BioStar 2010–Science in Exchange Meeting Abstracts 4th Congress on Regenerative Biology and Medicine October 13–15,2010 Stuttgart,Germany

1 ERBE Elektromedizin GmbH, Ernst-Simon straÕe 10, 72072 Tübingen, Germany, Markus.Enderle@erbe-med.de 2 Center for Regenerative Medicine and Department of Orthopaedic Surgery, Center for Medical Research, Tübingen, Germany OP 07 (E015) Potential role of waterjet technology in regenerative medicine

Background: Waterjet technology is an established method utilized in multiple medical disciplines for tissue dissection, cutting and cushioning. The first results using the waterjet in surgery were reported in 1982 for liver resection. Since then, waterjet dissection has been routinely applied in diverse surgical fields. This technology allows the application of an extremely thin laminar liquid jet at a preset pressure to the tissue surface. The precision of the waterjet is ideal for tissue selective and nerve sparing dissection and preparation: fibrous and collagenous tissue can be displaced, leaving blood vessels and nerves intact. A relatively new indication is the cushioning of the gastrointestinal wall by needleless pressure injection. Thereby, endoscopic resection procedures could be expanded to the treatment of extended lesions in en-bloc technique, concurrently limiting the depth of thermal injury.

Apart from its use during surgical interventions, the waterjet technology might also be useful in the field of regenerative medicine, serving as a transport vehicle for different substances, including biomaterials, and cells.

Initial experiments and further developmental steps: To this end, the waterjet system with special applicators plays the key role, supplemented by biomaterial and primary cells. Initial experiments on the transfer of cells in isotonic saline using waterjet technology look promising: depending on the cell line, the applied pressure and the diameter of the nozzle of the waterjet applicator, a survival rate of up to 100% could be achieved. However, for clinical application, new, deployable waterjet applicators have to be designed and corresponding hardware and software modifications made to the existing system. Furthermore, a suitable biomaterial has to be developed to exhibit a predefined viscosity in its fluid condition in order to be both sprayable and injectable, followed by gelation within a restricted time frame and it should adhere to the tissue's surface. Biocompatibility and the capability for resorption are further important prerequisites. The last of the three components are the primary cells for which the propagation and characterization of appropriate primary cells are further crucial steps. Using suitable in vitro/ex vivo/in vivo experiments as well as functional tests we optimize the individual components to fulfil the desired characteristics.

Summary and outlook: We develop an advanced therapeutic system for cell- and/or biomaterial-based local wound repare based for endoscopic use. We entertain the possibility to use the applicators in a spray-like fashion for the treatment of large lesions that would otherwise be accompanied by a functional restriction of the corresponding organ. The option to inject cells and/or biomaterial provides access to the reconstitution of functional defects by minimally invasive surgery and under endoscopic observation.

1 Charles University in Prague, 1st Faculty of Medicine, Institute of Anatomy, U Nemocnice 3, 12800 Prague 2, Czech Republic, bdvorankova@seznam.cz 2 Ludwig-Maximilians University, Faculty of Veterinary Medicine, München, Germany OP 09 (E009) Human adhesion/growth-regulatory galectins: inducers of transition of fibroblasts to myofibroblasts and of 3D extracellular matrix lattices

Cell therapy and tissue engineering are aiming to make use of stem cells. A key step to proper applications is to characterize the “niche” that maintains their stemness. The extracellular matrix is known to be an important part of this microenvironment. Studying the microenvironment in cancer stroma and wounded skin, we observed a high incidence of myofibroblasts and level of presence of an endogenous lectin, i.e. galectin-1 (for further details on lectins, please see [1]). To study the lectin's role, also in comparison to other members of this family, we used a panel of galectins incl. engineered variants and tested their influence on the transition of normal human dermal fibroblasts to myofibroblasts. Indeed, galectins were found to stimulate this transition, galectin-1 being the most potent. It is well known that TGF-beta activates the fibroblast – myofibroblast transition. The effect of galectin 1 was found to be independent on TGF-beta both substances have an additive effect. Examining cell features associated with galectin stimulation the galectin-exposed fibroblasts/myofibroblasts produce massively an extracellular scaffold rich in fibronectin and also galectin-1. When tested as substratum, it turned out to be beneficial for cultivation of keratinocytes without feeder cells. Interestingly, the keratinocytes cultured on this scaffold changed their phenotype and expressed keratin-19 (marker of epidermal stem cells). Giving these observations a perspective, the described approach could in long term be of use in tissue engineering and wound management.

This study was supported by Ministry of Education Youth and Sport of the Czech Republic, projects No. MSM0021620806.

1 Institute of Textile Technology and Process Engineering, Nonwoven Technologies, Körschtalstrasse 26, 73770 Denkendorf, Germany, cornelia.fano@itv-denkendorf.de 2 Herzchirurgische Klinik der Universität München, Germany OP 14 (D004) Development of textile scaffolds for tissue engineered heart valves

Introduction: In 2007, 3.000 aortic heart valve prostheses was implanted in Germany [1]. The risk of conventional valves: rejection, calcification or thrombosis created the need for a valve, without these adverse effects.

Material and Method: With the fiber spraying process of the ITV Denkendorf a new nonwoven scaffold of polyurethane was developed [2]. Its shape is that of a biological aortic valve, not to change the natural flow profile of the blood or destroying the platelets. Valve and aortic root are produced as a whole. At the Grosshadern Hospital the scaffold is seeded in vitro with human endothelial cells to prevent the risk of rejection, thrombosis and calcification [3].

To optimize the properties of the aortic root, various process parameters, forming die transversal (vStab), rotational speed (nStab), rotations of the spinning pump (nPumpe), spraying pressure (p), were considered. These parameters' effects on the tensile strength (F) were tested and investigated with SEM.

In order to prevent a separation of the valves from the aortic root, different forming dies were evaluated. Furthermore the valves bending and tensile strength were determined.

Results: The results of the aortic root showed for large vStab highest F and finer fibers. To prevent fiber ropes and to increase F, a high nStab should be set. If nPumpe is reduced, the fibers get finer and the nonwoven denser. When p is increased, fine, plaque-free fibers emerge.

Revising the forming dies, improved the pull-out strength out of the aortic root by 50%.

The bending trails showed that the porcine valves are more flexible than the PU- valves. Perfusion tests at the GroÕhadern Hospital demonstrated nevertheless smooth opening and closing of the valves.

The tensile test shows that the valve has the same strength as a human valve, but at a higher elongation.

Discussion: Fine small plaqued fibers and a high F of the aortic root can be gained choosing production parameters as low nPumpe, high p, nStab and vStab. The valves remain flexible and are opening and closing smoothly having the same strength but higher elongation than a human one.

Modification of the forming die prevents separation of valve and aortic root.

The Grosshadern Hospital showed that the surface of the prothesis can be settled with cells.

1 MPI for Colloids and Interfaces, Department of Biomaterials, Am Muehlenberg 1, Wissenschaftspark Golm, 14476 Potsdam, Germany, KrishnaPrasad.Kommareddy@mpikg.mpg.de 2 Center for Biomaterial development, Institute of Polymer Research, GKSS Research Center Geesthacht GmbH, Teltow., Germany 3 Institute of Biochemistry, Freie University, Berlin., Germany 4 Ludwig Boltzmann Institute of Osteologie, Hanusch Hospital of WGKK and AUVA, Vienna, Austria OP 16 (D034) Three-dimensional tissue growth in polymer scaffolds with different stiffness and in-vitro influence of BMP-2 on tissue formation in hydroxyapatite scaffolds

An understanding of how tissue forms within scaffolds is of fundamental importance for tissue engineering. Besides the surface properties, e.g. roughness, charge and chemistry, the stiffness and geometry of a scaffold play a major role in cell response and subsequent tissue formation. Recently, Rumpler et al. [1] demonstrated the influence of geometry by growing MC3T3-E1 pre-osteoblast cells in hydroxyapatite (HA) channels with various pore geometries. Although, total growth rate was found to be independent of shape, local growth rates were observed to be higher in the corners, i.e. concave regions of high curvature.

The above mentioned concept was then extended to 3D polymer scaffolds with different stiffness in the range of 74 to 310 MPa prepared from polyether urethanes (PEU), where the tissue formation of MC3T3-E1 pre-osteoblast cells were explored using different cell seeding conditions over 7 weeks. Tissue growth was observed by phase contrast microscopy. A two-stage behaviour of tissue formation kinetics was obtained in the PEU scaffolds [2]. A time delay (t0) in the tissue formation at the very beginning of the culture experiment was observed, which varied with material stiffness and cell seeding density. In later stages the tissue formation kinetics showed a similar growth behaviour as observed in HA scaffolds [1, 2]. This suggests, material properties no longer dominate and it is rather scaffold architecture that controls growth.

In another study, in-vitro application of growth factor (BMP-2) was tested during the culture experiment. Different BMP-2 induction times were probed during the MC3T3-E1 pre-osteoblast cultures (4 weeks) on hydroxyapatite scaffolds. It was observed that continuous in-vitro application of BMP-2 on pre-osteoblast cells kept them in proliferation phase for a few more days after the application, whereas control and short pulse BMP-2 treated samples showed lower proliferation. At later time points, differentiation of pre-osteoblast cells to mature osteoblasts overtook the proliferation and all treatment conditions showed similar tissue growth. This work gives insight into the tissue engineering aspects of in-vitro application of BMP-2 and its influence on tissue growth and differentiation of pre-osteoblasts, and in designing controlled release of growth factors in optimizing the tissue formation.

1 Max-Planck-Institute for Metals Research, New Materials and Biosystems, Heisenbergstr. 3, 70569 Stuttgart, Germany, thedieck@mf.mpg.de 2 South Westphalia University of Applied Sciences, Iserlohn, Germany 3 Section for Transplantation Immunology and Immunohematology, Center for Medical Research, University Medical Clinic, Tübingen, Germany OP 19 (A006) Regulation of Hematopoietic Stem Cell Behaviour by Matrix Elasticity

Hematopoietic stem cells (HSCs) reside in a niche within the bone marrow, which provides a specific environment, where the stem cells can expand while keeping their stem cell character. Proliferation outside of the niche finally leads to terminal differentiation. In vitro HSC expansion would be of great benefit for clinical application, but up to now adequate culture conditions for HSC expansion without differentiation do not exist. During the last years increasing evidence showed that not only biochemical signals but also matrix elasticity plays an important role in regulating cell fate and behaviour of mesenchymal and embryonic stem cells. The current study addresses the question if HSCs also respond to signals emerging from differences in matrix elasticity.

Although nothing is known about the influence of matrix elasticity on HSCs, there is some evidence that HSCs might respond to changes in the substrate stiffness. Osteoblasts, which are a major component of the endosteal HSCs niche, flatten during HSC mobilization in a mouse model upon adrenergic stimulation. We assumed that a flat osteoblast differs in its elasticity compared to a high osteoblast. To test this hypothesis, we developed an in vitro model for osteoblast flattening under adrenergic stimulation. The osteosarcoma cell line CAL-72 was used as a model for osteoblasts. In confocal microscopy a decrease of the cell monolayer height was observed upon treatment with the adrenergic agonist clenbuterol. A change in elasticity could be determined by atomic force microscopy. In order to investigate the influence of substrate elasticity on HSCs, established hematopoietic cell lines and hematopoietic stem and progenitor cells isolated from human umbilical cord blood were seeded onto fibronectin coated hydrogels with different elastic properties. Substrate elasticity clearly influenced cell morphology, adhesion and migration.

In summary, we could show that the substrate elasticity changes in a model of the endosteal HSC niche after adrenergic stimulation, which occurs during HSC mobilization. Furthermore we could observe that hematopoietic progenitor cells are able to sense substrate elasticity and integrate it into intracellular signals which modulate cell shape, adhesion and migration. Thus, matrix elasticity seems to be an important factor in the regulation of HSC retention in the endosteal stem cell niche and should be considered in attempts to propagate HSCs in vitro.

1 Medical University of Graz, Institute of Cell Biology, Histology and Embryology, Harrachgasse 21/7, 8010 Graz, Austria, julia.koenig@medunigraz.at 2 Department of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria 3 Centro di Ricerca E. Menni, Fondazione Poliambulanza, Istituto Ospedaliero, Brescia, Italy OP 20 (A012) Do mesenchymal cells from human placenta have angiogenic properties?

Objectives: Recent research in stem cell biology identified the human placenta as a new source of cells which show phenotypical similarities to bone marrow-derived mesenchymal stem cells (MSC) regarding their multipotent differentiation potential. In particular the amnion contains immunoprivileged cells of fetal origin, which are easily accessible and available in large supply.

Here, we aim to generate optimal isolation and characterization protocols of amnion derived mesenchymal cells (AMSC). We combine these methods with differentiation studies towards the endothelial lineage to create new therapies for vascular reformation.

Methods: For the isolation of AMSC, the amnion of human term placentas (n = 15) was manually separated from the chorion and treated with collagenase/DNase. Tissue for cryosections was collected from every placenta. AMSC were cultured in standard (DMEM with 15% FCS) or angiogenic conditions (endothelial cell medium ECM-2). They were characterized by the ability to take up DiI-AcLDL and to form networks in an angiogenesis assay (Matrigel assay). Both cultured cells and placental cryosections were examined immunohistochemically.

Results and Conclusions: AMSC express the common MSC markers CD73, CD105 and CD90 in situ as well as in vitro. They are negative for mature endothelial markers such as vWF and VE-Cadherin, however, they express VEGFR-2, which can also be found on vasculogenic and angiogenic precursor cells. Upon angiogenic culture, cells show an enhanced proliferation potential and viability compared to undifferentiated control cells. In addition, they change their morphology towards an endothelial, cobblestone-like phenotype and are able to take up DiI-AcLDL. Although they do not differentiate into mature endothelial cells as reflected by the absence of vWF and CD144, they form more stable and widespread networks than endothelial cells in the matrigel assay and seem to stabilize endothelial networks.

These results indicate angiogenic properties of AMSC which might be of therapeutic use in vascular biology.

1 University Hospital of Modena and Reggio Emilia, Laboratory of Cell Biology and Advanced Cancer Therapies, Oncology, Hematology and Respiratory Diseases Modena, Italy, massimo.dominici@unimore.it 2 CHOP, Philadelphia, USA OP 21 (A013) Focusing on the Borders Between Bone and Blood to Better Understand Stem Cells Based Regeneration

It has been proposed that areas which border both bone and marrow contain specific niches which are crucial for hematopoietic stem cell (HSC) engraftment (Calvi LM, 2003). Similarly, we have shown that bone regeneration takes place after BM transplantation in discrete districts close to bone within the so called bone lining cells compartment where early osteopoietic engraftment originates as well (Dominici et al. 2004 and 2008). Very little is known about the mechanisms that drive the restoration of these compartments following a bone marrow (BM) injury. Researchers have been focused almost entirely on how HSCs are regulated by their microenvironmental niches (Zhang J 2003), with little attention paid to the recovery of cellular constituents of these niches after use of cytotoxic conditioning regimens. Moreover, while several models have been proposed, the precise anatomic location of niches within the BM microenvironment is not well understood. Thus, focusing on ionizing radiation (IR) exposure, we asked how the niches may respond to specific stress and affect mesenchymal engraftment as well. Based on this animal model, we report a profound disruption of the BM microenvironment after IR exposure that leads to a survival and generation of osteoblast constituting the niche (Dominici et al. 2009). These complex events are associated with a megakaryocytes re-location close to the bone lining cell compartment capable to stimulate osteoblasts which are then keen to host transplanted donor HSC. Thus, following irradiation, a hematopoietic cell lineage contributes to both skeletal turnover and to HSC osteoblast niche homeostasis. These results provide critical insights into the mechanisms of recovery of stem cell niches after BM radioablation, and suggest novel means to manipulate the BM microenvironment to promote HSC and bone engraftment.

1 Fraunhofer IGB Stuttgart, Dept of Cell and Tissue Engineering, Nobelstr. 12, 70569 Stuttgart, Germany, steffen.koch@igb.fraunhofer.de 2 Fraunhofer Institute for Physical Measurement Techniques, Freiburg, Germany OP 24 (C006) Detection and Discrimination of cells and cell viability in Tissue Engineering by Raman micro-spectroscopy

Label-free and non-destructive methods for analysing different cell types and cell viability become more and more important in Tissue Engineering and Regenerative Medicine. Tissue Engineering and Regenerative Medicine deal with the support or replacement of damaged cells and tissue, mostly by autologous, primary cell material. Standard procedures in the production process of Tissue Engineering products are viability testings and a quality control at the end of the production process. Aim of this work is to show that Raman micro-spectroscopy can be used as a label-free and non-destructive tool to discriminate primary cells and cell lines as well as cell viability in vitro.

