Publications

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.

JTD Keywords: 3d architecture, alkaline-phosphatase, caco-2 cells, culture, drug development, efflux proteins, gene-expression, human-colon, intestinal absorption, intestinal models, microenvironment, paracellular transport, permeability, photopolymerization, villi, 3d architecture, 3d bioprinting, Drug development, In-vitro, Intestinal absorption, Intestinal models, Photopolymerization, Villi

Drug development is an ever-growing field, increasingly requesting reliable in vitro tools to speed up early screening phases, reducing the need for animal experiments. In oral delivery, understanding the absorption pattern of a new drug in the small intestine is paramount. Classical two-dimensional (2D) in vitro models are generally too simplistic and do not accurately represent native tissues. The main goal of this work was to develop an advanced three-dimensional (3D) in vitro intestinal model to test absorption in a more reliable manner, by better mimicking the native environment. The 3D model is composed of a collagen-based stromal layer with embedded fibroblasts mimicking the intestinal lamina propria and providing support for the epithelium, composed of enterocytes and mucus-secreting cells. An endothelial layer, surrogating the absorptive capillary network, is also present. The cellular crosstalk between the different cells present in the model is unveiled, disclosing key players, namely those involved in the contraction of collagen by fibroblasts. The developed 3D model presents lower levels of P-glycoprotein (P-gp) and Multidrug Resistance Protein 2 (MRP2) efflux transporters, which are normally overexpressed in traditional Caco-2 models, and are paramount in the absorption of many compounds. This, allied with transepithelial electrical resistance (TEER) values closer to physiological ranges, leads to improved and more reliable permeability outcomes, which are observed when comparing our results with in vivo data.

JTD Keywords: 3d intestinal model, drug absorption, drug development, endothelium, hydrogel, 3d intestinal model, 3d modeling, 3d models, 3d-modeling, Alkaline-phosphatase, Animal experiments, Biopharmaceutics classification, Caco-2 cells, Cell culture, Collagen, Collagen gel, Drug absorption, Drug development, Endothelium, Fibroblasts, Glycoproteins, Hydrogel, In-vitro, Matrix metalloproteinases, Membrane-permeability, Paracellular transport, Permeability, Single-pass vs., Speed up

One of the major problems faced by metallic implants is the high probability of bacterial infections, with significant consequences for the patient. In this work, a thermochemical treatment is proposed to obtain silver-doped calcium titanate coatings on the Ti surface to improve the bioactivity of porous 3D-printed Ti structures and simultaneously provide them with antibacterial properties. A complete characterization of the new coating, the study of the ion release and the analysis of its cytotoxicity were carried out together with evaluation of the natural apatite forming in simulated body fluid (SBF). Moreover, the antibacterial properties of the coatings were assessed against Pseudomona aeruginosa and Escherichia coli as gram-negative and Staphylococcus aureus and Staphylococcus epidermidis as gram-positive bacterial strains. Ag ions were integrated into the Ca titanate layer and Ag nanoparticles were formed within the entire 3D Ti surface. Ca and Ag ions were released from both porous and solid samples into the Hanks' solution for 48 h. The treated surfaces showed no cytotoxicity and an apatite layer precipitated on the entire porous surface when the samples were immersed in SBF. The release of Ag from the surface had a strong antibacterial effect and prevented bacterial adhesion and proliferation on the surface. Moreover, the nanostructured topography of the coating resulted also in a reduction of bacterial adhesion and proliferation, even in absence of Ag. In conclusion, the cost-effective approach here reported provided protection against the most predominant bacterial colonizers to the Ti porous implants, while maintaining their bioactivity.

JTD Keywords: 3d-printing, alkaline, antibacterial activity, arthroplasty, bacterial adhesion, biomaterials, generation, ions, nanoparticles, osseointegration, silver, surface-layer, titanium implants, toxicity, 3d-printing, Antibacterial activity, Biomaterials, Porous structures, Silver, Ti metal, Titanium implants

In this study gelatin (Gel) modified with calcium phosphate nanoparticles (SG5) and polycaprolactone (PCL) were used to prepare a 3D bi-layer scaffold by collecting electrospun PCL and gelatin/SG5 fibers separately in the same collector. The objective of this study was to combine the desired properties of PCL and Gel/SG5 in the same scaffold in order to enhance mineralization, thus improving the ability of the scaffold to bond to the bone tissue. The scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and the wide angle X-ray diffraction (WAXD) measurements confirmed that SG5 nanoparticles were successfully incorporated into the fibrous gelatin matrix. The composite Gel/SG5/PCL scaffold exhibited more enhanced mechanical properties than individual Gel and Gel/SG5 scaffolds. The presence of SG5 nanoparticles accelerated the nucleation and growth of apatite crystals on the surface of the composite Gel/SG5/PCL scaffold in simulated body fluid (SBF). The osteoblast response in vitro to developed electrospun scaffolds (PCL and Gel/SG5/PCL) was investigated by using normal human primary NHOst cell lines. NHOst cell culture studies showed that higher alkaline phosphatase (ALP) activity and better mineralization were obtained in the case of composite materials than in pure PCL scaffolds. The mechanically strong PCL scaffold served as a skeleton, while the Gel/SG5 fibers facilitated cell spreading and mineralization of the scaffold.

