Abstract

Engineering replacement organs is the next frontier in therapeutic technologies. Yet, the integration of innervation—critical for organ development, function, and homeostasis—remains underexplored. This review highlights the role of neural inputs in regulating critical organs including pancreas, liver, salivary gland, and spleen. We examine organ-specific neuroanatomy and emerging strategies to incorporate neuronal-axonal networks in engineered organs, drawing from innovations in scaffold design, multi-cell culture techniques, neural engineering, and biofabrication. Finally, we discuss tools for evaluating innervation across in vitro, preclinical, and clinical settings, advocating for innervation as a core design element in next-generation artificial organs.

The leap from tissue engineering to organ engineering

More than three decades after Joseph Murray performed the first human organ (kidney) transplantation, Y.C. Fung, a pioneer in the field of biomechanics, first introduced the concept of “tissue engineering” in 1985 in his proposal to the National Science Foundation (NSF)1. Tissue engineering was born out of the necessity to find a balance between the translational relevance of cellular-level findings and the experimental feasibility of fabricating a whole organ. From the days of the Vacanti mouse2, tissue engineering has made incredible progress over the last few decades, leading to multiple tissue-engineered medical products in clinical trials. Parallel advances in the field of biomaterials, 3D bioprinting and immunotherapy have encouraged biomedical engineers to leap from tissue engineering to whole organ engineering, thereby constantly closing the gap between experimental feasibility and clinical need to solve the organ shortage conundrum. Initial attempts at fabricating a whole organ followed a top-down approach wherein decellularized organs served as a scaffold to culture autologous cells3,4,5. The limited availability of cadaveric tissue/organs has been a major challenge for such top-down approaches to organ manufacturing5. On the other hand, bottom-up approaches to whole organ engineering involve fabricating the smallest structural/functional unit of the organ and using it as a building block to recreate the complex architecture, usually following additive manufacturing techniques like 3D bioprinting. Presently, it is possible to leverage stem cell biology and/or 3D printing to create miniaturized functional replicas of organs (called “organoids”) like liver and pancreas, as well as full-size transplantable versions of organs with comparatively simple/hollow architecture like skin, ear, and bladder. However, 3D printing a fully functional transplantable complex organ like a heart or kidney is still a “moonshot”. Among the multiple technological advancements necessary to fabricate a fully functional organ, researchers have already identified that achieving revascularization and reinnervation are two of the most critical prerequisites for ensuring survival and functionality of such bioengineered organs6.

Innervation has untapped potential in organ engineering

There has been significant progress in developing perfusable vascular networks in engineered organs, initially using top-down approaches of organ manufacturing and more recently across bottom-up approaches employing advanced 3D extrusion bioprinting techniques7. Further, several extensive reviews have been published that capture the current progress, challenges, as well as future perspectives of developing a vascularized bioengineered organ8,9,10. In contrast, innervation remains an untapped area in organ bioengineering, even though its implications in development, function, and regeneration are well established across multiple tissue/organ systems.

Innervation is key to organ growth, function, and repair

In more recent decades, the role of the nervous system in the involuntary regulation of internal organs has been expanded to virtually all other systems in the body, as contained in the autonomic nervous system (ANS) division of the peripheral nervous system (PNS). The ANS consists of sympathetic (“fight-or-flight”) and parasympathetic (“rest”) fibers that emanate from the central nervous system (CNS) and innervate organs through various pathways11,12. Preganglionic neurons of the sympathetic division emerge from paravertebral ganglia at the thoracic and mid-lumbar spinal cord and synapse at the sympathetic chain or the prevertebral ganglia with postganglionic fibers that ultimately innervate the effector organ. In contrast, parasympathetic preganglionic fibers originate from the brainstem or the sacral spinal cord and typically interact with the postganglionic component close to the target organ. Moreover, while acetylcholine (ACh) is the principal neurotransmitter between preganglionic and postganglionic fibers of any type, postganglionic sympathetic and parasympathetic nerves mainly employ norepinephrine (NE) and ACh, respectively, to communicate with and influence organs. The interaction between these efferent fibers (signal propagation towards effector organ/tissue, “motor”) and organs relies on afferent (sensory propagation towards CNS) inputs to the brain that provide information on environmental and physiological conditions in the organs (e.g., stretch receptors in the urinary bladder, baroreceptors for central blood pressure, thermoreceptors for skin blood flow)11,12. This is relayed to the autonomic nerves to regulate functions such as skin and muscle vasoconstriction, pancreatic secretions, urination, gut motility, and saliva production11,12,13. Variations in the density, sensitivity, and phenotypes of efferent and afferent inputs have also been related to various pathologies (e.g., gastrointestinal, pulmonary, cardiovascular diseases). Furthermore, although not extensively studied, innervation is increasingly being recognized as an essential component of organ development and regeneration13,14,15,16. For example, autonomic nerves contribute to organogenesis, wound healing, and tissue regrowth17 by preserving phenotypes and function in stem cell niches and presenting growth and transcription factors necessary for the maintenance of migrating cells in wounds16,18,19. In many instances, blood vessels and nerves follow the same paths, and their interactions are essential in pathfinding during development20,21,22. As a result, innervation is a crucial component of tissues and organs due to its role in development, functional regulation, modulation, and disease.

