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.
Immunotherapeutic treatments in head and neck cancer clinical trials include cancer vaccines targeting foreign viral antigens or mutational neoantigens derived from cancer-expressed proteins. Anti-tumor immune responses place cancer cells under selective pressure to lose or downregulate target antigens; therefore, vaccination against virus- or host- "driver" oncogenes are proposed as a strategy to overcome immune escape. Herein, we demonstrate the impact of immunogenic viral antigens on anti-tumor response and immune editing in MOC2-E6E7, a syngeneic murine oral cancer cell line expressing HPV-16 E6 and E7 oncoproteins. Using orthotopic syngeneic models, we observed tumor growth kinetics of MOC2-E6E7 is delayed in immunocompetent mice compared to parental MOC2 tumors. In contrast, tumor growth remained similar in mice lacking adaptive immunity. MOC2-E6E7 tumors demonstrated an "inflamed" or immune-activated tumor microenvironment and greater infiltration of CD8 T cells compared to MOC2. By real-time PCR, we detected downregulation of and genes in MOC2-E6E7 tumors only in immunocompetent mice, suggesting the loss of ectopic viral antigen expression due to immune editing. We then assessed the efficacy of a biomaterials-based mesoporous silica rod (MSR) cancer vaccine targeting HPV-16 E7 in our model. Vaccination induced robust infiltration of antigen-specific CD8 T cells, which led to tumor growth delay and modestly prolonged survival in MOC2-E6E7 tumors. Increased efficacy was seen in a separate head and neck cancer tumor model, mEER, which obligately expresses E7 antigen. Collectively, our data highlight the need for both immunogenicity and 'driver' status of target antigens to be considered in cancer vaccine design.
Connective tissue is one of the four major types of animal tissue and plays essential roles throughout the human body. Genetic factors, aging, and trauma all contribute to connective tissue dysfunction and motivate the need for strategies to promote healing and regeneration. The goal here is to link a fundamental understanding of connective tissues and their multiscale properties to better inform the design and translation of novel biomaterials to promote their regeneration. Major clinical problems in adipose tissue, cartilage, dermis, and tendon are discussed that inspire the need to replace native connective tissue with biomaterials. Then, multiscale structure-function relationships in native soft connective tissues that may be used to guide material design are detailed. Several biomaterials strategies to improve healing of these tissues that incorporate biologics and are biologic-free are reviewed. Finally, important guidance documents and standards (ASTM, FDA, and EMA) that are important to consider for translating new biomaterials into clinical practice are highligted.
Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for multiple disorders, but deficiency and dysregulation of T cells limit its utility. Here we report a biomaterial-based scaffold that mimics features of T cell lymphopoiesis in the bone marrow. The bone marrow cryogel (BMC) releases bone morphogenetic protein-2 to recruit stromal cells and presents the Notch ligand Delta-like ligand-4 to facilitate T cell lineage specification of mouse and human hematopoietic progenitor cells. BMCs subcutaneously injected in mice at the time of HSCT enhanced T cell progenitor seeding of the thymus, T cell neogenesis and diversification of the T cell receptor repertoire. Peripheral T cell reconstitution increased ~6-fold in mouse HSCT and ~2-fold in human xenogeneic HSCT. Furthermore, BMCs promoted donor CD4 regulatory T cell generation and improved survival after allogeneic HSCT. In comparison to adoptive transfer of T cell progenitors, BMCs increased donor chimerism, T cell generation and antigen-specific T cell responses to vaccination. BMCs may provide an off-the-shelf approach for enhancing T cell regeneration and mitigating graft-versus-host disease in HSCT.
