Reconstruction of auto-tissue-engineered lamellar cornea by dynamic culture for transplantation: a rabbit model

Zheng Wu, Qiang Zhou, Haoyun Duan, Xiaoran Wang, Jianhui Xiao, Hucheng Duan, Naiyang Li, Chaoyang Li, Pengxia Wan, Ying Liu, Yiyue Song, Chenjing Zhou, Zheqian Huang, Zhichong Wang, Zheng Wu, Qiang Zhou, Haoyun Duan, Xiaoran Wang, Jianhui Xiao, Hucheng Duan, Naiyang Li, Chaoyang Li, Pengxia Wan, Ying Liu, Yiyue Song, Chenjing Zhou, Zheqian Huang, Zhichong Wang

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. The construction process of auto-tissue-engineered…
Figure 1. The construction process of auto-tissue-engineered lamellar cornea.
(A) Submersion culture (5 days). (B) Perfusion culture (3 days). (C) Dynamic air-liquid interface culture (with perfusion culture on the bottom surface, 3 days). (D) Dynamic air-liquid interface culture (with a dry chamber on the bottom surface, 12 hours). (E) Macroscopic view of ATELC-Dynamic in each stage of the construction process.
Figure 2. The major components of the…
Figure 2. The major components of the basement membrane in APCS (↓: basement membrane components, green: collagen IV, laminin, fibronectin and collagen VII, red: collagen I).
Figure 3. The changes in histomorphology and…
Figure 3. The changes in histomorphology and DNA content during the construction process (▴: connection between neighbor cells, *: cellular nucleus,↓: desmosome).
(A) ATELC-Dynamic, after submersion culture. (B) ATELC-Dynamic, after perfusion culture. (C) ATELC-Dynamic, after dynamic air-liquid interface culture. (D) Negative control: ATELC-Static. (E) Positive control: NLC. (F) DNA contents of the NLC, ATELC-Dynamic, and ATELC-Static groups.
Figure 4. The differentiation phenotype and adhesion…
Figure 4. The differentiation phenotype and adhesion property of the epithelium in ATELC-Dynamic.
(A) The expressions of K3, P63, ABCG2 proteins. (B) The expressions of zonula occludens-1, desmocollin-2 and integrin β4 proteins. (C) The ultrastructures of tight junction, desmosome junction and hemidesmosome junction. (D) ELISA assay of the zonula occludens-1, desmocollin-2 and integrin β4 proteins.
Figure 5. Physiological functions of ATELC-Dynamic in…
Figure 5. Physiological functions of ATELC-Dynamic in vitro.
(A) Light transmittance over the 300–800 nm range of light wavelengths. (B) The areal modulus variation between 0 and 50 mmHg pressure differences.
Figure 6. Physiological function of ATELC-Dynamic in…
Figure 6. Physiological function of ATELC-Dynamic in a rabbit lamellar keratoplasty model.
(A) Postoperative observation of lamellar keratoplasty. (B) The light transmittance of transplanted lamellar cornea over the wavelength range of 300–800 nm at 20 days. (C) DiO-labeled seeding cells at 7 days, and the expression of collagen III at 20 days.

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