshi H, Inoue K, Ogura A Human induced pluripotent stem (iPS) cells have the potential to establish a new field of promising regenerative medicine. Therefore, the safety and the efficiency of iPS-derived cells must be tested rigorously using appropriate animal models before human trials can commence. Here, we report the establishment of rabbit iPS cells as the first human-type iPS cells generated from a small laboratory animal species. Using lentiviral vectors, four human reprogramming genes (c-MYC, KLF4, SOX2 and OCT3/4) were introduced successfully into adult rabbit liver and stomach cells. The resulting rabbit iPS cells closely resembled human iPS cells; they formed flattened colonies with sharp edges and proliferated indefinitely in the presence of bFGF. They expressed the endogenous pluripotency markers c-MYC, KLF4, SOX2, OCT3/4 and NANOG, while the introduced human genes were completely silenced. Using in vitro differentiating conditions, rabbit iPS cells readily differentiated into ectoderm, mesoderm and endoderm. They also formed teratomas containing a variety of tissues of all three germ layers in immunodeficient mice. Thus, the rabbit iPS cells fulfilled all of the requirements for the acquisition of the fully reprogrammed state and can be considered "true" iPS cells, which are very similar to their embryonic stem (ES) cell counterparts we recently generated. However, their global gene expression analysis revealed a slight, but rigid difference between these two types of rabbit pluripotent stem cells. The rabbit model should enable us to compare iPS cells and ES cells under the same standardized conditions in evaluating their ultimate feasibility for pluripotent cell-based regenerative medicine in humans. PMID: 20670936 [PubMed - as supplied by publisher] | | |
| Minimally-Invasive Implantation of Living Tissue Engineered Heart Valves A Comprehensive Approach From Autologous Vascular Cells to Stem Cells.
July 31, 2010 at 9:22 AM
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| | Minimally-Invasive Implantation of Living Tissue Engineered Heart Valves A Comprehensive Approach From Autologous Vascular Cells to Stem Cells. J Am Coll Cardiol. 2010 Aug 3;56(6):510-520 Authors: Schmidt D, Dijkman PE, Driessen-Mol A, Stenger R, Mariani C, Puolakka A, Rissanen M, Deichmann T, Odermatt B, Weber B, Emmert MY, Zund G, Baaijens FP, Hoerstrup SP OBJECTIVES: The aim of this study was to demonstrate the feasibility of combining the novel heart valve replacement technologies of: 1) tissue engineering; and 2) minimally-invasive implantation based on autologous cells and composite self-expandable biodegradable biomaterials. BACKGROUND: Minimally-invasive valve replacement procedures are rapidly evolving as alternative treatment option for patients with valvular heart disease. However, currently used valve substitutes are bioprosthetic and as such have limited durability. To overcome this limitation, tissue engineering technologies provide living autologous valve replacements with regeneration and growth potential. METHODS: Trileaflet heart valves fabricated from biodegradable synthetic scaffolds, integrated in self-expanding stents and seeded with autologous vascular or stem cells (bone marrow and peripheral blood), were generated in vitro using dynamic bioreactors. Subsequently, the tissue engineered heart valves (TEHV) were minimally-invasively implanted as pulmonary valve replacements in sheep. In vivo functionality was assessed by echocardiography and angiography up to 8 weeks. The tissue composition of explanted TEHV and corresponding control valves was analyzed. RESULTS: The transapical implantations were successful in all animals. The TEHV demonstrated in vivo functionality with mobile but thickened leaflets. Histology revealed layered neotissues with endothelialized surfaces. Quantitative extracellular matrix analysis at 8 weeks showed higher values for deoxyribonucleic acid, collagen, and glycosaminoglycans compared to native valves. Mechanical profiles demonstrated sufficient tissue strength, but less pliability independent of the cell source. CONCLUSIONS: This study demonstrates the principal feasibility of merging tissue engineering and minimally-invasive valve replacement technologies. Using adult stem cells is successful, enabling minimally-invasive cell harvest. Thus, this new technology may enable a valid alternative to current bioprosthetic devices. PMID: 20670763 [PubMed - as supplied by publisher] | | |
| Nanomedicine in Ophthalmology: The New Frontier.
July 31, 2010 at 9:22 AM
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| | Nanomedicine in Ophthalmology: The New Frontier. Am J Ophthalmol. 2010 Aug;150(2):144-162.e2 Authors: Zarbin MA, Montemagno C, Leary JF, Ritch R PURPOSE: To review the fields of nanotechnology and nanomedicine as they relate to the development of treatments for vision-threatening disorders. DESIGN: Perspective following literature review. METHODS: Analysis of relevant publications in the peer-reviewed scientific literature. RESULTS: Nanotechnology involves the creation and use of materials and devices at the size scale of intracellular structures and molecules and involves systems and constructs on the order of <100 nm. The aim of nanomedicine is the comprehensive monitoring, control, construction, repair, defense, and improvement of human biological systems at the molecular level, using engineered nanodevices and nanostructures, operating massively in parallel at the single cell level, ultimately to achieve medical benefit. The earliest impact of nanomedicine is likely to involve the areas of biopharmaceuticals (eg, drug delivery, drug discovery), implantable materials (eg, tissue regeneration scaffolds, bioresorbable materials), implantable devices (eg, intraocular pressure monitors, glaucoma drainage valves), and diagnostic tools (eg, genetic testing, imaging, intraocular pressure monitoring). Nanotechnology will bring about the development of regenerative medicine (ie, replacement and improvement of cells, tissues, and organs), ultrahigh-resolution in vivo imaging, microsensors and feedback devices, and artificial vision. "Regenerative nanomedicine," a new subfield of nanomedicine, uses nanoparticles containing gene transcription factors and other modulating molecules that allow for the reprogramming of cells in vivo. CONCLUSIONS: Nanotechnology already has been applied to the measurement and treatment of different disease states in ophthalmology (including early- and late-stage disease), and many additional innovations will occur during the next century. PMID: 20670739 [PubMed - as supplied by publisher] | | |
| Generating induced pluripotent stem cells from common marmoset (Callithrix jacchus) fetal liver cells using defined factors, including Lin28.
