|  |  |  | | | | | TE-RegenMed-StemCell feed | | | | | | | | | | | | | | | | | | | 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] | | | | | | | | | | | | | | | | | | | | | | | | | 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] | | | | | | | | | | | | | | | | | | | | | | | | | 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] | | | | | | | | | | | | | | | | | | | | | | | | | 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] | | | | | | | | | | | | | | | | | | | | | | | | | 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] | | | | | | | | | | | | | | | | | | | | | | | | | Regenerative medicine: Heart redevelopment. Nature. 2010 Sep 2;467(7311):39-40 Authors: Harvey RP PMID: 20811447 [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] | | | | | | | | | | | | | | | | | | | | | | | | | 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] | | | | | | | | | | | | | | | | | | | | | | | | | 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] | | | | | | | | | | | | | | | | | | | | | | | | | 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] | | | | | | | | | | | | | | | | | | | | | | | | | 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] | | | | | | | | | | | | | | | | | | | | | | | | | 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] | | | | | | | | | |  | |  | |  |
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