Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production.

Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment-with carriers derived from Aspergillus oryzae promoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry.

Characterization of an in vitro engineered ligament model.

In vivo tendon and ligament research can be limited by the difficultly of obtaining tissue samples that can be biochemically analyzed. In this study, we characterize the most widely used in vitro engineered ligament model. Despite previous works suggesting multiple passages change gene expression in 2D primary tenocytes, we found no relationship between passage number and expression of classical tendon fibroblast markers across different biological donors. When engineered into 3D ligaments, there was an increase in maximal tensile load between 7 and 14 days in culture, that corresponded with an increase in collagen content. By contrast, percent collagen increased logarithmically from Day 7 to Day 14, and this was similar to the increase in the modulus of the tissue. Importantly, there was no relationship between passage number and mechanical function or collagen content in the two independent donors tested. These results suggest that the model develops quickly and is reliable across differing passage numbers. This provides the field with the ability to 1) consistently determine functional changes of interventions out to passage number 10; and 2) to time interventions to the appropriate developmental stage: developing/regenerating (Day 7) or mature (Day 14) tissue.

Inhibiting JAK1, not NF-κB, reverses the effect of pro-inflammatory cytokines on engineered human ligament function.

The role of inflammation in chronic tendon/ligament injury is hotly debated. There is less debate about inflammation following acute injury. To better understand the effect of acute inflammation, in this study we developed a multi-cytokine model of inflammatory tendinitis. The combined treatment with TNF-α, IL-1ß, and IL-6, at dosages well below what are routinely used in vitro, decreased the mechanical properties and collagen content of engineered human ligaments. Treatment with this cytokine mixture resulted in an increase in phospho-NF-κB and MMP-1, did not affect procollagen production, and decreased STAT3 phosphorylation relative to controls. Using this more physiologically relevant model of acute inflammation, we inhibited NF-κB or JAK1 signaling in an attempt to reverse the negative effects of the cytokine mixture. Surprisingly, NF-κB inhibition led to an even greater decrease in mechanical function and collagen content. By contrast, inhibiting JAK1 led to an increase in mechanical properties, collagen content and thermal stability concomitant with a decrease in MMP-1. Our results suggest that inhibition of JAK1, not NF-κB, reverses the negative effects of pro-inflammatory cytokines on collagen content and mechanics in engineered human ligaments.

Spatial gene expression in the adult rat patellar tendon.

Tendons are dense connective tissues with relatively few cells which makes studying the molecular profile of the tissue challenging. There is not a consensus on the spatial location of various cell types within a tendon, nor the accompanying transcriptional profile. In the present study, we used two male rat patellar tendon samples for sequencing-based spatial transcriptomics to determine the gene expression profile. We integrated our data with a mouse Achilles single cell dataset to predict the cell type composition of the patellar tendon as a function of location within the tissue. The spatial location of the predicated cell types suggested that there were two populations of tendon fibroblasts, one located in the tendon midsubstance, while the other localized with red blood cells, pericytes, and immune cells to the tendon peripheral connective tissue. Of the highest expressed spatially variable genes, there were multiple genes with known function in tendon: Col1a1, Col1a2, Dcn, Fmod, Sparc, and Comp. Further, a novel spatially regulated gene (AABR07000398.1) with no known function was identified. The spatial gene expression of tendon associated genes (Scx, Thbs4, Tnmd, Can, Bgn, Lum, Adamts2, Lox, Ppib, Col2a1, Col3a1, Col6a2) was also visualized. Both patellar tendon samples had similar expression patterns for all these genes. This dataset provides new spatial insights into gene expression in a healthy tendon.

Multi-objective Bayesian algorithm automatically discovers low-cost high-growth serum-free media for cellular agriculture application.

In this work, we applied a multi-information source modeling technique to solve a multi-objective Bayesian optimization problem involving the simultaneous minimization of cost and maximization of growth for serum-free C2C12 cells using a hyper-volume improvement acquisition function. In sequential batches of custom media experiments designed using our Bayesian criteria, collected using multiple assays targeting different cellular growth dynamics, the algorithm learned to identify the trade-off relationship between long-term growth and cost. We were able to identify several media with >100% more growth of C2C12 cells than the control, as well as a medium with 23% more growth at only 62.5% of the cost of the control. These algorithmically generated media also maintained growth far past the study period, indicating the modeling approach approximates the cell growth well from an extremely limited data set.

