LSP1-myosin1e bimolecular complex regulates focal adhesion dynamics and cell migration.

Several cytoskeleton-associated proteins and signaling pathways work in concert to regulate actin cytoskeleton remodeling, cell adhesion, and migration. Although the leukocyte-specific protein 1 (LSP1) has been shown to interact with the actin cytoskeleton, its function in the regulation of actin cytoskeleton dynamics is, as yet, not fully understood. We have recently demonstrated that the bimolecular complex between LSP1 and myosin1e controls actin cytoskeleton remodeling during phagocytosis. In this study, we show that LSP1 downregulation severely impairs cell migration, lamellipodia formation, and focal adhesion dynamics in macrophages. Inhibition of the interaction between LSP1 and myosin1e also impairs these processes resulting in poorly motile cells, which are characterized by few and small lamellipodia. Furthermore, cells in which LSP1-myosin1e interaction is inhibited are typically associated with inefficient focal adhesion turnover. Collectively, our findings show that the LSP1-myosin1e bimolecular complex plays a pivotal role in the regulation of actin cytoskeleton remodeling and focal adhesion dynamics required for cell migration.

Engineering the Acoustic Response and Drug Loading Capacity of PBCA-Based Polymeric Microbubbles with Surfactants.

Gas-filled microbubbles (MB) are routinely used in the clinic as ultrasound contrast agents. MB are also increasingly explored as drug delivery vehicles based on their ultrasound stimuli-responsiveness and well-established shell functionalization routes. Broadening the range of MB properties can enhance their performance in both imaging and drug delivery applications. This can be promoted by systematically varying the reagents used in the synthesis of MB, which in the case of polymeric MB include surfactants. We therefore set out to study the effect of key surfactant characteristics, such as the chemical structure, molecular weight, and hydrophilic-lipophilic balance on the formation of poly(butyl cyanoacrylate) (PBCA) MB, as well as on their properties, including shell thickness, drug loading capacity, ultrasound contrast, and acoustic stability. Two different surfactant families (i.e., Triton X and Tween) were employed, which show opposite molecular weight vs hydrophilic-lipophilic balance trends. For both surfactant types, we found that the shell thickness of PBCA MB increased with higher-molecular-weight surfactants and that the resulting MB with thicker shells showed higher drug loading capacities and acoustic stability. Furthermore, the higher proportion of smaller polymer chains of the Triton X-based MB (as compared to those of the Tween-based ones) resulted in lower polymer entanglement, improving drug loading capacity and ultrasound contrast response. These findings open up new avenues to fine-tune the shell properties of polymer-based MB for enhanced ultrasound imaging and drug delivery applications.

Bone resorption and body reorganization during maturation induce maternal transfer of toxic metals in anguillid eels.

During their once-in-a-lifetime transoceanic spawning migration, anguillid eels do not feed, instead rely on energy stores to fuel the demands of locomotion and reproduction while they reorganize their bodies by depleting body reserves and building up gonadal tissue. Here we show how the European eel (Anguilla anguilla) breaks down its skeleton to redistribute phosphorus and calcium from hard to soft tissues during its sexual development. Using multiple analytical and imaging techniques, we characterize the spatial and temporal degradation of the skeletal framework from initial to final gonadal maturation and use elemental mass ratios in bone, muscle, liver, and gonadal tissue to determine the fluxes and fates of selected minerals and metals in the eels' bodies. We find that bone loss is more pronounced in females than in males and eventually may reach a point at which the mechanical stability of the skeleton is challenged. P and Ca are released and translocated from skeletal tissues to muscle and gonads, leaving both elements in constant proportion in remaining bone structures. The depletion of internal stores from hard and soft tissues during maturation-induced body reorganization is accompanied by the recirculation, translocation, and maternal transfer of potentially toxic metals from bone and muscle to the ovaries in gravid females, which may have direct deleterious effects on health and hinder the reproductive success of individuals of this critically endangered species.

Cryopreservation of amniotic membrane with and without glycerol additive.

