Gene delivery within hydrogel matrices can potentially direct mesenchymal stem cells (MSCs) towards a chondrogenic fate to promote regeneration of cartilage. Here, we investigated whether the mechanical properties of the hydrogel containing the gene delivery systems could enhance transfection and chondrogenic programming of primary human bone marrow-derived MSCs. We developed collagen-I-alginate interpenetrating polymer network hydrogels with tunable stiffness and adhesion properties. The hydrogels were activated with nanocomplexed SOX9 polynucleotides to direct chondrogenic differentiation of MSCs. MSCs transfected within the hydrogels showed higher expression of chondrogenic markers compared to MSCs transfected in 2D prior to encapsulation. The nanocomplex uptake and resulting expression of transfected SOX9 were jointly enhanced by increased stiffness and cell-adhesion ligand density in the hydrogels. Further, transfection of SOX9 effectively induced MSCs chondrogenesis and reduced markers of hypertrophy compared to control matrices. These findings highlight the importance of matrix stiffness and adhesion as design parameters in gene-activated matrices for regenerative medicine. STATEMENT OF SIGNIFICANCE: Gene-activated matrices (GAMs) are biodegradable polymer networks integrating gene therapies, and they are promising technologies for supporting tissue regeneration. Despite this interest, there is still limited information on how to rationally design these systems. Here, we provide a systematic study of the effect of matrix stiffness and cell adhesion ligands on gene transfer efficiency. We show that high stiffness and the presence of cell-binding sites promote transfection efficiency and that this result is related to more efficient internalization and trafficking of the gene therapies. GAMs with optimized mechanical properties can induce cartilage formation and result in tissues with better characteristics for articular cartilage tissue engineering as compared to previously described standard methods.
Targeted immunomodulation of dendritic cells (DCs) in vivo will enable manipulation of T-cell priming and amplification of anticancer immune responses, but a general strategy has been lacking. Here we show that DCs concentrated by a biomaterial can be metabolically labelled with azido groups in situ, which allows for their subsequent tracking and targeted modulation over time. Azido-labelled DCs were detected in lymph nodes for weeks, and could covalently capture dibenzocyclooctyne (DBCO)-bearing antigens and adjuvants via efficient Click chemistry for improved antigen-specific CD8 T-cell responses and antitumour efficacy. We also show that azido labelling of DCs allowed for in vitro and in vivo conjugation of DBCO-modified cytokines, including DBCO-IL-15/IL-15Rα, to improve priming of antigen-specific CD8 T cells. This DC labelling and targeted modulation technology provides an unprecedented strategy for manipulating DCs and regulating DC-T-cell interactions in vivo.
Synthetic antigen-presenting cells (APCs) are used to mediate scalable ex vivo T-cell expansion for adoptive cell therapy. Recently, we developed APC-mimetic scaffolds (APC-ms), which present signals to T cells in a physiological manner to mediate rapid and controlled T-cell expansion. APC-ms are composed of individual high-aspect-ratio silica microrods loaded with soluble mitogenic cues and coated with liposomes of defined compositions, to form supported lipid bilayers. Membrane-bound ligands for stimulation and co-stimulation of T-cell receptors are presented via the fluid, synthetic membranes, while mitogenic cues are released slowly from the microrods. In culture, interacting T cells assemble the individual APC-ms microrods into a biodegradable 3D matrix. Compared to conventional methods, APC-ms facilitates several-fold greater polyclonal T-cell expansion and improved antigen-specific enrichment of rare T-cell subpopulations. Here we provide a detailed protocol for APC-ms synthesis and use for human T-cell activation, and discuss important considerations for material design and T-cell co-culture. This protocol describes the facile assembly of APC-ms in ~4 h and rapid expansion or enrichment of relevant T-cell clones in <2 weeks, and is applicable for T-cell manufacturing and assay development.