Innervation is dispensable in transplants, essential in bioengineered grafts

Innervation is dispensable during orthotopic transplantation, but innervation with the recipient generally occurs over time. For instance, allogeneic whole organ transplantation usually involves denervated organs, and they can function without immediate neural integration (like the liver, heart, and kidney). They are eventually innervated and integrated with the host nervous system. This is primarily because such denervated organs receive neuroendocrine molecules essential for functioning through the vascular supply and this serves as a short-term substitute for physical rewiring with host nerves. Curiously, multiorgan sympathetic denervation has been proposed to combat cardiometabolic diseases when diet and exercise-based approaches have failed23, demonstrating the general disregard of the critical role of innervation in maintaining homeostasis and in organ pathophysiology23. However, artificial organs fabricated through organ engineering lack the cellular complexity, maturity, and matrix architecture to function like an adult whole organ. Organs are composed of multiple distinct tissue types, each comprising diverse cellular populations. Within a given organ, both the individual cell types and tissue types exhibit highly specific spatial organization, which is essential for proper organ development, maturation, and function. Reproducing this intricate cellular heterogeneity remains a significant challenge in current organ engineering approaches. In the following sections, we discuss the cellular complexity and architectural features of various organs and explore their implications for the development of effective innervated tissue engineering strategies. Moreover, precise spatiotemporal neural connections direct organogenesis as well as play a crucial role in organ function and regeneration (Fig. 1)24. Hence, although denervated orthotopic transplants can survive and function without immediate reconnection with the nervous system, innervation is a critical component of the organ biomanufacturing process, especially in bottom-up approaches that may lack an appropriately instructive extracellular matrix (ECM) scaffold.

Fig. 1: Neural implications in development, function and regeneration of organs.

Innervation regulates all facets of organ physiology. In this review, we focus on the role of neural input in the development, function, and regulation of some key organs in the body – pancreas, salivary glands, liver, and spleen, thereby establishing the necessity of incorporating preformed neuronal networks during biofabrication of artificial organs. The schematic here provides a snapshot of the multifaceted effects of innervation in various organs of the body. Figure created with professional scientific illustration services by Inmywork.

We have previously reviewed the role of innervation in regeneration and engineering of different muscle systems, along with surgical/engineering strategies that can be employed to promote re-innervation, thereby establishing innervation as the “missing link” in tissue engineering25. In the current article, we go a step further from tissue systems to expand upon the implications of innervation in organ biology, considering critical organs like the pancreas, liver, salivary gland, and spleen as examples. We further describe current tissue engineering solutions to develop pre-innervated organs. We conclude by reviewing clinically available technologies to monitor organ reinnervation and propose strategies to engineer organs with preformed neuronal-axonal networks.

Pancreatic innervation

The distribution of nerve fibers in the pancreas varies by species. In mice, pancreatic islets display abundant parasympathetic and sympathetic innervation, with direct associations primarily to α- and β-cells, while innervation of the exocrine compartment is much less pronounced26. Conversely, human islets show limited yet predominantly sympathetic innervation27. Further, most sympathetic fibers in the human pancreas align with smooth muscle cells of islet vasculature and do not interact directly with endocrine cells27.

