Aqueous humor outflow resistance is the primary determinant of intraocular pressure (IOP); increased outflow resistance is the basis for elevated IOP associated with glaucoma. In humans, IOP is primarily controlled by the outflow of the aqueous humor through the trabecular meshwork (TM). Researchers trying to screen new therapeutics for glaucoma, or elucidate TM cell behavior under various conditions, currently have the option of screening the response of TM cells cultured on flat two-dimensional plastic cell culture dishes, or screening the response of the TM using perfusion studies of donor eyes, which are expensive studies. Most in vitro studies of TM are over-simplified since there is a significant difference between the flat two-dimensional plastic surfaces on which the TM cells are traditionally cultured and the complex three- dimensional in vivo environment. The topographic features and biomechanic stresses present in vivo provide the cells with stimuli that influence their proliferation, migration, adhesion, and extracellular matrix (ECM) deposition. Our central hypothesis is that a novel constant flow perfusion system that utilizes a natural biopolymer scaffold with anisotropic pore structure to mimic more closely the trabecular meshwork cells' microenvironment can support TM cell growth and provide a platform that could be used for effective screening of various bioactive stimuli. From a biological and biomimetic point of view, natural polymers are desirable to use as biomaterial scaffolds, as their properties are very similar to the native ECM. Collagen, chondroitin sulfate (CS) and hyaluronic acid (HA) are the most predominant components of ECM of native TM. For this project, collagen-CS/HA scaffolds that have uniaxally aligned pores will be engineered to support TM cell growth. This proposal consists of three aims: (1) To determine the effect of hyaluronic acid and chondroitin sulfate on hTM cell growth and ECM production in our 3D collagen scaffolds; (2) To determine the influence of perfusion on the 3D hTM cell-seeded scaffolds; (3) To determine the effect of dexamethasone, TGF-?2, and Latrunculin B (Lat B) on the 3D hTM cell-seeded scaffolds under static and perfusion culture conditions. This 3D TM cell culture system will bring a huge advance to the field of glaucoma research and drug screening by establishing a system that allows the study of TM cells in an environment that more closely simulates their microenvironment, thus producing more physiologically relevant data. The potential impact this could ultimately have on patients is immense, as there are nearly 70 million people in the world that suffer from glaucoma. Furthermore, this research will provide exposure of graduate and undergraduate students to biomedical research at a university without an established bioengineering or biology department that has been traditionally focused on earth and energy sciences.
This proposal features an innovative approach for the culture of trabecular meshwork cells on 3D natural biopolymer scaffolds. The culture system used in this work will, for the first time, allow us to probe the influence of individual signals on these cells in a 3D environment, including specific ECM molecules (i.e. chondroitin sulfate and hyaluronic acid), perfusion rates, and bioactive drugs. To date there is no method by which the influence on the outflow resistance of the TM cells themselves and the ECM of the TM can be distinguished and the technology that we are developing here will be a strong step in this direction.
