Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

PURPOSES: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. METHODS: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. RESULTS: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). CONCLUSION: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Nonsebaceous lymphadenoma of salivary gland: report of a case with immunohistochemistry and review of the literature.

OBJECTIVE: Nonsebaceous lymphadenomas are rare benign neoplasms. We emphasize the role of immunohistochemistry and attempt to elucidate the pathogenesis by investigating the distribution of 2 transcription factors, MYC and BLIMP1. STUDY DESIGN: A 70-year-old man was evaluated for a 3-cm left parotid mass. Ultrasound-guided fine-needle aspiration biopsy findings were suggestive of a diagnosis of pleomorphic adenoma. A left superficial parotidectomy was performed, and based on histopathology a diagnosis of lymphadenoma, nonsebaceous type, was rendered. RESULTS: The tumor was positive for AE1/3, CKA, BclII, P63, CD79a, CD3, and MYC; focally positive for CK7 and epithelial membrane antigen; and negative for CD10, calponin, CD117, and BLIMP1. CONCLUSIONS: The rarity of nonsebaceous lymphadenoma and its superficial resemblance to commoner salivary gland tumors may present a diagnostic challenge for pathologists. The expression of MYC in the ductal component and the differentiation-related expression of PRDM1 in the superficial keratinizing layers point to a potential role for these 2 transcription factors in the pathogenesis of this neoplasm.

Intraoral carcinosarcoma on the floor of the mouth mimicking a benign lesion.

Carcinosarcoma is a rare malignant disease with aggressive behaviour rarely producing oral manifestations. This article reports a case of an intraoral carcinosarcoma affecting a 71-year-old black male; the diagnosis was made by histopathological and immunohistochemical analyses. Computed tomography scanning showed metastatic masses in the lungs. The patient was underwent a chemotherapy protocol regimen, but died as a consequence of the disease within 10 months of diagnosis. Distinctive characteristics of this presentation were the location of the lesion (floor of the mouth) and its clinical features resembling a benign lesion. A brief review of intraoral carcinosarcoma cases in the literature is also presented.

Microchimerism in labial salivary glands of hematopoietic stem cell transplanted patients.

OBJECTIVE: Microchimerism has been extensively investigated in autoimmune diseases, which display similarities with graft-vs-host disease. This study was conducted to investigate the presence of microchimerism in minor salivary glands of hematopoietic stem cell transplanted patients, one of the targets of graft-vs-host disease. METHODS: Labial salivary glands biopsy specimens from 11 stem cell transplanted patients were analysed. The samples were grouped in control (five specimens from a female-to-female transplantation) and study group (five glands from male-to-female transplantation). One male transplanted patient was used as a positive control. Fluorescence in situ hybridization with Y-chromosome probe and immunofluorescence with anticytokeratin AE1/AE3 and CD45 were used to identify Y-chromosome positive glandular epithelial cells from allogeneic hematopoietic stem cell transplanted patients. RESULTS: In the study group, all samples were positive to Y-chromosome and cytokeratin AE1/AE3, in agreement with the pattern exhibited by male labial salivary gland. None of the samples from control group were positive to Y-chromosome despite being positive to cytokeratin AE1/AE3. Positivity to CD45 was not relevant. CONCLUSION: Microchimerism in the labial salivary glands of sex-mismatched stem cell transplanted patients is a real phenomenon. Further studies are necessary to elucidate the impact of this phenomenon on the clinical status of stem cell transplanted patients.

In vivo behavior of complete human oral mucosa equivalents: characterization in athymic mice.

BACKGROUND AND OBJECTIVE: The interest in tissue engineering as a way to achieve repair of damaged body tissues has led to the carrying out of many studies whose results point to the potential effectiveness of these methods. In a previous study, we reported the obtaining of complete autologous oral mucosa equivalents (CAOMEs), characterized by oral immature keratinocytes and stem cells on an autologous plasma and fibroblast scaffold. The purpose of this study is to show their behavior in vivo, by using them as free grafts in experimental animals, and to demonstrate their potential capacity to regenerate oral mucosa. MATERIAL AND METHODS: We engineered CAOMEs, as previously described. All CAOMEs thus obtained were used as free grafts in nu/nu mice. To assess their evolution in vivo, we studied their histological and immunohistochemical features by using AE1/AE3 pancytokeratin, the 5/6 cytokeratin pair, cytokeratin 13, laminin 5, collagen IV, vimentin, p-63 and Ki-67, at 7, 14 and 21 d. RESULTS: The structure became progressively closer to that of oral mucosa samples. Cytokeratin 5/6 staining became increasingly intense in the basal and suprabasal layers, and cytokeratin 13 was exclusively positive in the superficial layers. The basal membrane was completed in 21 d. Vimentin showed a correct formation of the chorion. The increasingly positive staining of p-63 and Ki-67 indicated that the regeneration process was taking place. CONCLUSION: The present study shows the potential regenerative capacity of the CAOMEs by their ability to reach maturity similar to that seen in oral mucosa.

In vitro engineering of complete autologous oral mucosa equivalents: characterization of a novel scaffold.

