Sustainable Growth Factor Delivery through Affinity-Based

Chapter 24

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Sustainable Growth Factor Delivery through Affinity-Based Adsorption to starPEG-Heparin Hydrogels A. Zieris, S. Prokoph, K. R. Levental, P. B. Welzel, K. Chwalek, K. Schneider, U. Freudenberg, and C. Werner* Leibniz Institute of Polymer Research Dresden (IPF), Max Bergmann Center of Biomaterials Dresden (MBC) and Technische Universität Dresden (TUD), Center for Regenerative Therapies Dresden (CRTD), Hohe Strasse 6, 01069 Dresden, Germany *E-mail: [email protected]

Controlled delivery of growth factors is critically important in directing tissue regeneration, which motivates the development of customized biomaterials for growth factor provision. Following the lead of the natural extracellular matrix, reversible adsorption of growth factors to material building blocks possessing cytokine affinity is considered an advantageous design principle for that purpose. Based on this concept, a biohybrid hydrogel composed of star-shaped poly(ethylene-glycol) (starPEG) and heparin was developed. The presence of starPEG determines the structural and mechanical network characteristics, while heparin enables the reversible immobilization of growth factors and the covalent binding of cell adhesive peptides. By varying the molar ratio of starPEG to heparin during gel formation, different hydrogel types with gradated physical properties but constantly high heparin content were obtained. We show that the matrices bind and release various heparin-affine cytokines, including vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), bone morphogenetic protein 2 (BMP-2) and stromal-cell derived factor-1α (SDF-1α), independently of the network characteristics. Moreover, the material could be used for the parallel provision of different growth factor

© 2012 American Chemical Society In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


combinations over a broad range of concentrations. The cytokine delivery by modular starPEG-heparin hydrogels was proven to be effective in different in vitro and in vivo experiments, suggesting that the hydrogel platform will be an important tool in advancing regenerative therapies.

The interaction between proteins and biomaterials is often considered an undesired side effect in the application of various medical devices, causing activation of blood coagulation, biofouling, inflammation and infection. However, regenerative therapies now use biomaterials to locally deliver functional proteins, such as growth factors (often interchangeably used with the term “cytokines”), to stimulate growth, migration and differentiation of cells according to the requirements of therapeutical processes. Sustained release of growth factors from biomaterials can address problems such as ineffective or undefined dosing and unresponsiveness to cytokines, occurring with simple bolus injections. With the latter approach, growth factors often show a short half-life and poor bioactivity due to rapid diffusion, denaturation or degradation. Furthermore, as many of the applied proteins act on several tissues, severe adverse effects can occur if the signaling molecules are transported to adjacent sites. These issues highlight the need to control the spatio-temporal availability of bioactive growth factors in the context of specific therapies. Consequently, the presentation of cytokines from three-dimensional polymeric matrices has recently received increasing attention (1, 2). A simple and widely used approach is the conjugation of growth factors with polymers. This increases protein solubility and stability, reduces immunogenicity and prolongs the plasma half-life of the growth factor. For the synthesis of such conjugates, the polymer has to be stable and biocompatible as well as equipped with only one single reactive group at one terminal end to avoid protein crosslinking. Moreover, it should be attached by an approach that will lead to reproducible site-specific protein modification (3). Starting from the pioneering work of Davis et al. (4), a protein conjugation with poly(ethylene-glycol) (PEG), now called PEGylation, has emerged as an important growth factor delivery concept. PEGylated proteins are already on the market and in clinical development (5). In more advanced strategies, the conjugates can additionally be linked by bioresponsive elements or the proteins can be entrapped in polymeric micelles (3). However, regenerative approaches can additionally benefit from growth factor provision by biofunctional scaffolds, which can simultaneously mediate cell adhesion and direct a desired cellular localization and assembly. Physiologically, cells are closely associated with their highly hydrated environment, the extracellular matrix (ECM). Viscoelastic hydrogels, pioneered by Wichterle (6), are hydrophilic, highly water absorbent networks of synthetic and/or natural polymers that can mimic the structural character of the natural ECM (7). With these systems, growth factors can be either physically entrapped in the delivery matrix or chemically conjugated to the molecular scaffold, while their release is 526 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


