A bioartificial and vasculomorphic bone matrix-based organoid mimicking microanatomy of flat and short bones.

We engineered an in vitro model of bioartificial 3D bone organoid consistent with an anatomical and vascular microenvironment common to mammalian flat and short bones. To achieve this, we chose the decellularized-decalcified matrix of the adult male rat scapula, implemented with the reconstruction of its intrinsic vessels, obtained through an original intravascular perfusion with polylevolactic (PLLA), followed by coating of the PLLA-fabricated vascularization with rat tail collagen. As a result, the 3D bone and vascular geometry of the native bone cortical and cancellous compartments was reproduced, and the rat tail collagen-PLLA biomaterial could in vitro act as a surrogate of the perivascular extracellular matrix (ECM) around the wall of the biomaterial-reconstituted cancellous vessels. As a proof-of-concept of cell compatibility and site-dependent osteoinductive properties of this bioartificial 3D construct, we show that it in vitro leads to a time-dependent microtopographic positioning of rat mesenchymal stromal cells (MSCs), initiating an osteogenic fate in relation to the bone compartment. In addition, coating of PLLA-reconstructed vessels with rat tail collagen favored perivascular attachment and survival of MSC-like cells (mouse embryonic fibroblasts), confirming its potentiality as a perivascular stroma for triggering competence of seeded MSCs. Finally, in vivo radiographic topography of bone lesions in the human jaw and foot tarsus of subjects with primary osteoporosis revealed selective bone cortical versus cancellous involvement, suggesting usefulness of a human 3D bone organoid engineered with the same principles of our rat organoid, to in vitro investigate compartment-dependent activities of human MSC in flat and short bones under experimental osteoporotic challenge. We conclude that our 3D bioartificial construct offers a reliable replica of flat and short bones microanatomy, and promises to help in building a compartment-dependent mechanistic perspective of bone remodeling, including the microtopographic dysregulation of osteoporosis.

PCSK9 ablation attenuates Aß pathology, neuroinflammation and cognitive dysfunctions in 5XFAD mice.

BACKGROUND: Increasing evidence highlights the importance of novel players in Alzheimer's disease (AD) pathophysiology, including alterations of lipid metabolism and neuroinflammation. Indeed, a potential involvement of Proprotein convertase subtilisin/kexin type 9 (PCSK9) in AD has been recently postulated. Here, we first investigated the effects of PCSK9 on neuroinflammation in vitro. Then, we examined the impact of a genetic ablation of PCSK9 on cognitive performance in a severe mouse model of AD. Finally, in the same animals we evaluated the effect of PCSK9 loss on Aß pathology, neuroinflammation, and brain lipids. METHODS: For in vitro studies, U373 human astrocytoma cells were treated with Aß fibrils and human recombinant PCSK9. mRNA expression of the proinflammatory cytokines and inflammasome-related genes were evaluated by q-PCR, while MCP-1 secretion was measured by ELISA. For in vivo studies, the cognitive performance of a newly generated mouse line - obtained by crossing 5XFADHet with PCSK9KO mice - was tested by the Morris water maze test. After sacrifice, immunohistochemical analyses were performed to evaluate Aß plaque deposition, distribution and composition, BACE1 immunoreactivity, as well as microglia and astrocyte reactivity. Cholesterol and hydroxysterols levels in mouse brains were quantified by fluorometric and LC-MS/MS analyses, respectively. Statistical comparisons were performed according to one- or two-way ANOVA, two-way repeated measure ANOVA or Chi-square test. RESULTS: In vitro, PCSK9 significantly increased IL6, IL1B and TNFΑ mRNA levels in Aß fibrils-treated U373 cells, without influencing inflammasome gene expression, except for an increase in NLRC4 mRNA levels. In vivo, PCSK9 ablation in 5XFAD mice significantly improved the performance at the Morris water maze test; these changes were accompanied by a reduced corticohippocampal Aß burden without affecting plaque spatial/regional distribution and composition or global BACE1 expression. Furthermore, PCSK9 loss in 5XFAD mice induced decreased microgliosis and astrocyte reactivity in several brain regions. Conversely, knocking out PCSK9 had minimal impact on brain cholesterol and hydroxysterol levels. CONCLUSIONS: In vitro studies showed a pro-inflammatory effect of PCSK9. Consistently, in vivo data indicated a protective role of PCSK9 ablation against cognitive impairments, associated with improved Aß pathology and attenuated neuroinflammation in a severe mouse model of AD. PCSK9 may thus be considered a novel pharmacological target for the treatment of AD.

