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HUMAN WOUND REPAIR
I. Epidermal Regeneration
GEORGE ODLAND and RUSSELL ROSS
From the Departments of Medicine, Biological Structure, and
Pathology, University of Washington,Seattle, Washington 98105
ABSTRACT
A series of linearly incised superficial skin wounds was made on
the forearms of young adultmale volunteers. Wounds were sampled at
several intervals between 3 hr and 21 days afterwounding, for study
by light and electron microscopy. The light microscopic
observationsshow that regeneration of epidermis in human wounds
conforms chronologically to thatreported for the epidermis in
superficial wound repair in laboratory animals. It is furthershown
that "ruffling" of cell membranes characterizes the cells of the
migrating epidermisin early wound healing. This study reveals that
the basement lamina and hemidesmosomesare established by epidermis
in contact with the fibrin net at the base of early
wounds.Epidermal cells in the wound environment are shown to be
phagocytic. Analysis of thesubmicroscopic cytology of
differentiating and maturing regenerated epidermis reveals that,in
the sequence of events, the formation of filaments, basal lamina,
and desmosomes isfollowed chronologically by evolution of
keratohyalin granules and, subsequently, bykeratinization of the
surface epidermal elements. The entire sequence of migration,
differ-entiation, and ultimate keratinization in the superficial
wounds studied requires 3-5 daysfor completion.
INTRODUCTION
Recent advances in the knowledge of biochem-istry of healing
skin wounds have been attendedby informative analysis of the
submicroscopicstructure of regenerating connective tissue
com-ponents but not of the ultrastructure of regenerat-ing
mammalian epidermis (1). In man, the ab-sence of heavy pelage and,
therefore, a major hairfollicle source for epithelial regeneration
rendersepidermal repair somewhat distinctive from thatof hairy
mammals. To our knowledge, the ultra-structure of regenerating
connective tissue com-ponents has not been analyzed in man. In
orderto fill some of these gaps in information, butmore importantly
to relate the concomitant proc-esses of connective tissue and
epithelial repair,we have undertaken this study of repair of
incised
superficial skin wounds in man. This paper con-cerns itself with
the fine structure of the regenera-tion of human epidermis.
MATERIAL AND METHODS
Several series of superficial linear incisions weremade in the
skin of the flexor aspect of the forearmsof four young adult male
volunteers. Incisions weremade parallel to the long axis of the
forearm. Eachwound was 8-10 mm in length and about 0.5 mm indepth.
In no instance did the incision extend throughthe full thickness of
the dermal connective tissue.In a given subject, wounds were made
at differenttimes such that all wounds representing
differentperiods of repair could be sampled by biopsy at thesame
time and processed simultaneously. In eachseries of wounds in one
subject, individual wounds
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were spaced at least 3 cm apart. In several differentseries, the
wounds were evaluated at time intervalsof 3 and 12 hr, and 1, 2, 3,
5, 7, 14, and 21 daysafter wounding. With a high speed drill fitted
with askin biopsy punch of 2/1 mm diameter, two discs ofskin were
removed from each wound and fixed in1% osmium tetroxide buffered in
s-collidine (pH7.4) at 4°C for 2 hr. Some of the wounds were
post-fixed in neutral buffered formalin. Glutaraldehydefixation was
not employed because the originalblocks fixed in this agent did not
appear to be wellpreserved. The tissues were embedded in Epon
812.The blocks were remounted on aluminum chucks inorder to orient
them to provide full-thickness sectionsof the wound cut
perpendicular to the length of theoriginal incision. Sections 1-1)4
p in thickness weremade from all blocks for light microscopy. They
werestained by the method of Huber with basic fuchsinand methylene
blue (2). Thln sections prepared forelectron microscopy were cut at
thicknesses between600 and 1,000 A and were stained with uranyl
ace-tate solution followed by lead citrate (3). Specimenswere
examined in either a Siemens Elmiskop II, anRCA EMU 3-G, or an
AEI-EM6B electron micro-scope.
