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Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible.

Although branched and linear polyethylenimines (bPEIs and lPEIs) are gold standard transfectants, a systematic analysis of the effects of the preparation protocol of polyplexes and the composition of the transfection medium on their physicochemical behaviour and effectiveness in vitro have been much neglected, undermining in some way the identification of precise structure-function relationships. This work aimed to address these issues. bPEI/DNA and lPEI/DNA, prepared using two different modes of addition of reagents, gave rise to polyplexes with exactly the same chemical composition but differing in dimensions. Upon dilution in serum-free medium, the size of any kind of polyplex promptly rose over time while remained invariably stable in complete DMEM. Of note, the bigger the dimension of polyplexes (in the nano- to micrometer range), the greater their efficiency in vitro . Besides, centrifugal sedimentation of polyplexes displaying different dimensions to speed up and enhance their settling onto cells boosted transfection efficiencies. Conversely, transgene expression was significantly blunted in cells held upside-down and transfected, definitively pointing out the impact of gravitational sedimentation of polyplexes on their transfection efficiency. Overall, much more attention must be paid to the actual polyplex size that relies on the complexation conditions and the transfection medium.

Cardiovascular diseases (CVDs) account for the 31% of total death per year, making them the first cause of death in the world. Atherosclerosis is at the root of the most life-threatening CVDs. Vascular bypass/replacement surgery is the primary therapy for patients with atherosclerosis. The use of polymeric grafts for this application is still burdened by high-rate failure, mostly caused by thrombosis and neointima hyperplasia at the implantation site. As a solution for these problems, the fast re-establishment of a functional endothelial cell (EC) layer has been proposed, representing a strategy of crucial importance to reduce these adverse outcomes. Implant modifications using molecules and growth factors with the aim of speeding up the re-endothelialization process has been proposed over the last years. Collagen, by virtue of several favorable properties, has been widely studied for its application in vascular graft enrichment, mainly as a coating for vascular graft luminal surface and as a drug delivery system for the release of pro-endothelialization factors. Collagen coatings provide receptor-ligand binding sites for ECs on the graft surface and, at the same time, act as biological sealants, effectively reducing graft porosity. The development of collagen-based drug delivery systems, in which small-molecule and protein-based drugs are immobilized within a collagen scaffold in order to control their release for biomedical applications, has been widely explored. These systems help in protecting the biological activity of the loaded molecules while slowing their diffusion from collagen scaffolds, providing optimal effects on the targeted vascular cells. Moreover, collagen-based vascular tissue engineering substitutes, despite not showing yet optimal mechanical properties for their use in the therapy, have shown a high potential as physiologically relevant models for the study of cardiovascular therapeutic drugs and diseases. In this review, the current state of the art about the use of collagen-based strategies, mainly as a coating material for the functionalization of vascular graft luminal surface, as a drug delivery system for the release of pro-endothelialization factors, and as physiologically relevant vascular models, and the future trend in this field of research will be presented and discussed.

Collagen gels have been widely studied for applications in tissue engineering because of their biological implications. Considering their use as scaffolds for vascular tissue engineering, the main limitation has always been related to their low mechanical properties. During the process of in vitro self-assembly, which leads to collagen gelation, the size of the fibrils, their chemical interactions, as well as the resulting microstructure are regulated by three main experimental conditions: pH, ionic strength and temperature. In this work, these three parameters were modulated in order to increase the mechanical properties of collagen gels. The effects on the gelation process were assessed by turbidimetric and scanning electron microscopy analyses. Turbidity measurements showed that gelation was affected by all three factors and scanning electron images confirmed that major changes occurred at the microstructural level. Mechanical tests showed that the compressive and tensile moduli increased by four- and three-fold, respectively, compared to the control. Finally, viability tests confirmed that these gels are suitable as scaffolds for cellular adhesion and proliferation.

There has been a tremendous amount of research in the past decade to optimize the mechanical properties and degradation behavior of the biodegradable Mg alloy for orthopedic implant. Despite the feasibility of degrading implant, the lack of fundamental understanding about biocompatibility and underlying bone formation mechanism is currently limiting the use in clinical applications. Herein, we report the result of long-term clinical study and systematic investigation of bone formation mechanism of the biodegradable Mg-5wt%Ca-1wt%Zn alloy implant through simultaneous observation of changes in element composition and crystallinity within degrading interface at hierarchical levels. Controlled degradation of Mg-5wt%Ca-1wt%Zn alloy results in the formation of biomimicking calcification matrix at the degrading interface to initiate the bone formation process. This process facilitates early bone healing and allows the complete replacement of biodegradable Mg implant by the new bone within 1 y of implantation, as demonstrated in 53 cases of successful long-term clinical study.

