Exploring the Role of Spatial Confinement in Immune Cell Recruitment and Regeneration of Skin Wounds.

Microporous annealed particle (MAP) scaffolds are injectable granular materials comprised of micron sized hydrogel particles (microgels). The diameter of these microgels directly determines the size of the interconnected void space between particles where infiltrating or encapsulated cells reside. This tunable porosity allows the authors to use MAP scaffolds to study the impact of spatial confinement (SC) on both cellular behaviors and the host response to biomaterials. Despite previous studies showing that pore size and SC influence cellular phenotypes, including mitigating macrophage inflammatory response, there is still a gap in knowledge regarding how SC within a biomaterial modulates immune cell recruitment in vivo in wounds and implants. Thus, the immune cell profile within confined and unconfined biomaterials is studied using small (40 µm), medium (70 µm), and large (130 µm) diameter spherical microgels, respectively. This work uncovered that MAP scaffolds impart regenerative wound healing with an IgG1-biased Th2 response. MAP scaffolds made with large microgels promote a balanced pro-regenerative macrophage response, resulting in enhanced wound healing with mature collagen regeneration and reduced inflammation levels.

Void Volume Fraction of Granular Scaffolds.

Void volume fraction (VVF) is a global measurement frequently used to characterize the void space of granular scaffolds, yet there is no gold standard by which to measure VVF in practice. To study the relationship  between VVF and particles of varying size, form, and composition, a library of 3D simulated scaffolds is used. Results reveal that relative to particle count, VVF is a less predictable metric across replicate scaffolds. Simulated scaffolds are used to explores the relationship between microscope magnification and VVF, and recommendations are offered for optimizing the accuracy of approximating VVF using 2D microscope images. Lastly, VVF of hydrogel granular scaffolds is measured while varying four input parameters: image quality, magnification, analysis software, and intensity threshold. Results show that VVF is highly sensitive to these parameters. Overall, random packing produces variation in VVF among granular scaffolds comprising the same particle populations. Furthermore, while VVF is used to compare the porosity of granular materials within a study, VVF is a less reliable metric across studies that use different input parameters. VVF, a global measurement, cannot describe the dimensions of porosity within granular scaffolds, and the work supports the notion that more descriptors are necessary to sufficiently characterize void space.

Spatial Confinement Modulates Macrophage Response in Microporous Annealed Particle (MAP) Scaffolds.

Macrophages are essential in the initiation, maintenance, and transition of inflammatory processes such as foreign body response and wound healing. Mounting evidence suggests that physical factors also modulate macrophage activation. 2D in vitro systems demonstrate that constraining macrophages to small areas or channels modulates their phenotypes and changes their responses to known inflammatory agents such as lipopolysaccharide. However, how dimensionality and pore size affect macrophage phenotype is less explored. In this work, the change in macrophage M1/M2 polarization when confined in microporous annealed particle (MAP) scaffolds is studied. Particles sizes (40, 70, and 130 µm) are selected using outputs from software LOVAMAP that analyzes the characteristics of 3D pores in MAP gels. As the size of building block particle correlates with pore size inside the scaffolds, the three  types of scaffold allow us to study how the degree of spatial confinement modulates the behavior of embedded macrophages. Spatially confining macrophages in scaffolds with pore size on the scale of cells leads to a reduced level of the inflammatory response, which is correlated with a change in cell morphology and motility.

Exploring the Role of Spatial Confinement in Immune Cell Recruitment and Regeneration of Skin Wounds.

Microporous annealed particle (MAP) scaffolds are injectable granular materials comprised of micron sized hydrogel particles (microgels). The diameter of these microgels directly determines the size of the interconnected void space between particles where infiltrating or encapsulated cells reside. This tunable porosity allows us to use MAP scaffolds to study the impact of spatial confinement (SC) on both cellular behaviors and the host response to biomaterials. Despite previous studies showing that pore size and SC influence cellular phenotypes, including mitigating the macrophage inflammatory response, there is still a gap in knowledge regarding how SC within a biomaterial modulates immune cell recruitment in vivo in wounds and implants. Thus, we studied the immune cell profile within confined and unconfined biomaterials using small (40 µm), medium (70 µm), and large (130 µm) diameter spherical microgels, respectively. We discovered that MAP scaffolds imparted regenerative wound healing with an IgG1-biased Th2 response. MAP scaffolds generated from 130 µm diameter microgels have a median pore size that can accommodate ∼40 µm diameter spheres induced a more balanced pro-regenerative macrophage response and better wound healing outcomes with more mature collagen regeneration and reduced levels of inflammation.

