Establishment of a cultivated human conjunctival epithelium as an alternative tissue source for autologous corneal epithelial transplantation

PURPOSE: The corneal epithelium is essential for maintaining corneal transparency, and efforts have been made to develop improved techniques for corneal epithelial transplantation in patients with total limbal failure. We evaluated the suitability of transplanted cultivated human conjunctival epithelium (HCjE) as a corneal epithelium replacement in rabbits with total corneal and limbal deficiency. METHODS: HCjE cells, cultivated on human amniotic membrane (AM) to confluence and exposed to an air-liquid interface (air-lifted), were transplanted onto denuded rabbit corneas and monitored for 2 weeks. The cultivated HCjE sheet and the engrafted epithelium were analyzed by immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The transplanted HCjE remained transparent, smooth, and without epithelial defects during the follow-up period. Both the cultivated HCjE cells and the engrafted epithelium manifested five to six layers of stratified squamous epithelium similar in morphology to normal corneal epithelium. The basal cells expressed the putative stem cell markers (ABCG2 and P63) and hemidesmosome and desmosome component proteins. The cytokeratins (CK4, CK13, CK3, and CK12) and MUC4 were found in the engrafted epithelium. However, MUC5AC was not expressed. The results indicate that HCjE cultivated on AM has the potential to be used as an alternative corneal epithelium. CONCLUSIONS: The transplantation of cultivated HCjE sheets is a promising technique for the treatment of eyes with limbal failure.     

Clinical outcome of autologous cultivated limbal epithelium transplantation

PURPOSE: To report the clinical outcome of autologous cultivated limbal epithelial transplantation. METHODS: Eighty-six patients' records and their clinical photographs were reviewed for demographics, primary etiology, type of limbal transplantation, ocular surface stability, visual acuity, final outcome and possible factors affecting outcome and complications. RESULTS: Eighty-eight eyes of 86 patients with limbal stem cell deficiency (LSCD) underwent autologous cultivated limbal epithelium transplantation between March 2001 and May 2003, with a mean follow-up of 18.3 months. The etiology of LSCD was alkali burns in 64% patients. Sixty-one eyes had total LSCD. Thirty-two of the 88 eyes had undergone amniotic membrane transplantation and 10 eyes had previously undergone limbal transplantation with unfavorable outcome. Nineteen eyes underwent penetrating keratoplasty, of which 11 grafts survived at the final follow-up. Finally, 57 eyes (73.1%, 95% CI: 63.3-82.9) had a successful outcome with a stable ocular surface without conjunctivalization, 21 eyes (26.9%, 95%CI: 17.1-36.7) were considered failures and 10 patients were lost to follow-up. CONCLUSION: LSCD can be successfully treated by autologous cultivated limbal epithelium transplantation in majority of the cases.     

Ultrastructural study of conjunctival lymphoma

Summary The electron microscopic investigation of the conjunctival lymphoma of a male patient, aged 25 has shown that the tumour consists prevailingly of lymphoid elements. To a smaller extent, reticulum cells were revealed too. Of these, some were cells with lobated nuclei and multinuclear giant cells resembling the cells of Hodgkin and Sternberg-Reed type found in lymphogranulomatosis. The ultrastructural investigation supports the hyperplastic character of the conjunctival lymphoma.

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Zusammenfassung Die elektronenmikroskopische Untersuchung des Bindehautlymphoms eines 25jährigen Mannes zeigte, daß der größte Teil der Geschwulst aus lymphoiden Zellen besteht. In kleinerer Zahl sind Reticulumzellen zu finden. Zwischen diesen sind Zellen mit lobulärem Kern und auch Riesenzellen vorhanden. Diese letzten erinneren an den bei Lymphogranulomatose auffindbaren Hodgkin- und Sternberg-Reed-Zellen. Die elektronenmikroskopischen Untersuchungen unterstützen den hyperplasiogenen Charakter des Lymphoms.

