Alginate/human elastin-like polypeptide composite films with antioxidant properties for potential wound healing application

In this contribution we describe the preparation and characterization of a series of cross-linked films based on the combination of an elastin-derived biomimetic polypeptide (Human elastin-like polypeptide (HELP)) with alginate (ALG) to obtain a composite with enhanced properties. ALG/HELP composite films loaded with the hydrophobic natural antioxidant curcumin were prepared by solvent casting method followed by the cross-linking with calcium chloride. The compatibility between the two components as well as the final properties was evaluated. The micro-morphological study of films showed a homogeneous structure, but the film tensile strength decrease with HELP content and elongation at break was adversely affected by biopolymer addition. Spectroscopic and thermal analyses confirmed an interaction between ALG and HELP which also causes a modification in swelling kinetics and faster degradation. Moreover, the study of curcumin release showed a controlled delivery up to 10\u202fdays with a faster release rate in the presence of HELP. Human Dermal Fibroblasts (hDF) were used to test the in vitro cytocompatibility. The antioxidant activity correlated to the increase of HELP content suggested the applicability of these composites to develop smart biomaterials. Overall, these features indicated how this composite material has considerable potential as customizable platforms for various biomedical applications

HTLV-2 Induces Resistance to CCR5-Dependent HIV-1 Infection Via Selective PBMC Expression of CCL3L1

Background In HIV-1/HTLV-2 co-infected IDUs the CCL3/MIP-1alpha induction by HTLV-2 leads to HIV inhibition. CCL3 gene codes for CCL3/LD78alpha and CCL3L1/LD78beta isoforms. CCL3L1 binds more potently to CCR5 than any other chemokine. Possession of a CCL3L1 copy number lower than two (the population average for Europeans) is associated with markedly enhanced HIV/AIDS susceptibility. Here, we analysed the genotype frequency of CCL3L1 and its expression in 8 HTLV-2-infected/HIV-1exposed-seronegative (HTLV-2/HIV-1ESN) individuals, 7 LTNP-HIV-1/HTLV-2-co-infected and 8 LTNP-HIV-1mono-infected subjects

3D-printed scaffold composites for the stimuli-induced local delivery of bioactive adjuncts

Polysaccharide scaffolds have been successfully employed to reconstruct environments that sustain skin tissue regeneration after injuries. Three-dimensional (3D) advanced additive manufacturing technologies allow creating scaffolds with controlled and reproducible macro- and micro-structure that improve the quality of the restored tissue to favor spontaneous repair. However, when persistent inflammation occurs, the physiological tissue healing capacity is reduced, like in the presence of pathologies like diabetes, vascular diseases, chronic infection, and others. In these circumstances, the bioavailability of therapeutic adjuncts like the growth factors in addition to the standard treatments represents undoubtedly a promising strategy to accelerate the healing of skin lesions. Precisely designed polysaccharide scaffolds obtained by 3D printing represent a robust platform that can be further implemented with the controlled delivery of bioactive adjuncts. Human elastin-like polypeptides (HELPs) are stimuli-responsive biopolymers. Their structure allows the integration of domains endowed with biological functionality, making them attractive compounds to prepare composites with smart properties. In the present study, 3D-printed alginate and chitosan scaffolds were combined with the HELP components. The HELP biopolymer was fused to the epidermal growth factor (EGF) as the bioactive domain. Different constructs were prepared and the stimuli-responsive behavior as well as the biological activity were evaluated, suggesting that these smart bioactive composites are suitable to realize multifunctional dressings that sustain the local release of therapeutic adjuncts

The adaptation of lipid profile of human fibroblasts to alginate 2D films and 3D printed scaffolds

