Decellularized corneal lenticule embedded compressed collagen: toward a suturable collagenous construct for limbal reconstruction

Hong, Hyunsuk; Huh, Man-Il; Park, Sang Min; Lee, Kyoung‐Pil; Kim, Hong Kyun; Kim, Dong Sung

doi:10.1088/1758-5090/aad1a4

Cited by 21 publications

(20 citation statements)

References 76 publications

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“…Collagen gel (3 mg/mL, 35 mm × 15 mm × 3 mm) was prepared by neutralization of rat‐tail type I collagen (3.9 mg/mL in acetic acid, Corning) with 1 M NaOH solution and 10× Dulbecco's Modified Eagle Medium (DMEM; Hyclone) followed by gelation process in a cell culture incubator. The collagen compression process was performed as previously described (Hong et al, ). A compressive pressure was applied to the collagen gel with a customized compression machine with a linear movement of a top plate at a compression rate of <50 μm/s (Figure a‐i).…”

Section: Methodsmentioning

confidence: 99%

Grayscale mask‐assisted photochemical crosslinking for a dense collagen construct with stiffness gradient

et al. 2019

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Despite the potential of a collagen construct with a stiffness gradient for investigating cell–extracellular matrix (ECM) stiffness interaction or recapitulating an in vivo tissue interface, it has been developed in a limited way due to the low and poorly controllable mechanical properties of the collagen. This study proposes a novel fabrication process to achieve a compressed collagen construct with a stiffness gradient, named COSDIENT, at a level of ~ 1 MPa while maintaining in vivo ECM‐like dense collagen fibrillar structures. The COSDIENT was fabricated by collagen compression followed by grayscale mask‐assisted UV–riboflavin crosslinking. The collagen compression process enabled the remarkable increase in the stiffness of the collagen gel from ~ 1–10 kPa to ~ 1 MPa by physical compaction. The subsequent UV–riboflavin crosslinking with a continuous‐tone grayscale mask could simply generate a gradual change of UV irradiation followed by modulating riboflavin‐mediated crosslinking, thereby resulting in a continuous stiffness gradient with a range of 1.16–4.38 MPa in the single compressed collagen construct. The suggested grayscale mask‐assisted photochemical crosslinking had no effect on the physical and optical properties of the original compressed collagen construct, while inducing gradual changes of chemical bonds among collagen fibrils. A skin wound healing assay with epidermal keratinocytes was finally applied as an application example of the COSDIENT to examine the effect of stiffness on the skin keratinocyte behavior.

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Section: Methodsmentioning

confidence: 99%

Grayscale mask‐assisted photochemical crosslinking for a dense collagen construct with stiffness gradient

et al. 2019

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“…Suture retention near 0.6 N is enough for application in ophthalmic and microvascular surgeries. 30,33,34 Furthermore, to evaluate the cross-linking process on biomechanical properties of dbACM, we used glutaraldehyde with a concentration of 0.01% as a cross-linking agent. It was observed that cross-linking improved scaffold's resistance to collagenase in in vitro biodegradation tests.…”

Section: Discussionmentioning

confidence: 99%

Developing a Pro-Angiogenic Amniochorionic-Derived Decellularized Scaffold with Two Exposed Basement Membranes for Cultivating Endothelial Cells

Shariatzadeh

Shafiee

Tayebi

et al. 2021

Preprint

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Decellularized placental membrane has widely been used as scaffold and graft in tissue engineering and regenerative medicine. Exceptional pro-angiogenic and biomechanical properties and low immunogenicity have made the amniochorionic membrane a unique scaffold which provides enriched niche for cellular growth. Herein, an optimized combination of enzymatic solutions (based on Streptokinase) with mechanical scrapping is used to remove the amniotic epithelium and chorion trophoblastic layer, which results in exposing the basement membranes of both sides without their separation and subsequent damages to the in-between spongy layer. Biomechanical and biodegradability properties, endothelial proliferation capacity, and in-vivo pro-angiogenic capabilities of the scaffold were also evaluated. Histological staining and scanning electron microscope (SEM) demonstrated that the underlying amniotic and chorionic basement membranes remained intact while the epithelial and trophoblastic layers were entirely removed without considerable damage to basement membranes. The biomechanical evaluation showed that the scaffold is suturable. Proliferation assay and immunohistochemistry demonstrated that both side basement membranes could support growth of endothelial cells without altering endothelial characteristics. The dorsal skinfold chamber animal model indicated that both side basement membranes could promote angiogenesis. This bi-sided decellularized scaffold with two exposed surfaces for cultivating various cells would have potential applications in skin, cardiac, vascularized composite allografts, and microvascular tissue engineering.

