Jumping Exercise Combined With Collagen Supplementation Preserves Bone Mineral Density in Elite Cyclists.

This study assessed the effect of combined jump training and collagen supplementation on bone mineral density (BMD) in elite road-race cyclists. In this open-label, randomized study with two parallel groups, 36 young (21 ± 3 years) male (n = 8) and female (n = 28) elite road-race cyclists were allocated to either an intervention (INT: n = 18) or a no-treatment control (CON: n = 18) group. The 18-week intervention period, conducted during the off-season, comprised five 5-min bouts of jumping exercise per week, with each bout preceded by the ingestion of 15 g hydrolyzed collagen. Before and after the intervention, BMD of various skeletal sites and trabecular bone score of the lumbar spine were assessed by dual-energy X-ray absorptiometry, along with serum bone turnover markers procollagen Type I N propeptide and carboxy-terminal cross-linking telopeptide of Type I collagen. BMD of the femoral neck decreased in CON (from 0.789 ± 0.104 to 0.774 ± 0.095 g/cm2), while being preserved in INT (from 0.803 ± 0.058 to 0.809 ± 0.066 g/cm2; Time × Treatment, p < .01). No differences between treatments were observed for changes in BMD at the total hip, lumbar spine, and whole body (Time × Treatment, p > .05 for all). Trabecular bone score increased from 1.38 ± 0.08 to 1.40 ± 0.09 in CON and from 1.46 ± 0.08 to 1.47 ± 0.08 in INT, respectively (time effect: p < .01), with no differences between treatments (Time × Treatment: p = .33). Serum procollagen Type I N propeptide concentrations decreased to a similar extent in CON (83.6 ± 24.8 to 71.4 ± 23.1 ng/ml) and INT (82.8 ± 30.7 to 66.3 ± 30.6; time effect, p < .001; Time × Treatment, p = .22). Serum carboxy-terminal cross-linking telopeptide of Type I collagen concentrations did not change over time, with no differences between treatments (time effect, p = .08; Time × Treatment, p = .58). In conclusion, frequent short bouts of jumping exercise combined with collagen supplementation beneficially affects femoral neck BMD in elite road-race cyclists.

Towards Optimized Care After Bariatric Surgery by Physical Activity and Exercise Intervention: a Review.

Although there is growing evidence on the importance of physical activity and exercise intervention after bariatric surgery, it remains to be clarified as to why and how post-operative exercise intervention should be implemented. In this narrative and practically oriented review, it is explained why exercise interventions and physical activity are important after bariatric surgery, how to prescribe exercise and monitor physical activity and how and when physical fitness, muscle strength, fat (-free) mass and bone mineral density could be assessed during follow-up. It is suggested that the inclusion of physical activity and exercise training in the clinical follow-up trajectory could be of great benefit to bariatric surgery patients, since it leads to greater improvements in body composition, bone mineral density, muscle strength and physical fitness.

Improving the Diagnosis of Nonfunctional Overreaching and Overtraining Syndrome.

INTRODUCTION: This study aimed to simplify and optimize the distinction between nonfunctional overreaching (NFO) and overtraining syndrome (OTS) by developing a multivariate approach (discriminant analysis [DA]) including hormonal and psychological changes measured during the Training Optimization (TOP) test. METHODS: Sensitivity of previously defined cutoff values for hypothalamic-pituitary-adrenal axis hormonal changes were recalculated on a larger database (n = 100). Discriminant analysis including hormonal and psychological variables measured during the TOP test was used to discriminate between NFO and OTS and predict the diagnosis of new cases. RESULTS: Adrenocorticotrophic hormone (ACTH) and prolactin (PRL) responses to the second exercise test were most sensitive to NFO and OTS. Cutoff values for ACTH and PRL response to the second test (NFO > cutoff value (200%) > OTS), showed a sensitivity of 67% for ACTH and 93% for PRL in case of OTS and 74% for both ACTH and PRL in case of NFO. A DA including hormonal and psychological changes measured during the TOP test, resulted in the accurate diagnosis of NFO and OTS with 98% sensitivity. The ACTH and PRL responses to the first and second exercise tests and feeling of fatigue were the most discriminating variables. CONCLUSIONS: The ACTH and PRL responses during the TOP test are the most sensitive markers to discriminate between NFO and OTS. Discriminant analysis including hormonal and psychological responses during the TOP test, can be used to optimize the diagnosis of NFO and OTS.