     

Ultrastructural observations on the development of the human conjunctival epithelium

In human embryos with a gestation age of 8.6–22 weeks, the palpebral conjunctival epithelium was examined by transmission electron microscopy (TEM). During the gestation period studied, the structural integrity of the tissue is established by an increase in the quantity of tonofilaments, desmosomes, and hemidesmosomes as well as by the undulating appearance of the cell membranes, the widening of the intercellular space, and the development of cytoplasmic protrusions into it. The superficial cells display a chronological sequence in the elaboration of transport mechanisms. A precursor stage is described for hemidesmosome formation at the interface between the basal cell membrane and the conjunctival stroma.Presented in part at the 15th Annual Meeting of the European Club for Ophthalmic Fine Structure in Paris on 14 and 15 September 1987This study was performed under the support of a training grant in ophthalmic electron microscopy from Deutsche Forschungsgemeinschaft     

自体口腔黏膜上皮细胞培养眼表重建的实验研究

管计数、VEGF阳性表达量均最少.印迹细胞检杏:口腔黏膜细胞移植组角膜上皮未发现杯状细胞.结论 利用体外培养的自体口腔黏膜上皮细胞进行移植,可减少角膜新生血管的形成,其抑制新生血管的作用优于单纯羊膜移植抑制新生血管的作用.     

Ultrastructural localization of lectin-binding sites on the surface of the guinea pig conjunctival epithelium

The cell-surface binding sites of two lectins, concanavalin A (Con A) and wheat-germ agglutinin (WGA) in the guinea pig conjunctiva were investigated at the ultrastructural level by means of pre-embedding staining of the tissue with lectin-colloidal gold complexes. Wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, showed prominent and fairly uniform binding to the microvilli. The binding was markedly increased in the vicinity of the goblet cells, indicating that the same carbohydrate ligands were also present in the mucus. In contrast, no binding of concanavalin A, which recognizes mannose and N-glucose, was observed. The results suggest the presence of sialic acid and galactose as the constituent carbohydrates of glycoconjugates in the surface membrane of conjunctival epithelial cells as well as in the mucus produced by the goblet cells.This investigation was supported by grants from the Swedish Medical Research Council (Project 12X-734) and from the Research Committee of Östergbtlands Läns Landsting     

Ultrastructure of conjunctival epithelium replacing corneal epithelium

After corneas of mice had been totally denuded of their epithelium by the application of n-heptanol, the new epithelium which grew over the corneas was studied by electron microscopy at intervals up to 7 months. The purpose was to compare the basal attachment of the new cells, derived from conjunctiva, with that of true corneal epithelial cells growing on the same type of substratum, and studied previously. Goblet cells appeared after 2 weeks amid the squamous type of epithelial cells which had resurfaced the cornea in about 1 week. Goblet cells increased up to at least 6 weeks, but had decreased by 3 months. They persisted, however, for the entire 7 months of the study. Goblet cells had only a small area of contact with the basal lamina, and they had few desmosomes or hemidesmosomes. Basal cells of the squamous type had complex features of their basal attachment quite different from those of normal or repairing corneal epithelial cells studied previously. Flat cytoplasmic extensions of squamous cells underlay most of the goblet cell basal pole which therefore had only a small area on the basal lamina. Numerous filaments inserted into desmosomes and hemidesmosomes of squamous cells, and prominent bundles of these filaments lay just above the basal plasma membrane. They were orientated parallel to the radial axis of the cornea. Closely spaced corrugations of the basal plasma membrane were also orientated in this axis, as well as rows of hemidesmosomes. Even after a period of 7 months, the morphological features of conjunctival cells did not come to resemble those of normal corneal epithelium. The radial arrangement of fibers, hemidesmosome rows, and corrugations is interpreted as a reflection of the continued centripetal migration of the epithelium.     

Ultrastructural study of the corneal epithelium in the recurrent erosion syndrome.

