Chronic inflammation contributes to the pathogenesis of all muscular dystrophies. Inflammatory T cells damage muscle, while regulatory T cells (Tregs) promote regeneration. We hypothesized that providing anti-inflammatory cytokines in dystrophic muscle would promote proregenerative immune phenotypes and improve function. Primary T cells from dystrophic (mdx) mice responded appropriately to inflammatory or suppressive cytokines. Subsequently, interleukin-4 (IL-4)- or IL-10-conjugated gold nanoparticles (PA4, PA10) were injected into chronically injured, aged, mdx muscle. PA4 and PA10 increased T cell recruitment, with PA4 doubling CD4+/CD8- T cells versus controls. Further, 50% of CD4+/CD8- T cells were immunosuppressive Tregs following PA4, versus 20% in controls. Concomitant with Treg recruitment, muscles exhibited increased fiber area and fourfold increases in contraction force and velocity versus controls. The ability of PA4 to shift immune responses, and improve dystrophic muscle function, suggests that immunomodulatory treatment may benefit many genetically diverse muscular dystrophies, all of which share inflammatory pathology.
Most bacterial vaccines work for a subset of bacterial strains or require the modification of the antigen or isolation of the pathogen before vaccine development. Here we report injectable biomaterial vaccines that trigger potent humoral and T-cell responses to bacterial antigens by recruiting, reprogramming and releasing dendritic cells. The vaccines are assembled from regulatorily approved products and consist of a scaffold with absorbed granulocyte-macrophage colony-stimulating factor and CpG-rich oligonucleotides incorporating superparamagnetic microbeads coated with the broad-spectrum opsonin Fc-mannose-binding lectin for the magnetic capture of pathogen-associated molecular patterns from inactivated bacterial-cell-wall lysates. The vaccines protect mice against skin infection with methicillin-resistant Staphylococcus aureus, mice and pigs against septic shock from a lethal Escherichia coli challenge and, when loaded with pathogen-associated molecular patterns isolated from infected animals, uninfected animals against a challenge with different E. coli serotypes. The strong immunogenicity and low incidence of adverse events, a modular manufacturing process, and the use of components compatible with current good manufacturing practice could make this vaccine technology suitable for responding to bacterial pandemics and biothreats.
Current methods to obtain mesenchymal stem cells (MSCs) involve sampling, culturing, and expanding of primary MSCs from adipose, bone marrow, and umbilical cord tissues. However, the drawbacks are the limited numbers of total cells in MSC pools, and their decaying stemness during in vitro expansion. As an alternative resource, recent ceiling culture methods allow the generation of dedifferentiated fat cells (DFATs) from mature adipocytes. Nevertheless, this process of spontaneous dedifferentiation of mature adipocytes is laborious and time-consuming. This paper describes a modified protocol for in vitro dedifferentiation of adipocytes by employing an additional physical stimulation, which takes advantage of augmenting the stemness-related Wnt/β-catenin signaling. Specifically, this protocol utilizes a polyethylene glycol (PEG)-containing hypertonic medium to introduce extracellular physical stimulation to obtain higher efficiency and introduce a simpler procedure for adipocyte dedifferentiation.
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.
The COVID-19 pandemic has significantly impacted learning as many institutions switched to remote or hybrid instruction. An in-depth assessment of the risk of infection that considers environmental setting and mitigation strategies is needed to make safe and informed decisions regarding reopening university spaces. A quantitative model of infection probability that accounts for space-specific parameters is presented to enable assessment of the risk in reopening university spaces at given densities. The model uses the fraction of the campus population that are viral shedders, room capacity, face covering filtration efficiency, air exchange rate, room volume, and time spent in the space as parameters to calculate infection probabilities in teaching spaces, dining halls, dorms, and shared bathrooms. The model readily calculates infection probabilities in various university spaces, with face covering filtration efficiency and air exchange rate being among the dominant variables. When applied to university spaces, this model demonstrated that, under specific conditions that are feasible to implement, in-person classes could be held in large lecture halls with an infection risk over the semester <1%. Meal pick-ups from dining halls and usage of shared bathrooms in residential dormitories among small groups of students could also be accomplished with low risk. The results of applying this model to spaces at Harvard University (Cambridge and Allston campuses) and Stanford University are reported. Finally, a user-friendly web application was developed using this model to calculate infection probability following input of space-specific variables. The successful development of a quantitative model and its implementation through a web application may facilitate accurate assessments of infection risk in university spaces. However, since this model is thus far unvalidated, validation using infection rate and contact tracing data from university campuses will be crucial as such data becomes available at larger scales. In light of the impact of the COVID-19 pandemic on universities, this tool could provide crucial insight to students, faculty, and university officials in making informed decisions.