Primary chondrocytes and mesenchymal stem cells were isolated from porcine and human material. The data mining was done with Principal Component Analysis (PCA). For viability tests apoptosis and necrosis were induced by temperature stress. Cell death was verified by Annexin V/PI staining and Caspase activity assays (for apoptosis).

For quality control, Raman spectroscopy can be used for the determination of primary cells in use without labelling and under cell culture conditions.

For the testing of cell viability the Raman spectroscopic approach is promising and exceeds the results for the standard methods by the ability to discriminate between necrotic and apoptotic cell death. It can be shown that vital, necrotic and apoptotic cells are clearly separated in the PCA. Analysis of the loadings allows the picking of important spectral areas for this separation.

1 Maggiore della Carità Hospital, Eastern Piedmont University, Dept of Neurology, Corso Mazzini 18, 28100 Novara, Italy, mazzini.l@libero.it 2 Neuroscience Institute of the Cavalieri Ottolenghi Foundation, Turin, Italy 3 Department of Pediatrics, University of Turin, Italy 4 Stem Cell Transplantation and Cellular Therapy Laboratory, Division of Oncohaematology and Stem Cell Transplantation, ASO OIRM-S.Anna, Turin, Italy OP 28 (B005) Adult Stem cells and neurodegenerative diseases

Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common neurodegenerative disorders. Their etiology is unknown and despite different pathological hallmarks and classical clinical symptoms they all share neuronal loss. Available treatments provide symptomatic relief but none of them change can the course of the disease Stem cell therapy represents tool aimed at cell replacement and neuroprotection.

Mesenchymal Stem Cells (MSCs) are multipotent stem cells that are very attractive in view of a possible cell therapy approach in neurodegenerative diseases because of their great plasticity and their ability to provide the host tissue with growth factors or to modulate the host immune system.

The administration of bone marrow-derived mesenchymal stem cells (BM-MSCs) has led to beneficial effects in animal models for several neurodegenerative diseases. In AD mice BM-MSCs can modulate immune/inflammatory responses, ameliorate their pathophysiology, and improve the cognitive decline associated with Abeta deposits. In PD hMSCs have demonstrated neuroprotective effects on dopaminergic neurons through anti-inflammatory actions by the modulating microglial activation. Expanded MSCs can survive and migrate after transplantation in the lumbar spinal cord of SOD1G93A mice, where they prevent astrogliosis and microglial activation and delay ALS-related decrease in the number of motoneurons.

Encouraging data obtained with stem cells in animal models of neurodegenerative diseases led recently to the first clinical trials transplanting MSCs. We performed a Phase I trial in ALS for the assessment of the feasibility and toxicity of transplantation of autologous MSCs into the spinal cord. The trial was approved and monitored by the Italian Institute of Health and by the Ethics Committees. There was no immediate or delayed transplant related toxicity. Clinical, laboratory, and radiographic evaluations of the patients showed no serious transplant related adverse events also in the long term. Our study demonstrate for the first time the safety of MSC use after focal transplantation in the central nervous system. Therefore, our results represent a starting point for future studies in neurodegenerative diseases. The future for cell therapy is exciting and open a new scenario in the organization of clinical trials in neurodegenerative diseases. Many efforts should be addressed by clinicians to develop new small meaningful phase 1 clinical trial. Rigorously conducted and well managed designed controlled clinical trials might represent an hope for patients and an answer to their needs.

1 Gent University, Polymer Chemistry & Biomaterials Group, Krijgslaan 281 (Building S4), 9000 Gent, Belgium, Peter.Dubruel@UGent.be 2 Johannes Gutenberg University Mainz, Mainz, Germany 3 University of Pavia, Italy OP 33 (D019) Gelatin as promising cell-interactive ECM mimicking biomaterial

Gelatin has previously been widely applied as biomaterial. The interesting properties of this polymer are based on the fact that gelatin is derived from collagen, one of the major constituents of the extra cellular matrix.

In our research group, we have been focusing since many years on gelatin derivatives as cell-interactive biomaterials. The application of gelatin derivatives is required for biomedical applications since the biopolymer as such dissolves at body temperature. Various approaches have been developed in order to enable permanent or reversible cross-linking reactions using respectively (meth)acrylamide or disulfide functionalized gelatins. Starting from these materials, porous scaffolds were developed using an in-house developed cryogenic unit.

The materials developed proved to possess excellent biocompatibility properties as evidenced by cell adhesion/proliferation studies using a variety of cell lines and primary cells (fibroblasts, osteoblasts, endothelial cells, … ).

Moreover, preliminary gene expression studies (using immunofluorescence) indicated that endothelial cells were expressing after seeding on gelatin scaffolds typical genes including PECAM-1 and E-selectin (upon LPS stimulation).

Furthermore, studies in which electromagnetic or ultrasound stimulation of osteoblast seeded gelatin cryogels revealed the potential of this approach. increased the production of collagen, osteopontin, fibronectin, osteocalcin and osteonectin. The latter indicates the stimulation effects in terms of higher cell proliferation and a more pronounced extracellular matrix formation.

At present, we are evaluating in the framework of several national research projects alternative scaffolds production techniques including rapid prototyping and electrospinning for obtaining porous gelatin scaffolds.

1 NMI Natural and Medical Sciences Institute at the University of Tubingen, Regenerative Medicine I, Markwiesenstr. 55, 72770 Reutlingen, Germany, hanna.hartmann@nmi.de 2 NMI Natural and Medical Sciences Institute at the University of Tübingen, Biomaterials, Germany 3 INEB Instituto de Engenharia Biomédica, Divisão de Biomateriais, Universidade do Porto, Portugal OP 36 (D022) siRNA nanoparticles incorporated into polyelectrolyte multilayers for local delivery and controlled release of nanotherapeutics

Objectives: small interfering RNA (siRNA) specifically inhibits the synthesis of designated target proteins and can be harnessed to develop a new class of drugs that interfere with every desired disease-causing protein. To allow therapeutic application of siRNA we produce chitosan/siRNA nanoparticles for immobilization, stabilization and promoting cellular uptake of siRNA. Biodegradable polyelectrolyte multilayers (PEMs) have been explored as sophisticated coatings for controlled drug release and are built up in a layer-by-layer deposition technique, based on the deposition of polycations and polyanions. We aim to use siRNA nanoparticles incorporated in PEMs as a cross-sectional technology for the refinement of implants allowing us to deliver siRNA nanoparticles very locally and to ensure a controlled release.

Methods and Results: Our results show that the chitosan/siRNA particles possessed high stability as judged by gel retardation assays. Particle size and zeta potential were analysed by photon correlation spectroscopy whereby both parameters increased with higher chitosan ratio. PEMs were built up of natural polysaccharides and a regular deposition of PEMs and nanoparticles could be determined by quartz crystal micro balance (QCM) and fluorescence measurements. The constant release of siRNA from PEMs in different solutions was analysed by fluorescence quantification. In cell culture, PEMs containing nanoparticles showed low cytotoxicity as analysed by resazurin assays. The cellular uptake of nanoparticles resulted in a homogenous distribution in the cell cytoplasm as visualised by fluorescence microscopy.

Conclusions: Our results highlight that chitosan/siRNA nanoparticles incorporated in PEMs are an innovative drug delivery tool allowing biological functionalization of structural implants with controlled and sustained release, lower toxicity, and potentially better patient convenience over conventional formulations. Furthermore, it offers the possibility to control the location of nanoparticles with nanometer-scale precision and the amount of incorporated nanotherapeutics can simply be varied by the number of layers. Our aim for the future is to use this kind of biological refinement of implants as a cross-sectional technology, to bind siRNA as a bioactive molecule to a variety of medical implants and thereby to foster clinical therapy and regeneration.

Partly supported by BMBF, Germany.

1 Natural and Medical Sciences Institute at the University of Tuebingen, Regenerative Medicine I, Markwiesenstrasse 55, 72770 Reutlingen, Germany, beate.scholz@nmi.de 2 TETEC Tissue Engineering Technologies AG, Reutlingen, Germany 3 Cellendes GmbH, Tuebingen, Germany OP 37 (D015) Prevention of adverse angiogenesis by a novel albumin-based hydrogel for articular cartilage and intervertebral disc regeneration

Healthy mature articular cartilage and intervertebral discs (IVD) are devoid of blood vessels. However, blood vessels appear during pathophysiological conditions resulting in augmented degeneration and innervation by sensory neurons in both these tissues. Furthermore, in articular cartilage angiogenesis leads to the formation of bony outgrowths called osteophytes. We propose the adverse effects of angiogenesis under pathophysiological conditions should be considered when designing an optimized biomaterial for articular cartilage and IVD regeneration. Therefore, the purpose of the work was to evaluate an injectable polyethylene glycol-crosslinked albumin gel (AG) supplemented with hyaluronic acid as a matrix for autologous chondrocyte implantation with regard to its angiogenic properties.

In contrast to human endothelial cells, which were cultured on top of the AG, primary human articular chondrocytes encapsulated within the AG retained their viability. The AG did not release any diffusible toxic components. Furthermore, endothelial cells cultured on top of the AG did not adhere to the gel surface to a significant extent nor did they proliferate. Contrary to Matrigel, employed as positive control, the AG prevented endothelial chemoinvasion in transwell filter assays even in the presence of a chemotactic gradient of vascular endothelial growth factor (VEGF).

In the chick chorioallantoic membrane (CAM) assay, histochemical and immunohistochemical methods revealed a barrier function of the AG for blood vessels from the CAM, even when VEGF or VEGF-producing articular chondrocytes were encapsulated within the AG. These results could be confirmed by subcutaneous implantation of human IVD chondrocytes enclosed in the AG into immunodeficient mice. Whereas blood vessels could be found in the subcutaneous tissue adjacent to the AG, the AG itself remained devoid of blood vessels.

In summary, aiming at articular cartilage and IVD regeneration the albumin gel promises to be a beneficial implant matrix for chondrocytes simultaneously exhibiting non-permissive properties for adverse endothelial cells.

The studies were approved by the local ethics committee and informed consent was obtained from all individuals participating in the study. All animal experiments were approved by the Committee on Animal Care of the State of Baden-Wurttemberg (Germany).

1 Fraunhofer Institut für Grenzflächen- und Bioverfahrenstechnik, Zellsysteme, NobelstraÕe 12, 70569 Stuttgart, Germany, petra.kluger@igb.fraunhofer.de 2 Fraunhofer Institute for Production Technology IPT, Aachen, Germany 3 Fraunhofer-Institute for Biomedical Engineering IBMT, St. Ingbert, Germany OP 38 (D020) Development of high volume producible nano- and microstructured surfaces

Introduction: The investigation of nano- and microstructured surfaces is an emerging and important field in biomaterial research. Modified interfaces can be used to simulate tissue-like patterns. These altered materials are able to enhance cell proliferation and to identify structures that improve cell adhesion. Three dimensional structures are essential for the functional cultivation of primary cells and the optimization of implant interfaces. Structured surfaces were developed, produced and in cell cultivation experiments systematically analyzed for their applicability with different cell types.

Materials and Methods: All investigated surface structures were manufactured utilizing a process chain including mould making through micro machining or high volume replication using hot embossing and injection moulding. Concurrent with the ongoing structuring development, cell cultivation experiments have been conducted using polymeric test plates featuring interfaces of different nano- and microstructured surfaces. Investigated cells were primary human keratinocytes, fibroblasts, endothelial cells, Saos2 and SW-1353 cell line. The primary cells were isolated from human foreskin. Adhesion, morphology, proliferation and functionality of the different cell types were controlled by light-microscopy methods. The cells analyzed by cell proliferation tests and immunofluorescent stainings.

Results and Discussion: Using a new efficient process chain, we were able to significantly upscale the production of nano- and microstructured substrates. Topographies included parallel grooves of various cross sections and pitches, as well as knob-like surface textures of different geometries and spatial distributions. On a 4 cm x 4 cm substrate, 64 different surface structures could be screened. In extensive in vitro culture experiments, we analyzed the influence of these substrates on a variety of primary cells. We assessed cell cytoskeleton and adhesion profiles and found indication of various cell-substrate interactions. We further identified that the cells were able to migrate and proliferation, showing characteristic cell morphologies as well as alignment patterns.

Conclusion: Influence of topography on cellular behavior is essential for the improvement of biomaterials. Here we showed that structured surfaces can be manufactured using high volume production methods. These structured substrates are useful tools for screening and identifying cell-material interactions.

1 University Clinic of Tuebingen, Department of Internal Medicine II, Division of Hematology, Oncology, Immunology, Rheumatology, and Pneumology, Laboratory for Stem Cell Research, Bettenbau west, Ebene 2, Raum 580, Otfried-Müller-Str. 10, 72076 Tübingen, Germany, kavitha212@yahoo.com 2 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada PP 03 (F004) Novel Monoclonal Antibodies for the Isolation and Characterization of Bone Marrow Derived Mesenchymal Stem/Stromal Cells

Multipotent Mesenchymal Stem/Stromal Cells (MSCs) are attractive candidate cells for repair or regeneration of damaged tissues of mesenchymal origin. Conventionally, MSCs are functionally isolated by their property to adhere to the plastic surface in culture dishes. To gain more insight into the starting population, monoclonal antibodies (mAbs) were generated against the induced Pluripotent Stem Cell line iPS122 [Woltjen K et al.; Nature.2009;458, 766–770] and screened by flow cytometry for their selective recognition of CD271bright bone marrow (BM) MSCs as well as for their reactivity with cultured MSCs. One mAb selectively recognized primary CD271bright BM cells but not cultured MSCs. Other two mAbs did not react with CD271bright primary MSCs but detected surface antigens expressed on cultured MSCs. In contrast, three mAbs were less selective and reacted with both primary CD271bright BM cells and cultured MSCs. In conclusion, we generated new markers suitable for the prospective isolation of highly purified BM-MSCs as well as for the characterization of their cultured counterparts. These antibodies may be suitable tools for the isolation of MSCs and may be attractive markers in the field of regenerative medicine.

1 Department of Pediatric Surgery, University Children's Hospital Tuebingen, Hoppe-Seyler-Str.3, 72076 Tuebingen, Germany, heike.dettmann@med.uni-tuebingen.de 2 Institute of Anatomy, Center for Regenerative Biology and Medicine, University of Tuebingen, Tuebingen, Germany PP 06 (F008) Implantation of enteric nervous progenitors in the mouse model

Background: Hirschsprung's disease is characterized by the lack of ganglion cells in the distal colon. It occures in one of 5000 children and causes spastic obstruction of the affected segment. Although resection of the aganglionic bowel and colo-anal anastomosis enables stool passage, functional outcome of surgery is associated with functional problems in many patients.

Enteric nervous system (ENS) progenitor cells have been proposed as cell source for a cell based regenerative approach for Hirschsprung's disease.

Material and Methods: Murine (ENS) progenitor cells were isolated from postnatal gut of eGFP mice. After proliferation in vitro cells were either implanted into the rectum of NOD-SCIDIL2R-/IL2R mice in an undifferentiated state or after prior differentiation on a collagen cell carrier. Implanted cells were characterized by immunohistochemistry.

Results: Murine ENS progenitor cells could be isolated, proliferated and differentiated into neural and glial phenotypes from postnatal murine bowel. Implanted cells were viable for more than 12 weeks. Both, differentiated and undifferentiated cells maintained a constant neuronal phenotype after implantation into the mouse rectum.

Discussion: Implantation of enteric progenitor cells shows the potential of the isolated cells for a cell based therapeutic approach in neurogastrointestinal disorders. However, functionality and integration of the cells needs to be further characterized.

1 ZRM, Waldhörnlestrasse 22, 72072 Tübingen, Germany, seeyouchristine@web.de 2 Universtiy of Tübingen Medical Center, Tübingen, Germany 3 BG Center for Traumatology, Tübingen, Germany PP 08 (F011) The osteogenic differentiation of mesenchymal stromal cells correlates with their expression of CD146

Objectives: Mesenchymal stem cells (MSC) can be isolated in large quantities from bone marrow (bmMSC) or placenta (pMSC). The characteristics of MSC include plastic adherent growth, the expression of defined surface antigens and lack of others, and the differentiation capacity in multiple lineages. We observed that pMSC do not differentiate in osteoblast efficiently compared to bmMSC. Furthermore bmMSC express CD146 at pronounced intensity. Here we investigated if pMSC express CD146 and if this correlates with their differentiation capacities.

