Combined application of biosponges and an antifibrotic agent for the treatment of volumetric muscle loss.

Volumetric muscle loss (VML) causes irrecoverable loss of muscle mass and strength and results in permanent disability. VML injury shows extensive fibrosis, which impedes functional tissue regeneration. Our lab has created a biosponge scaffold composed of extracellular matrix (ECM) proteins (i.e., biosponge) that can enhance muscle regeneration and function following VML. In this work, a potent small molecule inhibitor of alpha v-subunit containing integrins known as IDL-2965 was incorporated into the biosponges for localized suppression of fibrosis post-VML. Our results demonstrate that local delivery of IDL-2965 via the biosponges attenuated the deposition of fibrotic tissue preceded by a downregulation of profibrotic genes in VML-injured muscles. The reduction in fibrotic tissue had no detrimental effects on muscle mass, function, size, or vascularity. Overall, these findings suggest that the codelivery of biosponges and IDL-2965 is a safe and effective strategy for the mitigation of fibrotic tissue deposition in VML-injured muscles.

The Role of Innate and Adaptive Immune Cells in Skeletal Muscle Regeneration.

Skeletal muscle regeneration is highly dependent on the inflammatory response. A wide variety of innate and adaptive immune cells orchestrate the complex process of muscle repair. This review provides information about the various types of immune cells and biomolecules that have been shown to mediate muscle regeneration following injury and degenerative diseases. Recently developed cell and drug-based immunomodulatory strategies are highlighted. An improved understanding of the immune response to injured and diseased skeletal muscle will be essential for the development of therapeutic strategies.

The impact of skeletal muscle contraction on CD146+Lin- pericytes.

Unaccustomed resistance exercise can initiate skeletal muscle remodeling and adaptive mechanisms that can confer protection from damage and enhanced strength with subsequent stimulation. The myofiber may provide the primary origin for adaptation, yet multiple mononuclear cell types within the surrounding connective tissue may also contribute. The purpose of this study was to evaluate the acute response of muscle-resident interstitial cells to contraction initiated by electrical stimulation (e-stim) and subsequently determine the contribution of pericytes to remodeling as a result of training. Mice were subjected to bilateral e-stim or sham treatment. Following a single session of e-stim, NG2+CD45-CD31- (NG2+Lin-) pericyte, CD146+Lin- pericyte, and PDGFRα+ fibroadipogenic progenitor cell quantity and function were evaluated via multiplex flow cytometry and targeted quantitative PCR. Relative quantity was not significantly altered 24 h postcontraction, yet unique gene signatures were observed for each cell population at 3 h postcontraction. CD146+Lin- pericytes appeared to be most responsive to contraction, and upregulation of genes related to immunomodulation and extracellular matrix remodeling was observed via RNA sequencing. Intramuscular injection of CD146+Lin- pericytes did not significantly increase myofiber size yet enhanced ECM remodeling and angiogenesis in response to repeated bouts of e-stim for 4 wk. The results from this study provide the first evidence that CD146+Lin- pericytes are responsive to skeletal muscle contraction and may contribute to the beneficial outcomes associated with exercise.

Impairment of early fracture healing by skeletal muscle trauma is restored by FK506.

BACKGROUND: Heightened local inflammation due to muscle trauma or disease is associated with impaired bone regeneration. METHODS: We hypothesized that FK506, an FDA approved immunomodulatory compound with neurotrophic and osteogenic effects, will rescue the early phase of fracture healing which is impaired by concomitant muscle trauma in male (~4 months old) Lewis rats. FK506 (1 mg/kg; i.p.) or saline was administered systemically for 14 days after an endogenously healing tibia osteotomy was created and fixed with an intermedullary pin, and the overlying tibialis anterior (TA) muscle was either left uninjured or incurred volumetric muscle loss injury (6 mm full thickness biopsy from middle third of the muscle). RESULTS: The salient observations of this study were that 1) concomitant TA muscle trauma impaired recovery of tibia mechanical properties 28 days post-injury, 2) FK506 administration rescued the recovery of tibia mechanical properties in the presence of concomitant TA muscle trauma but did not augment mechanical recovery of an isolated osteotomy (no muscle trauma), 3) T lymphocytes and macrophage presence within the traumatized musculature were heightened by trauma and attenuated by FK506 3 days post-injury, and 4) T lymphocyte but not macrophage presence within the fracture callus were attenuated by FK506 at 14 days post-injury. FK506 did not improve TA muscle isometric torque production CONCLUSION: Collectively, these findings support the administration of FK506 to ameliorate healing of fractures with severe muscle trauma comorbidity. The results suggest one potential mechanism of action is a reduction in local T lymphocytes within the injured musculoskeletal tissue, though other mechanisms to include direct osteogenic effects of FK506 require further investigation.

