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Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity.

ABSTRACT: Although bacterial cells are known to experience large forces from osmotic pressure differences and their local microenvironment, quantitative measurements of the mechanical properties of growing bacterial cells have been limited. We provide an experimental approach and theoretical framework for measuring the mechanical properties of live bacteria. We encapsulated bacteria in agarose with a user-defined stiffness, measured the growth rate of individual cells and fit data to a thin-shell mechanical model to extract the effective longitudinal Young's modulus of the cell envelope of Escherichia coli (50-150 MPa), Bacillus subtilis (100-200 MPa) and Pseudomonas aeruginosa (100-200 MPa). Our data provide estimates of cell wall stiffness similar to values obtained via the more labour-intensive technique of atomic force microscopy. To address physiological perturbations that produce changes in cellular mechanical properties, we tested the effect of A22-induced MreB depolymerization on the stiffness of E. coli. The effective longitudinal Young's modulus was not significantly affected by A22 treatment at short time scales, supporting a model in which the interactions between MreB and the cell wall persist on the same time scale as growth. Our technique therefore enables the rapid determination of how changes in genotype and biochemistry affect the mechanical properties of the bacterial envelope.

SUBMITTER: Tuson HH

PROVIDER: S-EPMC3359400 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity.

Tuson Hannah H HH Auer George K GK Renner Lars D LD Hasebe Mariko M Tropini Carolina C Salick Max M Crone Wendy C WC Gopinathan Ajay A Huang Kerwyn Casey KC Weibel Douglas B DB

Molecular microbiology 20120502 5

Although bacterial cells are known to experience large forces from osmotic pressure differences and their local microenvironment, quantitative measurements of the mechanical properties of growing bacterial cells have been limited. We provide an experimental approach and theoretical framework for measuring the mechanical properties of live bacteria. We encapsulated bacteria in agarose with a user-defined stiffness, measured the growth rate of individual cells and fit data to a thin-shell mechanic ...[more]

PMID: 22548341

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Project description:The precise mechanisms that lead to orthopedic implant failure are not well understood; it is believed that the micromechanical environment at the bone-implant interface regulates structural stability of an implant. In this work, we seek to understand how the 3D mechanical environment of an implant affects bone formation during early osteointegration. We employed two-photon lithography (TPL) direct laser writing to fabricate 3-dimensional rigid polymer scaffolds with tetrakaidecahedral periodic geometry, herewith referred to as nanolattices, whose strut dimensions were on the same order as osteoblasts' focal adhesions (?2?m) and pore sizes on the order of cell size, ?10?m. Some of these nanolattices were subsequently coated with thin conformal layers of Ti or W, and a final outer layer of 18nm-thick TiO2 was deposited on all samples to ensure biocompatibility. Nanomechanical experiments on each type of nanolattice revealed the range of stiffnesses of 0.7-100MPa. Osteoblast-like cells (SAOS-2) were seeded on each nanolattice, and their mechanosensitve response was explored by tracking mineral secretions and intracellular f-actin and vinculin concentrations after 2, 8 and 12days of cell culture in mineralization media. Experiments revealed that the most compliant nanolattices had ?20% more intracellular f-actin and ?40% more Ca and P secreted onto them than the stiffer nanolattices, where such cellular response was virtually indistinguishable. We constructed a simple phenomenological model that appears to capture the observed relation between scaffold stiffness and f-actin concentration. This model predicts a range of optimal scaffold stiffnesses for maximum f-actin concentration, which appears to be directly correlated with osteoblast-driven mineral deposition. This work suggests that three-dimensional scaffolds with titania-coated surfaces may provide an optimal microenvironment for cell growth when their stiffness is similar to that of cartilage (?0.5-3MPa). These findings help provide a greater understanding of osteoblast mechanosensitivity and may have profound implications in developing more effective and safer bone prostheses. STATEMENT OF SIGNIFICANCE:Creating prostheses that lead to optimal bone remodeling has been a challenge for more than two decades because of a lack of thorough knowledge of cell behavior in three-dimensional (3D) environments. Literature has shown that 2D substrate stiffness plays a significant role in determining cell behavior, however, limitations in fabrication techniques and difficulties in characterizing cell-scaffold interactions have limited our understanding of how 3D scaffolds' stiffness affects cell response. The present study shows that scaffold structural stiffness affects osteoblasts cellular response. Specifically this work shows that the cells grown on the most compliant nanolattices with a stiffness of 0.7MPa expressed ?20% higher concentration of intracellular f-actin and secreted ?40% more Ca and P compared with all other nanolattices. This suggests that bone scaffolds with a stiffness close to that of cartilage may serve as optimal 3D scaffolds for new synthetic bone graft materials.

Dataset Information

Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity.

Publications

Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity.

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