Session R65: Biomaterials: Structure, Function, Design IV

Biomimetic Mineralization via Polymer-Induced Liquid Precursors

The hallmark of biomineralization is the ability of organisms to form non-equilibrium crystal morphologies. Our group has been using in vitro model systems to examine the physicochemical mechanisms that might be involved in biomineralization, with emphasis on the role that biopolymers play. We have discovered that many of the long-standing enigmatic biomineral features can be reproduced in the beaker using a non-classical crystallization process we call the Polymer-Induced Liquid-Precursor (PILP) process. This process entails adding charged polymers to supersaturated salt solutions which then sequester ions/clusters/phases to induce phase separation of a hydrated amorphous mineral precursor. Interaction of these PILP colloids with various organic templates and matrices leads to a variety of non-equilibrium morphologies and composite textures, many of which emulate those seen in biominerals. We argue that a PILP type process may lie at the foundation of biomineralizing systems ranging from invertebrate exoskeletons to vertebrate bones and teeth. This argument is based on the diversity of features that can be emulated, and their distinct mineralogical signatures of formation mechanism, such as incorporation of high magnesium into calcite, transition bars and shrinkage patterns, and remnant colloidal textures within single-crystalline composites. Through this enhanced understanding of biomineralization processes, we are now developing biomimetic processing methods that enable the fabrication of hard-tissue biomaterials which emulate bone.   

Cell contraction induces long-ranged stress stiffening in the extracellular matrix

Animal cells in tissues are supported by biopolymer matrices, which typically exhibit highly nonlinear mechanical properties. While the linear elasticity of the matrix can significantly impact cell mechanics and functionality, it remains largely unknown how cells, in turn, affect the nonlinear mechanics of their surrounding matrix. Here, we show that living contractile cells are able to generate a massive stiffness gradient in three distinct 3D extracellular matrix model systems: collagen, fibrin, and Matrigel. We decipher this remarkable behavior by introducing nonlinear stress inference microscopy (NSIM), a technique to infer stress fields in a 3D matrix from nonlinear microrheology measurements with optical tweezers. Using NSIM and simulations, we reveal large long-ranged cell-generated stresses capable of buckling filaments in the matrix. These stresses give rise to the large spatial extent of the observed cell-induced matrix stiffness gradient, which can provide a mechanism for mechanical communication between cells.   

Force Transmission across the Endothelial Glycocalyx

The glycocalyx is an interfacial biomaterial found on the surface of a wide variety of cell types. This matrix plays an active role in defining the mechanical and biochemical environment of living cells. In addition, it is directly involved in the detection of mechanical stimuli in the cellular environment, such as the shear stress in the blood vessel wall. As an incredibly low-density soft material the glycocalyx has proved a challenging target to probe experimentally. In this work, we describe our efforts to measure the force transmission across the glycocalyx in the presence of time varying fluid flow. Strain in a soft deformable substrate is measured in the presence of sinusoidal fluid flow as a biomimetic glycocalyx comprised of hyaluronan and charged proteoglycans is grafted to its surface. We investigate the effect of these major structural elements of the glycocalyx on measured strain on the substrate. The results may point toward a potential physical mechanism for the involvement of the hyaluronan-rich glycocalyx in the detection of shear flow at the cell surface.
  

Visualizing the emergence of order during self-assembly of protein nanorods

Protein scaffolds, such as collagen fibrils, provide the template for generating macromolecular-inorganic hybrid structures. The production of synthetic protein templates would allow scientists to create materials with precise control over spatial arrangement and morphology. However, how to program intermolecular interactions to tune the order of synthetic protein templates is still not crystal.
In this presentation, I will share recent results in the effort to build 2D protein liquid crystal with long-range order from computationally designed protein nanorods. I further utilize in-situ video-rate, single-molecule atomic force microscopy to observe the assembly of these proteins into two-dimensional matrices. With this technique, I can directly observe dynamics of single-molecules, moving, reorienting, and generating extended crystalline order. With the help of molecular dynamic simulation, I am able to investigate how protein-protein interactions, protein-substrate interactions influence the overall assembly dynamics. Indeed, in many cases, it is entropy, both solute entropy and osmotic pressure, rather than enthalpy, formation of chemical bonds, to determine the phases of protein self-assembly matrix, from highly dense liquid to isotropic to liquid crystal to crystal.   