July 31, 2010 at 9:22 AM
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| | Generating induced pluripotent stem cells from common marmoset (Callithrix jacchus) fetal liver cells using defined factors, including Lin28. Genes Cells. 2010 Jul 28; Authors: Tomioka I, Maeda T, Shimada H, Kawai K, Okada Y, Igarashi H, Oiwa R, Iwasaki T, Aoki M, Kimura T, Shiozawa S, Shinohara H, Suemizu H, Sasaki E, Okano H Although embryonic stem (ES) cell-like induced pluripotent stem (iPS) cells have potential therapeutic applications in humans, they are also useful for creating genetically modified human disease models in nonhuman primates. In this study, we generated common marmoset iPS cells from fetal liver cells via the retrovirus-mediated introduction of six human transcription factors: Oct-3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28. Four to five weeks after introduction, several colonies resembling marmoset ES cells were observed and picked for further expansion in ES cell medium. Eight cell lines were established, and validation analyses of the marmoset iPS cells followed. We detected the expression of ES cell-specific surface markers. Reverse transcription-PCR showed that these iPS cells expressed endogenous Oct-3/4, Sox2, Klf4, c-Myc, Nanog and Lin28 genes, whereas all of the transgenes were silenced. Karyotype analysis showed that two of three iPS cell lines retained a normal karyotype after a 2-month culture. Both embryoid body and teratoma formation showed that marmoset iPS cells had the developmental potential to give rise to differentiated derivatives of all three primary germ layers. In summary, we generated marmoset iPS cells via the transduction of six transcription factors; this provides a powerful preclinical model for studies in regenerative medicine. PMID: 20670273 [PubMed - as supplied by publisher] | | |
| The roadmap to personalized medicine.
July 31, 2010 at 9:22 AM
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| | The roadmap to personalized medicine. Clin Transl Sci. 2008 Sep;1(2):93 Authors: Waldman SA, Terzic A PMID: 20443827 [PubMed - indexed for MEDLINE] | | |
| Age-Related Variation in Cell Density of Human Lumbar Intervertebral Disc.
July 31, 2010 at 6:38 AM
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| | Age-Related Variation in Cell Density of Human Lumbar Intervertebral Disc. Spine (Phila Pa 1976). 2010 Jul 27; Authors: Liebscher T, Haefeli M, Wuertz K, Nerlich AG, Boos N STUDY DESIGN.: Changes in cell density of endplate (EP), nucleus pulposus (NP), and anulus fibrosus (AF) during ageing were systematically investigated in defined regions of interest in complete human motion segments. OBJECTIVES.: To elucidate cell density and total cell number in distinct anatomic regions of the intervertebral disc; to test effects of gender, level and age on cell density; and to correlate changes in cell density with histologic signs of disc degeneration. SUMMARY OF BACKGROUND DATA.: The available information on the cell density within intervertebral discs and its age-related changes is sparse. This knowledge, however, is a crucial prerequisite for cell-based tissue engineering approaches of the intervertebral disc. METHODS.: In 49 complete cross-sections from lumbar motion segments (newborn to 86 years) from 22 specimens, cell density was determined by the Abercrombie method in EP, NP, and AF, and total cell number was counted per region of interest. RESULTS.: Cell density in EP, NP, and AF decreased significantly from 0 to 16 years with the main changes occuring from 0 to 3 years for NP and AF. No significant variations were observed thereafter. We found a significant correlation of cell density and histologic degeneration score between 0 and 1, but not for scores >1. Gender and disc level did not influence cell density. CONCLUSION.: This study provides data concerning the total number of cells in the various regions of the intervertebral disc for different age groups. This knowledge will be beneficial for cell-based treatment approaches, which may evolve in the future. PMID: 20671592 [PubMed - as supplied by publisher] | | |
| Minimally-Invasive Implantation of Living Tissue Engineered Heart Valves A Comprehensive Approach From Autologous Vascular Cells to Stem Cells.