determined by diffusion out of the network and/or matrix cleavage/degradation. In the context of these different strategies, physical growth factor adsorption to biomaterials, based on secondary chemical interactions, combines the advantages of a non-specific entrapment within the matrix and covalent conjugation to the scaffold. This approach can be used to provide cytokines which are either directly accessible when attached to the polymeric scaffold or which display their activity after release from the material (2). The concept of physical growth factor adsorption closely resembles the strategy within the natural ECM, where cytokines are reversibly immobilized to sulfated glycosaminoglycans (GAGs). This binding mainly occurs via spatially matching electrostatic interactions between negatively charged N- and O-sulfated groups of the GAGs and the basic lysine and arginine residues of the growth factors (8). This protects the cytokines from proteolytic cleavage and denaturation. Moreover, the dynamic protein binding and release by the GAGs also controls the cytokine diffusional distribution and presentation to cellular receptors (9). Consequently, by designing biomaterials with a certain growth factor affinity, such systems might allow for a prolonged delivery of highly bioactive proteins in the desired concentration regime. If biomaterials are to successfully support regenerative therapies, beyond effectively binding and releasing growth factors, they have to meet several additional requirements depending on the specific therapeutic approach. As with the natural ECM, key characteristics to control cell behavior are the physicochemical and mechanical properties of the scaffold combined with the provision of insoluble cues mediating cell adhesion and susceptibility to cellular matrix remodeling (10). Growth factor delivery systems designed for therapeutic approaches should ideally address all of these demands. Among the first materials developed according to this requirement, a PEG-based synthetic analogue of collageneous ECM for the delivery of bone morphogenetic protein 2 (BMP-2) has been produced (11). This scaffold was successfully applied to repair bone defects, and was constructed with a combination of RGDSP-peptides (in single-letter amino acid code) controlling cell adhesion and matrix metalloproteinase (MMP)-sensitive linkers for cell responsive degradation. However, developing successful matrix systems for therapeutic applications requires the dissection of both the viscoelastic properties and molecular signals that influence cellular behavior. So far, very few biomaterials allow for systematic and independent variation of mechanical and biomolecular characteristics (12). To address this, we now employ a mean field approach to analyze the force balance within biohybrid polymer networks to identify conditions that allow for decoupling biophysical and biochemical cues (see section “Design and key characteristics of starPEG-heparin hydrogels”). In addition, regenerative processes involving cell migration, proliferation and differentiation are governed by the temporal, spatial and concentration-dependent interplay of multiple growth factors. Functional biomaterials should therefore simultaneously or sequentially provide several different cytokines. The first single matrix system that was used to deliver multiple growth factors with distinct kinetics was based on alginate and could be successfully applied to promote vascularization by releasing vascular endothelial growth factor (VEGF) and 527 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


platelet-derived growth factor (PDGF) (13). Nevertheless, the integration of tunable mechanical and biofunctional material properties into one system that can be applied for the delivery of multiple cytokines remains a challenging but indispensable prerequisite to further enhance therapeutic effectiveness. We have developed a biohybrid hydrogel that addresses these key issues and offers a versatile platform of engineered biomaterials for regenerative therapies. It is formed by crosslinking amine functionalized star-shaped PEG (starPEG) and carbodiimide/N-hydroxysulfosuccinimide (EDC/s-NHS)-activated heparin (14). We report the design and main features of this system, specifically describing its potential for the defined and sustainable delivery of heparin-binding growth factors. An analytical strategy adapted to guide the matrix development is explained in detail. The applicability of the novel materials is demonstrated by examples highlighting the capability of the system to stimulate angiogenesis.

Design and Key Characteristics of starPEG-Heparin Hydrogels StarPEG-heparin hydrogels are formed using the synthetic polymer starPEG and the naturally occurring glycosaminoglycan heparin as building blocks (Figure 1). Such biohybrid materials combine the advantages of a structurally well-defined synthetic polymeric system, offering suitable mechanical properties, and the benefits of a naturally derived matrix, providing enhanced biofunctionality. PEG represents one of the most common synthetic polymers used for the design of biomaterials. Beside its excellent biocompatibility, the hydrophilic and uncharged character of PEG effectively prevents unspecific protein adsorption. Moreover, as PEG is a hydrolytically stable polymer with a good solubility in water and many different organic solvents, the possibility to easily modify its terminal functional groups opens up perspectives for a versatile PEG macromer chemistry (15).

