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In the field of biofabrication, biopolymer-based hydrogels are often used as bulk materials with defined structures or as bioinks. Despite their excellent biocompatibility, biopolymers need chemical modification to fulfill mechanical stability.
In this thesis, the primary alcohol of hyaluronic acid was oxidized using TEMPO/TCC oxidation to generate aldehyde groups without ring-opening mechanism of glycol cleavage using sodium periodate. For crosslinking reaction of the aldehyde groups, adipic acid dihydrazide was used as bivalent crosslinker for Schiff Base chemistry. This hydrogel system with fast and reversible crosslinking mechanism was used successfully as bulk hydrogel for chondrogenic differentiation with human mesenchymal stem cells (hMSC).
Gelatin was modified with pentenoic acid for crosslinking reaction via light controllable thiol-ene reaction, using thiolated 4-arm sPEG as multivalent crosslinker. Due to preservation of the thermo responsive property of gelatin by avoiding chain degradation during modification reaction, this gelatin-based hydrogel system was successfully processed via 3D printing with low polymer concentration. Good cell viability was achieved using hMSC in various concentrations after 3D bioprinting and chondrogenic differentiation showed promising results.
The aim of this thesis was the preparation of a biomaterial ink for the fabrication of chemically crosslinked hydrogel scaffolds with low micron sized features using melt electrowriting (MEW). By developing a functional polymeric material based on 2-alkyl-2-oxazine (Ozi) and 2-alkyl-2-oxazoline (Ox) homo- and copolymers in combination with Diels-Alder (DA)-based dynamic covalent chemistry, it was possible to achieve this goal. This marks an important step for the additive manufacturing technique melt electrowriting (MEW), as soft and hydrophilic structures become available for the first time. The use of dynamic covalent chemistry is a very elegant and efficient method for consolidating covalent crosslinking with melt processing. It was shown that the high chemical versatility of the Ox and Ozi chemistry offers great potential to control the processing parameters. The established platform offers straight forward potential for modification with biological cues and fluorescent markers. This is essential for advanced biological applications. The physical properties of the material are readily controlled and the potential for 4D-printing was highlighted as well. The developed hydrogel architectures are excellent candidates for 3D cell culture applications. In particular, the low internal strength of some of the scaffolds in combination with the tendency of such constructs to collapse into thin strings could be interesting for the cultivation of muscle or nerve cells. In this context it was also possible to show that MEW printed hydrogel scaffolds can withstand the aspiration and ejection through a cannula. This allows the application as scaffolds for the minimally invasive delivery of implants or functional tissue equivalent structures to various locations in the human body.
The implantation of any foreign material into the body automatically starts an immune reaction that serves as the first, mandatory step to regenerate tissue. The course of this initial immune reaction decides on the fate of the implant: either the biomaterial will be integrated into the host tissue to subsequently fulfill its intended function (e.g., tissue regeneration), or it will be repelled by fibrous encapsulation that determines the implant failure. Especially neutrophils and macrophages play major roles during this inflammatory response and hence mainly decide on the biomaterial's fate. For clinically relevant tissue engineering approaches, biomaterials may be designed in shape and morphology as well as in their surface functionality to improve the healing outcome, but also to trigger stem cell responses during the subsequent tissue regeneration phase.
The main focus of this thesis was to unravel the influence of scaffold characteristics, including scaffold morphology and surface functionality, on primary human innate immune cells (neutrophils and macrophages) and human mesenchymal stromal cells (hMSCs) to assess their in vitro immune response and tissue regeneration capacity, respectively. The fiber-based constructs were produced either via melt electrowriting (MEW), when the precise control over scaffold morphology was required, or via solution electrospinning (ES), when the scaffold design could be neglected. All the fiber-based scaffolds used throughout this thesis were composed of the polymer poly(ε caprolactone) (PCL).