July 31, 2010 at 6:38 AM
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| | Minimally-Invasive Implantation of Living Tissue Engineered Heart Valves A Comprehensive Approach From Autologous Vascular Cells to Stem Cells. J Am Coll Cardiol. 2010 Aug 3;56(6):510-520 Authors: Schmidt D, Dijkman PE, Driessen-Mol A, Stenger R, Mariani C, Puolakka A, Rissanen M, Deichmann T, Odermatt B, Weber B, Emmert MY, Zund G, Baaijens FP, Hoerstrup SP OBJECTIVES: The aim of this study was to demonstrate the feasibility of combining the novel heart valve replacement technologies of: 1) tissue engineering; and 2) minimally-invasive implantation based on autologous cells and composite self-expandable biodegradable biomaterials. BACKGROUND: Minimally-invasive valve replacement procedures are rapidly evolving as alternative treatment option for patients with valvular heart disease. However, currently used valve substitutes are bioprosthetic and as such have limited durability. To overcome this limitation, tissue engineering technologies provide living autologous valve replacements with regeneration and growth potential. METHODS: Trileaflet heart valves fabricated from biodegradable synthetic scaffolds, integrated in self-expanding stents and seeded with autologous vascular or stem cells (bone marrow and peripheral blood), were generated in vitro using dynamic bioreactors. Subsequently, the tissue engineered heart valves (TEHV) were minimally-invasively implanted as pulmonary valve replacements in sheep. In vivo functionality was assessed by echocardiography and angiography up to 8 weeks. The tissue composition of explanted TEHV and corresponding control valves was analyzed. RESULTS: The transapical implantations were successful in all animals. The TEHV demonstrated in vivo functionality with mobile but thickened leaflets. Histology revealed layered neotissues with endothelialized surfaces. Quantitative extracellular matrix analysis at 8 weeks showed higher values for deoxyribonucleic acid, collagen, and glycosaminoglycans compared to native valves. Mechanical profiles demonstrated sufficient tissue strength, but less pliability independent of the cell source. CONCLUSIONS: This study demonstrates the principal feasibility of merging tissue engineering and minimally-invasive valve replacement technologies. Using adult stem cells is successful, enabling minimally-invasive cell harvest. Thus, this new technology may enable a valid alternative to current bioprosthetic devices. PMID: 20670763 [PubMed - as supplied by publisher] | | |
| Hybrid PGS-PCL microfibrous scaffolds with improved mechanical and biological properties.
July 31, 2010 at 6:38 AM
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| | Hybrid PGS-PCL microfibrous scaffolds with improved mechanical and biological properties. J Tissue Eng Regen Med. 2010 Jul 28; Authors: Sant S, Hwang CM, Lee SH, Khademhosseini A Poly(glycerol sebacate) (PGS) is a biodegradable elastomer that has generated great interest as a scaffold material due to its desirable mechanical properties. However, the use of PGS in tissue engineering is limited by difficulties in casting micro- and nanofibrous structures, due to high temperatures and vacuum required for its curing and limited solubility of the cured polymer. In this paper, we developed microfibrous scaffolds made from blends of PGS and poly(epsilon-caprolactone) (PCL) using a standard electrospinning set-up. At a given PGS:PCL ratio, higher voltage resulted in significantly smaller fibre diameters (reduced from approximately 4 microm to 2.8 microm; p < 0.05). Further increase in voltage resulted in the fusion of fibres. Similarly, higher PGS concentrations in the polymer blend resulted in significantly increased fibre diameter (p < 0.01). We further compared the mechanical properties of electrospun PGS:PCL scaffolds with those made from PCL. Scaffolds with higher PGS concentrations showed higher elastic modulus (EM), ultimate tensile strength (UTS) and ultimate elongation (UE) (p < 0.01) without the need for thermal curing or photocrosslinking. Biological evaluation of these scaffolds showed significantly improved HUVEC attachment and proliferation compared to PCL-only scaffolds (p < 0.05). Thus, we have demonstrated that simple blends of PGS prepolymer with PCL can be used to fabricate microfibrous scaffolds with mechanical properties in the range of a human aortic valve leaflet. Copyright (c) 2010 John Wiley & Sons, Ltd. PMID: 20669260 [PubMed - as supplied by publisher] | | |
| Perfusion enhances solute transfer into the shell of hollow fiber membrane bioreactors for bone tissue engineering.
July 31, 2010 at 6:38 AM
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| | Perfusion enhances solute transfer into the shell of hollow fiber membrane bioreactors for bone tissue engineering. Int J Artif Organs. 2010 Jun;33(6):381-90 Authors: De Napoli IE, Catapano G Preparation of tissue engineered (TE) 3D constructs to repair large bone defects is limited by the difficult supply of nutrients and oxygen to cells in the innermost regions of constructs cultured in bioreactors. Poor oxygenation negatively affects cell viability and function. Bioreactor design optimization may help relieve these limitations. Bioreactors in which cells are cultured outside bundles of hollow fiber membranes (HFMBs) are structurally similar to natural bone. HFMB operation in pure diffusion has been reported to suffice for fibroblasts, but is deemed insufficient for bone cells. In this paper, the effect of perfusion flows in the cell compartment on solute transfer was investigated in HFMBs differing in design and operating conditions. HFMBs were designed and operated using values of non-dimensional groups that ensured solutes transfer towards the cell compartment mainly by diffusion; in the presence of low to high Starling flows; in the presence of pulsatile radial flows obtained by periodically stopping the solution flow leaving the bioreactor using a pinch valve. Distribution of matter in cell-free HFMBs was evaluated with tracer experiments in an optimized apparatus. Effectiveness of solute transfer to cell compartment was assessed based on the bioreactor response in terms of the shell volume actively involved in mass transfer (VMTA ) according to transport models developed specifically for the purpose. VMTA increased with increasing Starling flows. In the pulsatile radial flow mode, tracer concentration in the shell increased 3 times faster than at high Starling flows. This suggests that controlled perfusion flows in HFMBs might enable the engineering of large TE bone constructs. PMID: 20669143 [PubMed - in process] | | |
| A three-dimensional traction/torsion bioreactor system for tissue engineering.