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“…Hong et al reported a carrier for limbal epithelial stem cells based on embedding a single sheet of decellularized human cornea in a collagen gel that was further plastically compressed. 29 With this approach they increased the suturability and resistance to biodegradation of the construct.…”

Section: Discussionmentioning

confidence: 99%

“…43 Alternative strategies such as the one reported by Hong et al increased suturability by embedding a decellularized human corneal lenticule in a compressed collagen gel. 29 These authors also report the fabrication of anterior stromal equivalents by embedding keratocytes in a plastically compressed gel, composed of a collagen I and decellularized corneal ECM mixture, with epithelial cells on the surface. 44 In the approach we took, stacking the decellularized sheets with the normally unsuturable collagen gels provided a construct that could be sutured with conventional interrupted 10-0 sutures.…”

Section: Discussionmentioning

confidence: 99%

Engineering a Corneal Stromal Equivalent Using a Novel Multilayered Fabrication Assembly Technique

Fernández‐Pérez

Madden

Ahearne

2020

Tissue Engineering Part A

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To overcome the serious shortage of donor corneas for transplantation, alternatives based on tissue engineering need to be developed. Decellularized corneas are one potential alternative, but their densely packed collagen architecture inhibits recellularization in vitro. Therefore, a new rapid method of recellularizing these constructs to ensure high cellularity throughout the collagen scaffold is needed. In this study, we developed a novel method for fabricating corneal constructs by using decellularized porcine corneal sheets assembled using a bottom-up approach by layering multiple sheets between cell-laden collagen I hydrogel. Corneal lenticules were cut from porcine corneas by cryosectioning, then decellularized with detergents and air-dried for storage as sheets. Human corneal stromal cells were encapsulated in collagen I hydrogel and cast between the dried sheets. Constructs were cultured in serum-free medium supplemented with ascorbic acid and insulin for 2 weeks. Epithelial cells were then seeded on the surface and cultured for an additional week. Transparency, cell viability, and phenotype were analyzed by qPCR, histology, and immunofluorescence. Constructs without epithelial cells were sutured onto an ex vivo porcine cornea and cultured for 1 week. Lenticules were successfully decellularized, achieving dsDNA values of 13-1.2 ng/mg dry tissue, and were more resistant to degradation than the collagen I hydrogels. Constructs maintained high cell viability with a keratocyte-like phenotype with upregulation of keratocan, decorin, lumican, collagen I, ALDH3A1, and CD34 and the corneal epithelial cells stratified with a cobblestone morphology. The construct was amenable to surgical handling and no tearing occurred during suturing. After 7 days ex vivo, constructs were covered by a neoepithelium from the host porcine cells and integration into the host stroma was observed. This study describes a novel approach toward fabricating anterior corneal substitutes in a simple and rapid manner, obtaining mature and suturable constructs using only tissue-derived materials.

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Decellularized corneal lenticule embedded compressed collagen: toward a suturable collagenous construct for limbal reconstruction

Cited by 21 publications

References 76 publications

Grayscale mask‐assisted photochemical crosslinking for a dense collagen construct with stiffness gradient

Grayscale mask‐assisted photochemical crosslinking for a dense collagen construct with stiffness gradient

Developing a Pro-Angiogenic Amniochorionic-Derived Decellularized Scaffold with Two Exposed Basement Membranes for Cultivating Endothelial Cells

Engineering a Corneal Stromal Equivalent Using a Novel Multilayered Fabrication Assembly Technique

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