Forschergruppe 548 of the DFG
First application period:
Polysialic acid: Towards the evaluation of a new, bio-identical scaffold material
“If one way be better than another, you may be sure it is nature’s way”
[Aristotle, 4th century BC]
The rapidly growing knowledge and technical expertise in the field of transplantation medicine is paralleled by an urgent lack in organ availability and all projections indicate that this gap will continue to widen. A new interdisciplinary field “tissue engineering” has developed, attempting the de novo creation of organ substitutes. A major challenge in tissue engineering strategies resides in the reconstruction of the organs’ complex three-dimensional architecture. Numerous specialized zones are organized in a functional “mosaic” and are combined with extended capillary and neuronal networks.In vivo the development of organs essentially depends on the information provided by the three-dimensional system. However, normal cells in culture have lost the ability to orient in three dimensions and thus have lost the ability to reconstruct the anatomical structures of the organ. A way to circumvent this problem is by use of highly porous tissue growth supports (scaffolds) on which cells attach and proliferate. These carriers can be functionalized to serve as guides for cell growth and differentiation and as attractants for vessels and nerves. Scaffolding materials must be biocompatible but simultaneously provide the physico-chemical properties for being tailored into two- or three-dimensional objects. Finally scaffolds must be biodegradable in order to release an autonomous organ.
Inorganic ceramic materials have been proven to be useful in bone reconstitution efforts, while synthetic and natural polymers of which the degradation products can be removed by the natural circuits are the preferred materials in soft tissue engineering. Special emphasis has been put on bio-degradable polymers, because these materials offer the advantage of physical stability in different crafting processes and their multifunctionality enables the design of bioactive gradients. The major disadvantage of these materials is their uncontrolled degradation in the physiologic system. Once started, the degradation by hydrolysis can be hardly controlled and often leads into a deleterious cycle, ending with the destruction or rejection of the grafted organ/tissue.
We suggest the testing of polysialic acid towards putative scaffold material. The homopolymere of a2,8-linked sialic acid residues is optimized by nature for application in the vertebrate body. As a posttranslational modification of the neural cell adhesion molecule, polysialic acid is abundant during ontogenic development, becomes decreased after birth, but continues to be expressed in some regions of the adult brain. Polysialic acid is re-expressed in the regenerating nervous system and has been shown to be a reliable marker of neuronal stem cells.
From studies carried out in vivo and in vitro it is known that polysialic acid chains exhibit a pronounced self aggregation tendency, a feature, which is decisive for the shaping of solids. Moreover, the material has a long half-life in the circulation and has been successfully used in drug delivery systems to extend the life span of protease sensitive drugs (e.g. asparaginase; insulin). So far, no endogenous polysialic acid degrading activity has been observed in mammals. However, phage born endosialidases can be used for the specific degradation of polysialic acid. The short oligosaccharides released by enzymatic hydrolysis provide valuable nutrients for each animal cell. Most important in the context of this study, all enzymes involved in polysialic acid biosynthesis and degradation have been cloned in one of the applicants laboratory and can be used to set up a reaction chain for the recombinant production of natural and potentially also modified polysialic acid.
An interdisciplinary research group has formed in Hannover integrating researchers from university (the Medizinische Hochschule Hannover, the Universität Hannover) and non-university institutions (Deutsche Institut für Kautschuktechnologie e.V.). The consortium covers the wide range of expertise and technical competencies required to address the full scope of interdisciplinary questions in relation with the iterative evaluation of polysialic acid as scaffold materials in tissue engineering.
The project has been structured into six work packages (WP), which again represent interdisciplinary efforts in the cases of WP1, WP4, WP5 and WP6.
WP1 Studies combined in WP1 concentrate at the recombinant production of polysialic acids by enzyme reactor technology. Moreover, large scale purification of polysialic acid from natural sources following established protocols will guarantee stocks for pilot studies in all project parts. Purified enzyme systems from recombinant organisms will be supplied and the protocols for large scale production will be derived from these studies.
WP2 The central goal of WP2 is the chemical derivatization of polysialic acid in order to improve its properties with regard to optimized polymer processing, improvement of mechanical stability, and designed degradation properties. Organic and inorganic manipulations on the polymer are planed.
WP3 In WP3 aspects of solid fabrication will be studied on natural and chemically modified polysialic acids. Efforts will concentrate on the development of processes suited to generate fibers and nonwovens, specific rough films, and graduated porous sponges.
WP4 In WP4 a panel of advanced physico-chemical methods will be applied to determine basic polymer and surface characteristics in raw and crafted materials as generated in the other WPs. A special emphasis will be set on the investigation of materials in the presence of cell colonization. This process accompanying information will be constantly used for the refining of material properties.
WP5 Bio-medical properties of raw and crafted materials will be determined by a combination of gradually improved cellular and animal model systems and advanced molecular biological techniques (“tailor-made” microarrays). Materials will be tested before and after defined decoration with bioactive factors. The gradually increased sensitivity of these test systems shall enable a rapid and consequent selection of suited materials.
WP6 Conditions for the induced and controlled degradation of polysialic acid-based materials will be established in WP6. Studies are aimed at optimizing endosialidase-mediated degradation by fine tuning the cleavage characteristics of the enzyme by structure based mutagenesis approaches and by material-chemical techniques. These studies in addition point towards the development of controlled drug and/or cell delivery systems.