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

Abstract

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.

Keywords: Drug delivery; Electrospun nanofiber membranes; Expansion; Regenerative medicine; Subcritical CO(2); Three dimensional.

Fig. 1

Expansion and characterization of aligned nanofiber scaffolds. (a) Photographs of aligned PCL nanofiber mats after the first treatment of subcritical CO2 fluid (left) and raw PCL nanofiber mats (right). (b) Photographs of aligned PCL nanofiber mats after the second treatment (left) and raw PCL nanofiber mats (right). (c) Thickness of aligned PCL fiber mats after expanding once and twice. (d) The corresponding porosities of aligned PCL fiber mats after expanding once and twice. (e–h) SEM images showing cross-sectional morphologies of aligned PCL fiber mats before (e, f) expansion and after expansion in subcritical CO₂ fluid two times (g, h). The scale bar is 20 μm.

Fig. 2

Photographs of poly(vinylpyrrolidone) (PVP) nanofiber mats before (a, b) and after (c, d) expansion in subcritical CO₂ fluid. Due to the high hydrophilicity of PVP nanofibers, expanded PVP membranes were kept in the capped tube to prevent dissolving from water condensed from the surrounding air (c). The expanded membrane was taken out after the temperature of samples reached the room temperature (d).

Fig. 3

Expansion of coumarin 6-loaded PCL nanofiber scaffolds. (a) Photographs showing NaBH₄ expanded PCL fiber mats with coumarin 6 loading (NaBH₄) and CO₂ expanded PCL fiber mats with coumarin 6 loading (CO₂), (b) Top view of CO₂ liquid expanded PCL fiber mats with coumarin 6 loading (top left), NaBH₄ expanded PCL fiber mats with coumarin 6 loading (bottom left), PCL fiber mats with coumarin 6 loading (top right) and raw PCL fiber mats (bottom right). Insets: fluorescent images of each sample. (c) The fluorescence intensity quantified by Image J software.

Fig. 4

Expansion of LL37-loaded PCL nanofiber scaffolds using CO₂ fluid. (a) The in vitro release kinetics of LL 37 from expanded and unexpanded PCL fiber samples (Initial drug loading: 5 μg/mg). (b) Antibacterial performance of different fiber samples. PCL: unexpanded pristine PCL nanofiber membranes. PCL-LL37: LL37-loaded PCL nanofiber scaffolds.

Fig. 5

In vivo response of expanded nanofiber scaffolds with arrayed holes and traditional nanofiber mats. (a) H & E staining. Green dots indicate the boundary of cell filtrated area. (b) Masson’s trichrome staining. Green arrows indicate collagen deposition. (c) Highly magnified images in (a). (d) Highly magnified images in (a). Green arrows indicate giant cells. (e) Quantification of blood vessel formation per mm². (f) Quantification of giant cells per implant. For comparision, the result for taditional nanofiber mat was adapted from our previous published studies [24].

Fig. 6

Immunohistological staining of 3D expanded nanofiber scaffolds with arrayed holes and surrounding tissues against CD68 - a surface marker for pan macrophages, CD 206 – a surface marker for macrophages in M2 phase, and CCR7 – a surface marker for macrophages in M1 phase. The nanofiber scaffolds were subcutaneously implanted to rats for 1 week, 2 weeks, and 4 weeks.

Fig. 7

Quantification of immunhistological analysis of 3D expanded nanofiber scaffolds with arrayed holes and traditional nanofiber mats after subcutaneous implantation. a) CD 68, b) CCR 7 (M1), c) CD 206 (M2) immunpositve cells and d) ratio of number of CD163 positive cells (M2)/number of CCR7 positive cells (M1). The values were obtained by measuring six scaning images at 40× (objective lense) magnification for each specimen. For comparision, the result for taditional nanofiber mat was adapted from our previous published studies [24].

Fig. 8

Multinucleated giant cells after subcutaneous implantation of 3D expanded nanofiber scaffolds with punched holes. The rats were scarified at week 1, 2, and 4 after surgery. The multinucleated giant cells were stained against CD68 - a surface marker for pan macrophages, CD 206 – a surface marker for macrophages in M2 phase, and CCR7 – a surface marker for macrophages in M1 phase. Green arrows indicate multinucleated giant cells.

Fig. 9

The proposed schematic illustrating the cell infiltration and spatiotemporal distributions of M1 macrophages (yellow color), M2 (blue color) macrophages (top panel) and multinucleated giant cells (bottom panel) on the surface of traditional nanofiber mats and within expanded 3D nanofiber scaffolds with arrayed holes after subcutaneous implantation. The cell-infiltrated area is labeled in red. For comparison, the schematic of tradition nanofiber mats was drawn based on previous published results [24].