Biomechanics and wound healing in the cornea

Abstract

The biomechanical and wound healing properties of the cornea undermine the predictability and stability of refractive surgery and contribute to discrepancies between attempted and achieved visual outcomes after LASIK, surface ablation and other keratorefractive procedures. Furthermore, patients predisposed to biomechanical failure or abnormal wound healing can experience serious complications such as keratectasia or clinically significant corneal haze, and more effective means for the identification of such patients prior to surgery are needed. In this review, we describe the cornea as a complex structural composite material with pronounced anisotropy and heterogeneity, summarize current understanding of major biomechanical and reparative pathways that contribute to the corneal response to laser vision correction, and review the role of these processes in ectasia, intraocular pressure measurement artifact, diffuse lamellar keratitis (DLK) and corneal haze. The current understanding of differences in the corneal response after photorefractive keratectomy (PRK), LASIK and femtosecond-assisted LASIK are reviewed. Surgical and disease models that integrate corneal geometric data, substructural anatomy, elastic and viscoelastic material properties and wound healing behavior have the potential to improve clinical outcomes and minimize complications but depend on the identification of preoperative predictors of biomechanical and wound healing responses in individual patients.

Figure 1

An approach to biomechanical modeling of surgery and disease in the cornea. Disease is simulated by alteration of the substructural components or their material properties. Surgery is simulated by imposing an ablation profile or incisions. The model is optimized retrospectively by comparing model simulations to analogous experiments in tissue or clinical models. A model optimized with clinical data can then be used prospectively to design and evaluate patient-specific treatment algorithms.

Figure 2

Major biomechanical loading forces in the cornea and a model of biomechanical central flattening associated with disruption of central lamellar segments. A reduction in lamellar tension in the peripheral stroma reduces resistance to swelling and an acute expansion of peripheral stromal volume results (Dupps and Roberts, 2001; Roberts, 2000; Roberts, 2002). Interlamellar cohesive forces (Smolek, 1993) and collagen interweaving (Komai and Ushiki, 1991), whose distribution is greater in the anterior and peripheral stroma and is indicated by grey shading, provide a means of transmitting centripetal forces to underlying lamellae. Because the central portions of these lamellae constitute the immediate postoperative surface, flattening of the optical surface occurs, resulting in hyperopic shift. The degree of flattening is associated with the amount of peripheral thickening (Dupps and Roberts, 2001). This phenomenon is exemplified clinically by PTK-induced hyperopic shift but is important in any central keratectomy, including PRK and LASIK. Simultaneous elastic weakening of the residual stromal bed may occur (Guirao, 2005), and the threshold for inducing irreversible (plastic) or progressive (viscoelastic) steepening (or ectasia) is a matter of great clinical concern.

Figure 3

Experiments illustrating elastic and viscoelastic properties in a 7mm, full-thickness horizontal corneal strip from a 63 year old donor. Elliptical polarization allows visualization of non-homogeneous internal stresses. Progressive stretching of the sample (1, 2 and 3) and measurement of the induced load (stress) allows calculation of the elastic (Young’s) modulus from the slope of the stress-strain relationship. The relationship is nonlinear. A second experiment in which a constant displacement is imposed in the same sample demonstrates time-dependent stress relaxation, a viscoelastic property of biological soft tissues (4 and 5). Courtesy of W.J. Dupps, Jr., MD, PhD and T. Doehring, PhD.

Figure 4

Corneal wound healing cascade. This diagram provides a simplification of the corneal wound healing response. It can be described as a cascade, but many of the components noted here occur simultaneously or have temporal overlap and many other components that contribute are not depicted. Epithelial injury is the inciting event of wound healing in most cases. This injury can take the form of a scrape, incision, laser exposure (for example, the femtosecond laser), or other insult. The earliest observable stromal change following epithelial injury is the almost instantaneous programmed cell death (apoptosis) of underlying keratocyte cells. In this example, a section from a human cornea that underwent epithelial scrape prior to enucleation for choroidal melanoma is stained with the TUNEL assay to reveal keratocytes (arrows) undergoing apoptosis (500X mag.). Within a few hours, residual stromal keratocytes begin to undergo proliferation and migrate to restore stromal cellularity. The earliest mitosis in a cornea with a scrape injury is in the peripheral and posterior cornea. In this example, cells undergoing mitosis (arrowheads) are detected in a rabbit cornea at 24 hours after epithelial injury by performing immunocytochemistry for the Ki67 marker for mitosis (400X mag.). Within a few hours of injury, thousands of bone marrow-derived cells migrate into the cornea. These cells likely phagocytize remnants of dead cells and other debris, but may have other functions that have yet to be characterized. In this example, cells (arrows) expressing fluorescent green protein (FGP) can be seen migrating into the cornea at 24 hours after injury in a chimeric mouse in which only bone marrow-derived cells express the FGP marker. Depending on the type and extent of injury, myofibroblasts may be generated in the cornea. Current dogma is that these cells are derived from keratocytes that proliferate to form stromal fibroblasts, that then differentiate into myofibroblasts under the influence of transforming growth factor beta and other cytokines. However, studies have demonstrated that myofibroblasts in skin (Bhawan and Majno, 1989) and lung (Hashimoto et al., 2004) are derived from bone-marrow derived cells. Further work is needed to explore this possibility in the cornea. When myofibroblasts develop, they typically arise in stroma near the surface epithelium or epithelium that migrates into (or is placed ectopically within) stromal incisions – probably because the epithelium is a source of cytokines required for myofibroblast development. In the example provided, myofibroblasts (arrows) are detected using immunocytochemistry for the alpha smooth muscle actin marker in a rabbit cornea at one month after photorefractive keratectomy for 9 diopters of myopia. Corneas in which large numbers of myofibroblasts are generated develop haze, while those that do not remain clear–there tending to be a direct relationship between the level of haze and the density of myofibroblasts. Recent studies have demonstrated that surface irregularity (Netto et al., 2006), and likely associated abnormalities of the regenerated basement membrane (Netto et al., 2006; Stramer et al., 2003), are important factors in myofibroblast generation. There are also important species-related differences in the tendency to generate myofibroblasts. Rabbits have far greater tendency to develop myofibroblasts and corneal haze than humans and mice. The slit lamp photos of a clear cornea and a cornea with haze are both from humans (5X mag.).