Morphologic and histologic characterization of sheep and porcine TMJ as large animal models for tissue engineering applications.

OBJECTIVE: The aim of this study was to compare and characterize the structural and ultrastructural organization of the temporomandibular joint (TMJ) between two large animal models for use in the development of tissue engineering strategies. MATERIALS AND METHODS: Whole TMJs from sheep and pigs were evaluated with micro-computed tomography (µCT) for morphology and quantitative analyses of bone parameters. Histological examination was performed on the TMJ disc and its attachments to investigate regional distribution of collagen, elastin, and glycosaminoglycans (GAGs). RESULTS: µCT analyses demonstrate higher bone mineral density (BMD) in the temporal fossa compared to the mandibular condyle in both species, with this variable being significantly higher in sheep than pig. Quantitative morphometry of the trabecular condyle reveals no statistical differences between the species. Histology demonstrates similar structural organization of collagen and elastin between species. Elastin staining was nearly twofold greater in sheep than in the pig disc. Finally, Safranin-O staining for GAGs in the TMJ disc was localized to the intermediate zone in the sheep but was absent from the porcine disc. CONCLUSIONS: Our findings show some important differences in the pig and sheep TMJ µCT variables and histology and composition of the disc and discal attachment. These disparities likely reflect differences in masticatory and TMJ functional loading patterns between the two species and provide insights into large animal models towards human applications. CLINICAL RELEVANCE: As with the established pig model, the sheep is a suitable large animal model for TMJ research such as regenerative strategies, with specific considerations for design parameters appropriate for human-analog applications.

Starring or Supporting Role? Satellite Cells and Skeletal Muscle Fiber Size Regulation.

Recent loss-of-function studies show that satellite cell depletion does not promote sarcopenia or unloading-induced atrophy, and does not prevent regrowth. Although overload-induced muscle fiber hypertrophy is normally associated with satellite cell-mediated myonuclear accretion, hypertrophic adaptation proceeds in the absence of satellite cells in fully grown adult mice, but not in young growing mice. Emerging evidence also indicates that satellite cells play an important role in remodeling the extracellular matrix during hypertrophy.

Aged Muscle Demonstrates Fiber-Type Adaptations in Response to Mechanical Overload, in the Absence of Myofiber Hypertrophy, Independent of Satellite Cell Abundance.

Although sarcopenia, age-associated loss of muscle mass and strength, is neither accelerated nor exacerbated by depletion of muscle stem cells, satellite cells, we hypothesized that adaptation in sarcopenic muscle would be compromised. To test this hypothesis, we depleted satellite cells with tamoxifen treatment of Pax7(CreER)-DTA mice at 4 months of age, and 20 months later subjected the plantaris muscle to 2 weeks of mechanical overload. We found myofiber hypertrophy was impaired in aged mice regardless of satellite cell content. Even in the absence of growth, vehicle-treated mice mounted a regenerative response, not apparent in tamoxifen-treated mice. Further, myonuclear accretion occurred in the absence of growth, which was prevented by satellite cell depletion, demonstrating that myonuclear addition is insufficient to drive myofiber hypertrophy. Satellite cell depletion increased extracellular matrix content of aged muscle that was exacerbated by overload, potentially limiting myofiber growth. These results support the idea that satellite cells regulate the muscle environment, and that their loss during aging may contribute to fibrosis, particularly during periods of remodeling. Overload induced a fiber-type composition improvement, independent of satellite cells, suggesting that aged muscle is very responsive to exercise-induced enhancement in oxidative capacity, even with an impaired hypertrophic response.

Insulin-resistant subjects have normal angiogenic response to aerobic exercise training in skeletal muscle, but not in adipose tissue.

