Lung-Kun Yeh

Carbodiimide cross-linked amniotic membranes for cultivation of limbal epithelial cells.

In ophthalmic tissue engineering, amniotic membrane (AM) is one of the most prevalent natural matrices used for limbal epithelial cell (LEC) cultivation and transplantation. However, the application of AM as a scaffold is limited by its low biomechanical strength and rapid biodegradation. The present study reports the development of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross-linked AM as an LEC carrier. The collagenous tissue materials were modified with varying cross-linker concentrations (0-0.25 mmol EDC/mg AM) and were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), ninhydrin assays, electron microscopy, light transmission measurements, mechanical and in vitro degradation tests, as well as diffusion permeability and cell culture studies. Our results showed that chemical cross-linking approaches saturation at concentrations of 0.05 mmol EDC/mg AM. The formation of cross-links (i.e., amide bonds) in the samples treated with 0.05 mmol EDC/mg AM may cause significant aggregation of tropocollagen molecules and collagen microfibrils without affecting cell morphology of biological tissues. With the optimum concentration of 0.05 mmol EDC/mg AM, chemical cross-linker could significantly enhance the mechanical and thermal stability, optical transparency, and resistance to collagenase digestion. Continuous permeation of albumin through the cross-linked AM would be helpful to cell growth over the matrix surface. In addition, the EDC cross-linked samples were able to support LEC proliferation and preserve epithelial progenitor cells in vitro and in vivo. It is concluded that the AM cross-linked with 0.05 mmol EDC/mg AM may be a potential biomaterial for regenerative medicine.

Expression of angiogenesis-related factors in human corneas after cultivated oral mucosal epithelial transplantation.

PURPOSEnWe analyzed the expression of angiogenesis-related factors in corneal tissues that had undergone previously autologous cultivated oral mucosal epithelial transplantation (COMET).nnnMETHODSnSix eyes from four chemically- and two thermally-injured patients with limbal stem cell deficiency who received COMET to promote wound healing were studied retrospectively. Immunoconfocal microscopy was performed on corneal specimens from the patients after COMET, as well on normal corneas, conjunctiva, and oral mucosa for keratin 8, fibroblast growth factor-2 (FGF-2), VEGF, collagen XVIII (endostatin), pigment epithelium-derived factor (PEDF), soluble fms-like tyrosine kinase-1 (sFlt-1), tissue inhibitor of metalloproteinase-3 (TIMP-3), thrombospondin-1 (TSP-1), and interleukin-1 receptor antagonist (IL-1ra).nnnRESULTSnFGF-2, VEGF, endostatin, PEDF, and IL-1ra were detected in all the samples, with signals for FGF-2, VEGF, and IL-1ra localized to the full-thickness epithelial layer, as signals for endostatin limited to the basement membrane. Expression of PEDF varied in tissues, with a preferential expression in the suprabasal epithelial layer. FGF-2 and IL-1ra were abundantly expressed in the basal epithelial layer in specimens with increased stratification. Signals for sFlt-1, TIMP-3, and TSP-1 were detected in normal corneal epithelium, and in a specimen containing corneal epithelium, but were negative in all other specimens.nnnCONCLUSIONSnExpression of FGF-2, VEGF, PEDF, endostatin, and IL-1ra was similar in normal corneas, conjunctiva, oral mucosa, and corneas after COMET. Expression of sFlt-1, TIMP-3, and TSP-1 was limited to normal corneas and negative for other tissues. A lack of the aforementioned antiangiogenic factors may contribute to the peripheral corneal neovascularization seen after COMET.

Pediatric Microbial Keratitis in Taiwan: Clinical and Microbiological Profiles, 1998–2002 Versus 2008–2012

