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Ma D.H.-K.,Limbal Stem Cell Laboratory | Ma D.H.-K.,Chang Gung University | Yeh L.-K.,Limbal Stem Cell Laboratory | Chen H.-C.,Limbal Stem Cell Laboratory | And 4 more authors.
Molecular Vision | Year: 2014

Purpose: To understand whether the epithelial phenotype in total sclerocornea is corneal or conjunctival in origin. Methods: Four cases of total sclerocornea (male:female = 1:3; mean age = 5.4±4.3; 1-11 years old) who received penetrating keratoplasty (PKP) at our hospital between 2008 and 2011 were included. Corneal buttons obtained during PKP were used for transmission electron microscopy (TEM) as well as immunoconfocal microscopy for cytokeratins 3, 12, and 13, goblet cell mucin MUC5AC, connexin 43, stem cell markers p63 and ABCG2, laminin-5, and fibronectin. Results: After a mean follow-up period of 38.8±14.0 (12-54) months, the grafts remained clear in half of the patients. TEM examination revealed a markedly attenuated Bowman's layer in the scleralized corneas, with irregular and variably thinned collagen lamellar layers, and disorganization and random distribution of collagen fibrils, which were much larger in diameter compared with a normal cornea. Immunoconfocal microscopy showed that keratin 3 was expressed in all four patients, while p63, ABCG2, and MUC5AC were all absent. Cornea-specific keratin 12 was universally expressed in Patients 1 to 3, while mucosa (including conjunctiva)-specific keratin 13 was negative in these patients. Interestingly, keratin 12 and 13 were expressed in Patient 4 in a mutually exclusive manner. Linear expression of laminin-5 in the basement membrane zone and similar expression of fibronectin were observed. Conclusions: The epithelia in total sclerocornea are essentially corneal in phenotype, but in the event of massive corneal angiogenesis, invasion by the conjunctival epithelium is possible. © 2014 Molecular Vision.

Lai J.-Y.,Chang Gung University | Ma D.H.-K.,Limbal Stem Cell Laboratory | Ma D.H.-K.,Chang Gung University | Cheng H.-Y.,Chang Gung University | And 4 more authors.
Journal of Biomaterials Science, Polymer Edition | Year: 2010

Due to its innocuous nature, hyaluronic acid (HA) is one of the most commonly used biopolymers for ophthalmic applications. We recently developed a cell sheet delivery system using carbodiimide cross-linked HA carriers. Chemical cross-linking provides an improvement in stability of polymer gels, but probably causes toxic side-effects. The aim of this study was to investigate the ocular biocompatibility of HA hydrogels cross-linked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). HA discs without cross-linking and glutaraldehyde (GTA) cross-linked HA samples were used for comparison. The disc implants were inserted in the anterior chamber of rabbit eyes for 24 weeks and characterized by slit-lamp biomicroscopy, histology and scanning electron microscopy. The ophthalmic parameters obtained from biomicroscopic examinations were also scored to provide a quantitative grading system. Results of this study showed that the HA discs cross-linked with EDC had better ocular biocompatibility than those with GTA. The continued residence of GTA cross-linked HA implants in the intraocular cavity elicited severe tissue responses and significant foreign body reactions, whereas no adverse inflammatory reaction was observed after contact with non-cross-linked HA or EDC cross-linked HA samples. It is concluded that the cross-linking agent type gives influence on ocular biocompatibility of cell carriers and the EDC-HA hydrogel is an ideal candidate for use as an implantable material in cell sheet delivery applications. © 2010 Koninklijke Brill NV, Leiden.

Lai J.-Y.,Chang Gung University | Ma D.H.-K.,Limbal Stem Cell Laboratory | Ma D.H.-K.,Chang Gung University | Lai M.-H.,Chang Gung University | And 3 more authors.
PLoS ONE | Year: 2013

