|  |  |  | | | | | pubmed: "regenerative medici... | | | | | | | | | | | | | | | | | | | PTEN Loss Accelerates KrasG12D-Induced Pancreatic Cancer Development. Cancer Res. 2010 Aug 31; Authors: Hill R, Calvopina JH, Kim C, Wang Y, Dawson DW, Donahue TR, Dry S, Wu H KRAS mutations are found in approximately 90% of human pancreatic ductal adenocarcinomas (PDAC). However, mice genetically engineered to express Kras(G12D) from its endogenous locus develop PDACs only after a prolonged latency, indicating that other genetic events or pathway alterations are necessary for PDAC progression. The PTEN-controlled phosphatidylinositol 3-kinase (PI3K)/AKT signaling axis is dysregulated in later stages of PDAC. To better elucidate the role of PTEN/PI3K/AKT signaling in Kras(G12D)-induced PDAC development, we crossed Pten conditional knockout mice (Pten(lox/lox)) to mice with conditional activation of Kras(G12D). The resulting compound heterozygous mutant mice showed significantly accelerated development of acinar-to-ductal metaplasia (ADM), malignant pancreatic intraepithelial neoplasia (mPanIN), and PDAC within a year. Moreover, all mice with Kras(G12D) activation and Pten homozygous deletion succumbed to cancer by 3 weeks of age. Our data support a dosage-dependent role for PTEN, and the resulting dysregulation of the PI3K/AKT signaling axis, in both PDAC initiation and progression, and shed additional light on the signaling mechanisms that lead to the development of ADM and subsequent mPanIN and pancreatic cancer. Cancer Res; 70(18); OF1-11. (c)2010 AACR. PMID: 20807812 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | The chemistry of retinal transplantation: the influence of polymer scaffold properties on retinal cell adhesion and control. Br J Ophthalmol. 2010 Aug 31; Authors: Treharne AJ, Grossel MC, Lotery AJ, Thomson HA Age-related macular degeneration is the most common cause of blindness in the UK. Cellular replacement of retinal pigment epithelium cells is a potential therapeutic option to treat the cellular loss and dysfunction which is characteristic of age-related macular degeneration and other progressive retinopathies. A supportive scaffold, natural or artificial, may be required to facilitate cell delivery to the eye. Research to improve the biomimetic properties of such scaffolds, in order to optimise cell attachment and functionality following implantation, is ongoing. This short review will focus on the potential of biomaterials for ocular tissue engineering and how surface modification and the physical properties of these scaffolds can be tailored to help realise the full clinical potential of retinal pigment epithelium cell transplantation. PMID: 20807710 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Tissue Engineering of Flexor Tendons: The Effect of a Tissue Bioreactor on Adipoderived Stem Cell-Seeded and Fibroblast-Seeded Tendon Constructs. J Hand Surg Am. 2010 Sep;35(9):1466-1472 Authors: Angelidis IK, Thorfinn J, Connolly ID, Lindsey D, Pham HM, Chang J PURPOSE: Tissue-engineered flexor tendons could eventually be used for reconstruction of large tendon defects. The goal of this project was to examine the effect of a tissue bioreactor on the biomechanical properties of tendon constructs seeded with adipoderived stem cells (ASCs) and fibroblasts (Fs). METHODS: Rabbit rear paw flexor tendons were acellularized and seeded with ASCs or Fs. A custom bioreactor applied a cyclic mechanical load of 1.25 N at 1 cycle/minute for 5 days onto the tendon constructs. Three additional groups were used as controls: fresh tendons and tendons reseeded with either ASCs or Fs that were not exposed to the bioreactor treatment and were left in stationary incubation for 5 days. We compared the ultimate tensile stress (UTS) and elastic modulus (EM) of bioreactor-treated tendons with the unloaded control tendons and fresh tendons. Comparison across groups was assessed using one-way analysis of variance with the significance level set at p<.05. Pairwise comparison between the samples was determined by using the Tukey test. RESULTS: The UTS and EM values of bioreactor-treated tendons that were exposed to cyclic load were significantly higher than those of unloaded control tendons. Acellularized tendon constructs that were reseeded with ASCs and exposed to a cyclic load had a UTS of 66.76 MPa and an EM of 906.68 MPa; their unloaded equivalents had a UTS of 47.90 MPa and an EM of 715.57 MPa. Similar trends were found in the fibroblast-seeded tendon constructs that were exposed to the bioreactor treatment. The bioreactor-treated tendons approached the UTS and EM values of fresh tendons. Histologically, we found that cells reoriented themselves parallel to the direction of strain in response to cyclic strain. CONCLUSIONS: The application of cyclic strain on seeded tendon constructs that were treated with the bioreactor helped achieve a UTS and an EM comparable with those of fresh tendons. Bioreactor pretreatment and alternative cell lines, such as ASCs and Fs, might therefore contribute to the in vitro production of strong tendon material. PMID: 20807624 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Adhesive Substrates Modulate Activation and Stimulatory Capacity of Non-Obese Diabetic Mouse-Derived Dendritic Cells. Acta Biomater. 2010 Aug 28; Authors: Acharya AP, Dolgova NV, Xia CQ, Clare-Salzler MJ, Keselowsky BG It is known that adsorbed adhesive proteins on implanted biomaterials modulate inflammatory responses, however, modulation of dendritic cells (DCs) upon interaction with adhesive proteins has only begun to be characterized. Dendritic cells are specialized antigen presenting cells that modulate both innate and adaptive immune responses. Previously, we have shown that the activation and stimulatory capacity of DCs derived from C57BL6/j mice is differentially modulated by adhesive substrates. Here we extend investigation of adhesive substrate modulation of DC responses to consider the case where the DCs have maturational defects associated with diabetes. Understanding adhesive responses of DCs in diabetics is potentially important for immunotherapy and tissue engineering applications. In this work we use the non-obese diabetic (NOD) mouse, an established animal model for type 1 diabetes, to generate DCs (NOD-DCs). We demonstrate that NOD-DCs cultured on different adhesive substrates (collagen, fibrinogen, fibronectin, laminin, vitronectin, albumin, serum) respond with substrate-dependent modulation in surface expression of stimulatory molecule, MHC-II and costimulatory molecules CD80 and CD86 and, production of IL-12p40 and IL-10 cytokines. Furthermore, the capacity of NOD-DCs to stimulate CD4(+) T-cell proliferation and cytokine production (IL-4 and IFN-gamma) show substrate-dependent modulation. Specifically, NOD-DCs cultured on vitronectin induced the highest IL-12p40 production whereas collagen substrates induced highest IL-10 production. Dendritic cells cultured on collagen, fibrinogen and serum-coated substrates stimulated the highest CD4(+) T-cell proliferation. It was further determined that DCs cultured on vitronectin induced the highest percent population of IL-4 producing T-cells and DCs cultured on fibronectin substrate induced highest expression of IFN-gamma in T-cells. Pearson's correlation analysis revealed high correlations between T-cell proliferation and DC expression level of CD80, and the T-cell production level of IL-4 and DC production of IL-10. This demonstration of substrate-based control of NOD-DC activation and stimulatory capacity, distinct from non-diabetic B6-DC responses, helps establish the field of adhesive modulation of immune cell responses and informs the rational design of biomaterials for patients with type 1 diabetes. PMID: 20807596 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Role of wettability and nanoroughness on interactions between osteoblast and modified silicon surfaces. Acta Biomater. 2010 Aug 28; Authors: Padial-Molina M, Galindo-Moreno P, Fernández-Barbero JE, O'Valle F, Jódar-Reyes AB, Ortega-Vinuesa JL, Ramón-Torregrosa PJ Development of new biomaterials is a constant in Regenerative Medicine. Biomaterials surface properties, such as wettability, roughness, surface energy, surface charge, chemical functionalities and composition, are determinants for the cell and subsequent tissue behavior. Thus, the main aim of this study was to analyze the correlation between changes in wettability without topographical variations and the response of osteoblast-like cells. For this purpose, oxidized silicon surfaces were methylated to different degrees. Additionally, the influence of nanoroughness, and the subsequent effect of hysteresis on cell behavior, was also analyzed. In this case, oxidized silicon pieces were etched with caustic solutions to produce different degrees of nanoroughness. Axisymmetric Drop-Shape Analysis and Atomic Force Microscopy confirmed that the proposed surface treatments increase the nanometer roughness and/or the water contact angles. MG-63 osteoblast-like cells were cultured on altered surfaces for proliferation study, ultrastructural analysis, and immunocytochemical characterization. Nanometer or water contact angle increase enhanced osteoblast behavior in terms of cell morphology, proliferation, and immunophenotype, the effect provoked by methylation being more significant than that caused by nanoroughness. PMID: 20807595 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Critical role for white blood cell NAD(P)H oxidase mediated plasminogen activator inhibitor-1 oxidation and ventricular rupture following acute myocardial infarction. J Mol Cell Cardiol. 2010 Aug 28; Authors: Agarwal U, Zhou X, Weber K, Dadabayev AR, Penn MS Plasminogen Activator Inhibitor-1 (PAI-1) is an oxidant sensitive protease inhibitor that is inactivated by oxidation and has a critical role in ventricular remodeling post-myocardial infarction (MI). PAI-1 knockout (KO) mice die within 7days of myocardial infarction post MI due to increased plasmin activity leading to ventricular rupture. The goal of this study was to assess the relevant pathways of leukocyte-derived oxidants post-MI that alter PAI-1 activity. Transplantation of wild-type (WT) bone marrow into PAI-1 null mice prolonged survival after MI (WT marrow: 41.66% vs. PAI-1 KO marrow: 0% in PAI-1 KO mice at day 7 (p<0.02). To determine relevant enzyme systems, we transplanted marrow from mice with specific deletions relevant to leukocyte-derived oxidants (NAD(P)H Oxidase, iNOS, myeloperoxidase (MPO)) to determine which deletion controls PAI-1 oxidative inactivation and prolongs survival. MI was induced by ligation of the left anterior descending artery (LAD) and the incidence of cardiac rupture was monitored. PAI-1 KO transplanted with MPO KO, or iNOS KO bone marrow died within 9days after MI. PAI-1 KO mice transplanted with p47phox KO marrow exhibited prolonged survival 21days after MI (30% survival, p<0.03, n=10) compared to WT marrow (8.3%, n=12). Three days after MI, PAI-1 KO mice transplanted with p47phox KO marrow had increased PAI-1 activity and decreased nitration of PAI-1 in myocardial tissue compared to PAI-1 KO mice transplanted with WT marrow. These data suggest that modulating O(2)(*-) generation by NAD(P)H oxidase appears to be a therapeutically relevant target for increasing myocardial PAI-1 levels after MI whereas downstream enzymes like MPO and iNOS may not be. PMID: 20807543 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Regenerative medicine: The promises and perils of a promethean task. J Cell Mol Med. 2010 Aug 30; Authors: Moldovan NI PMID: 20807284 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | The Effect of Simulated Microgravity on Human Mesenchymal Stem Cells Cultured in an Osteogenic Differentiation System: A Bioinformatics Study. Tissue Eng Part A. 2010 Aug 31; Authors: Sheyn D, Pelled G, Netanely D, Domany E, Gazit D One proposed strategy for bone regeneration involves ex vivo tissue engineering, accomplished using bone-forming cells, biodegradable scaffolds, and dynamic culture systems, with the goal of three-dimensional tissue formation. Rotating wall vessel bioreactors generate simulated microgravity conditions ex vivo, which lead to cell aggregation. Human mesenchymal stem cells (hMSCs) have been extensively investigated and shown to possess the potential to differentiate into several cell lineages. The goal of the present study was to evaluate the effect of simulated microgravity on all genes expressed in hMSCs, with the underlying hypothesis that many important pathways are affected during culture within a rotating wall vessel system. Gene expression was analyzed using a whole genome microarray and clustering with the aid of the National Institutes of Health's Database for Annotation, Visualization and Integrated Discovery database and gene ontology analysis. Our analysis showed 882 genes that were downregulated and 505 genes that were upregulated after exposure to simulated microgravity. Gene ontology clustering revealed a wide variety of affected genes with respect to cell compartment, biological process, and signaling pathway clusters. The data sets showed significant decreases in osteogenic and chondrogenic gene expression and an increase in adipogenic gene expression, indicating that ex vivo adipose tissue engineering may benefit from simulated microgravity. This finding was supported by an adipogenic differentiation assay. These data are essential for further understanding of ex vivo tissue engineering using hMSCs. PMID: 20807102 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Reversal of experimental diabetes in mice by transplantation of neo-islets generated from human amnion-derived mesenchymal stromal cells using immuno-isolatory macrocapsules. Cytotherapy. 