A novel strategy to model and alleviate the first direct cell contact of the immune system with a peptide-bioactived fibrous material was presented in chapter 3 by treating the material with human neutrophil elastase (HNE) to imitate the neutrophil attack. The main focus of this study was put on the effect of HNE towards an RGDS-based peptide that was immobilized on the surface of a fibrous material to improve subsequent L929 cell adhesion. The elastase efficiently degraded the peptide-functionality, as evidenced by a decreased L929 cell adhesion, since the peptide integrated a specific HNE-cleavage site (AAPV-motif). A sacrificial hydrogel coating based on primary oxidized hyaluronic acid (proxHA), which dissolved within a few days after the neutrophil attack, provided an optimal protection of the peptide-bioactivated fibrous mesh, i.e, the hydrogel alleviated the neutrophil attack and largely ensured the biomaterial's integrity. Thus, according to these results, a means to protect the biomaterial is required to overcome the neutrophil attack.
Chapter 4 was based on the advancement of melt electrowriting (MEW) to improve the printing resolution of MEW scaffolds in terms of minimal inter-fiber distances and a concomitant high stacking precision. Initially, to gain a better MEW understanding, the influence of several parameters, including spinneret diameter, applied pressure, and collector velocity on mechanical properties, crystallinity, fiber diameter and fiber surface morphology was analyzed. Afterward, innovative MEW designs (e.g., box-, triangle-, round , and wall-shaped scaffolds) have been established by pushing the printing parameters to their physical limits. Further, the inter-fiber distance within a standardized box-structured scaffold was successfully reduced to 40 µm, while simultaneously a high stacking precision was maintained. In collaboration with a co-worker of my department (Tina Tylek, who performed all cell-based experiments in this study), these novel MEW scaffolds have been proven to facilitate human monocyte-derived macrophage polarization towards the regenerative M2 type in an elongation-driven manner with a more pronounced effect with decreasing pore sizes.
Finally, a pro-adipogenic platform for hMSCs was developed in chapter 5 using MEW scaffolds with immobilized, complex ECM proteins (e.g., human decellularized adipose tissue (DAT), laminin (LN), and fibronectin (FN)) to test for the adipogenic differentiation potential in vitro. Within this thesis, a special short-term adipogenic induction regime enabled to more thoroughly assess the intrinsic pro-adipogenic capacity of the composite biomaterials and prevented any possible masking by the commonly used long-term application of adipogenic differentiation reagents. The scaffolds with incorporated DAT consistently showed the highest adipogenic outcome and hence provided an adipo-inductive microenvironment for hMSCs, which holds great promise for applications in soft tissue regeneration.
Future studies should combine all three addressed projects in a more in vivo-related manner, comprising a co-cultivation setup of neutrophils, macrophages, and MSCs. The MEW-scaffold, particularly due to its ability to combine surface functionality and adjustable morphology, has been proven to be a successful approach for wound healing and paves the way for subsequent tissue regeneration.
The outcome of the innate immune response to biomaterials mainly determines whether the material will be incorporated in the body to fulfill its desired function or, when it gets encapsulated, will be rejected in the worst case. Macrophages are key players in this process, and their polarization state with either pro- (M1), anti-inflammatory (M2), or intermediate characteristics is crucial for deciding on the biomaterial’s fate. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates the proper healing and subsequent regeneration. Therefore, biomaterial-based polarization may aid in driving macrophages in the desired direction. However, the in vivo process is highly complex, and a mono-culture of macrophages in vitro displays only one part of the cellular system, but, to this date, there is a lack of established co-cultures to assess the immune response to biomaterials. Thus, this thesis aimed to establish a functional co-culture system of human macrophages and human mesenchymal stromal cells (hMSCs) to improve the assessment of the immune response to biomaterials in vitro. Together with macrophages, hMSCs are involved in tissue regeneration and inflammatory reactions and can modulate the immune response. In particular, endogenously derived hMSCs considerably contribute to the successful engrafting of biomaterials. This thesis focused on poly(ε-caprolactone) (PCL) fiber-based scaffolds produced by the technique of melt electrowriting (MEW) as biomaterial constructs. Via this fabrication technique, uniform, precisely ordered scaffolds varying in geometry and pore size have been created in-house.
To determine the impact of scaffold geometries and pore sizes on macrophages, mono-cultures incubated on scaffolds were conducted. As a pre-requisite to achieve a functional co-culture system on scaffolds, setups for direct and indirect systems in 2D have initially been established. These setups were analyzed for the capability of cell-cell communication. In parallel, a co-culture medium suitable for both cell types was defined, prior to the establishment of a step-by-step procedure for the co-cultivation of human macrophages and hMSCs on fiber-based scaffolds.
