The past decade has seen the rapid development of new strategies for the design of biodegradable macromolecular compounds, with properly suited architecture and tailored properties, functioning as ...temporary support for the engineering of living constructs in tissue regeneration applications. The purpose of this paper is to review recent research in the interdisciplinary field of tissue engineering, with particular regard to bone and cartilage tissues, aimed at the design, synthesis, evaluation and characterization of bioactive polymeric scaffolds guiding and promoting new tissue ingrowth. Current strategies in scaffold-guided tissue engineering approach, involving the most employed biodegradable polymers, either of natural or synthetic origin, will be reported underlying the role played by both material structure–property relationship and scaffold architecture. While there are many polymeric materials that may be employed for the regeneration of bone and cartilage tissue, we will focus specifically on those that have been more extensively applied, showing promising outcomes. Commonly exploited and innovative processing techniques for the fabrication of advanced tissue engineering scaffolds will be explored, highlighting theoretical principles and their potential in creating micro-nanostructures suitable for tissue regeneration applications.
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► Volatile/fixed oils were extracted by SFE-CO2 from Nigella sativa seeds of Turkey/Egypt. ► Essential oils were mainly composed by the oxygenated monoterpene thymoquinone. ► Fixed ...oils showed high level of unsaturated fatty acids, mainly essential 18:2 n−6. ► Volatile/fixed oils displayed interesting antituberculosis and antifungal activities. ► Results confirm N. sativa oils as a potential resource for food/pharmaceutical applications.
Nigella sativa L. (Ranunculaceae), commonly known as ‘black cumin’, is an erect herbaceous annual plant. N. sativa seeds have traditionally been used in folk medicine as a natural remedy for various diseases as well as a spice. The seeds contain both fixed and essential oils, proteins, alkaloids and saponins. Much of the biological activity of the seeds has been shown to be due to thymoquinone, the major component of the essential oil. The fixed oil is composed mainly of unsaturated fatty acids, including the unusual C20:2 eicosadienoic acid.
Isolation of volatile and fixed oils from N. sativa seed of Turkey and Egypt has been obtained by supercritical fractioned extraction with carbon dioxide. Extraction experiments were carried out at pressures of 90 and 300bar and temperature of 40°C. The extraction step performed at 90bar produced a volatile fraction mainly formed by tymoquinone (79÷86%) and o-cymene (5÷11%). The oil yield relative to this step of the process was 0.1÷0.3% by weight of the charge. The last extraction step at 300bar produced a fixed oil. The yield of this step was 21–26% by weight. The most represented fatty acids of fixed oil from N. sativa were 18:2 n−6 (54÷55%), 18:1 n−9 (22÷23%), 16:0 (12÷13%), 18:0 (3%), and 20:2 (2÷3%).
The volatile and fixed oils obtained from N. sativa were evaluated for the antibacterial activity by employing standard strains of Escherichia coli, Pseudomonas aeroginosa, Acinetobacer baumannii, Staphylococcus aureus, Enterococcus faecalis. In vitro antifungal activity of the derivatives against Candida albicans, C. tropicalis, and C. krusei were screened by using ketoconazole, and fluconazole as control agents. The anti-mycobacterium activity breakpoint concentration (μgmL−1) was determined against standard strains of Mycobacterium tuberculosis H37Rv and M. avium (ATCC 15769). The volatile and fixed oils displayed antimicrobial activity toward all of the standard (ATCC, RSKK) strains of the tested bacteria at MIC values of 8–64μgmL−1 and were revealed to be ineffective against isolated strains (MIC; >256μgmL−1). The volatile and fixed oils emerged as effective against the bacteria of M. avium with MIC values of 8μgmL−1. Moreover, all the extracts exhibited antifungal activity against C. albicans, C. tropicalis, and C. krusei at MIC values of 16–64μgmL−1.
Adeno-associated virus (AAV) has been used to direct gene transfer to a variety of tissues, including heart, liver, skeletal muscle, brain, kidney and lung, but it has not previously been shown to ...effectively target fibroblasts in vivo, including cardiac fibroblasts. We constructed expression cassettes using a modified periostin promoter to drive gene expression in a cardiac myofibroblast-like lineage, with only occasional spillover into cardiomyocyte-like cells. We compared AAV serotypes 6 and 9 and found robust gene expression when the vectors were delivered by systemic injection after myocardial infarction (MI), with little expression in healthy, non-infarcted mice. AAV9 provided expression in a greater number of cells than AAV6, with reporter gene expression visible in the cardiac infarct and border zones from 5 to 62 days post MI, as assessed by luciferase and Cre-activated green fluorescent protein expression. Although common myofibroblast markers were expressed in low abundance, most of the targeted cells expressed myosin IIb, an embryonic form of smooth muscle myosin heavy chain that has previously been associated with myofibroblasts after reperfused MI. This study is the first to demonstrate AAV-mediated expression in a potentially novel myofibroblast-like lineage in mouse hearts post MI and may open new avenues of gene therapy to treat patients surviving MI.
This study aims to evaluate the antifungal activity of Mentha spicata L. and Mentha pulegium L. from Sardinia and to assess their efficacy on virulence factors for Candida albicans, particularly on ...the inhibition of the germ tube formation. The major compounds of the essential oils were carvone (62.9%) for M. spicata and pulegone (86.2%) for M. pulegium. The essential oil from M. spicata showed a more preeminent effect against Cryptococcus neoformans and the dermatophytes Trichophyton rubrum and T. verrucosum (0.32 μL/mL). Both oils were very effective in inhibiting C. albicans germ tube formation, at doses well below their MIC (0.16 μL/mL).
Considering the great influence of tumour microenvironment on the development and metastasis of cancer progression, three dimensional (3D) culture models have become essential for improving cancer ...biology investigations as well as the development of novel anticancer drug screening approaches. The aim of this study was to develop a novel
in vitro
3D model of pancreas cancer based on a human pancreatic cancer cell line BxPC-3 and a microstructured hydrogel made of chitosan (mCS) or a polyelectrolyte complex (mPEC) between CS and poly(γ-glutamic acid) (γ-PGA). The investigated hydrogels well supported the adhesion and proliferation of the cultured cells that were able to colonize the interconnected porous network of the 3D porous microstructures. As shown by confocal laser scanning microscopy (CLSM) analysis, BxPC-3 cells cultured in microstructured hydrogels maintained the cancer features typical of the cell line, such as loss of cell polarity and duct-like structures differentiation, and formed multicellular spheroids that were not observed in two-dimensional (2D) monolayer culture. Fascin protein, investigated as a marker of tumor invasiveness by CLSM microscopy and fluorescent western blotting technique, was found overexpressed in 3D tumor engineered constructs, unlike what is observed in 2D culture. In addition, cells cultured in combination with 3D hydrogels overexpressed a set of cytokines associated with pancreatic cancer invasiveness, as assessed by means of luminex technology. The obtained results suggested that the developed microstructured hydrogel/cell constructs represent a promising 3D tumor engineered model with great potential for the development of
in vitro
investigations providing innovative insights into pancreas carcinogenesis processes.
Development of a novel
in vitro
3D model of pancreas cancer based on microstructured polyelectrolyte complex (mPEC) hydrogel.
Alzheimer's disease (AD) causes dementia in both young and old people affecting more than 40 million people worldwide. The two neuropathological hallmarks of the disease, amyloid beta (Aβ) plaques ...and neurofibrillary tangles consisting of protein tau are considered the major contributors to the disease. However, a more complete picture reveals significant neurodegeneration and decreased cell survival, neuroinflammation, changes in protein and energy homeostasis and alterations in lipid and cholesterol metabolism. In addition, gene and cell therapies for severe neurodegenerative disorders have recently improved technically in terms of safety and efficiency and have translated to the clinic showing encouraging results. Here, we review broadly current data within the field for potential targets that could modify AD through gene and cell therapy strategies. We envision that not only Aβ will be targeted in a disease‐modifying treatment strategy but rather that a combination of treatments, possibly at different intervention times may prove beneficial in curing this devastating disease. These include decreased tau pathology, neuronal growth factors to support neurons and modulation of neuroinflammation for an appropriate immune response. Furthermore, cell based therapies may represent potential strategies in the future.