Activity attenuates skeletal muscle fiber damage after ischemia and reperfusion.

INTRODUCTION: In this investigation we aimed to determine whether: (1) physical activity protects rat skeletal muscle from ischemia/reperfusion (I/R) injury; and (2) continued activity after I/R improves the rate of healing. METHODS: Rats were divided into sedentary or active (voluntary wheel running) groups. Active rats ran for 4 weeks before I/R or 4 weeks before plus 4 weeks after I/R. RESULTS: Activity before I/R resulted in 73.2% less muscle damage (Evans blue dye inclusion). Sedentary and active rats had a similar decline in neural-evoked (∼ 99%) and directly stimulated (∼ 70%) in vivo muscle torque, and a similar reduction in junctophilin 1. Active rats produced 19% and 15% greater neural-evoked torque compared with sedentary rats at 14 and 28 days postinjury, respectively, although the rate of recovery appeared similar. CONCLUSIONS: Activity protects against long-term muscle damage, but not short-term neural injury or excitation-contraction uncoupling. Continued activity neither accelerates nor hinders the rate of functional recovery.

Transplantation of devitalized muscle scaffolds is insufficient for appreciable de novo muscle fiber regeneration after volumetric muscle loss injury.

Volumetric muscle loss (VML) is a traumatic and functionally debilitating muscle injury with limited treatment options. Developmental regenerative therapies for the repair of VML typically comprise an ECM scaffold. In this study, we tested if the complete reliance on host cell migration to a devitalized muscle scaffold without myogenic cells is sufficient for de novo muscle fiber regeneration. Devitalized (muscle ECM with no living cells) and, as a positive control, vital minced muscle grafts were transplanted to a VML defect in the tibialis anterior muscle of Lewis rats. Eight weeks post-injury, devitalized grafts did not appreciably promote de novo muscle fiber regeneration within the defect area, and instead remodeled into a fibrotic tissue mass. In contrast, transplantation of vital minced muscle grafts promoted de novo muscle fiber regeneration. Notably, pax7+ cells were absent in remote regions of the defect site repaired with devitalized scaffolds. At 2 weeks post-injury, the devitalized grafts were unable to promote an anti-inflammatory phenotype, while vital grafts appeared to progress to a pro-regenerative inflammatory response. The putative macrophage phenotypes observed in vivo were supported in vitro, in which soluble factors released from vital grafts promoted an M2-like macrophage polarization, whereas devitalized grafts failed to do so. These observations indicate that although the remaining muscle mass serves as a source of myogenic cells in close proximity to the defect site, a devitalized scaffold without myogenic cells is inadequate to appreciably promote de novo muscle fiber regeneration throughout the VML defect.

Effect of recombinant human MG53 protein on tourniquet-induced ischemia-reperfusion injury in rat muscle.

INTRODUCTION: Skeletal muscle ischemia-reperfusion injury (I-R) is a complex injury process that includes damage to the sarcolemmal membrane, contributing to necrosis and apoptosis. MG53, a muscle-specific TRIM family protein, has been shown to be essential for regulating membrane repair and has been shown to be protective against cardiac I-R and various forms of skeletal muscle injury. The purpose of this study was to determine if recombinant human MG53 (rhMG53) administration offered protection against I-R. METHODS: rhMG53 was administered to rats immediately before tourniquet-induced ischemia and again immediately before reperfusion. Two days later muscle damage was assessed histologically. RESULTS: rhMG53 offered no protective effect, as evidenced primarily by similar Evans blue dye inclusion in the muscles of rats administered rhMG53 or saline. CONCLUSIONS: Administration of rhMG53 does not offer protection against I-R in rat skeletal muscle. Additional studies are required to determine if the lack of a response is species-specific.

Combined regenerative rehabilitation improves recovery following volumetric muscle loss injury in a rat model.

