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      Biofunctionalized Lysophosphatidic Acid/Silk Fibroin Film for Cornea Endothelial Cell Regeneration

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          Abstract

          Cornea endothelial cells (CEnCs) tissue engineering is a great challenge to repair diseased or damaged CEnCs and require an appropriate biomaterial to support cell proliferation and differentiation. Biomaterials for CEnCs tissue engineering require biocompatibility, tunable biodegradability, transparency, and suitable mechanical properties. Silk fibroin-based film (SF) is known to meet these factors, but construction of functionalized graft for bioengineering of cornea is still a challenge. Herein, lysophosphatidic acid (LPA) is used to maintain and increase the specific function of CEnCs. The LPA and SF composite film (LPA/SF) was fabricated in this study. Mechanical properties and in vitro studies were performed using a rabbit model to demonstrate the characters of LPA/SF. ATR-FTIR was characterized to identify chemical composition of the films. The morphological and physical properties were performed by SEM, AFM, transparency, and contact angle. Initial cell density and MTT were performed for adhesion and cell viability in the SF and LPA/SF film. Reverse transcription polymerase chain reactions (RT-PCR) and immunofluorescence were performed to examine gene and protein expression. The results showed that films were designed appropriately for CEnCs delivery. Compared to pristine SF, LPA/SF showed higher biocompatibility, cell viability, and expression of CEnCs specific genes and proteins. These indicate that LPA/SF, a new biomaterial, offers potential benefits for CEnCs tissue engineering for regeneration.

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          Most cited references 57

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          Descemet membrane endothelial keratoplasty (DMEK).

          To describe Descemet membrane endothelial keratoplasty (DMEK) with organ cultured Descemet membrane (DM) in a human cadaver eye model and a patient with Fuchs endothelial dystrophy. In 10 human cadaver eyes and 1 patient eye, a 3.5-mm clear corneal tunnel incision was made. The anterior chamber was filled with air, and the DM was stripped off from the posterior stroma. From organ-cultured donor corneo-scleral rims, 9.0-mm-diameter "DM rolls" were harvested. Each donor DM roll was inserted into a recipient anterior chamber, positioned onto the posterior stroma, and kept in position by completely filling the anterior chamber with air for 30 minutes. In all recipient eyes, the donor DM maintained its position after a 30-minute air-fill of the anterior chamber followed by an air-liquid exchange. In the patient's eye, 1 week after transplantation, best-corrected visual acuity was 1.0 (20/20) with the patient's preoperative refraction, and the endothelial cell density averaged 2350 cells/mm. DMEK may provide quick visual rehabilitation in the treatment of corneal endothelial disorders by transplantation of an organ-cultured DM transplanted through a clear corneal tunnel incision. DMEK may be a highly accessible procedure to corneal surgeons, because donor DM sheets can be prepared from preserved corneo-scleral rims.
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            Proliferative capacity of the corneal endothelium.

