Durable lymph-node expansion is associated with the efficacy of therapeutic vaccination.

Following immunization, lymph nodes dynamically expand and contract. The mechanical and cellular changes enabling the early-stage expansion of lymph nodes have been characterized, yet the durability of such responses and their implications for adaptive immunity and vaccine efficacy are unknown. Here, by leveraging high-frequency ultrasound imaging of the lymph nodes of mice, we report more potent and persistent lymph-node expansion for animals immunized with a mesoporous silica vaccine incorporating a model antigen than for animals given bolus immunization or standard vaccine formulations such as alum, and that durable and robust lymph-node expansion was associated with vaccine efficacy and adaptive immunity for 100 days post-vaccination in a mouse model of melanoma. Immunization altered the mechanical and extracellular-matrix properties of the lymph nodes, drove antigen-dependent proliferation of immune and stromal cells, and altered the transcriptional features of dendritic cells and inflammatory monocytes. Strategies that robustly maintain lymph-node expansion may result in enhanced vaccination outcomes.

Designed modular protein hydrogels for biofabrication.

Designing proteins that fold and assemble over different length scales provides a way to tailor the mechanical properties and biological performance of hydrogels. In this study, we designed modular proteins that self-assemble into fibrillar networks and, as a result, form hydrogel materials with novel properties. We incorporated distinct functionalities by connecting separate self-assembling (A block) and cell-binding (B block) domains into single macromolecules. The number of self-assembling domains affects the rigidity of the fibers and the final storage modulus G' of the materials. The mechanical properties of the hydrogels could be tuned over a broad range (G' = 0.1 - 10 kPa), making them suitable for the cultivation and differentiation of multiple cell types, including cortical neurons and human mesenchymal stem cells. Moreover, we confirmed the bioavailability of cell attachment domains in the hydrogels that can be further tailored for specific cell types or other biological applications. Finally, we demonstrate the versatility of the designed proteins for application in biofabrication as 3D scaffolds that support cell growth and guide their function. STATEMENT OF SIGNIFICANCE: Designed proteins that enable the decoupling of biophysical and biochemical properties within the final material could enable modular biomaterial engineering. In this context, we present a designed modular protein platform that integrates self-assembling domains (A blocks) and cell-binding domains (B blocks) within a single biopolymer. The linking of assembly domains and cell-binding domains this way provided independent tuning of mechanical properties and inclusion of biofunctional domains. We demonstrate the use of this platform for biofabrication, including neural cell culture and 3D printing of scaffolds for mesenchymal stem cell culture and differentiation. Overall, this work highlights how informed design of biopolymer sequences can enable the modular design of protein-based hydrogels with independently tunable biophysical and biochemical properties.

Metal-based porous hydrogels for highly conductive biomaterial scaffolds.

Multielectrode arrays are fabricated from thin films of highly conductive and ductile metals which cannot mimic the natural environment of biological tissues. These properties limit the conformability of the electrode to the underlying target tissue, and present challenges in developing seamless interfaces. By introducing porous, hydrogel materials that are embedded with metal additives, highly conductive hydrogels can be formed. Tuning the hydrogel composition, % volume and aspect ratio of different additive(s), and the processing conditions of these composite materials can alter the mechanical and electrical properties. The resulting materials have a high surface area, and can be used as biomaterial scaffolds to support the growth of macrophages for 5 days. Further optimization can enable the use of the materials for the electrodes in implantable arrays, or as living electrode platforms to study and modulate various cellular cultures. These advancements would benefit both in vivo and in vitro applications of tissue engineering.

Conductive Hydrogel Scaffolds for the 3D Localization and Orientation of Fibroblasts.

Dermal wounds and their healing are a collection of complex, multistep processes which are poorly recapitulated by existing 2D in vitro platforms. Biomaterial scaffolds that support the 3D growth of cell cultures can better resemble the native dermal environment, while bioelectronics has been used as a tool to modulate cell proliferation, differentiation, and migration. A porous conductive hydrogel scaffold which mimics the properties of dermis, while promoting the viability and growth of fibroblasts is described. As these scaffolds are also electrically conductive, the application of exogenous electrical stimulation directs the migration of cells across and/or through the material. The mechanical properties of the scaffold, as well as the amplitude and/or duration of the electrical pulses, are independently tunable and further influence the resulting fibroblast networks. This biomaterial platform may enable better recapitulation of wound healing and can be utilized to develop and screen therapeutic interventions.

