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Wound healing in chick embryos in vivo and in vitro
DEVELOPMENTAL
Wound
BIOLOGY,
Healing PAUL
1,
302-326 ( 19%)
in Chick WEISS
AND
Embryos
in Vivo and in Vitro””
A. GEDEON MATOLTSY
Rockefeller Institute,
New York, New York
Accepted May 20, 1959 INTRODUCTION
Wound healing is a rather elementary manifestation of morphogenesis and regeneration. Yet despite its superficial aspect of unity, the healing of a lesion is far from simple. It is the resultant of the combined activities of cells and ground substances from many sources. To be understood, the complex result-the restoration of tissue integrity-must be broken down into its tributary components, each of which must then be studied separately. The major components in the healing of skin were listed by P. Weiss in a recent symposium on the mechanisms of wound healing (Weiss, 1959) about as follows: ( 1) Retraction-mechanical widening, followed later by constriction, of the wound area. (2) Exudation -temporary closure of wound by a blood clot and other exudates. (3) Detachment-disconnection of the epithelial cells around the wound bed from their substratum. (4) Mobilization of the detached cells. (5) Migration-concentric advance of the mobilized cell front over the raw wound; also invasion of the underlying clot and exudate by fibroblasts from the surrounding connective tissue. (6) Proliferation-mitotic multiplication of cells at and behind the advancing front. (7) Deposition of ground substances and reorganization of the connective tissue bed. (8) Stoppage-fusion of the meeting wound edges, like-to-like. (9) Reattachment of the epidermal cells to the reconstituted boundary membrane. ( 10) Consolidation-cessation of reparative growth, followed by remodeling and reintegration of the 1 A preliminary report of the results appeared in Nature 180, 854 ( 1957). 2 The work was partly supported by a grant from the American Cancer Society.
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mended tissue in accordance with tensions, pressures, and physiological interrelations to the old tissues. This list is incomplete; moreover, each category, itself, can ho further subdivided. The very multiplicity of listings, however, illustrates the composite character of the phenomenon. This raises thr fundamental question of whether these manifold component processes are actuated by a common master drive or whether they are trrrly, independent and separable activities. For example, it has often been unplied that since injury entails both migration and growth, somca by-product of the local trauma, referred to as “wound hormone.” engenders a general tissue response of which cell shifts and cell ~nuitiplication would be joint expressions. This supposition is clearly con tradicted by the results described in this paper. As will be shown. the chick embryo cleanly dissociates migration both from the acrrte effects of wound trauma and from proliferation. During attempts to distort the feather tracts of chicks by excising epidermal patches from S-day-old embryos, in the expectation that the feather-forming epidermis would move over the denuded area ( Saunders and Weiss, 1950)) it was noted that in seven out of sixteen cases, the skin gap had remained open into late embryonic stlges. The present experiments following up this earlier observaticm in greater detail have confirmed that clean lesions made in thr, skin (or cornea) of young embryos do not start to heal until a critical age of approximately 10 days of incubation is reached, the drficicncy being due to failure of the migratory component of the healing process. The chick embryo thus furnishes a uniquely favorable object fol, the independent study of epithelial migration. MATERIALS
AND METHODS
Ywo classes of experiments were carried out: (A ) “in cil;o” rxprriments on embryos left to develop in the shell; and (b) “in oifro” experiments on isolated tissue fragments, cultured on plasma clots. Two types of surface epithelia were used: ( 1) epidermis, and (:! ) cornea; with or without injury of the underlying dermis or cornea] strcma. All embryos were White Leghorn stock obtained from a single source. Operations were carried out during the interval from the fourtir to the seventeenth day of incubation. A total of 270 embryos were operated on, 140 with skin wounds and 130 with cornea] wounds, The number of tissue cultures made was 185. Losses due to
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PAUL
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MATOLTSY
early mortality, to accidents during preparation and histological treatment, or to difficulty in identifying the site of the wound, left 25 embryos with skin wounds and 45 embryos with cornea1 wounds in viva, and 44 fragments in vitro, which could be fully evaluated. A. In Vivo Experiments Incubated eggs were prepared according to Price and Fowler (1940), with certain modifications. First, a pinhole is made in the shell over the air sac. Then, a circular cut is made in the shell over the embryo, measuring about 3/ inch across. (For this purpose, the edge of an Arkansas stone disk, mounted on a dental drill, proved to be superior to a circular saw.) After wetting the cut edge with Earle’s solution, the shell membrane is pierced. Through this second hole air enters between the shell membrane and the embryonic membranes and causes the embryo to sag away from the overlying cap of shell; space for this collapse is afforded by the escape of air from the air sac through the first hole. The shell cap is then removed and discarded. The operations proper were carried out through a rent in the chorioallantoic membrane varying in size with the age of the embryo, but usually not more than 1 mm in diameter. Skin lesions were always placed on the dorsal side of the neck; cornea1 lesions, in the area over the pupil. All cornea1 lesions, as well as skin lesions prior to the eighth day, were made by pinching with the tips of a sharply pointed forceps. Skin wounds after the eighth day were made by exposing the neck, bending it, and excising a small circular area with fine scissors. In the younger embryos, the wounds measured about 0.1 mm in diameter and 0.1 mm in depth, whereas in the older ones, they extended to a maximum of 1 mm and 0.5 mm, respectively. In most instances, the site of the wound was marked by a few carbon granules, which could later be identified in the histological preparations. After the operations, the shell holes were sealed with transparent Scotch tape. Despite the unsterile condition of the tape, no infections have occurred. The eggs were then returned to the incubator and sacrificed at various intervals. In one set of experiments involving the cornea, several drops of adult fowl plasma were applied to the wound surface as shield against contact with extraembryonic fluid.
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H. In Vitro Experiments Fragments of skin, approximately 1 mm square, were excised from the region of the neck or back of embryos of 4 days or older. The> fragments, supported on a piece of rayon-acetate netting, were placed in watch glass cultures, according to Fell (1957), on a medium consisting of five parts of adult chicken plasma mixed with three parts of 50% embryonic extract in Earle’s solution. Pricks were made with fine forceps after 1 day in culture, and the specimens were then fixed after 4-6 days more, including one transfer to fresh medium. Cornea in vitro was wounded immediately after explantation. The explant, resting on rayon-acetate netting on coagulated plasma, was then coated with additional blood plasma so that it became sandwiched between plasma layers. This procedure proved far superior to leaving the epithelium exposed. Explants from donors of 5-i-8 days of incubation age consisted of the whole eyes; from 8 to l-1 days, ot corneas with the attached lenses; and from still older embryos, s&l!, of corneas. These cultures were fixed 3 days after the operation. Embryos and cultures were fixed in Bouin’s solution, sectioned serially at 8 p, and stained in hematoxylin-Biebrich scarlet. In a number of cases the cornea was also prepared as whole rnomlt for determining mitotic distribution. TERMINOLOGY
For a description of the sequence of normal developmental stages of epidermis, see Matoltsy (1958). Tl le early epidermis consists of a basal layer of cuboidal cells and a top layer of flattened “periderm” cells. Toward the fourteenth day, multiple layers form, assuming squamous character by the sixteenth day. True, cornification is evident by the eighteenth day. The dermal layer is at first indistinct, b11t Clbout the eighth clay becomes rather clearly delineated against the underlying subdermal connective tissue. which has a much IOOSCI texture and sparser cell population. The cornea1 epithelium, at all stages used, consists of cuboidal cells, covered by a peridermal layer. It rests on a distinct stroma. bounded inwardly by a simple endothelium. The term “healing” will be used to designate epithelial migration over the wound bed. The wound will be considered as “healed”
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PAUL WEISS AND A. GEDEON MATOLTSY
after epithelial margins converging concentrically have closed over the wound bed and the epithelial sheet has regained unbroken continuity (represented as solid bars in Figs. 25). A wound which at the end of observation shows the epithelial border still at the original site of the incision without trace of migration, will be referred to as “nonhealing” (open bars in the graphs). Wounds between this stage and the terminal stage of complete closure will be referred to as “healing in progress” (cross-hatched bars). Cross sections through the wound area near plane C (Fig. 1) will be referred to as “central”; those near the margin at plane T, as “tangential.”
1 c
FIG. 1. tangential.
Diagram
of wound,
indicating
planes
of sectioning.
