Andrea Zieris

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.

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.

Two-tier hydrogel degradation to boost endothelial cell morphogenesis.

Cell-responsive degradation of biofunctional scaffold materials is required in many tissue engineering strategies and commonly achieved by the incorporation of protease-sensitive oligopeptide units. In extension of this approach, we combined protease-sensitive and -insensitive cleavage sites for the far-reaching control over degradation rates of starPEG-heparin hydrogel networks with orthogonally modulated elasticity, RGD presentation and VEGF delivery. Enzymatic cleavage was massively accelerated when the accessibility of the gels for proteases was increased through non-enzymatic cleavage of ester bonds. The impact of gel susceptibility to degradation was explored for the 3-dimensional ingrowth of human endothelial cells. Gels with accelerated degradation and VEGF release resulted in strongly enhanced endothelial cell invasion in vitro as well as blood vessel density in the chicken chorioallantoic membrane assay in vivo. Thus, combination of protease-sensitive and -insensitive cleavage sites can amplify the degradation of bioresponsive gel materials in ways that boost endothelial cell morphogenesis.

Heparin desulfation modulates VEGF release and angiogenesis in diabetic wounds.

While vascular endothelial growth factor (VEGF) has been shown to be one of the key players in wound healing by promoting angiogenesis current clinical applications of this growth factor to the wound environment are poorly controlled and not sustainable. Hydrogels made of sulfated glycosaminoglycans (GAG) allow for the sustained release of growth factors since GAGs engage in electrostatic complexation of biomolecules. In here, we explore a set of hydrogels formed of selectively desulfated heparin derivatives and star-shaped poly(ethylene glycol) with respect to VEGF binding and release and anticoagulant activity. As a proof of concept, supportive effects on migration and tube formation of human umbilical vein endothelial cells were studied in vitro and the promotion of wound healing was followed in genetically diabetic (db/db) mice. Our data demonstrate that the release of VEGF from the hydrogels is modulated in dependence on the GAG sulfation pattern. Hydrogels with low sulfate content (11% of initial heparin) were found to be superior in efficacy of VEGF administration, low anticoagulant activity and promotion of angiogenesis.

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.

Growth factor delivery from hydrogel particle aggregates to promote tubular regeneration after acute kidney injury

Local delivery of growth factors (GFs) can accelerate regeneration of injured tissue, but for many medical applications, injectable GF delivery systems are required for clinical success. Viscoelastic, injectable aggregates of micrometer-sized hydrogel particles made of multiarmed polyethylene glycol (starPEG) and heparin were prepared and tested for site-specific paracrine stimulation of tissue regeneration. Heparin was used as it binds, protects and releases numerous GFs. Hydrogel based delivery of basic fibroblast growth factor (bFGF) and murine epidermal growth factor (EGF) was monitored utilizing enzyme-linked immunosorbent assay (ELISA). bFGF was released slowly because of its high affinity to the heparin while the significantly higher release of the non-specific binding EGF was controlled by diffusion only. To investigate GF delivery in vivo, a hydrogel loaded with murine EGF or bFGF was injected subcapsularly into the left kidney of mice with experimental acute kidney injury caused by glycerol induced rhabdomyolysis. Visual examination confirmed sustained stability of the injected gel aggregates during the timescale of the experiment. The number of proliferating kidney tubular epithelial cells was quantified both in the injected kidney and the non-injected contralateral kidney. bFGF delivery from hydrogels induced a significant increase in cell proliferation in the injected kidney, although small effects were also seen in the non-injected kidney due to a systemic effect. EGF delivery strongly increased cell proliferation for both kidneys, but also showed a local effect on the injected kidney. The hydrogel without loaded GFs was used as a control and showed no increase in cell proliferation. Our results suggest that this novel starPEG-heparin hydrogel system can be an effective approach to deliver GFs locally.

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.