Uwe Freudenberg

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.

Glycosaminoglycan-based hydrogels to modulate heterocellular communication in in vitro angiogenesis models

Angiogenesis, the outgrowth of blood vessels, is crucial in development, disease and regeneration. Studying angiogenesis in vitro remains challenging because the capillary morphogenesis of endothelial cells (ECs) is controlled by multiple exogenous signals. Therefore, a set of in situ-forming starPEG-heparin hydrogels was used to identify matrix parameters and cellular interactions that best support EC morphogenesis. We showed that a particular type of soft, matrix metalloproteinase-degradable hydrogel containing covalently bound integrin ligands and reversibly conjugated pro-angiogenic growth factors could boost the development of highly branched, interconnected, and lumenized endothelial capillary networks. Using these effective matrix conditions, 3D heterocellular interactions of ECs with different mural cells were demonstrated that enabled EC network modulation and maintenance of stable vascular capillaries over periods of about one month in vitro. The approach was also shown to permit in vitro tumor vascularization experiments with unprecedented levels of control over both ECs and tumor cells. In total, the introduced 3D hydrogel co-culture system could offer unique options for dissecting and adjusting biochemical, biophysical, and cell-cell triggers in tissue-related vascularization models.

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.

The growth and differentiation of mesenchymal stem and progenitor cells cultured on aligned collagen matrices.

Cell-matrix interactions are paramount for the successful repair and regeneration of damaged and diseased tissue. Since many tissues have an anisotropic architecture, it has been proposed that aligned extracellular matrix (ECM) structures in particular could guide and support the differentiation of resident mesenchymal stem and progenitor cells (MSCs). We therefore created aligned collagen type I structures using a microfluidic set-up with the aim to assess their impact on MSC growth and differentiation. In addition, we refined our aligned collagen matrices by incorporating the glycosaminoglycan (GAG) heparin to demonstrate the versatility of the applied methodology to study multiple ECM components in a single system. Our reconstituted, aligned ECM structures maintained and allowed multilineage (osteogenic/adipogenic/chondrogenic) differentiation of MSCs. Most noticeable was the observation that during osteogenesis, aligned collagen substrates choreographed ordered matrix mineralization. Likewise, myotube assembly of C2C12 cells was profoundly influenced by aligned topographic features resulting in enhanced myotube organization and length. Our results shed light on the regulation of MSCs through directional ECM structures and demonstrate the versatility of these cell culture platforms for guiding the morphogenesis of tissue types with anisotropic structures.

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.

Aligned fibrillar collagen matrices obtained by shear flow deposition

Here we present a new technique to generate surface-bound collagen I fibril matrices with differing structural characteristics. Aligned collagen fibrils were deposited on planar substrates from collagen solutions streaming through a microfluidic channel system. Collagen solution concentration, degree of gelation, shear rate and pre-coating of the substrate were demonstrated to determine the orientation and density of the immobilized fibrils. The obtained matrices were imaged using confocal reflection microscopy and atomic force microscopy. Image analysis techniques were applied to evaluate collagen fibril orientation and coverage. As expected, the degree of collagen fibril orientation increased with increasing flow rates of the solution while the matrix density increased at higher collagen solution concentrations and on hydrophobic polymer pre-coatings. Additionally, length of the immobilized collagen fibrils increased with increasing solution concentration and gelation time.

Bio-responsive polymer hydrogels homeostatically regulate blood coagulation

Bio-responsive polymer architectures can empower medical therapies by engaging molecular feedback-response mechanisms resembling the homeostatic adaptation of living tissues to varying environmental constraints. Here we show that a blood coagulation-responsive hydrogel system can deliver heparin in amounts triggered by the environmental levels of thrombin, the key enzyme of the coagulation cascade, which—in turn—becomes inactivated due to released heparin. The bio-responsive hydrogel quantitatively quenches blood coagulation over several hours in the presence of pro-coagulant stimuli and during repeated incubation with fresh, non-anticoagulated blood. These features enable the introduced material to provide sustainable, autoregulated anticoagulation, addressing a key challenge of many medical therapies. Beyond that, the explored concept may facilitate the development of materials that allow the effective and controlled application of drugs and biomolecules.

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.