Driving electrochemical reactions at the microscale using CMOS microelectrode arrays.

Precise control of pH values at electrode interfaces enables the systematic investigation of pH-dependent processes by electrochemical means. In this work, we employed high-density complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs) as miniaturized systems to induce and confine electrochemical reactions in areas corresponding to the pitch of single electrodes (17.5 µm). First, we present a strategy for generating localized pH patterns on the surface of the CMOS MEA with unprecedented spatial resolution. Leveraging the versatile routing capabilities of the switch matrix beneath the CMOS MEA, we created arbitrary combinations of anodic and cathodic electrodes and hence pH patterns. Moreover, we utilized the system to produce polymeric surface patterns by additive and subtractive methods. For additive patterning, we controlled the in situ formation of polydopamine at the microelectrode surface through oxidation of free dopamine above a threshold pH > 8.5. For subtractive patterning, we removed cell-adhesive poly-L-lysine from the electrode surface and backfilled the voids with antifouling polymers. Such polymers were chosen to provide a proof-of-concept application of controlling neuronal growth via electrochemically-induced patterns on the CMOS MEA surface. Importantly, our platform is compatible with commercially available high-density MEAs and requires no custom equipment, rendering the findings generalizable and accessible.

Matrix viscoelasticity controls spatiotemporal tissue organization.

Biomolecular and physical cues of the extracellular matrix environment regulate collective cell dynamics and tissue patterning. Nonetheless, how the viscoelastic properties of the matrix regulate collective cell spatial and temporal organization is not fully understood. Here we show that the passive viscoelastic properties of the matrix encapsulating a spheroidal tissue of breast epithelial cells guide tissue proliferation in space and in time. Matrix viscoelasticity prompts symmetry breaking of the spheroid, leading to the formation of invading finger-like protrusions, YAP nuclear translocation and epithelial-to-mesenchymal transition both in vitro and in vivo in a Arp2/3-complex-dependent manner. Computational modelling of these observations allows us to establish a phase diagram relating morphological stability with matrix viscoelasticity, tissue viscosity, cell motility and cell division rate, which is experimentally validated by biochemical assays and in vitro experiments with an intestinal organoid. Altogether, this work highlights the role of stress relaxation mechanisms in tissue growth dynamics, a fundamental process in morphogenesis and oncogenesis.

Tunable Conductive Hydrogel Scaffolds for Neural Cell Differentiation.

Multielectrode arrays would benefit from intimate engagement with neural cells, but typical arrays do not present a physical environment that mimics that of neural tissues. It is hypothesized that a porous, conductive hydrogel scaffold with appropriate mechanical and conductive properties could support neural cells in 3D, while tunable electrical and mechanical properties could modulate the growth and differentiation of the cellular networks. By incorporating carbon nanomaterials into an alginate hydrogel matrix, and then freeze-drying the formulations, scaffolds which mimic neural tissue properties are formed. Neural progenitor cells (NPCs) incorporated in the scaffolds form neurite networks which span the material in 3D and differentiate into astrocytes and myelinating oligodendrocytes. Viscoelastic and more conductive scaffolds produce more dense neurite networks, with an increased percentage of astrocytes and higher myelination. Application of exogenous electrical stimulation to the scaffolds increases the percentage of astrocytes and the supporting cells localize differently with the surrounding neurons. The tunable biomaterial scaffolds can support neural cocultures for over 12 weeks, and enable a physiologically mimicking in vitro platform to study the formation of neuronal networks. As these materials have sufficient electrical properties to be used as electrodes in implantable arrays, they may allow for the creation of biohybrid neural interfaces and living electrodes.

Correction: Actuated 3D microgels for single cell mechanobiology.

Correction for 'Actuated 3D microgels for single cell mechanobiology' by Berna Özkale et al., Lab Chip, 2022, 22, 1962-1970, https://doi.org/10.1039/D2LC00203E.

