The tumor microenvironment (TME) is gaining increasing attention in oncology, as it is recognized to be functionally important during tumor development and progression. Tumors are heterogeneous tissues that, in addition to tumor cells, contain tumor-associated cell types such as immune cells, fibroblasts, and endothelial cells. These other cells, together with the specific extracellular matrix (ECM), create a permissive environment for tumor growth. While the influence of tumor-infiltrating cells and mechanical properties of the ECM in tumor invasion and progression have been studied separately, their interaction within the complex TME and the epithelial -to-mesenchymal transition (EMT) is still unclear. In this work, we develop a 3D co-culture model of lung adenocarcinoma cells and macrophages in an interpenetrating network hydrogel, to investigate the influence of the macrophage phenotype and ECM stiffness in the induction of EMT. Rising ECM stiffness increases both tumor cell proliferation and invasiveness. The presence of tumor-associated macrophages and the ECM stiffness jointly contribute to an invasive phenotype, and modulate the expression of key EMT-related markers. Overall, these findings support the utility of in vitro 3D cancer models that allow one to study interactions among key components of the TME.
Short peptides are the minimal modality of antigen recognized by cellular immunity and are therefore considered a safe and highly specific source of antigen for vaccination. Nevertheless, successful peptide immunotherapy is limited by the short half-life of peptide antigens in vivo as well as their weak immunogenicity. We recently reported a vaccine strategy based on dendritic cell-recruiting Mesoporous Silica Rod (MSR) scaffolds to enhance T-cell responses against subunit antigen. In this study, we investigated the effect of covalently conjugating peptide antigens to MSRs to increase their retention in the scaffolds. Using both stable thioether and reducible disulfide linkages, peptide conjugation greatly increased peptide loading compared to passive adsorption. In vitro, Bone Marrow derived Dendritic Cells (BMDCs) could present Ovalbumin (OVA)-derived peptides conjugated to MSRs and induce antigen-specific T-cell proliferation. Stable conjugation decreased presentation in vitro while reducible conjugation maintained levels of presentation as high as soluble peptide. Compared to soluble peptide, in vitro, expansion of OT-II T-cells was not affected by adsorption or stable conjugation to MSRs but was enhanced with reversible conjugation to MSRs. Both conjugation schemes increased peptide residence time in MSR scaffolds in vivo compared to standard bolus injections or a simple adsorption method. When MSR scaffolds loaded with GM-CSF and CpG-ODN were injected subcutaneously, recruited dendritic cells could present antigen in situ with the stable conjugation increasing presentation capacity. Overall, this simple conjugation approach could serve as a versatile platform to efficiently incorporate peptide antigens in MSR vaccines and potentiate cellular responses.
Existing strategies to enhance peptide immunogenicity for cancer vaccination generally require direct peptide alteration, which, beyond practical issues, may impact peptide presentation and result in vaccine variability. Here, we report a simple adsorption approach using polyethyleneimine (PEI) in a mesoporous silica microrod (MSR) vaccine to enhance antigen immunogenicity. The MSR-PEI vaccine significantly enhanced host dendritic cell activation and T-cell response over the existing MSR vaccine and bolus vaccine formulations. Impressively, a single injection of the MSR-PEI vaccine using an E7 peptide completely eradicated large, established TC-1 tumours in about 80% of mice and generated immunological memory. When immunized with a pool of B16F10 or CT26 neoantigens, the MSR-PEI vaccine eradicated established lung metastases, controlled tumour growth and synergized with anti-CTLA4 therapy. Our findings from three independent tumour models suggest that the MSR-PEI vaccine approach may serve as a facile and powerful multi-antigen platform to enable robust personalized cancer vaccination.