JTD Keywords: Bilayer fibrous scaffold, Ceramic nanoparticles, Electrospinning, Gelatin, Polycaprolactone, Biomechanics, Bone, Calcium phosphate, Cell culture, Electrospinning, Fourier transform infrared spectroscopy, Mechanical properties, Mineralogy, Nanoparticles, Phosphatases, Polycaprolactone, Scanning electron microscopy, X ray diffraction, Polycaprolactone, Alkaline phosphatase activity, Bone tissue engineering, Calcium phosphate nanoparticles, Ceramic nanoparticles, Fibrous scaffolds, Gelatin, Simulated body fluids, Wide-angle x-ray diffraction, Electrospuns, Scaffolds (biology), Electrospinning

Solution-mediated reactions due to ionic substitutions are increasingly explored as a strategy to improve the biological performance of calcium phosphate-based materials. Yet, cellular response to well-defined dynamic changes of the ionic extracellular environment has so far not been carefully studied in a biomaterials context. In this work, we present kinetic data on how osteoblast-like SAOS-2 cellular activity and calcium-deficient hydroxyapatite (CDHA) influenced extracellular pH as well as extracellular concentrations of calcium and phosphate in standard in vitro conditions. Since cells were grown on membranes permeable to ions and proteins, they could share the same aqueous environment with CDHA, but still be physically separated from the material. In such culture conditions, it was observed that gradual material-induced adsorption of calcium and phosphate from the medium had only minor influence on cellular proliferation and alkaline phosphatase activity, but that competition for calcium and phosphate between cells and the biomaterial delayed and reduced significantly the cellular capacity to deposit calcium in the extracellular matrix. The presented work thus gives insights into how and to what extent solution-mediated reactions can influence cellular response, and this will be necessary to take into account when interpreting CDHA performance both in vitro and in vivo.

JTD Keywords: Alkaline-phosphatase activity, Saos-2 cells, In-vitro, bone mineralization, Biological basis, Differentiation, Culture, Matrix, Proliferation, Topography

Polycrystalline Cu2O layers have been selectively grown by electrochemical anodization of polycrystalline Cu electrodes in an alkaline medium (pH 12.85). Uniform layers with thicknesses around 100 nm have been obtained. Using electrochemical impedance spectroscopy, it was concluded that the Cu2O films behave as a p-type semiconductor. The Mott-Schottky plot gives a value for the flat band potential of U-FB = -255 mV vs silver/silver chloride electrode (SSC), an estimated carrier density N-A = 6.1 x 10(17) cm(-3), and the space charge layer width was calculated to be W-SCL = 9 nm at a band bending of 120 mV. The electronic structure of the Cu vertical bar Cu2O vertical bar electrolyte interface was for the first time probed by in situ electrochemical tunneling spectroscopy. The use of in situ electrochemical scanning tunneling microscopy allows us to directly observed the valence band edge and determine its position against the absolute energy scale to be E-VB = -4.9 eV. Finally, we constructed a quantitative electronic diagram of the Cu vertical bar Cu2O vertical bar electrolyte interface, where the positions of the valence and conduction band edges are depicted, as well as the edge of the previously reported electronic subband.

JTD Keywords: 0.1 m NaOH, Electrochemical tunneling spectroscopy, Cuprous-oxide films, Anodic-oxidation, Electronic-structure, Alkaline-solution, Aqueous-solution, Initial-stages, Passive film, Thin-films

Surface topography is known to have an influence on osteoblast activity. However, in the case of bioactive materials, topographical changes can affect also ion exchange properties. This makes the problem more complex, since it is often difficult to separate the strictly topographical effects from the effects of ionic fluctuations in the medium. The scope of this paper is to analyze the simultaneous effect of topography and topography-mediated ion exchange on the initial cellular behavior of osteoblastic-like cells cultured on bioactive tissue engineering substrates. Two apatitic substrates with identical chemical composition but different micro/nanostructural features were obtained by low-temperature setting of a calcium phosphate cement. MG63 osteoblastic-like cells were cultured either in direct contact with the substrates or with their extracts. A strong and permanent decrease of calcium concentration in the culture medium, dependent on substrate topography, was detected. A major effect of the substrate microstructure on cell proliferation was observed, explained in part by the topography-mediated ion exchange, but not specifically by the ionic Ca(2+) fluctuations. Cell differentiation was strongly enhanced when cells were cultured on the finer substrate. This effect was not explained by the chemical modification of the medium, but rather suggested a strictly topographical effect.

JTD Keywords: Alkaline Phosphatase/metabolism, Bone Cements/pharmacology, Calcium/metabolism, Calcium Phosphates/pharmacology, Cell Adhesion/drug effects, Cell Differentiation/drug effects, Cell Proliferation/drug effects, Cell Shape/drug effects, Cells, Cultured, Culture Media, Durapatite/pharmacology, Humans, Interferometry, Ion Exchange, Materials Testing, Osteoblasts/ cytology/drug effects/enzymology/ultrastructure, Phosphorus/metabolism, Powders, Tissue Engineering, Tissue Scaffolds