7.3. INDUCTIVE STIMULATION PATTERNS IN CARDIAC TISSUE ENGINEERING 101

7.3.2MECHANICAL STIMULATION INDUCED BY MAGNETIC FIELD

The magnetic field has been attracting great interest as a tool for mechanical cell stimulation,although its signaling pattern within the stimulated cell is still not clear and poorly understood. Magnetic fields can protrude living cells since, unlike the electric field, they are not shielded by membrane potentials, and thus they can influence intracellular organelles. Also, due to their high penetration ability, magnetic fields can reach into deeper tissue layers when applied from a distance.

In stimulation settings, the magnetically mediated actuation “at distance” has a clear advantage compared to other stimulation setups since it enables highly controlled actuation both in vitro and in vivo. The target cell can be stimulated regardless of whether there are intervening structures, as long as these structures do not isolate the magnetic field. In addition, the magnetic field can be coupled with magnetically responsive particles that can be targeted to a specific cell or tissue site both in vitro and in vivo. Such coupling can easily and dynamically control the stress, applied directly by the magnetic particles to a desired area by varying the strength of the applied field.

Our group has recently developed magnetically responsive alginate scaffolds and tested their ability to provide means of a physical stimulation to living cells seeded within the scaffold (Fig. 7.5)

The nanocomposite alginate scaffolds were impregnated with magnetic iron-oxide nanoparticles (MNP), then seeded with bovine aortic endothelial cells, and the cell constructs has been exposed to an alternating magnetic field. The MNP-impregnated scaffolds were found to be more elastic compared to pristine scaffolds, while incorporation of MNP did not influence the macro-porosity structure of the scaffold (pore size and porosity) or their wetting extent by the culture medium. Endothelial cells cultivated within the magnetically-stimulated constructs showed significantly elevated metabolic activity during the stimulation period, most likely related to cell migration and re-organization into tube-like structures. Immunostaining and confocal microscopy examination on day 14 revealed that the magnetically-stimulated constructs, without supplementation of any angiogenic growth factors, contained vessel-like (loop) structures, while in the non-stimulated (control) scaffolds, the cells were mainly organized as sheets or aggregates (Fig. 7.5) [25].

It is still not clear what are the exact mechanisms acting on the cells within the nanocomposite scaffold under magnetic stimulation; however some speculations can be made. We showed that the impregnation process results in high density of MNP embedded within the scaffold wall, in close proximity and interacting with each other. Such direct NP interactions within the scaffold wall resemble the domain interactions existing in ferromagnetic material, leading to the phenomenon called magnetostriction. Magnetostriction causes the bulk materials to change their shape or dimensions during the process of magnetization, due to the domain interactions [26]. Such an effect would lead to overall scaffold contraction and consequently to cell stimulation, migration and organization into a tissue. This may explain the endothelial cell organization as observed in the magnetically-stimulated alginate scaffold. Although this hypothesis appears to be feasible, it needs further investigation and confirmation.

102 7. PERFUSION BIOREACTORS AND STIMULATION PATTERNS

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Figure 7.5: The promotion of in vitro vessel-like organization of endothelial cells in magnetically responsive alginate scaffolds. A. Scaffold morphology. Scanning electron microscopy (SEM) images of the (a) 1.4% (w/v) MNP-alginate and (b) non-magnetic alginate scaffolds. Note the presence of nanoparticles in MNP-alginate scaffold walls. B. Endothelial cell organization in MNP-alginate and alginate constructs, on day 14 post cell seeding. The cells are stained for F-actin (red) and nuclei (blue) (bar: 30 μm). C. Average loop number per image field counted on day 14 post cell seeding. A total of 25 randomly selected fields were analyzed per each group. Asterisks denote significant difference (by 2-way ANOVA),

***p < 0.005 (Bonferroni’s pos-hoc test was used for comparison between the groups). Reprinted with permission from [25].

7.3. INDUCTIVE STIMULATION PATTERNS IN CARDIAC TISSUE ENGINEERING 103

7.3.3ELECTRICAL STIMULATION

From the moment it is formed, the heart acts as a sequential contracting syncytium. Although each cardiomyocyte is able to contract spontaneously, the overall heart function is controlled by a group of specialized pacemaker cells. These cells stimulate the generation of cardiac impulse and trigger synchronous contraction. In cardiac tissue engineering, the addition of exogenous electrical stimulation has attributed to the reconstruction of an appropriate environment for tissue regeneration by mimicking the pacemaker activity during heart tissue development.

