On-Chip Fabrication of Colloidal Suprastructures by Assembly and Supramolecular Interlinking of Microgels.

In this report, a versatile method is demonstrated to create colloidal suprastructures by assembly and supramolecular interlinking of microgels using droplet-based microfluidics. The behavior of the microgels is systematically investigated to evaluate the influence of their concentration on their distribution between the continuous, the droplet phase, and the interface. At low concentrations, microgels are mainly localized at the water-oil interface whereas an excess of microgels results, following the complete coverage of the water-oil interface, in their distribution in the continuous phase. To stabilize the colloidal suprastructure, on-chip gelation is introduced by adding natural polyphenol tannic acid (TA) in the water phase. TA forms interparticle linking between the poly(N-vinylcaprolactam) (PVCL) microgels by supramolecular interactions. The combination of supramolecular interlinking with the variation of the microgel concentration in microfluidic droplets enables on-chip fabrication of defined colloidal suprastructures with morphologies ranging from colloidosomes to colloidal supraballs. The obtained supracolloidal structures exhibit a pH-responsive behavior with a disintegration at alkaline conditions within a scale of seconds. The destabilization process results from the deprotonation of phenolic groups and destruction of hydrogen bonds with PVCL chains at higher pH.

Actuation of Soft Thermoresponsive Hydrogels Mechanically Stimulates Osteogenesis in Human Mesenchymal Stem Cells without Biochemical Factors.

Mesenchymal stem cells (MSCs) have the potential to differentiate into multiple lineages and can be harvested relatively easily from adults, making them a promising cell source for regenerative therapies. While it is well-known how to consistently differentiate MSCs into adipose, chondrogenic, and osteogenic lineages by treatment with biochemical factors, the number of studies exploring how to achieve this with mechanical signals is limited. A relatively unexplored area is the effect of cyclic forces on the MSC differentiation. Recently, our group developed a thermoresponsive N-ethyl acrylamide/N-isopropylacrylamide (NIPAM/NEAM) hydrogel supplemented with gold nanorods that are able to convert near-infrared light into heat. Using light pulses allows for local hydrogel collapse and swelling with physiologically relevant force and frequency. In this study, MSCs are cultured on this hydrogel system with a patterned surface and exposed to intermittent or continuous actuation of the hydrogel for 3 days to study the effect of actuation on MSC differentiation. First, cells are harvested from the bone marrow of three donors and tested for their MSC phenotype, meeting the following criteria: the harvested cells are adherent and demonstrate a fibroblast-like bipolar morphology. They lack the expression of CD34 and CD45 but do express CD73, CD90, and CD105. Additionally, their differentiation potential into adipogenic, chondrogenic, and osteogenic lineages is validated by the addition of standardized differentiation media. Next, MSCs are exposed to intermittent or continuous actuation, which leads to a significantly enhanced cell spreading compared to nonactuated cells. Moreover, actuation results in nuclear translocation of Runt-related transcription factor 2 and the Yes-associated protein. Together, these results indicate that cyclic mechanical stimulation on a soft, ridged substrate modulates the MSC fate commitment in the direction of osteogenesis.

Cellular Architects at Work: Cells Building their Own Microgel Houses.

Microporous annealed particle (MAP) scaffolds are investigated for their application as injectable 3D constructs in the field of regenerative medicine and tissue repair. While available MAP scaffolds provide a stable interlinked matrix of microgels for cell culture, the infiltration depth and space for cells to grow inside the scaffolds is pre-determined by the void fraction during the assembly. In the case of MAP scaffolds fabricated from interlinked spherical microgels, a cellularity gradient can be observed with the highest cell density on the scaffold surface. Additionally, the interlinked microgel network limits the ability of cells to remodel their environment, which contradicts native tissue dynamics. In this work, a cell-induced interlinking method for MAP scaffold formation is established, which avoids the necessity of chemical crosslinkers and pre-engineered pores to achieve micro- or macropores in these 3D frameworks. This method enables cells to self-organize with microgels into dynamic tissue constructs, which can be further controlled by altering the microgel properties, the cell/microgel ratio, and well shape. To form a cell-induced interlinked scaffold, the cells are mixed with dextran-based microgels and function as a glue between the microgels, resulting in a more homogenous cell distribution throughout the scaffold with efficient cell-cell interactions.

