INJECTABLE HYBRID SYSTEM FOR STRONTIUM LOCAL DELIVERY TO PROMOTE BONE REGENERATION

In bone tissue regeneration strategies, injectable bone substitutes are very attractive since they can be applied with minimally invasive surgical procedures and can perfectly fill irregular defects created in cases of trauma, infection or tumor resection. These materials must combine adequate mechanical properties with the ability to induce new bone formation. Incorporating strontium (Sr) in bone substitute biomaterials may be a strategy to achieve high Sr concentrations, not in a systemic but in a local environment, taking advantage of the osteoanabolic and anti-osteoclastic activity of Sr, for the enhancement of new bone formation. In this context, the aim of the present work was to evaluate the response of a Sr-hybrid injectable system for bone regeneration, designed by our group, consisting of hydroxyapatite microspheres doped with Sr and an alginate vehicle crosslinked in situ with Sr, in an in vivo scenario. Two different animal models were used, rat (Wistar) and sheep (Merino Branco) critical sized bone defect. Non Sr-doped similar materials (Ca-hybrid) or empty defects were used as control. Sr-hybrid system led to an increased bone formation in both center and periphery of a rat critical sized defect compared to a non Sr–doped similar system, where new bone formation was restricted to the periphery. Moreover newly formed bone was identified as early as one week after its implantation in a sheep model. After eight weeks, the bone surrounded the microspheres, both in the periphery and in the center of the defect. Most importantly, the hybrid system provided a scaffold for cell migration and tissue ingrowth and offered structural support, as observed in both models. The effective improvement of local bone formation suggests that this might be a promising approach for bone regeneration, especially in osteoporotic conditions

Phenotypic and proliferative modulation of human mesenchymal stem cells via crosstalk with endothelial cells

AbstractThe purpose of this work was to investigate if a coculture system of human mesenchymal stem cells (hMSC) with endothelial cells (human umbilical vein endothelial cells, HUVEC) could modulate the phenotype and proliferation of harvested MSCs. In addition to previous investigations on the crosstalk between these two cell types, in the present work different relative cell ratios were analyzed for long, therapeutically relevant, culture periods. Moreover, MSCs osteogenic commitment was assessed in a non-osteogenic medium and in the presence of HUVECs through magnetic cell separation, cell quantification by flow cytometry, morphology by fluorescent microscopy, metabolic activity and gene expression of osteogenic markers. Collectively, the present findings demonstrate that, by coculturing MSCs with HUVECs, there was not only the promotion of osteogenic differentiation (and its enhancement, depending on the relative cell ratios used), but also a significant increase on MSCs proliferation. This augmentation in cell proliferation occurred independently of relative cell ratios, but was favored by higher relative amounts of HUVECs. Taken together, this data suggests that HUVECs not only modulate MSC phenotype but also their proliferation rate. Therefore, a coculture system of MSCs and HUVECs can a have a broad impact on bone tissue engineering approaches

Colorectal Cancer Triple Co-culture Spheroid Model to Assess the Biocompatibility and Anticancer Properties of Polymeric Nanoparticles

Colorectal cancer (CRC) is the third most common and the second deadliest type of cancer worldwide, urging the development of more comprehensive models and of more efficient treatments. Although the combination of nanotechnology with chemo- and immuno-therapy has represented a promising treatment approach, its translation to the clinic has been hampered by the absence of cellular models that can provide reliable and predictive knowledge about the in vivo efficiency of the formulation. Herein, a 3D model based on CRC multicellular tumor spheroids (MCTS) model was developed by combining epithelial colon cancer cells (HCT116), human intestinal fibroblasts and monocytes. The developed MCTS 3D model mimicked several tumor features with cells undergoing spatial organization and producing extracellular matrix, forming a mass of tissue with a necrotic core. Furthermore, monocytes were differentiated into macrophages with an anti-inflammatory, pro-tumor M2-like phenotype. For a combined chemoimmunotherapy effect, spermine-modified acetalated dextran nanoparticles (NPs) loaded with the chemotherapeutic Nutlin-3a (Nut3a) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were produced and tested in 2D cultures and in the MCTS 3D model. NPs were successfully taken-up by the cells in 2D, but in a significant less extent in the 3D model. However, these NPs were able to induce an anti-proliferative effect both in the 2D and in the 3D models. Moreover, Nut3a was able to partially shift the polarization of the macrophages present in the MCTS 3D model towards an anti-tumor M1-like phenotype. Overall, the developed MCTS 3D model showed to recapitulate key features of tumors, while representing a valuable model to assess the effect of combinatorial nano-therapeutic strategies in CRC. In addition, the developed NPs could represent a promising approach for CRC treatment.Peer reviewe

