Proteomic Modulation in TGF-ß-Treated Cholangiocytes Induced by Curcumin Nanoparticles.

Curcumin is a natural polyphenol that exhibits a variety of beneficial effects on health, including anti-inflammatory, antioxidant, and hepato-protective properties. Due to its poor water solubility and membrane permeability, in the present study, we prepared and characterized a water-stable, freely dispersible nanoformulation of curcumin. Although the potential of curcumin nanoformulations in the hepatic field has been studied, there are no investigations on their effect in fibrotic pathological conditions involving cholangiocytes. Exploiting an in vitro model of transforming growth factor-ß (TGF-ß)-stimulated cholangiocytes, we applied the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomic approaches to study the proteome modulation induced by curcumin nanoformulation. Our results confirmed the well-documented anti-inflammatory properties of this nutraceutic, highlighting the induction of programmed cell death as a mechanism to counteract the cellular damages induced by TGF-ß. Moreover, curcumin nanoformulation positively influenced the expression of several proteins involved in TGF-ß-mediated fibrosis. Given the crucial importance of deregulated cholangiocyte functions during cholangiopathies, our results provide the basis for a better understanding of the mechanisms associated with this pathology and could represent a rationale for the development of more targeted therapies.

Discrimination capability of pretest probability of stable coronary artery disease: a systematic review and meta-analysis suggesting how to improve validation procedures.

OBJECTIVE: Externally validated pretest probability models for risk stratification of subjects with chest pain and suspected stable coronary artery disease (CAD), determined through invasive coronary angiography or coronary CT angiography, are analysed to characterise the best validation procedures in terms of discriminatory ability, predictive variables and method completeness. DESIGN: Systematic review and meta-analysis. DATA SOURCES: Global Health (Ovid), Healthstar (Ovid) and MEDLINE (Ovid) searched on 22 April 2020. ELIGIBILITY CRITERIA: We included studies validating pretest models for the first-line assessment of patients with chest pain and suspected stable CAD. Reasons for exclusion: acute coronary syndrome, unstable chest pain, a history of myocardial infarction or previous revascularisation; models referring to diagnostic procedures different from the usual practices of the first-line assessment; univariable models; lack of quantitative discrimination capability. METHODS: Eligibility screening and review were performed independently by all the authors. Disagreements were resolved by consensus among all the authors. The quality assessment of studies conforms to the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2). A random effects meta-analysis of area under the receiver operating characteristic curve (AUC) values for each validated model was performed. RESULTS: 27 studies were included for a total of 15 models. Besides age, sex and symptom typicality, other risk factors are smoking, hypertension, diabetes mellitus and dyslipidaemia. Only one model considers genetic profile. AUC values range from 0.51 to 0.81. Significant heterogeneity (p<0.003) was found in all but two cases (p>0.12). Values of I2 >90% for most analyses and not significant meta-regression results undermined relevant interpretations. A detailed discussion of individual results was then carried out. CONCLUSIONS: We recommend a clearer statement of endpoints, their consistent measurement both in the derivation and validation phases, more comprehensive validation analyses and the enhancement of threshold validations to assess the effects of pretest models on clinical management. PROSPERO REGISTRATION NUMBER: CRD42019139388.

Systemic and vascular inflammation in an in-vitro model of central obesity.

Metabolic disorders due to over-nutrition are a major global health problem, often associated with obesity and related morbidities. Obesity is peculiar to humans, as it is associated with lifestyle and diet, and so difficult to reproduce in animal models. Here we describe a model of human central adiposity based on a 3-tissue system consisting of a series of interconnected fluidic modules. Given the causal link between obesity and systemic inflammation, we focused primarily on pro-inflammatory markers, examining the similarities and differences between the 3-tissue model and evidence from human studies in the literature. When challenged with high levels of adiposity, the in-vitro system manifests cardiovascular stress through expression of E-selectin and von Willebrand factor as well as systemic inflammation (expressing IL-6 and MCP-1) as observed in humans. Interestingly, most of the responses are dependent on the synergic interaction between adiposity and the presence of multiple tissue types. The set-up has the potential to reduce animal experiments in obesity research and may help unravel specific cellular mechanisms which underlie tissue response to nutritional overload.

