A scaffold vaccine to promote tumor antigen cross-presentation via sustained toll-like receptor-2 (TLR2) activation.

Cancer vaccination holds great promise for cancer treatment, but its effectiveness is hindered by suboptimal activation of CD8+ cytotoxic T lymphocytes, which are potent effectors to mediate anti-tumor immune responses. A possible solution is to switch antigen-presenting cells to present tumor antigens via the major histocompatibility complex class I (MHC-I) to CD8+ T cells - a process known as cross-presentation. To achieve this goal, we develop a three-dimensional (3D) scaffold vaccine to promote antigen cross-presentation by persisted toll-like receptor-2 (TLR2) activation after one injection. This vaccine comprises polysaccharide frameworks that "hook" TLR2 agonist (acGM) via tunable hydrophobic interactions and forms a 3D macroporous scaffold via click chemistry upon subcutaneous injection. Its retention-and-release of acGM enables sustained TLR2 activation in abundantly recruited dendritic cells in situ, inducing intracellular production of reactive oxygen species (ROS) in optimal kinetics that crucially promotes efficient antigen cross-presentation. The scaffold loaded with model antigen ovalbumin (OVA) or tumor specific antigen can generate potent immune responses against lung metastasis in B16-OVA-innoculated wild-type mice or spontaneous colorectal cancer in transgenic ApcMin/+ mice, respectively. Notably, it requires neither additional adjuvants nor external stimulation to function and can be adjusted to accommodate different antigens. The developed scaffold vaccine may represent a new, competent tool for next-generation personalized cancer vaccination.

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. Three-dimensional cellular materials may be useful to prolong the effect of cellular therapies and offer the possibility of creating structural volumetric constructs. Here, we report the manufacturing of shape-versatile fixed cell-based materials with immunomodulatory properties. Living cell aggregates with different shapes (spheres and centimeter-long fibers) were fixed using a method compatible with maintenance of structural integrity, robustness, and flexibility of three-dimensional constructs. The biological properties of living cells could be modulated before fixation, rendering an in vitro anti-inflammatory effect towards human macrophages, in line with a decreased activation of the NF-κB pathway that preponderantly correlated with the surface area of the materials. These findings were further corroborated in vivo in mouse skin wounds. Contact with fixed materials also reduced the proliferation of activated primary T lymphocytes, while promoting regulatory populations. We propose the fixation of cellular constructs as a versatile phenotypic stabilization method that can be easily implemented to prepare immunomodulatory materials with therapeutic potential. This article is protected by copyright. All rights reserved.

Photocrosslinkable microgels derived from human platelet lysates: injectable biomaterials for cardiac cell culture.

Cardiovascular diseases are a major global cause of morbidity and mortality, and they are often characterized by cardiomyocytes dead that ultimately leads to myocardial ischemia (MI). This condition replaces functional cardiac tissue with fibrotic scar tissue compromising heart function. Injectable systems for the in situ delivery of cells or molecules to assist during tissue repair have emerged as promising approaches for tissue engineering, particularly for myocardial repair. Methacryloyl platelet lysates (PLMA) have been employed for constructing full human-based 3D cell culture matrices and demonstrated potential for xeno-free applications. In this study, we propose using PLMA to produce microparticles (MPs) serving as anchors for cardiac and endothelial cells and ultimately as injectable systems for cardiac tissue repair. The herein reported PLMA MPs were produced by droplet microfluidics and showed great properties for cell attachment. More importantly, it is possible to show the capacity of PLMA MPs to serve as cell microcarriers even in the absence of animal-derived serum supplementation in the culture media.

Advances in Cell-Rich Inks for Biofabricating Living Architectures.

Advancing biofabrication toward manufacturing living constructs with well-defined architectures and increasingly biologically relevant cell densities is highly desired to mimic the biofunctionality of native human tissues. The formulation of tissue-like, cell-dense inks for biofabrication remains, however, challenging at various levels of the bioprinting process. Promising advances have been made toward this goal, achieving relatively high cell densities that surpass those found in conventional platforms, pushing the current boundaries closer to achieving tissue-like cell densities. On this focus, herein the overarching challenges in the bioprocessing of cell-rich living inks into clinically grade engineered tissues are discussed, as well as the most recent advances in cell-rich living ink formulations and their processing technologies are highlighted. Additionally, an overview of the foreseen developments in the field is provided and critically discussed.

