Poster Presentations

World Conference on Regenerative Medicine

Poster Presentations

Poster Presentations PP001

Phenotypic characterization of human B-cell & dendritic cell subsets in a humanized mouse model

M Ackermann1, M Kamprad1 and F Emmrich1 1 Universität Leipzig, Institute for Clinical Immunology, Leipzig, Germany Humanized mice with a functional human immune system would be very suitable for in vivo studies of human immunobiology. Recently, we have shown that transplantation of mononuclear cells from human cord blood into conditioned nonobese diabetic severe combined immunodeficiency mice leads to a long-lasting development of multiple lineages of lymphohaematopoietic cells. Humanized mice showed systemic blood cell repopulation including B cells, T-helper cells, cytotoxic T cells, NK cells, monocytes, granulocytes and CD34 + hematopoietic stem cells. Functional analysis of human cells demonstrated that B cells underwent normal class switching and produced T-cell-dependent antigen-specific immunoglobulins. Here, we investigate in detail the development of B cells and dendritic cells. Using flowcytometric analysis of peripheral blood, and single cell suspension of spleen and bone marrow, we described the presence of naive B cells, memory B cells, switched memory B cells and plasma cells. Plasmocytoid and myeloid dendritic cells were detectable. This humanized model is a powerful model system to study human immune function and should be suitable to serve as a preclinical model to investigate the activity of potential therapeutic compounds.

PP002

Amniotic membrane as a mesenchymal stem cell matrix for bladder augmentation in rats: preliminary results

J Adamowicz1, M Nowacki1, L Nazarewski1,2 and T Drewa1,3 1 Collegium Medicum Nicolaus Copernicus University, Department Of Tissue Engineering, Bydgoszcz, Poland 2 The Medical University of Warsaw, Poland, Department of General, Vascular and Transplant Surgery, Warsaw, Poland 3 Collegium Medicum, Nicolaus Copernicus University, Department of Urology, Bydgoszcz, Poland

Objective Autologus GI tract is used in standard surgical reconstruction of urinary tracts. Such treatment may have a reasonable proportion of complications. Research has been carried out on optimal materials for this type of treatment for many years. The use of autologus in vitro prepared graft could reduce complications that occur after standard urinary tract and bladder wall reconstruction. The aim of this work was to use mesenchymal stem cells (MSCs) cultured in a decontaminated human amniotic membrane as a matrix, in rat bladder wall reconstruction.

Materials & method Mesenchymal stem cells were delivered from Wistar rat femoral bone, the cells were then planted for 12 days into six-well plates; 1 × 106 cells in each well on feeder layer of human decontaminated amnion membrane put in Cellcrown – Scaffdex inserts. The human decontaminated amniotic membrane were derived from Eye Tissue Bank by SPSK nr 1 in Lublin county and were transported in Dulbecco’s Mod Eagle Medium (DMEM) plus glycerol. Cells were cultivated in DMEM/Ham’s F-12 (PAA) with 10% foetal bovine serum (PAA) with addition of basic fibroblast growth factor (Sigma)- and antibiotics (5 µg/ml amphothericin B; 100 U/ml penicilline 100 µg/ml; strepthomycine (in PAA) at a temperature of 37°C, 5% CO2 atmosphere. Such prepared MSC matrix was stained with a hematoxylin and eosin test, then sewed onto surgical patch sponge (Tachosil) to reinforce the prepared scaffold, and finally placed as a graft into prepared bladder walls of two Whistar rats. The bladder of two Whistar rats was augmented only with surgical patch sponge (Tachosil) graft, as a control group.

Results Augmentation with MSC matrix bladder graft was successful in all animals. No changes in urination and behavior in this group were observed. Control rats died, the first at 2 days after augmentation and the second 3 days after augmentation. The loss of bladder wall after surgical patch sponge (Tachosil) heomostatsis has been observed in autopsy. future science group

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Conclusion Preliminary results confirm the positive influence of augmentation with the MSCs cultured at the feeder layer of decontaminated human amniotic membrane matrix, and may probably apply in bladder wall reconstruction.

PP003

Collagen barrier membranes decrease osteoclastogenesis in murine bone marrow cultures

H Agis1,2, M Magdalenko1,2, K Stögerer1,2, G Watzek1,2 and R Gruber1,2 1 Medical University of Vienna, Department of Oral Surgery, Vienna, Austria 2 Austrian Cluster for Tissue Regeneration, Vienna, Austria

Objective Collagen barrier membranes (CBMs) can decrease resorption of bone grafts and are used for guided bone regeneration to support the process of graft consolidation. However, it remains unknown whether CBMs can affect the consolidation of bone grafts by controlling the differentiation of progenitor cells into bone-resorbing osteoclasts and bone-forming osteoblasts.

Materials & methods To gain insight into these cellular mechanisms, we performed in vitro bone marrow cultures on the surface of CBMs (Bio-Gide®, Geistlich Biomaterials) under conditions that favor osteoclastogenesis and osteoblastogenesis, respectively. Measures of osteoclastogenesis were based on the number of tartrate-resistant acid-phosphatase-positive (TRAP+) multinucleated cells and TRAP activity in the culture medium. The impact on osteoclast viability was determined utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Osteoblastogenesis was determined by alkaline-phosphatase activity in the supernatant and viability was studied using the MTT assay.

Results Here we report that the CBM reduces the number and activity of bone marrow progenitors into TRAP+ multinucleated cells, when compared with cells grown on the surface of culture plates. TRAP+ cells grown on both sides of the CBM appeared to be mono nucleated. Viability was not affected under these conditions. By contrast, the differentiation and vitality of bone marrow progenitors in terms of alkaline phosphatase activity and MTT was equivalent on the surface of CBMs and on the culture plate.

Conclusions Together, these in vitro findings demonstrate that CBMs can suppress the differentiation of bone marrow progenitors into multi nucleated osteoclasts, but not the differentiation of bone marrow progenitors into osteoblastic cells. These findings support the assumption that the potential beneficial effects of CBMs may be associated with an antiresorptive activity while bone formation is maintained during graft consolidation.

PP004

Clinical grade generation of human anti-adenovirus-cytotoxic T cells for adoptive immunotherapy

L Aïssi-Rothé1, V Decot1, V Venard2, H Jeulin2, A Salmon3, L Clement3, A Kennel4, C Mathieu4, C Cambouris5, G Rauser5, G Faure4, JF Stoltz1, P Bordigoni3 and D Bensoussan1 1 UTCT, CHU Brabois Nancy, Vandoeuvre-lès-Nancy, France 2 CHU Brabois Nancy, Virologie, Vandoeuvre-lès-Nancy, France 3 CHU Brabois Nancy, Unité de transplantation médulaire, Vandoeuvre-lès-Nancy, France 4 CHU Brabois Nancy, Immunologie, Vandoeuvre-lès-Nancy, France 5 Miltenyi Biotec, Bergisch Gladbach, Germany Adenovirus (ADV) infections represent one of the major causes of morbidity and mortality following hematopoietic stem cell transplantation, despite new antiviral treatment strategies. In fact, it has been demonstrated that a sufficient host T-cell response is essential to clear the virus.

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future science group



We describe here a complete clinical grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMCs) from seven healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 h with a synthetic peptide pool covering the ADV5 Hexon protein. IFN-g-secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01 ± 0.84 × 106 total nucleated cells was obtained. The isolated ADVspecific T cells were mainly CD4 + (mean = 56 ± 20.8%; yield = 51 ± 32.4%) but also CD8 + (mean = 42 ± 27%; yield = 56 ± 39.3%). Isolated cytotoxic T lymphocytes (CTLs) were cultured with IL-2 and autologous feeder cells in order to perform the functional quality controls. The ability of the amplified CTLs to secrete IFN-g and to proliferate upon restimulation with the Peptivator ADV5 Hexon was confirmed. A high cytotoxicity against autologous target cells (10:1) loaded with ADV5, ADV2 lysates or Peptivator ADV5 Hexon could be observed while a very low cytotoxicity was reported with nonloaded target cells. Finally, we could observe a decrease of 1.27 Log of the allogeneic reaction against non-HLA identical healthy donor PBMCs with CTLs compared with the PBMCs before selection. Good Manufacturing Practice-grade generation of ADV-specific T cells for adoptive immunotherapy could be achieved with a synthetic antigen. This technology presents the advantages to be fast, without any in vitro amplification before infusion, and to allow a good reactivity to propose immunotherapy in case of antiviral treatment failure.

PP005

New nondegradable polyurethane scaffolds for aortic valve tissue engineering

B Akra1, A Uhlig1, U Haas1, C Fano2, M Dauner2, C Schmitz1, B Meiser1, G Eissner1 and B Reichart1 1 Ludwigs-Maximillians-Universität, Grosshadern Medical Center, Department of Cardiac Surgery, Laboratory for Tissue Engineering, Munich, Germany 2 Institut für Textil- und Verfahrenstechnik, Denkendorf, Germany

Background The purpose of this study was to evaluate the cell colonization efficiency and the cell adhesion of human vascular cells seeded on newly designed polyurethane aortic valve scaffolds (PAVSs) under pulsatile flow conditions.

Materials & methods Human vascular fibroblasts (FBs) and endothelial cells (ECs) were isolated from saphenous vein segments and expanded in culture. PAVSs (n = 4) were primarily seeded with human FBs (mean 56.3 ± 6.8 × 106 cells) for 3 ± 0.5 days and secondarily seeded with FBs (mean 55.4 ± 7.18 × 106 cells) for 8 ± 1 days, followed by a colonization with ECs (mean 80.2 ± 22 × 106 cells) for 9.25 ± 1.75 days. Seeded PAVSs were then exposed for 24 h to pulsatile flow (V = 0.8 l/min, P = 40 mm Hg, n = 60 RPM) in a closed Perfusion System filled with M-199 medium (cell culture medium, Biochrom AG, Berlin, Germany). The temperature (mean 37.1 ± 0.4°C) was controlled and regulated simultaneously with a hot plate and a temperature sensor (IKA Works, Staufen, Germany). Samples were taken before (Subvalvular part of the Aorta) and after perfusion (cusp, subvalvular, valvular and supravalvular part of the aorta) and evaluated by scanning electron microscopy and immunohistochemical staining.

Results Scanning electron microscopy results showed a confluent cell layer on all PAVSs before perfusion. Immunohistochemical staining with CD31 (EC-antibody, DAKO GmbH, Germany) and P4hb (FB-antibody, Acris Antibodies GmbH, Germany) revealed a positive reaction of both cell layers before perfusion, whereas no or little CD31-positive reaction could be observed after perfusion. ECs were not able to resist to 24-h shear stress and detached from the PAVSs. The FBs layers were still confluent after perfusion.

