Bioconjugate Chem. 2007, 18, 695−701
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Immobilization of Aprotinin to Fibrinogen as a Novel Method for Controlling Degradation of Fibrin Gels Jason D. Smith,†,‡ Andrew Chen,† Lauren A. Ernst,‡ Alan S. Waggoner,‡ and Phil G. Campbell*,†,‡ Institute for Complex Engineered Systems and Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania. Received August 29, 2006; Revised Manuscript Received January 23, 2007
The goal of this work was to demonstrate that aprotinin conjugated to fibrinogen could (1) maintain its function and (2) control fibrin degradation. Using the chick chorioallantoic membrane (CAM) assay, we found that blood vessels did not directly invade fibrin constructs containing immobilized fibroblast growth factor-2. Because the fibrin quickly degraded within approximately 5 days, we hypothesized that controlling fibrinolysis may improve direct blood vessel invasion. Aprotinin, a protease inhibitor typically added to slow fibrinolysis, is a small protein and can diffuse out of the gel resulting in the loss of fibrinolysis protection. Therefore, using a novel synthesis strategy, aprotinin and a fluorescent reporter, Cy3, were chemically conjugated to fibrinogen. In vitro microplate absorbance assays showed that the conjugated aprotinin was able to inhibit plasmin-mediated fibrin degradation and that its activity was comparable to equimolar levels of soluble, nonconjugated aprotinin. Additionally, we found that fibrinolysis rates could be tuned by varying the level of conjugated aprotinin within the gel. The conjugated aprotinin also demonstrated functionality in vivo. In the chick CAM assay, fibrin gels containing conjugated aprotinin were approximately 5 times larger than gels containing soluble aprotinin after 4 days. Also, in support of our hypothesis, we found that immobilized aprotinin within fibrin gels demonstrated substantial blood vessel invasion.
INTRODUCTION Our laboratory studies the use of fibrin-based constructs for bone tissue engineering applications. Fibrin is the primary provisional extracellular matrix (ECM) protein in wound healing (1). Fibrin supports cell adhesion and proliferation (2, 3), can bind a number of growth factors, such as fibroblast growth factor-2 (FGF-2), with high specificity (4, 5), and can be actively remodeled and degraded by cells (6-8). For these reasons, fibrin is an attractive scaffolding material for tissue engineering applications (9-17). One of our laboratory’s focus areas is to develop strategies to induce blood vessel ingrowth into such fibrin-based constructs for translation to tissue engineering applications. Our basic in vivo model is the chick chorioallantoic membrane (CAM) assay, a commonly used in vivo angiogenesis assay to study blood vessel development as well as the angiogenic nature of tissue engineered constructs (9, 10, 18-28). However, our initial experiments indicated that the fibrin was completely degraded at the construct/CAM interface as the CAM is invading the construct. Therefore, it appears that vascularization does not directly occur into the construct but is secondary to the invading CAM. Therefore, we asked the following question: Could direct angiogenic invasion be induced into the construct by controlling fibrin degradation? In vivo, fibrin degradation, or fibrinolysis, occurs primarily through the serine protease plasmin (8, 18, 19). Aprotinin is a small (58 amino acids), potent competitive inhibitor for the active site of plasmin and is typically added to commercial fibrin glues to slow fibrinolysis (7, 20-22). Because of its small size, * To whom correspondence should be addressed. Phil G. Campbell, Carnegie Mellon University, 5000 Forbes Ave., 1201 Hamburg Hall, Pittsburgh, PA 15213; E-mail:
[email protected]. † Institute for Complex Engineered Systems. ‡ Molecular Biosensor and Imaging Center.
however, aprotinin can freely diffuse out of the fibrin gel in vivo and is therefore no longer able to protect fibrin from degradation. To circumvent this problem, we engineered a fused construct, directly linking aprotinin to fibrinogen, to immobilize aprotinin within the construct. Fibrinolysis would still be plasminmediated (and thus cell-mediated), but conjugated inhibitor levels would control the degradation rate. In this work, we conjugated aprotinin and a Cy3 fluorescent dye to fibrinogen through the use of a cysteine-based trifunctional cross-linker. Aprotinin was chosen due to its small size (