C, central;
T,
“Incubation age” is counted from the moment the fertilized eggs were placed in the incubator. In the case of tissue cultures, this term refers to the total number of days elapsed, both in vivo and in vitro, since the beginning of incubation. For brevity, we shall identify each case in the text by the sum of two numbers-the first indicating the incubation age at the time of the operation, and the second, the additional period from operation to fixation. RESULTS I. &JRVEY
The in vivo experiments have shown that wound closure in both skin and cornea is absent during the first half of the embryonic per-
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ERIHHYO
iod, the critical date being about the tenth day. Nonhealing prior to the critical stage is due to migratory failure, rather than to any impairment of growth and proliferation, both of which go on during the nonhealing, as well as the healing, period. This continued growth without migratory expansion during the nonhealing phase causes the epidermal cells at the wound edge to pile up instead of being spread over the denuded surface. In embryos kept beyond the nonhealing period into the second half of the embryonic term, healing starts
5
6
7
8
9
IO
12 13 14 I II Days of mcubatmn
-
I 18
FIG. 2. Summary tabulation of skin wounds in nioo. Open bars: cross-hatched bars: healing in progress; solid bars: healed.
nonhealing:
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PAUL
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MATOLTSY
spontaneously at the critical stage and runs its course much as in embryos operated during the later phase. In contrast to the in vizjo experiments, both skin and cornea1 fragments explanted in vitro at any of the tested stages (4 days of incubation and up) exhibited epithelial migration and wound closure either in process or completed. Therefore, the in viva failure of migration during the early period cannot be attributed to immobility of the cells. -I-
Days
FIG. 3.
SUImmary tabulation
6
FIG. 4.
Summary
7
8
tabulation
I
I
I
14
15
16
IO
-
I
I7
I8
I9
of incubation
of cornea1 wounds
9
-
II
12
of skin wounds
in uiuo. Symbols
13
14
in uitro.
I5
as in Fig. 2.
I6
Symbols
as in Fig. 2.
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HEALING
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A listing of all cases evaluated by detailed histological examination is given in Figs. 2-5, each bar representing the period of observation from the day of wounding to the day of sacrifice: solid, complete healing; open, decidedly cross-hatched, healing in nonhealing; progress. As can be seen from Figs. 2 (upper set ), 3. and 5, healing can br complete within 4 days after wounding in cico, and 3 days in I;itro. It is evident, therefore, that the incompleteness of healing in thra cross-hatched cases cannot be ascribed to insufficient observation time, but indicates a delayed start of the migratory process. Furthermore, according to Fig. 2, not a single case of a skin wound fixed prior to the twelfth day of incubation has shown even the beginning of migratory activity. We can, therefore, set the onset of this activit!-
E I :
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PAUL
WEISS
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MATOLTSY
about the tenth or eleventh day. All specimens observed after that date have been found with healing either in progress or completed. Of the specimens with cornea1 wounds in vivo (Fig. 3)) fixed on the thirteenth day, two are nonhealing and two with healing in progress. Considering the variability in the staging by incubation age and the complete healing of all cases by day 14, and allowing again a 3-day requirement for completing closure, we can set the critical age for inception of healing for the cornea likewise at around the tenth or eleventh day. In the series of twenty skin specimens wounded on the fifth day and carried for varying periods beyond the presumed critical stage (Fig. 2, lower set), of nine cases fixed on or after the fourteenth day, six had healed, two showed healing in progress, and only one was nonhealing. Allowing 3 days for regeneration time, it is evident, therefore, that migratory activity, which has been absent during the earlier phase, can start automatically about the eleventh day
FIG. 6. FIG. 7.
Healed skin wound in uiuo, 14 + 4 days. Magnification: X 230. Nonhealing skin wound, 5 + 7 days. Magnification: x 160.
WOUSD
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CHICK
E;\IHHYO
3 I 1
( 14 - :3), in accord with the results of the short-term espcriments with skin and cornea. On the other hand, it is also to be noted that of seven embryos in this series fixed after the eleventh day, but prior to day fourteen, only two were in the process of healing, whereas the other five had not yet started. This suggests that under these conditions, healing does not get underway until about the twelfth day. perhaps because of the longer postoperative period, which wdtl have amplified any slight developmental retardation. At the sarm time, this series proves conclusively the independence of migrator!, healing from the acute effects of wound trauma. The skin fragments wounded in z;itro (Fig. 4) all showed signs 01 healing, those observed prior to the critical in doe period as well as those studied later. The more extensive in dtro series with cornea (Fig. 5 ) likewise demonstrated that migratory wound closure can occur at all tested ages and be complete as early as on the seventh day of incubation. As will be described later, the intermediate group recorded here as unhealed has been accounted for by the presence of connective tissue obstructions resulting from the particular experimental trchniqucs used in these cases. II. DESCRIPTION OF CASES
.A. In Vim Experiments 1. Skin. Figure 6 shows the area of a wound made on the fourteenth day and allowed 4 days for healing ( l-1 + -J:days ) . One notes a slight condensation of the underlying connective tissue which has filled the dermal gap. The epidermal covering has been completeI) restored with signs of hyperplasia and hyperkeratinization. Thus. since 4 days has been sufficient for epidermal cells not only to cove1 the whole wound area, but to proliferate excessively, the time needed for sheer closure of a small wound may be no morP than Z-:3 cla)~s. This picture is characteristic of all later stages as well. It contrasts sharply with the results of operations in earlier stages to be described next. Figure 7 is representative of the seventeen unhealed cases tabulated in Fig. 2. It portrays a 5 + 7 day wound; as stated before. 7 t1ab.s is more than ample for complete wound coverage. Yet no healing has occurred and the lesion has persisted. (One edge of the wolmd is
PAUL
WEISS
AND
A. GEDEON
MATOLTSY
FIG. 8. Margin of nonhealing skin wound in vim, 5 + 4 days. Magnification: X 630. FIG. 9. Margin of nonhealing skin wound, 7 + 4 days. Magnification: x 600.
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marked by two carbon granules, which can be seen in the subdermal connective tissue. ) The loose subdermal connective tissue lies freely exposed. There is an accumulation of spindle cells in the exposed raw surface with condensation of the fibrous matrix, constituting a provisional wound cover. The dermis is distinct and terminates sharply at the former wound edge. The epidermis, likewise, c>ntls with a free margin at the same level. Although in this particular SWtion, the epidermal wound margin appears smooth and inactive: there are many places in which the cells at the free margin have undergone considerable growth and proliferation in sitrt. Figures 8 and 9 show at higher magnifications the free epidermal edges of two wounds of 5 + 4 days and 7 + 4 days, respectively. They show again the concentration of spindle cells on top of the subdermal connective tissue, the rather sharp delineation of the dermal edge of the wound (at arrows) without indications of dermal regerleration or epidermal shift. In addition, however, thev rcvcal mitotic activity in progress in both epidermal and dermal layers, as well as evidence of antecedent growth, which has resulted in the piling up of newly formed epithelial cells at the free margin in the form ot polyplike buds, folds, tubules, and cysts. These configurations, as ~~11 as the fact that the growing rims sometimes double back upon thcmselves, suggest that the proliferating peridermal and epidermal la! prs use their own sheets as substratum. Significantly correlated with this observation is the fact that in practically all cases of this group. the free margin has failed to adhere to the underlying subdermal tissue and protrudes freely over the edge of the dermis, as show,n in Figs. 8 and 9. This lack of adhesion to the connective tisslle substratum is further documented in Fig. 10, which represents a tangential section (T in Fig. 1) through the margin of a wound of 5 + 5 days. Roth the periderm and the epidermal layer, whose outlincls remain rather distinct, take part in proliferation, the former at timc,s more actively. Mitotic activity is folmcl botlr at and behind tilt? moving front. The rate is not perceptibly different from that in otllt‘l. parts df the skin. A cursory examination of total mounts of specimens of 5 + $3days fn Vi00 showed no crowditlg of niitotic figurc,s ill the vicinity of the wound. But no actlul counts 11avc~1we11 nl;~(jt~. During thcb nonhealing period, the cpidermal ~11s arc eitlrer iso diametric or, if elongated, normal to the surface, \vith conforming. that is spherical or oblate, nuclear shapes. After the critical age of I() days. when healing starts, the cells at tlic frctcx c>clge are found tcj
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PAUL
WEISS
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MATOLTSY
have become reoriented parallel to the surface so that the formerly thickened rim tapers, sometimes assuming wedge shape. This we consider as the morphological expression of the advance of the free edge over the raw wound (Fig. 11; 5 + 10 days). In general, the advancing edges of both the dermis and the epidermis are abreast. Whenever one was found ahead of the other, this
FIG. 10. Tangential section (see Fig. 1) through the margin of a nonhealing skin wound, 5 + 5 days. Magnification: x 190. Note lack of adhesion (gap) between marginal epithelium and connective tissue substratum. FIG. 11. Healing in progress in a skin wound in uioo, 5 + 10 days. Magnification: X 570. FIG. 12. Healed skin wound in uivo, 5 + 13 days, Magnification: x 190.