2010 Aug 31; Authors: Kadam SS, Sudhakar M, Nair PD, Bhonde RR Abstract Background aims. The ethical and biologic limitations with current sources of stem cells have resulted in a quest to look for alternative sources of multipotent stem cells of human origin. Amniotic membrane is of interest as a source of cells for regenerative medicine because of its ease of availability, plasticity and inexhaustible source that does not violate the sanctity of independent life. Although researchers have shown the stem cell-like potential of human amniotic epithelial cells, the mesenchymal part of amnion has remained less explored. Methods. We established a long-term culture of mesenchymal-like stem cells derived from full-term human amniotic membrane and their differentiation into functional pancreatic lineage. Results. The amnion-derived mesenchymal-like stem cells expressed various mesenchymal markers and demonstrated multilineage differentiation capacity. We also observed that these cells could form islet-like clusters (ILC) on exposure to serum-free defined media containing specific growth factor and differentiating agents. Differentiated ILC showed expression of human insulin, glucagon and somatostatin by immunocytochemistry, while quantitative reverse transcription/real-time-polymerase chain reaction (qRT-PCR) data demonstrated the expression of insulin, glucagon, somatostatin, Ngn3 and Isl1. Moreover, encapsulation of the ILC in polyurethane-polyvinyl pyrrolidone macrocapsules and their subsequent transplantation in experimental diabetic mice resulted in restoration of normoglycemia, indicating their ability to respond to high glucose without immunorejection. Conclusions. Our results demonstrate that amnion-derived mesenchymal stromal cells can undergo islet neogenesis, indicating amnion as an alternative source of islets for cell replacement therapy in diabetes. PMID: 20807019 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Elastic Properties of Induced Pluripotent Stem Cells. Tissue Eng Part A. 2010 Aug 31; Authors: Hammerick KE, Huang Z, Sun N, Lam MT, Prinz FB, Wu JC, Commons GW, Longaker M The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent cells, or iPSCs) has significantly changed the stem cell field. These cells have great promise for many clinical applications, including that of regenerative medicine. Our findings show that iPS cells can be derived from human adipose-derived stromal cells (hASCs), a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC), and also the gold standard human embryonic stem cell (hESC), we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness, and hence differences in material properties between cells. Human fibroblast and human ASC stiffness was also ascertained for comparison. Interestingly, cells exhibited a noticeable difference in stiffness. From least to most stiff, the order of cell stiffness was: hASC-iPSC, hESC, fibroblast-iPSC, fibroblasts, and lastly, as the stiffest cell, hASC. In comparing hASC-iPS cells to their origin cell, the hASC, the reprogrammed cell is significantly less stiff, indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell-culture-density, hence material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state. PMID: 20807017 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Dynamic Culturing of Smooth Muscle Cells in Tubular Poly(Trimethylene Carbonate) Scaffolds for Vascular Tissue Engineering. Tissue Eng Part A. 2010 Aug 31; Authors: Song Y, Wennink JW, Kamphuis MM, Sterk LM, Vermes I, Poot AA, Feijen J, Grijpma DW Porous, tubular, flexible and elastic poly(trimethylene carbonate) (PTMC) scaffolds (length 8 cm, inner diameter 3 mm) for vascular tissue engineering were prepared by means of a dip-coating and particulate leaching procedure. Using NaCl as porogen, scaffolds with an average pore size of 110 mum and a porosity of 85% were obtained. Before leaching the salt, the structures were made creep-resistant by means of crosslinking at 25 kGy gamma irradiation. To increase the efficiency of cell seeding, the scaffolds were provided with a micro-porous outer layer of 0.2 mm with an average pore size of 28 mum and a porosity of 65% (total wall thickness 1 mm). Human smooth muscle cells (SMCs) were seeded in these scaffolds with an efficiency of 43%, as determined after 24 h cell adhesion. SMCs were cultured in the scaffolds up to 14 days under stationary conditions or under pulsatile flow conditions in a bioreactor (pressure 70-130 mmHg, 69 pulsations/min, average wall shear rate 320 s<sup>-1</sup>). Although SMCs proliferated under both conditions, cell numbers were 3-5 times higher in case of dynamic culturing. This was qualitatively confirmed by means of histology. Also in terms of mechanical properties, the dynamically cultured constructs performed better than the statically cultured constructs. After culturing for 14 days, the maximum tensile strengths of the constructs, determined in the radial direction, had increased from 0.16 MPa (unseeded scaffold) to 0.48 MPa (dynamic culturing) and 0.38 MPa (static culturing). The results of this study indicate that a potentially useful medial layer for tissue-engineered vascular grafts can be prepared by dynamic culturing of human SMCs seeded in porous tubular PTMC scaffolds. PMID: 20807005 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Silver nanoparticle impregnated polycaprolactone scaffolds: optimization of antimicrobial and non-cytotoxic concentrations. Tissue Eng Part A. 2010 Aug 31; Authors: Madhavan RV, Rosemary M, Nandkumar MA, Krishnan KV, Krishnan LK Use of silver nanoparticles (SNPs) for control of implant associated infection is a promising strategy, if optimum antimicrobial yet nontoxic dose to mammalian cells is identified. This study was done to determine essential quantity of SNP, which stimulate antimicrobial activity without cytotoxicity, when immobilized on poly [epsilon-caprolactone] (PCL) scaffold proposed for vascular tissue engineering (VTE). During SNP synthesis and scaffold preparation nanoparticle aggregation was protected using poly (ethylene glycol) (PEG). Transmission electron microscopy was used to characterize SNP size and to detect its mobilization from scaffold to culture medium. Antimicrobial property of the SNP and its dose response was tested using both Gram positive and Gram negative bacteria by zone of inhibition assay (ZIA). Endothelial cells (EC), the main cell type required for VTE were grown on scaffolds to identify the nontoxic dose. After seeding EC on scaffolds; cell attachment, spreading, and viability/survival, were detected using specific markers by flow cytometric/fluorescence microscopic analysis. Real-time PCR detected effect of SNPs on mRNA expression of selected EC-specific functional proteins. Results suggest that even devoid of antibiotics in the medium, 0.1% (w/w) SNP on PCL scaffold is antimicrobial while nontoxic to EC at cellular and molecular level once cultured on the SNP-PCL scaffold. PMID: 20807004 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Spatial Control of Cell Adhesion and Patterning through Mussel-Inspired Surface Modification by Polydopamine. Langmuir. 2010 Aug 31; Authors: Ku SH, Lee JS, Park CB The spatial control and patterning of mammalian cells were achieved by using the universal adhesive property of mussel-inspired polydopamine (PDA). The self-polymerization of dopamine, a small molecule inspired by the DOPA motif of mussel foot proteins, resulted in the formation of a PDA adlayer when aqueous dopamine solution was continuously injected into poly(dimethylsiloxane) microchannels. We found that various cells (fibrosarcoma HT1080, mouse preosteoblast MC3T3-E1, and mouse fibroblast NIH-3T3) predominantly adhered to PDA-modified regions, maintaining their normal morphologies. The cells aligned in the direction of striped PDA patterns, and this tendency was not limited by the type of cell line. Because PDA modification does not require complex chemical reactions and is applicable to any type of material, it enables cell patterning in a simple and versatile manner as opposed to conventional methods based on the immobilization of adhesive proteins. The PDA-based method of cell patterning should be useful in many biomaterial research areas such as the fabrication of tissue engineering scaffolds, cell-based devices for drug screening, and the fundamental study of cell-material interactions. PMID: 20806924 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | The pseudokinase tribbles homolog 3 interacts with ATF4 to negatively regulate insulin exocytosis in human and mouse beta cells. J Clin Invest. 2010 Aug 2;120(8):2876-88 Authors: Liew CW, Bochenski J, Kawamori D, Hu J, Leech CA, Wanic K, Malecki M, Warram JH, Qi L, Krolewski AS, Kulkarni RN Insufficient insulin secretion and reduced pancreatic beta cell mass are hallmarks of type 2 diabetes (T2DM). Here, we confirm that a previously identified polymorphism (rs2295490/Q84R) in exon 2 of the pseudokinase-encoding gene tribbles 3 (TRB3) is associated with an increased risk for T2DM in 2 populations of people of mixed European descent. Carriers of the 84R allele had substantially reduced plasma levels of C-peptide, the product of proinsulin processing to insulin, suggesting a role for TRB3 in beta cell function. Overexpression of TRB3 84R in mouse beta cells, human islet cells, and the murine beta cell line MIN6 revealed reduced insulin exocytosis, associated with a marked reduction in docked insulin granules visualized by electron microscopy. Conversely, knockdown of TRB3 in MIN6 cells restored insulin secretion and expression of exocytosis genes. Further analysis in MIN6 cells demonstrated that TRB3 interacted with the transcription factor ATF4 and that this complex acted as a competitive inhibitor of cAMP response element-binding (CREB) transcription factor in the regulation of key exocytosis genes. In addition, the 84R TRB3 variant exhibited greater protein stability than wild-type TRB3 and increased binding affinity to Akt. Mice overexpressing TRB3 84R in beta cells displayed decreased beta cell mass, associated with reduced proliferation and enhanced apoptosis rates. These data link a missense polymorphism in human TRB3 to impaired insulin exocytosis and thus increased risk for T2DM. PMID: 20592469 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Long non-coding RNAs in nervous system function and disease. Brain Res. 2010 Jun 18;1338:20-35 Authors: Qureshi IA, Mattick JS, Mehler MF Central nervous system (CNS) development, homeostasis, stress responses, and plasticity are all mediated by epigenetic mechanisms that modulate gene expression and promote selective deployment of functional gene networks in response to complex profiles of interoceptive and environmental signals. Thus, not surprisingly, disruptions of these epigenetic processes are implicated in the pathogenesis of a spectrum of neurological and psychiatric diseases. Epigenetic mechanisms involve chromatin remodeling by relatively generic complexes that catalyze DNA methylation and various types of histone modifications. There is increasing evidence that these complexes are directed to their sites of action by long non-protein-coding RNAs (lncRNAs), of which there are tens if not hundreds of thousands specified in the genome. LncRNAs are transcribed in complex intergenic, overlapping and antisense patterns relative to adjacent protein-coding genes, suggesting that many lncRNAs regulate the expression of these genes. LncRNAs also participate in a wide array of subcellular processes, including the formation and function of cellular organelles. Most lncRNAs are transcribed in a developmentally regulated and cell type specific manner, particularly in the CNS, wherein over half of all lncRNAs are expressed. While the numerous biological functions of lncRNAs are yet to be characterized fully, a number of recent studies suggest that lnRNAs are important for mediating cell identity. This function seems to be especially important for generating the enormous array of regional neuronal and glial cell subtypes that are present in the CNS. Further studies have also begun to elucidate additional roles played by lncRNAs in CNS processes, including homeostasis, stress responses and plasticity. Herein, we review emerging evidence that highlights the expression and function of lncRNAs in the CNS and suggests that lncRNA deregulation is an important factor in various CNS pathologies including neurodevelopmental, neurodegenerative and neuroimmunological disorders, primary brain tumors, and psychiatric diseases. PMID: 20380817 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Deconstructing pancreas development to reconstruct human islets from pluripotent stem cells. Cell Stem Cell. 2010 Apr 2;6(4):300-8 Authors: McKnight KD, Wang P, Kim SK There is considerable excitement about harnessing the potential of human stem cells to replace pancreatic islets that are destroyed in type 1 diabetes mellitus. However, our current understanding of the mechanisms underlying pancreas and islet ontogeny has come largely from the powerful genetic, developmental, and embryological approaches available in nonhuman organisms. Successful islet reconstruction from human pluripotent cells will require greater attention to "deconstructing" human pancreas and islet developmental biology and consistent application of conditional genetics, lineage tracing, and cell purification to stem cell biology. PMID: 20362535 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Spatial regulation of human mesenchymal stem cell differentiation in engineered osteochondral constructs: effects of pre-differentiation, soluble factors and medium perfusion. Osteoarthritis Cartilage. 