In-Bath 3D Printing of Anisotropic Shape-Memory Cryogels Functionalized with Bone-Bioactive Nanoparticles.

Cryogels exhibit unique shape memory with full recovery and structural stability features after multiple injections. These constructs also possess enhanced cell permeability and nutrient diffusion when compared to typical bulk hydrogels. Volumetric processing of cryogels functionalized with nanosized units has potential to widen their biomedical applications, however this has remained challenging and relatively underexplored. In this study, we report a novel methodology that combines suspension 3D printing with directional freezing for the fabrication of nanocomposite cryogels with configurable anisotropy. When compared to conventional bulk or freeze-dried hydrogels, nanocomposite cryogel formulations exhibit excellent shape recovery (>95%) and higher pore connectivity. Suspension printing, assisted with a prechilled metal grid, was optimized to induce anisotropy. The addition of calcium- and phosphate-doped mesoporous silica nanoparticles into the cryogel matrix enhanced bioactivity toward orthopedic applications without hindering the printing process. Notably, the nanocomposite 3D printed cryogels exhibit injectable shape memory while also featuring a lamellar topography. The fabrication of these constructs was highly reproducible and exhibited potential for a cell-delivery injectable cryogel with no cytotoxicity to human-derived adipose stem cells. Hence, in this work, it was possible to combine a gravity defying 3D printed methodology with injectable and controlled anisotropic macroporous structures containing bioactive nanoparticles. This methodology ameliorates highly tunable injectable 3D printed anisotropic nanocomposite cryogels with a user-programmable degree of structural complexity.

Designing of a Multifunctional 3D-Printed Biomimetic Theragenerative Aerogel Scaffold via Mussel-Inspired Chemistry: Bioactive Glass Nanofiber-Incorporated Self-Assembled Silk Fibroin with Antibacterial, Antiosteosarcoma, and Osteoinductive Properties.

Biomaterial-mediated bone tissue engineering (BTE) offers an alternative, interesting approach for the restoration of damaged bone tissues in postsurgery osteosarcoma treatment. This study focused on synthesizing innovative composite inks, integrating self-assembled silk fibroin (SF), tannic acids (TA), and electrospun bioactive glass nanofibers 70SiO2-25CaO-5P2O5 (BGNF). By synergistically combining the unique characteristics of these three components through self-assembly and microextrusion-based three-dimensional (3D) printing, our goal was to produce durable and versatile aerogel-based 3D composite scaffolds. These scaffolds were designed to exhibit hierarchical porosity along with antibacterial, antiosteosarcoma, and bone regeneration properties. Taking inspiration from mussel foot protein attachment chemistry involving the coordination of dihydroxyphenylalanine (DOPA) amino acids with ferric ions (Fe3+), we synthesized a tris-complex catecholate-iron self-assembled composite gel. This gel formation occurred through the coordination of oxidized SF (SFO) with TA and polydopamine-modified BGNF (BGNF-PDA). The dynamic nature of the coordination ligand-metal bonds within the self-assembled SFO matrix provided excellent shear-thinning properties, allowing the SFO-TA-BGNF complex gel to be extruded through a nozzle, facilitating 3D printing into scaffolds with outstanding shape fidelity. Moreover, the developed composite aerogels exhibited multifaceted features, including NIR-triggered photothermal antibacterial and in vitro photothermal antiosteosarcoma properties. In vitro studies showcased their excellent biocompatibility and osteogenic features as seeded cells successfully differentiated into osteoblasts, promoting bone regeneration in 21 days. Through comprehensive characterizations and biological validations, our antibacterial scaffold demonstrated promise as an exceptional platform for concurrent bone regeneration and bone cancer therapy, setting the stage for their potential clinical application.

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.

Embedded 3D Bioprinting for Engineering Miniaturized In Vitro Tumor Models.

Embedded extrusion 3D bioprinting is a rapidly emerging additive manufacturing methodology that provides a precise spatial deposition of synthetic or natural-origin low-viscosity bioinks during the extrusion printing process. Such a strategy has to date unlocked the freeform extrusion biofabrication of complex micro-to-macro-scale living architectures for numerous applications, including tissue engineering and in vitro disease modeling. In this chapter, we describe a suspension bioprinting methodology leveraging a continuous viscoelastic biopolymer supporting bath functionalized with divalent calcium cations to enable a rapid processing of user-defined bioinks toward architecturally complex 3D in vitro tumor models. This highly simple and cost-effective viscoelastic supporting bath enables a full freeform biofabrication of cell-laden 3D tumor-mimetic architectures that exhibit structural stability in culture post-printing. The cytocompatibility of the supporting bath, its ease of removal from biofabricated living constructs, and its adaptability for processing different ECM-mimetic bioinks open avenues for multi-scale fabrication of numerous types of physiomimetic 3D tumor models for preclinical screening of candidate therapeutics.

