NeremRobert M.
Georgia Institute of Technology
Parker H. Petit Institute for Bioengineering and Bioscience, Building: Petit IBB Office: 1106 315 Ferst Drive, Atlanta GA 30332-0363, USA, robert.nerem@ibb.gatech.edu
Regenerative Medicine: Unlocking the Future
Although the basic concept of using a more biologic approach to the development of medical implants goes back to the first half of the 20th century, the modern era only began a quarter century ago. The initial focus was on replacement tissues, i.e. developing substitute tissues outside of the body for implantation into the body, with skin substitutes being some of the first targets. Although in the 1990s these had moved into commercial development, by the beginning of this decade commercial activity had for the most part encountered financial difficulties. Even while commercialization in this area was going through its “ups and downs,” the science was moving ahead. In the 1990s stem cell technology began to emerge and the focus on replacement evolved to include repair and regeneration with the result that today there are a variety of approaches being pursued. The interest in stem cells is because a major issue, whether one is talking about replacement, repair, or regeneration, is cell source. As exciting as the advances in the biology of stem cells and progenitor cells has been, regenerative medicine is more than stem cells. What is now becoming of interest to many in this field; however, is the concept of regeneration. The regenerative processes observed in species such as the newt and salamander have been replaced in the human by processes of inflammation and scar tissue formation. For the human, the extent to which we can jump start the regenerative process and supply the appropriate ingredients will dictate are success in achieving tissue and organ regeneration. It is clear, however, that no single approach will solve all problems; rather, each tissue and each pathologic condition are likely to require a different approach to obtain optimal results. Furthermore, the clinical translation of regenerative medicine will require more than scientific discovery. Regulatory agencies must develop appropriate processes regulating for safe and effective delivery of regenerative medicine strategies. Third party payers must provide the reimbursement to sustain promising approaches and reward regenerative medicine strategies that have the potential to significantly affect health care. With the industry appearing to have turned the corner and with the ever accelerating advances in the science, regenerative medicine has the potential to truly live up to the promise of delivering therapies for diseases, injuries, and disorders where currently patients have no options.
Regenerative Biology
WinklerJürgen
University Hospital Erlangen
Division of Molecular Neurology, Erlangen, Germany
Cellular Repair Strategies in Parkinson's Disease
Parkinson's disease (PD) is characterized by a progressive loss of distinct and well-defined neuronal populations in different neurotransmitter projection systems. The clinical course is slow and at present, therapeutic approaches improve motor symptoms only very limited without to halt or even reverse the progression. The pathological hallmark in PD is alpha-synuclein (ASN) aggregates observed in Lewy bodies and Lewy neurites. The dopaminergic deficit within the nigrostriatal projection resulting from neuronal degeneration within the substantia nigra pars compacta (SNpc) is associated with the motor symptoms in PD, bradykinesia, rigor, tremor and postural instability. Due to the loss of dopaminergic neurons within the SNpc PD has become an interesting target for cell-based restorative therapies. Generation and transplantation of dopaminergic cells into the nigostriatal system is intensively studied, however significant drawbacks have to be overcome before this approach may be implemented for PD paients.
Alternatively, one exciting idea for repair is the concept that one might be able to harness the adult brain's endogenous capacity for cell renewal. This is based on the observation that the adult mammalian brain has the capacity to generate new neurons in the subventricular zone (SVZ) and the hippocampus. Thus, adult neural precursor cells (aNPCs) may be recruited from these neurogenic sites toward regions affected by the disease. The ultimate goal is to differentiate aNPCs into the specific types that degenerate due to the PD underlying molecular and biochemical events.
Furthermore, the interaction between adult neurogenesis in the brain and the PD associated ASN aggregation and dopaminergic deficit may be viewed from an angle of a physiologically required cell renewal process. Impaired neurogenesis may contribute in the diseased brain to the development of specific symptoms in PD such as olfactory dysfunction and hippocampal dependent symptoms like anxiety, depression, novelty seeking and adaptive behaviors. In conjunction with this hypothesis, it is important to note that the initial central nervous ASN aggregates are observed within the olfactory bulb of PD brains and a decreased proliferation of aNPCs was described in the SVZ and the hippocampus of PD patients. Based on these concepts the seminar will focus on the interplay of adult neurogenesis with ASN pathology and the capacity of aNPCs to migrate towards regions with dopaminergic deficit. In summary, impaired adult neurogenesis may explain both some of the important non-motor symptoms preceding the motor deficit and offer the opportunity for a novel target for future restorative approaches in PD.
CanceddaRanieriTassoRobertaTortelliFedericoMastrogiacomoMaddalena
Dipartimento di Oncologia, Biologia e Genetica, Universita’ & Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi 10, 16132 Genova, Italy, ranieri.cancedda@unige.it
A Tissue Engineering Approach to Guided Bone Regeneration
A deeper comprehension of the nature of the stem/progenitor cells locally present or recruited in the bone regenerative niche and a better comprehension of their cross talk and interactions are required to elucidate cellular and molecular control mechanisms behind the bone formation/regeneration process. This is mandatory for the translation of the knowledge to the clinical application.
Bone marrow–derived mouse mesenchymal stem cells were seeded onto different porous ceramic scaffolds, and subcutaneously implanted in immunocompetent syngenic or immunodeficient mice for different times. We observed bone formation by cells of host origin within the implanted scaffold after 6–8 weeks. Seeded MSC appeared pivotal at the early stages of the tissue development. No bone formation was observed when the porous ceramic cubes were implanted without the addition of MSC. To investigate the role of seeded MSC in triggering and/or controlling the process, GFP+ MSC/scaffold constructs were implanted in syngenic, GFP- recipients. Implants were harvested after 3, 7, and 11 days, and collagenase-digested to generate single-cell suspensions. Recovered cells were sorted, based on GFP expression, in order to distinguish GFP+ implanted MSC and GFP- recruited cells. We identified two subgroups of cells, distinct for the expression of CD14 and CD45. CD14 + CD45+ double-positive cells (DP) presented characteristics similar to the cells that were not specifically recruited into implanted empty scaffolds. On the contrary, the number of CD14-CD45- double-negative cells (DN) progressively increased from 3 to 11 day implantation time. Day 7-DN cells were enriched in CD31+ endothelial cells, while day 11-DN cells were enriched in CD146+ cells and possessed osteogenic properties.
The origin of the bone cells and the ossification type were strictly dependent on the nature and commitment of the seeded cells. Indeed, MSC implants led to formation of bone of host origin through the activation of an endochondral ossification process while an intramembranous ossification directly performed by the seeded and implanted cells was observed in implants seeded with murine osteoblasts.
SchäferKarl-Herbert
University of Applied Sciences Kaiserslautern, Life Sciences, Department of Computer Sciences and Microsystems Technology, AmerikastraÕe, 66482 Zweibrücken, Germany, karl-herbert.schaefer@fh-kl.de
The enteric nervous system: a suitable source for autologeous neural stem cells
Evidence of neurogenesis in the postnatal human ENS brought new perspective for cell therapy and neural regeneration. Nestin-positive cells can be found within the adult enteric nervous system during the whole life span, even in aged people. These cells constitute a potential source for autologeous neural stem cells. The appendix as a part of the gastrointestinal tract which is easily accesible and removable might be the appropriate location with a sufficient amount enteric nervous tissue where these cells can easily be harvested. Appendices from adults and children from 6 month to 69 years were collected from appendectomies or right hemicolectomies for cancer surgery. From each appendix tissue samples were collected and processed for immunohistochemistry. The remaining tissue was enzymatically digested and myenteric and submucous plexus isolated. The isolated neural tissue could be harvested, dissociated and taken into culture. After 6 day in culture free floating neurospheres as well as neurons and glia cells were easily to discriminate. Neurospheres were dissociated and resuspended in an extracellular matrix gel to mimick the transplantation situation in vivo. Immunostaining for neuronal and glial markers were performed and cells analyzed. Sections from the parrafin blocks were stained for neuronal, glial and stem cell markers.
Using the appendix as a potential target opens up a new perspective which might lead to a relatively unproblematic harvest of neural stem cells without endangering the patient by risky surgical procedures or immunological incompetence. The neural stem cells can be used for both peripheral and central nervous system restoration.
Cell Based Therapies for Cardiovascular Applications
Cell based therapeutic concepts are focused on two mayor principles: a) de novo tissue creation (tissue engineering) for the repair/replacement, and b) cell transplantation for the regeneration of defective organ tissues. With regard to cardiovascular applications an array of new therapeutic options is under development and may revolutionize current treatment modalities. Here, we present research on cell based therapies addressing cardiovascular disorders such as congenital heart disease, as well as degenerative heart valve, vascular and myocardial disease.
Tissue engineering technologies are aiming at the creation of living replacements with the capacity of self-repair and growths. Ideally, these replacement are based on the patients own cells preventing the risk of immunological and infectious (e.g. zoonosis) complications. Such autologous tissue engineered constructs can be created by in vitro cell seeding onto biodegradable scaffold matrices. These scaffolds serve as an initial guiding structure for neo-tissue development before degradation. The in vitro tissue maturation can be enhanced by bioreactor technologies mimicking e.g. a physiological environment. The growth potential of tissue engineered living constructs was demonstrated in preclinical animal tests covering the full biological growth cycle. Several cell sources were investigated ranging from fully differentiated cells to various human stem cells. Bone marrow derived and blood derived cells were used for heart valve tissue engineering. Recently, prenatal stem cells which can be obtained during pregnancy before birth have been successfully utilized for cardiovascular tissue engineering, a technology which may allow the treatment of congenital heart malformations with autologous, growing replacement material directly after birth.
Cell transplantation using bone marrow and blood derived progenitor cells has been recently introduced to clinical use for the treatment of ischemic myocardial disease. Although showing some improvement of cardiac performance, the long-term benefits for patients as well as the underlying cellular mechanisms remain unclear. Mayor limitations as to current single cell transplantation are insufficient cell delivery, integration and function. In order to overcome these limitations, a new micro-scale tissue engineering approach using micro-tissue cell aggregates instead of single cells has been developed and is currently tested in animal models.
Medical Biotechnology
StallkampJanBrodeTobias
Fraunhofer Institut für Produktionstechnik und Automatisierung IPA, NobelstraÕe 12, 70569 Stuttgart, Germany, stallkamp@ipa.fraunhofer.de
Automation meets Medical Biotechnology
On the first view automation might seem a subject only for car or food production. But automation technologies offer a wide range of tools to ease and improve the work in biotechnical companies dealing with solutions for medical applications. Automation can essentially reduce time for processes e.g. for screening, make processes reproducible and stable and delivers accurate documentations. Moreover it is the core-technology to make biotechnical products available for everybody. In the near future automation might be a key to better, more reliable results or faster tracks from basic research to market.
Over the past fourteen years, the department for production and process automation at Fraunhofer IPA specialized in the development of solutions for automation of biotechnological processes. This paper will give an overview of traditional and new technologies in the field of automation technology using the example of a tissue factory and the potentials for research in medical biotechnology. It shows concepts for fast and precise handling methods, measurement and information technology as well as the current limitations of automation technology today.
Center for Regenerative Medicine and Department of Orthopaedic Surgery, Center for Medical Research, Tübingen, Germany
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.
ChristBruno
Martin-Luther-Universität Halle-Wittenberg, Klinik und Poliklinik für Innere Medizin I, Molekulare Hepatologie, Zentrum für Angewandte Medizinische und Humanbiologische Forschung (ZAMED), Heinrich-Damerow-StraÕe 1, 06907 Halle/Saale, Germany, bruno.christ@medizin.uni-halle.de
Stem cell-derived hepatocytes for the treatment of liver diseases
Hepatocytes execute most of the metabolic functions as the liver does as a whole. Therefore, hepatocyte transplantation is a feasible alternative to whole liver transplantation. This novel medicinal option of treatment is based on the assumption that transplanted hepatocytes integrate into the host liver, proliferate at the site of tissue integration and take over the hepatic synthetic capacity in the long-term range thus substituting for the diseased host liver tissue. However, clinical translation is hampered likely because of two major reasons: 1. Cadaveric donor livers for the isolation of hepatocytes are scarce if available at all and 2. The cells isolated from these marginal donor organs are largely of poor quality and quantity. Therefore, alternative cell sources have to be established to further prompt the clinical success of hepatocyte transplantation. Their multiple differentiation potential and nearly unlimited availability may render mesenchymal stem cells an attractive alternate resource. Recent studies demonstrated the potential of mesenchymal stem cells derived from various tissues to differentiate into hepatocyte-like cells in vitro continuing to feature specific hepatocyte functions after transplantation into the regenerating livers of mice or rats both under injury and non-injury conditions.
Charles University in Prague, 1st Faculty of Medicine, Institute of Anatomy, U Nemocnice 3, 12800 Prague 2, Czech Republic, bdvorankova@seznam.cz
Ludwig-Maximilians University, Faculty of Veterinary Medicine, München, Germany
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.
References
GabiusH-J. The Sugar Code. Fundamentals of glycosciences. Wiley-VCH: Weinheim, 2009.a-11
HammerTimoMüllerMarinaHöferDirk
Hohenstein Institute, Institute for Hygiene and Biotechnology, Schlosssteige 1, 74357 Boennigheim, Germany, t.hammer@hohenstein.de
Fibre-bound stem cells on scaffolds induce vascularization in the modified in vivo HET-CAM angiogenesis model
Angiogenesis is an important requirement for scaffold-based tissue engineering (TE), especially for large tissue volumes. Therefore, new strategies have to be developed that can promote angiogenesis in 3-D constructs of biodegradable polymeric scaffolds. In our study we functionalized the surface of PLA-textile implants with fibronectin and collagen by a customized padding process in order to achieve an extracellular-matrix-like environment, followed by cultivation of a dense layer of human adipose-derived stem cells (ADSC) on the fibre surface. In our approach, we thought to promote angiogenesis by fibre-bound stem cell delivery of growth factors. The presumed angiogenic effect was studied in the chick's chorioallantoic membrane assay, used as an in-vivo angiogenesis model (Hen's Egg Test, HET-CAM). Implants with fibre-bound stem cells (verum) were applied to the CAM and compared to implants seeded with fibroblasts (control) for the formation of new blood capillaries into the scaffold. After 4 days, a dense network of capillaries was observed in the verum. In contrast, fibroblast-covered implants were not vascularized. Whole mounts of the CAM and the implants were studied by microscopy, confirming the directed growth of blood vessels into the biofunctionalized scaffold. Whole mounts were further processed for immunohistochemistry. The formation of new capillaries was shown with antibodies against the intermediate filament desmin in whole mounts and in cross-sections of embedded CAM/implants. In a parallel experiment, the angiogenic effect was also achieved with fibres finished with vascular endothelial growth factor, VEGF. Our results show that fibre-bound stem cells induce vascularization. The modified HET-CAM test is an ideal in vivo model for the evaluation of angiogenic processes and for TE experiments with stem cells.
Biomaterials and Interfaces I
BoccacciniAldo R.
Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, 91058 Erlangen, Germany, aldo.boccaccini@ww.uni-erlangen.de
Multifunctional polymer/bioceramic composites for bone tissue engineering
Multifunctional scaffolds for bone tissue engineering are being developed combining biopolymers, bioactive ceramics (or bioactive glasses) and biomolecules [1,2]. After a summary of the state-of –the-art in the field of bone tissue scaffolds, including a discussion of the challenges ahead, our recent research results on bioactive silicate scaffolds coated and infiltrated with synthetic or natural biopolymers will be discussed. The fabrication of these scaffolds involves the standard replica technique [3] which leads to porous structures exhibiting a highly interconnected pore network (>90% porosity) and fully densified glass-ceramic struts. The coating of foam struts by a thin biodegradable polymer layer containing functionalised nanoscale inorganic particles increases the mechanical competence of scaffolds (compressive strength, work of fracture) and induces the formation of a biomimetic nanostructured hydroxyapatite surface layer upon immersion in relevant physiological fluids. Recent developments of model multifunctional scaffolds containing carbon nanotubes will be also discussed, describing the electrophoretic deposition technique developed to manipulate and control the deposition of electrically charged nanoscaled particles and other nanostructures on scaffold surfaces [4]. Nanostructuring 3D porous scaffolds has the benefit of mimicking the structural scale of proteins and it is thus an attractive approach to induce cell attachment and proliferation, leading to enhanced biological performance of the scaffolds [5]. Concepts for the development of multifunctional scaffolds based on biodegradable polymer/bioceramic composites will be finally discussed highlighting the relevance of biomaterial scaffolds in bone tissue engineering strategies. In this context, the application of bioactive glasses of new silicate compositions, doped with specific metallic ions, which are proposed for the controlled release of therapeutic ions in bone tissue engineering strategies, will be discussed and their potential effect on angiogenesis will be assessed based on preliminary in-vitro and in-vivo evidence.
