Glaucoma is a disease in which damage to the optic nerve leads to progressive, irreversible vision loss. The intraocular pressure (IOP) is the only modifiable risk factor for glaucoma and its lowering is considered a useful strategy for preventing or slowing down the progression of glaucomatous neuropathy. Elevated intraocular pressure associated with glaucoma is due to increased aqueous humor outflow resistance, primarily through the trabecular meshwork (TM) of the eye. Current in vitro models of the trabecular meshwork are oversimplified and do not capture the organized and complex three-dimensional nature of this tissue that consists primarily of collagen and glycoasaminoglycans. In this work, collagen and collagen-chondroitin sulfate (CS) scaffolds were fabricated via unidirectional freezing and lyophilization to induce the formation of aligned pores. Scaffolds were characterized by scanning electron microscopy, dynamic mechanical analysis, and a chondroitin sulfate quantification assay. Scaffold characterization confirmed the formation of aligned pores, and also that the CS was leaching out of the scaffolds over time. Primary porcine trabecular meshwork (TM) cells were seeded onto the surface of scaffolds and their gene expression, proliferation, viability, migration into the scaffolds, and morphology were examined. The TM cells were viable and proliferated 2 weeks after seeding. The cells migrated down into the internal scaffold structure and their morphology reflected the topography and alignment of the scaffold structure. This work is a promising step toward the development of a three dimensional in vitro model of the TM that can be used for testing of glaucoma pharmacological agents in future experimentation and to better our understanding of the trabecular meshwork and its complex physiology. Biotechnol. Bioeng. 2017;114: 915-923. © 2016 Wiley Periodicals, Inc.

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Glaucoma is a degenerative eye disease in which damage to the optic nerve leads to a characteristic loss of vision. The primary risk factor for glaucoma is an increased intraocular pressure that is caused by an imbalance of aqueous humor generation and subsequent drainage through the trabecular meshwork (TM) drainage system. The small size, donor tissue limitations, and high complexity of the TM make it difficult to research the relationship between the TM cells and their immediate environment. Thus, a biomaterial‐based approach may be more appropriate for research manipulations and in vitro drug development platforms. In this work, human TM (hTM) cells were cultured on various collagen scaffolds containing different glycosaminoglycans (GAGs) and different pore architectures to better understand how hTM cells respond to changes in their extracellular environment. Cellular response was measured by quantifying cellular proliferation and expression of an important extracellular matrix protein, fibronectin. The pore architecture of the scaffolds was altered using freeze‐casting technique to make both large and small pores that were aligned or with a non‐aligned random structure. The composition of the scaffolds was altered with the addition of chondroitin sulfate and/or hyaluronic acid. It was found that the hTM cells grown on large pore scaffolds proliferate more than those grown on small pores. There was an increase in the fibronectin expression with the incorporation of GAGs, and its morphology was changed by the underlying pore architecture. This work will help provide an insight into the behavior of hTM cells when introducing changes in their microenvironment.

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In the United States, $87 billion per year is spent on the care of diabetic ulcers alone. Although the pathophysiology of diabetic wound healing is multifaceted, high systemic levels of inflammation and increased reactive oxygen species are often implicated in the wound healing impairment. Zwitterionic materials have been demonstrated to reduce inflammation and increase extracellular matrix deposition in wound beds, and here, we demonstrate a fabrication method for photopolymerized zwitterionic hydrogels that also enables sustained drug delivery over time. A therapeutic molecule of interest that is examined in this work is cerium oxide nanoparticle tagged with microRNA-146a (CNP-miR146a) to combat both oxidative stress and inflammation. The hydrogels are composed of zwitterionic and nonzwitterionic monomers, and the hydrogel formation occurs in the absence of a crosslinker. The hydrogels exhibit a wide range of stiffness and mechanical properties depending on their monomer content. Additionally, these hydrogels exhibit sustained release of nanoparticles and proteins. Finally, when employed in an in vivo diabetic mouse wound healing model, the zwitterionic hydrogels alone and laden with the CNP-miR146a conjugate significantly improved the rate of diabetic wound healing. Overall, these materials have excellent potential to be used as a topical treatment for chronic diabetic wounds.

Glaucoma is the leading cause of irreversible blindness in the world, currently impacting 80 million people. Patients suffering from primary open-angle glaucoma experience aqueous humor accumulation within the eye causing an increase in intraocular pressure (IOP). The main cause of this rise in IOP is due to poor outflow of aqueous humor through the trabecular meshwork (TM), a tissue composed of collagen and glycosaminoglycans (GAGs) embedded with TM cells. The behavior of TM cells is impacted by their microenvironment, and studies conducted on two-dimensional plastic substrates do not necessarily reflect how TM cells would behave in their native setting. Here, we cultured human TM (hTM) cells on 3D biocompatible hydrogels composed of gelatin methacrylate (GelMA) incorporated with the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and hyaluronic acid (HA). Mechanical properties were quantified by storage moduli and viscosity data. Cellular response was measured by quantifying cellular proliferation and expression of an important extracellular matrix protein, fibronectin. We have shown substrate mechanical properties to impact hTM cell proliferation over 2 weeks. It was found that the incorporation of GAGs impacted cell proliferation and fibronectin expression in hTM cells. This work will help elucidate hTM cell response with changes in their microenvironment.

Glaucoma is a multifactorial progressive optic neuropathy characterized by the loss of retinal ganglion cells leading to irreversible blindness. It is the leading cause of global irreversible blindness and is currently affecting over 70 million people. Elevated intraocular pressure (IOP) is considered the only modifiable risk factor and is a target of numerous treatment modalities. Researchers have assigned this elevation of IOP to accumulation of extracellular matrix (ECM) components in the aqueous humor (AH) outflow pathway. The major drainage structure for AH outflow is the trabecular meshwork (TM). The ECM of the TM is important in regulating IOP in both normal and glaucomatous eyes. In this work, we have studied the role of exogeneous glycosaminoglycans (GAGs), glucocorticoids, and culture conditions on the expression of the ECM gene and proteins by human TM (hTM) cells cultured on biomaterial scaffolds. Gene and protein expression levels of elastin, laminin, and matrix metalloproteinase-2 (MMP-2) were evaluated using quantitative PCR and immunohistochemistry. Pressure gradient changes in cell-laden scaffolds in perfusion cultures were also monitored. Our findings show that GAGs and dexamethasone play an influencing role in hTM ECM turnover at both transcriptional and translational levels by altering expression levels of elastin, laminin, and MMP-2. Understanding the role of exogeneous factors on hTM cell behavior is helpful in gaining insights on glaucoma pathogenesis and ultimately pivotal in development of novel therapeutics against the disease.

The absence of quantitative in vitro cell-extracellular matrix models represents an important bottleneck for basic research and human health. Randomness of cellular distributions provides an opportunity for the development of a quantitative in vitro model. However, quantification of the randomness of random cell distributions is still lacking. In this paper, we have imaged cellular distributions in an alginate matrix using a multiview light-sheet microscope and developed quantification metrics of randomness by modeling it as a Poisson process, a process that has constant probability of occurring in space or time. Our light-sheet microscope can image more than 5 mm thick optically clear samples with 2.9 0.4 m μ ± depth-resolution. We applied our method to image fluorescently labeled human mesenchymal stem cells (hMSCs) embedded in an alginate matrix. Simulated randomness agrees well with the experiments. Quantification of distributions and validation by simulations will enable quantitative study of cell-matrix interactions in tissue models.