Tetsuya Kawakita

How Does Amniotic Membrane Work

Transplantation of amniotic membrane as a temporary or permanent graft promotes epithelial wound healing and exerts potent anti-inflammatory and anti-scarring effects on the ocular surface. These actions depend on the killing of allogeneic amniotic cells and preservation of the cytokine-containing matrix during the preparation of the amniotic membrane. This review describes how these actions inherently operate in utero and how amniotic membrane transplantation aims to recreate such a fetal environment to exert these actions by insulating the surgical site from the host environment. These actions also render the amniotic membrane a unique niche capable of expanding both epithelial and mesenchymal progenitor cells ex vivo, while maintaining their normal cell phenotypes. As a result, the amniotic membrane becomes an ideal substrate for engineering different types of ocular surface tissues for transplantation. Further studies investigating the exact molecular mechanism by which the amniotic membrane works will undoubtedly unravel additional applications in reconstruction and engineering of both ocular and nonocular tissues in the burgeoning field of regenerative medicine.

Functional lacrimal gland regeneration by transplantation of a bioengineered organ germ

The lacrimal gland has a multifaceted role in maintaining a homeostatic microenvironment for a healthy ocular surface via tear secretion. Dry-eye disease, which is caused by lacrimal gland dysfunction, is one of the most prevalent eye diseases that cause corneal epithelial damage and results in significant loss of vision and a reduction in the quality of life. Here we demonstrate orthotopic transplantation of bioengineered lacrimal gland germs into adult mice with an extra-orbital lacrimal gland defect, a mouse model that mimics the corneal epithelial damage caused by lacrimal gland dysfunction. The bioengineered lacrimal gland germs and harderian gland germs both develop in vivo and achieve sufficient physiological functionality, including tear production in response to nervous stimulation and ocular surface protection. This study demonstrates the potential for bioengineered organ replacement to functionally restore the lacrimal gland.

Prevalence and Risk Factors of Dry Eye Disease in Japan: Koumi Study

OBJECTIVE To estimate the prevalence and risk factors of dry eye disease (DED) in a rural setting in Japan. DESIGN Cross-sectional study. PARTICIPANTS We included 3294 subjects, aged ≥ 40 years who were in the residential registry for Koumi town. INTERVENTION Subjects in a rural mountain area, Koumi town, completed questionnaires designed to detect dry eye diagnosis and risk factors. MAIN OUTCOME MEASURES Clinically diagnosed DED was defined as the presence of a previous clinical diagnosis of DED by ophthalmologists or severe symptoms of DED (both dryness and irritation constantly or often). Current symptoms of DED and possible risk factors such as age, gender, educational history, smoking history, alcohol drinking history, height and weight, visual display terminal (VDT) use, and contact lens (CL) wear, and past/current history of certain common systemic diseases were the main outcome measures. We used logistic regression analysis to examine associations between DED and other demographic factors. RESULTS Of the 3294 eligible residents, 2791 residents (85%) completed the questionnaire. The percentage of women with a composite outcome of clinically diagnosed DED or severe symptoms (21.6%; 95% confidence interval [CI], 19.5-23.9) was higher than that of men (12.5%; 95% CI, 10.7-14.5; P<0.001). A low body mass index (BMI; odds ratio [OR], 2.07; 95% CI, 0.98-4.39), CL use (OR, 3.84; 95% CI, 1.46-10.10), and hypertension (HT) (OR, 1.39; 95% CI, 0.94-2.06) were risk factors for DED in men. Use of a VDT (OR, 2.33; 95% CI, 1.12-4.85), CL use (OR, 3.61; 95% CI, 2.13-6.10), and myocardial infarction or angina were the risk factors (OR, 2.64; 95% CI, 1.51-4.62), whereas high BMI was a preventive factor (OR, 0.69; 95% CI, 0.48-1.01) for DED in women. CONCLUSIONS Among a Japanese cohort, DED leading to a clinical diagnosis or severe symptoms is prevalent. Use of CLs was a common dry eye risk factor in both genders. The condition is more prevalent in men with low BMI, HT, and in women with myocardial infarction or angina and VDT use. Relevant measures directed against the modifiable risks may provide a positive impact on public health and quality of life of Japanese. FINANCIAL DISCLOSURE(S) The authors have no proprietary or commercial interest in any materials discussed in this article.

