Petra B. Welzel

A star-PEG-heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases

Biofunctional matrices for in vivo tissue engineering strategies must be modifiable in both biomolecular composition and mechanical characteristics. To address this challenge, we present a modular system of biohybrid hydrogels based on covalently cross-linked heparin and star-shaped poly(ethylene glycols) (star-PEG) in which network characteristics can be gradually varied while heparin contents remain constant. Mesh size, swelling and elastic moduli were shown to correlate well with the degree of gel component cross-linking. Additionally, secondary conversion of heparin within the biohybrid gels allowed the covalent attachment of cell adhesion mediating RGD peptides and the non-covalent binding of soluble mitogens such as FGF-2. We applied the biohybrid gels to demonstrate the impact of mechanical and biomolecular cues on primary nerve cells and neural stem cells. The results demonstrate the cell type-specific interplay of synergistic signaling events and the potential of biohybrid materials to selectively stimulate cell fate decisions. These findings suggest important future uses for this material in cell replacement based-therapies for neurodegenerative diseases.

FGF-2 and VEGF functionalization of starPEG―heparin hydrogels to modulate biomolecular and physical cues of angiogenesis

Tissue engineering therapies require biomaterials capable of encouraging an angiogenic response. To dissect the influence of different pro-angiogenic stimuli a set of starPEG-heparin hydrogels with varied physicochemical properties was used as a highly efficient reservoir and tunable delivery system for basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF). The engineered gel materials could be precisely tailored by decoupling the biomolecular functionalization from the variation of the viscoelastic matrix characteristics. Culture experiments with human umbilical vein endothelial cells (HUVECs) revealed the interplay of growth factor presentation, adhesive characteristics and elasticity of the gel matrices in triggering differential cellular behavior which allowed identifying effective pro-angiogenic conditions.

Dual independent delivery of pro-angiogenic growth factors from starPEG-heparin hydrogels.

Effective vascularization is a prerequisite for the success of various different tissue engineering concepts. While simultaneous administration of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) has been previously demonstrated to boost angiogenesis, the combined long-term delivery of both growth factors from biomaterials is still a major challenge. In this work, two important heparin binding cytokines were delivered in parallel from a modular starPEG (multi-armed polyethylene glycol)--heparin hydrogel system to human umbilical vein endothelial cells (HUVECs) grown in culture and in a chicken embryo chorioallantoic membrane (CAM) model. As the utilized gels contain high quantities of heparin, loading and subsequent release of both growth factors (as determined by radiolabeling studies and Enzyme-Linked Immunosorbent Assay [ELISA]) occurred independently from each other. The combined delivery of FGF-2 and VEGF through starPEG-heparin hydrogels resulted in pro-angiogenic effects in vitro (study of cell survival/proliferation, morphology and migration) and in vivo (quantification of CAM vascularization) being clearly superior over those of the administration of single factors. Consequently, the independent delivery of growth factor combinations by biohybrid starPEG-heparin matrices allows for the precise multifactorial control of cellular processes critically determining regeneration.

Geometry-Driven Cell Organization Determines Tissue Growths in Scaffold Pores: Consequences for Fibronectin Organization

To heal tissue defects, cells have to bridge gaps and generate new extracellular matrix (ECM). Macroporous scaffolds are frequently used to support the process of defect filling and thus foster tissue regeneration. Such biomaterials contain micro-voids (pores) that the cells fill with their own ECM over time. There is only limited knowledge on how pore geometry influences cell organization and matrix production, even though it is highly relevant for scaffold design. This study hypothesized that 1) a simple geometric description predicts cellular organization during pore filling at the cell level and that 2) pore closure results in a reorganization of ECM. Scaffolds with a broad distribution of pore sizes (macroporous starPEG-heparin cryogel) were used as a model system and seeded with primary fibroblasts. The strategies of cells to fill pores could be explained by a simple geometrical model considering cells as tensioned chords. The model matched qualitatively as well as quantitatively by means of cell number vs. open cross-sectional area for all pore sizes. The correlation between ECM location and cell position was higher when the pores were not filled with tissue (Pearson’s coefficient ρu200a=u200a0.45±0.01) and reduced once the pores were closed (ρu200a=u200a0.26±0.04) indicating a reorganization of the cell/ECM network. Scaffold pore size directed the time required for pore closure and furthermore impacted the organization of the fibronectin matrix. Understanding how cells fill micro-voids will help to design biomaterial scaffolds that support the endogenous healing process and thus allow a fast filling of tissue defects.

Interfacial Charge of Organic Thin Films Characterized by Streaming Potential and Streaming Current Measurements

Abstract Self-assembled monolayers of alkanethiol compounds chemisorbed on flat gold surfaces were characterized by streaming potential and streaming current measurements in aqueous electrolyte solutions using a novel microslit electrokinetic setup. The alkanethiols analyzed were terminated with different functional groups. Depending on the type of alkanethiol used different mechanisms were relevant for the generation of interfacial charge: dissociation for thiols bearing ionizable surface groups and preferential adsorption of ions for methyl-terminated thiols. In all investigated cases, the zeta potential calculated from the streaming potential was significantly lower than the zeta potential obtained from the streaming current. This was due to a contribution of the conductivity of the underlying gold substrate to the surface conductivity. Based on the data obtained for the zeta potential and the surface conductivity, the surface charge of acidically functionalized monolayers was concluded to be compensated almost completely in the stagnant layer whereas for methyl-terminated monolayers a considerable part of the countercharge is localized in the diffuse, hydrodynamically mobile part of the electric double layer.