2010 May;18(5):714-23 Authors: Grayson WL, Bhumiratana S, Grace Chao PH, Hung CT, Vunjak-Novakovic G OBJECTIVE: The objective of the study was to investigate the combined effects of three sets of regulatory factors: cell pre-differentiation, soluble factors and medium perfusion on spatial control of human mesenchymal stem cell (hMSC) differentiation into cells forming the cartilaginous and bone regions in engineered osteochondral constructs. DESIGN: Bone-marrow derived hMSCs were expanded in their undifferentiated state (UD) or pre-differentiated (PD) in monolayer culture, seeded into biphasic constructs by interfacing agarose gels and bone scaffolds and cultured for 5 weeks either statically (S) or in a bioreactor (BR) with perfusion of medium through the bone region. Each culture system was operated with medium containing either chondrogenic supplements (C) or a cocktail (Ck) of chondrogenic and osteogenic supplements. RESULTS: The formation of engineered cartilage in the gel region was most enhanced by using undifferentiated cells and chondrogenic medium, whereas the cartilaginous properties were negatively affected by using pre-differentiated cells or the combination of perfusion and cocktail medium. The formation of engineered bone in the porous scaffold region was most enhanced by using pre-differentiated cells, perfusion and cocktail medium. Perfusion also enhanced the integration of bone and cartilage regions. CONCLUSIONS: (1) Pre-differentiation of hMSCs before seeding on scaffold was beneficial for bone but not for cartilage formation. (2) The combination of medium perfusion and cocktail medium inhibited chondrogenesis of hMSCs. (3) Perfusion improved the cell and matrix distribution in the bone region and augmented the integration at the bone-cartilage interface. (4) Osteochondral grafts can be engineered by differentially regulating the culture conditions in the two regions of the scaffold seeded with hMSCs (hydrogel for cartilage, perfused porous scaffold for bone). PMID: 20175974 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | [Regeneration of the central nervous system using iPS cell-technologies] Rinsho Shinkeigaku. 2009 Nov;49(11):825-6 Authors: Okano H Induced pluripotent stem (iPS) cells are pluripotent stem cells directly reprogrammed from cultured mouse fibroblast by introducing Oct3/4, Sox2, c-Myc, and Klf4. Cells obtained using this technology, which allows the ethical issues and immunological rejection associated with embryonic stem (ES) cells to be avoided, might be a clinically useful source for cell replacement therapies. We found that secondary neurospheres (SNSs) generated from various mouse iPS cell showed their neural differentiation capacity and teratoma formation after transplantation into the brain of immunodeficient NOD/SCID mice. We found that origin (source of somatic cells) of the iPS cells are the crucial determinant for the potential tumorigenicity of iPS-derived neural stem/progenitor cells and that their tumorigenicity results from the persistent presence of undifferentiated cells within the SNSs. Surprisingly, SNSs derived from c-Myc minus iPS cells generated without drug selection showed robust tumorigenesis, in spite of their potential to contribute adult chimeric mice without tumor formation. Furthermore, we examined whether the transplantation of non-tumorigenic Nanog-iPS-derived SNSs into mouse spinal cord injury (SCI) model could promote locomotor function recovery. As a result, we found that properly pre-evaluated iPS clone-derived neural stem/progenitor cells may be a promising cell source for future transplantation therapy of SCI. PMID: 20030221 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | [Studies of gene therapy for Parkinson's disease] Rinsho Shinkeigaku. 2009 Nov;49(11):753-5 Authors: Mochizuki H Currently, four Phase I clinical trials are underway utilizing recombinant adeno-associated viral (rAAV) vectors for the treatment of Parkinson's disease. The vehicle used mainly for gene delivery to the human brain is rAAV vector, which is non-pathogenic and non-self-amplifying. At present, the gene therapy approach is not the best way for the treatment of PD patients, but we believe that the further progress is anticipated toward making this strategy a therapeutic option for PD in the future. This article will review currently ongoing clinical trials of PD gene therapy and then introduce our studies about the gene therapy for PD. PMID: 20030202 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Patterns of gene expression in a rabbit partial anterior cruciate ligament transection model: the potential role of mechanical forces. Am J Sports Med. 2010 Feb;38(2):348-56 Authors: Attia E, Brown H, Henshaw R, George S, Hannafin JA BACKGROUND: The inconsistency in healing after anterior cruciate ligament (ACL) repair has been attributed to ACL fibroblast cellular metabolism, lack of a sufficient vascular supply, and the inability to form a scar or scaffold after ligament rupture because of the uniqueness of the intra-articular environment. Hypotheses (1) Stress deprivation in the surgically transected ACL will increase matrix metalloproteinase (MMP) and alpha smooth muscle actin (alpha-SMA) expression. (2) Stress deprivation will decrease collagen expression. (3) The transected anteromedial bundle of the ACL will demonstrate a pattern of gene expression similar to the completely transected ACL, while gene expression profiles in the intact posterolateral bundle will be similar to the sham-operated controls. STUDY DESIGN: Controlled laboratory study. METHODS: Thirty-six New Zealand White rabbits underwent a partial ACL surgical transection separating the anteromedial (AM) and posterolateral (PL) bundles and transecting the AM bundle. Contralateral ACLs were either sham operated or completely transected. Ligament tissue was harvested at 1, 2, or 6 weeks after surgery, and real-time PCR was performed using primers for collagen I, collagen III, alpha-SMA, MMP-1, and MMP-13. RESULTS: At 1 week, a 28- and 29-fold increase in MMP-13 expression was seen in the complete transection and the transected AM bundle specimens when compared with sham-operated controls (P = .049, P = .018), respectively. There was no significant difference in MMP-13 between the sham controls and the intact PL bundle specimens. A 22- and 23-fold increase in alpha-SMA was seen (P = .03, P = .009) in the complete transection and transected AM bundle specimens, respectively, while no difference was seen between the intact PL bundle and controls. No significant differences were seen in collagen I (Col I) or collagen III (Col III) gene expression at 1 week. At 6 weeks, Col I expression increased 5-fold in complete transection samples (P = 3.9 x 10(-6)), 3-fold in transected AM samples (P = 3.3 x 10(-6)), and 2-fold in the intact PL bundle samples as compared with controls. alpha-SMA was increased 7.5-fold and 5-fold in complete transection and transected AM samples, respectively (P = .004, P = 2.2 x 10(-6)), while no significant change was seen in the intact PL bundle samples compared with controls. Complete transection specimens showed a 3-fold increase in MMP-1 expression. Col III increased 5.4-, 2.6-, and 2.4-fold in the complete transection, transected AM, and intact PL groups, respectively (P = .003, P = .004, P = .04). CONCLUSION: Partial or complete surgical transection of the rabbit ACL with resultant loss of mechanical stimuli results in an increase in MMP-13 and alpha-SMA expression at the early time point (1 week) and an increase in alpha-SMA, Col I, and Col III expression at the later time point (6 weeks). These data provide support for the hypothesis that there is a time-dependent alteration of anabolic and catabolic matrix gene expression after injury/loss of ligament integrity. Clinical Relevance Identification of pathways that respond to mechanical stress in the intact ACL and after surgical transection may permit development of novel therapies to alter healing of the partial ACL injury or to assist in the development of biomechanical active ''smart'' scaffolds for tissue-engineered ligament replacements. PMID: 19966107 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Ab initio calcineurin inhibitor-based monotherapy immunosuppression after liver transplantation reduces the risk for Pneumocystis jirovecii pneumonia. Transpl Infect Dis. 2010 Feb;12(1):11-5 Authors: Orlando G, Tariciotti L, Manzia TM, Gravante G, Sorge R, Manuelli M, Pisani F, Di Cocco P, Scelzo C, Burke GM, Soker S, Baiocchi L, Lerut J, Angelico M, Tisone G At the Tor Vergata University of Rome, ab initio calcineurin inhibitor-based monotherapy immunosuppression (IS) is the standard of treatment after liver transplantation (LT). As the net state of IS determines the onset of Pneumocystis jirovecii pneumonia (PCP), we hypothesized that, in the presence of weak impairment of the immune function, as determined by the above-mentioned IS, the host is not overexposed to the risk for PCP and consequently the specific anti-PCP prophylaxis is unnecessary. In a single-cohort descriptive study, we retrospectively investigated the incidence of PCP in 203 LT patients who did not receive anti-PCP prophylaxis because they were under monotherapy IS. The primary endpoint of the study was the incidence of PCP during the first 12 months following LT; secondary endpoints were the incidence of acute rejection requiring additional IS and of CMV infection. No cases of PCP were recorded. The incidence of CMV and acute rejection was 3.9% and 0.9%, respectively. Our data suggest that monotherapy IS after LT may nullify the risk for PCP even in the absence of any specific prophylaxis. PMID: 19744283 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Clump passaging and expansion of human embryonic and induced pluripotent stem cells on mouse embryonic fibroblast feeder cells. Curr Protoc Stem Cell Biol. 2010 Aug;Chapter 1:Unit1C.10 Authors: Hartung O, Huo H, Daley GQ, Schlaeger TM The ability of human embryonic stem cells (hESCs) to differentiate into essentially all somatic cell types has made them a valuable tool for studying human development and has positioned them for broad applications in toxicology, regenerative medicine, and drug discovery. This unit describes a protocol for the large-scale expansion and maintenance of hESCs in vitro. hESC cultures must maintain a balance between the cellular states of pluripotency and differentiation; thus, researchers must use care when growing these technically demanding cells. The culture system is based largely on the use of a proprietary serum-replacement product and basic fibroblast growth factor (bFGF), with mouse embryonic fibroblasts as a feeder layer. These conditions provide the basis for relatively inexpensive maintenance and expansion of hESCs, as well as their engineered counterparts, human induced pluripotent stem cells (hiPSCs). Curr. Protoc. Stem Cell Biol. 14:1C.10.1-1C.10.15. (c) 2010 by John Wiley & Sons, Inc. PMID: 20814935 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | [Interventional, intramyocardial stem cell therapy in ischemic cardiomyopathy: update 2010.] Herz. 2010 Jul 29; Authors: Bergmann MW, Jaquet K, Schneider C, Krause K, Ujeyl A, Kuck KH BACKGROUND: The intracoronary application of autologous bone marrow cells has proven hitherto to be safe but not sufficiently effective in patients with ischemic cardiomyopathy. The interventional application of cells injected directly into the myocardium represents one possible approach to improve effectiveness. TECHNIQUES: The NOGA method is based on the CARTO technique, which has been evaluated extensively for safety and feasibility in patients with heart failure. In a first step, an electrically and anatomically exact map of the left ventricle is obtained. Guided by this three-dimensional map direct injection of the cells into the ischemic area can be easily performed. CLINICAL STUDIES: Since its introduction in 2002 many studies have proven the safety, feasibility and effectiveness of NOGA-guided regenerative therapy to the left ventricle. While several studies also suggest effectiveness regarding various parameters of left ventricular function, no larger multicenter study is available to date. Such studies with also clinical endpoints are currently ongoing. CONCLUSION: The currently available data support, but do not yet prove, the hypothesis that intramyocardial stem cell therapy using NOGA-guided injection into the myocardium is safe and feasible in both acute and chronic ischemic cardiomyopathy. Ongoing trials will reveal whether this approach will become the standard form for applying cell therapy to the heart. PMID: 20814657 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Laser-based direct-write techniques for cell printing. Biofabrication. 