Fatiguing exercise reduces cellular passive Young's modulus in human vastus lateralis muscle.

Previous studies demonstrated that acute fatiguing exercise transiently reduces whole-muscle stiffness, which might contribute to increased risk of injury and impaired contractile performance. We sought to elucidate potential intracellular mechanisms underlying these reductions. To that end, the cellular passive Young's modulus was measured in muscle fibres from healthy, young males and females. Eight volunteers (four male and four female) completed unilateral, repeated maximal voluntary knee extensions until task failure, immediately followed by bilateral percutaneous needle muscle biopsy of the post-fatigued followed by the non-fatigued control vastus lateralis. Muscle samples were processed for mechanical assessment and separately for imaging and phosphoproteomics. Fibres were passively (pCa 8.0) stretched incrementally to 156% of initial sarcomere length to assess Young's modulus, calculated as the slope of the resulting stress-strain curve at short (sarcomere length = 2.4-3.0 µm) and long (sarcomere length = 3.2-3.8 µm) lengths. Titin phosphorylation was assessed by liquid chromatography followed by high-resolution mass spectrometry. The passive modulus was significantly reduced in post-fatigued versus control fibres from male, but not female, participants. Post-fatigued samples showed altered phosphorylation of five serine residues (four located within the elastic region of titin) but did not exhibit altered active tension or sarcomere ultrastructure. Collectively, these results suggest that acute fatigue is sufficient to alter phosphorylation of skeletal titin in multiple locations. We also found reductions in the passive modulus, consistent with prior reports in the literature investigating striated muscle stiffness. These results provide mechanistic insight contributing to the understanding of dynamic regulation of whole-muscle tissue mechanics in vivo. HIGHLIGHTS: What is the central question of this study? Previous studies have shown that skeletal muscle stiffness is reduced following a single bout of fatiguing exercise in whole muscle, but it is not known whether these changes manifest at the cellular level, and their potential mechanisms remain unexplored. What is the main finding and its importance? Fatiguing exercise reduces cellular stiffness in skeletal muscle from males but not females, suggesting that fatigue alters tissue compliance in a sex-dependent manner. The phosphorylation status of titin, a potential mediator of skeletal muscle cellular stiffness, is modified by fatiguing exercise. Previous studies have shown that passive skeletal muscle stiffness is reduced following a single bout of fatiguing exercise. Lower muscle passive stiffness following fatiguing exercise might increase risk for soft-tissue injury; however, the underlying mechanisms of this change are unclear. Our findings show that fatiguing exercise reduces the passive Young's modulus in skeletal muscle cells from males but not females, suggesting that intracellular proteins contribute to reduced muscle stiffness following repeated loading to task failure in a sex-dependent manner. The phosphorylation status of the intracellular protein titin is modified by fatiguing exercise in a way that might contribute to altered muscle stiffness after fatiguing exercise. These results provide important mechanistic insight that might help to explain why biological sex impacts the risk for soft-tissue injury with repeated or high-intensity mechanical loading in athletes and the risk of falls in older adults.

Production of Nanofibrillar Patterned Collagen for Tissue Engineering.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

Engineered nanofibrillar collagen with tunable biophysical properties for myogenic, endothelial, and osteogenic cell guidance.

A goal of regenerative engineering is the rational design of materials to restore the structure-function relationships that drive reparative programs in damaged tissues. Despite the widespread use of extracellular matrices for engineering tissues, their application has been limited by a narrow range of tunable features. The primary objective of this study is to develop a versatile platform for evaluating tissue-specific cellular interactions using Type I collagen scaffolds with highly tunable biophysical properties. The kinetics of collagen fibrillogenesis were modulated through a combination of varied shear rate and pH during neutralization, to achieve a broad range of fibril anisotropy, porosity, diameter, and storage modulus. The role that each of these properties play in guiding muscle, bone, and vascular cell types was comprehensively identified, and informed the in vitro generation of three distinct musculoskeletal engineered constructs. Myogenesis was highly regulated by smaller fibrils and larger storage moduli, endothelial inflammatory phenotype was predominantly guided by fibril anisotropy, and osteogenesis was enhanced by highly porous collagen with larger fibrils. This study introduces a novel approach for dynamically modulating Type I collagen materials and provides a robust platform for investigating cell-material interactions, offering insights for the future rational design of tissue-specific regenerative biomaterials. STATEMENT OF SIGNIFICANCE: The biophysical properties of regenerative materials facilitate key cell-substrate interactions that can guide the morphology, phenotype, and biological response of cells. In this study, we describe the fabrication of an engineered collagen hydrogel that can be modified to exhibit control over a wide range of biophysical features, including fibril organization and size, nanoscale porosity, and mechanics. We identified the unique combination of collagen features that optimally promote regenerative muscle, bone, and vascular cell types while also delineating the properties that hinder these same cellular responses. This study presents a highly accessible method to control the biophysical properties of collagen hydrogels that can be adapted for a broad range of tissue engineering and regenerative applications.

Age-associated functional healing of musculoskeletal trauma through regenerative engineering and rehabilitation.

Traumatic musculoskeletal injuries that lead to volumetric muscle loss (VML) are challenged by irreparable soft tissue damage, impaired regenerative ability, and reduced muscle function. Regenerative rehabilitation strategies involving the pairing of engineered therapeutics with exercise have guided considerable advances in the functional repair of skeletal muscle following VML. However, few studies evaluate the efficacy of regenerative rehabilitation across the lifespan. In the current study, young and aged mice are treated with an engineered muscle, consisting of nanofibrillar-aligned collagen laden with myogenic cells, in combination with voluntary running activity following a VML injury. Overall, young mice perform at higher running volumes and intensities compared to aged mice but exhibit similar volumes relative to age-matched baselines. Additionally, young mice are highly responsive to the dual treatment showing enhanced force production (p < 0.001), muscle mass (p < 0.05), and vascular density (p < 0.01) compared to age-matched controls. Aged mice display upregulation of circulating inflammatory cytokines and show no significant regenerative response to treatment, suggesting a diminished efficacy of regenerative rehabilitation in aged populations. These findings highlight the restorative potential of regenerative engineering and rehabilitation for the treatment of traumatic musculoskeletal injuries in young populations and the complimentary need for age-specific interventions and studies to serve broader patient demographics.

Decellularization Strategies for Regenerating Cardiac and Skeletal Muscle Tissues.

Cardiovascular disease is the leading cause of death worldwide and is associated with approximately 17.9 million deaths each year. Musculoskeletal conditions affect more than 1.71 billion people globally and are the leading cause of disability. These two areas represent a massive global health burden that is perpetuated by a lack of functionally restorative treatment options. The fields of regenerative medicine and tissue engineering offer great promise for the development of therapies to repair damaged or diseased tissues. Decellularized tissues and extracellular matrices are cornerstones of regenerative biomaterials and have been used clinically for decades and many have received FDA approval. In this review, we first discuss and compare methods used to produce decellularized tissues and ECMs from cardiac and skeletal muscle. We take a focused look at how different biophysical properties such as spatial topography, extracellular matrix composition, and mechanical characteristics influence cell behavior and function in the context of regenerative medicine. Lastly, we describe emerging research and forecast the future high impact applications of decellularized cardiac and skeletal muscle that will drive novel and effective regenerative therapies.