Innervation in pancreatic development

Pancreatic organogenesis in mice begins at embryonic day E 9.5, as buds emerge from the foregut endoderm, followed by epithelial branching and clustering of endocrine precursor cells (Fig. 2)28. Sympathetic neurons expressing vesicular monoamine transporter 2 (VMAT2) are detectable beginning E12.5 within the developing pancreatic bud (Fig. 2)29, when the neural crest cells around the pancreatic epithelial cells undergo neuronal differentiation[30](https://www.nature.com/articles/s41467-025-64801-4#ref-CR30 “Agerskov, R. H. & Nyeng, P. Innervation of the pancreas in development and disease. Development 151, https://doi.org/10.1242/dev.202254

(2024).“). These VMAT2+ axons become integrated with vasculature concurrent with postnatal islet maturation29.

Fig. 2: Timeline of innervation during development of organs – Illustration of the developmental timeline of innervation for the pancreas, salivary gland, liver, and spleen in a prenatal and postnatal context in rodents and human.

This captures the current state-of-the-art and indicates knowledge gaps in understanding the implications of innervation in organogenesis. (Figure created in BioRender - https://BioRender.com/ocgy6s0).

Sympathetic nerves critically shape the architecture of pancreatic islets during embryogenesis14. In human development, innervation from the celiac ganglion invades the pancreatic primordium by gestational week 6 (GW6), and by GW9, multiple sources—including the celiac plexus, vagus nerve, and superior mesenteric plexus—contribute to the innervation31. Nerve ending density then decreases by GW30–3232. Adult islet structure facilitates appropriate β-cell interactions and insulin secretion33, with deviations linked to diabetes34. Experimental denervation in neonatal mice disrupts typical α-cell localization around β-cell cores, while deletion of TrkA in sympathetic neurons results in disorganized islets with diminished cell–cell adhesion14. TrkA mutant mice lacking sympathetic innervation also manifest decreased NCAM and E-cadherin expression and altered islet positioning, highlighting innervation’s role in cell clustering and migration14.

Innervation in functional regulation of the pancreas

Although the pancreas can sustain function independent of direct innervation26, autonomic signaling orchestrates insulin release during the cephalic phase, sustains glucose tolerance, synchronizes islet activity, and modulates responses to hypoglycemia and diabetes14,35,36. Exogenous agonizts to muscarinic receptors and external stimulation of vagus nerve has been shown to promote insulin release whereas hypoglycemia also leads to parasympathetic activation and modulation of glucagon secretion14,35,36,37. Conversely, sympathetic stimulation suppresses glucose-stimulated insulin secretion and enhances glucagon output through norepinephrine (NE) signaling on islet adrenergic receptors35. Two regulatory mechanisms have been proposed: direct neurotransmitter release in mice and NE-dependent vascular contraction in humans, regulating islet blood flow and hormone distribution (“spillover”)27,38. However, the “spillover” does not account for the rapid breakdown of acetylcholine by acetylcholinesterase38. Altered innervation density is implicated in pancreatic disorders. Diabetic rats show early sympathetic fiber loss39, and insulitis in non-obese diabetic mice associates with reduced islet innervation and elevated neurotrophins that indicate the body’s marked attempts to promote nerve ingrowth and islet survival40.

Salivary gland innervation

The parotid gland receives parasympathetic nerves from the otic ganglion, whose preganglionic fibers originate in the inferior salivatory nucleus of the medulla15. Preganglionic nerves from the superior salivatory nucleus in the pons join the facial and lingual nerves to ultimately innervate the submandibular and sublingual glands via the submandibular ganglion15. Sympathetic innervation involves preganglionic fibers projecting to the superior cervical ganglia, with postganglionic axons traveling to salivary glands through the external carotid plexus15.

