Introduction: Neural-muscular tissue engineering in regenerative medicine requires the functional integration of motor neurons and muscle fibers in 3D scaffolds. Despite the development of co-culture models, synaptic efficiency and coordination of contractions often remain low. In these models, electrical stimulation at 1 Hz has led to a 45% increase in acetylcholine receptor (AChR) density and a 30% improvement in the synchronicity of muscle contractions . This functional gap has rendered many current models ineffective for therapeutic applications or drug screening. 3D models perform better than 2D cultures in forming and maintaining neuromuscular junctions . Engineered scaffolds and conductive hydrogels have improved muscle fiber alignment and enhanced synaptic transmission . Furthermore, neuromuscular organoids and microchips have enabled the study of neuromuscular diseases and precise drug screening . However, challenges such as incomplete synapse maturation and cellular heterogeneity highlight the need for engineered approaches with targeted stimulation . The importance of this topic lies in therapeutic applications such as neuromuscular graft transplantation, modeling degenerative diseases, drug screening, and even designing bioreactors for producing neurotrophic molecules . In this article, using an analytical review approach, ten recent key studies have been examined to determine the role of electrical signaling in enhancing the functional efficiency of neuron-muscle co-culture systems. This review focuses on the mechanisms of electrical stimulation, conductive scaffolds, and bioelectronic technologies, analyzing their role in synaptic maturation and neuromuscular tissue organization.
Methods: In this review study, the role of electrical stimulation in improving the performance of 3D neuromuscular models was analytically investigated by examining ten selected studies. These studies primarily involved co-culturing motor neurons derived from induced pluripotent stem cells (iPSC) and human muscle fibers in 3D scaffolds. Researchers used conductive hydrogels and engineered scaffolds to create an environment similar to natural tissue and optimize cell growth conditions. Electrical stimulation was applied regularly at a low frequency (1 Hz) to investigate its effects on synaptic maturation and coordination of muscle contractions. Functional parameters included the density and distribution of acetylcholine receptors (AChR), muscle fiber diameter and length, expression levels of muscle proteins (MHC and nAChR), and calcium transients. Mechanical responses were also evaluated using force measurement methods (contractile force measurement) and imaging with calcium indicators such as Fluo-4 or GCaMP.
In combined studies that utilized conductive scaffolds along with electrical stimulation, muscle fibers formed thicker, more organized, and with more uniform AChR clustering in the muscle fiber membrane region compared to models without stimulation. Calcium transients were rapid and strong, and the coordination of muscle contractions significantly increased. In models without stimulation, despite the presence of a 3D scaffold, incomplete synaptic maturation and scattered receptor distribution were observed, and weaker functional responses were presented.
Some studies used advanced bioelectronic technologies such as microelectrodes and opto-electronic scaffolds to apply targeted stimulation and precisely control neuronal activity. These technologies enabled quantitative assessment of synaptic maturation, tissue organization, and fiber alignment in organoid and microchip models.
The results indicate that integrating electrical stimulation with conductive scaffolds and 3D conditions leads to increased AChR receptor density, faster synapse maturation, improved contraction coordination, and enhanced neuromuscular efficiency. Consequently, these advancements will pave the way for the development of accurate drug models, production of transplantable grafts, and modeling of neuromuscular diseases such as ALS and SMA.
Results: The diameter of muscle fibers in the stimulated model increases, and the fibers are thicker and more mature, while in the unstimulated condition, the fiber diameter is smaller. The density of AChR receptors in the stimulated model is high, and their clustering is more regular and denser, but without stimulation, the receptor density remains low. The size of AChR clusters is larger in the presence of electrical stimulation, and a larger area of clusters is formed, while in the absence of stimulation, the clusters are smaller. The uniformity of AChR cluster distribution (lacunarity) is lower and the distribution is more uniform in the stimulated model, but in the unstimulated model, the dispersion of clusters is greater. The expression of muscle proteins such as MHC and nAChR is increased in the stimulated model, while without stimulation, the expression level of these proteins is lower. Calcium responses to neural stimulation are strong and rapid in the stimulated model, but in the unstimulated model, these responses are weak or absent.
Conclusion: Two-dimensional neuron-muscle co-culture models, although simpler and less expensive for initial testing or training in laboratory settings, have serious limitations; including insufficient formation of functional synapses, low coordination between neurons and iPSC-derived skeletal muscle fibers, and incompatibility with physiological conditions in the body. Consequently, these models have shown limited efficacy in areas such as drug screening, modeling neuromuscular diseases, and designing transplantable grafts. Recent studies have shown that the use of conductive scaffolds in 3D models leads to the regular orientation of muscle fibers (parallel alignment along scaffold axis), faster maturation of neuron-muscle connections, and increased coordination of tissue contractions, ultimately improving tissue function. These scaffolds are used in preclinical studies, the design of drug screening platforms, and the production of transplantable neuromuscular tissues [6,9]. Despite these advancements, challenges such as the limited durability of 3D models, cellular heterogeneity in co-cultures, and incomplete synaptic maturation have hindered the clinical translation of these technologies—including conductive scaffolds and electrical stimulation. With advancements in targeted electrical stimulation, precise control of cell differentiation, and bioelectronic technologies such as microelectrode arrays and optoelectronic scaffolds, the performance of 3D systems can be optimized and their efficiency increased [1,2,3]. These improvements enable the creation of neuromuscular organoids and cultivable organs, more accurate drug models, guided targeted cell differentiation, and enhanced efficiency of neuromuscular systems. Therefore, 3D co-cultures equipped with electrical stimulation, by increasing acetylcholine receptor density, promoting synaptic maturation, and improving functional coordination, are considered a promising solution for developing reliable and clinically translatable platforms in tissue engineering and modeling neuromuscular diseases such as ALS and SMA.