Reduced vessel density in adipose tissue and skeletal muscle is associated with obesity and may result in decreased perfusion, decreased oxygen consumption, and insulin resistance. In the presence of VEGFA, Angiopoietin-2 (Angpt2) and Angiopoietin-1 (Angpt1) are central determinants of angiogenesis, with greater Angpt2:Angpt1 ratios promoting angiogenesis. In skeletal muscle, exercise training stimulates angiogenesis and modulates transcription of VEGFA, Angpt1, and Angpt2. However, it remains unknown whether exercise training stimulates vessel growth in human adipose tissue, and it remains unknown whether adipose angiogenesis is mediated by angiopoietin signaling. We sought to determine whether insulin-resistant subjects would display an impaired angiogenic response to aerobic exercise training. Insulin-sensitive (IS, N = 12) and insulin-resistant (IR, N = 14) subjects had subcutaneous adipose and muscle (vastus lateralis) biopsies before and after 12 weeks of cycle ergometer training. In both tissues, we measured vessels and expression of pro-angiogenic genes. Exercise training did not increase insulin sensitivity in IR Subjects. In skeletal muscle, training resulted in increased vessels/muscle fiber and increased Angpt2:Angpt1 ratio in both IR and IS subjects. However, in adipose, exercise training only induced angiogenesis in IS subjects, likely due to chronic suppression of VEGFA expression in IR subjects. These results indicate that skeletal muscle of IR subjects exhibits a normal angiogenic response to exercise training. However, the same training regimen is insufficient to induce angiogenesis in adipose tissue of IR subjects, which may help to explain why we did not observe improved insulin sensitivity following aerobic training.

Blunted hypertrophic response in aged skeletal muscle is associated with decreased ribosome biogenesis.

The ability of skeletal muscle to hypertrophy in response to a growth stimulus is known to be compromised in older individuals. We hypothesized that a change in the expression of protein-encoding genes in response to a hypertrophic stimulus contributes to the blunted hypertrophy observed with aging. To test this hypothesis, we determined gene expression by microarray analysis of plantaris muscle from 5- and 25-mo-old mice subjected to 1, 3, 5, 7, 10, and 14 days of synergist ablation to induce hypertrophy. Overall, 1,607 genes were identified as being differentially expressed across the time course between young and old groups; however, the difference in gene expression was modest, with cluster analysis showing a similar pattern of expression between the two groups. Despite ribosome protein gene expression being higher in the aged group, ribosome biogenesis was significantly blunted in the skeletal muscle of aged mice compared with mice young in response to the hypertrophic stimulus (50% vs. 2.5-fold, respectively). The failure to upregulate pre-47S ribosomal RNA (rRNA) expression in muscle undergoing hypertrophy of old mice indicated that rDNA transcription by RNA polymerase I was impaired. Contrary to our hypothesis, the findings of the study suggest that impaired ribosome biogenesis was a primary factor underlying the blunted hypertrophic response observed in skeletal muscle of old mice rather than dramatic differences in the expression of protein-encoding genes. The diminished increase in total RNA, pre-47S rRNA, and 28S rRNA expression in aged muscle suggest that the primary dysfunction in ribosome biogenesis occurs at the level of rRNA transcription and processing.

Inducible depletion of satellite cells in adult, sedentary mice impairs muscle regenerative capacity without affecting sarcopenia.

A key determinant of geriatric frailty is sarcopenia, the age-associated loss of skeletal muscle mass and strength. Although the etiology of sarcopenia is unknown, the correlation during aging between the loss of activity of satellite cells, which are endogenous muscle stem cells, and impaired muscle regenerative capacity has led to the hypothesis that the loss of satellite cell activity is also a cause of sarcopenia. We tested this hypothesis in male sedentary mice by experimentally depleting satellite cells in young adult animals to a degree sufficient to impair regeneration throughout the rest of their lives. A detailed analysis of multiple muscles harvested at various time points during aging in different cohorts of these mice showed that the muscles were of normal size, despite low regenerative capacity, but did have increased fibrosis. These results suggest that lifelong reduction of satellite cells neither accelerated nor exacerbated sarcopenia and that satellite cells did not contribute to the maintenance of muscle size or fiber type composition during aging, but that their loss may contribute to age-related muscle fibrosis.

Engineered skeletal muscle units for repair of volumetric muscle loss in the tibialis anterior muscle of a rat.