Anti-Fibronectin Aptamer Modifies Blood Clot Pattern and Stimulates Osteogenesis: An Ex Vivo Study.

BACKGROUND: Scaffold (SCA) functionalization with aptamers (APT) provides adsorption of specific bioactive molecules on biomaterial surfaces. The aim of this study was to observe if SCA enriched with anti-fibronectin APT can favor coagulum (PhC) and osteoblasts (OSB) differentiation. METHODS: 20 µg of APT was functionalized on SCA by simple adsorption. For PhC formation, SCAs were inserted into rat calvaria defects for 17 h. Following proper transportation (buffer solution PB), OSBs (UMR-106 lineage) were seeded over PhC + SCAs with and without APT. Cells and PhC morphology, PhC cell population, protein labeling and gene expression were observed in different time points. RESULTS: The APT induced higher alkaline phosphatase and bone sialoprotein immunolabeling in OSB. Mesenchymal stem cells, leukocytes and lymphocytes cells were detected more in the APT group than when scaffolds were not functionalized. Additionally, an enriched and dense fibrin network and different cell types were observed, with more OSB and white blood cells in PhC formed on SCA with APT. The gene expression showed higher transforming growth factor beta 1 (TGF-b1) detection in SCA with APT. CONCLUSIONS: The SCA functionalization with fibronectin aptamers may alter key morphological and functional features of blood clot formation, and provides a selective expression of proteins related to osteo differentiation. Additionally, aptamers increase TGF-b1 gene expression, which is highly associated with improvements in regenerative therapies.

Woven bone formation and mineralization by rat mesenchymal stromal cells imply increased expression of the intermediate filament desmin.

Background: Disordered and hypomineralized woven bone formation by dysfunctional mesenchymal stromal cells (MSCs) characterize delayed fracture healing and endocrine -metabolic bone disorders like fibrous dysplasia and Paget disease of bone. To shed light on molecular players in osteoblast differentiation, woven bone formation, and mineralization by MSCs we looked at the intermediate filament desmin (DES) during the skeletogenic commitment of rat bone marrow MSCs (rBMSCs), where its bone-related action remains elusive. Results: Monolayer cultures of immunophenotypically- and morphologically - characterized, adult male rBMSCs showed co-localization of desmin (DES) with vimentin, F-actin, and runx2 in all cell morphotypes, each contributing to sparse and dense colonies. Proteomic analysis of these cells revealed a topologically-relevant interactome, focused on cytoskeletal and related enzymes//chaperone/signalling molecules linking DES to runx2 and alkaline phosphatase (ALP). Osteogenic differentiation led to mineralized woven bone nodules confined to dense colonies, significantly smaller and more circular with respect to controls. It significantly increased also colony-forming efficiency and the number of DES-immunoreactive dense colonies, and immunostaining of co-localized DES/runx-2 and DES/ALP. These data confirmed pre-osteoblastic and osteoblastic differentiation, woven bone formation, and mineralization, supporting DES as a player in the molecular pathway leading to the osteogenic fate of rBMSCs. Conclusion: Immunocytochemical and morphometric studies coupled with proteomic and bioinformatic analysis support the concept that DES may act as an upstream signal for the skeletogenic commitment of rBMSCs. Thus, we suggest that altered metabolism of osteoblasts, woven bone, and mineralization by dysfunctional BMSCs might early be revealed by changes in DES expression//levels. Non-union fractures and endocrine - metabolic bone disorders like fibrous dysplasia and Paget disease of bone might take advantage of this molecular evidence for their early diagnosis and follow-up.

