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, 11 (11), 986-94

Macroporous Nanowire Nanoelectronic Scaffolds for Synthetic Tissues

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Macroporous Nanowire Nanoelectronic Scaffolds for Synthetic Tissues

Bozhi Tian et al. Nat Mater.

Abstract

The development of three-dimensional (3D) synthetic biomaterials as structural and bioactive scaffolds is central to fields ranging from cellular biophysics to regenerative medicine. As of yet, these scaffolds cannot electrically probe the physicochemical and biological microenvironments throughout their 3D and macroporous interior, although this capability could have a marked impact in both electronics and biomaterials. Here, we address this challenge using macroporous, flexible and free-standing nanowire nanoelectronic scaffolds (nanoES), and their hybrids with synthetic or natural biomaterials. 3D macroporous nanoES mimic the structure of natural tissue scaffolds, and they were formed by self-organization of coplanar reticular networks with built-in strain and by manipulation of 2D mesh matrices. NanoES exhibited robust electronic properties and have been used alone or combined with other biomaterials as biocompatible extracellular scaffolds for 3D culture of neurons, cardiomyocytes and smooth muscle cells. Furthermore, we show the integrated sensory capability of the nanoES by real-time monitoring of the local electrical activity within 3D nanoES/cardiomyocyte constructs, the response of 3D-nanoES-based neural and cardiac tissue models to drugs, and distinct pH changes inside and outside tubular vascular smooth muscle constructs.