We present a methodology for building biologically inspired, soft microelectromechanical systems (MEMS) devices. Our strategy combines several advanced techniques including programmable colloidal self-assembly, light-harvesting with plasmonic nanotransducers, and in situ polymerization of compliant hydrogel mechanisms. We synthesize optomechanical microactuators using a template-assisted microfluidic approach in which gold nanorods coated with thermoresponsive poly(N-isopropylmethacrylamide) (pNIPMAM) polymer function as nanoscale building blocks. The resulting microactuators exhibit mechanical properties (4.8 ± 2.1 kPa stiffness) and performance metrics (relative stroke up to 0.3 and stress up to 10 kPa) that are comparable to that of bioengineered muscular constructs. Near-infrared (NIR) laser illumination provides effective spatiotemporal control over actuation (sub-micron spatial resolution at millisecond temporal resolution). Spatially modulated hydrogel photolithography guided by an experimentally validated finite element-based design methodology allows construction of compliant poly(ethylene glycol) diacrylate (PEGDA) mechanisms around the microactuators. We demonstrate the versatility of our approach by manufacturing a diverse array of microdevices including lever arms, continuum microrobots, and dexterous microgrippers. We present a microscale compression device that is developed for mechanical testing of three-dimensional biological samples such as spheroids under physiological conditions.
Materials that can mimic the fibrillar architecture of native extracellular matrix (ECM) while allowing for independent regulation of viscoelastic properties may serve as ideal, artificial ECM (aECM) to regulate cell functions. Here we describe an interpenetrating network of click-functionalized alginate, crosslinked with a combination of ionic and covalent crosslinking, and fibrillar collagen type I. Varying the mode and magnitude of crosslinking enables tunable stiffness and viscoelasticity, while altering neither the hydrogel's microscale architecture nor diffusional transport of molecules with molecular weight relevant to typical nutrients. Further, appropriately timing sequential ionic and covalent crosslinking permits self-assembly of collagen into fibrillar structures within the network. Culture of human mesenchymal stem cells (MSCs) in this mechanically-tunable ECM system revealed that MSC expression of immunomodulatory markers is differentially impacted by the viscoelasticity and stiffness of the matrix. Together, these results describe and validate a novel material system for investigating how viscoelastic mechanical properties of ECM regulate cellular behavior.
The clinical translation of regenerative therapy for the diseased heart, whether in the form of cells, macromolecules or small molecules, is hampered by several factors: the poor retention and short biological half-life of the therapeutic agent, the adverse side effects from systemic delivery, and difficulties with the administration of multiple doses. Here, we report the development and application of a therapeutic epicardial device that enables sustained and repeated administration of small molecules, macromolecules and cells directly to the epicardium via a polymer-based reservoir connected to a subcutaneous port. In a myocardial infarct rodent model, we show that repeated administration of cells over a four-week period using the epicardial reservoir provided functional benefits in ejection fraction, fractional shortening and stroke work, compared to a single injection of cells and to no treatment. The pre-clinical use of the therapeutic epicardial reservoir as a research model may enable insights into regenerative cardiac therapy, and assist the development of experimental therapies towards clinical use.
Persistence of inflammation, and associated limits in tissue regeneration, are believed to be due in part to the imbalance of M1 over M2 macrophages. Here, we hypothesized that providing a sustained source of an antiinflammatory polarizing cytokine would shift the balance of macrophages at a site of tissue damage to improve functional regeneration. Specifically, IL-4-conjugated gold nanoparticles (PA4) were injected into injured murine skeletal muscle, resulting in improved histology and an ∼40% increase in muscle force compared with mice treated with vehicle only. Macrophages were the predominant infiltrating immune cell, and treatment with PA4 resulted in an approximately twofold increase in the percentage of macrophages expressing the M2a phenotype and an approximately twofold decrease in M1 macrophages, compared with mice treated with vehicle only. Intramuscular injection of soluble IL-4 did not shift macrophage polarization or result in functional muscle improvements. Depletion of monocytes/macrophages eliminated the therapeutic effects of PA4, suggesting that improvement in muscle function was the result of M2-shifted macrophage polarization. The ability of PA4 to direct macrophage polarization in vivo may be beneficial in the treatment of many injuries and inflammatory diseases.
The past decade has witnessed the accelerating development of immunotherapies for cancer treatment. Immune checkpoint blockade therapies and chimeric antigen receptor (CAR)-T cell therapies have demonstrated clinical efficacy against a variety of cancers. However, issues including life-threatening off-target side effects, long processing times, limited patient responses and high cost still limit the clinical utility of cancer immunotherapies. Biomaterial carriers of these therapies, though, enable one to troubleshoot the delivery issues, amplify immunomodulatory effects, integrate the synergistic effect of different molecules and, more importantly, home and manipulate immune cells in vivo. In this Review, we will analyse thus-far developed immunomaterials for targeted modulation of dendritic cells, T cells, tumour-associated macrophages, myeloid-derived suppressor cells, B cells and natural killer cells, and summarize the promises and challenges of cell-targeted immunomodulation for cancer treatment.
