(781f) Design of Nanofiber Scaffolds for Regulating Cell Behaviors

Design of Nanofiber
Scaffolds for Regulating Cell Behaviors

Jingwei Xie,* Bing Ma

Marshall
Institute for Interdisciplinary Research and Center for Diagnostic Nanosystems,
Marshall University, Huntington, WV, 25755 USA

^*Correspondence
should be addressed to: xiej@marshall.edu

Introduction

Regenerative medine emerged in the 1980s and has
continuted to evolve as an exciting and interdisciplinary filed to develop
tissue constructs which can be used to restore, replace or regenerate defective
tissues or organs.¹ The most pormising strategy which have been
adopted for the creation of tissue constructs is to use a combination of cells
and materials typically in the form of scaffolds. The scaffolds provide the
structural support for cell attachment and subsequent tissue development. The
determination and control of cell-material interaction are very important to
ensure that the materials and host are compatible and function properly.² Previous
studies demonstrated that cell behaviors could be controlled by manipulating
the physical and chemical properties of the culture substrates.^3-5
These studies could provide important fundamental information on how to
regulate cell behaviros using these cues, however, they oversimplified the
scenario of microenvionment where cells usually live. Electrospun nanofibers
can be used to mimic the 3D archtecture of extracellular matric owing to its
high surface-to-volume ratio and fibrous morphology.⁶ Comparing to
other surface features, nanofibers are more physiological relevant. The aim of
present study is to design electrospun nanofiber scaffolds for regulating behaviors
of various cell types.

Materials and Methods

Nanofibers were produced
utilizing a standard electrospinning setup, described previously.^7,8
Poly(ε-caprolactone) (PCL) (Mw=80 kDa, Sigma-Aldrich, St. Louis, MO) was
dissolved in a solvent mixture consisting of dichloromethane (DCM) and N,
N-dimethylformamide (DMF) (Fisher Chemical, Waltham, MA) with a ratio of 4:1
(v/v) (at a concentration 10% (w/v)). Various collectors were employed to
generate nanofiber assemblies of different orders / patterns.

Fiber mats were firstly
treated with plasma. Subsequently they were immersed in 0.2 mg/mL of
dopamine.HCl of 10mM Tris buffer (pH 8.5) over 4 h and rinsed with water for
later use. Fibronectin conjugation to nanofibers was performed following
previous study. Specifically, the polydopamine-coated nanofibers were
transferred into fibronectin solution (50 µg/mL sodium phosphate buffer, pH
7.8) for overnight. The membrane was washed three times with PBs prior to cell
culture.

NIH3T3 cells were cultured at 37°C in a
95% air and 5% CO₂ atmosphere. Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% gentalmycine/streptomycin
was used as the cell culture medium and was changed every 2 days. Prior to
seeding the cells on the scaffold, cells were trypsinized and counted. Around
1×10⁴ cells were seeded on each scaffold. At 2 h, 4 h, and 24 h, the
live cells were stained with Calcein AM (Invitrogen) and F-actin was stained
with Alexa Fluor 488^® phalloidin (Invitrogen).

Results and Discussion

Promotion of robust cellular adhesion is
critical for the chronic success of implantable biomaterials, such as nanofiber
matrices.We modified
electrospun nanofiber scaffolds with various surface chemistries. The thickness of polydopamine coating can
be readily controlled by adjusting the concentration of dopamine and reaction
time indicated by TEM images. XPS analysis further confirms the coating.
Mechanical test shows the increase of stiffness after coating process. We show
that biomacromolecules like fibronectin can be directly immobilized on the
surface of polydopamine-coated, electrospun nanofibers, resulting in the increase
of NIH3T3 cell adhesion in the order of pristine nanofibers,
polydopamine-coated nanofibers, and fibronectin-immobilized,
polydopamine-coated nanofibers. Figure
1 shows live cell staining on seeded nanofiber scaffolds after incubation for
2, 4, and 24 h, indicating polydopamine coating and fibronectin immobilization
resulted in enhancement of NIH3T3 cell attachment, spreading and cytoskeletal
development.

Figure 1. Calcein AM staining of live cells cultured on random
nanofiber mats with different surface chemistries. PDA: polydopamine.

By utilizing a unique collector composed of a central
point electrode and a peripheral ring electrode during electrospinning prcess,
we sucessfully fabricated scaffolds consisint of radially-aligned nanofibers.
This novel class of scaffolds was able to present nanoscale topographic cues to
cultured fibroblasts, directing and enhancing their migration from the
periphery to the center.  

We also found that Schwann cells were
successfully organized and oriented by nanofibers on the surface of both
single- and double-layered scaffolds. Specifically, individual Schwann cells
were observed to be elongated parallel to the fiber axis of aligned nanofiber
scaffolds independent of nanofiber organization below the surface. Pre-seeding
nanofiber scaffolds with Schwann cells also increased neurite extension by
300-800% on all types of scaffolds tested. Neurite outgrowth on aligned
nanofiber scaffolds pre-seeded with Schwann cells was observed to run parallel
to the long axis of the underlying fibers, suggesting that topographical cues
presented by the nanofibers were effectively transmitted to the extending
neurites via Schwann cells. Taken together, these results suggest that
pre-seeding nanofiber scaffolds with Schwann cells could dramatically increase
their regenerative potential while preserving the topographical cues unique to
the aligned fibers.

By varying nanofiber densities, surface properties,
and culture supporters, we were able to control the direction of neurite
outgrowth projected from dorsal root ganlia, embryoid bodies, or dissociated
embryonic stem cells which were seeded on scaffolds made of uniaxially-aligned
nanofibers. In addition, we were capable of control the differentiation of
embryonic stem (ES) cells by utilizing nanofibers as a culture substrate. We
found that seeding a low density of dissociated ES cells onto uniaxially
aligned PCL nanofiber scaffolds could make ES cells differentiate into neurons
instead of astrocytes.

Conclusion

In summary, we have demonstrated the control over the
cell adhesion, cell migration, cell differentition, and neurite outgrowth using
designed electrospun nanofiber scaffolds. These scaffolds could be promising
for use in various tissue engineering applications.

References

1    
A. Khademhosseini, R.
Langer, J. Borenstein, and J. P. Vacanti, Proc. Natl. Acad. Sci. USA,
2006, 103, 2480.

2    
J. A. Hunt, Nat.
Mater., 2008, 7, 617.

3    
F. Guilak, D. M. Cohen,
B. T. Estes, J. M. Gimble, W. Liedtke, and C. S. Chen, Cell Stem Cell,
2009, 5, 17.

4    
R. J. McMurray, N.
Gadegaard, P. M. Tsimbouri, K. V. Burgess, L. E. McNamara, R. Tare, K.
Murawski, E. Kingham, R. O. C. Oreffo, and M. J. Dalby, Nat. Mater.,
2011, 10, 637.

5    
A. J. Engler, S. Sen,
H. L. Sweeney, D. E. Discher, Cell, 2006, 126, 645.

6    
J. Xie, X. Li, and Y.
Xia, Macromol. Rapid Commun., 2008, 29, 1775.

7    
J. Xie, S. M. Willerth,
X. Li, M. R. MacEwan, A. Rader, S. E. Sakiyama-Elbert, and Y. Xia, Biomaterials,
2009, 30, 354.

8    
J. Xie, M. R. MacEwan,
X. Li, S. E. Sakiyama-Elbert, and Y. Xia, ACS Nano, 2009, 3,
1151.