The aggregation of cytotoxic amyloid peptides (Aβ1-42) is widely recognised as the cause of brain tissue degeneration in Alzheimer’s disease (AD). Indeed, evidence indicates that the deposition of ...cytotoxic Aβ1-42 plaques formed through the gradual aggregation of Aβ1-42 monomers into fibrils determines the onset of AD. Thus, distinct Aβ1-42 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 Aβ1-42 aggregation. Among them, the KLVFF peptide sequence, which interacts with the hydrophobic region of Aβ16-20, has received widespread attention due to its ability to inhibit fibril formation of full-length Aβ1-42. 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 Aβ1-42 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 Aβ1-42 than that detected by linear KLVFF peptides.
The efficacy of stem-cell therapy depends on the ability of the transplanted cells to escape early immunological reactions and to be retained at the site of transplantation. The use of tissue ...engineering scaffolds or injectable biomaterials as carriers has been proposed, but they still present limitations linked to a reliable manufacturing process, surgical practice and clinical outcomes. Alginate microbeads are potential candidates for the encapsulation of mesenchymal stromal cells with the aim of providing a delivery carrier suitable for minimally-invasive and scaffold-free transplantation, tissue-adhesive properties and protection from the immune response. However, the formation of stable microbeads relies on the cross-linking of alginate with divalent calcium ions at concentrations that are toxic for the cells, making control over the beads’ size and a single-cell encapsulation unreliable. The present work demonstrates the efficiency of an innovative, high throughput, and reproducible microfluidic system to produce single-cell, calcium-free alginate coatings of human mesenchymal stromal cells. Among the various conditions tested, visible light and confocal microscopy following staining of the cell nuclei by DAPI showed that the microfluidic system yielded an optimal single-cell encapsulation of 2000 cells/min in 2% w/v alginate microcapsules of reproducible morphology and an average size of 28.2 ± 3.7 µm. The adhesive properties of the alginate microcapsules, the viability of the encapsulated cells and their ability to escape the alginate microcapsule were demonstrated by the relatively rapid adherence of the beads onto tissue culture plastic and the cells’ ability to gradually disrupt the microcapsule shell after 24 h and proliferate. To mimic the early inflammatory response upon transplantation, the encapsulated cells were exposed to proliferating macrophages at different cell seeding densities for up to 2 days and the protection effect of the microcapsule on the cells assessed by time-lapse microscopy showing a shielding effect for up to 48 h. This work underscores the potential of microfluidic systems to precisely encapsulate cells by good manufacturing practice standards while favouring cell retention on substrates, viability and proliferation upon transplantation.
Graphical Abstract
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.
Graphical abstract
The in vitro study of the properties of the human mesenchymal stem cells as well as their manipulation in culture for clinical purposes depends on the elimination of artefacts caused by the lack of ...their natural environment. It is now widely accepted that mesenchymal stem cells should be studied when they are organised as 3D spheroids rather than fibroblast-like colonies. Although this can be achieved with the use of some extracellular matrix proteins or by non-adherent conditions these suffer of significant limitations. The recent development of synthetic substrates resembling the physicochemical and biochemical properties of the adult stem cell niche has prompted questions about the role played by nanotopography and receptor-mediated adhesion. In the present paper, the influence of two types of substrates bearing the same nanostructure, but exposing either a non-specific or an integrin-specific binding motif was studied. Carboxybetaine-tethered hyperbranched poly(ɛ-lysine) dendrons showed that the hyperbranched structure was fundamental to induce spheroid formation, but these were forming more slowly, were of reduced size and less stable than those growing on substrates based on the same hyperbranched structures that had been functionalised at their uppermost branching generation by a laminin amino acid sequence, i.e. YIGSR. The study shows that both nanostructure and biorecognition need to be combined to achieve a substrate for stem cell spheroid formation as that observed in vivo in the adult stem cell niche.
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.
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.
Antibodies are the macromolecules of choice to ensure specific recognition of biomarkers in biological assays. However, they present a range of shortfalls including a relatively high production cost ...and limited tissue penetration. Peptides are relatively small molecules able to reproduce sequences of highly specific paratopes and, although they have less biospecificity than antibodies, they offer advantages like ease of synthesis, modifications of their amino acid sequences and tagging with fluorophores and other molecules required for detection. This work presents a strategy to design peptide sequences able to recognize the CD44 hyaluronic acid receptor present in the plasmalemma of a range of cells including human bone marrow stromal mesenchymal cells. The protocol of identification of the optimal amino acid sequence was based on the combination of rational design and in silico methodologies. This protocol led to the identification of two peptide sequences which were synthesized and tested on human bone marrow mesenchymal stromal cells (hBM-MSCs) for their ability to ensure specific binding to the CD44 receptor. Of the two peptides, one binds CD44 with sensitivity and selectivity, thus proving its potential to be used as a suitable alternative to this antibody in conventional immunostaining. In the context of regenerative medicine, the availability of this peptide could be harnessed to functionalize tissue engineering scaffolds to anchor stem cells as well as to be integrated into systems such as cell sorters to efficiently isolate MSCs from biological samples including various cell subpopulations. The data here reported can represent a model for developing peptide sequences able to recognize hBM-MSCs and other types of cells and for their integration in a range of biomedical applications.