FrancisL.et al.Acta Biomaterialia, 6. 2010; 2773–2786.a-13ChenQ. Z.et al.Biomaterials, 27. 2006; 2414–2425.a-14BoccacciniA. R.et al.J. Royal Society Interface, 7. 2010; S581–S613.a-15BoccacciniA. R.et al.Comp. Sci. Technol., 2010in press10.1016/j.compscitech.2010.06.002.a-16LendleinAndreas
Institute of Polymer Research, GKSS Research Center Geesthacht GmbH, KantstraÕe 55, 14513 Teltow, Germany, andreas.lendlein@gkss.de
Biomaterials for Regenerative Medicine
Different strategies are being pursued for regenerative therapies, including induced auto-regeneration, somatic cell therapy, and tissue engineering. Biomaterials play an essential role in the area of induced auto-regeneration, especially when critical defects have to be restored. The role of a biomaterial has to be that of an active participant with the task of facilitating, mimicking, and/or augmenting existing biological processes and essentially serving as a synthetic mimic of ECM. The complex requirements for such (multi)functional materials can not be completely fulfilled by the materials, which are well-established in clinical application to date. The challenge for the development of the next generation of biomaterials is the integration of biological principles and molecular recognition in material design. Therefore a knowledge-based system approach is required, which is based on a fundamental understanding of the interaction between materials and biological environments [1,2].
Polymers intended for in vivo applications have to fulfill the thermal, mechanical and functional demands of the specific application one of which is their biofunctionality. Therefore, the development of polymer systems, which enable tailoring of properties by variation of molecular parameters, is of paramount importance. Often biomaterials are designed to degrade in physiological environments [3], or are loaded with bioactive substances. The combination of different functions in one material is a scientific challenge [4,5]. Examples for such multifunctional polymers and their potential application in regenerative therapies will be presented [6].
ShastriV.P., LendleinA.MRS Bulletin, 2010; 35in press.a-18HofmannD., Entrialgo-CastañoM., KratzK., LendleinA.Adv. Mater, 2009; 21:3237–3245.a-19BehlM., RazzaqM., LendleinA.Adv. Mater, 2010published online on June 23, 201.a-20MironovVladimir
Medical University of South Carolina, Department of Regenerative Medicine and Cell Biology, MUSC South Carolina, USA, mironovv@musc.edu
Organ printing
Organ printing can be defined as a computer-aided robotic layer-by-layer additive biofabrication of 3D functional human tissue and organ constructs using self-assembling tissue spheroids as building blocks. The fundamental biophysical principle of organ printing technology is tissue spheroid fusion, driven by tissue surface tension. Organ printing includes three essential steps: (i) pre-processing or design of an “organ blueprint”; (ii) processing or bioprinting of 3D tissue and organ constructs using tissue spheroids as a “bioink”; and (iii) post-processing or accelerated tissue maturation. Using an analogy to text printing, it is possible to identify several critically important integral elements of organ printing technology: “blueprint” or computer-aided design of human organ construct in STL file, “bioink” or self-assembling tissue spheroids, “biopaper” or bioprocessible and biomimetic hydrogel, and a “bioprinter” or robotic dispensor. Organ printing represents a solid scaffold-free “bottom-up” modular approach in tissue engineering and a potentially superior alternative to the traditional solid scaffold-based “top-down” approach. Bioprinting a “built in” perfusable intraorgan branched vascular tree is critically important for maintaining the viability of bioprinted 3D thick human organ constructs. Combined employment of solid, mono-lumenal and multi-lumenal tissue spheroids is sufficient to bioengineer a complete intraorgan branched vascular tree. The feasibility of bioassembly of linear and branched segments of a vascular tree, using vascular tissue spheroids, has already been demonstrated. However, development of novel technologies for scalable biofabrication of tissue spheroids as well as new methods for accelerated tissue maturation are critical for successful clinical translation and commercialization of organ printing technology. The future bioassembly line for human organ bioprinting plants will include cell sorters, tissue spheroid biofabricators, robotic bioprinters and perfusion bioreactors integrated with novel devices for non-invasive and non-destructive biomonitoring of tissue and organ maturation and viability. In the short term, this emerging organ printing technology will be instrumental for robotic biofabrication of 3D models of vascularized human tissues and organs for modeling human diseases, drug discovery and toxicology research. In the long term, organ-printing technology can solve one of the most pressing clinical challenges: the shortage of human organs for transplantation.
Institute of Textile Technology and Process Engineering, Nonwoven Technologies, Körschtalstrasse 26, 73770 Denkendorf, Germany, cornelia.fano@itv-denkendorf.de
Herzchirurgische Klinik der Universität München, Germany
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.
GummertalJ. F.Cardiac surgery in Germany during 2007.a-25
DE 28 06 030 C2.a-26GulbinsH.Development of an artificial vessel lined with human vascular cells.a-27JustLothar1HauÕmannSusanne1MackAndreas1RohdeManfred2SchmidtTimo1
Institute of Anatomy, Experimental Embryology, Österbergstrasse 3, 72074 Tübingen, Germany, !just@anatom.uni-tuebingen.de
Helmholtz Centre for Infection Research, Braunschweig, Institute of Anatomy, Tübingen, Germany
A novel thin and mechanically stable collagen based scaffold for tissue engineering approaches
The biological function of adherent cell populations strongly depends on the physical and biochemical properties of extracellular matrix molecules. Therefore, numerous biocompatible cell carriers have been developed to specifically influence the cell attachment, proliferation, cellular differentiation and tissue formation for diverse cell culture applications and cell-based therapies.
In the present study, we evaluated the mechanical and the cell biological properties of a novel thin and planar collagen scaffold. The cell carrier is based on fibrillar bovine collagen I and exhibits a low material thicknesses coupled with a high mechanical stability as measured by tensile tests. Cell culture experiments with various cell populations demonstrated the in vitro biocompatibility of this scaffold and its impact on cell viability, proliferation and cell differentiation analysed by BrdU-proliferation assay, immunocytochemistry, WST-1 assay, live imaging and electron microscopy. Both, the mechanical characteristics and the in vitro biocompatibility of this collagen I carrier facilitate the engineering of thin transferable tissue constructs and thus opens new possibilities in the fields of cell culture techniques and regenerative medicine.
MPI for Colloids and Interfaces, Department of Biomaterials, Am Muehlenberg 1, Wissenschaftspark Golm, 14476 Potsdam, Germany, KrishnaPrasad.Kommareddy@mpikg.mpg.de
Center for Biomaterial development, Institute of Polymer Research, GKSS Research Center Geesthacht GmbH, Teltow., Germany
Institute of Biochemistry, Freie University, Berlin., Germany
Ludwig Boltzmann Institute of Osteologie, Hanusch Hospital of WGKK and AUVA, Vienna, Austria
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.
RumplerMet al.J R Soc Interface, 2008; 5,27:1173–1180.a-44a-46
SchröderTimm
Helmholtz Zentrum Muenchen–German Research Centre for Environmental Health (GmbH), Hematopoiesis Group, Institute of Stem Cell Research, Ingolstaedter LandstraÕe 1, 85764 München, Germany, timm.schroeder@helmholtz-muenchen.de
Tracking stem cells at the single cell level: New tools for old questions
Stem cell driven regenerative systems are highly complex and dynamic, consisting of larges number of different cells expressing many molecules controlling their fates. For example, millions of new cells of the more than 10 different blood lineages are being produced every second throughout lifetime. Despite intensive research, many long-standing questions in stem cell research remain unsolved. One major reason is the fact that stem cell systems are usually followed by analyzing the fate of populations of cells – rather than individual cells – at very few time points of an experiment, and without knowing their individual identities. Real-time tracking of individual cells in culture, tissues or whole organisms would be an extremely powerful approach to fully understand the developmental complexity of stem cell driven regeneration. We have therefore developed culture and imaging systems to follow the fate of individual cells over long periods of time. New software is programmed, helping to record and display the divisional history, position, properties etc. of all individual cells in a culture over many generations. Our approaches also allow the continuous long term quantification of protein expression levels in living stem cells. This novel kind of quantitative data of single cell behavior and molecule expression is used as the basis for the improved generation and falsification of models describing stem cell systems. I will discuss how we use these approaches to try to find answers for long standing questions in hematopoietic and embryonic stem cell research.
Migration of hematopoietic stem and progenitor cells
Hematopoietic stem and progenitor cells (HSPCs) are thought to reside mostly in specialized niches in the bone marrow (BM) compartment. Nonetheless, a considerable number of HSPCs constitutively migrates out of the BM and enters the blood. The residence time of individual HSPCs within the blood is short (in the order of minutes), implying that the turnover of HSPCs that enter and leave the bloodstream must be high. However, the ultimate fate and functional relevance of these circulating HSPCs is largely unknown. We have demonstrated that circulating BM-derived HSPCs with short- and long-term multilineage reconstitution capacity leave the blood and traffic constitutively to multiple non-lymphoid extramedullary tissues. Here, they reside for 1–2 days until entering draining lymphatics to return to the blood and eventually the BM. The egress of HSPCs from extramedullary tissues into lymph depends on sphingosine-1-phosphate (S1P) receptors, particularly S1P1. Under physiological conditions migratory HSPCs contribute to the continuous restoration of specialized hematopoietic cells that reside in peripheral tissues. Upon exposure to Toll-like receptor (TLR) agonists, migratory HSPCs proliferate locally within extramedullary tissues and generate innate immune effector cells. Thus, HSPCs can survey peripheral organs to replenish tissue-resident hematopoietic cells and act as a source of mature leukocytes during host defence against pathogens. In the context of chronic inflammatory diseases, migratory HSPCs could contribute to the local production of innate immune cells.
Max-Planck-Institute for Metals Research, New Materials and Biosystems, Heisenbergstr. 3, 70569 Stuttgart, Germany, thedieck@mf.mpg.de
South Westphalia University of Applied Sciences, Iserlohn, Germany
Section for Transplantation Immunology and Immunohematology, Center for Medical Research, University Medical Clinic, Tübingen, Germany
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.
Medical University of Graz, Institute of Cell Biology, Histology and Embryology, Harrachgasse 21/7, 8010 Graz, Austria, julia.koenig@medunigraz.at
Department of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria
Centro di Ricerca E. Menni, Fondazione Poliambulanza, Istituto Ospedaliero, Brescia, Italy
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.
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
CHOP, Philadelphia, USA
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.
Advanced Therapies
MacLellanW. Robb
Dept. of Medicine/Cardiology at UCLA, Los Angeles, USA, rmaclellan@mednet.ucla.edu
Therapeutic Potential of Human ISL-1 Positive Cardiovascular Progenitor Cells
One of the major limitations of the cardiovascular cell therapies tested thus far is the poorly characterized cell types used, which have limited capacity for robust cardiac differentiation. Although the recently described, Isl1+ multipotent cardiovascular progenitor cell (CPC) might overcome these limitations little is known regarding its biology and prevalence in human hearts or pluripotent stem cells. We have developed strategies to identify, isolate and clone human CPCs and have characterized their differentiation potential. To identify cell surface markers that can reliably identify this population of cells without genetic engineering, we performed a microarray analysis on Kdr+ cardiac progenitors isolated from mouse ES cells. Combinatorial testing of eight potential candidate cell surface markers revealed that a combination of Flt1 and Flt4 best identified the Isl-1+ CPCs in fetal human hearts or differentiating induced pluripotent stem cells (iPSC). Analysis of isolated undifferentiated Flt1+ /Flt4+ cells demonstrated expression of CPC-specific genes including Nkx2.5, Sox18, Mef2C and c-Myc, but no markers of differentiated cardiovascular cells. Purified CPCs developed spontaneously beating colonies after 10–12 days. Immunocytochemical staining of theses colonies identified cardiomyoctyes with cross-striations expressing Troponin C and sarcomeric actin (MF20). We also identified smooth muscle cells (SMCs) by immunostaining for alpha smooth muscle actin, h-caldesmon and smooth muscle myosin. Moreover, CPCs also differentiated into ECs expressing CD31, vWF and eNOS. CPCs can also stably engraft in vivo and we are currently testing their therapeutic efficacy. The capability of human CPCs to differentiate into all three cell types of the cardiovascular lineage suggests that this progenitor cell might be a good candidate for cell replacement therapies possessing the potential to more completely regenerate damaged myocardium, but with reduced oncogenic risk compared to more undifferentiated pluripotent stem cells.
SaarikoskiPamelaSierraMariaMagnussonMattiasSasidharanRajkumarZackJeromeGalicZoranMikkolaHanna
Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California Los Angeles, 615 Charles E Young Dr. S, Los Angeles, CA, USA
Towards generation of hematopoietic stem cells in a dish
Use of human pluripotent stem cells for generating patient-specific or HLA-matched hematopoietic stem cells (HSC) would greatly improve the treatment of inherited and acquired blood diseases. However, although several blood cell types can be generated from human ES (hES) cells or induced pluripotent stem (iPS) cells, so far the ability to generate multipotential, self-renewing HSCs have failed. In order to succeed, effort needs to be put on defining the conditions that could recapitulate embryonic HSC development in vitro, and on identifying cell-intrinsic regulators that could be used for programming functional HSCs. Our first goal was to define the molecular bottlenecks that limit the functionality of hES-derived HSPCs (hematopoietic stem/progenitor cells). We used a two-step differentiation system combining embryoid body differentiation and co-culture on bone marrow mesenchymal stem cell stroma, which facilitates a dramatic expansion of human fetal liver HSPCs that retain the HSC immunophenotype (CD34 + CD38-CD90 + CD45 + , herein referred to as CD90 + HSPC) and multipotency. Our data revealed that hES-cells form CD90 + HSPCs that differentiate into myeloid, erythroid and T-lymphoid cells. However, their major shortcoming was a lack of ability to self-renew/expand in culture, leading to their rapid exhaustion. Microarray analysis comparing hES-derived and freshly isolated or cultured fetal liver CD90 + HSPCs verified that a commitment toward the HSC fate had occurred, as many critical HSC transcription factors (SCL, RUNX1 etc.) were properly expressed. However, the hES-derived CD90 + HSPCs exhibited distinct molecular defects that were shared with long-term cultured (>5 weeks) fetal liver CD90 + HSPCs, suggesting that culture induced stress impacts their cell-intrinsic program. Furthermore, the hES-derived HSPCs also exhibited unique defects not found in cultured fetal liver HSPCs, including a profound downregulation of all HOX-A cluster genes, which are known regulators of HSC proliferation/self-renewal. Interestingly, poor induction of HOX-A genes was also seen in CD90 + HSPCS from early human placentas, implying that this defect may relate to an immature, embryonic nature of hES- and placenta derived HSPCs. In summary, our study has identified key molecular programs that distinguish the hES-derived HSPCs from their fully functional in vivo counterparts. These regulators emerge as candidates for programming the HSC fate in culture.
KochSteffen1PudlasM.1BolwienC.2WallesH.1
Fraunhofer IGB Stuttgart, Dept of Cell and Tissue Engineering, Nobelstr. 12, 70569 Stuttgart, Germany, steffen.koch@igb.fraunhofer.de
Fraunhofer Institute for Physical Measurement Techniques, Freiburg, Germany
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.
Schenke-LaylandKatja
Fraunhofer IGB Stuttgart, Dept of Cell and Tissue Engineering, Nobelstr. 12, 70569 Stuttgart, Germany, katja.schenke-layland@igb.fraunhofer.de
Multiphoton Imaging of Extracellular Matrix
In addition to cells, extracellular matrix (ECM) is one of the most important components of all tissue types in the human body. It consists of fibers and networks composed of structural proteins, such as collagen or elastin. Understanding the spatial distribution of these structures within tissues and organs often provides insight to their function and basic mechanisms of action. A detailed analysis of the miscellaneous elements of the ECM is of great value in gaining structural and diagnostic information, particularly with regard to efforts in the field of Regenerative Medicine. Multiphoton-induced autofluorescence microscopy and Second Harmonic Generation (SHG) imaging have become powerful methods for the artifact-free, nondestructive evaluation of deep-tissue cells and ECM structures in their native environment. In our studies we employed multiphoton-induced autofluorescence and SHG microscopy to identify the degree of order in collagen and elastic fiber orientation in native and processed, as well as healthy and diseased tissues and organs. SHG signal profiling was used to quantify ECM damage in various cardiovascular and exocrine tissues, as well as cartilage. Our data demonstrate that multiphoton-induced autofluorescence imaging and quantitative SHG signal profiling are useful techniques for detecting ECM damage in a variety of native and tissue-engineered tissues and organs.