Identification and analysis of 3D pores in packed particulate materials.

We took the classic 'guess the number of beans in a jar game' and amplified the research question. Rather than estimate the quantity of particles in the jar, we sought to characterize the spaces between them. Here we present an approach for delineating the pockets of empty space (three-dimensional pores) between packed particles, which are hotspots for activity in applications and natural phenomena that deal with particulate materials. We utilize techniques from graph theory to exploit information about particle configuration that allows us to locate important spatial landmarks within the void space. These landmarks are the basis for our pore segmentation, where we consider both interior pores as well as entrance and exit pores into and out of the structure. Our method is robust for particles of varying size, form, stiffness and configuration, which allows us to study and compare three-dimensional pores across a range of packed particle types. We report striking relationships between particles and pores that are described mathematically, and we offer a visual library of pore types. With a meaningful discretization of void space, we demonstrate that packed particles can be understood not by their solid space, but by their empty space.

Hydrogel microparticles for biomedical applications.

Hydrogel microparticles (HMPs) are promising for biomedical applications, ranging from the therapeutic delivery of cells and drugs to the production of scaffolds for tissue repair and bioinks for 3D printing. Biologics (cells and drugs) can be encapsulated into HMPs of predefined shapes and sizes using a variety of fabrication techniques (batch emulsion, microfluidics, lithography, electrohydrodynamic (EHD) spraying and mechanical fragmentation). HMPs can be formulated in suspensions to deliver therapeutics, as aggregates of particles (granular hydrogels) to form microporous scaffolds that promote cell infiltration or embedded within a bulk hydrogel to obtain multiscale behaviours. HMP suspensions and granular hydrogels can be injected for minimally invasive delivery of biologics, and they exhibit modular properties when comprised of mixtures of distinct HMP populations. In this Review, we discuss the fabrication techniques that are available for fabricating HMPs, as well as the multiscale behaviours of HMP systems and their functional properties, highlighting their advantages over traditional bulk hydrogels. Furthermore, we discuss applications of HMPs in the fields of cell delivery, drug delivery, scaffold design and biofabrication.

Granular hydrogels: emergent properties of jammed hydrogel microparticles and their applications in tissue repair and regeneration.

Granular hydrogels are emerging as a versatile and effective platform for tissue engineered constructs in regenerative medicine. The hydrogel microparticles (HMPs) that compose these materials exhibit particle jamming above a minimum packing fraction, which results in a bulk, yet dynamic, granular hydrogel scaffold. These injectable, microporous scaffolds possess self-assembling, shear-thinning, and self-healing properties. Recently, they have been utilized as cell cultures platforms and extracellular matrix mimics with remarkable success in promoting cellular infiltration and subsequent tissue remodeling in vivo. Furthermore, the modular nature of granular hydrogels accommodates heterogeneous HMP assembly, where varying HMPs have been fabricated to target distinct biological processes or deliver unique cargo. Such multifunctional materials offer enormous potential for capturing the structural and biofunctional complexity observed in native human tissue.

Improving Influenza Testing and Treatment in Hospitalized Children.

