Charge segregation in weakly ionized microgels.

We investigate microgels synthesized from N-isopropylacrylamide (NIPAM) copolymerized with a large mol% of acrylic acid, finding that when the acid groups are partially ionized at high temperatures, competition between ion-induced swelling and hydrophobic deswelling of poly(NIPAM) chains results in microphase separation. In cross-linked microgels, this manifests as a dramatic decrease in the ratio between the radius of gyration and the hydrodynamic radius to ∼0.2, indicating that almost all the mass of the microgel is concentrated near the particle center. We also observe a concurrent decrease of the polymer network length scale via small-angle neutron scattering, confirming the presence of a dense, deswollen core surrounded by a diffuse, charged periphery. We compare these results to those obtained for a system of charged ultralow-cross-linked microgels; the form factor shows a distinct peak at high q when the temperature exceeds a threshold value. We successfully fit the form factor to theory developed to describe scattering from weakly charged gels in poor solvents, and we tie this behavior to charge segregation in the case of the cross-linked microgels.

Dynamic assembly of ultrasoft colloidal networks enables cell invasion within restrictive fibrillar polymers.

In regenerative medicine, natural protein-based polymers offer enhanced endogenous bioactivity and potential for seamless integration with tissue, yet form weak hydrogels that lack the physical robustness required for surgical manipulation, making them difficult to apply in practice. The use of higher concentrations of protein, exogenous cross-linkers, and blending synthetic polymers has all been applied to form more mechanically robust networks. Each relies on generating a smaller network mesh size, which increases the elastic modulus and robustness, but critically inhibits cell spreading and migration, hampering tissue regeneration. Here we report two unique observations; first, that colloidal suspensions, at sufficiently high volume fraction (ϕ), dynamically assemble into a fully percolated 3D network within high-concentration protein polymers. Second, cells appear capable of leveraging these unique domains for highly efficient cell migration throughout the composite construct. In contrast to porogens, the particles in our system remain embedded within the bulk polymer, creating a network of particle-filled tunnels. Whereas this would normally physically restrict cell motility, when the particulate network is created using ultralow cross-linked microgels, the colloidal suspension displays viscous behavior on the same timescale as cell spreading and migration and thus enables efficient cell infiltration of the construct through the colloidal-filled tunnels.

A novel platelet lysate hydrogel for endothelial cell and mesenchymal stem cell-directed neovascularization.

UNLABELLED: Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration. STATEMENT OF SIGNIFICANCE: Innovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL hydrogel is ideally suited to serve as a cell delivery vehicle for MSCs injected into ischemic tissues to promote vascular regeneration, as demonstrated here in a murine model of hindlimb ischemia.

Molecular interference of fibrin's divalent polymerization mechanism enables modulation of multiscale material properties.

Protein based polymers provide an exciting and complex landscape for tunable natural biomaterials through modulation of molecular level interactions. Here we demonstrate the ability to modify protein polymer structural and mechanical properties at multiple length scales by molecular 'interference' of fibrin's native polymerization mechanism. We have previously reported that engagement of fibrin's polymerization 'hole b', also known as 'b-pockets', through PEGylated complementary 'knob B' mimics can increase fibrin network porosity but also, somewhat paradoxically, increase network stiffness. Here, we explore the possible mechanistic underpinning of this phenomenon through characterization of the effects of knob B-fibrin interaction at multiple length scales from molecular to bulk polymer. Despite its weak monovalent binding affinity for fibrin, addition of both knob B and PEGylated knob B at concentrations near the binding coefficient, Kd, increased fibrin network porosity, consistent with the reported role of knob B-hole b interactions in promoting lateral growth of fibrin fibers. Addition of PEGylated knob B decreases the extensibility of single fibrin fibers at concentrations near its Kd but increases extensibility of fibers at concentrations above its Kd. The data suggest this bimodal behavior is due to the individual contributions knob B, which decreases fiber extensibility, and PEG, which increase fiber extensibility. Taken together with laser trap-based microrheological and bulk rheological analyses of fibrin polymers, our data strongly suggests that hole b engagement increases in single fiber stiffness that translates to higher storage moduli of fibrin polymers despite their increased porosity. These data point to possible strategies for tuning fibrin polymer mechanical properties through modulation of single fiber mechanics.

Synergistic effects of particulate matter and substrate stiffness on epithelial-to-mesenchymal transition.

