Primary open-angle glaucoma (POAG), a leading cause of irreversible vision loss, is primarily associated with increased intraocular pressure (IOP), currently the only modifiable risk factor to slow vision impairment [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. By 2040, it is estimated that glaucoma will affect ∼112 million people worldwide [17]. In normal eyes, IOP stability is maintained via a delicate balance between the inflow and outflow of aqueous humor, which is regulated by active resistance in the conventional outflow pathway [18], [19], [20], [21], [22]. Disruptions in this equilibrium, an increase in the outflow resistance, can lead to increased IOP [22,23]. Transient fluctuations in IOP induce the juxtacanalicular tissue (JCT) and the inner wall endothelial cells of Schlemm's canal (SC) to modulate outflow resistance, a process believed to involve synergistic interactions [[20], [21], [22],24]. The adaptation primarily occurs via the remodeling of the extracellular matrix (ECM) [22,25,26].

Transient fluctuations in IOP [16,[27], [28], [29], [30], [31], [32], [33]] result in a dynamic biomechanical environment in the aqueous humor outflow pathway [34], [35], [36]. This dynamic setting notably influences the structure and mechanotransduction processes of the outflow tissues [37], [38], [39], as well as modulates the resistance to outflow [40]. Trabecular meshwork (TM) and JCT cells detect these IOP variations through mechanical stretching of their ECM and membranes [[19], [20], [21], [22],41], a result of active, bidirectional fluid-structure interaction (FSI) between the outflow tissues and the aqueous humor [24,42]. In response, TM cells undergo integrin-mediated cytoskeletal reorganization and exhibit changes in protein expression and phosphorylation [43], [44], [45], [46], [47], [48]. The ECM of the TM plays a critical role in adjusting the resistance to aqueous humor outflow, thereby governing IOP regulation [49], [50], [51], [52], [53], [54], [55], [56], [57]. Thus, TM cells are considered key players in controlling outflow facility and IOP levels. The stiffness of the ECM is crucial in directing cell migration and survival [58], [59], [60], with recent studies indicating that variations in ECM rigidity can prompt differentiation of mesenchymal stem cells into different cell types [61]. This underscores the significant impact of ECM properties on cellular behavior and tissue functionality, particularly within the context of glaucomatous changes in the TM.

Cellular functions reliant on adhesion are significantly impacted by changes in the composition and mechanical properties of the ECM. The ability of cells to sense and react to the stiffness of their substrate leads to diverse cellular behaviors, such as proliferation, differentiation, apoptosis, organization, and migration [57,60]. Additionally, cells demonstrate the capacity to modulate their stiffness in response to their underlying surface and other cues [46], a trait consistently observed in SC and TM cells [38,[62], [63], [64]]. In glaucoma, the mechanosensory abilities of TM cells, vital for regulating IOP, become impaired. Glaucomatous TM cells exhibit a diminished response to pressure changes, resulting in abnormal compliance and flow regions [65], [66], [67]. Elevated IOP in glaucoma subjects these cells to increased mechanical stresses. Atomic force microscopy (AFM) studies have disclosed significant differences in elastic moduli between ECM of normal and glaucomatous SC and TM cells with glaucomatous cells showing 20-fold larger stiffness compared to the normal counterpart [23,57,68]. The Young's modulus of SC endothelial cells also is larger in glaucomatous eyes compared to the normal eyes [69]. Glaucomatous TM cells, which are larger and stiffer than normal TM cells, are affected by age-related changes in the ECM's elastic fiber network [23,70]. These ECM modifications are further intensified in glaucoma, leading to observable ultrastructural alterations [11,49,54,68,71,72]. The stiffness of the ECM is a vital factor influencing TM cell behavior [62,65,73], as well as general cellular differentiation and functionality [59,60,74]. The relationship between SC cells and the JCT suggests that SC cells may become stiffer in glaucoma due to either a pathologically rigid substrate or increased sensitivity to ECM changes [68,75]. TM cells adjust their gene expression in response to shifts in substrate stiffness or architecture, as seen in genes like CTGF, SPARC, TGM2, and fibronectin [38,76]. The substrate's stiffness also affects fibronectin deposition patterns from TM cells and their cytoskeletal dynamics, including responses to latrunculin-B [62,63]. Furthermore, cellular spreading has been observed to be rigidity-dependent, with cells spreading more rapidly on stiffer substrates [77].