Regarding the scaffold morphologies tested within this thesis to improve M2-like polarization, box-shaped scaffolds outperformed triangular-, round- or disordered-shaped ones. Upon further investigation of scaffolds with box-shaped pores and precise inter-fiber spacing from 100 µm down to only 40 µm, decreasing pore sizes facilitated primary human macrophage elongation accompanied by their differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 µm. To the best of my knowledge, this was the first time that the elongation of human macrophages in a 3D environment has been correlated to their M2-like polarization. Thus, these results may set the stage for the design, the assessment, and the selection of new biomaterials, which can positively affect the tissue regeneration.
The cell communication of both cell types, detected via mitochondria exchange in direct and indirect co-cultures systems, took place in both directions, i.e., from hMSCs to macrophages and vice versa. Thereby, in direct co-culture, tunneling nanotubes enabled the transfer from one cell type to the respective other, while in indirect co-culture, a non-directional transfer through extracellular vesicles (EVs) released into the medium seemed likely. Moreover, the phagocytic activity of macrophages after 2D co-cultivation and hence immunomodulation by hMSCs increased with the highest phagocytic rate after 48 h being most pronounced in direct co-cultivation.
As the commonly used serum supplements for macrophages and hMSCs, i.e., human serum (hS) and fetal calf serum (FCS), respectively, failed to support the respective other cell type during prolonged cultivation, these sera were replaced by human platelet lysate (hPL), which has been proven to be the optimal supplement for the co-cultivation of human macrophages with hMSCs within this thesis. Thereby, the phenotype of both cell types, the distribution of both cell populations, the phagocytic activity of macrophages, and the gene expression profiles were maintained and comparable to the respective standard mono-culture conditions. This was even true when hPL was applied without the anticoagulant heparin in all cultures with macrophages, and therefore, heparin was omitted for further experiments comprising hPL and macrophages.
Accordingly, a step-by-step operating procedure for the co-cultivation on fiber-based scaffolds has been established comprising the setup for 3D cultivation as well as the description of methods for the analysis of phenotypical and molecular changes upon contact with the biomaterial. The evaluation of the macrophage response depending on the cultivation with or without hMSCs and either on scaffolds or on plastic surfaces has been successfully achieved and confirmed the functionality of the suggested procedures.
In conclusion, the functional co-culture system of human macrophages and hMSCs established here can now be employed to assess biomaterials in terms of the immune response in a more in vivo-related way. Moreover, specifically designed scaffolds used within the present thesis showed auspicious design criteria positively influencing the macrophage polarization towards the anti-inflammatory, pro-healing type and might be adaptable to other biomaterials in future approaches.
Hence, follow-up experiments should focus on the evaluation of the co-culture outcome on promising scaffolds, and the suggested operating procedures should be adjusted to further kinds of biomaterials, such as cements or hydrogels.
Aim of this thesis was the development of functionalizable hydrogel coatings for melt electrowritten PCL scaffolds and of bioprintable hydrogels for biofabrication.
Hydrogel coatings of melt electrowritten scaffolds enabled to control the surface hydrophilicity, thereby allowing cell-material interaction studies of biofunctionalized scaffolds in minimal protein adhesive environments. For this purpose, a hydrophilic star- shaped crosslinkable polymer was used and the coating conditions were optimized. Moreover, newly developed photosensitive scaffolds facilitated a time and pH independent biofunctionalization.
Bioprintable hydrogels for biofabrication were based on the allyl-functionalization of gelatin (GelAGE) and modified hyaluronic acid-products, to enable hydrogel crosslinking by means of the thiol-ene click chemistry. Optimization of GelAGE hydrogel properties was achieved through an in-depth analysis of the synthesis parameters, varying Ene:SH ratios, different crosslinking molecules and photoinitiators. Homogeneity of thiol-ene crosslinked networks was compared to free radical polymerized hydrogels and the applicability of GelAGE as bioink for extrusion-based bioprinting was investigated. Purely hyaluronic acid-based bioinks were hypothesized to maintain mechanical- and rheological properties, cell viabilities and the processability, upon further decreasing the overall hydrogel polymer and thiol content.
