Acrylonitrile and Pullulan Based Nanofiber Mats as Easily Accessible Scaffolds for 3D Skin Cell Models Containing Primary Cells

Abstract

(1) Background: Three-dimensional (3D) collagen I-based skin models are commonly used in drug development and substance testing but have major drawbacks such as batch-to-batch variations and ethical concerns. Recently, synthetic nanofibrous scaffolds created by electrospinning have received increasing interest as potential alternatives due to their morphological similarities to native collagen fibrils in size and orientation. The overall objective of this proof-of-concept study was to demonstrate the suitability of two synthetic polymers in creating electrospun scaffolds for 3D skin cell models. (2) Methods: Electrospun nanofiber mats were produced with (i) poly(acrylonitrile-co-methyl acrylate) (P(AN-MA)) and (ii) a blend of pullulan (Pul), poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) (Pul/PVA/PAA) and characterized by scanning electron microscopy (SEM) and diffuse reflectance infrared Fourier transform (DRIFT) spectra. Primary skin fibroblasts and keratinocytes were seeded onto the nanofiber mats and analyzed for phenotypic characteristics (phalloidin staining), viability (Presto Blue HS assay), proliferation (Ki-67 staining), distribution (H/E staining), responsiveness to biological stimuli (qPCR), and formation of skin-like structures (H/E staining). (3) Results: P(AN-MA) mats were more loosely packed than the Pul/PVA/PAA mats, concomitant with larger fiber diameter (340 nm ± 120 nm vs. 250 nm ± 120 nm, p < 0.0001). After sterilization and exposure to cell culture media for 28 days, P(AN-MA) mats showed significant adsorption of fetal calf serum (FCS) from the media into the fibers (DRIFT spectra) and increased fiber diameter (590 nm ± 290 nm, p < 0.0001). Skin fibroblasts were viable over time on both nanofiber mats, but suitable cell infiltration only occurred in the P(AN-MA) nanofiber mats. On P(AN-MA) mats, fibroblasts showed their characteristic spindle-like shape, produced a dermis-like structure, and responded well to TGFβ stimulation, with a significant increase in the mRNA expression of PAI1, COL1A1, and αSMA (all p < 0.05). Primary keratinocytes seeded on top of the dermis equivalent proliferated and formed a stratified epidermis-like structure. (4) Conclusion: P(AN-MA) and Pul/PVA/PAA are both biocompatible materials suitable for nanofiber mat production. P(AN-MA) mats hold greater potential as future 3D skin models due to enhanced cell compatibility (i.e., adsorption of FCS proteins), cell infiltration (i.e., increased pore size due to swelling behavior), and cell phenotype preservation. Thus, our proof-of-concept study shows an easy and robust process of producing electrospun scaffolds for 3D skin cell models made of P(AN-MA) nanofibers without the need for bioactive molecule attachments.

Review on applications of Pullulan in bone tissue engineering: Blends and composites with natural and synthetic polymers

“…This study focuses on combining different PUL and collagen molecular weights to create injectable hydrogels for skin healing using a novel method. 77,78 It was discovered that dialdehyde PUL and collagen are employed to create sophisticated scaffold materials.…”

Electrosprayed Core (Cellulose Acetate)–Shell (Polyvinylpyrrolidone) Nanoparticles for Smart Acetaminophen Delivery

“…One of the popular reasons for performing electrospinning is its ability to prepare core–shell [ 43 , 44 , 45 , 46 , 47 ], Janus [ 48 , 49 ], tri-layer core–shell [ 50 ], and tri-layer Janus nanofibers [ 51 ]; their combined structures; and fiber–particle hybrids in a single step and a straightforward manner [ 52 , 53 , 54 , 55 , 56 ]. Some of these electrospun structures are included in Figure 1 a–d, and some of them have been explored with regard to providing biphasic drug release (e.g., fast release phase I and sustained release phase II), including core–shell nanofibers ( Figure 1 a), Janus nanofibers ( Figure 1 b), tri-layer core–shell nanofibers ( Figure 1 c), and drug-coating nanofibers ( Figure 1 d) [ 35 ].…”

“…However, determining the best way to organize the soluble and insoluble polymers in a suitable nanostructure poses a big challenge to the researchers. One of the popular reasons for performing electrospinning is its ability to prepare core-shell [43][44][45][46][47], Janus [48,49], tri-layer core-shell [50], and tri-layer Janus nanofibers [51]; their combined structures; and fiber-particle hybrids in a single step and a straightforward manner [52][53][54][55][56]. Some of these electrospun structures are included in Figure 1a- One of the popular reasons for performing electrospinning is its ability to prepare core-shell [43][44][45][46][47], Janus [48,49], tri-layer core-shell [50], and tri-layer Janus nanofibers [51]; their combined structures; and fiber-particle hybrids in a single step and a straightforward manner [52][53][54][55][56].…”

“…One of the popular reasons for performing electrospinning is its ability to prepare core-shell [43][44][45][46][47], Janus [48,49], tri-layer core-shell [50], and tri-layer Janus nanofibers [51]; their combined structures; and fiber-particle hybrids in a single step and a straightforward manner [52][53][54][55][56]. Some of these electrospun structures are included in Figure 1a- One of the popular reasons for performing electrospinning is its ability to prepare core-shell [43][44][45][46][47], Janus [48,49], tri-layer core-shell [50], and tri-layer Janus nanofibers [51]; their combined structures; and fiber-particle hybrids in a single step and a straightforward manner [52][53][54][55][56]. Some of these electrospun structures are included in Figure 1a-d, and some of them have been explored with regard to providing biphasic drug release (e.g., fast release phase I and sustained release phase II), including core-shell nanofibers (Figure 1a), Janus nanofibers (Figure 1b), tri-layer core-shell nanofibers (Figure 1c), and drug-coating nanofibers (Figure 1d) [35].…”