Publications

Bone regeneration often fails due to implants/grafts lacking vascular supply, causing necrotic tissue and poor integration. Microsurgical techniques are used to overcome this issue, allowing the graft to anastomose. These techniques have limitations, including severe patient morbidity and current research focuses on stimulating angiogenesis in situ using growth factors, presenting limitations, such as a lack of control and increased costs. Non-biological stimuli are necessary to promote angiogenesis for successful bone constructs. Recent studies have reported that bioactive glass dissolution products, such as calcium-releasing nanoparticles, stimulate hMSCs to promote angiogenesis and new vasculature. Moreover, the effect of 3D microporosity has also been reported to be important for vascularisation in vivo. . Therefore, we used room-temperature extrusion 3D printing with polylactic acid (PLA) and calcium phosphate (CaP) based glass scaffolds, focusing on geometry and solvent displacement for scaffold recovery. Combining both methods enabled reproducible control of 3D structure, porosity, and surface topography. Scaffolds maintained calcium ion release at physiological levels and supported human mesenchymal stem cell proliferation. Scaffolds stimulated the secretion of vascular endothelial growth factor (VEGF) after 3 days of culture. Subcutaneous implantation in vivo indicated good scaffold integration and blood vessel infiltration as early as one week after. PLA-CaP scaffolds showed increased vessel maturation 4 weeks after implantation without vascular regression. Results show PLA/CaP-based glass scaffolds, made via controlled 3D printing, support angiogenesis and vessel maturation, promising improved vascularization for bone regeneration.

JTD Keywords: 3d-printed porosity, Angiogenic growth-factors, Bioceramic scaffolds, Bone angiogenesis, Calcium phosphat, Calcium-phosphate, Cells, Composite, Extracellular calcium, Functional-role, In-vitro, Inorganic trace-elements, Matri, Polylactic acid, Regeneration

There has been a huge demand for engineered skin tissues in the realms of both in vitro and in vivo applications. Selecting the right material scaffold is a critical consideration in making engineered skin tissues, since it should possess a good balance between elasticity and mechanical stability while promoting an adequate cell microenvironment to support both the dermal and the epidermal compartments of skin tissue. In this study, 3D-bioprinted norbornene-pullulan photocrosslinkable hydrogels were utilized as alternative scaffolds to produce epithelized dermal skin models. By employing visible light, 2.5 mm3 cell-laden hydrogels could be printed in 10 s. The thiol-ene photocrosslinking chemistry employed in this work enabled the formation of a well-defined extracellular matrix with orthogonal crosslinks, where encapsulated fibroblasts maintained high cellular viability rates. Through this method, an epidermal layer could be grown on top of the fibroblasts. The coexistence and interaction of human fibroblasts and keratinocytes were visualized by determining the expression of specific markers. This approach represents a promising starting point for the development of photocrosslinkable hydrogel-based human skin constructs by using thiol-ene norbornene chemistry, paving the way toward manufacture of complex in vitro models of human tissues.

JTD Keywords: Cells, Collagen, Differentiation, Gel, In-vitro, Light-based 3d bioprintin, Matrix, Mechanical-properties, Models, Photocrosslinkable hydrogels, Pullulan, Skin models

Biofilm formation by the pathobiont Haemophilus influenzae is associated with human nasopharynx colonization, otitis media in children, and chronic respiratory infections in adults suffering from chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD). beta-lactam and quinolone antibiotics are commonly used to treat these infections. However, considering the resistance of biofilm-resident bacteria to antibiotic -mediated killing, the use of antibiotics may be insufficient and require being replaced or complemented with novel strategies. Moreover, unlike the standard minimal inhibitory concentration assay used to assess antibacterial activity against planktonic cells, standardization of methods to evaluate anti-biofilm drug activity is limited. In this work, we detail a panel of protocols for systematic analysis of drug antimicrobial effect on bacterial biofilms, customized to evaluate drug effects against H. influenzae biofilms. Testing of two cinnamaldehyde analogs, (E)- trans-2-nonenal and (E)-3-decen-2-one, demonstrated their effectiveness in both H. influenzae inhibition of biofilm formation and eradication or preformed biofilms. Assay complementarity allowed quantifying the dynamics and extent of the inhibitory effects, also observed for ampicillin resistant clinical strains forming biofilms refractory to this antibiotic. Moreover, cinnamaldehyde analog encapsulation into poly(lactic-co-glycolic acid) (PLGA) polymeric nanoparticles allowed drug vehiculization while maintaining efficacy. Overall, we demonstrate the usefulness of cinnamaldehyde analogs against H. influenzae biofilms, present a test panel that can be easily adapted to a wide range of pathogens and drugs, and highlight the benefits of drug nanoencapsulation towards safe controlled release.

JTD Keywords: Anti-biofilm drugs, Antibodies, Biofilm, Cinnamaldehyde-analogs, Haemophilus influenzae, In-vitro, Maturation, Multimodal methods, Nanoformulation

Bacterial infection remains a significant problem associated with orthopaedic surgeries leading to surgical site infection (SSI). This unmet medical need can become an even greater complication when surgery is due to malignant bone tumor. In the present study, we evaluated in vitro titanium (Ti) implants subjected to gallium (Ga) and silver (Ag)-doped thermochemical treatment as strategy to prevent SSI and improve osteointegration in bone defects caused by diseases such as osteoporosis, bone tumor, or bone metastasis. Firstly, as Ga has been reported to be an osteoinductive and anti-resorptive agent, its performance in the mixture was proved by studying human mesenchymal stem cells (hMSC) and pre-osteoclasts (RAW264.7) behaviour. Then, the antibacterial potential provided by Ag was assessed by resembling "The Race for the Surface" between hMSC and Pseudomonas aeruginosa in two co-culture methods. Moreover, the presence of quorum sensing molecules in the co-culture was evaluated. The results highlighted the suitability of the mixture to induce osteodifferentiation and reduce osteoclastogenesis in vitro . Furthermore, the GaAg surface promoted strong survival rate and retained osteoinduction potential of hMSCs even after bacterial inoculation. Therefore, GaAg-modified titanium may be an ideal candidate to repair bone defects caused by excessive bone resorption, in addition to preventing SSI.

JTD Keywords: Antibacteria, Antibacterial, Bone resorption, Expression, Gallium, In-vitro, Ligan, Nf-kappa-b, Nfatc1, Osteoclast formation, Phosphatase, Receptor activator, Silver, Ti metal, Titanium

Human mesenchymal stromal cells (hMSCs) seeded on calcium phosphate (CaP) bioceramics are extensively explored in bone tissue engineering and have recently shown effective clinical outcomes. In previous pre-clinical studies, hMSCs-CaP-mediated bone formation was preceded by osteoclastogenesis at the implantation site. The current study evaluates to what extent phase composition of CaPs affects the osteoclast response and ultimately influence bone formation. To this end, four different CaP bioceramics were used, hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP) and two biphasic composites of HA/beta- TCP ratios of 60/40 and 20/80 respectively, for in vitro osteoclast differentiation and correlation with in vivo osteoclastogenesis and bone formation. All ceramics allowed osteoclast formation in vitro from mouse and human precursors, except for pure HA, which significantly impaired their maturation. Ectopic implantation alongside hMSCs in subcutis sites of nude mice revealed new bone formation at 8 weeks in all conditions with relative amounts for beta-TCP > biphasic CaPs > HA. Surprisingly, while hMSCs were essential for osteoinduction, their survival did not correlate with bone formation. By contrast, the degree of early osteoclastogenesis (2 weeks) seemed to define the extent of subsequent bone formation. Together, our findings suggest that the osteoclastic response could be used as a predictive marker in hMSC-CaPbased bone regeneration and strengthens the need to understand the underlying mechanisms for future biomaterial development. Statement of significance The combination of mesenchymal stromal cells (MSCs) and calcium phosphate (CaP) materials has demonstrated its safety and efficacy for bone regeneration in clinical trials, despite our insufficient understanding of the underlying biological mechanisms. Osteoclasts were previously suggested as key mediators between the early inflammatory phase following biomaterial implantation and the subsequent bone formation. Here we compared the affinity of osteoclasts for various CaP materials with different ratios of hydroxyapatite to beta-tricalcium phosphate. We found that osteoclast formation, both in vitro and at early stages in vivo, correlates with bone formation when the materials were implanted alongside MSCs in mice. Surprisingly, MSC survival did not correlate with bone formation, suggesting that the number or phenotype of osteoclasts formed was more important. (c) 2024 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

JTD Keywords: Acid phosphatase tartrate resistant isoenzyme, Animal, Animal cell, Animal experiment, Animal tissue, Animals, Article, Beta-tricalcium phosphate, Bioceramics, Biocompatible materials, Biomaterial, Bone, Bone development, Bone formation, Bone regeneration, Calcium phosphate, Calcium phosphate materials, Calcium phosphates, Cd14 antigen, Cell differentiation, Cell engineering, Cell maturation, Cell survival, Ceramics, Chemical composition, Controlled study, Correlation analysis, Correlation coefficient, Data correlation, Durapatite, Engraftment, Flowcharting, Human, Human cell, Human mesenchymal stromal cell, Human mesenchymal stromal cells, Humans, Hydroxyapatite, Hydroxyapatites, In vitro study, In vivo study, In-vitro, In-vivo, Mammals, Marrow stromal cells, Material composition, Material compositions, Mesenchymal stroma cell, Mesenchymal stromal cells, Mice, Mice, nude, Monocyte, Mouse, Nonhuman, Nude mouse, Ossification, Osteoclast, Osteoclastogenesis, Osteoclasts, Osteogenesis, Osteoinduction, Phase composition, Regeneration strategies, Resorption, Scaffolds, Stem-cells, Subcutaneous tissue, Tissue engineering, Transmission control protocol, Tri-calcium phosphates, Vimentin

Malaria, caused by different species of protists of the genus Plasmodium, remains among the most common causes of death due to parasitic diseases worldwide, mainly for children aged under 5. One of the main obstacles to malaria eradication is the speed with which the pathogen evolves resistance to the drug schemes developed against it. For this reason, it remains urgent to find innovative therapeutic strategies offering sufficient specificity against the parasite to minimize resistance evolution and drug side effects. In this context, nanotechnology-based approaches are now being explored for their use as antimalarial drug delivery platforms due to the wide range of advantages and tuneable properties that they offer. However, major challenges remain to be addressed to provide a cost-efficient and targeted therapeutic strategy contributing to malaria eradication. The present work contains a systematic review of nanotechnology-based antimalarial drug delivery systems generated during the last 10 years. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease

JTD Keywords: Adjuvant system, Antimalarial activities, Antimalarial agent, Antimalarial drug, Antimalarial drugs, Antimalarials, Artemisinin resistance, Causes of death, Child, Controlled drug delivery, Diseases, Drug delivery system, Drug delivery systems, Drug interactions, Drug side-effects, Drug-delivery, Experimental modelling, Heparan-sulfate, Human, Humans, In-vitro, Malaria, Malaria vaccine, Mannosylated liposomes, Medical nanotechnology, Models, theoretical, Nanocarriers, Nanomedicine, Nanotechnology, Parasite-, Parasitics, Plasmodium, Plasmodium-falciparum malaria, Red-blood-cells, Targeted delivery, Targeted drug delivery, Theoretical model, Therapeutic strategy

Microbial biofilms are complex three-dimensional structures where sessile microbes are embedded in a polymeric extracellular matrix. Their resistance toward the host immune system as well as to a diverse range of antimicrobial treatments poses a serious health and development threat, being in the top 10 global public health threats declared by the World Health Organization. In an effort to combat biofilm-related microbial infections, several strategies have been developed to independently eliminate biofilms or to complement conventional antibiotic therapies. However, their limitations leave room for other treatment alternatives, where the application of nanotechnology to biofilm eradication has gained significant relevance in recent years. Their small size, penetration efficiency, and the design flexibility that they present makes them a promising alternative for biofilm infection treatment, although they also present set-backs. This review aims to describe the main possibilities and limitations of nanomedicine against biofilms, while covering the main aspects of biofilm formation and study, and the current therapies for biofilm treatment. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

JTD Keywords: Anti-bacterial agents, Anti-infective agents, Antiinfective agent, Antimicrobial, Antimicrobials, Antimicrobials,bacteria,biofilm,infectious diseases,microorganism, Bacteria, Biofilm, Biofilm infections, Biofilms, Complex three dimensional structures, Diseases, Diverse range, Drug-delivery systems,in-vitro,cellular toxicity,nanoparticles,penetration,model,biocompatibility,perspectives,hyperthermia,diagnosi, Extracellular matrices, Global public health, Health risks, Infectious disease, Infectious diseases, Medical nanotechnology, Microbial biofilm, Microorganisms, Nanomedicine, Polymer, Polymers, Regulatory issues, Roadmap

A new generation of fully bioresorbable metallic Zn-based alloys could be used for stenting applications; however, the initial surface degradation delays stent re-endothelialization. Thus, this work proposes a dual strategy to control the corrosion and accelerate the endothelialization of ZnMg and ZnAg biodegradable alloys. First, a stable polycaprolactone (PCL) coating is obtained and followed by its functionalization with either linear RGD (Arg-Gly-Asp) or REDV (Arg-Glu-Asp-Val) peptides or a dual peptide-based platform combining both sequences (RGD-REDV). Scratching tests showed neither delamination nor detachment of the polymeric coating. Potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) measurements confirmed the corrosion resistance after PCL coating by revealing lower current density and higher absolute impedance values. X-ray photoelectron spectroscopy (XPS) and fluorescent microscopy confirmed the correct peptide immobilization onto PCL coated Zn alloys. The functionalized samples exhibited enhanced human umbilical vein endothelial cells (HUVEC) adhesion. The higher number of adhered cells to the functionalized surfaces with the RGD-REDV platform demonstrates the synergistic effect of combining both RGD and REDV sequences. Higher corrosion resistance together with enhanced endothelialization indicates that the dual functionalization of Zn alloys with PCL and peptide-based RGD-REDV platform holds great potential to overcome the clinical limitations of current biodegradable metal stents.

