Sex Specific Determinants in Osteoarthritis: A Systematic Review of Preclinical Studies

Osteoarthritis (OA) is a highly prevalent joint disease that primarily affects about 10% of the world's population over 60 years old. The purpose of this study is to systematically review the preclinical studies regarding sex differences in OA, with particular attention to the molecular aspect and gene expression, but also to the histopathological aspects. Three databases (PubMed, Scopus, and Web of Knowledge) were screened for eligible studies. In vitro and in vivo papers written in English, published in the last 11 years (2009-2020) were eligible. Participants were preclinical studies, including cell cultures and animal models of OA, evaluating sex differences. Independent extraction of articles and quality assessments were performed by two authors using predefined data fields and specific tools (Animals in Research Reporting In Vivo Experiments (ARRIVE) guideline and Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) tool). Twenty-three studies were included in the review: 4 in vitro studies, 18 in vivo studies, and 1 both in vitro and in vivo study. From in vitro works, sex differences were found in the gene expression of inflammatory molecules, hormonal receptors, and in responsiveness to hormonal stimulation. In vivo research showed a great heterogeneity of animal models mainly focused on the histopathological aspects rather than on the analysis of sex-related molecular mechanisms. This review highlights that many gaps in knowledge still exist; improvementsin the selection and reporting of animal models, the use of advanced in vitro models, and multiomics analyses might contribute to developing a personalized gender-based medicine

Involvement of Notch signaling in the osteogenic differentiation of human mesenchymal stem cells stimulated by pulsed electromagnetic fields

Biophysical stimulation with pulsed electromagnetic fields (PEMFs), used in clinics to promote bone repair, favour osteogenic differentiation in human mesenchymal stem cells (hMSCs), however their molecular mechanisms are not clarified. Notch is a pathway regulating cell fate decisions which play a role in skeletal development. Notch signaling is initiated by binding a Notch ligand to a cell surface Notch receptor, resulting in a cleavage of receptor and releasing Notch intracellular domain which translocates to the nucleus and activates transcription of nuclear Notch target genes, such as the Hes/Hey family.The aim of this study is to establish if the known PEMF-induced osteogenic effects may occur through the modulation of Notch pathway. Bone marrow hMSCs cultured in basal condition (control) and in osteoinductive medium (OM) for 28 days were unexposed or continuously exposed to PEMFs (75 Hz, 1.5 mT) (Igea, Carpi, Italy). To block Notch pathway, the Notch inhibitor DAPT was used to treat a series of hMSCs cultured in OM. At different time points (day 1,3,7,14,21,28), osteogenic markers (alkaline phosphatase activity, osteocalcin and matrix mineralization), mRNA expression of osteogenic transcription factors (Runx2, Dlx5, Osterix) as well as of Notch receptors (Notch1-4), their ligands (Jagged1, Dll1 and Dll4) and nuclear target genes (Hey1, Hey2, Hes1, Hes5) were analysed.Our results showed that osteogenic markers and transcription factors increased in OM compared to control and they were further stimulated by PEMFs. Notably, PEMFs significantly increased the expression of Notch4, Dll4, Hey1, Hes1 and Hes5 in the middle phase of differentiation in OM compared to control. In the presence of DAPT, osteogenic markers as well Hes1 and Hes5 expression were significantly inhibited, in unexposed and PEMF-exposed hMSCs. Hey1 was not inhibited by DAPT suggesting a possible regulation by other signaling pathway.These new findings show that PEMFs favor osteogenic differentiation acting through Notch pathway, adding important knowledge concerning the molecular mechanisms by which PEMFs can modulate osteogenesis. This work was supported by grants from IGEA, Carpi, Italy

Asthmatic Patients with Vitamin D Deficiency have Decreased Exacerbations after Vitamin Replacement

Background: Intervention studies with vitamin D in asthma are inconclusive for several reasons, such as inadequate dosing or duration of supplementation or uncontrolled baseline vitamin D status. Our aim was to evaluate the benefit of long term vitamin D add-on in asthmatic patients with actual vitamin D deficiency, that is a serum 25-hydroxy vitamin D (25-OHD ) below 20 ng/mL. Methods: Serum 25-OHD, asthma exacerbations, spirometry and inhaled corticosteroids (CS) dose were evaluated in a cohort of 119 asthmatic patients. Patients with deficiency were evaluated again after one year vitamin supplementation. Results: 25-OHD was low in 111 patients and was negatively related to exacerbations (p < 0.001), inhaled CS dose (p = 0.008) and asthma severity (p = 0.001). Deficiency was found in 90 patients, 55 of whom took the supplement regularly for one year, while 24 discontinued the study and 11 were not adherent. Patients with vitamin D deficiency after 12 months supplementation showed significant decrease of exacerbations (from 2.6 ± 1.2 to 1.6 ± 1.1, p < 0.001), circulating eosinophils (from 395 ± 330 to 272 ± 212 106/L, p < 0.001), and need of oral CS courses (from 35 to 20, p = 0.007) and improvement of airway obstruction. Conclusions: Asthma exacerbations are favored by vitamin D deficiency and decrease after long-term vitamin D replacement. Patients who are vitamin D deficient benefit from vitamin D supplementation