Monitoring bridge degradation using dynamic strain, acoustic emission and environmental data

This paper studies the long term structural behaviour of a Victorian railway viaduct under train loading and temperature variation. A multi-sensing, self-sustaining and remotely controlled data acquisition system combines fibre Bragg grating strain sensors with acoustic emission sensors for the study of both global dynamic deformation and local masonry deterioration. A statistical analysis of fibre Bragg grating signals reveals regions with permanent change in the dynamic deformation of the bridge over the last two years, whereas in other locations the deformation follows a seasonal cyclic pattern. In order to decouple changes in structural behaviour due to real mechanical damage from normal seasonal effect, the paper studies the ambient temperature effect on the dynamic deformation of the bridge, showing a clear linear dependence. In particular, when temperature increases, the dynamic strain due to train loading decreases uniformly in the longitudinal direction. In the transverse direction, where the thermal expansion is not constrained, the decrease is smaller. Decoupling damage from normal seasonal effect is of critical importance for the development of reliable early warning structural alert systems for infrastructure networks. The paper further studies local masonry deterioration at four critical location by combining data from the two sensing technologies: fibre optic and acoustic emission sensors.This work is being funded by the Lloyd’s Register Foundation, EPSRC and Innovate UK through the Data-Centric Engineering programme of the Alan Turing Institute and through the Cambridge Centre for Smart Infrastructure and Construction. Funding for the monitoring installation was provided by EPSRC under the Ref. EP/N021614/1 grant and by Innovate UK under the Ref. 920035 grant

Dynasore, a Dynamin Inhibitor, Inhibits Trypanosoma cruzi Entry into Peritoneal Macrophages

BACKGROUND: Trypanosoma cruzi is an intracellular parasite that, like some other intracellular pathogens, targets specific proteins of the host cell vesicular transport machinery, leading to a modulation of host cell processes that results in the generation of unique phagosomes. In mammalian cells, several molecules have been identified that selectively regulate the formation of endocytic transport vesicles and the fusion of such vesicles with appropriate acceptor membranes. Among these, the GTPase dynamin plays an important role in clathrin-mediated endocytosis, and it was recently found that dynamin can participate in a phagocytic process. METHODOLOGY/PRINCIPAL FINDINGS: We used a compound called dynasore that has the ability to block the GTPase activity of dynamin. Dynasore acts as a potent inhibitor of endocytic pathways by blocking coated vesicle formation within seconds of its addition. Here, we investigated whether dynamin is involved in the entry process of T. cruzi in phagocytic and non-phagocytic cells by using dynasore. In this aim, peritoneal macrophages and LLC-MK2 cells were treated with increasing concentrations of dynasore before interaction with trypomastigotes, amastigotes or epimastigotes. We observed that, in both cell lines, the parasite internalization was drastically diminished (by greater than 90% in LLC-MK2 cells and 70% in peritoneal macrophages) when we used 100 microM dynasore. The T. cruzi adhesion index, however, was unaffected in either cell line. Analyzing these interactions by scanning electron microscopy and comparing peritoneal macrophages to LLC-MK2 cells revealed differences in the stage at which cell entry was blocked. In LLC-MK2 cells, this blockade is observed earlier than it is in peritoneal macrophages. In LLC-MK2 cells, the parasites were only associated with cellular microvilli, whereas in peritoneal macrophages, trypomastigotes were not completely engulfed by a host cell plasma membrane. CONCLUSIONS/SIGNIFICANCE: Taken together our results demonstrate that dynamin is an essential molecule necessary for cell invasion and specifically parasitophorous vacuole formation by host cells during interaction with Trypanosoma cruzi

