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.

Functional recovery rarely occurs following injury or disease-induced degeneration within the central nervous system (CNS) due to the inhibitory environment and the limited capacity for neurogenesis. We are developing a strategy to simultaneously address neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative "living scaffolds" capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

Reestablishing cerebral connectivity is a critical part of restoring neuronal network integrity and brain function after trauma, stroke, and neurodegenerative diseases. Creating transplantable axon tracts in the laboratory is an unexplored strategy for overcoming the common barriers limiting axon regeneration in vivo, including growth-inhibiting factors and the limited outgrowth capacity of mature neurons in the brain. We describe the generation, phenotype, and connectivity of constrained threedimensional human axon tracts derived from brain organoids. These centimeter-long constructs are encased in an agarose shell that permits physical manipulation and are composed of discrete cellular regions spanned by axon tracts, mirroring the separation of cerebral gray and white matter. Features of cerebral cortex also are emulated, as evidenced by the presence of neurons with different cortical layer phenotypes. This engineered neural tissue represents a first step toward potentially reconstructing brain circuits by physically replacing neuronal populations and long-range axon tracts in the brain.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as "living scaffolds". Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based "living scaffolds". To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of <350 µm, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring <150 µm in diameter yet extending several cm in length. These engineered living scaffolds exhibited >97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based "living scaffolds" in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

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Parkinson′s disease (PD) affects 10 million patients worldwide, making it the second most prevalent neurodegenerative disease. Motor symptoms emerge from the loss of dopamine in the striatum after the death of dopaminergic neurons and the long-projecting axons of the nigrostriatal pathway. Current treatments, such as dopamine replacement, deep brain stimulation or cell therapies, disregard the loss of this pathway at the core of symptoms. We sought to address this by improving our tissue-engineered nigrostriatal pathway (TE-NSP) technology, which consists of a tubular hydrogel with a collagen/laminin core that encases an aggregate of dopaminergic neurons and their axons in a way that resembles the nigrostriatal pathway. These constructs can be implanted to replace the lost neurons and axons with fidelity to the pathway, and thus provide dopamine according to feedback from the host circuitry. While TE-NSPs have been traditionally fabricated with agarose, here we utilized a hyaluronic acid (HA) hydrogel to expand the functionality of the encasement and our control over its properties. Using rat ventral midbrain neurons, we found that TE-NSPs exhibited longer and faster neurite growth with HA relative to agarose, with no differences observed in electrically-evoked dopamine release. When transplanted, HA hydrogels reduced host neuron loss and inflammation around the implant compared to agarose, and the cells and axons within TE-NSPs survived and maintained their cytoarchitecture for at least 2 weeks.

Achieving full inclusion for people with disabilities in STEM is a matter of national security, economic prosperity, and equity. People with disabilities in STEM are underrepresented in postsecondary degrees and employment and they have higher unemployment rates and earn less. Inaction at the federal level has contributed to perpetuating these disparities. The federal government, especially through a signed law, could provide the funding and mandate to establish the institutional support, resources, and incentives needed so people with disabilities have equitable access to STEM and they can contribute to the scientific and technological innovation the U.S. needs to confront its great challenges. Congress has lately been working to bolster the country’s scientific and technological enterprise and to increase the diversity of our STEM workforce, through HR4521, the America COMPETES Act, and S1260, the U.S. Innovation and Competition Act. Some of these proposals are promising but fail to include provisions specific to people with disabilities. As Congress considers a HR4521/S1260 compromise bill, it has the opportunity to include programs that ensure the inclusion and promote the success of people with disabilities in STEM.

SummaryReestablishing cerebral connectivity is a critical part of restoring neuronal network integrity and brain function after trauma, stroke, and neurodegenerative diseases. Creating transplantable axon tracts in the laboratory is a novel strategy for overcoming the common barriers limiting axon regeneration in vivo, including growth-inhibiting factors and the limited outgrowth capacity of mature neurons in the brain. We describe the generation and phenotype of three-dimensional human axon tracts derived from cerebral organoid tissue. These centimeterlong constructs are encased in an agarose shell that permits physical manipulation and are composed of discrete cellular regions spanned by axon tracts and dendrites, mirroring the separation of grey and white matter in the brain. Features of cerebral cortex also are emulated, as evidenced by the presence of neurons with different cortical layer phenotypes. This engineered neural tissue has the translational potential to reconstruct brain circuits by physically replacing discrete cortical neuron populations as well as long-range axon tracts in the brain.