Skeletal muscle possesses a remarkable intrinsic capacity to regenerate following minor injuries, thanks to the activation of satellite cells—the resident muscle stem cells—which proliferate, express myogenic regulatory factors such as MyoD and myogenin, fuse, and rebuild damaged myofibers. This physiological mechanism reaches an insurmountable limit when tissue loss exceeds a critical threshold. Under these conditions, referred to in the literature as volumetric muscle loss (VML), the endogenous response proves entirely insufficient: the ???aged area becomes colonized by fibrous connective tissue, a permanent scar forms, and contractile function is not restored. When large portions of muscle become necrotic or are lost—as occurs in combat injuries or road accidents—the classical regenerative response is inadequate to repair the defect.
Available therapeutic options remain limited. Muscle flap transplants inevitably involve donor-site morbidity, while decellularized biological substitutes, although they recapitulate the composition of the native extracellular matrix, tend to resorb rapidly and, crucially, fail to provide topographical guidance to regenerating myofibers. This leads to the recruitment of randomly oriented fibers and consequent scar tissue formation. It is precisely this inability to reconstruct the anisotropic architecture of skeletal muscle—the parallel, strictly unidirectional geometry of myofibers—that represents the central unresolved challenge in muscle regenerative medicine. Addressing it requires scaffolds that are not only biocompatible but also capable of actively influencing cellular orientation.
Why fibroin and why nanofibers
Among natural biomaterials available for skeletal muscle tissue engineering, fibroin from Bombyx mori occupies a prominent position for reasons that extend far beyond simple biocompatibility. Its mechanical properties—high tensile strength and tunable elasticity depending on the degree of β-sheet crystallinity—enable the fabrication of scaffolds whose Young’s modulus approaches that of native skeletal muscle. Bombyx mori fibroin scaffolds support the formation of long, well-aligned myotubes in primary human myoblasts, and their Young’s modulus closely mimics that of normal skeletal muscle. This detail is critical: the stiffness of the mechanical environment in which myoblasts grow profoundly influences differentiation pathways, and scaffolds that are too stiff or too soft inhibit myotube maturation even in the presence of appropriate biochemical cues.
From a morphological standpoint, the ability to process fibroin into nanofibers via electrospinning makes it particularly suitable for replicating the nanostructure of the muscle extracellular matrix. Electrospinning technology enables the production of nanofibrous scaffolds that mimic the fibrous protein component of the extracellular matrix, whose morphology and composition influence cell adhesion, proliferation, and differentiation. Mature myofibers are surrounded by a network of collagen nanofibrils aligned axially: an electrospun fibroin scaffold with similarly oriented fibers is therefore not merely a passive support, but a structure that communicates in the topographical language of muscle cells.
Electrospinning as a tool for geometric control
The technological core of this approach is electrospinning, which uses a strong electrostatic field to stretch a jet of polymer solution—here, fibroin dissolved in formic acid or alkaline aqueous solution—into submicrometer-diameter filaments that deposit onto a collector. Electrospinning is a relatively simple process that allows the synthesis of highly complex three-dimensional fibrous structures, with good control over fiber alignment; the resulting scaffold achieves an architecture closely resembling that of the extracellular matrix, namely a high–aspect ratio nanofiber network.
The critical variable for muscle regeneration is fiber alignment, which can be controlled through collector geometry. A static flat collector produces random, disordered deposition, whereas a high-speed rotating cylindrical collector generates shear forces that align fibers along the axis of rotation. The result is a nanofibrous mat with parallel fibers, capable of exerting a contact guidance effect on seeded cells. It has been demonstrated that nanofiber alignment influences the arrangement and elongation of colonizing cells: engineering this parameter can guide cell growth to achieve a desired anisotropy.
Parameters governing fiber diameter—solution concentration, viscosity, applied voltage, nozzle-to-collector distance, and rotation speed—also modulate cell–substrate interactions. Studies on scaffolds with fiber diameters ranging from 300 nm to over 3 μm have shown that larger diameters better promote myoblast alignment and elongation, with significant upregulation of myogenin and myosin heavy chain (MHC), two key markers of terminal myogenic differentiation. Larger-diameter fibers support improved alignment, growth, and differentiation of myoblasts, associated with p38 MAPK phosphorylation and increased expression of myogenin and MHC.
