bluuebunny/crossref_metadata_2025 huggingface dataset:
Adaptation of the Mitral Valve to Chronic Ischemic Left Ventricular Failure in Swine
Authors: McIver Bryant V. , Hussain Samiya , Thourani Vinod , Padala Muralidhar
Published: July 2012

Mitral valve leaflets in patients with chronic heart failure are severely stiffened, larger and fibrotic; though the mechanisms underlying such biological remodeling are currently unknown. Chronic tethering of the valve leaflets by the enlarged left ventricle, and the presence of mitral regurgitation (MR), is hypothesized to trigger fibrotic pathways that induce such remodeling. In this study, we sought to develop a large animal model of chronic ischemic left ventricular failure in which moderate leaflet tethering and MR can be repeatedly induced, and the remodeling of the mitral valve can be assessed in vivo using echocardiography. Inferior wall myocardial infarction (IMI) was induced in 16 farm swine (30-35kg) using a percutaneous transfemoral approach. Coronary angiography was performed to selectively identify the marginal branches of the left circumflex artery perfusing the inferior left ventricular (LV) wall and the posterior papillary muscle (PPM), and then these regions were infarcted using a 2 mm balloon catheter and injection of 100% ethyl alcohol into the corresponding branch. Transthoracic 2D echocardiography (TTE) of the left heart was performed pre and post-IMI in all animals, at 4 weeks and at 8 weeks. MR percentage (MR jet/left atrial area), mitral leaflet length and thickness, and mitral annular geometry were measured. Leaflet length and thickness were measured at end diastole to assure measurements were taken in the maximal unloaded state. All animals survived the procedure without complications (0% mortality). At baseline, the swine had mild MR (10.7±8%) which significantly increased to 26.6±9% immediately post-op (p<0.05), declined slightly to 23.8±7.8% at 4 weeks, and increased to 32.2±13.9% at 8 weeks. Mitral annular diameter increased from 2.7±0.5 cm pre-op, to 3.1±0.5 cm post-op (p<0.05), then to 3.8±0.4 cm (p<0.005) and 3.9±0.6 cm (p<0.005), at 4 and 8 weeks, respectively. Posterior leaflet length was 1.8±0.2 cm pre-op, and then stabilized at 1.9±0.2 cm post-op, at 4 weeks, and at 8 weeks. Anterior leaflet length was 2.3±0.3 cm at baseline and increased with the increase in MR, to 2.5±0.4 cm post-op, 2.8±0.5 cm at 4 weeks (p<0.05), and 3.2±0.2 cm at 8 weeks (p<0.001). Posterior leaflet thickness increased from 0.36±0.05 cm at baseline, to 0.37±0.07 cm post-op, and then to 0.4±0.05 cm and 0.41±0.03 cm at 4 weeks and 8 weeks, respectively. Anterior leaflet thickness was 0.4±0.05 cm at baseline and post-operatively, and 0.47±0.03 cm (p<0.01) and 0.45±0.07 cm at 4 weeks and 8 weeks, respectively. Changes in mitral leaflet geometry correspond with changes in the mitral valvular apparatus after IMI in a chronic swine model. As the mitral annulus increases in diameter, significant lengthening is seen from the anterior leaflet, while the posterior leaflet remains relatively static and tethered. The anterior leaflet thickens by 4 weeks, while the posterior leaflet remains stable.
Distensibility of Decellularized and Glutaraldehyde-Preserved Aortic Full Roots as Evaluated by Computed Tomography
Authors: Honge Jesper L. , Mads B Kronborg Mads B Kronborg , Bottcher Morter , Norgaard Bjarne L , Hasenkam J Michael , Sten L Nielsen Sten L Nielsen
Published: July 2012

The aortic root is a dynamic structure and its distensibility is considered an important factor in native and prosthetic heart valve function. Glutaraldehyde-preserved (GLUT) valves have limited durability possibly due to increased tissue stiffening. Decellularized deoxycholic acid-treated (DOA) valves exhibit excellent long-term performance in the pulmonary position in humans possibly due to preservation of valve tissue distensibility. We investigated aortic distensibility in DOA (n=8) and GLUT (n=3) aortic root prostheses in 60 kg pigs 2 weeks after orthotopic implantation. Five pigs served as controls. Using a dual source computed tomography scanner (Somatom Flash, Siemens Medical Solution, Forcheim, Germany) the cross sectional area at the level of Sinus of Valsalva (SoV), sino-tubular junction (STJ), and ascending aorta (AA), respectively, was measured in both diastole and systole. Distensibility was defined as the change in area from diastole (RR 95%) to systole (RR 15%). For assessment of the prosthetic- independent aortic distensibility we performed the same measurements in the descending aorta (AD) in all animals. Data were analyzed using students t-test and reported as a mean±SD.
Native aortic distensibility was significant larger at the level of SoV (15.8%±4.9), STJ (46.7%±10.3), and AA (40.6%±9.0), compared with both DOA and GLUT aortic roots (p<0.05). No difference in distensibility between the DOA and GLUT aortic roots were observed: 8.8%±2.4 vs 6.2%±5.5 (SoV), 11.7%±3.0 vs 10.0%±7.1 (STJ), 14.9%±6.1 vs 11.8±1.7 (AA), and 15.6±1.5 vs 15.5±1.8 (DA) , respectively (p>0.05). There was no difference in mean distensibility between the three groups in the decending aorta. This is the first study to evaluate in-vivo distensibility of aortic root prostheses in vivo. Aortic root distensibility is reduced following implantation of DOA or GLUT prosthetic aortic valves. No difference was observed between DOA and GLUT valves. Long-term follow-up is needed in order to verify any changes in bioprosthetic aortic root distensibility over time.
The Role of Integrins on the Regulation of Contractility in Mitral Valve
Authors: Zamani Marzieh , Sarathchandra Padmini , Latif Najma , Yacoub Magdi H. , Chester Adrian H.
Published: July 2012