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.
Current treatment options for foot ulcers, a serious and prevalent complication of diabetes, remain nonspecific. In this Perspective, we present recent advances in understanding the pathophysiology of diabetic wound healing and the emergence of previously unidentified targets. We discuss wound dressings tailored to the diabetic wound environment currently under development.
The development of tough adhesive hydrogels has enabled unprecedented adhesion to wet and moving tissue surfaces throughout the body, but they are typically composed of nondegradable components. Here, a family of degradable tough adhesive hydrogels containing ≈90% water by incorporating covalently networked degradable crosslinkers and hydrolyzable ionically crosslinked main-chain polymers is developed. Mechanical toughness, adhesion, and degradation of these new formulations are tested in both accelerated in vitro conditions and up to 16 weeks in vivo. These degradable tough adhesives are engineered with equivalent mechanical and adhesive properties to nondegradable tough adhesives, capable of achieving stretches >20 times their initial length, fracture energies >6 kJ m-2 , and adhesion energies >1000 J m-2 . All degradable systems show complete degradation within 2 weeks under accelerated aging conditions in vitro and weeks to months in vivo depending on the degradable crosslinker selected. Excellent biocompatibility is observed for all groups after 1, 2, 4, 8, and 16 weeks of implantation, with minimal fibrous encapsulation and no signs of organ toxicity. On-demand removal of the adhesive is achieved with treatment of chemical agents which do not cause damage to underlying skin tissue in mice. The broad versatility of this family of adhesives provides the foundation for numerous in vivo indications.
Cancer nanomedicines were initially envisioned as magic bullets, travelling through the circulation to target tumours while sparing healthy tissues the toxicity of classic chemotherapy. While a limited number of nanomedicine therapies have resulted, the disappointing news is that major obstacles were overlooked in the nanoparticle's journey. However, some of these challenges may be turned into opportunities. Here, we discuss biological barriers to cancer nanomedicines and elaborate on two directions that the field is currently exploring to meet its initial expectations. The first strategy entails re-engineering cancer nanomedicines to prevent undesired interactions en route to the tumour. The second aims instead to leverage these obstacles into out-of-the-box diagnostic and therapeutic applications of nanomedicines, for cancer and beyond. Both paths require, among other developments, a deeper understanding of nano-bio interactions. We offer a forward look at how classic cancer nanomedicine may overcome its limitations while contributing to other areas of research.
Active biomaterials offer novel approaches to study mechanotransduction in mammalian cells. These material systems probe cellular responses by dynamically modulating their resistance to endogenous forces or applying exogenous forces on cells in a temporally controlled manner. Stimuli-responsive molecules, polymers, and nanoparticles embedded inside cytocompatible biopolymer networks transduce external signals such as light, heat, chemicals, and magnetic fields into changes in matrix elasticity (few kPa to tens of kPa) or forces (few pN to several μN) at the cell-material interface. The implementation of active biomaterials in mechanobiology has generated scientific knowledge and therapeutic potential relevant to a variety of conditions including but not limited to cancer metastasis, fibrosis, and tissue regeneration. We discuss the repertoire of cellular responses that can be studied using these platforms including receptor signaling as well as downstream events namely, cytoskeletal organization, nuclear shuttling of mechanosensitive transcriptional regulators, cell migration, and differentiation. We highlight recent advances in active biomaterials and comment on their future impact.
BACKGROUND/PURPOSE: We sought to test select properties of a novel, expandable bioadhesive composite that allows for enhanced adhesion control in liquid environments.
