Stabilized Chitosan Nanofibrous Membrane For Guided Tissue Engineering

Method: Nanofibrous membranes were prepared by electrospinning 5.5% wt/vol chitosan (71% DDA) in 70% trifluoroacetic acid-30% methylene chloride solution at 25 kV onto a non-stick aluminum foil target rotating at 8.4 rpm. Surface acylation reactions were performed in solutions contains 50(v/v)% pyridine and 50(v/v)% acetic anhydride or butyric anhydride for 1 h at 25 ^oC to stabilize the nanofibers. After reaction, the membranes were washed in distilled water to remove residue solvents and by-products. The stabilized membranes were characterized by immersion in PBS for stability, fourier transform infrared spectroscopy (FTIR) for acylation, and scanning electron microscope (SEM) for fiber morphology and diameter. Proliferation of osteoblast cells (SaOS-2) on membranes (n=5/membrane) was measured by the CellTitre Glow® (Promega) at 1, 3, and 5 days and compared to cast chitosan film controls. 

Result: SEM evaluation of the acyl-modified electrospun membranes revealed nanofiber diameters ~240 nm whereas the non-modified nanofibers were ~190 nm. Upon contacting with PBS, the untreated membranes dissolved within minutes due to the highly soluble trifluoroacetic acid – chitosan salt formed during electrospinning. Acylated treated membranes did not swell or dissolve after two weeks in PBS solution and retained nanofibrous structure in SEM images. FTIR results showed a strong carbonyl absorption peak at 1751 cm^-1, indicating successful acylation reactions. Osteoblasts proliferated by 82-102 % on both types of acylated membranes over the 5 day culture and there was no difference in growth between the acylated membranes or cast chitosan film controls. 

Conclusion: Our study identified surface acylation to be an effective strategy to stablize electrospun chitosan nanofibers. The acylated chitosan nanofibrous membranes were cytocompatible suggesting further investigation for guided tissue engineering.