Abstract-Various studies have identified a critical role for Notch signaling in cardiovascular development. In this and other systems, Notch receptors and ligands are expressed in regions that undergo epithelial-to-mesenchymal transformation. However, there is no direct evidence that Notch activation can induce mesenchymal transdifferentiation.In this study we show that Notch activation in endothelial cells results in morphological, phenotypic, and functional changes consistent with mesenchymal transformation. These changes include downregulation of endothelial markers (vascular endothelial [VE]-cadherin, Tie1, Tie2, platelet-endothelial cell adhesion molecule-1, and endothelial NO synthase), upregulation of mesenchymal markers (␣-smooth muscle actin, fibronectin, and platelet-derived growth factor receptors), and migration toward platelet-derived growth factor-BB. Notch-induced endothelial-to-mesenchymal transformation does not seem to require external regulation and is restricted to cells expressing activated Notch. Jagged1 stimulation of endothelial cells induces a similar mesenchymal transformation, and Jagged1, Notch1, and Notch4 are expressed in the ventricular outflow tract during stages of endocardial cushion formation. This is the first evidence that Jagged1-Notch interactions induce endothelial-to-mesenchymal transformation, and our findings suggest that Notch signaling may be required for proper endocardial cushion differentiation and/or vascular smooth muscle cell development. Key Words: endothelial-to-mesenchymal transformation Ⅲ Notch Ⅲ Jagged1 Ⅲ endocardial cushion T he Notch signaling pathway plays a critical role during development. Four mammalian Notch receptors (Notch1 through 4) and 5 Notch ligands (Delta-like [Dll]-1, Dll3, Dll4, Jagged1, and Jagged2) have been identified. Notch receptorligand interaction results in a series of proteolytic cleavages of the Notch receptor, producing a C-terminal intracellular fragment (NotchIC) that translocates to the nucleus. In the nucleus, NotchIC binds to the transcriptional repressor CBF1/ RBP-J, thereby derepressing or coactivating the expression of various lineage-specific genes. 1 Perturbation of the Notch pathway has been implicated in the pathogenesis of various cardiovascular diseases in humans. 2 Of interest, patients with Jagged1 mutations (Alagille syndrome) display congenital cardiovascular anomalies that seem to be secondary to faulty endocardial cushion formation. [3][4][5][6] In the mouse, Notch1-deficient embryos demonstrate severe vascular developmental defects, which are exacerbated in Notch1/Notch4 double-mutant embryos. 7 Constitutive activation of Notch4 also causes defects in vascular remodeling. 8,9 Mice that are rendered null for Jagged1 die from hemorrhage early during embryogenesis, whereas mice that are doubly heterozygous for a Jagged1-null allele and a Notch2 hypomorphic allele exhibit cardiac anomalies similar to those seen in Alagille syndrome. 10,11 Genes that lie downstream of Notch activation, such as the basic helix-loop...

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The ability to regulate stem cell proliferation and differentiation has relevance in numerous medical applications, including medical devices, tissue engineering, and regenerative medicine. To control cellular behavior at the biomaterial or scaffold interface, many studies have employed surface modifications that mimic the extracellular matrix. Strikingly absent is the immobilization of cell-surface ligands to the biomaterial surface. One cell-to-cell signaling pathway that has been shown to regulate tissue development and stem cell fate is the Notch pathway. Recently, the Notch signaling pathway was identified as a key regulator of epithelial differentiation. Utilizing this knowledge, we applied an affinity immobilization scheme designed to attach and orient the Notch ligand, Jagged-1, in an active conformation on a biomaterial surface. When epithelial stem cells were plated on the bound ligand, the Notch/CBF-1 signaling pathway was stimulated and the cells upregulated both intermediate- and late-stage differentiation markers. In addition, the ligand promoted tight clustering and extensive stratification. Soluble Jagged-1 showed no Notch/CBF-1 signaling and very little, if any, cell differentiating activity. The high potency of bound Jagged-1 suggests that modification of a surface with a Notch ligand presents a powerful method to control stem cell differentiation at the cell-biomaterial interface.

The hypothesis that ultrasound increases antibiotic transport through biofilms of Escherichia coli and Pseudomonas aeruginosa was investigated using colony biofilms. Biofilms grown on membrane filters were transferred to nutrient agar containing 50 μg/mL gentamicin. A smaller filter was placed on top of the biofilm and a blank concentration disk was situated atop the filter. Diffusion of antibiotic through the biofilms was allowed for 15, 30, or 45 min at 37°C. Some of these biofilms were exposed to 70 kHz ultrasound and others were not. Each concentration disk was then placed on a nutrient agar plate spread with a lawn of E. coli. The resulting zone of inhibition was used to calculate the amount of gentamicin that was transported through the biofilm into the disk. The E. coli and P. aeruginosa biofilms grown for 13 and 24 h were exposed to two different ultrasonic power densities. Ultrasonication significantly increased the transport of gentamicin through the biofilm. Insonation of biofilms of E. coli for 45 minutes more than doubled the amount of gentamicin compared to their non-insonated counterparts. For P. aeruginosa biofilms, no detectable gentamicin penetrated the biofilm within 45 min without ultrasound; however, when insonated (1.5 W/cm 2 ) for 45 min, the disks collected more than 0.45 μg of antibiotic. Ultrasonication significantly increased transport of gentamicin across biofilms that normally blocked or slowed gentamicin transport when not exposed to ultrasound. This enhanced transport may be partially responsible for the increased killing of biofilm bacteria exposed to combinations of antibiotic and ultrasound.

