In our previous studies, we have demonstrated effective regeneration of cartilage through the creation and application of layered cell sheets that combine both chondrocytes and synovial cells. In this study, we were able to demonstrate that cells derived from cell sheets can survive for long periods after transplantation into rat knee joints having osteochondral defects. We established a method for generating cell sheets from firefly luciferase-expressing chondrocytes obtained from transgenic Lewis rats, and carried out allogenic transplantation of these cell sheets into wild-type Lewis rats. We then administered luciferin and monitored the survival of the transplanted cells by using bioluminescence imaging (BLI). Our data showed that the transplanted cells survived and could be detected for more than 21 months, which was longer than expected. Furthermore, the BLI data showed that the transplanted cells remained in the knee joint and did not migrate to other parts of the body, thus confirming the safety of the cell sheets. In this study, we monitored the duration of survival of cell sheets composed of only chondrocytes, only synovial cells, or both chondrocytes and synovial cells, and found that all three types of cell sheets survived for an extended period of time.

A double polymeric nanolayer consisting of poly(N-isopropylacrylamide) (PIPAAm) and hydrophilic polyacrylamide (PAAm) was deposited on tissue culture polystyrene (TCPS) surfaces using electron beam irradiation to form a new temperature-responsive cell culture surface in which the basal hydrophilic PAAm component in the double polymeric layer promotes the hydration of the upper PIPAAm layer and induces rapid cell detachment compared to a conventional temperature-responsive cell culture surface, PIPAAmgrafted TCPS (PIPAAm-TCPS). Take-off angle-dependent X-ray photoelectron spectroscopy spectral analysis demonstrated that the grafted PIPAAm and PAAm components were located in the upper and basal regions of the double polymeric layer, respectively, suggesting that the double polymeric layer forms an inter-penetrating-network-like structure with PAAm at the basal portion of the PIPAAm grafted chains. The wettability of the temperature-responsive cell culture surfaces with the double polymeric layer tended to be more hydrophilic, with an increase in the basal PAAm graft density at a constant PIPAAm graft density. However, when the graft densities of the upper PIPAAm and basal PAAm were optimized, the resulting temperature-responsive cell culture surface with the double polymeric layer exhibited rapid cell detachment while maintaining cell adhesive character comparable to that of PIPAAm-TCPS. The cell adhesive character was altered from cell-adhesive to cell-repellent with increasing PAAm or PIPAAm graft density. The cell adhesive character of the temperature-responsive cell culture surfaces was relatively consistent with their contact angles. These results strongly suggest that the basal PAAm surface properties affect the degree of hydration and dehydration of the subsequently grafted PIPAAm. In addition, the roles of the hydrophilic component in accelerating cell detachment are further discussed in terms of the mobility of the grafted PIPAAm chains. Applications of this insight might be useful for designing temperature-responsive cell culture surfaces for achieving efficient cell culture and quick target cell detachment. Acta Biomaterialia j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l oc a t e / a c t a b i o m a t Please cite this article in press as: Akiyama Y et al. Accelerated cell-sheet recovery from a surface successively grafted with polyacrylamide and poly(Nisopropylacrylamide). Acta Biomater (2014), http://dx.

www.advmat.de 3635 Figure 2. Cell culture on thermoresponsive microtextured substrates. A) Phase contrast images of BCECs cultured on the substrates at 37°C. Black arrows indicate ridges on the microtextured surfaces. B) Phase contrast and C) fluorescent images of BCECs cultured on the substrates at 37°C for 1 week. D) Phase contrast and E) fluorescent images of BCECs detached from the substrates upon decreasing the temperature to 20°C. The cells have been stained with calcein-AM prior to cell detachment. The scale bars represent 100 lm.

Controlled laminar fluid flow, which is characterized by a decreased Reynolds number, Re, can be achieved in channels with micrometer dimensions. Under laminar fluid flow, flow streams in contact with each other within the microchannels mix only by lateral molecular diffusion of their molecules. Because of the excellent control that can be achieved over flow and mixing variables in small volumes, microfluidic channels are now popular for various investigations in chemistry and cell biology, [4] prompting many innovations in microfabrication. [5] Furthermore, microfluidic channels integrated into miniaturized analytical devices, called micro total analysis systems (lTASs) or lab-on-a-chip systems, [6] make it possible to realize full integration of analytical operations into microscale devices.

