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The effects of grafting during various stages of the hair growth cycle.

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The Effects of Grafting During Various Stages
of the Hair Growth Cycle
BARBARA H. SANFORD,’ HERMAN B. CHASE,2 SARAH B. CARROLL
AND CLAUDETTE T. ARSENAULT
Department of Surgery and Huntington Memorial Laboratories,
Hantard Medical School at Massachusetts General Hospital,
and the Biology Department, Brown University
ABSTRACT
Gross and microscopic observations of graft and surrounding area were
made after grafting during various stages of the hair growth cycle.
All grafts were successful although temporary disturbance was observed when
grafting was done during late anagen.
Biopsies showed epidermal breakdown, even in telogen grafts, with repair being
accomplished primarily by diversion of hair follicles to epidermal production. The
later i n anagen a graft was done, the less likely the follicles were to return to hair
production later.
Permanent loss of pigment was seen i n some grafts, particularly those done
i n late anagen. Loss of pigment cells or their attachments from scraping the graFt
undersurface is a possible explanation, as is loss or damage to melanocytes while
the follicles are producing epidermis.
Delays and irregularities in hair growth cycles were also observed in host skin
surrounding grafts. A n explanation involving loss of inhibitor during grafting is
proposed.
The authors feel that hair growth activity in either donor or host is not a n adequate explanation for graft rejection. It is also emphasized that hair growth is a poor
criterion for graft survival.
The relationship between skin grafting
and growth cycles has received scant attention except for the warning that “actively
growing” skin should be avoided because
of the danger of technical rejections due
to non-specific necrosis (Billingham et al.,
’54; Billingham, ’62). We felt that it
should be of some interest to study the
behavior of the graft and the surrounding
area after grafting during various stages
of the hair growth cycle.
Since plucking of club hairs initiates
new hair growth (Collins, ’18; David, ’34),
it is possible to perform grafts at known
stages of the hair growth cycle. The
terminology of Dry (’26) has been followed
with “anagen” referring to active proliferation, “catagen” to cessation of proliferation, and “telogen” to the resting stage.
Anagen has been further broken down into
six stages (Chase et al., ’51), referred to
as anagen I through anagen VI.
Anagen I is the period when cells of the
resting follicle go into cell division and
occurs about one day after plucking in the
45- to 50-day-old C3H mice used in these
experiments.
ANAT. REC., 252: 17-24.
About two days after plucking, there is
a downgrowth of the follicle around the
dermal papilla. This substage is called
anagen 11.
By three days after plucking the first
pigment cells appear in the bulb, and the
base of the follicle stretches downward.
This period is referred to as anagen 111.
Anagen IV is found four or five days
after plucking. At this time the hair shaft
is clearly defined, and the tip of the hair
extends to the base of the sebaceous gland.
The base of the follicle extends at least
500 c1 below the skin surface and usually
does not go any deeper.
During anagen V the hair tip reaches
the skin surface and the bulb is at its
maximum diameter. This stage is reached
about seven days after plucking. The skin
is visibly darkened by this time.
1 This work was supported in part by Public Health
Service Research Grant 1-501-FR-05182-01 from the
Division of Research Facilities and Resources and by
CRTY 5018(C3) and 4 CA 5018-08 from the National
Cancer Institute. This is publication no. 1205 of the
Cancer Commission of Harvard University.
2 This work was supported in part by United States
Public Health Service Grant CA-00592-13 to Brown
University and A.E.C. Contract AT(30-1)-2018 to
Brown University.
17
18
B. H. SANFORD, H. B. CHASE, S. B. CARROLL A N D C. T. ARSENAULT
Growth then continues through anagen
VI which lasts for eight or nine days. At
this time the critical formative stages have
already occurred, and this is predominantly
a period of rapid growt:h.
In this study, we were particularly concerned with the effects of grafting during
the various sub-stages of anagen as compared with telogen.
MATERIALS AN11 METHODS
Reciprocal grafts were performed between pairs of C3H/A mice of the same
sex at various stages of the hair growth
cycle. The cycle was controlled by plucking telogen hairs, thereby initiating growth
of the next hair generation. Males were
plucked at 45 days of aige and females at
50 days, just after comp:letion of the second
generation of hair growth (Eaton, ’59).
Telogen grafts were done 22 to 28 days
after plucking, when the C3H/A animals
had regained their normal coats. Timed
grafts were also done during various stages
of anagen and between anagen and
telogen.
The grafting procedure followed was
essentially that used in T. S. Hauschka’s
laboratory at Roswell Park Memorial Institute, Buffalo, N. Y. (Hauschka et al.,
’59) and involved placing a 1.8 centimeter
pinch graft upon the dorsal body wall
musculature. Both painniculus carnosus
and panniculus adiposus were removed
from the graft before it was applied. The
graft was rotated 90” and was secured to
the surrounding skin with Michel wound
clips. No dressings were required and
daily observations could therefore be made
throughout the healing period.
Biopsies were taken at regular intervals,
fixed i n 10% neutral lbuffered formalin,
embedded in paraffin, sectioned serially,
and stained with hematoxylin and eosin.
For further study some sections were also
stained with Masson’s Trichrome.
Mice were caged individually for the
first 3 to 4 weeks after grafting, during
which time observations of the graft and
surrounding area were made five times
weekly. After this period, they were caged
together in groups of 8 to 10 and observed
at weekly intervals.
OBSERVATIONS
Graft obseruations - gross
Regardless of the stage of the hair
growth cycle a t the time of grafting, all
grafts were essentially “takes” (table 1).
No contracture of the graft area was seen,
and some sideways hair (graft-oriented)
appeared in all cases (fig. 1). Hair was
sparse, however, on grafts done in late
anagen VI, and the majority of hairs on
such grafts were white. Predominantly
white hairs were seen in anagen V and
early anagen VI grafts and some white
hairs were occasionally seen on grafts done
during anagen I11 and IV, but the hair
density in most of these appeared to be
normal.
Most grafts done i n anagen V and VI
formed dark, dry “scabs” which dropped
off in the third week after grafting, exposing relatively healthy looking skin which
in many cases had already begun to appear
dark as a result of pigment in the hair
follicles. The later in anagen VI the grafts
were done, the more pronounced was the
disturbance; for example, most grafts done
eight days post-plucking eventually resulted in thick mixed agouti and white
sideways hair, while most grafts done 11
days post-plucking showed only sparse
white hairs.
A hair cycle was initiated i n the graft
after a variable lag period. The telogen
graft did not reach anagen V (obvious skin
darkening) for about 16 days. This represents a noticeable delay when compared
with plucked non-grafted controls which
reach a comparable stage in about six days.
Progress of the cycle was delayed (rather
than hastened) by taking a graft from a n
animal already in anagen, with the anagen
underway at the time of grafting being
aborted and a new anagen later beginning.
Graft observations - microscopic
Even in telogen grafts, the epidermis
sloughed and was replaced, mainly from
hair follicles within the graft (fig. 2). By
seven days post-grafting, epidermis could
be seen forming from sebaceous glands
and hair follicles, with islands of new
epidermis interspersed among areas where
epidermis was still absent. Around the
edges, epidermis could be seen invading,
19
GRAFTING AND HAIR GROWTH CYCLE
TABLE 1
Observations of grafts - (median days post-grafting; range given in parenthesis)
First
observation
of hair
Graft type
No. of
mice
Appearance
of “scab”
Non-grafted controls
20
none
Telogen
15
none
18( 18-21)
Thick sideways
pigmented hair
Anagen I and I1
8
none
15( 14-16)
Thick sideways
pigmented hair
Anagen I11
8
none
17( 12-18)
Thick sideways
pigmented hair,
usually mixed
with white
Anagen IV
6
none
17( 17-20)
Thick sideways
pigmented hair,
occasionally
mixed with
white
Anagen V
10
8( 7-9 )
14(11-16)
22(1446)
Usually thick
sideways mixed
pigmented and
white hair;
occasionally
sparse
Early anagen V1
13
9( 8-14)
15( 12-24)
18( 13-28)
Usually thick
sideways mixed
pigmented and
white hair;
occasionally
sparse
Late anagen VI
16
12(11-16)
17( 14-23)
25( 18-30)
Sparse, mostly
white, sideways
hair
Telogen to early
anagen VI
6
none
18( 17-18)
Thick sideways
pigmented hair
Early anagen VI
to telogen
6
14(14-15)
21(21-24)
Moderately thick,
mixed pigmented and
white hair
1
“Scab”
sloughed
9( 8-9 )
18(18-19)
End result
Thick sideways
pigmented hair
Hair also observed during first week after grafting but sloughed i n “scab.”
but this appeared limited to the marginal
wound area. No follicles were observed
in this migrating tissue. By 14 days after
grafting, follicles could be seen producing
hair again and appeared normal in structure and in density.
When grafting was performed during
late anagen, there was more delay in initiation of repair, but by the ninth day
marked involvement of follicles in epidermal replacement was seen (fig. 3 ) . The
“scab which was seen on these grafts con-
sisted largely of epidermal remnants, keratinized material and blood. Most of the
graft dermis remained intact during resurfacing by graft follicles. Again there
was some contribution from contiguous
epidermis in the marginal area.
By 14 days post-grafting, anagen V
grafts showed extensive epidermal hyperplasia, extending upward from follicular
remnants, with very few hair follicles remaining intact. Yet at 30 days a normally
dense population of healthy follicles, com-
20
B. H . SANFORD, H. B. CHASE, S. B. CARROLL A N D C. T. A R S E N A U L T
plete with sebaceous glands, had reappeared (fig. 4).
In contrast, anagen VI grafts showed
little or no return of normal hair follicles.
At 9 to 14 days post-grafting, biopsies
showed pronounced disturbance of the
dermal-epidermal rela.tionship with epithelial cells from the follicles extending
down as well as up and. losing contact with
the dermal papillae (fig. 5). By 30 days
post-grafting, the graft consisted of healthy
looking skin but was almost devoid of follicles and associated appendages (fig. 6).
OBSERVATIONS I1
Surrounding area
When an area of mouse skin is plucked,
the usual result is an1 even regrowth of
hair which is completed 17 to 19 days after
plucking (Chase et al., '51). Wounding,
without plucking, also tends to initiate hair
growth in the area surrounding the wound
(Argyris, ' 5 3 ) .
Animals grafted during early anagen
showed delay in initiation of hair growth
in the surrounding area (table 2). Among
those grafted during anagen I to I11 there
was often a patchy overall appearance,
with some areas remaiining pink and bald
even after other simultaneously plucked
sections of the dorsum of the same mouse
had thick hair growth. This patchiness varied in pattern from one animal to another,
and it was noteworthy that the growing
areas were irregular and lacked the usual
rounded appearance of normal growth
waves. Grafting during anagen IV did not
produce patchiness, but grafting during
anagen V or early anagen VI resulted in
prolonged patchiness in about half the
hosts. By late anagen VI, hair growth appeared to be far enough advanced to continue unaffected by the insult of grafting.
Telogen hosts produced hair in the surrounding area after a delay of several days
before entering active growth. In these
telogen hosts patchy hair growth in the
surrounding areas was sometimes seen,
particularly in animals grafted soon after
entering telogen. (Some follicles may still
have been in catagen.)
In contrast to the grafts themselves in
which hair cycles already in progress are
aborted, hair growth in the surrounding
areas apparently is determined by the time
of plucking rather than the time of grafting. Although a delay in the cycle was
regularly seen after grafting in early anagen, this was not of such magnitude as to
permit abortion of one cycle and initiation
of another.
TABLE 2
Observations on surrounding area. End result i n all cases was normal regrowth o f pigmented hairs.
(Median days post-grafting; range given i n parentheses)
Graft type
Non-grafted controls
z;zf
20
First
observation
of hair
9( 8-9 )
Observation of patchiness
None
15
13(13-14)
Prolonged patchiness seen in four hosts
grafted early in telogen
Anagen I and I1
8
13( 9-13)
Most showed some patchiness during first
week of hair growth
Anagen I11
a
7( 6-18)
Most patchy for week or more
Anagen IV
6
6( 5-7 )
None
Anagen V
10
Early anagen VI
13
Late anagen VI
16
continued to grow
Telogen to anagen VI
6
continued to grow
None
Anagen VI to telogen
6
15(14-15)
None
Telogen
3 ( 2-4
1
1( 1-2 )
4/10 showed patchiness for over two
weeks
6/13 were patchy for two weeks or more
None
GRAFTING A N D HAIR GROWTH CYCLE
DISCUSSION
The reconstruction which occurs within
a skin graft gives a clear picture of the
plasticity of the hair follicle. Even in the
telogen graft the original epidermis breaks
down and is replaced mainly by epithelial
cells from the follicle in a process resembling that reported by Bishop after wounding in man (Bishop, ’45). In telogen the
repair is quickly accomplished and the
follicle reverts back to its function as a
hair producing structure.
In early anagen, the follicle apparently
is also able to provide epidermis for the
graft surface and then reconvert to hair
production. As anagen proceeds, however,
it seems to become increasingly difficult
for the follicle to change its course without
contributing to its own destruction as a
hair-producing unit. By late anagen VI the
follicles break down completely during epidermal proliferation and lose contact with
the dermal papillae. Thus the graft is
ultimately reconstructed but only at the
expense of hair follicles and related structures. A contributory factor to the sparsity
of hair on such grafts may be physical
damage associated with removal of the
panniculus carnosus and panniculus adiposus from the graft; however, this is
somewhat unlikely since grafting done four
and five days post-plucking (at which time
follicles have already reached their ultimate depth in the skin) results in thick
hair growth.
When grafts with sparse white hairs
were replucked, the new hair growth was
again sparse and white indicating that the
failure to produce pigment was not a temporary effect. Further studies are under
way to determine the basis for this pigment
loss. Pigment cells or their attachments to
epithelium may have been irreversibly
damaged by vigorous scraping of the undersurface of anagen grafts. Other possible explanations include loss or damage
to melanocytes while the follicle is diverted to epidermal production or even
neoformation of follicles without incorporation of functional melanocytes.
The transient disturbance in late anagen
grafts may partly reflect the presence of a
more substantial “ghost” which must be
removed. Hairs on these grafts often continue to grow for a few days before this
21
hair cycle is aborted. Bleeding is also more
substantial in the more vascular hosts in
late anagen and doubtless contributes to
the formation of a “scab.” The increased
vascular requirements of late anagen may
also be a factor in graft behavior since a
temporary failure of blood supply to the
follicles at this time might well lead to
their degeneration.
Delays and irregularities in the hair
growth cycles after grafting are of interest
since it is generally recognized that it is
extremely difficult to interfere with a cycle
once it is under way. When an area is
plucked there is normally an even regrowth of hair; however, if a graft is
applied within the plucked area a patchy
appearance of hair is often observed.
Whereas plucking is assumed to initiate
growth in resting follicles by decreasing an
inhibitor which is then built up again during anagen, a further interference such as
grafting, wounding, or even another pretelogen plucking could disrupt the whole
sequence of events set in motion by the
reduced inhibitor. A low level of the inhibitor may be a major factor in initiation
of growth, but the level is not a factor in
subsequent events until at least catagen.
From grafting during telogen it is observed that the earlier the graft is made
the greater is the tendency toward patchiness of new growth in the surrounding
area. This observation could be interpreted
as a reflection of the inhibitor concentration which by late telogen is sufficiently
low so that the wound of grafting easily
leaches out the remaining small amount of
inhibitor in all the neighboring follicles.
In earlier telogen some follicles axe not yet
sufficiently near the critical threshold to be
influenced, at least at a distance, by this
wound in the skin (Chase, ’54).
From a technical point of view it is of
interest that there were no outright rejections as a result of grafting during anagen
and that hair growth stage should not,
therefore, be invoked as an explanation
for unexpected rejections. Variation in
hair growth stage is, however, an unnecessary variable which can easily be avoided
by plucking the experimental mice and
then waiting for them to return to telogen
before grafting. It should also be obvious
that hair growth, while a convenient graft
22
B. H. SANFORD, H. B. CHASE, S. B. CARROLL AND C. T . ARSENAULT
marker, is not a reliable criterion of graft
survival.
ACKNOWLEDGMENT
We wish to thank Mks. Yvonne Glen of
the Department of Experimental Biology,
Roswell Park Memorial Institute, Buffalo,
N. Y. for her assistance in the pilot study
for this project.
LITERATURE CITED
Argyris, T. S. 1953 Relationship between the
hair growth cycle and wound healing in the
mouse. Ph.D. thesis. Brown University, Providence, R. I.
Billingham, R. E. 1962 Free skin grafting in
mammals. I n : Transplantation of Tissues and
Cells, ed. R. E. Billingham and W. K. Silvers,
pp. 1-26. Wistar Institute Press, Philadelphia.
Billingham, R. E., L. Brent, P. B. Medawar and
E. M. Sparrow 1954 Quantitative studies on
tissue transplantation immunity. I. The survival times of skin homografts exchanged between members of different inbred strains of
mice. Proc. Roy. SOC.Lond., B, 143: 43-58.
Bishop, G. H. 1945 Regeneration after experimental removal of skin i n man. Am. J. Anat.,
76: 153-181.
Chase, H. B. 1954 Growth of the hair. Physiol.
Rev., 34: 113-126.
Chase, H. B., H. Rauch and V. W. Smith 1951
Critical stages of hair development and pigmentation i n the mouse. Physiol. Zool., 24:
1-10.
Collins, H. H. ‘1918 Studies of normal moult
and of artificially induced regeneration of pelage i n Peromyscus. J. Exp. Zool., 27: 73-99.
David, L. T. 1934 Studies in the expression of
genetic hairlessness in the house mouse (Mus
musculus) I, 11, 111, IV. J. Exp. Zool., 68: 501518.
Dry, F. W. 1926 The coat of the mouse (Mus
musculus). J. Genetics, 16: 287-340.
Eaton, G. J. 1959 Waves and patterns of hair
growth in the mouse. Ph.D. thesis. Brown University, Providence, R. I.
Hauschka, T. S., S. T. Grinnell, M. Meagher and
D. B. Amos 1959 Sex-linked incompatibility
of male skin and primary tumors transplanted
to isologous female mice. I n : Genetics and
Cancer, Texas University Press, Austin, Texas.
Pp. 271-294.
PLATE 1
EXPLANATION O F FIGURES
1
Dorsum of C3H mouse with anagen V isograft 60 days post-grafting,
showing thick predominantly white sideways hair.
2
Telogen isograft five days after grafting. Note marked epidermal
breakdown. Harris’ hematoxylin with eosin. X 225.
3
Anagen V isograft nine days after grafting. Follicles are providing
new epi,dermis for the graft surface. Notice the distinct epidermal
hyperpla.sia above the follicles. Harris’ hematoxylin with eosin. X 115.
4
Anagen V isograft 30 days after grafting. Hair follicles have reorganized and are again producing hair. Masson’s trichrome. X 115.
5
Late anagen VI isograft nine days after grafting. Hair follicles are
breaking down and losing contact with dermal papillae while contributing to epidermal repair. Harris’ hematoxylin with eosin. X 115.
6
Late anagen VI isograft 30 days post-grafting. Epidermal repair has
been completed but follicles are rarely seen. Harris’ hematoxylin
and eosin. x 115.
GRAFTING AND HAIR GROWTH CYCLE
Barbara H. Sanford, Herman B. Chase, Sarah B. Carroll and Claudette T. Arsenault
PLATE 1
23
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