Low magnetic field promotes recombinant human BMP-2-induced bone formation and influences orientation of trabeculae and bone marrow-derived stromal cells

Effects of high magnetic fields [MFs, ≥ 1 T (T)] on osteoblastic differentiation and the orientation of cells or matrix proteins have been reported. However, the effect of low MFs (< 1 T) on the orientation of bone formation is not well known. This study was performed to verify the effects of low MFs on osteoblastic differentiation, bone formation, and orientation of both cells and newly formed bone. An apparatus was prepared with two magnets (190 mT) aligned in parallel to generate a parallel MF. In vitro, bone marrow-derived stromal cells of rats were used to assess the effects of low MFs on cell orientation, osteoblastic differentiation, and mineralization. A bone morphogenetic protein (BMP)-2-induced ectopic bone model was used to elucidate the effect of low MFs on microstructural indices, trabecula orientation, and the apatite c-axis orientation of newly formed bone. Low MFs resulted in an increased ratio of cells oriented perpendicular to the direction of the MF and promoted osteoblastic differentiation in vitro. Moreover, in vivo analysis demonstrated that low MFs promoted bone formation and changed the orientation of trabeculae and apatite crystal in a direction perpendicular to the MF. These changes led to an increase in the mechanical strength of rhBMP-2-induced bone. These results suggest that the application of low MFs has potential to facilitate the regeneration of bone with sufficient mechanical strength by controlling the orientation of newly formed bone.Okada R., Yamato K., Kawakami M., et al. Low magnetic field promotes recombinant human BMP-2-induced bone formation and influences orientation of trabeculae and bone marrow-derived stromal cells. Bone Reports, 14, 100757. https://doi.org/10.1016/j.bonr.2021.100757

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

Gait Variability to Phenotype Common Orthopedic Gait Impairments Using Wearable Sensors

Mobility impairments are a common symptom of age-related degenerative diseases. Gait features can discriminate those with mobility disorders from healthy individuals, yet phenotyping specific pathologies remains challenging. This study aims to identify if gait parameters derived from two foot-mounted inertial measurement units (IMU) during the 6 min walk test (6MWT) can phenotype mobility impairment from different pathologies (Lumbar spinal stenosis (LSS)—neurogenic diseases, and knee osteoarthritis (KOA)—structural joint disease). Bilateral foot-mounted IMU data during the 6MWT were collected from patients with LSS and KOA and matched healthy controls (N = 30, 10 for each group). Eleven gait parameters representing four domains (pace, rhythm, asymmetry, variability) were derived for each minute of the 6MWT. In the entire 6MWT, gait parameters in all four domains distinguished between controls and both disease groups; however, the disease groups demonstrated no statistical differences, with a trend toward higher stride length variability in the LSS group (p = 0.057). Additional minute-by-minute comparisons identified stride length variability as a statistically significant marker between disease groups during the middle portion of 6WMT (3rd min: p ≤ 0.05; 4th min: p = 0.06). These findings demonstrate that gait variability measures are a potential biomarker to phenotype mobility impairment from different pathologies. Increased gait variability indicates loss of gait rhythmicity, a common feature in neurologic impairment of locomotor control, thus reflecting the underlying mechanism for the gait impairment in LSS. Findings from this work also identify the middle portion of the 6MWT as a potential window to detect subtle gait differences between individuals with different origins of gait impairment

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

C-C Motif Chemokine Ligand 2 Enhances Macrophage Chemotaxis, Osteogenesis, and Angiogenesis during the Inflammatory Phase of Bone Regeneration

Local cell therapy has recently gained attention for the treatment of joint diseases and fractures. Mesenchymal stem cells (MSCs) are not only involved in osteogenesis and angiogenesis, but they also have immunomodulatory functions, such as inducing macrophage migration during bone regeneration via macrophage crosstalk. C-C motif chemokine ligand 2 (CCL2), a known inflammatory mediator, is associated with the migration of macrophages during inflammation. This study examined the utility of CCL2 as a therapeutic target for local cell therapy. Using lentiviral vectors for rabbit MSCs, genetically modified CCL2 overexpressing MSCs were generated. Osteogenic differentiation assays were performed using MSCs with or without macrophages in co-culture, and cell migration assays were also performed. Additionally, co-cultures were performed with endothelial cells (ECs), and angiogenesis was evaluated using a tube formation assay. Overexpression of CCL2 did not affect bone formation under monoculture conditions but promoted chemotaxis and osteogenesis when co-cultured with macrophages. Furthermore, CCL2-overexpression promoted tube formation in co-culture with ECs. These results suggest that CCL2 induces macrophage chemotaxis and osteogenesis by promoting crosstalk between MSCs and macrophages; CCL2 also stimulates ECs to induce angiogenesis. These findings indicate that CCL2 may be a useful therapeutic target for local cell therapy in areas of bone loss

Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model

Abstract Background Continuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway. Methods We cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species—ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs. Results Exposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days. Conclusion This work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism