Diane Marinho, Cláudia Borowsky, Carlos Galia Ophthalmology Department - Hospital de Clínicas de Porto Alegre Federal University of Rio Grande do Sul - Porto Alegre - Brazil The authors have no financial interest in the subject matter of this poster CRYOPRESERVED vs FREEZE-DRIED AMNIOTIC MEMBRANE TRANSPLANTATION FOR CORNEAL EPITHELIAL DEFECTS IN RABBITS

Purpose The cryopreserved amniotic membrane (AM) is currently the most widely used in ophthalmological surgeries, and many papers have already been published on the use of this tissue 1,2,3,4. Few studies in the literature have used lyophilized AM, but all of them showed encouraging results 5,6. To compare the effectiveness of cryopreserved and freeze-dried (lyophilized) amniotic membrane transplantation in corneal epithelial defects in rabbits.

Methods Study design: Interventional experimental study. 26 eyes of 26 rabbits were submitted to a 7mm diameter epithelial defect and subsequently covered with human AM fixed with fibrin glue. The animals were randomized into 2 groups: – Group 1 received cryopreserved AM (13 eyes) – Group 2 received sterilized lyophilized AM (13 eyes)

The following criteria were evaluated: corneal reepithelialization rate of corneal reepithelialization by serial photographs on the 1st, 4th and 7th days postoperatively. Methods Reepithelialized area After the animals were sacrificed: anatomopathological exam of the corneas immunohistochemical analysis of the corneas Comparative scanning electron microscopy between cryopreserved and freeze-dried AM

Results Corneal reepithelialization rate Corneal reepithelialization rate Group 1 (cryopreserved AM): all 13 rabbits had their corneas completely re- epithelialized on the last day of postoperative examination Group 2 (lyophilized AM): 12 rabbits had their corneas completely re- epithelialized on the last day of postoperative examination Deepithelialized area marked with an AutoCAD program for calculation - rabbit 6 from Group 2 on the first (A),fourth (B) and seventh (C) days postoperatively A BC

corneal reepithelialization In each group the percentage of corneal reepithelialization was significantly higher between the 1st and 7th day postoperatively (p<0.001). There was no statistically significant difference between the groups (p=0.867), nor between the postoperative days when analyzed against each other – mean of the three moments (p=0.726). Results Percentual of healing First dayFourth daySeventh day

No significant difference between autolysis and epithelial vacuolization among the groups (p=0.064 and p=0.204, respectively). As to basement membrane integrity, when seen with PAS, all the samples had a preserved basement membrane Results of HE and PAS anatomopathological exams Rabbit 3 Group 1 HE 400x Rabbit 12 Group 1 PAS 400x

Immunohistochemistry Cytokeratin – marker of epithelial cells present in the corneal epithelium. Vimentin - mesenchymal cells marker. These cells are usually not present in the corneal tissue and their presence may indicate an inflammatory reaction or fibroses because it induces conjunctivalization. no significant differences were found between the two groups in any of the criteria evaluated. cytokeratin + staining (brownish color) observed in the corneal epithelium of rabbit 6 from Group 1 Vimentin + staining (reddish color) observed in the region close to the conjunctiva, connective tissue and muscle fibers of the ciliary body in the corneas of rabbit 4 from Group 2

Absence of the amniotic membrane epithelium in both samples. The preservation of basement membrane, with a homogeneous and continuous characteristic, also occurred in both membranes (lyophilized and cryopreserved). The chorionic side showed a typical appearance of collagen fibers in the two membranes. Lyophilized membrane was thinner, on average 25 microns, while the cryopreserved one measured around 35 microns. Scanning electron microscopy

Chorionic surface of cryopreserved AM Stroma, measuring about 20 microns Basement membrane measuring about 4.5 microns Lateral view of lyophilized AM

Conclusion Freeze-dried AM is as effective as the cryopreserved AM in treating deepithelialized corneas in rabbits. Anatomopathological and immunohistochemical exams showed similar results in both groups. Lyophilization should be considered as an alternative method of AM preservation because has the advantage of easy storage for a longer period of time, and the safety of a sterilized biomaterial.

References 1. Fernandes M, Sangwan VS, Rao GN. Amniotic Membrane Transplantation for ocular surface reconstruction. Cornea 2005 Aug; 24(6): Lee SH, Tseng SCG. Amniotic membrane transplantation for persistent epithelial defects with ulceration. Am J Ophthalmol 1997;123: Burman S, Tejwani S, Vemuganti GK, Gopinathan U, Sangwan V. Ophthalmic applications of preserved human amniotic membrane: A review of current indications. Cell Tissue Bank 2004 Nov; 5: Dua HS, Gomes JAP, King AJ, Maharajan VS. The amniotic membrane in ophthalmology. Surv Ophthalmol 2004 Jan-Feb;49(1): Nakamura T, Yoshitani M, Rigby H, Fullwood NJ, Ito W, Inatomi T, Sotozono C, Shimizu Y, Kinoshita S. Sterilized, freeze-dried amniotic membrane: a useful substrate for ocular surface reconstruction. Invest Ophthalmol Vis Sci 2004;45: Libera RD, Melo GB, Lima AS, Haapalainen EF, Cristovam P, Gomes JAP. Assessmet of the use of cryopreserved x freeze-dried amniotic membrane for reconstrution of ocular surface in rabbit model. Arq Br Oftalmol 2008; 71(5):