J. Embryo!, exp. Morph. Vol. 23, 1, pp. 219-236, 1970
Printed in Great Britain
219
The induction of
hair follicle formation in the adult hooded rat
by vibrissa dermal papillae
By R . F . O L I V E R 1
From the M.R.C. Unit for Research on the Experimental
Pathology of the Skin, The University of Birmingham
Hair follicles are essentially composed of two tissues. The inner epidermal
component, which gives rise to, among other products, the keratinized hair
shaft, is confluent with the surface epidermis and is ensheathed by the dermal
component which is confluent with the pars papillaris of the dermis.
A specialization of the dermal component is the dermal papilla which, in
follicles producing hair, is enclosed by the epidermal matrix of the hair bulb and
is connected to the dermal sheath by the papilla stalk.
Many authorities have considered that the dermal papilla is an essential
component of the hair follicle (reviews: Cohen, 1965; Oliver, 1969). It has been
suggested that the dermal papilla may be involved in both the induction of
follicle lengthening and hair growth during the proanagen phase (Chase, 1965)
of the hair cycle, a concept now justified by direct experimentation in the
vibrissa follicle at least (Oliver, 19676), and perhaps also in determining the
nature of the hair produced by a follicle.
It also seems likely that the developing dermal papilla is an important, if not
the primary, source of induction in follicle development during ontogeny
(Jacobson, 1966; Kollar, 1966, 1968). Indeed various authorities have considered that the dermal papilla is capable of inducing follicle formation in the
adult after wounding of the skin (Billingham, 1958; Billingham & Silvers, 1963).
In order to investigate these possibilities directly Cohen (1961) devised
techniques for isolating dermal papillae from excised bulbs from the comparatively large rodent vibrissa follicles, a possibility suggested by Billingham (1958).
Cohen (1961, 1964, 1965) has reported the results of implanting vibrissa
dermal papillae into ear dermis in the adult hooded rat. He has presented
persuasive evidence that where the vibrissa papillae contacted ear epidermis,
whether of follicular or non-follicular origin, they induced by 14 days the
development and growth of ear-type hairs. These papillae underwent a drastic
1
Author's address: Agricultural Research Council, Institute of Animal Physiology
Babraham, Cambridge, U.K.
220
R. F. OLIVER
shrinkage in size to ear follicle papilla proportions, involving a reduction in cell
number from hundreds down to tens of cells. However, where papillae did not
contact epidermis they remained the same size.
More recently Oliver (1968, 1969) has published preliminary accounts of
results of the implantation of vibrissa dermal papillae recombined with epidermis from three different regions of the body into ear dermis. This method was
used, in preference to implantation of papillae on their own, in the hope that it
would more effectively ensure papilla/epidermal contact and that the influence of
papillae on non-follicular epidermis could be evaluated. This report gives full
details of the above and further work.
MATERIALS AND METHODS
All operations were performed on 2- to 4-month-old animals from an inbred
strain of hooded rat. Anaesthesia was induced by the intraperitoneal injection
of a 10 x diluted solution of Nembutal (Abbott), 0-65 ml/100 g body weight.
Operative sequence
At each operation thin slices of skin were removed freehand with a scalpel
from one of the following sites after bathing the donor area with 70 % alcohol :
(i) the dorsum of the right ear. This provides ear epidermis which contains
FIGURE 1
All sections stained with Weigert's haematoxylin, alcian blue and Curtis's Ponceau S.
(A) Section of ear epidermis separated from its dermis with trypsin. Note presence
of follicular epidermis, including sebaceous gland cells, x 190.
(B) Section of afollicular scrotal sac epidermis separated from its dermis with
trypsin, x 160.
(C) Section of oral mucosa epithelium separated from its dermis with trypsin.
Note the compact cuticle and slight maceration of the basal cells some of which are
detached from the epithelial sheet, x 160.
(D) Section of a vibrissa dermal papilla (arrow) in ear dermis 33 days after implantation of dermal papillae alone. The papilla surface is entirely free of epidermal cells.
xllO.
(E-G) Sections of vibrissa dermal papilla/epidermis associations in ear at various
times after implantation.
(E) Implant of ear epidermis becoming locally organized around a single vibrissa
dermal papilla. 14 days, x 110.
(F) Short, stout hair follicle induced from ear epidermis by a single vibrissa dermal
papilla. Note the thick inner root sheath, fine hair shaft and papilla with many cells.
35 days. xllO.
(G) Base of a follicular structure developed from scrotal sac epidermis and incorporating three vibrissa dermal papillae which have fused to form a central 'pulp'.
Note the scalloped inner aspect of the matrix and the lack of recognizable hair
follicle epidermal elements. 21 days, x 80.
Induction of hair follicle formation
221
222
R. F. OLIVER
follicular epidermis, including sebaceous glands (Fig. 1A); (ii) the anterior
region of the stretched scrotum. This provides follicle-free scrotal sac epidermis
(Fig. 1B); (iii) the inner medial surface of the lower lip as far as the base of the
incisors. This provides keratinizing oral epithelium which is, of course, afollicular (Fig. 1C).
