Hyperbaric Oxygen Therapy Improves the Osteogenic and Vasculogenic Properties of Mesenchymal Stem Cells in the Presence of Inflammation In Vitro.

Hyperbaric oxygen (HBO) therapy has been reported to be beneficial for treating many conditions of inflammation-associated bone loss. The aim of this work was to in vitro investigate the effect of HBO in the course of osteogenesis of human Mesenchymal Stem Cells (MSCs) grown in a simulated pro-inflammatory environment. Cells were cultured with osteogenic differentiation factors in the presence or not of the pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α), and simultaneously exposed daily for 60 min, and up to 21 days, at 2,4 atmosphere absolute (ATA) and 100% O2. To elucidate osteogenic differentiation-dependent effects, cells were additionally pre-committed prior to treatments. Cell metabolic activity was evaluated by means of the MTT assay and DNA content quantification, whereas osteogenic and vasculogenic differentiation was assessed by quantification of extracellular calcium deposition and gene expression analysis. Metabolic activity and osteogenic properties of cells did not differ between HBO, high pressure (HB) alone, or high oxygen (HO) alone and control if cells were pre-differentiated to the osteogenic lineage. In contrast, when treatments started contextually to the osteogenic differentiation of the cells, a significant reduction in cell metabolic activity first, and in mineral deposition at later time points, were observed in the HBO-treated group. Interestingly, TNF-α supplementation determined a significant improvement in the osteogenic capacity of cells subjected to HBO, which was not observed in TNF-α-treated cells exposed to HB or HO alone. This study suggests that exposure of osteogenic-differentiating MSCs to HBO under in vitro simulated inflammatory conditions enhances differentiation towards the osteogenic phenotype, providing evidence of the potential application of HBO in all those processes requiring bone regeneration.

Age-dependent variations in the expression of myosin isoforms and myogenic factors during the involution of the proximal sesamoidean ligament of sheep.

In ungulates the stability of the fetlock joint is dependent on several muscles, which are exposed to high stress and strain. Among those muscles, the proximal sesamoidean ligament or PSL (also known as the suspensory ligament or Ruini's elasto-tendinous organ) is organized at birth in layers of muscle fibres alternated with abundant tendinous tissue that, during the postnatal development, becomes the predominant tissue. In this study we analysed the PSL of the sheep at the age of 1, 30 and 180 days and determined the expression of several genes which either (a) are markers of muscle fibre growth and maturation, or (b) play a role as signal molecules. We observed an accelerated maturation, as indicated by the transition of MyHC isoform expression towards the slow isoforms and a reduced regenerative potential indicated by the low Pax7 expression and the altered Wnt signalling. We also found a specific myogenic expression pattern of MyoD, Myf5 and Myogenin in the developing PSL and high mRNA levels of specific fibrogenic factors, as TGF-ß1, that, undoubtedly, stimulate the growth of connective tissue. Our observations confirmed, at molecular level, the peculiarity of the fast involution observed in PSL a muscle that undergoes a very specific active differentiation process during early development, which implies myofibres involution and their replacement with connective tissue.

Hyperbaric oxygen therapy ameliorates osteonecrosis in patients by modulating inflammation and oxidative stress.

Early stages of avascular necrosis of the femoral head (AVNFH) can be conservatively treated with hyperbaric oxygen therapy (HBOT). This study investigated how HBOT modulates inflammatory markers and reactive oxygen species (ROS) in patients with AVNFH. Twenty-three male patients were treated with two cycles of HBOT, 30 sessions each with a 30 days break between cycles. Each session consisted of 90 minutes of 100% inspired oxygen at 2.5 absolute atmospheres of pressure. Plasma levels of tumor necrosis factor alfa (TNF-α), interleukin 6 (IL-6), interleukin 1 beta (IL-1ß) and ROS production were measured before treatment (T0), after 15 and 30 HBOT sessions (T1 and T2), after the 30-day break (T3), and after 60 sessions (T4). Results showed a significant reduction in TNF-α and IL-6 plasma levels over time. This decrease in inflammatory markers mirrored observed reductions in bone marrow edema and reductions in patient self-reported pain.

Spirometry and oxidative stress after rebreather diving in warm water.

