Amniotic Membrane Extract Induces Limbal Stem Cell Proliferation and Accelerates Corneal Epithelium Healing
Objective: Corneal epithelium healing in corneal defects is due to limbal stem cells (LSCs) proliferation; so finding a drug for acceleration of these cells growth, is remarkable. In this study we are going to evaluate the effect of amniotic membrane extract (AME) on LSCs proliferation and finally corneal epithelium healing in rabbit model. Materials and Methods: The total protein concentration of AME was determined by Bradford assay. The quantitative amount of EGF, HGF, and KGF was detected by ELISA. Human LSCs, were cultured with different doses of AME (0.1-1 mg/ml) for 14 continues days to assess effective and cytotoxic dose. The LSC and corneal differentiation genes were assessed by Real Time PCR. 10 rabbit were selected and the corneal epithelium mechanically was removed in first day of experiment, then the left eye of each rabbit was treated with 1 mg/ml of AME. The right eye was as control group that were treated only with antibiotics. The Rabbits eyes were observed daily and scored by ophthalmologists. Finally after 2 weeks, 1 month and 3 months post exam were, biopsies from each eye were sent to Iranian Eye Bank for pathological investigation. Results: Our results determined that AME at concentration of 0.1-1mg/ml induced LSC proliferation (p≤0.05). Moreover cells grown in presence of 0.1and 1 mg/ml AME up-regulated ABCG2 and P63, which are LSCs markers and down- regulated K3, as corneal differentiation markers in compare with cells grown in presence of 10% fetal bovine serum (FBS) (p≤ 0.001). EGF because of having the lowest concentration variation compare to KGF and HGF was determined as the reliable marker for extract quality. Interestingly, in rabbits which were treated with AME, the time of epithelium healing decreased to 2-3 days with lower Edema, chemosis and injection in compare to rabbits in control group. No sign of pathologic abnormalities were observed in test and control groups in vivo. Conclusion: Our results suggest that amniotic membrane extract increase the LSCs proliferation without any differentiation and accelerate the corneal epithelium healing.