Methods: The pMSC were isolated from human term placenta and enriched via Percoll^® gradient centrifugation. The cells were cultured after isolation in GMP-conform media. Non-adherent cells were discarded. Plastic-adherent cells were characterized by their differentiation capacity into the osteogenic and adipogenic lingeages, their expression of characteristic surface antigens including CD73, CD90, CD105, and lack of CD14, CD34, CD45. Subsets of pMSC with different expression levels of CD146 were separated utilizing the MACS-technology. The osteogenic differentiation capacity of the CD146low and the CD146high fraction was investigated, and success of differentiation was studied by histochemistry.

Results: The pMSCs show a wide range of CD146 expression. The MACS-sorted CD146low-pMSC display a weak osteogentic differentation capacity, whereas the MACS-sorted CD146high-subset generated osteoblasts as visualized by strong von-Kossa staining.

Conclusion: The expression level of CD146 on MSC correlated with the ostegenic differentiation potential of the subsets. BmMSC express high amounts of CD146 and can be differentiated into osteoblast efficiently. High expression of CD146 by pMSCs correlated with a good osteogenic differentiation. Therefore, the CD146low-faction with it's weaker osteogenic potential may be utilized in applications were osteogenesis is undesirable.

This project was supported in part by grants from the BMBF.

1 Fraunhofer Institute for Interfacial Engineering and Biotechnology, Nobelstrasse 12, 70569 Stuttgart, Germany, Svenja.Hinderer@igb.fraunhofer.de 2 University of Würzburg, Würzburg, Germany PP 11 (F014) Angiogenetic structures in a 3-dimensional dynamic cultivation system

Introduction: Inducing angiogenesis in vitro is important to provide an adequate nutrition supply for cells in thicker tissues. Permanent shear stress causes up regulation of angiogenetic factors like VEGF (Vascular Endothelial Growth Factor) and is therefore an important parameter for vessel maturation. For this reason a dynamic 3D-test system represents a promising tool for inducing angiogenesis.

Materials and Methods: At the Fraunhofer IGB a bioreactor system has been established permitting a dynamic endothelial cell cultivation within a tubular structure in a collagen matrix. Investigated cells were human primary microvascular endothelial cells (mvEC) isolated from human foreskin. The morphology, proliferation and functionality of the cells were verified after six days of dynamic cultivation. Simultaneous to the bioreactor system a static experiment under the same conditions like 37° C and 5% CO₂ was realized. The specific endothelial cell markers CD31 and vWF (von Willebrand Factor) were tested by using immunohistochemical methods. Furthermore the definition of cell vitality worked out with a fluorescent life-death-staining. Sampling the cultivating medium every day enables a VEGF-ELISA for characterizing the cells and their angiogenetic activity.

Results: The cultivation of endothelial cells within a tubular structure in a collagen matrix under dynamic conditions was successful. First of all the cells proliferate und build a monolayer on the capillary surface. After six days, shear stress induced endothelial cell migration and formation of angiogenetic structures could be observed. A fluorescent staining of the endothelial cells within the collagen showed long chains of migrated cells based on the main capillary. Additionally this effect could be proofed by an increase of VEGF during the cultivation period. There was no VEGF detectable in the static test. The life-death-staining showed around 90% viability of the used cells. The endothelial cell markers CD31 and vWF could be visualized by immunohistochemical staining and light-microscopy methods.

Conclusion: Finally, the three-dimensional dynamic bioreactor system is a suitable tool for angiogenesis induction. Besides shear stress growth factors also play a crucial role in the angiogenesis process. Therefore future studies will include experiments with the application of growth factors like VEGF achieving directed angiogenetic structures.

1 Medical University Graz, Institute of Cell Biology, Histology and Embryology, Harrachgasse 21/7, 8010 Graz, Austria, karin.pekovits@medunigraz.at 2 Medical University Graz, Department of Dentistry and Maxillofacial Surgery, Graz, Austria PP 12 (F015) Different autogenous bone harvesting techniques: Influence on chip morphology, cell viability and differentiation status

Objectives: Autogenous bone is still regarded as the gold standard grafting material. Different harvesting methods for autogenous bone chips have been developed for bony augmentation in oral and maxillofacial surgery. The aim of the present study was to investigate the influence of harvesting techniques on chip morphology, viability and differentiation status of the outgrowing cells.

Material and Methods: A series of 40 bone specimens were obtained by either a manual bone scraper (MS) or a piezoelectric device (PD) during surgical removal of the third molar in the mandible. Morphology of the intra-orally harvested bone chips and the outgrowing cells were examined by scanning electron microscopy. A cytochemical test (alkaline phosphatase activity) and immunocytochemical stainings (osteocalcin, collagen type I, parathyroid hormone receptor, CD90, CD73, CD105, HLA-DR, CD34, CD45, STRO-1) as well as RT-PCR phenotyping were performed to determine cell viability and differentiation status.

Results: Regarding the weight of the bone chips, both harvesting methods showed equally successful results (two-sided paired t-test, p < 0.05). Scanning electron microscopy revealed a smooth bony surface using the PD, whereas a rougher ultrastructure was observed using the MS. In 90% of the MS and 80% of the PD samples, an outgrowth of adherent cells from the bone particles was detected after 7–18 days. RT-PCR analysis and staining with different monoclonal antibodies identified the cells as -osteoprogenitors.

Conclusions: The piezoelectric device and the manual bone scraper gave comparable results concerning the viability and differentiation status of the outgrowing cells. Both harvesting techniques are capable of collecting adequate amounts of bone chips and are equally useful for harvesting of autogenous bone for regenerative procedures in the clinical routine.

1 EMC microcollections GmbH, Combinatorial Chemistry, Sindelfinger Str. 3, 72070 Tuebingen, Germany, eickhoff@microcollections.de 2 Department of, Head & Neck Surgery, University Hospital of Tübingen, Tübingen, Germany PP 14 (F017) Control of regenerative biology at the inner ear by small molecules

Restrictions in the ability of hearing and thus to communicate affect profoundly the quality of life in nearly all vocational and social fields. Therefore defective hearing, affecting ten percent of the population of developed nations, is one of the main problems of health care in societies which are dependent on communication. The costs for the national economies caused by untreated defective hearing are presently estimated to amount to more than 75 billion Euros per year in Europe. In many cases, the reasons for defective hearing are related to the loss of sensory cells in the inner ear through ageing or loud noise. Currently, the only treatment offered is the use of hearing aids with often unsatisfactory results for the user. No curative therapy is available.

The aim of this project was the identification of low molecular weight substances which effect in the Corti's Organ a dedifferentiation to multipotent progenitor cells of the inner ear and lead thereby to restoration of hearing through de novo formation of sensory cells.

A set of 50 compounds from EMC's proprietary collection of 20,000 low molecular weight heterocyclic compounds has been selected by computational methods and has been screened in in-vitro model systems. A hit compound has been structurally optimized and tested in in-vivo models. The elucidation of the biological targets is still ongoing.

1 Fraunhofer IGB, Stuttgart, Nobelstr. 12, 70569 Stuttgart, Germany, dto@igb.fhg.de 2 Tierärztliche Hochschule Hannover, Hannover, Germany PP 16 (F019) In vitro culture of native jejunal segments for use as 3D intestinal testing system

Background: If new drug candidates are suitable for oral intake depends on their characteristics regarding their effectiveness, quality and safety, which have to be proven in preclinical trials.

Due to species specific differences, e.g. in metabolic processes, in vivo animal experiments, today's most precise way of developing intestinal uptake, can lead to inaccurate test results. Furthermore these experiments are ethically very critical.

Therefore it is still a need to establish new in vitro methods, which allow a better analysis of absorption processes at the intestinal barrier, preferably independent of species specific differences.

At the Fraunhofer IGB we developed a long time culture method for jejunal segments, which can be used as test system for evaluation of drugs and other substances regarding their oral bioavailability.

Methods: A segment of porcine or rat jejunum is explanted and transferred into a bioreactor system. During culture time it is perfused both through its blood vessel system and through its lumen.

After 6–10 days of culture the segment is compared to freshly explanted jejunal sements regarding cell vitality and functionality. For this purpose both undergo vitality tests and are characterized by histological and immunohistological (e.g. E-cadherin, CK AE, SMA, PCNA) staining. Comparative absorption studies for evaluation of their barrier function are in progress.

Results: Due to our specialized bioreactor module and our high supplemented media composition it was possible to cultivate native porcine and rat jejunal segments in vitro.

Histological and immunohistological staining showed markers of typical cell types of small intestine. These jejunal segments showed enhanced reepithelialisation of enterocytes over culture period. Undirected, but clearly recognizable peristaltic was shown up to 10 days in culture.

Perspectives: Our test system has to be evaluated by the use of different substances reflecting typical absorption mechanisms of the intestinal barrier, e.g. high and low permeable and efflux transit.

Also influences of chemicals and environmental parameters like temperature and pH on peristaltic in vitro could be tested in future.

A further interesting perspective is to culture jejunal segments of other species, especially human.

If the intestinal function in vitro is maintained comparable to the in vivo situation of the jejunum, this could outmatch and replace a lot of animal experiments.

1 Universitätsklinikum Tübingen, Innere Medizin III, Otfried-Müller-Str. 10, 72076 Tübingen, Germany, melanie.ziegler@med.uni-tuebingen.de 2 NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany PP 17 (F020) Bifunctional recombinant protein SDF-1-GPVI as a new therapeutic concept for improved vascular regeneration

Vascular regeneration after severe cardiovascular diseases such as myocardial infarction or myocarditis strongly depends on the recruitment and accumulation of hematopoietic progenitor cells. The interaction of platelets with these progenitors cells plays an important role in this process and is predominantly influenced by the platelet specific chemokine “stromal cell derived factor-1” (SDF-1) and its receptors CXCR4 and CXCR7. To improve the regeneration and healing process and therefore the patient's prognosis it is necessary to develop new biopharmaceutical therapies. Bifunctional proteins attract stem cells and accumulate them in the bone marrow or at damaged endothelial areas to enhance the healing process. For this reason we developed the novel bifunctional protein SDF-1-GPVI. This protein consists of the glycoprotein VI (GPVI) receptor and the chemokine “stromal cell derived factor-1” (SDF-1). GPVI serves as an anchor structure to bind to collagen which becomes exposed to the blood stream only after damage of the endothelium. The chemoattractants SDF-1 serves as the functional unit important for the progenitor cell attraction to the damaged endothelium.

ELISA studies proved that SDF-1-GPVI binds to the major collagen type I. FACS competition assays, chemotaxis assays and flow chamber adhesion experiments demonstrated the bifunctionality of the protein and the specific binding of SDF-1 to the CXCR4 receptor. Furthermore, colony forming studies provide evidence that SDF-1-GPVI coating improved the differentiation of CD34 + -cells to endothelial cells. In addition, the “chorioallantois membrane” (CAM) assays in chicken egg showed that SDF-1-GPVI has pro-angiogenic effects. Moreover, several ongoing in vivo studies show promising results indicating improved regeneration in SDF-1-GPVI treated mice.

Taken together the bifunctional protein SDF-1-GPVI could serve as a promising biopharmaceutical therapy concept to improve vascular regeneration and healing processes of damaged endothelium after cardiovascular diseases such as myocardial infarction.

1 NMI, Natural and Medical Sciences Institute at the University of Tuebingen, Regenerative Medicine I, Markwiesenstrasse 55, 72770 Reutlingen, Germany, yvonne.baumer@nmi.de 2 University Clinic of Tübingen, Department of Internal Medicine III, Tübingen, Germany 3 University Clinic of Tübingen, Department of Internal Medicine II, Tübingen, Germany PP 19 (F022) aCD133-GPVI: A bifunctional protein for improved stent performance and endothelial regeneration

Objectives: Restenosis and thrombosis are side effects after stent implantation associated with reduced or missing re-endothelialization of injured coronary arteries. To counteract this deleterious development we aim to improve local stem cell homing at the site of injury in order to support their differentiation into intact endothelium. We developed a bifunctional protein that binds to collagen presented at the lesion site via glycoprotein VI (GPVI)-binding domain and to stem/progenitor cells using aCD133-binding site.

Methods and Results: ELISA studies demonstrated that the aCD133-GPVI bifunctional protein binds to collagen type I comparably strong as the control GPVI-protein. Employing CD133-overexpressing cells and human CD34 + endothelial progenitor cells on a multi substrate array (MSA) spotted with 14 different extracellular matrix proteins, the bifunctionality and specificity of the aCD133-GPVI protein could be demonstrated. This specificity was substantiated in flow chamber adhesion experiments. Furthermore, we could detect endothelial like differentiation of CD34 + -cells on fusion protein coated surfaces by testing the colony forming ability and subsequent endothelial marker expression. Moreover, ongoing in vivo studies using intravital microscopy and a myocardial infarction model aim to evaluate the regenerative potential in aCD133-GPVI treated mice.

Conclusion: In conclusion, the aCD133-GPVI bifunctional protein is a promising construct to support re-endothelialization and therefore, reducing restenosis and thrombosis risk after stent implantation.

Supported by the German Ministry of Education and Research (BMBF) # 01GU0727 and 01GU0726.

1 Universidad de Granada, Histology, Avenida de Madrid 11, 10012 Granada, Spain, acampos@ugr.es 2 Hospital Universitario de Granada, Granada, Spain PP 20 (F023) Ex vivo epidermal maturation and differentiation in a bioengineered model of full-thickness human skin

Introduction: Several models of bioengineered human skin have been developed to the date using primary cell cultures and biomaterials. However, very few biomaterials are able to support and induce the efficient development and maturation of the bioengineered epidermis. In this work, we have generated an artificial substitute of the human skin using fibrin-agarose biomaterials and evaluated the level of differentiation of the epidermal layer.

Materials and Methods: First, we generated primary cell cultures of human keratinocytes and fibroblasts using small biopsies of healthy human skin. Then, an artificial dermis substitute was developed in the laboratory using fibrin-agarose biomaterials with human dermal fibroblasts subcultured within and epidermal keratinocytes on top. Air-liquid technique was used for 3 weeks. Immunofluorescence for pancytokeratin, filaggrin and involucrin was carried out to determine the differentiation status of the bioengineered epidermis.

Results and Discussion: Histological analysis of the artificial skin revealed the presence of a dermal substitute with numerous fibroblasts immersed in the fibrin-agarose mesh. In addition, an artificial epithelium with 7–8 cell layers was identified ex vivo, although the typical layers of the differentiated epithelium and the epithelial rete ridges of the normal skin were not found ex vivo. All the epithelium layers expressed pancytokeratin, filaggrin and involucrin, suggesting that these epidermal cells could be functional at this stage.

Conclusions: Our results suggest that the fibrin-agarose artificial skin model shows low levels of epithelial differentiation ex vivo. However, these cells are able to express some markers of mature epithelium from the first weeks of development.

Supported by GIT-CTS115.

1 University Clinic of Tübingen, Department of Internal Medicine II, Division of Hematology, Oncology, Immunology, Rheumatology, and Pulmonology, Otfried Müller Str. 10, 72076 Tübingen, Germany, m.sobiesiak@gmx.de 2 NMI Reutlingen, Regenerative Medicine II, Reutlingen, Germany 3 TETEC AG, Reutlingen, Germany PP 21 (F024) Human Bone Marrow CD271brightW5C5 + CD56 + Cells are Enriched for Progenitors with Increased Chondrocyte Differentiation Potential

Recently, we identified two morphologically distinct subpopulations in the human bone marrow (BM) that differ in their clonogenic potential and differentiation capacity. CD271brightTNAPdimCD56 + mesenchymal stromal cells (MSC) preferentially differentiated into chondrocytes and CD271brightTNAPbrightCD56- MSC exclusively differentiated into adipocytes. In this work we describe the antibody-defined W5C5 antigen as a novel and selective MSC marker. Clonogenic assays revealed that sorted CD271brightW5C5 + but not CD271brightW5C5- BM cells give rise to CFU-F. About 13 ± 4% of W5C5 + MSC coexpress the neural cell adhesion molecule CD56. Similar to CD271brightTNAPdimCD56 + MSC, CD271brightW5C5 + CD56 + MSC show a 2–3 fold increased CFU-F frequency compared to CD271brightW5C5 + CD56- cells. Upon differentiation into the chondrogenic lineage, CD56 + derived MSC showed increased mRNA expression of collagen type II, collagen type X and aggrecan compared to CD56- derived MSC. In addition, histochemical staining of chondrocytes derived from the CD56 + subset showed deposition of extracellular matrix proteins such as collagen type II and proteoglycans. In contrast, CD56- -derived chondrocytes did not show matrix deposition. The data show that effective chondrogenesis is preferentially induced in MSC derived from the CD271brightW5C5 + CD56 + subset. In conclusion, we demonstrate that chondrocyte progenitors are enriched in the CD271brightW5C5 + CD56 + BM subset. This population may therefore serve as an attractive source for autologous chondrocyte transplantation and treatment of spinal disc injuries.