WOUND
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was always the dermis, occasionally by as much as several cell lengths. Thus, as the epidermis advances, it does so invariably on :I substratum of dermal tissue, to which it is closely applied. None of these cases showed the cleft under the epidermal margin noted in the nonhealing phase. Tn all but one of seven cases of 5 + 1:3 or mart’ days, the process of healing has reached its completion with full restoration of epidermal and dermal continuity over the wolmd (Fig. 12; S + 1S days 1. In conclusion, therefore, all these cases demonstrate that whil(~ proliferation proceeds, migratory activity is suspended until at lrast the tenth day of incubation, but is automatically activated at that critical period regardless of how much time has elapsed sinccl thci infliction of the injury. 2. Cornecr. According to the chart, Fig. S, all cornea1 wounds rnad~~ on the tenth day or later were completely healed after 4 days. Figure 1:3 (16 + 4 days) is representative of this group. The formel lesion is smoothly covered, its original margin being indicated both by carbon granules and by plugs of epidermal cells which have fillet1 the shallow incision in the underlying stroma. As in the case of skin. the epithelial wound cover is hyperplastic, indicating that 2 or 3 days would be ample time for epidermis to spread over a wound of tlI(s given size. By contrast with these results, cornea injured during the earlier. nonhealing, phase shows the epithelial wound margins still at the place of the original cut if esamined by the tenth day, and sometimes even as late as the thirteenth day. Figure 1-I (9 + 4 days ‘1 illustrates a representative case. The stroma containing carbon markcLrs lies freely exposed; like subdermal connective tissue in the skill casts, it has become condensed at the surface. The epithclium has remainccl stagnant at the edge of the denuded area. .\s in thrh cast> of skin, it has continued to proliferate. But whereas the proliferating rim of the skin produced outward projections, the growth at the‘ cornc,al margin was directed inward. penetrating thcl lindrrlving stroma in the form of ramified cords, trlh~lles. md c),sts. Thrsra loc;~l infiltrations often reached considerable dimerlsious as (WIT ilS OII the fourth day after wounding ( Fig. 15, 9 +- 4 days ). Bccaus(l of 0111. else of carbon granules, which were sometimes found tancloscbd ilr thesr epithelial ingrowths, the possihility of an irritative c&act of th(l markcxrs had to he tested. Consequently, \I’(’ carrietl out ;I series of
316
PAUL
WEISS
AND
A. CEDEON
MATOLTSY
experiments in which carbon granules or other particles were dusted onto normal cornea1 surfaces. Histological sections of nine cases with carbon granules and one case each with chalk and iron, proved their inertness, as none of them had evoked any growth reaction. Thus, save for the different direction which the growth process at the wound margin takes in the two categories, the results are essentially the same for skin and cornea, migratory activity of the respective epithelia being absent prior to the critical stage about the tenth
FIG. 13. FIG. 14. x 190. FIG. 15. nification: X
x 100. Healed cornea wound in viuo, 16 + 4 days. Magnification: Nonhealing cornea wound in uiuo, 9 + 4 days. Magnification: Margin 190.
of nonhealing
cornea
wound
in uivo,
9 + 4 days.
Mag-
WOIIND
HEALISG
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l9CHHYO
317
day, but becoming fully operative thereafter. Tlic change from tllc’ nonhealing to the healing phase seems to bc sudden, rather than gradual. H. In Vitro Experiments 1. Skin. The fact that explants of skin can heal epithelial wounds its oifro has previously been demonstrated (hlatoltsy, 1955). In om present series (Fig. 4), wounds made in explants 1 day after esplantation, but prior to the tenth day, were not completely healed 4 days later, but healing was in progress in all of them. The fact that healing had begun seems more relevant than that it had not run its full course. The incompleteness of healing can readily be accounted for by mechanical inadequacies noted in our cultures, such as the detachment and retraction of the epidermal layer from its connective tissue bed and blockage or deflection of moving cell sheets by irregularities of the blood plasma clot. The result to be stressed.
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PAUL
WEISS
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MATOLTSY
t-herefore, is that contrary to the in viva experiments, even the youngest cases showed distinct evidence of epithelial migration over the wound, as well as active growth. Such healing in progress is exemplified in Fig. 16 (7 + 1 + 4 days), where the wedge-shaped epidermal front can be seen to have passed well beyond the carbon markings of the original wound border. Since the only completely
18. FIG. 19.
FIG.
Healed Healed
cornea wound in vitro, 13 + 3 days. Magnification: cornea wound in uitro, 4 + 3 days. Magnification:
x 214. x 425.
:3I9
E’K. 20. Healing in progress in a cornea w(~und in citro, 6 + :; (la)-s. Magnification: X 255. FIG. 21. a. Obstructed closure of cornea wocmtl in r;ifro fi + :3 dxya. \I;IKIlificntion: x 124. b. Same specimen at higher m~lfinification: >c 280.
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PAUL
WEISS
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A. GEDEON
MATOLTSY
healed wound among the eight skin explants (Fig, 17; 9 + 1 + 6 days; hyperplastic and excessively keratinized) was of an age which would have shown complete healing even in ~ivo, the results of this series, taken by themselves, cannot be considered crucial in establishing a sharp distinction between the in vivo and in vitro results. Yet, the subsequent experiments with cornea proved fully conclusive in this regard. 2. Cornea. Preliminary experiments revealed that far superior survival of the explanted eye tissues can be obtained if they are sandwiched between plasma layers. The wounds were made at the time of explantation before the outer plasma coat was applied. The portion of the clot that could thus settle in the lesion has at times blocked the union of the advancing wound margins. In other cases, a similar block resulted from the herniation of the lens through the lesion. Except for such diversions, attributable to the special in vitro conditions, cornea1 epithelium proved to be fully capable of both migration and growth at all stages investigated after the fourth day of incubation (see Fig. 5). This is in sharp contrast to the behavior of the same tissue in vivo (Fig. 3). Figures B-21 show samples of cornea1 epithelium after wounding in vitro. In Fig. 18 (13 + 3 days), the site of the lesion is identified by a deep scar in the stroma and a few scattered carbon markers. The stromal layers have not been fully restored. Epithelium, however, has completely covered the wound area and has filled the hiatus left by the incomplete reconstitution of the stroma. Whether the stromal defect is related to the early plugging of the lesion by blood plasma or to lack of resilience of the stroma at this particular stage, cannot be said. In older corneas the stroma scar is more prominent and has often been invaded by ramifying cords with tubular and cystic enlargements from hyperplastic nests of epithelium, such as that illustrated in Fig. 18. Otherwise, the process of wound healing has occurred in essentially the same manner as in the older in vivo cases. Seven corneas explanted on the fourth, fifth, or sixth days, wounded and allowed to proceed for 3 days, were found to have completely healed wounds. A sample case is presented in Fig. 19. The place of the injury is evident by the residual central indentation of the lens, which consequently has assumed the shape of a partial torus. The
WOUND
HE.ALINC
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lCx\IHHYO
32
I
cornea1 epithelium can he seen to have overgrown the wound area completely, filling hyperplastically the funnel-shaped median indentation of the underlying lens. The lens epithelium has also regenerated. The cornea1 stroma, however, has not yet extended over the whole wound region so that, in the center portion, the regeneratcatl cornea1 epithelium lies directly over the lens cpithelium. Paradoxically, in wounds made at stages intermediate betwetln thca sixth and tenth days, healing was listed as incomplete. though dcacidedly in progress (Fig. 5), but this turned out to havca Iwrn wholly incidental to the in z;itro conditions, as is illustrated in Figs. 20 and 21. Figurr, 20 (6 + 3 days) shows the wound edge of the cornc‘a moving in characteristic wedge shape over the exposed lens epithelium, the front of the cornea1 epithelium lagging slightly behind that of the stroma. As outlined earlier, the tangential orientation ot the cells alid of their nuclei is diagnostic for a freely advancing edge. None of the in ~ivo cases of comparable age have been fomid in similar activity. Figure 21, a and b, (6 + 3 days ), rcavcals why migration in C;itro often fails to effect complete wound closure. In this cast, the substance of the lens had erupted through the lesion into the, plasma clot, forming a plug which kept the wound edges apart ( Fig. 41, a). An enlarged picture at a slightly different level ( Fig. 21. 11) shows that the spindle cells of the stroma had likewise pourecl out into the surrounding plasma, leaving the free margin of tlw (@dermis reflected back upon itself. In other casts, a persistent stalk of blood plasma that had seeped into the wound at the time of the operation alld subsequently become populated by mesenchyme ceils From the eye, constituted a similar harrier to epithelial closure,. \\‘(I have no explanation why this occurrence was so frequent in thcx intermediate age group of this series, but in view of the different sizes, proportions, and mechanical properties of the tissues at difFcrent agt’s, one need not postulate more than a trivial constellatiotl of mechanical factors in that age group favorin
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PAUL WEISS AND A. GEDEON MATOLTSY
