Bioactive Collagen Peptides, Skin
Wrinkles and Dermal Matrix Synthesis
Skin Pharmacol Physiol 2014;27:113–119
tion with corticoids and/or antihistamines within 4 weeks prior
to thestart, medication with an anticoagulant, proneness to hy-
perpigmentation or hypertrophic scar formation, other system-
ic medication within 4 weeks prior to the start, systemic illness
of the subject at the beginning of the study, pregnancy or a pe-
riod of breast feeding, immunological disorders, severe disor-
ders within 6 months prior to the start, e.g. cancer, acute car-
diac and circulatory disorders, severe diabetes, alcohol and drug
abuse, participation in other studies with cosmetic products in
the test areas within 2 weeks prior to the start or during the
study, participation in a study with a pharmaceutical prepara-
tion within 4weeks prior to the start, intake of nutritional sup-
plements within 4 weeks prior to the start and, except for the
test products administered during the study, change in lifestyle
or eating habits during the study, treatment with leave-on prod-
ucts, oily or moisturizing skin-cleansing products on the arms,
or a change in the usual skin care routine, exposure to intensive
sunlight or artificial UV (solarium) light on the test sites within
1 week prior to the start or during the study, swimming, sauna
or intensive sport within 1 day prior to measurements, lack of
compliance, intellectual or mental inability to follow study in-
A s s e s s m e n t s
The test sites were the wrinkle area around the left eye (lateral
canthus) for eye wrinkle measurements and the inner aspect of the
right forearm for the suction blister biopsies used for the subse-
quent analysis of procollagen type I, elastin and fibrillin.
On every measurement day, the subjects had to expose their
uncovered test sites to the indoor climatic conditions (21.5
° C; 50%
relative humidity) for at least 30 min.
There were 4 measurement times for eye wrinkle assessments:
immediately before starting the product treatment (t
0 ), after 4 (t
and 8 weeks (t
2 ) of daily product intake, and 4 weeks after the last
3 , 4-week regression phase).
Suction blisters were generated before starting the treatment
0 ) and after 8 weeks (t
2 ) of intake.
In each case the subject’s compliance (dosage and way of in-
take) and tolerance towards the products were checked after 1, 4
and again after 6 weeks of intake.
In vivo Measurement of the Eye Wrinkle Volume
The influence of BCP on the eye wrinkle volume was measured
at the outer corner of the eye (lateral canthus) using the optical
3-dimensional in vivo measuring instrument Primos
(GF-Messtechnik GmbH). Three measurements were conducted
per test site. The size of the measurement area was 30 × 40 mm
The postbaseline measurements were performed using the overlay
function. For each subject, the original pictures of the reference
files at the baseline and the corresponding measurement files of the
postbaseline measurements were brought into congruence using
the 3-dimensional matching function. Height images were com-
puted according to the standard procedure using mathematical
filters. These height images were used to calculate the eye wrinkle
volumes (in cubic millimeters of one selected wrinkle). After man-
ual marking of the selected eye wrinkles, the volume was comput-
ed by the Primos software. This was performed for all 3 images that
were taken per test site and measurement point in time. Then, the
mean of the 3 single measurements was calculated for each test site
and point in time.
Suction Blister Biopsies
To quantify the amount of procollagen I, elastin and fibrillin in
the skin of the volunteers before and after treatment with BCP, the
so-called suction blister model was applied, according to Kiistala
 . Per point in time, 2 suction blisters of 7 mm in diameter were
generated in the test area. To induce the blisters, Plexiglas suction
chambers with 2 circular openings each with a diameter of 7 mm
were placed on the test site. A vacuum pressure of about 450–850
mbar was applied. The blisters were induced within 1.5–2.5 h. The
liquids of all suction blisters were collected using sterile hypoder-
mic syringes. The liquids were pooled, collected in cryovials at
° C and stored frozen at –80 ° C immediately after preparation.
The small wounds were covered with a wound occlusive and
healed completely and scar free within 6–10 days. On completion
of the study, the blister fluids were used for the quantitative analy-
sis of procollagen type I, elastin and fibrillin.
Enzyme-Linked Immunosorbent Assays for the Analysis of
Procollagen Type I, Elastin and Fibrillin
In vitro enzyme immunoassay kits were used for the quantita-
tive analysis of human procollagen type I (Takara Bio Inc., Japan),
human elastin (Cusabio Biotech, China) and human fibrillin-1
(Cusabio Biotech) in the suction blister fluids.
The tests were performed according to the respective instruc-
tion manual. Briefly, samples were diluted 1:
25 vol/vol with kit
sample buffers to reach concentrations within the range of the
standards. Every suction blister sample was measured in duplicate.
Moreover, the recovery of the enzyme immunoassay kits was ana-
lyzed, revealing recovery rates of more than 80% on average.
All data were tested for normal distribution by the 1-sample
Kolmogorov-Smirnov test. Statistically significant differences be-
tween both treatment groups were analyzed by the 2-sided inde-
pendent samples t test. The hypothesis of a normal distribution
was accepted when there was a p value >0.05. As for the differ-
ences between the treatment situations, for a p value <0.05 the dif-
ference was accepted as statistically significant.
The following treatment situations for wrinkle volume changes
were compared: treatment situations at points in time t
1 and t
2 (after 4 weeks and 8 weeks of treatment) and t
(4-week washout phase).
The results obtained from the blister suction fluids were ex-
pressed in relation to the initial situation and pairwise differences
were tested for statistical significance between both treatments.
R e s u l t s
Subjects and Dropouts
The results of 114 subjects were involved in data anal-
ysis. The subjects were between 45.0 and 65.4 years old
(55.6 ± 6.0), without statistically significant differences
between the treatment group and the placebo group
Verlag S. KARGER AG BASEL
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