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Cells Cultured on Porous, Fibrous Poly(Lactic-co-Glycolic Acid) Scaffolds:

The Effects of Fiber diameter on Cell Morphology and Viability

Daniel Margul, Dr. Sarah Richardson Burns, Jeffrey Hendricks,

and Dr. David C. Martin

Abstract

There is a growing need for the development of multifunctional, adaptive and biointegrated

prosthetic limbs for individuals who have lost limb function due to trauma, disease or congenital

disorders. Recent studies have focused on developing a new generation of assistive devices that

directly electronically interface with the patient’s nervous system. However, these devices will

not only require the creation of direct interfaces between the device and the nervous system but

will also require formation of an artificial interface between a robotic prosthetic and the bone,

skin, muscle, and connective tissue. Therefore, we will be investigating the possibility of using

nano and micro scale porous, fibrous polymer mats as scaffolds for culturing endothelial cells

toward development of a tissue scaffold that can serve as dermal tissue. The fibrous polymer

films will provide for robust, reliable mechanical integration between the device surface and the

skin of the individual. This novel tissue scaffold should also provide a barrier to bacterial

infection. We generated polymer fibers of systematically increasing diameters (50 nm-8 µm)

using electrospinning techniques that allowed for controlled variations in fibrous mat structure.

Electrospinning involves the application of an electric field to a solution jetted from a syringe tip.

The liquid jet is distorted in the electric field to create polymer nanofibers and microfibers.

Methodically changing the electrospinning parameters, including flow rate, voltage, distance

from the syringe tip to the substrate, and most importantly the solution concentration, allowed us

to create a gradient in the microstructure by changing the fiber diameter from the microscopic

level to the nanoscopic level. By using this process, we created films with controlled gradients

in diameter and porosity and determined the range of possible fiber diameters using optical

microscopy and scanning electron microscopy. In addition, the cell size, shape, and anisotropy

were examined and compared with cells grown on 2 dimensional tissue culture polystyrene

substrates.

1. Introduction

One of the focuses of the Martin Research Group (MRG) at the University of Michigan is

the development of a completely biointegrated, biomimetic prosthetic for the human body. This

device involves several biological/mechanical interactions including those between the device

and the skin, bone, nerves, and various connective tissues. The Martin Research is working on

the skin and nerve interactions with the device. The group’s previous research includes work on

improving the interfaces between electrodes and tissue, developing conductive polymers for

coating electrodes, and studying the topography and morphology of polymers.

Electrospinning is a moderately old technique used to create nano and micro diameter fibers

and particles. Recently, a variety of applications of this technique have developed in the tissue

engineering field with regard to the fabrication of scaffolds. Fibers or particles are created by

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applying a high voltage electric field to a solution in a syringe. The syringe sits inside a pump

that slowly pushes the solution through the needle. At the end of the syringe tip is an electrode

connected to a high voltage source. The electric field produced by the high voltage draws the

solution out into a shape commonly known as a Taylor cone. As the solution moves towards a

substrate, which is connected to a counter-electrode, it forms into fibers and/or particles. The

fibers that a produced are of similar diameter to fibers of the extra cellular matrix (ECM), which

makes mats of these fibers appealing as a tissue engineering scaffold. In addition, a variety of

proteins and chemicals can be added in order to increase biocompatibility or for drug delivery.

Poly(lactic-co-glycolic acid) PLGA is a common polymer to electrospin because it, in its

many varieties, has diverse biodegradability, good mechanical properties, good biocompatibility,

and Food and Drug Administration (FDA) approval for medical devices. In addition to being

biocompatible, electrospun PLGA scaffold mimics the random morphology of the ECM; making

it a very good polymer for the fabrication of tissue engineering scaffolds.

2. Materials and Methods

Sputter coating of cover slips

Plastic cover slips were placed into the vacuum chamber of a sputter coater with an Au/Pd

source. The covers slips were sputtered for approximately five minutes at ten milliamps. This

produced a thin electrically conductive coating on the surface of the cover slips. This coating

allowed the entire surface of the substrate to be charged by the counter electrode of the

electrospinning system.

