Harnessing Biomaterials to Amplify Immunity in Aged Mice through T Memory Stem Cells.

The durability of a protective immune response generated by a vaccine depends on its ability to induce long-term T cell immunity, which tends to decline in aging populations. The longest protection appears to arise from T memory stem cells (TMSCs) that confer high expandability and effector functions when challenged. Here we engineered artificial antigen presenting cells (aAPC) with optimized size, stiffness and activation signals to induce human and mouse CD8+ TMSCs in vitro. This platform was optimized as a vaccine booster of TMSCs (Vax-T) with prolonged release of small-molecule blockade of the glycogen synthase kinase-3ß together with target antigens. By using SARS-CoV-2 antigen as a model, we show that a single injection of Vax-T induces durable antigen-specific CD8+ TMSCs in young and aged mice, and generates humoral responses at a level stronger than or similar to soluble vaccines. This Vax-T approach can boost long-term immunity to fight infectious diseases, cancer, and other diseases.

Precise Engineering of Growth Factor Presentation Using Extracellular Microenvironment-Mimicking Microfluidic Microparticles.

One of the main challenges in tissue engineering is finding a way to deliver specific growth factors (GFs) with precise spatiotemporal control over their presentation. Here, we report a novel strategy for generating microscale carriers with enhanced affinity for high content loading suitable for the sustained and localized delivery of GFs. Our developed microparticles can be injected locally and sustainably release encapsulated growth factors for up to 28 days. Fine-tuning of particles' size, affinity, microstructures, and release kinetics is achieved using a microfluidic system along with bioconjugation techniques. We also describe an innovative 3D micromixer platform to control the formation of core-shell particles based on superaffinity using a polymer-peptide conjugate for further tuning of release kinetics and delayed degradation. Chitosan shells block the burst release of encapsulated GFs and enable their sustained delivery for up to 10 days. The matched release profiles and degradation provide the local tissues with biomimetic, developmental-biologic-compatible signals to maximize regenerative effects. The versatility of this approach is verified using three different therapeutic proteins, including human bone morphogenetic protein-2 (rhBMP-2), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1α). As in vivo morphogenesis is typically driven by the combined action of several growth factors, the proposed technique can be developed to generate a library of GF-loaded particles with designated release profiles.

A mechanical niche promotes the rejuvenation of blood stem cells.

Zhang et al.1 show that the mechanical properties of a three-dimensional (3D) hydrogel can enhance the secretion of niche factors from bone marrow stromal cells, which in turn promotes the maintenance of hematopoietic stem cells (HSCs) and reverses aging hallmarks in HSCs.

Systemic enhancement of antitumour immunity by peritumourally implanted immunomodulatory macroporous scaffolds.

A tumour microenvironment abundant in regulatory T (Treg) cells aids solid tumours to evade clearance by effector T cells. Systemic strategies to suppress Treg cells or to augment immunity can elicit autoimmune side effects, cytokine storms and other toxicities. Here we report the design, fabrication and therapeutic performance of a biodegradable macroporous scaffold, implanted peritumourally, that releases a small-molecule inhibitor of transforming growth factor ß to suppress Treg cells, chemokines to attract effector T cells and antibodies to stimulate them. In two mouse models of aggressive tumours, the implant boosted the recruitment and activation of effector T cells into the tumour and depleted it of Treg cells, which resulted in an 'immunological abscopal effect' on distant metastases and in the establishment of long-term memory that impeded tumour recurrence. We also show that the scaffold can be used to deliver tumour-antigen-specific T cells into the tumour. Peritumourally implanted immunomodulatory scaffolds may represent a general strategy to enhance T-cell immunity and avoid the toxicities of systemic therapies.

Cell-Taxi: Mesenchymal Cells Carry and Transport Clusters of Cancer Cells.

Cell clusters that collectively migrate from primary tumors appear to be far more potent in forming distant metastases than single cancer cells. A better understanding of the collective cell migration phenomenon and the involvement of various cell types during this process is needed. Here, an in vitro platform based on inverted-pyramidal microwells to follow and quantify the collective migration of hundreds of tumor cell clusters at once is developed. These results indicate that mesenchymal stromal cells (MSCs) or cancer-associated fibroblasts (CAFs) in the heterotypic tumor cell clusters may facilitate metastatic dissemination by transporting low-motile cancer cells in a Rac-dependent manner and that extracellular vesicles secreted by mesenchymal cells only play a minor role in this process. Furthermore, in vivo studies show that cancer cell spheroids containing MSCs or CAFs have faster spreading rates. These findings highlight the active role of co-traveling stromal cells in the collective migration of tumor cell clusters and may help in developing better-targeted therapies.

