The effect of prior long-term recellularization with keratocytes of decellularized porcine corneas implanted in a rabbit anterior lamellar keratoplasty model

Abstract

Decellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 μm thick decellularized lenticule against one that had been recellularized with human stromal cells after serum-free culture. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. Since there was little difference between acellular and cell-seeded scaffolds in our in vivo study, future scaffold development should use acellular controls to determine if cells are necessary.

Fig 1. Evaluation of porcine cornea decellularization.

A) Native cornea and B) decellularized scaffold, from left to right stained with, DAPI, H&E, Alcian blue or picrosirius red. Black scale bar = 500 μm, white = 250 μm. Quantification of native cornea and decellularized scaffold C) DNA, D) glycosaminoglycans and E) Collagen. p * < 0.05, p ** < 0.01, p *** < 0.001. F) Agarose gel electrophoresis of DNA from native cornea (N) and decellularized scaffold (D1 and D2). Ladder (L).

Fig 2. Scaffold optical clarity and light transmission.

A) Macroscopic optical clarity. Scale bar = 1 mm. B) Light transmittance spectra.

Fig 3. Analysis of the effectiveness of recellularizing scaffold with human keratocytes.

A) Longitudinal sections of recellularized scaffolds of keratocytes stained with DAPI nuclear staining following 14 or 28 days of serum culture (short and long expansion), or 14 days of serum culture then 14 days without serum (keratocytic expansion). Scale bar = 500 μm. B) Actin staining of keratocytes at a superficial depth (5 μm), and deep depth (100 μm). Scale bar = 200 μm. C) Quantification of number of keratocytes grouped by distance migrated from the surface of the scaffold. D) Cell quantification of recellularized scaffolds by total DNA (n.s. = not significant).

Fig 4. Gene expression analysis of keratocytes in the scaffold.

There is a significant difference for all gene expression compared with that of the keratocytic-expansion. (n = 3–4). * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 5. Phenotype of keratocytes in the scaffold.

Keratocyte markers, ALDH3A1 and keratocan. Fibrotic marker α-SMA. Nucleus, DAPI nuclear staining (Scale bar = 20 μm).

Fig 6. Confirmation of in vitro recellularization of porcine scaffold.

A) Human corneal epithelial cells on the scaffold surface (blue = nuclei stained with DAPI, green = F-actin). Scale bar = 50 μm. B) Rat DRG βIII-tubulin-positive neurites permeate the scaffold. (Scale bar = 100 μm).

Fig 7. Macroscopic appearance of scaffold in the rabbit eye.

Decellularized scaffold (left) with a recellularized scaffold (right). A & B) Day 0. C & D) 3 months post-implantation. At implantation the scaffold was an opaque white. At 3 months both the decellularized and recellularized implants showed some translucent areas, but overall transparency was not recovered and neovascularization had developed.

Fig 8. OCT of the rabbit eye.

A) Normal un-operated cornea. B) At the time of surgery, day 0. C) 3 months post-surgery. At day 0 the scaffold can be seen to be well-apposed with native tissue (white arrows) repairing a deep defect. At 3 months post-surgery an apparent regeneration area can also be seen anterior to the scaffold (black arrows).

Fig 9. Fluorescein staining of the rabbit eye.

A, C) 3 weeks and B, D) two months post-implantation. With both groups the epithelial barrier is not complete at the earlier time, but is intact later.

Fig 10. Rabbit cornea 3 months post-implantation stained for ECM content.

Representative images comparing a decellularized scaffold (left) with a recellularized scaffold (right). Picrosirius red staining (Scale bar = 200 μm) and immunostaining against collagen I, fibronectin and collagen III, with DAPI nuclear stain (Scale bar = 500 μm). Scaffold apposed with native tissue (white arrows) and regeneration area anterior to the scaffold (black arrows). The anterior regeneration area appears denser and the fibronectin more pronounced in the recellularized implant, but the other markers are similar. The Descemet’s membrane separation is a processing artefact.

Fig 11. Rabbit cornea 3 months post-implantation stained for cell distribution and phenotype.

Representative images of decellularized (left) and recellularized implant (right). A) H&E staining (Scale bar = 1 mm, 200 μm, low and high magnifications). Scaffold apposed with native tissue (white arrows) and regeneration area anterior to the scaffold (black arrows). B) Immunostaining for ALDH1A1 and, C) for α-SMA. B) and C) larger image is whole cornea with close-ups of anterior regenerating layer, central scaffold and posterior native cornea. (Scale bar = 500 μm, 50 μm, low and high magnifications).