2010 Jul 12;2(3):032001 Authors: Schiele NR, Corr DT, Huang Y, Raof NA, Xie Y, Chrisey DB Fabrication of cellular constructs with spatial control of cell location (+/-5 microm) is essential to the advancement of a wide range of applications including tissue engineering, stem cell and cancer research. Precise cell placement, especially of multiple cell types in co- or multi-cultures and in three dimensions, can enable research possibilities otherwise impossible, such as the cell-by-cell assembly of complex cellular constructs. Laser-based direct writing, a printing technique first utilized in electronics applications, has been adapted to transfer living cells and other biological materials (e.g., enzymes, proteins and bioceramics). Many different cell types have been printed using laser-based direct writing, and this technique offers significant improvements when compared to conventional cell patterning techniques. The predominance of work to date has not been in application of the technique, but rather focused on demonstrating the ability of direct writing to pattern living cells, in a spatially precise manner, while maintaining cellular viability. This paper reviews laser-based additive direct-write techniques for cell printing, and the various cell types successfully laser direct-written that have applications in tissue engineering, stem cell and cancer research are highlighted. A particular focus is paid to process dynamics modeling and process-induced cell injury during laser-based cell direct writing. PMID: 20814088 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | EGF and bFGF pre-treatment enhances neural specification and the response to neuronal commitment of MIAMI cells. Differentiation. 2010 Aug 31; Authors: Delcroix GJ, Curtis KM, Schiller PC, Montero-Menei CN AIMS: Multipotent mesenchymal stromal cells raise great interest for regenerative medicine studies. Some MSC subpopulations have the potential to undergo neural differentiation, including marrow isolated adult multilineage inducible (MIAMI) cells, which differentiate into neuron-like cells in a multi-step neurotrophin 3-dependent manner. Epidermal and basic fibroblast growth factors are often used in neuronal differentiation protocols for MSCs, but with a limited understanding of their role. In this study, we thoroughly assessed for the first time the capacity of these factors to enhance the neuronal differentiation of MSCs. MATERIALS AND METHODS: We have characterized MIAMI cell neuronal differentiation program in terms of stem cell molecule expression, cell cycle modifications, acquisition of a neuronal morphology and expression of neural and neuronal molecules in the absence and presence of an EGF-bFGF pre-treatment. RESULTS: EGF-bFGF pre-treatment down-regulated the expression of stemness markers Oct4A, Notch1 and Hes5, whereas neural/neuronal molecules Nestin, Pax6, Ngn2 and the neurotrophin receptor tyrosine kinase 1 and 3 were up-regulated. During differentiation, a sustained Erk phosphorylation in response to NT3 was observed, cells began to exit from the cell cycle and exhibit increased neurite-like extensions. In addition, neuronal beta3-tubulin and neurofilament expression was increased; an effect mediated via the Erk pathway. A slight pre-oligodendrocyte engagement was noted, and no default neurotransmitter phenotype was observed. Overall, mesodermal markers were unaffected or decreased, while neurogenic/adipogenic PPARgamma2 was increased. CONCLUSION: EGF and bFGF pre-treatment enhances neural specification and the response to neuronal commitment of MIAMI cells, further increasing their potential use in adult cell therapy of the nervous system. PMID: 20813449 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell. 2010 Sep 3;142(5):787-99 Authors: Kobayashi T, Yamaguchi T, Hamanaka S, Kato-Itoh M, Yamazaki Y, Ibata M, Sato H, Lee YS, Usui J, Knisely AS, Hirabayashi M, Nakauchi H The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment. PMID: 20813264 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Anti-CD47 Antibody Synergizes with Rituximab to Promote Phagocytosis and Eradicate Non-Hodgkin Lymphoma. Cell. 2010 Sep 3;142(5):699-713 Authors: Chao MP, Alizadeh AA, Tang C, Myklebust JH, Varghese B, Gill S, Jan M, Cha AC, Chan CK, Tan BT, Park CY, Zhao F, Kohrt HE, Malumbres R, Briones J, Gascoyne RD, Lossos IS, Levy R, Weissman IL, Majeti R Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative and must be combined with cytotoxic chemotherapy for clinical benefit. Here we report the eradication of human NHL solely with a monoclonal antibody therapy combining rituximab with a blocking anti-CD47 antibody. We identified increased expression of CD47 on human NHL cells and determined that higher CD47 expression independently predicted adverse clinical outcomes in multiple NHL subtypes. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab led to elimination of lymphoma and cure. These antibodies synergized through a mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers. PMID: 20813259 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Selection of reference genes for quantitative polymerase chain reaction studies in purified B cells from B cell chronic lymphocytic leukaemia patients. Br J Haematol. 2010 Aug 31; Authors: Valceckiene V, Kontenyte R, Jakubauskas A, Griskevicius L Summary The clinical heterogeneity of B-cell chronic lymphocytic leukaemia (B-CLL) makes it necessary to identify potent prognostic indicators to predict individual clinical course and select risk-adapted therapy. In recent years, numerous gene expression models have been suggested as prognostic factors of B-CLL. Today, quantitative polymerase chain reaction (qPCR) is a preferred method for rapid quantification of gene expression and validation of microarray data. The reliability of qPCR data is highly dependent on the use of appropriate reference genes for normalization. To date, no validated reference genes have been reported for the normalization of gene expression in B-CLL. Therefore, the present study was conducted to identify suitable reference genes for gene expression studies in CD19(+) B cells isolated from B-CLL patients' peripheral blood. The stability of ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, MRPL19, TBP and UBC genes was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper-1, which produced highly comparable results. Based on our results, B2M, HPRT1, and GUSB were found to be the most suitable reference genes for qPCR studies in B-CLL patients' peripheral blood B cells. PMID: 20813001 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | The Global Politics of Human Embryonic Stem Cell Science: Regenerative Medicine in Transition Herbert Gottweis , Brian Salter , Catherine Waldby . The Global Politics of Human Embryonic Stem Cell Science: Regenerative Medicine in Transition New York . Palgrave MacMillan . 2009 . 272 pages. ISBN 978-0230002630, Hardcover, $90.00 . Politics Life Sci. 2010 Mar;29(1):100-2 Authors: Blaser AW PMID: 20812808 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | In vitro Evaluation of Acellular Dermal Matrix as a Three-Dimensional Scaffold for Gingival Fibroblasts Seeding. J Periodontol. 2010 Sep 2; Authors: Maia LP, Novaes AB, Souza SL, Grisi MF, Taba M, Palioto DB Background: Tissue engineering principles could improve acellular dermal matrix (ADM) incorporation. The aim of this study was to verify if ADM is a suitable three-dimensional matrix for gingival fibroblasts and cancerous cells ingrowth; and also, if cultured medium conditioned in ADM affect cellular behavior. Methods: Canine gingival fibroblasts (CGF), human gingival fibroblasts (HGF) and murine melanoma cell line (B16F10) were seeded on AMD for up to 14 days. The following parameters were assessed: morphology and distribution of CGF, HGF and B16F10; CGF and HGF viability and the effect of ADM conditioned medium (CM) on CGF viability. Results: Epifluorescence revealed that CGF were unevenly distributed on ADM surface, showing no increase in cell number over the periods; HGF formed a monolayer on ADM surface, in a higher number at 14 days (p<0.05); B16F10 exhibited an increase in cell number within 7 days (p<0.05), and were mainly arranged in cell aggregates on ADM, forming a continuous layer at 14 days. A higher percentage of cells on ADM surface (p <0.05) compared to inside was observed for all cell types. MTT values indicated higher cell viability in samples cultured with HGF compared to CGF (p=0.024). A significantly lower cell viability for CGF grown in CM compared to cells grown in non conditioned medium was observed at 48 and 72 h (p <0.05). Conclusion: ADM is not suitable as a 3D matrix for gingival fibroblasts ingrowth. Gingival fibroblasts and highly proliferative cells as B16F10 can be only superficially located on ADM and CGF are negatively affected by culture medium conditioned in ADM, reducing its viability. PMID: 20812778 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Micropatterning and characterization of electrospun poly(epsilon-caprolactone)/gelatin nanofiber tissue scaffolds by femtosecond laser ablation for tissue engineering applications. Biotechnol Bioeng. 2010 Sep 1; Authors: Lim YC, Johnson J, Fei Z, Wu Y, Farson DF, Lannutti JJ, Choi HW, Lee LJ Experimental investigations aimed at assessing the effectiveness of femtosecond laser ablation for creating microscale features on electrospun poly(epsilon-caprolactone) (PCL)/gelatin nanofiber tissue scaffold capable of controlling cell distribution are described. Statistical comparisons of the fiber diameter and surface porosity on laser-machined and as-spun surface were made and results showed that laser ablation did not change the fiber surface morphology. The minimum feature size that could be created on electrospun nanofiber surfaces by direct-write ablation was measured over a range of laser pulse energies. The minimum feature size that could be created was limited only by the pore size of the scaffold surface. The chemical states of PCL/gelatin nanofiber surfaces were measured before and after femtosecond laser machining by Attenuated Total Reflectance Fourier Transform Infrared (ATR- FTIR) spectroscopy and x-ray photoelectron spectroscopy (XPS) and showed that laser machining produced no changes in the chemistry of the surface. In vitro, mES cells were cultured on as-spun surfaces and in laser-machined microwells. Cell densities were found to be statistically indistinguishable after one and two days of growth. Additionally, confocal microscope imaging confirmed that spreading of mES cells cultured within laser-machined microwells was constrained by the cavity walls, the expected and desired function of these cavities. The geometric constraint caused statistically significant smaller density of cells in microwells after three days of growth. It was concluded that femtosecond laser ablation is an effective process for microscale structuring of these electrospun nanofiber tissue scaffold surfaces. (c) 2010 Wiley Periodicals, Inc. PMID: 20812254 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Regenerative medicine: Heart redevelopment. Nature. 2010 Sep 2;467(7311):39-40 Authors: Harvey RP PMID: 20811447 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Tissue engineering: Vision restored. Nature. 