Ko-Hua Chen

Tissue-Engineered Human Corneal Endothelial Cell Sheet Transplantation in a Rabbit Model Using Functional Biomaterials

Background. This study was performed to investigate whether transplantation of bioengineered human corneal endothelial cell (HCEC) sheet grafts into corneas denuded of endothelium could restore corneal function and clarity in a rabbit model. Methods. After being labeled with PKH26 fluorescent dye, the adult HCECs derived from eye bank corneas were cultivated on the thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surfaces for 3 weeks at 37°C, and were harvested as transplantable cell sheets after incubation for 45 min at 20°C. Attached by gelatin hydrogel discs, the bioengineered cell monolayers were transplanted to rabbit corneas denuded of endothelium (HCEC sheet group). Traumatized rabbit corneas were served as controls. Postsurgical corneas underwent clinical observations and histological examinations for 6 months. Results. By transmission electron microscopy and Western blot analysis of zonula occludens-1 and Na+,K+-adenosine triphosphatase proteins, the structure and function of HCEC sheets resembled those of native corneal endothelium. After endothelial cells were removed, corneas of each group turned severe edematous and opaque. In the HCEC sheet groups, corneal clarity was gradually restored and corneal thickness was significantly less than that in the control groups (P<0.05). The attached PKH26-positive HCECs spread on rabbit Descemet’s membrane after receiving cell sheet grafts. Intraocular delivery of HCEC sheets by means of a minimally invasive technique (i.e., small-incision surgery using biodegradable hydrogels) demonstrated long-term graft integration with damaged corneas. Conclusions. These results indicate that using cultured HCECs and functional biomaterials, PNIPAAm and gelatin, an effective cell sheet-based therapy can be developed for the treatment of corneal endothelium deficiency.

Transforming growth factor-ß2 inhibition of corneal endothelial proliferation mediated by prostaglandin

Purpose. To determine the influence of Prostaglandin (PG) E2 on transforming growth factor (TGF)-ß2-mediated inhibitory effects on the proliferation of corneal endothelial cells (CE). Methods. The PGE2 and cell proliferation assays were performed using cultured rabbit corneal endothelium. A PGE2-specific enzyme immunoassay was used to check PGE2 synthesis in supernatants of cells cultured with and without added TGF-ß2 and/or indomethacin. To evaluate the inhibitory effects of PGE2 and TGF-ß2 on CE proliferation, the number of cells grown with exogenous PGE2, or TGF-ß2 with or without indomethacin pretreatment was determined. Results. TGF-ß2, 0.5 to 50 ng/ml, increased the PGE2 secretion of CE dose-dependently in a time-dependent manner. Indomethacin (=0.1mg/ml) inhibited this PGE2 secretion to a low level (around 5-10 ng/ml) in the presence or absence of exogenous TGF-ß2. Both exogenous TGF-ß2 and PGE2 inhibited CE proliferation dose-dependently over a wide range of concentrations. Indomethacin reversed the inhibitory effects of TGF-ß2 but not those of exogenous PGE2. In the medium supplemented with indomethacin, even in the presence of 50 ng/ml of TGF-ß2, CE growth did not differ from control cultures. Conclusions. TGF-ß2 stimulates PGE2 synthesis in CE and inhibits CE proliferation in a dose-dependent manner. Indomethacin extinguishes the inhibitory effects of TGF-ß2 on CE proliferation but not the effect of exogenous PGE2. These data suggest that the antiproliferative effects of TGF-ß2 on CE may be possibly due to TGF-ß2-induced synthesis of PG, most likely PGE2.

Identification of monoclinic calcium pyrophosphate dihydrate and hydroxyapatite in human sclera using Raman microspectroscopy.

Raman microspectroscopy was first used to determine the composition of a calcified plaque located at the pterygium‐excision site of a 51‐year‐old female patient’s left nasal sclera after surgery. It was unexpectedly found that the Raman spectrum of the calcified sample at 1149, 1108, 1049, 756, 517, 376 and 352/cm was similar to the Raman spectrum of monoclinic form of calcium pyrophosphate dihydrate (CPPD) crystal, but differed from the Raman spectrum of triclinic form of CPPD. An additional peak at 958/cm was also observed in the Raman spectrum of the calcified plaque, which was identical to the characteristic peak at 958/cm of hydroxyapatite (HA). This is the first study to report the spectral biodiagnosis of both monoclinic CPPD and HA co‐deposited in the calcified plaque of a patient with sclera dystrophic calcification using Raman microspectroscopy.

