A report of two cases

The TFOS DEWS II Pathophysiology Subcommittee reviewed the mechanisms involved in the initiation and perpetuation of dry eye disease. Its central mechanism is evaporative water loss leading to hyperosmolar tissue damage. Research in human disease and in animal models has shown that this, either directly or by inducing inflammation, causes a loss of both epithelial and goblet cells. The consequent decrease in surface wettability leads to early tear film breakup and amplifies hyperosmolarity via a Vicious Circle. Pain in dry eye is caused by tear hyperosmolarity, loss of lubrication, inflammatory mediators and neurosensory factors, while visual symptoms arise from tear and ocular surface irregularity. Increased friction targets damage to the lids and ocular surface, resulting in characteristic punctate epithelial keratitis, superior limbic keratoconjunctivitis, filamentary keratitis, lid parallel conjunctival folds, and lid wiper epitheliopathy. Hybrid dry eye disease, with features of both aqueous deficiency and increased evaporation, is common and efforts should be made to determine the relative contribution of each form to the total picture. To this end, practical methods are needed to measure tear evaporation in the clinic, and similarly, methods are needed to measure osmolarity at the tissue level across the ocular surface, to better determine the severity of dry eye. Areas for future research include the role of genetic mechanisms in non-Sjogren syndrome dry eye, the targeting of the terminal duct in meibomian gland disease and the influence of gaze dynamics and the closed eye state on tear stability and ocular surface inflammation.

TFOS DEWS II pathophysiology report

The past 20 years have witnessed ever-growing evidence that the mechanical properties of biological tissues, from nanoscale to macroscale dimensions, are fundamental for cellular behaviour and consequent tissue functionality. This knowledge, combined with previously known biochemical cues, has greatly advanced the field of biomaterial development, tissue engineering and regenerative medicine. It is now established that approaches to engineer biological tissues must integrate and approximate the mechanics, both static and dynamic, of native tissues. Nevertheless, the literature on the mechanical properties of biological tissues differs greatly in methodology, and the available data are widely dispersed. This Review gathers together the most important data on the stiffness of living tissues and discusses the intricacies of tissue stiffness from a materials perspective, highlighting the main challenges associated with engineering lifelike tissues and proposing a unified view of this as yet unreported topic. Emerging advances that might pave the way for the next decade’s take on bioengineered tissue stiffness are also presented, and differences and similarities between tissues in health and disease are discussed, along with various techniques for characterizing tissue stiffness at various dimensions from individual cells to organs. The complexity of biological tissue presents a challenge for engineering of mechanically compatible materials. In this Review, the stiffness of tissue components — from extracellular matrix and single cells to bulk tissue — is outlined, and how this understanding facilitates the engineering of materials with lifelike properties is discussed.

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The stiffness of living tissues and its implications for tissue engineering

Although three-dimensional (3D) bioprinting technology has gained much attention in the field of tissue engineering, there are still several significant engineering challenges to overcome, including lack of bioink with biocompatibility and printability. Here, we show a bioink created from silk fibroin (SF) for digital light processing (DLP) 3D bioprinting in tissue engineering applications. The SF-based bioink (Sil-MA) was produced by a methacrylation process using glycidyl methacrylate (GMA) during the fabrication of SF solution. The mechanical and rheological properties of Sil-MA hydrogel proved to be outstanding in experimental testing and can be modulated by varying the Sil-MA contents. This Sil-MA bioink allowed us to build highly complex organ structures, including the heart, vessel, brain, trachea and ear with excellent structural stability and reliable biocompatibility. Sil-MA bioink is well-suited for use in DLP printing process and could be applied to tissue and organ engineering depending on the specific biological requirements.

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Precisely printable and biocompatible silk fibroin bioink for digital light processing 3D printing

Light and optical techniques have made profound impacts on modern medicine, with numerous lasers and optical devices being currently used in clinical practice to assess health and treat disease. Recent advances in biomedical optics have enabled increasingly sophisticated technologies - in particular those that integrate photonics with nanotechnology, biomaterials and genetic engineering. In this Review, we revisit the fundamentals of light-matter interactions, describe the applications of light in imaging, diagnosis, therapy and surgery, overview their clinical use, and discuss the promise of emerging light-based technologies.

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Light in diagnosis, therapy and surgery.

The current standard of corneal diagnosis is structural analysis, by pachymetry1 and tomography,2 to measure corneal thickness and curvature. In addition to structure, the biomechanical properties of the cornea are also important indicators of corneal health. Keratoconus is a degenerative condition that involves a loss of corneal rigidity. Corneal ectasia, which can occur as a rare but serious complication of refractive surgery, results from a decrease in corneal stiffness. Corneal cross-linking is a procedure that increases the elastic modulus of the cornea to prevent ectasia.3–5 It has been used in the treatment of keratoconus and ectasia after refractive surgery. A noninvasive device that could measure corneal biomechanical properties3 would be highly useful in the clinic.6