PURPOSE: Amniotic membrane (AM) is an essential tool in ocular surface reconstruction. In this study, we analyzed the differential effects of glycerol and straight storage at - 80 °C for up to 6 months on the structural, biological, and mechanical properties of amniotic membrane (AM). METHODS: Human placentae of 11 different subjects were analyzed. AMs were stored at - 80 °C, either with a 1:1 mixture of Dulbecco's modified Eagle medium and glycerol (glycerol) or without any medium or additives (straight). Histological image analysis, tensile strength, cell viability, and basic fibroblast growth factor (bFGF) secretion were evaluated at 0.5, 1, 3, and 6 months. RESULTS: Histologically, neither glycerol nor straight storage significantly altered the epithelial or stromal structure of the AM. However, the cell number of the stroma was significantly reduced during the freezing process, independently of the storage method (p = 0.05-0.001). Tensile strength and Young's modulus were not influenced by the storage method, but longer storage periods significantly increased the tensile strength of the AMs (p = 0.028). Cell viability was higher in glycerol rather than straight AM samples for up to 3 months of storage (p = 0.047-0.03). Secretion of bFGF at 3 months of storage was significantly higher in glycerol versus straight frozen AM samples (p = 0.04). DISCUSSION: Glycerol led to higher cell viability and higher bFGF secretion for up to 3 months of AM storage. However, no significant differences between the two methods were observed at 6 months of storage at - 80 °C.

Proteome Profiling and Ultrastructural Characterization of the Human RCMH Cell Line: Myoblastic Properties and Suitability for Myopathological Studies.

Studying (neuro)muscular disorders is a major topic in biomedicine with a demand for suitable model systems. Continuous cell culture (in vitro) systems have several technical advantages over in vivo systems and became widely used tools for discovering physiological/pathophysiological mechanisms in muscle. In particular, myoblast cell lines are suitable model systems to study complex biochemical adaptations occurring in skeletal muscle and cellular responses to altered genetic/environmental conditions. Whereas most in vitro studies use extensively characterized murine C2C12 cells, a comprehensive description of an equivalent human cell line, not genetically manipulated for immortalization, is lacking. Therefore, we characterized human immortal myoblastic RCMH cells using scanning (SEM) and transmission electron microscopy (TEM) and proteomics. Among more than 6200 identified proteins we confirm the known expression of proteins important for muscle function. Comparing the RCMH proteome with two well-defined nonskeletal muscle cells lines (HeLa, U2OS) revealed a considerable enrichment of proteins important for muscle function. SEM/TEM confirmed the presence of agglomerates of cytoskeletal components/intermediate filaments and a prominent rough ER. In conclusion, our results indicate RMCH as a suitable in vitro model for investigating muscle function-related processes such as mechanical stress burden and mechanotransduction, EC coupling, cytoskeleton, muscle cell metabolism and development, and (ER-associated) myopathic disorders.

Effects of architecture on the stability of thermosensitive unimolecular micelles.

The influence of architecture on polymer interactions is investigated and differences between branched and linear copolymers are found. A comprehensive picture is drawn with the help of a fluorescence approach (using pyrene and 4HP as probe molecules) together with IR or NMR spectroscopy and X-ray/light scattering measurements. Five key aspects are addressed: (1) synergistic intramolecular complexation within miktoarm stars. The proximity of thermoresponsive poly(propylene oxide) (PPO) and poly(dimethylaminoethyl methacrylate) (PDMAEMA) within a miktoarm star leads to complexation between these weakly interacting partners. Consequently, the original properties of the constituents are lost, showing hydrophobic domains even at low temperatures, at which all homopolymers are water soluble. (2) Unimolecular micelles for miktoarm stars. The star does not exhibit intermolecular self-assembly in a large temperature range, showing unimers up to 55 °C. This behavior was traced back to a reduced interfacial tension between the PPO-PDMAEMA complex and water (PDMAEMA acts as a "microsurfactant"). (3) Unimolecular to multimolecular micelle transition for stars. The otherwise stable unimolecular micelles self-assemble above 55 °C. This aggregation is not driven by PPO segregation, but by collapse of residual PDMAEMA. This leads to micrometer-sized multilamellar vesicles stabilized by poly(ethylene oxide) (PEO). (4) Prevention of pronounced complexation within diblock copolymers. In contrast to the star copolymers, PPO and PDMAEMA adapt rather their homopolymer behavior within the diblock copolymers. Then they show their immanent LCST properties, as PDMAEMA turns insoluble at elevated temperatures, whereas PPO becomes hydrophobic below room temperature. (5) Two-step micellization for diblock copolymers. Upon heating of linear copolymers, the dehydration of PPO is followed by self-assembly into spherical micelles. An intermediate prevalence of unimolecular micelles is revealed in a small temperature window between PPO collapse and self-assembly of PEO-b-PPO. Also for PPO-b-PDMAEMA, PPO segregation prevails after initial weak complexation, leading to micelles with a PPO core. Considerable amounts of water are entrapped within the collapsed PDMAEMA domains above 55 °C (skin effect), preventing PPO-PDMAEMA complexation within precipitating PPO-b-PDMAEMA. Further, collapsed PDMAEMA is rather polar as sensed by pyrene and 4HP. In summary, advanced macromolecular architectures can lead to an unprecedented intramolecular self-assembly behavior, where internal complexation prevents intermolecular aggregation.