Rapid Prototyping of 3D-Printed AgNPs- and Nano-TiO2-Embedded Hydrogels as Novel Devices with Multiresponsive Antimicrobial Capability in Wound Healing.

Two antimicrobial agents such as silver nanoparticles (AgNPs) and titanium dioxide (TiO2) have been formulated with natural polysaccharides (chitosan or alginate) to develop innovative inks for the rapid, customizable, and extremely accurate manufacturing of 3D-printed scaffolds useful as dressings in the treatment of infected skin wounds. Suitable chemical-physical properties for the applicability of these innovative devices were demonstrated through the evaluation of water content (88-93%), mechanical strength (Young's modulus 0.23-0.6 MPa), elasticity, and morphology. The antimicrobial tests performed against Staphylococcus aureus and Pseudomonas aeruginosa demonstrated the antimicrobial activities against Gram+ and Gram- bacteria of AgNPs and TiO2 agents embedded in the chitosan (CH) or alginate (ALG) macroporous 3D hydrogels (AgNPs MIC starting from 5 µg/mL). The biocompatibility of chitosan was widely demonstrated using cell viability tests and was higher than that observed for alginate. Constructs containing AgNPs at 10 µg/mL concentration level did not significantly alter cell viability as well as the presence of titanium dioxide; cytotoxicity towards human fibroblasts was observed starting with an AgNPs concentration of 100 µg/mL. In conclusions, the 3D-printed dressings developed here are cheap, highly defined, easy to manufacture and further apply in personalized antimicrobial medicine applications.

Lactobacillus delbrueckii subsp. bulgaricus derivatives for 3D printed alginate/hyaluronic acid self-crosslinking hydrogels: Manufacturing and wound healing potential.

Derivatives [i.e. proteins and exopolysaccharides (EPS)] from Lactobacillus delbrueckii subsp. bulgaricus (LB) were extracted, characterized, and for the first time used in the production of novel self-crosslinking 3D printed alginate/hyaluronic acid (ALG/HA) hydrogels, as high-value functional biomaterials with therapeutic potentials in regenerative medicine applications. Derivatives coming from two different LB strains, LB1865 and LB1932, were tested in-vitro and compared for their cytotoxicity and effect on proliferation and migration on human fibroblast. EPS received particular attention as showing relevant dose-dependent cytocompatibility against the human fibroblast. The derivatives showed an ability to increase cell proliferation and migration, quantifiable between 10 and 20 % if compared to controls, with higher values for the derivatives obtained from the LB1932 strain. These were explained by liquid chromatography-mass spectrometry targeted protein biomarker analysis as a decrease in matrix-degrading and proapoptotic proteins, associated with an increase in collagen and antiapoptotic proteins production. LB1932 enriched hydrogel was found to be of benefit compared to control dressings, giving the more promising results as potential for in vivo skin wound healing tests.

3D Printed Chitosan/Alginate Hydrogels for the Controlled Release of Silver Sulfadiazine in Wound Healing Applications: Design, Characterization and Antimicrobial Activity.

The growing demand for personalized medicine requires innovation in drug manufacturing to combine versatility with automation. Here, three-dimensional (3D) printing was explored for the production of chitosan (CH)/alginate (ALG)-based hydrogels intended as active dressings for wound healing. ALG hydrogels were loaded with 0.75% w/v silver sulfadiazine (SSD), selected as a drug model commonly used for the therapeutic treatment of infected burn wounds, and four different 3D CH/ALG architectures were designed to modulate the release of this active compound. CH/ALG constructs were characterized by their water content, elasticity and porosity. ALG hydrogels (Young's modulus 0.582 ± 0.019 Mpa) were statistically different in terms of elasticity compared to CH (Young's modulus 0.365 ± 0.015 Mpa) but very similar in terms of swelling properties (water content in ALG: 93.18 ± 0.88% and in CH: 92.76 ± 1.17%). In vitro SSD release tests were performed by using vertical diffusion Franz cells, and statistically significant different behaviors in terms of the amount and kinetics of drugs released were observed as a function of the construct. Moreover, strong antimicrobial potency (100% of growth inhibition) against Staphylococcus aureus and Pseudomonas aeruginosa was demonstrated depending on the type of construct, offering a proof of concept that 3D printing techniques could be efficiently applied to the production of hydrogels for controlled drug delivery.

