TTOOWWAARRDDSS SSEELLEECCTTIIVVEE AADDHHEESSIIOONN OOFF MMEESSEENNCCHHYYMMAALL PPRROOGGEENNIITTOORR CCEELLLLSS FFRROOMM TTHHEE RRAATT BBOONNEE MMAARRRROOWW Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der naturwissenschaftlichen Fakultät IV (Chemie und Pharmazie) der Universität Regensburg vorgelegt von Breda Vogel aus Slowenien 2006 Die Arbeit wurde angeleitet von: Prof. Dr. Achim Göpferich (Universität Regensburg) und Prof. Dr. Michaela B. Schulz (Karl-Franzens Universität Graz) Promotionsgesuch eingereicht am: 26.04.2006 Mündliche Prüfung am: 24.05.2006 Prüfungsausschuss: Prof. Dr. S. Elz (Vorsitzender) Prof. Dr. A. Göpferich (Erstgutachter) Prof. Dr. M.B. Schulz (Zweitgutachter) Prof. Dr. J. Heilmann (Drittprüfer) 2 4 TTAABBLLEE OOFF CCOONNTTEENNTTSS Chapter 1 Introduction and goals of the thesis 7 Chapter 2 CD45-positive cells of hematopoietic origin enhance chondrogenic 39 marker gene expression in rat marrow stromal cells Chapter 3 Myeloid cells suppress in vitro osteogenic differentiation of rat marrow 59 stromal cells Chapter 4 Towards selective adhesion of marrow stromal cells: phenotype 83 characterization and adhesive properties of plastic adherent rat bone marrow cells Chapter 5 Pilot study to modulate integrin expression on rat MSC 107 Chapter 6 Tracing MSC phenotype: from bone marrow to several passages 117 Chapter 7 Influence of simvastatin and atorvastatin on osteogenic differentiation 141 of rat MSC Chapter 8 Summary and conclusion 153 Appendix Abbreviations 160 Curriculum vitae 162 List of publications 164 Acknowledgments 167 6 CCHHAAPPTTEERR 11 IINNTTRROODDUUCCTTIIOONN && GGOOAALLSS OOFF TTHHEE TTHHEESSIISS Breda Vogel INTRODUCTION _________________________________________________________ 9 MESENCHYMAL STEM / PROGENITOR CELLS (MSC) _____________________ 11 Multilineage differentiation potential & unlimited self-renewal_________________ 12 Surface marker characterization of MSC___________________________________ 14 Adhesion molecules _____________________________________________________ 17 Integrins_____________________________________________________________ 17 MSC in cell therapies____________________________________________________ 19 BONE __________________________________________________________________ 21 Bone cells______________________________________________________________ 22 Formation of the Skeleton________________________________________________ 22 MSC differentiation to osteoblasts_________________________________________ 23 Regulation of bone function ______________________________________________ 25 Bone Matrix ___________________________________________________________ 26 TISSUE ENGINEERING __________________________________________________ 28 Cells for tissue engineering_______________________________________________ 29 Scaffolds ______________________________________________________________ 30 Controlling cell-biomaterial interactions____________________________________ 30 Bone Tissue Engineering_________________________________________________ 32 GOALS OF THE THESIS _________________________________________________ 33 REFERENCES___________________________________________________________ 35 8 Chapter 1: Introduction and goals of the thesis IINNTTRROODDUUCCTTIIOONN The public has placed great hope in scientists, who “grow new organs and tissues in the labs” in the past decades. Nevertheless, the journey from the in vitro experiments to clinical applications of “off-the-shelf” tissues still presents numerous technical challenges; a native tissue is far more complex than just a mass of cells glued together, and the signals that regulate the extracellular matrix development in vivo are still poorly understood. Different test systems and animal models have been developed to qualify the intermediate in vitro results of tissue engineering. Whereas human individuals differ in age and many genetic determinants, rodent animal models (mouse, rat) can be manipulated to yield minimal intra-individual variation. Much characterization is needed, however, to find parallelisms between animal models and human systems. Whereas numerous biomaterials used as scaffolds show good in vivo performance, the most promising cell sources for tissue engineering are adult stem cells; they are present in every adult individual and give rise to differentiated cells of tissues or organs. Two main branches of stem cell research have been booming since the 1960s, when Friedenstein and his colleagues documented in vitro colony forming fibroblasts from the bone marrow, now called mesenchymal stem / progenitor cells (MSC). First, more detailed basic research deepens into “stemness”, differentiation potency and their molecular code, whereas, on the other hand, the more applicative other branch uses the advantages of MSC as cells for cellular therapies (gene delivery, immunosuppression) and neogenesis of mesenchymal tissues (tissue engineering). Both fields are strongly interdependent, providing useful information to the “sister” branch. Likewise, this thesis encompasses both approaches to stem cell research. The first phase included the characterization of MSC from the rat bone marrow, characterization of “accompanying” hematopoietic, cells and their influence on osteogenic differentiation. The outcomes of cell characterization lead us further to test for bone tissue engineering applications as a means of finding biomaterial for selective adhesion of pure MSC. The materials used as bone substitutes often have the disadvantage of causing fibrous tissue development at the interface with the surrounding tissue when implanted. This process follows the natural wound healing process of fibrin clot formation and inflammation by infiltration of macrophages and other cells of the immune system to the site of an implant1. Also in vitro, cells often fail to adhere or fail to differentiate and produce enough ECM when seeded onto raw biomaterials. To circumvent these issues, modification of the biomaterial surface can enable the adhesion of desired cells to the biomaterial surface and enhance their differentiation. One possibility is to covalently bind adhesion-promoting sequences, i.e. 9 Breda Vogel ligands for integrins, which are cell adhesion receptors, to the biomaterial surface and at the same time prevent nonspecific cell adhesion to the hydrophobic biomaterial surface using a hydrophilic poly-ethylene-glycol (PEG) layer. To this end, the ligands, for which receptors on the target cells are highly expressed, are bound to the biomaterial surface. Therefore, it is a prerequisite to determine which integrin receptors are actually expressed on “desired cells” (in our case rat MSC) and to which level. Then, the decision has to be made, which ligand will to be bound to the biomaterial surface, to ensure the desired adhesion and cell differentiation. Figure 1: The primary goal of the thesis was to discover integrins expressed only on MSC (e.g. Cell 1) to achieve selective surface immobilization and improved differentiation. 10