1

The Effect of LED Lights on Dental Pulp Derived Cells Plated on

Poly(3-hexylthiophene-2,5-diyl)

Abstract

Stem cells are widely studied due to the cells regenerative potential for treating

injured tissue. Dental pulp stem cells (DPSCs) are commonly studied because they

proliferate quickly and are easily accessible. However, current methods of inducing

differentiation are not ideal. Therefore, researchers are investigating factors that affect the

proliferation and differentiation of the cells in a way useful for tissue engineering.

Dental pulp derived cells (DPDCs), were plated on Poly(3-hexylthiophene-2,5-

diyl) (P3HT) and irradiated with red, blue, or green LED lights. Blue light irradiation

caused cell senescence, while red and green light promoted proliferation. Except the

TCP+blue, the control samples, grown on tissue culture plastic (TCP), proliferated at a

higher rate than the P3HT samples. The cells did not thrive on P3HT, however the P3HT

with lights produced a different effect. P3HT+red and P3HT+green had higher cell

proliferation and P3HT+blue had lower cell proliferation than the P3HT without light.

Analysis under SEM demonstrated biomineralization in every sample except for

P3HT without light. The photons released by the P3HT exposed to LEDs induced

calcification of the DPDCs. The results of this study contribute to the objective of

developing a safe and effective method of culturing DPSCs for transplant in vivo.

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Introduction

1.1- Dental Pulp Stem Cells

Stem cells are classified as nonspecialized, clonogenic cells with properties of

self-renewal and multi-lineage differentiation while having the ability to be isolated from

various parts of the body such as, bone marrow, brain, skin, muscle, and adipose tissue.1

Stem cells are primarily studied for their ability to regenerate tissue as an alternative to

other therapeutic processes to treat injured tissue.2

Human dental pulp stem cells (DPSCs) are an attractive source of stem cells for

oral tissue regeneration. The human dental pulp is rich with stem cells and the cells can

be efficiently and easily extracted from the dental pulp of extracted wisdom teeth unlike

other stem cells, which are usually extracted through an invasive process.3 DPSCs can be

used to regenerate dental tissue for tooth repair and have high proliferative potential,

which makes the cells easy to study.4 They were first discovered as the mystery precursor

of the odontoblasts used to repair dentin in teeth by Gronthos et al.5 They are studied for

their potential to differentiate into odontoblasts, osteoblasts, adipocytes, neural cells, and

chondrocytes.6 DPSCs must proliferate before they differentiate and generally begin

differentiation on day 21. The differentiation of DPSCs is determined by factors in the

cell and its environment such as: growth factors, signaling molecules, transcription

factors and extracellular matrix proteins.7 The cells release different markers, which allow

for characterization post-differentiation. For example, the osteogenic cells express gene

markers such as runt-related transcription factor 2 and osteocalcin, while odontogenic

cells express the marker desmoplakin.8 Dental pulp derived cells (DPDCs), used in this

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study, are a heterogeneous mixture of DPSCs and progenitor cells found in the dental

pulp.

Generally, scientists use dexamethasone (Dex) to induce differentiation in

DPSCs, however, Dex may be harmful to the human body.9 Finding a method that

quickly and safely results in the differentiation of DPSCs into the desired cell type for

tissue regeneration can be useful for clinical applications. The DPSCs have been studied

under many different environments to determine how different conditions affect the

growth and differentiation.

1.2- Photostimulation and P3HT

Photostimulation of cells in vitro has been shown to positively affect cell

proliferation and the rate of cell healing. 10,11 It is also used to induce stem cell

proliferation and differentiation. However, different wavelengths affect the cells

differently. The effects of red LED irradiation on dental pulp cells were determined by

Holder et al. to be beneficial for use for tissue regeneration. They determined that the red

LED lights increased the rates of growth, proliferation, metabolic activity, and mineral

deposition, all of which are cellular responses relevant to tissue repair. 12 Blue light was

shown by Wilner to inhibit human mesenchymal stem cells (hMSCs) proliferation and

differentiation. He concluded that blue light inhibited the cells metabolic processes

because of its higher energy.13 Green LED lights have been shown to stimulate

proliferation of human fibroblasts, but have not been studied in conjunction with stem

cells.14 Both blue and green lights have not been previously studied with DPSCs,

specifically. The objective of this study was to control the proliferation of the cells and

induce differentiation using the LEDs.

