Faculty Bibliography

Hydrolytically labile poly(ethylene glycol)-based hydrogels are fabricated via a Michael-type addition reaction between unsaturated acrylate moieties and nucleophilic thiols. Although these gels offer the advantage of selective, in situ polymerization and potential as biocompatible matrixes for cell and protein encapsulation, a thorough understanding of the complex structure-property relationships that control the macroscopic behaviors of these cross-linked networks before and during hydrolytic degradation does not exist. Therefore, in this work, a novel theoretical model is presented to describe the formation and hydrolytic degradation of the step-polymerized gels. The model accounts for variations in hydrolysis kinetics as well as structural effects such as precursor functionality and the presence of primary cycles or other structural nonidealities that lower the cross-linking efficiency of the networks. Comparison of model predictions and experimental data validate this methodology for optimizing biomaterial design and reveal that structural nonidealities play a key role in determining the degradation behavior of real cross-linked systems. Decreasing precursor concentration and functionality during network formation generate high concentrations of network nonidealities that ultimately lead to higher initial swelling ratios and faster apparent rates of degradation.

Clinical in vitro endothelialization has been shown to increase the patency of synthetic vascular grafts. The shear stress resistance of the cultured autologous endothelium represents a crucial cornerstone of the concept. We investigated whether an enrichment of the precoating matrix with adhesion sites can augment endothelial cell attachment. Adult human saphenous vein endothelial cells (AHSVECs) were seeded confluently ([58 +/- 11] x 10(3) AHSVECs/cm2) onto 10-cm-long ePTFE (expanded polytetrafluorethylene) vascular grafts (n = 24) precoated with commercial clinically approved fibrin gel (Tisseal) containing various concentrations of cross-linked RGD peptide (0.0, 4.0, 8.0, or 16.0 mg of RGD per milliliter of Tisseal fibrinogen component). Endothelialized grafts were postcultivated for 9 days before they were exposed to a pulsatile circulation model mimicking peak physiological shear stress conditions of the femoral artery (12 dyn/cm2; min/max, -60/+28 dyn/cm2). Cell loss after 24 h was quantitatively determined by image analysis of vital stains. Initial 24-h cell loss was 27.2 +/- 1.7% in grafts precoated with the non-RGD-enriched fibrin matrix. In contrast, cell loss was significantly less on fibrin containing 4.0 mg of RGD peptide per milliliter of Tisseal fibrinogen component (13.3 +/- 7.9%; p < 0.05). Cell loss on fibrin containing 8 and 16 mg of RGD per milliliter of Tisseal fibrinogen component was 41.0 +/- 27.4 and 43.0 +/- 23.2% (p > 0.05), respectively. We conclude that low concentrations of RGD peptide cross-linked into commercial fibrin matrices used for clinical in vitro lining of vascular grafts led to significantly increased endothelial cell retention. The failure of higher RGD concentrations to enhance endothelial cell attachment may be explained by competitive binding of endothelial cells to non-cross-linked RGD.

Fully synthetic polymers were used for the preparation of hydrogel beads and capsules, in a processing scheme that, originally designed for calcium alginate, was adapted to a "tandem" process, that is the combination a physical gelation with a chemical cross-linking. The polymers feature a Tetronic backbone (tetra armed Pluronics), which exhibits a reverse thermal gelation in water solutions within a physiological range of temperatures and pHs. The polymers bear terminal reactive groups that allow for a mild, but effective chemical cross-linking. Given an appropriate temperature jump, the thermal gelation provides a hardening kinetics similar to that of alginate. With slower kinetics, the chemical cross-linking then develops an irreversible and elastic gel structure, and determines its transport properties. In the present article this process has been optimized for the production of monodisperse, high elastic, hydrogel microbeads, and liquid-core microcapsules. We also show the feasibility of the use of liquid-core microcapsules in cell encapsulation. In preliminary experiments, CHO cells have been successfully encapsulated preserving their viability during the process and after incubation. The advantages of this process are mainly in the use of synthetic polymers, which provide great flexibility in the molecular design. This, in principle, allows for a precise tailoring of mechanical and transport properties and of bioactivity of the hydrogels, and also for a precise control in material purification.