Biological and proteomic characterization of a composite mesh for abdominal wall hernia treatment: Reference Study.

AIMS: The industrial development of a product requires performing a deep analysis to highlight its characteristics useful for future design. The clinical use of a product stimulates knowledge improvement about it in a constant effort of progress. This work shows the biological characterization of CMC composite mesh. CMC polypropylene prosthesis was seeded with Human fibroblast BJ. Samples (cells and medium) were collected at different time points in order to perform different analysis: inflammatory markers quantification; collagens immunohistochemistry; matrix metalloproteinases zimography; extracellular matrix proteomic profile. FINDINGS: CMC presented a good cell viability rate and cell growth during the 21 days. The inflammatory profile showed an initial secretion of anti-inflammatory IL-10 and a final increase of pro-inflammatory IL-6. Immunocytochemistry highlighted a similar Collagen type I/type III ratio. The proteomic analysis evidenced the ECM protein content profile composed, mainly, by collagens, fibronectin, laminin. MMPs resulted both expressed when in contact to mesh. CONCLUSIONS: CMC shows a good cell biocompatibility and growth. The increase of pro-inflammatory markers could stimulate proliferation, influencing the integration process in human body. Proteomics highlights the ECM modulation by CMC. An integrated investigation of these biological analyses with mechanical data should improve the design process of a new product. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2045-2052, 2017.

CREPE: mathematical model for crosstalking of endothelial cells and hepatocyte metabolism.

The liver shows a close coexistence between endothelial cells and hepatocytes (HepG2). Endothelial cells' main purpose is to protect (HepG2) from blood vessel shear stress, acting as a barrier, but experimental evidence suggests that they could also play a role in regulating (HepG2) glucose metabolism. A well-known singular effect in hepatocyte-endothelial co-cultures is the reduction of glucose consumption respect to (HepG2) in single culture. (HepG2) were shown to reduce their glucose consumption supporting energy needs of endothelial cells. Monti have studied the effects of endothelin-1 (Et-1) on Glucokinase activity in adult rat (HepG2). They observed a reduction in hepatocytes Glucokinase catalytic rate, which is dependent on Et-1 concentration. We developed crosstalking of endothelial cells and hepatocyte metabolism (CREPE) that is a mathematical model of the endothelin-1 mediated crosstalk between HepG2 and endothelial cells (human umbilical vein endothelial cells) in a traditional static co-culture system. CREPE was validated against experimental data, showing good agreement with them. CREPE can be a starting point to develop predictive tools on complex and highly interconnected environments.

PAM2 (piston assisted microsyringe): a new rapid prototyping technique for biofabrication of cell incorporated scaffolds.

Rapid prototyping techniques are widely used to fabricate well-defined three-dimensional structures of tissue homologs. The piston-assisted microsyringe (PAM2) is a rapid prototyping technology specifically developed for low-shear stress extrusion of viscous hydrogel solutions containing cells. In this article the working parameters of the system were established to guarantee the realization of spatially controlled hydrogel scaffolds. Moreover the shear stresses acting on the cell membrane during extrusion was investigated through a computational fluid-dynamic analysis. The computational models show that the shear stress on the cells is of the order of 100 Pa during the extrusion process. HepG2 cells encapsulated in alginate were then extruded into spatially organized hepatic lobule-like architectures and their viability and function were evaluated. The results show that the metabolic fingerprint of the cells is preserved with respect to controls and the cells are uniformly distributed through the gel scaffold.

Poly(ester urethane) guides for peripheral nerve regeneration.