METHODS: Rabbit fetuses (n = 23) underwent surgical creation of spina bifida on gestational day 22-25 (term 32-33 days). Defects were immediately covered with a two-component tough adhesive consisting of a hydrogel made of a double network of ionically crosslinked alginate and covalently crosslinked polyacrylamide linked to a bridging chitosan polymer adhesive. Animals were euthanized prior to term for different analyses, including hydraulic pressure testing.
RESULTS: Hydrogels remained adherent in 70% (16/23) of the recovered fetuses and in all of the last 14 fetuses as the procedure was optimized. Adherent hydrogels showed a median two-fold (IQR: 1.7-2.4) increase in area at euthanasia, with defect coverage confirmed by ultrasound and histology. The median maximum pressure to repair failure was 15 mmHg (IQR: 7.8-55.3), exceeding reported neonatal cerebrospinal fluid pressures.
CONCLUSIONS: This novel bioadhesive composite allows for selective, stable attachment of an alginate-polyacrylamide hydrogel to specific areas of the spina bifida defect in a fetal rabbit model, while the hydrogel expands with the defect over time. It could become a valuable alternative for the prenatal repair of spina bifida and possibly other congenital anomalies.
TYPE OF STUDY: N/A (animal and laboratory study).
LEVEL OF EVIDENCE: N/A (animal and laboratory study).
Human mesenchymal stromal cells (hMSCs) hold great promise in the treatment of inflammatory and immune diseases, due to their immunomodulatory capacity. Their therapeutic activity is often assessed measuring levels of expression of immunomodulatory genes such as indoleamine 2,3-dioxygenase 1 (IDO1) and real-time RT-qPCR is most predominantly the method of choice due to its high sensitivity and relative simplicity. Currently, multiple strategies are explored to promote hMSC-mediated immunomodulation, overlooking the effects they pose in the expression of genes commonly used as internal calibrators in real-time RT-qPCR analyses. However, variations in their expression could introduce significant errors in the evaluation of the therapeutic potential of hMSCs. This work investigates, for the first time, how some of these strategies - 3D encapsulation, the mechanical properties of the 3D matrix and inflammatory licensing - influence the expression of common reference genes in hMSCs. Both 3D encapsulation and inflammatory licensing alter significantly the expression of β-actin (ACTB) and Ubiquitin C (UBC), respectively. Using them as normalization factors leads to an erroneous assessment of IDO1 mRNA levels, therefore resulting in over or underestimation of the therapeutic potential of hMSCs. In contrast, the range of mechanical properties of the matrix encapsulating the cells did not significantly affect the expression of any of the reference genes studied. Moreover, we identify RPS13 and RPL30 as reference genes of choice under these particular experimental conditions. These results demonstrate the vital importance of validating the expression of reference genes to correctly assess the therapeutic potential of hMSCs by real-time RT-qPCR.
Poorly immunogenic tumors, including triple negative breast cancers (TNBCs), remain resistant to current immunotherapies, due in part to the difficulty of reprogramming the highly immunosuppressive tumor microenvironment (TME). Here we show that peritumorally injected, macroporous alginate gels loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF) for concentrating dendritic cells (DCs), CpG oligonucleotides, and a doxorubicin-iRGD conjugate enhance the immunogenic death of tumor cells, increase systemic tumor-specific CD8 + T cells, repolarize tumor-associated macrophages towards an inflammatory M1-like phenotype, and significantly improve antitumor efficacy against poorly immunogenic TNBCs. This system also prevents tumor recurrence after surgical resection and results in 100% metastasis-free survival upon re-challenge. This chemo-immunotherapy that concentrates DCs to present endogenous tumor antigens generated in situ may broadly serve as a facile platform to modulate the suppressive TME, and enable in situ personalized cancer vaccination.
