Torsional wave elastography to assess the mechanical properties of the cornea

Corneal mechanical changes are believed to occur before any visible structural alterations observed during routine clinical evaluation. This study proposed developing an elastography technique based on torsional waves (TWE) adapted to the specificities of the cornea. By measuring the displacements in the propagation plane perpendicular to the axis of the emitter, the effect of guided waves in platelike media was proven negligible. Ex vivo experiments were carried out on porcine corneal samples considering a group of control and one group of alkali burn treatment ( NH 4OH) that modified the mechanical properties. Phase speed was recovered as a function of intraocular pressure (IOP), and a Kelvin-Voigt rheological model was fitted to the dispersion curves to estimate viscoelastic parameters. A comparison with uniaxial tensile testing with thin-walled assumptions was also performed. Both shear elasticity and viscosity correlated positively with IOP, being the elasticity lower and the viscosity higher for the treated group. The viscoelastic parameters ranged from 21.33 to 63.17 kPa, and from 2.82 to 5.30 Pa s, for shear elasticity and viscosity, respectively. As far as the authors know, no other investigations have studied this mechanical plane under low strain ratios, typical of dynamic elastography in corneal tissue. TWE reflected mechanical properties changes after treatment, showing a high potential for clinical diagnosis due to its rapid performance time and paving the way for future in vivo studies.Ministerio de Educacion, Cultura y Deporte Grant DPI2017-83859-R DPI2014-51870-R UNGR15-CE-3664 EQC2018-004508-PSpanish Government DTS15/00093 PI16/00339Instituto de Salud Carlos III y Fondos FederJunta de Andalucia PI-0107-2017 PIN-0030-2017 IE2017-5537MCIN/AEI - European Social Fund "Investing in your future" PRE2018-086085Consejeria de economia, conocimiento, empresas y universidad SOMM17/6109/UGR B-TEP-026- IE2017-5537 P18-RT-1653European Commission SOMM17/6109/UGR B-TEP-026- IE2017-5537 P18-RT-165

The role of the P2X7 receptor in injury-induced calcium dynamics and cell migration in the corneal epithelium

Wound healing in the corneal epithelium is an essential process to maintain corneal clarity and organism health. The earliest events of cellular injury response include the release of nucleotides and the activation of P2 purinergic receptors. While the purinergic receptor P2X7 has been shown to promote cell migration, its role in corneal epithelial wound healing is still poorly understood. The goal of this work is to better understand the role of P2X7 in the injury response. We analyzed P2X7 expression after epithelial injury in rat corneal organ cultures and found that the receptor localizes to the leading edge of the corneal epithelium. However, overall mRNA and protein expression of P2X7 decreased after injury. Inhibition of P2X7 activation significantly delayed wound closure and prevented the leading edge-localization after injury. We found that P2X7 inhibition altered the wound-induced calcium wave in epithelial cells and altered the number and distribution of focal adhesions in the migrating cells. Live cell imaging of epithelial cells showed that P2X7 inhibition led to altered actin rearrangement, with thick actin bundles in the treated cells. In order to determine the importance of P2X7 in epithelial differentiation and stratified cell migration, we developed a stratified culture model. The cells in the stratified model expressed proliferative and differentiation markers similar to organ cultured corneas, as well as similar P2X7 expression and localization after injury. Together, these results show the importance of P2X7 in the overall purinergic response to injury, and provide tools to study P2X7 in stratified corneal cell migration. To determine if P2X7 may contribute to pathologic delayed wound healing in diseases such as type 2 diabetes, we analyzed P2X7 expression in diabetic human corneas and diabetic model rodent corneas. We showed that P2X7 expression is significantly elevated in unwounded diabetic corneas, and that wound healing is delayed in the rodent model. These data show that elevated P2X7 expression may contribute to the delayed healing in disease and may be a possible therapeutic target

Enzymatic degradation of the cornea to develop an experimental model for keratoconus: Biomechanical and optical characterisation

The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber. It is composed of five layers, in which the stroma is the thickest layer (approximately 90% of corneal thickness) that consists mainly of laminated collagen fibrils associated with proteoglycans. The cornea acts as the eye’s outermost lens that is accounted for approximately two-thirds of the eye's total optical power. Like other lenses, the cornea’s geometrical characteristics, such as the curvature, are important to maintain its functions for clear and stable vision. These geometrical characteristics are highly affected by the biomechanical properties of the cornea. For example, in keratoconus, the cornea is characterised by a progressive and localised thinning in corneal thickness, which is associated with a reduction in stiffness and other biomechanical properties. These alterations happen at the collagenous network, which is mainly responsible for the biomechanical features of the cornea, and are mostly attributed to genetic factors and abnormal enzymatic activity. Histological and biochemical studies suggested the role of amylase and collagenase activities in degradation of collagenous network and progression of keratoconus. However, the role of amylase and collagenase on biomechanical and optical properties have not been investigated. In this study, in vitro enzymatic degradation of porcine corneas was conducted with varying concentrations of α-amylase and collagenase (crude and purified) enzymes for different incubation periods. Several techniques, including atomic force microscopy, nanoindentation and optical coherence tomography, were utilised to assess the effect of the enzymes on biomechanical of corneal tissue at macroscale, microscale and nanoscale levels. Corneal transparency and absorption following enzymatic incubation were also measured using spectrophotometry. The biomechanical techniques that were utilised indicated that amylase and collagenase decrease corneal stiffness and thickness following incubation the corneas with amylase and collagenase. Further reduction in biomechanical properties and thickness of the corneas was found with increased enzymes concentrations and incubation periods. Corneal transparency was increased following incubation with the enzymes. The results suggest depletion of proteoglycans by amylase and digestion of collagen fibrils by collagenase. These results were used to propose an animal biomechanical model for keratoconus