2010 Sep 2;467(7311):8 Authors: PMID: 20811413 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Can C4d Immunostaining on Endomyocardial Biopsies Be Considered a Prognostic Biomarker in Heart Transplant Recipients? Transplantation. 2010 Aug 31; Authors: Fedrigo M, Gambino A, Tona F, Torregrossa G, Poli F, Benazzi E, Frigo A, Feltrin G, Toscano G, Caforio AP, Iliceto S, Valente M, Thiene G, Gerosa G, Angelini A BACKGROUND.: The aim of this study was to assess the significance of positive C4d capillary immunostaining of endomyocardial biopsies and its correlation to clinical outcome in adult heart transplant recipients. METHODS.: Nine hundred eighty-five endomyocardial biopsies from 107 heart transplant recipients were evaluated. Immunostaining for detection of intragraft C4d capillary deposition was performed on paraffin-embedded tissue using anti-human C4d polyclonal antibody. RESULTS.: Positive staining of C4d was present in 36 patients (34%) and antibody-mediated rejection in eight patients (7%). The patients were subdivided into four groups on the basis of their C4d, circulating antidonor antibodies (donor-specific antibodies [DSAs]), and graft function: group 1=C4d positive, DSA negative, and no graft dysfunction; group 2=C4d positive, DSA positive, and no graft dysfunction; group 3=C4d positive, DSA positive, and signs of graft dysfunction, and group 0 (control)=all negative. An higher mortality risk was found in C4d-positive patients, when compared with negative ones (unadjusted hazard ratios: group 1: 18, group 2: 61, and group 3: 32-fold risk; P<0.0001). CONCLUSIONS.: Antibody-mediated rejection is a complex and ongoing phenomenon with different phenotypic features. C4d positive predicts worse prognosis. C4d negative and DSA can be used as early mortality predictors in patients without signs of graft dysfunction. PMID: 20811321 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Islet Transplantation Using Donors After Cardiac Death: Report of the Japan Islet Transplantation Registry. Transplantation. 2010 Aug 31; Authors: Saito T, Gotoh M, Satomi S, Uemoto S, Kenmochi T, Itoh T, Kuroda Y, Yasunami Y, Matsumoto S, Teraoka S, BACKGROUND.: This report summarizes outcomes of islet transplantation employing donors after cardiac death (DCD) between 2004 and 2007 as reported to the Japan Islet Transplantation Registry. METHOD.: Sixty-five islet isolations were performed for 34 transplantations in 18 patients with insulin-dependent diabetes mellitus, including two patients who had prior kidney transplantation. All but one donor (64/65) was DCD at the time of harvesting. RESULTS.: Factors influencing criteria for islet release included duration of low blood pressure of the donor, cold ischemic time, and usage of Kyoto solution for preservation. Multivariate analysis selected usage of Kyoto solution as most important. Of the 18 recipients, 8, 4, and 6 recipients received 1, 2, and 3 islet infusions, respectively. Overall graft survival defined as C-peptide level more than or equal to 0.3 ng/mL was 76.5%, 47.1%, and 33.6% at 1, 2, and 3 years, respectively, whereas corresponding graft survival after multiple transplantations was 100%, 80.0%, and 57.1%, respectively. All recipients remained free of severe hypoglycemia while three achieved insulin independence for 14, 79, and 215 days. HbA1c levels and requirement of exogenous insulin were significantly improved in all patients. CONCLUSION.: Islet transplantation employing DCD can ameliorate severe hypoglycemic episodes, significantly improve HbA1c levels, sustain significant levels of C-peptide, and achieve insulin independence after multiple transplantations. Thus, DCD can be an important resource for islet transplantation if used under strict releasing criteria and in multiple transplantations, particularly in countries where heart-beating donors are not readily available. PMID: 20811319 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Tissue engineering in plastic surgery: a review. Plast Reconstr Surg. 2010 Sep;126(3):858-68 Authors: Wong VW, Rustad KC, Longaker MT, Gurtner GC Novel tissue- and organ-engineering strategies are needed to address the growing need for replacement biological parts. Collective progress in stem cell technology, biomaterials, engineering, and molecular medicine has advanced the state of regenerative medicine, yet many hurdles to clinical translation remain. Plastic surgeons are in an ideal position to capitalize on emerging technologies and will be at the forefront of transitioning basic science research into the clinical reconstructive arena. This review highlights fundamental principles of bioengineering, recent progress in tissue-specific engineering, and future directions for this exciting and rapidly evolving area of medicine. PMID: 20811219 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Depot-specific variation in the osteogenic and adipogenic potential of human adipose-derived stromal cells. Plast Reconstr Surg. 2010 Sep;126(3):822-34 Authors: Levi B, James AW, Glotzbach JP, Wan DC, Commons GW, Longaker MT BACKGROUND:: Adipose-derived stromal cells hold promise for use in tissue regeneration. However, multiple facets of their biology remain unclear. The authors examined the variations in osteogenesis and adipogenesis in adipose-derived stromal cells between subcutaneous fat depots and potential molecular causes. METHODS:: Adipose-derived stromal cells were isolated from human patients from subcutaneous fat depots, including arm, flank, thigh, and abdomen (n = 5 patients). Osteogenic and adipogenic differentiation was performed (alkaline phosphatase, alizarin red, and oil red O staining, and quantitative real-time polymerase chain reaction). Co-cultures were established to assess the paracrine effect of human adipose-derived stromal cells on mouse osteoblasts. Finally, HOX gene expression was analyzed by quantitative real-time polymerase chain reaction. RESULTS:: Subcutaneous fat depots retain markedly different osteogenic and adipogenic potentials. Osteogenesis was most robust in adipose-derived stromal cells from the flank and thigh, as compared with those from the arm and abdomen (p < 0.05 by all markers examined). This was accompanied by elevations of BMP4 and BMPR1B (p < 0.05 by all markers examined). The osteogenic advantage of cells from the flank and thigh was again observed when analyzing the paracrine effects of these cells. Conversely, those cells isolated from the flank had a lesser ability to undergo adipogenic differentiation. Adipose-associated HOX genes were less expressed in flank-derived adipose-derived stromal cells. CONCLUSIONS:: Variations exist between fat depots in terms of adipose-derived stromal cell osteogenic and adipogenic differentiation. Differences in HOX expression and bone morphogenetic protein signaling may underlie these observations. This study indicates that the choice of fat depot derivation of adipose-derived stromal cells may be an important one for future efforts in tissue engineering. PMID: 20811215 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Paracrine Interaction between Adipose-Derived Stromal Cells and Cranial Suture-Derived Mesenchymal Cells. Plast Reconstr Surg. 2010 Sep;126(3):806-21 Authors: James AW, Levi B, Commons GW, Glotzbach J, Longaker MT BACKGROUND:: Adipose-derived stromal cells are a potential cell source for the successful healing of skeletal defects. In this study, the authors sought to investigate the potential for cranial suture-derived mesenchymal cells to promote the osteogenic differentiation of adipose-derived stromal cells. Various reports have previously examined the unique in vitro attributes of suture-derived mesenchymal cells; this study sought to extend those findings. METHODS:: Suture-derived mesenchymal cells were isolated from wild-type mice (n = 30) from both fusing posterofrontal and patent sagittal sutures. Cells were placed in Transwell inserts with human adipose-derived stromal cells (n = 5 patients) with osteogenic differentiation medium with or without recombinant Noggin (10 to 400 ng/ml). Specific gene expression of osteogenic markers and Hedgehog pathway were assayed; standard osteogenic assays (alkaline phosphatase and alizarin red staining) were performed. All assays were performed in triplicate. RESULTS:: Both posterofrontal and sagittal suture-derived mesenchymal cells induced osteogenic differentiation of adipose-derived stromal cells (p < 0.05). Posterofrontal suture-derived mesenchymal cells induced adipose-derived stromal cell osteogenesis to a greater degree than sagittal suture-derived mesenchymal cells (p < 0.05). This was accompanied by an increase in bone morphogenetic protein expression (p < 0.05). Finally, recombinant Noggin mitigated the pro-osteogenic effects of co-culture accompanied by a reduction in Hedgehog signaling (p < 0.05). CONCLUSIONS:: Suture-derived mesenchymal cells secrete paracrine factors that induce osteogenic differentiation of multipotent stromal cells (human adipose-derived stromal cells). Cells derived from the fusing posterofrontal suture do this to a significantly greater degree than cells from the patent sagittal suture. Enhanced bone morphogenetic protein and Hedgehog signaling may underlie this paracrine effect. PMID: 20811214 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Effects of surfactant and gentle agitation on inkjet dispensing of living cells. Biofabrication. 2010 Jun;2(2):025003 Authors: Parsa S, Gupta M, Loizeau F, Cheung KC Inkjet dispensing is a promising method for patterning cells and biomaterials for tissue engineering applications. In a novel approach, this work uses a biocompatible surfactant to improve the reliability of droplet formation in piezoelectric drop-on-demand inkjet printing of Hep G2 hepatocytes onto hydrogels. During a long printing process, cell aggregation and sedimentation within the inkjet reservoir can lead to inconsistent printing results. In order to improve repeatability, the effects of gentle agitation on cell sedimentation and aggregation within the inkjet reservoir were also investigated. Cell viability and proliferation when printed onto prepared collagen substrates were assessed using live/dead staining and the Alamar Blue metabolic assay. The addition of 0.05% Pluronic as a surfactant did not reduce cell viability, which remained above 95% 2 days after printing. The surfactant improved the reliability of droplet formation. Although gentle stirring of the inkjet reservoir was sufficient to maintain a cell suspension and reduce sedimentation, aggregation within the suspension continued to affect printing performance over a 180 min printing period. PMID: 20811131 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Fabricating a pearl/PLGA composite scaffold by the low-temperature deposition manufacturing technique for bone tissue engineering. Biofabrication. 2010 Jun;2(2):025002 Authors: Xu M, Li Y, Suo H, Yan Y, Liu L, Wang Q, Ge Y, Xu Y Here we developed a composite scaffold of pearl/poly(lactic-co-glycolic acid) (pearl/PLGA) utilizing the low-temperature deposition manufacturing (LDM). LDM makes it possible to fabricate scaffolds with designed microstructure and macrostructure, while keeping the bioactivity of biomaterials by working at a low temperature. Process optimization was carried out to fabricate a mixture of pearl powder, PLGA and 1,4-dioxane with the designed hierarchical structures, and freeze-dried at a temperature of -40 degrees C. Scaffolds with square and designated bone shape were fabricated by following the 3D model. Marrow stem cells (MSCs) were seeded on the pearl/PLGA scaffold and then cultured in a rotating cell culture system. The adhesion, proliferation and differentiation of MSCs into osteoblasts were determined using scanning electronic microscopy, WST-1 assay, alkaline phosphatase activity assay, immunofluorescence staining and real-time reverse transcription polymerase chain reaction. The results showed that the composite scaffold had high porosity (81.98 +/- 3.75%), proper pore size (micropores: <10 microm; macropore: 495 +/- 54 microm) and mechanical property (compressive strength: 0.81 +/- 0.04 MPa; elastic modulus: 23.14 +/- 0.75 MPa). The pearl/PLGA scaffolds exhibited better biocompatibility and osteoconductivity compared with the tricalcium phosphate/PLGA scaffold. All these results indicate that the pearl/PLGA scaffolds fulfill the basic requirements of bone tissue engineering scaffold. PMID: 20811130 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Biofabrication to build the biology-device interface. Biofabrication. 