JTD Keywords: Binary alloys, Biodegradable metals, Bioresorbable, Cardiovascular applications, Cell adhesive peptides, Corrosion, Corrosion resistance, Corrosion resistant alloys, Corrosion resistant coatings, Degradation, Dual peptide-based platform, Electrochemical corrosion, Electrochemical impedance spectroscopy, Endothelial cells, Endothelialization, Functionalization, Functionalizations, In-vitro, Magnesium alloys, Metallics, Mg alloy, Peptides, Polycaprolactone coating, Polymer-coatings, Rgd-functionalization, Silver alloys, Stents, Surface, X ray photoelectron spectroscopy, Zinc, Zinc alloys, Zn alloys, Zn-based alloys

Laser surface micropatterning of dental-grade zirconia (3Y-TZP) was explored with the objective of providing defined linear patterns capable of guiding bone-cell response.A nanosecond (ns-) laser was employed to fabricate microgrooves on the surface of 3Y-TZP discs, yielding three different groove periodicities (i.e., 30, 50 and 100 µm). The resulting topography and surface damage were characterized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). X-Ray diffraction (XRD) and Raman spectroscopy techniques were employed to assess the hydrothermal degradation resistance of the modified topographies. Preliminary biological studies were conducted to evaluate adhesion (6 h) of human mesenchymal stem cells (hMSC) to the patterns in terms of cell number and morphology. Finally, Staphylococcus aureus adhesion (4 h) to the microgrooves was investigated.The surface analysis showed grooves of approximately 1.8 µm height that exhibited surface damage in the form of pile-up at the edge of the microgrooves, microcracks and cavities. Accelerated aging tests revealed a slight decrease of the hydrothermal degradation resistance after laser patterning, and the Raman mapping showed the presence of monoclinic phase heterogeneously distributed along the patterned surfaces. An increase of the hMSC area was identified on all the microgrooved surfaces, although only the 50 µm periodicity, which is closer to the cell size, significantly favored cell elongation and alignment along the grooves. A decrease in Staphylococcus aureus adhesion was observed on the investigated micropatterns.The study suggests that linear microgrooves of 50 µm periodicity may help in promoting hMSC adhesion and alignment, while reducing bacterial cell attachment.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

JTD Keywords: abutment material, alumina toughened zirconia, antibacterial, bacterial adhesion, biofilm growth, cell adhesion, dental implants, hydrothermal degradation, implant surfaces, in-vitro, laser patterning, osseointegration, osteogenic differentiation, part 1, surface topography, y-tzp ceramics, Antibacterial, Antibacterials, Bacteria, Bone, Cell adhesion, Cell culture, Cells adhesion, Ceramics, Chemistry, Degradation resistance, Dental implants, Dental material, Dental materials, Dental prostheses, Human, Human mesenchymal stem cells, Humans, Hydrothermal degradation, Laser patterning, Laser surface, Lasers, Low-temperature degradation, Materials testing, Microscopy, electron, scanning, Nanosecond lasers, Osseointegration, Piles, Scanning electron microscopy, Staphylococcus aureus, Stem cells, Surface analysis, Surface damages, Surface properties, Surface property, Surface topography, Topography, Yttrium, Zirconia, Zirconium

The Boston keratoprosthesis (BKPro) is a medical device used to restore vision in complicated cases of corneal blindness. This device is composed by a front plate of polymethylmethacrylate (PMMA) and a backplate usually made of titanium (Ti). Ti is an excellent biomaterial with numerous applications, although there are not many studies that address its interaction with ocular cells. In this regard, despite the good retention rates of the BKPro, two main complications compromise patients' vision and the viability of the prosthesis: imperfect adhesion of the corneal tissue to the upside of the backplate and infections. Thus, in this work, two topographies (smooth and rough) were generated on Ti samples and tested with or without functionalization with a dual peptide platform. This molecule consists of a branched structure that links two peptide moieties to address the main complications associated with BKPro: the well-known RGD peptide in its cyclic version (cRGD) as cell pro-adherent motif and the first 11 residues of lactoferrin (LF1-11) as antibacterial motif. Samples were physicochemically characterized, and their biological response was evaluated in vitro with human corneal keratocytes (HCKs) and against the gram-negative bacterial strain Pseudomonas aeruginosa. The physicochemical characterization allowed to verify the functionalization in a qualitative and quantitative manner. A higher amount of peptide was anchored to the rough surfaces. The studies performed using HCKs showed increased long-term proliferation on the functionalized samples. Gene expression was affected by topography and peptide functionalization. Roughness promoted α-smooth muscle actin (α-SMA) overexpression, and the coating notably increased the expression of extracellular matrix components (ECM). Such changes may favour the development of unwanted fibrosis, and thus, corneal haze. In contrast, the combination of the coating with a rough topography decreased the expression of α-SMA and ECM components, which would be desirable for the long-term success of the prosthesis. Regarding the antibacterial activity, the functionalized smooth and rough surfaces promoted the death of bacteria, as well as a perturbation in their wall definition and cellular morphology. Bacterial killing values were 58 % for smooth functionalised and 68 % for rough functionalised samples. In summary, this study suggests that the use of the dual peptide platform with cRGD and LF1-11 could be a good strategy to improve the in vitro and in vivo performance of the rough topography used in the commercial BKPro.Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.

JTD Keywords: binding, corneal blindness, differentiation, dual coating, iii collagen, in-vitro, infectious endophthalmitis, keratocyte, myofibroblast, peptide platform, proliferation, surface-roughness, titanium implant, Boston keratoprostheses, Corneal blindness, Dual coating, Gram-negative bacteria, Peptide platform, Titanium implant

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

Artemisinin, curcumin or quercetin, alone or in combination, were loaded in nutriosomes, special phospholipid vesicles enriched with Nutriose FM06®, a soluble dextrin with prebiotic activity, that makes these vesicles suitable for oral delivery. The resulting nutriosomes were sized between 93 and 146 nm, homogeneously dispersed, and had slightly negative zeta potential (around -8 mV). To improve their shelf life and storability over time, vesicle dispersions were freeze-dried and stored at 25 °C. Results confirmed that their main physico-chemical characteristics remained unchanged over a period of 12 months. Additionally, their size and polydispersity index did not undergo any significant variation after dilution with solutions at different pHs (1.2 and 7.0) and high ionic strength, mimicking the harsh conditions of the stomach and intestine. An in vitro study disclosed the delayed release of curcumin and quercetin from nutriosomes (∼53% at 48 h) while artemisinin was quickly released (∼100% at 48 h). Cytotoxicity assays using human colon adenocarcinoma cells (Caco-2) and human umbilical vein endothelial cells (HUVECs) proved the high biocompatibility of the prepared formulations. Finally, in vitro antimalarial activity tests, assessed against the 3D7 strain of Plasmodium falciparum, confirmed the effectiveness of nutriosomes in the delivery of curcumin and quercetin, which can be used as adjuvants in the antimalaria treatment. The efficacy of artemisinin was also confirmed but not improved. Overall results proved the possible use of these formulations as an accompanying treatment of malaria infections.Copyright © 2023. Published by Elsevier B.V.

JTD Keywords: artemisinin, delivery, flavonol, formulations, liposomes, malaria infections, nanomedicine, nutriose (r) fm06, oral administration, plasmodium falciparum, In-vitro, Liposomes, Malaria infections, Nanomedicine, Nutriose® fm06, Oral administration, Plasmodium falciparum

JTD Keywords: Bacterial biofilms, Device, Growth, In-vitro, Infections, Laboratory method, Lung model, Responses, Temperature, Time

Previous studies have shown hydrophilic/hydrophobic implant surfaces stimulate/hinder osseointegration. An analogous concept was applied here using common biological functional groups on a model surface to promote oral keratinocytes (OKs) proliferation and hemidesmosomes (HD) to extend implant lifespans through increased soft tissue attachment. However, it is unclear what physicochemistry stimulates HDs. Thus, common biological functional groups (NH2 , OH, and CH3 ) were functionalized on glass using silanization. Non-functionalized plasma-cleaned glass and H silanization were controls. Surface modifications were confirmed with X-ray photoelectron spectroscopy and water contact angle. The amount of bovine serum albumin (BSA) and fibrinogen, and BSA thickness, were assessed to understand how adsorbed protein properties were influenced by physicochemistry and may influence HDs. OKs proliferation was measured, and HDs were quantified with immunofluorescence for collagen XVII and integrin β4. Plasma-cleaned surfaces were the most hydrophilic group overall, while CH3 was the most hydrophobic and OH was the most hydrophilic among functionalized groups. Modification with the OH chemical group showed the highest OKs proliferation and HD expression. The OKs response on OH surfaces appeared to not correlate to the amount or thickness of adsorbed model proteins. These results reveal relevant surface physicochemical features to favor HDs and improve implant soft tissue attachment.© 2023 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.

JTD Keywords: attachment, chemistry, collagen, differentiation, epithelial-cells, hemidesmosome, implant, in-vitro, integrin, keratinocyte, mechanism, organosilane, physicochemistry, protein adsorption, Attachment, Cell-adhesion, Physicochemistry

The functionalization of 3D-printed poly-l-lactic acid (PLLA) and poly(l-lactic-co-ε-caprolactone) (PLCL) bioresorbable stents has been successfully achieved with linear RGDS and YIGSR peptides, as well as a dual platform containing both motifs within a single biomolecule.

JTD Keywords: adsorbed fibrinogen, chemistry, endothelialization, immobilization, platelets, plla, selectivity, surface, titanium, Absorbable implants, Cell adhesion, Endothelial cells, In-vitro hemocompatibility, Peptides, Polymers, Printing, three-dimensional, Stents, Tyrosyl-isoleucyl-glycyl-seryl-arginine

The low endogenous regenerative capacity of the heart,added tothe prevalence of cardiovascular diseases, triggered the advent ofcardiac tissue engineering in the last decades. The myocardial nicheplays a critical role in directing the function and fate of cardiomyocytes;therefore, engineering a biomimetic scaffold holds excellent promise.We produced an electroconductive cardiac patch of bacterial nanocellulose(BC) with polypyrrole nanoparticles (Ppy NPs) to mimic the naturalmyocardial microenvironment. BC offers a 3D interconnected fiber structurewith high flexibility, which is ideal for hosting Ppy nanoparticles.BC-Ppy composites were produced by decorating the network of BC fibers(65 & PLUSMN; 12 nm) with conductive Ppy nanoparticles (83 & PLUSMN; 8 nm).Ppy NPs effectively augment the conductivity, surface roughness, andthickness of BC composites despite reducing scaffolds' transparency.BC-Ppy composites were flexible (up to 10 mM Ppy), maintained theirintricate 3D extracellular matrix-like mesh structure in all Ppy concentrationstested, and displayed electrical conductivities in the range of nativecardiac tissue. Furthermore, these materials exhibit tensile strength,surface roughness, and wettability values appropriate for their finaluse as cardiac patches. In vitro experiments withcardiac fibroblasts and H9c2 cells confirmed the exceptional biocompatibilityof BC-Ppy composites. BC-Ppy scaffolds improved cell viability andattachment, promoting a desirable cardiomyoblast morphology. Biochemicalanalyses revealed that H9c2 cells showed different cardiomyocyte phenotypesand distinct levels of maturity depending on the amount of Ppy inthe substrate used. Specifically, the employment of BC-Ppy compositesdrives partial H9c2 differentiation toward a cardiomyocyte-like phenotype.The scaffolds increase the expression of functional cardiac markersin H9c2 cells, indicative of a higher differentiation efficiency,which is not observed with plain BC. Our results highlight the remarkablepotential use of BC-Ppy scaffolds as a cardiac patch in tissue regenerativetherapies.