A New ABCB1 Inhibitor Enhances the Anticancer Effect of Doxorubicin in Both In Vitro and In Vivo Models of NSCLC.

In tumors, the multi drug resistance phenomenon may occur through the efflux of chemotherapeutic drugs out of cancer cells, impeding their accumulation, and eventually reducing their toxicity. This process is mediated by transporters overexpressed in the plasma membranes of tumor cells, among which is the P-glycoprotein/multidrug resistance 1/ATP-binding cassette B1 (P-gp/MDR1/ABCB1). The aim of this study was to explore the effect of a new molecule, called AIF-1, on ABCB1 activity. In a cellular model of non-small cell lung cancer (NSCLC), AIF-1 significantly inhibited ABCB1 activity, which was evaluated by the fluorimetric measurement of the intracellular accumulation of calcein. AIF-1 also significantly increased the intracellular content of doxorubicin, which was evaluated by confocal microscopy and LC-MS/MS analysis. This effect translated to higher cytotoxicity of doxorubicin and reduced cellular proliferation. Finally, in a murine xenograft model, the tumor volume increased by 267% and 148% on average in mice treated with vehicle and doxorubicin alone, respectively. After the co-administration of doxorubicin with AIF-1, tumor volume increased by only 13.4%. In conclusion, these results suggest enhancement of the efficacy of the chemotherapeutic drug doxorubicin by AIF-1, laying the basis for the future development of new ABCB1 inhibitors for tumor treatment.

Development and single laboratory validation of a targeted liquid chromatography-triple quadrupole mass spectrometry-based method for the determination of insulin like growth factor-1 in different types of milk samples.

A simple and reliable targeted liquid chromatography-electrospray-tandem mass spectrometry (LC-MS/MS) method was developed and validated through the selection of two biomarker peptides for the identification and determination of bovine insulin like growth factor-1 (IGF-1) in milk samples. Two urea-based sample extraction procedures were tested. The validation results provided detection limits at the 1-5 ng IGF-1/mL level as a function of the milk matrix, precision ranged from 3 to 8% and the method accuracy in the different milk matrices was assured. Finally, IGF-1 was measured in milk samples obtained by treatment with eleven different technological processes: IGF-1 concentrations were spread over a wide range from 11.2 ± 0.3 ng/mL to 346 ± 8 ng/mL with a median of 57.0 ± 0.2 ng/mL. The highest amount of IGF-1 was found in fresh whole milk samples and no significant correlation was found between the total milk protein content and the IGF-1 concentration level.

Decellularization Detergents As Methodological Variables in Mass Spectrometry of Stromal Matrices.

Collagens, elastin, fibrillin, decorin, and laminin are key constituents of the extracellular matrix and basement membrane of mammalian organs. Thus, changes in their quantities may influence the mechanochemical regulation of resident cells. Since maintenance of a native stromal composition is a requirement for three-dimensional (3D) matrix-based recellularization techniques in tissue engineering, we studied the influence of the decellularization detergents on these proteins in porcine kidney, liver, pancreas, and skin. Using a quick thawing/quick microwave-assisted decellularization protocol and two different detergents, sodium dodecyl sulfate (SDS) vs Triton X-100 (TX100), at identical concentration, variations in matrix conservation of stromal proteins were detected by liquid chromatography-mass spectrometry coupled to light and scanning electron microscopies, in dependence on each detergent. In all organs tested except pancreas, collagens were retained to a statistically significant level using the TX100-based protocol. In contrast fibrillin, elastin (except in kidney), and decorin (only in liver) were better preserved with the SDS-dependent protocol. Irrespective of the detergent used, laminin always remained at an irrelevant level. Our results prompt attention to the type of detergent in organ decellularization, suggesting that its choice may influence morphoregulatory inputs peculiar to the type of 3D bioartificial mammalian organ to be reconstructed. Impact statement Simple change of the protocol's main detergent leads to a very substantial difference in the panel of the stromal proteins detected by qualitative and semiquantitative mass spectrometry in acellular porcine matrices. This remarkable methodological variable promises to yield proteomic reference panels in a number of different species-specific acellular matrices allowing for selective retainment of peculiar mechanochemical inputs, to differently address the development of the seeded cells in relation to the type of organ to be bioartificially reconstructed.