Modeling and Design of Adsorption Based Filters and the Bio-remediation of Heavy Metal Contaminated Water

I will discuss kinetic models of adsorption, as well as our mathematical models of such filters. These mathematical models have been developed in support of our interdisciplinary lab group and can be used in filter design. Our group conducts research into bio-remediation of heavy metal contaminated water via filtration. The filters are constructed out of biomass, such as spent tea leaves. The spent tea leaves are available in large quantities as a result of the industrial production of tea beverages. The heavy metals bond with the surfaces of the tea leaves (adsorption). I will compare the models' predictions to data obtained from computer simulations and experimentally by our lab group.   

Mucus and mucin biopolymer environments reduce the efficacy of polymyxin and fluoroquinolone antibiotics

Mucus, a biopolymer hydrogel that covers all wet epithelia, is one of the primary arenas for microbes in the body, including the microbiome and pathogenic microbes that can cause serious infections. Mucus has the potential to bind small molecules and influence bacterial physiology, two factors that might affect the efficacy of antibiotics. Despite this, the impact of mucus on antibiotic efficacy has not been thoroughly characterized. We investigated the efficacy of polymyxin and fluoroquinolone antibiotics against the opportunistic pathogen Pseudomonas aeruginosa in native mucus and purified mucin biopolymer environments. We found that mucus reduces the efficacy of polymyxin and fluoroquinolone antibiotics against P. aeruginosa. MUC5AC, MUC2, and MUC5B mucin biopolymers, the gel-forming components of mucus, are primary contributors to this reduced efficacy. Our findings highlight that inclusion of the biomaterial environmental context is an important consideration when evaluating antimicrobial efficacy in vitro.   

Morphological change of the phospholipid vesicle induced by actin polymerization of the artificial photosynthetic protocellular system.

We designed, built, and tested a light-harvesting encapsulated artificial organelle system that provides both a sustainable energy source and a means of controlling intracellular reactions. With the reconstitution into lipid vesicles, an ATP synthase and two photoconverters (plant-derived photosystem II [PSII] and bacteria-derived proteorhodopsin [PR]) enabled orchestration of ATP synthesis. Independent activation of the two photoconverters, which respond to different light wavelengths, allowed dynamic regulation of ATP synthesis. The resulting system was able to simulate a ubiquitous process in cells—cytoskeleton formation through ATP-dependent actin polymerization in a giant vesicle. Optical stimulation initiated ATP synthesis and induced ATP-dependent actin polymerization, leading to growth of three-dimensional actin filaments. Cytoskeleton formation and manipulation successfully induced morphological change of the outer membrane of the protocellular system by tuning the attraction–repulsion interactions between actin filaments and phospholipids.
  

Dual-gel Dual-porosity 3D ECM Mimics for Studying flow-induced mechanotransduction

Interstitial flow in the Extracellular Matrix (ECM) has been postulated to play a key role in regulating behaviors of cells. Among the cellular behaviors, cell migration is hypothesized to be influenced by interstitial flow via two mechanisms: autologous chemotaxis and integrin-mediated focal adhesion activation. It is commonly accepted that the effectiveness of the former depends on interstitial fluid velocity and the effectiveness of the latter depends on the flow-induced force on a cell, which relates to both interstitial fluid velocity and matrix permeability. The two mechanisms have never been rigorously verified using the traditional single hydrogel-based models due to the inevitable correlation between matrix permeability and stiffness. In this work, we developed a dual-gel dual-porosity 3D ECM mimics to address this challenge. With the new model, one can individually control the interstitial flow and matrix permeability without affecting the primary matrix stiffness. We also conduct preliminary studies on the efficiency of our model in studying the effect of interstitial flow on cancer cell migration.   