Hydrogel coatings: Highly structured PCL scaffolds were fabricated with MEW and subjected to coatings with six-armed star-shaped crosslinkable polymers (sP(EO-stat-PO)). Crosslinking results from the aqueous induced hydrolysis of reactive isocyanate groups (NCO) of sP(EO-stat-PO) and increased the surface hydrophilicity and provided a platform for biofunctionalizations in minimal protein adhesive environments. Not only the coating procedure was optimized with respect to sP(EO-stat-PO) concentrations and coating durations, instead scaffold pre-treatments were developed, which were fundamental to enhance the final hydrophilicity to completely avoid unspecific protein adsorption on sP(EO-stat-PO) coated scaffolds. The sP(EO-stat-PO) layer thickness of around 100 nm generally allows in vitro studies not only in dependence on the scaffold biofunctionalization but also on the scaffold architecture. The hydrogel coating extent was assessed via an indirect quantification of the NCO-hydrolysis products. Knowledge of NCO-hydrolysis kinetics enabled to achieve a balance of sufficiently coated scaffolds while maintaining the presence of NCO-groups that were exploited for subsequent biofunctionalizations. However, this time and pH dependent biofunctionalization was restricted to small biomolecules. In order to overcome this limitation and to couple high molecular weight biomolecules another reaction route was developed. This route was based on the photolysis of diazirine moieties and enabled a time and pH independent scaffold biofunctionalization with streptavidin and collagen type I. The fibril formation ability of collagen was used to obtain different collagen conformations on the scaffolds and a preliminary in vitro study demonstrated the applicability to investigate cell-material interactions.
The herein developed scaffolds could be applied to gain deeper insights into the fundamentals of cellular sensing. Especially the complexity by which cells sense e.g. collagen remain to be further elucidated. Therefore, different hierarchies of collagen-like conformations could be coupled to the scaffolds, e.g. gelatin or collagen-derived peptide sequences, and the activation of DDR receptors in dependence on the complexity of the coupled substances could be determined. Due to the strong streptavidin-biotin bond, streptavidin functionalized scaffolds could be applied as a versatile platform to allow immobilization of any biotinylated molecules.
Gelatin-based bioinks: First the GelAGE products were synthesized with respect to molecular weight distributions and amino acid composition integrity. A detailed study was conducted with varying molar ratios of reactants and synthesis durations and implied that gelatin degradation was most dominant for high alkaline synthesis conditions with long reaction times. Gelatin possesses multiple functionalizable groups and the predominant functionalization of amine groups was confirmed via different model substances and analyses. Polymer network homogeneity was proven for the GelAGE system compared to free radical polymerized hydrogels with GelMA. A detailed analysis of hydrogel compositions with varying functional group ratios and UV- or Vis-light photoinitiators was executed. The UV-initiator concentration is restricted due to cytotoxicity and potential cellular DNA damages upon UV-irradiation, whereas the more cytocompatible Vis- initiator system enabled mechanical stiffness tuning over a wide range by controlling the photoinitiator concentration at constant Ene:SH ratios and polymer weight percentages. Versatility of the GelAGE bioink for different AM techniques was proved by exploiting the thermo-gelling behavior of differently degraded GelAGE products for stereolithography and extrusion-based printing. Moreover, the viability of cell-laden GelAGE constructs was demonstrated for extrusion-based bioprinting. By applying different multifunctional thiol-macromolecular crosslinkers the mechanical and rheological properties improved concurrently to the processability. Importantly, lower thiol-crosslinker concentrations were required to yield superior mechanical strengths and physico-chemical properties of the hydrogels as compared to the small bis-thiol-crosslinker. Extrusion-based bioprinting with distinct encapsulated cells underlined the need for individual optimization of cell-laden hydrogel formulations.
Not only the viability of encapsulated cells in extrusion-based bioprinted constructs should be assessed, instead other parameters such as cell morphology or production of collagen or glycosaminoglycans should be considered as these represent some of the crucial prerequisites for cartilage Tissue Engineering applications. Moreover, these studies should be expanded to the stereolithographic approach and ultimately the versatility and cytocompatibility of formulations with macromolecular crosslinkers would be of interest. Macromolecular crosslinkers allowed reducing polymer weight percentages and amounts of thiol groups and are thus expected to contribute to increased cytocompatibility, especially in combination with the more cytocompatible Vis-initiator system, which remains to be elucidated.
