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Updated: May 28, 2026
A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse
Published on: November 17, 2016
[Photochemical tissue bonding technique for repairing limbal stem cell deficiency]
1Department of Burn and Plastic Surgery, Third People's Hospital, Institute of Trauma, Shanghai Jiaotong University School of Medicine, Shanghai 201900, PR China.
Objective:
To investigate the feasibility of photochemical tissue bonding (PTB) technique in repairing limbal stem cell (LSC) deficiency and the effect on cornea wound healing.
Methods:
LSCs were isolated from limbus of New Zealand rabbits by tissue block culture method, and then the LSCs of 2nd passage were cultured on de-epithelialized human amniotic membrane (HAM) for 3 weeks to prepare the HAM/LSC grafts. The LSC deficiency models of the left eyes were established by 0.5 mol/L NaOH in 24 New Zealand female rabbits, aged 3-4 months and weighing 1.5-2.0 kg. HAM/LSC grafts were used to repair the cornea wounds by sutures (suture group, n = 12) or by PTB technique (PTB group, n = 12). The gross was observed including the corneal transparency, erythema, and new blood vessel formation after surgery. At 3 and 28 days, the inflammatory cytokine of interleukin 1beta (IL-1beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) were assayed by ELISA method; and the amount of new blood vessels were quantified by immunohistochemistry staining at 28 days.
Results:
All animals survived to the end of the experiment. At 3 days, there was no obvious difference in the corneal transparency between 2 groups; at 28 days, the corneal transparency of PTB group was higher than that of suture group, and new blood vessels decreased. HE staining showed that mass inflammatory cells infiltrated between graft and cornea basal layer at 3 days, and no new blood vessel formed. inflammatory cells infiltration significantly decreased at 28 days in PTB group; the amount of new blood vessels was (2.0 +/- 0.8)/HP in PTB group and was (6.3 +/- 1.3)/HP in suture group, showing significant difference (t = 7.966, P = 0.002). At 28 days, the concentrations of inflammatory cytokine of IL-1beta, IL-6, and TNF-alpha in suture group were significantly higher than those in PTB group (P < 0.05); however, no significant differences were observed between 2 groups at 3 days (P > 0.05).
Conclusion:
PTB technique can be used to fix HAM/LSC grafts, which can decrease inflammatory cell infiltration and new vessel formation, and improve the outcomes when compared with suture technique.