In the 1970s, morphological evidence collected by electron microscopy linked mineral deposition ("calcification" or "mineralization") in newly-forming bone to membrane-encapsulated particles of a ...diameter of approximately 100 nm (50-200 nm) that were called "matrix vesicles". As the characterisation of these vesicles progressed towards their biochemical composition, the role of lipids in the biomineralization process appeared to be crucial. In particular, a group of cell-membrane phospholipids were identified as major players in the crystal formation process. Indeed, in the 1980s it became clear that phosphatidylserine, together with proteins of the annexin family, was among the most important molecules in binding calcium ions and that this phospholipid was involved in the regulation of the early stages of mineralization in vivo. During the same period of time, the number of surgical implantations of orthopaedic, dental and maxilo-facial devices requiring full integration with the treated bone prompted the study of new functionalization molecules able to establish a stable bonding with the mineral phase of the host tissue. In the late 1990 s studies started that aimed at exploiting the potential of calcium-binding phospholipids and, in particular, of the phosphatidylserine as functionalization molecules to improve the osteointegration of artificial implants. Later, papers have been published that show the potential of the phophatidylserine and phosphatidylserine-mimicking coating technology to promote calcification both in vitro and in vivo. The promising results support the future clinical application of these novel osteointegrative biomaterials.
The reconstruction of large segmental defects still represents a critical issue in the orthopedic field. The use of functionalized scaffolds able to create a magnetic environment is a fascinating ...option to guide the onset of regenerative processes. In the present study, a porous hydroxyapatite scaffold, incorporating superparamagnetic Fe
O
nanoparticles (MNPs), was implanted in a critical bone defect realized in sheep metatarsus. Superparamagnetic nanoparticles functionalized with hyperbranched poly(epsilon-Lysine) peptides and physically complexed with vascular endothelial growth factor (VEGF) where injected in situ to penetrate the magnetic scaffold. The scaffold was fixed with cylindrical permanent NdFeB magnets implanted proximally, and the magnetic forces generated by the magnets enabled the capture of the injected nanoparticles forming a VEGF gradient in its porosity. After 16 weeks, histomorphometric measurements were performed to quantify bone growth and bone-to-implant contact, while the mechanical properties of regenerated bone via an atomic force microscopy (AFM) analysis were investigated. The results showed increased bone regeneration at the magnetized interface; this regeneration was higher in the VEGF-MNP-treated group, while the nanomechanical behavior of the tissue was similar to the pattern of the magnetic field distribution. This new approach provides insights into the ability of magnetic technologies to stimulate bone formation, improving bone/scaffold interaction.
Abstract
Background
Alzheimer’s disease (AD) is the leading cause of dementia and loss of autonomy in the elderly, implying a progressive cognitive decline and limitation of social activities. The ...progressive aging of the population is expected to exacerbate this problem in the next decades. Therefore, there is an urgent need to develop quantitative diagnostic methodologies to assess the onset the disease and its progression especially in the initial phases.
Results
Here we describe a novel technology to extract one of the most important molecular biomarkers of AD (Aβ
1−42
) from a clinically-relevant volume − 100 µl – therein dispersed in a range of concentrations critical for AD early diagnosis. We demonstrate that it is possible to immunocapture Aβ
1−42
on 20 nm wide magnetic nanoparticles functionalized with hyperbranced KVLFF aptamers. Then, it is possible to transport them through microfluidic environments to a detection system where virtually all (~ 90%) the Aβ
1−42
molecules are concentrated in a dense plug of ca.50 nl. The technology is based on magnetic actuation by permanent magnets, specifically designed to generate high gradient magnetic fields. These fields, applied through submillimeter-wide channels, can concentrate, and confine magnetic nanoparticles (MNPs) into a droplet with an optimized shape that maximizes the probability of capturing highly diluted molecular biomarkers. These advancements are expected to provide efficient protocols for the concentration and manipulation of molecular biomarkers from clinical samples, enhancing the accuracy and the sensitivity of diagnostic technologies.
Conclusions
This easy to automate technology allows an efficient separation of AD molecular biomarkers from volumes of biological solutions complying with the current clinical protocols and, ultimately, leads to accurate measurements of biomarkers. The technology paves a new way for a quantitative AD diagnosis at the earliest stage and it is also adaptable for the biomarker analysis of other pathologies.
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