Figure 1. Design of the biohybrid starPEG-heparin hydrogel functionalized with adhesion ligands and growth factors. (see color insert) 528 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Heparin is a glycosaminoglycan structurally related to the growth factor-binding ECM component heparan sulfate (8). While heparin can be mass-produced at low costs, it offers similar cytokine binding-properties as heparan sulfate at a smaller structural variance of its shorter chains (16). In the system presented here, heparin was used as a building block to equip the hydrogels with its intrinsic biofunctionality. Heparin-binding growth factors, such as VEGF (17), BMP-2 (18), fibroblast growth factor 2 (FGF-2) (19) and stromal-cell derived factor-1α (SDF-1α) (20), can be reversibly bound to the GAG. The binding is mainly driven by electrostatic interactions of their basic residues with the sulfate groups of heparin. Moreover, by applying EDC/s-NHS chemistry, integrin-binding cell adhesion peptides, such as cyclic RGDYK peptides (RGD-peptide, in single-letter amino acid code), can be covalently coupled to the carboxylic acid groups of heparin. Conceptually, the network composition of starPEG-heparin matrices goes beyond classical strategies. Polymer-based hydrogels are often formed by interconnecting a polymeric building block with a short crosslinker. In contrast, starPEG-heparin gels follow a rational concept where starPEG functions as a flexible, structural building block, while heparin acts as a stiff, multifunctional crosslinker. The 14 kDa heparin used in these experiments carries up to ~ 24 carboxylic acid moieties (8). Consequently, by varying the molar ratio of starPEG to heparin (γ) from ~ 1.5 to 6, up to six four-arm starPEG molecules could be attached to form a dense meshwork. Applying a mean field approach, conditions could be identified, where the underlying expansion and retraction forces in the swollen hydrogels compensate each other in such a way that in a physiological situation, the concentration of the highly charged multifunctional crosslinker (i.e. heparin) stays nearly constant (Figure 2A). This key property of the starPEG-heparin matrices was theoretically predicted and experimentally verified (21, 22). As a consequence, when the molar ratio of starPEG to heparin (γ) was increased from 1.5 to 6, gel types with an enhanced starPEG content but a constant heparin concentration of 0.8 % (w/w) were obtained, while the molar ratio of the components defines the degree of crosslinking. The synthetic building block represents the component which critically determines the viscoelastic properties of the hydrogel matrices. Therefore, by increasing the content of starPEG, less hydrated (as indicated by a lower swelling degree, data not shown) and stiffer scaffolds (storage modulus varying from 1 to 14.8 kPa for γ = 1.5 to 6) could be produced (Figure 2B). This finding is related to a higher number of covalent crosslinks, thereby leading to the formation of a denser network with larger retraction forces, which is more rigid (higher storage modulus) and exhibits restricted water uptake (lower swelling). However, independent of the different viscoelastic characteristics of the networks, all different hydrogel types contain large and constant quantities of heparin (~ 8 mg/ml). This rationally designed characteristic of the hydrogels is crucial for a subsequent biomolecular functionalization, as it should principally allow for a decoupling of the structural parameters and the biofunctionality. Consequently, based on the constant heparin concentration in hydrogels with varying mechanical characteristics, similar amounts of RGD-peptides (~ 0.6 mol RGD/mol heparin) were attached to the different gel types (22) (Figure 2C). As 529 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


heparin also represents the basis for subsequent growth factor functionalization of the scaffold, the matrices might represent a system, where the cytokine delivery could be adapted independently of structural and mechanical material properties of the scaffold. Moreover, an advantage of the high heparin content within the gels is that the structural integrity of heparin can be preserved up to higher degrees of crosslinking, which might permit a rather unaffected interaction with several heparin-binding growth factors. Therefore, the developed starPEG-heparin hydrogels could represent a promising material for the independent and parallel delivery of multiple cytokines required to aid complex regenerative processes.