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Poly(3-hexylthiophene-2,5-diyl), or P3HT, an organic photovoltaic polymer, is

generally studied in association with solar cells because of its electron donating or

photosensitive ability. When light hits P3HT it is absorbed; if the light energy is higher

than the electron-binding energy of the P3HT, the polymer will release electrons. 15 P3HT

is known to be biocompatible, therefore we proposed it would be an effective scaffold for

the photostimulation of DPDCs. The growth of the cells on the P3HT was compared to

the growth of the cells on tissue culture plastic (TCP), to determine relative

characteristics.

1.3- Objectives

The effects of P3HT under each color of LED light with the DPDCs were studied

in hopes of identifying optimal conditions for achieving ideal responses for growing

tissue in vitro to be used for in vivo tissue repair. The hypothesis of this study was that

the cells irradiated by red light would proliferate at a higher rate than those exposed to

blue and green light. It was also predicted that the cells plated on the P3HT would

proliferate at a higher rate when exposed to the red and green light and at a lower rate

when exposed to the blue LEDs.

Materials and Methods

2.1- Preparing the Silicon Wafers

Silicon wafers of the [1,0,0] orientation (Wafer World Corporation) were

partitioned using a diamond-tipped cutter, a straight edge, and a ruler to create 1 cm2

squares. The wafers were later spin casted with P3HT to serve as a scaffold for the cells

to grow on. Cleaning the partitioned silicon wafers consisted of first sonicating in

methanol for 10 minutes in the Branson 3510 sonicator. The methanol was then discarded

5

and the wafers were washed with deionized water 3 times. The wafers were then treated

with a 3:1:1 ratio of deionized water, ammonium hydroxide, and hydrogen peroxide and

then boiled for 10 minutes. Next they were washed in a 3:1:1 ratio of deionized water,

sulfuric acid, and hydrogen peroxide and then boiled again for 10 minutes. P3HT is

hydrophobic, so the wafers needed a hydrophobic surface for the P3HT to attach. To

create a clean hydrophobic surface each wafer was immersed in a 3:1 ratio of deionized

water and hydrofluoric acid for 30 seconds prior to spin casting.

2.2- Creating P3HT solution and Spin Casting

90.26 mg of P3HT from Rieke Specialty Polymers was measured on a scale and

dissolved in 6 mL of chlorobenzene from Sigma Aldrich to create a 15.04 mg/mL

solution of P3HT. The solution was then heated at 70oC for 20 minutes in order to

dissolve the P3HT.

The wafers were spin casted (Photoresist Spinner 1PM101DR485) with 3-4 drops

of P3HT for 30 seconds at 2500 rpm to create thin, flat surfaces for the DPDCs to grow

on. To evaporate the chlorobenzene from the thin films and stabilize the polymer chains,

the wafers were annealed at 1800 C in an oil free vacuum oven at a pressure of 10-3 Torr

for 24 hours.

2.3- Characterizing the P3HT

The ultraviolet-visible (UV/Vis) spectrum of the P3HT was measured under the

spectrophotometer by spin casting the P3HT on a glass slide. Prior to spin casting, the

P3HT solution was heated to avoid aggregation of the solution and the glass slide was

cleaned by sonicating it in methanol for 10 minutes. 10 drops of the P3HT solution were

placed on the glass slide and then spin casted for 30 seconds at 2500 rpm. The glass slide

6

was chosen because it is transparent and can be read by the spectrophotometer. Using the

Evolution 220 Thermo Scientific UV-Visible Spectrophotometer a reading of a clean

glass slide was taken first and was subtracted from the reading of the P3HT film on the

glass slide. The results were then graphed to create an absorbance spectrum.

The P3HT was also characterized by measuring the thickness of the thin films

under the Ellipsometer.

2.4- Cell Culture and Media

The DPDCs used in this paper were obtained from anonymous waste tissue under

IRB approval (2007-6778). All cell work was done in a Class II A83 Bio Safety Cabinet

(Thermo Scientific Forma 1286) to keep the samples sterile and to keep the worker safe.

The cabinet was turned on 15 minutes prior to use to filter the air and 70% ethanol was

sprayed on everything that entered the cabinet to ensure sterilization and avoid

contamination.

The P3HT samples were grown in 12 well plates (Falcon, Corning Inc.) and the

controls were grown in P35 tissue culture dishes (Falcon, Corning Inc.). The growth areas

of the 12-well plate wells were coated with 600L of 2% agarose gel (Fisher) in

phosphate buffer solution (PBS). This agarose coating was used to force the cells to grow

on the P3HT scaffolds instead of the surrounding tissue culture plastic (TCP) growth area

of the 12-well plate well since the cells cannot attach to the agarose gel itself. After

coating the 12-well plate wells with agarose gel, the P3HT scaffolds were transferred to

each designated well.

