Coapplication of magnesium supplementation and vibration modulate macrophage polarization to attenuate sarcopenic muscle atrophy through PI3K/Akt/mTOR signaling pathway

Sarcopenia is an age-related geriatric syndrome characterized by the gradual loss of muscle mass and function. Low-magnitude high-frequency vibration (LMHFV) was shown to be beneficial to structural and functional outcomes of skeletal muscles, while magnesium (Mg) is a cofactor associated with better indices of skeletal muscle mass and strength. We hypothesized that LMHFV, Mg and their combinations could suppress inflammation and sarcopenic atrophy, promote myogenesis via PI3k/Akt/mTOR pathway in senescence-accelerated mouse P8 (SAMP8) mice and C2C12 myoblasts. Results showed that Mg treatment and LMHFV could significantly decrease inflammatory expression (C/EBPα and LYVE1) and modulate a CD206-positive M2 macrophage population at month four. Mg treatment also showed significant inhibitory effects on FOXO3, MuRF1 and MAFbx mRNA expression. Coapplication showed a synergistic effect on suppression of type I fiber atrophy, with significantly higher IGF-1, MyoD, MyoG mRNA (p < 0.05) and pAkt protein expression (p < 0.0001) during sarcopenia. In vitro inhibition of PI3K/Akt and mTOR abolished the enhancement effects on myotube formation and inhibited MRF mRNA and p85, Akt, pAkt and mTOR protein expressions. The present study demonstrated that the PI3K/Akt/mTOR pathway is the predominant regulatory mechanism through which LMHFV and Mg enhanced muscle regeneration and suppressed atrogene upregulation

Biophysical and nutritional combination treatment for myosteatosis in patients with sarcopenia: a study protocol for single-blinded randomised controlled trial

Introduction Sarcopenia is characterised by age-related loss of skeletal muscle and function and is associated with risks of adverse outcomes. The prevalence of sarcopenia increases due to ageing population and effective interventions is in need. Previous studies showed that β-hydroxy β-methylbutyrate (HMB) supplement and vibration treatment (VT) enhanced muscle quality, while the coapplication of the two interventions had further improved muscle mass and function in sarcopenic mice model. This study aims to investigate the efficacy of this combination treatment in combating sarcopenia in older people. The findings of this study will demonstrate the effect of combination treatment as an alternative for managing sarcopenia. Methods and analysis In this single-blinded randomised controlled trial, subjects will be screened based on the Asian Working Group for Sarcopenia (AWGS) 2019 definition. 200 subjects who are aged 65 or above and identified sarcopenic according to the AWGS algorithm will be recruited. They will be randomised to one of the following four groups: (1) Control+ONS; (2) HMB+ONS; (3) VT+ONS and (4) HMB+VT + ONS, where ONS stands for oral nutritional supplement. ONS will be taken in the form of protein formular once/day; HMB supplements will be 3 g/day; VT (35 Hz, 0.3 g, where g=gravitational acceleration) will be received for 20 mins/day and at least 3 days/week. The primary outcome assessments are muscle strength and function. Subjects will be assessed at baseline, 3-month and 6-month post treatment. Ethics and dissemination This study was approved by Joint CUHK-NTEC (The Chinese University of Hong Kong and New Territories East Cluster) Clinical Research Management Office (Ref: CRE-2022.223-T) and conformed to the Declaration of Helsinki. Trial results will be published in peer-reviewed journals and disseminated at academic conferences

Glycolytic reprogramming in macrophages and MSCs during inflammation

BackgroundDysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs).MethodsWe used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE.ResultsOur results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines.ConclusionThis study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation

Ultrasound as a stimulus for musculoskeletal disorders

Ultrasound is an inaudible form of acoustic sound wave at 20 kHz or above that is widely used in the medical field with applications including medical imaging and therapeutic stimulation. In therapeutic ultrasound, low-intensity pulsed ultrasound (LIPUS) is the most widely used and studied form that generally uses acoustic waves at an intensity of 30 mW/cm2, with 200 ms pulses and 1.5 MHz. In orthopaedic applications, it is used as a biophysical stimulus for musculoskeletal tissue repair to enhance tissue regeneration. LIPUS has been shown to enhance fracture healing by shortening the time to heal and reestablishment of mechanical properties through enhancing different phases of the healing process, including the inflammatory phase, callus formation, and callus remodelling phase. Reports from in vitro studies reveal insights in the mechanism through which acoustic stimulations activate cell surface integrins that, in turn, activate various mechanical transduction pathways including FAK (focal adhesion kinase), ERK (extracellular signal-regulated kinase), PI3K, and Akt. It is then followed by the production of cyclooxygenase 2 and prostaglandin E2 to stimulate further downstream angiogenic, osteogenic, and chondrogenic cytokines, explaining the different enhancements observed in animal and clinical studies. Furthermore, LIPUS has also been shown to have remarkable effects on mesenchymal stem cells (MSCs) in musculoskeletal injuries and tissue regeneration. The recruitment of MSCs to injury sites by LIPUS requires the SDF-1 (stromal cell derived factor-1)/CXCR-4 signalling axis. MSCs would then differentiate differently, and this is regulated by the presence of different cytokines, which determines their fates. Other musculoskeletal applications including bone–tendon junction healing, and distraction osteogenesis are also explored, and the results are promising. However, the use of LIPUS is controversial in treating osteoporosis, with negative findings in clinical settings, which may be attributable to the absence of an injury entry point for the acoustic signal to propagate, strong attenuation effect of cortical bone and the insufficient intensity for penetration, whereas in some animal studies it has proven effective

Inflammation and age-associated skeletal muscle deterioration (sarcopaenia)

Ageing is accompanied by chronic inflammatory responses due to elevated circulatory inflammatory cytokine production. Several inflammatory cytokines have been shown to be responsible for a decrease in muscle mass. However, little is known about the possible relationship between inflammation and sarcopaenia. This review aims to summarise the existing evidence about inflammation and sarcopaenia. Sarcopaenia is defined as an age-related decrease of muscle mass and/or muscle strength; it is caused by multiple factors, such as skeletal muscle atrophy, neuromuscular junction degeneration, hormone imbalance, cytokine imbalance, protein synthesis and proteolysis. Several inflammatory cytokines have been considered to promote muscle loss; C-reactive protein levels are significantly upregulated in sarcopaenia and sarcopenic obesity, and high levels of interleukin-6 are associated with reduced muscle mass and muscle strength (the administration of interleukin-6 could lead to a reduction in muscle mass). Up-regulation of tumour necrosis factor-α expression is also related to the development of sarcopaenia. Signalling pathways, such as protein kinase B/mammalian target of rapamycin, Janus kinase/signal transducer and activator of transcription-5 and signal transducer and activator of transcription 3 signalling, involved in muscle metabolism are regulated by insulin-like growth factor-1, tumour necrosis factor-α and interleukin-6 respectively. In conclusion, the inflammatory cytokines produced during chronic inflammation due to ageing, may influence their respective related pathways, thus leading to age-related muscle deterioration. The translational potential of this article: This review can provide more information for sarcopaenia medicine research in terms of anti-inflammation therapy

The role of osteocytes-specific molecular mechanism in regulation of mechanotransduction – A systematic review