Alginate Self-Crosslinking Ink for 3D Extrusion-Based Cryoprinting and Application for Epirubicin-HCl Delivery on MCF-7 Cells.

3D-printed hydrogels are particularly advantageous as drug-delivery platforms but their loading with water-soluble active compounds remains a challenge requiring the development of innovative inks. Here, we propose a new 3D extrusion-based approach that, by exploiting the internal gelation of the alginate, avoids the post-printing crosslinking process and allows the loading of epirubicin-HCl (EPI). The critical combinations of alginate, calcium carbonate and d-glucono-δ-lactone (GDL) combined with the scaffold production parameters (extrusion time, temperature, and curing time) were evaluated and discussed. The internal gelation in tandem with 3D extrusion allowed the preparation of alginate hydrogels with a complex shape and good handling properties. The dispersion of epirubicin-HCl in the hydrogel matrix confirmed the potential of this self-crosslinking alginate-based ink for the preparation of 3D-printed drug-delivery platforms. Drug release from 3D-printed hydrogels was monitored, and the cytotoxic activity was tested against MCF-7 cells. Finally, the change in the expression pattern of anti-apoptotic, pro-apoptotic, and autophagy protein markers was monitored by liquid-chromatography tandem-mass-spectrometry after exposure of MCF-7 to the EPI-loaded hydrogels.

3D-printed scaffold composites for the stimuli-induced local delivery of bioactive adjuncts.

Polysaccharide scaffolds have been successfully employed to reconstruct environments that sustain skin tissue regeneration after injuries. Three-dimensional (3D) advanced additive manufacturing technologies allow creating scaffolds with controlled and reproducible macro- and micro-structure that improve the quality of the restored tissue to favor spontaneous repair. However, when persistent inflammation occurs, the physiological tissue healing capacity is reduced, like in the presence of pathologies like diabetes, vascular diseases, chronic infection, and others. In these circumstances, the bioavailability of therapeutic adjuncts like the growth factors in addition to the standard treatments represents undoubtedly a promising strategy to accelerate the healing of skin lesions. Precisely designed polysaccharide scaffolds obtained by 3D printing represent a robust platform that can be further implemented with the controlled delivery of bioactive adjuncts. Human elastin-like polypeptides (HELPs) are stimuli-responsive biopolymers. Their structure allows the integration of domains endowed with biological functionality, making them attractive compounds to prepare composites with smart properties. In the present study, 3D-printed alginate and chitosan scaffolds were combined with the HELP components. The HELP biopolymer was fused to the epidermal growth factor (EGF) as the bioactive domain. Different constructs were prepared and the stimuli-responsive behavior as well as the biological activity were evaluated, suggesting that these smart bioactive composites are suitable to realize multifunctional dressings that sustain the local release of therapeutic adjuncts.

Cultured Horse Articular Chondrocytes in 3D-Printed Chitosan Scaffold With Hyaluronic Acid and Platelet Lysate.