Methods for Characterizing the Interaction of Growth Factors with starPEG-Heparin Hydrogels StarPEG-heparin hydrogels closely mimic the characteristics of the ECM by containing large quantities of heparin, which electrostatically binds and stabilizes numerous cytokines. To thoroughly characterize the interaction of growth factors with the hydrogels, several analytical methods have to be applied and compared (1). For this, fluorescently- or radioisotope (125I)-labeled proteins were observed by confocal laser scanning microscopy (CLSM) or detected by gamma counting respectively. Additionally, growth factors were quantified by amino acid analysis via high performance liquid chromatography (HPLC) and immunological detection by an enzyme-linked immunosorbent assay (ELISA). All of these approaches are based on distinct detection mechanisms and therefore, experimental parameters had to be adjusted to the requirements of the particular method (Table I). This included the potential presence of a protein label and the setting used to present the protein solution to the hydrogels. However, despite these experimental differences, the combination of all four analytical approaches allowed the characterization of the binding and release of the growth factors over a wide range of concentrations. The different methods were compared based on their ability to accurately and reliably quantify cytokine immobilization after adsorption and the diffusion-based release by starPEG-heparin hydrogels. Depending on the analytical approach, the protein could be directly detected in the gel network and/or in the supernatant of the surrounding medium. While all of the methods delivered the same qualitative results, the different experimental conditions showed quantitative differences (23). These discrepancies may arise from two effects. Firstly, an altered heparin-affinity was observed when a label had to be attached to the growth factors. Secondly, CLSM and HPLC were not compatible with the special immobilization chambers used to restrict non-specific protein binding to non-hydrogel surfaces. Only ELISA experiments could be performed using non-labeled protein and under conditions that minimized the contact area for non-specific protein interactions with ‘foreign’ glass or plastic surfaces. Due to these facts, in addition to its high sensitivity, ELISA was found to be the best performing assay for analyzing growth factor binding and release. For a more detailed discussion of the methodological optimization the reader is referred to (23). 530 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


531 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 2. Composition and key properties of different starPEG-heparin hydrogels. (A) Varying starPEG and constant heparin concentration in swollen gels matrices prepared with different molar starPEG/heparin ratios. (B) Gradual volumetric swelling characteristics of different hydrogel types depending on the starPEG content. (C) Constant RGD functionalization of different hydrogel types depending on the heparin content. All data are presented as mean ± root mean square deviation from n = 4. Adapted with kind permission from reference (22) (original Figure 2). Copyright 2012 John Wiley and Sons.

Table I. Experimental parameters used for growth factor binding and release studies with starPEG-heparin hydrogels. Due to higher sensitivity of these assays, cytokine release experiments were only performed via radiolabeling studies and ELISA. Adapted with kind permission from reference (23) (original Table 2). Copyright 2010 Springer Science and Business Media CLSM

radiolabeling (125I- ) studies

HPLC

ELISA

performance

well plate

immobilization chamber

well plate

immobilization chamber

analysis of protein

in gel and supernatant

in gel

in gel

in supernatant

protein labeled

yes

yes

no

no

sensitivity with particular setting

nanogram

picogramnanogram

nanogrammicrogram

picogram

Growth Factor Presentation by starPEG-Heparin Hydrogels Uptake and Release of Growth Factors Depending on the Physicochemical Network Properties To successfully support regenerative processes, it is important to realize that cellular behavior is critically influenced by interactions with the natural ECM. The physicochemical and structural properties of this network have been shown to have a major impact on cell fate (10). Therefore, in order to effectively advance therapeutic approaches for different tissues, biomaterials with distinct mechanical characteristics should be equally applicable for the delivery of functional signaling proteins. 532 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 3. Amount of hydrogel-immobilized or -released growth factor determined for gel types differing in their crosslinking degree. Left: amount of electrostatically bound (A) FGF-2, (B) VEGF, (C) SDF-1α or (D) BMP-2 per cm² scaffold area for the different gel types γ = 1.5; 3 or 6 (low, intermediate and high crosslinking degree). Proteins were adsorbed from 200 µl immobilization 533 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