Background: Osteocytes, composing over 90% of bone cells, are well known for their mechanosensing abilities. Aged osteocytes with impaired morphology and function are less efficient in mechanotransduction which will disrupt bone turnover leading to osteoporosis. The aim of this systematic review is to delineate the mechanotransduction mechanism at different stages in order to explore potential target for therapeutic drugs. Methods: A systematic literature search was performed in PubMed and Web of Science. Original animal, cell and clinical studies with available English full-text were included. Information was extracted from the included studies for review. Results: The 26 studies included in this review provided evidence that mechanical loading are sensed by osteocytes via various sensing proteins and transduced to different signaling molecules which later initiate various biochemical responses. Studies have shown that osteocyte plasma membrane and cytoskeletons are emerging key players in initiating mechanotransduction. Bone regulating genes expressions are altered in response to load sensed by osteocytes, but the genes involved different signaling pathways and the spatiotemporal expression pattern had made mechanotransduction mechanism complicated. Most of the included studies described the important role of osteocytes in pathways that regulate mechanosensing and bone remodeling. Conclusions: This systematic review provides an up-to-date insight to different steps of mechanotransduction. A better understanding of the mechanotransduction mechanism is beneficial in search of new potential treatment for osteoporotic patients. By delineating the unique morphology of osteocytes and their interconnected signaling network new targets can be discovered for drug development. Translational potential of this article: This systematic review provides an up-to-date sequential overview and highlights the different osteocyte-related pathways and signaling molecules during mechanotransduction. This allows a better understanding of mechanotransduction for future development of new therapeutic interventions to treat patients with impaired mechanosensitivity

Efficacy of low-magnitude high-frequency vibration (LMHFV) on musculoskeletal health of participants on wheelchair: a study protocol for a single-blinded randomised controlled study

Background Osteoporosis is an age-related disease with progressive loss of bone, leading to fragile bone. It is one of the major health issues in older adults and causes medical, social and economic impacts globally. Patients with osteoporosis have high risk of osteoporotic fractures. Low-magnitude high-frequency vibration (LMHFV) is a non-invasive biophysical intervention providing whole-body mechanical stimulation. Previous studies showed that LMHFV is beneficial to muscle strength, postural control, balancing ability, new bone formation, spinal bone mineral density (BMD) and blood circulation. During the LMHFV treatment, older adults need to stand upright on the platform for 20 min/day. However, some physically weak elderlies with poor musculoskeletal ability cannot stand for a long period. Therefore, the design of vibration platform is modified for the disabled patients to treat at sitting position and the efficacy of LMHFV on this group of elderlies will be verified. It is hypothesised that new design of LMHFV is beneficial to wheelchair users in terms of vertebral BMD, muscle health and musculoskeletal functions.Methods This study is a single-blinded randomised controlled trial to investigate the effect of LMHFV on vertebral BMD, muscle health, balancing ability and functional ability in wheelchair users (mainly on wheelchair for outdoor activities). Healthy elderlies aged 65 years or above with walking difficulties and using wheelchair are eligible. Exclusion criteria are those: (1) who cannot stand and walk independently, (2) who have vibration treatment before, (3) with malignancy, (4) with acute fractures or severe osteoarthritis, (5) with cardiovascular concern such as with pacemaker in situ, (6) with chronic inflammatory conditions known to affect muscle metabolism such as rheumatoid arthritis and (7) with high frequency of physical activities, such as participants who participated in regular exercise five times a week or more. Recruited participants will be randomised to either LMHFV or control group. Participant assigned to LMHFV group will receive LMHFV (35 Hz, 0.3g (g=gravitational acceleration), 20 min/day, at least three times/week) for 6 months. The primary outcome is BMD at the lumbar spine to be assessed by dual-energy X-ray absorptiometry that is clinically recommended for the diagnosis of osteoporosis. All primary and secondary outcome assessments for all groups will be performed in the investigators’ institute at baseline and 6 months post treatment.Discussion This study aims to investigate the effects of LMHFV on wheelchair users. The findings of this study will help to confirm the efficacy of LMHFV on vertebral BMD, muscle health, balancing ability and functional outcomes in wheelchair using elderlies. LMHFV therapy is an intervention strategy that is easy to implement at the community healthcare level or individually at home that has previously been proven to reduce fall risk and muscle strength at the lower limb. The ultimate goal is to improve their bone and muscle quality of wheelchair users, as well as enhancing their quality of life.Trial registration number ClinicalTrials.gov (NCT04180267)

Bone regeneration in inflammation with aging and cell-based immunomodulatory therapy

