Comparative Analysis of Traction Forces in Normal and Glaucomatous Trabecular Meshwork Cells within a 3D, Active Fluid-Structure Interaction Culture Environment.

This study presents a 3D in vitro cell culture model, meticulously 3D printed to replicate the conventional aqueous outflow pathway anatomical structure, facilitating the study of trabecular meshwork (TM) cellular responses under glaucomatous conditions. Glaucoma affects TM cell functionality, leading to extracellular matrix (ECM) stiffening, enhanced cell-ECM adhesion, and obstructed aqueous humor outflow. Our model, reconstructed from polyacrylamide gel with elastic moduli of 1.5 and 21.7 kPa, is based on serial block-face scanning electron microscopy images of the outflow pathway. It allows for quantifying 3D, depth-dependent, dynamic traction forces exerted by both normal and glaucomatous TM cells within an active fluid-structure interaction (FSI) environment. In our experimental design, we designed two scenarios: a control group with TM cells observed over 20 hours without flow (static setting), focusing on intrinsic cellular contractile forces, and a second scenario incorporating active FSI to evaluate its impact on traction forces (dynamic setting). Our observations revealed that active FSI results in higher traction forces (normal: 1.83-fold and glaucoma: 2.24-fold) and shear strains (normal: 1.81-fold and glaucoma: 2.41-fold), with stiffer substrates amplifying this effect. Glaucomatous cells consistently exhibited larger forces than normal cells. Increasing gel stiffness led to enhanced stress fiber formation in TM cells, particularly in glaucomatous cells. Exposure to active FSI dramatically altered actin organization in both normal and glaucomatous TM cells, particularly affecting cortical actin stress fiber arrangement. This model while preliminary offers a new method in understanding TM cell biomechanics and ECM stiffening in glaucoma, highlighting the importance of FSI in these processes. STATEMENT OF SIGNIFICANCE: : This pioneering project presents an advanced 3D in vitro model, meticulously replicating the human trabecular meshwork's anatomy for glaucoma research. It enables precise quantification of cellular forces in a dynamic fluid-structure interaction, a leap forward from existing 2D models. This advancement promises significant insights into trabecular meshwork cell biomechanics and the stiffening of the extracellular matrix in glaucoma, offering potential pathways for innovative treatments. This research is positioned at the forefront of ocular disease study, with implications that extend to broader biomedical applications.

Dynamic traction force in trabecular meshwork cells: A 2D culture model for normal and glaucomatous states.

Glaucoma, which is associated with intraocular pressure (IOP) elevation, results in trabecular meshwork (TM) cellular dysfunction, leading to increased rigidity of the extracellular matrix (ECM), larger adhesion forces between the TM cells and ECM, and higher resistance to aqueous humor drainage. TM cells sense the mechanical forces due to IOP dynamic and apply multidimensional forces on the ECM. Recognizing the importance of cellular forces in modulating various cellular activities and development, this study is aimed to develop a 2D in vitro cell culture model to calculate the 3D, depth-dependent, dynamic traction forces, tensile/compressive/shear strain of the normal and glaucomatous human TM cells within a deformable polyacrylamide (PAM) gel substrate. Normal and glaucomatous human TM cells were isolated, cultured, and seeded on top of the PAM gel substrate with embedded FluoSpheres, spanning elastic moduli of 1.5 to 80 kPa. Sixteen-hour post-seeding live confocal microscopy in an incubator was conducted to Z-stack image the 3D displacement map of the FluoSpheres within the PAM gels. Combined with the known PAM gel stiffness, we ascertained the 3D traction forces in the gel. Our results revealed meaningfully larger traction forces in the glaucomatous TM cells compared to the normal TM cells, reaching depths greater than 10-µm in the PAM gel substrate. Stress fibers in TM cells increased with gel rigidity, but diminished when stiffness rose from 20 to 80 kPa. The developed 2D cell culture model aids in understanding how altered mechanical properties in glaucoma impact TM cell behavior and aqueous humor outflow resistance. STATEMENT OF SIGNIFICANCE: Glaucoma, a leading cause of irreversible blindness, is intricately linked to elevated intraocular pressures and their subsequent cellular effects. The trabecular meshwork plays a pivotal role in this mechanism, particularly its interaction with the extracellular matrix. This research unveils an advanced 2D in vitro cell culture model that intricately maps the complex 3D forces exerted by trabecular meshwork cells on the extracellular matrix, offering unparalleled insights into the cellular biomechanics at play in both healthy and glaucomatous eyes. By discerning the changes in these forces across varying substrate stiffness levels, we bridge the gap in understanding between cellular mechanobiology and the onset of glaucoma. The findings stand as a beacon for potential therapeutic avenues, emphasizing the gravity of cellular/extracellular matrix interactions in glaucoma's pathogenesis and setting the stage for targeted interventions in its early stages.

Implementing new computational methods for the study of JCT and SC inner wall basement membrane biomechanics and hydrodynamics.

PURPOSE: The conventional aqueous outflow pathway, which includes the trabecular meshwork (TM), juxtacanalicular tissue (JCT), and the inner wall endothelium of Schlemm's canal (SC), regulates intraocular pressure (IOP) by controlling the aqueous humor outflow resistance. Despite its importance, our understanding of the biomechanics and hydrodynamics within this region remains limited. Fluid-structure interaction (FSI) offers a way to estimate the biomechanical properties of the JCT and SC under various loading and boundary conditions, providing valuable insights that are beyond the reach of current imaging techniques. METHODS: In this study, a normal human eye was fixed at a pressure of 7 mm Hg, and two radial wedges of the TM tissues, which included the SC inner wall basement membrane and JCT, were dissected, processed, and imaged using 3D serial block-face scanning electron microscopy (SBF-SEM). Four different sets of images were used to create 3D finite element (FE) models of the JCT and inner wall endothelial cells of SC with their basement membrane. The outer JCT portion was carefully removed as the outflow resistance is not in that region, leaving only the SCE inner wall and a few µm of the tissue, which does contain the resistance. An inverse iterative FE algorithm was then utilized to calculate the unloaded geometry of the JCT/SC complex at an aqueous humor pressure of 0 mm Hg. Then in the model, the intertrabecular spaces, pores, and giant vacuole contents were replaced by aqueous humor, and FSI was employed to pressurize the JCT/SC complex from 0 to 15 mm Hg. RESULTS: In the JCT/SC complex, the shear stress of the aqueous humor is not evenly distributed. Areas proximal to the inner wall of SC experience larger stresses, reaching up to 10 Pa, while those closer to the JCT undergo lower stresses, approximately 4 Pa. Within this complex, giant vacuoles with or without I-pore behave differently. Those without I-pores experience a more significant strain, around 14%, compared to those with I-pores, where the strain is roughly 9%. CONCLUSIONS: The distribution of aqueous humor wall shear stress is not uniform within the JCT/SC complex, which may contribute to our understanding of the underlying selective mechanisms in the pathway.