The effect of acute cocoa flavanol intake on the BOLD response and cognitive function in type 1 diabetes: a randomized, placebo-controlled, double-blinded cross-over pilot study.

RATIONALE: Type 1 diabetes (T1D), a chronic autoimmune disease, can result in cognitive dysfunction and is associated with vascular dysfunction. Cocoa flavanols (CFs) can stimulate nitric oxide-dependent vasodilation, resulting in enhanced hemodynamic responses and better cognitive function. OBJECTIVES: To investigate whether acute CF supplementation can improve cognitive function and hemodynamic responses in T1D. METHODS: In this randomized, double-blinded, cross-over pilot study, 11 patients with T1D and their healthy matched controls consumed CF (900 mg CF) and placebo (15 mg CF) 2 h before a flanker test. fMRI was used to measure blood oxygen level-dependent (BOLD) response during the cognitive test. Repeated measure ANOVAs were used to test the effects of CF and T1D on BOLD response and cognitive performance. RESULTS: CF improved reaction time on the flanker test and increased the BOLD response in the supramarginal gyrus parietal lobe and inferior frontal gyrus, compared to placebo, in both groups. In patients with T1D, cognitive performance was not deteriorated while the BOLD response was smaller in T1D compared to healthy controls in the subgyral temporal lobe and the cerebellum. CONCLUSIONS: Acute CF intake improved reaction time on the flanker test and increased the BOLD response in the activated brain areas in patients with T1D and their matched controls.

One-week cocoa flavanol intake increases prefrontal cortex oxygenation at rest and during moderate-intensity exercise in normoxia and hypoxia.

During exercise in hypoxia, O2 delivery to brain and muscle is compromised, and oxidative stress is elicited. Cocoa flavanols (CF) have antioxidant capacities and can increase blood flow by stimulating endothelial function. We aimed to examine the effects of 7-day CF intake on oxidative stress, nitric oxide production, and tissue oxygenation in response to exercise in normobaric hypoxia (14.3% O2). In a randomized, double-blind, cross-over study, 14 well-trained male cyclists completed four trials: exercise in normoxia or hypoxia, after 7-day CF or placebo intake. Flow-mediated dilation (FMD) was measured before intake of the last dose CF or placebo. One hundred minutes later, 20-min steady-state (SS; 45% V̇o2max) and 20-min time trial (TT) (cycling) were performed. Blood samples were taken. Prefrontal and muscular oxygenation was assessed by near-infrared spectroscopy. At baseline, FMD was increased by CF. Hypoxia increased exercise-induced elevations in lipid peroxidation and antioxidant capacity. CF suppressed exercise-induced lipid peroxidation but did not influence antioxidant capacity. At rest and during SS, prefrontal and muscular oxygenation was decreased by hypoxia. CF elevated prefrontal oxygenation but did not impact muscular oxygenation. During TT, hypoxia accelerated the exercise-induced decrease in prefrontal oxygenation, but not in muscular oxygenation. During TT, CF did not alter prefrontal and muscular oxygenation. CF did not change plasma nitrite, nitrate, and arginine:citrulline. During high-intensity exercise, CF improved neither tissue oxygenation nor performance in well-trained athletes. At rest and during moderate-intensity exercise, CF reduced exercise-induced lipid peroxidation and partially restored the hypoxia-induced decline in prefrontal oxygenation. NEW & NOTEWORTHY For the first time, we showed that CF had beneficial effects on endothelial function at rest, as well as on prefrontal oxygenation at rest and during moderate-intensity exercise, both in normoxia and hypoxia. Moreover, we showed that CF intake inhibited oxidative stress during exhaustive exercise in hypoxia.

