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Journal of the PGSS The Effect of Common Medicinal Compounds

The Effect of Common Medicinal Compounds on the Proliferation and Differentiation of C2C12 Myoblasts and 3T3 Fibroblasts

Nina Cheng, Christopher Gonzalez, Madison Greer, Urvi Gupta, Cindy Hsieh, Samantha Pcola, Mahima Reddy, Bliss Uribe, Panayiotis Vandris, Nicholas Yang

AbstractOne of the fastest-growing fields in science today, tissue engineering has shown great potential as an alternative for tissue repair in the body. This study examined the effects of various medicinal compounds on cell growth. Acetaminophen, dexamethasone, estradiol, ibuprofen, turmeric, and vitamin D were tested on proliferation and differentiation in C2C12 myoblasts and proliferation in 3T3 fibroblasts. Compounds were added to the variable groups in differing concentrations for both proliferation and differentiation testing. Cells tested for proliferation were then counted while those tested for differentiation were imaged. Data were analyzed via ANOVA and Dunnett’s tests. An inverse relationship was found for acetaminophen between concentration and cell proliferation. Dexamethasone produced a decrease in C2C12 proliferation at low (10 ng/mL) and high (100 ng/mL) doses, and a decrease in 3T3 proliferation at high doses (100 ng/mL). Cell proliferation was not statistically significant (using a threshold of p=0.05) at low estradiol doses, but a strong correlation was noted at the recommended dosage (100 ng/mL). A negative interaction was observed between ibuprofen and turmeric at low concentrations, which did not imply a synergistic effect. Both low (10 ng/mL) and high (100 ng/mL) doses of vitamin D had a negative effect on C2C12 growth and differentiation, but only the high dose had a significant effect on 3T3 proliferation. These results support existing clinically recommended doses aimed towards mitigating pain and fostering cell growth. However, because not all drugs and supplements yielded consistent effects on myotubular and fibroblastic development, it would be advisable for medical practitioners to gain a better understanding of individual and multiple drug interactions while prescribing therapy regimens utilizing stem cells.

I. Introduction

A. Tissue Engineering

Tissue engineering (TE) is one of the most interdisciplinary and fastest-growing fields in science and regenerative medicine. By developing and manipulating artificial implants, tissues, and genetically engineered cells, TE has great potential for supplementing and even replacing lost functionality or destroyed tissues.

Skeletal muscle (SM), a complex network of more than 600 individual muscles attached to the bones by tendons, functions as the body’s most abundant tissue and comprises approximately 45% of total adult body mass.1,2 Playing critical roles in biological functions such as movement and metabolism, SM also serves as reserves of amino acids and is integral for systemic glucose homeostasis.3,4

Somatic stem cells reside in SM and in surrounding non-muscle stem cell populations and are actively involved in the role of preserving and regenerating their host tissues. As a result, under many circumstances SM tissue is capable of complete regeneration after damage. However, in severe myopathic diseases, where muscle fibers do not function, this regenerative capacity is compromised, resulting in muscle weakness.5

Cheng, Gonzalez, Greer, Gupta, Hsieh, Pcola, Reddy, Uribe, Vandris, Yang

Compromise of the composition and function of SM has been shown to be strongly correlated with disease development and susceptibility. Sarcopenia, age-related declines in SM mass and strength prevalent in both men and women, may eventually lead to functional impairment and disability if left untreated.6 In addition, loss of SM mass, strength, and metabolism has been linked to both cardiovascular disease and cancer, currently the two leading causes of death in the United States. Studies have identified severe loss of muscle mass as a significant risk factor for mortality.7,8

Aside from SM, damage and failure of any tissue can be caused by various events and disorders, including trauma, infection, and disease. Although many patients can be effectively treated with transplanted tissues and organs, this process is often delayed and complicated by the shortage of donor organs and availability of tissues. Likewise, there is often not enough tissue within the patient to be harvested for the reconstructive procedure. Moreover, both approaches to treating organ damage are rarely able to restore full previous functionality.9 For these reasons, tissue engineers have looked to stem cells as a promising alternative in the field of regenerative medicine. Still, the ultimate efficacy and efficiency of tissue engineering relies on the generation of appropriate cells and the ability of those cells to execute specialized biological functions. As a result, it is especially important to understand how cell survivorship, proliferation, and differentiation are affected by the addition to the cellular microenvironment of chemical compounds, especially those that may make their way into the fluid compartments of the body after consumption of everyday medications. The aim of this study was to examine the effects of common medicinal compounds on cell growth.

B. Stem Cells and Cell Lines of Interest

Stem cells are undifferentiated cells that have the ability to replicate and to differentiate into specialized cells. They can be grouped into three subcategories based on potency: totipotent, pluripotent, and multipotent. Totipotent cells are able to differentiate and replicate continuously and also produce placental and embryonic cells. In other words, totipotent cells could hypothetically generate an entire organism. Pluripotent stem cells can produce cells from all three basic body layers, making possible the regeneration of specific cells and tissues (Figure 1). In addition, they can perpetually replicate. However, pluripotent cells cannot produce placental or embryonic cells, nor would they be able to generate an entire organism. Multipotent cells, like the other two types, can self-renew for long periods of time and can differentiate into specialized cells with specific functions. Cells of this potency, though, are limited to the types of cells into which they can differentiate.10 For example, bone marrow contains multipotent cells that can give rise to any type of blood cell, but not any other type of cell.

Figure 1: Development of Totipotent and Pluripotent Cells11


Mammals possess two general types of stem cells: embryonic stem cells and adult stem cells. Embryonic stem (ES) cells are found most prominently in blastocysts, or early-stage embryos. ES cells are considered to be pluripotent and provide valuable contributions to regenerative medicine research. However, the use of and experimentation with ES cells have been restricted by social perceptions of the ethical impropriety of the procedure. The most prominent objection concerns the necessary destruction of human embryos for the acquisition and derivation of ES cells.

Adult stem cells, on the other hand, are not defined by their origin, but are found in a multitude of different tissues, including the brain, bone marrow, blood, skin, and liver.12 Adult stem cells are multipotent and therefore are somewhat limited in the types of differentiation they can undergo. However, they do exhibit the characteristic of plasticity, meaning that they can differentiate into cells of other tissues.9 Similar to ES cells, adult stem cells are capable of self-renewal and self-maintenance for long periods of time. They also hold a number of advantages over ES cells; they are able to easily differentiate into specific cell lineages, do not require any feeder layers to grow, and can be obtained without the sacrifice of human embryos, thus avoiding the ethical concerns associated with the harvesting of ES cells.

C2C12 is a primary mouse myoblast cell line commonly used to study the behavior of stem-like cells in a variety of physiologic or in vitro conditions.13 Since its first derivation in 1977, C2C12 has become one of the most popular cell lines used in tissue engineering research as a powerful model of pluripotent cell behavior. Studies often focus on responses to scaffolds, growth factors, and physiological environments. These cells have the ability to differentiate into multinucleate myotubes and ultimately myofibers.14 This behavior is often used as an indicator of successful pluripotent response resulting from therapeutic intervention. Previous studies have shown that C2C12 cells treated with extract from regenerative cells derived from newts re-entered the cell cycle.15 This property makes C2C12 one of the few cell lines to be successful in differentiation.