Diabetic foot ulcers (DFUs) are a major complication of diabetes, and there is a critical need to develop novel cell- and tissue-based therapies to treat these chronic wounds. Induced pluripotent stem cells (iPSCs) offer a replenishing source of allogeneic and autologous cell types that may be beneficial to improve DFU wound-healing outcomes. However, the biologic potential of iPSC-derived cells to treat DFUs has not, to our knowledge, been investigated. Toward that goal, we have performed detailed characterization of iPSC-derived fibroblasts from both diabetic and nondiabetic patients. Significantly, gene array and functional analyses reveal that iPSC-derived fibroblasts from both patients with and those without diabetes are more similar to each other than were the primary cells from which they were derived. iPSC-derived fibroblasts showed improved migratory properties in 2-dimensional culture. iPSC-derived fibroblasts from DFUs displayed a unique biochemical composition and morphology when grown as 3-dimensional (3D), self-assembled extracellular matrix tissues, which were distinct from tissues fabricated using the parental DFU fibroblasts from which they were reprogrammed. In vivo transplantation of 3D tissues with iPSC-derived fibroblasts showed they persisted in the wound and facilitated diabetic wound closure compared with primary DFU fibroblasts. Taken together, our findings support the potential application of these iPSC-derived fibroblasts and 3D tissues to improve wound healing.-Kashpur, O., Smith, A., Gerami-Naini, B., Maione, A. G., Calabrese, R., Tellechea, A., Theocharidis, G., Liang, L., Pastar, I., Tomic-Canic, M., Mooney, D., Veves, A., Garlick, J. A. Differentiation of diabetic foot ulcer-derived induced pluripotent stem cells reveals distinct cellular and tissue phenotypes.
Degradable biomaterials aim to recapitulate the dynamic microenvironment that cells are naturally exposed to. By oxidizing the alginate polymer backbone, thereby rendering it susceptible to hydrolysis, and crosslinking it via norbornene-tetrazine click chemistry, we can control rheological, mechanical, and degradation properties of resulting hydrogels. Chemical modifications were confirmed by nuclear magnetic resonance (NMR) and the resulting mechanical properties measured by rheology and unconfined compression testing, demonstrating that these are both a function of norbornene coupling and oxidation state. The degradation behavior was verified by tracking mechanical and swelling behavior over time, showing that degradation could be decoupled from initial mechanical properties. The cell compatibility was assessed in 2D and 3D using a mouse pre-osteoblast cell line and testing morphology, proliferation, and viability. Cells attached, spread and proliferated in 2D and retained a round morphology and stable number in 3D, while maintaining high viability in both contexts over 7 days. Finally, oxidized and unoxidized control materials were implanted subcutaneously into the backs of C57/Bl6 mice, and recovered after 8 weeks. Histological staining revealed morphological differences and fibrous tissue infiltration only in oxidized materials. These materials with tunable and decoupled mechanical and degradation behavior could be useful in many tissue engineering applications.
Variations in a multitude of material microenvironmental properties have been observed across tissues in vivo, and these have profound effects on cell phenotype. Phenomenological experiments have suggested that certain of these features of the physical microenvironment, such as stiffness, could sensitize cells to other features; meanwhile, mechanistic studies have detailed a number of biophysical mechanisms for this sensing. However, the broad molecular consequences of these potentially complex and nonlinear interactions bridging from biophysical sensing to phenotype have not been systematically characterized, limiting the overall understanding and rational deployment of these biophysical cues. Here, we explore these interactions by employing a 3D cell culture system that allows for the independent control of culture substrate stiffness, stress relaxation, and adhesion ligand density to systematically explore the transcriptional programs affected by distinct combinations of biophysical parameters using RNA-seq. In mouse mesenchymal stem cells and human cortical neuron progenitors, we find dramatic coupling among these substrate properties, and that the relative contribution of each property to changes in gene expression varies with cell type. Motivated by the bioinformatic analysis, the stiffness of hydrogels encapsulating mouse mesenchymal stem cells was found to regulate the secretion of a wide range of cytokines, and to accordingly influence hematopoietic stem cell differentiation in a Transwell coculture model. These results give insights into how biophysical features are integrated by cells across distinct tissues and offer strategies to synthetic biologists and bioengineers for designing responses to a cell's biophysical environment.