JTD Keywords: bacterial nanocellulose, cardiac patches, conducting polymers, polypyrrole, Arrhythmias, Bacterial nanocellulose, Biomaterials, Cardiac patches, Cell therapy, Cellulose, Conductingpolymers, H9c2, In-vitro, Polymer, Polypyrrole, Scaffolds, Tissue, Tissue engineering, Viability

JTD Keywords: alignment, bioresorbable stents, cells, design, electrospinning, everolimus, impact, in-vitro, poly(l-lactic-co-e-caprolactone), proliferation, release, sirolimus, Scaffold topography, Solvent-cast direct-writing

Previously, functional coatings on 3D-printed titanium implants were developed to improve their biointegration by separately incorporating Ga and Ag on the biomaterial surface. Now, a thermochemical treatment modification is proposed to study the effect of their simultaneous incorporation. Different concentrations of AgNO3 and Ga(NO3)3 are evaluated, and the obtained surfaces are completely characterized. Ion release, cytotoxicity, and bioactivity studies complement the characterization. The provided antibacterial effect of the surfaces is analyzed, and cell response is assessed by the study of SaOS-2 cell adhesion, proliferation, and differentiation. The Ti surface doping is confirmed by the formation of Ga-containing Ca titanates and nanoparticles of metallic Ag within the titanate coating. The surfaces generated with all combinations of AgNO3 and Ga(NO3)3 concentrations show bioactivity. The bacterial assay confirms a strong bactericidal impact achieved by the effect of both Ga and Ag present on the surface, especially for Pseudomonas aeruginosa, one of the main pathogens involved in orthopedic implant failures. SaOS-2 cells adhere and proliferate on the Ga/Ag-doped Ti surfaces, and the presence of gallium favors cell differentiation. The dual effect of both metallic agents doping the titanium surface provides bioactivity while protecting the biomaterial from the most frequent pathogens in implantology.

JTD Keywords: 3d-printing, agent, antibacterial activity, bioactive ti, biomaterials, coatings, competition, cu, gallium, glasses, ions, metal, porous structures, promote osseointegration, silver, titanium implants, In-vitro, Porous structures, Titanium implants

Actual polymeric wound closure devices are not optimal for load-bearing applications due to the low mechanical properties and the risk of inflammation and bacterial infection mainly produced by multifil-ament and braided configurations. Biodegradable metallic Zn alloys are promising materials candidates; however, mechanical performance, corrosion behaviour, and biological response should be controlled in order to inhibit the risk of inflammation and bacterial infection. To this end, a Zn-2Ag (2 wt% Ag) alloy was processed by ECAP to evaluate the concurrent combined effect of grain refinement and Ag alloying on biodegradation and antibacterial activity. Two ECAP cycles were successfully applied to a Zn-2Ag alloy obtaining a homogeneous ultra-fine-grained structure in which nanoindentation maps suggested isotro-pic mechanical properties. Lower UTS and YS with higher elongation was reported after ECAP with similar corrosion rates as before processing. ECAP processed samples showed a homogeneous Ag+ release below the minimum inhibitory concentration for S. Aureus and no antibacterial effect was observed by diffusion. As expected, the presence of Ag in Zn-Ag alloys reduced bacterial attachment. Nevertheless, ECAP processed Zn-2Ag provided an excellent antibacterial activity after 3 h probably caused by the uniformly degraded and thus, non- stable, surface observed after bacterial adhesion.(c) 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

JTD Keywords: Behavior, Binary alloys, Biodegradable zinc-alloys, Biomaterials, Equal channel angular pressing, Grain-refinement, In-vitro degradation, Mg, Microstructure, Nanoindentation, Progress, Staphylococcus-aureus, Temperature superplasticity, Ultrafine-grained materials, Zinc alloys, Zn alloys

ConspectusDelivering biological effector molecules in cultured cells is of fundamental importance to any study or application in which the modulation of gene expression is required. Examples range from generating engineered cell lines for studying gene function to the engineering of cells for cell-based therapies such as CAR-T cells and gene-corrected stem cells for regenerative medicine. It remains a great challenge, however, to deliver biological effector molecules across the cell membrane with minimal adverse effects on cell viability and functionality. While viral vectors have been frequently used to introduce foreign nucleic acids into cells, their use is associated with safety concerns such as immunogenicity, high manufacturing cost, and limited cargo capacity.For photoporation, depending on the laser energy, membrane permeabilization happens either by local heating or by laser-induced water vapor nanobubbles (VNB). In our first study on this topic, we demonstrated that the physical force exerted by suddenly formed VNB leads to more efficient intracellular delivery as compared to mere heating. Next, we explored the use of different photothermal nanomaterials, finding that graphene quantum dots display enhanced thermal stability compared to the more traditionally used gold nanoparticles, hence providing the possibility to increase the delivery efficiency by repeated laser activation. To enable its use for the production of engineered therapeutic cells, it would be better if contact with cells with nondegradable nanoparticles is avoided as it poses toxicity and regulatory concerns. Therefore, we recently demonstrated that photoporation can be performed with biodegradable polydopamine nanoparticles as well. Alternatively, we demonstrated that nanoparticle contact can be avoided by embedding the photothermal nanoparticles in a substrate made from biocompatible electrospun nanofibers. With this variety of photoporation approaches, over the years we demonstrated the successful delivery of a broad variety of biologics (mRNA, siRNA, Cas9 ribonucleoproteins, nanobodies, etc.) in many different cell types, including hard-to-transfect cells such as T cells, embryonic stem cells, neurons, and macrophages.In this Account, we will first start with a brief introduction of the general concept and a historical development of photoporation. In the next two sections, we will extensively discuss the various types of photothermal nanomaterials which have been used for photoporation. We discriminate two types of photothermal nanomaterials: single nanostructures and composite nanostructures. The first one includes examples such as gold nanoparticles, graphene quantum dots, and polydopamine nanoparticles. The second type includes polymeric films and nanofibers containing photothermal nanoparticles as well as composite nanoscale biolistic nanostructures. A thorough discussion will be given for each type of photothermal nanomaterial, from its synthesis and characterization to its application in photoporation, with its advantages and disadvantages. In the final section, we will provide an overall discussion and elaborate on future perspectives.

JTD Keywords: cells, delivery, macromolecules, nanoparticles, Gold, Graphite, In-vitro, Metal nanoparticles, Nanostructures, Quantum dots

Introduction: Three-dimensional (3D) bioprinting is a promising technique for the development of neuronal in vitro models because it controls the deposition of materials and cells. Finding a biomaterial that supports neural differentiation in vitro while ensuring compatibility with the technique of 3D bioprinting of a self-standing construct is a challenge.Methods: In this study, gelatin methacryloyl (GelMA), methacrylated alginate (AlgMA), and hyaluronic acid (HA) were examined by exploiting their biocompatibility and tunable mechanical properties to resemble the extracellular matrix (ECM) and to create a suitable material for printing neural progenitor cells (NPCs), supporting their long-term differentiation. NPCs were printed and differentiated for up to 15 days, and cell viability and neuronal differentiation markers were assessed throughout the culture.Results and Discussion: This composite biomaterial presented the desired physical properties to mimic the ECM of the brain with high water intake, low stiffness, and slow degradation while allowing the printing of defined structures. The viability rates were maintained at approximately 80% at all time points. However, the levels of beta-III tubulin marker increased over time, demonstrating the compatibility of this biomaterial with neuronal cell culture and differentiation. Furthermore, these cells showed increased maturation with corresponding functional properties, which was also demonstrated by the formation of a neuronal network that was observed by recording spontaneous activity via Ca2+ imaging.

JTD Keywords: biomaterials, bioprinting, differentiation, in vitro models, neural progenitor cells, 2d, Biomaterials, Bioprinting, C17.2, Differentiation, Extracellular-matrix, Hydrogels, In vitro models, In-vitro, Neural progenitor cells, Neuronal models, Proliferation, Scaffolds, Stem-cells, Substrate stiffness

Lately, there has been an increasing demand for materials that could improve tissue regenerative therapies and provide antimicrobial effects. Similarly, there is a growing need to develop or modify biomaterials for the diagnosis and treatment of different pathologies. In this scenario, hydroxyapatite (HAp) appears as a bioceramic with extended functionalities. Nevertheless, there are certain disadvantages related to the mechanical properties and lack of antimicrobial capacity. To circumvent them, the doping of HAp with a variety of cationic ions is emerging as a good alterative due to the different biological roles of each ion. Among many elements, lanthanides are understudied despite their great potential in the biomedical field. For this reason, the present review focuses on the biological benefits of lanthanides and how their incorporation into HAp can alter its morphology and physical properties. A comprehensive section of the applications of lanthanides-substituted HAp nanoparticles (HAp NPs) is presented to unveil the potential biomedical uses of these systems. Finally, the need to study the tolerable and non-toxic percentages of substitution with these elements is highlighted.

JTD Keywords: biolabeling, biomedicine, biosensors, bone regeneration, calcium, cancer treatment, cationic ions, cell imaging, cerium, doped hap, hydroxyapatite, implants, in-vitro bioactivity, lanthanides-substitutions, lanthanidessubstitutions, nanoparticles, radiation synovectomy, sm-153 particulate hydroxyapatite, structural-characterization, theragnostics, theranostic nanoplatforms, Europium-doped hydroxyapatite, Hydroxyapatite, Theragnostics

Biomolecular and physical cues of the extracellular matrix environment regulate collective cell dynamics and tissue patterning. Nonetheless, how the viscoelastic properties of the matrix regulate collective cell spatial and temporal organization is not fully understood. Here we show that the passive viscoelastic properties of the matrix encapsulating a spheroidal tissue of breast epithelial cells guide tissue proliferation in space and in time. Matrix viscoelasticity prompts symmetry breaking of the spheroid, leading to the formation of invading finger-like protrusions, YAP nuclear translocation and epithelial-to-mesenchymal transition both in vitro and in vivo in a Arp2/3-complex-dependent manner. Computational modelling of these observations allows us to establish a phase diagram relating morphological stability with matrix viscoelasticity, tissue viscosity, cell motility and cell division rate, which is experimentally validated by biochemical assays and in vitro experiments with an intestinal organoid. Altogether, this work highlights the role of stress relaxation mechanisms in tissue growth dynamics, a fundamental process in morphogenesis and oncogenesis.© 2022. The Author(s), under exclusive licence to Springer Nature Limited.

JTD Keywords: in-vitro, migration, morphogenesis, stiffness, Intestinal stem-cell

Bioresorbable stents (BRS) are designed to provide initial sufficient mechanical support to prevent vessel recoil while being degraded until their complete resorption. Therefore, degradation rate of BRS plays a crucial role in successful stent performance. This work presents a complete study on the degradation of poly-llactic acid (PLLA) and poly(lactic-co-epsilon-caprolactone) (PLCL) stents fabricated by solvent-cast direct-writing (SC-DW) through two different accelerated assays: alkaline medium at 37 degrees C for 10 days and PBS at 50 degrees C for 4 months. On retrieval, degraded stents were characterized in terms of mass loss, molecular weight (Mw), thermal and mechanical properties. The results showed that under alkaline conditions, stents underwent surface erosion, whereas stents immersed in PBS at 50 degrees C experienced bulk degradation. M-n decrease was accurately described by the autocatalyzed kinetic model, with PLCL showing a degradation rate 1.5 times higher than PLLA. Additionally, stents were subjected to gamma-irradiation and ethylene oxide (EtO) sterilization. Whereas EtOsterilized stents remained structurally unaltered, gamma-irradiated stents presented severe deterioration as a result of extensive chain scission.