Varying the Stiffness and Diffusivity of Rod-Shaped Microgels Independently through Their Molecular Building Blocks.

Microgels are water-swollen, crosslinked polymers that are widely used as colloidal building blocks in scaffold materials for tissue engineering and regenerative medicine. Microgels can be controlled in their stiffness, degree of swelling, and mesh size depending on their polymer architecture, crosslink density, and fabrication method-all of which influence their function and interaction with the environment. Currently, there is a lack of understanding of how the polymer composition influences the internal structure of soft microgels and how this morphology affects specific biomedical applications. In this report, we systematically vary the architecture and molar mass of polyethylene glycol-acrylate (PEG-Ac) precursors, as well as their concentration and combination, to gain insight in the different parameters that affect the internal structure of rod-shaped microgels. We characterize the mechanical properties and diffusivity, as well as the conversion of acrylate groups during photopolymerization, in both bulk hydrogels and microgels produced from the PEG-Ac precursors. Furthermore, we investigate cell-microgel interaction, and we observe improved cell spreading on microgels with more accessible RGD peptide and with a stiffness in a range of 20 kPa to 50 kPa lead to better cell growth.

Cell Guiding Multicomponent Nanoyarn Tendon Scaffolds with Tunable Morphology and Flexibility.

Nanofibrous scaffolds are widely investigated for tendon tissue engineering due to their porous structure, high flexibility, and the ability to guide cells in a preferred direction. Previous research has shown that providing a microenvironment similar to in vivo settings improves tissue regeneration. Therefore, in this work, ingenious multicomponent nanoyarn scaffolds that mimic the fibrillar and tubular structures of tendons are developed for the first time through electrospinning and bundling nanoyarns followed by electrospinning of a nanofibrous shell around the bundle. Multicomponent nanoyarn scaffolds out of poly(ε-caprolactone) with varying porosity, density, and diameter were successfully produced by coelectrospinning with water-soluble poly(2-ethyl-2-oxazoline) as a sacrificial component. The diameter and fiber orientation of the nanoyarns were successfully tuned based on parameter-morphology models obtained by the design of experiments. Cyclic bending tests were performed, indicating that the flexibility of the multicomponent nanoyarn scaffolds depends on the morphology and can be tuned through controlling the number of nanoyarns in the bundle and the porosity. Indirect and direct cell culture tests using mouse and equine tendon cells revealed excellent cytocompatibility of the nanofibrous products and demonstrated the potential of the nanoyarns to guide the growing cells along the nanofiber direction, which is crucial for tendon tissue engineering.

Lifelike Transformative Materials for Biohybrid Implants: Inspired by Nature, Driven by Technology.

Today's living world is enriched with a myriad of natural biological designs, shaped by billions of years of evolution. Unraveling the construction rules of living organisms offers the potential to create new materials and systems for biomedicine. From the close examination of living organisms, several concepts emerge: hierarchy, pattern repetition, adaptation, and irreducible complexity. All these aspects must be tackled to develop transformative materials with lifelike behavior. This perspective article highlights recent progress in the development of transformative biohybrid systems for applications in the fields of tissue regeneration and biomedicine. Advances in computational simulations and data-driven predictions are also discussed. These tools enable the virtual high-throughput screening of implant design and performance before committing to fabrication, thus reducing the development time and cost of biomimetic and biohybrid constructs. The ongoing progress of imaging methods also constitutes an essential part of this matter in order to validate the computation models and enable longitudinal monitoring. Finally, the current challenges of lifelike biohybrid materials, including reproducibility, ethical considerations, and translation, are discussed. Advances in the development of lifelike materials will open new biomedical horizons, where perhaps what is currently envisioned as science fiction will become a science-driven reality in the future.

Transformative Materials to Create 3D Functional Human Tissue Models In Vitro in a Reproducible Manner.