Hyaluronic acid-based bioinks: Different molecular weight hyaluronic acid (HA) products were synthesized to bear ene- (HAPA) or thiol-functionalities (LHASH) to enable pure HA thiol-ene crosslinked hydrogels. Depending on the molecular weight of modified HA products, polymer weight percentages and Ene:SH ratios, a wide range of mechanical stiffness was covered. However, the manageability of high molecular weight HA (HHAPA) product solutions (HHAPA + LHASH) was restricted to 5.0 wt.-% as a consequence of the high viscosity. Based on the same HA thiol component (LHASH), hybrid hydrogels of HA with GelAGE were compared to pure HA hydrogels. Although the overall polymer weight percentage of HHAPA + LHASH hydrogels was significantly lowered compared to hybrid hydrogels (GelAGE + LHASH), similar mechanical and physico-chemical properties of pure HA hydrogels were determined with maintained Ene:SH ratios. Low viscous low molecular weight HA precursor solutions (LHAPA + LHASH) prevented the applicability for extrusion-based bioprinting, whereas the non-thermoresponsive HHAPA + LHASH system could be bioprinted with only one-fourth of the polymer content of hybrid formulations. The high viscous behavior of HHAPA + LHASH solutions, lower polymer weight percentages, decreased printing pressures and consequently declined shear stress during printing, were hypothesized to contribute to high cell viabilities in extrusion-based bioprinted constructs compared to the hybrid bioink.
The low molecular weight HA precursor formulation (LHAPA + LHASH) was not applicable for extrusion-based printing, but this system has potential for other AM techniques such as stereolithography. Similar to the GelAGE system a more detailed study on the functions of encapsulated cells would be useful to further develop this system. Moreover, the initiation with the Vis-initiator should be conducted.
Motivated by the great potential which is offered by the combination of additive manufacturing and tissue engineering, a novel polymeric bioink platform based on poly(2 oxazoline)s was developed which might help to further advance the young and upcoming field of biofabrication. In the present thesis, the synthesis as well as the characteristics of several diblock copolymers consisting of POx and POzi have been investigated with a special focus on their suitability as bioinks.
In general, the copolymerization of 2-oxazolines and 2-oxazines bearing different alkyl side chains was demonstrated to yield polymers in good agreement with the degree of polymerization aimed for and moderate to low dispersities.
For every diblock copolymer synthesized during the present study, a more or less pronounced dependency of the dynamic viscosity on temperature could be demonstrated. Diblock copolymers comprising a hydrophilic PMeOx block and a thermoresponsive PnPrOzi block showed temperature induced gelation above a degree of polymerization of 50 and a polymer concentration of 20 wt%. Such a behavior has never been described before for copolymers solely consisting of poly(cyclic imino ether)s.
Physically cross linked hydrogels based on POx b POzi copolymers exhibit reverse thermal gelation properties like described for solutions of PNiPAAm and Pluronic F127. However, by applying SANS, DLS, and SLS it could be demonstrated that the underlying gel formation mechanism is different for POx b POzi based hydrogels. It appears that polymersomes with low polydispersity are formed already at very low polymer concentrations of 6 mg/L. Increasing the polymer concentration resulted in the formation of a bicontinuous sponge like structure which might be formed due to the merger of several vesicles. For longer polymer chains a phase transition into a gyroid structure was postulated and corresponds well with the observed rheological data.
Stable hydrogels with an unusually high mechanical strength (G’ ~ 4 kPa) have been formed above TGel which could be adjusted over a range of 20 °C by changing the degree of polymerization if maintaining the symmetric polymer architecture. Variations of the chain ends revealed only a minor influence on TGel whereas the influence of the solvent should not be neglected as shown by a comparison of cell culture medium and MilliQ water.
Rotationally as well as oscillatory rheological measurements revealed a high suitability for printing as POx b POzi based hydrogels exhibit strong shear thinning behavior in combination with outstanding recovery properties after high shear stress.
Cell viability assays (WST-1) of PMeOx b PnPrOzi copolymers against NIH 3T3 fibroblasts and HaCat cells indicated that the polymers were well tolerated by the cells as no dose-dependent cytotoxicity could be observed after 24 h at non-gelling concentrations up to 100 g/L.