Cell sheet technology enables novel approaches to tissue engineering without the use of biodegradable scaffolds. Cell sheet technology consists of a temperature-responsive culture dish, which enables reversible cell adhesion to and detachment from the dish surface by controllable hydrophobicity of the surface. This allows for a non-invasive harvest of cultured cells as an intact monolayer cell sheet including deposited extra cellular matrices. The monolayer cell sheet can be transplanted to host tissues without using biodegradable scaffolds and sutures. Thick tissue constructs and patterned cell sheets using two or more kinds of cell source are also developed by means of layered cell sheets in vitro. This Progress Report summarizes temperature-controlled cell adhesion-detachment behavior and applications of the cell sheet technology to regeneration of cornea, periodontal ligament, bladder epithelia, oesophageal epithelia, myocardium, and liver.

To develop and characterize a new class of temperature-sensitive hydrogel microspheres composed Abstract of poly(N-isopropylacrylamide)/poly(ethylene glycol) diacrylate (PNIPAAm/PEG-DA). Methods: The PNIPAAm/PEG-DA hydrogel microspheres were fabricated in two aqueous systems as a result of polymer/polymer immiscibility. Both PNIPAAm and PEG-DA were used as the precursors; the PEG-DA was also used as a cross-linker for the formation of the hydrogel microspheres. Bovine serum albumin was used as the model protein drug to examine the effects of the thermo-responsive properties of the hydrogel microspheres on the release of a protein at two different temperatures (22°C and 37°C). Results: The hydrated PNIPAAm/PEG-DA hydrogel microspheres exhibited a swollen diameter of 50μm, with a narrow particle-size distribution. Scanning electron microscopy and environmental scanning electron microscopy observations revealed that, upon swelling, the resulting hydrogel microspheres had a regular spherical and rough surface morphology. The lower critical solution temperature (LCST) of the PNIPAAm/PEG-DA hydrogel microspheres was around 29.1°C, based on differential scanning calorimetric data. The release of BSA from the hydrogel microspheres at 37°C was slower than that at 22°C because of the thermo-responsive nature of PNIPAAm at temperatures above its LCST. Conclusions: We believe that these kinds of PNIPAAm/PEG-DA hydrogel microspheres may have wide applications as promising drug delivery systems, because of their intelligent nature upon external temperature change.

Engineering tissues in the subcutaneous space is an attractive method; however, hepatocyte survival has been transient due to insufficient vascular network formation. To establish a vascularized cavity, we created a polyethylene terephthalate mesh device coated with poly(vinylalcohol) that allowed for the gradual release of basic fibroblast growth factor (bFGF), a potent angiogenic factor. The efficacy of the bFGF-releasing device in inducing vascular network formation in the subcutaneous space was observed in mouse and rat studies. Isolated mouse hepatocytes transplanted into newly vascularized subcutaneous cavities allowed for persistent survival up to 120 days. In the absence of a vascularized compartment, the survival of the transplanted hepatocytes was markedly diminished. Functional maintenance of the engineered hepatic tissues was confirmed by high expression of liver-specific mR-NAs and proteins. These engineered hepatic tissues have the ability to take up inoculated compounds and express strong induction of drug-metabolizing enzymes, demonstrating functional relevance as a metabolic tissue. In conclusion, we have created a novel technology to engineer functionally active hepatic tissues in the subcutaneous space, which will likely facilitate hepatocyte-based therapies.

We used temperature-responsive culture dishes onto which the temperature-responsive polymer, poly(Nisopropylacrylamide), was covalently grafted for tissue engineering. Confluent cells harvested as intact sheets from these surfaces by simple temperature reduction can be transferred to various surfaces including additional culture dishes, other cell sheets, and tissues. In order to examine the maintenance of cell polarity, Madin-Darby canine kidney cells and human primary renal proximal tubule epithelial cells which had developed apical-basal cell polarity in culture, were subjected to cell sheet transfer. This functional and structural cell polarity, which is susceptible to treatment with trypsin, was examined by immunohistochemistry and transmission electron microscopy. Using our cell-sheet method, the noninvasive transfer of these cell sheets retaining typical distributions of Na + /K + -ATPase, GLUT-1, SGLT-1, aquaporin-1, neutral endopeptidase and dipeptidylendopeptidase IV, could be achieved. The transferred cell sheets also developed numerous microvilli and tight junctions at the apical and lateral membranes, respectively. For biochemical analysis, immunoblotting of occludin, a transmembrane protein that composes tight junctions, was conducted and results confirmed that occludin remained intact after cell sheet transfer. This two-dimensional cell sheet manipulation method promises to be useful for tissue engineering as well as in the investigation of epithelial cell polarity.