July 31, 2010 at 6:38 AM
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| | A three-dimensional traction/torsion bioreactor system for tissue engineering. Int J Artif Organs. 2010 Jun;33(6):362-9 Authors: Scaglione S, Zerega B, Badano R, Benatti U, Fato M, Quarto R Purpose: The aim of this study was to design, develop and validate a simple, compact bioreactor system for tissue engineering. The resulting bioreactor was designed to achieve ease-of-use and low costs for automated cell-culturing procedures onto three-dimensional scaffolds under controlled torsion/traction regimes. ?Methods: Highly porous poly-caprolactone-based scaffolds were used as substrates colonized by fibroblast cells (3T3 cell line). Constructs were placed within the cylindrical culture chamber, clumped at the ends and exposed to controlled sequences of torsional stimuli (forward/back-forward sequential cycles of 100 degrees from neutral position at a rate of 600 degrees /min) through a stepper-motor; working settings were defined via PC by an easy user-interface. Cell adhesion, morphology, cytoskeletal fiber orientation and gene expression of extracellular matrix proteins (collagen type I, tenascin C, collagen type III) were evaluated after three days of torsional stimulation in the bioreactor system. ?Results and Conclusions: The 3D bioreactor system was validated in terms of sterility, experimental reproducibility and flexibility. Cells adhered well onto the polymeric scaffolds. Collagen type I, tenascin C and collagen type III gene expression were significantly up-regulated when cells were cultured under torsion in the bioreactor for three days. In conclusion, we have developed a simple, efficient and versatile 3D cell-culture system to engineer ligament grafts. This system can be used either as a model to investigate mechanisms of tissue development or as a graft manufacturing system for possible clinical use in the field of regenerative medicine. PMID: 20669141 [PubMed - in process] | | |
| Effects of low concentrated BMP-7 administered by co-cultivation or plasmid transfection on human osteoarthritic chondrocytes.
July 31, 2010 at 6:38 AM
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| | Effects of low concentrated BMP-7 administered by co-cultivation or plasmid transfection on human osteoarthritic chondrocytes. Int J Artif Organs. 2010 Jun;33(6):339-47 Authors: Gavenis K, Schneider U, Wallich R, Mueller-Rath R, Schmidt-Rohlfing B, Andereya S Introduction: While BMP-7 has proven to be one of the most potent growth factors in cartilage tissue engineering, protein concentration and route of administration remain a matter of debate. Here we investigated the effects of a low concentration of BMP-7 on human osteoarthritic chondrocytes administered by protein co-cultivation and plasmid transfection. Methods: Freshly released (P0) or in vitro propagated chondrocytes (P2) were cultivated in a collagen type-I gel for 3 weeks in vitro or in nude mice. Seeded chondrocytes were treated with 50 ng/mL BMP-7 directly added to the medium or were subject to transient BMP-7 plasmid transfection prior to gel cultivation. Untreated specimens served as a control. After recovery, samples were investigated by histological and immunohistochemical staining and real-time PCR. Results: In vitro, collagen type-II protein production was enhanced, and it was stored mainly pericellularly. Collagen type-II and aggrecan gene expression were enhanced in both treatment groups. After nude mouse cultivation, col-II protein production was further enhanced, but specimens of the BMP-7 transfection group revealed a clustering of col-II positive cells. Gene expression was strongly upregulated, chondrocyte number was increased and the differentiated phenotype prevailed. In general, freshly released chondrocytes (P0) proved to be superior to chondrocytes pre-amplified in vitro (P2). Conclusions: Both BMP-7 co-cultivation and plasmid transfection of human osteoarthritic chondrocytes led to improved cartilage repair tissue. Nevertheless, the col-II distribution following BMP-7 co-cultivation was homogeneous, while samples produced by transient transfection revealed a col-II clustering. PMID: 20669139 [PubMed - in process] | | |
| Hypertrophy, gene expression, and beating of neonatal cardiac myocytes are affected by microdomain heterogeneity in 3D.
July 31, 2010 at 6:38 AM
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| | Hypertrophy, gene expression, and beating of neonatal cardiac myocytes are affected by microdomain heterogeneity in 3D. Biomed Microdevices. 2010 Jul 29; Authors: Curtis MW, Sharma S, Desai TA, Russell B Cardiac myocytes are known to be influenced by the rigidity and topography of their physical microenvironment. It was hypothesized that 3D heterogeneity introduced by purely physical microdomains regulates cardiac myocyte size and contraction. This was tested in vitro using polymeric microstructures (G' = 1.66 GPa) suspended with random orientation in 3D by a soft Matrigel matrix (G' = 22.9 Pa). After 10 days of culture, the presence of 100 mum-long microstructures in 3D gels induced fold increases in neonatal rat ventricular myocyte size (1.61 +/- 0.06, p < 0.01) and total protein/cell ratios (1.43 +/- 0.08, p < 0.05) that were comparable to those induced chemically by 50 muM phenylephrine treatment. Upon attachment to microstructures, individual myocytes also had larger cross-sectional areas (1.57 +/- 0.05, p < 0.01) and higher average rates of spontaneous contraction (2.01 +/- 0.08, p < 0.01) than unattached myocytes. Furthermore, the inclusion of microstructures in myocyte-seeded gels caused significant increases in the expression of beta-1 adrenergic receptor (beta1-AR, 1.19 +/- 0.01), cardiac ankyrin repeat protein (CARP, 1.26 +/- 0.02), and sarcoplasmic reticulum calcium-ATPase (SERCA2, 1.59 +/- 0.12, p < 0.05), genes implicated in hypertrophy and contractile activity. Together, the results demonstrate that cardiac myocyte behavior can be controlled through local 3D microdomains alone. This approach of defining physical cues as independent features may help to advance the elemental design considerations for scaffolds in cardiac tissue engineering and therapeutic microdevices. PMID: 20668947 [PubMed - as supplied by publisher] | | |
| Enhanced proteolytic degradation of molecularly engineered PEG hydrogels in response to MMP-1 and MMP-2.