Volumetric muscle loss (VML) injury causes irreversible deficits in muscle mass and function, often resulting in permanent disability. The current standard of care is physical therapy, but it is limited in mitigating functional deficits. We have previously optimized a rehabilitation technique using electrically stimulated eccentric contraction training (EST) that improved muscle mass, strength, and size in VML-injured rats. A biosponge scaffold composed of extracellular matrix proteins has previously enhanced muscle function postVML. This study aimed to determine whether combining a regenerative therapy (i.e., biosponge) with a novel rehabilitation technique (i.e., EST) could enhance recovery in a rat model of VML. A VML defect was created by removing ~20% of muscle mass from the tibialis anterior muscle in adult male Lewis rats. Experimental groups included VML-injured rats treated with biosponge with EST or biosponge alone (n = 6/group). EST was implemented 2 weeks postinjury at 150 Hz and was continued for 4 weeks. A linear increase in eccentric torque over 4 weeks showed the adaptability of the VML-injured muscle to EST. Combining biosponge with EST improved peak isometric torque by ~52% compared with biosponge treatment alone at 6 weeks postinjury. Application of EST increased MyoD gene expression and the percentage of large (>2000 µm2) type 2B myofibers but reduced fibrotic tissue deposition in VML-injured muscles. Together, these changes may provide the basis for improved torque production. This study demonstrates the potential for combined regenerative and rehabilitative therapy to improve muscle recovery following VML.

Extrusion-based 3D Bioprinting of Bioactive and Piezoelectric Scaffolds for Treating Critical Soft Tissue Wounds.

OBJECTIVE: This study focuses on developing bioactive piezoelectric scaffolds that could deliver bioelectrical cues to potentially treat injuries to soft tissues such as skeletal muscles and promote muscle regeneration. APPROACH: To address the underexplored aspect of bioelectrical cues in skeletal muscle tissue engineering (SMTE), we developed piezoelectric bioinks based on natural bioactive materials such as alginate, gelatin, and chitosan. Extrusion-based 3D bioprinting was utilized to develop scaffolds that mimic muscle stiffness and generate electrical stimulation when subjected to forces. The biocompatibility of these scaffolds was tested with C2C12 muscle cell line. RESULTS: The bioinks demonstrated suitable rheological properties for 3D bioprinting, resulting in high-resolution composite alginate-gelatin-chitosan scaffolds with good structural fidelity. The scaffolds exhibited a 42-60 kPa stiffness, similar to muscles. When a controlled force of 5 N was applied to the scaffolds at a constant frequency of 4 Hz, they generated electrical fields and impulses (charge), indicating their suitability as a standalone scaffold to generate electrical stimulation and instill bioelectrical cues in the wound region. The cell viability and proliferation test results confirm the scaffold's biocompatibility with C2C12s and the benefit of piezoelectricity in promoting muscle cell growth kinetics. Our study indicates that our piezoelectric bioinks and scaffolds offer promise as autonomous electrical stimulation-generating regenerative therapy for SMTE. INNOVATION: A novel approach for treating skeletal muscle wounds was introduced by developing a bioactive electroactive scaffold capable of autonomously generating electrical stimulation without stimulators and electrodes. This scaffold offers a unique approach to enhancing skeletal muscle regeneration through bioelectric cues, addressing a major gap in the SMTE, i.e., fibrotic tissue formation due to delayed muscle regeneration. CONCLUSION: A piezoelectric scaffold was developed, providing a promising solution for promoting skeletal muscle regeneration. This development can potentially address skeletal muscle injuries and offer a unique approach to facilitating skeletal muscle wound healing.

Electrically stimulated eccentric contraction training enhances muscle mass, function, and size following volumetric muscle loss.

Volumetric muscle loss (VML) overwhelms muscle's innate capacity for repair and can lead to permanent disability. The standard of care for VML injuries includes physical therapy, which can improve muscle function. The objective of this study was to develop and evaluate a rehabilitative therapy using electrically stimulated eccentric contraction training (EST) and determine the structural, biomolecular, and functional response of the VML-injured muscle. This study implemented EST using three different frequencies (50, 100, and 150 Hz) in VML-injured rats starting at 2 weeks postinjury. Four weeks of EST at 150 Hz showed a progressive increase in eccentric torque with an improvement in muscle mass (~39%), myofiber cross-sectional area, and peak isometric torque (~37.5%) relative to the untrained VML-injured sham group. EST at 150 Hz group also increased the number of large type 2B fibers (>5000 µm2 ). Elevated gene expression of markers associated with angiogenesis, myogenesis, neurogenesis, and an anti-inflammatory response was also observed. These results suggest that VML-injured muscles can respond and adapt to eccentric loading. The results of this study may aid in developing physical therapy regimens for traumatized muscles.

