ObjectiveOverweight and obesity are characterized by excess adiposity and systemic, chronic, low-grade inflammation, which is associated with several metabolic disorders. The aim of this study was to assess the feasibility and tolerability of beta-alanine supplementation and to explore the effects on cardiometabolic health and cardiovascular, hepatic, and renal function in adults with overweight and obesity.MethodsA total of 27 adults (44% female; mean [SD], age: 58 [10] years, BMI: 31.1 [2.9] kg/m2, hemoglobin A1c: 39.8 [4.3] mmol/mol) received beta-alanine (4.8 g/day) or a matched placebo for 3 months. Feasibility and tolerability outcomes included adherence, side effects, recruitment, attrition, and blinding, and exploratory outcomes included biochemical markers, blood pressures, and transthoracic echocardiography parameters. Data were analyzed using a Bayesian approach presented with 95% credible intervals (CrI).Results beta-alanine was well tolerated and adhered to (adherence: placebo, 0.91 [95% CrI: 0.84-0.95]; beta-alanine, 0.92 [95% CrI: 0.85-0.95]), and side effects remained at or below baseline throughout. The probability that beta-alanine supplementation affected cardiometabolic, cardiovascular, or clinical biochemical outcomes was low.ConclusionsSustained-release beta-alanine supplementation is well tolerated and adhered to in adults with overweight and obesity. Future research should consider more advanced metabolic conditions, which may benefit from longer duration supplementation.

Intermittent and continuous mechanical loads are known to influence osteogenic activity. The present study examines the effects of matched intermittent and continuous load in vitro on bone formation markers. MC3T3 (mouse pre-osteoblasts) were cultured and placed in a bioreactor to undergo continuous, intermittent, or unloading for 1, 3 and 12 days. Loading conditions were matched for magnitude, duration and frequency. At each time point the cells were analysed for alkaline phosphatase (ALP) activity, procollagen I N-terminal propeptide (PINP) and alizarin red staining (ARS). Intermittent load caused an increase in ALP activity across all time points compared to continuous loading (↑30% – 59%) and unloaded conditions (↑70% – 90%). PINP concentrations in intermittent load were lower compared to continuous load (↓112%) on day 3. However, no differences were observed between loading conditions at any other time point. No differences were observed for ARS between loading conditions. Intermittent load caused an increase in bone formation marker ALP, but not PINP, when compared to continuous loading and unloaded conditions. These findings expand our knowledge to aid the optimisation of exercise regimes for bone adaptation and provide additional tools for further analysis of osteogenesis in human cells.

Intermittent and continuous mechanical loads are known to influence osteogenic activity. The present study examines the effects of matched intermittent and continuous load in vitro on bone formation markers. MC3T3 (mouse pre-osteoblasts) were cultured and placed in a bioreactor to undergo continuous, intermittent, or unloading for 1, 3 and 12 days. Loading conditions were matched for magnitude, duration and frequency. Each time point was analysed for alkaline phosphatase (ALP) activity, procollagen 1 N-terminal propeptide (PINP) and alizarin red staining (ARS). Intermittent load caused an increase in ALP activity across all time points compared to continuous loading (↑30%-59%) and unloaded conditions (↑70%-90%). PINP concentrations from intermittent load were lower than continuous load (↓112%) on day 3. However, no differences were observed in PINP concentrations between loading conditions at other time points. No differences were observed for ARS between loading conditions. Intermittent load caused an increase in bone formation marker ALP, but not PINP, when compared to continuous loading and unloaded conditions. These findings further our knowledge in bone formation response and provide additional tools for the analysis of osteogenesis in vitro.

