Feasibility of corneal epithelial transplantation with polyethylene glycol hydrogel membrane as a carrier for limbal stem cell deficiency

Authors: Guo Yiyuan,  Xian Huimin,  Shereen Tan,  Qiang Fu,  Jin Xin,  Mark Daniel,  Greg.G. Qiao,  Zhang Hong
DOI: 10.3760/cma.j.cn115989-20201125-00794
Published 2022-12-10
Cite as Chin J Exp Ophthalmol, 2022, 40(12): 1125-1133.

Abstract                              [View PDF] [Read Full Text]

Objective

To investigate whether polyethylene glycol hydrogel films (PHFs) can be used as a carrier for the expansion of corneal epithelial cells (CECs) in vitro and whether PHFs can be used in the treatment of limbal stem cell deficiency (LSCD).

Methods

Sebacoyl chloride, dihydroxyl PCL and glycerol ethoxylate were used to synthesize PHFs.The thickness, transmittance and mechanical tensile properties of PHFs were measured.Four clean-grade New Zealand white rabbits were selected to culture primary limbal epithelial cells.The expression of keratin marker AE1/AE3 and stem cell marker p63 in the cultured cells were observed under a fluorescence microscope.The cells were divided into negative control group cultured with common cell culture solution, positive control group cultured with cell culture solution containing 100 μmol/L H2O2, and PHFs+ CECs group lined with PHFs cultured with common cell culture solution for 24 hours.The proliferation and apoptosis of cells in the three groups were observed by MTT and TUNEL staining, respectively.Fifteen clean-grade New Zealand white rabbits were divided into control group, PHFs group and PHFs+ CECs group by random number table method, with 5 rabbits in each group.LSCD model was constructed in the three groups.The control group was not given any treatment after modeling.In PHFs group, empty PHFs were placed on the corneal surface of rabbits.In PHFs+ CECs group, tissue-engineered grafts constructed with CECs after passage implanted on PHFs were placed on the corneal surface of rabbits.The corneal defect area of rabbits was detected and scored by fluorescein sodium staining.The histological characteristics of rabbits corneal epithelium was observed by hematoxylin-eosin staining.The use and care of animals complied with Guide for the Care and Use of Laboratory Animals by the U. S.National Research Council.The experimental protocol was approved by the Research and Clinical Trial Ethics Committee of The First Affiliated Hospital of Harbin Medical University (No.2021006).

Results

The synthetic PHFs were with a thickness ≤150 μm, a tensile strength about 6 MPa, and a transmittance over than 99% in the range of 400-700 nm.Most of the cells from primary culture of limbal tissue were positive for AE1/AE3 and p63.MTT test results showed that the A490 value of PHFs+ CECs group, negative control group and positive control group was 0.59±0.01, 0.65±0.07 and 0.06±0.04, respectively, showing a statistically significant overall difference (F=12.25, P<0.05). The A490 values of PHFs+ CECs group and negative control group were significantly higher than that of positive control group, and the differences were statistically significant (both at P<0.05). TUNEL test results showed that there was a significant difference in the TUNEL-positive cell rate among the three groups (F=13.45, P<0.05), and the rates of TUNEL-positive cells in PHFs+ CECs group and negative control group were significantly lower than that in positive control group (both at P<0.05). Fluorescein sodium staining results showed that with the extension of postoperative period, the corneal fluorescein sodium staining score of the three groups decreased, which decreased successively in control group, PHFs group and PHFs+ CECs group.Hematoxylin-eosin staining showed fewer irregularly shaped corneal epithelial cells in the control group, and sparse single layer of corneal epithelial cells in some areas of the PHFs group.In PHFs+ CECs group, the corneal epithelium coverage was the largest, and the cell layers increased to 3-5, and the cells were with regular morphology and in close arrangement.

Conclusions

PHFs have enough toughness, high transmittance and can expand corneal epithelium in vitro.PHFs are suitable for corneal epithelial transplantation and can promote the repair of corneal epithelium in rabbit model of LSCD.

Key words:

Epithelial cells, corneal; Limbal stem cell deficiency; Polyethylene glycol hydrogel film; Corneal epithelial cell regeneration; Corneal epithelial grafts

Contributor Information

Guo Yiyuan

Department of Ophthalmology, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China

Xian Huimin

Department of Cardiology, The 2nd Affiliated Hospital of Harbin Medical University, Harbin 150001, China

Shereen Tan

Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3001, Australia

Qiang Fu

Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3001, Australia

The School of Civil and Environmental Engineering, University of Technology Sydney, Sydney 2000, Australia

Jin Xin

Department of Ophthalmology, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China

Mark Daniel

Centre for Eye Research Australia (CERA), Royal Victorian Eye &

Ear Hospital, Melbourne 3002, Australia

Greg.G. Qiao

Department of Cardiology, The 2nd Affiliated Hospital of Harbin Medical University, Harbin 150001, China

Zhang Hong

Department of Ophthalmology, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China

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