JTD Keywords: Acid, Behavior, Bioresorbable stents, Copolymer, Hydrolytic degradation, In-vitro degradation, Mechanical-properties, Molecular-weight, Poly(l-lactide), Poly-l-lactic acid, Poly-l-lactide, Scaffolds, Solvent-cast direct-writing, Sterilization

The in vitro biological characterization of biomaterials is largely based on static cell cultures. However, for highly reactive biomaterials such as calcium-deficient hydroxyapatite (CDHA), this static environment has limitations. Drastic alterations in the ionic composition of the cell culture medium can negatively affect cell behavior, which can lead to misleading results or data that is difficult to interpret. This challenge could be addressed by a microfluidics-based approach (i.e. on-chip), which offers the opportunity to provide a continuous flow of cell culture medium and a potentially more physiologically relevant microenvironment. The aim of this work was to explore microfluidic technology for its potential to characterize CDHA, particularly in the context of inflammation. Two different CDHA substrates (chemically identical, but varying in microstructure) were integrated on-chip and subsequently evaluated. We demonstrated that the on-chip environment can avoid drastic ionic alterations and increase protein sorption, which was reflected in cell studies with RAW 264.7 macrophages. The cells grown on-chip showed a high cell viability and enhanced proliferation compared to cells maintained under static conditions. Whereas no clear differences in the secretion of tumor necrosis factor alpha (TNF-α) were found, variations in cell morphology suggested a more anti-inflammatory environment on-chip. In the second part of this study, the CDHA substrates were loaded with the drug Trolox. We showed that it is possible to characterize drug release on-chip and moreover demonstrated that Trolox affects the TNF-α secretion and morphology of RAW 264.7 ​cells. Overall, these results highlight the potential of microfluidics to evaluate (bioactive) biomaterials, both in pristine form and when drug-loaded. This is of particular interest for the latter case, as it allows the biological characterization and assessment of drug release to take place under the same dynamic in vitro environment.© 2022 The Authors.

JTD Keywords: alpha-tocopherol, antioxidant, biomaterials, calcium phosphate cement, culture, delivery, drug release, in vitro, in-vitro, ion, macrophage, on-chip, release, tool, Biomaterial, Calcium phosphate cement, Calcium-phosphate cements, Drug release, In vitro, Macrophage, On-chip

Tissue engineering is nowadays a powerful tool to restore damaged tissues and recover their normal functionality. Advantages over other current methods are well established, although a continuous evolution is still necessary to improve the final performance and the range of applications. Trends are nowadays focused on the development of multifunctional scaffolds with hierarchical structures and the capability to render a sustained delivery of bioactive molecules under an appropriate stimulus. Nanocomposites incorporating hydroxyapatite nanoparticles (HAp NPs) have a predominant role in bone tissue regeneration due to their high capacity to enhance osteoinduction, osteoconduction, and osteointegration, as well as their encapsulation efficiency and protection capability of bioactive agents. Selection of appropriated polymeric matrices is fundamental and consequently great efforts have been invested to increase the range of properties of available materials through copolymerization, blending, or combining structures constituted by different materials. Scaffolds can be obtained from different processes that differ in characteristics, such as texture or porosity. Probably, electrospinning has the greater relevance, since the obtained nanofiber membranes have a great similarity with the extracellular matrix and, in addition, they can easily incorporate functional and bioactive compounds. Coaxial and emulsion electrospinning processes appear ideal to generate complex systems able to incorporate highly different agents. The present review is mainly focused on the recent works performed with Hap-loaded scaffolds having at least one structural layer composed of core/shell nanofibers.

JTD Keywords: bone tissue, coaxial electrospinning, composite nanofibers, drug-release behavior, emulsion electrospinning, hydroxyapatite, in-vitro evaluation, mechanical-properties, osteogenic differentiation, pickering emulsions, protein adsorption, structured scaffolds, surface-initiated polymerization, tissue regeneration, Bone tissue, Coaxial electrospinning, Emulsion electrospinning, Hydroxyapatite, Multifunctional scaffolds, Poly(3-hydroxybutyrate) phb patches, Tissue regeneration

Chronic wounds infected by Pseudomonas aeruginosa and Staphylococcus aureus are a relevant health problem worldwide because these pathogens grow embedded in a network of polysaccharides, proteins, lipids, and extracellular DNA, named biofilm, that hinders the transport of antibiotics and increases their antimicrobial tolerance. It is necessary to investigate therapies that improve the penetrability and efficacy of antibiotics. In this context, our main objectives were to study the relationship between P. aeruginosa and S. aureus and how their relationship can affect the antimicrobial treatment and investigate whether functionalized silver nanoparticles can improve the antibiotic therapy. We used an optimized in vitro wound model that mimics an in vivo wound to co-culture P. aeruginosa and S. aureus biofilm. The in vitro wound biofilm was treated with antimicrobial combinatory therapies composed of antibiotics (gentamycin and ciprofloxacin) and biofilm-dispersing free or silver nanoparticles functionalized with enzymes (alpha-amylase, cellulase, DNase I, or proteinase K) to study their antibiofilm efficacy. The interaction and colocalization of P. aeruginosa and S. aureus in a wound-like biofilm were examined and detailed characterized by confocal and electronic microscopy. We demonstrated that antibiotic monotherapy is inefficient as it differentially affects the two bacterial species in the mixed biofilm, driving P. aeruginosa to overcome S. aureus when using ciprofloxacin and the contrary when using gentamicin. In contrast, dual-antibiotic therapy efficiently reduces both species while maintaining a balanced population. In addition, DNase I nanoparticle treatment had a potent antibiofilm effect, decreasing P. aeruginosa and S. aureus viability to 0.017 and 7.7%, respectively, in combined antibiotics. The results showed that using nanoparticles functionalized with DNase I enhanced the antimicrobial treatment, decreasing the bacterial viability more than using the antibiotics alone. The enzymes alpha-amylase and cellulase showed some antibiofilm effect but were less effective compared to the DNase I treatment. Proteinase K showed insignificant antibiofilm effect. Finally, we proposed a three-dimensional colocalization model consisting of S. aureus aggregates within the biofilm structure, which could be associated with the low efficacy of antibiofilm treatments on bacteria. Thus, designing a clinical treatment that combines antibiofilm enzymes and antibiotics may be essential to eliminating chronic wound infections.

JTD Keywords: antimicrobial therapies, biofilm, chronic infection, nanoparticle, Antimicrobial therapies, Biofilm, Chronic infection, In-vitro, Matrix, Model, Nanoparticle, Wound healing

The immunomodulatory potential of mycobacteria to be used for therapeutic purposes varies by species and culture conditions and is closely related to mycobacterial lipid composition. Although the lipids present in the mycobacterial cell wall are relevant, lipids are mainly stored in intracellular lipid inclusions (ILIs), which have emerged as a crucial structure in understanding mycobacteria-host interaction. Little is known about ILI ultrastructure, production, and composition in nonpathogenic species. In this study, we compared the lipid profiles of the nonpathogenic immunomodulatory agent Mycobacterium brumae during pellicle maturation under different culture conditions with qualitative and quantitative approaches by using high-resolution imaging and biochemical and composition analyses to understand ILI dynamics. The results showed wax esters, mainly in early stages of development, and acylglycerols in mature ILI composition, revealing changes in dynamics, amount, and morphometry, depending on pellicle maturation and the culture media used. Low-glycerol cultures induced ILIs with lower molecular weights which were smaller in size in comparison with the ILIs produced in glycerol-enriched media. The data also indicate the simple metabolic plasticity of lipid synthesis in M. brumae, as well as its high versatility in generating different lipid profiles. These findings provide an interesting way to enhance the production of key lipid structures via the simple modulation of cell culture conditions.

JTD Keywords: cell wall, electron microscopy, intrabacterial, lipid inclusions, mycobacterium, Bodies, Cell wall, Electron microscopy, Growth, In-vitro, Intrabacterial, Lipid inclusions, Mycobacterium, Prokaryotes, Triacylglycerol, Tuberculosis, Ultrastructural imaging, Virulence, Wax esters

One of humanity's greatest challenges is space exploration, which requires an in-depth analysis of the data continuously collected as a necessary input to fill technological gaps and move forward in several research sectors. Focusing on space crew healthcare, a critical issue to be addressed is tissue regeneration in extreme conditions. In general, it represents one of the hottest and most compelling goals of the scientific community and the development of suitable therapeutic strategies for the space environment is an urgent need for the safe planning of future long-term manned space missions. Osteopenia is a commonly diagnosed disease in astronauts due to the physiological adaptation to altered gravity conditions. In order to find specific solutions to bone damage in a reduced gravity environment, bone tissue engineering is gaining a growing interest. With the aim to critically investigate this topic, the here presented review reports and discusses bone tissue engineering scenarios in microgravity, from scaffolding to bioreactors. The literature analysis allowed to underline several key points, such as the need for (i) biomimetic composite scaffolds to better mimic the natural microarchitecture of bone tissue, (ii) uniform simulated microgravity levels for standardized experimental protocols to expose biological materials to the same testing conditions, and (iii) improved access to real microgravity for scientific research projects, supported by the so-called democratization of space.© 2022. The Author(s).

JTD Keywords: biomaterials, collagen/hydroxyapatite, composite scaffolds, in-vitro, mineralization, proliferation, regenerative medicine, stem-cells, vivo, Hydroxyapatite scaffolds

Background By 2016, signs of emergence of Plasmodium falciparum resistance to artemisinin and partner drugs were detected in the Greater Mekong Subregion. Recently, the independent evolution of artemisinin resistance has also been reported in Africa and South America. This alarming scenario calls for the urgent development of new antimalarials with novel modes of action. We investigated the interference with protein aggregation, which is potentially toxic for the cell and occurs abundantly in all Plasmodium stages, as a hitherto unexplored drug target in the pathogen. Results Attempts to exacerbate the P. falciparum proteome's propensity to aggregation by delivering endogenous aggregative peptides to in vitro cultures of this parasite did not significantly affect their growth. In contrast, protein aggregation inhibitors clearly reduced the pathogen's viability. One such compound, the bis(styrylpyridinium) salt YAT2150, exhibited potent antiplasmodial activity with an in vitro IC50 of 90 nM for chloroquine- and artemisinin-resistant lines, arresting asexual blood parasites at the trophozoite stage, as well as interfering with the development of both sexual and hepatic forms of Plasmodium. At its IC50, this compound is a powerful inhibitor of the aggregation of the model amyloid beta peptide fragment 1-40, and it reduces the amount of aggregated proteins in P. falciparum cultures, suggesting that the underlying antimalarial mechanism consists in a generalized impairment of proteostasis in the pathogen. YAT2150 has an easy, rapid, and inexpensive synthesis, and because it fluoresces when it accumulates in its main localization in the Plasmodium cytosol, it is a theranostic agent. Conclusions Inhibiting protein aggregation in Plasmodium significantly reduces the parasite's viability in vitro. Since YAT2150 belongs to a novel structural class of antiplasmodials with a mode of action that potentially targets multiple gene products, rapid evolution of resistance to this drug is unlikely to occur, making it a promising compound for the post-artemisinin era.

