Nanoscale modification of porous gelatin scaffolds with chondroitin sulfate for corneal stromal tissue engineering

Abstract

Recent studies reflect the importance of using naturally occurring biopolymers as three-dimensional corneal keratocyte scaffolds and suggest that the porous structure of gelatin materials may play an important role in controlling nutrient uptake. In the current study, the authors further consider the application of carbodiimide cross-linked porous gelatin as an alternative to collagen for corneal stromal tissue engineering. The authors developed corneal keratocyte scaffolds by nanoscale modification of porous gelatin materials with chondroitin sulfate (CS) using carbodiimide chemistry. Scanning electron microscopy/energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy showed that the amount of covalently incorporated polysaccharide was significantly increased when the CS concentration was increased from 0% to 1.25% (w/v). In addition, as demonstrated by dimethylmethylene blue assays, the CS content in these samples was in the range of 0.078–0.149 nmol per 10 mg scaffold. When compared with their counterparts without CS treatment, various CS-modified porous gelatin membranes exhibited higher levels of water content, light transmittance, and amount of permeated nutrients but possessed lower Young’s modulus and resistance against protease digestion. The hydrophilic and mechanical properties of scaffolds modified with 0.25% CS were comparable with those of native corneas. The samples from this group were biocompatible with the rabbit corneal keratocytes and showed enhanced proliferative and biosynthetic capacity of cultured cells. In summary, the authors found that the nanoscale-level modification has influence on the characteristics and cell-material interactions of CS-containing gelatin hydrogels. Porous membranes with a CS content of 0.112 ± 0.003 nmol per 10 mg scaffold may hold potential for use in corneal stromal tissue engineering.

Keywords:

• corneal keratocyte
• carbodiimide chemistry
• porous gelatin membranes
• stroma

Acknowledgments

This work was supported by grant NSC99-2221-E-182-008 from the National Science Council of the Republic of China and grant CMRPD190431 from Chang Gung Memorial Hospital.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary figures

Figure S1 Cross-linking index of porous gelatin scaffolds treated with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide as a function of cross-linking time.

Notes: An asterisk indicates statistically significant differences (*P < 0.05; n = 5) for the mean value of cross-linking index compared with the value at the previous time point.

Abbreviations: EDC, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride; NHS, N-hydroxysuccinimide.

Figure S2 Pore size of various porous gelatin scaffolds modified with chondroitin-4-sulfate.

Notes: Values are mean plus or minus standard deviation (n = 3); scaffold groups labeled according to chondroitin-4-sulfate concentration used (0%, 0.05%, 0.25%, or 1.25% (w/v)): GCS000, GCS005, GCS025, and GCS125.

Abbreviation: CS, chondroitin-4-sulfate.

Figure S3 Porosity of various porous gelatin scaffolds modified with chondroitin-4-sulfate.

Notes: Values are mean plus or minus standard deviation (n = 5); scaffold groups labeled according to chondroitin-4-sulfate concentration used (0%, 0.05%, 0.25%, or 1.25% (w/v)): GCS000, GCS005, GCS025, and GCS125.

Abbreviation: CS, chondroitin-4-sulfate.