High-density collagen patch prevents stricture after endoscopic circumferential submucosal dissection of the esophagus: a porcine model.

BACKGROUND AND AIMS: Extensive excision of the esophageal mucosa by endoscopic submucosal dissection (ESD) frequently evokes a luminal stricture. This study aimed to determine the efficacy of a high-density collagen patch for the prevention of esophageal stricture in extensive ESD. METHODS: Six pigs underwent circumferential esophageal ESD under general anesthesia. In 3 pigs, artificial ulcers were covered by 2 collagen patches. The other 3 pigs underwent circumferential ESD only. RESULTS: The 2 collagen patches were settled onto the ulcer surface using a general endoscope and instruments. The collagen patch-treated group showed significantly better patency rates on both the oral and anal sides of the wound area compared with the control group at day 14. The mucosal re-epithelization ratio was significantly promoted, and the extent of mucosal inflammation and fibrosis was significantly decreased with the collagen patch treatment in the wound area. The frequency of cells positive α-smooth muscle actin was significantly reduced in the collagen patch-treated group compared with the control group. CONCLUSIONS: We have established a high-density collagen device that can reduce the esophageal stricture associated with extensive ESD. This easy-to-handle device would be useful during superficial esophageal cancer treatment by ESD.

A new cell-free bandage-type artificial skin for cutaneous wounds.

Engineered skin substitutes are widely used in skin wound management. However, no currently available products satisfy all the criteria of usability in emergency situations, easy handling, and minimal scar formation. To overcome these shortcomings, we designed a cell-free bandage-type artificial skin, named "VitriBand" (VB), using adhesive film dressing, silicone-coated polyethylene terephthalate film, and collagen xerogel membrane defined as a dried collagen vitrigel membrane without free water. We analyzed its advantages over in-line products by comparing VB with hydrocolloid dressing and collagen sponge. For evaluation, mice inflicted with full-thickness skin defects were treated with VB, hydrocolloid dressing, and collagen sponge. A plastic film group treated only with adhesive film dressing and silicone-coated polyethylene terephthalate film, and a no treatment group were also compared. VB promoted epithelization while inhibiting the emergence of myofibroblasts and inflammation in the regenerating tissue more effectively than the plastic film, hydrocolloid dressing, and collagen sponge products. We have succeeded in establishing a cell-free bandage-type artificial skin that could serve as a promising first-line medical biomaterial for emergency treatment of skin injuries in various medical situations.

Epithelial-to-mesenchymal transition and slit function of mesothelial cells are regulated by the cross talk between mesothelial cells and endothelial cells.

Peritoneal dysfunction is a major factor leading to treatment failure of peritoneal dialysis (PD). However, the precise mechanism of the peritoneal diffusion changes related to PD remains to be elucidated. To this end, we have established a novel peritoneal diffusion model in vitro, which consists of a three-dimensional culture system using a collagen vitrigel membrane chamber and a fluid-stream generation system. This artificial peritoneal model revealed that high-glucose culture medium and fluid flow stress promoted the epithelial-mesenchymal transition (EMT) process of mesothelial cells and that endothelial cells inhibited this mesothelial EMT process. Mesothelial cells in the EMT state showed high expression of connective tissue growth factor and low expression of bone morphogenic protein-7, while non-EMT mesothelial cells showed the opposite expression pattern of these two proteins. In addition, these protein expressions were dependent on the presence of endothelial cells in the model. Our model revealed that the endothelial slit function was predominantly dependent on the covering surface area, while the mesothelial layer possessed a specific barrier function for small solutes independently of the surface area. Notably, a synergic barrier effect of mesothelial cells and endothelial cells was present with low-glucose pretreatment, but high-glucose pretreatment abolished this synergic effect. These findings suggest that the mesothelial slit function is not only regulated by the high-glucose-induced EMT process but is also affected by an endothelial paracrine effect. This peritoneal diffusion model could be a promising tool for the development of PD.

Transplantation of Human Corneal Endothelial Cells Cultured on Bio-Engineered Collagen Vitrigel in a Rabbit Model of Corneal Endothelial Dysfunction.

