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      Silk-Derived Protein Enhances Corneal Epithelial Migration, Adhesion, and Proliferation

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          Abstract

          Purpose

          The corneal surface is vulnerable to a myriad of traumatic insults including mechanical, chemical, and thermal injuries. The resulting trauma may render the naturally occurring regenerative properties of the cornea incapable of restoring a healthy epithelial surface, and may result in the loss of corneal transparency and vision. Healing of the corneal epithelium requires a complex cascade of biological processes that work to restore the tissue after injury. New therapeutic agents that act on the multiple steps of the corneal wound-healing process would offer a potential for improving patient outcomes. Here, a novel silk fibroin–derived protein (SDP) was studied for potential impacts on wound healing through studying an in vitro model.

          Methods

          Solubilized SDP, produced from the Bombyx mori silkworm cocoon, was added to human corneal limbal-epithelial (hCLE) cultures to evaluate the material's effects on epithelial cell migration, proliferation, and adhesion through the use of various scratch wound assays and flow chamber studies.

          Results

          Results indicated that the addition of SDP to culture increased hCLE migration rate by over 50%, and produced an approximate 60% increase in cell proliferation. This resulted in a nearly 30% enhancement of in vitro scratch wound closure time. In addition, cultures treated with SDP experienced increased cell-matrix focal adhesion formation by over 95% when compared to controls.

          Conclusions

          The addition of SDP to culture media significantly enhanced hCLE cell sheet migration, proliferation, and attachment when compared to untreated controls, and indicates SDP's potential utility as an ophthalmic therapeutic agent.

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          Most cited references32

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          Silk as a Biomaterial.

          Silks are fibrous proteins with remarkable mechanical properties produced in fiber form by silkworms and spiders. Silk fibers in the form of sutures have been used for centuries. Recently regenerated silk solutions have been used to form a variety of biomaterials, such as gels, sponges and films, for medical applications. Silks can be chemically modified through amino acid side chains to alter surface properties or to immobilize cellular growth factors. Molecular engineering of silk sequences has been used to modify silks with specific features, such as cell recognition or mineralization. The degradability of silk biomaterials can be related to the mode of processing and the corresponding content of beta sheet crystallinity. Several primary cells and cell lines have been successfully grown on different silk biomaterials to demonstrate a range of biological outcomes. Silk biomaterials are biocompatible when studied in vitro and in vivo. Silk scaffolds have been successfully used in wound healing and in tissue engineering of bone, cartilage, tendon and ligament tissues.
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            Vinculin controls focal adhesion formation by direct interactions with talin and actin

            Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.
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              Hierarchical assembly of cell-matrix adhesion complexes.

              The adhesion of cells to the extracellular matrix is a dynamic process, mediated by a series of cell-surface and matrix-associated molecules that interact with each other in a spatially and temporally regulated manner. These interactions play a major role in tissue formation, cellular migration and the induction of adhesion-mediated transmembrane signals. In this paper, we show that the formation of matrix adhesions is a hierarchical process, consisting of several sequential molecular events. One of the earliest steps in surface recognition is mediated, in some cells, by a 1 microm-thick cell-surface hyaluronan coat, which precedes the establishment of stable, cytoskeleton-associated adhesions. The earliest forms of these integrin-mediated contacts are dot-shaped FXs (focal complexes), which are formed under the protrusive lamellipodium of migrating cells. These adhesions recruit, sequentially, different anchor proteins that are involved in binding the actin cytoskeleton to the membrane. Conspicuous in its absence from FXs is zyxin, which is recruited to these sites only on retraction of the leading edge and the transformation of the FXs into a focal adhesion. Continuing application of force to focal adhesions results in the formation of fibrillar adhesions and reorganization of the extracellular matrix. The formation of these adhesions depends on actomyosin contractility and matrix pliability. Copyright 2004 Biochemical Society
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                Author and article information

                Journal
                Invest Ophthalmol Vis Sci
                Invest. Ophthalmol. Vis. Sci
                iovs
                Invest Ophthalmol Vis Sci
                IOVS
                Investigative Ophthalmology & Visual Science
                The Association for Research in Vision and Ophthalmology
                0146-0404
                1552-5783
                March 2017
                : 58
                : 3
                : 1425-1433
                Affiliations
                [1 ]Department of Biomedical Engineering, Cornell University, Ithaca, New York, United States
                [2 ]Department of Ophthalmology, Weill Cornell Medical College, New York, New York, United States
                [3 ]Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
                [4 ]Silk Technologies, Ltd., Plymouth, Minnesota, United States
                Author notes
                Correspondence: Mark I. Rosenblatt, Illinois Eye and Ear Infirmary, UIC Department of Ophthalmology, 1855 W. Taylor Street, MC 648, Chicago, IL 60612, USA; mrosenbl@ 123456uic.edu .
                Article
                iovs-57-15-52 IOVS-16-19957
                10.1167/iovs.16-19957
                6022413
                28257533
                97f23ece-7fc6-409d-bff0-0cf639a24bef
                Copyright 2017 The Authors

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

                History
                : 19 May 2016
                : 28 November 2016
                Categories
                Cornea

                corneal epithelium,cell migration,cell adhesion,corneal wound healing,silk protein

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