Cocoa Flavanol Supplementation and Exercise: A Systematic Review.

BACKGROUND: Cocoa flavanols (CFs) have antioxidant and anti-inflammatory capacities and can improve vascular function. It has recently been suggested that CF intake may improve exercise performance and recovery. This systematic review aimed to evaluate the literature on the effects of CF intake on exercise performance and recovery and exercise-induced changes in vascular function, cognitive function, oxidative stress, inflammation, and metabolic parameters. METHODS: Two electronic databases (Pubmed and Web of Science) were searched for studies examining the combination of CF intake and exercise in humans (up to 28 March 2017). Articles were included if the exact amount of CFs was mentioned. The methodological quality and level of bias of the 13 included studies was assessed according to the checklist for randomized controlled trials from the Dutch Cochrane center. RESULTS: Acute, sub-chronic (2 weeks) and chronic (3 months) CF intake reduced exercise-induced oxidative stress. Evidence on the effect of CF on exercise-induced inflammation and platelet activation was scarce. Acute CF intake reduced and tempered the exercise-induced increase in blood pressure in obese participants. Acute and sub-chronic CF intake altered fat and carbohydrate metabolism during exercise. Acute and sub-chronic CF intake did not have ergogenic effects in athletes, while chronic CF intake improved mitochondrial efficiency in untrained participants. While combining sub-chronic CF intake and exercise training improved cardiovascular risk factors and vascular function, evidence on the synergistic effects of CF and exercise training on oxidative stress, inflammation, and fat and glucose metabolism was lacking. CONCLUSION: CF intake may improve vascular function, reduce exercise-induced oxidative stress, and alter fat and carbohydrate utilization during exercise, but without affecting exercise performance. There is a strong need for future studies examining the synergetic effect of chronic CF intake and exercise training.

Can the Lamberts and Lambert Submaximal Cycle Test Reflect Overreaching in Professional Cyclists?

CONTEXT: The Lamberts and Lambert Submaximal Cycle Test (LSCT) consists of 3 stages during which cyclists cycle for 6 min at 60%, 6 min at 80%, and 3 min at 90% of their maximal heart rate, followed by 1-min recovery. PURPOSE: To determine if the LSCT is able to reflect a state of functional overreaching in professional female cyclists during an 8-d training camp and the following recovery days. METHODS: Six professional female cyclists performed an LSCT on days 1, 5, and 8 of the training camp and 3 d after the training camp. During each stage of the LSCT, power output and rating of perceived exertion (RPE) were determined. Training diaries and Profile of Mood States (POMS) were also completed. RESULTS: At the middle and the end of the training camp, increased power output during the 2nd and 3rd stages of the LSCT was accompanied with increased RPE during these stages and/or the inability to reach 90% of maximal heart rate. All athletes reported increased feelings of fatigue and muscle soreness, while changes in energy balance, calculated from the POMS, were less indicative of a state of overreaching. After 3 d of recovery, all parameters of the LSCT returned to baseline, indicating a state of functional overreaching during the training camp. CONCLUSION: The LSCT is able to reflect a state of overreaching in elite professional female cyclists during an 8-d training camp and the following recovery days.

Nutritional Supplements and the Brain.