In summary, copolymers consisting of POx and POzi significantly increased the accessible range of properties of POx based materials. In particular thermogelation of aqueous solutions of diblock copolymers comprising PMeOx and PnPrOzi was never described before for any copolymer consisting solely of POx or POzi. In combination with other characteristics, e.g. very good cytocompatibility at high polymer concentrations and comparably high mechanical strength, the formed hydrogels could be successfully used for 3D bioprinting. Although the results appear promising and the developed hydrogel is a serious bioink candidate, competition is tough and it remains an open question which system or systems will be used in the future.
This thesis concerned the quantification of cell adhesion molecules (CAM) in and on thin hydrogel films as surface modification of biomaterials. The established and well characterized, per se inert NCO-sP(EO-stat-PO) hydrogel system which allows the easy and reproducible bioactivation with peptides was used as basis for this thesis. Two methods can be used to functionalize the coatings. Ligands can either be mixed into the prepolymer solution in prior to layer formation (mix-in method), or freshly prepared coatings can be incubated with ligand solution (incubation method). Divided into three major parts, the first part of the thesis dealt with the concentration of ligands in the bulk hydrogel, whereas the second part of the thesis focused on the surface sensitive quantification of CAMs at the biointerface. The results were correlated with cell adhesion kinetics. The third part of this thesis investigated the biochemical and the structural mimicry of the extracellular matrix (ECM). ECM proteins were presented via sugar-lectin mediated binding and cell behavior on these surfaces was analyzed. Cell behavior on three-dimensional fibers with identical surface chemistry as the coatings in the previous sections of the thesis was analyzed and correlated with the amount of peptide used for bioactivation. Overall, the main question of this work was ‘How much?’ regarding maximal as well as optimal ligand concentrations for controlled cell-hydrogel interactions. The focus in the first practical part of this thesis was to analyze the amount of ligands in NCO-sP(EO-stat-PO) hydrogels using classical quantification methods. Coatings in 96-well plates as well as on glass were functionalized with GRGDS and 125I-YRGDS for radioisotopic detection (Chapter 3). Using the incubation method for functionalization, a maximal ligand binding using peptide concentrations of 600 µg/mL could be determined. When functionalization was introduced via the mix-in method, a clear tendency for higher ligand concentrations with increasing ligand to prepolymer ratio was observed, but no maximal ligand binding could be detected with a ligand to prepolymer ratio of 2/1 being the highest ratio investigated. This ratio of 2/1 was not exceeded to ensure that complete crosslinking of the hydrogel was not affected. In Chapter 4, a fluorinated amino acid and an iodinated peptide were immobilized to the hydrogels using the mix-in method and were detected by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). In these measurements, maximal ligand binding was detected for a ligand to prepolymer ratio of 1/1. Higher ligand to prepolymer ratios did not result in any significant increase in ligand concentrations in the surface near regions of the crosslinked hydrogels. To address the question of how many ligands were actually accessible for cell interaction at the interface, surface sensitive quantification methods were applied in the second part of this thesis. For the quantification with surface plasmon resonance (SPR) and surface acoustic wave technology (SAW) (Chapter 5), the hydrogel coating procedure needed to be transferred onto cystamine functionalized gold surfaces. Characterization with ellipsometry and atomic force microscopy (AFM) revealed inhomogeneous cystamine binding to the activated surfaces, which resulted in inhomogeneous coatings. Nevertheless, it could be shown that SPR as well as SAW were suitable methods for the surface sensitive quantification of the ligand concentration on NCO-sP(EO-stat-PO) hydrogels. Non-functionalized coatings resisted non-specific serum as well as streptavidin (SA) adsorption. Coatings functionalized with biocytin and GRGDSK-biotin introduced specific SA binding that was dependent on the biotin concentration at the surface. Additionally, enzyme linked immunosorbent assay (ELISA) and enzyme linked lectin assay (ELLA) (Chapter 6) were applied to coatings in 96-well plates and on glass. Coatings were functionalized with the model molecule biocytin, the biotinylated peptide GRGDSK-biotin, the ECM protein fibronectin (FN), as well as the carbohydrates N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc). All ligands