Clinical characteristics of conjunctivochalasis with or without aqueous tear deficiency

Aim: To show characteristic ocular surface findings caused by conjunctivochalasis (CCh) in dry eye patients with or without aqueous tear deficiency (ATD). Design: Comparative non-interventional cases. Patients and methods: Clinical data of five ATD patients without CCh (group A), eight CCh patients with ATD (group B), and eight CCh patients without ATD (group C) were retrospectively reviewed. Presence or absence of CCh was determined by fluorescein staining to outline tear meniscus and conjunctival folds with an enhancing filter. Dry eye symptoms, history of subconjunctival haemorrhage, meibum expression, tear break up time, fluorescein and rose bengal staining, and fluorescein clearance test, and other abnormal ocular surface findings were measured. Results: CCh patients were significantly older (p = 0.001). In pure ATD, the principal symptom of dryness became worse as the day progressed. In contrast, blurry vision, burning sensation, and dryness became worse during reading in all CCh patients (p = 0.0008) or worse in the morning upon awakening in the majority patients with CCh only (p = 0.02). Besides the interpalpebral exposure, which was noted in ATD, positive fluorescein or rose bengal staining was noted in the redundant conjunctival folds and the non-exposure zone in CCh (p = 0.0008). Redundant conjunctival folds were present in both lower and upper bulbar conjunctiva, obliterating both lower and upper tear meniscuses, and spatially correlated with anterior migration of the mucocutaneous junction in CCh. Delayed tear clearance was significantly more prevalent in CCh than ATD (p = 0.0008). Vigorous blinking worsened in CCh but not in ATD (p = 0.0008). Lacrimal puncta were swollen in groups B and C, but not in group A (p = 0.04). Conclusions: CCh is not restricted to the lower bulbar conjunctiva, and contributes to pathogenesis of dry eye by obliterating both lower and upper tear meniscus, causing unstable tear film and by creating delayed tear clearance. Dry eye symptoms were worsened by downgaze during reading and by vigorous blinking. Other characteristic signs including subconjunctival haemorrhage, swollen puncta, anterior migration of the mucocutaneous junction, and patterns of dye staining also help distinguish dry eye associated with CCh from that caused by ATD alone.

Intrastromal Invasion by Limbal Epithelial Cells Is Mediated by Epithelial-Mesenchymal Transition Activated by Air Exposure

Corneal epithelial stem cells are located in the basal layer of the limbus between the cornea and the conjunctiva. Regulation of these limbal epithelial progenitor cells by the stromal niche dictates corneal surface health. To further characterize this process, limbal explants were cultured at the air-fluid interface, termed air-lifting, to stimulate the niche. As compared to submerged cultures, air-lifting significantly promoted epithelial stratification, migration, proliferation, and intrastromal invasion by limbal epithelial cells. Epithelial intrastromal invasion was noted when the limbal, but not corneal, epithelium was recombined with the limbal stroma containing live, but not dead, cells. Invading limbal basal cells displayed up-regulated nuclear expression of p63 and Ki67, down-regulated E-cadherin and cornea-specific keratin 3, and switched expression of beta-catenin from intercellular junctions to the nucleus and cytoplasm, indicating the activation of the Wnt/beta-catenin pathway. Invaded cells isolated by collagenase from the stroma of air-lifted, but not submerged, explants showed vivid clonal growth on 3T3 fibroblast feeder layers and complete epithelial-mesenchymal transition by expressing nuclear p63 and cytoplasmic S100A4. These findings collectively suggest that epithelial-mesenchymal transition via the Wnt/beta-catenin pathway influences the fate of limbal epithelial cells, likely to be progenitor cells, between regeneration and fibrosis when the stromal niche is activated.

The use of human mesenchymal stem cell-derived feeder cells for the cultivation of transplantable epithelial sheets.