solution per cm² scaffold area. Right: cumulative amount of electrostatically bound (A) FGF-2, (B) VEGF, (C) SDF-1α or (D) BMP-2 released by the different gel matrices γ = 1.5; 3 or 6 (low, intermediate and high crosslinking degree). Proteins were released into 250 µl release medium per cm² scaffold area. All data are presented as mean ± root mean square deviation from n = 3. Adapted with kind permission from reference (24) (A and B, original Figure 2) and reference (22) (D, original Supporting Figure 2). Copyright 2010 Elsevier (24) and 2012 John Wiley and Sons (22). To address this need, the influence of the starPEG-heparin network structure on the binding and release of various growth factors was investigated for hydrogel types differing in their crosslinking degree (γ = 1.5, 3 and 6) and therefore in their mechanical properties (soft, intermediate and stiff networks, see Figure 2B) (22, 24). Figure 3 shows the results of these experiments for FGF-2 (A), VEGF (B), SDF-1α (C) and BMP-2 (D). For each of these proteins, it was demonstrated that similar quantities were bound by every scaffold independently of the gel type (Figure 3, left). Following growth factor immobilization by adsorption, the proteins were allowed to be released by diffusion from the hydrogels into the surrounding medium. Regarding the growth factor release kinetics (Figure 3, right), all proteins showed an initial burst release within the first hours. Such burst characteristics are often attributed to surface effects (25) and could have been caused by a protein fraction being entrapped in the meshwork but not bound specifically to heparin. However, after that, the protein continued to be released slowly over the course of the time period investigated. This suggests that the material has the potential to be used for applications that need long-term delivery profiles of growth factors. Moreover, comparable sequestered protein quantities were found for each scaffold independent of the gel type. The results demonstrate that the binding and release of the different growth factors is independent of the mechanical hydrogel properties. Considering the mesh sizes of the matrices, with large pores in the range of ~ 16 nm for γ = 1.5 and ~ 7 nm for γ = 6 (14), it becomes clear that cytokine diffusion is not affected by differences in the network structure, but that the protein immobilization and delivery correlates with the constant heparin concentration of the different scaffolds. Consequently, starPEG-heparin hydrogels could be used as growth factor storage systems, presenting these proteins independently of the particular structural and mechanical properties of the scaffolds. Uptake and Release of Growth Factors Depending on the Cytokine Concentration Besides the influence of the viscoelastic cell environment, tissue regeneration is controlled by the concentration-dependent interplay of various signaling proteins. Therefore, biomaterials should be able to deliver large quantities of growth factors as well as provide several cytokines in parallel. StarPEG-heparin hydrogels might be promising candidates for such an application, based on their high heparin content. 534 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Thus, as shown for the example of FGF-2 and/or VEGF, after evaluating the binding potential of starPEG-heparin hydrogels for high concentrations of single cytokines (Figure 4A), the possibility to deliver growth factor combinations was investigated (Figure 4B). For that, the immobilization and subsequent release of both proteins, introduced to the scaffolds either as single components or as combinations in different ratios, were analyzed for the gel with the intermediate crosslinking degree, γ = 3 (Figure 4B). When evaluating the capacity of the matrices to take up various amounts of FGF-2 or VEGF separately (Figure 4A) (24), it was shown that the immobilized quantities at a defined concentration were similar for both proteins. Moreover, there was a linear correlation between the concentration of the incubation solution and the amount of immobilized FGF-2 or VEGF within the gel. This indicates that saturation of binding was not reached within the concentration range monitored. This result correlates with estimates of the maximal storage capacity of the applied hydrogel system, based on the calculated heparin concentration within the swollen network, and HPLC-based analysis of immobilization experiments with high concentrations of growth factors. Even after incubation with 50 µg/ml protein, the molar ratio of heparin to growth factor was still 26:1 for FGF-2 and 62:1 for VEGF. Moreover, as reported for FGF-2 (26), each heparin molecule is able to interact with several cytokine molecules, so that a saturation of binding will occur only at concentrations much higher than those used in this study. In addition, it could be demonstrated that different combinations of FGF-2 and VEGF (Figure 4B) (27) can be bound to the matrices with the same efficiency as determined for the individual factors. Furthermore, the immobilized quantities of combinations of FGF-2 and VEGF at a defined concentration were found to be similar for both proteins. Besides an evaluation of the starPEG-heparin hydrogel binding ability for FGF-2 and VEGF, experiments on the release of each single protein and of different combinations were performed. Figure 4C illustrates the cumulative release of either FGF-2 (left) or VEGF (right) alone and of different combinations of both proteins. Irrespective of the immobilized concentration or the particular factor considered, the release curves show once again the typical burst within the first hours followed by a continuous release over time. Similar to the trends observed for FGF-2 and/or VEGF immobilization, a linear correlation between the amount of gel-bound growth factors and the quantities being released was found. Additionally, different combinations of FGF-2 and VEGF could be released by the matrices with the same efficiency as for the individual factors. The large excess of heparin appears to prevent any interference between the growth factors during their combined application. Moreover, an additional advantage of these starPEG-heparin hydrogels is the comparable release of both cytokines at a particular loading quantity. Given this finding, the FGF-2 and/or VEGF release characteristics can be adjusted by the initial amount of protein loaded, which in turn can be tuned over a wide range of concentrations.