Differentiation of myoblasts such as C2C12 forms the process of myogenesis, or the formation of muscular tissue (Figure 2). Single myoblasts are initially able to proliferate with ample space between them and to grow normally. As the cells become confluent, a reduction of the concentration of fibroblast growth factor causes the cells to first align and then differentiate.16 At this point, the cells cease to proliferate and begin expressing other genes necessary for differentiation.17 The cells then begin fusion into multinucleated fibers. Cell differentiation into myotubes can be qualitatively analyzed through imaging with an inverted microscope, as well as through quantitative analysis.18

Figure 2: Myogenesis19

3T3 cells are aggregated embryonic fibroblast cells derived from mice. Originally derived in 1962, they have become a standard immortal cell line. They are hypertriploid, meaning that they produce an excess of chromosomes; 30% of these cells have 68 chromosomes.20 These cells are often used in


transformation assays, cloned in a way to make them useful in studying acquired cell malignancies. Originally, fibroblast cells have high contact inhibition, but once transformed into a cell line, they lose this characteristic.21 These cells have been commonly used to model cells of the connective tissue compartments. They have been utilized to assess many cell behaviors, including proliferation, survivorship, wound healing, and extracellular matrix (ECM) synthesis and maintenance.

It is well known that physiological and environmental factors such as signaling molecules, temperature, pH levels, oxidative stress, mechanical stress, and minerals can affect cell behavior. Previous medical research has focused on the potential toxicities of medicinal compounds at the tissue or organ level. Recently, however, attention has mainly shifted to effects on the cellular level, specifically on therapeutic (progenitor/stem) cell populations, following exposure to medicinal compounds and other chemicals. The success of a therapeutic intervention utilizing tissue engineering ultimately depends upon the appropriate stem cell development behavior. Consequently, it would be prudent to assess the impacts of foreign chemical influences (i.e. medicinal compounds) on both seeded and host stem cell populations.

C. Variables Tested

The C2C12 and 3T3 cell lines were exposed to a number of different substances, namely acetaminophen, dexamethasone, estradiol, ibuprofen, turmeric, and vitamin D. The overarching purpose of this project was to investigate the way in which these frequently used pharmaceuticals would affect the differentiation and proliferation of C2C12 and proliferation of 3T3 cell lines. Since the murine cell lines used are standard laboratory models mimicking the cellular composition of human tissues, it was safe to conclude that the variables selected would have similar influences on human samples, making this project applicable to real-world clinical situations.

1. Acetaminophen

Acetaminophen (Figure 3), also known as paracetamol but more commonly known as the over-the-counter (OTC) remedy Tylenol, is one of the many painkillers prescribed to patients post-surgery and is often taken in conjunction with other painkillers. While its mechanism is still fairly unknown, acetaminophen is often placed under the category of painkillers known as non-opioid analgesics.22 This group is mainly composed of NSAIDs, non-steroidal anti-inflammatory drugs, including aspirin and ibuprofen. NSAIDs target and inhibit cyclooxygenase (COX) enzymes which are responsible for the formation of prostaglandins, lipid hormones that sensitize spinal neurons to pain and stimulate some inflammatory responses. However, research conflicts on the exact mechanism of action of acetaminophen, as its anti-inflammatory effect is not as potent as that of aspirin and other NSAIDs. This fact prompted the proposal of different classes of COX enzymes. Acetaminophen appears to inhibit COX-3, an impact that may not be as effective at high oxidation levels (i.e. muscle inflammation), and only selectively inhibits other COX enzymes.23 However, since prostaglandins are known to exist in almost all cells of the body, and acetaminophen’s uses are wide-ranging, including the treatment of pain, fever, allergies, cough, colds, and flu, its classification as an NSAID seems appropriate.24

Figure 3: Chemical Structure of Acetaminophen25

Many investigative studies have suggested the potential for acetaminophen to inhibit the growth of muscle cells under varying circumstances.26,27,28 Moreover, one study has specifically correlated the growth of myoblasts to the COX2 pathway.29 The choice to test acetaminophen’s effects on proliferation and differentiation looks to further investigate this hindering capability on the level of C2C12 cell lines.


More recent studies have also investigated acetaminophen’s effect on fibroblasts and interactions between the compound and cell type. One study, which was conducted on renal murine cells, points to the potential dual role of acetaminophen in both promoting proliferation of kidney fibroblasts when it is present in low doses and increasing cytotoxicity in high concentrations.30 Consequently, this experiment examines a different mice-derived fibroblast cell line, 3T3, to investigate how acetaminophen’s effects may support or contradict this previous study.

2. Dexamethasone

Dexamethasone (DEX) (Figure 4) is a small synthetic glucocorticoid. As an organic compound, it is classified as a 21-hydroxysteroid.31 DEX is used for its anti-inflammatory and immunosuppressive properties, as well as its ability to penetrate the central nervous system. It can be given orally, intranasally, topically, ophthalmically, and by injection. As a hydrophobic molecule, it crosses the cell membrane and binds to cytoplasmic glucocorticoid receptors. This ligand-receptor complex enters the nucleus and binds to DNA, modifying transcription, and by extension, protein synthesis. Specifically, DEX may increase the expression of lipocortins, a group of proteins that inhibit phospholipase A 2, which in turn diminishes the release of mediators of inflammation such as prostaglandins. DEX also inhibits leukocyte activity.

Treatment with glucocorticoids such as DEX has been associated with atrophy of skeletal muscle, but the mechanism remains unclear.32,33 DEX has previously been shown to reduce proliferation and inhibit protein synthesis of C2C12 myoblasts, along with producing dose-dependent increases in protein degradation and reductions in C2C12 myotube diameter.33

The effects of DEX and other such glucocorticoids on C2C12 development may be of particular interest to patients receiving transplants utilizing muscle stem cells. Anti-inflammatory drugs are usually taken post-surgery; if these drugs may be simultaneously hindering the growth and proliferation of the stem cells within the transplanted organ, the medical practitioner should reevaluate the pharmacological profile of the administered drugs to ensure that transplant integrity is being maintained. This experiment thus sought to investigate DEX and C2C12 in order to compare results to prior studies and to aid clinicians in their design of drug treatment regimens.

Based on current literature, DEX has not been studied in the context of 3T3 cell proliferation, so this study sought to identify any potential impacts of DEX on fibroblasts. It is important to determine whether the main type of cell secreting the ECM and preserving the structure of connective tissue is being affected by such a common anti-inflammatory medication.

Figure 4: Chemical Structure of Dexamethasone34

3. Estradiol

Estradiol (Figure 5), or 17β-estradiol, is the most biologically active form of the estrogen sex hormone. While it is essential in regulating and maintaining the menstrual female reproductive cycles and tissues, it


demonstrates significant effects in many other tissues as well, such as bone. However, the effect of estradiol on muscle cells, or at least the mechanism behind it, is not entirely understood. In terms of strength degeneration, declining levels of serum testosterone, the male sex hormone counterpart, have been shown to significantly contribute to muscle weakness in men. The same effect has been suggested for ovarian hormones in women, in that estrogens may play an important role in preserving muscle strength.35 Previous studies detailing the effects of estradiol on muscle weakness in rodent cell lines also support the above hypothesis of a positive relationship between the variables.36,37

Figure 5: Chemical Structure of Estradiol38

4. Ibuprofen

OTC NSAIDs such as the analgesic ibuprofen are known to inhibit myoblast differentiation and proliferation.39 Upon bodily injury, white blood cells collect at the site of the wound and initiate pangs of swelling, heat, redness, and pain. The antipyretic and pain-killing ibuprofen (Figure 6) controls the inflammatory response by preventing the synthesis of excess prostaglandins, compounds that signal to the body that an injury has occurred, by inhibiting the enzyme cyclooxygenase from converting arachidonic acid to prostaglandins.40 Recent studies have shown herbal medicines, such as neem and licorice, to be more effective than ibuprofen at suppressing inflammation in C2C12 cells.39

Figure 6: Chemical Structure of Ibuprofen41

As the popularity of combining common drugs and supplements to produce a greater cumulative therapeutic effect grows, reports concerning the unintended off-target effects of these drug interactions are rising as well. Though some reports warn of combined drug intoxication, many have found a synergy to be beneficial to the healing process of the body.42,43 For example, combinations of cytotoxic drugs during cancer and infection treatment require lower doses of each drug to obtain better therapeutic effects with less side effect toxicity (Figure 7). In addition, combinations of antibiotics yield increased efficacy through fewer side effects and reduced development of resistance.44


Figure 7: Combination of Drug A and Drug B45

5. Turmeric

Turmeric (Figure 8), a known natural remedy for muscle soreness and inflammation, contains the active ingredient curcumin responsible for pain relief. Turmeric is one of the most widely studied superfood. Turmeric offers a potent source of antioxidant-rich compounds, making it an effective antioxidant in the body. These curcuminoids have been found to be effective at donating their electrons to free radicals, thus neutralizing and stabilizing them without the creation of new free radicals. Because of turmeric’s efficacy in the body, studies have shown how curcumin is capable of enhancing muscle cell development and regeneration.46

Prior research has supported the hypothesis that taking turmeric eases muscle soreness and improves blood circulation by acting as an antioxidant in the body. Thus, these findings show that turmeric intake produces effects similar to those of ibuprofen with regard to muscle pain relief.

Figure 8: Chemical Structure of Turmeric47

This particular experiment was conducted to understand whether or not the mixture of turmeric and ibuprofen would create a synergistic effect—a combined effect greater than the sum of ibuprofen’s and turmeric’s individual impacts—on C2C12 growth. Synergy tends to exist when two drugs taken together at low doses create an effect similar to the two drugs taken individually at higher doses. Due to the similar effects of ibuprofen and turmeric on inflamed muscle, it was hypothesized that low doses of both turmeric and ibuprofen would yield similar effects on cell count and differentiation as high doses of each compound would individually. High doses of both ibuprofen and turmeric were hypothesized as being potent to the cells because of toxicity induced by drug hyperactivity. It is predicted that turmeric will be more favorable to cell proliferation and differentiation than ibuprofen.


6. Vitamin D

Vitamin D (Figure 9) is a fat-soluble secosteroid that is naturally found in food.48 It is also synthesized in the skin due to sunlight exposure and converted to its active form through enzymatic hydroxylation in the liver and kidneys. This vitamin is necessary for bone growth, bone remodeling, and healthy mineralization of bone. Vitamin D is also involved in skeletal muscle and the modification of cells.49 Due to its roles in the human body, vitamin D deficiency or surplus can greatly affect the way muscle cells develop and ultimately how well they function. Past studies have shown that vitamin D promotes proliferation of mice neural stem cells.50 Despite the different tissue type, vitamin D may elicit a similar effect in muscle stem cells. In addition, previous research suggests a correlation between vitamin D deficiency and myopathy; indeed, remediation of myopathy through vitamin D therapy has been shown to induce a positive effect on muscle health.[51] Vitamin D binds to receptors in cells, causing a series of cell processes pertaining to the homeostasis of intracellular calcium.51 Calcium is critical to fusion during myogenesis; because vitamin D directly affects calcium concentration within the cell, it is expected that there will be a relationship between vitamin D concentration and differentiation of C2C12 cells.52,53

Figure 9: Chemical Structure of Vitamin D354

II. Methods

A. Preparation of Medicinal Compounds

For all compounds, the volumes of compound added (μL) were considered to be negligible when compared to the total volume in each flask (5 mL) and each well (4 mL). All five experimental groups used a standard control group with no variable(s) added.

1. Acetaminophen

Acetaminophen is a widely available OTC drug and was purchased in its generic form. The recommended dosage was 15 mL per day. The average blood volume in humans was estimated to be five liters, giving a concentration of 15 mL per 5000 mL of blood, corresponding to 3 mL per 1000 mL of blood (or 3 μL per 1 mL and 15 per 5 mL). The 3 μL dosage was chosen for the middle concentration. Half the recommended dosage, 1.5 μL, was chosen for the low concentration. The high dosage was calculated based on the drug’s safety guidelines, which stated not to take more than four doses within a 24-hour time period. A 12 μL dosage was thus chosen for the high concentration.

2. Dexamethasone

High and low concentrations of DEX were used in the C2C12 and 3T3 proliferation experiments as well as in the C2C12 differentiation experiment. The recommended dosage for DEX based on prior literature


was determined to be 100 ng/mL.32,33 The high concentration was set at 100 ng/mL and the low concentration at 10 ng/mL, a ten-fold decrease in concentration.

3. Estradiol

Five different concentrations were used. The recommended dosage for estradiol was determined to be 100 ng/mL. The middle concentration was set at 100 ng/mL, the very low concentration at 1 ng/mL, the low concentration at 10 ng/mL, and the high concentration at 400 ng/ml. The very low dosage was representative of the incomplete removal by publicly owned treatment works of secreted endogenous estrogens.55The low and high dosages were representative of either a lower or higher dosage in comparison to the recommended dosage.

4. Ibuprofen and Turmeric

Three concentrations of both ibuprofen and turmeric were used: 0, low, and high. The recommended dosage of ibuprofen (50 mg) was used to calculate the low concentration of this variable (50 μg/mL), and the high concentration increased ten-fold from this value (500 μg/mL). The low concentration of ibuprofen reflects the recommended dosage of the variable, whereas the high concentration represents an overdose. As for turmeric, because the active ingredient curcumin comprised 10% of the 500-mg capsule, filtration was used to isolate curcumin from the rest of the capsule’s contents after dissolving the powder in 50 mL of distilled water. The low concentration of turmeric (5 μg/mL) was chosen because few people regularly use turmeric supplements as part of their diets. Therefore, the low concentration of turmeric, unlike that of ibuprofen, did not represent the recommended dosage (one capsule, or 50 mg). The high concentration of turmeric (500 μg/mL) was chosen to simulate a toxic level. Table 1 depicts all of the combinations of ibuprofen and turmeric used. Two treatments (a combination of high concentration of ibuprofen and low concentration of turmeric and a combination of low concentration of ibuprofen and high concentration of turmeric) were eliminated from the study due to time constraints. These two treatments were eliminated specifically because the study focused on looking for a synergistic effect and thus was interested in understanding how proliferation of cells exposed to low concentrations of both ibuprofen and turmeric compared to that of cells exposed to the control treatment and low and high concentrations of either ibuprofen or turmeric.

Table 1: Treatment Combinations of Ibuprofen and Turmeric

Ibuprofen→Turmeric↓

0 Low High

0 x x X

Low x x

High x X

5. Vitamin D

Both the low (10 ng/mL) and high (100 ng/mL) concentrations are higher than the recommended dose (1 mL per 5000 mL of blood, corresponding to 0.2 mL per 1000 mL, or 0.2 μL per 1 mL and 1 μL per 5 mL). The two concentrations were set at 10 and 100 ng/mL, ten-fold and hundred-fold increases in concentration, respectively. Studies have shown that consumption of large amounts of vitamin D can be harmful,56 so these concentrations were chosen in an attempt to quantify these deleterious effects.


Table 2: Treatment Levels for All Compounds

Compound Type Treatment Levels (ng/mL)

Acetaminophen 0 9.6 19.2 76.8

Dexamethasone 0 10 100

Estradiol 0 1 10 100 400

Ibuprofen*** 0 0 0 40,000 400,000 40,000 400,000

Turmeric*** 0 500 50,000 0 0 500 50,000

Vitamin D 0 10 100

***Ibuprofen and turmeric doses were added together.

Table 3: Experiment Types and Flask/Well Counts for All Compounds

Compound TypeExperiment Type

Proliferation Differentiation

Acetaminophen

8 T25 flasks total, 2 flasks for each treatment level (C2C12)

8 T25 flasks total, 2 flasks for each treatment level (3T3)

8 wells total, 2 wells for each treatment level (C2C12)

Dexamethasone



12 wells total, 4 wells for each treatment level (C2C12)

Estradiol 10 T25 flasks total, 2 flasks for each treatment level (C2C12) N/A

Ibuprofen + Turmeric


7 wells total, 1 well for each treatment level

Vitamin D



6 well total, 2 wells for each treatment level (C2C12)

B. Protocol


The proliferation experiments took place over a 2-day period. The differentiation experiments took place over a 6-day period.

1. Preparation of Cell Lines

Day 1

C2C12 (passage 14) and 3T3 (passage 7) cells were initially incubated in DMEM (Dulbecco’s Modified Eagle Media) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C and 5% CO2 in T75 flasks. The flasks were removed from incubation after 72 hours.

For all variable groups, two T75 flasks containing C2C12 cells were trypsinized. Each flask of cells was resuspended in 15 mL of 10% FBS media. The cell suspensions were pooled into a single T75 flask, yielding a total volume of 30 mL. For those variables tested on 3T3 cells, two T75 flasks containing 3T3 cells were trypsinized. Each flask of cells was resuspended in 5 mL of 10% FBS media. The cell suspensions were pooled into a single T75 flask, yielding a total volume of 10 mL.

Cell suspensions were created in T25 flasks for all compounds (Table 3). 4 mL of 10% FBS media was added to each T25 flask. 1 mL of the C2C12 cell suspension was added to each C2C12 flask. 1 mL of the 3T3 cell suspension was added to each 3T3 flask.

2 mL of 10% FBS media was added to each well, either on a 6-well or 12-well plate (Table 3). 2 mL of the C2C12 cell suspension was added to each C2C12 well. 2 mL of the 3T3 cell suspension was added to each 3T3 well.

Initial seeding of cells was estimated to be 150,000 cells in each C2C12 flask, 300,000 cells in each C2C12 well, 100,000 cells in each 3T3 flask, and 200,000 cells in each 3T3 well. The flasks and plates were incubated at 37°C and 5% CO2.

2. Addition of Medicinal Compounds

Day 2

The flasks and plates were removed from incubation after 24 hours. Each of the wells in the plates, a C2C12 flask, and a 3T3 flask were imaged using either a Nikon inverted microscope or EVOS system.

Stock (and sub-stock, when needed) solutions of each compound were then used to add the appropriate volumes to the flasks and wells to achieve the desired concentrations (Table 2). For example, with a total flask volume of 5 mL and using a 10 μg/mL sub-stock of DEX, no volume was added to the control flasks, 5 μL was added to the “low” flasks (10 ng/mL), and 50 μL was added to the “high” flasks (100 ng/mL).

The media was removed from each well plate. 4 mL of 1% FBS media was added to each well; subjecting cells to starvation media is a common method used to stimulate differentiation. The same procedure described above was then used to achieve the desired concentrations. The flasks and plates were incubated at 37°C and 5% CO2.

3. Cell Counting

Day 4

The flasks and plates were removed from incubation after 48 hours.


All flasks were imaged and then trypsinized. Each flask of cells was resuspended in 2 mL of 10% FBS media, yielding a total flask volume of 2.5 mL. Aliquots were transferred to a hemocytometer, and four cell counts were taken for each flask. More than 95% of cells were assumed to be alive based on attachment to surface, so trypan blue staining for differentiation between alive and dead cells was not used.

The media in each well was changed. The same volumes of each compound for each treatment level were then reapplied. The plates were incubated at 37°C and 5% CO2.

4. Plate Imaging

Day 7

The plates were removed from incubation after 96 hours. The media was removed from each well. Each well was washed with 2 mL of phosphate-buffered saline (PBS) and 2 mL of ice-cold 100% ethanol. After evaporation, 1.5 mL of 1% toluidine blue stain was then added to each well. The dye was removed after 30 seconds. Each well was then imaged.

C. Statistical Analysis

One-way ANOVA analysis was conducted on average cell counts across compounds and treatment levels, except ibuprofen and turmeric (which required Tukey’s tests to compare multiple groups’ means). ANOVA tests produced p-values, and 0.05 was used as the significance threshold. For statistically significant differences, a Dunnett’s post hoc test was conducted to localize the variation among the tested groups. Microsoft Excel was used to perform all statistical analyses.


III. Results

A. Acetaminophen

1. C2C12 Proliferation

0 9.6 19.2 76.80

20

40

60

80

100

120

140

160143

95

4436

Flask Dosage of Acetaminophen (ng/mL)

# of

Cel

ls p

er F

lask

(x 1

04)

Figure 10: Effect of Acetaminophen on C2C12 Proliferation

ANOVA yielded a significant difference between treatment levels at a p-value of 8.60E-15. Dunnett’s test revealed significant negative effects on cell population size for all variable groups (Figure 10). See Appendix A for all Dunnett's test results. In all graphs, error bars represent +/- 1 standard error.

The observed results of the C2C12 proliferation experiment (Figure 11) aligned well with the quantitative cell counts (Figure 10). The high-dosage flasks showed decreased cell survivorship and lower cell densities than the control and other variable groups. The small, round dark spots on the far-right photo below in Figure 11 (not to be confused with the bubbles seen in the other images) represent unadhered cells, indicating cell death.

Control Low Middle High

Figure 11: C2C12 flask images (100x) following two-day acetaminophen exposure


2. C2C12 Differentiation

Figure 12: C2C12 well images (100x) following five-day exposure to acetaminophen

Figure 12 shows a staggering contrast in the differentiation between wells treated with high dosages and the control group. The control group shows clear formation of elongated myotubes, indicating successful differentiation. The low-dosage group also has comparably successful myotube formation, indicating a minimal effect of low-concentration acetaminophen on the differentiation of C2C12 cell lines. The mid-dosage group’s level of differentiation visibly appears to be less successful, which may suggest that even a recommended dosage of acetaminophen can impede myoblastic growth in the body. The most significant contrast, however, can be seen in the high-dosage wells. Lack of myotube formation can be seen by the individual cells’ not being connected or elongated, which differs greatly from both the control and other variable groups. The variation in cell confluency (aside from direct differentiation inhibition) may well have been a major factor resulting low development of myotubes.

3. 3T3 Proliferation


0 9.6 19.2 76.80

5

10

15

20

25

30

35

18.75

23.44

30.31

15.63


# of

Cel

ls pe

r Fla

sk (x

104

)

Figure 13: Effect of Acetaminophen on 3T3 Proliferation

ANOVA yielded a significant difference between treatment levels at p-value 0.042. However, Dunnett’s test revealed no significant effects on cell population size for any variable groups (Figure 13).

The qualitative imaging results of the 3T3 proliferation experiment (Figure 14) appear to support the lack of significance in the results’ variation demonstrated by the Dunnett’s test (Figure 13). There was no visible contrast in cell densities among the variable groups and the control group. Moreover, there was no evidence that there was significant cell death since there was no observation of cells that were unadhered to the bottom of the flask.

Control Low Mid High

Figure 14: 3T3 flask images (100x) after two-day acetaminophen exposure B. Dexamethasone



0 10 1000

10

20

30

40

50

60

70

80

90

10090.63

47.19

19.38

Flask Dosage of Dexamethasone (ng/mL)

# of

Cel

ls pe

r Fla

sk (x

104

)

Figure 15: Effect of Dexamethasone on C2C12 Proliferation

ANOVA yielded a significant difference between treatment levels at p-value 5.57E-09. Dunnett’s test revealed significant negative effects on cell population size for both variable groups (Figure 15).



Figure 16: DEX-treated C2C12 well images (100x), stained with 1% toluidine blue

The qualitative imaging of the C2C12 flasks (Figure 16) do not support the quantitative results (Figure 15). Images for the control, low, and high flasks appeared virtually the same, even though quantitative results indicated a significant negative effect on cell population size for all treatment levels. One reason explaining this discrepancy may have been the length of the differentiation experiment. Cell counts were taken on Day 4, and well imaging was completed on Day 7. Even though drug was reapplied to wells on Day 4, the relatively long timespan over which incubation occurred may have masked any effect dexamethasone had on differentiation.



0 10 1000

10

20

30

40

50

60

46.2549.06

30.63


Tota

l # o

f Cel

ls pe

r Fla

sk (x

104

)

Figure 17: Effect of Dexamethasone on 3T3 Proliferation

ANOVA yielded a significant difference between treatment levels at p-value 9.17E-05. Dunnett’s test revealed a significant negative effect on cell population size only for the 100 ng/mL group (Figure 17).

Figure 18: DEX-treated 3T3 flask images (100x)

The qualitative imaging of the 3T3 flasks (Figure 17) appear to support the quantitative cell count results (Figure 18). There was no significant difference in cell population size between the control and low flasks, while a significant reduction in cell population size for the high flasks was observed. In the above images, the low and control flasks look virtually the same, while the high flask appears noticeably sparser in cells.

C. Estradiol



0 1 10 100 4000

50

100

150

200

250

300

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Figure 19: Effect of Estradiol on C2C12 Proliferation

ANOVA yielded a significant difference between treatment levels at p-value 1.54E-10. Dunnett’s test revealed a significant positive effect on cell population size only for the 100 ng/mL group (Figure 19).

Qualitative imaging supported this result, showing a significantly higher cell density in the medium flasks when compared to the control flasks (Figure 20).

Figure 20: Estradiol-treated C2C12 flask images (100x)

D. Ibuprofen + Turmeric



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Figure 21: Effects of Ibuprofen and Turmeric on C2C12 Proliferation

To determine if the difference between the groups exposed to low and high concentrations of ibuprofen and turmeric was statistically significant, both one-way and two-way ANOVA tests were utilized.

An overarching one-way ANOVA treating the experiment as a control and six variable groups yielded a p-value of 0.0054, indicating a significant difference between treatment levels (Figure 21).

Another one-way ANOVA determined whether there were significant differences within the groups exposed to 0, low, and high concentrations of ibuprofen and turmeric. Though no significant difference was found in the turmeric group, a p-value of 0.007 was generated for the ibuprofen group. At a significance threshold of 0.05, it can be concluded that significant differences exist between the groups treated with varying concentrations of ibuprofen.

Post hoc comparisons using Dunnett’s test and Tukey’s test localized the source of variation in the ibuprofen-exposed group. A post hoc Tukey’s test showed significant variation between the control group and the group exposed to low concentration of ibuprofen and no turmeric (p-value 0.016). In addition, Tukey’s test showed significant variation between groups exposed to low concentrations of both ibuprofen and turmeric and a low concentration of ibuprofen with no turmeric (p-value 0.0023) and between groups exposed to low and high concentrations of ibuprofen with no exposure to turmeric (p-value 0.0429). The Tukey test when applied to the entire data set revealed significant variation between the groups exposed to the low and high concentrations of ibuprofen in that the low concentration of ibuprofen was more beneficial to cell proliferation.

Two-way ANOVA determined whether the impact of the two independent variables, turmeric and ibuprofen, had a statistically significant effect on C2C12 proliferation. Since the low-high and high-low combinations of ibuprofen and turmeric were not tested, two two-way ANOVA tests were used to compare the low concentrations of both compounds to the control and high concentrations of both compounds to the control. The p-values generated for the 0-low two-way ANOVA and 0-high two-way ANOVA were


0.0029 (suggesting significant negative interaction) and 0.7958 (suggesting no significant interaction), respectively. 2. C2C12 Differentiation

Qualitatively speaking, a higher cell density exists for cells exposed to higher concentrations of turmeric and ibuprofen. A moderately higher differentiation density appears to exist for the low-low concentrations of turmeric and ibuprofen than for the low concentrations of either turmeric and ibuprofen (Figure 22). However, because high confluence was not reached, it was difficult to interpret the effects of varying compound concentrations with great confidence.