WolfeRussell J.LeleuxJardin A.AhsanTaby
Tulane University, Department of Biomedical Engineering, 535 Lindy Boggs Building, New Orleans, LA 70118, USA, tahsan@tulane.edu
Effect of Shear Stress Parameters on Differentiation of ESCs to Hematopoetic and Endothelial Phenotypes
Applying physical forces to embryonic stem cells can be used to study the effect of mechanical cues on signal transduction and stem cell fate. Previously, we have shown with embryonic stem cells that applied fluid shear stress can promote an endothelial phenotype. Last year, it was shown that shear stress in a similar model system can promote a hematopoetic phenotype. Given that a similar type of mechanical cue can promote differentiation to more than one phenotype, it is important to determine the parameters of the applied shear stress that may differentially regulate stem cell fate.
Differentiation towards endothelial and hematopoetic lineages includes stages which have previously been defined as the hemangioblast, hemogenic endothelium, and hematopoetic stem cell/progenitor. Characterization of these stages is based on a host of markers, including RUNX1, SCL, CD41, CD34, FLK1, VECAD, and PECAM. The transient expression of key markers and an evolving understanding of the differentiation process for these lineages require that a robust in vitro approach be utilized to determine the effect of the different parameters of applied shear stress on stem cell fate.
Using a parallel plate device to apply steady laminar fluid shear stress, we investigated the effects of shear stress magnitude, duration, and application at different stages of cellular differentiation. We have found that the application of shear stress for 1 day significantly (p < 0.05) increases both FLK1 and RUNX1 gene expression, classically indicative of endothelial and hematopoetic phenotypes, respectively. When shear stress was applied for 4 days, changes in gene expression for FLK1 and RUNX1 were more marked. However, if shear stress was applied for only 1 day after a 3 day delay, there was no longer a notable change in FLK1 expression. These results indicate that the shear stress-mediated promotion of an endothelial phenotype during the early differentiation of embryonic stem cells is lost within just a few days. As a result, slight changes in the state of the cells and/or changes in the parameters of applied physical cues can result in alternate stem cell fates.
Understanding the role of shear stress in the differentiation of endothelial and hematopoetic phenotypes will allow us to not only better understand the process of embryonic development, but to leverage in vitro the mechanisms of differentiation to generate endothelial and blood cells necessary for regenerative medicine therapies.
BrockbankKelvin
Cell and Tissue Systems Inc., Charleston, SC, USA, Kgbrockbankassoc@aol.com
Advanced Therapy Product Development and Biopreservation
Development of regenerative medicine products containing living cells and the importance of storage and transport strategy will be discussed. Short term preservation includes room temperature and refrigerated hypothermic storage methods. Long term preservation has two main two methods in use which employ either traditional freezing methods or ice-free vitrification. Both approaches have pros and cons and which is employed depends upon the final product. Vitrification avoids ice formation but has potential issues associated with cytotoxicity and cracking. Frozen cell suspensions and tissues have extensive extracellular and interstitial ice formation following traditional cryopreservation by freezing procedures. Such frozen specimens, particularly cell suspensions, may have excellent cell viability. In other cases viability may be very poor or cell viability can be good but the cells in the tissue may no longer operate as a functional unit or extensive extracellular matrix damage may be observed that correlates with poor performance in vivo. In these cases ice-free vitrification methods may have benefits. Improved post-preservation functions and matrix integrity have been observed in vitrified natural and engineered blood vessels, cartilage and heart valves. This presentation will conclude with examples of the impact that storage and transport strategies may have upon cost of manufacturing.
Maggiore della Carità Hospital, Eastern Piedmont University, Dept of Neurology, Corso Mazzini 18, 28100 Novara, Italy, mazzini.l@libero.it
Neuroscience Institute of the Cavalieri Ottolenghi Foundation, Turin, Italy
Department of Pediatrics, University of Turin, Italy
Stem Cell Transplantation and Cellular Therapy Laboratory, Division of Oncohaematology and Stem Cell Transplantation, ASO OIRM-S.Anna, Turin, Italy
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.
ShahKhalid
Massachusetts General Hospital Harvard Medical School, Department of Radiology and Neurology, MA 02129 Boston, USA, kshah@helix.mgh.harvard.edu
Engineered Diagnostic and Therapeutic Stem Cells for Cancer
Successful treatment of brain tumors remains one of the greatest challenges in oncology. The recognition that different stem cell types, including neural stem cells (NSC) can integrate appropriately throughout the mammalian brain following transplantation has unveiled new possibilities for their use in neural transplantation. Our laboratory has shown that different stem cell types home to sites of cerebral pathology and thus can be armed with therapeutic transgenes, a strategy that can be used to inhibit tumor growth by targeting angiogenesis or selectively inducing apoptosis in proliferating tumor cells in the brain.
Our research is based on simultaneously targeting cell death and proliferation pathways in an effort to eradicate gliomas using therapeutically engineered stem cells. We have engineered different stem cells types (i) to secrete therapeutic protein, S-TRAIL (secreted Tumor necrosis factor receptor-apoptosis inducing ligand) to specifically induce apoptosis in tumor cells and anti-angiogenic TSP-1 (thrombospondin-1) to inhibit tumor angiogenesis. These stem cells are then used to populate primary tumors and their secondary micro-invasive deposits in the brain. Inherently linked to the brain tumor therapy paradigm, we employ fluorescent/bioluminescent imaging markers and optical imaging techniques to track NSC, image apoptosis and changes in tumor volumes in real time in vivo. We also explore the use of microRNAs inhibitors to target brain tumor specific microRNAs and shRNAs to target proteins specifically over-expressed in brain tumors in combination with therapeutic stem cells.
SavkovicVukLindaMilkowaSimonJan-Christoph
Universität Leipzig, Translationszentrum für Regenerative Medizin, Philipp-Rosenthal-Str. 55, 4103 Leipzig, Germany, vsavkovic@trm.uni-leipzig.de
Applicative potential of stem cells from hair roots
Hair root is a complex system that not only gives rise to hair, but at the same time serves as a reservoir of adult stem cells, capable of differentiating into various precursors. The Outer Root Sheath (ORS) of the hair root gives rise to keratinocytes, melanocytes and fibroblasts. Beyond this standard understanding of the hair root stem cell epithelial commitment, multipotency has been shown by yielding neuronal precursors, sharing a common neural crest origin, from ORS. The accumulating data imply that the ORS stem cell pool is pluripotent, with a rising potency along the proximal-distal axis of the ORS. The base of the ORS gives rise to fibroblasts, the mid-part yields epithelial and neuronal precursors. The distal part, the bulge, harbours a pluripotent stem cell pool, containing embryonic neural crest stem cell-like cells, able to give rise to ectodermal precursors (neuronal and epithelial) and mesenchymal lineages (adipocytes, osteocytes, chondrocytes, smooth muscle). Some go as far as assuming totipotency within the hair root.
Clearly, next to the intriguing questions, developmental diversity of the hair root offers potential for regenerative therapies. Among various possibilities, we have utilised this potential for the treatment of Vitiligo.
Vitiligo is a local skin depigmentation disorder, due to either an impaired function or absence of melanocytes in epidermis. The disease is multi-factorial and can be coupled with autoimmune disorders and redox malfunction. The white patches, even though physiologically benign, bring about serious psychological disturbance and tremendously impact the life quality of the patient.
Conservative therapies for Vitiligo remain palliative and their effects are short-term. Transplanting melanocytes to the depigmented patches is proven to be a promising and efficient therapy and it has been moving towards non-invasive, low-sample-biopsy-based procedures.
Euroderm Biotech & Aesthetics, pioneers in the field of ORS-derived skin products, have successfully launched a keratinocyte-based skin transplant, Epidex®, cultivated from a small sample of autologous hair roots, into clinical trials and onto the market. By embedding ORS-derived melanocytes into an already functional skin transplant, our Group at Translational Centre for regenerative Medicine in Leipzig is working towards a promising autologous, ORS-derived, transplantation-based Advanced Therapy Medicinal Product, designed for the treatment of Vitiligo.
DaCostaJadersonCarrionMaria JuliaDa CostaDanielleSchillingLucasMarinovicDanielPortuguezMirnaRauppEduardoGaricocheaBernardoMachadoDenise
Pontificia Universidade Catolica do RS, Instituto do Cerebro, Av. Ipiranga 6690, Predio 60, 2o. andar, 90610-000 Porto Alegre, RS, Brasil, jcc@pucrs.br
Seizure frequency in patients with Mesial Temporal Lobe Epilepsy transplanted with autologous bone marrow mononuclear cells: Preliminary results
Temporal lobe epilepsy (TLE) is a highly prevalent syndrome in patients with seizures and it is usually refractory to drug treatment. Anatomical and physiological changes are often present in patients with TLE such as mesial temporal sclerosis (MTS) characterized by extensive cell loss, reactive gliosis, mossy fiber sprouting and neurogenesis in the hippocampus and para-hippocampal structures. Adult stem cells are proven to have the ability to regenerate neuronal tissues including hippocampal structures in experimental models (Costa-Ferro et al. Seizure 19: 84–92, 2010). The aim of this study is to verify the feasibility of such procedure and the therapeutic potential of bone marrow mononuclear cells (BMMC) transplantation on seizure control in patients with medically refractory mesial temporal lobe epilepsy (MTLE). Patients were diagnosed as MTLE following International League Against Epilepsy criteria. They were submitted to neurological evaluation, MRI study with Hippocampal volumetry, 72 hour- VideoEEG for epileptic seizure recording and laboratory tests. Also, patients were evaluated with a standardized neuropsychological test battery, focusing on verbal and nonverbal memory domains, consisting of the Wechsler Memory Scale–Revised, the Rey Auditory Verbal Learning Test, and the Rey Visual Design Learning Test. After informed consent and fulfilling all criteria, MTS, onset ictal zone and NPS tests congruence for the same side patients underwent autologous BMMSC transplant by selective posterior cerebral artery catheterism. This study was approved by Ethics Committee from our institution and National Committee on Ethics in Research. It is also registered on www.clinicaltrials.gov (NCT00919266).
Eight patients have already undergone this procedure. All patients were submitted to 3 and 6 months follow-up. After follow-up of 6 months, 3 patients (37,5%) were completely seizure free since transplant (Engel class 1a) and 3 patients (37,5%) had only auras (Engel class 1b). Two patients had > 75% seizure reduction (Engel class II). There were no complications related to the procedure. Given these data we believe that BMMSC autologous transplant for the treatment of MTLE is indeed feasible and seems to be safe so far. Seizure control achieved in this first experience gives us a very promising path in therapeutic potential of stem cells transplant in this population.
Supported by FINEP, CNPq and CAPES.
ShagidulinMuratNinaOnishchenkoMikhailKrasheninnikovIgorIljinskyNataliaMogeikoAlexeiLundupEugeniNemetsVictorSevastjanovSergeyGautier
Federal V. Shumakov Research Center of Transplantology and Artificial Organs, experimental transplantology and artificial organs department, Schukinskaya str. 1, 123182 Moscow, Russia, dr.shagidulin@mail.ru
Long-term surviving mesenchymal bone marrow stem cells as an active part of intracorporeal support units
Background: Working out of bioartificial support systems for the assistance of damaged organs is an actual problem of medicine. The using stem cells as the producer of bioregulatory peptides is to promote to decide this problem.
This investigation was undertaken for working out the prolonged functioning intracorporeal support unit (device) containing viable stem cells, attached on matrix.
Methods: Adult dogs (28–30 kg) were used as donors of multipotent mesenchymal stromal cells (MMSC). The survival of MMSC, immobilized on biodegradable heterogenous matrixes Sphero®GEL was investigated. Matrixes Sphero®GEL produced on the base of farm animals collagen, were grainy gel-like substances. MMSC were received by femur femoral bone washing with Hanks solution, which contained 200 mkg/ml of gentamycin and 250 un/ml of heparin. Suspension of MMSC was centrifuged and cell sedimentation was resuspended in lysing solution (114 mM NH4CI, 7.5 mM KHCO3, 100 mkM EDTA). MMSC were sown in Petri dishes and cultivated during 10 days with medium changing every 4 days at constant microscope control of cell viability. Suspension of autological MMSC was seeded on the matrix Sphero®GEL as 2×106–4×106cells/cm3 and in such set was transplanted into a small bowel mesentery of dogs in 10 days after their harvest. Proliferation and cell vitality of cells, seeded on the matrix were investigated in 30, 60 and 90 days. Morphological structures of MMSC and surrounding tissue were studied by using hematoxylin and eosin staining. Vessels were studied by using PAS staining.
Results: MMSC viability measured after isolation was: −94 ±2%. It were found out the viability and proliferative activity of MMSC on the matrixes and also neogenic plethoric vessels, growing through the biodegradable heterogenous matrixes Sphero®GEL at the all time of experiments, even in 90 days after transplantation of matrixes with MMSC into mesentery of experimental animals.
Conclusion: Our preliminary studies show that transplantation of matrix – immobilized MMSC is the effective method for long-term surviving of these cells in the intracorporeal support unit, which can be used for the assistance of damaged organs.
Johannes Gutenberg University Mainz, Mainz, Germany
University of Pavia, Italy
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.
GrollJürgen
DWI an der RWTH Aachen e.V., PauwelsstraÕe 8, 52074 Aachen, Germany, groll@dwi.rwth-aachen.de
Control of Nanofibre Surface Chemistry for Biomaterials and Tissue Engineering
Aside of basic biocompatibility, modern biomaterials are often specialized and tailored in their properties for a specific task. Nanofibres have gained interest because of their structural similarity to tissue components. However, control over their surface chemistry and thus cell adhesion has so far been limited.
We have developed a one-step preparation method for nanofibre-constructs with precise control over surface chemistry by the use of a functional prepolymer additive during electrospinning of biodegradable polyesters. The additive enriches at the surface during fibre preparation and minimizes protein adsoprtion as well as cell adhesion as demonstrated for human dermal fibroblasts and human mesenchymal stem cells. However, functionalization with cell-adhesion molecules such as peptides during the fibre preparation process results in specific cell adhesion due to interaction of cells with these signals [1,2].
We present fibre preparation and characterization as well as one application of this technology for a clinical problem. Congenital diaphragm hernias are severe threats for newborns for which no adequate treatment or material approach exists. Our nanofibre technology may be developed as sustainable and resorbable air-tight mesh for such treatments. For proof-of principle, we have used standard hernia meshes as mechanical support. Fibres were deposited on both sides of a plasma-activated Prolene® hernia mesh (Ethicon). Constructs were implanted into 18 female Chinchilla rabbits over an artificial defect of 1 cm in diameter in the lateral left diaphragm at the interface of tendon and muscle. Animals recovered well after operation, and explantation after 3 and 6 months showed good biocompatibility and no foreign body responses.
GasteierP., ReskaA., SchulteP., OffenhäuserA., MöllerM., GrollJ.Macromol. Biosci., 2007; 7:1010.a-86ButcherJonathan T.HockadayLauraKangKevinColangeloNicholasWuJunChuChih-Chang
Cornell University, Dept. of Biomedical Engineering Ithaca, NY, USA, jtb47@cornell.edu
High fidelity 3D tissue printing of scalable anatomically accurate living aortic valves
The engineering of a living heart valve replacement is a critical unmet need for growing children suffering from congenital heart defects. While great strides have been made over the past fifteen years, results to date are still inadequate for clinical use. A major unsolved challenge has been the inability to mimic the complex anatomical and material heterogeneity present in the native valve, which is critically important for normal function. Solid freeform fabrication (3D tissue printing) technology can generate three dimensional shapes with high precision, but its application to heart valves is challenging because of its non-self supporting geometry. To overcome this, we have developed a novel on-board 365 nm UV photocrosslinking system that is controlled directly with material deposition. Porcine aortic roots were scanned using microcomputed tomography, and the digital volumes converted into deposition tool files. The aortic wall and leaflet materials were determined via densitometry thresholds. Blends of 700 and 8000 MW polyethylene-glycol (PEG) hydrogel polymers were tested in uniaxial tension to establish a wide range of elastic
(τuts = [5,250] kPa, emax = [0.24,2.1]) material properties. Alternatively, direct printing of arbitrary 3D gradients comprising the two extremes was achieved using a novel dithering algorithm. Biomechanical testing of gradient hydrogels confirmed predicted material distribution.