Three-dimensional (3D) printing has gained popularity in tissue engineering and in the field of cartilage regeneration. This is due to its potential to generate scaffolds with spatial variation of cell distribution or mechanical properties, built with a variety of materials that can mimic complex tissue architecture. In the present study, horse articular chondrocytes were cultured for 2 and 4 weeks in 3D-printed chitosan (CH)-based scaffolds prepared with or without hyaluronic acid and in the presence of fetal bovine serum (FBS) or platelet lysate (PL). These 3D culture systems were analyzed in terms of their capability to maintain chondrocyte differentiation in vitro. This was achieved by evaluating cell morphology, immunohistochemistry (IHC), gene expression of relevant cartilage markers (collagen type II, aggrecan, and Sox9), and specific markers of dedifferentiated phenotype (collagen type I, Runx2). The morphological, histochemical, immunohistochemical, and molecular results demonstrated that the 3D CH scaffold is sufficiently porous to be colonized by primary chondrocytes. Thereby, it provides an optimal environment for the colonization and synthetic activity of chondrocytes during a long culture period where a higher rate of dedifferentiation can be generally observed. Enrichment with hyaluronic acid provides an optimal microenvironment for a more stable maintenance of the chondrocyte phenotype. The use of 3D CH scaffolds causes a further increase in the gene expression of most relevant ECM components when PL is added as a substitute for FBS in the medium. This indicates that the latter system enables a better maintenance of the chondrocyte phenotype, thereby highlighting a fair balance between proliferation and differentiation.

The adaptation of lipid profile of human fibroblasts to alginate 2D films and 3D printed scaffolds.

BACKGROUND: The investigation of the interactions between cells and active materials is pivotal in the emerging 3D printing-biomaterial application fields. Here, lipidomics has been used to explore the early impact of alginate (ALG) hydrogel architecture (2D films or 3D printed scaffolds) and the type of gelling agent (CaCl2 or FeCl3) on the lipid profile of human fibroblasts. METHODS: 2D and 3D ALG scaffolds were prepared and characterized in terms of water content, swelling, mechanical resistance and morphology before human fibroblast seeding (8 days). Using a liquid chromatography-triple quadrupole-tandem mass spectrometry approach, selected ceramides (CER), lysophosphatidylcholines (LPC), lysophosphatidic acids (LPA) and free fatty acids (FFA) were analyzed. RESULTS: The results showed a clear alteration in the CER expression profile depending of both the geometry and the gelling agent used to prepare the hydrogels. As for LPCs, the main parameter affecting their distribution is the scaffold architecture with a significant decrease in the relative expression levels of the species with higher chain length (C20 to C22) for 3D scaffolds compared to 2D films. In the case of FFAs and LPAs only slight differences were observed as a function of scaffold geometry or gelling agent. CONCLUSIONS: Variations in the cell membrane lipid profile were observed for 3D cell cultures compared to 2D and these data are consistent with activation processes occurring through the mutual interactions between fibroblasts and ALG support. These unknown physiologically relevant changes add insights into the discussion about the relationship between biomaterial and the variations of cell biological functions.

Microtopography of Immune Cells in Osteoporosis and Bone Lesions by Endocrine Disruptors.

Osteoporosis stems from an unbalance between bone mineral resorption and deposition. Among the numerous cellular players responsible for this unbalance bone marrow (BM) monocytes/macrophages, mast cells, T and B lymphocytes, and dendritic cells play a key role in regulating osteoclasts, osteoblasts, and their progenitor cells through interactions occurring in the context of the different bone compartments (cancellous and cortical). Therefore, the microtopography of immune cells inside trabecular and compact bone is expected to play a relevant role in setting initial sites of osteoporotic lesion. Indeed, in physiological conditions, each immune cell type preferentially occupies either endosteal, subendosteal, central, and/or perisinusoidal regions of the BM. However, in the presence of an activation, immune cells recirculate throughout these different microanatomical areas giving rise to a specific distribution. As a result, the trabeculae of the cancellous bone and endosteal free edge of the diaphyseal case emerge as the primary anatomical targets of their osteoporotic action. Immune cells may also transit from the BM to the depth of the compact bone, thanks to the efferent venous capillaries coursing in the Haversian and Volkmann canals. Consistently, the innermost parts of the osteons and the periosteum are later involved by their immunomodulatory action, becoming another site of mineral reabsorption in the course of an osteoporotic insult. The novelty of our updating is to highlight the microtopography of bone immune cells in the cancellous and cortical compartments in relation to the most consistent data on their action in bone remodeling, to offer a mechanist perspective useful to dissect their role in the osteoporotic process, including bone damage derived from the immunomodulatory effects of endocrine disrupting chemicals.