Numerous studies explored the influence of electrical stimulation on cardiac and other cell types [27, 28, 29, 30]. Studies performed on 2D cardiomyocyte cultures have revealed that the electrical stimulation (80–150 V, pulse duration of 5–10 ms, I < 5 mA, 1–5 Hz) induced cell enlargement, the development of more organized myofibrils, and greater expression of cardiac genes (ANF and MLC-2) [29]. Short-term electrical stimulation (60–120 min) of cardiomyocyte monolayers led to enhanced expression of Cx-43, an important gap junction protein, responsible for the mechanical and electrical communication between adjacent cells in the cardiac tissue [30].

Radisic and colleagues pioneered the application of electrical stimulation in cardiac cell constructs [3]. Electrical signals mimicking those in the native heart (rectangular pulses, 2ms, 5V/cm, 1Hz) were applied on neonatal cardiomyocytes seeded onto collagen constructs. Already after eight days of stimulation, the cardiac cells presented higher levels of gap junction proteins (Cx-43) expression compared to non-stimulated constructs. Electrical stimuli induced functional coupling, amplified contraction amplitude by a factor of 7, and there was also a significant level of ultrastructural differentiation [3]. Since these studies were performed under static conditions, the thickness of the engineered cardiac tissue was limited to 100 μm.

Additional works investigated the influence of electrical stimulation on endothelial cell behavior [31, 32]. Zhao et al reported that applied electric fields (EFs) of small physiological magnitude directly stimulate the production of vascular endothelial growth factor (VEGF) by endothelial cells in culture in the absence of any other cell type [32].

Recently, electrical stimulation has been implemented in tri-culture cell constructs (cardiomyocytes, endothelial cells, and fibroblasts) resulting in the formation of vascularized cardiac tissue [33].

Our group combined electrical stimulation with medium perfusion into one cultivation vessel to produce a thick functional cardiac patch (Fig. 7.6) [34].

For this, a custom-made electrical stimulator was integrated into the perfusion bioreactor described in Section 7.2.2. This was achieved by insertion of carbon rod electrodes between the two scaffold mesh holders. At first, the stimulation threshold for inducing a synchronous contraction in the cell constructs was determined under a microscope, by trial and error. Computer models of the electric fields (and current density) inside the bioreactor and the constructs were created. A successful stimulation of the cell construct in static cultivation mode was achieved at 6V with a current density of 74.4 mA/cm2, while in the bioreactor, with the carbon rod electrodes, 5V was sufficient to achieve the same current density. Already after four days under a continuous electrical

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Figure 7.6: Electric field stimulation integrated into perfusion bioreactor for cardiac tissue engineering. A. Bioreactor setup. B. Configuration of the carbon electrodes integrated into the bioreactor. C. 3D electric field model in bioreactor. (a) An illustration of the 3D configuration with the carbon electrodes in black and the cell constructs in blue. (b-d) The current density in the y (b), z (c), and x (d) directions (1 μV between the electrodes). D. The effect of the electrical stimulation on cell morphology and Cx-43 levels. (a, b) Confocal microscopy images of anti-α-sarcomeric actinin immunofluorescence (green) of the cell constructs in the bioreactor with (a) or without (b) electrical stimulation. To-Pro (To-Pro 3 Iodide) was used for nuclear staining (red). The electrically-stimulated cell constructs are those placed in between the carbon electrodes. (c, d) Confocal microscopy images of anti-Cx-43 (red), anti-α-sarcomeric actinin (green) immunofluorescence of the cell constructs in a bioreactor with (c) or without (d) electrical stimulation. The white arrows indicate positive staining of Cx-43 between adjacent cells. To-Pro was used for nuclear staining (blue). The electrically-stimulated cell constructs are those placed in between the carbon electrodes. (e) Representative Western blot analysis for Cx-43 expression after four days of cultivation in the bioreactor with or without electrical stimulation [34].