Immunomodulatory Microneedle Patch for Periodontal Tissue Regeneration.

Periodontal diseases are caused by microbial infection and the recruitment of destructive immune cells. Current therapies mainly deal with bacteria elimination, but the regeneration of periodontal tissues remains a challenge. Here we developed a modular microneedle (MN) patch that delivered both antibiotic and cytokines into the local gingival tissue to achieve immunomodulation and tissue regeneration. This MN patch included a quickly dissolvable gelatin membrane for an immediate release of tetracycline and biodegradable GelMA MNs that contained tetracycline-loaded poly(lactic-co-glycolic acid) nanoparticles and cytokine-loaded silica microparticles for a sustained release. Antibiotic release completely inhibited bacteria growth, and the release of IL-4 and TGF-ß induced the repolarization of anti-inflammatory macrophages and the formation of regulatory T cells in vitro. In vivo delivery of MN patch into periodontal tissues suppressed proinflammatory factors and promoted pro-regenerative signals and tissue healing, which demonstrated the therapeutic potential of local immunomodulation for tissue regeneration.

Engineered Delivery of Dental Stem-Cell-Derived Extracellular Vesicles for Periodontal Tissue Regeneration.

Periodontal disease begins as an inflammatory response to a bacterial biofilm deposited around the teeth, which over time leads to the destruction of tooth-supporting structures and consequently tooth loss. Conventional treatment strategies show limited efficacy in promoting regeneration of damaged periodontal tissues. Here, a delivery platform is developed for small extracellular vesicles (sEVs) derived from gingival mesenchymal stem cells (GMSCs) to treat periodontitis. EVs can achieve comparable therapeutic effects to their cells of origin. However, the short half-lives of EVs after their administration along with their rapid diffusion away from the delivery site necessitate frequent administration to achieve therapeutic benefits. To address these issues, "dual delivery" microparticles are engineered enabling microenvironment-sensitive release of EVs by metalloproteinases at the affected site along with antibiotics to suppress bacterial biofilm growth. GMSC sEVs are able to decrease the secretion of pro-inflammatory cytokines by monocytes/macrophages and T cells, suppress T-cell activation, and induce the formation of T regulatory cells (Tregs) in vitro and in a rat model of periodontal disease. One-time administration of immunomodulatory GMSC sEV-decorated microparticles leads to a significant improvement in regeneration of the damaged periodontal tissue. This approach will have potential clinical applications in the regeneration of a variety of tissues.

Biomaterial-based immunoengineering to fight COVID-19 and infectious diseases.

Infection by SARS-CoV-2 virus often induces the dysregulation of immune responses, tissue damage, and blood clotting. Engineered biomaterials from the nano- to the macroscale can provide targeted drug delivery, controlled drug release, local immunomodulation, enhanced immunity, and other desirable functions to coordinate appropriate immune responses and to repair tissues. Based on the understanding of COVID-19 disease progression and immune responses to SARS-CoV-2, we discuss possible immunotherapeutic strategies and highlight biomaterial approaches from the perspectives of preventive immunization, therapeutic immunomodulation, and tissue healing and regeneration. Successful development of biomaterial platforms for immunization and immunomodulation will not only benefit COVID-19 patients, but also have broad applications for a variety of infectious diseases.

Augmenting T-cell responses to tumors by in situ nanomanufacturing.

Recent innovations in immunoregulatory treatments have demonstrated both the impressive potential and vital role of T cells in fighting cancer. These treatments come at a cost, with systemic side effects including life-threatening autoimmunity and immune dysregulation the norm. Here, we developed an approach to locally synthesize immune therapies and in this way, avoid systemic toxicity. Rather than just encapsulating cytokines, we endowed our nanoparticles with transcriptional and translational machinery to make cytokines locally, in situ, and on demand (activated by light). We demonstrated the capabilities of these particles in vitro and in vivo, in a mouse model of melanoma, and showed that tumor-infiltrating T cells were more highly activated in the context of these "microfactory" particles that make the synthetic cytokine.

Engineered Hydrogels for Brain Tumor Culture and Therapy.