Cognitive function plays an important role in athletic performance, and it seems that brain functioning can be influenced by nutrition and dietary components. Thus, the central nervous system might be manipulated through changes in diet or supplementation with specific nutrients including branched-chain amino acids, tyrosine, carbohydrates, and caffeine. Despite some evidence that branched-chained amino acids can influence ratings of perceived exertion and mental performance, several well-controlled studies have failed to demonstrate a positive effect on exercise performance. Evidence of an ergogenic benefit of tyrosine supplementation during prolonged exercise is limited. There is evidence that mild dehydration can impair cognitive performance and mood. The beneficial effect of carbohydrate supplementation during prolonged exercise could relate to increased substrate delivery for the brain, with numerous studies indicating that hypoglycemia affects brain function and cognitive performance. Caffeine can enhance performance and reduce perception of effort during prolonged exercise and will influence specific reward centers of the brain. Plant products and herbal extracts such as polyphenols, ginseng, ginkgo biloba, etc. are marketed as supplements to enhance performance. In several animal studies, positive effects of these products were shown, however the literature on their effects on sports performance is scarce. Polyphenols have the potential to protect neurons against injury induced by neurotoxins, suppress neuroinflammation, and to promote memory, learning, and cognitive function. In general, there remains a need for controlled randomized studies with a strong design, sufficient statistical power, and well-defined outcome measures before "claims" on its beneficial effects on brain functioning can be established.

Acute cocoa Flavanols intake has minimal effects on exercise-induced oxidative stress and nitric oxide production in healthy cyclists: a randomized controlled trial.

BACKGROUND: Cocoa flavanols (CF) can stimulate vasodilation by improved nitric oxide (NO) synthesis and have antioxidant and anti-inflammatory capacities. This study aimed to examine whether acute CF intake can affect exercise-induced changes in antioxidant capacity, oxidative stress, inflammation and NO production, as well as exercise performance and recovery in well-trained cyclists. METHODS: Twelve well-trained male cyclists (mean ± SD age, VO2max: 30 ± 3 years, 63.0 ± 3.5 ml/kg/min) participated in this randomized, double-blind, cross over study. On 2 separate occasions, subjects performed two 30-min time trials 1.5 (TT1) and 3 (TT2) hours after CF (900 mg CF) or placebo (PL, 13 mg CF) intake, interposed by passive rest. Lactate, glucose, heartrate, rating of perceived exertion (RPE) and power output were measured during the TTs. Blood was drawn at baseline, before and after each TT and analyzed for epicatechin serum concentrations, trolox equivalent antioxidative capacity (TEAC), uric acid (UA), malonaldehyde (MDA), L-arginine/ADMA, citrulline, interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α plasma concentrations. Relative changes in blood markers and pacing strategy during TT were analysed by repeated measured ANOVA. TT performance was compared between PL and CF by paired t-test. RESULTS: Epicatechin concentrations were increased by CF intake. Exercise-induced increase in TEAC/UA was improved by CF intake (F(1) = 5.57; p = .038) (post-TT1: PL: 113.34 ± 3.9%, CF: 117.64 ± 3.96%, post-TT2: PL: 108.59 ± 3.95%, CF: 123.72 ± 7.4% to baseline), while exercise-induced increases in MDA, IL-1 and IL-6 were not affected by CF intake. TNF-α was unaltered by exercise and by CF. Exercise-induced decreases in L-arginine/ADMA and increases in citrulline were not affected by CF intake. TT1 and TT2 performance and exercise-induced physiological changes were unaffected by CF intake. CONCLUSION: Acute CF intake increased total antioxidant capacity in rest and during exercise, but did not affect exercise-induced lipid peroxidation, inflammation, nor NO production in healthy athletes. Acute CF intake did not improve TT performance and recovery. TRIAL REGISTRATION: ISRCTN32875, 21-11-2016, retrospectively registered.

Do Glucose and Caffeine Nasal Sprays Influence Exercise or Cognitive Performance?