BACKGROUND--This study describes the adhesion systems in the corneal epithelium in the recurrent erosion syndrome and the mechanisms by which binucleate and multinucleate cells are formed within the epithelium. METHODS--Twenty five samples of sliding epithelium were obtained from 23 patients and were examined by conventional light and transmission electron microscopy. RESULTS--The separation of the anchoring system occurred either below the level of the anchoring plaques or at the level of the epithelial cell membrane. Normal and degenerate polymorphonuclear leucocytes were found within and between the epithelial cells and within the anchoring layer. Binucleate and multinucleate cells were found within all the layers of the epithelium as were cysts containing degenerate cellular material. CONCLUSION--The presence of leucocytes and degenerate epithelial cells within the sliding epithelium suggests that these are the source of the metalloproteinases which cleave Bowman's layer below the anchoring system. The formation of binucleate and multinucleate giant cells does not appear to occur by fusion of adjacent cells, but rather by nuclear indentation and cleaving due to an abnormal microtubular system in the cytoskeleton.     

Dacryoadenoma. A unique tumor of the conjunctival epithelium

For 15 years, a 48-year-old woman had been aware of a pinkish translucent inferior epibulbar mass toward the fornix that was stationary in size. Light microscopic evaluation of the excised specimen showed that it was a gland-forming benign tumor. It originated from the surface epithelium by the downward invagination of tubular formations that underwent secondary and tertiary ramifications. The surface epithelium overlying the mass was not normal conjunctival squamous epithelium, but it was a modification in the form of a superficial layer of columnar to cuboidal cells surmounting several nuclei similar to the cells lining the subepithelial lumen-forming units. Electron microscopy showed that both the surface and tumor epithelium contained apical lacrimal-type zymogen granules and basilar whorls of rough-surfaced endoplasmic reticulum. Scattered myoepithelial cells were associated with the acinar-type epithelium of the tumor, and there were goblet cells intermixed. Neither light nor electron microscopy disclosed the presence of true ducts. The authors conclude that the lesion was a benign proliferation of lacrimal secretory-type cells and suggest the term "dacryoadenoma" for this previously undescribed entity.     

Cytologic evaluation of conjunctival epithelium after cataract surgery.

Although numerous studies have examined corneal epithelial wound healing, few reports describe conjunctival wound healing. Thus, we evaluated the conjunctival epithelium by brush cytology, in which a miniature brush was used to collect many cells relatively noninvasively. The temporal and superior conjunctivae were examined before and after cataract extraction on the first, third, seventh, and fourteenth days and then the first, second, and third months postoperatively, in a group of 20 patients (9 men and 11 women with a mean age of 69 years 3 months +/- 10 years). The cells collected were stained by the Papanicolaou method. Each postoperative specimen contained keratinized, polygonal, round, columnar, elongated, and inflammatory cells, as well as cells with nuclear alterations. No keratinized, elongated, or inflammatory cells were observed preoperatively. In contrast, columnar cells, which were present preoperatively, decreased during the wound healing process. Such nuclear changes as chromatin formation or the appearance of snake-like figures were observed in several patients postoperatively. These cellular changes may represent altered differentiation during the healing process. However, the conjunctival epithelium returned to normal within 2 months postoperatively in all patients.     