1 IdiPaz, Research, Castellana 261, 28046 Madrid, Spain, montecristo2007@mixmail.com 2 Biochemistry Unit. IdiPaz, Spain 3 Experimental Surgery Dept. IdiPaz, Spain 4 Donostia Hospital, Spain 5 CSIC, Spain PP 22 (F025) Progranuline promotes repair in cortical neurons in vitro under excitotocic conditions and its haploinsuficency increases soluble fractalkine levels in human with frontotemporal dementia

Progranulin (PGRN) is a widely glycosylated protein that is expressed in neurons and microglia. Mutations in PGRN casue autosomal dominant tau-negative frontotemporal lobe dementia –FTLD- in humans that leds to haploinsufficency. In the present work, we analyzed whether plasma progranuline levels and soluble fractalkine (levels in plasma) could be a systemic marker of FTD in patients with PGRN haploinsuficency.

We also evaluated glutamate levels by HPLC and several other standard biochemical markers including proinflammatory Stromal Cell Derivate Factor 1 Alpha (SDF1 Alpha) as well as fractalkine levels in plasma from FTD patients. Additionaly, we analyzed whether PGRN might induce PSA-NCAM in excytotoxic conditions in cortical neurons 7 DIV (Days “in vitro”).

We found that fractalkine soluble release may be useful systemic marker of frontotemporal dementia (FTD) in plasma samples with PGRN (Progranuline) haploinsufficency. In addition, we detected a strong correlation between glutamate and soluble fractalkine levels in plasma and reduced Acetylcholinesterase activity in plasma from FTD patients. On the other hand, we also reported for the first time that progranuline induces neurite length and upregulates Neural Cell Adhesion Molecule in glutamate cortical neurons treated at 7 DIV –Days in Vitro- under excitotoxic conditions. In conclusión, new pharmacological approaches that amilorate fractalkine release could delay clinic manifestations of systemic damage in clinical models of human frontotemporal dementia. Additionaly, Neural Cell Adhesion Molecule is a target of reparative effects of Porgranuline in neurons under excitotoxic conditions.

1 Danube University Krems, Center for Regenerative Medicine, Dr.-Karl-Dorrek-StraÕe 30, 3500 Krems, Austria, manuela.berger@donau-uni.ac.at 2 Croma Pharma GmbH, Leobendorf, Austria PP 23 (F026) Cross-linked hyaluronic acid as a scaffold for the treatment of cartilage defects

Due to its avascular nature, articular cartilage exhibits rather low intrinsic repair capacity. Current strategies for the treatment of cartilage defects are either based on the transplantation of autologous chondrocytes or mesenchymal stem cells seeded on biomaterials (matrix-associated autologous chondrocyte transplantation - MACT) or the implantation of an “empty” scaffold combined with marrow-stimulating techniques (e.g. microfracture). Employed biomaterials have to feature good biocompatibility and biodegradability, allow for proper cell adhesion and promote the establishment and stabilization of a chondrocytic phenotype of cells.

We examined the suitability of a porous three-dimensional matrix of cross-linked hyaluronic acid as a scaffold for cartilage regeneration. Scaffolds of different pore-sizes were produced by varying the extent of cross-linking and evaluated by electron microscopy. Dedifferentiated human articular chondrocytes were cultured in the scaffold with a pore-size of 40 to 100 μm for up to three weeks and metabolic activity and the expression of chondrocyte-specific genes were analyzed. Cells were retained well and showed homogenous distribution within the matrix. However, metabolic activity decreased during cultivation which might be attributed to the loss of cells due to matrix degradation or to a stop in cell proliferation. The latter might reflect a switch of the cellular genetic program from cell division to differentiation towards a chondrogenic phenotype indicated by the increased expression of chondrogenic markers (e.g. collagen 2). These results suggest that the investigated material has a chondroinductive effect on embedded cells.

1 Institute for Interfacial Engineering, Medical Interfacial Engineering, University of Stuttgart, Nobelstr. 12, 70569 Stuttgart, Germany, florian.groeber@igvt.uni-stuttgart.de 2 Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany PP 24 (E002) Development of a vascularised skin equivalent

Introduction: Due to the lack of an analogue for the dermal vascular system, current skin equivalents cannot be used to test the capacity of a given substance to penetrate through the skin into the bloodstream. The integration of such a vascular system into a skin equivalent would however amplify the possible applications in research fields like toxicity and efficacy testing, by providing a model for the critical barrier between the skin and the vascular system. Apart from that a vascularised skin equivalent can be used as an in vitro model for the investigation of basic processes in cancer research such as tumor angiogenesis and metastasis.

Objective: The objective of this study was to integrate an established and according to DIN ISO EN 10993-5 accredited skin equivalent into a natural vascularised matrix, based on an acellularised part of a porcine jejunum. The natural, vascularised matrix (BioVaSc) could already be used for the formation of renal and liver tissue and was successfully implanted into a patient as a trachea patch.

Results: After seeding of primary human keratinozytes and human fibroblasts on the vascularised matrix the construct was cultivated under submersed conditions for seven days and under air lift conditions for 12 days. Histological staining with hemalaun and eosin revealed a multi cellular layer of keratinozytes with a typical corneous layer on one side and adherent fibroblast on the other side.

Conclusion: In this work we could show that the natural vascularised matrix inoculated with human keratinozytes and fibroblasts was able to facilitate the formation of a functional skin equivalent.

1 University of Applied Sciences Esslingen, Biotechnologie, KanalstraÕe 33, 73728 Esslingen, Germany, Mareike.Schultze@hs-esslingen.de 2 nanoAnalytics GmbH, Münster 3 Institute of Pathology, University Hospital, Tübingen PP 25 (E003) Continuous survey of Calu-3 cells that differentiate in transfiltersystems

Cancer cell lines normally have lost their differentiation state in order to proliferate continuously. Therefore they are used as cell lines. Some cancer cell lines are able to redifferentiate in vitro and form epithelial barriers, like the colon cell line CaCo-2.

Here we report on a human airway epithelial cell line, Calu-3, cultured in transfiltersystems to build up a bronchoepithelial cell layer. The formation of the cellular barrier was monitored either by the measurement of transepithelial electrical resistance (TEER) day to day manually with EndOhm Voltohmmeter technology or continuously with automated impedance spectroscopy by cellZscope for two weeks. Finally the cell layers were prepared for transmission and light microscopy analysis.

Continuous measurement of impedance displayed several advantages: Monitoring from outside the incubation chamber, cultures could remain in the incubator and differentiation was not disturbed by a temperature and pH-shift. When cells had to be removed from the incubator for individual TEER measurements by EndOhm electrode a considerable decline of TEER was reported after each reading point. In addition the EndOhm was time-consuming.

After 4 days in culture, cells in cellZscope apparatus displayed TEER values of over 250 Ohm per cm² and differentiation was confirmed finally by morphological criteria. Cells formed a tight barrier of polarised cells with apical cilia. Tight junctions were formed between adjacent cells forming a tight junction belt visible in freeze-fracture preparations.

Incubation of differentiated cell layers with toxic samples or inflammation inducing agents was monitored and displayed more precise data with cellZscope compared to EndOhm.

These differentiated epithelia can now be combined with other cell lines in the basal compartment of the transfiltersystems to coculture assays for cytotoxicity testing.

1 University of Tuebingen, Center of Regenerative Medicine, WaldhörnlestraÕe 22, 72072 Tübingen, Germany, miriam.rothdiener@uni-tuebingen.de 2 BG-Trauma Center, Tübingen, Germany PP 26 (E004) Evaluation of Proliferation and Synthesis of osteoarthritic Chondrocytes with and without pericellular Matrix

The pericellular matrix (PCM) surrounding chondrocytes is responsible for structural and functional connections of the cell and its extracellular matrix (ECM) in articular cartilage. The PCM is considered to affect proliferation, maintenance of chondrocyte phenotype and matrix synthesis. The aim of this project is the evaluation of chondrocytes with intact PCM for biological therapy in osteoarthritic (OA) patients. The objectives of the current studies were to compare the proliferation of OA chondrocytes in monolayer culture and three-dimensional peptide hydrogel culture as well as expression and synthesis of matrix molecules in cells with and without PCM.

Chondrocytes with and without intact PCM were isolated enzymatically from articular knee cartilage of OA patients. The presence of functional important matrix components in intact PCM was demonstrated and gene expression analysis revealed a higher expression of matrix components and chondrogenic markers in chondrocytes with intact PCM immediately after isolation. In monolayer culture, the proliferation of chondrocytes was vastly superior with intact PCM. However, there was a loss of the spherical chondrocyte phenotype in cells with and without PCM in monolayer culture. In contrast, in peptide hydrogel culture chondrocytes maintained their typical phenotype. A higher cell proliferation and higher cell viability of chondrocytes with intact PCM in comparison to chondrocytes without PCM was observed in peptide hydrogel culture. In addition, after six weeks of cultivation in peptide hydrogel, chondrocytes with intact PCM had a higher matrix expression and synthesis compared to chondrocytes without intact PCM in hydrogel culture and to chondrocytes with intact PCM in monolayer culture.

These findings corroborate that the presence of PCM has an important influence on the metabolism of cultured chondrocytes.

1 University of Tuebingen, Center of Regenerative Medicine, WaldhörnlestraÕe 22, 72072 Tübingen, Germany, tino.felka@uni-tuebingen.de 2 BG-Trauma Center, Tübingen, Germany PP 27 (E005) Improvement of autologous chondrocyte transplantation by biomechanical stimulation

Biological reconstruction of articular cartilage utilizing autologous chondrocytes is applied nowadays mainly to younger patients with small defects. The amount of tissue used to isolate cells and therefore the number of chondrocytes is limited. In consequence, the expansion of chondrocytes maintaining their characteristic properties is limited as well, and may result in lower size, lower cell density or lower synthesis ability of in vitro generated implants.

The aim of this study is to develop a biomechanical stimulation procedure using the existing regenerative potential of chondrocytes more efficiently, and wide the spectra of applications of in vitro generated implants by varying their functional and structural characteristics.

Biomechanical stimulation with ElectroForce^® BioDynamic^® Test Instrument (Bose, Minnesota) will be used to enhance cell proliferation. In this procedure, chondrocytes seeded in a clinicaly established scaffold (Matricart^®, JoTech Hechingen) will be treated with several biomechanical extension protocols with different frequencies, amplitude and duration of time. In addition to the extension procedures, dynamic compression of cultured chondrcytes will also be applied to mimic physiological biomechanical stimuli, which lead to a stimulation of chondrocyte metabolism. Subsequent synthesis and deposition of proteins and proteoglycans have been shown to improve material properties of carrier systems like peptide hydrogels. A biomechanical loading machine (MIT, Boston) will be used to perform dynamic compression with varying parameters and collect computerized high-precision data. The verification of the formation of a mechanical resistant, hyaline-like extracellular matrix (ECM) is part of this study.

Biomechanical extension and dynamic compression can be combined to enhance both chondrocyte proliferation and matrix synthesis. In the future, biological reconstruction therapies of articular cartilage could be applicable to older patients or patients with bigger or more than one cartilage defects.

1 University of Applied Sciences Esslingen, Biotechnologie, KanalstraÕe 33,73728 Esslingen, Germany, bettina.weiss@hs-esslingen.de 2 Institute of Pathology, University Hospital, Tübingen, Germany PP 29 (E007) Cocultures of bronchoepithelial cells and immune cells in transfiltersystems to test nanoparticles

Calu-3 cells from bronchoepithelial carcinoma cells form tight barriers in transfilter systems. This differentiation was monitored by physical methods like TEER (transepithelial electrical resistance) measurement or impedance measurement (cellZscope), by morphological criteria (tight junction detection with electron microscopy or adherens proteins with fluorescence staining) and functional tests like transport studies of fluorescence beads. Differentiated Calu-3 cells grown in transfilter systems were combined with immune cells of the THP-1 cell line.

Titanium dioxide and silicium dioxide nanoparticles at different sizes and concentrations were applied to the apical compartment containing differentiated Calu-3 cells for 24 hours. In the lower compartment THP-1 cells were cultured simultaneously.

Supernatants of Calu-3 and THP-1 cells were collected separately and tested for cytokine expression (MCP-1 and interleukin-8). Finally Calu-3 cells have been prepared for morphological investigations with transmission and scanning electron microscopy or fluorescence microscopy.

Transepithelial transport was affected by the membrane type of the insert filters at a pore size of 0.4 μm. Nanoparticles with 50 nm diameters were incubated at four concentrations for 24 hours with Calu-3 cells in the cocultures. Supernatants of Calu-3 cells and THP-1 cells in the lower compartments were collected. Probes did not affect the release of the chemokine MCP-1 in Calu-3 or THP-1 cells. In contrast silicium dioxide particles with 50 nm cross section dimension displayed a concentration-dependant stimulation of MCP-1 release in Calu-3 cells.

Differentiated Calu-3 cells formed tight junctions seen on preparations for transmission electron microscopy and on freeze fracture preparations for scanning electron microscopy. Immunocytochemistry for adherens structures like Claudin-1, ZO-1 and Vinculin confirmed the results of TEER measurement.

The coculture system of differentiated epithelial cells in transfilters and immune cells in the basal compartment was applicable to test effects of nanoparticles on cytokine release and cytotoxicity.

Supported by a grant of the Baden-Württemberg Stiftung, Stuttgart.

1 Hochschule Lausitz (FH) Cell Biology and Tissue Engineering, GroÕenhainer StraÕe 57, 1968 Senftenberg, Germany, mario.lehmann@hs-lausitz.de 2 Hochschule Lausitz (FH) Department of Molecular Cell Biology, Germany PP 30 (E008) Human articular chondrocytes with extended life span: Genetic Engineering, Characterisation, and in vitro Tissue Formation

Monolayer cultures of chondrocytes as well as in vitro tissues using cells and scaffolds have been widely used to study normal and pathologic cartilage physiology. However, detailed studies have been hampered by a number of factors such as the scarce availability of human tissue, the limited proliferative capacity of cultured chondrocytes and the enormous donor dependent variances in the differentiation status of cells. It is frequently reported that primary chondrocytes in monolayer culture undergo a phenotypic drift including downregulation of cartilage markers like type II collagen and aggrecan. However, it is possible to reverse this dedifferentiation by culturing chondrocytes in a three-dimensional (3D) environment.

In order to establish a nontransformed human articular chondrocyte cell line possessing a remarkable extended life span, human articular chondrocytes were transfected with selected proliferation genes enhancing the replicative capacity. The transfection of the genes as well as the expression of markers characteristic for hyaline cartilage was verified at the mRNA level by RT-PCR and at the protein level by immunofluorescence. Genetically engineered chondrocytes were used to induce 3D-cartilage in vitro tissues without the aid of any scaffold by using the agar-overlay technique. Histological as well as immunohistochemical analyses of in vitro tissues were performed on cryosections.

A chondrocyte cell line which has the capacity to escape the Hayflick limit of cell proliferation was successfully established. Since this cell line displays a cartilage typical gene expression profile (type II collagen, aggrecan, and COMP) it might provide an ideal cell source for numerous donor independent studies.

In addition, the cells are able to form stable in vitro cartilage-like tissues whose cartilaginous differentiation potential is currently under investigation.

Especially the possibility to bypass the dilemma of donor variances nominates this human chondrocyte cell line as suitable basis for pharmacological test systems relevant to screen the physiological behaviour of cartilage cells in healthy tissue and in arthritic diseases, particularly in response to putative therapeutical agents. Moreover, the here established chondrocyte cell line could be a useful model system for studying both the metabolism of chondrocytes and their activity in a three-dimensional configuration known to modulate their phenotype.

This work was supported by the BMBF.

