Meniscus matrix morphological composition: age-dependent evaluation in a swine model

Menisci are fibro-cartilaginous structures interposed between femoral condyle and tibial plateau, which have multiple functions in the stifle joint: act as shock absorbers, bear loaders and allow joint stability, congruity and lubrication (Sweigart et al., 2004; Proffen et al., 2012). It is well known that meniscal injuries lead to osteoarthritis and for these reasons, menisci are considered important target of investigation. Their important role in the knee wellness is only equalled by their deficiency in proper self-repairing.Nowadays, the gold standard technique is not just to remove the damaged meniscus, but to rebuild it or to replace it. For these reasons, studies are necessary to increase the knowledge about these small but essential structures (Streuli, 1999; Deponti et al., 2013). Composition and morphology are basic fundamental information for the development of engineered meniscal substitutes (Di Giancamillo et al., 2014). The analysis of the morphological, structural and biochemical changes, which occur during growth of the normal menisci, represent the goal of the present study. For this purpose, menisci from adult (7-month old), young (1-month old), and neonates (stillbirths) pigs were collected. Cellularity and glycosamiglycans (GAGs) deposition were evaluated by ELISA, while Collagen-1 and Collagen-2 were investigated by immunohistochemistry and Western blot analyses. Cellularity (P<0.01, all comparisons) and Collagen-1 (P<0.05, neonatal-young vs adult) decreased from neonatal to adult stage while GAGs (P<0.01 neonatal vs young-adult) and Collagen-2 (P<0.01 neonatal-young vs adult) showed the opposite trend. Immunohistochemistry revealed similar changes occurring during animal growth thus revealing that cellular phenotype, cellularity and protein expression, as well as fibers aggregation in the matrix, are dissimilar in the three ages analysed categories. These changes reflect the progressive menisci maturation and hyper-specialisation. We observed the correlation between biochemical and phenotype properties of swine menisci follow age-dependent changes during growth: starting with an immature cellular and fiber pattern to the mature organised and differentiated adult menisci.Acknowledgments: This work was funded by the “Finanziamento Piano Sviluppo Ateneo - Linea 2A

A Systemic and Severe Infection via Cytomegalovirus and Other Herpesviruses in a Young Apparently Immunocompetent Patient: A Case Report

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Meniscal femoral and tibial surfaces characterization in the swine model.

Menisci are wedge-like structures interposed, in the knee joint, between the femoral and the tibial articular heads (Kohn et al. 1995; Greis et al. 2002). Improving the articular surface, the cellular nutrition and the articular lubrication, they are essential structures for the prevention of gonarthrosis (Proctor et al.1989; Makris et al. 2011). This study is focused upon the relationship between the contact forces at the femoral and tibial surfaces and the corresponding structure of these meniscal surfaces. For this purpose, 20 adult (~9 months old) female pigs (Landrace x Large white, average weight 75–90 kg; n=80 meniscal samples) were obtained from a local slaughterhouse and dissected to isolate the menisci. Swine meniscal samples were evaluated from morphological (Safranin-O, Sirius Red and collagen type I and II) (Di Giancamillo et al. 2014), biochemical (DNA and glycosaminoglycans, or GAGs, contents) and biomechanical (compression and traction tests) points of view at the level of femoral and tibial meniscal surfaces. Results revealed a characterization of the meniscus which is biomechanical-dependent.  The femoral surface, morphologically characterized by the interposition of radial and oblique fibers and biomechanically by the femoral condyles compression, sliding and rolling forces, shows a higher compressive modulus (p<0.05) and a greater amount of cells and GAGs deposition (p<0.01 for each analysis). On the other hand, results from traction test revealed a higher tensile modulus (p<0.05) in the tibial surface, characterized by a circumferential arrangement of the fibers and a poorer GAGs deposition and cellular distribution (p<0.01). Results (summarized in the figure 1) from this work suggest that a biphasic “femoral-to-tibial” scaffold that mimic the different behavior and composition of the two meniscal surfaces could be useful in the light of meniscal replacement.

Stifle anatomic, tomographic and biomechanical features of growing dogs affected by quadriceps contracture.

Quadriceps contracture is a debilitating and uncommon condition, mostly affecting young dogs. Itcan be congenital or acquired ) and is reported to induce muscular hypotrophy/fibrosis, progressive degenerative joint disease, bone hypoplasia and limb hyperextension. The aim of this study was to elucidate anatomic, tomographic and biomechanical features of stifles affected by quadriceps contracture.Seven 2-month-old dead Dobermann Pinschers with unilateral quadriceps contracture were included. Before gross anatomic evaluation, all stifles underwent Computed Tomography before and after intra-articular administration of iodinated contrast medium. Images were acquired in double positioning (stifle extension and flexion) to identify articular cartilage, Ossification Centres’ (OCs) and menisci abnormalities, which were compared between affected and unaffected limbs. In all affected limbs the stifle was back-turned, the distal femur was extra-rotated and the patella was luxated proximo-medially. Severe lack of physiological stifle movements (rolling, gliding, spinning) was observed, so that affected joints could not be flexed. The articular cartilage of the femur was flattened and irregular in thickness, the femoral trochlea was hypoplasic and sloping, the menisci were misshaped. The OC of the distal femur and proximal tibia were misshaped; the tibial plateau was oriented caudodorsally-cranioventrally and significantly smaller (P<0,05). Quadriceps contracture influenced stifle development. The action of quadriceps insertion on the tibia prevented normal development of the plateau, causing wedging and abnormal orientation. Constant compression also induced external rotation of the distal femur (unable to develop distally) and patellar luxation, ending up in genu recurvatum. Static compression was likely responsible for femoral trochlea hypoplasia, articular cartilage and meniscal deformation, due to the lack of physiological stifle movements.Quadriceps contracture induces severe alterations of stifle development in affected puppies. Histology, histochemistry and immunohistochemistry may better define the nature of such bone, cartilage and meniscal alterations