Biomaterials with tunable biophysical properties hold great potential for tissue engineering. The adaptive immune system plays an important role in bone regeneration. Our goal is to investigate the regeneration potential of cell-laden alginate hydrogels depending on the immune status of the animal model. Specifically, the regeneration potential of rat mesenchymal stromal cell (MSC)-laden, void-forming alginate hydrogels, with a stiffness optimized for osteogenic differentiation, is studied in 5-mm critical-sized femoral defects, in both T cell-deficient athymic Rowett Nude (RNU) rats and immunocompetent Sprague Dawley rats. Bone volume fraction, bone mineral density, and tissue mineral density are higher for athymic RNU nude rats 6 weeks postsurgery. In addition, these animals show a significantly higher number of total cells and cells with non-lymphocyte morphology at the defect site, while the number of cells with lymphocyte-like morphology is lower. Hydrogel degradation is slower and the remaining alginate fragments are surrounded by a thicker fibrous capsule. Ossification islands originating from alginate residues suggest that encapsulated MSCs differentiate into the osteogenic lineage and initiate the mineralization process. However, this effect is insufficient to fully bridge the bone defect in both animal models. Alginate hydrogels can be used to deliver MSCs and thereby recruit endogenous cells through paracrine signaling, but additional osteogenic stimuli are needed to regenerate critical-sized segmental femoral defects. Impact statement T cell-deficient athymic RNU nude rats are commonly used to evaluate the regeneration potential of biomaterials in combination with cells of human origin. In this study, we show that the effect of mesenchymal stromal cell (MSC)-laden alginate hydrogels on bone regeneration differs depending of the immune status of the animal model. Furthermore, while alginate hydrogels are interesting materials to investigate the effect tunable biophysical properties on cell response and regeneration, their use in combination with rat MSCs is insufficient to fully bridge critical-sized segmental femoral defects. For this purpose, additional osteogenic stimuli such as growth factor delivery are necessary.
Acute myeloid leukaemia (AML) is a malignancy of haematopoietic origin that has limited therapeutic options. The standard-of-care cytoreductive chemotherapy depletes AML cells to induce remission, but is infrequently curative. An immunosuppressive AML microenvironment in the bone marrow and the paucity of suitable immunotherapy targets limit the induction of effective immune responses. Here, in mouse models of AML, we show that a macroporous-biomaterial vaccine that delivers the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), the Toll-like-receptor-9 agonist cytosine-guanosine oligodeoxynucleotide and one or multiple leukaemia antigens (in the form of a defined peptide antigen, cell lysates or antigens sourced from AML cells recruited in vivo) induces local immune-cell infiltration and activated dendritic cells, evoking a potent anti-AML response. The biomaterial-based vaccine prevented the engraftment of AML cells when administered as a prophylactic and when combined with chemotherapy, and eradicated established AML even in the absence of a defined vaccine antigen. Biomaterial-based AML vaccination can induce potent immune responses, deplete AML cells and prevent disease relapse.
Dysregulated physical stresses are generated during tumorigenesis that affect the surrounding compliant tissues including adipocytes. However, the effect of physical stressors on the behavior of adipocytes and their cross-talk with tumor cells remain elusive. Here, we demonstrate that compression of cells, resulting from various types of physical stresses, can induce dedifferentiation of adipocytes via mechanically activating Wnt/β-catenin signaling. The compression-induced dedifferentiated adipocytes (CiDAs) have a distinct transcriptome profile, long-term self-renewal, and serial clonogenicity, but do not form teratomas. We then show that CiDAs notably enhance human mammary adenocarcinoma proliferation both in vitro and in a xenograft model, owing to myofibrogenesis of CiDAs in the tumor-conditioned environment. Collectively, our results highlight unique physical interplay in the tumor ecosystem; tumor-induced physical stresses stimulate de novo generation of CiDAs, which feedback to tumor growth.
