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Title: Regional Variations in the Histology of Porcine Skin

Neill J. Turner PhD1,2, Dominic Pezzone1, Stephen F. Badylak DVM PhD MD1,2, 3

1. McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh PA 15219


2. Department of Surgery, University of Pittsburgh, Pittsburgh PA, 15232
3. Department of Bioengineering, University of Pittsburgh, Pittsburgh PA, 15232

*Corresponding Author:
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Stephen F. Badylak, DVM, PhD, MD


Professor of Research
Department of Surgery
University of Pittsburgh
450 Technology Drive, Suite 300
Tissue Engineering Part C: Methods

Pittsburgh, PA 15219-3130
Phone (412) 624-5253
Fax (412) 624-5250
badylaks@upmc.edu

Running Title: Histology of porcine skin

Keywords: Extracellular Matrix, Skin, Histology, Scaffold


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Abstract

Porcine skin is commonly used as a model for human skin injury and as a source material for
biologic scaffold materials. Although remarkable similarities between porcine and human skin exist,
regional anatomic variations present in human skin are also present in porcine skin. The objective of the
present study was to evaluate the structure of porcine skin from 11 different anatomic regions in the
American Yorkshire Hamroc breed. Both qualitative and quantitative methods were used, with emphasis
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

upon epidermal and dermal thickness, hair follicle density and collagen and elastin composition and
distribution.

The results showed that significant regional differences in skin histology exist particularly with
respect to the thickness of the dermis and epidermis and the amount of collagen and elastin within each
Tissue Engineering Part C: Methods

tissue. Differences were also seen in the distribution of type I and type III collagen within the dermis.
Therefore while porcine skin shares many similarities with human skin, distinct regional differences in
composition and morphology exist. This study highlights the importance of appreciating these regional
differences to avoid misinterpretation of experimental results when using porcine skin as a human
analogue.
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Introduction

The skin is a complex organ serving important roles in thermoregulation and as a mechanical
and immunological barrier to disease. The structure and composition of human skin is not uniform
across the body and significant regional differences in thickness, presence of hair follicles and collagen
composition have been described (1-4). These differences are particularly important in relation to skin
grafts for which factors such as pigmentation, thickness and hair density become important
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

considerations for a successful cosmetic appearance (5-7).

Porcine skin is perhaps the most commonly used animal tissue in biomedical research and
regenerative medicine as a model for research and as a source material for biologic scaffold materials
(8-20). Previous studies have shown remarkable similarities between porcine and human skin with
Tissue Engineering Part C: Methods

regard to properties such as percutaneous absorption (21-23). However, it is important to realize that
the same regional variations present in human skin are also present in porcine skin. Significant
differences between the two species exist and careful consideration must be made in study design to
ensure the porcine skin model is an accurate representation of the equivalent human skin injury. While
the regional variations in human skin have been extensively evaluated (1-4, 22, 24-36), there is a paucity
of data on the regional variations in porcine skin structure and composition.

Since the introduction of dermal matrix in 1994, there has been a continual increase in the
surgical use of acellular tissue matrices as test materials and medical devices. Dermal matrices, derived
from allogeneic and xenogenic sources, are now commonly used for the treatment of extremity wounds,
such as diabetic or vascular ulcers or pressure wounds; the treatment of ventral hernias; and surgical
reconstruction of extremity trauma (14, 37-44). The composition of the dermis and the retention of
elastin and collagen fibers after decellularization impart mechanical and viscoelastic properties that
allow these dermal products to provide reinforcement to the surgical site, while promoting cellular
ingrowth and revascularization as the materials gradually degrade. While acellular dermal matrices have
shown notable success, a number of complications and failures have been reported(40, 45, 46). An
appreciation of the inherent regional variations in dermal thickness across the body may contribute to
improved practices in the preparation of acellular dermal matrices (ADM) and minimize some of the
variability found in these materials.
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To improve the reproducibility of animal experiments and minimize the variability in acellular
dermal products a better understanding of select factors in porcine skin and their variation with respect
to anatomic location is essential. For example, hair follicle density and epidermal thickness are
important mediators of percutaneous absorption and wound healing(21, 22, 24, 47-49); epidermal and
dermal thickness are important for the development of porcine burn models(12, 14); and collagen and
elastin content and distribution influence wound healing(6, 7, 50), scarring and the production of ADM
(51). Thus, the objective of the present study was to characterize the regional variations in porcine skin
from 11 different anatomic areas by both qualitative and quantitative methods, concentrating on
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

epidermal and dermal thickness, hair follicle density and collagen and elastin composition and
distribution.

Materials and methods

Preparation of porcine skin samples


Tissue Engineering Part C: Methods

Skin samples were obtained from six female domestic pigs, Sus scrofa, of standard American Yorkshire
Hamroc stock that had been euthanized as part of separate study, not involving the skin. The animals
were approx. 3-4 months of age, and weighed 120lbs ± 10lbs. Full thickness biopsies of skin,
approximately 5.0 x 8.0 cm were removed from 10 locations: anterior neck, inner ear, outer ear, rostral-,
mid- and caudal-belly, rostral-, mid- and caudal-back, lateral fore leg and lateral hind leg (Figure 1). Each
skin biopsy was pinned to a cork mounting board and stored frozen at -80oC until processed for
histomorphologic assessment.

