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Reactions of mammalian fetal tissues to injury. II. Skin

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REACTIONS OF MAMMALIAN F E T A L TISSUES
TO INJURY
11. SKIN
ARTHUR HESS
Department of A,natomy, Washingtm Unniuersity, S G ~ Oof
O ~Medicine,
S t Louis, Missouri
.
TEN FIGURES
The process of regeneration in lower forms, such as Amphibia, usually consists first of a dedifferentiation of the
cells, followed by formation and growth of a blastema and
differentiation of the regenerate (see Needham, ’52). Rose
( ’48) has described the dedifferentiation of epidermal cells
in Triturus and has concluded that these cells contribute the
major portion of a young regeneration blastema. “Dedifferentiation of cells to be reused in regeneration is not a conspicuous process in mammalian skin-healing, and has not
been extensively studied there” (Needham, ’52). Most previous studies of mammalian skin wounds have been performed
on postnatal young or adult animals. I n this investigation,
incisions were made in the skin of mammalian fetuses in an
attempt to determine if the process of dedifferentiation of
the cells in these “more primitive” mammalian forms plays
a more conspicuous role preceding repair than in postnatal
mammals.
MATERIAL AND METHODS
Pregnant guinea pigs were used. The surgical procedures
were the same as those previously described (Hess, ’53). The
age of the fetus was determined from the date of fertilization
‘This investigation waa supported in part by research grant B-341 from the
Institute of Neurological Diseases and Blindness of the National Institutes of
Health, Public Health Service.
435
436
ARTHUR HESS
to that of operation. The technique consisted essentially of
anaesthetizing pregnant guinea pigs, exposing the uterus,
and inserting scissors through the uterine wall. One blade
of the scissors was passed ventral to the vertebral column
and the other over the dorsal surface of the body of the fetus.
The entire vertebral column and overlying tissues were
severed. The effects of the operation on other tissues and
organs will form the basis of subsequent reports.
One hundred and 44 fetuses of 74 pregnant guinea pigs
were subjected to surgery. The unoperated siblings and
normal areas of skin of the operated animals served as controls. The mothers eventually died, aborted or resorbed their
operated young. However, the survival periods and duration
of the experiments were sufficient to allow for healing of the
skin wounds in many instances. The wound repair in 29
fetuses from 23 mothers will form the basis of this report.
The entire fetus was fixed in Bouin's fluid (saturated
aqueous solution of picric acid, 75 ml; 40% formaldehyde,
25 ml; glacial acetic acid, 5 ml). Pieces of the wound area
were then removed and serial sagittal or transverse paraffin
sections 15 p in thickness were prepared through the wound.
The sections were stained with hematoxylin and eosin, toluidine blue, or Bodian's protargol method.
RESULTS
There are at least two variables involved in describing the
results of the present investigation: (1) the age of the fetus
at the time of the operation and (2) the duration of the
experiment from the time of the operation to the recovery of
the fetus. It will be convenient to describe the healing process in relation to time and then to look for marked variations
from the time course of healing in relation to the age of the
fetus. The histories of the operated fetuses are presented
in table 1.
Process of wourcd healhag. The steps in the process of
wound healing have been summarized by Arey ('36) and
Localio, Casale, and Hinton ( '43). The first phase is that
437
SKIN WOUNDS I N MAMMALIAN FETUSES
of traumatic inflammation in which fluid is exuded, leucocytes
congregate, and a fibrin clot is formed after cessation of blood
flow. This is followed by a phase of destruction in which
macrophages and leucocytes free the wound of cellular debris.
These two phases compose a “lag period” in wound repair
and are followed by a phase of constructive changes or proliferation, which i s characterized by attempts at restoration
of epithelial continuity and production of connective tissue
TABLE 1
ANIMAL
NUMBER
AGE OP PETITS
DURATION O F
EXPERIMENT
AT OPERATION
H-CONSIDERED
“HEA~~ED”
days
days
(see tezt)
51
38
5ox
50 sag.
53x
53 sag.
36x
36 sag.
87
77
79
78
33x
33 sag.
75
62
56
57
66
63x
63 sag.
37
29
42
35x
35 sag.
34
24
49
1
1
1
1
1
1
2
2
4
4
4
4
4
4
43
5
5
6
6
6
6
6
6
7
7
7
8
12
12
45
46
46
46
47
47
46
46
37
41
42
43
45
45
40
37
38
37
38
46
46
47
48
41
47
47
45
47
47
H
H
H
H
H
H
H
H
H
H
435
ARTHUR HESS
fibrils. Epithelial continuity is achieved by cell movement,
apparently by amoebism, and aided by contraction of the
wound which serves to reduce the area of an open wound.
Connective tissue fibrils are produced after a proliferation
and infiltration of fibroblasts, other mesenchymal elements,
and capillaries into the wound area to form granulation tissue. The division of the schedule of wound repair into phases
is purely for convenience ; the processes are usually going on
simultaneously and there is much overlap.
F o r the present experiments, a wound is considered healed
if the epithelial layer is again continuous in all the serial
sections through the wound and if the dermal layers show the
presence of regenerated collagen fibers. Obviously, there are
two factors which can alter the schedule of the healing process in any individual animal and cause variations in the time
necessary for repair : (1) individual variation inherent in
the animal itself and ( 2 ) the extent of the wound inflicted in
these experiments. This latter factor, of course, cannot be
kept absolutely uniform in these experiments. The contraction of the tissues after severance of the vertebral column
and the consequent apposition of the wound surfaces are
factors over which there is no control. However, the extent of
the wound is fairly uniform for each animal since the method
of inflicting injury is the same in all cases. I n consequence,
the wounds of several animals heal at slightly different rates
so that the wounds of some animals that undergo rapid repair
can be considered “healed” after 4 days, while others are
still in the process of healing at this time.
The first day after the operation reveals a gaping open
wound with a fibrin clot present and not yet invaded or removed by phagocytes (fig. 1). I n the deeper layers of the
dermis, granulation tissue is forming and can be identified
by the presence of numerous nuclei of fibroblasts migrating
into the wound area. The epithelium has made no attempt
to cover the wound and the epithelial tongue, characteristic of
migrating Malpighian layer epithelial cells, is not present.
I n some animals, the old epithelial cells, traumatized by the
SKIN WOUNDS I N MAMMALIAN FETUSES
439
wound, are still present and not yet sloughed off. In adult
regeneration, Bishop ( ’45) has described a preliminary “precocious ” epithelization followed by the growth of the characteristic epithelial tongue. We have never seen evidence of
such a preliminary epithelial covering in the fetuses studied
here.
I n the two siblings of an experiment lasting two days, there
is poor healing of the wound. Granulation tissue is not yet
well-formed, the original clot is still present, and the epithelium has made no attempt a t migration.
After two days, which can be considered as the “lag
period,” the process of wound repair enters the phase of
proliferation. Constructive changes are effected very rapidly
so that by 4 days, the wounds of some animals can be considered “healed” and all the wounds have undergone marked
attempts a t repair.
In those wounds considered “healed, ” continuity has been
achieved in the epithelial layer. The epithelial cells over
the wound area frequently exhibit “overgrowth” so that
many more layers of epithelial cells are present than normally
(fig. 6). These cells later undergo remodeling and reduction
in degree of stratification. Frequently, the epithelial cells
can be seen migrating under the fibrin clot, which has been
somewhat reduced in extent by invading phagocytes and
fibroblasts and pushed to the surface of the wound by the
underlying regenerating tissues. The granulation tissue is
well developed and connective tissue fibers can be found
running between the fibroblast cell nuclei. In some animals
not yet “healed,” an extensive fibrin clot has prevented the
formation of granulation tissue. Nevertheless, the epithelial
tongue is quite extensive and a marked migration of epithelial cells has occurred, although continuity of the epithelial layer is not achieved. In some animals, apparently slow
healers, the epithelial tongues and granulation tissue are
well-formed and although not completed, the wound is well
on the way to repair.
440
ARTHUR HESS
After 4 days, a larger percentage of animals exhibits
wounds that are “healed.” At 5 days, the epithelial layer,
now complete, has undergone some remodeling but still consists of more layers than adjoining normal areas (fig. 2).
Granulation tissue is well developed. Connective tissue is
present in increasing amounts. I n one animal, a piece of
liver is displaced and located between the cut surfaces of
the skin, preventing continuity of the epithelial layers.
At 6 days, most of the operated animals exhibit “healed”
wounds. The granulation tissue is disappearing as such and
in its place are increasing amounts of connective tissue (fig.
3 ) . However, the remodeling process apparently is slower
and some wounds still present extensive granulation tissue
(figs. 7 and 8). I n one animal with a discontinuous epithelial
layer, the epithelial cells appear piled up at the wound margin,
an appearance that perhaps indicates that the granulation
tissue of this animal is not receptive to the migrating epithelial cells (see Bishop, ’45).
At 7 and 8 days, overgrown epithelial layers and granulation tissue are still present in “healed” wounds (fig. 4).
Wounds not yet healed nevertheless exhibit extensive epithelial migration and granulation tissue formation at this
time.
At 12 days, multiple epithelial layers and extensive granulation tissue are still present. It, therefore, appears that the
remodeling process (reduction of epithelial layers and removal of granulation tissue) persists for a long time. Collagen is deposited in ever increasing amounts until excessive
formation leads to the production of a scar in the extensive
wounds inflicted on the animals of the present experiments.
Regenerated hair follicles are never seen in the wound
area or in immediately adjacent areas (figs. 2, 3 and 4). I n
regions next to these areas, traumatized hair follicles, which
are devoid of pigment and not stained as intensely as unaffected hair follicles, can often be found (fig. 5). The contribution of the hair follicle to regenerating epithelial cells
has been emphasized by Bishop ( ’45). At times, a direct
441
S K I N WOUNDS IN MAMMALIAN FETUSES
connection between the cells of hair follicles apposed to the
wound area and the migrating cells of the regenerating epithelium can be seen (figs. 9 and 10).
E f e c t of age o f f e t u s o n wound healing. If table 1 be examined with emphasis on the age of the fetus a t operation, it
can be seen readily that as many old fetuses (over 45 days)
have wounds considered “healed” as young ones (under 45
days). I n addition, the wounds of the old fetuses do not necessarily take a longer time to heal than those of the young ones.
Thus, the time after the operation, rather than the age of the
fetus, is a far more significant factor for the determination
of wound healing.
DISCUSSION
The extensive wounds inflicted on the fetuses “heal” (according to the aforementioned histological standards) in at
least 4 days, and most are “healed” by 6 days. Incisions extending through the skin, subcutaneous tissue and abdominal
muscles of adult guinea pigs heal in 4-6 days (average 5
days) a t 70”-80”F. and 7-13 days (average 9 days) a t
10O0-11OoF. (Berman, Houser, and Kurtz, ’43). These incisions are sutured together and presumably made with a
scalpel. They are therefore limited in extent, espically when
compared to the extensive wounds inflicted by severance of
the vertebral column and overlying tissues of the fetus with
scissors as performed in the present study in which the wound
ends are not necessarily approximated and a gaping open
wound usually results. Yet the time necessary for healing
of these extensive wounds of the fetuses is the same as o r
even less than the limited incisions in adults. There are at
least 4 factors which could explain this:
1. The greater growth potential of the tissues of young
animals. Du Nouy (’16) has stressed the increased rapidity
of healing which is characteristic of younger individuals.
2. The embryonic tissue fluids liberated at the time of
wounding. The use of embryonic tissue extracts f o r the maintenance, proliferation, and growth of epithelial cells and fibroblasts in tissue culture attests to the fact that such juices
+
+
442
ARTHUR HESS
afford a favorable environment for the multiplication of these
cellular elements, despite the fact that embryonic tissue extracts are said not to contain any specific growth-promoting
hormone ( Carrel and Baker, ' 2 6 ) .
3. The hormonal infiuences on the fetus. Hormones have
been shown to influence the time of wound healing (see Arey,
' 3 6 ) and the hormones of pregnancy might well effect the
course of wound repair in a fetus. More recently, it has been
shown that gestation has a beneficial effect on experimental
skin homografts to the pregnant host (Valone, ' 5 2 ) .
4. The higher temperature of the environment of the fetus
and even perhaps of the fetus itself. Raising of the temperature accelerates wound healing and the beneficial effects of
sympathectomy on wound repair are supposed to be due to
the subsequent rise in temperature of the sympathectomized
part (Arey, ' 3 6 ) . However, Berman et al. ('43) believe that
high body temperatures retard wound healing. The investigations of Brooks and Ducan ('41) deal only with the effect of
temperature on infected wounds and inflammatory reactions
caused by turpentine.
The steps in the healing of fetal skin wounds, although more
rapid, are essentially the same as in postnatal animals. I n
these experiments, there certainly is no extensive change in
cell types, profound dedifferentiation of cells nor marked
variation in the repair process from that of postnatal animals.
I n addition, the observation that, a t least for the fetal ages
( 3 7 4 8 days) used here, the younger fetuses do not heal more
often or more rapidly than older ones seems to indicate that
the cells of the skin of the fetus are fully differentiated at
these ages, and although they may have a greater growth
potential than those of postnatal animals, they certainly do
not possess the ability of dedifferentiating to other more
primitive cell types. Goodwin ('46) has shown that in Triturus and Amblystoma the loss of powers of regeneration both
in ontogeny and in phylogeny is associated with loss of ability
to dedifferentiate. The ability of mammalian epithelial cells
to dedifferentiate is negligible a t all times, prenatally (at
SKIN WOUNDS IN MAMMALIAN FETUSES
443
least for the ages of fetuses employed here) and postnatally.
It thus seems that the phylogenetic influences on the state of
differentiation of cells and the capacity of cells to dedifferentiate are greater than the ontogenetic.
SUMMARY
The vertebral columns of guinea pig fetuses from 3 7 4 8
days of age were severed ilz utero. The skin wounds of 29
fetuses from 23 mothers were studied histologically. Attempts a t repair of the wound occurred in all cases and are
evident by 4 days after the operation. The epithelial layers
have migrated toward each other, extensive granulation tissue
has formed, and regenerated collagen fibers are present. By
4 days, some animals have continuous epithelium and many
regenerated collagen fibers present in the dermis ; these animals are considered “healed.” By 6 days, most of the animals are “healed.” For the extensive wounds inflicted, the
“healing” time is rather rapid. The probable factors accounting for this are discussed. However, the process of
wound healing in a mammalian fetus is the same as that in a
postnatal animal. The wounds of the younger fetuses (under
45 days of age) do not heal more often or more rapidly than
those of older (over 45 days of age) ones. The ability of
mammalian epithelial cells to dedifferentiate is negligible a t
all times, prenatally (at least for the ages of fetuses employed here) and postnatally. It is suggested that the
phylogenetic influences on the state of differentiation of cells
and the capacity of cells to dedifferentiate are greater than
the ontogenetic.
LITERATURE CITED
AREY,L. B. 1936 Wound healing. I’liysiol. Rev.. 26: 32’7-406.
J. K., A. D. ROUSEEA N D w. A. KURTZ 1943 Postoperative scrubbing
BERMAN,
in abdominal surgery. I. Experimental studies. Ann. Surg., lf7: 535543.
BISHOP, G. H. 1945 Regeneration after experimental removal of skin in man.
Am. J. Anat., 76: 153-181.
BROOKS,
B., AND G . D u ~ c a N 1941 The influence of temperature on wounds.
Ann. Surg., 114: 1069-1075.
444
ARTHUR HESS
CARREL, A., AND L. E. BAKER1926 The chemical nature of substances required for cell multiplication. J. Exp. Med., 4 4 : 503-521.
GOODWIN,
P. A. 1946 A comparison of regeneration rates and metamorphosis
in Triturus and Amblystoma. Growth, 10: 75-87.
IIEss, A. 1953 Reactions of mammalian fetal tissues t o injury. I. Surgical
technique. Anat. Rec., 119: 35-52.
LOCALIO,
S. A., W. CASALEA N D J. W. HINTON 1943 Wound healing-experimental
and statistical study. I. Historical. International Abstr. of Surg.
(Supp. t o Surg., Gyn. and Obstet.), 77: 369-375.
NEEDHAM,A. E. 1952 Regeneration and Wound-Healing. John Wiley and
Sons: New York. 152 pp.
DU NOUP, P. L. 1916 Cicatrization of wounds. 111. The relation between the
age of the patient, the area of the wound, and the index of cicatrization. J. Exp. Med., 24: 461-470.
ROSE, S. M. 1948 Epidermal dedifferentiation during blastema formation in
regenerating limbs of Triturus viridescens. J. Exp. Zool., 108: 337361.
VALONE,
J. A. 19.52 The effect of gestation on experimental skin homografts.
Plastic and Reoonstruc. Surg., 10: 354-364.
PLATE 1
EXPLANATION OF FIGURES
All photographs are of paraffin sections through the wound areas in guinea
pig fetal skin stained by hematoxylin and eosin and are X 100, except where
otherwise stated.
Animal no. 51, 45 days fetal age, one day after operation. A gaping open
wound is present with a fibrin clot; granulation tissue is beginning t o form.
The epithelium has made no attempt to cover the wound. X 45.
Animal no. 56, 38 days fetal age, 5 days after operation The epithelial layer
is complete and has more layers than normal. Granulation tissue is well
developed. No hair follicles are present.
Animal no. 63x, 46 days fetal age, 6 days after operation. The epithelital
layer is continuous and exhibits a marked ‘ ‘ overgrowth. ” Granulation tissue is decreasing in amount. NO hair follicles are present.
Animal no. 42, 41 days fetal age, 7 days after operation. “Overgrown”
complete epithelial layers and granulation tissue are present. No hair follicles
are present in the wound area.
Animal no. 77, 41 days fetal age, 4 days after operation. Region adjacent
t o wound area (not shown in photograph). Traumatized hair follicles are
to the left and are devoid of pigment and not stained as intensely as unaffected hair follicles.
PLATE 1
SKIN WOITWDS I N MAMMALIAN FETUSES
ARTHUK HESS
445
PLATE 2
EXPLANATTON O F FIGURES
All photographs are of paraffin sections tlirough the wound areas in guinea
pig fetal skin stained by lieniatoxylin and eosiii and are X 100, except where
otherwise stated.
6
Animal no. 7 7 , 4 1 days fetal age, 4 days a f t e r operation. Tlic epithelial
layer is coiitinuous and exhibits ‘ ‘overgrowth’ ’ of its layers.
7
Animal no. 37, 47 days fetal age, G days after operation. The cpitlirlial
layer is continuous in this region of the wound, but not in other parts of
tho wound (see figs, 9 and 1 0 ) . Extensive grnnulation tissue is still present
i n the wound area.
8 Animal no. 57, 37 days fetal age, G days a f t e r oper:ition. The epithelial
layers are not yet continuous and extensive granulation tissce is present.
9 ant1 10 Animal no. 37, 47 days fetal age, G days after operation. Oppositcl
cnds of the wound area. The wound is t o the right in figure 9 a n d to tlic
left in figure 10. A dircet connection exists between the cells of hair follicles appo:ed to the wound area and tlic migrating cells of the Iegencrating
epithelial tongue.
446
S K I N TVOUSUS I N MAMMALIAN FETUSES
PLATE 2
A K T H U R HESS
447
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