INTRODUCTION: Nasal spray (NAS) containing caffeine (CAF) or glucose (GLUC) activates sensory(motor) cortices. PURPOSE: To investigate the influence of CAF or GLUC NAS on exercise and cognitive performance. METHODS: Eleven male subjects (age 22 ± 2 y) performed a maximal cycle test and 2 familiarization and 3 experimental trials. Each trial included a 30-s Wingate test and a 30-min time-trial (TT) performance test interspersed by 15 min of rest. Before and after each exercise test a Stroop task was conducted. Placebo NAS with or without CAF or GLUC was provided before each exercise session and at each completed 25% of the TT. Exercise-performance, physiological, and cognitive measures were obtained. Magnitude-based inferences determined the likelihood that NAS solutions would be beneficial, trivial, or negative to exercise-performance measures based on the smallest worthwhile effect. Physiological and cognitive measures were analyzed using (non)parametric tests (P < .05). RESULTS: GLUC NAS substantially increased the average power output during the TT (very likely beneficial: 98%). No further worthwhile exercise-performance enhancements were found for both substances. In addition, no significant differences in physiological and cognitive measures were observed. In line with mouth rinsing, GLUC was shown to substantially enhance endurance performance, probably due to the activation of the olfactory pathway and/or extra-oral sweet-taste receptors. CONCLUSION: GLUC NAS enhances endurance performance, which indicates a novel administration route. The higher activity in sensory brain cortices probably elicited the ergogenic effect. However, no further physiological and cognitive changes occurred, indicating that higher doses of substrates might be required.

Bike Desks in the Classroom: Energy Expenditure, Physical Health, Cognitive Performance, Brain Functioning, and Academic Performance.

BACKGROUND: Physical activity is positively associated with physical health, cognitive performance, brain functioning and academic performance. The aim of this study is to investigate the influence of bike desks in the classroom on adolescents' energy expenditure, physical health, cognitive performance, brain functioning and academic performance. METHODS: Forty-four adolescents were randomly assigned to control group (CG) or intervention group (IG). During 5 months, the IG used a bike desk for 4 class hours/week. Energy expenditure was measured during 6 consecutive days. Anthropometric parameters, aerobic fitness, academic performance, cognitive performance and brain functioning were assessed before (T0) and after (T1) the intervention. RESULTS: Energy expenditure of the IG was significantly higher during the class hours in which they used the bike desks relative to normal class hours. The CG had a significantly higher BMI at T1 relative to T0 while this was not significantly different for the IG. Aerobic fitness was significantly better in the IG at T1 relative to T0. No significant effects on academic performance cognitive performance and brain functioning were observed. CONCLUSIONS: As the implementation of bike desks in the classroom did not interfere with adolescents' academic performance, this can be seen as an effective means of reducing in-class sedentary time and improving adolescents' physical health.

Bike Desks in the Office: Physical Health, Cognitive Function, Work Engagement, and Work Performance.

OBJECTIVE: The aim of this study was to examine the longitudinal effect of implementing bike desks in an office setting on physical health, cognition, and work parameters. METHODS: Physical health, cognitive function, work engagement, and work performance measured before (T0) and after (T2) the intervention period were compared between office workers who used the bike desk (IG, n = 22) and those who did not (CG, n = 16). RESULTS: The IG cycled approximately 98 minutes/week. The IG showed a significantly lower fat percentage and a trend toward a higher work engagement at T2 relative to T0, while this was not different for the CG. No effects on other parameters of health, cognition, or work performance were found. CONCLUSIONS: Providing bike desks in the office positively influences employees' fat percentage and could positively influence work engagement without compromising work performance.

Cycling on a Bike Desk Positively Influences Cognitive Performance.

PURPOSE: Cycling desks as a means to reduce sedentary time in the office has gained interest as excessive sitting has been associated with several health risks. However, the question rises if people will still be as efficient in performing their desk-based office work when combining this with stationary cycling. Therefore, the effect of cycling at 30% Wmax on typing, cognitive performance and brain activity was investigated. METHODS: After two familiarisation sessions, 23 participants performed a test battery [typing test, Rey auditory verbal learning test (RAVLT), Stroop test and Rosvold continuous performance test (RCPT)] with electroencephalography recording while cycling and sitting on a conventional chair. RESULTS: Typing performance, performance on the RAVLT and accuracy on the Stroop test and the RCPT did not differ between conditions. Reaction times on the Stroop test and the RCPT were shorter while cycling relative to sitting (p < 0.05). N200, P300, N450 and conflict SP latency and amplitude on the Stroop test and N200 and P300 on the RCPT did not differ between conditions. CONCLUSIONS: This study showed that typing performance and short-term memory are not deteriorated when people cycle at 30% Wmax. Furthermore, cycling had a positive effect on response speed across tasks requiring variable amounts of attention and inhibition.