1 Zentrum Medizin. Forschung (ZMF), Orthopädische Universitätsklinik, WaldhörnlestraÕe 22, 72072 Tübingen, Germany, aicher@uni-tuebingen.de 2 BGU, Tübingen, Germany 3 NMI, Reutlingen, Germany PP 31 (E010) Mesenchymal stem cell adherence profiling using bovine serum albumin-peptide conjugates

Mesenchymal stem cells (MSCs) have been identified in bone marrow (bmMSC) as well as in other tissues, including human placenta (pMSC). These cells are characterized by their phenotype, by expression of CD73, CD90 and CD105, by the absence of haematopoietic markers including CD34, CD45, CD11a, CD19, and HLA-DR, and by their ability to differentiate into three lineages: chondrocytes, osteoblasts and adipocytes. MSC's exceeding properties of self-renewal, proliferation and enormous plasticity have provided their advantages in specific tissue engineering. The success of tissue regeneration's attempt requires an optimal combination of carrier material and surrounding components of extracellular matrix (ECM). Most in vitro studies on cell-ECM-interactions are performed with whole ECM proteins. Using peptides instead yields several advantages, including low cost for production and the possibility of a GMP-conform synthesis. The purpose of this work was to investigate the attachment profile of bmMSC and pMSC to peptides derived from ECM proteins.

Proteins and peptides were pre-screened by MSA^™ and 17 peptides were selected for further investigation, coupled to albumin, spotted, and air-dried. The surface was sealed. Human bmMSCs, pMSCs, and fibroblast cells were expanded to reach 80% of confluence. The cells were detached from the culture plates, washed, and 5.0 x106 cells were resuspended in low glucose DMEM medium with 0,1% BSA in isotonic solution and then seeded on the assay plate. After 15 minutes of incubation, the non-adherent cells were washed away with PBS and the cells attached were recorded.

Using this attachment assay we identified 4 out of 17 peptides that facilitated attachment of MSC. Furthermore, a difference of adherence properties of bmMSCs compared to pMSCs to peptide-conjugates #4 and #17 was observed. pMSCs attached better to peptide-conjugate #4 compared to bmMSC, while bmMSCs have better binding to peptide #17 compared to pMSCs. The foreskin fibroblasts, as a control cell line, bind well to both types of the peptides, #4 and #17.

We conclude, that such adhesion profiles occur due to different receptor-mediated interactions of cells with the ECM peptide domains regulated by specific expression patterns of integrins.

This attachment assay may be further developed to eventually serve as a diagnostic tool and for the development of therapeutic reagents in regenerative medicine.

1 Nagoya University, Graduate School of Engineering, Room625, Build1, School of Engineering, Furocho, Chikusaku, 464-8603 Nagoya, Japan, kato-r@nubio.nagoya-u.ac.jp 2 Nikon Instruments Inc. PP 32 (E012) Cell Quality Estimation based on Bioinformatic Analysis of Non-stained Morphological Images for the Quality Control in Cell Production

For the industrialization of regenerative medicine, the technology for providing both higher safety assurance and efficient cell processing is strongly required. However, conventional and traditional experimental techniques were considered to be inappropriate for the continuous quality check in regenerative medicine. Since cells produced for therapy are limited and promised to be pure without any testing regents. In this aspect, image analysis is one of the few methodologies that could estimate the final condition of implanting cells after cell processing. There had been reports of such non-invasive cell evaluation strategies based on cell images. However, most of the image analysis has focused on few cell morphologies intentionally selected by experts, and there were no scientific reason to select such parameter.

In our research, we introduced bioinformatic analysis strategy in the image analysis of culturing cells to select the best combination for predicting cell quality. By successful combination strategy with the fully-automatic cell culture and monitoring system BioStationCT (Nikon Instruments Inc.), we established the strategy to establish cell quality prediction models using time-lapse phase contrast cell images (more than 4,000 images) of four different cell lines (fibroblasts, sarcomas, myoblasts, and keratinocytes) as a model of cell sources. From the image analysis combined with the bioinformatic parameter selection methodologies, we succeeded in predicting (1) contamination percentage of sarcoma cells in fibroblasts, (2) future rate of myoblast differentiation, and (3) contamination rate of fibroblasts in keratinocytes, from the early image of cultured cell images. We found that to recognize the differences in cell images (non-invasive and non-stained), multi-variant analysis of bioinformatics is significantly effective. We will report the advantages and tasks remaining in such image analysis for cell quality control.

1 HZI Helmholtz Center for infectious Research, MBIO, Inhoffenstr. 7, 38124 Braunschweig, Germany, ked@helmholtz-hzi.de 2 Institute for Clinical Transfusion Medicine, Städtisches Klinikum Braunschweig gGmbH, Germany 3 Institut für Schicht- und Oberflächentechnik, Fraunhofer Gesellschaft, Germany 4 Department of Hematology-Oncology, Städtisches Klinikum Braunschweig gGmbH, Germany 5 German collection of cell cultures and microorganism (DSMZ), Germany 6 Department of Human Genetics, Hannover Medical School, Germany 7 Department of Anatomy, University Tübingen, Germany PP 33 (E013) Completely-closed Bag Systems with modified plastic surfaces for growth of adherent cells, localized genetic modification, cell selection and cryo conservation

There are increasing numbers of studies investigating the application of therapeutic cells. A major problem for these studies is the safe standardized generation and cryo-conservation of these cells under GMP-compliant conditions. One approach is the generation of cells in a completely-closed bag system as we have shown for non-adherently growing dendritic cells.

We wanted to extend the advantages of the closed bag system to other aspects of cell generation: (1) growth of adherent cells, (2) site specific genetic modifications, (3) cell selection and (4) cryo-conservation.

For the modification of the plastic surface we used dielectric barrier discharge which allows the chemical modification of the inner bag surface by plasma at atmospheric pressure. We used different chemical precursors for the primary modification of the bags by plasma. The primary modification enabled the culture of adherently growing cells on such treated plastic surfaces, but it also allows secondary coupling of additional molecules for cell modification and selection.As a model for the specific localized genetic modification of cells, we used adenoviral vectors encoding for the EGFP- or dsRed-Protein. These vectors were linked to the surface by biotin-streptavidin based mechanisms and allowed localized transduction of target cells. The biotin-streptavidine system was also used for coupling of antibodies to cell surface molecules. In addition binding via Protein G could further enhance the coupling of antibodies to the surface allowing specific selection of target cells.In situ cryo-conservation of stem cells and from stem-cells differentiated cells could be performed directly on the modified plastic surfaces. For quality control the genetic stability of the cells were analyzed by STR-analysis and karyotyping.

1 Riga Stradins University, Department of Prosthodontics, Dzirciema 20, 1007 Riga, Latvia, antraragauska@inbox.lv 2 Riga Technical University, Riga Biomaterial Innovation and Development Center, Latvia PP 34 (D001) Morphology analysis of bonding interface of tooth tissue and restoration materials using SEM

Introduction: An important role for success rate of restorations plays bonding interface with tooth structure. Marginal leakage of restoration leads to secondary caries and replacement of restoration. It is important to achieve good bonding of dental materials with tooth structure.

Purpose: The aim of this study was to examine morphology of two esthetic dental restorative materials and their bonding with tooth tissues using SEM.

Material and Methods: Ten caries-free, sound human premolars, were selected and stored in 0.9% NaCl at room temperature. The teeth were randomly divided into 2 groups of 5 teeth in each group: I- MOD cavities restored with indirect leucite reinforced glass ceramic inlays (Finesse: Dentsply, Ceramco), II- MOD cavities restored with direct high viscosity hybrid composite fillings (Filtek P60: 3 M, ESPE). The restorations in both groups were bonded using same hydrophilic adhesive resin (Adper Single Bond 2: 3 M, ESPE). Ceramic inlays were cemented with resin luting agent (RelyX Adhesive Resin Cement: 3 M, ESPE).

For SEM analysis teeth were embedded in epoxy resin (Epoxide: Buhler) cilinders and left for 12 h setting. The teeth were sectioned vertically in a anterior-posterior aspect. Surfaces of sectioned teeth were polished with 20, 5, 3, 1 and 0.25 μm fine particles diamond pastes (Metadi: Buhler) and cleaned in distilled water for 10 min. The specimens were submited to demineralisation with 50% phosphoric acid for 4 min, followed by 10 min ultrasonication in distilled water and dried in a desiccator containing silica gel for 12 h. The surfaces were sputter-coated with a gold layer of 7 nm thick during 2 min with 25 mA pressure.

Results: Morphology of tooth tissues: dentin and enamel and morphology of ceramic material, resin luting agent and composite filling material were examined. Bonding between adhesive restoration material and tooth tissues was evaluated. The samples showed good adaptation of ceramic inlays, composite fillings with tooth tissues. In cases of composite fillings, hybrid layer was seen with demineralized dentin and resin tags. The average width of the adhesive layer on dentin was 5 μm. Examining proximal adaptation of filling, the filling material was observed to extend beyond cavity borders.

Pictures with ceramic materials, composite cement and tooth tissue morphology, and their bonding were obtained in ceramic inlay group. The average width of the adhesive layer on dentin was 5 μm, similar to composite group, the average width of cement layer was 100 μm. The cement layer width in proximal adaptation area was 100–200 μm.

Conclusion: SEM structure analysis shows good bonding of materials with enamel and dentin in both composite filling and porcelain inlay groups.

1 Department of biosciences and bioengineering IIT Bombay, Department of Biosciences and Bioengineering, Powai, 400076 Mumbai, India, amitanand22@iitb.ac.in 2 Department of Chemical Engineering, IIT Bombay, India PP 37 (D005) Fabrication of biocomposite membrane by electrospinning for guided bone regeneration

Objective: Morphology and physiochemical properties of scaffold play important role in in-vivo study. The objective of this study is to fabricate a flexible membrane by electrospinning with increased bioactivity and osteoconductivity that can act as conformal coating for the osteosynthesis. We hypothesize that the biomimetic electrospun nanofibrous scaffolds act as an osteoconductive material which will enhance osteointegration of the scaffold compared to pure nanofiber scaffolds.

Materials and Methods: For the preparation of electrospun nanofibrous scaffold, solution Poly-L-lactic acid (PLLA) and Gelatin (Gel) in 1,1,1,3,3-hexafluoro-2-propanol (HFP) were pumped to at a rate of 0.7 mL/h at the voltage of 12.5 kV. Mineralization of the scaffolds was done by alternate soaking in 0.5 M CaCl₂ and 0.3 M Na₂HPO₄ for 10 min. Characterization of scaffolds were done by scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), fourier transform infrared (FTIR), contact angle, and X-ray diffraction (XRD). MG63 cell adhesion and cell proliferation was evaluated by SEM and MTT assay respectively. Hemocompatibility of the scaffold was also examined.

Results: The SEM micrographs reveal that morphology of pure electrospun fibers were uniform and smooth compared to mineralized PLLA/Gel scaffolds. PLLA-Gel nanofibers can be successfully mineralized with hydroxyapatite. Deposition of HA on nanofibers can be confirmed by TEM, FTIR and XRD results show that apatite formed on the surface of scaffold is HA. Mineralization of the scaffold made the surface of the scaffold hydrophilic. Initial cellular assay showed excellent cell attachment and proliferation. Hemolysis assay reveals that the scaffolds are biocompatible and thus for the same scaffolds promising results for in vivo experiments are expected.

Conclusion: Above results show that the electrospinning is promising technique to fabricate scaffolds which mimics the architecture of extra cellular matrix (ECM) of bone. Alternate soaking method is a rapid method to deposit the hydroxyapatite on the nanofibrous scaffold. The functionalized electrospun nanofibrous scaffold is a promising candidate for guided bone regeneration.

1 Riga Technical University, Riga Biomaterials Innovation and Development Centre, Pulka 3/3, 1007 Riga, Latvia, salma.ilze@gmail.com 2 Riga Stradins University Institute of Stomatology, Latvia 3 Institute of Anatomy and Anthropology, Riga Stradins University, Latvia PP 43 (D011) Evaluation of bone regeneration after maxillary sinus floor elevation with Hap using Cone Beam volume tomography (CBVT) and histomorphological analysis

Introduction: The aim of this study was to evaluate the regeneration of bone after maxillary sinus floor elevation with Hap ceramic granules. Morphological analysis of biopsies from Hap/host tissue hybrid and residual alveolar bone and analysis of radiological outcomes was done.

Material and Methods: The properties of sintered ceramic were investigated by chemical compositions of high purity, X-ray diffraction, SEM, FT-IR methods. CBVT (Kavo) and trepan biopsies were evaluated in 20 patients six months after sinus floor elevation with synthetic Hap (RTU RBIAC) or Bioss (Geistlich) granules and dental implant insertion.

Routine histological method - staining with hematoxilin and eosin and evaluating with Leica BME microscopy was used for obtaining a review picture.

Biotin and streptavidin immunohistochemical method was used to detect growth factors - transforming growth factor β1 (TGF β1) and bone morphogenic protein 2 (BMP 2).

Results: Hap and Bioss granules were surrounded by new formed bone. Optical density of new formed bone is 2–2.5 times that optical density of the residual bone.

In biopsies biomaterial/host tissue hybrid consisted of small bone trabecules, fibrous tissue and granules of irregular shape without inflammatory cells. Degradation of Hap granules by activity of osteoclast like macrophages was observed. Evaluating the expression of TGF β1 and BMP 2 many positive structures were found.

Conclusions: Assessment of bone regenerate with CBVT and trepan biopsies may help in decision making for dental implant placement in the posterior maxilla. Expression of TGF β1 and BMP 2 have demonstrated the osseoconductive ability of biomaterials.

1 Leibniz Universität Hannover, Insitute of Inorganic Chemistry, Callinstr. 9, 30167 Hannover, Germany, anne.christel@acb.uni-hannover.de 2 Institute of Technical Chemistry, Leibniz University of Hannover, Germany 3 Department of Trauma Surgery, Hannover Medical School, Germany PP 44 (D012) Nanoparticles for multiple functionalization of scaffolds for stem cell engineering

Nanoparticles and coatings made from nanoporous silica are of great interest for applications in the field of stem cell research. As has been shown in many investigations [1], this material exhibits very good biocompatibility. Its main advantages are ordered pore networks, large pore volumes and high specific surface areas. The pores can be used for the delivery of bioactive agents, e.g. drugs. The surface exhibits reactive silanol groups which can easily be functionalized with biological molecules, for example with growth factors, recognition molecules etc. For this reason, nanoporous silica nanoparticles are interesting candidates for the specific and directed differentiation of stem cells. For the immobilization or binding of different biological molecules, appropriate linker systems have to be developed, the binding should not decrease their biological activity. Our work currently focuses on the immobilization of model proteins as ALP and on growth and differentiation factors as BMP2, FGF2 or TGF-β.

Different methods for the synthesis of nanoporous silica nanoparticles were tested. The obtained products were thoroughly characterized using a variety of analytical methods. The size of the particles was determined by dynamic light scattering and scanning electron microscopy to 40-110 nm. The latter method was also applied for morphology studies. Furthermore, sorption and X-ray diffraction measurements were performed.

For the immobilization of proteins on silica nanoparticles, a linker system was used which has been applied before for the binding of BMP2 and ALP to nanoporous silica coatings [2]. The linker consists of an aminosilane. The successful binding of the linker was confirmed by infrared spectroscopy. These derivatized nanoparticles were then incubated in different protein solutions for the immobilization. For the quantification of the immobilized BMP2, a standard ELISA assay was chosen. Since such a standard assay does not allow a direct quantification of the immobilized BMP2, the concentration of the protein was determined in the supernatant. The results show that the silica nanoparticles carrying the linker bind more BMP2 than those which do not.

1 Max Planck Institute for Colloids and Interfaces, Biomaterials, Am Mühlenberg, 1, 14476 Potsdam - Golm, Germany, cecile.bidan@mpikg.mpg.de 2 Ludwig Boltzmann Institute of Osteology, Wien, Austria PP 46 (D016) Geometric control of three-dimensional tissue growth

Identifying the mechanisms of tissue growth is fundamental to understand biological processes such as bone (re)modeling, bone and wound healing, and tumor growth. Cells have been shown to be sensitive not only to the biochemistry of their environment (BMP, growth factors) but also to physical characteristics of their surroundings. For example, stiffness (Engler et al. 2006), topology (Dalby et al. 2007) and geometry (Chen 1997) of the substrate determine cells fate through mechanotransduction (Vogel and Sheetz 2006). Lately, Rumpler and coworkers (Rumpler et al. 2008) seeded MC3T3-E1 cells on hydroxyapatite scaffolds containing pores of different convex sections (concave surfaces) to show that the geometry of three-dimensional channels influences bone tissue production. They concluded that the overall amount of tissue produced within a pore is independent of the shape but the distribution of tissue within a pore compares to a model based on a local growth rate proportional to curvature. The goal of the present work is to pursue the investigation on scaffolds with pores of non-convex sections (cross and star). Hydroxyapatite scaffolds are built with an indirect rapid prototyping technique using molds and incubated for 5 weeks with murine MC3T3-E1 pre-osteoblast cells. The amount of tissue produced in pores is followed in terms of projected tissue area (PTA) and measured twice a week using a phase contrast microscope. The first outcome reveals that (i) no growth occurs on convex surfaces, (ii) growth rate remains proportional to the local curvature on concave surfaces, (iii) the initial overall growth remains linear in non-convex shaped pores and compares with the previous model of curvature-driven growth (Rumpler et al. 2008), (iv) this growth rate is higher in non-convex shapes and depends on the geometry.