shifts the attention from the tissue itself to the conditions of its environment. Perhaps the suppression of healing in viva resulted from the exposure of the wound surface to embryonic fluid, as against blood plasma coagulum in vitro. To test this possibility, ten eyes on the fourth day of incubation were exposed in vivo, wounded as usual, and then coated with a drop of coagulating plasma. The embryos were otherwise treated as in the standard in tivo series and were sacrificed after 3 or 4 days. All of them proved to have developed adhesions between the cornea1 wound and the embryonic membranes, accompanied, in cases of perforating lesions, by a collapse of the lens. These transverse blocks, comparable to the ones observed in vitro, would, of course, have precluded a merger of the epithelial wound borders, even if migration had occurred. There was, however, no evidence that migratory activity had even been initiated. Thus, in this crucial regard, the coated corneas in vivo behaved no differently from the freely exposed corneas in vivo at comparable ages, and the outer plasma coat in the in vitro experiments can be discounted as a factor in explaining the marked difference between the in vitro and in vivo results. The in vitro experiments also rule out the assumption of any singular specific substratum as prerequisite for epithelial migration. For instance, in Fig, 20, the cornea1 epithelium moves over the surface of the advancing stroma, whereas in Fig. 19, it has migrated out in direct contact with the lens epithelium, without mediation of the stroma. Moreover, it was immaterial for the results whether the cornea had been explanted with or without underlying lens. DISCUSSION
It is not intended here to present a comprehensive review of the problem of wound healing in general. In line with our introductory comments about the need of breaking the complex process up into separable components, we simply intended to document the unique property of the young chick embryo of dissociating growth and proliferation from migratory activity. The experiments described have done little more than establish the reality of the phenomenon, exploring only in a very limited degree the variables that might be involved. In viuo, the only relevant variable was age. At 10 days of incubation age, some radical change appeared which seemed to endow
nonmigratory skin and cornea with the full capacity for migration. which they then retain throughout life. The crucial event is the inception of migration; for whether and when a wound is actually. closed, depends on incidental circumstances. Barring obstructions. medium-sized uncomplicated wounds in our experiments were fully, covered well within 23 days. For brevity, let us refer to the transition from migratory incapacity to full migratory ability as the “critical change,” and to the tenth day, at which it occrus, as the “critical stage.” What clues, then, do our experiments contain as to the nature of that critical change? First, since the critical stage is the sxw toI both skin and cornea, any structures, conditions, or processes not shared by both tissues can be dismissed as irrelevant. Since sizes. depth, and manner of application of the wormds varied in the different sets of experiments without altering the critical stage, thoscl variables can also be disregarded. The cleft beneath the epidermal margin in nonhealing skin wounds suggests nonadhesion as a possible explanation of migratory failruc. Yet, as no comparable gaps were observed in cornea1 wounds, whtrtl the epithelium at all stages seemed intimately applied to the stromal surface. the significance of this ch~e remains in doubt. Since in the skin experiments dermal regeneration likewise did not start until the critical stage and since the epidermal advance alwav~ remained confined to the area of new dermis, one might have suspected the primary migratory deficiency to lie in the dermis. On tht’ other hand, the cornea is devoid of any’ layer corresponding to dermis, and yet, prior to the critical stage. the epithelial cells fail to move over the present stromal substratum; moreover, after the critical stage they grow just as readily over lens epithelium as over tlrrir own stroma. However, the most crucial evidence against relating tlrcs critical change to a change of the substratum as such lies in the fact that the same epithelium that prior to the critical stage fails to mov.cl over the cornea1 stroma in r;i~jo, does so quite promptly in L;itro. 13!, the same token, the lack of migratory activity cannot he ascribed to an inherent immobility of the epithelial sheet. It has been pointed out in previous papers (e.g.. Weiss. 195~ ) that cell movement depends on three sets of variables: ( 1 ) the properties of the cell, particularly its siirfacc; ( 2) the phySic:ll :lJl(] chemical properties of the substratum; and (:3 1 the properties of the
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ambient medium. Since the facts just mentioned argue against any relevant changes at the critical stage in either the cell or the substratum, one must consider the possibility of a critical change in the fluid medium bathing cells and substratum. If this extraembryonic fluid were to be unfavorable to cell spreading prior to the tenth day, but on that day acquired the property of favoring cell spreading, and if this property is shared by the adult chicken plasma used in the in vitro experiments, the change in epithelial behavior, as well as its synchronism in skin and eye, would be brought one step nearer its explanation. Now, our critical stage of 10 days after incubation coincides with the period in which the major endocrine systems in the chick start to function (Willier, 1955). It is tempting, therefore, to relate the systemic change in wound-healing ability to hormonal influences, provided the latter are reflected in the composition and physicochemical properties of the extraembryonic fluids. The precocious healing ability in vitro would be accounted for by the presence of the full hormonal complement in the culture medium. A relation between the spreading ability of cells and the composition of embryonic juice has been suggested, and fetuin derived from calf embryos has been described as promoting cellular expansion in vitro (Puck, 1958). A promoting effect of glycoxyglycerol or batylsulfate on epithelial healing has likewise been reported (Bodman, 19588). Because of this, we injected into the extraembryonic cavity of embryos wounded on the fourth or fifth day varying amounts of these two substances in nontoxic concentrations. However, no perceptible initiation of wound healing could be registered 4 days later. This leaves the problem in the same fragmentary state as before, but perhaps indicates the direction in which further experimentation may be profitable. The fact that the extensive literature on embryonic grafts in the chick makes no mention of the inability of early skin to heal, is puzzling. Either it has escaped notice because the embryos were kept beyond the critical stage, or the grafting process itself introduces radically different conditions not duplicated in our experiments. Specific reference to embryonic skin migration in a context in which the occurrence of migration is highly relevant, is found in recent articles by Amprino and Camosso (1958) and Bell et al. (1959). Although some of their statements about healing age in
WOUND
HEALING
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limb epidermis are at variance with our experience with uncomplicated wounds in epithelia of other body regions, and some of their illustrations can readily be interpreted in terms of the criteria given in our present paper, we have no suggestion to offer for a resolution of the points of difference. SUMMARY
The faculty of chick embryos for healing skin and cornea1 wolmtls \laries significantly with age. After lesions in viuo, proliferation of epithelial cells proceeds, bitt migration over the wound is absent until at least the tenth day of in-cubation, at which date it automatically starts regardless of ho\\. much time has elapsed since the injury. This rules out any of thr, immediate traumatic effects of wounding as being causally related lo migration. When cultivated on a plasma clot in uitro, however, both skin and cornea fragments are capable of migratory wound healing at all ages. This proves that the early failure in z;ico is not due to intrinsic immobility of the epithelial cells; nor could it bt definitely conncctctl with cell-to-substratum relations. Tentatively, one could consider thcl critical change to occur in the humoral milieu. as the hormone SLY tern becomes active about that time. The remarkable property of dissociating growth and proliferation from migratory activity, exhibited during the first half of the developmental period, makes the chick embryo uniquely sllitablc fm of thr conmore penetrating analysis of the relative independence poncnts of wolmd healing. REFERENCES IL, and CAAIOSSO, RI. ( 1958). An&i spc’riownt:di dcllo svill~pp~~ dell’ala nell’embrione di polio. Wilhelm Roux Arch. EntlcicklIlnjisrnr,c/l. Or&ut. 150, 509541. BELL, E., and KAIGHN, M. E., and FESSENDEN, L. 11. (1959). The role of mty,(ldermal and ectodermal components in the tlrwlopment of the chick Iinlb Develop. L3iol. 1, 101-124. HODMAN, J. ( 1958). Personal communication. FELL, H. B. ( 1957). The effect of excess vitamin A 011 ctzltllres of ~unbryollic~ chicken skin explanted at different stages ot tlitt‘crcwtiation. Proc. Roy. SOC, B146, 242-256. AhlPHIXO,
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MATOLTSY, A. G. (1955). In vitro wound repair of adult hmxan skin. Anat. Record. 122, 581-588. NATOLTSY, A. G. ( 1958). Keratinization of embryonic skin. J. Invest. Dermatel. 31, 343-346. PRICE, J. W., and FOWLER, E. V. ( 1940). Eggshell cap method of incubating chick embryos. Science 91, 271-272. PUCK, T. T. ( 1958). Growth and genetics of somatic mammalian cells in u&o. J. Cellular Comp. Physiol. 52, Suppl. 1, 287-311. SAUNDERS, J. W., JR., and WEISS, P. (1950). Effects of removal of ectoderm on the origin and distribution of feather germs in the wing of the chick embryo. Anut. Record 108, 93. WEISS, P. (1958). Cell contact. Intern. Reu. Cytol. 7, 391423. WEISS, P. (1959). Biological aspects of wound healing. In “Wound Healing and Tissue Repair” (W. B. Patterson, ed.). Univ. of Chicago Press, Chicago, Illinois. WILLIER, B. H. ( 1955). Ontogeny of endocrine correlation. In “Analysis of Development” (B. H. Willier, P. Weiss, and V. Hamburger, eds.), pp. 574619. Saunders, Philadelphia, Pennsylvania.