Calcification of senile cataractous lens determined by Fourier transform infrared (FTIR) and Raman microspectroscopies

A calcified plaque on the surface of a senile cataractous lens (CL) isolated from a 79‐year‐old male patient was identified and its chemical composition quantified using Fourier transform infrared (FTIR) and confocal Raman microspectroscopies. The noncalcified area of the same CL and hydroxyapatite (HA) were selected as a control. Several unique absorption bands, at 960, 1034 and 1090 cm−1 assigned to the ν1 and ν3 stretching modes of phosphate and at 875 cm−1 attributed to carbonate, were clearly displayed in the infrared (IR) spectra of calcified plaque and HA. A peak at 961 cm−1 due to the ν1 stretching mode of phosphate was also evidenced in the Raman spectra of calcified plaque and HA. The calcified plaque formed within the lens protein was found to mainly consist of a mature HA, in which type‐A carbonate apatites (11.4%), type‐B carbonate apatites (55.6%) and liable surface carbonate ions (33.0%) were presented. A higher content of the liable carbonate implies that the calcification or mineralization in this calcified lens was incomplete and still in progress. Moreover, calcification seems not to influence the secondary structure of lens protein because both IR and Raman spectra for the lens protein in the noncalcified area and calcified plaque were similar. The result suggests that both microscopic FTIR and Raman spectroscopies were easy to perform and capable of determination of the chemical composition of a calcified CL.

Preliminary Identification of [beta]-Carotene in the Vitreous Asteroid Bodies by Micro-Raman Spectroscopy and HPLC Analysis

beta-carotene was first identified from the vitreous asteroid bodies (ABs) excised from one patient with asteroid hyalosis (AH) by confocal Raman microspectroscopy and was also verified by high performance liquid chromatography (HPLC). Two patients had been diagnosed with AH and intervened by surgical vitrectomy due to blurred vision. The morphology and components of both AB specimens were observed by optical microscopy and determined by using confocal Raman microspectroscopy and HPLC analysis, respectively. Surprisingly, two unique peaks at 1528 and 1157 cm(-1) were found in the Raman spectrum for the AB specimen of patient 1 alone, which were in close agreement with that of the Raman peaks at 1525 and 1158 cm(-1) for beta-carotene and/or lutein. However, HPLC analytical data clearly indicated that the retention time for the extracted sample from the AB specimen of patient 1 was observed at 13.685 min and just identical to that of beta-carotene (13.759 min) rather than lutein (2.978 min). In addition, the lack of any peak in the HPLC profile for the AB specimen of patient 2 also confirmed the absence of Raman peaks at 1525 and 1158 cm(-1). Thus this preliminary study strongly suggests that beta-carotene as a unique component of ABs was specifically detected from the AB specimen of one AH patient by using confocal Raman microspectroscopy and HPLC analysis.

Ocular hypersensitivitiy to topical vancomycin in a case of chronic endophthalmitis.

Purpose: To report a case of ocular allergy to topical vancomycin eye drops. Methods: Interventional case report. Results: A 76-year-old man presented with progressive itching, soreness, and burning sensations, photophobia, and tearing in his left eye 3 days after completing 2 weeks of treatment with 5% vancomycin eye drops. He had been suffering for the previous 2 years from chronic recurrent endophthalmitis and secondary glaucoma associated with cataract surgery in his left eye. He denied any history of drug or food allergy. Examination by slit-lamp microscope showed left periorbital skin erosive rash, marked hyperemia of conjunctiva, central epithelial defect, corneal stromal edema, and Descemet membrane striae. Vancomycin skin tests were positive. The patients symptoms resolved after discontinuing vancomycin eye drops and responded well to systemic corticosteroids, antihistamines, and topical corticosteroid treatments. Teicoplanin treated his endophthalmitis successfully. Conclusion: A case of hypersensitivity to topical vancamycin was reported. Patients who receive topical vancomycin should be informed of this possible adverse effect and instructed to contact their ophthalmologists if allergic reactions occur.

Laser-assisted subepithelial keratectomy for dry eye associated with soft contact lenses.

PURPOSE: To evaluate the efficacy and safety of laser‐assisted subepithelial keratectomy (LASEK) in myopic patients with preoperative dry eye associated with extended soft contact lens use. SETTING: Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan. METHODS: This nonrandomized prospective interventional case series study comprised 9 patients (18 eyes) with dry eye and keratoconjunctivitis associated with long‐term complicated soft contact lens use. Prior to LASEK surgery and for 1 year following surgery, measurements and comparisons were made of uncorrected visual acuity, best corrected visual acuity (BCVA), manifest refraction, symptoms, tear function, ocular surface abnormality, and corneal sensitivity. RESULTS: No significant differences (P>.05) in the mean results of the Schirmer test with anesthesia and tear breakup times before and after LASEK were evident. Corneal sensitivity recovered within 1 month after LASEK. No complications occurred during or after LASEK. Subepithelial haze and superficial punctate epithelial defects occurred in 1 eye, representing a loss of 1 Snellen line of BCVA. CONCLUSION: Laser‐assisted subepithelial keratomileusis in dry eyes and keratoconjunctivitis associated with complicated soft contact lens wearing was safe and efficacious.

Recurrent Corneal Epithelial Erosion as a Manifestation of Posner-Schlossman Syndrome

Purpose: To report a case of recurrent cornea abrasion as an early sign of glaucomacyclitic crisis (Posner-Schlossman syndrome). Methods: Observational case report: A 27-year-old man presented with recurrent corneal abrasion in his right eye. Result: Findings in this young man were consistent with a diagnosis of Posner-Schlossman syndrome. Conclusion: Glaucomatocyclitic crisis can manifest as recurrent corneal erosion, which we suggest should be included in a differential diagnosis of this condition. Intraocular pressure needs to be checked whenever Posner-Schlossman syndrome is suspected.