Skeletal Muscle Tissue Engineering: From Scaffolds to Functional Tissue

Author(s): Chidchaya Wankaew

Abstract

Severe skeletal muscle injuries resulting in volumetric muscle loss (VML) pose a persistent clinical challenge, as the intrinsic regenerative capacity of skeletal muscle is insufficient to support the regeneration of large-scale tissue loss and the recovery of functional deficits. In response, skeletal muscle tissue engineering (SMTE) has emerged as a promising regenerative approach aimed at reconstructing muscle structure and restoring function through diverse methodologies using biomaterials and bioengineering strategies such as hydrogels, electrospinning, bioprinting technologies, bioreactor-assisted systems, and extracellular matrix-based approaches. Despite the substantial progress SMTE has made, several limitations remain, including incomplete functional restoration, difficulty in replicating the structural and functional complexities of native skeletal muscle, and translational gaps in clinical applications. Current clinical evidence remains scarce and inconsistent, therefore further investigation and refinement of SMTE strategies are required to enhance functional integration for translational relevance. Overall, this review aims to provide a comprehensive evaluation of current skeletal muscle tissue engineering advancements and their potential for addressing volumetric muscle loss.

Keywords: Skeletal Muscle Tissue Engineering; Volumetric Muscle Loss; Biomaterials; Scaffolds; Electrospinning; Hydrogels; Bioprinting; Clinical Trials.

1. Introduction

Skeletal muscle injuries are a substantial and growing clinical burden due to the high prevalence of traumatic injuries, surgical interventions, and degenerative diseases¹. Skeletal muscle constitutes 40-45%² of total adult body mass and plays a crucial role in locomotion and movement³. While minor muscle damage can typically undergo the natural endogenous regeneration process, the intrinsic ability of  skeletal muscles to self-repair is limited upon severe injuries such as volumetric muscle loss (VML) due to chronic disease, congenital defects, tumor ablation, or traumatic injury (such as motor vehicle accidents, sports injuries, blast injuries or gunshot wounds) and are associated with long-term disability and reduced quality of life^1,4.

In a study by Corona et al. (2015), a retrospective analysis of disability records was conducted on two cohorts of U.S. military service members who have medically retired following combat-related extremity trauma. The orthopaedic group had severe open tibial fractures, and the other broader cohort included battlefield-injured personnel. The authors found that over 65% of disabilities involved muscle-related injuries, of which 92% were classified as VML⁵.

The extensive loss of myofibers, satellite cells, and extracellular matrix following VML overwhelms natural repair mechanisms. Existing surgical interventions, including autologous muscle transfer, remain insufficient to fully restore muscle mass and function, emphasizing the growing need for regenerative approaches that can restore functional skeletal muscle following severe injury⁶.

1.1. Skeletal Muscle Tissue Regeneration

Within the critical threshold, healthy muscles can perform endogenous regeneration⁷, in which the muscle progenitor cells (formed from satellite cells)³ are activated⁸ and undergo myogenesis, whilst self-renewing to sustain the pool of undifferentiated cells in the tissue niche⁹. Following proliferation, they differentiate into myoblasts¹⁰ that undergo fusion to generate new multinucleated myofibers¹¹. These fibers then integrate into mature, functional muscle tissue¹². However, in the case of volumetric muscle loss (VML), the intrinsic ability to regenerate by satellite cell activation is hindered. Failure of this self-repair mechanism can lead to the formation of non-functional fibrous tissue and degeneration of fatty muscle¹³. 

Figure 1. Normal skeletal muscle regeneration process: Following muscle fiber rupture, the satellite stem cells beneath the basal lamina are activated. These cells proliferate, differentiate into myogenic precursor cells and myoblasts, and subsequently fuse to form new multinucleated skeletal muscle fibers. This leads to the restoration of muscle structure and function. This figure was created using BioRender by Chidchaya Wankaew.

Current standard clinical approaches include implanting autologous muscle flaps known as free functional muscle transfer (FFMT)³. However, this procedure is limited by the risk of donor-site complications and incomplete restoration of muscle strength and function^14,15, potentially requiring it to be coupled with extended physical rehabilitation. As the field of skeletal muscle tissue engineering and regenerative medicine advances and overcomes the limitations of current treatment approaches, they possess a promising potential in enhancing muscle function and regeneration to achieve long-term functional recovery, ultimately improving quality of life.