Biomimetic sponges improve functional muscle recovery following composite trauma.

There is a dearth of therapies that are safe and effective for the treatment of volumetric muscle loss (VML), defined as the surgical or traumatic loss of muscle tissue, resulting in functional impairment. To address this gap in orthopedic care, we developed a porous sponge-like scaffold composed of extracellular matrix (ECM) proteins (e.g., gelatin, collagen, and laminin-111) and an immunosuppressant drug, FK-506. While the majority of VML injuries occur in orthopedic trauma cases, preclinical models typically study muscle injuries in isolation without a concomitant bone fracture. The goal of this study was to investigate the extent to which FK506 loaded biomimetic sponges support functional muscle regeneration and fracture healing in a composite trauma model involving VML injury to the tibialis anterior muscle and osteotomy (OST) to the tibia. In this model, implantation of the FK-506 loaded biomimetic sponges limited the extent of inflammation while increasing the total number of myofibers, mean myofiber cross-sectional area, myosin-to-collagen ratio, and peak isometric torque compared to untreated VML+OST muscles on Day 28. Although all tibia fractures were bridged by Day 28 post-injury, fracture healing was impaired in response to an adjacent VML injury. Sponge treatment increased bone callus volume, yet the bridged mineralized bone volume was not significantly different. Taken together, these results suggest that biomimetic sponges primarily benefitted muscle repair and may provide a promising therapy for traumatized muscle.

Laminin-111-Enriched Fibrin Hydrogels Enhance Functional Muscle Regeneration Following Trauma.

Volumetric muscle loss (VML) is the surgical or traumatic loss of skeletal muscle, which can cause loss of limb function or permanent disability. VML injuries overwhelms the endogenous regenerative capacity of skeletal muscle and results in poor functional healing outcomes. Currently, there are no approved tissue engineering treatments for VML injuries. In this study, fibrin hydrogels enriched with laminin-111 (LM-111; 50-450 µg/mL) were used for the treatment of VML of the tibialis anterior in a rat model. Treatment with fibrin hydrogel containing 450 µg/mL of LM-111 (FBN450) improved muscle regeneration following VML injury. FBN450 hydrogel treatment increased the relative proportion of contractile to fibrotic tissue as indicated by the myosin: collagen ratio on day 28 post-VML injury. FBN450 hydrogels also enhanced myogenic protein expression and increased the quantity of small to medium size myofibers (500-2000 µm2) as well as innervated myofibers. Improved contractile tissue deposition due to FBN450 hydrogel treatment resulted in a significant improvement (∼60%) in torque production at day 28 postinjury. Taken together, these results suggest that the acellular FBN450 hydrogels provide a promising therapeutic strategy for VML that is worthy of further investigation. Impact statement Muscle trauma accounts for 50-70% of total military injuries and complications involving muscle result in ∼80% of delayed amputations. The lack of a clinical standard of care for volumetric muscle loss (VML) injuries presents an opportunity to develop novel regenerative therapies and improve healing outcomes. Laminin-111-enriched fibrin hydrogel may provide a promising therapy for VML that is worthy of further investigation. The acellular nature of these hydrogels will allow for easy off the shelf access to critically injured patients and fewer regulatory hurdles during commercialization.

Co-delivery of fibrin-laminin hydrogel with mesenchymal stem cell spheroids supports skeletal muscle regeneration following trauma.

Volumetric muscle loss (VML) is traumatic or surgical loss of skeletal muscle with resultant functional impairment. Skeletal muscle's innate capacity for regeneration is lost with VML due to a critical loss of stem cells, extracellular matrix, and neuromuscular junctions. Consequences of VML include permanent disability or delayed amputations of the affected limb. Currently, a successful clinical therapy has not been identified. Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and their three-dimensional aggregation can further enhance therapeutic efficacy. In this study, MSC aggregation into spheroids was optimized in vitro based on cellular viability, spheroid size, and trophic factor secretion. The regenerative potential of the optimized MSC spheroid therapy was then investigated in a murine model of VML injury. Experimental groups included an untreated VML injury control, intramuscular injection of MSC spheroids, and MSC spheroids encapsulated in a fibrin-laminin hydrogel. Compared to the untreated VML group, the spheroid encapsulating hydrogel group enhanced myogenic marker (i.e., MyoD and myogenin) protein expression, improved muscle mass, increased presence of centrally nucleated myofibers as well as small fibers (<500 µm2 ), modulated pro- and anti-inflammatory macrophage marker expression (i.e., iNOS and Arginase), and increased the presence of CD146+ pericytes and CD31+ endothelial cells in the VML injured muscles. Future studies will evaluate the extent of functional recovery with the spheroid encapsulating hydrogel therapy.

