CO2-expanded nanofiber scaffolds maintain activity of encapsulated bioactive materials and promote cellular infiltration and positive host response.

Traditional electrospun nanofiber membranes were incapable of promoting cellular infiltration due to its intrinsic property (e.g., dense structure and small pore size) limiting their use in tissue regeneration. Herein, we report a simple and novel approach for expanding traditional nanofiber membranes from two-dimensional to three-dimensional (3D) with controlled thickness and porosity via depressurization of subcritical CO2 fluid. The expanded 3D nanofiber scaffolds formed layered structures and simultaneously maintained the aligned nanotopographic cues. The 3D scaffolds also retained the fluorescent intensity of encapsulated coumarin 6 and the antibacterial activity of encapsulated antimicrobial peptide LL-37. In addition, the expanded 3D nanofiber scaffolds with arrayed holes can significantly promote cellular infiltration and neotissue formation after subcutaneous implantation compared to traditional nanofiber membranes. Such scaffolds also significantly increased the blood vessel formation and the ratio of M2/M1 macrophages after subcutaneous implantation for 2 and 4 weeks compared to traditional nanofiber membranes. Together, the presented method holds great potential in the fabrication of functional 3D nanofiber scaffolds for various applications including engineering 3D in vitro tissue models, antimicrobial wound dressing, and repairing/regenerating tissues in vivo. STATEMENT OF SIGNIFICANCE: Electrospun nanofibers have been widely used in regenerative medicine due to its biomimicry property. However, most of studies are limited to the use of 2D electrospun nanofiber membranes. To the best of our knowledge, this article is the first instance of the transformation of traditional electrospun nanofiber membranes from 2D to 3D via depressurization of subcritical CO2 fluid. This method eliminates many issues associated with previous approaches such as necessitating the use of aqueous solutions and chemical reactions, multiple-step process, loss of the activity of encapsulated biological molecules, and unable to expand electrospun nanofiber mats made of hydrophilic polymers. Results indicate that these CO2 expanded nanofiber scaffolds can maintain the activity of encapsulated biological molecules. Further, the CO2 expanded nanofiber scaffolds with arrayed holes can greatly promote cellular infiltration, neovascularization, and positive host response after subcutaneous implantation in rats. The current work is the first study elucidating such a simple and novel strategy for fabrication of 3D nanofiber scaffolds.