JTD Keywords: amyloid pan-inhibitors, antimalarial drugs, malaria, plasmodium falciparum, protein aggregation, Amyloid formation, Amyloid pan-inhibitors, Antimalarial drugs, Colocalization, Cytosolic delivery, Derivatives, Disease, Drug, In-vitro, Malaria, Mechanism, Plasmodium falciparum, Polyglutamine, Protein aggregation, Yat2150

Most of the conventional in vitro models to test biomaterial-driven vascularization are too simplistic to recapitulate the complex interactions taking place in the actual cell microenvironment, which results in a poor prediction of the in vivo performance of the material. However, during the last decade, cell culture models based on microfluidic technology have allowed attaining unprecedented levels of tissue biomimicry. In this work, we propose a microfluidic-based 3D model to evaluate the effect of bioactive biomaterials capable of releasing signalling cues (such as ions or proteins) in the recruitment of endogenous endothelial progenitor cells, a key step in the vascularization process. The usability of the platform is demonstrated using experimentally-validated finite element models and migration and proliferation studies with rat endothelial progenitor cells (rEPCs) and bone marrow-derived rat mesenchymal stromal cells (BM-rMSCs). As a proof of concept of biomaterial evaluation, the response of rEPCs to an electrospun composite made of polylactic acid with calcium phosphates nanoparticles (PLA+CaP) was compared in a co-culture microenvironment with BM-rMSC to a regular PLA control. Our results show a significantly higher rEPCs migration and the upregulation of several pro-inflammatory and proangiogenic proteins in the case of the PLA+CaP. The effects of osteopontin (OPN) on the rEPCs migratory response were also studied using this platform, suggesting its important role in mediating their recruitment to a calcium-rich microenvironment. This new tool could be applied to screen the capacity of a variety of bioactive scaffolds to induce vascularization and accelerate the preclinical testing of biomaterials. STATEMENT OF SIGNIFICANCE: : For many years researchers have used neovascularization models to evaluate bioactive biomaterials both in vitro, with low predictive results due to their poor biomimicry and minimal control over cell cues such as spatiotemporal biomolecule signaling, and in vivo models, presenting drawbacks such as being highly costly, time-consuming, poor human extrapolation, and ethically controversial. We describe a compact microphysiological platform designed for the evaluation of proangiogenesis in biomaterials through the quantification of the level of sprouting in a mimicked endothelium able to react to gradients of biomaterial-released signals in a fibrin-based extracellular matrix. This model is a useful tool to perform preclinical trustworthy studies in tissue regeneration and to better understand the different elements involved in the complex process of vascularization.Copyright © 2022. Published by Elsevier Ltd.

JTD Keywords: angiogenesis, bioactive materials, bone regeneration, bone-formation, calcium-phosphate, extracellular calcium, in-vitro, interstitial flow, ion release, microfluidic model, signalling gradient, substitutes, tissue engineering, vascularization, vegf, Ion release, Mesenchymal stem-cells, Tissue engineering, Vascularization

Great advances in cancer treatment have been undertaken in the last years as a consequence of the development of new antitumoral drugs able to target cancer cells with decreasing side effects and a better understanding of the behavior of neoplastic cells during invasion and metastasis. Specifically, drug delivery systems (DDS) based on the use of hydroxyapatite nanoparticles (HAp NPs) are gaining attention and merit a comprehensive review focused on their potential applications. These are derived from the intrinsic properties of HAp (e.g., biocompatibility and biodegradability), together with the easy functionalization and easy control of porosity, crystallinity and morphology of HAp NPs. The capacity to tailor the properties of DLS based on HAp NPs has well-recognized advantages for the control of both drug loading and release. Furthermore, the functionalization of NPs allows a targeted uptake in tumoral cells while their rapid elimination by the reticuloendothelial system (RES) can be avoided. Advances in HAp NPs involve not only their use as drug nanocarriers but also their employment as nanosystems for magnetic hyperthermia therapy, gene delivery systems, adjuvants for cancer immunotherapy and nanoparticles for cell imaging.

JTD Keywords: antitumoral, cancer, cell imaging, controlled-release, drug-carrier, efficient drug-delivery, fatty-acid-metabolism, fe3o4 nanoparticles, gene delivery, hydroxyapatite, hyperthermia, immunotherapy, in-vitro, magnetic hydroxyapatite, nano-hydroxyapatite, protein adsorption, tumor-growth, Calcium-phosphate nanoparticles, Cancer, Immunotherapy

Type 1 Diabetes results from autoimmune response elicited against β-cell antigens. Nowadays, insulin injections remain the leading therapeutic option. However, injection treatment fails to emulate the highly dynamic insulin release that β-cells provide. 3D cell-laden microspheres have been proposed during the last years as a major platform for bioengineering insulin-secreting constructs for tissue graft implantation and a model for in vitro drug screening platforms. Current microsphere fabrication technologies have several drawbacks: the need for an oil phase containing surfactants, diameter inconsistency of the microspheres, and high time-consuming processes. These technologies have widely used alginate for its rapid gelation, high processability, and low cost. However, its low biocompatible properties do not provide effective cell attachment. This study proposes a high-throughput methodology using a 3D bioprinter that employs an ECM-like microenvironment for effective cell-laden microsphere production to overcome these limitations. Crosslinking the resulting microspheres with tannic acid prevents collagenase degradation and enhances spherical structural consistency while allowing the diffusion of nutrients and oxygen. The approach allows customization of microsphere diameter with extremely low variability. In conclusion, a novel bio-printing procedure is developed to fabricate large amounts of reproducible microspheres capable of secreting insulin in response to extracellular glucose stimuli.© 2022 The Authors. Advanced Materials Technologies published by Wiley‐VCH GmbH.

JTD Keywords: 3d bioprinter, beta-cell, biomaterial, collagen, encapsulation, mechanics, microspheres, survival, 3d bioprinter, ?-cell, Advanced material technologies, Biocompatibility, Cell encapsulations, Cells, Collagen, Cross-linking, Cytology, Drug delivery, Encapsulation, Fabrication, Flavonoids, Gelation, In-vitro, Insulin injections, Insulin release, Microspheres, Tannic acid, Tannins, Throughput, Tissue grafts, Type 1 diabetes, Β‐cell

The tumor extracellular matrix (ECM) plays a vital role in tumor progression and drug resistance. Previous studies have shown that breast tissue-derived matrices could be an important biomaterial to recreate the complexity of the tumor ECM. We have developed a method for decellularizing and delipidating a porcine breast tissue (TDM) compatible with hydrogel formation. The addition of gelatin methacrylamide and alginate allows this TDM to be bioprinted by itself with good printability, shape fidelity, and cytocompatibility. Furthermore, this bioink has been tuned to more closely recreate the breast tumor by incorporating collagen type I (Col1). Breast cancer cells (BCCs) proliferate in both TDM bioinks forming cell clusters and spheroids. The addition of Col1 improves the printability of the bioink as well as increases BCC proliferation and reduces doxorubicin sensitivity due to a downregulation of HSP90. TDM bioinks also allow a precise three-dimensional printing of scaffolds containing BCCs and stromal cells and could be used to fabricate artificial tumors. Taken together, we have proven that these novel bioinks are good candidates for biofabricating breast cancer models.

JTD Keywords: 3d in vitro cancer model, bioprinting, breast tissue, 3d in vitro cancer model, Bioink, Bioprinting, Breast tissue, Crosstalk, Decellularization, Extracellular-matrix, Growth, Hydrogels, In-vitro, Inhibition, Mechanical-properties, Metastasis, Proliferation

Heterotopic ossification (HO) is a condition triggered by an injury leading to the formation of mature lamellar bone in extraskeletal soft tissues. Despite being a frequent complication of orthopedic and trauma surgery, brain and spinal injury, the etiology of HO is poorly understood. The aim of this study is to evaluate the hypothesis that a sustained local ionic homeostatic imbalance (SLIHI) created by mineral formation during tissue calcification modulates inflammation to trigger HO. This evaluation also considers the role SLIHI could play for the design of cell-free, drug-free osteoinductive bone graft substitutes. The evaluation contains five main sections. The first section defines relevant concepts in the context of HO and provides a summary of proposed causes of HO. The second section starts with a detailed analysis of the occurrence and involvement of calcification in HO. It is followed by an explanation of the causes of calcification and its consequences. This allows to speculate on the potential chemical modulators of inflammation and triggers of HO. The end of this second section is devoted to in vitro mineralization tests used to predict the ectopic potential of materials. The third section reviews the biological cascade of events occurring during pathological and material-induced HO, and attempts to propose a quantitative timeline of HO formation. The fourth section looks at potential ways to control HO formation, either acting on SLIHI or on inflammation. Chemical, physical, and drug-based approaches are considered. Finally, the evaluation finishes with a critical assessment of the definition of osteoinduction.

JTD Keywords: apatite, beta-tricalcium phosphate, bone, bone graft, bone morphogenetic protein, demineralized bone-matrix, experimental myositis-ossificans, extracellular calcium, heterotopic ossification, in-vitro, inflammation, multinucleated giant-cells, osteoinduction, spinal-cord-injury, total hip-arthroplasty, traumatic brain-injury, Apatite, Calcium-sensing receptor, Osteoinduction

Over the most recent decades, the development of new biological platforms to study disease progression and drug efficacy has been of great interest due to the high increase in the rate of neurodegenerative diseases (NDDs). Therefore, blood-brain barrier (BBB) as an organ-on-a-chip (OoC) platform to mimic brain-barrier performance could offer a deeper understanding of NDDs as well as a very valuable tool for drug permeability testing for new treatments. A very attractive improvement of BBB-oC technology is the integration of detection systems to provide continuous monitoring of biomarkers in real time and a fully automated analysis of drug permeably, rendering more efficient platforms for commercialization. In this Perspective, an overview of the main BBB-oC configurations is introduced and a critical vision of the BBB-oC platforms integrating electronic read out systems is detailed, indicating the strengths and weaknesses of current devices, proposing the great potential for biosensors integration in BBB-oC. In this direction, we name potential biomarkers to monitor the evolution of NDDs related to the BBB and/or drug cytotoxicity using biosensor technology in BBB-oC.

JTD Keywords: biosensors, blood−brain barrier (bbb), neurodegenerative diseases (ndds), organ-on-a-chip (ooc), Bbb, Biosensors, Blood-brain barrier (bbb), Electrical-resistance, Electrochemical biosensors, Endothelial-cells, In-vitro model, Matrix metalloproteinases, Mechanisms, Neurodegenerative diseases (ndds), Organ-on-a-chip (ooc), Permeability, Stress, Transendothelial electrical resistance (teer), Transepithelial, Transepithelial/transendothelial electrical resistance (teer), Transport

Since the discovery that nanostructured surfaces were able to kill bacteria, many works have been published focusing on the design of nanopatterned surfaces with antimicrobial properties. Synthetic bone grafts, based on calcium phosphate (CaP) formulations, can greatly benefit from this discovery if adequate nanotopographies can be developed. However, CaP are reactive materials and experience ionic exchanges when placed into aqueous solutions which may in turn affect cell behaviour and complicate the interpretation of the bactericidal results. The present study explores the bactericidal potential of two nanopillared CaP prepared by hydrolysis of two different sizes of alpha-tricalcium phosphate (alpha-TCP) powders under biomimetic or hydrothermal conditions. A more lethal bactericidal response toward Pseudomonas aeruginosa (similar to 75% killing efficiency of adhered bacteria) was obtained from the hydrothermally treated CaP which consisted in a more irregular topography in terms of pillar size (radius: 20-60 nm), interpillar distances (100-1500 nm) and pillar distribution (pillar groups forming bouquets) than the biomimetically treated one (radius: 20-40 nm and interpillar distances: 50-200 nm with a homogeneous pillar distribution). The material reactivity was greatly influenced by the type of medium (nutrient-rich versus nutrient-free) and the presence or not of bacteria. A lower reactivity and superior bacterial attachment were observed in the nutrient-free medium while a lower attachment was observed for the nutrient rich medium which was explained by a superior reactivity of the material paired with the lower tendency of planktonic bacteria to adhere on surfaces in the presence of nutrients. Importantly, the ionic exchanges produced by the presence of materials were not toxic to planktonic cells. Thus, we can conclude that topography was the main contributor to mortality in the bacterial adhesion tests.

JTD Keywords: bactericidal, calcium deficient hydroxyapatite, calcium phosphates, nanopillars, pseudomonas aeruginosa, reactivity, Adhesion, Anti-bacterial agents, Antibacterial, Bacterial adhesion, Bactericidal, Biomaterials, Biomimetics, Calcium deficient hydroxyapatite, Calcium phosphates, Hydroxyapatite, In-vitro, Infections, Nanopillars, Nanostructures, Pseudomonas aeruginosa, Pseudomonas-aeruginosa, Reactivity, Recent progress, Silver, Topography, Transmission

Collagen VI-related dystrophies (COL6-RDs) are a group of rare congenital neuromuscular dystrophies that represent a continuum of overlapping clinical phenotypes that go from the milder Bethlem myopathy (BM) to the severe Ullrich congenital muscular dystrophy, for which there is no effective treatment. Mutations in one of the three Collagen VI genes alter the incorporation of this protein into the extracellular matrix (ECM), affecting the assembly and the structural integrity of the whole fibrillar network. Clinical hallmarks of COL6-RDs are secondary to the ECM disruption and include muscle weakness, proximal joint contractures, and distal hyperlaxity. Although some traits have been identified in patients’ ECMs, a correlation between the ECM features and the clinical phenotype has not been established, mainly due to the lack of predictive and reliable models of the pathology. Herein, we engineered a new personalized pre-clinical model of COL6-RDs using cell-derived matrices (CDMs) technology to better recapitulate the complexity of the native scenario. We found that CDMs from COL6-RD patients presented alterations in ECM structure and composition, showing a significantly decreased Collagen VI secretion, especially in the more severe phenotypes, and a decrease in Fibrillin-1 inclusion. Next, we examined the Collagen VI-mediated deposition of Fibronectin in the ECM, finding a higher alignment, length, width, and straightness than in patients with COL6-RDs. Overall, these results indicate that CDMs models are promising tools to explore the alterations that arise in the composition and fibrillar architecture due to mutations in Collagen VI genes, especially in early stages of matrix organization. Ultimately, CDMs derived from COL6-RD patients may become relevant pre-clinical models, which may help identifying novel biomarkers to be employed in the clinics and to investigate novel therapeutic targets and treatments. Copyright © 2022 Almici, Chiappini, López-Márquez, Badosa, Blázquez, Caballero, Montero, Natera-de Benito, Nascimento, Roldán, Lagunas, Jiménez-Mallebrera and Samitier.