Angiogenic signals can be matrix attached or freely diffusible. Here, the sixth Ig-like domain of L1 (L1Ig6), a ligand for alphavbeta3-integrin, was investigated. This domain was expressed as a fusion protein having a substrate sequence for factor XIII to enable covalent binding into three-dimensional fibrin matrices. Matrix-bound L1Ig6 induced endothelial cell (EC) process extension in vitro, which was associated with ligation and phosphorylation of alphavbeta3-integrin. VEGF-R2 and alphavbeta3 were observed to co-associate after stimulation with either L1Ig6 or VEGF-A165, whereas no co-association with bFGF-R was observed. Furthermore, VEGF-R2 was tyrosine phosphorylated after stimulation with L1Ig6, even in the absence of exogenous VEGF-A165, indicating close cooperation between VEGF-R2 and alphavbeta3. Angiogenesis was investigated in vivo by stimulating chicken chorioallantoic membranes (CAMs) with L1Ig6-modified matrices with or without co-incorporation of VEGF-A165 or bFGF. Matrix-immobilized L1Ig6 induced angiogenesis to a similar degree as VEGF-A165; co-stimulation with bFGF increased vascular branching, whereas VEGF-A165 did not. Matrix-immobilized L1Ig6 induced up-regulation of alphav in CAMs by a similar degree as stimulation with VEGF-A165, and this up-regulation was increased further by co-stimulation with matrix-bound L1Ig6 and VEGF-A165. alpha5 and beta1 levels were not increased. The similarity of action of matrix-bound L1Ig6 and soluble VEGF-A165 indicate a close link between specific ligation of alphavbeta3-integrin and VEGF-R2 and suggest the possible use of matrix-bound L1Ig6 in local therapeutic angiogenesis.

We have previously described a gelation process based on the occurrence of both physical and a chemical mechanisms ('tandem process'), in which a telechelic linear poly(propylene glycol)-bl-poly(ethylene glycol)-bl-poly(propylene glycol) is first thermally gelled and subsequently covalently cross-linked by the reaction of polymer end groups at the termini of the copolymer. The quick kinetics of the reverse thermal gelation and the harmless character of the Michael-type addition between two sets of terminal groups, acrylates on one set and thiols on the other, allows irreversibly cross-linked hydrogels to be obtained in a rapid and biocompatible fashion, even when gelation was conducted in direct contact with cells. This allows in principle for an application of the tandem process in cell encapsulation. In the present work, we have optimized the macromolecular architecture and functionality of the precursors for allowing the use of the tandem process in encapsulation devices designed for calcium alginate. The mechanical, diffusional and biocompatibility properties of these materials were characterized; the comparison of mass transport properties of the tandem gels with those of calcium alginate suggests a similar or even better immunoisolation effect.

With the rapid increase in approaches to pro- or anti-angiogenic therapy, new and effective methodologies for administration of cell-bound growth factors will be required. We sought to develop the natural hydrogel matrix fibrin as platform for extensive interactions and continuous signaling by the vascular morphogen ephrin-B2 that normally resides in the plasma membrane and requires multivalent presentation for ligation and activation of Eph receptors on apposing endothelial cell surfaces. Using fibrin and protein engineering technology to induce multivalent ligand presentation, a recombinant mutant ephrin-B2 receptor binding domain was covalently coupled to fibrin networks at variably high densities. The ability of fibrin-bound ephrin-B2 to act as ligand for endothelial cells was preserved, as demonstrated by a concomitant, dose-dependent increase of endothelial cell binding to engineered ephrin-B2-fibrin substrates in vitro. The therapeutic relevance of ephrin-B2-fibrin implant matrices was demonstrated by a local angiogenic response in the chick embryo chorioallontoic membrane evoked by the local and prolonged presentation of matrix-bound ephrin-B2 to tissue adjacing the implant. This new knowledge on biomimetic fibrin vehicles for precise local delivery of membrane-bound growth factor signals may help to elucidate specific biological growth factor function, and serve as starting point for development of new treatment strategies.