Volumetric muscle loss (VML) is the traumatic, degenerative, or surgical loss of muscle tissue, which may result in function loss and physical deformity. To date, clinical treatments for VML--the reflected muscle flap or transferred muscle graft--are limited by tissue availability and donor site morbidity. To address the need for more innovative skeletal muscle repair options, our laboratory has developed scaffoldless tissue-engineered skeletal muscle units (SMUs), multiphasic tissue constructs composed of engineered skeletal muscle with engineered bone-tendon ends, myotendinous junctions, and entheses, which in vitro can produce force both spontaneously and in response to electrical stimulation. Though phenotypically immature in vitro, we have shown that following 1 week of implantation in an ectopic site, our muscle constructs develop vascularization and innervation, an epimysium-like outer layer of connective tissue, an increase in myosin protein content, formation of myofibers, and increased force production. These findings suggest that our engineered muscle tissue survives implantation and develops the interfaces necessary to advance the phenotype toward adult muscle. The purpose of this study was to evaluate the potential of our SMUs to restore muscle tissue to sites of acute VML. Our results indicate that our SMUs continue to mature in vivo with longer recovery times and have the potential to repair VML sites by providing additional muscle fibers to damaged muscles. We conclude from this study that our SMUs have the potential to restore lost tissue volume in cases of acute VML.

Automatic Myonuclear Detection in Isolated Single Muscle Fibers Using Robust Ellipse Fitting and Sparse Representation.

Accurate and robust detection of myonuclei in isolated single muscle fibers is required to calculate myonuclear domain size. However, this task is challenging because: 1) shape and size variations of the nuclei, 2) overlapping nuclear clumps, and 3) multiple z-stack images with out-of-focus regions. In this paper, we have proposed a novel automatic detection algorithm to robustly quantify myonuclei in isolated single skeletal muscle fibers. The original z-stack images are first converted into one all-in-focus image using multi-focus image fusion. A sufficient number of ellipse fitting hypotheses are then generated from the myonuclei contour segments using heteroscedastic errors-in-variables (HEIV) regression. A set of representative training samples and a set of discriminative features are selected by a two-stage sparse model. The selected samples with representative features are utilized to train a classifier to select the best candidates. A modified inner geodesic distance based mean-shift clustering algorithm is used to produce the final nuclei detection results. The proposed method was extensively tested using 42 sets of z-stack images containing over 1,500 myonuclei. The method demonstrates excellent results that are better than current state-of-the-art approaches.

Isolation and Purification of Satellite Cells for Skeletal Muscle Tissue Engineering.

Engineered skeletal muscle holds promise as a source of graft tissue for the repair of traumatic injuries such as volumetric muscle loss. The resident skeletal muscle stem cell, the satellite cell, has been identified as an ideal progenitor for tissue engineering due to its role as an essential player in the potent skeletal muscle regeneration mechanism. A significant challenge facing tissue engineers, however, is the isolation of sufficiently large satellite cell populations with high purity. The two common isolation techniques, single fiber explant culture and enzymatic dissociation, can yield either a highly pure satellite cell population or a suitably large number or cells but fail to do both simultaneously. As a result, it is often necessary to use a purification technique such as pre-plating or cell sorting to enrich the satellite cell population post-isolation. Furthermore, the absence of complex chemical and biophysical cues influencing the in vivo satellite cell "niche" complicates the culture of isolated satellite cells. Techniques under investigation to maximize myogenic proliferation and differentiation in vitro are described in this article, along with current methods for isolating and purifying satellite cells.

Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy.

Our aim in the current study was to determine the necessity of satellite cells for long-term muscle growth and maintenance. We utilized a transgenic Pax7-DTA mouse model, allowing for the conditional depletion of > 90% of satellite cells with tamoxifen treatment. Synergist ablation surgery, where removal of synergist muscles places functional overload on the plantaris, was used to stimulate robust hypertrophy. Following 8 wk of overload, satellite cell-depleted muscle demonstrated an accumulation of extracellular matrix (ECM) and fibroblast expansion that resulted in reduced specific force of the plantaris. Although the early growth response was normal, an attenuation of hypertrophy measured by both muscle wet weight and fiber cross-sectional area occurred in satellite cell-depleted muscle. Isolated primary myogenic progenitor cells (MPCs) negatively regulated fibroblast ECM mRNA expression in vitro, suggesting a novel role for activated satellite cells/MPCs in muscle adaptation. These results provide evidence that satellite cells regulate the muscle environment during growth.

Engineering muscle constructs for the creation of functional engineered musculoskeletal tissue.