             N. Joyce (2003)
            Corneal endothelium is the single layer of cells forming a boundary between the corneal stroma and anterior chamber. The barrier and "pump" functions of the endothelium are responsible for maintaining corneal transparency by regulating stromal hydration. Morphological studies have demonstrated an age-related decrease in endothelial cell density and indicate that the endothelium in vivo either does not proliferate at all or proliferates at a rate that does not keep pace with the rate of cell loss. Lack of a robust proliferative response to cell loss makes the endothelium, at best, a fragile tissue. As a result of excessive cell loss due to accidental or surgical trauma, dystrophy, or disease, the endothelium may no longer effectively act as a barrier to fluid flow from the aqueous humor to the stroma. This loss of function can cause corneal edema, decreased corneal clarity, and loss of visual acuity, thus requiring corneal transplantation to restore normal vision. Studies from this and other laboratories indicate that corneal endothelium in vivo DOES possess proliferative capacity, but is arrested in G1-phase of the cell cycle. It appears that several intrinsic and extrinsic factors together contribute to maintain the endothelium in a non-replicative state. Ex vivo studies comparing cell cycle kinetics in wounded endothelium of young ( 50 years old) provide evidence that cells from older donors can enter and complete the cell cycle; however, the length of G1-phase appears to be longer and the cells require stronger mitogenic stimulation than cells from younger donors. In vivo conditions per se also contribute to maintenance of a non-replicative monolayer. Endothelial cells are apparently unable to respond to autocrine or paracrine stimulation even though they express mRNA and protein for a number of growth factors and their receptors. Exogenous transforming growth factor-beta (TGF-beta) and TGF-beta in aqueous humor suppress S-phase entry in cultured endothelial cells, suggesting that this cytokine could inhibit proliferation in vivo. In addition, cell-cell contact appears to inhibit endothelial cell proliferation during corneal development and to help maintain the mature endothelial monolayer in a non-proliferative state, in part, via the activity of p27kip1, a known G1-phase inhibitor. The fact that human corneal endothelium retains proliferative capacity has led to recent efforts to induce division and increase the density of these important cells. For example, recent studies have demonstrated that adult human corneal endothelial cells can be induced to grow in culture and then transplanted to recipient corneas ex vivo. The laboratory work that has been conducted up to now opens an exciting new door to the future. The time is right to apply the knowledge that has been gained regarding corneal endothelial cell proliferative capacity and regulation of its cell cycle to develop new therapies to treat patients at risk for vision loss due to low endothelial cells counts.
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              Silk film biomaterials for cornea tissue engineering.

              Biomaterials for corneal tissue engineering must demonstrate several critical features for potential utility in vivo, including transparency, mechanical integrity, biocompatibility and slow biodegradation. Silk film biomaterials were designed and characterized to meet these functional requirements. Silk protein films were used in a biomimetic approach to replicate corneal stromal tissue architecture. The films were 2 microm thick to emulate corneal collagen lamellae dimensions, and were surface patterned to guide cell alignment. To enhance trans-lamellar diffusion of nutrients and to promote cell-cell interaction, pores with 0.5-5.0 microm diameters were introduced into the silk films. Human and rabbit corneal fibroblast proliferation, alignment and corneal extracellular matrix expression on these films in both 2D and 3D cultures were demonstrated. The mechanical properties, optical clarity and surface patterned features of these films, combined with their ability to support corneal cell functions suggest that this new biomaterial system offers important potential benefits for corneal tissue regeneration.
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                Author and article information

                Journal
                Nanomaterials (Basel)
                Nanomaterials (Basel)
                nanomaterials
                Nanomaterials
                MDPI
                2079-4991
                30 April 2018
                May 2018
                : 8
                : 5
                Affiliations
                [1 ]Department of BIN Convergence Technology, Deokjin-gu, Jeonju-si, Jeollabuk-do 54896, Korea; zooheechoi@ 123456jbnu.ac.kr (J.H.C.); wjsgkdis@ 123456hanmail.net (H.J.); songje@ 123456jbnu.ac.kr (J.E.S.)
                [2 ]Department of Polymer Nano Science & Technology and Polymer BIN Research Center, Chonbuk National University, Deokjin-gu, Jeonju-si, Jeollabuk-do 54896, Korea
                [3 ]3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark—Parque de Ciência e Tecnologia, Zona Industrial de Gandra, 4805-017 Barco, Guimarães, Portugal; miguel.oliveira@ 123456dep.uminho.pt (J.M.O.); rgreis@ 123456dep.uminho.pt (R.L.R.)
                [4 ]ICVS/3B’s—PT Government Associated Laboratory, Braga/Guimarães, Portugal; The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Avepark, 4805-017 Barco, Guimarães, Portugal
                Author notes
                [* ]Correspondence: gskhang@ 123456jbnu.ac.kr ; Tel.: +82-63-270-2355
                Article
                nanomaterials-08-00290
                10.3390/nano8050290
                5977304
                29710848
                © 2018 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

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