A covalently crosslinked methacrylated (MA)-alginate cryogel vaccine has been previously shown to generate a potent response against murine melanoma, but is not mechanically robust and requires a large 16G needle for delivery. Here, covalent and ionic crosslinking of cryogels are combined with the hypothesis that this will result in a tough MA-alginate cryogel with improved injectability. All tough cryogels can be injected through a smaller, 18G needle without sustaining any damage, while covalently crosslinked-only cryogels break after injection. Cytosine-phosphodiester-guanine (CpG)-delivering tough cryogels effectively activate dendritic cells (DCs). Granulocyte macrophage colony-stimulating factor releasing tough cryogels recruit four times more DCs than blank gels by day 7 in vivo. The tough cryogel vaccine induces strong antigen-specific cytotoxic T-lymphocyte and humoral responses. These vaccines prevent tumor formation in 80% of mice inoculated with HER2/neu-overexpressing DD breast cancer cells. The MA-alginate tough cryogels provide a promising minimally invasive delivery platform for cancer vaccinations.
We present a novel ligand, 5-norbornene-2-nonanoic acid, which can be directly added during established quantum dot (QD) syntheses in organic solvents to generate "clickable" QDs at a few hundred nmol scale. This ligand has a carboxyl group at one terminus to bind to the surface of QDs and a norbornene group at the opposite end that enables straightforward phase transfer of QDs into aqueous solutions via efficient norbornene/tetrazine click chemistry. Our ligand system removes the traditional ligand-exchange step and can produce water-soluble QDs with a high quantum yield and a small hydrodynamic diameter of approximately 12 nm at an order of magnitude higher scale than previous methods. We demonstrate the effectiveness of our approach by incubating azido-functionalized CdSe/CdS QDs with 4T1 cancer cells that are metabolically labeled with a dibenzocyclooctyne-bearing unnatural sugar. The QDs exhibit high targeting efficiency and minimal nonspecific binding.
Controlled encapsulation and pairing of single cells within a confined 3D matrix can enable the replication of the highly ordered cellular structure of human tissues. Microgels with independently controlled compartments that can encapsulate cells within separately confined hydrogel matrices would provide precise control over the route of pairing single cells. Here, a one-step microfluidic method is presented to generate monodisperse multicompartment microgels that can be used as a 3D matrix to pair single cells in a highly biocompatible manner. A method is presented to induce microgels formation on chip, followed by direct extraction of the microgels from oil phase, thereby avoiding prolonged exposure of the microgels to the oil. It is further demonstrated that by entrapping stem cells with niche cells within separate but adjacent compartments of the microgels, it can create complex stem cell niche microenvironments in a controlled manner, which can serve as a useful tool for the study of cell-cell interactions. This microfluidic technique represents a significant step toward high-throughput single cells encapsulation and pairing for the study of intercellular communications at single cell level, which is of significant importance for cell biology, stem cell therapy, and tissue engineering.
Physical hydrogels with tunable stress-relaxation and excellent stress recovery are formed from anionic polyurethanes via addition of acids, monovalent salts, or divalent salts. Gel properties can be widely adjusted through pH, salt valence, salt concentration, and monomer composition. We propose and investigate a novel gelation mechanism based on a colloidal system interacting through charge repulsion and chrage shielding, allowing a broad use of the material, from acidic (pH 4-5.5) to pH-neutral hydrogels with Young's moduli ranging from 10 to 140 kPa.
Therapeutic ex vivo T-cell expansion is limited by low rates and T-cell products of limited functionality. Here we describe a system that mimics natural antigen-presenting cells (APCs) and consists of a fluid lipid bilayer supported by mesoporous silica micro-rods. The lipid bilayer presents membrane-bound cues for T-cell receptor stimulation and costimulation, while the micro-rods enable sustained release of soluble paracrine cues. Using anti-CD3, anti-CD28, and interleukin-2, we show that the APC-mimetic scaffolds (APC-ms) promote two- to tenfold greater polyclonal expansion of primary mouse and human T cells compared with commercial expansion beads (Dynabeads). The efficiency of expansion depends on the density of stimulatory cues and the amount of material in the starting culture. Following a single stimulation, APC-ms enables antigen-specific expansion of rare cytotoxic T-cell subpopulations at a greater magnitude than autologous monocyte-derived dendritic cells after 2 weeks. APC-ms support over fivefold greater expansion of restimulated CD19 CAR-T cells than Dynabeads, with similar efficacy in a xenograft lymphoma model.