The skin slices were incubated in 0-25 % trypsin (Nutritional Biochemicals
Corporation 1:300) for 20-35 min at 37 °C. Some slices were, in fact, incubated
for slightly longer periods which may well have affected the viability of the
epidermis, as is discussed below.
While the skin slices were incubating, the vibrissa follicles on the left upper lip
were exposed, the bulbar region of follicles removed and placed into Hanks's
solution and the lip stitched back into position as described previously (Cohen,
1961 ; Oliver, 1966). Dermal papillae were dissected from the bulbs and put into
fresh Hanks's solution, as described by Oliver (19676). From each rat 1-6 dermal papillae were isolated which were considered to be free of contaminating
epidermal matrix cells. At this stage the epidermis was cleanly separated from
the skin slices in Hanks's solution using watchmaker's forceps and cut into
rectangular sheets ca. 1-3 mm square.
Preparation of host site and implantation procedure
Using a pointed scalpel an L-shaped flap of skin was made then reflected on
the dorsum of the left ear. The horizontal plane of the incision was made in the
dermis, parallel to and between the skin surface and the ear cartilage.
In some of the earlier work the dermal papillae were wrapped in the epidermal
sheets prior to their implantation under the ear flap. However, in the majority of
implants the skin flap was reflected, the dermal papillae placed on the exposed
dermal bed and the appropriate epidermal sheet gently lowered on to the
papillae, basal surface down.
The skin flap was then carefully pulled back into position over the implants
and secured either with a single suture through the apex of the flap or with
Nobecutane (Duncan, Flockhart and Evans Ltd.).
Design of experiments
Details of animals used, number of papillae implanted and times of biopsy
after operation for each of the three types of recombination made are shown in
Tables 1-3. A total of 88 papillae were implanted into 25 rats: 28 papillae in
association with ear epidermis, 36 with scrotal sac epidermis and 19 with oral
epithelium. Five dermal papillae were implanted alone and, in addition, a
limited number of trypsinized but unseparated skin slices and sheets of epidermis
alone were also implanted under flaps in ear. All but three of the grafts were
autografts.
Induction of hair follicle
formation
223
Histology
Implant areas were fixed in buffered formalin or formolsaline fixatives. Serial
sections of the entire implant areas were cut at 8 /i and stained in a combination
of Weigert's haematoxylin, alcian blue and Curtis's Ponceau S.
RESULTS
While the experimental technique employed was essentially very simple, with
the object of ensuring papilla/epidermal contact within ear dermis, many of the
results cannot be interpreted in terms of this persisting anatomical relationship.
Two aspects of the results, summarized in Tables 1, 2 and 3, will therefore be
considered before presenting those results in which papillae were found in
various associations with epidermis.
(i) Number of dermal papillae recovered
Of a total of 88 dermal papillae implanted into ear skin, 76 were found with
certainty and a possible further three may have been present in the biopsy
material. This loss of papillae was not obviously related to duration of implantation since three papillae could not be accounted for in ears biopsied at
7 days (rats I, IX and X), while papillae were often fully accounted for at 35 and
42 days (e.g. rats XVl-XVIII).
(ii) Isolated dermal papillae
Of 83 papillae implanted in association with epidermis, 37, perhaps 39, were
found isolated in ear dermis, as were the five papillae implanted alone into ear.
Sometimes all of the implanted papillae in particular animals were found isolated
but in others some of the papillae were isolated while the remainder were either
unaccounted for or had formed associations with epidermis. Again isolation of
papillae was not related to duration of implantation. All of these isolated papillae were found to be entirely free of adherent epidermal cells (e.g. Fig. ID). In
two rats fusion of papillae had occurred. No obvious reduction in cell number of
the papillae was apparent, although some were compacted and showed a loss of
cellular cytoplasmic volume. One possible degenerated papilla was found (rat
XIII) but the rest appeared healthy and had acquired a capillary supply and
most had taken up the alcian blue stain in varying degrees of intensity. Interestingly three papillae were readily identified at 190 and 207 days after implantation (rats XIX, VIII). These papillae had not stained with alcian blue and
collagen fibres were present between the cells.
(iii) Dermal papillajepidermis associations
A total of 21, possibly 22, papilla/epidermis associations, each containing
from one to four papillae, were found (Tables 1-3).
224
R. F. OLIVER
The distinction has been made between those instances in which papillae had
formed associations with epidermis of questionable origin at the ear surface and
those which had formed intradermal associations with the epidermis with which
they had been implanted.
After implantation with ear epidermis (Table 1)
Both papillae recovered at 14 days were in intradermal contact with the
epidermis with which they had been implanted (rat II). However, one of these
papillae and its epidermis had degenerated. The other papilla was enclosed by
epidermis which was considerably thickened and contained suprabasal cell
divisions (Fig. IE). The inner aspect of this 'matrix' was irregular. Confluent
epidermis lateral to the matrix region was either largely degenerated or completely keratinized.
Table 1. Vibrissa dermal papilla/ear epidermis recombinants in ear skin
Animal
Time in
days
Comments on histology
(Numbers = number of papillae)
Number
of
papillae
in each
recombinant
Number of
papillae
recovered
Isolated
papillae
1
1
1
2
4
4
1
0
0
0
0
2
A
Papilla/surface
epidermis
associations
I
11
7
14
III
IV
V
21
28
35
2
1
1
2
4
5
VI
VJI
VIII
42
42
207
6
4
3
5 or 6
4
2
5 (2 fused)
4
2
] + + + +(?)
0
0
28
24/25
14
8/9
Papilla totals:
Intradermal
papilla/
epidermis
associations
0
0
0
1 (degenerated)
0
1+
2 (fused) +
0
4 (fused) + + +
0
0
1++++
1++++
0
0
0
+ = Epidermal matrix organized around papilla (e.g. Fig. 1 E).
+ + = Development of a 'follicular' structure (e.g. Fig. 1 G).
+ + + = Development of follicular epidermal elements (e.g. Fig. 2F).
+ -I- + + = Follicle producing hair (e.g. Fig. 1 F).
At 21 days two fused papillae were found in contact with the superficial ear
epidermis (rat 111). The epidermis was locally organized as a matrix around the
papillae and had an irregular inner aspect.
All four papillae in rat IV, at 28 days, had become fused and had organized
epidermis into a large 'follicular' structure around them. This structure, which
superficially resembled a developing feather follicle, was confluent with the
superficial epidermis but projected into the ear. Papillary cells extended through-
Induction of hair follicle formation
225
out the length of this elongated, cylindrical structure and again the inner aspect
of the matrix was irregular. The pattern of keratinization was similar to that of
superficial epidermis but considerably thicker keratin was being produced. The
base of this structure was of greatest interest since it showed, in greatly enlarged
form, some of those epidermal elements typical of a hair follicle. Peripherally,
vacuolated cells were recognized as outer root sheath bounding an inner root
sheath, the latter containing trichohyalin granules which are characteristically
found only in hair follicles. Nevertheless, hair keratin was not being produced
from the matrix cells, central to the inner root sheath, nor were there any convincing indications of sebaceous gland histogenesis.
At 35 days, in rat V, two follicles producing hair were found, in each of which
a single vibrissa dermal papilla had been obviously incorporated. Normal
anagen hair bulbs in the mid-dorsal region of the ear, where the implants were
made, contain papillae with between ca. 35 and 55 nuclei. Each of the above two
follicles had very large bulbs and papillae containing hundreds of nuclei but not,
apparently, a vascular supply. One of these follicles was cut transversely and the
other vertically (Fig. 1F). This latter follicle was much shorter than the longest
ear hair follicles but both were stouter than ear hair follicles. Notwithstanding
their comparatively large bulbs they were producing fine non-medullated hairs.
Obviously many of the matrix division products were becoming inner root
sheath rather than hair shaft since there were six cell layers to the inner root
sheath, four contributing to the Huxley layer, in these follicles. Ear hairs
generally have three cell layers comprising the inner root sheath and the much
larger vibrissa follicles have four layers. Interestingly, the inner root sheath in
these follicles extended up to the skin surface rather than disappearing below the
level of the sebaceous glands as in normal follicles. Sebaceous glands had not
fully developed; sebaceous cells were differentiating within small outgrowths
from the neck of each follicle but sebaceous ducts were not present.
At 42 days (rat YI) a follicle with a very large bulb was found but unfortunately the sections were folded in this region and while it seemed likely that it
contained a vibrissa papilla it was impossible to determine this with certainty.
With scrotal sac epidermis (Table 2)
A single papilla was found in contact with superficial epidermis at both 7 and
14 days (rats IX, XI). The local epidermis had formed a thickened matrix in
both of these associations. Also at 14 days (rat XII) three papillae were intradermally associated with the same implant of scrotal sac epidermis. The papillae
were discrete and each had organized a separate matrix.
Rat XIV, at 21 days, showed a structure, confluent with the surface epidermis
and containing three fused papillae as an inner dermal 'pulp', very similar to
that described for rat III. However, there were no indications of hair follicle
epidermal elements (Fig. 1G).
At 28 days (rat XV) three separate papilla/scrotal sac epidermis associations
226
R. F. OLIVER
were found within the ear dermis, two containing single papillae and one two
papillae. A matrix was organized around one of the single dermal papillae.
In the other two associations papillary cells were present over the greater
surface of the epidermis, which was keratinizing into an internal lumen, with
apparent attempts at matrix organization around the focal areas representing
the main papillary masses (Fig. 2 A). There was no development of recognizable
hair epidermal elements in any of these associations.
Table 2. Vibrissa dermal papilla/scrota! sac epidermis recombinants in ear skin
Animal
Number
of
papillae
in each Number of
Time in recompapillae
days
binant
recovered
IX
X
XI
7
7
14
XII
xm
XIV
XV
14
3
1
1
1
3
2
0
1
0
3
21
21
28
2
3
4
Oor 1
3
4
Comments on histology
(Numbers = number of papillae)
t
Isolated
papillae
1
0
0
0
0
Papilla/
surface
epidermis
associations
.1 +
0
1+
0
0
1 (degenerated?)
0
0
3 (fused)+ +
0
0
Intradermal
papilla/
epidermis
associations
0
0
0
0
1+
1+
1+
0
0
1+
1+
XVI
35
XVII
35
XVIII
42
XIX
J 90
Papilla totals:
5
5
0
0
6
5
2
36
6
5
1
30/31
6
5
1
13/14
0
0
0
5
2+
2(fused+ + + +
3 (fused)+ + + +
0
0
0
12
Legend See Table 1.
However, at 35 days (rat XVI), two intradermal structures were found which
were instantly recognizable as extraordinarily large although not perfectly
organized hair bulbs (Fig. 2B, C). One of these follicles contained two and the
largest three dermal papillae. In each follicle a large amount of epidermal matrix
was present around the papillae and giving rise to inner root sheath, hair cuticle
and hair keratin bounded by a layer of outer root sheath cells. The hair keratin
did not display the regular organization presented by naturally occuring hair
follicles. Neither of these two follicles showed the development of sebaceous
glands.
Induction of hair follicle formation
227
With oral epithelium (Table 3)
At 24 days (rat XX) two papillae were in intradermal association with oral
epithelium. As seen in rat XV the papillary cells enclosed most of the surface of
the thickened epithelium which was keratinizing into a central lumen. The
outer surface of the epithelium was locally lobulated and arranged as matrices
with a columnar basal layer around the two main papillary masses (Fig. 2D).
At 35 days (rat XXII) a follicle opening at the ear surface was seen which had
obviously incorporated a single vibrissa dermal papilla. In all essential respects
it was identical to the induced follicles described in rat V, and shown in Fig. 1F.
Also at 35 days (rat XXIII) were found two intradermal papilla/epithelial
associations, each probably containing two papillae. In one of these the papillary
tissue almost completely enveloped the considerably thickened epithelial mass
which, over its greater part, had a highly columnar outer basal layer. In regions
immediately central to the columnar layer, was a layer of cells arranged parallel
to the prominent basement membrane and central to this layer epithelial cells
were present as a stellate reticulum. Two foci of parakeratotic cells were also
present and confluent with a layer of keratinized cells bounding a keratin-filled
central lumen (Fig. 2E). Near to this implant were two separate areas of
intramembranous ossification.
In the second and larger of these two associations was a more familiar matrix
region, irregularly enveloping papillary tissue and giving rise to inner root sheath
which was bounded by a layer of typically vacuolated outer root sheath cells.
Intriguingly, the central core of this structure, capping the papillary tissue, was
present as an epithelial stellate reticulum which was separated from the inner
root sheath by a layer of parakeratotic cells (Fig. 2F). Hair shaft could not be
identified and there were no indications of sebaceous gland histogenesis.
(iv) Subsidiary experiments
Single implants of trypsinized but unseparated ear skin, scrotal sac skin and
oral mucosa were recovered at 42, 35 and 44 days respectively. The ear skin had
encysted but otherwise had a normal epidermis and follicles in catagen and
telogen. The scrotal sac implant, which had contacted the surface epidermis,
had an apparently normal non-pigmented epidermis without follicles, but had a
smoother than normal dermal/epidermal interface. The oral mucosa implant
had encysted, forming a distended sac packed with keratin. The thin, flattened
epithelium was non-pigmented and afollicular.
Implants were also made of ear epidermis alone (recovered at 42 days),
scrotal sac epidermis (recovered at 28, 35 and 42 days) and oral epithelium
(recovered at 24 days). Most of these presented a picture of disorganized intradermal cell masses, with no organization of a basal cell layer, although attempts
at keratinization were seen in one of the scrotal sac epidermis implants. Occasionally tongues of viable epidermal cells, confluent with the surface epidermis,
15
EMB 23
228
R. F. OLIVER
Induction of hair follicle formation
229
were seen protruding into the dermis but it could not be ascertained whether
they derived from native ear epidermis or from implanted epidermis.
Generally speaking the epidermis implanted with papillae, and not locally
organized by the papillae, also showed degenerative changes. However, cysts,
keratinizing internally and containing neither follicles nor sebaceous glands,
were seen formed from ear epidermis (rat VII), scrotal sac epidermis (rat XVI ;
Fig. 2B) and oral mucosa epithelium (rat XXIII; Fig. 2F).
(v) Other comments
No special study was made of the stage of the growth cycle of the ear hair
follicles at the time of operation. However, it is probable that most, if not all, of
the implant regions contained follicles in the resting phase since anagen follicles
are rarely seen in undisturbed ear skin taken from rats of the same age as those
used for operations. Apparently the implantation procedure coincided with or,
FIGURE 2
All sections stained with Weigert's haematoxylin, alcian blue and Curtis's Ponceau S,
(A-F) Sections of vibrissa dermal papilla/epidermis associations in ear dermis at
various times after implantation.
(A) Implant of scrotal sac epidermis keratinizing into a central lumen with localized
hyperplasia and attempts at matrix formation, with columnar basal cells, in
association with vibrissa papillary cells (left and especially right). 28 days, x 80.
(B) Huge, irregular hair bulb which has developed from scrotal sac epidermis and
three vibrissa dermal papillae, showing vacuolated outer root sheath cells (arrow)
and epidermal matrix and its differentiation products around the fused papillary
tissue. Note that scrotal sac epidermis unorganized by the papillae has formed a thinwalled cyst at left. 35 days, x 80.
(C) Oblique section through a large hair bulb which has developed from scrotal sac
epidermis and two vibrissa dermal papillae. Note the vacuolated outer root sheath
(top arrow), inner root sheath (middle arrow) and keratinizing hair (lower arrow).
35 days, x 80.
(D) Implant of oral mucosa epithelium which has thickened and is keratinizing into
a central lumen. Note localized attempts at matrix formation, with columnar basal
cells, in relation to vibrissa papillary tissue (at bottom and especially top). Suprabasal cell morphology and compact cuticle resemble normal mucosa epithelium.
24 days, x 80.
(E) Implant of oral mucosa epithelium surrounded by vibrissa papillary tissue. The
columnar basal cells are separated, at right and left, from an epithelial stellate
reticulum by a distinct intermediate cell layer. Note the two nidi of parakeratotic cells
confluent with the inner keratinized layer. 35 days, x 80.
(F) Implant of oral mucosa epithelium which has been organized by vibrissa papillary tissue at the top and has formed a confluent thin-walled epithelial cyst below.
At top are seen the typical hair follicle epidermal elements, outer root sheath (left
arrow) and inner root sheath (middle arrow), which enclose not developing hair shaft,
but a parakeratotic layer surrounding an inner epithelial stellate reticulum (right
arrow). The stellate reticulum contacts papillary tissue via a columnar basal cell layer
in other sections. 35 days, x 80.
15-2
230
R. F. O L I V E R
much more likely (Argyris & Trimble, 1964), precipitated a new hair growth
cycle in the implant areas for, at 7 days, follicles in the biopsied regions were in
proanagen and producing hair by 14 days. Local ear hair follicles were at their
maximum length at 21 and 28 days but most had entered catagen by 35 days
and, with some exceptions, were in telogen by 42 days.
Unfortunately little information was obtained of the wound healing process
which follows the implantation procedure used. Sections through the scab
present in rat IX showed that much of the original ear skin flap had been undergrown by epidermis from the wound edges and was being exfoliated. A similar
process may have occurred in other animals, especially where the epidermis
Table 3. Vibrissa dermal papilla/oral mucosa epithelium recombinants in ear skin
Animal
Number
of
papillae
in each
Time in recombinant
days
Comments on histology
(Numbers = number of papillae)
A
i
Number of
papillae
recovered
Isolated
papillae
Papilla/
surface
epidermis
associations
Intradermal
papilla/
epithelium
associations
XX
XXI
XXII
XXIII
24
28
35
35
3
3
4
4
3
2
4
4
1
2
3
0
0
0
1+ + + +
0
XXIV
42
5
4 or 5
0
19
17/18
4 or 5
(2 or 3 fused)
10/11
2+
0
0
2+
2+ + +
0
1
6
Papilla totals:
Legend See Table 1.
covering the implant area was devoid of hair follicles, and this is the only
reasonable explanation for the presence of dermal papillae immediately under
the surface epidermis.
Rigorous examination of all sections showed no indications of neoformation
of hair follicles except, of course, those follicles which had developed in association with the implanted vibrissa dermal papillae.
DISCUSSION
Fulfilment of experimental requirements
A proper investigation of the influence of vibrissa dermal papillae on epidermis essentially requires that viable papillae, known to be entirely free of
contaminating epidermal cells, are brought into uninterrupted contact with
viable epidermis in a site conducive for the growth of hair.
Ear dermis has been shown to support the growth and regeneration of
Induction of hair follicle formation
231
vibrissae, albeit of a stunted nature, from grafts of the bulbar and follicle wall
regions of vibrissa follicles (Cohen, 1961; Oliver, 1967 a).
The majority of the papilla implants were obviously viable since only one,
possibly two, out of 76, perhaps 79, recovered 7-207 days after implantation
showed degenerative changes.
There can also be little doubt that most, if not all, of the vibrissa dermal
papillae were uncontaminated with vibrissa epidermal cells. Oliver (19676) has
already observed that serial sections of vibrissa dermal papillae, dissected from
vibrissa bulbs by the same method used in this work, show the papillae to be
free of epidermal cells. Corroborative evidence is also convincingly available
from the present results. Of a total of 88 papillae implanted into ear skin,
including the five papillae implanted alone, 42, perhaps 44, were found isolated
in the dermis and all were entirely free of adherent epidermal cells. It would
appear unlikely that the 34 or 35 papillae found in association with epidermis,
often obviously graft epidermis, also happened to be papillae contaminated
with epidermal cells of vibrissal origin.
The fact that only 41 % of the papillae were found in association with epidermis raises another requirement necessary to validate the experiment: uninterrupted papilla contact with viable epidermis. It is highly possible that in some
instances papillae became displaced from under their epidermal sheets on
repositioning of the skin flap at operation. An alternative and perhaps additive
explanation is that the viability of some of the epidermal sheets may have been
impaired by too long exposure to the effects of trypsin. Billingham & Silvers
(1967) emphasize that trypsinization at 37 °C may result in variable degrees of
epidermal maceration, and thus the loss of germinal basal cells, as was occasionally seen in the present work. Such an effect may explain the degenerated
appearance of many of the intradermally sited implants of epidermis, whether
implanted alone or with papillae. This explanation gains credence from those
examples of epidermis which did manage to maintain some organization.
Presumably here epidermal viability was less impaired by the trypsin although
to what extent epidermal organization is dependent on a suitable substrate for
the basal layer cannot here be assessed (cf. Van Scott & Flaxman, 1968).
Twelve, but possibly only nine papillae (10-14 %), were not identified in the
biopsy material which may be accounted for by one or more of the following
reasons: non-inclusion in the biopsy area (although great care was taken to
avoid this eventuality), exfoliation from the skin with the skin flaps by epidermal
undergrowth during the wound healing process, degeneration and resorption
of papillae or incorporation of papillae into new ear-type follicles indistinguishable from native follicles. The fact that three papillae were not identified at
7 days strongly suggests that these at least were lost in the scab. Cohen (1964)
could only account for 9 out of 57 papillae implanted alone into ear and thought
that while some at least were exfoliated, others probably became incorporated
into new ear-type follicles.
232
R. F. OLIVER
Influence of vibrissa dermal papillae on epidermis
It has already been demonstrated that vibrissa dermal papillae, implanted
into the bases of the superficial halves of vibrissa follicles, induce lengthening of
the follicle and the growth of whiskers (Oliver, 19676). In the present studies
none of the implants of epidermal sheets alone showed any indication of follicle
organization, rather most showed degenerative changes. This emphasizes both
the profound influence of vibrissa papillae on epidermis and the pluripotential
characteristics of epidermis since not only can vibrissa papillae induce follicle
formation from ear epidermis but also from afollicular scrotal sac epidermis
and from oral epithelium.
A variety of papilla/epidermis associations were seen, some containing single
and others fused papillae. The development of follicles seemed to involve the
following incomplete sequence of events. The epidermis enveloped one or more
papillae to become locally organized into a matrix containing suprabasal
mitoses (7-14 days). The development of follicular structures, without elements
characteristic of hair follicles, then occurred (21 days), and by 28 days the
development of an outer root sheath separated from the matrix by an inner root
sheath was observed, but without the growth of hair. However, at 35 days,
examples were found of follicles producing either hair, or at least structures
resembling it, some with developing but non-functioning sebaceous glands.
Obviously time is an important factor in these events and in a previous publication (Oliver, 1968) I erroneously suggested that vibrissa papillae were unable to
induce follicle formation since there were no indications that this was occurring
up to 3 weeks after implantation.
The papillae in those follicles which incorporated single vibrissa papillae,
although obviously much larger than local ear follicle papillae, were smaller
than when implanted. This may have been due to the fact that they now did not
contain capillaries; the papillae may also have contributed to the new dermal
sheaths as seems to occur after implantation into vibrissa follicles.
These follicles also differed from ear follicles in that they were shorter and
stouter with comparatively large bulbs, yet producing fine, non-medullated
hairs. The first hairs produced during ontogenetic development may also be
non-medullated (Davidson & Hardy, 1952; Montagna, 1962). The presence of
the inner root sheath extending as far as the skin surface, associated with the
presence of non-functional sebaceous glands, would seem to relate to the suggestion that sebum may contain enzymes which normally effect the dissolution or
fragmentation of the inner root sheath at this level (Straile, 1965).
Comparatively huge intradermal follicles had developed from scrotal sac
epidermis and oral epithelium where they had incorporated two or more
papillae.
In other associations papillary tissue was seen to invest, rather than be invested
by, epidermis which had encysted. Even in these instances the papillae promoted
Induction of hair follicle formation
233
epidermal hyperplasia. The development of an epithelial stellate reticulum
adjacent to a highly columnar basal layer in two of the oral epithelia implants
associated with papillary tissue is most intriguing, since this configuration is
presented by the enamel organ of developing teeth. It would appear that here
papillary cells are promoting the expression of a potential in the mucosal
epithelium to form the epithelial contribution which is required for the development of teeth.
It is worth noting that isolated dermal papillae can persist for up to 207 days
in ear dermis; obviously their integrity is not dependent on contact with
epidermal cells.
Tract specificity
So far the results have only been considered in terms of the dermal papilla
and epidermis. However, it has been demonstrated that many characteristics
of the superficial epidermis in adult mammals, but not necessarily mucosal
epithelia, are determined and maintained by specific and persistent stimuli from
the underlying dermis (Billingham & Silvers, 1965, 1967, 1968).
Cohen (1965, 1969 a) has extended such findings to account for the determination of tract specificity in skin appendages (reviews: Cohen, 19696; Oliver,
1969). As mentioned previously Cohen considers that those vibrissa dermal
papillae implanted into ear skin which contact ear epidermis induce the formation of ear-type follicles by 14 days. He visualizes the induction of follicle
formation by the papillae and the determination of follicle type by the local
dermis. The present results showed no evidence of the development of ear-type
hairs nor of the shrinkage of vibrissa papillae to ear hair proportions where
they contacted epidermis. The characteristics of the follicles formed did not
readily implicate the local dermis or the epidermis which invested the papillae
as determinants of follicle specificity. Rather the size of the matrix of these
follicles, but not necessarily the size of the hair shaft produced, seemed to be
related to the amount of papillary tissue present. It is conceivable that in Cohen's
work cellular viability in the papillae was in some way reduced, before or at
implantation, so that follicles were formed with small papillae. However, it
may be relevant that at 14 days, when Cohen considered such follicles to be
forming, the local ear hair follicles, sometimes disorientated by the implantation
procedure, are in various stages of early anagen when they could perhaps be
interpreted as newly forming follicles.
Papilla/epidermis, and particularly oral epithelium, recombinants will need
to be left in ear dermis for much longer periods than reported here. Induced
follicles might then assume recognizable tract characteristics. It would also be
of great interest to discover whether the formation of an epithelial stellate
reticulum in oral epithelium/papilla recombinants is a reproducible phenomenon
and if so whether it is a terminal or an intermediary product. The behaviour of
recombinants could also be evaluated in non-skin environments.
234
R. F. O L I V E R
Refinement of present techniques could lead to more closely controlled
follicle formation in the adult. This would permit a more detailed causal analysis
of the factors involved in follicle formation and such problems as the determination of tract specificity than has been previously possible.
SUMMARY
1. The influence of vibrissa dermal papillae on epidermis of non-vibrissal
origin has been investigated in the adult hooded rat.
2. A method is described for preparing and implanting recombinants of
vibrissa dermal papillae and ear epidermis, afollicular scrotal sac epidermis and
oral epithelium into ear dermis.
3. A variety of dermal papilla/epidermis associations were found demonstrating a profound localized influence by dermal papillae on epidermis, including
formation of new hair follicles by 35 days.
4. It was thus conclusively shown that vibrissa dermal papillae can induce the
formation of hair follicles and hair growth, even from previously afollicular
epidermis.
5. Possible factors involved in determining the characteristics of these follicles
are discussed.
6. Dermal papillae isolated in ear dermis maintained their integrity for up to
207 days after implantation.
7. Epidermis from ear and scrotal sac skin and oral epithelium implanted
alone into ear generally appeared disorganized or degenerated, with no regeneration of hair follicles.
RÉSUMÉ
Induction de la formation de follicules pileux
chez le rat mantelé adulte par les papilles dermiques des vibrisses
1. L'influence des papilles dermiques des vibrisses sur de l'épiderme d'autre
origine a été étudiée chez le rat mantelé adulte.
2. On décrit une méthode de préparation et d'implantation, dans le derme
de l'oreille, de réassociations de papilles dermiques de vibrisses avec de l'épiderme de l'oreille, ou de l'épiderme afolliculaire du sac scrotal, ou encore de
J'épithélium buccal.
3. On constate que diverses associations papilles dermiques/épiderme révèlent une profonde influence localisée des papilles dermiques sur l'épiderme,
aboutissant, entre autres, à la formation de nouveaux follicules pileux au bout
de 35 jours.
4. On a donc montré de manière concluante que les papilles dermiques des
vibrisses peuvent induire la formation de follicules pileux et la croissance des
poils, même à partir d'épiderme d'origine afolliculaire.
5. Les facteurs éventuels impliqués dans la détermination des caractéristiques
de ces follicules sont discutés.
Induction of hair follicle formation
235
6. Des papilles dermiques isolées et implantées dans le derme de l'oreille,
gardent leur intégrité jusqu'à 207 jours après l'implantation.
7. L'épiderme de l'oreille et de la peau du sac scrotal ainsi que répithélium
buccal, implantés seuls dans l'oreille, apparaissent en général désorganisés ou en
dégénérescence, ne montrant aucune régénération des follicules pileux.
I would like to express my thanks to Dr C. N. D. Cruickshank, Director of the M.R.C. Unit
for Research on the Experimental Pathology of the Skin, The University of Birmingham, for
his valuable suggestions and Mr J. Cooper and Mr N. Newbold for their technical assistance.
REFERENCES
ARGYRIS, T. S. & TRIMBLE, M. E. (1964). On the mechanism of hair growth stimulation in
wound healing. Devi Biol. 9, 230-54.
BILLINGHAM, R. E. (1958). A reconsideration of the phenomenon of hair neogenesis, with
particular reference to the healing of cutaneous wounds in adult mammals. Jn The Biology
of Hair Growth. Ed. W. Montagna and R.A.Ellis, pp. 451-68. New York: Academic
Press.
BILLINGHAM, R. E. & SILVERS, W. K. (1963). The origin and conservation of epidermal
specificities. New Engl. J. Med. 268, 477-80, 539-45.
BILLINGHAM, R. E. & SILVERS, W. K. (1965). Some unsolved problems in the biology of skin.
In Biology of the Skin and Hair Growth. Ed. A. G. Lyneand B. F. Short, pp. 1-24. Sydney:
Angus and Robertson.
BILLINGHAM, R. E. & SILVERS, W. K. (1967). Studies on the conservation
of epidermal
specificities of skin and certain mucosas in adult mammals. J. exp. Med. 125, 429-46.
BILLINGHAM, R. E. & SILVERS, W. K. (1968). Dermoepidermal interactions and epithelial
specificity. In Epithelial-Mesenchymal Interactions. Ed. R. Fleischmajer and R. E.
Billingham, pp. 252-66. Baltimore: Williams and Wilkins.
CHASE, H. B. (1965). Cycles and waves of hair growth. In Biology of the Skin and Hair
Growth. Ed. A. G. Lyne and B. F. Short, pp. 461-65. Sydney: Angus and Robertson.
COHEN, J. (1961). The transplantation of individual rat and guinea-pig whisker papillae.
J. Embryo/, exp. Morph. 9, 117-27.
COHEN, J. (1964). Transplantation of hair papillae. Symp. zool. Soc. Lond. 12, 83-96.
C O H E N , J. (1965). T h e dermal papilla. In Biology
of the Skin
and Hair Growth E d . A. G .
Lyne and B. F. Short, pp. 183-99. Sydney: Angus and Robertson.
COHEN, J. (1969 A). Dermis, epidermis and dermal papilla interacting. In Hair Growth.
Ed. W. Montagna and R. L. Dobson, pp. 1-18. London: Pergamon Press.
COHEN, J. (19696). Interaction in the skin. Br. J. Derm. 81, Suppl. 3, 46-54.
DAVIDSON, P. & HARDY, M. H. (1952). The development of mouse vibrissae in vivo and in
vitro. J. Anat. 86, 342-56.
JACOBSON, C. M. (1966). A comparative study of the mechanisms by which X-irradiation
and genetic mutation cause loss of vibrissae in embryo mice. J. Embryol. exp. Morph. 16,
369-79.
KOLLAR, E. J. (1966). An in vitro study of hair and vibrissae development in embryonic mouse
skin. / . invest. Derm. 46, 254-62.
KOLLAR, E. J. (1968). The inhibition of vibrissa development in vitro by beta-2-thienylalanine.
J. invest. Derm. 50, 319-22.
MONTAGNA, W. (1962). The Structure and Function of Skin. New York: Academic Press.
OLIVER, R. F. (1966). Whisker growth after removal of the dermal papilla and lengths of
follicle in the hooded rat. / . Embryol. exp. Morph. 15, 331-47.
OLIVER, R. F. (1967a). Ectopic regeneration of whiskers in the hooded rat from implanted
lengths of vibrissa follicle wall. J. Embryol. exp. Morph. 17, 27-34.
OLIVER, R. F. (19676). The experimental induction of whisker growth in the hooded rat by
implantation of dermal papillae. / . Embryol. exp. Morph. 18, 43-5.1.
236
R. F. O L I V E R
OLIVER, R. F. (1968). The regeneration of vibrissae—a model for the study of dermal-epidermal interactions. In Epithelial-Mesenchymal Interactions. Ed. R. Fleischmajer and R. E.
Billingham, pp. 267-79. Baltimore: Williams and Wilkins.
OLIVER, R. F. (1969). The vibrissa dermal papilla and its influence on epidermal tissues. Br.
J. Derm. 81, Suppl. 3, 55-65.
STRAILE, W. E. (1965). Root sheath—dermal papilla relationships and the control of hair
growth. In Biology of the Skin and Hair Growth. Ed. A. G. Lyne and B. F. Short, pp. 35-57.
Sydney: Angus and Robertson.
VAN SCOTT, E. J. &FLAXMAN, B. A. (1968). Environmental control of epithelial cells in vivo
and in vitro. In Epithelial-Mesenchymal Interactions. Ed. R. Fleischmajer and R. E. Billingham, pp. 280-94. Baltimore: Williams and Wilkins.
{Manuscript received 31 March 1969)