Valve geometries were printed at 18–100% scale (10–25 cm diameter), covering a range of pediatric sizes. Layer specific and aggregate shape fidelity was quantified using a novel Boolean statistical algorithm. Engineered tissues containing PEG (700 or 8000 MW) or with a coating of up to 0.5 mg/ml type 1 collagen were seeded with porcine aortic valve interstitial cells (PAVIC) and cultured for up to 21 days. While cell viability did not vary significantly with hydrogel formulation (greater than 90% viable), PAVIC were significantly more spread on 8000 MW PEG and with collagen coating. Shape fidelity averaged 85–90% for printed valves, with the largest valve printing most accurately. The major locations of error were just below the sinotubular junction and at the base attachment zone.
Taken together, these results show that scalable 3D printing of anatomically accurate living aortic valves is feasible. Replicating these features is an important step to improved functional performance and long-term biological integration.
NMI Natural and Medical Sciences Institute at the University of Tubingen, Regenerative Medicine I, Markwiesenstr. 55, 72770 Reutlingen, Germany, hanna.hartmann@nmi.de
NMI Natural and Medical Sciences Institute at the University of Tübingen, Biomaterials, Germany
INEB Instituto de Engenharia Biomédica, Divisão de Biomateriais, Universidade do Porto, Portugal
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.
Natural and Medical Sciences Institute at the University of Tuebingen, Regenerative Medicine I, Markwiesenstrasse 55, 72770 Reutlingen, Germany, beate.scholz@nmi.de
TETEC Tissue Engineering Technologies AG, Reutlingen, Germany
Cellendes GmbH, Tuebingen, Germany
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).
Fraunhofer Institut für Grenzflächen- und Bioverfahrenstechnik, Zellsysteme, NobelstraÕe 12, 70569 Stuttgart, Germany, petra.kluger@igb.fraunhofer.de
Fraunhofer Institute for Production Technology IPT, Aachen, Germany
Fraunhofer-Institute for Biomedical Engineering IBMT, St. Ingbert, Germany
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.
Poster Presentations
Regenerative Biology
SchmidtTimoHauÕmannSusanneJustLothar
Institute of Anatomy, Experimental Embryology, Österbergstrasse 3, 72074 Tübingen, Germany, timoschmidt@gmx.de
A novel aerosol-based three-dimensional cell culture method
The field of regenerative medicine requires specific and standardized cell culture techniques to generate GMP-compliant three-dimensional tissue constructs. Culturing at the air-medium interface is one of the most successful techniques in tissue engineering to ensure vitality and functionality of tissues for long culture periods. Although roller-bottle and membrane cultures offer these conditions, their use is restricted due to the high induced shear stress and/or to the limitations in the cultivation of different shapes and sizes of tissue.
At the Center for Regenerative Biology and Medicine in Tübingen, we developed a novel cell bioreactor system based on ultrasonic culture medium nebulisation. Our bioreactor system bypasses the above-described disadvantages and thus offers complete new applications in tissue engineering. Ultrasonic nebulisation converts the liquid nutrient solution into a vapour phase and continuously envelops the biological material with nutrient aerosols. Thus, our technique is fundamentally different from cell culture systems, which submerge cells in culture medium.
Cell culture experiments using medium nebulisation demonstrated a high cell viability, proliferation and differentiation capacity. Our system does not only allow a high efficiency in nutrient and oxygen supply but also provides simultaneously a greater independency from tissue shape and size. For this reason, generated artificial tissues could be better adapted in a three-dimensional environment to the individual physiological and clinical requirements. In addition, this cell culture method is particularly suited to deliver drugs, toxins and/or nanoparticles in form of aerosols. As pharmacological test model this system provides the possibility to distribute these substrates in form of aerosols directly to their target cells, e.g. epithelial cells from skin, lung, or gastrointestinal mucosa.
RolletschekAlexandraZieglerCorneliaSchmitteckertStefanie
Karlsruhe Institute of Technology (KIT), Campus North, Institute for Biological Interfaces 1, H.-v.-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany, alexandra.rolletschek@kit.edu
Preparation of endothelial cells for co-cultivation with embryonic stem cell-derived cardiomyocytes
Differentiation experiments using embryonic stem (ES) cells are traditionally performed in conventional tissue culture plates by adding soluble growth factors and extra-cellular matrix (ECM) components to the medium. However, this approach does not consider the influence of a cell specific ECM composition. For engineering mature and long-term functional tissues in vitro, sophisticated cultivation systems are required that mimic the spatiotemporal regulation of tissues developing in vivo. It is well known that endothelial cells (ECs) in the heart exhibit the property of close anatomical and functional interaction with cardiomyocytes. They express and release a variety of auto- and paracrine agents (e.g. endothelins) as well as ECM proteins (fibronectin, collagens, laminin) which directly influence cardiac development and function. Therefore, the aim of our work was the establishment of a co-cultivation system of mouse ES cell-derived cardiomyocytes and ECs freshly isolated from the heart. Specifically, our studies focused on the influence of primary ECs on the differentiation and maturation of ES cell-derived cardiomyocytes. For the generation of cardiomyocytes ES cells were pre-differentiated into embryoid bodies (EBs) for 5 days and further cardiac differentiation was monitored for 2 weeks. ECs were isolated from hearts of E18.5 fetal mice. For comparison with primary ECs, we used endothelioma cell line eEND.2.
In order to optimize co-culture conditions different media compositions were tested and cell type specific markers were analysed by RT-PCR and immunocytochemistry (e.g. cardiac-specific troponin, actinin, and connexin43 as well as endothelial cell-specific endothelin, von Willebrand factor, and endothelin receptors). Transcript and protein data suggested that a mixed medium (ES cell differentiation/EC cultivation medium 1:1) was most suitable for co-cultivation experiments.
Co-cultivation of primary ECs and 5d EBs resulted in positive effects on the cardiogenic differentiation indicated by longer beating periods of ES cell-derived cardiomyocytes. In contrast, co-cultivation with endothelioma cell line eEnd.2 had no significant effect on cardiac ES cell differentiation.
Our future studies will focus on the influence of organ specific ECs on the further ES cell differentiation into mature cardiac phenotypes under physiological culture conditions such as shear stress.
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
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada
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.
GruppLarissaHajoschReinerNichterwitzSusanneSchlosshauerBurkhard
Natural and Medical Sciences Institute, Regenerative Medicine I, MarkwiesenstraÕe 55, 72770 Reutlingen, Germany, larissa.grupp@nmi.de
New nerve implantation method preserving the epineural tube
A novel epineural tube implantation paradigm in the adult rat was designed for the analysis of regulatory cell interactions in peripheral nerves and for the development of therapeutic implants. The aim was to allow the integration of synthetic regenerative structures and cells into the nerve interior while preserving an outer nerve tissue layer with a supportive vasculature.
The microsurgical technique allowed us to remove the interfascicular epineurium, leaving behind an epineural tube with an intact tissue wall of about 0.1–0.2 mm. The resulting tube was filled with hundreds of bioengineered bands of Büngner which were composed of resorbable polymer filaments seeded with Schwann cells. Alternatively, purified cells to be analyzed or different types of growth matrices were injected into the epineural tube.
Such manipulations will allow to generate and investigate concentration gradients of biological factors or to analyse cell-matrix interactions under defined conditions in a supportive in vivo environment. Our current aim is to evaluate bioengineered neural implants.
In summary, a microsurgical in vivo paradigm has been developed to address multiple aspects of peripheral nerve regeneration.
HajoschR, GruppL., NichterwitzS., SchlosshauerB.2010. A NOVEL MICROSURGICAL NERVE IMPLANTATION TECHNIQUE PRESERVING OUTER NERVE LAYERS. Journal of Neuroscience Methods, J. Neurosci. Meth., 189:205–209.FritzeOlafKallingerJanineSchleicherMartinaStockUlrich
University Hospital Tübingen, Dept. Thoracic and Cardiovascular Surgery, Silcherstr. 7/1, 72076 Tübingen, Germany, olaf.fritze@med.uni-tuebingen.de
Endothelial Progenitor Cells in Patients with Carotid Stenosis
Endothelial Progenitor Cells (EPC's) are primitive cells found in peripheral blood and bone marrow. In particular the potential of EPC's to differentiate into mature endothelial cells remain of high interest in regenerative medicine.
Objective of this study was the determination of EPC's kinetics in patients undergoing carotidal thromboendarterectomy (CTEA) and patch plasty.
20 CTEA patients were studied (15 male, mean 76 years). Peripheral blood samples were taken at 1 day preoperatively, 1, 3 and 5 days postoperatively. Peripheral Blood Mononuclear Cells (PBMC's) were isolated by Histopaque-1077 density centrifugation. Flow cytometrical analysis was performed for CD34, CD133, kinase insert domain receptor (KDR) and CD45. Furthermore ELISA testing was conducted for VEGF-R2, SDF-1α and G-CSF.
FACS analysis revealed 0.031 ± 0.016% (% of PBMCs) KDR+ cells and 0.052 ± 0.022% CD45-/CD34 + /CD133 + cells, preoperatively. A 33% decrease of CD45-/CD34 + /CD133 + cells was observed at day one after surgery. However, relative number (compared to initial preoperative values) of CD45-/CD34 + /CD133 + cells returned to values of 82% on day 3 and 94 % on day 5 postoperatively. A more profound up-regulation for CD45-/CD34 + /CD133 + cells was observed for diabetic (+47%) and male patients (+38% compared to females). No postoperative time-dependent differences were found for numbers of KDR+ cells and the amount of concentrations of VEGF-R2, SDF-1α and G-CSF by ELISA.
Determination of EPC's defined by CD45-/CD34 + /CD133 + labeling revealed a short-term decline with subsequent recovery after defined vascular injuries such as CTEA. Furthermore, we observed an up-regulation of EPC's for diabetics and males.
Department of Pediatric Surgery, University Children's Hospital Tuebingen, Hoppe-Seyler-Str.3, 72076 Tuebingen, Germany, heike.dettmann@med.uni-tuebingen.de
Institute of Anatomy, Center for Regenerative Biology and Medicine, University of Tuebingen, Tuebingen, Germany
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.
FreimarkDeniseEhlickeFranziskaDorresteijnAdriianCzermakPeter
University of Applied-Sciences Giessen-Friedberg, Institute of Biopharmaceutical Technology, Wiesenstrasse 14, 35390 Giessen, Germany, denise.freimark@tg.fh-giessen.de
Regeneration of intervertebral disc (IVD) using in vitro differentiated stem cells
Introduction: The degeneration of the IVD, particular of the nucleus pulposus (NP), causes low back pain. One possible method of treatment is cell-based therapy. In the degenerated IVD, the amount and vitality of NP cells is insufficient for application. In contrast to NP cells, vital human mesenchymal stem cells (hMSC) are available in sufficient amount in tissue. Furthermore, they avoid allorecognition and, therefore, circumvent immune response. For these reasons, our working group focuses on the applicability of hMSCs for IVD regeneration. hMSCs must be differentiated into NP cells prior to implementation, which requires a 3D environment. For a long time, NP cells are misleadingly dealt as chondrocytes because their protein expression profiles are similar. Thus, currently, NP cells are poorly characterized. Therefore, a method to distinguish NP cells (e.g. differentiated hMSCs) from undifferentiated hMSCs and chondrocytes has to be established.
Material and Methods: hMSCs were cultivated three-dimensional to form NP cells. To investigate the putative differentiation stimulating ability of several growth factors and components of the extracellular matrix (ECM), fluorescence immunostaining of NP-specific marker proteins was done.
Results and Conclusion: In all differentiation experiments with growth factors, NP-specific marker proteins were expressed. Data concerning the differentiation of hMSC under the influence of ECM components will be presented. The expression of NP-specific marker proteins indicates the ability of three growth factors to differentiate hMSCs into NP-like cells. In vivo, growth factors and the ECM form cellular niches to regulate the proliferation and differentiation of the embedded cells. Therefore, a stimulating influence of ECM components was presumed.
Universtiy of Tübingen Medical Center, Tübingen, Germany
BG Center for Traumatology, Tübingen, Germany
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.
AndreevaElenaBuravkovaLudmilaValyushkinaMariaLoginovVladimir
Institute of Biomedical Problems, Khoroshevskoe shosse 76a, 123007 Moscow, Russia, andreeva_er@mail.ru
MMSCs, expanded under different oxygen tension, improve regenerative process after experimental bone fracture
MMSCs have been recognized as a potential tool for the development of innovative therapeutic strategies because they are easy to generate, maintain, and expand in culture and because they can be potentially applied, due to their immune-privileged properties, in an allogeneic setting.
The effects of rat bone marrow multipotent mesenchymal stromal cells (MMSCs) infusion on bone callus formation in rats with fibula fracture after 14 and 30 days of healing were studied. MMSCs were preliminary expanded under normoxic (20% O2,) and hypoxic (5% O2) conditions along 2 passages. Before injection into experimental animals normoxic and hypoxic MMSCs dispalyed different morphology, but had similar viability and were positive on MMSC markers: CD90, 73, 105, CD44 and negative on blood borne markers CD45, CD14. 4 groups of Wistar male rats were used in experiment: 1 - control (spontaneous fracture repair), 2 - repair after the injection of serum-free culture medium in fracture site, 3 - repair after the injection of MMSCs (5×105 cells in 0,25 ml serum-free culture medium) cultured in normoxic conditions, 4 - repair after the injection of MMSCs (5×105 cells in 0,25 ml serum-free culture medium) expanded in reduced oxygen tension. A half of animals were sucrified after 14 and other part after 30 days of bone break regeneration. Histologic sections of bone callus were prepared and stained with standard histoligic dyes to reveal connective tissue, cartilage and bone. Bone callus square and the share of bone callus square occupied with each tissue were estimated using “SigmaScanPro” software.
After 14 days bone callus in all groups was consisted of cartilage, fibrous and reticulo-fibrous tissue. The amount of last one was significantly higher in group with MMSCs (5% O2). Bone callus square was 1,3 (5%O2) and 1,4 (20%O2) time larger (p < 0,05), than in control. The effectiveness of bone remodeling was evaluated as a ratio of bone callus square on days 14/30. On day 30 bone callus was more mature histologically and diameter of newly organised bone in was very close to initial in rats having MMSCs infusion. In groups w/o MSSCs bone callus was still apparently thickened.
Thus, injection of MMSCs, expanded in vitro under different oxygen tension, resulted in bigger amounts of newly synthesized tissues at early stages of bone callus formation and provided effective bone size restoration in rats with fibula fracture.
HarichandanAbhishekSivasubramaniyanKavithaGrimmSabrineCerabonaFlaviannaBühringHans-Jörg
University Clinic of Tübingen, Department of Internal Medicine II, Division of Hematology, Oncology, ImmunologyRheumatology, and Pneumology, Laboratory for Stem Cell Research, Otfried-Müller-Str. 10, 72076 Tübingen, Germany, abhishek.harichandan@gmail.com
Isolation and Characterization of Distinct Mesenchymal Stem Cell Subsets Based on the Expression of CD271, CD56, and SSEA-3
We have recently identified two distinct CD271brightTNAPdimCD56 + and CD271brightTNAPbrightCD56- MSC subsets in primary femur derived bone marrow (BM), which differ in their expression pattern and morphology as well as in their clonogenic and differentiation capacity. Here we show that CD271bright cells coexpress the embryonic stem cell marker SSEA-3 but not SSEA-4, TRA-1-60 or TRA-1-81. Analysis of the clonogenic and differentiation potential of FACS sorted CD271brightSSEA-3 + CD56 + , CD271brightSSEA-3 + CD56- and CD271brightSSEA-3-CD56 + populations revealed that colony forming units fibroblasts (CFU-F) assays were about 48-fold enriched in the CD271brightSSEA-3 + CD56- and CD271brightSSEA-3-CD56 +fractions and about 85-fold in the CD271brightSSEA-3 +CD56 + fraction. Differentiation of expanded cells derived from these subsets showed that adipocytes emerged exclusively from SSEA-3 + CD56- cells whereas the osteogenic differentiation potential was similar in all the studied subsets. The analysis of the chondrogenic differentiation potential of these subsets is in progress, but our preliminary data indicate that chondrocytes primarily emerge from CD271brightSSEA-3 + CD56 + cells. These data show an early commitment of these cell fractions into specific lineages. The isolated cells may promote the use of these cells as an attractive starting population for therapeutic applications.