Conclusion After 24 h perfusion, ECs detach from tissue-engineered cardiovascular prostheses preseeded with human FBs. FBs remain their ability to react to shear stress and maintain their confluence. ECs adhesion can be increased by splitting the ECs-colonization into two steps and/or by preconditioning the seeded PAVSs with low-flow conditions and gently raising the flow up to the required conditions.



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Quality control of human umbilical cord transdifferentiated endothelial cells by electron probe x-ray microanalysis

M Alaminos1, I Garzon1, M Vico1, PV Crespo1, B Perez-Köhler2, J Bujan2 and A Campos1 1 University of Granada, Histology, Granada, Spain 2 University of Alcala, Medical Specialities, Alcala De Henares-Madrid, Spain

Objectives Cell viability is one of the key parameters that should be evaluated in the context of cell therapy and regenerative medicine. In this work, we determined the cell viability of three types of cells for use in vascular regenerative medicine, including human umbilical cord vein endothelial cells (HUVECs), human Wharton’s Jelly stem cells (HWJSCs) and HWJSCs transdifferentiated to endothelial-like cells (Tr-HWJSCs), by quantitative electron probe x-ray microanalysis.

Materials & methods Primary cultures of HWJSCs and HUVECs were established from ten human umbilical cords using enzymatic methods. HWJSCs were cultured in Amniomax medium, whereas M199 medium supplemented with ECGF was used for HUVECs. To induce endothelial transdifferentiation of HWJSC, Amniomax medium was changed by M199 medium supplemented with ECGF. Quantitative electron probe x-ray microanalysis was carried out on the three cell types analyzed in this work by using a Philips XL30 scanning electron microscope with an energy-dispersive detector EDAX.

Results Microanalytical evaluation of all cell types revealed that the average ionic contents of Na, K, S, P, Ca, Mg and Cl (as mmol of each element per Kg of dry cell weight) were respectively, as follows: 53.91, 421.14, 32.13, 190.79, 10.20, 15.79 and 149.37 for HWJSC; 103.42, 466.16, 79.10, 296.85, 19.88, 32.65 and 263.05 for HUVEC; and 45.61, 429.00, 46.25, 232.40, 16.24, 21.40 and 169.00 for Tr-HWJSC. The K/Na index was 7.81 for HWJSC, 4.51 for HUVEC and 9.41 for Tr-HWJSC.

Conclusions Electron probe x-ray microanalysis was a sensitive technique to identify intracellular ionic profiles and, therefore, the cell viability of the different cell types. The specific microanalytical patterns found in this work suggest that transdifferentiated endothelial-like cells have high levels of cell survival, suggesting that these cells could be efficiently used in regenerative medicine for vascular tissue-engineering purposes. Financial & competing interests disclosure This work was supported by grants FIS PI061784 and PI061781.

PP007

In vitro evaluation of glass–glass ceramic thermoseed effects on proliferation of human osteosarcoma cell line after exposition to external magnetic fields

M Alcaide1, C Ramírez-Santillán1, M Feito1, E Ruíz-Hernández2, D Arcos2, M Vallet-Regí2 and M Portolés1 1 Universidad Complutense, Bioquímica y Biología Molecular I, Madrid, Spain 2 Universidad Complutense de Madrid, Química Inorgánica y Bioinorgánica, Madrid, Spain

Objective The thermoseeds for the cancer treatment by magnetic hyperthermia are designed to be implanted at the tumor site, increasing the local temperature to the hyperthermia interval (42–47°C) when exposed to an external magnetic field [1] . The aim of this study was to evaluate in vitro the effects of glass–glass ceramic G15–GC85 thermoseeds on the human osteosarcoma Saos-2 osteoblasts after exposition to external magnetic fields.

Materials & methods Magnetic implantable thermoseeds composed of a combination of a magnetic glass ceramic with sol-gel glasses were prepared by mixing a sol-gel glass (G) and a melt-derived glass (GC) [2] . These mixtures were thermally treated resulting in the final G15–GC85 magnetic glass-glass ceramic thermoseeds. The biocompatibility of this biomaterial has been recently demonstrated [3] . In order to know their possible application to bone cancer treatment, human osteosarcoma Saos-2 osteoblasts were cultured on 35 mm plastic S158

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Petri dishes and G15–GC85 thermoseeds were placed on the culture and exposed to an external magnetic field at 37ºC for different times and during consecutive sessions. The cell damage produced by these treatments and their effects on osteoblast proliferation were evaluated analyzing the cell number, viability, reactive oxygen species, cell cycle, apoptosis and lactate dehydrogenase leakage.

Results After increasing the local temperature (47ºC) in the human osteosarcoma Saos-2 osteoblast culture in contact with G15–GC85 thermoseeds for different times (20 and 40 min), a significant reduction in the cell number was observed. This effect depends on both time and number of consecutive sessions. The highest reduction of cell proliferation (from 1.8 × 106 to 68 × 103 cells/dish) was observed 6 days after two consecutive sessions of 40 min.

Conclusion G15–GC85 magnetic glass–glass ceramic thermoseeds can be considered as very promising candidates for bone cancer treatment.

References 1

Brezovich IA, Meredith RF: Practical aspects of ferromagnetic thermoseed hyperthermia. Radiol. Clin. North Am. 27(3), 589–602 (1989).

2

Ruiz-Hernández E, Serrano MC, Arcos D, Vallet-Regí M: Glass–glass ceramic thermoseeds for hyperthermic treatment of bone tumors. J. Biomed. Mater. Res. A 79(3), 533–543 (2006).

3

Serrano MC, Portolés MT, Pagani R et al.: In vitro positive biocompatibility evaluation of glass–glass ceramic thermoseeds for hyperthermic treatment of bone tumors. Tissue Eng. Part A 14(5), 617–627 (2008).

PP008

Isolation & culture of very small embryonic-like stem cells from murine bone marrow

A Heider1, M Cross2 and R Alt1 1 Universität Leipzig, Translational Centre for Regenerative Medicine, Leipzig, Germany 2 Universität Leipzig, Department of Haematology and Oncology, Leipzig, Germany

Objective Very small embryonic-like (VSEL) stem cells were first isolated from murine bone marrow and human umbilical cord blood by the group of M Ratajczak [1] . This rare population of adult stem cells possesses a differentiation potential into tissues from all three germ layers, indicative of a putatively pluripotent adult stem cell. Our aim is to establish VSEL stem cells for regenerative medicine by improving isolation and in vitro expansion of this rare stem cell population.

Results Preparations of murine bone marrow cells usually contain a contaminating population of small erythroid cells and dead cells, which overlaps with the much rarer population of similarly sized VSEL stem cells in the forward scatter – side scatter plot during fluorescenceactivated cell sorting analysis. We were able to improve the discrimination of dead cells, cell debris and enucleated erythroid cells during fluoresence activated cell sorting analysis and sorting by staining nucleated cells and dead cells with cell permeable Hoechst 33342 and propidium iodine (PI), respectively. In this way, we also avoid the need for hypotonic or ammonium chloride based lysis of the erythrocytes and reduce the amount of cell debris overall. Using 5 mg/ml Hoechst 33342, a concentration commonly used in the side population assay to isolate viable and functional hematopoietic stem cells, we sorted Sca-1+ lin- CD45- Hoechst+ PI- VSEL stem cells and plated them on C2C12 myoblast cells. Over a period of several days, we observed a marked expansion of Hoechst+ VSEL stem cells, followed by the appearance of multicellular, round spheres of 50–100 µm in diameter. Upon passaging into neuronal differentiation conditions, the spheres underwent a morphological change and formed adherent rosette structures, resembling neuronal differentiation from embryonic stem cells.

Conclusions An improved isolation protocol for VSEL stem cells from murine bone marrow yields VSEL stem cells that retain in vitro expansion and differentiation potential. The experience gained should facilitate the isolation and amplification of human VSEL stem cells, and their establishment for regenerative medicine.

Reference 1

Kucia M, Reca R, Campbell FR et al.: A population of very small embryonic-like (VSEL) CXCR4 (+) SSEA-1(+) Oct-4 + stem cells identified in adult bone marrow. Leukemia 20(5), 857–869 (2006).



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Improving bacterial cellulose for blood vessel replacement: functionalization with a chimeric protein containing a cellulose-binding module & an adhesion peptide

F Andrade1, R Costa2, R Soares2, L Domingues1and M Gama1 1 University of Minho, Institute for Biotechnology and Bioengineering – Centre of Biological Engineering, Braga, Portugal 2 University of Porto, Faculty of Medicine – Department of Biochemistry, Porto, Portugal

Background Cardiovascular disease is the main cause of mortality in the USA and Western countries, leading to a demand for new materials that can be used as substitutes for blood vessels. Bacterial cellulose (BC) is a material with excellent biocompatibility and mechanical properties, thus holding great potential for arterial replacement. To improve the adhesion of human microvascular endothelial cells (HMECs) to BC, chimeric proteins containing a cellulose-binding module (CBM) and an adhesion peptide (RGD or GRGDY) were produced and used to cover the cellulose fibers. The effects of these proteins were studied.

Materials & methods The attachment/viability and apoptosis were measured by the MTS and TUNEL methods, respectively. Confocal microscopy was used to analyze the morphology of microvascular cells grown on BC membrane. The visualization of cellulose fibers was also possible once the recombinant protein RGD-CBM was chemically linked to the fluorescein isothiocyanate. The formation of capillary-like structures by the endothelial cells was observed with optical microscopy. To evaluate the RGD effect on the migration of the cells through the BC, a migration chamber and an attractant were used to stimulate the cells to grow into the BC.

Results The results of the MTS test demonstrated that the recombinant proteins containing adhesion sequences were able to increase significantly the attachment of HMEC to BC surfaces, and TUNEL assay showed that after 24 h of adhesion, independently of the treatment, cells remained viable on the BC pellicle, even when less cells adhered to cellulose treated with the CBM or buffer comparing to RGD. The images by confocal microscopy showed that the cells on the BC treated with the RGD presented a more elongated morphology and proliferated on the pellicle, forming a confluent cell layer on the membrane after 7 days. The results showed that the RGD decreased the ingrowth of the HMEC cells through the BC and also stimulated the early assembly of endothelial cells into capillary-like structures.

Conclusions In this work, a new approach was successfully developed to functionalize the BC. The use of recombinant proteins containing a CBM domain, exhibiting high affinity and specificity for cellulose surfaces, allows the control on the interaction of this material with cells. The CBM may virtually be combined to any biologically active protein for the modification of cellulose-based materials, for in vitro or in vivo applications.