Wound
BIOLOGY,
Healing PAUL
1,
302-326 ( 19%)
in Chick WEISS
AND
Embryos
in Vivo and in Vitro””
A. GEDEON MATOLTSY
Rockefeller Institute,
New York, New York
Accepted May 20, 1959 INTRODUCTION
Wound healing is a rather elementary manifestation of morphogenesis and regeneration. Yet despite its superficial aspect of unity, the healing of a lesion is far from simple. It is the resultant of the combined activities of cells and ground substances from many sources. To be understood, the complex result-the restoration of tissue integrity-must be broken down into its tributary components, each of which must then be studied separately. The major components in the healing of skin were listed by P. Weiss in a recent symposium on the mechanisms of wound healing (Weiss, 1959) about as follows: ( 1) Retraction-mechanical widening, followed later by constriction, of the wound area. (2) Exudation -temporary closure of wound by a blood clot and other exudates. (3) Detachment-disconnection of the epithelial cells around the wound bed from their substratum. (4) Mobilization of the detached cells. (5) Migration-concentric advance of the mobilized cell front over the raw wound; also invasion of the underlying clot and exudate by fibroblasts from the surrounding connective tissue. (6) Proliferation-mitotic multiplication of cells at and behind the advancing front. (7) Deposition of ground substances and reorganization of the connective tissue bed. (8) Stoppage-fusion of the meeting wound edges, like-to-like. (9) Reattachment of the epidermal cells to the reconstituted boundary membrane. ( 10) Consolidation-cessation of reparative growth, followed by remodeling and reintegration of the 1 A preliminary report of the results appeared in Nature 180, 854 ( 1957). 2 The work was partly supported by a grant from the American Cancer Society.
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mended tissue in accordance with tensions, pressures, and physiological interrelations to the old tissues. This list is incomplete; moreover, each category, itself, can ho further subdivided. The very multiplicity of listings, however, illustrates the composite character of the phenomenon. This raises thr fundamental question of whether these manifold component processes are actuated by a common master drive or whether they are trrrly, independent and separable activities. For example, it has often been unplied that since injury entails both migration and growth, somca by-product of the local trauma, referred to as “wound hormone.” engenders a general tissue response of which cell shifts and cell ~nuitiplication would be joint expressions. This supposition is clearly con tradicted by the results described in this paper. As will be shown. the chick embryo cleanly dissociates migration both from the acrrte effects of wound trauma and from proliferation. During attempts to distort the feather tracts of chicks by excising epidermal patches from S-day-old embryos, in the expectation that the feather-forming epidermis would move over the denuded area ( Saunders and Weiss, 1950)) it was noted that in seven out of sixteen cases, the skin gap had remained open into late embryonic stlges. The present experiments following up this earlier observaticm in greater detail have confirmed that clean lesions made in thr, skin (or cornea) of young embryos do not start to heal until a critical age of approximately 10 days of incubation is reached, the drficicncy being due to failure of the migratory component of the healing process. The chick embryo thus furnishes a uniquely favorable object fol, the independent study of epithelial migration. MATERIALS
AND METHODS
Ywo classes of experiments were carried out: (A ) “in cil;o” rxprriments on embryos left to develop in the shell; and (b) “in oifro” experiments on isolated tissue fragments, cultured on plasma clots. Two types of surface epithelia were used: ( 1) epidermis, and (:! ) cornea; with or without injury of the underlying dermis or cornea] strcma. All embryos were White Leghorn stock obtained from a single source. Operations were carried out during the interval from the fourtir to the seventeenth day of incubation. A total of 270 embryos were operated on, 140 with skin wounds and 130 with cornea] wounds, The number of tissue cultures made was 185. Losses due to
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early mortality, to accidents during preparation and histological treatment, or to difficulty in identifying the site of the wound, left 25 embryos with skin wounds and 45 embryos with cornea1 wounds in viva, and 44 fragments in vitro, which could be fully evaluated. A. In Vivo Experiments Incubated eggs were prepared according to Price and Fowler (1940), with certain modifications. First, a pinhole is made in the shell over the air sac. Then, a circular cut is made in the shell over the embryo, measuring about 3/ inch across. (For this purpose, the edge of an Arkansas stone disk, mounted on a dental drill, proved to be superior to a circular saw.) After wetting the cut edge with Earle’s solution, the shell membrane is pierced. Through this second hole air enters between the shell membrane and the embryonic membranes and causes the embryo to sag away from the overlying cap of shell; space for this collapse is afforded by the escape of air from the air sac through the first hole. The shell cap is then removed and discarded. The operations proper were carried out through a rent in the chorioallantoic membrane varying in size with the age of the embryo, but usually not more than 1 mm in diameter. Skin lesions were always placed on the dorsal side of the neck; cornea1 lesions, in the area over the pupil. All cornea1 lesions, as well as skin lesions prior to the eighth day, were made by pinching with the tips of a sharply pointed forceps. Skin wounds after the eighth day were made by exposing the neck, bending it, and excising a small circular area with fine scissors. In the younger embryos, the wounds measured about 0.1 mm in diameter and 0.1 mm in depth, whereas in the older ones, they extended to a maximum of 1 mm and 0.5 mm, respectively. In most instances, the site of the wound was marked by a few carbon granules, which could later be identified in the histological preparations. After the operations, the shell holes were sealed with transparent Scotch tape. Despite the unsterile condition of the tape, no infections have occurred. The eggs were then returned to the incubator and sacrificed at various intervals. In one set of experiments involving the cornea, several drops of adult fowl plasma were applied to the wound surface as shield against contact with extraembryonic fluid.
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H. In Vitro Experiments Fragments of skin, approximately 1 mm square, were excised from the region of the neck or back of embryos of 4 days or older. The> fragments, supported on a piece of rayon-acetate netting, were placed in watch glass cultures, according to Fell (1957), on a medium consisting of five parts of adult chicken plasma mixed with three parts of 50% embryonic extract in Earle’s solution. Pricks were made with fine forceps after 1 day in culture, and the specimens were then fixed after 4-6 days more, including one transfer to fresh medium. Cornea in vitro was wounded immediately after explantation. The explant, resting on rayon-acetate netting on coagulated plasma, was then coated with additional blood plasma so that it became sandwiched between plasma layers. This procedure proved far superior to leaving the epithelium exposed. Explants from donors of 5-i-8 days of incubation age consisted of the whole eyes; from 8 to l-1 days, ot corneas with the attached lenses; and from still older embryos, s&l!, of corneas. These cultures were fixed 3 days after the operation. Embryos and cultures were fixed in Bouin’s solution, sectioned serially at 8 p, and stained in hematoxylin-Biebrich scarlet. In a number of cases the cornea was also prepared as whole rnomlt for determining mitotic distribution. TERMINOLOGY
For a description of the sequence of normal developmental stages of epidermis, see Matoltsy (1958). Tl le early epidermis consists of a basal layer of cuboidal cells and a top layer of flattened “periderm” cells. Toward the fourteenth day, multiple layers form, assuming squamous character by the sixteenth day. True, cornification is evident by the eighteenth day. The dermal layer is at first indistinct, b11t Clbout the eighth clay becomes rather clearly delineated against the underlying subdermal connective tissue. which has a much IOOSCI texture and sparser cell population. The cornea1 epithelium, at all stages used, consists of cuboidal cells, covered by a peridermal layer. It rests on a distinct stroma. bounded inwardly by a simple endothelium. The term “healing” will be used to designate epithelial migration over the wound bed. The wound will be considered as “healed”
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PAUL WEISS AND A. GEDEON MATOLTSY
after epithelial margins converging concentrically have closed over the wound bed and the epithelial sheet has regained unbroken continuity (represented as solid bars in Figs. 25). A wound which at the end of observation shows the epithelial border still at the original site of the incision without trace of migration, will be referred to as “nonhealing” (open bars in the graphs). Wounds between this stage and the terminal stage of complete closure will be referred to as “healing in progress” (cross-hatched bars). Cross sections through the wound area near plane C (Fig. 1) will be referred to as “central”; those near the margin at plane T, as “tangential.”
1 c
FIG. 1. tangential.
Diagram
of wound,
indicating
planes
of sectioning.