July 31, 2010 at 6:38 AM
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| | Enhanced proteolytic degradation of molecularly engineered PEG hydrogels in response to MMP-1 and MMP-2. Biomaterials. 2010 Jul 26; Authors: Patterson J, Hubbell JA Bioactive hydrogels formed by Michael-type addition reactions of end-functionalized poly(ethylene glycol) macromers with cysteine-containing peptides have been described as extracellular matrix mimetics and tissue engineering scaffolds. Although these materials have shown favorable behavior in vivo in tissue repair, we sought to develop materials formulations that would be more rapidly responsive to cell-induced enzymatic remodeling. In this study, protease-sensitive peptides that have increased k(cat) values were characterized and evaluated for their effects on gel degradability. Biochemical properties for soluble peptides and hydrogels were examined for matrix metalloproteinase (MMP)-1 and MMP-2. The most efficient peptide substrates in some cases overlap and in other cases differ between the two enzymes tested, and a range of k(cat) values was obtained. For each enzyme, hydrogels formed using the peptides with higher k(cat) values degraded faster than a reference with lower k(cat). Fibroblasts showed increased cell spreading and proliferation when cultured in 3D hydrogels with faster degrading peptides, and more cell invasion from aortic ring segments embedded in the hydrogels was observed. These faster degrading gels should provide matrices that are easier for cells to remodel and lead to increased cellular infiltration and potentially more robust healing in vivo. PMID: 20667588 [PubMed - as supplied by publisher] | | |
| Three-dimensional microstructured tissue scaffolds fabricated by two-photon laser scanning photolithography.
July 31, 2010 at 6:38 AM
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| | Three-dimensional microstructured tissue scaffolds fabricated by two-photon laser scanning photolithography. Biomaterials. 2010 Jul 25; Authors: Hsieh TM, Benjamin Ng CW, Narayanan K, Wan AC, Ying JY Current tissue engineering scaffolds fabricated via solvent casting and porogen leaching methods suffer from the lack of control over parameters such as interconnectivity and pore geometry, properties that are a function of the fabrication process. The progress of tissue engineering would thus benefit from the ability to design scaffolds that facilitate cell-cell interactions, and provide mass transfer characteristics necessary for good cell viability and function. In this research, we have developed two-photon laser scanning photolithography (TPLSP) for the fabrication of three-dimensional (3D) microstructured scaffolds with high resolution and fidelity. Modification of our two-photon setup allowed for a scan height of 30 mm and a scan speed of 30 mm/s, making it more amenable to scaffold fabrication. Scaffold production was adapted to computer-aided design (CAD)/computer-aided manufacturing (CAM) technology, to achieve the desired length scales from the submicron level and up. A commercially available photocurable resin that exhibited favorable ultraviolet-visible (UV-vis) transparency, cell compatibility and reproducibility in fabrication was used as the scaffold material. As a proof-of-concept, a microporous, cubic scaffold was fabricated for the purpose of hepatocyte culture. Primary hepatocytes could be uniformly seeded on these scaffolds as observed by confocal fluorescence microscopy. Albumin and urea assays demonstrated that hepatocytes cultured in the 3D scaffold maintained higher levels of liver-specific function over a period of 6 days as compared to the monolayer control. These results may be attributed to the high local concentration of soluble factors within the scaffold, which is important for maintaining the hepatocyte phenotype. Our study illustrates the potential of TPLSP as a new platform for the fabrication of designed, well-controlled, 3D microstructured tissue scaffolds. PMID: 20667410 [PubMed - as supplied by publisher] | | |
| Micro-Cavitary Hydrogel Mediating Phase Transfer Cell Culture for Cartilage Tissue Engineering.
July 31, 2010 at 6:38 AM
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| | Micro-Cavitary Hydrogel Mediating Phase Transfer Cell Culture for Cartilage Tissue Engineering. Tissue Eng Part A. 2010 Jul 28; Authors: Gong Y, Su K, Lau TT, Zhou R, Wang DA Hydrogels have been widely used as cell-laden vehicles for therapeutic transplantation in regenerative medicine. Although the advantages of biocompatibility and injectability for in situ grafting have made hydrogel a superior candidate in tissue engineering, there remain challenges in long-term efficacy of tissue development using hydrogel, especially when more sophisticated applications are demanded. The major bottleneck lies in environmental constraints for neo-tissue generation in the gel bulk such as proliferation of encapsulated cells (colonies) per se and also accommodation of their endogenously produced extracellular matrices (ECMs). In this study, we endeavor to develop an innovative tissue engineering system to overcome these drawbacks through a novel micro-cavitary hydrogel (MCG)-based scaffolding technology and a novel phase transfer cell culture (PTCC) strategy to enable phenotypically bona fide neo-tissue formation in an injectable artificial graft. For this purpose, microspherical cavities are created in cell-encapsulating hydrogel bulk via a retarded dissolution of co-encapsulated gelatin microspheres. Based on proliferation and affinity selection, the encapsulated cell colonies adjacent to the gel/cavity interface will spontaneously outgrow the hydrogel phase and sprout into cavities, enabling neo-tissue islets to fill up the voids and further expand throughout the whole system for full tissue regeneration. The design of MCG-PTCC strategy was elicited from an observation of a spontaneous dynamic outgrowth of chondrocytes from the edge of a cell-laden hydrogel construct over prolonged cultivation - a phenomenon named "edge flourish" (EF). This MCG-PTCC strategy potentially introduce a new application to hydrogels in the field of regenerative medicine through elevation of its role as a cell vehicle to a 3D-transplantable growth-guiding platform for further development of newly generated tissues that better fulfill the demanding criteria of scaffolds in therapeutic tissue regeneration. Keywords: Biomaterials; Hydrogel; Tissue Engineering; Phase Transfer Cell Culture; Regenerative Medicine. PMID: 20666616 [PubMed - as supplied by publisher] | | |
| Biodegradable Nanofibers-Reinforced Microfibrous Composite Scaffolds and the Bone Tissue Engineering.