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis, chronic obstructive pulmonary disease (COPD*), and tumorigenesis, have been increasing steadily over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that the lung "precursor cell"--the alveolar type II (ATII) epithelial cell--is central in the initiation and progression of pulmonary fibrosis. Specifically, ATII cells have been shown (Iwano et al. 2002) to be capable of undergoing an epithelial-to-mesenchymal transition (EMT). EMT, the de-differentiation of an epithelial cell into a mesenchymal cell, has been theorized to increase the number of extracellular matrix (ECM)-secreting mesenchymal cells, perpetuating fibrotic conditions and resulting in increased lung tissue stiffness. In addition, increased exposure to pollution and inhalation of particulate matter (PM) have been shown to be highly correlated with an increased incidence of pulmonary fibrosis. Although both of these events are involved in the progression of pulmonary fibrosis, the relationship between tissue stiffness, exposure to PM, and the initiation and course of EMT remains unclear. The hypothesis of this study was twofold: 1. That alveolar epithelial cells cultured on increasingly stiff substrates become increasingly contractile, leading to enhanced transforming growth factor beta (TGF-ß) activation and EMT; and 2. That exposure of alveolar epithelial cells to PM with an aerodynamic diameter ≤ 2.5 µm (PM2.5; also known as fine PM) results in enhanced cell contractility and EMT. Our study focused on the relationship between the micromechanical environment and external environmental stimuli on the phenotype of alveolar epithelial cells. This relationship was explored by first determining how increased tissue stiffness affects the regulation of fibronectin (Fn)-mediated EMT in ATII cells in vitro. We cultured ATII cells on substrates of increasing stiffness and evaluated changes in cell contractility and EMT. We found that stiff, but not soft, Fn substrates were able to induce EMT and that this event depended on a contractile phenotype of the cell and the subsequent activation of TGF-ß. In addition, we were able to show that activation or suppression of cell contractility by way of exogenous factors was sufficient to overcome the effect of substrate stiffness. Pulse-chase experiments indicated that the effect on cell contractility is dose- and time-dependent. In response to low levels of TGF-ß on soft surfaces, either added exogenously or produced through contraction induced by the stiffness agonist thrombin, cells initiate EMT; on removal of the TGF-ß, they revert to an epithelial phenotype. Overall, the results from this first part of our study identified matrix stiffness or cell contractility as critical targets for the control of EMT in fibrotic diseases. For the second part of our study, we wanted to investigate whether exposure to PM2.5, which might have higher toxicity than coarser PM because of its small size and large surface-to-mass ratio, altered the observed stiffness-mediated EMT. Again, we cultured ATII cells on increasingly stiff substrates with or without the addition of three concentrations of PM2.5. We found that exposure to PM2.5 was involved in increased stiffness-mediated EMT, as shown by increases in mesenchymal markers, cell contractility, and TGF-ß activation. Most notably, on substrates with an elastic modulus (E) of 8 kilopascals (kPa), a physiologically relevant range for pulmonary fibrosis, the addition of PM2.5 resulted in increased mesenchymal cells and EMT; these were not seen in the absence of the PM2.5. Overall, this study showed that there is a delicate balance between substrate stiffness, TGF-ß, and EMT. Furthermore, we showed that exposure to PM2.5 is able to further mediate this interaction. The higher levels of EMT seen with exposure to PM2.5 might have been a result of a positive feedback loop, in which enhanced exposure to PM2.5 through the loss of cell-cell junctions during the initial stages of EMT led to the cells being more susceptible to the effects of surrounding immune cells and inflammatory signals that can further activate TGF-ß and drive additional EMT progression. Overall, our work--showing increased cell contractility, TGF-ß activation, and EMT in response to substrate stiffness and PM2.5 exposure--highlights the importance of both the micromechanical and biochemical environments in lung disease. These findings suggest that already-fibrotic tissue might be more susceptible to further damage than healthy tissue when exposed to PM2.5.

Validity of predischarge measures for predicting time to harm in older adults.

BACKGROUND: Concern is often expressed about the ability of persons with cognitive impairment to manage safely after discharge home from hospital. Measures validated for predicting safety are required. PURPOSE: The purpose of this study was to determine whether two predischarge functional measures were valid for predicting time to incident of harm after discharge. METHOD: Participants (n = 47) were recruited from an inpatient rehabilitation unit. The Assessment of Motor and Process Skills (AMPS) and Cognitive Performance Test (CPT) were administered in hospital. Incident-of-harm outcome was measured by caregiver telephone questionnaire monthly for 6 months. FINDINGS: Compared with all independent variables, AMPS Process scale was the best single predictor of time to incident of harm (p = .01). CPT had a high specificity (91%) for identifying persons who did not have harm. IMPLICATIONS: Both AMPS and CPT demonstrated predictive validity for harm outcome over less predictive variables, such as comorbidities and activities-of-daily-living burden of care.