Rapamycin insensitive regulation of engineered ligament structure and function by IGF-1.

Following rupture, the anterior cruciate ligament (ACL) will not heal and therefore more than 400,000 surgical repairs are performed annually. Ligament engineering is one way to meet the increasing need for donor tissue to replace the native ligament; however, currently these tissues are too weak for this purpose. Treating engineered human ligaments with insulin-like growth factor-1 (IGF-1) improves the structure and function of these grafts. Since the anabolic effects of IGF-1 are largely mediated by rapamycin complex I (mTORC1), we used rapamycin to determine whether mTORC1 was necessary for the improvement in collagen content and mechanics of engineered ligaments. The effect of IGF-1 and rapamycin was determined independently and interactions between the two treatments were tested. Grafts were treated for 6 days before mechanical testing and analysis of collagen content. Following 8 days of treatment, mechanical properties increased 34% with IGF-1 and decreased 24.5% with rapamycin. Similarly, collagen content increased 63% with IGF-1 and decreased 36% with rapamycin. Interestingly, there was no interaction between IGF-1 and rapamycin, suggesting that IGF-1 was working in a largely mTORC1-independent manner. Acute treatment with IGF-1 did not alter procollagen synthesis in growth media, even though rapamycin decreased procollagen 55%. IGF-1 decreased collagen degradation 15%, whereas rapamycin increased collagen degradation 10%. Once again, there was no interaction between IGF-1 and rapamycin on collagen degradation. Together, these data suggest that growth factor-dependent increases in collagen synthesis are dependent on mTORC1 activity; however, IGF-1 improves human-engineered ligament mechanics and collagen content by decreasing collagen degradation in a rapamycin-independent manner. How the anticatabolic effects of IGF-1 are regulated have yet to be determined.NEW & NOTEWORTHY IGF-1 increases and rapamycin decreases mechanical and material properties of engineered human ligaments by regulating collagen content and concentration. There was no interaction between IGF-1 and rapamycin, suggesting that IGF-1 and rapamycin work independently. We found that IGF-1 improves collagen content by decreasing collagen degradation in a rapamycin-independent manner, whereas growth factor-dependent increases in collagen synthesis are blocked by rapamycin. These data may explain why interventions to increase IGF-1 have not helped rehabilitation.

The rat Achilles and patellar tendons have similar increases in mechanical properties but become transcriptionally divergent during postnatal development.

The molecular events that drive post-natal tendon development are poorly characterized. In this study, we profiled morphological, mechanical, and transcriptional changes in the rat Achilles and patellar tendon before walking (P7), shortly after onset of walking (P14), and at motor maturity (P28). The Achilles and patellar tendons increased collagen content and mechanical strength similarly throughout post-natal development. However, at P28 the patellar tendon tended to display a higher maximal tensile load (MTL) (P = 0.0524) than the Achilles tendon, but a similar ultimate tensile strength (UTS; load relative to cross-sectional area) probably due to its increased cross-sectional area during development. The tendons started transcriptionally similar, with overlapping PCA clusters at P7 and P14, before becoming transcriptionally distinct at P28. In both tendons, there was an increase in extracellular matrix (ECM) gene expression and a concomitant decrease in cell cycle and mitochondrial gene expression. The transcriptional divergence at P28 suggested that STAT signalling was lower in the patellar tendon where MTL increased the most. Treating engineered human ligaments with the STAT inhibitor itacitinib increased collagen content and MTL. Our results suggest that during post-natal development, cellular resources are initially allocated towards cell proliferation before shifting towards extracellular matrix development following the onset of mechanical load and provide potential targets for improving tendon function. KEY POINTS: Little is known about mechanisms of post-natal tendon growth. We characterized morphological, mechanical, and transcriptional changes that occur before (P7), and early (P14) and late after (P28) rats begin to walk. From P7 to P28, the Achilles tendon increased in length, whereas the patellar tendon increased in cross-sectional area. Mechanical and material properties of the Achilles and patellar tendon increased from P7 to P28. From P7 to P28, the Achilles and patellar tendons increased expression of ECM genes and decreased mitochondrial and cell cycle gene expression. Ribosomal gene expression also significantly decreased in the Achilles and tended to decrease in the patellar tendon. At P28, STAT1 signalling tended to be lower in the patellar tendon which had grown by increasing cross-sectional area and inhibiting STAT activation in vitro improved mechanical properties in engineered human ligaments.