PURPOSE To report the efficacy of human bone marrow-derived mesenchymal stem cells as a source of feeder cells for the cultivation of transplantable corneal epithelial cell sheets. METHODS Human mesenchymal stem cells (marrow adherent stem cells; MASCs) were cultured in alpha-modified Eagles medium with 10% serum and were treated with mitomycin C. Expression of cytokines in MASCs was confirmed by reverse transcription-polymerase chain reaction. Human limbal epithelial cells were cocultured with MASCs or 3T3 feeder cells to compare colony-forming efficiency (CFE). Limbal epithelial cells were cultured on MASCs or 3T3 feeder cells at the air-liquid interface to allow stratification, and stratified epithelial sheets were analyzed by immunohistochemistry against cytokeratin 3 (K3), K15, p63alpha, and ABCG2. Rabbit limbal epithelial cell sheets were cultivated with MASC feeder cells and transplanted to the ocular surface of the limbal-deficient rabbits. Epithelial grafts were observed by slit lamp microscopy for 4 weeks and then evaluated by histology and immunohistochemistry against K3 and K4. RESULTS MASC feeder cells expressed keratinocyte growth factor, hepatocyte growth factor, and N-cadherin. The CFE of human limbal epithelial cells was similar in MASC and 3T3 feeder groups. Stratified cell sheets were successfully cultivated with MASC feeder cells expressing K3, K15, p63alpha, and ABCG2. Transplanted epithelial sheets regenerated the corneal phenotype in limbal-deficient rabbits. CONCLUSIONS MASC-derived feeder cells are suitable for the engineering of epithelial sheets, avoiding the use of potentially hazardous xenologic feeder cells.

Transformation of α-glycine to γ-glycine

Transformation in a cell of the a form of glycine crystals to the γ form under high humidity was observed by an in situ observation method. The transformation of α-glycine to γ-glycine was found to be solvent-mediated phase transformation. The kinetic equation was obtained, and the calculated values were in good agreement with experimental values.

Porous Silk Fibroin Film as a Transparent Carrier for Cultivated Corneal Epithelial Sheets

Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin β1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium.

Stratified epithelial sheets engineered from a single adult murine corneal/limbal progenitor cell

The limbal region of the adult cornea contains stem cells which are ultimately responsible for regeneration of the corneal epithelium during wound repair. However, primarily‐isolated murine corneal/limbal epithelial cells rapidly senesce on plastic in a serum‐free low [Ca2+] medium, suggesting only transit amplifying cells are promoted. We developed a novel expansion method by seeding at a low cell density (<500 cells/cm2) and prolonging each culture time beyond the lifespan of transit amplifying cells (4 weeks). Expanded cells were uniformly small, negative to K12 keratin, but positive for p63 nuclear staining, and could be subcultured beyond 100 passages. After limiting dilution, one clone (TKE2) was selected that exhibited single cell clonal expansion with a doubling time of 34.2 hrs, and had normal karyotyping, but no anchorage‐independent growth. A single cell could be continually expanded to a confluent monolayer on denuded amniotic membrane and became stratified by exposing to the air‐medium interface. The resultant stratified epithelium expressed K14 keratin, involucrin, connexin 43 and p63, but not K12 keratin or Pax 6. However, expression of K12 could be up‐regulated by increasing extracellular calcium concentration and addition of foetal bovine serum (FBS) at P12, but less so at P85. Therefore, this murine lim‐bal/corneal epithelium‐derived progenitor cell line still retained the plasticity for adopting corneal lineage differentiation, could be useful for investigating limbal niche cues that may promote corneal epithelial fate decision.

Selenoprotein P Controls Oxidative Stress in Cornea

The ocular surface is always attacked by oxidative stress, and cornea epithelial cells are supposed to have their own recovery system against oxidative stress. Therefore we hypothesized that tears supply key molecules for preventing oxidative stress in cornea. The potential target key molecule we focused is selenoprotein P (SeP). SeP is a carrier of selenium, which is an essential trace element for many animals, for oxidative stress metabolism in the organism, and was extremely expressed in lacrimal gland. An experiment was performed with SeP eye drops in a rat dry eye model, prepared by removing the lacrimal glands. The anticipated improvement in corneal dry eye index and the suppression of oxidative stress markers were observed in SeP eye drop group. Furthermore, the concentration of SeP was significantly higher in dry eye patients compared with normal volunteers. Collectively, we concluded that tear SeP is a key molecule to protect the ocular surface cells against environmental oxidative stress.