The aim of this study was to investigate the prevalence and genetic diversity of Staphylococcus aureus and staphylococcal enterotoxins (SEs) in ready-to-eat foods marketed in Hanoi, Vietnam. Out of 212 samples tested, 45 were contaminated with this bacterium and 18 contained strains having classical SEs. Among different food products, the bacterial occurrence ranged between 12.5% and 35.4% and milk samples had the highest prevalence. The production of SEs varied according to the food types and 26 different ribotype patterns were observed among the 45 strains. Our dendogram analysis showed the existence of many clones without clear-cut affiliation to any particular food type indicating both diverse primary and secondary contamination sources. The SEs also occurred in a wide variety of genetically different S. aureus strains which might facilitate the bacterium with better ecological fitness to cause epidemics. It can be concluded that Vietnamese ready-to-eat foods pose a high risk of contamination by toxigenic S. aureus population urging the need of implementing proper hygienic practices.

Targeting cytotoxins or immunotoxins to tumor cell surface receptors represents a new approach for the treatment of cancers. We tested the antitumor activity of a cytotoxin (IL-4-PE) composed of an interleukin-4 (IL-4) targeting moiety and a truncated form of Pseudomonas exotoxin A against human biliary tract carcinoma (BTC). Immunohistochemical analysis showed that cultured BTC cell lines and cancerous epithelia in BTC tissue (e.g., gallbladder carcinoma, extraheaptic cholangiocarcinoma, and intrahepatic cholangiocarcinoma) expressed receptors for IL-4 in situ at high densities. However, normal epithelial cells in gallbladder and bile duct tissues did not express these IL-4 receptors. Eight BTC cell lines expressed IL-4R on the cell surface as determined by radiolabeled ligand binding assays. When these cells were treated with IL-4-PE, significant cytotoxicity was observed as determined by the inhibition of protein synthesis. The concentration of agent causing 50% inhibition of protein synthesis (IC 50 ) was found to be less than 10 ng/mL in 4 of the 8 BTC cell lines studied. The antitumor activity of IL-4-PE was assessed for human BTC cells implanted subcutaneously in immunodeficient mice. By intratumoral injection of IL-4-PE, complete disappearance of the established tumors was observed in 40% of animals. Intraperitoneal administration of IL-4-PE at tolerated doses to animals with peritoneally disseminated BTC exhibited significantly prolonged survival compared to untreated animals (>14 weeks vs. 5 weeks in treated and untreated mice, respectively). These results indicate that IL-4 receptor-targeted cytotoxin is a potent agent that may provide a new therapeutic option for BTC.

Background. Vibrio parahaemolyticus causes acute gastroenteritis and inflammations in humans. A variety of pathogenic bacteria can stimulate mitogen-activated protein kinases (MAPKs) in host cells. Phosphorylation of MAPKs leads to production of interleukin (IL)-8 and subsequently causes inflammations. Thus, MAPK cascades were strong candidates for the main signaling pathway of V. parahaemolyticus-induced acute inflammation

Several methods for management of anterior penile hypospadias are well known but no one is ideal or a standard way for treatment. Objective: Long term evaluation of urethral lengthening by three different techniques in management of anterior hypospadias Material & Methods: Thirty patients with anterior penile hypospadias were included in this study and subdivided into three groups; group (A) included 5 patients with coronal hypospadias and group B included 10 patients with subcoronal hypospadias. While group (C) included 15 patients with distal penile hypospadias (DPH),.Three different techniques were performed for urethral lengthening. Degloving of penile skin with mobilization of anterior urethra till the peno-scrotal junction in all patients with coronal hypospadias. Bulbar elongation technique through the perineum was done for all patients with subcoronal hypospadias. Double mobilization technique, including degloving with penile urethra mobilization and bulbar elongation, was performed in group C. All patients were followed for at least 2 years as regard: position and caliber of new meatus, presence or absence of penile curvature and the need for an additional maneuver for repair.