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The lymph node paracortex, also known as the T-cell zone, consists of a network of fibroblastic reticular cells (FRCs) that secrete chemokines to induce T-cell and dendritic cell trafficking into the paracortex. To model the lymph node paracortex, we utilized multi-channel microfluidic devices to engineer a 3D lymph node stromal network from human cultured FRCs embedded in a collagen I-fibrin hydrogel. In the hydrogel, the FRCs self-assembled into an interconnected network, secreted the extracellular matrix proteins entactin, collagen IV, and fibronectin, as well as expressed an array of immune cell trafficking chemokines. Although the engineered FRC network did not secrete characteristic CCR7-ligand chemokines (i.e. CCL19 and CCL21), human primary TNF-α matured monocyte-derived dendritic cells, CD45RA+T-cells, and CD45RA-T-cells migrated toward the lymph node stromal network to a greater extent than toward a blank hydrogel. Furthermore, the FRCs co-recruited dendritic cells and antigen-specific T-cells into the lymph node stromal network. This engineered lymph node stromal network may help evaluate how human dendritic cells and T-cells migrate into the lymph node paracortex via CCR7-independent mechanisms.

Mesenchymal stromal cells (MSCs) continue to be proposed for use in clinical trials to treat various diseases due to their therapeutic potential to pleiotropically influence endogenous regenerative processes, such as vasculogenesis. However, the functional heterogeneity of MSCs has hampered their clinical success and poses a significant manufacturing challenge with respect to MSC quality control. Here, we evaluated and qualified a quantitative bioassay based on an enhanced-throughput, microphysiological system to measure the specific paracrine bioactivity of MSCs to stimulate vasculogenesis as a measure of MSC potency. Using this novel bioassay, MSCs derived from multiple donors at different passages were co-cultured with human umbilical vein endothelial cells (HUVECs) and exhibited significant heterogeneity in vasculogenic potency between donors and cell passage. Using our microphysiological system (MPS)-based platform, we demonstrated that variations in MSC vasculogenic bioactivity were maintained when assayed across laboratories and operators. The differences in MSC vasculogenic bioactivity were also correlated with the baseline expression of several genes involved in vasculogenesis (hepatocyte growth factor (HGF), angiopoietin-1 (ANGPT)) or the production of matricellular proteins (fibronectin (FN), insulin-like growth factor-binding protein 7 (IGFBP7)). These findings emphasize the significant functional heterogeneity of MSCs in vasculogenic bioactivity and suggest that changes in baseline gene expression of vasculogenic or matricellular protein genes during manufacturing may affect this bioactivity. The development of a reliable and functionally relevant potency assay for measuring the specific vasculogenic bioactivity of manufactured MSCs will help to reliably assure their quality when used in appropriate clinical trials.

Mechanical stimulation (mechanotherapy) can promote skeletal muscle repair, but a lack of reproducible protocols and mechanistic understanding of the relation between mechanical cues and tissue regeneration limit progress in this field. To address these gaps, we developed a robotic device equipped with real-time force control and compatible with ultrasound imaging for tissue strain analysis. We investigated the hypothesis that specific mechanical loading improves tissue repair by modulating inflammatory responses that regulate skeletal muscle regeneration. We report that cyclic compressive loading within a specific range of forces substantially improves functional recovery of severely injured muscle in mice. This improvement is attributable in part to rapid clearance of neutrophil populations and neutrophil-mediated factors, which otherwise may impede myogenesis. Insights from this work will help advance therapeutic strategies for tissue regeneration broadly.

Mesenchymal stromal cells (MSCs) are increasingly combined with biomaterials to enhance their therapeutic properties, including their immunosuppressive function. However, clinical trials utilizing MSCs with or without biomaterials have shown limited success, potentially due to their functional heterogeneity across different donors and among different subpopulations of cells. Here, we evaluated the immunosuppressive capacity, as measured by the ability to reduce T-cell proliferation and activation, of interferon-gamma (IFN-γ)–licensed MSCs from multiple donors on fibrin and collagen hydrogels, the two most commonly utilized biomaterials in combination with MSCs in clinical trials worldwide according to ClinicalTrials.gov. Variations in the immunosuppressive capacity between IFN-γ–licensed MSC donors on the biomaterials correlated with the magnitude of indoleamine-2,3-dioxygenase activity. Immunosuppressive capacity of the IFN-γ–licensed MSCs depended on the α(V)/α(5) integrins when cultured on fibrin and on the α(2)/β(1) integrins when cultured on collagen. While all tested MSCs were nearly 100% positive for these integrins, sorted MSCs that expressed higher levels of α(V)/α(5) integrins demonstrated greater immunosuppressive capacity with IFN-γ licensing than MSCs that expressed lower levels of these integrins on fibrin. These findings were equivalent for MSCs sorted based on the α(2)/β(1) integrins on collagen. These results demonstrate the importance of integrin engagement to IFN-γ licensed MSC immunosuppressive capacity and that IFN-γ–licensed MSC subpopulations of varying immunosuppressive capacity can be identified by the magnitude of integrin expression specific to each biomaterial.

There are a growing number of globally approved products and clinical trials utilizing autologous and allogeneic therapeutic cells for applications in regenerative medicine and immunotherapies. However, there is a need to develop rapid and cost-effective methods for manufacturing therapeutically effective cells. Furthermore, the resulting manufactured cells may exhibit heterogeneities that result in mixed therapeutic outcomes. Engineering approaches that can provide distinct microenvironmental cues to these cells may be able to enhance the growth and characterization of these cell products. This mini-review describes strategies to potentially enhance the expansion of therapeutic cells with biomaterials and bioreactors, as well as to characterize the cell products with microphysiological systems. These systems can provide distinct cues to maintain the quality attributes of the cells and evaluate their function in physiologically relevant conditions.