could be successfully detected with antibodies or SA via ELISA or ELLA. Maximal GRGDSK-biotin binding to the hydrogel coatings on glass was achieved at a peptide to prepolymer ratio of 1/5, which was used as reference value in Chapter 8. Last but not least, cell adhesion (Chapter 7) was quantified depending on the GRGDS concentration on hydrogel coatings on glass. Maximal adhesion of primary human dermal fibroblast (HDF) was observed at GRGDS to prepolymer ratios of 1/5, when adherent cells were counted on life cell images. Quantification of adherent cells using the CASY® cell counter revealed maximal HDF adhesion at molar ligand to prepolymer ratios of 1/2. However, cell vitality detected by intracellular enzyme activities was not dependent on the GRGDS concentration. Cells which managed to adhere were vital regardless of the amount of ligands present. Additionally, adhesion of fibroblasts from the murine cell line NIH L929 was analyzed by counting on life cell images. These cells, being much smaller than the HDF cells, needed higher GRGDS to prepolymer ratios (2/1) for proper cell adhesion. All quantification methods applied to analyze hydrogels which were functionalized by the mix-in method in Chapter 3, 4, 6 and 7, were compared in Chapter 8. Radiodetection gave information about the ligand concentrations throughout the whole hydrogel and no maximal amount of ligands could be detected when increasing the peptide to prepolymer ratio. In contrast, XPS and TOF-SIMS which only penetrated the surface near regions of the coating, a maximal ligand binding to the hydrogel was detected for 1/1 ratios. SPR and SAW were not included in this comparison, as the coatings on gold need to be optimized first. The two surface sensitive quantification methods (ELISA and HDF adhesion) could give information about the quantity of peptide which was sterically available for SA or cell binding. With these methods, maximal SA and cell binding was detected at ratios of 1/5. These results underline the importance of carefully compare the different methods. Beside ligand quantification on hydrogels, the third part of this thesis was concerned with the biochemical and structural mimicry of the ECM by advanced ECM engineering to design biomimetic biomaterials that are better accepted by cells and tissue. The subject of Chapter 9 was the biomimetic and flexible presentation of the ECM protein FN. FN was attached via sugar-lectin mediated binding to NCO-sP(EO-stat-PO) hydrogels. The build-up of the covalently immobilized sugar poly-N-acetyllactosamine (polyLacNAc), the subsequent non-covalent binding of the fungal galectin His6CGL2, and FN could be elegantly proven by fluorescent staining on coatings which were functionalized with the sugar by micro contact printing (MCP). Further experiments were carried out on build-ups, where polyLacNAc was immobilized on the hydrogel by incubation. Optimal parameters for the layer build-up were determined by ELLA/ELISA. Only the complete build-up induced proper adhesion of HDFs. Compared to tissue culture polystyrene (TCPS), cells adhered and spread faster on the biomimetic surfaces. The flexible presentation of FN allowed HDFs to rearrange homogenously immobilized FN into fibrillar structures, which seemed not to be possible when FN was adsorbed on glass or covalently bound directly to the hydrogel coatings. This new approach of a flexible and biomimetic presentation of an ECM protein allows new ways to design biomaterials with best possible cell-material interactions. The work described in Chapter 10 focused on the structural mimicry of the fibrous ECM structures by electrospinning of synthetic, bioactive, and degradable fibers. Poly(D,L-lactide-co-glycolide) (PLGA) and NCO-sP(EO-stat-PO) were electrospun out of one solution in an easy one-step preparation resulting in fibers with an ultrathin inert hydrogel layer at the surface. By adding GRGDS to the solution prior to electrospinning, specifically interacting fibers could be obtained. In comparison to PLGA, the adsorption of bovine serum albumin (BSA) could be reduced by 99.2%. As a control, the non-active peptide GRGES was immobilized to the fiber. These fibers did not allow cell adhesion, showing that the integrity of the hydrogel coated fibers was not affected by the immobilization of peptides. HDF adhesion was obtained by functionalization with GRGDS, leading to the adhesion, spreading, and proliferation of HDFs. Also mesenchymal stem cells (MSC) could adhere to GRGDS functionalized fibers. Additionally, for ligand quantification, the ELISA technique was successfully transferred to fiber substrates. To highlight the potential of the approaches for the biochemical and structural mimicry of the ECM, the sugar polyLacNAc was immobilized on the PLGA/sP(EO-stat-PO) fibers followed by the subsequent layer build-up with His6CGL2 and FN. These fibers triggered HDF adhesion.