The mechanical properties of heart valves are modulated by the contractile response of valve interstitial cells (VICs). It is unknown how the contractile responses of VICs are translated to the extra cellular matrix to alter the stiffness of the valve tissue. We have studied the signalling mechanisms of integrin subunits and specifically α2β1 integrin, a predominant collagen binding molecule, in mediating the contractility of porcine mitral valve tissue and cells.
The expression of integrins was analysed by immunostaining of cells. The contractility of porcine mitral VICs in the presence and absence of a α2β1 integrin blocking antibody and the effect of different signalling inhibitors was analysed in a collagen gel. The effect of modulators of contractility was determined on mitral valve tissue incubated in organ baths which challenged with endothelin-1 (10-10-10-7M) following 24 hours incubation with α2β1 integrin blocking antibody. Immunohistochemistry confirmed positive staining for α2β1 integrin and the expression of α1, α2, α3 and β1 in the VICs. The α2β1 blocking antibody was able to significantly reduce the contraction of collagen gels from 63.6±7.8 to 23.4±4.3 % (p<0.001) of control (no cells in gel). PF573228, the inhibitor of focal adhesion kinase, caused a reduction of gel contraction by 30% of the control. The maximum contractile response of cusp tissue to ET-1 maintained under isometric conditions in tissue baths was significantly reduced from 9.5±5.0 to 3.0±2.5mN/g (p<0.001) wet weight by the presence of the α2β1 integrin blocking antibody. These results demonstrate a role for α2β1 integrin in linking the contractile response of valve cells to the extra cellular matrix and highlight further the complex interactions between the cells and the extra cellular matrix of the valve.
The Congenital Bicuspid Aortic Valve can Experience High Frequency Unsteady Shear Stresses on its Leaflet Surface
Authors: Yap Choon Hwai , Saikrishnan Neelakantan , Tamilselvan Gowthami , Vasilyev Nikolai , Yoganathan Ajit P.
Published: July 2012

Bicuspid aortic valve (BAV) is a common congenital malformation affecting 1-2% of the population and is highly correlated to early calcification of the valve leaflets. Two widely held hypothesis for this correlation are (1) altered shape of the BAV results in altered fluid mechanical environment, leading to pro-calcification biology responses, and (2) inherent genetic defects results in pre-disposition of the tissues to calcify. In the current study, we tested the first hypothesis with porcine valve models in an in vitro flow loop. One BAV model and one tricuspid aortic valve (TAV) model were constructed using healthy porcine AV leaflets and tested in a physiological pulsatile flow loop. Fluid velocities near the center of the aortic surface of the valve leaflets were measured with Laser Doppler Velocimetry at a spatial resolution of 89 microns, and ensemble average shear stresses were calculated at various time points in the cardiac cycle. Unsteadiness of flow near the valve leaflets was quantified with variance analysis and power spectral analysis. Particle Image Velocimetry was used to visualize flow fields downstream of the valves and in the sinuses. The leaflets of the BAV model experienced shear stresses on the aortic surface with magnitudes similar to that of the TAV. However, flow near the BAV leaflets had high frequency unsteadiness components, especially during mid- to late- systole, and had high cycle-to-cycle magnitude variability, indicating that shear stresses will have similar unsteadiness and magnitude variability. These are most likely due to the stenosis in the BAV and the skewed forward flow, which collided with the aorta wall. In conclusion, our study indicated that some BAVs could experience high frequency unsteadiness and cycle-to-cycle magnitude variability on the valve leaflets because of its geometry. We speculate that, together with genetic factors, such adverse mechanical force environment could play a role in causing early calcification in the BAV leaflets.
Shear- and Side-dependent microRNAs and Messenger RNAs in Aortic Valvular Endothelium
Authors: Holliday-Ankeny Casey J. , Ankeny Randall F. , Ferdous Zannatul , Nerem Robert M. , Jo Hanjoong
Published: July 2012