Mechanical checkpoint regulates monocyte differentiation in fibrotic niches.

Myelofibrosis is a progressive bone marrow malignancy associated with monocytosis, and is believed to promote the pathological remodelling of the extracellular matrix. Here we show that the mechanical properties of myelofibrosis, namely the liquid-to-solid properties (viscoelasticity) of the bone marrow, contribute to aberrant differentiation of monocytes. Human monocytes cultured in stiff, elastic hydrogels show proinflammatory polarization and differentiation towards dendritic cells, as opposed to those cultured in a viscoelastic matrix. This mechanically induced cell differentiation is blocked by inhibiting a myeloid-specific isoform of phosphoinositide 3-kinase, PI3K-γ. We further show that murine bone marrow with myelofibrosis has a significantly increased stiffness and unveil a positive correlation between myelofibrosis grading and viscoelasticity. Treatment with a PI3K-γ inhibitor in vivo reduced frequencies of monocyte and dendritic cell populations in murine bone marrow with myelofibrosis. Moreover, transcriptional changes driven by viscoelasticity are consistent with transcriptional profiles of myeloid cells in other human fibrotic diseases. These results demonstrate that a fibrotic bone marrow niche can physically promote a proinflammatory microenvironment.

Actuated 3D microgels for single cell mechanobiology.

We present a new cell culture technology for large-scale mechanobiology studies capable of generating and applying optically controlled uniform compression on single cells in 3D. Mesenchymal stem cells (MSCs) are individually encapsulated inside an optically triggered nanoactuator-alginate hybrid biomaterial using microfluidics, and the encapsulating network isotropically compresses the cell upon activation by light. The favorable biomolecular properties of alginate allow cell culture in vitro up to a week. The mechanically active microgels are capable of generating up to 15% compressive strain and forces reaching 400 nN. As a proof of concept, we demonstrate the use of the mechanically active cell culture system in mechanobiology by subjecting singly encapsulated MSCs to optically generated isotropic compression and monitoring changes in intracellular calcium intensity.

Materials for Implantable Surface Electrode Arrays: Current Status and Future Directions.

Surface electrode arrays are mainly fabricated from rigid or elastic materials, and precisely manipulated ductile metal films, which offer limited stretchability. However, the living tissues to which they are applied are nonlinear viscoelastic materials, which can undergo significant mechanical deformation in dynamic biological environments. Further, the same arrays and compositions are often repurposed for vastly different tissues rather than optimizing the materials and mechanical properties of the implant for the target application. By first characterizing the desired biological environment, and then designing a technology for a particular organ, surface electrode arrays may be more conformable, and offer better interfaces to tissues while causing less damage. Here, the various materials used in each component of a surface electrode array are first reviewed, and then electrically active implants in three specific biological systems, the nervous system, the muscular system, and skin, are described. Finally, the fabrication of next-generation surface arrays that overcome current limitations is discussed.

A Modular Biomaterial Scaffold-Based Vaccine Elicits Durable Adaptive Immunity to Subunit SARS-CoV-2 Antigens.

The coronavirus disease 2019 (COVID-19) pandemic demonstrates the importance of generating safe and efficacious vaccines that can be rapidly deployed against emerging pathogens. Subunit vaccines are considered among the safest, but proteins used in these typically lack strong immunogenicity, leading to poor immune responses. Here, a biomaterial COVID-19 vaccine based on a mesoporous silica rods (MSRs) platform is described. MSRs loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF), the toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid A (MPLA), and SARS-CoV-2 viral protein antigens slowly release their cargo and form subcutaneous scaffolds that locally recruit and activate antigen-presenting cells (APCs) for the generation of adaptive immunity. MSR-based vaccines generate robust and durable cellular and humoral responses against SARS-CoV-2 antigens, including the poorly immunogenic receptor binding domain (RBD) of the spike (S) protein. Persistent antibodies over the course of 8 months are found in all vaccine configurations tested and robust in vitro viral neutralization is observed both in a prime-boost and a single-dose regimen. These vaccines can be fully formulated ahead of time or stored lyophilized and reconstituted with an antigen mixture moments before injection, which can facilitate its rapid deployment against emerging SARS-CoV-2 variants or new pathogens. Together, the data show a promising COVID-19 vaccine candidate and a generally adaptable vaccine platform against infectious pathogens.

