Silvana Prokoph

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.

Sustained delivery of SDF-1α from heparin-based hydrogels to attract circulating pro-angiogenic cells.

Enrichment of progenitor cells in ischemic tissue has become a promising therapeutic strategy in the treatment of myocardial infarction. Towards this aim, we report a biology-inspired concept using sulfated glycosaminoglycans to sustainably generate chemokine gradients for the localized accumulation of early endothelial progenitor cells (eEPCs). StarPEG-heparin hydrogels, which have been previously demonstrated to support angiogenesis, were functionalized with SDF-1α, a potent chemoattractant known to act on EPCs. The gels were quantitatively shown to release the chemokine in amounts that are adjustable by the choice of loading concentrations and by matrix metalloprotease (MMP) mediated hydrogel cleavage. Transwell assays confirmed significantly enhanced migration of early EPCs towards concentration gradients of hydrogel-delivered SDF-1α in vitro. Subcutaneous implantation of SDF-1α-releasing gels in mice resulted in massive infiltration of early EPCs and subsequently improved vascularization. In conclusion, sustained delivery of SDF-1α from pro-angiogenic starPEG-heparin hydrogels can effectively attract early EPCs, offering a powerful means to trigger endogenous mechanisms of cardiac regeneration.

Defined Polymer–Peptide Conjugates to Form Cell‐Instructive starPEG–Heparin Matrices In Situ

Poly(ethylene glycol)-peptide- and glycosaminoglycan-peptide conjugates obtained by a regio-selective amino acid protection strategy are converted into cell-instructive hydrogel matrices capable of inducing morphogenesis in embedded human vascular endothelial cells and dorsal root ganglia.

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.

A novel, biased-like SDF-1 derivative acts synergistically with starPEG-based heparin hydrogels and improves eEPC migration in vitro

The CXC chemokine stromal cell-derived factor-1α (SDF-1α, CXCL12) has been proven to recruit CXCR4 positive stem and progenitor cells of different sources to defected heart sites, with significant clinical benefits. However, the rapid proteolytic inactivation by inflammation-related proteases, inaccurate drug delivery or inappropriate local concentrations belong to the largest disadvantages for feasible application. Herein, we present a switchable, biased-like SDF-1α variant, AAV-[S4V]-SDF-1α, whose distinct activity is coupled to the inflammation-associated presence of dipeptidylpeptidase-4 (DPP-4), which cleaves an alanine-alanine dipeptide from the precursor. We decorated starPEG-heparin hydrogels with our novel SDF-1α variant and tested them for immobilization efficiency, time-dependent protein release as well as mobilization of early endothelial progenitor cells (eEPCs) in vitro. We found higher migration rates compared to conventional SDF-1α. In summary, we provide a conceptual work on cooperative effects of enzymatically activatable SDF-1α and starPEG-heparin hydrogels.

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.

Quantifying the Effect of Covalently Immobilized Enzymes on Biofilm Formation by Atomic Force Microscopy‐Based Single‐Cell Force Spectroscopy

A novel atomic force microscopy-based single-cell force spectroscopy assay to quantify the adhesion of bacterial cells to surfaces was developed. The assay was applied to quantify the effect of two biofilm-degrading enzymes, the protease Subtilisin A and glycoside hydrolase cellulase, on the attachment of the biofilm-forming bacterial strain Cobetia marina. Insights on the mechanism of the initial adhesion and on the nature of the adhesion-mediating molecules were gained. The assay can be easily adapted to various other substrates, different bacterial strains and other fouling species (e.g., algae and diatoms).

Defined Geldrop Cultures Maintain Neural Precursor Cells

Distinct micro-environmental properties have been reported to be essential for maintenance of neural precursor cells (NPCs) within the adult brain. Due to high complexity and technical limitations, the natural niche can barely be studied systematically in vivo. By reconstituting selected environmental properties (adhesiveness, proteolytic degradability, and elasticity) in geldrop cultures, we show that NPCs can be maintained stably at high density over an extended period of time (up to 8 days). In both conventional systems, neurospheres and monolayer cultures, they would expand and (in the case of neurospheres) differentiate rapidly. Further, we report a critical dualism between matrix adhesiveness and degradability. Only if both features are functional NPCs stay proliferative. Lastly, Rho-associated protein kinase was identified as part of a pivotal intracellular signaling cascade controlling cell morphology in response to environmental cues inside geldrop cultures. Our findings demonstrate that simple manipulations of the microenvironment in vitro result in an important preservation of stemness features in the cultured precursor cells.