A Ketogenic Diet in Combination with Gemcitabine Mitigates Pancreatic Cancer-Associated Cachexia in Male and Female KPC Mice.

Cancer-associated cachexia (CAC) is a critical contributor to pancreatic ductal adenocarcinoma (PDAC) mortality. Thus, there is an urgent need for new strategies to mitigate PDAC-associated cachexia; and the exploration of dietary interventions is a critical component. We previously observed that a ketogenic diet (KD) combined with gemcitabine enhances overall survival in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. In this study, we investigated the effect and cellular mechanisms of a KD in combination with gemcitabine on the maintenance of skeletal muscle mass in KPC mice. For this purpose, male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD), a KD, a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. We observed that a KD or a KG-mitigated muscle strength declined over time and presented higher gastrocnemius weights compared CD-fed mice. Mechanistically, we observed sex-dependent effects of KG treatment, including the inhibition of autophagy, and increased phosphorylation levels of eIF2α in KG-treated KPC mice when compared to CG-treated mice. Our data suggest that a KG results in preservation of skeletal muscle mass. Additional research is warranted to explore whether this diet-treatment combination can be clinically effective in combating CAC in PDAC patients.

Time-dependent diffusion tensor imaging and diffusion modeling of age-related differences in the medial gastrocnemius and feasibility study of correlations to histopathology.

PURPOSE: Implement STEAM-DTI to model time-dependent diffusion eigenvalues using the random permeable barrier model (RPBM) to study age-related differences in the medial gastrocnemius (MG) muscle. Validate diffusion model-extracted fiber diameter for histological assessment. METHODS: Diffusion imaging at different diffusion times (Δ) was performed on seven young and six senior participants. Time-dependent diffusion eigenvalues (λ2 (t), λ3 (t), and D⊥ (t); average of λ2 (t) and λ3 (t)) were fit to the RPBM to extract tissue microstructure parameters. Biopsy of the MG tissue for histological assessment was performed on a subset of participants (four young, six senior). RESULTS: λ3 (t) was significantly higher in the senior cohort for the range of diffusion times. RPBM fits to λ2 (t) yielded fiber diameters in agreement to those from histology for both cohorts. The senior cohort had lower values of volume fraction of membranes, ζ, in fits to λ2 (t), λ3 (t), and D⊥ (t) (significant for fit to λ3 (t)). Fits of fiber diameter from RPBM to that from histology had the highest correlation for the fit to λ2 (t). CONCLUSION: The age-related patterns in λ2 (t) and λ3 (t) could tentatively be explained from RPBM fits; these patterns may potentially arise from a decrease in fiber asymmetry and an increase in permeability with age.

Co-Ingestion of Branched-Chain Amino Acids and Carbohydrate Stimulates Myofibrillar Protein Synthesis Following Resistance Exercise in Trained Young Men.

Branched-chain amino acids (BCAA) and carbohydrate (CHO) are commonly recommended postexercise supplements. However, no study has examined the interaction of CHO and BCAA ingestion on myofibrillar protein synthesis (MyoPS) rates following exercise. We aimed to determine the response of MyoPS to the co-ingestion of BCAA and CHO following an acute bout of resistance exercise. Ten resistance-trained young men completed two trials in counterbalanced order, ingesting isocaloric drinks containing either 30.6-g CHO plus 5.6-g BCAA (B + C) or 34.7-g CHO alone following a bout of unilateral, leg resistance exercise. MyoPS was measured postexercise with a primed, constant infusion of L-[ring13C6] phenylalanine and collection of muscle biopsies pre- and 4 hr postdrink ingestion. Blood samples were collected at time points before and after drink ingestion. Serum insulin concentrations increased to a similar extent in both trials (p > .05), peaking at 30 min postdrink ingestion. Plasma leucine (514 ± 34 nmol/L), isoleucine (282 ± 23 nmol/L), and valine (687 ± 33 nmol/L) concentrations peaked at 0.5 hr postdrink in B + C and remained elevated for 3 hr during exercise recovery. MyoPS was ∼15% greater (95% confidence interval [-0.002, 0.028], p = .039, Cohen's d = 0.63) in B + C (0.128%/hr ± 0.011%/hr) than CHO alone (0.115%/hr ± 0.011%/hr) over the 4 hr postexercise period. Co-ingestion of BCAA and CHO augments the acute response of MyoPS to resistance exercise in trained young males.