The DPDCs were first subcultured in a T-75 tissue culture flask at passage 5 using

a growth media of MEM alpha media (Gibco) containing 2 mM L-glutamine, 10% fetal

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bovine serum (HyClone), and 1% Penicillin-Streptomycin. When the cells reached 70-

80% confluency, they were detached from the T-75 flask using 0.1% trypsin. The cells

were then transferred to a centrifuge tube and centrifuged in the Fisher Scientific

Centrific Centrifuge (Model #023306) for 6 minutes at 1500 rpm. After aspirating the

supernatant, the cell pellet was resuspended in 4mL of media. A cell plating density of

2000 cells/cm2 was used for plating. The passage 6 cells were then transferred into the

incubator (Forma Scientific Automatic CO2 Incubator 3193) to grow at 37oC, 5% CO2

and 100% humidity.

The media was changed on the first day after plating and then every other day

following the first using the growth media aforementioned plus 200M L-ascorbic acid-

2-phosphate, and 10 mM - gylcerophosphate. Each time, media was aspirated with a

sterile Pasteur pipet attached to a vacuum pump and then replaced with, 1mL of media

for the P3HT samples and 2mL for the control samples in the P35 dishes.

2.5- Lights and Incubator Setup

The blue, red or green LED Lights were added after the first 24

hours of incubation. Each light enclosure was 8.0 cm above the cell

cultures and were in 40.64cm x 20.32cm x 2.54 cm boxes. The LEDs were

5m waterproof strip lighting with 300 SMD 3528 LEDs on a strip at 12 V

(Secst). Each single LED was 3.5x2.8 mm. The 5m light strip had an

intensity of 6000 luminous flux. The power source was a hp 6284A

DC Power Supply. All three LED strips were in a parallel circuit of

12V at 1.5A. (Figure 1)

Figure 1: Photo of incubator setup.

DPDCs under green LED lights are

shown on the top shelf, blue LED

lights on the middle shelf and red

LEDs on the bottom shelf.

8

To prevent the heat buildup from the LED light circuitry, aluminum was used in

the enclosures to act as a heat sink for the wires. As a result the temperature was

maintained at 37oC.

2.6- Cell Counting

The cells were counted at different time points of incubation to calculate the

proliferation rate of each sample. The media from the cells was aspirated and the cells

were washed with PBS and trypsinized for 5 minutes in the incubator at 37oC. Media was

added to the samples and they were transferred to microcentrifuge tubes and centrifuged

for 6 minutes at 1500rpm to uniformly disperse the cells. 10 L of each sample was

loaded into the Brite-line Hemocytometer and counted under an optical microscope.

2.7- SEM

To prepare samples for Scanning Electron Microscopy the cells needed to be

dehydrated. The media was first removed from the samples and they were washed twice

with PBS. The culture was then treated with 2% glutaraldehyde in PBS for 15 minutes.

The glutaraldehyde was washed out with PBS twice. To dehydrate the cells ethanol was

added to the cells in cycles. Every ten minutes a higher concentration of ethanol in PBS

was added then washed starting with 25% then, 50%, 70%, 95%, 100% and 100%. The

cells were then treated with hexamethyldisilazane (HMDS) for 10 minutes. The HMDS

completely dried out the cells and the samples were left to dry overnight.

2.8- Histochemistry

The cells were prepared for the Leica SP8X confocal microscope by fixing and

staining. The media was aspirated from the wells and the surface was washed gently with

PBS. The cells were fixed with 10% formalin for 15 minutes and then washed with PBS.

9

Next, the cells were treated with 0.4% Triton-X 100/PBS for 7.5 minutes in order to

permeabilize the cells. To stain the F-actin in the cells green, they were treated with a

1:200 dilution of Alexa Fluor 488 phalloidin in PBS for 20 minutes then aspirated and

washed with PBS. To stain the nuclei blue, the cells were treated with a 300 nM DAPI

solution in PBS for 5 minutes. The cells were then stored in PBS at 4oC until ready for

confocal microscopy analysis.

Results

3.1- Characterizing P3HT

Figure 2 shows the absorbance spectrum of P3HT taken under the UV/Vis

Spectrophotometer. The P3HT has an absorbance of 0.37 at a wavelength of 510 (green),

0.27 at a wavelength of 475 (blue) and 0.08 at a wavelength of 650 (red), which explains

its red fluorescence.

The thickness of the P3HT thin film was measured to be an average of 41.93 nm

using the ellipsometer (Rudolph AutoEl-ll).

Figure 2: Absorbance spectrum of P3HT taken on UV/Vis spectrophotometer. Shows a

peak at a green wavelength.

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3.2- DPDC Proliferation: Cell Count and Confocal Images

The cells were counted in two different trials: the first, on days 1,2,3, and 8, then

the second, on days 1,3,5,7, and 9. The results of the first trial, shown in Figure 3, depict

a large increase of control+red cells between days 3 and 8, therefore the second trial

(Figure 4) was performed to investigate the time point the cells suddenly increased. In

the first trial there is not much insight as to what happens between days 3 and 8 for all the

samples, however, the graph from trial two furthers the understanding . In trial 2 it is

clear that the control+red sample had a large increase between days 5 and 7 and then

grew at a much lower rate between days 7 and 9 with a resulting count lower than the

control. The first trial shows the control+red to have a larger count than the control but

does not take into account that the growth rate may begin to level. It is interesting to note

that the control+green cells in trial 2 decreased in number between days 7 and 9, unlike

any other sample. Both trials show expected relative growth of the samples under red and

blue light. The differences between the number of cells between trials can be accounted

to human error in the original plating density and is not significant in showing the relative

proliferation rates.

The DPDC growth with P3HT is shown in Figures 5 and 6. The DPDCs did not

grow relatively well on the P3HT, most likely due to the hydrophobic nature of the

polymer, however in both trials the red light helped the cells counteract the effect of the

P3HT and grow faster while the P3HT magnified the stunting effect of the blue light. The

red light causes a jump in the growth of the cells on the P3HT between days 7 and 9, two

days later than the jump in the control+red. It is unclear between the two trials whether

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the P3HT-light has a higher proliferation rate than the P3HT+green sample and should be

further investigated.

Images of the cells were taken under the confocal microscope on day 7 of

incubation (Figure 7). The nuclei are shown in blue via DAPI staining and show the

amount of cells in 40X magnification. The images support the proliferation data.

The aspect ratios of the nuclei of each sample were calculated using imagej.

(Figure 8) After calculating the standard deviations, none of the ratios were significantly

different, which showed that the varying conditions did not influence the size of the

nuclei.

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Trial 1 Trial 2

Figures 3-6: Graphs of cell counts using a hemocytometer.

Figure 3 (top left): Graph shows the proliferation rate of each sample during trial 1 on days 1,2,3 and 8.

Figure 4 (top right): Graph shows the proliferation rate of each samples during trial 2 on days 1,3,5,7 and 9.

Figure 5 (bottom left): Graph shows the proliferation rate of the effect of P3HT on the samples during trial 1 on days

1,2,3 and 8.

Figure 6 (bottom right): Graph shows the proliferation rate of the effect of P3HT on the samples during trial 2 on

days 1,3,5,7 and 9.

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Control Control+Red Control+Green Control+Blue

P3HT-Light P3HT+Red P3HT+Green P3HT+Blue

Figure 7: Confocal images of DPDC samples taken at 40X magnification on day 7 stained with Alexa Fluor 488 and

DAPI.

Figure 8: Aspect ratios of the nuclei of cells measured on imagej using the photos taken

on the confocal microscope. The graph shows no significant difference between the ratios

of each sample.

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3.3- SEM and EDX Results

The SEM and EDX analyses determine if the cells have biomineralized and if so,

to what extent. The resulting EDX spectra and pictures of the day 21 samples taken under

1000X magnification are shown in Figure 9. When a cell calcifies, it secretes calcium

from its extracellular matrix. 16 The cells showed signs of biomineralization under every

condition, except the P3HT -light.

By looking at the pictures it is apparent that the control, control+green, and the

control+red samples had the highest levels of calcification, the control+green, however,

had the most. The control+red sample had the fewest calcium deposits of the three and

tended to calcify in large clumps just like the P3HT+red.

Interestingly, the P3HT+blue sample had a lot of calcification relative to the

control+blue sample, which had very little and the P3HT light, which had none. This is

an indication that the photons released by the P3HT when hit with blue light may help the

cells differentiate. This was probably the case in every P3HT+light sample. The light

irradiation of the cells increases calcification, while the P3HT slows it down. Although in

the case of the P3HT+blue sample the effect of the light was more apparent than that of

the P3HT surface.

The P3HT+red and the P3HT+green samples showed similar results, however the

P3HT+red sample had more calcification. Both had clumps of calcium deposits, unlike

the control, which had widespread deposits. It is important to note that the P3HT+red

EDX spectrum shows traces of iron, chromium, and nickel, most likely due to the

shedding of a stainless steel tweezers contaminating the sample.

15

P3HT -Light

P3HT+Blue

P3HT+Green

P3HT+Red

TCP

TCP+Blue

TCP+Green

TCP+Red

Figure 9: EDX spectra (left), depicts elements present in the sample, with their

corresponding SEM photos to the right. The SEM photos (right) were taken of

day 21 samples under 1000X magnification.

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Discussion

During the first round of plating the incubator did not read the temperature

properly and the cells died. The incubator read 37oC, while the cells were sitting in 55oC

enclosures. The tremendous amount of heat caused a stress reaction in the cells and they

lifted off the plates.17 The heat came from the wires and built up inside the light

enclosures. To limit this heat aluminum panels were added to the tops of the enclosures to

act as a heat sink and the enclosures were raised to allow more room for air circulation.

After adjusting the light setup the incubator was monitored to ensure the temperature was

37oC throughout. Once regulated, we replated the cells and began round two of plating.

During round two we counted the cells (Trial 1) and because of the sudden increase in

control+red cells during Trial 1, we replated a third time to count again in order to learn

more about the growth patterns.

The red, blue, and green LED lights produced expected proliferation results with

regards to the DPDCs plated on TCP. As hypothesized, the blue lights had debilitating

effects on the cells, while the red and green lights promoted cell growth. The cells

growing on P3HT did not grow as well as those on TCP, which may be due to the

hydrophobic nature of P3HT. We did not predict that the P3HT would exaggerate the

effects of the lights on the cells. The cells plated on P3HT under red and green light grew

more successfully than the P3HT without light. Additionally, the P3HT magnified the

negative effects of the blue light. The blue light seemed to cause cell senescence on both

the P3HT and TCP, as indicated by the larger size of the cells in the confocal pictures and

the slow proliferation rates.

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While the blue light in conjunction with P3HT slowed down the cells

proliferation rate, it helped the cells calcify. Although they did not show exceptionally

high levels of calcification they had levels higher than the blue light alone and the P3HT

alone, which did not biomineralize at all. The photon released by the P3HT when struck

by light causes the cells to differentiate into either an osteoblast-like or odontoblast-like

cell lineage as shown by the phosphorus and calcium peaks in the EDX scans.

Although high rates of differentiation and proliferation are ideal, differentiation

and proliferation of stem cells does not happen simultaneously, first the cells proliferate

and then they differentiate. 18 We investigated the DPDCs under different conditions in

hopes of finding the ideal method to culture for use in medicine. While this is the

ultimate goal, this experiment serves as a preliminary step to any finalized ideal

procedure. We have not found any previous published papers detailing any work similar

to this experiment. It is even novel to plate cells on P3HT. The results from this

experiment will further the field of stem cell research and assist in the ultimate goal of

stem cell therapies.

Conclusion

To fully understand the properties of our DPDCs additional tests need to be

performed on the samples. In the future, qRT-PCR will be conducted at an early and late

time point to compare the genes expressed by the cells before and after the stimuli. It will

also determine what type of cell the DPSC differentiated into by looking at the

upregulated genes in each sample. Atomic Force Microscopy (AFM) will be executed to

get an accurate reading on the relative Youngs moduli of the cells. AFM was utilized for

18

this experiment, however the cells were too confluent and the readings were not clear-cut.

To obtain a clear reading, the AFM test will be run on day 4, before the cells are

confluent. The relative moduli can give insight to early differentiation of the samples. To

resolve the discrepancies between the 2 cell counts, the proliferation rate will be counted

again on the same time points as trial 2: 1,3,5,7, and 9.

After studying the cells under the environments detailed in this paper to

completion, the light stimuli will be adjusted to understand how the lights specifically

affect the cells. Instead of the lights running to full intensity throughout the experiment

the timing and intensities of the LEDs will be modified in different combinations.

Additionally, the molecular weight of the P3HT will be modified to determine the

significance of the P3HT and its effects on the DPDCs. Varying the different conditions

of the experiment will allow for a comprehensive understanding of the effect of LEDs on

DPDCs plated on P3HT and hopefully a result that will advance the field of regenerative

medicine.

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