Acute cocoa flavanol improves cerebral oxygenation without enhancing executive function at rest or after exercise.

Acute exercise-induced improvements in cognitive function are accompanied by increased (cerebral) blood flow and increased brain-derived neurotrophic factor (BDNF) levels. Acute cocoa flavanol (CF) intake may improve cognitive function, cerebral blood flow (in humans), and BNDF levels (in animals). This study investigated (i) the effect of CF intake in combination with exercise on cognitive function and (ii) cerebral hemodynamics and BDNF in response to CF intake and exercise. Twelve healthy men participated in this randomized, double-blind, crossover study. Participants performed a cognitive task (CT) at 100 min after acute 903-mg CF or placebo (PL) intake, followed by a 30-min time-trial. Immediately after this exercise, the same CT was performed. Prefrontal near-infrared spectroscopy was applied during CT and exercise to measure changes in oxygenated (ΔHbO2), deoxygenated (ΔHHb), and total haemoglobin (ΔHbtot) and blood samples were drawn and analyzed for BDNF. Reaction time was faster postexercise, but was not influenced by CF. ΔHbO2 during the resting CT was increased by CF, compared with PL. ΔHbO2, ΔHHb, and ΔHbtot increased in response to exercise without any effect of CF. During the postexercise cognitive task, there were no hemodynamic differences between CF or PL. Serum BDNF was increased by exercise, but was not influenced by CF. In conclusion, at rest, CF intake increased cerebral oxygenation, but not BDNF concentrations, and no impact on executive function was detected. This beneficial effect of CF on cerebral oxygenation at rest was overruled by the strong exercise-induced increases in cerebral perfusion and oxygenation.

Monitoring Physical and Cognitive Overload During a Training Camp in Professional Female Cyclists.

PURPOSE: High training loads combined with other stressors can lead to performance decrements. The time needed to recover determines the diagnosis of (non)-functional overreaching or the overtraining syndrome. The aim of this study was to describe the effects of an 8-day (intensified) training camp of professional female cyclists on physical and cognitive performance. METHODS: Nine subjects performed a 30-min time trial (TT), cognitive test, and Profile of Mood States questionnaire before, during, and after a training camp (49% increased training volume). On data collection, cyclists were classified as "overreached" (OR) or "adapted" (A) based on TT performance. Two-way repeated-measures analysis of variance was used to detect changes in physical and cognitive parameters. RESULTS: Five cyclists were described as OR based on decreased mean power output (MPO) (-7.03%) on day 8. Four cyclists were classified as A (increased MPO: +1.72%). MPO and maximal heart rate were significantly different between A and OR groups. A significant slower reaction time (RT) (+3.35%) was found in OR subjects, whereas RT decreased (-4.59%) in A subjects. The change in MPO was negatively correlated with change in RT in the cognitive test (R2 = .52). CONCLUSIONS: This study showed that the use of objective, inexpensive, and easy-to-interpret physical and cognitive tests can facilitate the monitoring of training adaptations in professional female athletes.

Metabolic studies of prostanozol with the uPA-SCID chimeric mouse model and human liver microsomes.