Encapsulation of pristine and silica-coated human adipose-derived mesenchymal stem cells in gelatin colloidal hydrogels for tissue engineering and bioprinting applications.

Colloidal gels assembled from gelatin nanoparticles (GNPs) as particulate building blocks show strong promise to solve challenges in cell delivery and biofabrication, such as low cell survival and limited spatial retention. These gels offer evident advantages to facilitate cell encapsulation, but research on this topic is still limited, which hampers our understanding of the relationship between the physicochemical and biological properties of cell-laden colloidal gels. Human adipose-derived mesenchymal stem cells were successfully encapsulated in gelatin colloidal gels and evaluated their mechanical and biological performance over 7 days. The cells dispersed well within the gels without compromising gel cohesiveness, remained viable, and spread throughout the gels. Cells partially coated with silica were introduced into these gels, which increased their storage moduli and decreased their self-healing capacity after 7 days. This finding demonstrates the ability to modulate gel stiffness by incorporating cells partially coated with silica, without altering the solid content or introducing additional particles. Our work presents an efficient method for cell encapsulation while preserving gel integrity, expanding the applicability of colloidal hydrogels for tissue engineering and bioprinting. Overall, our study contributes to the design of improved cell delivery systems and biofabrication techniques.

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.

Advances in screening hyperthermic nanomedicines in 3D tumor models.

Hyperthermic nanomedicines are particularly relevant for tackling human cancer, providing a valuable alternative to conventional therapeutics. The early-stage preclinical performance evaluation of such anti-cancer treatments is conventionally performed in flat 2D cell cultures that do not mimic the volumetric heat transfer occurring in human tumors. Recently, improvements in bioengineered 3D in vitro models have unlocked the opportunity to recapitulate major tumor microenvironment hallmarks and generate highly informative readouts that can contribute to accelerating the discovery and validation of efficient hyperthermic treatments. Leveraging on this, herein we aim to showcase the potential of engineered physiomimetic 3D tumor models for evaluating the preclinical efficacy of hyperthermic nanomedicines, featuring the main advantages and design considerations under diverse testing scenarios. The most recent applications of 3D tumor models for screening photo- and/or magnetic nanomedicines will be discussed, either as standalone systems or in combinatorial approaches with other anti-cancer therapeutics. We envision that breakthroughs toward developing multi-functional 3D platforms for hyperthermia onset and follow-up will contribute to a more expedited discovery of top-performing hyperthermic therapies in a preclinical setting before their in vivo screening.

A Supramolecular Injectable Methacryloyl Chitosan-Tricine-Based Hydrogel with 3D Printing Potential for Tissue Engineering Applications.

Printable hydrogels have attracted significant attention as versatile, tunable, and spatiotemporally controlled biomaterials for tissue engineering (TE) applications. Several chitosan-based systems are reported presenting low or no solubility in aqueous solutions at physiological pH. Herein, a novel neutrally charged, biomimetic, injectable, and cytocompatible dual-crosslinked (DC) hydrogel system based on a double functionalized chitosan (CHT) with methacryloyl and tricine moieties (CHTMA-Tricine), completely processable at physiological pH, with promising three-dimensional (3D) printing potential is presented. Tricine, an amino acid typically used in biomedicine, is capable of establishing supramolecular interactions (H-bonds) and is never explored as a hydrogel component for TE. CHTMA-Tricine hydrogels demonstrate significantly greater toughness (ranging from 656.5 ± 82.2 to 1067.5 ± 121.5 kJ m-3 ) compared to CHTMA hydrogels (ranging from 382.4 ± 44.1 to 680.8 ± 104.5 kJ m-3 ), highlighting the contribution of the supramolecular interactions for the overall reinforced 3D structure provided by tricine moieties. Cytocompatibility studies reveal that MC3T3-E1 pre-osteoblasts cells remain viable for 6 days when encapsulated in CHTMA-Tricine constructs, with semi-quantitative analysis showing ≈80% cell viability. This system's interesting viscoelastic properties allow the fabrication of multiple structures, which couple with a straightforward approach, will open doors for the design of advanced chitosan-based biomaterials through 3D bioprinting for TE.

