(251f) Covalent Conjugation of Transforming Growth Factor-beta1 to Fibrin Hydrogel for Tissue Engineering

Transforming Growth Factor-beta1 (TGF-beta1) is a multifunctional cytokine that participates in various physiological processes. Previous work showed that covalent conjugation of TGF-beta1 onto cell seeded tissue engineered scaffolds, either natural or synthetic, increased extracellular matrix (ECM) protein deposition and improved the mechanical properties of tissue constructs. Our present work aimed at incorporating TGF-beta1 into fibrin hydrogels for vascular tissue engineering. Previous studies showed that controlled release cannot be achieved by non-covalent incorporation into the hydrogels as approximately 50% of TGF-beta1 diffused out of the hydrogel within one day. In the present work, we developed a method to covalently conjugate mature TGF-beta1 into fibrin gel during polymerization. To this end, we engineered a fusion protein between a peptide that is recognized by Factor XIII and the TGF-beta1 sequence, termed as pTGF-beta. First, we optimized the position with the TGF-beta1 sequence where the peptide could be inserted without affecting the structure of native and processing of TGF-beta1. The fusion protein was expressed in CHO cells and purified by affinity and size exclusion chromatography. Proliferation of lung epithelial cells and promoter activity assays showed that pTGF-beta1 had similar biological activity as the native protein. Using immunoprecipitation assays we showed that pTGF-beta1 bound to fibrinogen in a Factor XIII dose dependent manner and could be released by the action of plasmin. Interestingly, the immobilized pTGF-beta1 exhibited enhanced biological activity as shown by luciferase promoter activity measurements. When mesenchymal stem cells were embedded in hydrogels with immobilized pTGF-beta1, hydrogel compaction increased significantly, suggesting that pTGF-beta1 enhanced the force generating ability of cells in a 3D context. Our current efforts focus on engineering the vascular media with fibrin hydrogels that are decorated with pTGF-beta1 in an attempt to improve ECM synthesis and the mechanical properties of these constructs for cardiovascular tissue engineering applications.