Bone is influenced by many factors such as genetics and mechanical loading, but the short-term physiological effects of these factors on bone (re)modelling are not well characterised. This study investigated the effects of endurance trainability phenotype, sex, and interval running training (7-week intervention) on bone collagen formation in rats using a deuterium oxide stable isotope tracer method. Bone samples of the femur diaphysis, proximal tibia, mid-shaft tibia, and distal tibia were collected after necropsy from forty-six 9 ± 3-month male and female rats selectively bred for yielding low (LRT) or high (HRT) responses to endurance training. Bone collagen proteins were isolated and hydrolysed, and fractional synthetic rates (FSRs) were determined by the incorporation of deuterium into protein-bound alanine via GC-pyrolysis-IRMS. There was a significant large main effect of phenotype at the femur site (p < 0.001; η2g = 0.473) with HRT rats showing greater bone collagen FSRs than LRT rats. There was a significant large main effect of phenotype (p = 0.008; η2g = 0.178) and a significant large main effect of sex (p = 0.005; η2g = 0.196) at the proximal site of the tibia with HRT rats showing greater bone collagen FSRs than LRT rats, and male rats showing greater bone collagen FSRs compared to female rats. There was a significant large main effect of training at the mid-shaft site of the tibia (p = 0.012; η2g = 0.159), with rats that underwent interval running training having greater bone collagen FSRs than control rats. Similarly, there was a significant large main effect of training at the distal site of the tibia (p = 0.050; η2g = 0.156), with rats in the interval running training group having greater bone collagen FSRs compared to rats in the control group. Collectively, this evidence highlights that bone responses to physiological effects are site-specific, indicating that interval running training has positive effects on bone collagen synthesis at the tibial mid-shaft and distal sites, whilst genetic factors affect bone collagen synthesis at the femur diaphysis (phenotype) and proximal tibia (phenotype and sex) in rats.

AbstractBackgroundSarcopenia is an age‐related muscle disease that increases the risk of falls, disabilities, and death. It is associated with increased muscle protein degradation driven by molecular signalling pathways including Akt and FOXO1. This study aims to identify genes, gene interactions, and molecular pathways and processes associated with muscle aging and exercise in older adults that remained undiscovered until now leveraging on an artificial intelligence approach called artificial neural network inference (ANNi).MethodsFour datasets reporting the profile of muscle transcriptome obtained by RNA‐seq of young (21–43 years) and older adults (63–79 years) were selected and retrieved from the Gene Expression Omnibus (GEO) data repository. Two datasets contained the transcriptome profiles associated to muscle aging and two the transcriptome linked to resistant exercise in older adults, the latter before and after 6 months of exercise training. Each dataset was individually analysed by ANNi based on a swarm neural network approach integrated into a deep learning model (Intelligent Omics). This allowed us to identify top 200 genes influencing (drivers) or being influenced (targets) by aging or exercise and the strongest interactions between such genes. Downstream gene ontology (GO) analysis of these 200 genes was performed using Metacore (Clarivate™) and the open‐source software, Metascape. To confirm the differential expression of the genes showing the strongest interactions, real‐time quantitative PCR (RT‐qPCR) was employed on human muscle biopsies obtained from eight young (25 ± 4 years) and eight older men (78 ± 7.6 years), partaking in a 6‐month resistance exercise training programme.ResultsCHAD, ZDBF2, USP54, and JAK2 were identified as the genes with the strongest interactions predicting aging, while SCFD1, KDM5D, EIF4A2, and NIPAL3 were the main interacting genes associated with long‐term exercise in older adults. RT‐qPCR confirmed significant upregulation of USP54 (P = 0.005), CHAD (P = 0.03), and ZDBF2 (P = 0.008) in the aging muscle, while exercise‐related genes were not differentially expressed (EIF4A2 P = 0.99, NIPAL3 P = 0.94, SCFD1 P = 0.94, and KDM5D P = 0.64). GO analysis related to skeletal muscle aging suggests enrichment of pathways linked to bone development (adj P‐value 0.006), immune response (adj P‐value <0.001), and apoptosis (adj P‐value 0.01). In older exercising adults, these were ECM remodelling (adj P‐value <0.001), protein folding (adj P‐value <0.001), and proteolysis (adj P‐value <0.001).ConclusionsUsing ANNi and RT‐qPCR, we identified three strongly interacting genes predicting muscle aging, ZDBF2, USP54, and CHAD. These findings can help to inform the design of nonpharmacological and pharmacological interventions that prevent or mitigate sarcopenia.