Shaken Baby Syndrome: Retinal Hemorrhaging. A Biomechanical Approach to Understanding the Mechanism of Causation

Shaken Baby Syndrome (SBS) is a form of abuse where typically an infant, age six months or less, is held and shaken. There may or may not be direct impact associated with this action. Further, there is very little agreement on the actual mechanism of SBS. Clinical studies are limited in showing the exact mechanism of injury and only offer postulations and qualitative descriptions. SBS has received much attention in the media, has resulted in a great deal of litigation and can be the source of unfounded accusations. Therefore, it is necessary to try to quantify the forces that may cause injury due to SBS. The physiology of infants makes injury due to SBS more likely. Infants have relatively large heads supported by weak necks that simply act as tethers (Prange et al., 2003). Therefore, there is minimal resistance to shaking. In addition, the cerebrospinal fluid (CSF) layer surrounding the infant\u27s brain is up to 10 mm thick as opposed to 1–2 mm in older children and adults (Morison, 2002). This thick layer reduces the resistance in rotation of the brain and can cause shearing injuries to the brain tissue. In addition, retinal hemorrhaging has been reported in SBS. The infant\u27s eyes have a vitreous that is typically more gelatinous and with a higher viscosity than in adult eyes. In addition, this vitreous is firmly attached to the retina and is difficult to remove (Levin, 2000). A preliminary parametric model of an infant eye will be presented so that resultant nodal retinal force of the posterior retina can be investigated and compared with a documented shaking frequency and a documented impact pulse. Retinal forces are then compared with various studies that investigate retinal detachment or adhesive strength. This eye model is built using a variety of material properties that have been reported for the sclero-cornea shell, choroids, retina, vitreous, aqueous, lens, ciliary, optic nerve, tendons, extra ocular muscles, optic nerve, and orbital fatty tissue. The geometry of the eye has been carefully optimized for this parametric model based on scaling to an infant from an adult using idealized eye globe geometry and transverse slice tracings of The Visible Human Project. This model shows promise in investigating the forces and kinematics of the infant eye exposed to harmonic shaking and further bolsters some of the few biomechanical studies investigating SBS. However, improvements are necessary to complete the eye model presented. Specifically, improvements on the mechanical properties for the components of the eye and especially the infant eye are needed. There is currently a deficit of biomechanical studies of the materials needed for the infant eye that is specifically geared for use in an explicit finite element code package. Conversions and adaptations of available materials are used in this first version of the infant eye model presented here and are in fair agreement with some of the clinical studies concerning SBS

Designing a Scaffold-Free Bio-Orthogonal Click Chemistry Method of Cell Assembly for Application in Tissue Engineering

Tissue engineering is a growing field of science that relies on the use of material chemistry, engineering, genetics, and cell biology to produce functional tissues for use in transplantation, drug testing and disease modelling. Presently, there is an urgent need for a technology which would enable assembly of cells into 3-dimensional multilayered tissues. Current cell-assembly technologies rely on biodegradable polymer scaffolds to assemble cells into 3D structures and to support the cell mass of the growing tissue. The presence of these materials in tissues, however, lowers the cell density and the process of scaffold biodegradation results in accumulation of monomer byproducts within the tissue. To overcome these issues we developed a scaffold free method of cell-assembly based on bio-orthogonal ligation reactions between oxyamine and ketone groups to form a stable oxime bond. The reaction is quick, specific and occurs under physiological conditions without a catalyst. To deliver the bio-orthogonal functionalities onto cell surfaces, ketone- and oxyamine- functionalized lipids were incorporated into liposomes which were subsequently fused with cell membranes. The surface engineered cells were assembled into three-dimensional tissues. Using this approach, we were able to produce functional cardiac and liver tissues with variable thicknesses and cell orientations for drug testing as well as the complex 3D co-cultures of stem cells to study stem cell differentiation. The rapid bio-orthogonal cell ligation process also enables assembly of cells into co-culture spheroids in flow, inside a microchannel. The introduction of a bi-functional oxyamine crosslinker molecule allowed for the rapid crosslinking of ketone-functionalized cells into 3D tissues. This bio-orthogonal click chemistry technology can be used with different cell types to produce customized tissues for applications in drug development and regenerative medicine