Figure 22: Ibuprofen- and turmeric-treated C2C12 flask images (100x)

E. Vitamin D



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Figure 23: Effect of Vitamin D on C2C12 Proliferation

ANOVA yielded a significant difference between treatment levels at p-value 9.13E-09. Dunnett’s test revealed significant negative effects on cell population size for both variable groups (Figure 23).



Figure 24: Vitamin D-treated C2C12 well images (100x)

From these images, it was concluded that vitamin D impeded the differentiation process. The wells that were not treated with vitamin D thrived; on Day 6, they exhibited visible myotubes and myofiber formation. The wells exposed to the low concentration did exhibit some myotubes, but these were more spread out, and the wells as a whole were less populated. The wells with a high concentration exhibited few to no cells present, and the few that existed had not undergone differentiation (Figure 24). The low cell confluency (aside from direct differentiation inhibition) may well have been a major factor resulting in the absence of myotubes.

It is evident that there may have been an error in the imaging of the cells, because the control and low Day 1 images appear to have no cells in their respective wells. However, in the Day 6 images, there are clearly cells that have differentiated or have begun to differentiate into myotubes.



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.Figure 25: Effect of Vitamin D on 3T3 Proliferation

ANOVA yielded a significant difference between treatment levels at p-value 6.33E-07. Dunnett’s test revealed a significant negative effect on cell population size only for the 100 ng/mL group (Figure 25).


IV. Discussion

A. Acetaminophen

The observed results of acetaminophen’s effect on C2C12 myoblast cells differed greatly from its observed effects on 3T3 fibroblasts. C2C12 results demonstrated a clear trend that as acetaminophen concentration increased, myoblastic cell proliferation decreased. The significance of these results was corroborated by ANOVA and Dunnett’s tests. The results are supported by previously cited reports that correlate acetaminophen with potential adverse effects on the growth of muscle cells. 25,28 Moreover, the differentiation results showed apparent differences between the high-dosage treated C2C12 cells and the control cells’ ability to form myotubes, aligning well with the quantitative results. The inability of the high-dosage group to form myotubes supports both the warning that taking OTC acetaminophen four times in a 24-hour period is detrimental and previous studies on acetaminophen’s effects on differentiation (as it showed clear debilitating effects on the formation of myotubes).

The 3T3 results presented an interesting correlation that while a steady increase in acetaminophen led to subsequent increases in proliferation of 3T3 cell populations, excessively high (i.e. overdose) levels of acetaminophen treatment caused slight decreases in population counts compared to the control. These results are in line with the findings of the previously cited study that demonstrated acetaminophen’s promotion of kidney fibroblast proliferation at low doses, but debilitation of growth at high doses.29

However, the lack of significance demonstrated by the Dunnett’s test eliminates the ability of these results to be confidently used to support this prior study.

B. Dexamethasone

This experiment supported the hypothesis regarding the effect of DEX on C2C12 myoblasts. The prediction was made that DEX would negatively affect cell proliferation as well as decrease myotube diameter in cell differentiation. Due to available resources and time constraints, a quantitative analysis of differentiation was not possible to further investigate the apparent inconsistencies between the qualitative imaging and cell counts. A decrease in cell proliferation of C2C12 myoblasts due to low and high dosages of DEX was observed. The experiment using the 3T3 fibroblasts demonstrated a lesser effect on cell proliferation. At a low dosage of DEX, little to no decrease in cell proliferation was observed. However, at a high dosage, the effect was more potent and showed a significant decrease in the number of 3T3 cells.

C. Estradiol

This experiment was successfully able to demonstrate a correlation between estradiol dosage and muscle cell proliferation. Findings suggested that while cell survivorship at low estrogen doses did not increase significantly, cell counts of those cultured in the presence of estradiol at a clinically recommended dosage peaked at more than 1.3 x 107 per mL before declining below the original control values at an overdose of estradiol. These results support not only the existing dosage values for clinically prescribed estradiol treatments, but also the role of estrogens in maintaining muscle strength, especially for older women. As moderate amounts of estradiol were shown to be beneficial for cell growth, this study further suggests the importance and impact of the role that hormones play in the development, maintenance, and treatment of muscle cells. It also suggests that these lower doses may not cause imbalances in therapeutic stem cell behavior. Moreover, the results also emphasize the importance of maintaining balance in cell signaling and hormones.

D. Ibuprofen and Turmeric


One-way ANOVAs, Dunnett’s tests, and Tukey tests (Appendix A, Table 10) consistently showed that statistically significant variation existed within the group exposed to only low and high concentrations of ibuprofen. In other words, the low concentration of ibuprofen (representative of recommended ibuprofen dosage) yielded the highest cell count when compared to the groups exposed to the control treatment and high concentration of ibuprofen. While one-way ANOVAs showed no statistically significant variation in the group exposed to varying concentrations of turmeric, two-way ANOVA test revealed an interaction between control and low concentrations of ibuprofen and turmeric.

Had a synergy existed in the experiment, C2C12 cells exposed to a combination of low concentrations of both turmeric and ibuprofen would have had a significantly higher proliferation than the cells exposed to the control treatment and low and high concentrations of either turmeric and ibuprofen. This interaction did not directly equate to a synergy, because two-way ANOVA implied that while C2C12 proliferation benefits from low concentrations of either ibuprofen or turmeric, combining low concentrations of both turmeric and ibuprofen hindered proliferation. Therefore, the hypothesis that the combination of taking low combinations of both turmeric and ibuprofen will yield a synergy-like effect on cell proliferation can be rejected.

In terms of differentiation, the hypothesis can be partially accepted, because cells exposed to low concentrations of both ibuprofen and turmeric yielded a higher C2C12 density of differentiation than the cells exposed to the control treatment and low concentrations of either compound. This difference in conclusions concerning differentiation and proliferation can serve as an area of improvement in future experimentation.

E. Vitamin D

Previous studies have noted the instrumental role of vitamin D in maintaining healthy muscle and bone and in aiding cell proliferation and differentiation.49,51 However, a negative correlation was observed between vitamin D concentration and cell survivorship. Experimental evidence supported a statistically significant negative effect on the C2C12 line at a dosage of 10 ng/mL, and large doses of vitamin D at 100 ng/mL had a highly toxic effect on both cell lines. While this experiment provided insight into cell development under various concentrations of vitamin D, further tests are necessary to understand the magnitude of this trend. Testing a wider range of concentrations, as well as testing effects on vitamin D-deficient cells in addition to healthy ones, may shed light on the relationship between this compound and cell growth.

F. Error Analysis

Errors in this experiment could have resulted from systematic error concerning uncertainty inherent in the gram scales and pipettes used to measure solid and liquid quantities. In addition, a lack of precision within experiments can be traced to procedural errors.

