Cellular microenvironments are important in coaxing cells to behave collectively as functional, structured
tissues. Important cues in this microenvironment are the chemical, mechanical and spatial
arrangement of the supporting matrix in the extracellular space. In engineered tissues, synthetic scaffolding
provides many of these microenvironmental cues. Key requirements are that synthetic scaffolds
should recapitulate the native three-dimensional (3D) hierarchical fibrillar structure, possess biomimetic
surface properties and demonstrate mechanical integrity, and in some tissues, anisotropy. Electrospinning
is a popular technique used to fabricate anisotropic nanofiber scaffolds. However, it suffers from
relatively low production rates and poor control of fiber alignment without substantial modifications to
the fiber collector mechanism. Additionally, many biomaterials are not amenable for fabrication via highvoltage
electrospinning methods. Hence, we reasoned that we could utilize rotary jet spinning (RJS) to
fabricate highly aligned hybrid protein-polymer with tunable chemical and physical properties. In this
study, we engineered highly aligned nanofiber constructs with robust fiber alignment from blends of the
proteins collagen and gelatin, and the polymer poly-ε-caprolactone via RJS and electrospinning. RJS-spun
fibers retain greater protein content on the surface and are also fabricated at a higher production rate
compared to those fabricated via electrospinning. We measured increased fiber diameter and viscosity,
and decreasing fiber alignment as protein content increased in RJS hybrid fibers. RJS nanofiber constructs
also demonstrate highly anisotropic mechanical properties mimicking several biological tissue types. We
demonstrate the bio-functionality of RJS scaffold fibers by testing their ability to support cell growth and
maturation with a variety of cell types. Our highly anisotropic RJS fibers are therefore able to support
cellular alignment, maturation and self-organization. The hybrid nanofiber constructs fabricated by RJS
therefore have the potential to be used as scaffold material for a wide variety of biological tissues and
organs, as an alternative to electrospinning