Abstract
To construct an auto-tissue-engineered lamellar cornea (ATELC) for transplantation, based on acellular porcine corneal stroma and autologous corneal limbal explants, a dynamic culture process, which composed of a submersion culture, a perfusion culture and a dynamic air-liquid interface culture, was performed using appropriate parameters. The results showed that the ATELC-Dynamic possessed histological structure and DNA content that were similar to native lamellar cornea (NLC, p>0.05). Compared to NLC, the protein contents of zonula occludens-1, desmocollin-2 and integrin β4 in ATELC-Dynamic reached 93%, 89% and 73%, respectively. The basal cells of ATELC-Dynamic showed a better differentiation phenotype (K3−, P63+, ABCG2+) compared with that of ATELC in static air-lift culture (ATELC-Static, K3+, P63−, ABCG2−). Accordingly, the cell-cloning efficiency of ATELC-Dynamic (9.72±3.5%) was significantly higher than that of ATELC-Static (2.13±1.46%, p<0.05). The levels of trans-epithelial electrical resistance, light transmittance and areal modulus variation in ATELC-Dynamic all reached those of NLC (p>0.05). Rabbit lamellar keratoplasty showed that the barrier function of ATELC-Dynamic was intact, and there were no signs of epithelial shedding or neovascularization. Furthermore, the ATELC-Dynamic group had similar optical properties and wound healing processes compared with the NLC group. Thus, the sequential dynamic culture process that was designed according to corneal physiological characteristics could successfully reconstruct an auto-lamellar cornea with favorable morphological characteristics and satisfactory physiological function.
Highlights
More than 10 million patients worldwide are in need of corneal transplants
It is unlikely that there will be a single ‘‘one-size-fits-all’’ corneal substitute for all indications [13]. When both the corneal epithelium and deep stroma were damaged simultaneously, a functional lamellar cornea properly constructed from corneal epithelial cells with thicker scaffold was required for treatment [14]
Static air-liquid interface culture, which was first introduced by Minami in 1993 [15], has been applied widely to induce epithelial differentiation during the corneal reconstruction process
Summary
More than 10 million patients worldwide are in need of corneal transplants. the shortage of corneal donors means that 1.5–2.0 million patients annually have untreated corneal blindness [1]. It is unlikely that there will be a single ‘‘one-size-fits-all’’ corneal substitute for all indications [13] When both the corneal epithelium and deep stroma were damaged simultaneously, a functional lamellar cornea properly constructed from corneal epithelial cells with thicker scaffold was required for treatment [14]. Static air-liquid interface culture, which was first introduced by Minami in 1993 [15], has been applied widely to induce epithelial differentiation during the corneal reconstruction process Using this process, 2–6 layers of stratified epithelium could be formed on different scaffolds [15,16,17,18,19,20,21,22], adhesive molecules and cell-cell junctions have been verified by immunofluorescence staining [17] and transmission electron microscopy [17,18,19]. The classical static culture method could promote epithelium growth and produce a favorable morphological outcome on amniotic membrane [23], compressed collagen [18], silk fibroin [24], corneal stromal lamella discs [25], and so on
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