Exploring the potential of all-aqueous immiscible systems for preparing complex biomaterials and cellular constructs.

All-aqueous immiscible systems derived from liquid-liquid phase separation of incompatible hydrophilic agents such as polymers and salts have found increasing interest in the biomedical and tissue engineering fields in the last few years. The unique characteristics of aqueous interfaces, namely their low interfacial tension and elevated permeability, as well as the non-toxic environment and high water content of the immiscible phases, confer to these systems optimal qualities for the development of biomaterials such as hydrogels and soft membranes, as well as for the preparation of in vitro tissues derived from cellular assembly. Here, we overview the main properties of these systems and present a critical review of recent strategies that have been used for the development of biomaterials with increased levels of complexity using all-aqueous immiscible phases and interfaces, and their potential as cell-confining environments for micropatterning approaches and the bioengineering of cell-rich structures. Importantly, due to the relatively recent emergence of these areas, several key design considerations are presented, in order to guide researchers in the field. Finally, the main present challenges, future directions, and adaptability to develop advanced materials with increased biomimicry and new potential applications are briefly evaluated.

Shape-Versatile Fixed Cellular Materials for Multiple Target Immunomodulation.

Therapeutic cells are usually administered as living agents, despite the risks of undesired cell migration and acquisition of unpredictable phenotypes. Additionally, most cell-based therapies rely on the administration of single cells, often associated with rapid in vivo clearance. 3D cellular materials may be useful to prolong the effect of cellular therapies and offer the possibility of creating structural volumetric constructs. Here, the manufacturing of shape-versatile fixed cell-based materials with immunomodulatory properties is reported. Living cell aggregates with different shapes (spheres and centimeter-long fibers) are fixed using a method compatible with maintenance of structural integrity, robustness, and flexibility of 3D constructs. The biological properties of living cells can be modulated before fixation, rendering an in vitro anti-inflammatory effect toward human macrophages, in line with a decreased activation of the nuclear factor kappa B (NF-κB) pathway that preponderantly correlated with the surface area of the materials. These findings are further corroborated in vivo in mouse skin wounds. Contact with fixed materials also reduces the proliferation of activated primary T lymphocytes, while promoting regulatory populations. The fixation of cellular constructs is proposed as a versatile phenotypic stabilization method that can be easily implemented to prepare immunomodulatory materials with therapeutic potential.

Encapsulated Mesenchymal Stromal Cells as Cyclic Providers of Immunomodulatory Secretomes: A Living on-Demand Delivery System.

The stimulation of mesenchymal stromal cells (MSCs) with inflammatory molecules is often used to boost their therapeutic effect. Prolonged exposure to inflammatory molecules has been explored to improve their action because MSCs therapies seem to be improved transiently with such stimuli. However, the possibility of cyclically stimulating MSCs to recover their optimized therapeutic potential is still to be elucidated, although the efficacy of cell-based therapies may be dependent on the ability to readapt to the relapse pathological conditions. Here, the response of MSCs, encapsulated in alginate hydrogels and cultured for 22 d, is explored using three different regimes: single, continuous, and intermittent stimulation with IFNγ. Exposure to IFNγ leads to a decrease in the secretion of IL-10, which is cyclically countered by IFNγ weaning. Conditioned media collected at different stages of pulsatile stimulation show an immunomodulatory potential toward macrophages, which directly correlates with IL-10 concentration in media. To understand whether the correlation between cyclic stimulation of MSCs and other biological actions can be observed, the effect on endothelial cells is studied, showcasing an overall modest influence on tube formation. Overall, the results describe the response of encapsulated MSCs to unusual pulsatile simulation regimens, exploring encapsulated MSCs as a living on-demand release system of tailored secretomes with recoverable immunomodulatory action.

Natural Polymer-Polyphenol Bioadhesive Coacervate with Stable Wet Adhesion, Antibacterial Activity, and On-Demand Detachment.

