Enzymatic incorporation of bidomain peptides into fibrin matrices for directed enhancement of three-dimensional in vitro neurite outgrowth and in vivo nerve regeneration

Citation

Schense, Jason Charles (1999) Enzymatic incorporation of bidomain peptides into fibrin matrices for directed enhancement of three-dimensional in vitro neurite outgrowth and in vivo nerve regeneration. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/2q3f-vw87. https://resolver.caltech.edu/CaltechETD:etd-02242008-092332

Abstract

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In this research, fibrin has been enzymatically modified using the transglutaminase, factor XIIIa, to incorporate bioactive domains from extracellular matrix and cell surface proteins. Fibrin was chosen as a base matrix as it is sensitive to cell-derived and cell-regulated protease activity. In order to improve the bioactive character of the fibrin, bi-domain peptides were designed where one domain contained a bioactive sequence and the other contained a factor XIIIa substrate sequence. These factor XIIIa substrates were derived from fibrinogen, [...]-plasmin inhibitor and a nonbiological substrate. Each of these peptides were then covalently incorporated into the fibrin during coagulation through the action of the enzyme, factor XIIIa, with the sequence from [...]-plasmin inhibitor incorporating at levels up to 8.2 mol peptide/mol fibrinogen.

Initially, these enzymatically modified fibrin gels were utilized in an academic study to probe the mechanisms involved in RGD-mediated three-dimensional cell migration. Two separate RGD sequences, one linear and one cyclic, were individually incorporated into fibrin matrices and the effect on neurite migration was measured, and it was shown that both RGD peptides have an adhesive-like, biphasic effect on cell migration. The density of peptide corresponding to maximal neurite outgrowth was lower for the cyclic than the linear peptide. However, since the cyclic RGD is a stronger binding sequence, it is likely that these two peptide densities represent a similar adhesive quality.

Development of enzymatically-modified fibrin matrices was also specifically directed towards enhancement of peripheral nerve regeneration. Peptides were individually incorporated in a concentration series and it was shown that these peptides, including RGD, HAV, IKVAV, YIGSR or RNIAEIIKDI, could improve neurite outgrowth by approximately 20%. A series of formulations were then tested, whereby multiple bioactive peptides were co-cross-linked into fibrin gels. One formulation, which contains the four laminin-derived sequences, RGD, IKVAV, YIGSR and RNIAEIIKDI, incorporated in equimolar densities of 1.7 mol/mol proved to enhance neurite outgrowth by 75% over unmodified fibrin. This formulation was then tested as a filler material for growth guides in peripheral nerve repair. Through these experiments, it was demonstrated that enzymatically modified fibrin is both nontoxic and capable of enhancing nerve regeneration.

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