Phthalate esters (PAEs) concentration pattern reflects dietary habitats (δ13C) in blood of Mediterranean loggerhead turtles (Caretta caretta)

Phthalic acid esters (PAEs) are classified as endocrine disruptors, but it remains unclear if they can enter the marine food-web and result in severe health effects for organisms. Loggerhead turtles (Caretta caretta) can be chronically exposed to PAEs by ingesting plastic debris, but no information is available about PAEs levels in blood, and how these concentrations are related to diet during different life stages. This paper investigated, for the first time, six PAEs in blood of 18 wild-caught Mediterranean loggerhead turtles throughout solid-phase extraction coupled with gas chromatography-ion trap/mass spectrometry. Stable isotope analyses of carbon and nitrogen were also performed to assess the resource use pattern of loggerhead turtles. DEHP (12-63 ng mL(-1)) and DBP (6-57 ng mL(-1)) were the most frequently represented PAEs, followed by DiBP, DMP, DEP and DOP. The total PAEs concentration was highest in three turtles (124-260 ng mL(-1)) whereas three other turtles had concentrations below the detection limit. PAEs were clustered in three groups according to concentration in all samples: DEHP in the first group, DBP, DEP, and DiBP in the second group, and DOP and DMP in the third group. The total phthalates concentration did not differ between large-sized (96.3 +/- 86.0 ng mL(-1)) and small-sized (67.1 +/- 34.2 ng mL(-1)) turtles (p < 0.001). However, DMP and DEP were found only in large-sized turtles and DiBP and DBP had higher concentrations in large-sized turtles. On the other hand, DEHP and DOP were found in both small- and large-sized turtles with similar concentrations, i.e. ~ 21.0/32.0 ng mL(-1) and ~ 7(.1)/9.9 ng mL(-1), respectively. Winsored robust models indicated that delta C-13 is a good predictor for DBP and DiBP concentrations (significant Akaike Information criterion weight, AIC(wt)). Our results indicate that blood is a good matrix to evaluate acute exposure to PAEs in marine turtles. Moreover, this approach is here suggested as a useful tool to explain the internal dose of PAEs in term of dietary habits (delta C-13), suggesting that all marine species at high trophic levels may be particularly exposed to PAEs, despite their different dietary habitats and levels of exposure

Electromagnetic fields counteract IL-1β during chondrogenesis in synovial bovine mesenchymal progenitor cells

Objective. Mesenchymal stem cells (MSCs) isolated from synovium and from synovial fluid, have shown a chondrogenesis potential suggesting that synovium is an excellent source of MSCs for cartilage regeneration. Electromagnetic fields (EMFs) display several effects on cartilage: increase the synthesis of proteoglycans (PGs), prevents the catabolic effect of the pro-inflammatory cytokine interleukin-1β (IL-1β), appear useful for the treatment of osteoarthritis. Our goal was to evaluate if the chondrogenic differentiation of synovial bovine mesenchymal progenitor cells, may be influenced by EMFs. Further, as chondrogenic differentiation of MSCs could be altered in an inflammatory environment and EMFs can counteract IL-1β activity, we also evaluated the role of EMFs during chondrogenic differentiation in the presence of IL-1β. Design. Synovial fluid was aspirated from the metacarpophalangeal joints of bovine. Synovial cells at the 3rd passage were centrifuged to obtain pellet cultures. Pellets were cultured in chondrogenic medium alone (control) or supplemented with 10 ng/ml TGF-β3 and/or 50 ng/ml IL-1β. The pellets were unexposed or exposed to EMF (75 Hz, 1.5 mT) (Igea, Carpi, Italy), during the whole period in culture (34 days). Alcian blue for sulphated glycosaminoglycans and immunostaining for type II collagen, were performed. PG synthesis was measured by radioactive 35S-sulphate incorporation. Results. Pellets cultured in the presence of TGF-β3 exhibited positive staining for type II collagen and Alcian blue, compared to control, indicating chondrogenic differentiation of synovial bovine mesenchymal progenitor cells. In the presence of IL-1β, type II collagen and Alcian blue staining dramatically decreased compared to TGF-β3 treatment alone. When pellets treated with both TGF-β3 and IL-1β were exposed to EMF, the histochemical staining for type II collagen and Alcian blue increased compared to EMF-unexposed pellets, suggesting that EMF might counteract the IL-1β effect. Biochemical analysis on PG synthesis confirmed histochemical data. Conclusions. The presence of inflammatory cytokines, such as IL-1β in human joints, may explain why existing methods of cartilage engineering repair strategies, that rely on the in situ differentiation of MSCs, fail to provide a reliably successful. Results of this study support the hypothesis that EMF treatment may favour chondrogenic differentiation in inflammatory conditions, suggesting a possible strategy for improving the clinical outcome of cartilage repair procedures