C, central;
T,
“Incubation age” is counted from the moment the fertilized eggs were placed in the incubator. In the case of tissue cultures, this term refers to the total number of days elapsed, both in vivo and in vitro, since the beginning of incubation. For brevity, we shall identify each case in the text by the sum of two numbers-the first indicating the incubation age at the time of the operation, and the second, the additional period from operation to fixation. RESULTS I. &JRVEY
The in vivo experiments have shown that wound closure in both skin and cornea is absent during the first half of the embryonic per-
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iod, the critical date being about the tenth day. Nonhealing prior to the critical stage is due to migratory failure, rather than to any impairment of growth and proliferation, both of which go on during the nonhealing, as well as the healing, period. This continued growth without migratory expansion during the nonhealing phase causes the epidermal cells at the wound edge to pile up instead of being spread over the denuded surface. In embryos kept beyond the nonhealing period into the second half of the embryonic term, healing starts
5
6
7
8
9
IO
12 13 14 I II Days of mcubatmn
-
I 18
FIG. 2. Summary tabulation of skin wounds in nioo. Open bars: cross-hatched bars: healing in progress; solid bars: healed.
nonhealing:
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spontaneously at the critical stage and runs its course much as in embryos operated during the later phase. In contrast to the in vizjo experiments, both skin and cornea1 fragments explanted in vitro at any of the tested stages (4 days of incubation and up) exhibited epithelial migration and wound closure either in process or completed. Therefore, the in viva failure of migration during the early period cannot be attributed to immobility of the cells. -I-
Days
FIG. 3.
SUImmary tabulation
6
FIG. 4.
Summary
7
8
tabulation
I
I
I
14
15
16
IO
-
I
I7
I8
I9
of incubation
of cornea1 wounds
9
-
II
12
of skin wounds
in uiuo. Symbols
13
14
in uitro.
I5
as in Fig. 2.
I6
Symbols
as in Fig. 2.
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A listing of all cases evaluated by detailed histological examination is given in Figs. 2-5, each bar representing the period of observation from the day of wounding to the day of sacrifice: solid, complete healing; open, decidedly cross-hatched, healing in nonhealing; progress. As can be seen from Figs. 2 (upper set ), 3. and 5, healing can br complete within 4 days after wounding in cico, and 3 days in I;itro. It is evident, therefore, that the incompleteness of healing in thra cross-hatched cases cannot be ascribed to insufficient observation time, but indicates a delayed start of the migratory process. Furthermore, according to Fig. 2, not a single case of a skin wound fixed prior to the twelfth day of incubation has shown even the beginning of migratory activity. We can, therefore, set the onset of this activit!-
E I :
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about the tenth or eleventh day. All specimens observed after that date have been found with healing either in progress or completed. Of the specimens with cornea1 wounds in vivo (Fig. 3)) fixed on the thirteenth day, two are nonhealing and two with healing in progress. Considering the variability in the staging by incubation age and the complete healing of all cases by day 14, and allowing again a 3-day requirement for completing closure, we can set the critical age for inception of healing for the cornea likewise at around the tenth or eleventh day. In the series of twenty skin specimens wounded on the fifth day and carried for varying periods beyond the presumed critical stage (Fig. 2, lower set), of nine cases fixed on or after the fourteenth day, six had healed, two showed healing in progress, and only one was nonhealing. Allowing 3 days for regeneration time, it is evident, therefore, that migratory activity, which has been absent during the earlier phase, can start automatically about the eleventh day
FIG. 6. FIG. 7.
Healed skin wound in uiuo, 14 + 4 days. Magnification: X 230. Nonhealing skin wound, 5 + 7 days. Magnification: x 160.
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( 14 - :3), in accord with the results of the short-term espcriments with skin and cornea. On the other hand, it is also to be noted that of seven embryos in this series fixed after the eleventh day, but prior to day fourteen, only two were in the process of healing, whereas the other five had not yet started. This suggests that under these conditions, healing does not get underway until about the twelfth day. perhaps because of the longer postoperative period, which wdtl have amplified any slight developmental retardation. At the sarm time, this series proves conclusively the independence of migrator!, healing from the acute effects of wound trauma. The skin fragments wounded in z;itro (Fig. 4) all showed signs 01 healing, those observed prior to the critical in doe period as well as those studied later. The more extensive in dtro series with cornea (Fig. 5 ) likewise demonstrated that migratory wound closure can occur at all tested ages and be complete as early as on the seventh day of incubation. As will be described later, the intermediate group recorded here as unhealed has been accounted for by the presence of connective tissue obstructions resulting from the particular experimental trchniqucs used in these cases. II. DESCRIPTION OF CASES
.A. In Vim Experiments 1. Skin. Figure 6 shows the area of a wound made on the fourteenth day and allowed 4 days for healing ( l-1 + -J:days ) . One notes a slight condensation of the underlying connective tissue which has filled the dermal gap. The epidermal covering has been completeI) restored with signs of hyperplasia and hyperkeratinization. Thus. since 4 days has been sufficient for epidermal cells not only to cove1 the whole wound area, but to proliferate excessively, the time needed for sheer closure of a small wound may be no morP than Z-:3 cla)~s. This picture is characteristic of all later stages as well. It contrasts sharply with the results of operations in earlier stages to be described next. Figure 7 is representative of the seventeen unhealed cases tabulated in Fig. 2. It portrays a 5 + 7 day wound; as stated before. 7 t1ab.s is more than ample for complete wound coverage. Yet no healing has occurred and the lesion has persisted. (One edge of the wolmd is
PAUL
WEISS
AND
A. GEDEON
MATOLTSY
FIG. 8. Margin of nonhealing skin wound in vim, 5 + 4 days. Magnification: X 630. FIG. 9. Margin of nonhealing skin wound, 7 + 4 days. Magnification: x 600.
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marked by two carbon granules, which can be seen in the subdermal connective tissue. ) The loose subdermal connective tissue lies freely exposed. There is an accumulation of spindle cells in the exposed raw surface with condensation of the fibrous matrix, constituting a provisional wound cover. The dermis is distinct and terminates sharply at the former wound edge. The epidermis, likewise, c>ntls with a free margin at the same level. Although in this particular SWtion, the epidermal wound margin appears smooth and inactive: there are many places in which the cells at the free margin have undergone considerable growth and proliferation in sitrt. Figures 8 and 9 show at higher magnifications the free epidermal edges of two wounds of 5 + 4 days and 7 + 4 days, respectively. They show again the concentration of spindle cells on top of the subdermal connective tissue, the rather sharp delineation of the dermal edge of the wound (at arrows) without indications of dermal regerleration or epidermal shift. In addition, however, thev rcvcal mitotic activity in progress in both epidermal and dermal layers, as well as evidence of antecedent growth, which has resulted in the piling up of newly formed epithelial cells at the free margin in the form ot polyplike buds, folds, tubules, and cysts. These configurations, as ~~11 as the fact that the growing rims sometimes double back upon thcmselves, suggest that the proliferating peridermal and epidermal la! prs use their own sheets as substratum. Significantly correlated with this observation is the fact that in practically all cases of this group. the free margin has failed to adhere to the underlying subdermal tissue and protrudes freely over the edge of the dermis, as show,n in Figs. 8 and 9. This lack of adhesion to the connective tisslle substratum is further documented in Fig. 10, which represents a tangential section (T in Fig. 1) through the margin of a wound of 5 + 5 days. Roth the periderm and the epidermal layer, whose outlincls remain rather distinct, take part in proliferation, the former at timc,s more actively. Mitotic activity is folmcl botlr at and behind tilt? moving front. The rate is not perceptibly different from that in otllt‘l. parts df the skin. A cursory examination of total mounts of specimens of 5 + $3days fn Vi00 showed no crowditlg of niitotic figurc,s ill the vicinity of the wound. But no actlul counts 11avc~1we11 nl;~(jt~. During thcb nonhealing period, the cpidermal ~11s arc eitlrer iso diametric or, if elongated, normal to the surface, \vith conforming. that is spherical or oblate, nuclear shapes. After the critical age of I() days. when healing starts, the cells at tlic frctcx c>clge are found tcj
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have become reoriented parallel to the surface so that the formerly thickened rim tapers, sometimes assuming wedge shape. This we consider as the morphological expression of the advance of the free edge over the raw wound (Fig. 11; 5 + 10 days). In general, the advancing edges of both the dermis and the epidermis are abreast. Whenever one was found ahead of the other, this
FIG. 10. Tangential section (see Fig. 1) through the margin of a nonhealing skin wound, 5 + 5 days. Magnification: x 190. Note lack of adhesion (gap) between marginal epithelium and connective tissue substratum. FIG. 11. Healing in progress in a skin wound in uioo, 5 + 10 days. Magnification: X 570. FIG. 12. Healed skin wound in uivo, 5 + 13 days, Magnification: x 190.
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was always the dermis, occasionally by as much as several cell lengths. Thus, as the epidermis advances, it does so invariably on :I substratum of dermal tissue, to which it is closely applied. None of these cases showed the cleft under the epidermal margin noted in the nonhealing phase. Tn all but one of seven cases of 5 + 1:3 or mart’ days, the process of healing has reached its completion with full restoration of epidermal and dermal continuity over the wolmd (Fig. 12; S + 1S days 1. In conclusion, therefore, all these cases demonstrate that whil(~ proliferation proceeds, migratory activity is suspended until at lrast the tenth day of incubation, but is automatically activated at that critical period regardless of how much time has elapsed sinccl thci infliction of the injury. 2. Cornecr. According to the chart, Fig. S, all cornea1 wounds rnad~~ on the tenth day or later were completely healed after 4 days. Figure 1:3 (16 + 4 days) is representative of this group. The formel lesion is smoothly covered, its original margin being indicated both by carbon granules and by plugs of epidermal cells which have fillet1 the shallow incision in the underlying stroma. As in the case of skin. the epithelial wound cover is hyperplastic, indicating that 2 or 3 days would be ample time for epidermis to spread over a wound of tlI(s given size. By contrast with these results, cornea injured during the earlier. nonhealing, phase shows the epithelial wound margins still at the place of the original cut if esamined by the tenth day, and sometimes even as late as the thirteenth day. Figure 1-I (9 + 4 days ‘1 illustrates a representative case. The stroma containing carbon markcLrs lies freely exposed; like subdermal connective tissue in the skill casts, it has become condensed at the surface. The epithclium has remainccl stagnant at the edge of the denuded area. .\s in thrh cast> of skin, it has continued to proliferate. But whereas the proliferating rim of the skin produced outward projections, the growth at the‘ cornc,al margin was directed inward. penetrating thcl lindrrlving stroma in the form of ramified cords, trlh~lles. md c),sts. Thrsra loc;~l infiltrations often reached considerable dimerlsious as (WIT ilS OII the fourth day after wounding ( Fig. 15, 9 +- 4 days ). Bccaus(l of 0111. else of carbon granules, which were sometimes found tancloscbd ilr thesr epithelial ingrowths, the possihility of an irritative c&act of th(l markcxrs had to he tested. Consequently, \I’(’ carrietl out ;I series of
316
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experiments in which carbon granules or other particles were dusted onto normal cornea1 surfaces. Histological sections of nine cases with carbon granules and one case each with chalk and iron, proved their inertness, as none of them had evoked any growth reaction. Thus, save for the different direction which the growth process at the wound margin takes in the two categories, the results are essentially the same for skin and cornea, migratory activity of the respective epithelia being absent prior to the critical stage about the tenth
FIG. 13. FIG. 14. x 190. FIG. 15. nification: X
x 100. Healed cornea wound in viuo, 16 + 4 days. Magnification: Nonhealing cornea wound in uiuo, 9 + 4 days. Magnification: Margin 190.
of nonhealing
cornea
wound
in uivo,
9 + 4 days.
Mag-
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HEALISG
IS
CHICK
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day, but becoming fully operative thereafter. Tlic change from tllc’ nonhealing to the healing phase seems to bc sudden, rather than gradual. H. In Vitro Experiments 1. Skin. The fact that explants of skin can heal epithelial wounds its oifro has previously been demonstrated (hlatoltsy, 1955). In om present series (Fig. 4), wounds made in explants 1 day after esplantation, but prior to the tenth day, were not completely healed 4 days later, but healing was in progress in all of them. The fact that healing had begun seems more relevant than that it had not run its full course. The incompleteness of healing can readily be accounted for by mechanical inadequacies noted in our cultures, such as the detachment and retraction of the epidermal layer from its connective tissue bed and blockage or deflection of moving cell sheets by irregularities of the blood plasma clot. The result to be stressed.
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t-herefore, is that contrary to the in viva experiments, even the youngest cases showed distinct evidence of epithelial migration over the wound, as well as active growth. Such healing in progress is exemplified in Fig. 16 (7 + 1 + 4 days), where the wedge-shaped epidermal front can be seen to have passed well beyond the carbon markings of the original wound border. Since the only completely
18. FIG. 19.
FIG.
Healed Healed
cornea wound in vitro, 13 + 3 days. Magnification: cornea wound in uitro, 4 + 3 days. Magnification:
x 214. x 425.
:3I9
E’K. 20. Healing in progress in a cornea w(~und in citro, 6 + :; (la)-s. Magnification: X 255. FIG. 21. a. Obstructed closure of cornea wocmtl in r;ifro fi + :3 dxya. \I;IKIlificntion: x 124. b. Same specimen at higher m~lfinification: >c 280.
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healed wound among the eight skin explants (Fig, 17; 9 + 1 + 6 days; hyperplastic and excessively keratinized) was of an age which would have shown complete healing even in ~ivo, the results of this series, taken by themselves, cannot be considered crucial in establishing a sharp distinction between the in vivo and in vitro results. Yet, the subsequent experiments with cornea proved fully conclusive in this regard. 2. Cornea. Preliminary experiments revealed that far superior survival of the explanted eye tissues can be obtained if they are sandwiched between plasma layers. The wounds were made at the time of explantation before the outer plasma coat was applied. The portion of the clot that could thus settle in the lesion has at times blocked the union of the advancing wound margins. In other cases, a similar block resulted from the herniation of the lens through the lesion. Except for such diversions, attributable to the special in vitro conditions, cornea1 epithelium proved to be fully capable of both migration and growth at all stages investigated after the fourth day of incubation (see Fig. 5). This is in sharp contrast to the behavior of the same tissue in vivo (Fig. 3). Figures B-21 show samples of cornea1 epithelium after wounding in vitro. In Fig. 18 (13 + 3 days), the site of the lesion is identified by a deep scar in the stroma and a few scattered carbon markers. The stromal layers have not been fully restored. Epithelium, however, has completely covered the wound area and has filled the hiatus left by the incomplete reconstitution of the stroma. Whether the stromal defect is related to the early plugging of the lesion by blood plasma or to lack of resilience of the stroma at this particular stage, cannot be said. In older corneas the stroma scar is more prominent and has often been invaded by ramifying cords with tubular and cystic enlargements from hyperplastic nests of epithelium, such as that illustrated in Fig. 18. Otherwise, the process of wound healing has occurred in essentially the same manner as in the older in vivo cases. Seven corneas explanted on the fourth, fifth, or sixth days, wounded and allowed to proceed for 3 days, were found to have completely healed wounds. A sample case is presented in Fig. 19. The place of the injury is evident by the residual central indentation of the lens, which consequently has assumed the shape of a partial torus. The
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cornea1 epithelium can he seen to have overgrown the wound area completely, filling hyperplastically the funnel-shaped median indentation of the underlying lens. The lens epithelium has also regenerated. The cornea1 stroma, however, has not yet extended over the whole wound region so that, in the center portion, the regeneratcatl cornea1 epithelium lies directly over the lens cpithelium. Paradoxically, in wounds made at stages intermediate betwetln thca sixth and tenth days, healing was listed as incomplete. though dcacidedly in progress (Fig. 5), but this turned out to havca Iwrn wholly incidental to the in z;itro conditions, as is illustrated in Figs. 20 and 21. Figurr, 20 (6 + 3 days) shows the wound edge of the cornc‘a moving in characteristic wedge shape over the exposed lens epithelium, the front of the cornea1 epithelium lagging slightly behind that of the stroma. As outlined earlier, the tangential orientation ot the cells alid of their nuclei is diagnostic for a freely advancing edge. None of the in ~ivo cases of comparable age have been fomid in similar activity. Figure 21, a and b, (6 + 3 days ), rcavcals why migration in C;itro often fails to effect complete wound closure. In this cast, the substance of the lens had erupted through the lesion into the, plasma clot, forming a plug which kept the wound edges apart ( Fig. 41, a). An enlarged picture at a slightly different level ( Fig. 21. 11) shows that the spindle cells of the stroma had likewise pourecl out into the surrounding plasma, leaving the free margin of tlw (@dermis reflected back upon itself. In other casts, a persistent stalk of blood plasma that had seeped into the wound at the time of the operation alld subsequently become populated by mesenchyme ceils From the eye, constituted a similar harrier to epithelial closure,. \\‘(I have no explanation why this occurrence was so frequent in thcx intermediate age group of this series, but in view of the different sizes, proportions, and mechanical properties of the tissues at difFcrent agt’s, one need not postulate more than a trivial constellatiotl of mechanical factors in that age group favorin
