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

How Does the Main Infective Stage of <em>T. cruzi</em> Enter and Avoid Degradation in Host Cells? A Description of the Pathways and Organelles Involved on These Processes

Trypanosoma cruzi, the etiological agent of Chagas disease, is an intracellular parasite that targets specific proteins of the host cell resulting in the generation of a unique parasitophorous vacuole (PV). As an intracellular parasite, T. cruzi interacts with cells from the mammalian host. Here we review aspects related with the binding of the main infective developmental stage (trypomastigote) to the host cell and its recognition by surface-exposed ligands/receptors. This process involves numerous signaling pathways and culminates in the entry of the parasite and modifications in both cells. The invasion of trypomastigotes occurs through multiple endocytic process, assembly of the PV, interaction of this vacuole with the endolysosomal system, lysis of the PV membrane, and multiplication of amastigotes within the cell in direct contact with host cell organelles

Leveling Up Hydrogels:Hybrid Systems in Tissue Engineering

Hydrogels can mimic several features of the cell native microenvironment and have been widely used as synthetic extracellular matrices (ECMs) in tissue engineering and regenerative medicine (TERM). However, some applications have specifications that hydrogels cannot efficiently fulfill on their own. Incorporating reinforcing structures like fibrous scaffolds or particles into hydrogels, as hybrid systems, is a promising strategy to improve their functionality. We describe recent advances in the fabrication and application of these hybrid systems, where structural properties and stimuli responsiveness of hydrogels are enhanced while their ECM-like features are preserved. Furthermore, we discuss how these systems can contribute to the development of more complex tissue engineered structures in the rapidly evolving field of TERM

Engineering modular half-antibody conjugated nanoparticles for targeting CD44v6-expressing cancer cells

Gastric cancer (GC) remains a major cause of death worldwide mainly because of the late detection in advanced stage. Recently, we proposed CD44v6 as a relevant marker for early detection of GC, opening new avenues for GC-targeted theranostics. Here, we designed a modular nanoscale system that selectively targets CD44v6-expressing GC cells by the site-oriented conjugation of a new-engineered CD44v6 half-antibody fragment to maleimide-modified polystyrene nanoparticles (PNPs) via an efficient bioorthogonal thiol-Michael addition click chemistry. PNPs with optimal particle size (200 nm) for crossing a developed biomimetic CD44v6-associated GC stromal model were further modified with a heterobifunctional maleimide crosslinker and click conjugated to the novel CD44v6 half-antibody fragment, obtained by chemical reduction of full antibody, without affecting its bioactivity. Collectively, our results confirmed the specific targeting ability of CD44v6-PNPs to CD44v6-expressing cells (1.65-fold higher than controls), highlighting the potential of CD44v6 half-antibody conjugated nanoparticles as promising and clinically relevant tools for the early diagnosis and therapy of GC. Additionally, the rational design of our nanoscale system may be explored for the development of several other nanotechnology-based disease-targeted approaches.This work was supported by Norte Portugal Regional Operational Programme (NORTE2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF) projects Norte-01-0145-FEDER-000012 and NORTE-07-0124-FEDER-000029, through COMPETE 2020-Operational Programme for Competitiveness and Internationalization (POCI) Portugal 2020 and Portuguese Foundation for Science and Technology (FCT) in the framework of the projects POCI-01-0145-FEDER-007274, POCI-01-0145-FEDER-016390, and PTDC/CTMNAN/120958/2010, B.N.L. doctoral grant (SFRH/BD/87400/2012) and postdoctoral grant (PTDC/MEC-GIN/29232/2017). R.F.P. was supported by Institute of Network Bioengineering for Healthy Aging (0245_IBEROS_1_E)

Cell-instructive pectin hydrogels crosslinked via thiol-norbornene photo-click chemistry for skin tissue engineering

Cell-instructive hydrogels are attractive for skin repair and regeneration, serving as interactive matrices to promote cell adhesion, cell-driven remodeling and de novo deposition of extracellular matrix compo nents. This paper describes the synthesis and photocrosslinking of cell-instructive pectin hydrogels using cell-degradable peptide crosslinkers and integrin-specific adhesive ligands. Protease-degradable hydro gels obtained by photoinitiated thiol-norbornene click chemistry are rapidly formed in the presence of dermal fibroblasts, exhibit tunable properties and are capable of modulating the behavior of embedded cells, including the cell spreading, hydrogel contraction and secretion of matrix metalloproteases. Keratinocytes seeded on top of fibroblast-loaded hydrogels are able to adhere and form a compact and dense layer of epidermis, mimicking the architecture of the native skin. Thiol-ene photocrosslinkable pec tin hydrogels support the in vitro formation of full-thickness skin and are thus a highly promising plat form for skin tissue engineering applications, including wound healing and in vitro testing modinfo:eu-repo/semantics/publishedVersio

Development of an improved 3D in vitro intestinal model to perform permeability studies of paracellular compounds

