Induced Formation and Maturation of Acetylcholine Receptor Clusters in a Defined 3D Bio-Artificial Muscle

Abstract

Dysfunction of neuromuscular junction (NMJ) is involved in a wide range of muscular diseases. The development of neuromuscular junction (NMJ) through which skeletal muscle is innervated requires the functional modulation of AchR clustering on myofibers. However, studies on AchR clustering in vitro are mostly done on monolayer muscle cell culture, which lacks a three-dimensional structure, a prominent limitation of the 2D system. To enable a better understanding of the structure-function correlation underlying skeletal muscle innervation, a muscle system with a well-defined geometry mimicking the in vivo muscular setting is needed. Here, we report a 3D bio-artificial muscle (BAM) bioengineered from GFP-transduced C3H murine myoblasts as a novel in vitro tissue-based model for muscle innervation studies. Our cell biological and molecular analysis showed that this BAM is structurally similar to in vivo muscle tissue and can reach the perinatal differentiation stage, higher than does 2D culture. Effective clustering and morphlogical maturation of AchRs on BAMs induced by agrin and laminin indicates the functional activity and plasticity of this BAM system toward innervation. Taken together, our results show that the BAM provides a favorable 3D environment that at least partially recapitulates real physiological skeletal muscle with regard to innervation. With a convenience of fabrication and manipulation, this 3D in vitro system offers a novel model for studying mechanisms underlying skeletal muscle innervation and testing therapeutic strategies for relevant nervous and muscular diseases.

Biomaterials for skeletal muscle tissue engineering

“…Following implantation at a site next to an injury, angiogenesis and anastomosis with the host vasculature were first observed; a subsequent transfer of the scaffold to the neighboring defect site, with the host vasculature still intact, was then demonstrated [18]. 3D, aligned tissue engineered myofibers have also exhibited acetylcholine receptor (AChR) clustering when treated with agrin or laminin [19,20] (Figure 2A) and this was shown to enhance subsequent innervation and angiogenesis when transplanted in vivo [20]. These engineered myofibers have also been cultured with motor neurons, leading to AChR formation at the interface of the two cell types; these constructs were then able to exhibit contraction when the neurons were activated with glutamic acid [21].…”

Design, evaluation, and application of engineered skeletal muscle

“…The fraction of myofibers expressing sarcomeres, muscle contractile units typically labeled by sarcomeric α-actinin (SAA) and myosin heavy chain (MyHC) antibodies, has been quantified to gauge myogenic differentiation [6, 7, 13, 58]. Additionally, the relative expression of MyHC isoforms (embryonic, neonatal, and adult) and types (I, IIa, IIx) has been analyzed to assess myofiber maturation and metabolic phenotype [78, 81, 83]. Additionally, the density and morphology of acetylcholine receptors have been identified through staining with bungarotoxin [59, 83].…”

“…Additionally, the relative expression of MyHC isoforms (embryonic, neonatal, and adult) and types (I, IIa, IIx) has been analyzed to assess myofiber maturation and metabolic phenotype [78, 81, 83]. Additionally, the density and morphology of acetylcholine receptors have been identified through staining with bungarotoxin [59, 83]. We recently reported a direct structural comparison of engineered muscle to native neonatal muscle fascicles revealing similar myofibril appearance (SAA + sarcomeric units), basal lamina composition and distribution (collagen IV and laminin along periphery of myofibers), peripheral epimysial-like connective tissue layer (vimentin + fibroblasts), and quiescent SC pool (Pax7 + cells) [6].…”

Endothelial Network Formation Within Human Tissue-Engineered Skeletal Muscle

“…In the absence of neural cells, BAMs have been shown to form mature AchR clusters by the addition of agrin and laminin. 46 In the absence of mature AchR clusters, BAMs can still be stimulated by electrical current, which results in increased protein synthesis, increased force production, and excitability. 5,47 The introduction of neural cells to a monolayer of myotubes resulted in the formation of neuromuscular-like junctions with clustering of acetylcholine receptors.…”