The Real-Time Validation of the Effectiveness of Third-Generation Hyperbranched Poly(ɛ-lysine) Dendrons-Modified KLVFF Sequences to Bind Amyloid-β1-42 Peptides Using an Optical Waveguide Light-Mode Spectroscopy System

The aggregation of cytotoxic amyloid peptides (Aβ(1-42)) is widely recognised as the cause of brain tissue degeneration in Alzheimer’s disease (AD). Indeed, evidence indicates that the deposition of cytotoxic Aβ(1-42) plaques formed through the gradual aggregation of Aβ(1-42) monomers into fibrils determines the onset of AD. Thus, distinct Aβ(1-42) inhibitors have been developed, and only recently, the use of short linear peptides has shown promising results by either preventing or reversing the process of Aβ(1-42) aggregation. Among them, the KLVFF peptide sequence, which interacts with the hydrophobic region of Aβ(16-20), has received widespread attention due to its ability to inhibit fibril formation of full-length Aβ(1-42). In this study, hyperbranched poly-L-lysine dendrons presenting sixteen KLVFF at their uppermost molecular branches were designed with the aim of providing the KLVFF sequence with a molecular scaffold able to increase its stability and of improving Aβ(1-42) fibril formation inhibitory effect. These high-purity branched KLVFF were used to functionalise the surface of the metal oxide chip of the optical waveguide lightmode spectroscopy sensor showing the more specific, accurate and rapid measurement of Aβ(1-42) than that detected by linear KLVFF peptides

A Substrate-Mimicking Basement Membrane Drives the Organization of Human Mesenchymal Stromal Cells and Endothelial Cells Into Perivascular Niche-Like Structures

Extracellular matrix-derived products (e.g. Matrigel) are widely used for in vitro cell cultures both as two-dimensional (2D) substrates and as three-dimensional (3D) encapsulation gels because of their ability to control cell phenotypes through biospecific cues. However, batch-to-batch variations, poor stability, cumbersome handling, and the relatively high costs strictly limit their use. Recently, a new substrate known as PhenoDrive-Y has been used as 2D coating of tissue culture plastic showing to direct the bone marrow mesenchymal stromal cells (MSCs) toward the formation of 3D spheroids. When organized into 3D spheroids, the MSCs expressed levels of pluripotency markers and of paracrine angiogenic activity higher than those of the MSCs adhering as fibroblast-like colonies on tissue culture plastic. The formation of the spheroids was attributed to the properties of this biomaterial that resemble the main features of the basement membrane by mimicking the mesh structure of collagen IV and by presenting the cells with orderly spaced laminin bioligands. In this study, PhenoDrive-Y was compared to Matrigel for its ability to drive the formation of perivascular stem cell niche-like structures in 2D co-culture conditions of human endothelial cells and adult bone marrow MSCs. Morphological analyses demonstrated that, when compared to Matrigel, PhenoDrive-Y led endothelial cells to sprout into a more consolidated tubular network and that the MSCs nestled as compact spheroids above the anastomotic areas of this network resemble more closely the histological features of the perivascular stem cell niche. A study of the expressions of relevant markers led to the identification of the pathways linking the PhenoDrive-Y biomimicking properties to the acquired histological features, demonstrating the enhanced levels of stemness, renewal potential, predisposition to migration, and paracrine activities of the MSCs

Development of scaffold-free vascularized pancreatic beta-islets in vitro models by the anchoring of cell lines to a bioligand-functionalized gelatine substrate

Bioengineered pancreatic β-islets have been widely advocated for the research and treatment of diabetes by offering both suitable cell culture models for the study of the pathology and the testing of new drugs and a therapy in those patients no longer responding to insulin administration and as an alternative to the shortage of donors for organ and islet transplantation. Unlike most of the studies published so far where pancreatic islets of pancreatic β-cells are encapsulated in hydrogels, this study demonstrate the formation of bioengineered pancreatic islets through cell anchoring to a gelatine-based biomaterial, PhenoDrive-Y, able to mimic the basement membrane of tissues. Through simple culture conditions, PhenoDrive-Y led human pancreatic β-cell lines and human umbilical endothelial cell lines to form organized structures closely resembling the natural vascularized pancreatic islets. When compared to gelatine, the cultures in presence of PhenoDrive-Y show higher degree of organization in tissue-like structures, a more pronounced endothelial sprouting and higher expression of typical cell markers. Noticeably, when challenged by hyperglycaemic conditions, the cells embedded in the PhenoDrive-Y assembled spheroids responded with higher levels of insulin production. In conclusion, the present work demonstrates the potential of PhenoDrive-Y as substrate for the development of bioengineered vascularized pancreatic islets and to be particularly suitable as a model for in vitro studies and testing of new therapeutics. [Figure: see text