Electrospinning

We used electrospun fibers using a KD Scientific Syringe pump KDS 100. BD 3ml syringes

were attached to EFD Precision stainless steel 15 gauge tips. The syringe was placed into the

pump and an electrode from a Hipotronics HV DC power supply. The powers supply produced

up to 12 kV at .1 mA. The solution was jetted to the sputtered cover slips.

Scanning Electron Microscopy

We imaged the surface and cross sections of the fibers in order to visualize the surface

morphology and internal 3-dimensional structure of the electrospun fibrous mats. Images were

obtained using a FEI Nova NanoLab Dualbeam FIB and Scanning Electron Microscope (SEM).

Prior to imaging, a thin film of gold was sputtered onto the surface of the fibers in order to make

them electrically conductive to dissipate charge. Photographs of fibers were taken at 500X,

1500X, 2500X, and 3500X.

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Optical Microscopy

We imaged the surface of the fibers in order to characterize the surface morphology and

structure of electrospun fibrous mats. Images were taken using a Nikon Optiphot POL with a

Spot RT digital camera.

Phase Contrast/Fluorescent Microscopy

We imaged the cells on the fibers using phase contrast/fluorescence microscopy. We used a

Nikon T2000 inverted light/fluorescence microscope with HG arc lamp, Hamamatsu CCD 16 bit

camera with Simple PCI imaging software (courtesy of Takayama Lab). We also used an

upright Olympus BX-51 with HG arc lamp, Olympus CCD camera, and Olympus imaging

software (University of Michigan Microscopy and Image Analysis Core Laboratory, MIL).

Cells

SH-SY5Y neuroblastoma-derived cells, GFP transfected epithelial cancer cells, and 3T3

fibroblasts were maintained in Dulbecco’s Modified-Eagle’s Media (DMEM with glucose, with

L-glutamine; Gibco/Invitrogen, Carlsbad, CA), Penn-Strep mixed antibiotic solution (dilute

1:100 in cell media; Gibco/Invitrogen) and 10% fetal bovine serum (FBS; Gibco/Invitrogen).

3. Results

3.1 Characterization the effects of changing electrospinning parameters

For this experiment, we used solutions made of 75:25 Poly(lactic-co-glycolic acid) in

Chloroform and 75:25 PLGA in Dichloromethane. We systematically altered the electrospinning

parameters one at a time in order to have a series of charts that characterize the results of

changing any single parameter. The parameters that were tested include voltage, solution

concentration, distance from the syringe tip to the substrate, flow rate, and syringe tip size.

By sequentially altering one parameter at a time, we were able to characterize the effect each

electrospinning parameter has on the fibers/beads that are created. We imaged each sample

using a Nikon Optiphot POL microscope and a SPOT RT digital camera, and we measured fiber

and bead diameter using Diagnostic Instrument’s SPOT software. We also described and looked

for trends in morphology.

Experiment 1: Vary Distance

Solution: PLGA (75/25) 15 % (w/v) in Chloroform

Conditions: Flow rate: .35mL/hr, Voltage: 10kV

Distance (cm) Description Fiber Width Trends

7 blobs 40 µm

9 thick fibers 7-8 µm fiber number of

11 medium fibers 5 µm thickness fibers

13 thinner fibers 4 µm

15 fibers: thin, few 3-5 µm

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We characterized the effects of varying the distance between the syringe tip and substrate.

We varied the distance from 7-15 cm with intervals of 2 centimeters. As a general trend, the

further away the substrate and syringe tip were, the thinner the fibers were. And it appeared that

the distance can be no less than 8-9 cm, because at short distances the solution does not have

enough time to dry before hitting the substrate. Also, as the substrate gets further away, less

fiber hit it, meaning that longer spinning times are necessary. Beads normally did not appear in

these samples.

