Severe shortage of donor corneas for transplantation, particularly in developing countries, has prompted the advancement of bioengineered tissue alternatives. Bioengineered corneas that can withstand transplantation while maintaining transparency and compatibility with host cells, and that are additionally amenable to standardized, low-cost mass production, are sought. In this study, a bioengineered porcine construct (BPC) was developed to function as a biodegradable scaffold to promote corneal stromal regeneration by host cells. Using high purity medical-grade type I collagen, high 18% collagen content and optimized EDC-NHS crosslinker ratio, BPCs were fabricated into hydrogel corneal implants with over 90% transparency and fourfold increase in strength and stiffness compared to previous versions. Remarkably, optical transparency was achieved despite the absence of collagen fibril organization at the nanoscale. In vitro testing indicated the BPC supported confluent human epithelial and stromal-derived mesenchymal stem cell populations. With a novel femtosecond laser-assisted corneal surgical model in rabbits, cell-free BPCs were implanted in vivo in the corneal stroma of 10 rabbits over an 8 week period. In vivo, transparency of implanted corneas was maintained throughout the postoperative period, while healing occurred rapidly without inflammation and without the use of postoperative steroids. BPC implants had a 100% retention rate at 8 weeks, when host stromal cells began to migrate into implants. Direct histochemical evidence of stromal tissue regeneration was observed by means of migrated host cells producing new collagen from within the implants. This study indicates that a cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate regenerative healing of the corneal stroma, and is compatible with human stem or organ-specific cells for future therapeutic applications as a stromal replacement for treating blinding disorders of the cornea.3

Inflammatory angiogenesis is the pathogenic mechanism of various sight-threatening eye diseases, among them corneal neovascularization. Current treatment options include steroids which have undesirable side effects, or anti-VEGF which has only limited efficacy. In an inflammatory environment, however, angiogenesis can be stimulated by numerous factors not directly targeted by anti-VEGF therapy. The aim of this study was to induce corneal inflammation leading to angiogenesis, and investigate the early, differential effects of steroid and anti-VEGF therapy at the cellular, tissue, and gene expression levels. Fifty-two Wistar rats received a single intrastromal corneal suture to induce a controlled inflammatory angiogenic response. Rats were subsequently treated with dexamethasone, rat specific anti-VEGF, or goat IgG (control), topically 4 times daily for 7 days. In vivo confocal microscopy of the cornea was performed longitudinally from 5 h up to 7 d to investigate morphology at the cellular and tissue-level. In vivo photographic vessel analysis and immunohistochemistry were also performed. RT-PCR for VEGF-A, FGF-2, IL-6, TNF-alpha, CXCL2, CCL2, CCL3 and DLL4 was performed at 24 h, and for VEGF-A, IL-6, TNF-alpha, FGF-2, CXCL2, CCL2, and CCL3 at 7 days. Early infiltration of CD11b + myeloid cells into the cornea at 5 h post-suture was delayed by both treatments relative to controls; however neither treatment was able to suppress accumulation of myeloid cells at day 2 or 7. Limbal vessel dilation was inhibited at 5 h by both treatments, but only dexamethasone showed sustained effect until day 2. Early macrophage recruitment was also suppressed by dexamethasone (but not by anti-VEGF) until day 2. Dexamethasone furthermore suppressed corneal neovascularization at day 7 by over 90%, whereas suppression by anti-VEGF was 14%. Despite differential suppression of vessel dilation, macrophage recruitment, and vascular invasion, anti-VEGF and dexamethasone both down-regulated VEGF-A and IL-6 expression at 24 h with sustained effect to 7 d. They also both down regulated FGF-2 and TNF-alpha at 24 h and CCL2 at 7 d. In conclusion, anti-angiogenic treatments influence early, pre-angiogenic tissue activity such as limbal vessel dilation, inflammatory cell infiltration of the stroma, and macrophage recruitment. Importantly, the differential effects of steroids and anti-VEGF treatment in suppressing neovascular growth could not be attributed to differential inhibition of several major angiogenic and inflammatory factors in the early pre-sprouting phase, including IL-6, VEGF-A, FGF-2, TNF-alpha, CCL2, CCL3, CXCL2, or DLL4.

Funding Agencies|Crown Princess Margaretas Foundation; County Council of Ostergotland; Swedish Research Council [2012-2472]

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BL thickness by a revised in vivo method was close to LM values in this study and to values reported in fixed tissue in other studies. The authors believe this revised method provides the most accurate estimates of BL thickness in vivo to date.