Investigating the effects of
Cissus quadrangularis and
IGF-I on the production of
collagen in rat fibroblast
cells
SRP Project Proposal
2010
Member:
Cheng Yong Jian
Member:
Er Yuan Zhi
Hwa Chong Institution (High School)
Mentor: Mrs. Har Hui Peng
Date: 22th October 2009
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Content
1. Introduction---------------------------------------------3
1.1 Background Information-------------------------3
1.2 Rational------------------------------------------------5
1.3 Hypothesis--------------------------------------------5
1.4 Objectives---------------------------------------------5
2. Project Details------------------------------------------6
2.1 Resources Required-------------------------------6
2.2 Outline of method----------------------------------8
2.3 Safety precautions-------------------------------10
2.4 Limitations------------------------------------------10
2.5 Time Line--------------------------------------------11
3. References---------------------------------------------11
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1. Introduction
1.1 Abstract
Tendons are mechanically responsible for transmitting muscle forces to bone and in doing so,
permit locomotion and joint stability…However, inappropriate physical training leads to tendon
overuse injuries (Wang, 2005). Tendinosis is a chronic form of tendinitis, which occurs when the
collagen that composes the tendon degenerates through repetitive motion and aging. Each
year, tens of thousands more U.S workers develop tendinosis (1999, The Bureau of Labor
Statistics). Experiments will be conducted to investigate the effects of Cissus quadrangularis
and IGF-I on the synthesis of collagen in mouse fibroblast cells. Masson Trichrome staining
would be used to identify if there has been an increase in the synthesis of collagen
1.2 Background Information
Fibroblast cells are embedded in the extracellular matrix of tendons, which is composed of
collagen, elastin, and proteoglycans, and young fibroblast cells synthesize and secrete the
matrix collagen, elastin, and proteoglycans (Erickson, 2002). Collagen is the main component of
tendons, with type I collagen is the most abundant type found in tendons (Erickson, 2002).
Tendons may degenerate due to repetitive motion and ageing (Summers, 2003). During the
degeneration process, collagen breaks down and the body will respond by increasing collagen
production (Erisson, 2004). Hence medications for tendon repair often contain compounds that
stimulate collagen production (Haukipuro, 1991). This project aims to study the ability of the
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herbal extract to enhance the production of collagen in mouse fibroblast cells. We hope that
our findings will pave the way for the discovery of the future treatment of tendon injuries.
Cissus quadrangularis, an ancient herb found in India, is said to cause an increase in collagen
production and aid the healing of join ailments (Jainu, M. and Mohan, KV., 2008). It is the active
ingredient found in a medication used to relieve joint pain and increase the rate of recovery from
tendon injuries (Cortes, B.P., Hingorani, L. and Thawani, V., 2004). In this project, the effect of extracts,
made using Cissus quadrangularis , on collagen production in mouse fibroblast will be studied.
Insulin-like growth factor 1 (IGF-I) is said to stimulate collagen production. It is a growth
hormone that has been shown to be an in-vitro stimulant of type I collagen synthesis, along
with insulin and ascorbic acid (Ivarsson, 1998). A study by (J.Tang, P.Liu, X.Wang, 2005) showed
that in vitro treatments of tenocytes with IGF-1 resulted in an increased production of type I
collagen. Also, studies have shown that production of type I collagen in human fibroblast cells is
known to be increased by IGF-1 (Jonsson, B., Johansson, G., Karlström, Ljunggren , Ljunghall and
Mallmin, 1980; Borg, Ivarsson, McWhirter and Rubin, 1998).
Current research is being done to maximize the healing of tendon injuries, and it was found that
healing is dependent on “early granulation of defects, maximizing collagen type I production,
organization, elasticity, and minimizing scar tissue formation” (Sutter, 2007). There are several
cell-based treatments of tendon injuries that are currently being investigated, and these include
bone marrow aspirate, platelet-rich plasma, and adult mesenchymal tissue-derived stem cells.
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This angle adopted by this project is different from the other studies in that instead of using
stem cells to re-grow tendons, mouse fibroblast cells will be treated with Insulin-like Growth
Factor 1 (IGF-1) and Cissus quadrangularis extracts to see if production of type I collagen can be
enhanced. No research has been done on the combined effects of IGF-1 and Cissus
quadrangularis. Previous research concerning the effects of IGF-1 on collagen production is
limited, but the positive results of a study described by Erickson (2002) will be used as a
reference for this project.
1.2 Rationale

We aim to investigate the effectiveness of Cissus quadrangularis and IGF-I to stimulate
the synthesis of collagen in mouse fibroblast cells to develop new treatments for
tendonitis.
1.3 Hypothesis

Cissus quadrangularis and IGF-I are both able to stimulate the synthesis of collagen in
rat fibroblast cells when used in isolation of combination to treat mouse fibroblast cells.
1.4 Objectives

Determine the effectiveness of Cissus quadragularis and IGF-I on the stimulation of
synthesis of collagen in mouse fibroblast cells
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2. Project Details
2.1 Resources Required
The resources we require are:

3T6 Swiss Albino (Mouse embryonic fibroblast cell line) Nuclear Lysate

Cissus quadragularis

IGF-1

Tissue culture flask

𝐶𝑂2 incubator

Variable volume micropipettes and pipette tips

Vortex mixer

Centrifuge machine

Spectrophotometer

Stock Solution A:
o Hematoxylin ----------------------------- 1 g
o 95% Alcohol ----------------------------- 100 ml

Stock Solution B:
o 29% Ferric chloride in water --------- 4 ml
o Distilled water ------------------------ 95 ml
o Hydrochloric acid, concentrated ---- 1ml

Biebrich Scarlet-Acid Fuchsin Solution:
o Biebrich scarlet, 1% aqueous --------- 90 ml
o Acid fuchsin, 1% aqueous --------------10 ml
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o Acetic acid, glacial --------------------- 1 ml

Phosphomolybdic-Phosphotungstic Acid Solution:
o 5% Phosphomolybdic acid ------------- 25 ml
o 5% Phosphotungstic acid -------------- 25 ml

Aniline Blue Solution:
o Aniline blue ------------------------------- 2.5 g
o Acetic acide, glacial --------------------- 2 ml
o Distilled water --------------------------- 100 ml

1% Acetic Acid Solution:
o Acetic acid, glacial ----------------------- 1 ml
o Distilled water ---------------------------- 99 ml

Sircol Assay Kit
o Dye reagent
o Alkali reagent
o Salt soluble collagen precipitating reagent
o Collagen standard (Concentration 1mg/ml)
o Capped 1.5ml capacity micro centrifuge tubes
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2.2 Outline of method
Here is an overview of our methods:
Cissus
quadrangularis
Preparation of rat fibroblast
cell culture
Rat fibroblast cell culture
Masson Trichrome
staining

IGF-I
Collagen assay
We will conduct our experiment in the Hwa Chong Institution Science Research Centre
Biology lab for a period of 7 months from Early January to Mid August. Mouse fibroblast
cells are cultured in a 𝐶𝑂2 chamber at 37
at a volume of 0.25ml/𝑐𝑚2 for a period of 5
days (Sircol Soluble collagen assay). A total of 4 sets of fibroblast cells will be cultured,
with Cissus quadrangularis added to one set, IGF-I added to another set, both IGF-I and
Cissus quadrangularis added to the third set, and lastly would be the control in which
nothing is added. The 4 sets will be incubated in the 𝐶𝑂2 chamber for a period of 1 week.

Masson Trichrome staining would be done on each of the cell cultures after the
incubation period to see if there has been any increase in the synthesis of collagen.
Deparaffinize the cell culture and rehydrate through 100% alcohol, 95% alcohol and 70%
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alcohol. Wash in distilled water, and stain in Weigert’s iron Hematoxylin working solution
for 10 minutes. Rinse in running warm tap water for 10 minutes, and then wash in
distilled water. Differentiate in phosphomolydic-phosphotungstic acid solution for 15
minutes. Transfer sections directly to aniline blue solution and stain for 5 to 10 minutes.
Rinse briefly in distilled water and differentiate in 1% acetic acid solution for 2 to 5
minutes. Wash in distilled water and dehydrate very quickly thorugh 95% ethyl alcohol
and absolute ethyl alcohol.

Collagen concentration would be determined using Sircol Soluble Collagen Assay. Add
1ml of Sircol Dye reagent and cap to each tube and cap all of them, mixing contents by
inverting. Place tubes in a mechanical shaker for 30 minutes. Transfer the tubes to a
micro centrifuge and spin the tubes at >10,000 x g for a 10 minute period. The unbound
dye solution is removed by carefully inverting and draining the tubes. Any remaining
droplets are removed from the tubes by gently tapping the inverted tube on a paper
tissue. Then, add 1 ml of the alkali reagent to each tube. Re-cap the tubes and release
the bound dye into solution. Wait for 10 minutes for the bound dye to dissolve. Set the
spectrophotometer’s wavelength to 540nm and set it to zero using water before
measuring absorbance of reagent blanks, collagen standards and the test samples.
Substrate the reagent blank reading from the standard and test sample readings. Plot
standards on graph and use the graph to calculate the collagen content of the test
samples.
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2.3 Safety Precautions
During our experiments, as we are working with cells with a biosafety level of 1, work is done in
a biosafety unit (Class 2A). Standard cell culture practices will be used when working in the
laboratory. All cell cultures and vessels used to contain them will be decontaminated using
chemical disinfectants or by steam autoclaving before use. Lab coats and latex gloves will be
worn. The laboratory work will be supervised by a lab staff with training in cell culture techniques.
2.4 Limitations
Throughout this project, we would encounter certain difficulties and limitations.
Problem
Reason
Unable to test on human fibroblast
As the Ministry of Education does not allow us to
conduct this experiment on human fibroblast
cells for safety reason, we are only able to
conduct this experiment on mouse fibroblast
cells, hence the results obtained in this
experiment may not be useful as it is not being
conducted on human cells
Mammalian cells
Mammalian cells require a CO2 incubator to be
cultured in, and our school lab does not have
such an incubator hence we may encounter a
problem in culturing these cells
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3.4 Time Line
Date
Activity
1st January 2010
Start of experiment
April 2010
Obtain 1st set of results
May 2010
1st Duplication of experiment
9th July 2010
Preparation for Project’s Day Semi-Finals
July 2010
2nd Duplication of experiment
21st August 2010
Tabulation, compilation, preparation for final
research paper and Project’s Day Finals
4. References
o Cissus Quadrangularis—The Best Kept Secret in Bodybuilding. (2007). Retrieved
September 28, 2009, from USPLabs Web site:
http://www.usplabsdirect.com/supercissus.html
o Erickson, L. (2002). Future treatments. Retrieved May 21, 2009, from
http://www.tendinosis.org/future.html
o Erickson, L. (2002). The Tendinosis Injury. Retrieved September 24, 2009, from
http://www.tendinosis.org/injury.html
o Huaux, F., Liu, T., McGarry, B., Phan, S. H., Ullenbruch, M. (2003). Dual roles of IL-4 in
lung injury and fibrosis. The Journal of Immunology, 170, 2083-2092.
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o Borg, T. K., Ivarsson, M., McWhirter, A., Rubin, K. (1998). Type I collagen synthesis in
cultured human fibroblasts regulation by cell spreading, platelet-derived growth factor
and interactions with collagen fibers. Matrix Biology. 16, 409-425.
o Daha, M. R., Geest, R. N., Lam, S., VanKooten, C., Verhagen, N. A. M. (2004). Secretion
of collagen type IV by human renal fibroblasts is increased by high glucose via a TGF-Bindependent pathway. Nephrol Dial Transplant, 19, 1694-1701.
o Leeson, C. R., Leeson, T. S., & Paparo, A. A. (1985). Connective tissue proper. Textbook of
Histology. Philidelphia: W. B. Saunders Company.
o Kanjanapothi, D., Panthong, A., Reutrakul, V., Supraditaporn, W., Taesotikul, T. (2007).
Analgesic, anti-inflammatory and venotonic effects of Cissus quadrangularis Linn.
Journal of Ethnopharmacology. 110, 264-270.
o Robertson, W. (1964). Metabolism of Collagen in Mammalian Tissues. Biophysical
Journal. 4, 1, 2, 93-106.
o Sutter, W. W. (2007). Autologous Cell-Based Therapy for Tendon and Ligament Injuries.
Clinical Techniques in Equine Practice. 6, 3, 198-208.
o Wang, J. H. C. (2006). Mechanobiology of tendon. Biomechanics. 39, 1563-1582.
o Wulff, S. (2004). Guide to special stains. Carpinteria, CA: Dako.
o
http://www.ihcworld.com/_protocols/special_stains/masson_trichrome.htm
o Jainu, M. and Mohan, KV. (June 8, 2008). Protective role of ascorbic acid isolated from
Cissus quadrangularis on NSAID induced toxicity through immunomodulating response
and growth factors expression
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o Ghahary, A., Houle, Kilani, T., Scott, G., Shen and Tredget, E., (2000), Vol. 17, No. 3,
Pages 167-176. Mannose-6-Phosphate/IGF-II Receptors Mediate the Effects of IGF-1Induced Latent Transforming Growth Factor β1 on Expression of Type I Collagen and
Collagenase in Dermal Fibroblasts
o Baroni, G., Benedetti, A., Casini, A., Folli, F., Gaglotti, G., Macarri, G., Marucci, L.,
Orlandoni, P., Perego, L., Ridolfi, F. and Sario, A. (1999) Insulin and Insulin-Like Growth
Factor-1 Stimulate Proliferation and Type I Collagen Accumulation by Human Hepatic
Stellate Cells: Differential Effects on Signal Transduction Pathways
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