Electrospun nanofibrous SF/P(LLA-CL) membrane: a potential substratum for endothelial keratoplasty

Abstract

Junzhao Chen,1,* Chenxi Yan,1,* Mengyu Zhu,1,* Qinke Yao,1 Chunyi Shao,1 Wenjuan Lu,1 Jing Wang,2 Xiumei Mo,2 Ping Gu,1 Yao Fu,1 Xianqun Fan1 1Department of Ophthalmology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 2Biomaterials and Tissue Engineering Laboratory, College of Chemistry and Chemical Engineering and Biotechnology, Donghua University, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: Cornea transplant technology has progressed markedly in recent decades, allowing surgeons to replace diseased corneal endothelium by a thin lamellar structure. A thin, transparent, biocompatible, tissue-engineered substratum with corneal endothelial cells for endothelial keratoplasty is currently of interest. Electrospinning a nanofibrous structure can simulate the extracellular matrix and have beneficial effects for cell culture. Silk fibroin (SF) has good biocompatibility but poor mechanical properties, while poly(L-lactic acid-co-Ɛ-caprolactone) (P(LLA-CL)) has good mechanical properties but poor biocompatibility. Blending SF with P(LLA-CL) can maintain the advantages of both these materials and overcome their disadvantages. Blended electrospun nanofibrous membranes may be suitable for regeneration of the corneal endothelium. The aim of this study was to produce a tissue-engineered construct suitable for endothelial keratoplasty.Methods: Five scaffolds containing different SF:P(LLA-CL) blended ratios (100:0, 75:25, 50:50, 25:75, 0:100) were manufactured. A human corneal endothelial (B4G12) cell line was cultured on the membranes. Light transmission, speed of cell adherence, cell viability (live-dead test), cell proliferation (Ki-67, BrdU staining), and cell monolayer formation were detected on membranes with the different blended ratios, and expression of some functional genes was also detected by real-time polymerase chain reaction.Results: Different blended ratios of scaffolds had different light transmittance properties. The 25:75 blended ratio membrane had the best transmittance among these scaffolds. All electrospun nanofibrous membranes showed improved speed of cell adherence when compared with the control group, especially when the P(LLA-CL) ratio increased. The 25:75 blended ratio membranes also had the highest cell proliferation. B4G12 cells could form a monolayer on all scaffolds, and most functional genes were also stably expressed on all scaffolds. Only two genes showed changes in expression.Conclusion: All blended ratios of SF:P(LLA-CL) scaffolds were evaluated and showed good biocompatibility for cell adherence and monolayer formation. Among them, the 25:75 blended ratio SF:P(LLA-CL) scaffold had the best transmittance and the highest cell proliferation. These attributes further the potential application of the SF:P(LLA-CL) scaffold for corneal endothelial transplantation. Keywords: silk fibroin, poly(L-lactic acid-co-Ɛ-caprolactone), B4G12, corneal endothelium, regeneration