Adam S. Zeiger

Stop-flow lithography for the production of shape-evolving degradable microgel particles.

Microgel particles capable of bulk degradation have been synthesized from a solution of diacrylated triblock copolymer composed of poly(ethylene glycol) and poly(lactic acid) in a microfluidic device using stop-flow lithography (SFL). It has been previously demonstrated that SFL can be used to fabricate particles with precise control over particle size and shape. Here, we have fabricated hydrogel particles of varying size and shape and examined their mass-loss and swelling behavior histologically and mechanically. We report that these features, as well as degradation behavior of the hydrogel particles may be tailored with SFL. By reducing the applied UV dose during fabrication, hydrogel particles can be made to exhibit a distinct deviation from the classical erosion profiles of bulk-degrading hydrogels. At higher UV doses, a saturation in cross-linking density occurs and bulk-degrading behavior is observed. Finally, we synthesized multifunctional composite particles, providing unique features not found in homogeneous hydrogels.

Photoelectrochemical complexes for solar energy conversion that chemically and autonomously regenerate

Naturally occurring photosynthetic systems use elaborate pathways of self-repair to limit the impact of photo-damage. Herein, we demonstrate a complex that mimics this process consisting of two recombinant proteins, phospholipids and a carbon nanotube. The components self-assemble into a configuration in which an array of lipid bilayers aggregate on the surface of the carbon nanotube, creating a platform for the attachment of light-converting proteins. The system can disassemble upon the addition of a surfactant and reassemble on its removal over an indefinite number of cycles. The assembly is thermodynamically meta-stable and can only transition reversibly if the rate of surfactant removal exceeds about 10−5 sec−1. Only in the assembled state do the complexes exhibit photoelectrochemical activity. We demonstrate a regeneration cycle that uses surfactant to switch between assembled and disassembled states, resulting in increased photo-conversion efficiency of more than 300% over 168 hours and an indefinite extension of the systems lifetime.

Probing the biomechanical contribution of the endothelium to lymphocyte migration: diapedesis by the path of least resistance

ABSTRACT Immune cell trafficking requires the frequent breaching of the endothelial barrier either directly through individual cells (‘transcellular’ route) or through the inter-endothelial junctions (‘paracellular’ route). What determines the loci or route of breaching events is an open question with important implications for overall barrier regulation. We hypothesized that basic biomechanical properties of the endothelium might serve as crucial determinants of this process. By altering junctional integrity, cytoskeletal morphology and, consequently, local endothelial cell stiffness of different vascular beds, we could modify the preferred route of diapedesis. In particular, high barrier function was associated with predominantly transcellular migration, whereas negative modulation of junctional integrity resulted in a switch to paracellular diapedesis. Furthermore, we showed that lymphocytes dynamically probe the underlying endothelium by extending invadosome-like protrusions (ILPs) into its surface that deform the nuclear lamina, distort actin filaments and ultimately breach the barrier. Fluorescence imaging and pharmacologic depletion of F-actin demonstrated that lymphocyte barrier breaching efficiency was inversely correlated with local endothelial F-actin density and stiffness. Taken together, these data support the hypothesis that lymphocytes are guided by the mechanical ‘path of least resistance’ as they transverse the endothelium, a process we term ‘tenertaxis’.

Pericyte actomyosin-mediated contraction at the cell?material interface can modulate the microvascular niche

Pericytes physically surround the capillary endothelium, contacting and communicating with associated vascular endothelial cells via cell-cell and cell-matrix contacts. Pericyte-endothelial cell interactions thus have the potential to modulate growth and function of the microvasculature. Here we employ the experimental finding that pericytes can buckle a freestanding, underlying membrane via actin-mediated contraction. Pericytes were cultured on deformable silicone substrata, and pericyte-generated wrinkles were imaged via both optical and atomic force microscopy (AFM). The local stiffness of subcellular domains both near and far from these wrinkles was investigated by using AFM-enabled nanoindentation to quantify effective elastic moduli. Substratum buckling contraction was quantified by the normalized change in length of initially flat regions of the substrata (corresponding to wrinkle contour lengths), and a model was used to relate local strain energies to pericyte contractile forces. The nature of pericyte-generated wrinkling and contractile protein-generated force transduction was further explored by the addition of pharmacological cytoskeletal inhibitors that affected contractile forces and the effective elastic moduli of pericyte domains. Actin-mediated forces are sufficient for pericytes to exert an average buckling contraction of 38% on the elastomeric substrata employed in these in vitro studies. Actomyosin-mediated contractile forces also act in vivo on the compliant environment of the microvasculature, including the basement membrane and other cells. Pericyte-generated substratum deformation can thus serve as a direct mechanical stimulus to adjacent vascular endothelial cells, and potentially alter the effective mechanical stiffness of nonlinear elastic extracellular matrices, to modulate pericyte-endothelial cell interactions that directly influence both physiologic and pathologic angiogenesis.