Hydrogels are under active development for controlled drug delivery, but their clinical translation is limited by low drug loading capacity, deficiencies in mechanical toughness and storage stability, and poor control over the drug release that often results in burst release and short release duration. This work reports a design of composite clay hydrogels, which simultaneously achieve a spectrum of mechanical, storage, and drug loading/releasing properties to address the critical needs from translational perspectives. The clay nanoparticles provide large surface areas to adsorb biological drugs, and assemble into microparticles that are physically trapped within and toughen hydrogel networks. The composite hydrogels demonstrate feasibility of storage, and extended release of large quantities of an insulin-like growth factor-1 mimetic protein (8 mg mL) over four weeks. The release rate is primarily governed by ionic exchange and can be upregulated by low pH, which is typical for injured tissues. A rodent model of Achilles tendon injury is used to demonstrate that the composite hydrogels allow for highly extended and localized release of biological drugs in vivo, while demonstrating biodegradation and biocompatibility. These attributes make the composite hydrogel a promising system for drug delivery and regenerative medicine.
Sustained, localized protein delivery can enhance the safety and activity of protein drugs in diverse disease settings. While hydrogel systems are widely studied as vehicles for protein delivery, they often suffer from rapid release of encapsulated cargo, leading to a narrow duration of therapy, and protein cargo can be denatured by incompatibility with the hydrogel crosslinking chemistry. In this work, we describe injectable nanocomposite hydrogels that are capable of sustained, bioactive, release of a variety of encapsulated proteins. Injectable and porous cryogels were formed by bio-orthogonal crosslinking of alginate using tetrazine-norbornene coupling. To provide sustained release from these hydrogels, protein cargo was pre-adsorbed to charged Laponite nanoparticles that were incorporated within the walls of the cryogels. The presence of Laponite particles substantially hindered the release of a number of proteins that otherwise showed burst release from these hydrogels. By modifying the Laponite content within the hydrogels, the kinetics of protein release could be precisely tuned. This versatile strategy to control protein release simplifies the design of hydrogel drug delivery systems.
STATEMENT OF SIGNIFICANCE: Here we present an injectable nanocomposite hydrogel for simple and versatile controlled release of therapeutic proteins. Protein release from hydrogels often requires first entrapping the protein in particles and embedding these particles within the hydrogel to allow controlled protein release. This pre-encapsulation process can be cumbersome, can damage the protein's activity, and must be optimized for each protein of interest. The strategy presented in this work simply premixes the protein with charged nanoparticles that bind strongly with the protein. These protein-laden particles are then placed within a hydrogel and slowly release the protein into the surrounding environment. Using this method, tunable release from an injectable hydrogel can be achieved for a variety of proteins. This strategy greatly simplifies the design of hydrogel systems for therapeutic protein release applications.
Mesenchymal stromal cells (MSCs) secrete paracrine factors that play crucial roles during tissue regeneration. Whether this paracrine function is influenced by the properties of biomaterials in general, and those used for cell delivery in particular, largely remains unexplored. Here, we investigated if three-dimensional culture in distinct microenvironments - nanoporous hydrogels (mean pore size ∼5 nm) and macroporous scaffolds (mean pore size ∼120 μm) - affects the secretion pattern of MSCs, and consequently leads to differential paracrine effects on target progenitor cells such as myoblasts. We report that compared to MSCs encapsulated in hydrogels, scaffold seeded MSCs show an enhanced secretion profile and exert beneficial paracrine effects on various myoblast functions including migration and proliferation. Additionally, we show that the heightened paracrine effects of scaffold seeded cells can in part be attributed to N-cadherin mediated cell-cell interactions during culture. In hydrogels, this physical interaction between cells is prevented by the encapsulating matrix. Functionally blocking N-cadherin negatively affected the secretion profile and paracrine effects of MSCs on myoblasts, with stronger effects observed for scaffold seeded compared to hydrogel encapsulated cells. Together, these findings demonstrate that the therapeutic potency of MSCs can be enhanced by biomaterials that promote cell-cell interactions.
Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its volume decreases, while its stiffness concomitantly increases. We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations, including varying substrate stiffness, cell spread area, and external osmotic pressure. The reduction of cell volume is a result of water efflux, which leads to a corresponding increase in intracellular molecular crowding. Furthermore, we find that changes in cell volume, and hence stiffness, alter stem-cell differentiation, regardless of the method by which these are induced. These observations reveal a surprising, previously unidentified relationship between cell stiffness and cell volume that strongly influences cell biology.
In-situ tissue regeneration aims to utilize the body's endogenous healing capacity through the recruitment of host stem or progenitor cells to an injury site. Stromal cell-derived factor-1α (SDF-1α) is widely discussed as a potent chemoattractant. Here we use a cell-free biomaterial-based approach to (i) deliver SDF-1α for the recruitment of endogenous bone marrow-derived stromal cells (BMSC) into a critical-sized segmental femoral defect in rats and to (ii) induce hydrogel stiffness-mediated osteogenic differentiation in-vivo. Ionically crosslinked alginate hydrogels with a stiffness optimized for osteogenic differentiation were used. Fast-degrading porogens were incorporated to impart a macroporous architecture that facilitates host cell invasion. Endogenous cell recruitment to the defect site was successfully triggered through the controlled release of SDF-1α. A trend for increased bone volume fraction (BV/TV) and a significantly higher bone mineral density (BMD) were observed for gels loaded with SDF-1α, compared to empty gels at two weeks. A trend was also observed, albeit not statistically significant, towards matrix stiffness influencing BV/TV and BMD at two weeks. However, over a six week time-frame, these effects were insufficient for bone bridging of a segmental femoral defect. While mechanical cues combined with ex-vivo cell encapsulation have been shown to have an effect in the regeneration of less demanding in-vivo models, such as cranial defects of nude rats, they are not sufficient for a SDF-1α mediated in-situ regeneration approach in segmental femoral defects of immunocompetent rats, suggesting that additional osteogenic cues may also be required.
STATEMENT OF SIGNIFICANCE: Stromal cell-derived factor-1α (SDF-1α) is a chemoattractant used to recruit host cells for tissue regeneration. The concept that matrix stiffness can direct mesenchymal stromal cell (MSC) differentiation into various lineages was described a decade ago using in-vitro experiments. Recently, alginate hydrogels with an optimized stiffness and ex-vivo encapsulated MSCs were shown to have an effect in the regeneration of skull defects of nude rats. Here, we apply this material system, loaded with SDF-1α and without encapsulated MSCs, to (i) recruit endogenous cells and (ii) induce stiffness-mediated osteogenic differentiation in-vivo, using as model system a load-bearing femoral defect in immunocompetent rats. While a cell-free approach is of great interest from a translational perspective, the current limitations are described.
Biomaterials have dramatically increased in functionality and complexity, allowing unprecedented control over the cells that interact with them. From these engineering advances arises the prospect of improved biomaterial-based therapies, yet practical constraints favour simplicity. Tools from the biology community are enabling high-resolution and high-throughput bioassays that, if incorporated into a biomaterial design framework, could help achieve unprecedented functionality while minimizing the complexity of designs by identifying the most important material parameters and biological outputs. However, to avoid data explosions and to effectively match the information content of an assay with the goal of the experiment, material screens and bioassays must be arranged in specific ways. By borrowing methods to design experiments and workflows from the bioprocess engineering community, we outline a framework for the incorporation of next-generation bioassays into biomaterials design to effectively optimize function while minimizing complexity. This framework can inspire biomaterials designs that maximize functionality and translatability.
Cartilage tissue equivalents formed from hydrogels containing chondrocytes could provide a solution for replacing damaged cartilage. Previous approaches have often utilized elastic hydrogels. However, elastic stresses may restrict cartilage matrix formation and alter the chondrocyte phenotype. Here we investigated the use of viscoelastic hydrogels, in which stresses are relaxed over time and which exhibit creep, for three-dimensional (3D) culture of chondrocytes. We found that faster relaxation promoted a striking increase in the volume of interconnected cartilage matrix formed by chondrocytes. In slower relaxing gels, restriction of cell volume expansion by elastic stresses led to increased secretion of IL-1β, which in turn drove strong up-regulation of genes associated with cartilage degradation and cell death. As no cell-adhesion ligands are presented by the hydrogels, these results reveal cell sensing of cell volume confinement as an adhesion-independent mechanism of mechanotransduction in 3D culture, and highlight stress relaxation as a key design parameter for cartilage tissue engineering.
DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.
Dental disease annually affects billions of patients, and while regenerative dentistry aims to heal dental tissue after injury, existing polymeric restorative materials, or fillings, do not directly participate in the healing process in a bioinstructive manner. There is a need for restorative materials that can support native functions of dental pulp stem cells (DPSCs), which are capable of regenerating dentin. A polymer microarray formed from commercially available monomers to rapidly identify materials that support DPSC adhesion is used. Based on these findings, thiol-ene chemistry is employed to achieve rapid light-curing and minimize residual monomer of the lead materials. Several triacrylate bulk polymers support DPSC adhesion, proliferation, and differentiation in vitro, and exhibit stiffness and tensile strength similar to existing dental materials. Conversely, materials composed of a trimethacrylate monomer or bisphenol A glycidyl methacrylate, which is a monomer standard in dental materials, do not support stem cell adhesion and negatively impact matrix and signaling pathways. Furthermore, thiol-ene polymerized triacrylates are used as permanent filling materials at the dentin-pulp interface in direct contact with irreversibly injured pulp tissue. These novel triacrylate-based biomaterials have potential to enable novel regenerative dental therapies in the clinic by both restoring teeth and providing a supportive niche for DPSCs.
Stem cells and their local microenvironment, or niche, communicate through mechanical cues to regulate cell fate and cell behaviour and to guide developmental processes. During embryonic development, mechanical forces are involved in patterning and organogenesis. The physical environment of pluripotent stem cells regulates their self-renewal and differentiation. Mechanical and physical cues are also important in adult tissues, where adult stem cells require physical interactions with the extracellular matrix to maintain their potency. In vitro, synthetic models of the stem cell niche can be used to precisely control and manipulate the biophysical and biochemical properties of the stem cell microenvironment and to examine how the mode and magnitude of mechanical cues, such as matrix stiffness or applied forces, direct stem cell differentiation and function. Fundamental insights into the mechanobiology of stem cells also inform the design of artificial niches to support stem cells for regenerative therapies.
Mechanical properties of the extracellular microenvironment are known to alter cellular behavior, such as spreading, proliferation or differentiation. Previous studies have primarily focused on studying the effect of matrix stiffness on cells using hydrogel substrates that exhibit purely elastic behavior. However, these studies have neglected a key property exhibited by the extracellular matrix (ECM) and various tissues; viscoelasticity and subsequent stress-relaxation. As muscle exhibits viscoelasticity, stress-relaxation could regulate myoblast behavior such as spreading and proliferation, but this has not been previously studied. In order to test the impact of stress relaxation on myoblasts, we created a set of two-dimensional RGD-modified alginate hydrogel substrates with varying initial elastic moduli and rates of relaxation. The spreading of myoblasts cultured on soft stress-relaxing substrates was found to be greater than cells on purely elastic substrates of the same initial elastic modulus. Additionally, the proliferation of myoblasts was greater on hydrogels that exhibited stress-relaxation, as compared to cells on elastic hydrogels of the same modulus. These findings highlight stress-relaxation as an important mechanical property in the design of a biomaterial system for the culture of myoblasts.
STATEMENT OF SIGNIFICANCE: This article investigates the effect of matrix stress-relaxation on spreading and proliferation of myoblasts by using tunable elastic and stress-relaxing alginate hydrogels substrates with different initial elastic moduli. Many past studies investigating the effect of mechanical properties on cell fate have neglected the viscoelastic behavior of extracellular matrices and various tissues and used hydrogels exhibiting purely elastic behavior. Muscle tissue is viscoelastic and exhibits stress-relaxation. Therefore, stress-relaxation could regulate myoblast behavior if it were to be incorporated into the design of hydrogel substrates. Altogether, we showed that stress-relaxation impacts myoblasts spreading and proliferation. These findings enable a better understanding of myoblast behavior on viscoelastic substrates and could lead to the design of more suitable substrates for myoblast expansion in vitro.