July 31, 2010 at 6:38 AM
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| | Biodegradable Nanofibers-Reinforced Microfibrous Composite Scaffolds and the Bone Tissue Engineering. Tissue Eng Part A. 2010 Jul 28; Authors: Martins A, Pinho ED, Correlo V, Faria S, Marques A, Reis RL, Neves NM Native bone extracellular matrix (ECM) is a complex hierarchical fibrous composite structure, resulting from the assembling of collagen fibrils at several length scales, ranging from the macro to the nanoscale. The combination of nanofibers within microfibers following conventional reinforcement methodologies seems to be a feasible solution to the rational design of highly functional synthetic ECM substitutes. The present work aims at the development of bone ECM inspired structures, conjugating electrospun chitosan (Cht) nanofibers within biodegradable polymeric microfibers (poly(butylene succinate) - PBS and PBS/Cht), assembled in a fiber mesh structure. The nanofibers-reinforced composite fiber mesh scaffolds were seeded with human bone marrow mesenchymal stem cells (hBMSCs) and cultured under osteogenic differentiation conditions. These nanofibers-reinforced composite scaffolds sustained ECM deposition and mineralization, mainly in the PBS/Cht-based fiber meshes, as depicted by the increased amount of calcium phosphates produced by the osteogenic differentiated hBMSCs. The osteogenic genotype of the cultured hBMSCs was confirmed by the expression of osteoblastic genes, namely Alkaline Phosphatase, Osteopontin, Bone Sialoprotein and Osteocalcin, and the transcription factors Runx2 and Osterix, all involved in different stages of the osteogenesis. These data represent the first report on the biological functionality of nanofibers-reinforced composite scaffolds, envisaging the applicability of the developed structures for bone tissue engineering. PMID: 20666612 [PubMed - as supplied by publisher] | | |
| Use of tissue engineered nerve grafts consisting of a chitosan/ poly(lactic-co-glycolic acid)-based scaffold included with bridging 50-mm dog sciatic nerve gaps.
July 31, 2010 at 6:38 AM
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| | Use of tissue engineered nerve grafts consisting of a chitosan/ poly(lactic-co-glycolic acid)-based scaffold included with bridging 50-mm dog sciatic nerve gaps. Tissue Eng Part A. 2010 Jul 28; Authors: Ding F, Wu J, Yang Y, Hu W, Zhu Q, Tang X, Liu J, Gu X Bone marrow mesenchymal cells (MSCs) have attracted increasing research interest due to their possible use as support cells for nerve tissue engineering approaches. We developed a novel design of tissue engineered nerve grafts consisting of a chitosan/poly(lactic-co-glycolic acid) (PLGA)-based neural scaffold included with autologous MSCs. The graft was used as an alternative to nerve autografts for bridging 50-mm-long gaps in dog sciatic nerve, and the repair outcome at 6 months post nerve grafting was evaluated by a combination of electrophysiological assessment, FluoroGold retrograde tracing, and histological investigation to regenerated nerve tissue and reinnervated target muscle. The experimental results indicated that introduction of autologous MSCs to the chitosan/PLGA-based neural scaffold promoted sciatic nerve regeneration and functional recovery, demonstrating significant efficacy that was, to a certain degree, close to that by nerve autografting, a gold standard for treating large peripheral nerve gaps, and better than that by grafting with the chitosan/PLGA-based scaffold alone. PMID: 20666610 [PubMed - as supplied by publisher] | | |
| Fluid Shear Stress Promotes an Endothelial-like Phenotype during the Early Differentiation of Embryonic Stem Cells.
July 31, 2010 at 6:38 AM
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| | Fluid Shear Stress Promotes an Endothelial-like Phenotype during the Early Differentiation of Embryonic Stem Cells. Tissue Eng Part A. 2010 Jul 28; Authors: Ahsan T, Nerem RM Stem and progenitor cells are emerging as a potential source for cell-based therapies, in which large homogenous populations of differentiated cells are frequently deemed necessary for efficacy. Methods focused on biochemical cues have not yet yielded the numbers of endothelial cells thought necessary for cardiovascular applications. Interest in alternate methods has prompted the study of physical cues on stem and progenitor cell differentiation. In this study, fluid-based shear stress, at levels comparable to those experienced by endothelial cells in large vessels, was applied during the first few days of mouse embryonic stem cell differentiation. After two days of applied shear stress, there were increases in cell proliferation and in protein expression of endothelial markers (FLK1, VECAD, and PECAM). Furthermore, treatment increased the number of FLK1+ cells from 1% to 40%, which were then capable of forming vessel-like structures in vitro. Thus, shear stress may be used to direct differentiation of embryonic stem cells towards an endothelial-like phenotype, helping to address the cell sourcing issue in cardiovascular regenerative medicine and tissue engineering. PMID: 20666609 [PubMed - as supplied by publisher] | | |
| Non-viral gene therapy strategies for keratinocytes, fibroblasts and endothelial progenitor cells for ex vivo gene transfer to skin wounds.