Substrate stiffness has been recognized as an important regulator of cell fate and function, but an understanding of the full extent of processes affected by stiffness is lacking as its transcriptome-wide effects have not been mapped. This limited understanding has restricted the contexts in which engineers can employ stiffness as an engineering design parameter. To address these limitations, we performed RNA-seq on mesenchymal stem cells (MSCs) cultured in alginate hydrogels over a range of moduli to broadly map the transcriptome-wide changes associated with stiffness sensing. We found a large number of stiffness-sensitive genes, and that many genes respond to stiffness in nonlinear ways. Informed by these differential expression results, we explored a hypothesis related to current MSC clinical activity, and found that stiffness can regulate the expression of MSC immunomodulatory markers in response to cytokine stimulation. Overall, these results reveal previously unknown features of MSC stiffness response and demonstrate the value of coupling -omics approaches with biophysical experiments.
BACKGROUND: Dendritic cells (DC) induce adaptive responses against foreign antigens, and play an essential role in maintaining peripheral tolerance to self-antigens. Therefore they are involved in preventing fatal autoimmunity. Selective delivery of antigens to immature DC via the endocytic DEC-205 receptor on their surface promotes antigen-specific T cell tolerance, both by recessive and dominant mechanisms. We provide evidence that the induction of antigen-specific T cell tolerance is not a unique property of CD11cCD8DEC-205 DCs.
METHODS: We employed a fusion between αDCIR2 antibodies and the highly encephalitogenic peptide 139-151 of myelin-derived proteolipid protein (PLP), to target CD11c CD8 DCs with a DEC-205-DCIR2 phenotype in vivo, and to substantially improve clinical symptoms in the PLP-induced model of experimental autoimmune encephalomyelitis (EAE).
RESULTS: Consistent with previous studies targeting other cell surface receptors, EAE protection mediated by αDCIR2-PLP fusion antibody (Ab) depended on an immature state of targeted DCIR2 DCs. The mechanism of αDCIR2-PLP mAb function included the deletion of IL-17- and IFN-γ-producing pathogenic T cells, as well as the enhancement of regulatory T (Treg) cell activity. In contrast to the effect of αDEC-205 fusion antibodies, which involves extrathymic induction of a Foxp3 Treg cell phenotype in naïve CD4Foxp3 T cells, treatment of animals with DCIR2 fusion antibodies resulted in antigen-specific activation and proliferative expansion of natural Foxp3 Treg cells.
CONCLUSIONS: These results suggest that multiple mechanisms can lead to the expansion of the Treg population, depending on the DC subset and receptor targeted.
Ischemic diseases, such as peripheral artery disease, affect millions of people worldwide. While CD4 T-cells regulate angiogenesis and myogenesis, it is not understood how the phenotype of these adaptive immune cells regulate these regenerative processes. The secreted factors from different types of CD4 T-cells (Th1, Th2, Th17, and Treg) were utilized in a series of in vitro assays and delivered from an injectable alginate biomaterial into a murine model of ischemia to study their effects on vascular and skeletal muscle regeneration. Conditioned medium from Th2 and Th17 T-cells enhanced angiogenesis in vitro and in vivo, in part by directly stimulating endothelial sprouting. Th1 conditioned medium induced vascular regression in vitro and provided no benefit to angiogenesis in vivo. Th1, Th2, and Th17 conditioned medium, to varying extents, enhanced muscle precursor cell proliferation and inhibited their differentiation in vitro, and prolonged early stages of muscle regeneration in vivo. Treg conditioned medium had a moderate or no effect on these processes in vitro and no discernible effect in vivo. These findings suggest that Th2 and Th17 T-cells may enhance angiogenesis and myogenesis in ischemic injuries, which may be useful in the design of immunomodulatory biomaterials to treat these diseases.