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.

Although skeletal muscle can naturally regenerate in response to minor injuries, more severe damage and myopathies can cause irreversible loss of muscle mass and function. Cell therapies, while promising, have not yet demonstrated consistent benefit, likely due to poor survival of delivered cells. Biomaterials can improve muscle regeneration by presenting chemical and physical cues to muscle cells that mimic the natural cascade of regeneration. This brief review describes strategies for muscle repair utilizing biomaterials that can provide signals to either transplanted or host muscle cells. These strategies range from approaches that utilize biomaterials alone to those that combine biomaterials with exogenous growth factors, ex vivo cultured cells, and extensive culture time.

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.

Recruitment of antigen-specific T cells promotes vascularization in ischemic injuries in mice., 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 TH2 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 TH2 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 TH2 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.

Oxygen status and tissue dimensionality are critical determinants of tumor angiogenesis, a hallmark of cancer and an enduring target for therapeutic intervention. However, it is unclear how these microenvironmental conditions interact to promote neovascularization, due in part to a lack of comprehensive, unbiased data sets describing tumor cell gene expression as a function of oxygen levels within three-dimensional (3D) culture. Here, we utilized alginate-based, oxygen-controlled 3D tumor models to study the interdependence of culture context and the hypoxia response. Microarray gene expression analysis of tumor cells cultured in 2D versus 3D under ambient or hypoxic conditions revealed striking interdependence between culture dimensionality and hypoxia response, which was mediated in part by pro-inflammatory signaling pathways. In particular, interleukin-8 (IL-8) emerged as a major player in the microenvironmental regulation of the hypoxia program. Notably, this interaction between dimensionality and oxygen status via IL-8 increased angiogenic sprouting in a 3D endothelial invasion assay. Taken together, our data suggest that pro-inflammatory pathways are critical regulators of tumor hypoxia response within 3D environments that ultimately impact tumor angiogenesis, potentially providing important therapeutic targets. Furthermore, these results highlight the importance of pathologically relevant tissue culture models to study the complex physical and chemical processes by which the cancer microenvironment mediates new vessel formation.

Biomaterials may improve outcomes of endothelial progenitor-based therapies for the treatment of ischemic cardiovascular disease, due to their ability to direct cell behavior. We hypothesized that local, sustained delivery of exogenous vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF) from alginate hydrogels could increase recruitment of systemically infused endothelial progenitors to ischemic tissue, and subsequent neovascularization. VEGF and SDF were found to enhance in vitro adhesion and migration of outgrowth endothelial cells (OECs) and circulating angiogenic cells (CACs), two populations of endothelial progenitors, by twofold to sixfold, and nearly doubled recruitment to both ischemic and nonischemic muscle tissue in vivo. Local delivery of VEGF and SDF to ischemic hind-limbs in combination with systemic CAC delivery significantly improved functional perfusion recovery over OEC delivery, or either treatment alone. Compared with OECs, CACs were more responsive to VEGF and SDF treatment, promoted in vitro endothelial sprout formation in a paracrine manner more potently, and demonstrated greater influence on infiltrating inflammatory cells in vivo. These studies demonstrate that accumulation of infused endothelial progenitors can be enriched using biomaterial-based delivery of VEGF and SDF, and emphasize the therapeutic benefit of using CACs for the treatment of ischemia.

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.

Both physiological and pathological tissue remodeling (e.g., during wound healing and cancer, respectively) require new blood vessel formation via angiogenesis, but the underlying microenvironmental mechanisms remain poorly defined due in part to the lack of biologically relevant in vitro models. Here, we present a biomaterials-based microfluidic 3D platform for analysis of endothelial sprouting in response to morphogen gradients. This system consists of three lithographically defined channels embedded in type I collagen hydrogels. A central channel is coated with endothelial cells, and two parallel side channels serve as a source and a sink for the steady-state generation of biochemical gradients. Gradients of vascular endothelial growth factor (VEGF) promoted sprouting, whereby endothelial cell responsiveness was markedly dependent on cell density and vessel geometry regardless of treatment conditions. These results point toward mechanical and/or autocrine mechanisms that may overwhelm pro-angiogenic paracrine signaling under certain conditions. To date, neither geometrical effects nor cell density have been considered critical determinants of angiogenesis in health and disease. This biomimetic vessel platform demonstrated utility for delineating hitherto underappreciated contributors of angiogenesis, and future studies may enable important new mechanistic insights that will inform anti-angiogenic cancer therapy.

Targeting small molecules to diseased tissues as therapy or diagnosis is a significant challenge in drug delivery. Drug-eluting devices implanted during invasive surgery allow the controlled presentation of drugs at the disease site, but cannot be modified once the surgery is complete. We demonstrate that bioorthogonal click chemistry can be used to target circulating small molecules to hydrogels resident intramuscularly in diseased tissues. We also demonstrate that small molecules can be repeatedly targeted to the diseased area over the course of at least one month. Finally, two bioorthogonal reactions were used to segregate two small molecules injected as a mixture to two separate locations in a mouse disease model. These results demonstrate that click chemistry can be used for pharmacological drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences–Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis., National Cancer Institute (U.S.) (Grant U54CA143874)