Cryo-Electrospinning Generates Highly Porous Fiber Scaffolds Which Improves Trabecular Meshwork Cell Infiltration.

Human trabecular meshwork is a sieve-like tissue with large pores, which plays a vital role in aqueous humor outflow. Dysfunction of this tissue can occur, which leads to glaucoma and permanent vision loss. Replacement of trabecular meshwork with a tissue-engineered device is the ultimate objective. This study aimed to create a biomimetic structure of trabecular meshwork using electrospinning. Conventional electrospinning was compared to cryogenic electrospinning, the latter being an adaptation of conventional electrospinning whereby dry ice is incorporated in the fiber collector system. The dry ice causes ice crystals to form in-between the fibers, increasing the inter-fiber spacing, which is retained following sublimation. Structural characterization demonstrated cryo-scaffolds to have closer recapitulation of the trabecular meshwork, in terms of pore size, porosity, and thickness. The attachment of a healthy, human trabecular meshwork cell line (NTM5) to the scaffold was not influenced by the fabrication method. The main objective was to assess cell infiltration. Cryo-scaffolds supported cell penetration deep within their structure after seven days, whereas cells remained on the outer surface for conventional scaffolds. This study demonstrates the suitability of cryogenic electrospinning for the close recapitulation of trabecular meshwork and its potential as a 3D in vitro model and, in time, a tissue-engineered device.

Morphological and biomechanical analyses of the human healthy and glaucomatous aqueous outflow pathway: Imaging-to-modeling.

BACKGROUND AND OBJECTIVE: Intraocular pressure (IOP) is maintained via a dynamic balance between the production of aqueous humor and its drainage through the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and Schlemm's canal (SC) endothelium of the conventional outflow pathway. Primary open angle glaucoma (POAG) is often associated with IOP elevation that occurs due to an abnormally high outflow resistance across the outflow pathway. Outflow tissues are viscoelastic and actively interact with aqueous humor dynamics through a two-way fluid-structure interaction coupling. While glaucoma affects the morphology and stiffness of the outflow tissues, their biomechanics and hydrodynamics in glaucoma eyes remain largely unknown. This research aims to develop an image-to-model method allowing the biomechanics and hydrodynamics of the conventional aqueous outflow pathway to be studied. METHODS: We used a combination of X-ray computed tomography and scanning electron microscopy to reconstruct high-fidelity, eye-specific, 3D microstructural finite element models of the healthy and glaucoma outflow tissues in cellularized and decellularized conditions. The viscoelastic TM/JCT/SC complex finite element models with embedded viscoelastic beam elements were subjected to a physiological IOP load boundary; the stresses/strains and the flow state were calculated using fluid-structure interaction and computational fluid dynamics. RESULTS: Based on the resultant hydrodynamics parameters across the outflow pathway, the primary site of outflow resistance in healthy eyes was in the JCT and immediate vicinity of the SC inner wall, while the majority of the outflow resistance in the glaucoma eyes occurred in the TM. The TM and JCT in the glaucoma eyes showed 1.32-fold and 1.13-fold larger beam thickness and smaller trabecular space size (2.24-fold and 1.50-fold) compared to the healthy eyes. CONCLUSIONS: Characterizing the accurate morphology of the outflow tissues may significantly contribute to constructing more accurate, robust, and reliable models, that can eventually help to better understand the dynamic IOP regulation, hydrodynamics of the aqueous humor, and outflow resistance dynamic in the human eyes. This model demonstrates proof of concept for determining changes to outflow resistance in healthy and glaucomatous tissues and thus may be utilized in larger cohorts of donor tissues where disease specificity, race, age, and gender of the eye donors may be accounted for.

A Review of Particle Size Analysis with X-ray CT.

Particle size and morphology analysis is a problem common to a wide range of applications, including additive manufacturing, geological and agricultural materials' characterisation, food manufacturing and pharmaceuticals. Here, we review the use of microfocus X-ray computed tomography (X-ray CT) for particle analysis. We give an overview of different sample preparation methods, image processing protocols, the morphology parameters that can be determined, and types of materials that are suitable for analysis of particle sizes using X-ray CT. The main conclusion is that size and shape parameters can be determined for particles larger than approximately 2 to 3 µm, given adequate resolution of the X-ray CT setup. Particles composed of high atomic number materials (Z > 40) require careful sample preparation to ensure X-ray transmission. Problems occur when particles with a broad range of sizes are closely packed together, or when particles are fused (sintered or cemented). The use of X-ray CT for particle size analysis promises to become increasingly widespread, offering measurements of size, shape, and porosity of large numbers of particles within one X-ray CT scan.

An Optimized Method to Decellularize Human Trabecular Meshwork.

Glaucoma is linked to raised intraocular pressure (IOP). The trabecular meshwork (TM) plays a major role in regulating IOP by enabling outflow of aqueous humor from the eye through its complex 3D structure. A lack of therapies targeting the dysfunctional TM highlights the need to develop biomimetic scaffolds that provide 3D in vitro models for glaucoma research or as implantable devices to regenerate TM tissue. To artificially mimic the TM's structure, we assessed methods for its decellularization and outline an optimized protocol for cell removal and structural retention. Using bovine TM, we trialed 2 lysing agents-Trypsin (0.05% v/v) and Ammonium Hydroxide (NH4OH; 2% v/v). Twenty-four hours in Trypsin caused significant structural changes. Shorter exposure (2 h) reduced this disruption whilst decellularizing the tissue (dsDNA 26 ± 14 ng/mL (control 1970 ± 146 ng/mL)). In contrast, NH4OH lysed all cells (dsDNA 25 ± 21 ng/mL), and the TM structure remained intact. For human TM, 2% v/v NH4OH similarly removed cells (dsDNA 52 ± 4 ng/mL (control 1965 ± 233 ng/mL)), and light microscopy and SEM presented no structural damage. X-ray computed tomography enabled a novel 3D reconstruction of decellularized human TM and observation of the tissue's intricate architecture. This study provides a new, validated method using NH4OH to decellularize delicate human TM without compromising tissue structure.