Fraunhofer Institute for Interfacial Engineering and Biotechnology, Nobelstrasse 12, 70569 Stuttgart, Germany, Svenja.Hinderer@igb.fraunhofer.de
University of Würzburg, Würzburg, Germany
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.
Medical University Graz, Institute of Cell Biology, Histology and Embryology, Harrachgasse 21/7, 8010 Graz, Austria, karin.pekovits@medunigraz.at
Medical University Graz, Department of Dentistry and Maxillofacial Surgery, Graz, Austria
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.
ZhangYingBareiÕPetraNothelferKatharinaMohrRolandHauÕmannSusanneJustLothar
Institute of Anatomy, Experimental Embryology, University Hospital, Österbergstrasse 3, 72074 Tübingen, Germany, hello_ying@yahoo.com
Influence of Wnt signalling on the proliferation and differentiation of neuronal progenitors from enteric nervous system
Neural stem and progenitor cells from the enteric nervous system (ENS) have been postulated as an appropriate cell source for treatment of specific neurogastrointestinal disorders such as hypo- or aganglionosis. An indispensable role for the embryonic and fetal development of vertebrate nervous system plays the Wnt signalling cascade. In the present study, we analyzed the influence of the canonic Wnt pathway on the proliferation and differentiation capacity of neuronal progenitors from enteric nervous system. Thus, neural enteric progenitors were isolated from murine (fetal or neonatal) and human neonatal gut tissues and subsequently cultured under proliferating cell culture conditions to induce the formation of neurosphere-like bodies. For pharmacological analysis single spheres were transferred into agarose coated 96-well plates and treated with different molecules that influencing the canonical Wnt pathway like Wnt receptor agonists, antagonists, or a GSK3 inhibitor. Our results demonstrate that the activation of the canonical Wnt signalling pathway strongly promotes the proliferation and neuronal differentiation of ENS progenitors. These finding opens new and improved cell expansion strategies in vitro which are necessary to obtain a sufficient stem and progenitor cell pool for future cell-based therapies.
Department of, Head & Neck Surgery, University Hospital of Tübingen, Tübingen, Germany
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.
HalbwirthFlorianNiculescu-MorzsaEugeniaZwicklHannesBergerManuelaBauerChristophNehrerStefan
Danube University Krems, Center for Regenerative Medicine, Dr.-Karl-Dorrek-StraÕe 30, 3500 Krems, Austria, florian.halbwirth@donau-uni.ac.at
Effect of mechano- stimulation on matrix-embedded human osteoarthritic chondrocytes
Purpose: The goal of this study was to determine the effect of cyclic mechanical stimulation on matrix-embedded human chondrocytes derived from osteoarthritic cartilage. For that purpose expression of genes known to be affected in diseased cartilage (MMP-3, MMP-13, collagen II, aggrecan) and its alteration upon mechanical stress was investigated via RT-PCR. In addition, matrix-embedded chondrocytes were histologically evaluated concerning a potential effect of mechanostimulation on cell morphology.
Methods: Human articular cartilage was obtained from osteoarthritis patients subjected to total knee arthroplasty. After isolation of the chondrocytes from cartilage tissue and an expansion under standard cell culture conditions for 14 to 20 days they were embedded in a collagen I-matrix. Following cultivation for 12 to 16 days, cell-seeded matrices were either mechanically stimulated (cyclic sinusoid compression regime for 4 days) or further cultivated without stimulus. For measurement of gene expression, chondrocytes were isolated and mRNA levels of the genes of interest were gathered via RT-PCR. Moreover, morphology and distribution of chondrocytes in the differentially treated matrices were histologically determined.
Results: Based on data from literature concerning the preferential targets of the respective proteinases, results were expressed as ratios of aggrecan to MMP-13 (MI13) and Col 2 to MMP-3 (MI3), respectively. Both, MI3 as well as MI13, significantly increased by mechanostimulation compared to control indicating an “anabolic shift”. Moreover, chondrocyte morphology proved to be drastically altered by appearing round rather than spindle-shaped in histological slices of stimulated compared to control matrices.
Conclusions: We got strong indications of a shift from catabolic towards anabolic transcription activity. Our findings further suggest the importance of mechanical stress for metabolism and function of chondrocytes and indicate that the supposed catabolic phenotype of matrix-embedded osteoarthritic chondrocytes might be reversible by mechanostimulation.
Tierärztliche Hochschule Hannover, Hannover, Germany
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.
NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany
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.
ArdjomandiNinaMaurerAndreasKalbacherHubertKleinChristianKohlerKonradReinertSiegmarAlexanderDorothea
University Tuebingen, Oral and Maxillofacial surgery, Osianderstr. 2–8, 72076 Tübingen, Germany, nina.ardjomandi@med.uni-tuebingen.de
Coating of polylactic acid scaffolds with different RGD peptides – promising candidates to enhance adhesion and differentiation of jaw periosteal cells?
Objective: The aim of our study was to generate a biofunctionalized 3D polylactic acid scaffold (OPLA) in order to enhance jaw periosteal cell (JPC) adhesion and differentiation into osteogenic tissue for applications in oral and maxillofacial surgery. Biofunctionalizing of the scaffolds was achieved by covalent peptide coating of OPLA matrices with different RGD peptides and coating variants.
Material and Methods: OPLA scaffolds were coated directly with linear and cyclic RGD-peptides and indirectly via a poly-L-lysin spacer (PLL). Coating efficiency was analyzed by amino acid analysis. 2D cell adhesion was evaluated by fluorescence microscopy. Proliferation of cell seeded scaffolds was measured by a colorimetric assay and was verified with 3D confocal laser scanning microscopy. Gene expression analysis was carried out using osteogenic and ECM pathway gene arrays.
Results: Coating efficiency varied depending on different peptides and coating variants. 17 nmol peptide/mg scaffold could be immobilized directly with linear GRGDS peptide compared to 11 nmol/mg scaffold and 9 nmol/mg scaffold of directly and indirectly (using the PLL spacer) coated linear and cyclic peptides. Cyclic and linear PLL peptides showed the highest adhesion capacity under 2D culture conditions. In contrast, 3D proliferation assays showed enhanced cell growth on OPLA directly and indirectly coated with linear peptides. Gene expression analysis of differentiated and undifferentiated JPCs seeded on OPLA scaffolds showed different patterns regarding ECM components, related integrins and osteogenic marker expression.
Conclusion: Indirect coating of linear RGD peptides via PLL and direct coating with cyclic peptides seem to be the best coating variant for jaw periosteal cells which might enhance cell adhesion and differentiation into bone tissue.
NMI, Natural and Medical Sciences Institute at the University of Tuebingen, Regenerative Medicine I, Markwiesenstrasse 55, 72770 Reutlingen, Germany, yvonne.baumer@nmi.de
University Clinic of Tübingen, Department of Internal Medicine III, Tübingen, Germany
University Clinic of Tübingen, Department of Internal Medicine II, Tübingen, Germany
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.
Universidad de Granada, Histology, Avenida de Madrid 11, 10012 Granada, Spain, acampos@ugr.es
Hospital Universitario de Granada, Granada, Spain
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.
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
NMI Reutlingen, Regenerative Medicine II, Reutlingen, Germany
TETEC AG, Reutlingen, Germany
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.
Biochemistry Unit. IdiPaz, Spain
Experimental Surgery Dept. IdiPaz, Spain
Donostia Hospital, Spain
CSIC, Spain
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.
Danube University Krems, Center for Regenerative Medicine, Dr.-Karl-Dorrek-StraÕe 30, 3500 Krems, Austria, manuela.berger@donau-uni.ac.at
Croma Pharma GmbH, Leobendorf, Austria
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.
Institute for Interfacial Engineering, Medical Interfacial Engineering, University of Stuttgart, Nobelstr. 12, 70569 Stuttgart, Germany, florian.groeber@igvt.uni-stuttgart.de
Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany
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.
University of Applied Sciences Esslingen, Biotechnologie, KanalstraÕe 33, 73728 Esslingen, Germany, Mareike.Schultze@hs-esslingen.de
nanoAnalytics GmbH, Münster
Institute of Pathology, University Hospital, Tübingen
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.
University of Tuebingen, Center of Regenerative Medicine, WaldhörnlestraÕe 22, 72072 Tübingen, Germany, miriam.rothdiener@uni-tuebingen.de
BG-Trauma Center, Tübingen, Germany
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.
University of Tuebingen, Center of Regenerative Medicine, WaldhörnlestraÕe 22, 72072 Tübingen, Germany, tino.felka@uni-tuebingen.de
BG-Trauma Center, Tübingen, Germany
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.
UV-induced mutations do not accumulate during in vitro proliferation of primary human melanocytes
Despite of all possible approaches, proliferation of human cells in vitro remains an artificial system in comparison to the cells' in vivo situation. High proliferation rates and cell expansion over passages in vitro hold the risk of an enrichment of cells containing DNA-damages or cells with pre-carcinogenic characteristics. The implantation of these mutated cells into a patient could lead to tumorigenic changes in the worst case. So, regarding tissue engineering issues, it is important to know how cells change during in vitro proliferation and if existing damages enrich or not. Furthermore, it matters if the cells' ability to repair DNA damages changes over time.
For human skin, UV-induced mutations are of special interest because skin is constantly exposed to UV-irradiation. Prominent UV-induced mutations are the CC-TT transition in the tumor suppressor gene p53 at codon 281/282, the CC-TT transition in mitochondrial DNA at codon 591/592 and the Common Deletion in the mitochondrial DNA. We established different real-time-PCR methods using SYBR Green technology and TaqMan probes to detect and quantify these mutations in primary human melanocytes. Therefore human skin samples were collected and isolation of melanocytes was performed. Later DNA-extraction was carried out every new passage. Furthermore repair capacity of these melanocytes was measured using the modified host cell reactivation assay.
None of the above mentioned mutations accumulated during in vitro proliferation and over passages. The measurement of DNA repair capacity in an early and a late passage resulted in a slightly better repair capacity in late passages compared to early passages. In conclusion, prolonged in vitro proliferation seems to have no harmful influence on the examined mutations and on repair capacity of melanocytes.
University of Applied Sciences Esslingen, Biotechnologie, KanalstraÕe 33,73728 Esslingen, Germany, bettina.weiss@hs-esslingen.de
Institute of Pathology, University Hospital, Tübingen, Germany
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.
Hochschule Lausitz (FH) Department of Molecular Cell Biology, Germany
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.
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.
Nagoya University, Graduate School of Engineering, Room625, Build1, School of Engineering, Furocho, Chikusaku, 464-8603 Nagoya, Japan, kato-r@nubio.nagoya-u.ac.jp
Nikon Instruments Inc.
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.
HZI Helmholtz Center for infectious Research, MBIO, Inhoffenstr. 7, 38124 Braunschweig, Germany, ked@helmholtz-hzi.de
Institute for Clinical Transfusion Medicine, Städtisches Klinikum Braunschweig gGmbH, Germany
Institut für Schicht- und Oberflächentechnik, Fraunhofer Gesellschaft, Germany
Department of Hematology-Oncology, Städtisches Klinikum Braunschweig gGmbH, Germany
German collection of cell cultures and microorganism (DSMZ), Germany
Department of Human Genetics, Hannover Medical School, Germany
Department of Anatomy, University Tübingen, Germany
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.
Biomaterials and Interfaces
RagauskaAntra1Berzina-CimdinaLiga2ApsePeteris1
Riga Stradins University, Department of Prosthodontics, Dzirciema 20, 1007 Riga, Latvia, antraragauska@inbox.lv
Riga Technical University, Riga Biomaterial Innovation and Development Center, Latvia
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.
GruppLarissaTetlingElkeSchlosshauerBurkhard
Natural and Medical Sciences Institute, Regenerative Medicine I, MarkwiesenstraÕe 55, 72770 Reutlingen, Germany, larissa.grupp@nmi.de
A gelatin-based adhesionbarrier prevents adhesion in an improved rat sidewall abrasion model
Formations of postoperative intra-abdominal adhesions are a severe problem in surgery. Apart from standard surgical procedures, a variety of different substances are available to prevent adhesions, but no universal method has been developed so far. For this purpose a spongy membrane consisting of cross-linked stabilized gelatin has been developed. The novel membrane was implanted to act as physical barrier – and thus to prevent adhesions – in an improved rat cecal - sidewall - abrasion model.
In a first step the most reliable method of the cecal – sidewall - abrasion model in the rat was defined between 4 approaches.
After that a randomized controlled study was conducted to prove the effectiveness of the gelatin membrane in reducing postoperative adhesion - formation after laparotomy in the most reliable abrasion model. All animals underwent a laparotomy with injuries at the abdominal wall and on the cecum to cause adhesions. The membranes were sutured onto the abdominal side wall defect in 24 animals to prevent their slipping or curling on the moist tissue and because of bowel movement. Another 24 rabbits served as a control group without any adhesion barrier being used. In all animals the two injured sides were fixed together by sutures.
Macroscopic evaluations of adhesion formation, tensiometry and histologic analysis after removing the sutures were used to determine the amount of adhesion after both 1 and 3 weeks.
The results showed that only severe injuries and a fixation of cecum at the abdominal wall can cause reliable adhesions to test adhesion-barriers.
In this improved in vivo model the gelatin-based membranes significantly reduced the number and severity of adhesions in the rat sidewall model. The implants are flexible, biocompatible and thus suitable for application to surgical sites for the prevention of adhesion formation.
BeerMeike
DWI an der RWTH Aachen e.V., cell culture, Pauwelsstr. 8, 52056 Aachen, Germany, beer@dwi.rwth-aachen.de
Electrospun fibre-meshes combining non-fouling properties with specific bioactivation as ECM-mimetic tissue-engineering scaffolds
Many tissue engineering approaches focus on the structural mimicry of the native environment of cells, the ECM consisting partly of protein fibres which provide a mechanical scaffold for cells. However, beyond structural design and degradability, the surface chemistry of biomaterials is a major criterion determining biocompatibility. 1) Unspecific protein adsorption, leading to immune responses, must be prevented. 2) Specifically interacting molecules (cell adhesion mediating peptides or whole ECM proteins) have to be presented on such inert surfaces preserving their biol. function. While bioactive signalling combined with minimization of non-specific protein adsorption is an advanced modification technique for flat surfaces, it is usually not accomplished for fibrous scaffolds. Here we present a one-step preparation method of fully synthetic bioactive degradable ECM-mimetic scaffolds consisting of hydrophilic fibres. This system includes a functional additive that transforms current polymers into fibres where non-specific protein adsorption is suppressed and specific bioactivation through covalently attached peptides allows controlled cell/scaffold interactions.
We add a functional amphiphilic macromolecule based on star-shaped PEO to poly(D,L-lactide-co-glycolide) (PLGA). The additive is a six armed star-shaped poly(ethylene oxide-stat-propylene oxide) with 80 % ethylene glycol content (MW 12 kDa) that bears reactive isocyanate groups at the six ends (NCO-sP(EO-co-PO)). Coatings prepared from NCO-sP(EO-co-PO) prevent the adsorption of proteins and the adhesion of cells (Groll et al., Langmuir 200).
JaiswalAmit1KadamSachin2SoniVivek1BellareJayesh2
Department of biosciences and bioengineering IIT Bombay, Department of Biosciences and Bioengineering, Powai, 400076 Mumbai, India, amitanand22@iitb.ac.in
Department of Chemical Engineering, IIT Bombay, India
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.