Brain tumors' severity ranges from benign to highly aggressive and invasive. Bioengineering tools can assist in understanding the pathophysiology of these tumors from outside the body and facilitate development of suitable antitumoral treatments. Here, we first describe the physiology and cellular composition of brain tumors. Then, we discuss the development of three-dimensional tissue models utilizing brain tumor cells. In particular, we highlight the role of hydrogels in providing a biomimetic support for the cells to grow into defined structures. Microscale technologies, such as electrospinning and bioprinting, and advanced cellular models aim to mimic the extracellular matrix and natural cellular localization in engineered tumor tissues. Lastly, we review current applications and prospects of hydrogels for therapeutic purposes, such as drug delivery and co-administration with other therapies. Through further development, hydrogels can serve as a reliable option for in vitro modeling and treatment of brain tumors for translational medicine.

T-cell activation is modulated by the 3D mechanical microenvironment.

T cells recognize mechanical forces through a variety of cellular pathways, including mechanical triggering of both the T-cell receptor (TCR) and integrin LFA-1. Here we show that T cells can recognize forces arising from the mechanical rigidity of the microenvironment. We fabricated 3D scaffold matrices with mechanical stiffness tuned to the range 4-40 kPa and engineered them to be microporous, independently of stiffness. We cultured T cells and antigen presenting cells within the matrices and studied T-cell activation by flow cytometry and live-cell imaging. We found that there was an augmentation of T-cell activation, proliferation, and migration speed in the context of mechanically stiffer 3D matrices as compared to softer materials. These results show that T cells can sense their 3D mechanical environment and alter both their potential for activation and their effector responses in different mechanical environments. A 3D scaffold of tunable stiffness and consistent microporosity offers a biomaterial advancement for both translational applications and reductionist studies on the impact of tissue microenvironmental factors on cellular behavior.

In situ bone tissue engineering using gene delivery nanocomplexes.

Gene delivery offers promising outcomes for functional recovery or regeneration of lost tissues at cellular and tissue levels. However, more efficient carriers are needed to safely and locally delivery of genetic materials. Herein, we demonstrate microfluidic-assisted synthesis of plasmid DNA (pDNA)-based nanocomplexe (NC) platforms for bone tissue regeneration. pDNA encoding human bone morphogenesis protein-2 (BMP-2) was used as a gene of interest. Formation and fine-tuning of nanocomplexes (NCs) between pDNA and chitosan (CS) as carriers were performed using a micromixer platform. Flow characteristics were adjusted to tune mixing time and consequently size, zeta potential, and compactness of assembled NCs. Subsequently, NCs were immobilized on a nanofibrous Poly(ε-caprolactone) (PCL) scaffold functionalized with metalloprotease-sensitive peptide (MMP-sensitive). This construct can provide an environmental-sensitive and localized gene delivery platform. Osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs) was studied using chemical and biological assays. The presented results converge to indicate a great potential of the developed methodology for in situ bone tissue engineering using immobilized microfluidic-synthesized gene delivery nanocomplexes, which is readily expandable in the field of regenerative nanomedicine. STATEMENT OF SIGNIFICANCE: In this study, we demonstrate microfluidic-assisted synthesis of plasmid DNA (pDNA)-based nanocomplexes (NCs) platforms for bone tissue regeneration. We used pDNA encoding human bone morphogenesis protein-2 (BMP-2) as the gene of interest. Using micromixer platform nanocomplexes (NCs) between pDNA and chitosan (CS) were fabricated and optimized. NCs were immobilized on a nanofibrous polycaprolactone scaffold functionalized with metalloprotease-sensitive peptide. In vitro and in vivo assays confirmed the osteogenic differentiation of mesenchymal stem cells (MSCs). The obtained data indicated great potential of the developed methodology for in situ bone tissue engineering using immobilized microfluidic-synthesized gene delivery nanocomplexes, which is readily expandable in the field of regenerative nanomedicine.

An engineered cell-laden adhesive hydrogel promotes craniofacial bone tissue regeneration in rats.

Cell-laden hydrogels are widely used in tissue engineering and regenerative medicine. However, many of these hydrogels are not optimized for use in the oral environment, where they are exposed to blood and saliva. To address these challenges, we engineered an alginate-based adhesive, photocrosslinkable, and osteoconductive hydrogel biomaterial (AdhHG) with tunable mechanical properties. The engineered hydrogel was used as an injectable mesenchymal stem cell (MSC) delivery vehicle for craniofacial bone tissue engineering applications. Subcutaneous implantation in mice confirmed the biodegradability, biocompatibility, and osteoconductivity of the hydrogel. In a well-established rat peri-implantitis model, application of the adhesive hydrogel encapsulating gingival mesenchymal stem cells (GMSCs) resulted in complete bone regeneration around ailing dental implants with peri-implant bone loss. Together, we have developed a distinct bioinspired adhesive hydrogel with tunable mechanical properties and biodegradability that effectively delivers patient-derived dental-derived MSCs. The hydrogel is photocrosslinkable and, due to the presence of MSC aggregates and hydroxyapatite microparticles, promotes bone regeneration for craniofacial tissue engineering applications.

