towards selective adhesion of mesenchymal progenitor cells from

168 Pages·2006·5.1 MB·English

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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)
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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
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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

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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.
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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.

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