Specifically, in the experiment looking for a synergistic effect between ibuprofen and turmeric, cell counts were consistent within flasks of one set but not between duplicates. Such inconsistencies in data concerning cell proliferation can be attributed to inconsistent exposure of cells to trypsin. Prolonged exposure of cells to this enzyme reduces cell count because trypsin exacerbates apoptosis by damaging cell membranes.57 Cell death due to over-trypsinization could have been prevented by washing the T25 cell flasks with PBS before and after each trypsinization to remove excess enzyme residue.

In addition, base-level cell counts were not taken. Therefore, conclusions concerning how proliferation changed over time could not be made. Also, treatments imposed on the cells may not have mirrored realistic conditions due to streamlined estimations of drug and supplement dosages.


Also, with the exceptions of DEX and estradiol, which were obtained in their pure forms, the variables tested were purchased OTC and contained inactive ingredients that could have indirectly affected the results of the experiments. The list of inactive ingredients in the impure compounds are listed below:

Acetaminophen: anhydrous citric acid, butylparaben, calcium sulfate, carrageenan, D&C red #33, FD&C red#40, flavor, glycerin, high fructose corn syrup, hydroxyethyl cellulose, microcrystalline cellulose, and carboxymethylcellulose sodium, propylene glycol, purified water, sodium benzoate, sorbitol solution, tribasic sodium phosphate

Ibuprofen: carnauba wax, colloidal silicon dioxide, croscarmellose sodium, hypromellose, lactose, magnesium stearate, microcrystalline cellulose, propylene glycol, titanium dioxide

Turmeric: It was assumed that filtration of the 100x turmeric solution resulting from a 500 mg (10% active ingredient curcumin) tablet dissolved in 50 mL of distilled water would isolate the inactive ingredient of turmeric root powder from the turmeric root extract containing curcuminoids. However, post-filtration testing for the presence of curcumin was not feasible due to time and financial constraints.

Vitamin D: glycerin, water, polysorbate 80, artificial flavor, caramel color, citric acid (antioxidant for vitamin D), sodium citrateG. Areas of Improvement and Expansion

To improve the project in the future, trials with more replicates would replace the duplicates in order to increase the precision of the results and offer assurance that observations of proliferation and differentiation are not due to random chance. Monitoring the cells in suspension at multiple time points would permit a more realistic understanding of how C2C12 and 3T3 cells proliferate into tissue. Had time permitted, 3T3 cells, which are capable of withstanding minor stress, would have been subjected to scratch tests to more closely mimic cells found in the site of an infection or wound in the body. Comparing stressed cells to healthy cells would have allowed for a stronger understanding of whether the medicinal compounds used in this experiment proved to be beneficial to wound healing and tissue regeneration.

To gain a better understanding of how tissue regeneration is impacted during surgery or scaffold-based regenerative medicine, compounds found in scaffolds or degradation products of scaffolds could be isolated and tested. In addition, to expand the current research horizon, hypothesized off-target effects of common drug and supplement combinations—two or more drugs taken together—can be verified in vitro with C2C12 and 3T3 work. To understand intracellular consequences of drugs, flow cytometry and assays such as ToxGlo could be used to measure cell size, newly synthesized DNA, and mitochondrial efficiency with great accuracy and precision. Using Western blots and real-time polymerase chain reactions (qPCR) to detect protein production and transcript levels, respectively, of specific immune factors being secreted by the cells would have allowed for a deeper analysis of whether or not cell immunity and thus vitality was strengthened post-drug exposure.

Perhaps the most significant project improvement would be to identify the actual typical in vivo exposure of each drug. Experimental exposures were selected to achieve a high-end estimate of this concentration. This estimation assumed that the drugs were readily absorbed into the bloodstream, and not significantly metabolized before reaching the tissue compartment. It is possible that the estimates might be reflective of a toddler or juvenile, though adult exposures are likely overestimated by as much as ten-fold. Further research into the tissue compartment exposure concentrations would be prudent.


V. Conclusion

All drugs and compounds studied in this experiment, at least at some of the tested dosages, appeared to have a statistically significant effect on either C2C12 or 3T3 proliferation. The medicinal compounds varied in their effect by concentration, some doses promoting cell proliferation, others reducing proliferation.

Estrogen and ibuprofen were shown to be beneficial to cell proliferation at moderate amounts and toxic at higher concentrations while acetaminophen, dexamethasone, and vitamin D proved to be harmful to tissue development as dosage concentration increased. These results verify that at recommended dosages, some commonly prescribed drugs may have the potential to mitigate pain and foster cell proliferation—for certain compounds, a higher concentration can have a harmful effect.

Dramatic negative effects on C2C12 differentiation were observed through qualitative analysis at the highest dosages of acetaminophen and vitamin D. No obvious effects were evident with dexamethasone exposures.

Implications from this experiment indicate that it would be prudent for regenerative medicine researchers to explore the impacts of medicinal compounds on stem cell populations, including those utilized to promote muscle or connective tissue regeneration.

VI. Acknowledgments


We would like to thank all those involved in PGSS who made this experience such an amazing one, giving us the opportunity to conduct an enriching team project, challenge ourselves inside and outside of the classroom, and broaden our horizons in science. We would especially like to extend our gratitude to the following people:

Dr. Barry LuokkalaMark KrotecEric DunkerleyLiti ZhangCameron BrezeDr. Carrie DoonanDr. Edwina KinchingtonNathan SendgikoskiDr. Phil CampbellCarnegie Mellon UniversityPGSS Faculty and StaffPGSS SponsorsFellow PGSS students

