Anna Marsano

Electrical stimulation systems for cardiac tissue engineering

We describe a protocol for tissue engineering of synchronously contractile cardiac constructs by culturing cardiac cells with the application of pulsatile electrical fields designed to mimic those present in the native heart. Tissue culture is conducted in a customized chamber built to allow for cultivation of (i) engineered three-dimensional (3D) cardiac tissue constructs, (ii) cell monolayers on flat substrates or (iii) cells on patterned substrates. This also allows for analysis of the individual and interactive effects of pulsatile electrical field stimulation and substrate topography on cell differentiation and assembly. The protocol is designed to allow for delivery of predictable electrical field stimuli to cells, monitoring environmental parameters, and assessment of cell and tissue responses. The duration of the protocol is 5 d for two-dimensional cultures and 10 d for 3D cultures.

Optimization of electrical stimulation parameters for cardiac tissue engineering.

In vitro application of pulsatile electrical stimulation to neonatal rat cardiomyocytes cultured on polymer scaffolds has been shown to improve the functional assembly of cells into contractile engineered cardiac tissues. However, to date, the conditions of electrical stimulation have not been optimized. We have systematically varied the electrode material, amplitude and frequency of stimulation to determine the conditions that are optimal for cardiac tissue engineering. Carbon electrodes, exhibiting the highest charge‐injection capacity and producing cardiac tissues with the best structural and contractile properties, were thus used in tissue engineering studies. Engineered cardiac tissues stimulated at 3 V/cm amplitude and 3 Hz frequency had the highest tissue density, the highest concentrations of cardiac troponin‐I and connexin‐43 and the best‐developed contractile behaviour. These findings contribute to defining bioreactor design specifications and electrical stimulation regime for cardiac tissue engineering. Copyright

Efficacy and mechanisms of vacuum-assisted closure (VAC) therapy in promoting wound healing: a rodent model

BACKGROUND The vacuum-assisted closure device (VAC) has revolutionised wound care, although molecular mechanisms are not well understood. We hypothesise that the VAC device induces production of pro-angiogenic factors and promotes formation of granulation tissue and healing. METHODS A novel rodent model of VAC wound healing was established. Excisional wounds were created on rat dorsa. Wounds were dressed with Tegaderm (control group), VAC Granulofoam and Tegaderm (special control group), or VAC Granulofoam, T.R.A.C. PAD((R)) with 125 mm Hg continuous negative pressure (VAC group). Wound closure rates were calculated as a percentage of initial wound sizes. Rats were sacrificed on postoperative days 3, 5 and 7; harvested tissues were processed for histology [haematoxylin & eosin (H&E), Massons trichrome, picrosirius red] and Western blot analysis (CD31, vascular endothelial growth factor, basic fibroblast growth factor). RESULTS Statistically significant wound closure rates were achieved in the experimental group at all measured time points: day 3, 28.1% (VAC) vs 8.2% (control) and 8.8% (special control) (ANOVA, P<0.0001); day 5, 45.3% (VAC) vs 23.7% (control) and 22.5% (special control) (ANOVA, P=0.0003); day 7, 54.4% (VAC) vs 43.0% (control) and 31.5% (special control) (ANOVA; P<0.0001). Morphological evaluation by Massons trichrome stain showed increased collagen organisation and wound maturation in the VAC group. These wounds also showed increased expression of vascular endothelial growth factor and fibroblast growth factor-2 on day 5 by Western blot analysis. CONCLUSION A small animal VAC wound model was established. Wounds treated with a VAC device showed accelerated wound closure rates, increased pro-angiogenic growth factor production and improved collagen deposition. Further application of this model may elucidate other mechanisms.

The effect of controlled expression of VEGF by transduced myoblasts in a cardiac patch on vascularization in a mouse model of myocardial infarction

Key requirements for cardiac tissue engineering include the maintenance of cell viability and function and the establishment of a perfusable vascular network in millimeters thick and compact cardiac constructs upon implantation. We investigated if these requirements can be met by providing an intrinsic vascularization stimulus (via sustained action of VEGF secreted at a controlled rate by transduced myoblasts) to a cardiac patch engineered under conditions of effective oxygen supply (via medium flow through channeled elastomeric scaffolds seeded with neonatal cardiomyocytes). We demonstrate that this combined approach resulted in increased viability, vascularization and functionality of the cardiac patch. After implantation in a mouse model of myocardial infarction, VEGF-expressing patches displayed significantly improved engraftment, survival and differentiation of cardiomyocytes, leading to greatly enhanced contractility as compared to controls not expressing VEGF. Controlled VEGF expression also mediated the formation of mature vascular networks, both within the engineered patches and in the underlying ischemic myocardium. We propose that this combined cell-biomaterial approach can be a promising strategy to engineer cardiac patches with intrinsic and extrinsic vascularization potential.

