Preservation Of Amniotic Membrane Research Articles

Ulinastatin Supplementation During Human Amniotic Membrane Preservation to Improve its Viability

ABSTRACTPurpose: The amniotic membrane (AM) is the transparent innermost layer of the placenta and it facilitates rapid wound healing in a diversity of ocular surface disorders. However, extended periods of cryopreservation before use induce significant impairment of cell viability due to oxidative stresses and inflammatory responses. We investigated the effect of supplementing ulinastatin (ULI), a known serine protease inhibitor, and relevant mechanisms of action in AM preservation solution through the hypothermic continuum on inflammatory and apoptotic signals and viability of AM tissue.Materials and methods: The expression of inflammatory signal factors, including high mobility group box 1 (HMGB1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and anti-TNF-inducible gene 6 (TSG-6) which is a TNF-α-inducible anti-inflammatory protein, and the expression of apoptotic signal factors, including caspase (Cas)-9 and Cas-8, the initiators, and Cas-3, the executioner caspase and Bax were analyzed with or without ULI during hypothermic preservation of human AM. Subsequently, the actual viability of human AM tissue was verified with or without ULI supplementation throughout hypothermic continuum (both hypothermic- and cryopreservation).Results: Hypothermic AM preservation with ULI for 48 h resulted in downregulated expression of cold-inducible inflammatory factors, including HMGB1 and NF-κB, as well as RIPK3. In addition, ULI suppressed apoptotic signals related with Cas-9, Cas-8, and Cas-3 under hypothermic conditions. Furthermore, ULI supplementation during hypothermic- and cryopreservation of AM significantly enhanced viability of AM tissue and amniotic epithelial cells.Conclusions: Supplementation of ULI during human AM preservation through the hypothermic continuum may be a feasible dual anti-inflammatory and anti-apoptotic strategy that enhances the viability of AM tissue.

Transplantation of Preserved Human Amniotic Membrane for Surface Reconstruction in Severely Damaged Rabbit Corneas

After n-heptanol removal of the total corneal epithelium and a limbal lamellar keratectomy, 23 rabbit eyes developed features of limbal stem cell deficiency including conjunctival epithelial ingrowth, vascularization and chronic inflammation. One month later, 10 control eyes received a total keratectomy, and 13 experimental eyes received additional transplantation of glycerin-preserved human amniotic membrane. In 3 months of follow-up, all control corneas were revascularized to the center with granuloma and retained a conjunctival epithelial phenotype. In contrast, five corneas in the experimental group became clear with either minimal or no vascularization; the rest had either mid peripheral (n = 5) or total (n = 3) vascularization and cloudier stroma. The success of corneal surface reconstruction correlated with the return of a cornea-like epithelial phenotype and the preservation of amniotic membrane, whereas the failure maintained a conjunctival epithelial phenotype and the amniotic membrane was either partially degraded or covered by host fibrovascular stroma. These results suggest that measures taken to facilitate epithelialization without allowing host fibrovascular ingrowth onto the amniotic membrane might prove this procedure clinically useful for ocular surface reconstruction.