A major function of the immune system is to detect threat from foreign invaders, tissue damage, or cancer and to mount a counter response that resolves the threat, restores homeostasis, and supplies immunological memory to prevent a second assault. Our increasing understanding of the immune system has opened up numerous avenues for modulating immune responses against infections, cancer, and autoimmunity. However, agents used for immunomodulation have been traditionally administered systemically via bolus injection, leading to unintended consequences by disrupting homeostasis at nontarget sites. Consequently, systemic hyperactivation and hypoactivation can result from bolus administration of immune-activators and immunosuppressants, respectively. Macroscale biomaterial scaffolds can instead be placed at the intended target site to provide both localized, controlled release of immunomodulatory agents and control over local immune cell trafficking and function, potentially maximizing therapeutic efficacy and limiting systemic exposure. These scaffolds have found utility in the area of cancer immunotherapy, especially cancer vaccination where controlled release of factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and the local presentation of tumor antigen and danger signals lead to the recruitment of immature dendritic cells and facilitate their activation and antigen presentation. These cells eventually migrate into secondary lymphoid organs where they prime tumor specific T cells for downstream tumor clearance. Scaffolds can also be used in adoptive T cell therapy to generate large numbers of potent antigen specific T cells or chimeric antigen receptor (CAR) T cells in vitro for subsequent delivery to patients. Macroscale biomaterial scaffolds have also found utility beyond cancer immunotherapy and have been developed to promote immune tolerance by regulatory T cell induction and to expedite tissue regeneration. The design of these macroscale biomaterial scaffolds considers their biocompatibility, biodegradability, mode of delivery, porosity, and kinetics of therapeutic cargo release. Consequently, the numerous approaches that have been developed to fabricate biomaterial scaffolds are aimed at tuning these parameters to achieve the desired therapeutic outcome. This Account will discuss the use of biomaterial scaffolds as niches for immunomodulation and will focus on (1) approaches that have been used to fabricate various biomaterial systems being employed as niches for immunomodulation and (2) how these biomaterial systems have been used to modulate immune responses, specifically in area of cancer immunotherapy, where we will discuss the role of macroscale biomaterial scaffolds for vaccination and in vitro T cell expansion. We will also briefly discuss the utility of biomaterial scaffolds beyond cancer, drawing examples from tolerance and tissue regeneration.
The therapeutic benefits of mesenchymal stromal cell (MSC) transplantation have been attributed to their secreted factors, including extracellular vesicles (EVs) and soluble factors. The potential of employing the MSC secretome as an alternative acellular approach to cell therapy is being investigated in various tissue injury indications, but EVs administered via bolus injections are rapidly sequestered and cleared. However, biomaterials offer delivery platforms to enhance EV retention rates and healing efficacy. In this review, we highlight the mechanisms underpinning the therapeutic effects of MSC-EVs and soluble factors as effectors of immunomodulation and tissue regeneration, conferred primarily via their nucleic acid and protein contents. We discuss how manipulating the cell culture microenvironment or genetic modification of MSCs can further augment the potency of their secretions. The most recent advances in the development of EV-functionalized biomaterials that mediate enhanced angiogenesis and cell survival, while attenuating inflammation and fibrosis, are presented. Finally, some technical challenges to be considered for the clinical translation of biomaterials carrying MSC-secreted bioactive cargo are discussed.
In cancer, lymph nodes (LNs) coordinate tumor antigen presentation necessary for effective antitumor immunity, both at the levels of local cellular interactions and tissue-level organization. In this review, we examine how LNs may be engineered to improve the therapeutic outcomes of cancer immunotherapy. At the cellular scale, targeting the LNs impacts the potency of cancer vaccines, immune checkpoint blockade, and adoptive cell transfer. On a tissue level, macro-scale biomaterials mimicking LN features can function as immune niches for cell reprogramming or delivery in vivo, or be utilized in vitro to enable preclinical testing of drugs and vaccines. We additionally review strategies to induce ectopic lymphoid sites reminiscent of LNs that may improve antitumor T cell priming.