Pre-clinical evaluation of soybean-based wound dressings and dermal substitute formulations in pig healing and non-healing in vivo models

In the last decade, a new class of natural biomaterials derived from de-fatted soybean flour processed by either thermoset or extraction procedures has been developed. These biomaterials uniquely combine adaptability to various clinical applications to proven tissue regeneration properties. In the present work, the biomaterials were formulated either as hydrogel or as paste formulation and their potential as wound dressing material or as dermal substitute was assessed by two in vivo models in pig skin: The healing full-thickness punch biopsy model and the non-healing full-thickness polytetrafluoroethylene (PTFE) chamber model. The results clearly show that collagen deposition is induced by the presence of these biomaterials. A unique pattern of early inflammatory response, eliciting neutrophils and controlling macrophage infiltration, is followed by tissue cell colonization of the wound bed with a significant deposition of collagen fibers. The study also highlighted the importance in the use of optimal formulations and appropriate handling upon implantation. In large size, non-healing wounds, wound dermis was best obtained with the paste formulation as hydrogels appeared to be too loose to ensure lasting scaffolding properties. On the contrary, packing of the granules during the application of paste reduced biomaterial degradation rate and prevent the penetration of newly vascularized tissue, thus impeding grafting of split-thickness autologous skin grafts on the dermal substitute base

Assessment of Cytocompatibility and AntiInflammatory (Inter)Actions of Genipin-Crosslinked Chitosan Powders

none5Chitosan is a polysaccharide commonly used, together with its derivatives, in the preparation of hydrogel formulations, scaffolds and films for tissue engineering applications. Chitosan can be used as such, but it is commonly stabilized by means of chemical crosslinkers. Genipin is one of the crosslinkers that has been considered that is a crystalline powder extracted from the fruit of Gardenia jasminoides and processed to obtain an aglycon compound. Genipin is gaining interest in biological applications because of its natural origin and anti-inflammatory actions. In this paper, the ability of chitosan-based materials crosslinked with genipin to exert anti-inflammation properties in applications such as bone regeneration was studied. Powders obtained from chitosan–genipin scaffolds have been tested in order to mimic the natural degradation processes occurring during biomaterials implantation in vivo. The results from osteoblast-like cells showed that specific combinations of chitosan and genipin stimulate high permissiveness towards cells, with higher performance than the pure chitosan. In parallel, evidences from monocyte-like cells showed that the crosslinker, genipin, seems to promote slowing of the monocyte-macrophage transition at morphological level. This suggests a sort of modularity of pro-inflammatory versus anti-inflammatory behavior of our chitosan-based biomaterials. Being both the cell types exposed to microscale powders, as an added value our results bring information on the cell–material interactions in the degradative dynamics of chitosan scaffold structures during the physiological resorption processes.noneSimona Dimida; Matteo Santin; Tiziano Verri; Amilcare Barca; Christian DemitriDimida, Simona; Santin, Matteo; Verri, Tiziano; Barca, Amilcare; Demitri, Christia

Dendrimeric Poly(Epsilon-Lysine) Delivery Systems for the Enhanced Permeability of Flurbiprofen across the Blood-Brain Barrier in Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive brain disorder and age-related disease characterised by abnormal accumulation of β-amyloid (Aβ). The development of drugs to combat AD is hampered by the lack of therapeutically-active molecules able to cross the blood-brain barrier (BBB). It is agreed that specifically-designed carriers, such as dendrimers, could support the drug penetration across the BBB. The aim of this study was to design biocompatible and biodegradable dendrimeric delivery systems able to carry Flurbiprofen (FP), as drug for AD treatment, across the BBB and liberate it at the target tissue. These dendrons were synthesised using solid-phase peptide synthesis method and characterised by mass spectrometry and fourier-transform infrared spectroscopy (FTIR). The results revealed successful synthesis of dendrons having FP been integrated during the synthesis at their branching ends. Cytotoxicity assays demonstrated the biocompatibility of the delivery systems, whereas HPLC analysis showed high percentages of permeability across an in vitro BBB model for FP-integrated dendrons. Results also revealed the efficiency of drug conjugates on the γ-secretase enzyme in target cells with evidence of eventual drug release by hydrolysis of the carrier. This study demonstrates that the coupling of FP to dendrimeric delivery systems can successfully be achieved during the synthesis of the poly(epsilon-lysine) macromolecules to improve the transport of the active drug across the BBB