Bioactive Self-Regulated Liquified Microcompartments to Bioengineer Bone-Like Microtissues.

Designing a microenvironment that drives autonomous stromal cell differentiation toward osteogenesis while recapitulating the complexity of bone tissue remains challenging. In the current study, bone-like microtissues are created using electrohydrodynamic atomization to form two distinct liquefied microcapsules (mCAPs): i) hydroxypyridinone (HOPO)-modified gelatin (GH mCAPs, 7.5% w/v), and ii) HOPO-modified gelatin and dopamine-modified gelatin (GH+GD mCAPs, 7.5%+1.5% w/v). The ability of HOPO to coordinate with iron ions at physiological pH allows the formation of a semipermeable micro-hydrogel shell. In turn, the dopamine affinity for calcium ions sets a bioactive milieu for bone-like microtissues. After 21 days post encapsulation, GH and GH+GD mCAPs potentiate autonomous osteogenic differentiation of mesenchymal stem cells accompanied by collagen type-I gene upregulation, increased alkaline phosphatase (ALP) expression, and formation of mineralized extracellular matrix. However, the GH+GD mCAPs show higher levels of osteogenic markers starting on day 14, translating into a more advanced and organized mineralized matrix. The GH+GD system also shows upregulation of the receptor activator of nuclear factor kappa-B ligand (RANK-L) gene, enabling the autonomous osteoclastic differentiation of monocytes. These catechol-based mCAPs offer a promising approach to designing multifunctional and autonomous bone-like microtissues to study in vitro bone-related processes at the cell-tissue interface, angiogenesis, and osteoclastogenesis.

Recent Developments in Layer-by-Layer Assembly for Drug Delivery and Tissue Engineering Applications.

Surfaces with biological functionalities are of great interest for biomaterials, tissue engineering, biophysics, and for controlling biological processes. The layer-by-layer (LbL) assembly is a highly versatile methodology introduced 30 years ago, which consists of assembling complementary polyelectrolytes or biomolecules in a stepwise manner to form thin self-assembled films. In view of its simplicity, compatibility with biological molecules, and adaptability to any kind of supporting material carrier, this technology has undergone major developments over the past decades. Specific applications have emerged in different biomedical fields owing to the possibility to load or immobilize biomolecules with preserved bioactivity, to use an extremely broad range of biomolecules and supporting carriers, and to modify the film's mechanical properties via crosslinking. In this review, the focus is on the recent developments regarding LbL films formed as 2D or 3D objects for applications in drug delivery and tissue engineering. Possible applications in the fields of vaccinology, 3D biomimetic tissue models, as well as bone and cardiovascular tissue engineering are highlighted. In addition, the most recent technological developments in the field of film construction, such as high-content liquid handling or machine learning, which are expected to open new perspectives in the future developments of LbL, are presented.

The European Polysaccharide Network of Excellence (EPNOE) research roadmap 2040: Advanced strategies for exploiting the vast potential of polysaccharides as renewable bioresources.

Polysaccharides are among the most abundant bioresources on earth and consequently need to play a pivotal role when addressing existential scientific challenges like climate change and the shift from fossil-based to sustainable biobased materials. The Research Roadmap 2040 of the European Polysaccharide Network of Excellence (EPNOE) provides an expert's view on how future research and development strategies need to evolve to fully exploit the vast potential of polysaccharides as renewable bioresources. It is addressed to academic researchers, companies, as well as policymakers and covers five strategic areas that are of great importance in the context of polysaccharide related research: (I) Materials & Engineering, (II) Food & Nutrition, (III) Biomedical Applications, (IV) Chemistry, Biology & Physics, and (V) Skills & Education. Each section summarizes the state of research, identifies challenges that are currently faced, project achievements and developments that are expected in the upcoming 20 years, and finally provides outlines on how future research activities need to evolve.

Effect of high cyclic hydrostatic pressure on osteogenesis of mesenchymal stem cells cultured in liquefied micro-compartments.

