Purpose: To report a detailed surgical procedure of tissue engineered endothelial keratoplasty (TEEK) in a rabbit model and its postoperative evaluation. Methods: TEEKs were prepared 7 days before transplantation by seeding human or rabbit corneal endothelial cells on either femtosecond laser-cut ultrathin human stromal lamellae (fs-UTSL) or femtosecond laser-cut human anterior lens capsule (fs-HALC). Thirty transplantations were performed on aphakic eyes. Recombinant tissue plasminogen activator (rTPA) was used throughout the surgery. The native endothelium was removed by full-surface scraping and central descemetorhexis. The transplantation was performed as a human Descemet's membrane endothelial keratoplasty. Controls included Descemetorhexis only and transplantation of carrier alone. Postoperative follow-up was performed by slit lamp and optical coherence tomography, followed by histology. Results: Controls remained oedematous. No fibrin occurred during surgery. All but three TEEKs adhered immediately. One/6 fs-UTSL and 9/16 fs-HALC cleared perfectly (p = 0.161). All failures could be explained by at least one of the following causes intraoperative bleeding, vitreous prolapsus, early partial detachment, postoperative irido corneal synechiea/angle closure. Presumed immune rejection was observed in three rabbits only after 4 weeks. Immunostaining with anti-human CD166 allowed to perfectly differentiate human cells from rabbit cells. In successful TEEK at 3 or 4 weeks, human cells formed a normal endothelium and started migrating outside the carrier. Conclusion: Though the transplantation of a TEEK in rabbits is a complex model with many causes of failure, established procedure including use of rTPA allows reliable preclinical study. In addition, we suggest that fs-HALC might be a potential carrier for TEEK.
PurposeOur patented corneal bioreactor (BR) improves the long‐term survival of human corneas ex vivo by restoring intraocular pressure and renewal of storage medium.AimTo use the BR to test the efficacy of eye drops containing devitalized ground amniotic membrane of umbilical cord extract (AMUCE) to treat deep stromal ulcerations. AMUCE contains high level of proteoglycans, collagen and physiological growth factors.MethodsOnly pairs of human corneas (with ECD > 800 cells/mm2) were used to obtain best possible controls. They were first stored for 2 weeks into the BR filled with CorneaMax (Eurobio, France), with 21 mmHg in the endothelial chamber to allow regeneration of a normal epithelium. Then, an 8.0 mm diameter calibrated epithelial ulcer was made with a spatula and a 6 mm diameter × 100 µm in depth was made with Excimer laser in the stroma. Using a specific BR lid allowing sterile handling, each cornea was treated topically 4 times daily either by the AMUCE eye drops (TBF, France) or by the vehicle (0.9% NaCl). Closure of the ulceration was monitored daily using fluorescein staining quantified by image analysis (FIJI) of macro‐zoom microscopy. After wound healing corneas were fixed for histology (HES) and immunolabeling of epithelial markers.ResultsThree pairs were used. Epithelium wound healing was obtained at D3, D9, D10 with AMUCE and D3, D9, D19 with the vehicle. Histology performed in ulcerations centre showed 3‐6 epithelial layers with AMUCE and 1‐4 with the vehicle. At D21, significant epithelial detachment was observed only for 2 corneas treated with the vehicle. For one pair, immunolabelling for E‐cadherin and Cytokeratin K3/K12 showed better epithelial differentiation with AMUCE than with vehicle. No evidence of stromal regeneration was observed in the 6 corneas.ConclusionsAMUCE has potential for a faster and lasting epithelium regeneration (stratification and maturation). Nevertheless, it has no impact on stromal regeneration in this model.