Perfusion seeding of channeled elastomeric scaffolds with myocytes and endothelial cells for cardiac tissue engineering

The requirements for engineering clinically sized cardiac constructs include medium perfusion (to maintain cell viability throughout the construct volume) and the protection of cardiac myocytes from hydrodynamic shear. To reconcile these conflicting requirements, we proposed the use of porous elastomeric scaffolds with an array of channels providing conduits for medium perfusion, and sized to provide efficient transport of oxygen to the cells, by a combination of convective flow and molecular diffusion over short distances between the channels. In this study, we investigate the conditions for perfusion seeding of channeled constructs with myocytes and endothelial cells without the gel carrier we previously used to lock the cells within the scaffold pores. We first established the flow parameters for perfusion seeding of porous elastomer scaffolds using the C2C12 myoblast line, and determined that a linear perfusion velocity of 1.0 mm/s resulted in seeding efficiency of 87% ± 26% within 2 hours. When applied to seeding of channeled scaffolds with neonatal rat cardiac myocytes, these conditions also resulted in high efficiency (77.2% ± 23.7%) of cell seeding. Uniform spatial cell distributions were obtained when scaffolds were stacked on top of one another in perfusion cartridges, effectively closing off the channels during perfusion seeding. Perfusion seeding of single scaffolds resulted in preferential cell attachment at the channel surfaces, and was employed for seeding scaffolds with rat aortic endothelial cells. We thus propose that these techniques can be utilized to engineer thick and compact cardiac constructs with parallel channels lined with endothelial cells.

Scaffold stiffness affects the contractile function of three-dimensional engineered cardiac constructs

We investigated the effects of the initial stiffness of a three‐dimensional elastomer scaffold—highly porous poly(glycerol sebacate)—on functional assembly of cardiomyocytes cultured with perfusion for 8 days. The polymer elasticity varied with the extent of polymer cross‐links, resulting in three different stiffness groups, with compressive modulus of 2.35 ± 0.03 (low), 5.28 ± 0.36 (medium), and 5.99 ± 0.40 (high) kPa. Laminin coating improved the efficiency of cell seeding (from 59 ± 15 to 90 ± 21%), resulting in markedly increased final cell density, construct contractility, and matrix deposition, likely because of enhanced cell interaction and spreading on scaffold surfaces. Compact tissue was formed in the low and medium stiffness groups, but not in the high stiffness group. In particular, the low stiffness group exhibited the greatest contraction amplitude in response to electric field pacing, and had the highest compressive modulus at the end of culture. A mathematical model was developed to establish a correlation between the contractile amplitude and the cell distribution within the scaffold. Taken together, our findings suggest that the contractile function of engineered cardiac constructs positively correlates with low compressive stiffness of the scaffold.

Channelled scaffolds for engineering myocardium with mechanical stimulation.

The characteristics of the matrix (composition, structure, mechanical properties) and external culture environment (pulsatile perfusion, physical stimulation) of the heart are important characteristics in the engineering of functional myocardial tissue. This study reports on the development of chitosan‐collagen scaffolds with micropores and an array of parallel channels (~ 200 µm in diameter) that were specifically designed for cardiac tissue engineering using mechanical stimulation. The scaffolds were designed to have similar structural and mechanical properties of those of native heart matrix. Scaffolds were seeded with neonatal rat heart cells and subjected to dynamic tensile stretch using a custom designed bioreactor. The channels enhanced oxygen transport and facilitated the establishment of cell connections within the construct. The myocardial patches (14 mm in diameter, 1–2 mm thick) consisted of metabolically active cells that began to contract synchronously after 3 days of culture. Mechanical stimulation with high tensile stress promoted cell alignment, elongation, and expression of connexin‐43 (Cx‐43). This study confirms the importance of scaffold design and mechanical stimulation for the formation of contractile cardiac constructs. Copyright

Alignment and elongation of human adipose-derived stem cells in response to direct-current electrical stimulation

In vivo, direct current electric fields are present during embryonic development and wound healing. In vitro, direct current (DC) electric fields induce directional cell migration and elongation. For the first time, we demonstrate that cultured human adipose tissue-derived stem cells (hASCs) respond to the presence of direct-current electric fields. Cells were stimulated for 2-4 hours with DC electric fields of 6 V/cm that were similar to those encountered in vivo post-injury. Upon stimulation, hASCs were observed to elongate and align perpendicularly to the applied electric field, disassemble gap junctions, and upregulate the expression of genes for connexin-43, thrombomodulin, vascular endothelial growth factor, and fibroblast growth factor. In separate related studies, human epicardial fat-derived stem cells (heASCs) were also observed to align and elongate. It is interesting that the morphological and phenotypic characteristics of mesenchymal stem cells derived both from liposuction aspirates and from cardiac fat can be modulated by direct current electric fields. In further studies, we will quantify the effects of the electrical fields in the context of wound healing.

Design of electrical stimulation bioreactors for cardiac tissue engineering

Electrical stimulation has been shown to improve functional assembly of cardiomyocytes in vitro for cardiac tissue engineering. Carbon electrodes were found in past studies to have the best current injection characteristics. The goal of this study was to develop rational experimental design principles for the electrodes and stimulation regime, in particular electrode configuration, electrode ageing, and stimulation amplitude. Carbon rod electrodes were compared via electrochemical impedance spectroscopy (EIS) and we identified a safety range of 0 to 8 V/cm by comparing excitation thresholds and maximum capture rates for neonatal rat cardiomyocytes cultured with electrical stimulation. We conclude with recommendations for studies involving carbon electrodes for cardiac tissue engineering.

Controlled Angiogenesis in the Heart by Cell-Based Expression of Specific Vascular Endothelial Growth Factor Levels

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.