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HA Biocompatible Nanofiber Mesh for Cell Binding
Studies Girma Endale, Raymond E. Turner, and
Michael F. Rubner Center for Materials Science
Engineering Massachusetts Institute of
Technology, Cambridge, MA 02139
Results
Introduction
Materials and Methods The sodium salt of
hyaluronic acid (HA) from Streptococcus was
purchased from Sigma-Aldrich and the viscosity
average molecular weight was determined to be
150, 000 grams/mole. HA preparations used in
this work were made from polymer dissolved in
sodium acetate buffer at pH 5.0, 4.0. or 3.0. In
some cases HA was dissolved in water free of
added salt. All chitosan used in the experiment
as medium molecular weight chitosan in acetate
buffer set at pH 4.1. Both solutions were used
at 0.1 gram . HA-chitosan multilayers were
made using a spin dipping protocol where the
multilayered polymer solutions were alternated
after 10 minutes emersion and each layer washed
in water for 2 minutes, 1 minutes, and 1 minute,
respectively. Hyaluronic acid was added to
finished HA-chitosan scaffolds as microdroplets,
spin-dipping, or as a spin-coating. Analysis of
HA on surfaces were carried out using the JEOL
Model JSM-6060 SEM, The P-10 Profilometer and
the Zeiss Axioplan Optical Microscope. Staining
of the multilayers where done using 10-3 M
methylene blue or alcian blue (8XG) dyes.?
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Dye Staining Intensity Versus pH of HA Top Layer
HA fabricated scaffolds can interact with cells
Research has shown that hyaluronic acid (HA), a
high molecular weight biocompatible and water
soluble polymer, has features which makes it
highly suitable for the biomaterials engineering
of SMART devices. Many cells are naturally
programmed to recognize HA. We determine the
optimal conditions needed to expose cell binding
regions of an approximately 150K HA fragment
embedded in a polyelectrolyte multilayer. The HA
binding regions identified by methylene blue
staining of the exposed carboxyl groups say
little about the helical nature of the region as
accesses by the poly-cationic dye, alcian blue.
The scaffolds we prepare are expected to be used
to study to binding and migratory behavior of
various cells containing Hyaluronic Acid Binding
Proteins or HABPs.
The optical microscope image of a 7 day DMEM
culture of BCE cells show that HA nanofiber mesh
and cable structures can link to cells in vitro.
This experiment demonstrated the electrospun HA
nanofiber composites may not interfere with the
HA binding sites recognized by HABPs.

Figure 6. The photograph on the left shows
alcian blue (top row) and methylene blue (bottom
row) staining of HA-chitosan multilayers produced
by spin-dipping. The photograph on the right
shows methylene blue staining (top row) and
alcian blue staining (bottom row) of HA-chitosan
multilayers produced by spin-coating.
Figure 9. Alcian blue stained HAchitosan
multilayers produced by spin dipping. The image
on the left is 0.1 HA prepared in water at pH
4.75 with the top layer at pH 4 and a height of
-3.5 bilayers. The image on the right was
prepared like the one on the left except only 3
bilayers with no added HA.
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HA-Chitosan Scaffolds with Added HA
Multilayer height as a Function of pH
HA-chitosan multilayers of varying thicknesses
were prepared. HA was added to the prepared
scaffolds with chitosan as the top layer. The
additional layer of HA was stained and washed
with alcian blue or methylene blue and imaged by
optical microscopy or SEM.
The height of HA-CH multilayers were determined
by profilometry and then stained with methylene
or alcian blue dye. After washing and drying. The
UV-VIS date was taken and plotted against
multilayer thickness.
Figure 12. Jannine Austrie et al, MIT-UROP-CC
Student Poster 2007
Figure 1. Hyaluronic acid (HA) is a linear
copolymer structure, consisting of alternating 1
and 3-linked 2-acetamido-2-deoxy-ß-D-glucose
(GlcNAc) and 1- and 4- linked ß-D-glucuronic acid
(GlcUA) residues (1). The distance between
adjacent carboxylic acid groups in the helical
twist is about 1 nm.
Discussion
This work demonstrates the feasibility of
fabricating biocompatible surfaces for cell
studies using hyaluronic acid (HA). Alcian blue
is the best dye for staining, but more
sophisticated staining techniques and site
specific agents are possible in collaborative
studies. It was previously shown that HA can
bind and stack the cationic dye, alcian blue,
when embedded in a gel matrix. These data show
that HA embedded in a multilayered matrix can
also bind and stack alcian blue dye (Turner and
Cowman, 1985). We have observed the binding of
Bovine capillary endothelial cells to HA fiber
mesh after several days in culture media. We
observed that the pH , multilayer thickness, and
the degree of hydration all play critical roles
in the surface structure of HA applied to
preformed HA-chitosan multilayers. More detailed
work on the staining by alcian blue can be
obtained by UV-VIS scanning of the slides.
Figure 3. HA is added as a top layer onto a
prepared HA-Chitosan bilayers. The left images
are stained with alcian blue (AB) and the right
images are stained with methylene blue (MB). The
thin films were tested for stability. The MB
diffused from the mesh after several hours while
the alcian blue continued to expose structures
after several days in water.
Creating HA Patterned Multilayered Scaffolds
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Figure 7. Profilometry results for HA-chitosan
bilayers.
Figure 10. SEM image of HA on a HA-chitosan
multilayer.
Figure 2. HA self-asssembly matrices attract
different types of cells under physiological
conditions. A major hyaluronic acid binding
protein (HABP) is CD44 (Toole, B.P. Turner, RE,
Banerjee, SD., Prog Clin Biol Res. 1993
383B437-44).
Figure 4. (Left) Methylene Blue staining of pH 4
HA top layer (micro-droplet) on a 3.5
multilayered substrate. The surface features of
the HA top layer appears to change with the
degree of hydration. (Right) HA multilayer
produced using HA only (no chitosan) on a glass
microscope slide.
Acknowledgements
This work would not have been possible without
the support of MITs Community college outreach
program. We would like to thank the students,
faculty members, and staff of CSME at MIT for
their support during this summer internship. A
special thanks to all members of the Rubner
Group.
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Figure 8. Profilometry results for HA-chitosan
bilayers
Figure 11. SEM image of HA on a HA-chitosan
multilayer.
Figure 3. This figure illustrates the research
strategies we use to create HA scaffolds for cell
binding studies. We alter the bilayer thickness
of the HA-chitosan and vary the pH of the HA top
layer. The top layer is added as a micro-droplet,
a spin dipping layer or as a spin coating. We
used two dyes, alcian blue and methylene blue to
assess the orientation of the HA suitable for
binding to cells containing HABP.
Figure 5. Alcian blue stained micro-droplet on
the surface of an HA-chitosan multilayer.
This work was supported by the CMSE Community
College Program, as part of the MRSEC Program of
the National Science Foundation under award
number DMR 02-13282. The Community College
Program gratefully acknowledges support from the
MIT Deans of Science and Engineering and from the
MIT VP for Research