Novel detergent for whole organ tissue engineering.

Whole organ tissue engineering for various organs, including the heart, lung, liver, and kidney, has demonstrated promising results for end-stage organ failure. However, the sodium dodecyl sulfate (SDS)-based protocol for standard decellularization has drawbacks such as clot formation in vascularized transplantation and poor cell engraftment in recellularization procedures. Preservation of the surface milieu of extracellular matrices (ECMs) might be crucial for organ generation based on decellularization/recellularization engineering. We examined a novel detergent, sodium lauryl ether sulfate (SLES), to determine whether it could overcome the drawbacks associated with SDS using rat heart and kidney. Both organs were perfused in an antegrade fashion with either SLES or SDS. Although immunohistochemistry for collagen I, IV, laminin, and fibronectin showed similar preservation in both detergents, morphological analysis using scanning electron microscopy and an assay of glycosaminoglycan content on ECMs showed that SLES-treated tissues had better-preserved ECMs than SDS-treated tissues. Mesenteric transplantation revealed SLES did not induce significant inflammation, as opposed to SDS. Platelet adhesion to decellularized tissues was significantly reduced with SLES. Overall, SLES could replace older detergents such as SDS in the decellularization process for generation of transplantable recellularized organs.

Effect of an artificial silk elastin-like protein on the migration and collagen production of mouse fibroblasts.

A silk elastin-like protein (SELP) is an artificial compound composing silk fibroin-like and elastin-like tandem repeats. The objective of this study is to evaluate the SELP effect on the migration, proliferation, and proteins production of L929 mouse fibroblasts. Upon culturing with different concentrations of SELP, the cells migration and their collagen production significantly enhanced in the SELP concentrations from 10(-3) to 10 µg/ml. However, irrespective of the SELP concentration, no difference in the production of fibronectin, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1α (SDF-1α) was observed. When the migration of mouse peritoneal macrophages by SELP was evaluated, significant enhancement of macrophages migration was observed in any concentration. It is concluded that the SELP has a potential to promote the migration of fibroblasts and macrophages, and the fibroblast collagen production.

p120-Catenin is essential for N-cadherin-mediated formation of proper junctional structure, thereby establishing cell polarity in epithelial cells.

The role of p120-catenin in the function of classical cadherins is still enigmatic despite various studies. To elucidate its role, we examined the effect of p120-catenin on the N-cadherin-mediated localization of junctional proteins in epithelial cells in this study. Cadherin-deficient MIA PaCa-2 epithelial cells did not show linear localization of tight junction proteins ZO-1 and occludin. When N-cadherin was expressed in these cells, however, the resultant transfectant cells revealed strong cell adhesion activity and linear localization of ZO-1, occludin, and N-cadherin in the lateral membrane. When the p120-catenin-binding site of N-cadherin was disrupted, the linear localization of ZO-1 and occludin disappeared, and the mutant N-cadherin became localized more diffusely in the transfectant, although the cell adhesion activity did not change much. Knockdown of p120-catenin also resulted in the very weak localization of ZO-1 and occludin. A similar effect of p120-catenin on the localization of junctional proteins was obtained under more dynamic conditions in a wound healing assay. Moreover, p120-catenin was essential for the regulation of centrosome orientation in this healing assay. Taken together, the present data indicate that p120-catenin is essential for N-cadherin-mediated formation of proper junctional structures and thereby the establishment of the cell polarity. Similar results were obtained when E-cadherin mutants comparable to those of N-cadherin were used, suggesting that p120-catenin plays the same role in the function of other classical cadherins.

The extracellular domains of E- and N-cadherin determine the scattered punctate localization in epithelial cells and the cytoplasmic domains modulate the localization.

The accumulation of classical cadherins is essential for their function, but the mechanism is poorly understood. Hence, we investigated the accumulation of E- and N-cadherin and the formation of cell junctions in epithelial cells. Immunostaining revealed a scattered dot-like accumulation of E- and N-cadherin throughout the lateral membrane in MDCK II and other epithelial cells. Mutant E-cadherin lacking the beta-catenin binding site accumulated granularly at cell-cell contact sites and showed weak cell aggregation activity in cadherin-deficient epithelial cells, MIA PaCa2 cells. Mutant E-cadherin lacking the p120-catenin binding site exhibited scattered punctate accumulation and strong cell adhesion activity in MIA PaCa2 cells. Electron microscopy demonstrated that MIA PaCa2 transfectants of E-cadherin containing beta-catenin binding site formed adherens junction, whereas E-cadherin lacking the binding site did not. Mutant N-cadherins showed accumulation properties similar to those of corresponding mutant E-cadherins. Moreover, wild type and mutant N-cadherin lacking the p120-catenin binding site showed subapical accumulation in polarized DLD-1 cells, whereas mutant N-cadherin lacking beta-catenin binding site did not. These results indicate that the extracellular domains of E- and N-cadherin determines the basic localization pattern, whereas the cytoplasmic domains modulate it thereby affects the cell adhesion activity, subapical accumulation, and the formation of adherens junction.