1.2. Methods in Skeletal Muscle Tissue Engineering

Tissue engineering strategies aim to create patient-specific muscle constructs that restore structure and function at the site of injury. As outlined by Cezar and Mooney⁵, the two approaches for engineering of skeletal muscle include in vitro engineering, maturing muscle tissues prior to transfer, and in situ engineering, delivering biomaterials and bioactive cues to stimulate later remodelling in the host environment. Biomaterials such as hydrogels, decellularized matrices, patterned scaffolds, and electrospun nanofibers can provide structural support, protect transplanted cells, and deliver physical, chemical, and mechanical cues that direct tissue regeneration. Interactions between these materials and cells through biochemical signaling by growth factors and other molecules, geometric patterning, mechanical stimulation, or electrical stimulation help to mimic microenvironmental cues experienced by the native muscle, which can enhance muscle maturation and functionality.

Scaffold design strives to achieve the highly aligned organization of myofibers that is essential for force generation and acts as constructs that provide guidance for cell alignment and maturation. Fiber-based scaffolds can be fabricated by techniques such as electrospinning, molding, and extrusion, which have shown to promote unidirectional myoblast alignment and fusion by providing topographical cues that mimic native muscle morphology¹⁶. Similarly, decellularized extracellular matrix (dECM) scaffolds preserve tissue-specific biochemical signals and structural complexity, supporting myogenic differentiation while also facilitating host integration upon implantation¹⁷. However, while these approaches improve structural organization, limitations remain in controlling construct geometry, cell distribution, and scalability for clinically relevant muscle volumes¹⁸.

To address these challenges, three-dimensional (3D) bioprinting has emerged as a powerful and versatile technique in skeletal muscle tissue engineering. Bioprinting enables precise, layer-by-layer deposition of cell-laden bioinks, allowing accurate spatial control over cell placement, scaffold geometry, and material composition¹⁹. Hydrogels such as gelatin methacryloyl (GelMA), alginate, fibrin, and polyethylene glycol-based systems are commonly used as bioinks due to their adjustable mechanical properties, cytocompatibility, and ability to be cross-linked during printing¹⁶. Importantly, bioprinting techniques aid the fabrication of aligned hydrogel fibers and bundled structures to replicate native muscle fascicles and hence promote myotube alignment and functional maturation²⁰. Furthermore, incorporating growth factors such as IGF-1, bFGF-2, and VEGF within bioprinted constructs allows local biochemical signaling which also enhances myogenesis, angiogenesis, and host tissue integration^21,22. Collectively, these bioprinting-based approaches display a significant advancement toward generating structurally organized and scalable skeletal muscle constructs that can address volumetric muscle loss.

Altogether, these emerging technologies in regenerative engineering converge Materials Science, Stem Cell Science, Developmental Biology, and Clinical Translation with the aim to regenerate complex musculoskeletal tissues and enable more effective repair of traumatic muscle loss and degenerative muscle diseases (Figure 2).

Figure 2. Techniques used in skeletal muscle tissue engineering. Illustrated are three common approaches: (1) electrospinning, which produces aligned nanofibrous scaffolds to guide myoblast orientation; (2) three-dimensional bioprinting, enabling controlled deposition of cell-laden bioinks to create organized muscle constructs; and (3) hydrogel-based systems, which provide a hydrated extracellular matrix-like environment to support cell differentiation and tissue remodelling. Together, these methods aim to promote myogenic differentiation and functional muscle regeneration. This figure was created using BioRender by Chidchaya Wankaew.