KrügerReinhardJoklitschkeDavidGbureckUweEwaldAndrea
Universitätsklinikum Würzburg, Department of Functional Materials in Medicine and Dentistry, Pleicher Wall 2, 97070 Würzburg, Germany, Reinhard.Krueger@fmz.uni-wuerzburg.de
Development and Cytocompatibility Testing of Trimagnesium Phosphate and Struvite Foams
In recent time Mg-phosphate ceramics have gained growing interest as biodegradable bone replacement materials. To mimic the structure of cancelous bone and to promote tissue repair mechanisms a highly porous structure with open cells is desired. Trimagnesium phosphate (Mg3(PO4)2) and struvite ((NH4)Mg(PO4)2 · 6H2O) scaffolds were developed in this study as open cell foams using the foam replica technique. The ceramic scaffolds were investigated for their mechanical performance, phase composition and cytocompatibility.
Reticulated polyurethane (PU) foam (30 ppi pore size) was used as template. The PU foam samples were dip coated with a ceramic slurry consisting of Mg3(PO4)2 · 8H2O powder in distilled water, TWEEN 20 as a surfactant and NH4OH (pH = 11). After the first infiltration excess slurry was removed by compressing the foams to 10% of their original height in a rolling process. By this only the struts of the PU scaffold were coated with slurry to obtain an open cell structure. Single coated green bodies were sintered at 1050°C to burn out the PU foam and consolidate the structure. By repeating of the infiltration, blowing out the excess slurry by an air gun and a second sintering at 1050°C, the compression strength was improved and cracks were filled as analyzed by SEM. Sintered re-infiltrated foams showed a diametric shrinkage of 66%. The relative density was 3.8%. The open cell structure of the PU templates was almost completely retained. XRD analysis revealed farringtonite (Mg3(PO4)2) as the only crystalline phase. Soaking the produced Mg3(PO4)2 scaffolds in ammonium phosphate solution ((NH4)3PO4) lead to successive transformation to struvite accompanied with mass increase of 88% (24 h). The transformation kinetics was investigated by XRD. Mechanical properties were analyzed by compression testing. Compression strength increased from 6.8 kPa after the first infiltration step to 14.1 kPa after the second infiltration and sintering. Transformation into struvite caused a further increase of strength to 31.1 kPa. Cracks observed in the farringtonite foams by SEM could no longer be found in the struvite foams.
The final scaffolds were tested for cytocompatibility using fibroblast cells. Cell number and cell activity (WST) were tested after 3 to 14 days. Cell activity and cell size increased on both materials with time. Cell number only increased on struvite, indicating an improved cytocompatibility compared to farringtonite.
Plasmachemical grafting of RGD peptides onto polymeric foils to improve the proliferation of cells
Since the discovery of the RGD peptide in 1984, several substrate materials have been RGD-modified to promote cell adhesion, especially for medical applications.
In principle, an RGD-tailoring of transparent polymeric foils should be advantageous for the development of biochips or microfluidic systems to analyse the vitality and metabolic activity of mammalian cells.
However, in order to overcome the surface inertness of the mostly used organic polymers like polycarbonate or cycloolefines, a suitable method for surface functionalization has to be found. Low pressure plasma technique is a straightforward method to introduce a chemical surface functionality of polymers.
The capability of this method to create carboxylic groups on the polymer was demonstrated by using acrylic acid as precursor. The plasma is depositing a thin layer containing carboxylic groups, which are detected by XPS. They serve as anchor groups for a further wet chemical grafting of biomolecules, such as RGD. To realize this aim, two reaction pathways with different bifunctional spacer chemistries were examined.
The first attempt for RGD coupling was done by using diamino-PEG. This spacer was covalently bound with one of its two NH2 groups to the carbodiimide-activated carboxylic groups of the surface via an amide bond.
The second one was carried out by using a heterobifunctional spacer (N-ɛ-Maleimido-caproic acid hydrazide), which contains a sulfhydryl reactive maleimide group on the one end and a carbonyl reactive hydrazide group on the other one. When using this spacer the successful covalent grafting of cysteine RGD could be demonstrated.
All modification steps were controlled by surface physico-chemical analysis methods: XPS was used to ensure covalent grafting after each step of the surface functionalization, contact angle measurements to characterize the wettability, and ellipsometry to measure the thickness of the functional layer.
Proliferation tests of suspended mammalian cells with a varying addition of foetal calf serum (FCS) were carried out in order to test the effect of surface grafted RGD peptides. It is shown that the proliferation is promoted by the RGD modified substrates especially in case of a low amount of FCS content. This does open the perspective to use serum free suspensions of cells avoiding the risk of any viral contamination.
Development of a dynamic intestinal tissue equivalent that enables the analysis of new drug candidates in vitro
Introduction: Nutrients and other orally administered substances are absorbed as far as possible over the small intestine epithelium into the systemic circulation. The testing of solubility and permeability of new substance classes is either possible by the use of complex tissue models which cannot be standardized or by the use of static cell culture models which reflect the conditions of the small intestine insufficiently.
For this reason more in vitro test systems must be developed, which allow a better validity for the human organism.
Methods: Comparing to a simplified 2D Caco-2 monolayer assay, a 3D dynamic intestinal tissue equivalent has been developed at the Fraunhofer Institute IGB (Stuttgart, Germany), which simulates the physiologic microenvironment of the small intestine in an enhanced way.
Therefore a decellularized collagen scaffold was reseeded with primary human endothelial cells and the epithelial cell line Caco-2. The co-culture system was then cultivated in a 2-chamber-bioreactor which provides an apical and basolateral flow of culture media for up to 14 days in the incubator.
The cells within the intestinal tissue model were characterized unsing antibody staining and by the analysis of their enzymatic activity. Additionally comparative absorption studies were performed between the common 2D Caco-2 assay and the enhanced tissue equivalent.
Results: Preliminary studies with co-cultivated Caco-2 cells and human microvascular endothelial cells showed an increasing paracellular permeability of the epithelial cells under dynamic culture conditions. Therefore an improved correlation of the generated transport data with the human organism could be proven by the use of low permeable substances. In addition the accomplishment of directed transport studies for the investigation of the transport protein p-gp by the use of Rhodamine 123 was possible and correlated with the data of the conventional Caco-2 assay.
Perspectives: Due to the remaining crypt region, the 3D structure of the collagen scaffold represents a protective mechanism for the reseeded cells against high shear stresses and could therefore enable the regeneration of the epithelial layer and multiple testing in the future. Furthermore it will be necessary to replace the Caco-2 cells against primary enterocytes of the small intestine to reach an advanced transfer of generated data to the human organism.
CuiJingKratzKarlHieblBernhardJungFriedrichLendleinAndreas
Center for Biomaterial Development, Institute of Polymer Research, GKSS Research Center Geesthacht GmbH and Berlin-Brandenburg-Center for Regenerative Therapies (BCRT), Kantstrasse 9, 14513 Teltow, Germany, jing.cui@gkss.de
Poly(n-butyl acrylate) networks with tailored mechanical properties designed as model substrates for mechano-responsive cells
Mechanical conditioning can serve as a potent tool to influence the behaviour of mechano-responsive cells during formation and regeneration of functional tissue. Besides external mechanical stimulation using specific bioreactors [1] or actively moving scaffolds [2], the stiffness of polymer-based substrates could affect cell adhesion, motility, differentiation or the tissue formation of MC3T3-E1 pre-osteoblasts [3].
Here, we introduce covalently crosslinked poly(n-butyl acrylate) networks (cPnBA) with adjustable mechanical properties designed for investigations with mechano-responsive cells. cPnBAs were synthesized via free radical polymerization, whereby the mechanical properties were tailored by varying the molar ratios of nBA monomer and polypropylene glycol dimethacrylate (PPGDMA) crosslinker in the starting material mixture [4]. An almost complete conversion was indicated by high gel content values. The purity of cPnBAs was confirmed by ATR-IR, 1H-NMR spectroscopy and gas chromatography, where no residual monomers or purification solvents could be observed. The Young's modulus increased systematically from 100 kPa to 9.61 MPa with rising PPGDMA-content, while the glass transition temperature increased from − 46°C to − 22°C. The surface roughness of cPnBAs decreased from 1.4 μm to 0.4 μm with increasing crosslink density, and the surface wettability of cPnBAs, represented by the advancing angle, decreased from 115° to 100°, while the hysteresis decreased from 75° to 50° indicating a more homogeneous surface. The surface wettability of cPnBAs was not affected by ethylene oxide sterilization.
The extracts of the sterilized cPnBAs were tested for cytotoxic effects with L929 cells. All tested samples were non-toxic. The functional integrity of cell membranes and mitochondrial activity stayed unaffected. The behaviour of primary human keratinocytes and smooth muscle cells was investigated in direct cell culture tests. Both cell types exhibited typical cell morphology after three days. Therefore the investigated polymer networks should be promising soft substrates for investigation of the interactions with mechano-responsive cells.
CuiJet al.Polym. Adv. Technol., 2010, 10.1002/pat.1733published online on 15.07.2010.a-265KommareddyKPet al.Biointerphases, 2010; 5,2:45–52.a-266CuiJet al.Polym. Adv. Technol., 2010submitted.a-267MuthChristine1Lee-ThedieckCornelia1SteinlCarolin2KleinGerd2SpatzJoachim1
Max Planck Institute for Metals Research, New Materials and Biosystems, Heisenbergstrasse 3, 70569 Stuttgart, Germany, muth@mf.mpg.de
Center for Medical Research, University Medical Clinic Tübingen, Germany
Towards an Artificial Stem Cell Niche: Interaction analysis of hematopoietic stem cells with nanostructured, biofunctionalized surfaces
The hematopoietic stem cell (HSC) niche in the bone marrow consists of a specialized population of cells, extracellular matrix (ECM) and soluble factors that play an essential role in regulating stem cell fate. It has been proposed that the ECM of the niche can provide specific adhesion signals which retain stem cells in their niche. Other ECM molecules of the bone marrow are capable of inducing cell proliferation, survival, migration and differentiation.
However, not only the composition of the ECM but also the geometric arrangement and the elasticity of the matrix seem to play an important role for stem cell behaviour. Successful expansion of potential HSCs ex vivo has not yet been fully accomplished mainly due to the fact that the contribution of these factors – composition of the ECM and their biophysical characters – has not been elucidated in detail.
The aim of our research is to develop a strategy to expand human cord blood derived HSCs by simulating the in vivo signals of the niche. For this purpose, we are optimizing a system based on a nanopattern embedded in a hydrogel presenting bioactive molecules to cells. This allows us to study the influence of spatial, chemical and mechanical parameters on cells. Hydrogel-supported gold nanopatterns with varying interparticle distances were produced using the transfer diblock copolymer micelle nanolithography technique. After biofunctionalization, cellular ligands were presented in a defined nano geometry on elastic substrates.
We could show that the critical adhesive interparticle distance for HSCs on RGD (integrin recognition sequence) functionalized hydrogels is 40 nm. HSCs remained spherical on the nanostructured surface and contacted the ligand via filapodia. Quantification of cell attachment to different adhesive ligands of the HSC niche revealed the highest HSC adhesion to fibronectin and osteopontin derived ligands as well as for bio-active peptides derived from the laminin alpha5 chain on nanostructured surfaces. Further investigations concerning the proliferation and differentiation of HSCs in contact with niche signals are currently in progress. A systematic ex vivo expansion would have far-reaching consequences for the clinical application of HSCs and could provide a basis for the development of a new generation of biomaterials for stem cell bioengineering.
Riga Technical University, Riga Biomaterials Innovation and Development Centre, Pulka 3/3, 1007 Riga, Latvia, salma.ilze@gmail.com
Riga Stradins University Institute of Stomatology, Latvia
Institute of Anatomy and Anthropology, Riga Stradins University, Latvia
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.
Leibniz Universität Hannover, Insitute of Inorganic Chemistry, Callinstr. 9, 30167 Hannover, Germany, anne.christel@acb.uni-hannover.de
Institute of Technical Chemistry, Leibniz University of Hannover, Germany
Department of Trauma Surgery, Hannover Medical School, Germany
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.
VogtJ. Ca-283
KnoellerTabea1HeldManuel1WilkommLina1JustLothar2SchallerHans-Eberhard1Rahmanian-SchwarzAfshin1
Center for Regenerative Biology and Medicine and Department of Plastic, Reconstructive, Hand and Burn Surgery, BG-Trauma Center, Eberhard Karls University Tuebingen, SchnarrenbergstraÕe 95, 72076 Tuebingen, Germany, tabea.knoeller@web.de
Institute of Anatomy, University of Tuebingen, Germany
A novel Collagen Cell Carrier (CCC) for in vivo application. An analysis of Biocompatibility in a rat model
Background: For tissue engineering apps, extra cellular matrices play an essential role. Based on in vitro results, in vivo investigations of materials are required for further establishment. The first step of in vivo evaluation should sustain the verification of biocompatibility, since many materials are unsuitable for medical application. In our previous in vitro studies on CCC as a new extra cellular matrix, we demonstrated an excellent cell proliferation and differentiation. Thus an animal study design should analyze the in vivo behaviors of this scaffold.
Methodology/Principal Findings: We implanted CCC in 48 adult male Lewis rats into subcutaneous pockets. After 1, 3, 6 and 12 weeks we took biopsies for histological evaluation regarding inflammatory reactions, encapsulation, formation of necrosis and biodegradation. Histological evaluation showed that there are no major signs for inflammation and encapsulation. Biodegradation was completed after 42 days.
Conclusions/Significance: Histological analysis validated the biocompatibility of CCC in vivo. By the absence of inflammatory reactions, encapsulation or necrosis formation in the surrounding tissue, CCC may be applied in further fields of in vivo investigations.
Max Planck Institute for Colloids and Interfaces, Biomaterials, Am Mühlenberg, 1, 14476 Potsdam - Golm, Germany, cecile.bidan@mpikg.mpg.de
Ludwig Boltzmann Institute of Osteology, Wien, Austria
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.
Universität Würzburg, Würzburg, Germany
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.
NolteAndreaWalkerTobiasSchneiderMartinaDenizOyaAvci-AdaliMeltemZiemerGerhardWendelHans
Universitätsklinikum Tübingen, Klinik für Kinder- und Jugendmedizin, klinisches Forschungslabor Kinderherzchirurgie, Calwerstr. 7/1, 72076 Tübingen, Germany, andrea.nolte@thg-lab.de
Functional siRNA delivering biomaterials for local gene silencing in intravascular applications
Although siRNAs have surpassed expectations in experiments to alter gene expression in vitro, the functional delivery of short interfering RNAs (siRNAs) is still the major obstacle for the in vivo application of RNA interference (RNAi). Therefore, delivery technologies need to be established for the local or systemic application of RNAi. In our recent work we describe an siRNA delivery system that would be readily adaptable for local intravascular applications in vivo.
We examined the transfection of gelatin coatings consisting of siRNA molecules complexed with branched 800 Da Polyethylenimine (PEI) to primary human endothelial cells. We tested various conditions like serum presence or absence in media during cultivation on coatings. Also different siRNA, PEI amounts and N/P ratios were tested and the transfection efficiency determined by flow cytometry. In addition cell viability was tested by MTT-assay. Furthermore we demonstrated the release of fluorescent labeled siRNA from the coatings.
Gelatin coatings consisting of PEI/siSELE complexes achieved a significant E-selectin receptor knockdown of around 70%. The transfection efficiency of the coatings was not reduced by the presence of serum in the media. Moreover, lower N/P ratios with higher amounts of siRNA enhanced the transfection efficiency but were more toxic. The release of siRNA from coatings can be characterized as a burst release.
Our data let suggest that this coating may be favourable for local in vivo RNAi applications when immobilized on balloons for percutaneous transluminal coronary angiography. Furthermore the preparation of this coating is simple and non toxic when administered in adequate quantities.
NMI Natural and Medical Sciences Institute at the University of Tübingen, Regenerative Medicine I, Markwiesenstr. 55, 72770 Reutlingen, Germany, hanna.hartmann@nmi.de
INEB Instituto de Engenharia Biomédica, Divisão de Biomateriais, Universidade do Porto, Portugal
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.
Natural and Medical Sciences Institute at the University of Tuebingen, Regenerative Medicine I, Markwiesenstr.55, 72770 Reutlingen, Germany, nadine.hoffmann@nmi.de
Interdisciplinary Nanoscience Center (iNANO) and Department of Molecular Biology, University of Aarhus, Denmark
Instituto de Engenharia Biomédica, Divisão de Biomateriais and Faculdade de Engenharia Universidade do Porto, Portugal
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.
