Gabi Guzman, Allison Meadows, Cheng Yong Jian, and Er Yuan Zhi
November 3, 2009
Investigating the effect of insulin-like growth factor-1 (IGF-1) and C. quadrangularis extract on
the collagen type I production of tendon fibroblasts
Problem: What is the effect of IGF-1 and Cissus quadrangularis extract on the collagen
production of fibroblasts? The chronic condition of tendinosis is caused by the deterioration of
collagen in tendons. The body is often not able to offset this deterioration, but if a treatment
could increase the collagen production of the fibroblasts, this production of new, healthy
collagen could possibly offset the deterioration.
Hypothesis: If fibroblasts are separately treated with IGF-1, C. quadrangularis, and a
combination of both, then IGF-1, C. quadrangularis, and the combination will each increase the
collagen type I production of the tendon fibroblasts. IGF-1 has previously been shown to
increase collagen production, so it will be used as a positive control. C. quadrangularis has
been suggested by anecdotal evidence to promote tendon health, and this is hypothesized to
be observable at the cell level by means of collagen production. Greater concentrations of C.
quadrangularis are hypothesized to increase the collagen production, until the concentration
becomes toxic. The combined effect of IGF-1 and C. quadrangularis should promote a greater
collagen production than either individual substance if each is shown to increase the collagen
production separately.
Independent variable – the treatment of the fibroblasts, either with IGF-1, C. quadrangularis,
or a combination of both; varying concentrations of C. quadrangularis
Dependent variable – the amount of collagen produced by the fibroblasts, measured
quantitatively by the Sircol™ Soluble Collagen Assay and qualitatively by ImageJ with Masson’s
trichome stain
Constants

ideally, the maturity of the fibroblast cells (young fibroblasts / fibrocytes?)

amount of fibroblast cells treated in each trial (confluence)

the source of the fibroblast cells (tendons, if possible)

environmental/lab conditions

10μg/ml concentration of IGF-1
Control – collagen production of untreated fibroblast cells; collagen production of fibroblasts
treated with vitamin C
Repeated Trials – each treatment will be tested (three?) times; partners in Singapore will also
treat insect cells with IGF-1, SuperCissus Rx™, and a combination of both treatments
Materials

Fibroblasts (from http://ccr.coriell.org)
o
o
o
o
These are human skin fibroblasts
Catalog ID: GM20944
$85.00
Used by Kim et al. (2000)

25cm Plastic tissue culture flasks

Dulbecco’s Modified Eagle’s Medium (from AOS)

Insulin-like Growth Factor-1 human (aka. somatomedin C)
o 50μg for $170.80
o From Sigma (p. 1090)

Cissus quadrangularis
o Seeds / potted plants
o Several containers
o Something for the vine to climb (?)

Biosafety cabinet and associated materials

CO2 incubator

Micropipettes and pipette tips

Vortex mixer (?)

Centrifuge machine

Spectrophotometer

Masson’s trichome stain materials
o
o
o
o
o
o
Bouin’s solution
Biebrich-scarlet-acid-fuchsin
Phosphomolybdic/phosphotungstic acid
Aniline blue
Acetic acid
Quality control (?)

Digital imaging microscope linked to ImageJ

Sircol Assay Kit
o
o
o
o
o
Dye reagent
Alkali reagent
Salt soluble collagen precipitating reagent
Collagen standard (Concentration 1mg/ml)
Capped 1.5ml capacity micro centrifuge tubes

OR – Human Collagen Type I ELISA assay kit

Autoclave
Procedure 1. Grow the C. quadrangularis
a. Space plants 38-45 cm apart
b. (Water timing?)
2. Extract C. quadrangularis using petroleum ether
a. Potu et al. (2008)
3. Establish the fibroblast culture
a. Transfer the fibroblasts to 25cm2 plastic culture flasks
b. Maintain in monolayer/multilayer (?) in Dulbecco’s Modified Eagle’s Medium
(DMEM) (supplemented with antibiotics?)
c. Culture in a CO2 chamber at 37 degrees C at a volume of 0.25ml/cm2
4. Routinely check cell growth using a microscope
5. Replace medium when it becomes exhausted
6. Prepare fibroblasts for treatment
a. Transfer cells to well plate with DMEM (and supplements?)
b. Incubate at 37 degrees C in a humidified atmosphere for three days to establish
the cells
7. Separately treat fibroblast cells with IGF-1, C. quadrangularis and a combination of both,
and include a control with no treatment for a total of 4 sets.
8. Stain the collagen using Masson’s trichome stain (Wulff, 2004)
a. Use Bouin’s solution as a mordant
b. Deparaffinize the cell culture and rehydrate through 100% alcohol, 95% alcohol,
and 70% alcohol (?)
c. Stain Nuclei with Weigert’s hematoxylin
d. Stain acidophilic tissue elements with Biebrich-scarlet-acid-fuchsin
e. Decolorize collagen fibers using phosphomolybdic/phosphotungstic acid
f. Stain collagen using aniline blue
g. Use 1% acetic acid to differentiate the tissue
9. Measure the resulting collagen production with the Sircol assay (or the ELISA assay?)
a. Add 1m of Sircol Dye reagent and cap to each tube and cap all of them, mixing
contents by inverting.
b. Place tubes in a mechanical shaker for 30 minutes.
c. Transfer the tuubes to a micro centrifuge and spin the tubes at >10,000 x g for a
10 minute period.
d. Carefully remove the unbound dye solution by inverting and draining the tubes.
e. Remove any remaining droplets by gently tapping the inverted tube on a paper
tissue.
f. Add 1ml of the alkali reagent to each tube.
g. Re-cap the tubes and release the bound dye into solution.
h. Wait 10 minutes for the bound dye to dissolve.
i. 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.
j. Substrate the reagent blank reading from the standard and test sample readings.
k. Plot graph standards and calculate the collagen content of the test samples.
10. Measure stained collagen using a digital imaging microscope linked to ImageJ
11. Repeat treatment and measurement (#?) more times.
12. Properly dispose of all cells and collagen
Safety: IGF-1 is a growth hormone that requires protective clothing and eyewear during use
because of its toxicity. The MSDS for IGF-1 can be found at
http://www.novozymes.com/nr/rdonlyres/017283D0-8889-4A04-BDAD-
D1459B9BF86A/0/IGF1MSDS.pdf. C. quadrangularis is nontoxic and has shown no serious
negative side effects. MSDS information for C. quadrangularis can be found at
http://www.vasuhealthcare.com/pdf/toxicity&safetyonCissusQuadrangularisBontoncapsule.pdf. The experiments will be conducted in biosafety units according to their
biosafety level. 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 sterile latex gloves will be
worn. The laboratory work will be supervised by a lab staff with training in cell culture
techniques.
References
Kim, S., Westphal, V., Srikrishna, G., Mehta, D. P., Peterson, S., Filiano, J., Karnes, P. S.,
Patterson, M. C., & Freeze, H. H. (2000). Dolichol phosphate mannose synthase (DPM1
mutations define congenital disorder of glycosylation Ie (CDG-Ie). Journal of Clinical
Investigations, 105, 191-198.
Wulff, S. (Ed.). (2004). Guide to special stains. Carpinteria, CA: Dako.
Statistics – Attach a cover sheet to your ED that answers the following questions:
1. What are you measuring and how are going to measure it?
2. What are you comparing?
3. Are you comparing with a control or is a control built into your
comparison?
4. How are you going to organize the data?
5. How many data points do you need to do an analysis?
6. What statistical test will you use and why?