OBJECTIVES: National guidelines recommend influenza testing for children hospitalized with influenza-like illness (ILI) during influenza season and treatment of those with confirmed influenza. Using quality improvement methods, we sought to increase influenza testing and treatment of children admitted to our hospital medicine service with ILI from 65% to 90% during the 2014-2015 influenza season. METHODS: We targeted several key drivers using multiple plan-do-study-act cycles. Interventions included awareness modules, biweekly flyers, and failure tracking. ILI admissions (fever plus respiratory symptoms) were reviewed weekly once surveillance data revealed elevated influenza activity. Appropriate testing and treatment of ILI was defined as influenza testing and/or treatment within 24 hours of admission unless a known cause other than influenza was present. We used statistical process control charts to track progress using established quality improvement methods. Appropriate testing and treatment was also assessed in the 2016-2017 influenza season by using similar methods, although no new interventions were introduced. RESULTS: For the 2014-2015 season, appropriate testing and treatment increased from a baseline mean of 65% to 91% within 3 months. For the 2016-2017 season, appropriate testing and treatment remained at a mean of 80% throughout the influenza season. CONCLUSIONS: Appropriate influenza testing and treatment increased to 90% in children with ILI during the 2014-2015 season. Improvements were sustained in a subsequent influenza season. Our initiative improved recognition of influenza and likely increased treatment opportunities. Future work should be focused on wider implementation and further reducing variation.

Determining duration of HER2-targeted therapy using stem cell extinction models.

INTRODUCTION: Trastuzumab dramatically improves survival in breast cancer patients whose tumor overexpresses HER2. A subpopulation of cells in human breast tumors has been identified with characteristics of cancer stem cells. These breast cancer stem-like cells (BCSCs) rely on HER2 signaling for self-renewal, suggesting that HER2-targeted therapy targets BCSCs even when the bulk of the tumor does not overexpress HER2. In order to guide clinical trials examining HER2-targeted therapy in the adjuvant setting, we propose a mathematical model to examine BCSC population dynamics and predict optimal duration of therapy. METHODS: Varying the susceptibility of BCSCs to HER2-targeted therapy, we quantify the average time to extinction of BCSCs. We expand our model using stochastic simulation to include the partially differentiated tumor cells (TCs) that represent bulk tumor population and examine effects of plasticity on required duration of therapy. RESULTS: Lower susceptibility of BCSCs and increased rates of dedifferentiation entail longer extinction times, indicating a need for prolonged administration of HER2-targeted therapy. We predict that even when therapy does not appreciably reduce tumor size in the advanced cancer setting, it will eventually eradicate the tumor in the adjuvant setting as long as there is at least a modest effect on BCSCs. CONCLUSIONS: We anticipate that our results will inform clinical trials of targeted therapies in planning the duration of therapy needed to eradicate BCSCs. Our predictions also address safety, as longer duration of therapy entails a greater potential impact on normal stem cells that may also be susceptible to stem cell-targeted therapies.

Predicted structures of agonist and antagonist bound complexes of adenosine A3 receptor.

We used the GEnSeMBLE Monte Carlo method to predict ensemble of the 20 best packings (helix rotations and tilts) based on the neutral total energy (E) from a vast number (10 trillion) of potential packings for each of the four subtypes of the adenosine G protein-coupled receptors (GPCRs), which are involved in many cytoprotective functions. We then used the DarwinDock Monte Carlo methods to predict the binding pose for the human A(3) adenosine receptor (hAA(3)R) for subtype selective agonists and antagonists. We found that all four A(3) agonists stabilize the 15th lowest conformation of apo-hAA(3)R while also binding strongly to the 1st and 3rd. In contrast the four A(3) antagonists stabilize the 2nd or 3rd lowest conformation. These results show that different ligands can stabilize different GPCR conformations, which will likely affect function, complicating the design of functionally unique ligands. Interestingly all agonists lead to a trans χ1 angle for W6.48 that experiments on other GPCRs associate with G-protein activation while all 20 apo-AA(3)R conformations have a W6.48 gauche+ χ1 angle associated experimentally with inactive GPCRs for other systems. Thus docking calculations have identified critical ligand-GPCR structures involved with activation. We found that the predicted binding site for selective agonist Cl-IB-MECA to the predicted structure of hAA(3)R shows favorable interactions to three subtype variable residues, I253(6.58), V169(EL2), and Q167(EL2), while the predicted structure for hAA(2A)R shows weakened to the corresponding amino acids: T256(6.58), E169(EL2), and L167(EL2), explaining the observed subtype selectivity.