2. Pre-clinical Studies on Skeletal Muscle Tissue Engineering

2.1. Electrospinning

In one study, researchers investigated whether aligned electrospun nanofibers composed of polycaprolactone blended with decellularized muscle ECM could improve regeneration in a mouse VML injury model. 28 days following injury, treated muscles showed significantly increased myosin heavy chain-positive fiber area and a higher MHC:collagen ratio compared with untreated controls (p < 0.05), although there was little improvement in force production of the whole muscle were detected at this time. These results suggest that aligned ECM-based nanofibrous scaffolds can improve the regenerative microenvironment and enhance formation of myofibers, but longer-term maturation and reinnervation may be required for full functional recovery²³. In another study, the authors developed multilayered electrospun polycaprolactone scaffolds coated with tendon-derived extracellular matrix to improve cell infiltration and matrix formation in musculoskeletal tissue engineering using in vitro human adipose-derived stem cells (hADSCs). It was observed that the hADSCs infiltrated the entire thickness of the scaffold within 28 days, and TDM-coated scaffolds showed a significantly higher total collagen content and increased type I collagen deposition compared to fibronectin- and PBS-coated controls (p < 0.05). Overall, the results show that using multilayered scaffolds together with tissue-specific extracellular matrix improves collagen formation while maintaining scaffold mechanical stability, making this approach useful for engineered skeletal muscle-tendon constructs²⁴. Furthermore, a study evaluated the use of an electrospun sandwich-like nanofibrous scaffold, composed of aligned outer layers and a bioactive inner layer, to guide skeletal muscle regeneration in a rat volumetric muscle injury model. Implanted scaffolds significantly enhanced muscle regeneration compared with untreated injuries, with regenerated myofibers showing improved alignment, increased myosin heavy chain-positive area, a reduction in fibrotic tissue, all alongside a shift toward pro-regenerative macrophage populations, and with these effects maintained for 4-8 weeks post-implantation. This demonstrates that electrospun scaffold constructs combining fiber alignment and layered bioactivity can modulate the injury microenvironment, highlighting electrospinning as a powerful strategy for advancing skeletal muscle tissue engineering²⁵. Researchers studied the potential of electrospun chitosan/poly(vinyl alcohol) nanofibrous scaffold seeded with mesenchymal stem cells in supporting skeletal muscle regeneration, using in vitro assays and in vivo implantation in a rabbit muscle defect model. The scaffold sustained cell viability for over 10 days with no significant cytotoxicity shown from MTT assays and exhibited a tensile strength of approximately 3.2(+/- 0.4) MPa that degraded gradually over around 20 days. Histological analysis following implantation also confirmed organized muscle-like tissue formation with minimal inflammatory response compared to untreated controls. Therefore, the results indicate that biodegradable electrospun chitosan/PVA scaffolds can provide a supportive microenvironment for cell survival and early muscle tissue formation²⁶.

