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We have previously shown that combined flexure and flow (CFF) augment engineered heart valve tissue formation using bone marrow-derived mesenchymal stem cells (MSC) seeded on polyglycolic acid (PGA)/poly-L-lactic acid (PLLA) blend nonwoven fibrous scaffolds (Engelmayr, et al., Biomaterials 2006; vol. 27 pp. 6083-95). In the present study, we sought to determine if these phenomena were reproducible at the organ level in a functional tri-leaflet valve. Tissue engineered valve constructs (TEVC) were fabricated using PGA/PLLA nonwoven fibrous scaffolds then seeded with MSCs. Tissue formation rates using both standard and augmented (using basic fibroblast growth factor [bFGF] and ascorbic acid-2-phosphate [AA2P]) media to enhance the overall production of collagen were evaluated, along with their relation to the local fluid flow fields. The resulting TEVCs were statically cultured for 3 weeks, followed by a 3 week dynamic culture period using our organ level bioreactor (Hildebrand et al., ABME, Vol. 32, pp. 1039-49, 2004) under approximated pulmonary artery conditions. Results indicated that supplemented media accelerated collagen formation (~185% increase in collagen mass/MSC compared to standard media), as well as increasing collagen mass production from 3.90 to 4.43 pg/cell/week from 3 to 6 weeks. Using augmented media, dynamic conditioning increased collagen mass production rate from 7.23 to 13.65 pg/cell/week (88.8%) during the dynamic culture period, along with greater preservation of net DNA. Moreover, when compared to our previous CFF study, organ level conditioning increased the collagen production rate from 4.76 to 6.42 pg/cell/week (35%). Newly conducted CFD studies of the CFF specimen flow patterns suggested that oscillatory surface shear stresses were surprisingly similar to a tri-leaflet valve. Overall, we found that the use of simulated pulmonary artery conditions resulted Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. substantially large collagen mass production levels and rates found in our earlier CFF study. Moreover, given the fact that the scaffolds underwent modest strains (~7% max) during either CFF or physiological conditioning, the oscillatory surface shear stresses estimated in both studies may play a substantial role in eliciting MSC collagen production in the highly dynamic engineered heart valve fluid mechanical environment. NIH Public Access

We have developed a unique microfluidic platform capable of capturing circulating endothelial progenitor cells (EPCs) by understanding surface chemistries and adhesion profiles. The surface of a variable-shear-stress microfluidic device was conjugated with 6 different antibodies [anti-CD34, -CD31, -vascular endothelial growth factor receptor-2 (VEGFR-2), -CD146, -CD45, and -von Willebrand factor (vWF)] designed to match the surface antigens on ovine peripheral blood-derived EPCs. Microfluidic analysis showed a shear-stress-dependent decrease in EPC adhesion on attached surface antigens. EPCs exhibited increased adhesion to antibodies against CD34, VEGFR-2, CD31, and CD146 compared to CD45, consistent with their endothelial cell-specific surface profile, when exposed to a minimum shear stress of 1.47 dyn/cm(2). Bone-marrow-derived mesenchymal stem cells and artery-derived endothelial and smooth muscle cells were used to demonstrate the specificity of the EPC microfluidic device. Coated hematopoietic specific-surface (CD45) and granular vWF antibodies, as well as uncoated bare glass and substrate (1% BSA), were utilized as controls. Microfluidic devices have been developed as an EPC capture platform using immobilized antibodies targeted as EPC surface antigens. This EPC chip may provide a new and effective tool for addressing challenges in cardiovascular disease and tissue engineering.