OBSERVATIONS
The regeneration of epidermis in mammals has
been described conventionally by histologists to
consist of three phases: mitosis, migration, and
differentiation. No attempt was made to evaluate
mitotic activity.
Light Microscopy
MIGRATION OF EPIDERMIS: Examination of
histological preparations reveals that the earliest
detectable change in the epidermis is a thickening
of the epidermis remaining at the wound margin
adjacent to the early clot. This thickening appears
to result from an increase in the volume of the
epidermal cells adjacent to the wound. At 12 hr,
there has been no apparent movement of epi-
thelium into the wound. At 24 hr, the cut edge
of the epidermis apposed to the clot shows dis-
tinctive cytoplasmic processes that project toward
the clot (Fig. ). Suprabasal cellular elements of
the thickened epidermis at the wound edge show
degenerative characteristics of decreased cell
volume and darkening of the cytoplasm (Fig. 1).
These degenerated cells appear to maintain con-
tact with viable-appearing elements of the epi-thelium. In the
interval between 24 and 48 hr the
epidermis has commenced migrating from theincised edge toward
the center of the wound. In
these small superficial wounds, migration usuallyappears to
proceed symmetrically from both sidestoward the center of the
wound; hence, in profile,the regenerating epidermis has the
appearance ofa pair of wedges directed towards each other atthe
middle of the wound (Fig. 2, insert). In someinstances, migration
from one side of the woundcommences in advance of that from the
otherside so that, at the time of sampling, there ap-pears to be
migration from only one side of thewound. At the leading edge of
the moving epi-thelial sheet which migrates across the
woundsurface, cells have a flat (squamous) contour whenseen in
cross-section. Toward the margin of thewound, away from the leading
edge, stratificationof the epithelial elements is more apparent
(in-serts, Figs. 1 and 2). The advancing epidermis ap-pears to
follow a plane defined by a fibrin netwhich in turn is enclosed by
serous exudate con-taining inflammatory cells. This plane lies
deepto the wound crust.
DIFFERENTIATION OF EPIDERMIS: Epithe-
lization of these small wounds is invariablycomplete by 3 days
and, often, by 2 days afterwounding. After wound closure, the newly
re-generated epidermis becomes highly stratified andthicker than
the normal surrounding epidermis(Fig. 3). Epidermal thickness
decreases to nearlynormal by the 5th-7th day after wounding.
On the 2nd-3rd day the cytoplasm of the re-generated epidermal
cells in the upper layers,near the center of the wound, has evolved
kerato-hyalin granules; this evidence of cellular differ-entiation
and subsequent keratinization appearsfirst at the periphery of the
regenerating epi-thelium near the original wound margin, anddoes
not appear in the center of the wound untilafter the epithelial
sheet has covered the entirewound and has become stratified (Fig.
3).
Electron Microscopy
MIGRATION OF EPIDERMIS: Electron mi-
croscopic analysis of the wound at the time ofearly epidermal
cell migration reveals a sub-stantial increase in the diameter of
cells at theadvancing margin. Ribosomes are relativelyprominent,
but intracellular filaments are sparse.Extensive pseudopodial
projections of cytoplasmat the cortex of such cells have a pale
appearancein comparison to the cytoplasm of unaffectedcells located
peripheral to the wound margin.The cytoplasmic projections contain
no organelles
136 THE JOURNAL OF CELL BIOLOGY VOLUME 39, 1968
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FIGuRE 1 Advancing margin of epidermal sheet in 24 hr old wound.
Basal surface is at left margin;serous exudate occupies lower right
corner of micrograph. Note relatively pale projections of cortical
cyto-plasm of advancing cells, especially at their surfaces
directed toward wound exudate. Numerous phago-cytic inclusions (p)
are seen in cytoplasm of migrating epidermal cells. )iark cell with
pycnotic nucleus (D)is interpreted to be degenerated. Arrows,
desmosomes. X 3,500.
Insert is a photomicrograph showing comparable region
(rectangle) in mirror-inlage relationship tofield depicted in
accompanying electron micrograph. In center, note tongue of
migrating epidermisextending beneath wound crust (left) from
thickened epidermis of wound margin (upper right). X 190.
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FIGURE 2 Migrating stratified epidermal sheet a few cells away
from the advancing edge of a 2 day oldwound. The basal surface lies
upon a mat of fibrin (F). Pale cortical cytoplasmic processes are
presentwithin the stratified epithelial mass. Note numerous
phagocytic inclusions (p) and degenerated cells (D)at the surface
of the epithelium in the wound crust. X 3,500.
Insert is a photomicrograph of a 1.5 ,u section from the same
specimen as that shown in the elec-tron micrograph (area of
rectangle). Note that symmetrical advancing epithelial wedges have
nearlymet at the base of the wound. X 190.
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FIGURE 3 Photomicrograph of the hyperplastic epidermis of a
newly epithelized 3 day old wound.Keratohyalin granules are seen in
the more differentiated upper strata beneath the crust near the
woundmargin, but not in the center of the wound. Arrows indicate
individual degenerated cells in basal andsuprabasal regions.
Careful examination may permit the viewer to perceive pale surface
projections ofcells at the basal surface and between the lower
strata of epidermal cells. The dark outlines of cells in theupper
strata near the center of the wound can be attributed to numerous
desmosomes. X 260.
except rosettes of free ribosomes and a scantydispersion of
tonofilaments. As the advancingedge of the epithelial sheet
subsequently movesacross the wound, these features of the
cellcytoplasm persist and the cells frequently havepale cytoplasmic
projections at their surfaces(Figs. 1 and 2). Pale projections of
the corticalcytoplasm prevail especially at the free surfaces ofthe
epithelial cells (Figs. 4 and 5) but are alsopresent between
stratified epidermal cells severalcells away from the advancing
margin (Figs. 2and 6). The size of the cellular projections
variesfrom that of ordinary microvilli to that of muchlarger blunt
processes, which appear to pervadethe intercellular space (Figs. 2,
5, 6) as well as toprotrude into the milieu of the wound
exudate.
The early migration of the epithelium takes
place in a milieu of homogeneous, dense exudatewhich is
interpreted to be a serous exudate (Fig.1). In the uppermost
portions of the clot thisexudate contains cell and tissue debris,
whereasbeneath the plane of the migrating epithelium theexudate
encloses fibrin strands. The fibrin subse-quently forms a loose
mesh and becomes anincreasingly predominant feature of the
extra-cellular matrix as the wound increases in age from24 to 72 hr
(see Figs. 5 and 7). In the early stagesof regeneration, the
homogeneous serous exudateappears to be the predominant component
ofthe milieu in immediate contact with theadvancing epithelium
(Figs. 4, 5). This exudate,together with strands of fibrin, is
readily observedwithin the markedly widened intercellular
spaceswhich characterize the advancing epithelial sheet
GEORGE ODLAND AND RUSSELL ROSS Human Wound Repair. 1. 139
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FIGURE 4 Cortical cytoplasmic process at advancing edge of
epidermal cell at tip of migrating epidermalsheet in 24 h wound.
Note paucity of filaments and organelles in process. Note that
process is in i-mediate proximity to exudate and erythrocytes in
exudate. Bits of fibrin appear at the lower right.X 10,500.
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FIGURE 5 24 hr wound. Cortical cytoplasmic projections at basal
surface of advancing margin of epi-dermis. This figure is a higher
magnification view of the cell appearing at the extreme lower left
corner ofFig. 1. Note paucity of structured elements in cell
processes; observe cytoplasmnic filaments, ribosomeclusters, and
phagocytic inclusions (p). Notefibrin strands in exudate at lower
left. Arrow indicates internalface of desmosome and associated
filaments. X 17,000.
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(Fig. 6). Occasionally inflammatory cells alsodistend the
intercellular spaces.
FORMATION OF BASEMENT LAMINA: The
only apparent initial support for the advancingmargin of the
migrating epithelial wedge con-sists of the desmosomes between
adjacent epi-dermal cells (Figs. I and 6). Subsequently, thebasal
surface of the epidermal sheet establishescontact with strands of
fibrin in the clot (Fig. 2).At such points the cytoplasm of the
cell appearsdifferentiated and contains small aggregates
offilaments in the otherwise watery cortical cyto-plasm. In these
regions the external milieu ismodified by the appearance of the
lucent zone ofbasement lamina structure at the site of
hemi-desmosomes. The basement lamina and hemi-desmosomes are formed
in the vicinity of fibrinand bear no observable relationship to
eithercollagen or fibroblasts. In those areas where thefibrin net
is discontinuous, the dark componentof the basement lamina may be
seen. With subse-quent replacement of the fibrin by newly
formedcollagen, the hemidesmosomes and basementlamina resume their
normal relationship to thecollagenous stroma (see Figs. 7 and
8).
PHAGOCYTIC INCLUSIONS IN REGENERAT-
ING EPIDERMAL CELLS: A striking character-istic of the
regenerating epithelial cells, which hasnot previously been
recognized in mammalianepidermis, is their capacity to phagocytize
par-ticulate debris from the milieu, including ele-ments of the
same density as the serous exudatetogether with bits of fibrin
which surround thecells (Figs. 2, 5, 6). The intracytoplasmic
in-clusions of fibrin and serum protein are commonlybounded by
membranes. In addition, large ag-gregates of melanin particles are
enclosed bymembranes in the cytoplasm of advancing epi-dermal
cells. A number of cells are seen to con-tain dense, matted
aggregates of tonofilamentswithout apparent attachment to
desmosomes atthe cell periphery.
DEGENERATION OF MIGRATING EPIDER-
MAL CELLS: Some epidermal cells degenerateduring the early stage
of wound repair. Thecharacteristics of cells interpreted to be
degen-
erating (Fig. 1) are pycnotic nuclei with clumped
chromatin, decreased cytoplasmic volume, ag-
gregation of previously differentiated intracellular
filaments, large numbers of ribosomes, and
structures which resemble either vacuoles or
mitochondria without demonstrable cristae. In-
dividual cells within the epidermal sheet mayshow these changes
in the early stages of woundepithelization, whereas most of the
adjacentcells possess the cytoplasmic characteristicsdescribed for
migrating epidermal cells. De-generating cells appear at the
advancing marginof the epithelial tongue (Fig. 1). They
commonlyappear at the surface of the epidermis in the origi-nal
wound margin and continue to appear, in3-day-old wounds, in the
basal and suprabasalregions of the hyperplastic epidermal
elementswhich characterize the fully but recently epithe-lized
wound surface (see Figs. 2 and 3).
DIFFERENTIATION: Cytological features in-terpreted as
differentiation of regenerating epi-dermal cells appear first
during the stage of migrationat a location several cells back from
the advancingepithelial edge, with the concomitant appearanceof
hemidesmosomes and basement lamina over-lying the fibrin network.
The basal epidermalcells evolve broadly developed cisternae of
roughendoplasmic reticulum and numerous clustersand aggregates of
ribosomes in spiral array (Fig.7). The mitochondria tend to have a
larger di-ameter than normally encountered in the epi-dermis, and
many of them are longer than usual.During this period an increasing
number oftonofilaments becomes apparent within thecytoplasm of the
cells.
In the hyperplastic epidermis of 3-4-day-old woundswhich have
completely reepithelized, the inter-cellular space becomes narrowed
to normal di-mensions (Figs. 3, 9, 10). In such wounds,
differ-entiation in the cells of the spinous layer, priorto their
maturation, is marked by the appearanceof many aggregates of three
to eight ribosomes,most of which lie free within the cytoplasm
(Fig.9). Subsequently the clusters of ribosomes are aless prominent
component of the cytoplasm incells which become increasingly filled
insteadwith cytoplasmic filaments, many of which formlarge parallel
strands (Fig. 10). During the earlystage of differentiation, the
epidermal cell showsincrements of tonofilaments in the cytoplasm,
andthe perinuclear region comes to be occupied byprominent
mitochondria with irregularly disposedcristae (Fig. 7). Significant
development of arough endoplasmic reticulum is rarely encoun-tered
in normal epidermis, but it is commonlyfound in the basal and
suprabasal regeneratingepidermal cells during differentiation.
Many of the cells which show signs of early
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FIGURE 6 4 hr wound. This micrograph reveals particularly the
exudate and fibrin strands betweenmigrating epidermal cells,
portions of three of which are shown. Phagocytic inclusions (p) are
aggregatesof matted tonofilaments (f) and exudate with bits of
fibrin. Arrow indicates desmosome. Note also palecortical cytoplasm
of cell in left center. X 15,000.
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FIGURE 7 This micrograph of the basal surface of epidermis in a
2 day old wound demonstrates theformation of hemidesmosomes and the
lucent component of the basement lamina (arrow) in relationshipto
the fibrin upon which the epithelial sheet migrates. It further
reveals features of early differentiationof the cytoplasm as
manifested by numerous large mitochondria, rough endoplasmic
reticulum, spiralarrays and clusters of rihosonmes, and fine
cytoplasmic filaments. X 23,500.
144 THE JOURNAL O CELL BIOLOGY VOLUME 39, 1968
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FIGURE 8 This micrograph of a 21 day old wound shows cytoplasm
of mature basal epidermal cells andalso hemidesmosomes (h) and
basement lamina (arrows) in the presence of regenerated collagen
and afibroblast (F). Bundles of tonofilaments (f) and free
ribosomes occupy the cytoplasm of the epidermal cells.Processes of
melanocytes (M) containing melanin particles pervade the
intercellular spaces. Comparewith Figs. S and 7. X 18,500.
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FIGUIIE 9 Early differentiation in lower strata of epidermal
cells near the original margin of a 2 day oldwound. This figure
depicts changes characteristic of early differentiation in the
basal and suprabasal celllayers. Filaments appear diffusely
scattered throughout the cytoplasm amidst mitochondria, clusters
ofribosomes, and some rough endoplasmic reticulum. Note convoluted
cell membranes and paucity ofdesmosomes (arrows). A small part of
the dense cytoplasm of a degenerated cell appears along the
rightmargin. X 8,600.
differentiation also possess a cortical zone con-taining a few
filaments, a few ribosomes, andsurface projections suggestive of
cortical cyto-plasmic changes described for the migratingepidermal
cells (Figs. 3, 7, 9). These features arecommonly seen in the basal
and suprabasalregion of the hyperplastic epidermis. A promi-nent
feature of the newly differentiating epidermalcells is the
increased number of desmosomes attheir profiles, particularly in
the upper spinouslayers (Figs. 3 and 11). No attempt has been
madeto quantitate these findings.
In the upper layers of newly differentiatingepidermis, there is
no stratum granulosum. Neitherkeratohyalin granules nor
membrane-coatinggranules are encountered (Fig. 11). The
surfacecells retain nuclear remnants as they becomeflattened
beneath the wound crust (Fig. 11).
Residua of phagocytic inclusions and aggre-gates of ribosomes
(and/or glycogen) are retainedwithin these "parakeratotic"
elements. Clumpsof densely stained tonofilaments are also
present.
Subsequent differentiation is manifested bykeratinization. It is
heralded by the appearance ofmembrane-coating granules in cells of
the upperspinous layers (Fig. 12). Keratohyalin granulesevolve in
the filament-filled epidermal cells. Theyappear to be smaller and
more numerous in eachcell than in the more normal epidermal
cellslocated distant from the area of regeneration(Fig. 3).
Overlying these spinous cells are anu-cleate cells of the stratum
corneum. These cellsare characterized by the normal
cytoplasmickeratin pattern and by the thick plasma mem-branes which
mark the morphologic end pointof epidermal cell differentiation
(Fig. 12). In the
146 THE JOURNAL OF CELL BIOLOGY VOLUME 39, 1968
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FIGURE 10 Portions of several maturing epidermal cells from the
upper spinous layers of hyperplasticepidermis of 3 day old wound.
Filaments tend to be aggregated in parallel strands and spare the
peri-nuclear region which is occupied by mitochondria and
endoplasmic reticulum. Mitochondria are largerand more numerous
than in mature cells. Note increased numbers of desmosomes at the
cell profile com-pared with those in Fig. 9. X 6,200.
present series, the 7-day-old wounds show re-generation and
differentiation to be complete andthe newly formed epidermis to be
indistinguish-able from normal epidermis, except for vestigesof
incompletely keratinized cells among the super-ficial layers of the
stratum corneum.
DISCUSSION
In their recent histologic analysis of epitheliza-tion of small
wounds in rabbit skin, Viziam et al.reviewed and clarified the
sequence of epidermalresponses to superficial wounds (4). They
pointedout that during the first 18 hr after wounding
nomicroscopically visible changes are present inthe epidermis
around the wound. However, by21 hr after wounding there is an
increase in boththe size of the cells of the Malpighian layers
aswell as in the number of cell layers. In their
material, epidermal cells commenced migratingacross the wound
surface after 24 hr, and woundepithelization was completed in 72
hr, at whichtime the newly regenerated epidermis coveringthe wound
was thickened and hyperplastic. Theystudied mitosis by the
techniques of colchicinearrest and radioautographic marking with
tri-tiated thymidine, and they concluded that epi-dermal cell
migration and mitosis accounted forthe cellular closure of the
wound and that con-tinued mitotic activity after cessation of
migrationaccounted for the thickened epidermis char-acterizing the
newly epithelized wound.
The observations afforded by the present elec-tron microscopic
analysis do not permit con-clusions regarding the role of mitosis.
However, itis clear that in human wounds the major eventsof
migration of epidermis and the thickening of
GEORGE ODLAND AND RUSSELL Ross Human Wound Repair. I. 147
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FIGURE 11 Upper strata of incompletely differentiated epidermis
near center of a 3 day old newlyepithelized wound. The region
depicted is comparable to that shown in the center of Fig. 6 just
below thewound crust. Note absence of keratohyalin granules and
membrane-coating granules in the cytoplasm ofthe cell shown in the
lower one-third of the micrograph. Observe numerous desmosomes
(arrows). Thecells in the upper two-thirds of the micrograph are
nucleated (N), "parakeratotic," incompletely keratin-ized squamae
with dense cytoplasmic filaments, aggregates of ribosomes and/or
glycogen, and thick,prominent cell membranes. X 20,500.
epidermis after wound closure correspond chrono-logically to the
events described histologically in
rabbit skin wounds by Viziam et al. (4).MIGRATION: This study
has revealed that
sometime between 12 and 24 hr after wounding,the epidermis
commences migration from themargin toward the center of the wound.
Themovement of the epithelial sheet is associated withdistinctive
changes occurring about the surface ofcells of the migrating
epidermis. The surfacephenomena are characterized by evolution
ofcytoplasmic processes at the cell cortex, whichare pale in
contrast to the normal filament-filledcytoplasm of epidermal
cells.
Comparable changes were observed at thebasal surface of
regenerating epidermal cells inamphibian wounds by Singer and
Salpeter (5).
The findings in the present study of human epi-dermis show that
these surface changes occur
on all surfaces of regenerating epidermal cells,and that they
are most prominent at those surfacesexposed to the wound exudate
during epidermalcell migration. Our analysis did not
includecontrols to eliminate the possibility that the vari-
ation in the size of the cortical projections is not afixation
artifact, but Singer and Salpeter notedcomparable changes in
amphibian epidermis fixedby different fixatives (5). The surface
changesare interpreted to be the "ruffling" undulationsof cell
membranes observed by cytologists in awide variety of epithelia and
connective tissuecells in culture. They are generally associated
withcell movement (6 and 7).
The epithelial sheet moves into an inflamma-
148 THE JOURNAL OF CELL BIOLOGY VOLUME 39, 1968
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FIGURE 12 Keratinizing elements of the upper strata of more
completely differentiated epidermis nearthe original wound margin
of a 3 day old wound. Note keratohyalin granules (kh) and filaments
and rela-tive paucity of ribosomes in cell at lower margin of
micrograph. Note membrane-coating granules incytoplasm and in
intercellular space (arrows). Normally keratinized anucleate cells
of stratum corneum(sc) have evolved above the newly formed granular
cell. X 18,000.
tory milieu, and our studies have shown that theadvancing cells
appear to be supported only bydesmosome contact between adjacent
epithelialcells. From histologic analyses various sug-
gestions have been offered regarding the ultimateplane for
epidermal regeneration: (a) that it isdefined by a band of
polymorphonuclear leuko-cytes in the lower portion of the wound
crust (4),(b) that it passes through dermal fibrous tissuebelow the
original wound surface (8), (c) that itis developed by epidermis
which enzymaticallydissolves its way through the wound clot
(9),
or (d) that it moves through a layer of fresh exudateunder the
scab (10). Electron microscopic analysis
of superficial wounds reveals that the advancingepidermis moves
initially through a serous exudatecontaining fibrin and some red
blood cells. In-flammatory cells do not regularly lie in
proximityto the advancing elements of the epidermis. The
viscosity of the exudate could not be determined,but the cells
appeared to be deformed only bystructured elements of the
exudate.
Wessels has pointed out that basal epidermalcells require
attachment to some form of physicalsubstrate in order to undergo
normal mitosis(11). It seems clear from the present morphologic
analysis that the migrating epidermal cellspromptly establish a
physical attachment to thefibrin substrate of the wound.
FORMATION OF BASEMENT LAMINA: Sub-
sequently, within this milieu, the formation of afibrin net
provides the first structural element ofthe temporary wound
architecture that appearsto be utilized by epidermal cells before
theyengage in the establishment of hemidesmosomesand a basement
lamina. This demonstration ofcontact of the epidermal sheet with
the fibrin nettends to strengthen morphologically the observa-
GEORGE ODLAND AND RUSSELL RSS Human Wound Repair. I. 149
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tions of Weiss and his colleagues (12) who pro-posed the concept
of "contact guidance," im-plying that early orientation of
epithelial ele-ments results from the linear orientation of
theirsubstrate. Hay and Revel (13) used electron mi-croscope
radioautographic methods to demonstrateapparent synthesis of
basement lamella proteinsby regenerating salamander epidermis.
Theirfindings may be relevant to the synthesis ofbasement lamina
protein by epidermal cells. Thebroader concept that epithelial
cells synthesizebasement lamina is reviewed and reinforced inthe
work of Pierce and colleagues who utilizedimmunofluorescent
techniques (14). Hence, it isof current interest to note in the
present study thatregenerating human epidermal cells appear
toproduce component structures of basementlamina in the absence of
fibroblasts and that theydo so at a time when the earliest signs of
differ-entiation appear in their cytoplasm; these signsare the
organelles generally associated withsynthesis and secretion of
extracellular proteins.
PHAGOCYTOSIS BY EPIDERMAL CELLS: A
phagocytic capacity of regenerating epidermis wasobserved by
Taban (15), who noticed liquefactionof a blood clot by the wound
epithelium in a sala-mander and who also observed debris of
varioustypes within the epidermal cells. Weber (16)noted epidermis
of regenerating amphibian skinto be phagocytic in the earliest days
after wound-ing and immediately after wound closure. Ourfindings
confirm and refine these observations andextend them to human
epidermis.
DIFFERENTIATION OF EPIDERMIS: In the
present study the cells a short distance back fromthe advancing
epidermal edge show characteris-tics interpreted as early
differentiation, even whilesome of the epidermal cells are
migrating and,according to other investigators, dividing. Theearly
differentiation is accompanied by synthesisof basement lamina and
by formation of newhemidesmosomes and of characteristic
intracellularfilaments. The markers of differentiation, appear-ance
of a rough endoplasmic reticulum and numer-ous free ribosome
clusters, were observed formerlyin regenerating amphibian epidermis
by Singer
and Salpeter (5).The thickening of epithelium newly covering
a
wound surface appears characteristic of 3-and
5-day-old wounds in the material presented herein.
The cellular kinetics in a similar epithelial thick-
ening in rabbit wounds (4) suggests that mitosis
continues at a high rate after wound closure, witha resultant
heaping up of cells still attuned tothe proliferative demands of
wound closure. Thepersistence of pale projections of the cortical
cyto-plasm of cells in the lower layers of epidermis atthis stage
also may be interpreted as response toproliferative demand. The
subsequent appearanceof increased numbers of desmosomes in the
upperlayers may reflect a residual cell surface activity,an
interpretation which is consonant with theconclusions of Vaughn and
Trinkaus (17) that theruffled membranes of epithelial cells are
particu-larly adhesive parts of the cell, and that stableadhesions
tended to be formed rapidly betweencontacting cells of epithelial
cell masses. Thoseauthors noted that the formation of these
adhesionswas associated with a cessation of membraneactivity.
Subsequent features of differentiation in thenewly regenerated
hyperplastic epidermis relateto stratification of cells and
evolution of increasingnumbers of filaments within cells in the
upperstrata of the epidermis. Those cells which flattenor become
squamous appear to do so without theinterposition of keratohyalin
granules. The result-ant squamous cell contains incompletely
con-solidated intracellular filaments in the presence ofvestiges of
cell organelles and condensed nuclearmaterial.
KERATINIZATION: Ultimate differentiationof the regenerating
epidermal cells proceeds nearthe wound margins before
epithelization is com-pleted. This event, keratinization, is
heralded inthe upper strata of the regenerating epidermal cellsby
the appearance of submicroscopic lamellargranules within the
cytoplasm (see membrane-coating granules; reference 18).
Concomitantly,small keratohyalin granules appear in the cyto-plasm
of the nucleated cells of the uppermost lay-ers, and the
characteristic structural image of fullykeratinized stratum corneum
is evolved. The ob-servations made after experimental disruption
ofthe chronology of epidermal cell differentiation bythe technique
of wounding permit the conclusionto be drawn that the appearance of
membrane-coating granules coincides with the evolution
ofkeratohyalin granules and, consequently, ofnormal
keratinization.
The authors would like to acknowledge with thanksthe laboratory
assistance of the following people:Dawn Bockus, Leslie Caldwell,
Janet Demorest,Judy Groombridge, James Huber, Franque Remiing-
150 THE JOURNAL OF CELL BIOLOGY VOLUME 39, 1968
-
ton, and Judy Reed. They would like to thank Mr.Johsel Namkung
for reproducing the plates and Mr.James Huber for preparing the
photomicrographsin Figs. 1-3.
This research was supported in part by the follow-
ing grants from the United States Public Health
Service: DE-01703, HE-02698, and AM-08368.Doctor Ross is the
holder of a Research CareerDevelopment Award from the United States
PublicHealth Service, grant No. DE-9053.
Received for publication 29 March 1968, and in revisedform 20
May 1968.
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