Biologically Engineered Protein-graft-Poly(Ethylene Glycol) Hydrogels: A Cell-Adhesive and Plasmin-Degradable Biosynthetic Material for Tissue Repair

The goal of the research presented in this dissertation was to create a biomimetic artificial material that exhibits functions of extracellular matrix relevant for improved nerve regeneration. To identify minimal factors necessary for neurite extension in a suitable model system, neural adhesion peptides were photoimmobilized on highly crosslinked poly(ethylene glycol)-based substrates that were otherwise non-adhesive. Neurons adhered in two-dimensional patterns for eleven hours, but no neurites extended. In contrast, human fibroblasts adhered and spread on regions with photoimmobilized RGDS oligopeptide, but not on RDGS peptide, suggesting that specific integrin-ligand binding accounted for fibroblast adhesion and spreading. To enable neurite extension and nerve regeneration in three dimensions, and to address the need for specifically cell adhesive and cell degradable materials for clinical applications in tissue repair in general, an artificial protein was recombinantly expressed and purified that consisted of a repeating amino acid sequence based on fibrinogen and antithrombin III. The artificial protein contained integrin-binding RGD sites, plasmin degradation sites, and heparin-binding sequences. Furthermore, the protein contained six cysteine residues as grafting sites for poly(ethylene glycol) diacrylate via Michael-type conjugate addition. The resulting protein-graft-poly(ethylene glycol)acrylates were crosslinked by photopolymerization to form hydrogels. Human fibroblasts attached to, invaded, and apparently proliferated in the artificial hydrogel matrices three-dimensionally. Fibroblast penetration was inhibited in a concentration-dependent manner by both soluble cyclo(RGDFV) peptide and aprotinin, a serine-protease inhibitor. Inhibition of fibroblast outgrowth by cyclic RGD peptide suggests that cellular integrins engaged in specific binding to RGD sites present in the artificial protein-graft-poly(ethylene glycol) hydrogels' protein core. Inhibition by aprotinin suggests that serine protease-mediated cleavage of the hydrogel matrix was the mode of cellular ingrowth. Although three-dimensional ingrowth of fibroblasts into protein-graft-poly(ethylene glycol) hydrogels occurred, only surface neurite outgrowth was observed from chick dorsal root ganglia. Neurite outgrowth depended on the concentration of matrix-bound heparin, suggesting that heparin was necessary to immobilize neuroactive adhesion- and/or growth factors in the hydrogels. Toward three-dimensional neurite outgrowth in protein-graft-poly(ethylene glycol) hydrogels, additional heparin-binding factors can be identified or designed for intentional immobilization in future experiments. Together, the above results show that specific biological functions can be harnessed by protein-graft-poly(ethylene glycol) hydrogels to serve as matrices for tissue repair and regeneration. In particular, the two design objectives, specific cell adhesion and degradability by cell-associated proteases, were fulfilled by the material. In the future, this and similar artificial protein-graft-poly(ethylene glycol) materials with varying protein elements for improved wound healing might serve as biosynthetic implant materials or wound dressings that degrade in synchrony with the formation of a variety of target tissues

Recombinant protein-co-PEG networks as cell-adhesive and proteolytically degradable hydrogel matrixes. Part II: biofunctional characteristics

We present here the biological performance in supporting tissue regeneration of hybrid hydrogels consisting of genetically engineered protein polymers that carry specific features of the natural extracellular matrix, cross-linked with reactive poly(ethylene glycol) (PEG). Specifically, the protein polymers contain the cell adhesion motif RGD, which mediates integrin receptor binding, and degradation sites for plasmin and matrix-metalloproteinases, both being proteases implicated in natural matrix remodeling. Biochemical assays as well as in vitro cell culture experiments confirmed the ability of these protein-PEG hydrogels to promote specific cellular adhesion and to exhibit degradability by the target enzymes. Cell culture experiments demonstrated that proteolytic sensitivity and suitable mechanical properties were critical for three-dimensional cell migration inside these synthetic matrixes. In vivo, protein-PEG matrixes were tested as a carrier of bone morphogenetic protein (rhBMP-2) to heal critical-sized defects in a rat calvarial defect model. The results underscore the importance of fine-tuning material properties of provisional therapeutic matrixes to induce cellular responses conducive to tissue repair. In particular, a lack of rhBMP or insufficient degradability of the protein-PEG matrix prevented healing of bone defects or remodeling and replacement of the artificial matrix. This work confirms the feasibility of attaining desired biological responses in vivo by engineering material properties through the design of single components at the molecular level. The combination of polymer science and recombinant DNA technology emerges as a powerful tool for the development of novel biomaterials