JTD Keywords: alpha-3 chain, binding, collagen vi related muscular dystrophy, decellularisation, decellularized matrices, deficiency, expression, extracellular matrix, fibroblasts, fibronectin, in vitro model, patient-derived ecms, skeletal-muscle, ullrich, Cell-derived matrices, Collagen, Collagen vi related muscular dystrophy, Decellularisation, Decellularization, Extracellular matrices, Extracellular matrix, Genes, In vitro model, In-vitro, In-vitro models, Matrix, Matrix model, Muscular dystrophy, Pathology, Patient-derived ecm, Patient-derived ecms, Pre-clinical

Poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) enhance the delivery of therapeutic enzymes for replacement therapy of lysosomal storage disorders. Previous studies examined NPs encapsulating or coated with enzymes, but these formulations have never been compared. We examined this using hyaluronidase (HAse), deficient in mucopolysaccharidosis IX, and acid sphingomyelinase (ASM), deficient in types A–B Niemann–Pick disease. Initial screening of size, PDI, ζ potential, and loading resulted in the selection of the Lactel II co-polymer vs. Lactel I or Resomer, and Pluronic F68 surfactant vs. PVA or DMAB. Enzyme input and addition of carrier protein were evaluated, rendering NPs having, e.g., 181 nm diameter, 0.15 PDI, −36 mV ζ potential, and 538 HAse molecules encapsulated per NP. Similar NPs were coated with enzyme, which reduced loading (e.g., 292 HAse molecules/NP). NPs were coated with targeting antibodies (> 122 molecules/NP), lyophilized for storage without alterations, and acceptably stable at physiological conditions. NPs were internalized, trafficked to lysosomes, released active enzyme at lysosomal conditions, and targeted both peripheral organs and the brain after i.v. administration in mice. While both formulations enhanced enzyme delivery compared to free enzyme, encapsulating NPs surpassed coated counterparts (18.4- vs. 4.3-fold enhancement in cells and 6.2- vs. 3-fold enhancement in brains), providing guidance for future applications.

JTD Keywords: active enzymes, encapsulation, enhanced delivery, enzyme therapeutics, formulation parameters, icam-1 targeting, icam-1-targeted nanocarriers, in vivo biodistribution, in-vitro, lysosomal delivery, model, oral delivery, plga nanoparticles, poly(lactic-co-glycolic acid) nanoparticles, protein therapeutics, surface loading, Acid sphingomyelinase, Enzyme therapeutics, Surface loading

Over the last decades, most of the strategies to improve the vascu-larization of biomaterials based on calcium phosphates (CaPs) arebased on the incorporation of well-known proangiogenic agents, suchas growth factors, despite their high cost and the complexity of deliv-ering safe and effective doses [1]. A promising alternative is the use ofinorganic elements that naturally occur within the body, namely me-tallic ions [2]. It has been shown that dissolution products of CaPs areable to induce vascularization [3,4], although the particular mechanismby which calcium stimulates this process is not very well understood,mainly due to the lack of suitable in vitro and in vivo models.In this work, we present a microphysiological system ( MPS) tostudy the role of calcium in neovascularization. The bone-healing mi-croenvironment was mimicked by 3D-culturing bone marrow ratmesenchymal stem cells (BM-rMSC) and rat endothelial progenitorcells (rEPC) either in mono or co-culture conditions. Migration assayswere performed in our proposed system, showing that calcium-enrichedmedia (10 mM) is only able to elicit a strong migratory response onendothelial progenitor cells when they are in co-culture conditions. Wealso show that calcium exerts a potent chemotactic effect on BM-rMSCand induces an increase in the osteopontin (OPN) secretion, a proteininvolved in chemotaxis and immune regulation [5]. Therefore, wepropose a novel mechanism by which calcium can stimulate endothelialprogenitor cell recruitment and subsequent vascularization and open upnew possibilities to test calcium-releasing biomaterials using MPS.

JTD Keywords: In-vitro

Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons.We used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation.We first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids.We show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.This work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926).Published by Elsevier B.V.

JTD Keywords: cell fusion, expression, fusion, ganglion-cells, in-vitro, mouse, müller glia, neural differentiation, organoids, regeneration, retina regeneration, stem cells, stromal cells, transplantation, 4',6 diamidino 2 phenylindole, 5' nucleotidase, Agarose, Alcohol, Arpe-19 cell line, Article, Beta catenin, Beta tubulin, Bone-marrow-cells, Bromophenol blue, Buffer, Calcium cell level, Calcium phosphate, Calretinin, Canonical wnt signaling, Cd34 antigen, Cell culture, Cell fusion, Cell viability, Coculture, Complementary dna, Confocal microscopy, Cornea transplantation, Cryopreservation, Cryoprotection, Crystal structure, Current clamp technique, Dimethyl sulfoxide, Dodecyl sulfate sodium, Edetic acid, Electrophysiology, Endoglin, Fetal bovine serum, Fibroblast growth factor 2, Flow cytometry, Fluorescence activated cell sorting, Fluorescence intensity, Glyceraldehyde 3 phosphate dehydrogenase, Glycerol, Glycine, Hoe 33342, Immunofluorescence, Immunohistochemistry, Incubation time, Interleukin 1beta, Lentivirus vector, Matrigel, Mercaptoethanol, Microinjection, Mueller cell, Müller glia, N methyl dextro aspartic acid, Nerve cell differentiation, Neural differentiation, Nitrogen, Nonhuman, Organoids, Paraffin, Paraffin embedding, Paraformaldehyde, Patch clamp technique, Penicillin derivative, Phenolsulfonphthalein, Phenotype, Phosphate buffered saline, Phosphoprotein phosphatase inhibitor, Polyacrylamide gel electrophoresis, Potassium chloride, Povidone iodine, Promoter region, Proteinase inhibitor, Real time polymerase chain reaction, Receptor type tyrosine protein phosphatase c, Restriction endonuclease, Retina, Retina dystrophy, Retina regeneration, Retinol, Rhodopsin, Rna extraction, Stem cell, Stem cells, Subcutaneous fat, Tunel assay, Visual impairment, Western blotting

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JTD Keywords: cancer, cells, cellular basis, delivery, encapsulation, in-vitro, inflammation, macrophage, methotrexate, pathogenesis, polymersome, polymersomes, synoviocyte, Arthritis, Rheumatoid-arthritis

The preparation, characterization, and controlled release of hydroxyapatite (HAp) nanopar-ticles loaded with streptomycin (STR) was studied. These nanoparticles are highly appropriate for the treatment of bacterial infections and are also promising for the treatment of cancer cells. The analyses involved scanning electron microscopy, dynamic light scattering (DLS) and Z-potential measurements, as well as infrared spectroscopy and X-ray diffraction. Both amorphous (ACP) and crystalline (cHAp) hydroxyapatite nanoparticles were considered since they differ in their release behavior (faster and slower for amorphous and crystalline particles, respectively). The encapsulated nanoparticles were finally incorporated into biodegradable and biocompatible polylactide (PLA) scaf-folds. The STR load was carried out following different pathways during the synthesis/precipitation of the nanoparticles (i.e., nucleation steps) and also by simple adsorption once the nanoparticles were formed. The loaded nanoparticles were biocompatible according to the study of the cytotoxicity of extracts using different cell lines. FTIR microspectroscopy was also employed to evaluate the cytotoxic effect on cancer cell lines of nanoparticles internalized by endocytosis. The results were promising when amorphous nanoparticles were employed. The nanoparticles loaded with STR increased their size and changed their superficial negative charge to positive. The nanoparticles’ crystallinity decreased, with the consequence that their crystal sizes reduced, when STR was incorporated into their structure. STR maintained its antibacterial activity, although it was reduced during the adsorption into the nanoparticles formed. The STR release was faster from the amorphous ACP nanoparticles and slower from the crystalline cHAp nanoparticles. However, in both cases, the STR release was slower when incorporated in calcium and phosphate during the synthesis. The biocompatibility of these nanoparticles was assayed by two approximations. When extracts from the nanoparticles were evaluated in cultures of cell lines, no cytotoxic damage was observed at concen-trations of less than 10 mg/mL. This demonstrated their biocompatibility. Another experiment using FTIR microspectroscopy evaluated the cytotoxic effect of nanoparticles internalized by endocytosis in cancer cells. The results demonstrated slight damage to the biomacromolecules when the cells were treated with ACP nanoparticles. Both ACP and cHAp nanoparticles were efficiently encapsulated in PLA electrospun matrices, providing functionality and bioactive properties. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: antibiotics, antimicrobial activity, behavior, cytotoxicity, delivery, drug, drug delivery, hydroxyapatite nanoparticles, in-vitro, mechanisms, mitochondria, polylactide, release, streptomycin, Antimicrobial activity, Cancer stem-cells, Cytotoxicity, Drug delivery, Hydroxyapatite nanoparticles, Polylactide, Streptomycin

With the currently available materials and technologies it is difficult to mimic the mechanical properties of soft living tissues. Additionally, another significant problem is the lack of information about the mechanical properties of these tissues. Alternatively, the use of phantoms offers a promising solution to simulate biological bodies. For this reason, to advance in the state-of-the-art a wide range of organs (e.g., liver, heart, kidney as well as brain) and hydrogels (e.g., agarose, polyvinyl alcohol –PVA–, Phytagel –PHY– and methacrylate gelatine –GelMA–) were tested regarding their mechanical properties. For that, viscoelastic behavior, hardness, as well as a non-linear elastic mechanical response were measured. It was seen that there was a significant difference among the results for the different mentioned soft tissues. Some of them appear to be more elastic than viscous as well as being softer or harder. With all this information in mind, a correlation between the mechanical properties of the organs and the different materials was performed. The next conclusions were drawn: (1) to mimic the liver, the best material is 1% wt agarose; (2) to mimic the heart, the best material is 2% wt agarose; (3) to mimic the kidney, the best material is 4% wt GelMA; and (4) to mimic the brain, the best materials are 4% wt GelMA and 1% wt agarose. Neither PVA nor PHY was selected to mimic any of the studied tissues. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: brain, composite hydrogel, dynamic mechanical analysis, elastography, hardness, hydrogels, in-vitro, liver, materials, mechanical-properties, mimicking, soft tissues, tissue scaffolding, viscoelasticity, warner-braztler shear test, warner–braztler shear test, Dynamic mechanical analysis, Hardness, Hydrogels, Materials, Mimicking, Soft tissues, Tissue scaffolding, Viscoelastic characterization, Viscoelasticity, Warner–braztler shear test

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

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (β-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic β-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of β-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to β-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significance: Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered β-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to β-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.