Biomimetic versus sintered macroporous calcium phosphate scaffolds enhanced bone regeneration and human mesenchymal stromal cell engraftment in calvarial defects.

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (ß-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic ß-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of ß-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to ß-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. STATEMENT OF SIGNIFICANCE: Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered ß-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to ß-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.

Viscoelastic surface electrode arrays to interface with viscoelastic tissues.

Living tissues are non-linearly elastic materials that exhibit viscoelasticity and plasticity. Man-made, implantable bioelectronic arrays mainly rely on rigid or elastic encapsulation materials and stiff films of ductile metals that can be manipulated with microscopic precision to offer reliable electrical properties. In this study, we have engineered a surface microelectrode array that replaces the traditional encapsulation and conductive components with viscoelastic materials. Our array overcomes previous limitations in matching the stiffness and relaxation behaviour of soft biological tissues by using hydrogels as the outer layers. We have introduced a hydrogel-based conductor made from an ionically conductive alginate matrix enhanced with carbon nanomaterials, which provide electrical percolation even at low loading fractions. Our combination of conducting and insulating viscoelastic materials, with top-down manufacturing, allows for the fabrication of electrode arrays compatible with standard electrophysiology platforms. Our arrays intimately conform to the convoluted surface of the heart or brain cortex and offer promising bioengineering applications for recording and stimulation.

Metabolic labeling and targeted modulation of dendritic cells.

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.

Microstructured thin-film electrode technology enables proof of concept of scalable, soft auditory brainstem implants.

Auditory brainstem implants (ABIs) provide sound awareness to deaf individuals who are not candidates for the cochlear implant. The ABI electrode array rests on the surface of the cochlear nucleus (CN) in the brainstem and delivers multichannel electrical stimulation. The complex anatomy and physiology of the CN, together with poor spatial selectivity of electrical stimulation and inherent stiffness of contemporary multichannel arrays, leads to only modest auditory outcomes among ABI users. Here, we hypothesized that a soft ABI could enhance biomechanical compatibility with the curved CN surface. We developed implantable ABIs that are compatible with surgical handling, conform to the curvature of the CN after placement, and deliver efficient electrical stimulation. The soft ABI array design relies on precise microstructuring of plastic-metal-plastic multilayers to enable mechanical compliance, patterning, and electrical function. We fabricated soft ABIs to the scale of mouse and human CN and validated them in vitro. Experiments in mice demonstrated that these implants reliably evoked auditory neural activity over 1 month in vivo. Evaluation in human cadaveric models confirmed compatibility after insertion using an endoscopic-assisted craniotomy surgery, ease of array positioning, and robustness and reliability of the soft electrodes. This neurotechnology offers an opportunity to treat deafness in patients who are not candidates for the cochlear implant, and the design and manufacturing principles are broadly applicable to implantable soft bioelectronics throughout the central and peripheral nervous system.

Programmable microencapsulation for enhanced mesenchymal stem cell persistence and immunomodulation.

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.

Multifunctional fibers for simultaneous optical, electrical and chemical interrogation of neural circuits in vivo.

Brain function depends on simultaneous electrical, chemical and mechanical signaling at the cellular level. This multiplicity has confounded efforts to simultaneously measure or modulate these diverse signals in vivo. Here we present fiber probes that allow for simultaneous optical stimulation, neural recording and drug delivery in behaving mice with high resolution. These fibers are fabricated from polymers by means of a thermal drawing process that allows for the integration of multiple materials and interrogation modalities into neural probes. Mechanical, electrical, optical and microfluidic measurements revealed high flexibility and functionality of the probes under bending deformation. Long-term in vivo recordings, optogenetic stimulation, drug perturbation and analysis of tissue response confirmed that our probes can form stable brain-machine interfaces for at least 2 months. We expect that our multifunctional fibers will permit more detailed manipulation and analysis of neural circuits deep in the brain of behaving animals than achievable before.