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Design of Artificial Modular Extracellular Matrices

Gräter, Stefan V. W.

German Title: Modulares Design künstlicher extrazellulärer Matrizes

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Abstract

Cellular functions such as cell growth, adhesion and differentiation are essentially controlled by the surrounding extracellular matrix (ECM). The mechanical, chemical and structural properties of the ECM are consequently crucial for the selection of cells at interfaces and the formation of tissues. The objective of this thesis was to develop an artificial ECM to determine and control the parameters influencing the crosstalk between cells and their surroundings on a molecular level. Artificial ECMs which mimic the natural environment of cells enable precise insights into cell-ECM crosstalk; ultimately, we aim to trigger the crosstalk, such that specific cell functions are provoked. To this end, a modular ECM system was developed, consisting of (i) poly(ethylene glycol) (PEG) as the basic material, (ii) gold nano-particles as the structuring component, and (iii) bioactive molecules which are immobilized on the basic material and on the nano-structure, to equip these modules with a biological function. The mechanical, structural, and chemical properties of the artificial ECM, as defined by the respective modules, can be tuned independently from one another, enabling the customized tailoring of the artificial ECM for specific applications. PEG hydrogels, used as both the basic material and first module of the artificial ECM, were chosen because of their resistance to protein adsorption, as well as their elastic and swelling properties, which partly mimic the hyaluronan material surrounding the cell membrane. A photo-initiated crosslinking reaction of PEG macromers was used to obtain hydrogels with well-controlled physical properties, characterized in terms of the gel content, swelling ratio, and mesh size. Elasticity at the nanoscale was assessed by an indentation method using atomic force microscopy (AFM). In this way, we were able to prepare hydrogel surfaces covering the biologically relevant range of elasticities. Structuring the hydrogel substrates with nanoscale gold patterns as the second module of the artificial ECM was achieved by means of a newly-developed transfer lithography method. Gold particles of a particular size, and separated by a defined distance were obtained by using block copolymer micelle nanolithography, which itself is restricted to solid, inorganic, and planar surfaces such as glass slides; the gold particles are transferred to polymers by means of a thiol-gold coupling scheme. Depending on the polymer to be gold-decorated, an appropriate thiol linker molecule was incubated on the gold-patterned glass surface, and crosslinked to the PEG hydrogel during polymerization. The transfer resulted in a complete and accurate transfer of the nano-pattern to the polymer surface. Cryo-electron microscopy was used for structural characterization of the resulting surfaces, including watercontaining soft hydrogels. 2 The transfer nano-lithography technique is the first method to successfully nanostructure soft and polymeric materials with metal structures on a large scale, and can in principle be applied to the structuring of any organic planar and non-planar surface. The structural properties of the artificial ECM, controlling, e. g., the clustering of receptors at adhesion sites of adhering cells, can be adjusted by choosing the particle size and distance of the original gold pattern. Another structural parameter can be introduced by the non-planarity of the surfaces. Hydrogel-based microchannels have been developed that were internally decorated with gold nanoparticles, resulting in nanopatterned, tube-shaped artificial ECMs surrounding the cell in three dimensions, mimicking, for example, blood vessels. As a third module of the artificial ECM, the nanostructured hydrogel surfaces were chemically modified to provide the cell with biofunctions. Proteins were coupled to the gold particles or the hydrogel surface via Ni(II)-NTA complexes, and peptides were coupled to the gold particles via thiol groups, or to the hydrogel surface via amino groups. The four different schemes were developed to specifically couple the bioactive molecules at well-defined orientations and in their native conformation to either the hydrogel surface or the gold moieties, without introducing either cytotoxicity or loss of biocompatibility. The selective functionalization was tested for representative biomolecules, the adhesion receptor-binding peptide RGD, the cell-cell adhesion protein L1, and the green fluorescent protein. This concept enables selective modification of the gold particles or the inter-particle surface by coupling virtually any biomolecule to the aforementioned domains of the artificial ECM. The functionality of the three different components of the artificial ECM was tested in cell experiments. Experiments using substrates with various inter-gold particle spacing, biofunctionalizations, and cell types, demonstrated the applicability of the artificial ECM as such. Most importantly, for the first time, nanopatterned hydrogels were shown by cryo-SEM to be deformed by the adhering fibroblasts, thereby revealing the direct crosstalk between the cell and the ECM mimic on the molecular level. In addition, the functionality of non-planar substrates for cell experiments was demonstrated by means of micro-channels. In conclusion, the modular artificial ECM, as developed in this research project, meets the mechanical, structural, and biological requirements necessary to serve as a versatile and adjustable tool to investigate and provoke specific cell-surface interactions. The artificial ECM provides a useful means by which to influence cell adhesion and function, thereby enabling systematic selection of cell types for biotechnological and medical applications.

