Chapter 3 Results
3.1 In Vivo Image Stacks of Human Skin
Chapter 3 Results
3.1 In Vivo Image Stacks of Human Skin
Using the harmonic generation biopsy system, a series of human skin images were
acquired in vivo starting from the stratum corneum to the upper reticular dermis. In the
image (a) to the image (e) of Fig. 2, all the layers of epidermis were clearly displayed by
the third harmonic generation signals. In the image (f), the second harmonic generation
signals showed the appearance of the papillary dermis distinctly. In the following
images, the epidermis seemed to diminish and the dermis increased their proportions of
image areas. Because the epidermis and dermis were showed by the THG and SHG
signals respectively, the interface of epidermis and dermis was clearly displayed.
Therefore, the structure of dermal papillae could be distinguished from the epidermis by
the SHG signals with fine resolution and surrounding THG signals.
3.2 In Vivo Analysis of Viable Epidermis Thickness
Although statistical significance of age group was found in ANOVA of the
thickness of viable epidermis excluding the depth of rete ridge (Fig. 7; p for age group =
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0.0107 and p for gender = 0.8920), there was no significance of age nor gender found in
the regression analysis of the thickness of viable epidermis excluding the depth of rete
ridge (Fig. 7; PexcludeRR for age = 0.3614 and PexcludeRR for gender = 0.5475). The
thicknesses excluding rete ridge for age groups 19-29, 30-59, and 60-79 years were
22.76 ± 2.73 ȝm, 27.50 ± 4.19 ȝm, and 24.76 ± 5.14 ȝm. No trend of decrease or
increase with age could be observed in Fig. 7. Therefore, the thickness of viable
epidermis excluding the depth of rete ridge did not increase or decrease with age. The
average and inter-subject standard deviation of the thickness excluding rete ridge from
all the subjects was 25.23 ȝm and 4.49 ȝm, and the coefficient of variance was 0.1781.
Statistical significance was not found in ANOVA of viable epidermis thickness
including the depth of rete ridge (Fig. 8; p for age group = 0.1592 and p for gender =
0.4282). However, the result showed significance of age in the regression analysis of
viable epidermis thickness including the depth of rete ridge (Fig. 8; PincludeRR for age =
0.0487 and PincludeRR for gender = 0.3396). A small and negative correlation was found
between age and the thickness of viable epidermis including the depth of rete ridge (R =
-0.293). The thicknesses including rete ridge for age groups 19-29, 30-59, and 60-79
years were 91.91 ± 17.56 ȝm, 83.40 ± 15.97 ȝm, and 79.10 ± 17.71 ȝm. The thicknesses
including rete ridge seemed to decrease with age, but differences between subjects were
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larger than the age-related decreases.
Both results together implied that not the thickness of viable epidermis but the depth
of rete ridge decreased with age. This implication would be confirmed later in the analysis
of the depth of dermal papilla zone, which was the same as the depth of rete ridge.
Fig. 7. Average thickness of viable epidermis (excluding rete ridge) versus age from the ventral forearms of 47 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 0.3614 (not statistically significant); P-value for gender = 0.5475 (not statistically significant). In ANOVA, p-value for age group = 0.0107 (statistically significant); p-value for gender = 0.8920 (not statistically significant).
Fig. 8. Average thickness of viable epidermis (including rete ridge) versus age from the ventral forearms of 47 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 0.0487 (statistically significant); P-value for gender = 0.3396 (not statistically significant). Correlation coefficient R to age = -0.293. In ANOVA, p-value for age group = 0.1592 (not statistically significant); p-value for gender = 0.4282 (not statistically significant). NS: not significant.
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3.3 In Vivo Analysis of Isolated Dermal Papilla
3.3.1 Height of Isolated Dermal Papilla
ANOVA showed statistical significances of age group for the three height
parameters, average, intra-subject standard deviation, and maximum of height,
respectively (Fig. 9-11; for age group, pave= 0.0003, pSD = 0.0051, and pmax = 0.0021),
but no significances of gender were found (Fig. 9-11; for gender, pave = 0.0975, pSD =
0.7703, and pmax = 0.5943). Regression analysis also showed significances between age
and the three height parameters, average, intra-subject standard deviation, and maximum
of height (Fig. 9-11; for age, Pave = 0.0003, PSD = 8.315ǘ10-5, and Pmax = 5.077ǘ10-5),
respectively, but gender only showed significances in the regression analysis of the
average height (for gender, Pave = 0.0285, PSD = 0.4806, and Pmax = 0.3165). The
intra-subject SD and the maximum of isolated dermal papilla height were negatively and
moderately correlated only to age (RSD = -0.540; Rmax = -0.551). The average of isolated
dermal papilla height was negatively and moderately correlated to both age and gender
(Rave = -0.563). The overall average for male subjects of the average height (18.91 ȝm)
was higher than that for female subjects (15.54 ȝm). The average heights for age groups
19-29, 30-59, and 60-79 years were 21.90 ± 5.98 ȝm, 15.01 ± 4.85 ȝm, and 14.25 ± 4.11
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ȝm. For respective age groups of females and males, the average heights for female age
groups 19-29, 30-59, and 60-79 years were 19.02 ± 4.77 ȝm, 14.97 ± 4.71 ȝm, and
13.40 ± 3.88 ȝm, and the average heights for male age groups 19-29, 30-59, and 60-79
years were 24.41 ± 6.06 ȝm, 15.10 ± 5.61 ȝm, and 15.38 ± 4.47 ȝm. The intra-subject
SDs of height for age groups 19-29, 30-59, and 60-79 years were 11.76 ± 4.37 ȝm, 8.10
± 5.14 ȝm, and 6.21 ± 2.69 ȝm. The maximum heights for subjects aged 19-29, 30-59,
and 60-79 years were 40.14 ± 11.30 ȝm, 28.69 ± 13.78 ȝm, and 23.98 ± 8.24 ȝm.
Fig. 9. Average of isolated dermal papilla height versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 0.0003 (statistically significant);
P-value for gender = 0.0285 (statistically significant). Correlation coefficient R to age and gender = -0.563.
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In ANOVA, p-value for age group = 0.0003 (statistically significant); p-value for gender = 0.0975 (not statistically significant). In two lower figures, data of female and male subjects were separately displayed.
Fig. 10. Intra-subject standard deviation of isolated dermal papilla height versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 8.315×10-5 (statistically significant); P-value for gender = 0.4806 (not statistically significant). Correlation coefficient R to age = -0.540. In ANOVA, p-value for age group = 0.0051 (statistically significant);
p-value for gender = 0.7703 (not statistically significant).
Fig. 11. Maximum of isolated dermal papilla height versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 5.077×10-5 (statistically significant); P-value for gender = 0.3165 (not statistically significant). Correlation coefficient R to age = -0.551. In ANOVA, p-value for age group = 0.0021 (statistically significant); p-value for gender = 0.5943 (not statistically significant).
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3.3.2 Volume of Isolated Dermal Papilla
ANOVA showed statistical significances of age group for the three volume
parameters, average, intra-subject standard deviation, and maximum of volume,
respectively (Fig. 12-14; for age group, pave = 0.0128, pSD = 0.0192, and pmax = 0.0067),
but no significances of gender were found (for gender, pave = 0.1604, pSD = 0.7484, and
pmax = 0.4859). There were statistical significances in the respective regression analyses
of age and the three volume parameters, average, intra-subject standard deviation, and
maximum of volume (Fig. 12-14; for age, Pave = 0.0037, PSD = 0.0006, and Pmax =
0.0005). The average, intra-subject SD, and maximum of isolated dermal papilla volume
were negatively related to age (Rave = -0.405; RSD = -0.482; Rmax = -0.482). In addition,
we found that none of them were correlated to gender with statistical significances (for
gender, Pave = 0.0930, PSD = 0.5912, and Pmax = 0.3244). The average volumes for age
groups 19-29, 30-59, and 60-79 years were 45667 ± 27010 ȝm3, 27147 ± 20758 ȝm3,
and 24291 ± 10794 ȝm3. The intra-subject SDs of volume for age groups 19-29, 30-59,
and 60-79 years were 49776 ± 28917 ȝm3, 33155 ± 30822 ȝm3, and 21219 ± 9538 ȝm3.
The maximum volumes for age groups 19-29, 30-59, and 60-79 years were 135624 ±
80970 ȝm3, 83118 ± 68373 ȝm3, and 58694 ± 23695 ȝm3.
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Fig. 12. Average of isolated dermal papilla volume versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 0.0037 (statistically significant); P-value for gender = 0.0930 (not statistically significant). Correlation coefficient R to age = -0.405. In ANOVA, p-value for age group = 0.0128 (statistically significant); p-value for gender = 0.1604 (not statistically significant).
Fig. 13. Intra-subject SD of isolated dermal papilla volume versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 0.0006 (statistically significant); P-value for gender = 0.5912 (not statistically significant). Correlation coefficient R to age = -0.482. In ANOVA, p-value for age group = 0.0192 (statistically significant); p-value for gender = 0.7484 (not statistically significant).
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Fig. 14. Maximum of isolated dermal papilla volume versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 0.0005 (statistically significant); P-value for gender = 0.3244 (not statistically significant). Correlation coefficient R to age = -0.482. In ANOVA, p-value for age group = 0.0067 (statistically significant); p-value for gender = 0.4859 (not statistically significant).
3.4 In Vivo Analysis of Dermal Papillae within Dermal Papilla Zone
3.4.1 Depth of Dermal Papilla Zone
ANOVA of the depth of dermal papilla zone (TDPZ) only showed the statistical
significance of age group (Fig. 15; p for age group = 0.0095 and p for gender = 0.2017).
The regression analysis was also statistically significant for age but not for gender (Fig.
15; P for age = 0.0009 and P for gender = 0.0989). The correlation was negative (R =
-0.457) for the depth of dermal papilla zone to age. The depths for age groups 19-29,
30-59, and 60-79 years were 74.22 ± 13.25 ȝm, 60.63 ± 16.32 ȝm, and 57.20 ± 14.29
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ȝm. The result of thinner dermal papilla zone in the aged skin indicated that the
dermal-epidermal junction flattened with aging.
Fig. 15. Depth of dermal papilla zone versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 0.0009 (statistically significant);
P-value for gender = 0.0989 (not statistically significant). Correlation coefficient R to age = -0.457. In ANOVA, p-value for age group = 0.0095 (statistically significant); p-value for gender = 0.2017 (not statistically significant).
3.4.2 Number Density of Dermal Papillae
No statistical significance of age group nor gender was found in ANOVA of the
number density of dermal papillae (DN; Fig. 16; p for age group = 0.8096 and p for
gender = 0.3678). Similarly, the regression analysis showed no statistical significance
for the number density of dermal papillae to age or gender (Fig. 16; P for age = 0.9503
and P for gender = 0.4749). Dermal papillae were not found to be more or fewer with
increasing age. The average and inter-subject standard deviation of number density from
all the subjects were 148.65 mm-2 and 52.06 mm-2, and the coefficient of variance was
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0.3502. The number densities for age groups 19-29, 30-59, and 60-79 years were 149.88
± 55.07 mm-2, 149.54 ± 49.58 mm-2, and 146.14 ± 55.85 mm-2, which were quite similar
for different age groups. In Fig. 16, no tendency of age-related changes could be
observed, and data were scattered in all age groups.
Fig. 16. Number density of dermal papillae versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 0.9503 (not statistically significant);
P-value for gender = 0.4749 (not statistically significant). In ANOVA, p-value for age group = 0.8096 (not statistically significant); p-value for gender = 0.3678 (not statistically significant). NS: not significant.
3.4.3 Dermal Papillae Volume per Unit Area
ANOVA of dermal papillae volume per unit area (DV) did not show statistical
significances of age group and gender (Fig. 17; p for age group = 0.4008 and p for
gender = 0.1561). There were no statistical significances in the regression analysis of
dermal papillae volume per unit area to age and gender (Fig. 17; P for age = 0.1471 and
P for gender = 0.1287). Therefore, the volume of dermal papillae within dermal papilla
zone per unit area did not increase or decrease with age. The volumes per unit area for
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age groups 19-29, 30-59, and 60-79 years were 39.09 ± 8.12 ȝm, 34.55 ± 10.76 ȝm, and
34.55 ± 12.04 ȝm. The average and inter-subject standard deviation of dermal papillae
volume per unit area from all the subjects were 35.97 ȝm and 10.42 ȝm, and the
coefficient of variance was 0.2897. In Fig. 17, no tendency of increasing or decreasing
with age could be observed, and data were scattered especially in elder groups.
Fig. 17. Dermal papillae volume per unit area versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 0.1471 (not statistically significant); P-value for gender = 0.1287 (not statistically significant). In ANOVA, p-value for age group = 0.4008 (not statistically significant); p-value for gender = 0.1561 (not statistically significant). NS: not significant.
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3.4.4 Average Volume per Dermal Papilla
In ANOVA of the average volume per dermal papilla (VDP), effects of age group
and gender were not statistically significant (Fig. 18; p for age group = 0.3120 and p for
gender = 0.0920). In the same way, no statistical significances of age and gender were
found in the regression analysis (Fig. 18; P for age = 0.5962 and P for gender = 0.1276).
The average volumes per dermal papilla for age groups 19-29, 30-59, and 60-79 years
were 282387 ± 80801 ȝm3, 248257 ± 100742 ȝm3, and 273554 ± 129590 ȝm3. The
average and inter-subject standard deviation of average volume per dermal papilla from
all the subjects were 266301 ȝm3 and 103472 ȝm3, and the coefficient of variance was
0.3886. Therefore, if the connected part of a dermal papilla was included in the volume
measurement, the average volume per dermal papilla did not decrease with age, which
was different from the decreasing volume with age in the analysis of isolated dermal
papilla.
It could be noticed that the average volume per dermal papilla (Vtotal / Ntotal) was
the dermal papillae volume per unit area (Vtotal / OAmax) divided by the number density
of dermal papillae (Ntotal / OAmax). The dermal papillae volume per unit area and the
number density of dermal papillae were not change with age, so it was reasonable that
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the average volume per dermal papilla was not found related to age.
Fig. 18. Average volume per dermal papilla versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 0.5962 (not statistically significant); P-value for gender = 0.1276 (not statistically significant). In ANOVA, p-value for age group = 0.3120 (not statistically significant); p-value for gender = 0.0920 (not statistically significant). NS: not significant.
3.4.5 Dermal Papillae Volume Ratio within Dermal Papilla Zone
ANOVA of the dermal papillae volume ratio within dermal papilla zone (RV)
showed statistical significance of age group (Fig. 19; p for age group = 0.0423 and p for
gender = 0.2307). We also found that the dermal papillae volume ratio within dermal
papilla zone was positively correlated to age with the significance (Fig. 19; P for age =
0.0143 and P for gender = 0.3796; R = 0.352). It meant that dermal papillae occupied
more proportion of volume in the dermal papilla zone as age increased. The volume
ratios for age groups 19-29, 30-59, and 60-79 years were 52.46 ± 4.26 %, 56.26 ± 5.49
%, and 59.18 ± 11.34 %.
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It could be noticed that the dermal papillae volume ratio within dermal papilla zone
(Vtotal / (OAmax × TDPZ)) was the dermal papillae volume per unit area (Vtotal / OAmax)
divided by the depth of dermal papilla zone (TDPZ). Previously, it was found that the
dermal papillae volume per unit area was not related to age but the depth of dermal
papilla zone decreased with increasing age, so the increase with age of the dermal
papillae volume ratio within dermal papilla zone was quite reasonable.
Fig. 19. Dermal papillae volume ratio within dermal papilla zone versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 0.0143 (statistically significant); P-value for gender = 0.3796 (not statistically significant). Correlation coefficient R to age = 0.352. In ANOVA, p-value for age group = 0.0423 (statistically significant); p-value for gender
= 0.2307 (not statistically significant).
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3.4.6 3D Interdigitation Index
The statistical significance of age group was found in ANOVA of the 3D
interdigitation index (I3D; Fig. 20; p for age group = 0.0013 and p for gender = 0.9041).
Similarly, it was showed that the correlation between the 3D interdigitation index and
age was negative and moderate with statistical significance (Fig. 20; P for age =
2.035×10-5 and P for gender = 0.6453; R = -0.578). The indexes for age groups 19-29,
30-59, and 60-79 years were 2.734 ± 0.378, 2.389 ± 0.554, and 2.089 ± 0.291. The
interface area of dermal papillae became smaller with advancing age, which revealed
the undulation of dermal-epidermal junction was smaller in the older skin.
Fig. 20. 3D interdigitation index versus age from the ventral forearms of 48 Asian subjects (skin phototype III & IV). In linear regression analysis, P-value for age = 2.035 10-5 (statistically significant); P-value for gender = 0.6453 (not statistically significant). Correlation coefficient R to age = -0.578. In ANOVA, p-value for age group = 0.0013 (statistically significant); p-value for gender = 0.9041 (not statistically significant).
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