A cell-extraction protocol yielding an esophagus acellular matrix (EAM) scaffold for use in tissue engineering of an esophagus, including hypotonic lysis, multiple detergent cell extraction steps, and nucleic acid digestion, was developed in a rat model. Histological techniques, burst pressure studies, in vitro esophageal epithelial cell seeding, and in vivo implantation were used to assess cell extraction, extracellular matrix (ECM) preservation, and biocompatibility. Microscopy demonstrated that cell extraction protocols using sodium dodecyl sulfate (SDS) (0.5%, wt/vol) as a detergent resulted in cell-free EAM with retained ECM protein collagen, elastin, laminin, and fibronectin. Burst pressure studies indicated a loss of tensile strength in EAMs, but at intraluminal pressures that were unlikely to affect in vivo application. In vitro cell seeding studies exhibited epithelial cell proliferation with stratification similar to native esophagi after 11 days, and subcutaneously implanted EAMs displayed neovascularization and a minimal inflammatory response after 30 days of implantation. This study presents an esophagus acellular matrix tissue scaffold with preserved ECM proteins, biomechanical properties, and the ability to support esophageal cell proliferation to serve as the foundation for a tissue-engineered esophagus.

SUMMARYInfection of implanted medical devices by Gram-positive organisms such as Staphylococcus ssp. is a serious concern in the biomaterial community. In this research the application of low frequency ultrasound to enhance the activity of vancomycin against implanted Staphylococcus epidermidis biofilms was examined. Polyethylene disks covered with a biofilm of S. epidermidis were implanted subcutaneously in rabbits on both sides of their spine. The rabbits received systemic vancomycin for the duration of the experiment. Following 24 h of recovery, one disk was insonated for 24 or 48 h while the other was a control. Disks were removed and viable bacteria counted. At 24 h of insonation, there was no difference in viable counts between control and insonated biofilms, while at 48 h of insonation there were statistically fewer viable bacteria in the insonated biofilm. The S. epidermidis biofilms responded favorably to combinations of ultrasound and vancomycin, but longer treatment times are required for this Gram-positive organism than was observed previously for a Gram-negative species.

Low-frequency ultrasound has been investigated as an adjuvant to antimicrobial therapy, targeted at both planktonic and biofilm (sessile) organisms. Our previous work showed that ultrasound (US) effectively enhances the bactericidal activity of certain antibiotics against planktonic cultures (Pitt et al., 1994;Rediske et al., 1999) and in vitro biofilms (Johnson et al., 1998;Qian et al., 1999) and in vivo biofilms (Carmen et al., 2004b(Carmen et al., , 2005Rediske et al., 2000) of gram-positive and gram-negative bacteria. Ultrasound was shown to increase the transport of antibiotics through biofilms (Carmen et al., 2004a) which could account for some (or all) of the enhanced antibiotic activity against insonated biofilms; but such a mechanism could not account for US-enhanced antibiotic activity in planktonic cultures which have no extensive exopolymer matrix to retard antibiotic transport.Because this ultrasonic enhancement of antibiotic activity operates on both planktonic and sessile bacteria, we posit that US does more than simply increase the transport of antibiotic to the cells; ultrasound is postulated to increase uptake of antibiotic into the cells by rendering the cell membrane more permeable to the antibiotic. To examine this postulate, we must first review how ultrasound interacts with cells.Bacterial cells are fairly transparent to ultrasound; that is, ultrasonic waves go right through cells with little absorption, scattering or other interaction. However, the pressure oscillations of ultrasound produce size oscillations in any gas bubbles in the liquid (Brennen, 1995). These bubbles range in size from approximately 1 mm to 100 mm in diameter (Brennen, 1995). The oscillations of bubbles, called cavitation, are generally divided into "stable" and "collapse" types of cavitation. Stable cavitation is the low intensity oscillation of the bubbles without complete collapse of the bubble, while collapse cavitation occurs at higher intensity levels and lower frequencies wherein these bubbles collapse and violently accelerate the fluid around them. During bubble collapse, adiabatic heating of the gas produces very high temperature, produces free radicals, generates very high liquid shear force, and generates a shock wave as the collapsing spherical wall slams into itself (Brennen, 1995). With a sufficient number of collapse cavitation events, cell membranes

The oesophagus acellular matrix (EAM) tissue-scaffold has the potential to serve as the foundation for a tissue-engineered oesophagus for repair of ablative defects. Similar to all collagen-based biomaterials, the EAM is subject to enzymatic degradation in vivo. The introduction of exogenous crosslinks to collagen molecules via glutaraldehyde (Glu) is the most accepted method of stabilizing collagen biomaterials, but fixation with Glu incurs adverse effects. Genipin (Gp), a naturally occurring crosslinking agent, has shown to be effective at improving the stability of collagen-based biomaterials with less cytotoxicity and reduced in vivo inflammatory responses than Glu. The aim of this study was to show that crosslinking with Gp improves the stability of the EAM while maintaining minimal biological reactivity and preserving EAM regeneration potential in a rat model. EAMs were crosslinked with Gp and Glu. Uncrosslinked EAMs served as controls. Denaturation temperature measurement and burst-pressure measurement after enzymatic degradation assays were used to determine the effectiveness of crosslinking on in vitro stability. Subcutaneous allograft implantation and oesophageal epithelial cell-seeding studies assessed the crosslinking effects on biological reactivity and regeneration potential, respectively. Both Gp and Glu improved EAM stability. After 30 days of implantation, the EAM elicited a minimal inflammatory response and crosslinking did not increase inflammation. Gp-crosslinked EAMs supported epithelial adhesion and proliferation while Glu-crosslinked EAMs did not. Gp improves the stability of the EAM while maintaining minimal biological reactivity and preserving EAM epithelial proliferation capacity, yielding a tissue scaffold that may form the basis of a durable and biocompatible tissue-engineered oesophagus.