PP010

A novel light-emitting photosensitive technique to select & purify cell populations for use in diagnostics & therapeutic applications

K Andrews1 and J Hunt1 University of Liverpool, UKCTE, UKBioTEC, Clinical Engineering, School of Clinical Sciences, LINSET, Liverpool, UK

1

Objectives Regenerative medicine increasingly requires accurate diagnostic techniques combined with cell-based therapies. However, cells sought for these applications are often rare in number and difficult to obtain/isolate, resulting in patients undergoing complicated, expensive and invasive procedures. These methods are time consuming with variable clinical success rates, largely due to poor or nonexistent cell-purification techniques. The aim of this research was to develop a novel light-emitting photosensitive technique, based on patent US 7354733B2, capable of selecting targeted cells for diagnostics and eventual use in specific therapeutic applications.

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Materials & methods Bioactive nanostructured materials, utilizing silver photochemistry were coated onto microscope slides (Cellect Biotech Ltd, UK). Cells, including blood, fibroblasts and stem cells, were specifically labeled with selected markers including CD3, 11b, 14, 19, 29, 34 and 133. These monoclonal antibody techniques in combination with an HRP enzyme catalyzed a chemiluminescent (light emitting) reaction, quantified using chemiluminescence emission assays. The cells were placed into contact with the photographic surfaces and nontoxic developer solution added. The targeted cells (i.e., tagged with specific markers), through a photographic exposure process, were bound to the nanosurfaces. Any unlabelled cells remained unbound and were removed in a wash step. Slides with attached cells were analyzed using fluorescence-activated cell sorting, scanning electron microscope and laser scanning confocal microscopy.

Results Specific attachment of the labeled cells to the underlying photoreactive surfaces was optimized. Photographically exposed halos on the cell-attached slides confirmed that the nature of the binding was specific through the light emitting reaction between cells and underlying photosensitive nanostructures. This technique was successful with a range of cells and avoided the inherent problems found in the existing cell targeting/sorting techniques. Cells remained viable throughout the process, allowing the possibility of culturing the cells further after purification and eliminating any negative effects upon the patient from treatment/damage to the cells as can result with currently available methods.

Conclusions Use of the developed light-emitting photosensitive cell-based technique allows for the successful purification of specific cell types and their subpopulations. This novel process holds great potential for clinical use, for a wide range of diagnostic and cell therapy applications.

PP011

Encapsulated mesenchymal stem cells to counteract tissue rejection

S Baden1,2,3, M Zenke1, L Elling4, J Groll5, A Sechi1 and S Neuss2 1 RWTH Aachen, Institute for Biomedical Engineering, Department of Cell Biology and Helmholtz Institute for Biomedical Engineering, Aachen, Germany 2 RWTH Aachen, University Hospital, Institute of Pathology, Aachen, Germany 3 RWTH Aachen, Interdisciplinary Center for Clinical Research, IZKF ‘Biomat‘, Aachen, Germany 4 RWTH Aachen, Institute for Biomedical Engineering, Department of Biomaterials, Aachen, Germany 5 RWTH Aachen, Department of Textile and Macromolecular Chemistry and DWI, Aachen, Germany Mesenchymal stem cells (MSCs) are particularly promising multipotent cells for use in autologous regenerative medicine. Clinical interest stems from the observations that MSCs display anti-inflammatory and immune-regulatory responses via complex mechanisms allowing for allogenous treatments. Recent reports described the immunosuppressive properties of MSCs, yet these studies are controversal and further studies are important to evaluate mechanisms underlying the immune-regulatory effects of MSCs in more detail. Furthermore, co-transplanted MSCs are able to counteract rejection of transplanted cells and/or organs. However, one of the difficulties is that MSCs, directly after injection, start to migrate and differentiate. Therefore, our aim is to encapsulate MSCs to prevent migration and to inhibit differentiation, but to ensure sustained self-renewal for a prolonged immunomodulatory activity. The interactions between MSCs and cells of the immune system, in particular dendritic cells and T cells, are analyzed by cytokine secretion, gene expression and the expression of surface markers. To immobilize MSCs we are establishing a 3D-construct composed of a nonadhesive hydrogel (star-PEG) and an extracellular matrix protein linked by poly-N-acetyllactosamine and galectin. We are performing systematic screening assays to analyze MSC behavior on these 3D biomaterial constructs regarding cell adhesion, morphology, vitality, proliferation, cytoxicity and apoptosis. In summary, we are developing an innovative approach to encapsulate MSCs. The MSCs surrounding 3D biomaterial construct have to mimic the ‘MSC niche’, supporting self-renewal and avoiding differentiation. This biohybrid system should then be used in future therapies to inhibit the immune response against transplanted cells and/or organs.



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The use of cell stress markers to inform on tissue-engineered product quality

M-O Baradez1, G Chennel1, F Pu2, J Hunt2, N Harris1, H Abhedi1 and D Marshall1 1 LGC, Molecular and Cell Biology, Teddington, UK 2 University of Liverpool, School of Clinical Sciences, Liverpool, UK

Objective Advancements in the field of tissue engineering (TE) are driving the need for measurement techniques that can assess the ‘quality’ of cells within manufactured living constructs. This presents many challenges as the cells are often maintained in a 3D supporting matrix, which makes measurement of even simple characteristics such as viability, morphology and cell stress difficult. In addition, many approaches for monitoring TE constructs involve destructive techniques not suitable for TE product batch characterization. As part of the European Framework 6 consortium A Systems Approach to Tissue Engineering Processes and Products (STEPS) we have identified, optimized and applied inferential techniques to measure cellular stress in TE products. These measurement techniques can be applied to improve the control over TE product quality from manufacture and handling through to shipment and storage.

Materials & methods Human dermal fibroblasts were seeded onto collagen scaffolds and maintained at a range of pH and temperature conditions in either static or perfusion bioreactor for up to 4 weeks. Samples were taken at regular intervals and analyzed for cell metabolic activity using a Nova biomedical Bioprofile 400, the production of a panel of markers associated with cell stress using a range of fluorescent assays and the release of VEGF using enzyme-linked immunosorbent assay.

Results & conclusion Cell metabolic activity remained constant in collagen scaffolds grown in both static and perfusion bioreactors when the samples were maintained at 37oC. Changes in temperature to 21oC and 4oC, to represent product storage conditions, resulted in a significant reduction in cell metabolism to baseline levels. Changes in temperature and pH resulted in rapid production of cell stress markers that could be correlated and used to profile the level of stress to which the cells were exposed. Analysis of marker expression patterns showed that specific profiles could be attributed to different growth, temperature and pH conditions. Taken together, these results show that monitoring cellular stress in TE products using a comprehensive library of stress patterns can inform upon the quality status of TE products during both manufacture and shipping.

PP013

Identification of self-renewal pathways in breast cancer stem cells

PM Bareiss1, H Neubauer2, H-G Kopp1, E Solomayer2, D Wallwiener2, F Fend3, A Staebler3, L Kanz1, T Fehm2 and C Lengerke1 1 Universität Tübingen, Medical Center II, Hematology & Oncology, Tuebingen, Germany 2 Universität Tübingen, Department of Obstetrics and Gynaecology, Tuebingen, Germany 3 Universität Tübingen, Department of Pathology, Tuebingen, Germany Cancer stem cells (CSCs) or cancer-initiating cells are rare tumor cells capable of initiation and propagation of the malignant disease. Like stem cells in other normal or diseased compartments, CSCs display specific stem cell features (e.g., slow proliferation rate, dormancy and expression of protein pumps such as multidrug resistance [MDR]). These characteristics can be used for isolating them from the tumor mass and convey to them biological properties that partly mediate their resistance to conventional genotoxic therapy and perhaps mediate later disease relapses. In breast cancer, CSCs can be isolated by CD24 - /CD44 + staining and/or their high aldehyde dehydrogenase activity. Recent studies report expression of the embryonic stem cell regulator Oct-4 in human tumor samples, including breast and lung cancer. Furthermore, Oct-4 downregulation was shown to have a functional role in CD133 + cells derived from cell lines, inducing their differentiation into attached epithelial-like cells [1] . However, Hu et al. demonstrated in a murine lung carcinoma as well as a human breast cancer cell line, that silencing of Oct-4 results in cell apoptosis [2] . While much is known on the function of Oct-4 in embryonic stem cells, the precise role of Oct-4 during tumorigenesis still needs to be explored in more detail.

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In our laboratory we confirmed the expression of two Oct-4 isoforms by semi-quantitative PCR in several primary breast and lung tumor samples as well as cell lines (including MCF-7, SKBR3, H209, H1963, A549 and HCC-78). Oct-4 expression was enhanced in the putative breast CSC compartment. In primary breast cancer samples, significant differences in CSC activity were documented in different patient samples using CD24 - /CD44 + staining and assessment of aldehyde dehydrogenase activity, as well as functional analyses by in vitro mammosphere formation assays. Oct-4 and ALDH1 expression was also detected on protein level, by immunohistochemistry analysis. Mammospheres could be differentiated into epithelial and/ or myoepithelial cells following specific protocols, which overall provides us an experimental framework for studies on molecular pathways governing stem cell self-renewal versus differentiation. Future efforts will concentrate on using this model to understand the molecular regulation of Oct-4 in breast CSCs and its function and role in breast CSC tumorigenicity.

References 1

Chen YC, Hsu HS, Chen YW et al.: Oct-4 expression maintained cancer stem-like properties in lung cancer-derived CD133-positive cells. PLoS One 3(7), E2637 (2008).

2

Hu C, Wu Y, Wan Y, Wang Q, Song J: Introduction of hIGF-1 gene into bone marrow stromal cells and its effects on the cell’s biological behaviors. Cell Transplant. 17(9), 1067–1081 (2008).

PP014

Characterization of a pluripotent mesenchymal stem cell subtype from bone marrow & its regenerative effects in vivo

E Bauer1 and A Stolzing1 Fraunhofer IZI, Cell Therapy, Stem Cell Biology, Leipzig, Germany

1

Bone marrow is composed of a very heterogeneous population of cells and its regenerative potential has been reported in several studies. Due to different cultivation methods, we observed different types of stem cells that varied in morphology but where similar to mesenchymal stem cells. The aim of the study was to characterize these different stem cell types, to find the population with the highest regenerative potential and to characterize their pluripotency status. Therefore we used bone marrow from C57BL/6 mice that was cultivated via two different systems, first in a suspension cell culture system in which cell culture supernatant was transferred to a new dish every day within a time span of several days and second a conventional cell culture system. Differences can be seen between both culturing systems. While pluripotent cell aggregates could be observed in the primary cultures of both methods, they seem to become depleted during the transfer steps. To get more insights, we tested the proliferation- and differentiation potential as well as cytokine levels and telomerase activity in the different stem cell subtypes and compared them to classical culture expanded mesenchymal stem cells. For investigation of the above mentioned regenerative potential, all the obtained stem cell types were used for transplantations in an Alzheimer’s disease mouse model.