INTRODUCTION: Hyperbaric oxygen (HBO2) therapy and use of enriched air can result in oxidative injury affecting the brain, lungs and eyes. HBO2 exposure during diving can lead to a decrease in respiratory parameters. However, the possible effects of acute exposure to oxygen-enriched diving on subsequent spirometric performance and oxidative state in humans have not been recently described recently. We aim to investigate possible effects of acute (i) hyperbaric and (ii) hyperbaric hyperoxic exposure using scuba or closed-circuit rebreather (CCR) on subsequent spirometry and to assess the role of oxidative state after hyperoxic diving. METHODS: Spirometry and urine samples were obtained from six well-trained divers (males, mean ± SD, age: 43.33 ± 9.16 years; weight: 79.00 ± 4.90 kg; height: 1.77 ± 0.07 meters) before (CTRL) and after a dive breathing air, and after a dive using CCR (PO2 1.4). In the crossover design (two dives separated by six hours) each subject performed a 20-minute session of light underwater exercise at a depth of 15 meters in warm water (31-32°C). We measured urinary 8-isoprostane and 8-OH-2-deoxyguanosine evaluating lipid and DNA oxidative damages. RESULTS: Different breathing conditions (air vs. CCR) did not significantly affect spirometry. A significant increase of 8-OH-dG (1.85 ± 0.66 vs. 4.35 ± 2.12; P ⟨ 0.05) and 8-isoprostane (1.35 ± 0.20 vs. 2.59 ± 0.61; P ⟨ 0.05) levels after CCR dive with respect to the CTRL was observed. Subjects did not have any ill effects during diving. CONCLUSIONS: Subjects using CCR showed elevated oxidative stress, but this did not correlate with a reduction in pulmonary function.

Effects of the Ketogenic diet in overweight divers breathing Enriched Air Nitrox.

Central Nervous System Oxygen Toxicity (CNS-OT) is one of the most harmful effects of Enriched Air Nitrox (EAN) diving. Protective factors of the Ketogenic Diet (KD) are antioxidant activity, the prevention of mitochondrial damage and anti-inflammatory mechanisms. We aimed to investigate if a short-term KD may reduce oxidative stress and inflammation during an hyperoxic dive. Samples from six overweight divers (mean ± SD, age: 55.2 ± 4.96 years; BMI: 26.7 ± 0.86 kg/m2) were obtained a) before and after a dive breathing Enriched Air Nitrox and performing 20-minute mild underwater exercise, b) after a dive (same conditions) performed after 7 days of KD. We measured urinary 8-isoprostane and 8-OH-2-deoxyguanosine and plasmatic IL-1ß, IL-6 and TNF-α levels. The KD was successful in causing weight loss (3.20 ± 1.31 Kgs, p < 0.01) and in limiting lipid peroxidation (3.63 ± 1.16 vs. 1.11 ± 0.22; p < 0.01) and inflammatory response (IL-1ß = 105.7 ± 25.52 vs. 57.03 ± 16.32, p < 0.05; IL-6 = 28.91 ± 4.351 vs. 14.08 ± 1.74, p < 0.001; TNF-α = 78.01 ± 7.69 vs. 64.68 ± 14.56, p < 0.05). A short-term KD seems to be effective in weight loss, in decreasing inflammation and protective towards lipid peroxidation during hyperoxic diving.

Internist, anesthesiologist and surgeon use of ketogenic diet.

Ketogenic diet is being increasingly utilized in recent decades because of its success as an effective tool for short and intermediate-term weight loss. Promoting physiological ketosis from a drastically low carbohydrate diet is the fundamental basis for this diet regime. Though debated, these diets have been demonstrated to be effective, at least in the short- to medium terms, to manage excess weight, hyperlipidemia, and other cardiovascular risk factors. We reviewed the cardiovascular, metabolic, anesthetic, and postsurgical profiles in the literature and summarized technical issues of anesthesia and surgery along with long-term changes from published papers. Doubts with ketogenic diet were raised due to possible renal damage caused by significant excretion of nitrogen found in animal models, the effects of acidosis, and the concerns of increasing triglycerides and cholesterol levels. Though current literature supports the efficacy of very low carbohydrate keto-diets their potential negative effects on renal function and acidosis are debated. An increase in nitrogen excretion during protein metabolism in the postoperative period could lead to renal damage. Research on the value of ketogenic diets is emerging because of its value in weight loss and in managing other pathologies.

Review on hyperbaric oxygen treatment in femoral head necrosis.

BACKGROUND: Femoral head necrosis (FHN) is a common invalidating disease with an unclear etiology and pathophysiology that affects middle-aged people. FHN may lead to joint collapse and require invasive treatments. Because of its clinical and socioeconomic significance, an early diagnosis, staging and appropriate treatment are required. Unfortunately, to date a unique algorithm for the treatment of FHN has not been defined. OBJECTIVE: In this report we summarize the Tenth European Consensus Conference on hyperbaric medicine (April 2016, France), during which experts from Europe revised the list of accepted indications for hyperbaric oxygen (HBO2) treatment, including FHN. METHODS: In this report all aspects of osteonecrosis discussed during the meeting have been considered: pathophysiology,clinical presentation, standard management, HBO2 therapy and evidence-based review of its efficacy. All observations are based on a thorough review of the best available research and evidence-based medicine. CONCLUSIONS: The Consensus Conference in Lille established FHN as an indication for HBO2 therapy and suggested a protocols guideline to adopt for this pathology.