Background Circulating biomarkers of bone formation and resorption are widely used in exercise metabolism research, but their responses to exercise are not clear. This study aimed to quantify group responses and inter-individual variability of P1NP and β-CTX-1 after prolonged, continuous running (60–120 min at 65–75% V̇O 2max ) in young healthy adult males using individual participant data (IPD) meta-analysis. Methods The protocol was designed following PRISMA-IPD guidelines and was pre-registered on the Open Science Framework prior to implementation ( https://osf.io/y69nd ). Changes in P1NP and β-CTX-1 relative to baseline were measured during, immediately after, and in the hours and days following exercise. Typical hourly and daily variations were estimated from P1NP and β-CTX-1 changes relative to baseline in non-exercise (control) conditions. Group responses and inter-individual variability were quantified with estimates of the mean and standard deviation of the difference, and the proportion of participants exhibiting an increased response. Models were conducted within a Bayesian framework with random intercepts to account for systematic variation across studies. Results P1NP levels increased during and immediately after running, when the proportion of response was close to 100% (75% CrI: 99 to 100%). P1NP levels returned to baseline levels within 1 h and over the next 4 days, showing comparable mean and standard deviation of the difference with typical hourly (0.1 ± 7.6 ng·mL −1 ) and daily (− 0.4 ± 5.7 ng·mL −1 ) variation values. β-CTX-1 levels decreased during and up to 4 h after running with distributions comparable to typical hourly variation (− 0.13 ± 0.11 ng·mL −1 ). There was no evidence of changes in β-CTX-1 levels during the 4 days after the running bout, when distributions were also similar between the running data and typical daily variation (− 0.03 ± 0.10 ng·mL −1 ). Conclusion Transient increases in P1NP were likely biological artefacts (e.g., connective tissue leakage) and not reflective of bone formation. Comparable small decreases in β-CTX-1 identified in both control and running data, suggested that these changes were due to the markers’ circadian rhythm and not the running intervention. Hence, prolonged continuous treadmill running did not elicit bone responses, as determined by P1NP and β-CTX-1, in this population.

Type-2 diabetes (T2D) is characterised by a dysregulation of metabolism, including skeletal muscle insulin resistance, mitochondrial dysfunction, and oxidative stress. Reactive species, such as methylglyoxal (MGO) and 4-hydroxynonenal (4-HNE), positively associate with T2D disease severity and can directly interfere with insulin signalling and glucose uptake in skeletal muscle by modifying cellular proteins. The multifunctional dipeptide carnosine, and its rate-limiting precursor β-alanine, have recently been shown to improve glycaemic control in humans and rodents with diabetes. However, the precise mechanisms are unclear and research in human skeletal muscle is limited. Herein, we present novel findings in primary human T2D and lean healthy control (LHC) skeletal muscle cells. Cells were differentiated to myotubes, and treated with 10 mM carnosine, 10 mM β-alanine, or control for 4-days. T2D cells had reduced ATP-linked and maximal respiration compared with LHC cells (p = 0.016 and p = 0.005). Treatment with 10 mM carnosine significantly increased insulin-stimulated glucose uptake in T2D cells (p = 0.047); with no effect in LHC cells. Insulin-stimulation increased MGO-modified proteins in T2D cells by 47%; treatment with carnosine attenuated this increase to 9.7% (p = 0.011). There was no effect treatment on cell viability or expression of other proteins. These findings suggest that the beneficial effects of carnosine on glycaemic control may be explained by its scavenging actions in human skeletal muscle.

Healthy skeletal muscle can regenerate after ischaemic, mechanical, or toxin-induced injury, but ageing impairs that regeneration potential. This has been largely attributed to dysfunctional satellite cells and reduced myogenic capacity. Understanding which signalling pathways are associated with reduced myogenesis and impaired muscle regeneration can provide valuable information about the mechanisms driving muscle ageing and prompt the development of new therapies. To investigate this, we developed a high-throughput in vitro model to assess muscle regeneration in chemically injured C2C12 and human myotube-derived young and aged myoblast cultures. We observed a reduced regeneration capacity of aged cells, as indicated by an attenuated recovery towards preinjury myotube size and myogenic fusion index at the end of the regeneration period, in comparison with younger muscle cells that were fully recovered. RNA-sequencing data showed significant enrichment of KEGG signalling pathways, PI3K-Akt, and downregulation of GO processes associated with muscle development, differentiation, and contraction in aged but not in young muscle cells. Data presented here suggest that repair in response to in vitro injury is impaired in aged vs. young muscle cells. Our study establishes a framework that enables further understanding of the factors underlying impaired muscle regeneration in older age.

