Extensive muscle defect injuries, often resulting from trauma or tumor ablation, present a significant clinical challenge. Current reconstructive surgical procedures, such as autologous muscle flap transfers, are limited by the availability of grafts and donor site morbidity. Bioengineering implantable skeletal muscle constructs to restore normal function represents a substantial advance in treating these injuries. Tissue engineering strategies aim to recapitulate the structural organization of native skeletal muscle, particularly the uniaxial alignment of muscle cells crucial for contractile properties and force generation. Various techniques, including mechanical stimulation, anchors, electrical stimulation, and micro-patterned scaffolds, have been employed to induce cellular alignment in bioengineered constructs. Co-culturing with endothelial cells and fibroblasts has also improved construct survival and vascularization. While 3D bioprinting offers precise control over construct architecture, integrating the constructs with the host nervous system remains a challenge. Native skeletal muscle is innervated via the peripheral nervous system, forming NMJs essential for muscle survival, development, maturation, and contraction. Denervation leads to muscle atrophy and loss of contractility, highlighting the need for rapid integration with the host nervous system for bioengineered constructs. This study aims to address this by developing human skeletal muscle constructs with integrated neural cells, hypothesizing that the cellular interactions would promote muscle maturation and facilitate rapid integration with host nerve tissues. Pre-forming NMJs within the construct is also expected to increase long-term survival and maturation before complete innervation.

The literature extensively covers strategies for bioengineering skeletal muscle tissue, focusing on achieving uniaxial alignment of muscle cells to mimic native tissue structure and function. Studies have explored various methods to achieve this alignment, including mechanical stimulation, electrical stimulation, and micro-patterned scaffolds. The importance of vascularization for long-term survival and function of engineered muscle tissue is also well-established, with several studies demonstrating improved outcomes with co-culture of muscle cells with endothelial cells and fibroblasts. However, relatively fewer studies have focused on integrating neural cells into engineered muscle constructs and demonstrating functional improvement in vivo. The critical role of NMJs in muscle development, maturation, and function has been widely reported, yet the development of strategies to accelerate innervation of implanted constructs remains an area requiring further investigation. Existing 3D co-culture systems incorporating muscle and motor neurons have largely served as in vitro models for NMJ-related diseases. The potential impact of neurotrophic factors and neurotransmitters released from neural components on muscle development and NMJ formation is also a subject of ongoing research.

This study utilized human muscle progenitor cells (hMPCs) and human neural stem cells (hNSCs) to create neural cell-integrated 3D bioprinted skeletal muscle constructs. Initially, the optimal ratio of hMPCs to hNSCs was determined in a 2D co-culture system by evaluating myotube formation, long-term maintenance, and NMJ formation at different ratios. Immunofluorescence staining for myosin heavy chain (MHC), MyoD, myogenin, glial fibrillary acidic protein (GFAP), beta-III tubulin (βIIIT), neurofilament (NF), and acetylcholine receptor (AChR) were employed to assess myogenic and neuronal differentiation. The optimal ratio (300:1 hMPCs:hNSCs) was then used to create 3D bioprinted constructs using a custom bioprinting system, employing fibrinogen-based composite hydrogel, sacrificial acellular bioink, and supporting polycaprolactone (PCL) bioink. The constructs were evaluated in vitro for cell viability, myotube formation, NMJ formation, and functional NMJ activity (calcium uptake imaging). A rat model of tibialis anterior (TA) muscle defect injury was used for in vivo evaluation. Constructs (MPC only and MPC+NSC) were implanted into the defect sites, and functional outcomes (muscle weight, tetanic force), histological examination (H&E, Masson's trichrome staining), and immunofluorescent analyses (MHC, HLA, vWF, α-SMA, NF, AChR) were performed at 4 and 8 weeks post-implantation. Statistical analysis (ANOVA, Student's t-test) was used to compare different groups. Human MPCs were isolated from human gracilis muscle biopsies. Human NSCs were obtained commercially and cultured according to the manufacturer's instructions. Detailed protocols for cell culture, bioink preparation, 3D bioprinting, in vitro and in vivo evaluation, and statistical analysis are provided in the Methods section of the paper.

In 2D co-culture, the optimal ratio of 300:1 hMPCs:hNSCs significantly increased myotube formation, length, diameter, and striation compared to hMPCs alone. The co-culture also enhanced neuronal differentiation and NMJ formation. In 3D bioprinted constructs, the MPC+NSC group showed significantly higher cell viability, myotube density, and myotube length compared to the MPC only group. The hNSCs differentiated into neurons and glial cells, and NMJs were observed in the MPC+NSC group. In vivo studies using a rat TA muscle defect model showed that the MPC+NSC group exhibited significantly faster restoration of muscle weight and function compared to the MPC only group at 4 weeks post-implantation. At 8 weeks, both MPC only and MPC+NSC groups restored muscle volume to levels comparable to sham controls. However, the tetanic muscle force in the MPC+NSC group was significantly greater than the MPC only group, indicating improved functional recovery. Histological analysis showed organized muscle fiber formation and reduced fibrosis in both groups compared to the non-treated group. Immunofluorescence confirmed that implanted hMPCs contributed to new myofibers. Notably, the MPC+NSC group demonstrated a significantly greater number of NMJs and AChR clusters compared to the MPC only group. Vascularization was comparable between the MPC only and MPC+NSC groups.

This study's findings strongly support the hypothesis that integrating neural cells into 3D bioprinted skeletal muscle constructs accelerates functional muscle regeneration. The enhanced myofiber formation, improved survival, and increased NMJ formation observed in vitro are consistent with the known roles of neurotrophic factors and neurotransmitters in muscle development and maturation. The superior functional restoration and increased NMJ formation seen in vivo in the MPC+NSC group demonstrate the clinical significance of neural cell integration. The rapid recovery of muscle weight and function in the MPC+NSC group at 4 weeks post-implantation highlights the potential to significantly shorten the recovery time following muscle injury. The results suggest a mechanism whereby neurotrophic factors and neurotransmitters released from the integrated neural cells promote muscle cell differentiation, survival, and NMJ formation, contributing to the accelerated regeneration observed. These findings advance the field of musculoskeletal tissue engineering by providing a novel strategy to improve the functional outcome of bioengineered muscle constructs.

This study demonstrates that integrating neural cells into 3D bioprinted skeletal muscle constructs enhances myogenesis, promotes NMJ formation, and accelerates functional muscle regeneration in vivo. The improved outcomes in both in vitro and in vivo studies suggest this approach as a promising therapeutic strategy for repairing extensive skeletal muscle defects. Future studies should investigate alternative neural cell sources, further optimize the bioprinting process to enhance vascularization, and explore the long-term functional outcomes in larger animal models and eventually clinical trials. Furthermore, in-depth investigation of the molecular mechanisms underlying the observed effects would be beneficial.

The study utilized an immunocompromised rat model to minimize immune rejection of human-derived cells. The results may not fully reflect the response in immunocompetent animals or humans. The study's duration was limited to 8 weeks post-implantation, leaving open the question of long-term functional outcomes. Further research should explore the potential limitations of the hNSC source and consider alternative methods to achieve similar effects. While the study included functional assessment of muscle force, direct assessment of contractility and other parameters of muscle function may offer a more comprehensive understanding of the construct's capabilities.