Although the mechanisms of geometry sensing by cells are still unclear, a qualitative model for tissue growth can still be proposed. Computer simulations based on curvature-driven growth reproduce the observed qualitative behaviour in both convex and non-convex geometries and the estimated growth rates fit with the experimental results. Theory and simulation compared with observation and experimental data support the approach that geometry can be used to optimize tissue growth in scaffolds for tissue engineering.

1 Fraunhofer IGB, Zellsysteme, NobelstraÕe 12, 70569 Stuttgart, Germany, simone.wurster@igb.fraunhofer.de 2 Universität Würzburg, Würzburg, Germany PP 47 (D018) Effect of plasma-treated polystyrene surfaces on human dermal microvascular endothelial cells

Introduction: For the engineering of in vitro tissues one of the basic principles is culturing of primary cells. Chemical modifications of specific biomaterials may improve cell culture conditions. In this work, simple chemical functionalizations were carried out using plasma treatment to modify the polystyrene surface with different functional groups. These substrates were used to evaluate the influence of chemical stimuli to human dermal microvascular endothelial cells (mECs).

Materials and Methods: To obtain changes in surface chemistry, CO₂-plasma, NH₃-plasma and acrylic acid plasma were applied. X-ray photoelectron spectroscopy (XPS), colorimetric methods and contact angle measurements were performed to distinguish the chemical surface modification. Analyses of substrate-specific cell behaviour were carried out using mECs, derived from biopsies of different body regions. These cells were seeded on the modified substrates and cultured over 72 h. To evaluate cell morphology, migration and proliferation on the diverse substrates, light and scanning electron microscopy, WST-1 cell proliferation assay and immunocytochemical staining were performed after 72 h.

Results: The surface modification by plasma treatment could be verified with XPS and the colorimetric methods. For the mECs seeded on the chemically functionalized substrates differences in proliferation could be observed. WST-1 cell proliferation assays showed significantly higher cell activities on polystyrene surfaces modified with amine groups (150.4%) compared to routine tissue culture substrates (100%). In contrast, when cultured on substrates functionalized with carboxy groups (86.9%) the proliferation rates decreased significantly. Cells cultured on CO₂-plasma-treated surfaces showed a proliferative activity of 102.5%, which is comparable to routine tissue culture substrates. Immunocytochemical staining revealed comparable expression patterns of the endothelial cell-specific markers CD31, CD144 and vWF on all endothelial cells that were cultured on the modified surfaces.

Conclusion: In this work, it was possible to create well-defined advanced cell culture substrates with the capability to significantly impact the in vitro behaviour of mECs. Further experiments should be carried out to examine the impact of plasma-modified substrates on mECs in co-culture with other cell types. Therefore co-cultures of fibroblasts and endothelial cells will be investigated after cultivation on the substrates.

1 NMI Natural and Medical Sciences Institute at the University of Tübingen, Regenerative Medicine I, Markwiesenstr. 55, 72770 Reutlingen, Germany, hanna.hartmann@nmi.de 2 INEB Instituto de Engenharia Biomédica, Divisão de Biomateriais, Universidade do Porto, Portugal PP 49 (D023) Towards inhibition of fibrotic incapsulation of implants using siRNA

Objectives: RNA interference is a highly specific mechanism of gene silencing induced by small interfering RNA (siRNA). These small oligonucleotides initiate the sequence-specific cleavage of messenger RNA (mRNA). RNA interference can be harnessed to develop a new class of drugs that interfere with every desired disease-causing or disease-promoting gene, thereby inhibiting the synthesis of designated target proteins.

We aim to use siRNA as a tool for biological functionalisation of implants. One example for the application of our concept is the inhibition of undesired fibrotic encapsulation of implants by down-regulating cellular collagen secretion. Inhibition of fibrotic encapsulation can, among other things, be important for accurate performance of implanted biosensors.

Methods: To overcome extracellular and intracellular barriers that restrict therapeutic application of siRNA, we utilize a biomaterial carrier – imidazole grafted chitosan (CHimi). Subjects of our work are electrostatic binding of siRNA to the biomaterial and characterisation of the resulting nanoparticles using photon correlation spectroscopy (size and zeta potential). Furthermore, in cell culture we analyse particle uptake by microscopy and subsequent gene knockdown using real time quantitative reverse transcription (qRT)-PCR. In addition, collagen synthesis is quantified photometrically using the sirius red stain.

Results: Fibrosis is characterised by enhanced synthesis and unorganized aggregation of collagen. In cell culture experiments we are able to effectively down-regulate a gene important for collagen secretion (reduced mRNA and protein level) and to modify collagen secretion. Usage of CHimi in certain ratio to siRNA allows the formation of small particles with size in the nanometer range and positive zeta potential. Moreover, these particles are efficiently taken up into fibroblasts and produce a significant reduction of the targeted gene.

Conclusions: Our results highlight the potential of chitosan/siRNA nanoparticles to inhibit a fundamental fibrotic pathway. Our aim for the future is to use biological functionalisation of implants as a cross-sectional technology and to bind siRNA as bioactive molecule to a variety of medical implants. We thereby hope to foster clinical application of biosensors and other medical products.

1 Natural and Medical Sciences Institute at the University of Tuebingen, Regenerative Medicine I, Markwiesenstr.55, 72770 Reutlingen, Germany, nadine.hoffmann@nmi.de 2 Interdisciplinary Nanoscience Center (iNANO) and Department of Molecular Biology, University of Aarhus, Denmark 3 Instituto de Engenharia Biomédica, Divisão de Biomateriais and Faculdade de Engenharia Universidade do Porto, Portugal PP 50 (D024) Differential side-effects caused by siRNA nanoparticles in neuronal and glial cells

Objectives: RNA interference (RNAi) is a technique of highly specific gene silencing. Within the last decade RNAi has become one of the most important and promising tools in developing new classes of drugs and exploring signaling pathways. Even though small interfering RNA (siRNA) has become a standard tool, various non-specific side-effects caused by siRNA have been observed. For future therapeutic application of siRNA in the regenerating nervous system, we investigated potential side-effects in glial and neuronal cells.

Methods: Non-targeting fluorescence-labelled siRNA was transfected via lipofectamine (LF) or chitosan/siRNA-nanoparticles (NP) into a) the neuronal cell line PC12 and b) primary glial Schwann cells (SC). By fluorescence microscopy the transfection rate was monitored. Different parameters including cell viability (Resazurin assay), cell proliferation (BrdU assay) and cell migration were quantified. By using RT-PCR, mRNA levels of inducible nitric oxide synthase (iNOS) and 2′-5′-oligoadenylate synthetase 1 (oas1), two toll-like-receptor (TLR)-mediated inflammatory response genes, were determined. All assays were performed 24 hours after transfection.

Results: siRNA was successfully taken up by both cell types as judged from qualitative microscopy. SC viability remained unaffected by transfection, whereas their proliferation rate decreased significantly after NP transfection. SC migration which is crucial during nerve regeneration in the peripheral nervous system, was slightly affected by siRNA transfection. As an indicator of inflammatory activation, iNOS expression was analyzed. Neither of both siRNA transfection regimes altered mRNA levels of iNOS compared to untreated cells. In contrast to SCs, the neuronal cell line PC12 showed a reduced cell viability and proliferation rate following LF and NP transfection. As in SCs, no activation of inflammatory response genes was evident.

Conclusions: Our results reveal differential responses in glial and neuronal cells after siRNA transfection independent of the two selected siRNA formulations. However, inflammatory response genes were never affected. In order to make use of the therapeutic potential of siRNA for the regeneration of the nervous system in the future, it will be important to exclude non-specific effects in addition to inflammatory responses which had been in focus in the past.

1 Fraunhofer IGB, Zellsysteme, Nobelstr. 12, 70569 Stuttgart, Germany, esther.novosel@igb.fraunhofer.de 2 Fraunhofer IAP, Potsdam, Germany 3 Universität Stuttgart IGVT, Stuttgart, Germany 4 Universität Würzburg, Würzburg, Germany PP 53 (D027) Characterization of endothelial cell-biomaterial interaction on newly developed 3D-printable polymer surfaces for vascular grafts

Introduction: Medical implants need to fulfill a variety of requirements regarding long term duration, design, function and biocompatibility. In our work, polymer materials were designed for the buildup of an artificial 3-dimensional (3D) blood vessel system using a 3D-inkjet printing process. The wanted properties of the cured 3D scaffolds like flexibility were setup. Materials are being developed, synthesized and in cell cultivation experiments systematically analyzed for their applicability with endothelial cells.

Materials and Methods: Different types of α,ω-hydroxy-oligoethers have been synthesized and characterized. Viscosity and surface tension were adapted to the needs of the printing process. Concurrent with the ongoing polymer development, cell cultivation experiments have been conducted using the α,ω-hydroxy-oligoethers as a substrate. Investigated cell types were human primary microvascular endothelial cells and primary human umbilical vein endothelial cells. All applied cells were seeded on the new developed substrates. The adhesion, morphology, proliferation and functionality of the endothelial cell were controlled 24 h and 48 h after seeding by light-microscopy methods and WST-1 cell proliferation assay. Additionally, assays were performed to detect the Low density lipoprotein - uptake activity of the cells. After 48 h cultivation immunohisto-chemical stainings against specific endothelial antigens like CD31, von Willebrand factor and VE-Cadherin were performed. Differences in gene expression patterns of eNOS, VEGFR-2, VE-Cadherin, CD31 and vWF were evaluated by RT- PCR.

Results: The synthesis of the 3D-printable polymers with the designated viscosity, surface tension, flexibility and biocompatibility was successful. The results of the WST-1 assay showed for all produced materials an average percentage of viable cells that varies from 60 to 90%. On these polymers, cell-surface interactions have been observed including characteristic endothelial cell morphologies and proliferation patterns. The endothelial cell markers could be visualized by immuno-histochemical stainings. Cell functionality was proved by LDL uptake of vital endothel cells on the material surface. In parallel the expression of endothel cell specific markers was analyzed by RT-PCR.

Conclusion: Finally, outcome of the cell-polymer interaction study demonstrates that our new 3D-printable polymers could be a suitable material for vascular grafts.

1 Saarland University, Faculty of Medicine, Department of Trauma-, Hand- and Reconsctructive Surgery, Kirrbereger StraÕe, Building 61.4, 66421 Homburg, Germany, lisaschipf@yahoo.de 2 INM-Leibniz Institute for New Materials, Saarbrücken, Germany PP 54 (D028) Cell compatibility of micro- and nanostructured alumina surfaces prepared by chemical vapor deposition

Background: A lot of progress has been made in the development of permanent bone implants, but a reliable and stable osseointegration is still a great challenge. Clinically observed fibrosis, micro movements and osteolysis are the predominant complications. It is our goal to structure Al/Al₂O₃-coatings in a defined way in the nano- and micrometer range to create cell selective topographies without changing the surface chemistry. Such a coating might be useful to increase the adhesion of osteoblasts and to decrease the adhesion of fibroblasts in order to develop a cell selective surface.

Materials/Methods: Al/Al₂O₃-nanowires with a mean diameter between 20 and 30 nm were deposited on glass carriers by means of chemical vapour deposition (CVD) in different densities (ultra high density (UHD), high density (HD), medium density (MD) and low density (LD)).

The influence of the produced topographies alone on cell compatibility was examined in vitro using normal human dermal fibroblasts (NHDF) and human osteoblasts (HOB) (n = 5) compared to glass controls. After incubation (48 h) the cell density and the mean cell surface were analyzed and the results were normalized to the controls.

Cell vitality on the surfaces was analyzed applying the WST test. The results were tested in terms of significance (p < 0.05) by applying an ANOVA or a t-test, respectively.

Results:

It was possible to deposit Al/Al₂O₃-biphasic nanowires in different densities depending on the deposition time on planar glass carriers by means of CVD.

Conclusion: Our results show a reduced adhesion of NHDF compared to HOB determined by the surface topography alone. Therefore, CVD coatings might be useful to create cell selective coatings for implant material applications. In the future we will additionally modify the topography of Al/Al₂O₃-nanowires deposited in different densities by applying a pulsed nanosecond laser leading to a defined melting of the nanowires.

1 Dept. Thoracic, Cardiac and Vascular Surgery, University Hospital, Silcherstr. 7/1, 72076 Tübingen, Germany, martina.schleicher@med.uni-tuebingen.de 2 Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany 3 Section of Medical Materials and Technology, Dept. of Prosthodontics, University Hospital, Tübingen, Germany 4 Dept. Evolutionary Biology of Invertebrates, Institut of Evolution and Ecology, Eberhard Karls University Tübingen, Germany 5 Surgery of congenital heart defects and pediatric cardio-surgery, Children Hospital, University Hospital, Tübingen, Germany PP 58 (D035) Endothelialization of oligonucleotide coated diX AM surfaces for in vivo tissue engineering

Introduction: Current limitations of in vitro tissue engineering include long in vitro culturing, the accompanied risk of infection and required cost intensive infrastructures. Ongoing research therefore turned toward development of spontaneously in vivo endothelializing implants. Objective of this study was the creation of cell adhesive DNA-oligonucleotide coatings on implant surfaces. DNA is an intriguing coating material as it is naturally occurring in the body, non-immunogenic and can be chemically synthesized easily and rapidly.

Methods: DNA-oligonucleotides were immobilized on amino-parylene (diX AM) coated polypropylene dishes. Coupled oligonucleotides were exposed to physiologic shear stress conditions (10 N/m²) and incubated under dynamic conditions with cells of an immortalized mouse EPC line, HUVECs and human saphenous vein endothelial cells. Biocompatibility was investigated by incubation with blood, granulocytes and thrombocytes and by determination of released thrombogenic and immunogenic factors.

Results: The immobilization of oligonucleotides on diX AM coated surfaces is extremely stable and withstands shear stress characteristic for native heart valves. Oligonucleotides enhance cell adhesion under continuous flow conditions significantly. Oligonucleotide coated surfaces showed low thrombogenicity and excellent haemocompatibility. Incubation with human serum showed no oligonucleotide degradation after 72 h.

Conclusion: Surface immobilization of oligonucleotides can facilitate manufacturing of an “off-the-shelf” heart valve for in vivo endothelialization. Additionally immobilization of oligonucleotides on other types of implants opens new opportunities for biocompatible coatings enhancing the capability of incorporation in surrounding tissue.

1 Max Planck Institute for Metals Research, Department New Materials and Biosystems, Heisenbergstr. 3, 70569 Stuttgart, Germany, eva.altrock@mf.mpg.de 2 Section for Transplantation Immunology and Immunohematology, Center for Medical Research, University Medical Clinic, Tübingen, Germany PP 63 (A002) Lipid raft formation of hematopoietic stem cells in response to nano-patterned substrates

Hematopoietic stem cells (HSCs) give rise to all kind of blood cells. In vivo they reside in stem cell niches, where they can proliferate while keeping their stem cell character. All attempts to expand HSCs in vitro by deciphering the optimal culture medium supplemented with soluble factors failed so far. It became increasingly clear that cell adhesion is a prerequisite for HSCs to maintain stem cell properties. In this regard integrins play a major role. They mediate cell matrix contacts and activate signalling pathways upon ligand binding. However, not only the nature of the ligands presented, but also the arrangement of the ligands on the nanoscale is able to influence cells. Thus, we investigated which is the critical distance between integrin ligands to induce the formation of lipid rafts, which serve as platforms for receptor clustering during cell signalling, in HSCs.

Gold nano-patterns were produced by diblock copolymer micellar nanolithography and functionalized with the integrin ligand RGD. As control, fibronectin was homogeneously adsorbed to surfaces. The hematopoietic cell line KG1a or hematopoietic stem and progenitor cells (HSPCs) purified from umbilical cord blood were seeded onto the substrates and lipid raft formation and expression of integrins, CD34 and CD133 were monitored by confocal microscopy.