Healthy skeletal muscle can regenerate after ischemic, mechanical or toxin-induced injury. However, age-related changes in skeletal muscle cells and their niche may impair the regeneration potential. While strategies to regenerate muscle are being developed, in vitro models to investigate the regenerative potential after injury in aged versus young muscle cells are lacking. PURPOSE: This study aims to develop in vitro muscle models of injury and regeneration using cells from older and young donors. METHODS: Differentiated C2C12 murine and human myoblasts from old and young donors were injured after exposure to 12% barium chloride for 6 h. Regeneration was assessed by morphological analysis including myotube width, number of nuclei and fusion index. For that cells were stained with phalloidin and DAPI to label the cytoskeleton and nuclei of muscle cells, respectively. Images were acquired with the Leica fluorescence microscope and analysed using ImageJ. Timepoints were pre-injury (control), post-injury, end of proliferation and end of differentiation. RESULTS: The number of myonuclei post-injury remained unaltered compared to pre-injury among all cell lines (p > 0.05). Three days post-injury, nuclei number reached significance only in murine (p = 0.0006) and young (p = 0.03) muscle cells, whereas aged myotubes showed a significant increase in the total number of myonuclei at the end of the regeneration period (p = 0.01). Myotube diameter at the end of the regeneration recovered to pre-injury state in C2C12 and young but was significantly smaller in aged cells (p = 0.04). CONCLUSION: Delayed peak in proliferation and significantly smaller myotubes in aged compared to young and murine muscle cells at the end of the experiment could be attributed to declined proliferation rate of resident satellite cells that causes suspended myogenesis and regenerative response in older muscle cells. Barium chloride injury model is an easily reproducible in vitro approach that could be applied to study age and/or disease-related regenerative changes in different cell lines. KEYWORDS: regeneration, injury, skeletal muscle, ageing

Skeletal muscle is a highly adaptive tissue that responds to different stimuli such as exercise and supplements. However, age-related changes in muscle fibres can modify such adaptive responses with negative impacts on muscle health. Several genes and molecular mechanisms have been identified, but the full array remains incomplete. PURPOSE: To identify the most influenced genes associated with (i) age-related changes in skeletal muscle; (ii) long term exercise in older adults and (iii) supplement administration in older adults. METHODS: Six publicly available datasets were selected from the Gene Expression Omnibus data repository. Inclusion criteria were transcriptome data from the skeletal muscle of aged healthy adults on chronic exercise training and dietary supplementation with a duration of 12 weeks minimum. Artificial intelligence based - data mining was used to identify enriched gene transcripts. Enriched gene transcripts were then fed into an artificial neural network inference (ANNI) algorithM to identify the interactions between these key genes. The top100 genes with the strongest interactions were selected as the drivers and as the most influenced genes of the network (FDR < 0.05). The interactome map and Gene Ontology pathways were identified by Cytoscape and STRING software, respectively. RESULTS: The most influenced genes linked to skeletal muscle ageing were CHAD, CDKN1A, LOXL4 and ARHGAP, while in chronic exercise training were SCFD1, SRSF10, KDMD5D and MID2. In long-term supplement administration, these were CD163, SLC6A2, DTX3 and UMPS. Gene validation by real-time quantitative PCR is about to be performed. The most enriched GO pathways related to skeletal muscle ageing were bone development, cyclin activity and DNA repair pathways, exercise was protein binding, mRNA metabolism and chromatin organization and in supplement administration were transmembrane transporters, nucleotide metabolism and anti-inflammatory response pathways. CONCLUSIONS: Novel genes observed by ANNI analysis as the most influenced suggest new pathways regulated by age-related changes and chronic resistance exercise and dietary supplementation in skeletal muscle of older adults. Our findings could represent new metabolic and regulatory pathways as therapeutic targets in ageing skeletal muscle.

The development of safe and practical strategies to prevent weakening of bone tissue is vital, yet attempts to achieve this have been hindered by a lack of understanding of the short-term (days-weeks) physiology of bone collagen turnover. To address this, we have developed a method to quantify bone collagen synthesis in vivo, using deuterium oxide (D2 O) tracer incorporation techniques combined with gas chromatography pyrolysis isotope-ratio mass spectrometry (GC-pyrolysis-IRMS). Forty-six male and female rats from a selectively bred model ingested D2 O for 3 weeks. Femur diaphyses (FEM), tibia proximal (T-PRO), and distal (T-DIS) epiphyses-metaphyses and tibia mid-shaft diaphyses (T-MID) were obtained from all rats after necropsy. After demineralisation, collagen proteins were isolated and hydrolysed and collagen fractional synthetic rates (FSRs) determined by incorporation of deuterium into protein-bound alanine via GC-pyrolysis-IRMS. The collagen FSR for the FEM (0.131 ± 0.078%/day; 95% CI [0.106-0.156]) was greater than the FSR at T-MID (0.055 ± 0.049%/day; 95% CI [0.040-0.070]; p < 0.001). The T-PRO site had the highest FSR (0.203 ± 0.123%/day; 95% CI [0.166-0.241]) and T-DIS the lowest (0.027 ± 0.015%/day; 95% CI [0.022-0.031]). The three tibial sites exhibited different FSRs (p < 0.001). Herein, we have developed a sensitive method to quantify in vivo bone collagen synthesis and identified site-specific rates of synthesis, which could be applicable to studies of human bone collagen turnover.