Reduced taste sensitivity to 6-n-propylthiouracil (PROP), a genetic trait regarded as a general index for oral chemosensory perception, has been associated with a calorie-rich food preference and ...lower circulating endocannabinoid levels in participants with normal weight (NW), which suggests an adaptive mechanism to maintain a lean phenotype. In this study, we assessed whether participants with obesity (OB) show different patterns of plasma endocannabinoids and lipid metabolism biomarkers from those of NW, with further categorization based on their PROP sensitivity. NW and OB were classified by their PROP taster status as non-tasters (NT), medium-tasters (MT) and supertasters (ST). The blood samples were analysed for plasma endocannabinoids, nonesterified fatty acids (NEFA) and retinol, which have been associated to metabolic syndrome. In OB, we found a higher BMI and lower circulating endocannabinoids in ST vs. OB NT. However, OB ST showed lower circulating NEFA and retinol levels, which suggested a more favourable lipid metabolism and body fat distribution than those of OB NT. We confirmed lower plasma endocannabinoid levels in NW NT than in NW ST. These data suggest that PROP taste sensitivity determines metabolic changes and ultimately body mass composition differently in OB and NW.
The multifunctional CD36 scavenger receptor facilitates fatty acid (FA) uptake and oxidation and it has been involved in the pathophysiology related to dysfunctional FA metabolism. The common variant ...in the
gene,
(A/G), whose allele A is characterized by a reduced protein expression, has been associated with taste sensitivity to and preference for fat. We therefore aimed at evaluating whether the
polymorphism may influence fatty acid metabolism and endocannabinoid biosynthesis in normal weight (NW) and obese (OB) subjects. Red blood cell (RBC) fatty acid composition, and plasma endocannabinoid levels were determined. In NW subjects with AA genotype was found a marked reduction of RBC saturated fatty acids and palmitic/linoleic ratio (PA/LA), considered as
lipogenesis (DNL) biomarkers. Remarkably, to the reduction of DNL biomarkers corresponded an increase of omega-6 index, an indirect marker of the impact on fatty acid metabolism of dietary omega-6 fatty acids, endocannabinoid levels and a higher waist/hip ratio. The presence of the G allele was instead associated with increased endocannabinoid plasma levels and a trend for increased waist/hip ratio in obese subjects, even though exhibited decreased BMI with respect to those with AA genotype. These data indicate that the
polymorphism,
, leads to a distinct metabolic pattern in NW and in OB subjects. Therefore, their determination may be crucial in developing personalized therapeutic strategies for ameliorating dyslipidemia and other metabolic disorders.
AAV9 is a powerful gene delivery vehicle capable of providing long-term gene expression in a variety of cell types, particularly cardiomyocytes. The use of AAV-delivery for RNA interference is an ...intense area of research, but a comprehensive analysis of knockdown in cardiac and liver tissues after systemic delivery of AAV9 has yet to be reported. We sought to address this question by using AAV9 to deliver a short-hairpin RNA targeting the enhanced green fluorescent protein (GFP) in transgenic mice that constitutively overexpress GFP in all tissues. The expression cassette was initially tested in vitro and we demonstrated a 61% reduction in mRNA and a 90% reduction in GFP protein in dual-transfected 293 cells. Next, the expression cassette was packaged as single-stranded genomes in AAV9 capsids to test cardiac GFP knockdown with several doses ranging from 1.8×10(10) to 1.8×10(11) viral genomes per mouse and a dose-dependent response was obtained. We then analyzed GFP expression in both heart and liver after delivery of 4.4×10(11) viral genomes per mouse. We found that while cardiac knockdown was highly efficient, with a 77% reduction in GFP mRNA and a 71% reduction in protein versus control-treated mice, there was no change in liver expression. This was despite a 4.5-fold greater number of viral genomes in the liver than in the heart. This study demonstrates that single-stranded AAV9 vectors expressing shRNA can be used to achieve highly efficient cardiac-selective knockdown of GFP expression that is sustained for at least 7 weeks after the systemic injection of 8 day old mice, with no change in liver expression and no evidence of liver damage despite high viral genome presence in the liver.
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