Ketogenic Diets and Mitochondrial Function: Benefits for Aging But Not for Athletes.

As humans age, we lose skeletal muscle mass, even in the absence of disease (sarcopenia), increasing the risk of death. Low mitochondrial mass and activity contributes to sarcopenia. It is our hypothesis that a ketogenic diet improves skeletal muscle mitochondrial mass and function when they have declined because of aging or disease, but not in athletes where mitochondrial quality is high.

Assessing Edible Filamentous Fungal Carriers as Cell Supports for Growth of Yeast and Cultivated Meat.

The growth and activity of adherent cells can be enabled or enhanced through attachment to a solid surface. For food and beverage production processes, these solid supports should be food-grade, low-cost, and biocompatible with the cell of interest. Solid supports that are edible can be a part of the final product, thus simplifying downstream operations in the production of fermented beverages and lab grown meat. We provide proof of concept that edible filamentous fungal pellets can function as a solid support by assessing the attachment and growth of two model cell types: yeast, and myoblast cells. The filamentous fungus Aspergillus oryzae was cultured to produce pellets with 0.9 mm diameter. These fugal pellets were inactivated by heat or chemical methods and characterized physicochemically. Chemically inactivated pellets had the lowest dry mass and were the most hydrophobic. Scanning electron microscope images showed that both yeast and myoblast cells naturally adhered to the fungal pellets. Over 48 h of incubation, immobilized yeast increased five-fold on active pellets and six-fold on heat-inactivated pellets. Myoblast cells proliferated best on heat-treated pellets, where viable cell activity increased almost two-fold, whereas on chemically inactivated pellets myoblasts did not increase in the cell mass. These results support the use of filamentous fungi as a novel cell immobilization biomaterial for food technology applications.

2-month ketogenic diet preferentially alters skeletal muscle and augments cognitive function in middle aged female mice.

The effect of a ketogenic diet (KD) on middle aged female mice is poorly understood as most of this work have been conducted in young female mice or diseased models. We have previously shown that an isocaloric KD started at middle age in male mice results in enhanced mitochondrial mass and function after 2 months on diet and improved cognitive behavior after being on diet for 14 months when compared with their control diet (CD) fed counterparts. Here, we aimed to investigate the effect of an isocaloric 2-month KD or CD on healthy 14-month-old female mice. At 16 months of age cognitive behavior tests were performed and then serum, skeletal muscle, cortex, and hippocampal tissues were collected for biochemical analysis. Two months on a KD resulted in enhanced cognitive behavior associated with anxiety, memory, and willingness to explore. The improved neurocognitive function was associated with increased PGC1α protein in the gastrocnemius (GTN) muscle and nuclear fraction. The KD resulted in a tissue specific increase in mitochondrial mass and kynurenine aminotransferase (KAT) levels in the GTN and soleus muscles, with a corresponding decrease in kynurenine and increase in kynurenic acid levels in serum. With KAT proteins being responsible for converting kynurenine into kynurenic acid, which is unable to cross the blood brain barrier and be turned into quinolinic acid-a potent neurotoxin, this study provides a potential mechanism of crosstalk between muscle and brain in mice on a KD that may contribute to improved cognitive function in middle-aged female mice.

Spent media analysis suggests cultivated meat media will require species and cell type optimization.

Cell culture media design is perhaps the most significant hurdle currently facing the commercialization of cultivated meat as an alternative source of dietary protein. Since media optimization for a specific culture system requires a significant amount of effort and investment, a major question remaining is whether media formulations can be easily shared across multiple production schemes for cells of different species and lineages. Here, we perform spent medium analysis to compare the specific nutrient utilization of primary embryonic chicken muscle precursor cells and fibroblasts to the murine C2C12 myoblast cell line. We demonstrate that these related cell types have significantly different nutrient utilization patterns collectively and on a per-cell basis, and that many components of conventional media do not appear to be depleted by the cells. Namely, glucose was not consumed as rapidly nor as completely by the chicken muscle precursors compared to other cells overall, and there were significant differences in specific consumption rates for several other key nutrients over the first day of culture. Ultimately, our results indicate that no one medium is likely ideal and cost effective to culture multiple cell types and that novel methods to streamline media optimization efforts will be important for the industry to develop.

Dominant-negative p53-overexpression in skeletal muscle induces cell death and fiber atrophy in rats.