Macromolecular Crowding Directs Extracellular Matrix Organization and Mesenchymal Stem Cell Behavior

Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro.

Why the dish makes a difference: Quantitative comparison of polystyrene culture surfaces

There is wide anecdotal recognition that biological cell viability and behavior can vary significantly as a function of the source of commercial tissue culture polystyrene (TCPS) culture vessels to which those cells adhere. However, this marked material dependency is typically resolved by selecting and then consistently using the same manufacturers product - following protocol - rather than by investigating the material properties that may be responsible for such experimental variation. Here, we quantified several physical properties of TCPS surfaces obtained from a wide range of commercial sources and processing steps, through the use of atomic force microscopy (AFM)-based imaging and analysis, goniometry and protein adsorption quantification. We identify qualitative differences in surface features, as well as quantitative differences in surface roughness and wettability that cannot be attributed solely to differences in surface chemistry. We also find significant differences in cell morphology and proliferation among cells cultured on different TCPS surfaces, and resolve a correlation between nanoscale surface roughness and cell proliferation rate for both cell types considered. Interestingly, AFM images of living adherent cells on these nanotextured surfaces demonstrate direct interactions between cellular protrusions and topographically distinct features. These results illustrate and quantify the significant differences in material surface properties among these ubiquitous materials, allowing us to better understand why the dish can make a difference in biological experiments.

Tuning adhesion failure strength for tissue-specific applications

Soft tissue adhesives are employed to repair and seal many different organs, which range in both tissue surface chemistry and mechanical challenges during organ function. This complexity motivates the development of tunable adhesive materials with high resistance to uniaxial or multiaxial loads dictated by a specific organ environment. Co-polymeric hydrogels comprising aminated star polyethylene glycol and dextran aldehyde (PEG:dextran) are materials exhibiting physico-chemical properties that can be modified to achieve this organ- and tissue-specific adhesion performance. Here we report that resistance to failure under specific loading conditions, as well as tissue response at the adhesive material-tissue interface, can be modulated through regulation of the number and density of adhesive aldehyde groups. We find that atomic force microscopy (AFM) can characterize the material aldehyde density available for tissue interaction, and in this way enable rapid, informed material choice. Further, the correlation between AFM quantification of nanoscale unbinding forces with macroscale measurements of adhesion strength by uniaxial tension or multiaxial burst pressure allows the design of materials with specific cohesion and adhesion strengths. However, failure strength alone does not predict optimal in vivo reactivity. Thus, we demonstrate that the development of adhesive materials is significantly enabled when experiments are integrated along length scales to consider organ chemistry and mechanical loading states concurrently with adhesive material properties and tissue response.

Molecular Modeling of the Axial and Circumferential Elastic Moduli of Tubulin

Microtubules play a number of important mechanical roles in almost all cell types in nearly all major phylogenetic trees. We have used a molecular mechanics approach to perform tensile tests on individual tubulin monomers and determined values for the axial and circumferential moduli for all currently known complete sequences. The axial elastic moduli, in vacuo, were found to be 1.25 GPa and 1.34 GPa for α- and β-bovine tubulin monomers. In the circumferential direction, these moduli were 378 MPa for α- and 460 MPa for β-structures. Using bovine tubulin as a template, 269 homologous tubulin structures were also subjected to simulated tensile loads yielding an average axial elastic modulus of 1.10 ± 0.14 GPa for α-tubulin structures and 1.39 ± 0.68 GPa for β-tubulin. Circumferentially the α- and β-moduli were 936 ± 216 MPa and 658 ± 134 MPa, respectively. Our primary finding is that that the axial elastic modulus of tubulin diminishes as the length of the monomer increases. However, in the circumferential direction, no correlation exists. These predicted anisotropies and scale dependencies may assist in interpreting the macroscale behavior of microtubules during mitosis or cell growth. Additionally, an intergenomic approach to investigating the mechanical properties of proteins may provide a way to elucidate the evolutionary mechanical constraints imposed by nature upon individual subcellular components.