JTD Keywords: beta-tricalcium phosphate, bone regeneration, calcium deficient hydroxyapatite, differentiation, engraftment, human bone marrow mesenchymal stromal cells, hydroxyapatite scaffolds, in-vitro, inhibition, osteogenesis, osteoinduction, stem-cells, surface-topography, tissue, Animals, Beta-tricalcium phosphate, Biomimetics, Bone regeneration, Calcium deficient hydroxyapatite, Calcium phosphate, Calcium phosphates, Cell differentiation, Engraftment, Human bone marrow mesenchymal stromal cells, Humans, Mesenchymal stem cells, Mice, Mice, nude, Osteogenesis, Tissue scaffolds

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient’s immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and β-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

JTD Keywords: expression, in-vitro, mouse model, muscle, mutations, phenotype, quantification, sarcolemma, therapy, 3' untranslated regions, Cells, cultured, Crispr-cas systems, Cytoskeletal proteins, Drug discovery, Dystroglycans, Dystrophin, Gene editing, Hek293 cells, Humans, Muscular dystrophy, duchenne, Myoblasts, Myogenic regulatory factor 5, Primary cell culture, Sarcoglycans, Utrophin, Utrophin up-regulation

A plethora of applications using polysaccharides have been developed in recent years due to their availability as well as their frequent nontoxicity and biodegradability. These polymers are usually obtained from renewable sources or are byproducts of industrial processes, thus, their use is collaborative in waste management and shows promise for an enhanced sustainable circular economy. Regarding the development of novel delivery systems for biotherapeutics, the potential of polysaccharides is attractive for the previously mentioned properties and also for the possibility of chemical modification of their structures, their ability to form matrixes of diverse architectures and mechanical properties, as well as for their ability to maintain bioactivity following incorporation of the biomolecules into the matrix. Biotherapeutics, such as proteins, growth factors, gene vectors, enzymes, hormones, DNA/RNA, and antibodies are currently in use as major therapeutics in a wide range of pathologies. In the present review, we summarize recent progress in the development of polysaccharide-based hydrogels of diverse nature, alone or in combination with other polymers or drug delivery systems, which have been implemented in the delivery of biotherapeutics in the pharmaceutical and biomedical fields. © 2021 American Chemical Society.

JTD Keywords: biodegradable dextran hydrogels, biotherapeutics, bone morphogenetic protein-2, carrageenan-based hydrogels, chitosan-based hydrogels, controlled delivery, controlled-release, cross-linked hydrogels, growth-factor delivery, hydrogels, in-vitro characterization, polysaccharides, self-healing hydrogel, stimuli-responsiveness, tissue engineering, Antibodies, Bioactivity, Biodegradability, Biomedical fields, Biomolecules, Biotherapeutics, Chemical modification, Circular economy, Controlled delivery, Controlled drug delivery, Delivery systems, Drug delivery system, Functional polymers, Hyaluronic-acid hydrogels, Hydrogels, Industrial processs, Polysaccharides, Recent progress, Renewable sources, Stimuli-responsiveness, Targeted drug delivery, Tissue engineering, Waste management

The generation of human mini-organs, the so-called organoids, is one of the biggest scientific advances in regenerative medicine. This technology exploits traditional three-dimensional culture techniques that support cell-autonomous self-organization responses of stem cells to derive micrometer to millimeter size versions of human organs. The convergence of the organoid technology with organ transplantation is still in its infancy but this alliance is expected to open new venues to change the way we conduct both transplant and organoid research. In this Forum we provide a summary on early achievements facilitating organoid derivation and culture. We further discuss on early advances of organoid transplantation also offering a comprehensive overview of current limitations and challenges to instruct organoid maturation. We expect that this Forum sets the ground for initial discussions between stem cell biologists, bioengineers, and the transplant community to better direct organoid basic research to advance the organ transplantation field.

JTD Keywords: in-vitro, matrix, mice, organoids, regenerative medicine, vivo, Intestinal stem-cell, Organoids, Regenerative medicine

Atmospheric pressure plasma jets have been shown to impact several cancer cell lines, both in vitro and in vivo. These effects are based on the biochemistry of the reactive oxygen and nitrogen species generated by plasmas in physiological liquids, referred to as plasma-conditioned liquids. Plasma-conditioned media are efficient in the generation of reactive species, inducing selective cancer cell death. However, the concentration of reactive species generated by plasma in the cell culture media of different cell types can be highly variable, complicating the ability to draw precise conclusions due to the differential sensitivity of different cells to reactive species. Here, we compared the effects of direct and indirect plasma treatment on non-malignant bone cells (hOBs and hMSCs) and bone cancer cells (SaOs-2s and MG63s) by treating the cells directly or exposing them to previously treated cell culture medium. Biological effects were correlated with the concentrations of reactive species generated in the liquid. A linear increase in reactive species in the cell culture medium was observed with increased plasma treatment time independent of the volume treated. Values up to 700 µM for H2O2 and 140 µM of NO2− were attained in 2 mL after 15 min of plasma treatment in AdvDMEM cell culture media. Selectivity towards bone cancer cells was observed after both direct and indirect plasma treatments, leading to a decrease in bone cancer cell viability at 72 h to 30% for the longest plasma treatment times while maintaining the survival of non-malignant cells. Therefore, plasma-conditioned media may represent the basis for a potentially novel non-invasive technique for bone cancer therapy.

JTD Keywords: expression, in-vitro, jet, mechanisms, nitrate, nitrite, osteosarcoma cells, reactive oxygen, Cold atmospheric plasma

Biomimetic calcium-deficient hydroxyapatite (CDHA) as a bioactive material exhibits exceptional intrinsic osteoinductive and osteogenic properties because of its nanostructure and composition, which promote a favorable microenvironment. Its high reactivity has been hypothesized to play a relevant role in the in vivo performance, mediated by the interaction with the biological fluids, which is amplified by its high specific surface area. Paradoxically, this high reactivity is also behind the in vitro cytotoxicity of this material, especially pro-nounced in static conditions. The present work explores the structural and physicochemical changes that CDHA undergoes in contact with physiological fluids and to investigate its interaction with proteins. Calcium-deficient hydroxyapatite discs with different micro/nanostructures, coarse (C) and fine (F), were exposed to cell-free complete culture medium over extended periods of time: 1, 7, 14, 21, 28, and 50 days. Precipitate formation was not observed in any of the materials in contact with the physiological fluid, which would indicate that the ionic exchanges were linked to incorporation into the crystal structure of CDHA or in the hydrated layer. In fact, CDHA experienced a maturation process, with a progressive increase in crystallinity and the Ca/P ratio, accompanied by an uptake of Mg and a B-type carbonation process, with a gradual propagation into the core of the samples. However, the reactivity of biomimetic hydroxyapatite was highly dependent on the specific surface area and was amplified in nanosized needle-like crystal structures (F), whereas in coarse specimens the ionic exchanges were restricted to the surface, with low penetration in the material bulk. In addition to showing a higher protein adsorption on F substrates, the proteomics study revealed the existence of protein selectivity to-ward F or C microstructures, as well as the capability of CDHA, and more remarkably of F-CDHA, to concentrate specific proteins from the culture medium. Finally, a substantial improvement in the material's ability to support cell proliferation was observed after the CDHA maturation process.

JTD Keywords: calcium phosphates, ion exchange, nanostructure, protein adsorption, Biological-systems, Biomaterials, Biomimetic hydroxyapatites, Biomimetics, Bone-formation, Calcium deficient hydroxyapatite, Calcium phosphate, Calcium phosphates, Cell proliferation, Crystal structure, Crystallinity, Crystals structures, Culture medium, Growth, High reactivity, Hydroxyapatite, In-vitro, Ion exchange, Ionic exchange, Molecular biology, Nanocrystalline apatites, Nanostructure, Nanostructures, Octacalcium phosphate, Physicochemical studies, Physiological fluids, Physiology, Protein adsorption, Proteins, Proteomic studies, Raman spectroscopy, Serum-albumin, Specific surface area

Currently, three major circumstances threaten the management of bacterial infections: increasing antimicrobial resistance, expansion of chronic biofilm-associated infections, and lack of an appropriate approach to treat them. To date, the development of accelerated drug susceptibility testing of biofilms and of new antibiofouling systems has not been achieved despite the availability of different methodologies. There is a need for easy-to-use methods of testing the antibiotic susceptibility of bacteria that form biofilms and for screening new possible antibiofilm strategies. Herein, we present a microfluidic platform with an integrated interdigitated sensor (BiofilmChip). This new device allows an irreversible and homogeneous attachment of bacterial cells of clinical origin, even directly from clinical specimens, and the biofilms grown can be monitored by confocal microscopy or electrical impedance spectroscopy. The device proved to be suitable to study polymicrobial communities, as well as to measure the effect of antimicrobials on biofilms without introducing disturbances due to manipulation, thus better mimicking real-life clinical situations. Our results demonstrate that BiofilmChip is a straightforward tool for antimicrobial biofilm susceptibility testing that could be easily implemented in routine clinical laboratories.

JTD Keywords: cells, model, resistance, shear, technology, In-vitro

Pseudomonas aeruginosa biofilms and the capacity of the bacterium to coexist and interact with a broad range of microorganisms have a substantial clinical impact. This review focuses on the main traits of P. aeruginosa biofilms, such as the structural composition and regulatory networks involved, placing particular emphasis on the clinical challenges they represent in terms of antimicrobial susceptibility and biofilm infection clearance. Furthermore, the ability of P. aeruginosa to grow together with other microorganisms is a significant pathogenic attribute with clinical relevance; hence, the main microbial interactions of Pseudomonas are especially highlighted and detailed throughout this review. This article also explores the infections caused by single and polymicrobial biofilms of P. aeruginosa and the current models used to recreate them under laboratory conditions. Finally, the antimicrobial and antibiofilm strategies developed against P. aeruginosa mono and multispecies biofilms are detailed at the end of this review.

JTD Keywords: aeruginosa models, antibiotic-resistance, antimicrobials, bacterial biofilms, biofilms, c-di-gmp, chronic infections, enterococcus-faecalis, extracellular dna, in-vitro, lectin pa-iil, p, p. aeruginosa models, polymicrobial, polymicrobial interactions, staphylococcus-aureus, Antimicrobials, Biofilms, Chronic infections, P. aeruginosa models, Polymicrobial, Pseudomonas aeruginosa, Urinary-tract-infection

For decades, fetal bovine serum (FBS) has been used routinely for culturing many cell types, based on its empirically demonstrated effects on cell growth, and the lack of suitable non-xenogeneic alternatives. The FBS-based culture media do not represent the human physiological conditions, and can compromise biomimicry of preclinical models. To recapitulate in vitro the features of human bone and bone cancer, we investigated the effects of human serum and human platelet lysate on modeling osteogenesis, osteoclastogenesis, and bone cancer in two-dimensional (2D) and three-dimensional (3D) settings. For monitoring tumor growth within tissue-engineered bone in a non-destructive fashion, we generated cancer cell lines expressing and secreting luciferase. Culture media containing human serum enhanced osteogenesis and osteoclasts differentiation, and provided a more realistic in vitro mimic of human cancer cell proliferation. When human serum was used for building 3D engineered bone, the tissue recapitulated bone homeostasis and response to bisphosphonates observed in native bone. We found disparities in cell behavior and drug responses between the metastatic and primary cancer cells cultured in the bone niche, with the effectiveness of bisphosphonates observed only in metastatic models. Overall, these data support the utility of human serum for bioengineering of bone and bone cancers.

JTD Keywords: 3d cancer models, 3rs, alpha tnf-alpha, culture, cypridina luciferase, ewings-sarcoma, ewing’s sarcoma, human platelet lysate, human serum, human tumor, in-vitro, osteogenic differentiation, stem-cells, zoledronic acid, 3d cancer models, 3rs, Cypridina luciferase, Ewing's sarcoma, Ewing’s sarcoma, Fetal bovine serum, Human serum

Mesenchymal stromal cell (MSC)-based cell therapy in acute respiratory diseases is based on MSC secretion of paracrine factors. Several strategies have proposed to improve this are being explored including pre-conditioning the MSCs prior to administration. We here propose a strategy for improving the therapeutic efficacy of MSCs based on cell preconditioning by growing them in native extracellular matrix (ECM) derived from the lung. To this end, a bioink with tunable stiffness based on decellularized porcine lung ECM hydrogels was developed and characterized. The bioink was suitable for 3D culturing of lung-resident MSCs without the need for additional chemical or physical crosslinking. MSCs showed good viability, and contraction assays showed the existence of cell–matrix interactions in the bioprinted scaffolds. Adhesion capacity and length of the focal adhesions formed were increased for the cells cultured within the lung hydrogel scaffolds. Also, there was more than a 20-fold increase of the expression of the CXCR4 receptor in the 3D-cultured cells compared to the cells cultured in plastic. Secretion of cytokines when cultured in an in vitro model of lung injury showed a decreased secretion of pro-inflammatory mediators for the cells cultured in the 3D scaffolds. Moreover, the morphology of the harvested cells was markedly different with respect to conventionally (2D) cultured MSCs. In conclusion, the developed bioink can be used to bioprint structures aimed to improve preconditioning MSCs for therapeutic purposes.

