Mary Beth Browning

Multilayer vascular grafts based on collagen-mimetic proteins.

A major roadblock in the development of an off-the-shelf, small-caliber vascular graft is achieving rapid endothelialization of the conduit while minimizing the risk of thrombosis, intimal hyperplasia, and mechanical failure. To address this need, a collagen-mimetic protein derived from group A Streptococcus, Scl2.28 (Scl2), was conjugated into a poly(ethylene glycol) (PEG) hydrogel to generate bioactive hydrogels that bind to endothelial cells (ECs) and resist platelet adhesion. The PEG-Scl2 hydrogel was then reinforced with an electrospun polyurethane mesh to achieve suitable biomechanical properties. In the current study, initial evaluation of this multilayer design as a potential off-the-shelf graft was conducted. First, electrospinning parameters were varied to achieve composite burst pressure, compliance, and suture retention strength that matched reported values of saphenous vein autografts. Composite stability following drying, sterilization, and physiological conditioning under pulsatile flow was then demonstrated. Scl2 bioactivity was also maintained after drying and sterilization as indicated by EC adhesion and spreading. Evaluation of platelet adhesion, aggregation, and activation indicated that PEG-Scl2 hydrogels had minimal platelet interactions and thus appear to provide a thromboresistant blood contacting layer. Finally, evaluation of EC migration speed demonstrated that PEG-Scl2 hydrogels promoted higher migration speeds than PEG-collagen analogs and that migration speed was readily tuned by altering protein concentration. Collectively, these results indicate that this multilayer design warrants further investigation and may have the potential to improve on current synthetic options.

Determination of the in vivo degradation mechanism of PEGDA hydrogels

Poly(ethylene glycol) (PEG) hydrogels are one of the most extensively utilized biomaterials systems due to their established biocompatibility and highly tunable properties. It is widely acknowledged that traditional acrylate-derivatized PEG (PEGDA) hydrogels are susceptible to slow degradation in vivo and are therefore unsuitable for long-term implantable applications. However, there is speculation whether the observed degradation is due to hydrolysis of endgroup acrylate esters or oxidation of the ether backbone, both of which are possible in the foreign body response to implanted devices. PEG diacrylamide (PEGDAA) is a polyether-based hydrogel system with similar properties to PEGDA but with amide linkages in place of the acrylate esters. This provides a hydrolytically-stable control that can be used to isolate the relative contributions of hydrolysis and oxidation to the in vivo degradation of PEGDA. Here we show that PEGDAA hydrogels remained stable over 12 weeks of subcutaneous implantation in a rat model while PEGDA hydrogels underwent significant degradation as indicated by both increased swelling ratio and decreased modulus. As PEGDA and PEGDAA have similar susceptibility to oxidation, these results demonstrate for the first time that the primary in vivo degradation mechanism of PEGDA is hydrolysis of the endgroup acrylate esters. Additionally, the maintenance of PEGDAA hydrogel properties in vivo indicates their suitability for long-term implants. These studies serve to elucidate key information about a widely used biomaterial system to allow for better implantable device design and to provide a biostable replacement option for PEGDA in applications that require long-term stability.

Development of a Biostable Replacement for PEGDA Hydrogels

The exceptional tunability of poly(ethylene glycol) (PEG) hydrogel chemical, mechanical, and biological properties enables their successful use in a wide range of biomedical applications. Although PEG diacrylate (PEGDA) hydrogels are often used as nondegradable controls in short-term in vitro studies, it is widely acknowledged that the hydrolytically labile esters formed upon acrylation of the PEG diol make them susceptible to slow degradation in vivo. A PEG hydrogel system that maintains the desirable properties of PEGDA while improving biostability would be valuable in preventing degradation-related failure of gel-based devices in long-term in vivo applications. To this end, PEG diacrylamide (PEGDAA) hydrogels were synthesized and characterized in quantitative comparison to traditional PEGDA hydrogels. It was found that PEGDAA hydrogel modulus and swelling can be tuned over a similar range and to comparable degrees as PEGDA hydrogels with changes in macromer molecular weight and concentration. Additionally, PEGDAA cytocompatibility, low cell adhesion, and capacity for incorporation of bioactivity were analogous to that of PEGDA. In vitro hydrolytic degradation studies showed that the amide-based PEGDAA had significantly increased biostability relative to PEGDA. Overall, these findings indicate that PEGDAA hydrogels are a suitable replacement for PEGDA hydrogels with enhanced hydrolytic resistance. In addition, these studies provide a quantitative measure of the hydrolytic degradation rate of PEGDA hydrogels which was previously lacking in the literature.

