The presentation of biological active factors on the surface of biomedical devices has emerged as a promising strategy to enhance host healing responses to implanted devices [6
]. Nonetheless, these biomimetic approaches have elicited marginal improvements in in vivo
functional performance [12
]. We hypothesized that these marginal healing responses result from uncontrolled signaling responses at the tissue-implant interface arising from unregulated or sub-optimal integrin binding. While model biomaterial surfaces to control ligand presentation (e.g., self-assembled monolayers on gold or silicon) have been extensively studied, the development of robust coating technologies for tunable presentation of bioactive factors on materials approved for biomedical implantation has been particularly challenging. Recent bioactive implant surface treatments on Ti, including porous hydroxyapatite, collagen I, and calcium phosphate co-precipitated with various other biological ligands, have augmented aspects of bone healing compared to unmodified Ti in various animal models [27
]. However, these strategies lack control over protein adsorption, ligand presentation and density, and surface stability. The utility of this brush system in this study is the level of precise control over bioactive ligand density and presentation and cellular integrin interaction in a physiological environment.
In the present work, we grafted clinical-grade titanium implants with a robust non-fouling polymer coating functionalized with controlled densities of ligands of varying integrin specificity to examine the role of integrin binding specificity on tissue responses to implanted devices. Our results demonstrate that conferring integrin binding specificity to biomedical implants regulates the osteoblastic differentiation of bone marrow-derived progenitor cells and significantly enhances in vivo bone healing and implant functional osseointegration. This work provides the first experimental demonstration that in vivo healing response can be finely tuned by engineering bioadhesive cues on synthetic biomedical material surfaces, and provides novel insights into the role of integrin receptors in directing specific signaling pathways and osteogenic cell functions. Importantly, this biomolecular strategy is based on surface engineering a robust non-fouling polymer coating on clinical-grade titanium, and therefore is applicable to existing biomedical implants. The integrin-specific biomaterial surfaces significantly enhanced in vivo implant integration and fixation compared to the current clinical standard (unmodified titanium) as well as biomimetic RGD-based surface treatments. This integrin-specific enhancement of functional integration may be potentially even greater since the FNIII7–10 surface density tethered to the polymer coating was below saturation. Given the central role of integrin receptors in the maintenance and repair of numerous tissues, we expect that this strategy of conveying integrin specificity will enhance healing responses and integration of other biomedical implanted devices.
We attribute the enhanced bone tissue formation and functional osseointegration of the α5
-specific titanium implants to increased recruitment of osteoprogenitor cells and differentiation into osteoblasts at the tissue-implant interface. As demonstrated in the in vitro
analyses (), α5
-mediated adhesion upregulated osteoblastic gene and protein expression and matrix mineralization in marrow-derived progenitor cells. The α5
integrin is the central fibronectin receptor, and its expression has been associated with increased mineralization of osteosarcoma and calvarial osteoblast cells [27
]. This study provides further evidence for directed α5
-mediated osteogenic differentiation as well as additional associated signaling through specific residues of FAK. In addition to cells directly interacting with the bioadhesive ligands on the implant surface, paracrine factors secreted at the tissue-interface could contribute to the pro-osteogenic healing response by recruiting additional osteoprogenitors and/or promoting osteoblastic differentiation in neighboring cells.
No differences in bone-implant contact or functional osseointegration were observed between RGD-tethered and unmodified titanium implants. We attribute the suboptimal host healing responses to these implants to reduced osteoblastic differentiation and bone formation. These two surfaces present adhesive ligands that primarily bind αV
. Osteogenic cell adhesion to the short synthetic RGD is primarily mediated by this integrin (). Similarly, cell adhesion to the unmodified titanium is mediated by RGD-containing proteins (e.g., fibrinogen, vitronectin [30
]) that adsorb non-specifically to titanium and support αV
-mediated adhesion. As demonstrated in the in vitro
analyses () and previous work (15), binding of αV
suppresses osteoblastic differentiation. Moreover, α v
-overexpressing osteoblasts exhibited impeded mineralization capacity due to suboptimal integrin-matrix interactions, JNK activity, and matrix protein expression [31
]. In addition to reduced osteoblastic differentiation, it is possible that presentation of αV
-selective ligands reduces overall bone formation by enhancing osteoclastic activity. Integrin αV
is a major component of podosomes in the sealing zones of resorptive pits [32
], and αV
antagonists reduce bone resorption by reducing osteoclast activity [33
]. However, we did not observe accumulation of multi-nucleated cells at the bone tissue-implant interface, suggesting that osteoclastic responses were not the dominant mechanism. Using the unique integrin-specific ligand presentation system in this study, we have obtained mechanistic insights into the functional roles of integrins in directing osteogenic behavior of stem-like stromal cells both in vitro
, and, for the first time, in vivo
A major contribution of the present study is the application of a stable non-fouling polymer coating that can be precisely engineered to present bioactive factors as a general strategy to convey biofunctionality to existing clinical devices. This approach is directly translatable to metal oxides and ceramic materials, and the tethering scheme is amenable to other protein ligands, including growth factors, antibodies, and enzymes, as well as aminated nucleic acids, carbohydrates, and lipids. Moreover, we have established the high physiological stability of this polymer brush system, affording long-term direct in vivo functional comparison of bioactive ligands. Consequently, we have been able to demonstrate in this study an integrin-specific mechanism for the regulation of progenitor cell differentiation into osteoblasts and in vivo enhancement of bone healing and implant osseointegration.