Biomimetic sponges improve muscle structure and function following volumetric muscle loss.

Skeletal muscle is inept in regenerating after traumatic injuries such as volumetric muscle loss (VML) due to significant loss of various cellular and acellular components. Currently, there are no approved therapies for the treatment of muscle tissue following trauma. In this study, biomimetic sponges composed of gelatin, collagen, laminin-111, and FK-506 were used for the treatment of VML in a rodent model. We observed that biomimetic sponge treatment improved muscle structure and function while modulating inflammation and limiting the extent of fibrotic tissue deposition. Specifically, sponge treatment increased the total number of myofibers, type 2B fiber cross-sectional area, myosin: collagen ratio, myofibers with central nuclei, and peak isometric torque compared to untreated VML injured muscles. As an acellular scaffold, biomimetic sponges may provide a promising clinical therapy for VML.

Laminin-111 Improves the Anabolic Response to Mechanical Load in Aged Skeletal Muscle.

Anabolic resistance to a mechanical stimulus may contribute to the loss of skeletal muscle mass observed with age. In this study, young and aged mice were injected with saline or human LM-111 (1 mg/kg). One week later, the myotendinous junction of the gastrocnemius muscle was removed via myotenectomy (MTE), thus placing a chronic mechanical stimulus on the remaining plantaris muscle for 2 weeks. LM-111 increased α7B integrin protein expression and clustering of the α7B integrin near DAPI+ nuclei in aged muscle in response to MTE. LM-111 reduced CD11b+ immune cells, enhanced repair, and improved the growth response to loading in aged plantaris muscle. These results suggest that LM-111 may represent a novel therapeutic approach to prevent and/or treat sarcopenia.

Bio-conjugation of platelet-rich plasma and alginate through carbodiimide chemistry for injectable hydrogel therapies.

Alginate is a highly tailorable, biocompatible polymer whose properties can be tuned to mimic the properties of native nucleus pulposus (NP) tissue. Platelet-rich plasma (PRP) is a highly accessible, inexpensive, and readily available mix of pro-regenerative factors. By functionalizing alginate with PRP, a mechanically optimized, bioactive alginate NP analogue may stimulate NP cells to proliferate and accumulate matrix over a longer period of time than if the PRP were solely encapsulated within the hydrogel. In this study, PRP was chemically bound to alginate using carbodiimide chemistry and mechanically, physically, and cytologically compared to plain alginate as well as alginate containing free-floating lyophilized PRP. The alginates were mechanically and physically characterized; PRP-conjugated alginate had similar mechanical properties to controls and had the benefit of retained PRP proteins within the hydrogel. Human nucleus pulposus cells (hNPCs) were seeded within the modified alginates and cultured for 14 days. Quantification data of glycosaminoglycans suggests that PRP-incorporated alginate has the potential to increase ECM production within the characterized alginate constructs, and that PRP-functionalized alginate can retain protein within the hydrogel over time. This is the first study to functionalize the milieu of PRP proteins onto alginate and characterize the mechanical and physical properties of the modified alginates. This study also incorporates hNPCs into the characterized PRP-modified alginates to observe phenotypic maintenance when encapsulated within the in situ gelling constructs.

Aligned nanofibers of decellularized muscle extracellular matrix for volumetric muscle loss.

Volumetric muscle loss (VML) is a traumatic loss of muscle tissue that results in chronic functional impairment. When injured, skeletal muscle is capable of small-scale repair; however, regenerative capacities are lost with VML due to a critical loss stem cells and extracellular matrix (ECM). Consequences of VML include either long-term disability or delayed amputations of the affected limb. While the prevalence of VML is substantial, currently a successful clinical therapy has not been identified. In a previous study, an electrospun composed of polycaprolactone (PCL) and decellularized-ECM (D-ECM) supported satellite cell-mediated myogenic activity in vitro. In this study, we investigate the extent to which this electrospun scaffold can support functional muscle regeneration in a murine model of VML. Experimental groups included no treatment, pure PCL treated, and PCL:D-ECM (50:50 blend) treated VML defects. The PCL:D-ECM scaffold treated VML muscles supported increased activity of anti-inflammatory M2 macrophages (arginase+ ) at Day 28, compared to other experimental groups. Increased myofiber (MHC+ ) regeneration was observed histologically at both Days 7 and 28 post-trauma in blend scaffold treated group compared to PCL treated and untreated groups. However, improvements in muscle weights and force production were not observed. Future studies would evaluate muscle function at longer time-points post-VML injury to allow sufficient time for reinnervation of regenerated muscle fibers.