PP015

Preclinical toxicity assessment of anti-human CD4 monoclonal antibodies MAX.16H5 used for experimental tolerance-induction strategies in preclinical regenerative medicine studies on human monocytes

K Bauer1,2 and M Braun1,2 1 Universität Leipzig, Translational Centre for Regenerative Medicine, Leipzig, Germany 2 Universität Leipzig, Institute for Clinical Immunology, Leipzig, Germany

Aim To assess the immunological effect of the mouse monoclonal anti-human CD4 antibody MAX.16H5 on human CD4-positive monocytes, ex vivo biological functions of MAX.16H5 was evaluated using human monocytes.



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Materials & methods Binding, CD4 modulation, and intracellular calcium release as indicators of human monocyte stimulation during CD4 and native or acid modified MAX.16H5 interaction were assessed using standard flow cytometric procedures.

Results Acid treatment of MAX.16H5 (pH3) did not alter the ability of the monoclonal antibody to bind or modulate CD4 on human monocytes. Acid modified MAX.16H5 resulted in the loss of anti-CD4-induced intracellular calcium signaling, and the ability of MAX.16H5 to inhibit bacterial lipopolysaccharide (LPS) induced TNF-a secretion.

Discussion Adaptive immunity arises when the body is exposed to various antigens, thus developing a defence that is specific to a particular antigen. CD4-mediated signaling in T-lymphocytes is well described, while little is known regarding which CD4 induced all response of human monocytes. We investigated the immunological effect and ex vivo biological functions of the mouse monoclonal anti-human CD4 antibody MAX.16H5 on human CD4-positive monocytes. Binding, CD4 modulation and intracellular calcium release as indicators of monocyte stimulation during CD4 interaction with native or acid modified MAX.16H5 were assessed using standard flow cytometric procedures. In addition, inhibition of LPS-induced TNF-a secretion was determined by ELISA. Acid treatment of Max.16H5 (pH 3) did not alter the ability of the monoclonal antibody to bind to CD4, or to modulate CD4 on human monocytes.

Conclusion Acid modification of MAX.16H5 may result in the loss of anti-CD4-induced intracellular calcium signaling and the ability of MAX.16H5 to inhibit bacterial LPS induced TNF-a secretion. Our results indicate that MAX.16H5 interacts with CD4 receptor through epitope recognition. Additionally, our data suggest that a second receptor of anti-CD4 complex may play an essential role for the induction of a biological response. These findings provide insight into the immunotoxicological potencies of clinically applied anti-CD4 antibodies currently used in tolerance induction in liver transplantation.

PP016

New end points for developmental osteotoxicity in the embryonic stem cell test

L Baumgartner1 and NI zur Nieden1,2 1 Fraunhofer IZI, Applied Stem Cell Technologies, Leipzig, Germany 2 University of California Riverside, Department of Cell Biology & Neuroscience and Stem Cell Center, Riverside, CA, USA

Objective Osteogenic birth defects, malformation or teratogenic events result from impaired differentiation processes during embryogenesis. These can, for instance, be caused when the mother is exposed to teratogenic chemicals during pregnancy. The embryonic stem cell test (EST) by Spielmann et al. is currently the only validated in vitro embryotoxicity assay that truly refrains from using animals [1] . Although the validated classic EST may identify inhibitory effects caused by test chemicals in cardiac tissue, most teratogenic compounds are known to influence skeletal, limb or CNS development. The aim of this study was therefore to find new end points for the EST that allow for the determination of toxic effects on osteogenic development in particular. On that account, we examined the influence of chemical substances on the osteogenic differentiation potential of embryonic stem cells (ESCs) comparing three different bone specific end points.

Materials & methods Different concentrations of test chemicals with known effects on osteogenic development were added to the cell cultures to determine their toxic potential in vitro. The evaluation of results was based upon different end points determined in two cell lines, the cytotoxicity in 3T3 fibroblasts (adult effect), the cytotoxicity of the chemical on ESCs and the inhibition of osteogenic differentiation in ESCs. Cytotoxicity was determined with the help of the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. To evaluate osteogenic differentiation, the expression of the bone-specific marker osteocalcin was determined by means of quantitative PCR, an established end point for osteotoxicity in the EST [2] . Furthermore, the suitability of a previously established calcium assay as well as a morphometric image analysis as new end points was characterized.

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Results The cytotoxicity data demonstrated that ESCs are more sensitive to toxic agents than the fully differentiated adult cells. We observed different gene-expression profiles for osteocalcin caused by compound treatment. Furthermore, we were able to advocate the calcium assay as well as the image analysis as new suitable end points to study osteotoxic effects.

Conclusion Our results prove that the EST, with the characterized specific end points, appears to be auspicious as screening assay to evaluate developmental osteotoxicity based on the most important mechanisms in embryotoxicity, cytotoxicity and differentiation as well as on differences in sensitivity between adult and embryonic tissues.

References 1

Spielmann H, Pohl I, Doring B, Liebsch M, Moldenhauer F: The embryonic stem cell test, an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. In vitro Toxicol. 10(1), 119–127 (1997).

2

zur Nieden NI, Kempka G, Ahr HJ: Toxicol. Appl. Pharmacol. 194(3), 257–269 (2004).

PP017

Influence of donor & recipient age on bone marrow mononuclear cell treatment after experimental stroke

M Bojko1, M Lorenz1, C Voigt1, A Kranz1, M Kamprad2, F Emmrich1,2,3, J Boltze1,3 and D-C Wagner1 1 Fraunhofer IZI, Leipzig, Germany 2 Universität Leipzig, Institute for Clinical Immunology, Leipzig, Germany 3 Universität Leipzig, Translational Centre for Regenerative Medicine, Leipzig, Germany

Objectives Bone marrow mononuclear cell (BM-MNC) therapy after experimental stroke has been tested in different studies ultimately aiming to establish an autologous model of cell therapy. Animals and cells used for these purposes are commonly young and healthy. Stroke patients, however, are usually older and suffer from underlying diseases. Cells donated by these patients within an autologous therapeutic approach are consequently not comparable to cells donated by young and healthy organisms. This study aimed to investigate the effects of age and comorbidity on therapeutic efficacy of BM-MNC administration following stroke.

Methods A total of 40 old (20 months old) and 30 young adult (3 months old) male spontaneously hypertensive rats underwent permanent middle cerebral artery occlusion (MCAO). Both young and old animals were randomly assigned to one of the following groups: intravenous transplantation of 8 × 106/kg body weight (young/old subjects) (a/b) human juvenile BM-MNC (n = 10/10), (c/d) human aged BM-MNC (n = 10/10) or (e/f) infusion of vehicle solution (n = 10/20). Mortality within the experiment was a = 2, b = 3, c = 2, d = 3, e = 2 and f = 2. After MCAO, functional recovery was consecutively measured using multiple independent behavioral tests. Infarct size and hemispheric atrophy were measured on days 1, 8, 32 and 63 after MCAO using MRI. All treatment and analysis procedures were performed in blinded settings.

Results All animals showed severe deficits in BeamWalk, modified Neurological Severity Score and LadderRung test after MCAO. Even though performance in all groups improved until day 64, there was a persistence of chronic deficits. Infarct size correlated positively with impairment in all behavioral tests on each day of measurement. Despite past accounts of effective BM-MNC therapy we found no significant differences in therapeutic outcome between treated and control groups. Furthermore, we found no differences in outcome between animals who received either young or aged BM-MNC. Reason for absence of therapeutic success could be the preaggrieved brain of the spontaneously hypertensive rat not responding to cell therapy. However, increase of animal number would be necessary to validate these observations.



S165



Biotechnological construction of a native porcine esophagus matrix scaffold

A Boldt1, S Ossmann2, F Emmrich1,3, S Dhein2, U Sack1,3 and H Till4 1 Universität Leipzig, TRM Leipzig, Leipzig, Germany 2 Herzzentrum Leipzig, Clinic for Cardiac Surgery, Leipzig, Germany 3 Fraunhofer IZI, Leipzig, Germany 4 Universität Leipzig, Clinic for Pediatric Surgery, Leipzig, Germany

Objective In esophagus atresia, previous solutions (e.g., stomach and colon interposition, esophagus elongation) to heal children carry serious disadvantages and high therapeutic costs. Therefore, we propose a new therapeutic concept by the development of native decellularized esophagus matrix scaffolds.

Methods Porcine esophagi were harvested and the cells were removed by hypotonic lysis in different sodium dodecyl sulfate solutions in static and perfusion systems. All decellularized scaffolds were digested enzymatically to remove remaining DNA/RNA. Subsequently, esophagial cells were isolated from mice and reseeded for 10 days on the scaffolds. Histological (Azan, HE, nuclear fast red) and immunohistological staining methods (collagen III, IV, fibronectin and elastin) providing the evidence of successful cell adhesion and preservation of extracellular matrix proteins in esophagus scaffold.

Results Using a bioreactor system we could observe an excellent decellularization of porcine esophagus by perfusion with sodium dodecyl sulfate solution for 5 days. The decellularized matrix scaffold exhibited an excellent geometrical arrangement without any cells, cell debris or chromatin remnants and extracellular matrix proteins were preserved (collagen III, IV, elastin and fibronectin), as compared with a native esophagus. The DNA content of the matrix-scaffolds was decreased approximately 98% relative to a native esophagus (2.0 ± 0.4 ng/µl [with enzymatic digestion] vs 6.0 ± 0.5 ng/µl [without enzymatic digestion] vs 76.6 ± 15.1 ng/µl [original esophagus]). Moreover, the scaffolds could be reseeded with esophagial mice cells.

Conclusion In this study we developed a natural esophagus matrix scaffold with preserved extracellular matrix proteins and ability to reseed with cells of a foreign species. We provide the foundation for the development of an esophagus xenograft.

PP019

Thrombogenic & hemolytic activity of acellular esophagus matrix scaffolds

A Boldt1, H Schneider2,3, K Schröck2,3, F Emmrich2,3,4, H Till5, U Sack2,3,4 and M Kamprad2,3,4 1 Universität Leipzig, TRM Leipzig, Leipzig, Germany 2 Universität Leipzig, TRM Leipzig, Leipzig, Germany 3 Universität Leipzig, Institute for Clinical Immunology and Transfusion Medicine, Leipzig, Germany 4 Fraunhofer IZI, Leipzig, Germany 5 Universität Leipzig, Clinic for Pediatric Surgery, Leipzig, Germany

Objective We prepared an acellular esophagus matrix scaffold for use as xenograft in esophagus atresia. Lowering the risk of postoperative complications, thrombolytic and hematolytic activities have been investigated in vitro prior testing in animal models.