Aortic valve (AV) disease is a major cause of cardiovascular-linked deaths globally. In addition, AV disease is a strong risk factor for additional cardiovascular events; however, the mechanism by which it initiates and progresses is not well-understood. We hypothesize that low and oscillatory flow is present on the fibrosa side of the AV and stimulates ECs to differentially regulate microRNA (miRNA) and mRNAs and influence AV disease progression. This hypothesis was tested employing both in vitro and in vivo approaches, high throughput microarray and pathway analyses, as well as a variety of functional assays. First, we isolated and characterized side-dependent, human aortic valvular endothelial cells (HAVECs) isolated from transplant recipient AVs. We found that HAVECs express both endothelial cell markers (VE-Cadherin, vWF, and PECAM) as well as smooth muscle cell markers (SMA and basic calponin). Further, HAVECs align in the direction of the flow as well as uptake acetylated LDL. Using microarray analysis on sheared, side-specific HAVECs, we identified side- and shear-induced changes in miRNA and mRNA expression profiles. More specifically, we identified over 1000 shear-responsive mRNAs which showed robust validation (93% of those tested). We then used Ingenuity Pathway Analysis to identify key miRNAs, including those with many relationships to other genes (for example, thrombospondin and IκB) and those that are members of over-represented pathways and processes (for example, sulfur metabolism). Furthermore, we validated five shear-sensitive miRNAs: miR-139-3p, miR-148a, miR-187, miR-192, and miR-486-5p and one side-dependent miRNA, miR-370. To prioritize these miRNAs, we performed in silico analysis to group these key miRNAs by cellular functions related to AV disease (including tissue remodeling, inflammation, and calcification). Additional miRNAs of interest (including miR-7 and miR-506) were determined through analysis of overrepresented miRNA binding sequences in the shear-sensitive mRNA array. Next, to compare our in vitro HAVEC results in vivo, we developed a method to isolate endothelial-enriched, side-dependent total RNA and identify and validate side-dependent (fibrosa vs. ventricularis) miRNAs in porcine aortic valvular endothelium. From this analysis, we discovered and validated eight side-dependent miRNAs in porcine endothelial-enriched AV RNA, including one miRNA previously identified in vitro, miR-486-5p, as well as a shear-responsive miRNA cluster, miR-199a/214. Through microarray studies and in silico analysis, we have prioritized key miRNAs which may serve as master regulators of AV disease. Better understanding of AV biology and disease in terms of gene-regulation under different hemodynamic conditions will facilitate the design of a tissue-engineered valve and provide alternative treatment options.
Modeling Flow-Responsive Gene Regulation In Native Valve Tissue
Authors: Siyahian Arpi , Chalajour Fariba , Ma Xiaoyuan , Hanley Frank L. , Riemer R. Kirk
Published: July 2012

A variety of approaches to heart valve tissue engineering are underway by many groups of investigators. We believe that a thorough understanding of the biophysical and biomolecular interactions that mediate normal leaflet homeostasis will inform improved approaches to the engineering of truly regenerative heart valve grafts. However, our understanding of the fundamental biology of normal valve tissue is still quite limited. This informational gap has led us to develop an ex vivo system for long-term valve culture in order to study the mechanobiology of native tissue. Using this system, we have cultured trileaflet rat heart valves with and without flow-induced valve cycling. We report here the preliminary findings about the effects of both ex vivo culture and of valve cycling on pulmonary leaflet cell gene expression following 7 days of flow culture. Expression of leaflet mRNA was queried using Affymetrix whole genome microarrays and the results analyzed using GeneSpring, EXPLAIN and MetaCore (GeneGO) software. Our analysis revealed 2147 genes whose expression was altered under flow conditions, and 1918 genes under static conditions. 1501 genes were common to both conditions. The effects of culture on gene expression affected multiple pathways, but the most active gene groups involved pathways for cytoskeletal remodeling, development, and cell adhesion. The genes involved in these pathways were altered in both culture conditions. Changes in cytoskeletal and ECM remodeling genes were more prominent in the static condition, while those involved in VEGF signaling were seen under flow condition. Our previously reported histological analyses of cultured rat pulmonary valve leaflet tissue revealed that flow promotes the maintenance of tissue integrity while the lack of flow leads to extracellular matrix remodeling and fibrinoid tissue formation. These gene expression analyses confirm the expected role of flow in maintaining leaflet tissue integrity as evidenced by its induction of VEGF signaling genes, while the lack of flow induces changes in genes involved in extracellular matrix and cytoskeletal remodeling.
Computational Structural Biomechanical Models to Guide Tissue Engineered Heart Valve Leaflet Fabrication
Authors: D'Amore Antonio , Bayoumi Ahmed S. , Amoroso Nicholas , Wagner William R. , Mayer John , Sacks Michael S.
Published: July 2012

A computational structural deterministic modeling strategy has been developed and experimentally validated to (1) assist tissue engineering scaffold fabrication, and as a consequence to improve in vivo scaffolds performances, and (2) provide a better understanding of cellular mechanical and metabolic response to local micro-structural deformations of the extracellular matrix (ECM). Image analysis software was developed and tested on electrospun poly (ester urethane) urea (PEUU) scaffolds, collagen gels, decellularized tissues. The algorithm analyzed SEM and multi-photon images (maximum imaging penetration depth: 160 µm) providing a full 3D characterization of engineered constructs morphology (n ≥ 6). The detected material topologies were adopted to generate statistically equivalent scaffold biomechanical models minimizing the difference between the real material and network model architectural features. The mechanical response at the macro scale was fully characterized by stress control biaxial tests (n ≥ 6). The experimental biaxial response was used to calibrate a Finite Element Model able to predict, for a given material topology, the mechanical response at both organ (cm), cells (100 µm) and fiber (1 µm) levels. Scaffold networks models were imported in Abaqus, Yeoh strain energy with incompressibility hypothesis and t2d2h elements were adopted. Stress vs. strain prediction was produced for four different scaffold groups: isotropic ES-PEUU, anisotropic ES-PEUU, Vascular Smooth Muscle Cells integrated PEUU, Polystyrene micro-spheres integrated (10 µm diameter) proving the flexibility of the modeling approach. At mesoscopic level nuclear aspect ratio vs. strain curve for the rat VSMCs embedded into the scaffold was produced and compared with previous experimental findings. At the microscopic level the single fiber initial shear modulus was quantified from the strain energy function material parameters, and compared with Atomic Force Microscopy measurements on single PEUU fibers. The developed generalist modeling approach bridges scaffold fabrication parameters, micro architecture, and organ level - cell level mechanical response.
Inflammation Drives Endothelial-to-Mesenchymal Transition and Interstitial Calcification in 3D in Vitro Culture
Authors: Richards Jennifer , Farrar Emily , Mahler Gretchen , Butcher Jonathan
Published: July 2012