KrastevRumenEdelmannMarkusHossfeldSusanneFreudigmannChristianSchröppelBirgitBurkhardtClausBenzKarin
NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen, Biomaterials, MarkwiesenstraÕe 55, 72770 Reutlingen, Germany, rumen.krastev@nmi.de
Human primary chondrocyte cell interactions with synthetic polyelectrolyte multilayer films
Contemporary biomaterials have to prevent or promote cellular adhesion and viability. Coatings with desired interaction to the cells have potential for the application in the field of biomaterials. The optimal cell adhesion and cell viability on materials depends on a complex combination of different parameters, like surface charge, roughness or hydrophilicity1. A polymeric system which shows perspective properties as a biomaterial coating is the polyelectrolyte multilayer thin film (PEM). These coatings are easy built from biocompatible materials in a precisely controlled manner using the Layer-by-Layer (LbL) technique, based on alternative deposition of polycations and polyanions2 on the surfaces of the biomaterials.
Present work aims to understand the adhesion and viability of human primary chondrocyte cells on PEM formed from synthetic polyelectrolytes. PEM with different thickness and surface electrical charge were used. This was controlled by adsorbing different number of polymer layers and using coatings with polycation or polyanion as a capping layer. The adsorption of serum proteins on the interaction with the cells was also proved. The study has aimed some of the conditions for cell adhesion and viability to be defined and the mechanism of interaction between the PEM and cells to be elucidated. The build up of PEM was followed by quartz crystal microbalance (QCM), fluorescence spectrometry and scanning electron microscopy (SEM). Human chondrocytes were used as cell material. The cell adhesion was analysed by quantifying the number of adhered cells with fluorescent DAPI. The cell growth was monitored by optical microscopy and with alamar blue assay for up to 7 days.
The human chondrocytes show higher adhesion on PEM compared to that of the control sample. Cells show high viability after seven days. The presence of serum proteins in the cell culture media influences the interaction between the cells and PEM. Cell adhesion depends on the thickness of the PEM. An influence of the surface charge of the PEM on cell adhesion and viability was observed. PEMs are an innovative system to be used as coatings for implants. They allow biological functionalization of biomaterials with low cytotoxicity. The coatings are of particular interest in dental or orthopaedic implantology, tissue engineering or targeted drug delivery.
RichertL.et al. Biomacromolecules, 2002; 3:1170.
DecherG.Science, 1997; 3:1232.NovoselEsther1MeyerWolfdietrich2WegenerMichael2KrügerHartmut2BorchersKirsten1KlugerPetra1TovarGünter3WallesHeike4HirthThomas3
Fraunhofer IGB, Zellsysteme, Nobelstr. 12, 70569 Stuttgart, Germany, esther.novosel@igb.fraunhofer.de
Fraunhofer IAP, Potsdam, Germany
Universität Stuttgart IGVT, Stuttgart, Germany
Universität Würzburg, Würzburg, Germany
Covalent heparin immobilization on new developed 3D-printable biomaterials
Introduction: The design of materials for use at the blood interface in vascular prostheses requires an understanding of the characteristics for surface endothelialization and for inhibition of undesirable platelet interactions. In our study, new 3D-inkjet printable polymer materials were designed for the buildup of an artificial 3-dimensional (3D) blood vessel. To improve the inadequate interaction between polymer and cells, material modification by immobilization of haemocompatible heparin sulphate (HS) was being developed.
Materials and Methods: Different types of a-o-hydroxy-oligoethers with amino endgroups have been synthesized and characterized by X-ray Photoelectron Spectroscopy (XPS) and methylorange assay. HS was covalently immobilized onto the aminated surfaces using an amide linkage. The carboxylic acid groups of heparin (HS–COOH) were first activated by EDC and NHS to form an N-succinimide ester. The activation reaction was conducted in a pH 4.5 MES buffer solution for 3h at room temperature. The amino groups on the outer surface of the coating were then reacted with the activated carboxylic groups of the heparin to form stable amide bonds. After the reaction, the surfaces were extensively washed with 2 M NaCl solution, and finally with DI water to completely remove the unbound heparin. The amount of bound heparin was quantified by XPS and photometrically by toluidine-blue test.
Results: The synthesis of the 3D-printable polymers with the designated amino endgroups was successful. The results of the XPS measurement showed for all produced materials that surface nitrogen concentration increases 1,5% in comparison to the control material. Additionally the results of the methylorange assay confirmed the presence of amino groups on the surface. A successfull heparin immobilization on the polymer surface was shown by the increase in surface sulfur content after HS immobilization by XPS. Furthermore an increasement of carboxy groups of the HS in comparison to unfunctionalized control material were detected by tolidine-blue test photometrically.
Conclusion: New 3D-printable polymers could be a suitable material for vascular grafts. Haemocompatible heparin was successfully immobilized onto the outer surface of the aminated polymers using EDC/NHS as coupling agents. The reported coating is expected to provide an anti-coagulant property at the blood/polymer interface. Further experiments are necessary to investigate the bioactivity of immobilized heparin.
Fraunhofer IAP, Potsdam, Germany
Universität Stuttgart IGVT, Stuttgart, Germany
Universität Würzburg, Würzburg, Germany
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.
Saarland University, Faculty of Medicine, Department of Trauma-, Hand- and Reconsctructive Surgery, Kirrbereger StraÕe, Building 61.4, 66421 Homburg, Germany, lisaschipf@yahoo.de
INM-Leibniz Institute for New Materials, Saarbrücken, Germany
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.
Cell density and cell surface of NHDF were significantly reduced on MD, HD and UHD compared to HOB on the corresponding surfaces.
The WST-test revealed a significantly reduced vitality on MD, HD and UHD compared to glass and to LD for NHDF and HOB. Between both cell types no differences could be detected.
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.
Controlled Assembly of Hepatocytes in Hydrogel Microwells
Creating liver-like tissue structures in vitro has important applications in basic pathobiology studies and pharmaceutical drug development. Bioartificial liver devices also holds promise to provide therapeutic solutions for liver tissue repair. In efforts to develop liver tissue constructs/models over the past years, the main strategies that have been investigated so far include the use of micropatterned culture systems and construction of three dimensional (3-D) cell aggregates with or without scaffolds. And these studies suggest the importance of cellular spatial organization and appropriate 3-D extracellular microenvironment in maintaining the hepatocyte viability and function in vitro.
In this study, we present a method to culture hepatocytes in agarose hydrogel microwells fabricated through a micromolding technique. Hydrogel microwells have been developed using polyethylene glycol materials in the past for generation of 3-D embryonic cell aggregates with controllable sizes. To create agarose microwells, PDMS molds were fabricated through a silicone master and placed on top of heated agarose solutions. After curing the agaorse by lowing the temperature, hydrogel microwells were generated and light microscopy shows uniform microwell structures having variable diameters (100, 200 and 500 microns) can be obtained. This facile method for fabricating hydrogel microwells requires no chemicals and photoirradiation process, offering advantages in cell culture applications.
The agarose microwells were used to culture HepG2 cells. It is discovered that the HepG2 adhered to the dented regions in microwells, and the cells may form 2-D or 3-D aggregates depending on the microwell size and cell seeding density. Our study suggests that the topographical features of hydrogel microwells may guide the hepatocytes behavior and provide a strategy to control the cell-cell contact and spatial arrangement essential for supporting hepatocyte functions in vitro. The microwell culture systems will be further investigated to engineer liver tissue models for use in basic and applied research activities.
KhetaniSRet al. Microscale culture of human liver cells for drug development. Nat Biotechnol., 200.
EhlertNina
Leibniz Universität Hannover, Institut für Anorganische Chemie, CallinstraÕe 9, 30167 Hannover, Germany, nina.ehlert@acb.uni-hannover.de
Amino-modified silica surfaces efficiently immobilize Bone Morphogenetic Protein 2 (BMP2) for medical purposes
Due to its ability to induce de novo bone formation, the differentiation factor Bone Morphogenetic Protein 2 (BMP2) is often used for enhanced integration of bone implants. With the aim to reduce possible high-dose side effects and to lower the costs, in this way targeting an affordable implant, we developed a simple and fast method for the immobilization of BMP2 on silica-based surfaces using silane linkers which carry amino or epoxy functions. We put special emphasis on the influence of the nanoscale surface topography of the silica layer. Therefore we chose glass (for control experiments) and Bioverit® II (as a typical implant base material) as support materials and coated these substrates with unstructured or nanoporous amorphous silica layers for comparison. Immobilized BMP2 was quantified by two different methods: by ELISA and by a cell-based assay for active BMP2, these tests probe for immunologically and biologically active BMP2, respectively. The results show that the amino functionalization is better suited for immobilizing the protein. Strikingly, a considerably higher amount of BMP2 could be immobilized on coated Bioverit® II surfaces as compared to coated glass substrates, which was presumably due to the macroscopic roughness of the Bioverit® II substrates. In addition, it was found that the nanoporous silica coatings on Bioverit® II substrates were able to bind more BMP2 than the unstructured ones.
HochEva
University of Stuttgart, Institute for Interfacial Engineering, NobelstraÕe 12, 70569 Stuttgart, Germany, eva.hoch@igb.fraunhofer.de
Evaluation of Gelatin-Based Cell Substrates and 3D Microstructuring by Multiphoton Polymerization
Introduction: The generation of functional tissues on appropriate substrates with well-defined properties has been a major topic in recent scientific research. A promising approach to mimic the behavior of tissues in vitro is the use of 3D polymeric hydrogels. Besides synthetic polymers, e.g. PEG, biological materials such as gelatin are at focus for the preparation of hydrogels for tissue engineering approaches due to their ability to promote cell adhesion by Arg-Gly-Asp-like sequences.
In the present study we describe the development of gelatin-based hydrogels and their characterization with regard to controllable mechanical properties and applicability for preparation of 3D microstructured substrates using multiphoton polymerization (MPP). Both, planar and microstructured gelatin hydrogels were evaluated for their use as cell substrates for primary porcine chondrocytes.
Materials and Methods: Gelatin methacrylamide (Gel-MA) was prepared by reaction of gelatin with methacrylic anhydride. The methacrylation was characterized using 1H-NMR- and IR-spectroscopy; the degree of substitution was determined by a method using trinitrobenzene-sulfonic acid. Hydrogels were prepared by photoinduced radical crosslinking of solubilized Gel-MA in the presence of a photoinitiator. Swelling characteristics and dynamic shear oscillation measurements were used to distinguish the viscoelastic properties of the hydrogels depending on the concentration of Gel-MA. The fabrication of 3D microstructured Gel-MA hydrogels was done by multiphoton polymerization.
Cell response of porcine articular chondrocytes to planar and microstructured hydrogels was evaluated by characterization of cell adhesion, morphology, proliferation and immunohistochemical staining against the cytoskeleton.
Results: We will present results concerning the derivatization of gelatin, 3D substrate preparation by photoinduced and multiphoton polymerization and physico-chemical characteristics of the gelatin-based hydrogels. Moreover we will showcase the reactions of porcine chondrocytes towards planar as well as microstructured substrates of different mechanical properties.
Conclusion: Material studies and cell-substrate interaction studies are in the scope of basic research investigations as well as applied science and development. We aim to develop material systems for assembly of 3D structured soft tissue scaffolds and for optimized biomaterial interfaces for tissue engineering or implants.
Dept. Thoracic, Cardiac and Vascular Surgery, University Hospital, Silcherstr. 7/1, 72076 Tübingen, Germany, martina.schleicher@med.uni-tuebingen.de
Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany
Section of Medical Materials and Technology, Dept. of Prosthodontics, University Hospital, Tübingen, Germany
Dept. Evolutionary Biology of Invertebrates, Institut of Evolution and Ecology, Eberhard Karls University Tübingen, Germany
Surgery of congenital heart defects and pediatric cardio-surgery, Children Hospital, University Hospital, Tübingen, Germany
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.
StuckensenKaiGbureckUwe
University of Würzburg, Universitätsklinik Würzburg, Department for Functional Materials in Medicine and Dentistry, Pleicherwall 2, 97070 Würzburg, Germany, kai.stuckensen@fmz.uni-wuerzburg.de
Biphasic layered scaffolds with anisotropic mineralization behavior for osteochondral tissue replacement
Biphasic layered scaffolds with an anisotropic mineralization behavior were obtained through a spray-coating-process. The synthesis was carried out using wet-chemical precursors, whereas the mineralization process occurred simultaneously to the ionotropic gelation of the biopolymer alginate. The mineralization took place biomimetically through the precipitation of planar brushite crystals (CaHPO4 · 2H2O) directed by the structure of the organic matrix. This composite structure allegorizes the subchondral zone of cartilage, a chondral zone replacement was formed through an organic matrix which exhibited a monolithic structure together with the subchondral zone.
Alginate solutions were prepared using 3% Protanal LF 10/60, where (NH4) · 2HPO4 was supplemented in variable concentrations. A 1.5 molar CaCl2 solution was used for ionic cross-linking. The solutions were processed layer-wise by airbrush guns spraying the phosphate containing biopolymer solution and the calcium solution alternately to generate a multi-layer structure. After several layers of mineralized biocomposite had formed, further layers were preceded with a non-phosphate containing biopolymer solution and the final structures were compressed unidirectional. The films were then transferred into a calcium chloride solution. After being washed, the specimen were subsequently dehydrated in acetone and covalently cross-linked in a bath consisting of 10% 1,6-hexamethylene diisocyanate and 90% acetone.
The spatial mineralization behavior of the specimen was investigated by X-ray diffraction analysis. Compared with randomly oriented brushite powder, the (0|0)-crystal planes of the brushite crystals within the scaffolds were arranged parallel to the sample surface at a high extent. Consequently an anisotropic mineralization can be concluded. Structures produced with molar hydrogenphosphate concentrations from 0.05 mol/l to 0.3 mol/l were analyzed regarding their tensile strength and elastic modulus using rectangular specimen with dimensions of 13 mm x 60 mm and an average thickness of 0.2 mm. The tensile strength decreased linearly from 4.9 MPa at 0.05 mol/l of hydrogenphosphate to 3.5 MPa at 0.3 mol/l. According to these results, the elastic modulus increased linearly from 63.5 MPa, in the case of highest mineral content, to 325 MPa, at the lowest mineral content. Thus a higher degree composite mineralization yields in this case a lower tensile strength and elastic modulus, respectively.
Characterization of Agarose Gels in an ElectroForce® Test Instrument
The objective of this work was to employ the Bose ElectroForce® testing platform with Dynamic Mechanical Analysis (DMA) software to apply mechanical stimuli and evaluate the mechanical properties of agarose gels. Dr. Murakami and Dr. Sawae from the Kyushu Institute of Technology provided the agarose samples to investigate the mechanical behavior of low concentration gels at high frequencies. 1% and 2% (wt.) agarose type VII gels were produced from type VII powder using a standard gelling protocol supplied by Dr. Murakami. Test specimens, cut from the bulk gels, were approximately 18 mm in diameter and 2.5 mm in height. 50 mm diameter solid platens were used to secure the sample. Due to the low loads, a 10 N load cell was used. DMA consisted of a frequency sweep from 0.1 Hz to 100 Hz at a range of mean displacements. These mean amplitudes, as measured by stretch ratio, ranged from 0.99 to 0.70, where the stretch ratio, λ, is defined as λ = l/Lo where l is the loaded height and Lo is the unloaded height. The displacement amplitude was ± 0.5% of the unloaded height. The contribution to the complex modulus at lower frequencies is nearly equal, while the storage modulus dominates at higher frequencies, indicating that less energy is dissipated at higher frequencies. Responses at different mean stretch ratios were also evaluated. Tan Î′ was measured for the 1% gel. There was a much larger phase shift at lower frequencies. Complex modulus at different λ, provided insight into the material behavior. As the mean stretch ratio is decreased, the modulus of that material is significantly increased. It was also found that the material behaves differently depending upon pre-stretch. As the stretch ratio is decreased, the contribution by the loss modulus to the complex modulus is reduced. Similar results could be seen for the 2% gel with different magnitudes. Tan Î′ for the 2% gel is less than 1% gels at all most mean stretch ratios tested. The complex modulus of the 2% gel is greater than the modulus for the 1% gel. 3D-graphs can be developed displaying tan Î′ as a function of both mean stretch ratio and frequency. Incorporating tan Î′ into a mathematical model would further strengthen the predictive capabilities. With this knowledge, scaffold design parameters and clinical application of gels can be understood prior to development. These experiments demonstrate that the ElectroForce® 3200 test system operates at the desired high frequencies while measuring small loads with precision.