Methods Esophagei were harvested from adult pigs and decellularized by rinsing in hypotonic sodium dodecyl sulfate solution. After washing, to remove sodium dodecyl sulfate, acellular tissue was sterilized and cut into pieces of 0.25 cm². The scaffold was incubated with human thrombocytes for 0, 30, 120 and 240 min. Thrombogenic activity was determined by fluorescence-activated cell sorting analysis of CD62P, an activation-dependent surface marker (negative controls without tissue, positive control = 10 µM ADP). Furthermore, for investigation of hemolytic activity, human whole blood (obtained from healthy volunteer donors and diluted in phosphate buffered saline) was incubated with scaffold material (0.25 cm²) for 0, 2, 4 and 24 h. The rate of hemolysis

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was investigated by measuring optical density (at 405 nm) of free hemoglobin in the supernatant. Blood either diluted in water (positive) or in phosphate buffered saline (negative) served as controls. All following data are ratios in percentage and therefore do not require any units.

Results Fluorescence-activated cell sorting analysis of CD62P did not reveal increased thrombogenic activity of acellular matrix scaffolds (after 0 h = 113 ± 9 to 4 h = 73 ± 11, n = 4) as compared with negative controls (0 h = 100 to 4 h = 42 ± 5; n = 4). In contrast, in ADP-stimulated positive control, CD62P signal was increased approximately twofold (0 h = 192 ± 12 to 4 h = 234 ± 25; n = 4). In addition, we could demonstrate that esophagus matrix scaffolds did not exhibit increased hemolytic activity (0 h = 4.80 ± 0.25 to 24 h = 21.63 ±5.64, n = 3) as compared with negative control (0 h = 3.99 ± 0.12 to 24 h = 11.31 ± 0.99; n = 3;positive control: 0 h = 100.00 ± 0.0 to 24 h = 88.76 ± 1.70; n = 3).

Conclusion We established a preclinical esophagus xenograft model based on acellular matrix scaffolds. The decellularized esophagus did not exhibit increased thrombogenic and hemolytic activities. These findings are essential for further surgical animal experiments because adverse effects can be estimated before starting graft transplantation.

PP020

Oriented immobilization of epidermal growth factor for tissue engineering applications

C Boucher1,2, B Liberelle1, G St-Laurent2, M Loignon2, M Jolicoeur1, Y Durocher2 and G De Crescenzo1 1 École Polytechnique de Montreal, Chemical Engineering, Montreal, Canada 2 National Research Council Canada, Animal Cell Technology Group, Bioprocess Center, Montreal, Canada

Objective Development and validation of a novel approach using a de novo designed heterodimerization peptide pair (E and K coils peptides) for the oriented immobilization of EGF on any type of aminated surfaces for tissue engineering applications.

Materials & methods Production & purification Production of N-terminally coil-and His-tagged EGF was performed in HEK293 cells. The fusion protein was purified by immobilized metal-affinity chromatography.

Bioactivity The ability of the fusion protein to promote A431 cell surface EGF receptors autophosphorylation was assayed in vitro (western blot).

Grafting procedures Covalent grafting of Kcoil peptides was characterized by surface plasmon resonance, ellipsometry and contact angle measurements. Tethering of purified Ecoil-tagged EGF was performed by simple incubation on Kcoil-derivatized surfaces. Quantification was achieved by MicroBCA protein assay kit. A-431 cell adhesion and receptor autophosphorylation assays were performed.

Results The fusion protein was efficiently secreted in cell medium and purified by affinity chromatography. Soluble purified Ecoil-EGF was as active as untagged EGF, as both were characterized by similar EC50. When captured to biosensor surfaces via coiled-coil interactions, Ecoil-EGF was demonstrated to bind to the artificially dimerized extracellular domain of EGF receptor in a highly stable fashion with strong apparent affinity. Aminated glass surfaces were functionalized with Kcoil and characterized by ellipsometry and contact angle measurements. The surfaces were shown to be able to recruit Ecoil-tagged EGF (115 ± 8 pmol/cm2) without any unspecific adsorption. EGF-receptor autophosphorylation assays demonstrated that grafted EGF, through coiled-coil interactions, allowed for a prolonged (>5 h) and more intense EGF-receptor phosphorylation when compared with that observed with soluble or randomly coupled EGF. For cell adhesion, our approach was more efficient than random EGF grafting, respectively, 65 versus 35% increase compared with no increase for control. Together, these results show that coiled-coil immobilization of EGF is more efficient than soluble EGF supply or nonoriented EGF immobilization.



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Conclusion We have developed an easy, versatile and controllable approach to graft bioactive proteins in an oriented fashion on any type of aminated surfaces. The E/K coiled-coil capture strategy presents a great interest to graft any coil fusion growth factor for tissue engineering and regenerative medicine purposes.

PP021

How to tame Her2/Neu-overexpressing tumors

M Buchold1, A Hintersdorf1, I Lindner1, N Hemdan2, W Gerdes2 and G Birkenmeier1 1 Universität Leipzig, Institute of Biochemistry, Leipzig, Germany 2 Fraunhofer IZI, Project Service Team, Leipzig, Germany

Objective Tumors that overexpress the growth factor receptor Her2/Neu grow rapidly and metastasize in vivo. Targeting this receptor is part of a new strategy to combat cancer growth. Growth factors stimulate cell growth by increasing the glucose uptake and its oxidation primarily via the aerobic glycolytic pathway. This provides sufficient energy, NADPH and ribose-phosphates for diverse anabolic reactions and diminishes reactive oxygen species formation along mitochondrial oxidative pathway. An increased glycolytic flux is accompanied by enhanced formation of the very reactive compound, methyl glyoxal, in a nonenzymatic process. To control methyl glyoxal production, many tumor cells upregulate methyl glyoxal-degrading enzymes, called glyoxalases. Accordingly, we hypothesize that inhibiting of glyoxalases would kill tumor cells by a suicide mechanism elicited by high levels of the reactive aldehyde. We recently found that the polyphenol curcumin is a strong inhibitor of the glyoxalase 1. We therefore carried out experiments to acertain whether curcumin can inhibit tumor growth. We treated mouse 3T3-fibroblasts, genetically engineered to overexpress the human Her2/Neu receptor, with curcumin in vitro and in vivo.

Results Curcumin inhibited the proliferation of mouse 3T3/Her2/Neu-fibroblasts and diminished the expression of Her2/Neu receptor along with a modulation of the expression of glyoxalase 1. Inoculation of 3T3/Her2/Neu-fibroblasts into nude mice elicited strong tumor growth, which could be significantly blocked by curcumin supplements in the food.

Conclusion Since curcumin is a nontoxic plant compound we have a significant interest in demonstrating whether it might by therapeutically applicable to treat Her2/Neu-overexpressing tumors in human.

PP022

Environmental reprogramming of adult chondrocytes in 3D matrices

BM Bubmann1, A Glodschei1, T Fernandez Muinos2, A Grodzinsky3, J Meisel4 and CE Semino1,2 1 Universität Leipzig, Translational Centre for Regenerative Medicine, Leipzig, Germany 2 Universidad Ramon Llull , Institut Químic de Sarrià, Barcelona/Spain, Spain 3 Massachusetts Institute of Technology, Center for Biomedical Engineering, Cambridge, MA, USA 4 Berufsgenossenschaftliche Kliniken Bergmannstrost, Klinik für Neurochirurgie, Halle, Germany The aim of this study is to investigate the regenerative potential of human chondrocyte samples from nucleus pulposum and anulus fibrosis. Our plan is to environmentally reprogram these cells, expand and then redifferentiate them back into chondrocytes for the therapeutic application of lumbar disc degeneration. We have recently developed a simple and reliable method to dedifferentiate and reprogram adult cells into primitive multipotential derivates of the same cellular lineage [1] . The method consists basically in culturing the cells into soft gels for days or weeks in the presence of special media conditions. Moreover, we have recently used bovine new born calf chondrocytes to test the feasibility of our project. In this case, the isolated cells were dedifferentiated by culturing them in culture flasks for five passages and partially re-differentiated into chondrocyte-likecells after culturing for up to 30 days in the 3D microenvironment. These constructs develop autonomously into cartilage-like tissue indicating that our working hypothesis could be correct with human chondrocyte samples.

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Based on our results, cells isolated from patient samples will be expanded and then cultured in our 3D peptide matrix. Cells from the developing constructs will be analyzed by immunohistological techniques and molecularbiological methods. The process of dedifferentiation will be characterized by methylation studies of specific genes, which could indicate to modifications within gene expression. We propose that by reculturing the differentiated chondrocytes (fibroblasts) in this biomimetic scaffold will promote the cells to re-differentiate into normal and functional chondrocytes, producing appropriate quantities of collagen type II and aggrecan, reconstituting the biomechanical properties of the degenerated disc.

Reference 1

Quintana L, Muiños TF, Genove E, Del Mar Olmos M, Borrós S, Semino CE: Early tissue patterning recreated by mouse embryonic fibroblasts in a three-dimensional environment. Tissue Eng. Part A 15(1), 45–54 (2009).

PP023

Microarray gene expression analysis of a mouse model of distraction osteogenesis

C Chang1,2, J Helm2, MA Mello2, Y Song2, G Jiang2, J Onodera2, S Janjanin2, R Mauck2, J Triffitt1, P Hulley1 and R Tuan2 1 Nuffield Orthopaedic Centre/University of Oxford, Botnar Research Centre, Oxford, UK 2 National Institutes of Health, NIAMS, Bethesda, MD, USA

Objectives Distraction osteogenesis (DO) is a bone regeneration process used for limb lengthening, involving surgical osteotomy and mechanical distraction. The unique mechanical and biological environment within the regenerating gap in DO drives the events that produce new bone, but the specific cellular and molecular mechanisms responsible for this process are still largely unknown. To gain insights into the molecular events involved in this mechanically stimulated bone regeneration, microarray-based analysis was performed in a mouse model to describe the global gene expression profile throughout the time course of DO.

Materials & methods Surgery was performed on mice to install a custom-designed external fixator on the right tibia, which was then osteotomized. After a 7‑day latency period, lengthening was applied for a 7‑day distraction phase, and further healing (consolidation) was allowed. A group of nondistracted fractures was also produced. x-ray images and RNA and histology samples were taken throughout the latency, distraction and consolidation phases of the process. Histological and immunofluorescence staining was performed on tissue from the osteotomy site. Microarray hybridization was performed using Affymetrix technology, and gene expression was analyzed with EDGE, TIGR MeV, and Ingenuity Pathways Analysis software platforms (San Francisco, CA, USA).