Magnetic force microscopy (MFM) has been demonstrated as valuable technique for the characterization of magnetic nanomaterials. To be analyzed by MFM techniques, nanomaterials are generally deposited on flat substrates, resulting in an additional contrast in MFM images due to unavoidable heterogeneous electrostatic tip-sample interactions, which cannot be easily distinguished from the magnetic one. In order to correctly interpret MFM data, a method to remove the electrostatic contributions from MFM images is needed. In this work, we propose a new MFM technique, called controlled magnetization MFM (CM-MFM), based on the in situ control of the probe magnetization state, which allows the evaluation and the elimination of electrostatic contribution in MFM images. The effectiveness of the technique is demonstrated through a challenging case study, i.e., the analysis of superparamagnetic nanoparticles in absence of applied external magnetic field. Our CM-MFM technique allowed us to acquire magnetic images depurated of the electrostatic contributions, which revealed that the magnetic field generated by the tip is sufficient to completely orient the superparamagnetic nanoparticles and that the magnetic tip-sample interaction is describable through simple models once the electrostatic artifacts are removed.

Electronic waste (e-waste) is becoming a serious problem that impacts the environment due to its fast-growing volume. This rise is linked to high electronic and electrical equipment production to meet the increasing demand for high-end electronic devices. Conventional e-waste recycling approaches, including hydrometallurgy and pyrometallurgy, often involve substantial water and energy consumption and the generation of by-products, such as the emission of toxic gases or hazardous effluents. Within this context, solvometallurgy has emerged as a compelling alternative, whereby green non-toxic non-aqueous solvents, namely deep eutectic solvents (DESs), are used to extract and recover the metals with minimal water and harsh acid/base chemical use. The current study presents the solvo-leaching results of critical and strategic metals, i.e., copper (Cu) and nickel (Ni), and precious metals, i.e., gold (Au), from waste printed circuit boards (PCBs). Five different DESs were tested at mild conditions, namely at a temperature of 65 °C, a stirring speed of 300 rpm, a solid/liquid ratio of 10 g/L, and in the presence of iodine (I2) for 96 h. Among the different solvents tested, the one consisting of choline chloride (ChCl), acetic acid (AA), and I2 emerged as the optimal solvent, leading to the selective extraction of 99% of Cu, 92% of Ni, and 90% of Au from the PCB powder.

Biodegradable metallic materials represent a potential step‐change technology that may revolutionize the treatment of broken bones. Implants made with biodegradable metals are significantly stronger than their polymer counterparts and fully biodegradable in vivo, removing the need for secondary surgery or long‐term complications. Here, it is shown how clinically approved Mg alloy promotes improved bone repair using an integrated state of the art fetal mouse metatarsal assay coupled with in vivo preclinical studies, second harmonic generation, secretome array analysis, perfusion bioreactor, and high‐resolution 3D confocal imaging of vasculature within skeletal tissue, to reveal a vascular‐mediated pro‐osteogenic mechanism controlling enhanced tissue regeneration. The optimized mechanical properties and corrosion rate of the Mg alloy lead to a controlled release of metallic Mg, Ca, and Zn ions at a rate that facilitates both angiogenesis and coupled osteogenesis for better bone healing, without causing adverse effects at the implantation site. The findings from this study support ongoing development and refinement of biodegradable metal systems to act as crucial portal technologies with significant potential to improve many clinical applications. An integrated state‐of‐the‐art in vivo and in vitro approach reveals clinically approved Mg5Ca1Zn samples stimulate accelerated bone healing by releasing anabolic metallic ions into the surrounding tissues to enhance the growth of type H blood vessels, which localize at active sites of bone remodeling and actively recruit Osterix‐positive osteoprogenitors to the degrading implant site.

The clinical demand for tissue-engineered vascular grafts is still rising, and there are many challenges that need to be overcome, in particular, to obtain functional small-diameter grafts. The many advances made in cell culture, biomaterials, manufacturing techniques, and tissue engineering methods have led to various promising solutions for vascular graft production, with available options able to recapitulate both biological and mechanical properties of native blood vessels. Due to the rising interest in materials with bioactive potentials, materials from natural sources have also recently gained more attention for vascular tissue engineering, and new strategies have been developed to solve the disadvantages related to their use. In this review, the progress made in tissue-engineered vascular graft production is discussed. We highlight, in particular, the use of natural materials as scaffolds for vascular tissue engineering.

Collagen is a widely investigated extracellular matrix material with extensive potentials in the field of tissue engineering. This protocol describes a method to prepare reconstituted collagen that can be ready-to-use, storable and suitable for further in vitro and in vivo investigations. Type I collagen was extracted from rat tail tendons and processed in acetic acid solution to obtain sterile soluble collagen. At first, crude collagen was dissolved in acetic acid, then frozen at −20 °C and lyophilized to obtain a sponge, which could be stored at −80 °C. Lyophilized collagen was then dispersed in acetic acid to obtain a sterile solution of collagen at targeted concentrations. The whole low-cost process from the extraction to the final sterile solution takes around 2–3 weeks. The collagen solution, once neutralized, has the potential to be used to produce gels or scaffolds, to deposit thin films on supports and to develop drug delivery systems.