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33 Desler, M. M., Jones, S. J., Smith, C. W., & Woods, T. L. (1996). Effects of dexamethasone and anabolic agents on proliferation and protein synthesis and degradation in C2C12 myogenic cells. Journal of Animal Science, 74(6), 1265–1273. http://www.ncbi.nlm.nih.gov/pubmed/8791198 34 Dexamethasone Drug Information. (2008, July 30). Retrieved July 28, 2016, from http://www.rxlist.com/dexamethasone-drug.html35 Lowe, D. A., Baltgalvis, K. A., & Greising, S. M. (2010). Mechanisms behind Estrogen’s Beneficial Effect on Muscle Strength in Females. Exercise and Sport Sciences Reviews, 38(2), 61–67. http://doi.org/10.1097/JES.0b013e3181d496bc36 Moran, A. L., Warren, G. L., & Lowe, D. A. (2005). Soleus and EDL muscle contractility across the lifespan of female C57BL/6 mice. Experimental Gerontology, 40(12), 966–975. http://doi.org/10.1016/j.exger.2005.09.00537 Felicio, L. S., Nelson, J. F., & Finch, C. E. (1984). Longitudinal studies of estrous cyclicity in aging C57BL/6J mice: II. Cessation of cyclicity and the duration of persistent vaginal cornification. Biology of Reproduction, 31(3), 446–453. http://www.ncbi.nlm.nih.gov/pubmed/6541508/ 38 Estrasorb (estradiol topical emulsion). (2014, July) Retrieved July 28, 2016, from https://dailymed.nlm.nih.gov/dailymed/archives/fdaDrugInfo.cfm?archiveid=14786739 Kang, J. J., Samad, M. A., Kim, K. S., & Bae, S. (2014). Comparative anti-inflammatory effects of anti-arthritic herbal medicines and ibuprofen. Natural Product Communications, 9(9), 1351–1356. http://www.ncbi.nlm.nih.gov/pubmed/2591880940 How does ibuprofen work? (n.d.). Retrieved July 28, 2016, from http://www.rsc.org/learn-chemistry/resources/chemistry-in-your-cupboard/nurofen/2 41 Hydrocodone Bitartrate and Ibuprofen. (2014, August 1). Retrieved July 28, 2016, from http://medlibrary.org/lib/rx/meds/hydrocodone-bitartrate-and-ibuprofen-9/42 Breitinger, H.-G. (2012). Drug Synergy - Mechanisms and Methods of Analysis. Retrieved July 28, 2016, from http://cdn.intechopen.com/pdfs-wm/28118.pdf 43 Tallarida, R. J. (2011). Quantitative Methods for Assessing Drug Synergism. Genes & Cancer, 2(11), 1003–1008. http://doi.org/10.1177/1947601912440575 44 Polydrug Use. (2014). Retrieved July 28, 2016, from https://comorbidity.edu.au/sites/default/files/Polydrug%20Use.pdf45 Drug Synergy. (n.d.). Retrieved July 28, 2016, from http://www.graphpad.com/support/faqid/99146 Thaloor, D., Miller, K. J., Gephart, J., Mitchell, P. O., & Pavlath, G. K. (1999). Systemic administration of the NF-kappaB inhibitor curcumin stimulates muscle regeneration after traumatic injury. The American Journal of Physiology, 277(2 Pt 1), C320–329. http://www.ncbi.nlm.nih.gov/pubmed/10444409 47 Joshi, K. (2012, March 31). Turmeric: Uses & Health Benefits. Retrieved July 28, 2016, from https://www.sunzu.com/articles/turmeric-uses-health-benefits-176013/48 Pérez-López, F. R. (2007). Vitamin D: the secosteroid hormone and human reproduction. Gynecological Endocrinology, 23(1), 13–24. http://www.ncbi.nlm.nih.gov/pubmed/17484507 49 Human Vitamin and Mineral Requirements: Vitamin D. (2002). Retrieved July 28, 2016, from http://www.fao.org/docrep/004/Y2809E/y2809e0e.htm50-J., Zhao, G., & Li, G.-Y. (2015). Role of vitamin D in regulating the neural stem cells of mouse model with multiple sclerosis. European Review for Medical and Pharmacological Sciences, 19(21), 4004–4011, http://www.ncbi.nlm.nih.gov/pubmed/2659282151 Ceglia, L. (2009). Vitamin D and Its Role in Skeletal Muscle. Current Opinion in Clinical Nutrition and Metabolic Care, 12(6), 628–633. http://doi.org/10.1097/MCO.0b013e328331c707 52 Przybylski, R. J., MacBride, R. G., & Kirby, A. C. (1989). Calcium regulation of skeletal myogenesis. I. Cell content critical to myotube formation. In Vitro Cellular & Developmental Biology: Journal of the Tissue Culture Association, 25(9), 830–838. http://www.ncbi.nlm.nih.gov/pubmed/2507513 53 Hall, A. C., & Juckett, M. B. (2013). The Role of Vitamin D in Hematologic Disease and Stem Cell Transplantation. Nutrients, 5(6), 2206–2221. http://doi.org/10.3390/nu5062206 54 Helmenstine, A. M. (n.d.). Cholecalciferol - Vitamin D3 Chemical Structure. Retrieved July 28, 2016, from http://chemistry.about.com/od/factsstructures/ig/Chemical-Structures---C/Cholecalciferol---Vitamin- D3.htm


55 Barrett, J. R. (2009). Endocrine Disruptors : Estrogens in a Bottle? Environmental Health Perspectives, 117(6), A241. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2702426/ 56 Vitamin D: Fact Sheet for Health Professionals. (2016, February 11). Retrieved July 28, 2016, from https://ods.od.nih.gov/factsheets/VitaminD-HealthProfessional/57 Cell Dissociation with Trypsin. (n.d.). Retrieved July 28, 2016, from http://www.sigmaaldrich.com/technical-documents/articles/biology/cell-dissociation-with-trypsin.html58 Abdi, H., & Williams, L., J. (2010). Tukey’s Honestly Significant Difference (HSD) Test. Retrieved July 27, 2016, from https://www.utdallas.edu/~herve/abdi-HSD2010-pretty.pdf

VIII. Appendix A: Dunnett’s and Tukey’s Test Value Tables

A. Acetaminophen

Table 4: C2C12 Proliferation


Dunnett’s Test Value Significance based on Dunnett’s test critical value (p<0.05,

CV=2.88; p<0.01, CV=4.00)

Low (9.6) 8.239 Significant

Middle (19.6) 17.021 Significant

High (76.8) 15.961 Significant

Table 5: 3T3 Proliferation



CV=2.88; p<0.01, CV=4.00)

Low (9.6) 0.915 Not Significant

Middle (19.6) 2.257 Not Significant

High (76.8) 0.610 Not Significant

B. Dexamethasone



Dunnett's Test Value Significance based on Dunnett’s test critical value (p<0.05,

CV=2.67; p<0.01, CV=3.77)

Low (10) 6.265 Significant

High (100) 10.278 Significant





CV=2.67; p<0.01, CV=3.77)

Low (10) 0.774 Not Significant


C. Estradiol


Flask Dosage of Estradiol (ng/mL)


CV=3.02; p<0.01, CV=4.17)

Very Low (1) 0.342 Not Significant

Low (10) 2.054 Not Significant

Middle (100) 8.699 Significant

High (400) 0.262 Not Significant

D. Ibuprofen + Turmeric

Table 9: C2C12 Proliferation (only Ibuprofen)

Flask Dosage of Ibuprofen (ng/mL)


CV=3.80; p<0.01, CV=5.62***)

40,000 3.894 Significant

400,000 0.483 Not Significant

***Critical values were extrapolated

Table 10: C2C12 proliferation (Ibuprofen and Turmeric - Tukey’s Test***)

Compared Groups |Ma-Ma’| Significance based on Tukey’s test critical value (p<.05,

HSD[0.05]=23.16; p<0.01, HSD[0.01]=31.78)

M1 vs. M2 32.25 Significant

M1 vs. M3 4 Not Significant

M2 vs. M3 28.25 Significant


***A Tukey’s test was used to determine which groups in the experiment differed.58 M1 represents the control, and M2 and M3 represent the low and high concentrations of ibuprofen, respectively. The honest significant difference (HSD) represents the absolute difference between any two sample means required for significance at a given level. If the absolute value of the difference of means is more than the HSD value, then the comparison is considered statistically significant.

E. Vitamin D




CV=2.67; p<0.01, CV=3.77)

Low (10) 3.826 Significant





CV=2.67; p<0.01, CV=3.77)

Low (10) 0.881 Not significant


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