Recreating human tissues and organs in the petri dish to establish models as tools in biomedical sciences has gained momentum. These models can provide insight into mechanisms of human physiology, disease onset, and progression, and improve drug target validation, as well as the development of new medical therapeutics. Transformative materials play an important role in this evolution, as they can be programmed to direct cell behavior and fate by controlling the activity of bioactive molecules and material properties. Using nature as an inspiration, scientists are creating materials that incorporate specific biological processes observed during human organogenesis and tissue regeneration. This article presents the reader with state-of-the-art developments in the field of in vitro tissue engineering and the challenges related to the design, production, and translation of these transformative materials. Advances regarding (stem) cell sources, expansion, and differentiation, and how novel responsive materials, automated and large-scale fabrication processes, culture conditions, in situ monitoring systems, and computer simulations are required to create functional human tissue models that are relevant and efficient for drug discovery, are described. This paper illustrates how these different technologies need to converge to generate in vitro life-like human tissue models that provide a platform to answer health-based scientific questions.

Microgels as Platforms for Antibody-Mediated Cytokine Scavenging.

Therapeutic antibodies are the key treatment option for various cytokine-mediated diseases, such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. However, systemic injection of these antibodies can cause side effects and suppress the immune system. Moreover, clearance of therapeutic antibodies from the blood is limiting their efficacy. Here, water-swollen microgels are produced with a size of 25 µm using droplet-based microfluidics. The microgels are functionalized with TNFα antibodies to locally scavenge the pro-inflammatory cytokine TNFα. Homogeneous distribution of TNFα-antibodies is shown throughout the microgel network and demonstrates specific antibody-antigen binding using confocal microscopy and FLIM-FRET measurements. Due to the large internal accessibility of the microgel network, its capacity to bind TNFα is extremely high. At a TNFα concentration of 2.5 µg mL-1 , the microgels are able to scavenge 88% of the cytokine. Cell culture experiments reveal the therapeutic potential of these microgels by protecting HT29 colorectal adenocarcinoma cells from TNFα toxicity and resulting in a significant reduction of COX II and IL8 production of the cells. When the microgels are incubated with stimulated human macrophages, to mimic the in vivo situation of inflammatory bowel disease, the microgels scavenge almost all TNFα that is produced by the cells.

Nonspherical ultrasound microbubbles.

Surface tension provides microbubbles (MB) with a perfect spherical shape. Here, we demonstrate that MB can be engineered to be nonspherical, endowing them with unique features for biomedical applications. Anisotropic MB were generated via one-dimensionally stretching spherical poly(butyl cyanoacrylate) MB above their glass transition temperature. Compared to their spherical counterparts, nonspherical polymeric MB displayed superior performance in multiple ways, including i) increased margination behavior in blood vessel-like flow chambers, ii) reduced macrophage uptake in vitro, iii) prolonged circulation time in vivo, and iv) enhanced blood-brain barrier (BBB) permeation in vivo upon combination with transcranial focused ultrasound (FUS). Our studies identify shape as a design parameter in the MB landscape, and they provide a rational and robust framework for further exploring the application of anisotropic MB for ultrasound-enhanced drug delivery and imaging applications.

Liposome manufacturing under continuous flow conditions: towards a fully integrated set-up with in-line control of critical quality attributes.

Continuous flow manufacturing (CFM) has shown remarkable advantages in the industrial-scale production of drug-loaded nanomedicines, including mRNA-based COVID-19 vaccines. Thus far, CFM research in nanomedicine has mainly focused on the initial particle formation step, while post-formation production steps are hardly ever integrated. The opportunity to implement in-line quality control of critical quality attributes merits closer investigation. Here, we designed and tested a CFM setup for the manufacturing of liposomal nanomedicines that can potentially encompass all manufacturing steps in an end-to-end system. Our main aim was to elucidate the key composition and process parameters that affect the physicochemical characteristics of the liposomes. Total flow rate, lipid concentration and residence time of the liposomes in a high ethanol environment (i.e., above 20% v/v) emerged as critical parameters to tailor liposome size between 80 and 150 nm. After liposome formation, the pressure and the surface area of the filter in the ultrafiltration unit were critical parameters in the process of clearing the dispersion from residual ethanol. As a final step, we integrated in-line measurement of liposome size and residual ethanol content. Such in-line measurements allow for real-time monitoring and in-process adjustment of key composition and process parameters.