Targeted photothermal lysing of E. Coli via a geometrically-modified Au-binding M13 virus

Increasing antibiotic resistance (ABR) in bacteria is a continuing public threat to human health. Antibody functionalized nanoparticles (NPs) in conjunction with photothermal lysis have been successful in targeting and eradicating these pathogens. Yet, synthesis of antibodies is an expensive and time-consuming process. Alternative approaches capable of selectively delivering photothermally-active NPs to ABR bacteria are essential. In our work, a geometrically-modified, Au-binding M13 bacteriophage offers a novel solution to this challenge. Rod-shaped and spherically-shaped viral templates were created via chloroform interaction with the filamentous bacteriophage. Decorated with Au NPs, both of these scaffolds were capable of targeting E.Coli bacteria via attachment of exposed minor coat protein to the pathogen. Bactericidal studies of rods and spheroids were executed under a range of 532 nm laser irradiation conditions: power (0 - 60 mW) and time (0 - 20 min). Using geometrically-modified M13 scaffolds up to 65% of E.Coli were killed within 20 min. Through genetic modification of the minor coat protein the bactericide effects of these templates could be extended to target other pathogens. These findings advance efforts to engineer versatile and low-cost strategies to tackle ABR.   

Gold Binding by Design: A Combined Theoretical/Experimental Study of Biohybrid Systems

Biomolecular-inorganic hybrid materials have many potential applications in medicine, electronics, and nanotechnology. It is therefore of great interest to design peptides that have predictable and controllable binding to inorganic surfaces, requiring a detailed understanding of biotic/abiotic interactions. Computational models have provided key insight into the underlying intermolecular interactions in such systems. However, owing to limited computational resources, these studies are often performed using periodic models where care must be taken to understand the effects of model size and periodicity, as well as any possible additional limitations in the method. Here we perform DFT calculations exploring the binding interactions between amino acid residues and the Au(111) surface. Results are presented in the context of experimental results from bacterial display studies of engineered E. coli binding to gold surfaces with applications including development of viable E. coli/AuNP hybrid systems and microbial fuel cells.   

A Comparative Study on Interspecies Blood Rheology

Blood is a complex suspension of red blood cells (RBCs), white blood cells, and platelets in an aqueous plasma phase with several dissolved proteins. Despite a consistent makeup, with minor changes in constituent sizes and volume fractions, blood from different species exhibits significantly different rheological signatures. Blood from some species including horse, pig, and human exhibit a yield stress at low shear rates, while blood from other species including cow, sheep, and guinea pig exhibit no yield stress. This is attributed to the presence or absence of microstructural RBC aggregates at low shear rates. Moreover, blood samples across all species may exhibit unique transient behavior which enables insight into how blood evolved to the specific organism. In this work, we present new steady and transient rheological data on blood from several species. We show that a previously developed thixotropy model can be used to model blood rheology across species and discuss the similarities and differences. This study improves the understanding of how blood changes across species, which is important for drug clinical testing and elucidates the connection between physiology and blood rheology.   

Early onset of kinetic roughening due to finite step width in hematin crystallization

Crystallization is an example of a highly non-equilibrium process, in which the flows of mass and energy are governed by dynamic structures comprising a two-dimensional interface between adjacent three-dimensional semi-spaces. During crystal growth, the structure of its interface with the growth medium dictates the molecular mechanism of solute incorporation, the response of the growth dynamics to temperature and composition gradients, the action of impurities and dopants, and, ultimately, the crystal perfection. Interfaces that are smooth at equilibrium may become rough during growth at elevated supersaturation. We observed a smooth to rough transition during the growth of hematin crystals from a biomimetic mixed organic-aqueous solvent. Hematin crystallization is the main pathway employed by malaria parasites to sequester toxic hematin, released during hemoglobin digestion; its inhibition is considered the most successful target for antimalarial drugs. We show that the transition occurs at a supersaturation significantly lower than that predicted by published criteria.