High Human Papillomavirus DNA loads in Inflammatory Middle Ear Diseases

Background. Previous studies reported human papillomaviruses (HPVs) in middle ear tumors, whereas these viruses have been poorly investigated in chronic inflammatory middle ear diseases. The purpose of this study was to investigate HPVs in non-tumor middle ear diseases, including chronic otitis media (COM). Methods. COM specimens (n=52), including chronic suppurative otitis media (CSOM) (n=38) and cholesteatoma (COMC) (n=14), as well as normal middle ear specimens (NME) (n=56) were analyzed. HPV DNA sequences and DNA loads were analyzed by quantitative PCR. HPV genotyping was performed by direct sequencing of the amplimers. Results. HPV DNA was detected in 23% (12/52) of COM and in 30.4% (17/56) NME (p>0.05). Specifically, HPV DNA sequences were revealed in 26.3% (10/38) of CSOM and in 14.3% (2/14) COMC (p>.05). Interestingly, the HPV DNA load was higher in COMC (mean 7.47 copy/cell) than in CSOM (mean 1.02 copy/cell), and NME (mean 1.18 copy/cell) (P=.03 and P=.017 versus CSOM and NME, respectively). HPV16 and HPV18 were the main genotypes detected in COMC, CSOM and NME. Conclusions. This data indicates that HPV-positive CSOM and COMC are generally associated with higher viral DNA loads as compared to NME. In addition, for the first time, HPVs were detected in normal middle ear mucosa specimens. This result suggests that NME is an additional epithelial tissue that can be HPV infected

Effect of electrode distance in grid electrode: Numerical models and in vitro tests

Electrochemotherapy is an emerging local treatment for the management of superficial tumors and, among these, also chest wall recurrences from breast cancer. Generally, the treatment of this peculiar type of tumor requires the coverage of large skin areas. In these cases, electrochemotherapy treatment by means of standard small size needle electrodes (an array of 0.73 cm spaced needles, which covers an area of 1.5 cm2) is time-consuming and can allow an inhomogeneous coverage of the target area. We have previously designed grid devices suitable for treating an area ranging from 12 to 200 cm2. In this study, we propose different approaches to study advantages and drawbacks of a grid device with needles positioned 2 cm apart. The described approach includes a numerical evaluation to estimate electric field intensity, followed by an experimental quantification of electroporation on a cell culture. The electric field generated in a conductive medium has been studied by means of 3-dimensional numerical models with varying needle pair distance from 1 to 2 cm. In particular, the electric field evaluation shows that the electric field intensity with varying needle distance is comparable in the area in the middle of the 2 electrodes. Differently, near needles, the electric field intensity increases with the increasing electrode distance and supply voltage. The computational results have been correlated with experimental ones obtained in vitro on cell culture. In particular, electroporation effect has been assessed on human breast cancer cell line MCF7, cultured in monolayer. The use of 2-cm distant needles, supplied by 2000 V, produced an electroporation effect in the whole area comprised between the electrodes. Areas of cell culture where reversible and irreversible electroporation occurred were identified under microscope by using fluorescent dyes. The coupling of computation and experimental results could be helpful to evaluate the effect of the needle distance on the electric field intensity in cell cultures in terms of reversible or irreversible electroporation

Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E2 and cytokine production in human osteoarthritic synovial fibroblasts

Objective. Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro-inflammatory mediators, including cytokines and lipid mediators of inflammation (1). Previous studies show that electromagnetic fields (EMFs) may represent a potential therapeutical approach to limit cartilage degradation and to control inflammation associated to OA, and that they may act through the adenosine pathway (2). On this basis the aim of this study was to investigate if EMFs might modulate inflammatory activities of human SFs derived from OA patients (OASFs) and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. Design. SFs obtained from OA patients, undergoing total hip joint replacement surgery, were exposed to EMFs (1.5 mT; 75 Hz) for 24 hours. In control and EMF-exposed cells, ARs were evaluated by western blotting, quantitative real-time RT-PCR and saturation binding experiments and cAMP levels were measured by a specific assay. In the absence and in the presence of interleukin-1β (IL-1β), used as a pro-inflammatory stimulus, prostaglandin E2 (PGE2), cytokine and matrix degrading enzyme production was evaluated in OASFs exposed to EMFs and treated with selective adenosine receptor agonists and antagonists. Results. EMF exposure induced a selective increase in A2A and A3 ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active in EMF-exposed cells. In IL-1β-treated OASFs, functional data obtained in the presence of  A2A and A3 adenosine agonists and antagonists showed that EMFs inhibit the release of (PGE2) and of the proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), whilst stimulate the release of interleukin-10 (IL-10), an antinflammatory cytokine. Further, results show that these effects appear to be mediated by the EMF-induced upregulation of A2A and A3 ARs. No effects of EMFs or ARs have been observed on matrix degrading enzymes production. Conclusions: EMFs display anti-inflammatory effects in human OASFs and these EMF-induced .ffects are in part mediated by the adenosine pathway, specifically by the A2A and A3 ARs activation. Taken together, these results suggest that SFs could represent potential therapeutic targets cells for EMF treatment and open new clinical perspectives to the control of inflammation associated to joint diseases. 1. Martel-Pelletier J et al. Eklem Hastalik Cerrahisi. 2010; 21(1):2-14. 2. De Mattei M et al. Osteoarthritis Cartilage. 2009; 17(2):252-262