2.2.Hydrogels

A study investigated gelatin-genipin hydrogels surface patterned as biomaterial scaffolds in guiding myogenic cell alignment, differentiation, and regeneration both in vitro and following implantation in mouse models by engineering it to match native skeletal muscle stiffness of around 13 kPa. The hydrogels supported aligned myotube formation and myogenic differentiation in vitro, exhibited high biocompatibility with minimal inflammatory response in vivo, and slow, controlled biodegradation over several weeks, while implantation under the skin and into partially ablated muscle did not impair endogenous muscle regeneration when compared with controls. Altogether, the findings suggest that mechanically tuned, patterned gelatin-genipin hydrogels can provide a stable microenvironment for skeletal muscle organisation and regeneration, illustrating its potential as biomaterial scaffolds for skeletal muscle tissue engineering²⁷. In another study, researchers examined whether human gingival mesenchymal stem cells (GMSCs) enclosed within RGD-functionalised alginate hydrogels could undergo myogenic differentiation and support skeletal muscle formation in vitro following subcutaneous implantation in a mouse model. GMSCs exhibited significantly higher expression of myogenic markers (MyoD, Myf5, and Myogenin) than bone marrow MSCs (p < 0.05), with optimal differentiation observed in hydrogels of intermediate stiffness (10-16 kPa), and in vivo implantation resulted in muscle-like tissue formation accompanied by a significant increase in CD31-positive microvessel density (p < 0.05). Overall, this study highlights that combining stem cell delivery with biomaterials with controlled biochemical and mechanical cues can enhance myogenesis and vascularisation²⁸. Moreover, the use of nanoengineered myogenic scaffolds for skeletal tissue engineering was investigated in vitro through developing nanoengineered GelMA hydrogels that incorporate nanoclays to allow sustained and controlled release of insulin-like growth factor-1 (IGF-1) for improved muscle progenitor cell proliferation and myogenic differentiation. It was found that nanoclay-functionalised scaffolds sustained IGF-1 retention over several days compared with GelMA alone and led to increased cell proliferation (~1.5-2 fold), higher myogenic marker expression (MyoD and Myogenin), and enhanced myotube formation and alignment in vitro (p < 0.05). Overall, these findings imply that nanoengineered scaffolds enabling controlled growth factor presentation can significantly improve myogenic outcomes, supporting their potential use in skeletal muscle repair and regenerative tissue-engineering strategies²⁹. On top of that, a study evaluated a thiolated hyaluronic acid-chondroitin sulfate (HA-CS) hydrogel cross-linked with PEGDA as an implantable scaffold to promote skeletal muscle regeneration in a mouse quadriceps volumetric muscle loss (VML) model. HA-CS hydrogels supported C2C12 myoblast proliferation and enhanced regulation of myogenic markers (MyoD, MyoG, MYH8). After 4 weeks in vivo, hydrogel-treated mice showed integration with host tissue, increased Pax7+ satellite cell migration, angiogenesis, and improved treadmill performance compared to autograft controls alongside a significant reduction in scar tissue relative to untreated defects. These results indicate that HA-CS hydrogels can accelerate muscle regeneration, cellular infiltration, and functional recovery following severe muscle loss, highlighting their potential as a regenerative engineering strategy for skeletal muscle tissue repair³⁰. Lastly, in another study, researchers tested a wet tissue adhesive dextran-aldehyde/gelatin powder hydrogel as an injectable scaffold to support skeletal muscle regeneration in a mouse volumetric muscle loss model by modulating the local microenvironment. Post-implantation, muscles treated with the hydrogel exhibited significantly enhanced regeneration with reduced fibrosis and increased angiogenesis compared to untreated controls, demonstrating improved structural repair and tissue infiltration. These findings suggest that adhesive hydrogels combining adhesion and scaffold support can enhance vascular and cellular regeneration in skeletal muscle injuries, offering a promising hydrogel strategy for SMTE³¹.

2.3. Bioprinting

A study was conducted on the use of 3D bioprinting with GelMA-alginate composite bioinks using C2C12 myoblast model (in vitro) in fabricating functional skeletal muscle constructs. Systematically varying alginate concentration (0-8% w/v) and crosslinking strategy (UV alone versus dual ionic + UV crosslinking) was used to optimise printability and myogenic outcomes. The researchers found that constructs composed of 10% GelMA and 8% alginate with dual crosslinking exhibited significantly higher compressive modulus (>200 kPa), improved cell viability post-printing, and ~50% greater metabolic activity over 7 days compared with 6% alginate constructs (p < 0.05), alongside enhanced myotube formation and desmin-positive alignment by day 12. Overall, the results show that tuning bioink mechanical properties and crosslinking mechanisms can substantially promote myoblast proliferation, differentiation, and functional maturation in bioprinted skeletal muscle tissues, highlighting the potential of engineered hydrogel bioinks for muscle tissue engineering applications³². Additionally, researchers investigated whether 3D-bioprinted skeletal muscle constructs composed of human muscle progenitor cells embedded into fibrin-based bioink could restore structure and function when implanted into volumetric muscle loss (VML) injuries in mice. Eight weeks after implantation, the bioprinted muscle constructs restored up to 82% of native tetanic force, showed significantly increased myofiber density, vascularization, and innervation, and reduced fibrotic tissue formation compared with untreated VML controls. These findings indicate that bioprinted, cell-laden muscle constructs can integrate with host tissue and substantially recover muscle function, highlighting bioprinting as a promising strategy for treating severe skeletal muscle injuries³³. In another study, researchers evaluated an in situ bioprinting approach using a handheld printer to directly deposit a VEGF-eluting GelMA-Laponite hydrogel (“Muscle Ink”) into mouse volumetric muscle loss defects to promote functional muscle regeneration. 8 weeks post-injury, mice that were treated with VEGF-loaded Muscle Ink benefited from significantly improved functional recovery, achieving near-baseline running speeds and a two-fold increase in running distance, increased CD31 capillary density (p = 0.0013), and markedly reduced collagen deposition compared with untreated and VEGF-negative controls. These results signify that in vivo bioprinting of bioactive hydrogels enabling controlled growth factor delivery improved functional recovery, underlining its potential for skeletal muscle tissue engineering³⁴. Similarly, researchers also investigated whether muscle-mimetic tissues fabricated by microvalve-assisted coaxial 3D bioprinting could restore structure and function in a rat tibialis anterior VML model. It was found that implanted artificial muscle tissue showed vigorous myotube formation and spontaneous contraction in vitro. Whereas, in vivo implantation resulted in generation of force, upon electrical induction, that reached approximately 65-70% of native muscle levels, alongside increased myosin heavy chain-positive fiber density and organized fascicle-like structure compared to untreated defects. Altogether, this study suggests that biomimetic, 3D bioprinted constructs are capable of partially restoring muscle function³⁵. Lastly, a study examined the outcome of 3D bioprinted GelMA/HAMA hydrogels with incorporated MXene nanoparticles to see if it could enhance myogenesis and functional regeneration in a mouse volumetric muscle loss (VML) model. In vivo, constructs that contained MXene had a significant increase in regenerated myofiber area (~1.8 fold vs. hydrogel-only controls, p < 0.01). Moreover, the construct also showed upregulated myogenic gene expression (MyoD, MyoG, and Myh4 were all >2-fold, p < 0.05), reduced inflammatory cell infiltration, and histologic confirmations of better-aligned muscle fibers at the defect site. Overall, these findings demonstrate that electrically active nanocomposite bioinks can promote muscle regeneration and control inflammation, highlighting a promising bioprinting-based strategy for SMTE³⁶.