July 31, 2010 at 6:38 AM
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| | Non-viral gene therapy strategies for keratinocytes, fibroblasts and endothelial progenitor cells for ex vivo gene transfer to skin wounds. Tissue Eng Part C Methods. 2010 Jul 28; Authors: Dickens S, Van den Berge S, Hendrickx B, Verdonck K, Luttun A, Vranckx J In search for the most accurate non-viral gene transfer technique in epidermal and dermal supportive extracellular matrix studies, we investigated the efficiency of late generation liposomal transfection reagents and nucleofection of fibroblasts, endothelial progenitor cells and keratinocytes as essential representatives of the healing skin wound. Fibroblasts, keratinocytes and endothelial progenitor cells were grown under serum-reduced conditions and manipulated according to the optimized manufacturing protocols in vitro. Fugene(R) HD, EffecteneTM, PEI, and Lipofectin(R) were compared to Amaxa Nucleofection(R). A GFP-encoding reporter gene plasmid was incorporated and visualized by green fluorescence-activated cell sorting. Normal cell morphology was observed after transfection or nucleofection. For keratinocyte cell cultures, Fugene(R) HD resulted in highest transgene expression in human (41%) and porcine keratinocytes (42%). For endothelial progenitor cells, EffecteneTM was most successful for human derived cells (42%) whereas for porcine cells Nucleofection(R) was optimal (32%). However for fibroblasts, Nucleofection(R) resulted in highest transfection rates in human (50%) and porcine derived fibroblasts (60%). For specific epidermal cell studies Fugene(R) HD is preferable as gene transfer method, while EffecteneTM appears to be the most optimal agent for pro-angiogenic studies. When transfecting with Nucleofection(R), fibroblasts are the best gene carriers for overall ex vivo gene transfer strategies in wound healing or skin tissue engineering. PMID: 20666605 [PubMed - as supplied by publisher] | | |
| Cyclic Tensile Culture Promotes Fibroblastic Differentiation of Marrow Stromal Cells Encapsulated in Poly(Ethylene Glycol)-Based Hydrogels.
July 31, 2010 at 6:38 AM
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| | Cyclic Tensile Culture Promotes Fibroblastic Differentiation of Marrow Stromal Cells Encapsulated in Poly(Ethylene Glycol)-Based Hydrogels. Tissue Eng Part A. 2010 Jul 28; Authors: Doroski DM, Levenston ME, Temenoff JS To inform future efforts in tendon/ligament tissue engineering, our laboratory has developed a well-controlled model system with the ability to alter both external tensile loading parameters and local biochemical cues to better understand marrow stromal cell differentiation in response to both stimuli concurrently. In particular, the synthetic, poly(ethylene glycol)-based hydrogel material oligo(poly(ethylene glycol) fumarate) (OPF) has been explored as a cell carrier for this system. This biomaterial can be tailored to present covalently incorporated bioactive moieties and can be loaded in our custom cyclic tensile bioreactor for up to 28 days with no loss of material integrity. Human marrow stromal cells encapsulated in these OPF hydrogels were cultured (21 days) under cyclic tensile strain (10%, 1 Hz, 3 h of strain followed by 3 h without) or at 0% strain. No difference was observed in cell number due to mechanical stimulation or across time (n = 4), with cells remaining viable (n = 4) through 21 days. Cyclic strain significantly upregulated all tendon/ligament fibroblastic genes examined (collagen I, collagen III, and tenascin-C) by day 21 (n >/= 6), whereas genes for other pathways (osteogenic, chondrogenic, and adipogenic) did not increase. After 21 days, the presence of collagen I and tenascin-C was observed via immunostaining (n = 2). This study demonstrates the utility of this hydrogel/bioreactor system as a versatile, yet well-controlled, model environment to study marrow stromal cell differentiation toward the tendon/ligament phenotype under a variety of conditions. PMID: 20666585 [PubMed - as supplied by publisher] | | |
| Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.
July 31, 2010 at 6:38 AM
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| | Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med. 2010 Jul;16(7):814-20 Authors: Uygun BE, Soto-Gutierrez A, Yagi H, Izamis ML, Guzzardi MA, Shulman C, Milwid J, Kobayashi N, Tilles A, Berthiaume F, Hertl M, Nahmias Y, Yarmush ML, Uygun K Orthotopic liver transplantation is the only available treatment for severe liver failure, but it is currently limited by organ shortage. One technical challenge that has thus far limited the development of a tissue-engineered liver graft is oxygen and nutrient transport. Here we demonstrate a novel approach to generate transplantable liver grafts using decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network, allowing efficient recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for in vitro culture. The recellularized graft supports liver-specific function including albumin secretion, urea synthesis and cytochrome P450 expression at comparable levels to normal liver in vitro. The recellularized liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage. These results provide a proof of principle for the generation of a transplantable liver graft as a potential treatment for liver disease. PMID: 20543851 [PubMed - indexed for MEDLINE] | | |
| Tissue-level modeling of xenobiotic metabolism in liver: An emerging tool for enabling clinical translational research.
July 31, 2010 at 6:38 AM
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| | Tissue-level modeling of xenobiotic metabolism in liver: An emerging tool for enabling clinical translational research. Clin Transl Sci. 2009 Jun;2(3):228-37 Authors: Lerapetritou MG, Georgopoulos PG, Roth CM, Androulakis LP This review summarizes some of the recent developments and identifies critical challenges associated with in vitro and in silico representations of the liver and assesses the translational potential of these models in the quest of rationalizing the process of evaluating drug efficacy and toxicity. It discusses a wide range of research efforts that have produced, during recent years, quantitative descriptions and conceptual as well as computational models of hepatic processes such as biotransport and biotransformation, intra- and intercellular signal transduction, detoxification, etc. The above mentioned research efforts cover multiple scales of biological organization, from molecule-molecule interactions to reaction network and cellular and histological dynamics, and have resulted in a rapidly evolving knowledge base for a "systems biology of the liver." Virtual organ/organism formulations represent integrative implementations of particular elements of this knowledge base, usually oriented toward the study of specific biological endpoints, and provide frameworks for translating the systems biology concepts into computational tools for quantitative prediction of responses to stressors and hypothesis generation for experimental design. PMID: 20443896 [PubMed - indexed for MEDLINE] | | |
| A computer model of engineered cardiac monolayers.
