From Design to Production: Applications of Polymer and Protein Nanofibers for Regenerating Tissue

Abstract

Designing wound dressings that restore cutaneous wounds to their original, healthy structure remains a clinical and engineering challenge. In the absence of external intervention, adult wound healing is an inherently slow and inefficient process. It often leads to scar formation and a stiffening in tissue mechanics due in part to the disruption of scaffolding proteins making up the extracellular matrix (ECM) of the skin. Within the ECM, the glycoprotein fibronectin (FN) is critical to maintaining the structural integrity and spatial organization of cells and tissues during wound healing. During development, fibrillar FN is highly expressed in fetal skin, a feature that also corresponds with scarless healing after injury. During aging, FN is broken down and replaced by fibrillar collagen. When adult skin is wounded, the regenerated tissue forms a dense collagen-rich matrix, or scar. We hypothesize engineered fibrillar FN nanofibers embedded within an injury site can improve wound healing rate and reduce scar formation in vivo. However, this is a challenging task because the currently available manufacturing techniques amenable to protein fiber engineering have limited production rates. Thus, to test this hypothesis we employ a new method of polymer nanofiber production using the Rotary Jet-Spinning (RJS) and define how this system can generate the precise control over the chemistry, fiber geometry, and network orientation of protein fibers that can be used to recapitulate the microscale architecture of native ECM. The RJS is an ideal system for this study. It consists of a perforated reservoir rotating at high speeds to propel a liquid, polymeric jet out of the reservoir orifice that stretches, dries and eventually solidifies to form nanoscale fibers. While it is known that the RJS enables the mass production of nanostructured fibers by centrifugal forces, methods to control topography and surface morphology of the formed fibers are unknown. In an effort to control fiber properties when building synthetic ECM, we developed a scaling framework complemented by a semi-analytic and numerical approach to characterize the regimes of nanofiber production, leading to a theoretical model for the fiber diameter consistent with experimental observations. In addition, our study yielded a phase diagram for the design of continuous nanofibers as a function of process parameters with implications for the morphological quality of fibers. Because ECM analogues should mimic geometry and surface topology of the native proteins, we also asked which parameters can be tuned during production to control fiber morphology. We developed and tested a mathematical model that describes how the competition between fluid instability and solvent removal in RJS regulates the degree of beading in fibers. The RJS was used to vary experimental parameters showing that fiber beading can be reduced by increasing solvent volatility, solution viscosity, and spinning velocity. After gaining a thorough understanding of the mechanisms necessary to control fiber topology, we hypothesized that RJS could also be used to control molecular orientation within fibers. We aim to create wound dressings composed of unfolded, polymerized FN nanofibers to be used to accelerate cutaneous wound healing and reduce scar formation in vivo. We describe a fluid dynamics model of shear induced fibrillogenesis using an RJS system to manufacture the FN nanofibers. The formed fibers are applied as dressings, which improve the quality of healing in a murine model of wound healing. Wounds healed with the FN dressings exhibited enhanced hair follicle and fat deposit regeneration, epidermal thinning, and a decrease in tissue stiffness, suggesting that these scaffolds effectively restore tissue structure and functional architecture. Collectively, our data suggests that FN-based fiber scaffolds may contribute to regeneration of a healthy, elastic skin tissue structure.