PURPOSEnTo identify the change in clinical and microbiological profiles of pediatric microbial keratitis in Taiwan between 1998-2002 and 2008-2012.nnnDESIGNnRetrospective, observational study.nnnMETHODnsetting: Chang Gung Memorial Hospital, a referral center in Taiwan. patient population: We retrospectively reviewed the medical records of 68 eyes of 67 children aged 16 years or younger who were diagnosed with microbial keratitis and treated at our hospital between July 2008 and December 2012. main outcomes and measures: Predisposing factors, isolated organisms, antibiotic susceptibility, and clinical outcomes. The findings were compared with the results of our previous study conducted between July 1998 and December 2002.nnnRESULTSnAs in 1998-2002, the leading risk factor for microbial keratitis during 2008-2012 was contact lens use, and the infection rate significantly increased from 40.7% to 52.9% (Pxa0= .024), which was mainly attributable to the recent increase in the rate of orthokeratology-related keratitis from 9.9% to 19.1% (Pxa0= .011). Pseudomonas aeruginosa remained the most commonly isolated organism (30.6%), but the number of isolated coagulase-negative Staphylococcus cases increased significantly in the 2008-2012 cases (Pxa0= .04). Antibiotic susceptibility of organisms did not change significantly between the 2 study periods. By using multiple linear stepwise regression analysis, we found that gram-negative bacterial infection played a crucial role in poor visual outcome.nnnCONCLUSIONSnContact lens-related microbial keratitis increased in Taiwanese children over time, especially because of the use of overnight orthokeratology. Clinicians must understand the infection background and pay further attention to contact lens use in pediatric patients.

Wnt/β-catenin signaling modulates corneal epithelium stratification via inhibition of Bmp4 during mouse development

The development of organs with an epithelial parenchyma relies on reciprocal mesenchymal-epithelial communication. Mouse corneal epithelium stratification is the consequence of a coordinated developmental process based on mesenchymal-epithelial interactions. The molecular mechanism underlying these interactions remains unclear. The Wnt/β-catenin signaling pathway is involved in fundamental aspects of development through the regulation of various growth factors. Here, we show that conditional ablation of either β-catenin (Ctnnb1cKO) or co-receptors Lrp5/6 (Lrp5/6cKO) in corneal stromal cells results in precocious stratification of the corneal epithelium. By contrast, ectopic expression of a murine Ctnnb1 gain-of-function mutant (Ctnnb1cGOF) retards corneal epithelium stratification. We also discovered that Bmp4 is upregulated in the absence of β-catenin in keratocytes, which further triggers ERK1/2 (Mapk3/1) and Smad1/5 phosphorylation and enhances transcription factor p63 (Trp63) expression in mouse corneal basal epithelial cells and in a human corneal epithelial cell line (HTCE). Interestingly, mouse neonates given a subconjunctival BMP4 injection displayed a phenotype resembling that of Ctnnb1cKO. Conditional ablation of Bmp4 eradicates the phenotype produced in Ctnnb1cKO mice. Furthermore, ChIP and promoter-luciferase assays show that β-catenin binds to and suppresses Bmp4 promoter activity. These data support the concept that cross-talk between the Wnt/β-catenin/Bmp4 axis (in the stromal mesenchyme) and Bmp4/p63 signaling (in the epithelium) plays a pivotal role in epithelial stratification during corneal morphogenesis. Summary: In mice, the canonical Wnt signaling pathway regulates Bmp4 signaling pathway activation in corneal keratocytes to modulate corneal epithelial stratification during development.

Staphylococcus aureus Keratitis: A Review of Hospital Cases

Background Methicillin-resistant Staphylococcus aureus (MRSA) infection is an important public health issue. The study aimed to characterize the patient demographics, clinical features, antibiotic susceptibility, and clinical outcomes of keratitis caused by S. aureus, and to make a comparison between MRSA and methicillin-sensitive S. aureus (MSSA) isolates. Methodology/Principal findings Patients (nu200a=u200a59) with culture-proven S. aureus keratitis treated in Chang Gung Memorial Hospital between January 1, 2006, and December 31, 2010, were included in our study. Patients demographic and clinical data were retrospectively reviewed. Twenty-six MRSA (44%) and 33 MSSA (56%) isolates were collected. The MRSA keratitis was significantly more common among the patients with healthcare exposure (Pu200a=u200a0.038), but 46.2% (12/26) of patients with MRSA keratitis were considered to have community-associated infections. All isolates were susceptible to vancomycin. MRSA isolates were significantly more resistant to clindamycin, erythromycin, and sulfamethoxazole/trimethoprim. Ocular surface disease was a significant risk factor for MRSA keratitis (Pu200a=u200a0.011). Visual outcome did not differ significantly between the MRSA and MSSA groups. However, age (Bu200a=u200a0.01, Pu200a=u200a0.035, 95% confidence interval [CI]: 0.001–0.019) and visual acuity at presentation (Bu200a=u200a0.749, P<0.001, 95% CI: 0.573–0.926) were significantly correlated with visual outcome. Conclusions/Significance Ocular surface disease is an important predisposing factor for S. aureus keratitis, especially for MRSA infections. Advanced age and poor visual acuity at presentation are important prognostic indicators for poor visual outcome in S. aureus keratitis. Oxacillin resistance may not be a significant prognostic indicator.