Figure 4. Amount of hydrogel-immobilized (top) and/or -released (bottom) FGF-2 and VEGF determined for different protein concentrations. (A) Uptake of single FGF-2 or VEGF in dependence on the protein concentration (0.5-50 µg/ml) in the immobilization medium; linear regression, R² (FGF-2) = 0.99999; R² (VEGF) = 0.99999. (B) Amount of electrostatically bound FGF-2 and/or VEGF per cm² scaffold area for different protein concentrations. Proteins were adsorbed from 200 µl immobilization solution per cm² scaffold area. (C) Cumulative amount of electrostatically bound FGF-2 (left) or VEGF (right) released by gels which were loaded with either single cytokines (dashed lines) or different combinations of FGF-2 and VEGF (continuous lines). Proteins were released into 250 µl release medium per cm² scaffold area. All data are presented as mean ± root mean square deviation from n = 3. Adapted with kind permission from reference (24) (A, original Figure 2) and reference (27) (B and C, original Figure 1). Copyright 2010 Elsevier (24) and 2011 Elsevier (27).

Growth Factor Delivery by starPEG-Heparin Hydrogels To Modulate a Specific Cell Behavior In the context of regenerative medicine, the aim of growth factor delivery by biofunctional materials is to trigger a therapeutically relevant cell behavior. To illustrate the potential of cytokine administration by starPEG-heparin hydrogels 536 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


for controlling cell fate decisions, cell culture experiments with an array of customized materials varying in their elastic characteristics and cell adhesive properties were performed.

Figure 5. In vitro identification of pro-angiogenic conditions offered by VEGF delivery from starPEG-heparin hydrogels of different physical and biomolecuar characteristics. Endothelial cells elongate to form a network of cord-like structures on starPEG-heparin hydrogels with independently varying VEGF and RGD incorporation and storage modulus (turquoise frames, arrows indicate elongated cells). Representative confocal immunofluorescence images of CD31 (green, endothelial cell marker), actin (red), and DAPI (blue) staining of HUVECs plated for 20-24 hours on different scaffolds. Adapted with kind permission from reference (22) (original Figure 3). Copyright 2012 John Wiley and Sons. (see color insert)

As angiogenesis, the formation of new blood vessels from pre-existing capillaries, represents a key process for the regeneration of almost any tissue, it has to be inducible under a variety of different biophysical and biochemical properties of the cellular environment (28). StarPEG-heparin hydrogels were used to identify various pro-angiogenic conditions that were able to support morphological changes of endothelial cells indicative of in vitro capillary-like network formation (Figure 5, highlighted in turquoise, for exact quantification see reference (22)). In this example, a VEGF delivery from soft and intermediately elastic hydrogels (γ = 1.5 and 3) that were less adhesive (functionalized with low RGD concentrations) preferentially led to the development of an elongated phenotype. On more adhesive gels, cell spreading and the formation of a monolayer were observed. With VEGF released by stiffer hydrogels (γ = 6), endothelial cells required a higher RGD concentration to induce adequate cell 537 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


adhesion and the formation of cord-like structures. In summary, with the gradual and independent variation of synergistically acting matrix characteristics, it could be shown that multiple sets of effective combinations of VEGF release, together with an appropriate network stiffness and adhesion ligand density can support the development of cord-like structures as it is required for blood vessel formation in distinct cellular environments within different tissues (22). StarPEG-heparin hydrogels have also been applied as a culture carrier system to direct human mesenchymal stem cell (MSC) differentiation in the context of tissue engineering for bone regeneration. Similar to the natural situation being advantageous to the development of an osteogenic phenotype (29), the delivery of BMP-2 from cell adhesive, rather stiff hydrogels was found to preferentially promote MSC differentiation into this lineage (22).