Bone resident cells are constantly subjected to a range of distinct mechanical loadings, which generates a complex microenvironment. In particular, hydrostatic pressure (HP) has a key impact on modulation of cell function and fate determination. Although HP is a constant mechanical stimulus, its role in regulating the osteogenesis process within a defined 3D microenvironment has not been comprehensively elucidated. Perceiving how environmental factors regulate the differentiation of stem cells is essential for expanding their regenerative potential. Inspired by the mechanical environment of bone, this study attempted to investigate the influence of different ranges of cyclic HP on human adipose-derived mesenchymal stem cells (MSCs) encapsulated within a compartmentalized liquefied microenvironment. Taking advantage of the liquefied environment of microcapsules, MSCs were exposed to cyclic HP of 5 or 50 MPa, 3 times/week at 37 °C. Biological tests using fluorescence staining of F-actin filaments showed a noticeable improvement in cell-cell interactions and cellular network formation of MSCs. These observations were more pronounced in osteogenic (OST) condition, as confirmed by fluorescent staining of vinculin. More interestingly, there was a significant increase in alkaline phosphatase activity of MSCs exposed to 50 MPa magnitude of HP, even in the absence of osteoinductive factors. In addition, a greater staining area of both osteopontin and hydroxyapatite was detected in the 50 MPa/OST group. These findings highlight the benefit of hydrostatic pressure to regulate osteogenesis of MSCs as well as the importance of employing simultaneous biochemical and mechanical stimulation to accelerate the osteogenic potential of MSCs for biomedical purposes.

In situ generated hemostatic adhesives: From mechanisms of action to recent advances and applications.

Conventional surgical closure techniques, such as sutures, clips, or skin closure strips, may not always provide optimal wound closure and may require invasive procedures, which can result in potential post-surgical complications. As result, there is a growing demand for innovative solutions to achieve superior wound closure and improve patient outcomes. To overcome the abovementioned issues, in situ generated hemostatic adhesives/sealants have emerged as a promising alternative, offering a targeted, controllable, and minimally invasive procedure for a wide variety of medical applications. The aim of this review is to provide a comprehensive overview of the mechanisms of action and recent advances of in situ generated hemostatic adhesives, particularly protein-based, thermoresponsive, bioinspired, and photocrosslinkable formulations, as well as the design challenges that must be addressed. Overall, this review aims to enhance a comprehensive understanding of the latest advancements of in situ generated hemostatic adhesives and their mechanisms of action, with the objective of promoting further research in this field.

Advanced 3D Printing Strategies for the Controlled Delivery of Growth Factors.

The controlled delivery of growth factors (GFs) from tissue engineered constructs represents a promising strategy to improve tissue repair and regeneration. However, despite their established key role in tissue regeneration, the use of GFs is limited by their short half-life in the in vivo environment, their dose-dependent effectiveness, and their space- and time-dependent activity. Promising results have been obtained both in vitro and in vivo in animal models. Nevertheless, the clinical application of tissue engineered constructs releasing GFs is still challenging due to the several limitations and risks associated with their use. 3D printing and bioprinting, by allowing the microprecise spatial deposition of multiple materials and the fabrication of complex geometries with high resolution, offer advanced strategies for an optimal release of GFs from tissue engineered constructs. This review summarizes the strategies that have been employed to include GFs and their delivery system into biomaterials used for 3D printing applications to optimize their controlled release and to improve both the in vitro and in vivo regeneration processes. The approaches adopted to overcome the above-mentioned limitations are presented, showing the potential of the technology of 3D printing to get one step closer to clinical applications.

Novel Insights on the Role of Epigenetics in Androgen Receptor's Expression in Prostate Cancer.

The androgens/androgen receptor (AR) axis is the main therapeutic target in prostate cancer (PCa). However, while initially responsive, a subset of tumors loses AR expression through mechanisms putatively associated with epigenetic modifications. In this study, we assessed the link between the presence of CpG methylation in the 5'UTR and promoter regions of AR and loss of AR expression. Hence, we characterized and compared the methylation signature at CpG resolution of these regulatory regions in vitro, both at basal levels and following treatment with 5-aza-2-deoxycytidine (DAC) alone, or in combination with Trichostatin A (TSA). Our results showed heterogeneity in the methylation signature of AR negative cell lines and pinpointed the proximal promoter region as the most consistently methylated site in DU-145. Furthermore, this region was extremely resistant to the demethylating effects of DAC and was only significantly demethylated upon concomitant treatment with TSA. Nevertheless, no AR re-expression was detected at the mRNA or protein level. Importantly, after treatment, there was a significant increase in repressive histone marks at AR region 1 in DU-145 cells. Altogether, our data indicate that AR region 1 genomic availability is crucial for AR expression and that the inhibition of histone methyltransferases might hold promise for AR re-expression.