2.4. Other Methods in SMTE  

Researchers investigated how the extracellular matrix protein Tenascin-C (TnC) regulates muscle stem cell (MuSC) maintenance and regenerative capacity using Tenascin-C knockout mice subjected to barium chloride-induced muscle injury. The loss of TnC led to an approximate 50% reduction in the number of Pax7+ MuSC in adult muscle, impaired self-renewal, and significantly reduced MuSC migration, while over 80% of MuSC expressed the TnC receptor Annexin A2 and treatment with soluble TnC partially improved stem cell maintenance and migratory defects. The findings suggest that microenvironmental ECM cues such as TnC are critical regulators of MuSC function and hence it is important to incorporate bioactive ECM signaling components into skeletal muscle tissue-engineering strategies to enhance stem cell maintenance and regeneration³⁷.

In skeletal muscle tissue engineering, bioreactors are specialised in vitro culture systems designed to regulate the physical and biochemical conditions necessary for cells to produce three-dimensional tissue constructs for regenerative applications^38,39. Bioreactors actively control parameters such as nutrient delivery, oxygen transport, and waste removal, all of which are critical for maintaining cell viability and supporting tissue growth in thick constructs⁴⁰. Perfusion bioreactors operate by continuously circulating culture medium through porous scaffolds⁴¹. A study investigated three-dimensional skeletal muscle-like tissue constructs in perfusion-based bioreactors, in which the tissues were engineered by culturing myoblasts within microgroove collagen scaffolds under perfusion-based bioreactor conditions to improve tissue thickness, alignment, and maturation compared with static culture. The perfusion culture allowed thick, viable muscle constructs that reached several hundred micrometres in thickness and had significantly higher myosin heavy chain expression (several-fold increase), increased extracellular matrix deposition, and improved cell viability relative to static conditions, to form alongside highly aligned, multi-layered myotube bundles. Overall, these results support combining scaffold micro-topography with perfusion bioreactors as it quantitatively enhances muscle tissue thickness, alignment, and molecular maturation, which can be essential to generating functionally relevant engineered skeletal muscle for regenerative applications⁴².

Table 1. Summary of pre-clinical studies on SMTE:

Citation	Method used	Preclinical model	Main outcome
(Patel et al. 2020)	Aligned electrospun PCL-containing nanofibers blended with decellularized muscle ECM.	Mouse (VML model)	28 days post-injury, treated muscles showed significantly increased myosin heavy chain-positive fiber area and higher MHC:collagen ratio compared with untreated controls (p < 0.05). Minimal improvement in whole-muscle force.
(Chainani et al. 2013)	Multilayered electrospun polycaprolactone (PCL) scaffold coated with tendon-derived ECM	In vitro human adipose-derived stem cells (hADSCs)	Human adipose-derived stem cells infiltrated the full scaffold thickness by 28 days. TDM-coated scaffolds showed significantly higher total collagen content and increased type I collagen deposition compared with controls (p < 0.05).
(Jin et al. 2025)	Sandwich-like electrospun nanofibrous scaffold (with aligned outer layers and a bioactive inner layer)	Rat (volumetric muscle injury model)	Scaffold implantation increased aligned myofibers, elevated MHC-positive area, reduced fibrosis, and a shift toward pro-regenerative macrophage populations. Effects persisted for 4-8 weeks post-implantation.
(Kheradmandi et al. 2016)	Electrospun chitosan/PVA nanofibrous scaffold seeded with MSCs	Rabbit (muscle defect model)	Scaffold maintained cell viability >10 days (no significant cytotoxicity), tensile strength ~3.2 (+/- 0.4) MPa with gradual degradation ~20 days, and histology showed organized muscle-like tissue formation with minimal inflammation compared with controls.
(Gattazzo et al. 2018)	Surface-patterned gelatin-genipin hydrogel tuned to ~13 kPa	Mouse	Hydrogels supported aligned myotube formation in vitro, showed high biocompatibility and slow biodegradation in vivo, and did not impair endogenous regeneration when implanted subcutaneously or into partially ablated muscle.
(Ansari et al. 2016)	RGD-functionalised alginate hydrogels with enclosed human GMSCs	Mouse (subcutaneous implantation)	GMSCs showed higher MyoD, Myf5, and Myogenin expression than BM-MSCs (p < 0.05), optimal differentiation at 10-16 kPa, and implantation increased CD31-positive microvessel density (p < 0.05).
(Quint et al. 2022)	Nanoengineered GelMA hydrogels with nanoclays for controlled IGF-1 release	In vitro myogenic progenitor cell model	Nanoclay scaffolds sustained IGF-1 retention and increased proliferation by around 1.5-2-fold, elevated MyoD and Myogenin expression, and enhanced aligned myotube formation (p < 0.05).
(Narayanan et al. 2020)	Thiolated hyaluronic acid-chondroitin sulfate (HA-CS) hydrogel cross-linked with PEGDA	Mouse (quadriceps VML model)	Enhanced regulation of myogenic markers (MyoD, MyoG, MYH8) and C2C12 myoblast proliferation. After 4 weeks, treated muscles showed integration with host tissue, increased Pax7+ satellite cell migration, angiogenesis, reduced scar tissue, and improved treadmill performance compared with autograft controls.
(Lee et al. 2024)	Injectable dextran-aldehyde/gelatin adhesive powder hydrogel	Mouse (VML model)	Hydrogel treatment resulted in significantly reduced fibrosis and increased angiogenesis compared with untreated controls, indicating enhanced structural regeneration and tissue infiltration.
(Seyedmahmoud et al. 2019)	3D bioprinting with GelMA-alginate bioinks with encapsulated C2C12 myoblasts	In vitro murine myoblast model (C2C12)	Constructs with 10% GelMA and 8% alginate (dual crosslinking) showed compressive modulus >200 kPa, ~50% higher metabolic activity over 7 days compared with 6% alginate constructs (p < 0.05), and improved myotube alignment by day 12.
(Kim et al. 2018)	3D bioprinted muscle constructs of fibrin-based bioink with embedded human progenitor cells	Mouse (VML model)	At 8 weeks, constructs restored up to 82% of native tetanic force, with increased myofiber density, vascularisation, innervation, and reduced fibrosis compared with untreated controls.
(Quint et al. 2021)	In situ bioprinting to deposit VEGF-eluting GelMA-Laponite hydrogel (“Muscle Ink”)	Mouse (VML model)	At 8 weeks, treated mice achieved near-baseline running speed, ~2-fold increase in running distance, increased CD31+ capillary density (p = 0.0013), and reduced collagen deposition compared with controls.
(Lee et al. 2022)	Microvalve-assisted coaxial 3D bioprinting (muscle-mimetic tissue)	Rat (tibialis anterior VML model)	Implanted constructs generated electrically induced force reaching ~65-70% of native muscle, with increased MHC-positive fiber density and organized fascicle-like structure.
(Jo et al. 2024)	3D bioprinted GelMA/HAMA hydrogels incorporating MXene nanoparticles	Mouse (VML model)	Constructs containing MXene produced a ~1.8-fold increase in regenerated myofiber area compared with hydrogel-only controls (p < 0.01), >2-fold upregulation of myogenic genes (MyoD, MyoG, and Myh4, p < 0.05), reduced inflammatory cell infiltration, and improved alignment of regenerated muscle fibers in vivo.
(Cecchini et al. 2025)	ECM protein Tenascin-C	Tenascin-C knockout mice (with barium chloride-induced muscle injury)	Loss of TnC caused ~50% reduction in Pax7+ MuSC numbers, impaired self-renewal, and reduced MuSC migration. Whereas treatment with soluble TnC treatment partially improved stem cell maintenance and migratory defects.
(Cerino et al. 2016)	Perfusion bioreactor culture of myoblasts in microgroove collagen scaffolds	In vitro myoblast-based engineered tissue	Perfusion culture produced constructs several hundred micrometres thick with several-fold higher myosin heavy chain expression, increased ECM deposition, improved cell viability, and highly aligned multilayered myotubes compared to static culture.