Volumetric muscle loss (VML) is a disabling condition in which current clinical procedures are suboptimal. The field of tissue engineering has many promising strategies for the creation of functional skeletal muscle in vitro. However, there are still two key limitations that prevent it from becoming a solution for treating VML. First, engineered muscle tissue must be biocompatible to facilitate muscle tissue regrowth without generating an immune response. Second, engineered muscle constructs must be scaled up to facilitate replacement of clinically relevant volumes of tissue (centimeters in diameter). There are currently no tissue engineering strategies to produce tissue constructs that are both biocompatible and large enough to facilitate clinical repair. However, recent advances in tissue engineering using synthetic scaffolds, native scaffolds, or scaffold-free approaches may lead to a solution for repair of VML injuries.

Automated fiber-type-specific cross-sectional area assessment and myonuclei counting in skeletal muscle.

Skeletal muscle is an exceptionally adaptive tissue that compromises 40% of mammalian body mass. Skeletal muscle functions in locomotion, but also plays important roles in thermogenesis and metabolic homeostasis. Thus characterizing the structural and functional properties of skeletal muscle is important in many facets of biomedical research, ranging from myopathies to rehabilitation sciences to exercise interventions aimed at improving quality of life in the face of chronic disease and aging. In this paper, we focus on automated quantification of three important morphological features of muscle: 1) muscle fiber-type composition; 2) muscle fiber-type-specific cross-sectional area, and 3) myonuclear content and location. We experimentally prove that the proposed automated image analysis approaches for fiber-type-specific assessments and automated myonuclei counting are fast, accurate, and reliable.

Muscle-specific substrate use during cycle exercise at 1 G: implications for astronaut muscle health.

INTRODUCTION: Studies of real and simulated microgravity exposure show the lower limb muscles atrophy to the greatest extent, with the calf muscles being most affected and most difficult to target with exercise countermeasures. This ground-based study examined the metabolic involvement of the thigh and calf muscles during two cycle exercise protocols (moderate and high intensity) central to the exercise countermeasures program on the International Space Station. METHODS: Intramuscular glycogen and triglyceride levels were quantified in the vastus lateralis and soleus muscles before and after a moderate (current ISS prescription: 45 min at 55% VO(2max), 131 +/- 12 W) and high (proposed ISS prescription: 8 x 30-s intervals at 150% VO(2max), 459 +/- 34 W) intensity cycle exercise bout in nine individuals. RESULTS: During moderate intensity cycling, glycogen was significantly reduced in the vastus lateralis (114 +/- 27 mmol x kg(-1) dry weight) and remained unchanged in the soleus. High intensity cycling significantly reduced glycogen in both muscles, but the vastus lateralis (151 +/- 25 mmol x kg(-1) dry weight) used significantly more (-160%) than the soleus (59 +/- 11 mmol x kg(-1) dry weight). Intramuscular triglycerides were unchanged in both muscles at both intensities. DISCUSSION: These findings, coupled with other ground-based studies, provide strong support for high intensity cycling being a more appropriate component of the ISS prescription for upper and lower leg skeletal muscle health and cardiorespiratory fitness, although additional exercise paradigms that target the calf are warranted. These muscle-specific findings should be considered when designing exercise strategies for combating conditions of sarcopenia and muscle wasting on Earth.

Time course of gene expression during mouse skeletal muscle hypertrophy.

The purpose of this study was to perform a comprehensive transcriptome analysis during skeletal muscle hypertrophy to identify signaling pathways that are operative throughout the hypertrophic response. Global gene expression patterns were determined from microarray results on days 1, 3, 5, 7, 10, and 14 during plantaris muscle hypertrophy induced by synergist ablation in adult mice. Principal component analysis and the number of differentially expressed genes (cutoffs ≥2-fold increase or ≥50% decrease compared with control muscle) revealed three gene expression patterns during overload-induced hypertrophy: early (1 day), intermediate (3, 5, and 7 days), and late (10 and 14 days) patterns. Based on the robust changes in total RNA content and in the number of differentially expressed genes, we focused our attention on the intermediate gene expression pattern. Ingenuity Pathway Analysis revealed a downregulation of genes encoding components of the branched-chain amino acid degradation pathway during hypertrophy. Among these genes, five were predicted by Ingenuity Pathway Analysis or previously shown to be regulated by the transcription factor Kruppel-like factor-15, which was also downregulated during hypertrophy. Moreover, the integrin-linked kinase signaling pathway was activated during hypertrophy, and the downregulation of muscle-specific micro-RNA-1 correlated with the upregulation of five predicted targets associated with the integrin-linked kinase pathway. In conclusion, we identified two novel pathways that may be involved in muscle hypertrophy, as well as two upstream regulators (Kruppel-like factor-15 and micro-RNA-1) that provide targets for future studies investigating the importance of these pathways in muscle hypertrophy.