Three-dimensional in vitro model of bone metastases of neuroblastoma as a tool for pharmacological evaluations.

In vitro metastatic models are foreseen to introduce a breakthrough in the field of preclinical screening of more functional small-molecule pharmaceuticals and biologics. To achieve this goal, the complexity of current in vitro systems requests an appropriate upgrade to approach the three-dimensional (3D) in vivo metastatic disease. Here, we explored the potential of our 3D ß-tricalcium phosphate (ß-TCP) model of neuroblastoma bone metastasis for drug toxicity assessment. Tailor-made scaffolds with interconnected channels were produced by combining 3D printing and slip casting method. The organization of neuroblastoma cells into a mesenchymal stromal cell (MSC) network, cultured under bioactive conditions provided by ß-TCP, was monitored by two-photon microscopy. Deposition of extracellular matrix protein Collagen I by MSCs and persistent growth of tumor cells confirmed the cell-supportive performance of our 3D model. When different neuroblastoma cells were treated with conventional chemotherapeutics, the ß-TCP model provided the necessary reproducibility and accuracy of experimental readouts. Drug efficacy evaluation was done for 3D and 2D cell cultures, highlighting the need for a higher dose of chemotherapeutics under 3D conditions to achieve the expected cytotoxicity in tumor cells. Our results confirm the importance of 3D geometry in driving native connectivity between nonmalignant and tumor cells and sustain ß-TCP scaffolds as a reliable and affordable drug screening platform for use in the early stages of drug discovery.

Hybrid ultrasound and photoacoustic contrast agent designs combining metal phthalocyanines and PBCA microbubbles.

Photoacoustic (PA) imaging is an emerging diagnostic technology that combines the penetration depth of ultrasound (US) imaging and the contrast resolution of optical imaging. Although PA imaging can visualize several endogenous chromophores to obtain clinically-relevant information, multiple applications require the administration of external contrast agents. Metal phthalocyanines have strong PA properties and chemical stability, but their extreme hydrophobicity requires their encapsulation in delivery systems for biomedical applications. Hence, we developed hybrid US/PA contrast agents by encapsulating metal phthalocyanines in poly(butyl cyanoacrylate) microbubbles (PBCA MB), which display acoustic response and ability to efficiently load hydrophobic drugs. Six different metal chromophores were loaded in PBCA MB, showing greater encapsulation efficiency with higher chromophore hydrophobicity. Notably, while the US response of the MB was unaffected by the loading of the chromophores, the PA characteristics varied greatly. Among the different formulations, MB loaded with zinc and cobalt naphthalocyanines showed the strongest PA contrast, as a result of high encapsulation efficiencies and tunable optical properties. The strong US and PA contrast signals of the formulations were preserved in biological environment, as demonstrated by in vitro imaging in serum and whole blood, and ex vivo imaging in deceased mice. Taken together, these findings highlight the advantages of combining highly hydrophobic PA contrast agents and polymeric MB for the development of contrast agents for hybrid US/PA imaging, where different types of information (structural, functional, or potentially molecular) can be acquired by combining both imaging modalities.

