Shockwave fractures treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5’-triphosphate (ATP) release-induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c-fos and c-jun mRNA was determined with real-time reverse transcription polymerase chain reaction and Western blotting. P2X7-siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK sig naling in shockwave-induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (~7 μM) of ATP from hMSCs. Shockwaves and exogenous ATP induced c-fos and c-jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation.

The rac1 GTPase promotes oxidative stress through reactive oxygen species (ROS) production, whereas the DNA repair enzyme and transcriptional regulator redox factor-1 (ref-1) protects against cell death due to oxidative stimuli. However, the function of ref-1 in regulating intracellular oxidative stress, particularly that induced by rac1, has not been defined. We production), NF-k kB activation, and apoptosis, and also mitigated rac1-regulated H 2 O 2 production and NF-k kB transcriptional activity. We conclude that inhibition of oxidative stress is another mechanism by which ref-1 protects against apoptosis, and that this is achieved through modulation of cytoplasmic rac1-regulated ROS generation. This suggests a novel extra-nuclear function of ref-1.

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Diabetes is associated with an increased risk of cardio-vascular disease. A decrease in the number and functionality of endothelial progenitor cells (EPCs) leads to reduced endothelial repair and the development of cardiovascular disease. The aim of the present study was to explore the effect and underlying mechanisms of nuclear factor erythroid 2-related factor 2 (Nrf2) on EPC dysfunction caused by diabetic mellitus. The biological functions of EPCs in streptozotocin-induced diabetic mice were evaluated, including migration, proliferation, angiogenesis and the secretion of vascular endothelial growth factor (VEGF), stromal-derived growth factor (SDF) and nitric oxide (NO). Oxidative stress levels in diabetic EPCs were also assessed by detecting intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA). EPC senescence was evaluated by measuring p16 and b-gal expression and observing the senescence-associated secretory phenotype. In addition, the function of EPCs and level of oxidative stress were assessed following Nrf2 silencing or activation. Nrf2 silencing resulted in a decrease of EPC biological functions, accelerated cell senescence and increased oxidative stress, as indicated by ROS and MDA upregulation accompanied with decreased SOD activity. Furthermore, Nrf2 silencing inhibited migration, proliferation and secretion in EPCs, while it increased oxidative stress and cell senescence. Nrf2 activation protected diabetic EPCs against the effects of oxidative stress and cell senescence, ameliorating the biological dysfunction of EPCs derived from mice with diabetes. In conclusion, Nrf2 overexpression protected against oxidative stress-induced functional damage in EPCs derived from diabetic mice by regulating cell senescence.

Background The Lancet Commission on Global Surgery established the Three Delays framework, categorising delays in accessing timely surgical care into delays in seeking care (First Delay), reaching care (Second Delay), and receiving care (Third Delay). Globally, knowledge gaps regarding delays for fracture care, and the lack of large prospective studies informed the rationale for our international observational study. We investigated delays in hospital admission as a surrogate for accessing timely fracture care and explored factors associated with delayed hospital admission. MethodsIn this prospective observational substudy of the ongoing International Orthopaedic Multicenter Study in Fracture Care (INORMUS), we enrolled patients with fracture across 49 hospitals in 18 low-income and middle-income countries, categorised into the regions of China, Africa, India, south and east Asia, and Latin America. Eligible patients were aged 18 years or older and had been admitted to a hospital within 3 months of sustaining an orthopaedic trauma. We collected demographic injury data and time to hospital admission. Our primary outcome was the number of patients with open and closed fractures who were delayed in their admission to a treating hospital. Delays for patients with open fractures were defined as being more than 2 h from the time of injury (in accordance with the Lancet Commission on Global Surgery) and for those with closed fractures as being a delay of more than 24 h. Secondary outcomes were reasons for delay for all patients with either open or closed fractures who were delayed for more than 24 h. We did logistic regression analyses to identify risk factors of delays of more than 2 h in patients with open fractures and delays of more than 24 h in patients with closed fractures. Logistic regressions were adjusted for region, age, employment, urban living, health insurance, interfacility referral, method of transportation, number of fractures, mechanism of injury, and fracture location. We further calculated adjusted relative risk (RR) from adjusted odds ratios, adjusted for the same variables. This study was registered with ClinicalTrials.gov, NCT02150980, and is ongoing. Findings Between April 3, 2014, and May 10, 2019, we enrolled 31 255 patients with fractures, with a median age of 45 years (IQR 31-62), of whom 19 937 (63•8%) were men, and 14 524 (46•5%) had lower limb fractures, making them the most common fractures. Of 5256 patients with open fractures, 3778 (71•9%) were not admitted to hospital within 2 h. Of 25 999 patients with closed fractures, 7141 (27•5%) were delayed by more than 24 h. Of all regions, Latin America had the greatest proportions of patients with delays (173 [88•7%] of 195 patients with open fractures; 426 [44•7%] of 952 with closed fractures). Among patients delayed by more than 24 h, the most common reason for delays were interfacility referrals (3755 [47•7%] of 7875) and Third Delays (cumulatively interfacility referral and delay in emergency department: 3974 [50•5%]), while Second Delays ...

Tissue engineering allows the restoration of pathologically damaged tissues such as cartilage and bone using bio-scaffolds containing functionally active cells. Bone-marrow-derived mesenchymal stem cells (BMSCs) are a promising source of cells for tissue engineering due to their multilineage differentiation potential. However, proliferative and osteogenic abilities of BMSCs, and quantity of stem cells decreases in the bone marrow in aged population. We cultured BMSCs isolated from rats of various ages and evaluated their morphology, activity, and differentiation potential. Cultured BMSCs formed monolayer of fibroblast-like cells and maintained their characteristic morphology for 7-10 generations. Flow cytometry showed that aging of the cultured cell population correlated with the decrease in the expression of mesenchymal and hematopoietic surface markers, such as CD44, CD45, CD90, and CD29. We detected strong correlation between the age of BMSC donor and ALP activity in BMSC culture induced with low doses of dexamethasone and vitamin C. Cells from 2- and 6-week-old donor SD rats exhibited markedly increased ALP activity that coincided with increased bone content and strong positive staining of mineralized nodules. In contrast, BMSCs isolated from 10-month-old donors showed the lowest ALP activity, and decreased bone content and mineralized nodules formation. Our results demonstrate that the increase in donor age negatively affects proliferation and differentiation capacity of BMSCs in culture.

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