Abstract Aging of the global population increases the incidence of osteoporosis and associated fragility fractures, significantly impacting patient quality of life and healthcare costs. The acute inflammatory reaction is essential to initiate healing after injury. However, aging is associated with “inflammaging”, referring to the presence of systemic low-level chronic inflammation. Chronic inflammation impairs the initiation of bone regeneration in elderly patients. This review examines current knowledge of the bone regeneration process and potential immunomodulatory therapies to facilitate bone healing in inflammaging. Aged macrophages show increased sensitivity and responsiveness to inflammatory signals. While M1 macrophages are activated during the acute inflammatory response, proper resolution of the inflammatory phase involves repolarizing pro-inflammatory M1 macrophages to an anti-inflammatory M2 phenotype associated with tissue regeneration. In aging, persistent chronic inflammation resulting from the failure of M1 to M2 repolarization leads to increased osteoclast activation and decreased osteoblast formation, thus increasing bone resorption and decreasing bone formation during healing. Inflammaging can impair the ability of stem cells to support bone regeneration and contributes to the decline in bone mass and strength that occurs with aging. Therefore, modulating inflammaging is a promising approach for improving bone health in the aging population. Mesenchymal stem cells (MSCs) possess immunomodulatory properties that may benefit bone regeneration in inflammation. Preconditioning MSCs with pro-inflammatory cytokines affects MSCs’ secretory profile and osteogenic ability. MSCs cultured under hypoxic conditions show increased proliferation rates and secretion of growth factors. Resolution of inflammation via local delivery of anti-inflammatory cytokines is also a potential therapy for bone regeneration in inflammaging. Scaffolds containing anti-inflammatory cytokines, unaltered MSCs, and genetically modified MSCs can also have therapeutic potential. MSC exosomes can increase the migration of MSCs to the fracture site and enhance osteogenic differentiation and angiogenesis. In conclusion, inflammaging can impair the proper initiation of bone regeneration in the elderly. Modulating inflammaging is a promising approach for improving compromised bone healing in the aging population

Low intensity pulsed ultrasound enhanced mesenchymal stem cell recruitment through stromal derived factor-1 signaling in fracture healing.

Low intensity pulsed ultrasound (LIPUS) has been proven effective in promoting fracture healing but the underlying mechanisms are not fully depicted. We examined the effect of LIPUS on the recruitment of mesenchymal stem cells (MSCs) and the pivotal role of stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4) pathway in response to LIPUS stimulation, which are essential factors in bone fracture healing. For in vitro study, isolated rat MSCs were divided into control or LIPUS group. LIPUS treatment was given 20 minutes/day at 37 °C for 3 days. Control group received sham LIPUS treatment. After treatment, intracellular CXCR4 mRNA, SDF-1 mRNA and secreted SDF-1 protein levels were quantified, and MSCs migration was evaluated with or without blocking SDF-1/CXCR4 pathway by AMD3100. For in vivo study, fractured 8-week-old young rats received intracardiac administration of MSCs were assigned to LIPUS treatment, LIPUS+AMD3100 treatment or vehicle control group. The migration of transplanted MSC to the fracture site was investigated by ex vivo fluorescent imaging. SDF-1 protein levels at fracture site and in serum were examined. Fracture healing parameters, including callus morphology, micro-architecture of the callus and biomechanical properties of the healing bone were investigated. The in vitro results showed that LIPUS upregulated SDF-1 and CXCR4 expressions in MSCs, and elevated SDF-1 protein level in the conditioned medium. MSCs migration was promoted by LIPUS and partially inhibited by AMD3100. In vivo study demonstrated that LIPUS promoted MSCs migration to the fracture site, which was associated with an increase of local and serum SDF-1 level, the changes in callus formation, and the improvement of callus microarchitecture and mechanical properties; whereas the blockade of SDF-1/CXCR4 signaling attenuated the LIPUS effects on the fractured bones. These results suggested SDF-1 mediated MSCs migration might be one of the crucial mechanisms through which LIPUS exerted influence on fracture healing