Results Radiography showed that the custom fixators produced effective bone lengthening. Extensive new bone formation and the presence of collagen type I was evident with histology and immunostaining. From the microarray data, genes with significantly altered expression profiles over the DO time course were identified and grouped into biological functions and signaling pathways. These groups were mainly related to connective tissue disorders and development, cell cycle and movement, inflammatory responses, neuronal signaling, and cytokine and growth factor activity. For selected panels of genes (i.e., key factors in osteogenesis and angiogenesis), heat maps displayed individual gene expression levels at different phases of DO. In comparison to distraction, the fracture microarray data showed greater involvement of immune and metabolic functions.

Conclusions Our results shed light on the complicated processes involved in DO, a unique and exquisite example of de novo adult tissue regeneration. Further analysis of the action of the identified candidate genes may lead to a better understanding of the underlying mechanisms that will lead to improvement of DO and other forms of bone regeneration.



S169



Biofunctionalized silk fibers drive axonal regeneration in retinal ganglion cells

T Claudepierre1, C Wittmer2, F Pfrieger3, P Wiedemann1, D Kaplan2 and C Egles4 1 Universität Leipzig, Faculty of Medicine, Ophthalmology Department, Leipzig, Germany 2 Tufts University, Department of Biomedical Engineering, Medford, OR, USA 3 CNRS , Institute of Cellular and Integrative Neurosciences UPR 3212, Strasbourg, France 4 Tufts University, Division of Cancer Biology and Tissue Engineering, Boston, MA, USA

Objectives Traumatic optic neuropathy is a severe complication of a head or face trauma. Impairment or loss of vision due to optic nerve injury occurs in approximately 10% of patients with cranio–facial fractures and are irreversible in case of axotomy. Glial scar, inflammatory response and cell death are some of the many surrounding factors barring the growth cone to cross the lesion site; in addition to the fact that axotomized retinal ganglion cells (RGCs) are unable to switch to a regenerative state and degenerate. To circumvent this cascade of events, we aim to develop a physical guide that will topically deliver molecules of interest, helping to restore the regenerative state of RGCs, and could be used to promote vision recovery following optic nerve lesion.

Methods We selected silk fibers prepared from silkworm (Bombyx mori) for their biocompatibility and tested, in different culture conditions, regenerative abilities of purified RGCs at the contact of this biomaterial.

Results In a first step we used our model of pure primary RGCs culture to identify factors that promote axon regeneration in vitro. We analyzed the effect of different growth factors on survival and neurite elongation of pure RGCs preparation from postnatal rat retina. Among all factors tested, we demonstrated that brain-derived neurotrophic factor promotes survival of RGCs whereas cilliary neurotrophic factor (CNTF) acts both on survival and axon elongation. We then tested the ability of silk fibers to serve as a support for growth cones and demonstrated that neurites elongate preferentially on silk fibers. Moreover, direction of growth was influenced by the silk network. Silk fibers were then biofunctionalized with growth factors and orientated in order to obtain a directing path of silk fibers loaded with brain-derived neurotrophic factor and CNTF. In a minimal culture medium we then observed that RGCs exhibit strong survival and axonal growth along silk fibers loaded with CNTF.

Conclusions Our results demonstrate the properties of silk fibers to serve as a guide and a reservoir for therapeutic molecules that can be released topically at lesion site. To date, CNTF is the most promising factor we tested. Development of a 3D silk guide to study the neuroregenerative properties of CNTF in vivo would be of main interest in the treatment of optic nerve lesions after facial trauma.

PP025

Characterization of c-kit+ cells in human left ventricular aneurism tissue

K Dergilev1, A Gmyzina1, K Rubina2, V Sysoeva2, Z Tsokolaeva1, T Rahmat-Zade1, R Akchurin1 and Y Parfyonova1 1 Russian Cardiology Research Centre, Angiogenesis Laboratory, Moscow, Russian Federation 2 Lomonosov Moscow State University, Faculty of Medicine, Moscow, Russian Federation

Introduction Stem cell transplantation appears to be a prospective therapy for the repair of damaged myocardium. In the past few years it has been established that the heart contains a reservoir of stem and progenitor cells (c-kit+, sca-1+ and isl-1+) having the capability for ex vivo and in vivo differentiation toward the vascular and cardiac lineages and showing cardiac regeneration potential in vivo following injection in the infracted myocardium. It was shown that cardiac stem cells (CSCs) could be isolated from small biopsies of human myocardium and expanded ex vivo, suggesting a possibility of new therapeutic strategies for cardiac repair based on the regeneration potential of CSCs.

Objective The aim of the present study is to characterize c-kit+ cells in the tissue of chronic left ventricular aneurism.

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Materials & methods We screened 15 samples of aneurysm tissue harvested during surgical aneurysm resection and analyzed samples immunohistochemically and by flow cytometry.

Results The amount of c-kit+ cells was approximately 3300/cm3 of tissue. Co-staining experiments demonstrated that c-kit+ cells were of nonhematopoietic origin, since they did not express CD34 or CD45. Flow cytometry confirmed that c-kit+ cells in aneurysm tissue were largely CD34-, it also showed that they were negative for a cocktail of blood lineage markers such as Lin1. However, we identified two populations of c-kit+ cells: approximately 60% of cells were c-kit+ CD45 +, which might populate the heart via circulation and another one was CD45-, which might contain distinct human resident CSCs. Human c-kit+ cells were located in fibrous, muscle and adipose parts of aneurysm tissue. They localized mainly in fibrous tissue nearby large vessels; however, no co-staining with endothelial cells was observed. The majority of c-kit+ cells expressed another stem cell marker MDR1, although they were negative for the marker of cell proliferation Ki67, nor did they express markers of differentiation in cardiac (Nkx 2.5, Gata-4, Mef2c and a-actinin), endothelial (Ets1, CD105 and vW) and smooth muscle (Gata6 and SMA) lineages. Approximately 20% of c-kit+ cells expressed cyclin-dependent kinase inhibitor p21. We observed samples of aneurysm tissue with clusters of cells, however, they were also negative for markers of cardiomyocyte differentiation. Using magnetic cell sorting c-kit+ cells could be successfully isolated from aneurysm tissue and expanded in vitro.

Conclusion It thus appears that aneurysm tissue could be an alternative source of autologous CSCs.

PP026

Analysis of wear particles released from surface-modified CoCr alloys

C Díaz1, JA García1, S Mändl1,2 and J Lutz1,2,3 1 Asociacion De La Industria Navarra, Reserch and Development, Cordovilla-Pamplona, Spain 2 Leibniz-Institut für Oberflächenmodifizierung, Leipzig, Germany 3 Universität Leipzig, Translational Centre for Regenerative Medicine, Leipzig, Germany

Introduction For a long time, ceramic–metal and polymer–metal prostheses have been widely used in some hip and knee prosthesis, together with advanced metal-on-metal prostheses made from CoCr alloys. Despite a significant lower particle generation for the last material group, a finite release rate of microscopic and nanoscopic particles, which are toxic inside the human body, is still observed. The size distribution and the chemical characteristics of these particles are still under investigation.

Objective Thermochemical surface modification should be a promising technology to improve the biocompatibility and bioactivity of such already approved biomaterials.

Methods Oxygen plasma immersion ion implantation (PI3) and thermal oxidation at different temperatures were studied in relation to improve the wear rate in CoCr alloys. The wear rate was analyzed by ball-on-disk tests in Hank’s Buffered Salt Solution and by calotest technique.

Results The microstructure of the surface, roughness, contact angle and results in adhesion and abrasion were studied to obtain conclusions about influences of the surface modification and providing detailed modeling. Detection of wear particles after ball-on-disk tests in Hank’s Buffered Salt Solution was carried out by scanning electron microscopy–energy dispersive spectrometer technique in order to determine their size distribution and composition as a function of surface modification.

Conclusions The reported results indicate that PI3 significantly reduces the wear rate and number of toxic particles in CoCr alloy used in human body implants, while a similar size distribution was observed.



S171



Differentiation of murine embryonic stem cells to osteoclasts is favored in low glucose conditions

A Dienelt1 and N zur Nieden1,2 1 Fraunhofer IZI, Group Applied Stem Cell Technologies, Leipzig, Germany 2 University of California Riverside, Department of Cell Biology & Neuroscience and Stem Cell Center, Riverside, Germany

Objective High blood glucose levels caused by diabetes mellitus can lead to osteoporosis in the adult, which is thought to be the result of reduced osteoblast function, thus affecting the proper deposition of new bone matrix. To date, what role the matrix resorbing osteoclast plays in the development of this disease in diabetic patients remains controversial. It is a fact that bone formation and turnover is a complex interplay between osteoblasts and osteoclasts. Osteoclast commitment relies on osteoblast surface interactions via RANKL and secretion of macrophage colony stimulating factor (MCSF). The respective receptors, RANK and MCSFR, can be found on the surface of osteoclasts and their progenitors. Against this background it was the aim of this study to analyze the effect of d-glucose on the differentiation of bone tissue using murine embryonic stem cells as an in vitro model.

Materials & methods In order to better understand the effect of glucose on bone tissue development, murine embryonic stem cells were differentiated for 30 days in media containing either low (1.0 g/l, physiological) or high (4.5 g/l, diabetic) concentrations of d-glucose. At selected time points the cells were lysed, RNA was isolated and analyzed for osteoclast and osteoblast markers by multiplex real-time PCR. In addition, the enzyme activities of both alkaline and tartrate resistant acidic phosphatase, which are specific for osteoblasts and osteoclasts, were measured and the amount of calcium deposited into the matrix was determined.

Results Cells differentiated in low glucose media showed an increased matrix calcification and differing enzyme activities compared with high glucose cultivated cells. Differences in the expression of osteoblast and osteoclast markers and diverse expression profiles of MCSF/MCSFR and RANK/RANKL mRNA were also found.

Conclusion The conducted study suggests that the maturation state of the osteoblast and osteoclast lineages is impaired in hyperglycemic conditions equivalent to diabetic blood glucose concentrations. The lower presence of mature osteoblasts and osteoclasts in hyperglycemic conditions may be due to an altered interaction between precursors of both cell types, which can be concluded from the expression profiles of the interaction markers MCSF/MCSFR and RANK/RANKL. Ultimately, our results show that low glucose concentrations favor the development of both osteoblasts and osteoclasts from murine embryonic stem cells.