A biocompatible and elastomeric PU was synthesized from low-molecular-weight PCL as macrodiol, CMD as chain extender and HDI as chain linker for applications in the field of peripheral nerve repair. PU cast films supported in vitro attachment and proliferation of NOBEC. The in vitro adhesion and proliferation of S5Y5 neuroblastoma cells on the inner surface of uncoated, gelatin- and PL-coated PU guides were compared. Due to their superior in vitro performance, PL-coated PU guides were tested in vivo for the repair of 1.8 cm-long defects in rat sciatic nerves. The progressive regeneration was confirmed by EMG and histological analysis showing the presence of regenerating fibers in the distal stumps.

Melt-extruded guides for peripheral nerve regeneration. Part I: poly(epsilon-caprolactone).

Melt-extruded guides for peripheral nerve repair based on poly(epsilon-caprolactone) (PCL) were realised and their physico-chemical properties were evaluated. Preliminarily, PCL cast films were found to support the attachment and proliferation of Neonatal Olfactory Bulb Ensheating Cells (NOBEC). S5Y5 neuroblastoma cells were cultured inside PCL guides in their uncoated form or coated with a non-specific adhesion protein (gelatin) and a specific peptide for nerve regeneration (poly(L-lysine)). Coating increased cell density (gelatin) and/or the cell density rate on substrates (poly(L-lysine); gelatin) as compared to uncoated guides. Various in vivo tests were carried out for the repair of small (0.5 cm), medium (1.5 cm) and long (4.5 cm) size defects in the peripheral nerves of Wistar rats. For the small nerve defects, uncoated and coated PCL guides were tested. Results from in vivo tests were subjected to histological examination after 45 days, 6 and 8 months postoperative for small, medium and large defects, respectively. Regeneration was found for small and medium size defects. For 0.5 cm defects, the coating did not affect regeneration significantly. Grip-tests also evidenced functional recovery for the 1.5 cm-long defects treated with PCL guides, after 6 months from implantation. On the other hand, mechanical stiffness of PCL conduits impaired the repair of 4.5 cm-long defects in 8-month period: the lack of flexibility of the guide to rat movements caused its detachment from the implant site. The research showed that PCL guides can be used for the successful repair of small and medium size nerve defects, with possible improvements by suitable bio-mimetic coatings.

PAM-microfabricated polyurethane scaffolds: in vivo and in vitro preliminary studies.

Polymeric scaffolds were realised with linear degradable PU in the form of square, hexagonal and octagonal grids. They were characterised in terms of their mechanical properties. Analysis shows that the mechanical properties of the scaffolds depend on their geometries which are easily modulated using PAM. In vitro biological assays showed that PU promotes the adhesion and proliferation of fibroblast cells and that cell activities are better on PU scaffolds than on PU films. In vivo implantation of PU and PLGA scaffolds and PU films demonstrated that the scaffolds are completely resorbed after three months with a slight inflammatory response, while the PU film was still present after six months with an intense granulomatous reaction.

Chitosan/gelatin blends for biomedical applications.

Blends between chitosan (CS) and gelatin (G) with various compositions (CS/G 0/100 20/80, 40/60, 60/40, 100/0 w/w) were produced as candidate materials for biomedical applications. Dehydro-thermal crosslinking was adopted to promote the formation of amide and ester bonds between the macromolecules ((CS/G)-t). The effect of composition and crosslinking on the physico-chemical properties of the samples was evaluated by scanning electron microscopy, thermogravimetry, contact angle measurements, dissolution and swelling tests. Mechanical properties of (CS/G)-t samples were also determined through stress-strain and creep-recovery tests. The elastic moduli of dry blend samples showed a positive deviation from the additive law of the in-series model, because of interactions and/or chemical bonds between components. The comparison between the elastic moduli of wet samples and those of different human tissues showed that (CS/G)-t substrates can be suitable for soft-tissue reconstruction. (CS/G)-t two-dimensional scaffolds were fabricated by micro-molding, based on the use of a polydimethylsiloxane mould to create patterns with micro-scale resolution on cast films. Biocompatibility of (CS/G)-t samples was studied by means of cell tests using NIH-3T3 fibroblasts. Finally, the evaluation of the affinity of (CS/G)-t samples towards neuroblastoma cells adhesion and proliferation was performed, showing promising results for the blend containing 80 wt % gelatin.