Most of our current knowledge on the mechanical properties of the cornea came from modeling7 or ex vivo experimental studies performed with stress–strain tests,8 dynamic rheometry, and other mechanical tests.9,10 As the compelling clinical need of biomechanical information has increased,6 various in vivo techniques have been under active development. The ocular response analyzer11 (ORA; Reichert, DePew, NY) uses an air puff to induce pressure on the corneal surface and optically measures the time-dependent deformation of the cornea. However, its sensitivity and clinical usefulness remain questionable.12–14 Several elastography techniques are currently under development.15–18 Ultrasound has long been investigated,19 but thus far is difficult to use in vivo because of its low sensitivity and resolution. However, more sophisticated techniques such as quantitative ultrasound spectroscopy continue to be investigated.20

In this context, we have recently developed a novel technique termed Brillouin optical microscopy that can measure the viscoelastic properties by probing the hypersonic acoustic waves inherently present in the sample.21 Like ultrasound spectroscopy, Brillouin microscopy can determine intrinsic viscoelastic properties decoupled from the structural information and applied pressure. In contrast, it can measure the local acoustic properties with much higher spatial resolution and sensitivity, and the measurement is performed optically without the need for acoustic transducers or physical contact with the cornea. Vaughan and Randall22 have measured the Brillouin spectra of excised cornea and lens. The conventional Brillouin instruments had extremely slow data acquisition, requiring 10 to 60 minutes to obtain a single Brillouin spectrum. Brillouin microscopy drastically improved the data acquisition time to less than a second,23 allowing spatially resolved analysis.

We report our investigation of corneal biomechanics using Brillouin microscopy. For this work, we constructed a tabletop instrument combining a simple confocal microscope with a high-speed spectrometer. Using Brillouin microscopy on fresh bovine whole eyes ex vivo, we showed, to our knowledge for the first time in situ, the ability to image corneal elasticity in three dimensions and measure the depth-dependent variation of elastic modulus within the cornea. We also demonstrate the feasibility of monitoring riboflavin-assisted corneal cross-linking procedures, using Brillouin microscopy.

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Brillouin optical microscopy for corneal biomechanics.

Cataract in the Adult Eye Preferred Practice Pattern

PURPOSE. Corneal collagen crosslinking (CXL) is designed to halt the progression of keratoconus and corneal ectasia by inducing corneal stiffening. However, it currently is difficult to monitor and evaluate CXL outcome objectively due to the lack of suitable methods to characterize corneal mechanical properties. We validated noncontact Brillouin microscopy to quantify corneal mechanical properties before and after CXL. METHODS. CXL was performed on fresh porcine eyes using various presoaking times and light doses, with or without epithelial debridement. From Brillouin maps of corneal elastic modulus, stiffness and average modulus of anterior, middle, and posterior stroma were analyzed. Corneal stiffening index (CSI) was introduced as a metric to compare the mechanical efficacy of a given CXL protocol with respect to the standard protocol (30-minute riboflavin presoak, 3 mW/cm 2 ultraviolet illumination for 30 minutes). RESULTS. Brillouin corneal stiffness increased significantly (P 0.98). Compared to the standard epioff procedure, a typical epi-on procedure resulted in a third of stiffness increase in porcine corneas (CSI ¼ 33).

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Brillouin microscopy of collagen crosslinking: noncontact depth-dependent analysis of corneal elastic modulus.

Objective To study the safety and efficacy of topical bevacizumab in the treatment of corneal neovascularization (NV). Design In a prospective, open-label, noncomparative study, 10 eyes from 10 patients with stable corneal NV were treated with topical bevacizumab, 1.0%, for 3 weeks and followed up for up to 24 weeks. Main Outcome Measures The primary safety variables were the occurrence of ocular and systemic adverse events throughout the course of the study. The primary efficacy variables were neovascular area, the area of the corneal vessels themselves; vessel caliber, the mean diameter of the corneal vessels; and invasion area, the fraction of the total corneal area covered by the vessels. Results From baseline visit to the last follow-up visit, mean reductions were 47.1% (standard deviation [SD], 36.7%) for neovascular area, 54.1% (SD, 28.1%) for vessel caliber, and 12.2% (SD, 42.0%) for invasion area. The decreases in neovascular area and vessel caliber were statistically significant ( P  = .001 and P P  = .19). Visual acuity and central corneal thickness showed no significant changes. Topical bevacizumab was well tolerated with no adverse events. Conclusions Short-term topical bevacizumab therapy reduces the severity of corneal NV without local or systemic adverse effects. Application to Clinical Practice Topical bevacizumab provides an alternative therapy in the treatment of stable corneal NV. Trial Registration clinicaltrials.gov Identifier:NCT00559936

Topical bevacizumab in the treatment of corneal neovascularization: results of a prospective, open-label, noncomparative study.

Purpose.
Loss of corneal strength is a central feature of keratoconus progression. However, it is currently difficult to measure corneal mechanical changes noninvasively. The objective of this study is to evaluate if Brillouin optical microscopy can differentiate the mechanical properties of keratoconic corneas versus healthy corneas ex vivo.

Roberto Pineda

Papers

Brillouin optical microscopy for corneal biomechanics.

Cataract in the Adult Eye Preferred Practice Pattern

Brillouin microscopy of collagen crosslinking: noncontact depth-dependent analysis of corneal elastic modulus.

Topical bevacizumab in the treatment of corneal neovascularization: results of a prospective, open-label, noncomparative study.

Biomechanical characterization of keratoconus corneas ex vivo with Brillouin microscopy.