Quantification of hair follicle density

Two 8.0 mm punch biopsies were obtained from each skin sample to assess hair follicle density. The
biopsies were taken from random areas across each skin sample. The biopsies were the viewed under a
stereo dissecting microscope (Nikon Instruments, Melville, NY) and the number of individual hair
follicles per biopsy recorded. Hair follicle density was then calculated as number of hair follicle per cm2.

Morphometric analysis

Morphometric analysis was performed on paraffin embedded tissue sections stained with
hematoxylin and eosin, Masson’s Trichrome, Verhoeff’s vanGieson and Herovici’s polychrome stains.
Briefly, the frozen skin samples were thawed at 4oC overnight and two 2.0 cm x 1.0 cm full thickness
biopsies were cut from random areas of the skin sample. For each skin sample there was at least 1 cm of
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tissue separating the two biopsy sites to ensure that the biopsies were representative of the entire skin
sample. The biopsies were pinned to a cork mount and fixed in 10% neutral buffered formalin for 48hrs
before being processed for routine paraffin embedding. Following embedding, 5um thick sections were
cut and stained with either hematoxylin and eosin, Masson’s Trichrome, Verhoeff’s vanGieson and
Herovici’s polychrome stains.

Stained slides were viewed using an axio-observer Z1 microscope (Carl Zeiss Microscopy,
Thornwood, NY) at x100 or x200 magnification. Measurements of total skin thickness, and individual
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

measurements of the corneum, epidermis and dermis were obtained using Axiovision software (Carl
Zeiss Microscopy) with measurements calibrated to the microscope and objectives used to obtain the
images. The number of cell layers of the corneum, total number of Langerhans cells per field and total
number of melanocytes per field were also recorded in x1000 magnification in 6 representative fields of
view per specimen.
Tissue Engineering Part C: Methods

Assessment of collagen composition

Collagen composition and organization, was assessed using slides stained with Herovici’s
polychrome and focused on collagen types I and III. Herovici’s polychrome staining was performed using
a modified protocol developed in our laboratory (52), compared to that first described by Herovici
(1963)(53). Images of stained slides at x200 magnification were obtained using a Nuance FX
multispectral imaging system (Perkins Elmer, Cambridge MA) connected to a standard light microscope
(Nikon Eclipse E600, Nikon Instruments). A spectral library for Herovici’s polychrome was prepared using
control slides and comprised the unique spectra of acid fucshin (purple/red), methyl blue (blue) and
picric acid (yellow).

Following spectral unmixing, the individual color components of the image were quantified
using image analysis software built in to the Nuance FX system. Color thresholding of Herovici’s
polychrome stained samples was used to identify areas corresponding to type I and type III collagen
within the image. The resulting thresholding values were saved with the spectral library and used to
analyze all subsequent images in this study. For each skin sample a region of interest was defined by the
epidermal: dermal and dermal: subcutis borders. The percentage of image pixels corresponding to either
type I or type III collagen following spectral unmixing and color thresholding was recorded for three
fields of view per sample with each sample repeated in duplicate.

Assessment of elastin composition


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Elastin composition and organization was assessed using two methods. The first method was a
histologic assessment and quantification using Verhoeff’s vanGieson staining to label the elastin fibers
within the dermis and epidermis. The second method used a colorimetric assay (Fastin, Biocolor Life
Sciences, County Antrim, UK) to quantify the elastin content extracted from skin samples.

Verhoeff’s vanGieson staining was performed using standard histologic methods resulting in red
staining collagen and black staining elastin fibers. Images of stained slides at x200 magnification were
obtained using an axio-observer microscope and axiovision acquisition software (Carl Zeiss Microscopy).
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

The captured images were imported into Photoshop CS2 (Adobe Systems Inc, San Jose, CA) and the
replace color tool was used to remove the red collagen staining from the image to leave only the black
staining of the elastin fibers. These corrected images were then imported into ImageJ (54) where a
region of interest was defined by the epidermis and dermal: subcutis border. The image was
thresholded and the percentage of black and white pixels calculated using a macro supplied with
Tissue Engineering Part C: Methods

ImageJ. The percentage of black pixels was taken to represent the percentage of elastic tissue within the
skin section.

The colorimetric assay for elastin quantification utilized 5,10,15,20-tetraphenyl-21H,23H-


porphine dye (TPPS) to analyze solubilized elastin polypeptides extracted from the skin samples. For
each skin sample, one 8.0 mm punch biopsy was obtained and lyophilized until completely dry. The mass
of the dried tissue samples was recorded and then the tissue samples were solubilized in 750l 0.25M
Oxalic acid at 100oC for 60 minutes. The specimens were then centrifuged at 10,000 rpm for 10 minutes
and the supernatant retained for analysis. This solubilization step was repeated a further 2 times on the
residual tissue to ensure complete elastin extraction. Quantification of the solubilized elastin was
performed using the Fastin Elastin Assay (Biocolor Life Sciences) following the manufacturer’s
recommended protocol with elastin content expressed as g elastin/mg of dry weight tissue.

