Collagen scaffolds, one of the most used biomaterials in corneal tissue engineering, are frequently crosslinked to improve mechanical properties, enzyme tolerance, and thermal stability. Crosslinkers such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) are compatible with tissues but provide low crosslinking density and reduced mechanical properties. Conversely, crosslinkers such as glutaraldehyde (GTA) can generate mechanically more robust scaffolds; however, they can also induce greater toxicity. Herein, we evaluated the effectivity of double-crosslinking with both EDC and GTA together with the capability of sodium metabisulfite (SM) and sodium borohydride (SB) to neutralize the toxicity and restore biocompatibility after crosslinking. The EDC-crosslinked collagen scaffolds were treated with different concentrations of GTA. To neutralize the free unreacted aldehyde groups, scaffolds were treated with SM or SB. The chemistry involved in these reactions together with the mechanical and functional properties of the collagen scaffolds was evaluated. The viability of the cells grown on the scaffolds was studied using different corneal cell types. The effect of each type of scaffold treatment on human monocyte differentiation was evaluated. One-way ANOVA was used for statistical analysis. The addition of GTA as a double-crosslinking agent significantly improved the mechanical properties and enzymatic stability of the EDC crosslinked collagen scaffold. GTA decreased cell biocompatibility but this effect was reversed by treatment with SB or SM. These agents did not affect the mechanical properties, enzymatic stability, or transparency of the double-crosslinked scaffold. Contact of monocytes with the different scaffolds did not trigger their differentiation into activated macrophages. Our results demonstrate that GTA improves the mechanical properties of EDC crosslinked scaffolds in a dose-dependent manner, and that subsequent treatment with SB or SM partially restores biocompatibility. This novel manufacturing approach would facilitate the translation of collagen-based artificial corneas to the clinical setting.

Research from JAMA Ophthalmology — The Boston Keratoprosthesis in Herpetic Keratitis

Purpose: To review clinical aspects and cellular and molecular steps in the development of long-term glaucoma after corneal surgery or acute trauma—especially the pivotal role of tumor necrosis factor alpha (TNF-α), the rapidity of the secondary damage to the retinal ganglion cells, and the clinical promise of early antiinflammatory intervention. Methods: A series of laboratory studies on post-injury and post-surgery glaucoma have been compared to clinical outcome studies on the subject, focusing particularly on the vulnerability of the retinal ganglion cells. Alkali burn to the cornea of mice and rabbits served as the main experimental model. TNF-α titer, ganglion cell apoptosis, and depletion of optic nerve axons have been examined. Anti-TNF-α antibodies or corticosteroids have been used to protect the retinal ganglion cells. Intraocular pressure (IOP) postburn was recorded by manometric methods. Results: In animals with alkali burn to the cornea, damage to the retina can occur within 24 to 72 hours. This is not because of a direct pH change posteriorly—the alkali is effectively buffered at the iris–lens level. Rather, TNF-α (and other inflammatory cytokines), generated anteriorly, rapidly diffuses posteriorly to cause apoptosis of the ganglion cells. During this time, the IOP remains much lower than the reported values required to cause ganglion cell damage. The TNF-α antibody infliximab or corticosteroids, if administered promptly, are markedly protective of the ganglion cells. Conclusions: A rapidly initiated, inflammatory (TNF-α mediated), IOP-independent pathway to glaucoma, resulting from acute anterior segment trauma or surgery, has been identified in laboratory studies. Prompt prophylactic treatment with antiinflammatory agents has been shown to be markedly neuroprotective of retinal ganglion cells, presumably capable of reducing the risk of late glaucoma.

Millions of people worldwide are bilaterally blind due to corneal diseases including infectious etiologies, trauma, and chemical injuries. While corneal transplantation can successfully restore sight in many, corneal graft survival decreases in eyes with chronic inflammation and corneal vascularization. Additionally, the availability of donor cornea material can be limited, especially in underdeveloped countries where corneal blindness may also be highly prevalent. Development of methods to create and implant an artificial cornea (keratoprosthesis) may be the only option for patients whose eye disease is not suitable for corneal transplantation or who live in regions where corneal transplantation is not possible. The Boston Keratoprosthesis (B-KPro) is the most commonly implanted keratoprosthesis worldwide, having restored vision in thousands of patients. This article describes the initial design of the B-KPro and the modifications that have been made over many years. Additionally, some of the complications of surgical implantation and long-term care challenges, particularly complicating inflammation and glaucoma, are discussed.