Decellularized extracellular matrix gelloids support mesenchymal stem cell growth and function in vitro.

Volumetric muscle loss (VML) injuries are irrecoverable due to a significant loss of regenerative elements, persistent inflammation, extensive fibrosis, and functional impairment. When used in isolation, previous stem cell and biomaterial-based therapies have failed to regenerate skeletal muscle at clinically relevant levels. The extracellular matrix (ECM) microenvironment is crucial for the viability, stemness, and differentiation of stem cells. Decellularized-ECM (D-ECM) scaffolds are at the forefront of ongoing research to develop a viable therapy for VML. Due to the retention of key ECM components, D-ECM scaffolds provide an excellent substrate for the adhesion and migration of several cell types. Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and are currently under investigation in clinical trials for a wide range of medical conditions. However, a major limitation to the use of MSCs in clinical applications is their poor viability at the site of transplantation. In this study, we have fabricated spherical scaffolds composed of gelatin and skeletal muscle D-ECM for the adhesion and delivery of MSCs to the site of VML injury. These spherical scaffolds termed "gelloids" supported MSC survival, expansion, trophic factor secretion, immunomodulation, and myogenic protein expression in vitro. Future studies would determine the therapeutic efficacy of this approach in a murine model of VML injury.

Biomaterial and stem cell-based strategies for skeletal muscle regeneration.

Adult skeletal muscle can regenerate effectively after mild physical or chemical insult. Muscle trauma or disease can overwhelm this innate capacity for regeneration and result in heightened inflammation and fibrotic tissue deposition resulting in loss of structure and function. Recent studies have focused on biomaterial and stem cell-based therapies to promote skeletal muscle regeneration following injury and disease. Many stem cell populations besides satellite cells are implicated in muscle regeneration. These stem cells include but are not limited to mesenchymal stem cells, adipose-derived stem cells, hematopoietic stem cells, pericytes, fibroadipogenic progenitors, side population cells, and CD133+ stem cells. However, several challenges associated with their isolation, availability, delivery, survival, engraftment, and differentiation have been reported in recent studies. While acellular scaffolds offer a relatively safe and potentially off-the-shelf solution to cell-based therapies, they are often unable to stimulate host cell migration and activity to a level that would result in clinically meaningful regeneration of traumatized muscle. Combining stem cells and biomaterials may offer a viable therapeutic strategy that may overcome the limitations associated with these therapies when they are used in isolation. In this article, we review the stem cell populations that can stimulate muscle regeneration in vitro and in vivo. We also discuss the regenerative potential of combination therapies that utilize both stem cell and biomaterials for the treatment of skeletal muscle injury and disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1246-1262, 2019.

Regulation of Myogenic Activity by Substrate and Electrical Stimulation In Vitro.

Skeletal muscle has a remarkable regenerative capacity in response to mild injury. However, when muscle is severely injured, muscle regeneration is impaired due to the loss of muscle-resident stem cells, known as satellite cells. Fibrotic tissue, primarily comprising collagen I (COL), is deposited with this critical loss of muscle. In recent studies, supplementation of laminin (LM)-111 has been shown to improve skeletal muscle regeneration in several models of disease and injury. Additionally, electrical stimulation (E-stim) has been investigated as a possible rehabilitation therapy to improve muscle's functional recovery. This study investigated the role of E-stim and substrate in regulating myogenic response. C2C12 myoblasts were allowed to differentiate into myotubes on COL- and LM-coated polydimethylsiloxane molds. The myotubes were subjected to E-stim and compared with nonstimulated controls. While E-stim resulted in increased myogenic activity, irrespective of substrate, LM supported increased proliferation and uniform distribution of C2C12 myoblasts. In addition, C2C12 myoblasts cultured on LM showed higher Sirtuin 1, mammalian target of rapamycin, desmin, nitric oxide, and vascular endothelial growth factor expression. Taken together, these results suggest that an LM substrate is more conducive to myoblast growth and differentiation in response to E-stim in vitro.