Objectives: Menstrual cups come in a range of shapes, sizes, and firmnesses, but unlike tampons are not categorized in any way. With these factors having an impact on product leaks and comfort, as well as being linked to illness and injury, women need the same level of transparency when purchasing a menstrual cup. The comparison of physical and mechanical properties of menstrual cups will be the first step to achieve this. Methods: In October 2020, 14 popular and highly rated menstrual cups underwent quantitative comparison in laboratory settings (the United Kingdom), and they were compared in terms of their dimensions, volume, and compressive strength (firmness) using the Instron Universal Testing System. The overall designs were compared including shape, material, and features. Results: Although all the products in this comparison were marketed to women below 30 years of age having never given birth, total volume varied from 18.88 mL to 38.14 mL, and compressive load to compress the menstrual cup 50% (±0.5%) maximum diameter varied from 3.39 N to 13.92 N. Conclusions: Women are not sufficiently informed when choosing a menstrual cup. With no correlation between menstrual cup size, shape, and its volume, or material, shape, and its firmness, consumers cannot estimate which menstrual cup might be most suitable, and incorrect choice could cause injury. Transparency is needed across menstrual cup brands. With this and further regulation, women will make an informed decision to choose the correct menstrual cup and minimize injury. This work recommends firmness categories, ranging from ‘very soft’ to ‘very firm’ as a first step.

Exercise is a non-pharmacological intervention that can enhance bone regeneration and improve the management of bone conditions like osteoporosis or metastatic bone cancer. Therefore, it is gaining increasing importance in an emerging area of regenerative medicine—regenerative rehabilitation (RR). Osteocytes are mechanosensitive and secretory bone cells that orchestrate bone anabolism and hence postulated to be an attractive target of regenerative exercise interventions. However, the human osteocyte signalling pathways and processes evoked upon exercise remain to be fully identified. Making use of a computer-controlled bioreactor that mimics exercise and the latest omics approaches, RNA sequencing (RNA-seq) and tandem liquid chromatography-mass spectrometry (LC-MS), we mapped the transcriptome and secretome of mechanically stretched human osteocytic cells. We discovered that a single bout of cyclic stretch activated network processes and signalling pathways likely to modulate bone regeneration and cancer. Furthermore, a comparison between the transcriptome and secretome of stretched human and mouse osteocytic cells revealed dissimilar results, despite both species sharing evolutionarily conserved signalling pathways. These findings suggest that osteocytes can be targeted by exercise-driven RR protocols aiming to modulate bone regeneration or metastatic bone cancer.

We evaluated whether insulin could stimulate β-alanine uptake by skeletal muscle cells in vitro. Mouse myoblasts (C2C12) (n = 3 wells per condition) were cultured with β-alanine (350 or 700 µmol·L−1), with insulin (100 µU·mL−1) either added to the media or not. Insulin stimulated the β-alanine uptake at the lower (350 µmol·L−1) but not higher (700 µmol·L−1) β-alanine concentration in culture medium, indicating that transporter saturation might blunt the stimulatory effects of insulin.