In emergency medicine, blood lactate levels are commonly measured to assess the severity and response to treatment of hypoperfusion-related diseases (e.g., sepsis, trauma, cardiac arrest). Clinical blood lactate testing is conducted with laboratory analyzers, leading to a delay of 3 h between triage and lactate result. Here, a fluorescence-based blood lactate assay, which can be utilized for bedside testing, based on measuring the hydrogen peroxide generated by the enzymatic oxidation of lactate is described. To establish a hydrogen peroxide assay, near-infrared cyanine derivatives are screened and sulfo-cyanine 7 is identified as a new horseradish peroxidase (HRP) substrate, which loses its fluorescence in presence of HRP and hydrogen peroxide. As hydrogen peroxide is rapidly cleared by erythrocytic catalase and glutathione peroxidase, sulfo-cyanine 7, HRP, and lactate oxidase are encapsulated in a liposomal reaction compartment. In lactate-spiked bovine whole blood, the newly developed lactate assay exhibits a linear response in a clinically relevant range after 10 min. Substituting lactate oxidase with glucose and alcohol oxidase allows for blood glucose, ethanol, and methanol biosensing, respectively. This easy-to-use, rapid, and versatile assay may be useful for the quantification of a variety of enzymatically oxidizable metabolites, drugs, and toxic substances in blood and potentially other biological fluids.
Mesenchymal stromal cells (MSCs) modulate immune cells to ameliorate multiple inflammatory pathologies. Biophysical signals that regulate this process are poorly defined. By engineering hydrogels with tunable biophysical parameters relevant to bone marrow where MSCs naturally reside, we show that soft extracellular matrix maximizes the ability of MSCs to produce paracrine factors that have been implicated in monocyte production and chemotaxis upon inflammatory stimulation by tumor necrosis factor-α (TNFα). Soft matrix increases clustering of TNF receptors, thereby enhancing NF-κB activation and downstream gene expression. Actin polymerization and lipid rafts, but not myosin-II contractility, regulate mechanosensitive activation of MSCs by TNFα. We functionally demonstrate that human MSCs primed with TNFα in soft matrix enhance production of human monocytes in marrow of xenografted mice and increase trafficking of monocytes via CCL2. The results suggest the importance of biophysical signaling in tuning inflammatory activation of stromal cells to control the innate immune system.
Allotransplantation offers the potential to restore the anatomy and function of injured tissues and organs, but typically requires life-long, systemic administration of immunosuppressive drugs to prevent rejection, which can result in serious complications. Targeting the immunosuppressive drug to the graft favors local tissue concentration versus systemic drug exposure and end-organ toxicity. This could reduce the overall dose and dosing frequency of immunosuppressive drugs, and improve the safety and efficacy of treatment. Here, we developed dibenzocyclooctyne (DBCO)-modified prodrugs of the immunosuppressive drugs tacrolimus, rapamycin and mycophenolic acid, and demonstrated their targeted conjugation both in vitro and in vivo to azido-modified hydrogels via Click chemistry. Such azido-modified hydrogels placed in transplanted tissues enable sustained local release of drugs, and could be repeatedly refilled with systemically administered acid-labile prodrugs after drug exhaustion. Thus, clickable prodrugs with degradable linkers provide new possibilities for graft targeted immunosuppression in the context of allotransplantation.
Cells encounter complex environments in vivo where they interact with the extracellular matrix, neighboring cells, and soluble cues, which together influence their fate and function. However, the interplay of these interactions and their collective impact on the regenerative effects of mesenchymal stromal cells (MSCs) remains insufficiently explored. Here, we show that 3D culture in microporous (~125 μm) hydrogels that passively promote cell-cell interactions sensitizes MSCs to growth factors, particularly to IGF-1. IGF-1 enhances MSC paracrine secretion activity, and application of secreted factors to myoblasts potently stimulates their migration and differentiation. In contrast, the paracrine activity of MSCs encapsulated in nanoporous (~10 nm) hydrogels remain unchanged. Blocking N-cadherin on MSCs abrogates the stimulatory effects of IGF-1 in microporous but not nanoporous hydrogels. The role of N-cadherin in regulating MSC function is further clarified by functionalizing alginates with the HAVDI peptide sequence that is derived from the extracellular domain of N-cadherin and that acts to mimic cell-cell interactions. MSCs encapsulated in nanoporous HAVDI-gels, but not in gels functionalized with a scrambled sequence, show heightened paracrine activity in response to IGF-1. These findings reveal how interactions with the matrix, neighboring cells, and soluble factors impact and maximize the regenerative potential of MSCs.