Calcific aortic stenosis is a serious pathology that accounts for 43% of those suffering from heart valve disease. Valve disease initially presents with inflammation and endothelial dysfunction, which can lead to an endothelial-to-mesenchymal (EndMT) shift in the endothelial layer of the valve. It is not yet well understood how valve endothelial cells (VEC) regulate valve interstitial cells (VIC) in inflammatory or osteogenic environments. We here present a 3D culture system that models cellular responses and interactions when exposed to different environmental conditions. Porcine aortic VEC and/or VIC were cultured in mechanically constrained 3D type I collagen hydrogels for up to 14 days. Early disease studies introduced different dosages of TNFα to VEC on gel surfaces for up to 48 hours. EndMT was assessed via real time PCR and cell invasion within the matrix. Apoptosis and proliferation in VEC via TUNEL and anti-BrdU IHC staining were also evaluated in early inflammatory conditions. Later early-stage disease studies introduced osteogenic and inflammatory environments to 3D VIC/VEC gels through the addition of osteogenic differentiation factors into the culture media (OGM), or 30 ng/ml TNFα. Calcium deposition within the matrix was measured via Alizarin Red staining. Real time PCR was used to evaluate the expression of calcification-related genes such as osteocalcin and runx-2.
Early inflammatory conditions as induced by addition of TNFα for 48 hours stimulated EndMT-like VEC activation, including matrix invasion and upregulation of αSMA and snail. Addition of TNFα increases VEC proliferation in a dose dependent manner. TNFα does not significantly increase apoptosis, even at the highest dosage levels. VIC gels cultured in OGM for 14 days significantly calcified and expressed osteocalcin and runx-2. Co-culture with VEC inhibited both matrix calcium deposition and osteoblastic differentiation. When 30 ng/ml TNFα was added to VIC 3D cultures, TNFa induced increased matrix calcium deposition in 14 days, which was then mitigated by co-culture with VEC. The inhibitory effect of VEC on VIC calcification in OGM was blocked when TNFα was introduced to the culture. These results demonstrate that inflammatory microenvironments induce calcification in valve interstitial cells. There is a protective role for the valve endothelium in VIC calcification, which could be inhibited by EndMT induced by the inflammatory conditions found in diseased valves. We found that apoptosis is not a driving factor in inflammatory activation of VEC or osteogenic differentiation of VIC. Targeting TNFα and EndMT signaling could be important therapeutic strategies. More generally, targeting VEC is a promising approach for early treatment of valve disease. This co-culture 3D system is a powerful tool to elucidate mechanisms of valve disease.
Design of a Novel Ex Vivo Bioreactor to Investigate the Effect of Pressure Induced Stretch on Aortic Valve Biology
Authors: Arjunon Sivakkumar , Dinh Anh-thi , Rathan Swetha , Ajit Yoganathan Ajit Yoganathan
Published: July 2012

A variety of ex vivo bioreactors have been developed to study the mechanobiology of valves and valve substitutes. Especially, bioreactors to mimic the individual mechanical stimuli (stretch, pressure and shear), have been well established. Going forward, it is essential to characterize the structural and biological changes in the aortic valve when subjected to a combination of these stimuli. Especially, since it is expected that a synchronized exposure to combined mechanical stimuli has a significantly different effect than the effect caused by individual stimulus. To this end, a novel bioreactor was designed, fabricated and validated to study the effect of stretch induced by the physiological transvalvular pressure gradient on fresh porcine aortic valve. The ex vivo bioreactor developed in the current study can maintain consistent physiological loading conditions with independent control of the flow rate, pressure and frequency to reproduce clinically relevant mechanical conditions. The design consists of a linear actuator system driving the culture media through the valve at a prescribed flow rate. The pressure levels on either side of the valve can be controlled by using a compliance chamber on the aortic side which is connected to a compressed air circuit. The linear drive system has a piston arrangement at its end, and drives the media in a rigid cylindrical ventricle section. The media returns to the ventricular side though a compliant channel during the reverse motion of the linear actuator. The entire setup is placed inside an incubator maintained at 370 C and 5% CO2. The flow and pressure variations were observed to be physiologically accurate and consistent over the time period of the experiment. Other salient features include low volume of the entire setup, and replaceable valve mounting mechanism to accommodate different sized valves. To ensure that the valve cells are healthy and native phenotype is maintained in the bioreactor, porcine aortic valves were mounted to the stents and subjected to normal physiological conditions of 80/120mmHg pressure, 5 LPM flow rate of Dulbecco’s modified eagle media(DMEM). The entire setup was placed inside an incubator. After 48 hours of culture, Movat Pentachrome stain was done to observe the tissue morphology, and DAPI stain was done to assess the cell viability. Fresh porcine AV leaflets were used as controls. The results were comparable to fresh controls, and indicated that the bioreactor is capable of preserving leaflet morphology and cell viability when cultured under normal physiological conditions for 48 hours.
Decellularized Tissue Engineered Heart Valves: Infiltration, Inflammation and Regeneration?
Authors: Sanders Bart , Driessen-Mol Anita , Dijkman Petra E. , Hoerstrup Simon P. , Baaijens Frank P.T.
Published: July 2012