Fibrin-Based Hydrogels with Reactive Amphiphilic Copolymers for Mechanical Adjustments Allow for Capillary Formation in 2D and 3D Environments.

This study focuses on enhancing controllable fibrin-based hydrogels for tissue engineering, addressing existing weaknesses. By integrating a novel copolymer, we improved the foundation for cell-based angiogenesis with adaptable structural features. Tissue engineering often faces challenges like waste disposal and nutrient supply beyond the 200 µm diffusion limit. Angiogenesis breaks through this limitation, allowing the construction of larger constructs. Our innovative scaffold combination significantly boosts angiogenesis, resulting in longer branches and more capillary network junctions. The copolymer attached to fibrin fibers enables precise adjustment of hydrogel mechanical dynamic properties for specific applications. Our material proves effective for angiogenesis, even under suppression factors like suramin. In our study, we prepared fibrin-based hydrogels with and without the copolymer PVP12400-co-GMA10mol%. Using a co-culture system of human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), we analyzed angiogenetic behavior on and within the modified hydrogels. Capillary-like structures were reproducibly formed on different surfaces, demonstrating the general feasibility of three-dimensional endothelial cell networks in fibrin-based hydrogels. This highlights the biomaterial's suitability for in vitro pre-vascularization of biohybrid implants.

Impact of Fibrin Gel Architecture on Hepatocyte Growth Factor Release and Its Role in Modulating Cell Behavior for Tissue Regeneration.

A novel scaffold design has been created to enhance tissue engineering and regenerative medicine by optimizing the controlled, prolonged release of Hepatocyte Growth Factor (HGF), a powerful chemoattractant for endogenous mesenchymal stem cells. We present a new stacked scaffold that is made up of three different fibrin gel layers, each of which has HGF integrated into the matrix. The design attempts to preserve HGF's regenerative properties for long periods of time, which is necessary for complex tissue regeneration. These multi-layered fibrin gels have been mechanically evaluated using rheometry, and their degradation behavior has been studied using D-Dimer ELISA. Understanding the kinetics of HGF release from this novel scaffold configuration is essential for understanding HGF's long-term sustained bioactivity. A range of cell-based tests were carried out to verify the functionality of HGF following extended incorporation. These tests included 2-photon microscopy using phalloidin staining to examine cellular morphology, SEM analysis for scaffold-cell interactions, and scratch and scatter assays to assess migration and motility. The analyses show that the novel stacking scaffold promotes vital cellular processes for tissue regeneration in addition to supporting HGF's bioactivity. This scaffold design was developed for in situ tissue engineering. Using the body as a bioreactor, the scaffold should recruit mesenchymal stem cells from their niche, thus combining the regenerative abilities of HGF and MSCs to promote tissue remodeling and wound repair.

A Dexamethasone-Loaded Polymeric Electrospun Construct as a Tubular Cardiovascular Implant.

Cardiovascular tissue engineering is providing many solutions to cardiovascular diseases. The complex disease demands necessitating tissue-engineered constructs with enhanced functionality. In this study, we are presenting the production of a dexamethasone (DEX)-loaded electrospun tubular polymeric poly(l-lactide) (PLA) or poly(d,l-lactide-co-glycolide) (PLGA) construct which contains iPSC-CMs (induced pluripotent stem cell cardiomyocytes), HUVSMCs (human umbilical vein smooth muscle cells), and HUVECs (human umbilical vein endothelial cells) embedded in fibrin gel. The electrospun tube diameter was calculated, as well as the DEX release for 50 days for 2 different DEX concentrations. Furthermore, we investigated the influence of the polymer composition and concentration on the function of the fibrin gels by imaging and quantification of CD31, alpha-smooth muscle actin (αSMA), collagen I (col I), sarcomeric alpha actinin (SAA), and Connexin 43 (Cx43). We evaluated the cytotoxicity and cell proliferation of HUVECs and HUVSMCs cultivated in PLA and PLGA polymeric sheets. The immunohistochemistry results showed efficient iPSC-CM marker expression, while the HUVEC toxicity was higher than the respective HUVSMC value. In total, our study emphasizes the combination of fibrin gel and electrospinning in a functionalized construct, which includes three cell types and provides useful insights of the DEX release and cytotoxicity in a tissue engineering perspective.

