Introduction: Fibronectin (Fn) is an essential extracellular matrix protein that is assembled from its monomeric form in solution in the cytosol to an insoluble fibrous network as it is extruded by the cell into the extracellular space. This polymerization process is not well understood and difficult to recapitulate in a laboratory environment, even using alternate means such as electrospinning. Successful reconstitution methods for Fn fibers outside of the tissue environment rely on surface tension as a driving polymerization force[1],[2]. The limitations of this method are the length scale and quantity of Fn that can be obtained, such that scalability is low. In this work, we present a microfluidic technique that takes advantage of the polymerization method of silk fibroin (SF) in order to cross-link Fn into a continuous spin to improve fiber yields while not altering the biological effects of Fn.
Materials and Methods: A tiered microfluidic device (Figure 1) designed for the spinning of pure SF solutions was adapted by modifying the spinning solution[3]. Concentrated solutions of SF (>70 mg/ml) and Fn (>3 mg/ml) were mixed at various ratios to create a range of protein densities in extruded fibers. Flow through the microfluidic device induces elongation of protein chains leading to hydrogen bonding into hierarchical secondary structures. Extrusion into an organic solvent dehydrates the assembled protein fiber to finalize assembly of protein strands in a mature fiber. Fibers were tested for cellular interactivity and structural effects and compared to Fn[1] and SF properties[3], respectively.
Results and Discussion: Continuous extrusion was observed for fibers with mass ratios of Fn:SF ranging from 1:1600 to 1:10. Fiber diameters decreased proportionately from the low Fn:SF ratio (30-50 micron) to the high ratio (1-5 micron), shown in Figure 2. Fiber diameters of Fn fibers extruded using the surface tension methodologies range from hundreds of nanometers to 1-3 microns, demonstrating the shift from a predominantly silk-like fiber morphology to that closer resembling pure Fn fibers. Functional degrees of cellular adherence are observed in the spun fibers shown in Figure 3 even at the minimal level of Fn incorporation (1:1600, fluorescently labeled – red), which is not a capability of SF fibers alone. FTIR results demonstrate that the interaction of the SF and Fn solutions occurs at the secondary structure level. This is supported by the result obtained where SF solution was diluted to deprive it of sufficient protein strands to form physical cross-links and, therefore, no fiber. When Fn is added at minimal concentrations, protein fiber production resumes.
Conclusion: These results demonstrate that this method for spinning Fn fibers allows for the creation of a physically cross-linked fiber with composite properties that vary depending on the mixing ratio of the spinning solution. This method provides a means to create large quantities of Fn fibers using a simple microfluidic approach that previously could not be achieved with existing methods.
References:
[1] Klotzsch E, Smith ML, Kubow KE, Muntwyler S, Little WC, Beyeler F, et al. Fibronectin forms the most extensible biological fibers displaying switchable force-exposed cryptic binding sites. PNAS. 2009;106:18267-72.
[2] Feinberg AW, Parker KK. Surface-initiated assembly of protein nanofabrics. Nano letters. 2010;10:2184-91.
[3] Kinahan ME, Filippidi E, Koster S, Hu X, Evans HM, Pfohl T, et al. Tunable silk: using microfluidics to fabricate silk fibers with controllable properties. Biomacromolecules. 2011;12:1504-11.