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.

Annealing High Aspect Ratio Microgels into Macroporous 3D Scaffolds Allows for Higher Porosities and Effective Cell Migration.

Growing millimeter-scaled functional tissue remains a major challenge in the field of tissue engineering. Therefore, microporous annealed particles (MAPs) are emerging as promising porous biomaterials that are formed by assembly of microgel building blocks. To further vary the pore size and increase overall MAP porosity of mechanically stable scaffolds, rod-shaped microgels with high aspect ratios up to 20 are chemically interlinked into highly porous scaffolds. Polyethylene glycol based microgels (width 10 µm, lengths up to 200 µm) are produced via in-mold polymerization and covalently interlinked into stable 3D scaffolds via epoxy-amine chemistry. For the first time, MAP porosities can be enhanced by increasing the microgel aspect ratio (mean pore sizes ranging from 39 to 82 µm, porosities from 65 to 90%). These porosities are significantly higher compared to constructs made from spherical or lower aspect ratio rod-shaped microgels. Rapid filling of the pores by either murine or primary human fibroblasts is ensured as cells migrate and grow extensively into these scaffolds. Overall, this study demonstrates that highly porous, stable macroporous hydrogels can be achieved with a very low partial volume of synthetic, high aspect ratio microgels, leading to large empty volumes available for cell ingrowth and cell-cell interactions.

Controlled Covalent Self-Assembly of a Homopolymer for Multiscale Materials Engineering.

Polymer self-assembly is a crucial process in materials engineering. Currently, almost all polymer self-assembly is limited to non-covalent bonding methods, even though these methods have drawbacks as they require complicated synthesis techniques and produce relatively unstable structures. Here, a novel mechanism of covalent polymer self-assembly is discovered and employed to address drawbacks of non-covalent polymer self-assembly. A simple ketone homopolymer is found to self-assemble into nano- to macroscale hydrogels during covalent crosslinking. In contrast to non-covalent self-assembly, the covalent self-assembly is independent of and unaffected by solvent conditions (e.g., polarity and ionic strength) and does not require additional agents, e.g., organic solvents and surfactants. The covalent polymer self-assembly is subjected to a new mechanism of control by tuning the covalent crosslinking rate. This leads to nanogels with an unprecedented and tightly controlled range of dimensions from less than 10 nm to above 100 nm. Moreover, the crosslinking rate also regulates the assembly behavior of microgels fabricated by microfluidics. The microgels self-assemble into granular fibers, which is 3D printed into stable porous scaffolds. The novel covalent polymer assembly method has enormous potential to revolutionize multiscale materials fabrication for applications in drug delivery, tissue engineering, and many other fields.

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.

Interlinked Macroporous 3D Scaffolds from Microgel Rods.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

High Macromolecular Crowding in Liposomes from Microfluidics.

The intracellular environment is crowded with macromolecules that influence biochemical equilibria and biomacromolecule diffusion. The incorporation of such crowding in synthetic cells would be needed to mimic the biochemistry of living cells. However, only a few methods provide crowded artificial cells, moreover providing cells with either heterogeneous size and composition or containing a significant oil fraction. Therefore, a method that generates monodisperse liposomes with minimal oil content and tunable macromolecular crowding using polydimethylsiloxane (PDMS)-based microfluidics is presented. Lipid stabilized water-in-oil-in-water emulsions that are stable for at least several months and with a high macromolecular crowder concentration that can be controlled with the external osmolality are formed. A crucial feature is that the oil phase can be removed using high flow conditions at any point after production, providing the highly crowded liposomes. Genetically encoded macromolecular crowding sensors show that the high level of macromolecular crowding in the emulsions is fully retained throughout the generation of minimal-oil lipid bilayers. This modular and robust platform will serve the study of biochemistry under physiologically relevant crowding conditions.