A new approach towards extracorporeal gas exchange and first in vitro results.

OBJECTIVES: Extracorporeal life support (ECLS) pertains to therapeutic and prophylactic techniques utilized in a wide range of medical applications, with severe pulmonary diseases being the most prominent cases. Over the past decades, little progress has been made in advancing the basic principles and properties of gas exchangers. Here, in an unconventional approach, dialysis hollow fibers are handled with silicone to create a purely diffusive coating that prevents plasma leakage and promotes gas exchange. METHODS: Commercial dialyzers of varying surface area and fiber diameter have been coated with silicone, to determine the impact of each parameter on performance. The impermeability of the silicone layer has been validated by pressurization and imaging methods. SEM images have revealed a homogeneous silicone film coating the lumen of the capillaries, while fluid dynamic investigations have confirmed its purely diffusive nature. RESULTS: The hemodynamic behavior and the gas exchange efficiency of the silicone-coated prototypes have been investigated in vitro with porcine blood under various operating conditions. Their performance has been found to be similar to that of a commercial PMP oxygenator. CONCLUSIONS: This novel class of gas exchangers is characterized by high versatility and expeditious manufacturing. Intraoperability between conventional ECLS systems and dialysis machines broadens the range of application infinitely. Ultimately, long-term clinical applicability ought to be determined over in vivo animal investigations.

3D geometry orchestrates the transcriptional landscape of metastatic neuroblastoma cells in a multicellular in vitro bone model.

A key challenge for the discovery of novel molecular targets and therapeutics against pediatric bone metastatic disease is the lack of bona fide in vitro cell models. Here, we show that a beta-tricalcium phosphate (ß-TCP) multicellular 3D in vitro bone microtissue model reconstitutes key phenotypic and transcriptional patterns of native metastatic tumor cells while promoting their stemness and proinvasive features. Comparing planar with interconnected channeled scaffolds, we identified geometry as a dominant orchestrator of proangiogenic traits in neuroblastoma tumor cells. On the other hand, the ß-TCP-determined gene signature was DNA replication related. Jointly, the geometry and chemical impact of ß-TCP revealed a prometastatic landscape of the engineered tumor microenvironment. The proposed 3D multicellular in vitro model of pediatric bone metastatic disease may advance further analysis of the molecular, genetic and metabolic bases of the disease and allow more efficient preclinical target validations.

Development of a Silk Fibroin-Small Intestinal Submucosa Small-Diameter Vascular Graft with Sequential VEGF and TGF-ß1 Inhibitor Delivery for In Situ Tissue Engineering.

Proper endothelialization and limited collagen deposition on the luminal surface after graft implantation plays a crucial role to prevent the occurrence of stenosis. To achieve these conditions, a biodegradable graft with adequate mechanical properties and the ability to sequentially deliver therapeutic agents isfabricated. In this study, a dual-release system is constructed through coaxial electrospinning by incorporating recombinant human vascular endothelial growth factor (VEGF) and transforming growth factor ß1 (TGF-ß1) inhibitor into silk fibroin (SF) nanofibers to form a bioactive membrane. The functionalized SF membrane as the inner layer of the graft is characterized by the release profile, cell proliferation and protein expression. It presents excellent biocompatibility and biodegradation, facilitating cell attachment, proliferation, and infiltration. The core-shell structure enables rapid VEGF release within 10 days and sustained plasmid delivery for 21 days. A 2.0-mm-diameter vascular graft is fabricated by integrating the SF membrane with decellularized porcine small intestinal submucosa (SIS), aiming to facilitate the integration process under a stable extracellular matrix structure. The bioengineered graft is functionalized with the sequential administration of VEGF and TGF-ß1, and with the reinforced and compatible mechanical properties, thereby offers an orchestrated solution for stenosis with potential for in situ vascular tissue engineering applications.

Silk Fibroin as Adjuvant in the Fabrication of Mechanically Stable Fibrin Biocomposites.