Automated image analysis of skeletal muscle fiber cross-sectional area.

Morphological characteristics of muscle fibers, such as fiber size, are critical factors that determine the health and function of the muscle. However, at this time, quantification of muscle fiber cross-sectional area is still a manual or, at best, a semiautomated process. This process is labor intensive, time consuming, and prone to errors, leading to high interobserver variability. We have developed and validated an automatic image segmentation algorithm and compared it directly with commercially available semiautomatic software currently considered state of the art. The proposed automatic segmentation algorithm was evaluated against a semiautomatic method with manual annotation using 35 randomly selected cross-sectional muscle histochemical images. The proposed algorithm begins with ridge detection to enhance the muscle fiber boundaries, followed by robust seed detection based on concave area identification to find initial seeds for muscle fibers. The final muscle fiber boundaries are automatically delineated using a gradient vector flow deformable model. Our automatic approach is accurate and represents a significant advancement in efficiency; quantification of fiber area in muscle cross sections was reduced from 25-40 min/image to 15 s/image, while accommodating common quantification obstacles including morphological variation (e.g., heterogeneity in fiber size and fibrosis) and technical artifacts (e.g., processing defects and poor staining quality). Automatic quantification of muscle fiber cross-sectional area using the proposed method is a powerful tool that will increase sensitivity, objectivity, and efficiency in measuring muscle adaptation.

Association of fibromyalgia with altered skeletal muscle characteristics which may contribute to postexertional fatigue in postmenopausal women.

OBJECTIVE: To identify muscle physiologic properties that may contribute to postexertional fatigue and malaise in women with fibromyalgia (FM). METHODS: Healthy postmenopausal women with (n = 11) and without (n = 11) FM, ages 51-70 years, participated in this study. Physical characteristics and responses to self-reported questionnaires were evaluated. Strength loss and tissue oxygenation in response to a fatiguing exercise protocol were used to quantify fatigability and the local muscle hemodynamic profile. Muscle biopsies were performed to assess between-group differences in baseline muscle properties using histochemical, immunohistochemical, and electron microscopic analyses. RESULTS: There was no significant difference between healthy controls and FM patients in muscle fatigue in response to exercise. However, self-reported fatigue and pain were correlated with prolonged loss of strength following 12 minutes of recovery in patients with FM. Although there was no difference in percent succinate dehydrogenase (SDH)-positive (type I) and SDH-negative (type II) fibers or in mean fiber cross-sectional area between groups, FM patients exhibited greater variability in fiber size and altered fiber size distribution. In healthy controls only, fatigue resistance was strongly correlated with the size of SDH-positive fibers and hemoglobin oxygenation. In contrast, FM patients with the highest percentage of SDH-positive fibers recovered strength most effectively, and this was correlated with capillary density. However, overall, capillary density was lower in the FM group. CONCLUSION: Peripheral mechanisms, i.e., altered muscle fiber size distribution and decreased capillary density, may contribute to postexertional fatigue in FM. Understanding of these defects in fibromyalgic muscle may provide valuable insight with regard to treatment.

Satellite cell depletion does not inhibit adult skeletal muscle regrowth following unloading-induced atrophy.

