Structure of Bovine Skin and Hair Root

A Scanning Electron Microscope Investigation

by Matthias Wagner and David G. Bailey

Abstract
A great deal of research has been done on the structure of human and ovine skin and hair,
but the literature provides only limited information on bovine skin and hair tissue at the
level of scanning electron microscopy.

Details of normally hidden surface structures of bovine skin epidermis and of the different
cell types associated with the hair root were obtained by scanning electron microscopy
using simple sample preparation techniques. These indude the pulling of hairs after acidic
incubation, modification of hair roots by trypsin or mechanical action, and freece-fracturing
of skin samples. The details presented reveal the hair root sheath as a highly organised
and differentiated structure of five various cell layers, and confirm the general structure of
bovine skin, hair root and epidermis as found in other mammals.

Introduction
Skin is composed of the dermis, a three-dimensional weave of collagen fibers converted to
leather in the tanning process, and the covering epidermis with its appendices, the hairs.
Both hair and epidermis are keratinized structures destroyed in the alkaline sulfide-lime
process, used in tanning for unhairing. The search for alternative sulfid-free processes,
which may act in a different way, requires therefore detailed knowledge of the structure of
the skin, prirnarily the hair root and the epidermis. Although a great deal of research has
been done on human or bovine skin,1,2,3,4 the literature provides only limited information on
bovine skin and hair tissue at the level of scanning electron microscopy. This research,
performed primarily with the scanning electron microscope, expands this area of
knowledge in bovine animals.

The epidermis is the outermost layer of the skin. It consists of a variety af cell layers, which
are produced in the germinative basal cell region located at the base of the dermis. The
descendents of these cells move to the surface of the epidermis while undergoing
morphological changes and keratinization. At the final stage they form a horny layer on the
surface of the epidermis. This is a continuous process with new cells, produced by the
germinative layer, replacing the horny layer as it breaks into flakes and sloughs off.5 The
surface of the cells is enlarged through fingerlike protrusions called microvilli. These
structures stabilize the contact of the cells within the epidermis, as well as between the
cells in the epidermis and the tissue below. In the latter case the microvilli are enlarged.6

Hair is comprised not only of the visible dead hair shaft above the skin but also its actively
growing root zone inside the skin. This hidden base of the hair is surrounded by its root
sheath revealing an astonishing differentiated and complex structure. This general
structure is found in the hair of all mammals (Figure 1).1,3
Figure 1.-Schematic drawing of the hair follicle;
single cell Iayers are emphasized through different shadings.

At its base the hair is shaped like a bell (hair bulb), surrounding a small dermal papilla,
which belongs to tile dermis. Its function is the nutrition of the growing cells of the hair. The
cells next to the papilla are not differentiated and represent the germinative zone of the
hair, induding the root sheath. Depending on their position in the bulb, the cell
descendents of this zone differentiate in ring-like patterns into different types of cells
forming the hair and its surrounding tissue. This tissue consists of 5 different concen-tric
layers. The first three innermost layers are united in the Inner Root Sheath (IRS). Directly
in contact with the cuticle of the hair is the cuticle of the IRS (IRS-Cuticle), fol-lowed by the
next two layers, Huxley's Layer (FIu) and Henle's Layer (He). Emerging from the same
differentiation zone the Companion Layer (CL) is the next ceIl layer within the root sheath.7
This layer is also called the inner-most layer of the Outer Root Sheath or middle root
sheath.9 The outermost layer of the root sheath is the Outer Root Sheath (ORS),
surrounding the other layers and the hair, not only in the lower part of the hair follicle, but
also in the hair channel, changing into the epidermis at its upper end.

The hair root, like the epidermis, is separated from the grain by a non-cellular thin layer of
the basal lamina (basement membrane), built up by different proteins.6 After removing the
epidermis during tanning, the grain is transformed into the surfacc of the leather. Only a
small amount of research has been done to investigate the part of the skin that might
undergo this transformation.

Experimental
Sample Preparation of Skin, Epidermis, and Hairs

Frozen pieces of steer hide (black angus, fresh frozen) were thawed and incubated
overnight at 31C in 1000 % float of 200 mM acetic acid, and 2% (w/v) sodium chloride,
0.02 % (v/v) Aracit KL (TFL USA/Canada).11 Pieces of hide were cut with a razor blade
after the incubation and prepared as SEM samples. Freeze-fracturing of acidic incubated
skin (thin cut) was done using two forceps after immersing the skin in liquid nitrogen.

Following acidic incubation, epidermis samples for the SEM investigation were obtained by
scraping them loose from the hide sample.

To observe the hair root structures, hairs were pulled out manually after the incubation and
dyed with p-dimethy-laminocinnamaldehyde (DACA) by immersing them in a 1 % (w/v)
DACA solution made in 0.5 M hydrochloric acid.12 Individual hairs showing the red dyed
IRS and the white-appearing ORS were selected. For the examination of the root sheath
structure the root sheaths of some hairs were gently disrupted with a scalpel blade, so that
hidden parts became visible. After that the hairs were prepared as SEM samples. Other
hair roots were washed several times with buffer (50 mM TRIS, 100 mM sodium chioride,
0.02 % sodium EDTA, pH 8.5) and incubated for 15 min. in 0.1 % (w/v) trypsin (Sigma-
Aldrich/USA), resolved in the buffer.2 Thereafter the hairs were prepared as SEM samples.

Preparation of SEM samples

Samples were fixed in 1 % (v/v) glutaraldehyde, 0.1 M imi-dazole-HCl buffer, pH 6.5, for at
least 1 h, and washed twice with 0.1 M imidazole-HCl buffer, pH 6.5. Thereafter the
samples were incubated for 2 h in 2 % (w/v) osmium tetroxide in 0.1 M imidazole-HCl
buffer, pH 6.5. After washing the samples with deionized water, they were stepwise
dehydrated in 50% (v/v), 80% (v/v), absolute ethanol. Drying was done in a carbon dioxide
critical point drying apparatus (Denton Vacuum Inc., Cherry Hill, NJ).

The samples were mounted on aluminum specimen stubs using colloidal silver adhesive
paint (Electron Microscopy Sciences, Ft. Washington, PA). Mounted samples were coated
with a thin layer of gold by low voltage DC sputtering (Plasma Science, Lorton, VA).

Scanning Electron Microscopy

Observations were made using a Model JSM840A scanning electron microscope (JEOL,
USA), operated in the secondary electron imaging mode and integrated with an Imix
workstation (Princeton Gamma-Tech, NJ), for collecting digital images.

Results and discussion

Structures of the Epidermis
Figure 2 shows the isolated epidermis including its underside with the single cells visible
underneath. Depending on their position on the epidermis the cells possess different
shapes. The cells at the bottom of the epidermis are more round to cubic. They represent
the germinative layer. Their descendants produced by cell divisions (see arrow) migrate to
the surface of the epidermis and undergo morphological changes. At the surface they are
flattened into an oblong to polygonal form. The thickness of bovine epidermis is around 25
- 40 mm. Depending on the species of mammal, epidermal thickness varies also with its
position on the body (human: upper arm 55 mm, palm 800 mm,13 mouse: 10 -23 mm,14
giraffe: 100 mm15).

FIGURE 2. - Scanning Electron Micrograph of bovine epidermis; epidermis loosened after acidic
incubation of skin; magnification: '2000, length of bar: 10 mm. Arrow marks dividing cell.

The complete surface of the epidermis cells appears rough (Figure 2) due to the protruding
fingerlike structures of the cell membrane called "microvilli".6 They enlarge the contact
area between the cells and stabilize the tissue connection. The microvilli of the cells of the
germinative layer directed towards the underside of the epidermis are especially greatly
enlarged, anchoring the epidermis to the grain.

Interface between epidermis and corium

After lifting the epidermis from the grain (Figure 3), the underside of the epidermis can be
seen revealing fine grooves built up presumably by alignment of the microvilli of the
germinative layer of the epidermis. In the grain layer, larger collagen fibers can be
recognized. At the top of the grain layer a network of fine fibrils is found.5,10,16,17 The nature
of the proteins building these structures is unknown. The space between the fibrils is
around 0.6 to 2.1 mm. This seems to be the right size to form a complementary interface
enabling the reception of the larger microvilli of the underside of the epidermis. After
mechanical removal of the epidermis, the fibrils at the grain surface seem to be squeezed
together yielding a rather uniform surface (data not shown). As the surface of leather
consists of fine fibrils packed close together,10 the fibrils seen in Figure 3 may represent
the layer of the skin that is transformed during tanning into the enamel of the leather.
Figure 3. - Scanning Electron Micrograph of bovine skin, cut after acidic incubation. Epidermis has
separated from the grain revealing its surface structure, magnification: '700, length of bar: 25 mm.
Epi = epidermis; G = grain.

Layers of the root sheath

The Inner Root Sheath (IRS) consists of three different cell layers (cf. Figure 1). Its
innermost layer is the cuticle (IRS-Cu; Figure 4). It is in direct contact with the cuticle of the
hair and consists of only a single cell layer. Its cells represent the smallest cells of the hair,
with a thickness below 1 mm. They are shaped and arranged like scales.

Figure 4. - Scanning Electron Micrograph of the root sheath of bovine hair, section of the Cuticle of
the IRS with its surrounding tissue. Bovine skin, freeze-fractured after acidic incubation,
magnification, 3000, length of bar: 5 mm. He Henle's = Layer, keratinized; Hu = Huxley's Layer,
keratinized; IRS-Cu = Cuticle of the Inner Root Sheath, keratinized.
The cuticle is followed by two single-cell-layers built up by cells of similar structure: the
inner Huxley's Layer and the outer Henle's Layer. These layers consist of elongated and
lanceolate shaped cells covering the smooth surface of the IRS-Cuticle (Figure 5).

One feature of the three layers of the IRS is the keratinization that occurs at different
times.18 This process is responsible for the hard, bark-like structure of these cell layers.
The physiological meaning of this process is unknown. The first layer that is keratinized is
the Henle's Layer (Figure 6). The next layer to be keratinized is the IRS-Cuticle. At the
stage represented in Figure 6 keratinization as can be found in Figure 4 still has not
occurred in the cells of the IRS-Cuticle. The Huxley's Layer between these layers is still
showing the fibrillar structure of soft, non-keratinized tis-sue. After keratinization is
completed the hard, bark-like structure of the Huxley's Layer and Henle's Layer can be
found as seen in Figure 4.18

Figure 5. - Scanning Electron Micrograph of bovine hair, showing the IRS-Cuticle, and
Henle's and Huxley's Layers. Hair treated 15 min with trypsin after acidic incubation;
magnification: '500, length of bar: 25 mm. IRS-Cu = Cuticle of the Inner Root Sheath;
H = hair; He = Henle's Layer; Hu = Huxley's Layer.

The Companion Layer (CL) surrounds the IRS (cf. Figure 1). It can be seen in longitudinal
section next to the Henle's Layer as a thinner, brighter layer (Figure 6). lt consists of one
layer of flattened, rectangular shaped cells (Figure 7).
Figure 6. - Scanning Electron Micrograph of the root sheath of bovine hair, showing different stages
of keratinization in the single cell layers. Bovine skin, freeze-fracture after acidic incubation;
magnification: ,3000, Iength of bar: 5 mm. CL = Companion Layer; Re = Henle's Layer, keratinized; Hu
= Huxley's Layer, non-keratinized; IRS-Cu = Cuticle of the Inner Root Sheath, non-keratinized.

The Outer Root Sheath (ORS) is the outermost layer of the hair root (cf. Figure 1). lt is in
direct contact with the CL and consists of round to cubic cells (Figure 7). The ORS
surrounds not only the lower parts of the hair follicle (Figure 7) but also the upper part of
the hair channel. There it becomes continuous with the epidermis, representing one entity
together with the ORS. The change of the ORS from the upper part of the hair channel into
the epidermis covering the grain layer (G) of the dermis can be seen in Figure 8.

Hair growth
Hair growth has its beginnings in cell divisions of the non-differentiated cells surrounding
the dermal papilla. The descendants differentiate and divide further into the different cells
of the hair, into the precursor of the three layers of the IRS, including the Companion
Layer, and the ORS. The production of cells leads to the continuous movement of the hair
towards and above the epidermis. The IRS with its three layers and the CL follows this
movement.7 The only layer of the root sheath that is nearly stationary is the ORS.
Therefore a kind of gliding has to occur during hair growth, at the interface between the
Companion Layer and the ORS.
At a level around the attachment site of the hair muscle the cells of the Henle's and the
Huxley's Layer start to disintegrate 3,18,19 producing a small crevice filled with cell debris
and sealed by sebum. The cells of the IRS-Cuticle stay on the hair up to the sufface of the
epidermis, where the loosened cells are shed or sloughed off.

Figure 7. - Scanning Electron Micrograph of isolated bovine hair with the different cell layers
of the root sheath. Hair after acidic incubation; magnification: '250, length of bar: 50 mm.
CL = Companion Layer; R = hair; IRS = Inner Root Sheath; ORS = Outer Root Sheath

Conclusions
The Scanning Electron Microscopic Method is a good technique for showing surface
structures of parts of the skin and the hair that are normally hidden. These results were
obtained in combination with relatively simple sample preparation techniques. These
methods were able to provide more information than usually obtained from the thin slice
preparation usually used in light microscopy or transmission electron microscopy.
Specifically this included the pulling of hairs after an acidic incubation, modification of hair
roots by trypsin or mechanical action, and freeze-fracturing of skin samples that all
provided better access to these surface structures.

The resulting micrographs confirmed the same general structure of bovine skin, hair root
and epidermis as found in earlier investigations of other species. They revealed the root
sheath as a highly organized and differentiated structure of five layers. These layers are
arranged in a ring-like pattern and comprise the Inner Root Sheath - consisting of the
Cuticle, Huxley's Layer and Henle's Layer -, Companion Layer, and Outer Root Sheath.
Each layer consists of cells that differ from one neighbouring cell layer to the other. The
single cell types of bovine origin are presented in the Scanning Electron Micrographs.
The data show morphological targets for research on alternative, sulfide-free dehairing
method

Figure 8. - Scanning Electron Micrograph of cut bovine skin, section of epidermis and grain.
magnification: '200, length of bar: 100mm. Epi = epidermis; G = grain.

Acknowledgement
The authors would like to acknowledge Peter Cooke, ERRC Core Research Unit, for help
in handling the scanning electron microscope.
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