3. Clinical Trials on Skeletal Muscle Tissue Engineering

A clinical cohort study investigated whether implanting acellular ECM bioscaffolds in 13 patients with volumetric muscle loss (VML) could improve strength and range of motion as a tissue engineering approach. The clinical cohort consisted of 13 adult patients (11 males, 2 females) of ages ranging from 27 to 66 years old with chronic VML injuries primarily due to traumatic extremity injuries, including motor vehicle accidents and blast-related trauma. The patients were recruited only if they were at least 6 months post-injury, had persistent functional deficits despite standard surgical repair and rehabilitation, and measurably limited strength or range of motion in the injured limb compared to the contralateral side. The participants underwent surgery to implant an acellular extracellular matrix bioscaffold, followed by an individualized rehabilitation program. 6 months following implantation, the patients showed an average strength improvement of 37.3% (p < 0.05) and a 27.1% increase in range of motion (p < 0.05) compared with pre-surgical measures, with histologic evidence of vascularized and innervated skeletal muscle formation at the injury site. This provides early clinical evidence that acellular ECM scaffolds can promote site-appropriate remodeling and meaningful functional recovery in human muscle defects⁴³. Similarly, another clinical and preclinical study investigated the use of a porcine urinary bladder-derived extracellular matrix (ECM) biologic scaffold and implanted it into volumetric muscle loss (VML) defects in both animals and five male human patients aged 27-37 years to determine whether it supports constructive tissue remodeling. The inclusion criteria were to be at least 6 months post-injury, to have at least 25% structural and functional deficit relative to the contralateral limb, and to have previously undergone standard treatments without satisfactory outcomes. All patients had lost approximately 58-90% of normal muscle volume in the affected area prior to ECM scaffold implantation, according to MRI and CT imaging assessments. The results showed that ECM implantation was linked with mobilization and accumulation of perivascular stem cells and de novo formation of skeletal muscle cells, with functional improvement observed in 3 out of 5 patients. The results show that acellular scaffolds can serve as an effective microenvironment promoting endogenous regeneration and functional recovery in severe muscle loss¹⁷. A longitudinal clinical case series investigated whether surgical implantation of an acellular extracellular matrix (ECM) bioscaffold for volumetric muscle loss (VML) leads to measurable neuromuscular and functional improvements, using nerve conduction studies (NCS) and electromyography (EMG) to measure outcomes. The cohort consisted of 8 adults that had a mean age of 32 years with chronic VML who all had to have persistent functional deficits following traumatic muscle injury, be at least 6 months post-injury, and had electrodiagnostic evidence of neuromuscular impairment at the injury site. The researchers found that 5 out of 8 participants (62.5%) showed improvements in NCS amplitude and/or EMG motor unit recruitment. One participant showed an 80% increase in tibialis anterior muscle strength and electrodiagnostic improvements were concurrent with clinically observed strength gains, while 3 participants showed no measurable electrodiagnostic change. Overall, the findings provide clinical evidence that ECM bioscaffold implantation in SMTE can support partial neuromuscular recovery and functional improvement in human VML⁴⁴.

