Nanowired three-dimensional cardiac patches

Abstract

Engineered cardiac patches for treating damaged heart tissues after a heart attack are normally produced by seeding heart cells within three-dimensional porous biomaterial scaffolds. These biomaterials, which are usually made of either biological polymers such as alginate or synthetic polymers such as poly(lactic acid) (PLA), help cells organize into functioning tissues, but poor conductivity of these materials limits the ability of the patch to contract strongly as a unit. Here, we show that incorporating gold nanowires within alginate scaffolds can bridge the electrically resistant pore walls of alginate and improve electrical communication between adjacent cardiac cells. Tissues grown on these composite matrices were thicker and better aligned than those grown on pristine alginate and when electrically stimulated, the cells in these tissues contracted synchronously. Furthermore, higher levels of the proteins involved in muscle contraction and electrical coupling are detected in the composite matrices. It is expected that the integration of conducting nanowires within three-dimensional scaffolds may improve the therapeutic value of current cardiac patches.

Figure 1. Schematic overview of 3D nanowired cardiac tissue

a, Isolated cardiomyocytes are cultured in either pristine alginate or Alg-NW composites. Insets highlight the components of the engineered tissue: cardiac cells (red), alginate pore wall (blue) and gold nanowires (yellow). b, Whereas cardiomyocytes in pristine alginate scaffolds (top) typically form only small clusters that beat asynchronously and with random polarization, Alg-NW scaffolds (bottom) could exhibit synchronization across scaffold walls, throughout the entire scaffold. Colors, contour lines, and arrow represent the spatial and temporal evolution of signal maximum. c, Cardiomyocytes cultured in alginate scaffolds (top) form small beating clusters, but synchronously-beating cardiomyocytes in Alg-NW composites (bottom) have the potential to form organized cardiac-like tissue.

Figure 2. Incorporation of NWs within alginate scaffolds

a, and b, TEM images of a typical distribution of gold nanowires. The NWs exhibited an average length of ~1 μm and an average diameter of 30 nm. c, and d, SEM revealed that the NWs (1 mg/mL) assembled within the pore walls of the scaffold into star-shaped structures with a total length scale of 5 μm. The assembled wires were distributed homogenously within the matrix (c) with a distance of approximately 5 μm from each other (d).

Figure 3. Increased electrical conductivity of alginate by incorporation of NWs

a, Spatial conductivity was measured by conductive probe atomic force microscopy (C-AFM). The ITO slide served a backside contact, while the conductive AFM probe was used to simultaneously measure surface topography and conductance through the film. b, The equivalent circuit can be represented by capacitors (alginate) and resistors (NWs) connected in parallel. c, Topographic mapping revealed NWs protruding from the composite alginate thin film (5×5 μm). d, Spatial conductivity within the Alg-NW film as measured by C-AFM. Current spikes were measured at the location of the NWs. e, Current measured at the NWs (red) increased with bias voltage over the range −1 to 1V, while negligible current passed through NW-free regions of the alginate film (blue) over that same range. f, Overall impedance of the scaffold biomaterial before and after modification with NWs. Thin layers of Alg-NW or pure alginate films were pressed between two ITO glass slides. These slides served as electrodes and were used to apply an AC bias with frequency swept between 1 MHz and 10 Hz. At frequencies near DC, the impedance of the composite membrane was much lower than that of the pure film.

Figure 4. Cardiac cell organization within the 3D scaffold

a– d, H&E stained sections of thin sections of the engineered tissues on day 8 revealed a thick tissue in the NW scaffold (a, b) while the engineered tissue in the pristine scaffolds revealed non-continuous tissue separated by pore walls (c, d). NWs are seen within the pore walls of a relatively empty region of a scaffold (black dots indicated by yellow arrows) e, In a sparsely populated region, wires within the wall (black dots indicated by yellow arrows) were in close proximity to cell aggregates (red asterisks) (f). g–h, Immunostaining of the cell seeded scaffolds on day 8 revealed pervasive troponin I expression (red) within the Alg-NW scaffold (g), while less staining was observed in the aggregates in the un-modified scaffolds (h). i, Connexin 43 gap junction protein was found between cardiomyocytes in the NW-containing scaffolds (green dots indicated by white arrows). Nuclei are colored in blue. j, Quantification of connexin 43 protein expression by Western blot. k. Quantification of sarcomeric actinin protein expression by Western blot. Scale bars are 200 μm (a,c) or 20 μm (b,d,e–i).

Figure 5. Calcium transient propagation within the engineered tissues

Calcium transient was assessed at specified points (white circles) by monitoring calcium dye fluorescence (green) a, Sites monitored in pristine scaffold where site I is the stimulation point. b, Calcium transients were only observed at the stimulation point in the unmodified scaffold. F/F₀ refers to measured fluorescence normalized to background fluorescence. c, Sites monitored in an Alg-NW scaffold. The stimulation point was 2 mm diagonally to the lower left of point I (i.e. off the figure). The white arrow represents the direction of propagation. d, Calcium transients were observed at all points. e, Comparison of the initial time courses of single signals from sites I–V in panel d. f. Quantification of calcium transients (by relative fluorescence) from all samples (n=6 in each group). Bars represent signal maximum transient 2 mm from the stimulation site (F_2mm), normalized to the signal maximum at the simulation site (F_stim). Scale bar in a, c = 100 μm.