Alginate/human elastin-like polypeptide composite films with antioxidant properties for potential wound healing application.

In this contribution we describe the preparation and characterization of a series of cross-linked films based on the combination of an elastin-derived biomimetic polypeptide (Human Elastin-Like Polypeptide, HELP) with alginate (ALG) to obtain a composite with enhanced properties. ALG/HELP composite films loaded with the hydrophobic natural antioxidant curcumin were prepared by solvent casting method followed by the cross-linking with calcium chloride. The compatibility between the two components as well as the final properties was evaluated. The micro-morphological study of films showed a homogeneous structure, but the film tensile strength decrease with HELP content and elongation at break was adversely affected by biopolymer addition. Spectroscopic and thermal analyses confirmed an interaction between ALG and HELP which also causes a modification in swelling kinetics and faster degradation. Moreover, the study of curcumin release showed a controlled delivery up to 10 days with a faster release rate in the presence of HELP. Human Dermal Fibroblasts (hDF) were used to test the in vitro cytocompatibility. The antioxidant activity correlated to the increase of HELP content suggested the applicability of these composites to develop smart biomaterials. Overall, these features indicated how this composite material has considerable potential as customizable platforms for various biomedical applications.

Three-Dimensional (3D) Printed Silver Nanoparticles/Alginate/Nanocrystalline Cellulose Hydrogels: Study of the Antimicrobial and Cytotoxicity Efficacy.

Here, a formulation of silver nanoparticles (AgNPs) and two natural polymers such as alginate (ALG) and nanocrystalline cellulose (CNC) was developed for the 3D printing of scaffolds with large surface area, improved mechanical resistance and sustained capabilities to promote antimicrobial and cytotoxic effects. Mechanical resistance, water content, morphological characterization and silver distribution of the scaffolds were provided. As for applications, a comparable antimicrobial potency against S. aureus and P. aeruginosa was demonstrated by in vitro tests as function of the AgNP concentration in the scaffold (Minimal Inhibitory Concentration value: 10 mg/mL). By reusing the 3D system the antimicrobial efficacy was demonstrated over at least three applications. The cytotoxicity effects caused by administration of AgNPs to hepatocellular carcinoma (HepG2) cell culture through ALG and ALG/CNC scaffold were discussed as a function of time and dose. Finally, the liquid chromatography-mass spectrometry (LC-MS) technique was used for targeted analysis of pro-apoptotic initiation and executioner caspases, anti-apoptotic and proliferative proteins and the hepatocyte growth factor, and provided insights about molecular mechanisms involved in cell death induction.

3D-printed chitosan scaffolds modified with D-(+) raffinose and enriched with type IV collagen to improve epithelial cell colonization.

Tissue regeneration often requires the use of biocompatible resorbable scaffolds to support the ingrowth of cells from neighboring tissues into a localized tissue defect. Such scaffolds must possess surface molecular cues that stimulate cells to populate the device, the first necessary condition for the formation of a healthy tissue. Chitosan is a natural polymer that has long been tested in biomedical applications because of its high biocompatibility, which can be further increased by modifying its formulation, e.g. adding D-(+) raffinose. We used this formulation in an ad hoc designed 3D printer to create regularly ordered scaffolds, which we then enriched with type IV collagen, an isoform of collagen that is exclusively found in basement membranes. Human epithelial A549 cells were then seeded on control scaffolds or on scaffolds coated with collagen, which was precipitated, or on scaffolds first collagenized and then exposed to either UVB or UVC radiation. Observations by the transmission light microscope, confocal microscope after staining with calcein-AM/propidium iodide, and by environmental scanning electron microscope revealed that collagen-enriched UV-treated scaffolds promoted the attachment of a higher number of cells, which covered a more extensive area of the scaffold, as also confirmed by alamar blue viability assay. Together these data confirm that coating 3D-printed scaffolds made of D-(+) raffinose-modified chitosan with type IV collagen and exposing them to UV light sensibly increases the cell compatibility of scaffolds, making them a better candidate to serve as a tool for the regeneration of epithelia.

