Electrospun poly(N-isopropyl acrylamide)/poly(caprolactone) fibers for the generation of anisotropic cell sheets

Abstract

Cell alignment in muscle, nervous tissue, and cartilage is requisite for proper tissue function; however, cell sheeting techniques using the thermosensitive polymer poly(N-isopropyl acrylamide) (PNIPAAm) can only produce anisotropic cell sheets with delicate and resource-intensive modifications. We hypothesized that electrospinning, a relatively simple and inexpensive technique to generate aligned polymer fibers, could be used to fabricate anisotropic PNIPAAm and poly(caprolactone) (PCL) blended surfaces that both support cell viability and permit cell sheet detachment via PNIPAAm dissolution. Aligned electrospun PNIPAAm/PCL fibers (0%, 25%, 50%, 75%, 90%, and 100% PNIPAAm) were electrospun and characterized. Fibers ranged in diameter from 1-3 μm, and all fibers had an orientation index greater than 0.65. Fourier transform infrared spectroscopy was used to confirm the relative content of PNIPAAm and PCL. For advancing water contact angle and mass loss studies, only high PNIPAAm-content fibers (75% and greater) exhibited, temperature-dependent properties like 100% PNIPAAm fibers, whereas 25% and 50% PNIPAAm fibers behaved similarly to PCL-only fibers. 3T3 fibroblasts seeded on all PNIPAAm/PCL fibers had high cell viability and spreading except for the 100% PNIPAAm fibers. Cell sheet detachment by incubation with cold medium was successful only for 90% PNIPAAm fibers, which had a sufficient amount of PCL to allow cell attachment and spreading but not enough to prevent detachment upon PNIPAAm dissolution. This study demonstrates the feasibility of using anisotropic electrospun PNIPAAm/PCL fibers to generate aligned cell sheets that can potentially better recapitulate anisotropic architecture to achieve proper tissue function.

Fig. 1

PNIPAAm/PCL fibers. (A) SEM images of electrospun PNIPAAm/PCL fibers. Percentage in upper left indicates PNIPAAm content. Scalebars (black bars, bottom left) are 10 μm. (B) Average fiber diameter determined using DiameterJ. (C) Fiber orientation index determined using OrientationJ. *p < 0.05 compared to 0% PNIPAAm fibers.

Fig. 2

Chemical structures of (A) PCL and (B) PNIPAAm. (C) FTIR spectra of PNIPAAm/PCL fibers. Dashed lines indicate absorption peaks.

Fig. 3

PNIPAAm dissolution from PNIPAAm/PCL fibers (A) PNIPAAm/PCL mass loss in water. PNIPAAm/PCL fiber (B) area percent change and (C) axes (relative to fiber direction) length percent change following PNIPAAm dissolution. # p < 0.05 compared to all other groups.

Fig. 4

(A) Advancing water contact angle on PNIPAAm/PCL fibers. (B) Representative images of water droplet on fibers. White dashed lines indicate fiber edge. * p < 0.05 compared to 0% PNIPAAm fibers. & p < 0.05 compared to 100% PNIPAAm fibers.

Fig. 5

Cell viability and cell alignment on PNIPAAm/PCL fibers. (A) Cell viability relative to 0% PNIPAAm fibers determined by MTS assay. * p < 0.05 compared to 0% PNIPAAm fibers. (B) Representative images of fibroblasts seeded on PNIPAAm/PCL fibers with actin (left) and actin/DAPI overlays (right). Scalebars are 200 μm. (C) Insets from 90% PNIPAAm images, as indicated by white dashed-box in (B). Scalebar is 50 μm. * p < 0.05 compared to 0% PNIPAAm fibers.

Fig. 6

Cell sheet detachment. (A) Cell sheets detached from 90% PNIPAAm fibers using room temperature medium; scalebar is 1 cm. (B, C) Cell sheet viability was confirmed with calcein, AM live-staining. (D, E) Corresponding phase contrast images of (B, C); white arrows indicate residual PCL. Scalebars are (B, D) 400 μm and (C, E) 200 μm.