2010 Jun;2(2):022002 Authors: Liu Y, Kim E, Ghodssi R, Rubloff GW, Culver JN, Bentley WE, Payne GF The last century witnessed spectacular advances in both microelectronics and biotechnology yet there was little synergy between the two. A challenge to their integration is that biological and electronic systems are constructed using divergent fabrication paradigms. Biology fabricates bottom-up with labile components, while microelectronic devices are fabricated top-down using methods that are 'bio-incompatible'. Biofabrication-the use of biological materials and mechanisms for construction-offers the opportunity to span these fabrication paradigms by providing convergent approaches for building the bio-device interface. Integral to biofabrication are stimuli-responsive materials (e.g. film-forming polysaccharides) that allow directed assembly under near physiological conditions in response to device-imposed signals. Biomolecular engineering, through recombinant technology, allows biological components to be endowed with information for assembly (e.g. encoded in a protein's amino acid sequence). Finally, self-assembly and enzymatic assembly provide the mechanisms for construction over a hierarchy of length scales. Here, we review recent advances in the use of biofabrication to build the bio-device interface. We anticipate that the biofabrication toolbox will expand over the next decade as more researchers enlist the unique construction capabilities of biology. Further, we look forward to observing the application of this toolbox to create devices that can better diagnose disease, detect pathogens and discover drugs. Finally, we expect that biofabrication will enable the effective interfacing of biology with electronics to create implantable devices for personalized and regenerative medicine. PMID: 20811128 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Tissue engineering by self-assembly and bio-printing of living cells. Biofabrication. 2010 Jun;2(2):022001 Authors: Jakab K, Norotte C, Marga F, Murphy K, Vunjak-Novakovic G, Forgacs G Biofabrication of living structures with desired topology and functionality requires the interdisciplinary effort of practitioners of the physical, life and engineering sciences. Such efforts are being undertaken in many laboratories around the world. Numerous approaches are pursued, such as those based on the use of natural or artificial scaffolds, decellularized cadaveric extracellular matrices and, most lately, bioprinting. To be successful in this endeavor, it is crucial to provide in vitro micro-environmental clues for the cells resembling those in the organism. Therefore, scaffolds, populated with differentiated cells or stem cells, of increasing complexity and sophistication are being fabricated. However, no matter how sophisticated scaffolds are, they can cause problems stemming from their degradation, eliciting immunogenic reactions and other a priori unforeseen complications. It is also being realized that ultimately the best approach might be to rely on the self-assembly and self-organizing properties of cells and tissues and the innate regenerative capability of the organism itself, not just simply prepare tissue and organ structures in vitro followed by their implantation. Here we briefly review the different strategies for the fabrication of three-dimensional biological structures, in particular bioprinting. We detail a fully biological, scaffoldless, print-based engineering approach that uses self-assembling multicellular units as bio-ink particles and employs early developmental morphogenetic principles, such as cell sorting and tissue fusion. PMID: 20811127 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Biomatrices and biomaterials for future developments of bioprinting and biofabrication. Biofabrication. 2010 Mar;2(1):014110 Authors: Nakamura M, Iwanaga S, Henmi C, Arai K, Nishiyama Y The next step beyond conventional scaffold-based tissue engineering is cell-based direct biofabrication techniques. In industrial processes, various three-dimensional (3D) prototype models have been fabricated using several different rapid prototyping methods, such as stereo-lithography, 3D printing and laser sintering, as well as others, in which a variety of chemical materials are utilized. However, with direct cell-based biofabrication, only biocompatible materials can be used, and the manufacturing process must be performed under biocompatible and physiological conditions. We have developed a direct 3D cell printing system using inkjet and gelation techniques with inkjet droplets, and found that it had good potential to construct 3D structures with multiple types of cells. With this system, we have used alginate and fibrin hydrogel materials, each of which has advantages and disadvantages. Herein, we discuss the roles of hydrogel for biofabrication and show that further developments in biofabrication technology with biomatrices will play a major part, as will developments in manufacturing technology. It is important to explore suitable biomatrices as the next key step in biofabrication techniques. PMID: 20811125 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Bio rapid prototyping by extruding/aspirating/refilling thermoreversible hydrogel. Biofabrication. 2010 Mar;2(1):014108 Authors: Iwami K, Noda T, Ishida K, Morishima K, Nakamura M, Umeda N This paper reports a method for rapid prototyping of cell tissues, which is based on a system that extrudes, aspirates and refills a mixture of cells and thermoreversible hydrogel as a scaffold. In the extruding mode, a cell-mixed scaffold solution in the sol state is extruded from a cooled micronozzle into a temperature-controlled substrate, which keeps the scaffold in the gel state. In the aspiration mode, the opposite process is performed by Bernoulli suction. In the refilling mode, the solution is extruded into a groove created in the aspiration mode. The minimum width of extruded hydrogel pattern is 114 +/- 15 microm by employing a nozzle of diameter 100 microm, and that of aspirated groove was 355 +/- 10 microm using a 500 microm-diameter nozzle. Gum arabic is mixed with the scaffold solution to avoid peeling-off of the gel pattern from the substrate. Patterning of Sf-9 cell tissue is demonstrated, and the stability of the patterned cell is investigated. This system offers a procedure for rapid prototyping and local modification of cell scaffolds for tissue engineering. PMID: 20811123 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | A droplet-based building block approach for bladder smooth muscle cell (SMC) proliferation. Biofabrication. 2010 Mar;2(1):014105 Authors: Xu F, Moon SJ, Emre AE, Turali ES, Song YS, Hacking SA, Nagatomi J, Demirci U Tissue engineering based on building blocks is an emerging method to fabricate 3D tissue constructs. This method requires depositing and assembling building blocks (cell-laden microgels) at high throughput. The current technologies (e.g., molding and photolithography) to fabricate microgels have throughput challenges and provide limited control over building block properties (e.g., cell density). The cell-encapsulating droplet generation technique has potential to address these challenges. In this study, we monitored individual building blocks for viability, proliferation and cell density. The results showed that (i) SMCs can be encapsulated in collagen droplets with high viability (>94.2 +/- 3.2%) for four cases of initial number of cells per building block (i.e. 7 +/- 2, 16 +/- 2, 26 +/- 3 and 37 +/- 3 cells/building block). (ii) Encapsulated SMCs can proliferate in building blocks at rates that are consistent (1.49 +/- 0.29) across all four cases, compared to that of the controls. (iii) By assembling these building blocks, we created an SMC patch (5 mm x 5 mm x 20 microm), which was cultured for 51 days forming a 3D tissue-like construct. The histology of the cultured patch was compared to that of a native rat bladder. These results indicate the potential of creating 3D tissue models at high throughput in vitro using building blocks. PMID: 20811120 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Laser printing of cells into 3D scaffolds. Biofabrication. 2010 Mar;2(1):014104 Authors: Ovsianikov A, Gruene M, Pflaum M, Koch L, Maiorana F, Wilhelmi M, Haverich A, Chichkov B One of the most promising approaches in tissue engineering is the application of 3D scaffolds, which provide cell support and guidance in the initial tissue formation stage. The porosity of the scaffold and internal pore organization influence cell migration and play a major role in its biodegradation dynamics, nutrient diffusion and mechanical stability. In order to control cell migration and cellular interactions within the scaffold, novel technologies capable of producing 3D structures in accordance with predefined design are required. The two-photon polymerization (2PP) technique, used in this report for the fabrication of scaffolds, allows the realization of arbitrary 3D structures with submicron spatial resolution. Highly porous 3D scaffolds, produced by 2PP of acrylated poly(ethylene glycol), are seeded with cells by means of laser-induced forward transfer (LIFT). In this laser printing approach, a propulsive force, resulting from laser-induced shock wave, is used to propel individual cells or cell groups from a donor substrate towards the receiver substrate. We demonstrate that with this technique printing of multiple cell types into 3D scaffolds is possible. Combination of LIFT and 2PP provides a route for the realization of 3D multicellular tissue constructs and artificial ECM engineered on the microscale. PMID: 20811119 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Bioprinting by laser-induced forward transfer for tissue engineering applications: jet formation modeling. Biofabrication. 2010 Mar;2(1):014103 Authors: Mézel C, Souquet A, Hallo L, Guillemot F In this paper, a nanosecond LIFT process is analyzed both from experimental and modeling points of view. Experimental results are first presented and compared to simple estimates obtained from physical analysis, i.e. energy balance, jump relations and analytical pocket dynamics. Then a self-consistent 2D axisymmetric modeling strategy is presented. It is shown that data accessible from experiments, i.e. jet diameter and velocity, can be reproduced. Moreover, some specific mechanisms involved in the rear-surface deformation and jet formation may be described by some scales of hydrodynamic process, i.e. shock waves propagation and expansion waves, as a consequence of the laser heating. It shows that the LIFT process is essentially driven by hydrodynamics and thermal transfer, and that a coupled approach including self-consistent laser energy deposition, heating by thermal conduction and specific models for matter is required. PMID: 20811118 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Combining electrospinning and fused deposition modeling for the fabrication of a hybrid vascular graft. Biofabrication. 2010 Mar;2(1):014102 Authors: Centola M, Rainer A, Spadaccio C, De Porcellinis S, Genovese JA, Trombetta M Tissue engineering of blood vessels is a promising strategy in regenerative medicine with a broad spectrum of potential applications. However, many hurdles for tissue-engineered vascular grafts, such as poor mechanical properties, thrombogenicity and cell over-growth inside the construct, need to be overcome prior to the clinical application. To surmount these shortcomings, we developed a poly-l-lactide (PLLA)/poly-epsilon-caprolactone (PCL) scaffold releasing heparin by a combination of electrospinning and fused deposition modeling technique. PLLA/heparin scaffolds were produced by electrospinning in tubular shape and then fused deposition modeling was used to armor the tube with a single coil of PCL on the outer layer to improve mechanical properties. Scaffolds were then seeded with human mesenchymal stem cells (hMSCs) and assayed in terms of morphology, mechanical tensile strength, cell viability and differentiation. This particular scaffold design allowed the generation of both a drug delivery system amenable to surmount thrombogenic issues and a microenvironment able to induce endothelial differentiation. At the same time, the PCL external coiling improved mechanical resistance of the microfibrous scaffold. By the combination of two notable techniques in biofabrication-electrospinning and FDM-and exploiting the biological effects of heparin, we developed an ad hoc differentiating device for hMSCs seeding, able to induce differentiation into vascular endothelium. PMID: 20811117 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Bioprinting is coming of age: report from the International Conference on Bioprinting and Biofabrication in Bordeaux (3B'09). Biofabrication. 