The Cullen Laboratory at the University of Pennsylvania has over a decade of experience engineering living neural scaffolds of varying dimensions (ultralong to microscale) that are employed to repair the nervous system following trauma or neurodegenerative disease. Historically, the projects in lab have ranged from pathway reconstruction in the brain to long distance axon regeneration in peripheral nerves, with testing done in small and large animal models. Upon joining the Cullen Lab as a postdoc, I (Das) wanted to combine the biomaterial and nanotechnology experience obtained during my doctoral studies with the lab’s long-standing expertise on neural regeneration. Dr. Cullen and I often discussed the idea that even though the nervous system is intricately linked to virtually every other system in the body, most non-neural tissue engineering endeavors gave little consideration to innervation, and just assumed that constructs would properly “wire up” after implantation. As I began to vigorously study innervation, I quickly learned that it was much more critical – and complicated – than I had initially thought. Indeed, innervation plays a crucial role in tissue and organ development, maturation, function, and regulation, and hence should be considered as an essential component throughout the entire process, from biofabrication to implementation. We recently published a detailed review elaborating the importance of innervation – using cardiac, smooth, or skeletal muscle as examples – and showcasing different methods to promote graft innervation as well as techniques to generate pre-innervated tissue engineered constructs1. Exploring the role of innervation in engineering living tissues is now an additional focus area of the Cullen Lab across numerous organ systems in the body.
The current study published in Communications Biology builds on these concepts, and was focused on studying the role of pre-innervation in skeletal muscle regeneration2. Volumetric Muscle Loss (VML) is defined as loss of skeletal muscle beyond the inherent regenerative capacity of the body. VML is associated with progressive motor axotomy, and reestablishing appropriate innervation remains one of the biggest unmet challenges for both autologous grafts as well as tissue engineered muscle. We addressed 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. The presence of motor neurons significantly enhanced maturation of skeletal myocytes in vitro and the topographical alignment provided by the nanofibers augmented formation of aligned neuromuscular bundles.
Next, we implanted the cell laden nanofiber sheets in the Tibialis Anterior (TA) muscle of athymic rats by creating a VML deficit 1 cm long, 5mm wide and 3mm deep (roughly a 20% volume loss). To approximately fill the defect size, we then implanted 3 stacked acellular or cell laden sheets per animal, and used Ultrasound imaging to monitor muscle volume over 3-weeks. While Ultrasound is commonly used in clinical cases of VML, to our knowledge this is the first report using this modality in a rodent VML model. Being cheap, rapid, and portable, Ultrasound can be a great alternative to monitor muscle volume in small animals for labs that do not have more sophisticated imaging capabilities such as MRI. Using Ultrasound, we observed that pre-innervated bioscaffolds significantly facilitated TA volume recovery by 3 weeks.
Immunohistochemical analysis of the graft area as well as remaining host muscle was performed through extensive confocal imaging. Our implanted cells survived for at least 3-weeks and we observed that pre-innervated bioscaffolds significantly increased the density of muscle stem cells or satellite cells near the injury site. Satellite cells play a key role in myogenesis and the fact that pre-innervation augmented their proliferation is very exciting. Graft revascularization is another critical part of survival and integration of tissue engineered implants. Although at the 1-week time point there was no endothelial cell migration within the implanted sheets, we observed significant revascularization within pre-innervated bioscaffolds by 3-weeks. Next, we assessed the co-labeling of nicotinic Acetylcholine Receptors (nAchRs) – forming pretzel-shaped clusters to constitute motor end plates – with a presynaptic marker to identify the points of innervation on the muscle, referred to as Neuromuscular Junctions (NMJs). We found that the pre-innervated group had the most NMJs as compared to other groups at both 1- and 3-weeks after VML repair.
These promising findings in the field of skeletal muscle repair should stimulate further research into developing pre-innervated tissue engineered constructs for applications in smooth muscle as well as cardiac muscle tissue engineering. In future work, such engineered nerve-muscle complexes may also be fabricated using cells derived from adult human stem cell sources (e.g., iPSCs), thereby making them translational as autologous, personalized tissue engineered constructs. These pro-regenerative effects can potentially lead to enhanced functional neuromuscular regeneration, thereby improving the levels of functional recovery in afflicted patients.
Poster Image - Confocal microscopic image of a longitudinal section of TA muscle showing survival of our implanted cells (Myocytes - green; Axons - purple) on nanofiber sheets stained with Laminin (red).
Acknowledgement - We thank Ms. Melanie Hilman for her help with the Figure summarizing the findings.
1. Das, S., Gordián-Vélez, W.J., Ledebur, H.C. et al. Innervation: the missing link for biofabricated tissues and organs. npj Regen Med 5, 11 (2020). https://doi.org/10.1038/s41536-020-0096-1.
2. Das, S., Browne, K.D., Laimo, F.A. et al. Pre-innervated tissue-engineered muscle promotes a pro-regenerative microenvironment following volumetric muscle loss. Commun Biol 3, 330 (2020). https://doi.org/10.1038/s42003-020-1056-4.