Background-In the vasculature, the angiotensin type 2 (AT 2 ) receptor (AT 2 R) exerts antiproliferative, antifibrotic, and proapoptotic effects. Normal adult animals have low AT 2 R expression; however, vascular injury and exposure to proinflammatory cytokines augment AT 2 R levels. We hypothesized that AT 2 R expression increases during initiation and progression of atherosclerosis. Methods and Results-Atherosclerotic

Background-Optimal cell sources and scaffold-cell interactions remain unanswered questions for tissue engineering of heart valves. We assessed the effect of different protein precoatings on a single scaffold type (elastomeric poly (glycerol sebacate)) with a single cell source (endothelial progenitor cells). Methods and Results-Elastomeric poly (glycerol sebacate) scaffolds were precoated with laminin, fibronectin, fibrin, collagen types I/III, or elastin. Characterized ovine peripheral blood endothelial progenitor cells were seeded onto scaffolds for 3 days followed by 14 days incubation. Endothelial progenitor cells were CD31 ϩ , vWF ϩ , and ␣-SMA

Background— Valvular endothelial cells and circulating endothelial progenitor cells (EPCs) can undergo apparent phenotypic change from endothelial to mesenchymal cell type. Here we investigated whether EPCs can promote extracellular matrix formation in tissue engineering scaffolds in response to transforming growth factor (TGF)-β1. Method and Results— Characterized ovine peripheral blood EPCs were seeded onto poly (glycolic acid)/poly (4-hydroxybutyrate) scaffolds for 5 days. After seeding at 2×10 6 cells/cm 2 , scaffolds were incubated for 5 days in a roller bottle, with or without the addition of TGF-β1. After seeding at 15×10 6 cells/cm 2 , scaffolds were incubated for 10 days in a roller bottle with or without the addition of TGF-β1 for the first 5 days. Using immunofluorescence and Western blotting, we demonstrated that EPCs initially exhibit an endothelial phenotype (ie, CD31 + , von Willebrand factor + , and α–smooth muscle actin (SMA) − ) and can undergo a phenotypic change toward mesenchymal transformation (ie, CD31 + and α-SMA + ) in response to TGF-β1. Scanning electron microscopy and histology revealed enhanced tissue formation in EPC-TGF-β1 scaffolds. In both the 10- and 15-day experiments, EPC-TGF-β1 scaffolds exhibited a trend of increased DNA content compared with unstimulated EPC scaffolds. TGF-β1–mediated endothelial to mesenchymal transformation correlated with enhanced expression of laminin and fibronectin within scaffolds evidenced by Western blotting. Strong expression of tropoelastin was observed in response to TGF-β1 equal to that in the unstimulated EPC. In the 15-day experiments, TGF-β1–stimulated scaffolds revealed dramatically enhanced collagen production (types I and III) and incorporated more 5-bromodeoxyuridine and TUNEL staining compared with unstimulated controls. Conclusions— Stimulation of EPC-seeded tissue engineering scaffolds with TGF-β1 in vitro resulted in a more organized cellular architecture with glycoprotein, collagen, and elastin synthesis, and thus noninvasively isolated EPCs coupled with the pleiotropic actions of TGF-β1 could offer new strategies to guide tissue formation in engineered cardiac valves.

No abstract

Heart valve disease is a significant medical problem worldwide. Current treatment for heart valve disease is heart valve replacement. State of the art replacement heart valves are less than ideal and are associated with significant complications. Using the basic principles of tissue engineering, promising alternatives to current replacement heart valves are being developed. Significant progress has been made in the development of a tissue-engineered semilunar heart valve substitute. Advancements include the development of different potential cell sources and cell-seeding techniques; advancements in matrix and scaffold development and in polymer chemistry fabrication; and the development of a variety of bioreactors, which are biomimetic devices used to modulate the development of tissue-engineered neotissue in vitro through the application of biochemical and biomechanical stimuli. This review addresses the need for a tissue-engineered alternative to the current heart valve replacement options. The basics of heart valve structure and function, heart valve disease, and currently available heart valve replacements are discussed. The last 10 years of investigation into a tissue-engineered heart valve as well as current developments are reviewed. Finally, the early clinical applications of cardiovascular tissue engineering are presented.