Ischemic diseases are a leading cause of mortality and can result in autoamputation of lower limbs. We explored the hypothesis that implantation of an antigen-releasing scaffold, in animals previously vaccinated with the same antigen, can concentrate T2 T cells and enhance vascularization of ischemic tissue. This approach may be clinically relevant, as all persons receiving childhood vaccines recommended by the Centers for Disease Control and Prevention have vaccines that contain aluminum, a T2 adjuvant. To test the hypothesis, mice with hindlimb ischemia, previously vaccinated with ovalbumin (OVA) and aluminum, received OVA-releasing scaffolds. Vaccinated mice receiving OVA-releasing scaffolds locally concentrated antigen-specific T2 T cells in the surrounding ischemic tissue. This resulted in local angiogenesis, increased perfusion in ischemic limbs, and reduced necrosis and enhanced regenerating myofibers in the muscle. These findings support the premise that antigen depots may provide a treatment for ischemic diseases in patients previously vaccinated with aluminum-containing adjuvants.
Wound infections cause inflammation, tissue damage and delayed healing that can lead to invasive infection and even death. The efficacy of systemic antibiotics is limited due to poor tissue penetration that is especially a problem in burn and blast wounds where the microcirculation is disrupted. Topical administration of antimicrobials is an attractive approach because it prevents infection and avoids systemic toxicity, while hydrogels are an appealing vehicle for topical drug delivery. They are easy to apply to the wound site by being injectable, the drug release properties can be controlled and their many characteristics, such as biodegradation, mechanical strength, and chemical and biological response to stimuli can be tailored. Hydrogels also create a moist wound environment that is beneficial for healing. The purpose of this study was to formulate an agarose hydrogel that contains high concentrations of minocycline or gentamicin and study its characteristics. Subsequently, the minocycline agarose hydrogel was tested in a porcine burn model and its effect as a prophylactic treatment was studied. The results demonstrated that 0.5 % agarose in water was the optimal concentration in terms of viscosity and pH. Bench testing at room temperature demonstrated that both antibiotics remained stable in the hydrogel for at least 7 days and both antibiotics demonstrated sustained release over the time of the experiment. The porcine burn experiment showed that prophylactic treatment with the agarose minocycline hydrogel decreased the burn depth and reduced the number of bacteria as efficiently as the commonly used silver sulfadiazine cream.
Inspired by embryonic wound closure, we present mechanically active dressings to accelerate wound healing. Conventional dressings passively aid healing by maintaining moisture at wound sites. Recent developments have focused on drug and cell delivery to drive a healing process, but these methods are often complicated by drug side effects, sophisticated fabrication, and high cost. Here, we present novel active adhesive dressings consisting of thermoresponsive tough adhesive hydrogels that combine high stretchability, toughness, tissue adhesion, and antimicrobial function. They adhere strongly to the skin and actively contract wounds, in response to exposure to the skin temperature. In vitro and in vivo studies demonstrate their efficacy in accelerating and supporting skin wound healing. Finite element models validate and refine the wound contraction process enabled by these active adhesive dressings. This mechanobiological approach opens new avenues for wound management and may find broad utility in applications ranging from regenerative medicine to soft robotics.
Enzymatically-degradable materials recapitulate the dynamic and reciprocal interactions between cells and their native microenvironment by allowing cells to actively shape the degradation process. In order to engineer a synthetic 3D environment enabling cells to orchestrate the degradation of the surrounding material, norbornene-modified alginate was crosslinked with two different peptide crosslinkers susceptible to cleavage by matrix metalloproteinases using UV-initiated thiol-ene chemistry. Resulting hydrogels were characterized for their initial mechanical and rheological properties, and their degradation behavior was measured by tracking changes in wet weight upon enzyme incubation. This process was found to be a function of the crosslinker type and enzyme concentration, indicating that degradation kinetics could be controlled and tuned. When mouse embryonic fibroblasts were encapsulated in 3D, cell number remained constant and viability was high in all materials, while cell spreading and extensive filopodia formation was observed only in the degradable gels, not in non-degradable controls. After implanting hydrogels into the backs of C57/Bl6 mice for 8 weeks, histological stainings of recovered gel remnants and surrounding tissue revealed higher tissue and cell infiltration into degradable materials compared to non-degradable controls. This alginate-based material platform with cell-empowered enzymatic degradation could prove useful in diverse tissue engineering contexts, such as regeneration and drug delivery.