Fibrin is a very attractive material for the development of tissue-engineered scaffolds due to its exceptional bioactivity, versatility in the fabrication, affinity to cell mediators; and the possibility to isolate it from blood plasma, making it autologous. However, fibrin application is greatly limited due to its low mechanical properties, fast degradation, and strong contraction in the presence of cells. In this study, we present a new strategy to overcome these drawbacks by combining it with another natural polymer: silk fibroin. Specifically, we fabricated biocomposites of fibrin (5 mg/mL) and silk fibroin (0.1, 0.5 and 1% w/w) by using a dual injection system, followed by ethanol annealing. The shear elastic modulus increased from 23 ± 5 Pa from fibrin alone, to 67 ± 22 Pa for fibrin/silk fibroin 0.1%, 241 ± 67 Pa for fibrin/silk fibroin 0.5% and 456 ± 32 Pa for fibrin/silk fibroin 1%. After culturing for 27 days with strong contractile cells (primary human arterial smooth muscle cells), fibrin/silk fibroin 0.5% and fibrin/silk fibroin 1% featured minimal cell-mediated contraction (ca. 15 and 5% respectively) in contrast with the large surface loss of the pure fibrin scaffolds (ca. 95%). Additionally, the composites enabled the formation of a proper endothelial cell layer after culturing with human primary endothelial cells under standard culture conditions. Overall, the fibrin/silk fibroin composites, manufactured within this study by a simple and scalable biofabrication approach, offer a promising avenue to boost the applicability of fibrin in tissue engineering.

Physiological Mineralization during In Vitro Osteogenesis in a Biomimetic Spheroid Culture Model.

Bone health-targeting drug development strategies still largely rely on inferior 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings. MC3T3-E1 pre-osteoblasts were cultured in α-MEM with 10% FCS, at 37 °C and 5% CO2 for up to 28 days, in non-adherent V-shaped plates to form uniformly sized 3D spheroids. Osteogenic differentiation was induced by 10 mM ß-glycerophosphate and 50 µg/mL ascorbic acid. Mineralization stages were assessed through studying expression of marker genes, alkaline phosphatase activity, and calcium deposition by histochemistry. Mineralization quality was evaluated by Fourier transformed infrared (FTIR) and scanning electron microscopic (SEM) analyses and quantified by micro-CT analyses. Expression profiles of selected early- and late-stage osteoblast differentiation markers indicated a well-developed 3D biomineralization process with strongly upregulated Col1a1, Bglap and Alpl mRNA levels and type I collagen- and osteocalcin-positive immunohistochemistry (IHC). A dynamic biomineralization process with increasing mineral densities was observed during the second half of the culture period. SEM-Energy-Dispersive X-ray analyses (EDX) and FTIR ultimately confirmed a native bone-like hydroxyapatite mineral deposition ex vivo. We thus established a robust and versatile biomimetic, and high-throughput compatible, cost-efficient spheroid culture model with a native bone-like mineralization for improved pharmacological ex vivo screenings.

Biohybrid elastin-like venous valve with potential for in situ tissue engineering.

Chronic venous insufficiency (CVI) is a leading vascular disease whose clinical manifestations include varicose veins, edemas, venous ulcers, and venous hypertension, among others. Therapies targeting this medical issue are scarce, and so far, no single venous valve prosthesis is clinically available. Herein, we have designed a bi-leaflet transcatheter venous valve that consists of (i) elastin-like recombinamers, (ii) a textile mesh reinforcement, and (iii) a bioabsorbable magnesium stent structure. Mechanical characterization of the resulting biohybrid elastin-like venous valves (EVV) showed an anisotropic behavior equivalent to the native bovine saphenous vein valves and mechanical strength suitable for vascular implantation. The EVV also featured minimal hemolysis and platelet adhesion, besides actively supporting endothelialization in vitro, thus setting the basis for its application as an in situ tissue engineering implant. In addition, the hydrodynamic testing in a pulsatile bioreactor demonstrated excellent hemodynamic valve performance, with minimal regurgitation (<10%) and pressure drop (<5 mmHg). No stagnation points were detected and an in vitro simulated transcatheter delivery showed the ability of the venous valve to withstand the implantation procedure. These results present a promising concept of a biohybrid transcatheter venous valve as an off-the-shelf implant, with great potential to provide clinical solutions for CVI treatment.

