(A-2) Sprayable biopolymer improves surgical handling of corneal endothelial cell grafts

Dysfunction of corneal endothelial cells (CECs), the innermost layer of the cornea, causes hazy, unfocused vision. Mitigation of CEC dysfunction requires corneal allograft transplantation, 30,000 of which occur each year in the United States. Since 2000, the field has pivoted from using full thickness to partial thickness transplants of only the endothelium; the latest procedure is Descemet’s membrane endothelial keratoplasty (DMEK). DMEK allografts are 5 to 10-micron layers of human donor tissue comprising the endothelium and its basement membrane. Compared to alternative techniques, the graft’s lack of corneal stroma confers superior vision outcomes, but also causes DMEK grafts to scroll spontaneously once isolated from the cornea. Thus, unlike grafts with a redundant stromal layer, DMEK grafts must be unscrolled after injection to the anterior chamber. This is a challenging and mechanically rough task that causes damage to delicate corneal endothelial cells. In addition, the DMEK donor pool is comparatively narrow because tissue from young donors (< 40 y.o.) scrolls especially tightly and is thus typically disqualified.

Our objective is to develop a temporary support material to protect DMEK tissue and reduce its scrolling during transplantation. The material must: decrease tissue scrolling while remaining flexible enough for loading in a surgical tool; adhere to the endothelial side of the cornea in a thin layer; not cause undue damage to the corneal endothelial cells; and rapidly degrade. A material that meets these functional requirements would help improve the surgical handleability of DMEK grafts and widen the donor pool for corneal endothelium replacement.

In general, our approach to meeting the functional requirements was to use spray application of a modified gelatin solution followed by brief photopolymerization. First, we determined the impact of light exposure at various wavelengths (each at 30 mW/cm²) on donor CEC viability using Calcein AM staining followed by fluorescence imaging and image analysis (FIJI Trainable Weka Segmentation). A mixture of methacrylated fish-derived gelatin (fGelMA, 245 mg/mL) and photoinitiator (LAP, 100 mg/mL) was applied to donor corneal tissue in a fine spray using a cosmetic-grade airbrush (loading volume 10-20 mL), then exposed to violet light (410 nm, 30 mW/cm²) for 104 seconds. The thickness of the resulting biomaterial layer was determined using Optical Coherence Tomography (OCT). In addition, DMEK tissue with applied biomaterial was isolated from the donor cornea using an 8-mm donor trephine punch and microsurgical instrumentation to peel the graft from the stroma. Handling and resistance to scrolling were assessed qualitatively by Iowa Lion’s Eye Bank staff (GS). Corneal endothelial cell viability after biomaterial application and handling was evaluated using Calcein AM staining and image analysis with FIJI. Degradation rate was also determined for larger biomaterial samples (12.6 mL, 1 mm thickness) over the course of two weeks in sterile PBS at 37 C. All tissue used during this study had been deemed unsuitable for transplantation due to reasons unrelated to endothelial pathology. Research consent was obtained for all tissues used in this study.

Exposure to light in the visible range did not cause significant corneal endothelial cell loss in donor tissue; viability for wavelengths as low as 410 nm were within 5% of the unexposed control (Figure 1). At the same intensity, exposure to 320 nm (UV) light caused approximately 10% cell loss compared to the controls, which was statistically significant (p< 0.05). We therefore elected to utilize low-wavelength visible light (violet, 410 nm) as the light source in all subsequent experiments.

Application of 10 µL of the pre-biomaterial liquid at a spray distance of 7.5 cm followed by violet light exposure for 104 seconds resulted in a final biomaterial coating that covered approximately 150 mm² of tissue at a center thickness of 140 mm and edge thickness of 65 mm. This biomaterial coating decreased scrolling by a factor of 2.4 (e.g., increased the width of the tissue from 3.4 to 8.0 mm), did not interfere with graft loading into an injector, and seemed to qualitatively improve handleability of the graft by making it spontaneously unscroll rather than scroll. Importantly, application of the biomaterial coating caused minimal endothelial cell loss compared to untreated tissue. During the first three days of incubation in PBS, biomaterial samples degraded rapidly, losing approximately 25% of their starting mass. Slower degradation followed for the subsequent week: only about 60% of original biomaterial mass remained after 10 days. Based on these results, we approximate that a 150 mm-thick biomaterial coating would fully degrade within 2 days. 

The biomaterial and coating approach we developed here in pilot form successfully met the functional requirements of resisting tissue scrolling, adhering to the cornea in a thin layer, not causing undue damage to corneal endothelial cells, and degrading rapidly. Further replicates are needed to confirm our findings and optimize the spray-on material and application method to maximize reproducibility for potential commercialization. Our anti-scrolling biomaterial spray technique, if utilized by eye banks in collaboration with corneal surgeons, could have a global positive impact in that it could increase the accessibility of DMEK procedures by improving tissue handling and widening the DMEK donor pool.

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