Faculty Bibliography

A quantitative structure-reactivity relationship for the Michael-type addition of thiols onto acrylates was determined. Several thiol-containing peptides were investigated by examining the correlation between the second-order rate constant of their addition onto PEG-diacrylate and the pK(a) of the thiols within a peptide. By introducing charged amino acids in close proximity to a cysteine, the pK(a) of the thiol was systematically modulated by electrostatic interactions. Positive charges from the amino acid arginine decreased the pK(a) of the thiol and accelerated the reaction with acrylates while negative charges from aspartic acids showed the opposite effect. A linear correlation between thiolate concentrations and kinetic constants was found, confirming the role of thiolates as the reactive species in this Michael-type reaction. The relevant factors influencing the reactivity were the sign and the number of the neighboring charges, while the position of these charges had little effect on reactivity. These results provide a basis for the rational design of peptides, where the kinetics and thus selectivity of protein/peptide conjugation with polymeric structures via Michael-type addition reactions can be controlled.

PEG-diacrylamide was synthesized and utilized to make materials for tissue engineering. Acrylamide groups readily react with thiol groups, and peptides containing a single thiol group were coupled to the PEG-diacrylamide in aqueous solution at room temperature in about 2 h. If only a portion of the acrylamide groups were targeted for reaction with peptide, sufficient amounts of PEG-diacrylamide remained to be polymerized by free-radical mechanisms via photoinitiation. The photopolymerization step can be performed in contact with cells, providing a means to produce bioactive scaffolds for tissue engineering. The photopolymerization conditions and precursor composition greatly affect the stiffness of the materials, which subsequently affected cell spreading. The kinetics and extent of cell spreading on the bioactive materials were measured and compared to cell spreading on tissue culture polystyrene. Although the PEG materials resist protein adsorption, the experiments suggest that the cells can secrete extracellular matrix that can adhere to the gels.

The goal of this work was to develop a growth factor delivery system for use in nerve regeneration that would provide localized release of beta-nerve growth factor (beta-NGF) and other members of the neurotrophin family in a controlled manner. Although beta-NGF does not bind heparin with high affinity, we postulated that a basic domain found at the surface of native beta-NGF could interact with heparin and slow its diffusion from a heparin-containing delivery system. To test this hypothesis, we used a heparin-containing fibrin-based cell ingrowth matrix consisting of three components, namely an immobilized heparin-binding peptide, heparin and a neurotrophin with low heparin-binding affinity. The heparin-binding peptide contained a factor XIIIa substrate and was covalently cross-linked to fibrin matrices during polymerization. This cross-linked heparin-binding peptide served to immobilize heparin within the matrix, and this immobilized heparin interacted with the neurotrophin and slowed the passive release of the growth factor from the matrix. The ability of heparin-containing fibrin matrices, with a high excess of heparin-binding sites, to slow the diffusion-based release of beta-NGF from fibrin matrices was measured in the absence of cells. Conditions that provided for slow diffusion-based release of beta-NGF, brain-derived neurotrophic factor, and neurotrophin-3 were tested in an assay of neurite extension from dorsal root ganglia to determine the ability of the delivery system to release active growth factor. The results demonstrated that neurotrophins, interacting with fibrin matrices containing a large molar excess of heparin relative to growth factor, enhanced neurite extension by up to 100% relative to unmodified fibrin. In the absence of the delivery system, free neurotrophins within the fibrin matrix did not enhance neurite extension. The results suggest that these matrices could serve as therapeutic materials to enhance peripheral nerve regeneration through nerve guide tubes and may have more general usefulness in tissue engineering for the delivery of non-heparin-binding growth factors.

OBJECTIVE:In tissue engineering, three-dimensional biodegradable scaffolds are generally used as a basic structure for cell anchorage, cell proliferation and cell differentiation. The currently used biodegradable scaffolds in cardiovascular tissue engineering are potentially immunogenic, they show toxic degradation and inflammatory reactions. The aim of this study is to establish a new three-dimensional cell culture system within cells achieve uniform distribution and quick tissue development and with no toxic degradation or inflammatory reactions. METHODS:Human aortic tissue is harvested from the ascending aorta in the operation room and worked up to pure human myofibroblasts cultures. These human myofibroblasts cultures are suspended in fibrinogen solution and seeded into 6-well culture plates for cell development for 4 weeks and supplemented with different concentrations of aprotinin. Hydroxyproline assay and histological studies were performed to evaluate the tissue development in these fibrin gel structures. RESULTS:The light microscopy and the transmission electron microscopy studies for tissue development based on the three-dimensional fibrin gel structures showed homogenous cell growth and confluent collagen production. No toxic degradation or inflammatory reactions could be detected. Furthermore, fibrin gel myofibroblasts structures dissolved within 2 days in medium without aprotinin, but medium supplemented with higher concentration of aprotinin retained the three-dimensional structure and had a higher collagen content (P<0.005) and a better tissue development. CONCLUSIONS:A three-dimensional fibrin gel structure can serve as a useful scaffold for tissue engineering with controlled degradation, excellent seeding effects and good tissue development.

The goal of this work was to develop a growth factor delivery system for use in wound healing that would provide localized release of heparin-binding growth factors in a biomimetic manner, such that release occurs primarily in response to cell-associated enzymatic activity during healing. A key element of the drug delivery system was a bi-domain peptide with an N-terminal transglutaminase substrate and a C-terminal heparin-binding domain, based on antithrombin III. The bi-domain peptide was covalently cross-linked to fibrin matrices during coagulation by the transglutaminase activity of factor XIIIa and served to immobilize heparin electrostatically to the matrix, which in turn immobilized the heparin-binding growth factor and slowed its passive release from the matrix. Basic fibroblast growth factor (bFGF) was considered as an example of a heparin-binding growth factor, and cell culture experimentation was performed in the context of peripheral nerve regeneration. A mathematical model was developed to determine the conditions where passive release of bFGF would be slow, such that active release could dominate. These conditions were tested in an assay of neurite extension from dorsal root ganglia to determine the ability of the delivery system to release bioactive growth factor in response to cell-mediated processes. The results demonstrated that bFGF, immobilized within fibrin containing a 500-fold molar excess of immobilized heparin relative to bFGF, enhanced neurite extension by up to about 100% relative to unmodified fibrin. A variety of control experiments demonstrate that all components of the release system are necessary and that the bi-domain peptide must be covalently bound to the fibrin matrix. The results thus suggest that these matrices could serve as therapeutic materials to enhance peripheral nerve regeneration through nerve guide tubes and may have more general usefulness in tissue engineering.