In hematopoietic cells, lipid rafts form within the first 5 minutes upon contact with a homogeneously adhesive surface. The rafts are concentrated at the bottom of the cells. This effect increases over time. A similar phenomenon was observed for the integrin alpha 5 chain in KG1a cells. The integrin beta 1 chain was distributed over the entire cell surface in KG1a cells. In polarized primary HSPCs it was concentrated in the uropod like the lipid rafts. CD133 was mainly localized in the uropod in polarized cells, but in 17% of the cells, which showed a strongly elongated phenotype, CD133 was concentrated at the leading edge, opposite to the rafts.On nano-structured substrates functionalized with RGD similar effects could be observed. The critical distance between RGD ligands for hematopoietic cell adhesion and lipid raft formation seems to be at 30 nm.

In conclusion, we could show that lipid raft formation during hematopoietic cell adhesion is relatively fast.The rafts as well as integrins and CD133 reorganize during cell adhesion and polarization.30 nm seems to be the critical distance between RGD ligands for adhesion and lipid raft formation.

1 University of Tübingen, Section for Transplantation, Immunology and Immunohematology, Waldhörnlestr. 22, 72072 Tübingen, Germany, carolin.steinl@uni-tuebingen.de 2 NMI Reutlingen, Germany PP 64 (A003) Matrix metalloproteinase 8 impairs adhesion of hematopoietic stem cells in the endosteal stem cell niche

Hematopoietic stem and progenitor cells (HSPCs) can be found within the bone marrow in an endosteal stem cell niche which is mainly composed of osteoblasts and their secreted extracellular matrix (ECM). In the process of induced stem cell mobilization several proteases including neutrophil elastase, cathepsin G and matrix metalloproteinase (MMP)-9 can contribute to the release of HSPCs from their niches, but the precise mechanism of mobilization is still not known. In our present study we characterized the expression and secretion of further members of the MMP family in the context of the endosteal stem cell niche.

RT-PCR analysis, Western blotting and immunofluorescence staining revealed that several gelatinases, collagenase and stromelysines including MMP-8 are expressed by human osteoblasts. ELISA analysis comparing cord blood serum with normal blood serum disclosed elevated protein levels of MMP-8 as well as for MMP-9. The influence of these two MMPs on the interaction of HSPCs with osteoblasts was quantified by cell-cell adhesion assays with labeled HSPCs. After binding of CD34 + HSPCs to primary osteoblasts, activated MMPs were added. These assays unveiled a strong reduction of HSPC cell attachment to osteoblasts after incubation with activated MMP-8 and - 9 compared to non-activated MMPs. A detailed analysis of the ECM protein family of laminins by RT-PCR, immunoprecipitation and immunofluorescence staining revealed the expression of the laminin isoform LM 511/521 by human osteoblast. Proteolytic digestion of LM 511/521 by MMP-8 or MMP-9 drastically reduced adhesion of HSPCs to this laminin isoform. However, the MMP-8 processed laminin-511/521 isoform had a strong migration-stimulating effect on HSPCs. Additionally CXCL12alpha, an important chemokine in HSPC migration and mobilisation was tested as a substrate for MMP-8. MMP-9, used as a positive control, showed an N-terminal reduction of four amino acids using MALDI-TOF analysis. In contrast, activated MMP-8 is able to proteolytically remove specifically three amino acids at the N-terminal end of CXCL12alpha.

In summary we provide strong evidence that the matrix metalloproteinase MMP-8 which can be released from granulocytes, but also from osteoblasts, can drastically reduce binding of HSPCs to osteoblasts. Furthermore, proteolytic degradation of ECM components and chemokines by MMPs might support mobilization and migration of HSPC out of the niche.

1 Fraunhofer EMB, Cell Differentiation, Paul-Ehrlich-Str. 1-3, 23562 Lübeck, Germany, bjoern.boehmert@emb.fraunhofer.de 2 Department of Gynecology and Obstetrics, University of Schleswig-Holstein, Campus Luebeck, Germany PP 65 (A004) Oocyte-like cells derived from rat pancreatic stem cells show morphologic and functional similiarities to native female germ cells

Pancreatic stem cells derived from rat express typical stem cell markers like Oct4, Nestin and SSEA1 and have the ability to differentiate into all three germ layers. Clonal cell lines of these pancreatic stem cells show similar characteristics but also remarkable differences: one clonal cell line proliferates in suspension by forming aggregates which fuse to tissue like structures, so called “Tissue bodies”. These tissue bodies constantly release cells from their surface which resemble mammalian oocytes in morphological as well as genetic properties and are therefore called “oocyte like cells” (OLC). We could show the expression of oocyte specific markers like Zona Pelucida Protein 3, SSEA1 and SCP3 on the transcriptional and translational level by real time PCR and immuncytochemistry.

Further investigations show that oocyte like cells form follicle like structures by recruiting their surrounding cells and produce the sex steroid estradiol.

We were able to observe the attachment of sperm, derived from rat epididymis, to the outer margin of oocyte-like cells showing a prosperous interaction of the putative oocyte and the male germ cell.

The findings of this study could be used to establish an In vitro model system for oogenesis and follicular maturation.

1 Universität Stuttgart, IGVT, Nobelstr. 12, 70569 Stuttgart, Germany, c.kleinhans@gmx.de 2 Fraunhofer IGB, Stuttgart, Germany 3 Universität Würzburg, Würzburg, Germany PP 68 (A008) Impact of new functionalized biomaterials on the adhesion and proliferation of human mesenchymal stem cells

Introduction: Tissue Engineering is an interdisciplinary research field that aims to support or replace diseased human tissues. One prerequisite is the combination of synthetic and living components. A major problem of Tissue Engineering is the terminal differentiation and therefore restrictive in vitro proliferation of cells in an adult organism. Alternative cell sources are adult stem cells like human mesenchymal stem cells (hMSCs). Their advantages are the high proliferation and differentiation capacity. We designed new biomaterials with amino- or carboxyfunctionalized surfaces to optimize stem cells' proliferation and differentiation conditions.

Materials and Methods: Polymer surfaces were functionalized with amino or carboxy groups by low pressure plasma. Successful functionalisation was proved by X-ray photoelectron spectroscopy (XPS) and colorimetric methods. The substrates were analyzed by contact angle measurement. HMSCs isolated from cancellous bone biopsies were seeded on the functionalized substrates. Adhesion, morphology, proliferation and functionality of hMSCs were investigated by light-microscopy methods, WST-1 cell proliferation- and fluorescein diacetat-propidiumiodide assays (FDA/PI). The cell substrate interaction was observed by scanning electron microscopy (SEM) and immunohistochemical staining of Actin-Vinculin. Furthermore immunohisto-chemical stainings against specific hMSCs markers were performed.

Results: The functionalisation of the plasma treated surfaces was successful. A significant increase of amino and carboxy groups on the substrates, after the application of low pressure plasma, was demonstrated by XPS-measurements and colometric analyses. The WST-1- as well as FDA/PI assay revealed a different proliferation rate of hMSCs on the modified surfaces compared to the commercial available tissue culture substrates, which served as a positive control. The formation of focal contacts could be observed on all surfaces. Additionally the expression of specific MSC markers could be detected.

Conclusion: This work verified an influence of surface modification with amino and carboxy groups on the adhesion and proliferation of hMSCs. Further improvements in the substrates topography as well as further investigations in differentiation conditions are necessary to provide an efficient in vitro system for Tissue Engineering.

1 University Clinic of Tübingen, Department of Internal Medicine II, Division of Hematology, Oncology, Immunology, Rheumatology and Pulmonology, Otfried-Mueller Str.10, 72076 Tübingen, Germany, nel.petkova@gmail.com 2 University Clinic of Tübingen, Department of Urology, Tübingen, Germany PP 69 (A009) Isolation and surface marker characterization of epithelial and stromal stem cells in benign prostate tissue

Prostate cancer is one of the most frequent diagnosed cancers in men. In the search for novel targets to diagnose and to effectively eliminate prostate cancer (stem) cells, surface antigens need to be identified that are either selectively expressed or overexpressed in tumor cells. To discriminate between cancer and benign prostate (stem) cells the specific phenotypic features of these cell types must be determined. In this work we pursued the prospective identification of epithelial and mesenchymal stem cells of benign prostate tissue using a large panel of our monoclonal antibody collection in combination with commercially available antibody conjugates, including antibodies against the integrins CD49a, CD49b, CD49f, and against the stem cell markers CD34, SSEA-4, and TNAP (Tissue non-specific alkaline phosphatase). Using flow cytometry and colony forming assays, we investigated the clonogenic potential of prospectively isolated candidate stem cell populations. Our preliminary results indicate that prostate mesenchymal stem cells express the phenotype CD49a(bright)CD34 + TNAP + , whereas epithelial stem cells are CD49b(bright)CD49f + SSEA-4 + . In conclusion, we developed novel procedures to isolate mesenchymal and epithelial stem cell subsets from benign prostate tissue. The knowledge of the phenotypic features of benign prostate stem/progenitor cells will contribute to identify aberrantly expressed tumor antigens on malignant prostate cells, which represent candidate targets for therapeutic treatment.

1 Leibniz Universität Hannover, Institut für Technische Chemie, Callinstr. 5, 30167 Hannover, Germany, kasper@iftc.uni-hannover.de 2 Medizinische Hochschule Hannover, Klinik für Frauenheilkunde und Geburtshilfe, Hannover, Germany 3 Medizinische Hochschule Hannover, Klinik für Immunologie und Rheumatologie, Hannover, Germany PP 70 (A010) Shaping the Microenvironment of Umbilical Cord-derived Mesenchymal Stem Cell-like Cells

Bone marrow-derived mesenchymal stem or stromal cells (BM-MSCs) have been proven to display a high potential for cell-based therapies and tissue engineering applications because of their multilineage differentiation potential and their immunomodulatory properties. However, bone marrow presents several disadvantages, namely low frequency of MSCs, high donor-dependent variations in quality and the isolation procedure is painful and implies the risk of infection. In search of alternative sources of MSCs, the umbilical cord (UC) tissue gained more and more attention. Since the UC is discarded after birth, the cells are easily accessible without ethical concerns. We isolated a population of plastic-adherent mesenchymal stem cell-like cells from human UC-tissue, which exhibit a high proliferative potential and express several MSC markers, including CD44, CD73, CD90 and CD105 (negative for CD31, CD34 and CD45). Furthermore, the cells display multilineage differentiation potential.

The aim of this study was to shape the microenvironment of the cells with regard to different oxygen concentrations and cell-cell interactions with immune cells. Therefore, the oxygen consumption, as well as the metabolic activity and HIF-1α target gene expression were determined. In addition, immunomodulatory properties of MSC-like cells were analyzed by direct and indirect co-culture experiments using peripheral blood mononuclear cells (PBMC) in CFSE-based proliferation assays.

Our study revealed that UC-derived MSC-like cells consume 2-3 times less oxygen under hypoxic conditions (1.5% O₂, 2.5% O₂ and 5% O₂) as compared to 21% O₂ control. Hypoxic culture conditions caused stabilization of HIF-1α protein and subsequent regulation of its target genes, involved in glucose metabolism. Moreover, UC-derived MSC-like cells showed increased proliferation at 2.5% O₂.

Furthermore, our results indicate immunomodulatory properties of MSC-like cells which did not induce proliferation of allogeneic PBMCs in vitro. Additionally, co-culturing of MSC-like cells and PHA-stimulated PBMCs in direct or in transwell co-culture experiments led to a decrease of PBMC proliferation compared to PHA-stimulated control PBMCs.

1 University of Tuebingen, ZMF, Section for Transplantation Immunology, Med.Klinik II, Waldhörnlestrasse 22, 72072 Tübingen, Germany, nicole.staudt@medizin.uni-tuebingen.de 2 Center for Regenerative Medicine and Department of Orthopedic Surgery, Center for Medical Research Center, Tübingen, Germany 3 Medical and Natural Sciences Research Center, Tübingen, Germany 4 Department of Immunology, Institute for Cell Biology, University of Tübingen, Germany 5 Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil 6 Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA PP 71 (A011) Cathepsin X is activated by cathepsin L, inactivates the chemokine SDF-1 and reduces adhesion of hematopoietic stem and progenitor cells to osteoblasts

Hematopoietic stem and progenitor cells (HSPC) are known to reside in specialized niches at the endosteum in the trabecular bone. It is well established that proteases can take part in the cytokine-induced mobilization process. However, migratory processes such as the regular trafficking and induced mobilization of HSPC are not fully understood.

In the present study we showed that the osteoblast-secreted activated cathepsin X is able to reduce the direct interaction of HSPC with human bone-forming osteoblasts.

Immature cathepsin X is also bound to the cell surface of human osteoblasts. Knocking-down endogenous cathepsin X in osteoblasts with siRNA and subsequent HSPC adhesion studies led to a significant increase of HSPC binding to the adherent cells confirming its proteolytic influence on HSPC adhesion. In this context we studied the activation of cathepsin X and elucidated with different biochemical methods that cathepsin X can be activated by cathepsin L, a protease that is known to get secreted by activated osteoclasts.

Applying MALDI-TOF analysis we showed that the chemokine SDF-1, which is secreted by bone marrow stromal cells, can be readily digested with the carboxymonopeptidase cathepsin X. SDF-1 is a highly potent chemoattractant and a mediator of cell adhesion for HSPC. Migration assays with cathepsin X-digested SDF-1 showed a significant decrease in migration of HSPC compared to the non-truncated chemokine indicating that the protease is capable to inactivate SDF-1α. Furthermore, cathepsin X can convert the other isoform SDF-1β to SDF-1α.

Current studies focus on the interaction between HSPC and their niche, especially the involvement of secreted proteases of the cathepsin family, their regulation and their extracellular substrate specificity to investigate the trafficking of HSPC in more detail.

1 University Hospital Tübingen, Department of Radiology, WaldhörnlestraÕe 22, 72072 Tübingen, Germany, r.bantleon@uni-tuebingen.de 2 University Hospital Tübingen, Department of Diagnostic and Interventional Radiology 3 University Hospital Tübingen, Institute of Clinical and Experimental Transfusion Medicine 4 University Hospital Tübingen, Department of Thoracic Cardiac and Vascular Surgery 5 University of Tübingen, Department of Medical Biometry 6 University Hospital Tübingen, Departments of Orthopaedics 7 University of Tübingen, Institute of Pathology PP 72 (A014) Nanoparticles and Polyethylenimine Modulate the Adhesion of human Mesenchymal Stem Cells to Endothelium

Purpose: Aim of the study was to evaluate the influence of labeling procedures using clinically approved small particles of iron oxide (SPIO) with or without transfection reagent (TA) on adhesion of human mesenchymal stem cells (MSCs) to endothelium in a flow chamber model.

Materials and Methods: Adult human MSCs were isolated from the bone marrow of patients undergoing orthopedic operations. The MSC populations, characterized by flow cytometry and in vitro differentiation, were treated with 200 μg iron/ml SPIO or with 60 μg iron/ml SPIO (Resovist^®, Schering) alone or in combination with the TA (JetPEI^®, polythylenimine, Polyplus Transfection) for four hours. Directly after labeling the adhesion of MSC on confluent human endothelial cell layers was tested and quantified in a dynamic flow model using small slides (μ-Slide I, Collagen IV, IBIDI) and software-supported phase contrast microscopy (Axiovert, Zeiss, Oberkochen). Quantification of the cellular total iron load, determination of the cellular viability, mixed lymphocyte reaction (MLR) and electron microscopy were performed additionally.

Results: Treatment of MSCs with iron nanoparticles plus polyethylenimine increased the adhesion capacity of MSCs, whereas treatment of MSCs with iron nanoparticles alone had no significant influence, but treatment with polyethylenimine alone decreased the adhesion capacity of MSCs. Blocking of adhesion receptors CD44 and CXCR4 with antibodies reduced the adhesion significantly in the control group and the nanoparticles plus polyethylenimine group, but not in the polyethylenimine alone group. Treatment of MSCs had no effect on lymphocyte proliferation in the MLR. Additionally, labeling of MSCs did not affect the differentiation capacity of MSCs in vitro.

Conclusion: Treatment with nanoparticles and/or polyethylenimine modulates the adhesion of human MSCs to Endothelium.

These engineering processes could be the first steps towards the production of customized stem cell grafts with adjusted adhesion and homing properties.