Nano-in-Micro Dual Delivery Platform for Chronic Wound Healing Applications.

Here, we developed a combinatorial delivery platform for chronic wound healing applications. A microfluidic system was utilized to form a series of biopolymer-based microparticles with enhanced affinity to encapsulate and deliver vascular endothelial growth factor (VEGF). Presence of heparin into the structure can significantly increase the encapsulation efficiency up to 95% and lower the release rate of encapsulated VEGF. Our in vitro results demonstrated that sustained release of VEGF from microparticles can promote capillary network formation and sprouting of endothelial cells in 2D and 3D microenvironments. These engineered microparticles can also encapsulate antibiotic-loaded nanoparticles to offer a dual delivery system able to fight bacterial infection while promoting angiogenesis. We believe this highly tunable drug delivery platform can be used alone or in combination with other wound care products to improve the wound healing process and promote tissue regeneration.

Injectable Drug-Releasing Microporous Annealed Particle Scaffolds for Treating Myocardial Infarction.

Intramyocardial injection of hydrogels offers great potential for treating myocardial infarction (MI) in a minimally invasive manner. However, traditional bulk hydrogels generally lack microporous structures to support rapid tissue ingrowth and biochemical signals to prevent fibrotic remodeling toward heart failure. To address such challenges, a novel drug-releasing microporous annealed particle (drugMAP) system is developed by encapsulating hydrophobic drug-loaded nanoparticles into microgel building blocks via microfluidic manufacturing. By modulating nanoparticle hydrophilicity and pregel solution viscosity, drugMAP building blocks are generated with consistent and homogeneous encapsulation of nanoparticles. In addition, the complementary effects of forskolin (F) and Repsox (R) on the functional modulations of cardiomyocytes, fibroblasts, and endothelial cells in vitro are demonstrated. After that, both hydrophobic drugs (F and R) are loaded into drugMAP to generate FR/drugMAP for MI therapy in a rat model. The intramyocardial injection of MAP gel improves left ventricular functions, which are further enhanced by FR/drugMAP treatment with increased angiogenesis and reduced fibrosis and inflammatory response. This drugMAP platform represents a new generation of microgel particles for MI therapy and will have broad applications in regenerative medicine and disease therapy.

Augmentation of T-Cell Activation by Oscillatory Forces and Engineered Antigen-Presenting Cells.

Activation of T cells by antigen presenting cells (APCs) initiates their proliferation, cytokine production, and killing of infected or cancerous cells. We and others have shown that T-cell receptors require mechanical forces for triggering, and these forces arise during the interaction of T cells with APCs. Efficient activation of T cells in vitro is necessary for clinical applications. In this paper, we studied the impact of combining mechanical, oscillatory movements provided by an orbital shaker with soft, biocompatible, artificial APCs (aAPCs) of various sizes and amounts of antigen. We showed that these aAPCs allow for testing the strength of signal delivered to T cells, and enabled us to confirm that that absolute amounts of antigen engaged by the T cell are more important for activation than the density of antigen. We also found that when our aAPCs interact with T cells in the context of an oscillatory mechanoenvironment, they roughly double antigenic signal strength, compared to conventional, static culture. Combining these effects, our aAPCs significantly outperformed the commonly used Dynabeads. We finally demonstrated that tuning the signal strength down to a submaximal "sweet spot" allows for robust expansion of induced regulatory T cells. In conclusion, augmenting engineered aAPCs with mechanical forces offers a novel approach for tuning of T-cell activation and differentiation.

Hierarchically Patterned Polydopamine-Containing Membranes for Periodontal Tissue Engineering.