Experiment 2: Vary Flow Rate

Solution: PLGA (75/25) 15 % (w/v) in Chloroform

Conditions: Distance: 11 cm, Voltage: 10kV

Flow Rate (mL/hr) Description Fiber Width Trends

0.75 thick fibers, some beads 5-8 µm

0.65 thick fibers, few beads 4-5 µm

0.55 thinner fibers, very few beads 3-5 µm fiber number of number of

0.45 thinner fibers no beads 3-4 µm thickness fibers beads

0.35 thinner fibers 2-3 µm

0.25 thinner fibers (swirls) 2 µm

0.2 thinnest fibers (swirls) 2 µm

In this experiment, we characterized the effects of varying the flow rate of the solution out

of the syringe. We varied the flow rate from .2-.75 mL/hr. General trends that were noticed

included fiber thickness increases as the flow rate increases, the number of fibers that accumulate

on the substrate increase as flow rate increases, and the number of beads that appear increase as

flow rate increases. The morphologies that we saw ranged from beads on a string at higher flow

rates to thin fibers at the lower flow rates. We also noticed a swirl morphology at very low flow

rates, which is appears in several forms that look like ribbons, like overlapping connected circles,

or like a sinusoidal curve.

Experiment 3: Vary Voltage

Solution: PLGA (75/25) 15 % (w/v) in Chloroform

Conditions: Distance: 11 cm, Flow Rate: .45 mL/hr

Voltage (kV) Description Fiber Width Trends

≤ 6 few fibers develop NA

7 thin fibers 1-2 µm

8 slightly thicker fibers 2-6 µm fiber number of

9 slightly thicker fibers 3-6 µm thickness fibers

10 thicker fibers 6-7 µm

11 thick fibers (unstable cone) 3-7 µm

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In this experiment, we varied the voltage from the high voltage power supply. The voltages

tested ranged from 6 to 11 kV at intervals of 1 kV. The most important trends were that fiber

thickness increased as voltage increased, and higher voltages are associated with faster fiber

accumulation. Also at voltage of 6 kV or less, few fibers appeared and a Taylor cone barely

appeared at the end of the syringe. At voltages above 10 kV, the Taylor cone was very unstable,

sometimes splitting into two cones.

After testing these three parameters (distance, flow rate, and voltage) with PLGA in

chloroform, we tested them again using Dichloromethane, and the same general trends were

observed.

Experiment 4: Vary Solution Concentration

Solution: PLGA (75/25) in Chloroform

Conditions: Distance: 11 cm, Flow Rate: .45 mL/ hr, Voltage: 10 kV,

Temperature 22˚ C, Humidity 28%

Concentration (% w/v) Description Fiber Width Bead Width Trends

7.5 blobs NA 12-120 µm

15 beads on a string 1 µm 10-20 µm Number of Fiber

30 only fibers 2-5 µm NA Beads Thickness

40 thick fibers 6-14 µm NA

In this experiment, we created four different solutions with different concentrations of

PLGA dissolved in the chloroform. The most significant trends were that the number of beads

and particles decreased as concentration increased and that fiber thickness increased as

concentration increases. In addition, when the concentration gets below 8 or 9 % (w/v), blobs

start to appear. Fibers begin to form once the concentration is greater than 12 % (w/v); however,

these fibers exhibit the beads on a string morphology. Once the concentration is above 20%,

only fibers appear.

Experiment 5: Vary syringe tip size

Solution: PLGA (75/25) 15 % (w/v) in Chloroform

Conditions: Distance: 11 cm, Flow Rate: .45mL/hr, Voltage 10kV

Tip Gauge Description Fiber Width Trends

15 Beads on a string <1 µm Fiber Number

23 More beads on a string <1 µm Width of beads

30 Most beads on a string <2 µm

In this experiment, we jetted three identical solutions with different syringe tips. We used

15, 23 and 30 gauge tips. Larger tip sizes (smaller gauges) create thinner fibers with fewer

beads, while smaller tip sizes (larger gauges) produce thicker fibers with more beads.

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Experiment 6: Cold Solution vs. Heated/Homogenized solution

Solution: PLGA (75/25) 15 % (w/v) in Chloroform

Conditions: Distance: 11cm, Flow Rate: .77mL/hr, Voltage: 10kV

In this experiment, we spun samples from the same solution before and after

heating/homogenization. As expected, the fibers made from the solution that came out of the

refrigerator had a variable thickness, which is most probably due to the lack of homogeneity of

the solution. The warmer solution that was homogenized produced thin fibers with the beads on

a string morphology. We are unsure about the effects of temperature at this point, and question

will be investigated in the future.

After completing the above analysis, we came to the conclusion that even if all the

parameters mentioned above are held constant, there will be variance in data from day to day.

We discovered that the cause of this is humidity, which can have a large effect on morphology,

fiber size, and bead size. Although we have not quantitatively measured the effects of humidity,

we noticed that when the humidity is over 60% in the lab, it is difficult to get thin fiber or small

particles. Since the vapor pressure is higher when humidity is high, the solvent in the solution

cannot evaporate as easily, which causes samples created on different days to have different

morphologies, fiber diameters, and particle diameters. Generally speaking, lower humidity is

ideal for creating fibers with diameters in the nanometer range.

3.2 Using Dyes and creating layered mats

In this experiment, we used solutions of 20% 75:25 Poly(lactic-co-glycolic acid) in

Chloroform mixed with various dyes. Dyes tested include red food coloring, blue food coloring,

green food coloring, Trypan blue, Multiple Stain, and Nuclear Fast Red.

The first part of this experiment involved testing the dyes to see if they went into solution

and seeing how visible they made the fibers under light microscopy. We also tested different

concentrations of dye in order to find what concentration was necessary to make the dye visible

to the naked eye and under the microscope.

Dye Result

Red Food Coloring Visible

Blue Food Coloring Visible

Green Food Coloring Visible

Trypan Blue Cannot see dye

Multiple Stain Visible, many particles form

Nuclear Fast Red visible, large particles form

Solution Description

Heated/Homogenized solution (60°C) Fibers with few beads

Refrigerated Solution (~10°C) Large beads on a string, variable thickness

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We concluded that blue, red, and green food dyes work the best as they are all visible and

only did not cause notable changes in the samples’ morphologies. Nuclear Fast Red and

Multiple Stain both visibly colored the fibers, but they caused changes in morphology. Finally,

Trypan blue did not successfully dye the sample. We also came to the conclusion that 5 drops of

food dye per every 2 mL of PLGA solution was the best amount of dye.

The second part of this experiment involved using the dyes to visually represent layered

mats. First, we jetted a mat from a solution that was dyed with red food coloring. We

electrospun the solution until the mat was thick enough that the substrate could not be seen

through the mat. Then we jetted a second solution containing a blue food coloring on top of the

red fibers until we could only see the blue fibers. This was to prove that it is possible to layer

mats on top of each other. Applications of this could involve a vertical gradient of fiber

diameter, putting a certain chemical in only part of the mat, or even layer particles and fibers.

Red Fibers (Bottom Layer of Mat) Blue Fibers (Top Layer of Mat)

Vertical cross section of the layer of blue fibers

on top of the layer of red fibers

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3.3 Masking substrates in order to create patterned mats

For this experiment, we used solutions of 20% 75:25 Poly(lactic-co-glycolic

acid) in Chloroform mixed with red and blue food coloring. The concentration

of food coloring was 5 drops/ 2 mL of solution.

This experiment involved developing a method to create a mat with a pattern. Specifically,

we wanted a design that had to concentric squares. The outer one would have one color of

fibers, while the fibers of the inner square would be another color (Figure 3.3a). The difficult

part of this experiment, was finding a way to prevent random fibers from going to one area,

while encouraging the fibers to go to specific areas on the substrate. We discovered that this

could be done by using a cover slip with a square hole in the center to mask the substrate (Figure

3.3b). While using the outer mask, we jetted the fibers for the middle of the pattern, and, by

using the square cut out from the coverslip as a second mask (Figure 3.3c), we surrounded the

middle square with fibers of another color. The use of masks prevents layering of fibers, but can

prevent the different layers from overlapping at their boundaries

Figure 3.3a

Figure 3.3b: Figure 3.3c:

Outer Mask Inner Mask

Figure 3.3d Figure 3.3e

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As seen in Figure 3.3d and 3.3e, the different layers do not always overlap at the edges. The

reason for this can be explained in Figure 3.3f. In this figure, the mask is lying on top of the

substrate. There is an area near where the edge of the

mask meets the substrate where fibers do not want to

go. Since the height is not uniform between the mask

and the substrate, a shadow area develops where the

fibers do not appear. We found that by moving the

cover slip mask about a millimeter further away from

where we were spinning fibers allowed the fibers

from different sections to overlap. We did this while

creating the fibrous mat in Figure 3.3e, where there is

some overlap between the two fibrous mats.

3.4 Establishing a horizontal gradient in fiber diameter

This experiment was the final product of the masking experiments and previous experiments

involving variation of electrospinning parameters. Again we used 75:25 Poly(lactic-co-glycolic

acid) in chloroform, but this time we used three concentrations: 35 % (w/v), 25 % (w/v), and

17.5 % (w/v). The other parameters follow in Figure 3.4a.

Concentration 17.5 % (w/v) 25 % (w/v) 35 % (w/v)

Voltage 9 kV 10 kV 12 kV

Flow Rate .45 mL/hr .55 mL/hr .65 mL/hr

Distance 13 cm 11 cm 9 cm

Figure 3.4a

In order to create the gradient, we electrospun these three solutions onto different areas of

the cover slip. In order to prevent each new layer of fibers from covering up the others, we

applied the masking techniques explained in section. For the first mat, which was made with the

35 % (w/v) solution, we covered up the right 2/3 of the substrate with a cover slip. Once this

section was completed, the right and left 1/3 of the substrate were masked with cover slips, and

we electrospun the 25 % (w/v) solution into the middle section. Finally, we covered the left 2/3

of the substrate and electrospun the 17.5% (w/v) solution. This created a gradient across the mat

that went from thick fibers on the left side to thinner fibers on the right side.

In Figure 3.4b, we measured the fiber diameters of

many fibers inside the mats we created with the

gradient. We then calculated an approximate µ ± 2σ

for each solution. Since electrospun fibers are created

randomly, the mean and standard deviation are

approximations that will vary from sample to sample.

In addition, there are many outliers.

Mask

Fibers

Shadow Area

without fibers

Solution Fiber Diameter (µ ± 2σ)

35 % (w/v) 4.5 ± 2 µm

25 % (w/v) 2.25 ± 1.5 µm

17.5 % (w/v) 1.5 ± 1.5 µm

Figure 3.4b

Figure 3.3f

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3.5 Analyzing the growth of epithelial cells across the gradient

In this experiment, we took the samples created in the previous experiment and grew three

types of epithelial cells on the surface: SY5Y neuroblastoma-derived cells, 3T3 Fibroblasts, and

GFP transfected epithelial cancer cells. We cultured epithelial cells on the mats that have a

horizontal gradient in fiber diameter (section 3.4). We then imaged the results using fluorescent

microscopy in order to examine the cell size, shape, and anisotropy.

500x 1500X 3500X

Figure 3.4c SEM images of fibers

35 % (w/v)

25 % (w/v)

17.5 % (w/v)

Control Small Fibers Medium Fibers Large Fibers

Figure 3.5a: SY5Y neuroblastoma-derived cells

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The SY5Y cells seem to prefer the medium and thick fibers, as more Actin is present in the

cells that were cultured on the thicker fibers, which means that the cells adhere better the fibers

with larger diameter fibers.

The shape of the 3T3 cells grown on the smaller fibers look more like those in the control;

however, as they are fibroblasts, the function of these cells involves changing their shape in

order to fit into their environment. This means that the cells on the larger fibers developed more

like they would in living tissue.

The GFP transfected epithelial cancer cells seem to prefer the thick fibers as well; there is

more Actin present in the fibers that were cultured on the thicker fibers, which means that the

cells spread out more and adhere better to the fibers with larger diameters.

3.6 Fibers spun simultaneously with particles

In the pursuit of creating a more porous scaffold that cells might prefer over a plain fibrous

scaffold, we worked to electrospin fibers and particles simultaneously. We predicted that we

could get the particles to stack up on each other, creating an uneven surface for the fibers to rest

on. By using two syringes, we were able to spin 10 % (w/v) and 20 % (w/v) solutions at the

same time using one pump and only one voltage source. The positive electrode was connected to

both syringe tips by using a piece of copper wire. (Figure 3.6a)

Control Small Fibers Medium Fibers Large Fibers

Figure 3.5b: 3T3 Fibroblasts

Control Small Fibers Medium Fibers Large Fibers

Figure 3.5c: GFP transfected epithelial cancer cells

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In order to visualize the results, we electrospun clear PLGA fiber simultaneously with blue

particles. As seen in images Figure 3.6b and Figure 3.6c, the fibers and particles were

successfully electrospun together.

We also took SEM mages of mats made of just fibers from the 20% solution in order to

compare the general morphological differences between fibers and fibers electrospun with

particles. In image ZRZ on the left, we only electrospun fibers, and the general structure is

fibrous and flat. In image ZQy on the right, we electrospun the fibers and particles

simultaneously, and the surface is more porous and has much more three dimensional structure.

Figure 3.6a: Electrospinning using

two syringes to simultaneously create

fibers and particles

Figure 3.6b: Clear fibers and blue particles Figure 3.6c: Vertical cross section

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In addition, Figures 3.6d and 3.6e are SEM images that show examples of the large pours

that formed inside these mats. Figure 3.6f was taken at a 55° angle and Figure 3.6g is a vertical

cross section of the mat; this allowed us to see into the mat in order to verify that these mats were

as porous as we predicted.

Figure 3.6d: PLGA fibers under SEM Figure 3.6e: PLGA fibers and particles under SEM

Figure 3.6f: image at a 55° angle Figure 3.6g: vertical cross section

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These 3T3 fibroblasts were cultured on various areas on the scaffold, with mixed results.

Unfortunately, the method used creates a scaffold that does not have a uniform distribution of

fibers and beads; this causes the variety of results seen in this image. The images on the right

side indicate that in some areas the 3T3 cells appear to fit into the scaffold like they do in the

human body; however, the other images indicate that this is not always the case.

4. Conclusions

We have shown how to manipulate electrospinning to produce tissue engineering scaffolds

with fiber diameters from less than 1 µm up to 7 µm. Higher voltages, more concentrated

solutions, faster flow rates, and shorter distances between the tip of the syringe and the substrate

are all associated with producing fibers with larger diameters. In addition we also noticed that

humidity has a large impact on the process of electrospinning in general. When the humidity is

over 60%, it is especially difficult to produce smaller diameter fibers, and fibers of all diameters

are formed with enlargements and holes.

We have also demonstrated that patterned fibrous tissue engineering scaffolds can be

produced by using masks made of cover slips while electrospinning. We also determined that

the process is limited by the fact that it leaves an area directly next to the mask about 1 mm thick

where no fibers appear.

Finally, we have shown that cells cultured on fibrous tissue engineering scaffolds spread out

and adhere better to fibers that have fiber diameters in the 3-7 µm range than they adhere to

thinner fibers. 3T3 Fibroblasts, SH-SY5Y neuroblastoma-derived cells, and GFP transfected

epithelial cells all appear to favor thicker fibers.

3.6h: Cells 3T3 cells grown on fibers and particles

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The results from culturing cells on a tissue engineering scaffold consisting of fibers and

particles were inconclusive. The morphology of the scaffold was too varied to determine how

cell growth is effected. More experimentation would be necessary in order to make a more

uniform surface in order to draw any conclusions.

5. Future Directions

The fiber diameter experiment should be conducted again in order to verify the results. It

may also be useful to use fibers that contain a fluorescent protein or polymer in order to better

visualize the results. The method for creating scaffolds consisting of fibers and particles needs to

be more thoroughly developed before any conclusions may be drawn.

6. Acknowledgements

This project was funded by General Electric, Motorola, and the University of Michigan

Undergraduate Research Opportunities program as part of the 2006 Engineering & Physical

Sciences Fellowship. In addition, the project was also supported by the NIH, NSF, University of

Michigan College of Engineering. In addition, the support of Armo MURI on Bio-Integrated

Structural and Neural Prosthetic Materials is also appreciated. The author would also like to

thank Dr. Mohammad Reza Abidian for his guidance and support.

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