(500f) Migration Dynamics of Mouse C2C12 Myoblasts On Single Suspended Fiber Mimicking ECM Fibril Beam Stiffness (N/m)

Migration
Dynamics of Mouse C₂C₁₂ Myoblasts on Single Suspended
Fiber Mimicking ECM Fibril Beam Stiffness (N/m)

Sean Meehan¹. Kevin Sheets²,
Amrinder S. Nain^1,2,   Department
of Mechanical Engineering¹, School of Biomedical Engineering and
Sciences², Virginia Tech, Blacksburg VA

Introduction

In vitro
testing platforms are one of the particularly promising approaches to
characterize the dynamic roles of biophysical and biochemical cues on cellular
growth, development, and behavior.  Numerous
studies have shown that mechanical stimuli in a cell's external environment can
have a profound effect on migration, proliferation, differentiation, and
apoptosis.  Nanoscale topographical
features have been shown to not only increase migration and directional
persistence time, but also decrease proliferation in human endothelial cells [1]. 
Additionally, it has also been shown that mesenchymal stem cell
differentiation can be influenced using custom topographical and geometric cues
[2,3]. 
In addition to topographical cues, the stiffness (measured in N/m²)
of the commonly used flat substrate
used for cell studies has been shown to have a significant effect on cellular
migration and differentiation.  As little
as a four kilopascal change in stiffness can elicit significant changes in
cellular proliferation, migration speed, and differentiation in both single
cells [4] and confluent sheets [5].  However,
flat substrates do not exhibit many of the fiber specific qualities that cells
encounter when interacting with the native extra cellular matrix (ECM), which
is a three dimensional, dense, fibrous mesh immediately surrounding the
cell.   Cells on the ECM fibers attach,
spread and eventually migrate with the fiber beam stiffness (measured in N/m)
playing an integral role in cellular behavior. 
Furthermore, it has been shown that the mechanical properties of the
extracellular matrix are tissue-specific, and these different properties can
dictate migration speed, cell's ability to remodel the ECM, and many other
important cellular behaviors [6,7].  Hence, it is obvious that the mechanical
properties of the ECM, especially stiffness (both modulus and beam stiffness),
have an important role in the life and behavior of cells within the body.  However, creating a mechanistically tunable
system to easily explore these properties is a challenging task due to lack of
understanding regarding cell-ECM relationships and difficulties to fabricate
precise nano-micro fibrous scaffolds of native proteins mimicking the native
environment.

In
order to explore this relationship between cellular activity and fiber
substrate, a novel testing platform is presented in this study.  Using a combination of the previously
reported STEP (Spinneret Based Tunable Engineering Parameters) fiber
manufacturing platform [8-11] with soft-lithography based micron scale PDMS
pillars, we explore the link between cellular behavior and fiber beam substrate
stiffness.  The importance of this study
lies in the unique ability of the STEP platform to provide precise control over
fiber diameter and spatial deposition, thus providing a tunable mechanistic
environment to explore cellular behavior. 
Current studies using electrospinning platforms have been used to study
cell behavior on a collection-mat of fibers. However, single cell-fiber studies
using electrospinning platforms are hard to accomplish due to lack of control
on fiber alignment, spacing and diameter. [12] In this study, the role of
substrate fiber beam stiffness (N/m) on cellular behavior, particularly
migration, is envisioned to provide new insights into the dynamics of cellular
polarizability, focal adhesion complex and cytoskeleton arrangement, and
cellular mechanics as a whole. 

Methods
and Materials

Standard
photolithography practices were used to create SU-8 2025 (Microchem) molds for
the fabrication of micropillars.  In
short, the SU-8 photoresist was spun onto a 4 inch diameter silicon wafer
(University Wafers), prebaked, exposed using a mask depicting pillar size and
placement, and then developed using SU-8 Developer (Microchem).  Molds were silanized and then coated with
Sylgard 184 PDMS and cured at 80^oC for 1 hour.  The thin PDMS films with attached pillars
were then ?glued? to glass substrates using uncured PDMS as an adhesive, and
then were cured again at 80^oC. 
Deposition of single nano-scale polystyrene (PS 8% w/w) fibers was
performed using the previously reported STEP technique, ensuring even spacing
and homogenous fiber parameters.  A small
amount of epoxy adhesive was used to ensure fixed boundaries at each end of the
fibers.  Scanning Electron Microscope
(SEM) images were taken to verify fiber homogeneity and fixed boundaries (Fig
1).  C₂C₁₂ Mouse
Myoblast cells were seeded onto the fibers and migration was characterized
using time lapse microscopy (Fig 2(A)). 
After time lapse studies were completed, samples were immunostained for
F-actin stress fibers, nuclei, and focal adhesions.  Scanning electron microscope images were also
taken of some samples for complete characterization of micropillar and
nanofiber geometry.

Figure 1: SEM image showing fiber and micro-scale pillars
with fixed boundaries.  Arrows indicate
suspended fiber

Results
and Discussion

Overall,
cells were largely observed to migrate away from the center of the fiber, where
fiber beam stiffness is lowest, with average migration speed decreasing near
the edges of the fiber, where fiber stiffness is the highest (Fig 2(B)).  Suspended fibers were characterized for
stiffness using an Atomic Force Microscope. 
Preliminary data has shown that fiber stiffness is highly variable along
the length of the fiber (Figure (2(C)). 
Cells were observed to attach to single fibers and elongate along the
fiber axis with immunostaining studies showing that focal adhesion sites
concentrated at the poles of the cells with a higher level of focal adhesion at
the leading edge of the cell.  F-actin
stress fibers were found to be localized along the periphery of the cell with a
higher concentration at the leading edge of the cell.  The nucleus was found to be stretched along
the fiber axis and enclosed within stress fibers (Fig 2(D)).  This phenotype suggests a high level of
polarizability of cells on the single fibers, with migration occurring in a
single direction until an external stimuli (decreasing fiber beam stiffness)
causes a change in direction. Quantification of the leading and trailing edge
spatio-temporal dynamics is of immense interest and part of our future
efforts.  

Figure 2:  A)
Phase image of a cell traveling along a single fiber.  B) Average
cellular migration speed as a function of distance from the center of the fiber
(where stiffness is lowest) C) Average fiber stiffness along the fiber.  Stiffness dramatically increases near the
pillars  D) Immunostained image showing
f-actin (red), nuclei (blue), and focal adhesions (green)

Conclusions

The
complete characterization of the influence of fiber substrate stiffness on
cellular migratory behavior will be an important step in understanding many
complex processes occurring within cells. 
The use of the STEP technique with simple photolithography techniques
provides suspended single fibers that mimic single ECM fibrils in order to
provide conditions that are as similar to in
vivo conditions as possible.  This
technique allows for accurate measurement of cellular migration while
maintaining simple, low cost fabrication as well as the possibility of
modification to mimic the natural microenvironment of cells in various
conditions.  Cells attached on single
suspended fibers were observed to preferentially migrate towards the stiffest
region of the fiber with migration speed decreasing with increase in substrate
stiffness.  Additionally, immunostaining
showed a high level of polarization and organization of F-actin stress fibers
along the fiber axis.  The complete
characterization of migratory behavior on this combined system will allow for a
more complete view of the in vivo
interaction of cells with the extra cellular matrix and it's relation to
cellular mechanics as a whole, which will be invaluable in the design of
accurate wound healing scaffolds, drug testing platforms and design of accurate
tissue engineering scaffolds. 

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