Periodontitis is a common chronic inflammatory disease that affects tooth-supporting tissues. We engineer a multifunctional periodontal membrane for the guided tissue regeneration of lost periodontal tissues. The major drawback of current periodontal membranes is the lack of tissue regeneration properties. Here, a series of nanofibrous membranes based on poly(ε-caprolactone) with tunable biochemical and biophysical properties were developed for periodontal tissue regeneration. The engineered membranes were surface coated using biomimetic polydopamine to promote the adhesion of therapeutic proteins and cells. We demonstrate successful cellular localization on the surface of the engineered membrane by morphological patterning. Polydopamine accelerates osteogenic differentiation of dental-derived stem cells by promoting hydroxyapatite mineralization. Such multiscale designs can mimic the complex extracellular environment of periodontal tissue and serve as functional tissue constructs for periodontal regeneration. In a periodontal defect model in rats, our engineered periodontal membrane successfully promoted the regeneration of periodontal tissue and bone repair. Altogether, our data demonstrate that our biomimetic membranes have potential as protein/cell delivery platforms for periodontal tissue engineering.

Polyserotonin Nanoparticles as Multifunctional Materials for Biomedical Applications.

Serotonin-based nanoparticles represent a class of previously unexplored multifunctional nanoplatforms with potential biomedical applications. Serotonin, under basic conditions, self-assembles into monodisperse nanoparticles via autoxidation of serotonin monomers. To demonstrate potential applications of polyserotonin nanoparticles for cancer therapeutics, we show that these particles are biocompatible, exhibit photothermal effects when exposed to near-infrared radiation, and load the chemotherapeutic drug doxorubicin, releasing it contextually and responsively in specific microenvironments. Quantum mechanical and molecular dynamics simulations were performed to interrogate the interactions between surface-adsorbed drug molecules and polyserotonin nanoparticles. To investigate the potential of polyserotonin nanoparticles for in vivo targeting, we explored their nano-bio interfaces by conducting protein corona experiments. Polyserotonin nanoparticles had reduced surface-protein interactions under biological conditions compared to polydopamine nanoparticles, a similar polymer material widely investigated for related applications. These findings suggest that serotonin-based nanoparticles have advantages as drug-delivery platforms for synergistic chemo- and photothermal therapy associated with limited nonspecific interactions.

Mechanobiological Mimicry of Helper T Lymphocytes to Evaluate Cell-Biomaterials Crosstalk.

The unique properties of immune cells have inspired many efforts in engineering advanced biomaterials capable of mimicking their behaviors. However, an inclusive model capable of mimicking immune cells in different situations remains lacking. Such models can provide invaluable data for understanding immune-biomaterial crosstalk. Inspired by CD4+ T cells, polymeric microparticles with physicochemical properties similar to naïve and active T cells are engineered. A lipid coating is applied to enhance their resemblance and provide a platform for conjugation of desired antibodies. A novel dual gelation approach is used to tune the elastic modulus and flexibility of particles, which also leads to elongated circulation times. Furthermore, the model is enriched with magnetic particles so that magnetotaxis resembles the chemotaxis of cells. Also, interleukin-2, a proliferation booster, and interferon-γ cytokines are loaded into the particles to manipulate the fates of killer T cells and mesenchymal stem cells, respectively. The penetration of these particles into 3D environments is studied to provide in vitro models of immune-biomaterials crosstalk. This biomimicry model enables optimization of design parameters required for engineering more efficient drug carriers and serves as a potent replica for understanding the mechanical behavior of immune cells.

Cytokine Secreting Microparticles Engineer the Fate and the Effector Functions of T-Cells.

T-cell immunotherapy is a promising approach for cancer, infection, and autoimmune diseases. However, significant challenges hamper its therapeutic potential, including insufficient activation, delivery, and clonal expansion of T-cells into the tumor environment. To facilitate T-cell activation and differentiation in vitro, core-shell microparticles are developed for sustained delivery of cytokines. These particles are enriched by heparin to enable a steady release of interleukin-2 (IL-2), the major T-cell growth factor, over 10+ d. The controlled delivery of cytokines is used to steer lineage specification of cultured T-cells. This approach enables differentiation of T-cells into central memory and effector memory subsets. It is shown that the sustained release of stromal cell-derived factor 1α could accelerate T-cell migration. It is demonstrated that CD4+ T-cells could be induced to high concentrations of regulatory T-cells through controlled release of IL-2 and transforming growth factor beta. It is found that CD8+ T-cells that received IL-2 from microparticles are more likely to gain effector functions as compared with traditional administration of IL-2. Culture of T-cells within 3D scaffolds that contain IL-2-secreting microparticles enhances proliferation as compared with traditional, 2D approaches. This yield a new method to control the fate of T-cells and ultimately to new strategies for immune therapy.