Medical adhesives are emerging as an important clinical tool as adjuvants for sutures and staples in wound closure and healing and in the achievement of hemostasis. However, clinical adhesives combining cytocompatibility, as well as strong and stable adhesion in physiological conditions, are still in demand. Herein, a mussel-inspired strategy is explored to produce adhesive coacervates using tannic acid (TA) and methacrylate pullulan (PUL-MA). TA|PUL-MA coacervates mainly comprise van der Waals forces and hydrophobic interactions. The methacrylic groups in the PUL backbone increase the number of interactions in the adhesives matrix, resulting in enhanced cohesion and adhesion strength (72.7 Jm-2), compared to the non-methacrylated coacervate. The adhesive properties are kept in physiologic-mimetic solutions (72.8 Jm-2) for 72 h. The photopolymerization of TA|PUL-MA enables the on-demand detachment of the adhesive. The poor cytocompatibility associated with the use of phenolic groups is here circumvented by mixing reactive oxygen species-degrading enzyme in the adhesive coacervate. This addition does not hamper the adhesive character of the materials, nor their anti-microbial or hemostatic properties. This affordable and straightforward methodology, together with the tailorable adhesivity even in wet environments, high cytocompatibility, and anti-bacterial activity, enables foresee TA|PUL-MA as a promising ready-to-use bioadhesive for biomedical applications.

An Intracellular Metabolic Signature as a Potential Donor-Independent Marker of the Osteogenic Differentiation of Adipose Tissue Mesenchymal Stem Cells.

This paper describes an untargeted NMR metabolomics study to identify potential intracellular donor-dependent and donor-independent metabolic markers of proliferation and osteogenic differentiation of human adipose mesenchymal stem cells (hAMSCs). The hAMSCs of two donors with distinct proliferating/osteogenic characteristics were fully characterized regarding their polar endometabolome during proliferation and osteogenesis. An 18-metabolites signature (including changes in alanine, aspartate, proline, tyrosine, ATP, and ADP, among others) was suggested to be potentially descriptive of cell proliferation, independently of the donor. In addition, a set of 11 metabolites was proposed to compose a possible donor-independent signature of osteogenesis, mostly involving changes in taurine, glutathione, methylguanidine, adenosine, inosine, uridine, and creatine/phosphocreatine, choline/phosphocholine and ethanolamine/phosphocholine ratios. The proposed signatures were validated for a third donor, although they require further validation in a larger donor cohort. We believe that this proof of concept paves the way to exploit metabolic markers to monitor (and potentially predict) cell proliferation and the osteogenic ability of different donors.

Cell Response in Free-Packed Granular Systems.

The study of the interactions of living adherent cells with mechanically stable (visco)elastic materials enables understanding and exploitation of physiological phenomena mediated by cell-extracellular communication. Insights into the interaction of cells and surrounding objects with different stability patterns upon cell contact might unveil biological responses to engineer innovative applications. Here, we hypothesize that the efficiency of cell attachment, spreading, and movement across a free-packed granular bed of microparticles depends on the microparticle diameter, raising the possibility of a necessary minimum traction force for the reinforcement of cell-particle bonds and long-term cell adhesion. The results suggest that microparticles with diameters of 14-20 µm are prone to cell-mediated mobility, holding the potential of inducing early cell detachment, while objects with diameters from 38 to 85 µm enable long-lasting cell adhesion and proliferation. An in silico hybrid particle-based model that addresses the time-dependent biological mechanisms of cell adhesion is proposed, providing inspiration for engineering platforms to address healthcare-related challenges.

All-Aqueous Freeform Fabrication of Perfusable Self-Standing Soft Compartments.

Compartmentalized structures obtained in all-aqueous settings have shown promising properties as cell encapsulation devices, as well as reactors for trans-membrane chemical reactions. While most approaches focus on the preparation of spherical devices, advances on the production of complex architectures have been enabled by the interfacial stability conferred by emulsion systems, namely mild aqueous two-phase systems (ATPS), or non-equilibrated analogues. However, the application of non-spherical structures has mostly been reported while keeping the fabricated materials at a stable interface, limiting the free-standing character, mobility and transposition of the obtained structures to different setups. Here, the fabrication of self-standing, malleable and perfusable tubular systems through all-aqueous interfacial assembly is shown, culminating in the preparation of independent objects with stability and homogeneity after disruption of the polymer-based aqueous separating system. Those hollow structures can be fabricated with a variety of widths, and rapidly printed as long structures at flow rates of 15 mm s-1 . The materials are used as compartments for cell culture, showcasing high cytocompatibility, and can be tailored to promote cell adhesion. Such structures may find application in fields that benefit from freeform tubular structures, including the biomedical field with, for example, cell encapsulation, and benchtop preparation of microfluidic devices.

Endo- and Exometabolome Crosstalk in Mesenchymal Stem Cells Undergoing Osteogenic Differentiation.