Background: The investigation of the interactions between cells and active materials is pivotal in the emerging 3D printing-biomaterial application fields. Here, lipidomics has been used to explore the early impact of alginate (ALG) hydrogel architecture (2D films or 3D printed scaffolds) and the type of gelling agent (CaCl2 or FeCl3) on the lipid profile of human fibroblasts. Methods: 2D and 3D ALG scaffolds were prepared and characterized in terms of water content, swelling, mechanical resistance and morphology before human fibroblast seeding (8 days). Using a liquid chromatography-triple quadrupole-tandem mass spectrometry approach, selected ceramides (CER), lysophosphatidylcholines (LPC), lysophosphatidic acids (LPA) and free fatty acids (FFA) were analyzed. Results: The results showed a clear alteration in the CER expression profile depending of both the geometry and the gelling agent used to prepare the hydrogels. As for LPCs, the main parameter affecting their distribution is the scaffold architecture with a significant decrease in the relative expression levels of the species with higher chain length (C20 to C22) for 3D scaffolds compared to 2D films. In the case of FFAs and LPAs only slight differences were observed as a function of scaffold geometry or gelling agent. Conclusions: Variations in the cell membrane lipid profile were observed for 3D cell cultures compared to 2D and these data are consistent with activation processes occurring through the mutual interactions between fibroblasts and ALG support. These unknown physiologically relevant changes add insights into the discussion about the relationship between biomaterial and the variations of cell biological functions

Evaluation of Fructooligosaccharides and Inulins as Potentially Health Benefiting Food Ingredients by HPAEC-PED and MALDI-TOF MS

This paper describes the complementarity of high-performance anion exchange chromatography coupled with pulsed electrochemical detection (HPAEC-PED) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) to evaluate commercial available fructans (fructooligosaccharides (FOS) and inulins), having different degrees of polymerization (DP) which are usually employed by food industry as functional ingredients either for their prebiotic properties or as a fat replacer, giving a fat-like mouth feel and texture. The developed HPAEC-PED methods are able to analyze FOS (fructans with DP 3–10) and inulins (DP ranging from 3 to 80) with a good resolution and relatively short retention times to evaluate structural differences between fructooligosaccharide and inulins and the possible presence of inulooligosaccharides as well as of branching. To characterize FOS and inulin at different degrees of polymerization and to assure correct molecular assignment, MALDI-TOF MS analysis was also investigated. The 2,5-dihydroxy benzoic acid (2,5-DHB) was found to be the best matrix for FOS analysis as Actilight and Raftilose P95 products, while 3-aminoquinoline (3-AQ) seems to be the best matrix for inulin with higher DP. The applicability of the optimized methods to the identification and determination of FOS contained in a symbiotic milk as well as a type of inulin added as functional ingredient to a cooked ham is demonstrated

Aptamer-Mediated Selective Protein Affinity to Improve Scaffold Biocompatibility

Protein adsorption on surfaces occurs shortly after scaffold insertion. This process is of pivotal importance to achieve therapeutic success in tissue engineering (TE), and favorable proteins should be adsorbed at the interface without unfolding to preserve their structure and function. Protein misfolding at the interface is a common phenomenon, which can impair cell adhesion and scaffold colonization. Many efforts have been done to improve scaffold biocompatibility by ameliorating protein adsorption, but with poor results. In the present chapter, we propose the use of a novel class of molecules, aptamers, to improve scaffold biocompatibility. Aptamers are small, single stranded oligonucleotides, which specifically bind to a target molecule: they work as antibodies, but without many of the drawbacks associated to the use of antibodies. We propose to immobilize aptamers on scaffolds to retain specific proteins, acting as docking points to guide cell activity. In particular, we show the results obtained by enriching different polymeric scaffolds with aptamers against human fibronectin, a naturally abundant protein in tissues, which plays a pivotal role in cell adhesion. We demonstrate that scaffold enrichment with aptamers lead to a better colonization of the substrate from cells. The results we obtained pave the way to the possibility of further investigating the role of aptamers as useful molecules to improve scaffold biocompatibility in the contest of tissue engineering

Three-Dimensional (3D) Printed Silver Nanoparticles/Alginate/Nanocrystalline Cellulose Hydrogels: Study of the Antimicrobial and Cytotoxicity Efficacy

