Collagen Nanoyarns: Hierarchical Three-Dimensional Biomaterial Constructs

Hierarchical fibrous scaffolds (HFS) consist of nanoscale fibers arranged in larger macroscale structures, much in the same pattern as in native tissue such as tendon and bone. Creation of continuous macroscale nanofiber yarns has been made possible using modified electrospinning set-ups that combine electrospinning with techniques such as twisting, drawing, and winding. In this paper, a modified electrospinning setup was used to create continuous yarns of twisted type I collagen nanofibers, also known as collagen nanoyarns (CNY), from collagen solution prepared in acetic acid. Fabricated CNYs were cross-linked and characterized using SEM imaging and mechanical testing, while denaturation of collagen and dissolution of the scaffolds were assessed using circular dichroism (CD) and UV-vis spectroscopy, respectively. HeLa cells were then cultured on the nanoyarns for 24 h to assess cell adhesion on the scaffolds. Scanning electron micrographs revealed a twisted nanofiber morphology with an average nanofiber diameter of 213 ± 60 nm and a yarn diameter of 372 ± 23 μm that shrank by 35% after covalent cross-linking. Structural denaturation assessment of native collagen using circular dichroism (CD) spectroscopy showed that 60% of the triple-helical collagen content in CNYs was retained. Cross-linking of CNYs significantly improved their mechanical properties as well as stability in buffered saline with no sign of degradation for 14 days. In addition, CNY strength and stiffness increased significantly with cross-linking although in the wet state, significant loss in these properties, with a corresponding increase in elasticity, was observed. HeLa cells cultured on cross-linked CNYs for 24 h adhered to the yarn surface and oriented along the nanofiber alignment axis, displaying the characteristic spindle-like morphology of cells grown on surfaces with aligned topography. Collectively, the results demonstrate the promising potential of collagen nanoyarns as a new class of shapable biomaterial scaffold and building block for generating macroscale fiber-based tissues.