3D Optoelectronic Scaffold with Impregnated Silicon Nanowires for Leadless Bioelectrical Modulation with Sub-Cellular Resolution

Bioelectricity is gaining more and more interest due to its ability to modulate tissue regeneration and function. However, although substrate bound micro and nanoelectrodes are extremely useful for bioelectronic modulation of cells cultured in vitro in 2D monolayers, they lack the ability to interface cells that are inside a 3D engineered tissue construct. Thus, many leadless technologies are being developed. One of the most promising approaches for leadless electrical modulation with high spatial resolution is the use of optoelectronic nanomaterials that can transduce optical illumination to an electrical output. Free standing silicon nanowires (SiNWs) are ideal for minimally invasive intracellular biointerface as they are soft, flexible, and thus can spontaneously internalize into many different cell types. However, introducing theses silicon-based leadless probes into the inner volume of an engineered tissue without disrupting its integrity is challenging. <br/>To address this challenge, we developed an electronic scaffold (e-scaffold)- a novel 3D macroporous scaffold from alginate and/or collagen, with impregnated optoelectronic SiNWs. The e-scaffold can be comprised of different ratios of alginate and collagen, so that cellular interactions can be enhanced towards cell-scaffold (high collagen low alginate) or cell-cell (high alginate low collagen) interactions. Moreover, by using high alginate e-scaffolds and coating impregnated SiNWs with fibronectin, cell-Si interactions may be enhanced. During the development of engineered tissue constructs, the SiNWs within the soft and biodegradable scaffold are free to interface with the cells and form extra- and intracellular biointerfaces. Thereafter, a spinning disc confocal with integrated FRAP modulus can be used to apply local optoelectronic modulation of cells baring or interfacing SiNWs. <br/>We found that cells grown within the e-scaffold were able to integrate and proliferate within the 3D environment. Moreover, the cells were interfaced with SiNWs regardless to their volumetric locations. On the other hand, when SiNWs were seeded onto cells in regular scaffolds, only the superficial cells (top, bottom and perimeter of the scaffold) were interfaced with SiNWs, while intra-volumetric cells were not interfaced. <br/>When intracellular SiNWs were optically modulated, cells exhibited an immediate intracellular local electrical response as indicated via calcium imaging. This demonstrates the utility of the e-scaffold as a platform for 3D engineered tissue with bioelectronic interrogation capabilities that is free standing, 3D accessible, spatially and temporally precise, and versatile. Moreover, when using standard bioelectronics, a finite number of electrical interrogation sites must be hard wired and their locations must be pre-determined during fabrication. However, the use of free standing SiNWs without any hard wire requirements, allows us to homogenously distribute the SiNWs within the e-scaffold so that numerous bioelectronic interfaces are formed without any limitation on the number or locations of the biointerfaces. <br/>Interestingly, cells were found to electrically couple to one another, as electrical modulation of a specific cell, resulted in calcium flux from the stimulated cell to neighboring cells. Thus, we decided to approach a long-lasting debate of whether cardiomyocytes and myofibroblast form heterocellular electrical coupling in the heart. We have previously tackled this question in vivo, by modulating a single myofibroblast within a viable contacting heart ex vivo. Interestingly, we found that such coupling was not established, opposing our in vitro 2D monolayered cells in which they did couple. In this study we investigated whether they electrically couple in a 3D engineered cardiac tissue and found no such electrical coupling. These findings suggest that the 3D engineered cardiac tissue is much better and comprehensive model of the cardiac tissue than 2D monolayered cells.