Advancing Key Gaps in the Knowledge of Plasmodium vivax Cryptic Infections Using Humanized Mouse Models and Organs-on-Chips

Plasmodium vivax is the most widely distributed human malaria parasite representing 36.3% of disease burden in the South-East Asia region and the most predominant species in the region of the Americas. Recent estimates indicate that 3.3 billion of people are under risk of infection with circa 7 million clinical cases reported each year. This burden is certainly underestimated as the vast majority of chronic infections are asymptomatic. For centuries, it has been widely accepted that the only source of cryptic parasites is the liver dormant stages known as hypnozoites. However, recent evidence indicates that niches outside the liver, in particular in the spleen and the bone marrow, can represent a major source of cryptic chronic erythrocytic infections. The origin of such chronic infections is highly controversial as many key knowledge gaps remain unanswered. Yet, as parasites in these niches seem to be sheltered from immune response and antimalarial drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Due to ethical and technical considerations, working with the liver, bone marrow and spleen from natural infections is very difficult. Recent advances in the development of humanized mouse models and organs-on-a-chip models, offer novel technological frontiers to study human diseases, vaccine validation and drug discovery. Here, we review current data of these frontier technologies in malaria, highlighting major challenges ahead to study P. vivax cryptic niches, which perpetuate transmission and burden

Vascular units as advanced living materials for bottom-up engineering of perfusable 3D microvascular networks

The timely establishment of functional neo-vasculature is pivotal for successful tissue development and regeneration, remaining a central challenge in tissue engineering. In this study, we present a novel (micro)vascularization strategy that explores the use of specialized "vascular units" (VUs) as building blocks to initiate blood vessel formation and create perfusable, stroma-embedded 3D microvascular networks from the bottom-up. We demonstrate that VUs composed of endothelial progenitor cells and organ-specific fibroblasts exhibit high angiogenic potential when embedded in fibrin hydrogels. This leads to the formation of VUs-derived capillaries, which fuse with adjacent capillaries to form stable microvascular beds within a supportive, extracellular matrix-rich fibroblastic microenvironment. Using a custom-designed biomimetic fibrin-based vessel-on-chip (VoC), we show that VUs-derived capillaries can inosculate with endothelialized microfluidic channels in the VoC and become perfused. Moreover, VUs can establish capillary bridges between channels, extending the microvascular network throughout the entire device. When VUs and intestinal organoids (IOs) are combined within the VoC, the VUs-derived capillaries and the intestinal fibroblasts progressively reach and envelop the IOs. This promotes the formation of a supportive vascularized stroma around multiple IOs in a single device. These findings underscore the remarkable potential of VUs as building blocks for engineering microvascular networks, with versatile applications spanning from regenerative medicine to advanced in vitro models

Design, synthesis and antiparasitic evaluation of click phospholipids

A library of seventeen novel ether phospholipid analogues, containing 5-membered heterocyclic rings (1,2,3-triazolyl, isoxazolyl, 1,3,4-oxadiazolyl and 1,2,4-oxadiazolyl) in the lipid portion were designed and synthesized aiming to identify optimised miltefosine analogues. The compounds were evaluated for their in vitro antiparasitic activity against Leishmania infantum and Leishmania donovani intracellular amastigotes, against Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the substituents of the heterocyclic ring (tail) and the oligomethylene spacer between the head group and the heterocyclic ring was found to affect the activity and toxicity of these compounds leading to a significantly improved understanding of their structure\u2013activity relationships. The early ADMET profile of the new derivatives did not reveal major liabilities for the potent compounds. The 1,2,3-triazole derivative 27 substituted by a decyl tail, an undecyl spacer and a choline head group exhibited broad spectrum antiparasitic activity. It possessed low micromolar activity against the intracellular amastigotes of two L. infantum strains and T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes, while its cytotoxicity concentration (CC50) against THP-1 macrophages ranged between 50 and 100 \ub5M. Altogether, our work paves the way for the development of improved ether phospholipid derivatives to control neglected tropical diseases