Innervation in salivary gland development

During mouse submandibular gland development, the oral epithelium infiltrates neural crest–derived mesenchyme at E11, establishes a single duct by E12, and branches extensively by E14 (Fig. 2)41. Early parasympathetic innervation from the submandibular ganglion aligns with epithelial branching of the submandibular gland, guiding axonal trajectories42. Likewise, parotid duct branching initiates when otic ganglion–derived nerves reach the epithelium15. Parasympathetic nerves maintain progenitor epithelial populations, as removal of the submandibular ganglion reduces cytokeratin-5/−15 positive progenitor cells and developmental end bud formation43. These deficits are mimicked by inhibiting acetylcholine (ACh) signaling, and reversed with ACh analogs, confirming the role of parasympathetic innervation-derived ACh in maintaining epithelial progenitor populations43. In addition, Neurturin blockade impairs parasympathetic-mediated ductal tubulogenesis, with vasoactive intestinal peptide identified as the key promoter (Fig. 2)44. Critical windows exist for parasympathetic or sympathetic interventions: early postnatal (48 h after birth) parasympathectomy impedes myoepithelial differentiation in acinar buds, whereas later timing spares acinar maturation and gland size45. On the other hand, neonatal sympathectomy reduces acinar size and granule content, and adult interventions shrink parotid glands while altering protein output46,47 (Fig. 2).

In humans, parasympathetic neurons drive salivary branching and morphogenesis from early stages, whereas sympathetic associations are delayed until later development48,49. The submandibular ganglion forms around GW6, with innervation of parenchyma and duct by GW848,50.

Innervation in functional regulation of the salivary gland

Autonomic nerves control salivary gland output by modulating acinar contraction, saliva composition, and local blood flow51. Parasympathetic stimulation dominates serous and mucous secretion in major glands. Sympathetic signals primarily drive protein exocytosis from acinar cells, though parasympathetic input also contributes15,51.

Hepatic innervation

The extrinsic neuroanatomy of the liver is generally conserved across species. However, there is considerable species-wise variation in the distribution of intrahepatic nerves that make up the intrinsic neuroanatomy of a liver52,53. For example, rats and mice only present an extrinsic supply of sympathetic nerves limited around the portal triad, whereas in humans’ sympathetic efferent branches extend deep inside the hepatic lobules and has direct contact with hepatocytes52. This lack of direct sympathetic innervation within rat hepatic lobules is compensated by the abundance of cellular gap junctions between rat hepatocytes54.

Innervation in hepatic development

Spatiotemporal patterns of development of hepatic innervation varies widely among species and is mostly unclear. However, previously reported developmental studies indicate that innervation plays a crucial role in liver morphogenesis and stabilization of portal tract structures. In mice, innervation first appears around the primitive extra-hepatic biliary ducts at E17.5 and slowly extends towards the liver throughout the initial postnatal term. Axon outgrowth within the liver is guided by ectopic expression of NGF by murine biliary epithelial cells (Fig. 2)55. A separate study on mouse hepatic innervation observed initial traces of neuronal cell bodies near the hepatic hilus at E16.5 that spread towards the periphery by P28 (Fig. 2)56. In humans, innervation begins in the developing liver much earlier as compared to mice. Initial evidence of nerve fibers is seen as early as gestational week 7-8 near the hilum. Subsequently, by week 12, the portal tract receives the bulk of PGP9.5-expressing nerves. Intrasinusoidal innervation in humans is only observed beginning at 28 weeks (Fig. 2)57.

Innervation in functional regulation of liver

Hepatic afferent nerves express a myriad of chemoreceptors that act as sensors for osmoreception, glucose and fatty acid levels, relaying information back the CNS58. Efferent pathways within the liver regulate the hepatic vasculature, bile production, metabolism and maintain circadian rhythms via secretion of a variety of neurotransmitters (aminergic, cholinergic, peptidergic and nitrergic). Efferent pathways have also been implicated in liver repair, regeneration, as well as in pathology58.