Translation of abstract (English)

Cellular functions such as cell growth, adhesion and differentiation are essentially controlled by the surrounding extracellular matrix (ECM). The mechanical, chemical and structural properties of the ECM are consequently crucial for the selection of cells at interfaces and the formation of tissues. The objective of this thesis was to develop an artificial ECM to determine and control the parameters influencing the crosstalk between cells and their surroundings on a molecular level. Artificial ECMs which mimic the natural environment of cells enable precise insights into cell-ECM crosstalk; ultimately, we aim to trigger the crosstalk, such that specific cell functions are provoked. To this end, a modular ECM system was developed, consisting of (i) poly(ethylene glycol) (PEG) as the basic material, (ii) gold nano-particles as the structuring component, and (iii) bioactive molecules which are immobilized on the basic material and on the nano-structure, to equip these modules with a biological function. The mechanical, structural, and chemical properties of the artificial ECM, as defined by the respective modules, can be tuned independently from one another, enabling the customized tailoring of the artificial ECM for specific applications. PEG hydrogels, used as both the basic material and first module of the artificial ECM, were chosen because of their resistance to protein adsorption, as well as their elastic and swelling properties, which partly mimic the hyaluronan material surrounding the cell membrane. A photo-initiated crosslinking reaction of PEG macromers was used to obtain hydrogels with well-controlled physical properties, characterized in terms of the gel content, swelling ratio, and mesh size. Elasticity at the nanoscale was assessed by an indentation method using atomic force microscopy (AFM). In this way, we were able to prepare hydrogel surfaces covering the biologically relevant range of elasticities. Structuring the hydrogel substrates with nanoscale gold patterns as the second module of the artificial ECM was achieved by means of a newly-developed transfer lithography method. Gold particles of a particular size, and separated by a defined distance were obtained by using block copolymer micelle nanolithography, which itself is restricted to solid, inorganic, and planar surfaces such as glass slides; the gold particles are transferred to polymers by means of a thiol-gold coupling scheme. Depending on the polymer to be gold-decorated, an appropriate thiol linker molecule was incubated on the gold-patterned glass surface, and crosslinked to the PEG hydrogel during polymerization. The transfer resulted in a complete and accurate transfer of the nano-pattern to the polymer surface. Cryo-electron microscopy was used for structural characterization of the resulting surfaces, including watercontaining soft hydrogels. 2 The transfer nano-lithography technique is the first method to successfully nanostructure soft and polymeric materials with metal structures on a large scale, and can in principle be applied to the structuring of any organic planar and non-planar surface. The structural properties of the artificial ECM, controlling, e. g., the clustering of receptors at adhesion sites of adhering cells, can be adjusted by choosing the particle size and distance of the original gold pattern. Another structural parameter can be introduced by the non-planarity of the surfaces. Hydrogel-based microchannels have been developed that were internally decorated with gold nanoparticles, resulting in nanopatterned, tube-shaped artificial ECMs surrounding the cell in three dimensions, mimicking, for example, blood vessels. As a third module of the artificial ECM, the nanostructured hydrogel surfaces were chemically modified to provide the cell with biofunctions. Proteins were coupled to the gold particles or the hydrogel surface via Ni(II)-NTA complexes, and peptides were coupled to the gold particles via thiol groups, or to the hydrogel surface via amino groups. The four different schemes were developed to specifically couple the bioactive molecules at well-defined orientations and in their native conformation to either the hydrogel surface or the gold moieties, without introducing either cytotoxicity or loss of biocompatibility. The selective functionalization was tested for representative biomolecules, the adhesion receptor-binding peptide RGD, the cell-cell adhesion protein L1, and the green fluorescent protein. This concept enables selective modification of the gold particles or the inter-particle surface by coupling virtually any biomolecule to the aforementioned domains of the artificial ECM. The functionality of the three different components of the artificial ECM was tested in cell experiments. Experiments using substrates with various inter-gold particle spacing, biofunctionalizations, and cell types, demonstrated the applicability of the artificial ECM as such. Most importantly, for the first time, nanopatterned hydrogels were shown by cryo-SEM to be deformed by the adhering fibroblasts, thereby revealing the direct crosstalk between the cell and the ECM mimic on the molecular level. In addition, the functionality of non-planar substrates for cell experiments was demonstrated by means of micro-channels. In conclusion, the modular artificial ECM, as developed in this research project, meets the mechanical, structural, and biological requirements necessary to serve as a versatile and adjustable tool to investigate and provoke specific cell-surface interactions. The artificial ECM provides a useful means by which to influence cell adhesion and function, thereby enabling systematic selection of cell types for biotechnological and medical applications.

Item Type: Dissertation
Supervisor: Dr. Joachim P. Spatz, Prof.
Date of thesis defense: 19 July 2006
Date Deposited: 16 Jan 2007 12:05
Date: 2006
Faculties / Institutes: Fakultät für Chemie und Geowissenschaften > Institute of Physical Chemistry
Subjects: 540 Chemistry and allied sciences
Controlled Keywords: Tissue Engineering, Nanolithographie
Uncontrolled Keywords: cell adhesion , extracellular matrix
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