This paper describes, for the first time to our knowledge, a lipidome and exometabolome characterization of osteogenic differentiation for human adipose tissue stem cells (hAMSCs) using nuclear magnetic resonance (NMR) spectroscopy. The holistic nature of NMR enabled the time-course evolution of cholesterol, mono- and polyunsaturated fatty acids (including ω-6 and ω-3 fatty acids), several phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens), and mono- and triglycerides to be followed. Lipid changes occurred almost exclusively between days 1 and 7, followed by a tendency for lipidome stabilization after day 7. On average, phospholipids and longer and more unsaturated fatty acids increased up to day 7, probably related to plasma membrane fluidity. Articulation of lipidome changes with previously reported polar endometabolome profiling and with exometabolome changes reported here in the same cells, enabled important correlations to be established during hAMSC osteogenic differentiation. Our results supported hypotheses related to the dynamics of membrane remodelling, anti-oxidative mechanisms, protein synthesis, and energy metabolism. Importantly, the observation of specific up-taken or excreted metabolites paves the way for the identification of potential osteoinductive metabolites useful for optimized osteogenic protocols.

NMR Metabolomics Assessment of Osteogenic Differentiation of Adipose-Tissue-Derived Mesenchymal Stem Cells.

This Article presents, for the first time to our knowledge, an untargeted nuclear magnetic resonance (NMR) metabolomic characterization of the polar intracellular metabolic adaptations of human adipose-derived mesenchymal stem cells during osteogenic differentiation. The use of mesenchymal stem cells (MSCs) for bone regeneration is a promising alternative to conventional bone grafts, and untargeted metabolomics may unveil novel metabolic information on the osteogenic differentiation of MSCs, allowing their behavior to be understood and monitored/guided toward effective therapies. Our results unveiled statistically relevant changes in the levels of just over 30 identified metabolites, illustrating a highly dynamic process with significant variations throughout the whole 21-day period of osteogenic differentiation, mainly involving amino acid metabolism and protein synthesis; energy metabolism and the roles of glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation; cell membrane metabolism; nucleotide metabolism (including the specific involvement of O-glycosylation intermediates and NAD+); and metabolic players in protective antioxidative mechanisms (such as glutathione and specific amino acids). Different metabolic stages are proposed and are supported by putative biochemical explanations for the metabolite changes observed. This work lays the groundwork for the use of untargeted NMR metabolomics to find potential metabolic markers of osteogenic differentiation efficacy.

Metabolomic Applications in Stem Cell Research: a Review.

This review describes the use of metabolomics to study stem cell (SC) characteristics and function, excluding SCs in cancer research, suited to a fully dedicated text. The interest in employing metabolomics in SC research has consistently grown and emphasis is, here, given to developments reported in the past five years. This text informs on the existing methodologies and their complementarity regarding the information provided, comprising untargeted/targeted approaches, which couple mass spectrometry or nuclear magnetic resonance spectroscopy with multivariate analysis (and, in some cases, pathway analysis and integration with other omics), and more specific analytical approaches, namely isotope tracing to highlight particular metabolic pathways, or in tandem microscopic strategies to pinpoint characteristics within a single cell. The bulk of this review covers the existing applications in various aspects of mesenchymal SC behavior, followed by pluripotent and neural SCs, with a few reports addressing other SC types. Some of the central ideas investigated comprise the metabolic/biological impacts of different tissue/donor sources and differentiation conditions, including the importance of considering 3D culture environments, mechanical cues and/or media enrichment to guide differentiation into specific lineages. Metabolomic analysis has considered cell endometabolomes and exometabolomes (fingerprinting and footprinting, respectively), having measured both lipid species and polar metabolites involved in a variety of metabolic pathways. This review clearly demonstrates the current enticing promise of metabolomics in significantly contributing towards a deeper knowledge on SC behavior, and the discovery of new biomarkers of SC function with potential translation to in vivo clinical practice.

Engineering Strategies for Allogeneic Solid Tissue Acceptance.

Advances in allogeneic transplantation of solid organs and tissues depend on our understanding of mechanisms that mediate the prevention of graft rejection. For the past decades, clinical practice has established guidelines to prevent allograft rejection, which mostly rely on the intake of nontargeted immunosuppressants as the gold standard. However, such lifelong regimens have been reported to trigger severe morbidities and commonly fail in preventing late allograft loss. In this review, the biology of allogeneic rejection and self-tolerance is analyzed, as well as the mechanisms of cellular-based therapeutics driving suppression and/or tolerance. Bioinspired engineering strategies that take advantage of cells, biomaterials, or combinations thereof to prevent allograft rejection are addressed, as well as biological mechanisms that drive their efficacy.