Autologous cultivated conjunctival transplantation for pterygium surgery

PURPOSE: To investigate the safety and efficacy of autologous cultivated conjunctival transplantation for the treatment of primary pterygium. DESIGN: Prospective nonrandomized clinical trial. METHODS: Forty patients with primary pterygium were recruited. Excision of the pterygium was followed by reconstruction using a serum-free derived autologous cultivated conjunctival equivalent in 22 patients (group A) and conventional denuded amniotic membrane transplantation in 18 patients (group B). In group A patients, conjunctival epithelial equivalents were constructed by the ex vivo expansion of conjunctival epithelial cells on human amniotic membranes (HAM). The main outcome measures were conjunctival epithelialization, recurrence, survival analysis, and incidence of complications. RESULTS: The mean follow-up was 14.1 +/- 3.9 months (range, 12 to 25 months). Complete epithelialization was achieved within five days after surgery in group A patients compared with approximately three weeks for group B patients. The proportion of patients that had true recurrences was 22.7% in group A and 25.0% in group B. The mean time to recurrence was 4.8 +/- 1.6 months in group A and 5.0 +/- 2.9 months in group B. No ocular complications were noted in group A, while one eye (6.0%) in group B developed scleral necrosis associated with a persistent epithelial defect. CONCLUSIONS: Transplantation of an autologous cultivated conjunctival epithelial sheet facilitated early postoperative epithelialization and recovery, and may aid in preventing serious complications associated with simple denuded HAM transplantation, such as scleral necrosis and secondary infection. This may provide a novel method for conjunctival epithelial replacement in the treatment of ocular surface disorders.     

Unusual structure of rat conjunctival epithelium. Light and electron microscopy

The conjunctiva of the adult Sprague-Dawley rat was studied by light microscopy of 3 micron glycol methacrylate sections of whole eyes with lids and by electron microscopy of conjunctiva from the lower fornix. Rat conjunctiva is unique among species studied. All the superficial epithelial cells are squamous cells rather than polyhedral or columnar cells. Furthermore, the goblet cells are aggregated into clusters rather than distributed randomly throughout the epithelium. These clusters are not found at the lid margin or limbus, but are present in the palpebral and bulbar conjunctivae and achieve maximal size and number near the fornix. The stratified squamous epithelium is typical, composed of a layer of basal cells, an intermediate zone of wing cells, and an upper zone of several layers of squamous cells. Dividing cells are seen only in the basal layer. Occasional mononuclear leukocytes are found in the basal and intermediate layers. The goblet cell clusters are largely composed of columnar cells. Goblet cells predominate, but there are also occasional tuft cells, characterized by thick microvilli at their apices. Basal cells form only an incomplete layer beneath the columnar cells, which in places span the entire epithelium. The conjunctiva of the adult rat has few cells with potential for immunological activity. It does not contain appreciable numbers of plasma cells, and lymphoid follicles are absent.     

Keratinization of the bulbar conjunctival epithelium in superior limbic keratoconjunctivitis in humans. An electron microscopic study.

A prominent histological feature of superior limbic keratoconjunctivitis is the presence of keratinization of the superior bulbar conjunctiva. Ultrastructurally the affected conjunctival epithelium shows all of the characteristics of keratinization as described in skin. There is a superficial layer of horny cells with reduced organelles and thickened cell membrane. In the intermediate layers, the cells contain an increased number of cytoplasmic filaments which are sometimes aggregated into dense masses, keratohyalin granules, both in the cytoplasm and in the nucleus, membrane coating granules, lysosomes and various other types of membranous structures.     

Autologous cultivated conjunctival transplantation for recurrent viral papillomata

PURPOSE: To describe the use of an autologous serum-free derived cultivated conjunctival epithelial equivalent in the treatment of extensive recurrent viral papillomata. DESIGN: Interventional case report. METHODS: A 10-year-old child with extensive recurrent viral papillomata involving the superior and inferior tarsal, forniceal, and bulbar conjunctiva underwent surgical excision of all diseased areas and double freeze-thaw cryotherapy. Autologous serum-free cultivated conjunctival equivalents were used to reconstruct the ocular surface and conjunctival fornices. RESULTS: Almost complete epithelialization was achieved within 5 days postoperatively. A good cosmetic and functional result was obtained, and no recurrences or cicatricial complications developed during 12-month follow-up. CONCLUSION: Transplantation of autologous cultivated conjunctiva was effectively used in the reconstruction of the ocular surface after extensive excision of recurrent viral papillomata. This modality of treatment may be useful in the treatment of ocular surface disorders in which extensive conjunctival and fornix reconstruction is required.