Swine cortical and cancellous bone: histomorphometric and densitometric characterisation

Introduction: Swine bone morphology, composition and remodelling are similar to humans’, therefore they are considered good models in bone-related research. They have been used for several studies involving bone growth, bone and cartilage fractures and femoral head osteonecrosis. Nevertheless, the literature about pig normal bone features is incomplete. This work aims to fill the literature gaps on the microarchitecture and Bone Mineral Density (BMD) of swine femoral diaphysis and distal epiphysis and tibial plateau and diaphysis.Materials and methods: Five hind limbs were collected from slaughtered 80-100 kg pigs. Microscopic analysis of cortical and cancellous bone from middle/distal femur and proximal/middle tibia was performed to determine basic histomorphometric parameters at different sites. Dual-energy X-Rays Absorptiometry was also employed to evaluate BMD. ANOVA and correlation between BMD, bone area (BA) and cortical thickness were performed.Results and discussion: Diaphyseal cortical bone was mostly plexiform both in the tibia and the femur; primary/secondary osteons without clear organization were also found. Mean values for bone area, bone perimeter, trabecular width, number and separation and BMD at different anatomical sites were defined. No significant difference was found for these values at different anatomical sites. BMD proved to be positively correlated with cortical thickness (r=0,80; p<0,01). Despite the small sample size, these results seem homogeneous. They could therefore represent reference values for normal bone parameters in pigs. Applied anatomy and regenerative medicine, in fact, demand very precise information about bone micromorphology, composition and density to provide reliable indication in bone substitutes building. Moreover, since the interpretation of bone abnormalities is based on mastering normal bone characteristics, the definition of reference parameters is mandatory to avoid misinterpretation and allow comparative evaluation.Conclusion: The results of this study, although preliminary, may be considered a dependable starting point for the definition of normal bone features in pigs

The Orexin-A serum levels are strongly modulated by physical activity intervention in diabetes mellitus patients

The Orexin-A (hypocretin-1) is a neuropeptide secreted by neurons in the lateral hypothalamus. This protein regulates physiological and behavioural processes that have an essential impact on energy balance and metabolic status, physical activity, blood glucose levels, and food intake. Furthermore, that orexin-A regulates insulin sensitivity, energy expenditure and metabolic rate and is involved in immune processes and then regulate inflammatory response, with an anti-inflammatory action. Diabetes mellitus (T2DM) is a worldwide health problem associated with obesity and sedentary lifestyle. High glycaemic levels and lipid serum profile, low col-HDL, or hypertension and increased body mass index (BMI) are significantly associated with increased T2DM risk and with increased cardiovascular mortality and morbidity in T2DM patients. For these reasons the aim of this study is to evaluate the biochemical and anthropometric parameters, orexin-A levels by ELISA test and western blotting analysis, and inflammatory cytokines levels such as TNF-a, IL-8 and IL-10 by ELISA test in subjects affected by diabetes mellitus following an accurate physical activity program at baseline, after 3 months and after 6 months. We found that there is a ameliorate of many anthropometric and biochemical parameters; furthermore, there is a statistical increase of orexin-A serum levels already after 3 months compared to baseline in T2DM subjects and also there is a strongly modulation in inflammatory cytokines expression. These found indicates that the physical activity has beneficial effects not only on anthropometric and biochemical parameters but also on orexin-A levels, and then on CNS

The beneficial effects of physical activity and weight loss on human colorectal carcinoma cell lines