Cave canem: HBO2 therapy efficacy on Capnocytophaga canimorsus infections: a case series.

BACKGROUND: Dog bites are a frequent injury, but the incidence and type of lesions vary across countries. Although only few patients develop complications, the treatment of advanced injuries has a considerable medical, social and economic impact. A frequently isolated pathogen in dog bite wounds is Capnocytophaga canimorsus, a bacterium that can cause sepsis or meningitis. Hyperbaric oxygen (HBO2) therapy has been shown to be useful in treating anaerobic infections, most likely because it creates an inhospitable environment for the bacterium and enhances the patient's immune response. AIM: We present a case series of C. canimorsus infections treated with HBO2 in adjunction to antibiotic therapy. Furthermore, we tested the in vitro activity of ceftaroline against C. canimorsus, alone and in association with hyperbaric oxygen therapy. METHODS: We included nine (9) patients admitted to the surgery department of "A. Cardarelli" Hospital (Naples) after dog bite, from 2010 to 2016. All were initially treated with antibiotics and required transfer to the intensive care unit due to worsening conditions. C. canimorsus was isolated from wounds, and HBO2 therapy was administered in adjunction to antibiotics, until clinical improvement and microbiological test negativity. We tested the activity of hyperbaric oxygen therapy in adjunction to ceftaroline on cultured plates with C. canimorsus versus ceftaroline alone. Minimal inhibitory concentration was evaluated. CONCLUSIONS: Our findings confirm the utility of HBO2 therapy after biting injuries. Indeed, increased oxygen supply to the wound (as well as in vitro) may be toxic for bacteria, can improve healing and may improve the effectiveness of antibiotics.

Hyperbaric oxygen therapy modulates serum OPG/RANKL in femoral head necrosis patients.

Hyperbaric oxygen therapy (HBOT) has beneficial effects on avascular necrosis of femoral head (ANFH), but its mechanism of action is still unclear. We investigated if HBOT upregulates serum osteoprotegerin (OPG) and/or inhibits osteoclast activation. 23 patients with unilateral ANFH at stage I, II and III consented to the study: the patients received standard HBOT. Serum OPG levels were obtained at the beginning of HBOT (T0), after 15 sessions (T1), 30 sessions (T2), after a 30-day break (T3), and after 60 sessions (T4). Magnetic resonance imaging (MRI) was obtained at T0 and about one year from the end of HBO treatments. Lesion size was compared between pre- and post-HBOT. 19 patients completed the study. HBOT reduced pain symptoms in all patients. HBOT significantly reduced lesion size in all stage I and II patients and in 2 of 11 stage III patients. HBOT increased serum OPG levels but receptor activator of nuclear factor kappa-B ligand (RANKL) levels did not change.

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7.

BACKGROUND: Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs. METHODS: Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme-substrate and protein-protein interaction were analyzed by molecular docking and surface plasmon resonance analysis. RESULTS: Oxidation of the CP is fast (k+1>10(3)M(-1)s(-1)), however the rate of reduction by GSH is slow (k'+2=12.6M(-1)s(-1)) even though molecular docking indicates a strong GSH-GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+1>10(3)M(-1)s(-1)), but not by Trx. By surface plasmon resonance analysis, a KD=5.2µM was calculated for PDI-GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo. CONCLUSIONS: GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates. GENERAL SIGNIFICANCE: In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.

Band 3 tyr-phosphorylation in human erythrocytes from non-pregnant and pregnant women.

Pregnancy is associated with changes in circulating red blood cells, mainly involving band 3 protein and membrane lipid peroxidation. Membrane band 3 is a multifunctional protein containing four Tyr-phosphorylatable residues which modulate the physiological status of erythrocytes by regulating glycolysis, cell shape and membrane transport. Erythrocytes from nine pregnant and 12 age-matched non-pregnant healthy women were subjected to oxidative and hyperosmotic stress conditions and the extent of band 3 Tyr-phosphorylation and membrane Syk recruitment as a membrane marker were evaluated. Results indicated that, in pregnancy, red blood cells show a decrease in band 3 Tyr-phosphorylation and a clear-cut rearrangement of band 3 protein within the membrane. In fact, band 3 shows a decrease in high molecular weight aggregates (HMWA), with different subdivision between Triton-soluble and -insoluble compartments, and an increase in proteolytic fragments. In conclusion, it is demonstrated that pregnancy is associated with membrane adjustments which reduce the sensitivity of erythrocytes to both oxidative and osmotic stress. Band 3 Tyr-phosphorylation is proposed as a new parameter in the evaluation of erythrocyte membrane arrangement.