J. Anat. (1981), 133, 1, pp.

19-35 19
With 14 figures
Printed in Great Britain

Outer root sheath keratinization in anagen

and catagen of the mammalian hair follicle. A seventh distinct
type of keratinization in the hair follicle: trichilemmal
keratinization
HERMANN PINKUS*, TOSHIRO IWASAKIt AND
YUTAKA MISHIMAt
* Department of Dermatology, Wayne State University School of Medicine,
Detroit, Michigan 48201 and
t Departnment of Dermatology, Kobe University School of Medicine,
Kobe, Japan 650
(Accepted 9 September 1980)
INTRODUCTION
Keratinization processes of the hair and its inner root sheath have been thoroughly
investigated for many years and documented in journals, monographs and con-
ference reports (e.g. Montagna & Parakkal, 1974; Kobori & Montagna, 1976;
Brown & Crounse, 1980). On the other hand, little attention has been paid to
keratinization of the outer root sheath in the region below the opening of the
sebaceous duct. The only author who investigated outer root sheath keratinization
in the early phase of histological research was Maurer, who in 1895 published a
monograph on the epidermis and its derivatives, feathers and hairs, of many species.
Among mammals, he studied monotremes and higher animals including man. One
of his discoveries was keratinization of the outer root sheath, which he illustrated
(Fig. I) and described in hair follicles of Ornithorhynchus, Dasypus, Manis, cat,
dog and man.
Maurer found keratinization of the outer root sheath in two situations. He
examined one of the large hairs of Ornithorhynchus in the telogen phase and found
a very long club of the hair directly bordering on the external root sheath. Between
the two there was a thick, corrugated horny layer, which was the product of the
root sheath and extended upward to the duct of the sebaceous gland. At the lower
end, the outer cells of the hair shaft were slightly brush-like and extended into the
outer root sheath. Higher up, the hair had a definite cuticle.
In all the other species, Maurer examined growing (anagen) hairs. He described
hairs of Dasypus and found that the inner root sheath ended below the opening of
the sebaceous gland. Above this point, the outer root sheath showed increase of cells
and keratinization. Maurer claimed that he found this formation in all hair follicles
of adult mammals which he examined, including man. The keratinizing portion of
the outer root sheath could be short or longer. It always ended at the opening of the
sebaceous gland and started close to the upper end of the inner root sheath, which
usually was below the sebaceous duct, although in a 14 days old dog it extended
somewhat higher.
In Ornithorhynchus, the keratinizing material of the outer root sheath was
stained bluish-green with a mixture of borax-carmine, Delafield's haematoxylin, and
20 H. PINKUS, T. IWASAKI AND Y. MISHIMA
Th(fi
3
j.

It

7'
Fig. 1. Reproduction of the upper half of Plate 9 of Maurer's (1895) monograph. Longitudinal
sections of hair follicles, upper portion with opening to skin surface. H, Hair; R, cortex; m,
medulla, o, cuticle; HS, internal root sheath; Hw, outer root sheath above and below the
opening of the sebaceous gland (t); c, densely keratinized portion below opening of sebaceous
gland. (1) Man, scalp. (2) Half grown cat, pelage hair of upper lip. (3) Old cat, vibrissa of upper
lip. (4) Fourteen days old dog, hair of back. (5) Ornithorhynchus, adult, back, telogen hair.

acid fuchsin-picric acid. The staining procedure was not specified in the other
species, but presumably was the same.
Oyama (1904), following Stohr's suggestion, examined the development of the
pelage of the white mouse, comparing it with his teacher's results in human vellus
hair (Stohr, 1903). He disagreed with Maurer's interpretation. He claimed that the
A
layer of horny cells containing small rudimentary nuclei, which was found below
the opening of the sebaceous duct and extended downward almost to the level of the
bulge, intercalating itself between inner and outer root sheaths, was a remnant of
hair canal cells, which originated earlier, before the hair developed. According to a
footnote in Oyama's paper, Stohr informed him that hair canal cells were also
present in the root sheath of adult human hairs.
This was a definite misinterpretation ofMaurer's results. Haircanal cells originate in
the intra-epidermal portion of the hair follicle, above the opening of the sebaceous duct,

Fig. 1. Light micrographs showing trichilemmal keratinization (T) in anagen. (A) Human scalp
hair stained with Giemsa solution. H, hair; I, inner root sheath stained dark and breaking up,
one flake resting on keratinized trichilemma higher up. (B) Canine hair follicle stained with
haematoxylin and eosin. Hair is not present in this section. E, epidermoid keratinization of
infundibular epithelium; I, inner root sheath. Arrows point to pocket-like structure. x 940.
 .-@:'w;ot .i!
Trichilemmal keratinization 21

-p

:..... r..... 11:14W, ::

.5.". .....-
22 H. PINKUS, T. IWASAKI AND Y. MISHIMA
 Trichilemmal keratinization 23
and they keratinize with keratohyalin formation (Stohr, 1903; Pinkus, 1958). They
survive as part of the infundibulum of the adult follicle, while Maurer's layer is present
in the isthmus of the follicle below the sebaceous duct. Stohr's authority, however,
apparently prevailed, since no later author has referred to Maurer's findings.
Auber (1952), in his classic monograph on the sheep hair follicle, extensively
reviewed the literature, but admitted that he had no access to Maurer's work. He
quoted Oyama (1904) and Stohr (1903). Auber, who evidently was more interested
in the hair and its inner root sheath, described in sheep the distal region of the outer
root sheath, extending upward from the end of the inner root sheath, as follows.
The area is marked by a system of irregular transverse corrugations which project
into the follicle lumen in an axiodistal direction. The lumen of the most distal
follicle region, in which the fibre lies more loosely than it does more proximally, is
lined with stratum corneum. At a level between the half and the upper third of this
region one or several sebaceous glands open into the follicle lumen. The structure
of the outer root sheath approaches conditions very similar to those of the epidermis
proper and is particularly noticeable from the level above the distal limit of the
inner root sheath. The granular layer is more distinct than in the epidermis proper.
Auber's drawn illustration shows the fused Henle-Huxley layer film passing directly
into the stratum corneum of the very prominent corrugations of the outer root sheath.
F. Pinkus (1927), in his extensive work on anatomy of the human skin, barely
mentioned partial keratinization of the outer root sheath below the sebaceous duct
and made no reference to Maurer's findings. Later German authors (Hoepke, 1927;
Horstmann, 1957; Zaun, 1968) did not mention them. In modern American litera-
ture, Montagna (1956, 1962) has stated that the inner cells of the outer root sheath
became hyalinized and seemed to undergo partial keratinization about halfway up
the follicle. He quoted Gibbs' (1938) work on Trichosurus. The latest edition of
Montagna & Parakkal's book (1974) does not mention the subject. It was only in
1968 that Holmes and H. Pinkus independently called attention to keratinization of
the outer root sheath in those portions of the human hair follicle in which it is not
apposed to the inner root sheath. These portions are the stretch of anagen hair
between the upper end of the inner root sheath and the opening of the sebaceous
duct, and the sac of epithelium surrounding the lower end of the catagen hair, after
the inner root sheath has ceased to be formed (Pinkus, 1980). Pinkus (1969) termed
this process trichilemmal keratinization and called attention to its identity with
Maurer's stratum corneum of the outer root sheath.
Trichilemmal keratinization is the end product of the outer root sheath (tri-
chilemma) and therefore is not derived from the hair matrix, but from stratified
squamous epithelium. It transforms stratified squamous epithelium into non-
nucleated keratinized cells without the formation of a keratohyalin layer. Only
occasional small keratohyalin droplets are seen with the light microscope. The cells

Fig. 2. Light micrographs showing catagen hair follicles. (A) Human scalp hair stained with
van Gieson's haematoxylin-acid fuchsin-picric acid. The hair shaft is separated from the
keratinized inner root sheath by a cleft but it adheres to the non-keratinized outer root sheath,
which is surrounded by the dark staining vitreous membrane. At the lower end, the hair comes
to a pointed end within the club formed by keratinizing cells of the outer root sheath, which
thin out in the region where the inner root sheath has its lower margin. Below the hair club is
the collapsed remnant of the outer root sheath (Montagna's hair germ). (B) Canine hair, stained
PASHPA. Tangential section of hair club arising from the trichilemma. Below it, the hair germ
with rudimentary dermal papilla. x 420.
24 H. PINKUS, T. IWASAKI AND Y. MISHIMA

Fig. 3. Electron micrograph showing longitudinal section of the initiation point of trichilemmal
keratinization in anagen hair of dog (trichilemmal pocket-like structure (arrow)). As outer root
sheath (ORS) cells move toward the hair shaft, they become flattened with the thickening of
cell membrane. Remaining keratinized inner root sheath (KIRS) cell layers are also seen over-
lapping with keratinized outer root sheath cell layers (KORS). x 6300.
Fig. 4. Electron micrograph, at higher magnification, of innermost cells of the outer root sheath
 Trichilemmal keratinization 25
of the outer root sheath swell and transform individually, thus producing an uneven
border outline. Nuclei may persist for a short distance in the keratinized layer. Cell
borders between the keratinized cells are outlined by PAS-positive substance. The
keratin stains yellow with picric acid, whitish-green with thioflavin T under fluores-
cent illumination, and deep red with rhodamin-B (Pinkus, Mehregan, Rahbari &
Krobock, 1980 a). In sections stained with a combination of PAS, iron-haematoxylin
and picric acid (PASHPA), low power examination shows a brownish tinge due to
the red cell borders surrounding yellow bulky cell bodies. Histochemical reactions
for SH- and S-S groups (Burnett-Seligman) are positive (Pinkus, 1969).
That trichilemmal keratin is formed in two separate regions of the hair follicle at
different stages of the hair cycle is remarkable, and invited electron microscopic
study for more details. Parakkal (1970) has described the morphogenesis of the club
hair in mice and rats. It is obvious from his paper that the dimensions of the thin
hairs in these small rodents are not quite comparable to the human follicle on which
our light microscopic studies have been done. It seemed important to study the
thicker hairs of man, since the problems involved also have a bearing on the histo-
genesis of tumours and cysts of the human hair follicle. It is, however, difficult to
obtain human catagen follicles. The scalp, which is the most desirable source, con-
tains only about 1 % catagen hairs, and they are almost impossible to locate in speci-
mens obtained from the scalp. We therefore decided to investigate dog hair, which in
its dimensions and structure resembles human hair closely. Besides, the constant
shedding of most races of dogs promised a higher percentage of catagen hairs.

MATERIALS AND METHODS

Material was taken from six adult mongrel dogs: three were male and three
female. Dorsal skin of each dog was removed under systemic anaesthesia by 25 mg/kg
of sodium pentobarbital. Specimens were fixed in 10 % neutral formalin and embedded
in paraffin. They were then stained with haematoxylin and eosin and with PASHPA.
Human skin was obtained after local injection of lidocain solution. The specimens
were fixed in 10 % neutral formalin and embedded in paraffin. Ten micron sections
were stained with haematoxylin and eosin, with van Gieson's mixture of haemat-
oxylin and picrofuchsin, and with acid orcein and Giemsa solution (Pinkus &
Hunter, 1960).
For electron microscopic study, slices of tissue were fixed in 2 % glutaraldehyde
(0 1 M phosphate buffer pH 7 4) and post-fixed in 1 % osmium tetroxide (0 1 M
phosphate buffer pH 7 4). The slices were transferred to ascending concentrations
of ethanol and embedded in Epon 812. Embedded tissues were cut by MT-1 ultra-
microtome and double-stained with uranyl acetate and lead nitrate. Specimens were
examined by JEOL 1OOS electron microscope.
For the acid phosphatase activity, materials were fixed in Karnovsky's solution
for 2 hours, then cut into slices of 60-80 microns by vibratome. Slices were incubated
in Gomori's solution at room temperature for 15-20 minutes and post-fixed in 1 %
osmium tetroxide.
LIGHT MICROSCOPIC DATA
Anagen hairs
Longitudinal sections of dog and human hair follicles resemble each other closely.
In anagen, the hair and its inner root sheath arise from the matrix and show their
26 H. PINKUS, T. IWASAKI AND Y. MISHIMA

Fig. 5. Trichilemmal zone of dog showing unique ladder-like membrane coating granules
(LMCG) enveloping narrow elongated area of keratinized outer root sheath cell layers (KORS)
interdigitating with keratinizing outer root sheath cells (ORS). x 9200.
Fig. 6. Ladder-like membrane coating granules exhibit strong acid phosphatase activity by the
deposition of reaction product, lead phosphate (arrow) at the mid-portion of the trichilemmal
zone in anagen hair in dog. x 52500.
 Trichilemmal keratinization 27
six different types of keratinization, i.e. medulla, cortex, and cuticle of the hair;
cuticle, Huxley's and Henle's layer of the inner root sheath. The hair is closely
surrounded by the inner root sheath up to a point below the level of the sebaceous
duct opening where (Fig. 1 A, B) it breaks up in Straile's (1962) 'zone of sloughing'
and the hair continues upward without covering. At the same level, the inner (axial)
surface of the outer root sheath (trichilemma) is freed from its close apposition to
Henle's layer and begins to show evidence of keratinization. The trichilemmal
keratin becomes thicker and coats the follicular side of the ledge formed by the
sebaceous duct as it traverses the follicular wall in oblique fashion. On the side of
the sebaceous duct, this ledge is covered by a more epidermoid type of keratin with
a keratohyalin layer, which is the specific way of sebaceous duct keratinization.
This type of keratinization (Knutson, 1974) continues upward and lines the in-
fundibulum (pilosebaceous canal).
The upper end of the inner root sheath can have a variety of configurations.
Cornified inner root sheath stains strikingly blue with methylene blue or toluidine
blue. In sections stained with Giemsa solution, it suddenly or gradually loses this
staining reaction at the upper end and continues without colour until it breaks up
completely. Auber (1952), Montagna & Parakkal (1974), and Straile (1962) have
attributed this to keratolytic enzymes in the zone of sloughing. The shape of the
upper end is either a thin edge, free within the hair canal, or is closely apposed to
the inner surface of the trichilemma. Rarely in human material, much more com-
monly in the dog, the free edge appears to be sharply bent outward (Fig. 1 B), and
hooked into the trichilemmal keratin, its end pointing downward. The explanation
of this peculiar configuration will be discussed later.
If one follows the interface between inner root sheath and trichilemma downward
(proximally), one sees flattening of trichilemmal cells, but only faint indications of
keratinization for a short distance. The keratinizing tendencies of trichilemma seem
to be inhibited by the adjacent inner root sheath.
Catagen hairs
When the hair stops growing and undergoes the involutionary stages leading to
telogen, the hair papilla loses its metachromasia and shrinks. The hair matrix
retracts and mitosis stops (Fig. 2). Formation of the inner root sheath, which
originates close to the neck of the papilla, ceases first, while matrix cells of the cortex
continue their keratogenous process and elongate greatly, lifting the lower end of
the inner root sheath upwards (Braun-Falco & Kint, 1965; Pinkus, 1980). The
question of persistence or cessation of hair cuticle formation seems to have been
answered by staining human catagen and telogen hairs with toluidine blue and
Rhodamin B at pH 3'6 (Pinkus, Mehregan, Rahbari & Krobock, 1980a). This
mixture stains the cuticle of the hair light blue and reveals a light blue border all
around the periphery of the lower end of catagen and telogen hairs. At the same
time, it reveals a bright red colour of the club material which surrounds the lower
part of the hair, that part which always has been described as 'brush-like' kera-
tinized fibres extending downward and outward between the cells of the trichilemmal
sac.
Inspection of sections of human catagen hairs, without preconceived assumptions,
indicates that this red fringe, which envelops the lower end of the dying hair and
fixes it firmly to the surrounding cells of the trichilemma, has its origin in the
trichilemmal cells. It is the stratum corneum of the outer root sheath in Maurer's
28 H. PINKUS, T. IWASAKI AND Y. MISHIMA

DRS

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,+ ,W. . , '? Y , : \ ;; sy4,lis
?XX w ; p:!Js ^M, w~~~~~~~~~~~1W4c
 Trichilemmal keratinization 29
sense, which converges on to and holds secure the telogen hair as it slowly moves
upward in the shrinking transient portion of the lower hair follicle and eventually
comes to rest at the level of the bulge of the permanent portion (Pinkus, 1980). The
question of whether this material has relations to persisting inner root sheath was
answeied negatively (Pinkus, 1969) by staining with thioflavin T and examination
with fluorescent illumination. Thioflavin T stains keratinized inner root sheath a
brilliant yellow and shows the retraction of the lower end upwards. Trichilemmal
keratin, on the other hand, whether at the follicular isthmus in anagen or around
the lower end of the hair in telogen, shows up with a bright whitish blue tinge.

ELECTRON MICROSCOPIC DATA

Anagen hair of dogs
At the proximal end of the zone of sloughing, where the keratinized Henle's layer
is in close contact with the outer root sheath, there is no evidence of trichilemmal
keratinization, but the innermost trichilemmal cells are flattened and form com-
plicated interdigitations with the more peripheral cells of the outer root sheath, with
which they are connected by desmosomes. The plasma membranes are thick. No
membrane coating granules or keratohyalin granules or droplets are seen. The
peripheral cells of the outer root sheath contain numerous mitochondria and endo-
plasmic reticulum. A little higher up along the hair follicle, the innermost cells of
the outer root sheath are very electron-dense and are apt to form a pocket-like
configuration (Fig. 3) into which inner root sheath material appears to invaginate.
This trichilemmal pocket-like structure is often seen on dog hairs, while it is rarely
seen on human scalp hairs (Pinkus, 1969). Its formation will be discussed later.
Transverse sections of the follicle in this region show the outer root sheath to consist
of two to three nucleated layers containing some bundles of tonofilaments. Then
follows a layer of cells of decreased diameter having pyknotic nuclei and inter-
digitating with protruding extensions of the electron-dense keratinized layer. The
mid-portion of the trichilemmal zone contains occasional small round keratohyalin
droplets (Fig. 4), not closely associated with the numerous tonofilament bundles,
and many membrane coating granules in addition to a few mitochondria. Apposed
to the thick electron-dense protrusions of the keratinized cells, the membrane
coating granules (Fig. 5), identified by their strong acid phosphatase activity (Fig. 6),
assume a unique ladder-like configuration (Pinkus, Iwasaki & Mishima, 1980b).

Fig. 7. Low magnification electron micrograph of club hair of dog having trichilemmal keratin-
ization showing interdigitation of keratinizing and keratinized outer root sheath cells without
distinct ladder-like membrane coating granules and keratohyalin droplets. However, unique
irregular honeycomb-like infolding (arrow) of the cell membrane and remaining desmosome-
tonofilament complexes (DT) are seen at the interface among the keratinizing outer root sheath
cells (ORS). x 9980.
Fig. 8. Higher magnification of honeycomb-like infolding of the outer root sheath cell mem-
brane in the club hair of dog. x 42700.
Fig. 9. Interface between keratinizing outer root sheath cells exhibits a unique structure
resulting from thickening of cell membrane in dog. x 65 800.
30 H. PINKUS, T. IWASAKI AND Y. MISHIMA
p*..

Fig. 10. Transitional keratinizing outer root sheath cells (TORS) are seen at the interface
between completely keratinized and keratinizing cells of the sheath in the dog. x 26300.
Fig. 11. Membrane coating granules (MCG) here form a peculiar compound structure in the
cytoplasm of keratinizing outer root sheath cells of the dog without forming a distinct ladder-
like pattern and without distinct acid phosphatase activity. x 21000.
 Trichilemmal keratinization 31
Catagen hair of dogs
A somewhat similar configuration is seen in longitudinal and transverse sections
of the catagen stage of hair growth. Multiple layers of outer root sheath contain
nuclei, tonofilaments, mitochondria, and glycogen granules. They border on electron-
dense keratinized cells in a complicated manner characterized by many irregular
interdigitations of the two cell layers (Figs. 7-9). There are no ladder-like membrane
coating granules or distinct keratohyalin droplets. However, there are complicated
infoldings of the plasma membrane of the non-keratinized cells, and there are
numerous desmosomes between non-keratinized and keratinized cells. Transitional
cells of the outer root sheath (Fig. 10) are encountered, which are of intermediate
electron density and contain traces of tonofilament bundles and glycogen. Occasional
peculiar membrane coating granules are recognized (Fig. 11).
These findings are illustrated diagrammatically in Figures 12 and 13 (Pinkus et al.
1 980b).
DISCUSSION
Electron microscopic examination of dog hair leaves little doubt that the outer
root sheath keratinizes in anagen and catagen in a very special fashion. The inter-
digitation of pre-keratinized and keratinized cells is perhaps the most prominent
feature, together with high electron density of the keratinized cells that does not
permit recognition of details in their cytoplasm. The fact that no, or only a few,
small keratohyalin droplets are present in non-keratinized cells seems to exclude
the presence of a 'keratin pattern' that according to Brody (1964) results from an
interplay of tonofilaments and keratohyalin in epidermal keratinization. The pre-
sence of complicated infoldings of plasma membranes provides a highly charac-
teristic picture to the border zone of trichilemmal keratinization. This seems to
correlate with the uneven border and the individualistic transition of cells seen in
light microscopy.
Anagen hair
The data concerning the isthmus of the anagen follicle seem to support the view
expressed by Auber (1952), Straile (1962) and Montagna & Parakkal (1974) that
enzymic action contributes to the sloughing of the inner root sheath. The strongly
acid phosphatase-active 'ladder-like' membrane coating granules seen in this 'zone
of sloughing' suggest that they are the site of formation of digestive enzymes. They
are absent in the otherwise similar trichilemma of the catagen club hair.
One feature of the anagen hair of dogs requires some discussion. That is the
peculiar hooking-in of the distal end of the inner root sheath into the pocket-like
configuration of the outer sheath. This feature, frequently seen in dog hair, is
encountered occasionally in human scalp hairs and is illustrated in Figure 6 of the
paper by Pinkus (1969). It was seen rnore frequently in human beard hair by Gunther
(1895) and was described in his doctoral thesis. It seems that the upper edge of the
inner root sheath adhered to the outer root sheath, and as the hair and inner root
sheath kept moving upward, was itself inverted downward. If correct, this explana-
tion would make the condition very temporary and terminated by disintegration of
the inner root sheath.
If one compares the data given in the text and illustrations of those authors who
have studied the thin hairs of rodents (Oyama, 1904; Braun-Falco & Kint, 1965;
 32 H. PINKUS, T. IWASAKI AND Y. MISHIMA

r.)
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 Trichilemmal keratinization 33
Parakkal, 1970; Montagna & Parakkal, 1974) one appreciates that the differences
described may derive from the facts that the hair and its sheath consist of relatively
few cells and cannot be expected to develop all the details seen in a study of larger
hairs.
Catagen hair
The findings of strong desmosomal connections between the non-keratinized and
keratinized cells of the outer root sheath in the club region of the catagen hair are
convincing evidence of the derivation of the 'brush-like' periphery of the hair club
from the surrounding trichilemmal cells, rather than from the subsiding lower end
of the hair. The processes leading to and following cessation of hair growth have
attracted the attention of numerous investigators since the middle of the nineteenth
century and have been described and analysed in great detail. F. Pinkus, in his
monograph of 1927, eliminated several untenable older concepts, reviewed the work
of von Ebner (1876), Garcia (1891), and several others, and co-ordinated what was
known at that time. In more recent years, the hair cycle has been investigated more
commonly in animals, especially rodents, than in men. The only modern study of
human catagen was that of Kligman (1959). We concern ourselves here entirely with
human hair.
In catagen begins the formation of the hair club, which in its mature state will
anchor the telogen hair in the trichilemmal sac at the level of the bulge. Most
authors have described the club as being formed by the keratinizing cells of the
dying hair, which remains bulbous and from which keratinized fibres extend radially
between the surrounding living cells of the outer root sheath. F. Pinkus stated that
in the final stages keratinizing cells of hair and keratinizing cells of the outer root
sheath mix to form an indissoluble knot. Our observations show that the lower end
of the hair itself is not bulbous, but of the same, or possibly smaller diameter as the
hair shaft above it. It becomes surrounded by cells of the outer root sheath under-
going trichilemmal keratinization, which converge on to and fuse with the cortex.
The often described 'brush' consists of keratinizing cells of the outer root sheath,
which become elongated rather than flattened just as they do in the zone of sloughing
of the anagen follicle.
This trichilemmal club is less voluminous higher up and comes to a thin margin
at the point where the inner root sheath has its lower end (Fig. 14). At this level, in
longitudinal section, the hair is surrounded by a slit, which in three dimensions is a
moat (Montagna, 1956, p. 179). The inner wall of the moat is the cuticle of the hair,
the outer wall is the keratinized inner root sheath, which often adheres tightly to
the luminal surface of the outer root sheath and prevents its keratinization. Tri-
chilemmal keratin is again present higher up in the follicle, above the upper end
of the inner root sheath.
In conclusion, our findings support and amplify Maurer's data on keratinization
of the outer root sheath of the hair follicle in anagen and catagen, and contribute
electron microscopic data suggesting the source of keratolytic enzymes in the zone
of sloughing.
Fig. 12. Diagrammatic drawing summarizing our findings on trichilemmal keratinization in the
canine anagen hair.
Fig. 13. Diagrammatic drawing summarizing our findings on trichilemmal keratinization in
the canine club hair.
'2 ANA I.3
34 H. PINKUS, T. IWASAKI AND Y. MISHIMA

Fig. 14. Human scalp hair in catagen. Lower end of hair cortex contains a few melanin granules
and is enveloped in trichilemmal keratin forming the club. The remnants of the inner root
sheath, stained partly dark by Giemsa solution, are visible at either side of the hair where it
emerges from the club. They form Montagna's moat by separating from the cuticle covered
shaft, have a sharp lower border and a pointed tip. The inner root sheath borders on flattened
cells with dark nuclei of the non-keratinized outer root sheath (arrows). Acid orcein-Giemsa
stain. x420.
SUMMARY
Trichilemmal keratinization, first described by Maurer in 1895 and rediscovered
by Holmes (1968) and Pinkus (1968) converts the stratified epithelium of the outer
root sheath into anuclear keratin without an intervening keratohyalin layer. It is a
distinct seventh type of keratinization in the hair follicle, not derived from the hair
matrix, it occurs wherevrer outer root sheath is not apposed to inner root sheath, in
anagen in the zone of sloughing just below the opening of the sebaceous duct, in
catagen in the trichilemmal sac surrounding the lower end of the dying hair shaft
where it forms the club of the telogen hair. Electron microscopic study of the thick
hairs of dogs (but not the tiny hairs of rodents) reveals intricate infoldings of non-
keratinized and keratinized cells. It also shows unique ladder-like membrane coating
granules in anagen, which are strongly acid phosphatase-positive and are suggested
to be the source of enzyme involved with disintegration of the inner root sheath.
 Trichilemmal keratinization 35
REFERENCES
AUBER, L. (1952). The anatomy of follicles producing wool fibres, with special reference to keratinization.
Transactions of the Royal Society of Edinburgh 62, 191-254.
BRAUN-FALCO, 0. & KINT, A. (1965). Zur Dynamik der Katagenphase; eine experimentelle Studie an
Albino Ratten. Archiv far klinische und experimentelle Dermatologie 223, 1-15.
BRODY, I. (1964). Observations on the fine structure of the horny layer in the normal human epidermis.
Journal of Investigative Dermatology 42, 27-31.
BROWN, A. C. & CROUNSE, R. G. (1980). Hair, Trace Elements and Human Illness. New York: Praeger.
EBNER, V. VON (1876). Mikroskopische Studien uber Wachsthum und Wechsel der Haare. Sitzungs-
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