Figures

Figure 1
Figure 1. Integrating nanoelectronics with cells and tissue
Conventional bulk electronics are distinct from biological systems in composition, structural hierarchy, mechanics and function. Their electrical coupling at the tissue/organ level is usually limited to the tissue surface, where only boundary or global information can be gleaned unless invasive approaches are used. We have introduced a new concept by creating an integrated system from the discrete electronic and biological building blocks (e.g., semiconductor nanowires, molecular precursors of polymers and single cells). Three biomimetic and bottom-up steps have been designed: A) patterning, metallization and epoxy passivation for single nanowire FETs. B) forming 3D nanowire FET matrices (nanoelectric scaffolds) by self- or manual organization and hybridization with traditional extracellular matrices, and C) incorporation of cells and growth of synthetic tissue via biological processes. Yellow dots: nanowire components, blue ribbons: metal and epoxy interconnects, green ribbons: traditional extracellular matrices, pink: cells.
Figure 2
Figure 2. Macroporous and flexible nanowire nanoES
a, Device fabrication schematics. I: Reticular nanowire FET devices. II: mesh nanowire FET devices. Light blue: silicon oxide substrates, blue: nickel sacrificial layers, green: nanoES, yellow dots: individual nanowire FETs. b, 3D reconstructed confocal fluorescence images of reticular nanoES viewed along y (I) and x (II) axes. The scaffold was labeled with rhodamine 6G. The overall size of the structure, x-y-z = 300-400-200 μm. Solid and dashed open magenta boxes indicate two nanowire FET devices located on different planes along x axis. Scale bars, 20 μm. c, SEM image of a single kinked nanowire FET within a reticular scaffold, showing (1) kinked nanowires, (2) metallic interconnects (magenta lines) (3) the SU-8 backbone. Scale bar, 2 μm. d, Photograph of a mesh device, showing (1) nanowires, (2) metal interconnects, (3) SU-8 structural elements. The circle indicates the position of a single nanowire FET. Scale bar, 2 mm. e, Photograph of a partially rolled-up mesh device. Scale bar, 5 mm. f, SEM image of a loosely packed mesh nanoES, showing the macroporous structure. Scale bar, 100 μm. g, Histograms of nanowire FET conductance and sensitivity in one typical mesh nanoES. The conductance and sensitivity were measured in water-gate configuration without rolling. The device yield for this mesh nanoES is 95 %. h, Water-gate sensitivity and conductance of a nanowire FET device during the rolling process in a mesh device. Upper panel, schematic of nanowire FET position (yellow dot) during rolling process; 0-6 denote the number of turns. i, Relative change in conductance and sensitivity of 14 nanowire FETs evenly distributed throughout a fully rolled-up mesh device. Upper panel, schematic of NFWET position (yellow dots). In (h) and (i) the thicknesses of the tubular structures have been exaggerated for schematic clarity.
Figure 3
Figure 3. Geometry control by design in nanoES
(a, b) Basic design and structural subunit for simulation. a, Top-down view of the entire subunit. Blue ribbons are stressed metal lines with SU-8 passivation. Red lines are single SU-8 ribbons without residual stress. b, Cross-sectional views of those two key structural elements used for simulation. c, Plot of projected (on x-y plane) length versus height (in z direction) for the vertical blue ribbon in (a) as determined from the simulation. Open red squares with error bars are experimental data recorded in air for point A and B in (a). The simulation of the bending of the subunit model for the reticular structure was carried out using the commercial finite element software ABAQUS. Inset shows a 3D view of the simulated structure, and the scale bar shows different height in z direction. d, Schematic showing the integration of periodic reticular-device domains (light blue) into a flexible mesh (green). In individual reticular domains, the 3D device positions relative to the global flexible mesh can be controlled by their geometry designs (a-c). (e, f) Design patterns (I) and experimental data (II) for two reticular units. SU8, metal and nanowires are shown in blue, pink and yellow in e. Changing the structure of the connecting feature (white arrows) between adjacent device units during pattern design (I) yields controlled variations in 3D positioning of the nanowire FETs, which can be further tuned by the stress in the metal connections. In these experiments, the device positions are 40 μm (e,II) and 23 μm (f,II) above the mesh plane. Scale bars in e and f, 20 μm.
Figure 4
Figure 4. Hybrid macroporous nanoelectronic scaffolds
a, Confocal fluorescence micrograph of a hybrid reticular nanoES/collagen matrix. Green (Fluorescein isothiocyanate): collagen type-I, organge (rhodamine 6G): epoxy ribbons. White arrow marks the position of nanowire. Scale bar, 10 μm. b, SEM images of mesh nanoES/alginate scaffold, top (I) and side (II) views. The epoxy ribbons from nanoES are false-colored in brown for clarity. Scale bars, 200 μm (I) and 100 μm (II). c, A bright-field optical micrograph of the folded scaffold, showing multilayered structures of PLGA and nanoelectronic interconnects. Inset shows the photograph of the hybrid sheet before folding. A sheet of PLGA fibers with diameters of ~ 1-3 μm was deposited on both sides of the device. No damage or reduction of device yield was observed following this deposition. Scale bars, 200 μm and 5 mm (inset). d, Relative changes in nanowire FET sensitivity over time in culture (37 °C; 5 % CO2, supplemented Neurobasal™ medium). n = 5; data are means ± SD.
Figure 5
Figure 5. 3D cell culture and electrical sensing in nanoES
a and b, 3D reconstructed confocal images of rat hippocampal neurons after 2 week culture in Matrigel™ on reticular nanoES. Red (Alexa Fluor® 546): neuronal β-tubulin, yellow (rhodamine 6G): epoxy ribbons. The metal interconnects are false-colored in blue, and are imaged in reflected light mode. White arrow highlights a neurite passing through a ring-like structure supporting a NFWET. Dimensions in (a), x: 317 μm; y: 317 μm; z: 100 μm; in (b), x: 127 μm; y: 127 μm; z: 68 μm. c, Confocal fluorescence images of synthetic cardiac patch. II and III, zoomed-in view of the upper and lower dashed regions in I, showing metal interconnects, SU-8 scaffold (arrows in II), electrospun PLGA fibers (arrows in III). Scale bar, 40 μm. d, Epi-fluorescence image of the surface of the cardiac patch. Green (Alexa Fluor® 488): α-actinin, blue (Hoechst 34580): cell nuclei. The position of the source-drain electrodes is outlined with dashed lines. Scale bar, 40 μm. e, Percentage of viable hippocampal neurons cultured in nanoES/Matrigel™ vs Matrigel™. Cell viability was evaluated with a LIVE/DEAD cytotoxicity assay. Cells were counted from 3D reconstructed confocal fluorescence photomicrographs. n = 6; data are means ± SD. Differences between groups were very small although statistically significant (p < 0.05). f, MTS cytotoxicity assay of cardiomyocytes evaluated using the MTS assay. n = 6; data are means ± SD. Differences between groups were very small although statistically significant (p < 0.05). g, Conductance versus time traces recorded from a single nanowire FET before (black) and after (blue) applying norepinephrine. h, Multiplex electrical recording of extracellular field potentials from 4 nanowire FETs (a-d) in a mesh nanoES. I. Data are conductance versus time traces of a single spike recorded at each nanowire FET.
Figure 6
Figure 6. Synthetic vascular construct enabled for sensing
a, Schematic of the synthesis of smooth muscle nanoES. The upper panels are side views, while the lower ones are either top views (I and II) or zoom-in view (III). Grey: mesh nanoES, blue fibers: collagenous matrix secreted by HASMC, yellow dots: nanowire FETs, pink: HASMC. b, I. Photograph of a single HASMC sheet cultured with sodium L-ascorbate on a nanoES. II. Zoomed-in view of the dashed area in (I), showing metallic interconnects macroscopically integrated with cellular sheet. c, Photograph of the vascular construct after rolling into a tube and maturation in a culture chamber for 3 weeks. d, I. Micro-computed tomograph of a tubular construct segment. II. Zoomed-in view of (I). Arrows mark the individual nanowire FET-containing layers of the rolled construct. Scale bar, 1 mm. e, Hematoxylin & eosin (panel I) and Masson Trichrome (panel II; collagen is blue) stained sections (~ 6 μm thick) cut perpendicular to the tube axis; lumen regions are labeled. Arrows mark the positions of SU-8 ribbons of the nanoES. Scale bars, 50 μm. f, Changes in conductance over time for two nanowire FET devices located in the outermost (red) and innermost (blue) layers. Inset: schematic of the experimental set-up. Outer tubing delivered bathing solutions with varying pH (red dashed lines and arrows); inner tubing delivered solutions fixed pH (blue dashed lines and arrows).

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