Cell sheet-mediated tissue regeneration is a promising approach for corneal reconstruction. However, the fragility of bioengineered corneal endothelial cell (CEC) monolayers allows us to take advantage of cross-linked porous gelatin hydrogels as cell sheet carriers for intraocular delivery. The aim of this study was to further investigate the effects of biopolymer concentrations (5-15 wt%) on the characteristic and safety of hydrogel discs fabricated by a simple stirring process combined with freeze-drying method. Results of scanning electron microscopy, porosity measurements, and ninhydrin assays showed that, with increasing solid content, the pore size, porosity, and cross-linking index of carbodiimide treated samples significantly decreased from 508±30 to 292±42 μm, 59.8±1.1 to 33.2±1.9%, and 56.2±1.6 to 34.3±1.8%, respectively. The variation in biopolymer concentrations and degrees of cross-linking greatly affects the Young's modulus and swelling ratio of the gelatin carriers. Differential scanning calorimetry measurements and glucose permeation studies indicated that for the samples with a highest solid content, the highest pore wall thickness and the lowest fraction of mobile water may inhibit solute transport. When the biopolymer concentration is in the range of 5-10 wt%, the hydrogels have high freezable water content (0.89-0.93) and concentration of permeated glucose (591.3-615.5 μg/ml). These features are beneficial to the in vitro cultivation of CECs without limiting proliferation and changing expression of ion channel and pump genes such as ATP1A1, VDAC2, and AQP1. In vivo studies by analyzing the rabbit CEC morphology and count also demonstrate that the implanted gelatin discs with the highest solid content may cause unfavorable tissue-material interactions. It is concluded that the characteristics of cross-linked porous gelatin hydrogel carriers and their triggered biological responses are in relation to biopolymer concentration effects. © 2013 Lai et al.

Lai J.-Y.,Chang Gung University | Cheng H.-Y.,Chang Gung University | Hui-Kang Ma D.,Limbal Stem Cell Laboratory | Hui-Kang Ma D.,Chang Gung University
PLoS ONE | Year: 2015

Hyaluronic acid (HA) is a linear polysaccharide naturally found in the eye and therefore is one of the most promising biomaterials for corneal endothelial regenerative medicine. This study reports, for the first time, the development of overrun-processed porous HA hydrogels for corneal endothelial cell (CEC) sheet transplantation and tissue engineering applications. The hydrogel carriers were characterized to examine their structures and functions. Evaluations of carbodiimide cross-linked air-dried and freeze-dried HA samples were conducted simultaneously for comparison. The results indicated that during the fabrication of freeze-dried HA discs, a technique of introducing gas bubbles in the aqueous biopolymer solutions can be used to enlarge pore structure and prevent dense surface skin formation. Among all the groups studied, the overrun-processed porous HA carriers show the greatest biological stability, the highest freezable water content and glucose permeability, and the minimized adverse effects on ionic pump function of rabbit CECs. After transfer and attachment of bioengineered CEC sheets to the overrun-processed HA hydrogel carriers, the therapeutic efficacy of cell/ biopolymer constructs was tested using a rabbit model with corneal endothelial dysfunction. Clinical observations including slit-lamp biomicroscopy, specular microscopy, and corneal thickness measurements showed that the construct implants can regenerate corneal endothelium and restore corneal transparency at 4 weeks postoperatively. Our findings suggest that cell sheet transplantation using overrun-processed porous HA hydrogels offers a new way to reconstruct the posterior corneal surface and improve endothelial tissue function. Copyright: © 2015 Lai et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Lai J.-Y.,Chang Gung University | Lue S.J.,Chang Gung University | Cheng H.-Y.,Chang Gung University | Ma D.H.-K.,Limbal Stem Cell Laboratory | Ma D.H.-K.,Chang Gung University
Journal of Biomedical Nanotechnology | Year: 2013

Carbodiimide cross-linked amniotic membrane (AM) can potentially serve as an artificial corneal epithelial stem cell niche in ocular surface wound healing. For the first time, this study was performed to investigate the relationship between nano-structure and functionality of carbodiimide cross-linked AM tissues as limbal epithelial cell (LEC) scaffold biomaterials. The triple-helical molecular conformation of AM collagen was checked after chemical treatment for varying cross-linking durations (1-4 h). Our data indicated that the unraveling of the helical structure into a more random globular state is accompanied by an increase in the cross-linking index of AM samples. The cross-linker-mediated alterations in tissue ultrastructure and substrate nanotopography of these proteinaceous matrices were confirmed by transmission electron and atomic force microscopy studies. With increasing treatment time, the chemically cross-linked AM possessed larger nanofiber diameter and exhibited rougher texture. Marked increases in the water content, light transmittance, and resistance to enzymatic degradation were found, probably due to collagen fibril aggregation in biological tissues. All the test AM materials were not toxic to the human corneal epithelial cell cultures and retained anti-inflammatory activity, indicating the tolerability and safety of carbodiimide (i.e., a zero-length cross-linker). In addition, the enhanced LEC growth and increased p63 and ABCG2 gene expressions were significantly noted on the AM samples with greater cross-linking degree. In summary, the findings reported in this paper suggest that a specific limbal epithelial stem cell-biomaterial interaction may occur in response to biophysical cue such as nanostructure of carbodiimide cross-linked AM matrix. Copyright © 2013 American Scientific Publishers. All rights reserved.

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