Decellularized native biological tissue is currently investigated for its use as heart valve tissue engineering scaffold. Important features are the intact native matrix structure and retained mechanical properties. Unfortunately, these materials lack sufficient cell infiltration, reducing its remodeling potential. As shown in our recent ovine study (see abstract A. Driessen-Mol), in vitro cultured decellularized tissue-engineered heart valves facilitate complete cell infiltration, even after only 5 hours in vivo. This rapid cell infiltration is essential for remodeling. In general, all implanted biological materials evoke an inflammatory response, resulting in undesired chronic inflammation and/or fibrosis or in desired regeneration. Macrophage phenotype (M1/M2) is demonstrated to play an essential role in regulating the delicate balance between inflammation and regeneration. More in-depth insights underlying the process of cell infiltration and subsequent host inflammation response is crucial to achieve regeneration and is studied here using an in vitro model system. For this in vitro study, we used an adapted setup of the IBIDI flow chamber system and simulated native pulmonary conditions. A mix of mono- and polynuclear cells was isolated from fresh ovine blood and added to the IBIDI system to circulate along inserted pieces of tissues. Ovine tissue-engineered patches were cultured according to our protocols for heart valve tissue engineering. In short, ovine vascular cells were isolated and cultured for 4 weeks on a PGA-P4HB scaffold. The patches were decellularized afterwards. Cell infiltration into both decellularized ovine tissue-engineered patches and decellularized native ovine pulmonary leaflets was studied up to 5 hours. Thereafter, the tissues were fixed, stained with DAPI and visualized by confocal microscopy. Tremendous cell infiltration was observed at the edges in the decellularized tissue-engineered patches, whereas no clear cell infiltration was observed for the decellularized native leaflets. These first preliminary in vitro results are indicative of the host body’s capacity to rapidly infiltrate decellularized tissue-engineered matrices with inflammation-associated blood cells. Further research for cell type identification and tissue regenerative capacity is ongoing.

The European Union’s Seventh Framework Program is acknowledged for funding this study.
Fibronectin Coating Triggers In Vivo Recellularization of Tissue-Engineered Aortic Conduits
Authors: Assmann Alexander , Delfs Christofer , Horstkötter Kim , Munakata Hiroshi , Poudel-Bochmann Bhawana , Huynh Khon , Stoldt Volker , Lichtenberg Artur , Akhyari Payam
Published: July 2012

Strategies that aim at an acceleration of in vivo neoendothelialization and medial repopulation are warranted to increase the integrity and functionality of tissue-engineered cardiovascular implants. Proactive coating of decellularized implants represents a promising approach, however, little is known about the in vivo fate of coated agents over time. Detergent-decellularized aortic rat conduits (n = 24) were coated with covalently Alexa488-labelled (green emission) fibronectin (FN; 50 µg/ml, 24 hours) and implanted in the infrarenal aorta of wildtype Wistar rats (Group A; n = 12). Uncoated implants served as controls (Group B; n = 12). Before implantation and at postoperative day 1 and week 1, 4 and 8, fluorescence-based detection of FN coating was performed. Cellular repopulation was examined by histology and immunohistochemistry.
All rats survived without clinical or sonographic signs of lower body malperfusion. Confocal microscopy of the aortic conduits revealed bright green FN fluorescence before and 1 day after implantation on the luminal as well as on the adventitial surface. The signal intensity decreased after 1 week, but was still present after 4 and 8 weeks. Four weeks after the operation, the luminal surface in the perianastomotic regions of Group A was completely neoendothelialized (vWF+) and a myofibroblast hyperplasia (αSMA+) with increased ratio of intima-to-media (I/M) thickness occurred. After 8 weeks I/M was significantly increased in Group A versus Group B (p < 0.01). At the same time point, medial repopulation starting at the adventitial zone was observed in group A, while only marginal repopulation occurred in group B (p < 0.001). In both groups vonKossa staining revealed sparse medial calcification and staining against inflammatory cell markers (CD3 & CD68) was negative. In our standardized rat transplantation model, a biofunctional protein coating of cardiovascular implants in the systemic circulation proved feasible and persistent up to 8 weeks. FN surface coating of aortic conduits induced a significantly increased medial recellularization, originating from the adventitial surface. The role of intimal hyperplasia and the relevance thereof needs further investigation.
Development of In-Body Tissue-Engineered, Completely Autologous Valve (BIOVALVE)
Authors: Takewa Yoshiaki , Nakayama Yasuhide , Yamanami Masashi , Kishimoto Yuichiro , Arakawa Mamoru , Matsui Yuichi , Kanda Keiichi , Yaku Hitoshi , Tajikawa Tsutomu , Ohba Kenkichi , Taenaka Yoshiyuki , Tatsumi Eisuke
Published: July 2012

Using simple, safe, and economical in-body tissue engineering, autologous valved conduits (BIOVALVEs) with the sinus of Valsalva and without any artificial support materials were developed in animal recipients’ bodies. In this study, the feasibility of theBIOVALVE as an aortic valve was evaluated in a goat model. BIOVALVEs were prepared by 2-month embedding of the molds, assembled using 2 types of specially designed plastic rods, in the dorsal subcutaneous spaces of goats. One rod had 3 projections, resembling the protrusions of the sinus of Valsalva. Completely autologous connective tissue BIOVALVEs with 3 leaflets in the inner side of the conduit with the sinus of Valsalva were obtained after removing the molds from both terminals of the harvested implants with complete encapsulation. The BIOVALVE leaflets had appropriate strength and elastic characteristics similar to those of native aortic valves; thus, a robust conduit was formed. Tight valvular coaptation and sufficient open orifice area were observed in vitro. BIOVALVEs (n=3) were implanted in the specially designed apico-aortic bypass for 2 months as a pilot study under the systemic circulation. Postoperative echocardiogram and angiogram showed smooth movement of the leaflets with little regurgitation (2.6 ±1.1 L/min). The α-SMA–positive cells migrated in the tissue of the conduit significantly with rich angiogenesis and expanded toward the leaflet tip. At the sinus portions, marked elastic fibers were formed. The luminal surface was covered with thin pseudointima without thrombus formation. Completely autologous BIOVALVEs with robust and elastic characteristics satisfied the higher requirements of systemic circulation in goats for 2 months with the potential for valvular tissue regeneration.
A Pulmonary Valved Conduit of Porcine SIS Remodels into Native Tissue in an Ovine Model
Authors: Fallon Anna , Goodchild Traci , Gilbert Christian , Matheny Robert
Published: July 2012

