The Distribution of TIMPs in Cryo–Preserved and Lyophilized Amniotic Membrane

Abstract

Abstract: : Purpose: To investigate the change of TIMPs in cryo–preserved amniotic membrane(AM) according to preservation time and to compare it with those in lyophilized AM Methods: 2 of each Fresh AM, cryo–preserved AM for 3,6,12 months, and 4 of lyophilized AM(AmniSite–Cornea®, Bioland, Korea) were used. Total RNA was extracted in amniotic epithelial cells harvested with dispase II or in stromal cells in each AM. The distribution of TIMP1and TIMP2 was evaluated using RNA–PCR kit (Shiga, Japan). TIMP1, TIMP2 were measured in a ratio of density of TIMP to density of betha actin using Image analyzer(Vilberlourmat) and densitometry(Tina 2.0) in each AM. Results: The density ratio of epithelial and stromal TIMP1 was 1.56, 1.51,1.59, 1.11 and 1.21, 3.74, 2.67, 2.01 in fresh AM, 3–months AM, 6–months AM, and 12 months–AM. The density ratio of epithelial and stromal TIMP2 was 0.55, 0.83, 0.80, 1.04 and 0.1, 0.57, 0.11, 0.47 in fresh AM, 3–months AM, 6–months AM, and 12 months–AM. The average density ratio of stromal TIMP 1 and TIMP2 in lyophilized–AM were 1.44 and 0.095, respectively. There was no statistically significant difference of TIMP 1 and 2 between in cryo–preserved and in lyophilized AM(p>0.05, Mann–Whitney U test) although TIMP 1 and 2 tended to be lower in lyophilized AM than those in cryo–preserved AM. Conclusions: Cryo–preservation time seemed not to effect on density of TIMP I and 2 till 1 year and the density of TIMPs might be not affected by cryo–preservation or lyophilization, suggesting that the anti–angiogenic effect of amniotic membrane may not be affected with preservation method or preservation time.

Keywords: cornea: basic science