(632g) Single Nanofiber Structural Stiffness Directly Affects Cellular Migration and Cytoskeletal Response of C2C12 Cells

Introduction

Cell substrate interactions are an important part of
many biological processes, including wound healing, stem cell differentiation,
and immune response [1,2]. 
Both the cytoskeletal arrangement and migration characteristics respond to
physical and chemical cues from the cell substrate in each of these processes. 
The substrate that cells are interacting with while in vivo is the
extracellular matrix (ECM), a dense fibrous mesh that serves as the support
structure for tissues [3?5]. 
Previous  in vitro studies have adopted the use of both fibrous
constructs and gels made of collagen, fibrin, or other biocompatible materials
in order to try to match the substrate interactions that are seen between the
cell and ECM.  However, there has been little research into the effects of the
local stiffness of the microarchitecture that makes up these fibrous substrates
on cell behavior, despite the fact that the overall material modulus of the
substrate has been shown to alter cellular behaviors, including cytoskeletal arrangement
and migration characteristics [6?8]. 
In this study the previously reported STEP technique (Spinneret based Tunable
Engineering Parameters) has been utilized to create evenly spaced single
suspended nanofibers on which the effects of the gradient of fiber structural stiffness
has been explored [9,10].
These studies in understanding single cell mechanics will aid in design of nanostructured
biomaterials.

Methods and Materials

Aligned, evenly spaced single polystyrene nanofibers
were deposited on a substrate consisting of two polydimethysiloxane (PDMS)
blocks fixed to a glass slide.  Before fiber deposition, PDMS constructs were
coated with uncured PDMS to act as an adhesive.  Fibers were coated with 2
µg/ml fibronectin overnight before C2C12 mouse myoblasts were cultured on the
fibers.  Cells adhered to single nanofibers and migrated along the fiber
length.  Time lapse imaging was performed to allow for migration speed and cell
spreading characterization.  Cells were fixed using 4% paraformaldehyde and
immunostained for focal adhesions, f-Actin stress fibers, and the nucleus. 
Characterization of cell length, focal adhesion cluster length, and nuclear
shape index was performed using immunostained images.  

Results and Discussion

It has been found that cells sense and respond to
the changing gradient of structural stiffness that is present along a single
nanofiber.  Cell length and focal adhesion cluster length have been shown to
increase with increasing structural stiffness, while nuclear shape index has
been shown to decrease with increasing structural stiffness [11]  Additionally,
preliminary results have shown that cells reach their fully spread state and
begin migrating in less time on areas of higher structural stiffness.  These
modifications to the cytoskeletal arrangement and cellular behavior in turn
lead to a decrease in average cellular migration speed at higher structural
stiffness areas as shown in Figure 1.

Figure 1.  A)  Typical suspended nanofiber
substrate consisting of a glass coverslip with fibers deposited over and fixed
to two PDMS blocks.  B)  Migration speed reaction to structural stiffness. 
Average migration speed decreases with increasing structural stiffness.  C) 
Time for cells to reach their fully spread state and begin migrating decreases
with increasing structural stiffness.    D)  Nucleus length and width
respectively increase and decrease with increasing structural stiffness. 

Conclusions

The results found in this work indicate that cells
are employing a substrate stiffness sensing mechanism that goes beyond bulk
material stiffness sensing.  Cells are shown to respond to structural stiffness
by modulating their cytoskeletal characteristics both in magnitude and
temporally leading a change in migratory behavior.  These results add to the
current understanding of single cell mechanics and aid in design of nanostructured
biomaterials.

References

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In Review