Reconstruction of the pulmonary valve and outflow tract is frequently needed to repair congenital defects. Current substitutes lead to graft failure and reoperation due to calcification, shrinkage, progressive insufficiency or transvalvular gradients, and relative growth of the patient compared to the valve. CorMatrix extracellular matrix (ECM), derived from decellularized, non-crosslinked small intestine submucosa (SIS) is used for general cardiac repairs and regenerates into normal cardiac tissue with growth potential. Previously, we showed that an ECM pulmonary valve leaflet remodeled into a neo-leaflet histologically similar to native valve architecture. In this study we used an ECM valved conduit for pulmonary valve replacement in an ovine model to demonstrate its potential to remodel into native tissue. A trileaflet valved conduit was produced from CorMatrix ECM sutured into a tube then intussuscepted to form a tube within a tube. At three equidistant points the inner tube was sutured to the outer tube forming three leaflets to guide unidirectional flow with physiologic opening and closing mechanics. Under cardiopulmonary bypass the ovine pulmonary valve and pulmonary artery section was removed and replaced with the ECM valved conduit. Valve function was evaluated by echocardiography post-operatively and at bi-monthly intervals until euthanasia at 3, 5, 8, and 12 months. Histological evaluation included H and E, Movat pentachrome, von Kossa, anti-CD31, and anti-eNOS. Our echocardiography results show that a pulmonary valve constructed from ECM opens and closes completely without regurgitation or stenosis for 12 months. Grossly, explanted valves appeared similar to native valves and were remodeling after 3 months with further progression to native morphology after 5, 8 and 12 months. Histological examination showed diffuse cellular infiltration by 3 months. At 5 months, collagen organization was increased and glycosaminoglycans were distributed throughout the middle of the leaflet. At 3 months, SEM and eNOS staining demonstrated a confluent and functional endothelial lining on the pulmonary artery and hinge regions of the valve. At 5 months, this lining extended to the center of the leaflet with confluent areas at the leaflet tip. At 8 and 12 months, a tri-layered structure similar to native valve architecture was demonstrated histologically by a Movat stain with a confluent endothelial lining demonstrated by eNOS and CD31 staining. The von Kossa stain showed an absence of calcific deposits at all time points except occasionally at the suture. These results demonstrate the potential of a CorMatrix ECM pulmonary valve to remodel into endothelialized tissue that is indistinguishable from the host’s native valve both grossly and histologically. Such a regenerated valve would be expected to improve patient outcomes since it remodels into native tissue with growth potential.
Finite Element Modeling of Transcatheter Aortic Valve Replacement
Authors: Azadani Ali N. , Chitsaz Sam , Wisneski Andrew , Lui Natalie , Guccione Julius M. , Ge Liang , Tseng Elaine E.
Published: July 2012

Transcatheter aortic valve replacement (TAVR) has revolutionized treatment for inoperable and high risk surgical patients with severe symptomatic aortic stenosis. Transcatheter aortic valves (TAVs) are deployed within the native diseased valve without sutures to secure them within the annulus. Oversizing of TAVs with respect to the annulus size is required to achieve appropriate anchoring. Optimal TAV function requires expansion of the frame to its nominal dimension. However, clinically TAVR results routinely in incomplete expansion of the stent frame. We have previously demonstrated that significant under-expansion results in suboptimal TAV function with impaired coaptation of TAV leaflets, but precise characteristics of TAV leaflets and frame after implantation have been poorly studied. The aim of this study was to determine the effect of TAV under-expansion as observed clinically on stress distribution and magnitude in the TAV stent and leaflets using finite element (FE) modeling. A computer aided design (CAD) model of a TAV was developed based on the 23mm Edwards-SAPIEN design and used to create a finite element (FE) model. The 3D model consists of a stent, three pericardial leaflets, a clamp compression unit and an expandable balloon. Large deformation FE simulations were conducted to model the TAVR procedure, including TAV crimping followed by balloon-expansion to 17, 21, and 23mm. Stress distribution on the stent and leaflets were determined. As the post-inflation diameter increased, the von Mises stress on the TAV stent decreased. The maximum von Mises stresses of stent after expansion to 17, 21, and 23mm were 365, 346, and 262 MPa, respectively. However, unlike the stent, the leaflet stress increased as the post-inflation diameter increased. The peak von Mises stresses after expansion were 1.5, 1.3, and 2.4 MPa, respectively. We present the first FE simulation which was developed to model the TAVR procedure from crimping and balloon inflation of the TAV. Stress on stent and leaflets after implantation is dependent on the internal diameter of the inflated stent. While stress on the stent decreases with increasing TAV expansion, stress on the leaflets increases. FE modeling can be further applied evaluate whether a specific TAV size and design is optimal for a specific patient.
Prevalence of Patients With Severe Aortic Stenosis, Low Flow And Preserved Ejection Fraction: Results From a Cath-Lab Data Base.
Authors: Mauro Toniolo , Andrea Rossi , Mariantonietta Cicoira
Published: July 2012