Hydrogels with patterned biophysical and biochemical properties have found increasing attention in the biomaterials community. In this work, we explore alginate-based materials with two orthogonal crosslinking mechanisms: the spontaneous Diels-Alder reaction and the ultraviolet light-initiated thiol-ene reaction. Combining these mechanisms in one material and spatially restricting the location of the latter using photomasks, enables the formation of dual-crosslinked hydrogels with patterns in stiffness, biomolecule presentation and degradation, granting local control over cell behavior. Patterns in stiffness are characterized morphologically by confocal microscopy and mechanically by uniaxial compression and microindentation measurement. Mouse embryonic fibroblasts seeded on stiffness-patterned substrates attach preferably and attain a spread morphology on stiff compared to soft regions. Human mesenchymal stem cells demonstrate preferential adipogenic differentiation on soft surfaces and osteogenic differentiation on stiff surfaces. Patterns in biomolecule presentation reveal favored attachment of mouse pre-osteoblasts on stripe regions, where thiolated cell-adhesive biomolecules have been coupled. Patterns in degradation are visualized by microindentation measurement following collagenase exposure. Patterned tissue infiltration into degradable regions on the surface is discernible in n=5/12 samples, when these materials are implanted subcutaneously into the backs of mice. Taken together, these results demonstrate that our hydrogel system with patterns in biophysical and biochemical properties enables the study of how environmental cues affect multiple cell behaviors in vitro and could be applied to guide endogenous tissue growth in diverse healing scenarios in vivo. STATEMENT OF SIGNIFICANCE: Hydrogels with patterns in biophysical and biochemical properties have been explored in the biomaterials community in order to spatially control or guide cell behavior. In our alginate-based system, we demonstrate the effect of local substrate stiffness and biomolecule presentation on the in vitro cell attachment, morphology, migration and differentiation behavior of two different mouse cell lines and human primary cells. Additionally, the effect of degradation patterns on the in vivo tissue infiltration is analyzed following subcutaneous implantation into a mouse model. The achievement of patterned tissue infiltration following the hydrogel template represents an important step towards guiding endogenous healing responses, thus inviting application in various tissue engineering contexts.
Substantial research over the past two decades has established that extracellular matrix (ECM) elasticity, or stiffness, affects fundamental cellular processes, including spreading, growth, proliferation, migration, differentiation and organoid formation. Linearly elastic polyacrylamide hydrogels and polydimethylsiloxane (PDMS) elastomers coated with ECM proteins are widely used to assess the role of stiffness, and results from such experiments are often assumed to reproduce the effect of the mechanical environment experienced by cells in vivo. However, tissues and ECMs are not linearly elastic materials-they exhibit far more complex mechanical behaviours, including viscoelasticity (a time-dependent response to loading or deformation), as well as mechanical plasticity and nonlinear elasticity. Here we review the complex mechanical behaviours of tissues and ECMs, discuss the effect of ECM viscoelasticity on cells, and describe the potential use of viscoelastic biomaterials in regenerative medicine. Recent work has revealed that matrix viscoelasticity regulates these same fundamental cell processes, and can promote behaviours that are not observed with elastic hydrogels in both two- and three-dimensional culture microenvironments. These findings have provided insights into cell-matrix interactions and how these interactions differentially modulate mechano-sensitive molecular pathways in cells. Moreover, these results suggest design guidelines for the next generation of biomaterials, with the goal of matching tissue and ECM mechanics for in vitro tissue models and applications in regenerative medicine.
Current preclinical studies in drug development utilize high-throughput in vitro screens to identify drug leads, followed by both in vitro and in vivo models to predict lead candidates' pharmacokinetic and pharmacodynamic properties. The goal of these studies is to reduce the number of lead drug candidates down to the most likely to succeed in later human clinical trials. However, only 1 in 10 drug candidates that emerge from preclinical studies will succeed and become an approved therapeutic. Lack of efficacy or undetected toxicity represents roughly 75% of the causes for these failures, despite these parameters being the primary exclusion criteria in preclinical studies. Recently, advances in both biology and engineering have created new tools for constructing new preclinical models. These models can complement those used in current preclinical studies by helping to create more realistic representations of human tissues in vitro and in vivo. In this review, we describe current preclinical models to identify their value and limitations and then discuss select areas of research where improvements in preclinical models are particularly needed to advance drug development. Following this, we discuss design considerations for constructing preclinical models and then highlight recent advances in these efforts. Taken together, we aim to review the advances as of 2020 surrounding the prospect of biological and engineering tools for adding enhanced biological relevance to preclinical studies to aid in the challenges of failed drug candidates and the burden this poses on the drug development enterprise and thus healthcare.