BACKGROUND AND OBJECTIVE: Restoration of oral mucosa defects by means of in vitro-cultured equivalents has become a valid alternative in the field of oral and periodontics surgery. Although different techniques have been described, none has been able to provide an equivalent with an autologous scaffold for the epithelium. The purpose of this study was to obtain complete autologous oral mucosa equivalents (CAOME) using the patient's own fibroblasts and plasma and to characterize these equivalents both morphologically and immunohistochemically. MATERIAL AND METHODS: We acquired cell types (keratinocytes and fibroblasts) from the same mucosal samples, which were taken from healthy patients who underwent oral surgery. To construct the CAOME, a small sample of blood was obtained from the patient and subsequently processed to obtain a fibrin glue scaffold. All CAOME thus obtained were stained using the standard hematoxylin and eosin method to study their morphological characteristics. To establish the type of cells in the epithelial layer, CAOME were stained with pancytokeratin AE1/AE3, cytokeratins 5/6 and 13, p-63 and Ki-67. Finally, laminin 5 and collagen IV were used to reveal the presence of a basal membrane. RESULTS: The CAOME featured a monolayer of cube-shaped epithelial cells similar to that found on the basal layer of the oral mucosa. Close to the epithelial layer lay the fibrin and fibroblasts-embedded scaffold. The CAOME was positive to pancytokeratin AE1/AE3, cytokeratin 5/6 and p-63. No reaction was found to cytokeratin 13 and Ki-67. There was staining to laminin 5 but not to collagen IV. CONCLUSIONS: It is possible to engineer a CAOME with an epithelium of basal-like and immature keratinocytes, which could potentially reconstruct in vivo loss of tissue.

Merkel cell carcinoma of the alveolar mucosa in a young adult: a rare case report.

Merkel cell carcinoma (MCC) is an extremely rare and aggressive primary neuroendocrine neoplasm of the skin with a poor prognosis. It occurs mainly in the sixth and seventh decades of life and is usually associated with damage caused by over exposure to the sun. Its occurrence in intraoral mucosal sites is rare, and we know of only six reported cases. We report a rare case of MCC that arose in the alveolar mucosa of a young adult.

Adenomatoid odontogenic tumor concomitant with cystic complex odontoma: case report.

This case report describes a 10-year-old female patient with an adenomatoid odontogenic tumor developing together with a cystic complex odontoma. This occurrence is considered very unusual. Immunohistochemical detection of cytokeratins AE1/AE3, CK5, CK8, CK10, CK14, CK19 and Ki-67 was performed.

Existence of small slow-cycling Langerhans cells in the limbal basal epithelium that express ABCG2.

Despite the obvious importance of limbal stem cells in corneal homeostasis and tumorigenesis, little is known about their specific biological characteristics. The purpose of this study was to characterize limbal slow-cycling cells based on the expression of ABCG2 and major histocompatibility complex (MHC) class II and the cell size. Wistar rats were daily injected with 5-bromo-2-deoxyuridine (BrdU) at a dose of 5 mg/100 g for 2 weeks. After 4-week BrdU-free period, corneal tissues were excised, and immunofluorescence staining for ABCG2, BrdU, and MHC class II was performed by confocal microscopy. In another series, corneal tissues of normal rat were double immunostained for ABCG2, keratin 14, keratin 3, CD11c, and MHC class II. In addition, limbal, peripheral and central corneal epithelial sheets were isolated by Dispase II digestion and dissociated into single cell by trypsin digestion and cytospin preparations were double immunostained for ABCG2 and MHC class II. The cell size and nucleus-to-cytoplasm (N/C) ratio of limbal ABCG2+ cells were analyzed and compared with those of cells from other zones. BrdU label-retaining cells (LRCs) with expression of ABCG2 were found in the limbal epithelial basal layer, but not in other parts of the cornea. Approximately 20% of these cells were MHC class II positive. All MHC class II+ cells in the corneal epithelium were positive for CD11c, a marker for dendritic cells (DCs). Double labeling with ABCG2 and keratin 14 showed that nearly four-fifth of limbal ABCG2+ cells were positive for keratin 14 but negative for keratin 3, exhibiting an undifferentiated epithelial cell lineage. Cytospin sample analysis revealed the presence of a distinct population of smaller ABCG2+ cells with expression of MHC class II with a larger N/C ratio in the limbal epithelium. A new population of small slow-cycling cells with large N/C ratio has been found to express ABCG2 in the limbal epithelial basal layer. Some of these cells normally express MHC class II antigen. These findings may have important implications for our understanding of the characteristics of limbal slow-cycling cells.

RCE1 corneal epithelial cell line: its variability on phenotype expression and differential response to growth factors.

BACKGROUND: By serial transfer of rabbit corneal epithelial cells, the spontaneous RCE1 cell line was previously established. These cells mimic the stage-dependent differentiation of the corresponding cell type. METHODS: RCE1 cells were cultured either on plastic culture dishes or on collagen rafts to compare the epithelial ultrastructure after growth on these substrata. Phenotypic variability was studied after subcloning of cells. The differentiation ability of each subclone was determined by Western blot with antibodies against the differentiation-linked keratin pair K3/K12 and by measuring LDH activity and LDH isozymes in cytosolic extracts. The proliferative response of RCE1 cells to EGF, TGFalpha, amphiregulin, bFGF or IL-6 was determined under serum-free culture conditions. RESULTS: Cells grown on collagen rafts formed 5- to 7-layered epithelia with characteristics closer to those found in normal corneal epithelium than cells cultivated on plastic substrata, which formed 3- to 5-layered epithelia. Subcloning experiments demonstrated that every proliferative cell is able to grow and constitute stratified epithelia expressing K3/K12 keratins. LDH levels in RCE1 epithelia were similar to those of cultured or freshly harvested corneal epithelia; however, they showed a slightly altered LDH isozyme set, with prevalence of LDH-3 isoform. Whereas EGF and TGF-alpha were equipotent, amphiregulin elicited a 4-fold lower proliferative response. Also, bFGF was 10-fold less mitogenic than EGF, and IL-6 had the lowest effect with an ED(50) 20-fold lower than EGF. CONCLUSIONS: The results demonstrate that every RCE1 proliferative cell has the ability to generate epithelial sheets. We conclude that EGF and TGF-alpha are the major effectors of RCE1 cell proliferation.

Amniotic membrane immobilized poly(vinyl alcohol) hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium.

Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis.