Published on: Mar 3, 2016
Transcripts - Nagurney_Summer_2014_Final_Paper_OfficialDoc
N-Cadherin: β-Catenin Signaling Leading to Maintenance or Differentiation of Cell
Populations in Response to Different Diameter Electrospun Fibers
Rebecca A. Nagurney
Abstract--Non-union fractures, bone fusions, osteotomies and
bone cancer are just some of the reasons that a person may need
a bone graft. Bone grafts are procedures used to replace and
help repair bone. Autogeneous bone grafts come from the
patient’s bone, most commonly the hip bone. The problems with
these grafts are limited supply in addition to pain and morbidity
associated with the harvesting surgeries. Allogeneic bone grafts
come from cadavers and these grafts can also cause
complications. Allogeneic bone grafts have lost the mechanical
integrity neededto grow bone, and they also have a higherrisk of
disease transmission and immune rejection.
Designing a scaffold for bone growth would reduce the need
for autogeneous and allogeneic bone grafts which are not always
the ideal option. β-Catenin is a protein that is involved in cell
adhesion through cell-to-cell adherens junctions and is also a
regulator of bone formation. Cells are influenced by chemical
factors but can also be influenced by surface topography and
Electrospun fibers mimic the extracellular matrix of bone.
Growing cells on electrospun fiber surfaces can influence β-
Catenin translocation to the nucleus and promote osteogenesis.
Electrospinning uses a high voltage source to charge a solution,
evaporate off the solvent, and stream fibers to a grounded source.
The fibers being made can vary in diameterbased on the solution
viscosity, the distance from the syringe to the grounded source, or
the voltage applied.
In this project, electrospun fibers were made out of Poly
methyl methacralate (PMMA) Nitromethane solution and spun
on glass coverslips spin coated with PolyHEMA (PHEMA)
solution. Glass coverslips were also spin coated with a PMMA
Nitromethane solution and not coated with fibers. MC3T3 cells
were seeded and grown until confluent onto the fibers and onto
the no-fiber surfaces. A β-Catenin assay using
immunofluorescence was usedto show the locations and amounts
of β-Catenin present on fiber and no-fiber surfaces. An increase
in nuclear β-Catenin on fiber surfaces relative to the no-fiber
surfaces would show that the fibers promote bone growth.
Keywords—nanofibers, electrospinning, osteogenic
Problems with bone can happen at any age and can include
breaking a bone, bones that do not heal, bone cancer, and more.
Each year there are over two million patients or medical
procedures performed relating to bone replacement,
reconstruction, or repair.1 Many procedures include using
allogeneic or autogeneous bone grafts. Allogeneic bone grafts
pose the rise of infection and immune rejection because they
come from cadavers. Autogeneous bone grafts can come from
the patient’s bone; however, these grafts can be painful and
have negative effects on the site from which the bone was
Designing bone scaffolds would reduce the need for
autogeneous and allogeneic bone grafts. While designing and
testing scaffolds,scientists must keep in mind that they must be
biocompatible with the body, provide adequate mechanical
strength, and be porous.2 The biodegradability and the
osteoinductive capabilities of the scaffold must also be
examined. If the scaffolds degrade over time, patients would
not need another surgical procedure to remove the scaffold
once the area is healed.2 If the material is biodegradable over
time, this can sacrifice the mechanical integrity that is needed
for bone growth. Osteoinductive capability of the scaffold
material is also important because growth factors may be
needed to control cell differentiation.
The purpose ofthis research is to determine the effects that
different diameter electrospun fibers have on beta-catenin
translocation to the nucleus by comparing beta-catenin levels in
cells on fibers and not on fibers. This is important because an
increase in beta-catenin in the nucleus would lead to an
increase in osteogenic differentiation. Cells were grown on
Poly methyl methacrylate (PMMA) electrospun fibers and on
PMMA spin coated coverslips. PMMA is a biocompatible
material that is used in bone cement and is not biodegradable.
Because cells will be grown on the same material (PMMA), the
only change will be in the surface on which the cells are grown.
Therefore, the results recorded fromfibers and no fibers will be
due to the change in the surface, not the material.
II. LITERATURE REVIEW
Surface topography has been studied and shown to affect
cell differentiation. When developing a scaffold for bone
tissue engineering,the effects that the surface geometry has on
cells must be studied. Geometric cues can have strong effects
on the fate of cells and is comparable to chemical factors. A
study was conducted that tested the significance of scaffold
geometry on cell differentiation. Mesenchymal stem cells
(MSCs) were grown on flat surfaces and on straight and
random nanofiber surfaces. The nanofiber surfaces directed
the cell differentiation toward osteogeneis even though the
medium in which the cells were grown had chemical factors
that should have directed the cells into adipocytes.3 Surfaces
that are convex tend to direct MSC differentiation toward
adipogenesis while concave surfaces direct differentiation
toward osteogenesis. Surfaces that force cell shape into
rectangles also tend to direct MSC differentiation towards
osteogenesis as aspect ratios increase.4 Electrospun fibers are
long and thin like rectangles and therefore can influence cells
to develop into bone cells.
Along with shape, stiffness of a substrate can also impact
the fate of cells. Stiff substrates tend to direct stem cells to
differentiate into bone cells. The field of biomedical
engineering is holistic. Because of this,scientists must think of
the many ways in which cells can be affected when designing
bone scaffolds. Substrate geometry and stiffness can be
studied, but other factors such as porosity and topography
must be recognized for the possible influences that they may
exhibit on cell growth and differentiation.5 Bone scaffold
designs must be examined because the slightest change in
porosity, stiffness, size, topography, or geometry can direct
cells to differentiate into a different lineage.
Electrospinning is a technique that uses a high voltage
source to charge a solution as it is being pushed through a
syringe. The drip of solution at the tip of the syringe forms a
Taylor cone. The solution streams out, the solvent evaporates
off and the fibers travel to and are collected on a grounded
source. Conditions such as temperature and humidity can
affect the fibers that are being spun. High humidity results in
straight fibers while low humidity tends to produce curly
fibers. The temperature and humidity can also change the
polymer to spray instead of stream to the grounded source.
Fibers can be spun until the desired density is reached that
would mimic the extracellular matrix.
Fibers can be electrospun to have different diameters.
Small diameter fibers allow more cell-to-cell adherens
junctions to form. Larger diameter fibers allow cells to wrap
around the fibers and create fewer adherens junctions.
Adherens junctions are cell-to-cell contacts that connect
transmembrane proteins known as cadherins with actin
filaments. There are many different types of cadherins such as
E (endothelial), N (neural), O (osteoblast), P (placental), and
VE (vascular endothelial) Cadherin. The cadherins interact
and connect with cytoplasmic proteins called catenins. An
alpha-beta-catenin complex forms and those proteins then
connect to the actin filaments.6 This creates a structural and
biochemical connection between cells. This will in turn affect
the organizational development of the cells.7
Beta-Catenin is one catenin that connects to the cadherins
in adherens junctions. Beta-Catenin can be found at the plasma
membrane, free in the cytoplasm, or in the cell nucleus. While
beta-catenin is connected in the adherens junctions, the levels
of free beta-catenin in the cytoplasmare kept low. When beta-
catenin travels to the nucleus, it can affect transcription. There
are many different sites where beta-catenin can be
phosphorylated which would trigger its translocation to the cell
nucleus. Phosphorylation controls the stability of the beta-
catenin connection at the plasma membrane to cadherins in
adherens junctions. For example, phosphorylation at tyrosine
142 increases transcription activity of beta-catenin by breaking
the catenin-cadherin connection. This then leads to
translocation to the cell nucleus and gene transcription. 8
Glass coverslips were spin coated with 100μL of 30% w/v
PolyHEMA (PHEMA) 95:5 EtOH:H2O solution at 5000 rpm
and a 2% w/v PMMA Nitromethane solution at 2500 rpm.
Fibers were spun onto the PHEMA coverslips for 2 minutes at
5.5 mL/hr. while applying 13 kV. Fibers were not spun onto
the 2% w/v PMMA coated coverslips because they were used
as the control.
Some problems that occurred were that the conditions such
as temperature and humidity did not stay constant while
electrospinning fibers on different days. The temperature and
humidity affected the distance that the syringe needed to be
placed in relation to the grounded source. The distance needed
to be changed to prevent fluffy fibers. Fluffy fibers are not
ideal because they disconnect from the coverslips during cell
seeding and incubation. To tackle this, various heating
methods were used. The coverslips with fibers were heated on
a hot plate at 75°C for 1 minute in the center of the hot plate.
The edges ofthe coverslips coated with fibers were also heated
at 200°C. This created a border around the coverslip of melted
fibers that would prevent them fromlifting off of the coverslip.
Another method used to prevent the fibers from lifting off of
the coverslips was UVgluing. The glue was placed on the four
edges of the coverslips and quickly dried to prevent the glue
from bleeding in to the remainder of the fibers on the cover
slip. The method that produced the best results was heating the
edges at 200°C. This method did not prevent all of the fibers
from lifting off but it held down most of the fiber mat. This
heating method was used to complete the experimentation.
Another issue that must be addressed is the PHEMA
solution not evenly coating the glass cover slips. PHEMA is
spin coated onto the cover slips so that the cells adhere and
grow on the fibers and not on the glass cover slips. When the
cells are seeded on to the cover slips, pockets can be seen
where the glass is exposed. The cells will settle into these
pockets and grow in there instead of on the fibers. The
PHEMA is spin coated on to the glass cover slips at 5000 rpm.
To try and fix this PHEMA problem, the speed at which the
cover slips were spin coated was reduced to 3000 rpm. Results
have yet to be recorded for those upcoming trials using the
3000 rpm spin coated PHEMA solution.
MC3T3-E1 S4 cells were grown and fed every 2 days
using α MEM 1X media. They are an osteoblast precursor cell
line that is derived from mouse calvarias (skulls). When the
cells were confluent, they were seeded onto the fiber and no
fiber cover slips in a 6 well plate. The cells were grown until
confluent on the fiber and no fiber surfaces.
Immunofluorescence was then performed to see the
translocation of beta-catenin on the fiber and no fiber surfaces.
Beta-Catenin (E-5) and cadherin primary antibodies were
used to perform the beta-catenin assay using
immunofluorescence. The secondary antibodies chosen for the
immunofluorescence were Anti-Mouse IgG Dylight 488 and
cy5 antibodies. Phalloidin and DAPI were used to stain the
cell’s actin and DNA respectively. Phalloidin is a highly toxic
small molecule that is taken from mushrooms. DAPI is also
toxic and it binds to the adenine thymine (A-T) regions in
DNA in the nucleus. Once the immunofluorescence was
complete, the cover slips were mounted onto glass slides and
viewed under the microscope. Expected results would show
that beta-catenin would be highly concentrated at the plasma
membrane in adherens junctions on the control surfaces. Beta-
catenin would be expected to be more concentrated in the
nucleus for the cells seeded onto the fiber surfaces.
The MC3T3-E1 S4 cells were imaged using differential
interference contrast (DIC) and fluorescence microscopy. The
cell nuclei are in purple, actin is red, cadherins are blue and
beta-catenin is green. Because there were many focal planes,
the cells seeded and grown onto the PMMA fibers were
difficult to image. Figure 1 shows the separate color channels
for the cells. The four channels were overlapped to be viewed
all at once when imaging the cells.
The results for the immunofluorescence staining showed
what was expected for the control coverslips which is that the
beta-catenin would be at the plasma membrane held in
adherens junctions. Figure 2 shows the cells grown onto the
control coverslips without fibers. The beta-catenin, stained
green, is highly concentrated at the cell’s plasma membrane
which can be seen by the large amount of green. This is
because the cells are allowed adhere, spread out and make
many cell-to-cell contacts on the flat PMMA surface.
The cells seeded and grown on the PMMA fibers are
imaged below in Figures 3 and 4. Some beta-catenin can be
seen in the nucleus in Figure 3, but beta-catenin can also be
seen outside nucleus oriented along the fiber in Figure 4. More
trials will need to be performed to understand why the beta-
catenin is found oriented along the fiber without any cell-to-
Figure 2: MC3T3-E1 S4 cells grown on glass
coverslips spin coated with 2% PMMA Nitromethane
Figure 1: This figure shows the four separate color
channels. Top Left: purple stained nuclei. Bottom
Left: blue stained cadherin, Top Right: red stained
actin, Bottom Right: green stained beta-catenin.
Figure 4:MC3T3-E1 S4 cells grown on 35% w/v
PMMA fibers. Beta-catenin (green) can be seen
oriented along the fibers.
Figure 3: MC3T3-E1 S4 cells grown on 35% w/v
PMMA fibers. Beta-catenin (green) can be seen in and
near the nucleus.
V. FUTURE WORK
This REU has served as an excellent introduction to upper
level research. I intend to use my REU opportunity as the start
of my honors thesis work and I plan to continue this research
through the fall semester and until I graduate. My future work
will consist of electrospinning smaller diameter fibers. This
will allow me to compare the translocation of beta-catenin for
cells grown on large and cells grown on small diameter fibers.
I have already begun to spin smaller diameter fibers and was
confronted with beading issues on the fibers. The PMMA
solution was switch to a 90:10 Nitromethane: DMF solvent and
this reduced the amount of beading.
Immunofluorescence will be performed again when cells are
confluent on the different diameter fibers and control
coverslips. One change for the immunofluorescence will be
that an additional antibody (Y142) will be used to stain for
nuclear beta-catenin. The beta-catenin antibody used in
experimentation this summer only stained for total beta-
A western blot will be performed to detect the intensity of
nuclear beta-catenin using the Y142 antibody in the cells
grown on fibers versus the cells grown on the flat PMMA
control surface. Western blots separate proteins based on their
molecular weight and the band intensity is shown by color.
The results that will be obtained by this future work for the
remainder of the summer will direct the pathway for my future
research with beta-catenin and its influence on cell osteogenic
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