Table 2. Summary of clinical trials on SMTE:

Citation	Patient cohort information	Method	Main outcome
(Dziki et al. 2016)	13 adult patients (11 males, 2 females) of ages ranging from 27 to 66 years old with chronic VML from traumatic extremity injuries. Required at least 6 months or over post-injury with persistent functional deficits despite standard repair and rehabilitation.	Surgical implantation of acellular extracellular matrix (ECM) bioscaffold followed by individualized rehabilitation	At 6 months post-implantation, patients showed a mean strength increase of 37.3% (p < 0.05) and a 27.1% improvement in range of motion (p < 0.05), with histological evidence of vascularized and innervated skeletal muscle formation at the injury site.
(Sicari et al. 2014)	5 male patients aged 27-37 years with chronic VML. Required at least 6 months post-injury, have 25% or more functional and structural deficit, and failed prior treatments.	Implantation of porcine urinary bladder-derived ECM scaffold into VML defects	3 out of 5 patients showed functional improvement alongside evidence of perivascular stem cell mobilization and de novo skeletal muscle formation.
(Han et al. 2016)	8 adult patients (with a mean age of 32 years) with chronic VML and electrodiagnostic evidence of neuromuscular impairment. Required at least 6 months post-traumatic injury with persistent deficits.	Surgical implantation of acellular ECM bioscaffold with outcome assessment using nerve conduction studies (NCS) and electromyography (EMG)	5 out of 8 (62.5%) patients showed improved NCS amplitude and/or EMG motor unit recruitment. One patient exhibited an 80% increase in tibialis anterior strength, while 3 patients showed no measurable electrodiagnostic improvement.

4. Challenges and Future Directions

Despite the promising outcomes reported in the pre-clinical studies reviewed in this paper, a key challenge for skeletal muscle tissue engineering (SMTE) lies in translating experimental success into consistent functional recovery. Many of the approaches examined demonstrate clear improvements in myofiber alignment, cellular organisation, and vascularisation, yet these outcomes do not consistently correspond to full restoration of muscle strength or neuromuscular integration. This highlights a central limitation within current SMTE research, which is that inferring regenerative success from histological or biochemical markers alone is insufficient. Ultimately, future applications should build on the strategies explored in this paper by placing greater emphasis on functional integration and long-term muscle performance as primary measures of success.

A further challenge is the imbalance between the data obtained from extensive pre-clinical research and limited clinical trials. Amongst the few clinical trials examined in this review providing promising evidence that acellular extracellular matrix scaffolds can support muscle regeneration and functional improvement in patients with volumetric muscle loss, the outcomes remain inconsistent across individuals. This variability reflects the complexities of human injury compared with controlled experimental models due to factors such as differences in injury severity, timing of intervention, and rehabilitation protocols. Moving forward, SMTE should focus on increasing the number of clinical trials and strengthening the clinical evidence base through standardised clinical trial designs and more consistent assessment of functional outcomes.

Finally, the research explored in this paper reiterates the difficulty of replicating the highly organised, vascularised, and functional nature of native skeletal muscle for SMTE methods.

Advanced strategies such as bioprinting, nanoengineered scaffolds, and perfusion-based bioreactors offer clear advantages in promoting tissue alignment, maturation, and cell viability. However, skeletal muscle is a highly organised, vascularised, and innervated tissue, and many current approaches often address only individual aspects of muscle regeneration rather than achieving fully integrated tissue repair. Future SMTE developments are therefore likely to depend on further integrating these strategies, combining biomaterial design, controlled mechanical environments, and biological signalling to more closely replicate native muscle structure and function.

5. Conclusion

Skeletal muscle tissue engineering is a rapidly evolving field with significant potential to address lasting clinical challenges of volumetric muscle loss. The studies reviewed show that biomaterial-based scaffolds, bioprinted constructs, and bioreactor-enhanced systems can promote myogenic differentiation, tissue organisation, and partial functional recovery. Importantly, early clinical findings further suggest that these strategies may translate into meaningful improvements for patients, although current outcomes remain variable and limited in scope. Overall, while it is indicated that SMTE has yet to achieve complete functional muscle restoration, the research discussed reflects a promising direction toward more effective regenerative therapies, stressing the importance of continued translational research and functional evaluation of tissue constructs.

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