Artificial cornea preparation using collagen/chondroitin sulfate/fibroblast growth factor composite film / 中国组织工程研究

ABSTRACT

BACKGROUND:The traditional corneal scaffolds exhibit poor strength and biological compatibility. Little is reported on the artificial cornea prepared by collagen and chondroitin sulfate (CS), which consist of the natural corneal tissue. OBJECTIVE:To prepare the collagen/CS/fibroblast growth factor (FGF) composite artificial cornea with slow-release growth factor, high strength and light transmittance, as well as good biocompatibility. METHODS:Regenerated collagen films were prepared by 1％, 5％, 10％ collagen solutions using flow casting method, and the regenerated collagen film with the best bioactivity that was prepared by 5％ collagen solution was screened through a biomechanical test. Then, the CS/collagen composite film was achieved by cross-linking the CS (2, 20, 80 g/L) with collagen by using N-(3-Dimethylaminopropyl)- N'S-ethylcarbodimide hydrochloride-N-Hydroxysuccinimide. The composite film made of 20 g/L CS was confirmed to have the best transparency, which was used to be mixed with 5, 25, 50 mg/L FGF in PBS for 24 hours to prepare the collagen/CS/FGF composite films. ELISA method was used to detect the FGF level in the supernatant. Afterwards, corneal epithelial cells were co-cultured with regenerated collagen film, collagen/CS composite film and collagen/CS/FGF composite film, respectively. After 48 hours of co-culture, cell proliferation was detected by MTT method, based on which we could screen the optimal collagen/CS/FGF composite film. After co-culture with the collagen/CS/FGF composite film for 48 and 72 hours, cell morphology was observed by confocal microscope and scanning electron microscope, respectively. RESULTS AND CONCLUSION:The release amount of FGF from the composite films was dependent on the initial loading amount of FGF. Meanwhile, FGF released slowly from the three kinds of composite films, and the release amount was 11％, 23％, 30％ at 72 hours after culture, in accordance with the pharmacokinetic process. MTT findings indicated that the optimal loading concentration of FGF was 25 mg/L. Under the microscope, the collagen/CS/FGF composite film promoted the adhesion, growth and proliferation of corneal epithelial cells. To conclude, the collagen/CS/FGF composite film is expected to be an ideal scaffold material for artificial cornea preparation.