2010 Mar;2(1):010201 Authors: Guillemot F, Mironov V, Nakamura M Abstract The International Conference on Bioprinting and Biofabrication in Bordeaux (3B'09) demonstrated that the field of bioprinting and biofabrication continues to evolve. The increasing number and broadening geography of participants, the emergence of new exciting bioprinting technologies, and the attraction of young investigators indicates the strong growth potential of this emerging field. Bioprinting can be defined as the use of computer-aided transfer processes for patterning and assembling living and non-living materials with a prescribed 2D or 3D organization in order to produce bio-engineered structures serving in regenerative medicine, pharmacokinetic and basic cell biology studies. The use of bioprinting technology for biofabrication of in vitro assay has been shown to be a realistic short-term application. At the same time, the principal feasibility of bioprinting vascularized human organs as well as in vivo bioprinting has been demonstrated. The bioprinting of complex 3D human tissues and constructs in vitro and especially in vivo are exciting, but long-term, applications. It was decided that the 5th International Conference on Bioprinting and Biofabrication would be held in Philadelphia, USA in October 2010. The specially appointed 'Eploratory Committee' will consider the possibility of turning the growing bioprinting community into a more organized entity by creating a new bioprinting and biofabrication society. The new journal Biofabrication was also presented at 3B'09. This is an important milestone per se which provides additional objective evidence that the bioprinting and biofabrication field is consolidating and maturing. Thus, it is safe to state that bioprinting technology is coming of age. 1. Back to Europe The International Conference on Bioprinting and Biofabrication in Bordeaux (3B'09), France, 6-8 July 2009, was held after several international meetings which had been organized previously. The First International Workshop on Bioprinting and Biopatterning [1] was held at the University of Manchester (UK) in September 2004 and was organized by Professor Brian Derby (University of Manchester), Douglas B Chrisey (Naval Research Laboratory, Washington, USA), Richard K Everett (ONR Global, London) and Nuno Reis (Universidade de Beira Interior, Covilha, Portugal). The Second International Workshop on Bioprinting, Biopatterning and Bioassembly was chaired by Vladimir Mironov in 2005 in Charleston (USA) [2]. The Third International Symposium on Bioprinting and Biofabrication was held in Kawasaki (Japan) in November 2006 and was organized by Professor Makoto Nakamura (University of Toyama, Japan). After three years without an international meeting, Fabien Guillemot (INSERM, University of Bordeaux, France) and Professor Makoto Nakamura decided to organize the International Conference on Bioprinting and Biofabrication (3B'09) [3] in Bordeaux, the wine capital of the world. This 4th international meeting on bioprinting was endorsed by TERMIS (Tissue Engineering and Regenerative Medicine International Society) and sponsored by the clusters Advanced Materials in Aquitaine, Materials in Bordeaux and Route des Lasers, INSERM and the Aquitaine Regional Council. It took place at the Burdigala Hotel in a very pleasant and friendly atmosphere where new players, especially the young, were welcome and were given the opportunity to discuss their work. The balanced geographical representation was another important feature of the 3B'09 conference. Indeed, more than 65 scientists and engineers from 11 countries (Belgium, France, Germany, Italy, Japan, Poland, Portugal, Romania, The Netherlands, UK, USA) attended this conference, which included five oral presentation sessions, one poster session and social events organized at the Bordeaux City Hall and at the Château Giscours. 2. Scientific program The scientific program was established to highlight the latest developments associated with bioprinting technologies and biofabrication approaches. Indeed, since the early pioneering works of Thomas Boland (Clemson University, USA) and Vladimir Mironov [4], the bioprinting scientific community has been evolving, bringing together physicists, biologists and physicians [5, 6]. Consequently, the oral presentation sessions covered: (1) the latest developments in bioprinting technologies; (2) the potential to combine the bioprinting process with other biofabrication and rapid prototyping methods; (3) matrices and biomaterials for bioprinting and biofabrication; (4) methods for designing, modeling and biomechanically evaluating 3D constructs; (5) developmental biology and tissue engineering. This scientific schedule emphasized some of the main areas that have to be connected prior to envisaging real applications in regenerative medicine, pharmacokinetic and toxicological studies, where high throughput and high-resolution bioprinting technologies are required. Thus, it is now obvious that, in addition to core bioprinting technologies, biomaterial and bioink properties, rapid prototyping approaches and basic cell biology have to be taken into account within a single perspective. Consequently, while the initial definition of our burgeoning field was formulated at the First International Workshop on Bioprinting and Biopatterning in Manchester (UK) as 'the use of material transfer processes for patterning and assembling biologically relevant materials, molecules, cells, tissues, and biodegradable biomaterials with a prescribed organization to accomplish one or more biological functions', we propose to enlarge this definition to 'the use of computer-aided transfer processes for patterning and assembling living and non-living materials with a prescribed 2D or 3D organization in order to produce bio-engineered structures serving in regenerative medicine, pharmacokinetic and basic cell biology studies'. Regarding the poster session, three young scientists were awarded a bottle of Château Giscours wine for their impressive work. Laureates were Virginie Kériquel in vivo high-throughput biological laser printing of nano-hydroxyapatite in mice calvaria critical sized defects: preliminary results) [7], Kayo Sakaue (Integration of 3D-micro tissue models for creation of tissue chip) [8] and Alberto Rainer (Regeneration of osteochondral segment via mesenchymal stem cells culturing on 3D rapid prototyped scaffolds) [9]. 3. On bioprinting technologies The 3B'09 conference was an excellent opportunity to perform an in-depth review of bioprinting technologies (jet-based and extrusion methods) through a number of exciting talks given by both academics and industrialists. Recent advances in ink-jet bioprinting technologies were reviewed by Professor Thomas Boland and supplemented mainly by the presentations given by Professor Makoto Nakamura [10, 11] and Professor Brian Derby. Regarding laser-based technologies, a series of talks dealing with experimental and modeling approaches of laser-assisted bioprinting (LAB) [12] were given by members of Fabien Guillemot [13] and Boris Chichkov's groups [14] from The University of Bordeaux and Lazer Zentrum in Hannover (Germany), respectively. Utkan Demirci (Harvard University, USA) introduced a new ultrasonic wave-based bioprinting technology, the so-called layer-by-layer 3D tissue epitaxy by cell-laden hydrogel droplets [15] while Hedges and Wirth from Germany presented an aerosol jet bioprinting technology and its applications in surface biofunctionalization [16]. Besides the above-mentioned jet-based bioprinting techniques, many presentations were related to 3D plotting by extrusion processes. Giovani Vozzi's group (University of Pisa, Italy) presented several talks dealing with the micro-fabrication of two- and three-dimensional structures by pressure-assisted micro-syringe (PAM) [17]. Moreover, processing considerations for the 3D plotting of thermoplastic scaffolds were presented by Kim Ragaert (Ghent University, Belgium) [18] and Hendrik John (Sys-Eng, Germany) while Kentaro Iwami (Tokyo University, Japan) described for the first time rapid prototyping by extruding/aspirating/refilling thermoreversible hydrogel [19]. Finally, additional 3D biofabrication methods were presented. Matsusaki et al (Japan) introduced possible, layer-by-layer, short-term applications of bioprinting for biofabrication of tissue chips. Frasca et al (University of Paris-Diderot, France) demonstrated how nanotechnology (magnetic nanoparticles) can be employed in magnetic force-driven tissue engineering. In summary, while each of these technologies displays specific properties such as high resolution, high throughput, low price, bio-safety, etc, it seems obvious that achieving more advanced applications in tissue engineering will require a combination of these tools, and thus a combination of their performances. Moreover, to print human organs, highly integrative approaches should be set up; these should include the development of new biomaterials, the improvement of reverse-engineering and rapid prototyping methods, intensification of scientific gateways between developmental biology and tissue engineering [20] (as introduced by Gabor Forgacs, University of Missouri, USA) [21], in particular with the help of numerical modeling. All these perspectives were discussed during the conference and are reported in the following sections. 4. Scaffold or not scaffold, that is the question! One of the controversial topics of discussion during the conference was the definition of 'scaffold'. Synthetic biodegradable scaffold is considered to be a fundamental principle of the traditional scaffold-based or 'top down' tissue engineering approach [22]. The emerging modular or 'bottom-up' approach is sometimes called 'scaffold-free' [23] or 'scaffold-less'. Some participants insisted that scaffold according to definition is a temporal and removable support. (ABSTRACT TRUNCATED) PMID: 20811115 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | The role of printing parameters and scaffold biopolymer properties in the efficacy of a new hybrid nano-bioprinting system. Biofabrication. 2009 Sep;1(3):035003 Authors: Buyukhatipoglu K, Jo W, Sun W, Clyne AM We created a hybrid nano-bioprinting system, which combines the initial patterning capabilities of direct cell writing with the active patterning capabilities of superparamagnetic nanoparticles. Biofabrication conditions, including printing parameters and scaffold biopolymer properties, may affect cell viability, nanoparticle manipulation and patterning capabilities. Nanoparticles were printed under varied conditions either in the biopolymer or loaded inside cells. Cell viability, alginate viscosity, nanoparticle movement and printing resolution were measured. We now show that while nanoparticles decreased cell viability, nozzle size had no significant effect. High printing pressure decreased cell viability, but viability loss was not accentuated by nanoparticles. High nanoparticle concentrations increased alginate viscosity at higher alginate concentrations. Nanoparticle velocity in response to a magnetic field was a function of nanoparticle diameter and scaffold viscosity, which agreed with a mathematical model of nanoparticle movement. Finally, the nano-bioprinting system resolution and patterning precision were not affected by nanoparticles in the prepolymer solution. These data suggest that nanoparticle incorporation in solid freeform fabrication does not change biofabrication parameters unless high nanoparticle concentrations are used. Future work includes developing vascularized tissue engineering constructs using the nano-bioprinting system. PMID: 20811107 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | A brief review of dispensing-based rapid prototyping techniques in tissue scaffold fabrication: role of modeling on scaffold properties prediction. Biofabrication. 2009 Sep;1(3):032001 Authors: Li MG, Tian XY, Chen XB Artificial scaffolds play vital roles in tissue engineering as they provide a supportive environment for cell attachment, proliferation and differentiation during tissue formation. Fabrication of tissue scaffolds is thus of fundamental importance for tissue engineering. Of the variety of scaffold fabrication techniques available, rapid prototyping (RP) methods have attracted a great deal of attention in recent years. This method can improve conventional scaffold fabrication by controlling scaffold microstructure, incorporating cells into scaffolds and regulating cell distribution. All of these contribute towards the ultimate goal of tissue engineering: functional tissues or organs. Dispensing is typically used in different RP techniques to implement the layer-by-layer fabrication process. This article reviews RP methods in tissue scaffold fabrication, with emphasis on dispensing-based techniques, and analyzes the effects of different process factors on fabrication performance, including flow rate, pore size and porosity, and mechanical cell damage that can occur in the bio-manufacturing process. PMID: 20811104 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | During EPO or anemia challenge, erythroid progenitor cells transit through a selectively expandable proerythroblast pool. Blood. 