The tumor suppressor p53 is thought to play a key role in the maintenance of cell size and homeostasis, but relatively little is known about its role in skeletal muscle. Based on its ability to suppress cell growth, we hypothesized that inhibiting the function of wild-type p53 through the overexpression of a dominant-negative p53 mutant (DDp53) could result in muscle fiber hypertrophy. To test this hypothesis, we electroporated adult rat tibialis anterior muscles with DDp53 and collected the tissue three weeks later. We confirmed successful overexpression of DDp53 on a histological and biochemical level and found pronounced changes to muscle architecture, metabolism, and molecular signaling. Muscle mass, fiber cross-sectional area, and fiber diameter significantly decreased with DDp53 overexpression. We found histopathological changes in DDp53 transfected muscle which were accompanied by increased levels of proteins that are associated with membrane damage and repair. In addition, DDp53 decreased oxidative phosphorylation complex I and V protein levels, and despite its negative effects on muscle mass and fiber size, caused an increase in muscle protein synthesis as assessed via the SUnSET technique. Interestingly, the increase in muscle protein synthesis was concomitant with a decrease in phospho-S6K1 (Thr389). Furthermore, the muscle wasting in the DDp53 electroporated leg was accompanied by a decrease in global protein ubiquitination and an increase in proteasome activity. In conclusion, overexpression of a dominant-negative p53 mutant in skeletal muscle results in decreased muscle mass, myofiber size, histological muscle damage, a metabolic phenotype, and perturbed homeostasis between muscle protein synthesis and degradation.

Multi-information source Bayesian optimization of culture media for cellular agriculture.

Culture media used in industrial bioprocessing and the emerging field of cellular agriculture is difficult to optimize due to the lack of rigorous mathematical models of cell growth and culture conditions, as well as the complexity of the design space. Rapid growth assays are inaccurate yet convenient, while robust measures of cell number can be time-consuming to the point of limiting experimentation. In this study, we optimized a cell culture media with 14 components using a multi-information source Bayesian optimization algorithm that locates optimal media conditions based on an iterative refinement of an uncertainty-weighted desirability function. As a model system, we utilized murine C2C12 cells, using AlamarBlue, LIVE stain, and trypan blue exclusion cell counting assays to determine cell number. Using this experimental optimization algorithm, we were able to design media with 181% more cells than a common commercial variant with a similar economic cost, while doing so in 38% fewer experiments than an efficient design-of-experiments method. The optimal medium generalized well to long-term growth up to four passages of C2C12 cells, indicating the multi-information source assay improved measurement robustness relative to rapid growth assays alone.

Myofibrillar protein synthesis rates are increased in chronically exercised skeletal muscle despite decreased anabolic signaling.

The molecular responses to acute resistance exercise are well characterized. However, how cellular signals change over time to modulate chronic adaptations to more prolonged exercise training is less well understood. We investigated anabolic signaling and muscle protein synthesis rates at several time points after acute and chronic eccentric loading. Adult rat tibialis anterior muscle was stimulated for six sets of ten repetitions, and the muscle was collected at 0 h, 6 h, 18 h and 48 h. In the last group of animals, 48 h after the first exercise bout a second bout was conducted, and the muscle was collected 6 h later (54 h total). In a second experiment, rats were exposed to four exercise sessions over the course of 2 weeks. Anabolic signaling increased robustly 6 h after the first bout returning to baseline between 18 and 48 h. Interestingly, 6 h after the second bout mTORC1 activity was significantly lower than following the first bout. In the chronically exercised rats, we found baseline anabolic signaling was decreased, whereas myofibrillar protein synthesis (MPS) was substantially increased, 48 h after the last bout of exercise. The increase in MPS occurred in the absence of changes to muscle fiber size or mass. In conclusion, we find that anabolic signaling is already diminished after the second bout of acute resistance type exercise. Further, chronic exposure to resistance type exercise training results in decreased basal anabolic signaling but increased overall MPS rates.

Scleraxis and collagen I expression increase following pilot isometric loading experiments in a rodent model of patellar tendinopathy.