PP028

Wnt5a enhances osteogenic specification in murine embryonic stem cells

H Ding1, B Kuske1 and N zur Nieden1,2 1 Fraunhofer IZI, AG Stem Cell Technologies, Leipzig, Germany, Germany 2 University of California Riverside, Department for Cell Biology & Neuroscience and Stem Cell Center, Riverside, CA, USA

Objective Embryonic stem cells (ESCs) self-renew indefinitely and contribute to derivatives of all three germ layers. It has been demonstrated that murine ESCs can be effectively differentiated to osteoblasts with vitamin D3. This offers a powerful approach to study mechanisms implicated in the earliest stages of bone cell differentiation in vitro. Wnt family members are secreted-glycoproteins that play an important role in development and cell growth. We previously found that Wnt5a can support specific stages of the osteogenic process during ESC differentiation. Here, we have attempted to explore how Wnt5a is involved in osteogenesis.

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Materials & methods Murine ESCs were differentiated to osteoblasts with medium supplemented with 1a, 25-(OH)2 vitamin D3, ascorbic acid and b-glycerophosphate. To study the effect of Wnt5a on b-catenin (CatnB), a stable ESC line was established that integrates a green fluorescent protein reporter responding to CatnB mediated transcriptional activation in the nucleus. RNA samples and protein samples were taken during differentiation and analyzed by reverse transcription PCR or western blot, respectively. Matrix-incorporated calcium was quantified with the purple substrate Arsenazo III.

Results Until now, several sub-pathways were reported to be involved in Wnt5a downstream regulation. We investigated the RNA expression of the perspective Wnt5a downstream targets by reverse transcription PCR. It showed that most of these genes have special expression patterns during the first 11 days of differentiation. The nuclear activity of CatnB was downregulated by Wnt5a between differentiation days 5–10, which may correspond to the enhancement of osteogenesis. Furthermore, blocking the prospective Wnt5a downstream targets – calmodulin dependent protein kinase II (CamKII), c-Jun terminal kinase (JNK) and protein kinase C (PKC) pathways with – specific inhibitors, upregulated CatnB protein expression levels in the nucleus. As a result, osteogenesis as determined by calcium assay was decreased.

Conclusion These findings indicate that Wnt5a can enhance osteogenesis by activating CamKII, JNK and PKC sub-pathways during mouse ESC differentiation.

PP029

Pluripotent stem cells are highly susceptible targets for syngeneic, allogeneic & xenogeneic natural killer cells

R Dressel1, J Nolte2, L Elsner1, K Guan3, G Hasenfub3 and W Engel2 1 Universität Göttingen, Cellular and Molecular Immunology, Göttingen, Germany 2 Universität Göttingen, Human Genetics, Göttingen, Germany 3 Universität Göttingen, Cardiology and Pneumology, Göttingen, Germany

Objectives Multipotent adult germ line stem cells (maGSCs) and induced pluripotent stem cells are potentially new sources of cells for stem cell transplantation therapies. maGSCs could be used to generate autologous cells for various therapeutic purposes that are expected to be tolerated by the recipient. However, the effects of the immune system on these new pluripotent cells have not been investigated.

Materials & methods We have compared the susceptibility of maGSCs (maGSC Stra8, maGSC 129/Sv, maGSC FVB and maGSC C57BL), iPSCs, embryonic stem cells (ESCs;ESC Stra8, ESC 129/Sv R1, ESC FVB and ESC C57BL), and F9 teratocarcinoma cells to lymphokine-activated killer and natural killer (NK) cells. The expression of ligands for NK receptors has been determined by flow cytometry.

Results All stem cell lines have been shown to be pluripotent by teratoma growth assays in immunodeficient mice. All cell lines were efficiently killed by syngeneic, allogeneic and xenogeneic killer cells. When analyzed by flow cytometry, all stem cell lines lacked major histocompatibility complex class I molecules, which can interact with inhibitory NK receptors. Instead, most of them expressed ligands for the activating NK receptors NKG2D and DNAM-1. The expressed NKG2D ligands belong to the RAE-1 family. The DNAM-1 ligands CD112 and CD155 were both found to be expressed. All stem cell lines were negative for ligands of the activating 2B4 and NKp46 receptors. In the RAE-1 positive cell lines, the killing could be partly inhibited by a soluble NKG2D protein indicating a functional role for these molecules on stem cells as target structures for NK cells. During undirected differentiation as embryoid bodies in hanging drops the ligands for activating NK receptors were downregulated and the cells became resistant to lymphokine-activated killer cell-mediated killing.

Conclusions A high susceptibility to NK cells might constitute a common feature of pluripotent stem cells that could affect transplantation therapies due to rejection even of syngeneic stem cells. However, it could also offer a strategy to deplete pluripotent cells before grafting of in vitro differentiated cells in order to avoid teratoma formation.



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Preparation of resorbable ceramic scaffold materials with defined shape & bone-like morphology

M Dressler1, K Winter1 and J Liese1 1 Universität Leipzig, Translational Centre for Regenerative Medicine, Leipzig, Germany Shaping of bioceramic materials is an important technological step during preparation of implant or scaffold materials. In the present work, rapid prototyping (3D printing) was used to prepare porous bioceramic materials with a defined porosity and pore shape. The ceramic used was a resorbable material (Ca10[K/Na](PO4)7) [1] . The outer contour of the prepared bioceramics was designed using micro-computed tomography derived 3D data from the jar bone of minipigs. The prepared specimens had the same shape as the scanned region on the jarbone of minipigs. Hence, the prepared samples can be tightly put onto the jar bone when implanted. The results show that a manifold of different data transfer stages have to be completed in order to produce bioceramic samples with both a defined porous structure and defined outer contours. A suitable data transfer route was established in order to obtain data files with sufficient resolution and reasonable data sizes.

Reference 1

Georg Berger: Federal Institute for Materials Research and Testing. Berlin (2009).

PP031

Hyaluronic acid scaffolds for chondrogenesis of mesenchymal stem cell by controlled delivery of TGF-b1

C Du1, S Gao1, ACA Wan1 and JY Ying1 Institute of Bioengineering and Nanotechnology, Cell and Tissue Engineering, Singapore

1

Human bone marrow-derived mesenchymal stem cells (hMSCs) have strong potential in regeneration of musculoskeletal tissues including cartilage and bone. The microenvironment, comprising of scaffold and soluble factors, plays a pivotal role in determining the efficacy of cartilage tissue regeneration from MSCs. Previous attempts to engineer cartilage using hMSCs have depended on in vitro predifferentiation in order to form chondrogenic engineered constructs. Such techniques greatly increase the time of implant fabrication and the differentiated cells suffer from loss of phenotype upon withdrawal from chondrogenic medium. In this study, hyaluronic acid hydrogel constructs seeded with MSCs were cultured in the presence or absence of TGF-b1 for 4 weeks. TGF-b1 encapsulated in poly(lactide-co-glycolide) microspheres was incorporated in the hydrogel, remained bioactive in the long term and was released in a controlled fashion. At the same time, the specimens were implanted into the subcutaneous pocket of severe combined immunodeficiency mice and harvested after 4 weeks. Chondrospecific mRNA expression was analyzed using reverse transcriptase PCR. Corresponding extracellular matrix synthesis was demonstrated using immunohistochemical staining. Results show that chondrospecific marker molecules such as collagen type II and type X, cartilage oligomeric matrix protein and aggrecan obviously expressed after 4 weeks in vitro and in vivo compared with control hydrogels without TGF-b1 microspheres. Cartilage matrix proteins such as collagen type II were found within the extracellular matrix. Moreover, a significant downregulation of type X collagen expression was observed in our system, indicating that hyaluronic acid hydrogel with TGF-b1 inhibits the further differentiation of MSCs into hypertrophic chondrocytes. These findings underline the potential of a tissue-engineered construct that incorporates sustained delivery of TGF-b1 to induce differentiation of hMSCs into chondrocytes in situ, using injectable hyaluronic acid scaffolds.

PP032

Regeneration of intervertebral disc using in vitro differentiated stem cells

F Ehlicke1, D Freimark1 and P Czermak1,2 1 Fachhochschule Giessen-Friedberg, Institute of Biopharmaceutical Technology, Giessen, Germany 2 Kansas State University, Department of Chemical Engineering, Manhattan, KS, USA

Objective The degeneration of the intervertebral disc (IVD), particularly the nucleus pulposus (NP), is one reason for low back pain, which is a common disease in modern society. In cases of IVD protrusion in the late phase of IVD degeneration, one possible method of treatment is cell-based therapy. Therefore, adequate cells are needed. Besides the auxiliary operation itself to extract autologous NP S174

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cells, one main problem in using these cells is their insufficient amount and vitality resulting from the progressing IVD degeneration. In contrast to NP cells, vital human bone marrow-derived mesenchymal stem cells (hMSCs) are available in sufficient amounts in tissue. Besides the immune privilege of the avascular structured IVD itself, hMSCs avoid allorecognition and, therefore, circumvent the immune response. For these reasons, at the Institute of Biopharmaceutical Technology (IBPT, Frankfurt, Germany) we focus on the application of hMSCs for IVD regeneration. hMSCs must be differentiated to NP cells prior to implementation, which requires a 3D environment as NP cells dedifferentiate when growing in a monolayer [1,2] . In further experiments, the differentiation grade of hMSCs for efficient therapy has to be identified. For a long time NP cells have been misleadingly dealt with as chondrocytes as their protein expression profile is similar to that of chondrocytes [3] . Thus, currently, NP cells are poorly characterized. Therefore, a method to distinguish NP cells (e.g., differentiated hMSCs) from undifferentiated stem cells and chondrocytes has to be established.

Materials & methods 3D differentiation of hMSCs was carried out in a hydrogel for 21 days to form NP cells. Various stimuli were investigated regarding their applicability to differentiate hMSCs to NP cells. To evaluate the success of this method, NP-specific marker proteins, especially components of the extracellular matrix surrounding the NP cells (e.g., collagen II), were detected by immunostaining.

Results & conclusion The feasibility of immunostaining to detect NP-specific marker proteins in differentiated hMSCs was proved by previous experiments with pellet cultivations using the differentiation-inducing growth factor TGF-b3. Data concerning the differentiation of hMSCs in a 3D hydrogel under the influence of various stimuli (e.g., other growth factors or components of the extracellular matrix) will be presented.

References 1

Aulthouse AL, Beck M, Griffey E et al.: Expression of the human chondrocyte phenotype in vitro. In Vitro Cell. Dev. Biol. 25(7), 659–668 (1989).

2

Darling EM, Athanasiou KA: Rapid phenotypic changes in passaged articular chondrocyte subpopulations. J. Orthop. Res. 23(2), 425–432 (2005).