Characterization of the modified chitosan membrane cross-linked with genipin for the cultured corneal epithelial cells.

OBJECTIVESnTo modify a chitosan membrane (CM) by cross-linking the chitosan with genipin, a naturally occurring cross-linker extracted from Gardenia jasminoides fructus, with the aim of developing a new cell culture support and to observe the phenotypes of cultured human corneal epithelial cells (HCECs) on genipin-cross-linked chitosan membrane (GCM).nnnMETHODSnWe tested the cross-linking characteristics and mechanical strength of the GCM. CMs modified by cross-linking with different concentrations of genipin were prepared to investigate the rate of membrane degradation. The biocompatibility of the GCMs was investigated by determining the viability of HCECs cultured on them in vitro. The morphology of the HCECs cultured on CM or GCM was analyzed by confocal microscopy and scanning electron microscopy (SEM). Immunocytochemical staining was conducted to determine the phenotypes of the cultured cells.nnnRESULTSnThe fixation index of the GCM was 31 ± 3% after treatment of CM with 0.5mM genipin. A stress-strain test showed that the GCM could tolerate three times the mechanical force of noncross-linked CM. The biodegradation rate of GCM was much slower than for CM. A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that cell viability was not affected by cross-linking with 5.0mM genipin. SEM showed that the cultured HCECs adhered to and grew well on the surface of the GCM. Immunocytochemical staining showed keratin 3 (K3) and connexin 43 (Cx-43) immunoreactive HCECs on the GCM and their proliferative ability was not significantly affected by strong immunoreactivity of Ki-67 and p63 markers.nnnCONCLUSIONSnGCM has potential as a scaffold for corneal epithelium in ocular surface surgery and greater mechanical strength and slower degradation than unmodified CM.

Clinical Characteristics of Posterior Staphylomas in Myopic Eyes With Axial Length Shorter Than 26.5 Millimeters

PURPOSEnTo describe the features of posterior staphylomas in myopic eyes with an axial length shorter than 26.5xa0mm causing visual loss in old age.nnnDESIGNnRetrospective, observational case series.nnnMETHODSnAfter reviewing cases of staphyloma, we isolated 16 eyes from 10 patients with an axial length <26.5xa0mm for further analysis. These patients had all undergone fundus photography and spectral-domain optical coherence tomography (SDOCT) and some had undergone 3-dimensional magnetic resonance imaging (3D-MRI).nnnRESULTSnThe mean age of the patients was 70.0 ± 6.3 years (mean ± standard deviation; range 59-81). The mean axial length of the eyes with a posterior staphyloma was 25.14 ± 0.77xa0mm and the mean refractive error of the affected eyes wasxa0-4.28 ± 2.65 diopters. The median visual acuity was 20/60. All eyes presented with diffuse chorioretinal atrophy, which resembles pathologic myopic maculopathy. Fuchs spots previously were detected in 3 eyes (19%) and lacquer cracks in 2 (12%). SDOCT imaging of the macula showed decreased choroidal thickness in all eyes. A macular retinoschisis was detected in 5 eyes (31%), decreased ellipsoid zone reflectivity in 4 (25%), foveal retinal detachment in 2 (12%), a dome-shaped macula in 1 (6%), a choroidal neovascularization-related subretinal scar in 3 (19%), and loss of ellipsoid zone and outer nuclear layer in 3 (19%). The 3D-MRI scans from 8 eyes showed posterior staphylomas, lateral angulation, or protrusion in all eyes. No eye featured a tilted disc syndrome.nnnCONCLUSIONSnPosterior staphylomas in myopic eyes with an axial length shorter than 26.5xa0mm exhibit features resembling pathologic myopic maculopathy. This type of staphyloma might cause visual impairment in elderly patients and can be considered atypical forms ofxa0pathologic myopia in old age.

