A tissue-engineered human trabecular meshwork hydrogel for advanced glaucoma disease modeling

Abstract

PurposeAbnormal human trabecular meshwork (HTM) cell function and extracellular matrix (ECM) remodeling contribute to HTM stiffening in primary open-angle glaucoma (POAG). Most current cellular HTM model systems do not sufficiently replicate the complex native three dimensional (3D) cell-ECM interface, which makes them less than ideal to investigate POAG pathology. Tissue-engineered protein-based hydrogels are ideally positioned to overcome shortcomings of current models. Here, we report a novel biomimetic HTM hydrogel and test its utility as a POAG disease model.MethodsHTM hydrogels were engineered by mixing normal donor-derived HTM cells with collagen type I, elastin-like polypeptide and hyaluronic acid, each containing photoactive functional groups, followed by UV light-activated free-radical crosslinking. Glaucomatous conditions were induced with dexamethasone (DEX), and therapeutic effects of the Rho-associated kinase (ROCK) inhibitor Y27632 on cytoskeletal organization and tissue-level function, contingent on HTM cell-ECM interactions, were assessed.ResultsDEX exposure increased HTM hydrogel contractility, f-actin and alpha smooth muscle actin abundance and rearrangement, ECM remodeling, and fibronectin and collagen type IV deposition, all contributing to HTM hydrogel condensation and stiffening consistent with recent data from normal vs. glaucomatous HTM tissue. Y27632 treatment produced precisely the opposite effects and attenuated the DEX-induced pathologic changes, resulting in HTM hydrogel relaxation and softening.ConclusionsWe have developed a biomimetic HTM hydrogel model for detailed investigation of 3D cell-ECM interactions under normal and simulated glaucomatous conditions. Its bidirectional responsiveness to pharmaceutical challenge and rescue suggests promising potential to serve as screening platform for new POAG treatments with focus on HTM biomechanics.

YAP/TAZ mediate TGFβ2-induced Schlemm’s canal cell dysfunction

“…Therefore, we hypothesized that TGFβ2 may increase HSC cell contractility, which could be reduced by YAP/TAZ inhibition. To test this hypothesis, we encapsulated HSC cells in ECM hydrogels - a method developed in our laboratory to characterize HTM cell behavior 26, 30, 43 - and treated the constructs with TGFβ2, either alone or in combination with VP, to assess the level of hydrogel contraction at 5 d. TGFβ2-treated HSC hydrogels exhibited significantly greater contraction compared to controls (62.79% of controls; Fig. 5A,B ), consistent with our previous studies using HTM cells 26, 30, 43 .…”

“…Thirty microliters of the hydrogel solution were pipetted onto a Surfasil (Fisher Scientific)-coated 18 × 18-mm square glass coverslip followed by placing a regular 12-mm round glass coverslip onto the hydrogels. Constructs were crosslinked by exposure to UV light (OmniCure S1500 UV Spot Curing System; Excelitas Technologies, Mississauga, Ontario, Canada) at 320-500 nm, 2.2 W/cm 2 for 5 s, as previously described 26, 30, 43 . The hydrogel-adhered coverslips were removed with fine-tipped tweezers and placed in 24-well culture plates (Corning; Thermo Fisher Scientific).…”

“…Excelitas Technologies, Mississauga, Ontario, Canada) at 320-500 nm, 2.2 W/cm 2 for 5 s, as previously described 26,30,43 . The hydrogel-adhered coverslips were removed with fine-tipped tweezers and placed in 24-well culture plates (Corning; Thermo Fisher Scientific).…”

Melt Electrowriting of Scaffolds with a Porosity Gradient to Mimic the Matrix Structure of the Human Trabecular Meshwork

“…Such changes in the porosity are a consequence of alterations in the morphology and the mechanical properties of the cell-matrix layers in the HTM. Studies have shown that the elastic modulus of healthy HTM, measured locally using AFM, is ~4 kPa, whereas in glaucomatous HTM, this value increases [11][12][13][14][15].…”

“…The proposed model mimicked in vivo outflow physiology: it was responsive to latrunculin-B in a dose-dependent manner [8], and a pathological state with increased ECM accumulation and decreased tissue permeability could be induced by treatment with steroids [19] or with the fibrotic agent TGFβ-2 [18], and counteracted by a ROCK inhibitor [18, 19]. In other studies, the model based on fibrillar hydrogels of collagen/elastin-like peptides was used [13], and the pathological state was successfully induced by dexamethasone, and attenuated by ROCK inhibitor, as revealed by the increased contractility, fibronectin deposition, and hydrogel stiffening. Collagen and collagen/chondroitin sulfate scaffolds obtained via freeze-drying were also used to build in vitro HTM models.…”