Page 49 - ASME_NEMB_2016_Program

TRACK 4 TRACK 4 Technical Program

understood. Here we reveal that cell traction force exerted on extracellular characterized in our lab (Whisler et al., 2014) and we run experiments with
matrix directs and signals metastatic-like dispersion and malignant transfor- cells encapsulated in the fibrin matrix at two densities: (i) a cell density that
mation of cancerous HCT-8 cell colonies in vitro. Our cell culture studies on facilitates vascular network formation, and (ii) a much lower density where
gels evidence that the initiation of metastatic-like dispersion of HCT-8 col- vascular networks do no form. To systematically vary the cross-link density,
onies depends on both gel stiffness and colony size: dispersion occurs for we polymerize fibrin in the presence of different concentration of factor XIII,
cell colonies on stiff gels but not on soft ones; on stiff gels smaller colonies a well-known cross-linker for human fibrin gel. Such cross-linker variations
disperse statistically earlier than larger ones. Traction force microscopy stud- encompass a physiologically relevant level and three progressively decreas-
ies show that the traction force is inherent to the gel stiffness and colony ing values. Fluorescent beads are also introduced and tethered in the fibrin
size, indicating that there exists a traction force threshold at the pre-disper- mesh. Displacement data are obtained through tracking of beads from the
sion stage above which the cell colonies disperse into individual malignant stressed configuration to the relaxed, stress-free configuration. The latter
cells and below which the cell colonies remain cohesive. The finding under- is imposed by treatment with Cytochalasin D to inhibit cell contractility. A
scores the importance of cellular forces for regulating cancer metastasis and 3D displacement map is then extracted around the cells contained in each
progression of malignancy of cells, and opens up a unique avenue for the region of interest. These displacement maps evidence higher bulk displace-
development of anti-metastasis therapies. ment values after relaxing contractility as the cross-link density decreases.
The mean of the bead displacements around single cells in the case of the
lowest cross-link density fibrin was indeed about 30% higher than in the
case of the highest cross-link density. However, in terms of vascular network
4-3: TISSUE MECHANICS
formation, preliminary results indicate that the lowest cross-link density is not
optimal, as vascular networks forms with fewer branches and the structure is
Harris 11:30am - 1:00pm more planar. This might suggest that gels with very low cross-linking do not
provide enough support for cell protrusions and connections in early vascu-
logenesis. However, a deeper investigation on the strain that each cell feels
Session Organizer: Xianqiao Wang, University of Georgia, Athens, is required to confirm that. We also plan Brownian dynamics simulation of 3D
GA, United States fiber networks (Mak et al., 2014) to interpret these results, particularly taking
into account fiber nanomechanics and cross-linking, thus mimicking the
11:30am Non-linear elasticity and relaxation in polymer net- fibrin gel microarchitecture. We explore the multiscale mechanical factors
works and soft tissues in the extracellular matrix that govern the propagation of force signals and
regulate vascular tissue formation and maintenance.
Keynote. NEMB2016-5939 Acknowledgements:
Marie Curie International Outgoing Fellowship within the 7th European Com-
munity Framework Program Grant PIOF-GA-2013-625500
Paul Janmey, Univ Of Pennsylvania, Philadelphia, PA, United States References:
Whisler, J. A., Chen, M. B., and Kamm, R. D. (2014). Tissue Eng. Part C. Meth-
The stiffness of tissues in which cells are embedded has effects on cell ods 20, 543–52.
structure and function that can act independently of or override chemical Mak, M., Kamm, R. D., and Zaman, M. H. (2014). PLoS Comput. Biol. 10,
stimuli. Most measurements of tissue stiffness report elastic moduli mea- e1003959.
sured at a single frequency and at a low strain, but tissues and the cells
within them are subjected strains that often exceed the range of linear visco- 12:20pm Mechanical Reinforcement of Proteins with Polymer
elasticity. Rheologic measurements of liver, brain, and adipose tissues over Conjugation
a range of shear, compressive, and elongational strains show that the visco-
elastic response of these tissues differs from that of synthetic hydrogels that
have similar elastic moduli when measured in the linear range. The shear Technical Presentation. NEMB2016-5995
moduli of soft tissues generally decrease with increasing shear or elonga-
tional strain, but they strongly increase under uniaxial compression. This Elizabeth P. DeBenedictis, Elham Hamed, Sinan Keten, North-
response requires contributions from both cells and the extracellular matrix. western University, Evanston, IL, United States
In contrast, networks of ECM constructs such as crosslinked collagen or
fibrin soften under compression, but strongly increase shear modulus when Many proteins are subject to mechanical stress in vivo, have load-bearing
deformed in extension. The mechanisms leading to the unusual strain-de- and load-sensing functions, and structures sensitive to external forces. Me-
pendent rheology of soft tissues and fibrous networks are not explained chanical reinforcement of proteins is highly relevant to tissue engineering,
by current models of polymer mechanics, but appear to relate to local and immunology, biosensing, biomaterials, and drug delivery. Conjugating poly
global volume conservation within the networks and tissues. ethylene glycol (PEG) to peptides, also known as PEGylation, is proven to in-
crease the thermodynamical stability of peptides, and has been successfully
12:00pm The Role Of Fibrin Cross-Linking On Forces Among applied to prolong the lifetime of peptide-based vaccines and therapeutic
Endothelial Cells In Vasculogenesis agents. While it is known that protein structure and function can be altered
by mechanical stress, whether PEGylation can reinforce proteins against
Technical Presentation. NEMB2016-6133 mechanical unfolding remains to be ascertained. In this work, all-atomis-
tic Molecular Dynamics simulations of alpha-helical peptides undergoing
Andrea Malandrino, Massachusetts Institute of Technology / Insti- constant tensile forces to induce unfolding are employed to assess protein
stability. With mechanical stresses ranging over an order of magnitude, our
tute for Bioengineering of Catalonia, Cambrige, MA, United States, results show that PEGylated alpha-helices consistently require a longer time
Michael Mak, Roger D. Kamm, Massachusetts Institute of Technol- to unfold compared with their native counterparts, indicating that polymer
ogy, Cambridge, MA, United States conjugation provides a mechanical reinforcement effect. Three regimes can
be distinguished for force-induced unfolding response of the alpha-helix:
Mechanics of soft fibrous matrices fundamentally relates to cell behavior in small, intermediate, and large force regimes, with the PEG stabilization effect
development and disease. Such matrices have important properties, such being most pronounced for small forces. Our results illustrate that unfolding
as strain-stiffening, or viscoelasticity, that emerge from their composition, progresses with backbone H-bonds breaking, which is followed by the ex-
dynamics, and structural organization. One of the key components in the posed polar groups forming H-bonds with the surrounding water molecules.
structural organization is the covalent cross-linking of the fiber mesh. In In PEGylated peptides, the likelihood of hydrogen bond exchange between
the present work, we study how the cross-link density within a fibrin gel af- the backbone and surrounding water molecules is reduced, which leads 49
fects the 3D force exchanges and distant mechano-communication among to greater unfolding times. PEGylation is found to increase the unfolding
endothelial cells. We use an in vitro model of vasculogenesis previously time through two mechanisms. We see that first, the unfolding rate of a he-