PURPOSE: To evaluate the effect of transplanting bioengineered corneal endothelial grafts in a rabbit model of corneal endothelial failure. METHODS: Human corneal endothelial cells (HCECs) were seeded on a vitrigel carrier. After Descemet's membrane was removed from the eyes of rabbits, transplantation was done with a vitrigel/HCEC graft or vitrigel alone without cells, or the eyes were left untreated. Slit lamp examinations and measurement of the central corneal thickness (CCT) were performed for 14 days postoperatively. RESULTS: HCECs cultured on vitrigel were strongly positive for ZO-1 and Na+/K+ ATPase. On day 14, the cornea showed mild edema and the pupil margins were visible through the grafts in the vitrigel/HCEC graft group. HCECs completely covered the grafts on day 14. In contrast, there was severe corneal edema and the pupil margins were undetectable on day 14 after transplantation of the vitrigel carrier alone or no transplantation. Proliferation of host cells was not observed in these groups. On day 14, the mean CCT was significantly thinner in the vitrigel/HCEC graft group than in the other two groups (p = 0.0008). CONCLUSIONS: Transplantation of a vitrigel/HCEC graft was effective for reducing the corneal thickness and restoring corneal transparency, suggesting the usefulness of vitrigel as a carrier for corneal endothelial cells.

Development of an oxygenation culture method for activating the liver-specific functions of HepG2 cells utilizing a collagen vitrigel membrane chamber.

We recently developed a collagen vitrigel membrane (CVM) chamber possessing a scaffold composed of high-density collagen fibrils. In this study, we first confirmed that the advantage of CVM chamber in comparison to the traditional culture chamber with porous polyethylene terephthalate membrane is to preserve a culture medium poured in its inside even though the under side is not a liquid phase but solid and gas phases. Subsequently, we designed three different culture systems to grow HepG2 cells in a culture medium (liquid phase) on the CVM which the under side is a culture medium, a plastic surface (solid phase) or 5 % CO2 in air (gas phase) and aimed to develop a brief culture method useful for activating the liver-specific functions and analyzing the pharmacokinetics of fluorescein diacetate. HepG2 cells cultured for 2 days on the liquid-solid interface and subsequently for 1 day on the liquid-gas interface represented excellent cell viability and morphology in comparison to the others, and remarkably improved albumin secretion and urea synthesis to almost the same level of freshly isolated human hepatocytes and CYP3A4 activity to about half the level of differentiated HepaRG cells. Also, the cells rapidly absorbed fluorescein diacetate, distributed it in cytosol, metabolized it into fluorescein, and speedily excreted fluorescein into both bile canaliculus-like networks and extracellular solution. These data suggest that hepatic structure and functions of monolayered HepG2 cells can be induced within a day after the oxygenation from beneath the CVM.

Development and evaluation of porcine atelocollagen vitrigel membrane with a spherical curve and transplantable artificial corneal endothelial grafts.

PURPOSE: To develop a collagen vitrigel (CV) optimized as a corneal endothelial cell (CEC) carrier and create an artificial corneal endothelial graft. METHODS: We first developed a flat-shaped collagen vitrigel for regenerative medicine (CV-RM) using porcine atelocollagen and ultraviolet (UV) irradiation. The optimal UV amount was determined by measuring the CV-RM transparency under various irradiating conditions. The collagen vitrigel for corneal endothelial regenerative treatment (CV-CERT), a transparent porcine atelocollagen with a curved shape, was made using spherically curved molds and UV irradiation. The membrane permeability of the CV-CERT was tested in vitro. The biocompatibility, transparency, and adhesiveness of the CV-CERT were evaluated in rabbit eyes. We also developed a culture technique for distributing human CECs on the curved CV-CERT. RESULTS: The optimal amount of UV irradiation for CV-RM transparency was 2400 mJ/cm(2). Membrane permeability of CV-CERT at day 5 was higher than that of commercially available CV (P = 0.032). The CV-CERT was transparent and biocompatible in rabbit corneas for up to 4 months. The CV-CERT remained attached to the rabbit corneal posterior surface, whereas the flat-shaped CV-RM, differing only in shape from the CV-CERT, dislocated soon after surgery. Human CECs seeded on the CV-CERT using our technique were evenly distributed with a single layer structure and a mean cell density of 2650 ± 100 cells/mm(2). CONCLUSIONS: We developed a transparent and biocompatible porcine-derived atelocollagen vitrigel membrane with a spherical curvature. A transplantable artificial endothelial graft was created by combining cultured human CECs and the CV-CERT.