Resident muscle stem cells, known as satellite cells, are thought to be the main mediators of skeletal muscle plasticity. Satellite cells are activated, replicate, and fuse into existing muscle fibers in response to both muscle injury and mechanical load. It is generally well-accepted that satellite cells participate in postnatal growth, hypertrophy, and muscle regeneration following injury; however, their role in muscle regrowth following an atrophic stimulus remains equivocal. The current study employed a genetic mouse model (Pax7-DTA) that allowed for the effective depletion of >90% of satellite cells in adult muscle upon the administration of tamoxifen. Vehicle and tamoxifen-treated young adult female mice were either hindlimb suspended for 14 days to induce muscle atrophy or hindlimb suspended for 14 days followed by 14 days of reloading to allow regrowth, or they remained ambulatory for the duration of the experimental protocol. Additionally, 5-bromo-2'-deoxyuridine (BrdU) was added to the drinking water to track cell proliferation. Soleus muscle atrophy, as measured by whole muscle wet weight, fiber cross-sectional area, and single-fiber width, occurred in response to suspension and did not differ between satellite cell-depleted and control muscles. Furthermore, the depletion of satellite cells did not attenuate muscle mass or force recovery during the 14-day reloading period, suggesting that satellite cells are not required for muscle regrowth. Myonuclear number was not altered during either the suspension or the reloading period in soleus muscle fibers from vehicle-treated or satellite cell-depleted animals. Thus, myonuclear domain size was reduced following suspension due to decreased cytoplasmic volume and was completely restored following reloading, independent of the presence of satellite cells. These results provide convincing evidence that satellite cells are not required for muscle regrowth following atrophy and that, instead, the myonuclear domain size changes as myofibers adapt.

The effect of pre-exercise carbohydrate supplementation on anaerobic exercise performance in adolescent males.

Carbohydrate (CHO) consumption before anaerobic exercise was studied in 13 adolescent boys (15.2 ± 0.9 yrs). A within subjects design was employed where subjects consumed a 22% CHO or volume-matched placebo (PL) beverage 30-min before anaerobic exercise on two separate days. Exercise consisted of a Wingate Anaerobic Test (WAnT), ten by 10-s-sprints, and a second WAnT. Fatigue index and peak power (PP) were similar while mean power (MP) was higher (p < .025) in CHO trial; however this difference was ascribed to initial WAnT performance. PP and MP for the 10-s sprints were similar between trials. Intravenous blood glucose and insulin concentrations were higher (p < .05) in the CHO trial while lactate and catecholamine concentrations were similar. Improved performance on a single WAnT was apparent with CHO consumption before exercise; however, this strategy did not attenuate fatigue over time in adolescent boys.

Influence of acetaminophen and ibuprofen on skeletal muscle adaptations to resistance exercise in older adults.

Evidence suggests that consumption of over-the-counter cyclooxygenase (COX) inhibitors may interfere with the positive effects that resistance exercise training has on reversing sarcopenia in older adults. This study examined the influence of acetaminophen or ibuprofen consumption on muscle mass and strength during 12 wk of knee extensor progressive resistance exercise training in older adults. Thirty-six individuals were randomly assigned to one of three groups and consumed the COX-inhibiting drugs in double-blind placebo-controlled fashion: placebo (67 ± 2 yr; n = 12), acetaminophen (64 ± 1 yr; n = 11; 4 g/day), and ibuprofen (64 ± 1 yr; n = 13; 1.2 g/day). Compliance with the resistance training program (100%) and drug consumption (via digital video observation, 94%), and resistance training intensity were similar (P > 0.05) for all three groups. Drug consumption unexpectedly increased muscle volume (acetaminophen: 109 ± 14 cm(3), 12.5%; ibuprofen: 84 ± 10 cm(3), 10.9%) and muscle strength (acetaminophen: 19 ± 2 kg; ibuprofen: 19 ± 2 kg) to a greater extent (P < 0.05) than placebo (muscle volume: 69 ± 12 cm(3), 8.6%; muscle strength: 15 ± 2 kg), when controlling for initial muscle size and strength. Follow-up analysis of muscle biopsies taken from the vastus lateralis before and after training showed muscle protein content, muscle water content, and myosin heavy chain distribution were not influenced (P > 0.05) by drug consumption. Similarly, muscle content of the two known enzymes potentially targeted by the drugs, COX-1 and -2, was not influenced (P > 0.05) by drug consumption, although resistance training did result in a drug-independent increase in COX-1 (32 ± 8%; P < 0.05). Drug consumption did not influence the size of the nonresistance-trained hamstring muscles (P > 0.05). Over-the-counter doses of acetaminophen or ibuprofen, when consumed in combination with resistance training, do not inhibit and appear to enhance muscle hypertrophy and strength gains in older adults. The present findings coupled with previous short-term exercise studies provide convincing evidence that the COX pathway(s) are involved in the regulation of muscle protein turnover and muscle mass in humans.