Statistical Analysis

For all quantitative assays, for which data passed tests for normality and equal variance, two way
ANOVA was performed to determine differences between sample sites and if any difference existed
between animals. If no difference between animals was found, a one way ANOVA with Tukey’s post hoc
test was performed to identify specific difference between body sites. Where data failed tests for
normality, a Kruskal-Wallis one way analysis of variance on ranks was performed. A value of P≤0.05 was
Tissue Engineering Part C: Methods
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)
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CA).
considered significant. All tests were performed using SigmaPlot software (Systat Software Inc, San Jose
7
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Results

A two way ANOVA showed no significant differences between animals for all statistical tests.

Histology of porcine skin

Porcine skin samples were evaluated by histologic methods on paraffin embedded sections stained with
Hematoxylin and Eosin and Masson’s Trichrome. A keratinized outer layer and epidermal and dermal
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

layers could be clearly identified in all specimens. It was not possible to discern a distinct papillary or
reticular layer within the dermis by H&E or Masson’s staining. All skin samples were absent of eccrine
glands although occasional sebaceous glands and apocrine glands were present surrounding hair
follicles. Representative images of skin structure from each body site are shown in Figure 2.

The keratinized layer of the epidermis at each body site generally consisted of between 5 and 17
Tissue Engineering Part C: Methods

cell layers with the average thickness being 18.88 m ± 2.58 m. The keratinized layer was thickest in
the hind leg and thinnest in the mid back with average thicknesses of 24.37 m and 15.41 m
respectively. However, counting the number of cell layers showed that the caudal belly skin had the
most layers with 15.93 ± 0.44 while the mid back had the smallest number of layers with 4.97 ± 1.01
(Fig. 3A). While there were significant differences in the thickness and number of cell layers of the
corneum between different body sites (see supplemental data), comparing related body sites inner vs
outer ear; belly; back; and fore vs hind leg showed no significant differences in overall thickness (Fig. 2B)
but there were differences in the number of cell layers in caudal belly vs mid and rostral belly skin; all
the back skin areas; and the inner vs outer ear. The epidermis in all samples had a clearly defined
stratum germinativum although the stratum spinosum and stratum granulosum were less distinct. Rete
ridges were not always present in the skin samples, and where present had a periodicity of
approximately 200m. There were notable changes in epidermal thickness across the different body
sites. The epidermis was thickest in the fore and hind legs, with average thicknesses of 86.06 m and
91.74 m respectively, and thinnest in the mid back (average thickness 45.82 m). Comparing related
body sites showed no differences between inner and outer ear or between the fore and hind legs. There
were differences between rostral belly and caudal belly skin, with the epidermis becoming thinner in the
caudal region. The caudal epidermis on the back was significantly thicker than the rostral back
epidermis, while the epidermis in the mid back was the thinnest of all the skin samples tested (Fig 2C).
Results of statistical comparisons between all body sites can be found in supplemental data.
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Quantification of the number of Langerhans cells, the antigen presenting cells of the epidermis,
and melanocytes, the pigment producing cells of the epidermis, showed differences in the cellular
composition of the epidermis between body sites (Fig. 3C & D). The number of Langerhans cells was
highest in the mid belly with an average of 16.57 ± 2.15 cells per field and lowest in the outer ear and
caudal back with 3.33 ± 1.01 and 3.20 ± 0.67 cells per field respectively. Comparing related body sites
showed differences in the number of Langerhans cells between the mid belly, rostral belly and caudal
belly and the inner vs outer ear. There were also differences between unrelated body sites which can be
found in supplemental data. Quantification of the number of melanocytes showed much less variability
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

between skin locations, although differences were still apparent. In general, the number of melanocytes
per field varied between 1 and 3 cells per field with the hind leg having the highest number of cells with
an average of 2.07 ± 0.43 cells per field, other body sites generally average 1 cell per field. Consequently
there were no statistical differences in melanocyte counts between different body sites or within related
body sites except between the fore and hind legs. Complete statistical analysis can be found in the
Tissue Engineering Part C: Methods

supplemental data.

The dermis showed the greatest variability in thickness between the different body sites (Fig
2D). The dermis of the inner ear was the thinnest with an average thickness of 796.33 m while the
dermis in the caudal back was the thickest averaging 1687.67 m, more than twice as thick as the
dermis of the inner ear. Comparing related body sites showed that in general the thickness of the dermis
increased rostro-caudally. While there was no difference in thickness between the inner and outer ear
or between the rostral, mid and caudal belly, the dermis increased in thickness between the rostral and
caudal back and between the fore and hind legs (Fig. 2D). Results of statistical comparisons between all
body sites can be found in supplemental data.

Despite these changes in dermal thickness, the histologic appearance of the dermis varied little
between the different body sites. In general, the dermis was composed of dense interlacing bands of
fibrous tissue (Fig 2A). These fibers appeared to give a cross-hatch appearance to the dermis with some
fibers arranged longitudinally and others transversely. The dermis was relatively acellular in comparison
to the epidermis, with individual cells dispersed throughout the dermis. Occasional clusters of cells were
visible, corresponding to hair follicles, blood vessels or apocrine glands. Blood vessels were most evident
in the upper dermis while apocrine glands were typically observed at the junction between the dermis
and subcutaneous fat.
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Analysis of hair follicle density

Porcine skin is a preferred in vitro model for the study of percutaneous absorption (11, 55). Hair
follicle density is an important modulator of absorption (56) and is therefore an important consideration
when designing percutaneous absorption experiments. In addition, the presence of hair follicles and the
stem cell population associated with them may influence the rate and extent of wound healing(47-49).
Hair follicles in the skin samples were sparsely arranged as single follicles rather than in clusters of two
or more follicles. The density of hair follicles varied greatly across the different body sites (Fig. 4). The
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

highest density of hair follicles was in the skin of the outer ear with 42 ± 11 follicles per cm2. The lowest
density was in the belly skin with the caudal belly having only 4 ± 2 follicles per cm2. There was no
significant difference in the follicle density between the different belly skin sites, or between the
different sites on the back. However, there was consistently more than double the hair follicle density
on the back versus the belly (Fig. 4). No difference was seen between the fore and hind legs. The
Tissue Engineering Part C: Methods

greatest difference was seen between the inner and outer ear. Full details of the significant differences
observed between all sampling sites can be found in the supplemental data.

Analysis of type I and type III collagen content

Collagen in porcine skin has a similar composition to human collagen (57) and elicits a minimal
immune response making it ideal for production of ADMs. The mechanical properties of the skin depend
largely on the organization of the collagen fibers within the skin, while elasticity and extensibility are
determined in part by the proportions of type I and type III collagen present. Given the use of porcine
skin as an experimental model and for production of ADMs, an understanding of the arrangement and
proportion of these fibers is important.

The collagen composition of the dermis in the different skin samples was assessed by semi-
quantitative assessment of Herovici’s polychrome staining. A representative example of Herovici stained
skin is shown in figure 5. While it was not possible to discern the papillary and reticular layers of the
dermis by H&E or Masson’s trichrome, staining with Herovici’s polychrome clearly identified two distinct
layers in the dermis based on their collagen content. Immediately below the epidermis, the dermis was
predominately composed of type III collagen with little type I collagen visible. Below this layer, type I
collagen predominately in the dermis as large diameter fibers (Fig. 5A). Using image analysis and color
thresholding(52) the dermis was isolated and the collagen composition quantified. Type III collagen
content averaged 10.10% ± 0.78% of the dermal ECM in all samples (Fig. 5B). There were no significant
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differences in type III collagen content between related body sites but differences were seen between
the lower jaw versus caudal belly, fore leg and hind leg, and between the hind leg versus inner ear, outer
ear, rostral back and mid belly. Greater variation was seen in in the type I collagen content which ranged
from 23.46 % in the rostral belly to 29.05% in the mid back (Fig. 5B). Expressing these values as a ratio
showed that porcine skin typically has a collagen I:III ratio of approx. 2.5:1. While significant differences
in type I collagen content were seen between multiple body sites (see supplementary data), comparing
related body sites show no difference in type I collagen content between inner and outer ear, fore and
hind leg or between belly skin samples. A difference was seen between rostral back and mid back skin.
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Analysis of elastin content

Elastin provides recoil to the skin allowing it to recover when pinched or compressed. The
elastin fibers provide an important signaling network and may play a role in wound healing and repair,
particularly mediating the extent of scar tissue formation (58, 59). Porcine tissue is known to have a high
Tissue Engineering Part C: Methods

elastin content (60) and it has been suggested that the elastin content in ADMs may be important for
improved elastin regeneration following burn injury.

Elastin content in the different skin samples was assessed by two methods. First skin samples
were stained with Verhoeff’s van Gieson to visualize the elastic fibres (Fig. 6A). This staining was then
quantified by image analysis (Fig. 6B). Second, biopsy samples from the skin sites were digested in oxalic
acid and the elastin isolated and quantified using a biochemical assay (Fig 6C). Comparison of the semi-
quantitative image analysis and quantitative elastin assay showed a similar pattern of elastin content,
although some differences in quantification were seen. The greatest difference between the two assays
was in the ear skin which had the lowest percentage elastin by image analysis, but the highest elastin
content when quantified biochemically.

In general, the elastin content of the skin samples was low, ranging between 4.24% and 9.8% of
the dermis by image analysis or 2.45 and 11.30 g/mg by elastin assay. Staining with Verhoeff’s van
Gieson stain showed that the elastin fibers were found in the highest density in the upper dermis
appearing as long thin diameter fibres (Fig. 6A). Image analysis showed elastin content was highest in
the caudal belly with elastin content decreasing rostrally. There was no difference in elastin content in
skin samples from the back or between the fore and hind legs or inner and outer ear.
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Quantitative assessment using the elastin assay appeared more sensitive and detected greater
differences between the body sites. Elastin content was highest in the outer ear compared to all other
body sites. The caudal belly skin also contained high levels of elastin and was higher than rostral or mid
belly skin, which themselves were not different from each other. Differences in elastin content were
also seen between the fore and hind legs and the rostral, mid and caudal back.

Discussion

The histology of porcine skin from 11 different anatomic sites was studied. While other studies
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

have investigated the characteristics of porcine skin, these studies have focused on specific aspects such
as the enzymatic properties and collagen fiber alignment or have been limited to analysis of skin from a
specific location (9, 11, 34, 61-65). The present study shows that significant regional differences in skin
histology exist between various body sites and suggests that consideration regarding anatomic location
should be made when selecting porcine skin as an analogue for human skin either for in vivo studies or
Tissue Engineering Part C: Methods

in the development of biologic scaffolds derived from decellularized dermis.

The pig has been utilized in numerous studies involving wound healing, the study of burns and
scalds and scar tissue formation (12, 14, 50, 66, 67). In addition, porcine skin, and porcine dermis in
particular, have been used in the production of biologic scaffolds and used as surgical meshes for hernia
repair and in the treatment of burn injuries, although their success has been mixed (16-18, 37, 41, 43,
46, 68). The use of porcine skin as an experimental model or as a source of biologic scaffolds has
resulted from investigations into the properties of skin of domestic animals which showed that on all the
animals studied, the pig offered the most appropriate model (34). However, it has been shown that
regional differences exist in the composition and morphology of human skin from different body sites
and it is therefore likely that similar variations exist in porcine skin. Determining the appropriateness of
porcine skin as an analogue for human skin should logically factor in these regional differences and any
experimental model should match the morphology and composition of the likely human application site.
As such, a comparison or the results of this study to the properties of human skin is warranted.

The average hair follicle density in the porcine skin samples ranged from approx. 4 hairs/cm2 in
the caudal belly to 42 hairs/cm2 in the outer ear. In contrast, human skin, with the exception of the
head, typically ranges between 14 and 32 hairs/cm2 (28). While this is similar to the hair follicle density
found in the porcine skin, the greater variability on the pig skin samples suggests that notable site to site
variation may exist. The average thickness of the keratinized layer of porcine skin in the present study
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was approx. 18.88 m ranging between 15.41 m and 24.37 m and consisted of between 5 and 17 cell
layers. In comparison, while the keratinized layer of human skin typically ranges between 6 and 19 m
depending on body site and shows a strong similarity to the porcine skin (1, 29, 31), the human corneum
has been shown to contain between 6 and 30 cell layers depending on region(1, 31) with the skin of the
genitals, face and neck having the least number of layers, while areas such as the palms and heels may
have between 40 and 90 layers (31). Comparing the viable epidermis thickness shows some similarities
to human skin but also notable differences. Reported thicknesses of human epidermis vary by body site
with reported thicknesses of 70 m and 82 m in the shoulder and buttock respectively (29) and
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

generally between 50 and 120m (34). Comparing similar body sites to those used in the present study,
human adult (23-53 years) epidermal thickness in the neck, abdomen, back, anterior arm and anterior
leg are reported as 130 m, 179.3 m, 97 m, 113 m and 143.9 m respectively (25). In comparison,
the same measurements in the porcine skin samples were, 51.7 mm, 64.9 mm, 45.8 mm, 86.1 mm and
91.7 mm for the lower jaw, mid belly, mid back, fore and hind legs respectively, considerably thinner
Tissue Engineering Part C: Methods

than the comparative human location. The epidermal thickness in pigs has been reported by others to
be between 30 and 140 m (34, 69). Langerhans cells are important immune mediator cells of the
epidermis. In general they are thought to comprise 3% of the epidermal cells, but the results of the
present study show distinct variations in the distribution of these cells by body location(70). The highest
concentration of Langerhans cells was found in the mid belly skin and inner ear and these differences in
distribution may indicate the relative pathogenic insult each region is exposed to (3, 4, 71). Melanocytes
provide pigmentation to the skin preventing UV light damage to the hypodermis. The American Yorksire
Hamroc breed of pigs used in this study are a large white pig strain with little skin pigmentation, as a
result the numbers of melanocytes within the pig skin was low with only 1 to 3 melanocytes per field. In
humans melanocytes make up between 5 and 10% of the basal epithelial cells with around 1000 to 2000
melanocytes per square millimeter of skin(24, 72, 73). The highest concentration of melanocytes in the
pig skin samples was found in the hind leg and this may be related to the hamroc breeding stock which
has a characteristic black head and hindquarters.

Similar regional differences also exist in dermal thickness. In humans, the dermis typically ranges
from around 1.9mm in the back to approx. 1mm in other body regions (25, 30). The porcine skin samples
in the present study had a dermal thickness ranging from approx. 1.7mm in the caudal back to 0.7mm
in the outer ear showing great similarity to the regional patterning in humans, although they remained
thinner than the equivalent human site. One possible explanation for the differences in epidermal and
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dermal thickness may be related to age of the sample population. The skin samples used in this study all
came from pigs between 3 and 4 months of age, the typical age for use for in vivo studies. Previous
studies have shown that, in terms of organ maturity, pigs at 4 months of age are analogous to humans in
their mid-teens (74, 75). Therefore, the skin samples in this study may not have reached full maturity
and would likely be thinner than adult skin. In humans, epidermal and dermal thickness has been shown
to vary significantly as a consequence of age in multiple body sites (25, 29). In addition, the present
study investigated only one strain of pig, American Yorkshire Hamroc, and the regional variations in skin
morphology in this strain may differ considerably from other pig breeds commonly used for in vivo
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

studies.

Together, the results on porcine skin thickness and hair follicle density provide important
information for studies such as percutaneous absorption where regional differences in skin morphology
may influence absorption rates. Previous studies have shown the importance of hair follicle density on
Tissue Engineering Part C: Methods

the penetration of topically applied substances both in humans and pigs, while regional differences in
skin morphology have also been shown to influence percutaneous absorption. The results of the present
study support these previous studies demonstrating that while on average porcine skin is remarkably
similar to human skin, notable differences can exist on a site by site comparison. Differences in the
thickness of the dermis and epidermis within related body sites in the pig may also influence the
production of biologic scaffolds derived from porcine dermis. Typically, these scaffolds are prepared
from the skin on the back between the shoulders and the hips, with the epidermal and subcutaneous fat
layer mechanically delaminated to a uniform thickness (51). In the present study, the dermis increased
in thickness between the rostral and caudal back samples while the epidermis showed a variable
thickness being thicker in the caudal back and thinnest in the mid back. This variability in thickness may
influence the preparation of biologic scaffolds as the mechanical delamination process may result in
tissues from different depths in the dermis being retained. As a result, there may be differences in
mechanical properties, degradation rate, and composition of the final scaffolds as a result of
composition differences in the starting material.

Collagen and elastin content of the dermis both showed differences in distribution within the
dermis as well as variations between different body sites. Most notable was the presence of a
predominately type III collagen layer in the upper dermis beneath the epidermis. While the porcine skin
samples had no distinct papillary or reticular layering to the dermis, the presence of this collagen layer
indicates that differences in the composition of the dermis exist that are similar to those found in
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humans. The dense, predominantly type I collagen, deep dermis in the porcine skin samples showed
many similarities to the architecture described by others with large fiber bundles arranged in a complex
meshwork (62, 64), although the ratio of type I:III collagen in the porcine dermis was slightly lower than
has been reported to human skin (52). Elastin content showed the greatest variation between skin
samples. Previous studies have shown that porcine skin has a distinct elastin network that is interwoven
with the collagen fibers in the dermis. The present study showed that the elastin component comprised
between approx. 4 and 10% of the dermis when quantified by image analysis. Interestingly, Hult and
Goltz (1965) showed that human dermis contained approx. 4.16 to 9.4% elastin when quantified by
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

enzymatic digestion(35) suggesting that the composition of porcine skin closely resembles that of
humans, although others have report much higher concentrations (60).

In conclusion, the pig arguably presents the most suitable experimental model for use in
research studies on human skin. The results of the present study show that while porcine skin shares
Tissue Engineering Part C: Methods

many similarities with human skin, in the American Yorkshire Hamroc breed, distinct regional
differences in composition and morphology exist. However, many body sites on the pig share similarities
to human skin with the skin in the rostral and mid back regions being closest in thickness, hair follicle
density and composition to human skin. The results on skin thickness and hair follicle density also
provide important information for studies such as percutaneous absorption. Moreover, the variability in
back skin thickness may influence the preparation of biologic scaffolds where variable dermal thickness
may alter the mechanical properties, degradation rate, and composition of the final product. To avoid
misinterpretation of experimental results that utilize porcine skin, care should be taken to appreciate
these regional differences.
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Acknowledgments

We would like to thank McGowan histology lab for preparation of the histologic slides.
20
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Figure Legends
21
Tissue Engineering Part C: Methods
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Figure 1: Diagram illustrating the locations on the body where skin samples were obtained.
22
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23
Tissue Engineering Part C: Methods
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Scale bar = 100m.


average thickness (± SEM) of the stratum corneum (B), epidermis (C) and dermis (D) for each body site.
Figure 2: Summary of the histologic appearance of porcine skin from different body locations (A) and the
24
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20m
and the average number of Langerhans cells (B) and melanocytes (C) for each body site. Scale bar =
Figure 3: Summary of the histologic appearance of porcine epidermis from different body locations (A)
26
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Figure 4: The average number (± SEM) of hair follicles per cm2 for each body site tested.
27
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staining (A) was quantified at each body site and the percentage staining (± SEM) was recorded (B).
Figure 5: Quantification of Herovici’s polychrome staining for type I and type III collagen. Representative
29
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30
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Figure 6: Quantification of elastin staining within porcine skin samples. Skin samples were stained with
Verhoeff’s vanGieson stain (A) and percentage elastin content (± SEM) was quantified by image analysis
(B). In addition tissue samples were solubilized and the elastin extracted and quantified in g/mg tissue
(± SEM) by colorimetric assay (C).
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32

Lower Inner Outer Rostral Rostral Mid Mid Caudal Caudal Fore Hind
Jaw Ear Ear Belly Back Belly Back Belly Back Leg Leg
Lower Jaw No No No No Yes No No No Yes Yes
Inner Ear No No No No No No No No No Yes
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Outer Ear No No No No No No No No No Yes


Rostral
Belly No No No No No Yes No No No No
Rostral
Back No No No No Yes No No No Yes Yes
Mid Belly Yes No No No Yes Yes No No No No
Mid Back No No No Yes No Yes Yes No Yes Yes
Tissue Engineering Part C: Methods

Caudal
Belly No No No No No No Yes No No Yes
Caudal
Back No No No No No No No No No Yes
Fore Leg Yes No No No Yes No Yes No No No
Hind Leg Yes Yes Yes No Yes No Yes Yes Yes No

Table 1: Comparison of the keratinized layer thickness indicating significant differences between body locations, Yes = P≤0.05.
as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro
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Lower Inner Outer Rostral Rostral Mid Mid Caudal Caudal Fore Hind
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Jaw Ear Ear Belly Back Belly Back Belly Back Leg Leg
Lower Jaw Yes Yes Yes No Yes No No Yes Yes Yes
Inner Ear Yes No No Yes No Yes Yes No No No
Outer Ear Yes No No Yes No Yes Yes No No No
Rostral
Belly Yes No No Yes No Yes Yes No No No
Rostral
Tissue Engineering Part C: Methods

Back No Yes Yes Yes No No No Yes Yes Yes


Mid Belly Yes No No No No Yes Yes No Yes Yes
Mid Back No Yes Yes Yes No Yes No Yes Yes Yes
Caudal
Belly No Yes Yes Yes No Yes No Yes Yes Yes
Caudal
Back Yes No No No Yes No Yes Yes No Yes
Fore Leg Yes No No No Yes Yes Yes Yes No No
Hind Leg Yes No No No Yes Yes Yes Yes Yes No

Table 2: Comparison of epidermal thickness indicating significant differences between body locations, Yes = P≤0.05.
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34

Lower Inner Outer Rostral Rostral Mid Mid Caudal Caudal Fore Hind
Jaw Ear Ear Belly Back Belly Back Belly Back Leg Leg
Lower Jaw Yes Yes Yes No Yes Yes No Yes No Yes
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Inner Ear Yes No No Yes No Yes No Yes Yes Yes


Outer Ear Yes No No Yes Yes Yes Yes Yes Yes Yes
Rostral
Belly Yes No No Yes No Yes No Yes Yes Yes
Rostral
Back No Yes Yes Yes Yes No No Yes No No
Tissue Engineering Part C: Methods

Mid Belly Yes No Yes No Yes Yes No Yes Yes Yes


Mid Back Yes Yes Yes Yes No Yes Yes No No No
Caudal
Belly No No Yes No No No Yes Yes No Yes
Caudal
Back Yes Yes Yes Yes Yes Yes No Yes Yes No
Fore Leg No Yes Yes Yes No Yes No No Yes Yes
Hind Leg Yes Yes Yes Yes No Yes No Yes No Yes

Table 3: Comparison of dermal thickness indicating significant differences between body locations, Yes = P≤0.05.
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Lower Inner Outer Rostral Rostral Mid Mid Caudal Caudal Fore Hind
Jaw Ear Ear Belly Back Belly Back Belly Back Leg Leg
Lower Jaw Yes Yes No No Yes Yes Yes Yes No No
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Inner Ear Yes Yes Yes Yes Yes Yes Yes No Yes Yes
Outer Ear Yes Yes No No No Yes Yes Yes No No
Rostral
No Yes No No No Yes Yes Yes No No
Belly
Rostral
No Yes No No No Yes Yes Yes No No
Back
Tissue Engineering Part C: Methods

Mid Belly Yes Yes No No No Yes Yes Yes No No


Mid Back Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Caudal
No Yes Yes Yes Yes Yes Yes Yes Yes Yes
Belly
Caudal
Yes No Yes Yes Yes Yes Yes Yes Yes Yes
Back
Fore Leg No Yes No No No No Yes Yes Yes No
Hind Leg No Yes No No No No Yes Yes Yes No

Table 4: Comparison of the number of keratinized layers indicating significant differences between body locations, Yes = P≤0.05.
as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro Page 36 of 43

36

Lower Inner Outer Rostral Rostral Mid Mid Caudal Caudal Fore Hind
Jaw Ear Ear Belly Back Belly Back Belly Back Leg Leg
Lower Jaw Yes No No No Yes No No No No Yes
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Inner Ear Yes Yes Yes Yes Yes Yes Yes Yes Yes No
Outer Ear No Yes No No Yes No No No Yes Yes
Rostral
No Yes No No Yes No No No Yes Yes
Belly
Rostral
No Yes No No Yes No No No No Yes
Back
Tissue Engineering Part C: Methods

Mid Belly Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Mid Back No Yes No No No Yes No No Yes Yes
Caudal
No Yes No No No Yes No No Yes Yes
Belly
Caudal
No Yes No No No Yes No No Yes Yes
Back
Fore Leg No Yes Yes Yes No Yes Yes Yes Yes No
Hind Leg Yes No Yes Yes Yes Yes Yes Yes No

Table 5: Comparison of the number of Langerhans cells indicating significant differences between body locations, Yes = P≤0.05.
as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro
Page 37 of 43

37

Lower Inner Outer Rostral Rostral Mid Mid Caudal Caudal Fore Hind
Jaw Ear Ear Belly Back Belly Back Belly Back Leg Leg
Lower Jaw No No No No No No No No No Yes
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Inner Ear No No No No No No No No No Yes