1 NMI Natural and Medical Sciences Institute at the University of Tuebingen, MarkwiesenstraÕe 55, 72770 Reutlingen, Germany, christian.freudigmann@nmi.de 2 TETEC Tissue Engineering Technologies AG, Reutlingen, Germany PP 74 (C001) Non-destructive methods for monitoring the vitality of chondrocyte-based medicinal products

European regulatory bodies have recently outlined stringent quality control guidelines for the production of cell-based medicinal products (CBMPs). CBMPs are now subject to a central marketing authorization by the EMEA. The characterization of a CBMP must encompass all components present and the combined final product. For products containing cells together with scaffolds, as frequently in tissue engineering products, the required characterization is challenging. The direct access to cells is not possible without destroying the product; however cells have to be characterized in terms of identity, purity, viability, and potency. This highlights the need for non-destructive quantitative methods to describe relevant biological activities.

In this study we evaluated the suitability of several methods to describe the vitality of chondrocytes in monolayer cultures and in three-dimensional scaffolds as used in autologous chondrocyte transplantation for the treatment of cartilage defects. Metabolic parameters and cell viability were analyzed.

Viability of cultures was determined indirectly by the measurement of lactate dehydrogenase (LDH) activity in the culture medium. LDH is released into the medium upon cell damage or lysis. The activity of the released enzyme corresponds to the number of dead cells. To conclude from the measured LDH activity to the number of dead cells in the culture, the intracellular LDH activity of the cells has to be determined. A mean LDH activity of 0.19 U/106 cells (sd 0.04) was determined for human chondrocytes.

The metabolic activity was analyzed by the consumption of glucose. Glucose concentrations in the culture medium were measured and consumption rates calculated. Already after 24 hours a decrease of glucose could be detected.

Here we presented quantitative non-destructive assays which are suitable for the quality control of CBMPs, as shown exemplarily with chondrocytes used for ACT.

All assays can be performed using culture supernatant and results are obtained with a few hours. This is an important aspect because the assays have to be integrated into the production process. These assays could represent significant elements towards regulatory compliant manufacturing of CBMPs meeting specific quality criteria.

Acknowledgements

We would like to acknowledge the German Ministry for Education and Research (BMBF) for financial support (FKZ 0315579B).

1 Eberhard Karls University, Department of Plastic, Reconstructive, Hand and Burn Surgery, BG-Trauma Center, Schnarrenbergstrasse 95, 72076 Tuebingen, Germany, Manuel.Held@student.uni-tuebingen.de 2 Institute of Anatomy, University of Tuebingen, Germany PP 75 (C002) A novel Collagen Cell Carrier (CCC) for the regeneration and reconstruction of skin and soft tissue: An in vivo evaluation.

Background: Skin defects caused by burns, venous and diabetic ulcers or acute injury are still difficult to handle. Thus, there is a high need for functional and cost-effective skin substitutes. In our previous in vitro studies on Collagen Cell Carrier (CCC) as a new extra cellular matrix, we demonstrated an excellent cell proliferation and differentiation. The first in vivo studies showed a good biocompatibility and biodegradation of this matrix. For this reason we developed a rat model study to analyze its suitability as a skin substitution device in superficial wounds.

Methodology/Principal Findings: CCC was used as wound coverage on simulated superficial wounds in 14 adult male Lewis rats. For comparison, we covered in the same amount of animals superficial wounds using Suprathel and Biobrane. Subsequently, we evaluated in a twelve week analysing period speed of healing, skin elasticity, blood flow and oxygen level of the skin. As the analysis has shown, the mean time of wound healing of the lesions covered with CCC is comparable to the time of wound healing achieved in Suprathel and Biobrane covered wounds. We also could not find significant differences in skin elasticity between these groups.

Conclusions/Significance: As our rat study was able to show CCC displays comparable wound healing and elasticity values to already established skin substitution devices. Further investigations will help to determine the role of CCC in regenerative and reconstructive medicine.

1 Eberhard-Karls-Universität, Department of Urology, Hoppe-Seyler-StraÕe 3, 72076 Tübingen, Germany, gerhard.feil@med.uni-tuebingen.de 2 Allg. öffentl. Bezirkskrankenhaus Hall in Tirol, Austria PP 77 (C004) Biocompatibility of fibrin glue as stabilizer for tissue-engineered matrix-free urothelium

Objectives: For tissue-engineering of urothelial cell-matrix implants appropriate biodegradable matrices are a continued challenge concerning biocompatibility, stability, and degradation in vivo. To overcome these problems, autologous urothelium generated in vitro without matrices or scaffolds might be an alternative. However, matrix-free urothelium (MFU) is unstable and thus has to be stabilized for reconstructive surgery, e.g. of the urethra. Aim of the study was to prove 1) fibrin glue as a stabilization factor concerning influence on the viability of fibrin glue-sprayed human urothelial cells (HUCs) and for surgical manipulation of MFUs sprayed with fibrin glue and 2) to investigate the outcome of transplanted MFUs sprayed with fibrin glue in a pilot nude rat model.

Methods: The influence of fibrin glue on the viability of proliferating and confluent monolayer HUC cultures was analyzed with the metabolic WST-1 assay. Seven enzymatically detached MFUs established from three different primary HUC lines were sprayed with fibrin glue and investigated for mechanical stability. For verifying the outcome in vivo, MFUs were sprayed with fibrin glue and sutured on the musculus rectus abdominis of athymic rats. For in vivo tracking, HUCs have been labelled with fluorescent PKH26 cell linker. Transplants were examined histologically and immunologically for epithelial pancytokeratin (AE1/AE3) after 7 days.

Results: Viability of fibrin glue-sprayed HUC cultures both in the proliferative and confluent phase reached up to 62% and 89%, respectively, of the control group at day seven. MFUs sprayed with fibrin glue after detachment demonstrated a good mechanical stability compared to unsprayed MFUs. The fibrin glue-sprayed MFUs were well handable with surgical instruments. In performed cryosections of MFUs at day 7 after transplantation the integration in the target tissue and the epithelial phenotype could be demonstrated. Fibrin glue was nearly degradated. There was no inflammatory reaction.

Conclusions: Clinical application of tissue-engineered MFU requires stabilization factors due to its mechanical instability. Spraying with fibrin glue enhanced the mechanical stability of MFUs so that they could be well manipulated with surgical instruments. The impact of fibrin glue on the vitality of HUCs was considerably low. These findings suggest fibrin glue as biocompatible stabilizer for urothelial constructs generated in vitro.

1 University Stuttgart IGVT, Medical Interfacial Engineering (MGVT), NobelstraÕe 12, 70569 Stuttgart, Germany, iris.dally@igb.fraunhofer.de 2 Fraunhofer Institute for Interfacial and Biotechnology IGB, Stuttgart, Germany 3 Klinik Schillerhöhe, Gerlingen, Germany PP 78 (C005) In vitro development of a vascularised tracheal patch to restore airway defects after resection

Background: Airway defects occurring after cranial pneumonectomy because of e.g. cancer, are associated with persistent contamination and difficulties in the residual lung. Therapeutic interventions are limited, and the outcome for the patient is often fatal. A huge variety of graft materials and transplantation approaches have been applied to generate a clinically applicable tracheal substitute. The most of them include drawbacks like: lacking quality and quantity of autologous grafts, immunogenicity of allogenic grafts, and loosening of alloplastic implants. Vascularisation remains a critical obstacle in engineering thicker, metabolically demanding organs of sufficient size. We developed techniques to generate a 3-D, nonimmunogenic, bioartifical, human tissue with an innate vascularization, which is sufficient to meet the implant's demand for oxygen and nutrients.

Methods: A porcine jejunal segment, consisting of the intestinal lumen, the capillary bed and an arterial and venous pedicle, was obtained from a 3-month-old pig for scaffold generation. The next steps have been realized in our certified GMP manufacturing unit according to German Drug Act. For chemical decellularization, the arterial pedicle was connected to a circulating bioreactor system and perfused with desoxycholate solution. Afterwards the matrixes were washed with PBS and incubated in DNAse to remove porcine DNA. The acellular vascular structures were reseeded with endothelial cells and the scaffold lumen with autologous fibroblasts and muscle cells. The engineered graft was cultivated in vitro in a computer-controlled bioreactor system.

Results: Histological analyses confirmed the complete decellularization, only cellular and DNA residues were detectable by H&E staining and Feulgen reaction. Elimination of desoxycholate was analysed and values of endotoxins were GMP conform. The injected cells repopulated the decellularized vascular structures and expressed characteristic vascular surface markers CD 31 and vWF. Cellular metabolic activity of 3D-construct was proven by functional tests.

Perspectives: This project unlocks the door for the clinical application of bioengineered airway tissue in pathologies. To check the biocompatibility of the matrix animal experiments will be conducted. The next step of the project will be achieving the manufacturing license in accordance with the German Drug Act.

1 NMI Reutlingen, Regenerative Medicine II, Markwiesenstr. 55, 72770 Reutlingen, Germany, jana.mueller@nmi.de 2 TETEC Tissue Engineering Technologies AG, Reutlingen, Germany PP 80 (B002) Chondrogenic differentiation of mesenchymal stromal precursor cells (MSCs) in the absence of dexamethasone

Objective: The aim of our studies is the future application of MSCs in regeneration of degenerated intervertebral discs. Bone marrow-derived MSCs were differentiated into the chondrogenic lineage in vitro, to estimate the functionality of injectable hydrogels as cell carrier systems which are able to deliver and anchor the cells within the disc tissue. Chondrogenic differentiation is inducible by a standard medium first published in 1982 by Johnstone and colleagues. Beside TGFÕ3 as inducer, it contains dexamethasone which however increases the risk of steroid hormone related untowarded effects. Therefore it is advantageous to minimize the addition of biologically active compounds. In this study we analyze the potential for chondrogenic differentiation after excluding dexamethasone from the culture medium.

Material and methods: MSCs were extracted from bone marrow (provided by BG Trauma Clinic Tübingen) of patients via density gradient centrifugation and were expanded in monolayer. All procedures were approved by the local ethics committee. MSCs were embedded in gelatin hydrogels for chondrogenic differentiation. Chondrogenesis was induced by medium containing 10 ng/ml TGFÕ3. Specifically, 0.1 μM dexamethasone was added to, respectively, excluded from the medium. After culturing for four and nine weeks chondrocyte markers, such as collagen type II and aggrecan were analyzed via RT-qPCR and localized by histochemical staining to determine the state of differentiation.

Results: Chondrogenically differentiated MSCs embedded in gelatin hydrogels produced typical chondrocyte markers. Importantly, in the absence of dexamethasone the quality of chondrogenic differentiation was not diminished as judged by expression of the markers.

Conclusion: For clinical applications it is not sufficient to have an injectable cell system, but also to use differentiation protocols which can be approved by the regulatory agencies. Therefore the employment of biologically active components that increase the risks of side-effects has to be minimized. We could show that dexamethasone, a component of the standard differentiation medium, is not required for a successful chondrogenic differentiation of MSCs. Our established model is one first step towards the clinical application of differentiated MSCs in disc regeneration.

1 Universidad de Granada, Histology, Avenida de Madrid 11, 10012 Granada, Spain, acampos@ugr.es 2 Hospital Universitario de Granada, Granada, Spain PP 81 (B003) Expression of chondral differentiation genes as indicators of quality control in human chondrocytes cultured for cell therapy

Introduction: Potential cell differentiation is one of the most important parameters influencing the clinical results of cell therapy using autologous cultured chondrocytes. However, the specific changes in chondral differentiation capability that happen on cultured human chondrocytes along time in culture remain unknown. In this study we analyzed the expression of several key chondral differentiation genes to select the most appropriate passages for use in cell therapy.

Methods: Human hyaline chondrocytes were isolated from small biopsies of the articular surface of the knee joint using collagenase digestion at 37° for 4 h. Primary cell cultures were established using DMEM medium supplemented with 10% fetal serum and growth factors. Comprehensive gene expression analysis was carried out using Affymetrix Human Genome U133 plus 2.0 arrays on 8 successive cell passages, and genes associated to chondral differentiation were selected.

Results and discussion: Our gene expression analysis of markers of chondral differentiation showed several differences among the successive cell passages analyzed here. On the one hand, several markers of chondral differentiation became significantly upregulated in the third passage, including aggrecan, chondroitin-sulfate and type II-collagen. On the other hand, the gene encoding for aggrecanase, which plays an important role on aggrecan degradation and remodeling, was downregulated in chondrocytes corresponding to the third cell passage. These findings show that cells at the third passage could be functionally appropriate for use in cell therapy and tissue engineering of the articular cartilage.

Conclusions: These findings show that cells at the third passage could be functionally appropriate for use in cell therapy and tissue engineering of the articular cartilage.

Supported by GIT- CTS-115.

1 Universidad de Granada, Histology, Avenida de Madrid 11, 10012 Granada, Spain, acampos@ugr.es 2 Hospital Universitario de Malaga, Spain 3 Hospital de Viseu, Portugal PP 82 (B004) Gene expression and K/Na index as indicators of cell viability in transdifferentiated human endothelial cells cultured for clinical use

Introduction: Cell viability is one of the most important parameters influencing the clinical results of cell therapy. In the present study we investigated gene expression and K/Na index as indicators of cell viability of endothelial-like cells generated for tissue engineering as result of human Wharton's Jelly stem cells (HWJSC) transdifferentiation.

Material and Methods: Primary cultures of HWJSC were established from 10 human umbilical cords using enzymatic methods. HWJSC were cultured in Amniomax medium. To induce the endothelial transdifferentiation of the HWJSC, these cells were cultured in M199 medium supplemented with ECGF. Then, total RNA was extracted from transdifferentiated endothelial-like cells (Tr-ELC)) and a comprehensive gene expression analysis was carried out by using Affymetrix Human Genome U133 plus 2.0 arrays. Cytoscape and BiNGO programs were applied. To analyze the K/Na index, we quantified the ionic contents of Na, and K by electron probe X-ray microanalysis using a Philips XL30 scanning electron microscope with an energy-dispersive detector EDAX. In both studies HWJSC were use as a control.

Results and discussion: The analysis of the genes demonstrated differences between specific pathways and gene functions in both types of cells (Tr-HWJSC and HWJSC). The results were: structural component of the cell (36,5%/38,5%), growth and proliferation (31, 7%/30%) apoptosis and cell death (0%/3,1%) and other functions (30,1%/21,2%). K/Na index was 1.6 higher in Tr-HWJSC than in HWJSC.

Conclusion: Gene expression and K/Na index found in this work suggest that transdifferentiated human endothelial cells show the highest levels of cell survival, which supports the idea of using these cells for vascular tissue engineering purposes.

Supported by GIT-CTS115.

1 Fraunhofer EMB, Zelldifferenzierung, Paul-Ehrlich-Str. 1-3, 23552 Lübeck, Germany, sabine.nagel@emb.fraunhofer.de 2 Department of Plastic and Hand Surgery, Burn Centre, Lübeck, Germany PP 83 (B006) Multipotent human sweat gland-derived cells accelerate skin regeneration in a mouse model

In mammalian skin, stem cells reside in the hair follicle bulge areas, epidermis, dermis, subcutaneous adipose tissues and skin appendages such as sweat glands and sebaceous glands. Since it is already shown that other glandular tissues like pancreas and salivary glands are sources of mulitpotent stem cells, we characterised sweat gland-derived cells, which we have identified as a source of stem cells with multilineage differentiation potential. Stem cells from human sweat glands showed a high proliferation activity and remarkable self-renewal in vitro. This stem cell population is characterized by expression of the stem cell associated marker filament Nestin in more than 80% of the cells. Expression of further stem cell markers like Oct-4, Nucleostemin, KLF4 and Nanog could be shown on mRNA and protein level. The differentiation potential crosses lineage boundaries into cell types of ectoderm (e.g. PGP 9.5, NSE and NF), mesoderm (e.g. αSMA, AP und PPARγ) and endoderm (e.g. Amylase and Albumin). Additionally, sweat gland-derived cells expressed the epithelial markers CK14, CK15, CK18 and CK19. The isolation method of sweat gland-derived cells based on mechanical and enzymatic digestion of human axillary and abdominal skin, isolation of sweat glands and their cultivation on collagen-coated cell culture dishes. The first results of an in vivo mouse model with a second-degree burn of the skin suggest that cells derived from sweat glands are capable to accelerate skin regeneration. It turned out that these cells ameliorated re-epithelialisation and vascularisation. Therefore, sweat glands promise to provide an easily accessible and abundantly available, autologous source of adult stem cells for skin replacement therapies.