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shifts the attention from the tissue itself to the conditions of its environment. Perhaps the suppression of healing in viva resulted from the exposure of the wound surface to embryonic fluid, as against blood plasma coagulum in vitro. To test this possibility, ten eyes on the fourth day of incubation were exposed in vivo, wounded as usual, and then coated with a drop of coagulating plasma. The embryos were otherwise treated as in the standard in tivo series and were sacrificed after 3 or 4 days. All of them proved to have developed adhesions between the cornea1 wound and the embryonic membranes, accompanied, in cases of perforating lesions, by a collapse of the lens. These transverse blocks, comparable to the ones observed in vitro, would, of course, have precluded a merger of the epithelial wound borders, even if migration had occurred. There was, however, no evidence that migratory activity had even been initiated. Thus, in this crucial regard, the coated corneas in vivo behaved no differently from the freely exposed corneas in vivo at comparable ages, and the outer plasma coat in the in vitro experiments can be discounted as a factor in explaining the marked difference between the in vitro and in vivo results. The in vitro experiments also rule out the assumption of any singular specific substratum as prerequisite for epithelial migration. For instance, in Fig, 20, the cornea1 epithelium moves over the surface of the advancing stroma, whereas in Fig. 19, it has migrated out in direct contact with the lens epithelium, without mediation of the stroma. Moreover, it was immaterial for the results whether the cornea had been explanted with or without underlying lens. DISCUSSION
It is not intended here to present a comprehensive review of the problem of wound healing in general. In line with our introductory comments about the need of breaking the complex process up into separable components, we simply intended to document the unique property of the young chick embryo of dissociating growth and proliferation from migratory activity. The experiments described have done little more than establish the reality of the phenomenon, exploring only in a very limited degree the variables that might be involved. In viuo, the only relevant variable was age. At 10 days of incubation age, some radical change appeared which seemed to endow
nonmigratory skin and cornea with the full capacity for migration. which they then retain throughout life. The crucial event is the inception of migration; for whether and when a wound is actually. closed, depends on incidental circumstances. Barring obstructions. medium-sized uncomplicated wounds in our experiments were fully, covered well within 23 days. For brevity, let us refer to the transition from migratory incapacity to full migratory ability as the “critical change,” and to the tenth day, at which it occrus, as the “critical stage.” What clues, then, do our experiments contain as to the nature of that critical change? First, since the critical stage is the sxw toI both skin and cornea, any structures, conditions, or processes not shared by both tissues can be dismissed as irrelevant. Since sizes. depth, and manner of application of the wormds varied in the different sets of experiments without altering the critical stage, thoscl variables can also be disregarded. The cleft beneath the epidermal margin in nonhealing skin wounds suggests nonadhesion as a possible explanation of migratory failruc. Yet, as no comparable gaps were observed in cornea1 wounds, whtrtl the epithelium at all stages seemed intimately applied to the stromal surface. the significance of this ch~e remains in doubt. Since in the skin experiments dermal regeneration likewise did not start until the critical stage and since the epidermal advance alwav~ remained confined to the area of new dermis, one might have suspected the primary migratory deficiency to lie in the dermis. On tht’ other hand, the cornea is devoid of any’ layer corresponding to dermis, and yet, prior to the critical stage. the epithelial cells fail to move over the present stromal substratum; moreover, after the critical stage they grow just as readily over lens epithelium as over tlrrir own stroma. However, the most crucial evidence against relating tlrcs critical change to a change of the substratum as such lies in the fact that the same epithelium that prior to the critical stage fails to mov.cl over the cornea1 stroma in r;i~jo, does so quite promptly in L;itro. 13!, the same token, the lack of migratory activity cannot he ascribed to an inherent immobility of the epithelial sheet. It has been pointed out in previous papers (e.g.. Weiss. 195~ ) that cell movement depends on three sets of variables: ( 1 ) the properties of the cell, particularly its siirfacc; ( 2) the phySic:ll :lJl(] chemical properties of the substratum; and (:3 1 the properties of the
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ambient medium. Since the facts just mentioned argue against any relevant changes at the critical stage in either the cell or the substratum, one must consider the possibility of a critical change in the fluid medium bathing cells and substratum. If this extraembryonic fluid were to be unfavorable to cell spreading prior to the tenth day, but on that day acquired the property of favoring cell spreading, and if this property is shared by the adult chicken plasma used in the in vitro experiments, the change in epithelial behavior, as well as its synchronism in skin and eye, would be brought one step nearer its explanation. Now, our critical stage of 10 days after incubation coincides with the period in which the major endocrine systems in the chick start to function (Willier, 1955). It is tempting, therefore, to relate the systemic change in wound-healing ability to hormonal influences, provided the latter are reflected in the composition and physicochemical properties of the extraembryonic fluids. The precocious healing ability in vitro would be accounted for by the presence of the full hormonal complement in the culture medium. A relation between the spreading ability of cells and the composition of embryonic juice has been suggested, and fetuin derived from calf embryos has been described as promoting cellular expansion in vitro (Puck, 1958). A promoting effect of glycoxyglycerol or batylsulfate on epithelial healing has likewise been reported (Bodman, 19588). Because of this, we injected into the extraembryonic cavity of embryos wounded on the fourth or fifth day varying amounts of these two substances in nontoxic concentrations. However, no perceptible initiation of wound healing could be registered 4 days later. This leaves the problem in the same fragmentary state as before, but perhaps indicates the direction in which further experimentation may be profitable. The fact that the extensive literature on embryonic grafts in the chick makes no mention of the inability of early skin to heal, is puzzling. Either it has escaped notice because the embryos were kept beyond the critical stage, or the grafting process itself introduces radically different conditions not duplicated in our experiments. Specific reference to embryonic skin migration in a context in which the occurrence of migration is highly relevant, is found in recent articles by Amprino and Camosso (1958) and Bell et al. (1959). Although some of their statements about healing age in
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HEALING
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limb epidermis are at variance with our experience with uncomplicated wounds in epithelia of other body regions, and some of their illustrations can readily be interpreted in terms of the criteria given in our present paper, we have no suggestion to offer for a resolution of the points of difference. SUMMARY
The faculty of chick embryos for healing skin and cornea1 wolmtls \laries significantly with age. After lesions in viuo, proliferation of epithelial cells proceeds, bitt migration over the wound is absent until at least the tenth day of in-cubation, at which date it automatically starts regardless of ho\\. much time has elapsed since the injury. This rules out any of thr, immediate traumatic effects of wounding as being causally related lo migration. When cultivated on a plasma clot in uitro, however, both skin and cornea fragments are capable of migratory wound healing at all ages. This proves that the early failure in z;ico is not due to intrinsic immobility of the epithelial cells; nor could it bt definitely conncctctl with cell-to-substratum relations. Tentatively, one could consider thcl critical change to occur in the humoral milieu. as the hormone SLY tern becomes active about that time. The remarkable property of dissociating growth and proliferation from migratory activity, exhibited during the first half of the developmental period, makes the chick embryo uniquely sllitablc fm of thr conmore penetrating analysis of the relative independence poncnts of wolmd healing. REFERENCES IL, and CAAIOSSO, RI. ( 1958). An&i spc’riownt:di dcllo svill~pp~~ dell’ala nell’embrione di polio. Wilhelm Roux Arch. EntlcicklIlnjisrnr,c/l. Or&ut. 150, 509541. BELL, E., and KAIGHN, M. E., and FESSENDEN, L. 11. (1959). The role of mty,(ldermal and ectodermal components in the tlrwlopment of the chick Iinlb Develop. L3iol. 1, 101-124. HODMAN, J. ( 1958). Personal communication. FELL, H. B. ( 1957). The effect of excess vitamin A 011 ctzltllres of ~unbryollic~ chicken skin explanted at different stages ot tlitt‘crcwtiation. Proc. Roy. SOC, B146, 242-256. AhlPHIXO,
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MATOLTSY, A. G. (1955). In vitro wound repair of adult hmxan skin. Anat. Record. 122, 581-588. NATOLTSY, A. G. ( 1958). Keratinization of embryonic skin. J. Invest. Dermatel. 31, 343-346. PRICE, J. W., and FOWLER, E. V. ( 1940). Eggshell cap method of incubating chick embryos. Science 91, 271-272. PUCK, T. T. ( 1958). Growth and genetics of somatic mammalian cells in u&o. J. Cellular Comp. Physiol. 52, Suppl. 1, 287-311. SAUNDERS, J. W., JR., and WEISS, P. (1950). Effects of removal of ectoderm on the origin and distribution of feather germs in the wing of the chick embryo. Anut. Record 108, 93. WEISS, P. (1958). Cell contact. Intern. Reu. Cytol. 7, 391423. WEISS, P. (1959). Biological aspects of wound healing. In “Wound Healing and Tissue Repair” (W. B. Patterson, ed.). Univ. of Chicago Press, Chicago, Illinois. WILLIER, B. H. ( 1955). Ontogeny of endocrine correlation. In “Analysis of Development” (B. H. Willier, P. Weiss, and V. Hamburger, eds.), pp. 574619. Saunders, Philadelphia, Pennsylvania.