Fabrication of Quasi-2D Shape-Tailored Microparticles using Wettability Contrast-Based Platforms.

The ability to fabricate materials with ultrathin architectures enables the breakthrough of low-dimensional structures with high surface area that showcase distinctive properties from their bulk counterparts. They are exploited in a wide range of fields, including energy harvesting, catalysis, and biomedicine. Despite such versatility, the fine tuning of the lateral dimensions and geometry of these structures remains challenging. Prepatterned platforms gain significant attention as enabling technologies to process materials with highly controlled shapes and dimensions. Herein, different nanometer-thick particles of various lateral sizes and geometries (e.g., squares, circles, triangles, hexagons) are processed with high precision and definition, taking advantage of the wettability contrast of oleophilic-oleophobic patterned surfaces. Quasi-2D polymeric microparticles with high shape- and size-fidelity can be retrieved as freestanding objects in a single step. These structures show cell-mediated pliability, and their integration in gravity-enforced human adipose-derived stem cell spheroids leads to an enhanced metabolic activity and a modulated secretion of proangiogenic factors.

One-Step All-Aqueous Interfacial Assembly of Robust Membranes for Long-Term Encapsulation and Culture of Adherent Stem/Stromal Cells.

The therapeutic effectiveness and biological relevance of technologies based on adherent cells depend on platforms that enable long-term culture in controlled environments. Liquid-core capsules have been suggested as semipermeable moieties with spatial homogeneity due to the high mobility of all components in their core. The lack of cell-adhesive sites in liquid-core structures often hampers their use as platforms for stem cell-based technologies for long-term survival and cell-directed self-organization. Here, the one-step fast formation of robust polymeric capsules formed by interfacial complexation of oppositely charged polyelectrolytes in an all-aqueous environment, compatible with the simultaneous encapsulation of mesenchymal stem/stromal cells (MSCs) and microcarriers, is described. The adhesion of umbilical cord MSCs to polymeric microcarriers enables their aggregation and culture for more than 21 days in capsules prepared either manually by dropwise addition, or by scalable electrohydrodynamic atomization, generating robust and stable capsules. Cell aggregation and secretion overtime can be tailored by providing cells with static or dynamic (bioreactor) environments.

Synthesis and characterization of scaffolds produced under mild conditions based on oxidized cashew gums and carboxyethyl chitosan.

This study describes the development of scaffolds based on carboxyethyl chitosan (CEC) and different oxidized cashew gums (CGOx) for tissue engineering (TE) applications. After the physico-chemical characterizations of CEC and CGOx (oxidation degree of 20, 35 and 50%), these macromolecules were used for producing the CGOx-CEC hydrogels through a Schiff base reaction, in the absence of any crosslinking agent. The CGOx-CEC scaffolds obtained after a freeze-drying process were characterized for their morphology, mechanical properties, swelling ability, degradation, and porosity. Those revealed to be highly porous (25-65%), and showed a stable swelling behavior, as well as degradation properties in the absence of enzymes. The use of the cashew gum with higher degree of oxidation led to scaffolds with higher crosslinking densities and increased compressive modulus. None of the hydrogels show cytotoxicity during the 14 days of incubation. Considering all the properties mentioned, these scaffolds are excellent candidates for soft tissue regeneration, owing to the use of eco-friendly starting materials and the easy tuning of their properties.

Strategies for re-vascularization and promotion of angiogenesis in trauma and disease.

The maintenance of a healthy vascular system is essential to ensure the proper function of all organs of the human body. While macrovessels have the main role of blood transportation from the heart to all tissues, microvessels, in particular capillaries, are responsible for maintaining tissues' functionality by providing oxygen, nutrients and waste exchanges. Occlusion of blood vessels due to atherosclerotic plaque accumulation remains the leading cause of mortality across the world. Autologous vein and artery grafts bypassing are the current gold standard surgical procedures to substitute primarily obstructed vascular structures. Ischemic scenarios that condition blood supply in downstream tissues may arise from blockage phenomena, as well as from other disease or events leading to trauma. The (i) great demand for new vascular substitutes, arising from both the limited availability of healthy autologous vessels, as well as the shortcomings associated with small-diameter synthetic vascular grafts, and (ii) the challenging induction of the formation of adequate and stable microvasculature are current driving forces for the growing interest in the development of bioinspired strategies to ensure the proper function of vasculature in all its dimensional scales. Here, a critical review of well-established technologies and recent biotechnological advances to substitute or regenerate the vascular system is provided.

