Cell adhesion to the extracellular matrix through cell-surface integrin receptors is essential to development, wound healing, and tissue remodeling and therefore represents a central theme in the design of bioactive surfaces that successfully interface with the body. This is especially significant in the areas of integrative implant coatings and tissue engineering, since adhesion triggers signals that regulate cell cycle progression and differentiation in multiple cellular systems.

This research project focuses on establishing a molecular strategy for engineering biomimetic surfaces that promote bone formation and osseointegration. The objective is to engineer bioactive hybrid surfaces that support osteoblastic differentiation and promote osseointegration by targeting specific integrin receptors that are critical to osteoblast function. The central hypothesis of this project is that the controlled presentation of type I collagen and fibronectin binding domains onto well-defined substrates will result in integrin-specific bioadhesive surfaces that support osteoblastic differentiation, matrix mineralization, and osseointegration.

We tested this central hypothesis by designing and characterizing a collagen-mimetic peptide that specifically targets the α₂β₁ integrin by incorporating the necessary primary, secondary, and tertiary conformations. The integrin α₂β₁ recognizes the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif in residues 502-507 of the α1[I] chain of type I collagen. Integrin recognition is entirely dependent on the triple-helical conformation of the ligand similar to that of native collagen. Our first study focuses on engineering α₂β₁-specific bioadhesive surfaces by immobilizing our triple-helical collagen-mimetic peptide incorporating the GFOGER binding sequence onto model non-adhesive substrates. Circular dichroism spectroscopy verified that this peptide adopts a stable triple-helical conformation in solution. Passively adsorbed GFOGER-peptide exhibited dose-dependent HT1080 cell adhesion and spreading comparable to that observed on type I collagen. Subsequent antibody blocking conditions verified the involvement of integrin α₂β₁ in these adhesion events. Focal adhesion formation was observed by immunofluorescent staining for α₂β₁ and vinculin on MC3T3-E1 cells. Model functionalized surfaces were then engineered using three complementary peptide tethering schemes. These peptide-functionalized substrates supported α₂β₁-mediated cell adhesion and focal adhesion assembly. Our results demonstrate that this peptide is active in an immobilized conformation and may be applied as a surface modification agent to promote α₂β₁–specific cell adhesion.

Several studies indicate that the α₂β₁ integrin interaction with type I collagen is a crucial signal for the induction of osteoblastic differentiation and matrix mineralization. Our next study demonstrates that the α₂β₁ integrin-specific GFOGER-peptide triggers the activation of focal adhesion kinase (FAK) and alkaline phosphatase in MC3T3-E1 murine immature osteoblast-like cells - two proteins that have been implicated in the osteoblastic differentiation pathway. These GFOGER-peptide surfaces also support the expression of multiple osteoblast-specific genes, including osteocalcin and bone sialoprotein, and induce calcification and matrix mineralization in a manner similar to type I collagen, suggesting that this triple-helical peptide represents a promising surface modification strategy for the design of collagen-mimetic bioadhesive surfaces that support osteoblastic differentiation and bone formation.

Implant osseointegration, defined as close bone apposition and functional fixation, is a prerequisite for clinical success in orthopaedic and dental applications, many of which are restricted by implant loosening.(Pilliar, 2005), (Anderson, 2001) Our strategy to improve osseointegration of titanium implants focuses on presenting the GFOGER collagen-mimetic peptide that triggers α₂β₁ cellular integrin receptor binding, which is crucial for bone mineral deposition. Titanium surfaces presenting integrin-specific GFOGER-peptide trigger osteoblastic differentiation in primary rat bone marrow stromal cells, including bone-specific gene expression, alkaline phosphatase activity, and mineral deposition, leading enhanced osteoblastic function compared to unmodified orthopaedic-grade titanium. Furthermore, this integrin-targeted surface coating significantly improved peri-implant bone regeneration and mechanical osseointegration compared to untreated titanium in a rat tibia cortical bone implant model. Faster integration of these GFOGER coated implants would result in sooner and more reliable loading in a clinical setting, improving device function and patient outcomes. This study establishes a simple, single-step biologically active implant coating that enhances bone repair and titanium implant integration for clinical orthopaedic and dental applications. The objective of our next study was to engineer bioactive hybrid surfaces that control cell function by mimicking integrin-ECM interactions. We target two specific integrins essential to differentiation in several cell systems – the type I collagen (COL-I) receptor α₂β₁ and the fibronectin (FN) receptor α5β1 – by tethering varying densities of a collagen-mimetic peptide and a recombinant fragment of FN onto non-adhesive supports.

The wide range of controlled mixed ligand densities generated by this process demonstrates the feasibility of generating integrin-specific hybrid surfaces. Results indicate increased cell adhesion and synergistic activation of FAK, which underscore the advantage of specifically targeting more than one integrin implicated in a particular signaling pathway and downstream cellular effect. Proliferation rate results confirm that the enhanced signaling effects of mixed ligand surfaces translate to downstream cellular responses. This study suggests that, instead of focusing on a single integrin-ligand interaction, in some cases it may be advantageous to consider the interplay of multiple integrins implicated in a desired cell response and their combined effect on downstream cellular signals.

Extracellular matrix proteins are also attractive biomimetic targets for functionalizing orthopaedic implant surfaces in order to promote healing, bone formation, and implant fixation. Again, we target two specific integrins essential to differentiation in osteoblast cells – the type I collagen (COL-I) receptor α₂β₁ and the fibronectin (FN) receptor α5β1 – using the GFOGER triple helical peptide and the recombinant FNIII7-10 fibronectin fragment. This final study compares the osseointegrative potential of these single-component integrin-specific peptides to a mixed surface treatment presenting both peptides. We also examine the efficacy of the biomimetic integrin-targeted peptides compared to their native matrix proteins as implant coating treatments.

The in vivo results indicate that either of the integrin-targeted peptide treatments is sufficient to improve bone formation and implant mechanical integration compared to unmodified titanium. These biomimetic peptides also show improved osseointegration over the native matrix proteins, fibronectin and type I collagen. However, the combination treatment of both biomimetic peptides did not confer any osseointegrative advantage over the single-component coatings.

This thesis proposes a specific biomolecular strategy to engineer implant surfaces that enhance bone formation and osseointegration. We designed and evaluated a collagen mimetic peptide as an α₂β₁ integrin-specific surface modification agent for biomaterials, implant surface treatments, and tissue engineering scaffolds. This peptide was verified in the osteoblast cell model, but may be applied to several other cell systems that express α₂β₁, including platelets, epithelial cells, fibroblasts, chondrocytes, endothelial cells, and lymphocytes. We also established the extent to which the presentation of multiple integrin-binding ligands synergize to enhance intracellular signaling. This allows for the rational engineering of optimal biospecific surfaces for implant coatings and tissue engineering scaffolds. Finally, by analyzing the osseointegrative properties of these bioinspired materials, we have established the potential of this biomimetic ligand approach as a beneficial surface treatment for orthopaedic implants. As a whole, this project has established a targeted biomolecular surface engineering strategy for designing and optimizing biologically active implant coatings and grafting substrates that enhance implant bone repair.