2010 Sep 1; Authors: Dev A, Fang J, Sathyanarayana P, Pradeep A, Emerson C, Wojchowski DM Investigations of bone marrow (BM) erythroblast development are important for clinical concerns, but are hindered by progenitor cell and tissue availability. We've therefore sought to more specifically define dynamics, and key regulators, of the formation of developing BM erythroid cell cohorts. A unique Kit(neg)CD71(high)Ter119(neg) "stage-E2" proerythroblast pool first is described which (unlike its Kit(pos) "stage-E1" progenitors, or maturing Ter119(pos) "stage-E3" progeny) proved to selectively expand ~7-fold upon EPO challenge. During short-term bone marrow transplantation, stage E2 proerythroblasts additionally proved to be a predominantly expanded progenitor pool within spleen. This E1-->E2-->E3 erythroid erythroid series reproducibly formed ex vivo, enabling further characterizations. Expansion, in part, involved E1 cell hyper-proliferation together with rapid E2 conversion plus E2 stage- restricted BCL2 expression. Possible EPO/EPOR proerythroblast stage-specific events were further investigated in mice expressing minimal EPOR alleles: For a hypomorphic EPOR-HM allele, major defects in erythroblast development occurred selectively at stage-E2. In addition, stage-E2 cells proved to interact productively with primary BM stromal cells in ways which enhanced both survival, and late-stage development. Overall, findings reveal a novel transitional proerythroblast compartment that deploys unique expansion devices. PMID: 20810925 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Rab11 and Its Effector Rab Coupling Protein Contribute to the Trafficking of {beta}1 Integrins during Axon Growth in Adult Dorsal Root Ganglion Neurons and PC12 Cells. J Neurosci. 2010 Sep 1;30(35):11654-69 Authors: Eva R, Dassie E, Caswell PT, Dick G, Ffrench-Constant C, Norman JC, Fawcett JW Integrins play an important part in axon growth, but integrin traffic in neurons is poorly understood. Expression of the tenascin-C-binding integrin alpha9 promotes axon regeneration. We have therefore studied the mechanism by which alpha9 integrin and its partner beta1 are trafficked along axons and at the growth cone using adult DRG neurons and PC12 cells. We have focused on the small GTPase Rab11 and its effector Rab coupling protein (RCP), as they are involved in the long-range trafficking of beta1 integrins in other cells. Rab11 colocalizes with alpha9 and other alpha integrins and with beta1 integrin in growth cones and axons, and immunopurified Rab11 vesicles contain alpha9 and beta1. Endocytosed beta1 integrins traffic via Rab11. However, Rab11 vesicles in axons are generally static, and alpha9 integrins undergo bouts of movement during which they leave the Rab11 compartment. In growth cones, alpha9 and beta1 overlap with RCP, particularly at the growth cone periphery. We show that beta1 integrin trafficking during neurite outgrowth involves Rab11 and RCP, and that manipulation of these molecules alters surface integrin levels and axon growth, and can be used to enhance alpha9 integrin-dependent neurite outgrowth. Our data suggest that manipulation of trafficking via Rab11 and RCP could be a useful strategy for promoting integrin-dependent axonal regeneration. PMID: 20810886 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Suppression of NF-{kappa}B Increases Bone Formation and Ameliorates Osteopenia in Ovariectomized Mice. Endocrinology. 2010 Sep 1; Authors: Alles N, Soysa NS, Hayashi J, Khan M, Shimoda A, Shimokawa H, Ritzeler O, Akiyoshi K, Aoki K, Ohya K Bone degenerative diseases, including osteoporosis, impair the fine balance between osteoclast bone resorption and osteoblast bone formation. Single-agent therapy for anabolic and anticatabolic effects is attractive as a drug target to ameliorate such conditions. Inhibition of nuclear factor (NF)-kappaB reduces the osteoclast bone resorption. The role of NF-kappaB inhibitors on osteoblasts and bone formation, however, is minimal and not well investigated. Using an established NF-kappaB inhibitor named S1627, we demonstrated that inhibition of NF-kappaB increases osteoblast differentiation and bone formation in vitro by up-regulating the mRNAs of osteoblast-specific genes like type I collagen, alkaline phosphatase, and osteopontin. In addition, S1627 was able to increase bone formation and repair bone defect in a murine calvarial defect model. To determine the effect of NF-kappaB on a model of osteoporosis, we injected two doses of inhibitor (25 and 50 mg/kg . d) twice a day in sham-operated or ovariectomized 12-wk-old mice and killed them after 4 wk. The anabolic effect of S1627 on trabecular bone was determined by micro focal computed tomography and histomorphometry. Bone mineral density of inhibitor-treated ovariectomized animals was significantly increased compared with sham-operated mice. Osteoblast-related indices like osteoblast surface, mineral apposition rate, and bone formation rate were increased in S1627-treated animals in a dose-dependent manner. NF-kappaB inhibition by S1627 increased the trabecular bone volume in ovariectomized mice. Furthermore, S1627 could inhibit the osteoclast number, and osteoclast surface to bone surface. In vitro osteoclastogenesis and bone resorbing activity were dose-dependently reduced by NF-kappaB inhibitor S1627. Taken collectively, our results suggest that NF-kappaB inhibitors are effective in treating bone-related diseases due to their dual anabolic and antiresorptive activities. PMID: 20810563 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | The primordium of a biological joint replacement: Coupling of two stem cell pathways in biphasic ultrarapid compressed gel niches. J Craniomaxillofac Surg. 2010 Aug 30; Authors: Brady MA, Sivananthan S, Mudera V, Liu Q, Wiltfang J, Warnke PH The impaired temporomandibular joint might be the first to benefit from applied tissue engineering techniques because it is small and tissue growth in larger amounts is challenging. Bone and cartilage require different competing environmental conditions to be cultivated in vitro. But coupling both the osteogenic and cartilaginous pathways of mesenchymal stem cell differentiation in homeostasis will be a key essential to grow osteochondral constructs or even the first biological joint replacement in the future. The aim of this study was to test a single source biomaterial and a single source cell type to engineer a biphasic osteochondral construct in vitro for future in vivo implantation. Ultrarapid tissue engineering techniques were used to create the biphasic matrix and primary human mesenchymal stem cells (MSCs) preconditioned in osteogenic and chondrogenic media were then seeded in opposite portions of the hyper-hydrated collagen gel in order to further substantiate the respective bone-like and cartilage-like layers thus potentially customising the collagen scaffold according to patient needs in regards to future biological joint replacements. After incubation for 7 days to allow cell growth and differentiation, mineralization of the bone-like layer was demonstrated using von Kossa staining and biochemical bone markers. The cartilage-like layer was demonstrated using alcian blue staining and biochemical cartilage markers. Integration of the bone-like and cartilage-like layers to simulate a tidemark layer was achieved through partial setting of the gels. Cell tracking was used to further confirm the establishment of distinct cartilage-like and bone-like areas within the single construct. This is the first report of one homogeneous human MSC population differentiating into dissimilar "bone-like" and "cartilage-like" zones hosted in a biphasic ultrarapid compressed gel phase niche and mimicking a primordial joint-like structure. PMID: 20810288 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Expansion of human articular chondrocytes and formation of tissue-engineered cartilage: A step towards exploring a potential use of matrix-induced cell therapy. Tissue Cell. 2010 Aug 30; Authors: Munirah S, Samsudin OC, Aminuddin BS, Ruszymah BH Monolayer culture expansion remains as a fundamental step to acquire sufficient number of cells for 3D constructs formation. It has been well-documented that cell expansion is however accompanied by cellular dedifferentiation. In order to promote cell growth and circumvent cellular dedifferentiation, we evaluated the effects of Transforming Growth Factor Beta-2 (TGF-beta2), Insulin-like Growth Factor-I (IGF-I) and basic Fibroblast Growth Factor (bFGF) combination on articular chondrocytes culture and 'chondrocytes-fibrin' construct formation. Chondrocytes were serially cultured in: (1) F12:DMEM+10% Foetal Bovine Serum (FBS) with growth factors (FD10GFs), (2) F12:DMEM+2%FBS with the growth factors (FD2GFs) and, (3) F12:DMEM+10%FBS without growth factors (FD) as control. Cultured chondrocytes were evaluated by means of growth kinetics parameters, cell cycle analysis, quantitative phenotypic expression of collagen type II, aggrecan core protein sox-9 and collagen type I and, immunochemistry technique. Harvested chondrocytes were incorporated with plasma-derived fibrin and were polymerized to form the 3D constructs and implanted subcutaneously at the dorsum of athymic nude mice for eight (8) weeks. Resulted constructs were assigned for gross inspections and microscopic evaluation using standard histochemicals staining, immunochemistry technique and, quantitative phenotypic expression of cartilage markers to reassure cartilaginous tissue formation. Growth kinetics performance of chondrocytes cultured in three (3) types of culture media from the most to least was in the following order: FD10GFs>FD2GFs>FD. Following growth kinetics analysis, we decided to use FD10GFs and FD (control) for further evaluation and 'chondrocytes-fibrin' constructs formation. Chondrocytes cultured in FD10GFs preserved the normal diploid state (2c) with no evidence of aneuploidy, haploidy or tetraploidy. Expression of cartilage-specific markers namely collagen type II, aggrecan core protein and sox-9 were significantly higher in FD10GFs when compared to control. After implantation, 'chondrocytes-fibrin' constructs exhibited firm, white, smooth and glistening cartilage-like properties. FD10GFs constructs formed better quality cartilage-like tissue than FD constructs in term of overall cartilaginous tissue formation, cells organization and extracellular matrix distribution in the specimens. Cartilaginous tissue formation was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan was confirmed by positive Safranin O staining. Collagen type II exhibited immunopositivity at the pericellular and inter-territorial matrix area. Chondrogenic properties of the construct were further confirmed by the expression of genes encoding collagen type II, aggrecan core protein and sox9. In conclusion, FD10GFs promotes the proliferation of chondrocytes and formation of good quality 'chondrocytes-fibrin' constructs which may have potential use of matrix-induced cell implantation. PMID: 20810142 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Mechanical Strain Stabilizes Reconstituted Collagen Fibrils against Enzymatic Degradation by Mammalian Collagenase Matrix Metalloproteinase 8 (MMP-8). PLoS One. 2010;5(8): Authors: Flynn BP, Bhole AP, Saeidi N, Liles M, Dimarzio CA, Ruberti JW BACKGROUND: Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain. METHODOLOGY/PRINCIPAL FINDINGS: The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded. CONCLUSIONS/SIGNIFICANCE: In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen. PMID: 20808784 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | siRNA Nanoparticle Functionalization of Nanostructured Scaffolds Enables Controlled Multilineage Differentiation of Stem Cells. Mol Ther. 2010 Aug 31; Authors: Andersen MO, Nygaard JV, Burns JS, Raarup MK, Nyengaard JR, Bünger C, Besenbacher F, Howard KA, Kassem M, Kjems J The creation of complex tissues and organs is the ultimate goal in tissue engineering. Engineered morphogenesis necessitates spatially controlled development of multiple cell types within a scaffold implant. We present a novel method to achieve this by adhering nanoparticles containing different small-interfering RNAs (siRNAs) into nanostructured scaffolds. This allows spatial retention of the RNAs within nanopores until their cellular delivery. The released siRNAs were capable of gene silencing BCL2L2 and TRIB2, in mesenchymal stem cells (MSCs), enhancing osteogenic and adipogenic differentiation, respectively. This approach for enhancing a single type of differentiation is immediately applicable to all areas of tissue engineering. Different nanoparticles localized to spatially distinct locations within a single implant allowed two different tissue types to develop in controllable areas of an implant. As a consequence of this, we predict that complex tissues and organs can be engineered by the in situ development of multiple cell types guided by spatially restricted nanoparticles. PMID: 20808289 [PubMed - as supplied by publisher] | | | | | | | | | |  | |  | |  |
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