Recent studies have demonstrated that obesity is a significant risk factor for the development of several malignancies such as cancer. Colorectal cancer is among the most common cancers worldwide and is strong linked to obesity. A healthy lifestyle, characterized by hypocaloric diet and physical activity, is important to reduce a chronic inflammation, oxidative stress and metabolic disorders typical of obesity (Messina et al, 2018; Messina et al, 2017; Messina et al, 2015). It is well known that the chronic inflammation state and oxidative stress are responsible for the aging and development of many diseases, such as cancer. Dysregulation of cytokine’s secretion probably participates in the establishment of cancer in obese patients. The aim of this study is to analyse the effects of sera from obese patients subjected to a physical activity program before and after weight loss on cell viability, apoptosis and oxidative stress in HCT116 carcinoma cell line treated for 24, 48 and 72 hours through MTT test. We analysed the expression of cytokines in HCT116 cells. We found that sera from obese after physical activity intervention compared to treatment with sera from obese patients before physical activity intervention reduce the survival rate of HCT116 cells through induction of apoptosis and oxidative stress. Finally, we found a reduction of mRNA levels corresponding to the pro-inflammatory IL-6 and IL-8 cytokines together with an increase of the anti-inflammatory IL-10 cytokine. We can conclude that the physical activity has numerous beneficial effects also in colorectal cancer cell, indeed the physical activity and weight loss in obese subjects have an inhibitory and anti-inflammatory effects in a short period on carcinoma cell line

Executive Functions in Overweight and Obese Treatment-Seeking Patients: Cross-Sectional Data and Longitudinal Perspectives

Background: Recent evidence suggests that a higher body weight may be linked to cognitive impairment in different domains involving executive/frontal functioning. However, challenging results are also available. Accordingly, our study was designed to verify whether (i) poor executive functions are related to a higher body weight and (ii) executive functioning could contribute to weight loss in treatment-seeking overweight and obese patients. Methods: We examined general executive functioning, inhibitory control, verbal fluency, and psychomotor speed in a sample including 104 overweight and obese patients. Forty-eight normal-weight subjects participated in the study as controls. Results: Univariate Analysis of Variance showed that obese patients obtained lower scores than overweight and normal-weight subjects in all executive measures, except for errors in the Stroop test. However, when sociodemographic variables entered the model as covariates, no between-group difference was detected. Furthermore, an adjusted multiple linear regression model highlighted no relationship between weight loss and executive scores at baseline. Conclusions: Our results provide further evidence for the lack of association between obesity and the executive domains investigated. Conflicting findings from previous literature may likely be due to the unchecked confounding effects exerted by sociodemographic variables and inclusion/exclusion criteria

Proof-of-Concept Study on the Use of Tangerine-Derived Nanovesicles as siRNA Delivery Vehicles toward Colorectal Cancer Cell Line SW480

In the last years, the field of nanomedicine and drug delivery has grown exponentially, providing new platforms to carry therapeutic agents into the target sites. Extracellular vesicles (EVs) are ready-to-use, biocompatible, and non-toxic nanoparticles that are revolutionizing the field of drug delivery. EVs are involved in cell-cell communication and mediate many physiological and pathological processes by transferring their bioactive cargo to target cells. Recently, nanovesicles from plants (PDNVs) are raising the interest of the scientific community due to their high yield and biocompatibility. This study aims to evaluate whether PDNVs may be used as drug delivery systems. We isolated and characterized nanovesicles from tangerine juice (TNVs) that were comparable to mammalian EVs in size and morphology. TNVs carry the traditional EV marker HSP70 and, as demonstrated by metabolomic analysis, contain flavonoids, organic acids, and limonoids. TNVs were loaded with DDHD1-siRNA through electroporation, obtaining a loading efficiency of 13%. We found that the DDHD1-siRNA complex TNVs were able to deliver DDHD1-siRNA to human colorectal cancer cells, inhibiting the target expression by about 60%. This study represents a proof of concept for the use of PDNVs as vehicles of RNA interference (RNAi) toward mammalian cells

Spectrum of mutations and long-term clinical outcomes in genetic chylomicronemia syndromes

OBJECTIVE: Familial chylomicronemia syndrome (FCS) and multifactorial chylomicronemia syndrome (MCS) are the prototypes of monogenic and polygenic conditions underlying genetically based severe hypertriglyceridemia. These conditions have been only partially investigated so that a systematic comparison of their characteristics remains incomplete. We aim to compare genetic profiles and clinical outcomes in FCS and MCS. Approach and Results: Thirty-two patients with severe hypertriglyceridemia (triglyceride >1000 mg/dL despite lipid-lowering treatments with or without history of acute pancreatitis) were enrolled. Rare and common variants were screened using a panel of 18 triglyceride-raising genes, including the canonical LPL, APOC2, APOA5, GP1HBP1, and LMF1. Clinical information was collected retrospectively for a median period of 44 months. Across the study population, 37.5% were classified as FCS due to the presence of biallelic, rare mutations and 59.4% as MCS due to homozygosity for nonpathogenic or heterozygosity for pathogenic variants in canonical genes, as well as for rare and low frequency variants in noncanonical genes. As compared with MCS, FCS patients showed a lower age of hypertriglyceridemia onset, higher levels of on-treatment triglycerides, and 3-fold higher incidence rate of acute pancreatitis. CONCLUSIONS: Our data indicate that the genetic architecture and natural history of FCS and MCS are different. FCS expressed the most severe clinical phenotype as determined by resistance to triglyceride-lowering medications and higher incidence of acute pancreatitis episodes. The most common genetic abnormality underlying FCS was represented by biallelic mutations in LPL while APOA5 variants, in combination with high rare polygenic burden, were the most frequent genotype of M