PurposeThe deswelling of a corneal graft at the end of storage in the eye bank is essential before perforating or deep anterior lamellar keratoplasty to allow good donor/recipient congruence and to accelerate visual recovery. In France, CorneaJet (Eurobio, France) containing 5% Dextran T500 is the most commonly used medium, but its deswelling kinetics is not referenced and the duration of 48 or 72 hr is chosen purely by habit while the increase in endothelial toxicity over time is documented.AimTo determine the corneal deswelling kinetics in CorneaJet.Methods12 corneas discarded by our eye bank were used. They had an endothelial cell density at reception (day 2 to day 4) of 1713 ± 389 cell/mm2 (mean ± SD). All had been stored in organoculture in CorneaMax medium at 31°C for at least 1 week. The corneas were immersed in 50 ml vials of CorneaJet (same batch) and were stored at 31°C. Central corneal thickness (CCT in µm) and central corneal volume (on an 8mm disc, CCV8 in mm3) were measured by optical coherence tomography (OCT, Casia I, Tomey, Japan). Transparency was evaluated on a prototype measuring contrast transmission (relative scale in %). Measurements were performed before immersion in CorneaJet and repeated at hour (H)3, H6, H9, H12, H18, H24, H30, H36 and H48.ResultsAll data were expressed as mean ± SD respectively at the different times. CCT: 1284 ± 249; 1031 ± 233; 897 ± 22; 773 ± 160; 741 ± 123; 673 ± 75; 626 ± 27; 638 ± 41; 645 ± 49; 652 ± 55. CCV8: 73 ± 9; 61 ± 9; 53 ± 9; 47 ± 8; 44 ± 6; 40 ± 4; 38 ± 3; 37 ± 3; 37 ± 3 and 37 ± 3. Transparency: 13 ± 4; 19 ± 5; 24 ± 6; 29 ± 6; 33 ± 7; 40 ± 7 41 ± 7; 44 ± 9; 45 ± 8 and 44 ± 11.ConclusionsCorneal deswelling in CorneaJet is rapid during the first 12 hr and reaches a plateau at 20 hr. After 20 hr, transparency and thickness are ideal for surgery.
Purpose: The corneas preserved in bioreactor (BR) had been shown to have not only a better endothelial viability, but also a more differentiated and stratified epithelium than corneas preserved in organoculture. Purpose: By using BR, we would analyse the respective contribution of corneal (C), limbal (L), and conjunctival (Conj) epithelia in corneal epithelial regeneration. Methods: Five pairs of corneas from body donation to Science were used with a death‐to‐collection time < 20 h. A 3‐ to 5‐mm‐wide conjunctival flange was kept intact. Five patterns were set up by fully mechanical removal of 1, 2, or 3 epithelia: C‐L + Conj+, C‐L‐Conj+, C‐L + Conj‐, C + L‐Conj‐, C‐L‐Conj‐ (control) n = 2 for each pattern. The limbus was destroyed by scraping and thermocoagulation. Corneas were then kept in BR (21 mmHg, 2.5 μl/min of Corneamax Eurobio, 31°C) for 3 weeks to allow epithelial regeneration. The epithelium was then analysed using immunofluorescence (IF) on flat mounted cornea by targeting CK12 (corneal epithelium) and CK15 (limbal epithelium). Cell nuclei were counterstained with DAPI. Corneal transparency was quantified using a transparometer. Results: No epithelium was reconstituted in the controls. In the other 4 models including the C‐L‐Conj+ group, the cornea was transparent and covered by a pluristratified corneal epithelium, characterized by CK12 expression. Conclusions: In this BR model, conjunctival epithelial cells allowed the regeneration of a typical corneal epithelium in model C‐L‐Conj+. The corneal epithelium can also migrate to the limbus and conjunctiva. We hypothesize that all 3 ocular surface epithelia (including the conj) contain stem cells or progenitors capable of migrating throughout the cornea and restoring the corneal epithelium independently of each other. The main difference between our ex vivo model and in vivo situation is absence of neovascularization. This suggests that the main cause of limbic insufficiency is due to the loss of the anti‐angiogenic barrier rather than the loss of limbic stem cells.
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