Hepatic afferent neurons lining the portal vasculature can detect the osmolarity of fluids through specific ion channel receptors. Decreased osmolarity, triggered by water intake, is sensed by hepatic afferents and transmitted via dorsal root ganglia to efferent sympathetic fibers that increase norepinephrine production and blood pressure (pressure reflex)59. Further, the liver maintains ionic concentration (like Na+) of fluids through the hepatorenal reflex that involves decreased renal sympathetic activity in response to increased blood Na+ concentration, thereby leading to urinary excretion of Na+60. The liver plays a central role in sensing glucose and lipid levels and subsequently modulating glucose and lipid metabolism. A gradual decrease in glucose levels in the portal vein is detected by GLUT2 glucose transporters that activate vagal afferents, leading to an increase in food uptake61. On the other hand, vagal efferent activity induces secretion of Hepatic Insulin Sensitizing Substance (HISS) from the liver that promotes glucose storage in skeletal muscle, decreases glucose production and increases glycogenesis62. Stimulation of the sympathetic splanchnic nerve results in an opposite effect, increasing glucose production and decreasing glycogenesis63. Autonomic input regulates overall lipid metabolism in the liver through monitoring levels of free fatty acids (especially linoleic acid and triglycerides) by vagal afferents, leading to reduced food intake, whereas decreasing the secretion of lipids (VLDL and Apo-B) by hepatocytes following sympathetic stimulation64. Liver regeneration following vagotomy is delayed due to decreased activity of aspartate transcarbamoylase65. On the other hand, the sympathetic nervous system has been shown to inhibit the activity of hepatic oval cells (HOC), which are the resident stem cells and promote the proliferation of fibrogenic hepatic stellate cells (HSC), which are involved in the progression of disease like cirrhosis66.

Spleen innervation

Sympathetic postganglionic axons reach the spleen via the splenic nerve, with their cell bodies in prevertebral ganglia. These fibers accompany splenic artery branches targeting especially the white pulp and occasionally marginal zones67,68. These axons interface with preganglionic fibers within the greater splanchnic nerve coming from the thoracic spinal cord (T9-T11)69. Parasympathetic innervation of the spleen is reportedly minimal and still under debate70,71.

Innervation in spleen development

In rats, noradrenergic fibers emerge in the white pulp at birth, proliferating alongside splenic compartment growth. These fibers encircle developing follicles, organizing within the Periarteriolar lymphoid sheaths (PALS) structure as lymphocytes are redistributed72. The pattern of noradrenergic innervation develops steadily from day 13 onward73 (Fig. 2). In humans, the spleen is primarily innervated by the splenic nerve, composed of sympathetic noradrenergic fibers originating from the celiac ganglion, suprarenal ganglia, and thoracic sympathetic chain. The sympathetic neurons are responsible for regulating immune responses74. Spleen has few parasympathetic neurons, but the parasympathetic innervation of the spleen is poorly understood74.

Innervation in functional regulation of the spleen

Sympathetic innervation of the spleen contributes to innate immune regulation, affecting activity of natural killer cells, dendritic cells, macrophages, and other types of leukocytes75. During acute inflammation, NE released from sympathetic axons binds β-adrenergic receptors on macrophages, suppressing TNF-α production in response to endotoxin exposure76,77. Stress-induced sympathetic signals decrease cytokine output, while splenic nerve resection blocks this immune suppression78.

Neurological damage can disrupt splenic innervation, resulting in elevated NE, increased splenocyte apoptosis, impaired antibody synthesis, and greater infection susceptibility79. Such persistent immune suppression is a consequence of a hyperactive anti-inflammatory reflex circuit below the spinal cord injury site, resulting from chronic maladaptive plasticity80.

Criticial design components for innervated organ biomanufacturing

The multi-tissue composition, diverse physiochemical properties, intricate architecture, and range of functions displayed by an organ determine critical design criteria for organ biomanufacturing, like choice of fabrication technique(s), cells, and appropriate scaffolding materials. As organs are exponentially more complex in form and function as compared to tissues, conventional tissue engineering techniques do not automatically translate into engineering whole artificial organs.

Biofabrication technique

It is essential for organ manufacturing technologies to have the capacity to integrate heterogeneous cell types and multiple materials to recapitulate native organ geometries, constituents, and functions81. Organ manufacturing technologies vary in degree of automation, ranging from fully automated multi-nozzle rapid prototyping and additive molding as to more manually intensive top-down approaches like decellularization/recellularization of ECM-based matrix. Commonly used strategies include extrusion-based printing, inkjet printing, laser-assisted bioprinting,