Development of Novel Aligned Collagen Fibril Coated Substrates for Mimicking Corneal Tissue Structures

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2020-01-23

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Abstract

Scarring of the cornea is a leading cause of blindness and decreased vision quality globally. Although it is known that wound healing in the cornea is a complex process that is regulated by both soluble biochemical cues and insoluble biophysical cues present in the corneal stroma, our understanding of which factors lead to stromal fibrosis and corneal haze instead of normal wound repair is limited. Previous studies have suggested that the unique arrangement of aligned collagen fibrils into sheets of lamellae in the corneal stroma not only is important for the structural integrity and transparency of the cornea, but is also important in modulating the behavior of keratocytes, which are specialized cells residing in the corneal stroma, that are responsible for wound repair. Thus, the overall goal of this thesis was to develop methods to fabricate substrates coated with aligned Type I collagen fibrils that mimic the normal corneal stroma. In the first part of this work, a simple low-cost, high-throughput microfluidic method was developed for coating glass substrates with aligned fibrils of Type I collagen that were easily integrated into a novel PDMS-ring microwell tissue culture system that allowed high-resolution imaging of keratocyte behavior and morphology. While the density of the collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion, the degree of fibril alignment was observed to be independent of the perfusion shear rate. It was determined that when the density of aligned collagen fibrils was high, primary normal rabbit keratocytes (NRK) elongated and co-aligned with the underlying collagen fibrils when cultured in the presence of platelet derived growth factor (PDGF). In contrast, NRKs showed no preferential orientation when cultured on substrates with a low density of aligned collagen fibrils. In the second part of this work, these substrates were used to observe how NRK respond to the simultaneous exposure to topographical, compositional, and soluble cues. In specific, it was demonstrated that substrates coated with fibronectin had a stimulating effect on NRK morphology as evidenced by a change in cell shape, stress fiber formation, and expression of alpha smooth muscle actin (α-SMA). The magnitude of these effects appeared to depend upon whether the NRK were adhered to flat, rigid glass or on aligned collagen fibrils. However, when the NRK were simultaneously exposed to adsorbed fibronectin and a soluble growth factor such as PDGF, the PDGF appeared to be the more potent cue as evidenced by their elongated morphology and reduced amounts of α-SMA expression. In the final part of this work, a novel method for creating well-defined micropatterns of proteins onto the aligned collagen fibrils was developed. This method allows for fabricating protein patterns with a wide range of sizes (50 – 1500 um) and for multiple proteins to be deposited onto a single substrate. To demonstrate that such line patterns could influence keratocyte behavior, line patterns of PDGF were deposited onto glass substrates and resulted in an increased density of NRK adhering to the patterns of PDGF. In summary, the fabrication methods developed here provide a novel platform for investigating how keratocytes simultaneously integrate the topographical cues provided by aligned collagen fibrils in the presence of various biochemical cues (i.e. secreted proteins, hormones, growth factors) during the wound healing process.

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Tumors, Plasma desorption mass spectrometry, Fibronectins, Microfluidics, Cornea

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