Here, a formulation of silver nanoparticles (AgNPs) and two natural polymers such as alginate (ALG) and nanocrystalline cellulose (CNC) was developed for the 3D printing of scaffolds with large surface area, improved mechanical resistance and sustained capabilities to promote antimicrobial and cytotoxic effects. Mechanical resistance, water content, morphological characterization and silver distribution of the scaffolds were provided. As for applications, a comparable antimicrobial potency against S. aureus and P. aeruginosa was demonstrated by in vitro tests as function of the AgNP concentration in the scaffold (Minimal Inhibitory Concentration value: 10 mg/mL). By reusing the 3D system the antimicrobial efficacy was demonstrated over at least three applications. The cytotoxicity effects caused by administration of AgNPs to hepatocellular carcinoma (HepG2) cell culture through ALG and ALG/CNC scaffold were discussed as a function of time and dose. Finally, the liquid chromatography-mass spectrometry (LC-MS) technique was used for targeted analysis of pro-apoptotic initiation and executioner caspases, anti-apoptotic and proliferative proteins and the hepatocyte growth factor, and provided insights about molecular mechanisms involved in cell death induction

Chitosan Hydrogels for Chondroitin Sulphate Controlled Release: An Analytical Characterization

This paper provides an analytical characterization of chitosan scaffolds obtained by freeze-gelation toward the uptake and the controlled release of chondroitin sulphate (CS), as cartilage repair agent, under different pH conditions. Scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and liquid chromatography-UV spectrophotometry (LC-UV) techniques were exploited to obtain qualitative and quantitative descriptions of polymer and drug behaviour in the biomaterial. As for morphology, SEM analysis allowed the evaluation of scaffold porosity in terms of pore size and distribution both at the surface (Feret diameter 58±19 μm) and on the cross section (Feret diameter 106±51 μm). LC and ATR-FTIR evidenced a pH-dependent CS loading and release behaviour, strongly highlighting the role of electrostatic forces on chitosan/chondroitin sulphate interactions

PCSK9 ablation attenuates Aβ pathology, neuroinflammation and cognitive dysfunctions in 5XFAD mice

Background: Increasing evidence highlights the importance of novel players in Alzheimer's disease (AD) pathophysiology, including alterations of lipid metabolism and neuroinflammation. Indeed, a potential involvement of Proprotein convertase subtilisin/kexin type 9 (PCSK9) in AD has been recently postulated. Here, we first investigated the effects of PCSK9 on neuroinflammation in vitro. Then, we examined the impact of a genetic ablation of PCSK9 on cognitive performance in a severe mouse model of AD. Finally, in the same animals we evaluated the effect of PCSK9 loss on Aβ pathology, neuroinflammation, and brain lipids. Methods: For in vitro studies, U373 human astrocytoma cells were treated with Aβ fibrils and human recombinant PCSK9. mRNA expression of the proinflammatory cytokines and inflammasome-related genes were evaluated by q-PCR, while MCP-1 secretion was measured by ELISA. For in vivo studies, the cognitive performance of a newly generated mouse line - obtained by crossing 5XFADHet with PCSK9KO mice – was tested by the Morris water maze test. After sacrifice, immunohistochemical analyses were performed to evaluate Aβ plaque deposition, distribution and composition, BACE1 immunoreactivity, as well as microglia and astrocyte reactivity. Cholesterol and hydroxysterols levels in mouse brains were quantified by fluorometric and LC-MS/MS analyses, respectively. Statistical comparisons were performed according to one- or two-way ANOVA, two-way repeated measure ANOVA or Chi-square test. Results: In vitro, PCSK9 significantly increased IL6, IL1B and TNFΑ mRNA levels in Aβ fibrils-treated U373 cells, without influencing inflammasome gene expression, except for an increase in NLRC4 mRNA levels. In vivo, PCSK9 ablation in 5XFAD mice significantly improved the performance at the Morris water maze test; these changes were accompanied by a reduced corticohippocampal Aβ burden without affecting plaque spatial/regional distribution and composition or global BACE1 expression. Furthermore, PCSK9 loss in 5XFAD mice induced decreased microgliosis and astrocyte reactivity in several brain regions. Conversely, knocking out PCSK9 had minimal impact on brain cholesterol and hydroxysterol levels. Conclusions: In vitro studies showed a pro-inflammatory effect of PCSK9. Consistently, in vivo data indicated a protective role of PCSK9 ablation against cognitive impairments, associated with improved Aβ pathology and attenuated neuroinflammation in a severe mouse model of AD. PCSK9 may thus be considered a novel pharmacological target for the treatment of AD