Recent echocardiographic studies described that almost 30% of patients with severe aortic valve stenosis on the basis of aortic valve area may paradoxically have a relatively low mean gradient despite a preserved left ventricular ejection fraction (EF). However the existence of this pathologic entity has been questioned mainly for the lack of invasive data. We aimed to describe the prevalence of patients with severely reduced aortic valve area and low gradient from a consecutive series of patients with aortic stenosis and normal EF undergoing cardiac catheterization. Sixty one consecutive patients with invasively measured aortic valve area < 0,6 cmq/mq (AHA/ACC definition for severe aortic stenosis) and EF> 50% formed the study population. Each patient underwent to right and left heart catheterization for a comprehensive invasive hemodynamic evaluation. Aortic valve area was measured by Gorlin formula. Cardiac output was measured by thermodilution or Fick method. Low mean gradient was defined < 30 mmHg. 16 % of patients were characterized by low GM despite severely reduced aortic valve area. Patients with low GM were characterized by significantly higher aortic valve area (0.47±0.09 vs 0.36±0.09 cm2/m2; p=0.0008) but similar left ventricular stroke volume (SV) (65±22 vs 65±17 ml; p=0.9) and cardiac output (4.8±1.1 vs 4.7±1.0; p=0.7). The prevalence of low flow (defined as SV < 35 ml/ m2) was similar between groups (50% vs 43%; p=0.3). There was no difference in term of age (78±10 vs 79±11 years; p=0.6), female gender (50% vs 48%; p=0.5), body surface area (1.79±0.4 vs 1.80±0.4; p=0.8), pulmonary artery systolic pressures (37±9 vs 35±11 mmHg; p=0.8), LV end diastolic pressure (16±4 vs 20±7; p=0.1) and mean wedge pressure (17±7 vs 15±7; p=0.2). Patients with low GM showed a higher mean AO pressure (111±14 vs 93±14; p=0.009) but similar level of aortic distensibility (0.78±0.3 vs 0.9±0.4 ml/mmHg; p=0.3). This invasive study confirms that a substantial percent of patients may have a low GM despite a severely reduced aortic valve area and normal EF. It should be acknowledge that the barely perception of this pathologic entity might have reduced the likelihood of patients to undergo catheterization leading to underestimation of the prevalence of this condition.
Characterization of Early Porcine Aortic Valve Disease
Authors: Sider Krista L. , Kwong Andrea V. , Wei Bowen , Simmons Craig A.
Published: July 2012

In calcific aortic valve disease (CAVD), pathological differentiation of valve interstitial cells (VICs) and lesion formation occur focally in the fibrosa layer. We have shown that VIC pathological differentiation in vitro is sensitive to matrix stiffness. We therefore hypothesized that focal changes in the stiffness of the fibrosa with CAVD progression correlate with alterations in the extracellular matrix (ECM) and pathological cell phenotypes in vivo. Twenty-four male Yorkshire pigs were fed either a control or experimental diet supplemented with 12% lard and 1.5% cholesterol for 2 or 5 months (n = 6 per group). Effective moduli were measured in focal regions of the fibrosa (n = 295) and ventricularis (n = 142) layers of intact right coronary aortic valve leaflets by micropipette aspiration. Histology was conducted within radial center sections and selected focal test locations within the fibrosa from each leaflet to characterize local ECM and cell phenotypes. Early stage disease was induced by the experimental diet, as evidenced by more significant proteoglycan-rich lesions (~10 - 200 µm thick) onlayed on the fibrosa (p < 0.05, controlling for genetic variability). Interestingly, lipid deposition was seen in only 28% of regions with early lesions, and 15% of regions without early lesions. No osteoblastic or macrophage cells were present in control or experimental valves, with only slight myofibroblast presence at the base of some 5 month lesions. Chondrogenic Sox9-positive cells were observed extensively and were positively correlated with proteoglycan content (p < 0.0001). Regions within the fibrosa were significantly stiffer than those in the ventricularis in both control and experimental valves (p <0.001). Although there were no significant differences between the control and experimental overall tissue moduli at these early time points, modulus heterogeneity did increase with disease. Furthermore, lesions in the fibrosa had lower moduli than non-lesion fibrosa locations (p < 0.06). These soft lesions contained significantly more proteoglycan (p <0.001) and Sox9 expression (p = 0.014) than normal fibrosa tissue. In conclusion, ECM remodelling can occur in a porcine model of early CAVD in the absence of lipid deposition, inflammatory cells, osteoblasts, or myofibroblasts, but with significant proteoglycan-rich lesion and chondrogenic cell presence. Mechanically soft proteoglycan-rich lesions may support chondrogenic VIC differentiation, providing new insights into early CAVD pathogenesis.
Biomarketing strategy and tactics 101 PART II of III
Authors: Dogramatzis Dimitris
Published: July 2012