JTD Keywords: 3d bioprinting, acute lung injury, adhesion, collagen, differentiation, dimension, elastic properties, extracellular matrix, hydrogels, in-vitro, mechanical-properties, mesenchymal stromal cells, microenvironment, potentiate, tissue engineering, 3d bioprinting, Acute lung injury, Extracellular matrix, Hydrogels, Mesenchymal stromal cells, Stem-cells, Tissue engineering

Amphiphilic block co-polymer nanoparticles are interesting candidates for drug delivery as a result of their unique properties such as the size, modularity, biocompatibility and drug loading capacity. They can be rapidly formulated in a nanoprecipitation process based on self-assembly, resulting in kinetically locked nanostructures. The control over this step allows us to obtain nanoparticles with tailor-made properties without modification of the co-polymer building blocks. Furthermore, a reproducible and controlled formulation supports better predictability of a batch effectiveness in preclinical tests. Herein, we compared the formulation of PLGA-PEG nanoparticles using the typical manual bulk mixing and a microfluidic chip-assisted nanoprecipitation. The particle size tunability and controllability in a hydrodynamic flow focusing device was demonstrated to be greater than in the manual dropwise addition method. We also analyzed particle size and encapsulation of fluorescent compounds, using the common bulk analysis and advanced microscopy techniques: Transmission Electron Microscopy and Total Internal Reflection Microscopy, to reveal the heterogeneities occurred in the formulated nanoparticles. Finally, we performed in vitro evaluation of obtained NPs using MCF-7 cell line. Our results show how the microfluidic formulation improves the fine control over the resulting nanoparticles, without compromising any appealing property of PLGA nanoparticle. The combination of microfluidic formulation with advanced analysis methods, looking at the single particle level, can improve the understanding of the NP properties, heterogeneities and performance.

JTD Keywords: controlled-release, doxorubicin, encapsulation, functional nanoparticles, nanoprecipitation, pharmacokinetics, polymeric nanoparticles, shape, surface-chemistry, Breast neoplasms, Drug carriers, Drug delivery systems, Female, Humans, In-vitro, Mcf-7 cells, Microfluidics, Nanoparticles, Polyesters, Polyethylene glycol-poly(lactide-co-glycolide), Polyethylene glycols, Polymers

BACKGROUND: The embryo implantation process is crucial for the correct establishment and progress of pregnancy. During implantation, the blastocyst trophectoderm cells attach to the epithelium of the endometrium, triggering intense cell-to-cell crosstalk that leads to trophoblast outgrowth, invasion of the endometrial tissue, and formation of the placenta. However, this process, which is vital for embryo and foetal development in utero, is still elusive to experimentation because of its inaccessibility. Experimental implantation is cumbersome and impractical in adult animal models and is inconceivable in humans. OBJECTIVE AND RATIONALE: A number of custom experimental solutions have been proposed to recreate different stages of the implantation process in vitro, by combining a human embryo (or a human embryo surrogate) and endometrial cells (or a surrogate for the endometrial tissue). In vitro models allow rapid high-throughput interrogation of embryos and cells, and efficient screening of molecules, such as cytokines, drugs, or transcription factors, that control embryo implantation and the receptivity of the endometrium. However, the broad selection of available in vitro systems makes it complicated to decide which system best fits the needs of a specific experiment or scientific question. To orient the reader, this review will explore the experimental options proposed in the literature, and classify them into amenable categories based on the embryo/cell pairs employed. The goal is to give an overview of the tools available to study the complex process of human embryo implantation, and explain the differences between them, including the advantages and disadvantages of each system. SEARCH METHODS: We performed a comprehensive review of the literature to come up with different categories that mimic the different stages of embryo implantation in vitro, ranging from initial blastocyst apposition to later stages of trophoblast invasion or gastrulation. We will also review recent breakthrough advances on stem cells and organoids, assembling embryo-like structures and endometrial tissues. OUTCOMES: We highlight the most relevant systems and describe the most significant experiments. We focus on in vitro systems that have contributed to the study of human reproduction by discovering molecules that control implantation, including hormones, signalling molecules, transcription factors and cytokines. WIDER IMPLICATIONS: The momentum of this field is growing thanks to the use of stem cells to build embryo-like structures and endometrial tissues, and the use of bioengineering to extend the life of embryos in culture. We propose to merge bioengineering methods derived from the fields of stem cells and reproduction to develop new systems covering a wider window of the implantation process.

JTD Keywords: in vitro models, blastocyst, blastocyst-like structures, early-pregnancy, endometrial cells, epidermal-growth-factor, gene-expression, implantation, in vitro models, in-vitro model, indian hedgehog, organoids, receptivity, self-organization, spheroids, trophoblast, trophoblast invasion, uterine receptivity, Blastocyst, Blastocyst-like structures, Early-pregnancy, Endometrial cells, Endometrial stromal cells, Epidermal-growth-factor, Gene-expression, Implantation, In vitro models, In-vitro model, Indian hedgehog, Organoids, Receptivity, Self-organization, Spheroids, Trophoblast, Trophoblast invasion, Uterine receptivity

Significance: The incidence of chronic wounds is increasing due to our aging population and the augment of people afflicted with diabetes. With the extended knowledge on the biological mechanisms underlying these diseases, there is a novel influx of medical technologies into the conventional wound care market. Recent Advances: Several nanotechnologies have been developed demonstrating unique characteristics that address specific problems related to wound repair mechanisms. In this review, we focus on the most recently developed nanotechnology-based therapeutic agents and evaluate the efficacy of each treatment in in vivo diabetic models of chronic wound healing. Critical Issues: Despite the development of potential biomaterials and nanotechnology-based applications for wound healing, this scientific knowledge is not translated into an increase of commercially available wound healing products containing nanomaterials. Future Directions: Further studies are critical to provide insights into how scientific evidences from nanotechnology-based therapies can be applied in the clinical setting.

JTD Keywords: chronic, diabetes, liposomes, nanofibers, nanoparticles, Chronic, Chronic wound, Diabetes, Diabetic wound, Diabetic-rats, Dressings, Drug mechanism, Extracellular-matrix, Growth-factor, Human, In-vitro, Liposome, Liposomes, Mesenchymal stem-cells, Metal nanoparticle, Nanofiber, Nanofibers, Nanofibrous scaffolds, Nanoparticles, Nanotechnology, Nonhuman, Polyester, Polymer, Polysaccharide, Priority journal, Protein, Review, Self assembled protein nanoparticle, Silk fibroin, Skin wounds, Wound healing, Wound healing promoting agent

In recent years considerable progress has been made in the development of faithful procedures for the differentiation of human pluripotent stem cells (hPSCs). An important step in this direction has also been the derivation of organoids. This technology generally relies on traditional three-dimensional culture techniques that exploit cell-autonomous self-organization responses of hPSCs with minimal control over the external inputs supplied to the system. The convergence of stem cell biology and bioengineering offers the possibility to provide these stimuli in a controlled fashion, resulting in the development of naturally inspired approaches to overcome major limitations of this nascent technology. Based on the current developments, we emphasize the achievements and ongoing challenges of bringing together hPSC organoid differentiation, bioengineering and ethics. This Review underlines the need for providing engineering solutions to gain control of self-organization and functionality of hPSC-derived organoids. We expect that this knowledge will guide the community to generate higher-grade hPSC-derived organoids for further applications in developmental biology, drug screening, disease modelling and personalized medicine. This Review provides an overview of bioengineering technologies that can be harnessed to facilitate the culture, self-organization and functionality of human pluripotent stem cell-derived organoids.

JTD Keywords: Differentiation, Embryonic-tissues, Extracellular-matrix, In-vitro, Kidney organoids, Model, Neural-tube, Pluripotent stem-cells, Reconstitution, Self-organization

© 2020 American Chemical Society. The performance of supramolecular nanocarriers as drug delivery systems depends on their stability in the complex and dynamic biological media. After administration, nanocarriers are challenged by physiological barriers such as shear stress and proteins present in blood, endothelial wall, extracellular matrix, and eventually cancer cell membrane. While early disassembly will result in a premature drug release, extreme stability of the nanocarriers can lead to poor drug release and low efficiency. Therefore, comprehensive understanding of the stability and assembly state of supramolecular carriers in each stage of delivery is the key factor for the rational design of these systems. One of the main challenges is that current 2D in vitro models do not provide exhaustive information, as they fail to recapitulate the 3D tumor microenvironment. This deficiency in the 2D model complexity is the main reason for the differences observed in vivo when testing the performance of supramolecular nanocarriers. Herein, we present a real-time monitoring study of self-assembled micelles stability and extravasation, combining spectral confocal microscopy and a microfluidic cancer-on-a-chip. The combination of advanced imaging and a reliable 3D model allows tracking of micelle disassembly by following the spectral properties of the amphiphiles in space and time during the crucial steps of drug delivery. The spectrally active micelles were introduced under flow and their position and conformation continuously followed by spectral imaging during the crossing of barriers, revealing the interplay between carrier structure, micellar stability, and extravasation. Integrating the ability of the micelles to change their fluorescent properties when disassembled, spectral confocal imaging and 3D microfluidic tumor blood vessel-on-a-chip resulted in the establishment of a robust testing platform suitable for real-time imaging and evaluation of supramolecular drug delivery carrier's stability.

JTD Keywords: cancer-on-a-chip, complex, delivery, endothelial-cells, in-vitro, microfluidic, model, nanoparticle, penetration, shear-stress, stability, supramolecular, Cancer-on-a-chip, Cell-culture, Micelle, Microfluidic, Nanoparticle, Stability, Supramolecular

Movement of skeletal-muscle fibers is generated by the coordinated action of several cells taking part within the locomotion circuit (motoneurons, sensory-neurons, Schwann cells, astrocytes, microglia, and muscle-cells). Failures in any part of this circuit could impede or hinder coordinated muscle movement and cause a neuromuscular disease (NMD) or determine its severity. Studying fragments of the circuit cannot provide a comprehensive and complete view of the pathological process. We trace the historic developments of studies focused on in-vitro modeling of the spinal-locomotion circuit and how bioengineered innovative technologies show advantages for an accurate mimicking of physiological conditions of spinal-locomotion circuit. New developments on compartmentalized microfluidic culture systems (cμFCS), the use of human induced pluripotent stem cells (hiPSCs) and 3D cell-cultures are analyzed. We finally address limitations of current study models and three main challenges on neuromuscular studies: (i) mimic the whole spinal-locomotion circuit including all cell-types involved and the evaluation of independent and interdependent roles of each one; (ii) mimic the neurodegenerative response of mature neurons in-vitro as it occurs in-vivo; and (iii) develop, tune, implement, and combine cμFCS, hiPSC, and 3D-culture technologies to ultimately create patient-specific complete, translational, and reliable NMD in-vitro model. Overcoming these challenges would significantly facilitate understanding the events taking place in NMDs and accelerate the process of finding new therapies.

JTD Keywords: 3D-culture, Compartmentalized microfluidic culture systems (cμFCS), HiPSC, In-vitro models, Neuromuscular circuit

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

We present a method for using long-term organotypic slice co-cultures of the entorhino-hippocampal formation to analyze the axon-regenerative properties of a determined compound. The culture method is based on the membrane interphase method, which is easy to perform and is generally reproducible. The degree of axonal regeneration after treatment in lesioned cultures can be seen directly using green fluorescent protein (GFP) transgenic mice or by axon tracing and histological methods. Possible changes in cell morphology after pharmacological treatment can be determined easily by focal in vitro electroporation. The well-preserved cytoarchitectonics in the co-culture facilitate the analysis of identified cells or regenerating axons. The protocol takes up to a month.

JTD Keywords: Cajal-retzius cells, Green-fluorescent-protein, In-vitro model, Rat hippocampus, Nervous-tissue, Brain-slices, Dentate gyrus, Gene-transfer, Cultures, Damage

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid beta peptide (A beta) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, A beta(1-42). Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant beta-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for A beta(1-42) fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different A beta(1-42) species on Ca2+ homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca2+ that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca2+ increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.

JTD Keywords: AFM, Alzheimers-disease, Chondroitin sulfate, Heparan-sulfate, Lipid-bilayers, Beta-peptide, In-vitro, Neurodegenerative diseases, Extracellular-matrix, Prion protein