Compositional control of poly(ethylene glycol) hydrogel modulus independent of mesh size

Poly(ethylene glycol) (PEG) hydrogels are of great interest in tissue engineering because of their established biocompatibility, high permeability, and tunable material properties. However, rational design of PEG hydrogel scaffold properties has been inhibited by the interdependence of key material properties such as modulus and mesh size. This study examined the effect of an acrylated 4-arm PEG cross-linker on gel modulus and mesh size as a means of inducing local increases in cross-link density to decouple these two parameters. It was determined that adding the 4-arm PEG cross-linker to PEG hydrogels resulted in statistically significant increases in both tensile and compressive modulus while having minimal effects on overall gel mesh size. The incorporation of the 4-arm PEG cross-linker also broadened the range of achievable mechanical properties. This study provides the methodology to independently tune PEG hydrogel modulus and mesh size, which may be utilized in future investigations of the individual and combined effects of PEG hydrogel modulus and mesh size on cell behavior and viability. It also presents a more finely tunable hydrogel scaffold with utility in a broad range of tissue engineering applications.

Bioactive hydrogels with enhanced initial and sustained cell interactions

The highly tunable properties of poly(ethylene glycol) (PEG)-based hydrogel systems permit their use in a wide array of regenerative medicine and drug delivery applications. One of the most valuable properties of PEG hydrogels is their intrinsic resistance to protein adsorption and cell adhesion, as it allows for a controlled introduction of desired bioactive factors including proteins, peptides, and drugs. Acrylate-PEG-N-hydroxysuccinimide (Acr-PEG-NHS) is widely utilized as a PEG linker to functionalize bioactive factors with photo-cross-linkable groups. This enables their facile incorporation into PEG hydrogel networks or the use of PEGylation strategies for drug delivery. However, PEG linkers can sterically block integrin binding sites on functionalized proteins and reduce cell-material interactions. In this study we demonstrate that reducing the density of PEG linkers on protein backbones during functionalization results in significantly improved cell adhesion and spreading to bioactive hydrogels. However, this reduction in functionalization density also increases protein loss from the matrix over time due to ester hydrolysis of the Acr-PEG-NHS linkers. To address this, a novel PEG linker, acrylamide-PEG-isocyanate (Aam-PEG-I), with enhanced hydrolytic stability was synthesized. It was found that decreasing functionalization density with Aam-PEG-I resulted in comparable increases in cell adhesion and spreading to Acr-PEG-NHS systems while maintaining protein and bioactivity levels within the hydrogel network over a significantly longer time frame. Thus, Aam-PEG-I provides a new option for protein functionalization for use in a wide range of applications that improves initial and sustained cell-material interactions to enhance control of bioactivity.

Drying and storage effects on poly(ethylene glycol) hydrogel mechanical properties and bioactivity.

Hydrogels based on poly(ethylene glycol) (PEG) are increasingly used in biomedical applications because of their ability to control cell-material interactions by tuning hydrogel physical and biological properties. Evaluation of stability after drying and storage are critical in creating an off-the-shelf biomaterial that functions in vivo according to original specifications. However, there has not been a study that systematically investigates the effects of different drying conditions on hydrogel compositional variables. In the first part of this study, PEG-diacrylate hydrogels underwent common processing procedures (vacuum-drying, lyophilizing, hydrating then vacuum-drying), and the effect of this processing on the mechanical properties and swelling ratios was measured. Significant changes in compressive modulus, tensile modulus, and swelling ratio only occurred for select processed hydrogels. No consistent trends were observed after processing for any of the formulations tested. The effect of storage conditions on cell adhesion and spreading on collagen- and streptococcal collagen-like protein (Scl2-2)-PEG-diacrylamide hydrogels was then evaluated to characterize bioactivity retention after storage. Dry storage conditions preserved bioactivity after 6 weeks of storage; whereas, storage in PBS significantly reduced bioactivity. This loss of bioactivity was attributed to ester hydrolysis of the protein linker, acrylate-PEG-N-hydroxysuccinimide. These studies demonstrate that these processing methods and dry storage conditions may be used to prepare bioactive PEG hydrogel scaffolds with recoverable functionality after storage.