Recent advances in the design of implantable insulin secreting heterocellular islet organoids.

Islet transplantation has proved one of the most remarkable transmissions from an experimental curiosity into a routine clinical application for the treatment of type I diabetes (T1D). Current efforts for taking this technology one-step further are now focusing on overcoming islet donor shortage, engraftment, prolonged islet availability, post-transplant vascularization, and coming up with new strategies to eliminate lifelong immunosuppression. To this end, insulin secreting 3D cell clusters composed of different types of cells, also referred as heterocellular islet organoids, spheroids, or pseudoislets, have been engineered to overcome the challenges encountered by the current islet transplantation protocols. ß-cells or native islets are accompanied by helper cells, also referred to as accessory cells, to generate a cell cluster that is not only able to accurately secrete insulin in response to glucose, but also superior in terms of other key features (e.g. maintaining a vasculature, longer durability in vivo and not necessitating immunosuppression after transplantation). Over the past decade, numerous 3D cell culture techniques have been integrated to create an engineered heterocellular islet organoid that addresses current obstacles. Here, we first discuss the different cell types used to prepare heterocellular organoids for islet transplantation and their contribution to the organoids design. We then introduce various cell culture techniques that are incorporated to prepare a fully functional and insulin secreting organoids with select features. Finally, we discuss the challenges and present a future outlook for improving clinical outcomes of islet transplantation.

Efficient Single-Dose Induction of Osteogenic Differentiation of Stem Cells Using Multi-Bioactive Hybrid Nanocarriers.

Bone regeneration requires the presence of specific factors to induce the differentiation of stem cells into osteoblasts. These factors induce osteogenesis by stimulating the expression of bone-related proteins, bone cell proliferation and differentiation. Herein, bioactive mesoporous silica nanoparticles are doped with calcium and phosphate ions while the porous network is loaded with dexamethasone (MSN-CaPDex). The bioactive MSN-CaPDex nanocarriers are prepared without affecting the narrow size distribution, pore structure, and morphology of the MSNs, while incorporating multi-stimuli, complementary ionic/biochemical bioactive mediators. The bioactive nanocarriers induce osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) after a single-dose administration, and without the need for further soluble osteogenic factors, in contrast to the standard continuous stimulation provided by osteogenic medium. The hBM-MSCs exhibit several biomarkers of osteogenic differentiation, including alkaline phosphatase peaking at early time points, secretion of osteopontin and osteocalcin, and deposition of a calcium-rich matrix. Overall, by inducing the osteogenic differentiation of stem cells with a single-dose administration and without requiring repeated osteogenic supplementation, the newly synthesized multi-bioactive hybrid nanocarrier shows great potential for bone tissue engineering applications.

Leachable-Free Fabrication of Hydrogel Foams Enabling Homogeneous Viability of Encapsulated Cells in Large-Volume Constructs.

The popularity of cell-laden injectable hydrogels has steeply increased due to their compatibility with minimally invasive surgical procedures. However, the diffusion of indispensable molecules for cell survival through bulk hydrogel structures, particularly oxygen, is often limited to micrometric distances, often hampering cell viability or uniform tissue formation in constructs with clinically relevant sizes. The introduction of micropores in hydrogels or the use of oxygen-generating materials has enabled combining advantages of porous 3D scaffolds with the injectability properties of in situ-solidifying hydrogels. Here, cell-laden injectable gelatin methacryloyl (GelMA) foams are fabricated using a single polymer formulation. Air bubbles are introduced into GelMA solutions using a simple-to-implement method based on pulling/pushing the solution through a syringe. Human mesenchymal stem cells derived from the adipose tissue (hASCs) cultured in bulk hydrogels (diameter c.a. 5 mm) show low permanence in the core of the materials and stain for factors associated to hypoxia (hypoxia-inducible factor-1 alpha (HIF-1α)) after 7 days of culture. In opposition, cells cultured in optimized foams do not stain for HIF-1α, show high permanence, homogeneous viability, and consistent phenotype in the whole depth of the biomaterials, while secreting increased amounts of regenerative growth factors to the surrounding medium.