PLA/Hydroxyapatite scaffolds exhibit in vitro immunological inertness and promote robust osteogenic differentiation of human mesenchymal stem cells without osteogenic stimuli.

Bone defects stand out as one of the greatest challenges of reconstructive surgery. Fused deposition modelling (FDM) allows for the printing of 3D scaffolds tailored to the morphology and size of bone damage in a patient-specific and high-precision manner. However, FDM still suffers from the lack of materials capable of efficiently supporting osteogenesis. In this study, we developed 3D-printed porous scaffolds composed of polylactic acid/hydroxyapatite (PLA/HA) composites with high ceramic contents (above 20%, w/w) by FDM. The mechanical properties of the PLA/HA scaffolds were compatible with those of trabecular bone. In vitro degradation tests revealed that HA can neutralize the acidification effect caused by PLA degradation, while simultaneously releasing calcium and phosphate ions. Importantly, 3D-printed PLA/HA did not induce the upregulation of activation markers nor the expression of inflammatory cytokines in dendritic cells thus exhibiting no immune-stimulatory properties in vitro. Evaluations using human mesenchymal stem cells (MSC) showed that pure PLA scaffolds exerted an osteoconductive effect, whereas PLA/HA scaffolds efficiently induced osteogenic differentiation of MSC even in the absence of any classical osteogenic stimuli. Our findings indicate that 3D-printed PLA scaffolds loaded with high concentrations of HA are most suitable for future applications in bone tissue engineering.

Enhanced Stable Cavitation and Nonlinear Acoustic Properties of Poly(butyl cyanoacrylate) Polymeric Microbubbles after Bioconjugation.

Microbubbles (MB) are used as ultrasound (US) contrast agents in clinical settings because of their ability to oscillate upon exposure to acoustic pulses and generate nonlinear responses with a stable cavitation profile. Polymeric MB have recently attracted increasing attention as molecular imaging probes and drug delivery agents based on their tailorable acoustic responses, high drug loading capacity, and surface functionalization capabilities. While many of these applications require MB to be functionalized with biological ligands, the impact of bioconjugation on polymeric MB cavitation and acoustic properties remains poorly understood. Hence, we here evaluated the effects of MB shell hydrolysis and subsequent streptavidin conjugation on the acoustic behavior of poly(butyl cyanoacrylate) (PBCA) MB. We show that upon biofunctionalization, MB display higher acoustic stability, stronger stable cavitation, and enhanced second harmonic generation. Furthermore, functionalized MB preserve the binding capabilities of streptavidin conjugated on their surface. These findings provide insights into the effects of bioconjugation chemistry on polymeric MB acoustic properties, and they contribute to improving the performance of polymer-based US imaging and theranostic agents.

Influence of microgel architecture and oil polarity on stabilization of emulsions by stimuli-sensitive core-shell poly(N-isopropylacrylamide-co-methacrylic acid) microgels: Mickering versus Pickering behavior?

Charged poly(N-isopropylacrylamide-co-methacrylic acid) [P(NiPAM-co-MAA)] microgels can stabilize thermo- and pH-sensitive emulsions. By placing charged units at different locations in the microgels and comparing the emulsion properties, we demonstrate that their behaviors as emulsion stabilizers are very different from molecular surfactants and rigid Pickering stabilizers. The results show that the stabilization of the emulsions is independent of electrostatic repulsion although the presence and location of charges are relevant. Apparently, the charges facilitate emulsion stabilization via the extent of swelling and deformability of the microgels. The stabilization of these emulsions is linked to the swelling and structure of the microgels at the oil-water interface, which depends not only on the presence of charged moieties and on solvent polarity but also on the microgel (core-shell) morphology. Therefore, the internal soft and porous structure of microgels is important, and these features make microgel-stabilized emulsions characteristically different from classical, rigid-particle-stabilized Pickering emulsions, the stability of which depends on the surface properties of the particles.