Figure 6. Effects of growth factor provision by starPEG-heparin scaffolds on vascularization in the chicken embryo CAM assay. (A and B) Representative images of the CAM vascularization in response to starPEG-heparin hydrogels with single FGF-2 or VEGF and a combination FGF-2 and VEGF (A) and the untreated CAM which served as a control (B) (scale bar 1 mm). (C) Quantification of the relative CAM vascularization in relation to starPEG-heparin hydrogels with single FGF-2 or VEGF and a combination of FGF-2 and VEGF. Data are presented as mean ± root mean square deviation from n = 5-16 (* indicates p < 0.05; analysis of variance). Reproduced with kind permission from reference (27). Copyright 2011 Elsevier. 538 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


After analyzing the effect of a growth factor administration by materials with tunable mechanical and biofunctional properties, the influence of concentration-dependent cytokine release and the provision of multiple growth factors was investigated. Both in in vitro migration assays and during subcutaneous implantation in mice, SDF-1α release from starPEG-heparin hydrogels could generate a growth factor gradient to induce site-directed attaction of early endothelial progenitor cells (eEPCs) towards SDF-1α (30). eEPC migration to ischemic tissues, with the incorporation of the cells into new capillaries and their release of intrinsic factors that promote angiogenesis, is a promising strategy for cardiac regeneration (31). Moreover, we applied starPEG-heparin hydrogels for a combined delivery of FGF-2 and VEGF with the intention to support blood vessel formation (27). A parallel cytokine provision resulted in superior pro-angiogenic effects in vitro (enhanced endothelial cell survival/proliferation, morphology and migration) compared to the administration of single factors. We studied these effects further using in vivo experiments. Growth factor-functionalized hydrogels (γ = 3) were transferred to the developing chorioallantoic membrane (CAM) of fertilized chicken eggs (Figure 6). These onplants were surrounded by an increased number of allantoic vessels that looped towards the gel with the delivery of any angiogenic cytokine (A and C), compared to an untreated site (B). However, once again the combined provision of FGF-2 and VEGF enhanced the CAM vascularization most effectively (Figure 6 and reference (27)). As a conclusion, starPEG-heparin hydrogels providing controlled release of various heparin-affine cytokines can be successfully applied in different tissue engineering approaches. The possibility to decouple the mechanical properties from the biofunctionalization is advantageous to adjust multiple different material parameters, with the option to deliver high quantities or several growth factors in parallel being of particular benefit for regenerative concepts.

Summary and Conclusion For a successful strategy in regenerative medicine, therapeutically relevant growth factors need to be actively administered to the tissue of interest by biomaterials. The reversible adsorption of growth factors to materials that are composed of a building block with an intrinsic cytokine affinity, such as starPEG-heparin hydrogels, are ideal for this purpose. Different hydrogel types with distinct mechanical characteristics but a constant heparin content could be produced by varying the molar ratio of starPEG to heparin upon network formation. As heparin represents the basis for the growth factor interaction with the scaffolds, the matrices were found to bind and release various heparin-affine cytokines independently of the network stiffness and structural properties of the different gel types. Moreover, based on the high heparin content of the hydrogels, the material could be utilized for a modular delivery of growth factor combinations over a broad range of concentrations. Using this system, different in vitro and in vivo cell experiments demonstrated the suitability of cytokine delivery by distinct starPEG-heparin hydrogels to support tissue engineering 539 In Proteins at Interfaces III State of the Art; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

approaches. In future, useful extensions of the introduced cytokine release system could be achieved by the inclusion of peptide units to control gel degradation (by incorporation of matrix metalloproteinase-sensitive peptide sequences) (32), and by selective heparin desulfation to modulate the affinity of the hydrogel to different growth factors. In conclusion, starPEG-heparin hydrogels with independently adaptable physical and biomolecular composition demonstrated to be able to provide time-resolved multi-factor delivery of various growth factors. These results provide valuable new options for therapeutic tissue engineering concepts.


Acknowledgments We would like to thank Tina Lenk (Leibniz Institute of Polymer Research Dresden) for radiolabeling of growth factors and Milauscha Grimmer (Leibniz Institute of Polymer Research Dresden) for performing HPLC analysis. U.F. and C.W. were supported by the Deutsche Forschungsgemeinschaft through grants WE 2539-7/1 and FOR/EXC999, and by the Leibniz Association. S.P., K.R.L., K.C. and C.W. were supported by the Seventh Framework Programme of the European Union through the Integrated Project ANGIOSCAFF. A.Z. was supported by the Dresden International Graduate School for Biomedicine and Bioengineering.

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