Anat Perets

Micropatterning of three-dimensional electrospun polyurethane vascular grafts.

The uniform alignment of endothelial cells inside small-diameter synthetic grafts can be directed by surface topographies such as microgrooves and microfibers to recapitulate the flow-induced elongation and alignment of natural endothelium. These surface micropatterns may also promote directional migration and potentially improve anastomotic ingrowth of endothelial cells inside the synthetic grafts. In this paper, we developed electrospinning and spin casting techniques to pattern the luminal surface of small-diameter polyurethane (PU) grafts with microfibers and microgrooves, respectively, and evaluated endothelial cell orientation on these surface micropatterns. Tracks of circumferentially oriented microfibers were generated by electrospinning PU onto a mandrel rotated at high velocity, whereas longitudinal tracks of microgrooves were generated by spin casting PU over a rotating poly(dimethylsiloxane) mold. We found that both PU grafts possessed longitudinal Youngs moduli in the range of 0.43 ± 0.04 to 2.00 ± 0.40 MPa, comparable with values obtained from native artery. Endothelial cells seeded onto the grafts formed confluent monolayers with individual cells exhibiting elongated morphology parallel to the micropatterns. The cells were phenotypically similar to natural endothelium as assessed by the expression of the endothelial cell-specific marker, vascular endothelial cell cadherin. In addition, the cells were also responsive to stimulation with the pro-inflammatory cytokine tumor necrosis factor-α as assessed by the inducible expression of intercellular adhesion molecule-1. These results demonstrate that our micropatterned PU grafts possessed longitudinal Youngs moduli in the same range as native vascular tissue and were capable of promoting the formation of aligned and cytokine-responsive endothelial monolayers.

Textile-templated electrospun anisotropic scaffolds for regenerative cardiac tissue engineering

For patients with end-stage heart disease, the access to heart transplantation is limited due to the shortage of donor organs and to the potential for rejection of the donated organ. Therefore, current studies focus on bioengineering approaches for creating biomimetic cardiac patches that will assist in restoring cardiac function, by repairing and/or regenerating the intrinsically anisotropic myocardium. In this paper we present a simplified, straightforward approach for creating bioactive anisotropic cardiac patches, based on a combination of bioengineering and textile-manufacturing techniques in concert with nano-biotechnology based tissue-engineering stratagems. Using knitted conventional textiles, made of cotton or polyester yarns as template targets, we successfully electrospun anisotropic three-dimensional scaffolds from poly(lactic-co-glycolic) acid (PLGA), and thermoplastic polycarbonate-urethane (PCU, Bionate(®)). The surface topography and mechanical properties of textile-templated anisotropic scaffolds significantly differed from those of scaffolds electrospun from the same materials onto conventional 2-D flat-target electrospun scaffolds. Anisotropic textile-templated scaffolds electrospun from both PLGA and PCU, supported the adhesion and proliferation of H9C2 cardiac myoblasts cell line, and guided the cardiac tissue-like anisotropic organization of these cells in vitro. All cell-seeded PCU scaffolds exhibited mechanical properties comparable to those of a human heart, but only the cells on the polyester-templated scaffolds exhibited prolonged spontaneous synchronous contractility on the entire engineered construct for 10 days in vitro at a near physiologic frequency of ∼120 bpm. Taken together, the methods described here take advantage of straightforward established textile manufacturing strategies as an efficient and cost-effective approach to engineering 3D anisotropic, elastomeric PCU scaffolds that can serve as a cardiac patch.

Alimentary ‘green’ proteins as electrospun scaffolds for skin regenerative engineering

As a potential alternative to currently available skin substitutes and wound dressings, we explored the use of bioactive scaffolds made of plant‐derived proteins. We hypothesized that ‘green’ materials, derived from renewable and biodegradable natural sources, may confer bioactive properties to enhance wound healing and tissue regeneration. We optimized and characterized fibrous scaffolds electrospun from soy protein isolate (SPI) with addition of 0.05% poly(ethylene oxide) (PEO) dissolved in 1,1,1,3,3,3‐hexafluoro‐2‐propanol, and from corn zein dissolved in glacial acetic acid. Fibrous mats electrospun from either of these plant proteins remained intact without further cross‐linking, possessing a skin‐like pliability. Soy‐derived scaffolds supported the adhesion and proliferation of cultured primary human dermal fibroblasts. Using targeted PCR arrays and qPCR validation, we found similar gene expression profiles of fibroblasts cultured for 2 and 24 h on SPI substrates and on collagen type I at both time points. On both substrates there was a pronounced time‐dependent upregulation of several genes related to ECM deposition remodelling, including MMP‐10, MMP‐1, collagen VII, integrin‐α2 and laminin‐β3, indicating that both plant‐ and animal‐derived materials induce similar responses from the cells after initial adhesion, degrading substrate proteins and depositing extracellular matrix in a ‘normal’ remodelling process. These results suggest that ‘green’ proteins, such as soy and zein, are promising as a platform for organotypic skin equivalent culture, as well as implantable scaffolds for skin regeneration. Future studies will determine specific mechanisms of their interaction with skin cells and their efficacy in wound‐healing applications. Copyright

Fibronectin-mediated upregulation of α5β1 integrin and cell adhesion during differentiation of mouse embryonic stem cells.

