Evaluation of an Improved Chitosan Scaffold Cross-Linked With Polyvinyl Alcohol and Amine Coupling Through 1-Ethyl-3-(3-Dimethyl Aminopropyl)-Carbodiimide (EDC) and 2 N-Hydroxysuccinimide (NHS) for Corneal Applications

Abstract

Background: Corneal blindness resulting from various medical conditions affects millions worldwide. The rapid developing tissue engineering field offers design of a scaffold with mechanical properties and transparency similar to that of the natural cornea.

Aim: The present study aimed at to prepare and investigate the properties of PVA/chitosan blended scaffold by further cross-linking with 1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) and 2 N-Hydroxysuccinimide (NHS) as potential in vitro carrier for human limbal stem cells delivery.

Material and methods: Acetic acid dissolved chitosan was added to PVA solution, uniformly mixed with a homogenizer until the mixture was in a colloidal state, followed by H₂SO₄ and formaldehyde added and the sample was allowed to cool, subsequently it was poured into a tube and heated in an oven at 60°C for 50 minutes. Finally, samples were soaked in a cross-linking bath with EDC, NHS and NaOH in H₂O/EtOH for 24 h consecutively stirred to cross-link the polymeric chains, reduce degradation. After soaking in the bath, the samples were carefully washed with 2% glycine aqueous solution several times to remove the remaining amount of cross-linkers, followed by washed with water to remove residual agents. Later the cross-linked scaffold subjected for various characterization and biological experiments.

Results: After viscosity measurement, the scaffold was observed by Fourier transform infrared (FT-IR). The water absorbency of PVA/Chitosan was increased 361% by swelling. Compression testing demonstrated that by increasing the amount of chitosan, the strength of the scaffold could be increased to 16×10-1 MPa. Our degradation results revealed by mass loss using equation shows that scaffold degraded gradually imply slow degradation. In vitro tests showed good cell proliferation and growth in the scaffold. Our assay results confirmed that the membrane could increase the cells adhesion and growth on the substrate.

Conclusion: Hence, we strongly believe the use of this improved PVA/chitosan scaffold has potential to cut down the disadvantages of the human amniotic membrane (HAM) for corneal epithelium in ocular surface surgery and greater mechanical strength in future after successful experimentation with clinical trials.

Keywords: Biocompatibility; Chitosan; Corneal cells; PVA; Poly (vinyl alcohol); Tissue engineering.

Figure 1

FTIR spectra of the PVA/chitosan cross-linked (a) and HAM scaffolds (b)

Figure 2

Swelling as a function of PVA and chitosan scaffold at various composition percentages (%) over the period (1-24 h). Graphical data are presented as mean (n = 3) ± standard deviation of three independent experiments

Figure 3

Measurement rate of breakdown of PVA/chitosan scaffolds. Dry weight ratio or degradation behaviour (%) of PVA and chitosan/PVA hydrogels was measured using PBS at different time points. Graphical data are presented as mean (n = 3) ± standard deviation of three independent experiments

Figure 4

H&E staining of cultured HCEC in HAM and PVA/Chitosan scaffolds

Figure 5

Semi-quantitative RT-PCR for SC-associated markers ABCG2 (379 bp), differentiation-associated markers, K3 (145 bp), and connexin 43 (154 bp) expressed by corneal epithelial cells (a, b, and c); A 100 bp DNA ladder is shown in the first left lane. GAPDH, a housekeeping gene, was used as an internal control (d)

Figure 6

PCR amplification for AMPs expression by human corneal epithelial cells. Semi-quantitative RT-PCR for AMP-associated markers hBD1 (215 bp), hBD2 (204 bp) and hBD3 (205 bp) expressed by corneal epithelial cells (a, b, and c); A 100 bp DNA ladder is shown in the first left lane. GAPDH, a housekeeping gene, was used as an internal control (d)