Innervation plays a pivotal role as a driver of tissue and organ development as well as a means for their functional control and modulation. Therefore, innervation should be carefully considered throughout the process of biofabrication of engineered tissues and organs. Unfortunately, innervation has generally been overlooked in most non-neural tissue engineering applications, in part due to the intrinsic complexity of building organs containing heterogeneous native cell types and structures. To achieve proper innervation of engineered tissues and organs, specific host axon populations typically need to be precisely driven to appropriate location(s) within the construct, often over long distances. As such, neural tissue engineering and/or axon guidance strategies should be a necessary adjunct to most organogenesis endeavors across multiple tissue and organ systems. To address this challenge, our team is actively building axon-based “living scaffolds” that may physically wire in during organ development in bioreactors and/or serve as a substrate to effectively drive targeted long-distance growth and integration of host axons after implantation. This article reviews the neuroanatomy and the role of innervation in the functional regulation of cardiac, skeletal, and smooth muscle tissue and highlights potential strategies to promote innervation of biofabricated engineered muscles, as well as the use of “living scaffolds” in this endeavor for both in vitro and in vivo applications. We assert that innervation should be included as a necessary component for tissue and organ biofabrication, and that strategies to orchestrate host axonal integration are advantageous to ensure proper function, tolerance, assimilation, and bio-regulation with the recipient post-implant.

The present study describes the fabrication of polyaniline-silk fibroin (PASF) nanocomposite-based nerve conduits and their subsequent implantation in a rat sciatic nerve injury model for peripheral nerve regeneration. This is the first in vivo study of polyaniline-based nerve conduits describing the safety and efficacy of the conduits in treating peripheral nerve injuries. The nanocomposite was synthesized by electrospinning a mixture of silk fibroin protein and polyaniline wherein the silk nanofibers were observed to be uniformly coated with polyaniline nanoparticles. Tubular shaped nerve conduits were subsequently formed by multiple rolling of the electrospun sheet over a stainless steel mandrel. The conduits were characterized in vitro for their physico-chemical properties as well as their compatibility with rat Schwann cells. Upon implantation in a 10 mm sciatic nerve injury model, the conduits were evaluated for their neuro-regenerative potential through extensive electrophysiological studies and monitoring of gait pattern over a course of 12 months. Gross examination, histological and ultra-structure analyses of the conduits and the regenerated nerve were also performed to evaluate morphological regeneration of transected nerve. PASF nanocomposite conduits seeded with Schwann cell (cell seeded PASF) exhibited excellent nerve conduction velocity (NCV) (50 m s ⁻¹ ), compound muscle action potential (CMAP) (12.8 mV), motor unit potential (MUP) (124 μ V), growth of healthy tissue along the nerve gap and thick myelination of axons 12 months after implantation indicating enhanced neuro-regeneration. The excellent functional recovery achieved by animals implanted with cell seeded PASF conduits (86.2% NCV; 80.00% CMAP; 76.07% MUP) are superior to outcomes achieved previously with similar electrically conductive conduits. We believe that the present study would encourage further research in developing electrically active neural implants using synthetic conducting polymers and the in vivo applications of the same.

Volumetric muscle loss (VML) is the traumatic or surgical loss of skeletal muscle beyond the inherent regenerative capacity of the body, generally leading to severe functional deficit. Formation of appropriate somato-motor innervations remains one of the biggest challenges for both autologous grafts as well as tissue-engineered muscle constructs. We aim to address this challenge by developing pre-innervated tissue-engineered muscle comprised of long aligned networks of spinal motor neurons and skeletal myocytes on aligned nanofibrous scaffolds. Motor neurons led to enhanced differentiation and maturation of skeletal myocytes in vitro. These pre-innervated tissue-engineered muscle constructs when implanted in a rat VML model significantly increased satellite cell density, neuromuscular junction maintenance, graft revascularization, and muscle volume over three weeks as compared to myocyte-only constructs and nanofiber scaffolds alone. These pro-regenerative effects may enhance functional neuromuscular regeneration following VML, thereby improving the levels of functional recovery following these devastating injuries.

Intracortical neural microelectrodes, which can directly interface with local neural microcircuits with high spatial and temporal resolution, are critical for neuroscience research, emerging clinical applications, and brain computer interfaces (BCI). However, clinical applications of these devices remain limited mostly by their inability to mitigate inflammatory reactions and support dense neuronal survival at their interfaces. Herein we report the development of microelectrodes primarily composed of extracellular matrix (ECM) proteins, which act as a bio-compatible and an electrochemical interface between the microelectrodes and physiological solution. These ECM-microelectrodes are batch fabricated using a novel combination of micro-transfer-molding and excimer laser micromachining to exhibit final dimensions comparable to those of commercial silicon-based microelectrodes. These are further integrated with a removable insertion stent which aids in intracortical implantation. Results from electrochemical models and in vivo recordings from the rat’s cortex indicate that ECM encapsulations have no significant effect on the electrochemical impedance characteristics of ECM-microelectrodes at neurologically relevant frequencies. ECM-microelectrodes are found to support a dense layer of neuronal somata and neurites on the electrode surface with high neuronal viability and exhibited markedly diminished neuroinflammation and glial scarring in early chronic experiments in rats.