The small intestine is the primary site of drug absorption following oral administration, making paramount the proper monitoring of the absorption process. In vitro tools to predict intestinal absorption are particularly important in preclinical drug development since they are less laborious and cost-intensive and raise less ethical considerations compared to in vivo studies. The Caco-2 model is considered the gold standard of in vitro intestinal models regarding the prediction of absorption of orally delivered compounds. However, this model presents several drawbacks, such as the expression of tighter tight junctions, not being suitable to perform permeability of paracellular compounds. Besides, cells are representative of only one intestinal cell type, without considering the role of non-absorptive cells on the absorption pathway of drugs. In the present study, we developed a new three-dimensional (3D) intestinal model that aims to bridge the gap between in vitro tools and animal studies. Our 3D model comprises a collagen layer with human intestinal fibroblasts (HIFs) embedded, mimicking the intestinal lamina propria and providing 3D support for the epithelium, composed of Caco-2 cells and mucus-producing HT29-MTX cells, creating a model that can better resemble, both in terms of composition and regarding the outcomes of drug permeability when testing paracellular compounds, the human small intestine. The optimization of the collagen layer with HIFs was performed, testing different collagen concentrations and HIF seeding densities in order to avoid collagen contraction before day 14, maintaining HIF metabolically active inside the collagen disks during time in culture. HIF morphology and extracellular matrix (ECM) deposition were assessed, confirming that fibroblasts presented a normal and healthy elongated shape and secreted fibronectin and laminin, remodeling the collagen matrix. Regarding the epithelial layer, transepithelial electrical resistance (TEER) values decreased when cells were in the 3D configuration, comparing with the 2D analogs (Caco-2 and coculture of Caco-2+HT29-MTX models), becoming more similar with in vivo values. The permeability assay with fluorescein isothiocyanate (FITC)-Dextran 4 kDa showed that absorption in the 3D models is significantly higher than that in the 2D models, confirming the importance of using a more biorelevant model when testing the paracellular permeability of compounds

The shape of our gut: Dissecting its impact on drug absorption in a 3D bioprinted intestinal model

The small intestine is a complex organ with a characteristic architecture and a major site for drug and nutrient absorption. The three-dimensional (3D) topography organized in finger-like protrusions called villi increases surface area remarkably, granting a more efficient absorption process. The intestinal mucosa, where this process occurs, is a multilayered and multicell-type tissue barrier. In vitro intestinal models are routinely used to study different physiological and pathological processes in the gut, including compound absorption. Still, standard models are typically two-dimensional (2D) and represent only the epithelial barrier, lacking the cues offered by the 3D architecture and the stromal components present in vivo, often leading to inaccurate results. In this work, we studied the impact of the 3D architecture of the gut on drug transport using a bioprinted 3D model of the intestinal mucosa containing both the epithelial and the stromal compartments. Human intestinal fibroblasts were embedded in a previously optimized hydrogel bioink, and enterocytes and goblet cells were seeded on top to mimic the intestinal mucosa. The embedded fibroblasts thrived inside the hydrogel, remodeling the surrounding extracellular matrix. The epithelial cells fully covered the hydrogel scaffolds and formed a uniform cell layer with barrier properties close to in vivo. In particular, the villus-like model revealed overall increased permeability compared to a flat counterpart composed by the same hydrogel and cells. In addition, the efflux activity of the P-glycoprotein (P-gp) transporter was significantly reduced in the villus-like scaffold compared to a flat model, and the genetic expression of other drugs transporters was, in general, more relevant in the villus-like model. Globally, this study corroborates that the presence of the 3D architecture promotes a more physiological differentiation of the epithelial barrier, providing more accurate data on drug absorbance measurements.Copyright © 2023. Published by Elsevier B.V

Calcium phosphate-alginate microspheres as enzyme delivery matrices

The present study concerns the preparation and initial characterisation of novel calcium titanium phosphate-alginate (CTPalginate) and hydroxyapatite-alginate (HAp-alginate) microspheres, which are intended to be used as enzyme delivery matrices and bone regeneration templates. Microspheres were prepared using different concentrations of polymer solution (1% and 3% w/v) and different ceramic-to-polymer solution ratios (0.1, 0.2 and 0.4 w/w). Ceramic powders were characterised using X-ray diffraction, laser granulometry, Brunauer, Emmel and Teller (BET) method for the determination of surface area, zeta potential and Fourier transform infrared spectroscopy (FT-IR). Alginate was characterised using high performance size exclusion chromatography. The methodology followed in this investigation enabled the preparation of homogeneous microspheres with a uniform size. Studies on the immobilisation and release of the therapeutic enzyme glucocerebrosidase, employed in the treatment of Gaucher disease, were also performed. The enzyme was incorporated into the ceramic-alginate matrix before gel formation in two different ways: preadsorbed onto the ceramic particles or dispersed in the polymeric matrix. The two strategies resulted in distinct release profiles. Slow release was obtained after adsorption of the enzyme to the ceramic powders, prior to preparation of the microspheres. An initial fast release was achieved when the enzyme and the ceramic particles were dispersed in the alginate solution before producing the microspheres. The latter profile is very similar to that of alginate microspheres. The different patterns of enzyme release increase the range of possible applications of the system investigated in this work.info:eu-repo/semantics/publishedVersio

Violence and alcoholism in the family: how are the children affected?

Alcohol Alcohol. 1998 Jan-Feb;33(1):42-6. Violence and alcoholism in the family: how are the children affected? Malpique C, Barrias P, Morais L, Salgado M, Pinto Da Costa I, Rodriques M. SourceChild and Adolescent Psychiatry Department, Hospital Central de Criancas Maria Pia, Porto, Portugal. Abstract We made an evaluation of how children and adolescents are affected if they live in a family environment where violence associated with alcoholism is a feature. Interviews with 20 families and the use of psychological tests on their children were performed in this study. The study has demonstrated the existence of psychopathological disturbances in those families' children, whose immaturity and insecurity were expressed by aggressive behaviour or by depressive manifestations. It also became evident that there was a transgenerational alcoholism-violence frequency

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