Embryonic stem (ES) cells have a broad potential application in regenerative medicine and can be differentiated into cells of all three germ layers. Adhesion of ES cells to extracellular matrix (ECM) proteins is essential for the differentiation pathway; Cell-ECM adhesion is mediated by integrins that have the ability to activate many intracellular signaling pathways. Therefore, we hypothesize that the expression and function of integrin receptors is a critical step in ES differentiation. Using functional cell adhesion assays, our study demonstrates that α5β1 is a major functional integrin receptor expressed on the cell surface of undifferentiated mouse ES-D3 cells, which showed significantly higher binding to fibronectin as compared to collagens. This adhesion was specific mediated by integrin α5β1 as evident from the inhibition with a disintegrin selective for this particular integrin. Differentiation of ES-D3 cells on fibronectin or on a collagen type1/fibronectin matrix, caused further selective up-regulation of the α5β1 integrin. Differentiation of the cells, as evaluated by immunofluorescence, FACS analysis and quantitative RT-PCR, was accompanied by the upregulation of mesenchymal (Flk1, isolectin B4, α-SMA, vimentin) and endodermal markers (FoxA2, SOX 17, cytokeratin) in parallel to increased expression of α5β1 integrin. Taken together, the data indicate that fibronectin-mediated, upregulation of α5β1 integrin and adhesion of ES-D3 cells to specific ECM molecules are linked to early stages of mouse embryonic stem cells commitment to meso-endodermal differentiation.

Quantitative Assessment of Neuronal Differentiation in Three-dimensional Collagen Gels Using Enhanced Green Fluorescence Protein Expressing PC12 Pheochromocytoma Cells

There is a paucity of quantitative methods for evaluating the morphological differentiation of neuronal cells in a three-dimensional (3-D) system to assist in quality control of neural tissue engineering constructs for use in reparative medicine. Neuronal cells tend to aggregate in the 3-D scaffolds, hindering the application of two-dimensional (2-D) morphological methods to quantitate neuronal differentiation. To address this problem, we developed a stable transfectant green fluorescence protein (GFP)-PC12 neuronal cell model, in which the differentiation process in 3-D can be monitored with high sensitivity by fluorescence microscopy. Under 2-D conditions, the green cells showed collagen adherence, round morphology, proliferation properties, expression of the nerve growth factor (NGF) receptors TrkA and p75NTR, stimulation of extracellular signal-regulated kinase phosphorylation by NGF and were able to differentiate in a dose-dependent manner upon NGF treatment, like wild-type (wt)-PC12 cells. When grown within 3-D collagen gels, upon NGF treatment, the GFP-PC12 cells differentiated, expressing long neurite outgrowths. We describe here a new validated method to measure NGF-induced differentiation in 3-D. Having properties similar to those of wt-PC12 and an ability to grow and differentiate in 3-D structures, these highly visualized GFP-expressing PC12 cells may serve as an ideal model for investigating various aspects of differentiation to serve in neural engineering.

Textile-templated electrospun anisotropic scaffolds for tissue engineering and regenerative medicine

Cardiovascular diseases, specifically myocardial infarction and end-stage heart failure represent some of the major pathologies that threaten human life. Here we present a novel approach for a bioactive cardiac patch based on a combination of biomedical and textile manufacturing techniques in concert with nano-biotechnology based tissue-engineering stratagems. The technological goal is to create BioNanoTextiles™ (BNT) by using “conventional” fabrics as templates for creating three-dimensional nanofibrous scaffolds. Electrospinning nanofibrous scaffolds templated after “ordinary” textiles is a novel way to create complex-patterned, 3-D scaffolds intrinsically mimicking some of the anisotropic structural features of the ventricular walls extracellular matrix. In preliminary studies, we established that this approach will yield anisotropic 3-D scaffolds with mechanical properties dependent upon the yarn type of the textile-templates. These scaffolds are biocompatible, as inferred from their support of H9C2 cardiac myoblast adhesion which promotes their proliferation as well as cardiac-like anisotropic organization. The use of textile manufacturing strategies will enhance the complexity of the 3-D scaffold structures and enable their commercialization, while providing an opportunity for the textile industry to advance established “low-tech” manufacturing technologies into the realm of “high-tech” BioNanoTextiles.

Designing Intelligent Polymeric Scaffolds for Tissue Engineering: Blending and Co-Electrospinning Synthetic and Natural Polymers

There is a growing interest in blending natural and synthetic polymers as biomaterials for generating complex fibrous scaffolds for tissue engineering purposes. In this talk we will report on co-electrospinning binary and tertiary blends of gelatin (denatured collagen) with either a conductive polymer, polyaniline (PANi), or with a mixture of polylactic acid / polyglycolic acid, and elastin (PGE) Finally, we will demonstrate the usefulness of elastin-based fibrous scaffolds for pulmonary tissue engineering