Role of SH2-containing tyrosine phosphatase Shp2 in mouse corneal epithelial stratification.

PURPOSEnShp2 protein tyrosine phosphatase mediates a wide variety of receptor tyrosine kinases (RTK) cell signaling. Herein, we investigate the role of Shp2 in corneal morphogenesis and homeostasis.nnnMETHODSnShp2 was conditionally knocked out (Shp2(cko)) in Krt14-rtTA;tet-O-Cre;Shp2(f/f) triple transgenic mice administrated with doxycycline (Dox) from postnatal day 1 (P1) to P10, P15, and P25, respectively. In addition, corneal epithelial debridement was performed in adult (P42) mice treated with or without Dox for 8 days (from P42-P50). Mouse eyes were then subjected to histology and immunohistochemistry.nnnRESULTSnShp2(cko) revealed impaired stratification of conjunctival and corneal epithelia during morphogenesis. Likewise, Shp2(cko) failed to restore epithelial stratification after a corneal epithelial wound in adult Shp2(cko). At the cellular level, the ratio of proliferating cell nuclear antigen (PCNA-positive)/total basal cells remained unchanged, but cells in G2/M (survivin-positive) phase was significantly increased in Shp2(cko) as compared with those in the control littermate. Interestingly, deltaN-p63 (ΔNp63) expression and the asymmetric division of the basal cells were coincidentally dampened in Shp2(cko). Transmission electron microscopic study showed that desmosome and hemidesmosome densities were reduced in the corneal epithelium of Shp2(cko). Immunohistochemistry also demonstrated that expression of E-cadherin/β-catenin junction and laminin-β1 was extensively downregulated in Shp2(cko). On the other hand, corneal epithelium lacking Shp2 remained positive for K14, Pax-6, and keratin 12 (K12), suggesting that Shp2 was dispensable for the corneal epithelial-type differentiation.nnnCONCLUSIONSnThese data argued that Shp2 deficiency predominantly impacted p63-dependent cell division and cell adhesive ability, which resulted in the impairment of stratification during corneal epithelial development and wound healing.

Risk factors associated with the development of retinopathy 10 yr after the diagnosis of juvenile-onset type 1 diabetes in Taiwan: a cohort study from the CGJDES.

The aim of this study was to examine risk factors associated with the development of diabetic retinopathy (DR) 10u2009yr after the diagnosis of juvenile‐onset type 1 diabetes in Taiwan.

Colored corn starch dust explosion-related ocular injuries at a Taiwan water park: A preliminary report from a single medical center

Purpose: To elucidate the manifestations of ocular injuries in the colored corn starch dust explosion at a Taiwan water park. Methods: This is a retrospective, non-comparative, consecutive-interventional case series. Fifty explosion-injury patients on 27 June 2015 treated at Chang-Gung Memorial Hospital, Linkou, were included. Thorough ophthalmic examinations were based on emergent triage and consecutive ophthalmological consultations. Multiple ocular and systemic parameters were assessed. Results: Of the 100 eyes in the 50 cases reviewed, 22 cases were male and 28 cases were female. The mean age was 22.08 ± 4.64 years, and the mean burn total body surface area (TBSA) of patients was 45.92 ± 20.30%. Of the 50 patients, 20 had Grade 1 ocular burns, and the others were without ocular involvement. Two of the 20 cases that presented Grade 1 ocular burns died within 1 month due to other systemic complications. The most common ocular manifestations among those with ocular injuries included periocular swelling (75%), followed by conjunctival chemosis (65%), conjunctival hyperemia (50%), singed eyelashes (20%), cornea epithelial defects (10%), and punctate keratopathy (5%). It is worth mentioning that one patient developed herpes simplex keratitis due to stress 3 weeks after being burned. Half of the 50 patients had facial burns. Specifically, the patients with a greater TBSA presented more significant ocular-burn manifestations than those patients with lower TBSA. Conclusion: Prompt ophthalmologic consultations are particularly necessary for mass burn-casualty patients with facial burns, inhalation injuries, and greater TBSA. The inspection and control of all ignition sources and the manipulation of dust with low concentrations and in an open space are crucial factors to prevent future dust explosions.