The American Marketing Association defines marketing as the activity, set of institutions, and processes for creating, communicating, delivering, and exchanging offerings that have value for customers, clients, partners, and society at large. According to the Pharmaceutical Research and Manufacturers of America appropriate marketing of medicines ensures that patients have access to the products they need and that the products are used correctly for maximum patient benefit. The most important promotional tools for biopharmaceutical firms are 1) personal selling, 2) advertising, 3) public relations and publicity, and 4) web promotion. Part I of this three-part article focuses on the nature of the biopharmaceutical marketingâ€™s four Pâ€™s, the importance of marketing strategy, the conduct of environmental analysis,Â  and maket segmentation. Part II delves into the processes of targeting and positioning, marketing planning, as well as biopharmaceutical branding. Part III completes the series by focusing on the push and pull promotional strategies, advertising, selling, and biopharmaceutical web and social marketing.
Mayo â€œnaysâ€: The Supreme Court says no to patenting laws of nature
Authors: Camacho Jennifer A.
Published: July 2012

On March 20, 2012, the U.S. Supreme Court handed down its decision in Mayo Collaborative Services, et al v. Prometheus Laboratories, Inc (â€œMayoâ€) and ended an eight-year legal battle over patents covering processes for determining patient-specific dosing for a thiopurine drug to treat autoimmune diseases.Â  In a unanimous decision, the Court held that the claimed processes are not patent-eligible subject matter under 35 U.S.C. Â§101 of the U.S. patent laws, and overturned the decision of the Court of Appeals for the Federal Circuit.The Supreme Court decision in Mayo established that the machine-or-transformation test is not the definitive test for determining the patent-eligibility of process claims, including process claims that embody laws of nature or natural phenomena.Â  In its analysis, the Court determined considered whether the claims were drawn to patent eligible subject matter as provided under 35 U.S.C. Â§101 of the U.S. patent laws, or patent ineligible subject matter excepted from Â§101.Â  The Court held that the process claims were essentially drawn to the laws of nature themselves and thus fell into the laws-of-nature exception to Â§101.Â  The process claims did not cover patent-eligible processes of applying certain laws of nature.Â  This decision has clear implications for the biotechnology industry that go beyond diagnostics and personalized medicine. As such, biotechnology companies should consider re-evaluating their patent position and adapting their patent strategies in view of Mayo.
The Fall of the Moche: A Critique of Claims for South America’s First State
Authors: Quilter Jeffrey , Koons Michele L.
Published: July 2012

The Moche archaeological culture of the North Coast of Peru has been reified into a political system and claimed as the first state in South America. While some recent scholarship has reduced the size of the proposed state, the idea of Moche as a distinct political, social, or ethnic entity remains. In this article we demonstrate that even by the “classic” neoevolutionary theory of the 1960s—1980s the criteria for Moche statehood were not met. We suggest that neoevolutionary models for one or more Moche states are inadequate for understanding an archaeological culture that endured for more than six centuries, and we offer suggestions for directions for future research.
Bone Chemistry at Cerro Oreja: A Stable Isotope Perspective on the Development of a Regional Economy in the Moche Valley, Peru During the Early Intermediate Period
Authors: Lambert Patricia M. , Gagnon Celeste Marie , Billman Brian R. , Katzenberg M. Anne , Carcelén José , Tykot Robert H.
Published: July 2012

In this paper we test the hypothesis that an intensification of maize production preceded the development of a regional Moche political economy in the Moche Valley of north coastal Peru during the Early Intermediate period (400 B.C.—A.D. 600). To do so we analyze stable isotopic signatures of 48 bone apatite and 17 tooth enamel samples from human remains recovered from the site of Cerro Oreja, a large urban and ceremonial center in the Moche Valley. These remains date to the Guañape, Salinar, or Gallinazo phases and provide a diachronic picture of subsistence before the appearance of the Southern Moche state. The most notable patterns identified in the study include a lack of significant change in δ13Capatitevalues from the Guañape to Satinar phases, followed by a significant enrichment in δ13Capatitevalues from the Salinar to Gallinazo phases. Several lines of evidence, including archaeological context, dental data, and comparative carbon stable isotope data from experimental animal studies and studies of archaeological human remains support the interpretation that the observed13C enrichment in stable isotope values in the Gallinazo phase primarily reflects maize intensification. The stable isotope data from Cerro Oreja thus suggest that a shift in subsistence toward a highly productive and storable crop may have served as an important precursor to state development during the Early Intermediate period in the Moche Valley.
Virtual Singular Scattering of Electromagnetic Waves in Transformation Media Concept
Authors: Barabanenkov M. Y. , Barabanenkov Y. N. , Nikitov S. A.
Published: July 2012

If a scatterer and an observation point (receive) both approach the so-called near field zone of a source of electromagnetic waves, the scattering process becomes singular one which is mathematically attributed to the spatial singularity of the free space Green function at the origin. Starting from less well known property of left-handed material slab to transfer the singularity of the free space Green function by implementing coordinate transformation, we present a phenomenon of virtual singular scattering of electromagnetic wave on an inhomogeneity located in the volume of left – handed material slab. Virtual singular scattering means that a scatterer is situated only virtually in the near field zone of a source, being, in fact, positioned in the far field zone. Such a situation is realized if a scatterer is embedded into a flat Veselago’s lens and approaches the lens’s inner focus because a slab of Veselago medium produces virtual sources inside and behind the slab and virtual scatterer (as a source of secondary waves) from both slab sides. Considering a line-like dielectric scatterer we demonstrate that the scattering efficiency is proportional to product of singular quasistatic parts of two empty space Green functions that means a multiplicative quasistatic singularity of the Green function for a slab of inhomogeneous Veselago medium. We calculate a resonance value of the scattering amplitude in the regime similar to the known Mie resonance scattering.