The effect of mechanical load on tendinopathic tissue is usually studied in the context of identifying mechanisms responsible for tendon degradation. However, loading is also one of the most common treatments for tendinopathy. It is therefore possible that different loads result in different cellular responses within a tendon. To test this hypothesis, we first established a rodent model of tendinopathy that has a transcriptional signature similar to human tendinopathy. Tendinopathy was modeled in the rat by producing a lesion in the central core of the patellar tendon using a biopsy punch, followed by two weeks to allow scar formation. We performed 3' Tag RNA-Seq to identify genes that were differentially expressed between the native and scarred rat patellar tendon. Genes involved in extracellular matrix (ECM) structure and turnover were increased, mitochondrial genes were decreased, and there was no inflammatory signature in the tendinopathic tissue. These transcriptional changes phenocopy previously published whole transcriptome analysis in human tendinopathy. After validating the model, the initial response to injury and loading was determined. Two weeks after creation of the patellar tendon lesion, the tendon was loaded using either 4 × 30s isometric or a time-under-tension matched (360 × 0.33s) dynamic protocol. Injured +/- loading and contralateral control tendons were collected eighteen hours after loading, RNA was extracted, and gene expression was quantified using qRT-PCR of the scar with or without loading. The expression of scleraxis and type I collagen increased following isometric loading relative to those loaded dynamically. By contrast, the expression of type II collagen increased in the dynamic samples relative to those loaded isometrically. These data suggest that dynamic loading of a central core tendon injury increases fibrocartilage markers, whereas long isometric loads stimulate markers of tendon regeneration.

Sex differences in systemic bone and muscle loss following femur fracture in mice.

Fracture induces systemic bone loss in mice and humans, and a first (index) fracture increases the risk of future fracture at any skeletal site more in men than women. The etiology of this sex difference is unknown, but fracture may induces a greater systemic bone loss response in men. Also sex differences in systemic muscle loss after fracture have not been examined. We investigated sex differences in systemic bone and muscle loss after transverse femur fracture in 3-month-old male and female C57BL/6 J mice. Whole-body and regional bone mineral content and density (BMC and BMD), trabecular and cortical bone microstructure, muscle contractile force, muscle mass, and muscle fiber size were quantified at multiple time points postfracture. Serum concentrations of inflammatory cytokines (IL-1ß, IL-6, and TNF-α) were measured 1-day postfracture. One day postfracture, IL-6 and Il-1B were elevated in fracture mice of both sexes, but TNF-α was only elevated in male fracture mice. Fracture reduced BMC, BMD, and trabecular bone microstructural properties in both sexes 2 weeks postfracture, but declines were greater in males. Muscle contractile force, mass, and fiber size decreased primarily in the fractured limb at 2 weeks postfracture and females showed a trend toward greater muscle loss. Bone and muscle properties recovered by 6 weeks postfracture. Overall, postfracture systemic bone loss is greater in men, which may contribute to sex differences in subsequent fracture risk. In both sexes, muscle loss is primarily confined to the injured limb and fracture may induce greater inflammation in males.

Cannabidiol Does Not Impact Acute Anabolic or Inflammatory Signaling in Skeletal Muscle In Vitro.

Background: Cannabidiol (CBD) is becoming increasingly popular for the treatment of clinical conditions including as an aid for muscle recovery. Previous work demonstrated that CBD exhibited mild effects on skeletal muscle, with a tendency to increase anabolic signaling and decrease inflammatory signaling. Methods: To gain mechanistic insight and extend these findings, we conducted a set of experiments using C2C12 myotubes. Results: Increasing the dose of CBD (1-5 µM) provided with insulin-like growth factor 1 (IGF-1) showed no effect on anabolic signaling through mTORC1 (S6K1 [Thr389], p=0.27; rpS6 [Ser240/244], p=0.81; or 4E-BP1 [Thr37/46], p=0.87). Similarly, inflammatory signaling through nuclear factor kappa B (NF-κB) (p105, p=0.88; p50, p=0.93; or phosphorylated p65 [Ser536], p=0.84) in response to tumor necrosis factor α (TNFα) was unaffected by CBD (2.5 µM), whereas dioscin, a natural product that blocks NF-κB signaling, reduced p105 and phosphorylated p65 (Ser536) compared with the TNFα and the TNFα + CBD condition (p<0.01 and p<0.05, respectively). Finally, cannabinoid receptor type 1 (CB1) receptor levels were measured in C2C12 cells, murine skeletal muscle, cortex, and hippocampus. Although CB1 was not detectable in muscle cells or muscle tissue, high levels were observed in brain tissue. Conclusion: In conclusion, CBD does not directly modulate anabolic or inflammatory signaling in myotubes in vitro, which can likely be explained by the lack of functional receptors.