Macroporous starPEG-heparin cryogels.

Macroporous scaffolds with adaptable mechanical and biomolecular properties can be instrumental in enabling cell-based therapies. To meet these requirements, a cryostructuration method was adapted to prepare spongy hydrogels based on chemically cross-linked star-shaped poly(ethylene glycol) (starPEG) and heparin. Subzero temperature treatment of the gel forming reaction mixtures and subsequent lyophilization of the incompletely frozen gels resulted in macroporous biohybrid cryogels showing rapid swelling, porosity of up to 92% with interconnected large pores (30-180 μm), low bulk stiffness, and high mechanical stability upon compression. The applicability of the cryogel scaffolds was investigated using human umbilical vein endothelial cells. Cell attachment and three-dimensional spreading resulted in evenly distributed viable cells within the macroporous starPEG-heparin materials, demonstrating the significant translational potential of the developed three-dimensional cell carriers.

Tailoring uptake and release of ATP by dendritic glycopolymer/PNIPAAm hydrogel hybrids: first approaches towards multicompartment release systems

A multicompartment release system is described which combines the advantages of dendritic architectures and hydrogels to enhance the desired delivery features in complex biological compartments. Here, a hydrogel hosts dendritic glycopolymers as nanocontainers and a delivery system for drug molecules. The dendritic glycopolymer used consists of a hyperbranched poly(ethylene imine) with a maltose shell and acts as a host for the guest molecule adenosine triphosphate disodium salt hydrate (ATP). The ATP uptake and release from the dendritic host have been elucidated in detail with dependence on the dendritic glycostructure and pH. The complex interactions within the three components ATP, dendritic glycopolymer and hydrogel have been evaluated and could be fine-tuned. A selective release at pH 5.4–7.4 only of ATP from the multicompartment release system ATP@dendritic glycopolymer@hydrogel has been achieved when a boronic acid containing hydrogel was used which allowed chemical binding between the maltose units from the dendritic glycopolymer and the boronic acid (BA) units in the hydrogel. However, when using a hydrogel without BA units, simultaneous release of ATP and the dendritic glycopolymer scaffold from the ATP@dendritic glycopolymer@hydrogel multicompartment release system is observed in the pH range 2–7.4. This multicompartment release system can be applied in complex biological environments with changing pH values and has potential in biomedical applications and sensory devices.

Biohybrid Networks of Selectively Desulfated Glycosaminoglycans for Tunable Growth Factor Delivery

Sulfation patterns of glycosaminoglycans (GAG) govern the electrostatic complexation of biomolecules and thus allow for modulating the release profiles of growth factors from GAG-based hydrogels. To explore options related to this, selectively desulfated heparin derivatives were prepared, thoroughly characterized, and covalently converted with star-shaped poly(ethylene glycol) into binary polymer networks. The impact of the GAG sulfation pattern on the network characteristics of the obtained hydrogels was theoretically evaluated by mean field methods and experimentally analyzed by rheometry and swelling measurements. Sulfation-dependent differences of reactivity and miscibility of the heparin derivatives were shown to determine network formation. A theory-based design concept for customizing growth factor affinity and physical characteristics was introduced and validated by quantifying the release of fibroblast growth factor 2 from a set of biohybrid gels. The resulting new class of cell-instructive polymer matrices with tunable GAG sulfation will be instrumental for multiple applications in biotechnology and medicine.

Analytical approaches to uptake and release of hydrogel-associated FGF-2.

Strategies to control the delivery of growth factors are critically important in the design of advanced biomaterials. In this study we investigated the binding and release of fibroblast growth factor 2 (FGF-2) to/from a biohybrid hydrogel matrix by four independent analytical methods: radioisotope and fluorescence labeling, amino acid analysis and Enzyme-Linked Immunosorbent Assays (ELISA). The compared analyses provided qualitatively similar uptake characteristics while the results of the FGF-2 quantification strongly depended on the particular experimental conditions. The release kinetics of FGF-2 from the gels could be monitored sensitively by 125I labeling and by ELISA-techniques. The latter method was concluded to be advantageous since it permits the application of unmodified (“native”) growth factors.

Sources of error in Langmuir trough measurements: Wilhelmy plate effects and surface curvature

Abstract When characterising so-called insoluble monolayers at the air/water interface by means of a film balance, it is customary to calculate the surface area solely from the geometric trough dimensions. In this paper we show that use of a Wilhelmy plate made of filter paper gives rise to an additional surface area. Ignoring this area increment may lead to a non-negligible systematic error which results in underrating the area per molecule, with an error becoming more severe upon higher compression. We also consider an increase of the surface area due to the curvature of its meniscus at the periphery of the trough. The practical significance of these errors will be assessed and appropriate remedies to eliminate fallacious data are suggested. Finally we discuss possible consequences regarding previously published results.