July 31, 2010 at 6:38 AM
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| | A computer model of engineered cardiac monolayers. Biophys J. 2010 May 19;98(9):1762-71 Authors: Kim JM, Bursac N, Henriquez CS Engineered monolayers created using microabrasion and micropatterning methods have provided a simplified in vitro system to study the effects of anisotropy and fiber direction on electrical propagation. Interpreting the behavior in these culture systems has often been performed using classical computer models with continuous properties. However, such models do not account for the effects of random cell shapes, cell orientations, and cleft spaces inherent in these monolayers on the resulting wavefront conduction. This work presents a novel methodology for modeling a monolayer of cardiac tissue in which the factors governing cell shape, cell-to-cell coupling, and degree of cleft space are not constant but rather are treated as spatially random with assigned distributions. This modeling approach makes it possible to simulate wavefront propagation in a manner analogous to performing experiments on engineered monolayer tissues. Simulated results are compared to previously published measured data from monolayers used to investigate the role of cellular architecture on conduction velocities and anisotropy ratios. We also present an estimate for obtaining the electrical properties from these networks and demonstrate how variations in the discrete cellular architecture affect the macroscopic conductivities. The simulations support the common assumption that under normal ranges of coupling strength, tissues with relatively uniform distributions of cell shapes and connectivity can be represented using continuous models with conductivities derived from random discrete cellular architecture using either global or local estimates. The results also reveal that in the presence of abrupt changes in cell orientation, local estimates of tissue properties predict smoother changes in conductivity that may not adequately predict the discrete nature of propagation at the transition sites. PMID: 20441739 [PubMed - indexed for MEDLINE] | | |
| Application of acellular dermal matrix for intestinal elongation in animal models.
July 31, 2010 at 6:38 AM
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| | Application of acellular dermal matrix for intestinal elongation in animal models. World J Gastroenterol. 2010 Apr 28;16(16):2023-7 Authors: Xu HM, Wang ZJ, Han JG, Ma HC, Zhao B, Zhao BC AIM: To investigate the efficacy of acellular dermal matrix (ADM) for intestinal elongation in animal models. METHODS: Japanese white big-ear rabbits (n = 9) and Wuzhishan miniature pigs (n = 5) were used in the study. Home-made and commercial ADM materials were used as grafts, respectively. A 3-cm long graft was interposed in continuity with the small bowel and a side-to-side anastomosis, distal to the graft about 3-4 cm, was performed. The animals were sacrificed at 2 wk, 4 wk, 8 wk and 3 mo after surgery and the histological changes were evaluated under light microscope and electron microscope. RESULTS: The animals survived after the operation with no evidence of peritonitis and sepsis. Severe adhesions were found between the graft and surrounding intestine. The grafts were completely absorbed within postoperative two or three months except one. Histological observation showed inflammation in the grafts with fibrinoid necroses, infiltration of a large amount of neutrophils and leukomonocytes, and the degree varied in different stages. The neointestine with well-formed structures was not observed in the study. CONCLUSION: It is not suitable to use acellular dermal matrix alone as a scaffold for the intestinal elongation in animal models. PMID: 20419841 [PubMed - indexed for MEDLINE] | | |
| Low oxygen tension and synthetic nanogratings improve the uniformity and stemness of human mesenchymal stem cell layer.
July 31, 2010 at 6:38 AM
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| | Low oxygen tension and synthetic nanogratings improve the uniformity and stemness of human mesenchymal stem cell layer. Mol Ther. 2010 May;18(5):1010-8 Authors: Zhao F, Veldhuis JJ, Duan Y, Yang Y, Christoforou N, Ma T, Leong KW A free-standing, robust cell sheet comprising aligned human mesenchymal stem cells (hMSCs) offers many interesting opportunities for tissue reconstruction. As a first step toward this goal, a confluent, uniform hMSC layer with a high degree of alignment and stemness maintenance needs to be created. Hypothesizing that topographical cue and a physiologically relevant low-oxygen condition could promote the formation of such an hMSC layer, we studied the culture of hMSCs on synthetic nanogratings (350 nm width and 700 nm pitch) and either under 2 or 20% O(2). Culturing hMSCs on the nanogratings highly aligned the cells, but it tended to create patchy layers and accentuate the hMSC differentiation. The 2% O(2) improved the alignment and uniformity of hMSCs, and reduced their differentiation. Over a 14-day culture period, hMSCs in 2% O(2) showed uniform connexon distribution, secreted abundant extracellular matrix (ECM) proteins, and displayed a high progenicity. After 21-day culture on nanogratings, hMSCs exposed to 2% O(2) maintained a higher viability and differentiation capacity. This study established that a 2% O(2) culture condition could restrict the differentiation of hMSCs cultured on nanopatterns, thereby setting the foundation to fabricate a uniformly aligned hMSC sheet for different regenerative medicine applications. PMID: 20179678 [PubMed - indexed for MEDLINE] | | | | 
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