Mesenchymal stem cell (MSC) therapies demonstrate particular promise in ameliorating diseases of immune dysregulation but are hampered by short in vivo cell persistence and inconsistencies in phenotype. Here, we demonstrate that biomaterial encapsulation into alginate using a microfluidic device could substantially increase in vivo MSC persistence after intravenous (i.v.) injection. A combination of cell cluster formation and subsequent cross-linking with polylysine led to an increase in injected MSC half-life by more than an order of magnitude. These modifications extended persistence even in the presence of innate and adaptive immunity-mediated clearance. Licensing of encapsulated MSCs with inflammatory cytokine pretransplantation increased expression of immunomodulatory-associated genes, and licensed encapsulates promoted repopulation of recipient blood and bone marrow with allogeneic donor cells after sublethal irradiation by a ∼2-fold increase. The ability of microgel encapsulation to sustain MSC survival and increase overall immunomodulatory capacity may be applicable for improving MSC therapies in general.
Microvascular muscle transfer is the gold standard for reanimation following chronic facial nerve paralysis, however, despite the regenerative capacity of peripheral motor axons, poor reinnervation often results in sub-optimal function. We hypothesized that injection of alginate hydrogels releasing growth factors directly into donor tissue would promote reinnervation, muscle regeneration, and function. A murine model of sciatic nerve ligation and neurorrhaphy was first used to assess the ability of gel delivery of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) to promote functional reinnervation. VEGF + IGF-1 gel delivery to aged mice resulted in prolonged ability to control toe movement, increased toe spreading, and improved static sciatic index score, indicative of improved sciatic nerve and neuromuscular junction function. Further, a 26% increase in muscle fiber area, and 2.8 and 3.0-fold increases in muscle contraction force and velocity, respectively, were found compared to blank alginate in the murine model. This strategy was subsequently tested in a rabbit model of craniofacial gracilis muscle transplantation. Electromyography demonstrated a 71% increase in compound muscle action potential 9 weeks after transplantation following treatment with VEGF + IGF-1 alginate, compared to blank alginate in the rabbit model. Improving functional innervation in transplanted muscle via a hydrogel source of growth factors may enhance the therapeutic outcomes of facial palsy treatments and, more broadly, muscle transplantations.
Recent innovations highlight the integration of diverse materials with synthetic and biological hydrogels. Examples include brain-machine interfaces, tissue regeneration, and soft ionic devices. Existing methods of strong adhesion mostly focus on the chemistry of bonds and the mechanics of dissipation, but largely overlook the molecular topology of connection. Here, we highlight the significance of molecular topology by designing a specific bond-stitch topology. The bond-stitch topology achieves strong adhesion between preformed hydrogels and various materials, where the hydrogels have no functional groups for chemical coupling, and the adhered materials have functional groups on the surface. The adhesion principle requires a species of polymer chains to form bond with a material through complementary functional groups, and form a network in situ that stitches with the polymer network of a hydrogel. We study the physics and chemistry of this topology, and describe its potential applications in medicine and engineering.
The key to understanding, harnessing, and manipulating natural biological processes for the benefit of tissue engineering lies in providing a controllable dynamic environment for tissue development in vitro while being able to track cell activity in real time. This work presents a multi-channel bioreactor specifically designed to enable on-line imaging of fluorescently labeled cells embedded in replicated 3D engineered constructs subjected to different flow conditions. The images are acquired in 3D using a standard upright confocal microscope and further analyzed and quantified by computer vision. The platform is used to characterize and quantify the pace and directionality of angiogenic processes induced by flow. The presented apparatus bears considerable potential to advance scientific research, from basic research pursuing the effect of flow versus static conditions on 3D scaffolds and cell types, to clinically oriented modeling in drug screening and cytotoxicity assays.