BACKGROUND:Diabetes is a major public health issue and there is a need to develop low-cost, novel interventions to prevent or reduce disease progression. Growing evidence shows that supplementation with carnosine, or its rate-limiting precursor β-alanine, can ameliorate aspects of the metabolic dysregulation that occurs in diabetes. There is, however, a need to develop a better understanding of the magnitude of effect and the factors associated with positive outcomes. The purpose of this systematic review and meta-analysis is to evaluate the effect of carnosine or β-alanine supplementation on markers of glycaemic control and insulin resistance in humans and animals.METHODS:We will perform a systematic search for randomised and non-randomised controlled trials. Studies will be retrieved by searching electronic databases, clinical trial registers, author review, and cross-referencing. Primary outcomes include changes in (i) fasting glucose, (ii) glycated haemoglobin, and (iii) 2-h glucose following a glucose tolerance test. A set of additional outcomes includes other markers of glycaemic control and insulin resistance. Risk of bias (RoB) will be assessed using the Cochrane RoB 2.0 tool (human studies) and the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) RoB tool (animal studies). Confidence in the cumulative evidence will be assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. All meta-analyses will be conducted within a Bayesian framework, providing a flexible modelling approach to account for uncertainty in model parameters and underlying structures within the data.DISCUSSION:By including all available human and animal data, we will provide the most comprehensive overview on the topic to date. The results will have implications for those working in prediabetes, diabetes, and metabolic health in general and may lead to the development of new treatment approaches.DISSEMINATION:Study results will be presented at a professional conference and published in a peer-reviewed journal.SYSTEMATIC REVIEW REGISTRATION:CRD42020191588.

To test whether high circulating insulin concentrations influence the transport of β-alanine into skeletal muscle at either saturating or subsaturating β-alanine concentrations, we conducted two experiments whereby β-alanine and insulin concentrations were controlled. In experiment 1, 12 men received supraphysiological amounts of β-alanine intravenously (0.11 g·kg-1·min-1 for 150 min), with or without insulin infusion. β-Alanine and carnosine were measured in muscle before and 30 min after infusion. Blood samples were taken throughout the infusion protocol for plasma insulin and β-alanine analyses. β-Alanine content in 24-h urine was assessed. In experiment 2, six men ingested typical doses of β-alanine (10 mg/kg) before insulin infusion or no infusion. β-Alanine was assessed in muscle before and 120 min following ingestion. In experiment 1, no differences between conditions were shown for plasma β-alanine, muscle β-alanine, muscle carnosine and urinary β-alanine concentrations (all P > 0.05). In experiment 2, no differences between conditions were shown for plasma β-alanine or muscle β-alanine concentrations (all P > 0.05). Hyperinsulinemia did not increase β-alanine uptake by skeletal muscle cells, neither when substrate concentrations exceed the Vmax of β-alanine transporter TauT nor when it was below saturation. These results suggest that increasing insulin concentration is not necessary to maximize β-alanine transport into muscle following β-alanine intake.

Osteocytes are secretory bone cells that regulate bone homeostasis and for this reason, are often coined as the “brain of the bone”. In vitro studies demonstrated that mechanically stimulated osteocytes release interleukins and growth factors that help coordinating bone formation and resorption, however, their secretome remains largely unknown. PURPOSE: To investigate WNT signalling and the secretome of mouse and human osteocyte-like cells. Insights from this study could help to devise informed therapeutic exercise regimen e.g. aiming to preserve bone mass across ageing or accelerate bone fracture healing. METHODS: The murine MLO-Y4 (Kerafast) cell line was cultured according to Kerafast instructions. Human adipose stem cells (ATCC® PCS -500-011™) were expanded and differentiated into osteocyte-like cells (hOC) according to ATCC instructions. Cells were cultured in a computer-controlled bioreactor (Flexcell Int) for mechanical loading (3.4%, 2Hz, 5h). Static cultures were used as control. Relative expression of 84 key genes of the WNT signalling pathway (Sabiosciences) was quantified by RT-qPCR. Relative protein expression was estimated by western blotting. The secretome was analysed by quantitative mass spectrometry (TripleTOF 6600, SCIEX) using SWATH and IDA and processed using OneOmics (SCIEX) software. RESULTS: The relative gene expression remained unchanged in mechanically MLO-Y4 and hOC. Regarding protein quantification, active β-catenin and Cyclin D1 showed an up-regulation trend in mechanically stimulated MLO-Y4 but this was not statistically significant. A total of 917 proteins were identified in the MLO-Y4 secretome, ~12% present exclusively under mechanical active conditions. The secretome obtained under loading contained 14 cyclin-dependent kinases (CDKs) including CDK6, a critical regulator of osteoblasts and osteoclasts differentiation. A total of 329 proteins were identified in the supernatant of hOC, ~9% present exclusively under mechanical stimulation. Unlike MLO-Y4, no CDKs were identified in this cell type. The small ubiquitin-like modifier (SUMO) 2 and 3 were present in the secretomes of mechanically loaded MLO-Y4 and hOC. CONCLUSION: Mechanically stimulated osteocyte-like cells secrete a specific set of proteins which could impact bone health and regeneration.