Contact guidance and the myogenic program
When a myoblast is seeded onto an aligned nanofibrous substrate, its cytoskeleton senses the physical constraints of the topography and responds by reorganizing actin and myosin filaments parallel to the fibers. This phenomenon—contact guidance—is not merely geometric: it triggers a mechanotransductive signaling cascade that converges on the activation of myogenic regulatory factors. The topographical signal is thus translated into biological information, promoting the progression from proliferating myoblasts to terminally differentiating cells.
The functional capabilities of skeletal muscle are tightly linked to its well-organized microstructure, composed of parallel aligned myotubes. In cases of extensive muscle loss, endogenous regenerative capacity is hindered by scar tissue formation, which disrupts native muscle architecture and ultimately leads to severe functional impairment. Aligned fibroin nanofiber scaffolds intervene upstream in this process by providing the topographical environment necessary for proper deployment of the myogenic program. In culture on aligned fibroin nanofiber substrates, C2C12 myoblasts and primary human myoblasts exhibit a consistent progression: within the first days of differentiation, downregulation of Pax7—a marker of quiescent satellite cells—is observed alongside upregulation of MyoD and myogenin. With prolonged culture, elongated cells fuse to form multinucleated myotubes which, under optimal conditions, begin to display transverse sarcomeric striation, an unequivocal sign of contractile apparatus assembly. Bombyx mori fibroin scaffolds support the formation of long, well-aligned myotubes, and immunofluorescence along with quantitative gene expression analysis reveals that myotube formation varies across different scaffolds, confirming that three-dimensional structural properties—not merely fibroin’s chemical composition—determine the outcome of myotube maturation.
Composite architectures
Current research extends well beyond simple electrospun mats. One of the most promising developments is the core–shell structure, in which a core of aligned fibroin nanofibrous yarns—often combined with polycaprolactone and conductive polyaniline—is encapsulated within a photopolymerizable hydrogel shell. Core–shell scaffolds combine the aligned nanofibrous core, which guides myoblast alignment and differentiation, with a hydrogel shell that provides a suitable three-dimensional environment for nutrient exchange and mechanical protection. In this configuration, the anisotropic core delivers directional cues while the hydrogel recreates a more physiological microenvironment, ensuring permeability to nutrients and waste products.
On the front of chemical bioactivation, click chemistry has enabled precise functionalization of nanofiber surfaces with signaling molecules. This approach allows biomolecules to be attached to scaffold architectures in a controlled manner, enhancing efficiency and functionality while promoting tissue regeneration through reactions that involve simple starting conditions, readily available reagents, and non-toxic or solvent-free environments. Growth factors such as IGF-1 or HGF can thus be immobilized on the fibroin surface in an oriented fashion, ensuring localized and sustained release near myogenic cells without the pharmacokinetic limitations of systemic delivery.
From In Vitro Culture to In Vivo Evaluation
Translating these constructs from in vitro studies to animal models is the step that determines their true translational potential. Murine VML models—typically generated by partial resection of the tibialis anterior or gastrocnemius—serve as the standard testing ground. In these models, implantation of fibroin scaffolds with aligned nanofibers and seeding with primary myoblasts or satellite cells has yielded encouraging results: measurable increases in myofiber cross-sectional area, higher density of MHC-positive fibers in the ???? area compared to untreated controls, and attenuation of the inflammatory response through macrophage polarization toward the pro-regenerative M2 phenotype. Electrospun fibroin scaffolds are capable of regulating macrophage polarization and promoting neovascularization, both key factors in reconstructing volumetric muscle loss defects.
Vascularization remains one of the main obstacles to the successful integration of large tissue-engineered constructs: without vascular supply, central cells undergo ischemic necrosis. Sericin—the companion protein of fibroin, often removed during degumming—has shown, in some composite formulations, the ability to promote local angiogenesis. It thus integrates synergistically with the fibroin structural component to support long-term construct survival.