Anabolic androgenic steroids are prohibited by the World Anti-Doping Agency because of their adverse health and performance enhancing effects. Effective control of their misuse by detection in urine requires knowledge about their metabolism. In case of designer steroids, ethical objections limit the use of human volunteers to perform excretion studies. Therefore the suitability of alternative models needs to be investigated. In this study pooled human liver microsomes (HLM) and an uPA(+/+)-SCID chimeric mouse model were used to examine the metabolism of the designer steroid prostanozol as a reference standard. Metabolites were detected by GC-MS (full scan) and LC-MS/MS (precursor ion scan). In total twenty-four prostanozol metabolites were detected with the in vitro and in vivo metabolism studies, which could be grouped into two broad classes, those with a 17-hydroxy- and those with a 17-keto-substituent. Major first phase metabolic sites were tentatively identified as C-3'; C-4 and C-16. Moreover, 3'- and 16ß-hydroxy-17-ketoprostanozol could be unequivocally identified, since authentic reference material was available, in both models. Comparison with published data from humans showed a good correlation, except for phase II metabolism. As metabolites were in contrast to the human studies predominantly present in the free fraction. Two types of metabolites ((di)hydroxylated prostanozol metabolites) that have not been described before could be confirmed in a real positive doping control sample. Hence, the results provide further evidence for the applicability of chimeric mice and HLM to perform metabolism studies of designer steroids.

Guidelines to Classify Female Subject Groups in Sport-Science Research.

AIM: To review current cycling-related sport-science literature to formulate guidelines to classify female subject groups and to compare this classification system for female subject groups with the classification system for male subject groups. METHODS: A database of 82 papers that described female subject groups containing information on preexperimental maximal cycle-protocol designs, terminology, biometrical and physiological parameters, and cycling experience was analyzed. Subject groups were divided into performance levels (PLs), according to the nomenclature. Body mass, body-mass index, maximal oxygen consumption (VO2max), peak power output (PPO), and training status were compared between PLs and between female and male PLs. RESULTS: Five female PLs were defined, representing untrained, active, trained, well-trained, and professional female subjects. VO2max and PPO significantly increased with PL, except for PL3 and PL4 (P < .01). For each PL, significant differences were observed in absolute and relative VO2max and PPO between male and female subject groups. Relative VO2max is the most cited parameter for female subject groups and is proposed as the principal parameter to classify the groups. CONCLUSION: This systematic review shows the large variety in the description of female subject groups in the existing literature. The authors propose a standardized preexperimental testing protocol and guidelines to classify female subject groups into 5 PLs based on relative VO2max, relative PPO, training status, absolute VO2max, and absolute PPO.

Endothelial Network Formation Within Human Tissue-Engineered Skeletal Muscle.

The size of in vitro engineered skeletal muscle tissue is limited due to the lack of a vascular network in vitro. In this article, we report tissue-engineered skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. We extend our bioartificial muscle (BAM) model by coculturing human muscle progenitor cells with human umbilical vein endothelial cells (HUVECs) in a fibrin extracellular matrix (ECM). First, the optimal medium conditions for coculturing myoblasts with HUVECs were determined in a fusion assay. Endothelial growth medium proved to be the best compromise for the coculture, without affecting the myoblast fusion index. Second, both cell types were cocultured in a BAM maintained under tension to stimulate myofiber alignment. We then tested different total cell numbers containing 50% HUVECs and found that BAMs with a total cell number of 2 × 10(6) resulted in well-aligned and densely packed myofibers while allowing for improved interspersed endothelial network formation. Third, we compared different myoblast-HUVEC ratios. Including higher numbers of myoblasts improved endothelial network formation at lower total cell density; however, improvement of network characteristics reached a plateau when 1 × 10(6) or more myoblasts were present. Finally, addition of Matrigel to the fibrin ECM did not enhance overall myofiber and endothelial network formation. Therefore, in our BAM model, we suggest the use of a fibrin extracellular matrix containing 2 × 10(6) cells of which 50-70% are muscle cells. Optimizing these coculture conditions allows for a physiologically more relevant muscle model and paves the way toward engineering of larger in vitro muscle constructs.