Hydrogel substrate stress-relaxation regulates the spreading and proliferation of mouse myoblasts.

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.

Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation.

DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.

Advances in Therapeutic Cancer Vaccines.

Therapeutic cancer vaccines aim to induce durable antitumor immunity that is capable of systemic protection against tumor recurrence or metastatic disease. Many approaches to therapeutic cancer vaccines have been explored, with varying levels of success. However, with the exception of Sipuleucel T, an ex vivo dendritic cell vaccine for prostate cancer, no therapeutic cancer vaccine has yet shown clinical efficacy in phase 3 randomized trials. Though disappointing, lessons learned from these studies have suggested new strategies to improve cancer vaccines. The clinical success of checkpoint blockade has underscored the role of peripheral tolerance mechanisms in limiting vaccine responses and highlighted the potential for combination therapies. Recent advances in transcriptome sequencing, computational modeling, and material engineering further suggest new opportunities to intensify cancer vaccines. This review will discuss the major approaches to therapeutic cancer vaccination and explore recent advances that inform the design of the next generation of cancer vaccines.

The effect of surface modification of mesoporous silica micro-rod scaffold on immune cell activation and infiltration.

Biomaterial scaffold based vaccines show significant potential in generating potent antigen-specific immunity. However, the role of the scaffold surface chemistry in initiating and modulating the immune response is not well understood. In this study, a mesoporous silica micro-rod (MSR) scaffold was modified with PEG, PEG-RGD and PEG-RDG groups. PEG modification significantly enhanced BMDC activation marker up-regulation and IL-1ß production in vitro, and innate immune cell infiltration in vivo. PEG-RGD MSRs and PEG-RDG MSRs displayed decreased inflammation compared to PEG MSRs, and the effect was not RGD specific. Finally, the Nlrp3 inflammasome was found to be necessary for MSR stimulated IL-1ß production in vitro and played a key role in regulating immune cell infiltration in vivo. These findings suggest that simply modulating the surface chemistry of a scaffold can regulate its immune cell infiltration profile and have implications for the design and development of new material based vaccines.

Injectable, spontaneously assembling, inorganic scaffolds modulate immune cells in vivo and increase vaccine efficacy.

Implanting materials in the body to program host immune cells is a promising alternative to transplantation of cells manipulated ex vivo to direct an immune response, but doing so requires a surgical procedure. Here we demonstrate that high-aspect-ratio, mesoporous silica rods (MSRs) injected with a needle spontaneously assemble in vivo to form macroporous structures that provide a 3D cellular microenvironment for host immune cells. In mice, substantial numbers of dendritic cells are recruited to the pores between the scaffold rods. The recruitment of dendritic cells and their subsequent homing to lymph nodes can be modulated by sustained release of inflammatory signals and adjuvants from the scaffold. Moreover, injection of an MSR-based vaccine formulation enhances systemic helper T cells TH1 and TH2 serum antibody and cytotoxic T-cell levels compared to bolus controls. These findings suggest that injectable MSRs may serve as a multifunctional vaccine platform to modulate host immune cell function and provoke adaptive immune responses.

Cell-friendly inverse opal-like hydrogels for a spatially separated co-culture system.

Three-dimensional macroporous scaffolds have extensively been studied for cell-based tissue engineering but their use is mostly limited to mechanical support for cell adhesion and growth on the surface of macropores. Here, a templated fabrication method is described to prepare cell-friendly inverse opal-like hydrogels (IOHs) allowing both cell encapsulation within the hydrogel matrix and cell seeding on the surface of macropores. Ionically crosslinked alginate microbeads and photocrosslinkable biocompatible polymers are used as a sacrificial template and as a matrix, respectively. The alginate microbeads are easily removed by a chelating agent, with minimal toxicity for the encapsulated cells during template removal. The outer surface of macropores in IOHs can also provide a space for cell adherence. The cells encapsulated or attached in IOHs are able to remain viable and to proliferate over time. The elastic modulus and cell-adhesion properties of IOHs can be easily controlled and tuned. Finally, it is demonstrated that IOH can be used to co-culture two distinct cell populations in different spatial positions. This cell-friendly IOH system provides a 3D scaffold for organizing different cell types in a controllable microenvironment to investigate biological processes such as stem cell niches or tumor microenvironments.

Effect of pore structure of macroporous poly(lactide-co-glycolide) scaffolds on the in vivo enrichment of dendritic cells.

The in vivo enrichment of dendritic cells (DCs) in implanted macroporous scaffolds is an emerging strategy to modulate the adaptive immune system. The pore architecture is potentially one of the key factors in controlling enrichment of DCs. However, there have been few studies examining the effects of scaffold pore structure on in vivo DC enrichment. Here we present the effects of surface porosity, pore size, and pore volume of macroporous poly(lactide-co-glycolide) (PLG) scaffolds encapsulating granulocyte macrophage colony-stimulating factor (GM-CSF), an inflammatory chemoattractant, on the in vivo enrichment of DCs. Although in vitro cell seeding studies using PLG scaffolds without GM-CSF showed higher cell infiltration in scaffolds with higher surface porosity, in vivo results revealed higher DC enrichment in GM-CSF loaded PLG scaffolds with lower surface porosity despite a similar level of GM-CSF released. The diminished compressive modulus of high surface porosity scaffolds compared to low surface porosity scaffolds lead to the significant shrinkage of these scaffolds in vivo, suggesting that the mechanical strength of scaffolds was critical to maintain a porous structure in vivo for accumulating DCs. The pore volume was also found to be important in total number of recruited cells and DCs in vivo. Varying the pore size significantly impacted the total number of cells, but similar numbers of DCs were found as long as the pore size was above 10-32 µm. Collectively, these results suggested that one can modulate in vivo enrichment of DCs by altering the pore architecture and mechanical properties of PLG scaffolds.

Materials based tumor immunotherapy vaccines.

Immunotherapy is a promising approach for treating cancer. However, there are limitations inherent to current approaches which may be addressed by integrating them with biomaterial-based strategies. Material platforms have been fabricated to interact with immune cells through spatially controlled and temporally controlled delivery of immune modulators and to promote immune cell crosstalk. Particle vaccines have been developed to specifically target and deliver agents to organs, cells and subcellular compartments. These strategies have been shown to generate antigen-specific CTL responses and, in some cases, tumor regression. Therefore, collaboration between immunology and materials engineering is likely to result in the creation of strong vaccines to combat cancer in the future.