Outer Ear No No No No No No No No No Yes
Rostral
No No No No No No No No No Yes
Belly
Rostral
No No No No No No No No No Yes
Back
Tissue Engineering Part C: Methods

Mid Belly No No No No No No No No No Yes


Mid Back No No No No No No Yes No No No
Caudal
No No No No No No No No No Yes
Belly
Caudal
No No No No No No No No No Yes
Back
Fore Leg No No No No No No No No No Yes
Hind Leg Yes Yes Yes Yes Yes Yes No Yes Yes Yes

Table 6: Comparison of the number of melanocytes indicating significant differences between body locations, Yes = P≤0.05.
as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro Page 38 of 43

38
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Lower Inner Outer Rostral Rostral Mid Mid Caudal Caudal Fore Hind
Jaw Ear Ear Belly Back Belly Back Belly Back Leg Leg
Lower Jaw No Yes Yes No Yes No Yes No No No
Inner Ear No Yes No No No No Yes No Yes No
Outer Ear Yes Yes Yes Yes Yes Yes Yes Yes No Yes
Rostral
Tissue Engineering Part C: Methods

Belly Yes No Yes Yes No Yes No Yes Yes Yes


Rostral
Back No No Yes Yes Yes No Yes No No No
Mid Belly Yes No Yes No Yes Yes No Yes Yes Yes
Mid Back No No Yes Yes No Yes Yes No No No
Caudal
Belly Yes Yes Yes No Yes No Yes Yes Yes Yes
Caudal
Back No No Yes Yes No Yes No Yes No No
Fore Leg No Yes No Yes No Yes No Yes No No
Hind Leg No No Yes Yes No Yes No Yes No No

Table 7: Comparison of hair follicle density indicating significant differences between body locations, Yes = P≤0.05.
as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro
Page 39 of 43

39

Lower Inner Outer Rostral Rostral Mid Mid Caudal Caudal Fore Hind
Jaw Ear Ear Belly Back Belly Back Belly Back Leg Leg
Lower Jaw Yes Yes No No Yes Yes No Yes Yes Yes
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Inner Ear Yes No Yes No No No No No No No


Outer Ear Yes No Yes No No No No No No No
Rostral
Belly No Yes Yes No No Yes No Yes Yes Yes
Rostral
Back No No No No Yes Yes No No No No
Tissue Engineering Part C: Methods

Mid Belly Yes No No No Yes No No No No NO


Mid Back Yes No No Yes Yes No Yes No No No
Caudal
Belly No No No No No No Yes No No No
Caudal
Back Yes No No Yes No No No No No No
Fore Leg Yes No No Yes No No No No No No
Hind Leg Yes No No Yes No No No No No No

Table 8: Comparison of Type I collagen content indicating significant differences between body locations, Yes = P≤0.05.
as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro Page 40 of 43

40

Lower Inner Outer Rostral Rostral Mid Mid Caudal Caudal Fore Hind
Jaw Ear Ear Belly Back Belly Back Belly Back Leg Leg
Lower Jaw No No No No No No Yes No Yes Yes
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Inner Ear No No No No No No No No No Yes


Outer Ear No No No No No No No No No Yes
Rostral
Belly No No No No No No No No No No
Rostral
Back No No No No No No No No No Yes
Tissue Engineering Part C: Methods

Mid Belly No No No No No No No No No Yes


Mid Back No No No No No No No No No No
Caudal
Belly Yes No No No No No No No No No
Caudal
Back No No No No No No No No No No
Fore Leg Yes No No No No No No No No No
Hind Leg Yes Yes Yes No Yes Yes No No No No

Table 9: Comparison of Type III collagen content indicating significant differences between body locations, Yes = P≤0.05.
as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro
Page 41 of 43

41

Lower Inner Outer Rostral Rostral Mid Mid Caudal Caudal Fore Hind
Jaw Ear Ear Belly Back Belly Back Belly Back Leg Leg
Lower Jaw Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)

Inner Ear Yes Yes Yes Yes Yes Yes No Yes Yes Yes
Outer Ear Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Rostral
Belly Yes Yes Yes Yes No No Yes No Yes No
Rostral
Back Yes Yes Yes Yes Yes Yes Yes Yes No Yes
Tissue Engineering Part C: Methods

Mid Belly Yes Yes Yes No Yes No Yes No Yes Yes


Mid Back Yes Yes Yes No Yes No Yes No Yes Yes
Caudal
Belly Yes No Yes Yes Yes Yes Yes Yes Yes Yes
Caudal
Back Yes Yes Yes No Yes No No Yes Yes No
Fore Leg Yes Yes Yes Yes No Yes Yes Yes Yes Yes
Hind Leg Yes Yes Yes No Yes Yes Yes Yes No Yes

Table 10: Comparison of elastin content, calculated by elastin assay, indicating significant differences between body locations, Yes = P≤0.05.
Tissue Engineering Part C: Methods
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)
as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro
Page 42 of 43

42
Tissue Engineering Part C: Methods
Regional Variations in the Histology of Porcine Skin (doi: 10.1089/ten.TEC.2014.0246)
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 43 of 43

43