Cells apply forces in multiple dimensions on their substrates, influencing not just the immediate cell-ECM interface but also penetrating deeper into the ECM via depth-dependent traction forces [78]. Traction force microscopy (TFM), a method to quantify these cellular forces, involves tracking the displacement of markers like FluoSpheres within a deformable polyacrylamide (PAM) gel [74,79,80]. In environments with increased extracellular stiffness, cells typically exert larger traction forces and form more extensive adhesion contacts with the matrix [78], enhancing both mechanical stability and cellular signaling. Such stiff settings also affect various cellular activities, including spreading, migration, and the differentiation of stem cells [61,74,[81], [82], [83], [84]]. SC endothelial cells, akin to airway smooth muscle cells, are capable of modulating contractile stress and adjusting stiffness across significant distances, an essential function for coping with the complex mechanical environment of the conventional outflow pathway [85]. The dynamics of ECM interaction and cellular contractility vary markedly between 2D and 3D environments [86], [87], [88]. While 2D TFM is limited to measuring tangential force components, omitting a comprehensive view of biomechanical stress and strain [89], [90], [91], 3D TFM offers a more complete depiction of cellular forces, encompassing rotational and perpendicular elements [92], [93], [94]. Our objective in this study is to utilize 3D TFM to gain a more precise and comprehensive understanding of the forces exerted by TM cells. This approach promises to deepen our knowledge of cellular mechanics across different dimensional contexts, a crucial aspect for understanding the complex biomechanics involved in various physiological and pathological conditions [79,[95], [96], [97], [98], [99]].

In our prior publication [78], we developed a 2D in vitro model to analyze the traction forces exerted by normal and glaucomatous human TM cells. Utilizing PAM gel substrates with stiffness levels ranging from 1.5 to 81.5 kPa, we established a clear relationship between cellular traction forces and the stiffness of the ECM surrogate, the PAM gel. A significant finding was that glaucomatous TM cells generated higher traction forces compared to normal cells. In our current study, we emphasize several advancements and distinctions from our prior work and the broader research landscape:

While our previous 2D in vitro cell culture model [78] provided efficient imaging and PAM gel reconstruction, it did not fully capture the natural complexity of the 3D TM structure. To address this, we have moved to a sophisticated 3D-printed in vitro model of the human conventional outflow pathway, meticulously reconstructed from serial block-face scanning electron microscopy (SBF-SEM) images provided by Dr. Haiyan Gong's laboratory [100,101]. This advanced model allows for a more accurate examination of the dynamic traction forces exerted by both normal and glaucomatous TM cells. Crucially, it incorporates a realistic active FSI environment, mirroring the dynamic interplay between aqueous humor, cells, and the ECM in three dimensions. The incorporation of modeled flow into this 3D system significantly augments the physiological relevance of our study, bringing us closer to understanding the true biomechanical environment of the TM in the context of glaucoma.

In our current study, we have adopted modified 3D PIV and 3D TFM algorithms, recently developed and validated in our lab [78]. These algorithms will track the spatial coordinates (X, Y, and Z) of FluoSpheres within the 3D PAM gels. These modified algorithms differ from traditional PIV/TFM by tracking individual FluoSpheres instead of voxels, avoiding the errors associated with digital volume correlation [78,99] or finite element methods [102]. The precision in mapping 3D displacements offered by this algorithm is a substantial improvement, ensuring enhanced accuracy in our results. By accurately tracking these displacements, we gain a more detailed understanding of the dynamic biomechanical environment within the gel, reflecting the nuanced interactions between the cells and their ECM substrate. This level of precision in mapping cellular-induced strains is pivotal for a deeper understanding of the mechanobiology involved in normal and glaucomatous TM cells.

While previous efforts have been made to develop 3D TM cell culture models [103], [104], [105], these have primarily concentrated on static properties, operating under non-flow conditions and not exploring how cells respond to substrates with varying stiffness levels. We understand that the conventional aqueous outflow pathway operates in an active FSI environment, characterized by continuous interactions between the aqueous humor and the cells/ ECM. That said, our model represents a significant progression as it closely mimics the physiological outflow pathway. It does so by encapsulating the dynamic interaction between “aqueous humor” flow and TM structure, thereby modifying both the biomechanical and biochemical environment in a way that more accurately reflects in vivo conditions. This approach allows for a comprehensive examination of how TM cells react within a constantly changing environment, akin to what they would encounter within the eye, thus providing deeper insights into the complexities of glaucoma pathology and TM cell functionality.

A significant knowledge gap exists in understanding the active FSI between cells and the ECM across various elastic moduli. The primary goal of this study was to develop an anatomically precise 3D in vitro cell culture model, meticulously based on SBF-SEM images. In this model, normal and glaucomatous human TM cells were actual and seeded within 3D-printed PAM gels of varying stiffness levels. We then embark on calculating the resultant 3D dynamic traction forces within these cells, situated in an active FSI environment. This is achieved by monitoring the interaction between flow and the 3D gel walls, as well as the TM cells’ membranes, over a sustained period of 20 hours. Moreover, the model we have developed presents a unique opportunity to assess the effectiveness of various drugs in altering cell-ECM interactions. This could pave the way for new methods to modify outflow resistance and, subsequently, IOP, potentially revolutionizing glaucoma treatment. By more accurately simulating physiological conditions, our study aims to reveal new mechanisms and therapeutic targets that could profoundly influence the management of glaucoma in clinical settings.