The composition of the extracellular matrix (ECM) is believed to play a role in heart valve disease, and is highly relevant to the design of heart valve tissue engineering scaffolds, yet the interaction of valvular interstitial cells (VICs) with the ECM environment has not been well characterized. Thus, the transformation of VICs to an osteoblast-like phenotype was quantified in VICs cultured on different types of ECM coatings. The results show that the number and size of calcific nodules formed in VIC cultures, as well as the expression of the mineralization markers alkaline phosphatase (ALP) and CBFa1, were highly dependent upon the composition of the culture surface. In fact, VICs cultured on certain ECM components, namely collagen and fibronectin, were resistant to calcification, even upon treatment with several mineralization-inducing growth factors. Meanwhile, cultures of VICs on fibrin, laminin, and heparin coatings not only had a high number of calcified nodules, but also elevated levels of ALP and CBFa1. Nodule composition analysis revealed the presence of multiple types of mineralization, including hydroxyapatite. Although apoptotic and necrotic cells were more concentrated in nodules than in other parts of the VIC cultures, the nodules contained a strong majority population of viable cells. By demonstrating this ECM-dependence of VIC calcification, we aim to identify appropriate biomaterial environments for heart valve tissue engineering as well as elucidate mechanisms of valvular disease.

f Biofilm formation by Pseudomonas aeruginosa has been implicated in the pathology of chronic wounds. Both the D and L isoforms of tryptophan inhibited P. aeruginosa biofilm formation on tissue culture plates, with an equimolar ratio of D and L isoforms producing the greatest inhibitory effect. Addition of D-/L-tryptophan to existing biofilms inhibited further biofilm growth and caused partial biofilm disassembly. Tryptophan significantly increased swimming motility, which may be responsible in part for diminished biofilm formation by P. aeruginosa.

Disruption of the extracellular matrix (ECM) is frequently found in calcific aortic valve disease (CAVD), yet the role of ECM components in valvular interstitial cell (VIC) function and dysfunction remains poorly understood. This study examines the contributions of exogenous and endogenous hyaluronic acid (HA), in both two-dimensional (2D) and 3D environments, in regulating the phenotype and calcification of VICs. VIC calcification was first assessed in a 2D setting in which the cells were exposed to different molecular weights of exogenous HA presented in either an immobilized or soluble form. Delivery of HA suppressed nodule formation in a molecular weight-dependent manner, while blocking VIC recognition of HA via an antibody to CD44 abolished these nodule-suppressive effects and stimulated other hallmarks of valvular dysfunction. These 2D results were then validated in a more physiologically-relevant setting, using an approach that allowed the characterization of VIC phenotype in response to HA alterations in the native 3D environment. In this approach, leaflet organ cultures were analyzed following treatment with anti-CD44 or with hyaluronidase to specifically remove HA. Disruption of VIC-HA interactions upregulated markers of VIC disease and induced leaflet mineralization. Similarly, HA-deficient leaflets exhibited numerous hallmarks of CAVD, including increased VIC proliferation, apoptosis, increased expression of disease-related markers, and mineralization. These findings suggest that VIC-HA interactions are crucial in maintaining a healthy VIC phenotype. Identification of ECM components that can regulate VIC phenotype and function has significant implications for understanding of native valve disease, investigating possible treatments, and designing new biomaterials for valve tissue engineering.

BackgroundExtracellular matrix (ECM) disarray is found in calcific aortic valvular disease (CAVD), yet much remains to be learned about the role of individual ECM components in valvular interstitial cell (VIC) function and dysfunction. Previous clinical analyses have shown that calcification is associated with decreased collagen content, while previous in vitro work has suggested that the presence of collagen attenuates the responsiveness of VICs to pro-calcific stimuli. The current study uses whole leaflet cultures to examine the contributions of endogenous collagen in regulating the phenotype and calcification of VICs.MethodsA “top-down” approach was used to characterize changes in VIC phenotype in response to collagen alterations in the native 3D environment. Collagen-deficient leaflets were created via enzymatic treatment and cultured statically for six days in vitro. After culture, leaflets were harvested for analysis of DNA, proliferation, apoptosis, ECM composition, calcification, and gene/protein expression.ResultsIn general, disruption of collagen was associated with increased expression of disease markers by VICs in whole organ leaflet culture. Compared to intact control leaflets, collagen-deficient leaflets demonstrated increased VIC proliferation and apoptosis, increased expression of disease-related markers such as alpha-smooth muscle actin, alkaline phosphatase, and osteocalcin, and an increase in calcification as evidenced by positive von Kossa staining.ConclusionsThese results indicate that disruption of the endogenous collagen structure in aortic valves is sufficient to stimulate pathological consequences in valve leaflet cultures, thereby highlighting the importance of collagen and the valve extracellular matrix in general in maintaining homeostasis of the valve phenotype.

A method for the construction of an array of fuel cells wherein each cell is 200 nm in diameter is presented. Electrodeposition of Pt-Cu nanowires inside the cylindrical pores of an Anodisc filter membrane and the subsequent dealloying of the Cu by soaking the filter in fuming nitric acid for several hours are used to construct an array of porous platinum electrodes. About 10(9) electrically isolated cylindrical porous electrodes, each 200 nm in diameter, are formed in this manner. Utilizing two arrays of porous electrodes with a polymer electrolyte membrane or an electrolyte support matrix sandwiched between, an array of nano fuel cells is produced. This method of producing an array of coplanar fuel cells allows for the series connection of fuel cells outside the array and eliminates the need for fuel and air manifolds, greatly reducing the overall system complexity. Initial prototypes utilizing an aqueous solution of NaBH(4) as a fuel have produced power densities of ca. 1 mW/cm(2) based on an estimate of the area of the current collectors in contact with the nano-fuel-cell array and have demonstrated the ability to wire bundles of fuel cells either in parallel or in series.