Development of in vivo methods of measuring hair growth
Chu K.H.T1
Department of Physiology, Faculty of Medicine, National University of Singapore
10 Kent Ridge Road, Singapore 117546
ABSTRACT
BiolynTM Hair Serum synthesized by Lynk Biotechnologies Pte Ltd has shown great
capacity in treating alopecia in men and women, caused mainly by the hormone androgen
dihydrotestosterone. Methods of in vivo testing of hair growth need to be developed to evaluate the
various fractions present within the active plant extract included in the hair serum. Non-polar and
polar fractions were obtained by fractionation, and these were tested on male white mice of age two
weeks. Male mice were shaved, and lotions containing various fractions were applied on the mice.
Hair length was the main factor of growth considered, followed by general hair growth appearance
of the mice. Three experiments were carried out to obtain optimized results. Experimental results
show that the non-polar dark fraction was more effective than other fractions in achieving an
increased rate of hair growth, while the polar fraction showed least effectiveness.
INTRODUCTION
Androgenetic alopecia, or hair loss mediated by the presence of the androgen
dihydrotestosterone, is the most common form of alopecia in men and women (Thiedke, 2003). In
males, excessive testosterone is being converted to DHT, which attaches to the androgen receptors
in the hair follicles, causing hair thinning. Some people have a higher number of androgen receptors
in their hair follicles, and this results in greater hair loss.
To address the problems of hair loss, Lynk Biotechnologies Pte Ltd synthesized the
TM
Biolyn Hair Serum, which is a 3-in-1 formula functioning as an immune modulator, endocrine
modulator and also provides nutrients to the hair follicles. To further improve the hair serum, it is
imperative to find out which components of the active plant extract used in the preparation of the
serum is most effective. As such, methods of in vivo testing of hair growth using male white mice as
animal models need to be developed.
Hair growth is a complex concept which embraces many dimensions: duration of the hair
growth-cycle, longitudinal and diameter growth, hair density and pigment content (Barth, 1986). As
such, various techniques of growth measurement have been discovered. These include the duration
of the growth-cycle, kinetic studies of the hair root, length and diameter measurements including
autoradiography, compound measurements – the Trichogram and ‘pluckability’ (Barth, 1986).
Length and diameter measurements can be made using primitive yet effective methods such as a
1
Student
hand lens or modified microscope, or improved methods such as the capillary tube. Regular
photography of a defined area can be also used as a direct measurement of growth.
This report focuses on the experiments conducted to develop in vivo methods of testing the
rate of hair growth, using male white mice as animal models.
METHODS
Animal models were used to aid in the in vivo study of hair growth. All mice were clean
shaved before used. From the fractionation of the active plant extract, polar and non polar fractions
were obtained. The non-polar fraction was being separated into two components – The dark
component and the light component. Concentrations of fractions are taken in percentage (w/v). The
fractions collected were made into lotions to facilitate application to the shaved parts of the mice.
Each experiment was carried out for 14 – 21 days and was modified from the preceding one in order
to optimize the results obtained. In all, three experiments were carried out.
Experiment one
Two sets of 12 mice were used in the first experiment. Labeling of mice was done by ear hole
punching. For set one, enough lotion to cover the entire shaved area was applied to each mouse
according to the assigned fractions. The lotion was rubbed onto each mouse for 5 minutes, left on
for 5 minutes, and then washed off. Washing was carried out to the control mice as well, even
though no lotion was applied. This process was repeated daily.
For set two, the steps taken in step one were repeated, except that the lotion was left
overnight for 24 hours before being washed off. For both sets of mice, care was taken to ensure that
the application time each day was approximately the same, to ensure that the time lapse between
each application was consistent. At the end of 18 and 21 days, hair was plucked from each mouse,
and the average length recorded. Visual monitoring of the hair growth was also carried out on a
weekly basis.
Experiment two
Four concentrations of each fraction were used, namely 0.3%, 1%, 2% and 3%. For each
concentration, two mice were used instead of one, as in experiment one. Holes were punched in ears
as in experiment one for identification. Also, application was made to half of the shaved region, and
the other half was left untouched. The untouched region served as an internal control. The lotion
was rubbed onto the shaved region for five minutes, left on for five minutes, and then washed off.
As in the above experiment, hair was plucked from each mouse after 18 days, and the average
length recorded.
Experiment three
For this experiment, twenty-five mice were utilized. Four lotions (total, polar, non-polar dark
and non-polar light fractions) were used. The percentage of active plant extract in each lotion was
standardized to 1%. Five mice were used per lotion, and five mice were used as external controls.
The base and the test lotion were applied to the left and right flank regions respectively. A
small circle was denoted on each mouse using picric acid, and care was ensured such that the
application of the lotion was limited to only the small marked area. Centre dorsal region was the
internal control. For the external controls, only the base was applied to the right side of the mouse.
All applied lotion was rubbed on for five minutes, left on for another five minutes before being
washed off. As with the previous experiments, hair was plucked from each region after 14 days.
Hair measurements
Manual measurement of plucked hair was carried out with tweezers holding the fine hair against
a ruler, with the aid of a magnification lamp. Random samples of 10 strands of hair were measured,
and the average length was computed. Hair length was measured in millimeters.
RESULTS
Experiment one
Visual monitoring of the growth of the mouse fur on each mouse was made once every seven
days. The qualitative analysis for Set one is presented in Table 1. Observations that show positive
results are marked in bold.
FRACTION APPLIED
Polar 0.3%
Polar 1.0%
Polar 3.0%
Non-polar dark 0.3%
Non-polar dark 1.0%
Non-polar dark 3.0%
Non-polar light 0.3%
Non-polar light 1.0%
Non-polar light 3.0%
OBSERVATIONS IN COMPARISON TO CONTROL AFTER 14 DAYS
Similar to control
Less hair growth than control
Thick hair at top section, other areas have fine growth
Thick band of hair across middle section. Remaining sections have thicker
hair than control
Thick uniform growth across shaved area
Thick hair growth for most shaved regions, except for the bottom section
where there’s fine growth
Even distribution of hair growth, hair is thicker than control
Hair growth at the top section (similar to control), remaining regions are bare
Similar to control
Table 1: Observations of Set one after 14 days
Hair lengths measurements showed that the non-polar fractions have displayed positive
growth.
Experiment two
The hair measurements of mice used in experiment two showed that the hair length of the control
regions and test regions are similar. The faults in this experiment will be discussed later. As such,
the results are not taken into consideration for the final conclusions.
Experiment three
Hair was plucked only once, at the end of the entire experimental period of 14 days, in contrast to
experiment one, where hair was plucked from the mice twice. This is due to the small test region on
the mouse as described in the methodology. To prevent uneven growth after plucking the hair for
the first time, hair length is only measured once in this experiment. Hair length measurements
showed positive results for non-polar dark and total fractions.
DISCUSSION
Experiment one
This experiment has no internal controls, only three external controls. The absence of
internal controls posed a problem as each mice had their own innate rate of hair growth. Also, since
there was only one mouse per experimental fraction, there was difficulty in concluding if the results
obtained were due to the effectiveness of that particular fraction, or due to other innate factors
within the mouse such as genetic differences.
Nevertheless, from Tables 1 and 2, it can be seen clearly that the non-polar dark fraction has
more significant results than the other fractions. At this point, it is not possible to differentiate
successfully between the effectiveness of different concentrations of non-polar dark fraction. Nonpolar light fraction of 0.3% shows positive growth, but non-polar light fraction 3.0% shows
negative growth. As such, the successfulness of non-polar light fraction is not conclusive at this
point of time.
Experiment two
The main aim of experiment two was to introduce the idea of an internal control for each mouse,
and to use two mice for each fraction to reduce errors caused by innate differences in rate of hair
growth within mice. As such, no external control mice were used. However, no visible differences
were seen during the course of the entire 18 days of experiment. This was further confirmed when
the measurement of hair showed no distinct difference between the control and test regions.
It was later realized that the chemicals applied to the test region could have exerted an effect
in the surrounding area. Since the control region was just beside the test region, the rate of hair
growth in the control region was probably affected by the hair lotion applied.
Experiment three
Experiment three was carefully designed to avoid the errors in both experiment one and two.
Both internal and external controls were employed in this experiment. The test region was restricted
to only a very small area, to minimize the spreading effect of the chemicals. To prove that any
change in rate of growth of mouse hair was due to the active plant extract in the lotion, the base
ingredients of the lotion (excluding the plant extract) was applied to a very small area, further away
from the test region. The centre region of the mouse was taken to be the internal control.
REFERENCES
C. Carolyn Thiedke (2003). Alopecia in Women. American Family Physician 67