MAB1345 Sigma-AldrichAnti-Collagen Type VII Antibody, CT, clone LH7.2

PURPOSE: The purpose of this study was to evaluate sutureless keratoplasty using a chemically-defined bioadhesive (CDB) made from food or medical additives. METHODS: Sutureless automated lamellar therapeutic keratoplasty (ALTK) using a CDB was performed on three rabbit eyes. Allogenic lamellar graft was transplanted onto the recipient bed using either suture fixation or a sutureless technique using the CDB. Slit-lamp examination was performed at selected intervals to evaluate the grade of epithelialization and the corneal clarity. The rabbits were killed at 90 days after operation and the eyes processed for histology, electron microscopic examination, and immunohistochemistry for cytokeratins and cell junction-related proteins. RESULTS: Sutureless keratoplasty was successfully performed with appropriate handling time before the CDB gelatinized. All the glued grafts were rapidly epithelialized within 7 days, and thereafter remained clear and attached for 90 days. Histologic and ultrastructural findings on the sutureless group showed the normal feature of stromal and epithelial cells and the grafts to be closely adhered with no inflammatory or scarring changes on the interface. Immunohistochemistry of the epithelial cells on the sutureless group revealed a similar expression pattern to the control group. CONCLUSIONS: These results demonstrate that sutureless keratoplasty using the CDB is easy to perform, with reliable attachment and no fear of toxic effects or disease transmissions. The authors expect the CDB to become a major choice for corneal treatment, especially in lamellar keratoplasty, posterior keratoplasty, and amniotic membrane transplantation on corneas.

To evaluate the efficacy and safety of a novel sutureless transplantation of bioadhesive-coated, sterilized, freeze-dried amniotic membrane (FD-AM) for ocular surface reconstruction.A bioadhesive-coated, freeze-dried amniotic membrane was made by freeze drying the denuded AM in a vacuum, applying the minimum amount of fibrin glue (mixture of fibrinogen and thrombin) necessary to retain adhesion on the chorionic side, and sterilizing it by gamma-radiation. The resultant AM was characterized for its biological and morphologic properties by immunohistochemical and electron microscopic examination. In addition, fibrin glue-coated, freeze-dried (FCFD) AM was transplanted onto a rabbit scleral surface without sutures, to examine its biocompatibility.Immunohistochemistry of the FCFD-AM revealed that fibrinogen existed on its chorionic side, and the process of applying fibrin glue did not affect its biological and morphologic properties. Moreover, electron microscopic examination of the chorionic side of the FCFD-AM revealed tiny microfibrils (which are probably fibrinogen protofibrils), and showed that the epithelial surface of FCFD-AM consisted of intact basal lamina similar to that of FD-AM. FCFD-AM transplantation was very easily performed, and the graft adhered to the bare sclera immediately. Though the fibrinogen naturally biodegraded within 2 weeks, the FCFD-AM remained for at least 12 weeks after transplantation. Epithelialization on the FCFD-AM was achieved within 2 weeks, as was the case with FD-AM transplantation. The conjunctival epithelium on the FCFD-AM was well stratified and not keratinized, suggesting that FCFD-AM supports normal cell differentiation.The FCFD-AM retained most of the biological characteristics of FD-AM. Consequently, this sutureless method of transplantation of FCFD-AM is safe, simple, and useful for ocular surface reconstruction.

Human calcium-activated chloride channel 2 (hCLCA2) belongs to a family of multifunctional proteins and is localised mainly in basal cells of squamous epithelia. However, its function is still not fully understood. Relative amounts of hCLCA2 were analysed using real-time PCR in several human epithelial tissues and tissues expressing high amounts were identified. These tissues then underwent double immunolabelling with anti-hCLCA2 antibodies and antibodies against the adhesion molecules integrin beta4 and collagen VII and were visualised by fluorescence microscopy. Real-time PCR found hCLCA2 gene expression to be primarily associated with stratified squamous epithelia. Subsequent immunohistochemistry clearly demonstrated colocalisation between hCLCA2 and integrin beta4. This study reports on a possible underlying relationship between hCLCA2 and stratified epithelia and the close association of hCLCA2 with basal cell adhesion molecules in normal tissue, suggesting it may play an important role in basal cell attachment in stratified epithelia.

Cultured skin substitutes have become useful as adjunctive treatments for excised, full-thickness burns, but no skin substitutes have the anatomy and physiology of native skin. Hypothetically, deficiencies of structure and function may result, in part, from nutritional deficiencies in culture media. To address this hypothesis, vitamin C was titrated at 0.0, 0.01, 0.1, and 1.0 mM in a cultured skin substitute model on filter inserts. Cultured skin substitute inserts were evaluated at 2 and 5 wk for viability by incorporation of 5-bromo-2'-deoxyuridine (BrdU) and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion. Subsequently, cultured skin substitute grafts consisting of cultured human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates were incubated for 5 wk in media containing 0.0 mM or 0.1 mM vitamin C, and then grafted to athymic mice. Cultured skin substitutes (n = 3 per group) were evaluated in vitro at 2 wk of incubation for collagen IV, collagen VII, and laminin 5, and through 5 wk for epidermal barrier by surface electrical capacitance. Cultured skin substitutes were grafted to full-thickness wounds in athymic mice (n = 8 per group), evaluated for surface electrical capacitance through 6 wk, and scored for percentage original wound area through 8 wk and for HLA-ABC-positive wounds at 8 wk after grafting. The data show that incubation of cultured skin substitutes in medium containing vitamin C results in greater viability (higher BrdU and MTT), more complete basement membrane development at 2 wk, and better epidermal barrier (lower surface electrical capacitance) at 5 wk in vitro. After grafting, cultured skin substitutes with vitamin C developed functional epidermal barrier earlier, had less wound contraction, and had more HLA-positive wounds at 8 wk than without vitamin C. These results suggest that incubation of cultured skin substitutes in medium containing vitamin C extends cellular viability, promotes formation of epidermal barrier in vitro, and promotes engraftment. Improved anatomy and physiology of cultured skin substitutes that result from nutritional factors in culture media may be expected to improve efficacy in treatment of full-thickness skin wounds.

We have performed an immunohistochemical study of intact adherent layers of human long-term bone marrow cultures (hLTBMC) in order to characterize the cell types present. Our panel of antibodies was selected to demonstrate various mesenchymal and haemopoietically derived cell types and to assess the presence of molecules of potential importance as adhesive ligands between haemopoietic cells and stroma. Subpopulations of fibroblasts and macrophages were identified which differed in immunophenotype. We were able to demonstrate modulation of fibroblast and extra-cellular matrix immunophenotypes between 2 and 12 weeks in culture. Stromal cells and matrix expressed a wide variety of antigens of potential importance in haemopoietic cell adhesion, but no ICAM-1, 2 or 3 could be demonstrated to correspond to the strong LFA-1 expression seen in haemopoietic precursor cells. No localization of antigen expression by stromal elements was found to account for the formation of haemopoietic foci at particular sites. However, granulocyte-predominant foci preferentially occupied the interstices and margins of structures which appeared to be vascular arrays.