3

Steck E, Bertram H, Abel R, Chen B, Winter A, Richter W: Induction of intervertebral disc-like cells from adult mesenchymal stem cells. Stem Cells 23(3), 403–411 (2005).

PP033

The bench to bedside legal framework of regenerative medicine

T Faltus1 1 Universität Leipzig, TRM Leipzig, CELLT, Leipzig, Germany Stem cell and tissue engineering research as major topics of regenerative medicine need a reliable legal framework. Contrary to public opinion there are many opportunities for such research in Germany and the EU, even if the German Embryo Protection Act and the Stem Cells Act still prohibits the use of totipotent embryonic cells and developmental stages to gain pluripotent embryonic stem cells. However, considering the great hopes that stem cell research holds, the German lawmaker amended the national provisions in this area. Due to this change there are now many possibilities for international cooperation in stem cell research between foreign and Germany-based researchers and of course for research in Germany. However, the above-mentioned amendment of the German stem cell law coincided with unexpected results of stem cell research in the field of the creation of ethically unloaded stem cells by techniques of reprogramming. However, the German Stem Cell Act contains a subsidiary provision that states that the import of human embryonic stem cells to Germany is not permitted if there is a scientific alternative for the use of human embryonic stem cells. Therefore, if the further import of embryonic stem cells is legally cut off there is the probability that further stem cell research in Germany flags. Due to legal restrictions for Germany-based researchers, the import of human embryonic stem cells is the only possibility of getting access to these cells. For this reason, the legal (and scientific) status of reprogrammed stem cells must be clarified. The presentation will show the regulatory and legal scope stem cell researchers have and focus on the possibilities of purchasing embryonic stem cells in Germany. The presentation will also pay attention to the frequent questions as to what kind of international collaborations in Germany-based stem cell research are possible. Furthermore, the talk will show that reprogramming research is permissible in Germany as long as you gain pluripotent stem cells. Finally, the aspects of market authorization for regenerative medicine products will be explained.



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Directed differentiation of human embryonic stem cells to functional hepatocyte on nanofiber scaffold

This abstract has been withdrawn

PP035

Effects of nitric oxide on mesenchymal stem cells with regard to cell therapy

T Felka1, R Schäfer2, P De Zwart2, B Rolauffs2 and WK Aicher2 1 Universität Tübingen, ZRM, Tuebingen, Germany 2 Universität Tübingen, IKET, Tuebingen, Germany

Objectives Mesenchymal stem cells (MSCs) are attracting increasing interest as a strategy for cell therapies. However, the injured tissue is often accompanied by inflammatory processes, which yield the production of nitric oxide (NO). In this study we investigated the effect of NO on the metabolism of MSCs.

Materials & methods Human bone marrow-derived MSCs (n ≥ 3) were cultured in a foetal calf serum-free, good manufacturing practise-compatible medium and characterized according to the defined minimal criteria of multipotent MSCs. After treatment with the NO-donor sodium nitroprusside (SNP), viability was investigated using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-(MTT) assay. mRNA expression and protein expression were analyzed by gene array, quantitative real-time PCR or western blot analysis.

Results Low concentrations of SNP (5–50 µM) yielded a slight increase in the viability of MSCs, whereas concentrations of 100 µM–5 mM resulted in a plateau-like decrease with a 50% viability at 1.5 mM SNP. Gene array analysis indicating an activation of networks associated with apoptosis or even cancer at concentrations of 1.5 mM SNP. Western blot analyses implicated an effect of SNP via MEK- and ERK-mediated signaling pathways.

Conclusion Nitric oxide yielded concentration-dependent positive and negative effects on the metabolism of MSCs. Higher concentrations of NO caused by chronic inflammation will probably affect transplanted MSCs in a proapoptotical manner. Before one can consider clinical applications of MSCs, further investigations are necessary to avoid a development of MSCs into cells with tumoric phenotype. Financial & competing interests disclosure This project was supported in part by a BMBF grant.

PP036

Automated multipexed assays for standardized cytocompatibility testing of biomaterials

M Ferreira1, W Jahnen-Dechent2 and S Neuss1 1 RTWH Aachen, Institute of Pathology, Aachen, Germany 2 RWTH Aachen, Institute for Biomedical Engineering, Department of Biointerface, Aachen, Germany Life science laboratories benefit today from automation by increasing the throughput, while freeing human resources. Minimization of errors, faster work and improvement of reproducibility are other important advantages. Besides, there is an enormous interest for improved high-throughput cell-based screening platforms for use in preclinical trials for tissue engineering. In our study, we developed an automated multiplexed method of analysis of cytocompatibility testing of biomaterials. A combination of three independent test kits for cell viability (CellTiterBlue®), cytotoxicity (CytotoxOne™) and apoptosis (ApoOne®, all Promega, Madison, WI, USA) was adapted to the EpMotion 5070 robotic platform (Eppendorf, Hamberg, Germany). The cytotoxicity analysis of biomaterials included collagen, nitrocellulose and nylon in different textures. Despite all recent developments of robotic liquid handling platforms there are still some drawbacks: pipetting issues, biological variation effects and user intervention were optimized in this study. S176

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In the context of these studies, significant differences in precision and speed between manual and robotic functions were detected. Results proved the reliability and flexibility of the robotic platform allowing multiple researchers to use it without user variability. The established protocol combines and automates three different assay chemistries with high accuracy, speed and with minimal deck/instrumentation requirements. The multiplexed cell-based assay can be performed in a 96-well format with high reproducibility allowing for compatibility testing of biomaterials in tissue-engineering applications. As the demand for standardization and efficiency of biomaterial cytotoxicity screening tests increases within the scientific community this development of new highly reproducible automated protocols may be more widely applicable.

PP037

Production of scaffolds for tissue engineering applying two-photon polymerization

S Fiedler1, R Schade1, G Hildebrand1, K Liefeith1, M Schnabelrauch2, A Berg2 and R Wyrwa2 1 Institut für Bioprozess- und Analysenmesstechnik (iba) e.V., Biowerkstoffe, Heilbad Heiligenstadt, Germany 2 INNOVENT e.V., Jena, Germany

Objective Two-photon polymerization (2PP) is an innovative technique among the rapid prototyping methods to produce microstructured 3D features with custom designed shape [1] . This technique offers a considerable potential to develop tissue-engineering scaffolds for osteochondral implants based on biocompatible polymers for bone and joint regeneration. Here, we tested the 2PP of several polymer precursors for the bone phase and hydrogel forming biopolymers for the cartilage phase of osteochondral implants.

Materials & methods A Ti:saphire laser (Tsunami) emitting pulsed near-infrared light is employed to initiate 2PP reactions. Scaffolds were produced applying the 2PP-device M3DL (Laserzentrum, Hannover, Germany) and precision stages from Aerotech (Nuernberg, Germany). For polymerization in organic solvents the photoinitiators Igracure ®369 and Irgacure ®651 (Ciba) were applied and for polymerization in aqueous solution the photoinitiators rose bengal/triethanolamine [2] and Irgacure ®369 (after conversion into its hydrochloride) were used. Several methacrylated oligo(lactide-co-caprolactone)s and polyglycerins as materials for the bone phase were tested for their ability to be polymerized and 3D structured by 2PP. For the cartilage Phase hyaluronic acid, chondroitin sulfate and gelatin (all methacrylated) were chosen. Poly(ethylene glycol)-diacrylate (PEG-DA, Aldrich) was applied as matrix component.

Results & conclusion Polyglycerins have shown a good ability for processing by 2PP. Although methacrylated biopolymers (hyaluronic acid, chondroitin sulfate and gelatin) could not be polymerized efficiently in aqueous solution, it was possible to polymerize them into a matrix of 20% (w/w) poly(ethylene glycol)-diacrylate in water. Scaffolds (500 × 500 µm) with regular 3D-structures (‘woodpiles’, STL-formats) were produced. Pore sizes were varied from 10 to 120 µm. The biocompatibility of materials and 3D structures of the scaffolds will be investigated in cell culture applying osteoblasts and chondrocytes, respectively.

References 1

Lee K-S, Kim RH, Yang, Park SH: Advances in 3D nano/microfabrication using two photon initiated polymerization. Prog. Polym. Sci. 33, 631–681 (2008).

2

Basu S, Campagnola PJ: Properties of crosslinked protein matrices for tissue engineering applications synthesized by multiphoton excitation. J. Biomed. Mater. Res. A 71, 359–368 (2004).

PP038

Long bone growth depends on the ability of periosteum cells to generate stress

J Foolen1, CC van Donkelaar1 and K Ito1 1 Eindhoven University of Technology, Biomedical Engineering, Eindhoven, The Netherlands

Introduction Long bones grow faster when the fibrous periosteum (PO), which connects the proximal with the distal epiphysis, is cut circumferentially. We recently falsified the theory that tension in intact PO restricts growth in the epiphyseal plates; PO tension in the direction of bone growth is too low to have such effect, even at ages of high bone growth rates [1] . An alternative theory is that cutting the PO future science group


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alleviates tension in PO cells, which subsequently change the expression of growth-modulating factors [2] . Indeed, extended growth after removing the PO was inhibited when chick tibiotarsi were cultured in conditioned medium obtained from PO cells grown on glass substrates [3] . We postulate that this inhibition is PO-cell tension dependent, and test this hypothesis by culturing bones in conditioned medium, derived from PO cells cultured on substrates with different stiffness.

Materials & methods

PO/PC free PO/PC intact Length ratio (stripped/unstripped) [-]


1.50 * 1.25

*

* * *

1.00 Polyacrylamide gels of various stiffness (3, 14, 21, 48 and 80 kPa) were prepared on cover slips and coated with Matrigel. A PO cell population was obtained after 6 days of culturing 0.75 PO tissue in a well-plate. PO cells were passaged and seeded on the gels. After 5 days, medium was replaced by 4-ml serum0.50 Control 3 kPa 14 kPa 21 kPa 48 kPa 80 kPa free Dulbecco’s Modified Eagle Medium (Invitrogen, Carlsbad, medium Canada). After 24 h, conditioned medium was collected and Gel stiffness added to dissected tiobiotarsi with intact PO, or where PO was removed. Tibiotarsi were harvested after 3 days and stained PP038 Figure 1. with alizarin red (bone) and alcian blue (cartilage), and distal epiphysis length was measured from images taken through a stereomicroscope. Length was determined along the midline of the cartilage, from the midpoint of the tarsal curve to the midpoint of the border with the bone shaft [3] .

Results Epiphysis growth with conditioned medium decreased with increasing substrate stiffness (R 2 = 0.30; p