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Joana Witt, Jana Dietrich, Gerd Geerling, Sonja Mertsch, Stefan Schrader, Kristina Spaniol; Tissue engineered conjunctival substitute on the basis of decellularized porcine conjunctiva. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6253.
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© ARVO (1962-2015); The Authors (2016-present)
Various biomaterials are currently in the focus of research to develop alternatives to the amniotic membrane for ocular surface reconstruction. We have recently shown that porcine decellularized conjunctiva (PDC) is a suitable matrix for the repair of conjunctival damages. Here, we compare two culture methods to produce a multilayered, stem cell rich epithelium on PDC that persists after transplantation into a conjunctival defect in a rabbit model.
For culture method A, human conjunctival epithelial cells (HCEC) were co-cultured with 3T3 cells for 10-12 days and seeded on PDC. For culture method B, a human conjunctival explant (HCEx) was cultured directly on PDC. After 7, 14 and 24 days, PDC were fixed for (immuno)-histological staining. After determining the ideal method and cultivation duration, epithelialized PDC was transplanted into a bulbar conjunctival defect in rabbits (n=6). PDC without cells were used as control (n=6). At day 0, 3 and 10, lissamine green staining was performed to assess epithelialization. After euthanasia at day 10, rabbit eyes were fixed for (immuno)-histological analysis.
The thickness of the epithelium on PDC was significantly higher after cultivation with method B in comparison to method A from day 14 on (all p values <0.0001). Immunohistological, higher count of epithelia cells expressing Ki67 and progenitor cell markers CK15, p63α and ABCG2 was seen using method B compared to A. MUC5AC positive cells were exclusively found using method B from day 14 on (1.0±0.15%). Rabbit conjunctivas grafted with PDC+HCEx showed no lissamine green staining at day 0, 3 or 10 whereas the control group showed a strong green staining of the graft at all time points. The engrafted PDC+HCEx showed 3.56±0.44% goblet cells and the count of p63α, CK15 and ABCG2 positive cells were not significant different to native conjunctiva (all p>0.05).
The direct cultivation of explant tissue on PDC resulted in a thicker and better stratified epithelium with higher preservation of progenitor cell properties compared to the conventional culture method A. We were able to generate a conjunctival graft which maintained a stable, multilayered epithelium with high preservation of stem cell properties and secretory capacity after 10 days in a conjunctival defect. Hence, PDC might offer an easily available carrier for HCEC in patients with extended conjunctival stem cell loss.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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