Electrospun Pectin-Polyhydroxybutyrate Nanofibers for Retinal Tissue Engineering

Abstract

Natural polysaccharide pectin has for the first time been grafted with polyhydroxybutyrate (PHB) via ring-opening polymerization of β-butyrolactone. This copolymer, pectin-polyhydroxybutyrate (pec-PHB), was blended with PHB in various proportions and electrospun to produce nanofibers that exhibited uniform and bead-free nanostructures, suggesting the miscibility of PHB and pec-PHB. These nanofiber blends exhibited reduced fiber diameters from 499 to 336-426 nm and water contact angles from 123.8 to 88.2° on incorporation of pec-PHB. They also displayed 39-335% enhancement of elongation at break relative to pristine PHB nanofibers. pec-PHB nanofibers were found to be noncytotoxic and biocompatible. Human retinal pigmented epithelium (ARPE-19) cells were seeded onto pristine PHB and pec-PHB nanofibers as scaffold and showed good proliferation. Higher proportions of pec-PHB (pec-PHB10 and pec-PHB20) yielded higher densities of cells with similar characteristics to normal RPE cells. We propose, therefore, that nanofibers of pec-PHB have significant potential as retinal tissue engineering scaffold materials.

Figure 1

Ring-opening polymerization of β-butyrolactone using pectin as initiator to yield pec-PHB.

Figure 2

FTIR spectra of the precursors (pectin and PHB) and copolymer pec-PHB.

Figure 3

SEM images (7500×) of the electrospun fibers (PHB, pec-PHB2, pec-PHB5, pec-PHB10, pec-PHB20) and their respective fiber diameters. Values plotted are means ± standard deviations, with three replicates taken per data point. ^abcd Values with different superscript letters are significantly different (p < 0.05).

Figure 4

Water contact angles of electrospun fibers: PHB, pec-PHB2, pec-PHB5, pec-PHB10, and pec-PHB20. Values plotted are means ± standard deviations, with three replicates taken per data point. ^abcd Values with different superscript letters are significantly different (p < 0.05).

Figure 5

DSC curves of the electrospun fibers: PHB, pec-PHB2, pec-PHB5, pec-PHB10, and pec-PHB20.

Figure 6

MTT assay indicated comparable cell attachment densities on all groups on days 1 and 3. Values plotted are means ± standard deviations, with three replicates taken per data point. One-way ANOVA (Tukey post hoc test) indicated that there was no significant difference between any groups.

Figure 7

(A) Immunofluorescence images of ARPE-19 cells on different PHB nanofiber scaffolds stained with rhodamine phalloidin (red) to label F-actin and 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei; the scale bar is 10 μm. (B) SEM images (330×) of proliferating ARPE-19 cells on PHB scaffolds 24 h after seeding.