SCREENED: A Multistage Model of Thyroid Gland Function for Screening Endocrine-Disrupting Chemicals in a Biologically Sex-Specific Manner.

Endocrine disruptors (EDs) are chemicals that contribute to health problems by interfering with the physiological production and target effects of hormones, with proven impacts on a number of endocrine systems including the thyroid gland. Exposure to EDs has also been associated with impairment of the reproductive system and incidence in occurrence of obesity, type 2 diabetes, and cardiovascular diseases during ageing. SCREENED aims at developing in vitro assays based on rodent and human thyroid cells organized in three different three-dimensional (3D) constructs. Due to different levels of anatomical complexity, each of these constructs has the potential to increasingly mimic the structure and function of the native thyroid gland, ultimately achieving relevant features of its 3D organization including: 1) a 3D organoid based on stem cell-derived thyrocytes, 2) a 3D organoid based on a decellularized thyroid lobe stromal matrix repopulated with stem cell-derived thyrocytes, and 3) a bioprinted organoid based on stem cell-derived thyrocytes able to mimic the spatial and geometrical features of a native thyroid gland. These 3D constructs will be hosted in a modular microbioreactor equipped with innovative sensing technology and enabling precise control of cell culture conditions. New superparamagnetic biocompatible and biomimetic particles will be used to produce "magnetic cells" to support precise spatiotemporal homing of the cells in the 3D decellularized and bioprinted constructs. Finally, these 3D constructs will be used to screen the effect of EDs on the thyroid function in a unique biological sex-specific manner. Their performance will be assessed individually, in comparison with each other, and against in vivo studies. The resulting 3D assays are expected to yield responses to low doses of different EDs, with sensitivity and specificity higher than that of classical 2D in vitro assays and animal models. Supporting the "Adverse Outcome Pathway" concept, proteogenomic analysis and biological computational modelling of the underlying mode of action of the tested EDs will be pursued to gain a mechanistic understanding of the chain of events from exposure to adverse toxic effects on thyroid function. For future uptake, SCREENED will engage discussion with relevant stakeholder groups, including regulatory bodies and industry, to ensure that the assays will fit with purposes of ED safety assessment. In this project review, we will briefly discuss the current state of the art in cellular assays of EDs and how our project aims at further advancing the field of cellular assays for EDs interfering with the thyroid gland.

Validation of RP-HPLC method to assess the compatibility of metoclopramide and midazolam intravenous mixture used in patients with cancer.

BACKGROUND: The delivery of intravenous medication by continuous infusion is necessary and widespread for treatment of patients with advanced cancer. Few scientific papers have focused on assessment of the chemical compatibility of these therapeutic mixtures. An analytical assessment of the physical and chemical compatibility of these combinations is needed. OBJECTIVES: To determine the chemical and physical compatibility of binary mixtures of metoclopramide (MET) and midazolam (MID). METHODS: Mixtures of drugs were prepared under aseptic conditions in 0.9% sodium chloride at concentrations used in our clinical practice for continuous infusion. The samples were stored in polyethylene bags at room temperature in the presence of light for 15 days. Chemical compatibility was evaluated by high-performance liquid chromatography (HPLC). Physical compatibility was tested by visual inspection (for evidence of precipitation and colour change) and by pH determination. RESULTS: No changes in colour, precipitation of components, measurable losses of volume or notable changes in pH were seen. The combinations tested were compatible for 15 days (retained >95% of their initial concentration). CONCLUSIONS: This study confirms the analytical compatibility of MET and MID, when mixed in 0.9% sodium chloride at concentrations employed in our clinical practice.