Fabrication of Hyaluronic Acid-Dextran Hydrogel Microparticles for Tissue Engineering Applications
Spherical microcarriers based on various polymer materials have been widely used for culturing mammalian cells and engineering tissue constructs. In tissue engineering applications in particular, microcarriers may offer a way to control the cellular spatial organization within tens to hundreds micrometers, a range that is critical for induction of cellular communications and tissue morphogenesis. In addition, growing cells on microcarrier scaffolds can potentially make the in vitro culture scalable and the resulting micro-tissues injectable for minimal invasive transplantation. Recently we developed a hydrogel material via crosslinking functionalized hyaluronic acid (HA) and dextran. This composite matrix demonstrates tunable degradability and mechanical properties as well as good biocompatibility in vivo.
In this study, we describe a method to fabricate HA-dextran hydrogel microspheres of adjustable sizes for use as micro-scaffolds to support cell growth and microscale tissue formation. The fabrication process employs electrostatic force to first induce liquid droplets and then the phase separation between polyethylene glycol (PEG) and dextran to facilitate hydrog ed from a syringe pump as liquid droplets, which were collected and crosslinked by dextran solutions containing dextran-aldehyde. The phase separation between PEG and dextran helped to maintain the shape of droplets during the crosslinking reaction. Light microscopy studies show that the resulting hydrogel particles are spherical in shape with a narrow size distribution. By varying the concentration of HA and dexrtan components, the diameter of the resulting hydrogel microspheres can be tuned within the range of 50 microns up to 1 mm. After being freeze-dried, fibrous, HA-dextran microspheres exhibit interconnected structures under SEM. Further studies will be carried out to culture hepatocyte and endothelial cells using these new degradable HA-dextran hydrogel microspheres to engineering micro-tissues for therapeutic purposes.
Adult Stem Cells 1 – basics
BuravkovaLudmilaAndreevaElenaRylovaJulia
Institute of Biomedical Problems RAS, Cell Physiology, Khoroshevskoye sh. 76-A, 123007 Moscow, Russia, buravkova@wolf.ru
O2-mediated MMSCs plasticity
High plasticity of multipotential mesenchymal stromal cells (MMSCs) is the outcome of multiple physical and chemical environmental factors. Oxygen is fundamental for life and its concentration is an important signal for virtually all cellular processes The question arises – how does oxygen regulate the functional status of progenitor cells? It is well known that the oxygen tension in MMSCs tissue niches comprises 5–7% and in sites of tissue regeneration is even lower – 05–3%. Thus, it may be assumed, that routinely used in laboratory practice 20% O2 in cell culture medium should induce oxidative stress and nonspecific selection of progenitor cells from heterogenous MMSCs popupalation, isolated from different tissue sources. Recently we clearly demonstrated that permanent expansion of MMSCs during several passages under reduced oxygen tension had altered cell morphology, induced proliferation and slowed down osteogenic, adipogenic ans chondrogenic differentiation without affecting telomerase activity. The profile of cytokines and growth factors, especially VEGF, is also strongly governed by O2 concentration therewith the expression of main CD markers does not altered in low O2. Differential gene expression analysis of hypoxic MMSCs showed up-regulation of genes associated with cell signaling, proliferation, and cell metabolism and down regulation of genes responsible for cytoskeleton and exptracellular matrix proteins. Revealed changes were reversible that confirmed the physiological effect of reduced O2 on MMSCs plasticity possibly both in vitro and in situ. Based on these findings we have developed new approaches for cell product preparation and got encouraging data on these cells application in bone callus formation after bone break in rats and human HSC-MMSCs coculture. Up to now some questions concerning the effects of low oxygen on MMSCs pecularities are not still solved: in particular – does low O2 provide the specific selection of MMSCs subpopulation or just supports the growth of particular type of MMSc. Also the impact of extracellular components of MMSCs niches in O2-mediated MMSCs plasticity should be clarified. Thus far, the oxygen concentration essentially contributes to MMSCs physiology and should be taking into account in the setting of protocol for cellular therapy.
Max Planck Institute for Metals Research, Department New Materials and Biosystems, Heisenbergstr. 3, 70569 Stuttgart, Germany, eva.altrock@mf.mpg.de
Section for Transplantation Immunology and Immunohematology, Center for Medical Research, University Medical Clinic, Tübingen, Germany
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.
University of Tübingen, Section for Transplantation, Immunology and Immunohematology, Waldhörnlestr. 22, 72072 Tübingen, Germany, carolin.steinl@uni-tuebingen.de
NMI Reutlingen, Germany
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.
Department of Gynecology and Obstetrics, University of Schleswig-Holstein, Campus Luebeck, Germany
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.
Mammary gland: a source of multipotent adult stem/progenitor cells
In the field of stem cell-based therapy, it is a matter of common knowledge that adult stem cell sources are of great interest. Various tissues are considered by the researchers' community as potential sources of stem cells. However, the application of stem cells for cell-based therapy requires an accessible tissue for cell isolation and a proliferating stem cell population with the ability to differentiate into the desired lineages.
Here we present the first steps of isolation and characterization of primary human mammary gland stem cells acquired from mammary gland specimens removed during a breast reduction operation. Using common known techniques like quantitative Real-Time PCR and immunohistochemistry, we were able to demonstrate the stemness of the cells and their differentiation potential over several passages.
The outgrowth of the cells from lobular structures acquired by enzymatic digestion was monitored by Time-Lapse-Microscopy. Similar to the outgrowth of pancreatic stem cells from pancreatic acini, the 3D-structures formed by the isolated cells melt on the bottom of the cell culture labware after attachment. The outcoming cells build up a stable cell population with a characteristic strong spindle-shaped morphology.
The cells showed the expression of stem cell markers (e.g. Nestin) and differentiation markers (e.g. neurofilaments, smooth muscle actin alpha, cytokeratin 18, von-Willebrandt-Factor) on both transcriptional and translational levels. The proliferative capacity was furthermore verified by the strong expression of the proliferation-marker Ki-67. Moreover, we were able to generate proliferating stem cell populations from single cell clones.
Consequently, we propose that this source of stem cells might be an interesting alternative for clinical applications due to their easy accessibility, abundance and their good stem cell behavior.
FeilGerhardBaumannSimonSchäferRichardSchäferJochenMöhleRobertStenzlArnulfSievertKarl-Dietrich
Eberhard-Karls-Universität, Department of Urology, Hoppe-Seyler-StraÕe 3, 72076 Tübingen, Germany, gerhard.feil@med.uni-tuebingen.de
Human mesenchymal stem cells transplanted into rectus abdominis muscle of athymic rats differentiate into skeletal muscle and show neuromuscular junction
Objectives: Urinary incontinence is associated with damaged sphincteric myofibers or with an age-dependent decrease of myofibers. Since mesenchymal stem cells (MSCs) can differentiate into muscle cells in vitro, autologous MSCs might be an option for a functional treatment of urinary incontinence. The aim of the study was to investigate survival and myogenic differentiation of transplanted human MSCs in a rat model and to demonstrate a linkage to the host's nerve system.
Methods: Human MSCs were isolated from bone marrow aspirates. To induce myogenic differentiation in vitro, human MSCs were exposed to 5-azacytidine (AZA) in passage (P) 1. Native MSCs in P1 and P3 as well as AZA-exposed MSCs of subsequent P2 or P3 were directly injected into the rectus abdominis muscle of athymic rats. For in vivo tracking MSCs were labeled with PKH26 cell linker. Survival and myogenic differentiation of MSCs in rat muscle tissue was monitored histologically from 4 days up to 16 weeks after cell injection. The muscle markers desmin and skeletal muscle myosin heavy chain were immunologically detected. Innervation of newly formed skeletal muscle was investigated by staining cryosections with alpha-bungarotoxin conjugate that binds to the acetylcholine receptors of motor end plates.
Results: Native and AZA-exposed MSCs of all passages could be demonstrated in all animals investigated. Histology of animals in the short-term experiments up to 8 days revealed well-defined clusters and beginning migration of transplanted MSCs in the rectus abdominis muscle. After 4 and 8 weeks of cell injection, a continuous dissemination of transplanted MSCs was detected. Histology of animals in the long-term study revealed PKH26-positive myofibers that were in parallel with the native skeletal muscle fibers. Immunohistochemistry for myogenic desmin demonstrated striated myofibers. Skeletal muscle myosin was expressed in PKH26-positive myofibers. Staining for acetylcholine receptors showed motor end plates adjacent to newly formed PKH26-positive myofibers.
Conclusions: The data revealed a sequence of histogenesis of human MSCs into myfibers in vivo that were well integrated into the host muscle tissue. Junction to the nerve system of the newly formed muscle indicates a functional integration of transplanted and myogenically differentiated MSCs. Thus, regeneration of rhabdosphincter muscle based on autologous adult stem cells in patients suffering from urinary incontinence might be possible.
Universität Stuttgart, IGVT, Nobelstr. 12, 70569 Stuttgart, Germany, c.kleinhans@gmx.de
Fraunhofer IGB, Stuttgart, Germany
Universität Würzburg, Würzburg, Germany
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.
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
University Clinic of Tübingen, Department of Urology, Tübingen, Germany
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.
Leibniz Universität Hannover, Institut für Technische Chemie, Callinstr. 5, 30167 Hannover, Germany, kasper@iftc.uni-hannover.de
Medizinische Hochschule Hannover, Klinik für Frauenheilkunde und Geburtshilfe, Hannover, Germany
Medizinische Hochschule Hannover, Klinik für Immunologie und Rheumatologie, Hannover, Germany
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.
University of Tuebingen, ZMF, Section for Transplantation Immunology, Med.Klinik II, Waldhörnlestrasse 22, 72072 Tübingen, Germany, nicole.staudt@medizin.uni-tuebingen.de
Center for Regenerative Medicine and Department of Orthopedic Surgery, Center for Medical Research Center, Tübingen, Germany
Medical and Natural Sciences Research Center, Tübingen, Germany
Department of Immunology, Institute for Cell Biology, University of Tübingen, Germany
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil
Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
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.
University Hospital Tübingen, Department of Radiology, WaldhörnlestraÕe 22, 72072 Tübingen, Germany, r.bantleon@uni-tuebingen.de
University Hospital Tübingen, Department of Diagnostic and Interventional Radiology
University Hospital Tübingen, Institute of Clinical and Experimental Transfusion Medicine
University Hospital Tübingen, Department of Thoracic Cardiac and Vascular Surgery
University of Tübingen, Department of Medical Biometry
University Hospital Tübingen, Departments of Orthopaedics
University of Tübingen, Institute of Pathology
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.
Electrophysiolgical comparison of native and progenitor cell derived enteric neurons
The enteric nervous system (ENS) is a complex network of interconnected ganglia (neuronal and glia cells) within the wall of the gastrointestinal tract.
Failures in gut colonization during embryogenesis can lead to local peristaltic dysfunction. Hirschsprung's disease (HSCR) is one of the most common defects in newborns (1:5000), which leads to life threatening intestinal obstruction. At present replacing the affected area with a stoma (artificial anus) is the only treatment available. New possible approaches include implantation of artificial ENS sections newly-created from progenitor cells directly derived from the HSCR patients.
The basis of neuronal activity is a complex interplay between different sets of ion channels. Using various electrophysiological approaches from patch clamp to microelectrode array (MEA) and thereby covering single cell to network levels we collect and compare electrophysiological data in vitro from native ENS neurons and progenitor derived neurons from our project partners. The aim is to improve neuronal differentiation and to mimic native ENS neurons including functional network behaviour, particularly with regard to the usability as ENS transplant. Furthermore this approach could be expanded to other neurodegenerations like Parkinsons disease.
NMI Natural and Medical Sciences Institute at the University of Tuebingen, MarkwiesenstraÕe 55, 72770 Reutlingen, Germany, christian.freudigmann@nmi.de
TETEC Tissue Engineering Technologies AG, Reutlingen, Germany
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).
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
Institute of Anatomy, University of Tuebingen, Germany
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.
GMP conform manufacturing process of an autologous melanocyte graft
Vitiligo is a chronic disorder which leads to the depigmentation of the skin. The cause can be attributed to the degeneration or malfunction of the melanin producing cells, the so called melanocytes. Knowledge about the ultimate cause of this disorder is, at present, still very incomplete. Amongst others, hypotheses were proposed which postulate a genetic predisposition or autoimmune effects in vitiligo patients.
Nevertheless, with a lack of the necessary skin protection against UV-radiation, the risk of vitiligo patients for developing cancer is increased in the melanocyte-free areas.
Depending on the stage of disease there are different therapy methods which lead to a repigmentation of the affected skin areas.
At the Fraunhofer IGB we have developed an isolation and proliferation process for patient-own, autologous melanocytes within our certified GMP manufacturing unit (according to the German Drug Law). For this purpose, the melanocytes are first isolated from a skin biopsy of the patient and then cultivated over 21 days. During their cultivation we have verified the cellular identity over the expression of melanocyte specific markers (HMB45, MelanA and Vimentin). To ensure the quality of our product, we also control different parameters like the cellular vitality, endotoxin level and sterility (according to the Ph. Eur. 2.6.1) during the manufacturing process.
Finally, the cells are harvested and transported at a defined cell concentration as a suspension to the operation site. There the melanocytes are transplanted into the pigment-free skin areas of the patient. A manufacturing authorisation has already been applied for with the responsible local authority, which will allow the use of autologous melanocyte transplants in clinical studies. There we want to analyse, if our melanocyte grafts promote the repigmentation of the skin and the melanocyte grow for longer periods.
Eberhard-Karls-Universität, Department of Urology, Hoppe-Seyler-StraÕe 3, 72076 Tübingen, Germany, gerhard.feil@med.uni-tuebingen.de
Allg. öffentl. Bezirkskrankenhaus Hall in Tirol, Austria
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.
University Stuttgart IGVT, Medical Interfacial Engineering (MGVT), NobelstraÕe 12, 70569 Stuttgart, Germany, iris.dally@igb.fraunhofer.de
Fraunhofer Institute for Interfacial and Biotechnology IGB, Stuttgart, Germany
Klinik Schillerhöhe, Gerlingen, Germany
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.
von der RuhrJuergen
Institute of Anatomy Tuebingen, Tissue Engineering, Österberg Str.3, 72074 Tübingen, Germany, jruhr@anatom.uni-tuebingen.de
Drug eluting stents with shikonin: a tool for vascular regeneration and impeding of re-stenosis?
The Incidence rate for cardiovascular diseases is one of the highest and so it's closely associated to one of the most common causes of death in our society. State of the art intervention for stenosis is grafting a stent to maintain the blood-flow. An unwelcome side-effect from stenting however is a severe re-stenotic risk due to hyperplasia which derives from fast proliferating cells migrating from the intima to the intraluminal side. The use of DES (drug eluting stents) with anti proliferating agents (e.g. Paclitaxel, Sirolimus) should reduce this risk by inhibiting cell proliferation. Unfortunately regenerative processes are affected similarly thus limiting the application. In search of a selectively acting drug we focused on Shikonin a pharmaceutical which is known for its regenerative potential since it is applied for a long time in TCM (traditional Chinese medicine). Current evaluations from Shikonin actually revealed likewise anti proliferative characteristics. Therefore we here investigated the impact of Shikonin as a selectively acting drug on the responsible cells for re-stenosis in vitro and in vivo. In vitro our studies revealed such a differential dose dependant effect of Shikonin on different cell types. First in vivo data in rabbits suggest contradictory effects such as more neointimal growth however also a higher re-endothelialization rate.
When previous in vitro data is validated further from second in vivo trials a beneficial therapeutic tool for cardiovascular therapy could be obtained.
NMI Reutlingen, Regenerative Medicine II, Markwiesenstr. 55, 72770 Reutlingen, Germany, jana.mueller@nmi.de
TETEC Tissue Engineering Technologies AG, Reutlingen, Germany
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.
Universidad de Granada, Histology, Avenida de Madrid 11, 10012 Granada, Spain, acampos@ugr.es
Hospital Universitario de Granada, Granada, Spain
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.
Universidad de Granada, Histology, Avenida de Madrid 11, 10012 Granada, Spain, acampos@ugr.es
Hospital Universitario de Malaga, Spain
Hospital de Viseu, Portugal
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.
Department of Plastic and Hand Surgery, Burn Centre, Lübeck, Germany
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.